Biology of Hoplia philanthus (Col., Scarabaeidae, Melolonthinae): A ...

POPULATION ECOLOGY

Biology of Hoplia philanthus (Col., Scarabaeidae, Melolonthinae): A New and Severe Pest in Belgian Turf MINSHAD ALI ANSARI,1,2 HANS CASTEELS,1 LUC TIRRY,3

AND

MAURICE MOENS1,3

Environ. Entomol. 35(6): 1500Ð1507 (2006)

ABSTRACT We studied the adult behavior, oviposition, and larval and pupal development of Hoplia philanthus Fu¨ essly (Scarabaeidae: Melolonthinae) in the Þeld and laboratory in Belgium. Adult emergence was observed in the Þrst week of June 2000, 2001, and 2002, peaked ⬇2 wk later, and continued until the last week of the month. The average sex ratio of emerging adults was 1.3:1 (male and females) during the collection period. Adults were observed feeding mainly on leaves of Betula utilis variety jacquemontii (B. u. Doorenbos) and Carpinus betulus L. During the day, H. philanthus adults were most active at 1400 hours (GMT ⫹ 1.00). Oviposition started in the last week of June and lasted until the end of July. Each female deposited ⬇25Ð 40 white eggs at a depth of 10 Ð15 cm in soil. Eggs hatched 28 ⫾ 5 d after being laid at an average monthly soil temperature of 18.1⬚C. Three larval instars could be discerned by head capsule width; all larval instars fed on grass roots. The Þrst-instar larvae were found in the last week of July 2000; second instars appeared mostly in the second week of September 2000 and could be found until May 2001. Third instars were found from the second week of June 2001 until April 2002. Pupae could be found from the Þrst week of May until the end of the month. The duration of the pupal stage was 28 ⫾ 5 d at an average monthly soil temperature of 16.5⬚C. According to these observations, H. philanthus has a 2-yr life cycle. KEY WORDS Hoplia philanthus, Scarabaeidae, turfgrass, white grub

A number of scarabaeid beetles, such as Hoplia philanthus Fu¨ essly (Scarabaeidae: Melolonthinae), the European cockchafer, Melolontha melolontha L., the garden chafer, Phyllopertha horticola L., the summer chafer, Amphimallon solstitialis L., and the brown chafer, Serica brunnea L., are pests in Belgium. The four latter species are present at low population densities and rarely cause economically important damage. H. philanthus, sometimes called the Welsh chafer, however, has become an economically important pest of turf and pastures (Ansari et al. 2003b). Its presence was Þrst noted during a survey in the surroundings of Ghent (Casteels and De Clercq 1998). Since then, it has spread continuously and has reached pest status in a large part of Flanders. Elsewhere in Europe, problems of H. philanthus are reported from Germany (Ehlers 2000), Poland (Bunalski 1995), Spain (Baraud 1992, Mico´ et al. 2003), The Netherlands (Vlug 2001), and the United Kingdom (Gratwick 1992). At the moment, millions of dollars are spent each year for the control of the white grubs and for renewing or replacing damaged turf (Klein et al. 2000). The larvae of H. philanthus feed on underground parts of 1 Institute for Agricultural and Fisheries Research, Burg. Van Gansberghelaan 96, B-9820 Merelbeke, Belgium. 2 Corresponding author: Department of Biological Science, University of Wales, Swansea SA2 8PP, UK (e-mail: m.a.ansari@ swan.ac.uk). 3 Laboratory of Agrozoology, Department of Crop Protection, Ghent University, Coupure Links 653, B-9000 Gent, Belgium.

various plants, including turf, herbaceous ornamentals, nursery trees, and shrubs (Ansari 2004). Heavily infested turf can often be lifted from the soil or rolled back like a carpet because the bigger part of the root system has been consumed. Secondary damage from foraging insectivorous birds and mammals often causes further disruption to the turf surface. Adults feed on the foliage of various plants but generally cause little damage. Little information is available on the biology and distribution of H. philanthus. To develop a control or management strategy for H. philanthus, more information is needed on its life cycle and ecology. Therefore, we observed the species in the Þeld and in the laboratory. Materials and Methods Observation Site. The Þeld studies were conducted in the period 2000 Ð2002 in a heavily infested lawn (⬇0.5 ha) at the Research Station for Ornamental Plants (PCS) at Destelbergen, province of East Flanders. The lawn was composed of a mixture of perennial ryegrass (Lolium perenne L.) and bentgrass (Agrostis tenuis Sibth.) on a sandy soil (89.9% sand, 6.6% silt, 3.5% clay; 3.02% organic matter; pH 4.52). Ornamental trees [Carpinus betulus L., Betula utilis variety jacquemontii (B. u. Doorenbos) and Liquidambar styraciflua L.], shrubs [Thuja plicata Donn ex D. Don, Picea sp., Cupressocyparis leylandii (Jacks.

0046-225X/06/1500Ð1507$04.00/0 䉷 2006 Entomological Society of America

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ALI ANSARI ET AL.: BIOLOGY OF H. philanthus

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Fig. 1. Environmental conditions at observation site (lawn consisted of perennial ryegrass and bentgrass). The monthly mean maximum, minimum, and average air temperature and the average soil temperature at 10, 20, and 50 cm depth were recorded over the observation period or obtained from the local weather station. Daily rainfall totals (cm).

Dallimori) Dallimori, Viburnum plicatum Thunb., and V. opulus L.], and bushes (Rhododendron sp. and Erica carnea L.) bordered the lawn. Temperature at the site varied greatly within a day. The monthly mean maximum, minimum, and average air temperature and the average soil temperature at 10, 20, and 50 cm depth were recorded over the observation period at the study site or obtained from the local weather station (Fig. 1).

Field Population. To estimate the initial population density of larvae at the observation site, for each observation date (5, 15, and 26 June 2000), 15 plugs (30 by 30 by 20 cm) were randomly taken by cutting through the turf and thatch on three sides. Grubs were carefully collected from the upper 5Ð20 cm of soil, below the thatch layer, identiÞed to the genus level by means of the raster pattern on the ventral side of the pygidium (in the genus Hoplia, spines are irregu-

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larly implanted) (Niklas 1974). The grubs were put back in the soil plug, which was returned to its original place. Adult Emergence. Adult beetles were identiÞed to species level using identiÞcation keys by Freude et al. (1969). Counting of emergence holes was evaluated as a method to determine adult emergence. Although holes made by earthworms were also present in the observation site, the emergence holes of H. philanthus could be distinguished from the previous one by the fact that the adults scattered soil on one side of the emergence hole, whereas earthworms piled up their casting all around holes. Emergence holes could also be distinguished from entrance holes made by the female beetles for oviposition, by observing how soil was scattered around the hole. Emergence holes were always clean on one side of the hole with soil scattered around the other side, whereas entrance holes were clogged with soil and had some soil scattered all around the hole. Twenty-Þve (1.0 by 1.0 m) observation plots were randomly established on the lawn. On each observation date, Þve plots were randomly selected, and the total number of emergence holes in these plots was counted. Observations were done on 2, 6, 12, 20, and 30 June 2000; 1, 8, 15, 22, and 29 June 2001, and 4, 11, 18, 25, and 30 June 2002. Adult Activity. Trees and shrubs located at the edge of the study site (25 by 15-m area) were observed for the presence of H. philanthus adults on 14, 15, and 16 June 2000, 2001, and 2002. Observations were made from 1000 to 2000 hours (GMT ⫹ 1). All observed adults were collected by hand net and stored per time period of 2 h in separate petri dishes (9 cm diameter) containing 70% ethanol, which were wrapped in ParaÞlm. In the laboratory, adults were separated by sex to get an estimate of the sex ratio. The number of males and females per square meter was recorded per time period. For a more exact determination of the sex ratio, 150 pupae of H. philanthus were collected from the observation site in the Þrst week of May 2005 and transferred to plastic containers (11 cm diameter by 8 cm height) Þlled with 350 g sandy soil (14% moisture). Ten pupae were placed in each container and were kept at 22 ⫾ 2⬚C. The development of pupa into adults was observed at 3-d intervals. Oviposition. To determine the oviposition period, 7Ð10 d after Þrst adult emergence, adults were collected from the turf and shrub border at the observation site (on 7 June 2000, 11 June 2001, and 10 June 2002). Adults (n ⫽ 20) were placed in each of three cages (1.5 by 1.5 by 1.5 m), which were open at the bottom, placed on the ground, and Þlled with an additional layer of 20 cm of soil. The cages were closed at the top and the four sides by an iron mesh to prevent adult escaping. Commercially produced sod of ryegrass (L. perenne), 3.0 Ð 4.0 cm thick, were placed on the soil surface and watered. In each cage, a 1.0-mhigh, pot grown C. betulus was administered as food source for the adults.

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To determine the oviposition period and egg hatch, Þve soil samples (5Ð10 cm depth and 10 cm diameter) were taken around entrance holes and searched for eggs every 3Ð 4 d. Eggs were collected from the soil and counted. Simultaneously, the oviposition was observed at the observation site by taking sod of grass as described in the section on the Þeld population. The soil was inspected for eggs at 4-d intervals in June 2000, 2001, and 2002. Eggs. Egg hatch was monitored in the cage experiment. Groups of 10 eggs were collected from soil with a camel hair brush, transferred to petri dishes (9 cm diameter), covered with moist soil, closed with a lid, and kept in the dark at 18 ⫾ 2⬚C and 90% RH. Care was taken to minimize mortality caused by manipulation. The number of hatched or collapsed eggs was recorded every day. The egg sizes were measured upon collection. Observations started on 24 June 2002, i.e., when the Þrst adults laid eggs under the sod of grass. Larval Development. To study the larval development, 10 plots (2.0 by 2.0 m) separated by 0.5-m buffers were established at the observation site in June 2000. One soil plug (15 by 15 by 10 Ð50 cm range of depths) was taken from each of the 10 plots with a shovel at bimonthly intervals from June 2000 until July 2002. In each of the plots, larvae from different size were present, indicating the presence of overlapping generations. On each sampling date, 25 individuals were collected from each larval size that was encountered and brought to the laboratory. Head capsule width was measured from widest point to widest point under dissecting microscope to differentiate the larval instars. The number of larvae sampled varied according to the period and the instars present. The location where each larva was found (depth below the thatch layer), larval migration in the soil, overwintering, and natural larval mortality were recorded; soil temperature at 10, 20, and 50 cm depth was measured (Fig. 1). Statistical Analysis. The number of H. philanthus adults collected at different times from the observation site was compared by analysis of variance (ANOVA). Means were separated with TukeyÕs test (SPSS 2003). To compare adult emergence between years, the number of emergence holes observed in different plots on the observation site was averaged to determine mean adult emergence/m2.

Results and Discussion Field Population. The density of H. philanthus larvae in the Þeld varied from 75 to 450/m2 at different months of the year. At the start of the observations (early June 2000), different larval instars and adults were found; eggs were detected only at the end of June. IdentiÞcation of the grubs to the genus level showed that only larvae of the genus Hoplia were present. All adult beetles were identiÞed as H. philanthus. Adult Emergence. Overall, we did not Þnd signiÞcant differences in total number of emergence holes among the 3 yr (F ⫽ 0.657; df ⫽ 2,42; P ⬎ 0.524).

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Fig. 2. Emergence of H. philanthus adults from a lawn at different days in June 2000, 2001, and 2002. Bars with different letters represent means that are signiÞcantly different based on Tukey test (P ⬍ 0.05); error bars represent SEM.

However, there were signiÞcant differences between the number of emergence holes at different dates in June 2000 (F ⫽ 45.9; df ⫽ 4,10; P ⬍ 0.001), June 2001 (F ⫽ 22.2; df ⫽ 4,10; P ⬍ 0.001), and June 2002 (F ⫽ 29.8; df ⫽ 4,10; P ⬍ 0.001). In 2000, 2001, and 2002, the Þrst H. philanthus adult emergence hole was observed on 2 June, 1 June, and 4 June, respectively. Each year, adult emergence increased until 12, 15, and 18 June and decreased until completion by the end of the month (Fig. 2). Because scarab beetles often tend to reinfest their site of origin because of favorable conditions (environmental and biological) for oviposition, the observation of adult emergence sites and patterns might be useful to predict where turf damage by the larvae of the next generation is most likely going to increase. A similar conclusion with respect to the oriental beetle, Exomala orientalis (Waterhouse), in golf courses was drawn by Choo et al. (2002). Adult Activity. Overall, the number of males caught per square meter (F ⫽ 0.34; df ⫽ 2,51; P ⬍ 0.710) and

females (F ⫽ 0.62 df ⫽ 2,51; P ⬍ 0.542) was not signiÞcantly different among the years in 2000, 2001, and 2002. The timing at which males (F ⫽ 6.0; df ⫽ 5,12; P ⬍ 0.005) and females (F ⫽ 3.2; df ⫽ 5,12; P ⬍ 0.046) were collected was signiÞcantly different in 2000. However, males (F ⫽ 2.80; df ⫽ 5,12; P ⬎ 0.067) and females (F ⫽ 0.81; df ⫽ 5,12; P ⬎ 0.565) were not signiÞcantly different in 2001. In 2002, the numbers of collected males were signiÞcantly different at different hours of the day (F ⫽ 4.4; df ⫽ 2,12; P ⬍ 0.017), but the number of females was not signiÞcant (F ⫽ 1.7; df ⫽ 2,12; P ⬍ 0.20; Fig. 3). The number of adults collected in the period 14 Ð16 June in 2000, 2001, and 2002 was plotted as the mean number of adults collected every 2 h, with the replications being the 3 d in the month (Fig. 3). The greatest number of adults was collected at 1400 hours. In general, more males than females were collected. Of 298 adults collected, 67.1% were males and 32.9% were females.

Fig. 3. Average number of H. philanthus adults collected on shrubs and trees surrounding a lawn. Adults were collected every 2 h during the period 14Ð16 June 2000, 2001, and 2002.

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Of 150 pupae collected from the observation site, 143 adult beetles emerged from rearing containers, and 58% were males and 42% were females, which is a sex ratio of ⬃1.3:1. The difference with the sex ratio observed in the Þeld might be caused by a different migratory behavior between the sexes. Observation of adult activity throughout the study showed that emergence of adults mainly occurred during the day. Temperature conditions inßuenced foraging activity, with adult beetles becoming more active at 1400 hours. We could conÞrm some of the observations made by Facundo et al. (1999) on E. orientalis. In Belgium, little beetle activity was observed when air temperature was ⬍14⬚C and when days were overcast. There is no information available on mating activity of H. philanthus beetles. However, during the observation period, mating was observed on surrounding shrubs and trees from 900 to 1600 hours. It lasted ⬇2 min and was repeated several times. We also noticed that males of H. philanthus often ride on the dorsum of the females and tried copulating repeatedly. Females that were not receptive to copulation tried to escape when males mounted them. In 2000, 2001, and 2002, the Þrst mating was observed on 7, 9, and 11 June, respectively. Additional studies on the sexual behavior of H. philanthus are necessary for understanding the life cycle of H. philanthus. No study has been conducted on the feeding behavior of H. philanthus adults. During our observation, we frequently observed H. philanthus beetles feeding on leaves of B. utilis variety jacquemontii, and C. betulus, causing moderate damage to plants. However, other scarab species such as Adoretus tenuimaculatus (Waterhouse) (Lee et al. 1998), Ectinohoplia rufipes (Motschulsky) (Choo et al. 1999), and Popillia japonica (Newman) (Fleming 1972) feed heavily on the leaves and ßowers of trees and shrubs. The signiÞcance of the damage caused by the adult beetle of H. philanthus remains unknown and warrants further study. Oviposition. On their release in the cages, the beetles stayed a few hours on the cage net or on the C. betulus leaves, but eventually buried deep into the soil. The following days, the beetles moved out of the soil, spent a few hours on the plant leaves, and Þnally returned to the soil. Oviposition started during the last week of June 2000 depending on the age of the beetle and was completed the last week of July 2000. Oviposition started around 1000 hours and was most common in the afternoon at 1400 hours. Gravid females generally oviposited alone, although sometimes two females were found on a single entrance hole. The female deposited a cluster of 25Ð 40 eggs in the same hole at a depth of 10 Ð15 cm below the thatch layer. The depth of oviposition seemed to depend on the moisture content of the soil; if the water content of the soil was near to its Þeld capacity, ⬎90% of the eggs were laid in the upper 10 cm. In the last week of July in each of the 3 yr, the females stopped laying eggs and reduced feeding and general activity. During oviposition, the mean maximum temperature was 22.1, 23,

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and 22.7⬚C in June and July, the mean minimum temperature was 12.8, 13.8, and 13.6⬚C, and the average mean temperature was 17.1, 17.8, and 18.1⬚C for 2000, 2001, and 2002, respectively. During the different years, the data on oviposition obtained at the observation site were similar to those observed in the cages. Females were sexually mature after 7Ð10 d of feeding and ßew to deposit eggs in soil. Eggs. Eggs are glossy, milky-white, and oval. The average length of the eggs was 1.9 ⫾ 0.15 mm (n ⫽ 50), and the average width was 1.6 ⫾ 0.10 mm (n ⫽ 50). Before hatching, larvae are visible through the chorion of mature eggs. Preliminary observations conducted in the laboratory in June and July 2000 on eggs kept in petri dishes showed that high moisture was necessary for eclosion (data not shown); this is similar to what they need in their natural habitats. The average egg development time was 28 ⫾ 5.0 (n ⫽ 50) days. More than 95% of the eggs hatched under these conditions. Larval Development. On 26 June 2000, the Þrst eggs were found in the soil at the observation site. By 24 July of that year, only a few eggs remained, while the population was mainly composed of Þrst instars (L1). Their average head capsule width was 1.1 ⫾ 0.5 mm (n ⫽ 125). In the second week of September 2000, L1s had molted to L2s. The average head capsule width was 2.2 ⫾ 0.20 mm (n ⫽ 450). With decreasing soil temperature, L2 larvae descended into the soil where they spent the winter (December and January) below the frost line (below 5Ð10 cm deep). In March (spring) 2001, as soil temperature increased, L2 larvae began their upward migration. By late March to early April, the overwintering L2 larvae had moved close to the grass roots and resumed feeding. L2 larvae moulted to third instar (L3) by June 2001; their average head capsule width was 3.0 ⫾ 0.25 (n ⫽ 550). This is the phase in the grubsÕ life cycle during which it feeds most actively on grass roots and causes the most important damage. In late October and early November 2001, L3 larvae migrated deeper into the soil. In late March 2002 overwintering L3 larvae became active and moved toward the soil surface for a brief feeding period. At this time, minor turf damage occurred as the mature larvae soon stopped feeding to initiate pupation in May 2002. The life span of the third-instar larvae was ⬎330 d. From these studies and observations, it could be concluded that in Belgium H. philanthus has one generation per 2 yr with three larval stages (Fig. 4). Through continuous soil sampling during the larval development in plots, we observed larval migration to different levels in the soil. The depth at which the larvae could be found depended on the soil temperature in late autumn, winter, and early spring and mainly on soil moisture during the rest of the year. Second- and third-instar larvae were found as deep as 50 cm in the soil during the winter (Fig. 5). However, during the grub sampling, we observed that ⬎50% of the larvae were present at a depth of 10 cm (Fig. 5). We did not estimate the larval population below 20and 50-cm soil depth. Further studies on larval distribution and percentage of soil moisture at different

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Fig. 4. Life cycle of H. philanthus in lawn at the Research Station for Ornamental Plants (PCS), Destelbergen, under natural conditions showing the presence of multiple generations in 3 consecutive sampling yr. Plain block: adults emerged in June 2000. Horizontal lines: generation started from eggs laid by adults in June 2000. Vertical lines: generation started from eggs laid by adults in June 1999. Multi-blocks: generation started from eggs laid by adults which emerged in June 2001. Cross lines: generation started from eggs laid by adults which emerged in June 2002.

levels in the soil are planned. Both second- and thirdinstar larvae of H. philanthus overwintered. High numbers of larvae survived in winter because of their vertical migration (the soil does not usually freeze at ⬍50 cm; Fig. 5). A preliminary observation indicated that larvae could resist soil temperatures below 0⬚C for several days. When observing 150 larvae left to overwinter in 2000, 2001, and 2002 at 7Ð10 cm depth under the thatch layer for 250 h in frozen soil in the Þeld, an average larval survival rate of ⬎90% was recorded for L2 and 80% for L3 larvae. An average survival of 98% was observed for larvae kept at 50 cm depth. All the L2 and L3 resumed feeding within 24 h when moved in DecemberÐFebruary from the open Þeld to the laboratory at 20⬚C.

Pupae. Pupae were present in the soil from the Þrst week of May 2002 to the end of that month; no pupae were found later than May. Initially, pupae were pale yellow; but turn brownish-yellow with aging. They were ⬇14.0 ⫾ 2.0 mm long and 4.0 ⫾ 1.0 mm wide (n ⫽ 80). In May 2002, at an average soil temperature of 16.5⬚C, the development from pupa to adult took 28 ⫾ 5 d under Þeld conditions. Approximately 80% of the pupae were found in the most superÞcial layer (0 Ð10 cm) and 20% in the layer (10 Ð20 cm). Mortality Factors. No parasitized eggs were found at the observation site. During our study, we collected several naturally infected larvae of H. philanthus from observation sites (lawns, sport turf, and pastures) and

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Fig. 5. Presence of H. philanthus larvae in the soil over a 2-yr period. Clear bars indicate the number of larvae in the Þrst 10 cm of an 0.1-m2 area; dark bars indicate average maximal depth (in cm) at which larvae were found data average of at least two samplings per month.

isolated entomopathogenic nematodes and Hyphomycetes fungi. An entomopathogenic fungus of the genus Metarhizium infected a total of 10% of the larvae collected and was identiÞed as M. anisopliae variety anisopliae (Metschnikoff) Sorokin (Ansari et al. 2004a). A second fungus species was identiÞed as Beauveria bassiana (Balsamo) Vuillemin (Humber 1997). In addition to fungi, entomopathogenic nematodes of the genus Heterorhabditis (Nematoda: Heterorhabditidae) were also isolated from infected larvae of both H. philanthus and M. melolontha; the nematodes were identiÞed as H. bacteriophora (Poinar) (Ansari et al. 2003a). Recently, we isolated Steinernema glaseri (Steiner) (Ansari et al. 2005) from naturally infected third-instar H. philanthus in football Þeld in Eeklo, province of East Flanders. In conclusion, the studies and observations conducted show that in Belgium one H. philanthus generation takes 2 yr; the life cycle is spread over 3 calendar yr. The life cycle of scarabs varies considerably with climate and species. Species belonging to the subfamily Melolonthinae, tribe Sericini usually have a 1-yr life cycle, but some species in more temperate climates have a 2-yr life cycle (Ritcher 1957). The European cockchafer or May beetle, M. melolontha, has a 4-yr life cycle (Keller et al. 1999), especially in temperate regions. In North America, many species of Phyllophaga have a 2- or 3-yr life cycle (Ritcher 1957). To our knowledge, there has been no extensive study of the life cycle of H. philanthus. However, Freude et al. (1969) have mentioned a 2-yr development. The larvae of H. philanthus are a serious pest of turfgrass. Soil insecticides are still the primary means for turfgrass managers, growers, and millions of homeowners for white grubs control (Vlug 1996). However, because in Belgium, this type of control is not allowed in turf, alternative products will be needed. For ex-

ample, use of traps might help to reduce beetle population and plant damage, although this has to be conÞrmed in Þeld studies Þrst. Cultural control methods that could be deployed by home gardeners (e.g., trap cropping, companion planting, repellent mulches) warrant further study. Also, more detailed studies of the biology (adult fertility and longevity and host range) of this species are needed. Recently, biological control has been examined as control tool for H. philanthus. Experiments with entomopathogenic nematodes (Ansari et al. 2003b), fungi (Ansari et al. 2004a), and their combined use (Ansari et al. 2004b) have been carried out in laboratory, greenhouse and under Þeld condition (Ansari et al. 2006) against third-instar H. philanthus. Results from combined application had an additive to synergistic effect on larval mortality. This new approach may offer a powerful and reliable tool for biological control of H. philanthus. Acknowledgments We thank H. Van De Sype and J. Witters for very valuable help with the Þeldwork. We also thank the Research Station for Ornamental Plant Production (PCS), Destelbergen, Belgium, for providing access to a Þeld heavily infected with H. philantus.

References Cited Ansari, M. A. 2004. Biological control of Hoplia philanthus (Coleoptera: Scarabaeidae) with entomopathogenic nematodes and fungi. PhD dissertation, Ghent University, Ghent, Belgium. Ansari, M. A., P. K. Long, and M. Moens. 2003a. Heterorhabditis bacteriophora (Heterorhabditidae: Rhabditida), parasitic in natural populations of white grubs (Coleoptera: Scarabaeidae) in Belgium. Russ. J. Nematol. 11: 57Ð59.

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