Warm water induces apoptosis, gonadal degeneration, and germ cell loss in subadult pejerrey Odontesthes bonariensis (Pisces, Atheriniformes)

Abstract Recent studies have shown that exposure to warm water can trigger gonadal degeneration and germ cell loss in fish of both sexes, but the mechanism behind this pathology is still not understood. This study was designed to characterize this process histologically and determine whether apoptosis plays any role during high temperature-induced gonadal cell degeneration in subadult pejerrey (Odontesthes bonariensis). For this purpose, fish were reared continuously at constant temperatures of 24 degrees C (control) and 29 degrees C (prolonged heat stress) or exposed for 36 h to 31 degrees C and then returned to 24 degrees C (short heat stress). Gonads were sampled at various times (hours, days, weeks) after the start of the experiment and were analyzed by light microscopy and stereometry for histological integrity/degeneration and germ cell counts, as well as by acridine orange fluorescence microscopy, TUNEL, and caspase activity assay for histochemical and biochemical signs of apoptosis. The results clearly implicate apoptosis in heat-induced somatic and germ cell degeneration in pejerrey and revealed that the dynamics and severity of this process were proportional to the magnitude of the thermal stress. Even a 36-h exposure to 31 degrees C induced significant increases in caspase-3 activity and number of apoptotic cells in both sexes, but males were shown to be more sensitive to heat stress than females.     

Comparative survival and growth of embryos,larvae, and juveniles of pejerrey Odontesthes bonariensis and O. hatcheri* at different salinities

The hatching of fertilized eggs and the survival and growth of larvae and juveniles of the inland‐water atherinids Odontesthes bonariensis (Valenciennes 1835) and O. hatcheri (Eigenmann 1909) were examined at salinities of 0, 5, 10, 20, and 30 ppt. In addition, a limited study compared the salinity responses of O. bonariensis eggs and larvae from different origins. Overall, embryos, larvae, and juveniles of both species were euryhaline, although best survival and growth rates were obtained at the intermediate salinities. Survival of O. bonariensis at 0 ppt varied from very good to very poor. Comparison of the salinity responses of eggs and larvae of O. bonariensis from the current Japanese strain with newly introduced strains from three locations in Argentina did not reveal a clearly superior strain for freshwater culture. In general, O. hatcheri showed higher survival and growth rates and better adaptability to fresh water compared with O. bonariensis. Although both species are commonly regarded as freshwater species, the results of this study emphasize the importance of millimolar quantities of salts in the rearing water for improved survival and growth.     

Effects of short periods of warm water fluctuations on reproductive endocrine axis of the pejerrey (Odontesthes bonariensis) spawning

The aim of this study was to assess fluctuations in daily water temperature in Chascomús Lagoon during one year, and to evaluate whether the highest temperature recorded during pejerrey spawning season can produce an endocrine disruption on brain-pituitary-gonads axis. Fish were subjected to daily temperature fluctuations: 17 °C to 19 °C (reproductive control), 19 °C to 25 °C, and 19 °C to 27 °C. After 8 days, ten fish per treatment were sacrificed and gene expression of gonadotropin-releasing hormone (GnRH-I, GnRH-II, GnRH-III), gonadotropin subunits-β (FSH-β, LH-β), glycoprotein hormone-α (GPH-α), gonadotropin receptors (FSH-R, LH-R), and gonadal aromatase (cyp19a1a) was analyzed. Also, plasma levels of sexual steroids and gonadal reproductive status were studied. Fish exposed to high temperature fluctuations quit spawning, presenting clear signs of gonadal regression. Fish recovered its spawning activity 11 weeks after heat treatment. At endocrine level, GnRH-I and FSH-β in both sexes, LH-β and GPH-α in males and FSH-R, LH-R and cyp19a1a in females decreased significantly in treated fish. Also, a strong reduction in plasma sex steroid levels was found for both sexes. This study demonstrated that pulses of warm water in natural environment during pejerrey spawning season can disrupt all levels of the reproductive axis, impairing reproduction.     

Vulnerability of pejerrey Odontesthes bonariensis populations to climate change in pampean lakes of Argentina

The vulnerability of the pejerrey Odontesthes bonariensis population in Lake Chasicó was assessed under different climate change conditions. During the sampling period, the water temperature was adequate for fish reproduction and to sustain an adequate sex ratio. Climate-driven higher temperatures, however, may severely distort population structure and cause drastic reduction or local extinction of stocks. Lake Chasicó can be classified as eutrophic with clear waters and cyanobacteria that regularly cause fish mortality were identified as Nodularia spumigena and Oscillatoria sp. Global warming may strengthen the effects of eutrophication (e.g. toxic blooms or anoxia). Since many Cyanophyta species tolerate higher temperatures better than other algae, toxic blooms could increase. Furthermore, cyanobacteria have low nutritional value and could decouple the low-diversity food web. Lake Chasicó has currently the salinity optimum (c. 20) for the development of the early life-history stages of O. bonariensis. Climate change, however, is likely to amplify the intensity of droughts or inundations. Floods can endanger O. bonariensis development due to its sub-optimal growth at low salinity and droughts could increase lake salinity and also temperature and nutrient concentration. In order to reduce some of the effects of climate change on the O. bonariensis population in Lake Chasicó, integrated basin management based on an eco-hydrological approach is proposed.     

Heat-induced germ cell deficiency in the teleosts Odontesthes bonariensis and Patagonina hatcheri

Pathological or experimental elevation of testicular temperature is known to trigger degeneration and disappearance of germ cells in scrotal mammals. In contrast, there are no reports of heat-induced germ cell deficiency in males of non-scrotal mammals and other vertebrate taxa, nor in females of any species. This study describes the induction of germ cell deficiency up to complete sterility in the teleosts Patagonina hatcheri and Odontesthes bonariensis by rearing larvae and juveniles for prolonged periods at incipient lethal, high temperatures (27-28.5 and 29 degrees C, respectively). It was shown that female germ cells are also heat-sensitive and disappear under high temperatures. The potential implications of these findings for physiological, ecological, and environmental studies and the usefulness of heat-induced germ cell-deficient fish in research and animal production are discussed.     

Nucleolar variation in response to nutritional condition in juvenile pejerrey Odontesthes bonariensis (Valenciennes)

The variations of the nucleolar characteristics size and number were studied in the gill and liver of Odontesthes bonariensis under different feeding regimes in order to test the hypothesis that functional genomes at the nucleolus, which determine variation of size (heterogeneity) in juvenile fishes, are modulated by environmental factors such as food availability. The programme that controls genetic changes of the nucleolar characteristics of the diploid cells of O. bonariensis was different from that described in the diploid cells of cyprinid fishes. No variations of the mean nucleolar number per nucleus were observed in liver or gill cells in relation to the feeding regime. Conversely, significant reduction of the mean nucleolar volume per nucleus ( V _MN) and change in the shape of the frequency distribution of the total nucleolar volume per cell was evident in both cell types in response to starvation or food restriction. Tissue specificity was observed in relation to the absolute values of the nucleolar characteristics; however, the relative behaviour was comparable in digestive and non-digestive tissues. The time-course reduction of V _MN in gill and liver cells of restrictively fed fish followed a negative exponential. A good correlation between V _MN and the condition index of the fish ( K ) was observed after long-term food restriction; however, a faster response of V _MN was observed after short-term fasting events. Results supported the stated hypothesis and showed nucleolar activity as a direct, rapid and sensitive indicator of the nutritional status of the fish.     

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.     

Relation between the reproductive status and somatolactin cell activity in the pituitary of pejerrey,Odontesthes bonariensis (Atheriniformes)

The aim of this study was to analyze the relationship between somatolactin (SL) expressing cells and the reproductive status in a multiple spawning fish, the pejerrey Odontesthes bonariensis. Somatolactin cells were identified in adults of both sexes by immunocytochemistry using a heterologous piscine antiserum. The area of the cells that showed immunoreactivity to SL (ir-SL) was compared in specimens with different degrees of reproductive activity as inferred from histological examination of the gonads and calculation of the gonadosomatic index (GSI %). The results showed a significant difference between the area of ir-SL cells of resting/regressing (62.9 +/- 2.1 micron 2) and sexually active/vitellogenic (76.8 +/- 2.3 micron 2) females and a significant positive correlation between the ir-SL cellular area and the GSI % (P < 0.01 in both cases). In males, the correlation between the area of ir-SL cells and the GSI % was not statistically significant. However, in those animals with the highest GSI % values, the ir-SL cells appeared more numerous and showed an increase in the immunostained area when compared to individuals with lower GSI % values. The present in morphological observations are in accordance with biochemical data obtained from other species and support the assumption that SL might be involved in the regulation of reproduction in fish.     

Hepatocyte nuclear size and nutritional condition of larval pejerrey, Odontesthes bonariensis (Cuvier et Valenciennes)

This paper presents evidence that the size of the nuclei of parenchymal cells of the liver is correlated with the nutritional status of first-feeding pejerrey larvae, Odontesthes bonariensis (Cuvier et Valenciennes). Kariometry revealed that larvae which successfully began exogenous feeding maintained the large nuclear sizes attained during the period of endogenous feeding. In contrast, nuclei of fasted larvae shrank gradually and lowest values were attained before starvation death. The degree of shrinkage seemed to be dependent on rearing temperature; reduction in nuclear area was faster and more pronounced at higher temperatures. In addition, mean hepatocyte nuclear areas of larger as well as of triploid fish are presented. The results suggest that hepatocyte nuclear size might constitute an alternative indicator for the identification of starving condition in field larval specimens.     

Dynamics and histological characteristics of gonadal sex differentiation in pejerrey (Odontesthes bonariensis) at feminizing and masculinizing temperatures

This study evaluated the effects of different temperatures on the histological process of sex differentiation in the pejerrey Odontesthes bonariensis, a fish with marked temperature-dependent sex determination (TSD), at feminizing, neutral, and masculinizing temperatures. Fish reared at three temperatures (17 degrees C, 24 degrees C, and 29 degrees C) from hatching were sampled weekly until 11 weeks and their gonads were examined by histology. The percentages of females at 17 degrees C, 24 degrees C, and 29 degrees C were 100%, 73%, and 0%, respectively. Sex differentiation occurred earlier and at a smaller body size at higher temperatures in both sexes. The first signs of ovarian differentiation were observed at 4 and 7 weeks at 24 degrees C and 17 degrees C, respectively, and those of testicular differentiation at 4 and 7 weeks at 29 degrees C and 24 degrees C, respectively. Body or gonadal growth rates before sex differentiation were not proportional to temperature and showed no sexual dimorphism at 24 degrees C, where both sexes were present. Thus, differential growth rate is probably not a factor in TSD or histological sex differentiation in pejerrey. Blood vessels were formed before sex differentiation in both sexes and at all temperatures, and may be important for sex differentiation. No signs of intersexuality were found in any of the groups, and this characterizes pejerrey as the differentiated type of gonochorist even at feminizing and masculinizing temperatures. Ovaries were formed by the same histological processes at feminizing (17 degrees C) and neutral (24 degrees C) temperatures and without any pathological features such as germ cell degeneration. The process of testicular formation was generally similar at 24 degrees C and 29 degrees C, but some fish at 29 degrees C had widespread germ cell degeneration before sex differentiation. This suggests that pathological processes leading to germ cell death, such as heat-induced dysfunction of the supporting somatic cells, could be involved in masculinization of the genetic females at high temperatures.     

Thermal threshold and histological process of heat-induced sterility in adult pejerrey (Odontesthes bonariensis): a comparative analysis of laboratory and wild specimens

Elevated water temperature has been found to cause gonadal degeneration in fish, including the partial or complete loss of germinal elements, and might impair fertility and reproductive performance. Germ cell-deficient and even completely sterile pejerrey Odontesthes bonariensis have been found in two lagoons in Argentina, and exposure to warm water is one of the possible causes of these abnormalities. This experiment was conducted (a) to compare the histological characteristics of the abnormal gonads from wild pejerrey with those of animals exposed to heat in the laboratory and (b) to examine whether short-term pulses of heat similar to diurnal temperature variations in natural environments during summer can trigger gonadal cell degeneration in adult pejerrey. Wild fish with gonadal abnormalities were obtained from the San Miguel del Monte and Lacombe Lagoons (Buenos Aires Province, Argentina). Laboratory specimens were obtained by exposure of adult pejerrey to five thermal regimes (constant 24 degrees and 29 degrees C and 12-h cycles of 27 degrees -31 degrees, 28 degrees -30 degrees, and 28 degrees -31 degrees C) for up to 16 wk. Germ cell-deficient specimens for histological comparison with wild animals were also obtained by exposing larvae and juveniles for 8-12 wk to 29 degrees C and rearing until they became adults. The histological characteristics of the abnormal gonads of wild pejerrey closely resembled those of fish partially or completely sterilized by high water temperature in the laboratory. The results indicate that fluctuating (high) thermal regimes triggered germ cell disappearance in a manner comparable to a constant temperature of 29 degrees C. These results support the notion that high temperature during unusually warm summers might trigger germ cell degeneration and could be the cause of the observed gonadal abnormalities in wild pejerrey.     

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.     

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°C, FPT), both sexes (25°C, MixPT), and only males (29°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. Mol. Reprod. Dev. 77: 521–532, 2010. © 2010 Wiley‐Liss, Inc.     

Fatty acid composition of wild Odontesthes bonariensis (Valenciennes 1835) larvae: implications on lipid metabolism and trophic relationships

The fatty acid composition of the pejerrey Odontesthes bonariensis larvae was studied to elucidate potential dietary relationships. Their principal fatty acids were characteristic for membrane lipids of aquatic organisms. The fatty acid composition varied little throughout seasons, with high proportions of 22 : 6(n‐3) (27% of total fatty acids), which is biosynthesized de novo from dietary precursor fatty acids and/or accumulated from the diet. Other major fatty acids were 16 : 0 and 18 : 0. The diatom‐typical 16 : 1(n‐7) and other dietary fatty acids (zooplankton and microplankton) are not reflected in the larvae, thus limiting the use of fatty acids as trophic markers for food web relationships of atherinopsids.