Characterization of gonadal development phases and maturation mechanisms in male Pacific whiteleg shrimp (Litopenaeus vannamei)

A comprehensive understanding of gonad development in the Pacific whiteleg shrimp, Litopenaeus vannamei, is vital for cultivating high-quality male broodstock and seedlings. The aim of this study was to clarify the characteristic changes that transpire during discrete developmental phases in male L. vannamei gonads via morphological, histological, biochemical, and molecular analyses. The gonadal development of male L. vannamei is categorized into four distinct phases during sexual maturation based on observations of external morphological traits. During phase I, male gonads complete gonadal differentiation but have yet to initiate development. Phase II signifies rapid development and initiation of reproductive functions; concurrently, a subtle whitish ampulla emerges at the base of the fifth pereiopod, concomitant with structural differentiation in the vas deferens. Seminiferous tubules and germ cells appear in the testis, and a primary spermatophore layer forms in the medial vas deferens, although mature sperm masses are absent. Phases III and IV primarily involve enhancing individual reproductive abilities. In phase III, a white spermatophore structure develops at the base of the fifth pereiopod, and the first pair of pleopods form the petasma. In Phase IV, the ampulla becomes fuller, and the spermatophore can be expelled artificially. Our analysis indicated that in phase II, the mRNA expression levels of the crustacean hyperglycemic hormone family (CHHs), which includes CHH isoforms (CHH1 and CHH2), a gonadal-inhibiting hormone (GIH), and a molting-inhibiting hormone (MIH), decreased within the eyestalk. During phase III, despite the presence of immature sperm in the vas deferens and a reduction in serum hyperglycemic hormone levels, gonadal development approached a state of maturity. In phase IV, mature sperm are present in the medial vas deferens, indicating that during gonad maturation, the mRNA expression levels of CHHs and insulin-like androgenic gland hormone (IAG) significantly increase. Commencing from phase II, a prominent shift occurs in the transcription levels of testicular genes. Analysis via weighted gene coexpression network (WGCNA) and functional enrichment demonstrated the upregulation of pathways pertinent to the cell cycle, RNA transport, and meiosis, juxtaposed with the downregulation of pathways linked to lysosomes. This study provides key insights into the reproductive biology and maturation of L. vannamei males.