The genetic-based sterile insect technique (SIT) is an effective and environmentally safe strategy to diminish populations of agricultural and horticultural insect pests. Functional characterization of genes related to male fertility can enhance the genetic-based SIT. Tssk1 has been involved to control male fertility in both mammals and insects. Moreover, Tektin1 has also been revealed to influence male fertility in both human and mammals. These findings suggested that Tssk1 and Tektin1 identified from Bactrocera dorsalis could be required for male fertility in B. dorsalis. In this study, expression profiles of these two genes were studied at different developmental stages and in various tissues of adult males. Remarkably, it was found that Tssk1 and Tektin1 were highly expressed in the testis of mature adult males of B. dorsalis. Furthermore, Tssk1 and Tektin1 genes were downregulated by using the RNA interference (RNAi) method. Fertility assays including egg laying, hatching, and spermatozoa count were also performed to investigate male fertility of B. dorsalis. Results showed that knockdown of Tssk1 and Tektin1 caused male sterility up to 58.99% and 64.49%, respectively. As expected, the total numbers of spermatozoa were also significantly reduced by 65.83% and 73.9%, respectively. These results suggested that male sterility was happened wing to the low number of spermatozoa. In conclusion, we demonstrate that Tssk1 and Tektin1 are the novel agents that could be used to enhance the genetic-based SIT, or their double-stranded RNA (dsRNA) can be used as biopesticides to control the population of B. dorsalis.

RNAi based sterile insect technique (SIT) is an authentic insect management approach but requires proper target genes. During this study, spermless males were developed by interfering with germ cell differentiation and azoospermia related genes. Data demonstrates significant reductions in the target genes expressions (boul, zpg, dsx ^M , fzo and gas8) after oral dsRNAs administration. Knock down of target genes significantly affected the reproductive ability of males and reduced egg-hatching as compared to the control group. Furthermore, different combinations of selected gene dsRNAs (boul + zpg, boul + dsx ^M and zpg + dsx ^M ) were made, which resulted up to 85.40% of male sterility. The most effective combination was selected to prepare different concentrations of dsRNA, 250, 500, 750 and 1000 ng/μl, that caused 18.97%, 38.68%, 58.02% and 85.40% male sterility, respectively. Subsequently, 1000 ng/μl of the same combination of ds-RNAs was used against differently aged adult flies (1, 5, 7, 10 days) which lead to 85.40%, 31.42%, 21.76% and 9.90% male sterility, respectively. SIT developed in this study showed that, boul + zpg combination of dsRNA feeding for 6 hours significantly reduced the number of spermatozoa and viability of sperm in 1-day-old B. dorsalis flies. In short, this study provides an effective SIT technique for long-term B. dorsalis management.

Panonychus citri is one of the most damaging pests of horticultural crops. Conventional control of this pest population through pesticides has led to the enhanced pest resistance. Management of P. citri population through RNAi, is still largely unknown. In oviparous organisms, fabrication and development of yolk protein play a vital role in the reproduction. Vitellin (Vn) is the source of eggs storage that helps in proper functioning of Vitellogenin (Vg) and Vitellogenin receptor (VgR). VgR is very compulsory protein for the development of Vg into oocytes. In the current study, Vg (PcVg) and VgR (PcVgR) genes were studied and their expressions at different developmental stages were quantified by RT-qPCR. Females treated with dsRNA of PcVg and PcVgR genes exhibited reduction in gene expression. Down regulation of target genes significantly effected oviposition and reduced the egg laying capacity up to 48% as compared to control (ds-egfp). Synergistic effect of target gene’s dsRNA was also accessed that reduced the egg laying up to 60.42%. Furthermore, combination of target dsRNA on deutonymph and protonymph also resulted in 67% and 70% reduction in eggs, respectively. Synergistic effect of dsRNA at 1000 ng/ul resulted in longer life span as compared to control treatments. This study suggests to develop a new strategy of P. citri population control by reducing its reproduction.

Chitin synthase 1 (CHS1) is an essential gene regulating chitin during different developmental stages of arthropods. In the current study, we explored for the first time the role of CHS1 gene regulation in the citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), by silencing its expression using (RNA interference) RNAi-based strategies. The results reveal that P. citri tested in different developmental stages, including larvae, protonymphs, deutonymphs, and adults fed on sweet orange leaves dipped in various concentrations (200, 400, 600, and 800 ng/μL) of dsRNA-PcCHS1, resulted in a continuous reduction in their gene expression, and the extent of transcript knockdown was positively correlated with the concentration of dsRNA. Concentration–mortality response assays revealed a mortality of more than 50% among all the studied developmental stages, except for adulthood. Furthermore, the target gene dsRNA-PcCHS1 treatment of larvae, protonymphs, deutonymphs, and females at a treatment rate of 800 ng/mL of dsRNA significantly decreased the egg-laying rates by 48.50%, 43.79%, 54%, and 39%, respectively, and the hatching rates were also considerably reduced by 64.70%, 70%, 64%, and 52.90%, respectively. Moreover, using the leaf dip method, we found that the RNA interference effectively reduced the PcCHS1 transcript levels by 42.50% and 42.06% in the eggs and adults, respectively. The results of this study demonstrate that the RNAi of PcCHS1 can dramatically reduce the survival and fecundity of P. citri, but the dsRNA concentrations and developmental stages can significantly influence the RNAi effects. These findings indicate the potential utility of the PcCHS1 gene in causing developmental irregularities, which could aid in the development of effective and novel RNAi-based strategies for controlling P. citri.

Natural pathogen pressure is an important factor that shapes the host immune defense mechanism. The current study primarily aimed to explore the molecular basis of the natural immune defense mechanism of a sporadic pest, Gryllus bimaculatus, during swarming by constructing cDNA libraries of the female mid-gut, male mid-gut, testes, and ovaries. The Illumina HiSeq platform generated an average of 7.9 G, 11.77 G, 10.07 G, and 10.07 G bases of outputs from the male mid-gut, female mid-gut, testes, and ovaries and libraries, respectively. The transcriptome of two-spotted field crickets was assembled into 233,172 UniGenes, which yielded approximately 163.58 million reads. On the other hand, there were 43,055 genes in common that were shared among all the biological samples. Gene Ontology analysis successfully annotated 492 immune-related genes, which comprised mainly Pattern Recognition Receptors (62 genes), Signal modulators (57 genes), Signal transduction (214 genes), Effectors (36 genes), and another immune-related 123 genes. In summary, the identified wide range of immune-related genes from G. bimaculatus indicates the existence of a sophisticated and specialized broad spectrum immune mechanism against invading pathogens, which provides, for the first time, insights into the molecular mechanism of disease resistance among two-spotted field crickets.