Arginine vasotocin V1a2 receptor and GnRH-I co-localize in preoptic neurons of the sex changing grouper, Epinephelus adscensionis.

The arginine vasotocin/vasopressin (AVT/AVP) and gonadotropin releasing hormone (GnRH) systems are known to control sexual behaviors and reproduction, respectively, in different vertebrate groups. However, a direct functional connection between these two neuroendocrine systems has not been demonstrated for any vertebrate species. Therefore, the objective of this research was to test the hypothesis that AVT acts on the GnRH system via an AVT V1a receptor in a sex changing grouper species, the rock hind, Epinephelus adscensionis. AVT V1a2 receptors were co-localized with GnRH-I on neurons in the preoptic anterior hypothalamus identifying a structural linkage between the AVT system and GnRH-I. Transcripts for avt, gnrh-I, and two AVT receptor subtypes (v1a1 and v1a2) were isolated and characterized for E. adscensionis and their expression was measured in males and females by q-RT-PCR. Translation of V1a-type cDNA sequences revealed two distinct forms of the AVT V1a receptor in E. adscensionis brain similar to those reported for other species. The observation of significantly higher gnrh-I mRNA in the POA+H of rock hind males as compared to females suggests differential regulation of the gnrh-I transcripts in the two sexes of this protogynous species. In male E. adscensionis, but not in females, a negative relationship was seen between plasma 11-ketotestosterone (11-KT) and the v1a1 receptor mRNA levels in the POA+H, while a positive trend was observed between 11-KT and v1a2 receptor mRNA levels, indicating that these receptor forms may be differentially regulated.

Distribution and habitat associations of billfish and swordfish larvae across mesoscale features in the Gulf of Mexico.

Ichthyoplankton surveys were conducted in surface waters of the northern Gulf of Mexico (NGoM) over a three-year period (2006-2008) to determine the relative value of this region as early life habitat of sailfish (Istiophorus platypterus), blue marlin (Makaira nigricans), white marlin (Kajikia albida), and swordfish (Xiphias gladius). Sailfish were the dominant billfish collected in summer surveys, and larvae were present at 37.5% of the stations sampled. Blue marlin and white marlin larvae were present at 25.0% and 4.6% of the stations sampled, respectively, while swordfish occurred at 17.2% of the stations. Areas of peak production were detected and maximum density estimates for sailfish (22.09 larvae 1000 m(-2)) were significantly higher than the three other species: blue marlin (9.62 larvae 1000 m(-2)), white marlin (5.44 larvae 1000 m(-2)), and swordfish (4.67 larvae 1000 m(-2)). The distribution and abundance of billfish and swordfish larvae varied spatially and temporally, and several environmental variables (sea surface temperature, salinity, sea surface height, distance to the Loop Current, current velocity, water depth, and Sargassum biomass) were deemed to be influential variables in generalized additive models (GAMs). Mesoscale features in the NGoM affected the distribution and abundance of billfish and swordfish larvae, with densities typically higher in frontal zones or areas proximal to the Loop Current. Habitat suitability of all four species was strongly linked to physicochemical attributes of the water masses they inhabited, and observed abundance was higher in slope waters with lower sea surface temperature and higher salinity. Our results highlight the value of the NGoM as early life habitat of billfishes and swordfish, and represent valuable baseline data for evaluating anthropogenic effects (i.e., Deepwater Horizon oil spill) on the Atlantic billfish and swordfish populations.

The distribution of an AVT V1a receptor in the brain of a sex changing fish, Epinephelus adscensionis.

The present study describes the distribution of an arginine vasotocin (AVT) V1a receptor (AVTr) throughout the brain of a sex-changing grouper, rock hind Epinephelus adscensionis. The objectives of this study were to describe the AVTr distribution in the brain of rock hind for potential linkages of the AVT hormone system with sex-specific behaviors observed in this species and to examine sex-specific differences that might exist. An antibody was designed for rock hind AVTr against the deduced amino acid sequence for the third intracellular loop. Protein expression, identified with immunohistochemistry showed high concordance with mRNA expression, identified with in situ hybridization. AVTr protein and mRNA expression was widely distributed throughout the brain, indicating that AVT may act as a neuromodulator via this V1a receptor subtype. AVTr protein and mRNA were present in regions associated with behavior, reproduction and spatial learning, as well as sensory functions such as vision, olfaction and lateral line sensory processing. We observed high AVTr expression in granular cell formations in the internal cellular layer of olfactory bulbs, torus longitudinalis, granular layer of the corpus cerebellum, valvula of the cerebellum, nuclei of the lateral and posterior recesses, and granular eminence. High protein and mRNA expression was also observed in the preoptic area, anterior hypothalamus, and habenular nucleus. No obvious sex differences were noted in any region of the rock hind brain.

Behavior, color change and time for sexual inversion in the protogynous grouper (Epinephelus adscensionis).

Hermaphroditism, associated with territoriality and dominance behavior, is common in the marine environment. While male sex-specific coloration patterns have been documented in groupers, particularly during the spawning season, few data regarding social structure and the context for these color displays are available. In the present study, we define the social structure and male typical behavior of rock hind (Epinephelus adscensionis) in the wild. In addition, we detail the captive conditions and time period necessary to induce the onset of the sex-specific coloration and sexual change. At six oil production platform locations in the Gulf of Mexico, rock hind social group size and typical male rock hind social behavior were documented. We observed a rapid temporary color display in rock hind that could be turned on and off within three seconds and was used for confronting territory intruders and displays of aggression towards females. The male-specific "tuxedo" pattern consists of a bright yellow tail, a body with alternating dark brown and white patches and a dark bar extending from the upper mandible to the operculum. Identification and size ranges of male, female and intersex fish collected from oil platforms were determined in conjunction with gonadal histology. Rock hind social order is haremic with one dominant male defending a territory and a linear dominance hierarchy among individuals. In five captive experiments, the largest remaining female rock hind displayed the male specific color pattern within 32d after dominant male removal from the social group. To our knowledge, this is the first evidence in a grouper species of color patterning used to display territoriality and dominance outside of spawning aggregations. The behavioral paradigm described here is a key advance that will enable mechanistic studies of this complex sex change process.

The novel finding of four distinct prepro-IGF-I E domains in a perciform fish, Sciaenops ocellatus, during ontogeny.

In fishes, insulin-like growth factor-I (IGF-I) stimulates growth and differentiation but also plays a role in a number of other processes including osmoregulation, metabolism, immune response and reproduction. This study presents the cDNA encoding multiple prepro-IGF-I transcripts obtained from red drum, Sciaenopsocellatus, and examines differential expression in select adult tissues and during ontogeny. Four distinct transcripts were sequenced which were identical in the coding region for the signal (132 bp) and mature (204 bp) peptides but differed in the coding region of the E peptide by the exclusion of 117 (Ea-1), 81 (Ea-2) or 36 (Ea-3) bp compared to the 222 bp present in Ea-4. Analysis of the pertinent portion of the genomic sequence of this gene suggests that the transcripts are a result of alternative splicing. This is the first report of the expression of all four known prepro-IGF-I transcripts in a teleost other than a salmonid. The deduced amino acid sequences exhibited 70-95% identity with teleosts and somewhat lower identity to other vertebrates (60-75%). Three of the 4 transcripts (Ea-2, Ea-3, Ea-4) were expressed in the liver, ovary, spleen, gall bladder, brain, red muscle, pancreas and spinal cord of adults. Only the Ea-4 transcript was expressed in adult stomach tissue while no signal was detected in pituitary, retina, intestine, adipose or white muscle. In contrast, all 4 transcripts were expressed throughout ontogeny. The apparent expression of the Ea-1 transcript only during the larval stage may indicate a developmental role for this E peptide in red drum.

Cholecystokinin: molecular cloning and immunohistochemical localization in the gastrointestinal tract of larval red drum, Sciaenops ocellatus (L.).

The current study sought to clarify the role of cholecystokinin (CCK) in the digestion of larval red drum (Sciaenops ocellatus) in order to better characterize the processes limiting the utilization of microparticulate diets at first feeding. The red drum CCK cDNA, isolated from adult anterior intestine and pyloric caeca, contains a 414 base pair (bp) open reading frame encoding a deduced amino acid sequence of 138 residues which is highly similar to preprocholecystokinin from other vertebrates. The mature CCK octapeptide has the same amino acid sequence as that found in mammals and in Atlantic herring (Clupea harengus). Tissue distribution analysis of adult and juvenile red drum using primers specific for red drum CCK mRNA revealed bright bands in samples from the brain, pyloric caeca, anterior intestine, and gonad with fainter bands seen in all other tissues. Immunohistochemical analysis of larval red drum showed that CCK-immunoreactive (CCK-IR) cells were present as early as 3 days post hatch (DPH) in some fish and were present in all fish by 6 DPH. CCK-IR cells were found in the anterior midgut in early larvae and had spread to the first bend of the gut by day 6. In older larvae (18+ DPH), CCK-IR cells were found in large numbers in the anterior intestine and in the developing pyloric caeca. The sequence and distribution of CCK mRNA along with the presence of CCK-IR cells in early red drum larvae suggest that CCK is present and may be capable of regulating pancreatic secretion in early red drum larvae.

The onset of cortisol synthesis and the stress response is independent of changes in CYP11B or CYP21 mRNA levels in larval red drum (Sciaenops ocellatus).

Although cortisol plays an important role in teleost development, the onset of cortisol production and the cortisol stress response in teleosts remain poorly understood. Here we have reported basal cortisol levels and the development of the cortisol stress response in larval red drum (Sciaenops ocellatus). We isolated partial nucleic acid sequences encoding two key corticosteroidogenic enzymes, CYP11B and CYP21 and assessed ontogenetic patterns of their mRNA levels relative to basal and stress-induced cortisol production. Basal cortisol was first detected 3 days post-hatch (DPH) and reached a maximum at 9 DPH. Cortisol did not increase in response to an acute stressor prior to 6 DPH. From 6 DPH forward, stress caused significant increases in larval cortisol content. Stress-induced cortisol levels in 6-9 DPH larvae were highest 1h post-stress. In larvae 11 DPH and older, the highest cortisol measurements occurred 0.5h post-stress. Elevated cortisol was still evident after 3h in 6 DPH larvae. From 11 DPH onward, basal cortisol levels were reestablished in larvae by 1h post-stress. CYP11B and CYP21 transcripts were detected in red drum 12h prior to hatching and in all post-hatch larvae examined. Changes in CYP11B and CYP21 mRNA levels did not occur in association with the ontogenetic appearance of cortisol, or the onset of the stress response. As larvae developed, the dynamics of the cortisol stress response matured from a low magnitude, slow recovery response, to a response similar to that observed in juvenile and adult fish.

Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia.

The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups.

Interrenal and thyroid development in red drum (Sciaenops ocellatus): effects of nursery environment on larval growth and cortisol concentration during settlement.

Red drum settle into shallow seagrass meadows during the larval stage. Day-night cycles in these habitats result in marked diel temperature and dissolved oxygen (DO) cycles, and it is possible that extreme fluctuations influence endocrine development and growth of larvae. Here, we described red drum interrenal and thyroid ontogeny and determine responses to environmental stimuli with special emphasis on settlement to explore possible role of hormones as mediator of directive environmental factors. This study detected an early activation of thyroid and interrenal axis during the yolk-sac phase and a second activation of the thyroid starting at settlement size to the end of the larval period. Whole-body l-thyroxine (T4) and 3-5-3'-triiodo-l-thyronine (T3) showed a sharp decline at the juvenile stage. In contrast, cortisol steadily declines during the larval phase to a minimum before the end of the larval period. Older settlement-size larvae exposed to a strong stimulus increased whole body cortisol. In contrast, new settlers showed a minor cortisol rise suggesting changes on stress responsiveness during the ontogeny of the species. Additionally, settlement-size larvae exposed to various environmentally realistic temperature or DO fluctuations showed no difference in growth compared to fish grown under stable conditions (control). However, growth rate was significantly reduced in DO cycled fish with prolonged exposure to hypoxia. No differences were found in whole-body cortisol levels in the reduced growth treatment groups, suggesting that growth retardation was not related to a cortisol-mediated stress response. In moderate DO and temperature treatment groups, cortisol showed wider fluctuations than control groups during the night time that were not related to stress.