Resistance to Bacillus thuringiensis CryIA delta-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration.

The Bacillus thuringiensis toxin-binding properties of midgut epithelial cells from two strains of Heliothis virescens were compared. One H. virescens strains (YHD2) which was selected against CryIAc toxin had over 10,000-fold resistance to CryIAc toxin relative to the susceptible strain and was cross-resistant to CryIAa and CryIAb. The second H. virescens strain (YDK) was susceptible to these toxins in the order CryIAc > CryIAb > CryIAa. Receptor-binding properties of CryIAa, CryIAb, and CryIAc toxins were compared between the susceptible and resistant strains. Saturation and competition-binding experiments were performed with brush border membrane vesicles prepared from midguts of the susceptible and resistant insects and 125I-labeled toxins. In the susceptible strain, saturable, specific, and high-affinity binding of all three toxins was observed. The relative binding-site concentration was directly correlated with toxicity (CryIAc > CryIAb > CryIAa). In the resistant strains, the binding affinities of CryIAb and CryIAc were similar to that observed with the susceptible strain and ony minor differences in binding-site concentration (Bmax) were observed. The major difference between the two strains was the total lack of binding of CryIAa toxin to the brush border membrane vesicles of the resistant strain. Heterologous competition-binding experiments and ligand blot analysis supported the hypothesis that there were multiple binding sites for the toxins. On the basis of results of the present study, we propose that alterations in binding proteins shared by all three toxins are a major factor in resistance. This suggests that not all receptors of CryIAc might be involved in toxic function.     

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.     

Influence of the host plant on occluded virus production and lethal infectivity of a baculovirus

Intra- and inter-specific effects of cotton, soybean, and clover on the time until death of Helicoverpa zea (Boddie) and Heliothis virescens (F.) larvae lethally infected with H. zea nucleopolyhedrovirus (HzSNPV) were evaluated in the laboratory. In the first test, on second instar only, the time until death of lethally infected larvae of both species differed with the plant tissues (vegetative or reproductive) and plant species. The total viral activity produced per larva in LC(50) units (occluded viral bodies (OBs) per larva/LC(50) in OBs/mm(2) of diet surface) was greater from H. virescens larvae fed vegetative than reproductive tissues of all host plants, but from H. zea virus production was greater only when fed vegetative tissue of soybean. In a second test that compared second and fourth instar H. virescens on cotton, total viral activity from larvae treated in both instars was greater when fed vegetative than reproductive tissues. Results of these tests suggest that the ability of host plants to influence baculovirus disease is more complex than previously believed. When examining the epizootic potential of a baculovirus, more attention must be given to the effects of the host plant on the insect-virus interactions.     

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.     

Susceptibility of bollworm and tobacco budworm (Lepidoptera: Noctuidae) to Cry2Ab2 insecticidal protein

Susceptibilities of 82 bollworm, Helicoverpa zea (Boddie), and 44 tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), populations to Cry2Ab2 protein were measured in diet incorporated assays at the University of Arkansas from 2002 to 2005. Resulting data were used to calculate overall (pooled data) estimates of species susceptibility for future benchmarks of resistance. Variabilities among populations also were studied by comparing regressions for individual populations and calculating mean susceptibilities for different subgroups of the colonies studied. Individual lethal concentration (LC50) estimates for nine laboratory, seven laboratory-cross, and 28 field populations of H. virescens varied up to 48-fold when adjusted for the response of the most susceptible laboratory colony studied. Mean susceptibilities of all laboratory, laboratory-cross, or field colonies varied only two-fold. When grouped by host plants, populations collected on tobacco, Nicotiana tabacum (L.), seemed to be less susceptible than those collected on other host plants. Individual LC50 values for 82 laboratory, laboratory-cross and field populations of H. zea varied up to 37-fold. Mean LC50 values of all laboratory, laboratory-cross, or field populations varied only three-fold. Susceptibilities of populations from Bollgard cotton were up to four-fold less than those from Bacillus thuringiensis corn, Zea mays L. Field populations collected during late season were generally less susceptible than those collected early in the season. Across the two species, H. zea was less sensitive to Cry2Ab2 than H. virescens. Both species seem to be less sensitive to Cry2Ab2 than to CrylAc.     

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.     

Relative resistance of transgenic tomato tissues expressing high levels of tobacco anionic peroxidase to different insect species.

Different parts of genetically transformed tomato (Lycopersicon esculentum L.) plants that express the tobacco anionic peroxidase were compared for insect resistance with corresponding wild type plants. Leaf feeding by first instar Helicoverpa zea and Manduca sexta was often significantly reduced on intact transgenic plants and/or leaf disks compared to wild type plants, but the effect could depend on leaf age. Leaves of transgenic plants were generally as susceptible to feeding damage by third instar Helicoverpa zea (Boddie) and Manduca sexta (L.) as wild type plants. Green fruit was equally susceptible to third instar larvae of H. zea in both type plants, but fruit of transgenic plants were more resistant to first instar larvae as indicated by significantly greater mortality. Basal stem sections were more resistant to neonate larvae of H. zea and adults of Carpophilus lugubris Murray compared to wild type plants as indicated by significantly greater mortality and/or reduced feeding damage. Thus, tobacco anionic peroxidase activity can increase plant resistance to insects in tomato, a plant species closely related to the original source plant species, when expressed at sufficiently high levels. However, the degree of resistance is dependent on the size of insect and plant tissue involved.     

Susceptibilities of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) populations to Cry1Ac insecticidal protein

Susceptibilities of bollworm, Helicoverpa zea (Boddie) and tobacco budworm, Heliothis virescens (F.) to Cry1Ac were measured via a diet-incorporated assay with MPV II at the University of Arkansas during 2002-2004. Lethal concentration-mortality (LC50) estimates of five laboratory, seven laboratory-cross, and 10 field populations of H. virescens varied 12-fold. Pooled susceptibilities of H. virescens across all laboratory and field populations varied five-fold. The LC50 estimates for H. virescens were higher than those reported by previous research before the introduction of transgenic crops. However, the ratio of susceptibility of laboratory and field populations was similar, suggesting no change in overall species susceptibility. Individual LC50 estimates of five laboratory, nine laboratory-cross, and 57 field populations of H. zea varied over 130-fold. Pooled susceptibilities across laboratory and field populations varied widely. Among the field populations, colonies from non-Bacillus thuringiensis (Bt) crops were generally more susceptible than those from Bt crops. Across the Bt crops expressing Cry protein, colonies from Bollgard (Monsanto Company) cotton had lower susceptibility to CrylAc than those from Bt corn and those from non-Bt crops.     

Differences in cuticular lipid composition of the antennae of Helicoverpa zea, Heliothis virescens, and Manduca sexta

Analyses of the hexane washes of antennae, forelegs and whole bodies of Helicoverpa zea, Heliothis virescens, and Manduca sexta revealed notable differences in the components of the cuticular coatings of each species. Most striking were the differences between the cuticular coatings of male and female antennae of both H. zea and H. virescens. Novel esters of short-chain acids (C2-C4) and long-chain secondary alcohols (C25-C32) were identified in the hexane washes of the male antenna and forelegs of H. zea and H. virescens. These compounds were found in only small amounts or were completely absent on the female antennae of both species. In H. zea, butyrates of 7- and 8-pentacosanol and 8- and 9-heptacosanol were found, whereas, in the foreleg extracts of H. virescens, acetates and propionates were detected in addition to butyrates. While cholesterol is a major component of antennal washes (10-15%), only traces were found in the foreleg extracts. Although the composition of the cuticular coating of M. sexta differed greatly from that of the other two species, the extractable coatings of the antennae of male and female M. sexta were nearly identical.     

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.     

Development,survival and fitness performance of Helicoverpa zea (Lepidoptera: Noctuidae) in MON810 Bt field corn

Helicoverpa zea (Boddie) development, survival, and feeding injury in MON810 transgenic ears of field corn (Zea mays L.) expressing Bacillus thuringiensis variety kurstaki (Bt) Cry1Ab endotoxins were compared with non-Bt ears at four geographic locations over two growing seasons. Expression of Cry1Ab endotoxin resulted in overall reductions in the percentage of damaged ears by 33% and in the amount of kernels consumed by 60%. Bt-induced effects varied significantly among locations, partly because of the overall level and timing of H. zea infestations, condition of silk tissue at the time of egg hatch, and the possible effects of plant stress. Larvae feeding on Bt ears produced scattered, discontinuous patches of partially consumed kernels, which were arranged more linearly than the compact feeding patterns in non-Bt ears. The feeding patterns suggest that larvae in Bt ears are moving about sampling kernels more frequently than larvae in non-Bt ears. Because not all kernels express the same level of endotoxin, the spatial heterogeneity of toxin distribution within Bt ears may provide an opportunity for development of behavioral responses in H. zea to avoid toxin. MON810 corn suppressed the establishment and development of H. zea to late instars by at least 75%. This level of control is considered a moderate dose, which may increase the risk of resistance development in areas where MON810 corn is widely adopted and H. zea overwinters successfully. Sublethal effects of MON810 corn resulted in prolonged larval and prepupal development, smaller pupae, and reduced fecundity of H. zea. The moderate dose effects and the spatial heterogeneity of toxin distribution among kernels could increase the additive genetic variance for both physiological and behavioral resistance in H. zea populations. Implications of localized population suppression are discussed.     

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.     

Central role of hemocytes in Autographa californica M nucleopolyhedrovirus pathogenesis in Heliothis virescens and Helicoverpa zea

Autographa californica M nucleopolyhedrovirus (AcMNPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between AcMNPV pathogenesis in Heliothis virescens and Helicoverpa zea, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of H. zea, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in H. virescens. Further, H. zea hemocytes, unlike those of H. virescens, were highly resistant to AcMNPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes-by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci-together with the host's melanization response, formed the basis of H. zea's resistance to fatal infection by AcMNPV.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.     

Life history traits of Helicoverpa zea (Lepidoptera: Noctuidae) on non-Bt and Bt transgenic corn hybrids in eastern North Carolina.

Transgenic varieties of field corn that express the CrylAb B. thuringiensis (Bt) toxin in ear tissue present the potential of reducing ear feeding by the corn earworm, Helicoverpa zea (Lepidoptera: Noctuidae), and for reducing the size of populations of the insect infesting other host crops. Life history parameters of H. zea feeding on ears of conventional and Bt field corn varieties were measured in field plots in eastern North Carolina in 1997 and 1998. Transformation events investigated were Mon-810 and Bt-11. Bt corn was found to cause a steady mortality of larvae during development, but permitted approximately 15-40% survival to the prepupal stage compared with non-Bt corn. Mortality of prepupae and pupae from Bt corn was also higher than from non-Bt corn, reducing overall adult production by 65-95%. The larvae that did survive grew more slowly on Bt than on non-Bt corn, and produced pupae that weighed 33% less. Pupation and adult eclosion were delayed by 6-10 d by feeding on Bt corn ears. Corn varieties expressing Bt in ear tissue have the potential to reduce H. zea ear feeding by up to 80%, and the potential to reduce populations emerging from ear-stage corn fields to infest cotton, soybean and other crops by around 75%. To have a measurable effect on area-wide populations, Bt corn varieties would need to be planted in large proportions of corn fields. Extensive planting of varieties such as those tested here, having only moderate effects on H. zea, would raise concerns about rapid evolution of resistance.     

The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens

Retrotransposon-mediated disruption of the BtR-4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to high levels of resistance in the YHD2 strain to Cry1Ac toxin from Bacillus thuringiensis. This suggests that HevCaLP functions as a Cry1Ac toxin receptor on the surface of midgut cells in susceptible larvae and that the BtR-4 gene disruption eliminates this protein in resistant larvae. However, Cry1Ac toxin binding to HevCaLP is yet to be reported. We used the polymerase chain reaction and immunoblotting as tools to discriminate between individual H. virescens larval midguts from susceptible (YDK) and resistant (CXC, KCBhyb, and YHD2-B) strains according to their BtR-4 gene disruption genotype and phenotype. This approach allowed us to test the correlation between BtR-4 gene disruption, lack of HevCaLP, and altered Cry1A toxin binding. Toxin-binding assays using brush border membrane vesicles revealed that a wild-type BtR-4 allele is necessary for HevCaLP production and Cry1Aa toxin binding, while most of Cry1Ab and Cry1Ac binding was independent of the BtR-4 genotype. Moreover, toxin competition experiments show that KCBhyb midguts lacking HevCaLP are more similar to midguts of the original YHD2 strain than to the current YHD2-B strain. This resolves discrepancies in published studies of Cry1A binding in YHD2 and supports our earlier suggestion that a separate genetic change occurred in YHD2 after appearance of the cadherin disruption, conferring even higher resistance in the resulting YHD2-B strain as well as a large reduction in Cry1Ab and Cry1Ac binding.     

Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor

Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.