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

Abstract

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 us to establish 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 establishing the molecular basis to identify 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 has allowed the complementation and confirmation of the pre-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 were characterized, namely large granulocytes and small granulocytes. Using functional analysis it was demonstrated that both types of cells showed the 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 phagocytic activity increased according to the increase of the infection, mainly in autumn; whereas, respiratory burst (ROS) and NO decreased when the coccidia infection increase. The NO production decline was particularly notorious in low infected octopuses, but also in the heaviest individuals. In addition, a similar pattern in the cellular immune defense was observed in wild octopuses and in those reared in floating cages. In both cases, the phagocytic ability increase with the level of infection, but respiratory burst and NO decreased. Furthermore, NO production was significantly lower in wild octopuses than in those reared in floated cages, suggesting that the stressful culture conditions and

coccidia infection acts synergistically, and triggers a high cytotoxic response in those octopuses 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 for the identification of 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. In 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 proteins filamin, fascin and peroxiredoxin are highlighted because of their implication in the octopus immune defense. Considering the information obtained in this study, there is evidenced that coccidiosis by A. octopiana affects 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 the 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.