DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L.) larvae

Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. To test the assays, neonate larvae were fed droplets containing a known concentration of L. dispar nuclear polyhedrosis virus and observed for up to 10 days to determine the percentage of infected larvae. The average percent mortalities were 88.0, 60.7, 26.0, and 5.3% for larvae fed droplets containing 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) polyhedral inclusion bodies (PIBs) per ml, respectively. Other larvae treated with the same virus concentrations were frozen at 2, 4, and 6 days postinoculation and examined by the hybridization techniques. The average percentage of slot blots containing viral DNA equaled 81.0, 58.0, 18.0, and 6.0% for larvae blotted 4 days after treatment with 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) PIBs per ml, respectively, and 89.9, 52.1, 26.6, and 6.0%, respectively at 6 days postinoculation. Thus, the hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.     

DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L.) larvae.

Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. To test the assays, neonate larvae were fed droplets containing a known concentration of L. dispar nuclear polyhedrosis virus and observed for up to 10 days to determine the percentage of infected larvae. The average percent mortalities were 88.0, 60.7, 26.0, and 5.3% for larvae fed droplets containing 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) polyhedral inclusion bodies (PIBs) per ml, respectively. Other larvae treated with the same virus concentrations were frozen at 2, 4, and 6 days postinoculation and examined by the hybridization techniques. The average percentage of slot blots containing viral DNA equaled 81.0, 58.0, 18.0, and 6.0% for larvae blotted 4 days after treatment with 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) PIBs per ml, respectively, and 89.9, 52.1, 26.6, and 6.0%, respectively at 6 days postinoculation. Thus, the hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.     

绿原酸提高舞毒蛾核型多角体病毒(LdNPV)的致病力

【目的】明确绿原酸(chlorogenic acid,CA)对舞毒蛾核型多角体病毒Lymantria dispar necleopolyhydrovirus(Ld NPV)致病力的影响,为舞毒蛾的防治提供参考依据。【方法】采用食料给毒法进行生物测定,测定舞毒蛾2龄幼虫对单独Ld NPV及添加绿原酸的Ld NPV(CA+Ld NPV)的剂量及致死时间响应。【结果】CA+Ld NPV与单独Ld NPV对舞毒蛾2龄幼虫的剂量及时间响应间有显著差异,二者对舞毒蛾2龄幼虫的致死中浓度(LC50)分别为161.8 OBs/μL(95%的置信区间为105.6~235.3 OBs/μL)和264.4 OBs/μL(95%的置信区间为178.6~384.0 OBs/μL),前者对舞毒蛾的致病力较后者强。CA+Ld NPV对舞毒蛾2龄幼虫的致死中时间(LT_(50))较Ld NPV的短,当Ld NPV浓度为590 OBs/μL,CA+Ld NPV及Ld NPV对舞毒蛾2龄幼虫的LT_(50)分别为9.9 d和12.3 d;当Ld NPV浓度为5 900 OBs/μL时,则LT_(50)分别为6.9 d和8.0 d。绿原酸降低了Ld NPV对舞毒蛾幼虫的致死中浓度,缩短了致死中时间。【结论】绿原酸可提高Ld NPV对舞毒蛾2龄幼虫的致病力,其机理有待进一步研究。     

A field release of genetically engineered gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (LdNPV)

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A beta-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for its presence using a colorimetric assay. In 1993, LdGEV-infected gypsy moths were released in a forested plot in Massachusetts to test for spread and persistence. A similar forested plot 2 km away served as a control. For 3 years (1993-1995), gypsy moths were established in the two plots in Massachusetts to serve as test and control populations. Each week, larvae were collected from both plots. These field-collected larvae were reared individually, checked for mortality, and then tested for the presence of beta-galactosidase. Other gypsy moth larvae were confined on LdGEV-contaminated foliage for 1 week and then treated as the field-collected larvae. The LdGEV was sought in bark, litter, and soil samples collected from each plot. To verify the presence of the LdGEV, polymerase chain reaction, slot blot DNA hybridization, and restriction enzyme analysis were also used on larval samples. Field-collected larvae infected with the engineered virus were recovered in the release plot in 1993, but not in subsequent years; no field-collected larvae from the control plot contained the engineered virus. Larvae confined on LdGEV-contaminated foliage were killed by the virus. No LdGEV was recovered from bark, litter, or soil samples from either of the plots.     

Tebufenozide targeted against codling moth (Lepidoptera: Tortricidae) adults, eggs, and larvae

The effectiveness of tebufenozide applied against the adult, egg, and larvae of codling moth, Cydia pomonella (L.), was evaluated. Significant reductions in fecundity and egg hatch occurred after 1-h and 24-h exposures of females and 24-h exposures of males-only to residues in plastic plates. A significant reduction in egg hatch was also found after a 1-h exposure of males. The ovicidal effects of tebufenozide in field trials did not significantly differ for eggs laid on residues or treated topically. Corrected egg mortality exceeded 95% for cohorts laid <130 degree-days after sprays were applied. Fecundity and egg hatch were measured after either a 24-h exposure of moths or a 10-d exposure of moths and eggs on apple trees. Significant reductions in both fecundity and fertility occurred compared with an untreated control up to 7 d in the 24-h assays and accounted for 60-70% fewer larvae produced per mated female. The mean numbers of larvae produced per mated female after the 10-d exposure were reduced 100-75% in assays started on day 0-21 after the spray application compared with the untreated control. Fecundity was significantly reduced for 7 d and egg hatch was reduced for the entire 21-d test period in these trials. Residues had a 14-d half-life on apple foliage. Residues applied to foliage or to foliage and fruit did not significantly increase the proportion of uninjured fruit compared with the untreated control in bioassays where neonates were placed on foliage 10 cm from fruit. However, the proportion of injured fruits with shallow stings versus deep entries was higher on the treated versus the untreated plants. Field applications of tebufenozide reduced fruit injury >90% when applied early during emergence. Plots treated after the beginning of egg hatch had a larger proportion of injured fruits with shallow larval feeding.     

Biomodal patterns of mortality from nuclear polyhedrosis virus in gypsy moth (Lymantria dispar) populations

A bimodal temporal pattern of mortality caused by the nuclear polyhedrosis virus (NPV) was observed in nine gypsy moth (Lymantria dispar) populations of varying densities. In all cases, peak mortality from NPV occurred during the second wave (late larval instars) and the highest mortality occurred in high density populations. Patterns of NPV mortality were established several weeks before being expressed. There was no discernible correlation between weekly mortality rates and temperature, rainfall, or total solar radiation. The bimodality was also apparent in NPV contamination on foliage which was measured by bioassay. A similar pattern was observed in the laboratory among larvae reared in groups from field-collected egg masses and from eggs artificially contaminated with NPV from a laboratory population. As in field populations, the period of low mortality from NPV between the two waves occurred when most larvae were late third and fourth instars. Larvae reared individually did not exhibit the second wave of mortality.     

Laboratory environment effects on the reproduction and mortality of adult screwworm (Diptera: Calliphoridae)

The New World screwworm, Cochliomyia hominivorax Coquerel, is mass reared for screwworm eradication initiatives that use the sterile insect technique. New methods for rearing have helped to reduce the cost of the eradication program. We examined the effect and interaction of three temperatures (24.5, 29.5 and 34.5 degrees C), two diets (2% spray-dried blood plus 0.05% vitamins and corn syrup carrageenan) and three population densities (300, 400, and 500 flies/cage) on egg production, egg hatch, number of observable fertilized eggs, mortality (male and female) and ovarian development. The three population densities did not affect any of the parameters monitored. Using the protein diet increased egg production at all temperatures. Diet did not affect egg hatch or female mortality. Male mortality was significantly greater when fed the protein diet and reared at 24.5 degrees C and 34.5 degrees C. Egg hatch was significantly less when the flies were reared at 34.5 degrees C. When exposed to high temperatures (37 degrees C and 40 degrees C) egg production, egg hatch, fertility and mortality were adversely affected. At the higher temperatures, yolk did not adequately form during oogenesis. When compared to the normal rearing photoperiod (12 L:12 D), short photoperiod (1 L:23 D) increased egg production, egg hatch and fertility but lowered mortality.     

Utilization of the polymerase chain reaction in the diagnosis of nuclear polyhedrosis virus infections of gypsy moth (Lymantria dispar, Lep., Lymantriidae) populations

Abstract:  Three specific DNA probes were used for the detection of the nuclear polyhedrosis (NPV) virus of Lymantria dispar ( Ld NPV) genome. Two of these probes, H2 and H3 were obtained by classical cloning method and one (TR6) by polymerase chain reaction (PCR). These probes, used individually or in a pool in the standard slot–blot hybridizations, were able to detect 10⁹ genome copies. By performing 35 cycles of PCR amplification before hybridization with primers specific to Ld NPV genome on DNA extracted from infected larvae, the sensitivity of the hybridization technique was increased, so that as little as 10 copies of the Ld NPV genome could be detected. Using these methods, L. dispar naturally infected by Ld NPV were identified among field populations in Canada and in the United States near the eastern Canadian border. Using a combination of PCR and hybridization, Ld NPV contamination of egg masses were also detected. By disinfecting the eggs with sodium hypochlorite prior to PCR amplification and hybridization, it was also demonstrated that transmission of viral infection in the natural populations is mainly caused by external contamination of the egg and is unlikely to occur through the transovarial route.     

Failure of white-tailed deer, Odocoileus virginianus L., to sustain a population of cattle ticks, Boophilus annulatus (Say), through successive generations

Cattle ticks, Boophilus annulatus (Say), previously reared only on cattle, were placed on 3 white-tailed deer, Odocoileus virginianus L. Ticks were maintained through successive generations solely on the same deer as they aged (3, 6, and 9 mo of age) and received repeated challenges (0, 1, and 2 previous challenges). Cattle were infested simultaneously to assess tick viability and provide a comparison of tick numbers, female weight, egg mass weight, and egg hatch. The initial infestation (3,000 larvae/animal) produced a mean of 12.7 and 506.7 females from deer and cattle, respectively. Ticks recovered from deer weighed less, laid smaller egg masses, and had lower egg hatchability than cattle-reared ticks. A second infestation (3,000 larvae/animal) produced a 6.3-fold reduction in tick numbers on deer (means = 2.0 females/deer), whereas the number on cattle increased (means = 578.0 females/calf). Ticks reared on the deer were again smaller, laid fewer eggs, and had lower egg hatch, although differences were not significant. A third infestation of deer (1,900 larvae/deer) produced only 1 engorged female tick and no viable eggs, thus eliminating the population of deer-reared ticks within 3 generations. Results of the study suggest that a population of B. annulatus will not be sustained indefinitely through time solely on deer; thus, efforts to reduce deer populations severely as a means of eradicating ticks are unnecessary.     

Introduced pathogens follow the invasion front of a spreading alien host

1.?When an invasive species first colonizes an area, there is an interval before any host-specific natural enemies arrive at the new location. Population densities of newly invading species are low, and the spatial and temporal interactions between spreading invasive species and specific natural enemies that follow are poorly understood. 2.?We measured infection rates of two introduced host-specific pathogens, the entomophthoralean fungus Entomophaga maimaiga and the baculovirus Lymantria dispar nucleopolyhedrovirus (LdNPV), occurring in spreading populations of an invasive forest defoliator, L. dispar (gypsy moth), in central Wisconsin. 3.?Over 3 years, we found that host density was closely associated with the presence and prevalence of both pathogens. The fungal and viral pathogens differed in the sensitivity of their response as E. maimaiga was present in lower-density host population than LdNPV. 4.?We examined the relationship between weather conditions and infection prevalence and found that activity of both the fungus and virus was strongly seasonally influenced by temperature and rainfall or temperature alone, respectively. 5.?Purposeful releases of pathogens (median distances of study sites from release sites were 65·2 km for E. maimaiga and 25·6 km for LdNPV) were not associated with pathogen prevalence. 6.?A generalist fly parasitoid, Compsilura concinnata, also killed L. dispar larvae collected from the study sites, and parasitism was greater when infection by pathogens was lower. 7.?Our results demonstrated that although infection levels were low in newly established host populations, host-specific pathogens had already moved into host populations close behind advancing populations of an invasive host; thus, spreading hosts were released from these enemies for only a relatively short time.     

Effects of disodium octaborate tetrahydrate on survival, behavior, and egg viability of adult muscoid flies (Diptera: Muscidae).

Disodium octaborate tetrahydrate (Na2B8O13(.)4H2O) was mixed with sugar and fed to adult Musca domestica L. and Fannia canicularis (L.) to determine concentration-mortality relationships. LC50s (48-h exposure) were 5.7% for M. domestica and 1.0% for F. canicularis. Rates of 1 and 2% were used to test effects on M. domestica mortality and egg hatch over an 8-d period. Reduced egg hatch was evident after 1 d of feeding on the treated mixtures and was greatest (less than 10% egg hatch) after flies fed only on treated mixtures for 2 d. A partial rebound in egg hatch occurred after 3-4 d of feeding on treated diet. Sperm motility in females fed treated sugar was apparently normal. Fertile egg placed on treated poultry manure did not hatch, indicating embryonic death, which also may have been involved in the low hatch of eggs observed from treated flies. When flies were exposed to treated sugar for 2 d then returned to untreated diet, delayed mortality effects and reduced egg hatch persisted for at least 3 d. Behavioral assays (feeding) with M. domestica demonstrated that flies rejected borate-sugar mixtures in favor of sugar alone when the concentration of borate was greater than 2%. Given a choice of treated and untreated poultry manure for oviposition, flies also rejected the treated manure. The potential of borates in adult bait formulations or applied to developmental substrates for fly control is discussed.     

Fungi associated with eggs and first-instar larvae of the European corn borer

Fungi isolated from field-collected egg masses of the European corn borer, Ostrinia nubilalis, were identified as Alternaria spp., A. porri, Fusarium spp., Fusarium oxysporum, Beauveria bassiana, Mucor spp., and an unidentified yeast. Most fungi were associated with predator injury to the egg mass. Bioassay of fungi on egg masses, however, showed that Alternaria spp. and A. porri reduced the hatch of both injured and uninjured egg masses, and Mucor sp. reduced the hatch only when the egg mass was injured.     

Evaluation of a nonconventional insecticide and appropriate application timing for destruction of gypsy moth (Lepidoptera: Lymantriidae) egg masses

Two field studies were conducted in 2001-2002 and 2003 to evaluate the effectiveness and appropriate application timing of Golden Pest Spray Oil (GPSO) for destruction of gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), egg masses in Wisconsin. GPSO is a commercially available, registered pesticide that is predominantly comprised of a soybean-oil base (93%); its primary mode of action is by means of suffocation. Because gypsy moth spends the majority (>75%) of its life cycle in the egg stage (August-April), the potential utility of this product by arborists, city foresters, landscapers, and homeowners is high, especially because GPSO is a United States Environmental Protection Agency registered, nonconventional pesticide that is considered relatively nontoxic. When GPSO was applied at a 1:1 ratio with water, >96% control of gypsy moth egg masses was achieved, regardless of application timing (October, 3 d before egg hatch).     

Density-related variation in vertical transmission of a virus in the African armyworm

Larvae of the African armyworm, Spodoptera exempta, are darker and more resistant to baculovirus infection when reared in groups (gregarious form) compared to being reared singly (solitary form). Lepidoptera that survive virus challenge as larvae could potentially retain a sublethal virus infection which is then transmitted vertically to the next generation. Here we examine whether gregarious and solitary forms of the armyworm differ in the costs of surviving virus infection and in their capacity to transmit an active baculovirus infection to their offspring. Pupae of larvae reared gregariously that survived virus challenge weighed significantly less than uninfected individuals, but this was not so for those reared solitarily. This did not, however, translate into differences in fecundity, at least under laboratory conditions. As found in previous studies, pre-oviposition period was shorter for solitary than gregarious insects, and it was also shorter for females that had been challenged with virus as larvae. Both the prevalence of egg batches containing larvae that died from nucleopolyhedrovirus (NPV) infection and the proportion of infected larvae within each egg batch were significantly increased (approximately doubled) when parental moths were previously challenged with the virus during their larval state. This demonstrates that horizontal transmission in one generation can elevate vertical transmission to the next generation. Moreover, prevalence of overt infection in the offspring generation was two to three times greater when parental moths were reared solitarily as larvae than when reared gregariously. Disease prevalence and proportional infection were both independent of the sex of the infected parent and whether or not the egg batch was surface-sterilized to remove potential contaminants. This suggests that the eggs are infected internally (transovarial) rather than externally (transovum). These results help to shed light on the observed temporal pattern of virus epizootics in eastern Africa.     

Hatching Response of Meloidogyne incognita acrita to Electric Shock

The influence of electric shock on hatch of Meloidogyne incognita acrita from egg masses taken from roots of ''Acala SJ-I'' cotton (Gossypium hirsutum L.) was studied. Egg masses in tap water were individually placed between the tips of needle electrodes 1 mm apart and exposed to potentials of l, 10, 20, and 60 vdc/mm at 1, 1, 1, and 86 milliamperes dc, respectively, for periods of 2 and 60 seconds. Hatched larvae were counted at five-day intervals for 60 days. Of the eight treatment combinations used, six gave a greater egg hatch than the control. The largest hatch, 520 percent greater than the control, resulted from exposure to 1 vdc/mm for 60 seconds; 60 vdc/mm for 2 and 60 sec decreased egg hatch 11 and 94 percent of the untreated control. Hatched larvae from all treatments except the 60 vdc/mm, 60-second exposure were infective and reproduced on young cotton plants in a glasshouse.     

Genotypic variation and presence of rare genotypes among Douglas-fir tussock moth multicapsid nucleopolyhedrovirus (OpMNPV) isolates in British Columbia

The Douglas-fir tussock moth (Orgyia pseudotsugata) multicapsid nucleopolyhedrovirus (OpMNPV) is periodically applied to suppress Douglas-fir tussock moth populations in British Columbia and in the western United States. The strain of OpMNPV in the product currently used for suppression is not genetically distinct from naturally occurring OpMNPV. To separate the mortality caused by the applied virus from that caused by the naturally occurring virus, a rare and genetically distinct strain of OpMNPV must be applied. To learn more about the genotypic diversity of OpMNPV populations in BC and to identify rare strains in this region, viral DNA was extracted from larvae reared from 208 field-collected egg masses found in five geographic regions of British Columbia and subjected to REN analysis. Nine, 12, and 9 different genotypes were detected using PstI, SalI, and HindIII, respectively. When the PstI, SalI, and HindIII profiles for each pure (single strain) isolate were grouped and considered as a combined PstI-SalI-HindIII genotype, 23 different genotypes were identified among 185 isolates. Nine rare OpMNPV genotypes were selected as ideal candidates for use as a potential 'marker strain' to accurately determine the efficacy of the treatment.     

Gypsy moth (Lymantria dispar) larval development and survival to pupation on diet plus extractables from green ash foliage

Gypsy moth, Lymantria dispar L., rate of larval development, molting, pupal weight and survival were studied on an artificial diet containing different concentrations of green ash ethyl acetate extractables (EtOAc Exts). Insects were reared on experimental diets from egg to pupa. Addition of EtOAc Exts to artificial diet significantly prolonged larval development and reduced their survival compared to larvae reared on control diet. Weights of pupae were significantly reduced when larvae were reared on diet containing the lowest dosage of EtOAc Exts (i.e., 0.01%) versus on control diet. EtOAc Exts in diet (e.g., 0.01, 0.06 and 0.2%) frequently caused incomplete ecdysis which invariably resulted in larval death. Impaired feeding, locomotion and excretion are likely causes of death. The combination of these results with our earlier findings of repellents and feeding deterrents against gypsy moth larvae (GML) in green ash foliage shows that the non-host status of green ash to the highly polyphagous GML involves three orders of chemical defense: repellents, feeding deterrents and inhibitors of nutritional and developmental physiology. As the insect becomes sequentially exposed to these orders of defense, it incurs higher costs because the adverse effects become less reversible.     

Bacteria associated with eggs and first-instar larvae of the european corn borer: Isolation techniques and pathogenicity

Egg masses of the European corn borer, Ostrinia nubilalis, were collected from caged corn plants to study the bacterial diseases of egg masses and first-instar larvae. In 1972, a reduction in the percentage hatch, indicative of a disease epizootic, was noted among the first-generation egg masses. Bioassays of the bacteria on first-instar larvae and egg masses showed that Bacillus thuringiensis var. kurstaki was pathogenic only to larvae. Bacillus megaterium was active primarily against the egg stage.     

Field Assessment of Adhesion and Hatch ofChrysoperlaEggs Mechanically Applied in Liquid Carriers

A mechanical technique was evaluated for releasing green lacewing eggs in liquid suspensions. Deposited eggs were enclosed within a circle of nonpoisonous adhesive to protect them from predation and to prevent escape of hatched larvae. Released eggs were monitored daily for 5 days after release by measuring three response variables: adhesion rate of eggs to foliage, hatch rate of eggs, and “yield” of larvae from discharged eggs; “yield” was the product of egg adhesion and egg hatch. Factors tested were: egg conditioning prior to release (incubated or refrigerated), carrier (distilled water or commercial carrier solution), application technique (mechanical or hand application), and row facing (North or South). Release technique did not significantly effect egg hatch on any day. Conditioning eggs prior to release had the greatest effect on hatch of eggs and resulting yield of larvae during the 5-day monitoring period. Carrier had a significant effect on adhesion of eggs to leaves and hatch of eggs. Commercial carrier solution increased egg adhesion but decreased egg hatch compared to water. Overall mean yield of larvae from incubated eggs distributed mechanically was not significantly different for eggs suspended in water (36.4% on day 5 post-release) and for eggs suspended in commercial carrier solution (36.1% on day 5 post-release). Hand-applied eggs had a higher hatch and subsequent yield of larvae than mechanically released eggs; however, the hand technique was labor intensive.     

Oviposition behavior of Edovum puttleri, reared on two hosts, Leptinotarsa decemlineata and L. texana

Female Edovum puttleri Grissell [Hymenoptera: Eulophidae], reared from eggs of Leptinotarsa decemlineata (Say) or Leptinotarsa texana Schaeffer [Coleoptera: Chrysomelidae], were videotaped as they attacked egg masses of L. decemlineata containing 20 host eggs. We identified 15 components of ovipositional behavior. Parasitoids reared on L. texana attacked and oviposited in significantly more host eggs than did females reared on L. decemlineata. Ethometric analyses of behavioral transitions and a clustering analysis of 34 behavioral parameters showed that females reared on L. texana attacked the host egg mass in a different manner than those reared from L. decemlineata. It was concluded that differences were associated with the host species upon which they were reared. Contrary to previous reports, mortality of unparasitized hosts was caused by an ovipositor probe of short duration, which was not related to host-feeding.