Larval survival and plant injury of Cry1F-susceptible, -resistant,and -heterozygous fall armyworm (Lepidoptera: Noctuidae) on non-Bt and Bt corn containing single or pyramided genes

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target of transgenic corn, Zea mays L., expressing Bacillus thuringiensis (Bt) proteins in both North and South America. A highly Cry1F-resistant strain of S. frugiperda was established from a field collection in Puerto Rico in 2011. In this study, three greenhouse trials were conducted to evaluate larval survival and leaf injury of Cry1F-susceptible, -resistant, and -heterozygous genotypes of S. frugiperda on whole plants of five non-Bt and eight Bt corn hybrids. The Bt corn products included two single-gene Bt corn hybrids containing Herculex^®I (Cry1F) and YieldGard^® (Cry1Ab) traits and six pyramided Bt corn hybrids representing four traits: Genuity^® VT Double Pro™, Genuity^®VT Triple Pro™, Genuity^® SmartStax™, and Agrisure^® Viptera™ 3111. In each trial, neonates of S. frugiperda were placed into the plant whorls at vegetative plant stages (V6–V10). Larvae of the three insect genotypes on non-Bt corn hybrids survived well and caused serious plant injury. Cry1Ab corn was ineffective against all three insect genotypes. On Cry1F corn plants, resistant larvae survived on 72.9% plants after 12–15 d and caused a leaf injury rating (Davis' 1 to 9 scales) of 5.7 after 7 d and 7.6 after 12–15 d. Both the larval survivorship and leaf injury rates of the resistant larvae on Cry1F corn plants were not significantly different from those observed on non-Bt corn hybrids. In contrast, no live larvae and little or no leaf injury were observed on the Cry1F corn plants that were infested with susceptible or heterozygous genotypes, or on the pyramided Bt plants. The results demonstrated that the Cry1F-resistant S. frugiperda was highly resistant to whole plants of Cry1F corn and the resistance was recessive. Hybrids that contained one of the four pyramided Bt traits were effective for managing the Cry1F resistance in S. frugiperda.     

Frequency of Bacillus thuringiensis Cry1A.105 resistance alleles in field populations of the fall armyworm,Spodoptera frugiperda,in Louisiana and Florida

Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major pest of many crops and a cross-crop target of transgenic maize, cotton, and soybean containing Bacillus thuringiensis (Bt) genes. Some of the current Bt maize products for controlling lepidopteran species contain the Bt event MON 89034. The objective of this study was to determine the frequency of resistance alleles in field populations of S. frugiperda collected from Louisiana and Florida, U.S. to Cry1A.105, one of the two Bt genes in MON 89034. A total of 150 F₂ two-parent families of S. frugiperda were established using single-pair mating of field-collected individuals in 2011, which included 79 families from two locations in Louisiana and 71 families from one location in Florida. F₂ screen was conducted to detect resistance alleles in these families to Cry1A.105 protein in maize plants. Four out of the 79 Louisiana and 14 out of the 71 Florida families were identified to possess resistance alleles to the Cry1A.105 maize plants. Thus, the corresponding frequency of resistance alleles to Cry1A.105 maize was estimated to be 0.0158 with a 95% credibility interval (CI) of 0.0052–0.0323 for the Louisiana populations and 0.0559 with a 95% CI of 0.0319–0.0868 for the Florida populations. The resistant families survived on whole Cry1A.105 maize plants and demonstrated a significant level (>116-fold) of resistance to the Cry1A.105 protein in a diet-incorporated bioassay. These findings suggest that resistance allele frequency in S. frugiperda to single-gene Cry1A.105 maize in the U.S. southeast region apparently is not rare, most likely due to the selection of Cry1F resistance and its cross-resistance to Cry1A.105.     

Damage and survivorship of fall armyworm (Lepidoptera: Noctuidae) on transgenic field corn expressing Bacillus thuringiensis Cry proteins

Field corn, Zea mays L., plants expressing Cry1Ab and Cry1F insecticidal crystal (Cry) proteins of Bacillus thuringiensis (Bt) Berliner are planted on considerable acreage across the Southern region of the United States. The fall armyworm, Spodoptera frugiperda (J.E. Smith), is an economically important pest during the mid-to-late season on non-Bt and some commercial Bt corn hybrids. The objective of this study was to quantify foliar injury and survivorship of fall armyworm on transgenic corn lines expressing Cry1Ab or Cry1F Bt proteins. Corn lines/hybrids expressing Cry1Ab, Cry1F, and a conventional non-Bt cultivar were evaluated against artificial infestations of fall armyworm in field trials. Larvae (second instars) of fall armyworm were placed on corn plants (V8-V10 stages). Leaf injury ratings were recorded 14 d after infestation. Hybrids expressing Cry1F had significantly lower feeding injury ratings than non-Bt corn plants. Development and survivorship of fall armyworm on Bt corn lines/hybrids were also evaluated in no-choice laboratory assays by offering freshly harvested corn leaf tissue to third instars. Transgenic corn hybrids expressing Cry1Ab or Cry1F significantly reduced growth, development, and survivorship of fall armyworm compared to those offered non-Bt corn tissue. However, 25-76% of third instars offered Bt corn leaf tissues successfully pupated and emerged as adults. These results suggest Cry1Ab has limited effects on fall armyworm; whereas Cry1F demonstrated significant reductions in foliar injury and lower survivorship compared to that on non-Bt corn tissues. Although fall armyworm is not considered a primary target for insect resistance management by the U.S. Environmental Protection Agency, these levels of survivorship could impact selection pressures across the farmscape, especially when considering that transgenic Bt cotton cultivars express similar Cry (Cry1Ac or Cry1F) proteins.     

Field-evolved resistance to Cry1F maize by Spodoptera frugiperda (Lepidoptera: Noctuidae) in Brazil

The Cry1F protein from Bacillus thuringiensis Berliner expressed in event TC1507 maize (Zea mays L.) was one of the most effective ways to control Spodoptera frugiperda (J. E. Smith) in Brazil. After reports of reduced effectiveness of this Bt maize event in some areas of Brazil, research was undertaken to investigate if damage to Cry1F maize was caused by resistant S. frugiperda. Additional investigations were conducted to evaluate the genetic basis of the resistance and to test if Cry1F resistant S. frugiperda selected from populations of different regions of Brazil share the same resistance locus by using complementation tests. Neonate larvae of S. frugiperda collected from TC1507 maize fields with damage in Western Bahia region in 2011 were able to survive on Cry1F maize plants under laboratory conditions and subsequently produced normal adults. Survival of Cry1F-susceptible S. frugiperda on non-Bt maize was significantly higher in leaf than plant bioassays. Resistance ratio in diet overlay bioassays was >5000-fold. A discriminating concentration of 2000 ng cm⁻² of Cry1F protein was defined for monitoring the frequency of resistance of S. frugiperda to Cry1F. Cry1F resistant S. frugiperda showed a recessive autosomal inheritance for alleles involved in resistance to Cry1F protein. In complementation tests, the resistant population from Western Bahia was crossed with the other seven resistant populations collected from different States of Brazil. F₁ larvae from each cross had the same survival at discriminating concentration of 2000 ng cm⁻² of Cry1F protein, indicating that the resistance alleles in each population were likely at the same locus. Therefore, implementation of resistance management strategies is urgent to prolong the lifetime of Cry1F for controlling S. frugiperda in Brazil.     

Pest resistance to Cry1Ab Bt maize: Field resistance,contributing factors and lessons from South Africa

This paper documents the historical development of resistance of the African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) to Bt maize (Zea mays L.). This pest was one of the first to evolve resistance to Bt maize expressing Cry1Ab protein. A time-line of events and contributing factors are presented, from the commencement of efficacy testing through to the present situation, where the Cry1Ab toxin has lost its efficacy against B. fusca at many localities throughout the maize producing region, and single-gene Bt maize events often require insecticide treatments for which farmers are compensated. Significant levels of pest survival on Bt maize was observed in the first season after commercial release in 1998 and confirmed seven years later. Reduced selection pressure on the target pest is the objective of insect resistance management (IRM), and strategies to accomplish this should receive highest priority. Where resistance is prevalent, the only viable options to reduce selection pressure are withdrawal of the product and/or enforcement of high-dose/refuge requirements. The latter action may however be of no value under conditions where resistance is prevalent, since the value of refugia to an IRM strategy may be compromised. Remedial actions taken in South Africa included the propagation and enforcement of refuge compliance followed by the release of pyramided maize hybrids in 2011. These pyramids combine Cry1A.105 and Cry2Ab2 toxin-producing transgenes, replacing the ineffective single-transgene. However, it remains uncertain if cross-resistance occurs between Cry1A.105/Cry2Ab2 and the closely related Cry1Ab toxin, and for how long this pyramided event will endure. Cultivation of Cry1Ab-expressing hybrids continues in areas where resistance levels have been confirmed to be high. In retrospect, this case provides lessons regarding IRM, not only in South Africa, but wherever Bt crops are being introduced.     

Susceptibility of Cry1Ab-susceptible and -resistant sugarcane borer to transgenic corn plants containing single or pyramided Bacillus thuringiensis genes

Multiple independent trials were conducted to evaluate the performance of Cry1Ab-susceptible (Cry1Ab-SS), -heterozygous (Cry1Ab-RS), and -resistant (Cry1Ab-RR) genotypes of the sugarcane borer, Diatraea saccharalis (F.), on eight commercial hybrids and six experimental corn lines. The commercial varieties included two non-Bt and six Bt corn hybrids that expressed a single Bt protein (either Cry1Ab or Cry1F) targeting above-ground lepidopteran pests. The six experimental lines consisted of two non-Bt and four Bt corn lines, two expressing just the Cry1Ab protein and two containing the pyramided-genes Cry1A.105 and Cry2Ab2 (event MON 89034). Larval mortality on non-Bt corn leaf tissue ranged from 6 to 45% after 12 d across insect genotypes. The 12 d mortality of Cry1Ab-SS on leaf tissue of commercial Cry1Ab or Cry1F corn was 96-100%, whereas it was 80-96% for Cry1Ab-RS and 68-78% for Cry1Ab-RR. On intact plants, 39-64% of larvae survived on non-Bt corn plants after 21-25 d. Larval survivorship on intact plants of commercial Cry1Ab or Cry1F corn was 0-8.1% for Cry1Ab-SS, 1.3-34% for Cry1Ab-RS, and 19-51% for Cry1Ab-RR. Larvae of Cry1Ab-RR and -RS also caused significant plant injury to most of the commercial Bt corn hybrids, especially to the Cry1Ab corn. Cry1Ab resistance in D. saccharalis was incompletely dominant on commercial Bt corn hybrids. However, both experimental lines with pyramided genes of Cry1A.105 and Cry2Ab2 provided complete control of all three insect genotypes in both leaf tissue and intact plant tests. Results of this study suggest that MON 89034 should offer a means for Bt resistance management in D. saccharalis.     

No fitness costs associated with resistance of Busseola fusca (Lepidoptera: Noctuidae) to genetically modified Bt maize

A concern regarding planting of Bt crops is that their widespread cultivation could lead to evolution of insect resistance to Bt toxins. In South Africa, the noctuid maize stem borer (Busseola fusca [Fuller]), is resistant to Bt maize (Zea mays L.; MON810) which produces Cry1Ab protein. The presence of fitness costs in resistant populations could be a valuable component of resistance management since the non-Bt maize refuge may select against resistance. The aim of the study was to determine if there are fitness costs associated with Bt resistance of B. fusca. Life history parameters were compared between individuals of a Bt maize resistant B. fusca population when feeding on Bt or non-Bt maize. Similar comparisons were done using a control population. Field collected larvae as well as their F₁-generation were used in the study. The following parameters were compared: pupal mass, moth longevity, fecundity, fertility, larval mass and survival, and sex ratio. Except for LT50-values, no fitness costs were associated with the resistance trait in the highly resistant B. fusca population. The absence of fitness costs and presence of resistant populations may promote the use of a multi-gene strategy which would be expected to impact negatively on fitness.     

Functional dominance of different aged larvae of Bt-resistant Spodoptera frugiperda (Lepidoptera: Noctuidae) on transgenic maize expressing Vip3Aa20 protein

Fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is the main target pest of transgenic maize expressing insecticidal proteins from Bacillus thuringiensis Berliner (Bt) in Brazil. To optimize resistance management strategies, we evaluated the functional dominance of different aged larvae of Bt-resistant FAW on Vip3Aa20 maize. We measured the survival and development of Vip3Aa20-resistant, -heterozygote, and -susceptible strains on MIR162 (expressing Vip3Aa20) and Bt11 × MIR162 × GA21 (expressing Vip3Aa20 and Cry1Ab) maize. The resistant strain, from neonate to sixth instar, showed more than 72% survival on Vip3Aa20 maize. From surviving larvae, more than 64 and 54% developed to pupae and adults, respectively. In contrast, heterozygote and susceptible strains showed no larval survival up to fourth instar, and less than 25% larval survival in the fifth and sixth instar on Vip3Aa20 maize. These larvae produced less than 21% of pupae and adults. The development time of FAW strains from neonate-to-adult exposed to Vip3Aa20 maize was similar; however, the resistant strain showed an increase of ∼ 2 d when compared to those fed only non-Bt maize. In summary, the resistance of S. frugiperda to Vip3Aa20 maize is functionally recessive from neonate up to fourth instar larvae. However, high larval survival of resistant strain and some survival of heterozygote larvae in advanced instars on Vip3Aa20 maize were observed. These results will be important for designing insect resistance management to Bt maize plants expressing Vip3Aa20 protein in Brazil.     

Prevalence of Helicoverpa zea (Lepidoptera: Noctuidae) on late season volunteer corn in Mississippi: Implications on Bt resistance management

The southern United States has a long growing period between corn, Zea mays L., harvest and first winter frost, so volunteer corn which germinates after harvest has a growing period sufficient for corn earworm, Helicoverpa zea (Boddie) and fall armyworm, Spodoptera frugiperda (J. E. Smith) to feed on these plants. However, lower air temperatures can limit larval development on late season volunteer corn and thereby successful pupation. Here we explore the suitability of late season volunteer corn for larval development and the potential contribution of H. zea larvae to the overwintering population. Our survey revealed the occurrence of volunteer corn in high densities, with monthly mean densities ranging from 56,000 to 143,000 plants ha⁻¹. H. zea larvae were found feeding on both vegetative and reproductive stage plants while S. frugiperda were only found on vegetative stage plants. An analysis of H. zea growing degree day (GDD) accumulations based on Mississippi weather data from 1980 to 2010 revealed that sufficient GDD to reach prepupation would always be accumulated before first frost if oviposition occurred by 9 September, with the probability of successful pupation decreasing rapidly thereafter. However, most of the H. zea larvae were oviposited after this, and could not reach pupation. Because S. frugiperda cannot overwinter in Mississippi, their ability to pupate was not examined. Low suitability of whorl stage corn for H. zea development coupled with low larval densities during this stage effectively diminish the number of larvae that complete development on late season volunteer transgenic corn expressing genes from the soil bacterium, Bacillus thuringiensis (Bt). This limits the Bt resistance risk posed by larvae developing on late season volunteer corn in all but the most southern locations in the US.     

A preliminary study on the effects of a transgenic corn event on the non-target pest Dalbulus maidis (Hemiptera: Cicadellidae)

The inclusion of the cry gene in corn may produce direct effects on non-target pests. Our research was focused on the relationship between Bt corn germplasm, expressing the cry1F protein to control the fall armyworm [Spodoptera frugiperda (Noctuidae)], and a non-target pest, the corn leafhopper [Dalbulus maidis (Cicadellidae)]. The aim of this contribution was to elucidate if Bt corn plants have influence on the oviposition preference of the leafhopper and to evaluate the effect of the transgenic plant on the hatching rate of egg. Female corn leafhoppers were released in cages each containing two potted plants in the V2 stage: a Bt germplasm and the corresponding isogenic hybrid. Laid eggs were counted and the number of hatched nymphs recorded. D. maidis females oviposited and laid more eggs in Bt plants. The egg hatching rate was negatively affected by the Bt germplasm. In addition, a field study was conducted in order to determine the abundance of D. maidis adults in Bt corn and the corresponding non-Bt isoline. Two corn plots sown with the same germplasms as used in the laboratory bioassays were sampled weekly. In the field, the population of the corn leafhopper was higher in the Bt corn plot than in the non-Bt isoline. Possible hypotheses for the differences in abundance of the vector in the field are: a) that pleiotropic effects of Bt corn could attract adults; b) the existence of a possible direct competition between the corn leafhopper and the target pest in order to utilize the whorls of corn plants as refuge and feeding sites, so the high populations of the vector could be due to the large supply of healthy whorls in the transgenic plot; and/or c) a differential attack of natural enemies occurring in non-Bt plots.     

Identification and Expression of Two Novel Cytochrome P450 Genes,CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Cnaphalocrocis medinalis Güenée can cause severe losses in rice. Cytochrome P450s play crucial roles in the metabolism of allelochemicals in herbivorous insects. Two novel P450 cDNAs, CYP6CV1 and CYP9A38, were cloned from the midgut of C. medinalis. CYP6CV1 encodes a protein of 500 amino acid residues, while CYP9A38-predicted protein has 531 amino acid residues. Both cDNA-predicted proteins contain the conserved functional domains for all P450s. Phylogenetic analyses showed that CYP6CV1 is grouped in the cluster containing CYP6B members, while CYP9A38 is in the cluster including CYP9 members. However, both clusters are contained in the same higher lineage. Homologous analysis revealed that CYP6CV1 is most similar to CYP6B8, CYP6B7, CYP6B6, CYP6B2, and CYP6B4 with the highest amino acid identity of 41%. CYP9A38 is closest to CYP9A17, CYP9A21, CYP9A20, and CYP9A19 with the highest amino acid identity of 66%. Studies of temporal expression profiles revealed that CYP9A38 showed a steady increase in mRNA level during the five instar stages, but a low-expression level in pupae, and then presented at a high-expression level again in adults. Similar expression patterns were obtained with CYP6CV1. In the fifth instar larvae, CYP6CV1 was mainly expressed in midgut and fat bodies, whereas CYP9A38 was mainly expressed in midgut. Expression studies also revealed a 3.20-fold over-expression of CYP6CV1 and 3.54-fold over-expression of CYP9A38 after larval exposure to host rice resistance. Our results suggest that both CYP6CV1 and CYP9A38 may be involved in detoxification of rice phytochemicals.