Coccidiosis and molecular basis of the immune response of common octopus (Octopus vulgaris Cuvier, 1797)

Resumo

The common octopus, Octopus vulgaris Cuvier, 1797, is one of the most important species in worldwide fisheries and aquaculture. Galicia is the pioneer Autonomic Community in octopus culture, which is considered one of the most important alternative resources to diversify the aquaculture. One of the main constraints in this activity is the diseases caused by several pathogens. Therefore, in order to control and eradicate the main diseases, such as coccidiosis caused by Aggregata octopiana, it is highly important to develop studies focused on knowing the octopus immune response against pathogens. Those studies will allow establishing the basis to develop strategies towards an appropriate sanitary practice in octopus aquaculture. Furthermore, supplementary studies of genes involved in immune response will contribute to establish the molecular basis for identifying and select octopuses resistant against the coccidia infection. Hence, the first study of the common octopus immune response and their interaction with the infection by the coccidia A. octopiana is herein presented. The molecular characterization of A. octopiana from NE Atlantic (Ria of Vigo) using 18S rRNA gene allowed complementing and confirming the yet existing morphological description. Likewise, the molecular characterization of A. eberthi that infects Sepia officinalis was also performed. The new sequences obtained were compared with the only sequences of A. octopiana and A. eberthi available in GenBak from the Adriatic Sea (Croatia). The low genetic divergence between A. eberthi species indicates that these coccidia infect two different populations of S. officinalis. In contrast, the high genetic divergence between A. octopiana from NE Atlantic and Adriatic Sea indicates that they correspond to different coccidia species. Therefore, according to previous morphological descriptions, host specificity and the molecular data herein obtained, A. octopiana from NE Atlantic (Ria of Vigo) is considered as the valid species. The studies conducted through microscopy and flow cytometry allowed to characterize the hemocytes present in the octopus hemolymph. Two sub-populations or types of hemocytes named large granulocytes and small granulocytes were characterized. Through functional analysis it was demonstrated that both types of cells showed ability to develop defensive activities in the organism. However, phagocytic ability and respiratory burst were higher in large granulocytes than in small ones. Nitric oxide (NO) production was measured in the total hemocytic population following challenge with zymosan, LPS and PMA in a time course. The highest NO production was reached after 3 h of incubation. There was confirmed that cellular immune defense is affected by the level of A. octopiana infection. The number of hemocytes decreased in highly infected octopuses. The phagocytic activity increased according to the increase of the infection whereas, respiratory burst was reduced. Nonetheless, any relationship between NO production and level of infection was found. In addition, a similar pattern in the cellular immune defense was observed in wild octopuses and reared in floating cages. In both cases, the phagocytic ability tended to increase with the level of infection, but respiratory burst decreased. However, NO production was significantly higher in wild octopuses than those reared in floated cages, suggesting than wild octopuses have a better immune response than those reared in floating cages. The transcriptomic study of the hemocytes from O. vulgaris by construction of cDNA library using a high-throughput sequencing method, allowed identifying important immune pathways such as NFkB, complement, Toll-Like Receptors (TLR) and apoptosis. From the present study, most of the immune genes identified are reported for the first time in cephalopods. The transcriptome of hemocytes from octopuses harboring high and low infection by A. octopiana were compared. A total of 539 genes were found differentially expressed between both levels of infection. Q-PCR analysis of genes selected according to their importance in the host-pathogen interaction confirmed the previous expression pattern and corroborated the results obtained by the high-throughput sequencing. The proteomic study of the octopus hemolymph, 42 significant spots were found in hemocytes from octopuses harboring high and low infection by A. octopiana. These spots were statistically analyzed by principal component analysis, from which 7 proteins are herein suggested as candidates of putative resistance biomarkers against the coccidia infection. Particularly, the protein filamin, fascin and peroxiredoxin are highlighted because their implication in the octopus immune defense. Considering the information obtained in this study, here is evidenced that coccidiosis by A. octopiana affect to the proper functioning of the octopus cellular immune response. Phagocytosis is stimulated by the infection, however respiratory burst is suppressed. The molecular evidence agreed with functional assays. The respiratory burst reduction results in a down-regulation of antioxidant genes at both trancriptomic and proteomic level. Likewise, the increase in phagocytic ability of hemocytes is consistent with the significant up-regulation of proteins like filamin and fascin (both related to phagocytosis) in highly infected octopuses. Therefore, the results exposed in the present work provide the first molecular insights into the molecular basis of host-pathogen relationship between O. vulgaris and A. octopiana.