Influence of sewage discharge on dissolved oxygen concentration and fish diversity in the Girado stream and Chascomús lake.

Hypoxic aquatic environments have increased in recent decades mainly by human activities that generate pollution. The objective of this work was to study the influence of the discharge of sewage effluents from Chascomús city on the concentration of dissolved oxygen (DO) in the Girado stream and its connection with Chascomús lake and analyze the impact on fish biodiversity. Four sites were selected to measure DO and temperature weekly for one year: discharge of sewage effluent, union of the sewage effluent with the Girado stream, Girado stream and Chascomús lake. Also, ichthyological samplings were carried out in a presumed hypoxic zone and a normoxic zone of the Girado stream. The results showed that there were two hypoxic zones corresponding to the sewage effluent and the union of the effluent with the Girado stream (~3 mg l-1). Also, two normoxic zones were identified, the course of the Girado stream and the union with Chascomús lake (~ 8 mg l-1). In these areas the water temperature varied according to the seasonality of the region (from 10 to 30 °C) without differences between zones. It is important to note that these areas received a constant flow of sewage effluent from Chascomús city (~ 885,600 l per day). In the fish sampling carried out in the 4 seasons of the year, 14 typical species of the Pampas lakes were captured with species more tolerant to low DO. Pejerrey were captured in a small number in both sites but with no signs of abnormalities in the gonads.

Modulation of miR-429 during osmotic stress in the silverside Odontesthes humensis.

Silverside fish inhabit marine coastal waters, coastal lagoons, and estuarine regions in southern South America. Although silversides are not fully adapted to freshwater, they can tolerate a wide range of salinity variations. MicroRNAs (miRNAs) are a class of ∼22 nucleotide noncoding RNAs, which are crucial regulators of gene expression at post-transcriptional level. Current data indicate that miRNAs biogenesis is altered by situations of environmental stress, thereby altering the expression of target mRNAs. Foremost, the silversides were acutely exposed to 30 g.L-1 of salt to reveal in which tissue miR-429 could be differentially expressed. Thus, fish were acclimated to freshwater (0 g.L-1) and to brackish water (10 g.L-1), and then exposed to opposite salinity treatment. Here, we reveal that miR-429, a gill-enriched miRNA, emerges as a prime osmoregulator in silversides. Taken together, our findings suggest that miR-429 is an endogenous regulator of osmotic stress, which may be developed as a biomarker to assist silverside aquaculture.

The cellular basis of compensatory muscle growth in the teleost Odontesthes bonariensis.

This study evaluates white muscle growth and in vivo cell proliferation during a fasting and refeeding trial, using pejerrey (Odontesthes bonariensis) as animal model, in order to better understand the cellular basis governing catch-up growth. Experiments consisted of two groups of fish, a control group continuously fed ad libitum, and a group fasted for 2 weeks and then fed for another 2 weeks. We examined how the formation of new muscle fibers and their increase in size were related to muscle precursor cell (MPC) proliferation under both experimental conditions. During fasting, the number of 5-ethynyl-2'-deoxyuridine-positive (EdU+) cells decreased along with myogenic regulatory factor (MRF) mRNA levels related to myoblast proliferation and differentiation, and the muscle stem cell marker Pax7 mRNA level increased. Analysis of myomere cross-sectional area, distribution of muscle fiber sizes and number of fibers per myomere showed that muscle hypertrophy but not hyperplasia was inhibited during fasting. Both higher igf2 mRNA level and the persistence of cell proliferation could be supporting new myofiber formation. In contrast, an exacerbated MPC proliferation occurred during catch-up growth, and this increase in cell number could be contributing to the growth of both pre-existing and newly formed small fibers. The findings that some MPCs proliferate during fasting and that muscle growth mechanisms, hyperplasia and hypertrophy are differentially regulated could help to explain why re-fed fish could grow at faster rates, and why they return to the lost growth trajectory.

Mitochondrial genome architecture and phylogenetic relationships of Odontesthes argentinensis within Atherinomorpha.

Silversides are a widely distributed group across South America, with several species occupying marine, freshwater and estuarine environments. Several authors suggest main transitions among these environments took place during Pleistocene, and were accompanied with rapid speciation events. This scenario produced very limited genetic and morphological differentiation among the species. However, most of these surveys have an incomplete coverage of the intraspecific genetic diversity of the taxa studied. In this work, we reconstructed six mitochondrial genomes of O. argentinensis using transcriptomic data, and used them-in combination with several nuclear markers retrieved from the same transcriptomes-to explore the effect of additional coverage of intraspecific diversity of this species in phylogenetic reconstructions. Unlike previous works, phylogenetic analyses failed to identify O. argentinensis as a monophyletic group in relation with closely related taxa. Our results suggest that several species of the genus, especially those related to O. argentinensis, need further taxonomic revision.

Effects on Ultrastructure, Composition and Specific Surface Area of the Gills of Odontesthes bonariensis Under Subchronic Glyphosate-Based Herbicide Exposure.

Gills represent one of the major sites of gas exchange of fish, consequently they are in continuous close contact with the aquatic environment and its pollutants. In the present study the effects on gills of pejerrey fish, Odontesthes bonariensis, under glyphosate-based herbicide subchronic exposure were analyzed. Adult animals were exposed to sublethal concentrations of a glyphosate-based commercial formulation (1 and 10 PMG mg L-1, PMG: glyphosate active ingredient) for 15 days, while control group was maintained in rearing water. Ultrastructural changes in gills were observed by scanning electron microscopy (SEM). The composition of the surface epithelium and specific surface area were determined by energy dispersive spectroscopy (EDS) and N2 (g) adsorption-desorption isotherms, respectively. The herbicide exposure induced severe alterations in gill ultrastructure, as shown in the SEM micrographs. Accordingly, an increase in surface area of the gills of exposed animals was determined. These results support that gills parameters of freshwater fish are sensitive morphological biomarkers for glyphosate exposure.

Cooling of pejerrey Odontesthes bonariensis (Teleostei, Atherinidae) embryos at sub-zero temperatures.

Pejerrey fish (Odontesthes bonariensis) is a seasonal multiple spawner with great economic importance and an adequate species for Aquaculture. For these reasons, it is necessary to apply biotechnologies to optimize its reproduction in captivity. In this context, the aim of this work was to develop a cooling protocol for pejerrey embryos at sub-zero temperatures. Two cryoprotective solutions (CSs: S1 and S2), two cooling curves (a fast and a slow one) and two storage temperatures (-14 and -20 °C) were evaluated for 1 h. High percentages of embryo survival (80-100%) were obtained in all cases. In particular, for cooling at -14 °C, the most suitable protocol was the slow temperature decrease in combination with S1 (2.5 M methanol, 1.4 M Me2SO, 0.3 M sucrose, and 0.08 M NaCl). The hatching rate (86.67 ± 11.55%) and the larval survival observed did not differ from those of the control group, and about 30% of normal-looking larvae were obtained. Besides, the slow cooling was also the best way to reach -20 °C, obtaining a hatching rate of around 60%. However, all the larvae had different kind of malformations. Finally, in order to improve the results obtained at -20 °C, the CSs were incorporated into the embryos by microinjection. In this case, it was observed that the most convenient combination was the microinjection of S2 (same composition as S1 but without Me2SO) in the perivitelline space followed by rapid cooling. Although the hatching rate was not improved (67.93 ± 8.31%), the microinjection allowed to obtain at least 4.5% normal-looking larvae. These results showed that the cooling of pejerrey embryos at zub-zero temperatures was feasible. Moreover, the microinjection of cryoprotectants within the pejerrey O.bonariensis embryos was employed for the first time in this species.

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.

Ghrelin and NUCB2/Nesfatin-1 Co-Localization With Digestive Enzymes in the Intestine of Pejerrey (Odontesthes bonariensis).

Ghrelin (orexigenic) and nesfatin-1 (anorexigenic) are two peptides with opposing actions on food intake regulation and are mainly expressed in the hypothalamus and gut of mammals and fish. Both are involved in the regulation of a wide range of physiological processes in vertebrates, including metabolism, growth, and reproduction. However, the anatomical relationship between these peptides and the nutrient assimilation processes are not well understood. Thus, the aim of this work was to determine the localization of ghrelin, nesfatin-1, and several enzymes involved in the digestive process (lipoprotein lipase, aminopeptidase A, trypsin, and sucrase-isomaltase) in the intestine of pejerrey (Odontesthes bonariensis), a species with commercial importance in South America. We observed co-localization of ghrelin and nesfatin-1 in enteroendocrine cells, absorptive cells, and in cells of the lamina propia. Approximately half of the cells displaying ghrelin-like immunoreactivity co-localized the NUCB2/nesfatin-1-like signal. In addition, both peptides showed co-localization with lipoprotein lipase, aminopeptidase A, trypsin, or sucrase-isomaltase. All digestive enzymes except for aminopeptidase A and trypsin, showed high co-localization (68-88%) with both ghrelin-like and NUCB2/nesfatin-1-like signals in absorptive, enteroendocrine, and lamina propria cells. Together, our results provide immunohistochemical evidence supporting a role for both ghrelin and NUCB2/nesfatin-1 in the regulation of nutrient assimilation in fish. Anat Rec, 302:973-982, 2019. © 2018 Wiley Periodicals, Inc.

Sex hormone binding globulin during an annual reproductive cycle in the hepatopancreas and ovary of pejerrey (Odontesthes bonariensis).

In the present study, we determined the hepatopancratic shbg transcript abundance and ovarian immunoreactive Shbg (ir-Shbg) localization in pejerrey females at different gonadal stages during an annual ovarian cycle. In the hepatopancreas, shbg expression remains was constant in pre-vitellogenic stages, decreased at final vitellogenesis to increase again in final maturation and atretic stages to previous levels at post-vitellogenic stages. Related to this, also we found a negative significant relation between sex steroid and shbg expression. On the other hand, in the ovary we found ir-Shbg inside of cortical alveoli, from previtellogenic stages to final maturation. This localization of Shbg in a teleost fish ovary suggests a new role for Shbg in oocytes, that may also extend the oocyte fertilization/development process.

Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish.

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey.

Kisspeptins and their receptors in the brain-pituitary-gonadal axis of Odonthestes bonariensis: Their relationship with gametogenesis along the reproductive cycle.

In vertebrates, the reproduction is controlled by the brain-pituitary-gonadal (BPG) axis and kisspeptin has emerged as a key player of this axis. In this study, we analyzed changes in the expression levels of kiss1, kiss2, and their receptors, kissr2 and kissr3 during gametogenesis in the BPG axis of feral Odontesthes bonariensis. In females, levels of brain kiss1 showed an increase at final maturation (Fm), while kiss2 levels were shown to be high at primary growth (Pg) stage, with no differences in the expression of their receptors. In the pituitary, kiss1 and kiss2 peaked at the cortical alveoli (Ca) stage, and kissr3 at initial vitellogenesis. In parallel, there was an increase of kiss1, kissr2 and kissr3 in the ovary during the Ca stage and both receptors again at Fm stage. In males, the four genes were highly expressed in the brain at the arrested (A) stage. In the pituitary, kiss2 peaked at spermatogonial (SG) and spermatocytary (SC) stages; while kissr3 reached a peak at the spermiogenic stage (SP). In testes, kiss1 and kiss2 significantly increased during the SG and SC stages; meanwhile, kissr2 increased at SG and SC, whereas kissr3 levels were significantly high at SC and SP stages. Taken together these results showed that the kisspeptin system in pejerrey is expressed in the three levels of the BPG axis with different expression profiles during the gonadal cycle. These findings pointed that kisspeptins have different roles in gametogenesis in this species.

Exposure to E2 and EE2 environmental concentrations affect different components of the Brain-Pituitary-Gonadal axis in pejerrey fish (Odontesthes bonariensis).

The present study focuses on the effects of E2 and EE2 environmental concentrations on different components of the reproductive axis of pejerrey (Odontesthes bonariensis), a native fish species from Pampas lakes of Argentina. The results obtained demonstrated that E2 and EE2 separate or mixed, could disrupt key pathways of the pejerrey Brain-Pituitary-Gonadal axis. First, it was observed that at the brain level, gnrh-III and cyp19a1b mRNA expression increased significantly in the exposed fish. Secondly, in the pituitary fshb and lhb mRNA expression levels, the study did not show any differences between treated and control groups. Thirdly, fshr and lhcgr transcript levels showed a significant decrease at testicular level. Nevertheless, testosterone plasmatic levels remained unchanged in exposed fish. In addition, in a histological analysis, it was possible to find pyknotic nuclei in estrogen only on treated fish testis linked to a reduction in the GSI index and a decrease in the length of spermatogenic lobules. All these findings highlighted the fact that environmental concentrations of E2, EE2 and their mixture disrupted the endocrine-reproductive axis of pejerrey, being the testis the main direct target.

Sex hormone binding globulin: Expression throughout early development and adult pejerrey fish, Odontesthes bonariensis.

Sex hormone binding globulin (Shbg) is a plasma glycoprotein that binds and transports steroids in the blood of all vertebrate classes apart from birds. In the present study we characterized shbg from pejerrey, a fish species with a well characterized temperature-dependent sex determination. The pejerrey shbg mRNA comprises 1185bp encoding for a 395 amino acid Shbg precursor protein that includes a leader sequence for secretion. Relative quantification of shbg transcript abundance revealed expression early in development coinciding with the sex-determining period and probably in association with temperature leading to male determination. The hepatopancreas was the main site of shbg expression, which varied according to the sex cycle in females. It was also expressed in gills, gonads, gut and taste buds during both larval stages and in adult fish. The presence of Shbg in organs in close contact with the environment such as gills, pseudobranchs, gut and taste buds suggests that these are potential sources of uptake or release of steroids/xenosteroids to and from the aquatic environment.

Kisspeptin system in pejerrey fish (Odontesthes bonariensis). Characterization and gene expression pattern during early developmental stages.

In vertebrates, kisspeptins and their receptors are known to be related to puberty onset and gonadal maturation, however, there are few studies concerning their role in early development. Here, we characterize the kisspeptin system in the pejerrey, Odontesthes bonariensis, a fish with strong temperature-dependent sex determination. We reconstructed the phylogenetic history of the two ligands (kiss1 and kiss 2) and two receptors (kissr2 and kissr3) in pejerrey in the context of recent classifications of bony fishes, determined their tissue distribution and documented the early expression pattern of these ligands and receptors. Phylogenetic analysis of these gene families clearly resolved the percomorph clade and grouped pejerrey with Beloniformes. Paralogous sets of genes putatively arising from the teleost-specific genome duplication event (3R) were not detected. Kisspeptins and their receptors showed a wide tissue distribution in adult pejerrey, including tissues not related to reproduction. In larvae reared at 24°C, the four kisspeptin elements were expressed in the head from week 1 to week 8 of life, with no differences in transcript levels. Larvae kept at a female-producing temperature (17°C) did not show statistically significant differences in the transcript levels of all analyzed genes during the sex determination/differentiation period; however, in those larvae raised at male producing temperature (29°C), kiss2 levels were increased at week 4 after hatching. These results showed that all members of the kisspeptin system are expressed at this early period, and the increase of kiss2 transcripts at week 4 could be interpreted as it would be related to the differentiation of the brain-pituitary axis in male development.

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.

Genotoxic Effects Induced by Cd(+2), Cr(+6), Cu(+2) in the Gill and Liver of Odontesthes bonariensis (Piscies, Atherinopsidae).

Genotoxic effects of Cd(+2), Cr(+6), and Cu(+2) on the gill and liver of the Argentinean Silverside (Odontesthes bonariensis) were studied using the comet assay and in relation with the metal tissue accumulation. Fish were exposed to three waterborne concentrations of each metal for 2 and 16 days. Genotoxicity was assessed by the single cell gel electrophoresis (comet assay). After 2 days, significant increase of the genetic damage index (GDI) was only observed in the gill of fish exposed to Cr(+6) and Cu(+2), and the LOECs were 2160 nM and 921.1 nM, respectively. The gill LOEC for Cd(+2) by 16 days was 9.4 nM. In the liver, LOECs were obtained only for Cd(+2) and Cr(+6) and were 9.4 and 2160 nM, respectively. The three metals were able to induce genotoxic effects at environmentally relevant concentrations and the gill was the most sensitive organ.

Populations of Odontesthes (Teleostei: Atheriniformes) in the Andean region of Southern South America: body shape and hybrid individuals

The original distribution area of the Patagonian 'pejerrey' Odontesthes hatcheri has been subjected to the introduction of a related species; the Bonaerensean 'pejerrey' Odontesthes bonariensis. This species currently coexists with O. hatcheri in lakes and reservoirs, and can interbreed and produce fertile hybrid offspring. The purposes of this study were; a) the extensive sampling of Patagonian and Andean-Cuyan populations of pejerrey, b) the species identification according to taxonomic key, c) validation of taxonomic results on the basis of mitochondrial DNA composition, and d) applying morphometric analysis to explore the effects of hybridization and environmental conditions on body shape. Cytochrome b sequence analysis showed a high degree of genetic divergence between species and low intraspecific variation in O. hatcheri. Geometric Morphometric Analyses detected shape differences in agreement with diagnostic characteristics of each species. Putative hybrids exhibiting intermediate diagnostic characteristics were identified by Geometric Morphometric Analysis. Significant regressions between body shape and total phosphorus and altitude were found, suggesting a dependence on trophic web structure. This multi-level approach suggests the introgression of O. bonariensis into several O. hatcheri populations throughout Patagonia. Managers should take this into account when considering further exotic introductions into regions where non-native fishes have not yet become established.(AU)

La distribución original del 'pejerrey' patagónico Odontesthes hatcheri ha sido sometida en las últimas décadas a la introducción de una especie relacionada; el 'pejerrey' Bonaerense Odontesthes bonariensis. Ambas especies coexisten actualmente en algunos lagos y embalses debido a prácticas de siembra y pueden cruzarse y producir progenie híbrida y fértil. Los propósitos de este estudio fueron a) un amplio muestreo de las poblaciones patagónicas y andino-cuyanas del pejerrey, b) la identificación de las especies de acuerdo con la clave taxonómica, c) la validación de los resultados taxonómicos sobre la base de la composición del ADN mitocondrial y d) aplicar el análisis morfométrico para explorar los efectos de la hibridización y las condiciones ambientales sobre la forma corporal. El análisis de la secuencia del Citocromo b mostró un alto grado de divergencia genética entre ambas especies y una muy baja variación intraespecífica en O. hatcheri. El análisis de la Morfometría Geométrica detectó diferencias de forma coincidentes con las características diagnósticas de cada especie. Presuntos híbridos exhibiendo características diagnósticas intermedias fueron identificados por el análisis de la Morfometría Geométrica. Regresiones significativas entre la forma corporal y la concentración total de fósforo y la altitud fueron halladas, sugiriendo una dependencia con la estructura de la trama trófica. Este enfoque múltiple sugiere la introgresión de genes de O. bonariensis dentro de varias poblaciones de O. hatcheri a lo largo de la Patagonia. Las autoridades de aplicación deberían tomar en cuenta estos riesgos al momento de considerar nuevas introducciones de especies exóticas en regiones donde estas especies no se encuentren previamente establecidas.(AU)

Populations of Odontesthes (Teleostei: Atheriniformes) in the Andean region of Southern South America: body shape and hybrid individuals

The original distribution area of the Patagonian 'pejerrey' Odontesthes hatcheri has been subjected to the introduction of a related species; the Bonaerensean 'pejerrey' Odontesthes bonariensis. This species currently coexists with O. hatcheri in lakes and reservoirs, and can interbreed and produce fertile hybrid offspring. The purposes of this study were; a) the extensive sampling of Patagonian and Andean-Cuyan populations of pejerrey, b) the species identification according to taxonomic key, c) validation of taxonomic results on the basis of mitochondrial DNA composition, and d) applying morphometric analysis to explore the effects of hybridization and environmental conditions on body shape. Cytochrome b sequence analysis showed a high degree of genetic divergence between species and low intraspecific variation in O. hatcheri. Geometric Morphometric Analyses detected shape differences in agreement with diagnostic characteristics of each species. Putative hybrids exhibiting intermediate diagnostic characteristics were identified by Geometric Morphometric Analysis. Significant regressions between body shape and total phosphorus and altitude were found, suggesting a dependence on trophic web structure. This multi-level approach suggests the introgression of O. bonariensis into several O. hatcheri populations throughout Patagonia. Managers should take this into account when considering further exotic introductions into regions where non-native fishes have not yet become established.

La distribución original del 'pejerrey' patagónico Odontesthes hatcheri ha sido sometida en las últimas décadas a la introducción de una especie relacionada; el 'pejerrey' Bonaerense Odontesthes bonariensis. Ambas especies coexisten actualmente en algunos lagos y embalses debido a prácticas de siembra y pueden cruzarse y producir progenie híbrida y fértil. Los propósitos de este estudio fueron a) un amplio muestreo de las poblaciones patagónicas y andino-cuyanas del pejerrey, b) la identificación de las especies de acuerdo con la clave taxonómica, c) la validación de los resultados taxonómicos sobre la base de la composición del ADN mitocondrial y d) aplicar el análisis morfométrico para explorar los efectos de la hibridización y las condiciones ambientales sobre la forma corporal. El análisis de la secuencia del Citocromo b mostró un alto grado de divergencia genética entre ambas especies y una muy baja variación intraespecífica en O. hatcheri. El análisis de la Morfometría Geométrica detectó diferencias de forma coincidentes con las características diagnósticas de cada especie. Presuntos híbridos exhibiendo características diagnósticas intermedias fueron identificados por el análisis de la Morfometría Geométrica. Regresiones significativas entre la forma corporal y la concentración total de fósforo y la altitud fueron halladas, sugiriendo una dependencia con la estructura de la trama trófica. Este enfoque múltiple sugiere la introgresión de genes de O. bonariensis dentro de varias poblaciones de O. hatcheri a lo largo de la Patagonia. Las autoridades de aplicación deberían tomar en cuenta estos riesgos al momento de considerar nuevas introducciones de especies exóticas en regiones donde estas especies no se encuentren previamente establecidas.

Seasonal changes and endocrine regulation of pejerrey (Odontesthes bonariensis) spermatogenesis in the wild.

The endocrine mechanisms that regulate spermatogenesis and their interaction with environmental cues have been poorly studied compared with oogenesis in fish. The aim of this work was to study the spermatogenesis in pejerrey under the influence of photoperiod and water temperature fluctuation in the wild, evaluating the transcript levels of brain Gnrh variants and cyp19a1b, pituitary Gth subunits, gonadal Gth receptors, 11ß-hsd, and 11-KT plasma levels. Males at spermiogenic stage were observed during spring and autumn, under a photoperiod above 11h of light and a water temperature below 23 °C. Most arrested males were observed in summer when water temperatures increased above 23 °C. Males at spermatogonial stage were mainly observed in autumn, while most males at spermatocytary stage were caught in winter. An increase of gnrh-I, cyp19a1b, fshb, gpha and 11ß-hsd transcripts and 11-KT plasma levels was observed during spermatogonial and/or spermatocytary stage (early spermatogenesis). The spermiogenic stage was associated to the maximum gnrh-I gene expression level and a significant increase of Gth receptors transcripts, being this fact more evident for lhcgr. During this last gonadal stage, cyp19a1b transcript level remained high, while fshb mRNA and 11-KT plasma levels showed a significant decreased compared to that occurred at the spermatocytary stage. Also, gphα and 11ß-hsd gene expression levels fell during spermiation up to similar values to those observed in arrested males. A significant correlation between 11-KT and gnrh-I, cyp19a1b, gphα, fshb, 11ß-hsd transcripts, and the number of spermatocytes was observed during spermatogenesis. All these findings suggested that in pejerrey, the spermatocyte proliferation occurs mainly during winter under the stimulation of 11-KT induced by FSH through the stimulation of specific enzymes, including the 11ß-hsd while spermiation occurs after photoperiod increase and with temperatures of the water below 23 °C, through the stimulation of gnrh-I, cyp19a1b and lhcgr.

Effects of 5α-dihydrotestosterone on expression of genes related to steroidogenesis and spermatogenesis during the sex determination and differentiation periods of the pejerrey, Odontesthes bonariensis.

Sex steroid hormones are important players in the control of sex differentiation by regulating gonadal development in teleosts. Although estrogens are clearly associated with the ovarian differentiation in teleosts, the effects of androgens on early gonadal development are still a matter of debate. Traditionally, 11-ketotestosterone (11-KT) is considered the major androgen in fish; however, 5α-dihydrotestosterone (5α-DHT), the most potent androgen in tetrapods, was recently found in fish testis and plasma, but its physiological role is still unknown. In this context, the expression of genes associated with steroidogenesis and spermatogenesis, body growth and sex differentiation were assessed in Odontesthes bonariensis larvae fed with food supplemented with two doses of 5α-DHT (0.1 and 10µg/g of food) from hatching to 6weeks of age. At the lowest dose, 5α-DHT treated larvae showed an estrogenic gene expression pattern, with low hsd11b2 and high cyp19a1a and er2 expression levels with no differences in sex ratio. At the highest dose, 5α-DHT produced a male-shifted sex ratio and the larvae exhibited a gene expression profile characteristic of an advancement of spermatogenesis, with inhibition of amh and stimulation of ndrg3. No differences were observed in somatic growth. These results suggest that in this species, 5α-DHT could have a role on sex differentiation and its effects can differ according to the dose.