In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2–1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density‐gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral‐like particles (~100–180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%–99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow‐out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread.
Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45-60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.
Using Penaeus monodon as the model, we demonstrated the molecular changes and the mechanism of thelycal-dependent sperm modification resulting in an enhanced acrosome reaction (AR) response. Attention was paid to the modification of the sperm plasma membrane which was mediated through an adsorption or removal of sperm peripheral and integral membrane proteins as indicated by the different profiles of these proteins in spermatophore (S) and thelycal (T) sperm. In vitro adsorption of Alexa-488 conjugated T proteins onto the entire S-sperm surface confirmed protein transfer in a time-dependent manner. Specific anchoring of 83 and 140 kDa proteins to sperm peripheral proteins as well as 53/55 and 60 kDa proteins to sperm lipids was demonstrated. Apart from membrane modification, a substantial increase in protein tyrosine phosphorylation was shown to be closely associated with T-dependent sperm modification event. The physiological significance of this sperm modification in enhancing sperm AR response, which required at least 3 days of T residence in order for the sperm to gain a complete AR response, was also elucidated.
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