Understanding the cephalopod immune system based on functional and molecular evidence

11 páginas, 6 figurasCephalopods have the most advanced circulatory and nervous system among the mollusks. Recently, they have been included in the European directive which state that suffering and pain should be minimized in cephalopods used in experimentation. The knowledge about cephalopod welfare is still limited and several gaps are yet to be filled, especially in reference to pathogens, pathologies and immune response of these mollusks. In light of the requirements of the normative, in addition to the ecologic and economic importance of cephalopods, in this review we update the work published to date concerning cephalopod immune system. Significant advances have been reached in relation to the characterization of haemocytes and defensive mechanisms comprising cellular and humoral factors mainly, but not limited, in species of high economic value like Sepia officinalis and Octopus vulgaris. Moreover, the improvement of molecular approaches has helped to discover several immune-related genes/proteins. These immune genes/proteins include antimicrobial peptides, phenoloxidases, antioxidant enzymes, serine protease inhibitor, lipopolysaccharide-induced TNF-α factor, Toll-like receptors, lectins, even clusters of differentiation among others. Most of them have been found in haemocytes but also in gills and digestive gland, and the characterization as well as their precise role in the immune response of cephalopods is still pending to be elucidated. The assessment of immune parameters in cephalopods exposed to contaminants is just starting, but the negative impact of some pollutants on the immune response of the common octopus has been reported. This review summarizes the current status of our knowledge about the cephalopod immune system that seems to be far from simply. On the contrary, the advances gained to date point out a complex innate immunity in cephalopodsPeer reviewe

Análisis de la expresión de genes de respuesta inmune durante el desarrollo ontogénico de paralarvas de pulpo Octopus vulgaris criadas en cautividad

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.0,000

Reproducción del pulpo octopus bimaculatus Verrill, 1883 en Bahía de los Ángeles, Baja California, México

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

266 páginasThe 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 andcoccidia 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.Peer reviewe

Molecular phylogenetic analysis of the coccidian cephalopod parasites Aggregata octopiana and Aggregata eberthi (Apicomplexa: Aggregatidae) from the NE Atlantic coast using 18S rRNA sequences

8 páginas, 2 figuras, 1 tablaThe coccidia genus Aggregata is responsible for intestinal coccidiosis in wild and cultivated cephalopods. Two coccidia species, Aggregata octopiana, (infecting the common octopus Octopus vulgaris), and A. eberthi, (infecting the cuttlefish Sepia officinalis), are identified in European waters. Extensive investigation of their morphology resulted in a redescription of A. octopiana in octopuses from the NE Atlantic Coast (NW Spain) thus clarifying confusing descriptions recorded in the past. The present study sequenced the 18S rRNA gene in A. octopiana and A. eberthi from the NE Atlantic coast in order to assess their taxonomic and phylogenetic status. Phylogenetic analyses revealed conspecific genetic differences (2.5%) in 18S rRNA sequences between A. eberthi from the Ria of Vigo (NW Spain) and the Adriatic Sea. Larger congeneric differences (15.9%) were observed between A. octopiana samples from the same two areas, which suggest the existence of two species. Based on previous morphological evidence, host specificity data, and new molecular phylogenetic analyses, we suggest that A. octopiana from the Ria of Vigo is the valid type species.This work has been funded by a research grant from the Galician Council Xunta de Galicia (10PXIB402116PR)Peer reviewe

Immune response of Octopus vulgaris against the infection by the gastrointestinal parasite Aggregata octopiana

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Immune parameters in the common octopus (Octopus vulgaris Cuvier, 1797) naturally infected by the gastrointestinal protozoan parasite Aggregata octopiana

7 pages, 2 tables, 3 figuresHealth 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 responseThis work was supported by the Galician Council Xunta de Galicia (10PXIB402116P) that also allowed the collection of samples. S.C.M. was funded by a CONACyT grantPeer reviewe

Pathogens and immune response of cephalopods

9 páginas, 1 tablaCephalopod mollusks are an important marine resource for fisheries, and have received marked attention for studies on organismal biology; they are also good candidates for aquaculture. Wild and reared cephalopods are affected by a wide variety of pathogens, mainly bacteria, protozoa and metazoan parasites. Cephalopods do not have acquired immunity and immunological memory; therefore vaccination cannot be used to protect them against infectious diseases. Their defense mechanisms rely only on their innate immunity. In this review, we will summarize and update knowledge on the most common pathogens, the diseases they cause, and on symbionts. In addition, we provide a general overview of the cephalopod immune system, response to pathogens with a short discussion on the gene expression involved in the immune response by these animals.This work has been partly funded by a research grant from the Galician Council Xunta de Galicia (10PXIB402116PR). Sheila Castellanos-Martínez has been supported by a scholarship from CONACyT.Peer reviewe

The role of DNA methylation on Octopus vulgaris development and their perspectives

7 páginas, 2 figuras, 3 tablas.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.-- This Document is Protected by copyright and was first published by Frontiers. All rights reserved. it is reproduced with permissionDNA 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 aquacultureThis work was funded by Xunta de Galicia (Grupos con Potencial Crecimiento, GPC2013-011, and FONDOS FEDER: “Unha maneira de facer Europa” and by Xunta de Galicia (10PXIB402116PR). Sheila Castellanos-Martínez is funded by a CONACyT grant.Peer reviewe