basic-methods-for-the-study-of-reproductive-ecology-of-fish-in-aquaria

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

Tokyo University of Marine Science and Technology

To investigate the role of estrogen in the serial-sex changing fish Trimma okinawae, we isolated complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms from adult ovary and brain (termed P450aromA and P450aromB, respectively). Sequence and phylogenic analyses showed that the goby P450arom forms belong to two separate CYP19 subfamilies. Transient expression of these cDNAs in HEK293 cells caused conversion of exogenous testosterone to estradiol-17beta. RT-PCR showed that P450aromA was expressed in the brain, spleen, testis and ovary. P450aromB was expressed in the brain, liver, testis and ovary. In situ hybridization studies showed that P450aromA mRNA, but not P450aromB mRNA, was present in both ovary and testis. Positive signals were restricted to granulosa cells of vitellogenic follicles and interstitial cells of mature testis. Ovarian expression of both P450arom genes during the spawning cycle was examined by quantitative real-time RT-PCR. P450aromA transcripts increased during vitellogenesis and decreased prior to spawning. In contrast, P450aromB transcripts were barely detectable and did not correlate with ovarian development. These findings suggest that P450aromA, but not P450aromB, is involved in regulating ovarian vitellogenesis in goby.

Characterization of two types of cytochrome P450 aromatase in the serial-sex changing gobiid fish, Trimma okinawae.

In order to obtain basic information about the role played by endogenous sex hormones in bringing about sex changes in the serial-sex changing gobiid fish Trimma okinawae, the gonadal structure of male and female phases were observed histologically. Steroid-producing cells (SPC; Leydig cells in a testis) were observed ultrastructurally in the ovaries and testes of both female-phase and male-phase fish. In addition, gonadal expression of P450 cholesterol side-chain-cleavage (scc) was examined immunohistochemically. Gonads of fish in female and male phases were observed to have both ovaries and testes simultaneously. Female-phase fish had matured with many developed vitellogenic oocytes, while male-phase individuals had immature ovaries with many numbers of previtellogenic oocytes at the perinucleolus stage. Testes of fish in different sexual phases had active spermatogenic germ cells. Organellae of SPC in the ovaries of female-phase fish had active structures of steroid production. In contrast, SPC in the ovaries of male-phase fish did not show active structures of steroid production. Immunopositive reactions against the scc antibody in the ovaries of female-phase fish were very strong, but immunoreactions in the ovaries of male-phase fish were very weak. In the testis, moderate immunopositive signals were obtained from dual-phase male/females.

Gonadal structure of the serial-sex changing gobiid fish Trimma okinawae.

The role of aromatase (Arom) in the process of bi-directional sex change in the gobiid fish Trimma okinawae was investigated by immunohistochemical methods. Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. In each sexual phase of females, the 2nd (2DF-M) and 4th (4DF-M) days after initiation of sex change to male, males, and the 2nd (2DM-F), 4th (4DM-F) and 6th (6DM-F) days after the initiation of reversion from male to female, ovarian and testicular histological observations were made. During the female, 2DF-M, 4DF-M and 6DM-F phases, the ovary contained vitellogenic and previtellogenic oocytes, compared with previtellogenic oocytes in the other phases. Although sperm was found in the testis in every phase, sperm ducts were apparent in the male phase, but not the female phase. Arom immunoreactivity was detected in the interstitial cells between the oocytes in all phases. On the other hand, it was localized in the thecal and granulosa cells of the follicular layer enclosing the oocytes in the female, 2DF-M, 4DF-M and 6DM-F phases. Activity of Arom in the thecal and granulosa cells is thought to be important for the development of oocytes and subsequent sex change.

Aromatase immunoreactivity and the role of enzymes in steroid pathways for inducing sex change in the hermaphrodite gobiid fish Trimma okinawae.

Involvement of Ad4BP/SF-1 was investigated using the serial sex changing goby Trimma okinawae. First, a cDNA encoding Ad4BP/SF-1 was cloned from ovarian follicles. The open reading frame of goby Ad4BP/SF-1 encodes a protein of 489 amino acids. Ad4BP/SF-1 was expressed in gonadal tissues, brain, and kidney. Second, transcript levels of Ad4BP/SF-1 were measured in the ovary and testis of the same individuals. Expressions were related to sexual phases. Moreover, ovarian expression of Ad4BP/SF-1 increased during vitellogenesis and declined sharply during the post-vitellogenic period. Finally, the expression profile of Ad4BP/SF-1 was measured during serial sex change in the ovary. Ad4BP/SF-1 increased in parallel with the onset of the female-phase and decreased as female became male. Therefore, Ad4BP/SF-1 probably acts as an important indirect regulator of oocyte growth and maturation at female-phase of serial sex changing gobiid fish T. okinawae.

Molecular cloning and expression of Ad4BP/SF-1 in the serial sex changing gobiid fish, Trimma okinawae.

Sex-changing fish Trimma okinawae can change its sex back and forth from male to female and then to male serially, depending on the social status in the harem. T. okinawae is well equipped to respond to its social status by possessing both ovarian and testicular tissues even though only one gonad remains active at one time. Here we investigated the involvement of gonadotropins in sex change by determining the changes in gonadotropin receptor (GtHR) gene expression during the onset of sex change from female to male and male to female. The expression of the GtHR was found to be confined to the active gonad of the corresponding sexual phase. During the sex-change from female to male, initially the ovary had high levels of FSHR and LHR, which eventually went up in the testicular tissue if the fish was bigger. Changing of the gonads started with switching of GtHR expression discernible within 8-12 h of the visual cue. Further in vitro culture of the transitional gonads with a supply of exogenous gonadotropin (human chorionic gonadotropin) revealed that the to-be-active gonad acquired the ability to produce the corresponding sex hormone within 1 d of the activation of GtHR. Conversely, the to-be-regressed gonad did not respond to the exogenous gonadotropin. Our findings show that the gonads of successive sex-changing fish possess the intrinsic mechanism to respond to the social cue differentially. Additionally, this location switching of GtHR expression also could substantiate the importance of the hypothalamo-pituitary-gonadotropic axis.

Sex change in the Gobiid fish is mediated through rapid switching of gonadotropin receptors from ovarian to testicular portion or vice versa.

Despite numerous endocrine studies on sex change in teleost, no general mechanism that mediates sex change has emerged. The gobiid fish, Trimma okinawae, can change sex in both directions repeatedly. This phenomenon of sex change in goby assigns it as an excellent animal model to elucidate the understanding mechanisms of sex change. In hermaphrodite fishes, estrogen plays a particularly important role in natural and experimentally induced sex change. To investigate the role of estrogen in the serial-sex changing fish T. okinawae, we cloned and analyzed the 5'-flanking regions of P450arom genes from goby genome DNA. Both regions have consensus sequences of TATA, CRE and ERE. Ad4 binding site was restricted in the region of P450aromA. These findings indicate that different regulators control the expression of the two P450arom genes.

Promoter analysis of two aromatase genes in the serial-sex changing gobiid fish, Trimma okinawae.

The gobiid fish Trimma okinawae changes its sex bi-directionally according to its social status. Morphological changes in the urinogenital papillae (UGP) of this fish have been reported during sex change. However, there have been no detailed observations of such changes. Here, we histologically examined the UGP structure of male- and female-phase fish. UGPs of fish in female and male phase contained both oviducts and sperm ducts. Both ducts were coalesced into one duct within the posterior region of the UGP. Female-phase fish had many longitudinal folds in the hypertrophied tunica mucosa of the oviduct, which was found to be responsible for the transport of eggs and the removal of follicular cells from the oocyte. In contrast, male-phase fish had an immature oviduct and a mature sperm duct in the UGP. In the male-phase fish, the co-existence of spermatozoa and fibrillar secretions was observed in the sperm duct during spermiation.

Histological observation of the urogenital papillae in the bi-directional sex-changing gobiid fish, Trimma okinawae.

In this study, we measured the accumulation of tributyltin (TBT) in wharf roach (Ligia exotica Roux) and examined the species' ability to be used for TBT biomonitoring in coastal environments. In an exposure test, wharf roach were exposed to TBT via diet for 2d. TBT was accumulated in wharf roach, and its metabolite dibutyltin was detected. The concentrations of these compounds gradually decreased during the depuration period, but they were still detected 12d after exposure ceased (TBT 290±140ng/g; dibutyltin 1280±430ng/g). The biological half-life of TBT in wharf roach was estimated to be about 4d. In a field study conducted in 2011-2012, wharf roach were collected from 15 coastal sites in Japan and 3 sites in Manado, Indonesia. TBT was detected in both Japanese and Indonesian samples. The highest concentration of TBT was found in wharf roach collected at Bitung ferry port, Manado (57.9±16.5ng/g), which is close to a shipyard, and the highest concentration at a Japanese site was 12.3±6.2ng/g. Thus, we were able to detect organotins in the coastal environments by testing wharf roach, suggesting that L. exotica might serve as a good bioindicator for monitoring organotin pollution.

Accumulation of organotins in wharf roach (Ligia exotica Roux) and its ability to serve as a biomonitoring species for coastal pollution.

Size-advantage and low-density models have been used to explain how mating systems favor hermaphroditism or gonochorism. However, these models do not indicate historical transitions in sexuality. Here, we investigate the evolution of bidirectional sex change and gonochorism by phylogenetic analysis using the mitochondrial gene of the gobiids Trimma (31 species), Priolepis (eight species), and Trimmatom (two species). Trimma and Priolepis formed a clade within the sister group Trimmatom. Gonadal histology and rearing experiments revealed that Trimma marinae, Trimma nasa, and Trimmatom spp. were gonochoric, whereas all other Trimma and Priolepis spp. were bidirectional sex changers or inferred ones. A maximum-likelihood reconstruction analysis demonstrated that the common ancestor of the three genera was gonochoristic. Bidirectional sex change probably evolved from gonochorism in a common ancestor of Trimma and Priolepis. As the gonads of bidirectional sex changers simultaneously contain mature ovarian and immature testicular components or vice versa, individuals are always potentially capable of functioning as females or males, respectively. Monogamy under low-density conditions may have been the ecological condition for the evolution of bidirectional sex change in a common ancestor. As T. marinae and T. nasa are a monophyletic group, gonochorism should have evolved from bidirectional sex change in a common ancestor.

Tomoki Sunobe

Laboratory of Fish Behavioral Ecology,
Tateyama Station,
Field Science Center,
Laboratory of Fish Behavioral Ecology, Tateyama Station, Field Science Center

Tokyo University of Marine Science and Technology

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Tomoki Sunobe

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Tokyo University of Marine Science and Technology

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Laboratory of Fish Behavioral Ecology,
Tateyama Station,
Field Science Center,
Laboratory of Fish Behavioral Ecology, Tateyama Station, Field Science Center