The Kinome of Pacific Oyster Crassostrea gigas, Its Expression during Development and in Response to Environmental Factors

Oysters play an important role in estuarine and coastal marine habitats, where the majority of humans live. In these ecosystems, environmental degradation is substantial, and oysters must cope with highly dynamic and stressful environmental constraints during their lives in the intertidal zone. The availability of the genome sequence of the Pacific oyster Crassostrea gigas represents a unique opportunity for a comprehensive assessment of the signal transduction pathways that the species has developed to deal with this unique habitat. We performed an in silico analysis to identify, annotate and classify protein kinases in C. gigas, according to their kinase domain taxonomy classification, and compared with kinome already described in other animal species. The C. gigas kinome consists of 371 protein kinases, making it closely related to the sea urchin kinome, which has 353 protein kinases. The absence of gene redundancy in some groups of the C. gigas kinome may simplify functional studies of protein kinases. Through data mining of transcriptomes in C. gigas, we identified part of the kinome which may be central during development and may play a role in response to various environmental factors. Overall, this work contributes to a better understanding of key sensing pathways that may be central for adaptation to a highly dynamic marine environment.

Faisabilité biologique de la production de larves d'huîtres creuses Crassostrea gigas à partir d'eau de mer du site de Leucate. Convention de recherche Ifremer/Région Languedoc-Roussillon - Rapport final.

Afin de vérifier sur un site régional la présence d'une qualité d'eau de mer permettant la réalisation d'élevages larvaires d'huîtres creuses Crassostrea gigas (et/ou d'huîtres plates Ostrea edulis) sur une base régulière tout au long de l'année, une série d'élevages comparatifs a été réalisée à la Station IFREMER de Palavas, de mars 1995 à septembre 1995. Le matériel biologique nécessaire à cette comparaison est constitué de larves d'huîtres creuses Crassostrea gigas et en algues unicellulaires provenant de l'éc1oserie commerciale de la SATMAR. Les résultats obtenus démontrent la capacité de l'eau de mer du site étudié sur Leucate à supporter la production de larves d'huîtres creuses compétentes à se métamorphoser et à se fixer. Cette étude s'inscrit dans la démarche de la Région Languedoc-Roussillon et du CEPRALMAR visant à favoriser l'implantation d'une écloserie de production de Mollusques Bivalves sur le littoral méditerranéen.

Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections

Since 2008, massive mortality events of Pacific oysters (Crassostrea gigas) have been reported worldwide and these disease events are often associated with Ostreid herpesvirus type 1 (OsHV-1). Epidemiological field studies have also reported oyster age and other pathogens of the Vibrio genus are contributing factors to this syndrome. We undertook a controlled laboratory experiment to simultaneously investigate survival and immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Our data corroborates epidemiological studies that juveniles are more susceptible to OsHV-1, whereas adults are more susceptible to Vibrio. We measured the expression of 102 immune-genes by high-throughput RT-qPCR, which revealed oysters have different transcriptional responses to OsHV-1 and Vibrio. The transcriptional response in the early stages of OsHV-1 infection involved genes related to apoptosis and the interferon-pathway. Transcriptional response to Vibrio infection involved antimicrobial peptides, heat shock proteins and galectins. Interestingly, oysters in the later stages of OsHV-1 infection had a transcriptional response that resembled an antibacterial response, which is suggestive of the oyster's microbiome causing secondary infections (dysbiosis-driven pathology). This study provides molecular evidence that oysters can mount distinct immune response to viral and bacterial pathogens and these responses differ depending on the age of the host.

Salinity influences disease-induced mortality of the oyster Crassostrea gigas and infectivity of the ostreid herpesvirus 1 (OsHV-1)

Mortality of young Pacific oysters Crassostrea gigas associated with the ostreid herpesvirus 1 (OsHV-1) is occurring worldwide. Here, we examined for the first time the effect of salinity on OsHV-1 transmission and disease-related mortality of C. gigas, as well as salinity-related effects on the pathogen itself. To obtain donors for OsHV-1 transmission, we transferred laboratory-raised oysters to an estuary during a disease outbreak and then back to the laboratory. Oysters that tested OsHV-1 positive were placed in seawater tanks (35‰, 21°C). Water from these tanks was used to infect naïve oysters in 2 experimental setups: (1) oysters acclimated or non-acclimated to a salinity of 10, 15, 25 and 35‰ and (2) oysters acclimated to a salinity of 25‰; the latter were exposed to OsHV-1 water diluted to a salinity of 10 or 25‰. The survival of oysters exposed to OsHV-1 water and acclimated to a salinity of 10‰ was >95%, compared to only 43 to 73% survival in oysters acclimated to higher salinities (Expt 1), reflecting differences in the levels of OsHV-1 DNA and viral gene expression (Expts 1 and 2). However, the survival of their non-acclimated counterparts was only 23% (Expt 2), and the levels of OsHV-1 DNA and the expression of 4 viral genes were low (Expt 1). Thus, OsHV-1 may not have been the ultimate cause of mortality in non-acclimated oysters weakened by a salinity shock. It appears that reducing disease risk by means of low salinity is unlikely in the field.

Resposta metabólica da ostra Crassostrea gigas na presença do parasita Amyloodinium ocellatum

Dissertação de Mestrado, Aquacultura e Pescas, Especialização em Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Transport of Pirarucu Arapaima gigas juveniles in plastic bag

Este estudo examinou as respostas de estresse de juvenis de pirarucu transportados em sistema fechado. Pirarucu (Arapaima gigas) é um peixe nativo da bacia Amazônica, da família Osteoglossidae que possui respiração aérea obrigatória. Foi realizado um transporte de curta duração (6 h) em sacos de polietileno inflados com ar atmosférico (grupo ar) ou com oxigênio puro (grupo oxi). O oxigênio dissolvido foi o único parâmetro de qualidade da água que apresentou diferença estatística entre os grupos, e como esperado, o oxigênio estava supersaturado para o grupo oxi. Não houve mortalidade após o transporte em ambos os grupos. Os peixes se alimentaram 36 h após o transporte e apresentaram um consumo de ração habitual após 72 h. As respostas fisiológicas foram semelhantes nos dois grupos. O cortisol não apresentou mudança significativa durante o período de amostragem. Ao contrário da maioria das espécies, os valores de cortisol se apresentaram inalterados nos dois grupos durante a amostragem, enquanto a glicose teve um aumento significativo até 12 h após o transporte. Os resultados mostram que o transporte de pirarucu em sacos de polietileno pode ser realizado com ar atmosférico ou oxigênio puro, uma vez que as respostas de estresse, a qualidade da água e o comportamento alimentar após 36 h foram similar entre os grupos.

Occurrence of OsHV-1 in Crassostrea gigas cultured in Ireland during an exceptionally warm summer. Selection of less susceptible oysters

The occurrence of OsHV-1, a herpes virus causing mass mortality in the Pacific oyster Crassostrea gigas was investigated with the aim to select individuals with different susceptibility to the infection. Naïve spat transferred to infected areas and juveniles currently being grown at those sites were analyzed using molecular and histology approaches. The survey period distinguishes itself by very warm temperatures reaching up to 3.5°C above the average. The virus was not detected in the virus free area although a spread of the disease could be expected due to high temperatures. Overall mortality, prevalence of infection and viral load was higher in spat confirming the higher susceptibility in early life stages. OsHV-1 and oyster mortality were detected in naïve spat after 15 days of cohabitation with infected animals. Although, infection was associated with mortality in spat, the high seawater temperatures could also be the direct cause of mortality at the warmest site. One stock of juveniles suffered an event of abnormal mortality that was significantly associated with OsHV-1 infection. Those animals were infected with a previously undescribed microvariant whereas the other stocks were infected with OsHV-1 μVar. Cell lesions due to the infection were observed by histology and true infections were corroborated by in situ hybridization. Survivors from the natural outbreak were exposed to OsHV-1 μVar by intramuscular injection and were compared to naïve animals. The survival rate in previously exposed animals was significantly higher than in naïve oysters. Results derived from this study allowed the selection of animals that might possess interesting characteristics for future analysis on OsHV-1 resistance.

Lethal dose and clinical signs of Aeromonas hydrophila in Arapaima gigas (Arapaimidae), the giant fish from Amazon.

Aeromonas hydrophila is causing substantial economic losses in world aquaculture. This study determined the tolerance limit (LD50-96h) of A. hydrophila in Arapaima gigas, and also investigated the clinical signs after intradermal inoculation.

Efeitos in vitro da Mentha piperita (Lamiaceae) em Dawestrema spp. (Monogenoidea) e toxicidade aguda em Arapaima gigas (Arapaimidae).

Este estudo avaliou o efeito in vitro do óleo essencial de Mentha piperita L. sobre monogenoideas e sua ação tóxica para Arapaima gigas.

Respostas de estresse em pirarucu (Arapaima gigas) durante práticas de rotina em piscicultura.

O objetivo deste trabalho foi analisar as respostas de estresse em pirarucu quando submetido a práticas comuns em sistema de criação, como o transporte, o adensamento e a exposição a altas concentrações de amônia.

Uso de sal durante o transporte de juvenis (1kg) de pirarucu (Arapaima gigas).

O pirarucu é um peixe nativo da bacia Amazônica cuja criação vem sendo estudada em algumas partes do Brasil. O objetivo desse trabalho foi testar o sal de cozinha como mitigador de estresse durante o transporte de juvenis de pirarucu (1 kg). Para isso, os peixes foram transportados em dois diferentes sistemas: caixas sem adição de oxigênio (transporte aberto) e sacos plásticos com injeção de oxigênio e lacrado (transporte fechado).

Net uptake of dissolved free amino acids by the giant clam, Tridacna maxima: alternative sources of energy and nitrogen?

The role of dissolved free amino acids (DFAA) in nitrogen and energy budgets was investigated for the giant clam, Tridacna maxima, growing under field conditions at One Tree Island, at the southern end of the Great Barrier Reef, Australia. Giant clams (121.5-143.7 mm in shell length) took up neutral, acidic and basic amino acids. The rates of net uptake of DFAA did not differ between light and dark, nor for clams growing under normal or slightly enriched ammonium concentrations. Calculations based on the net uptake concentrations typical of the maximum concentrations of DFAA found in coral reef waters (similar to 0.1 mu M)revealed that DFAA could only contribute 0.1% and 1% of the energy and nitrogen demands of giant clams, respectively. These results suggest that DFAA does not supply significant amounts of energy or nitrogen for giant clams or their symbionts.

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neo-goniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H, curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in H. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.

An architectonic comparison of the ventrobasal complex of two Megachiropteran and one Microchiropteran bat: implications for the evolution of Chiroptera

The architectonic features of the thalamic ventrobasal complex (Vb) of two species of Megachiropteran (Grey-headed flying fox, Pteropus poliocephalus, and the Eastern tube-nosed bat, Nyctimene robinsoni) are compared with those of a Microchiropteran (Australian ghost bat, Macroderma gigas). The somatosensory system was chosen for comparison as it represents a sensory system that has undergone analogous modifications in both Chiropteran lineages (the evolution of the wing). The components of Vb were examined as there are taxon-specific features in this region of the brain. Within the Megachiropteran Vb, four subnuclei were recognized: the ventral posterior medial (VPM), the ventral posterior lateral (VPL), the ventral posterior inferior (VPI), and the basal ventral medial (VMb). In the ghost bat only VPM and VPL were identified with certainty. No VPI was evident in the ghost bat, however a putative VMb was observed. Vb of the ghost bat also lacked the arcuate lamina, which distinguishes VPM from VPL in the Megachiropterans and many other mammals. These taxon-specific differences lend support to the proposal that the order Chiroptera has a diphyletic origin.

Spermatozoal ultrastructure of spiny oysters (Spondylidae, Bivalvia) including a comparison with other bivalves

Sperm ultrastructure in three representative species of the marine bivalve family Spondylidae (spiny or thorny oysters) is examined and compared with available data on other bivalves, especially other families of the subclass Pteriomorphia. Spondylid spermatozoa are of the externally fertilizing aquasperm. type (ect-aquasperm). The acrosomal vesicle is conical with a deep basal invagination extending almost the full length of the vesicle. Vesicle contents are divisible into an inner, highly electron-dense anterior layer and a less dense posterior layer. The anterior layer is folded back on itself posteriorly and exhibits radiating plates (best developed peripherally). The vesicle rests on, and is partially embedded in, an extensive granular deposit of subacrosomal. material at the nuclear apex. This deposit extends partly into acrosomal vesicle invagination and also fills a broad depression in the anterior of the nucleus. No pre-formed axial rod (perforatorium) is present. The nucleus is round-pyriform and its contents coarsely fibrogranular. At the base of the nucleus, four broad depressions partially accommodate the midpiece mitochondria. The midpiece consists the four spherical mitochondria and the proximal and distal centrioles. The centrioles are arranged at approximately 90degrees to each other, and each consists of nine, angularly-oriented, microtubular triplets embedded in a granular matrix. A short, periodically banded rootlet connects the proximal centriole to the nuclear fossa, whereas the distal centriole, which forms the basal body to the flagellar axoneme, is anchored to the plasma membrane by nine terminally forked satellite fibres. Extensive deposits of putative glycogen rosettes surround the centrioles and mitochondria. The flagellum consists of a 9+2 axoneme sheathed by the plasma membrane. Spondylid spermatozoa strongly resemble those of the Pectinidae, further confirming the traditional view (based on comparative anatomy and shell morphology) of a close relationship between the Spondylidae and the Pectinidae. Differences in acrosomal shape and dimensions were noted between the three species examined, indicating potential taxonomic utility for comparative sperm ultrastructure within the Spondylidae.