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Health of wild and reared Octopus vulgaris is threatened by the most dangerous parasite for the octopus, the protozoan coccidia Aggregata octopiana. This host-parasite relationship was studied to analyze the effect of A. octopiana on the cellular immune parameters of O. vulgaris. A. octopiana sporocysts infecting the digestive tract of octopuses were counted in order to confirm two octopus groups of infection: 1) healthy (low parasite load, without histological caecum damage) and 2) sick (high parasite load and strong histological caecum damage). Cellular defense parameters (phagocytosis, respiratory burst (ROS) and nitric oxide (NO) production) were measured in the octopus hemolymph. In addition, i) infection degree (total parasitic infection or distributed by groups of infection (sick/healthy)), ii) octopus origin (wild or reared in floating cages), biometric data (sex, length, weight, maturity) and iii) season of collection were tested to know their contribution to the octopus cellular response. Results showed that season of collection and total parasitic infection were the most important factors affecting the phagocytic ability of hemocytes. Phagocytosis increased accordingly to the infection intensity and was particularly higher in Autumn (P < .01) relative to Winter and Spring. ROS (P < .01) and NO (P < .1) production decreased with the A. octopiana infection increase. Related to biometric data, a markedly decrease in NO was observed in heaviest octopuses (P < .01). Comparing wild and reared octopuses, the cytotoxic activity notably decreased in the former group. The present results evidenced for the first time that the intensity of infection by A. octopiana severely weaken the octopus cellular immune response

Background: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. Results: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. Conclusion: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers

DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP), firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern changes with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since no significant effect was revealed in the analyses of molecular variance (AMOVA) performed. Our observations highlight the importance of epigenetic mechanisms in the first developmental steps of the common octopus and opens new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for aquacultu

El pulpo común es una especie de gran importancia comercial, considerada como una especie emergente en acuicultura. En este trabajo se analizó el nivel de expresión de los genes inmunes TLR, C1q, Galectina, PGRP, LITAF, SERPIN, PRDX y Caspasa 3 mediante PCR cuantitativa (q-PCR) en embriones y paralarvas de O. vulgaris de edades 0, 10, 20 y 34 días. Adicionalmente, se infectaron paralarvas de 22 días con bacterias patógenas vivas Vibrio lentus y V. splendidus a 1h, 4h y 24h. El estudio del desarrollo del sistema inmune de estas paralarvas ayudará a identificar factores claves para la supervivencia y cultivo del pulpo común. Durante el desarrollo ontogénico, los embriones mostraron el menor nivel de expresión de PGRP, Caspasa 3 y PRDX. Por el contrario, C1q, Galectina y LITAF se observaron visiblemente expresados. C1q, TLR y SERPIN fueron los genes que presentaron mayor nivel de expresión en Pa0D. A partir de Pa10D se observó un notable incremento en la expresión de C1q, Galectina, PGRP y LITAF. La expresión de Caspasa3 se incrementó gradualmente desde Em. V. lentus y V. splendidus inducen un notable incremento de la expresión de C1q y PRDX entre 1h y 4h post infección. Sin embargo, durante las primeras horas de infección se observó una disminución de la expresión de Galectina, TLR, PGRP y LITAF. Particularmente la infección por V. lentus produjo una disminución de la expresión de SERPIN. Los resultados obtenidos sugieren que la capacidad del sistema inmune de reconocer patógenos y evitar infecciones es significativamente activo desde los estadíos de paralarvas recién eclosionadas. Sin embargo, se observó un aumento significativo de los genes seleccionados a partir de Pa10D., The common octopus is a species of high commercial interest and nowadays is considered as an emergent species in aquaculture. The mRNA expression level of immune-related genes (TLR, C1q, Galectin, PGRP, LITAF, SERPIN, PRDX and Caspase 3) was analyzed by Real time qPCR on embryos and paralarvae of O. vulgaris at age of 0, 10, 20 and 34 days. Additionally, paralarvae of 22 days were challenged with live Vibrio lentus and V. splendidus during a time course (1 h, 4 h and 24 h). This analysis will allow a better understanding of the developmental of immune system of these paralarvae, which will help to identify key factors for survival and culture of the common octopus. Regarding ontogeny, Em showed the lowest expression of PGRP, Caspase 3 and PRDX. In contrast, C1q, Galectin and LITAF were visibly expressed. C1q, TLR and SERPIN were highly expressed in Pa0D. A notably increase in the expression of C1q, Galectin, PGRP and LITAF began from Pa10D. Caspase 3 expression was gradually increased from Em. V. lentus and V. splendidus induced a notable expression of C1q and PRDX at 1 h and 4 h post infection (p. i.). Nevertheless, they markedly suppress the activation of Galectin, TLR, PGRP and LITAF during the first hours p.i.. Particularly, V. lentus suppressed the expression of SERPIN. The present results suggest that the ability of immune system to recognize pathogens and avoid infections is a priority in recently hatched paralarvae. However, a noticeable increase of gene expression was observed from Pa10D.

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 c, 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