Insulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females. A pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis. IIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.

Sterile insect release technique (SIT) is effective for eradicating quarantine insects including various tephritid fruit flies. When SIT is used for fruit flies, it is challenging to remove females from sterile males due to oviposition-associated piercing damage. This study developed a sex transition technique by feeding double-stranded RNA (dsRNA) specific to a sex-determining gene, Transformer-2 (Zs-Tra2) of the striped fruit fly, Zeugodacus scutellata. Zs-Tra2 is homologous to other fruit fly orthologs. It is highly expressed in female adults. RNA interference (RNAi) of Zs-Tra2 by injecting or feeding its specific dsRNA to larvae significantly increased male ratio. Recombinant Escherichia coli cells expressing dsRNA specific to Zs-Tra2 were prepared and used to feed larvae to suppress Zs-Tra2 gene expression levels. When these recombinant bacteria were fed to larvae during the entire feeding stage, the test population was significantly male-biased. Some females treated with such recombinant E. coli exhibited mosaic morphological characters such as the presence of male-specific abdominal setae in females. This study proposes a novel technique by feeding dsRNA specific to Transformer-2 to reduce female production during mass-rearing of tephritid males for SIT.

Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones ofSpodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12^thcarbon position. A desaturase ofS.exigua(SexiDES5) was proposed to have dual functions by forming double bonds at the 11^thand 12^thcarbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion ofSexiDES5using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components inS.exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.

Several endocrine signals mediate mosquito egg development, including 20-hydroxyecdysone (20E). This study reports on prostaglandin E₂ (PGE₂) as an additional, but core, mediator of oogenesis in a human disease-vectoring mosquito, Aedes albopictus. Injection of aspirin (an inhibitor of cyclooxygenase (COX)) after blood-feeding (BF) inhibited oogenesis by preventing nurse cell dumping into a growing oocyte. The inhibitory effect was rescued by PGE₂ addition. PGE₂ was found to be rich in nurse cells and follicular epithelium after BF. RNA interference (RNAi) treatments of PG biosynthetic genes, including PLA₂ and two COX-like peroxidases, prevented egg development. Interestingly, 20E treatment significantly increased the expressions of PG biosynthetic genes, while the RNAi of Shade (which is a 20E biosynthetic gene) expression prevented inducible expressions after BF. Furthermore, RNAi treatments of PGE₂ receptor genes suppressed egg production, even under PGE₂. These results suggest that a signaling pathway of BF-20E-PGE₂ is required for early vitellogenesis in the mosquito.

Several endocrine signals are known to mediate mosquito egg development including insulin-like peptide, 20-hydroxyecdysone, and juvenile hormone. The objective of this study was to determine the effects of prostaglandin E ₂ (PGE ₂ ) as an additional mediator of oogenesis in the mosquitoes, Aedes albopictus and Anopheles gambiae . The injection of aspirin (an inhibitor of cyclooxygenase) shortly after blood-feeding significantly inhibited egg development at choriogenesis in a dose-dependent manner in Ae. albopictus . Moreover, oral administration of aspirin to An. albopictus and An. gambiae also inhibited egg production. The aspirin treatment suppressed expression of the genes ( Yellow-g and Yellow-g2 ) associated with exochorion darkening and led to the production of a malformed egg shell in Ae. albopictus . These inhibitory effects of aspirin on egg development were rescued by the addition of PGE ₂ , confirming the specificity of aspirin in inhibiting prostaglandin production. To validate these results, we identified a putative PGE ₂ receptor (Aa-PGE ₂ R) in Ae. albopictus. Aa-PGE ₂ R expression was highly inducible in adult ovary after blood-feeding. RNA interference of Aa-PGE ₂ R expression resulted in the significant suppression of choriogenesis similar to aspirin treatment, where the addition of PGE ₂ to Aa-PGE ₂ R -silenced females failed to rescue egg production. Together, these results suggest that PG synthesis and signaling are required for egg development across diverse mosquito taxa. Author Summary: Progstaglandins (PGs) play crucial roles in mediating various physiological processes in insects. Aspirin (ASP) inhibits PG biosynthesis and has been used as an anti-inflammatory drug. ASP injection or feeding to mosquitoes of Aedes albopictus or Anopheles gambiae significantly inhibits egg production at chorion formation. This led to significant reduction in fecundity and egg hatchability. PG signal is interrupted by RNA interference (RNAi) of PGE ₂ receptor. The RNAi treatment also gave a similar damage to females in egg production as seen in ASP treatment. Thus, PG signal is required for egg production of these mosquitoes. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information file.