Rainbow Trout Gill Research Articles

Acute toxicity of the cationic surfactant C12‐benzalkonium in different bioassays: How test design affects bioavailability and effect concentrations

Using an ion-exchange-based solid-phase microextraction (SPME) method, the freely dissolved concentrations of C12-benzalkonium were measured in different toxicity assays, including 1) immobilization of Daphnia magna in the presence or absence of dissolved humic acid; 2) mortality of Lumbriculus variegatus in the presence or absence of a suspension of Organisation for Economic Co-Operation and Development (OECD) sediment; 3) photosystem II inhibition of green algae Chlorella vulgaris; and 4) viability of in vitro rainbow trout gill cell line (RTgill-W1) in the presence or absence of serum proteins. Furthermore, the loss from chemical adsorption to the different test vessels used in these tests was also determined. The C12-benzalkonium sorption isotherms to the different sorbent phases were established as well. Our results show that the freely dissolved concentration is a better indicator of the actual exposure concentration than the nominal or total concentration in most test assays. Daphnia was the most sensitive species to C12-benzalkonium. The acute Daphnia and Lumbriculus tests both showed no enhanced toxicity from possible ingestion of sorbed C12-benzalkonium in comparison with water-only exposure, which is in accordance with the equilibrium partitioning theory. Moreover, the present study demonstrates that commonly used sorbent phases can strongly affect bioavailability and observed effect concentrations for C12-benzalkonium. Even stronger effects of decreased actual exposure concentrations resulting from sorption to test vessels, cells, and sorbent phases can be expected for more hydrophobic cationic surfactants.

Critical body residues, Michaelis–Menten analysis of bioaccumulation, lethality and behaviour as endpoints of waterborne Ni toxicity in two teleosts

Traditionally, water quality guidelines/criteria are based on lethality tests where results are expressed as a function of waterborne concentrations (e.g. LC50). However, there is growing interest in the use of uptake and binding relationships, such as biotic ligand models (BLM), and in bioaccumulation parameters, such as critical body residue values (e.g. CBR50), to predict metal toxicity in aquatic organisms. Nevertheless, all these approaches only protect species against physiological death (e.g. mortality, failed recruitment), and do not consider ecological death which can occur at much lower concentrations when the animal cannot perform normal behaviours essential for survival. Therefore, we investigated acute (96h) Ni toxicity in two freshwater fish species, the round goby (Neogobius melanostomus) and rainbow trout (Oncorhynchus mykiss) and compared LC, BLM, and CBR parameters for various organs, as well as behavioural responses (spontaneous activity). In general, round goby were more sensitive. Ni bioaccumulation displayed Michaelis-Menten kinetics in most tissues, and round goby gills had lower Kd (higher binding affinity) but similar Bmax (binding site density) values relative to rainbow trout gills. Round goby also accumulated more Ni than did trout in most tissues at a given exposure concentration. Organ-specific 96h acute CBR values tended to be higher in round goby but 96h acute CBR50 and CBR10 values in the gills were very similar in the two species. In contrast, LC50 and LC10 values were significantly higher in rainbow trout. With respect to BLM parameters, gill log KNiBL values for bioaccumulation were higher by 0.4-0.8 log units than the log KNiBL values for toxicity in both species, and both values were higher in goby (more sensitive). Round goby were also more sensitive with respect to the behavioural response, exhibiting a significant decline of 63-75% in movements per minute at Ni concentrations at and above only 8% of the LC50 value; trout exhibited no clear behavioural response. Across species, diverse behavioral responses may be more closely related to tissue Ni burdens than to waterborne Ni concentrations. To our knowledge, this is the first study to link Ni bioaccumulation with behavioural endpoints. In future it would be beneficial to expand these analyses to a wider range of species to determine whether Ni bioaccumulation, specifically in the gills, gut and whole fish, may be a good predictor of behavioural changes from metal exposure; which in the wild can lead to ecological death.

Tissue-specific responses of oxidative stress biomarkers and antioxidant defenses in rainbow trout Oncorhynchus mykiss during a vaccination against furunculosis

The present study was conducted to evaluate the effects of vaccination against furunculosis on responses of oxidative stress and antioxidant defenses in rainbow trout Oncorhynchus mykiss muscle, gills, liver, and brain tissues. The oxidative stress markers (malondialdehyde and carbonyl derivatives of protein oxidative destruction levels), antioxidant defenses (superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase), and total antioxidant capacity in different tissues of rainbow trout were measured. Our data showed that exposure of trout to vaccine against furunculosis produced changes (either increase or decrease) in oxidative stress and antioxidant enzymes responses, and these responses showed marked organ differences, associated with tissue patterns. Our study demonstrated that vaccinated trout showed alteration in antioxidant defenses and oxidative stress responses, with higher severity in the liver, compared with other tissues. Our data also suggest that vaccination against furunculosis induced lipid peroxidation in gill and liver tissues. However, muscle and brain tissue are capable of restoring its pro- and antioxidant balance after vaccination.

Transcriptomic responses in rainbow trout gills upon infection with viral hemorrhagic septicemia virus (VHSV)

It has been previously demonstrated that even though the fin bases constitute the main portal of entry of viral hemorrhagic septicemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss), an important number of chemokine genes are up-regulated in the gills upon bath exposure to the virus. Because chemokines mediate the recruitment of leukocytes through the action of specific chemokine receptors, in the current study, we have studied the transcription of several immune genes in response to a VHSV bath infection in the gills, focusing both on chemokine receptor genes and on genes characteristic of distinct leukocyte populations such as IgM, IgD, IgT, CD4, CD8, perforin and MHC-II. We have studied the response to the virus in naïve fish as well as in fish that had been previously intramuscularly (i.m.) injected with a VHSV DNA vaccine. Additionally, we have sorted both IgM+ and CD8+ cells from the gills of naïve and infected animals to study some of these up-regulated genes in specific leukocyte populations. Our results indicate that despite the low replication level, VHSV provokes an up-regulation of IgM, IgT, CD3 and perforin transcription together with the up-regulation of CCR7, CCR9, CXCR3B and CXCR4 mRNA levels. Interestingly, MHC-II mRNA was up-regulated and CCR7 was down-modulated in IgM+ cells from infected gills, whereas perforin, CCR7 and CXCR4 mRNA levels were higher in sorted CD8+ cells from infected animals. Surprisingly, when fish had been previously injected with either the empty plasmid or the VHSV DNA vaccine, these up-regulations in immune gene transcription were no longer observed. Our results point to the gills as an important site for innate and acquired viral defense.

The effect of pH on the toxicity of fatty acids and fatty acid amides to rainbow trout gill cells

Harmful algal blooms (HABs) expose aquatic organisms to multiple physical and chemical stressors during an acute time period. Algal toxins themselves may be altered by water chemistry parameters affecting their bioavailability and resultant toxicity. The purpose of this study was to determine the effects of two abiotic parameters (pH, inorganic metal salts) on the toxicity of fatty acid amides and fatty acids, two classes of lipids produced by harmful algae, including the golden alga, Prymnesium parvum, that are toxic to aquatic organisms. Rainbow trout gill cells were used as a model of the fish gill and exposed to single compounds and mixtures of compounds along with variations in pH level and concentration of inorganic metal salts. We employed artificial neural networks (ANNs) and standard ANOVA statistical analysis to examine and predict the effects of these abiotic parameters on the toxicity of fatty acid amides and fatty acids. Our results demonstrate that increasing pH levels increases the toxicity of fatty acid amides and inhibits the toxicity of fatty acids. This phenomenon is reversed at lower pH levels. Exposing gill cells to complex mixtures of chemical factors resulted in dramatic increases in toxicity compared to tests of single compounds for both the fatty acid amides and fatty acids. These findings highlight the potential of physicochemical factors to affect the toxicity of chemicals released during algal blooms and demonstrate drastic differences in the effect of pH on fatty acid amides and fatty acids.

Effects of salinity acclimation on the endocrine disruption and acute toxicity of bifenthrin in freshwater and euryhaline strains of Oncorhynchus mykiss

The pyrethroid insecticide bifenthrin is frequently detected at ng/L concentrations in tributaries of the San Francisco Bay Delta. The estuary is also experiencing increasing salinity through climate change and water redirection. To evaluate the impacts of hypersaline conditions on bifenthrin toxicity in anadromous salmonids of the San Francisco Bay Delta (CA, USA), a 14-d laboratory exposure was performed using 2 strains of Oncorhynchus mykiss (rainbow trout and steelhead) acclimated to freshwater and to 8 g/L and 17 g/L salinity. The fish were then exposed to nominal concentrations of 0 µg/L, 0.1 µg/L, and 1.5 µg/L bifenthrin. Rainbow trout exhibited significant mortality following exposure to 1.5 µg/L (1.07 ± 0.35 µg/L measured) bifenthrin in freshwater. Elevated levels of Na⁺ /K⁺ adenosine triphosphatase α1A mRNA subunit expression was observed in the gill of rainbow trout acclimated to hypersaline conditions relative to freshwater animals. No significant difference was noted in Na⁺ /K⁺ adenosine triphosphatase subunit levels in brains of either strain in freshwater or hypersaline conditions. Likewise, significant differences were not observed in plasma vitellogenin or steroid hormone concentrations in either strain whether maintained in freshwater or saltwater. Saltwater acclimation significantly reduced nicotinamide adenine dinucleotide phosphate-catalyzed biotransformation of bifenthrin in liver microsomes of rainbow trout but not of steelhead. The present study showed that, relative to steelhead, rainbow trout have different responses to bifenthrin acute toxicity as well as different rates of hepatic bifenthrin biotransformation and regulation of Na⁺ /K⁺ adenosine triphosphatase subunits in gills. These data indicate that significant differences exist between the strains and that animal life history may have important effects on the susceptibility of each strain to environmental contaminants.

Transcriptome Profiling of Gill Tissue in Regionally Bred and Globally Farmed Rainbow Trout Strains Reveals Different Strategies for Coping with Thermal Stress

Thermal stress can pose a major challenge to salmonid fish. A 4x44K oligonucleotide microarray approach was used to screen for genetically determined variations of a temperature stress response during acclimation in fish gills, a highly specialized and complex organ responsible for gas and electrolyte exchange as well as excretion. The comparison addressed transcriptional changes in the local breeding strain BORN and imported (TCO) rainbow trout after graded 2-week acclimation to 8 and 23°C. Besides well-characterized mediators of thermoregulation such as genes encoding cold-inducible RNA-binding protein and heat shock proteins, the present microarray study suggests several new candidate genes commonly regulated in gills of the two trout lines. Having identified the differential expression of thermoregulated genes as duplicated paralogues, they were subsequently validated in a gill cell model. Moreover, the comparison of transcriptome profiles provides evidence for distinctively employed expression patterns. The induction of genes encoding factors of the early innate immunity in BORN trout upon warming contrasts with the increased expression of adaptive immune genes in import trout. Cold acclimation induced genes assigned to the functional categories "cell death" and "ion channel activity" in import trout, but repressed "lipid metabolism." This manuscript provides an overview of the genes of the multifunctional gills in rainbow trout that are mandated after temperature change, suggesting links between the different temperature-dependent pathways and gene networks.

A multi-assay screening approach for assessment of endocrine-active contaminants in wastewater effluent samples

Environmental agencies must monitor an ever increasing range of contaminants of emerging concern, including endocrine disrupting compounds (EDCs). An alternative to using ultra-trace chemical analysis of samples for EDCs is to test for biological activity using in vitro screening assays, then use these assay results to direct analytical chemistry approaches. In this study, we used both analytical approaches and in vitro bioassays to characterize the EDCs present in treated wastewater from four wastewater treatment plants (WWTPs) in Ontario, Canada. Estrogen-mediated activity was assessed using a yeast estrogenicity screening (YES) assay. An in vitro competitive binding assay was used to assess capacity to interfere with binding of the thyroid hormone, thyroxine (T4) to the recombinant human thyroid hormone transport protein, transthyretin (i.e. hTTR). An in vitro binding assay with a rat peroxisome proliferator responsive element transfected into a rainbow trout gill cell line was used to evaluate binding and subsequent gene expression via the peroxisome proliferator activated receptor (PPAR). Analyses of a suite of contaminants known to be EDCs in extracts from treated wastewater were conducted using either gas chromatography with mass spectrometry (GC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS). Estrogenic activity was detected in the YES assay only in those extracts that contained detectable amounts of estradiol (E2). There was a positive relationship between the degree of response in the T4-hTTR assay and the amounts of polybrominated diphenyl ether (PBDE) congeners 47 and 99, triclosan and the PBDE metabolite, 4-OH-BDE17. Several wastewater extracts gave a positive response in the PPAR assay, but these responses were not correlated with the amounts of any of the EDCs analyzed by LC-MS/MS. Overall, these data indicate that a step-wise approach is feasible using a combination of in vitro testing and instrumental analysis to monitor for EDCs in wastewater and other environmental matrixes.

Strain variability in fatty acid composition of Chattonella marina (Raphidophyceae) and its relation to differing ichthyotoxicity toward rainbow trout gill cells.

Lipid profiles of three strains (Mexico, Australia, Japan) of Chattonella marina (Subrahmanyan) Hara et Chihara were studied under defined growth (phosphate, light, and growth phase) and harvest (intact and ruptured cells) conditions. Triacylglycerol levels were always <2%, sterols <7%, free fatty acids varied between 2 and 33%, and polar lipids were the most abundant lipid class (>51% of total lipids). The major fatty acids in C.marina were palmitic (16:0), eicosapentaenoic (EPA, 20:5ω3), octadecatetraenoic (18:4ω3), myristic (14:0), and palmitoleic (16:1ω7c) acids. Higher levels of EPA were found in ruptured cells (21.4-29.4%) compared to intact cells (8.5-25.3%). In general, Japanese N-118 C.marina was the highest producer of EPA (14.3-29.4%), and Mexican CMCV-1 the lowest producer (7.9-27.1%). Algal cultures, free fatty acids from C.marina, and the two aldehydes 2E,4E-decadienal and 2E,4E-heptadienal (suspected fatty acid-derived products) were tested against the rainbow trout fish gill cell line RTgill-W1. The configuration of fatty acids plays an important role in ichthyotoxicity. Free fatty acid fractions, obtained by base saponification of total lipids from C.marina showed a potent toxicity toward gill cells (median lethal concentration, LC50 (at 1h) of 0.44μg·mL(-1) in light conditions, with a complete loss of viability at >3.2μg·mL(-1) ). Live cultures of Mexican C.marina were less toxic than Japanese and Australian strains. This difference could be related to differing EPA content, superoxide anion production, and cell fragility. The aldehydes 2E,4E-decadienal and 2E,4E-heptadienal also showed high impact on gill cell viability, with LC50 (at 1h) of 0.34 and 0.36μg·mL(-1) , respectively. Superoxide anion production was highest in Australian strain CMPL01, followed by Japanese N-118 and Mexican CMCV-1 strains. Ruptured cells showed higher production of superoxide anion compared to intact cells (e.g., 19 vs. 9.5pmol·cell(-1) ·hr(-1) for CMPL01, respectively). Our results indicate that C.marina is more ichthyotoxic after cell disruption and when switching from dark to light conditions, possibly associated with a higher production of superoxide anion and EPA, which may be quickly oxidized to produce more toxic derivates, such as aldehydes.

Application of LDH-Release Assay to Cellular-Level Evaluation of the Toxic Potential of Harmful Algal Species

Lactate dehydrogenase (LDH)-release assay was applied to estimate the toxic potential of harmful algal species at the cellular level. African green monkey kidney (Vero), yellowtail fin epithelia (MJF), and rainbow trout gill (RTgill-W1) cells were used as target cells. A live cell suspension of Karenia mikimotoi (SUO-1) induced the release of LDH from these cell lines, while the activity of another strain, FUK, was much lower. The cell-free culture supernatants and ruptured cell suspensions of both strains of K. mikimotoi were less effective on LDH-release assay. Exposure experiments against abalone and shrimp revealed that SUO-1 showed much stronger lethal effects on these organisms than FUK. Among six phytoplankton species, three species known to be harmful algal species induced the release of LDH to different extents depending on the cell line, whereas the other three species, known to be non-toxic, showed no effects on any cell lines. These results suggest that LDH-release assay is a useful micro-plate assay for estimation of the toxic potential of harmful phytoplankton.

Sensitivity of ventilation and brain metabolism to ammonia exposure in rainbow trout,Oncorhynchus mykiss

Ammonia has been documented as a respiratory gas that stimulates ventilation, and is sensed by peripheral neuroepithelial cells (NECs) in the gills in ammoniotelic rainbow trout. However, the hyperventilatory response is abolished in trout chronically exposed (1+ months) to high environmental ammonia [HEA; 250 μmol l(-1) (NH4)2SO4]. This study investigates whether the brain is involved in the acute sensitivity of ventilation to ammonia, and whether changes in brain metabolism are related to the loss of hyperventilatory responses in trout chronically exposed to HEA ('HEA trout'). Hyperventilation (via increased ventilatory amplitude rather than rate) and increased total ammonia concentration ([TAmm]) in brain tissue were induced in parallel by acute HEA exposure in control trout in a concentration-series experiment [500, 750 and 1000 μmol l(-1) (NH4)2SO4], but these inductions were abolished in HEA trout. Ventilation was correlated more closely to [TAmm] in brain rather than to [TAmm] in plasma or cerebrospinal fluid. The close correlation of hyperventilation and increased brain [TAmm] also occurred in control trout acutely exposed to HEA in a time-series analysis [500 μmol l(-1) (NH4)2SO4; 15, 30, 45 and 60 min], as well as in a methionine sulfoxamine (MSOX) pre-injection experiment [to inhibit glutamine synthetase (GSase)]. These correlations consistently suggest that brain [TAmm] is involved in the hyperventilatory responses to ammonia in trout. The MSOX treatments, together with measurements of GSase activity, TAmm, glutamine and glutamate concentrations in brain tissue, were conducted in both the control and HEA trout. These experiments revealed that GSase plays an important role in transferring ammonia to glutamate to make glutamine in trout brain, thereby attenuating the elevation of brain [TAmm] following HEA exposure, and that glutamate concentration is reduced in HEA trout. The mRNAs for the ammonia channel proteins Rhbg, Rhcg1 and Rhcg2 were expressed in trout brain, and the expression of Rhbg and Rhcg2 increased in HEA trout, potentially as a mechanism to facilitate the efflux of ammonia. In summary, the brain appears to be involved in the sensitivity of ventilation to ammonia, and brain ammonia levels are regulated metabolically in trout.

Histopathological effects of waterborne copper nanoparticles and copper sulphate on the organs of rainbow trout (Oncorhynchus mykiss)

It is unclear whether copper nanoparticles are more toxic than traditional forms of dissolved copper. This study aimed to describe the pathologies in gill, gut, liver, kidney, brain and muscle of juvenile rainbow trout, Oncorhynchus mykiss, exposed in triplicate to either a control (no added Cu), 20 or 100μgl−1 of either dissolved Cu (as CuSO4) or Cu-NPs (mean primary particle size of 87±27nm) in a semi-static waterborne exposure regime. Fish were sampled at days 0, 4, and 10 for histology. All treatments caused organ injuries, and the kinds of pathologies observed with Cu-NPs were broadly of the same type as CuSO4 including: hyperplasia, aneurisms, and necrosis in the secondary lamellae of the gills; swelling of goblet cells, necrosis in the mucosa layer and vacuole formation in the gut; hepatitis-like injury and cells with pyknotic nuclei in the liver; damage to the epithelium of some renal tubules and increased Bowman's space in the kidney. In the brain, some mild changes were observed in the nerve cell bodies in the telencephalon, alteration in the thickness of the mesencephalon layers, and enlargement of blood vessel on the ventral surface of the cerebellum. Changes in the proportional area of muscle fibres were observed in skeletal muscle. Overall the data showed that pathology from CuSO4 and Cu-NPs were of similar types, but there were some material-type effects in the severity or incidence of injuries with Cu-NPs causing more injury in the intestine, liver and brain than the equivalent concentration of CuSO4 by the end of the experiment, but in the gill and muscle CuSO4 caused more pathology.

The rad1 gene in Rainbow Trout (Oncorhynchus mykiss) is highly conserved and may express proteins from non-canonical spliced isoforms

Cell-cycle checkpoint proteins maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. RAD1 is a checkpoint protein involved in the sensing of damaged DNA and is a part of the 9-1-1 complex. In this project rainbow trout rad1 (rtrad1) was cloned, sequenced, expressed as a recombinant protein and anti-rtRAD1 antibodies were developed. RAD1 protein levels were characterized in various rainbow trout tissues. It was determined that an 840bp open-reading frame encodes 279 aa with a predicted protein size of 31kDa. The rtRAD1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify three non-canonical splice variants of rtrad1, two of which are capable of forming functional proteins. The rad1 splice variant that encodes an 18kDa protein appears to be abundant in rainbow trout spleen, heart and gill tissue and in the RTgill-W1 cell-line. Based on the genomic rtrad1 sequence the splice variants contain only partial exons which are consistent with the splicing of rad1 variants in mammals. This is the first time that rad1 has been fully characterized in a fish species.

The contribution of fatty acid amides to Prymnesium parvum Carter toxicity

It is well known that toxic blooms of Prymnesium parvum result in widespread fish mortalities. An assemblage of fatty acid amides (FAAs) has been shown to be toxic metabolites of P. parvum. Several compounds and compound classes have been implicated in P. parvum toxicity, but unlike FAAs, thus far none have been shown to accumulate to lethal levels at ichthyotoxic bloom events. The purpose of this study is to characterize the hemolytic activity, cytotoxicity, and ichthyotoxicity of the fatty acid amides identified from P. parvum using mammalian red blood cells, mouse neuroblastoma cells, and rainbow trout gill cells. We also investigate the effect of abiotic factors (divalent cations and pH) on fatty acid amide toxicity. Samples from P. parvum bloom events with fish kills were analyzed in order to demonstrate that fatty acid amides are found in lethal levels at fish kills. Hemolytic and cytoxicity assays were completed with the introduction of abiotic factors to assess fatty acid amides toxicity. Our results show that fatty acid amides are detected in toxicologically significant quantities in samples from at least one fish kill event. We also show that the toxicity of oleamide and linoleamide in cell culture is increased in the presence of divalent cations and increasing pH, demonstrating that multiple abiotic factors affect the toxicity of fatty acid amides. Fatty acid amides possess the hemolytic and cytotoxic properties previously attributed to P. parvum toxins throughout history. After fatty acid amides have been released into the environment, multiple abiotic factors can increase their toxicity.

First isolation and characterization of Chryseobacterium shigense from rainbow trout

BackgroundThere have been an increasing number of infections in fish associated with different species of Chryseobacterium, being considered potentially emerging pathogens. Nevertheless the knowledge of the diversity of species associated with fish disease is partial due to the problems for a correct identification at the species level based exclusively on phenotypic laboratory methods.ResultsChryseobacterium shigense was isolated from the liver, kidney and gills of diseased rainbow trout in different disease episodes that occurred in a fish farm between May 2008 and June 2009. Identity of the isolates was confirmed by 16 S rRNA gene sequencing and phenotypic characterization. Isolates represented a single strain as determined by random amplified polymorphic DNA analysis.ConclusionsThis is the first description of the recovery of C. shigense from clinical specimens in trout, a very different habitat to fresh lactic acid beverage where it was initially isolated.

Atorvastatin up-regulate toxicologically relevant genes in rainbow trout gills

There are large and increasing discharges of statins into the aquatic environment. Statins are cholesterol-lowering pharmaceuticals, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, an enzyme in the cholesterol synthesis pathway. Earlier studies have shown that statins will affect the expression of a range of genes in mammalian tissues and this group of pharmaceuticals has also been shown to affect membrane transporters. Changes in gene expression and ion transport in aquatic organisms may have dramatic consequences for the individual. The aim of the present study was to clarify whether waterborne exposure to a selected statin, atorvastatin, would affect gene expression in rainbow trout (Oncorhynchus mykiss) gill or liver or ion regulation in gills. Juvenile rainbow trout were exposed to two atorvastatin acid and atorvastatin lactone concentrations for 7 days (nominal concentrations 200 ng L(-1) and 10 μg L(-1)). The exposures caused up-regulated gene expression in gill, not liver, and only at the lowest concentration. Genes involved in membrane transport (pgp, mrp1), oxidative stress response (sod, mt), apoptosis (bax) and biotransformation (sult2b) were differentially expressed whereas the expression of genes involved in cholesterol biosynthesis (hmgr, fdps) or peroxisomal proliferation (ppar) were not affected. There were no significant changes in gill Na(+)/K(+) ATPase activity following exposure to atorvastatin. The pattern of differentially expressed genes in rainbow trout gills differ from responses previously observed in mammalian tissues following statin exposure.

Secretory IgD has an evolutionarily conserved role in respiratory mucosal defense (160.18)

Abstract Recently, we discovered secreted IgD in rainbow trout, which is produced by a novel exon-splicing mechanism. Molecular and cellular analysis revealed that secIgD plasma cells were distributed within the primary and secondary immune tissues. The concentration of serum IgD (2-80µg/ml) was ~400x lower than serum IgM (800-9000µg/ml). Two dominant secIgD heavy chain isoforms were identified in serum of trout which were associated with antibody light chains to form 240 or 370 kDa IgD-monomers. Antigen-specific secIgD was produced in response to vaccination with either TI or TD antigens. Interestingly, the distribution of antigen-specific IgD plasma cells within mucosal tissues revealed that they were preferentially located in the gills of rainbow trout. Analysis of gill mucosal secretions revealed that secIgD was the dominant antibody isotype, which contrasts the profile observed in serum. However, our data suggests that the number of IgD plasma cells in the gills of rainbow trout could not account for the elevated IgD concentration observed in the gill mucus. We hypothesize that serum IgD is preferentially transported across the gill epithelium to neutralize pathogens at this important site. The receptor responsible for trans-epithelial transport of IgD has proved elusive. However, the analogous distribution of IgD plasma cells and secreted IgD in the respiratory mucosa of trout and mammals would suggest that IgD provides an evolutionarily conserved function within this tissue.

Subcellular differences in handling Cu excess in three freshwater fish species contributes greatly to their differences in sensitivity to Cu

Since changes in metal distribution among tissues and subcellular fractions can provide insights in metal toxicity and tolerance, we investigated this partitioning of Cu in gill and liver tissue of rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). These fish species are known to differ in their sensitivity to Cu exposure with gibel carp being the most tolerant and rainbow trout the most sensitive. After an exposure to 50μg/l (0.79μM) Cu for 24h, 3 days, 1 week and 1 month, gills and liver of control and exposed fish were submitted to a differential centrifugation procedure. Interestingly, there was a difference in accumulated Cu in the three fish species, even in control fishes. Where the liver of rainbow trout showed extremely high Cu concentrations under control conditions, the amount of Cu accumulated in their gills was much less than in common and gibel carp. At the subcellular level, the gills of rainbow trout appeared to distribute the additional Cu exclusively in the biologically active metal pool (BAM; contains heat-denaturable fraction and organelle fraction). A similar response could be seen in gill tissue of common carp, although the percentage of Cu in the BAM of common carp was lower compared to rainbow trout. Gill tissue of gibel carp accumulated more Cu in the biologically inactive metal pool (BIM compared to BAM; contains heat-stable fraction and metal-rich granule fraction). The liver of rainbow trout seemed much more adequate in handling the excess Cu (compared to its gills), since the storage of Cu in the BIM increased. Furthermore, the high % of Cu in the metal-rich granule fraction and heat-stable fraction in the liver of common carp and especially gibel carp together with the better Cu handling in gill tissue, pointed out the ability of the carp species to minimize the disadvantages related to Cu stress. The differences in Cu distribution at the subcellular level of gills and liver of these fish species strongly reflects their capacity to handle Cu excess and is one of the greatest contributors to their difference in sensitivity to Cu.

Characterization of rainbow trout gonad, brain and gill deep cDNA repertoires using a Roche 454-Titanium sequencing approach

Rainbow trout, Oncorhynchus mykiss, is an important aquaculture species worldwide and, in addition to being of commercial interest, it is also a research model organism of considerable scientific importance. Because of the lack of a whole genome sequence in that species, transcriptomic analyses of this species have often been hindered. Using next-generation sequencing (NGS) technologies, we sought to fill these informational gaps. Here, using Roche 454-Titanium technology, we provide new tissue-specific cDNA repertoires from several rainbow trout tissues. Non-normalized cDNA libraries were constructed from testis, ovary, brain and gill rainbow trout tissue samples, and these different libraries were sequenced in 10 separate half-runs of 454-Titanium. Overall, we produced a total of 3million quality sequences with an average size of 328bp, representing more than 1Gb of expressed sequence information. These sequences have been combined with all publicly available rainbow trout sequences, resulting in a total of 242,187 clusters of putative transcript groups and 22,373 singletons. To identify the predominantly expressed genes in different tissues of interest, we developed a Digital Differential Display (DDD) approach. This approach allowed us to characterize the genes that are predominantly expressed within each tissue of interest. Of these genes, some were already known to be tissue-specific, thereby validating our approach. Many others, however, were novel candidates, demonstrating the usefulness of our strategy and of such tissue-specific resources. This new sequence information, acquired using NGS 454-Titanium technology, deeply enriched our current knowledge of the expressed genes in rainbow trout through the identification of an increased number of tissue-specific sequences. This identification allowed a precise cDNA tissue repertoire to be characterized in several important rainbow trout tissues. The rainbow trout contig browser can be accessed at the following publicly available web site (http://www.sigenae.org/).

Long-term storage and impedance-based water toxicity testing capabilities of fluidic biochips seeded with RTgill-W1 cells

Rainbow trout gill epithelial cells (RTgill-W1) are used in a cell-based biosensor that can respond within one hour to toxic chemicals that have the potential to contaminate drinking water supplies. RTgill-W1 cells seeded on enclosed fluidic biochips and monitored using electric cell-substrate impedance sensing (ECIS) technology responded to 18 out of the 18 toxic chemicals tested within one hour of exposure. Nine of these chemical responses were within established concentration ranges specified by the U.S. Army for comparison of toxicity sensors for field application. The RTgill-W1 cells remain viable on the biochips at ambient carbon dioxide levels at 6°C for 78weeks without media changes. RTgill-W1 biochips stored in this manner were challenged with 9.4μM sodium pentachlorophenate (PCP), a benchmark toxicant, and impedance responses were significant (p<0.001) for all storage times tested. This poikilothermic cell line has toxicant sensitivity comparable to a mammalian cell line (bovine lung microvessel endothelial cells (BLMVECs)) that was tested on fluidic biochips with the same chemicals. In order to remain viable, the BLMVEC biochips required media replenishments 3 times per week while being maintained at 37°C. The ability of RTgill-W1 biochips to maintain monolayer integrity without media replenishments for 78weeks, combined with their chemical sensitivity and rapid response time, make them excellent candidates for use in low cost, maintenance-free field-portable biosensors.