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Abstract MicroRNAs are non-coding RNAs, which widely participate in biological processes. In recent years, Siniperca chuatsi rhabdovirus (SCRV) has caused mass mortality in Chinese perch (Siniperca chuatsi). To identify specific miRNAs involved in SCRV infection, deep sequencing of microRNA on Chinese perch brain cell line (CPB) with or without SCRV infection were performed at 6 and 12 h post of infection (hpi). Totally 382 miRNAs were identified, including 217 known miRNA aligned with zebrafish miRNAs and 165 novel miRNAs by MiRDeep2 program. Of which 15 and 35 differentially-expressed miRNAs were determined respectively to 6 and 12 hpi. Nine miRNAs were selected randomly from the differentially-expressed miRNAs and validated by quantitative real-time PCR (qRT-PCR). These results were consistent with the microRNA sequencing results. Besides, target genes of 98 differentially-expressed miRNAs were predicted. Three of miRNAs (miR-122, miR-214, miR-135a) were selected, and its effects were analyzed in CPC cells transfected with appropriate miRNA mimics/inhibitors to evaluate its regulation effects by qRT-PCR and western blot. The results demonstrated that miR-214 inhibited the replication of SCRV, while miR-122 promoted the replication of SCRV and there was no correlation between the miR-135a and SCRV replication. These results will pave a new way for the development of effective strategies against the SCRV infection. Highlights • 382 miRNAs were identified by high throughput sequencing. • MiR-122 can stimulate SCRV replication. • MiR-122 can't target SCRV genome. [ABSTRACT FROM AUTHOR]

Background: The impact of nutritional status and diet composition on mitochondrial oxidative phosphorylation (OXPHOS) in fish remains largely unknown. To identify biomarkers of interest in nutritional studies, herein we obtained a deep-coverage transcriptome by 454 pyrosequencing of liver and skeletal muscle cDNA normalised libraries from long-term starved gilthead sea bream (Sparus aurata) and fish fed different diets. Results: After clean-up of high-throughput deep sequencing reads, 699,991 and 555,031 high-quality reads allowed de novo assembly of liver and skeletal muscle sequences, respectively (average length: 374 and 441 bp; total megabases: 262 and 245 Mbp). An additional incremental assembly was completed by integrating data from both tissues (hybrid assembly). Assembly of hybrid, liver and skeletal muscle transcriptomes yielded, respectively, 19,530, 11,545 and 10,599 isotigs (average length: 1330, 1208 and 1390 bp, respectively) that were grouped into 15,954, 10,033 and 9189 isogroups. Following annotation, hybrid transcriptomic data were used to construct an oligonucleotide microarray to analyse nutritional regulation of the expression of 129 genes involved in OXPHOS in S. aurata. Starvation upregulated cytochrome c oxidase components and other key OXPHOS genes in the liver, which exhibited higher sensitive to food deprivation than the skeletal muscle. However, diet composition affected OXPHOS in the skeletal muscle to a greater extent than in the liver: most of genes upregulated under starvation presented higher expression among fish fed a high carbohydrate/low protein diet. Conclusions: Our findings indicate that the expression of coenzyme Q-binding protein (COQ10), cytochrome c oxidase subunit 6A2 (COX6A2) and ADP/ATP translocase 3 (SLC25A6) in the liver, and cytochrome c oxidase subunit 5B isoform 1 (COX5B1) in the liver and the skeletal muscle, are sensitive markers of the nutritional condition that may be relevant to assess the effect of changes in the feeding regime and diet composition on fish farming. [ABSTRACT FROM AUTHOR]

Thermoregulation in ectothermic animals is influenced by the ability to effectively respond to thermal variations. While it is known that ectotherms are affected by thermal changes, it remains unknown whether physiological and/or metabolic traits are impacted by modifications to the thermal environment. Our research provides key evidence that fish ectotherms are highly influenced by thermal variability during development, which leads to important modifications at several metabolic levels (e.g., growth trajectories, microstructural alterations, muscle injuries, and molecular mechanisms). In Atlantic salmon ( Salmo salar), a wide thermal range (ΔT 6.4°C) during development (posthatch larvae to juveniles) was associated with increases in key thermal performance measures for survival and growth trajectory. Other metabolic traits were also significantly influenced, such as size, muscle cellularity, and molecular growth regulators possibly affected by adaptive processes. In contrast, a restricted thermal range (ΔT 1.4°C) was detrimental to growth, survival, and cellular microstructure as muscle growth could not keep pace with increased metabolic demands. These findings provide a possible basic explanation for the effects of thermal environment during growth. In conclusion, our results highlight the key role of thermal range amplitude on survival and on interactions with major metabolism-regulating processes that have positive adaptive effects for organisms. [ABSTRACT FROM AUTHOR]

We provide cytogenetic data for the threatened species Gymnogeophagus setequedas, and the first record of that species collected in the Iguaçu River, within the Iguaçu National Park's area of environmental preservation, which is an unexpected occurrence for that species. We verified a diploid number of 2n = 48 chromosomes (4sm + 24st + 20a) and the presence of heterochromatin in centromeric and pericentromeric regions, which are conserved characters in the Geophagini. The multiple nucleolar organizer regions observed in G. setequedas are considered to be apomorphic characters in the Geophagini, whereas the simple 5S rDNA cistrons located interstitially on the long arm of subtelocentric chromosomes represent a plesiomorphic character. Because G. setequedas is a threatened species that occurs in lotic waters, we recommend the maintenance of undammed environments within its known area of distribution. [ABSTRACT FROM AUTHOR]

Synchronous activity in populations of neurons potentially encodes special stimulus features. Selective readout of either synchronous or asynchronous activity allows formation of two streams of information processing. Theoretical work predicts that such a synchrony code is a fundamental feature of populations of spiking neurons if they operate in specific noise and stimulus regimes. Here we experimentally test the theoretical predictions by quantifying and comparing neuronal response properties in tuberous and ampullary electroreceptor afferents of the weakly electric fish Apteronotus leptorhynchus. These related systems show similar levels of synchronous activity, but only in the more irregularly firing tuberous afferents a synchrony code is established, whereas in the more regularly firing ampullary afferents it is not. The mere existence of synchronous activity is thus not sufficient for a synchrony code. Single-cell features such as the irregularity of spiking and the frequency dependence of the neuron's transfer function determine whether synchronous spikes possess a distinct meaning for the encoding of time-dependent signals. [ABSTRACT FROM AUTHOR]

In the last few decades, MALDI-TOF MS has become a useful technique not only in proteomics, but also as a fast and specific tool for whole cell analysis through intact cell mass spectrometry (IC-MS). The present study evaluated IC-MS as a novel tool for the detection of distinct patterns that can be observed after exposure to a certain toxin or concentration by utilizing the eukaryotic fish cell line RTL-W1. Two different viability assays were performed to define the range for IC-MS investigations, each of which employing copper sulfate, acridine, and β-naphthoflavone (BNF) as model compounds for several classes of environmental toxins. The IC-MS of RTL-W1 cells revealed not only specific spectral patterns for the various toxins, but also that the concentration used had an effect on RTL-W1 profiles. After the exposure with copper sulfate and acridine, the spectra of RTL-W1 showed a significant increase of certain peaks in the higher mass range ( m/ z >7000), which is probably attributed to the apoptosis of RTL-W1. On the contrary, exposure to BNF showed a distinct change of ion abundances only in the lower mass range ( m/ z <7000). Furthermore, a set of mass peaks could be identified as a specific biomarker for a single toxin treatment, so IC-MS demonstrates a new method for the distinction of toxic effects in fish cells. Due to fast sample preparation and high throughput, IC-MS offers great potential for ecotoxicological studies to investigate cellular effects of different substances and complex environmental samples. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Histochemical properties of the mucus cells in oesophagus and stomach in a teleost, black tetra ( Gymnocorymbus ternetzi), are described and compared. These cells were sac-shaped, very numerous, and embraced by ordinary epithelial cells with highly variable shape, throughout the entire length of oesophagus. The stomach luminal epithelial cells were filled with mucin apically, whereas the gastric gland cells lacked mucin. When treated with alcian blue (pH 2.5) before periodic acid-Schiff, the oesophagus mucin displayed a colour between blue and magenta, whereas it was strongly magenta in stomach. After high iron diamine followed by alcian blue (pH 2.5), the oesophagus mucin displayed a clean blue colour, whereas the stomach mucin was uncoloured. The present results for the lectin affinities in mucus cells in oesophagus of black tetra were similar to those of stomach and together suggest that these mucins contain significant amounts of N-acetylglucosamines, galactose- N-acetylgalactosamines sequences, and probably some sialic acid in terminal positions attached to the N-acetylglucosamine, but the mucin seems nearly or entirely to lack glucose and mannose. In addition, all its N-acetylgalactosamines in oesophagus, unlike in stomach, seem to be attached to galactose, as these amines were not coloured by DBA in oesophagus, but intensely coloured by PNA. Together the present results suggest that the oesophagus mucin in black tetra contains both neutral and carboxylated glycoproteins, but lacks sulphated glycoproteins, whereas the stomach mucin contains neutral glycoproteins only. The present results for black tetra suggest that the large amounts of oesophageal mucus and its increased toughness caused by anions may largely compensate for the lack of salivary glands. The stomach mucin lacks anions, a feature which may enhance the flow of the mucus and therefore also its ability to clean and protect the stomach wall against the gastric juice. [ABSTRACT FROM AUTHOR]

Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage, and mate choice and have played important roles in speciation. Here, we review studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve-associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns. [ABSTRACT FROM AUTHOR]

An off-gel non-denaturing isoelectric focusing (IEF) method was developed to separate uranium-biomolecule complexes from biological samples as a first step in a multidimensional metalloproteomic approach. Analysis of a synthetic uranium-bovine serum albumin complex demonstrated the focusing ability of the liquid-phase IEF method and the preservation of most of the uranium-protein interactions. The developed method was applied to gill cytosol prepared from zebrafish ( Danio rerio) exposed to depleted uranium. The results were compared in terms of resolution, recovery, and protein identities with those obtained by in-gel IEF using an immobilized pH gradient gel strip. [ABSTRACT FROM AUTHOR]

Abstract: Accurate and controlled methods to measure physiological stress are crucial to effectively monitor and assess the health of wildlife populations and evaluate resilience to external stressors. Glucocorticoids, particularly cortisol, are frequently used to measure stress in fish. While measurements of cortisol concentrations provide a powerful indicator of physiological stress, there are important considerations in accurately measuring and interpreting results. We assessed methods to capture and sample wild populations of salmonids and evaluated potential biases from sampling disturbance. We present results of a stress series and suggest approaches to mitigate bias associated with sampling disturbance. Studies on physiological stress in salmonids often focus on particular life stages (e.g. outward migration to marine waters, return migration to freshwater systems), or processes (e.g. fisheries interactions, spawning success), characterized by dramatic physiological challenges related to the developmental stage of the fish and the external environment. Such pressures influence baseline cortisol levels and complicate efforts to interpret the effects of additional external stressors. We present a profile for naturally occurring shifts in cortisol levels at migration, reproductive maturation, spawning, and senescence. This profile provides a crucial baseline for use as reference in evaluating physiological stress in Pacific salmon during crucial life stages. Our findings provide guidance for sampling wild salmonids and highlight the need for caution in interpreting cortisol in the context of physical challenges and physiological developments relevant to their complex life history. [Copyright &y& Elsevier]

The present study was conducted to evaluate the safety of long-term dietary curcumin at doses 0.5 and 1 % in Anabas testudineus employing hematological and cytological techniques. The fish were fed with curcumin-supplemented feed for 6 months. Fine blood smears were prepared and subjected to three different staining techniques. The erythrocyte micronucleus frequency (MN) and the cytometric measurements of erythrocytes were determined. Blood from the control and treated fish was subjected to the assessment of several hematological parameters. Also, DNA fragmentation assay on hepatocytes was conducted. The results showed that hemoglobin content, RBC count and hematocrit increased in the curcumin-fed fish compared to control, whereas WBC count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were unaffected. WBC/RBC ratio was lower in the case of curcumin-treated fish. The cytometric measurements revealed no change in the erythrocytes and their nuclei after curcumin treatment. DNA fragmentation assay revealed intact DNA in curcumin-fed group, ruling out the possibility of curcumin-induced DNA damage. The positive control group showed a significant increase in MN frequency compared to negative control and curcumin-fed groups. In fact, the MN frequency decreased in 1 % curcumin-fed group compared to the negative control and 0.5 % curcumin groups. All these indicated a state of well-being of the curcumin-treated fish. Therefore, it is concluded that curcumin could be used as a safe feed ingredient to improve the growth of finfish in aquaculture. [ABSTRACT FROM AUTHOR]

In cytosol from liver of pacu, Piaractus mesopotamicus, a hypoxia-tolerant fish that dwells in Pantanal, we found an enzyme activity capable of modulating the alkenal 4-hydroxy-2-nonenal (HNE) by conjugating it with glutathione (GST-HNE activity). HNE is a downstream metabolite from the oxidation of polyunsaturated fatty acids by reactive oxygen species arisen from mitochondria of animal cells. HNE production may increase more intensively under oxidative stress. Harmful effects to cell survival may occur when HNE increases over 10 M. Pacus submitted to hypoxia in July (cold season in Pantanal) showed 40 % less of this GST-HNE conjugating activity in their liver cytosol. Injecting pacus subjected to hypoxia during the cold season with a summer physiological dose of melatonin caused their liver cytosolic GST-HNE activity to increase up to the levels found in the warm season. From October to March (warm season in Pantanal), pacus are prone to oxidative stress particularly during potamodromous active oxygen-demanding swimming, when they migrate up rivers to spawn. Thus, our findings point out that the higher levels of melatonin in circulation during the summer are important to avoid the increase of 4-HNE inside liver cells of this fish species. [ABSTRACT FROM AUTHOR]

Abstract: The presence and distribution of apoptotic cells during thymus development and in adult were studied by in situ end-labelling of fragmented DNA in three temperate species carp (Cyprinus carpio), sea bass (Dicentrarchus labrax) and dusky grouper (Epinephelus marginatus) and in the adult thymus of three Antarctic species belonging to the genus Trematomus spp. During thymus development some few isolated apoptotic cell (AC) firstly appeared in the central–external part of the organ (carp: 5 days ph; sea bass: 35 days ph grouper: 43 days ph). Initially the cells were isolated and then increased in number and aggregated in small groups in the outer-cortical region of the thymus larvae. The high density of apoptotic cells was observed in the junction between cortex and medulla from its appearance (border between cortex and medulla, BCM). ACs decreased in number in juveniles and adult as well as the ACs average diameter. In late juveniles and in adulthood, the apoptosis were restricted to the cortex. In Antarctic species the thymus is highly adapted to low temperature (high vascularisation to effort the circulation of glycoproteins enriched plasma and strongly compact parenchyma). The apoptosis process was more extended (4–7 fold) as compare with the thymus of temperate species, even if the distribution of ACs was similar in all examined species. Data suggested a common process of T lymphocyte negative-selection in BCM of thymus during the ontogeny. The selection process seems to be still active in adult polar fish, but restricted mainly in the cortex zone. [Copyright &y& Elsevier]

Background and purpose: Heavy metals are non-degradable pollutants that could accumulate in marine animals including fish. The aim of this study was to determine the levels of six heavy metals (Zn, Cu, Ni, Pb, Cd and Hg) in edible tissue of Caspian carp (Cyprinus Carpio). We also studied the bioaccumulation factor (BFA) and target hazard quotients (THQ) of this fish in the southern Iranian Caspian Sea coast. Materials and methods: Twenty eight samples of fish were collected from seine and trawl during winter and spring 2010. Heavy metals were prepared (using Freeze dryer) by digestion and extraction process and then analyzed using atomic absorption equipped with flame, graphite and cold vapor system. Results: Results showed that Zinc (185±57) and Cu (3.68±0.30) µg/g.dw were seen more amongst the contaminants in the edible tissue of the Caspian carp. Concentration of Hg was 0.50±0.17 µg/g.dw and levels of Cd and Pb were below the limit of detection. BAF of metals were Zn> Cu> Hg. Conclusion: Ni, Cd and Pb in the Caspian carp muscle were lower that standard levels, but concentration of Hg was higher than the standard levels recommended by WHO, and ERL and ERM limits. Also, THQ revealed low levels of all metals and hazard index (HI) was found lower than critical level. It is believed that consumption of 142 gram of Caspian carp muscle per week does not have any prohibition for adults. [ABSTRACT FROM AUTHOR]

Abstract: Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities. [Copyright &y& Elsevier]

Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, Rana grylio virus (RGV, an iridovirus) thymidine kinase (TK) gene and viral envelope protein 53R gene were chosen as targets for foreign gene insertion. ΔTK-RGV and Δ53R-RGV, two recombinant RGV, expressing enhanced green fluorescence protein (EGFP) were constructed and analyzed in Epithelioma papulosum cyprinid (EPC) cells. The EGFP gene which fused to the virus major capsid protein (MCP) promoter p50 was inserted into TK and 53R gene loci of RGV, respectively. Cells infected with these two recombinant viruses not only displayed plaques, but also emitted strong green fluorescence under fluorescence microscope, providing a simple method for selection and purification of recombinant viruses. ΔTK-RGV was purified by seven successive rounds of plaque isolation and could be stably propagated in EPC cells. All of the plaques produced by the purified recombinant virus emitted green fluorescence. However, Δ53R-RGV was hard to be purified even through twenty rounds of plaque isolation. The purified recombinant virus ΔTK-RGV was verified by PCR analysis and Western blotting. These results showed EGFP was expressed in ΔTK-RGV infected cells. Furthermore, one-step growth curves and electron microscopy revealed that infection with recombinant ΔTK-RGV and wild-type RGV are similar. Therefore, RGV was demonstrated could be as a viral vector for foreign gene expression in fish cells. [Copyright &y& Elsevier]

During development, the embryos and larvae of the starfish Asterina pectinifera possess a single type of mesenchyme cell. The aim of this study was to determine the patterns of behavior of mesenchyme cells during the formation of various organs. To this end, we used a monoclonal antibody (mesenchyme cell marker) to identify the distribution patterns and numbers of mesenchyme cells. Our results revealed the following: (i) mesenchyme cell behavior differs in the formation of different organs, showing temporal variations and an uneven pattern of distribution; and (ii) mesenchyme cells continue to be generated throughout development, and their numbers are tightly regulated in proportion to total cell numbers. [ABSTRACT FROM AUTHOR]

The aim of the present study was to analyze the ultrastructural aspects of spermatogenesis in Phalloceros caudimaculatus, during cell proliferation. The parenchyma is organized morphologically as lobular restricted spermatogonial testis. Spermiogenesis in this species is characterized by four morphological stages of development (spermatids S1 through S4). The mature spermatids and spermatozoa heads are situated at the periphery of the cyst surrounded by the cytoplasm of the Sertoli cells. The germ cell's ultrastructure and spermatogenesis in P. caudimaculatus are very similar to that of other poeciliids. [ABSTRACT FROM AUTHOR]

Teleost fish have recently been implemented as suitable model organisms to study vertebrate development, in particular skeletogenesis. In vitro cell systems derived from fish bone have been successfully established, although their development has been hampered by the limited availability of fish serum to supplement culture medium. Commercially available sera are mostly of mammalian origin and thus not necessarily adequate to fish cell growth. The main objective of this work was to compare proliferative and mineralogenic potential of bovine and fish sera using fish bone-derived cell lines VSa13 and VSa16. Fish serum was shown to (i) strongly stimulate cell proliferation in an apparent dose-dependent and cell type-specific manner, (ii) induce morphological changes, and (iii) enhance extracellular matrix mineralization of bone cells, although cytotoxic for fish osteoblast-like cells at the concentration tested. To better understand mechanisms underlying mineralogenic effect of fish serum in fish chondrocytes, expression of several mineralization-related genes was evaluated by qPCR. Regulation of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP2) gene expression was modified upon culture with fish serum in a way compatible with an early onset and an increase in mineralization. In conclusion, fish serum was shown to be more adequate to proliferation and differentiation/mineralization of fish bone-derived cells. [ABSTRACT FROM AUTHOR]

Abstract: In fish, amphibians and mammals, gap junctions of some cells allow passage of elongate molecules as large as 18kDa, while excluding smaller, less elongate molecules. Fluorescently labeled Calmodulin (17kDa) and fluorescently labeled Troponin-C (18kDa), when microinjected into oocytes of Danio rerio, Xenopus laevis or Mus domestica, were able to transit the gap junctions between these oocytes and the granulosa cells which surrounded them. Co-microinjected with these Ca2+-binding proteins, Texas-red-labeled dextran (10kDa) remained in the microinjected cell. Osteocalcin (6kDa), also a Ca2+-binding protein, but with a wide “V” shape proved unable to transit these gap junctions. Calmodulin, but not Troponin-C, was able to transit gap junctions of gonadotropin treated WB cells in culture. We show evidence that molecules as large as 18kDa can pass through some vertebrate gap junctions, both homologous and heterologous, and that it is primarily molecular configuration which governs gap junctional permeability. [Copyright &y& Elsevier]

Abstract: Ichthyophthirius multifiliis, more commonly known as ‘white spot’ is recognised as one of the most pathogenic diseases of wild and cultured freshwater fish. In trout hatchery systems infections can quickly establish and result in high mortality if left unregulated. Current control centres around the use of regular bath treatments with formalin, however, treatments are not always effective and are labour intensive. Environmentally safe, non-chemical alternatives are required. This paper describes the performance of a mechanical system developed to remove cysts from commercial trout raceways. The system consists of two parts: a specially designed suction head connected to a pump that is used to vacuum the bottom of hatchery raceways, and a low-adhesion polymer raceway lining. Following tests on a series of potential linings, the most efficacious product in preventing settlement of cysts was used to line three raceways in a commercial rainbow trout (Oncorhynchus mykiss) hatchery. Over a period of three months, lined raceways were vacuumed on a daily basis. The three control raceways were maintained according to the farms normal husbandry procedures. Approximately thirty fish were sampled from each raceway at fortnightly intervals, and the abundance of the parasite in each raceway determined. The mechanical system led to a significant reduction in the abundance of the parasite in the test raceways, with a mean abundance of 361.88±260.75 parasites per fish in control raceways compared with 1.64±1.59 parasites per fish in the test raceways at the peak of the infection. Additionally fish survival was significantly higher in test raceways over control, with a mean of 84.5% of the stock surviving in the test raceways compared with only 70.6% in the controls by trial end. [Copyright &y& Elsevier]

Abstract: The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV), Pike-perch iridovirus (PPIV), European catfish virus (ECV), European sheatfish virus (ESV), EHNV, Doctor fish virus (DFV), Guppy virus 6 (GF6), short-finned eel virus (SERV) and Rana esculenta virus Italy 282/102 (REV 282/102). Each isolate was titrated in five cell lines: bluegill fry (BF-2), epithelioma papulosum cyprini (EPC), chinook salmon embryo (CHSE-214) rainbow trout gonad (RTG-2) and fathead minnow (FHM), and incubated at 10, 15, 20, 24 and 28°C for two weeks. BF-2, EPC and CHSE-214 cells performed well and titers obtained in the three cell lines were similar, whereas FHM and RTG-2 cells consistently produced lower titers than the other cell lines at all temperatures. The optimal temperature for propagating the isolates collectively to high titers in vivo was 24°C. Additionally, three established methods for re-isolation of virus from EHNV-infected organ material were compared. Challenged fish were sampled twice weekly and 7 organs were processed separately according to the three methods. Samples incubated on BF-2 cells at 22°C for 2weeks+1week sub-cultivation (method 1) provided more positive results than the other 2 methods and when using the EPC cell line. Virus was most frequently isolated from the kidney, followed by brain, muscle, heart, liver, gills and lastly spleen. [Copyright &y& Elsevier]

Abstract: The establishment and partial characterization of continuous cell lines from Epinephelus quoyanus are described. Three new cell lines, designated as RGB, RGG and RGH, were established from brain, gill and heart tissue, respectively, of the rockfish grouper E. quoyanus. Since their initiation in December of 2006, these cell lines have been subcultured in Leibovitz''s L-15 medium with 10% fetal bovine serum for more than 80 passages at 30 °C. The morphologies of RGB and RGG cells were epithelioid, while RGH was predominantly fibroblast-like. Propagation of these cell lines was serum-dependent, with a low plating efficiency (<4%). Karyotyping analysis indicated that the modal chromosome number was 48 for each cell type. The RGB and RGH cells were susceptible to grouper nervous necrosis virus (GNNV) but only yielded titers of 104.8 and 104.6 TCID50 ml−1, respectively. Moreover, RGB was susceptible to hard clam reovirus (HCRV) and yielded titers of 107. 2 TCID50 ml−1. All three cell lines were also sensitive to the extracellular products of Photobacterium damselae ssp. piscicida (Ph.d.p.) and produced significant fluorescent signals after transfection with pEGFP-C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity among the cell lines and E. quoyanus. Our results suggest that the three cell lines provide a valuable tool for transgenic studies and the investigation of the extracellular products of Ph.d.p. [Copyright &y& Elsevier]

Abstract: Incomplete eye migration is one of the major problems in intensive production of juvenile Atlantic halibut. More than 60% of an average juvenile population reared according to best practice suffers from this abnormality. In commercial production, these fish are discharged and represent a substantial economic loss and a large welfare problem. In the present investigation it is demonstrated that by controlling diurnal light and darkness periods together with a meal based feeding regime, incomplete eye migration can be dramatically reduced in production systems for Atlantic halibut. Control groups were reared under continuous light conditions, whereas the experimental groups were given 7 h of darkness and 17 h of light during a 24 hour cycle, in a period lasting from 12 to 35 days post first-feeding. Otherwise both groups were reared under continuous light conditions. All larvae were fed short time enriched Artemia supplied two times daily. The experimental conditions did not affect the overall growth or survival up to day 85 after first feeding. However, 27±3% of the fry reared under continuous light conditions had complete eye migration, whereas in juveniles reared under shifting light and darkness conditions, complete eye migration was 85±7%. These results represent a major improvement in production systems for Atlantic halibut juveniles. [Copyright &y& Elsevier]

Epulopiscium sp. type B, a member of the Firmicutes, is a large (up to 300 μm), cigar-shaped bacterial symbiont of surgeonfish that propagates itself by forming multiple intracellular offspring. This unusual form of reproduction is an apparent modification of a developmental program used by some Firmicutes to produce an endospore. At the onset of offspring formation, the Epulopiscium cell divides at both poles. The polar cells are engulfed by the larger mother cell and grow within the mother cell. At the final stages of development, the Epulopiscium mother cell lyses. Here we describe changes in Epulopiscium cell structure, focusing on mother cell DNA replication and cell death. DNA replication was examined by labeling cells with the nucleotide analog bromodeoxyuridine. As expected, DNA replication occurs in the developing offspring. However, well after passage of genetic information from parent to offspring is complete, DNA within the mother cell continues to replicate. Using fluorescence microscopy, we found that near the end of the offspring growth cycle, mother cell DNA disintegrates. The mother cell membrane and wall deteriorate as well. DNA replication within this terminally differentiated cell indicates the importance of mother cell nucleoids in cell maintenance and the development of offspring. The synchronized timing of mother cell deterioration within a population suggests that the Epulopiscium mother cell undergoes a programmed cell death. The programmed death of the mother cell may allow for the timely release of resources accumulated in the mother cell to provide nutrients to populations of these intestinal microbes and their host. [ABSTRACT FROM AUTHOR]

Chemokines are a superfamily of cytokines responsible for regulating cell migration under both inflammatory and physiological conditions. CC chemokines are the largest subfamily of chemokines, with 28 members in humans. A subject of intense study in mammalian species, the known functional roles of CC chemokines ligands in both developmental and disease conditions continue to expand. They are also an important family for the study of gene copy number variation and tandem duplication in mammalian species. However, little is known regarding the evolutionary origin and status of these ligands in primitive vertebrates such as teleost fish. In this paper, we review the evolution of the teleost fish CC chemokine gene family, noting evidence of widespread tandem gene duplications and examining the implications of this phenomenon on immune diversity. Through extensive phylogenetic analysis of the CC chemokine sets of four teleost species, zebrafish, catfish, rainbow trout, and Atlantic salmon, we identified seven large groups of CC chemokines. It appeared that several major groups of CC chemokines are highly related including the CCL19/21/25 group, the CCL20 group, CCL27/28 group, and the fish-specific group. In the three remaining groups that contained the largest number of members, the CCL17/22 group, the MIP group, and the MCP group, similarities among species members were obscured by rapid, tandem duplications that may contribute to immune diversity. [ABSTRACT FROM AUTHOR]

A scanning electron microscopic study of the skin and eye of UV-B radiated ayu Plecoglossus altivelis(age 30 days, mean ± s. e. total length: 16·25 ± 0·11 mm) under laboratory condition showed marked changes when compared with the control fish without UV-B radiation. The exposure of the fish to the radiation resulted in the destruction of microridges in the epidermis and exposed neuromast cells of the skin. Domed protrusions were also more common in the skin of UV-B radiated fish than in the control fish. The appearance of mucus in both groups was different. In the control skin the mucus was spread over a wide area whereas in the treated fish the mucus was concentrated in a small area. The anastomozing structures of the microridges of the eyes were lost in UV-B radiated fish and the microridges themselves were fewer in number, fragmented, and aggregated. Mucus cells, prominently visible in the control fish, were distorted in the treated fish. Cell contours were irregular in UV-B radiated fish and cell to cell contacts had been lost in this group. [ABSTRACT FROM AUTHOR]

Thyroid proliferative lesions are rather common in bony fishes but disagreement exists in the fish pathology community concerning diagnosticcriteria for hyperplastic versus neoplastic lesions. To simplify the diagnosis of proliferative thyroid lesions and to reduce confusion regarding lesioninterpretation, we propose specific criteria for distinguishing hyperplastic from neoplastic lesions. Development of these criteria was based on theexamination of a large series of proliferative lesions from Japanese medaka (Oryzias latipes), lesions from other small fish species, and a reexaminationof the 97 cases of proliferative thyroid lesions from bony fishes deposited in the Registry of Tumors in Lower Animals. Specific diagnostic criteriaare provided for all lesion categories including follicular cell hyperplasia (simple, nodular, or ectopic), adenoma (papillary or solid), and carcinoma(well-or poorly differentiated). These criteria should assist fish pathologists in describing and categorizing naturally occurring proliferative lesions from wild fishes, lesions that develop in laboratory fishes due to suboptimal culture practices or water quality, those in fishes used in toxicologicalassays, and captive aquarium fishes. [ABSTRACT FROM AUTHOR]

Abnormal oocytes were detected in the ovaries of triploid yellowtail flounder of three years of age. Triploidy in these females had been induced artificially by hydrostatic pressure shock. Binucleate oocytes were found in the ovaries of six different triploid females. These cells were always small and in a primary growth stage. Cytoplasmic vacuoles, presumed to be due to nucleoplasmic extrusion, was another abnormal feature observed in two primary growth oocytes, each seen in a separate female. The appearance of binucleate oocytes in triploid females, not previously noted in diploid yellowtail flounder, is proposed to be an ephemeral phenomenon associated with the triploid condition. [ABSTRACT FROM AUTHOR]

Tissues of Penaeus japonicus were primary cultured. Among them, dividing cells in varied shapes were found in primary cultured lymphoid cells and ovarian cells. Cell division processes of 18 lymphoid cells and 18 ovarian cells were studied. It was found that, at metaphase, aligned chromosomes of two dividing lymphoid cells and one dividing ovarian cell rotated 90° and these dividing cells cleaved along the deduced original spindle axis, which suggest that these cells may have carried on asymmetric division-like cell division. These results prove that the culture conditions used are suitable for cultured lymphoid cells and ovarian cells of P. japonicus to proliferate in vitro and that cultured lymphoid cells and ovarian cells still have the ability to carry on cell division. These findings will give a light to the study of continuous shrimp cell line establishment and will also provide us with the knowledge of shrimp cell division. [Copyright &y& Elsevier]

Methodology was developed for presenting to fish cells in culture whole-water samples without extraction and used to evaluate the toxicity to a rainbow trout gill cell line, RTgill-W1, of more than 30 whole-water samples collected from a paper mill over approximately a year of operation. Presentation to cells was achieved by adding to water samples the amounts of salts, galactose and sodium pyruvate, as solids, that were necessary to give concentrations and osmolality of the basal growth medium, Leibovitz''s L-15. Cell viability was measured with three fluorescent indicator dyes: alamar Blue™ for metabolism, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for membrane function, and neutral red for lysosomal activity. Eighteen samples were tested with the Daphnia lethality bioassay and 11 of these were toxic. None of these were judged cytotoxic to RTgill-W1. Sixteen samples were tested with the rainbow trout lethality bioassay and only one was toxic. This sample was also the only sample that was cytotoxic to RTgill-W1. Therefore, these methods for presenting water samples and measuring their cytotoxicity to RTgill-W1 are a promising substitute for toxicity tests of industrial effluent with rainbow trout but not with Daphnia. [Copyright &y& Elsevier]

The cytology of development and aging of the liver and pancreas in domestic carp was studied in a large sample of EM and LM sections from larvae juveniles 1–10 year old adults and 15 year old experimentally stunted fish weighing 20 g and of 110–120 mm TL. The primordium of the two glands appears in embryos 3–4 days after fertilization as buds from the gut wall. Formation of the hepatic tubules is first seen in larvae 10 days after fertilization. Two types of hepatocytes are discernable: one with a pale cytoplasm and the other with a dense opaque cytoplasm; both extend microvilli into the sinusoidal space of Disse and bear intracellular bile canaliculi. With maturation and branching of the hepatic tubules a complicated hepatic muralium is formed. The pancreatic cells develop in 5–6 mm TL embryos primarily along the blood capillaries and in 10–11 day old larvae exocrine A and B cells are seen prominent by their zymogen granules and spires and stacks of rough endoplasmic reticulum. Ripening of the Islets of Langerhans begin in larvae of 10 mm TL. By 16 mm TL two types of endocrine cells are visible. With aging three processes are recognizable in the liver: (1) increased branching of the blood net displaces the hepatic units and complicates the structure of the liver parenchyma; (2) a gradual increase in lipid vacuoles and debris deposition in secretory cells takes place; and (3) the number of melano-macrophage cells and melano-macrophage centers gradually increases. In 15-year-old experimentally stunted fish (110–120 mm TL) the liver and pancreas resemble those of juvenile fish appearing much healthier than those of 8–10 year old large carp from commercial ponds. These findings provide evidence that aging is not a simple time-dependent mechanism but is also strongly modulated by environmental factors. [ABSTRACT FROM AUTHOR]

Provides information on a study that investigated the effects of nonsteroidal anti-inflammatory drugs on the heat shock-induced heat shock proteins expression in fish cell line CHSE-214. Methodology of the study; Results and discussion on the study.

Presents a study suggesting that the fish E587 glycoprotein participates in axonal fasciculation and the age-related order of ganglion cell axons in the goldfish retina. Enrichment of the E587 at axon/axon contact sites; In vitro perturbation of axon fasciculation with antibodies against E587 antigen.

Examines the association between endothelial cells and rodlet cells (RC) in the head kidney of carp and discusses the function of RC. Theories that have been proposed concerning the nature and function of RC; Locations of RC within the carp; Connections between RC and the epithelium.

Abstract Aeromonas hydrophila can enter fish cells and exist as intracellular parasites. Phase‐contrast and confocal microscopy were used to examine morphological changes and various cytoskeletal components of infected fish cells. Four fish cell lines were included in this study: (1) AS, (2) BF2, (3) CHSE‐214, and (4) EPC cells. Virulent but not avirulent strains of A. hydrophila PPD 134/91 invaded fish cells, causing morphological changes, and inducing microfilament (F‐actin) rearrangement. Morphological changes were observed in all infected fish cell lines and could be classified into three different stages. In stage I, the cells became detached from each other and pointed ends were observed. In stage II, tubular cytoplasmic extensions formed at contact points connecting neighbouring cells. The monolayers formed a satellite‐like organization and became less confluent. Finally (stage III), cells were heavily infected with bacteria, and bacteria containing vacuoles occupied most of the cells. They eventually detached and lysed. Rearrangement of F‐actin was observed as local polymerization (actin clouds) in stage I and massive reorganization in stage III of infection. Actin clouds could have been induced by A. hydrophila for ‘assisted' uptake into the cells. The massive reorganization of actin in stage III may be due to products released by the bacteria and the growth of vacuoles. Pretreatment of fish cells with the microfilament inhibitors such as cytochalasins induced a similar effect. There were little if any rearrangements in intermediate and microtubule filaments during bacterial entry (stages I and II). These results suggest that A. hydrophila may bind to the surface and trigger a signal to the microfilament which then generates the force necessary for bacterial uptake. [ABSTRACT FROM AUTHOR]

Focuses on the use of immuno-histochemical technique to demonstrate the transition form of a squamous cell carcinoma in a mirror carp, Cyprinus carpio L. Evaluation of the papillomatous elements in carinomas; Application of immunohistochemical methods on normal and neoplastic fish tissues; Characterization of the different cell types.

The composition of leukocytes in the Patagonian toothfish (Dissostichus eleginoidesSmitt, 1898), caught in the Scotia Sea at a depth of more than 1000 m, was studied. Cells with various morphofunctional characteristics and cell structure were found. Lymphocytes were predominant, followed by, in decreasing order, eosinophils, monocytes, blast forms and neutrophils. The composition of leukocytes in the Patagonian toothfish consists of the same types of leukocytes as the earlier studied Antarctic toothfish (Dissostichus mawsoniNorman, 1937), but the percentage of white blood cells in the leukocyte formula differs between the two species. [ABSTRACT FROM PUBLISHER]

An abstract of the article "Fish Microsomal CYP450 Activities and Endocrine Disruption Screening," by Antony Strong, Andrew Deacon, Andrew McEwen, Guy Webber, and Stuart Wood is presented. Information on the metabolic activity of the major CYP450 enzymes in fish liver microsomes are presented, which was conducted as part of a validation of the use of fish in endocrine disruption screening studies. The data of which are used in comparison to those in rat liver microsomes.