Dual RNA-seq Identifies Virulence Factors in Mesanophrys sp. and Immune Defenses in Portunus trituberculatus Hemocytes.

PURPOSE: Mesanophrys sp. is a scuticociliate parasite associated with severe hemocyte damage in the swimming crab Portunus trituberculatus, but the molecular basis of parasite-hemocyte interaction remains incompletely understood. This study aimed to characterize host and parasite transcriptomic responses during in vitro interaction and to evaluate the potential involvement of parasite proteolytic activity in hemocyte damage.

METHODS

An in vitro Mesanophrys sp.-hemocyte co-culture model was established, and dual RNA-seq was used to simultaneously profile parasite and crab hemocyte transcriptomes at early (18 h) and late (72 h) interaction stages. Otherwise, gelatin zymography was used to detect gelatinolytic activity in parasite lysates, and protease inhibitor assays were conducted to assess the potential contribution of protease activity to hemocyte survival and morphology during co-culture.

RESULTS

Dual RNA-seq revealed induction of hemocyte innate immune signaling, including Toll-like receptor pathway components and downstream adaptor and MAPK-related genes, along with broad stress- and metabolism-associated responses. In the parasite, differentially expressed genes were enriched for several upregulated putative secreted proteases, including cysteine proteases, thimet oligopeptidase 1 and oligopeptidase B-like peptidases previously implicated in protozoan virulence. Gelatin zymography detected four reproducible gelatinolytic bands in parasite lysates, with distinct inhibitor sensitivity profiles, including EDTA-sensitive, PMSF-sensitive, PMSF- and leupeptin-sensitive, and leupeptin- and E-64-sensitive activities. These gelatinolytic bands showed near-neutral pH-dependent activity patterns, and the strongest temperature-dependent signals were observed at 12 °C, consistent with the low-temperature growth advantages reported for Mesanophrys sp. In co-culture assays, selected protease inhibitors, particularly leupeptin and E-64, were associated with higher hemocyte survival and preservation of hemocyte morphology at early time points, although these effects decreased after 72 h.

CONCLUSION

These results suggest that parasite proteases are candidate factors potentially associated with hemocyte damage during Mesanophrys sp.-hemocyte interaction in vitro. This study provides a dual transcriptomic resource for further investigation of crab-parasite interactions, but in vivo validation will be required to define the roles of these candidate proteases during infection.