Effects of Recombinant Baculoviruses on Three Nontarget Heliothine Predators

Genetically engineered baculoviruses, relative to their wild-type progenitors, have successfully improved the time-to-kill of these arthropod-specific biopesticides. Beneficial arthropods that prey on targeted pest insects are likely the first nontarget organisms to be adversely affected by the applications of such biopesticides. The goals of this project were to assess potential risks of the recombinant baculoviruses on Solenopsis invicta, Geocoris punctipes, and Hippodamia convergens, all of which are common predators of heliothines in Texas cotton. Four recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV), one Helicoverpa zea nuclear polyhedrosis virus (HzNPV), and two corresponding wild-type NPVs were used in this risk assessment study. Risks associated with these baculoviruses were determined by possible shifts in predator life history traits (rate of food consumption, travel speed, fecundity, and survival) when fed prey infected with recombinant viruses compared to prey infected with wild-type viruses or to healthy prey. We also tested for possible transmission of these viruses by predators using the polymerase chain reaction (PCR). No significant shifts in life history characteristics were detected in predators fed Heliothis virescens larvae infected with any of the seven viruses. Viral DNA was discovered using PCR in 2.3% of fire ant workers, but not from any of the queens or eggs. In G. punctipes, 13.4% of adults and 0.5% of eggs scored positive for viruses. Twelve percent of H. convergens adults were found PCR positive. Residency in all three predators tested provides a pathway which could increase the persistence of recombinant viral particles in the environment and thus may produce an indeterminable amount of risk associated with their inadvertent movement.     

Signal Sequence and Promoter Effects on the Efficacy of Toxin-Expressing Baculoviruses as Biopesticides

Baculovirus recombinants expressing a neurotoxin gene,tox34,from the straw itch mitePyemotes triticihave been previously shown to paralyze or kill insects approximately 50% faster than wild-type. We constructed a series of recombinants of the baculovirusAutographa californicanucleopolyhedrovirus which expressedtox34with different signal sequences or were controlled by different promoters to evaluate their influence on toxin expression in cell culture and in insects. Heterologous signal sequences provided no significant increase in the overall levels of the maturetox34gene product, Tox34, secreted into the tissue culture media from infected cells and no improvement in the time required for paralysis of insect hosts. The time required for paralysis was promoter-dependent; the late 6.9K DNA binding protein gene promoter was generally the most effective promoter, although an insect HSP70 promoter was equally or more effective in one of the species.     

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.     

Serial Selection for Resistance to a Wild-Type and to a Genetically Modified Nucleopolyhedrovirus in Trichoplusia ni

In previous work, cabbage loopers (Trichoplusia ni) evolved 22-fold resistance to the single nucleocapsid nucleopolyhedrovirus of T. ni (TnSNPV) after 26 generations of selection with the virus. The goal of the present study was to determine if T. ni could evolve resistance to the recombinant Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV-AaIT) that expresses an insect specific neurotoxin and to determine if it was influenced by prior development of resistance to TnSNPV. To answer these questions, the T. ni line that had been exposed to TnSNPV was divided into two sublines at generation 27. One of them was serially selected for resistance to AcMNPV-AaIT (subline TnSNPV/AcMNPV-AaIT), while the other one was mock infected with distilled water (subline TnSNPV/H₂O). The same was done with the line that was used as a control from generations 1 to 26 (subline H₂O/AcMNPV-AaIT and subline H₂O/H₂O). After 17 generations of selection with AcMNPV-AaIT, T. ni that had not been previously exposed to TnSNPV evolved only twofold resistance to AcMNPV-AaIT. However, those that had been selected with TnSNPV evolved fourfold resistance to AcMNPV-AaIT. Exposure to AcMNPV-AaIT conferred cross-resistance to TnSNPV in only one subline, subline H₂O/AcMNPV-AaIT. Resistance to AcMNPV-AaIT did not affect the developmental time, pupal weight, egg production, or percentage of egg hatch of T. ni.     

Greenhouse Evaluation of Dose– and Time–Mortality Relationships of Two Nucleopolyhedroviruses for the Control of Beet Armyworm, Spodoptera exigua, on Chrysanthemum

Dose– and time–mortality relationships of baculoviruses in pest insects are important for the determination of effective spraying regimes. A series of experiments with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV) against synchronized populations of S. exigua larvae in greenhouse chrysanthemum was conducted. Dose– and time–mortality relationships of different virus concentrations and S. exigua target stages were determined and the area foliage consumption was measured. Crop injury was greatly reduced when S. exigua were controlled as second or third instar larvae, whereas virus applications against fourth instar larvae could not prevent considerable crop injury, even at high concentrations. SeMNPV was approximately 10 times as infectious as AcMNPV when applied on greenhouse chrysanthemum. The relative virulence of AcMNPV and SeMNPV corresponded reasonably well with previously published laboratory bioassay data. SeMNPV killed second and fourth instar S. exigua larvae approximately 12 h faster than did AcMNPV in chrysanthemum, but no difference in speed of action was found for third instar larvae. The relative speed of action of AcMNPV and SeMNPV determined in chrysanthemum and in laboratory bioassays did not correspond for third instar S. exigua larvae; laboratory bioassay data can therefore not simply be extrapolated to the crop level.     

Effect of nucleopolyhedrovirus concentration in soil on viral transport to cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) plants

Soil-to-plant abiotic transport of a recombinant nucleopolyhedrovirus (HzSNPV.LqhIT2) was studied to quantify the proportion of different concentrations of soil virus transported to specific parts of cotton plants under controlled greenhouse conditions; these results were related to transport in the field where wind, rain, and soil type were not controlled. Under conducive precipitation conditions in the greenhouse, the estimated number of viral occlusion bodies (OB) transported ranged from 7 OB (to the top third of the plant, 40–60 cm above the soil, at the low virus concentration, 250 OB/g soil) to 629 OB (to the bottom third of the plant, 0–20 cm, at the high virus concentration, 12,500 OB/g soil). Under conducive wind conditions in the greenhouse, the estimated number of OB transported ranged from 8 OB (to the top third of the plant at the low concentration) to 94 OB (to the bottom third of the plant at the high concentration). The overall proportion of OB transported from soil to plant was greatest, ranging from 2.1–6.2  ×  10⁻⁶, from the lowest soil concentration to the lowest 40 cm of the plant. Only 5 × 10⁻⁸ of the soil OB were transported from the high-concentration soil to a height of 40–60 cm on the plants. In the field experiment, the estimated number of OB on each cotton plant depended on the concentration of OB in soil in June and July, but this effect was no longer significant in August. There were significantly more OB on the lower third of plants than on the top third in July, but not in June or August. Significantly more OB were detected on cotton leaves than on buds or squares in July, and there were more OB on leaves than on buds, squares, bracts, or bolls in August. The amount of HzSNPV.LqhIT2 naturally transported from soil to cotton plants was sufficient to infect 6–11% (low to high soil concentration) of first instar Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) in June, 2–6% in July, and 1–3% in August. These results fill gaps in understanding NPV epizootiology that are important to biological control and risk assessment.     

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five™) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth (growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five™ cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium (LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of Proteases to Improve the Insecticidal Activity of Baculoviruses

Basement membranes that surround the tissues of lepidopterous larvae act as potential barriers to baculovirus movement and establishment of systemic infection. Hence, one potential approach to improving the insecticidal activity of baculoviruses is to perforate or eliminate the basement membranes of their hosts, thereby facilitating the process of infection. Toward this end, we constructed six recombinant clones of Autographa californica nucleopolyhedrovirus (AcMNPV) that express three proteases that digest basement membrane proteins: rat stromelysin-1, human gelatinase A, and flesh fly (Sarcophaga peregrina) cathepsin L. Expression of these proteases was directed from either the ie-1 promoter (in AcIE1TV3.STR1, AcIE1TV3.GEL, and AcIE1TV3.ScathL) or the p6.9 promoter (in AcMLF9.STR1, AcMLF9.GEL, and AcMLF9.ScathL). Recombinant proteases were detected in the culture medium of cells infected with recombinant viruses by either zymography or azocoll assay. AcMLF9.STR1 and AcMLF9.ScathL caused premature cuticular melanization of 5th instar Heliothis virescens. Melanization and fragmentation of internal tissues were observed in half of the larvae infected with AcMLF9.ScathL and not at all in larvae infected with AcMLF9.STR1 or wild-type AcMNPV. Lethal-concentration bioassays revealed no significant differences in virulence toward H. virescens among the protease-expressing recombinants and wild-type AcMNPV. However, in survival-time bioassays, AcMLF9.ScathL killed H. virescens approximately 30% faster than AcMLF9.LqhIT2, a virus expressing an insect-selective scorpion neurotoxin from the p6.9 promoter. Larvae infected with AcMLF9.ScathL consumed approximately 26-fold less lettuce than wild-type virus-infected larvae. These results highlight the potential of improving baculovirus efficacy through the expression of proteases.     

Impact of Soil Applications ofBeauveria bassianaandMariannaeasp. on Nontarget Forest Arthropods

Replicated plots were established in pear thrips-infested sugar maple stands. They were treated withBeauveria bassianaas an emulsifiable concentrate and a nutrient-based granular formulation and withMariannaeasp. as a wettable powder. Postapplication invertebrates moving across plots were collected daily for 10 days in pitfall traps. Collected individuals were identified, held for 10 days, and then evaluated for the presence of fungal infection and other potential pathogenic microorganisms. A total of 3615 invertebrates were collected and identified. Of the invertebrate population in treated plots that fell into pitfall traps, 2.8% became infected with eitherB. bassianaorMariannaeasp. The fungi applied also occur naturally in sugar maple forest soils; therefore, it was impossible to confirm whether the infection we observed in treated plots was a result of contact with the spores we applied. Because we obtained infection among invertebrates collected from control plots, it is likely that these fungi do attack invertebrates under natural forest conditions. Of the 2584 invertebrates examined further for obligate internal pathogens, 2.5% were infected with entomophthoralean fungi, protozoans, and/or nematodes. Results suggest thatB. bassianaandMariannaeasp. could be applied to forest soil without a significant negative impact on the forest-dwelling invertebrate population.     

Effect of dietary selenium supplementation on resistance to baculovirus infection

We have examined the effects of dietary selenium (Se) supplementation on larval growth and immunocompetence of the lepidopteran pest, the cabbage looper, Trichoplusia ni. Supplementation of the diet of T. ni larvae with 10–20 ppm Se resulted in a 1 day delay in pupation. The effects of the addition and/or removal of dietary Se on total Se bioaccumulation and sequestration were determined by neutron activation analysis of pupae. Early penultimate instar larvae moved from selenium containing diet to basal diet lost total pupal Se content down to the level of those fed basal diet. Conversely, larvae moved from basal diet to diet containing additional Se rapidly attained pupal Se levels comparable to larvae fed Se throughout larval development. Therefore, dietary Se is rapidly accumulated or lost during larval development, but significant amounts are sequestered from diet into pupae. Larvae were reared on diet supplemented with 5 or 10 ppm Se until the onset of the penultimate instar then infected per os with increasing concentrations of the fatal baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Larvae fed Se in the penultimate and ultimate instars were more resistant to viral infection than larvae not fed Se in the final instars. This study indicates that dietary Se levels rapidly impact Se assimilation and sequestration and that tissue Se levels are an important factor in resistance to AcMNPV infection in larval T. ni.     

The effect of age of parents on the progeny of diapaused and non-diapaused Heliothis zea

The effect of parental age at oviposition on the offspring of diapaused and non-diapaused females of Heliothis zea (Boddie) (Lepidoptera: Noctuidae) was examined under laboratory conditions. Diapause was induced in the laboratory by reducing day lengths and temperatures. The number of eggs per female, duration of the egg stage, percent hatch, duration of the larval stage, percent pupation and four-day pupal weights of progeny from diapaused and non-diapaused adults were determined. Significantly reduced egg production and increased duration of egg and larval stages occurred in offspring of diapaused females, but no effect on hatching and pupation percentages and pupal weights were observed. The possible effects of these differences on reproductive potential and length of generation are discussed.

---

Résumé Nous avons examiné au laboratoire l'influence de l'age des parents au moment de la ponte sur la descendance des femelles d'Heliothis zea Boddie diapausantes ou non. La réduction de la longueur du jour et de la température avait induit la diapause au laboratoire. L'étude a porté sur le nombre d'oeufs par femelle, la durée de l'incubation des oeufs, le taux d'éclosion, la durée du développement larvaire, le taux de nymphose et le poids des chrysalides de 4 jours chez les descendants d'adultes diapausantes ou non. Chez les descendants des femelles diapausantes, la production d'oeufs est significativement réduite; de même, l'incubation des oeufs et le développement larvaire sont significativement prolongés; mais on n'a observé aucun effet tant sur les taux d'éclosion et de nymphose que sur le poids des chrysalides. La discussion porte sur les influences envisageables de des différences dans le potentiel reproducteur et la durée des générations.

     

The surface hydrocarbons of larval Heliothis virescens and Helicoverpa zea

Third instar larvae of Heliothis virescens and Helicoverpa zea could be distinguished based on the hydrocarbons of their surface lipids. Hydrocarbons were the major components of the surface lipids and a distinctive capillary gas chromatographic profile could be obtained from a hexane extract of the surface lipids of a single larva. Analysis of hexane extracts of the surface lipids by capillary gas chromatography-mass spectrometry (CGC-MS) showed several obvious differences between the two species: (1) in their gas chromatographic profiles; (2) in the presence of a major alkene, hentriacontene, only in H. zea; (3) in H. virescens the CGC-MS peak with a KI of approximately 2860 was 2-methyloctacosane, but in H. zea it was a mixture of 4-methyloctacosane plus 9,13- and 8,12-dimethyloctacosanes; and (4) in the methyl branch positions of dimethyl-branched alkanes with carbon backbones of C₃₁, C₃₃, C₃₅, C₄₅, C₄₇, C₄₉ and C₅₁. The methyl branch points of H. virescens dimethylalkanes were separated by seven or nine methylenes, while in H. zea the methyl branch points of the dimethylalkanes were separated by three and sometimes five methylenes.     

Populations of Predaceous Natural Enemies Developing on Insect-Resistant and Susceptible Tomato in North Carolina

Naturally occurring populations of immature and adultGeocoris punctipes,adultColeomegilla maculataand immature coccinellids were monitored on field-grown tomato lines susceptible and resistant toManduca sextaandHelicoverpa zea. Helicoverpa zeaandHeliothis virescenseggs and small larvae that serve as prey for these predators also were monitored. MoreH. zeaandH. virescenseggs and small larvae were found on resistant than on susceptible plant lines. However, similar populations of largeH. zeaandH. virescenslarvae were found on resistant and susceptible plants. The number of adultGeocoris punctipes,adultColeomegilla maculataand immature coccinellids on resistant plants was always as high or higher than the number on susceptible plants. The data demonstrate no incompatibility of host-plant resistance with biological control provided by these predaceous insects, but indicate that the number ofG. punctipesand coccinellids required to provide effective biological control may develop too late in the season to be of practical value. Large populations of stilt bugs (Jalysus wickhami,Hemiptera: Berytidae) and spiders were observed to occur earlier in the growing season than eitherG. punctipesor coccinellids and may be a significant source of mortality forH. zeaeggs and small larvae.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Modified Behavior in Baculovirus-Infected Lepidopteran Larvae and Its Impact on the Spatial Distribution of Inoculum

Quantifying the rate of dispersal of target insects when infected with a disease agent will aid the development of biorational pest control programs. The effect of nucleopolyhedrovirus (NPV) infection on the mobility of second and fourth instarMamestra brassicaelarvae was investigated in the laboratory and field. NPV infection altered larval mobility, with the changes in behavior varying with the timecourse of infection. Diseased larvae moved three to five times further than healthy ones during the middle stages of infection. By the 7th day postinfection diseased larvae were less mobile than healthy counterparts. The same pattern of modified behavior was observed in both instars. Fourth instar larvae moved further than second instars under laboratory and field conditions. In the field, infected larvae tended to die on the apex of the cabbage leaves. Bioassay of the leaves showed a linear decrease in inoculum from central to peripheral plants within the plots, which occurred to the same extent for second and fourth instars. Leaves from plots where infected fourth instar larvae had been introduced had higher inoculum density than those from plots with second instars.     

Induced tolerance of neonate Heliothis zea to host plant allelochemicals and carbaryl following incubation of eggs on foliage of Lycopersicon hirsutum f. glabratum

Summary Incubation of Heliothis zea (Boddie) eggs on foliage of Lycopersicon hirsutum f. glabratum C.H. Mull (accession PI 134417) results in neonates with elevated levels of tolerance to the toxic effects of PI 134417 foliage attributable to 2-tridecanone found in the glandular trichomes which abound on that foliage. The neonates from such eggs are also shown to have elevated levels of tolerance to the carbamate insecticide carbaryl. Incubation of eggs in an atmosphere containing 2-tridecanone similarly produced elevated levels of tolerance to 2-tridecanone among resulting neonates, indicating that 2-tridecanone is the likely inducing agent and that exposure to 2-tridecanone vapor, which is known to emanate from PI 134417 foliage, is sufficient for induction. Analysis of the cytochrome P-450 content in gut microsomes of fifth instar larvae indicated that exposure of larvae to 2-tridecanone in artificial diet or to PI 134417 foliage resulted in significantly elevated levels of cytochrome P-450 relative to larvae fed diet without 2-tridecanone or foliage of L. esculentum which contains no 2-tridecanone. In addition, removal of the glandular trichomes from PI 134417 foliage eliminated the ability of that foliage to induce elevated levels of cytochrome P-450. These results provide circumstantial evidence that cytochrome P-450 may be involved in the induced tolerance to xenobiotics among neonates from eggs exposed to 2-tridecanone or PI 134417 foliage.Support for this research was provided by the USDA Competitive Research Grants Program in Biological Stress under Grant No. 83-CRCR-1-1241 and Grant No. 85-CRCR-1-1615, and the North Carolina Agricultural Research Service. Paper No. 10856 of Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC, USA 27650. Use of trade names does not imply endorsement of products named nor criticisms of similar ones not mentioned     

Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae

It is of interest to understand what kinds of physiological and biochemical changes occur in insects if the homeostasis of trehalose in the hemolymph is disrupted by the infection with a recombinant baculovirus containing a secretory-trehalase gene. For this purpose, two recombinant non-occluded Autographa california multicapsid nucleopolyhedroviruses (AcMNPVs), vTREVL and vERTVL, containing a trehalase cDNA of the mealworm beetle, Tenebrio molitor, were constructed. The trehalase cDNA was inserted in the sense orientation downstream of the polyhedrin promoter for vTREVL, and in the anitsense orientation for vERTVL. The active trehelase of T. molitor was found outside of cells when SF-9 cells or larvae of the cabbage armyworm, Mamestra brassicae, were infected with vTREVL. In the hemolymph of vTREVL-infected larvae, expression of the active trehelase was followed by disappearance of trehalose and appearance of glucose. However, the mortality time of virus-infected 5th instar larvae increased in the following order: AcMNPV C6 (wild-type virus) ≤ vERTVL < vTREVL. The symptoms (the browning and liquefying of the host body) of NPV infection were moderated considerably in vTREVL-infected larvae.     

Susceptibility of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) to Vip3A insecticidal protein expressed in VipCot™ cotton

Susceptibility of laboratory and field colonies of Helicoverpa zea (Boddie) and Heliothis virescens F. to Vip3A insecticidal protein was studied in diet incorporation and diet overlay assays from 2004 to 2008. Responses of field populations were compared to paired responses of University of Arkansas laboratory susceptible H. zea (LabZA) and H. virescens (LabVR) colonies. After 7 d of exposure, observations were made on number of dead larvae (M) and the number of larvae alive but remaining as first instars (L1). Regression estimates using M (LC₅₀) and M plus L1 (MIC₅₀) data were developed for laboratory and field populations. Susceptibility of laboratory and field populations exposed to Vip3A varied among different batches of protein used over the study period. Within the same batch of Vip3A protein, susceptibilities of laboratory colonies of both species (LabZA and LabVR) were similar. Field colonies were significantly more susceptible to Vip3A than the respective reference colonies of both species. Within field populations, susceptibility to Vip3A varied up to 75-fold in H. zea and 132-fold in H. virescens in LC₅₀ estimates. Variabilities in MIC₅₀s were up to 59- and 11-fold for H. zea and H. virescens, respectively.     

Transmission of NPV in uniform- and mixed-age populations ofHeliothis zea [Lep: Noctuidae] on caged soybean

Transmission of a nuclear polyhedrosis virus (NPV) was examined in uniformaged and uniform- vs mixed-age populations ofHeliothis zea (Boddie) on caged soybean. Larval collections revealed viral disease outbreaks occurred in all treatments following release of infected larvae (Primary infected larvae). Transmission of NPV in uniform-aged populations was related to the density of primary infected larvae released in the population but not to the size at death of primary infected larvae (P<0.05). In mixed-age populations horizontal transmission in the oldest larvae in the population was equal to that in uniform-aged populations, providing that primary infected larvae in the mixed-aged population were all the age of the oldest noninfected cohorts. As the mixed-age population aged, transmission increased and was generally higher than that in the uniform-aged populations. Transmission was also higher when primary infected larvae were medium sized at death than when small or large at death. The concentration of virus deposited on foliage and in soil after all larvae had died on plants was related to density of primary infected larvae released. In soil, but not on foliage, the virus concentration was related to the size at death of primary infected larvae released. This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement No. CRSR-2-1000.     

Response ofHelicoverpa spp. (Lep.: Noctuidae) and their natural enemies to petroleum spray oil in cotton in Australia

Refined petroleum products have been used since last century for the control of phytophagous arthropod pests. The response ofHelicoverpa spp. and its natural enemies to petroleum oil sprays on cotton was investigated under laboratory choice/no-choice and small plot field conditions at the Australian Cotton Research Institute at Narrabri in New South Wales. In oviposition choice and no-choice tests, petroleum oil sprays suppressed oviposition inHelicoverpa punctigera (Wallengren) but notH. armigera (Hubner) (Lepidoptera: Noctuidae).H. punctigera females laid about seven times as many eggs on the control (water sprayed) cotton plants as on either petroleum oil spray alone or oil mixed with Kelgum. HoweverH. armigera females laid the same number of eggs on both treated and control plants. In field trials, the number of eggs per metre per sample date found on the plots treated with petroleum oil spray was 3.83 compared with 6.29 and 7.82 eggs respectively on plots treated with conventional insecticide sprays and the unsprayed plots. The number of larvae found on plots treated with petroleum oil sprays was 2.9 times higher than the conventional insecticide sprayed plot, however, this was 1.6 times fewer than the unsprayed plot. The cotton yields from plots treated with petroleum oil sprays was 3.70 bales/ha compared with 7.32 and 2.69 bales respectively from the conventional insecticide treated and the unsprayed plots. Petroleum oil sprays had little or no effect on predatory beetles, bugs, lacewings and spiders.

---

Résumé Des produits de pétrole raffiné ont été utilisés depuis le siècle dernier pour lutter contre les arthropodes ravageurs phytophages. La réponse deHelicoverpa spp. et des ses ennemis naturels à des pulvérisations d'huile de pétrole sur cotonnier a été étudiée en laboratoire dans des expériences de choix et d'absence de choix ainsi que dans de petites parcelles en plein champ à l'Institut de Recherche australien sur le coton (Narrabri, Nouvelles Galles du Sud). Dans des expériences de choix/non choix concernant l'oviposition, les pulvérisations d'huile de pétrole empêchaient la ponte d'Helicoverpa punctigera (Wallengren) mais pas deH. armigera (Hubner) (Lep.: Noctuidae). Les femelles deH. punctigera pondent environ 7 fois plus d'oeufs sur les plants de coton témoins (pulvérisés avec de l'eau) que sur les plants traités à l'huile de pétrole seule ou en mélange avec du Kelgum. Par contre,H. armigera pond le même nombre d'oeufs sur les plants traités ou témoins. Dans les essais en plein champ, les parcelles pulvérisées avec de l'huile de pétrole comportent significativement moins deHelicoverpa et présentent une récolte plus importante que celles non traitées; cependant le nombre de chenilles est plus élevé et la récolte moindre que dans les parcelles traitées avec des insecticides. Les pulvérisations d'huile de pétrole ont un effet faible ou nul sur la faune auxiliaire.