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The present study aimed to know the effect of the administration of Debaryomyces hansenii yeast on growth, development and skeletal deformities, through the quantification of molecular and morphophysiological indicators in longfin yellowtail larvae. Larvae from the yeast group were fed with the rotifers and Artemia metanaupli half enriched with D. hansenii (50% of live prey) and half enriched with Origreen® (50% of live prey) and were compared to control (100% Origreen®) from 5 to 30 days post-hatching (DPH). Each treatment had two methodological replicates. Studies on bone mineralization, skeletal deformities, intestinal histological analysis, digestive enzyme activity, differential gene expression (RT-qPCR) and transcriptome analysis (RNA-Seq) were performed in whole larvae. Results showed that larvae fed D. hansenii encapsulated within enriched live preys had higher survival and growth, higher intestinal mucin secretion, as well as higher activity of alkaline phosphatase, pepsin and α-amylase. The degree of bone mineralization in the cranial and caudal fin complex was higher in larvae fed D. hansenii and a lower incidence of deformities at the vertebral column was also observed. In general, the gene expression throughout the development fluctuated between the control group and the yeast treatment; however, at 30 DPH, a higher expression of the bone morphogenetic protein type 2 (bmp2), collagen type 1α1 (col1α1) and proliferating cell nuclear antigen (pcna) genes was detected in larvae fed with yeast. The transcriptomic analyses using RNA-Seq revealed that the main genes related to bone mineralization degree and digestive tract maturation, were overrepresented in the D. hansenii treatment, 1,25-dihydroxyvitamin D (3) 24-hydroxylase (cyp24), cytochrome P450 family 27 subfamily A (cyp27a), protein 5 related to the low-density lipoprotein receptor (lrp5), myocyte-specific enhancing factor 2C (mef2), enterokinase (entk), pepsin and alkaline phosphatase. Based on the results, S. rivoliana larvae supplemented with the yeast D. hansenii presented higher growth and survival, a higher degree of maturation of the digestive tract, a higher degree of bone mineralization and a reduction in skeletal deformities, for which the continuous use of yeast is recommended as a food supplement to larvae from 5 days post-hatching onwards. info:eu-repo/semantics/acceptedVersion

Zebrafish (Danio rerio) is a well-established vertebrate model in ecotoxicology research that responds to a wide range of xenobiotics such as pesticides, drugs, and endocrine-disrupting compounds. The epigenome can interact with the environment and transform internal and/or external signals into phenotypic responses through changes in gene transcription. Environmental exposures can also generate epigenetic variations in offspring even by indirect exposure. In this review, we address the advantages of using zebrafish as an experimental animal model to study transgenerational epigenetic processes upon exposure to xenobiotics. We focused mostly on DNA methylation, although studies on post-translational modifications of histones, and non-coding RNAs related to xenobiotic exposure in zebrafish are also discussed. A revision of the methods used to study epigenetic changes in zebrafish revealed the relevance and reproducibility for epigenetics-related research. PubMed and Google Scholar databases were consulted for original research articles published from 2013 to date, by using six keywords: zebrafish, epigenetics, exposure, parental, transgenerational, and F2. From 499 articles identified, 92 were considered, of which 14 were selected as included F2 and epigenetic mechanisms. Current knowledge regarding the effect of xenobiotics on DNA methylation, histone modifications, and changes in non-coding RNAs expressed in F2 is summarized, along with key experimental design considerations to characterize transgenerational effects.

Muscle growth is regulated by several factors including the growth differentiation factor 8, known as myostatin, an inhibitor of myocyte differentiation and proliferation. Research on myostatin regulation was already conducted to improve growth rates in farmed animals, including aquatic species. To explore the effects of myostatin inactivation in a commercial marine fish (spotted rose snapper, Lutjanus guttatus) in vivo, we induced post-transcriptional silencing (knockdown) of myostatin-1 (mstn-1) by injecting dsiRNA directly into the muscle of juvenile fish (87 days post-hatch) using a commercial polymer as vehicle. Results show a significant decrease in mstn-1 expression starting at 2 days after injection and for up to 5 days. Knockdown of mstn-1 caused muscle fiber hypertrophy (but not hyperplasia); however, there were no significant changes in weight or length. Although still experimental, this study provides evidence that temporary knockdown of mstn-1 in a commercial marine fish in vivo promotes fiber hypertrophy and therefore could potentially help grow-out programmes in fish aquaculture.

The fem-1 gene in Caenorhabditis elegans is involved in sex differentiation; it is specifically required for all aspects of male development. In this study, the full-length cDNA of the fem-1 (Pvfem-1) gene was isolated from the Pacific whiteleg shrimp Penaeus vannamei. The Pvfem-1 transcript is 3778 nt long and encodes a putative protein (PvFEM-1) of 638 amino acids that presented eight ankyrin repeats. The translated protein showed a significant (P 0.05) structural similitude by superposition with C. elegans FEM-1 protein. Pvfem-1 expression was evaluated by qPCR and in situ hybridization (ISH) during embryogenesis, larval development, and gonads of both genders in subadult and adult life stages. Pvfem-1 was found expressed in brain, intestine, hepatopancreas, and in the gonads of both genders in subadults and adults when quantified by RT-qPCR. A significant finding was the discovery of a natural antisense transcript (NAT) of Pvfem-1 by ISH. It was present in the oocyte nucleus of subadult female shrimp gonads but was not seen within oocytes from adult females, although it was detected in follicular cells, suggesting a possible post-transcriptional regulation of Pvfem-1 in female gonad. Conversely, in males, no NAT was observed, and Pvfem-1 was found expressed in spermatogonia of both, subadult and adult shrimps indicating a function in male sexual differentiation and gametes generation. This study represents the first step for future functional analysis that is expected to contribute to clarifying the role of Pvfem-1 in sex differentiation and determination.

Diet modulates the gut microbiota and is one of the main factors promoting obesity and overweight. In the present study, we investigated the effect of a high-fat diet (HFD) on the gut micr...

Five experimental diets with different carbohydrates/lipid ratio (CHO:L, 0.75, 1.28, 2.10, 2.52, 4.63) were formulated. Fish were randomly assigned by triplicate (10 fish per tank, 0.50 ± 0.01 g). Growth was registered every 15 days until end of the experiment (45 days). Samples were taken for analysis of chemical carcass composition, blood chemistry, glycogen and lipid liver content, digestive and metabolic enzyme activities. Results showed that survival, growth performance parameters and plasma glucose were not affected by treatments (p > 0.05). Lipids of carcass and liver, as well as triglycerides and plasma cholesterol increase significantly as CHO:L ratio decreased. While an inversely proportional tendency was observed for carcass protein and liver glycogen (p 0.05). Finally, Hexokinase (HK), glucokinase (GK), phosphoenol pyruvate carboxykinase (PEPCK) and fructose‐1,6‐biphosphase (FBPase) showed high regulation by carbohydrates up to the CHO:L ratio of 2.10 (p

The chihuil sea catfish (Bagre panamensis) is endemic of the Eastern Pacific and is a species of fishery importance in the Mexican Pacific. The complete mitochondrial genome of Bagre panamensis has been assembled from Illumina sequencing data. The circular genome was 16,714 bp in lengh, and consist of 13 protein-coding, two ribosomal RNAs (rRNAs), and 22 transfer RNA (tRNA) genes. Base composition is 30.8% A, 26.6% T, 28.2% C, and 14.4% G, and 42.6% GC content. Protein-coding genes present two start codon (ATG and GTG) and eight stop codon (TAA, TCT, CCT, TTA, CAT, AAT, ATT, and TAG). The control region possesses the highest A + T (64.4%) and lowest G + C content (35.6%) among all mitochondrial regions. These data would contribute to the evolutionary studies of related taxa.

The mitochondrion genome of Occidentarius platypogon was assembled from Illumina short reads, and consisted of 16,714 base pairs, with 13 protein-coding, two ribosomal RNAs (rRNAs), and 22 transfer RNA (tRNA) genes. Base composition is 30.7% A, 26.4% T, 28.5% C, and 14.4% G, and 42.9% GC content. Two start codon (ATG and GTG) and seven stop codon (TAA, ACT, CCT, TTA, CAT, AAT, and TAG) patterns were found in protein-coding genes. Control region presented the highest A + T (64%) and lowest G + C content (35.7%) among all mitochondrial regions.

Genomic resources for Platyhelminthes of the class Monogenea are scarce, despite the diversity of these parasites, some species of which are highly pathogenic to their fish hosts. This work aimed to generate de novo-assembled transcriptomes of two monogenean species, Scutogyrus longicornis (Dactylogyridae) and Rhabdosynochus viridisi (Diplectanidae), providing a protocol for cDNA library preparation with low input samples used in single cell transcriptomics. This allowed us to work with sub-microgram amounts of total RNA with success. These transcriptomes consist of 25,696 and 47,187 putative proteins, respectively, which were further annotated according to the Swiss-Prot, Pfam, GO, KEGG, and COG databases. The completeness values of these transcriptomes evaluated with BUSCO against Metazoa databases were 54.1% and 73%, respectively, which is in the range of other monogenean species. Among the annotations, a large number of terms related to G-protein-coupled receptors (GPCRs) were found. We identified 109 GPCR-like sequences in R. viridisi, and 102 in S. longicornis, including family members specific for Platyhelminthes. Rhodopsin was the largest family according to GRAFS classification. Two putative melatonin receptors found in S. longicornis represent the first record of this group of proteins in parasitic Platyhelminthes. Forty GPCRs of R. viridisi and 32 of S. longicornis that were absent in Vertebrata might be potential drug targets. The present study provides the first publicly available transcriptomes for monogeneans of the subclass Monopisthocotylea, which can serve as useful genomic datasets for functional genomic research of this important group of parasites.Assemblage de novo du transcriptome et identification des récepteurs couplés aux protéines G (RCPG) chez deux espèces de Monogènes parasites de poissons.Les ressources génomiques pour les Plathelminthes de la classe Monogenea sont rares, malgré la diversité de ces parasites dont certaines espèces sont hautement pathogènes pour leurs hôtes poissons. Ce travail visait à générer des transcriptomes assemblés de novo pour deux espèces de monogènes, Scutogyrus longicornis (Dactylogyridae) et Rhabdosynochus viridisi (Diplectanidae), fournissant un protocole pour la préparation de la bibliothèque d’ADNc avec des échantillons à faible apport utilisés en transcriptomique unicellulaire, ce qui a permis de travailler avec des quantités inférieures au microgramme d’ARN total avec succès. Ces transcriptomes se composent de 25 696 et 47 187 protéines putatives, respectivement, qui ont ensuite été annotées selon les bases de données Swiss-Prot, Pfam, GO, KEGG et COG. L’exhaustivité de ces transcriptomes évaluée avec BUSCO par rapport aux bases de données des Métazoaires était respectivement de 54,1 % et 73 %, ce qui est dans la gamme des autres espèces de monogènes. Parmi les annotations, un grand nombre de termes liés aux récepteurs couplés aux protéines G (RCPG) ont été trouvés. Nous avons identifié 109 séquences de type RCPG chez R. viridisi et 102 chez S. longicornis, y compris des membres de la famille spécifiques de Platyhelminthes. La rhodopsine était la plus grande famille selon la classification GRAFS. Deux récepteurs putatifs de la mélatonine trouvés chez S. longicornis représentent le premier signalement de ce groupe de protéines chez les Plathelminthes parasites. Quarante RCPG de R. viridisi et 32 de S. longicornis, qui sont absents chez les Vertébrés, pourraient être des cibles médicamenteuses potentielles. La présente sont fournit les premiers transcriptomes accessibles au public pour les monogènes de la sous-classe Monopisthocotylea, qui peuvent servir d’ensembles de données génomiques utiles pour la recherche génomique fonctionnelle de cet important groupe de parasites.

Normal development involves the interplay of genetic and epigenetic regulatory mechanisms. Pax6 is an eye-selector factor responsible for initiating the regulatory cascade for the development of the eyes. For the olive ridley sea turtle (Lepidochelys olivacea), a threatened species, eye malformations have been reported. In order to study the DNA methylation status of the putative promoter of the Pax6 gene in embryos with ocular malformations, an exploratory study was carried out in which DNA was isolated from embryos with anophthalmia, microphthalmia, and cyclopia, as well as from their normal counterparts. The 5'-flanking region from the Pax6 gene was isolated, showing two CpG islands (CGIs). The methylation status of CGIs in malformed embryos was compared with that of normal embryos by bisulfite sequencing. Putative transcription factor binding sites and regulatory features were identified. Methylation patterns were observed in both CpG and non-CpG contexts, and were unique for each malformed embryo; in the CpG context, an embryo with cyclopia showed a methylated cytosine upstream the CGI-1 not present in other embryos, an embryo with left anophthalmia presented two methylated cytosines in the CGI-1, whereas an embryo with left anophthalmia and right microphthalmia showed two methylated cytosines in the CGI-2. Normal embryos did not show methylated cytosines in the CGI-1, but one of them showed one methylcytosine in the CGI-2. Methylated transcription factor-binding sites may affect Pax6 expression associated to the cellular response to environmental compounds and hypoxia, signal transduction, cell cycle, lens physiology and development, as well as the transcription rate. Although preliminary, these results suggest that embryos with ocular malformations present unique DNA methylation patterns in the putative promoter of the Pax6 gene in L. olivacea, and probably those subtle, random changes in the methylation status can cause (at least in part) the aberrant phenotypes observed in these embryos.

The rose spotted snapper is an important fishery resource with potential for farming in several Latin American countries. Since transcriptomic information was not available for this species, we generated a reference transcriptome from eleven tissues (brain, heart, gonad, liver, gut, muscle, white skin, dark skin, and visceral fat) using Illumina HiSeq2000 platform. Transcriptome assembly and annotation were performed with Trinity and Trinotate respectively; 267,601 contigs were assembled, average contig length was 845 bp; 85,677 putative protein sequences were identified. Sixty‐six per cent of them showed a significant match to the SwissProt Database. From this assembly, we selected growth/atrophy‐related genes to investigate the effect of dietary protein source on gene expression. Pacap expression was measured in the brain; igf1, igf2 and ghr were measured in liver; and mtor, igf1, igf2, myod1, myod2, myogenin, foxo3a, foxo3b, murf1, mafbx, mstn1 and mstn2 were measured in muscle. A feeding trial with juveniles was performed, in which three diets were formulated, a fishmeal‐based diet (FM), a terrestrial animal protein diet (PPM) with porcine meat meal and poultry by‐products meal, and a vegetable diet (VEG) with a blend of soybean protein concentrate and corn gluten. A reduced expression of somatotropic and myogenic genes in fish fed the VEG diet was observed; however, expression of atrophy genes did not increase with this diet suggesting that atrophy mechanisms were not involved. These results indicate that protein from a vegetable source does not provide proper amino acid requirements to juvenile fish, decreasing expression of myogenic and other growth‐related genes.

The effects of feed deprivation were evaluated for 1 week and 2 weeks in Lutjanus guttatus juveniles. A significant reduction in body mass was observed in both feed deprivation schemes, as well as in hepatosomatic, viscerosomatic and mesenteric fat indexes. The composition of fasted fish was characterized by a decrease in lipid content; the liver displayed an intense reduction of lipid reserves in both fasted groups, and increased expression of the lysosomal acid lipase. 1 week after re-feeding, both experimental groups showed an increase in specific growth rate, feed intake and feed conversion ratio. A recovery in hepatic lipid reserves was observed, and the expression of the lysosomal acid lipase decreased, although lipid content in both groups was still significantly lower than in control groups. Hepatic expression of the growth hormone receptor decreased after fasting, and remained low even after the fish were fed, whereas the expression of insulin-like growth factor 1 and 2 increased after fasting and decreased in both groups when fish were fed again. Altogether, these results showed a partial compensatory growth response in L. guttatus juveniles after fasting, with enhanced growth rates and improved feed efficiency. Fish used stored lipid reserves as the main energy source, and the expression of growth-related and lipid mobilization marker genes in the liver showed similar patterns in both fasting schemes. Based on the results, we suggest as much as 1-week fasting intervals during grow-out programmes to reduce visceral fat and increase growth rate in this species.

Implementation of massive sequencing techniques in aquaculture has led to increase genomic resources of different species and have generated useful specific markers to improve their management. In this regard, transcriptomic approaches have been widely used to easily obtain gene expression profiles and understand the regulation of molecular pathways of interest in aspects as development and nutrition. The three spot cichlid (Amphilophus trimaculatus) is a native fish from South Mexico, with commercial regional importance and aquaculture potential. However, there is no genomic information available for the species that could allow the analysis in physiological processes of metabolic pathways to contribute or improve its culture. Hence, a comprehensive transcriptome of three different developmental stages: early (3–5 dpf larvae), mid (8–13 dpf larvae) and late (16–25 dpf larvae), were obtained using Illumina Hi-seq 2000 platform. De novo assembly generated 237,164 transcripts grouped in 196,186 genes with an average contig length of 778 bp and an N50 value of 1546 bp. Conceptual translation yielded in 78,883 proteins, of which 35,033 are complete proteins. By analyzing the transcriptomic profile and differential expression among developmental stages, we identified nutritional markers involved in digestive enzyme, metabolic hormone, and organogenesis of the digestive system. Likewise, in order to confirm the transcriptomic results obtained, histological analyzes were performed using hematoxylin-eosin (H&E) staining, finding that the most important morphological changes occur in the mid stage (endo-exptrophic larvae, 8 dpf and exotrophic larvae, 13 dpf), coinciding with the maximum expressions of the candidate genes for nutritional markers.

The Devil Ray (Mobula thurstoni) is a species with global distribution and is an important species in conservation terms, here we present its complete mitochondrial genome assembled with Illumina sequencing data. The circular genome was 17,610 bp in length, and consists of 13 protein-coding, two ribosomal RNAs (rRNAs), and 22 transfer RNA (tRNA) genes. Base composition is 30.7% A, 29.1% T, 26.5% C, and 13.7% G, and 40.2% GC content. Protein-coding genes present two start codon (ATG and GUG) and seven stop codon (UAA, AUA, UUU, UUA, AAU, CCU, and UAG). The control region possesses the highest A + T (66.6%) content among all mitochondrial regions. These data would contribute to the evolutionary studies of this genus, where there has been recent reclassification.

Jellyfish are non-model organisms that have brought the scientific community's attention over the last decades since the frequency and intensity of these species blooms around the world have increased significantly. The occurrence of blooms has been hypothesized due to the rise in seawater temperature associated with climate change. This fact has promoted an increased interest in studying jellyfish metabolic responses to face thermal stress and maintain cell homeostasis. In this study, the response of the cannonball jellyfish Stomolophus meleagris to short-term exposure to different temperatures was evaluated. The survival and respiratory rates, and the concentration of glucose, L-lactate, glycogen, ATP, and total soluble protein in the umbrella of adult jellyfish were measured. Low survival rates at extreme temperatures (18 and 33 °C) and an increase in the jellyfish respiratory rates were observed as seawater temperature raised. Rapid changes were detected in energy substrates such as glucose and glycogen that significantly varied at extreme temperatures and longer exposure periods. L-lactate levels increased after 2 h of exposure, and then remained steady at longer times of exposure. Soluble protein concentration decreased significantly in jellyfish exposed to extreme temperatures, and ATP increased after exposure to non-optimal temperatures (18, 28, and 33 °C). These results suggest that S. meleagris faces temperature changes through rapid metabolic responses that use energetic reserves, activate anaerobic metabolism, and display a remarkable ability to synthesize ATP and maintain energy, even at long exposure.

The Pacific Whiteleg shrimp Litopenaeus vannamei is one of the most important aquaculture species globally, for which monosex female culture is desirable due to its higher weight at harvest. For this purpose, the development and validation of biotechnologies directed to manipulate sexual differentiation requires a verification protocol based on molecular sex markers in the early life stages where no secondary sexual characteristics are displayed. In the present study, the sequences of a 542 bp putatively sex-linked locus containing three single nucleotide polymorphisms (SNP) followed the homogametic (male, n = 8) and heterogametic (female, n = 4) sex-determination system. The locus is probably not located in previously reported sex-associated linkage groups. A post-PCR high resolution melt (HRM) analysis was designed to amplify and differentiate homozygotes from heterozygotes of the SNP in the locus. The expected patterns in a ZZ/ZW genetic system was confirmed in wild and cultivated shrimp samples (n = 168). These results are of interest for sex detection and for future detailed studies on the sex-determination system in L. vannamei.

Selective breeding of shrimp has major potential to enhance production traits, including growth and disease resistance. Genetic characterization of broodstock populations is a key element of breeding programs, as it enables decisions on inbreeding restrictions, family structure, and the potential use of genomic selection. Single Nucleotide Polymorphisms (SNPs) are suitable genetic markers for this purpose. A set of SNPs was developed to characterize commercial breeding stocks in Mexico. Individuals from local and imported lines were selected for sequencing using the nextRAD technique, resulting in the identification of 2619 SNPs. Genetic structure analysis showed three to five genetic groups of Ecuadorian and Mexican origins. A subset of 1231 SNPs has potential for stock identification and management. Further, three SNPs were identified as candidate sex-linked markers. The role of SNPs possibly associated with genes related to traits of importance to shrimp farming, such as growth and immune response, should be further investigated.

White spot syndrome virus (WSSV) has a ∼300 kb double-stranded DNA genome. It originated in China, spread rapidly through shrimp farms in Asia, and subsequently to America. This study determined complete genome sequences for nine historic WSSV strains isolated from Pacific white shrimp (Litopenaeus vannamei) captured in farm ponds in northwest Mexico (Sinaloa and Nayarit). Genomic DNA was captured by an amplification method using overlapping long-range PCR and sequencing by Ion Torrent-PGM. Complete genome sequences were assembled (length range 255-290 kb) and comparative genome analysis with WSSV strains revealed substantial deletions (3 and 10 kb in two regions) in seven strains, with two strains differing from the rest. Phylogenetic analysis identified that the WSSV strains from the northern area of the state of Sinaloa clustered with strains from China (LC1, LC10, DVI) and Korea (ACF2, ACF4), while those from the southern region of the state of Nayarit (AC1 and JP) differed from both of those and from strains found in Taiwan and Thailand. Our data offer insights into the diversity of the WSSV genome in one country and their divergent origin, suggest that it entered Mexico via multiple routes and that specific genome regions can accommodate substantial deletions without compromising viability.

The Pacific white shrimp Penaeus vannamei is the most cultured shrimp species around the world. Because females grow larger than males, the culture of 'only females' is of great interest, but knowledge on sex determination and differentiation is required for producing only females. In an effort to obtain information associated with reproduction in P. vannamei, transcriptomic data from female gonads was generated, and partial sequences of a transcript were identified as Sex-lethal (Sxl). Its characterization indicated that, differently from other penaeids in which this gene has been isolated, there are six isoforms of the Sxl transcript in P. vannamei (PvanSxl 1-6). These isoforms result from alternative splicing at three splice sites (SS1, SS2, SS3). The first splice-site is unique to P. vannamei, as it has not been reported for other Arthropod species; the second splice-site (SS2) is common among crustaceans, and the third splice-site (SS3) is also unique to P. vannamei and when spliced-out, it is always together with SS2. All isoforms are expressed during embryogenesis as well as gametogenesis of both genders. The two shorter isoforms, PvanSxl-5 and PvanSxl-6, which result from the splicing of SS2 and SS3, were found mostly expressed in adult testis, but PvanSxl-6 was also expressed in oocytes during gametogenesis. During oogenesis, the second largest isoform, PvanSxl-2, which splices-out only SS1, and PvanSxl-4 that splices-out SS1 and SS2 were highly expressed. These two isoforms were also highly expressed during embryonic development. In situ hybridization allowed pinpointing more specifically the cells where the PvanSxl transcripts were expressed. During embryogenesis, hybridization was observed from the one-cell stage embryo to late gastrula. In the female gonad in previtellogenesis, hybridization occurred in the nucleus of oocytes, whereas in secondary vitellogenesis the transcript also hybridized cytoplasmic granules and cortical crypts. Finally, in situ hybridization corroborated the expression of PvanSxl also in the male gonad during spermatogenesis, mostly occurring in the cytoplasm from spermatogonia and spermatocytes.

Muscle development involves coordinated molecular events leading to cell proliferation, fusion, differentiation, sarcomere assembly, and myofibrogenesis. However, under physiological or pathological stress, energy requirements and secretion of glucocorticoids increase, resulting in muscle atrophy because of the depletion of energy reserves. Glucocorticoids induce muscular atrophy by two main mechanisms, protein degradation through the ubiquitin-proteasome system, and inhibition of protein synthesis through the negative regulation of the IGF1-Akt-mTOR signaling pathway. Other signaling pathways (such as the myostatin-activin-smad pathway) involved in muscle atrophy by glucocorticoid exposure are unclear. In fish, the role of glucocorticoids in muscle atrophy has not been fully elucidated. The aim of the present study was to evaluate the mechanisms of muscle atrophy induced by a synthetic glucocorticoid (dexamethasone, DEX) in an ex vivo muscle culture system of a marine fish (Lutjanus guttatus). Results showed that DEX was able to induce the expression of myostatin-1, and the expression of the transcription factor foxo3b. Myostatin-1 silencing by RNAi produced a decrease in the expression of foxo3b and murf1, and increased the expression of mtor, myod-2 and myogenin. These results suggest that in fish skeletal muscle, myostatin-1 signaling participates in glucocorticoid-induced muscle wasting through the negative regulation of genes involved in muscle growth, such as mtor, myod-2 and myogenin, and the induction of atrophy genes like foxo3b and murf1.

The white spot syndrome virus (WSSV) remains the most devastating viral pathogen of shrimp culture worldwide. Gene silencing by RNA interference (RNAi) using double stranded RNA (dsRNA) has been considered a powerful tool for conferring protection against WSSV when viral genes are silenced, as documented in several shrimp species. However, this effect is not long lasting. Our results provide the first evidence that long-term silencing of the LvRab7 endogen produced antiviral effect against WSSV, which endured at least 21 d after dsRNA treatment (dat). Until now, the most efficient way to implement RNAi with dsRNA into the shrimp is by injection. Consequently, its application to broodstock in hatcheries is possible, minimizing the risk of vertical transmission of the virus. We show that the expression of Rab7 in hemocytes is lowest at 2 dat and finally recovers to basal status. In contrast, in gills and pleopods, gene expression silencing continued for at least 21 d. We challenged Litopenaeus vannamei broodstock with WSSV at 7, 14, or 21 dat reaching mortality rates of 0, 40, and 27%, respectively. In conclusion, the LvRab7 gene silencing is progressive and effective against WSSV. However, further studies are necessary to elucidate the functions of Rab7 in shrimp cells before applying this methodology at a commercial level.

The lion-paw, Nodipecten subnodosus is one of three scallop species commercially exploited on the west coast of the Peninsula of Baja California. Because nothing is known about sex determination and sexual differentiation in hermaphrodite scallops, in the present work, a global transcriptomic analysis was performed in two early developmental stages, settling eyed-larvae and spat, as well as in three tissues (undifferentiated gonad, digestive gland, and adductor muscle). Over 27 million Illumina paired-end reads were obtained through the MiSeq platform. After processing the reads a total of 243,774 transcripts were assembled with an N50 of 980 and an average length of 775nt. A total of 43,252 proteins were inferred and 36,103 transcripts had at least one homolog in the SwissProt database according to a blastx search. After differential expression analyses and GO annotations it was possible to identify several sex-related genes in the scallop, including one known to be involved in the sex determination pathway of the hermaphrodite model organism Caenorhabditis elegans, N. subnodosus-sex1 (Ns-sex1). Other interesting sex determination and differentiation genes were Ns-dmrta2, Ns-sox9, Ns-wnt4, Ns-doa, Ns-ovo, Ns-vir, among others. Most of these genes were mainly expressed in the testis region, suggesting their participation in male gonad region sex differentiation. These results represent the first available information on the genetics of sex determination and differentiation in a functional hermaphrodite scallop.

ATP-Binding Cassette (ABC) transporters are proteins that actively mediate the transport of a wide variety of molecules, including drugs. Thus, in parasitology, ABC transporters have gained attention as potential targets for therapeutic drugs. Among the parasitic Platyhelminthes, ABC transporters have been identified and classified in a few species of Trematoda and Cestoda but not in Monogenea. Monogeneans are mainly ectoparasites of marine and freshwater fish, although they can also be found on other aquatic organisms. Severe epizootics caused by monogeneans have been reported around the world, mainly in confined and/or overcrowded fish. The purpose of this study was to identify the ABC transporters in four species of monogeneans (Gyrodactylus salaris, Protopolystoma xenopodis, Eudiplozoon nipponicum and Neobenedenia melleni) for which genomic resources are publicly available. For comparative purposes, ABC transporters were also identified in endoparasitic (Schistosoma mansoni and Echinococcus granulosus) and free-living (Macrostomun lignano and Schmidtea mediterranea) platyhelminths. Thirty-two putative ABC transporters were identified in the genome of G. salaris, 40 in the genome of P. xenopodis, 46 in the transcriptome of E. nipponicum and 9 in a rather limited ESTs set available for N. melleni. Of the eight ABC subfamilies (A-H) known in metazoans, subfamily H was the only one not found in any monogenean species. In contrast, ABCC was the best represented subfamily. Phylogenetic analyses showed a few cases of one-to-one orthologous relationships, which agree with results from other metazoan species. We found some monogenean ABC members related to subfamilies B, C and G involved in drug resistance in humans. This information may be useful for future functional studies on ABC transporters in monogeneans.

The increased use of massive sequencing technologies has enabled the identification of several genes known to be involved in different mechanisms associated with reproduction that so far have only been studied in vertebrates and other model invertebrate species. In order to further investigate the genes involved in Litopenaeus vannamei reproduction, cDNA and SSH libraries derived from female eyestalk and gonad were produced, allowing the identification of expressed sequences tags (ESTs) that potentially have a role in the regulation of gonadal maturation. In the present study, different transcripts involved in reproduction were identified and a number of them were characterized as full-length. These transcripts were evaluated in males and females in order to establish their tissue expression profiles during developmental stages (juvenile, subadult and adult), and in the case of females, their possible association with gonad maturation was assessed through expression analysis of vitellogenin. The results indicated that the expression of vitellogenin receptor (vtgr) and minichromosome maintenance (mcm) family members in the female gonad suggest an important role during previtellogenesis. Additionally, the expression profiles of genes such as famet, igfbp and gpcr in brain tissues suggest an interaction between the insulin/insulin-like growth factor signaling pathway (IIS) and methyl farnesoate (MF) biosynthesis for control of reproduction. Furthermore, the specific expression pattern of farnesoic acid O-methyltransferase suggests that final synthesis of MF is carried out in different target tissues, where it is regulated by esterase enzymes under a tissue-specific hormonal control. Finally, the presence of a vertebrate type steroid receptor in hepatopancreas and intestine besides being highly expressed in female gonads, suggest a role of that receptor during sexual maturation.

This study explored whether Crassostrea gigas oysters can be used as a bioindicator of white spot syndrome virus (WSSV) in shrimp farm water canals. Bioassays showed that C. gigas can accumulate WSSV in their gills and digestive glands but do not become infected, either by exposure to seawater containing WSSV or by cohabitation with infected shrimp. The use of a WSSV nested PCR to screen oysters placed in water canals at the entry of a shrimp farm allowed WSSV to be detected 16 d prior to the disease occurring. The finding that C. gigas can concentrate small amounts of WSSV present in seawater without being harmed makes it an ideal sentinel species at shrimp farms.

The mitochondrion genome of Occidentarius platypogon was assembled from Illumina short reads, and consisted of 16,714 base pairs, with 13 protein-coding, two ribosomal RNAs (rRNAs), and 22 transfer RNA (tRNA) genes. Base composition is 30.7% A, 26.4% T, 28.5% C, and 14.4% G, and 42.9% GC content. Two start codon (ATG and GTG) and seven stop codon (TAA, ACT, CCT, TTA, CAT, AAT, and TAG) patterns were found in protein-coding genes. Control region presented the highest A + T (64%) and lowest G + C content (35.7%) among all mitochondrial regions.

The crustacean hyperglycemic hormone (CHH) family is an important group of neuropeptides involved in controlling growth, reproduction, and stress response in decapod species. In this study, a new gene containing 4 exons-3 introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions was isolated from Litopenaeus vannamei. Two full length transcripts of this CHH were isolated from eyestalk and pericardial tissue of males and females using rapid amplification of cDNA ends (RACE). Transcripts sequences were 1578bp in length in males pericardial tissues and in males and females eyestalk with 100% identity, but the transcript isolated from females pericardial tissues was shorter (974bp). The differences in transcripts length is a result of two polyadenylation sites present in the 3'UTR resulting in two transcription termination signals. Transcript sequences encoded one unique protein that can be classified as type I CHH subfamily because of the 4 exons and 3 introns structure, although the CPRP region is not-well conserved and there is no amidation in the C-terminal of the deduced amino acid sequence. Furthermore, there is a glycine inserted in the mature peptide not at position 12 as in type II CHHs but after amino acid 31 and the phylogenetic analysis did not group the peptide within type I, but closer to type II CHHs. We demonstrated by endpoint-PCR, qPCR, and in situ hybridization (ISH), that this gene is expressed in neuroendocrine organs known to express CHHs in penaeid shrimp, including X-organ and optic nerve in eyestalk, supraesophageal ganglion (SoG), but it is also expressed in other organs as gill, gut, pericardial cavity, as well as in terminal ampoule or spermatophore and vas deferens of males.

International audience; In addition to the reported single cDNA sequence encoding vitellogenin in the Pacific oyster Crassostrea gigas (referred as Cg-Vtg1), we isolated and characterized the 3′ end of a novel vitellogenin (Vtg) in C. gigas, named Cg-Vtg2. The transcript size is up to 6 kb long and includes a D-type von Willebrand domain, characteristic of most vitellogenins, but not occurring in Cg-Vtg1. The expression of Cg-Vtg2 was found restricted to follicular cells in maturing and ripe female gonads. Quantitative expression analysis of both vitellogenins showed a closely correlated transcription pattern during the female gametogenic cycle, with the highest expression during the ripe (mature) stage, and a declining expression, to almost undetectable levels, in post-spawning (resting) stage. Levels of vitellogenin mRNA associates with the quantity of ingested food. Mature female oysters fed either a 2% or a 12% algal biomass per female biomass, showed a 2.9-fold increase in expression of Cg-Vtg1 and a 3.7-fold increase of Cg-Vtg2 expression levels in those females fed the higher level of algae, suggesting that Vtg's could be used as quantitative indicator of vitellogenesis. Our results suggest that vitellogenins are synthesized during the late period of vitellogenesis, when most of the yolk has already been incorporated into maturing oocytes.

Prevalence of the protozoan Perkinsus spp. in the gills of the pleasure oyster Crassostrea corteziensis from two estuaries in Nayarit, Mexico, was measured. The protozoan was identified by PCR amplification of the internal transcribed spacer (ITS) region of the rDNA of Perkinsus spp. The pathogen was found in 92% of oysters from Boca de Camichín and 77% of oysters from Pozo Chino. ITS sequences characterized from C. corteziensis showed 96–100% similarity to Perkinsus marinus. The most frequent ITS sequence, (GenBank JQ266236) had 100% identity with the ITS locus of P. marinus from New Jersey, Maryland, South Carolina, and Texas, and the second most frequent observed sequence (GenBank JQ266240) was 100% identical to ITS sequences of P. marinus from New ersey, South Carolina, Louisiana, and Bahía Kino, Sonora, Mexico. The 14 sequences from the non-transcribed spacer (NTS) showed 98% similarity to P. marinus from Texas. The most frequent polymorphism identified was at nucleotide 446 of the ITS region, however, the NTS showed the highest nucleotide diversity, thereby suggesting that this region is suitable for genotype identification. Moreover, the most conserved ITS marker is better for species-specific diagnosis. Both the ITS and NTS sequences of P. marinus obtained from C. corteziensis were grouped in two clades, identifying two allelic variants of P. marinus

International audience; For non-model species, as many used for aquaculture, with minimal or no genomic information, relative quantification of gene expression studies requires preliminary research including the isolation of potential reference genes and the identification of those stably expressed under the biological conditions of interest. Here we report on the isolation of five partial gene sequences from gonad tissue cDNA in the functional hermaphrodite scallop Nodipecten subnodosus to be evaluated as reference genes: 18S-rRNA, riboprotein l8 (rp-l8), actin-β (act-β), elongation factor 1α (ef-1α) and alpha-tubulin-α (tub-α). We found that 18S-rRNA was stably expressed independently of the priming method used to reverse transcribe RNA to cDNA, oligo-dT or random hexamer. Stability analysis for the five putative reference genes with geNorm and NormFinder indicated that 18S together with rp-l8 were the most stable genes for normalization of gene expression during gonad development in both, male and female sexual regions of the hermaphrodite N. subnodosus. The least stable gene was tub-α, showing a biased expression profile between sexual regions of the gonad, therefore this gene was analyzed thereafter as a target gene together with vitellogenin (vit) and a DEAD-box RNA helicase (dbx) gene. Relative expression, estimated by normalization with the combination of 18S and rp-l8 as reference genes, indicated that as gonad development advanced two of the target genes were up-regulated, tub-α in the male region and vit in the female region. Whereas an increased expression was expected during development for vit for its known role in vitellogenesis, the increased expression of tub-α in the male sexual region was unexpected, and pointed toward this gene being a testis-specific α-tubulin isotype. Further analyses of gene expression among tissues indicated that tub-α is specifically and highly expressed in the male gonad, although expression in adductor muscle was also observed at significantly lower levels. The existence of testis specific α- and β-tubulins has been previously reported in other taxa, relating their function to sperm axoneme formation. Tissue-specific tubulin genes, particularly their promoters, have recently found an application as native promoters for transgene tissue-specific expression in research and reproductive control of insect plagues. The third target gene, a putative member of the DEAD-box RNA helicase family (dbx), showed no changes in expression during gonad development or between sexual regions, therefore it was chosen to discuss the different statistical inferences resulting from the arbitrary use of 'randomly chosen' reference genes when normalizing gene expression.

The aim of this study was to evaluate the use of metallothionein (MT) concentrations in tissues of the clam Megapitaria squalida as biomarkers of environmental cadmium (Cd) pollution from phosphorite enrichments in the marine environment, which resulted from mining activities in La Paz Bay, Baja California Sur, Mexico. Cd and MT were quantified in gills, digestive gland, and kidney of clams exposed to 0.2 or 0.5 mg Cd l−1 for 10, 20, or 30 days. In addition, clams from four strategically selected natural sites of La Paz Bay were collected for analysis. In tissues of bioassayed and untreated clams, the gradient of Cd concentrations was digestive gland >> gills > kidney, whereas that of MT was digestive gland > gills > kidney. Digestive gland of the clams exposed to 0.5 mg Cd l−1 for 30 days showed the highest concentrations of Cd (16.3 ± 3.9 μg Cd g−1). The highest statistically significant MT concentrations were found in digestive gland at 10 days of exposure to Cd. In the untreated clams, one of the highest Cd concentrations, but not MT levels, was found in digestive glands of the organisms collected from the area close to phosphorite mining activities. For environmental monitoring, MT levels in digestive gland can be used as a first approximation of the presence of high levels of divalent metals in the environment. However, in this study, MT levels did not correlate with high Cd levels in clams that had been collected from areas associated with phosphorite enrichment.

This study evaluated organismal toxicity, cytotoxicity, and genotoxicity and the filtration rate in response to different concentrations of subchronic lindane (gamma-hexachlorocyclohexane [gamma-HCH]), exposure (12 d) in adult Pacific oysters Crassostrea gigas. Oysters were exposed in vivo in laboratory aquaria to 10 different concentrations (0.0-10.0 mg/L) of gamma-HCH. The median lethal concentration (LC50) after 12 d was calculated as 2.22 mg/L. Cytotoxic effects were observed in hemocytes, where the mean cell viability was significantly decreased at 1.0 mg/L of gamma-HCH after 12 d. Genotoxicity of gamma-HCH measured by single cell gel electrophoresis assay, in hemocytes was evident at 0.7 mg/L of gamma-HCH after 12 d. After 4 h of exposure to gamma-HCH, filtration rates were reduced compared with controls to 65.8 and 38.2% at concentrations of 0.3 and 0.7 mg/L, respectively, and after 11 d of exposure, filtration rates were reduced to 60.4 and 30.9% at concentrations of 0.1 mg/L and higher. These results show the subchronic effects of gamma-HCH at different concentrations and effect sensitivities are categorized as filtration rate < genotoxicity < cytotoxicity < mortality. The relevance of integral toxicity evaluation, considering different endpoints from molecular, cellular, and individual levels is discussed.