Changes in Trace Metals in Hemolymph of Baculovirus-Infected Noctuid Larvae

We studied how biologically relevant trace metals (i.e., micronutrients) in the hemolymph of larval Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) changed in response to per os baculovirus infection, larval development, and injection of heat-killed bacteria. Concentrations of hemolymph Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, and Zn were measured using inductively coupled plasma-mass spectrometry. H. virescens larvae exhibited greater fluctuations in hemolymph trace metal levels in response to baculovirus infection and development than did H. zea larvae. H. zea single nucleopolyhedrosis virus infection significantly altered the levels of Cu, Fe, Mg, Mn, Mo, and Zn in fourth instar H. virescens larvae. Conversely, in fifth instar H. virescens and both H. zea instar infections, no metal levels were significantly different between infected and uninfected larvae. In fourth instar H. virescens hemolymph, Cu, Fe, Mo, and Zn increased during development. Cu, Fe, Mg, Mn, Mo, and Zn levels changed significantly during development in fifth instar H. virescens as well as both H. zea instars. Based on this analysis, metals were identified whose levels changed during development in both species and during the immune response of H. virescens larvae.     

Effect of Campoletis sonorensis ichnovirus cys-motif proteins on Heliothis virescens larval development

Polydnaviruses are obligate symbionts of some parasitic hymenopteran wasps responsible for modifying the physiology of their host lepidopteran larvae to benefit the endoparasite. Injection of Campoletis sonorensis ichnovirus (CsIV) into Heliothis virescens larvae alters larval growth, development and immunity but genes responsible for alterations of host physiology are not well described. Recent studies of polydnavirus genomes establish that these genomes encode families of related genes expressed in parasitized larvae. Here we evaluate five members of the CsIV cys-motif gene family for their ability to inhibit growth and development of lepidopteran larvae. To study the function of cys-motif proteins, recombinant proteins were produced from baculovirus expression vectors and injected or fed to H. virescens larvae in diet. rVHv1.1 was identified as the most potent protein tested causing a significant reduction in growth of H. virescens and Spodoptera exigua larvae. H. virescens larvae ingesting this protein also exhibited delayed development, reductions in pupation and increased mortality. Increased mortality was associated with chronic sub-lethal baculovirus infections. Taken together, these data indicate that the cys-motif proteins have pleiotropic effects on lepidopteran physiology affecting both development and immunity.     

Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests

Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with CrylAc. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S.frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30-80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S.frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20-69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.     

Insect resistance management for Syngenta's VipCot transgenic cotton

Syngenta is seeking commercial registration for VipCot cotton, a pyramided transgenic cotton trait that expresses two insecticidal proteins derived from Bacillus thuringiensis Vip3A and Cry1Ab. Both proteins are highly effective against two key cotton pests, Helicoverpa zea cotton bollworm; and Heliothis virescens, tobacco budworm. To investigate the role of VipCot cotton in delaying the development of resistance in these pests to transgenic Bt traits, Syngenta has performed studies to determine the dose of proteins expressed in VipCot and evaluate the potential for cross-resistance between the component proteins. Following United States Environmental Protection Agency (US EPA) high dose methods 1 and 4, VipCot was shown to express a high dose of proteins for H. zea and H. virescens. VipCot was also confirmed to express a high dose of proteins for H. zea through US EPA Method 5. Additionally, all the data collected to date verify a lack of cross-resistance between Vip3A and Cry proteins. These two key pieces of information indicate that VipCot cotton should be very durable under the currently mandated high dose plus refuge insect resistance management strategy.     

Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae)

A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster.     

Comparative activity of baculoviruses against the codling moth Cydia pomonella and three other tortricid pests of tree fruit

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54-93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC(50) values for each virus. The LC(50)s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm(2), 1.77 x 10(3) occlusion bodies (OBs)/mm(2), and 3.05 x 10(3)OBs/mm(2), respectively. The LT(50) determined for AfMNPV using an approximate LC(95) of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.     

Enzymatic decomposition of elicitors of plant volatiles in Heliothis virescens and Helicoverpa zea

Feeding by larvae of Heliothis virescens induces cotton, corn and tobacco plants to release blends of volatile organic compounds that differ in constituent proportions from blends released when Helicoverpa zea larvae feed on the same plant species. The same elicitors (and analogs) of plant biosynthesis and release of volatiles, originally identified in oral secretions of Spodoptera exigua larvae, were also found in oral secretions of H. virescens and H. zea. However, relative amounts of these compounds, particularly N-(17-hydroxylinolenoyl)-L-glutamine (volicitin), 17-hydroxylinolenic acid, and N-linolenoyl-L-glutamine, varied among batches of oral secretions, more so in H. virescens than in H. zea. This variation was due to cleavage of the amide bond of the fatty acid-amino acid conjugates by an enzyme, or enzymes, originating in the midgut. The enzymatic activity in guts of H. virescens was significantly greater than that found in guts of H. zea. Furthermore, H. zea frass contains N-linolenoyl-L-glutamine in more than 0.1% wet weight, while this conjugate comprises only 0.003% wet weight in H. virescens frass. These results indicated that physiological differences between these two species affect the proportions of volicitin and its analogs in the caterpillars. Whether this causes different proportions of volatiles to be released by plants damaged by each caterpillar species is yet to be determined.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Ascovirus infectivity and effects of infection on the growth and development of noctuid larvae

The infectivity per os and by inoculation of ascoviruses isolated from Heliothis zea, Spodoptera frugiperda, and Trichopulsia ni and the effects of infection by these viruses on host growth and development were studied in the species from which these viruses were originally isolated, or in the case of the H. zea isolate, in H. virescens. Mortality caused by all three viral isolates averaged less than 15% in third instars fed doses of 10 to 10(5) viral vesicles per larva incorporated into diet, whereas inoculation with as few as 10 viral vesicles consistently yielded mortality rates greater than 90%. In tests where groups of 10 larvae were inoculated sequentially with a minuten pin that had been inserted into the hemocoel of an infected larva and then dried for up to 24 hr, mortality rates were consistently greater than 90%. The latter results suggest that ascoviruses in the field may be vectored by insect parasites. The studies of the effects of infection on larval growth and development demonstrated a marked loss of appetite and a decrease in weight gain within 24-48 hr of infection in all three isolates. Third instars inoculated at a weight of ca. 15 mg gained little weight after infection and had difficulty molting, but survived in an arrested state of development for 2-5 weeks. Controls pupated within 5-7 days after attaining weights, depending on the host species, in the range of 300-500 mg. Arrested development and lack of weight gain appear to be due to decreased feeding, whereas the long survival of infected larvae is likely due to the limited destruction of host tissues.     

Infectivity and reproductive potential of the Oswego strain of Heterorhabditis bacteriophora associated with life stages of the clover root curculio,Sitona hispidulus

The infectivity and reproductive potential of the entomopathogenic nematode Heterorhabditis bacteriophora (Oswego strain), at different concentrations, was studied. Seventy to 80.0% mortality to late instar larvae of the clover root curculio, Sitona hispidulus, and 40.0-76.0% mortality to pupae, was observed at concentrations of 15-100 infective juveniles. There were no significant differences in mortality among nematode concentrations. LC(50) levels of 4.0 and 21.4 nematodes were determined for clover root curculio larvae and pupae, respectively. Nematodes did not cause significant mortality to adult or first instar clover root curculio. H. bacteriophora was able to complete its development and reproduce in 74.0-95.0% of clover root curculio late instar larvae and pupae. Reproductive potential in curculio larvae and pupae ranged from 0 to 7040 infective juveniles per host. Larvae exposed to 100 nematodes had a reproductive potential significantly higher than in those larvae exposed to 15 and 50 nematodes. Reproductive potential in pupae decreased with an increased nematode dose, indicating potential crowding effects. Host larval and pupal mass were positively correlated with nematode progeny production.     

Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L

Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs/larva) and a dosage of about 2000OBs/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the spermatocytes and nurse cells in the young oocytes were infected in the larval rudiments of testis and ovary, respectively. The mating of infected females with uninfected males resulted in significant reduction in fecundity (P < 0.01) and hatching of eggs (P < 0.001) due to transovarial transmission of BmNPV. Mating tests of uninfected females and infected males also confirmed venereal transmission as there was a significant reduction in hatching of eggs (P < 0.01). Further, among the F1 hybrid offspring (infected female x uninfected male) that were infected transovarially, larval progeny died at first and second instar stages, whereas those infected venereally developed acute lethal infection late and died by the end of third and fourth instar stage. PCR amplification and sequencing of 473bp of immediate early-1 (ie-1) gene of BmNPV isolated from the viral-infected parent and the F1 offspring confirmed that the viral infection is vertically transmitted to the progeny.     

Juvenile hormone analogs greatly increase the production of a nucleopolyhedrovirus

The commercial production of baculovirus insecticides is limited by the need to produce the virus in living insects. The influence of juvenile hormone analogs (JHA) on the growth and survival of Spodoptera exigua larvae placed on treated diet in the fifth instar was examined. Weight increases observed in methoprene- and fenoxycarb-treated larvae were over three-fold greater than that of control insects, whereas other compounds resulted in lower weight gains (pyriproxyfen) or highly variable responses (hydroprene). Approximately 90% and 70% of fenoxycarb and methoprene-treated larvae, respectively, molted to a supernumerary sixth instar and attained a final weight at 8–10 days post-treatment that was approximately double the maximum weight observed in control larvae. Inoculation of fenoxycarb and methoprene-treated sixth instars with a nucleopolyhedrovirus (SeMNPV) resulted in 2.4- or 2.9-fold increases in final weights, compared to control larvae inoculated in the fifth instar. The total yield of SeMNPV occlusion bodies (OBs) per larva was 2.7- and 2.9-fold greater in fenoxycarb- and methoprene-treated larvae, respectively, compared to fifth instar controls. A significant but small increase in the yield of OBs/mg larval weight was observed in fenoxycarb-treated insects but not in the methoprene treatment. The LC₅₀ value of OBs harvested from fenoxycarb-treated insects was slightly higher than that of OBs from control insects, whereas no such difference was observed in OBs from methoprene-treated insects. We conclude that appropriate use of JHA technology is likely to provide considerable benefits for the mass production of baculoviruses.     

Survivorship of Helicoverpa zea and Heliothis virescens on cotton plant structures expressing a Bacillus thuringiensis vegetative insecticidal protein

A series of tests quantified bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), larval survival on plant structures of a nontransgenic cotton (Gossypium hirsutum L.), 'Coker 312', and two transgenic cottons expressing Vip3A protein or both Vip3A + CrylAb proteins (VipCot). Vegetative and reproductive structures including terminal leaves, flower bud (square) bracts, whole debracted squares, flower petals, flower anthers, and intact capsules (bolls) were harvested from plants in field plots. Each structure was infested with 2-d-old larvae from one of the two heliothine species. Larvae were allowed to feed for 96 h on fresh tissue. Survivorship at 96 h after infestation was significantly lower on all structures of Vip3A and VipCot cotton lines compared with similar structures of Coker 312. VipCot plant structures generally resulted in lower larval survivorship compared with similar structures of the Vip3A cotton line. H. zea survivorship ranged from 4 to 28% and from 1 to 18% on Vip3A and VipCot plant structures, respectively. H. virescens survivorship ranged from 10 to 43% and from 2 to 12% on Vip3A and VipCot plant structures, respectively. H. virescens survivorship was higher on VIP3A plant structures compared with that for H. zea on similar structures. These differences between species were not observed on plants from the cotton line expressing VipCot proteins. The results for these plant structures demonstrate that the combination of proteins expressed in VipCot cotton lines are more effective than Vip3A cotton lines against this heliothine complex.     

In vivo production of recombinant protein by a baculovirus vector inoculated perorally to the prefinal instar larvae of Bombyx mori L. (Lep., Bombycidae) aided by an optical brightener, Tinopal UNPA-GX

Abstract: Budded particles of nucleopolyhedrovirus (NPV) were found to infect perorally the 4th (prefinal) instar larvae of Bombyx mori L. that were treated by an optical brightener, Tinopal UNPA-GX (Tinopal). Host larvae were fed a diet containing 0.3% (w/w) Tinopal on day 1 in the 4th instar and then fed a diet contaminated by budded particles of NPV (1.0 × 10⁶ TCID₅₀ U/larva) that was pathogenic to B . mori (BmNPV) on day 2 (inoculation schedule 1). Another set of host larvae was fed a diet containing BmNPV budded particles (2.5 × 10⁶ TCID₅₀ U/larva) together with 0.3% (w/w) Tinopal on day 1 in the 4th instar (inoculation schedule 2). Host larvae treated by both schedules died of viral infection. The operation of schedule 2 is simpler than that of schedule 1, although the former required higher doses of the virus for satisfactory infection. We inoculated a baculovirus vector carrying human serum albumin (HSA) gene into 4th instar B . mori larvae by schedule 1. Recombinant HSA was detected in the homogenate of host larvae 4 days after inoculation. The peroral inoculation of BmNPV budded particles aided by Tinopal may thus lead to industrial pharmaceutical production using a baculovirus vector for large numbers of insect hosts.     

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.     

The influence of two endoparasitic wasps, <Emphasis Type="Italic">Hyposoter didymator</Emphasis> and <Emphasis Type="Italic">Chelonus inanitus</Emphasis>, on the growth and food consumption of their host larva <Emphasis Type="Italic">Spodoptera littoralis</Emphasis>

The influence of parasitism by Hyposoter didymator (Thunberg; Hymenoptera: Ichneumonidae) and Chelonus inanitus (Linnaeus) (Hymenoptera: Braconidae) on the growth and food consumption of their host Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) was studied in the laboratory. Parasitised larvae consumed significantly less artificial diet than unparasitised ones. Egg parasitisation by C. inanitus affected host larval consumption from the second day after emergence and it was significantly different from that of unparasitised ones. H. didymator, however, started to reduce larval consumption 4 days after parasitisation on the third instar host larvae. The overall reduction achieved by the larval endoparasitoid H. didymator is higher than that caused by the egg-larval endoparasitoid C. inanitus. The final body weight of a parasitised host larva by H. didymator and C. inanitus was only 6.7 and 13.0% of the maximum weight of an unparasitised sixth instar larva respectively. Moreover, parasitised larvae never reached the last instar. Results indicated that parasitised larvae might cause considerable less damage to the host plant than unparasitised ones.     

TMOF-like factor controls the biosynthesis of serine proteases in the larval gut of Heliothis virescens.

Proteolytic enzyme biosynthesis in the midgut of the 4th instar larva of Heliothis virescens is cyclical. Protease activity increases immediately after the molt from the 3rd to the 4th instar larvae and declines just before the molt into the 5th instar. Characterization of the midgut proteases using soybean tryspin inhibitor (SBTI) Bowman Birk Inhibitor (BBI) 4-(2-aminoethyl)benzensulfonylfluoride (AEBSF) and N-tosyl-L-phenylalanine chloromethylketone (TPCK) indicate that protease activity is mostly trypsinlike (80%) with a small amount of chymotrypsinlike activity (20%). Injections of late 3rd and 4th instar larval hemolymph into H. virescens larvae inhibited tryspin biosynthesis in the larval midgut. Similar results were obtained when highly purified 4th instar larval hemolymph that crossreacted with Aea-TMOF antisurm using ELISA was injected into 2nd instar larvae. Injections of Aea-TMOF and its analogues into 2nd instar, and Aea-TMOF alone into 4th instar larvae stopped trypsin biosynthesis 24 and 48 h after the injections, respectively. Injections of 4th instar H. virescens larval hemolymph into female Aedes aegypti that took a blood meal stopped trypsin biosynthesis and egg development. These results show that the biosynthesis of trypsin-like enzymes in the midgut of a lepidoptera is modulated with a hemolymph circulating TMOF-like factor that is closely related to Aea-TMOF. Arch.     

Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer

Laboratory, greenhouse and field studies were conducted to characterize the insecticidal properties of genetically altered forms of Autographa californica (Speyer) nucleopolyhedrovirus (AcNPV) and Helicoverpa zea (Boddie) NPV (HzNPV) against selected heliothine species. The altered viruses each contained a chimeric 0.8-kb fragment encoding the insect-specific, sodium channel neurotoxin from the Algerian scorpion Androctonus australis Hector (AaIT, hence recombinant viruses designated Ac-AaIT and Hz-AaIT). Based on LD50 values, results from diet-overlay bioassays showed Ac-AaIT and Hz-AaIT to be equally virulent against larval tobacco budworm, Heliothis virescens (F.), but Hz-AaIT averaged 1,335-fold greater bioactivity than Ac-AaIT against larval cotton bollworm, Helicoverpa zea (Boddie). Hz-AaIT killed larvae of both heliothine species at rates significantly faster than those imparted by HzNPV (viral LT50 values averaged 2.5 and 5.6 d, respectively). In greenhouse studies, foliar sprays of Ac-AaIT and Hz-AaIT were equally effective in controlling H. virescens on cotton; however, Hz-AaIT provided control of H. zea on cotton at a level superior to that of Ac-AaIT. For example, after three weekly sessions of foliar application and H. zea artificial infestation, cotton treated with Ac-AaIT or Hz-AaIT at 10 x 10(11) occulsion bodies (OB)/ha averaged 2.5 and 16.2 nondamaged flower buds per plant, respectively. Another greenhouse study conducted against heliothine species on cotton showed that the quicker killing speed exhibited by Hz-AaIT led to improved plant protection versus HzNPV. Finally, results from three field trials demonstrated that Hz-AaIT at 5-12 x 10(11) OB/ha provided control of the heliothine complex in cotton at levels slightly better than Bacillus thuringiensis, equal to the macrolide, spinosad, and only slightly less than that of selected pyrethroid and carbamate insecticides. Overall, results from these studies indicate that, because of host range differences between the two wild-type viruses, HzNPV is the better vectoring agent (versus AcNPV) for designing recombinant clones as insecticides targeted at the multi-species heliothine complex. Further, these studies suggest that if appropriately tailored for the pest complex, recombinant NPVs may be very effective, insect-specific approaches to managing pests in many cropping scenarios. Possible Hz-AaIT deployment strategies for control of heliothine species on conventional and transgenic cotton varieties are discussed.