Hemocyte cell types of the Cortes Geoduck, Panopea globosa (Dall 1898), from the Gulf of California, Mexico.

The geoduck Panopea globosa is an endemic and economic valuable species from the Mexican Northwest coast whose biology has been little studied. No information exists about their hemocytes to date, which is highly important to assess the welfare of wild and cultured organisms. In this study, hemocytes of adult P. globosa were characterized at the morphological, ultrastructural and functional level. The mean number of hemocytes in the hemolymph of P. globosa was 6 × 105 ± 2 × 105 cells mL-1. The cells were identified as granulocytes (Gr) and hyalinocytes (H). The former accounted for 28% of adhered cells in the hemolymph, measured 6-18 µm, showed numerous basophilic granules in the cytoplasm, with round and eccentric nuclei, and a nucleus:cytoplasm ratio of 0.44 ± 0.01. Hyalinocytes were the most abundant cells in the hemolymph of P. globosa (72% adhered cells) and were subdivided, according to their size, in small (Hs) 4-12 µm and large (HL) 6-18 µm. Hyalinocytes were eosinophilic round or ovoid cells with a central or eccentric nucleus, few or no granules in the cytoplasm and similar nucleus:cytoplasm ratio (Hs: 0.63 and HL: 061). Lysosomes and lipids were observed in Gr, while carbohydrates were the most abundant energy substrate in H. Both hemocytic cell types, mainly Gr, were capable to ingest particles and yield superoxide (P > 0.05). The present study shows for the first time the cell types, abundance and immune activities of hemocytes present in the hemolymph of P. globosa. This information provides a useful baseline to carry out further research on the cellular immune response of the clam to potential pathogens or changes in environmental factors.

Selection of reliable reference genes for RT-qPCR studies in Octopus vulgaris paralarvae during development and immune-stimulation.

The common octopus, Octopus vulgaris is a new candidate species for aquaculture. However, rearing of octopus paralarvae is hampered by high mortality and poor growth rates that impede its entire culture. The study of genes involved in the octopus development and immune response capability could help to understand the key of paralarvae survival and thus, to complete the octopus life cycle. Quantitative real-time PCR (RT-qPCR) is the most frequently tool used to quantify the gene expression because of specificity and sensitivity. However, reliability of RT-qPCR requires the selection of appropriate normalization genes whose expression must be stable across the different experimental conditions of the study. Hence, the aim of the present work is to evaluate the stability of six candidate genes: ß-actin (ACT), elongation factor 1-α (EF), ubiquitin (UBI), ß-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GADPH) and ribosomal RNA 18 (18S) in order to select the best reference gene. The stability of gene expression was analyzed using geNorm, NormFinder and Bestkeeper, in octopus paralarvae of seven developmental stages (embryo, paralarvae of 0, 10, 15, 20, 30 and 34days) and paralarvae of 20days after challenge with Vibrio lentus and Vibrio splendidus. The results were validated by measuring the expression of PGRP, a stimuli-specific gene. Our results showed UBI, EF and 18S as the most suitable reference genes during development of octopus paralarvae, and UBI, ACT and 18S for bacterial infection. These results provide a basis for further studies exploring molecular mechanism of their development and innate immune defense.

Understanding the cephalopod immune system based on functional and molecular evidence.

Cephalopods 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 cephalopods.

Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 µm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 µm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.