Bt-susceptible Strain Research Articles

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

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.

Performance of Field-Collected Spodoptera frugiperda (Lepidoptera: Noctuidae) Strains Exposed to Different Transgenic and Refuge Maize Hybrids in Argentina.

Spodoptera frugiperda (J. E. Smith) is one of the major pests of maize in Argentina. The main tool for its control is the use of genetically modified maize hybrids expressing Bacillus thuringiensis (Bt) insecticidal proteins. Maize growers in Argentina initially controlled this pest with Bt maize that expressed a single Bt protein (Cry1Ab or Cry1F). Currently it is necessary to plant maize cultivars that produce two Bt proteins to achieve the satisfactory control. Recently, Cry1F field-evolved resistant populations of this species were confirmed in Argentina. The objective of this study was to evaluate the performance of S. frugiperda field-collected strains on different Bt and non-Bt maize hybrids. Strains were collected from non-Bt maize (T1), Agrisure TDMax (T2), Agrisure Viptera (T3), Agrisure Viptera 3110 (T4), Genuity VT Triple Pro (T5), and Power Core (T6). Three experiments were performed to 1) determine the survivorship and reproduction of field-collected larvae (F0) from Bt maize hybrids, 2) evaluate Cry1F resistance using an F1 screen, and 3) assess the performance of F1 strains on different maize hybrids. In the F0, the survivorship from larva to adult ranged from 0 to 63%. We obtained adults from only the T1, T2, T5, and T6 strains with no significant differences in the reproductive parameters. Continuously rearing F1 larvae on their collected hosts affected larval duration, which was significantly shorter for a known-laboratory Bt-susceptible strain than the field-collected strains. Our results support the existence of Cry1F-resistance alleles in S. frugiperda field populations in Argentina.

Better cold tolerance of Bt-resistant Spodoptera exigua strain and the corresponding cold-tolerant mechanism

Spodoptera exigua is a secondary target pest of Bt cotton commercialized in China. With the continuous adoption of Bt cotton, populations of S. exigua have gradually increased. However, the cold tolerance ability of Bt-resistant S. exigua and the effect of continuous Bt diet on anti-cold materials are unknown. In our study, it was found that Bt-resistant S. exigua (Bt10) developed better with shorter larval and pupal duration and higher pupation rate compared to CK at the suboptimal low temperature. The supercooling points and freezing points of the Bt-resistant S. exigua strain were determined, and body water content and anti-cold materials such as total sugar, trehalose and glycogen, glycerol and fat were examined to explore the effect of Bt toxin on overwintering and on population increase. The results showed that the supercooling point and the freezing point of the Bt-resistant S. exigua pupae were both significantly lower than that of the Bt-susceptible strain. No difference was found in the body water content of pupae and adults between the two strains. Total sugar content of the Bt-resistant strain at both the pupal and adult stages was higher than that of the susceptible strain at the corresponding stages, and glycogen content of the Bt-resistant strain at the larval stage was higher than that of the susceptible larval S. exigua. Fat content of the Bt-resistant larvae, pupae and adults was for each higher than that of the susceptible strain, but the difference was not significant except for that of the 3rd instar larvae. Glycerol content of the Bt-resistant strain at larval, pupal and adult stages was for each higher than that of the corresponding life stages of the susceptible strain. It can be seen that more glycerol was accumulated in Bt-resistant S. exigua. The results indicate that Bt-resistant S. exigua has better cold tolerance. The contents of the anti-freeze substances of progeny, especially glycerol, were increased after previous generations were continuously fed on Bt protein, which means that the Bt-resistant secondary target pests could more easily overcome the overwinter season and become a source of crop damage the following year.

Fitness costs of Cry1F resistance in two populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from Puerto Rico and Florida

The development of resistance in target pest populations is a threat to the sustainability of transgenic crops expressing Bacillus thuringiensis (Bt) proteins. Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target pest of Bt maize in North and South America. This insect is the first target pest that has developed field resistance to Bt maize at multiple locations in these regions. The objective of this study was to assess the fitness costs associated with the Cry1F resistance in two populations of S. frugiperda collected from Puerto Rico (RR-PR) and Florida (RR-FL). In the study, fitness costs were evaluated by comparing survival, growth, and developmental time of seven populations of S. frugiperda on (1) non-Bt meridic diet and (2) non-Bt maize leaf tissue and non-Bt diet. The seven populations were RR-PR, RR-FL, a Bt-susceptible strain (Bt-SS), and four F1 populations developed from reciprocal crosses between Bt-SS and the two resistant populations. Biological parameters measured were neonate-to-adult survivorship, neonate-to-adult developmental time, 10day larval weight on non-Bt maize leaf tissue, pupal weight, and sex ratios. Results of the study show that the Cry1F resistance in both RR-PR and RR-FL was associated with considerable fitness costs, especially for the Florida population. Compared to the Bt-susceptible population, RR-PR showed an average of 61.1% reduction in larval weight, 20.4% less in neonate-to-adult survivorship, and 3.7days delay in neonate-to-adult developmental time. These fitness costs for RR-FL were 66.9%, 31.7% and 4.4days, respectively. The fitness costs of RR-PR and RR-FL appeared to be non-recessive. The results indicate that a diversified genetic basis may exist for the Cry1F resistance in S. frugiperda.

Resistance to Bacillus thuringiensis in the cabbage looper ( Trichoplusia ni) increases susceptibility to a nucleopolyhedrovirus

Cabbage loopers, Trichoplusia ni, are pests in many agricultural settings including vegetable greenhouses in British Columbia (Canada), where microbial insecticides based on Bacillus thuringiensis ( Bt) toxins are commonly used. Frequent use of these insecticides has led to resistance in some populations. An alternative microbial control is the multiple nucleopolyhedrovirus of the alfalfa looper ( Autographa californica), AcMNPV which occurs naturally, but at low frequencies in T. ni populations. Bioassays show that T. ni resistant to Bt were twice as susceptible to AcMNPV as were individuals from the Bt-susceptible strain and AcMNPV could be complementary in a resistance management program for T. ni.

Growth and development of Bacillus thuringiensis Cry1Ab‐susceptible and Cry1Ab‐resistant sugarcane borer on diet and conventional maize plants

AbstractThe sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), is a dominant maize borer pest and a major target of Bacillus thuringiensis (Bt)‐maize in Louisiana and the Gulf Coast area of Texas (USA). Growth and development of D. saccharalis on non‐toxic diet, diet treated with three low concentrations (0.01, 0.05, and 0.1 μg g−1) of Cry1Ab toxin, and on non‐Bt maize plants were compared for five insect genotypes: a Bt‐susceptible strain (BT‐SS), a Cry1Ab‐resistant strain (BT‐RR), a back‐crossed and re‐selected resistant strain (BT‐R’R’), and two F1 progeny of the BT‐SS and BT‐R’R’ strains. Fitness of the five genotypes was examined by infesting neonates on diet with/without Cry1Ab toxin in the laboratory and on intact non‐Bt maize plants in the greenhouse. Biological parameters measured were neonate‐to‐pupa development time and pupation rate, larval survival, larval and pupal weight, and sex ratio. Larvae of BT‐SS and BT‐R’R’ on non‐toxic diet and non‐Bt maize plants grew normally and there were no significant differences between the two strains in all measured parameters, suggesting a lack‐of‐fitness cost of the Cry1Ab resistance in D. saccharalis. Except for the development time on non‐Bt diet, all other parameters on both non‐Bt diet and non‐Bt maize plants were similar among the five genotypes. Larval development of BT‐SS was significantly affected on diet treated with Cry1Ab toxin at 0.05 and 0.1 μg g−1, whereas the effect to BT‐RR and BT‐R’R’ was not significant. Pupal weight and sex ratio reared on Cry1Ab‐diet were similar and there were no significant differences among the five genotypes. Neonate‐to‐pupation rate decreased as Cry1Ab concentrations increased but the decrease was more significant for BT‐SS than for the other four genotypes. The lack‐of‐fitness costs of Bt resistance in D. saccharalis imply a greater challenge in managing Bt resistance for this maize borer species.

Novel pink bollworm resistance to the Bt toxin Cry 1Ac: effects on mating, oviposition, larval development and survival.

Bt cotton plants are genetically engineered to produce insecticidal toxins from the Bacillus thuringiensis (Bt) Berliner (Bacillales: Bacillaceae) bacterium and target key lepidopteran pests. In all previous strains of pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) selected in the laboratory for resistance to insecticidal Cry1Ac toxin using an artificial diet containing the toxin, resistance to Cry1Ac and to Bt cotton is linked to three cadherin alleles (r1, r2, and r3). In contrast, the BG(4) pink bollworm strain was selected for resistance to Bt cotton by feeding larvae for four days in each of 42 generations on bolls of ‘NuCOTN33B®’ that expressed Cry1Ac toxin. After additional selection for eleven generations on Cry1Ac-incorporated diet, the susceptibility to Cry1Ac, fecundity, egg viability, and mating of this strain (Bt4R) was compared with the unselected Cry1Ac-susceptible parent strain. Some larvae of the Bt4R strain survived on diet containing ≥ 10 µg Cry1Ac per milliliter artificial diet, but none survived on transgenic cotton bolls. In contrast to strains selected exclusively on Cry1Ac diet, some survival of progeny of reciprocal moth crosses of Bt4R resistant and Bt-susceptible strains occurred on Cry1Ac-treated diet, suggesting differences in levels of dominance. The Bt4R resistant strain does not have the r1, r2, or r3 mutant cadherin genes as do all previous strains of pink bollworm selected on Cry1Ac-treated artificial diet. The combined results suggest a mechanism of resistance to Cry1Ac that is different from previously described cadherin mutations.

F2 screen for resistance to a Bacillus thuringiensis-maize hybrid in the sugarcane borer (Lepidoptera: Crambidae)

A novel F2 screening technique was developed for detecting resistance in sugarcane borer, Diatraea saccharalis (F.), to transgenic Bacillus thuringiensis (Bt)-maize expressing the Cry1Ab insecticidal protein. The F2 screening method involved (i) collecting larvae from maize fields; (ii) establishing two-parent families; (iii) screening F2 neonates for survival on Bt-maize leaf tissues; and (iv) confirming resistance on commercial Bt-maize plants. With the F2 screening method, 213 iso-line families of D. saccharalis were established from field collections in northeast Louisiana, USA and were screened for Bt resistance. One family was confirmed to carry a major Bt resistance allele(s). In a laboratory bioassay, larval mortality of the Bt-resistant D. saccharalis on Bt-maize leaf tissues was significantly lower than that of a Bt-susceptible strain. This Bt-resistant D. saccharalis population is the first corn stalk borer species that has completed larval development on commercial Bt-maize. The F2 screening protocol developed in this study could be modified for detecting Bt resistance alleles in other similar corn stalk borers, such as the European corn borer, Ostrinia nubilalis (Hübner), and the southwestern corn borer, D. grandiosella Dyar.

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.

CDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins

Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase-like proteins from both Bt-susceptible RC688 s and Bt-resistant HD198 r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HE XXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51–52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688 s and HD198 r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp 185→Glu 185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt-susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688 s and AF034484 for resistant strain HD198 r).

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.