Active Microbiota of Penaeus stylirostris Larvae: Partially Shaped via Vertical and Horizontal Transmissions and Larval Ontogeny.

During their entire lifecycle, mariculture animals are farmed in water that contains various microorganisms with which they are in close associations. Microbial exchanges between the animals and their surrounding water can occur. However, little is known about the interactions between shrimp larvae and water, and more especially, about larval bacterial selection and microbiota modulation across ontogeny. To address this gap, using HiSeq sequencing targeting the V4 region of the 16S rRNA molecule, we investigated the active prokaryotic diversity and structure of healthy Penaeus stylirostris larvae and seawater. Comparisons between different larval stages revealed evidence of stage-specific microbiotas and biomarkers, a core microbiota common to all stages, and shared taxa between successive stages, suggesting vertical transmission of bacterial taxa. Comparisons between stage-specific microbiotas and core microbiotas with water storages highlighted that many taxa associated with the larvae were originally present in the natural seawater, underlining horizontal transmission of bacteria from water to larvae. As some of these lineages became active at specific larval stages, we suggest that larvae were able to modulate their microbiota. This study provides insight into larvae-microbiota interactions at the larval stage scale.

Microbial biomarker detection in shrimp larvae rearing water as putative bio-surveillance proxies in shrimp aquaculture.

Background: Aquacultured animals are reared in water hosting various microorganisms with which they are in close relationships during their whole lifecycle as some of these microorganisms can be involved in their host's health or physiology. In aquaculture hatcheries, understanding the interactions existing between the natural seawater microbiota, the rearing water microbiota, the larval stage and the larval health status, may allow the establishment of microbial proxies to monitor the rearing ecosystems. Indeed, these proxies could help to define the optimal microbiota for shrimp larval development and could ultimately help microbial management. Methods: In this context, we monitored the daily composition of the active microbiota of the rearing water in a hatchery of the Pacific blue shrimp Penaeus stylirostris. Two distinct rearing conditions were analyzed; one with antibiotics added to the rearing water and one without antibiotics. During this rearing, healthy larvae with a high survival rate and unhealthy larvae with a high mortality rate were observed. Using HiSeq sequencing of the V4 region of the 16S rRNA gene of the water microbiota, coupled with zootechnical and statistical analysis, we aimed to distinguish the microbial taxa related to high mortality rates at a given larval stage. Results: We highlight that the active microbiota of the rearing water is highly dynamic whatever the larval survival rate. A clear distinction of the microbial composition is shown between the water harboring heathy larvae reared with antibiotics versus the unhealthy larvae reared without antibiotics. However, it is hard to untangle the effects of the antibiotic addition and of the larval death on the active microbiota of the rearing water. Various active taxa of the rearing water are specific to a given larval stage and survival rate except for the zoea with a good survival rate. Comparing these communities to those of the lagoon, it appears that many taxa were originally detected in the natural seawater. This highlights the great importance of the microbial composition of the lagoon on the rearing water microbiota. Considering the larval stage and larval survival we highlight that several genera: Nautella, Leisingera, Ruegerira, Alconivorax, Marinobacter and Tenacibaculum, could be beneficial for the larval survival and may, in the rearing water, overcome the r-strategist microorganisms and/or putative pathogens. Members of these genera might also act as probiotics for the larvae. Marivita, Aestuariicocccus, HIMB11 and Nioella, appeared to be unfavorable for the larval survival and could be associated with upcoming and occurring larval mortalities. All these specific biomarkers of healthy or unhealthy larvae, could be used as early routine detection proxies in the natural seawater and then during the first days of larval rearing, and might help to manage the rearing water microbiota and to select beneficial microorganisms for the larvae.

Functional Diversification of Oyster Big Defensins Generates Antimicrobial Specificity and Synergy against Members of the Microbiota.

Big defensins are two-domain antimicrobial peptides (AMPs) that have highly diversified in mollusks. Cg-BigDefs are expressed by immune cells in the oyster Crassostrea gigas, and their expression is dampened during the Pacific Oyster Mortality Syndrome (POMS), which evolves toward fatal bacteremia. We evaluated whether Cg-BigDefs contribute to the control of oyster-associated microbial communities. Two Cg-BigDefs that are representative of molecular diversity within the peptide family, namely Cg-BigDef1 and Cg-BigDef5, were characterized by gene cloning and synthesized by solid-phase peptide synthesis and native chemical ligation. Synthetic peptides were tested for antibacterial activity against a collection of culturable bacteria belonging to the oyster microbiota, characterized by 16S sequencing and MALDI Biotyping. We first tested the potential of Cg-BigDefs to control the oyster microbiota by injecting synthetic Cg-BigDef1 into oyster tissues and analyzing microbiota dynamics over 24 h by 16S metabarcoding. Cg-BigDef1 induced a significant shift in oyster microbiota ß-diversity after 6 h and 24 h, prompting us to investigate antimicrobial activities in vitro against members of the oyster microbiota. Both Cg-BigDef1 and Cg-BigDef5 were active at a high salt concentration (400 mM NaCl) and showed broad spectra of activity against bacteria associated with C. gigas pathologies. Antimicrobial specificity was observed for both molecules at an intra- and inter-genera level. Remarkably, antimicrobial spectra of Cg-BigDef1 and Cg-BigDef5 were complementary, and peptides acted synergistically. Overall, we found that primary sequence diversification of Cg-BigDefs has generated specificity and synergy and extended the spectrum of activity of this peptide family.

Microbiota of the Rearing Water of Penaeus stylirostris Larvae Influenced by Lagoon Seawater and Specific Key Microbial Lineages of Larval Stage and Survival.

Aquacultured animals are reared in water, where they interact with microorganisms which can be involved in their development, immunity, and disease. It is therefore interesting to study the rearing water microbiota, especially in the hatcheries of the Pacific blue shrimp Penaeus stylirostris, where larval mass mortalities occur. In this study, using HiSeq sequencing of the V4 region of the 16S rRNA molecule coupled with zootechnical and chemical analyses, we investigated whether any microbial lineages could be associated with certain mortality rates at a given larval stage. Our results indicate that the active microbiota of the rearing water was highly dynamic throughout the rearing process, with distinct communities influenced by progressive water eutrophication, larval stage, and survival rate. Our data also highlighted the role of the lagoon seawater on the rearing water microbiome, as many operational taxonomic units (OTUs) specific to a given larval stage and survival rate were detected in the primary reservoir which contained the lagoon water. We also identified biomarkers specific to water eutrophication, with Alteromonadaceae, Vibrionaceae, and Methylophilaceae, respectively, linked to increases in ammonia, nitrogen, and soluble reactive phosphate, or to increases in colored dissolved organic matter in the rearing water; other biomarkers were specific to certain larval stages and survival rates. Indeed, the Marinobacteraceae were specific to the Nauplii, and the Thalassospiraceae and Saprospiraceae to the Zoea Good condition; when mortality occurred, the Litoricolaceae were specific to the Zoea Bad, Microbacteraceae to the Mysis Bad, and Methylophilaceae to the Mysis Worst condition. Thus, these biomarkers might be used as potential early warning sentinels in water storage to infer the evolution of larval rearing to improve shrimp larval rearing. IMPORTANCE In New Caledonia, rearing of P. stylirostris is one of the main economic activities; unfortunately, mass larval mortalities cause important production decreases, involving major economic losses for the farmers and the Territory. This phenomenon, which has occurred at any larval stage over the past decade, is poorly understood. The significance of our research is in the identification of biomarkers specific to larval stage and survival rate, with some of these biomarkers being already present in the lagoon water. This enhances the role of the lagoon on the active microbiota of the rearing water at various larval stages and survival rates. Together, our results help us understand which active microbial communities are present in the rearing water according to larval stage and health. This might lead to broader impacts on hatcheries by helping to develop useful tools for using the water-lagoon, reservoir, or rearing-to test for the presence of these biomarkers as an early monitoring strategy.

Ontogenetical development of branchial chambers of Litopenaeus vannamei (Boone, 1931) and their involvement in osmoregulation: ionocytes and Na+/K+-ATPase.

Branchial chambers constitute the main osmoregulatory site in almost all decapod crustaceans. However, few studies have been devoted to elucidate the cellular function of specific cells in every osmoregulatory structure of the branchial chambers. In decapod crustaceans, it is well-known that the osmoregulatory function is localized in specific structures that progressively specialize from early developmental stages while specific molecular mechanisms occur. In this study, we found that although the structures developed progressively during the larval and postlarval stages, before reaching juvenile or adult morphology, the osmoregulatory capabilities of Litopenaeus vannamei were gradually established only during the development of branchiostegites and epipodites, but not gills. The cellular structures of the branchial chambers observed during the larval phase do not present the typical ultrastructure of ionocytes, neither Na+/K+-ATPase expression, likely indicating that pleura, branchiostegites, or bud gills do not participate in osmoregulation. During early postlarval stages, the lack of Na+/K+-ATPase immunoreactivity of the ionocytes from the branchiostegites and epipodites suggests that they are immature ionocytes (ionocytes type I). It could be inferred from IIF and TEM results that epipodites and branchiostegites are involved in iono-osmoregulation from PL15, while gills and pleura do not participate in this function.

The Active Microbiota of the Eggs and the Nauplii of the Pacific Blue Shrimp Litopenaeus stylirostris Partially Shaped by a Potential Vertical Transmission.

The many ecological niches present in an organism harbor distinct microorganisms called microbiota. Different factors can influence the establishment of these commensal microbial communities. In a previous article, we have concluded that some bacterial lineages associated with the early larval stages of the Pacific blue shrimp Litopenaeus stylirostris could be acquired from the breeders via a potential vertical transmission. The present study was conducted in order to investigate this hypothesis. Using HiSeq sequencing of the V4 region of 16S rRNA gene, we analyzed the active microbiota associated with the eggs and the nauplii of L. stylirsotris as well as with the reproductive organs of their breeders. Microbial communities associated with the rearing water were also considered to discriminate environmental microbial lineages. Using these analyses, we highlight a set of core bacterial families present in all samples and composed of members of Colwelliaceae, Alteromonadaceae, Pseudoalteromonadaceae, Saccharospirillaceae, Oceanospirillaceae, Vibrionaceae, Burkholderiaceae, Rhodobacteraceae, Flavobacteraceae, and Corynebacteriaceae; showing the importance of the environment in the establishment of the larval microbiota. We also present specific bacteria affiliated to the Arcobacteraceae, Rhodobacteraceae, Comamonadaceae, and Colwelliaceae families, which were only found in the breeders and their offspring strengthening the hypothesis of a potential vertical transmission shaping the active microbiota of the eggs and the nauplii of L. stylirostris.

Potential lineage transmission within the active microbiota of the eggs and the nauplii of the shrimp Litopenaeus stylirostris: possible influence of the rearing water and more.

BACKGROUND: Microbial communities associated with animals are known to be key elements in the development of their hosts. In marine environments, these communities are largely under the influence of the surrounding water. In aquaculture, understanding the interactions existing between the microbiotas of farmed species and their rearing environment could help establish precise bacterial management. METHOD: In light of these facts, we studied the active microbial communities associated with the eggs and the nauplii of the Pacific blue shrimp (Litopenaeus stylirostris) and their rearing water. All samples were collected in September 2018, November 2018 and February 2019. After RNA extractions, two distinct Illumina HiSeq sequencings were performed. Due to different sequencing depths and in order to compare samples, data were normalized using the Count Per Million method. RESULTS: We found a core microbiota made of taxa related to Aestuariibacter, Alteromonas, Vibrio, SAR11, HIMB11, AEGEAN 169 marine group and Candidatus Endobugula associated with all the samples indicating that these bacterial communities could be transferred from the water to the animals. We also highlighted specific bacterial taxa in the eggs and the nauplii affiliated to Pseudomonas, Corynebacterium, Acinetobacter, Labrenzia, Rothia, Thalassolituus, Marinobacter, Aureispira, Oleiphilus, Profundimonas and Marinobacterium genera suggesting a possible prokaryotic vertical transmission from the breeders to their offspring. This study is the first to focus on the active microbiota associated with early developmental stages of a farmed shrimp species and could serve as a basis to comprehend the microbial interactions involved throughout the whole rearing process.

Whole-Genome Sequence of Pseudoalteromonas sp. NC201, a Probiotic Strain for Litopenaeus stylirostris Hatcheries in New Caledonia.

The marine bacterium Pseudoalteromonas sp. strain NC201 has shown probiotic potential in Litopenaeus stylirostris rearing. In this study, the complete genome of NC201 was sequenced. This genome consists of a chromosome (4.13 Mb) and a chromid (1.24 Mb). The genome contains gene clusters coding for antibacterial peptides and secondary metabolites.

The effects of acute transfer to freshwater on ion transporters of the pharyngeal cavity in European seabass (Dicentrarchus labrax).

Gene expression of key ion transporters (the Na+/K+-ATPase NKA, the Na+, K+-2Cl- cotransporter NKCC1, and CFTR) in the gills, opercular inner epithelium, and pseudobranch of European seabass juveniles (Dicentrarchus labrax) were studied after acute transfer up to 4 days from seawater (SW) to freshwater (FW). The functional remodeling of these organs was also studied. Handling stress (SW to SW transfer) rapidly induced a transcript level decrease for the three ion transporters in the gills and operculum. NKA and CFTR relative expression level were stable, but in the pseudobranch, NKCC1 transcript levels increased (up to 2.4-fold). Transfer to FW induced even more organ-specific responses. In the gills, a 1.8-fold increase for NKA transcript levels occurs within 4 days post transfer with also a general decrease for CFTR and NKCC1. In the operculum, transcript levels are only slightly modified. In the pseudobranch, there is a transient NKCC1 increase followed by 0.6-fold decrease and 0.8-fold CFTR decrease. FW transfer also induced a density decrease for the opercular ionocytes and goblet cells. Therefore, gills and operculum display similar trends in SW-fish but have different responses in FW-transferred fish. Also, the pseudobranch presents contrasting response both in SW and in FW, most probably due to the high density of a cell type that is morphologically and functionally different compared to the typical gill-type ionocyte. This pseudobranch-type ionocyte could be involved in blood acid-base regulation masking a minor osmotic regulatory capacity of this organ compared to the gills.

Nigritoxin is a bacterial toxin for crustaceans and insects.

The Tetraconata (Pancrustacea) concept proposes that insects are more closely related to aquatic crustaceans than to terrestrial centipedes or millipedes. The question therefore arises whether insects have kept crustacean-specific genetic traits that could be targeted by specific toxins. Here we show that a toxin (nigritoxin), originally identified in a bacterial pathogen of shrimp, is lethal for organisms within the Tetraconata and non-toxic to other animals. X-ray crystallography reveals that nigritoxin possesses a new protein fold of the α/ß type. The nigritoxin N-terminal domain is essential for cellular translocation and likely encodes specificity for Tetraconata. Once internalized by eukaryotic cells, nigritoxin induces apoptotic cell death through structural features that are localized in the C-terminal domain of the protein. We propose that nigritoxin will be an effective means to identify a Tetraconata evolutionarily conserved pathway and speculate that nigritoxin holds promise as an insecticidal protein.

Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): Deciphering the role of the Na(+)/K(+)-ATPase.

The role of the main ion transporting enzyme Na+/K+-ATPase in osmoregulation processes was investigated in Litopenaeus stylirostris. The development and localization of the osmoregulation sites were studied during ontogenesis by immunodetection of Na(+)K(+)-ATPase using monoclonal antibodies and transmission electron microscopy (TEM). Osmoregulation sites were identified as the pleurae and branchiostegites in the zoeae and mysis stages. In the subsequent post-metamorphic stages the osmoregulatory function was mainly located in the epipodites and branchiostegites and osmotic regulation was later detected in the gills. The presence of ionocytes and microvilli in these tissues confirmed their role in ionic processes. The complete open reading frame of the mRNA coding for the α-subunit of Na+K+-ATPase was characterized in L. stylirostris. The resulting 3092-bp cDNA (LsNKA) encodes a putative 1011-amino-acid protein with a predicted molecular mass of 112.3kDa. The inferred amino acid sequence revealed that the putative protein possesses the main structural characteristics of the Na+K+-ATPase α-subunits. Quantitative RT-PCR analyses indicated that LsNKA transcripts did not significantly vary between the different developmental stages. The number of transcripts was about 2.5-fold higher in the epipodites and gills than in any other tissues tested in juveniles. A reverse genetic approach was finally implemented to study the role of LsNKA in vivo. Knockdown of LsNKA expression by gene-specific dsRNA injection led to an increase of shrimp mortality following an abrupt salinity change compared to control animals. These data strongly suggest that LsNKA plays an important role in osmoregulation when the shrimp are challenged by changing salinities.

Osmoregulation in larvae and juveniles of two recently separated Macrobrachium species: Expression patterns of ion transporter genes.

In this comparative study, osmoregulatory mechanisms were analyzed in two closely related species of palaemonid shrimp from Brazil, Macrobrachium pantanalense and Macrobrachium amazonicum. A previous investigation showed that all postembryonic stages of M. pantanalense from inland waters of the Pantanal are able to hyper-osmoregulate in fresh water, while this species was not able to hypo-osmoregulate at high salinities. In M. amazonicum originating from the Amazon estuary, in contrast, all stages are able to hypo-osmoregulate, but only first-stage larvae, late juveniles and adults are able to hyper-osmoregulate in fresh water. The underlying molecular mechanisms of these physiological differences have not been known. We therefore investigated the expression patterns of three ion transporters (NKA α-subunit, VHA B-subunit and NHE3) following differential salinity acclimation in different ontogenetic stages (stage-V larvae, juveniles) of both species. Larval NKAα expression was at both salinities significantly higher in M. pantanalense than in M. amazonicum, whereas no difference was noted in juveniles. VHA was also more expressed in larvae of M. pantanalense than in those of M. amazonicum. When NHE3 expression is compared between the larvae of the two species, further salinity-related differences were observed, with generally higher expression in the inland species. Overall, a high expression of ion pumps in M. pantanalense suggests an evolutionary key role of these transporters in freshwater invasion.

Differential distribution of V-type H(+)-ATPase and Na (+)/K (+)-ATPase in the branchial chamber of the palaemonid shrimp Macrobrachium amazonicum.

V-H(+)-ATPase and Na(+)/K(+)-ATPase were localized in the gills and branchiostegites of M. amazonicum and the effects of salinity on the branchial chamber ultrastructure and on the localization of transporters were investigated. Gills present septal and pillar cells. In freshwater (FW), the apical surface of pillar cells is amplified by extensive evaginations associated with mitochondria. V-H(+)-ATPase immunofluorescence was localized in the membranes of the apical evaginations and in clustered subapical areas of pillar cells, suggesting labeling of intracellular vesicle membranes. Na(+)/K(+)-ATPase labeling was restricted to the septal cells. No difference in immunostaining was recorded for both proteins according to salinity (FW vs. 25 PSU). In the branchiostegite, both V-H(+)-ATPase and Na(+)/K(+)-ATPase immunofluorescence were localized in the same cells of the internal epithelium. Immunogold revealed that V-H(+)-ATPase was localized in apical evaginations and in electron-dense areas throughout the inner epithelium, while Na(+)/K(+)-ATPase occurred densely along the basal infoldings of the cytoplasmic membrane. Our results suggest that morphologically different cell types within the gill lamellae may also be functionally specialized. We propose that, in FW, pillar cells expressing V-H(+)-ATPase absorb ions (Cl(-), Na(+)) that are transported either directly to the hemolymph space or through a junctional complex to the septal cells, which may be responsible for active Na(+) delivery to the hemolymph through Na(+)/K(+)-ATPase. This suggests a functional link between septal and pillar cells in osmoregulation. When shrimps are transferred to FW, gill and branchiostegite epithelia undergo ultrastructural changes, most probably resulting from their involvement in osmoregulatory processes.

Adaptation to freshwater in the palaemonid shrimp Macrobrachium amazonicum: comparative ontogeny of osmoregulatory organs.

The ontogeny of osmoregulatory organs was studied in two geographically isolated populations of the palaemonid shrimp Macrobrachium amazonicum, one originating from the Amazon estuary (A) and the other from inland waters of the Pantanal (P) in northeastern and southwestern Brazil, respectively. A previous investigation had shown that the estuarine population is able to hypo-osmoregulate in seawater, whereas the hololimnetic inland population has lost this physiological function. In the present study, the structural development of the branchial chamber and excretory glands and the presence of Na(+)/K(+)-ATPase (NKA) were compared between populations and between larval and juvenile stages after exposure to two salinities representing hypo- and hypertonic environments. In the newly hatched zoea I stage of both populations, gills were absent and NKA was localized along the inner epithelium of the branchiostegite. In intermediate (zoea V) and late larval stages (decapodids), significant differences between the two populations were observed in gill development and NKA expression. In juveniles, NKA was detected in the gills and branchiostegite, with no differences between populations. At all developmental stages and in both populations, NKA was present in the antennal glands upon hatching. The strong hypo-osmoregulatory capacity of the early developmental stages in population A could be linked to ion transport along the inner side of the branchiostegite; this seemed to be absent or weak in population P. The presence of fully functional gills expressing NKA appears to be essential for efficient hyper-osmoregulation in late developmental stages during successful freshwater adaptation and colonization.

The ClC-3 chloride channel and osmoregulation in the European sea bass, Dicentrarchus labrax.

Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na(+)/K(+)-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10 min to 30 days. In gills, NKA-α1 subunit expression transiently increased from 10 min and reached a stabilized intermediate expression level after 24 h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50 %) 1 h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30 % higher 7 days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-α1 subunit expression was followed by significantly higher stable levels from 24 h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30 days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.

Osmoregulatory response to low salinities in the European sea bass embryos: a multi-site approach.

Embryonic osmoregulation effected by embryonic ionocytes in the European sea bass Dicentrarchus labrax has been studied at several sites, including the yolk sac membrane, the first gill slits and the gut ionocytes. D. labrax embryos, spawned in seawater (SW) (39 ‰), were exposed to dilute seawater (DSW) (5 ‰) during 48 h, from stage 10 pairs of somites (10S) to hatching time (HT). Control embryos originating from the same spawn were maintained in SW. Both SW and DSW embryos were examined after 24- and 48-h exposure. Nanoosmometric measurements of the embryonic fluids osmolality suggest that late embryos are confronted with the variations in external salinity and that they were able to slightly regulate their osmolality. Immunolocalization of Na⁺/K⁺ ATPase, NKCC and CFTR has shown that DSW-exposed embryos can limit ion losses due to compensatory physiological mechanisms. CFTR and NKCC were not observed in DSW embryos in the yolk sac ionocytes and in the tegumentary ionocytes of the gill slits. The quantification of mRNA indicated that NKA, NKCC1 and CFTR transcript levels increased from stage 10S to stage HT. At stage HT, following 48 h of DSW- or SW-exposure, different responses were observed according to salinity. These results, when compared to those obtained in D. labrax juveniles and adults long-term exposed to fresh water (FW), show that in embryos the physiological response following a short-term DSW exposure is different. The mechanisms of hyper-osmoregulation observed in D. labrax embryos, although not fully efficient, allow their survival for several days in DSW.

Expression and Localization of Aquaporin 1a in the Sea-Bass (Dicentrarchus labrax) during Ontogeny.

The successful establishment of a species in a given habitat depends on the ability of each of its developing stages to adapt to the environment. In order to understand this process we have studied the adaptation of a euryhaline fish, the sea-bass Dicentrarchus labrax, to various salinities during its ontogeny. The expression and localization of Aquaporin 1a (AQP1a) mRNA and protein were determined in different osmoregulatory tissues. In larvae, the sites of AQP1a expression are variable and they shift according to age, implying functional changes. In juveniles after metamorphosis (D32-D48 post-hatch, 15-25 mm) and in pre-adults, an increase in AQP1a transcript abundance was noted in the digestive tract, and the AQP1a location was observed in the intestine. In juveniles (D87-D100 post-hatch, 38-48 mm), the transcript levels of AQP1a in the digestive tract and in the kidney were higher in sea water (SW) than at lower salinity. These observations, in agreement with existing models, suggest that in SW-acclimated fish, the imbibed water is absorbed via AQP1a through the digestive tract, particularly the intestine and the rectum. In addition, AQP1a may play a role in water reabsorption in the kidney. These mechanisms compensate dehydration in SW, and they contribute to the adaptation of juveniles to salinity changes during sea-lagoon migrations. These results contribute to the interpretation of the adaptation of populations to habitats where salinity varies.

Cellular and molecular osmoregulatory responses to cadmium exposure in Gammarus fossarum (Crustacea, Amphipoda).

Osmoregulation represents a reliable indicator of the physiological state of crustaceans. It is mainly effected in gills via Na(+)/K(+)-ATPase (NKA) providing the major driving force for ion transport. In the present study conducted in the freshwater amphipod Gammarus fossarum, the impact of an exposure to 15 µg Cd L(-1) for 3 and 7d was investigated on the haemolymph osmolality (HO), gill structure, NKA localization in gills and its relative expression. In Cd-exposed G. fossarum, mean HO significantly decreased compared to controls. In animals exposed for 3 and 7d, high inter-individual variations in HO values were noted, resulting in their separation into unimpacted, slightly impacted and impacted animals. In unimpacted individuals, gills retained their organization, showing a thicker gill epithelium than in controls; NKA fluorescence was continuously observed along the gill epithelium and was distributed on a broader area than in controls. In slightly impacted individuals, a thinner epithelium, a slight collapse of the gill and a lower NKA fluorescence were observed compared to unimpacted specimens. In impacted individuals, dramatic alterations of the gill structure, including hyperplasia and alteration of the pillars, resulting in the collapse of the gill and the disappearance of the haemolymphatic canals were observed, as well as very limited NKA fluorescence. Therefore, the degree of gill alteration and the intensity of NKA fluorescence observed in the different groups were correlated with their respective HO levels. The relative amount of the NKA α-subunit mRNA significantly increased in specimens exposed to Cd for 3d compared to controls, and then returned to control level after 7d. The relationships between the changes in HO values, NKA immunostaining and mRNA relative expression are discussed. These results confirm that HO represents a valuable biomarker to evaluate crustacean health, and they underline the interest to assess individual responses to contaminants.

Microarray-based identification of gonad transcripts differentially expressed between lines of Pacific oyster selected to be resistant or susceptible to summer mortality.

Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event, using a cDNA microarray that we designed. ANOVA analysis revealed that 34 genes were differentially expressed between R and S lines on four dates preceding the mortality event. Annotation of some of these genes highlights reproduction and its allocation and antioxidant defenses as the main pathways that operate differentially between R and S lines. This transcriptional analysis provides new indications to define markers for quantitative trait loci searches and functional studies and evaluate the potential role of each gene in the resistance to summer mortality.

Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase.

BACKGROUND: Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. DESCRIPTION: In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. CONCLUSION: A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism.