to Nitrogenous Compounds and - Springer Link

P1: GMX Journal of Chemical Ecology [joec]

pp799-joec-461509

March 17, 2003

20:23

Style file version June 28th, 2002

C 2003) Journal of Chemical Ecology, Vol. 29, No. 4, April 2003 (°

OVIPOSITION RESPONSE OF Chironomus tepperi TO NITROGENOUS COMPOUNDS AND BIOEXTRACTS IN TWO-CHOICE LABORATORY TESTS

M. M. STEVENS,∗ G. N. WARREN, and B. D. BRAYSHER New South Wales Agriculture and Cooperative Research Centre for Sustainable Rice Production Yanco Agricultural Institute Private Mail Bag, Yanco, New South Wales 2703 Australia (Received February 19, 2002; accepted December 14, 2002)

Abstract—Two-choice laboratory tests were used to investigate the oviposition response of Chironomus tepperi to a range of nitrogenous compounds and crude bioextracts. Responses to nitrogenous compounds varied in response to concentration. Ammonium nitrate did not influence oviposition at concentrations from 2 to 12 mg/liter. Hydroxylamine hydrochloride increased oviposition at 6 mg/liter, but had no effect at either 2 or 12 mg/liter. Sodium nitrate reduced oviposition at 2 mg/liter relative to the controls, but had no significant effect at 6 or 12 mg/liter. C. tepperi responded to many of the crude bioextracts, strongly avoiding oviposition in solutions containing homogenized chironomid larvae (1 final instar/100 ml; C. tepperi or Polypedilum nubiferum), and avoiding solutions conditioned by conspecific larvae at concentrations down to the equivalent of 1 final instar/100 ml over 24 hr. Homogenates of adult conspecifics had no effect on oviposition site selection. Homogenates of larval Culex annulirostris (Culicidae) deterred oviposition, but only at high concentrations (3 final instars/100 ml). Our results demonstrate that chemical cues from larval populations deter oviposition by C. tepperi females searching for newly flooded habitats where larval competition will be minimized. Key Words—Diptera, Chironomidae, midges, Chironomus tepperi, oviposition, bioextracts, rice, Australia. INTRODUCTION

The Chironomidae, or nonbiting midges, are the most widely distributed and abundant insects inhabiting freshwater ecosystems (Pinder, 1986; Cranston, 1995). Larval chironomid communities logically develop in response to two processes: ∗

To whom correspondence should be addressed. E-mail: [email protected]

911 C 2003 Plenum Publishing Corporation 0098-0331/03/0400-0911/0 °


912

pp799-joec-461509

March 17, 2003

20:23


STEVENS, WARREN, AND BRAYSHER

the recruitment of larvae derived from eggs laid by airborne female midges, and the subsequent survival of these larvae that may or may not be suited to a particular aquatic environment. Considerable effort has been directed towards characterizing chironomid assemblages associated with different environments, and numerous studies, many of them laboratory-based, have examined the effects of physicochemical parameters on the survival of different chironomid species. Little attention has been given to factors that may influence oviposition site selection by female midges, and thereby influence larval recruitment. In contrast, the factors that influence oviposition site selection by mosquitoes (Diptera: Culicidae) have been extensively researched. Female mosquitoes respond, either positively or negatively, to a range of interacting physical and chemical stimuli (Bentley and Day, 1989). Oliver (1971) remarked that little was understood about directional oviposition flight and oviposition site selection in chironomids, and comparatively few new data on oviposition site selection have been generated since publication of his review. Williams et al. (1987) found that Chironomus riparius Meigen avoid oviposition in water containing high concentrations of cadmium, but that the avoidance response is fairly insensitive and does not lead to females avoiding solutions containing sufficient cadmium to cause acute toxicity to early instars. Similarly, Dauble and Skalski (1983) found that Tanytarsus dissimilis (Johannsen) readily oviposit in solutions containing acutely toxic levels of coal liquid derivatives, but that egg mass size is reduced. Xue et al. (1994) found that ovipositing Glyptotendipes paripes Edwards do not discriminate between water from four different sources, despite the fact that egg hatchability is significantly higher in lake water. These studies indicate that some chironomids have the ability to detect chemical cues that identify unfavorable oviposition sites, but support the view of Davies (1976) that the level of discrimination is generally poor. Visual cues also may be significant in oviposition site selection by chironomids. Frouz (1997) found that two species of terrestrial chironomids oviposit preferentially in open areas and areas of low vegetation. Harrison and Hildrew (1998) found that oviposition site selection by Cricotopus sylvestris (Fabricius) and Microtendipes pedellus (de Geer) favored shaded areas along lake margins, despite conditions in more open areas appearing to favor larval survival. Chironomus tepperi Skuse is a native Australian chironomid that rapidly exploits newly flooded environments. It is an important food source for breeding waterfowl (Maher and Carpenter, 1984; Crome, 1986) and is also a serious pest of establishing rice crops in southern New South Wales (Stevens, 1998). In small experimental rice fields, C. tepperi has a single generation. Oviposition generally occurs only during the first few days after the fields are flooded, with a single adult emergence peak occurring approximately 17 days later. Females do not oviposit back into the fields from which they have emerged (Stevens, 1994). This study was conducted as a first step towards determining why C. tepperi oviposit only into newly flooded rice fields and avoid older fields even if they have


pp799-joec-461509

March 17, 2003

OVIPOSITION RESPONSE OF MIDGES

20:23


913

only been flooded for a relatively short period. Ikeshoji et al. (1980) found that nutrients (in the form of organic or inorganic fertilizers) increased chironomid production from rice fields in Japan, and consequently we tested a range of nitrogenous compounds to determine their possible influence on oviposition site selection. We also tested a range of bioextracts (larval and adult insect homogenates, as well as water conditioned by C. tepperi larvae) to determine whether chemical cues originating from organisms already present in aquatic environments may influence oviposition behavior.

METHODS AND MATERIALS

Rearing and Collection of C. tepperi. Egg masses of C. tepperi were collected from temporary pools at Yanco Agricultural Institute (34◦ 37’S 146◦ 26’E) and cultured using the technique of Stevens (1992). Aquaria (20-liter capacity) containing 10 liters of 1 × Martin’s rearing solution, (1 × MRS) (Martin et al., 1980) were lined with paper tissue prior to the addition of 3–5 C. tepperi egg masses. A small quantity of K9 goldfish food (Friskies Pet Care, Melbourne, Australia) was added, and the aquarium was covered with plastic film. Aquaria were maintained at 25 ± 1◦ C with a 15L:9D photoperiod. Aeration was provided to each aquarium by a hypodermic needle attached to an aquarium aerator by plastic tubing. Additional food was provided every 2–3 days during larval development. When adult emergence began, the plastic film was removed and replaced with a gauze-covered pyramidal frame. A 2-liter plastic jar was fitted at the apex of the frame for the collection of adults. Test Procedure. Two-choice oviposition tests were conducted using empty 20liter capacity glass aquaria (40 × 23 × 23 cm deep). Two translucent polystyrene cups (model B260, 315 ml capacity, 90 mm diam. at the rim; Polarcup Australia Limited, Sydney, Australia) were placed in each aquarium 5 cm from either end. One of the cups was filled to a depth of 45 mm (20 mm below the rim) with 200 ml of the control solution, while the other cup was filled to the same depth with the test solution/suspension being assessed. Several droplets of sucrose solution were placed on the walls of the aquarium midway between the cups as a food source. Adults were anesthetized using food-grade carbon dioxide (BOC Gases Australia, Sydney, Australia), and 15 males and 15 females were removed from the collection jar and placed in a 9-cm-diam. glass Petri dish between the two test solutions. The aquarium was covered with plastic film and placed in a controlled temperature room (25 ± 1◦ C, 15L:9D). Aquaria were placed at right angles to the horizontal fluorescent lighting system to eliminate the possibility of a phototactic response influencing the choice of oviposition site, and the positioning of test and control solutions at either end of the aquaria was randomized across replicates.


914

pp799-joec-461509

March 17, 2003

20:23



The number of egg masses deposited by the female midges in control and test solutions was counted 24 hr after establishment. Replicates in which no egg masses were laid were discarded, since oviposition failure could occur as a consequence of an inadequate preoviposition period for a particular group of midges or, alternatively, as a consequence of excessive anesthesia. Replication of the test for each solution/suspension continued until 15 replicates were obtained, in each of which at least one egg mass was deposited. Test Solutions/Suspensions. An initial test was conducted using 2 × MRS (i.e., double salt concentrations) against 1 × MRS to determine if there was a simple response to total dissolved solids. When no significant response was obtained, the test was repeated using 3 nitrogenous compounds [ammonium nitrate (NH4 NO3 ), hydroxylamine hydrochloride (NH2 OH.HCl), sodium nitrate (NaNO3 ), all Univar grade, Ajax Chemicals Ltd, Sydney, Australia]. Each compound was tested separately at three rates (2, 6, and 12 mg/liter). Compounds were dissolved in 1 × MRS, and 1 × MRS without supplementary nitrogen was used as the reference solution in the control cups. Oviposition tests were also conducted using 10 different bioextracts prepared with either larval or adult C. tepperi, larval Polypedilum nubiferum (Skuse) (Chironomidae), or larval Culex annulirostris Skuse (Culicidae). Eight of these bioextracts were prepared by homogenizing either adult or larval insects in 1 × MRS, while the remaining two were 1 × MRS conditioned by C. tepperi larvae. Larval and adult C. tepperi were obtained from the laboratory cultures described previously. P. nubiferum larvae were also cultured in the laboratory from locally collected eggs by using a technique based on that described by Hatakeyama (1988). Larval Cx annulirostris were collected from drainage channels at Yanco Agricultural Institute. All larvae used to produce bioextracts were final instars and were washed in distilled water before being manually ground in a 2-ml-capacity glass tissue homogenizer containing a small volume of 1 × MRS. The homogenate was washed into a 200-ml volumetric flask and diluted with 1 × MRS to achieve the desired test concentration. Test concentrations for larval homogenates were: C. tepperi and P. nubiferum, each 1 larva/100 ml; Cx annulirostris, 1 and 3 larvae/100 ml. Adult C. tepperi homogenates were prepared in the same way; however, the adults were not washed prior to grinding. Concentrations for C. tepperi adult homogenates were 1 and 5 adults/100 ml, with homogenates of male and female adults tested separately. Test solutions conditioned by larval C. tepperi were prepared by placing 50 washed final instars into a 1-liter glass beaker containing 500 ml of 1 × MRS. The beaker was placed in to a controlled temperature room (25 ± 1◦ C, 15L:9D) with aeration, but without any food being provided. After 24 hr, the rearing solution was poured off and used immediately in the oviposition tests. This conditioned rearing solution was tested at two concentrations: 10 larvae/100 ml (i.e., undiluted), and diluted with fresh 1 × MRS to 1 larva/100 ml. In all cases, fresh


pp799-joec-461509

March 17, 2003

20:23


915


1 × MRS was used in the reference (control) cups. Test concentrations for conditioned rearing solution were chosen to reflect common C. tepperi densities in establishing rice crops, which often exceed 2,000 larvae/m2 and can reach 13,000 larvae/m2 in favorable seasons (Stevens, unpublished data). In water 5 cm deep, 10 larvae/100 ml and 1 larva/100 ml equate, respectively, to population densities of 5,000 and 500 larvae/m2 . Statistical Analysis. As the data were counts and did not follow normal distributions, it was inappropriate to use t tests to determine statistical significance. Instead, data were assumed to follow Poisson distributions. This assumption did not hold for several data sets; however, this was resolved by incorporating overdispersion parameters in the model. For each data set, a separate Poisson generalized linear model with a loglink function (McCullagh and Nelder, 1989; Schall, 1991) was fitted in ASREML (Gilmour et al., 2001) with an overdispersion parameter if required. Due to possible blocking effects associated with the replication procedure, a random effect for pairs was also fitted. RESULTS

Results from the tests of 2 × MRS and the three nitrogenous compounds are given in Table 1. Doubling the concentration of salts in 1 × MRS did not result in a significant change in oviposition preference, indicating that slight changes in total dissolved solids (from 75 to 150 mg/liter) do not affect oviposition site selection. Ammonium nitrate did not affect oviposition site choice at any of the concentrations evaluated, although the 6 mg/liter concentration did attract more oviposition than either higher or lower concentrations. Similar results were obtained with hydroxylamine hydrochloride, where more egg masses were laid in the 6 mg/liter concentration relative to the 1 × MRS control (P < 0.02). The higher and lower concentrations did not affect oviposition site selection. Sodium nitrate acted as a slight oviposition deterrent at 2 mg/liter (P < 0.05), but had no effect at higher concentrations. Table 2 shows the oviposition response of C. tepperi to bioextracts and conditioned 1 × MRS. Bioextracts containing homogenized C. tepperi or P. nubiferum larvae strongly deterred oviposition by C. tepperi (P < 0.001), with between 89% and 93% of all egg masses produced being laid in control cups. Homogenized Cx annulirostris larvae also deterred oviposition (P < 0.05), but only at the higher concentration of 3 final instars/100 ml. 1 × MRS conditioned by the presence of C. tepperi larvae over a 24-hr period also acted as an oviposition deterrent, even when diluted to a concentration equivalent to 1 larva/100 ml. In contrast, homogenates of adult C. tepperi had no effect on oviposition site choice (P > 0.05), regardless of the sex of the adults used in the homogenate or their concentration.

1.07 ± 0.28 0.93 ± 0.28 1.60 ± 0.21 1.80 ± 0.51 1.40 ± 0.25 1.73 ± 0.33 1.00 ± 0.34 1.00 ± 0.24 1.27 ± 0.25 1.80 ± 0.40 63.6 58.8 31.4 52.6 61.8 29.7 65.9 64.3 40.6 43.7

% of egg masses in control nsdb nsd nsd nsd nsd P < 0.02 nsd P < 0.05 nsd nsd

Significance

stimulates deters -

Treatment effect (stimulates/deters)

916

March 17, 2003 20:23

Mean of 15 replicates. Replicates with no oviposition in either cup were discarded. nsd, no significant difference (P > 0.05).

1.87 ± 0.54 1.33 ± 0.29 0.73 ± 0.30 2.00 ± 0.48 2.27 ± 0.61 0.73 ± 0.21 1.93 ± 0.53 1.80 ± 0.28 0.87 ± 0.29 1.40 ± 0.48

2 × MRS NH4 NO3 2 mg/liter NH4 NO3 6 mg/liter NH4 NO3 12 mg/liter NH2 OH.HCl 2 mg/liter NH2 OH.HCl 6 mg/liter NH2 OH.HCl 12 mg/liter NaNO3 2 mg/liter NaNO3 6 mg/liter NaNO3 12 mg/liter

treatment

pp799-joec-461509

a b

control (1 × MRS)

Treatment

Egg masses (mean ± SE)a

Journal of Chemical Ecology [joec]

TABLE 1. OVIPOSITION RESPONSE OF Chironomus tepperi TO 2 × MARTIN’S REARING SOLUTION (2 × MRS) AND DIFFERENT CONCENTRATIONS OF THREE NITROGENOUS COMPOUNDS IN TWO-CHOICE LABORATORY TESTS

P1: GMX Style file version June 28th, 2002


89.5 92.3 85.3 72.2 57.6 65.4 42.9 44.4 57.9 64.6

< 0.001 < 0.001 < 0.01 < 0.05 nsdb nsd nsd nsd nsd P < 0.05 P P P P

Significance

deters deters deters deters deters

Treatment effect (stimulates/deters)

March 17, 2003

Mean of 15 replicates. Replicates with no oviposition in either cup were discarded. nsd, no significant difference (P > 0.05).

0.27 ± 0.15 0.20 ± 0.11 0.33 ± 0.21 0.67 ± 0.23 0.93 ± 0.27 0.60 ± 0.16 1.60 ± 0.34 1.00 ± 0.20 1.60 ± 0.34 1.87 ± 0.32

2.27 ± 0.42 2.40 ± 0.53 1.93 ± 0.34 1.73 ± 0.34 1.27 ± 0.27 1.13 ± 0.26 1.20 ± 0.30 0.80 ± 0.24 2.20 ± 0.38 3.40 ± 0.58

C. tepperi, 1 larva/100 ml, homogenized P. nubiferum, 1 larva/100 ml, homogenized C. tepperi, 10 larvae/100 ml, conditioned C. tepperi, 1 larva/100 ml, conditioned C. tepperi, 1 adult/100 ml, homogenized C. tepperi, 5 adults/100 ml, homogenized C. tepperi, 1 adult/100 ml, homogenized C. tepperi, 5 adults/100 ml, homogenized Cx annulirostris, 1 larva/100 ml, homogenized Cx annulirostris, 3 larvae/100 ml, homogenized

% of egg masses in control

pp799-joec-461509

a b

treatment

control

Treatment

Egg masses (mean ± SE)a

Journal of Chemical Ecology [joec]

TABLE 2. OVIPOSITION RESPONSE OF Chironomus tepperi TO CRUDE BIOEXTRACTS AND REARING SOLUTION CONDITIONED BY CONSPECIFIC LARVAE IN TWO-CHOICE LABORATORY TESTS

P1: GMX 20:23



917


pp799-joec-461509

March 17, 2003

918

20:23


STEVENS, WARREN, AND BRAYSHER DISCUSSION

C. tepperi appears to have a complex oviposition response to nitrogenous compounds, and our results for these materials are difficult to interpret. 1 × MRS contains no nitrogen compounds (Martin et al., 1980). Low levels of added nitrate (as NaNO3 ) deterred oviposition (P < 0.05), while higher concentrations had no significant effect. Addition of hydroxylamine stimulated oviposition (P < 0.02), but only at the middle concentration tested—higher and lower concentrations did not elicit a significant response. Our data suggest that responses to nitrogenous compounds are subtle and may only occur over limited concentration ranges. The response of ovipositing C. tepperi to the bioextracts we tested was less ambiguous. C. tepperi avoided oviposition in 1 × MRS containing homogenized conspecific larvae and similarly avoided 1 × MRS conditioned by conspecific larvae, even when the conditioned 1 × MRS was diluted to provide a concentration equivalent of 1 larva/100 ml over 24 hr. Failure of homogenized adult C. tepperi suspended in 1 × MRS to elicit a response from ovipositing females demonstrates that the larval factor or factors causing the deterrent effect are lost prior to adult emergence. Homogenized P. nubiferum larvae produced an extremely strong avoidance response (P < 0.001) by ovipositing C. tepperi, and although water conditioned by P. nubiferum was not evaluated in this study, this result indicates a factor responsible for deterring oviposition in C. tepperi is present in at least some other chironomids. This was anticipated, since if the deterrent effect was triggered only by the presence of conspecific larvae, multiple discrete cohorts of C. tepperi would be expected in the field, with recolonization occurring after each successive cohort emerged. An ongoing deterrent effect caused by species such as P. nubiferum (which is present in fields throughout the rice season) would be necessary to ensure C. tepperi females restrict oviposition to newly flooded habitats, rather than to habitats characterized purely by an absence of conspecific larvae. Our results indicate that C. tepperi has developed a sensitive method of oviposition site selection based on detection of compounds produced by larval conspecifics, other chironomid larvae, and possibly other aquatic dipterans. The ability to identify newly flooded habitats that are not yet inhabited by other chironomids would be of particular value to a specialized colonist species such as C. tepperi, as the larvae resulting from oviposition into such habitats would be exposed to minimal competition during development. C. tepperi has been recognized as having several other characteristics of particular value to colonist midge species, including rapid development, wide thermal tolerance, protandry (Stevens, 1998), and the capacity to reproduce without the formation of mating swarms (Martin and Porter, 1977). Our study indicates that oviposition site selection in at least some chironomids may be more complex than previously thought. Although the results appear to be


pp799-joec-461509

March 17, 2003

20:23


919


the first experimental evidence to support the idea that oviposition site selection in colonist chironomid species may be mediated by the presence of larvae, this possibility has been hinted at by other workers. Jones (1974) found that recruitment of Paraborniella tonnoiri Freeman was lower when conspecific larvae were present, and in some cases there was no new recruitment until all members of the previous cohort had emerged. Paterson and Cameron (1982) studied the pitcher plant chironomid, Metriocnemus knabi Coquillett, and suggested that ovipositing females could detect the density of larvae already in the pitchers. McLachlan and Cantrell (1980) found that Chironomus imicola Kieffer was a poor colonizer of ponds containing larvae of Polypedilum vanderplanki Hinton, but readily colonized artificial ponds where no larvae were present. Extensive studies have been conducted on the colonization of waterbodies by chironomids, and some of these have identified colonist taxa that appear to have only a single cohort in newly flooded environments. Matˇena (1990) found that C. melanescens Keyl and C. piger Strenzke rapidly colonized experimental ponds, but are soon replaced by other taxa. Whether or not these and other colonist chironomid species respond to oviposition cues produced by chironomid larvae remains to be determined. Identification of the compounds responsible for C. tepperi oviposition deterrence in our short-range laboratory tests will allow field experiments to be conducted on a larger spatial scale and could potentially lead to the development of novel oviposition deterrents for use in situations where control of colonist chironomid midges is required. Acknowledgments—Dr. David James (Washington State University) and Dr. Brian Cullis (NSW Agriculture) are thanked for providing comments on an early draft of the manuscript. Funding for this study was provided by the Rural Industries Research and Development Corporation and the Cooperative Research Centre for Sustainable Rice Production.

REFERENCES BENTLEY, M. D. and DAY, J. F. 1989. Chemical ecology and behavioural aspects of mosquito oviposition. Annu. Rev. Entomol. 34:401–421. CRANSTON, P. S. 1995. Introduction, pp. 1–7, in P. D. Armitage, P. S. Cranston and L. C. V. Pinder (eds.). The Chironomidae. Biology and Ecology of Non-Biting Midges. Chapman and Hall, London. CROME, F. H. J. 1986. Australian waterfowl do not necessarily breed on a rising water level. Aust. Wildlife Res. 13:461–480. DAUBLE, D. D. and SKALSKI, J. R. 1983. Oviposition of Tanytarsus dissimilis (Diptera: Chironomidae) in avoidance trials with coal liquid water-soluble components. Environ. Entomol. 12:1733–1736. DAVIES, B. R. 1976. The dispersal of Chironomidae larvae: a review. J. Entomol. Soc. South. Afr. 39:39–62. FROUZ, J. 1997. The effect of vegetation patterns on oviposition habitat preference: a driving mechanism in terrestrial chironomid (Diptera: Chironomidae) succession? Res. Pop. Ecol. 39:207–213. GILMOUR, A. R., CULLIS, B. R., WELHAM, S. J., and THOMPSON, R. 2001. ASREML Reference Manual. Biometric Bulletin No. 3, NSW Agriculture, Orange, Australia.


920

pp799-joec-461509

March 17, 2003

20:23



HARRISON, S. S. C. and HILDREW, A. G. 1998. Distribution dynamics of epilithic insects in a lake littoral. Arch. Hydrobiol. 143:275–293. HATAKEYAMA, S. 1988. Chronic effects of Cu on reproduction of Polypedilum nubifer (Chironomidae) through water and food. Ecotoxicol. Environ. Safety 16:1–10. IKESHOJI, T., ISEKI, A., KADOSAWA, T., and MATSUMOTO, Y. 1980. Emergence of chironomid midges in four differently fertilized rice paddies. Japan. J. Sanit. Zool. 31:201–208. JONES, R. E. 1974. The effects of size selective predation and environmental variation on the distribution and abundance of a chironomid Paraborniella tonnoiri Freeman. Aust. J. Zool. 22:71–89. MAHER, M. and CARPENTER, S. M. 1984. Benthic studies of waterfowl breeding habitat in southwestern New South Wales. II. Chironomid populations. Aust. J. Mar. Freshwater. Res. 35:97–110. MATEˇ NA, J. 1990. Succession of Chironomus Meigen species (Diptera, Chironomidae) in newly filled ponds. Int. Rev. Ges. Hydrobiol. 75:45–57. MARTIN, J. and PORTER, D. L. 1977. Laboratory biology of the rice midge, Chironomus tepperi Skuse (Diptera: Nematocera): Mating behaviour, productivity and attempts at hybridization. J. Aust. Entomol. Soc. 16:411–416. MARTIN, J., KUVANGKADILOK, C., PEART, D. H., and LEE, B. T. O. 1980. Multiple sex determining regions in a group of related Chironomus species (Diptera: Chironomidae). Heredity 44:367–382. MCCULLAGH, P. and NELDER, J. A. 1989. Generalised Linear Models, 2nd edn. Chapman and Hall, London, United Kingdom. MCLACHLAN, A. J. and CANTRELL, M. A. 1980. Survival strategies in tropical rain pools. Oecologia (Berlin) 47:344–351. OLIVER, D. R. 1971. Life history of the Chironomidae. Annu. Rev. Entomol. 16:211–230. PATERSON, C. G. and CAMERON, C. J. 1982. Seasonal dynamics and ecological strategies of the pitcher plant chironomid, Metriocnemus knabi Coq. (Diptera: Chironomidae), in southeast New Brunswick. Can. J. Zool. 60:3075–3083. PINDER, L. C. V. 1986. Biology of freshwater Chironomidae. Annu. Rev. Entomol. 31:1–23. SCHALL, R. 1991. Estimation in generalised linear models with random effects. Biometrika 78:719–727. STEVENS, M. M. 1992. Toxicity of organophosphorus insecticides to fourth-instar larvae of Chironomus tepperi Skuse (Diptera: Chironomidae). J. Aust. Entomol. Soc. 31:335–337. STEVENS, M. M. 1994. Emergence phenology of Chironomus tepperi Skuse and Procladius paludicola Skuse (Diptera: Chironomidae) during rice crop establishment in southern New South Wales. Aust. J. Exp. Agric. 34:1051–1056. STEVENS, M. M. 1998. Development and survival of Chironomus tepperi Skuse (Diptera: Chironomidae) at a range of constant temperatures. Aquat. Insects 20:181–188. WILLIAMS, K. A., GREEN, D. W. J., PASCOE, D., and GOWER, D. E. 1987. Effect of cadmium on oviposition and egg viability in Chironomus riparius (Diptera: Chironomidae). Bull. Environ. Contam. Toxicol. 38:86–90. XUE, R.-D, ALI, A., and LOBINSKE, R. J. 1994. Oviposition, hatching, and age composition of a pestiferous midge, Glyptotendipes paripes (Diptera: Chironomidae). J. Am. Mosq. Control Assoc. 10:24–28.