Gill Cells Research Articles

PeBiToSens™: A Platform for PBT Screening of Fragrance Ingredients Without Animal Testing.

The determination of persistence (P), bioaccumulation (B) and toxicity (T) plays a central role in the environmental assessment of chemicals. Persistence is typically evaluated via standard microbial biodegradation tests. Bioaccumulation refers to the accumulation of chemicals in organisms and is usually assessed in fish exposed to the test chemical. Toxicity is determined at three trophic levels, with fish toxicity as the highest trophic level assessed. Thus, animal tests are classically needed for both B and T assessment. In vitro systems based on fish liver cells or liver S9 fractions ('RT-S9 assay') have been recently adopted by OECD to measure the biotransformation rates for the chemicals for B assessment. Biotransformation drives clearance from the body and reduces bioaccumulation. For T assessment, an assay based on in vitro toxicity on fish gill cells has been established ('RTgill-W1 assay'). Here we summarize our findings indicating that these tests are highly predictive for fragrance ingredients, and show with two case studies of our latest new registered substances how we apply these tests in particular during development and also for chemical registration. This platform of tests (PeBiToSens™) could fully replace animal tests in ecotoxicological assessment and is key in the Givaudan Safe by Design™ approach to develop safer and environmentally compatible novel fragrance ingredients.

Genome-wide analysis of MicroRNA-messenger RNA interactome in ex-vivo gill filaments, Anguilla japonica

BackgroundGills of euryhaline fishes possess great physiological and structural plasticity to adapt to large changes in external osmolality and to participate in ion uptake/excretion, which is essential for the re-establishment of fluid and electrolyte homeostasis. The osmoregulatory plasticity of gills provides an excellent model to study the role of microRNAs (miRs) in adaptive osmotic responses. The present study is to characterize an ex-vivo gill filament culture and using omics approach, to decipher the interaction between tonicity-responsive miRs and gene targets, in orchestrating the osmotic stress-induced responses.ResultsEx-vivo gill filament culture was exposed to Leibovitz’s L-15 medium (300 mOsmol l− 1) or the medium with an adjusted osmolality of 600 mOsmol l− 1 for 4, 8 and 24 h. Hypertonic responsive genes, including osmotic stress transcriptional factor, Na+/Cl−-taurine transporter, Na+/H+ exchange regulatory cofactor, cystic fibrosis transmembrane regulator, inward rectifying K+ channel, Na+/K+-ATPase, and calcium-transporting ATPase were significantly upregulated, while the hypo-osmotic gene, V-type proton ATPase was downregulated. The data illustrated that the ex-vivo gill filament culture exhibited distinctive responses to hyperosmotic challenge. In the hyperosmotic treatment, four key factors (i.e. drosha RNase III endonuclease, exportin-5, dicer ribonuclease III and argonaute-2) involved in miR biogenesis were dysregulated (P < 0.05). Transcriptome and miR-sequencing of gill filament samples at 4 and 8 h were conducted and two downregulated miRs, miR-29b-3p and miR-200b-3p were identified. An inhibition of miR-29b-3p and miR-200b-3p in primary gill cell culture led to an upregulation of 100 and 93 gene transcripts, respectively. Commonly upregulated gene transcripts from the hyperosmotic experiments and miR-inhibition studies, were overlaid, in which two miR-29b-3p target-genes [Krueppel-like factor 4 (klf4), Homeobox protein Meis2] and one miR-200b-3p target-gene (slc17a5) were identified. Integrated miR-mRNA-omics analysis revealed the specific binding of miR-29b-3p on Klf4 and miR-200b-3p on slc17a5. The target-genes are known to regulate differentiation of gill ionocytes and cellular osmolality.ConclusionsIn this study, we have characterized the hypo-osmoregulatory responses and unraveled the modulation of miR-biogenesis factors/the dysregulation of miRs, using ex-vivo gill filament culture. MicroRNA-messenger RNA interactome analysis of miR-29b-3p and miR-200b-3p revealed the gene targets are essential for osmotic stress responses.

MEMS biosensor for monitoring water toxicity based on quartz crystal microbalance.

This paper presents the use of a commercial quartz crystal microbalance (QCM) to investigate live-cell activity in water-based toxic solutions. The QCM used in this research has a resonant frequency of 10 MHz and consists of an AT-cut quartz crystal with gold electrodes on both sides. This QCM was transformed into a functional biosensor by integrating with polydimethylsiloxane culturing chambers. Rainbow trout gill epithelial cells were cultured on the resonators as a sensorial layer. The fluctuation of the resonant frequency, due to the change of cell morphology and adhesion, is an indicator of water toxicity. The shift in the resonant frequency provides information about the viability of the cells after exposure to toxicants. The toxicity result shows distinct responses after exposing cells to 0.526 μM of pentachlorophenol (PCP) solution, which is the Military Exposure Guidelines concentration. This research demonstrated that the QCM is sensitive to a low concentration of PCP and no further modification of the QCM surface was required.

Comparative Therapeutic Effects of Natural Compounds Against Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae).

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers.

Green biolubricant infused slippery surfaces to combat marine biofouling

HypothesisMarine biofouling is a global, longstanding problem for maritime industries and coastal areas arising from the attachment of fouling organisms onto solid immersed surfaces. Slippery Liquid Infused Porous Surfaces (SLIPS) have recently shown promising capacity to combat marine biofouling. In most SLIPS coatings, the lubricant is a silicone/fluorinated-based synthetic component that may not be fully compatible with the marine life. We hypothesized that eco-friendly biolubricants could be used to replace synthetic lubricants in SLIPS for marine anti-fouling. ExperimentsWe developed SLIPS coatings using oleic acid (OA) and methyl oleate (MO) as infusing phases. The infusion efficiency was verified with confocal microscopy, surface spectroscopy, wetting efficiency, and nanocontact mechanics. Using green mussels as a model organism, we tested the anti-fouling performance of the biolubricant infused SLIPS and verified its non-cytotoxicity against fish gill cells. FindingsWe find that UV-treated PDMS infused with MO gives the most uniform infused film, in agreement with the lowest interfacial energy among all surface/biolubricants produced. These surfaces exhibit efficient anti-fouling properties, as defined by the lowest number of mussel adhesive threads attached to the surface as well as by the smallest surface/thread adhesion strength. We find a direct correlation between anti-fouling performance and the substrate/biolubricant interfacial energy.

Effects of Water Acidification on Senegalese Sole Solea senegalensis Health Status and Metabolic Rate: Implications for Immune Responses and Energy Use

Increasing water CO2, aquatic hypercapnia, leads to higher physiological pCO2 levels in fish, resulting in an acidosis and compensatory acid-base regulatory response. Senegalese sole is currently farmed in super-intensive recirculating water systems where significant accumulation of CO2 in the water may occur. Moreover, anthropogenic releases of CO2 into the atmosphere are linked to ocean acidification. The present study was designed to assess the effects of acute (4 and 24 h) and prolonged exposure (4 weeks) to CO2 driven acidification (i.e., pH 7.9, 7.6, and 7.3) from normocapnic seawater (pH 8.1) on the innate immune status, gill acid-base ion transporter expression and metabolic rate of juvenile Senegalese sole. The acute exposure to severe hypercapnia clearly affected gill physiology as observed by an increase of NHE3b positive ionocytes and a decrease of cell shape factor. Nonetheless only small physiological adjustments were observed at the systemic level with (1) a modulation of both plasma and skin humoral parameters and (2) an increased expression of HIF-1 expression pointing to an adjustment to the acidic environment even after a short period (i.e., hours). On the other hand, upon prolonged exposure, the expression of several pro-inflammatory and stress related genes was amplified and gill cell shape factor was aggravated with the continued increase of NHE3b positive ionocytes, ultimately impacting fish growth. While these findings indicate limited effects on energy use, deteriorating immune system conditions suggest that Senegalese sole is vulnerable to changes in CO2 and may be affected in aquaculture where a pH drop is more prominent. Further studies are required to investigate how larval and adult Senegalese sole are affected by changes in CO2.

Molecular characterization and expression analysis of two crucial MAPKs- jnk1 and erk1 as cellular signal transducers in Labeo rohita in response to PAMPs stimulation and pathogenic invasion.

Mitogen-activated protein kinases (MAPKs) are crucial Ser/Thr protein kinases that play important roles in innate immunity by converting extracellular stimuli into a wide range of cellular responses, including the production of cytokines. In this study, two MAPK genes, jnk1 and erk1, were cloned and characterized in rohu (Labeo rohita), a commercially important freshwater fish species in the Indian subcontinent. In healthy rohu, both jnk1 and erk1 gene expressions were highest in the spleen as compared to gill, liver, blood and kidney tissues. In vitro stimulation of the L. rohita gill (LRG) cell line with γ-D-glutamyl-meso-diaminopimelic acid, muramyl dipeptide and polyinosinic: polycytidylic acid (poly I:C) resulted in significantly enhanced expressions of jnk1 and erk1 genes. In the in vivo experiments, jnk1 and erk1 gene expressions were also enhanced in lipopolysaccharides and poly I:C-treatment. Infection of rohu fingerlings with Aeromonas hydrophila and Bacillus subtilis revealed significantly enhanced expressions of the jnk1 and erk1 genes in all of the tested organs/tissues. Together these results imply the important role of jnk1 and erk1 genes in fish during pathogenic invasion and diseases.

Ultrastructure of intracytoplasmic Rickettsia-like infection of the gills of the teleost Archosargus probatocephalus (Sparidae) in northeastern Brazil.

A histopathological survey was conducted to investigate the presence of microparasites in fish Archosargus probatocephalus in a river near Maceió, Brazil. Light microscope observations of fragments of gill showed the presence of small cysts containing numerous myxospores that were morphologically identified as Henneguya. Transmission electron microscopy observations further revealed several gill cells containing groups of prokaryotic cells within large cytoplasmic vacuoles. Each infected host cell displayed a single vacuole containing a variable number of Rickettsia-like cells (up to 11), some of which presented the dumbbell shape characteristic of binary fission. The Rickettsia-like cells were pleomorphic, without a nucleus and with chromatin dispersed in the cytoplasm. They had a thin electron-dense wall of Gram-negative type. The morphology of these prokaryotic was similar to those of the order Rickettsiales and was described as a Rickettsia-like organism. Histopathological evaluation showed that several vacuole membranes had a lysed appearance. Some had ruptured, thus allowing direct contact between the Rickettsia-like organism and the cytoplasm of the host cell. The rupturing of the branchial epithelium may have contributed towards reduction of the surface area of the gills, but it is not possible to say that this was the cause of the host's death.

ABC transporters in gills of rainbow trout (Oncorhynchus mykiss).

Fish gills are a structurally and functionally complex organ at the interface between the organism and the aquatic environment. Gill functions include the transfer of organic molecules, both natural ones and xenobiotic compounds. Whether the branchial exchange of organic molecules involves active transporters is currently not known. Here, we investigated the presence, diversity and functional activity of ATP-binding cassette (ABC) transporters in gills of juvenile rainbow trout. By means of RT-qPCR, gene transcripts of members from the abcb, abcc and abcg subfamilies were identified. Comparisons with mRNA profiles from trout liver and kidney revealed that ABC transporters known to have an apical localization in polarized epithelia, especially abcc2 and abcb1, were under-represented in the gills. In contrast, ABC transporters with mainly basolateral localization showed comparable gene transcript levels in the three organs. The most prominent ABC transporter in gills was an abcb subfamily member, which was annotated as abcb5 based on the synteny and phylogeny. Functional in vivo assays pointed to a role of branchial ABC transporters in branchial solute exchange. We further assessed the utility of primary gill cell cultures to characterize transporter-mediated branchial exchange of organic molecules, by examining ABC transporter gene transcript patterns and functional activity in primary cultures. The gill cultures displayed functional transport activity, but the ABC mRNA expression patterns were different to those of the intact gills. Overall, the findings of this study provide evidence for the presence of functional ABC transporter activity in gills of fish.

Novel Insights into the Role of Copper in Critical Life Stages of Oysters Revealed by High-Resolution NanoSIMS Imaging.

Copper (Cu) is a key trace element for many biological processes, but there is little available information regarding its requirements and functions in critical life stages of marine bivalves, during which dramatic morphological and physiological changes occur. In this study, the ontogeny pattern of Cu accumulation across the life history of oysters was explored for the first time and the distributions of Cu in oysters at critical life stages (pediveliger and early settled spat) were in situ mapped by nanoscale secondary ion mass spectrometry (NanoSIMS) with high lateral resolution. We first demonstrated that the late pelagic stage to early settled stage was the critical stage requiring Cu during oyster development. NanoSIMS imaging revealed a significant elevation of intercellular Cu levels along with mitochondrial calcium overload and obvious structural degradation of mitochondria in velar cells at the pediveliger stage, implying the possible role of Cu in cell apoptosis of the velum during larval metamorphosis. Furthermore, an obvious enrichment of Cu together with calcium was observed in the nucleus and mitochondria of ciliated cells of gill tissue at the early settled stage. Their accumulation in the gill cells was significantly higher than that in the juveniles, indicating the potential role of Cu in sustaining the fast growth of the gill filament and the concomitant acceleration of energy metabolism during early benthic development. Our findings offer new insights into the understanding of the interactions between trace metals and marine bivalves.

CYTOTOXIC AND GENOTOXIC EFFECT OF OXYTETRACYCLINE ON FISH CYPRINUS CARPIO AFTER ACUTE EXPOSURE

Objective: Cytotoxicity in blood cells and genotoxic alteration in gill, liver, and kidney of Cyprinus carpio acutely (96 h) exposed to 80 mg/L oxytetracycline (OTC) including a control (non-exposed group) were evaluated in the present study. Genotoxic endpoints reflecting different types of genetic damage in cells of the liver, gill, and kidney were also determined by analysis of nuclear and cytoplasmic abnormalities.&#x0D; Methods: C. carpio was divided into two groups, one control and others treated with 80 mg/L OTC. After 96 h, sampling was done and slides were prepared for different tissues as well as for measuring polychromatic erythrocyte (PCE) frequency in blood. Slides were scored for micronucleus, nuclear abnormalities, swollen cells, and vacuolated cytoplasm.&#x0D; Results: The hereby data obtained showed a higher and significant increase in the genotoxic effect in all the tissues tested. Furthermore, gill cells showed the highest genotoxic effect followed by liver and kidney, while PCE frequency increases up to 72 h of exposure, on the other hand, a significant decrease in the value was observed at 96 h of exposure.&#x0D; Conclusion: The present study revealed that OTC has cytotoxic and genotoxic effect on different organs and blood cells of C. carpio at this concentration and suggests gill as sensitive tissue for genotoxic assessment.

Cytogenotoxic effects of fullerene C60 in the freshwater teleostean fish, Anabas testudineus (Bloch, 1792)

In recent years, carbon nanomaterials, including fullerene C60 is regarded as the building block in nanotechnology because of its widespread use in medicine, industry, cosmetics and commercial products. Despite the special properties, several reports have raised public health concerns due to the unknown and practically unexplored toxic effects of nanomaterials. However, there have been relatively few studies regarding the genotoxic responses of fullerene C60in vivo. Genotoxic effects of DMSO-solublized C60 nanomaterial suspension at sublethal concentrations (5 and 10 mg/L) were investigated on adult freshwater fish, Anabas testudineus using micronucleus and comet assays. An assessment of micronucleus induction showed severe cytoplasmic and nuclear abnormalities in erythrocytes, gill and liver cells. Abnormalities in cytoplasm were identified as formation of sticky cells, vacuolated cytoplasm, cytoplasmic degeneration, echinocyte, acanthocyte, anisochromatic cells and abnormal erythrocyte membrane. The nuclear abnormalities included micronucleus, binucleated cells, nuclear buds, irregular nucleus, vacuolated, notched and serrated nucleus in the erythrocytes compared to the control groups. Similarly, significant increase (P < 0.05) in micronucleus frequencies were observed in gill and liver cells. The high frequency of micronucleus was observed in the gill cells followed by liver and erythrocytes, respectively, at both sublethal concentrations, and the severity was duration and concentration-dependent. In comet assay, significant increase (P < 0.05) in DNA damage was observed using the comet parameter, percent tail DNA. The highest level of comet damage with grade 3 was observed in blood, gill and liver cells on increase in duration and concentration when compared to the respective control groups. Thus the results revealed that fullerene C60 nanomaterials may pose risk to aquatic organisms, especially fish, by the induction of genotoxicity. Further studies are warranted to provide new insights on the mechanisms and consequences of C60 nanomaterials interactions with biological membranes.

In vitro propagation of tilapia lake virus in cell lines developed from Oreochromis mossambicus.

Tilapia lake virus (TiLV)-suspected samples of tilapia were collected from grow-out ponds located with clinical signs and mortality ranged from 5% to 50%. The reverse transcription-polymerase chain reaction (RT-PCR) assay revealed the presence of TiLV in the disease outbreak ponds. Cell lines were developed from heart, gill and eye of Mozambique tilapia and characterized. Morphologically, these cell lines are composed of epithelioid cells. The optimum growth of these cells was observed at 28°C and 20% concentration of FBS. After cryopreservation, 70%-90% of cells were found to be viable. The cells of all three cell lines were found to be positive to fibronectin and pancytokeratin. PCR amplification of 16S rRNA and COI of O.mossambicus confirmed the origin of these cell lines from O.mossambicus. Heart and gill cell lines were found to be highly susceptible to TiLV and found to be useful for its isolation from infected fish samples. The experimental infection was carried out in O.niloticus and O.mossambicus using the TiLV propagated in susceptible cell lines. The RT-PCR results revealed the presence of TiLV in brain, gill, liver, kidney, spleen, eye, muscle, intestine and heart of experimentally infected O.niloticus and O.mossambicus.

Participation of Na+/K+-ATPase and aquaporins in the uptake of water during moult processes in the shrimp Palaemon argentinus (Nobili, 1901).

Due to the presence of the exoskeleton, the moult cycle is a required event in the life of crustaceans. In order for the exoskeleton to be replaced, it is necessary for these animals to uptake water from the environment for their body tissues during the late pre-moult, ecdysis and in the early post-moult for the expansion of the new cuticle. The mechanisms and organs used to uptake water in these events are not yet completely clear. In this study, we investigated the participation of aquaporins and Na+/K+-ATPase in cells of two potential organs responsible for the uptake of water (gills and gut) at three different stages of the moult cycle in freshwater shrimp Palaemon argentinus. We showed the participation of these two proteins with different functional patterns in gills and intestinal cells as water uptake pathways for moult and early post-moult. Our results indicate that Na+/K+-ATPase promotes the necessary osmotic gradient in the gills for water uptake through the gut cells during the pre-moult. This process, in turn, remains active during the post-moult stage with the addition of water influx through the gill cells.

Immune response of channel catfish (Ictalurus punctatus) against Ichthyophthirius multifiliis post vaccination using DNA vaccines encoding immobilization antigens

The channel catfish (Ictalurus punctatus) immune response against Ichthyophthirius multifiliis (Ich) after vaccination using plasmid DNA vaccines pcDNA3.1-IAg52a and pcDNA3.1-IAg52b, encoding Ich immobilization antigen genes was studied. Parasite infection level, serum anti-Ich antibodies level, fish mortality after theront challenge, and immune-related gene expression were measured. After in vitro transfection of walking catfish gill cells (G1b) with both pcDNA3.1-IAg52a and pcDNA3.1-IAg52b, antigens IAG52A and IAG52B were detected. During the vaccination trial, 76-fold increase in the Iag52b gene expression was observed in the vaccinated fish group h4 post vaccination. Administration of DNA vaccines by IM injection induced significant gene up-regulation in the head kidney, including immunoglobulin M (IgM), cluster of differentiation 4 (CD4), major histocompatibility I (MHC I), and T cell receptor α (TcR-α) from h4 to d5 post immunization. Fish vaccinated with DNA vaccines or theronts showed increased gene expression of the cytokine interferon (IFN-γ), complement component 3 (C3), and toll-like receptor-1 (TLR-1). Anti-Ich antibodies were detected in fish received pcDNA3.1-IAg52a, pcDNA3.1-IAg52b and the combination of both vaccines d10 post vaccination. Fish vaccinated with pcDNA3.1-IAg52b showed mild parasite infection level, partial survival (20%) and longer mean day to death (MDD) after theront challenge. By contrast, a heavy parasite load, 0% survival and short MDD were observed in the sham vaccinated control fish that received pcDNA3.1 (plasmid without genes encoding Ich immobilization antigen). Further research is needed to improve DNA vaccines for Ich that can induce strong protective immunity in fish. Suggested studies include improved transfection efficiency, use of appropriate adjuvants and including additional parasite antigen genes in the plasmid.

Identification of cells expressing two peptidoglycan recognition proteins in the gill of the vent mussel, Bathymodiolus septemdierum

In symbiotic systems in which symbionts are transmitted horizontally, hosts must accept symbionts from the environment while defending themselves against invading pathogenic microorganisms. How they distinguish pathogens from symbionts and how the latter evade host immune defences are not clearly understood. Recognition of foreign materials is one of the most critical steps in stimulating immune responses, and pattern recognition receptors (PRRs) play vital roles in this process. In this study, we focused on a group of highly conserved PRRs, peptidoglycan recognition proteins (PGRPs), in the deep-sea mussel, Bathymodiolus septemdierum, which harbours chemosynthetic bacteria in their gill epithelial cells. We isolated B. septemdierum PGRP genes BsPGRP-S and BsPGRP-L, which encode a short- and a long-type PGRP, respectively. The short-type PGRP has a signal peptide and was expressed in the asymbiotic goblet mucous cells in the gill epithelium, whereas the long-type PGRP was predicted to include a transmembrane domain and was expressed in gill bacteriocytes. Based on these findings, we hypothesize that the secreted and transmembrane PGRPs are engaged in host defence against pathogenic bacteria and/or in the regulation of symbiosis via different cellular localizations and mechanisms.

From DNA to ecological performance: Effects of anthropogenic noise on a reef-building mussel

Responses of marine invertebrates to anthropogenic noise are insufficiently known, impeding our understanding of ecosystemic impacts of noise and the development of mitigation strategies. We show that the blue mussel, Mytilus edulis, is negatively affected by ship-noise playbacks across different levels of biological organization. We take a novel mechanistic multi-method approach testing and employing established ecotoxicological techniques (i.e. Comet Assay and oxidative stress tests) in combination with behavioral and physiological biomarkers. We evidence, for the first time in marine species, noise-induced changes in DNA integrity (six-fold higher DNA single strand-breaks in haemocytes and gill epithelial cells) and oxidative stress (68% increased TBARS in gill cells). We further identify physiological and behavioral changes (12% reduced oxygen consumption, 60% increase in valve gape, 84% reduced filtration rate) in noise-exposed mussels. By employing established ecotoxicological techniques we highlight impacts not only on the organismal level, but also on ecological performance. When investigating species that produce little visually obvious responses to anthropogenic noise, the above mentioned endpoints are key to revealing sublethal effects of noise and thus enable a better understanding of how this emerging, but often overlooked stressor, affects animals without complex behaviors. Our integrated approach to noise research can be used as a model for other invertebrate species and faunal groups, and inform the development of effective methods for assessing and monitoring noise impacts. Given the observed negative effects, noise should be considered a potential confounding factor in studies involving other stressors.

IMPACT OF WHITE SPOT SYNDROME VIRUS DISEASE ON GILLS IN SHRIMP, PENAEUS MONODON

Protozoan parasites cause problems in Penaeid shrimp culture and major impact on the shrimp farming industry.White spot syndrome virus (WSSV) is a major shrimp pathogen that also infects Penaeus monodon species was selected. Histological changes in the gill cells were studied. Histological techniques using paraffin embeded tissues as well as frozen tissues were used for identification of WSSV infection. Histological manifestation such as gill cells could be detected. The gill cells were marked and characteristic of WSSV infections were observed. The present investigation was focused on the virus infecting shrimp. The microscopic examinations of the histological preparation are presently used to detect WSSV zoothamnium.

Prymnesium parvum differentially triggers sublethal fish antioxidant responses in vitro among salinity and nutrient conditions

Significant fish kills have been attributed to Prymnesium parvum in coastal and inland waters around the world. However, specific mechanisms responsible for adverse outcomes resulting from this harmful algal bloom (HAB) species remain unclear, though the gill has previously been identified as an important target organ. In the present study, an in vitro approach was used to examine cytotoxicity and antioxidant responses in fish liver (Hepa-E1 and PLHC-1) and gill (G1B and RTgill-W1) cell lines, following exposure to P. parvum grown at different salinities and nutrient concentrations, which can influence the magnitude of acute toxicity. Cultures from high salinity compromised survival of hepatic cell lines exposed to high dilutions, whereas no significant cytotoxicity was observed for gill cell lines. With respect to control groups, catalase showed significant activity in both gill cell lines, especially RTgill-W1, following exposure to high salinity cultures. High levels of superoxide dismutase were measured in Hepa-E1 cells exposed to all experimental treatment combinations and in RTgill-W1 cells following exposure to high salinity conditions, with respect to non-exposed cells Glutathione peroxidase activity was also detected at significant levels in Hepa-E1 cells after exposure to cultures from high salinity and the low salinity X low nutrients. Slight GPx increases were only observed in PLHC-1 and G1B exposed to P. parvum grown at high salinity. These results suggest that: 1. specific combinations of salinity and nutrient levels may contribute to production and potency of P. parvum toxins resulting in sub-lethal effects, and 2. sub-lethal responses are more prominent than cytotoxicity, and that oxidative stress may be a significant adverse effect of toxins produced by P. parvum.

Regulation of glyceraldehyde-3-phosphate dehydrogenase by hypoxia inducible factor 1 in the white shrimp Litopenaeus vannamei during hypoxia and reoxygenation

Hypoxia is a frequent source of stress in the estuarine habitat of the white shrimp Litopenaeus vannamei. During hypoxia, L. vannamei gill cells rely more heavily on anaerobic glycolysis to obtain ATP. This is mediated by transcriptional up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The hypoxia inducible factor 1 (HIF-1) is an important transcriptional activator of several glycolytic enzymes during hypoxia in diverse animals, including crustaceans. In this work, we cloned and sequenced a fragment corresponding to the 5′ flank of the GAPDH gene and identified a putative HIF-1 binding site, as well as sites for other transcription factors involved in the hypoxia signaling pathway. To investigate the role of HIF-1 in GAPDH regulation, we simultaneously injected double-stranded RNA (dsRNA) into shrimp to silence HIF-1α and HIF-1β under normoxia, hypoxia, and hypoxia followed by reoxygenation, and then measured gill HIF-1α, HIF-1β expression, GAPDH expression and activity, and glucose and lactate concentrations at 0, 3, 24 and 48 h. During normoxia, HIF-1 silencing induced up-regulation of GAPDH transcripts and activity, suggesting that expression is down-regulated via HIF-1 under these conditions. In contrast, HIF-1 silencing during hypoxia abolished the increases in GAPDH expression and activity, glucose and lactate concentrations. Finally, HIF-1 silencing during hypoxia-reoxygenation prevented the increase in GAPDH expression, however, those changes were not reflected in GAPDH activity and lactate accumulation. Altogether, these results indicate that GAPDH and glycolysis are transcriptionally regulated by HIF-1 in gills of white shrimp.