Identification and functional characterization of doublesex gene in the testis of Spodoptera litura

Abstract

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific Sldsx and female-specific Sldsx isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.

Functional characterization of the cis‐regulatory region for the vitellogenin gene in Plutella xylostella

“…We also found some interesting binding sites such as dsx F and BrC ‐Z2 in PxVgP2 . The dsx gene plays an essential role in sex development and differentiation in Drosophila melanogaster (Shukla and Nagaraju, ), B. mori (Sakai et al ., ), S. litura (Du et al ., ) and P. xylostella (Wang et al ., ). The transcripts of dsx undergo sex‐specific alternative splicing, which produces a female‐specific isoform ( Pxdsx F ) or a male‐specific isoform ( Pxdsx M ) in P. xylostella (Wang et al ., ).…”

P-element Somatic Inhibitor Protein Binding a Target Sequence in dsx Pre-mRNA Conserved in Bombyx mori and Spodoptera litura

“…From an evolutionary perspective, the four conserved KH_1 motifs in SlPSI and CE1 in female specific splicing of Sldsx provide strong evidence of conserved structures for essential components of the sex determination process in Lepidoptera [23]. Furthermore, the conserved interaction of SlPSI and CE1 indicates functional conservation of essential components in the sex determination process in Lepidoptera.…”

Positional Relationships among Male Reproductive Organs in Insects

“…Similarly, in Drosophila melanogaster Meigen, there was a significant reduction in the testes size after five successive matings [25]. In general, testis development is closely related to spermatogenesis [26], and testis size usually increases as spermatogenesis becomes more active [27]. However, the relationship between testis size and spermatogenesis depends on the species and developmental stage.…”