Gills de novo assembly reveals oxidative stress, unfolded protein, and immune response on red cusk-eel (Genypterus chilensis) under thermal stress.

The heat waves on the South Pacific coast could lead to thermal stress in native fish. The red cusk-eel (Genypterus chilensis) is relevant for Chilean artisanal fisheries and aquaculture diversification. This study examined the effect of high-temperature stress in the gills of G. chilensis in control (14 °C) and high-temperature stress (19 °C) conditions. High-temperature stress induces a significant increase in gills cortisol levels. Additionally, oxidative damage was observed in gills (protein carbonylation and lipoperoxidation). RNA-seq data was used to build the first transcriptome assembly of gills in this species (23,656 annotated transcripts). A total of 1138 down-regulated and 1531 up-regulated transcripts were observed in response to high-temperature stress in gills. The enrichment analysis showed immune response and replication enriched processes (on down-regulated transcripts), and processes related to the folding of proteins, endoplasmic reticulum, and transporter activity (on up-regulated transcripts). The present study showed how gills could be affected by high-temperature stress.

First record of cannibalism in the giant mottled eel Anguilla marmorata (Actinopterygii: Anguilliformes) as revealed by DNA barcoding

Although the giant mottled eel, Anguilla marmorata Quoy & Gaimard, 1824, is widely distributed in the Indo-Pacific region, few ecological studies have been conducted on the species. We investigated the stomach contents of A. marmorata visually and used the DNA-barcode region of the mitochondrial cytochrome c oxidase subunit 1 (CO1) to confirm the species identification. The stomach content analysis revealed that teleosts and crustaceans are the major prey items of A. marmorata. Interestingly, the stomach content of one of the specimens, which was 1029 mm in total length (TL), contained an eel-like fish identified as A. marmorata measuring 510 mm in TL. This study is the first to record cannibalism in the diet of A. marmorata. Although the diet of anguillid eels is generally selective for a single prey species, larger eels are more likely to adopt a diverse feeding habit that includes cannibalism in the tropical river ecosystems.(AU)

DNA barcoding of a tropical anguillid eel, Anguilla bicolor (Actinopterygii: Anguilliformes), in Indo-Pacific region and notes on its population structure

The tropical anguillid eel, Anguilla bicolor McCelland, 1844, includes two subspecies, Anguilla bicolor bicolor McCelland, 1844 and Anguilla bicolor pacifica Schmidt, 1928, and is distributed across the Indo-Pacific region. Although A. bicolor is widely distributed and recognized as an important fish resource in the Indo-Pacific region, few studies have been conducted on its genetic variation and population structure. DNA barcoding of A. bicolor specimens collected in the Indo-Pacific region was carried out in this study using mitochondrial cytochrome c oxidase subunit I. Anguilla bicolor was found to diverge genetically, which supported its classification into two different subspecies. In addition, our study showed that A. bicolor bicolor had two genetically distinct populations/groups, and these different populations co-occur geographically in Indonesia and Malaysia in the eastern Indian Ocean. Our findings suggest that the eel larvae might be transported from at least two geographically different spawning grounds in the Indian Ocean, and then recruited to and settled in the same habitats in Indonesian and Malaysian waters. The molecular evidence calls for further research on the life history, stock assessment and protection of the populations of A. bicolor bicolor in Indonesia and Malaysia.(AU)

DNA barcoding of a tropical anguillid eel, Anguilla bicolor (Actinopterygii: Anguilliformes), in Indo-Pacific region and notes on its population structure

The tropical anguillid eel, Anguilla bicolor McCelland, 1844, includes two subspecies, Anguilla bicolor bicolor McCelland, 1844 and Anguilla bicolor pacifica Schmidt, 1928, and is distributed across the Indo-Pacific region. Although A. bicolor is widely distributed and recognized as an important fish resource in the Indo-Pacific region, few studies have been conducted on its genetic variation and population structure. DNA barcoding of A. bicolor specimens collected in the Indo-Pacific region was carried out in this study using mitochondrial cytochrome c oxidase subunit I. Anguilla bicolor was found to diverge genetically, which supported its classification into two different subspecies. In addition, our study showed that A. bicolor bicolor had two genetically distinct populations/groups, and these different populations co-occur geographically in Indonesia and Malaysia in the eastern Indian Ocean. Our findings suggest that the eel larvae might be transported from at least two geographically different spawning grounds in the Indian Ocean, and then recruited to and settled in the same habitats in Indonesian and Malaysian waters. The molecular evidence calls for further research on the life history, stock assessment and protection of the populations of A. bicolor bicolor in Indonesia and Malaysia.

Physiological and molecular responses to thermal stress in red cusk-eel (Genypterus chilensis) juveniles reveals atrophy and oxidative damage in skeletal muscle.

The red cusk-eel (Genypterus chilensis) is a native species with strong potential to support Chilean aquaculture diversification. Environmental stressors, such as temperature, may generate important effects in fish physiology with negative impact. However, no information exists on the effects of thermal stress in Genypterus species or how this stressor affects the skeletal muscle. The present study evaluated for the first time the effect of high temperature stress in red cusk-eel juveniles to determine changes in plasmatic markers of stress (cortisol, glucose and lactate dehydrogenase (LDH)), the transcriptional effect in skeletal muscle genes related to (i) heat shock protein response (hsp60 and hsp70), (ii) muscle atrophy and growth (foxo1, foxo3, fbxo32, murf-1, myod1 and ddit4), and (iii) oxidative stress (cat, sod1 and gpx1), and evaluate the DNA damage (AP sites) and peroxidative damage (lipid peroxidation (HNE proteins)) in this tissue. Thermal stress generates a significant increase in plasmatic levels of cortisol, glucose and LDH activity and induced heat shock protein transcripts in muscle. We also observed an upregulation of atrophy-related genes (foxo1, foxo3 and fbxo32) and a significant modulation of growth-related genes (myod1 and ddit4). Thermal stress induced oxidative stress in skeletal muscle, as represented by the upregulation of antioxidant genes (cat and sod1) and a significant increase in DNA damage and lipid peroxidation. The present study provides the first physiological and molecular information of the effects of thermal stress on skeletal muscle in a Genypterus species, which should be considered in a climate change scenario.

Identification and Evaluation of Long Noncoding RNAs in Response to Handling Stress in Red Cusk-Eel (Genypterus chilensis) via RNA-seq.

The red cusk-eel (Genypterus chilensis) is a native species with strong potential to support Chilean aquaculture diversification. Under commercial conditions, fish are exposed to several stressors. To date, little is known about the mechanism involved in the stress response of red cusk-eel, and there is no information related to the regulation mediated by long noncoding RNAs (lncRNAs). The objective of this work was to identify for the first time the lncRNAs in the transcriptome of G. chilensis and to evaluate the differential expression levels of lncRNAs in the liver, head kidney, and skeletal muscle in response to handling stress. We used previously published transcriptome data to identify the lncRNAs by applying a series of filters based on annotation information in several databases to discard coding sequences. We identified a total of 14,614 putative lncRNAs in the transcriptome of red cusk-eel, providing a useful lncRNA reference resource to be used in future studies. We evaluated their differential expression in response to handling stress in the liver, head kidney, and skeletal muscle, identifying 112, 323, and 108 differentially expressed lncRNAs, respectively. The results suggest that handling stress in red cusk-eel generate an altered metabolic status in liver, altered immune response in head kidney, and skeletal muscle atrophy through an important coding and noncoding gene network. This is the first study that identifies lncRNAs in Genypterus genus and that evaluates the relation between handling stress and lncRNAs in teleost fish, thereby providing valuable information regarding noncoding responses to stress in Genypterus species.

RNA-seq analysis of the head-kidney transcriptome response to handling-stress in the red cusk-eel (Genypterus chilensis).

Stress is a primary contributing factor of fish disease and mortality in aquaculture. We have previously reported that the red cusk-eel (Genypterus chilensis), an important farmed marine fish, demonstrates a handling-stress response that results in increased juvenile mortality, which is mainly associated with skeletal muscle atrophy and liver steatosis. To better understand the systemic effects of stress on red cusk-eel immune-related gene expression, the present study assessed the transcriptomic head-kidney response to handling-stress. The RNA sequencing generated a total of 61,655,525 paired-end reads from control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,840 transcripts and created a reference transcriptome with a N50 of 1426bp. Reads mapped onto the assembled reference transcriptome resulted in the identification of 569 up-regulated and 513 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation of the biological processes, like response to stress, response to biotic stimulus, and immune response. Conversely, a significant down-regulation of biological processes is associated with metabolic processes. These results were validated by RT-qPCR analysis for nine candidate genes involved in the immune response. The present data demonstrated that short term stress promotes the immune innate response in the marine teleost G. chilensis. This study is an important step towards understanding the immune adaptive response to stress in non-model teleost species.

Cadmium accumulation and metallothionein gene expression in the liver of swamp eel (Monopterus albus) collected from the Mae Sot District, Tak Province, Thailand.

Cadmium (Cd) is produced mainly as a by-product of zinc mining. In Thailand, the largest zinc mine is located in the Mae Sot district, Tak Province. Samples of Monopterus albus were collected from paddy fields in 4 sites, three downstream and one upstream from the zinc mine. The upstream site was considered to be uncontaminated while the three downstream sites were considered to be contaminated with Cd. Studies on the accumulation level of cadmium were conducted on the liver of the fish using the atomic absorption spectrophotometer technique. The metallothionein (MT) gene expression level in the liver, as a potential biomarker for long-term Cd exposure in their natural habitat, was also assessed. The level of hepatic MT gene expression was performed by quantitative real-time PCR. The result showed that Cd accumulation in the liver was much higher in swamp eels collected from the downstream sites when compared to those collected from the upstream site. The hepatic MT level in the upstream site was 0.75-fold, while the other three downstream sites were 0.36-, 4.44- and 0.94-fold. There is no parallel correlation between hepatic cadmium levels and hepatic MT gene expression. This study then suggests that MT gene expression biomarkers might be not suitable for swamp eels with prolonged exposure to Cd.

The European Eel NCCß Gene Encodes a Thiazide-resistant Na-Cl Cotransporter.

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCß is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCß from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCß is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCß is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCß exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.

Phylogenetic relationships of twenty Gymnothorax species based on cytochrome b sequence data.

To study the phylogenetic relationships of the genus Gymnothorax (moray eels) distributed in South China Sea, polymerase chain reactions were performed, and the amplification products were sequenced by cloning into the PMD18T-vector (TaKaRa). The entire gene sequences encoding cytochrome b (1140 bp) for 16 Gymnothorax (G. flavimarginatus, G. meleagris, G. undulates, G. reticularis, G. reevesi, G. melanospilus, G. rueppeliae, G. javanicus, G. chilospilus, G. pseudothyrsoideus, G. fimbriatus, G. hepaticus, G. berndti, G. curostus, G. favagineus, and G. margaritophorus) were obtained. Four additional Gymnothorax sequences from GenBank were also included. The nucleotide composition, genetic distances, and base substitution saturation analysis were calculated using the MEGA 5.0 Software. Phylogenetic analysis was performed using maximum-parsimony, maximum-likelihood (ML), and neighbor-joining (NJ). The results were as follows: 1) base-substitution saturation analysis suggested that both in third codon positions, and the full-length cytochrome b data set, Ts are not saturated, but Tv substitutions may be saturated, 2) the genus Gymnothorax, native to the South China Sea, is divided into four distinct clades, with two clades in the NJ and ML trees, and 3) according to our experimental data, G. melanospilus (Bleeker, 1855) and G. favagineus (Bloch and Schneider, 1801) are the same species.

Chromosomes of Gymnothorax funebris and the karyotypical differentiation within Gymnothorax (Anguilliformes: Muraenidae).

Cytogenetic studies in Gymnothorax funebris revealed a diploid chromosome number 2n = 42 (6 metacentrics, 4 submetacentrics, and 32 acrocentrics, FN = 52). The results obtained are novel and similar to those previously described for species belonging to Muraenidae family. The conventional karyotype is also novel and divergent from other species of the genus Gymnothorax, where a higher proportion of metacentric chromosomes predominate. The data are reported and discussed considering the cytotaxonomy of the genus. These results strongly support the current view that chromosomal alterations such as centric fusion and Robertsonian's translocations have an important role in the evolution of this group.

Genetic evidence for panmixia of Japanese eel (Anguilla japonica) populations in China.

The Japanese eel population has dramatically declined since the 1970s. In order to conserve this species, the background genetic structure affecting these populations should be well documented. Previous genetic studies of this species have produced seemingly conflicting results, ranging from no detectable heterogeneity to small, but statistically significant variance. This study investigates the population structure of Japanese glass eels collected from 10 localities in China in 2009 using a mitochondrial DNA (mtDNA) control region and 19 polymorphic microsatellite loci. Results revealed evidence of low genetic differentiation using both mtDNA (FST = 0.001, P = 0.291) and microsatellite data (FST = 0.003, P = 0.008). Pairwise F-statistic values generated from mtDNA and microsatellite DNA were similar, showing little evidence of significant genetic differentiation. The minimum spanning haplotype network constructed using mtDNA control regions produced no clear phylogeographic structure. The Mantel test revealed no significant correlation with distances for both mtDNA and microsatellite DNA. Therefore, our results suggest a panmictic population of Japanese eels in China, which should be conserved as a single management unit.

Novel tetranucleotide microsatellite markers for Chinese beard eel (Cirrhimuraena chinensis Kaup).

In this study, we characterized 12 novel polymorphic microsatellite loci for Chinese beard eel (Cirrhimuraena chinensis Kaup) from a tetranucleotide microsatellite-enriched library. Loci screened on a sample of 37 individuals from Xiamen wild stocks revealed 8 to 24 alleles per locus, with a mean of 13.83 over all loci. Observed and expected heterozygosities ranged from 0.270 to 0.944 and 0.439 to 0.942, respectively. These efficient genetic markers thus provide useful tools in the study of the population genetics and phylogeography of Chinese beard eel.

Cytogenetical studies in five Atlantic Anguilliformes fishes

The order Anguilliformes comprises 15 families, 141 genera and 791 fish species. Eight families had at least one karyotyped species, with a prevalence of 2n = 38 chromosomes and high fundamental numbers (FN). The only exception to this pattern is the family Muraenidae, in which the eight species analyzed presented 2n = 42 chromosomes. Despite of the large number of Anguilliformes species, karyotypic reports are available for only a few representatives. In the present work, a species of Ophichthidae, Myrichthys ocellatus (2n = 38; 8m+14sm+10st+6a; FN = 70) and four species of Muraenidae, Enchelycore nigricans (2n = 42; 6m+8sm+12st+16a; FN = 68), Gymnothorax miliaris (2n = 42; 14m+18sm+10st; FN = 84), G. vicinus (2n = 42; 8m+6sm+28a; FN = 56) and Muraena pavonina (2n = 42; 6m+4sm+32a; FN = 52), collected along the Northeastern coast of Brazil and around the St Peter and St Paul Archipelago were analyzed. Typical large metacentric chromosomes were observed in all species. Conspicuous polymorphic heterochromatic regions were observed at the centromeres of most chromosomes and at single ribosomal sites. The data obtained for Ophichthidae corroborate the hypothesis of a karyotypic diversification mainly due to pericentric inversions and Robertsonian rearrangements, while the identification of constant chromosome numbers in Muraenidae (2n = 42) suggests a karyotype diversification through pericentric inversions and heterochromatin processes.

XX:XY sex chromosome system with X heterochromatinization: an early stage of sex chromosome differentiation in the Neotropic electric eel Eigenmannia virescens.

An early stage of sex chromosome differentiation is reported to occur in the electric eel Eigenmannia virescens (Pisces, Sternopygidae) from populations of two tributaries of the Paraná river system (Brazil). Cytogenetic studies carried out in the two populations showed that the Mogi-Guaçu population is characterized by 2n = 38 chromosomes and undifferentiated sex chromosomes and the Tietê population presents 2n = 38 both for males and females and an XX:XY sex chromosome system. The X-chromosome is acrocentric, easily recognized by the presence of a conspicuous heterochromatin block in its distal portion; the Y-chromosome is probably one of the medium sized acrocentrics present in the male karyotype. BrdU induced R-bands of the two populations did not reveal any difference in the euchromatic regions of the chromosomes. AluI and HaeIII restriction enzyme digestion patterns and chromomycin A3 staining of the X-chromosome are presented. The possible role of heterochromatinization in the evolution of sex chromosomes in fish is discussed.