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Cotton, Gossypium hirsutum (L.), plants expressing insecticidal crystal (Cry) proteins of Bacillus thuringiensis (Bt) Berliner are planted on significant acreage across the southern region of the United States. Fall armyworm, Spodoptera frugiperda (J. E. Smith), can be a significant cotton pest in some years, but this species has not been a primary target of Bt cotton technologies. The objective of this study was to quantify fall armyworm larval survivorship and fruiting form injury on transgenic cotton lines expressing Cry1Ac (Bollgard), Cry1Ac+Cry2Ab (Bollgard II), and Cry1Ac+Cry1F (WideStrike) Bt proteins. Larval survivorship and fruiting form damage of fall armyworm on Bollgard, Bollgard II, WideStrike, and non-Bt (control) cotton lines were evaluated in no-choice field studies. Fall armyworm (third instars) were placed on flower buds (squares), white flowers, and bolls, enclosed within a nylon mesh exclusion cage, and evaluated at selected intervals after infestation. Exposure of fall armyworm larvae to Bollgard cotton lines generally resulted in no significant effects on survivorship compared with larvae exposed to the non-Bt cotton line. Survivorship and plant injury by fall armyworm on Bollgard II cotton lines was variable compared with that on non-Bt cotton lines, and significant differences between treatments were inconsistent. Fall armyworm had significantly lower survivorship and caused less plant injury on WideStrike cotton lines than on non-Bt cotton lines across all plant structures. Development and survivorship of fall armyworm larvae on these cotton lines also were evaluated in no-choice laboratory assays by offering the previously described fruiting forms to third instars. Bollgard II and WideStrike cotton lines significantly reduced fall armyworm development and survivorship compared with those larvae offered non-Bt tissue. These results suggest that differences exist among selected Bt cotton technologies in their performance against fall armyworm., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The sugarcane borer, Diatraea saccharalis (F.), is a major target species of transgenic corn expressing Bacillus thuringiensis (Bt) proteins in South America and the U.S. mid-south region. In this study, the fitness of seven insect genotypes of D. saccharalis were assayed on non-toxic diet, which included a Cry1Ab-susceptible strain (SS-2009), two Cry1Ab-resistant strains (RR-43A(BC), RR-L5B(BC)), and four F1 hybrids (F1-R43A(m)S(f), F1-R43A(f)S(m), F1-R5B(m)S(f), and F1-R5B(f)S(m)). The F1 hybrids were generated by reciprocal crosses of SS-2009 with RR-43ABC and RR-L5BBC, respectively. Biological parameters measured were neonate-to-pupa survivorship, neonate-to-pupa development time, pupal mass, pupa-to-adult emergence rate, and progeny (neonates) production. The overall performance of the two resistant strains and the four F1 genotypes was either similar or better than SS-2009 for all biological parameters measured, suggesting a lack of fitness costs associated with the Cry1Ab resistance traits in both RR-43A(BC) and RR-L5B(BC). In addition, resistance stability was evaluated by measuring the Cry1Ab susceptibility of RR-43A(BC) and RR-L5B(BC) in the absence of selection pressure. Laboratory bioassays showed that larval mortality of the two resistant strains did not significantly increase after selection pressure was removed for 16 generations across all Cry1Ab concentrations assayed. The results provide valuable information on assessing resistance risk and developing effective management strategies for the sustainable use of Bt corn technology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes., (Published 2012. This article is a U.S. Government work and is in the public domain in the USA.)

Transgenic crops that produce Bacillus thuringiensis (Bt) toxins are grown widely for pest control, but insect adaptation can reduce their efficacy. The genetically modified Bt toxins Cry1AbMod and Cry1AcMod were designed to counter insect resistance to native Bt toxins Cry1Ab and Cry1Ac. Previous results suggested that the modified toxins would be effective only if resistance was linked with mutations in genes encoding toxin-binding cadherin proteins. Here we report evidence from five major crop pests refuting this hypothesis. Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of Plutella xylostella and Ostrinia nubilalis in which resistance was not linked with cadherin mutations. Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains.

The sugarcane borer, Diatraea saccharalis, is a major target pest of transgenic corn expressing Bacillus thuringiensis (Bt) proteins (i.e., Cry1Ab) in South America and the mid-southern region of the United States. Evolution of insecticide resistance in such target pests is a major threat to the durability of transgenic Bt crops. Understanding the pests' resistance mechanisms will facilitate development of effective strategies for delaying or countering resistance. Alterations in expression of cadherin- and alkaline phosphatase (ALP) have been associated with Bt resistance in several species of pest insects. In this study, neither the activity nor gene regulation of ALP was associated with Cry1Ab resistance in D. saccharalis. Total ALP enzymatic activity was similar between Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-RR) strains of D. saccharalis. In addition, expression levels of three ALP genes were also similar between Cry1Ab-SS and -RR, and cDNA sequences did not differ between susceptible and resistant larvae. In contrast, altered expression of a midgut cadherin (DsCAD1) was associated with the Cry1Ab resistance. Whereas cDNA sequences of DsCAD1 were identical between the two strains, the transcript abundance of DsCAD1 was significantly lower in Cry1Ab-RR. To verify the involvement of DsCAD1 in susceptibility to Cry1Ab, RNA interference (RNAi) was employed to knock-down DsCAD1 expression in the susceptible larvae. Down-regulation of DsCAD1 expression by RNAi was functionally correlated with a decrease in Cry1Ab susceptibility. These results suggest that down-regulation of DsCAD1 is associated with resistance to Cry1Ab in D. saccharalis.

Aminopeptidase N (APN) proteins located at the midgut epithelium of some lepidopteran species have been implicated as receptors for insecticidal proteins from Bacillus thuringiensis. cDNAs of three APN isoforms, DsAPN1, DsAPN2, and DsAPN3, from Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-RR) strains of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), were identified and sequenced using reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA end (5' RACE). The characteristic APN sequence features were derived from deduced amino acid sequences of the cloned cDNAs. cDNA sequences of the three APN genes were identical between the Cry1Ab-SS and -RR strains. However, total APN proteolytic activity and gene expression of the three APNs from Cry1Ab-RR larvae were significantly lower than those of the Cry1Ab-SS strain. RNA interference (RNAi) was employed using an oral droplet feeding technique for the three APNs of the Cry1Ab-SS strain. Down-regulating expressions of the three APN genes by RNAi were corresponding to the reductions in the specific APN activity. In addition, silencing of all three APNs in D. saccharalis in vivo by RNAi resulted in a decrease in Cry1Ab susceptibility. Our results showed that reduction in expression of the three APNs is functionally associated with the Cry1Ab resistance in D. saccharalis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Transgenic Bacillus thuringiensis (Bt) corn, Zea mays L., has been widely used to manage a corn borer complex in the mid-southern region of the United States. The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), has become a dominant cornstalk boring species in some areas of this region, especially in Louisiana. Therefore, management of sugarcane borer resistance to Bt corn is critical to ensure the long-term sustainability of Bt corn for the region. This study screened 280 two-parent family-lines of sugarcane borer from four geographical populations in Louisiana during 2005 to determine whether Bt resistance allele frequency in sugarcane borer is sufficiently low to meet the rare resistance assumption of the current "high dose/refuge" resistance management strategy for Bt corn. These sugarcane borer family-lines were examined for Bt resistance by using novel F2 screening procedures. No major Bt resistance alleles were detected in these four populations. The estimated frequency of major Bt resistance alleles was < 0.0027, with a 95% probability and a detection power of 94%. The estimated minor resistance allele frequency was 0.0063, with a 95% CI of 0.0025-0.0117. During a previous study, a major Bt resistance allele was detected in one individual from 213 family-lines of another Louisiana population of sugarcane borer. Combining these data with the current screen, the frequency of major Bt resistance alleles across the five populations was 0.001, with a 95% credibility interval of 0.0001-0.0028 and a detection power of 95%. Major Bt resistance allele frequencies in Louisiana sugarcane borer populations seem to be low, and they should support the rare resistance allele requirement of the high dose/refuge strategy.

A Louisiana strain of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), was selected for resistance to the CrylAb protein of Bacillus thuringiensis (Bt) by using an F2 screening procedure. Survival of Bt-resistant, -susceptible, and -heterozygous genotypes of sugarcane borer was evaluated on vegetative and reproductive stages of five non-Bt and seven Bt field corn, Zea mays L., hybrids in a greenhouse study. Larval survival was recorded 21 d after infestation of neonates on potted plants. Larval survival across the three sugarcane borer genotypes and five non-Bt corn hybrids after 21 d ranged from 23.6 +/- 5.2% (mean +/- SEM) to 57.5 +/- 5.2%. Mean survival of Cry1Ab-resistant larvae on vegetative and reproductive plant stages was 12 and 21%, respectively. During the vegetative stages, all seven Bt corn hybrids were highly efficacious against Cry1Ab-susceptible and -heterozygous genotypes of sugarcane borer, with a larval survival rate of <2% for the Bt-susceptible genotype and < or =5% for the heterozygotes. However, 8-18% of the heterozygous genotype survived on reproductive stage plants for four of the seven Bt corn hybrids tested. The variation in performance of Bt corn cultivars at vegetative and reproductive growth stages against Cry1Ab resistant sugarcane borer suggests differential seasonal expression that may hasten resistance in the field. Bt corn hybrids expressing a "high dose" for European corn borer, Ostrinia nubilalis (Hübner), may not produce a sufficient high dose for the sugarcane borer.

Transgenic maize, Zea mays L., expressing the Bacillus thuringiensis (Bt) CrylAb toxin has been planted to extensive areas across the United States and several other countries, but no resistance has been documented in field populations oflepidopteran target pests. This article describes the first report of resistance alleles to commercially available Cry1Ab Bt maize in a Louisiana population of sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae). Two hundred thirteen two-parent isolines of D. saccharalis were screened for Cry1Ab resistance on Bt maize leaf tissue using an F2 screening technique. Larvae representing three isolines survived >15 d on Bt tissue in the F2 generation. The second generation backcross progeny (B1F2) derived from isoline 52 completed larval development on Bt maize in the greenhouse. Segregation and resistance frequency analysis associated with isoline 52 suggested that Bt resistance is probably determined by a nearly completely recessive allele at a single locus. With this assumption, the estimated resistance allele frequency in this population is 0.0023, within a 95% confidence interval of 0.0003-0.0064.

One field strain each of the European corn borer, Ostrinia nubilalis (Hübner); southwestern corn borer, Diatraea grandiosella Dyar; and sugarcane borer, Diatraea saccharalis (F.); were collected from cornfields in northeastern Louisiana. Susceptibilities of the field strain and a corresponding laboratory strain of the three borer species to Cry1Ab protein in DK69-70 Bacillus thuringiensis (Bt) corn hybrid were determined by exposing neonates to intact leaf tissues from whorl stage plants or by feeding neonates or third instars on a meridic diet treated with different concentrations of Cry1lAb protein extracted from Bt corn leaves. Mortality and growth of larvae were evaluated after 2 and 4 d posttreatment in the bioassays by using intact leaf tissues or after 7 d in the bioassays by using diet incorporating Cry1Ab protein. D. saccharalis was the least susceptible species to Cry1Ab protein among the three species, followed by D. grandiosella, whereas O. nubilalis was most susceptible. The 2-d mortality of D. saccharalis neonates on intact Bt leaf tissues was lower than that of O. nubilalis and D. grandiosella. All neonates of O. nubilalis were killed on the diet treated with Cry1Ab protein at 0.5 and 1 mg/kg. The mortality of D. grandiosella was > 75% at 1 mg/kg, but it was < 6% for D. saccharalis at 1 mg/kg. The LC50 values of D. saccharalis were 3- and 11-fold higher than those of D. grandiosella and O. nubilalis, respectively. The LC90 values of D. saccharalis were 8- and 32-fold higher than those of D. grandiosella and O. nubilalis, respectively. Larval growth of the three species on Cry1Ab-treated diet was inhibited, but the inhibition was greater for O. nubilalis and D. grandiosella than for D. saccharalis. The lower susceptibility of D. saccharalis to Cry1Ab protein suggests that it is necessary to verify if a high-dose Bt corn for O. nubilalis and D. grandiosella is also a high dose for D. saccharalis.

The efficacy of transgenic corn hybrids expressing an insecticidal Bacillus thuringiensis (Bt) delta-endotoxin from different transformation events was evaluated in field corn, Zea mays L., against the southwestern corn borer, Diatraea grandiosella Dyar, and sugarcane borer, Diatraea saccharalis (F.). Susceptibilities of neonates and third instars were determined on Bt and non-Bt corn plants (V6 and R1 stages) in field plots and corn leaf tissue feeding exposure in laboratory bioassays. Bt corn hybrids associated with MON810 and CBH351 transformation events sustained significantly less injury by southwestern corn borer and sugarcane borer during mid-whorl stage infestations compared with their respective non-Bt hybrid equivalents. Southwestern corn borer and sugarcane borer feeding injury to ear leaf-sheath and husk tissues during the silking stage of corn was significantly reduced in MON810 and CBH351 Bt corn compared with their respective non-Bt hybrids. However, resistance levels to feeding injury in Bt hybrids associated with the MON810 event were significantly higher than that in the hybrid associated with the CBH351 event. Southwestern corn borer and sugarcane borer caused more feeding injury to husk tissue than to ear leaf-sheath tissue in both Bt and non-Bt hybrids infested during the silking stage. Laboratory performance of the MON810 event against southwestern corn borer and sugarcane borer varied among hybrids associated with the same event. Third instars of southwestern corn borer were highly susceptible to MON810 Bt corn hybrids in leaf tissue experiments. However, sugarcane borer larvae were susceptible to the MON810 event only in one of the Bt hybrids evaluated. Sugarcane borer mortality was significantly lower after 96 h of feeding exposure on CBH351 Bt corn leaf tissue than on MON810 Bt corn leaf tissue. Plant resistance to southwestern corn borer and sugarcane borer increased as plants matured, independent of the presence of a Bt construct. These results are essential to estimate the importance of Bt transgenic corn in areas of southern United States and other areas where mixed populations of southwestern corn borer and sugarcane borer are predominant and cause severe damage to corn production.