Entomopathogenic nematodes

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COST European cooperation in the field of | scientific and technical research

European Commission

COST 819 Entomopathogenic nematodes

SURVIVAL OF ENTOMOPATHOGENIC NEMATODES

EUR 18855 EN

EUROPEAN COMMISSION

Edith Cresson, Member of the Commission responsible for research, innovation, education, training and youth DG XII/B.1 — RTD actions: Cooperation with non-member countries and international organisations — European Economic Area, COST, Eureka and international organisations Contact: Ms F. Coudert European Commission, rue de la Loi 200 (SDME 1/39) B-1049 Brussels — Tel. (32-2) 29-65507; fax (32-2) 29-64239

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A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server (http://europa.eu.int). Cataloguing data can be found at the end of this publication. Luxembourg: Office for Official Publications of the European Communities, 1999 ISBN 92-828-6885-0 © European Communities, 1999 Reproduction is authorised provided the source is acknowledged. Printed in Belgium PRINTED ON WHITE CHLORINE-FREE PAPER

Cover page: Desiccated Steinernema feltiae, tS-6 strain, in coiled position (courtesy of Mr Aharon Solomon, ARO, Volcani Centre, Bet Dagan, Israel).

COST European cooperation in the field of scientific and technical research

European Commission

m

COST 819 Entomopathogenic nematodes

SURVIVAL OF ENTOMOPATHOGENIC NEMATODES Proceedings of the workshop held at Horticulture Research International, Wellesbourne, Warwick, United Kingdom 24 April 1998

Edited by I. Glazer, P. Richardson, N. Boemare and F. Coudert

Directorate-General Science, Research and Development

1999

EUR 18855 EN

Wright), biophysical (by J. H. Crowe) and molecular aspects of environmental stresses (by K-O. Holmström). The possible links between stress resistance and EPN longevity was discussed by A. Burnell. In the second part, scientists involved directly in different aspects of research related to EPN survival presented their current data and findings. I am confident that the presentations and discussions during the workshop, as well as the wealth ofinformation and ideas included in the present proceedings, will provide the basis for further advancement in understanding of survival mechanisms of EPNs. This will enable to improve EPNs persistence and efficacy as biological control agents. I would like to thank: Paul Richardson and his team at HRI, Wellesbourne, for the excellent local organization, Robin Bedding for chairing the 2nd session, the WG3 coordinator Christine Griffin, the COST-819 Chairman Noel Boemare and secretary Françoise Coudert for helping me with the invitations and administrative aspects and finally all the speakers of whose contributions made this workshop a success. Bet Dagan, Israel, September 8, 1998 Itamar Glazer, Workshop Convenor

LIST of PARTICIPANTS1 Robin BEDDING Mark BLIGHT Noël BOEMARE Mari BOFF John BROWNE Brigitte BRUNEL Ann BURNELL Shulong CHEN Jozef COOSEM ANS Françoise COUDERT John CROWE Ralf-Udo EHLERS Marion FISCHER LE SAUX Paul FITTERS Andrås FODOR Bertold FRIDLENDER Karin GERBER Lonne GERRITSEN Itamar GLAZER David GLEN Christine GRIFFIN Jüerg GRUNDER Nigel HAGUE Richou HAN Birgit HASS Abdelaziz HEDDI Barry HOLLAND Kjell-Ove HOLMSTROM Bill HOMINICK Magdalena JAWORSKA Zsuzsanna KISS Elisabeth KOSCHI ER Igor KRAMER JieLIU Steve LONG Maurice MOENS Alun MORGAN Otto NIELSEN Philippe NORMAND Orsolya ORAVECZ Horolan PANJAV Mavji PATEL Roland PERRY Arne PETERS

CSIRO, Australia University of Paris, France University of Montpellier, France IPO-DLO, The Netherlands National University of Ireland INRA, France National University of Ireland Rijksstation Nematologie Entomologie, Belgium Katholieke University Leuven, Belgium COST Secretariat, Brussels University of California, USA Institut fur Phytopathologie, Kiel, Germany University of Montpellier, France National University of Ireland Eotvos University, Hungary Bio Integrated Technology, Italy Intitut fur Phytomedizin, Austria IPO-DLO, Netherlands Volcani Centre, Israel IACR, Bristol, UK National University of Ireland Federal Research Station, Switzerland University of Reading, UK Christian-Albert University of Kiel, Germany Berlin, Germany INSA, Lyon, France University of Paris, France Uppsala Genetic Centre, Sweden CAB International, UK Academy of Agriculture, Poland Eotvos University, Hungary University of Agricultural Sciences, Austria Swiss Federal Research Station, Switzerland Oregon State University, USA HRI Wellesbourne, UK Rijksstation Nematologie Entomologie, Belgium HRI Wellesbourne, UK Royal Vet. & Agrie. University, Denmark INSA, Lyon, France Eotvos University, Hungary Eotvos University, Hungary Imperial College, London, UK IACR, Rothamsted, UK Institute for Phytopathologie, Germany

Addresses of participants are detailed in the annual report of 1998, and addresses of the speakers are given under the title of each chapter.

Holger PHILIPSEN Andrea PIMENTA Lihong QIU Alex REID Nick RENN Manuele RICCI Paul RICHARDSON Solveig SALINAS-HAUKELAND Cándido SANTIAGO-ALVAREZ Danny SEGAL Nelson SIMÕES Aharon SOLOMON Erko STACKEBRANDT Marek TOMALAK Karolien VAN DEN BERGH Rick VAN DER PAS Tibor VELLAI Deena WELCH Denis WRIGHT

Royal Vet. & Agrie. University, Denmark University of Paris, France CSIRO, Australia CAB International, UK Central Science Laboratory, York, UK Bio Integrated Technology, Italy HRI Wellesbourne, UK Crop Research Institute, Norway Universidad de Cordoba, Spain Tel-Aviv University, Israel Universidade dos Acores, Portugal University of Jerusalem, Israel DSM, Germany Institute of Plant Protection, Poland Katholieke University Leuven, Belgium Koppert B.V., The Netherlands Eotvos University, Hungary HRI Wellesbourne, UK Imperial College, London, UK

Survival of Terrestrial Organisms Roland N. Perry Entomology and Nematology Department, IACR-Rothamsted, Harpenden, Herts. AL5 2JQ, UK. Few environments are consistently favorable and organisms from several groups, including bacteria, fungi, plants and animals, can survive in a dormant state which considerably prolongs the organism's life span and enables it to withstand the rigorous of a fluctuating regime. Dormancy can be separated into diapause and quiescence. Diapause is a state of arrested development and development does not recommence until specific requirements have been satisfied, even if suitable environmental conditions return. Quiescence is a facultative response, involving lowered metabolism, to unpredictable unfavorable environmental conditions and is readily reversible when favorable conditions return; if the stress persists, some organisms can enter a state of cryptobiosis where there is no measurable metabolism. Extreme environmental conditions include absence of water, high temperature, cooling, lack of oxygen and osmotic stress; the types of quiescence induced in organisms by these conditions are termed anhydrobiosis, thermobiosis, cryobiosis, anoxybiosis and osmobiosis, respectively. Most information on dormancy relates to anhydrobiotic survival and this introductory review will focus mainly on this aspect. In many species, survival involves the ability to withstand a combination of environmental extremes; to some extent this is to be expected, as many environmental stresses, such as drought, temperature extremes and increased osmotic pressure, involve removal of body water. Adverse conditions for development also include lack of food; the ability of organisms to enter a dormant state ensures survival during periods of food scarcity and has developed to ensure synchrony with seasonal hosts, especially in parasitic organisms such as the plant-parasitic cyst nematodes (Perry, 1989). The over-wintering diapause of certain species of insects is another example of this aspect of host-parasite interactions. Among non-parasitic terrestrial organisms there is frequently a specialized stage to ensure survival in the absence of food; the dauer larva, a survival stage common to many free-living rhabditid nematodes including Caenorhabditis elegans, is an example of this survival strategy. As population densities of C. elegans increase, the rise in dauer-forming pherornone concentration and the decrease in the food signal as the food supply is exhausted act on the first stage juvenile, resulting in an altered physiology and developmental potential and

culminating in the formation of a third stage dauer larva (Riddle & Albert, 1997). Water availability is one of the most important environmental factors affecting habitat selection and population dynamics of terrestrial organisms. The ability of some terrestrial organisms to survive in a desiccated state is remarkable considering that all living organisms consist of approximately 75% water and for higher animals, such as mammals, the loss of ca 20% water is lethal. Studies on anhydrobiosis originated with Leeuwenhoek's observations in 1702 on the abilities of desiccated animals (probably tardigrades and rotifers) to revive on the addition of water. This apparent resurrection was even cited, during the 19th century, as evidence in support of the theory of 'spontaneous generation'. Forty years after Leeuwenhoek, the Reverend Turbeville Needham described how apparently

lifeless

organisms (now known to be the plant-parasitic nematode Anguina tritici) from wheat galls revived upon the addition of water and some species of plant-parasitic nematodes provide the best examples of long-term survival of environmental extremes. Dormant nematodes are frequently more resistant to control measures, such as nematicides, and this has generated much research interest, resulting in more extensive information about the survival strategies of nematodes (see Evans & Perry, 1976, and Womersley et al., 1998, for reviews) than any other group of terrestrial organisms. Many species show behavioural and/or morphological adaptations to prevent the loss of body water. For example, snails survive dry periods by withdrawing into their shells, some species of diplopods roll up into a ball and some species of earthworms respond to water stress by coiling and secreting a coating of mucus. However, these mechanisms frequently only enable the organism to retain body water and are similar in function to the retention of moulted cuticles as sheaths by the nematodes Heterorhabditis megidis (Menti et al. 1997) and Rotylenchulus reniformis (Gaur & Perry, 1991), which slow the rate of drying of the enclosed nematode but only enable it to survive for the period of time that water loss is slowed. The majority of soil-dwelling nematodes require slow rates of evaporative water loss to become anhydrobiotic and, although slow drying is assured by the physical nature of the soil itself, many species have structural and behavioural attributes which provide additional control of water loss. In those species able to survive anhydrobiotic cryptobiosis, the mechanisms to slow the rate of drying are accompanied by additional adaptations which enable individuals to survive for periods of time far in excess of the period for which water loss can be slowed. Controlling the rate of water loss provides sufficient time for these organisms to undergo biochemical and morphological changes which preserve functional integrity and enable X

survival in the absence of body water. Individual fourth stage juveniles (J4s) of Ditylenchus dipsaci are capable of withstanding rapid dehydration and prolonged periods of desiccation because they have an intrinsic ability to control water loss through a decrease in permeability of the cuticle as it dries (Perry, 1977). In addition, D. dipsaci exhibits behavioural adaptations which enhance control of water loss: they swarm from senescing host tissue and form aggregates, known as eelworm wool , which comprise almost exclusively J4s and, as dehydration proceeds, the nematodes coil before becoming inactive. However, although a controlled rate of water loss may be necessary for survival, it does not, by itself, enable a species to survive prolonged periods of desiccation. One characteristic which is common to many anhydrobiotic organisms, and which may be central to their desiccation survival, is the capacity to synthesize trehalose. Trehalose is thought to preserve structural and functional integrity through the physical replacement of structural water as it is removed during drying. This facilitates the stabilization of biomolecules and biomembranes, as discussed by Crowe (this volume). The accumulation of trehalose by bakers' yeast (Saccharomyces cerevisiae) has been positively correlated with an increased ability to withstand desiccation, and mutants which have lost the ability to synthesise trehalose are unable to survive drying. Genetic engineering has been used to produce transgenic plants, expressing genes for trehalose production, which show increased resistance to drought stress (Holmstrom et al., this volume). In general, free-living mycophagous nematodes preferentially store trehalose at the expense of glycogen and lipid reserves; J4s of D. dipsaci can also sequester trehalose but not at the expense of lipid reserves as other carbohydrates like myo-inositol and ribitol may play important roles (Womersley, 1987). It is possible that genetic engineering for trehalose production by entomopathogenic nematodes may enhance considerably their abilities to survive desiccation (Vellai et al., this volume). However, dauer infective juveniles of the entomopathogenic nematode Steinernema carpocapsae produce trehalose (ca 1% dry wt.) but show no ability to enter cryptobiosis, surviving only as quiescent anhydrobiotes at high relative humidities (Womersley, 1990); thus, the mere ability to synthesise trehalose may not be sufficient to enable organisms to survive. With the completion of the C. elegans genome sequence in 1998, there is the potential to examine the genetic basis of survival strategies of nematodes and to obtain more detailed information about the associated biochemical adaptations. Trehalose is present in the perivitelline fluid of eggs of many cyst nematodes, such as Globodera rostochiensis. This trehalose-containing fluid, together with the eggshell and the cyst wall, surrounds and protects the unhatched second stage juvenile (J2) from desiccation.

Alteration of the eggshell permeability characteristics through the action of natural 'hatching factors' and the concomitant loss of exogenous trehalose from the egg results in an increased susceptibility of the unhatched J2 to desiccation (Perry, 1983). Exogenous application of desiccation protectants is of major importance to enhance the efficacy of organisms used for biological control. The entomophthoralean fungi Zoophthora radkans and Erynia neoaphidis are of considerable interest as biological control agents for the diamondback moth and aphids, respectively. To achieve successful agricultural usage, techniques have been evaluated for drying and storing pathogenic fungi (Pell et al, 1998). Sugars, such as trehalose, maltose, glucose and dextrose, and polyethylene glycol have been used as protectants to enhance desiccation survival. Sugars and polyols also have roles as cryoprotectants or antifreezes, and the production of natural cryoprotectants, such as trehalose, is known to enhance the supercooling abilities of arthropods (Bale, 1989). There are two main strategies by which cold tolerant organisms are able to survive freezing temperatures. They are either freezing-tolerant, surviving ice formation in their tissues, or they are freezing-susceptible, avoiding ice nucleation and maintaining their body fluids as liquid at temperatures well below their melting point by supercooling. Freezing-tolerant animals are usually thought only to survive ice formation in their body cavity and extracellular spaces; intracellular freezing is generally fatal. However, survival of intracellular freezing has been reported in the Antarctic nematode Panagrolaimus davidi (Wharton & Ferns, 1995). Freeze avoidance occurs in the eggs of G. rostochiensis and P. davidi, where the eggshell prevents inoculative freezing (Perry & Wharton, 1995; Wharton & Ramlov, 1995), and in the infective juveniles of the entomopathogenic nematode, Heterorhabditis zealandica, and the soil-dwelling stage of the animal-parasitic nematode, Trichostrongylas colubriformis, where inoculative freezing is prevented by a sheath (Wharton, 1995). Some nematodes are able to survive temperatures as low as -80"C, a temperature at which metabolism is likely to have ceased. High temperature causes destruction of proteins so survival of temperatures above the range at which metabolism operates appears unlikely; however, some organisms are heat tolerant, surviving probably by the production of heat-shock proteins that protect proteins against denaturation (Parseli & Lindquist, 1994). Heat treatment at a sub-lethal temperature produces a subsequent increased tolerance to heat stress in both

Heterorhabditis

bacteriophora (Selvän et al., 1996) and C. elegans (Snutch & Baillie, 1983) and heat-tolerant strains of entomopathogenic nematodes have also been isolated from high temperature

10

habitats (Glazer et al., 1996). It has been possible to outline only briefly the important mechanisms utilised by terrestrial organisms, especially nematodes, to survive the rigours of environmental extremes. In the context of the subject area of this conference session, 'The Survival of Entomopathogenic Nematodes', it is evident that their survival attributes are unremarkable; clearly, if they had the survival abilities of D. dipsaci they would be better suited for commercial use as control agents for insect pests! The use of entomopathogenic nematodes for control purposes, especially in spray formulations, will require abilities to survive drying far in excess of the natural attributes of current commercial strains. In addition, for more widespread use in areas where temperatures are extreme, strains will have to be identified which are able to survive high or low temperature stresses. The identification and selection of strains tolerant to environmental extremes (Solomon et al. and Ricci & Fridlender, this volume), the use of genetic engineering techniques to enhance tolerance (Vellai et al., this volume) and the development of delivery strategies which protect the nematodes from lethal environmental stresses (Wright, this volume) are all important areas of research which are needed to achieve more widespread and effective use of entomopathogenic nematodes for pest management.

References Bale, J.S. (1989). Cold hardiness and overwintering of insects. Agricultural Zoology Reviews 3, 157-192. Evans, A.A.F. & Perry, R.N. (1976). Survival Strategies in Nematodes. In: N.A. Croll (ed.) The Organisation of Nematodes. Academic Press, New York & London, pp. 383 424. Gaur, H.S. & Perry, R.N. (1991). The role of the moulted cuticles in the desiccation survival of adults of Rotylenchulus reniformis. Revue de N^natologie 14, 491-496. Glazer, I., Kozodoi, E., Hashmi, G. & Gaugler, R. (1996). Biological characteristics of the entomopathogenic nematode Heterorhabditis sp: a heat tolerant isolate from Israel. Nematologica 42, 481-492. Leeuwenhoek, A. van (1702). On certain animalcules found in the sediment of gutters of the roofs of houses. Letter 144 in The Select Works of Antony van Leeuwenhoek, Volume 2. Samuel Hoole, London, pp. 207-213. Menti H., Wright, D.J. & Perry, R.N. (1997). Desiccation survival of populations of the entomopathogenic nematodes Steinernema feltiae and Heterorhabditis megidis from Greece and the UK. Journal of Helminthology, 71, 41-46. II

Needham, J.T. (1743). A letter concerning certain chalky tubulous concretions, called malm, with some observations on the farina of Red Lily, and of worms discovered in smutty corn. Philosophical Transactions 42, 634-641. Parseli, D.A. & Lindquist, S. (1994). Heat shock proteins and stress tolerance. In: Morimoto, R.I., Tissires, A. and Georgopoulos, C. (eds) The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 457-495. Pell, J.K., Barker, A .D.P., Clark, S.J., Wilding, N. & Alderson, P.G. (1998). Use of a novel sporulation monitor to quantify the effects of formulation and storage on conidiation by dried mycelia of the entomopathogenic fungus Zoophthora radkans. Biocontrol Science and Technology 8, 13-21. Perry, R.N. (1977). The water dynamics of stages of Ditylenchus dipsaci and

D.

myceliophagus during desiccation and rehydration. Parasitology 75, 45 70. Perry, R.N. (1983).

The effect of potato root diffusate on the desiccation survival of

unhatched juveniles of Globodera rostochiensis. Revue de N ynatologie 6,99 102. Perry, R.N. (1989). Dormancy and hatching of nematode eggs. Parasitology

Today 5,

377-383. Perry, R.N. & Wharton, D.A . (1985). Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode, Globodera rostochiensis. International Journal for Parasitology 15, 441-445. Riddle, D.L. & Albert, P.S. (1997). Genetic and environmental regulation of dauer larvae. In Riddle, D.L., Blumenthal, Τ, Meyer, B.M. and Preiss, J.R. (eds) C. elegans II. Cold Spring Harbor Press, Cold Spring Harbor, pp. 739-768. Selvän, S., Grewal, P.S., Leustek, T. & Gaugier, R. (1996). Heat shock enhances thermotolerance of infective juvenile insect-parasitic nematodes Heterorhabditis bacteriophora (Rhabditida, Heterorhabditidae). Experientia 52, 727-730. Snutch, T.P. & Baillie, D.L. (1983). A lterations in the pattern of gene expression following heat shock in the nematode Caenorhabditis elegans. Canadian Journal of Biochemistry and Cell Biology 61, 480-487. Wharton, D.A . (1995). Cold tolerance strategies in nematodes. Biological Reviews 70, 161-185. Wharton, D.A . & Ferns, D.J. (1995). Survival of intracellular freezing by the A ntarctic nematode Panagrolaimus davidi. Journal of Experimental Biology 198, 1381 -1387. Wharton, D.A . & Ramlov, H. (1995). Differential scanning calorimetry studies on the cysts 12

of the potato cyst nematode Globodera rostochiensis during freezing and melting. Journal of Experimental Biology 198, 2551-2555. Womersley, C.Z. (1987). A re-evaluation of strategies employed by nematode anhydrobiotes in relation to their natural environment. In: Veech, J. and Dickson, D.W. (eds) Vistas on Nematology. Society of Nematologists, Hyattsville, pp. 165-173. Womersley,

C.Z.

(1990).

entomopathogenic

Dehydration

nematodes.

survival

In:

Gaugler

and R.

anhydrobioic and

Kaya,

potential H.K.

of (eds)

Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, pp. 117-130. Womersley, C.Z., Wharton, D.A. & Higa, L.M. (1998). Survival biology. In: Perry, R.N. & Wright, D.J. (eds)

The Physiology

and Biochemistry

of Free-Living

Plant-Parasitic Nematodes. CAB International, Wallingford (in press).

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and

Anhydrobiosis: The Water Replacement Hypothesis John H. Crowe and Lois M. Crowe Section of Molecular and Cellular Biology, University of California, Davis, CA 95616

It is well known that water is required for maintenance of the integrity of biological membranes and proteins. Nevertheless, many organisms are capable of surviving removal of virtually all of their body water. The dry organisms, said to be in a state of "anhydrobiosis" may persist without water for decades, and in some cases centuries (Crowe and Clegg, 1973; Crowe et al. 1992; Potts, 1994). When rehydrated, they rapidly resume active metabolism, often within minutes.

Such organisms often contain large quantities of sugars and sugar alcohols, the

presence of which appears to be associated with their survival in the dry state (Clegg. 1965, 1986; Madin and Crowe, 1975; Eleutherio et ai, 1993; Martin et al., 1986; van Laere, 1989; Potts, 1994). Many anhydrobiotic organisms, including, for example, fungal spores, yeast cells, certain soil-dwelling animals, cysts of the brine shrimp, Artemia, and the desert resurrection plant contain large quantities (as much as 20% of the dry weight) of trehalose (see Crowe et ai, 1984 and 1992 for references). Over twenty years ago (see Crowe and Clegg, 1973) we suggested that these molecules might replace the water around polar residues in labile macromolecular assemblages such as membranes, thus stabilizing these structures in the absence of appreciable amounts of water. We discuss here the evidence that has subsequently accumulated in support of this "water replacement" hypothesis. In the present paper we will concentrate primarily on membranes. However, many of the comments below apply to water soluble proteins, and probably other macromolecules and organelles since there is reason to believe that water replacement occurs in vivo, based on studies of the properties and behavior of water in the encysted embryos of Anemia using nuclear magnetic resonance, microwave dielectrics, and quasi-elastic neutron scattering (Clegg, 1986).

Two stress vectors appear to be involved in destabilizing membranes during drying: fusion and lipid phase transitions. A. Fusion. If membranes are dried without sugars, they can be seen with electron microscopy to undergo extensive fusion. On the other hand, if they are dried in the presence of sucrose or trehalose fusion is completely inhibited.

A remarkably small

amount of the sugars is required to stop fusion— as little as 0.1 g trehalose/g lipid. But much more— as much as 1 g trehalose/g lipid is required to stop leakage of water soluble contents of the vesicles. Thus, it appears that at least one other stress vector is involved. B. Lipid Phase Transitions. The polar headgroups of phospholipids are hydrated; about 10 water molecules are associated with a typical phosphatidylcholine (PC) headgroup. The physical state of this water is not well understood, but their removal has profound consequences for the physical state of the bilayer (reviewed in Crowe et al., 1988). They spatially separate the polar headgroups, and when they are removed, the packing density of the headgroups increases. This increased packing, in turn, leads to increased opportunities for van der Waals' interactions among the hydrocarbon chains. As a result, the temperature at which the chains melt to form the liquid crystalline phase (Tm) increases. For example, fully hydrated egg PC has a transition temperature of about -7°C. When this phospholipid is fully dehydrated Tm rises to about 70° C. Thus, it is in gel phase at room temperature when it is dry, and will pass through the phase transition when it is rehydrated. The significance of this phase transition during rehydration is that when phospholipids pass through such transitions the bilayer becomes transiently leaky (Crowe et al, 1989). Thus, the leakage that normally accompanies this transition must be avoided if the contents of membrane vesicles and whole cells are to be retained. During drying this need not be a problem since Tm is not affected until all the bulk water has been removed. But during rehydration it is a serious problem; the membranes are placed in water and will undergo the phase transition in the presence of excess bulk water. When phospholipids are dried in the presence of sucrose or trehalose Tm is depressed to a remarkable degree. In the case of egg PC mentioned about, Tm is driven down as low as -20°C— at least 10° lower than Tm for the fully hydrated lipid and about 90°C lower than Tm for the lipid

16

dried without trehalose. Thus, such membranes are in liquid crystalline phase, even though they are dry, and will not pass through a phase transition during rehydration. This mechanism has been shown to apply to intact cells (Crowe et ai, 1989; Hoekstra et al, 1992; Leslie et ai, 1994, 1995) as well as the liposomes (reviewed in Crowe and Crowe, 1992) with which it was first described.

For instance, dry yeast cells are known to require

rehydration at elevated temperatures, above about 40°C. Leslie et al (1994, 1995) have shown that membrane lipids in the dry yeast cells have a phase transition between 30-38°C. If the cells are rehydrated at lower temperatures, they leak their contents and are killed during the rehydration. But if they are hydrated at 40°C or warmer they do not leak. Trehalose in the cells, Leslie et al. (1995) established, depresses Tm from about 70°C to 30-38°C. Vitrification. A currently popular suggestion concerning the mechanism of stabilization of biomolecules in the dry state is vitrification, as described by Levine and Slade (1992). Thus, we undertook studies to establish whether vitrification is indeed required for stabilization of dry membranes and found that vitrification is indeed required. It is essential that membranes be maintained in the glassy state during long term storage (Crowe et al, 1994; Sun et al, 1996). For instance, the glass transition temperature (Tg) for sucrose with a residual water content of about 0.02 g water/ g dry wgt is about 35°C. Sun et al. (1996) reported that when liposomes were dried in the presence of sucrose to such a water content, they retained essentially all their contents unless the dry preparations were heated about 35°C.

Above this temperature, the

liposomes underwent massive fusion and leaked their contents to the surrounding medium. The strong correlation between fusion and leakage suggests that the role of vitrification is to immobilize the vesicles, thus limiting fusion.

In keeping with this suggestion is that if the

vesicles were dried in the presence of the sugars to vast excess, and then heated above Tg, leakage and fusion were still strongly inhibited.

In this case, the sugar can act as a spacer

between the vesicles, even at temperatures well above Tg.

17

Direct Interaction. Six categories of evidence suggest that the interaction between sugar and phospholipid is a direct interaction: 1. Infrared Spectroscopy. Studies with this method showthat in dry phospholipids in the presence of trehalose bands assigned to the phosphate head group are strongly affected; the P=0 asymmetric stretch is displaced to lower frequencies. Addition of water to the dry lipid has much the same effect. Further, bands assigned to -OH vibrations in trehalose are also affected by the presence of the lipid (Crowe et al., 1984). The sonicated DPPC vesicles used as a model above illustrate this effect. The dry vesicles have a P=0 asymmetric stretch centered on 1260 cm" , a high frequency in keeping with the fact that they were kept extremely dry. Addition of water results in a decrease in vibrational frequency of this band to 1230 cm"' due to hydrogen bonding of water molecules to the phosphate. Trehalose drives the frequency down to 1235 cm"1 before the sample is heated. After it is heated past Tm there is a further decline in frequency to 1222 cm'1, in keeping with the decrease in calorimetrie Tm following heating. 2. Retention of Water by Trehalose.

A corollary of the finding that the

residual water content of dry lipid-trehalose mixtures does not change with increasing amounts of trehalose (Crowe et al., 1987) is that the presence of the lipid inhibits water binding by trehalose. It is evident in the data that at low mass ratios of trehalose to lipid the trehalose binds far less water than does the same amount of trehalose in the absence of the lipid Crowe et al., 1987). We suggest that this effect is due to an interaction between the water binding -OH groups on the trehalose and the phosphate head group. 3. Competition for Phosphate by Other Molecules.

Europium is known to form an ionic linkage to the phosphate of membrane

phospholipids (Strauss and Hauser, 1986). If sugars interact with the same site, as we suggested above, Europium would be expected to compete with the sugar. Strauss and Hauser (1986) have shown that this is the case; addition of Europium to phospholipid vesicles abolishes the ability of sucrose to preserve them. Subsequently, we have extended these findings to other lipids and other sugars, with similar results (Anchordoguy et al, 1987). Along the same lines, Nakagaki et al. (1992) showed that when DPPC is dried with trehalose, Tm of the phospholipid is about 60°C. However, when the dry preparation is heated above Tm and then cooled, T,„ is seen to decrease to

IS

24°C, good agreement with previous results (Crowe and Crowe, 1988). When small amounts of water were then added, the trehalose was seen with solid state NMR to exhibit increased mobility, while the added water appeared to be bound to the phosphate. Nakagaki et al. (1992) interpreted this result to mean that the added water displaced trehalose from its binding site on the membrane phospholipid. 4. Nuclear Magnetic Resonance. Using solid state NMR Lee et al. (1989) have shown that the phosphate head group of phospholipids dried in the presence of trehalose is rigidly held, while the hydrocarbon chains appear to be freely mobile.

These

findings are clearly consistent with the suggestion that the trehalose interacts with the phosphate. 5. Molecular Modeling. Rudolph et al. (1990) have produced molecular models of DPPC and trehalose which suggest that hydrogen bonding of trehalose to the phosphate is sterically feasible. Their models also demand that the bilayer be expanded to accommodate the trehalose, a finding that is consonant with depression of Tm by the sugar. 6. Comparative Effects of Sugars. One of the predictions of the water replacement hypothesis is that as the molecular weight of the added sugar is increased, a point should be reached where the sugar is too big to intercalate between the polar headgroups. Recent evidence shows that this is indeed the case (Crowe et al, 1996). When DPPC was dried in the presence of glucose, Tm was seen on the first heating to be about 40°C— approximately equal to Tm for the fully hydrated phospholipid. When the lipid was heated repeatedly, Tm did not change. With trehalose, by contrast, Tm was about 60°C on the first heating, but decreased to 24°C after a heating above 60°C. This depressed Tm is stable so long as the phospholipid is not kept below 24°C for very long. But when such samples were chilled to 4°C and held there, Tm was seen to rise to 60°C within a few minutes. DPPC dried with the trisaccharide raffinose also gives a Tm of about 60°C on the first heating, but once the hydrocarbon chains are melted Tm decreases to a remarkable 17°C.

This preparation is

metastable, however, and reverts immediately to a Tm of 60°C when it is chilled below 17°C. The tetrasaccharide stachyose showed no effect on Tm of dry DPPC, despite repeated heating, even to temperatures well over 100°C. With polymers larger than stachyose, no effect on Tm was observed.

1')

In summary of these results, the monosaccharide glucose— the smallest sugar tested— depresses Tm immediately, but with the larger sugars the hydrocarbon chains must be melted before the maximal effect is obtained. We interpret this effect to mean that glucose is able to penetrate between the polar headgroups of gel phase DPPC, while larger sugars cannot do so. When the chains are melted, the larger disaccharides and trisaccharides can penetrate and thus depress Tm. As one might predict, the magnitude of the depression of Tm depends on the size of the sugar. However, the association between the disaccharides and trisaccharides and the bilayer is metastable; trehalose is expelled from the bilayer within minutes below 24°C, and raffinose is expelled immediately when the samples are chilled below 17°C. Does Trehalose Have Special Properties?

We showed some time ago that trehalose is

among the most effective sugars tested at stabilizing liposomes during drying (reviewed in Crowe and Crowe, 1992), although other sugars later proved to be equally effective, particularly at high concentrations.

There has been considerable confusion on this point.

At elevated

concentrations the differences between the sugars tend to disappear, leading to confusion about their relative effectiveness. Nevertheless, numerous workers have reported that trehalose seems to have special abilities in preserving dry and frozen biological materials. Recently, we found that bacteria freeze-dried in the presence of trehalose showed remarkably high survival immediately after freeze drying (Leslie et al, 1994). Furthermore, we found that the bacteria freeze-dried with trehalose retained a high viability even after long exposure to moist air. By contrast, when the bacteria were freeze-dried with sucrose they showed lower initial survival and when they were exposed to moist air, viability deceased rapidly. Using liposomes as a model, we attempted to find a mechanism for the results obtained with bacteria. As with the bacteria, the liposomes exposed to 58% relative humidity rapidly leaked their contents when they were dried with sucrose, but not when they were dried with trehalose. Measurements on fusion of the liposomes showed that they had undergone extensive fusion in the moist air when dried with sucrose, but not with trehalose.

20

120

-120 0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

G WATER/G DRY WEIGHT SUGAR

Fig. 1. State diagram for trehalose compared with that for sucrose. Adapted from Crowe et al., 1996. Examination of the state diagram for trehalose (Crowe, L. et al., 1996), provides an explanation for this effect. Tg for trehalose is much higher than that for sucrose (Fig. 1), in qualitative agreement with previous results of Green and Angeli (1989). [Green and Angeli reported a significantly lower Tg than we found, almost certainly due to an under-estimate of the water contents of their samples.]

As a result, one would expect that addition of small amounts

of water to sucrose by adsorption in moist air would decrease Tg to below the storage temperature, while at the same water content Tg for trehalose would be above the storage temperature. This proved to be the case. Furthermore, Aldous et al. (1995) have suggested an additional interesting property of trehalose, which we were able to confirm. They suggested that since the crystalline structure of trehalose is a dihydrate some of the sugar might, during adsorption of water vapor, be converted to the crystalline dihydrate, thus sparing the remaining trehalose from contact with the water. This suggestion emerged as correct; with addition of

21

small amounts of water the crystalline dihydrate immediately appeared, and Tg for the remaining glassy sugar remained unexpectedly high. We point out, however, that the elevated Tg seen in trehalose is not anomalous, as has been claimed (Green and Angeli, 1989). Indeed, trehalose lies at the end of a continuum of sugars that show increasing T2, although the basis for this effect is not understood.

References Aldous, B.J., Auffret, A.D., and Franks, F. 1995. The crystallisation of hydrates from amorphous carbohydrates. Cryo-Lett. 16:181-186. Anchordoguy, T.J., Rudolph, A.S., Carpenter, J.F., and Crowe, J.H. 1987. Modes of interaction of cryoprotectants with membrane

phospholipids

during

freezing.

Cryobiology

24:324-331. Carpenter, J.F. 1994. Interactions of stabilizers with proteins during freezing and drying. In: Formulation And Delivery of Proteins and Peptides, Washington D.C. American Chemical Society, p. 134-147. Clegg, J.S. 1965. The origin of trehalose and its significance during the formation of encysted dormant embryos of Artemia salina. Comp. Biochem. Physiol. 14:135-143. Clegg, J.S. 1986. The physical properties and metabolic status of Artemia cysts at low water contents: the "water replacement hypothesis".

In: Membranes, Metabolism and Dry

Organisms, edited by A. Carl Leopold, New York: Cornell University Press. Crowe, J.H. and Clegg, J.S. 1973. Anhydrobiosis. Stroudsburg: Dowden, Hutchison and Ross. Crowe, J.H., Spargo, B.J., and Crowe, L.M. 1987. Preservation of dry liposomes does not require retention of residual water. Proc. Nat. Acad. Sci. 84:1537-1540. Crowe, J.H., Leslie, S.B., and Crowe, L.M. 1994. Is vitrification sufficient to preserve liposomes during freeze-drying? Cryobiology 31:355-366. Crowe, J.H., Hoekstra, F.Α., and Crowe, L.M. 1989. Membrane phase transitions are responsible for imbibitional damage in dry pollen. Proc. Nat. Acad. Sci. 86:520-523.

22

Crowe, J.H. and Crowe, L.M. 1992. Preservation of liposomes by freeze drying. In: Liposome Technology, 2nd Edition, edited by Gregoriadis, G. Boca Raton, FL: CRC Press. Crowe, J.H., Hoekstra, F .Α., Nguyen, K.H.N., and Crowe, L.M. 1996. Is vitrification involved in depression of the phase transition temperature in dry phospholipids? Biochim. Biophys. Acta 1280:187-196. Crowe, L.M. and Crowe, J.H. 1989. Effects of water and carbohydrates on membrane fluidity. In: Physiol ogical Regul ation of Membrane Fl uidity, edited by Aloia, R.C. New York: Alan R. Liss. Crowe, J.H., Crowe, L.M., and Chapman, D. 1984. Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223:701-703. Crowe, L.M., Mouradian, R., Crowe, J.H., Jackson, S.A., and Womersley, C. 1984. Effects of carbohydrates on membrane stability at low water activities. Biochim. Biophys. Acta 769:141-150. Crowe, J.H., Crowe, L.M., and Jackson, S.A. 1983. Preservation of structural and functional activity in lyophilized sarcoplasmic reticulum. Arch. Biochem.Biophys. 220:477-484. Crowe, J.H., Crowe, L.M., and Hoeskstra, F .A. 1989. Phase transitions and permeability changes in dry membranes during rehydration. Mini review. J. Bioenergética and Biomembranes 21:77-91. Crowe, L.M., Reid, D.S., and Crowe, J.H. 1996. Is trehalose special for preserving dry biomaterials? Biophys. J. 71: 2087-2093. Crowe, L.M. and Crowe, J.H. 1988. Trehalose and dry dipalmitoylphosphatidylcholine revisited. Biochim. Biophys. Acta 946:193-201. Eleutherio, E.C.A., de Araújo, P.S., and Panek, A.D. 1993. Role of the trehalose carrier in dehydration

resistance

of

Saccharomyces

cerevisiae.

Biochim.

Biophys.

Acta

1156:263-266. Green, J.L. and Angell, CA. 1989. Phase relations and vitrification in saccharide-water solutions and the trehalose anomaly. Journal ofPhys. Chem. 93:2880-2882.

23

Bryant, G. and Wolfe, J. 1992. Interfacial forces in cryobiology and anhydrobiology. Cryo-Lett. 13:23-36. Hoekstra, F .Α., Crowe, J.Η., and Crowe, L.M. 1992. Germination and ion leakage are linked with phase transitions of membrane lipids during imbibition of Typha l atifol ia pollen. Physiol. Plant. 84:29-34. Lee, C.W.B., Das Gupta, S.K., Mattai, J., Shipley, G.G., Abdel-Mageed, O.H., Makriyannis, Α., and Griffin, R.G. 1989. Characterization of the Llambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction. Biochemistry 28:5000-5009. Leslie, S.B., Israeli, E., Lighthart, Β., Crowe, J.H., and Crowe, L.M. 1995. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appi. Env. Microbiol. 61:3592-3597. Leslie, S.B., Teter, S.A., Crowe, L.M., and Crowe, J.H. 1994. Trehalose lowers membrane phase transitions in dry yeast cells. Biochim. Biophys. Acta 1192:7-13. Levine, Η. and Siade, L. 1992. Another view of trehalose for drying and stabilizing biological materials. BioPharm 5:36-40. Madin, K.A.C, and Crowe, J.H. 1975. Anhydrobiosis in nematodes: carbohydrate and lipid metabolism during dehydration. J. Exp. Zool. 193:335-342. Martin, M.C., Diaz, L.A., Manzanal, Μ.B., and Hardisson, C. 1986. Role of trehalose in the spores of Streptomyces. FEMS Microb. Letts. 35:49-54. Nakagaki, M., Hágase, H., and Ueda, H. 1992. Stabilization of the lamellar structure of phosphatidylcholine by complex formation with trehalose. J. Membrane Sci. 73:173-180. Potts, M. 1994. Desiccation tolerance of prokaryotes. Microbiol . Rev. 58:755-805. Prestrelski, S.J., Arakawa, T., and Carpenter, J.F . 1993. Separation of

freezing-and

drying-induced denaturation of lyophilized proteins using stress-specific stabilization. II. Structural studies using infrared spectroscopy. Arch. Biochem. Biophys. 303(2):465-473. Strauss, G. and Hauser, H. 1986. Stabilization of lipid bilayer vesicles by sucrose during freezing. Proc. Nat. Acad. Sci. 83:2422-2426.

24

Sun, W.Q., Leopold, A.C., Crowe, L.M., and Crowe, J.H. 1996. Stability of dry liposomes in sugar glasses. Biophys. J. 70(4): 1769-1776. Van Laere, A. 1989. Trehalose, reserve and/or stress metabolite? FEMS Microbiology Reviews 63:201-210.

25

Enhanced protection against environmental stress in plants transformed with the trehalose or glycine betaine biosynthesis pathways from yeast and Escherichia coli, respectively. Holmström K-O. 1 *, Mäntylä E. 2 , Mandal Α.', Koch Κ.3, Londesborough J . 4 , Palva ET.5andWelinB.2

' Dept. of Natural Sciences, University of Skövde, Box 408, S-541 28 Skavde, Sweden 2

Dept. of Plant Biology, Uppsala Genetic Center, Swedish University of Agricultural Sciences, Box 7080, S-750 07 Uppsala, Sweden.

3

Dept. of Food Science, Swedish University of Agricultural Sciences, Box 7051, S-750 07 Uppsala, Sweden. 4 5

VTT Biotechnology and Food Research, Box 1501, FIN-02044 VTT, Finland.

Dept. of Biosciences, Div. of Genetics, Box 56, FIN-00014 University of Helsinki, Finland. Corresponding author. E-mail: [email protected]

Summary: The fact that seemingly very low concentrations of betaine and trehalose can improve stress tolerance implies a different mode of action than merely an osmoprotective effect. Nevertheless, it cannot be excluded that the concentration of the osmoprotectants is enough to enhance stress resistance if compartmentalised or localised to specific target molecules. Even though the underlying molecular mechanisms for stress protection provided by betaine and trehalose are still unknown, our data show that osmoprotectants can be synthesised in plants without perturbing physiological and developmental processes. Furthermore, our results clearly demonstrate the advantage of using stress-induced regulatory sequences for the expression of stress-related genes at appropriates times, i.e. when needed. The result support our original notion that introduction of genes related to stress in other living organisms is a promising approach to enhance environmental stress-resistance in higher plants. Introduction In most living organisms environmental stresses like salinity, drought and freezing ultimately lead to withdrawal of water from the living cells. A decline in cellular water content induces many different physiological and biochemical modification such as changes in cell structure, hormone levels, gene expression and sugar content. In response to water stress many organisms accumulate small osmotically active organic solutes such as amino acids and derivatives thereof, betaines, polyhydroxyl alcohols

(polyols)

and

sugars.

Such

compounds

(usually

referred

to

as

osmolytes,

osmoprotectants or compatible solutes) enable the organism to maintain cellular water balance, protect biological membranes, enzyme activity and metabolic processes vital for the survival of the organism. Water deficiency is one of the major limiting factors in crop production, and breeding towards improved drought tolerance in crop plants has a high priority in agriculture. In recent years, transgenic plants transformed with one or two genes involved in osmoregulation have given substantial proofs of the potential to genetically engineer plant stress resistance. Tarczynski and colleagues (1993) first demonstrated the possibility to enhance salt tolerance, by the transfer of mannitol production into tobacco. Another sugar, fructan has been shown to enhance drought resistance in transgenic tobacco (Pilon-Smits et al, 1995). Kishor et al. (1995) increased the production of the amino acid proline resulting in enhanced tolerance of the transgenic to both salt and drought.

The simplicity of the biosynthesis pathways for the two osmoprotccting molecules, glycine betaine and trehalose, encouraged us to separately introduce the corresponding genes into tobacco, a plant not accumulating these substances in response to water stress, in order to study the effects on stress tolerance.

Transfer of the Escherichia coli glycine betaine biosynthesis pathway to tobacco results in improved salt tolerance. Many organisms accumulating betaine inhabit saline and arid areas and accumulate the compound in response to drought and salinity (Csonka, 1989; Rhodes and Hanson, 1993). Several plant species, including important crops, are however incapable of synthesising glycine betaine. Betaine is thought to protect the plant by acting as an osmolyte maintaining the water balance between the plant cell and the environment and by stabilising

macromolecules during cellular dehydration

and at high salt

concentrations (Papageorgiou and Murata, 1995). Betaine has also been shown to accumulate in response to low and high temperature stress in higher plants, where it might play a role in protecting membranes and/or protein complexes (Yang et al, 1996; Zhao et ai. 1992).

2X

In the bacteria Escherichia coli a 62 kDa membrane associated choline dehydrogenase (CDH) first oxidises choline into betaine aldehyde and subsequently betaine aldehyde into betaine. The second step of the process is further catalysed by a 53 kDa soluble betaine aldehyde dehydrogenase (BADH), only oxidising betaine aldehyde into betaine. As betaine aldehyde is toxic to the cell it seems reasonable to assume that the existence of both enzymes is to ensure efficient oxidation of toxic betaine aldehyde into the neutral betaine. The two genes betA and betB encoding the CDH and BADH, have been cloned and characterised (Lamark et al, 1991). In a recent study the first enzyme of the E. coli betaine biosynthesis pathway, CDH, was introduced into tobacco (Lilius et al, 1996). Transgenic plants expressing betA showed improved salt tolerance as seen from in vitro growth studies. Furthermore, introduction of the gene codA encoding a choline oxidase from the soil bacterium Arthrobacter globiformis, and targeting the corresponding polypeptide into chloroplasts of transgenic Arabidopsis thaliana plants, increased tolerance to salt stress (Hayashi et al, 1997). In the same study it was shown that betaine-producing transgenic plants exhibited increased tolerance to inhibition of the photosynthesis. In a previous study we have described the successful introduction of the second enzyme of the E. colt betaine pathway, BetB, into tobacco (Holmstrim et al., 1994). However, the enzyme produced, BADH, can only oxidise the second enzymatic step in the betaine biosynthesis pathway, converting betaine aldehyde into betaine, but is incapable of oxidising choline into betaine aldehyde, the first step of the pathway. We have recently described the introduction of the first enzyme, CDH, into BADH-producing lines, thereby completing the E. coli biosynthesis pathway in transgenic tobacco (Holmström et al. submitted A). By crossing a transgenic plant expressing betA, producing glycine betaine to 35 nmol g"1 fresh weight (FW), with a plant expressing betB, not producing glycine betaine, we obtained offspring producing both enzymes in the same transgenic plant, and we could show that betaine accumulation increased two- to three-fold compared to transgenics only expressing betA. The concentration of betaine found in leaves of our transgenics, was in the range of 40-80 nmol g"' FW.

29

The accumulation of betaine found in transgenics producing CDH alone, was sufficient to give enhanced tolerance to salt stress as seen from whole plant studies (Figure 1). In order to elucidate underlying stress-protecting mechanisms generated by production of glycine betaine we measured the activity of photosystem II (PS II). In crossings producing both the CDH and the BA DH enzymes, salinity did not affect PS II activity of leaf discs as such, however, in combination with high light a 40% reduction in O2 evolution was observed for both transgenic and wild-type tobacco plants (Figure 2). Leaf discs subjected to salt stress and high light were thereafter transferred to normal growth medium at low light for 24 hours. PS II activity was measured after 13 hours and 24 hours of recovery at low light. Both betA as well as betAxbetB transformed tobacco plants recovered to PS II activities prior to stress treatment whereas wild-type plants did not. Thus, it implicates that the protection of betaine at the whole plant level might be improved recovery from photoinhibition during the salt treatment.

% Fresh weight 30 —

Τ

25 —

#·'->-

20 —

>;

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15 — '

CDH1 ...

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WT Λ s· Τ* i JsSÜ'5-Ä

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^^#\^£ 0 %g¿& --'%^

CDH1

CDH2

Plant line

Figure 1. Enhanced salt stress tolerance in green-house grown CDH-producing tobacco. Seeds were germinated in vitro on solidified MS-media, and seedlings were thereafter planted in perlite soil and grown in the green-house. A fter one week of growth in the green-house, plants were subjected to salt stress by watering with 200 mM of NaCl. After two weeks of stress treatment non-transformed control tobacco (WT) and two independent transgenic CDH-producing lines, CDH1 and CDH2, were harvested and FW was measured. Upper row in both photos represent the same line grown under non-stressed conditions.

30

The betaine concentration found in our transgenic tobacco is probably not high enough to generate an osmolyte effect in the cell. The increased tolerance towards salt stress is therefore likely due to other effects than osmoregulation. The protection of betaine could include stabilising protein complexes and membranes. In support of this is our finding that PS II seems to be protected at stress conditions by the elevated levels of betaine in the transgenic plants. A similar result was also obtained by producing betaine in Arabidopsis chloroplasts (Hayashi et al, 1997). It is noteworthy that the ten times lower concentrations of betaine found in our study gives similar effects seen in Arabidopsis plants transformed with the codA gene. Our results demonstrate that cytoplasmic synthesis of glycine betaine gives protection in a similar manner as the chloroplast targeted production in Arabidopsis (Hayashi et al, 1997).

Relative photosynthetic capacity in % 100

-

90

-

80 70 60

-

50

-

40

L I

Τ

ιτ

0

14

·'■'■]

30 20 10 0

14

23

14

23

CDH

23

CDHxBADH

Plant line

Figure 2. Glycine bet aine-producing t ransgenics have increased recovery from photoinhibition imposed by salt st ress and high light . Leaf discs were taken from green-house grown plants and exposed to salt stress (200 mM N aCl) under high light (390 μπιοί m"2 s"1) for 7 hours. Discs were then transferred to water for 14 or 23 hours at low light (30 μτηοΐ m'2 s"1) for recovery. The capacity of photosynthesis was measured directly after stress relief, indicated in the figure by 0 h, and after 14 hours and 23 hours of recovery. Photosynthetic capacity was plotted as percentage compared to unstressed plants from the same lines or wild-type. Non-transformed wild-type tobacco (WT), a betA (CDH) transformant and Fi progeny from a cross between betA (CDH) and betB (BADH).

31

Although crosses from CDH and BADH transformed tobacco lines accumulate higher amounts of betaine than CDH producing lines, no direct evidence for improved stress protection was seen. Thus it seems, that even relatively low concentrations of betaine can improve the stress tolerance in photosynthesising organisms by protecting protein complexes involved in the photosynthetic processes. In conclusion, we have demonstrated that genetic engineering of glycine betaine production in tobacco is associated with increased salt-tress tolerance and that this partly is based on improved protection of the photosynthetic apparatus.

Transfer of the yeast trehalose biosynthesis pathway into tobacco offer enhanced drought tolerance. Simple sugars, most notably sucrose and trehalose, have been shown to stabilise biomaterials of various composition and origin. In nature, trehalose is synthesised in response to stress by such diverse organisms as bacteria, fungi, algae, insects, invertebrates and lower plants (Elbein, 1974). In yeast the biosynthesis is a two-step process where the enzyme trehalose-6-phosphate

synthase

(TPS1)

condenses

UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate (Tre6P) and this intermediate is further converted to trehalose by a second enzyme trehalose-6-phosphate phosphatase (TPS2). It has long been believed that higher plants lack the capacity to produce trehalose as accumulation of the disaccharide never has been confirmed in higher vascular plants, except desiccation tolerant angiosperms like Myrothamnus flabellifolia (Bianchi et al, 1993; Drennan et al, 1993). In most plants the substrate molecules for trehalose synthesis, glucose-6-phosphate and UDP-glucose, are present as components of the sugar metabolism, indicating the availability of substrate for production of trehalose in higher plants.

To test whether also trehalose production would render plants improved stress tolerance we introduced the yeast gene TPS J under control of the promoter of the light-stimulated Arabidopsis Rubisco small subunit gene RbcSIA (Holmstrøm et al, 1996) into tobacco. Transgenic plants producing the TPS 1-subunit did accumulate trehalose, whereas the

32

intermediate product, trehalose-6-phosphate, could not be detected, suggesting that tobacco harbours endogenous phosphatases that can convert trehalose-6-phosphate into trehalose. When submitted to drought stress, the trehalose-producing plants did exhibit a dramatically enhanced tolerance at all developmental stages. However, many transgenic plants also presented varying degrees of stunted growth, and sometimes an altered leaf phenotype. Similar growth aberrations have been shown in two other independent reports with transgenic plants harbouring the E. coli otsA and yeast TPS1 genes under control of the cauliflower mosaic virus 35S promoter (Goddijn et al, 1997; Romero et ai, 1997).

In order to identify and circumvent the growth aberrations seen in transgenic tobacco expressing only the first enzyme TPS1, we chose two different approaches. First, we simultaneously introduced both genes for yeast trehalose biosynthesis, TPS1 and TPS2. As was the case of the first experiment both the TPS J and TPS2 coding sequences were fused to the Arabidopsis promoter pRbcSIA, resulting in a light-stimulated, constitutive, production of the enzymes (Holmström et al, submitted Β). In contrast to plants producing TPS1 alone, overproduction of TPS2 together with TPS1 corrected all anomalies, and greenhouse grown transgenic tobacco demonstrated a normal development indistinguishable from that of wild-type tobacco (Figure 3). Furthermore, these transgenic tobacco exhibited normal growth and retained an enhanced drought resistant phenotype, which is therefore not linked to the previously observed altered growth phenotype.

Our second approach to circumvent negative growth effects in trehalose producing transgenic plants constitutively producing only TPS1, involved engineering the trehalose production to be strictly stress-responsive by utilising the drought-responsive Arabidopsis promoter pRabI8 (Ling & Palva, 1992). This resulted in transgenic tobacco plants where expression of TPSJ was activated when plants were subjected to drought stress, followed by a production of trehalose and an enhanced drought resistance. Transgenic plants harbouring a double construct consisting of the inducible TPS 1-fusion together with the light-stimulated TPS2 were also generated (Holmström et al, submitted Β). Neither plants harbouring the single nor the double inducible gene

33

construct showed any deviation from the normal growth phenotype, in contrast to transgenics with the constitutive enzyme production (Figure 3).

Figure 3. Growth phenotype of green-house grown trehalose accumulating transgenic tobacco. Plant representing greenhouse-grown transgenic tobacco (SRI) producing TPS1 constitutively resulting in a stunted growth phenotype, RBC-1. Growth problems were corrected by simultaneous production of TPS 1 and TPS2, both fused to the RbcSJA promoter, RBC-2. If trehalose accumulation was regulated by the stress-induced promoter Rabl8, no growth aberration was seen in either plants producing TPS1 (RAB-1) alone or co-producing TPS1 and TPS2 (RAB-2), as compared to wild-type or vector-transformed control tobacco (112). Transfer of the yeast trehalose biosynthesis pathway into tobacco resulted in low trehalose production sufficient to give a dramatic enhancement of drought resistance as compared to control plants. The amount of trehalose accumulating in the drought resistant phenotype (Table 1) is only a fraction of trehalose accumulation in some cryptobiotic plants (M

et al, 1995). As in the case of mannitol (Tarczynski et al,

1993) these levels are too low to cause any osmotic adjustment and much lower than the concentrations obtained for other compatible solutes, like proline and fructan (Kishor et al, 1995, Pilon-Smits et al, 1995), giving an enhanced osmoprotection in transgenic plants.

34

Construct

Line number

Trehalose mg/g fresh w

Control, Empty vector

(112:3)

0.00

Drought-induced, TPS1

(955:10)

1.95

Drought-induced, TPS 1

(955:25A)

2.00

Drought-induced, TPS1 +TPS2

(965:5)

2.68

Drought-induced, TPS1 + TPS2

(965:22)

3.07

Light-stimulated, TPS1

(952:4)

1.25

Light-stimulated, TPS1

(952:23)

1.67

Light-stimulated, TPS1 +TPS2

(962:10)

1.97

Light-stimulated, TPS1 + TPS2

(962:13)

1.34

Table 1. Trehalose accumulation in TPSl-producing in vitro grown tobacco exhibiting enhanced drought stress. To verify that the four different constructs resulted in trehalose production, leaf extracts were analysed by HPAEC-PED for accumulation of trehalose. Transgenics harbouring the drought-induced chimeric genes were drought stressed 20 hours at 28 -s-C prior to the extraction. Data represent one plant with improved drought tolerance in a DLT test. ND, Not detected.

When transgenic tobacco grown in vitro were subjected to elevated light intensities and drought stress, trehalose transformants were visibly unharmed while leaves of control plants rapidly lost their turgor and bleached (Figure 4). This selective bleaching of leaves of control plants is presumably an indication of secondary stress symptoms, linked to the dehydration of the plant tissue. Transgenic trehalose-producing plants showed no signs of drought-stress during this test, neither did they show any visible sign of bleaching. This indicates that the chlorophyll bleaching in control plants most likely was a result of oxidative stress, brought about by the combination of light and water stress.

35

^^^^>^fi

HWI

ïS s f

Hw*-

ìtSs

NS-VC

TJ8

till

jTj

Λ

VC

RAB-1

RAB-2

Figure 4. Enhanced drought resistance of in vitro grown transgenic tobacco producing trehalose. Tobacco (Ti) plants were subjected to drought stress, by removal of the lids from the glass jars where plants were grown, and a moderate light stress (320 μπιοί m" s" ) for 60 h. Three plants from each construct was tested. Already after 30 minutes leaves of vector-transformed control tobacco exhibited loss of turgor. At the end of the experiment, most leaves of the vector-controls were completely dry with nearly all of the chlorophyll bleached out. Transgenic seedlings containing different TPS-constructs demonstrated no signs of water deficit under these conditions. NS-VC; unstressed vector control, VC; vector control, RAB-1; transgenic tobacco harbouring stress-inducible TPS1, RAB-2; transgenic tobacco harbouring stress-inducible TPS1 and constitutive production of TPS2. To verify whether the improved drought tolerance was a result of reduced water loss by lowering of cell osmotic potential, according to the theory of osmoregulation, water potential (Ψ\ν) and osmotic (^Fs) potential was monitored during drought stress treatment of in vitro grown transgenic seedlings. During the first 80 minutes of our experiment, drought stress did neither affect the plant water potential nor the osmotic potential in transgenic tobacco where TPS1 production was regulated by the RABI'8 promoter. In constitutive trehalose-producing transgenic lines both VFW and Ts did decline but to a much lesser extent as compared to control plants (Figure 5). Interestingly, during these 80 minutes control plants visibly lost turgor very rapidly while no turgor loss was observed in trehalose producing lines, a result supported by the Ψν/ measurements.

36

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— · — R AB - 2 22

Ü

RAB -205

I RB CS-2 10B

Π RAB -2 22

I RB CS-210A

Figure 5. Changes in water potential ( w ) a n d osmotic potential ( s) of detached leaves from trehalose accumulating transgenic tobacco during drought, a) Detached leaf test showing changes in fresh weight during air-drying of six weeks old in vitro grown tobacco. Drought treatment was terminated after 80 minutes of drying, when samples were prepared from the leaf tissue for water potential and osmotic potential measurements. Plants used in the study were transformed lines constitutively producing both TPS1 and TPS2, RBC-2:10, two lines with drought-induced TPS1 and constitutive production of TPS2, RAB-2:05 and RAB-2:22, plus an empty vector control = VC. b) Water potential ( w ) values of tobacco leaves before the onset of the drought treatment (0 minutes), after 30 and 80 minutes of exposure to air-drying. Values are means of 4 independent measurements. Plant material used in the study were tobacco lines harbouring constitutively produced TPS 1 and TPS2, RBC-2:10A and RBC-2:10B. Lines with drought-induced TPS1 and constitutive production of TPS2, are labelled RAB-2:05 and RAB-2:22 and an empty vector transformed control line, VC. c) Osmotic potential ( s) values of samples from the same plants and time points as in (b). Values are the mean of three independent measurements for each timepoint. From this study it can be concluded that trehalose does not seem to exert its protective function in transgenic plants through en masse accumulation in a way that would cause osmotic adjustment of the cell sap. The decrease in Ψ$ observed in control plants is

37

probably

due to osmotic

adjustment

by

accumulation

of other osmolytes, or

concentration of the cell sap upon loss of water, or most likely, a combination of both these factors. It is clear that enhanced drought resistance in trehalose producing tobacco is depending on an ameliorated water retention rather than on a lowering of the cell water potential by osmotic adjustment of the cell sap.

Our results suggest that it is not the low trehalose accumulation per se that leads to the growth aberrations seen in transgenic plants with constitutive expression of IPS!,

but

rather the production of the sugar, and/or processing of the intermediate product Tre6P. This is also in agreement with studies in soybean where trehalose added to the growth medium was taken up by the plant and accumulating to high levels ( 8 % DW), without any detectable impairment of growth or health (M

et al., 1995).

Expressed sequence tags (ESTs) from Arabidopsis and rice with high homology to the yeast genes TPSI and TPS2, as well as their E. coli homologues otsA and otsB, have recently been published in DNA databases, indicating possible trehalose production in these plants. Goddijn and colleagues (1997) also demonstrated that wild-type tobacco plants did indeed accumulate trehalose when the trehalase-blocker validamycin A was added to the growth medium. Furthermore, it has recently been shown that Arabidopsis harbour functional TPSI AtTPPA

and AtTPPB

and TPS2 homologues, AtTPSl

(Blllzquez et al. 1998),

(Vogel et al. 1998). Interestingly, AtTPSl'was

non-specific manner while AtTPPA

and AtTPPB

expressed in a

predominantly were expressed in

young seedlings and flowers. Both genes are expressed at low levels, which together with the fact that trehalase has been shown to be active in higher plants (M

et al,

1995), may explain why trehalose accumulation has not been found previously. As trehalose is present at infinitesimal amounts it is likely that the sugar do not serve as an osmoprotectant by itself in wildtype plants, but may serve other purposes yet to be understood.

Although no trehalose-6-phosphate could be detected in transgenic plants expressing the TPSI gene alone (Holmstrøm et al, 1996) it is intriguing that over-production of the TPSI-subunit have such unexpected developmental consequences when the Tre6P is processed by endogenous enzymes in tobacco. Production of trehalose may therefore

3S

directly or indirectly be involved in several signalling events affecting for example plant sugar metabolism, carbohydrate-mediated feedback or photosynthesis. This possible interference with regulation of plant metabolism is completely avoided when production of the TPSI-subunit is controlled by a stress-induced promoter, as can be seen in transgenic plants harbouring TPSI under control of the drought-induced

Rabl8

promoter. References Bianchi G, Gamba A , Limiroli R, Pozzi N, Elster R, Salamini F, Bartels D. 1993. The unusual sugar composition in leaves of the resurrection plant Myrothamnus flabellifolia. Physiologia Plantarum, 87, 223-226. Bbzquez M A , Santos E. Flores C L, Martinezzapater J M, Salinas J, Gancedo C. (1998) Isolation and molecular characterization of the A rabidopsis Tpsl gene, encoding trehalose-6-phosphate synthase. Plant Journal 13, 685-689. Csonka L N, Hanson A D . 1991. Prokaryotic osmoregulation: genetics and physiology. Annual Review of Microbiology 45, 569-606. Drennan Ρ M, Smith Μ Τ, Goldsworthy D, van Staden J. 1993. The occurrence of trehalose in the leaves of the desiccation-tolerant angiosperm Myrothamnus flabellifolius. Journal of Plant Physiology, 142, 493-496. Elbein A . (1974) The metabolism of alpha-alpha-trehalose. Adv. Carbohydr. Chem., 30, 227-256. Eleutherio E C A , A raújo Ρ S, Panek A D. (1993) Protective role of trehalose during heat stress in Saccharomyces cerevisiae. Cryobiology, 30, 591-596. Goddijn O J M, Verwoerd T C, Voogd E, Kruiwagen Ρ W Η Η, Degraaf Ρ Τ Η M , Poels J, Vandun Κ, Ponstein A S, Damm B, Pen J. (1997) Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants. Plant Physiology, 113, 181-190. Hayashi H, Mustardy L, Deshnium P, Ida M, Murata Ν. 1997. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase - accumulation of glycine betaine and enhanced tolerance to salt and cold stress. Plant Journal 12, 133-42. Holmström K-O, Mäntylä E, Welin B, Mandal A , Palva E T. (1996) Drought tolerance in tobacco. Nature, 379, 683-684.

39

Holmström K-O, Somersalo S, Mandai A , Palva E T, Welin Β. Submitted. Improved tolerance to low temperature and salinity in transgenic tobacco producing glycine betaine. Holmström K-O, Welin B, Mandai A , Kristiansdottir I, Teeri Τ Η, Lamark Τ, Ström A R, Palva E T. (1994) Production of the Escherichia coli betaine-aldehyde dehydrogenase, an enzyme required for the synthesis of the osmoprotectant glycine betaine, in transgenic plants. Plant Journal, 6, 749-758. Holmström K-O.1*, Mäntylä E Holmström K-O, Welin Β, Mäntylä E, Ahmadzadeh A , Mandal A , Koch K, Palva E T. Submitted. Enhanced drought resistance in transgenic tobacco with stress-induced trehalose production. Kishor Ρ Β Κ, Hong Ζ, Miao G Η, Hu C A A, Verma D P S . (1995) Overexpression of DELTA-l-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiology, 108, 1387-1394. Lamark T, Kaasen I, Eshoo M W, Falkenberg Ρ, McDougall J, Stnm A R. 1991. DNA sequence and analysis of the bel genes encoding the osmoregulatory choline-glycine betaine pathway of Escherichia coli. Molecular Microbiology 5, 1049-64. Lilius G, Holmberg Ν, Β D low L. 1996. Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase. Biotechnology 14, 177-80. Ling V, Palva E T. (1992) The expression of a røo-related gene, rabl8, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh. Plant Molecular Biology, 20, 951-962. M

Boller Τ, Wiemken Α. (1995) Effects of validamycin A , a potent trehalase inhibitor, and phytohormones on trehalose metabolism in roots and root nodules of soybean and cowpea. Planta, 197, 362-368.

Papageorgiou G C, Murata Ν. 1995. The unusually strong stabilising effects of glycine betaine on the structure and function of the oxygen-evolving Photosystem II complex. Photosynthesis Research, 44, 243-252 Pilon-Smits E A H, Ebskamp M J M, Paul M J, Jeuken M J W, Weisbeek Ρ J, Smeekens S C M. (1995) Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiology, 107, 125-130.

40

Rhodes D, Hanson AD. 1993. Quaternary ammonium and tertiary

sulfonium

compounds in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 44, 357-84. Romero C, Belles J M, Vaya J L, Serrano R, Culianezmacia F A. (1997) Expression of the yeast trehalose 6 phosphate synthase gene in transgenic tobacco plants: Pleiotropic phenotypes include drought tolerance. Planta, 201, 293-297. Tarczynski M C, Jensen R G, Bohnert FI J. (1993) Stress protection of transgenic tobacco by production of the osmolyte mannitol. Sciences, 259, 508-510. Vogel

G,

Aeschbacher

R

A,

M

Boller

Τ,

Wiemken

Α.

1998.

Trehalose-6-phosphate phosphatases from Arabidopsis thaliana - identification by functional complementation of the yeast TPS2 mutant. Plant Journal 13, 673-683 Yang G, Rhodes D, Joly R J. 1996. Effects of high temperature on membrane stability and

chlorophyll

fluorescence

in glycine betaine-deficient

and

glycine

betaine-containing maize lines. Australian Journal of Plant Physiology 23, 437-43. Zhao Y, Aspinall D, Paleg L G. 1992. Protection of membrane integrity in Medicago sativa L. by glycine betaine against the effects of freezing. Journal of Plant Physiology 140, 541-3.

4!

Survival of Entomopathogenic Nematodes in Relation to Commercial Application Denis J. Wright, Mavji N. Patel and Judy M. Mason Department of Biology, Imperial College of Science, Technology & Medicine Silwood Park, Ascot, Berkshire, SL5 7PY, UK Abstract: Two aspects of the survival of infective juveniles (IJs) of entomopathogenic rhabditid nematodes are considered: energy reserves in relation to infectivity during storage, and the requirements for IJs to be applied successfully as sprays against apparent and cryptic insect pests on foliage. It seems clear that the survivorship of IJs of entomopathogenic nematodes during storage is largely a product of their metabolic rate and their initial levels of energy reserves. Various studies have demonstrated the relationship between the rate of consumption of neutral lipids (primarily triacylglycerols) and the maintenance and subsequent decline in infectivity and survival of IJs. Glycogen also appears to have a significant role in the maintenance of infectivity in several Steinernema species and in Steinernema carpocapsae it acts as the key energy store in aged IJs. Apart from the quantity, the quality (fatty acid composition) of the neutral lipids in freshly-produced IJs is also known to vary considerably between batches and between different production systems and may influence the effectiveness of commercial products. Where IJs are exposed to the full rigors of the environment as foliar sprays, few control successes have been reported and these have been mainly against cryptic pests. The abiotic factors generally identified as limiting for IJs in aboveground environments are UV radiation, desiccation and temperature. The selection of temperature-tolerant species/strains with a high level of infectivity against the target pest is clearly advantageous. However, improvements in spray application technology and spray formulations are also critical. Such developments would help to nullify the effects of UV and desiccation and increase the rate of infection by nematodes, thus making control of at least some foliar pests attainable. In addition, screening of nematodes for their ability to osmoregulate under the hyperosmotic conditions which are likely to develop in spray droplets prior to desiccation may be of greater value than screening for desiccation tolerance per se.

1. GENERAL INTRODUCTION The infective, third stage juvenile (IJ) of rhabditid entomopathogenic nematodes is a non-feeding, phenotypically distinct (dauer) survival stage whose ability to locate and infect the host can be related to the level of its energy reserves during storage (Lewis et al., 1992; Patel et al., 1997; Fitters et al., 1997). Since the effective Storage life (maintenance of infectivity) of IJs of different species of entomopathogenic nematodes produced commercially as bioinsecticides (Georgis, 1992) varies from one or two months to more than a year, studies

on the synthesis and utilization of their energy reserves may provide insights which will assist in the maintenance and enhancement of nematode "quality" during production and storage. The use of Us as bioinsecticides has been restricted largely to soil insects (Klein, 1990), where the nematodes are afforded a degree of protection from environmental factors. In contrast, their success against foliar pests, where Us are exposed to the full rigours of the environment, has been limited although some efficacy has been reported against cryptic pests (e.g. Begley. 1990; Williams and MacDonald, 1995). The major abiotic factors identified as affecting the IJs in aboveground environments are UV radiation, desiccation and temperature. This paper reviews current knowledge of energy reserves and their utilization in IJs of entomopathogenic nematodes and nematode survival in relation to foliar application.

2. ENERGY STORES Triacylglycerol neutral lipids are generally accepted to be the major energy reserves in free-living nematodes including the IJs of plant and animal parasitic species (Barrett and Wright, 1998). Several studies have investigated the relationship between the rate of consumption of lipids in IJs of steinernematid and heterorhabditid species and the maintenance and subsequent decline in their infectivity and survival (Lewis et al., 1995; Patel et al., 1997). For example, when IJs were stored in water at 25°C, S. carpocapsae and S. riobrave (=S. riobravis) survived less well (120 - 135 days) than S. feltiae and S. glaseri (>450 days), the decline in neutral lipids in the last three species correlating closely with the observed decline in infectivity and survivorship (Patel et al., 1997). Intraspecific variation in survivorship can be predicted to occur since the quantity and quality (fatty acid composition) of lipids in freshly-produced nematodes can vary considerably between batches and between different production systems. This may at least in part explain the observed variation in the stability and efficacy of commercially-produced (in vitro) nematodes (Gaugler and Georgis, 1992; Womersley, 1993). For example, Abu Hatab et al. (1998) found that IJs of S. glaseri cultured in a natural host, Popillia japónica, contained significantly more triacylglycerols compared with nematodes reared in a factitious host, Galleria mellonella or in solid or liquid media (Table 1). Differences in specific fatty acids were less consistent between the culture systems but polyunsaturated fatty acids (particularly

44

linoleic) formed a significantly lower percentage of total triacylglycerol fatty acids in in vivo compared with in vitro nematodes. There was very close agreement in the fatty acid composition of G. mellonella-culturea S. glaseri with an earlier study (Patel and Wright, 1997a; Table 1). In nematodes and other helminths, unsaturated fatty acids have been reported to be predominant (Barrett, 1981). This certainly appears to be the case for G«//e/7«-produced Heterorhabditis megidis and H. bacteriophora, where oleic acid (C18:ln-9) was found to be the major fatty acid by Selvän et al. (1993a,b) and Menti (1997). Reports that the majority of fatty acids were saturated in Gn//miJ-produced Steinernema species (Selvän et al., 1993a,b) have not been confirmed (Fodor et al., 1994; Wijbenga and Rodgers, 1994; Patel and Wright, 1997a; Abu Hatab et al., 1998; see Table 1). In all of the latter studies, the dominant fatty acids found were oleic and linoleic (C18:2n-6). The relatively poor survivorship of Heterorhabditis compared with Steinernema species cannot, therefore, be related necessarily to overall levels of fatty acid saturation (Patel and Wright, 1997a) although the possible effects of more subtle interspecific differences in fatty acid composition remain to be investigated.

45

Table 1. Culture media and triacylglycerol fatty acids of Steinernema glaseri. Mean % of triacylglycerol fatty acids Fatty acid

Galleria

Popitlia

mellonetla

japónica

Solid media

Liquid media

20 (22)

5

10

8

7(7)

10

8

2

50 (46)

50

29

20

7(8)

7

45

57

Saturated

30 (33)

19

18

10

Polyunsaturated

13(15)

18

48

67

Triacylglycerol (% dry wt)

43

50

42

38

16:0 (palmitic) 18:0 (stearic) 18:ln-9 (oleic) 18:2n-6 (linoleic)

Based on Abu Hatab et al. (1998). Data in parentheses from Patel & Wright (1997a).

The other significant energy reserve found in nematodes is glycogen, which tends to predominate in animal parasitic species where the host environment can have a low partial pressure of oxygen (Wright, 1998; Barrett and Wright, 1998) although aerobic utilization of glycogen has been demonstrated in one free-living species (Cooper and Van Gundy, 1970). While glycogen has been known to occur in appreciable amounts in some entomopathogenic nematodes for some years (Selvän et al., 1993), its functional significance has been less clear. Utilization of glycogen by IJs of S. carpocapsae, S. glaseri and Heterorhabditis bacteriophora can be inferred from the data of Lewis et al. (1995). Subsequent work confirmed that the glycogen content of S. carpocapsae, S. glaseri, S. feltiae and S. riobrave declined during storage and suggested that glycogen may play a significant if secondary role to neutral lipids in the maintenance of infectivity in these species (Patel and Wright, 1997b). In addition, glycogen rather than neutral lipids appears to act as the most important energy store in aged IJs of S. carpocapsae (c. 60 days-old at 20°C), high levels of infectivity being maintained despite very low neutral lipid reserves (Patel & Wright, 1997b; Wright et al., 1997).

46

The metabolic mechanisms which regulate glycogen utilization during storage are as yet unknown. The survival of S. carpocapsae for > 40 days under anoxic conditions (Burman & Pye, 1980) demonstrates its ability to use anaerobic metabolism but the requirement for oxygen for long-term survival in Steinernema spp (Kung et al., 1990) also indicates the essentially aerobic nature of these nematodes. Studies on key glycolytic and oxidative enzymes in S. carpocapsae indicate metabolism typical of a facultative aerobe (Shih et al., 1996). In aged IJs of the latter species which contain appreciable glycogen but little neutral lipid (see above), the glycolysis inhibitor iodoacetamide was shown to reversibly block nematode infection of Galleria larvae (Patel and Wright, 1997b). From the above studies on energy stores, it is clear that survivorship of IJs of entomopathogenic nematodes is a function of their metabolic rate and their initial levels of energy reserves. For example, IJs of the relatively short-lived S. riobrave and S. carpocapsae can be classified as 'fast' and IJs of the much longer-lived S.feltiae and S. glaseri as 'slow' in relation to lipid utilization (Patel et al., 1997). The same classification can be given for glycogen utilization except that S. glaseri falls into the 'fast' category (Patel and Wright (1997b). This apparent anomaly is explained when the levels of energy reserves for the above Steinernema species are compared per nematode rather than as a % of dry weight (Table 2), the glycogen content per IJ of S. glaseri being 5 and 6-fold greater than S. carpocapsae and S. riobrave respectively. Table 2. Mean neutral lipid and glycogen content of freshly-emerged infective juveniles (IJ) of four Steinernema species (adapted from Patel Species

Neutral lipid ng/IJ

Glycogen ng/IJ

et al., 1997; Patel and Wright, 1997b). Neutral lipid % dry wt

Glycogen % dry wt

S. carpocapsae

35

15

31

13

S. riobrave

47

12

31

8

S. feltiae

52

35

24

16

S. glaseri

115

77

26

18

47

Differences in nematode foraging behaviour may account to some extent for the greater levels of neutral lipid and glycogen per IJ in S. feltiae and S. glaseri (both "cruising forager" species) compared with S. carpocapsae which has many of the characteristics of a "sit-and-waif forager (Lewis et ai, 1992; Campbell and Gaugler, 1993; Patel and Wright, 1996). However, differences in nematode activity during storage are unlikely to be as important as the basal metabolic rate in determining their shelf-life. Studies on infective juveniles (J2) of the potato cyst nematode Globodera rostochiensis showed, for example, that when movement was completely but reversibly inhibited by the oximecarbamate 'nematicide' oxamyl consumption of neutral lipid was only reduced by c. 30% over a 35 day period when compared with untreated controls (Wright et al., 1989).

3. SURVIVAL AND FOLIAR SPRAYS The IJs of some species or isolates of entomopathogenic nematodes are better able than others to tolerate, within defined limits, the major abiotic factors (temperature, desiccation and UV radiation) to which they are exposed on foliage (Bauer et al., 1995; Mason and Wright, 1997). Bauer et al. (1995) also found that survival/infectivity of in v;'vo-produced IJs of S. carpocapsae on foliage was greater compared with those produced in vitro. Such differences may be related to their neutral lipid (Section 2) or to their phospholipid composition (see Abu Hatab et ai, 1998 for effects of culture media on phospholipid fatty acids in S. glaseri), the latter potentially influencing membrane permeability and thus the nematode's water relations (see below). However, the extent to which nematodes need to be selected or engineered (e.g. Burnell and Dowds, 1996) for tolerance to environmental extremes on foliage is less certain and if we do select are temperature, desiccation and UV the only factors that should be considered? Clearly, for a defined pest problem, the nematodes used should be highly infective at the ambient temperatures encountered (Mason and Wright, 1997). However, the ability to survive desiccation on foliage presupposes 1) the desiccated nematodes remain infective following ingestion and rehydration in the insect gut (which remains unproven), or 2) that rehydration of IJs at dew formation or following rainfall would enhance insect control significantly. We suggest that it is more important to ensure that the rate of drying of the spray droplets (and

48

thus desiccation of IJs) is reduced as far as possible through, for example, improved spray formulations. Certainly, the use of antidesiccants has been shown to improve the efficacy of S. carpocapsae control of several foliar pests on cotton (Glazer et al., 1992). Rather than the ability to survive desiccation per se, the osmoregulatory ability of IJs may be more critical, as the osmotic pressure of the spray solution increases during evaporative loss of water from the leaf.

Nothing is known of osmoregulation in

entomopathogenic nematodes (Glazer, 1996). In general, the ability of free-living soil species and stages of nematodes to regulate in hyperosmotic conditions is less than their ability to regulate in hyposmotic ones (Wright, 1998), although IJs of entomopathogenic species are likely to encounter a considerable increase osmotic pressure on entry into the host insect. Heterorhabditis

bacteriophora and S. glaseri are reported to survive exposure to

hyperosmotic salt concentrations equivalent to soils with high salinities (Thurston et al., 1994) but whether these species merely osmoconform, with reduced movement, or osmoregulate and retain normal movement and infectivity under such conditions is less certain. In this respect, there may be some correlation between the ability to osmoregulate and the ability to survive desiccation since the latter is generally accepted to involve a passive component to reduce the rate of water loss (Womersley, 1990). Thus, greater osmoregulatory ability could help account for the greater insect control reported with foliar sprays of a nematode strain better able to survive desiccation compared with a strain which was relatively poor at desiccation survival (Glazer and Navron, 1990). When the effects of UV radiation on six isolates of Steinernema and Heterorhabditis species were examined using a solar simulator (as opposed to UV lamps with specific bandwidths) all isolates were found to retain appreciable activity against larvae of the diamondback moth, Plutella xylostella for up to 20 min. at 30 °C (Mason and Wright, 1997). Since the level of solar irradiation used in this test was approximately equivalent to equatorial sunlight at midday at an altitude of 1000 m, the above results were considered encouraging. Under such conditions, osmotic and desiccation effects were probably more limiting than UV for exposure periods of more than 20 min. Thus, rather than looking for UV tolerance, we would argue that efforts should be concentrated on reducing nematode exposure through the

49

development of improved spray formulations (with UV screens) and, in particular, improved spray systems. In general, entomopathogenic nematodes have been applied using standard spraying equipment. While this may be adequate for soil applications, the more rigorous conditions required for success against foliar pests (see above) requires more controlled applications (Mason et al., 1998). Specifically, there is a clear case for improving spray application technology to optimise the placement of IJs in relation to the pest (e.g. underside of foliage), and thus increase the rate of infection and reduce the impact of many if not all of the above abiotic factors. Ideally, spray applications of nematodes should also coincide with the onset of more favourable environmental conditions (e.g. dusk, days with high relative humidity) and/or periods of greatest pest activity. Recent work has suggested that the development of spinning disc application technology for nematodes holds some promise (Lello et al., 1996; Mason et al., 1998) but has also highlighted the variation in insect control that can occur between different standard fan and full cone hydraulic nozzles (Lello et al., 1996). Further studies on both types of spraying technology are certainly justified. The development of low volume application systems would have the economic benefit of reducing the amount of nematodes required in foliar sprays (Lello et al, 1996).

4. CONCLUSION Current knowledge of energy stores in IJs suggests that considerable improvements in the 'quality' of nematode products are achievable. It should also be possible, with the selection of suitable species or strains of nematodes and, more importantly, the development of improved

spray application technologies, to greatly increase their efficacy against

aboveground insect pests

Acknowledgements - Our research on entomopathogenic nematodes has been supported by The Leverhulme Trust, the Biotechnology and Biological Sciences Research Council (UK), CABI Biosciences and Biosys Inc.

50

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GEORGIS, R. (1992) Present and future prospects for entomopathogenic nematode products. Biocontrol Science and Technology 2, 83-99. GLAZER, I. (1996). Survival mechanisms of entomopathogenic nematodes. Biocontrol Science and Technology 6, 373-378. GLAZER, I., KLEIN, M., NAVON, A. & NAKACHE, Y. (1992). Comparison of efficacy of entomopathogenic nematodes combined with antidesiccants applied by canopy sprays against three cotton pests (Lepidoptera: Noctuidae). Journal of Economic Entomology 85, 1636-1641. GLAZER, I. & NAVON, A. (1990). Activity and persistence of entomoparasitic nematodes tested against Heliothis armigera (Lepidoptera: Noctuidae). Journal of Economic Entomology 83, 1795-1800. KLEIN, M.G. (1990). Efficacy against soil-inhabiting insects. In Entomopathogenic nematodes in biological control (Gaugler, R. and Kaya, H.K, eds), pp. 195-214, CRC Press Inc., Boca Raton, FL, USA. KUNG, S-P., GAUGLER, R. & KAYA, H.K. (1991). Effects of soil temperature, moisture and relative humidity on entomopathogenic nematode persistence. Journal of Invertebrate Pathology 57, 242-249. LELLO, E.R., PATEL, M.N., MATTHEWS, G.A. & WRIGHT, D.J. (1996). Application technology for entomopathogenic nematodes against foliar pests. Crop Protection 15, 567-574. LEWIS, E.E., SELVÄN, S., CAMPBELL, J.F. & GAUGLER, R. (1995). Changes in foraging behaviour during the infective stage of entomopathogenic nematodes. Parasitology 110, 583-590. LEWIS, E.E., GAUGLER, R. & HARRISON, R. (1992). Entomopathogenic nematode host finding: Response to contact cues by cruise and ambush foragers. Parasitology 105, 309-315. MASON, J.M., MATTHEWS, G.A. & WRIGHT, D.J. (1998). Appraisal of spinning disc technology for the application of entomopathogenic nematodes. Crop Protection (in press). MASON, J.M. & WRIGHT, D.J. (1997). Potential for the control of Pattella xylostella larvae with entomopathogenic nematodes. Journal of Invertebrate Pathology 70, 234-242.

52

MENTI, H. (1997). Comparative biology o/Steinernema feltiae and Heterorhabditis megidis from Greece and the UK. Ph.D. Thesis, Imperial College of Science, Technology & Medicine, University of London. PATEL, M.N., STOLINSKI, M. & WRIGHT, D.J. (1997). Neutral lipids and the assessment of infectivity in entomopathogenic nematodes: observations on four Steinernema species. Parasitology 114, 489-496. PATEL, M.N. & WRIGHT, D.J. (1996). The influence of neuroactive pesticides on the behaviour of entomopathogenic nematodes. Journal of Helminthology 70, 53-61. PATEL, M.N. & WRIGHT, D.J. (1997a). Fatty acid composition of neutral lipids energy reserves in infective juveniles of entomopathogenic nematodes. Comparative Biochemistry and Physiology 118B, 341-348. PATEL, M.N. & WRIGHT, D.J. (1997b). Glycogen: its importance in the infectivity of aged Steinernema carpocapsae. Parasitology 114, 591-596. SELVÄN, S., GAUGLER, R. & GREWAL, P.S. (1993a). Water contant and fatty acid composition of infective juvenile entomopathogenic nematodes during storage. Journal of Parasitology 79, 510-516. SELVÄN, S., GAUGLER, R. & LEWIS, E.E. (1993b). Biochemical energy reserves of entomopathogenic nematodes. Journal of Parasitology 79, 167-172. SHIH, J.J.M., PLATZER, E.G., THOMPSON, S.N. & CARROLL, E.J., JR. (1996). Characterization of key glycolytic and oxidative enzymes in Steinernema carpocapsae. Journal ofNematology 28, 431 -441. THURSTON, G.S., YANSONG, N. AND KAYA, H.K. (1994). Influence of salinity on survival and infectivity of entomopathogenic nematodes. Journal ofNematology 26, 345-351. WIJBENGA, J. & RODGERS, P.B. (1994). Lipid content of insect parasitic nematodes. Bulletin ofIOBC/WPRS 17, 155-158. WILLIAMS, E.C. & MACDONALD, O.C. (1995). Critical factors required by the nematode Steinernema feltiae for the control of the leafminers Liriomyza huidobrensis, Liriomyza bryoniae and Chromatomyia syngenesiae. Annals of Applied Biology 127, 329-341.

53

WOMERSLEY, C. (1990). Dehydration survival and anhydrobiotic potential. In Entomopathogenic nematodes in biological control (ed. Gaugler, R. & Kaya, H.K.), pp. 117-137. CRC Press Inc., Boca Raton, FL, USA. WRIGHT, D.J. (1998). Respiratory physiology, nitrogen excretion and osmotic and ionic regulation. In The Physiology of Free-living and Plant Parasitic Nematodes (eds Perry, R.N. & Wright, D.J.), Chapter 5, CAB International, Wallingford, UK (in press). WRIGHT, D.J., GREWAL, P.S. & STOLINSKI, M. (1997). Relative importance of neutral lipids and glycogen as energy stores in dauer larvae of two entomopathogenic nematodes, Steinernema carpocapsae and Steinernema feltiae. Comparative Biochemistry & Physiology 118B, 269-273. WRIGHT, D.J., ROBERTS, I.T.J. & EVANS, S.G. (1989). Effect of the nematicide oxamyl on lipid utilization and infectivity in Globodera rostochiensis. Parasitology 98, 151-154.

54

Is there a Link Between Stress Resistance and Longevity In Heterorhabditis Infective Juveniles? Ann M. Burnell, Sean A. O'Leary and Colin M. Stack Biology Department, National University of Ireland, Maynooth, Co. Kildare, Ireland. Summary: In the free living nematode Caenorhabditis elegans the age-1 phenotype is characterised by a large increase in post reproductive adult longevity and is also associated, in adults, with increased superoxide dismutase and catalase activities, increased tolerance to hydrogen peroxide and to paraquat and with increased tolerance to high temperatures and UV irradiation. We have isolated paraquat resistant mutants in the entomopathogenic nematode (EPN) Heterorhabditis megidis and we find that the infective juveniles (IJs) of these mutant lines have increased longevity, increased tolerance to UV irradiation and increased desiccation tolerance. Recent molecular studies show that the age-1 phenotype of C. elegans results from the expression in adult worms of genes which are normally activated only during entry into the long lived dauer juvenile (DJ) stage. The extension in IJ longevity which we detect in these paraquat resistant mutants of H. megidis supports the theory that increased resistance to reactive oxygen radicals leads to increased longevity and suggests that while there may be strong selection pressure for increased IJ longevity in natural populations of//, megidis, phenotypic improvement of IJ longevity can nevertheless be achieved by chemical mutagenesis. 1. INTRODUCTION A common feature of the life cycle of almost all multicellular organisms is the progressive decline in the efficiency of various physiological factors once the reproductive of life is over. Thus it has been postulated that genes which extend post-reproductive life span will not be selected by natural selection because such genes do not enhance the evolutionary fitness of their carriers (Medawar, 1952). Nevertheless it appears that there are genes, in particular, the stress-response genes, whose products have a large impact on the ageing process.

Thus

resistance to environmental stress, especially oxidative stress, has been frequently hypothesised to play a role in longevity (see review by Sohal, 1993).

2. THE FREE RADICAL THEORY OF AGEING The free radical theory of ageing proposes that oxygen radicals produced as a by product of metabolism inflict cellular damage at the molecular level and that this damage significantly contributes to the ageing process. According to this theory, increased resistance to oxidative stress, either by increased prevention of the initial damage or by an increased rate of repair of

the damage leads to increased longevity. Evidence supporting the free radical theory of ageing has been obtained in studies with Drosophila melanogaster and Caenorhabditis elegans.

C elegans mutants with increased

adult longevity have been isolated by Klass (1983). One of these mutants which resulted in an increase in mean lifespan of the hermaphrodites of 70% and in the maximal life span of 100% at 20 °C was further characterised by Friedman and Johnson (1988a, 1998b). They found that the life extension phenotype segregated as a single gene, designated the age-1 gene. The increase in life expectancy of the age-1 phenotype is due to an increase in the length of the post reproductive life.

Other life history traits such as rate of development, length of the

reproductive period, food uptake, movement and behaviour remain unchanged. The age-1 phenotype was subsequently found to be associated with increased levels of catalase and superoxide dismutase, enzymes which provide protection from oxidative damage and age-1 adult nematodes were also found to be resistant to high concentrations of hydrogen peroxide and of the superoxide generating drug methyl viologen (paraquat) (Larsen, 1993; Van Fleteren, 1993). Similarly, a line of D. melanogaster selected for increased longevity was also found to have increased activity of catalase and superoxide dismutase and to be more tolerant to paraquat (Dudas and Arking, 1995).

Further analysis of C. elegans age-1 mutants demonstrated that these were also more thermotolerant (Lithgow et al, 1995) and more resistant to UV irradiation (Murakami and Johnston, 1996). Mutations in some other C elegans genes, viz. daf-2, daf-23 (daf = dauer formation defective) and spe-26 (spe = spermatogenesis defective) also extend lifespan and confer a set of stress resistance phenotypes. A hypothesis was proposed that age-1 may be a co-ordinate regulator of a range of stress response genes that shift cellular physiology towards maintenance (Lithgow et al, 1995; Lithgow and Kirkwood, 1996)

3. PARAQUAT RESISTANT MUTANTS IN HETERORHABDITIS Since the age-J mutants of C. elegans which were isolated on the basis of their longevity were subsequently shown to be resistant to high concentrations of paraquat and since long lived lines of D. melanogaster were also resistant to paraquat, we tested the possibility that paraquat resistance might be used as a screen to isolate Heterorhabditis strains with increased longevity of the infective juvenile (IJ) stage. The mutant screen which we used was based on the ability of mutagenised IJs of H megidis to develop to hermaphrodites following exposure to 100 mM 56

paraquat for two days. development.

Under these conditions IJs of H. megidis UK211 fail to resume

Three paraquat resistant mutants were isolated. Two of these mutant lines

displayed increased longevity at 20 C, surviving for up to 120 and 140 days respectively, whereas the parental strain survived for up to 100 days (Fig. 1). The third mutant line displayed increased survival during storage at 20 C as compared to the parental strain, but did not survive for a greater period of time. In addition, the paraquat resistant lines had increased levels of superoxide dismutase activity (Table 1) and they were more resistant to UV radiation (Fig. 2). Thus the extension in IJ longevity which we detect in these paraquat resistant mutants of H megidis supports the theory that increased resistance to reactive oxygen radicals leads to increased longevity.

The increase in maximal longevity of 20 - 40% which we detected in the DJs of the paraquat resistant mutants is substantially less than the 100% increase in maximal life span detected in the age-1 hermaphrodites of C. elegans. No studies have been reported in C. elegans on the longevity of the DJ stage of any of the lifespan extension mutants, nor is it known whether the increased tolerance to UV exposure and temperature which was detected in

age-1

hermaphrodites also occurs in the DJ stage. The nematode DJ is a non feeding, long lived, developmentally arrested dispersal and/or infective stage. In contrast to other organisms in which genes which extend post reproductive longevity are likely to be selected against, it possible that there exists, in nature, a positive selection pressure for IJ/DJ longevity in EPN and in other parasitic and free living nematodes. Our data indicate, however, that while there may be strong selection pressure for increased IJ longevity in natural populations of H. megidis phenotypic improvement of this trait can still be achieved by chemical mutagenesis.

57

% Sur vi\ al

Time (days)

Fig. 1. Longevity of IJs of the parental strain (WT) and the paraquat resistant mutant lines (6.1, 6.2 and 6.4) of H megidis UK211 stored in water at 20 °C. Each point is the mean ± SE of six replicates.

Fig 2. The tolerance of IJs of the paraquat resistant mutant lines (6.1, 6.2 and 6.4) and the parental strain (WT) of H megidis UK 211 to UV irradiation for three min. Each point is the mean ± SE of six replicates. Mean survival values with the same letter are not significantly different (Duncan's multiple range test; ρ < 0.05).

5S

80 7060-



>

't ss

Fig 3. The desiccation tolerance of IJs of the paraquat resistant mutant lines (6.1, 6.2 and 6.4) and the parental strain (WT) of //. megidis UK 211. IJ survival was measured following exposure to 98% relative humidity at 20 °C for seven days. Each point is the mean ± SE of five replicates. Mean survival values with the same letter are not significantly different (Duncan's multiple range test; ρ < 0.05).

Table 1. Superoxide dismutase (SOD) activity* of freshly harvested IJs and aged IJs (stored in water at 20 °C for four weeks) of the paraquat resistant mutant lines (6.1, 6.2 and 6.4) and the parental strain (WT) of H. megidis UK 211. Each point is the mean ± S E of three replicates.

Nematode Strain WT

6.1

6.2

6.4

Freshly harvested

10.7 ±0.09

14.1 ±1.8

15.5 ±1.3

13.9± 1.2

Aged for 4 weeks

3.1 ±0.5

4.4 ± 0.4

4.2 ±0.8

3.8± 1.1

*units mg protein"

59

4. THE MOLECULAR IDENTITY OF THE C. elegans age-1 GENE Two temperature sensitive daf genes of C. elegans, daf-2 and daf-23, confer adult longevity phenotypes if the nematodes are shifted to the nonpermissive temperature at the J4 stage (Kenyon et α/.1993; Larsen et al, 1995). The age-1 gene of C elegans has been cloned by Morris et al. (1996) and it was found to map to the same genetic locus as the daf-23 gene (so the daf-23 gene is now assigned to the age-1 locus). The age-1 gene codes for a homologue of the catalytic subunit of mammalian phosphatidylinositol (PI) 3-kinase which is postulated to function as part of a protein kinase signalling cascade. The daf-2 gene (which also yields a longevity phenotype) codes for an insulin-like receptor gene (Kimura et al, 1997). This DAF-2 insulin-like receptor most probably mediates interactions the AGE-1 PI 3-kinase which acts in the same genetic pathway as daf-2. Genetic evidence indicates that the C. elegans gene daf-16 acts downstream of age-1. daf-16 codes for a DNA transcription factor (Lin et al. 1997; Ogg et al. 1997). Thus, in C. elegans, Age-1 seems to be part of an insulin-like signalling complex and signalling in this Age-l/Daf-16 pathway seems to be decisive in choosing which of two alternative developmental pathways is switched on in developing larvae: reproductive development and active metabolism or entry into the long lived non reproductive dauer larval stage. In the age-1 phenotype these two alternative states seem to be coupled, leading to a dauer-like longevity of adult worms.

5. CONCLUSIONS The post reproductive longevity detected in age-1 and daf-2 mutants of C. elegans appears to result from the expression in the adult of longevity mechanisms that are normally associated only with dauer juvenile formation. No studies have been reported in C. elegans on the longevity of the DJ stage of any of these lifespan extension mutants. We have isolated paraquat resistant mutants in H. megidis and we find that the IJs of these mutant lines have increased longevity, and increased tolerance to UV irradiation and increased desiccation tolerance. The extension in IJ longevity which we detect in these paraquat resistant mutants of H. megidis supports the theory that increased resistance to reactive oxygen radicals lead to increased longevity. Although there may be strong selection pressure for increased longevity of the infective stage juveniles in natural populations of H. megidis our data indicate that phenotypic improvement of this trait can nevertheless be achieved by chemical mutagenesis.

Acknowledgements: This work was carried out with the financial support of the European Union (STD CT 940273). 60

Refernces Dudas, S.P. & Arking, R. (1995). A coordinate upregulation of antioxidant gene activities is associated with delayed onset of senescence in a long-lived strain of Drosophila. Journal of Gerontology: Biological Sciences 50A, Bl 17-B127. Friedman, D.B. and Johnston, T.E. (1988a). A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. Genetics 118, 75-86. Friedman, D.B. and Johnston, T.E. (1988b). Three mutants that extend both the mean and maximum lifespan of the nematode Caenorhabditis elegans define the age-1 gene. Journal of Gerontology: Biological Sciences 43, B270-B276 Kenyon, C , Chang, J., Gensch, E, Rudner, A. and Tabtlang, R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature 366, 461-464. Kimura, K.D., Tissenbaum, FLA., Liu, Y. and Ruvkun, G. (1997).

daf-2, an insulin

receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science, 277, 942-946. Klass, M.R. (1983).

A method for the isolation of longevity mutants in the nematode

Caenorhabditis elegans and initial results. Mechanisms of Ageing and Development 6, 279-286. Larsen, P.A. (1993). Aging and resistance to oxidative damage in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the USA 90, 8905-8909. Larsen. P.A., Albert, P.S. and Riddle, D.L. (1995). Genes that regulate both development and longevity in Caenorhabditis elegans. Genetics 139, 1567-1583. Lin, K. Dormán, J.B., Rodan, A. and Kenyon, C. (1997). daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Nature 278, 1319-1322. Lithgow, G.J., White, T.M., Melov, S. and Johnston, T.E. (1995). Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress. Proceedings of the National Academy of Sciences of the USA 92, 7540-7544. Lithgow, G.J. and Kirkwood, T.B.L. (1996). Mechanisms and evolution of aging. Science 273, 80. Medawar, P.B. (1952). An Unsolved Problem of Biology. H.K. Lewis, London. Morris, J.Z., Tissenbaum, H.A. and Ruvkun, G. (1996). A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382, 536-539.

61

Murakami, S. and Johnston, T.J. (1996). A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans. Genetics 143, 1207-1218. Ogg, S., Paradis, S., Gottlieb, S., Patterson, G.I., Lee, L., Tissenbaum, H.A. and Ruvkun, G. (1997). The fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature, 389, 994-999. Sohal, R.S. (1993). The free radical theory of aging: an appraisal of the current status. Aging and Clinical Experimental Research 5,3-17. Van Fleteren, J.R. (1993).

Oxidative stress and ageing in Caenorhabditis

Biochemical Journal 292, 605-608.

62

elegans.

Desiccation Survival of Infective Juveniles of Steinernematid Nematodes Mavji N. Patel & Denis J. Wright Applied Entomology and Nematology Research Group, Department of Biology, Imperial College of Science, Technology and Medicine, Silwood Park, Ascot, Berkshire SL5 7PY, UK.

Summary: How well do infective juveniles (IJs) of entomopathogenic nematodes survive desiccation? This has been a question of great importance since the moment they were first used in biological control. However, we still lack an integrated answer to this question. The information so far gained has been fragmented and has mainly been restricted exclusively to one species (namely Steinernema carpocapsae). The study presented in this paper took a comparative approach in trying to answer the above question. The first part of the study looked at the relative survival ability of the four species, Steinernema carpocapsae, S. riobravis, S.feltiae and 5. glaseri, followed by studies on cuticle ultrastructure and preliminary biochemical studies to establish if active mechanisms were present to promote survival during desiccation.

1. GENERAL INTRODUCTION

Infective juveniles (IJs) of entomopathogenic nematodes are not normally exposed to desiccating conditions (Womersley, 1990). The soil environment provides adequate moisture, and even when the soil water potential falls to below -1.0 MPa, the relative humidity in the soil is still above 99% (Stirling, 1991). However, there are instances when moisture can be a limiting factor. For example, the most common method of applying nematodes to control soil insects is to spray them onto the soil surface. It is at this point that they are most prone to rapid drying, particularly when applied to relatively dry soil. At the other extreme, foliar applications, which are increasingly being considered against a number of pest insects, pose an obvious risk to nematode survival (Lello et al., 1996; Mason & Wright, 1997; Mason et al., 1998).

Furthermore, many commercial

nematode formulations partially desiccate the nematodes to improve survival and shelf life. Therefore, an understanding of how IJs can survive desiccation is important in order to select the appropriate species, and would provide information on how survival characteristics could be improved upon possibly by genetic manipulation.

This paper describes an investigation which (i) assessed the ability of four species of Steinernema (S. carpocapsae, S. riobravis, S. feltiae and S. glaseri) to survive desiccation and (ii) examined the likely mechanisms involved in surviving desiccation. Each section starts with a

review of the relevant literature, followed by a summary of the results. A detailed presentation of the work described in this paper can be found in Patel (1997), Patel et al. (1997a) and Patel & Wright (1998). 2. ASSESSING DESICCATION SURVIVAL

2.1 Introduction A number of studies have investigated the desiccation survival ability of steinernematid nematodes but, except for a few (Campbell & Gaugler, 1991a; Kung et al., 1991; Baur et al., 1995; Menti et al., 1997), most have concentrated exclusively on S. carpocapsae (e.g., Simons & Poinar. 1973; Ishibashi et al., 1987; Womersley, 1990; Glazer, 1992). The general finding has been that under slow drying conditions, S. carpocapsae (various strains) can survive for appreciable lengths of time. In addition, S. carpocapsae (strain All) survives desiccation better than S. glaseri (Kung et al. 1991) and S. riobravis (Baur et al., 1995). Behavioural adaptations, such as coiling has been induced in S. glaseri exposed to elevated RHs (97%), and in S. carpocapsae (strain All) on slow drying model substrates (Womersley, 1990), but in general, coiling has not been consistenti)· observed during desiccation and does not correlate with survival. Clumping has been found to aid the survival of S. carpocapsae IJs (strain Agrotis) when the IJs are desiccated in large aggregates (>1000 IJs) (Simons & Poinar, 1973).

However, it is not known whether such behavioural

responses occur naturally, particularly since aggregation and clumping may not be in direct response to dehydration (Womersley et al., 1998). Survival of individual IJs of S. carpocapsae exposed at 97% RH has been shown to be greater than that of Rotylenchulus reniformis, Aphelenchus avenae or Ditylenchus myceliophagus, although survival declined rapidly at lower RHs (Womersley, 1990). Womersley (1990) suggested that the sheath of IJs of entomopathogenic nematodes might help to reduce the rate of water loss during desiccation. However, Campbell & Gaugler (1991a) and Patel et al. (1997a) did not find evidence for the role of the sheath in desiccation survival in S. carpocapsae, although Campbell & Gaugler (1991a) found it to be important in the survival of Heterorhabditis bacteriophora. In the latter species, the sheath is more closely associated with the IJ body whereas in the case of S. carpocapsae it is more loosely fitting and so may not have a role in protection since it is readily lost (Campbell & Gaugler, 1991b; Campbell & Gaugler, 1992; Patel & Wright, 1998; section 3). 2.2 Survival of IJs of steinernematid nematodes Most of the methods which have been used to assess the ability of IJs to survive desiccation have involved drying large aggregates of nematodes (e.g., Simons & Poinar, 1973; Ishibashi et al., 1987; Kung et al., 1991; Glazer, 1992), and since the protective value of aggregation is well known, the level of survival recorded may have been unrealistic. Furthermore, the inconsistency between 64

different studies in the methods used has made comparing different nematode species difficult. While it is important to study survival characteristics under the conditions which the nematodes are normally exposed to, these are often difficult to reproduce in laboratory experiments and in most cases, particularly that of entomopathogenic nematodes, it is more useful to obtain comparative information on how well one species survives relative to others. Desiccating nematodes on glass slides or on an alternative non-absorbent surface has been found to be an effective method for investigating the ability of nematodes, particularly plant-parasitic species, to survive desiccation stress, and to study mechanisms of survival (e.g., Ellenby, 1968; Perry, 1977; Womersley & Ching, 1989; Ibrahim & Perry, 1993). The technique has been use to compare Greek and UK isolates of S. feltiae and H megidis (Menti et al., 1997) and we have used it to compare four commercial species of Steinernema (Patel et al., 1997a). More recently, Mason & Wright (1997) used it to screen Malaysian isolates of entomopathogenic nematodes to select the most suitable for use against larvae of the diamondback moth, Ρ lute I la xylostella. The most striking result obtained in our study (Patel et al., 1997a) was the superior survival of newly emerged S. carpocapsae compared with the other species (Figure 1A). However, aged IJs (newly emerged IJs were stored at 25°C in distilled water for 75-days) of S. carpocapsae were not able to tolerate desiccation as much as newly emerged IJs (Figure IB). The difference in survival ability of the four species is illustrated clearly when the IJs were desiccated at 80% RH (Figure 2). Determining water content by interference microscopy was found to a reliable and consistent method, more so than estimating wet and dry weights. The latter relies on drying large numbers of nematodes and estimating wet weight, which is not always accurate, whereas in contrast, interference microscopy can be used to directly measure the water content of single nematodes. The drying profiles for newly emerged IJs desiccated at 80% RH clearly showed that the superior survival ability of S. carpocapsae was a result of its lower rate of water loss (Figure 3A), and that the demise of this property in older IJs resulted in its poor survival (Figure 3B). The differences in the rate of water loss between the four species and between newly emerged and aged IJs directed us to take a closer look at the cuticle. Since, the nematodes had been 'fast dried', there was unlikely to have been time for biochemical adjustments and therefore, a difference in cuticle permeability was the most obvious explanation for the above result.

65

Figure 1. Fifty-percent survival times (S50 ±95% confidence interval) of (A ) newly emerged and (B) aged (75-day-old) infective juveniles desiccated on glass slides at a range of relative humidities.

Β

—Τ

S. carpucapta«

—·—

S. rwhravis

♦ -m-

S-ghMri S. fettine



—Τ'

-=»=£ Relative humidity (*/·)

Rel an ve humidity (%)

Figure 2. Survival (% ±SE) of (A ) newly emerged and (Β) aged (75-day-old) infective juveniles desiccated on glass slides at 80% relative humidity.

Β

£ X S. S.

■—Τ— —·— —♦— —·—

carptKopsae riohravis glasen felliae

Time (min) Time (min)

66

S. çarpocaftst, i'. riobravis .V giaurri S. felttav

Figure 3. Water contents (% ±SE) of (A ) newly emerged and (Β) aged (75-day-old) infective juveniles desiccated on glass slides at 80% relative humidity.

Β

■ ii carpocapsa* • S. riobruM* ■ S. glabri ■ S.feltia«

ï

3. C U T I C L E U L T R A S T R U C T U R E

3.1 The nematode cuticle—structure and function The nematode cuticle or integument is a highly ordered extracellular multilayered structure, which varies in different species and between developmental stages of the same species (Inglis, 1983; Wright, 1987, 1991; Bird & Bird, 1991). The cuticle is repeatedly shed and replaced during the life cycle as the nematode makes the transition between the four larval stages and the adult in a series of moults (Bird & Bird, 1991). The cuticle is a dynamic structure, which protects the nematode from the external environment, and acts as an exoskeleton, preserving the structural integrity of the nematode body while protecting the internal organs. The functions of the cuticle are therefore three-fold, being concerned with permeability, movement and growth (see Bird & Bird, 1991 for review).

The thickness of the nematode cuticle varies considerably, ranging usually

between 0-2 Tm and 50 Tm (Bird & Bird, 1991). It is generally agreed that the cuticle consists of a three-zoned or layered structure covered by a trilaminar epicuticle1 These zones are referred to by Bird & Bird (1991), from the surface inward, as the cortical, median, and basal zones. However, the literature is rife with various permutations of this terminology and a number of workers have subdivided one or more of these zones (Table 1 summarises the most frequently used nomenclature). Furthermore, there is much confusion over which layer or zone of the cuticle one set of nomenclature is describing.

For example, Maggenti (1981) uses the terms exocuticle,

mesocuticle and endocuticle, respectively, for cortical, median, and basal zones, but his description of the mesocuticle encompasses that of the median and basal layers, as defined by Bird & Bird 67

(1991). Therefore, the 'endocuticle' of Maggenti (1981) is not equivalent to the basal layer as defined by Bird & Bird (1991) but it does agree with description of the basal layer given by Peixoto & De Souza (1994). Most of what we know about nematode cuticle structure and function stems from work on animal-parasitic nematodes, and the popular C. elegans (see Bird & Bird, 1991 for review). I shall not attempt to provide details on cuticle synthesis and moulting as this is better obtained from other sources (e.g., Maggenti, 1981; Bird & Bird, 1991; Kennedy, 1991). However, there is very little information available on the function of the different layers of the cuticle. Regarding chemical composition, the cuticle consists of three groups of cuticular proteins: (1) collagens; (2) cuticlin; and (3) surface-associated proteins and glycoproteins (Politz & Philipp, 1992), with lipids forming the major non-proteinaceous components of the cuticle. Table 1. Nomenclature used to describe the zones or layers in the cuticle of a typical nematode.

Wright (1987); Bird & Bird (1991)

Maggenti (1981)

surface coat trilaminar epicuticle

Surface coat trilaminar epicuticle

cortical zone

Exocuticle

Peixoto & De Souza (1994)

Martinez & De Souza (1997)

surface coat trilaminar epicuticle externa! cortical layer

surface coat trilaminar epicuticle cortical layer

internal cortical layer median zone

intermediate layer

medial layer

fibrous layer basal layer

fibrous layer basal layer

Mesocuticle basal zone Endocuticle

The epicuticle and the surface coat are two consistent features of the cuticle. The epicuticle is a trilaminar structure consisting of two electron dense layers separated by a less electron dense layer (Maggenti, 1981). Other workers have described its structure as resembling that of biological membranes (Martinez & De Souza, 1995). For example, Gounaris et al. (1996) described the epicuticle of the muscle stage larvae of Trichinella spiralis as being composed of two extensive sheets of lipid in a non-bilayer configuration, which overlay a conventional lipid bilayer. However, 68

Wright (1987) argues that the analogy with biological membranes is confusing.

Locke (1982)

suggested that the descriptive term 'envelope' is more appropriate considering the function of the cuticle, although there has been reluctance to adopt this term and reference to 'membrane-like' or "membrane' continues (e.g., Martinez & De Souza, 1995; Gounaris et al., 1996). Obviously, reference to the epicuticle, as a membrane structure will continue to be controversial. The epicuticle is composed mainly of protein (collagen) and the upper surface is enriched with lipids (neutral and polar) and carbohydrates (Bird & Bird, 1991; Gounaris et al, 1996). Dunphy & Webster (1987) working with S. carpocapsae, suggested that the lipids in the epicuticle may have a role in regulating permeability and protecting the nematode against encapsulation by insect host haemocytes. On the surface of the epicuticle, a fuzzy coating is often visualised in electron micrographs. The fuzzy coating or surface coat is composed of sugars of mucin-like proteins and its thickness varies between different nematode species, for example, it is ca. 5 nm in second stage juveniles of A. tritici and ca. 15 nm in the infective larvae of T. spiralis (Bird, 1988 and references therein). The epidermis and/or secretory-excretory glands (Bird & Bird, 1991; Spiegel & McClure, 1995) secrete the surface coat. With regard to function, four suggestions have been put forward: regulation of cuticle permeability, protection against abiotic and biotic factors, lubrication to facilitate locomotion and, in the case of animal-parasites, as an antigenic surface (Brown et al, 1971; Dunphy & Webster, 1987; Grove et al, 1987; Bird et al, 1988; Bird & Bird, 1991, Maizels et al, 1993; Spiegel & McClure, 1995). Of all the cuticle layers, the epicuticle is thought to provide the major permeability barrier (Wright & Newall, 1980; Bird & Bird, 1991). The cortical zone lies just below the epicuticle and is relatively consistent in form throughout the Nematoda (Maggenti, 1981). Under the transmission electron microscope (TEM) it appears as an amorphous and often electron dense layer (Bird & Bird, 1991). In some parasitic nematodes, the cortical zone is divided into the external cortical layer, which has no visible structure, and the internal cortical layer, which is characterized by radial striations (Maggenti, 1981). The structure of the median layer of the cuticle is much more variable than the other layers. It may appear as a fluid-filled layer, as a layer containing proteinaceous rods or struts surrounded by fluids or, in some species, it may be almost impossible to differentiate from the basal zone (Wright, 1991). The basal zone or striated layer contains layers of collagenous fibres arranged in various ways within a matrix, which gives the cuticle strength and flexibility (Inglis, 1983). Maggenti (1981) refers to a layer termed the endocuticle, which is not present in all nematode cuticles and hence very little is understood about its structure or function. It is common throughout the Adenophorea and in some members of the secernentean order Rhabditida. The basal layer is fibroid in nature but the level of 69

Organization is not as complex as that seen in the striated layer (basal layer) and it is often disorganised (Maggenti, 1981). Jackson & Bradbury (1970) identified such a layer in the cuticle of S. glaseri IJs. 3.2 A comparison of cuticle structure of steinernematid infective juveniles The differences we found in survival and the rate of water loss during desiccation at 80% RH between S. carpocapsae and the other three species suggested that the ultrastructure of the cuticle might provide some explanation for this (Patel & Wright, 1998). Transmission electron microscopy of newly emerged IJs of the four species showed that the thickness of the cuticle (sections taken at anterior end) was proportional to the size ratio (length to width) of the IJ (Table 2). We divided the nematode cuticle into four layers: epicuticle, combined cortical and median layer, striated layer and the fibrous mat (Figure 4). The proportion that each layer constituted to the total thickness is shown in Table 2. Steinernema carpocapsae, which survives desiccation on glass slides better than the other three species (see above), had the thinnest epicuticle which may indicate that either the epicuticle is not the main regulator of cuticle permeability (with regard to water) or that the chemical composition of the epicuticle rather than its thickness is functionally more important. The most obvious difference in the cuticle of S. carpocapsae as compared with the other three species is that the striated layer in the cuticle of newly emerged IJs is proportionately greater. The striated layer provides structural support to the cuticle and therefore its greater thickness in the cuticle of S. carpocapsae may be functionally related to nictation behaviour. The thickness of the cuticle of aged IJs of S. carpocapsae was greater (+50%) than that of newly emerged IJs (Table 2): this was due to a greater than 100% increase in the combined cortical and median layer.

Two possible explanations for this increase are: (i) new material was

synthesized; (ii) the fluid content of this layer increased due to an increase in the permeability of the outer layers of the cuticle. The latter seems more likely since aged IJs of S. carpocapsae have a greater rate of water loss when desiccated at 80% RH as compared with newly emerged IJs, which suggests that a deterioration occurs in the permeability barrier e.g., degeneration of the epidermis resulting in a reduced ability to osmoregulate; comprise in the integrity of the epicuticle. Alternatively, the limited amount energy reserves available in older IJs of S. carpocapsae and S. riobravis (Patel et al, 1997b) might have reduced the efficiency of energy dependent processes such as osmoregulation. The sheath of S. glaseri was thicker than that of S. feltiae (Table 2), which may explain why IJs of 5. glaseri do not lose their sheaths as readily as S. feltiae, i.e. a thicker cuticle would be stronger and therefore less likely to tear. In both species, the epicuticle and the striated layer are present and even possibly the fibrous mat. Furthermore, the sheath did not appear to be physically 70

attached to the cuticle. It is possible that in IJs of steinernematid nematodes the sheath is an evolutionary relic and serves no functional purpose.

I

Figure 4.

li*î

High power TEM section of a newly emerged exsheathed infective juvenile of

Steinernema feltiae; section taken from the anterior portion of the nematode. Scale bar = 0.1 μτη. ep=epicuticle, ccm=combined cortical and median layer, sl=striated layer, fm=fibrous mat, ed=epidermis. The fibrous mat is the layer indicated between the two solid lines.

71

Table 2. Thickness of layers of the cuticle of newly emerged and 80-day-old infective juveniles of Steinernema carpocapsae, and newly emerged infective juveniles of S. riobravis, S. feltiae and S. glaseri measured from TEM sections taken from the anterior end of the nematode. S. carpocapsae Newly 80-day-old emerged Nematode size ratio (L/W)1 Cuticle thickness as a % of nematode radius Cuticle layer (nm)3 M total thickness epicuticle cortical and median striated layer fibrous mat

2

., /

23-2

4-5

6-7

408 (5) 18 (31) 200

273 16 85

±33 ±3 ±2

165 10

± 3 4 (60) ±0-6 (4)

*

Sheath IT

22-3

166 19

S. riobravis

S. glaseri

22-2

32-6

26-3

5-3

81

4-7

± 0.4 ±2 ±2

(4) (49)

312 ± 2 4 30 ±2 103 ± 1 0

±00 ±3

(41) (5)

157 ± 1 4 19 ±0-3

*

S. feltiae

*

(10) (33) (50) (6)

374 ± 2 8 33 ± 5 136 ± 1 1

(9) (36)

458 ± 4 5 34 ± 6 182 ± 19

(40)

185 ± 12 15 ±0-5

(49) 216 ± 11 (4) 1') ± 4

(47) (4)

181

±20

273

(6)

±20

__,__,.

Nematode radius measured at anterior part of nematode (between 50-100 Tm along the nematode

body). 3

Mean thickness (nm) ± SE; n = 4-5 IJs; values in parentheses are percentages of total cuticle

thickness. 4

Total cuticle thickness excludes sheath. * Sheathed IJs were not available. 4. METABOLIC INFLUENCES ON DESICCATION SURVIVAL

4.1 Introduction The mechanisms by which nematodes use to survive dehydration are summarised in Table 3. They can either be behavioural and/or physical or biochemical. The rate of water loss is critical to survival (Crowe & Madin, 1975), with rates of water loss in excess of 3% h" usually resulting in reduced survival. Slow dehydration allows time for the necessary behavioural and biochemical adjustments needed if the nematode is to enter a state of anhydrobiosis (Barrett, 1991).

72

Table 3. Mechanisms used by nematodes to survive dehydration stress. Behavioural and Physical

Physiological and Biochemical

Cuticle/sheath (e.g., moulted cuticles)

Synthesis of protectants (e.g., trehalose, glycerol, antioxidants)

Coiling Aggregation/clumping

Alterations to membrane phospholipids? (e.g., increasing the degree of unsaturation of constituent fatty acids or changes in phospholipid species)

4.2 Biochemical mechanisms . The biochemical mechanisms of desiccation survival in nematodes are poorly understood. One biochemical change that has been reported in anhydrobiotic nematodes is the accumulation of polyols/and or sugars, which act as protectants of biological membranes and intracellular proteins during dehydration (e.g., Madin & Crowe, 1975; Clegg et al,

1982; Womersley, 1981a,b;

Womersley, 1990; Barrett, 1991; Behm, 1997). For example, Madin & Crowe (1975) found that in the mycophagous nematode, Aphelenchus avenae, lipid and glycogen levels declined while glycerol and trehalose accumulated during dehydration. The authors showed a strong correlation between survival of the nematode in dry air and the production of these two compounds.

Similar

observations, in some cases involving different compounds, have been made in other nematode species (Womersley & Smith, 1981; Womersley et al, 1982; Higa & Womersley, 1993). Trehalose has been the main focus of research since it is present in high concentrations in a number of organisms capable of anhydrobiosis (e.g., bacteria, yeast, nematodes and brine shrimps) and because of this, Crowe et al (1992) concluded that the physical principles governing the stability of dry materials may be universal. During dehydration, proteins can be protected by trehalose in two main ways: it can replace 'bound' water and reduce the reaction of dried glucose with amino-acid side chains of proteins (known as the 'browning' or Maillard reaction) (Behm, 1997). There are two mechanisms by which trehalose is thought to stabilise membranes (Hoekstra et al, 1997). The first involves the direct interaction between the hydroxyl groups of trehalose and the phosphate of phospholipids, which acts to replace water normally associated with the phospholipid bilayer. The end result is a depression in the phase transition temperature (Tm) of the dry phospholipids, maintaining membrane fluidity and keeping the bilayer in the liquid crystalline state, thus preventing the transition to the gel phase, which would otherwise result in loss of membrane integrity during dehydration (see Crowe et al, 1992). Secondly, trehalose can reduce Tm of dry phospholipids by forming glass (vitrification). 73

This is an indirect interaction, whereby the phospholipids are trapped in a sugar glass, a supersaturated and thermodynamically unstable solid solution (Sun et al, 1996). However, recent evidence suggests that vitrification alone is not sufficient to depress Tm in dry phospholipids (Crowe et al, 1996). The functional role of glycerol does not parallel that of trehalose particularly since it is fusogenic in dry membrane systems (e.g., Gekko & Timasheff, 1981; Crowe et al, 1984). However, the vitrification of carbohydrates during dehydration may perturb the damaging effects of glycerol in anhydrobiotes known to accumulate this polyol (C.Z. Womersley, pers. coram.). The Tm can also be depressed by increasing the degree of unsaturation in the fatty acids of the phospholipids (Crowe et al, 1992).

Increasing the unsaturation of the phospholipid acyl chains increases the

fluidity of the bilayer through increased spacing of the phospholipid head groups.

Such a

mechanism has been found to operate in some xerophytic plants (e.g., Hoekstra et al, 1992; Navariizzo et al, 1995) but has yet to be demonstrated in nematodes. The universal view that trehalose is a pivotal factor in anhydrobiotic survival has recently come into question. Higa & Womersley (1993) argued that the reported synthesis of trehalose and glycerol during dehydration in large aggregates of A. avenae (Madin & Crowe, 1975) may have been a consequence of the experimental protocol rather than the result of changes that occur under more natural conditions. Higa & Womersley (1993) used smaller aggregates of A. avenae and found that even though large quantities of trehalose were produced during preconditioning at 97% RH, the nematodes showed only a limited ability to survive direct transfer to relative humidities of 75% and below. In contrast, exposing nematodes to gradual dehydration resulted in more of the nematodes surviving, suggesting that rate of water loss was more critical in survival than the presence of trehalose. The rate of water loss has also been shown to be paramount in the survival of the mycophagous nematode Ditylenchus myceliophagus (Womersley, 1988). The influence of the dehydration regime or experimental protocol on trehalose content has also been demonstrated in other anhydrobiotic organisms (Higa & Womersley, 1993). With regards to entomopathogenic nematodes, our knowledge of survival mechanisms is very poor. This is somewhat surprising bearing in mind the commercial importance of these nematodes, although this very attribute may explain the gap in current knowledge. The entomopathogenic nematode industry is relatively small and the production costs and small market share has meant that companies have not been able to afford investment into research and development which does not yield immediate returns.

The little amount of research that has been done using

entomopathogenic nematodes has provided evidence to suggest that the survival mechanisms may be similar to those implied in other nematode groups. Womersley (1990) found that when IJs of S. carpocapsae (strain All) were slow-dried at 97% RH on 1% agarose, levels of lipid and glycogen 74

declined while trehalose levels increased from 0-2% dry wt in fully hydrated nematodes to 7-0% dry wt, a level comparable with that found in other anhydrobiotes. However, there have been no other published studies which confirm these observations or which have taken the work further. 4.3 The role of lipids and carbohydrates in desiccation survival Here we describe a preliminary study which attempted to define the role of lipids and carbohydrates in the desiccation survival ability of IJs of entomopathogenic nematodes. The question we proposed was: is the utilization of lipids and glycogen a prerequisite for enhanced desiccation survival? The experiment involved pre-incubating newly emerged IJs of S. carpocapsae with or without the glycolytic inhibitor iodoacetamide (Patel & Wright, 1997a), and then preconditioning the IJs at ca. 97% RH for 72 h under aerobic or anaerobic conditions.

The

anaerobic treatment was intended to block the utilization of lipids (θ-oxidation is an aerobic process) but not carbohydrates and the inhibitor treatment was intended to reduce glycogen utilization.

A combined inhibitor-anaerobic treatment would in principle reduce glycogen

utilization and block θ-oxidation. An anaerobic environment was created inside a vacuum desiccator continuously purged with oxygen-free nitrogen (Figure 5) and all solutions were bubbled through with nitrogen. Newly emerged IJs of 5. carpocapsae were incubated in buffered distilled water or buffered iodoacetamide (10"4 or 10~3 M) for 48 h at 20 °C. The nematodes were then concentrated in fresh distilled water and transferred to a glass dish lined with Parafiini (the dish was rotated at 45° to spread out the nematodes), and conditioned at 97% RH for 72 h at 23 °C either in an aerobic or anaerobic environment. After conditioning, a sample of nematodes from each treatment was desiccated at 80% RH on glass slides and survival determined after set periods of desiccation. The remainder were immediately freeze-dried and sonicated.

The samples were then subjected to a comprehensive biochemical analysis: (I)

Glycogen content was determined (Patel & Wright, 1997a).

(II)

Other sugars and polyols were analysed using a Dionex Chromatograph (Higa & Womersley, 1993).

(III)

The lipids were fractionated by HPLC to separate and quantify the individual phospholipid species. An analysis of the fatty acid composition of different lipid classes had been carried out on undesiccated IJs (Patel & Wright, 1997b,c), but apart from the preliminary work by Womersley (1990), the fatty acid composition of the different phospholipid species in desiccated/anhydrobiotic IJs had not been determined. 75

The experimental analysis is still in progress and we expect to publish our findings this year. Figure 5. Experimental apparatus used to create an anaerobic environment. Humidity chambers were placed inside the vacuum desiccators and nitrogen gas was continuously passed through during the preconditioning period.

N;

5_Z

]

ι

ν-

■χ

Water trap

υ

y Vacuum desiccators

5. CONCLUSION

The focus must be on understanding how IJs of entomopathogenic nematodes survive in the various habitats in which they are used. This means reliably assessing how one species survives stresses such as desiccation relative to others, and determining the mechanisms involved. In our study, we compared how individual nematodes survived before going on to explore the mechanisms that may be responsible for some species surviving better than others. The next step will be to establish a genetic basis for the results that we have obtained.

Acknowledgements—We thank Dr Roland Perry (BBSRC IACR-Rothamsted) for help with the interference microscopy and Professor Christopher Womersley (University of Hawai'i) for help with some of the biochemical analysis. The research was supported by the Biotechnology and Biological Sciences Research Council (UK), Biosys Inc. (formally of Columbia, MD, USA) and CAB International Institute of Parasitology (now CABI Biosciences).

76

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C. (1990). Dehydration survival and anhydrobiotic potential. In Entomopathogenic

nematodes in biological control (ed. Gaugler, R. & Kaya, H.K.), pp. 117-137. CRC Press Inc., Boca Raton, FL, USA. WOMERSLEY,

C. & CHING, C. (1989). Natural dehydration regimes as a prerequisite for the

successful induction of anhydrobiosis in the nematode Rotylenchulus reniformis. Journal of Experimental Biology 143, 359-372. WOMERSLEY,

C.Z.,

HIGA,

L.M. & WHARTON, D.H. (1998). Survival biology. In The physiology and

biochemistry of free-living and plant-parasitic nematodes (ed. Perry, R.N. & Wright, D.J.). CAB International, Wallingford, Oxon, UK. In press. WOMERSLEY,

C. & SMITH, L. (1981). Anhydrobiosis in nematodes - 1 . The role of glycerol

myo-inositol and trehalose during desiccation. Comparative Biochemistry and Physiology 70B, 579-586. WOMERSLEY,

C,

THOMPSON,

S.N. & SMITH, L. (1982). Anhydrobiosis in nematodes II:

Carbohydrate and lipid analysis in undesiccated and desiccate nematodes. Journal of Nematology 14, 145-153. WRIGHT,

D.J. & NEWALL, D.R. (1980). Osmotic and ionic regulation in nematodes. In Nematodes

as biological models Vol 2 (ed. Zuckerman, B.M.), pp. 143-164. Academic Press Inc., New York, NY, USA. WRIGHT,

K.A. (1987). The nematode's cuticle - its surface and the epidermis: Function, homology,

analogy - a current consensus. Journal of Parasitology 73, 1077-1083. WRIGHT,

K.A. (1991). Nematoda. In Microscopic anatomy of invertebrates: Aschelminthes, Vol 4

(ed. Harrison, F.W. & Ruppert, E.E.), pp. 196-218. Wiley-Liss, New York, NY, USA.

Physiological and beavioural adaptation of Steinernema feltiae to desiccation stress. Aharon Solomon*, Han Paperna* and Itamar Glazer **. * Department of Animal Sciences, Faculty of Agriculture of the Hebrew University of Jerusalem, Rehovot 76100, Israel, and ** Department ofNematology, ARO, The Volcani Center, Bet Dagan, 50250, Israel.

Summary: In the present study we compared the desiccation tolerance of three strains of Steinernema feltiae (IS-6, IS-15 ,SF). All strains can survive desiccation at 75% or 85% RH only after a slow dehydration regime at 97% RH for 3 days, 23°C It is suggested that slow dehydration of the infective-juveniles (IJs) induces a quiescent anhydrobiosis state which enabled their survival at the low humidity. The IS-6 strain isolated from the desert region of Israel exhibited the best desiccation tolerance. The second best tolerance was observed in the IS-15 strain isolated from the northern part of Israel, while the poorest tolerance was exhibited by the SF strain, which was obtained from Germany. The higher desiccation tolerance of the IS-6 and IS-15 strains was associated with a dispersal response of aggregated IJs at the slow dehydration regime, which was followed by entrance into a state of anhydrobiosis as individually coiled IJs. Whereas IJs of the SF strain remained in a clump formation. The IS-6 strain was further used to determine the optimal conditions that induce anhydrobiosis, recovery and changes in trehalose and glycogen levels during those processes. An increase in trehalose levels were determined in IJs after the 3 day's exposure to 97% RH (from 0.3 to 0.6 Trehalose g/Protein g), while an opposite trend was observed in glycogen concentrations (from 0.09 to 0.02 Trehalose g/Protein g). Upen rehydration glycogen levels returned to its original concentration after 24h, while trehalose reached only to 50% from the original concentration (0.16 Trehalose g/Protein g) by the same time. Survival of individual IJs at 85% RH was positively correlated to the initial pre-conditioned clump size (ranging from 70 to 7700 IJs) (r=0.875, P«0.05). The same recovery rates of pre-conditioned IJs exposed to 85% RH over a period of 12 days, were obtained with either direct immersion in distilled water or following a 24 hours exposure to 100% RH prior to immersion in water. No significant differences were observed in virulence and ability to penetrate Tenebrio molitor larvae between desiccated IJs (5 days at 85% RH) and non-desiccated nematodes. INTRODUCTION Infective-juveniles (IJs) of steinernematid and heterorabditid nematodes are currently used as biological control agents, but their sensitivity to desiccation and other environmental constraints reduces their field efficacy (Kaya & Gaugler, 1993). Poor stability in storage is another factor limiting nematode expansion beyond niche markets (Friedman, 1990). Anhydrobiosis is a general term for a reversible

physiologically arrested state of dormancy that results from the absence of water. The following traits broadly define the characteristics of anhydrobiotes (Barrett, 1991): true anhydrobiotes can lose up to 95-98% of their body water and in anhydrobiosis they have virtually no metabolism, thereby conserving energy reserves. Nematodes in the anhydrobiotic state

are highly resistant to extreme environmental conditions

(Demeure & Freckman, 1981). Entomopathogenic nematodes are capable only of a shallow level of dormancy described as quiescent anhydrobiosis (Womersley, 1990). In the present study we compared the desiccation tolerance of three different strains of Steinernema feltiae and determined in one selected desiccation tolerant strain (i) the anhydrobiosis induction conditions affecting the desiccation survival (ii) changes in trehalose and glecogen levels during the anhydrobiosis process (iii) the influence of desiccation on the infectivity and virulence of this entomopathogenic nematode.

MATERIALS AND METHODS Nematode culture In the present study, IJs of three different strains ofSteinernema feltiae, IS-6, IS-15 and SF were studied. The IS-6 strain was isolated from the soil of a citrus orchard in the Negev, a semi-arid region in Israel. The IS-15 strain was isolated from the soil of an olive orchard in Galilee, in the northern part of Israel. The SF strain was obtained from the laboratory of Dr. Ralf-Udo Ehlers, north of Hamburg near Kiel, Germany. The new isolates were recovered from soil samples using the last-instar larvae of the greater wax moth, Galleria mellonella, in a live baiting technique (Fan & Hominick, 1991). Nematodes were reared in larvae of G. mellonella as described by Dutky et al. (1964). The emerging IJs were stored for 3-4 weeks in 250 ml distilled water in culture flasks at 5°C prior to use in the experiments. Desiccation Survival Comparison of desiccation tolerance of S. feltiae strains Relative humidity (RH) levels were controlled in sealed desiccators with 60 ml of saturated salt solutions at 23°C as follows: K2SO4 for 97% RH, KCl for 85% RH, and NaCl for 75% RH (Winston & Bates, 1960). Relative humidity of 100% was established with 90 ml distilled water. For the desiccation experiments, 20 μΐ of distilled water containing 660±50 IJs from the different strains, were placed on the surface of a cap of an Eppendorf tube. The excess water was removed with a tip of a X4

filter paper (Whatman No. 1). Under these conditions, the nematodes formed an aggregated clump on the Eppendorf cap. Caps were placed on petri-dishes (5 cm diameter) and immediately transferred to the desiccators with the different RH. The desiccation treatments included: (;') fast dehydration - direct exposure to 85% or 75% RH for 3 days; (;'/) slow dehydration- initial exposure for 3 days at 97% RH (pre-conditioning treatment) followed by exposure to 85% or 75% RH for 3 more days. Nematode survival was determined daily following rehydration by exposure to 100% RH for 24 h, prior to immersion in distilled water for another period of 24 h. Nematode viability was scored by counting active IJs or by motility following a gentle prodding with a hair probe under an inverted microscope. Each treatment was replicated three times. Factors affecting desiccation survival of the IS-6 strain: (i) The behaviuoral response of aggregated IJs in the slow dehydration regime was monitored after 4, 20, 44, and 72 h. Samples were taken from the desiccators and nematode behaviour was observed under the inverted microscope. Numbers of coiled vs non-coiled individual IJs were determined in clumps of

70±5, 1550±50 and

7700±500 IJs and distribution of nematodes was recorded. Three replicates were examined from each desiccation time. The behavioural response to 97% RH was also monitored in the IS-15 and SF strains, but was not quantified. (//) The effect of the initial clump size (70±5, 600±25, and 7700±500 IJs) on IJs survival was determined after conditioning the different clumps at 97% RH for 3 days followed by exposure to 85% RH over a period of 9 days at 23±1°C Each treatment was replicated five times. A possible correlation between desiccation survival of IJs and the initial pre-conditioned clump size was determined in a different experiment, by exposing six clumps of different

sizes (70±5, 660±50, 1660±36, 4400±300, 5200±390, and

7700±500 IJs) to 85% RH for a fixed period of 3 days. Their recovery rates were determined as described above. Each treatment was replicated five times. (///') To determine the effect of the rehydration regime on nematode recovery from the anhydrobiotic state, IJs were either rehydrated directly in distilled water overnight or exposed to 100% RH for 24 h prior to immersion in distilled water at 23±1°C. In this

85

experiment, 4400±300 IJs were used to create the initial clump of each sub-sample. Each treatment was replicated five times.

Extraction of trehalose and glycogen Samples of 200 mg of IJs of the IS-6 strain were taken after been exposed to 97% RH for 4, 8, 24, 48, 72 h and after rehydration of 4, 8, 12 and 24h in water. The nematodes were dipped in liquid nitrogen and kept frozen until use. Each sample was boiled in 1 ml of boiling distilled water at 100°C for 10 min, and were homogenized in liquid nitrogen with a mortar and a pestle. The homogenates were centrifuge at 14,000g (30 min, 4°C), and the supernatant was stored in liquid nitrogen until used. Protein concentration in each sample was determined with the Lowry method (Lowry et al, 195 U using bovine serum albumin (sigma, Israel) as a standard. Enzymatic reactions Protein denatoration and solution clarification was preformed on each supernatant befure the enzymatic hydrolysis, with Carrez-I solution (3.6% of Potassium ferry-cyanide) and Carrez- II solution (7.2% of Zinc sulfate) in 0.5M NaOFI pH=7.8 (Boeringer-Mannheim, 1986). Trehalose was assayed with trehalase (Sigma, Israel) as followes: 50 μΐ of extract was incubated for 1 h at 30°C in a toatl volume of 250 μΐ with 150 μΐ 25 niM sodiom acetate, pi 1=5.7 and 0.27 U/ml trehalase. Glycogen was assayed with 2 U/ml amyloglycosidase (sigma) as follwes: 125 μΐ of extract was incubated for 2h at 37°C in a total volume of 250 μΐ with 120 μΐ 25 mM sodiom acetate, pH=4.5 (Koning et al, 1992; Blazquez e/α/., 1994). Determination of treahalose and glycogen concentration In each sample trehalose and glycogen concentration were calculted from the formed glucose, and were expressed per gram soluble protein. Glucose was quantified using glucose oxidase-peroxidase kit (trinder) of Sigma, Israel. In calculating the trehalose and glycogen concentration, corrections were made for the glucose concentration in the non-enzymatic treated supernatant. Effect of desiccation on the IS-6 infectivity and pathogenicity Rehydrated IJs from a desiccation treatment (pre-conditioning followed by 5 days exposure to 85% RH) and non-desiccated IJs were exposed to the last-instar larvae of the meal-worm Tenebrio molitor in 100 ml plastic containers filled with 1 lg moist vermiculite (30% water content). In each container, 14 400±3025 IJs and 13 S6

larvae of T. molitor were placed. Ten containers for each treatment were incubated at 23±1°C Nematode-free containers with insect larvae were used as controls. Total mortality of the insects was recorded 72 h after exposure to the nematodes. Ten cadavers from each treatment were dissected under an inverted microscope after 48 h, and the numbers of nematodes which had invaded the insect were counted. SEM examination of desiccated IJs of the IS-6 strain For observation with scanning electron microscopy (SEM), dehydrated infective-juveniles as described above were coated directly with gold as described by Crowe et al. (1978) and were examined with a Jeol ISM-5140C Statistical analysis Statistical analysis was performed with the SAS 6.04 software package. To compare survival curves, a linear curve of survival by time was fitted for each treatment, after performing a logit transformation on the survival data [log (p/l-p)} with ρ = percentage of surviving IJs. The model was fitted by GLM, using the repeated measurements from each desiccator. The slopes of the curves were compared overall by a F-test and in pairs by Γ-test. In addition, the percentages of survival at the end of each experiment were compared by one-way Anova after arcsine transformation. In the 75% RH treatment, the change in survival rates over time did not fit the logit model, therefore we used a two-way analysis of variance with repeated measurements after arcsin transformation. Recovery rates of

IJs in the

different rehydration regimes were also compared by the latter statistical test. The correlation between desiccation survival of IJs and the initial clump size was measured by the Pearson correlation coefficient. A semi-logarithmic regression was fitted to the survival rate after arcsin transformation. After back-transforming the regression equation, a prediction equation was determined for percentage of survival. Mean numbers of nematodes recovered in the insect cadaver were compared over two treatments by the Wilcoxon-Mann-Whitney two-sample test. Chi-square values were calculated to compare coiling rates of IJs for the different clump sizes and survival of insects in the pathogenicity experiments. RESULTS When IJs of the three different strains were exposed directly to a fast-dehydration regime, complete mortality was recorded with all strains after 24 h of exposure. However, after a pre-conditioning treatment, all strains survived at 75% and 85% RFI. 87

Survival curves of pre-conditioned IJs of the different strains in 85% RH at 23°C are presented in Fig. 1. By day 3, the SF strain exhibited the poorest survival rate (24.7±9.3% SE, n=3), compared to the IS-6 and IS-15 strains (63.3±4.7% and 52.3±1.7% n=3, respectively) (F=8.76, d.f=2, PO.05).

> Í, 3 GO

1

2

Time (days) Fig. 1. Survival curves of pre-conditioned infective juveniles (IJs) of three different strains of Steinernema feltiae (SF, IS-15, and IS-6) in 85% RH at 23°C (n=3; error bars show ± S.E. [initial clump size of 660±50 IJ] ).

The IS-6 strain was the only strain that survived 48 to 72 h of exposure to 75% RH (F=12.9, d.f=2, P«0.05) (Fig. 2).

■ SF

i ■ IS-15J ! D IS-6

Time (days) Fig. 2. Survival rates of pre-conditioned IJs of three different strains of Steinernema feltiae (SF, IS-15, IS-6) in 75% RH at 23°C (n=3; error bars= ± S.E. [initial clump sizeof660±50IJ]). Factors affecting desiccation survival of the IS-6 strain (i) When IJs were exposed in clumps of 1550 and 7700 IJs to 97% RFI for 3 days, they exhibited a characteristic pattern of behaviour: after 4 h of exposure they were active and aggregated in a ball-shaped formation. Within 20 h they started to disperse from the clump on the exposed surface; by this time only 5% (N=147) of IJs from both clumps were found coiled (/>>0.05). A fter 44 h of exposure, 64.4% (N=720) and 33% (N=210) of the IJs from the large (7700 IJs) and small clumps (1550 IJs) were found individually coiled, respectively (P0.05), and 0% mortality of the control group, n=10). Numbers of post-desiccated and non-desiccated nematodes that penetrated the insect cadaver were also similar (104±26 SE vs 117±17, n=10) (P>0.05)

8

12

16

24

Time (h) Fig 9. Accumulation of glycogen in IJs of Steinernema feltiae IS-6 strain during recovery from desiccation (n=2; error bars= ± SD).

0.7 τ-

Ι 0.5

8

12

16

20

24

Time (h) Fig 10. Trehalose levels in IJs of Steinernema feltiae IS-6 strain during recover)' from desiccation (n=3; error bars= ± SD). DISCUSSION We demonstrated that a quiescent anhydrobiosis state can be induced in different strains of S. feltiae after exposure to 97% RH for 72 h. These findings are in agreement with previous results for other steinernematid species (Popiel et al, 1989; 93

Womersley, 1990) and demonstrate the importance of the slow dehydration rate as a prerequisite

for

induction

of

anhydrobiosis.

Womersley

(1990)

classified

steinernematid nematodes as slow dehydration strategists, as they inhabit the upper soil profile where the rates of water loss are moderate. Patel et al. (1997) improved the success of survival of S. feltiae at 80% RH by allowing them to dry slowly on 1% agarose. Womersley (1987, 1990) and Barrett (1991) reviewed the importance of slow rates of water loss in different species of nematodes in modulating metabolic and biochemical processes crucial for the successful induction of a state of anhydrobiosis. In the present work, it has been shown that strains differ in their ability to survive desiccation. The IS-6 strain, which obtained from the desert region of Israel, exhibited the highest desiccation tolerance, indicating adaptation to the harsh environmental constraints in the desert habitat. In addition, a difference in the behavioural response to desiccation was also observed among these strains. The higher desiccation tolerance of the IS-6 and IS-15 strains was associated with a dispersal response at 97% RH and entrance into anhydrobiosis as individuals in a coiling position, whereas poor desiccation tolerance was characteristic of the SF strain, which remained in the clump formation at 97% RH after 3 days. The ability to disperse may be attributed either to physiological differences such as synthesis of trehalose or stress proteins, and intrinsic mechanisms for control of water loss. Which is probably slower in the IS-6 and IS-15 strain and faster in the strain with the poorer desiccation tolerance. Those assumptions, however, still needs to be verified. Patel et al. (1997) and Menti et al. (1997) presented evidence of differences in the rate of water loss between species and strains of steinernematid nematodes which were related to survival rates. Those studies, however, were conducted under direct exposure to lower relative humidity regimes (0-80% RH). We found that coiling of individual IS-6 IJs was proportional to the increase in dehydration time at the 97% RH treatment. Patel et al. (1997) also observed large proportions of coiled S. feltiae (UK76 strain) in slow drying conditions on 1% agarose. The physical response of coiling is thought to reduce the surface area of the nematode body exposed to the dry environment and to subsequently reduce the rate of evaporative water loss (Bird & Buttrose, 1974). Our results demonstrated a high positive correlation between the initial clump size of pre-conditioned IJs of IS-6 strain

94

and desiccation survival. Further more higher significant coiling rate of 70% was observed in the large clump (7700 IJs) after 72h. This result strongly suggests the importance of the clump size on the coiling behaviour and thus aiding survival of IJs in dry conditions. Although this strain dispersed completely from the clump, we suggest that the clump formation is important for the reduction of water loss rates. As the initial clump size increases the surface area which the nematodes are exposed is reduced allowing slower evaporative water loss from the 'ball formation' exposed to the dry environment. This enables the IJs to undergo the appropriate physiological changes necessary to withstand low relative humidity conditions. Ishibashi et al (1987) also demonstrated that an increase (by weight) in the IJs clump size enhanced the success of the survival of S. carpocapsae (DD-136), but no numbers of nematodes were given. It is expected that under natural conditions, when IJs migrate out of infected insect cadavers and are exposed to lower relative humidity in the soil environment, they will aggregate in clumps. Whether the clump formation occurs among entomopathogenic nematodes under natural field conditions has yet to be determined. In the present work, gradual rehydration of desiccated IJs at 100% RH,

prior to

immersion in water, did not affect their survival. When anhydrobiotic dry organisms are rehydrated directly in water, their cells membranes can undergo a lipid phase transition (from gel phase to liquid crystalline) and may be expected to leak their contents during rehydration (Womersley, 1981; Crowe & Crowe, 1992) and, therefore, influence the survival of the organisms. Thus, we expected that the gradual rehydration at 100% RH will enhanced the viability of the IJs. Our present results can be explain by the fact that the pre-conditioning treatment was sufficient to induce the proper physiological changes needed for protection from the low relative humidity condition. One of those changes is an increase in trehalose levels, as was reported in other anhydrobiotic nematodes during the dehydration process (Womersley 1987, 1990; Crowe & Crowe 1992). We also found a increase in trehalose level in the IS-6 strain during the pre-conditioning treatment. It is well known that trehalose can preserve the liquid crystalline phase of dry lipids and can therefore prevent the cell leakage described above (Crowe & Crowe 1992). We suggest that the decrease in glycogen levels during the pre-conditioning period is probably in order to supply the needed metabolites for the trehalose synthesis. Upon rehydration glycogen returned to it's original concentration after 24h, while trehalose by the same

95

time reached only to 50% from the initial original. This results suggest that during the rehydration process trehalose probably is used as an energy source or is hydrolyzed for the glycogeneogenesis process. Although it has been shown that desiccation and rehydration processes are highly energy costing (Storey et al, 1982), we demonstrated here that the

virulence and

invasion ability of nematode was not hampered by the anhydrobiotic process. Our present data imply, indirectly, that the symbiotic bacterium is not affected by the low relative humidity conditions inside the desiccated IJs. Kung et al. (1991) demonstrated a significant decrease in pathogenicity of S. carpocapsae and 5. glaseri to Galleria mellonella after 4 days of direct exposure to 90% RH. The latter regime was probably too stressful for the IJs, and resulted in a reduction in their pathogenicity.

REFERENCES Barrett, J. (1991). Anhydrobiotic nematodes. Agricultural Zoology Reviews, 4: 161-176. Bird, A. F. & Buttrose, M. S. (1974). Ultrastructural changes in the nematode Anguina tritici associated with anhydrobiosis. Journal of ultrastructural Research, 48: 177-189. Blazquez Μ. Α., Gancedo J. M. and Gancedo C. (1994). Use of Yarrowia lipolytka hcxokinasc for the quantitive determination of trchaIosc-6-phosphate. FEBS Microbiology letters, 121: 223-228. Boehringer-Mannheim, GmbH (1986). Methods of biochemical analysis and food analysis using test combinations. Boehringer-Mannheim Biochemical, Mannheim, FRG. Crowe, J. H. & Crowe L. M. (1982). Inducing of anhydrobiosis: Membrane changes during drying. Cryobiology, 19:317-328. Crowe, J. H. & Crowe, L. M. (1992). Membrane integrity in anhydrobiotic organisms: Towards a mechanism for stabilizing dry cells. In : Somerso, G. N., Osmond, C Β. & Bolis, C L. (Eds). Water and life. Berlin, Heidelberg, Germany, Springer-Verlag : 87-103. Crowe, J. H., & Lambert, D. T. & Crowe, L. M. (1978). Ultrastructural and freeze fracture studies on anhydrobiotic nematodes. In: Crowe, J. H. & Clegg J. S. (Eds). Dry biological systems. New York, USA, Academic Press : 23-51.

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De-Koning W., and Van Dam K. (1992). A method for the determination of changes of glycolytic metabolites in yeast on a subsecound time scale using extraction at neutral pH. Analytical biochemistry, 204: 118-123 Demeure, Y. & Freckman, D. W. (1981). Recent advances in the study of anhydrobiotic nematodes. In:

Zuckerman, B. M. & Rohde, R. A. (Eds). Plant parasitic

nematodes. New York, USA, Academic Press : Vol 3, 205-226. Dutky, S. R., Thompson, J. V. & Cantwell, G. E. (1964). A technique for the mass propagation of the DD-136 nematode. Journal of Insect Pathology, 5: 417-422. Fan, X. & Hominick, W. M. (1991). Efficiency of the Galleria (wax moth) baiting technique for recovering infective (Steinernematidae

stages of entomopathogenic

and Heterorhabditidae)

from

rhabditids

sand and soil. Revue de

Neonatologie, 14:381-387. Friedman, M. J. (1990). Commercial production and development. In : Gaugler, R. & Kaya, H. K. (Eds). Entomopathogenic nematodes in biological control. Boca Raton, FL, USA, CRC Press : 153-157. Glazer, I. & Orion, D. (1983). Studies on anhydrobiosis of Pratylenchus thornei. Journal of Nematology, 15:333-338. Ishibashi, N., Tojo, S. & Hatate, H. (1987). Desiccation survival ofSteinernema feltiae str. DD-136 and possible desiccation protectants for foliage appliccation. In : Ishibashi, N. (Ed.). Recent advances in biological control of insects pests by entomogenous nematodes in Japan. Ministry of Education, Japan, Grant no. 59860005 : 139-144. Kaya, H. K. & Gaugler, R. (1993). Entomopathogenic nematodes. Annual Review of Entomology, 38: 181-206. Kung, S. P., Gaugler, R. & Kaya H. K. (1991). Effects of soil temperature, moisture and relative humidity

on entomopathogenic

nematode persistence. Journal

of

Invertebrate Pathology, 57: 242-249. Lowry. O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin reagent. Journal of Biological Chemistry, 193: 265-275. Menti, H., Wright, D. J. & Perry, R. N. (1997). Desiccation survival of populations of the entomopathogenic nematodes Steinernema feltiae and

Heterorhabditis megidis

from Greece and the UK. Journal ofHelminthology, 71: 41-46.

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Patel, M. N., Perry, R. N. & Wright, D. J. (1997). Desiccation survival and water contents of entomopathogenic nematodes, Steinernema spp. (Rhabditida: Steinernematidae). International Journal for Parasitology, 27: 61-70. Popiel, I., Glazer, I. & Vasques, E. M. (1989). Desiccation survival of Steinernema feltiae infective juveniles. Journal of Nematology, 21: 580. Storey, R. M. J., Glazer,I. & Orion, D. (1982). Lipid utilization by starved anhydrobiotic individuales of Pratylenchus thornei. Nematologica, 28: 373-378. Winston, P. W. & Bates, D. H. (1960). Saturated salt solutions for the control of humidity in biological research. Ecology, 41: 232-237. Womersley, C

(1981). Biochemical and physiological aspects of anhydrobiosis.

Comparative Biochemistry and Physiology, 70: 669-678. Womersley, C (1987). A réévaluation of strategies employed by nematode anhydrobiotes in relation to their natural environment. In: Veech, J. A. & Dickson, D. W. (Eds). Vistas on Nematology. Society of Nematologists, Hyattsville, MD, USA: 165-173. Womersley, C (1990). Dehydration survival and anhydrobiotic potential. In : Gaugler, R. & Kaya, H. K. (Eds). Entomopathogenic nematodes in biological control. Boca Raton, FL,USA, CRC Press : 117-137.

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Survival of the non-indigenous nematode Steinernema glaseri in The Netherlands. Lonne J. M. Gerritsen, Gerrie L. Wiegers & Peter H. Smits Research Institute for Plant Protection (IPO-DLO), Binnenhaven 5, P.O. Box 9060, NL6700GW Wageningen, The Netherlands.

Three different field trials were performed, on three locations, in three different years, to test the biological control of white grubs with the entomopathogenic nematode Steinernema glaseri. S. glaseri is not a native species in The Netherlands. To check survival of this nematode species soil samples were taken one year after application of the nematodes. In none of the three field trials S. glaseri could be detected in the soil when using the waxmoth trap method. In one trial natural populations of S. feltiae and Heterorhabditis megidis could be detected. This shows that S. glaseri cannot survive for a long period under natural conditions in Dutch soil. Therefore, release of S. glaseri will be of no risk to the Dutch natural fauna. SUMMARY:

INTRODUCTION

Surveys have shown that entomopathogenic nematodes are common and widely distributed in many parts of the world (Mracek, 1980; Akhurst and Brooks 1984; Akhurst and Bedding, 1986, Griffin et al, 1991). Some species occur in a large part of the world while others only occur under specific geographical, climatic and environmental conditions. For instance, Heterorhabditis bacteriophora occurs in Australia, the USA and Europe, while the Irish Heterorhabditis strain has only been isolated in Ireland and Norway (Smits et al, 1991; Gerritsen and Haukeland, unpublished results). Since some species are more pathogenic to certain insects than other species, it might be of interest to use other nematode species than the native species for biological control. For instance, Melolontha melolontha is a pest insect in The Netherlands. Steinernema glaseri performs better against M. melolontha than the native Dutch nematode species, S. feltiae and

H megidis (Smits, unpublished results). Therefore introduction of S.

glaseri in The Netherlands would be of interest for biological control of M melolontha. To determine the risks of introduction of a non-native nematode species the survival of S. glaseri in The Netherlands was determined.

MATERIAL AND METHODS NEMATODES

In 1994 and 1995 S. glaseri #326 was obtained from Biosys, USA, as a commercial product. Between 1995 and 1996 these nematodes were cultured twice yearly on last instar larvae of waxmoth (Galleria mellonella) at 25°C, according to methods of Dutky et al. (1964), and were stored in tap water in the dark at 10°C In 1994 and 1995 the commercial product was used in the trials while in 1996 nematodes produced on waxmoth larvae were used.

FIELD TRIAL

Three different field trials were performed, on three locations, in three different years. All trials were performed in summer to test biological control of white grubs with entomopathogenic nematodes. Trial 1 was done in 1994 on grass in a cemetery, trial 2 was done in 1995 in an apple orchard, around the apple trees and trial 3 was done in 1996 on the other side of the same orchard on the grass between the trees. All trials were on sandy soil. Trial 1 contained plots of 1 by 2 m on which, per m2, 0.25 or 0.5 million nematodes were sprayed (5 plots per treatment). Trial 2 contained plots of 2.5 by 2.5 m with an apple tree in the middle, on which, per m2, 0.25 million nematodes were sprayed (3 plots per treatment). Trial 3 contained plots of 2 by 2m, on which, per m2, 0.25 million nematodes were sprayed (6 plots per treatment). In all trials control plots were sprayed with water only. All plots in all trials were afterwards watered with 101 water per plot. SAMPLING

One year after application of the nematodes soil samples were taken to check survival of the nematodes. The samples taken were 5cm diameter and 5cm deep. In trial 1 four samples were taken per plot, in trials 2 and 3 three samples per plot. In trial 1 two samples per plot were put together in a plastic container and 5 waxmoth larvae were added (total 10 waxmoth larvae per plot). To samples from two control plots S. glaseri were added to check if nematodes could be detected with this method. In trials 2 and 3 all three samples were put together and 5 waxmoth larvae were added. Samples were put at 25°C in the dark. After

100

one week waxmoth larvae were taken from the soil and mortality was assessed. Dead larvae were put in a Petri dish with moist filter paper and were put at 25°C in the dark. These larvae were checked every other day for nematode production. Nematodes produced in these larvae were identified morphologically. In trial 1, per plot, one extra sample was taken. From 100ml soil from this sample nematodes were extracted according to Oostenbrink's elutriator method (s'Jacob and van Bezooijen, 1984). To one sample S. glaseri was added to the soil after sampling, this to check if the elutriator method could detected these nematodes.

Table 1. Survival of S. glaseri in three field trials. Trial

Treatment (nematodes/m )

% Wax moth with nematodes

nematode species

S. feltiae + H.megidis 10 250,000 1 S. feltiae 2 500,000 1 S. feltiae 2 0 1 S. glaseri 100 0 + S. glaseri* 1 0 250,000 2 0 0 2 0 250,000 3 0 0 5 * = S. glaseri was added to the soil after sampling nt = not tested

S. glaseri recovered from elutriator 0 0 0 many nt nt nt nt

RESULTS

In none of the three field trials S. glaseri could be detected in the soil one year after release of the nematodes (table 1). When nematodes were added to the soil after sampling, the nematodes could be detected, showing that both methods were suitable to detect S. glaseri. In trial 1 also natural populations of S. feltiae and H megidis could be detected when using the waxmoth trap. The samples coming from the Oostenbrink's elutriator method were only checked for the presence of S. glaseri not for other entomopathogenic nematodes.

101

DISCUSSION AND CONCLUSIONS

Gaugler et al. (1992) checked survival of S. glaseri in North Carolina. They could only isolate these nematodes in the south and one of their conclusions was that S. glaseri is intolerant to temperate climates and cold temperature. Poinar & Kozodoi (1988) regarded S. glaseri as a neotropical species. Results from our experiments support these conclusions: S. glaseri does not survive under the Dutch temperate climate conditions. S. glaseri can be used in biological control of grubs in The Netherlands but only in inundative release. Classical biological control, i.e. release of a new natural enemy, will be of no use since S. glaseri survives only for a short period and can not establish itself in the Dutch soil. As a consequence, the nematodes will not survive long enough to become a major pest to beneficial insects. Therefore, release of S. glaseri is of no risk to the Dutch natural fauna.

REFERENCES

Akhurst, R. J. & Brooks, W. M., 1984. The distribution of entomophilic nematodes (Heterorhabditidae and Steinernematidae) in North Carolina. J. Invert. Pathol., 44 : 140-145. Akhurst, R. J. & Bedding, R. Α., 1986. Natural occurrence of insect pathogenic nematodes (Steinernematidae and Heterorhabditidae) in soil in A ustralia. J. of the A utralian entomol. Soc, 25 : 241-244. Dutky, S. R., Thomson, J. V. & Cantwell, G. E., 1962. A technique for mass rearing the greater wax moth (Lepidoptera: Galleriidae). Proc. Entomol. Soc. Wash., 64 : 56-58. Gaugler,R., Campbell, J. F., Selvän, S. & Lewis, E. E., 1992. Large-scale inoculative releases of the entomopathogenic nematode Steinernema glaseri: A ssesment 50 years later. Biological control, 2 : 181-187. Griffin, C. T., Moore, F. J. & Downes, M. J., 1991. Occurrence of insect-parasitic nematodes (Steinernematidae, Heterorhabditidae) in the republic of Ireland. Nematologica, 37 : 92-100. Mracek, Z., 1980. The use of 'Galleria traps' for obtaining nematode parasites of insects in Czechoslovakia. A cta Entomologica Bohemoslovaca, 77 : 378-382.

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Poinar, G. O. & Kozodoi, E. M., 1988. Neoaplectana glaseri and N. anomali: Sibling species or parallelism? Rev. Nematol., 11 : 13-19. s' Jacob, J. J. & van Bezooijen, J., 1983. A manual for practical work in nematology. Int. Agricultural Centre, Wageningen, The Netherlands. Smits, P. H., Groenen, J. T. M. & de Raay, G., 1991. Characterization of Heterorhabditis isolates using DNA restriction fragment length polymorphism. Revue Nematol., 14 : 445-453.

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Transgenic Nematodes Carrying A Cloned Stress Resistance Gene From Yeast Tibor Vellai1, A. Molnar2, L. Lakatos2, Zs. Bánfalvi2, A. Fodor1, Gy. Sáringer3. 'Department of Genetics, Eötvös University, H-1088 Budapest, Múzeum krt. 4/A, Hungary; 2AgroBiotechnology Research Centre of the Hungarian Ministry of Agriculture, H-2101 Gödöllo, Szent-Györgyi Albert utca 4, Hungary; institute of Plant Protection, Pannon University of Agricultural Sciences, Georgikon Faculty of Agriculture, H-8360 Keszthely, Deák Ferene utca 57. Introduction As a prominent example of the biological pest control, several strains of the entomopathogenic nematode species (EPN; Heterorhabditis spp. and Steinernema spp.) could be used generally against the wide spectrum of insect pests. These nematodes can be cultured industrially in large quantities and sprayed on cultivated plants replacing some chemical insecticides. However, the agricultural application of EPNs has been limited by their extreme sensitivity to desiccation due to high salinity and drought. One approach of getting desiccation tolerant EPNs is the rather traditional: to (i) isolate trough resistant individuals from the nature or (2) follow a kind of selection protocol. Glazer et al. has recently isolated Steinernema feltiae individuals from a desert and developed a strain (called IS6). IS6 has been characterised it thoughtfully

(Glazer, personal

communication). In spite of the encouraging discovery of IS6, there are, however, natural limits of this approach, since the degree of desiccation tolerance of many other nematodes species, such as plant parasitic Pratylenchus and Meloidogyne species proved higher (with orders of magnitude) than those of even the most tolerant EPN strains. Another approach is to produce transgenic nematode carrying genes responsible from desiccation resistance from another organism. One of the osmoprotectant molecules of key importance accumulated in a number of organisms in response to desiccation stress is the disaccharide trehalose (Crowe et al., 1992), which is known to stabilise the dehydrated lipid membranes efficiently. In trehalose biosynthetic process, trehalose-6-phosphate

synthase (TPSI)

converts UDP- and

ADP-glucose, as well as glucose-6-phosphate, to trehalose-6-phosphate

that is

dephosphorilated subsequently to trehalose by trehalose-6-phosphate synthase (TPS2, Kaasen et al, 1992; Vuoiro et al, 1993). Herein the authors report the first successful attempt to introduce the desiccation tolerance gene TPSI of the yeast in to the entomopathogenic nematode Steinernema feltiae. transgenically. Materials and methods Nematode strains and culture Caenorhabditis elegans were cultured on NGM agar plates seeded with the Escherichia coli strain OP50 as described (Brenner, 1974, Suiston and Hodgkin, 1988). Both the wild type (strain N2 Bristol) and dauer constitutive (Daf-c) mutant (CB1372, Riddle, 1977, Riddle et al, 1981) strains used in this work were originated from Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. Steinernema feltiae strain scp. (Tomalak, 1998) was kindly provided by Dr. Marek Tomalak (Poznan, Poland) and cultured on Woots agar plates seeded with X. bovienii bacteria isolated from strain Vija Norway by Emília Szállás in our laboratory. We found that all S. feltiae strains can be cultured very well on this bacteria. Experiments were later repeated on S. feltiae strain Nyíregyháza (isolated by A. Fodor in Hungary). Isolation and cloning of TPSI gene from yeast The genomic DNA from yeast was isolated as described by Hoffman and Wintston (1987), and 100 ng was used for a subsequent PCR reaction to amplify TPSI. 200-200 ng of the TRESEN and TREASE primers were used for PCR. The sequences of these TPS 1 specific primers were the followings: TRESEN containing a Sad restriction site , 5' GAG CTC ATG ACT ACG GAT AAC GCT AAG G 3'; TREASE containing a Kpnl site , 5' GGT ACC GGT TCA TCA GTT TTT GGT GG 3'. A denaturation cycle for 2 min. at 94°C was carried out prior to 30 cycles that were performed; each cycle consisted of 1 min. denaturation at 94°C, 1 min. annealing at 50°C, and 3 min. elongation at 72°C The 1.5 kb PCR product was isolated from agarose gel with a QIAquick gel extraction kit and ligated into a EcoR Vdigested Bluescript SK(-) vector. Testing of intact function of the cloned TPSI in E.coli The Bluescipt cloning vector containing the PCR fragment of 1.5 kb was digested with Sad and PstI restriction enzymes, and the insert was subsequently treated with T4 DNA polimerase. The fragment was inserted into a PstI digested pKK233-i E. coli vector that was transformed into E. coil DH5a cells. Control and transformed DH5a cells were 106

cultured in media supplemented with 0.4 and 0.6 M NaCl, respectively. Their growth rate under different osmotic conditions were compared with each other. Construction ofhspl6-2 - TPSI containing expression vector The 1.5 kb large insert from the Bluescript vector double-digested with KpnI-SacI enzymes was inserted into the Kpnl, Sad sites of polylinker region of the nematode expression vector pPD49.78 (Fire et al. 1990). We termed the construction pAM/C-02. Microscopy The microscope used for transformation was a Zeiss Axiovert 135 equipped with differential interference contrast optics, a free sliding oil cushion stage, and a Narashige micromanipulator system. The phenotypes of the nematodes were examined using a Leitz Wild M3Z (types-S) (stereo microscope with 16-40 X magnification. Transformation ofC.elegans The microinjection mediated marker rescue experiments (Mello et al, 1991) were done to identify the expression pattern of hspl6-2 promoter. The DNA solution was injected into the distal arm of each side of the gonad of adult wild type hermaphrodites. Bacterial cultures containing plasmid DNAs (pRF4, DR277 and pPD49.78) were grown in 5 ml 2XTY selective media for 16 hours. The plasmid DNA solution was prepared by alkali lysis and further purified by LiCl precipitation (Spence et al, 1990). The DNA solutions for injection were prepared as described by Fire (1986). The plasmid DNA of co-transformation markers of pRF4 and of DR277, was similarly prepared, and was used at a concentration of 20 ugml' 1 . This is an empirical experience, that co-transformation occur frequently in C. elegans, which provide an option to identify transformants to be searched for visually. The pRF4 was a pUC19 based plasmid DNA containing the cloned C. elegans ro/-6~(sul006), a dominant allele resulting in a characteristic roller (Rol) phenotype late larval and adult C. elegans (Brenner, 1974) was used as one of the co-transformation markers. (Both the homozygous and the heterozygous animals for this allele roll to the right as they move, and could easily be recognised amongst a mass of wild type ones). Another co-transformation markers we used was plasmid DR 277 (kindly provided by D. L. Riddle, University of Missouri-Columbia, USA) containing daf-7(+) allele. DR 277 is capable of rescuing mutants homozygous for recessive alleles of daf-7. While the animals homozygous for any recessive daf-7 allele, such as the thermosensitive daf-7 (el372) (Reference strain: CB1372) performing a dauer constitutive (Daf-c) phenotype, (which

107

means, that they grow to dauer larvae at non-permissive temperature, 25°C but develop to adults at 15°C, Riddle, 1977, Riddle et al, 1981, Swanson and Riddle, 1981). Generally 20-30 CB1372

hermaphrodites (which, of course, had been grown at permissive

temperature) were injected in each experiment. Three to four days thereafter animals of Rol (or Non-Daf-c) phenotypes were scored for amongst the Fl progeny (grown at 25 °C) of the injected hermaphrodites. The rescued

worms were maintained on NGM agar plates

(Suiston and Hodgkin, 1988) seeded with the E. coli strain OP50 at 25 °C Nematodes of the Rol (or Non-Dauer) phenotype were transferred individually to new plates to establish permanent transformed lines. The methods of transformation of S. feltiae was carried out similarly as described above. In this case, however, young females were injected, and then mated. The animals were maintained on Woots agar plates seeded with X. bovienii Vija Norway bacteria. Because there has been no co-transformation marker gene available, transgenic S. feltiae Fl progenies were selected directly on the basis of their increased osmotic tolerance. All the offspring of transformed females were washed into M9 buffer supplemented with 0.6 M NaCl, and the survival transgenic ones were isolated one day later. Staining for ß-galactosidase activity in situ Transgenic animals were tested for lacZ expression by a heat shock consisted of two 2-h exposures at 33°C, which were separated by a 30-min recovery period at 20°C, on NGM plates seeded with bacteria. Worms were subsequently allowed to recover for 15 min at 20°C before being washed off by M9. The animals were transferred into acetone and methanol for 5 min, respectively, and incubated in a histochemical stain containing X-gal (Fire et al. 1990) for 1 h at 37DC Staining was generally visible after only 15 min of incubation. RESULTS The intact function of the isolated TPSI was tested in E. coli DH5 alpha derivative as follows: in the presence of TSP1 (cloned into the bacterial vector pKK223-3) cells of three independent transformed lines, unlike to those of TPSI-free control, proved tolerant to and growing in a liquid medium of relatively high molarity of NaCl. (Fig. 1).

108

2.5

2 1.5 -· D O

1 0.5

οχ-

Fig. l.a: The function of TPSI tested in E. coli. The growth rate of one control and three independent DH5 alpha lines transformed with TSP1 (cloned into the bacterial vector pKK223-3), in minimum liquid media supplemented with 0.4 M of NaCl. The transformant lines grew a little better. Series 1: E. coli DH5a, Line No. 1, control; 0— Series 2: E. coli DH5a, Line No.2 transformant, expressing TPS 1 ; Δ— Series 3: E. coli DH5cc, Line No.3 transformant, expressing TPSI; X— Series 4: E. coli DH5a, Line No.4 transformant, expressing TPSI.

c c

Fig. l.b.: The function of'TPSI tested in E. coli. The growth rate of one control and three independent DH5 alpha lines transformed with TSP1 (cloned into the bacterial vector pKK223-3), in minimum liquid media supplemented with 0.6 M of NaCl. Only the transformant lines grew. Series 1: E. coli DH5a, Line N o . l , control; 0— Series 2: E. coli DH5a, Line No. 2 transformant, expressing TPSI; V— Series 3: E. coli DH5cc, Line No. 3 transformant, expressing TPSI; X— Series 4: E. coli DH5a, Line No. 4 transformant, expressing TPSI.

109

The presence of

the TP1 in the transformed nematodes were detected by Southern

hybridization (Fig. 2).

Fig. 2: Southern gel of 11 transformed C. elegans of Roi phenotypes. One proved co-transformant expressing TPSI. Left lane: DNA molecular weight standard. The expression pattern of the inducible promoter driving the TPSI in nematodes was determined as follows: C. elegans hermaphrodites were co-transformed with the pPD49.78 base vector carrying lacZ reporter gene and the pRF4 vector set up with a cloned dominant morphological marker gene, rol-6(sul006). Transformants of ROL phenotype were stained for Beta gal. All proved co-transformants and stained for Beta gal in the pharynx as well as in body muscle and hypodermal cells. (Fig. 3, A). In contrast to previous observations (Stringham et al, 1992), lacZ expression was observed in several embryos as well. (Fig. 3,b, c, d, e & f). Later on, we increased the frequency of transformation by using DR227 (daf-7*), as a co-transformation vector, and daf-7(el372) mutants (of thermosensitive dauer constitutive phenotype) were injected. Transformants of DAF-7(+) phenotypes having continuously been tested for desiccation tolerance.

110

Fig 3: The expression pattern of the heat shock driven TPSI gene in nematodes was monitored as follows: C. elegans hermaphrodites were co-transformed with the pPD49.78 vector carrying lacZ reporter gene and rol-6(sul006)as marker gene. Transformants showing the roller phenotype were stained for Beta gal activity, a: Staining was found in the pharynx as well as in body muscle and hypodermic cells, b, c, d, eandf: In contrast to previous observations (Stringham et al, 1992), lacZ expression was observed in several embryos as well. Sometimes all, sometimes only a few cells of the embryos express the gene.

112

Fig. 4: A spectacular increase of the degree of osmotic tolerance, supposedly due to trehalose overproduction, was demonstrated in transgenic S. feltiae adults. At 0.7 M NaCl all control females died (Fig. 4 , SI) while all transgenic ones were alive (Fig 4 , S2). The LD50 for contols was between 0.3 and 0.4 M of NaCl while that for transgenic adults was between 0.7 and 0.8M, respectively (see Fig. 6 & 7).

113

There was no technical difficulties for applying this method directly to heterorhabditids or steinernematides; the point is to use the appropriate co-transformation marker. A spectacular increase of the degree of osmotic tolerance, supposedly due to trehalose overproduction, was demonstrated in transgenic S. feltiae adults. At 0.7 M NaCl all control females died (Fig. 5 , SI) while all transgenic ones were alive (Fig 5 , S2). The LDjo of CaCL for controls was between 0.1 and 0.2 M of NaCl while that for transgenic adults was between 0.4 and 0.5M, respectively (Fig. 6). The LD50 of NaCl for controls was between 0.3 and 0.4 M while that for transgenic adults was between 0.7 and 0.8M, respectively (Fig. 7). A dramatic increase in desiccation tolerance, could also be observed in transgenic S. feltiae adults and infective juveniles on 1% agarose pads. As expected, transgenic nematodes carrying TPSI grew slower than wild-type. The retarded growth rate of the transgenic nematodes seems to minimize the risk of using them as bioinsectides in the practice.

ε

NaCl concentration (M)

Fig. 5: Average survival of control and transformant C. elegans adult hermaphrodites in M9 buffer supplemented with NaCl up to different concentrations. D Series 1 : Control N2 wild type adults; £ Series 2: Co-transformants of Rol phenotypes expressing TPS 1.

114

il

ε

CaCIj concentration (M)

Fig. 6: Average survival of control and transformant S. feltiae adult hermaphrodites in M9 buffer supplemented with CaCb up to different concentrations. -

Series 1 : Control S1 : wild type S. feltiae females; Series 2: Co-transformants S2 S. feltiae females expressing TPSI.

—u ~B

0

°-1 02 03 , u



0.4

nc

°· 5

T

._

0.6

0.7

^

o.i

0.9

NaCl concentration (M)

Fig. 7: Average survival of control and transformant S. feltiae adult hermaphrodites in M9 buffer supplemented with NaCl up to different concentrations. □ Series 1 : Control S1 : wild type S. feltiae females; — -B— Series 2: Co-transformants S2 S. feltiae females expressing TPSI.

115

DISCUSSION The first successful genetic transformation of an EPN, H. bacteriophora HP88 was made first by Hashmi et al. (1996) demonstrating that the C. elegans rol-6 (sul006) can be used as a co-transformation marker and that the transgenic hsp-16 gene was expressed in body musculature, hypodermis, and pharyngeal muscles. We proved that this technique can be adopted to steinemematids. One of the well characterised desiccation tolerant gene is the trehaloze-phosphate synthase 1 (TPSI) in yeast (McDougall et al, 1993). TPSI was first transgenically introduced into tobacco (Holström et al, 1996) resulting in desiccation tolerant plants. In spite that it was published, the authors did not make the cloned gene available for us. Therefore we had to isolate and clone it. into the bacterial vector pKK223-3.'fhe intact function of the isolated and cloned TPSI was tested in E. coli DH5 αϊ derivatives and it was found that only transformed lines proved tolerant to high molarity of NaCl. (Fig. 1). The transgenic and desiccation tolerant tobaccos in the Holstrom et al. (1996) experiments were rather crippled transgenic plants, probably due to the aphysiologically high level of the constitutively synthesised trehalose. To avoid negative consequences, we have cloned TPSI into tiiepPD49.78 nematode expression vector (kindly provided by A. Fire) that carries a heat-inducible promoter (upstream to the polycloning site) derived from C. elegans.The presence of

the TP IS in the co-transformant nematodes was proved

by

Southern hybridisation (Fig. 2) It was found that at least one of 10 transformant showing Rol or 4 Non-Daf (c) phenotypes expressed TPSI as well. The expression pattern of the inducible promoter driving the TPSI gave some new information, since it was found in contrast to previous observations (Stringham et al, 1992), lacZ expression was observed in several embryos as well. (Fig. 3,b, c, d, e & f). There was not any technical difficulty when the tranformation technique was adopted to heterorhabditids or steinernematides; however, appropriate co-transformation marker will have to be developed. The pleiotropic effect of the expression of TPSI on the osmotic tolerance can be detercted quantitatively. We found that the osmotic shock caused by CaC^ is more drastic that that caused by NaCl. A significant increase of the degree in osmotic tolerance toward both CaCl and NaCl respectively, were unambigoulsy demonstrated (supposedly due to trehalose overproduction) in transgenic S. feltiae adults. At 0.7 M NaCl all control females died (Fig. 4 , SI) while all transgenic ones were alive (Fig 4 , S2). A dramatic increase in

116

desiccation tolerance, could also be observed in transgenic S. feltiae adults and infective juveniles on 1% agarose pads. The transgenic approach for getting transgenic EPN strains of elevated desiccation tolerance approach has advantages and disadvantages. The advantage is that if the experiment is successful, we can obtain an organism of higly elevated degree of desiccation tolarance. The disadvatages are that (1) people are worried about anything which is transgenic, which may cause registration problems in the future; (2) the existence of a transgenic individual does not mean the existence of a frangente strain or line, since the trans genes in nematodes usually found as extrachromosomal arrays, which may easily be lost. Recently Fritz Müller and his colleagues (Müller, personal communication) have isolated a C elegans homologue of the plant LEA proteins (Ce-led). Plant LEA-proteins are constitutively expressed in seeds and can be induced by various stress conditions (including drought stress) in other parts of the plant body. LEA proteins are thought to confer desiccation tolerance to the plants, particularly to seeds. The C elegans homologue (Ce-lea) may have a similar function. Its expression is developmentally regulated: Ce-LEA is absent from early embryos and first appears in a few specific cells of the comma stage. Later, during larval stages and in adults it is mainly expressed in the excretory system, but weak expression is also seen and in hypodermic cells and some other non-identified cells. Adults show additional Ce-LEA expression in the spermatheca. Strong induction of Ce-lca expression is observed when C. elegans worms are exposed to stress condition, such as desiccation and high salt concentration. Highest expression, however, is seen in dauer larvae, where Ce-lea is expressed in all cells of the body. By using different daf mutants we could show that the high dauer-specific Ce-lea expression is related to and depending on the dauer pathway. In an alternative series of experiments we plan to use the Ce-lea promotor for the expression of tps-1 in transgenic dauers or stress induced C. elegans worms. In a pilot experiment, a Ce-lea: :gfp fusion construct was inducible and highly expressed in stress induced worms and in dauer larvae. If the results with C. elegans turn out to be satisfying, we will search for the Ce-lea homologues in Heterorhabditis bacteriophora and Steinernema feltiae, and test their promotors for inducibility under stress conditions and in the infectious dauer larvae.

17

References

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94. Crowe, J. H , Hoekstra, F. A. and Crowe, L. M. Anhydrobiosis. (1992).Æ Rev. Physiol 54, 579-599. Fire, A. (1986). Integrative transformation of Caenorhabditis elegans. EMBO J. 5, 2673-2680. Fire, A, Harrison, S. W. and Dixon, D. K. (1990). A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93, 189-198. Hashmi, S, Hasmi, G. and Gaugler, R. (1995). Genetic transformation of entomopathogenic nematode by microinjection J. Inverebr.Pathol. 66,293-296. Hoffman, C. S. and Wintson, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267- 272. Holmström, K.-O, Mantyla, E , Welin, B , Mandai, A, Palva, T , Tunnela, O. E. and Londesborough, J. (1996).Drought tolerance in tobacco. Nature 379, 683-684. Kaasen, I , Falkenberg, Ρ , Styrvokl, Ο. E. and Strom, A. R (1992). Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF. J. Bacteriol. 174,889-898. Kay, R. J , Boissy, R. J , Russnak, R. H. and Candido, Ε. P. (1986). Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally. Mol. Cell. Biol. 6, 3134-3143. McDougall, J , Kaasen, I. and Strom, A. R. (1993). A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant. FEMS Microbiol. Let 107,25-30 Mello, C. C , Kramer, J. M , Stinchcomb, D. and Ambros, V. (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959-3970. Riddle, D. L. (1977). A genetic pathway for dauer larva formation in Caenorhabditis elegans. Stadler Genet. Symp. 9, 101-120.

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Riddle, D U , Swanson, M. M. and Albert, P. S. (1981). Interacting genes in nematode dauer larva formation. Nature 290, 668-671. Spence, A. M , Coulson, A. and Hodgkin, J. (1990). The product offem-1, a nematode sex-determining gene, contains a motif found in cell cycle control proteins and receptors for cell-cell interaction. Cell 60, 981-990. Stringham, E. G, Dixon, D. K, Jones, D. and Candido, Ε. P. (1992). Temporal and spatial expression patterns of the small heat shock (hspl6) genes in transgenic Caenorhabditis elegans. Mol. Biol. Cell 3, 221-233. Suiston, J. E. and Hodgkin, J. (1988). Methods. In The nematode Caenorhabditis elegans, (ed. W.B.Wood) pp 587-606. New York: Cold Spring Harbor Laboratory. Swanson, M. M. and Riddle, D. L. (1981). Critical periods in the development of the Caenorhabditis elegans dauer larva. Dev. Biol. 84, 27-40. Tomalak, M. (1998). Analysis ofSteinernema feltiae mutants. In: COST 819 Genetic and molecular biology of entomopathogenic nematodes, Proceedings of the workshop held at the Centre de researches agronomiques de rovance-Alpes-Cote d'Azur Institute national de la reserche agronomique 1 to 5 April, 1997, Antibes, France. EUR 1826 European Communities, 1998. ISBN 92-828-3410-7. pp. 125-134. Vuorio, O , Kalkkinen, N. and Londesborough J. (1993). Cloning of two related genes encoding the 56-kDa and 123-kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae. Eur. J. Biochem. 216, 849-861.

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Development of a Cold Active Entomopathogenic Nematode Based Product Manuele Ricci and Bertold Fridlender Bio Integrated Technology S.r.l. Pantalla di Todi (PG), Italy

SUMMARY: No commercially available product based on entomopathogenic nematodes are active at temperature below 12°C On the other hand, larvae of the insect pest Otiorrhynchus sulcatus develop well during winter and are capable to feed at temperatures as low as 3°C The biocontrol of these type of insects with entomopathogenic nematodes will require a strain that is active at temperatures below 12 0 Candaslowas5°C Several nematode isolates were screened for cold activity against insects. Mainly Steinernematids were found in soil samples above 1500 m. on the Italian Alps. Their activity was measured on potted plants with peat to which larvae of O. sulcatus were added, conditions that are very similar to real field environment. From 11 Steinernematid isolates tested at 12°C, four potential candidates giving insect mortality between 50 to 90% were found. Among them only one isolate showed a very activity at very low temperatures. This isolate was efficient as a bioinsecticide even at 3°C By genetic analysis it has been found to belong to the species Steinernema kraussei. In an in vivo production test, the identified cold active isolate showed an optimal growing temperature at 15°C However, in a liquid fermentation assay, the isolate grew very well at 20°C It was concluded that the cold active isolate described is a good candidate for controlling O. sulcatus larvae at low temperatures. INTRODUCTION Otiorrhynchus sulcatus F. (Coleóptera, Curculionidae) is considered one of the major worldwide insect pest. The larvae feed on the root system of several plant species (mainly ornamentals and nursery stock) producing its consequent decay and very significant economic losses. The most effective strategy today to prevent damage is to spray adult insects with chemical pesticides. However, this is a difficult task because they hide during daytime and are not controlled by the materials used. Several chemicals with good activity against larval stages, such as aldrin and dieldrin, have been withdrawn from the market in the last years due to their negative effect on the environment (Moorhouse et al, 1992). As a result of this, no real good and safe pesticides are presently available for the control of this insect. The capability of entomopathogenic nematodes to effectively control the larvae of this pest is very well documented, however, no commercially available products based on entomopathogenic nematodes that are effective at temperature below 12-13°C

are available (Shirocki and Hague, 1994). At these

temperatures, the insect larvae are still active under the soil.

It has been reported that they can feed

even at temperatures as low as 3°C (Smith, 1932). Biocontrol of this insect at these low temperatures will require a nematode that is active at these rather extreme environmental conditions (Berry et al, 1997) Even though several researchers have focused their attention on cold active nematodes, they have failed to find an effective isolate that works at temperatures below 12° in experimental conditions that closely resembles the real field environment (Gwynn, 1994; Mason and Hominick, 1995; Griffin and Downes 1991; Grewal et al 1994; Westermann and Van Zeeland 1989; Miduturi et al. 1994; Eculica et al. 1997; Steiner 1996). The aim of this research was to find an active nematode isolate for the biocontrol of insect pests that are responsible for crop losses at temperatures between 3 to 12°C MATERIALS AND METHODS Insects Galleria mellonella larvae: Larvae were biweekly supplied by Ruggeri S.P.A, Bologna, Italy and stored at 10°C until use. Their weight ranged between 150-300 mg. O. sulcatus larvae: They were reared in potted plants in the greenhouse. Weight of larvae used on bioassays was always between 15-45 mg. Tenebrio molitor larvae: They were reared on a mix of flours. Weight of larvae used on bioassays was always 100 ± 5 mg. Always fresh larvae were used. Balaninus elephas larvae: Larvae were collected from infested chestnut. After collection, the larvae were stored for 1 day at 15°C in soil coming from the area of the trees in which the larvae were collected.

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Nematodes Heterorhabditis Heterorhabditis. megidis (Larvanem product), H. bacteriophora HI1007-M strain, H. sp. HDO strain, //. megidis Tenebrio strain, H. sp. Eugeni strain, H. sp. HNHI strain, H. bacteriophora NJ strain and H. megidis HSH2 strain. The Heterorhabditis were multiplied by in vivo production using the last instar G. mellonella larvae according to the method of Dutky et α/.(1964). The nematodes were then stored at 10°C and used within 4 weeks from insect final emergence. Steinernematids 5. feltiae UK strain and 11 out of the 45 nematode strains found on the Alps around 1500 meters above sea level denominated: N0035, N0091, N0087, N0094, N0093, N0089, N0104, N0080, N0048, N0073 and NO 130, were used. They were multiplied by in vivo production using the last instar G. mellonella larvae according to the method of Dutky et α/.(1964). The nematodes were then stored at 5°C and used within 8 weeks from insect final emergence. Tenebrio-Assay This assay was developed as an alternative to the more complicated Otiorrhynchus-Assay. Thirty larvae of T. molitor of about 100 mg each, were challenged with 1500 nematodes in a 250 c.c. plastic box with 50 gr. of peat at 75% humidity. The insects were adapted at the incubation temperature for at least 24 hours before the treatment. The boxes were incubated at constant temperature and after the incubation time, larval mortality was scored. Control treatments with only water were always included. Otiorrhynchus-Assay Ten larvae of O. sulcatus were challenged with 6400 nematodes in a 1000 c.c. plastic pot filled with peat and containing a 2 month old Impatiens sp. plant. The insects were adapted at the incubation temperature for at least 24 hours before the treatment. The pots were incubated at constant temperature and after the incubation time, larval mortality was scored.. Control treatments with only water were always included.

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Balaninus-Assay Ten larvae of the B. elephas (Coleóptera: Curculionidae) were challenged with 6400 nematodes in 1 liter pots (about 500,000 nematodes/m2) filled with soil collected from the area under the chestnut trees in where the larvae were collected. The pots were incubated at 8°C and larval mortality assessed 21 days after treatment (DAT). Control treatments with only water were always included. Field trials Three litres pots with strawberry plants and ten O. sulcatus larvae were treated with 125,000 and 500,000 DJs/m2 respectively. Larval mortality was assessed 24 DAT. There were 5 reps per treatment in a complete randomised block. The soil temperatures were recorded hourly with a data logger. Control treatment with S. feltiae UK strain and water were always included. In Vivo (G. mellonella) nematode production The in vivo production of the cold active isolate (N0093) was done at different temperatures in parallel with S. feltiae UK strain using G. mellonella larvae according to the method of Dutky et al (1964). Three replicates of five G. mellonella larvae were challenged with 100 infective nematodes each. The temperatures used were 10-15-20 and 25°C All the emerging nematodes were regularly collected. The parameters studied were the number of nematodes produced per mg of insect and the length of the entire production. In vitro (liquid fermentation) nematode production at 20°C Shake flasks containing a liquid media were inoculated first with the symbiotic bacteria and 20 hours later with monoxenized nematodes. The cold active isolate N0093 was compared with S. feltiae UK strain. After 2 weeks at 20°C the final concentration of DJs were evaluated. In order to ascertain the cold activity of the liquid produced nematodes at 20°C, an Otiorrhynchus-Assay incubated at 5°C and assessed 21 DAT was performed with these nematodes in comparison to those produced in vivo. Species determination of the cold active isolate N0093 A PCR AFLP analysis has been performed by Dr. Alex Reid CABI BIOSCIENCE St. Albans, UK (Reidera/, 1997).

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RESULTS AND DISCUSSION Screening of nematodes at 12°C using Tenebrio-Assay This assay was performed in order to see if this technique could substitute the more complicated Otiorrhynchus-Assay. The first screening was done with Heterorhabditid strains and a sample of Steinernematid

5. feltiae UK strain was included as direct control for comparison purposes. The

second screening was done with Steinernematid isolates. The screening on both cases was done at 12°C + 1. Larval mortality was scored at 7 and 14 DAT and was corrected according to the Abbott's formula. On Table 1 are reported the results obtained for the screening with Heterorhabditid strains. It can be seen that at 7 DAT the control Steinernematid S. feltiae UK strain already gives almost a complete control of the insects. The best Heterorhabditid strain at 7 DAT was //. megidis Larvanem (47.41 % larval mortality). However at 14 DAT there were five isolates that gave more then 70% larval mortality (//. megidis Larvanem, H. megidis Tenebrio, h. sp. Eugeni, H. sp. HNHI and H. megidis HSH2). On Table 2 are reported the results obtained with Steinernematid isolates. Apart from the control S. feltiae UK strain, other 4 isolates, namely N0094, N0093, N0089 and N0035 gave more than 80% larval mortality. None of the isolates showed a significant increase of activity at 14 DAT.

Table 1 Screening of Heterorhabditis nematodes at 12°C using Tenebrio-Assay Nematode SfUK Hm Larvanem HI-1007-M H.sp. HDO Hm Tenebrio H. sp. Eugeni H sp. HNHI HbNJ HmHSH2

(%) Larval Mortality'* 1 + St. Err. at 7 DAT 99.26 + 0.25 47.41 +3.12 2.96 ± 0.99 0.00 ±0.00 27.04 ± 4.04 10.37 + 0.86 22.96 + 2.79 0.37 + 0.12 27.78 + 3.11

(%) Larval Mortality 1 * 1 + St. Err. at 14 DAT 99.63 ±0.12 85.91 ±1.86 68.83 ± 2.76 40.20 ±2.59 77.02 + 3.28 71.43 + 2.69 74.76 ±2.13 56.18 ±2.37 87.40 ± 1.92

Corrected according to the Abbott's formula

I 25

Table 2 Screening of Steinernematids nematodes at 12°C using Tenebrio-Assay Nematode Sf UK N0091 N0094 N0087 N0093 N0089 N0130 N0080 N0035 N0048 N0073 N0104

(%) Larval Mortality'*' ± St. Err. at 7 DAT 98.89 + 1.11 31.12 ± 15.56 86.67 ± 8.89 45.00 ± 18.34 91.67 ± 1.67 94.44 ± 1.11 30.00 + 11.11 32.22 ± 0.00 100.00 ± 0.00 8.33 ± 7.22 6.11 ± 6.11 42.22 ± 22.22

(%) Larval Mortality'* 1 ± St. Err. at 14 DAT 98.89 ± 1.12 48.55 ± 9.23 99.44 ± 0.56 59.16 + 9.73 92.18 ± 1.07 96.1 ± 1.65 47 4 4 ± 8.12 63.33 ± 0.00 100.00 ± 0.00 28.38 ± 20.51 18.35 ±16.10 56.33 ± 17.01

'*' Corrected according to the Abbott's formula Screening of nematodes at 12°C using Otiorrhynchus-Assay This assay was performed always in parallel to the Tenebrio-Assay in order to find out the correlation existing between the two methods. The first screening was done with Heterorhabditid strains in which S. feltiae UK strain was included as control. The second screening was done with Steinernematid isolates. The screening in both cases was done at 12°C +1. Larval mortality was scored at 14 DAT and was corrected according to the Abbott's formula. On Table 3 are reported the results of the screening with Heterorhabditid strains. As expected, the larval mortality of this insect is lower than the one seen in the assay with larvae of T. molitor. Even S. feltiae UK strain gave only 61.39% mortality. Among the Heterorhabditid strains the best were //. megidis Larvanem, H. sp. Eugeni and H. megidis HSH2. On Table 4 are reported the results of the screening with Steinernematid isolates. Three isolates (N0093, N0089 and N0035) gave good performance against O. sulcatus larvae. These same isolates were within the four active ones shown for the Tenebrio-Assay (Table 2).

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Table 3 Screening of Heterorhabditis nematodes at 12°C using Otiorrhynchus-Assay Nematode Sf UK Hm Larvanem HI-1007-M H. sp. HDO Hm Tenebrio H. sp. Eugeni Hsp. HNHI HbNJ Hm HSH2

(%) Larval Mortality'* 1 ± St. Err. at 14 DAT 61.39 + 10.02 65.98+ 5.44 30.27 ± 18.23 11.75 ± 3.00 38.78+ 1.32 52.19 ± 9.14 39.12+ 6.44 22.86 ± 11.20 55.47 ± 3.75

'*' Corrected according to the Abbott's formula Table 4 Screening of Steinernematids nematodes at 12°C using Otiorrhynchus-Assay Nematode SfUK N0091 N0094 N0087 N0093 N0089 N0130 N0080 N0035 N0048 N0073 N0104

(%) Larval Mortality'* 1 ± St. Err. at 14 DAT 57.89 ± 0.00 21.05 ± 1.67 28.07 ± 15.00 17.54 ± 8.33 85.96 ± 3.33 87.72 ± 5.00 3.51 ± 1.67 17.54 + 18.34 63.16 ±11.67 8.77 ± 0.00 10.53 ± 1.67 17.54 ± 11.67

'*' Corrected according to the Abbott's formula Correlation among Tenebrio and Otiorrhynchus-Assay The Tenebrio-Assay was performed in order to see if it could substitute the more complicated Otiorrhynchus-Assay. By plotting the results obtained with both assays (Table 5) is it possible to see that the Tenebrio-Assay at 14 DAT correlates well with the Otiorrhynchus-Assay, both with Heterorhabditis and Steinernematids isolates. This means that it is possible to screen several nematode isolates for cold activity against O. sulcatus larvae using the more simple Tenebrio-Assay.

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Table 5 Correlation between Tenebrio and Otiorrhynchus Assays at 12°C and at different incubation times

Heterorhabditids Heterorhabditids Steinernematids Steinernematids

Equation

R5

y = 0.72 χ - 2.13

0.3009

y = 0.80 χ - 39.39

0.7191

y = 0.96 χ - 11.50

0.4302

y = 0.91 χ - 34.90

0.87.80

Bioassays

Nematode

Tenebrio-Assay 7 DAT and Otiorrhynchus-Assay 14 DAT Tenebrio-Assay 14 DAT and Otiorrhynchus-Assay 14 DAT Tenebrio-Assay 7 DAT and Otiorrhynchus-Assay 14 DAT Tenebrio-Assay 14 DAT and Otiorrhynchus-Assay 14 DAT

Screening at 5°C of selected nematodes tested with the Otiorrhynchus-Assay The nematodes (Steinernematids) that gave higher mortality at 12°C (N0093, N0089 and N0035) were tested with the Otiorrhynchus-Assay at 5°C S. feltiae UK strain as well as a water treatment were included as controls. Larval mortality (corrected according to the Abbott's formula) was scored at 21 and 35 DAT. On Table 6 are reported the results of the screening. The isolate N0093 shows a larval mortality of about 84% already after 21 DAT. After two more weeks the isolate N0093 did not significantly improve its performance. At 35 DAT none of the other isolates could reach a larval mortality higher than 50%. Isolate N0093 keeps the same higher level of activity already shown at 21 DAT. Table 6 Otiorrhynchus-Assay at 5°C with selected Steinernematids Nematode SfUK N0093 N0089 N0035

(%) Larval Mortality'*1 ± St. Err. at 5°C at 21 DAT 33.33 ± 5.00 84.62 ± 2.89 48.72 ± 7.07 23.08 ± 6.45

(%) Larval Mortality'*1 + St. Err. at 5°C at 35 DAT 50.00+ 6.50 86.11 ± 2.50 38.89 ± 9.60 33.33 ±11.00

'*' Corrected according to the Abbott's formula

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Response of the selected isolates at different temperatures Only the most promising N0093 and N0089 cold active isolates were tested. The method used was the Otiorrhynchus-Assay with a dose corresponding to 125,000 nematodes/m". Different temperatures and incubation times were used. At the temperature of 20°C the efficacy was measured at 7 and 14 DAT; while when incubated at either 12 or 5°C the efficacy was measured at 14 and 28 DAT. All the treatments were done simultaneously. 5. feltiae UK strain and water treatments were included as control. Larval mortality was corrected according to the Abbott's formula. The results obtained are shown in Table 7. At 20°C all the three nematodes performed in the same efficient manner at 7 DAT. At 14 DAT the isolate N0089 reached the larval mortality values of about 90% and was superior in activity than N0093 and S. feltiae UK strain. At 12°C, isolate N0093 and N0089 were significantly better than S. feltiae UK strain when measured at 14 DAT while at 28 DAT N0093 was the best ranking isolate reaching about 85% insect mortality. At 5°C isolate N0093, kept its high efficacy and was able to kill about 70% of the insects already at 14 DAT in comparison to only about 20% to 30% shown by the two other isolates. This same pattern was kept at 28 DAT Table 7 Response of the selected isolates at different temperatures and different exposure times, with 125,000 nematodes/m2, using Otiorrhynchus-Assay Nematode SfUK N0093 N0089

Temperature 20°C 20°C 20°C

SfUK N0093 N0089

12°C 12°C 12°C

(%) Larval Mortality'* 1 ± St. Err. at 7 DAT 80.77+ 3.70 84.61 ± 9.80 73.08 ±13.35

(%) Larval Mortality'* 1 ±St. Err. at 14 DAT 77.27 ± 3.70 77.27+ 7.41 90.91 + 7.41

-

30.43+ 7.41 73.91 + 0.00 69.56 ± 9.80

55.00 ±11.11 85.00 ± 6.41 65.00 ± 7.41

20.00+ 3.70 68.00 ± 3.70 32.00+13.35

41.67 ±13.35 79.17 ± 3.70 54.17+ 3.70

Corrected according to the Abbott's formula

SfUK N0093 N0089

5°C 5°C 5°C

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(%) Larval Mortality'* 1 ±St. Err. at 21 DAT

-

Larval mortality and invasion rate of N0093 isolate at 3°C The efficacy of isolate N0093 was tested even at temperature of 3°C using the Otiorrhynchus-Assay for 7, 14, 21 and 28 DAT. After each exposure time the larval mortality (corrected according to the Abbott's formula) was recorded and all the larvae (dead and alive) were washed and incubated for 3 more days at 20°C The larval mortality (corrected according to the Abbott's formula) was recorded again and all the dead larvae were opened for the evaluation of nematode invasion expressed as number of nematodes per insect larva. Three classes of nematodes were recorded: Dauer Juveniles and fourth stage (DJ/J4) together, males and females. As it can be seen in Figure 1, at 3°C only about 20% of the insects died 7 DAT. The insect mortality reached 40% at 14 DAT, 55% at 21 DAT and more then 60% after 28 DAT. After 3 more days of incubation at 20°C there was an increase of about 30% constant for all the four incubation periods. This indicates that after an exposure of only 7 days at 3°C a significant number of nematodes penetrate the host resulting in 50% insect mortality. If these parasitized insects are further incubated for three more days at temperature of 20°C the insect mortality reaches values of 80 to 90%. The total number of nematodes (Figure 2) found in each insect cadaver exposed at 3°C for 7 DAT is about of 30 specimens (2.3% of the total nematode population). The number of invading nematodes did not increase with time and it suggests that only about 2-3% of the total nematode population penetrates the host very quickly when incubated at 3°C After 7 days at 3°C plus another 3 at 20°C most of the nematodes were in the juvenile stages (DJ or J4) while after 14, 21 or 28 days at 3°C plus another 3 days at 20°C there was a predominance of adults. These results suggest that the isolate N0093 can develop at this extremely low temperature. This was confirmed during the experiment by opening some insects that were present in spare pots prepared for the specific objective.

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Figure 1: Larval mortality caused by N0093 isolate at 3°C at different exposure times using Otiorrhynchus-A ssay Mortality after different DAT at 3°C with and without an additional 3 days incubation at 20°C

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Efficacy of the selected isolates against larvae of B. elephas at cold temperature In order to test the efficacy of the selected cold active isolates N0093 and N0098, they were tested together with 5. feltiae UK strain, against mature larvae of B. elephas, an insect pest of chestnut that causes damage during the autumn-winter seasons. Table 8 shows that at 8°C the isolate N0093 caused an insect mortality of about 70% 21 DAT in comparison to 40% for isolate N0089 and only 25% S. feltiae UK strain. Table 8 Efficacy at 8°C of the selected isolates at 21 DAT against larvae of Balaninus elephas (COLEÓPTERA; CURCULIONIDAE) Nematode SfUK N0093 N0089

(%) Larval Mortality'* 1 ±St. Err. at 21 DAT 24.14 ± 5.00 68.97 ± 6.29 37.93 ± 6.45

Corrected according to the Abbott's formula

Winter outdoor trial An outdoor trial was done in February 1998. Only the most promising N0093 and N0089 cold active isolates were tested. Three litres pots with 1 year old strawberry plants and ten O. sulcatus larvae were treated with 125,000 or 500,000 DJs/m2. Larval mortality was assessed 24 DAT. The results shown in Table 9 show that only the isolate N0093 at a dose of 500,000 DJs/m2 gives a significant insect control effect (about 70% insect mortality) 24 DAT. The temperatures recorded during the trial were very low (Table 10). During only 8% of the time the temperature was above 8°C and for very short intervals of time (11-12°C was reached only twice and for no more then 1 hour each time). On the other hand, 46% of the time the temperature was below 3°C

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Table 9 Winter outdoor trial with hvo doses of the selected isolates on potted strawberry plants infected with O. sulcatus larvae at 24 DAT Treatment Water Control Sf UK Low dose* SfUK High d o s e " N0089 Low dose N0089 High dose N0093 Low dose N0093 High dose

(%) Larval Mortality ± St. Err. at 5°C at 21 DAT 18 ± 4.89 34 ± 11.64 34 ± 8.11 3 4 + 3.99 46 ±12.06 42 ± 3.73 72 ± 9.68

*Low dose = 125,000 nematodes/m' **High dose = 500,000 nematodes/m2

Table 10 Temperature frequency during the outdoor trial Temperature (°C) -1/0 0/1 1 11 2/3 3/4 4/5 5/6 6/7 7/8 8/9 9 / 10 10/11 11/12

Frequency (%) 14 10 9 13 11 10 9 10 6 3 3 1 1

In Vivo nematode production This experiment has been done in order to study the reproduction rate in vivo in G. mellonella larvae of the cold active isolate N0093 in comparison to S. feltiae UK strain. Table 11 shows that the isolate N0093 reproduce very well at 15°C while no reproduction is observed at25°C

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Table 11 In Vivo (G. mellonella) nematode production at different temperatures expressed as number of nematodes/mg of insect + St. Err.

10°C 15°C 20°C 25°C

N0093

S. feltiae UK strain

6+/-1 136 days* 207 +/- 37 111 days* 34 +/- 20 69 days* 0

7+/-4 136 days* 160+/-26 92 days* 121 +/-44 45 days* 125+/-29 38 days*

* Length of production In vitro (liquid fermentation) nematode production Table 12 shows the final concentration of DJs obtained after 2 weeks at 20°C The isolate N0093 was able to reproduce well under these conditions. In order to ascertain the cold activity of the liquid produced nematodes at 20°C, an Otiorrhynchus-Assay has been performed comparing these nematodes to those produced in vivo at 5°C for 21 DAT (Table 13). The ability of the liquid produced nematodes N0093 to kill O. sulcatus larvae at 5°C is comparable to those produced in vivo. Table 12 Liquid nematode production N0093

S. feltiae UK strain

(DJs/ml of media ± St. Err.)

(DJs/ml of media ± St. Err.)

150,000+/-24,000

110,000+/-6,700

Table 13 Efficacy of the liquid produced cold active nematode at 5°C for 21 DAT compare to the one produced in vivo, using Otiorrhynchus Assay. Treatment N0093 in vivo production N0093 in liquid production S. feltiae In vivo production S. feltiae in liquid production

(%) Larval Mortality'* 1 ± St. Err. at 5°C at 21 DAT 69.23 ± 4.08 61.54 ±11.09 25.64 ± 4.79 17.95 ± 4.08

'*' Corrected according to the Abbott's formula

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Species determination of the cold active isolate N0093 From the studies done by Dr. Alex Reid, it was concluded that isolate N0093 belongs to the S. kraussei species.

CONCLUSIONS: A unique nematode strain very active at temperatures as low as 3-5°C and belonging to the S. kraussei species has been isolated from a natural environment (Italian Alps, 1500 a.s.l.) This strain shows a much better activity at low temperatures than any other species reported in the literature. More over, its capabilities to perform well in the cold has been demonstrated in experimental conditions very similar to actual field environments. The field data reported confirmed these observations. The use of this new strain will allow for the biocontrol of susceptible insects in areas in which the average soil temperature is below 12°C So far no products based on entomopathogenic nematodes are available for this type of applications. Its activity against larvae of O. sulcatus , a real target pest that causes damage in nature at very low temperatures, has been demonstrated in the field. Other nematode strains reported in the literature with cold activity have used model insect such as G. mellonella or T. molitor or other Lepidopteran species. These insects are much more sensitive to nematodes than O. sulcatus. Several authors also have used artificial environmental conditions such as petri dishes with paper or sand or columns with sand in order to demonstrate activity of nematodes at low temperatures. In this study a system containing all the natural elements needed for the control of O. sulcatus by entomopathogenic nematodes has been used. Pots containing non-sterile peat, fertilizers and the plants attacked by the pest were used. The organisms present in this mix are similar to the ones present in normal soil and are those able to interfere with the nematode activity. They could be represented by fungi, bacteria or insects (Collembola) that could kill nematodes. The presence of a root system could also confuse nematodes due to their carbon dioxide emission. Under this environmental conditions, and at an application dose of 500,000 nematodes/m2, (recommended commercial dose for other products), isolate N0093 retained 80% insect killing efficacy even at 5°C This suggests that this specific strain could be used in the biocontrol of sensitive insect pests during late autumn or early spring or even during mild winter soil temperatures.

135

The selected cold active nematode strain has a relatively fast mode of action. A t 5°C the selected nematode can control over 80% of the insect population in just 2-3 weeks. In this manner they are able to prevent serious damage to the treated crops. The selected cold-active isolate is active not only at a constant temperature of 3°C but also at a variable soil temperature with an average around 3°C (see field trial). However, its capability to control O. sulcatus larvae is retained even at temperatures as high 20°C. Other isolates selected on the Italian A lps such as N0089, N0035 and N0094 are also good as cold active nematodes. However, they do not reach the wide range of temperature activity shown by isolate N0093 and did not reach the efficacy of this isolate at temperatures of 5°C or lower. Isolate N0093 has been genetically characterized and has been the subject of a recently filed patent. It is expected that its unique characteristics will allow for the development of a new bioinsecticide product.

References:

Berry, R.E., Liu, J. and Groth, E. 1997. Efficacy and persistence of Heterorhabditis marelatus (Rhabditida: Heterorhabditidae) against root weevils (Coleóptera: Curculionidae) in strawberry. Environmental Entomology. 26(2): 465-470. Dutky, S. R., Thompson, J. V. & Cantwell, G. E. 1964. A technique for mass propagation on the -DD-136 nematode. Journal of Insect Pathology, 6: 417-422. Eculica, J.F., S. Becvar, Ζ. Mracek and P. Kindlmann 1997. Laboratory evaluation of control of the European corn borer, Ostrinia nubilalis (HB) (Lep, Pyralidae) by nematodes of the genus Steinernema (Nematoda, Steinernematidae) at low temperature. Journal of Applied Entomology-Zeitshrift fur Angewandte Entomologie 121: 407-409. Griffin, CT. and Downes, M.J. 1991. Low temperature activity in Heterorhabditis sp. (Nematoda: Heterorhabditidae). Nematologica. 37, 83-91. Grewal, P.S., Selvän, S. and Gaugler, R. 1994. Thermal adaptation of entomopathogenic nematodes: Niche breadth for infection, establishment, and reproduction. J. therm. Biol. 19, 245-253. Gwynn, R.L. 1994. Development of cold active nematodes for insect pest control. PhD. Thesis, Department of Agriculture, Reading University, UK, 214 p. Mason, J.M. and Hominick, W.M. 1995. The effect of temperature on infection, development and reproduction of heterorhabditis. Journal ofHelminthology. 69, 337-345.

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Miduturi J.S., R. De Clercq, H. Casteels and A. De Grisse 1994. Effect of temperature on the infectivity of entomopathogenic nematodes against black vine weevil (Otiorrhynchus sulcatus F.) Parasitica. 50(3-4): 103-108. Moorhouse, E.R., Charnley, A.K., and Gillespie, A.T. 1992. A review of the biology and control of the vine weevil, Otiorrhynchus sulcatus (Coleóptera: Curculionidae). Annals of applied Biology 121:431-454. Reid A.P., Hominick, W.M. & Briscoe, B.R. (1997). Molecular taxonomy and phylogeny of entomopathogenic nematode species (Rhabditida: Steinernematidae) by RFLP analysis of the ITS region of the ribosomal DNA repeat unit. Systematic Parasitology, 37, 187-193. Shirocki, A. and Hague, N.G.M. 1994. The effect of temperature on the susceptibility of the black vine weevil, Otiorrhynchus sulcatus, to different isolates of Steinernema and Heterorhabditis. In: Burnell, A.M., Ehlers, R.U. and Masson, J.P. (edit.): Genetics of entomopathogenic nematode-bacterium complexes. 222. Brussels. Smith, F.F. 1932. Biology and control of the black vine weevil. U.S. Dep. Agric.Tech. Bull. 325. Steiner, W.A., 1996. Dispersal and host finding ability of entomopathogenic nematodes at low temperatures. Nematologica. 42: 243-261. Westermann, P.R., and Van Zeeland, M.G., 1989. Comparison of Heterorhabditis isolates at low temperatures. Med. Fac. Landbouww. Riiksuniv. Gent, 54: 1115-1124.

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Breeding oí Steinernema Feltiae For UV Resistance Marek Tomalak Department of Biocontrol and Quarantine, Institute of Plant Protection Miczurina 20, 60-318 Poznan, Poland

SUMMARY: Susceptibility of entomopathogenic nematodes to ultraviolet (UV) can be a serious limiting factor in their field application against pest insects. Infective juveniles are paralyzed and killed within a short period of time after exposure to UV radiation. As the phenotypic variation of this character is very narrow in natural populations, genetic selection cannot be considered here as a potentially effective method for the nematode improvement. Searching for UV resistant mutants seems to be much more promising approach. Phenotypic analysis of Steinernema feltiae mutants revealed that a proportion of infective juveniles homozygous for pn36 mutation in the Sfrol-1 gene survived prolonged exposure to UV, successfully infected Galleria mellonella and reproduced in its hemocoel, thereafter. Subsequent detailed breeding study showed that the increased UV resistance was inherited in the offspring of UV treated survivors. However, the penetrance of the responsible gene is not complete, as both the susceptible and resistant infective juveniles are segregated in the following generations. The increased UV resistance is apparently caused by a recessive mutation in an individual gene. This could be a specific mutation in Sfrol-1 gene or in a separate gene closely linked to the Sfrol-1 gene. Cross breeding with a series of other morphological mutants showed that the increased UV resistance could be easily transferred to other populations within the S. feltiae species. Although the examined mutant allele improved the nematode survival after UV treatment, infectivity of juveniles to insect hosts was reduced.

INTRODUCTION Nematodes present a limited tolerance to UV radiation. Particularly harmful is the short UV light of 254 nm wavelength. A number of negative effects such as reduction of survival, motility, host invasion and reproduction have been observed in plant parasitic nematodes (Green & Webster, 1965; Green & Plumb, 1967). Among entomopathogenic species Steinernema carpocapsae showed to be less sensitive to UV radiation than Heterorhabditis bacteriophora, however, in both species reduced pathogenicity, lower reproductive potential, partial or complete inactivation and premature death are frequent effects of the nematodes' exposure to UV (Gaugler & Boush, 1978; Gaugler et al, 1989; Gaugler et al, 1992). This causes that the

nematode susceptibility to UV is considered as an important factor limiting their field efficacy (Gaugler & Boush, 1978; Gaugler et al., 1992; Smits, 1996). Improvement of S. carpocapsae resistance/tolerance to UV has been attempted by the use of UV protectants (Nickle & Shapiro, 1992) and selection (Gaugler et al, 1989). The latter approach could not, however, be effective as natural variation of the trait was low among tested geographically distant populations of S. carpocapsae. Nevertheless, high heritability of UV tolerance observed in H. bacteriophora suggests that selection for improvement of this trait can still be possible in other species (Glazer et. al, 1991). Mutagenesis and identification of UV resistant/tolerant mutants could be another possible approach with the unquestionable advantage for relatively easy transfer of the improved trait to any other strain within the species. A series of mutants presenting increased UV tolerance has been recently found in Caenorhabditis elegans (Duhon et al, 1996; Murakami & Johnson, 1996) Our preliminary screening of available populations of Steinernema feltiae revealed that a proportion of infective juveniles homozygous forpn36 mutation in the roller Sfrol-1 gene survived prolonged exposure to UV, successfully infected Galleria mellonella and reproduced in its hemocoel, thereafter, while a similar dose of radiation was absolutely lethal to other strains.

The main objective of the research reported here was to provide further details of the observed phenomenon and to explain the genetic basis for UV resistance/tolerance in the identified S. feltiae mutant.

MATERIALS AND METHODS The UV resistance/tolerance screening was conducted on 19 strains of S. feltiae, including 4 fresh isolates (PLO; PLI; PL2; PL4), 10 laboratory strains (ScP; SN; UK76; SF1; OBSIII; JHF; V25A; M103; Te Anau; Canterbury) and 15 mutant strains carrying individual mutations in the following genes: dpy-l(pn7, pnll, pn29, pn31, pn33, pn34), dpy-2 (pn35), seg-I(pn!2), unc-l(pnl3, pnl5, pn28, pn37), rol-l(pn¡6, pn36) and vab-l(pnl) (Tomalak, 1994; 1997; 1998).

140

The screening was performed in 0 9 cm Petri dishes with 5 mm layer of 2% agar. Some 1000 IJs in ΙΟΟμΙ H2O were pipetted onto the agar surface. After pipetting the juveniles, dishes remained open for 60 minutes to facilitate drying out the excess of water and to allow nematodes to disperse over the agar surface. Subsequently, the nematodes were subjected to UV light of 254 nm wavelength, generated by Philips TUV 30W tube, placed 30 cm above the dishes. The exposure time was 0, 3, 6, 9, 12, 15, 18 and 24 min. for estimation of a standard minimum exposure time in which all individuals were killed, and 15 minutes for all subsequent screening tests. After the exposure all nematodes were washed off from the agar surface and left in distilled water at 20±2°C Survival of UV treated individuals was evaluated on day 2 and 7 after the exposure.

Since at the chosen standard minimum exposure time (15 min.) only a small proportion of nematodes from a single mutant strain (rol-l(pn36)) (Tomalak, 1998) survived the treatment, individual survivors were subjected to further breeding. After individual backcrosses to wild-type ScP strain in vitro and subsequent injection of the resulted Fl juveniles into G. mellonella, the obtained populations of IJs were again exposed to UV. All nematodes which survived 7 days after the exposure were injected into G. mellonella to produce homozygous lines with respect to the allele controlling the UV resistance/tolerance. The method of in vitro I in vivo breeding was as described earlier for other S. feltiae mutants (Tomalak, 1994; 1997).

In order to examine the possibility of transfer of the UV resistance/tolerance allele to other S. feltiae strains a series of crosses between individuals from the tolerant pn36 population and morphological and behavioral mutants, such as

seg-l(pnl2),

vab-l(pn32), vab-2(pnl), vab-3(pn41), dpy-l(pn7), dpy-l(pn31), dpy-2(pn35) and unc-l(pnl3) was made according to the method described earlier for breeding of double mutants (Tomalak, 1997; Tomalak & Mracek, 1998).

The UV resistant/tolerant mutant populations were examined for their ability to infect Galleria mellonella and Bradysia paupera on filter paper and in composted bark peat-moss substrate, respectively. Three G. mellonella or 10 B. paupera larvae were exposed to 100 IJ of the nematode for 24 h at 20±1°C Subsequently, all insects were 141

dissected and both the number of infected insects and proportion of infecting nematodes were counted. All tests were performed in 6 replicates.

RESULTS AND DISCUSSION Determination of a standard lethal UV dose Wild-type infective juveniles of S. feltiae (ScP strain) showed to be very sensitive to the 254 nm UV radiation. As short as 3 min. exposure caused paralysis of a proportion of nematodes immediately after the experiment. The process of dying was, however, much longer. After 2 days dead individuals could be observed only in populations exposed for 6 min. and longer. The recorded nematode mortality was 43.3% for 6 min. exposure, 97.7% for 9 min., 99.8% for 12 min., and 100% for longer exposures. A difference in mortality rates was found between plates where nematodes dispersed evenly over the agar surface and those with distinct clumps or multi-layer aggregations of IJs. The latter group could survive somewhat longer exposure. Mortality rates observed here were 18.8, 94.4, 99.5 and 100% for 6, 9, 12 min. and longer, respectively. By the day 7 survival of nematodes exposed to UV was further dramatically reduced. The 9 min. exposure was lethal to all individuals. Recorded mortality rates were 79.0% for 3 min. and 100% for all longer exposures of dispersing nematodes. Aggregating juveniles survived better. Mortality recorded here was 52.4% for 3 min. exposure, 78.1% for 6 min. and 100% for all longer exposures. These results can be explain by a relatively low ability of short wave radiation to penetrate deep into objects. External layers of aggregating nematodes apparently protected individuals hidden underneath. However, this protection was not completely effective as most individuals continuously moved within the aggregation.

It is clear that S. feltiae does not differ from other nematodes in its susceptibility to UV radiation. Important to note is, however, the extended period of time before the lethal effect could be recorded in the whole population. This observation suggests that in practical terms even a short exposure to the sun and UV radiation during field

142

application

can

have

a

long-term

effect

on the

nematode

survival

and

entomopathogenic potential in the soil. Resistance/tolerance ofSteinernema feltiae strains to UV at 15 min. exposure Based on results of the previous experiment with ScP strain, 15 min. exposure time of UV radiation has been chosen as a minimum standard dose which supposed to kill all nematodes. Subsequent tests with 4 fresh isolates, 10 laboratory strains and 15 mutant strains of S. feltiae confirmed validity of this choice. The only exception was Sfrol-1 (pn36) strain. In many plates with this strain individual live IJs were repeatedly isolated 7 days after the 15 min. exposure to UV. All pn36 mutants present characteristic roller phenotype which makes them easy to distinguish from wild-type and other mutant strains.

Determination of the genetic basis for UV resistance/tolerance in Steinernema feltiae Additional tests allowed us to isolate over 54 UV survivors out of some 1 χ IO5 examined pn36 IJs. The UV resistance/tolerance character was also segregated by F2 offspring of crosses between these nematodes and wild-type individuals from ScP strain. From populations presumably homozygous for the allele in question and subjected to subsequent UV treatment 2.2-3.1% individuals survived the exposure. Further breeding within the UV survivors group did not improve overall resistance/tolerance to UV of the offspring populations. All subsequent generations presented similar survivorship after exposure to UV radiation of 254 nm. These observations suggest that the improved UV resistance/tolerance could be a result of a recessive mutation in an individual gene. Penetrance of this mutation is, however, very law, as the characteristic phenotype can be observed in only a small proportion of individuals homozygous for the examined allele. Interestingly, all survivors presented distinct roller phenotype. Whether the observed UV resistance/tolerance results from mutation within the Sfrol-1 gene or it occurs in a separate, but closely linked to Sfrol-1 gene remains to be investigated in the nearest future. During our breeding of mutants and double mutants for homozygosity UV tolerant morphologically wild-type individuals were occasionally isolated from

143

various populations. Unfortunately, they all were lost due to inability of infective juveniles to recover for further development or due to inability of adults to mate and/or reproduce in vitro.

Transfer of 'UV resistance/tolerance allele' between Steinernema feltiae strains If the described character is controlled by individual mutation it should be easily transferred to any population within S. feltiae species. Examination of homozygous double mutant populations obtained from crosses between the UV resistant/tolerant individuals from pn36 roller strain and other morphological mutants revealed that the UV resistance/tolerance can be transferred to other strains by the means of genetic recombination. However, this trait was not universally expressed in all tested recombinants. Populations of double mutants carrying pn7, pn31, pn35 and pnl3 alleles did not show any increase in tolerance to UV radiation, while those with pnl2, pnl and pn41 alleles yielded 1.8 - 2.9% individuals which could survive 15 min. exposure for a period longer than 7 days. The difference in expression of the examined character could result from epistasis between tested alleles. In tested crosses pn7,pn31,pn35

and pnl3 alleles were epistatic to the UV resistance/tolerance allele,

while the latter was epistatic io pnl2, pnl and pn-ll alleles.

Effect of'UV resistance/tolerance allele' on S. feltiae infectivity to insects From the practical point of view it would be interesting to know how well the newly acquired character protects nematodes from the loss of activity and infectivity to insect

hosts. Effects

of

the

identified

UV

resistance/tolerance

allele

on

entomopathogenic activity against G. mellonella and B. paupera have been presented in Fig. 1 and 2, respectively.

144

D % infected insects

ro

i ■ %infecting nemáodes

3

■D

> 100-

-σ mc 60 4—

O

40-

*-» ?nfe 0 o

b

Di 1 SP

ιΟ) OL

b

τ

τ á

LK76

pn36

ùL pi36U/(-)

pn3cU/(+)

Fig. 1. Infectivity of Steinernema feltiae wild-type and mutant strains to Galleria mellonella larvae.

In tests with G. mellonella pn36 mutants were generally less effective than wild-type strains ScP and UK76, both in the proportion of infected insects and the proportion of infecting nematodes. Before UV treatment no difference was shown between pn36 populations with, and without the UV resistance/tolerance alleles. However, the UV treatment significantly reduced the prevalence of infection by the survivors (pn36UV+), while the proportion of infecting nematodes remained almost unchanged.

D% infected insects

"S 3 Ό >

C

■ % infecting nemat odes 80 η 6 0

τ-

τ

τ

_3I

1

Ζ- 40 2 20 „ i


n36UV (-)

pn36UV (+)

Fig. 2. Infectivity of Steinernema fel tiae wild-type and mutant strains to Bradysia paupera larvae (** significantly different).

145

Contrary to results from the previous experiment infectivity of wild-type and pn36 mutant strains to B. paupera larvae was similar. Here again before UV treatment no difference

was

found

between

roller

mutants

with,

and

without

UV

resistance/tolerance alleles. The UV exposure reduced, however, infectivity of survivors, with respect to both the proportion of infected insects and proportion of infecting nematodes, with only the first value significantly different from the rest.

Results of these experiments indicate that in untreated with UV populations of mutants the presence of UV resistance/tolerance alleles does not change the nematode infectivity to insect hosts. However, in spite of improved survival after the exposure to UV radiation, survivors are significantly less infective to insects than unexposed mutants and wild-type individuals. This may suggest that at least some characters crucial to the host finding or recognition are damaged by the UV treatment.

CONCLUSIONS The increased UV resistance/tolerance observed in S. feltiae is apparently caused by a mutation in an individual gene. This could be a specific mutation in Sfrol-1 gene or in a separate gene closely linked to the Sfrol-1 gene. Penetrance of this mutation is, however, low as in subsequent generations only 2.2 3.1% of homozygous IJs present the UV resistant/tolerant phenotype. The UV resistance/tolerance allele can be easily transferred to other S. feltiae populations by the means of genetic recombination, but it becomes phenotypically expressed in only some of them. Although the described mutation improves survival of IJs after the exposure to UV, the survivors' infectivity to insect hosts can be significantly reduced. Due to its low penetrance the described mutation seems to have rather limited potential for improvement of entomopathogenic nematodes. Nevertheless, further

146

examination of factors involved in its expression may help in understanding mechanisms of the nematode resistance/tolerance to UV radiation.

References Duhon, S.A., Murakami, S. & Johnson, T.E. 1996. Direct isolation of longevity mutants in the nematode Caenorhabditis elegans. Developmental Genetics. 18: 144-153. Gaugler, R., Bednarek, A. & Campbell, J.F. 1992. Ultraviolet inactivation of heterorhabditid and steinernematid nematodes. Journal of Invertebrate Pathology, 59: 155-160. Gaugler, R. & Boush, G.M. 1978. Effects of ultraviolet radiation and sunlight on the entomogenous nematode, Neoaplectana carpocapsae. Journal of Invertebrate Pathology, 32: 291-296. Gaugler, R., McGuire, T. & Campbell, J. 1989. Genetic variability among strains of the entomopathogenic nematode Steinernema feltiae. Journal of Nematology, 21:247-253. Glazer, I., Campbell, J.F. & Segal, D. 1991. Genetics of the nematode Heterorhabditis bacteriophora strain HP88: the diversity of beneficial traits. Journal of Nematology, 23: 324-333. Green, CD. & Plumb, S. 1967. The effect of ultra-violet radiation on the invasion, survival and fertility of larvae of Lleterodera rostochiensis. Nematologica, 13: 186-190. Green, CD. & Webster, J.M. 1965. The effect of ultraviolet radiation on the stem and bulb nematode Ditylenchus dipsaci (Kuhn). Nematologica, 11: 638-642. Murakami, S. & Johnson, T.e. 1996. A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans. Genetics, 143: 1207-1218. Nickle, W.R. & Shapiro, M. 1992. Use of a stilbene brightener, Tinopal LPW, as a radiation protectant for Steinernema carpocapsae. Journal ofNematology, 24: 371-373. Smits, P.H. 1996. Post-application persistence of entomopathogenic nematodes. Biocontrol Science and Technology, 6: 379-387. 147

Tomalak, M. 1994. Phenotypic and genetic characterization of dumpy infective juvenile mutant in Steinernema feltiae

(Rhabditida: Steinernematidae).

Fundamental and Applied Nematology, 17:485-495. Tomalak, M. 1997. New morphological variants of infective juveniles associated with mutations in four sex-linked genes of Steinernema feltiae

(Filipjev)

(Nematoda: Steinernematidae. Fundamental and Applied Nematology, 20: 541-550. Tomalak, M. 1998. Analysis of Steinernema feltiae mutants. Genetic and Molecular Biology of entomopathogenic Nematodes. Cost 819. Report EUR, in press. Tomalak, M. & Mracek, Z. 1998. Scanning electron microscope study on morphological modifications of lateral fields in infective juveniles of mutant Steinernema feltiae (Filipjev) (Nematoda: Steinernematidae). Fundamental and Applied Nematology, 21: 89-94.

14S

The relationship between energy metabolism and survival of the infective juveniles of Steinernema carpocapsae under unstressed-aerobic and anaerobic conditions Lihong Qiu & Robin Bedding Division of Entomology, CSIRO

Introduction The infective juveniles (IJs) of entomopathogenic nematodes (ENs) are non-feeding and so, unlike most other animals, they are unable to compensate for energy consumption with energy intake, depending solely on the energy reserve materials (ERMs) for energy supply. ERMs are likely to be one of the key limitations on the life-span of IJs as well as important factors influencing infectivity. Indeed, it has been suggested that glycogen is an important source of energy for maintaining infectivity of the aged IJs (Patel & Wright, 1997).

Currently, our understanding of the process of the energy metabolism of IJs is rather limited with many questions remaining unanswered. For instance, what are the key ERMs of the IJs; how do they consume these materials; what are the effects of environmental factors on this process, and what is the relationship between the consumption of ERMs and the survival and infectivity of the nematodes? Answers to these questions will not only increase our understanding of the general biology of these organisms but also provide us valuable information to improve the EPN based biopesticides products. This paper is intended to address these questions for IJs of Steinernema carpocapsae under both unstressed-aerobic and anaerobic conditions.

Materials and methods Nematodes: The infective juveniles of the entomopathogenic nematode Steinernema carpocapsae Agriotos were produced on a semi solid medium containing egg, corn flour, oil, yeast etc. in flasks or in self aerating trays (Bedding, 1981; Bedding et al, 1991). Pure, clean infective juveniles, which are essential for accurate measurement of mean dry weight and biochemical analysis, were obtained by firstly incubating the IJs in 0.4% formalin solution for 2 hours on a shaker to kill most non-infective nematodes then having them actively migrate through a layer of milk filter to remove all medium debris and dead nematodes.

Experimental design: To investigate the energy metabolism of the IJs under aerobic unstressed conditions and the relationship between the consumption of ERMs and the survival and infectivity of the IJs, freshly harvested and purified IJs were suspended in tap water at a density of 0.2 million/ml. 200 ml of such suspensions were incubated in 500 ml flasks at 28°C on an orbital shaker at 100 rpm. Samples were taken weekly for biochemical analysis and the measurement of the mean dry weight (MDW), survival rate and infectivity.

To investigate the energy metabolism and survival of the IJs under anaerobic conditions, the nematodes were diluted to 0.1 million/ml in M9 buffer in a 500 ml reagent bottle equipped with a Quickfit cap (Bibby Sterilin Ltd, England). Pure nitrogen was bubbled into the bottle through a long needle inserted into the bottle via the septum on the Quickfit at a rate of about 100 ml/min to achieve absolute anaerobic conditions and maintain the suspension of the nematodes. Another short needle with a smaller ID was insert into the bottle via the septum to allow the exit of nitrogen. Samples were taken for analysing the biochemical composition and assessing the survival rate, MDW etc. at designated time intervals.

Survival rate of the IJs: The survival rates of the IJs under aerobic conditions were assessed by comparing the density of IJs at the time of examination to the initial density while compensating for water vapour lost during incubation. The percentage survival of the IJs

150

under anaerobic conditions was assessed by directly counting the surviving and dead IJs after allowing for revival in M9 buffer. Mean Dry weight: MDW of the IJs was measured by filtering 0.6 to 1 million IJs on predried and preweighed Watman No. 1 (ID 42mm) filter papers then drying in an oven at 75°C for 3 hours. Infectivity: The infectivities of the IJs were assayed using the following two protocols: 1. Exposure of 50 mealworm (Tenebrio molitor) larvae to 5000 IJs in moist peat moss at 23°C for 4 days then assessing mealworm mortality (3 replicates for each treatment); 2. Exposing single Galleria larvae to 100 IJs in tubes of pre-washed and sterile sand for 2 days at 23 °C prior to dissecting the cadavers in Ringer solution and counting the numbers of established nematodes (10 replicates for each treatment).

Biochemical analysis: The lipid levels of the IJs were analysed by hydrolysing all hydrolysable fat into fatty acids then esterifying the fatty acids into methyl esters followed by analysing with gas chromatography. Glycogen and proteins were determined colorimetrically using anthrone (Mokrasch, 1954) and Commassie brilliant blue (Bradford, 1976) respectively. Trehalose, lactate and other mono and di-saccharides were analysed by direct silylation of the dried nematode samples then analysing using GC (Brown, unpublished).

Results MDW and biochemical composition of the freshly harvested IJs: Freshly harvested IJs of S. carpocapsae produced and purified using the methods describe above have a MDW of 74.3 + 0.9ng containing 8.16 ± 0.07% glycogen, 25.5 ± 0.4% lipids, 49.9+2.9% protein and 2.17+0.02% trehalose. Trehalose was the only sugar found present in significant quantity in the nematode, Fig. 1. Except for trehalose and glycerol which accounted for 0.3 to 0.5% dry weight, no other polyol and mono- or di-saccharide was found present in the nematodes in significant amounts.

151

ï. co

e co

2

CD C

ω

CL

I

ω -Q

£ c

o

77ÅW\V7Å

l

I

^ T^ >i £¿¿1



¿1 VTA τ

10 different dissected larvae

Fig. 3 : Number of penetrated nematodes per housfly larvae (n = 10). Treatment : 5 χ IO4 nematodes per 5 larvae

The same conclusion could not be drawn from the number of hatched flies out of the pupae : 47% untreated pupae hatched in contrast to 37% hatched treated pupae. Big variations were found in the number of nematodes penetrated in the pupae. Numbers varied from 5 till 110 nematodes per pupae treated with 104 nematodes. In one pupae active adult nematodes were found; otherwise infective juveniles were present. Most infective juveniles were inactive or dead, in general these nematodes were clustered as inactive "wool" of nematodes.

165

o o o

■α

120

4-·

cu

E o c

TD Φ

OJ

E 13 C

0J

I iiili L■III I

60 CD C CD Q.

m™mffiW

I

%

12

dissected pupa Fig. 4 : Number of penetrated nematodes per housfly pupa (n = 12). Treatment : 5 χ 104 nematodes per 5 pupae

No differences were observed related to the treatment of full grown, just hatched flies, which could take up water inoculated with juveniles.

Discussion and Conclusion House fly larvae of 2 days old. The dead rate shows a sigmoid curve : first a short lag phase, by the dead of the larvae, followed by an exponential phase, at the moment of the dead of most of the well developed larvae. Two days after the start of the experiment 50% of the larvae were dead as well for the treatment as for the control. Renn et al. (1985) obtained the same results e.g. mortality of 32% after 48 hours for the control objects. Probably this phenomenon can be explained by a lack of food for the 2 days old fly larvae. However, the high mortality in this short period makes it impossible to draw conclusions about the influence of Steinernema on the 2 days old larval stage.

166

Five davs old larvae. Infested larvae with the Steinernema carpocapsae didn't show discoloration as the symbiotic bacterium Xenorhabdus nematophilus do not cause pigmentation. The significantly different LT50 value indicates an increased dead rate caused by the nematodes. Although larvae were in contact with the same amount of infective juveniles, the number of nematodes in the insect larvae was largely different (Fig. 4). Pupae. The number of hatched flies was quite similar for treated and untreated pupae. Important is to mention that most of the pupae were infested but the infective juveniles were inactive, clustered together, with lack of adults (Fig. 5). In the soil aggregation is a typical symptom indicating stress situation (Ishibashi and Kondo, 1990). Renn et al. (1985) reported a complete lack of pupal infection and explained this phenomenon by the inhibitive structure of the spiracula. Experiments on chicken manure. Infection of fly larvae inoculated in chicken manure by the infective juveniles failed completely. Probably the conditions of the manure are not favourable for nematode survival. The same observation was done by Georgis et al, 1987 who mentioned a nematode dead rate of 100%, 24 hours after nematode supply to the manure. To analyse this problem, different characteristics of the chicken manure were analysed: pH, humidity, temperature, uric acid content. The pH range was between 8.6 and 7.9, the humidity varied from 37% for one source of chicken manure to 60% for another origin, the temperature was between 18° and 23°C : none of these factors have a negative influence on nematode survival. The uric acid was 1,9% and this is proven to be harmful to the nematodes. Localisation of fly larvae and pupae in chicken manure. A second problem is the location of the fly larvae and pupae in the chicken manure to solve the question how far nematodes have to migrate through the manure to reach fly larvae.

167

Five days old larvae were placed either on top or in the middle of a 8 cm high container, filled with chicken manure, humidity 37%, pH 8,6 (30 larvae per pot, four replicates). Larvae inoculated in the top layer moved down 6,6 cm (max. possible 8 cm) and pupated larvae 1,7 cm. Those larvae placed in the middle moved up 29% ± 22% and down 71% ± 22%. In contrast pupated larvae moved up 88% ± 9% and moved down 12% ± 9%. Larvae had a tendency to move down to the more wet conditions whereas pupae had a clear tendency to move up to the dryer part. Other authors (Renn et al, 1985 and Kettle, 1990) did the same observations on larvae: e.g. migration to dryer parts in the substrate to pupate. Steinernema carpocapsae was characterised as a more sessile nematode, not actively migrating with poor migrating capacity (Gaugler et al, 1989, Georgis et al, 1987). From this observations it was clear that the localisation of the fly larvae is unsuitable for Steinernema carpocapsae, applied on the upper layer of the manure. On the other hand, the localisation of Steinernema applied in the upper part of the manure is on the right good place for infection of pupae, but pupae seemed to be less susceptible for Steinernema carpocapsae "in vitro".

References CIBA, 1993. Exhibit, biological larvicide. Ciba Groot-Bijgaarden, Belgium, 26 p. Gaugler, R., Mc Guire, T. & Cambell, J., 1989. Genetic variability among strains of the entomopathogenic nematode Steinernema feltiae. Journal of Nematology 21,247. Georgis, R., Mullens, B. & Meyer, J., 1987. Survival and movement of insect parasitic nematodes in poultry manure and their infectivity against Musca domestica. Journal ofNematology 19(3), 292-295. Ishibashi,

N.

&

Kondo,

E.,

1990.

Behavior

of

infective

juveniles.

In:

Entomopathogenic nematodes in biological control. (GAUGLER, R. & KAYA, H. Eds.), CRC Press, Boca Raton, FL, USA, 139-150. Kettle, D., 1990. Muscidae (Houseflies, stableflies). In: Medical and Veterinary Entomology. C.A.B. International, 223-240.

168

Mullens, Β., Meyer, J. & Cyr, T., 1987. Infectivity of insect-parasitic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae) for larvae of some manure-breeding flies (Diptera: Muscidae). Environmental Entomology 16, 769-773. Renn, Ν., Barson, G. & Richardson, P., 1985. Preliminary laboratory tests with two species of entomophilic nematodes for control of Musca domestica in intensive animal units. Annals of Applied Biology 106, 229-233. Stoftelen, R., 1994. Biologische bestrijding van insekten met de entomopathogene nematode Steinernema carpocapsae W. (Rhabditida: Steinernematidae). Thesis, Katholieke Universiteit Leuven, 97p.

169

Posters presented at the workshop

Improved infectivity of cold-stored nematodes against black vine weevil P. F. L. Fitters1, C.T. Griffin1 and R. Dunne2 Department of Biology, National University of Ireland, Maynooth, Ireland " AGASC Experimental Station, Kinsealy, Ireland Insect parasitic nematodes (Heterorhabditis sp.) give good control of black vine weevil at temperatures above 12-14° C, but not in autumn when the temperatures drop and the larvae are most damaging. Our work aims to improve the prospects for control of this pest at low temperatures through cold conditioning of the nematodes. Previous work has shown that improved infectivity against Galleria mellonella in sand tests was obtained by prolonged cold storage. In this experiment cold-stored nematodes were tested against black vine weevil larvae at 9° C. Heterorhabditis nematodes (UK211) were stored for up to 16 weeks at 9° C. At bi-weekly intervals they were used against vine weevil larvae in potted Primula in potting compost at 9° C. Vine weevil mortality increased from 8.4% using freshly produced nematodes, to 26.7 % using nematodes that had been cold stored for 12 weeks. In the same period, the number of nematodes that were able to invade the vine weevil larvae increased almost three fold.

Influence of mineral fertilization on entomopathogenic nematodes in meadow soil M. Jaworska and D. Ropek Department of Agricultural Environment Protection, Academy of Agriculture, 31-120 Cracow, Poland Entomopathogenic nematodes (EN) are found in a range of soil habitats and are exposed to many abiotic and biotic factors. Among them, humidity, soil texture, potential hosts and antagonists are of particular importance. Mineral fertilization should also be included in the list of essential factors affecting nematode activity. This aspect was studied on fertilized meadows where experiments were carried out either under static fertilizer conditions or where different rates of ammonium nitrate and urea were applied. In every case half of each plot was limed. The EN populations (Steinernema feltiae and Heterorhabditis spp.) were studied in 1996 and 1997, after the first and the second cut. Nematodes were present in the majority of plots but were absent in those fertilized with the highest rate of nitrogen (480 kg/ha). Mineral fertilization significantly influenced the pathogenicity of the nematodes. Many potential hosts, including the larvae of beetles and flies, were present in the soil. Nematodes of the family Mermithidae were most frequently found in limed meadows.

Evaluation of cold-active nematodes for the biological control of vine weevils in strawberries P.N. Richardson1, S.J. Long1, D.B. Hay2 & D.M. Welch1 Entomological Sciences Department, Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK ~ Department of Educational Sciences, University of Surrey, Guildford, GU2 5XH, UK In Europe, the vine weevil (Otiorhynchus sulcatus) is a major pest of soft fruit, hardy nursery stock and glasshouse ornamentals. The main damage is caused by larvae feeding on the roots; adults feed on leaves and also contaminate mechanically harvested soft fruit. In the UK, strawberry production (4494 hectares worth £55M, MAFF Statistics for 1996) is severely affected by vine weevil larvae. In most outdoor situations, persistent pesticides such as carbofuran and chlorpyrifos are only partially successful in controlling O. sulcatus. Commercial preparations of entomopathogenic nematodes (Steinernema and Heterorhabditis spp.) are used to control vine weevil larvae in strawberries but they are ineffective below 14 C In laboratory bioassays, some UK isolates of Steinernema spp. parasitised weevil larvae at 2 - 6 C; here we describe two trials to assess their potential for the biological control of O. sulcatus. In the first trial, three isolates of indigenous cold-active nematodes (5. feltiae L128; S. kraussei L17 and L137) were mass-produced in vitro and their efficacy compared with that of two commercial products (S. feltiae 'Nemasys', S. carpocapsae 'Exhibit'). Each nematode was applied at two rates (15000 or 30000 infective larvae per 4 litre pot of horticultural compost), outdoors in mid-January, to strawberry plants that had each been inoculated with 40 weevil eggs in October. Two weeks (February) and eight weeks (March) after application, half of the plants were examined for live or parasitised O. sulcatus larvae. Recovery of parasitised larvae was higher in pots treated with L17 or L137 than it was in pots treated with L128, 'Nemasys' or 'Exhibit'. No parasitised larvae were found in the pots treated with 'Exhibit'. By February, adult nematodes had developed in parasitised hosts; some progeny were second stage larvae. By March, most of the nematodes were infective larvae ready to disperse and invade other hosts.

The following winter, the most effective isolate (LI 37) was tested further. Strawberry plants were inoculated with 20 vine weevil eggs in September; the pots were sunk into field soil in November. In December, nematodes were applied at two rates (S. carpocapsae 'Exhibit') or three rates (S. kraussei LI37). In March, the pots were searched for weevil larvae and nematode persistence and infectivity were assessed. There were no significant differences in the numbers of unparasitised weevil larvae found in untreated pots compared with pots treated with S. carpocapsae. There were highly significant differences in the numbers of unparasitised weevil larvae found in untreated pots and those treated with S. kraussei. The mean soil temperature at a depth of 10 cm (December - March) was 2.7 C.

As a potential control agent for over-wintering vine weevils, S. kraussei out-performed S. feltiae and S. carpocapsae. Further trials are required to determine the efficacy of S. kraussei applied as a drench, or through drip irrigation systems, either to peat bag-grown or field-grown strawberries. A late-autumn application of S. kraussei may result in recycling and dispersal of nematodes from parasitised weevil larvae and reduce the need for further treatment in the spring.

178

Biological control of Hylobius abietis I

S. H. Salinas and E. Christiansen

2

Norwegian Crop Research Institute, Plant Protection Centre, Dept. of Entomology and Nematology. Fellesbygget, NO-1432 Aas, Norway " Norwegian Forest Research Institute, Higskoleveien 12, NO-1432 Aas, Norway In Norway, the trend for reducing and even banning certain insecticides continues and has led to increased interest in alternative control methods. It is expected that within a few years fewer effective insecticides will be available for the control of the Large Pine Weevil, Hylobius abietis L. (Coleóptera, Curculionidae). In Sweden, the authorities have decided to outlaw the use of all insecticides in forestry by the year 2000. In the southern part of Scandinavia, H. abietis is the most serious pest of conifers in restocking areas. If young conifers are left unprotected, approximately 30% of all plants will succumb to pine weevil damage in the first year after planting, and damage will increase in the following 2-3 years (Anon., 1978).

Currently treatment of

transplants with the insecticide permethrin is the common control measure employed in Norway. Several alternative control methods, often related to physical protection of the stems, have been investigated.

Entomopathogenic nematodes have been tried with

success in Sweden (Burman et al, 1979) and in some other countries where H abietis is a problem (Eidt et al, 1995; Collins & Evans, 1989). However, the potential for entomopathogenic nematodes as control agents against this forest pest has yet to be realised. In Norway, work involving MSc. students has just begun to investigate this potential.

In a recent study, the susceptibility of H abietis larvae and adults to

Steinernema feltiae and S. carpocapsae was investigated in the laboratory and in an outdoor insectary. Results showed that S. feltiae was more suitable for control under simulated field conditions (Burls, 1997). In a second, ongoing study, results indicate that horizontal movement of S. feltiae is rather slow. A field experiment is planned for this spring, including nematode treatments of transplants (against adult weevils) and stumps (against developing larvae). In this work, imported commercial S. feltiae will be

used. In future experiments, Norwegian isolates of Steinernema and Heterorhabditis will be tested for their potential against this pest.

The natural occurrence and

distribution of entomopathogenic nematodes in forest soils will also be examined.

References Anon. (1978). "Snyttbaggeutredningen". National Board of Forestry. Jönköping, 1-161 (In

Swedish)

Burman, M. & Pye, A.E. (1979). Ann. Entomol. Fennici 45, 88 Burls, K.O. (1997). MSc. Thesis (In Norwegian), Department of Forest Sciences, Agricultural

University of Norway, N-1432 Aas, Norway.

Collins, S.A. & Evans, H.F. (1989). Section Regionale Ouest Paleartique. Versailles, France2-4

Sept. 1987. OILB p.17-18.

Eidt, D.C, Zervos, S., Pye, A.E. & Finney-Crawley, J.R. (1995).C