Gill Cells Research Articles

Assessment of cytotoxicity in gills of fish Labeo rohita reared in untreated and treated sewage water.

The unregulated discharge of untreated municipal sewage water to the natural water bodies is a major threat to the aquatic ecosystems. In the present study, the fingerlings of Labeo rohita were exposed to treated sewage water and 1/10th of LC50 and 1/20th of LC50 of untreated sewage water (UT) obtained from sewage water treatment plant, Ludhiana, India. After determining 96-h LC50 value of UT, fingerlings were divided into four groups: control, treated, 1/10th of LC50 UT and 1/20th of LC50 UT and exposed for a period of 2months. Our study revealed that 1/10th LC50 UT and 1/20th LC50 UT groups had significant reduction in body weight, total body length, standard body length and gills somatic index in comparison with the control and treated groups. Histopathological alterations in cellular structure of gills such as ruptured primary lamellae, broken secondary lamellae, distorted chloride cells and goblet cells, missing lamellae and disintegrated lamellar epithelium were also observed in 1/10th LC50 UT and 1/20th LC50 UT groups. A significantly high frequency of micronucleated (MN) cells was observed in 1/10th LC50 UT and 1/20th LC50 UT groups along with the presence of binucleated cells (BN), elongated nuclei (EN) and nuclear buds in gill cells. Genotoxic nature of UT was further confirmed from significantly high values of genetic damage index (GDI) and percentage (%) DNA damage in gill cells of fingerlings exposed to sub-lethal concentrations of UT. The study concluded that untreated sewage water has potential to induce anatomical and physiological defects in gills cells and the severity of toxicity increases with the increase in duration of exposure.

The Atlantic Salmon Gill Transcriptome Response in a Natural Outbreak of Salmon Gill Pox Virus Infection Reveals New Biomarkers of Gill Pathology and Suppression of Mucosal Defense

The salmon gill poxvirus (SGPV) is a large DNA virus that infects gill epithelial cells in Atlantic salmon and is associated with acute high mortality disease outbreaks in aquaculture. The pathological effects of SGPV infection include gill epithelial apoptosis in the acute phase of the disease and hyperplasia of gill epithelial cells in surviving fish, causing damage to the gill respiratory surface. In this study, we sampled gills from Atlantic salmon presmolts during a natural outbreak of SGPV disease (SGPVD). Samples covered the early phase of infection, the acute mortality phase, the resolving phase of the disease and control fish from the same group and facility. Mortality, the presence and level of SGPV and gill epithelial apoptosis were clearly associated. The gene expression pattern in the acute phase of SGPVD was in tune with the pathological findings and revealed novel transcript-based disease biomarkers, including pro-apoptotic and proliferative genes, along with changes in expression of ion channels and mucins. The innate antiviral response was strongly upregulated in infected gills and chemokine expression was altered. The regenerating phase did not reveal adaptive immune activity within the study period, but several immune effector genes involved in mucosal protection were downregulated into the late phase, indicating that SGPV infection could compromise mucosal defense. These data provide novel insight into the infection mechanisms and host interaction of SGPV.

Pharmacological and nutritional modulation of autophagy in a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1.

Autophagy is involved in the modulation of nutrition, immunity, and disease in humans and animals. To understand the impact of autophagy modulation on a rainbow trout gill cell line, RTgill-W1, treatments including reduced nutrition (2% fetal bovine serum compared with 10% control), rapamycin, 3-methyladenine, deoxynivalenol, and chloroquine were tested. Western blot and immunofluorescence were used to detect microtubule-associated protein 1A/1B-light chain protein and quantitative polymerase chain reaction was used to detect the expression of 10 autophagy-related genes. At 3-d post-treatment, reduced nutrition significantly (p < 0.05) increased autophagy while deoxynivalenol significantly (p < 0.01) suppressed it compared to controls. These phenomena were confirmed by using immunofluorescence to detect the number of autophagosomes in RTgill-W1. Chloroquine is critical to allow observation of microtubule-associated protein 1A/1B-light chain protein in this model. The commonly used autophagy-modulating chemicals rapamycin and 3-methyladenine either activated or suppressed microtubule-associated protein 1A/1B-light chain protein, respectively, as expected from the literature, but did not act in a consistently significant manner. Expression of five of the 10 Atg genes, including lc3, gabarap, atg4, atg7, and atg12, were altered in a similar pattern to microtubule-associated protein 1A/1B-light chain protein. The consistent trend of autophagy-related gene upregulation including becn1, lc3, gabarap, and atg9 following treatment with 3-methyladenine and chloroquine is suggestive of a novel feedback regulation in the autophagy machinery.

Polarized Trout Epithelial Cells Regulate Transepithelial Electrical Resistance, Gene Expression, and the Phosphoproteome in Response to Viral Infection.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1.

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p<.05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.

Cellular oxygen consumption, ROS production and ROS defense in two different size-classes of an Amazonian obligate air-breathing fish (Arapaima gigas).

In air-breathing fish a reduction of gill surface area reduces the danger of losing oxygen taken up in the air-breathing organ (ABO) to hypoxic water, but it also reduces the surface area available for ion exchange, so that ion regulation may at least in part be transferred to other organs, like the kidney or the gut. In the air-breathing Arapaima gigas, gill lamellae regress as development proceeds, and starting as a water-breathing embryo Arapaima turns into an obligate air-breathing fish with proceeding development, suggesting that ion regulation is shifted away from the gills as the fish grows. In Arapaima the kidney projects medially into the ABO and thus, probably a unique situation among fishes, is in close contact to the gas of the ABO. We therefore hypothesized that the kidney would be predestined to adopt an increased importance for ion homeostasis, because the elevated ATP turnover connected to ion transport can easily be met by aerobic metabolism based on the excellent oxygen supply directly from the ABO. We also hypothesized that in gill tissue the reduced ion regulatory activity should result in a reduced metabolic activity. High metabolic activity and exposure to high oxygen tensions are connected to the production of reactive oxygen species (ROS), therefore the tissues exposed to these conditions should have a high ROS defense capacity. Using in vitro studies, we assessed metabolic activity and ROS production of gill, kidney and ABO tissue, and determined the activity of ROS degrading enzymes in small (~ 5g, 2–3 weeks old) and larger (~ 670 g, 3–4 months old) A. gigas. Comparing the three tissues revealed that kidney tissue oxygen uptake by far exceeded the uptake measured in gill tissue or ABO. ROS production was particularly high in gill tissue, and all three tissues had a high capacity to degrade ROS. Gill tissue was characterized by high activities of enzymes involved in the glutathione pathway to degrade ROS. By contrast, the tissues of the ABO and in particular the kidney were characterized by high catalase activities, revealing different, tissue-specific strategies in ROS defense in this species. Overall the differences in the activity of cells taken from small and larger fish were not as pronounced as expected, while at the tissue level the metabolic activity of kidney cells by far exceeded the activity of ABO and gill cells.

Heavy metal bioaccumulation, oxidative stress and genotoxicity in African catfish Clarias gariepinus from Orontes river.

One of the major problems affecting the ecosystem health is the exposure of heavy metals due to anthropogenic activities. These exposures may induce toxiogenetic damage and carcinogenesis in aquatic organism. DNA damage biomarkers for fish species detect genotoxic parameters for ecological risk assessment. In the present study, the effect of heavy metals (Cr, Cd, Cu, Fe and Mn) on DNA damage and oxidative stress of Clarias gariepinus was examined by Comet assay at Reference site and two different sampling sites of the Orontes River. Moreover, the relationship between DNA damage and physiological response to oxidative stress caused in C. gariepinus was investigated by sampling seasonal water and fish samples for one year. In this study, Cr, Cd and Cu in water column of Orontes River and the concentrations of Fe and Mn in liver tissue of C. gariepinus were highly exceed the maximum permissible limit set by EPA (1999, 2016) WHO (1989) and TEG (2012) that can cause adverse effects on environment health. SOD activities in liver tissues were significantly higher than those in muscle tissues. SOD, CAT activity and MDA levels of both Site 1 and Site 2 at Orontes River were higher than those of the Reference site. In result of the COMET analysis, the highest levels of DNA damage were found at gill and liver tissues in Site 2 (17.746 ± 1.072% T-DNA; 16.014 ± 0.710% T-DNA respectively) at Orontes river. A higher level of DNA damage in gill cells compared to liver cells was observed in all regions of the Orontes River. In Principal component analysis (PCA), the heavy metals Cd, Cr and Cu (in the water column) and MDA in liver and muscle revealed strong contribution to the observed DNA damage that were scattered opposite to each other's along the principal components. Moreover, correlations between parameters revealed a positive significant relationship between Cd, Cr and Cu in water and DNA damage levels in C. gariepinus. Pearson correlation analysis detected a positive relationship between MDA, CAT and SOD and DNA damage levels in African catfish. Consequently, this study exposed genotoxic damage and oxidative stress of C. gariepinus due to metal pollution in Orontes River and lead to the better understanding of genotoxicty, oxidative stress and heavy metal relationships.

Mechanisms of pH-Dependent Uptake of Ionizable Organic Chemicals by Fish from Oil Sands Process-Affected Water (OSPW).

Uptake and effects of ionizable organic chemicals (IOCs) that are weak acids in aqueous solution by fish can differ as a function of pH. While the pH-dependent behavior of select IOCs is well-understood, complex mixtures of IOCs, e.g., from oil sands process-affected water (OSPW), have not yet been studied systematically. Here, we established an in vitro screening method using the rainbow trout gill cell line, RTgill-W1, to investigate pH-dependent cytotoxicity and permeation of IOCs across cultured epithelia using ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UPLC-HRMS). The assay was benchmarked using model chemicals and technical mixtures, and then used to characterize fractions and reconstituted extracts of field-collected OSPW. Significant pH-dependent cytotoxicity of individual IOCs, acidic fractions, and reconstituted extracts of OSPW was observed. In vitro data were in good agreement with data from a 96 h in vivo exposure experiment with juvenile rainbow trout. Permeation of some IOCs from OSPW was mediated by active transport, as revealed by studies in which inhibitors of these active transport mechanisms were applied. We conclude that the RTgill-W1 in vitro assay is useful for the screening of pH-dependent uptake of IOCs in fish, and has applications for in vitro-in vivo extrapolation, and prioritization of chemicals in nontarget screenings.

Effect of heavy metals on toxicogenetic damage of European eels Anguilla anguilla.

Aquatic organisms are exposed to a variety of contaminants such as heavy metals introduced into the environment as a consequence of anthropogenic activities that usually cause genotoxic damage in aquatic organisms. DNA damage biomarkers for fish species detect genotoxic parameters for ecological risk assessment. In the present study, the effect of heavy metals (Cd, Cr, Cu, Fe, Ni, Pb, Zn, Co, and Mn) on DNA damage of Anguilla anguilla was examined by comet assay at reference site and three different sampling sites of the Orontes River. The relative mean heavy metal concentrations in water column were in the order of Fe>Cr>Pb>Zn>Ni>Cu>Mn>Cd>Co in all the sampling sites. Cr, Cd, and Pb concentrations at all sampling sites were above the values allowed by the EPA (1999, 2016). With these results, negative effects of Cr, Cd, and Pb appeared on ecosystem health. The comet assay showed a higher level of DNA damage in the gill cells in comparison with the liver cells of A. anguilla. The highest level of DNA damage as %T-DNA, tail moment, and tail migration in gill cells were 20.007 ± 1.744 %; 2.899 ± 0.341 μm, and 12.383 ± 01.040 TMi and 20.172 ± 1.944 %, 2.559 ± 0.265 μm, and 10.763 ± 0.910 TMi at Site 2 and Site 3, respectively. The correlations between heavy metals and DNA damage parameters revealed that both Cu and Co in water showed significant negative correlations (p < 0.05) with DNA damage levels. Consequently, this study revealed the genotoxic damage of A. anguilla due to pollution in Orontes River and lead to the better understanding of genotoxicity and heavy metal relationships.

Türkiye'deki Ekogenotoksikolojik Çalışmalarda Balık Mikroçekirdek Sonuçlarının Değerlendirilmesi

In recent years, ecotoxicological studies have been conducted around the world, especially in aquatic ecosystems. It is noteworthy that genotoxicity tests have been frequently used in the biomonitoring of pollution. The present study evaluates genotoxicity tests including comet assays, micronucleus assays, fluorescent in-situ hybridization assays, chromosomal aberration assays, and sister chromatid exchange assays used in fish species for biomonitoring of environmental pollution in Turkey. Thirty-six ecotoxicological studies, which aimed to investigate possible genotoxic damage in fish species between the years 1970 and 2016, were analyzed. As a result of the literature review, it was concluded that the largest number of studies were done in 2011. There were micronucleus assays in 83.33%, comet assays in 30.55%, chromosomal aberration assays in 5.55%, and sister chromatid exchange experiments in 2.77% of the studies. Most of the micronucleus experiments were performed in eco-genotoxicological studies. Our results show that there were no statistically significant differences (p&amp;gt;0.05) between the erythrocyte and gill samples in the micronucleus results from the studies. This study is the first assessment of the use of genotoxicity tests in ecotoxicological studies in Turkey. Considering the principle of refinement in 4 R theory our results show that the use of gill cells instead of peripheral erythrocytes would be more appropriate in micronucleus assays.

Lymphocystis Disease Virus (Iridoviridae) Enters Flounder (Paralichthys olivaceus) Gill Cells via a Caveolae-Mediated Endocytosis Mechanism Facilitated by Viral Receptors.

In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder (Paralichthys olivaceus) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.

Effect-Based Approach to Assess Nanostructured Cellulose Sponge Removal Efficacy of Zinc Ions from Seawater to Prevent Ecological Risks.

To encourage the applicability of nano-adsorbent materials for heavy metal ion removal from seawater and limit any potential side effects for marine organisms, an ecotoxicological evaluation based on a biological effect-based approach is presented. ZnCl2 (10 mg L−1) contaminated artificial seawater (ASW) was treated with newly developed eco-friendly cellulose-based nanosponges (CNS) (1.25 g L−1 for 2 h), and the cellular and tissue responses of marine mussel Mytilus galloprovincialis were measured before and after CNS treatment. A control group (ASW only) and a negative control group (CNS in ASW) were also tested. Methods: A significant recovery of Zn-induced damages in circulating immune and gill cells and mantle edges was observed in mussels exposed after CNS treatment. Genetic and chromosomal damages reversed to control levels in mussels’ gill cells (DNA integrity level, nuclear abnormalities and apoptotic cells) and hemocytes (micronuclei), in which a recovery of lysosomal membrane stability (LMS) was also observed. Damage to syphons, loss of cilia by mantle edge epithelial cells and an increase in mucous cells in ZnCl2-exposed mussels were absent in specimens after CNS treatment, in which the mantle histology resembled that of the controls. No effects were observed in mussels exposed to CNS alone. As further proof of CNS’ ability to remove Zn(II) from ASW, a significant reduction of >90% of Zn levels in ASW after CNS treatment was observed (from 6.006 to 0.510 mg L−1). Ecotoxicological evaluation confirmed the ability of CNS to remove Zn from ASW by showing a full recovery of Zn-induced toxicological responses to the levels of mussels exposed to ASW only (controls). An effect-based approach was thus proven to be useful in order to further support the environmentally safe (ecosafety) application of CNS for heavy metal removal from seawater.

Isolation of the Tephrosia vogelii extract and rotenoids and their toxicity in the RTgill-W1 trout cell line and in zebrafish embryos

This study focused on identifying the rotenoids from the Tephrosia vogelli plant (fish-poison-bean), investigating the toxic potency of a crude T. vogelii extract and individual rotenoids (tephrosin, deguelin and rotenone) in vitro and in vivo and assessing the mode of action. A trout (Onychorynhis mykiss) gill epithelial cell line (RTgill-W1) was used to determine the cytotoxicity of rotenoids and effects on cell metabolism. Zebrafish (Danio rerio) aged from 3 h post fertilization (hpf) to 72 hpf were used for testing the developmental toxicity. The crude T. vogelii plant extract significantly decreased the cellular metabolic activity and was cytotoxic at lower concentrations (5 and 10 nM, respectively), while tephrosin, deguelin and rotenone showed these effects at concentrations ≥ 50 nM. The crude T. Vogelli extract had the highest toxic potency and induced adverse health effects in zebrafish including deformities and mortality at the lowest concentration (5 nM) compared to rotenone (10 nM) and deguelin and tephrosin (50 nM). These results indicate that the crude T. Vogelii extracts are highly potent and the bioactivity of these extracts warrant further investigation for their potential use to treat parasites in human and veterinary medicine and as a natural alternative to pesticides.

Screening of Chilean fish-killing microalgae using a gill cell-based assay

Fish-killing algal species are responsible for important global economic losses to the finfish industry. Chile is the world’s second-largest salmon exporter, and fish-killing algal blooms have widely impacted its production in the last decades. The lack of standardized analytical methods to quantify and characterize the so-called “ichthyotoxins” has hindered our understanding of the underlying ichthyotoxic modes of action. The novel application of a highly sensitive and reproducible fish RTgill-W1 cell line-based assay has allowed significant progress in the field. In this study, the ichthyotoxic potency of the main microalgae species, which has been reported in fish-killing events in the historical Chilean monitoring programs, was assessed. The dinoflagellate Karenia selliformis was the most ichthyotoxic species against the RTgill-W1 (cell viability down to 8%), representing the major threat for the local salmon industry. In comparison, the raphidophyte Heterosigma akashiwo and the dinoflagellate Prorocentrum micans were the least toxic (gill cell viability down to 81 and 89%, respectively). Importantly, ichthyotoxic flagellates were more toxic to fish gill cells upon rupture. These results have important implications for the mitigation and management of algal blooms by the salmon industry.

Labeo rohita Mx1 exhibits the critical structural motifs of the family of large GTPases of mammals and is activated by rhabdovirus vaccination and bacterial RNA stimulations

Myxovirus resistance (Mx) proteins belonging to the dynamin superfamily of high molecular weight GTPases exist in various isoforms and play crucial role in innate immunity. In addition to the isoforms, Mx1 also plays important role in exerting its anti-viral actions against a broad range of animal RNA viruses. In rohu (Labeo rohita), mx1 full-length cDNA sequence consists of 2440 nucleotides (nt) encoding 628 amino acids (aa) polypeptide of 71.289 kDa. Structurally, it belongs to the family of large GTPases with one DYNc domain (13-257aa) comprising of dynamin family motifs (LPRGSGIVTR) and the tripartite GTP-binding motifs (GDQSSGKS, DLPG and TKPD) at the N-terminal and one GED domain (537-628aa) at C-terminus. Rohu Mx1 is closely related to zebrafish Mx1 and is widely expressed in gill, liver, kidney, spleen and blood. In response to rhabdovirus vaccinations, poly I:C stimulation and bacterial infections, mx1 gene expression in rohu was significantly (p < 0.05) induced in majority of the tested organs/tissues. Stimulation of rohu gill cell line with bacterial RNA also induced mx1 gene expression. Together these data suggest the important role of Mx1 in innate immunity in rohu against wide spectrum of fish pathogens.

Caspase-8 in Labeo rohita is evolutionary conserved and is activated in Aeromonas hydrophila and Edwardsiella tarda infection and rhabdovirus vaccination

BackgroundCaspase-8, a member of the family of conserved cysteine proteases, plays a crucial role in the initiation phase of the apoptotic death-signaling cascade and thereby attracts interest for its study across the animal species including fish. In India, rohu (Labeo rohita) is an important freshwater fish species; thus, this study on caspase-8 was undertaken to investigate its role during pathogenic invasion.ResultsThe complete cDNA sequence of Labeo rohita caspase-8 (Lrcasp8) consisted of 1746 bp nucleotides (nt) having an ORF of 1440 nt encoding a polypeptide of 480 amino acid (aa) residues with the molecular mass of ∼ 54.8 kDa. Structurally, Lrcasp8 comprised two DED domains (DED11-77aa and DED297-174aa) and one CASc domain230-476aa. Within the CASc domain, various putative motifs, viz., a large subunit (p20237-360aa), a small subunit (p10389-474aa), and a penta-peptide (QACQG354-358aa) active site, were identified. The secondary structure of Lrcasp8 protein comprised seventeen α-helices, eleven β-strands, and twenty-nine coils. Phylogenetically, it is closely related to common carp caspase-8 and exhibits significant (p < 0.05) similarity (88.3%) and identity (78.7 %) in their amino acid sequence. The tissue-specific expression of Lrcasp8 has been analyzed by quantitative real-time PCR assay, and it revealed the highest expression (~ 23-fold) in the blood and lowest in the spleen. In Aeromonas hydrophila and Edwardsiella tarda infection and rhabdovirus vaccination, caspase-8 gene expression in rohu fingerlings was significantly (p <0.05) induced in various organs/tissues. Infection of the Labeo rohita gill cells with A. hydrophila resulted in apoptosis and cell death with the induction of caspase-8 gene expression.ConclusionThis is the first report on the identification and structural characterization of caspase-8 cDNA and predicted protein and the analysis of caspase-8 gene expression in Labeo rohita following Aeromonas hydrophila and Edwardsiella tarda infections and rhabdovirus vaccinations. The data in this article together suggest the critical role of caspase-8 during infection and apoptosis in Labeo rohita.

Unfolding the Mysteries of Oxygen Sensing ‐ A Comprehensive Analysis of the Hypoxic Response in Zebrafish Gills One Cell at a Time via Single Cell RNA Sequencing

All vertebrates “sense” changes in O2 through specialized cells, called chemoreceptors, which interact with the peripheral and central nervous systems to maintain O2 homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability. Therefore, it is vital for them to readily detect changes in PO2 and make appropriate respiratory and cardiovascular adjustments. Teleost fish, such as zebrafish (Danio rerio), have O2 chemoreceptors, called neuroepithelial cells (NECs), present in the gill epithelium and undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. NECs have also been shown to increase in number and size following chronic hypoxia. Here we present a comprehensive analysis of the effects of hypoxia on gill cells including NECs through a transcriptomic study. Using the transgenic line ETvmat2:GFP, zebrafish were acclimated to normoxia (PO2 = 160 mmHg) or severe hypoxia (PO2 = 35 mmHg ) and maintained for 14 days. Isolated cells from the distal ends of gill filaments, areas rich in NECs, were analyzed using single cell RNA sequencing (scRNA‐seq) analysis. Four normoxia (control) and four hypoxia‐treated biological samples were individually labelled before scRNA‐seq library construction using the 10X Genomics chromium technology. A total of 45,000 gill cells were consequently sequenced on a NextSeq500 (Illumina) sequencer with a read depth of 17,000 per cell. Our preliminary results separated 21 cell clusters and identified one cluster to be NECs based on gene expression. Highly differentially expressed genes in NECs included tph1a encoding tryptophan hydroxylase 1, the rate limiting enzyme in 5‐HT biosynthesis, and sv2 encoding synaptic vesicle 2 protein. Other differentially expressed genes in NECs are involved in mitochondrial oxidative metabolism and ATP synthesis. Moreover, differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of chronic hypoxia. NECs in the hypoxia group showed a high expression of genes involved in stress‐regulated polyubiquitination in contrast to the control group. The transcriptomic change implies a change in dynamics of the NEC population. We are the first group to conduct a single cell transcriptomic study in zebrafish gill cells. Our study is expected to provide a complete atlas for the hypoxic response in single gill cells and serve as a platform for future work on peripheral oxygen sensing.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).

Carp edema virus in ornamental fish farming in india: a potential threat to koi carps but not to co-cultured Indian major carp or goldfish

Koi sleepy disease caused by carp edema virus (CEV) of pox virus is a potential carp killer and poses a constant threat to co-cultured fish. Only limited studies are available on its occurrence from the eastern part of India. Here, we report a large-scale mortality of koi carps ( Cyprinuscarpio koi )in ornamental fish farm of Choudwar, Cuttack, Odisha. The fish showed clinical signs of ulcers on body, patchy haemorrhages, massive necrosis of gills, and observed sleeping at the bottom of tanks before death. However, the goldfish maintained in the same tanks did not show any clinical signs or mortality. We noticed Trichodina infestation in the gills of affected animals and secondary bacterium Aeromonashydrophila in kidney tissue. The gills, liver and kidneys of koi carp were found positive for carp edema virus (CEV) (Poxviridae) in PCR followed by sequencing and blast search, and phylogenetic analysis. the damages to the gills and kidney along with the presence of intracytoplasmic eosinophilic inclusions in gill epithelial cells upon histopathology suggested viral association. Experimental challenge with CEV failed to reproduce the disease in Indian major carp and goldfish, the commonly co-cultured fish species with koi carp, though, the disease was reproduced in koi carp. To our best knowledge, it is the first report of CEV infection from the farm of the eastern part of the country. Another mortality incidence in ornamental shop retailer in the same locality also revealed the presence of CEV in koi carp and pointed possibly towards its wide spread. Observations from this study suggest the need for CEV disease surveillance in the country

Hyperosmolality Induces Nuclear Translocation of Osmotic Stress Transcription Factor 1 in Oreochromis Mossambicus Cells

Euryhaline fish tolerate a wide range of environmental salinity by employing molecular mechanisms for coping with the associated osmotic stress. We have previously shown that osmotic stress transcription factor 1 (OSTF1) is part of these mechanisms. OSTF1 is transiently and rapidly upregulated in gill epithelial cells of tilapia (Oreochromis mossambicus) exposed to hyperosmolality. Hyperosmotic induction of OSTF1 was shown in tilapia gills, a response that was reproduced in the tilapia OmB cell brain fibroblast cell line (OmB). OSTF1 shares the signature sequence of the TSC‐22 family predicting that it is a transcription factor. If in fact OSTF1 is a transcription factor, we hypothesize it will localize to the nucleus during hyperosmotic stress. Using molecular cloning, OSTF1 was tagged with enhanced green fluorescent protein (EGFP) at either the C‐ or N‐terminus. Using fluorescent microscopy we showed that the fusion proteins were retained in the cytosol under iso‐osmotic conditions. To evaluate potential nuclear translocation of OSTF1 during hyperosmotic stress, we subjected OmB cells expressing the OSTF1:EGFP fusion protein to hyperosmotic media and imaged at time intervals from 5 minutes to 4 hours using a Leica Dmi8 microscope. At four hours and 650 mOsmol/kg, subcellular localization quantified by LASX image analysis (Leica) demonstrated that OSTF1:EGFP was mostly localized to the nucleus. This result supports our hypothesis that OSTF1 is indeed an osmotically inducible transcription factor. Current work evaluates the persistency of nuclear localization of OSTF1 during sustained hyperosmotic stress and isosmotic recovery.Support or Funding InformationThis study was funded by a grant from NSF (IOS‐1656371).

Functional trait thermal acclimation differs across three species of mid-Atlantic harmful algae

Characterizing the thermal niche of harmful algae is crucial for understanding and projecting the effects of future climate change on harmful algal blooms. The effects of 6 different temperatures (18–32 °C) on the growth, photophysiology, and toxicity were examined in the dinoflagellate Karlodinium veneficum, and the raphidophytes, Heterosigma akashiwo and Chattonella subsalsa from the Delaware Inland Bays (DIB). K. veneficum and H. akashiwo had skewed unimodal growth patterns, with temperature optima (Topt) at 28.6 and 27.3 °C respectively and an upper thermal niche limit of 32 °C. In contrast, C. subsalsa growth increased linearly with temperature, suggesting Topt and upper thermal boundaries >32 °C. K. veneficum photosystem II (PSII) photochemical efficiency remained stable across all temperatures, while H. akashiwo PSII efficiency declined at higher temperature and C. subsalsa was susceptible to low temperature (~18 °C) photoinactivation. Cell toxicity thermal response was species-specific such that K. veneficum toxicity increased with temperature above Topt. Raphidophyte toxicity peaked at 25–28 °C and was in close agreement with Topt for growth in H. akashiwo but below C. subsalsa maximal growth. The mode of toxicity was markedly different between the dinoflagellate and the raphidophytes such that K. veneficum had greater hemolytic activity while the raphidophytes had pronounced fish gill cell toxicity. These results and patterns of natural abundance for these algae in the DIB suggest that continued ocean warming may contribute to C. subsalsa bloom formation while possibly promoting highly toxic blooms of K. veneficum.