Effects of phthalates on marine organisms: cytotoxicity and genotoxicity of mono-(2-ethylhexyl)-phthalate (MEHP) on European sea bass (Dicentrarchus labrax) embryonic cell line.

INTRODUCTION: Mono-(2-ethylhexyl) phthalate (MEHP) represents a toxicological risk for marine organisms due to its widespread presence in aquatic environments. METHODS: MEHP effects on cell viability, cell death and genotoxicity were investigated on the DLEC cell line, derived from early embryos of the European sea bass Dicentrarchus labrax L. RESULTS: A dose-dependent cytotoxic effect, with no induction of necrotic process, except at its highest concentration, was observed. Moreover, chromosomal instability was detected, both in binucleated and mononucleated cells, coupled with a minor inhibition of cell proliferation, whereas genomic instability was not revealed. To our knowledge, the overall results suggest the first evidence of a possible aneugenic effect of this compound in the DLEC cell line, that is the induction of chromosomal loss events without the induction of primary DNA damage. CONCLUSIONS: MEHP should be considered more harmful than its parent compound DEHP, because it induces genomic instability in the DLEC cell line without triggering cell death.

Fish muscle hydrolysate obtained using largemouth bass Micropterus salmoides digestive enzymes improves largemouth bass performance in its larval stages.

The present study utilized digestives tracts from adult largemouth bass (LMB) to hydrolyze Bighead carp muscle and obtain an optimal profile of muscle protein hydrolysates that would be easily assimilated within the primitive digestive tract of larval LMB. Specifically, muscle protein source was digested for the larva using the fully developed digestive system of the same species. The objectives of this study were: 1) to develop an optimal in vitro methodology for carp muscle hydrolysis using LMB endogenous digestive enzymes, and 2) to evaluate the effect of dietary inclusion of the carp muscle protein hydrolysate on LMB growth, survival, occurrence of skeletal deformities, and whole-body free amino acid composition. The study found that the in vitro hydrolysis method using carp intact muscle and LMB digestive tracts incubated at both acid and alkaline pH (to mimic digestive process of LMB) yielded a wide range of low molecular weight fractions (peptides), as opposed to the non-hydrolyzed muscle protein or muscle treated only with acid pH or alkaline pH without enzymes from LMB digestive tracts, which were comprised of large molecular weight fractions (polypeptides above 150 kDa). Overall, the dietary inclusion of the carp muscle hydrolysate improved growth performance of larval LMB in terms of final average weight, weight gain, DGC, SGR, and body length after 21 days of feeding compared to fish that received the diet based on non-hydrolyzed carp muscle. The study also found that hydrolysate-based feed significantly reduced skeletal deformities. The positive growth performance presented by fish in the hydrolysate-fed group possibly resulted from matching the specific requirements of the larvae with respect to their digestive organ development, levels of digestive enzymes present in the gut, and nutritional requirements.

Optimization of vitrification factors for embryo cryopreservation of kelp grouper (Epinephelus moara).

Cryopreservation of marine fish embryos causes to severe cryogenic damage, and to date, adults have not been reared from embryos that were cryopreserved. Here, we optimized vitrification factors to improve the survival and hatching rate of kelp grouper (Epinephelus moara) embryos after cryopreservation. We screened the effects of 11 vitrification solution concentrations (25-50%) on the survival rate of embryos at four developmental stages (16S, 18S, 22S, TB). We investigated the effects of different equilibration time (25-45min) on the survival rate and the influence of vitrification solutions on embryonic volume. In addition, we tested the effects of treating embryos at five different developmental stages (4-6S, 16S, 22S, TB, HB) with different vitrification solutions (35% PMG3S and 35% PMG3T), prechilling temperature (-5 °C and 4 °C) and prechilling time. In total, 9855 embryos were cryopreserved at 10 developmental stages, from optic capsule stage to pre-hatch stage. We found that kelp grouper embryos performed best at equilibration time of 30 min. Embryos at the tail-bud stage exhibited greater tolerance to vitrification than other stages. Vitrification solutions that contained sucrose showed better survival rates compared to embryos treated with vitrification solutions containing trehalose. Pre-chilling treatment improved viability before freezing, but did not improve viability after freezing. In the most optimal condition we identified in this study, the average survival, normal development and malformation rates of cryopreserved embryos were 6.32%, 2.36% and 3.49%, and 39.85% of the surviving embryos that were cryopreserved hatched. The hatched larvae gradually died at day 12 of cultivation, where the longest surviving individuals lived for 16 days. This study provides valuable data for improving survival and hatching rate of cryopreserved grouper embryos, and provides references for further exploring techniques in fish embryo cryopreservation.

RNA seq- and DEG-based comparison of developmental toxicity in fish embryos of two species exposed to Iranian heavy crude oil.

To determine and compare the toxic effects of Iranian heavy crude oil (IHCO) on the embryonic development of two fish species, we examined transcriptome profiles using RNA-seq. The assembled contigs were 66,070 unigenes in olive flounder embryos and 76,498 unigenes in spotted seabass embryos. In the differential gene expression (DEG) profiles, olive flounder embryos showed different up- and down-regulated patterns than spotted seabass embryos in response to fresh IHCO (FIHCO) and weathered IHCO (WIHCO). In this work, we categorized DEG profiles into six pathways: ribosome, oxidative phosphorylation, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cardiac muscle contraction, validating the expression patterns of 13 DEGs using real-time quantitative RT-PCR. The expression of the CYP1A, CYP1B1, and CYP1C1 genes in spotted seabass embryos was higher than in olive flounder embryos, whereas genes related to cell processing, development, and the immune system showed the opposite trend. Orthologous gene cluster analysis showed that olive flounder embryos were sensitive (fold change of genes with cutoff P<0.05) to both FIHCO and WIHCO, but spotted seabass embryos exhibited higher sensitivity to WIHCO than FIHCO, indicating that species-specific differences are likely to be reflected in population levels after oil spills. Overall, our study provides new insight on the different embryonic susceptibilities of two marine fish species to FIHCO and WIHCO and a better understanding of the underlying molecular mechanisms via RNA-seq and DEGs.

Tissue localization of piscidin host-defense peptides during striped bass (Morone saxatilis) development.

Infectious diseases are a major cause of larval mortality in finfish aquaculture. Understanding ontogeny of the fish immune system and thus developmental timing of protective immune tissues and cells, may help to decrease serious losses of larval fishes when they are particularly vulnerable to infection. One component of the innate immune system of fishes is the host-defense peptides, which include the piscidins. Piscidins are small, amphipathic, α-helical peptides with a broad-spectrum of action against viral, bacterial, fungal, and protozoan pathogens. We describe for the first time the cellular and tissue localization of three different piscidins (1, 3, and 4) during striped bass (Morone saxatilis) larval ontogeny using immunofluorescent histochemistry. From 16 days post hatch to 12 months of age, piscidin staining was observed in cells of the epithelial tissues of gill, digestive tract, and skin, mainly in mast cells. Staining was also seen in presumptive hematopoietic cells in the head kidney. The three piscidins showed variable cellular and tissue staining patterns, possibly relating to differences in tissue susceptibility or pathogen specificity. This furthers our observation that the piscidins are not a monolithic family of antimicrobials, but that different AMPs have different (more specialized) functions. Furthermore, no immunofluorescent staining of piscidins was observed in post-vitellogenic oocytes, embryos, or larvae from hatch to 14 days post hatch, indicating that this critical component of the innate immune system is inactive in pre-hatch and young larval striped bass.

Differential Toxicokinetics Determines the Sensitivity of Two Marine Embryonic Fish Exposed to Iranian Heavy Crude Oil.

Interspecific difference in the developmental toxicity of crude oil to embryonic fish allows the prediction of injury extent to a number of resident fish species in oil spill sites. This study clarifies the comparative developmental effects of Iranian heavy crude oil (IHCO) on the differences of biouptake and toxic sensitivity between embryonic spotted sea bass (Lateolabrax maculates) and olive flounder (Paralichthys olivaceus). From 24 h after exposure to IHCO, several morphological defects were observed in both species of embryonic fish, including pericardial edema, dorsal curvature of the trunk, developmental delay, and reduced finfolds. The severity of defects was greater in flounder compared to that in sea bass. While flounder embryos accumulated higher embryo PAH concentrations than sea bass, the former showed significantly lower levels of CYP1A expression. Although bioconcentration ratios were similar between the two species for some PAHs, phenanthrenes and dibenzothiophenes showed selectively higher bioconcentration ratios in flounder, suggesting that this species has a reduced metabolic capacity for these compounds. While consistent with a conserved cardiotoxic mechanism for petrogenic PAHs across diverse marine and freshwater species, these findings indicate that species-specific differences in toxicokinetics can be an important factor underlying species' sensitivity to crude oil.

Wnt4 in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides): identification and expression.

Wnt4 (Wingless-type MMTV integration site family member 4) has been demonstrated to play critical roles in ovarian development in mammals, but its function in fish reproduction is still unclear. In the present study, two full-length wnt4 cDNA sequences (named wnt4a and wnt4b) were cloned from the orange-spotted grouper (Epinephelus coioides). Amino acid alignment analysis showed that both orange-spotted grouper Wnt4s proteins had the typical characteristics of the Wnt family. RT-PCR revealed that both wnt4a and wnt4b were highly expressed in the ovaries of the orange-spotted grouper. Temporal expression profiles of both wnt4 genes during embryonic and ovarian development were examined. The expressions of wnt4a and wnt4b genes were first detected at the embryonic morula stage, but the gens showed different expression patterns. During ovarian development, high expression of wnt4a was observed in the ovarian lumen formation and gonium proliferation stage, while wnt4b exhibited strong expression in the early developmental stage of oocytes. Taken together, the present study indicates that the two wnt4 genes are involved in the regulation of ovarian development in the orange-spotted grouper.

Genetic inactivation of European sea bass (Dicentrarchus labrax L.) eggs using UV-irradiation: observations and perspectives.

Androgenesis is a form of uniparental reproduction leading to progenies inheriting only the paternal set of chromosomes. It has been achieved with variable success in a number of freshwater species and can be attained by artificial fertilization of genetically inactivated eggs following exposure to gamma (γ), X-ray or UV irradiation (haploid androgenesis) and by restoration of diploidy by suppression of mitosis using a pressure or thermal shock. The conditions for the genetic inactivation of the maternal genome in the European sea bass (Dicentrarchus labrax L.) were explored using different combinations of UV irradiation levels and durations. UV treatments significantly affected embryo survival and generated a wide range of developmental abnormalities. Despite the wide range of UV doses tested (from 7.2 to 720 mJ x cm(-2)), only one dose (60 mJ x cm(-2) x min(-1) with 1 min irradiation) resulted in a small percentage (14%) of haploid larvae at hatching in the initial trials as verified by flow cytometry. Microsatellite marker analyses of three further batches of larvae produced by using this UV treatment showed a majority of larvae with variable levels of paternal and maternal contributions and only one larva displaying pure paternal inheritance. The results are discussed also in the context of an assessment of the UV-absorbance characteristics of egg extracts in this species that revealed the presence of gadusol, a compound structurally related to mycosporine-like amino acids (MAAs) with known UV-screening properties.

Ontogenesis of the HPI axis and molecular regulation of the cortisol stress response during early development in Dicentrarchus labrax.

The cortisol stress response and the molecular programming of the corticoid axis were characterized for the first time during early ontogeny in a Mediterranean marine teleost, the European sea bass (Dicentrarchus labrax). Sea bass embryos, pre-larvae and larvae at specific points of development were exposed to acute stressors and the temporal patterns of cortisol whole body concentrations and the expression of genes involved in corticosteroid biosynthesis, degradation and signaling were determined. Expression of genes (gr1, gr2, mr, crf) involved into the corticoid response regulation combined with histological data indicated that, although a cortisol stress response is evident for the first time around first feeding, a pattern becomes established in larvae at flexion until the formation of all fins. Moreover, mRNA transcript levels of 11ß-hydroxylase and 11ß-hsd2 showed a strong correlation with the whole body cortisol concentrations. Concluding, our data reveal the presence of an adaptive mechanism in European sea bass at early ontogeny enabling to cope with external stressful stimuli and provide a better insight into the onset and regulation of the stress response in this species.

Ovary transcriptome profiling via artificial intelligence reveals a transcriptomic fingerprint predicting egg quality in striped bass, Morone saxatilis.

Inherited gene transcripts deposited in oocytes direct early embryonic development in all vertebrates, but transcript profiles indicative of embryo developmental competence have not previously been identified. We employed artificial intelligence to model profiles of maternal ovary gene expression and their relationship to egg quality, evaluated as production of viable mid-blastula stage embryos, in the striped bass (Morone saxatilis), a farmed species with serious egg quality problems. In models developed using artificial neural networks (ANNs) and supervised machine learning, collective changes in the expression of a limited suite of genes (233) representing <2% of the queried ovary transcriptome explained >90% of the eventual variance in embryo survival. Egg quality related to minor changes in gene expression (<0.2-fold), with most individual transcripts making a small contribution (<1%) to the overall prediction of egg quality. These findings indicate that the predictive power of the transcriptome as regards egg quality resides not in levels of individual genes, but rather in the collective, coordinated expression of a suite of transcripts constituting a transcriptomic "fingerprint". Correlation analyses of the corresponding candidate genes indicated that dysfunction of the ubiquitin-26S proteasome, COP9 signalosome, and subsequent control of the cell cycle engenders embryonic developmental incompetence. The affected gene networks are centrally involved in regulation of early development in all vertebrates, including humans. By assessing collective levels of the relevant ovarian transcripts via ANNs we were able, for the first time in any vertebrate, to accurately predict the subsequent embryo developmental potential of eggs from individual females. Our results show that the transcriptomic fingerprint evidencing developmental dysfunction is highly predictive of, and therefore likely to regulate, egg quality, a biologically complex trait crucial to reproductive fitness.

Osmoregulatory response to low salinities in the European sea bass embryos: a multi-site approach.

Embryonic osmoregulation effected by embryonic ionocytes in the European sea bass Dicentrarchus labrax has been studied at several sites, including the yolk sac membrane, the first gill slits and the gut ionocytes. D. labrax embryos, spawned in seawater (SW) (39 ‰), were exposed to dilute seawater (DSW) (5 ‰) during 48 h, from stage 10 pairs of somites (10S) to hatching time (HT). Control embryos originating from the same spawn were maintained in SW. Both SW and DSW embryos were examined after 24- and 48-h exposure. Nanoosmometric measurements of the embryonic fluids osmolality suggest that late embryos are confronted with the variations in external salinity and that they were able to slightly regulate their osmolality. Immunolocalization of Na⁺/K⁺ ATPase, NKCC and CFTR has shown that DSW-exposed embryos can limit ion losses due to compensatory physiological mechanisms. CFTR and NKCC were not observed in DSW embryos in the yolk sac ionocytes and in the tegumentary ionocytes of the gill slits. The quantification of mRNA indicated that NKA, NKCC1 and CFTR transcript levels increased from stage 10S to stage HT. At stage HT, following 48 h of DSW- or SW-exposure, different responses were observed according to salinity. These results, when compared to those obtained in D. labrax juveniles and adults long-term exposed to fresh water (FW), show that in embryos the physiological response following a short-term DSW exposure is different. The mechanisms of hyper-osmoregulation observed in D. labrax embryos, although not fully efficient, allow their survival for several days in DSW.

Oxidative stress associated with paternal care in smallmouth bass (Micropterus dolomieu).

In species that provide parental care, care for offspring is often accompanied by an increase in locomotor activity and a decrease in feeding opportunities which can negatively impact endogenous energy reserves. Depletion of parental energy stores and declines in nutritional condition can cause physiological disturbances, such as an imbalance between free radical production and available antioxidants, known as oxidative stress. Using the teleost smallmouth bass (Micropterus dolomieu) as a model, we tested if the energetic challenge associated with sole paternal care was associated with oxidative stress. Blood samples from parental males were collected throughout parental care, during egg, embryo, and larval stages of offspring development, and assayed for both antioxidant capacity and oxidative damage. A reduction in oxygen radical absorbance capacity was observed during the parental care period, indicating a decrease in resistance to oxidative stress. Although no change was observed in the reduced:total thiol ratio, a significant increase in the concentration of both oxidized and total thiols occurred during the parental care period. No increase in the oxidative stress markers 8-hydroxy-2-deoxyguanosine, protein carbonyls and lipid peroxides was observed. We concluded that oxidative stress did not occur as a result of parental care in the male smallmouth bass. This study provides evidence that participation in energetically taxing activities, such as parental care, can result in a decrease in antioxidant resources, but may not always result in oxidative stress.

Orange-spotted grouper (Epinephelus coioides) orexin: molecular cloning, tissue expression, ontogeny, daily rhythm and regulation of NPY gene expression.

Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro.

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality.

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na(+) /K(+) -ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na(+) /K(+) /2Cl⁻ cotransporters (NKCCs) and the chloride channel "cystic fibrosis transmembrane regulator" (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.

Embryonic occurrence of ionocytes in the sea bass Dicentrarchus labrax.

Because of the permeability of the chorion, sea bass embryos are exposed to seawater before hatching and hence require precocious osmoregulatory processes. Several studies of other species have demonstrated the existence of ion-transporting cells located on the yolk sac membrane of embryos. In these cells, called ionocytes, ion movements are controlled by a pool of transmembrane proteins. Among them, the Na(+)/K(+)-ATPase, an abundant driving enzyme, has been used to reveal the presence or absence of ionocytes. We have immunostained the Na(+)/K(+)-ATPase in sea-bass embryos and shown the presence of the first ionocytes on the yolk sac membrane at stage 12 somites and the occurrence of ionocytes at other sites before hatching. Ionocytes located on the first gill slits have been identified at stage 14 somites. Primitive enteric ionocytes have also been detected at stage 14 somites in the mid and posterior gut. The presence of these cells might be related to the early opening of the gut to perivitelline fluids, both anteriorly by the gill slits and posteriorly by the anus. The role of embryonic ionocytes in osmoregulation before hatching is discussed.

Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax).

The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), beta-actin (two regions of beta-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), beta2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization.

Early development of the digestive tract (pharynx and gut) in the embryos and pre-larvae of the European sea bass Dicentrarchus labrax.

The European sea bass Dicentrarchus labrax is a marine teleost important in Mediterranean aquaculture. The development of the entire digestive tract of D. labrax, including the pharynx, was investigated from early embryonic development to day 5 post hatching (dph), when the mouth opens. The digestive tract is initialized at stage 12 somites independently from two distinct infoldings of the endodermal sheet. In the pharyngeal region, the anterior infolding forms the pharynx and the first gill slits at stage 25 somites. The other three gill arches and slits are formed between 1 and 5 dph. Posteriorly, in the gut tube region, a posterior infolding forms the foregut, midgut and hindgut. The anus opens before hatching, at stage 28 somites. Associated organs (liver, pancreas and gall bladder) are all discernable from 3 dph. Some aspects of the development of the two independent initial infoldings seem original compared with data in the literature. These results are discussed and compared with embryonic and post-embryonic development patterns in other teleosts.

Lipoprotein lipase (LPL) is highly expressed and active in the ovary of European sea bass (Dicentrarchus labrax L.), during gonadal development.

The oocytes of many fish species accumulate high amounts of neutral lipids as a caloric reserve for embryonic and larval development. We propose that lipoprotein lipase (LPL, EC 3.1.1.34) plays an important role in supplying the oocytes with fatty acids and we have cloned its cDNA from the ovary of sea bass, and determined the patterns of LPL activity and LPL mRNA expression in the ovary. The cDNA obtained was 3051 bp long with an open reading frame encoding 518 amino acids. The amino acid sequence has a high similarity and shows similar structural features to LPL of other species. Northern blot analysis revealed LPL expression in adipose tissue and gonads only. LPL activity and LPL mRNA expression in the ovary was very high in fish with a gonadosomatic index (GSI) above 5, coinciding with the appearance of a high number of lipid droplets in the ooplasm. The LPL mRNA expression was localised to the follicle cells surrounding the oocyte. Our results suggest that LPL is likely to play an important role in the incorporation of neutral lipids into the oocytes, and that follicle cells, in addition to participating in steroidogenesis, also may be important in building up oocyte lipid reserves.

Comparative analysis of Hox paralog group 2 gene expression during Nile tilapia (Oreochromis niloticus) embryonic development.

The hindbrain and pharyngeal arch-derived structures of vertebrates are determined, at least in part, by Hox paralog group 2 genes. In sarcopterygians, the Hoxa2 gene alone appears to specify structures derived from the second pharyngeal arch (PA2), while in zebrafish (Danio rerio), either of the two Hox PG2 genes, hoxa2b or hoxb2a, can specify PA2-derived structures. We previously reported three Hox PG2 genes in striped bass (Morone saxatilis), including hoxa2a, hoxa2b, and hoxb2a and observed that only HoxA cluster genes are expressed in PA2, indicative that they function alone or together to specify PA2. In this paper, we present the cloning and expression analysis of Nile tilapia (Oreochromis niloticus) Hox PG2 genes and show that all three genes are expressed in the hindbrain and in PA2. The expression of hoxb2a in PA2 was unexpected given the close phylogenetic relationship of Nile tilapia and striped bass, both of which are members of the order Perciformes. A reanalysis of striped bass hoxb2a expression demonstrated that it is expressed in PA2 with nearly the same temporal and spatial expression pattern as its Nile tilapia ortholog. Further, we determined that Nile tilapia and striped bass hoxa2a orthologs are expressed in PA2 well beyond the onset of chondrogenesis whereas neither hoxa2b nor hoxb2a expression persist until this stage, which, according to previous hypotheses, suggests that hoxa2a orthologs in these two species function alone as selector genes of PA2 identity.

Sex hormone-binding globulin expression in sea bass (Dicentrarchus labrax L.) throughout development and the reproductive season.

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in the blood of vertebrate species, including fish, and regulates the bioavailability and metabolic clearance of these steroids. Liver is the major site of plasma SHBG synthesis, while an SHBG homologue, known as the androgen-binding protein, is produced in testes. When shbg gene expression was examined throughout European sea bass (Dicentrarchus labrax L.) development, SHBG mRNA was clearly detectable at 7 days post-fertilization and persisted throughout embryonic development. In male and female sea bass, the liver is the principal site of shbg gene expression, as determined by SHBG mRNA analyses. Immunoreactive SHBG is present in the liver and villous stroma of the intestine in both sexes. It is also present in the interstitial space between testicular lobules, and the connective tissue surrounding the ovary in the non-reproductive season and around post-vitellogenic oocytes. Plasma SHBG levels were measured over a 10-month period as male sea bass undergo sexual maturation. Immature females of the same age were also studied over the same time interval. The mean+/-S.E.M. plasma SHBG levels in 2-year-old males and females are lower (80+/-15nM and 82+/-16nM, respectively) during the winter reproductive season (December-March) than the spring (April-June) months (144+/-32nM and 193+/-18nM, respectively). In both sexes, plasma SHBG levels start to decline 1-2 months before the reproductive season, coincident with a period of rapid weight gain, while increases after the reproductive season are not accompanied by significant changes in body weight. In addition, plasma SHBG in triploid (sterile) and diploid (fertile) male sea bass do not differ during the first spawning season. These data suggest that the decrease in plasma SHBG levels during sexual maturation in sea bass is related to nutritional or metabolic effects in relation to water temperatures and food intake, rather than changes in gonadal sex steroid production.