To delay evolution of pest resistance to transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt), the "pyramid" strategy uses plants that produce two or more toxins that kill the same pest. We conducted laboratory diet experiments with the cotton bollworm, Helicoverpa armigera, to evaluate cross-resistance and interactions between two toxins in pyramided Bt cotton (Cry1Ac and Cry2Ab). Selection with Cry1Ac for 125 generations produced 1000-fold resistance to Cry1Ac and 6.8-fold cross-resistance to Cry2Ab. Selection with Cry2Ab for 29 generations caused 5.6-fold resistance to Cry2Ab and 61-fold cross-resistance to Cry1Ac. Without exposure to Bt toxins, resistance to both toxins decreased. For each of the four resistant strains examined, 67 to 100% of the combinations of Cry1Ac and Cry2Ab tested yielded higher than expected mortality, reflecting synergism between these two toxins. Results showing minor cross-resistance to Cry2Ab caused by selection with Cry1Ac and synergism between these two toxins against resistant insects suggest that plants producing both toxins could prolong the efficacy of Bt cotton against this pest in China. Including toxins against which no cross-resistance occurs and integrating Bt cotton with other control tactics could also increase the sustainability of management strategies.

Developing oocytes accumulate plentiful yolk protein during oogenesis through receptor-mediated endocytosis. The vitellogenin receptor (VgR), belonging to the low-density lipoprotein receptor (LDLR) family, regulates the absorption of yolk protein. In this work, the full-length vitellogenin receptor (HaVgR) in the cotton bollworm Helicoverpa armigera was identified, encoding a 1817 residue protein. Sequence alignment revealed that the sequence of HaVgR contained all of the conservative structural motifs of LDLR family members, and phylogenetic analysis indicated that HaVgR had a high identity among Lepidoptera and was distinct from that of other insects. Consistent with other insects, HaVgR was specifically expressed in ovarian tissue. The developmental expression pattern showed that HaVgR was first transcribed in the newly metamorphosed female adults, reached a peak in 2-day-old adults and then declined. Western blot analysis also revealed an ovarian-specific and developing expression pattern, which was consistent with the HaVgR mRNA transcription. Moreover, RNAi-mediated HaVgR knockdown strongly reduced the VgR expression in both the mRNA and protein levels, which inhibited the yolk protein deposition in the ovaries, led to the dramatic accumulation of vitellogenin and the up-regulation of HaVg expression in hemolymph, and eventually resulted in a declined fecundity. Together, all of these findings demonstrate that HaVgR is a specific receptor in uptake and transportation of yolk protein for the maturation of oocytes and that it plays a critical role in female reproduction.

Insects in the same taxonomic group generally have similar responses to light at various wavelengths in the laboratory. However, there is lack of direct evidence of between-group differences in insect responses to various light wavelengths under field conditions. During 2014 and 2015, we evaluated the relative attractiveness of LEDs with 19 single wavelengths to three pest orders and four natural predator orders in cotton fields. The average numbers of Lepidoptera, Hemiptera, Coleoptera, and total pests captured by traps with a 395-nm LED wavelength were higher than those for all others, except 440-nm wavelength captured the largest number of Hemiptera in 2015. For natural enemies, the average numbers of Coleoptera, Neuroptera, and total natural enemies were the largest in traps with a 572-nm LED wavelength, except 538-nm wavelength captured the largest number of Coleoptera in 2014. In general, the ratio of pests to natural enemies captured in the 395-nm wavelength LED trap was significantly more than all others. These results demonstrated that insects in different taxonomic groups have significantly different responses to light at various wavelengths under field conditions; these results will provide insights for in-depth studies on insect phototaxis and guide the long-term monitoring of insects in different groups.

A pyramid strategy combining the Cry1A and Cry2A toxins in Bt crops has been widely used throughout the world to delay pest adaption to transgenic crops and broaden the insecticidal spectrum. Midgut membrane-bound cadherin (CAD), aminopeptidase-N (APN) and alkaline phosphatase (ALP) are important for Cry1A toxicity in some lepidopteran larvae, but the proteins that bind Cry2A in the midgut of target insects and their role in the Cry2A mechanism of action are still unclear. In this study, we found that heterologously expressed CAD, APN4 and ALP2 peptides from the midgut of Helicoverpa armigera could bind to the Cry2Aa toxin with a high affinity. Additionally, the efficiency of Cry2Aa insecticidal activity against H. armigera larvae was obviously reduced after the genes encoding these proteins were silenced with specific siRNAs: CAD- and ALP2-silenced larvae showed significantly similar reductions in mortality due to the Cry2Aa toxin (41.67% and 43.06%, respectively), whereas a larger reduction in mortality was observed in APN4-silenced larvae (61.11%) than in controls. These results suggest that CAD, APN4 and ALP2 are involved in the mechanism of action of Cry2Aa in H. armigera and may play important functional roles in the toxicity of the Cry2Aa toxin.