Comparative behavioral ecotoxicology of Inland Silverside larvae exposed to pyrethroids across a salinity gradient

Pyrethroids, a class of commonly used insecticides, are frequently detected in aquatic environments, including estuaries. The influence that salinity has on organism physiology and the partitioning of hydrophobic chemicals, such as pyrethroids, has driven interest in how toxicity changes in saltwater compared to freshwater. Early life exposures in fish to pyrethroids cause toxicity at environmentally relevant concentrations, which can alter behavior. Behavior is a highly sensitive endpoint that influences overall organism fitness and can be used to detect toxicity of environmentally relevant concentrations of aquatic pollutants. Inland Silversides (Menidia beryllina), a commonly used euryhaline model fish species, were exposed from 5 days post fertilization (~1-day pre-hatch) for 96 h to six pyrethroids: bifenthrin, cyfluthrin, cyhalothrin, cypermethrin, esfenvalerate and permethrin. Exposures were conducted at three salinities relevant to brackish, estuarine habitat (0.5, 2, and 6 PSU) and across 3 concentrations, either 0.1, 1, 10, and/or 100 ng/L, plus a control. After exposure, Inland Silversides underwent a behavioral assay in which larval fish were subjected to a dark and light cycle stimuli to determine behavioral toxicity. Assessment of total distanced moved and thigmotaxis (wall hugging), used to measure hyper/hypoactivity and anxiety like behavior, respectively, demonstrate that even at the lowest concentration of 0.1 ng/L pyrethroids can induce behavioral changes at all salinities. We found that toxicity decreased as salinity increased for all pyrethroids except permethrin. Additionally, we found evidence to suggest that the relationship between log KOW and thigmotaxis is altered between the lower and highest salinities

Studying interaction between the Pacific oyster, Crassostrea gigas, and Ostreid herpesvirus type 1

Ostreid herpesvirus type 1 (OsHV-1) can be considered one of the major infectious agents in Pacific oysters, Crassostrea gigas, in France and all around the world. Differences in terms of mortality among infected animals were reported suggesting a genetic basis of the susceptibility to the OsHV-1 infection. In this context, the main objective of this thesis was to understand the interactions between Pacific oysters and OsHV-1, in particular, the molecular basis of the viral cycle. Results showed that the virus is able to replicate in the host regardless of its stage of development or its susceptibility. However, multiplication kinetics is faster in the most susceptible individuals compared to less susceptible ones. After an active replication phase, it would appear that the virus is no detectable in survival individuals. This observation suggests (i) a remission with elimination of the virus or (ii) virus persistence without detectable symptoms. These results highlight the ability of the virus circulating in the host without causing mortality. These individuals can excrete viral particles and interfere with the infection process in the field. Genes that could be involved in antiviral defenses were also identified in Pacific oysters (IFI44, IAP, glypican, …). All these results represent a first contribution to the understanding of OsHV-1 cycle in Pacific oysters, particularly at the molecular level.Le virus ostreid herpesvirus type 1 (OsHV-1), peut être considéré comme un des agents infectieux majeurs affectant les élevages d’huîtres creuses, Crassostrea gigas, en France et dans le monde. Des différences de mortalités ont été observées au sein de cette espèce lors de l’infection virale laissant suspecter une base génétique de la sensibilité à la maladie. Dans ce contexte, l’objectif principal du travail de thèse était de mieux comprendre les interactions entre l’huître creuse et OsHV-1, et plus particulièrement, les bases moléculaires du cycle viral. Les résultats obtenus montrent que le virus est capable de se répliquer chez l’hôte quel que soit son stade de développement et sa sensibilité à l’infection. Cependant, la cinétique de multiplication est plus rapide chez des individus les plus sensibles comparés aux animaux moins sensibles. Il apparaît également que chez les individus survivants suite à l’infection, le virus n’est plus détectable après une phase de réplication active. Cette observation laisse suspecter (i) une rémission avec une élimination du virus ou (ii) une persistance du virus sans symptômes détectables. Ces résultats mettent en lumière la possibilité du virus de circuler au sein des individus ne présentant pas de signes particuliers de maladie. Ces individus peuvent excréter des particules virales et intervenir ainsi dans le processus d’infection en milieu naturel. Les travaux réalisés ont également permis d’identifier des gènes d’intérêt chez l’huître creuse, pouvant intervenir dans les mécanismes de défense anti-viraux (IFI44, IAP, glypican, …). L’ensemble de ces résultats représente une contribution à la compréhension du cycle d’OsHV-1 et des réponses de l’hôte, plus particulièrement au niveau moléculaire

Quantification of genes expressed during an in vitro infection of haemocytes from Ostrea edulis with parasites Bonamia ostreae by Real times PCR

Oyster fanning represents a significant economic part of the French aquaculture production. This production is approximately about 150 000 tons of shells a year [Buestel, 2004]. However the development ofthis activity can be limited by infectious diseases (virus, protozoan or bacteria). Flat oysters suffered from two epizooties caused by two parasitic di seases: martieilliosis and the bonamiosis. These diseases appeared at the end of the Sixties and Seventies respectively. These diseases had a negative impact on the economy because they decrease the production of Flat oysters by 90% between 1979 and .1980. [Buestel 2004, Grizel 1985] The means of fights against bonamiosis are limited and mainly rely on a preventive approach. In fact, it is impossible to use the vaccination especially because they do not have abodes. But it is also impossible to use treatments for invertebrate because they are product in open systems on the wild. The bonamiosis is a notifiable disease listed by the The World Organisation for Animal Health (OIE) as weil as by European Union. That implies a surveillance of stocks and restrictions of transfer in order to avoid the contamination of healthy zones free. In addition another approach is based on the obtention of resistant animaIs through a genetic selection. Thanks to that it is possible to obtain a better rate of survival in the zones where the parasite is endemic [Naciri et Al.; 1998]. In this context, a better understanding of defence mechanisms developed by the flat oyster against parasite and interaction between oyster and parasite is needed. These data are necessary for the conservation of the flat oyster and to the restoring of flat oyster on our coasts. The objective of this work was to study in vitro the expression of genes of haemocytes of flat oyster in contact with some purified parasite Bonamia ostreae. In a first step, our study has consisted in standardizing the Real Time Quantitative PCR in order to obtain curves standards for quantity the expression of genes of Ostrea edulis. After a session dedicated to bibliographical researches, we will see material and methods presented in the second part. Lastly results will be exposed and discussed

Transcriptomic study of 39 ostreid herpesvirus 1 genes during an experimental infection

Massive mortality outbreaks have been reported in France since 2008 among Pacific oysters, Crassostrea gigas, with the detection of a particular OsHV-1 variant called μVar. Virus infection can be induced in healthy spat in experimental conditions allowing to better understand the disease process, including viral gene expression. Although gene expression of other herpesviruses has been widely studied, we provide the first study following viral gene expression of OsHV-1 over time. In this context, an in vivo transcriptomic study targeting 39 OsHV-1 genes was carried out during an experimental infection of Pacific oyster spat. For the first time, several OsHV-1 mRNAs were detected by real-time PCR at 0 h, 2 h, 4 h, 18 h, 26 h and 42 h post injection. Several transcripts were detected at 2 h post infection and at 18 h post infection for all selected ORFs. Quantification of virus gene expression at different times of infection was also carried out using an oyster housekeeping gene, Elongation factor. Developing an OsHV-1-specific reverse transcriptase real time PCR targeting 39 viral gene appears a new tool in terms of diagnosis and can be used to complement viral DNA detection in order to monitor viral replication

Detection and distribution of ostreid herpesvirus 1 in experimentally infected Pacific oyster spat

High mortality rates are reported in spat and larvae of Pacific oyster Crassostrea gigas and associated with ostreid herpesvirus 1 (OsHV-1) detection in France. Although the viral infection has been experimentally reproduced in oyster larvae and spat, little knowledge is currently available concerning the viral entry and its distribution in organs and tissues. This study compares OsHV-1 DNA and RNA detection and localization in experimentally infected oysters using two virus doses: a low dose that did not induce any mortality and a high dose inducing high mortality. Real time PCR demonstrated significant differences in terms of viral DNA amounts between the two virus doses. RNA transcripts were detected in oysters receiving the highest dose of viral suspension whereas no transcript was observed in oysters injected with the low dose. This study also allowed observing kinetics of viral DNA and RNA detection in different tissues of oyster spat. Finally, viral detection was significantly different in function of tissues (p < 0.005), time (p < 0.005) with an interaction between tissues and time (p < 0.005) for each probe

In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas

Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection. Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein leve

Salinity Changes the Dynamics of Pyrethroid Toxicity in Terms of Behavioral Effects on Newly Hatched Delta Smelt Larvae

Salinity can interact with organic compounds and modulate their toxicity. Studies have shown that the fraction of pyrethroid insecticides in the aqueous phase increases with increasing salinity, potentially increasing the risk of exposure for aquatic organisms at higher salinities. In the San Francisco Bay Delta (SFBD) estuary, pyrethroid concentrations increase during the rainy season, coinciding with the spawning season of Delta Smelt (Hypomesus transpacificus), an endangered, endemic fish. Furthermore, salinity intrusion in the SFBD is exacerbated by global climate change, which may change the dynamics of pyrethroid toxicity on aquatic animals. Therefore, examining the effect of salinity on the sublethal toxicity of pyrethroids is essential for risk assessments, especially during the early life stages of estuarine fishes. To address this, we investigated behavioral effects of permethrin and bifenthrin at three environmentally relevant concentrations across a salinity gradient (0.5, 2 and 6 PSU) on Delta Smelt yolk-sac larvae. Our results suggest that environmentally relevant concentrations of pyrethroids can perturb Delta Smelt larvae behavior even at the lowest concentrations (&lt;1 ng/L) and that salinity can change the dynamic of pyrethroid toxicity in terms of behavioral effects, especially for bifenthrin, where salinity was positively correlated with anti-thigmotaxis at each concentration

Investigation of Molecular Pathogen Screening Assays for Use in Delta Smelt

Pathogen surveillance must be part of any population supplementation or reintroduction program for the conservation of threatened and endangered species. The unintended transmission of pathogens can have devastating effects on these already at-risk populations or the natural ecosystem at large. In the San Francisco Estuary (estuary), abundance of the endemic Delta Smelt (Hypomesus transpacificus) has declined to the point where regulatory managers are preparing to augment the wild population using fish propagated in a hatchery to prevent species extinction. Although disease is not an overt cause of population decline, comprehensive pathogen presence and prevalence data are lacking. Here, we performed a pilot study that applied molecular assays originally developed in salmonids to assess the presence of a wide variety of pathogens in the gill tissue of cultured and wild Delta Smelt—as well as cultured fish—deployed in enclosures in the estuary. We found the assays to be highly sensitive, and observed positive detections of a single pathogen, Ichthyophthirius multifiliis, in 13% of cultured Delta Smelt. We also detected ten other pathogens at very low levels in cultured, enclosure-deployed, and wild Delta Smelt that likely represent the ambient pathogen composition in the estuary (as opposed to actual infection). Our results corroborate previous work that cultured Delta Smelt do not appear to present a high risk for pathogen transmission during population supplementation or reintroduction. However, the molecular pathogen screening assays tested here have great utility as an early warning system indicator of when further diagnostic testing might be necessary to limit the extent and frequency of disease outbreaks; their utility will be further increased once they are customized for Delta Smelt

Expression de gènes viraux de l'herpès virus OsHV-1 sous sa forme µVar chez l'huître creuse, Crassostrea gigas

Le premier cas d'infection à virus de type herpès a été rapporté en 1972, aux Etats Unis, chez l'huître américaine, Crassostrea virginica (Farley et al, 1972). A partir de 1991, des mortalités massives associées à la détection de virus de type herpes ont été observées chez plusieurs espèces de bivalves marins, à différents stades de développement (essentiellement chez les larves et le naissain) et dans différentes régions du monde. Sur la base d’observation en microscopie électronique à transmission, ces virus présentaient des caractéristiques morphologiques identiques à celles des Herpesvirus (Roizman, 1982). Les connaissances acquises concernant la taille, la structure et le séquençage du génome d’un virus infectant les larves d’huîtres creuses en France ont confirmé l'hypothèse que ce virus, appelé Ostreid herpesvirus 1 appartient à l’ordre des Herpesviales et à la famille des Malacoherpesviridae (Le Deuff & Renault, 1999 ; Davison et al., en 2005 ; Davison et al., 2009). Le séquençage complet a montré que la structure de l'ADN viral est comparable à l'herpès simplex virus de type 1, HSV-1. La taille du génome d'OsHV-1 est estimée à 207 kpb et comprend de 124 cadres de lecture (ORFs). Depuis 2008, des épisodes de surmortalités ont été rapportés dans différentes régions de production d’huîtres creuses, C. gigas, en France, en Irlande et au Royaume Uni. Les données disponibles suggèrent que le virus est une cause prépondérante avec la détection un génotype viral particulier, appelé OsHV-1 µVar à partir de 2008 (Segarra et al. 2010). Bien que les facteurs climatiques seuls n’apparaissent pas comme une cause suffisante pour expliquer le phénomène, les surmortalités sont bien saisonnières en termes d’apparition. Une augmentation rapide de la température de l’eau au printemps a été clairement identifiée comme un facteur de risque majeur. Pour diminuer les effets de ce virus, les moyens de lutte sont relativement restreints. Au vue des conditions d'élevage des huîtres en milieu ouvert, l'utilisation de traitements médicamenteux reste inadaptée. De plus, l'absence de mémoire immunitaire chez les invertébrés ne permet pas d'utiliser la vaccination pour combattre l’infection virale. Ainsi, les moyens de lutte reposent essentiellement sur (1) la surveillance des stocks et des transferts afin de limiter la propagation de la maladie et (2) sur le développement d'animaux résistants à l'infection se traduisant par une meilleure survie. Ces différentes approches nécessitent des outils de diagnostic adaptés, une bonne connaissance du cycle viral, et plus particulièrement des interactions entre l’hôte, le virus et l’environnement. Dans ce contexte, il apparaît indispensable de mieux connaître l'expression des gènes viraux au cours de l'infection chez l'huître creuse, Crassostrea gigas. Il a été choisi de suivre l’expression de gènes viraux lors d’une infection expérimentale. En effet, très récemment, Schikorski et al. (2011) ont développé un protocole d’infection expérimentale rendant possible la reproduction de l'infection virale en laboratoire chez le naissain d'huîtres creuses. D'autre part, le séquençage complet d'OsHV-1 (Davison et al., 2005) permettait de sélectionner des gènes d’intérêt ( 41 gènes candidats). Afin d'étudier la cinétique d'expression des gènes sélectionnés, la PCR en temps réel à été optimisé pour chacun couple d'amorces. La présente étude a été réalisée chez des huîtres de moins d’un an appartenant de 2 familles présentant un comportement contrastées en terme de résistance à l’infection en condition expérimentale : une famille résistante (10% de mortalité 6 jours post infection) et une famille sensible (100% de mortalité 3 jours post infection). Suite à l'infection, un fragment de manteau a été prélevé sur 12 individus pour chaque condition et chaque temps de prélèvement. Les temps de prélèvements post injection sélectionnés étaient : 0h, 4h, 8h, 12h et 26h. Les premiers résultats obtenus en PCR en temps réel mettent en évidence la détection de gènes très précoces mais également plus tardifs. Dans cette étude, après 26h d'infection, les 41 gènes viraux semblent tous être exprimés chez la famille sensible. Ainsi, l'expression des gènes d'OsHV-1 semble avoir une cinétique similaire à ceux des herpesvirus de vertébrés. En effet, l’expression des gènes viraux chez les herpersvirus comporte trois phases au cours de l’infection : (1) des gènes sont exprimés très précocement avec synthèse de protéines activatrices, (2) d’autres gènes précocement avec la synthèse d’enzymatiques impliquées dans la réplication dont l'ADN polymérase et (3) et enfin un troisième groupe de gènes tardivement avec la synthèse des composants protéiques de la capside et des glycoprotéines d'enveloppe. Ainsi, cette étude a permis de détecter la présence d'ARN transcrits durant une infection expérimentale à OsHV-1 chez le naissain d'huître creuse Crassostrea gigas. Enfin, ceci confirme la réplication du virus au sein même de son hôte et par conséquent renseigne sur le cycle de vie viral d'OsHV-1

Salinity Changes the Dynamics of Pyrethroid Toxicity in Terms of Behavioral Effects on Newly Hatched Delta Smelt Larvae

Salinity can interact with organic compounds and modulate their toxicity. Studies have shown that the fraction of pyrethroid insecticides in the aqueous phase increases with increasing salinity, potentially increasing the risk of exposure for aquatic organisms at higher salinities. In the San Francisco Bay Delta (SFBD) estuary, pyrethroid concentrations increase during the rainy season, coinciding with the spawning season of Delta Smelt (Hypomesus transpacificus), an endangered, endemic fish. Furthermore, salinity intrusion in the SFBD is exacerbated by global climate change, which may change the dynamics of pyrethroid toxicity on aquatic animals. Therefore, examining the effect of salinity on the sublethal toxicity of pyrethroids is essential for risk assessments, especially during the early life stages of estuarine fishes. To address this, we investigated behavioral effects of permethrin and bifenthrin at three environmentally relevant concentrations across a salinity gradient (0.5, 2 and 6 PSU) on Delta Smelt yolk-sac larvae. Our results suggest that environmentally relevant concentrations of pyrethroids can perturb Delta Smelt larvae behavior even at the lowest concentrations (&lt;1 ng/L) and that salinity can change the dynamic of pyrethroid toxicity in terms of behavioral effects, especially for bifenthrin, where salinity was positively correlated with anti-thigmotaxis at each concentration