Minimizing the time to evaluate pheromone-mediated reduction of aggressive behavior in Mozambique tilapia (Oreochromis mossambicus).

Some cichlid fishes release urine-containing chemical cues that lower aggression in their opponents. Bioassays to identify the aggression-modulating pheromone include assessing the effect of urine fractions on the behavior towards a mirror image or in interactions with another male. However, many of these methods can be time-consuming and require many fish. The objective of the present study was to assess the behavior of male Mozambique tilapia (Oreochromis mossambicus) towards male urine using two methods with the intent of simplifying the bioassays: aggression towards a mirror image (mirror assay) and real opponents in which the urogenital papilla was tied using surgical silk to prevent urination. The results confirm the aggression-reducing effect of dominant male urine in both experimental approaches. Ten minutes of biting or 15 min of tail-beating behaviors in the mirror assay, or 5 min of opercular expansion or 15 min of lateral display in interactions with real opponents were necessary to detect a statistically significant reduction in aggressive behavior towards dominant male urine. We also found that males with subordinate status had lower latency to initiate aggressive behaviors towards the mirror than dominants in the same condition, even though fish had been isolated for 1 week. However, no such differences in latency were found in the real opponent assay. We conclude that 5 min of opercular expansion behavior in real opponent fights or 10 min of biting behavior in the mirror assay are the shortest times necessary to test aggressive behavior in urine fractions in bioassay-guided identification of pheromones.

Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii.

The genome evolution of Antarctic notothenioids has been modulated by their extreme environment over millennia and more recently by human-caused constraints such as overfishing and climate change. Here we investigated the characteristics of the immune system in Notothenia rossii and how it responds to 8 h immersion in viral (Poly I:C, polyinosinic: polycytidylic acid) and bacterial (LPS, lipopolysaccharide) proxies. Blood plasma antiprotease activity and haematocrit were reduced in Poly I:C-treated fish only, while plasma protein, lysozyme activity and cortisol were unchanged with both treatments. The skin and duodenum transcriptomes responded strongly to the treatments, unlike the liver and spleen which had a mild response. Furthermore, the skin transcriptome responded most to the bacterial proxy (cell adhesion, metabolism and immune response processes) and the duodenum (metabolism, response to stress, regulation of intracellular signal transduction, and immune system responses) to the viral proxy. The differential tissue response to the two proxy challenges is indicative of immune specialisation of the duodenum and the skin towards pathogens. NOD-like and C-type lectin receptors may be central in recognising LPS and Poly I:C. Other antimicrobial compounds such as iron and selenium-related genes are essential defence mechanisms to protect the host from sepsis. In conclusion, our study revealed a specific response of two immune barrier tissue, the skin and duodenum, in Notothenia rossii when exposed to pathogen proxies by immersion, and this may represent an adaptation to pathogen infective strategies.

The physiological effect of polystyrene nanoplastic particles on fish and human fibroblasts.

Numerous studies have identified the detrimental effects for the biosphere of large plastic debris, the effect of microplastics (MPs) and nanoplastics (NPs) is less clear. The skin is the first point of contact with NPs, and skin fibroblasts have a vital role in maintaining skin structure and function. Here, a comparative approach is taken using three fibroblast cell lines from the zebrafish (SJD.1), human male newborn (BJ-5ta) and female adult (HDF/TERT164) and their response to polystyrene NP (PS-NPs) exposure is characterized. Cells were exposed to environmentally relevant PS-NP sizes (50, 500 and 1000 nm) and concentrations (0.001 to 10 µg/ml) and their uptake (1000 nm), and effect on cell viability, proliferation, migration, reactive oxygen species (ROS) production, apoptosis, alkaline phosphatase (ALP) and acid phosphatase (AP) determined. All fibroblasts took up PS-NPs, and a relationship between PS-NP particle size and concentration and the inhibition of proliferation and cell migration was identified. The inhibitory effect of PS-NPs on proliferation was more pronounced for human skin fibroblasts. The presence of PS-NPs negatively affected fibroblast migration in a time-, size- and concentration-dependent manner with larger PS-NPs at higher concentrations causing a more significant inhibition of cell migration, with human fibroblasts being the most affected. No major changes were detected in ROS production or apoptosis in NP challenged fibroblasts. While the ALP activity was increased in all fibroblast cell lines, only fish fibroblasts showed a significant increase in AP activity. The heterogeneous response of fibroblasts induced by PS-NPs was clearly revealed by the segregation of HDF, BJ.5ta and SJD.1 fibroblasts in principal component analysis. Our results demonstrate that PS-NP exposure adversely affected cellular processes in a cell-type and dose-specific manner in distinct fibroblast cell lines, emphasizing the need for further exploration of NP interactions with different cell types to better understand potential implications for human health.

Biosensors Based on Stanniocalcin-1 Protein Antibodies Thin Films for Prostate Cancer Diagnosis.

Prostate cancer is one of the most prevalent tumors in men, accounting for about 7.3% of cancer deaths. Although there are several strategies for diagnosing prostate cancer, these are only accurate when the tumor is already at a very advanced stage, so early diagnosis is essential. Stanniocalcin 1 (STC1) is a secreted glycoprotein, which has been suggested as a tumor marker as its increased expression is associated with the development and/or progression of different types of malignant tumors. In this work, an electronic tongue (ET) prototype, based on a set of four sensors prepared from thin films that included STC1 antibodies for detecting prostate cancer, was developed. In the preparation of the thin films, polyelectrolytes of polyallylamine hydrochloride, polystyrene sulfonate of sodium and polyethyleneimine, and the biomolecules chitosan, protein A, and STC1 antibody were used. These films were deposited on quartz lamellae and on solid supports using layer-on-layer and self-assembly techniques. The deposition of the films was analyzed by ultraviolet-visible spectroscopy, and the detection of STC1 in aqueous solutions of PBS was analyzed by impedance spectroscopy. The impedance data were statistically analyzed using principal component analysis. The ETs formed by the four sensors and the three best sensors could detect the antigen at concentrations in the range from 5 × 10-11 to 5 × 10-4 M. They showed a linear dependence with the logarithm of the antigen concentration and a sensitivity of 5371 ± 820 and 4863 ± 634 per decade of concentration, respectively. Finally, the results allow us to conclude that this prototype can advance to the calibration phase with patient samples.

The fusion gene hsf5-rnf43 in Nile tilapia: A potential regulator in the maintenance of testis function and sexual differentiation.

We identified that the genes heat shock transcription factor 5 (hsf5) and ring finger protein 43 (rnf43) happened fusion in Nile tilapia (Oreochromis niloticus), called hsf5-rnf43, and provided the characteristic and functional analysis of hsf5-rnf43 gene in fish for the first time. Analysis of spatiotemporal expression showed that hsf5-rnf43 was specifically expressed in the testis and located in primary spermatocytes of adult Nile tilapia and gradually increased during testis development from 5 to 180 days after hatching. We also found DNA methylation regulated sex-biased expression of hsf5-rnf43 in the early development of Nile tilapia, and was affected by high temperature during the thermosensitive period of Nile tilapia sex differentiation. Therefore, we first reported that the fusion gene hsf5-rnf43 was sex-biased expressed in the testis regulated by DNA methylation and affected by high temperature, which may be involved in the maintenance of testis function and sex differentiation of Nile tilapia.

Bile acids as putative social signals in Mozambique tilapia (Oreochromis mossambicus).

Chemical cues provide potential mates with information about reproductive status and resource-holding potential. In the Mozambique tilapia (Oreochromis mossambicus), males can distinguish female reproductive status through chemical cues, and accessibility of males to females depends on their position in the hierarchy, determined in part by chemical cues. Here, we hypothesized that tilapia faecal cues are attractive to conspecifics once released into the water. C18 solid-phase extracts of faeces from dominant males and pre-ovulatory females evoked stronger olfactory epithelium electrical responses (EOG) than, respectively, subordinate males and post-spawning females. Mass spectrometry of the reverse-phase C18 high-performance liquid chromatography fractions of these extracts with highest EOG, identified by amino acids and bile acids. Faeces from pre-ovulatory females contain significantly higher concentrations of cholic acid (CA) and taurocholic acid (TCH) than both post-spawning females and males. A pool of amino acids had no effect on aggression or attraction in males. However, males were attracted to the scent of pre-ovulatory female faeces, as well as CA and TCH, when applied separately. This attraction was accompanied by increased digging behaviour compared to the odour of post-spawning females. CA and TCH exert their action through separate receptor mechanisms. These findings are consistent with a role for faeces - and bile acids therein - in chemical communication in this species, acting as an attractant for males to reproductive females.

Core microbiome profiles and their modification by environmental, biological, and rearing factors in aquaculture hatcheries.

16S rRNA gene sequencing and bacteria- and genus-specific quantitative PCR was used to profile microbial communities and their associated functions in water, live feed (microalgae, Artemia, and rotifer), and European sea bass and gilthead sea bream larvae from hatcheries in Greece and Italy. The transfer to larvae of genus containing potential pathogens of fish was more likely with Artemia and rotifer than with microalgae or water, irrespective of geographic location. The presence of potentially pathogenic bacteria (Vibrio and Pseudoalteromonas) in the core microbiota of water, live feed, and fish larvae, the enrichment of different bacterial resistance pathways and biofilm formation, and the overall low beneficial bacteria load during larval ontogeny emphasizes the risk for disease outbreaks. The present data characterizing microbiota in commercial aquaculture hatcheries provides a baseline for the design of strategies to manage disease and to model or remediate potential adverse environmental impacts.

New Vectors of TTX Analogues in the North Atlantic Coast: The Edible Crabs Afruca tangeri and Carcinus maenas.

Tetrodotoxin (TTX) and its analogues are naturally occurring toxins historically responsible for human poisoning fatalities in Eastern Asia. It is typically linked to the consumption of pufferfish and, to a lesser extent, marine gastropods and crabs. In the scope of a comprehensive project to understand the prevalence of emergent toxins in edible marine organisms, we report, for the first time, the detection of TTX analogues in the soft tissues of edible crabs, the European fiddler crab (Afruca tangeri) and green crab (Carcinus maenas), harvested in southern Portugal. No TTX was detected in the analyzed samples. However, three TTX analogues were detected-an unknown TTX epimer, deoxyTTX, and trideoxyTTX. These three analogues were found in the European fiddler crab while only trideoxyTTX was found in the green crab, suggesting that the accumulation of TTX analogues might be influenced by the crabs' different feeding ecology. These results highlight the need to widely monitor TTX and its analogues in edible marine species in order to provide adequate information to the European Food Safety Authority and to protect consumers.

Tissue accumulation of tetrodotoxin (TTX) and analogues in trumpet shell Charonia lampas.

Tetrodotoxin (TTX) is a potent neurotoxin responsible for a human intoxication event in Spain associated with the consumption of trumpet shell Charonia lampas. In Europe, TTX is not regulated or monitored, and there is little knowledge about its presence in seafood. Here, we investigated the tissue distribution of TTX and analogues in three specimens of trumpet shell C. lampas bought in a market in southern Portugal. Toxin concentration was above the EFSA recommended limit in the non-edible tissues of all specimens and within the limit in the edible tissues of two specimens. 4,9-AnhydroTTX and 13 additional TTX analogues were detected in tissues, the most abundant being anhydrotrideoxyTTX and trideoxyTTX. These results suggest that although thorough evisceration may lower the amount of TTX consumed, it may not be sufficient to ensure consumer safety. Regular monitoring of TTX and analogues in trumpet shell and other edible gastropods is therefore recommended to avoid poisoning incidents.

LC-HRMS Profiling of Paralytic Shellfish Toxins in Mytilus galloprovincialis after a Gymnodinium catenatum Bloom.

Saxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels' whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs' profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicity.

Population genomics of an icefish reveals mechanisms of glacier-driven adaptive radiation in Antarctic notothenioids.

BACKGROUND: Antarctica harbors the bulk of the species diversity of the dominant teleost fish suborder-Notothenioidei. However, the forces that shape their evolution are still under debate. RESULTS: We sequenced the genome of an icefish, Chionodraco hamatus, and used population genomics and demographic modelling of sequenced genomes of 52 C. hamatus individuals collected mainly from two East Antarctic regions to investigate the factors driving speciation. Results revealed four icefish populations with clear reproduction separation were established 15 to 50 kya (kilo years ago) during the last glacial maxima (LGM). Selection sweeps in genes involving immune responses, cardiovascular development, and photoperception occurred differentially among the populations and were correlated with population-specific microbial communities and acquisition of distinct morphological features in the icefish taxa. Population and species-specific antifreeze glycoprotein gene expansion and glacial cycle-paced duplication/degeneration of the zona pellucida protein gene families indicated fluctuating thermal environments and periodic influence of glacial cycles on notothenioid divergence. CONCLUSIONS: We revealed a series of genomic evidence indicating differential adaptation of C. hamatus populations and notothenioid species divergence in the extreme and unique marine environment. We conclude that geographic separation and adaptation to heterogeneous pathogen, oxygen, and light conditions of local habitats, periodically shaped by the glacial cycles, were the key drivers propelling species diversity in Antarctica.

Long non-coding RNAs are involved in immune resistance to Aeromonas hydrophila in black carp (Mylopharyngodon piceus).

A growing number of studies identified long non-coding RNAs (lncRNAs) to be closely associated with immune function through the regulation of immune cell differentiation and immune cell effector function. Here we tested whether lncRNAs are involved in immune function in black carp (Mylopharyngodon piceus) through the exposure to Aeromonas hydrophila and analysis of the spleen gene expression response using RNA-seq. A total of 9036 lncRNAs were identified with high confidence. Differential expression analysis identified a total of 3558 DElncRNAs (Differential expression lncRNA) involved in A. hydrophila infection and 4526 target genes corresponding to DElncRNAs. After screening 4526 target genes in the InnateDB database, a total of 150 immunity genes were identified. After GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of the obtained immunity genes, the Toll-like receptor (TLR) signaling pathway, TLR2, TLR3, TLR5, and TLR8 were identified as particularly significant in A. hydrophyla-resistant black carp. At the same time, the Ras signaling pathway was particularly enriched in the spleen of susceptible black carp. Analysis of PPI (protein-protein interaction) networks of the obtained immune genes identified SRC (SRC Proto-Oncogene), MYD88 (Myeloid differentiation primary response 88), MAPK3 (Mitogen-Activated Protein Kinase 3), MYC (MYC Proto-Oncogene) as main hub genes regulated by lncRNA and possibly mediating a mechanism of susceptibility to bacteria. These results establish a functional role of lncRNAs and a mechanistic base for the immune response in black carp resistant to A. hydrophila.

Toll-Like Receptor Evolution: Does Temperature Matter?

Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and are an ancient and well-conserved group of pattern recognition receptors (PRRs). The isolation of the Antarctic continent and its unique teleost fish and microbiota prompted the present investigation into Tlr evolution. Gene homologues of tlr members in teleosts from temperate regions were present in the genome of Antarctic Nototheniidae and the non-Antarctic sister lineage Bovichtidae. Overall, in Nototheniidae apart from D. mawsoni, no major tlr gene family expansion or contraction occurred. Instead, lineage and species-specific changes in the ectodomain and LRR of Tlrs occurred, particularly in the Tlr11 superfamily that is well represented in fish. Positive selective pressure and associated sequence modifications in the TLR ectodomain and within the leucine-rich repeats (LRR), important for pathogen recognition, occurred in Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23 presumably associated with the unique Antarctic microbiota. Exposure to lipopolysaccharide (Escherichia coli O111:B4) Gram negative bacteria did not modify tlr gene expression in N. rossii head-kidney or anterior intestine, although increased water temperature (+4°C) had a significant effect.

Proteome dataset of sea bass (Dicentrarchus labrax) skin-scales exposed to fluoxetine and estradiol.

Contamination of aquatic ecosystems with anthropogenic pollutants, including pharmaceutical drugs, is a major concern worldwide. Aquatic organisms such as fish are particularly at risk of exposure to pollutants. The surface of fish is the first point of contact with pollutants, but few studies have considered the impact of pollutants on the skin-scale barrier. The present proteome data are the basis of the findings discussed in the associated research article "Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol" [1]. Juvenile sea bass were exposed by intraperitoneal injections to: a) the antidepressant fluoxetine (FLX), a widely prescribed psychotropic drug and an emerging pollutant; b) the natural estrogen 17ß-estradiol (E2) and c) the vehicle, coconut oil (control). The scale proteome of fish exposed to these compounds for 5 days was analysed using quantitative label-free proteomics technology SWATH-MS (sequential windowed data-independent acquisition of the total high-resolution-mass spectra). The proteome data generated was submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020983. LC-MS data from pooled protein extracts from the scales of all experimental groups was acquired using information-dependent acquisition (IDA) and 1,254 proteins were identified by searching against the sea bass genome database. 715 proteins were quantified by SWATH acquisition, and 213 proteins had modified levels (p < 0.05) between the E2- or FLX-exposed fish compared to the control. The main biological processes and KEGG pathways affected by E2 or FLX treatments were identified using Cytoscape/ClueGO enrichment analyses.

Quantitation Overcoming Matrix Effects of Lipophilic Toxins in Mytilus galloprovincialis by Liquid Chromatography-Full Scan High Resolution Mass Spectrometry Analysis (LC-HR-MS).

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.

Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol.

Teleost fish skin-scales are essential for protection and homeostasis and the largest tissue in direct contact with the environment, but their potential as early indicators of pollutant exposure are hampered by limited knowledge about this model. This study evaluated multi-level impacts of in vivo exposure of European sea bass to fluoxetine (FLX, a selective serotonin-reuptake inhibitor and an emerging pollutant) and 17ß-estradiol (E2, a natural hormone and representative of diverse estrogenic endocrine-disrupting pollutants). Exposed fish had significantly increased circulating levels of FLX and its active metabolite nor-FLX that, in contrast to E2, did not have estrogenic effects on most fish plasma and scale indicators. Quantitative proteomics using SWATH-MS identified 985 proteins in the scale total proteome. 213 proteins were significantly modified 5 days after exposure to E2 or FLX and 31 were common to both treatments and responded in the same way. Common biological processes significantly affected by both treatments were protein turnover and cytoskeleton reorganization. E2 specifically up-regulated proteins related to protein production and degradation and down-regulated the cytoskeleton/extracellular matrix and innate immune proteins. FLX caused both up- and down-regulation of protein synthesis and energy metabolism. Multiple estrogen and serotonin receptor and transporter transcripts were altered in sea bass scales after E2 and/or FLX exposure, revealing complex disruptive effects in estrogen/serotonin responsiveness, which may account for the partially overlapping effects of E2 and FLX on the proteome. A large number (103) of FLX-specifically regulated proteins indicated numerous actions independent of estrogen signalling. This study provides the first quantitative proteome of the fish skin-scale barrier, elucidates routes of action and biochemical and molecular signatures of E2 or FLX-exposure and identifies potential physiological consequences and candidate biomarkers of pollutant exposure, for monitoring and risk assessment.

Factors Driving Bacterial Microbiota of Eggs from Commercial Hatcheries of European Seabass and Gilthead Seabream.

A comprehensive understanding of how bacterial community abundance changes in fishes during their lifecycle and the role of the microbiota on health and production is still lacking. From this perspective, the egg bacterial communities of two commercially farmed species, the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata), from different aquaculture sites were compared, and the potential effect of broodstock water microbiota and disinfectants on the egg microbiota was evaluated. Moreover, 16S ribosomal RNA gene sequencing was used to profile the bacterial communities of the eggs and broodstock water from three commercial hatcheries. Proteobacteria were the most common and dominant phyla across the samples (49.7% on average). Vibrio sp. was the most highly represented genus (7.1%), followed by Glaciecola (4.8%), Pseudoalteromonas (4.4%), and Colwellia (4.2%), in eggs and water across the sites. Routinely used iodine-based disinfectants slightly reduced the eggs' bacterial load but did not significantly change their composition. Site, species, and type of sample (eggs or water) drove the microbial community structure and influenced microbiome functional profiles. The egg and seawater microbiome composition differed in abundance but shared similar functional profiles. The strong impact of site and species on egg bacterial communities indicates that disease management needs to be site-specific and highlights the need for species- and site-specific optimization of disinfection protocols.

A new subfamily of ionotropic glutamate receptors unique to the echinoderms with putative sensory role.

Chemosensation is a critical signalling process in animals and especially important in sea cucumbers, a group of ecologically and economically important marine echinoderms (class Holothuroidea), which lack audio and visual organs and rely on chemical sensing for survival, feeding and reproduction. The ionotropic receptors are a recently identified family of chemosensory receptors in insects and other protostomes, related to the ionotropic glutamate receptor family (iGluR), a large family of membrane receptors in metazoan. Here we characterize the echinoderm iGluR subunits and consider their possible role in chemical communication in sea cucumbers. Sequence similarity searches revealed that sea cucumbers have in general a higher number of iGluR subunits when compared to other echinoderms. Phylogenetic analysis and sequence comparisons revealed GluH as a specific iGluR subfamily present in all echinoderms. Homologues of the vertebrate GluA (aka α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA), GluK (aka kainate) and GluD (aka delta) were also identified. The GluN (aka N-methyl-d-aspartate, NMDA) as well as the invertebrate deuterostome subfamily GluF (aka phi) are absent in echinoderms. The echinoderm GluH subfamily shares conserved structural protein organization with vertebrate iGluRs and the ligand binding domain (LBD) is the most conserved region; genome analysis indicates evolution via lineage and species-specific tandem gene duplications. GluH genes (named Grih) are the most highly expressed iGluRs subunit genes in tissues in the sea cucumber Holothuria arguinesis, with Griha1, Griha2 and Griha5 exclusively expressed in tentacles, making them candidates to have a chemosensory role in this species. The multiple GluH subunits may provide alternative receptor assembly combinations, thus expanding the functional possibilities and widening the range of compounds detected during aggregation and spawning in echinoderms.

Galanin isoforms by alternative splicing: Structure, expression, and immunohistochemical location in the gonads of European sea bass.

Galanin (Gal) is a neuropeptide with multiple functions that is widely expressed in the central and peripheral nervous systems of vertebrates. Anatomical and functional evidence suggests a possible role in regulating reproduction in fishes. To test this possibility, we have isolated and characterized two gal alternative transcripts in European sea bass (Dicentrarchus labrax) that encode two prepropeptides, respectively of 29 (gal_MT853221) and 53 (gal_MT853222) amino acids. The two gal transcripts are highly expressed in brain, pituitary and gonads, and appear to be differentially regulated in males and females. In males, gal_MT853222 in the hypothalamus and gal_MT853221 in the pituitary were downregulated with the progression of spermatogenesis (stages I-III). Both transcripts are downregulated in testicles of 1-year (precocious) and 2-year spermiating males compared to immature fish of the same age. Gal peptides and receptors are expressed throughout ovarian development in the hypothalamic-pituitary-gonadal (HPG) axis of females. In the testis, immunoreactive Gal-29 and Gal-53 peptides were detected in blood vessels and Leydig cells during the spermatogenesis stages I-III but Gal immunostaining was barely undetected in more advanced stages. In the ovary, both peptides localized in interstitial cells and blood vessels and in theca cells surrounding the maturing oocytes. The immunolocalization of galanin in Leydig and theca cells suggests a possible role in steroid production regulation. The different pattern of gal expression and Gal localization in the testis and ovary may suggest the possibility that androgens and estrogens may also regulate Gal gene transcription and translation. Altogether, this study showed evidence for the possible involvement of locally produced Gal in gametogenesis and that its production is differentially regulated in male and female gonads.