Sex reversal of pejerrey (Odontesthes bonariensis), a species with temperature-dependent sex determination, in a seminatural environment.

This study examined the changes in sex ratios and sex reversal rates in pejerrey Odontesthes bonariensis that occur with the progression of the spawning season in a seminatural setting. Four groups of hatchery-produced pejerrey larvae were stocked in floating cages in La Salada de Monasterio lake (Pampas region), a natural habitat of this species, and reared from hatching beyond gonadal sex determination with minimum human interference. Cage 1 was stocked at the beginning of the spring spawning season and the other cages were stocked with monthly delays until cage 4 in early summer. The genotypic (amhy+, XY/YY; amhy-, XX) and phenotypic (testis, male; ovary, female) sex ratios and proportions of genotype/phenotype mismatched individuals were estimated and their relation to water temperature and daylength during the experiment was analysed by generalized linear modelling. Water temperature varied between 11 and 30.5°C, and daylength duration between 11 h 22 min and 14 h 35 min. Sex genotyping revealed nearly balanced sex ratios of XY/YY (46%-49.1%) and XX (50.9%-54%) fish in cages 2-4 whereas the genotypic sex ratio in cage 1 was clearly biased towards XY/YY fish (60.6%). Phenotypic males ranged from 42% to 54.4% in cages 1-3. Cage 4, in turn, had significantly more phenotypic males (66%). The percentage of XX males (phenotypic male/genotypic female) was 23.1% in cage 1, decreased to a minimum of 5.4% in cage 2 and gradually increased in cages 3 and 4 to a maximum of 40.7% in the latter. The percentages of XY/YY females (phenotypic female/genotypic male) were highest in cage 1 (30%) and decreased progressively in the other cages to a significantly lower value (4.3%) in cage 4. These results generally support the findings of laboratory studies on the effect of temperature on the sex determination of this species and also provide novel evidence of a XX genotype-specific masculinizing effect of short daylength.

Phenotypic/genotypic sex mismatches and temperature-dependent sex determination in a wild population of an Old World atherinid, the cobaltcap silverside Hypoatherina tsurugae.

Several New World atheriniforms have been recognized as temperature-dependent sex determined (TSD) and yet possess a genotypic sex determinant (amhy) which is primarily functional at mid-range temperatures. In contrast, little is known about the sex determination in Old World atheriniforms, even though such knowledge is crucial to understand the evolution of sex determination mechanisms in fishes and to model the effects of global warming and climate change on their populations. This study examined the effects of water temperature on sex determination of an Old World atheriniform, the cobaltcap silverside Hypoatherina tsurugae, in which we recently described an amhy homologue. We first assessed the occurrence of phenotypic/genotypic sex mismatches in wild specimens from Tokyo Bay for three years (2014-2016) and used otolith analysis to estimate their birth dates and approximate thermal history during the presumptive period of sex determination. Phenotypic sex ratios became progressively biased towards males (47.3%-78.2%) during the period and were associated with year-to-year increases in the frequency of XX-males (7.3%-52.0%) and decreases in XY/YY-females (14.5%-0%). The breeding season had similar length but was delayed by about 1 month per year between 2014 and 2016, causing larvae to experience higher temperatures during the period of sex determination from year to year. Larval rearing experiments confirmed increased likelihood of feminization and masculinization at low and high temperatures, respectively. The results suggest that cobaltcap silverside has TSD, or more specifically the coexistence of genotypic and environmental sex determinants, and that it affects sex ratios in wild populations.

Crowding stress during the period of sex determination causes masculinization in pejerrey Odontesthes bonariensis, a fish with temperature-dependent sex determination.

The pejerrey is an atherinopsid species from South America that presents a combination of genotypic and environmental (temperature-dependent) sex determination whereby low and high temperatures induce feminization and masculinization, respectively. Masculinization involves a heat-induced stress response leading to increased circulating cortisol and androgens. We tested whether crowding would elicit a similar response as high temperature and affect the sex ratios of pejerrey. Larvae with XX and XY genotypes were reared at 15, 62 and 250 larvae/L in 0.4, 1.6, and 6.4 L containers during a period considered critical for sex determination at 25 °C, a mixed-sex promoting temperature. Fish were analysed at 3-7 weeks for whole-body cortisol and 11-ketotestosterone (11-KT) titer and hydroxy-steroid dehydrogenase (hsd11b2) mRNA transcript abundance, and after completion of gonadal sex differentiation (10-14 weeks) for determination of phenotypic and genotypic sex mismatches. Crowding was associated with depressed growth, higher cortisol and 11-KT titers, increased hsd11b2 transcription, and increased frequency of masculinization compared to intermediate and/or low rearing densities. Perceived crowding (by rearing in containers with mirror-finish, reflecting walls) also caused masculinization. These results suggest the possibility that other environmental factors besides temperature can also affect sex determination in pejerrey and that a stress response leading to increased cortisol and androgen levels, which is potentially perceived by the brain, may be a common feature among different forms of environmental sex determination in this species.

Evidence of a landlocked reproducing population of the marine pejerrey Odontesthes argentinensis (Actinopterygii; Atherinopsidae).

In South America, the order Atheriniformes includes the monophyletic genus Odontesthes with 20 species that inhabit freshwater, estuarine and coastal environments. Pejerrey Odontesthes argentinensis is widely distributed in coastal and estuarine areas of the Atlantic Ocean and is known to foray into estuaries of river systems, particularly in conditions of elevated salinity. However, to our knowledge, a landlocked self-sustaining population has never been recorded. In this study, we examined the pejerrey population of Salada de Pedro Luro Lake (south-east of Buenos Aires Province, Argentina) to clarify its taxonomic identity. An integrative taxonomic analysis based on traditional meristic, landmark-based morphometrics and genetic techniques suggests that the Salada de Pedro Luro pejerrey population represents a novel case of physiological and morphological adaptation of a marine pejerrey species to a landlocked environment and emphasises the environmental plasticity of this group of fishes.

Expression profiles of amhy and major sex-related genes during gonadal sex differentiation and their relation with genotypic and temperature-dependent sex determination in pejerrey Odontesthes bonariensis.

To shed light on the mechanisms of and interactions of GSD and TSD in pejerrey, we investigated how the transcriptional profiles of amhy and amha are affected by feminizing (17 °C) and masculinizing (29 °C) temperatures during the critical period of sex determination/differentiation and their relation with the expression profiles of AMH receptor type II (amhrII), gonadal aromatase (cyp19a1a), and 11 beta-hydroxysteroid dehydrogenase 2 (hsd11b2). Careful consideration of the results of this study and all information currently available for this species, including similar analyzes for an intermediate, mixed-sex promoting temperature (25 °C), suggests a model for genotypic/temperature-dependent sex determination and gonadal sex differentiation that involves a) cyp19a1a-dependent, developmentally-programmed ovarian development as the default state that becomes self-sustaining in the absence of a potent and timely masculinizing stimulus, b) early, developmentally-programmed amhy expression and high temperature as masculinization signals that antagonize the putative female pathway by suppressing cyp19a1a expression, c) increasing stress response, cortisol, and the synthesis of the masculinizing androgen 11-keto-testosterone via hsd11b2 with increasing temperature that is important for masculinization in both genotypes but particularly so in XX individuals, and d) an endocrine network with positive/negative feedback mechanisms that ensure fidelity of the male/female pathway once started. The proposed model, albeit tentative and non-all inclusive, accounts for the continuum of responses, from all-females at low temperatures to all-males at high temperatures and for the balanced-, genotype-linked sex ratios obtained at intermediate temperatures, and therefore supports the coexistence of TSD and GSD in pejerrey across the range of viable temperatures for this species.

Optimization of protocols for microinjection-based delivery of cryoprotective agents into Japanese whiting Sillago japonica embryos.

Microinjection has proven useful for introduction of low-permeability cryoprotective agents (CPAs) into fish eggs or embryos for cryopreservation. In this work, we examined the suitable conditions for single or combined microinjection into the perivitelline space (PS) and the yolk mass (YM) of embryos of the Japanese whiting, an alternative marine fish model for embryo cryopreservation studies. The parameters examined were injection volume, CPA type and concentration, vehicle (diluent), and suitable developmental stage. Somites and tail elongation embryos tolerated single or combined injection with 2.1 and 15.6 nl in the PS and YM, respectively, whereas earlier embryonic stages tolerated only up to 8.2 nl in the YM. The injected solutions diffused rapidly throughout the PS and YM and remained contained within each compartment unless in the case of structural damage caused by injection of larger volumes. Yamamoto solution was marginally better as a vehicle for microinjection of CPAs than fish Ringer and phosphate buffer saline whereas » artificial sea water was clearly unsuitable. Ethylene glycol was well tolerated by embryos in all developmental stages whereas 1, 2-propylene glycol was suitable only for early embryonic stages. Overall, microinjection was efficient in delivering high loads of CPAs inside whiting embryos more swiftly than previously obtained for this species by immersion-based impregnation protocols. Embryos microinjected with CPAs showed a decrease in embryo nucleation temperature and an increase in chilling tolerance. CPA-microinjected embryos will provide valuable materials to optimize the remaining parameters that are critical for successful cryopreservation such as cooling and warming strategies.

Alterations in gene expression during fasting-induced atresia of early secondary ovarian follicles of coho salmon, Oncorhynchus kisutch.

Molecular processes that either regulate ovarian atresia or are consequences of atresia are poorly understood in teleost fishes. We hypothesized that feed restriction that perturbs normal ovarian growth and induces follicular atresia would alter ovarian gene expression patterns. Previtellogenic, two-year old coho salmon (Oncorhynchus kisutch) were subjected to prolonged fasting to induce atresia or maintained on a normal feeding schedule that would promote continued ovarian development. To identify genes that were specifically up- or down-regulated during oocyte growth in healthy, growing fish compared to fasted fish, reciprocal suppression subtractive hybridization (SSH) cDNA libraries were generated using ovaries from fed and fasted animals. Differential expression of genes identified by SSH was confirmed with quantitative PCR. The SSH library representing genes elevated in ovaries of fed fish relative to those of fasted fish contained steroidogenesis-related genes (e.g., hydroxy-delta-5-steroid dehydrogenase), Tgf-beta superfamily members (e.g., anti-Mullerian hormone) and cytoskeletal intermediate filament proteins (e.g., type I keratin s8). Overall, these genes were associated with steroid production, cell proliferation and differentiation, and ovarian epithelialization. The library representing genes elevated in ovaries of fasted fish relative to fed fish contained genes associated with apoptosis (e.g., programmed cell death protein 4), cortical alveoli (e.g., alveolin), the zona pellucida (e.g., zona pellucida protein c), and microtubules (e.g., microtubule associated protein tau). Elevated expression of this suite of genes was likely associated with the initiation of atresia and/or a reduced rate of follicle development in response to fasting. This study revealed ovarian genes involved in normal early secondary oocyte growth and potential early markers of atresia.

Diversity and Convergence of Sex-Determination Mechanisms in Teleost Fish.

Sexual reproduction is prevalent across diverse taxa. However, sex-determination mechanisms are so diverse that even closely related species often differ in sex-determination systems. Teleost fish is a taxonomic group with frequent turnovers of sex-determining mechanisms and thus provides us with great opportunities to investigate the molecular and evolutionary mechanisms underlying the turnover of sex-determining systems. Here, we compile recent studies on the diversity of sex-determination mechanisms in fish. We demonstrate that genes in the TGF-ß signaling pathway are frequently used for master sex-determining (MSD) genes. MSD genes arise via two main mechanisms, duplication-and-transposition and allelic mutations, with a few exceptions. We also demonstrate that temperature influences sex determination in many fish species, even those with sex chromosomes, with higher temperatures inducing differentiation into males in most cases. Finally, we review theoretical models for the turnover of sex-determining mechanisms and discuss what questions remain elusive.

Fish reproduction in a warming world: vulnerable points in hormone regulation from sex determination to spawning.

Reproduction in fishes is sensitive to temperature. Elevated temperatures and anomalous 'heat waves' associated with climate change have the potential to impact fish reproductive performance and, in some cases, even induce sex reversals. Here we examine how thermal sensitivity in the hormone pathways regulating reproduction provides a framework for understanding impacts of warmer conditions on fish reproduction. Such effects will differ depending on evolved variation in temperature sensitivity of endocrine pathways regulating reproductive processes of sex determination/differentiation, gametogenesis and spawning, as well as how developmental timing of those processes varies with reproductive ecology. For fish populations unable to shift geographical range, persistence under future climates may require changes in temperature responsiveness of the hormone pathways regulating reproductive processes. How thermal sensitivity in those hormone pathways varies among populations and species, how those pathways generate temperature maxima for reproduction, and how rapidly reproductive thermal tolerances can change via adaptation or transgenerational plasticity will shape which fishes are most at risk for impaired reproduction under rising temperatures. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Temperature- and genotype-dependent stress response and activation of the hypothalamus-pituitary-interrenal axis during temperature-induced sex reversal in pejerrey Odontesthes bonariensis, a species with genotypic and environmental sex determination.

In the pejerrey Odontesthes bonariensis (Atheriniformes, Atherinopsidae), exposure to high and low temperatures during the critical period of sex determination (CPSD) induce testicular and ovarian differentiation, respectively, regardless of the presence or not of the sex determining gene amhy, which is crucial for testis formation only at intermediate, sexually neutral temperatures. In this study we explored the existence of genotype-specific signaling of Crh (Corticotropin Releasing Hormone) family genes and their associated carrier protein, receptors, and other stress-related genes in response to temperature during the CPSD and the potential involvement of the central nervous system via the hypothalamus-pituitary-interrenal (HPI) axis in the sex determination of this species. The Crh family genes crhb, uts1, ucn3, the receptor crhr1 and the stress-related genes gr1, gr2, nr3c2 were transiently upregulated in the heads of pejerrey larvae during the CPSD by high temperature alone or in combination with other factors. Only crhr2 transcript abundance was not influenced by temperature but independently by time and genotype. In most cases, mRNA abundance was higher in the XX heads compared to that of XY individuals. The mRNAs of some of these genes were localized in the hypothalamus of pejerrey larvae during the CPSD. XX larvae also showed higher whole-body cortisol titers than the XY, downregulation of cyp19a1a and upregulation of the testis-related genes amhy/amha in trunks (gonads) and were 100% masculinized at the high temperature. In contrast, at the low temperature, crhbp and avt were upregulated in the heads, particularly the former in XY larvae. cyp19a1a and amhy/amha were up- and downregulated, respectively, in the gonads, and fish were 100% feminized. Signaling via the HPI axis was observed simultaneously with the first molecular signs of ongoing sex determination/differentiation in the gonads. Overall, the results strongly suggest a temperature-dependent, genotype-specific regulatory action of the brain involving the Crh family of stress-related genes on the process of environmental sex determination of pejerrey.

The spatiotemporal expression of multiple coho salmon ovarian connexin genes and their hormonal regulation in vitro during oogenesis.

BACKGROUND: Throughout oogenesis, cell-cell communication via gap junctions (GJs) between oocytes and surrounding follicle cells (theca and granulosa cells), and/or amongst follicle cells is required for successful follicular development. To gain a fundamental understanding of ovarian GJs in teleosts, gene transcripts encoding GJ proteins, connexins (cx), were identified in the coho salmon, Oncorhynchus kisutch, ovary. The spatiotemporal expression of four ovarian cx transcripts was assessed, as well as their potential regulation by follicle-stimulating hormone (FSH), luteinizing hormone (LH) and insulin-like growth factor 1 (IGF1). METHODS: Salmonid ovarian transcriptomes were mined for cx genes. Four gene transcripts designated cx30.9, cx34.3, cx43.2, and cx44.9 were identified. Changes in gene expression across major stages of oogenesis were determined with real-time, quantitative RT-PCR (qPCR) and cx transcripts were localized to specific ovary cell-types by in situ hybridization. Further, salmon ovarian follicles were cultured with various concentrations of FSH, LH and IGF1 and effects of each hormone on cx gene expression were determined by qPCR. RESULTS: Transcripts for cx30.9 and cx44.9 were highly expressed at the perinucleolus (PN)-stage and decreased thereafter. In contrast, transcripts for cx34.3 and cx43.2 were low at the PN-stage and increased during later stages of oogenesis, peaking at the mid vitellogenic (VIT)-stage and maturing (MAT)-stage, respectively. In situ hybridization revealed that transcripts for cx34.3 were only detected in granulosa cells, but other cx transcripts were detected in both oocytes and follicle cells. Transcripts for cx30.9 and cx44.9 were down-regulated by FSH and IGF1 at the lipid droplet (LD)-stage, whereas transcripts for cx34.3 were up-regulated by FSH and IGF1 at the LD-stage, and LH and IGF1 at the late VIT-stage. Transcripts for cx43.2 were down-regulated by IGF1 at the late VIT-stage and showed no response to gonadotropins. CONCLUSION: Our findings demonstrate the presence and hormonal regulation of four different cx transcripts in the salmon ovary. Differences in the spatiotemporal expression profile and hormonal regulation of these cx transcripts likely relate to their different roles during ovarian follicle differentiation and development.

Disruption of the salmon reproductive endocrine axis through prolonged nutritional stress: changes in circulating hormone levels and transcripts for ovarian genes involved in steroidogenesis and apoptosis.

Mechanisms regulating the normal progression of ovarian follicular growth versus onset of atresia in fishes are poorly understood. To gain a better understanding of these processes, we exposed immature female coho salmon (Oncorhynchus kisutch) to prolonged fasting to induce follicular atresia and monitored body growth, development of the ovarian follicles, changes in reproductive hormones, and transcripts for ovarian genes. Prolonged fasting reduced body and ovary weight and increased the appearance of atretic follicles relative to normally fed controls. Endocrine analyses showed that fasting reduced plasma insulin-like growth factor 1 (IGF1), estradiol-17ß (E2), and pituitary, but not plasma, levels of follicle-stimulating hormone (FSH). Transcripts for ovarian fsh receptor (fshr) and steroidogenesis-related genes, such as steroidogenic acute regulatory protein (star), 3ß-hydroxysteroid dehydrogenase (hsd3b), and P450 aromatase (cyp19a1a) were significantly lower in fasted fish. Ovarian expression of apoptosis-related genes, such as Fas-associated death domain (fadd), caspase 8 (casp8), caspase 3 (casp3), and caspase 9 (casp9) were significantly elevated in fasted fish compared to fed fish, indicating that apoptosis is involved in the process of atresia in this species. Interestingly, some genes such as fadd, casp8, casp3, and hsd3b, were differentially expressed prior to increases in the number of atretic follicles and reductions in hormone levels induced by fasting, and may therefore have potential as early indicators of atresia. Together these results suggest that prolonged nutritional stress may disrupt the reproductive system and induce follicular atresia in part via reductions in ovarian IGF and FSH signaling, and downstream effects on steroidogenesis-related genes and E2 production.

Where the Ends Meet: An Overview of Sex Determination in Atheriniform Fishes.

Atheriniform fishes have recently emerged as attractive models for evolutionary, ecological, and molecular/physiological studies on sex determination. Many species in this group have marked temperature-dependent sex determination (TSD) and yet many species also have a sex determinant gene that provides a strong drive for male differentiation. Thus, in these species the 2 forms of sex determination that were once considered to be mutually exclusive, environmental (ESD) and genotypic (GSD) sex determination, can coexist at environmentally relevant conditions. Here, we review the current knowledge on sex determination in atheriniform fishes with emphasis on the molecular and physiological mechanisms of ESD and GSD, the coexistence and cross-talk between these 2 mechanisms, the possibility of extragonadal transduction of environmental information and/or extragonadal onset of sex determination, and the results of field studies applying novel tools such as otolith increment analysis and molecular markers of genetic sex developed for selected New World and Old World atheriniform species. We also discuss the existence of molecular and histological mechanisms to prevent the discrepant differentiation in parts of the gonads because of ambiguous or conflicting environmental and genetic signals and particularly the possibility that the female is the default state in these species.

Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences.

Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this 'double-duplex invasion', a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the alpha-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G-C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.

Flatfishes colonised freshwater environments by acquisition of various DHA biosynthetic pathways.

The colonisation of freshwater environments by marine fishes has historically been considered a result of adaptation to low osmolality. However, most marine fishes cannot synthesise the physiologically indispensable fatty acid, docosahexaenoic acid (DHA), due to incomplete DHA biosynthetic pathways, which must be adapted to survive in freshwater environments where DHA is poor relative to marine environments. By analysing DHA biosynthetic pathways of one marine and three freshwater-dependent species from the flatfish family Achiridae, we revealed that functions of fatty acid metabolising enzymes have uniquely and independently evolved by multi-functionalisation or neofunctionalisation in each freshwater species, such that every functional combination of the enzymes has converged to generate complete and functional DHA biosynthetic pathways. Our results demonstrate the elaborate patchwork of fatty acid metabolism and the importance of acquiring DHA biosynthetic function in order for fish to cross the nutritional barrier at the mouth of rivers and colonise freshwater environments.

Origin of high fidelity in target-sequence recognition by PNA-Ce(IV)/EDTA combinations as site-selective DNA cutters.

Double-duplex invasion of pseudocomplementary peptide nucleic acid (pcPNA) is one of the most important strategies for recognizing a specific site in double-stranded DNA (Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11804-11808). This strategy has recently been used to develop artificial restriction DNA cutters (ARCUTs) for site-selective scission of double-stranded DNA, in which a hot spot formed by double-duplex invasion of PNA was hydrolyzed by Ce(IV)/EDTA (Nat. Protoc. 2008, 3, 655-662). The present paper shows how and where the target sequence in double-stranded DNA is recognized by the PNA-Ce(IV)/EDTA combinations for site-selective scission. The mismatch-recognizing activities in both the invasion process and the whole scission process are evaluated. When both pcPNA additives are completely complementary to each strand of the DNA, site-selective scission is the most efficient, as expected. Upon exchange of one DNA base pair at the invasion site with another base pair, which introduces mismatches between the pcPNAs and the DNA, the site-selective scission by the ARCUT is notably diminished. Mismatches in (or near) the central double-invasion region are especially fatal, showing that Watson-Crick pairings of the DNA bases in this region with the pcPNA strands are essential for precise recognition of the target sequence. Both gel-shift assays and melting temperature measurements on the double-duplex invasion process have confirmed that the fidelity in this process primarily governs the fidelity of the DNA scission. According to these systematic analyses, the typical ARCUT involving two 15-mer pcPNAs precisely recognizes 14-16 base pairs in substrate DNA. This remarkable fidelity is accomplished at rather high salt concentrations that are similar to the values in cells.

Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA.

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000 bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.

Environmental regulation of sex determination in fishes: Insights from Atheriniformes.

Sex determination is the first step toward the establishment of phenotypic sex in most vertebrates. Aquatic poikilotherms such as teleost fishes exhibit a high diversity of sex-determination mechanisms and gonadal phenotypes that are remarkably plastic and responsive to a variety of environmental factors (e.g., water temperature, pH, salinity, photoperiod, population density). This chapter reviews current knowledge of genotypic and environmental sex determination systems in fishes with special reference to Atheriniformes-one of the best-characterized taxa in this field-and offers perspectives to guide and stimulate further research.

Spatiotemporal Correlations between amh and cyp19a1a Transcript Expression and Apoptosis during Gonadal Sex Differentiation of Pejerrey, Odontesthes bonariensis.

Sex determination in pejerrey is genetically prescribed by the Y chromosome-linked anti-müllerian hormone amhy but is also strongly influenced by water temperature during the critical period of sex determination. Its gonadal differentiation is characterized by a cephalocaudal and left-to-right histological gradient in both sexes that presumably helps prevent discrepant intersex development in different regions of the gonads in response to ambiguous thermal and genetic stimuli, but the relation of this gradient to molecular processes of sex differentiation is unknown. In this study, we investigated the spatiotemporal expression patterns of amh, gonadal aromatase (cyp19a1a), and apoptosis in relation to the histological gradient in ovaries and testes at an intermediate, sexually neutral temperature. The location and timing of expression of amh, cyp19a1a, and apoptosis seemed to be highly coordinated with the time of gonadal sex differentiation and the histological gradient of gonadal sex differentiation. Apoptosis occurred predominantly in the anterior region of the right gonads and is surmised to be a process to delay differentiation in this area compared to the left gonad, possibly as a means to ensure uniform development in both gonads. Aromatase expression early during development was noted even in putative XY males, supporting the notion of primacy of female development in pejerrey gonads. Thus, apoptosis may be particularly important to prevent discrepant gonadal differentiation in XY individuals where genetic pro-male (amhy), pro-female (cyp19a1a), and thermal stimuli may antagonize.

The Duplicated Y-specific amhy Gene Is Conserved and Linked to Maleness in Silversides of the Genus Odontesthes.

Sex-determining genes have been successively isolated in several teleosts. In Odontesthes hatcheri and O. bonariensis, the amhy gene has been identified as a master sex-determining gene. However, whether this gene is conserved along related species is still unknown. In this study, the presence of amhy and its association with phenotypic sex was analyzed in 10 species of Odontesthes genus. The primer sets from O. hatcheri that amplify both amhs successfully generated fragments that correspond to amha and amhy in all species. The full sequences of amhy and amha isolated for four key species revealed higher identity values among presumptive amhy, including the 0.5 Kbp insertion in the third intron and amhy-specific insertions/deletions. Amha was present in all specimens, regardless of species and sex, whereas amhy was amplified in most but not all phenotypic males. Complete association between amhy-homologue with maleness was found in O. argentinensis, O. incisa, O. mauleanum, O. perugiae, O. piquava, O. regia, and O. smitti, whereas O. humensis, O. mirinensis, and O. nigricans showed varied degrees of phenotypic/genotypic sex mismatch. The conservation of amhy gene in Odontesthes provide an interesting framework to study the evolution and the ecological interactions of genotypic and environmental sex determination in this group.