Pejerrey Odontesthes Bonariensis Research Articles

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.

Gonadal development in pejerrey (Odontesthes bonariensis) during spawning season in relation with sex steroids and temperature variation in Gómez lake (Pampas region, Argentina).

Gómez lake (34 ° 39 'S 61 ° 01' W) is a typical shallow lake of Pampas region placed in the upper area of the Salado river. The most abundant fish species in this lake is the pejerrey (Odontesthes bonariensis) valued due to the quality of its flesh and its attractiveness as a game fish. The aim of this study was to describe for the first time in this pejerrey wild population the gonadal stages during three consecutive spawning seasons (August to December) in relation with sexual steroids and temperature in this lake. In general, pejerrey gonadal development, the gonadosomatic index and the plasma levels of estradiol and testosterone varied in relation to air temperature. During the sampling period, pejerrey females started to ovulate in early August (winter), with a peak in October and ending in December with some of them with atretic oocytes. For males, it was possible to find spermiating animals during the whole spawning season and some arrested animals only in December. Our results confirm the relationship with pejerrey maturation and temperature and can be useful for decision making in the management of this natural resource.

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.

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.

Pituitary-thyroid axis development during the larval-juvenile transition in the pejerrey Odontesthes bonariensis.

The morphological development of the thyroid gland of pejerrey Odontesthes bonariensis during larval-juvenile transition was studied and related to whole-body concentrations of thyroxine (T4 ) and tri-iodothyronine (T3 ). A complementary (c)DNA fragment of the thyroid-stimulating hormone β-subunit (tshb) was sequenced and transcript levels quantified during this period. Follicles with eosinophilic and T4 -immunoreactive colloids were detected at hatching together with tshb transcript levels and whole-body concentrations of T4 and T3 hormones. Thyroid follicles were located in the subpharyngeal region associated with the ventral aorta below the hyoid bone. Follicle structure switched from the rounded form at hatching to oval in juveniles. Significant increase of follicle number per larva, mean colloidal area and total colloidal area was observed throughout development with maximum values at the end of the larval-juvenile transition. A significant decrease of tshb expression together with a significant increase in T4 and T3 whole-body concentrations was observed prior to achieving the juvenile phenotype. These results are in accordance with a negative feedback regulation of tshb expression by thyroid hormones and a possible association between thyroid hormone levels and the acceleration of metabolic processes necessary to complete metamorphosis.

Growth hormone receptors in the atherinid Odontesthes bonariensis: characterization and expression profile after fasting-refeeding and growth hormone administration.

In order to improve the understanding of pejerrey Odontesthes bonariensis, growth hormone (Gh)-insulin-like growth factor-1(Igf1) axis, O. bonariensis growth hormone receptor type 1 (ghr1) and type 2 (ghr2) mRNA sequences were obtained. Both transcripts were ubiquitously expressed except in kidney, encephalon and anterior intestine. Alternative transcripts of both receptors were found in muscle. Interestingly, two different ghr2 transcripts with alternative polyadenylation (APA) sites located in the long 3' untranslated region (UTR-APA) were also found in liver. Hepatic ghr1, ghr2 and insulin-like growth factor type 1 (igf1) transcript levels were examined under two different metabolic conditions. In the first experimental condition, fish were fasted for 2 weeks and then re-fed for another 2 weeks. Despite igf1 mRNA relative expression did not show significant differences under the experimental period of time examined, both ghr transcripts decreased their expression levels after the fasting period and returned to their control levels after re-feeding. In the second treatment, recombinant O. bonariensis growth hormone (r-pjGh) was orally administered once a week. After 4 weeks of treatment, liver igf1, ghr1 and ghr2 mRNA relative expression increased (13, 4·5 and 2·1 fold, P < 0·05) compared to control values. These results add novel information to the growth hormone-insulin-like growth factor system in teleosts.

Influence of pejerreyOdontesthes bonariensis(Valenciennes, 1835) broodstock age on gamete quality, reproductive performance and plasma sex steroid levels during the spawning season

This study evaluated the effect of pejerrey Odontesthes bonariensis (Valenciennes, 1835) broodstock age on gamete and spawning quality and its relationship with sex steroid plasma levels. Sperm was analysed using a computer-assisted sperm analysis (CASA) developed for pejerrey. Semen samples were taken from all fish from mid age groups (5- and 7-year fish), but it was not possible to obtain stripped semen samples from all fish of younger (3-year) or older (10-year) groups. The highest relative sperm weight and sperm concentration were recorded in 5- and 7-year groups respectively, while viability was not different between age groups. It was not possible to identify an effect of age on sperm quality by CASA. Non-viable spawned eggs were obtained in the 3-year group and, the relative fecundity and the relative number of fertilized eggs decreased with age in other groups. Fertilization and hatching rates showed the highest values in the 10-year group. However, the estimated number of hatched larvae was similar in 5-, 7- and 10-year fish. A clear effect of age on 11 ketotestosterone (11-KT) and estradiol (E2) levels was identified, with the highest values in the 5- and 7-year groups. These results might be related to the onset of puberty in the 3-year group and ageing in the 10-year group. Considering that the estimated number of larvae obtained was similar among age groups, the results of this study suggest that broodstock maintenance cost could be reduced by using mid-age fish rather than older fish in pejerrey hatcheries.

Surrogate production of eggs and sperm by intrapapillary transplantation of germ cells in cytoablated adult fish.

Germ cell transplantation (GCT) is a promising assisted reproductive technology for the conservation and propagation of endangered and valuable genetic resources. In teleost fish, GCT in adult gonads has been achieved only in male recipients, limiting greatly the usefulness of this technique in situations where both sexes need equal and timely attention for conservation and/or propagation. Here we describe a simplified GCT approach that ultimately leads to production of donor-derived eggs and sperm in considerably short time. Donor germ cells isolated from young pejerrey Odontesthes bonariensis (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey O. hatcheri recipients whose gonads have been depleted of endogenous GCs by heat (26°C) and chemical treatment (four doses of Busulfan at 30 mg/kg and 40 mg/kg for females and males, respectively). Transplanted spermatogonial and oogonial cells were able to recolonize the recipients' gonads and produce functional donor origin eggs and sperm within 7 months from the GCT. We confirmed the presence of donor-derived gametes by PCR in 17% and 5% of the surrogate O. hatcheri fathers and mothers, respectively. The crosses between surrogate fathers and O. bonariensis mothers yielded 12.6–39.7% pure O. bonariensis and that between a surrogate mother and an O. bonariensis father yielded 52.2% pure O. bonariensis offspring. Our findings confirm that transplantation of germ cells into sexually competent adult fish by non-surgical methods allows the production of functional donor-derived eggs and sperm in a considerably short time. The methods described here could play a vital role in conservation and rapid propagation of endangered fish genetic resources.

Benzocaine and clove oil as anesthetics for pejerrey (Odontesthes bonariensis) fingerlings

Pejerrey (Odontesthes bonariensis) is a native species from Rio Grande do Sul, Uruguay and Argentina where it is of great economic importance for artisanal fishing. One difficulty in laboratory research with pejerrey is related to its sensitivity, as it presents higher basal cortisol levels than other freshwater species. For this reason, the aim of this work was to evaluate the efficiency of benzocaine and clove oil as anesthetics for pejerrey fingerlings. Two experiments were done where fingerlings (57±7.8mm and 1.1±0.44g) were exposed to benzocaine with concentrations between 40mgL-1 and 120mgL-1 and to clove oil with concentrations between 12mgL-1 and 75mgL-1. Survival, anesthesia induction time and recovery time for each pharmaceutics were evaluated. Both benzocaine and clove oil pharmaceutics showed efficiency as anesthetics for pejerrey fingerlings, with negative correlation between the dose of anesthetics and the anesthesia induction time. For benzocaine, the concentrations between 80mgL-1 and 100mgL-1 showed better results, as for clove oil the optimal concentrations were between 25mgL-1 and 50mgL-1. On the other hand, the anesthesia recovery time did not present significant variation on the different concentrations of the tested products. The tested products are highly metabolizable by pejerrey.

Thermal and Endocrine Regulation of Gonadal Apoptosis during Sex Differentiation in Pejerrey Odontesthes bonariensis

The fate of the differentiating gonads in pejerrey Odontesthesbonariensis is determined by the environmental water temperature experienced by the larvae during the critical period of sex determination. We previously reported a link between apoptosis, temperature and sex differentiation in this species. To clarify this link, we subjected larvae to thermal and endocrine treatments between hatching and the onset of histological sex differentiation of the gonads and assessed the patterns of gonadal development and apoptosis by light microscopic histology and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, respectively. Apoptotic labeling was widespread among somatic cells of the anterior region of the right gonads of fish reared at the male-producing temperature (MPT) and part of the fish at the mixed sex-producing temperature prior to sex differentiation. In contrast, TUNEL-positive cells were rarely observed in gonads at the female-producing temperature (FPT). Administration of exogenous estrogen completely prevented MPT-induced masculinization, induced feminization and reduced gonadal apoptosis, whereas an aromatase inhibitor (fadrozole) induced TUNEL signals in the gonads of FPT-reared larvae. These results provide strong evidence that apoptosis in somatic cells in the right lobe of the gonads might play a key role in testicular differentiation in pejerrey and that estrogens are involved in the regulation of this process.

Presence of 11-ketotestosterone in pre-differentiated male gonads of Odontesthes bonariensis

The involvement of androgens during sex differentiation period was investigated in the pejerrey Odontesthes bonariensis, by classic biochemical studies and gonadal histology. We studied in particular whether the enzyme activities involved in 11-oxygenated androgen production were active in a gonadal/peritoneum complex (GPC) of very small larvae exposed to masculinizing temperatures previous to morphological sex differentiation (5weeks post-hatching). The GPC was incubated with 17-hydroxyprogesterone ((3)H-17P), and the presence of 11-KT as major metabolite in early gonads undergoing masculine pathway after temperature treatment exposure is reported. 11-KT was identified by thin-layer chromatography and high-pressure liquid chromatography. The present results show that 11-KT is produced at very early stages of testis development in pejerrey, being this androgen one of the main mediators of the masculinization induced by temperature treatment at the gonad level.

Extrahypophyseal expression of gonadotropin subunits in pejerrey Odontesthes bonariensis and effects of high water temperatures on their expression

It has been traditionally accepted that the gonadotropins (GtHs), follicle stimulating hormone (FSH) and luteinizing hormone (LH), are synthesized and secreted only by the pituitary. However, the presence of theses hormones in extrapituitary tissues has been demonstrated in mammals, and more recently also in fish. In this study, we cloned the cDNAs and characterized the expression of FSH-β, LH-β, and glycoprotein hormone α ( GPH-α) subunits from brain and gonads of male and female pejerrey Odontesthes bonariensis at different stages of gonadal maturation. In situ hybridization revealed that, in addition to their classical location in pituitary cells, the three GtH transcripts were also located in the gonads. FSH-β and GPH-α subunits were found in the cytoplasm of oogonia, previtellogenic and vitellogenic oocytes in ovaries. LH-β expression was detected in previtellogenic and vitellogenic oocytes but not in oogonia. In males, the three subunits were expressed in spermatogonia and to a lesser extent in spermatocytes. Exposure of fish to high water temperatures that impair pejerrey reproduction also induced a decrease of extrahypophyseal expression of GtH subunits.

Undifferentiated Gonads of Pejerrey ( Odontesthes bonariensis ) Larvae Exposed to Masculinizing Temperatures Produce 11-ketotestosterone when Incubated with a Tritiated Precursor

Introduction: The involvement of androgens as early mediators of testis differentiation in fish is still a matter of debate [1]. Larval gonadal size limits the possibility to establish whether an early production of 11-oxigenated androgens precedes testicular differentiation. In a previous study we reported the expression of genes involved in androgen synthesis (i.e. cyp11a y la cyp11b) in larval trunks [2] and the production of 11-ketotestosterone was reported in larval trunks by EIA [3]. In this context the aim of the present work was to study the androgen synthesis capacity of gonads and peritoneum of Odontesthes bonariensis in larvae exposed to masculinizing temperatures (29oC) before morphological differentiation of the testis. First we validate the nature of androgens produced by adult testis, liver and spleen and then we studied the androgen production in larval gonads at 29oC. Methods: Pejerrey larvae were raise for 5 weeks at 29oC in order to obtain a whole male population. At 5 that moment, 100 fish were sampled and individually immerse in ice-cold water until movement ceased. Due to technological constrains in taking then apart, the gonads and peritoneum were dissected together under binocular microscope and pooled in ice cold L-15 medium. Adult testis at the onset of spermatogenesis, spleen and liver were also obtained and placed on L-15 medium. Tissue samples were incubated for 18 hs in 500 μL of L-15 medium in the presence of tritium labeled precursors. 17P was used for gonad and peritoneum, and A4 for spleen and liver. The reaction was arrested by ethanol addition to 80% followed by tissue disaggregation using a glass-glass grinder. The supernatant was evaporated under nitrogen until 20% its original volume qws reached and then extracted three times with dichloromethane (DM): methanol (M) (9DM:1M). This fraction was re-suspended and applied to a silica TLC plate, cleaned by a non-polar mobile phase run and then resolved by using benzene acetone (2:1). The resolved radioactive steroids were revealed by autoradiography, the characteristic Rf computed and radioactive zones isolated by scratching the silica followed by extraction using three rounds of 9DM: 1M. The bands extracts corresponding to 11-ketotestosterone (11-KT) and 11β-hydroxyandrostenedione (11β-OHA4) were separately analyzed by HPLC using a RP-C18 column and 0.5 mL fractions were obtained and their radioactivity content determined by scintillation counting. The 11β-OHA4 obtained from kidney extracts was analyzed by co-crystallization to constant specific activity.

Some Like it Hot: Temperature Selection during the Critical Period of Thermolabile Sex Determination in Pejerrey ( Odontesthes bonariensis )

Introduction: Many fish species have thermolabile sex determination (TSD) and the sex ratios of wild populations may be influenced by the thermal conditions during the reproductive season. The pejerrey (Odontesthes bonariensis) is an Atherinopsid fish that shows a remarkable level of TSD at environmentally relevant temperatures [1]. Thus, gonadal sex is determined between 1 and 5 weeks after hatching and the frequency of males varies from 0% at temperatures below 19°C to 100% at 29°C [2]. As a first step to model the effects of temperature on the sex ratios of wild populations and to clarify the adaptive significance of TSD, we examined the thermal preference of pejerrey larvae during the time of gonadal sex determination and how sex ratios vary under a situation where larvae can freely select the temperature. Methods: Newly hatched pejerrey larvae were stocked in a horizontal thermal gradient consisting of five interconnected aquaria kept at constant temperatures of 17, 20, 23, 26, and 29°C. Larvae were able to move freely through the entire gradient. Similar amounts of live food (Artemia nauplii) were delivered to each aquarium three times a day regardless of the presence of larvae. Larval behavior was monitored 24-hour/day for 5 weeks using overhead cameras and the number of larvae and time spent at each temperature was calculated. Sex ratios were analyzed at the end of the experiments by light histology. Results and Conclusion: The behavioral analysis revealed a preference for high, masculinizing temperatures (26-29°C) and the resulting sex ratios were highly male-biased (80-100%). We also observed a diurnal rhythm of temperature selection, with fish preferring higher temperatures at night compared to daytime. These results provide important clues to understand the ecology of TSD for this species and predict the sex ratios of wild populations as a function of temperature. They could be also useful for modeling the effects of global warming on pejerrey resources.

Temperature effects on sex differentiation of the reciprocal hybrids of Odontesthes bonariensis and Odontesthes hatcheri (Atherinopsidae)

The pejerrey Odontesthes bonariensis (Valenciennes 1835) and the Patagonian pejerrey Odontesthes hatcheri (Eigenmann 1909) are Atherinopsid species with commercial importance and potential for aquaculture. The hybrids of the two species are viable but their mode of sex determination is unknown. This study examined the gonadal histology and sex ratios of reciprocal hybrids that were reared at 15, 17, 21, 25 or 29 °C during the sex differentiation period. The genetic sex of hybrids from O. hatcheri fathers was inferred from a sex-linked SNP marker. Both hybrids showed female-biased sex ratios at the lowest temperature, female-biased to balanced sex ratios at intermediate temperatures and balanced or male-biased sex ratios at 29 °C, but unlike in purebred O. bonariensis, the lowest and highest temperatures did not yield monosex populations. The proportion of females in the offspring was affected more by parental genome than by hybrid combination. Female hybrids bearing the O. hatcheri Y chromosome showed temporary arrest of ovarian development that was rescued in adults. These results reveal strong interactions between genotype and temperature for sex determination and differentiation of the hybrids and provide important clues to understand the mechanisms of sex determination in these species.

An immunohistochemical study of the gut neuroendocrine system in juvenile pejerrey Odontesthes bonariensis (Valenciennes)

In this study, several neuropeptides were identified by immunohistochemistry in neuroendocrine cells (NEC) located in the gut epithelium and nerve cell bodies of the enteric nervous system of pejerrey Odontesthes bonariensis, a species that is a promising candidate for intensive aquaculture. The neuropeptides involved in orexigenic or anorexigenic action, i.e. gastrin, cholecystokinin-8, neuropeptide Y and calcitonin gene-related peptide (CGRP), displayed a significantly higher number of immunoreactive NECs in the anterior intestine, suggesting that this region of the gut plays an important role in the peripheral control of food intake. On the other hand, leu-enkephalin and vasoactive intestinal peptide (VIP), both associated with the modulation of the enteric immune system, showed no significant variations in the mean value of immunopositive NECs between the anterior and posterior intestine. This may indicate that their activity is required at a similar level along the entire gut. In addition, CGRP and VIP-immunoreactive neurons and nerve fibres were observed in the myenteric plexus, which might exert synergistic effects with the neuropeptides immunolocalized in NECs.

Effect of three fungicides on in vitro growth of Fusarium solani and Fusarium semitectum, parasitic on Odontesthes bonariensis eggs

An in vitro evaluation of the effect of three fungicides on the growth rate (GR) of Fusarium solani and Fusarium semitectum was performed. The tested fungicides, commonly used in fish farming, were sodium chloride (NaCl), methanol-stabilised formaldehyde (F) and malachite green (MG). Both Fusarium species were isolated from eggs of the Argentinean pejerrey Odontesthes bonariensis (Valenciennes, 1835). The concentrations of NaCl and MG at which growth of both species was inhibited were higher than the respective mean 96h median lethal concentrations (LC50-96h) for silverside eggs. In the case of F, the growth inhibition (GI) of F. solani was achieved also at concentrations higher than the respective LC50-96h. Only for F. semitectum a complete GI was observed at a concentration lower than the LC50-96h. This indicates that the efficacy of these substances is not adequate for treatment of fungal infections of silverside eggs.

Molecular cloning and expression analysis of Fshr and Lhr in relation to Fshb and Lhb subunits during the period of temperature‐dependent sex determination in pejerrey Odontesthes bonariensis

In this study, we cloned and characterized the follicle stimulating hormone receptor (Fshr) and luteinizing hormone receptor (Lhr) cDNAs of pejerrey Odontesthes bonariensis, a species with temperature-dependent sex determination (TSD), and analyzed their expression in relation to Fshb and Lhb subunits during gonadogenesis at temperatures producing only females (17 degrees C, FPT), both sexes (25 degrees C, MixPT), and only males (29 degrees C, MPT). The pejerrey Fshr cDNA had 3,069 bp for a mature protein of 694 amino acids (aa) and a signal peptide of 22 aa; the Lhr cDNA had 2,936 bp for a mature protein of 676 aa and a signal peptide of 25 aa. With the exception of Lhr in fish at the MPT, all genes showed significant increases and/or peaks of expression before histological differentiation of the gonads regardless of temperature. Larvae at the FPT had lower Fshb and Lhb but higher Lhr expression during the TSD period than those at the MPT; a clear pattern could not be ascertained for Fshr. At the MixPT, Fshb, Lhb, and Lhr mRNA increased in approximately half of the fish during TSD and sex differentiation and the sex ratio was 55.2% male. Based on the above results, it is suggested that animals with high Fshb and Lhb and low Lhr values represent putative males. These evidences, together with other studies, suggest that temperature may signal through the pituitary (differential expression of Fshb and Lhb) down to the gonads (differential expression of Lhr), probably affecting the regulation of steroidogenesis during the TSD process of pejerrey.

Assessment of condition in pejerrey Odontesthes bonariensis (Atheriniformes: Atherinopsidae) populations: which index works best?

The pejerrey Odontesthes bonariensis is the most important target species in temperate freshwater fisheries of Argentina, and assessment of condition has been a regular practice and common diagnostic tool. Most pejerrey fishery studies have used Fulton's (K) index, without testing whether underlying assumptions or requirements were met. We analyzed and contrasted the applicability of K, Kn and Wr indices to assess condition status in several pejerrey populations inhabiting Pampean lakes. Our results showed that whereas Wr and Kn displayed significant condition changes across length at some study lakes, Kn portrayed a small range of variation. We also noted that pejerrey maximum length and size structure strongly varied among populations probably due to the characteristics of trophic niche changes through lifespan, depending on lake limnological characteristics and zooplankton availability. We conclude that the K index should be disregarded in those cases where populations show allometric growth and size ranges strongly vary. In turn, the Kn index appears to be only appropriate for regular within population assessment, being difficult to apply when comparisons between populations are needed and when they exhibit different weight-length relationship slopes. Finally, the Wr index should be strongly preferred if the objective is to perform comparisons between pejerrey populations, particularly when population structure is not well known, stocking has been used for population recovery, lakes are strongly supported by limnological changes, data are limited to only one or few samplings and metaphoetesis is suspected in pejerrey populations.

Germ Cell Transplantation Using Sexually Competent Fish: An Approach for Rapid Propagation of Endangered and Valuable Germlines

The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC) proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT) in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae), were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg) and high water temperature (25°C) treatments. The observation of the donor cells' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2–13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species.