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.

Cooling of Siamese fighting fish Betta splendens (Teleostei, Osphronemidae) embryos at low temperatures.

The Siamese fighting fish (Betta splendens) has great importance as an ornamental aquarium fish as well as laboratory model species. Due to its rapid development, a cooling-embryo protocol could provide some advantages in their transportation, embryonic synchronization, and optimization of hatcheries. In this context, this work aimed to develop a protocol to storage B. splendens embryos at two temperatures (5 and 14 °C), testing three cryoprotective solutions (S1: 0.5 M sucrose, 1.5 M methanol; S2: 0.25 M sucrose, 0.75 M methanol; and S3: 0.125 M sucrose, 0.375 M methanol) and evaluating the quality of the larvae obtained. Moreover, a method to isolate the embryos from the bubble nest constructed by the male and to incubate them without parental care was applied in this study. The cooling assays were done using embryos of 24-h-post-fertilization at 26 °C and the results demonstrated that it is possible to store these embryos deprived of cryoprotectants at 5 °C for at least 6-h without negative effects. Meanwhile, S2 and S3 were the most suitable solutions for its storage for 9-h at 5 °C or 24-h at 14 °C, obtaining 77% hatching and 52% normal larvae in the first case or 88% hatching and 81% larvae with mild abnormalities in the second one. Indeed, type and frequency of larval abnormalities were evaluated and, remarkably, a partial recovery was described on malformed larvae from embryo cooled at 14 °C. Finally, this work is the first report about the cooling of B. splendens embryos and establishes the conditions for further studies on this field with this species.

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.

Effects of global warming on fish reproductive endocrine axis, with special emphasis in pejerrey Odontesthes bonariensis.

The ongoing of global warming trend has led to an increase in temperature of several water bodies. Reproduction in fish, compared with other physiological processes, only occurs in a bounded temperature range; therefore, small changes in water temperature could significantly affect this process. This review provides evidence that fish reproduction may be directly affected by further global warming and that abnormal high water temperature impairs the expression of important genes throughout the brain-pituitary-gonad axis. In all fishes studied, gonads seem to be the organ more readily damaged by heat treatments through the inhibition of the gene expression and subsequent synthesis of different gonadal steroidogenic enzymes. In view of the feedback role of sex steroids upon the synthesis and release of GnRH and GtHs in fish, it is possible that the inhibition observed at brain and pituitary levels in treated fish is consequence of the sharp decrease in plasma steroids levels. Results of in vitro studies on the inhibition of pejerrey gonad aromatase expression by high temperature corroborate that ovary functions are directly disrupted by high temperature independently of the brain-pituitary axis. For the reproductive responses obtained in laboratory fish studies, it is plausible to predict changes in the timing and magnitude of reproductive activity or even the total failure of spawning season may occur in warm years, reducing annual reproductive output and affecting future populations.

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-ß, LH-ß, 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-ß and GPH-α subunits were found in the cytoplasm of oogonia, previtellogenic and vitellogenic oocytes in ovaries. LH-ß 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.

Gonadotropin-releasing hormone neuronal development during the sensitive period of temperature sex determination in the pejerrey fish, Odontesthes bonariensis.

The development of gonadotropin-releasing hormone (GnRH) neurons was studied in relation to the sensitive period of thermolabile sex determination in the pejerrey Odontesthes bonariensis, an atherinid fish from South America. Fish were raised from hatching at three different temperatures: 17 degrees C (100% females), 24 degrees C (70% females), and 29 degrees C (100% males). Three groups of immunoreactive GnRH (ir-GnRH) neurons were identified at the terminal nerve ganglion (TNG), the midbrain tegmentum (MT), and the preoptic area (POA). Immunoreactive GnRH (ir-GnRH) neurons were identified in the TNG at hatching (day 0) and in the MT at day 3 at all the experimental temperatures. In the POA ir-GnRH neurons were identified in the nucleus preopticus periventricularis simultaneously with the first appearance of ir-GnRH fibers in the pituitary on days 11, 14, and 17 for larvae kept at 29, 24, and 17 degrees C, respectively. The number of ir-GnRH neurons in the TNG did not show any statistical difference between temperatures. The number of ir-GnRH neurons in the MT increased in number during the experiment for larvae kept at 17 and 24 degrees C but decreased between days 17 and 31 in larvae kept at 29 degrees C. The number of ir-GnRH neurons in the POA increased during development with a peak at day 28 for all temperatures studied and the magnitude of this peak showed a correlation with incubation temperature. These results reinforce the notion that the hypothalamus-pituitary-gonadal axis is active during sex determination in pejerrey suggesting a possible role of the central nervous system and GnRH in this process. It is also suggested that GnRH neurons located in the preoptic area might be the physiological transducers of temperature during the temperature sensitive period in this species.

Ontogenetic development and neuroanatomical localization of growth hormone-releasing hormone (GHRH) in the brain and pituitary gland of pejerrey fish Odontesthes bonariensis.

The presence and distribution of growth hormone-releasing hormone (GHRH) were studied by immunocytochemistry in adult and developing pejerrey fish, Odontesthes bonariensis (Atheriniformes). A few perikarya and fibers with immunoreactivity to GHRH (ir-GHRH) were identified in the olfactory bulbs at hatching. One week later, scattered ir-GHRH cell bodies were observed in the preoptic area and some fibers were detected entering the pituitary gland. Isolated ir-GHRH perikarya were revealed in the hypothalamus and in the medulla oblongata (MO) 3 weeks after hatching. Seven weeks after hatching, ir-GHRH cells were also identified in the nucleus of the lateral lemniscus and the cerebellum. Both nuclei presented strong ir-GHRH projections extending rostro-ventrally. At 11 weeks after hatching another group of ir-GHRH cells were revealed in the midbrain tegmentum. After that time the pattern of distribution of ir-GHRH structures remained unchanged. At 1 week after hatching and later, the pituitary gland consistently revealed ir-GHRH cells and fibers mainly in the proximalis pars distalis and in a minor proportion of the pars intermedia since week 1. The pineal gland showed ir-GHRH cells projecting into the pineal lumen, at week 6 after hatching and later. The pineal stalk and the subcomissural organ also presented ir-GHRH structures. Additionally, ir-GHRH material was found from week 3 to the adult stage in the following extraneural organs: gills, gut, kidney and hepatopancreas. These results represent the anatomical substrate for understanding the physiology of GHRH peptide in pejerrey, adding information on the ontogeny of neural structures expressing GHRH.

Ultrastructural study on thyroid glands of Bufo arenarum larvae kept in potassium perchlorate solution

The ultrastructure of thyroid glands in Bufo arenarum tadpoles was studied with inhibited thyroid function. After hatching, larvae were kept in potassium perchlorate solution, a goitrogenic substance known to prevent absorption of iodine from water or food, resulting in impaired thyroid hormone synthesis. Increased thyroid gland volume and follicular cell height were observed in treated larvae. The changes in size and volume of follicular cells in experimental tadpoles appeared to be mainly a function of the overstimulation of proteinpoietic apparatus. The cell shows a conspicuous development of endoplasmic reticulum and Golgi complex, as well as an increase in number of mitochondria and colloid droplets. These observations in follicular thyroid cells from Bufo arenarum, obtained under such experimental conditions, were interpreted in terms of interruption of negative feedback mechanisms involving the decrease in the thyroxine circulatory level, and therefore the increase in hypothalamo-hypophyseal endocrine secretory stimulation.

Ultrastructural study on thyroid glands of Bufo arenarum larvae kept in potassium perchlorate solution

The ultrastructure of thyroid glands in Bufo arenarum tadpoles was studied with inhibited thyroid function. After hatching, larvae were kept in potassium perchlorate solution, a goitrogenic substance known to prevent absorption of iodine from water or food, resulting in impaired thyroid hormone synthesis. Increased thyroid gland volume and follicular cell height were observed in treated larvae. The changes in size and volume of follicular cells in experimental tadpoles appeared to be mainly a function of the overstimulation of proteinpoietic apparatus. The cell shows a conspicuous development of endoplasmic reticulum and Golgi complex, as well as an increase in number of mitochondria and colloid droplets. These observations in follicular thyroid cells from Bufo arenarum, obtained under such experimental conditions, were interpreted in terms of interruption of negative feedback mechanisms involving the decrease in the thyroxine circulatory level, and therefore the increase in hypothalamo-hypophyseal endocrine secretory stimulation.(AU)