Identification and profiling of Bactrocera dorsalis microRNAs and their potential roles in regulating the developmental transitions of egg hatching, molting, pupation and adult eclosion.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs (18-25 nt) that are involved in many physiological processes including development, cancer, immunity, apoptosis and host-microbe interactions through post-transcriptional regulation of gene expression. In this study, we measured the profile of small RNAs over the developmental transitions of the oriental fruit fly Bactrocera dorsalis from egg hatching, molting, and pupation to adult eclosion. We identified 250 miRNAs, including 83 known and 167 novel miRNAs, and 47 isomiRNAs. In addition, we identified the miRNAs differentially expressed over the developmental transitions. Interestingly, the miR-309 cluster, the miR-2 cluster/family and the let-7 cluster were among these differentially expressed miRNAs, suggesting a role in the regulation of egg hatching, molting and pupation/adult eclosion, respectively. Moreover, a detailed analysis of the temporal expression patterns of 14 highly expressed miRNAs in the pupal stage revealed three types of expression profiles. Furthermore, injection of a miR-100 mimic in the 3rd instar larvae resulted in a significant decrease in pupation and adult eclosion rates, whereas injection of a miR-317 antagomir resulted in a significant decrease in the pupation rate and a decrease in the pupation time, indicating that miR-100 and miR-317 are involved in the process of pupation. Finally, injection of a miR-100/miR-285 mimic or antagomir in pupae resulted in a significant decrease in the eclosion rate and a significant increase in the prevalence of a partial eclosion phenotype, implying the involvement of miR-100 and miR-285 in the process of adult eclosion. This study identified critical miRNAs involved in the transitions of this important holometabolic model and pest insect B. dorsalis from egg hatching to adult eclosion, thus providing a useful resource for exploring the regulatory role of miRNAs during insect post-embryonic development.

Molecular Characterization and Expression Profiling of Nuclear Receptor Gene Families in Oriental Fruit Fly, Bactrocera Dorsalis (Hendel).

The oriental fruit fly (Bactrocera dorsalis) is a pest that causes large economic losses in the fruit and vegetable industry, so its control is a major challenge. Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that directly combine with DNA to regulate the expression of downstream target genes. NRs are closely associated with multiple physiological processes such as metabolism, reproduction, and development. Through sequence searches and analysis, we identified 21 B. dorsalis NR genes, all of which contained at least one of the two characteristic binding domains. On the basis of the conserved sequences and phylogenetic relationships, we divided the 21 NR genes into seven subfamilies. All members of the NR0 subfamily and BdHR83, which belonged to the NR2E group, lacked ligand-binding domains. The BdDSF and BdHR51, which also belonged to the NR2Egroup, and BdE78 (which belonged to the NR1E group) all lacked DNA-binding domains. The BdDSF and BdHR83 sequences were incomplete, and were not successfully amplified. Development- and tissue-specific expression profiling demonstrated that the transcript levels of the 19 NR genes varied considerably among eggs, larva, pupae, and adults, as well as among larval and adult male and female tissues. Our results will contribute to a better understanding of NR evolution and expand our knowledge of B. dorsalis physiology.

The Transcription Factor MafB Regulates the Susceptibility of Bactrocera dorsalis to Abamectin via GSTz2.

Pesticide resistance is a serious problem that poses a major challenge to pest control. One of the most potent resistance mechanisms is the overexpression of genes coding for detoxification enzymes. The expression of detoxification genes is regulated by a series of transcription factors. Previous studies have revealed that the increased expression of detoxification genes contributes to the insecticide tolerance of Bactrocera dorsalis. Our objective was thus to identify the transcription factors involved in this process. Temporal expression profiles showed that the transcription factor MafB and detoxification genes were expressed highly in the fat body. Further analysis showed that the expression of MafB, GSTz2, and CYP473A3 was induced by abamectin. Disruption of the MafB transcription factor through RNA interference decreased the transcript levels of GSTz2 and CYP473A3 and increased the susceptibility to abamectin significantly. Direct silencing of the expression of GSTz2 also increased susceptibility to abamectin, while CYP473A3 did not. In conclusion, these results suggest that the expression of GSTz2 and CYP473A3 was regulated by the transcription factor MafB, and the up-regulation of GSTz2 via MafB decreased the susceptibility of B. dorsalis to abamectin.

The alternative splicing of BdTai and its involvement in the development of Bactrocera dorsalis (Hendel).

Interest in insect metamorphosis has primarily focused on juvenile hormone (JH) and ecdysone. Compared to ecdysone signaling, the molecular action of JH is less well established because Methoprene-tolerant (Met) as the JH receptor has been identified until recently. In vitro studies have indicated that Met forms an active JH-dependent complex with one partner protein, Taiman (Tai). However, the related studies on Tai's role in insect metamorphosis are very limited. In this study, five Tai isoforms differing in C-terminal region are identified from the oriental fruit fly Bactrocera dorsalis, an important worldwide pest infesting fruits and vegetables. The spatiotemporal expression pattern analysis indicates that BdTai-A and BdTai-B are highly expressed in early larvae while BdTai-D is more abundant in middle-late larvae. Meanwhile, in vivo methoprene stress leads to dramatic expression pattern fluctuation of BdTai isoforms. The subsequent reverse genetic study reveal that all Tai isoforms (denoted as "Tai-core") depletion in larvae stage of B. dorsalis produce precocious larvae-pupae development, i.e. shortened pupation process and miniature pupae. Further knockdown of individual Tai isoform show that silence of BdTai-E causes the same phenotype as of BdTai-core RNAi. The current data suggest that BdTai-E is involved in transducing the JH signal that represses metamorphosis. Besides, isoforms should be considered when studying Tai functions.