Electron probe microanalysis and gene expression analysis of melanization caused by summer atrophy virus in the Akoya pearl oyster (Pinctada fucata)

Abstract

The Akoya pearl oyster, Pinctada fucata, is the major species used for pearl culture in Japan. However, a massive mortality of juvenile and mantle atrophy of adult P. fucata have occurred during summer in Japan's major pearl production areas since 2019, and the symptoms were named “summer atrophy,” where a brown substance was deposited on the inner surface of their shell and mainly at the edge of the mantle. Summer atrophy is caused by a non-enveloped virus with a diameter of <100 nm. This study aimed to reveal whether the deposits are melanin and determine the gene expression in the mantle with the deposition through an infectious experiment. The body fluid of diseased P.fucata containing the causative virus was inoculated into the adductor muscle of three-year-old P. fucata. P. fucata were sampled at 0 (before injection), 1, 3, 6, 10, 14, 21, and 35 days post-injection. First, an electron probe microanalyzer was used to observe the detailed structure and material composition of the deposits of experimentally and spontaneously diseased P. fucata. Protuberances and wrinkled structures differed from a normal inner surface shell. Cluster analysis of these elemental composition ratios revealed that the brown deposits formed a cluster with the melanin standard, a prismatic layer of Mytilus galloprovincialis. Therefore, the brown deposits were considered to be melanin. The gene expression of infected mantles was analyzed by quantitative PCR. RNA was extracted from the mantle and body fluids, and the gene expression of shell matrix protein genes (msi31 and msi60), tyrosinase genes involved in melanin formation (OT47 and pfty1), and superoxide dismutase (SOD), which is an indicator of reactive oxygen species, was analyzed. A general linear model was created using the presence of deposits as an objective variable, with virus gene copy numbers in the body fluid and mantle and expression levels of the above five genes as explanatory variables. Then, an optimal model was selected. As a result, OT47, msi60, and SOD were selected as explanatory variables. The results suggest that virus infection induced the up-regulation of SOD and tyrosinase, inducing the deposits on the inner surface of shells in summer atrophy. Melanization is known in many animals and plays a major role in immunity, particularly in invertebrates. In bivalves, melanization caused by bacterial diseases has been reported, but this is the first report of melanization caused by viral diseases.