Paula A. Price

F2 screen for resistance to Bacillus thuringiensis Cry2Ab2-maize in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) from the southern United States.

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target pest of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established using single-pair mating of field individuals collected from seven locations in four states of the southern U.S.: Texas, Louisiana, Georgia, and Florida. The objective of the investigation was to detect resistance alleles in field populations to Cry2Ab2, a common Bt protein produced in transgenic maize and cotton. For each F2 family, 128 F2 neonates were screened on leaf tissue of Cry2Ab2 maize plants in the laboratory. A conservative estimate of the frequency of major Cry2Ab2 resistance alleles in S. frugiperda from the four states was 0.0023 with a 95% credibility interval of 0.0003-0.0064. In addition, six families were considered to likely possess minor resistance alleles at a frequency of 0.0082 with a 95% credibility interval of 0.0033-0.0152. One F2 family from Georgia (GA-15) was confirmed to possess a major resistance allele to the Cry2Ab2 protein. Larvae from this family survived well on whole maize plants expressing Cry2Ab2 protein and demonstrated a significant level (>15-fold) of resistance when fed with the same protein incorporated in a meridic diet. The detection of the major resistance allele along with the relatively abundant minor resistance alleles revealed in this study may have important implications for resistance management.

Cry3Bb1-Resistant Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte) Does Not Exhibit Cross-Resistance to DvSnf7 dsRNA

Background and Methodology There is a continuing need to express new insect control compounds in transgenic maize against western corn rootworm, Diabrotica virgifera virgifera (LeConte) (WCR). In this study three experiments were conducted to determine cross-resistance between the new insecticidal DvSnf7 dsRNA, and Bacillus thuringiensis (Bt) Cry3Bb1; used to control WCR since 2003, with field-evolved resistance being reported. Laboratory susceptible and Cry3Bb1-resistant WCR were evaluated against DvSnf7 dsRNA in larval diet-incorporation bioassays. Additionally, the susceptibility of seven field and one field-derived WCR populations to DvSnf7 (and Cry3Bb1) was assessed in larval diet-overlay bioassays. Finally, beetle emergence of laboratory susceptible and Cry3Bb1-resistant WCR was evaluated with maize plants in the greenhouse expressing Cry3Bb1, Cry34Ab1/Cry35Ab1, or DvSnf7 dsRNA singly, or in combination. Principal Findings and Conclusions The Cry3Bb1-resistant colony had slight but significantly (2.7-fold; P<0.05) decreased susceptibility to DvSnf7 compared to the susceptible colony, but when repeated using a field-derived WCR population selected for reduced Cry3Bb1 susceptibility, there was no significant difference (P<0.05) in DvSnf7 susceptibility compared to that same susceptible population. Additionally, this 2.7-fold difference in susceptibility falls within the range of DvSnf7 susceptibility among the seven field populations tested. Additionally, there was no correlation between susceptibility to DvSnf7 and Cry3Bb1 for all populations evaluated. In greenhouse studies, there were no significant differences (P<0.05) between beetle emergence of susceptible and Cry3Bb1-resistant colonies on DvSnf7 and Cry34Ab1/Cry35Ab1, and between DvSnf7 and MON 87411 (DvSnf7 + Cry3Bb1) for the Cry3Bb1-resistant colony. These results demonstrate no cross-resistance between DvSnf7 and Cry3Bb1 against WCR. Therefore, pyramiding DvSnf7 with Bt proteins such as Cry3Bb1 and Cry34Ab1/Cry35Ab1 will provide a valuable IRM tool against WCR that will increase the durability of these Bt proteins. These results also illustrate the importance of using appropriate bioassay methods when characterizing field-evolved resistant WCR populations.

Fitness costs and inheritance of Cry2Ab2 resistance in Spodoptera frugiperda (J.E. Smith)

Cry2Ab2 is a Bacillus thuringiensis (Bt) protein expressed in transgenic corn and cotton targeting above-ground lepidopteran pests including the fall armyworm, Spodoptera frugiperda (J.E. Smith). The objective of this study was to characterize fitness costs and inheritance of Cry2Ab2 resistance in S. frugiperda. To determine if fitness costs were associated with the resistance, life history parameters (larval survival, growth, development and egg production) of Cry2Ab2-resistant, -susceptible, and two reciprocal F1 colonies of S. frugiperda were assayed on non-toxic diet and non-Bt corn leaf tissue. The results showed that there were no significant differences among the four insect colonies for all the biological parameters measured with few exceptions, suggesting that the resistance in the colony was not associated with significant fitness costs in the test conditions. To examine the inheritance of resistance, susceptibilities of the resistant and susceptible parents, as well as eight additional colonies generated from various genetic crosses, were assayed using Cry2Ab2-treated diet and Cry2Ab2 corn leaf tissue. The Cry2Ab2 resistance in S. frugiperda in the colony was inherited as a single autosomal recessive or incompletely recessive gene. The results of the study suggest a potential risk of resistance development in S. frugiperda to the Cry2Ab2 protein and thus effective management strategies should be implemented for the sustainable use of the Bt corn technology for pest management.

Performance of Cry1Ab-susceptible and -heterozygous resistant populations of sugarcane borer in sequential feedings on non-Bt and Bt maize plant tissue

A seed mix strategy has been used to provide refuge to susceptible insects for resistance management in planting transgenic maize expressing Bacillus thuringiensis Berliner (Bt) proteins. To determine whether larval movement in a seed mix planting creates favorable conditions for resistant heterozygotes of a target pest, performance of Cry1Ab‐susceptible (SS) and ‐heterozygous resistant (RS) populations of the sugarcane borer, Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), was evaluated in sequential feeding on non‐Bt and Cry1Ab (event MON 810) maize plant tissue. For each insect population, nine feeding sequences were employed. SS and RS feeding on non‐Bt plants for their entire larval stages survived well and >60% of the adult pairs produced viable eggs, with an average of 269 progeny per female, whereas none of the two populations on Bt maize plants survived to the pupal stage. SS larvae could not develop to adults if the larvae fed on non‐Bt plants for ≤15 days and then moved to Bt plants. In contrast, 4.2–29.2% of RS larvae that fed on non‐Bt plants for ≥9 days and then moved to Bt plants developed to adults, and 63.6% of pairs of these adults produced viable eggs, with an average of 185 progeny per female. For SS larvae that fed on Bt plants for 1 or 2 days and then moved to non‐Bt plants, few larvae developed to adults with varied emergence times, whereas 28.1 and 13.5% RS larvae feeding on Bt plants for 1 and 2 days, respectively, successfully developed to adults; 43.8% of pairs of these adults produced viable eggs, with an average of 220 progeny per female. For the case of the single Bt gene maize plants (event MON 810), the results suggest that RS insects may have advantages in survival and reproduction over SS if RS larvae hatch and feed on Bt plants during the first 1 or 2 days and then move to non‐Bt plants. This advantage is less for RS larvae that hatch and feed on non‐Bt plants first and then move to Bt plants, unless the larval movement occurs in the later stages (e.g., fourth or fifth instars).

Fitness costs of Vip3A resistance in Spodoptera frugiperda on different hosts: Fitness costs of Vip3A resistance in S. frugiperda

BACKGROUND Fall armyworm, Spodoptera frugiperda, is a major target pest of the Vip3A protein. The fitness of Bacillus thuringiensis (Bt)-resistant insects on refuges is an important factor in determining the speed of resistance development. Fitness costs associated with Bt resistance can vary among host plants. Here, we provide the first experimental evaluation of the interactions between non-Bt hosts and fitness costs of Vip3A resistance in S. frugiperda. RESULTS Laboratory bioassays showed that survivorship, pupal weight, sex ratio, developmental time, fecundity, net reproductive rate, and intrinsic rate were not different among Vip3A-resistant (RR), -susceptible (SS) and -heterozygous (R♂ S♀ and R♀ S♂ ) strains of S. frugiperda on non-Bt corn, non-Bt cotton, and non-Bt diet. R♂ S♀ and R♀ S♂ also showed no differences on sorghum relative to SS. However, compared to SS, RR on sorghum showed significant reduction in pupal weight, and took longer time to develop to pupa and adult. CONCLUSION These findings suggest that recessive fitness costs of reduced pupal weight and growth rate were detected at the individual level in the Vip3A resistant populations of S. frugiperda on sorghum. However, at the population level in terms of net reproductive rate and intrinsic rate, fitness costs of Vip3A resistance was not evident in S. frugiperda.

Performance of Bt-susceptible and -heterozygous genotypes of Spodoptera frugiperda (J.E. Smith) possessing single- or dual-gene resistance alleles in sequential feedings of non-Bt and Cry1A.105/Cry2Ab2 maize leaf tissues

We simulated larval feeding behavior in seed blends of non-Bt and Bt maize to determine if seed blends create more favorable conditions for heterozygous-resistant insects over their Bt-susceptible counterparts. Survival, growth, development, and progeny production of four genotypes of the fall armyworm, Spodoptera frugiperda, Bt-susceptible (aabb), Cry1A.105 heterozygous resistant (Aabb), Cry2Ab2 heterozygous resistant (aaBb), and Cry1A.105/Cry2Ab2 heterozygous resistant (AaBb), were evaluated in eight feeding sequences (Seq 1-8) of non-Bt and MON89034 Bt maize leaf tissue expressing the Cry1A.105 and Cry2Ab2 proteins. We report variation in the performance of the four genotypes across the feeding sequences and biological parameters measured. Three heterozygous genotypes generally outperformed the susceptible genotype in larval survival, pupation rate, pupal weight, and progeny production. The performance was greater for Aabb over aaBb, AaBb over Aabb or aaBb, in two of the feeding sequences. The findings of this study could have important implications in assessing the risk of seed blends as refuge plantings for Bt crop resistance management where resistance in the target pest is not functionally recessive.