Daniel A. Medesani

Ovarian growth in the crab Chasmagnathus granulata induced by hormones and neuroregulators throughout the year. In vivo and in vitro studies

Abstract The hormones 17α-hydroxyprogesterone and juvenile hormone III (JHIII), as well as the dopaminergic antagonist spiperone, were assayed both in vivo and in vitro on adult females of the estuarine crab Chasmagnathus granulata at different times of the year. All the chemicals were administered both by injection and by incorporation into the food. Each of these in vivo assays lasted 1 month, and they were done during three different periods of the year, in accordance with the reproductive cycle of this crab (pre-, post- and reproductive periods). Spiperone and progesterone were always administered at a dose of 10 −8 mol/crab, while JHIII at 10 −7 mol/crab. The assays done during the reproductive period (spring and summer) yielded a significantly higher gonadosomatic index (GSI) than that of the concurrent control for all the chemicals assayed, whether injected or incorporated into the food. During the pre-reproductive period (winter), only progesterone and JHIII increased the GSI, while for the post-reproductive period (autumn), only spiperone and progesterone did so. These differences may be related to the phases of the annual reproductive cycle that is synchronized by both external and internal stimuli. In vitro assays that involved a 24-h incubation of isolated pieces of ovary from females sampled during the reproductive period were also done. A significant increase of leucine uptake by the ovary fragments was observed by merely adding either progesterone or JHIII to the incubation medium, but a similar effect of spiperone was only observed when thoracic ganglion (TG) was also added. Spiperone was also able to reverse the inhibitory effect caused by eyestalk tissue (ET) added to medium. The results of these in vitro assays support the hypothesis, previously suggested, about the inhibitory role of dopamine in crustacean reproduction acting on neuroendocrine organs such as TG and ET.

Effects of lead and cadmium on hatching of the southern king crab Lithodes santolla (Decapoda, Anomura)

Summary Eggs of the king crab Lithodes santolla were exposed to cadmium (0.2 and 2 mg/1) and lead (0.16 and 1.6 mg/1) for 30 days. Concentrations used represented 1/10 and 1 of the respective LC50 values for zoea I of the studied species. Proportion of larvae hatching, meantime of eclosion and proportion of abnormal larvae were recorded. Abnormalities detected were hydropsy; small size; and atrophy of dorsal, rostral and telson spines, as well as atrophy of pereiopods and telson setae. The mean time of eclosion was inversely correlated to the incidence of structural abnormalities such as atrophy of spines and setae. These results suggest that both cadmium and lead caused an early eclosion of underdeveloped larvae, but while cadmium caused also a significant increase in hatching rate, lead produced a significant decrease in the proportion of larvae hatching.

In vitro effects of both dopaminergic and enkephalinergic antagonists on the ovarian growth of Cherax quadricarinatus (Decapoda, Parastacidae), at different periods of the reproductive cycle

The in vitro effect of both spiperone (dopaminergic antagonist) and naloxone (enkephalinergic antagonist), was assayed on small pieces of ovary dissected from C. quadricarinatus females, with the eventual addition of some neuroendocrine organs, such as thoracic ganglion or eyestalk tissue. The incorporation of tritiated leucine by the ovary was measured in order to estimate the ovarian growth. During the post-reproductive period, both mentioned antagonists were able to significantly stimulate the ovary in the presence of thoracic ganglion, but did not produce any significant effect in the preparation containing ovary and eyestalk tissue, or only ovary. No significant effects of the assayed antagonists were noted during the pre-reproductive period. These results were in accordance with previous models describing the neuroendocrine control of crustacean reproduction, and represent new findings about the hormonal context in different periods of the reproductive cycle of crayfish. Besides, by means of the experimental combination of the tested antagonists with dopamine or met-enkephalin, a new model dealing with the interaction of these two neurotransmitters on the hormonal secretion of thoracic ganglion has been proposed.

Acute and chronic effects of cadmium on blood homeostasis of an estuarine crab, Chasmagnathus granulata, and the modifying effect of salinity

Whole body oxygen consumption and some hemolymph parameters such as pH, partial pressure of gases, level of ions and lactate were measured in the estuarine crab Chasmagnathus granulata after both acute (96 h) and chronic (2 weeks) exposure to cadmium at concentrations ranging from 0.4 to 6.3 mg/l. In all instances, the crabs developed hemolymph acidosis, but no respiratory (increased PCO2) or lactate increases were evident. Hemolymph levels of sodium and calcium were always increased by cadmium exposure. The chronic toxicity of cadmium was enhanced at 12 per mil salinity, even causing a significantly higher mortality in comparison with the higher salinity (30 per mil ) used. A general metabolic arrest took place at 12 per mil salinity in the crabs chronically exposed to cadmium, as indicated by decreases of oxygen consumption and PCO2, an increase of PO2, along with no changes in lactate levels. These imbalances were associated with severe necrosis and telangiectasia in the respiratory gills, probably leading to respiratory impairment and finally histotoxic hypoxia and death of the animals.

Induction of ovarian growth in the red claw crayfish, Cherax quadricarinatus, by the enkephalinergic antagonist naloxone: in vivo and in vitro studies

Summary The effects of spiperone and domperidone (dopaminergic antagonists), as well as naloxone (enkephalinergic antagonist) on ovarian growth of the crayfish Cherax quadricarinatus, were evaluated during the non-reproductive period. In vivo assays were carried out by administering these compounds by adding them to the crayfish food, and subsequently measuring the oocyte diameter (oocytes in secondary vitellogenesis). Only naloxone produced a significantly (p <0.05) higher oocyte diameter than that of the controls, suggesting that this antagonist was blocking the effects of endogenous enkcephalins on the secretion of one or more neurohormones involved in crustacean reproduction. To test this hypothesis, an in vitro incubation of ovarian pieces with thoracic ganglion (TG), a source of the gonad stimulating hormone (GSH), or eyestalk tissue (ET), the source of the gonad inhibiting hormone (GIH), with or without naloxone added to the incubation medium, was done. A significant increase of ovarian growth was observed when TG was present. Moreover, the addition of naloxone to this preparation was able to further increase the ovarian growth, presumably by preventing enkephalinergic inhibition of GSH secretion.

Levels of ions and organic metabolites in the adult freshwater crayfish, Cherax quadricarinatus, exposed to different salinities

Adult male crayfish, Cherax quadricarinatus, were exposed to the salinities of 0 (control), 7.5, 15, 25 and 35 g L−1 for 3 weeks. Survival, oxygen consumption rate, hemolymphatic levels of glucose, lactate, sodium, potassium, pH, and free amino acids (FAA), as well as FAA concentration in muscle, were determined. Survival was significantly reduced (p < 0.05) only at the highest salinity assayed, together with a significant (p < 0.05) increase of glucose and decrease of lactate. Concerning ions, a hyper-regulation of both hemolymphatic sodium and potassium was maintained up to a salinity of 15 g L−1, at which hemolymph became isoionic with ambient water. A certain degree of ionic hypo-regulation was also noted. Hemolymph pH levels decreased as salinity increased from 0 to 15 g L−1. FAA content in muscle showed an increase parallel to that of hemolymphatic sodium, in accordance with their expected function in the isosmotic regulation of muscular cells.

In vitro effect of methyl farnesoate on the vitellogenin content of ovary and hepatopancreas, in the crayfish Cherax quadricarinatus

Vitellogenin levels were determined in pieces of either ovary or hepatopancreas taken from females of the crayfish Cherax quadricarinatus during both early pre-reproductive and reproductive periods, and exposed in vitro to 0.15, 1.5, or 15 µM of the juvenoid methyl farnesoate (MF). A significant accumulation of vitellogenin was seen in ovaries treated with 15 µM of MF during the early pre-reproductive period. Besides, protein synthesis, measured as tritiated leucine incorporation, was correspondingly increased in ovaries taken during both pre- and post-reproductive periods and exposed to 15 µM of MF. On the other hand, no stimulating effect of MF on the vitellogenin content of the hepatopancreas was seen in any period. These results, taken together, support the hypothesis of the endogenous vitellogenin synthesis stimulated by MF during the early ovarian maturation. However, this hormone was not able to stimulate vitellogenin synthesis in the ovary during the reproductive period.

Effects of atrazine on ovarian growth, in the estuarine crab Neohelice granulata

Atrazine, a herbicide that is intensively used in Argentina, was assayed to evaluate the alteration of reproduction in a wild species of crustaceans. Adult females of the estuarine crab Neohelice granulata were exposed to formulated atrazine during the 3-month pre-reproductive period. Three atrazine concentrations (0.03, 0.3 and 3mg/L) were assayed, together with a water dilution control. At the end of the exposure period, several variables concerning the energetic status of animals were measured, such as weight gain, glycemia, and energy reserves in both muscle and hepatopancreas. The gonadosomatic index (GSI) was also determined, as well as the proportion and relative area of each oocyte type in histological sections. Besides, the total content of vitellogenin proteins (Vg) in both ovary and hepatopancreas was measured. A significant (p<0.05) decrease of glycogen content was observed in muscle, while a significant (p<0.05) lower area of both previtellogenic and vitellogenic oocytes was verified in the ovary by effect of atrazine, in correspondence with a Vg content significantly (p<0.05) diminished in the ovary and augmented in the hepatopancreas. Besides, a higher proportion of previtellogenic oocytes were seen by effect of atrazine. Taken together, these results indicate a clear reduction and delay in the ovarian growth of the studied species, during the period at which the ovary normally grows up prior to spawning. On the other hand, a decrease of Vg content was observed at 0.3 and 3mg/L of atrazine after 24-h in vitro assays carried out with ovarian explants, providing additional evidence about the inhibitory role of this herbicide on the ovarian growth. These results encourage future research on deleterious effects of atrazine on crustacean reproduction.

Induction of vitellogenesis by 17-hydroxyprogesterone and methyl farnesoate during post-reproductive period, in the estuarine crab Neohelice granulata

Adult female crabs (Neohelice granulata) were treated during the post-reproductive period with both 17-hydroxyprogesterone (17PG) and methyl farnesoate (MF). During the 4-week in vivo assay, animals were fed hormone-enriched (17PG or MF) pellets twice a week, at a dose of 2 nmol/g of body weight. Additionally, at the middle of the in vivo experiment, ovarian explants were incubated in vitro with each hormone at a concentration of 15 μM for 24 h. At the end of the in vivo assay, both the gonadosomatic index and the vitellogenic protein content (Vg) of crabs treated with either 17PG or MF were significantly higher (p < 0.05) than the values of the concurrent controls. In addition, the hemolymphatic vitellogenin level of both hormonally treated groups was higher. At the end of the in vitro assay, ovary explants incubated with 17PG had a significantly higher (p < 0.05) protein synthesis and content of Vg proteins than controls. Ovary explants incubated with MF had similar levels to controls (p > 0.05). The involvement of these hormones in the regulation of ovarian growth during the reproductive cycle of N. granulata is discussed.

Effects of atrazine on vitellogenesis, steroid levels and lipid peroxidation, in female red swamp crayfish Procambarus clarkii

Atrazine, a widely use herbicide, has been classified as a potential endocrine disruptor, especially for freshwater species. In this study, we tested the hypothesis that atrazine can affect reproduction in crayfish through dysregulation of vitellogenin expression and hormone synthesis. Adult female crayfish (Procambarus clarkii) were exposed during one month to atrazine at concentrations of either 1 or 5 mg/L. At the end of the exposure, ovaries, hepatopancreas, and hemolymph samples were harvested for analysis of vitellogenin expression and steroid hormone levels. Ovarian tissue was also sampled for both biochemical and histological analyses. Our results show that atrazine-exposed crayfish had a lower expression of vitellogenin in the ovary and hepatopancreas, as well as smaller oocytes, and reduced vitellogenin content in the ovary. Despite these effects, circulating levels of estradiol increased in females exposed to 5 mg/L of atrazine, showing that the inhibiting effect of atrazine on vitellogenin production was not related to a lower secretion of sexual steroids. Instead, some early stimulating effects of estradiol on vitellogenesis could have occurred, particularly in the hepatopancreas. On the other hand, atrazine caused a higher metabolic effort, in terms of lactate production, presumably triggered to provide the energy needed to face the unspecific stress produced by the herbicide. Lipid peroxidation was not affected by atrazine, but glutathione levels were significantly increased.