Physiological Alterations in Deletion Mutants of Two Insulin-Like Peptides Encoded in Maruca vitrata Using CRISPR/Cas9.

Most insect species encode multiple insulin-like peptides (ILPs) that exhibit functional overlaps in mediating physiological processes such as development and reproduction. Why do they need multiple ILPs? To address this question, we tested a hypothesis of the requirement of multiple ILPs by generating mutants lacking individual ILP genes using the CRISPR/Cas9 technology. Two ILPs (ILP1 and ILP2) in the legume pod borer, Maruca vitrata, mediate similar physiological processes such as hemolymph sugar level, larval development, and adult reproduction. Individual knock-out mutants (ΔILP1 and ΔILP2) were generated. They showed successful development from larvae to adults. However, they suffered from high hemolymph sugar levels by enhancing trehalose titers in the hemolymph. The hyperglycemic effect was more evident in ΔILP2 mutants than in ΔILP1 mutants. Both mutants showed increased expression of trehalose-6-phosphate synthase but suppressed expression of trehalase. These mutants also showed altered expression patterns of insulin signaling components. Expression levels of insulin receptor and Akt genes were upregulated, while those of FOXO and Target of rapamycin genes were downregulated in these mutants. These alterations of signal components resulted in significant retardation of immature development and reduced body sizes. ΔILP1 or ΔILP2 females exhibited poor oocyte development. Bromo-uridine incorporation was much reduced at the germarium of ovarioles of these mutants compared with wild females. Expression of the vitellogenin gene was also reduced in these mutants. Furthermore, males of these deletion mutants showed impaired reproductive activities when they mated with wild-type females. These results suggest that both ILPs are required for mediating larval development and adult reproduction in M. vitrata.

A Mixture of Bacillus thuringiensis subsp. israelensis With Xenorhabdus nematophila -Cultured Broth Enhances Toxicity Against Mosquitoes Aedes albopictus and Culex pipiens pallens (Diptera: Culicidae).

Xenorhabdus and Photorhabdus spp. (Enterobacteriaceae) can synthesize and release secondary metabolites that play crucial roles in their pathogenicity by suppressing the immunity of target insects. The insect immunity contributes to defense against the pathogenicity of Bacillus thuringiensis (Bt). This study tested a hypothesis that bacterial immunosuppresants could enhance the susceptibility of mosquitoes ( Aedes albopictus and Culex pipiens pallens ) to Bt. Three symbiotic bacteria [ X. nematophila (Xn), X. hominickii (Xh), and P. temperata temperata (Ptt)] were cultured in nutrient broth to allow them to produce secondary metabolites. Bacillus thuringiensis israelensis (BtI) was highly toxic to both culicid mosquitoes with median lethal concentration (LC 50 , spores/ml) of 2.9 × 10 5 and 2.2 × 10 5 at 16 h after treatment, respectively. Addition of each bacteria-cultured broth enhanced BtI toxicity to these mosquito larvae. The LC 50 values of BtI to Ae. albopictus larvae were reduced to 1.5 × 10 5 in Xn mixture, 1.7 × 10 5 in Xh mixture, and 1.9 × 10 5 in Ptt mixture. The LC 50 values of BtI to Cx. pipiens pallens larvae were also reduced to 1.2 × 10 5 in Xn mixture, 1.3 × 10 5 in Xh mixture, and 1.5 × 10 5 in Ptt mixture. Adding benzylideneacetone or oxindole produced from Xn and Ptt also enhanced BtI toxicities to these mosquito larvae. Based on these results, we developed a new mosquitocidal Bt formulation called "Dip-Kill" consisting of 80% Xn-cultured broth, 10% BtI (10 10 spores/ml), and 10% preservative. Dip-Kill at 1,000 ppm was superior to a commercial BtI product at its recommended dose.

Population genetic structure of Aphis glycines.

The soybean aphid (Aphis glycines Matsumura) is an invasive pest of cultivated soybean (Glycine max L.) in North America. After the initial invasion in 2000, the aphid has quickly spread across most of the United States and Canada, suggesting large-scale dispersal and rapid adaptation to new environments. Using microsatellite markers from closely related species, we compared the genetic diversity and the amount of genetic differentiation within and among 2 South Korean and 10 North American populations. Overall allelic polymorphism was low, never exceeding four alleles per locus. However, differences in genetic diversity were seen among South Korean and North American populations in terms of heterozygote excesses and genotypic richness. Within North America, two populations (Michigan and Ontario), had lower genetic diversities and exhibited high genetic differentiation compared with the remaining eight populations. The earlier collection time of Michigan and Ontario samples explained the genetic differences better than geographic subdivisions. These data indicate a pattern of small colonizing populations on soybeans, followed by rapid clonal amplification and subsequent large-scale dispersal across North America.