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

On the life cycle of Aggregata eberthi and observations on Aggregata octopiana (Apicomplexa, Aggregatidae) from Galicia (NE Atlantic)

Stages of merogony of a coccidian parasite were commonly observed during histological examination of the digestive tract of 7 crustacean species from Galician coastal waters. Study of the fine structure of these merogonial stages by transmission electron microscopy revealed the presence of a typical apicomplexan apical complex. Newly hatched Sepia officinalis obtained from egg masses, cultured in 3 separate tanks, were fed with three parasitised prawn species in infection experiments. Cuttlefish fed with Palaemon elegans and P. adpersus developed sexual stages and sporocysts diagnostic of Aggregata eberthi, identifying these prawns as new intermediate hosts of this parasite. No infection was found in Sepia fed with P. serratus, suggesting that this prawn could be an intermediate host of another species of Aggregata. By experimental infections of cultured cuttlefish, achieved here for the first time, the complete life cycle of A. eberthi in Spanish NE Atlantic waters was established

S.E.M. study of the plerocercoid larval Phyllobothrium sp. (Tetraphyllidae, Phyllobothriidae) and Nybelinia lingualis (Trypanorhyncha, Tentaculariidae), cestode parasite in Octopus vulgaris (Mollusca, Cephalopoda) off Vigo estuary

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Ultrastructural and molecular characterization of Aggregata spp (Frenzel, 1885) in Octopus vulgaris (Cuvier, 1797) from central Mediterranean

The work focused on ultrastructural and molecular characterization of protozoan parasites of the genus Aggregata (Apicomplexa, Aggregatidae) in specimens of the common octopus Octopus vulgaris obtained from Ionian and Tyrrhenian seas (Southern Italy, Central Mediterranean). Aggregata spp. are reported to parasitize cephalopod species and to be responsible of severe pathologies, which seem to be mostly evident in cultured cephalopods. In the Central Mediterranean the parasite is known to occur with high prevalence, however its taxonomic status in this area is still under debate. Following detection and purification of parasitic stages from octopus tissues, genomic DNA was extracted from Aggregata sporocysts. 18S rRNA gene of the parasite was amplified by PCR using specific primers and the sequences obtained were compared to those available from Aggregata octopiana type species infecting O. vulgaris in Northeastern Atlantic. Samples for Transmission Electron Microscopy (TEM) were treated using standardized protocols specifically designed for the processing of Aggregata spp. parasites. Histological preparations obtained from samples of infected oesphagus, crop, caecum, intestine and mantle of O. vulgaris showed the presence of both gamogonic and sporogonic stages of Aggregata and allowed the use of a semiquantitative method to compare the intensity of infection per each sampled locality. Information from histology was integrated with the analysis of parasite ultrastructure by TEM, which allowed the observation of important taxonomic features to distinguish between different species of Aggregata

Epigenetic DNA methylation mediating octopus vulgaris early development: effect of essential fatty acids enriched diet

The common octopus, Octopus vulgaris, is a good candidate for aquaculture but a sustainable production is still unviable due to an almost total mortality during the paralarvae stage. DNA methylation regulates gene expression in the eukaryotic genome, and has been shown to exhibit plasticity throughout O. vulgaris life cycle, changing profiles from paralarvae to adult stages. This pattern of methylation could be sensitive to small alterations in nutritional and environmental conditions during the species early development, thus impacting on its health, growth and survival. In this sense, a full understanding of the epigenetic mechanisms operating during O. vulgaris development would contribute to optimizing the culture conditions for this species. Paralarvae of O. vulgaris were cultured over 28 days post-hatching (dph) using two different Artemia sp. based diets: control and a long chain polyunsaturated fatty acids (LC-PUFA) enriched diet. The effect of the diets on the paralarvae DNA global methylation was analyzed by Methyl-Sensitive Amplification Polymorphism (MSAP) and global 5-methylcytosine enzyme-linked immunosorbent assay (ELISA) approaches. The analysis of different methylation states over the time revealed a global demethylation phenomena occurring along O. vulgaris early development being directly driven by the age of the paralarvae. A gradual decline in methylated loci (hemimethylated, internal cytosine methylated, and hypermethylated) parallel to a progressive gain in non-methylated (NMT) loci toward the later sampling points was verified regardless of the diet provided and demonstrate a pre-established and well-defined demethylation program during its early development, involving a 20% of the MSAP loci. In addition, a differential behavior between diets was also observed at 20 dph, with a LC-PUFA supplementation effect over the methylation profiles. The present results show significant differences on the paralarvae methylation profiles during its development and a diet effect on these changes. It is characterized by a process of demethylation of the genome at the paralarvae stage and the influence of diet to favor this methylation loss.Ministerio de Economía y Competitividad | Ref. AGL20134910-C02-2RXunta de Galicia | Ref. ED481A-2015/44