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.

Aggregata bathytherma sp. nov. (Apicomplexa:Aggregatidae), a new coccidian parasite associated with a deep-sea hydrothermal vent octopus.

Aggregata bathytherma sp. nov. is described from the digestive tract of Vulcanoctopus hydrothermalis, a deep-sea octopus recently discovered associated with hydrothermal vents in the northeast Pacific Ocean. Oocysts typically are spherical in shape, sometimes irregular, 163 to 356 microm in length, and 219 to 313 microm in width. Each oocyst contains from 50 to over 200 sporocysts. Sporocysts measure 27 to 32 microm in longest diameter. The cyst wall is smooth and 1 microm thick. Each sporocyst typically contains 14 to 17 sporozoites, 49 microm in length. Histological lesions associated with the presence of A. bathytherma include rupture of the basal membrane and detachment of the epithelial cells. In heavily infected areas, most of the tissue of the host digestive tract is replaced by parasites. A. bathytherma is the first Aggregata species described from a host that lives in association with hydrothermal vents, and the third species of Aggregata from eastern North Pacific waters.

Differentially expressed genes of the carpet shell clam Ruditapes decussatus against Perkinsus olseni.

Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host-pathogen interactions in R. decussatus.

Characterization of a C3 and a factor B-like in the carpet-shell clam, Ruditapes decussatus.

The alternative pathway is considered to be the most ancient route for activation of the complement system. Herein, we report the characterization of C3 and factor B-like proteins in the clam Ruditapes decussatus, termed Rd-C3 and Rd-Bf-like. The Rd-C3 is a three-chain protein, similar to other protoC3 proteins, and the Rd-Bf-like is composed of two complement control protein modules (CCP domains) that differ from other described Bf proteins. The inoculation of clams with live bacteria did not result in induction of these functions, but inhibited the expression of Rd-C3 and Rd-Bf-like.

Functional and molecular immune response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria.

The effect of live bacteria (Micrococcus lysodeikticus and Vibrio anguillarum), and PAMPs (poly I:C, zymosan, LPS, LTA and CpG) on the production of intermediate toxic radicals (respiratory burst activity and production of nitric oxide) and mytilin B, myticin C and lysozyme gene expression was studied in vivo and in vitro. In vitro, bacteria were able to modulate the haemocytes' respiratory burst activity, being significantly increased after 6 h of incubation. The effect of pathogen-associated molecular patterns (PAMPs) was also studied. Zymosan produced an increase of the PMA-mediated response but an inhibition of the zymosan-mediated response. A significant increase of nitric oxide production was found at all the sampled time points (1, 3 and 6 h) in comparison with controls on both, the Gram-positive and Gram-negative bacteria. The in vivo responses measured on haemocytes after M. lysodeikticus injection were faster than those induced by V. anguillarum. However, V. anguillarum induced stronger in vitro effects. Mytilin B, myticin C and lysozyme in vitro gene expression, occurred at short times after infection. The maximum in vitro expression was detected 3 h post-infection. The differences between M. lysodeikticus and V. anguillarum in different measured parameters may suggest that different signalling pathways might be involved. Moreover, among all assayed PAMPs, LPS elicited the highest response.

Global impact of diet and temperature over aquaculture of Octopus vulgaris paralarvae from a transcriptomic approach.

Common octopus, Octopus vulgaris, is an economically important cephalopod species. However, its rearing under captivity is currently challenged by massive mortalities previous to their juvenile stage due to nutritional and environmental factors. Dissecting the genetic basis and regulatory mechanism behind this mortality requires genomic background knowledge. A transcriptomic sequencing of 10 dph octopus paralarvae from different experimental conditions was constructed via RNA-seq. A total of 613,767,530 raw reads were filtered and de novo assembled into 363,527 contigs of which 82,513 were annotated in UniProt carrying also their GO and KEGG information. Differential gene expression analysis was carried out on paralarvae reared under different diet regimes and temperatures, also including wild paralarvae. Genes related to lipid metabolism exhibited higher transcriptional levels in individuals whose diet includes crustacean zoeas, which had an impact over their development and immune response capability. High temperature induces acclimation processes at the time that increase metabolic demands and oxidative stress. Wild individuals show an expression profile unexpectedly similar to Artemia fed individuals. Proteomic results support the hypothesis revealed by transcriptional analysis. The comparative study of the O. vulgaris transcriptomic profiles allowed the identification of genes that deserve to be further studied as candidates for biomarkers of development and health. The results obtained here on the transcriptional variations of genes caused by diet and temperature will provide new perspectives in understanding the molecular mechanisms behind nutritional and temperature requirements of common octopus that will open new opportunities to deepen in paralarvae rearing requirements.

Ultrastructural aspects of hepatic coccidiosis caused by Goussia lusca n. sp. (Apicomplexa: Coccidia) infecting Trisopterus luscus (Gadidae) from the NE Atlantic Ocean.

Goussia lusca n. sp. is described from the liver of pouting Trisopterus luscus from the NE Atlantic Ocean in Ibero-Atlantic Portuguese and Spanish waters. Mature oocysts were 31.7 (28.8 to 35.4) microm in diameter. Each oocyst contained 4 ellipsoidal sporocysts arranged in an aleatory position, and measuring approximately 13.7 x 9.2 microm. Each sporocyst contained 2 sporozoites. Ultrastructurally, the sporocyst wall consisted of a dense inner layer 115 nm thick, transversely striated, regularly intercalated by thin grooves with electron-lucent spaces, and separated from the outer layer by a thin, light (electron-lucent) space. The outer layer was multilamellated and consisted of parallel dense bands alternating with light spaces. These lamellae formed filamentous extensions of the wall. The dehiscence suture, a characteristic feature of the genus, was present in the sporocysts. No external clinical signs were observed in the host fish. Parasites observed in the liver tissue were often enveloped in a yellowish-brown matrix, generally known as 'yellow bodies'. Sometimes sporocysts were observed in direct contact with the liver cells. Parasites in degeneration and aggregations of amylopectin granules were frequently observed surrounded by host inflammatory cells. In severe infections, we observed large agglomerations of oocysts encapsulated by layers of concentrically arranged connective tissue forming large granulomas, which caused significant replacement of the host liver parenchyma by the parasite.

Observations on associated histopathology with Aggregata octopiana infection (Protista: Apicomplexa) in Octopus vulgaris.

Gamogony and sporogony of Aggregata octopiana were commonly observed during histological examination of the digestive tract of wild Octopus vulgaris from Ria de Vigo (NW Spain). A. octopiana infected noncuticularized caecum and intestine, and cuticularized oesophagus and crop. Infection was also observed in the gills and in covering mesenterium, mainly of the digestive gland and gonad. Histological and ultrastructural lesions associated with A. octopiana included host cell hypertrophy with nuclear displacement, inflammation, phagocytosis, ulceration and destruction of organ architecture. The possible existence of a malabsorption syndrome in the host is deduced.

Macroparasites in cetaceans stranded on the northwestern Spanish Atlantic coast.

An extensive parasitological survey was carried out during autopsy of 80 cetaceans representing 8 species within 4 families (Delphinus delphis, Stenella coeruleoalba, Tursiops truncatus, Grampus griseus, Globicephala melas, Kogia breviceps, Phocoena phocoena and Megaptera novaeangliae) collected on the northwestern Spanish Atlantic coast from February 1991 to October 1996. Two species of tetraphyllidean cestodes (Phyllobothrium delphini and Monorygma grimaldii), 2 ascaridoid nematodes (Anisakis simplex and A. physeteris), a single spirurid nematode (Crassicauda magna), 4 rhabditidiform nematodes (Halocercus delphini, H. invaginatus, Halocercus spp. and Stenurus globicephalae), a single polymorphynae acanthocephalan (Bolbosoma sp.), and 2 amphipods (Isocyamus delphini and Cyamus boopis) were found. This paper presents 6 new geographic records of macroparasites from cetaceans in temperate Atlanto-Iberian waters. A total of 11 component parasite species were found, mainly parasitizing the blubber, mesentery and stomach of cetaceans. Cetaceans harboured a suite of 4 generalist and 8 specialist species.

Long-term recording of gastric ulcers in cetaceans stranded on the Galician (NW Spain) coast.

Long-term (1990-1996) results of recording gastric ulcers in the common dolphin are presented for the NW Spanish Atlantic coast. The occurrence and abundance of cetacean carcasses (belonging to 10 species) with gastric ulcers are also given and discussed. Ulcerations were detected in 17.2% of the animals examined, with 15% for common dolphins and 0 to 29.4% in other cetacean species. A positive relation was noted between ulcer counts and length and maturity of Delphinus delphis. Clusters of the nematode Anisakis simplex could be seen embedded in the gastric ulcers of 3 common dolphins. It could be concluded that gastric ulcers are non-fatal lesions in cetaceans stranded in NW Spain.

Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa).

Octopus vulgaris infected with Aggregata octopiana were collected from an open-water culture system in the Ría of Aldán (NW Spain). Digestive tract infection values were determined with the use of a Neubauer chamber by counting the number of A. octopiana sporocysts. After determining enzyme activity values by the colorimetric Api-Zym system Biomerieux, one representative enzyme of glycosidases, peptid hydrolases and phosphoric hydrolases showing high activity was spectrophotometrically analysed. The enzymes were maltase and leucine-aminopeptidase (LAP) involved in the absorption process, and acid phosphatase, a lysosomic enzyme, respectively. Enzymatic activity of maltase and LAP decreased significantly, with increased sporocyst counts. However, acid phosphatase activity increased with severity of infection, indicating the presence of degradative enzymes from phagocytic cells in the infected area. A detrimental effect on gastrointestinal function may result from a decrease or malfunction of absorption enzymes. The results suggest a malabsorption syndrome resulting from parasitic infection.

Larval nematodes (Spiruroidea: Cystidicolidae) in Octopus vulgaris (Mollusca: Cephalopoda: Octopodidae) from the northeastern Atlantic Ocean.

Larval nematode parasites (Spiruroidea: Cystidicolidae) are recorded for the first time in Octopus vulgaris Cuvier, 1797 in the northeastern Atlantic Ocean. Prevalence was 16% and mean intensity was 1.46 worms/host. Body length of larval nematodes ranges from 8.3 to 9.3 mm, with a distance from the anterior end to nerve ring from 187.5 to 200 microm, and to excretory pore 194.6-350 microm. Anatomical characteristics, such as deirid, nerve ring, cephalic alae, excretory pore, pseudolabia amphids, sclerotized protuberance, and anus, examined using light microscopy (LM) or scanning electron microscopy (SEM), are illustrated. The nematode was designed as a cystidicolid "Type A" larva. The hemocytic infiltration present in the host tissue around the nematode capsule and the mechanical compression in the infected organs denote the pathogenicity of this nematode. In the study area, O. vulgaris may play the role of an intermediate or paratenic host in the nematode life cycle.

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.

Naso-pharyngeal mites Halarachne halichoeri (Allman, 1847) in Grey seals stranded on the NW Spanish Atlantic Coast.

In North Atlantic European waters, the nasal mite Halarachne halichoeri has been described affecting Grey seals (Halichoerus grypus) producing different levels of respiratory disease. This study provides data on the prevalence, clinical signs and produced macro-pathology of this parasite mite infecting juvenile wild Grey seals stranded in North-Western Spanish coast. Among the 25 seals examined during the study, a total of 19 had nasal mites in their respiratory upper ways, including adult and larval stages. This represented a percentage of prevalence of 76 ± 8.37. All the live positive seals presented a typical clinical symptomatology associated to upper respiratory tract infections. In dead positive seals, a light to intense sinusitis could be diagnosed macroscopically. The presence of the parasite in the nasal sinuses appears as the primary cause of the high respiratory tract symptomatology presented in most of the juvenile seals stranded in the north coast of Spain. Ultrastructural characterization by scanning electronic microscopy (SEM) has confirmed the taxonomic status of the mite. This identification of H. halichoeri represents the first description of the occurrence of this parasite in Southern Europe.

Ultrastructure of Goussia cruciata (Apicomplexa: Coccidia) infecting the liver of horse mackerel, Trachurus trachurus (L.), from Ibero-Atlantic waters.

The ultrastructure of developmental stages of Goussia cruciata and the pathology they cause in the liver of Trachurus trachurus (Teleostei: Carangidae) caught off the Galician (North-West Spain) and Portuguese North Atlantic coasts are described. Each oocyst contained four ellipsoidal sporocysts, with two sporozoites. The sporocyst wall consisted of a thick and dense inner layer with transverse striations and a multi-lamellated outer layer formed by parallel dense internal bands alternating with lighter areas. The lamellae formed filamentous extensions of the wall. The sporocyst wall striation period was smaller than that observed in G. clupearum, which has a similar habitat. The dehiscence suture, characteristic of the genus, was present in the sporocysts of G. cruciata. The sporocysts were arranged in a symmetrical and characteristic cross shape. A large number of sporocysts with sporozoites were observed in direct contact with host liver cells. No macroscopic lesions were observed. In heavily infected fish, aggregations of oocysts were often enveloped in a 'yellow body' composed of amylopectin granules derived from the parasite and necrotic or aggregated host cells. Degenerating parasites were frequently observed in liver tissue. Host inflammatory cells were accumulated near some oocysts. The ultrastructure of the parasite, together with its strict host specificity, confirmed G. cruciata as a separate and valid species.

Aggregata andresi n. sp. (Apicomplexa: Aggregatidae) from the ommastrephid squid Martialia hyadesi in the SW Atlantic Ocean and some general remarks on Aggregata spp. in cephalopod hosts.

A new species of coccidian, Aggregata andresi, is described from the digestive tract of the flying squid Martialia hyadesi, an ommastrephid squid that lives in cold subantarctic waters in the Southwest Atlantic. Gamogonic and sporogonic stages were observed in the digestive tract of 96.5% studied hosts. Oocysts were ovoid to subspheroid in shape, 170-590 microm in length and 200-530 microm in width. Each oocyst contained approximately 45,000 sporocysts. Sporocysts measured 9.5-10 microm in length and 8-8.5 microm in width. The surface of the sporocyst wall was smooth and the cyst wall thick. Each sporocyst contained three sporozoites measuring 16-20 microm in length and 2-2.5 microm width. A. andresi is the second species of Aggregata to be reported from a nerito-oceanic cephalopod host.

Anisakid infection in the European shag Phalacrocorax aristotelis aristotelis.

A total of 100 specimens of the European shag Phalacrocorax aristotelis aristotelis were examined for anisakid infections in the south-east North Atlantic. The taxonomic status of the anisakid nematodes was studied by structural analysis using light and scanning electron microscopy. Two species, Contracaecum septentrionale and C. rudolphii A (Nematoda: Ascaridoidea), occurred in the European shag with high values of prevalence and mean intensities of infection. These constitute new host records for both parasites, widening their known geographical distribution. Although the histopathology associated with the infections indicated that the anisakids did not have the potential to cause bird death, the parasites are capable of contributing to and hastening avian mortality in the case of heavily-parasitized cormorants and when stressed by xenobiotics and/or through a synergetic effect by other bioagressors.

Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective.

A total of 2,673 fresh specimens of cephalopod and fish representing 35 species were obtained from commercial local fisheries in Galician waters (NW Spain). They were examined for anisakid nematodes by digestion of the muscle and elution of the viscera and whole body cavity. All larval nematodes recovered were identified by light microscopy and multilocus electrophoresis as belonging to the species Anisakis simplex sensu stricto and A. pegreffii. Encysted larvae mostly occurred in the viscera but were also found in the flesh of squid and fish. Demographic values for larval nematodes are discussed in relation to host preferences and the ecological niche of both anisakid species at the sampling area. Primary recommendations are also expressed concerning the effects of current fishing and aquaculture practices on the Anisakis problem.

Aggregata sagittata n. sp. (Apicomplexa: Aggregatidae), a coccidian parasite from the European flying squid Todarodes sagittatus (Mollusca: Cephalopoda).

A new species of the coccidian genus Aggregata Frenzel, 1885 (Apicomplexa, Aggregatidae) is described from the digestive tract of Todarodes sagittatus (Lamarck), an ommastrephid squid that lives in temperate waters in the northeastern Atlantic Ocean. Using light microscopy. oöcysts were 225-333 microm in length and 220-323 microm in width. Each oöcyst contained 600-1,000 sporocysts. Sporocysts were 16-18 microm in length and 15-17 microm in width, with a smooth surface and thick wall. Each sporocyst contained 4-8 sporozoites each with a length of 11-13 microm. This paper presents the description of a new Agregata species in a nerito-oceanic flying squid. Moreover. it extends the distribution of Aggregata to oceanic cephalopod host species. A. sagittata represents the third named species in the genus Aggregata in European waters and the seventh worldwide.