MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate various biological processes including sexual dimorphism. The oriental fruit fly Bactrocera dorsalis is one of the most destructive agricultural insect pests in many Asian countries. However, no miRNAs have been identified from the separate sex and gonads to elucidate sex gonad differentiation in B. dorsalis. In this study, we constructed four small RNA libraries from whole body of females, males (except ovaries and testes) and ovaries, testes of B. dorsalis for deep sequencing. The data analysis revealed 183 known and 120 novel miRNAs from these libraries. 18 female-biased and 16 male-biased miRNAs that may be involved in sexual differentiation were found by comparing the miRNA expression profiles in the four libraries. Using a bioinformatic approach, we predicted doublesex (dsx) as a target gene of the female-biased miR-989-3p which is considered as the key switch gene in the sex determination of tephritid insects. This study reveals the first miRNA profile related to the sex differentiation and gives a first insight into sex differences in miRNA expression of B. dorsalis which could facilitate studies of the reproductive organ specific roles of miRNAs.

Insect gut microbiota has been reported to participate in regulating host multiple biological processes including metabolism and reproduction. However, the corresponding molecular mechanisms remain largely unknown. Recent studies suggest that microRNAs (miRNAs) are involved in complex interactions between the gut microbiota and the host. Here, we used next-generation sequencing technology to characterize miRNA and mRNA expression profiles and construct the miRNA-gene regulatory network in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis. A total of 3016 differentially expressed genes (DEGs) and 18 differentially expressed miR-NAs (DEMs) were identified. Based on the integrated analysis of miRNA and mRNA sequencing data, 229 negatively correlated miRNA-gene pairs were identified from the miRNA-mRNA network. Gene ontology enrichment analysis indicated that DEMs could target several genes involved in the metabolic process, oxidation-reduction process, oogenesis, and insulin signaling pathway. Finally, real-time quantitative polymerase chain reaction further verified the accuracy of RNA sequencing results. In conclusion, our study provides the profiles of miRNA and mRNA expressions under antibiotics treatment and provides an insight into the roles of miRNAs and their target genes in the interaction between the gut microbiota and its host.

Increasing evidence indicates that miRNAs play crucial regulatory roles in various physiological processes of insects, including systemic metabolism. However, the molecular mechanisms of how specific miRNAs regulate energy metabolic homeostasis remain largely unknown. In the present study, we found that an evolutionarily conserved miR-275/305 cluster was essential for maintaining energy metabolic homeostasis in response to dietary yeast stimulation in Bactrocera dorsalis. Depletion of miR-275 and miR-305 by the CRISPR/Cas9 system significantly reduced triglyceride and glycogen contents, elevated total sugar levels, and impaired flight capacity. Combined in vivo and in vitro experiments, we demonstrated that miR-275 and miR-305 can bind to the 3’UTR regions of SLC2A1 and GLIS2 to repress their expression, respectively. RNAi-mediated knockdown of these two genes partially rescued metabolic phenotypes caused by inhibiting miR-275 and miR-305. Furthermore, we further illustrated that the miR-275/305 cluster acting as a regulator of the metabolic axis was controlled by the insulin signaling pathway. In conclusion, our work combined genetic and physiological approaches to clarify the molecular mechanism of metabolic homeostasis in response to different dietary stimulations and provided a reference for deciphering the potential targets of physiologically important miRNAs in a non-model organism.

Background Bactrocera dorsalis is a destructive polyphagous and highly invasive insect pest of tropical and subtropical species of fruit and vegetable crops. The sterile insect technique (SIT) has been used for decades to control insect pests of agricultural, veterinary, and human health importance. Irradiation of pupae in SIT can reduce the ecological fitness of the sterile insects. Our previous study has shown that a gut bacterial strain BD177 that could restore ecological fitness by promoting host food intake and metabolic activities. Results Using long-read sequence technologies, we assembled the complete genome of K. michiganensis BD177 strain. The complete genome of K. michiganensis BD177 comprises one circular chromosome and four plasmids with a GC content of 55.03%. The pan-genome analysis was performed on 119 genomes (strain BD177 genome and 118 out of 128 published Klebsiella sp. genomes since ten were discarded). The pan-genome includes a total of 49305 gene clusters, a small number of 858 core genes, and a high number of accessory (10566) genes. Pan-genome and average nucleotide identity (ANI) analysis showed that BD177 is more similar to the type strain K. michiganensis DSM2544, while away from the type strain K. oxytoca ATCC13182. Comparative genome analysis with 21 K. oxytoca and 12 K. michiganensis strains, identified 213 unique genes, several of them related to amino acid metabolism, metabolism of cofactors and vitamins, and xenobiotics biodegradation and metabolism in BD177 genome. Conclusions Phylogenomics analysis reclassified strain BD177 as a member of the species K. michiganensis. Comparative genome analysis suggested that K. michiganensis BD177 has the strain-specific ability to provide three essential amino acids (phenylalanine, tryptophan and methionine) and two vitamins B (folate and riboflavin) to B. dorsalis. The clear classification status of BD177 strain and identification of unique genetic characteristics may contribute to expanding our understanding of the symbiotic relationship of gut microbiota and B. dorsalis.

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an invasive and polyphagous pest of horticultural crops, and it can cause huge economic losses in agricultural production. The rapid development of CRISPR/Cas9 gene editing technology has provided new opportunities for the scientific control of agricultural pests. Here, we explore the applicability of the B. dorsalis sex peptide receptor (Bdspr) as a target gene for the CRISPR/Cas9-based sterile insect technique (SIT) in B. dorsalis. We screened two high-efficient single guide RNAs (sgRNAs) for gene editing. The results showed that both mutation efficiency and germline transmission rate were 100% in the surviving G0 females (8/8) from injected embryos, and that 75% of mosaically mutated G0 females (6/8) were sterile. The 50% of heterozygous G1 females (4/8) could not lay eggs; 100% of eggs laid by them could not survive; and 62.5% of individual females (5/8) had abnormal ovaries. These results indicate that Bdspr plays an important role in regulating fertility, egg viability, and ovary development in female B. dorsalis, suggesting that the spr gene can be used for CRISPR/Cas9-based SIT in B. dorsalis.