Factors affecting entomopathogenic nematodes (Steinernematidae) for control of overwintering codling moth (Lepidoptera: Tortricidae) in fruit bins

Fruit bins infested with diapausing codling moth larvae, Cydia pomonella (L.), are a potential source of reinfestation of orchards and may jeopardize the success of mating disruption programs and other control strategies. Entomopathogenic nematodes (EPNs) were tested as a potential means of control that could be applied at the time bins are submerged in dump tanks. Diapausing cocooned codling moth larvae in miniature fruit bins were highly susceptible to infective juveniles (IJs) of Steinernema carpocapsae (Weiser) and Steinernema feltiae (Filipjev) in a series of experiments. Cocooned larvae are significantly more susceptible to infection than are pupae. Experimental treatment of bins in suspensions of laboratory produced S. feltiae ranging from 10 to 100 IJs/ml of water with wetting agent (Silwet L77) resulted in 51-92% mortality. The use of adjuvants to increase penetration of hibernacula and retard desiccation of S. feltiae in fruit bins resulted in improved efficacy. The combination of a wetting agent (Silwet L77) and humectant (Stockosorb) with 10 S. feltiae IJs/ml in low and high humidity resulted in 92-95% mortality of cocooned codling moth larvae versus 46-57% mortality at the same IJ concentration without adjuvants. Immersion of infested bins in suspensions of commercially produced nematodes ranging from 10 to 50 IJs/ml water with wetting agent in an experimental packing line resulted in mortality in cocooned codling moth larvae of 45-87 and 56 - 85% for S. feltiae and S. carpocapsae, respectively. Our results indicate that EPNs provide an alternative nonchemical means of control that could be applied at the time bins are submerged in dump tanks at the packing house for flotation of fruit.     

Control of Codling Moth,Cydia pomonella (Lepidoptera: Tortricidae), with Steinernema carpocapsae: Effects of Supplemental Wetting and Pupation Site on Infection Rate

Infection of cocooned codling moth (cydia pomonella) larvae by the entomopathogenic nematode Steinernema carpocapsae was studied in three field experiments. Factors that varied within or between experiments included method of application, type of substrate containing cocooned larvae, time when nematodes were applied, seasonal effects, and supplemental wetting before or after nematode application. Conventional air-blast sprayer applications of 0.5–5.0 million infective juveniles (IJs)/tree in fall resulted in ca. 30% mortality of larvae in cardboard trap bands, whereas hand-gun application (2 million IJs/tree) produced mortality of ca. 70%. Application in the evening caused higher larval mortality than application in the morning when no supplemental wetting was used after treatments. Morning and evening applications caused equivalent larval mortality when a postwetting treatment was included. In a trial conducted in midsummer, supplemental wetting, either before or after hand-gun application of 1 million IJs/tree, enhanced nematode-produced mortality. Mortality approached 100% if both pre- and postwetting was used. Larvae in exposed cocoons on apple wood were infected at a higher rate (86%) than those on wood in less exposed positions (73%) or in nonperforated cardboard (72%). Mortality rates for larvae in perforated cardboard were intermediate (77%). Application volumes used to deliver nematodes slightly enhanced infection rate of larvae in some substrates but not others. In one trial, parasitism of codling moth by the wasp Mastrus ridibundus (Ichneumonidae) was negatively correlated with nematode infection of codling moth larvae. Dissections showed that ca. 10% of larvae infected by nematodes had been attacked by the wasp.     

The influence of humidity on the effect of Steinernema feltiae against diapausing codling moth larvae (Cydia pomonella L.) (Lepidoptera: Tortricidae)

Codling moth (CM) is a serious and global pest of pome fruit. It overwinters in cryptic habitats as cocooned diapausing larvae. Field trials with the entomopathogenic nematode Steinernema feltiae (Rhabditida: Steinernematidae) report control of diapausing CM of up to 70%, but results are variable. The objective of this study was to define environmental conditions favouring the performance of the nematodes. Cocooned larvae were more susceptible than non-cocooned larvae. S. feltiae was unable to infect CM at a water activity (aw-values) < or = 0.9. Mortality of cocooned larvae was reported at lower aw-values than of non-cocooned larvae. Exposure time and impact of external relative humidity (RH) was studied. Mortality of cocooned larvae did not further increase after half an hour of exposure, whereas the mortality increased with increasing exposure time in non-cocooned larvae. LC50 and LC90 considerably decreased with increasing RH. The influence of the relative humidity was less pronounced when surpassing 80% than the effect of the volume of applied water. When S. feltiae was formulated in a surfactant-polymer-formulation (SPF), mortality significantly increased when compared to application in water.     

Differences between the pathogenic processes induced by Steinernema and Heterorhabditis (Nemata: Rhabditida) in Pseudaletia unipuncta (Insecta: Lepidoptera)

Larvae of Pseudaletia unipuncta are moderately susceptible to infections caused by entomopathogenic nematodes, being a desirable host to study pathogenic processes caused by Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema glaseri and their associated bacteria. The ability of the infective stage of these nematodes to invade hosts is quite different. S. carpocapsae invades the highest number of insects and presents the highest penetration rate, followed by H. bacteriophora. Regression analysis between the number of insects parasitized and the number of IJs counted per insect, over time, showed a high correlation for S. carpocapsae whereas for H. bacteriophora it was low. Dose-response was most evident at a concentration below 100 IJs per insect on H. bacteriophora, whereas on S. carpocapsae it was found for doses ranging from 100 to 2,000 IJs. Student's t test analysis of dose-response showed parallel, yet unequal, slopes for both strains of H. bacteriophora, whereas distinct regressions were obtained for S. carpocapsae and S. glaseri, thus, evidencing each species develop a distinct pathogenic process. Insects injected with Photorhabdus luminescens died within 50 h after injection, whereas those treated with X. nematophila died much later. Moreover, the mortality in insects exposed to H. bacteriophora complex and injected with P. luminescens was close, but insects injected with bacteria died faster. Insect mortality in treatments with complexes S. carpocapsae and S. glaseri was significantly higher than that which was observed in insects injected with symbiotic bacteria.     

Laboratory Evaluation of Seven Pakistani Strains of Entomopathogenic Nematodes Against a Stored Grain Insect Pest, Pulse beetle Callosobruchus chinensis (L.)

Seven Pakistani strains of entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis were tested against last instar and adult stages of the pulse beetle, Callosobruchus chinensis (L.). These nematodes included Steinernema pakistanense Shahina, Anis, Reid and Maqbool (Ham 10 strain); S. asiaticum Anis, Shahina, Reid and Rowe (211 strain); S. abbasi Elawad, Ahmad and Reid (507 strain); S. siamkayai Stock, Somsook and Reid (157 strain); S. feltiae Filipjev (A05 strains); Heterorhabditis bacteriophora Poinar (1743 strain); and H. indica Poinar, Karunakar and David (HAM-64 strain). Activity of all strains was determined at four different nematode densities in Petri dishes and in concrete containers. A significant nematode density effect was detected for all nematode species tested. Overall, Heterorhabditis bacteriophora, S. siamkayai, and S. pakistanense were among those that showed the highest virulence to pulse beetle larvae and adults. For all nematode species, the last larval stage of the pulse beetle seems to be more susceptible than the adult. LC(50) values in Petri dish and concrete containers were 14-340 IJs/larvae and 41-441 IJs/larvae, respectively, and 59-1376 IJs/adult and 170-684/adult, respectively.     

Efficacy of entomopathogenic nematodes against neonate larvae of <Emphasis Type="Italic">Capnodis tenebrionis</Emphasis> (L.) (Coleoptera: Buprestidae) in laboratory trials

The efficacy of five entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae was tested against the neonate larvae of Capnodis tenebrionis. The nematode strains screened included two of Steinernema carpocapsae (Exhibit and M137), and one each of S. feltiae (S6), S. arenarium (S2), and Heterorhanditis bacteriophora (P4). Exposure of neonate larvae of Capnodis to 10 and 150 infective juveniles (IJs) per larva (equivalent to 3 and 48 IJs/cm² respectively) in test tubes with sterile sand, resulted in mortality between 60–91% and 96–100%, respectively. At a concentration of 150 IJs/larva, all of the nematode strains were highly virulent. Both S. carpocapsae strains (Exhibit and M137) caused infection and mortality to larvae more quickly than the other strains. However, at a lower concentration assay (10 IJs/larva), S. arenarium was the most virulent strain. The penetration rate as an indicator of entomopathogenic nematode infection was also evaluated. The highest value was recorded for S. arenarium (36%), followed by H. bacteriophora (30.6%), S. feltiae (23.1%), and S. carpocapsae (20.7%).     

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.     

Influence of nematode age and culture conditions on morphological and physiological parameters in the bacterial vesicle of Steinernema carpocapsae (Nematoda: Steinernematidae)

Steinernema spp. third-stage infective juveniles (IJs) play a key role in the symbiotic partnership between these entomopathogenic nematodes and Xenorhabdus bacteria. Recent studies suggest that Steinernema carpocapsae IJs contribute to the nutrition and growth of their symbionts in the colonization site (vesicle) [Martens, E.C. and Goodrich-Blair, H., 2005. The S. carpocapsae intestinal vesicle contains a sub-cellular structure with which Xenorhabdus nematophila associates during colonization initiation. Cellular Microbiol. 7, 1723-1735.]. However, the morphological and physiological interactions between Xenorhabdus symbionts and Steinernema IJs are not understood in depth. This study was undertaken to assess the influence of culture conditions and IJ age on the structure, nutrition, and symbiont load (colonization level) of S. carpocapsae vesicles. Our observations indicate the vesicles of axenic IJs are shorter and wider than those of colonized IJs. Moreover, as colonized IJs age the vesicle becomes shorter and narrower and bacterial load declines. The colonization proficiency of several bacterial metabolic mutants was compared between two cultivation conditions: in vitro on lipid agar and in vivo in Galleria mellonella insects. Colonization defects were generally less severe in IJs cultivated in vivo versus those cultivated in vitro. However, IJs from both cultivation conditions exhibited similar declining bacterial load over time. These results suggest that although the vesicle forms in the absence of bacteria, the presence of symbionts within the vesicle may influence its fine structure. Moreover, these studies provide further evidence in support of the concept that the conditions under which steinernematid nematodes are cultivated and stored affect the nutritive content of the vesicle and the bacterial load, and therefore have an impact on the quality of the nematodes for their application as biological control agents.     

Entomopathogenic Nematodes for Control of Codling Moth,Cydia pomonella(Lepidoptera: Tortricidae): Effect of Nematode Species, Concentration, Temperature, and Humidity

The susceptibility of codling moth diapausing larvae to three entomopathogenic nematode species was assessed in the laboratory using a bioassay system that employed cocooned larvae within cardboard strips. The LC₅₀values forSteinernema carpocapsae, S. riobrave,andHeterorhabditis bacteriophorawere 4.7, 4.8, and 6.0 infective juveniles/cm², respectively. When a discriminating concentration of 10 infective juveniles/cm²of each of the three nematode species was evaluated at 15, 20, 25, and 30°C,S. carpocapsaewas the most effective nematode with mortalities ranging from 66 to 90%. Mortalities produced byS. riobraveandH. bacteriophoraat the four temperatures were 2–94 and 25–69%, respectively. Studies were also conducted to test infectivity at 10, 35, and 40°C. No mortality was produced by any of the nematode species at 10°C.S. riobravewas the most infective nematode at 35°C producing 68% mortality which was more than twice that observed forS. carpocapsaeorH. bacteriophora.Codling moth larvae treated with 10 infective juveniles/cm²ofS. carpocapsaeand kept in 95+% RH at 25°C for 0–24 h followed by incubation at 25–35% RH indicated that more than 3 h in high humidity was needed to attain 50% mortality. Trials ofS. carpocapsae, S. riobrave,andH. bacteriophoraat 50 infective juveniles/cm²against cocooned larvae on pear and apple logs resulted in reductions of codling moth adult emergence of 83, 31, and 43%, respectively, relative to control emergence. Trials of the three entomopathogenic nematodes at 50 infective juveniles/cm²against cocooned larvae in leaf litter resulted in 99 (S. carpocapsae), 80 (S. riobrave), and 83% (H. bacteriophora) mortality, respectively. Our results indicate good potential of entomopathogenic nematodes, especiallyS. carpocapsae,for codling moth control under a variety of environmental conditions.     

Effectiveness of different species of entomopathogenic nematodes for biocontrol of the Mediterranean flatheaded rootborer, Capnodis tenebrionis (Linné) (Coleoptera: Buprestidae) in potted peach tree

The susceptibility of larvae of the Mediterranean flatheaded rootborer (Capnodis tenebrionis) to 13 isolates of entomopathogenic nematodes was examined using GF-677 potted trees (peachxalmond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of autochthonous nematodes to control C. tenebrionis larvae inside the roots is discussed.     

Susceptibility of the strawberry crown moth (Lepidoptera: Sesiidae) to entomopathogenic nematodes

The objective of this study was to determine the susceptibility of the strawberry crown moth, Synanthedon bibionipennis (Boisduval) (Lepidoptera: Sesiidae) larvae to two species of entomopathogenic nematodes. The entomopathogenic nematodes Steinernema carpocapsae (Weiser) strain Agriotos and Heterorhabditis bacteriophora (Steiner) strain Oswego were evaluated in laboratory soil bioassays and the field. Both nematode species were highly infective in the laboratory bioassays. Last instars were extremely susceptible to nematode infection in the laboratory, even in the protected environment inside the strawberry (Fragaria x ananassa Duch.) crown. Infectivity in the laboratory was 96 and 94% for S. carpocapsae and H. bacteriophora, respectively. Field applications in late fall (October) were less effective with S. carpocapsae and H. bacteriophora, resulting in 51 and 33% infection, respectively. Larval mortality in the field from both nematode treatments was significantly greater than the control, but treatments were substantially less efficacious than in the laboratory. Soil temperature after nematode applications in the field (11 degrees C mean daily temperature) was below minimum establishment temperatures for both nematode species for a majority of the post-application period. It is clear from laboratory data that strawberry crown moth larvae are extremely susceptible to nematode infection. Improved control in the field is likely if nematode applications are made in late summer to early fall when larvae are present in the soil and soil temperatures are more favorable for nematode infection.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Field Efficacy of Entomopathogenic Nematodes against the Sugar-beet Weevil Temnorhinus ( = Conorrhynchus ) mendicus Gyll. (Coleoptera: Curculionidae)

In 1992 and 1993, the field effectiveness of Heterorhabditis sp. (NL-HL81 strain), H. bacteriophora (HP 88 strain) and Steinernema carpocapsae ('All' strain) against the larvae of Temnorhinus mendicus Gyll. was assessed. The biological tests were compared with two chemical treatments (cypermethrin or deltamethrin) and one untreated control. In 1992, S. carpocapsae gave better results than Heterorhabditis sp. in reducing the percentage of infested roots, as compared with the untreated sample and the chemical one; similarly, the irrigated control gave the best results. In 1993, three concentrations of entomopathogenic nematodes (EPNs) were tested: 0.250 106 infective juveniles (IJs) m - 2, 0.125 106 IJs m - 2 and 0.075 106 IJs m - 2. The different numbers of EPNs did not give very different results from each other; however, H. bacteriophora at 0.075 106 IJs m - 2 was the least effective. In general, cypermethrin was more effective than deltamethrin, but one treatment with EPNs followed by irrigation was always more effective than two chemical applications.     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.     

Biological Control of the Leafminer Liriomyza trifolii (Burgess): Implications for Intraguild Predation between Diglyphus begini Ashmead and Steinernema carpocapsae (Weiser)

Studies were conducted on the combined use of the eulophid parasitoid wasp Diglyphus begini Ashmead and the entomopathogenic nematode Steinernema carpocapsae (Weiser) for control of the leafminer Liriomyza trifolii (Burgess) on chrysanthemums. Several factors indicated that these two agents were suitable for combined use: adult D. begini were not susceptible to nematode infection, leafminer larvae parasitized by the wasp were less susceptible to nematode infection, adult wasps detected and tended to avoid ovipositing on nematode-infected leafminer larvae, nematode-infected larvae served as host-feeding sources for the adult wasps, and nematodes showed equal orientation toward paralyzed/parasitized leafminer larvae and healthy leafminer larvae. However, interspecific interference and intraguild predation (IGP) between the agents were found. Infection of D. begini larval stages by nematodes was seen in petri dishes and in intact leaf mines. The presence of nematodes in mines with wasp eggs decreased the chance of wasp survival to adulthood. IGP may be minimized through proper timing of natural enemy releases.     

Rapid age-related changes in infection behavior of entomopathogenic nematodes

Nonfeeding infective juvenile (IJ) entomopathogenic nematodes (EPNs) are used as biological agents to control soil-dwelling insects, but poor storage stability remains an obstacle to their widespread acceptance by distributors and growers as well as a frustration to researchers. Age is one factor contributing to variability in EPN efficacy. We hypothesized that age effects on the infectiousness of IJs would be evident within the length of time necessary for IJs to infect a host. The penetration behavior of "young" (<1-wk-old) and "old" (2- to 4-wk-old) Heterorhabditis bacteriophora (GPS 11 strain), Steinernema carpocapsae (All strain), and Steinernema feltiae (UK strain) IJs was evaluated during 5 "exposure periods" to the larvae of the wax moth, Galleria mellonella. Individual larvae were exposed to nematode-infested soil for exposure periods of 4, 8, 16, 32, and 64 hr. Cadavers were dissected after 72 hr, and the IJs that penetrated the larvae were counted. Larval mortality did not differ significantly between 72- and 144-hr "observation periods," or points at which larval mortality was noted, for any age class or species. However, age and species effects were noted in G. mellonella mortality and nematode penetration during shorter time periods. Initial mortality caused by S. carpocapsae and H. bacteriophora IJs declined with nematode age but increased with S. feltiae IJ age. Young S. carpocapsae IJs penetrated G. mellonella larvae at higher rates than old members of the species (27-45% vs. 1-4%). Conversely, old S. feltiae IJs had higher penetration rates than young IJs (approximately 8 to 57% vs. 4 to approximately 31%), whereas H. bacteriophora IJs had very low penetration rates regardless of age (3-5.6%). Our results show that the effect of age on IJ infectiousness can be detected in IJs aged only 2 wk by a 4-hr exposure period to G. mellonella. These results have important implications for storage and application of EPNs and suggest the possibility of shortening the time required to detect nematodes in the soil.     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.     

Acclimation of entomopathogenic nematodes to novel temperatures: trehalose accumulation and the acquisition of thermotolerance

The effect of thermal acclimation on trehalose accumulation and the acquisition of thermotolerance was studied in three species of entomopathogenic nematodes adapted to either cold or warm temperatures. All three Steinernema species accumulated trehalose when acclimated at either 5 or 35 degrees C, but the amount of trehalose accumulation differed by species and temperature. The trehalose content of the cold adapted Steinernema feltiae increased by 350 and 182%, of intermediate Steinernema carpocapsae by 146 and 122% and of warm adapted Steinernema riobrave by 30 and 87% over the initial level (18.25, 27.24 and 23.97 microg trehalose/mg dry weight, respectively) during acclimation at 5 and 35 degrees C, respectively. Warm and cold acclimation enhanced heat (40 degrees C for 8h) and freezing (-20 degrees C for 4h) tolerance of S. carpocapsae and the enhanced tolerance was positively correlated with the increased trehalose levels. Warm and cold acclimation also enhanced heat but not freezing tolerance of S. feltiae and the enhanced heat tolerance was positively correlated with the increased trehalose levels. In contrast, warm and cold acclimation enhanced the freezing but not heat tolerance of S. riobrave, and increased freezing tolerance of only warm acclimated S. riobrave was positively correlated with the increased trehalose levels. The effect of acclimation on maintenance of original virulence by either heat or freeze stressed nematodes against the wax moth Galleria mellonella larvae was temperature dependent and differed among species. During freezing stress, both cold and warm acclimated S. carpocapsae (84%) and during heat stress, only warm acclimated S. carpocapsae (95%) maintained significantly higher original virulence than the non-acclimated (36 and 47%, respectively) nematodes. Both cold and warm acclimated S. feltiae maintained significantly higher original virulence (69%) than the non-acclimated S. feltiae (0%) during heat but not freezing stress. In contrast, both warm and cold acclimated S. riobrave maintained significantly higher virulence (41%) than the non-acclimated (14%) nematodes during freezing, but not during heat stress. Our data indicate that trehalose accumulation is not only a cold associated phenomenon but is a general response of nematodes to thermal stress. However, the extent of enhanced thermal stress tolerance conferred by the accumulated trehalose differs with nematode species.     

小卷蛾斯氏线虫侵染对草地贪夜蛾幼虫天然免疫反应的影响

【目的】探讨昆虫病原线虫小卷蛾斯氏线虫Steinernema carpocapsae All侵染对草地贪夜蛾Spodoptera frugiperda幼虫天然免疫反应的影响。【方法】借助倒置显微镜观察和鉴定草地贪夜蛾幼虫的血细胞类型,并对小卷蛾斯氏线虫侵染后不同时间的草地贪夜蛾幼虫血细胞总数目进行统计;通过倒置显微镜观察草地贪夜蛾幼虫对侵入的小卷蛾斯氏线虫的包囊反应;利用倒置荧光显微镜观察小卷蛾斯氏线虫侵染后的草地贪夜蛾幼虫血细胞对金黄色葡萄球菌Staphylococcus aureus的吞噬活性;检测小卷蛾斯氏线虫侵染后的草地贪夜蛾幼虫血淋巴中酚氧化酶(phenoloxidase, PO)活性、体内抗菌肽基因相对表达水平以及血浆的抗菌活性。【结果】从草地贪夜蛾幼虫体内共发现5种不同类型的血细胞,分别为原血细胞、粒细胞、类绛色细胞、珠血细胞和浆血细胞。注射1 μL侵染期(infective juveniles, IJs)小卷蛾斯氏线虫(3 IJs/μL)后9和12 h,草地贪夜蛾幼虫的血细胞总数目显著增多。草地贪夜蛾幼虫的血细胞不能包囊活的以及冷处死的小卷蛾斯氏线虫,但可以包囊热处死的线虫。活的小卷蛾斯氏线虫会显著抑制草地贪夜蛾幼虫血细胞对金黄色葡萄球菌的吞噬活性,但冷处死和热处死的线虫不能。注射1 μL(3 IJs/μL)小卷蛾斯氏线虫后,草地贪夜蛾幼虫血淋巴PO活性总体呈“下降 升高 下降”变化趋势;体内抗菌肽基因Attacin-A2, Attacin-B1, Cecropin-B3, Cecropin-D, Gallerimycin, Gloverin-3以及Lebocin-2的表达水平在线虫侵染后12 h时显著上调,24 h时恢复到对照水平或低于对照水平;血淋巴抗菌活性水平在小卷蛾斯氏线虫侵染后12 h时显著升高,24 h时与对照无显著差异。【结论】小卷蛾斯氏线虫在侵入早期会抑制草地贪夜蛾幼虫的天然免疫反应来建立感染;随后草地贪夜蛾的免疫系统会被激活试图抵御小卷蛾斯氏线虫的侵染;后期随着线虫的成功定殖,草地贪夜蛾的免疫系统最终被抑制或破坏。本研究所得结果为进一步揭示线虫 草地贪夜蛾的免疫互作机理奠定了基础,也为改善昆虫病原线虫对草地贪夜蛾的防治效果提供了理论依据。     

Influence of humidity and a surfactant-polymer-formulation on the control potential of the entomopathogenic nematode <Emphasis Type="Italic">Steinernema feltiae</Emphasis> against diapausing codling moth larvae (<Emphasis Type="Italic">Cydia pomonella</Emphasis> L.) (Lepidoptera: Tortricidae)

The codling moth (Cydia pomonella L.) is a serious pest of pome fruit. Diapausing cocooned larvae overwinter in cryptic habitats in the soil or in the bark of infested trees. The entomopathogenic nematode Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) is used to control diapausing codling moth larvae. The objective of this study was to define environmental conditions favouring the performance of the nematodes. Cocooned larvae were more susceptible than non-cocooned larvae. Susceptibility of pupae was low. To determine the influence of decreasing water activity (a_w-value) on the activity of the nematodes, mortality of codling moth larvae and Galleria mellonella L. were tested in sand-sodium-polyacrylate mixtures of variable water activity. S. feltiae was able to infect both insects at a_w-values >0.9. Cocooned larvae of both insects died at lower a_w-values than non-cocooned larvae. Mortality of cocooned larvae did not further increase after half an hour of exposure to nematodes, whereas the mortality of non-cocooned larvae increased with increasing exposure time. LC₅₀ and LC₉₀ considerably decrease with increasing RH. The negative influence of the relative humidity (macro environment) was less important than the effect of the water activity in the bark substrate (micro environment). The micro environment can be manipulated by applying S. feltiae with higher volumes of water. A surfactant-polymer-formulation significantly increased nematode efficacy and can buffer detrimental environmental effects.