Molecular identification of Amyloodinium ocellatum infection in European sea bass (Dicentrarchus labrax) and expression of immune-related genes during natural infection.

Accumulated evidences indicated that in the Mediterranean area, amyloodiniosis represents a major hindrance for semi-intensive marine aquaculture causing extremely high mortalities in lagoon-type based rearing sites. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish, that live within its ecological range. In the present study, A. ocellatum was collected from naturally infected European sea bass (D. labrax) during an outbreak in July 2016 from Pila (RO), Italy. Further, parasite samples were collected from European sea bass experimentally infected in laboratory by using a strain of A. ocellatum from Pila, Italy. Genomic DNA was isolated from infected gills and molecular analysis was performed using specific primers of A. ocellatum AO18SF and AO18SR. They amplified consistently and yielded 248 bp specific amplicon of A. ocellatum, that was also confirmed using sequencing and NCBI Blast analysis. The sequence was edited and submitted to NCBI GenBank with the accession number KY474336.1. The evolution tree was developed by neighbor joining phylogenetic analysis and indicated that the tree detected two groups of A. ocellatum. One group contains A. ocellatum from all the oceanic isolates, while the other contains isolate from the laboratory experimental infection. A. ocellatum from natural infected sample clustered well with their high bootstrap value counterparts from other isolates which are closely related to Mediterranean sea isolates. Further, multiple sequence alignment was performed using the CLUSTALW and indicated that sequenced AO was conserved with all the Mediterranean isolates. The results seem to be encouraging and on the other hand, the immune response of European sea bass to the parasite is still poorly understood. Therefore, further research will be conducted to determine the European sea bass immune related gene response (innate immunity, adaptive immunity, and stress) post A. ocellatum infection, using RNA-seq to generate transcriptomic data for gills and spleen, will be discussed. Altogether, the results on parasite identification and host immune-related genes will allow for a better understanding of immunity in European sea bass against A. ocellatum, carrying out detailed functional analysis of these genes and developing strategies aimed at an efficient immune protection against this infection.