Genes on the Y chromosome play important roles in male sex determination and development. The identification of Y-chromosome-specific genes not only provides a theoretical basis for the study of male reproductive development, but also offers genetic control targets for agricultural pests. However, Y-chromosome genes are rarely characterized due to their high repeatability and high heterochromatinization, especially in the oriental fruit fly. In this study, 1 011 Y-chromosome-specific candidate sequences were screened from 2 to 4 h Bactrocera dorsalis embryo datasets with the chromosome quotient method, 6 of which were identified as Y-chromosome-specific sequences by polymerase chain reaction, including typo-gyf, a 19 126-bp DNA sequence containing a 575-amino acid open reading frame. Testicular deformation and a significant reduction in sperm number were observed after typo-gyf knockdown with RNA interference in embryos. After typo-gyf knockout with clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 in the embryonic stage, the sex ratio of the emergent adults was unbalanced, with far more females than males. A genotype analysis of these females with the Y-chromosome gene MoY revealed no sex reversal. Typo-gyf knockout led to the death of XY individuals in the embryonic stage. We conclude that typo-gyf is an essential gene for male survival, and is also involved in testicular development and spermatogenesis. The identification of typo-gyf and its functional verification provide insight into the roles of Y-chromosome genes in male development.

Regulation of male sexual differentiation by a Y chromosome-linked male determining factor (M-factor) is one of a diverse array of sex determination mechanisms found in insects. By deep sequencing of small RNAs from Bactrocera dorsalis early embryos, we identified an autosomal-derived microRNA, miR-1-3p, that has predicted target sites in the transformer gene ( Bdtra ) required for female sex determination. We further demonstrate by both in vitro and in vivo tests that miR-1-3p suppresses Bdtra expression. Injection of a miR-1-3p mimic in early embryos results in 87–92% phenotypic males, whereas knockdown of miR-1-3p by an inhibitor results in 67–77% phenotypic females. Finally, CRISPR/Cas9-mediated knockout of miR-1-3p results in the expression of female-specific splice variants of Bdtra and doublesex ( Bddsx ), and induced sex reversal of XY individuals into phenotypic females. These results indicate that miR-1-3p is required for male sex determination in early embryogenesis in B. dorsalis as an intermediate male determiner.

: The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most devastating and highly invasive agricultural pests world-wide, resulting in severe economic loss. Thus, it is of great interest to understand the transcriptional changes that occur during the activation of its zygotic genome at the early stages of embryonic development, especially the expression of genes involved in sex determination and the cellularization processes. In this study, we applied Illumina sequencing to identify B. dorsalis sex determination genes and early zygotic genes by analyzing transcripts from three early embryonic stages at 0–1, 2–4, and 5–8 h post-oviposition, which include the initiation of sex determination and cellularization. These tests generated 13,489 unigenes with an average length of 2185 bp. In total, 1683, 3201 and 3134 unigenes had significant changes in expression levels at times after oviposition including at 2–4 h versus 0–1 h, 5–8 h versus 0–1 h, and 5–8 h versus 2–4 h, respectively. Clusters of gene orthology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed throughout embryonic development to better understand the functions of differentially expressed unigenes. We observed that the RNA binding and spliceosome pathways were highly enriched and overrepresented during the early stage of embryogenesis. Additionally, transcripts for 21 sex-determination and three cellularization genes were identified, and expression pattern analysis revealed that the majority of these genes were highly expressed during embryogenesis. This study is the first assembly performed for B. dorsalis based on Illumina next-generation sequencing technology during embryogenesis. Our data should contribute significantly to the fundamental understanding of sex determination and early embryogenesis in tephritid fruit flies, and provide gene promoter and effector gene candidates for transgenic pest-management strategies for these economically important species.

MicroRNAs (miRNAs) regulate various biological processes during insect development; however, their role in larval–pupal development in oriental fruit fly, Bactrocera dorsalis (Hendel) remains unknown. In the current study, we address the biological function of a conserved miRNA, Bdo‐Let‐7 in the regulation of BdE75 gene, which belongs to the ecdysone signaling pathway and participates in the larval–pupal development in B. dorsalis . Using dual luciferase reporter assay in HEK293T cells we show that Bdo‐Let‐7 miRNA interacts with the 3′ untranslated region of BdE75 gene and suppresses its expression. The Bdo‐Let‐7 and BdE75 are also co‐expressed in the larval–pupal stages and in different tissues of B. dorsalis . In in vivo experiments, the injection of Bdo‐Let‐7 agomir and antagomir in third instar larvae down‐ and up‐regulated the expression of BdE75 , respectively. The 20‐hydroxyecdysone (20E) injection assay shows that 20E up‐regulated the expression of Bdo‐Let‐7 on the 5th day of the larvae. Moreover, abnormal pupation and eclosion were observed after larval Bdo‐Let‐7 antagomir injection. Based on these results, we show that Bdo‐Let‐7 regulates the ecdysone signaling pathway through the exact dose of BdE75 gene, and is indispensable for normal larval–pupal development in B. dorsalis .