In this study, we aimed to encapsulate the sizable double-stranded DNA (dsDNA, 3.9 kbp) into a small-sized infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP; T = 1) and compared the changes in capsid structure between dsDNA-filled VLP and empty VLP. Based on our encapsulation protocol, IHHNV-VLP was able to load dsDNA at an efficiency of 30–40% (w/w) into its cavity. Structural analysis revealed two subclasses of IHHNV-VLP, so-called empty and dsDNA-filled VLPs. The three-dimensional (3D) structure of the empty VLP produced in E. coli was similar to that of the empty IHHNV-VLP produced in Sf9 insect cells. The size of the dsDNA-filled VLP was slightly bigger (50 Å) than its empty VLP counterpart; however, the capsid structure was drastically altered. The capsid was about 1.5-fold thicker due to the thickening of the capsid interior, presumably from DNA–capsid interaction evident from capsid protrusions or nodules on the interior surface. In addition, the morphological changes of the capsid exterior were particularly observed in the vicinity of the five-fold axes, where the counter-clockwise twisting of the “tripod” structure at the vertex of the five-fold channel was evident, resulting in a widening of the channel’s opening. Whether these capsid changes are similar to virion capsid maturation in the host cells remains to be investigated. Nevertheless, the ability of IHHNV-VLP to encapsulate the sizable dsDNA has opened up the opportunity to package a dsDNA vector that can insert exogenous genes and target susceptible shrimp cells in order to halt viral infection.

Background and Aim: Cholesterol (CHO) sequestration from the sperm plasma membrane is a fundamental event during mammalian sperm capacitation, but the molecular mechanisms underlying capacitation-like sperm activation in brachyuran crabs remain unknown. Although Niemann–Pick type C2 (NPC2), a high-affinity CHO-binding protein, has been implicated in sperm maturation in penaeid shrimp, its occurrence and physiological role in brachyuran species have not been investigated. This study characterized the Portunus pelagicus NPC2 (PpNPC2) gene, determined its localization in the male reproductive tract, and evaluated its role in capacitation-like sperm remodeling during reproductive tract transit in CHO-dependent capacitation. Materials and Methods: The PpNPC2 sequence was identified and analyzed using comparative molecular and phylogenetic approaches. Localization of PpNPC2 in the testis and spermatic duct was examined by immunohistochemistry. Recombinant PpNPC2 protein was produced in Escherichia coli and functionally evaluated using isolated testicular sperm. Membrane CHO content was quantified by high-performance thin-layer chromatography and Filipin III fluorescence staining, whereas sperm morphology and protein tyrosine phosphorylation were assessed using light microscopy, immunofluorescence, and Western blotting. Results: PpNPC2 encoded a conserved NPC2 protein that clustered phylogenetically with crustacean NPC2 homologs and was predominantly localized in epithelial cells of the testis and spermatic duct. During sperm transit, membrane CHO decreased by approximately 32%, accompanied by progressive transformation of round testicular sperm into characteristic snowman-like spermatic duct sperm. Recombinant PpNPC2 reproduced these physiological changes by promoting CHO sequestration, inducing time- and concentration-dependent morphological remodeling, and significantly increasing protein tyrosine phosphorylation by up to 2.33-fold (p < 0.01). These findings indicate that PpNPC2 functions as a CHO-sequestering protein that initiates capacitation-like biochemical and structural remodeling during sperm maturation. Conclusion: This study provides the first molecular and functional characterization of NPC2 in a brachyuran crab and demonstrates that NPC2-mediated CHO sequestration is a conserved mechanism driving capacitation-like sperm activation during reproductive tract transit. These findings expand current understanding of reproductive biology in marine crustaceans and provide a molecular foundation for future studies on sperm preservation, assisted reproduction, and crustacean aquaculture. Keywords: Brachyuran crab, cholesterol sequestration, external fertilization, Niemann–Pick type C2, Portunus pelagicus, protein tyrosine phosphorylation, sperm capacitation-like process, sperm remodeling.