Bt-resistant Strains Research Articles

Evaluating local plant species for effective fall armyworm management strategies in Taiwan

BackgroundThe emergence of Spodoptera frugiperda (fall armyworm; FAW) in the world has raised concerns regarding its impact on crop production, particularly on corn and sorghum. While chemical control and Bt crops have been effective in managing FAW damage, the development of pesticide-resistant and Bt-resistant strains necessitates alternative control methods. The push-pull farming system has gained attention, but direct utilization of African plant species in Taiwan faces challenges due to invasive potential and climatic disparities. Therefore, identifying and evaluating suitable local plant species, such as Napier grass (Pennisetum purpureum), Desmodium species, and signal grass (Brachiaria brizantha), is crucial for implementing effective FAW management strategies in Taiwan.ResultsIn screening fifty Napier grass germplasms, all demonstrated an antibiotic effect, reducing leaf consumption compared to corn. Notably, thirty-five germplasms exhibited robust antibiotic traits, decreasing FAW consumption and increasing mortality rates. Three Napier grass germplasms also attracted more female moths for oviposition. Further evaluation of selected Napier grass germplasms and signal grass demonstrated efficacy in reducing FAW larval weight and survival duration. Additionally, Desmodium species, particularly D. uncinatum, showed promising toxicity against FAW larvae.ConclusionOur findings support the effectiveness of selected Napier grass germplasms and signal grass as pull plants, and highlight the potential of D. uncinatum as a push plant in FAW management strategies in Taiwan.

Comparison of Gut Microbial Community between Bt-Resistant and Susceptible Strains of Ostrinia furnacalis

Bacillus thuringiensis is an effective entomopathogen, and its crystal toxin expressed in transgenic crops has been widely used for pest control. However, insect resistance risk is the main threat to the continued successful utility of Bt crops. Several studies reported the role of midgut microbiota in Bt resistance, but the mechanism remains controversial. In the present study, using high-throughput sequencing of the bacterial 16S ribosomal RNA gene, we surveyed the midgut bacterial flora of Ostrinia furnacalis from one Bt-susceptible (ACB-BtS) and two Bt-resistant (ACB-AbR and ACB-FR) strains and explored the mortality of O. furnacalis after eliminating the gut bacteria. Gut bacterial diversity in Bt-resistant strains was significantly lower in Bt-resistant than in Bt-susceptible strains. Ordination analyses and statistical tests showed that the bacterial community of ACB-AbR was distinguishable from ACB-BtS. The genus Halomonas was dominated in ACB-BtS, but the unclassified_Enterobacteriaceae was the most enriched genus in ACB-AbR and ACB-FR. Furthermore, interactions of the bacterial community are more complex in Bt-resistant strains than in Bt-susceptible strains. Moreover, the mortalities of ACB-AbR and ACB-BtS strains treated by the Cry1Ab toxin were significantly reduced after eliminating the gut bacteria. Our findings suggest that Bt stressors structured in the midgut bacterial community and the microbiota have the potential to regulate the Bt-induced killing mechanism.

Comparison of the co-occurrence patterns of the gut microbial community between Bt-susceptible and Bt-resistant strains of the rice stem borer, Chilo suppressalis

Comparison of the co-occurrence patterns of the gut microbial community between Bt-susceptible and Bt-resistant strains of the rice stem borer, Chilo suppressalis

Relative fitness of susceptible and Cry1A.105/Cry2Ab2-single-/dual-protein-resistant Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) on non-Bt diet and a diet containing a low concentration of two proteins.

Helicoverpa zea (Boddie) is a destructive agricultural pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1A.105 and Cry2Ab2 are two Bt proteins expressed in a widely planted maize event MON 89034. In this study, two tests (Test-I and Test-II) were conducted to evaluate the relative fitness of Bt-susceptible and -resistant H. zea on non-Bt diet (Test-I and Test-II) and a diet containing a mix of Cry1A.105 and Cry2Ab2 at a low concentration (Test-II only). Insect populations evaluated in Test-I were two Bt-susceptible strains and three Bt-resistant strains (a single-protein Cry1A.105-, a single-protein Cry2Ab2-, and a dual-protein Cry1A.105/Cry2Ab2-resistant strains). Test-II analyzed the same two susceptible strains, three backcrossed-and-reselected Cry1A.105/Cry2Ab2-single-/dual-protein-resistant strains, and three F1 heterozygous strains. Measurements of life table parameters showed that neither the single- nor dual-protein Cry1A.105/Cry2Ab2 resistance in H. zea was associated with fitness costs under the test conditions. The single Cry protein resistances at a concentration of a mix of Cry1A.105 and Cry2Ab2 that resulted in a zero net reproductive rate for the two susceptible strains were functionally incomplete recessive or codominant, and the dual-protein resistance was completely dominant. The lack of fitness costs could be a factor contributing to the rapid revolution of resistance to the Cry proteins in this species. Data generated from this study should aid our understanding of Cry protein resistance evolution and help in refining IRM programs for H. zea.

Map-based cloning and functional analysis revealed ABCC2 is responsible for Cry1Ac toxin resistance in Bombyx mori.

Bt toxins are parasporal crystals produced by Bacillus thuringiensis (Bt).They have specific killing activity against various insects and have been widely used to control agricultural pests. However, their widespread use has developed the resistance of many target insects. To maintain the sustainable use of Bt products, the resistance mechanism of insects to Bt toxins must be fully clarified. In this study, Bt-resistant and Bt-susceptible silkworm strains were used to construct genetic populations, and the genetic pattern of silkworm resistance to Cry1Ac toxin was determined. Sequence-tagged sitemolecular marker technology was used to finely map the resistance gene and to draw a molecular genetic linkage map, and the two closest markers were T1590 and T1581, indicating the resistance gene located in the 155 kb genetic region. After analyzing the sequence of the predicted gene in the genetic region, an ATP binding cassette transporter (ABCC2) was identified as the candidate gene. Molecular modeling and protein-protein docking result showed that a tyrosine insertion in the mutant ABCC2 might be responsible for the interaction between Cry1Ac and ABCC2. Moreover, CRISPR/Cas9-mediated genome editing technology was used to knockout ABCC2 gene. The homozygous mutant ABCC2 silkworm was resistant to Cry1Ac toxin, which indicated ABCC2 is the key gene that controls silkworm resistance to Cry1Ac toxin. The results have laid the foundation for elucidating the molecular resistance mechanism of silkworms to Cry1Ac toxin and could provide a theoretical basis for the biological control of lepidopteran pests.

Comparative Transcriptome Analysis of Bt Resistant and Susceptible Strains in Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae)

The evolution of target pest population resistance to Bt toxins is the most relevant threat to the sustainability of Bt technology, thus it is necessary to clarify insect resistance mechanisms. Firstly, the resistance level of Asian corn borer was determined by bioassay. After 28 generations selection in the lab, the Cry1Ie-resistant strain (ACB-IeR) developed more than 862-fold resistance to Cry1Ie, and the Cry1F-resistant strain (ACB-FR) developed 961-fold resistance to Cry1F. The results show that long-term exposure to Bt toxins can lead to resistance. Then, we compared the differential expression genes (DEGs) of ACB-FR and ACB-IeR with susceptible strain (ACB-BtS), and analyzed GO function and KEGG pathway through transcriptome sequencing. The comparison showed that in Bt-resistant strains, many genes have a significant down-regulated trend. Several Bt-resistance candidate genes were differentially expressed in both resistant strains. Furthermore, the DEGs were verified by RT-qPCR and showed similar trend. These results provide candidate genes for further research on the Bt resistance mechanism.

HearNPV susceptibility in Helicoverpa armigera and Helicoverpa punctigera strains resistant to Bt toxins Cry1Ac, Cry2Ab, and Vip3Aa

Genetically engineered crops expressing insecticidal toxins from Bacillus thuringiensis (Bt) have improved the management of targeted lepidopteran pests and reduced the use of insecticide sprays. These benefits explain an increasing adoption of Bt crops worldwide, intensifying the selection pressure on target species and the risk of resistance. Nucleopolyhedroviruses (NPVs) are effective bioinsecticides against numerous important lepidopteran pests. If Bt-resistant insects are shown to be susceptible to NPVs then these bioinsecticides could be a valuable component of Insecticide Resistance Management (IRM) strategies for Bt crops. We assessed the effectiveness of a Helicoverpa nucleopolyhedrovirus (HearNPV) against several different Bt-resistant strains. Utilising a droplet feeding bioassay we confirmed susceptibility to HearNPV in Helicoverpa punctigera and Helicoverpa armigera larvae resistant to the Bt toxins Cry1Ac, Cry2Ab, and Vip3A. Dual resistant H. punctigera, (Cry1Ac/Cry2Ab, and Cry2Ab/Vip3A) and dual resistant H. armigera (Cry2Ab/Vip3A) were also susceptible to HearNPV. Regardless of their specific resistance profile, Bt-resistant larvae displayed statistically similar lethal concentration (LC50) and lethal time (LT50) responses to HearNPV when compared to Bt-sensitive control insects. These results indicate that Bt-resistant H. armigera and H. punctigera are not cross-resistant to HearNPV. Consequently, the use of HearNPV against these pests may be a valuable tool to an IRM strategy for controlling Bt-resistant populations.

Fused: a promising molecular target for an RNAi-based strategy to manage Bt resistance in Plutella xylostella (L.)

Bacillus thuringiensis (Bt), a naturally occurring entomopathogenic soil bacterium, has been the active ingredient of sprayable Bt biopesticides for over a century. Insecticidal Bt Cry proteins are particularly well suited for use as plant-incorporated protectants in transgenic crops (Bt crops) due to their specificity against insect pests and safety for non-target organisms. The sustainability of these Bt products, however, has been challenged by the development of resistance in the field. RNA interference (RNAi), a species-specific control alternative that has been deregulated and commercialized in the USA and Canada, provides a new mode of action to complement the existing Bt products. Based on our preliminary research, we hypothesized that pyramiding/integrating Bt with RNAi can address/manage resistance issues related to Bt traits. To examine this overarching hypothesis, we (1) cloned and characterized a serine/threonine kinase gene (fused) of the Hedgehog (Hh) signaling pathway in the diamondback moth, Plutella xylostella (L.), a global superpest; (2) profiled Pxfused expression in Bt-resistant P. xylostella strains; and (3) investigated the involvement of Pxfused in Bt Cry1Ac resistance in P. xylostella. Pxfused expression was elevated ubiquitously in all Bt-resistant strains, and silencing of Pxfused led to larval and pupal mortality in both Cry1Ac-susceptible and -resistant strains, suggesting that Pxfused is a potential target for RNAi-based resistance management. Taken together, our results not only identify a molecular target to control a devastating lepidopteran pest, but also shed light on a novel resistance management strategy through the integration of two biotechnological techniques with distinct modes of action.

Low risk of resistance evolution of Spodoptera frugiperda to chlorfenapyr in Brazil

Insecticide resistance is serious problem for Spodoptera frugiperda (Lepidoptera: Noctuidae). Due to intense selection pressure, the evolution of S. frugiperda resistance to major insecticides groups and Bt proteins has already been documented. Therefore, studies to preserve the lifetime of insecticides with unique modes of action such as chlorfenapyr are important in insect resistance management programs. For implementing a proactive resistance management program of S. frugiperda to chlorfenapyr, studies were conducted with field populations collected from major corn-growing regions in Brazil from 2016 to 2018. We aimed to (i) characterize and monitor the susceptibility of field populations, (ii) estimate the frequency of resistance alleles and (iii) evaluate cross-resistance patterns between insecticides and Bt-resistant strains of S. frugiperda to chlorfenapyr. Baseline susceptibility of field population showed a LC50 ranged from 18.3 to 25.1 µg ml−1. The survival rate at the diagnostic concentration ranged from 0 to 8.4%. Using the F2 screen method, the overall estimated resistance allele frequency was 0.0003. No cross-resistance was observed between chlorfenapyr and other insecticides and Bt proteins. Results demonstrated a high susceptibility, low frequency of resistance alleles and lack of cross-resistance. Thus, we concluded there is a low risk of resistance evolution of S. frugiperda to chlorfenapyr in Brazil using the current pest management strategies.

Effective dominance of resistance of Spodoptera frugiperda to Bt maize and cotton varieties: implications for resistance management.

The resistance of fall armyworm (FAW), Spodoptera frugiperda, has been characterized to some Cry and Vip3A proteins of Bacillus thuringiensis (Bt) expressed in transgenic maize in Brazil. Here we evaluated the effective dominance of resistance based on the survival of neonates from selected Bt-resistant, heterozygous, and susceptible (Sus) strains of FAW on different Bt maize and cotton varieties. High survival of strains resistant to the Cry1F (HX-R), Cry1A.105/Cry2Ab (VT-R) and Cry1A.105/Cry2Ab/Cry1F (PW-R) proteins was detected on Herculex, YieldGard VT PRO and PowerCore maize. Our Vip3A-resistant strain (Vip-R) exhibited high survival on Herculex, Agrisure Viptera and Agrisure Viptera 3 maize. However, the heterozygous from HX-R × Sus, VT-R × Sus, PW-R × Sus and Vip-R × Sus had complete mortality on YieldGard VT PRO, PowerCore, Agrisure Viptera, and Agrisure Viptera 3, whereas the HX-R × Sus and Vip-R × Sus strains survived on Herculex maize. On Bt cotton, the HX-R, VT-R and PW-R strains exhibited high survival on Bollgard II. All resistant strains survived on WideStrike, but only PW-R and Vip-R × Sus survived on TwinLink. Our study provides useful data to aid in the understanding of the effectiveness of the refuge strategy for Insect Resistance Management of Bt plants.

Fitness costs and stability of Cry1Fa resistance in Brazilian populations of Spodoptera frugiperda.

The presence of fitness costs of resistance to Bacillus thuringiensis (Bt) insecticidal proteins in insect populations may delay or even reverse the local selection of insect resistance to Bt transgenic crops, and deserves rigorous investigation. Here we assessed the fitness costs associated with Cry1Fa resistance in two strains of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), derived from field collections in different Brazilian regions and further selected in the laboratory for high levels of resistance to Cry1Fa using leaves of TC1507 corn. Fitness components were compared using paired resistant and susceptible strains with similar genetic backgrounds and F1 generations from reciprocal crosses, all of them reared on non-transgenic corn leaves. No apparent life history costs in the larval stage were observed in the Bt-resistant strains. Moreover, the resistance remained stable for seven generations in the absence of selection, with no decrease in the proportion of resistant individuals. Larval respiration rates were also similar between resistant and susceptible homozygotes, and heterozygotes displayed respiration rates and demographic performance equal or superior to those of susceptible homozygotes. In combination, these results indicate the lack of strong fitness costs associated with resistance to Cry1Fa in the fall armyworm strains studied. These findings suggest that Cry1Fa resistance in S. frugiperda populations is unlikely to be counterselected in Cry1Fa-free environments. © 2016 Society of Chemical Industry.

Effects of Resistance to Bt Cotton on Diapause in the Pink Bollworm,Pectinophora gossypiella

Fitness costs associated with resistance to Bacillus thuringiensis (Bt) crops are expected to delay the evolution of resistance. In a previous study where pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae), larvae overwintered in outdoor insectaries, individuals from Bt-resistant strains had lower survival than individuals from Bt-susceptible strains or F1 progeny from crosses between resistant and susceptible adults. To investigate the physiological basis of such recessive cost, diapause duration was experimentally manipulated in the laboratory. Compared to a Bt-susceptible strain and F1 progeny, we hypothesized that Bt-resistant strains could exhibit a lower propensity or intensity of diapause, faster weight loss during overwintering, lower initial weight of diapausing larvae, and reduced longevity of moths emerging from diapause. Results were as expected for initial weight of diapausing larvae and longevity of overwintered male moths or female moths remaining in diapause for a short period. However, a higher diapause induction and intensity and slower weight loss occurred in F1 progeny and Bt-resistant strains than in a Bt-susceptible strain. Moreover, F1 progeny had greater overwintering survival than the Bt-resistant and Bt-susceptible strains, and F1 female moths had the greatest longevity after sustaining long diapausing periods. All of these unexpected results may be explained by pleiotropic effects of resistance to Bt cotton that increased the strength of diapause in the F1 progeny and Bt-resistant strains. Incomplete resistance was reflected in disadvantages suffered by Bt-resistant individuals feeding on a Bt diet instead of a non-Bt diet, including lower diapause propensity, lower diapause intensity and reduced longevity of overwintered male moths. While this study suggests that the evolution of resistance to Bt cotton and feeding on a Bt diet in Bt-resistant individuals have pervasive effects on several traits associated with diapause, further field experiments are needed to elucidate the basis of the overwintering cost in the pink bollworm.

Cadherin-Based Resistance to <I>Bacillus thuringiensis</I> Cotton in Hybrid Strains of Pink Bollworm: Fitness Costs and Incomplete Resistance

Recessive resistance to Bacillus thuringiensis (Bt) cotton, Gossypium hirsutum L., in laboratory-selected strains of pink bollworm, Pectinophora gossypiella (Saunders), is associated with three resistance alleles (r1, r2, and r3) of a cadherin gene. Previous experiments based on measurement of fitness components in Bt-resistant and Bt-susceptible strains revealed that fitness costs and incomplete resistance are associated with resistance. Here, we used two hybrid strains of pink bollworm, each containing a mixture of susceptible and resistant individuals, and polymerase chain reaction (PCR) amplifications to test the association between cadherin genotype and fitness components for individuals sharing a common genetic background. All survivors on Bt cotton had two r alleles, confirming that recessive cadherin alleles are tightly linked with resistance to Bt cotton. On non-Bt cotton, significantly greater developmental time for rr than ss larvae indicated a recessive fitness cost, but costs did not affect survival or pupal weight. Incomplete resistance was manifested as longer developmental time, lower survival, and smaller pupal weight in rr individuals developing on Bt cotton compared with non-Bt cotton. As in previous experiments, no significant variation in performance on Bt cotton was detected among rr genotypes. However, a meta-analysis of data from seven experiments revealed that survival on Bt cotton relative to non-Bt cotton was lower in r2r3 and higher in r1r2 compared with the other rr genotypes. Assessment of fitness components associated with cadherin genotypes in hybrid strains of pink bollworm confirms that recessive resistance to Bt cotton is associated with recessive fitness costs and incomplete resistance.

Analysis of midgut proteinases from Bacillus thuringiensis-susceptible and -resistant Heliothis virescens (Lepidoptera: Noctuidae)

Insects with altered proteinases can avoid intoxication by Bacillus thuringiensis (Bt) toxins. Therefore, proteinase activities from gut extracts of Bt-susceptible (YDK) and -resistant (YHD2-B, CXC and KCBhyb) Heliothis virescens strains were compared. The overall pH of gut extracts from YDK and CXC were statistically similar (9.56 and 9.62, respectively), while the pH of extracts from KCBhyb and YHD2-B were significantly more alkaline (9.81 and 10.0, respectively). Gut extracts from YHD2-B and CXC larvae processed Cry1Ac and Cry2Aa protoxin slower than extracts from YDK larvae, suggesting that differences in proteolysis contribute to resistance in these strains. Casein zymogram analysis of gut extracts revealed both qualitative and quantitative differences in caseinolytic activities among all strains, but the overall caseinolytic activity of YHD2-B gut extract was lower. Kinetic microplate assays with a trypsin substrate ( l-BApNA) demonstrated that proteinases in YDK gut extract had increased alkaline pH optima compared to resistant strains YHD2-B, CXC and KCBhyb. Gut extracts from YHD2-B had reduced trypsin-like activity, and activity blots indicated that YHD2-B had lost a trypsin-like proteinase activity. In assays with a chymotrypsin substrate (SAAPFpNA), enzymes from all Bt-resistant strains had increased pH optima, especially those from KCBhyb. Activity blots indicated that CXC had lost a chymotrypsin-like proteinase activity. Because serine proteinases are a critical component of Bt toxin mode of action, these differences may contribute to decreased toxicity in the Bt-resistant strains.

Cry toxin mode of action in susceptible and resistant Heliothis virescens larvae

Many pest insect species are effectively controlled by Bacillus thuringiensis (Bt) Cry toxins delivered in plants and biopesticides. Since the insect midgut epithelium contains receptors and other molecules that determine Bt toxicity, characterization of these molecules is necessary for sustained usage of Bt toxins. Studies of Bt susceptible and resistant strains of Heliothis virescens have provided insights into resistance mechanisms and toxin receptors. For example, the first gene identified as involved in high levels of Cry1Ac resistance in H. virescens encodes a cadherin-like protein, a functional Cry1A receptor in Lepidoptera. This manuscript discusses the most updated information on the mode of action of Cry1A toxins obtained from the characterization of resistant mechanisms in H. virescens strains. Our studies are focused on biochemical and molecular comparison of a susceptible and three resistant H. virescens strains to identify alterations that correlate with toxin resistance. Following this approach we have been able to identify an alkaline phosphatase (HvALP) as a potential receptor and tested the utility of this protein as a marker for resistance to Cry1Ac. Comparison of brush border proteomes from susceptible and resistant larvae has allowed us to identify additional molecules directly involved in the toxicity process.

Association Between Resistance to Bt Cotton and Cadherin Genotype in Pink Bollworm

Two strains of pink bollworm, Pectinophora gossypiella (Saunders), each derived in 1997 from a different field population, were selected for resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac in the laboratory. One strain (MOV97-R) originated from Mohave Valley in western Arizona; the other strain (SAF97-R) was from Safford in eastern Arizona. Relative to a susceptible laboratory strain, Cry1Ac resistance ratios were 1700 for MOV97-R and 520 for SAF97-R. For the two resistant strains, larval survival did not differ between non-Bt cotton and transgenic cotton producing CrylAc. In contrast, larval survival on Bt cotton was 0% for the two unselected parent strains from which the resistant strains were derived. Previously identified resistance (r) alleles of a cadherin gene (BtR) occurred in both resistant strains: r1 and r3 in MOV97-R, and r1 and r2 in SAF97-R. The frequency of individuals carrying two r alleles (rr) was 1.0 in the two resistant strains and 0.02 in each of the two unselected parent strains. Furthermore, in two hybrid strains with a mixture of susceptible (s) and r alleles at the BtR locus, all survivors on Bt cotton had two r alleles. The results show that resistance to Cry1Ac-producing Bt cotton is associated with recessive r alleles at the BtR locus in the strains of pink bollworm tested here. In conjunction with previous results from two other Bt-resistant strains of pink bollworm (APHIS-98R and AZP-R), results reported here identify the cadherin locus as the leading candidate for molecular monitoring of pink bollworm resistance to Bt cotton.

Is the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae?

Bacillus thuringiensis endotoxins (Bt-toxins) are the most important biopesticides used in controlling insect pests and vectors of diseases. The emergence of widespread resistance to Bt in some insect species is a serious threat to agricultural production. Analysis of Bt-resistant and susceptible laboratory strains of Helicoverpa armigera revealed elevated immune responses involving increased melanization and the presence of a soluble toxin-binding glycoprotein in the hemolymph and gut lumen of the resistant strain. We propose a resistance mechanism against toxins based on a systemic immune-induction that can be transmitted to the next generation by a maternal effect.

Identification of a Gene Associated with Bt Resistance in Heliothis virescens

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used for pest control. Bt-resistant insect strains have been studied, but the molecular basis of resistance has remained elusive. Here, we show that disruption of a cadherin-superfamily gene by retrotransposon-mediated insertion was linked to high levels of resistance to the Bt toxin Cry1Ac in the cotton pest Heliothis virescens. Monitoring the early phases of Bt resistance evolution in the field has been viewed as crucial but extremely difficult, especially when resistance is recessive. Our findings enable efficient DNA-based screening for resistant heterozygotes by directly detecting the recessive allele.

CDNAs for a Chymotrypsinogen-like Protein from Two Strains of Plodia interpunctella

Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt( entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P. interpunctella.