Inhibition of Oocyte Maturation in the Estuarine Crab Neohelice Granulata, by the Effect of Anti-Inflammatory Drugs.

The drugs ibuprofen and diclofenac were assessed in vivo on adult females of the estuarine crab Neohelice granulata. In a first, preliminary assay comprising 60-d, a significant (p < 0.05) lower content of total vitellogenic proteins was detected in the ovary at 10 mg/L of each drug. In a second 90-d assay, comprising the exposure of crabs to 5 mg/L of each drug during the entire pre-reproductive period, a significant (p < 0.05) decrease in the proportion of vitellogenic oocytes was observed by effect of diclofenac. The same effect was also observed in a third assay only comprising the last month of the pre-reproductive period, at 5 mg/L of diclofenac, and also at a mixture of both drugs; besides, this mixture significantly (p < 0.05) increased the proportion of reabsorbed vitellogenic oocytes. The obtained results indicate that the effect of diclofenac is critical at the final stage of ovarian maturation, when the participation of prostaglandins is relevant.

In vitro Interference of a Glyphosate Commercial Formulation with the Stimulation of Ovarian Maturation by Progesterone, in the Estuarine Crab Neohelice granulata.

This study was aimed at evaluating the possible interference of formulated glyphosate to the endocrine control of ovarian growth exerted by progesterone, in the estuarine crab Neohelice granulata. The addition of Roundup Ultramax® (0.2 mg/L of glyphosate in the incubation medium) was able to potentiate the stimulating effect of progesterone on total vitellogenic protein (Vg) content of ovarian pieces from crabs. Moreover, the sole addition of mifepristone (antagonist of progesterone receptors) was able to produce a decrement of the Vg content, which was not reverted by the addition of Roundup. A similar result was confirmed by means of histological analysis, which showed that mifepristone, both alone and in combination with Roundup, inhibited ovarian maturation, while Roundup alone increased it, in terms of a higher proportion of vitellogenic oocytes. We conclude that Roundup could stimulate the progesterone secretion exerted by the ovary and/or could act as a partial agonist of this hormone in the same tissue.

Imbalances in the male reproductive function of the estuarine crab Neohelice granulata, caused by glyphosate.

The effect of glyphosate, both pure and formulated (Roundup Ultramax®), was evaluated on males of the estuarine crab Neohelice granulata, by means of both in vivo and in vitro assays. The in vivo assays comprised the exposure for 30 d to 1 mg/L of the herbicide, until finally assessing weight gain, levels of energy reserves, sperm number per spermatophore, proportion of abnormal spermatophores, and sperm viability. At the end of this assay, significant (p < 0.05) decrease in weight gain and muscle protein levels was detected by effect of both pure and formulated glyphosate. In spermatophores from the vas deferens, a significant (p < 0.05) decrease of the sperm count was observed by effect of Roundup, while a significant incidence (p < 0.05) of abnormal spermatophores was observed either with glyphosate or with Roundup treatment. No changes were seen in the spermatophore area or in vas deferens secretions. Since no sperm mortality was induced by the formulated herbicide, we propose a probable inhibiting effect on spermatogenesis might explain the observed sperm count decrease. In this sense, an in vitro assay was designed by incubating testes and vasa deferentia with Roundup, in order to corroborate the possible interference of glyphosate with the secretion of the androgenic gland hormone that controls the spermatogenesis, in the presence or absence of the androgenic gland. Although the herbicide per se was able to reduce the sperm count to some extent, the increase in the number of spermatozoa/spermatophore produced by the co-incubation with the androgenic gland was completely reverted by the addition of Roundup (1 mg/L of glyphosate a.e.), suggesting that an inhibition on the secretion and/or transduction of the androgenic gland hormone could be taking place.

Hard-shell mating in Neohelice granulata: the role of ecdysone in female receptivity and mate attraction.

Most brachyuran females become receptive during the intermolt period, a condition considered "derived". However, as far as we know, studies testing the existence and function of pheromones in decapods are based on species which have mating linked to molting, a condition considered as "ancestral". For the first time, we studied some physiological and morphological processes involved in Neohelice granulata intermolt female crabs becoming receptive and potentially attracting males. We found that receptive females have mobile vulvae opercula due to a softening process of the cuticle hinge which showed lower calcium levels compared to the hinge of unreceptive females. Local softening of the hinge was stimulated by a low concentration of ecdysone during the intermolt period. A putative pheromone liberated by receptive females to attract males is presumed to be released through the mobile vulvae and not through the urine.

Effects of glyphosate on sperm quality of the estuarine crab Neohelice granulata, under the "One Health" perspective.

This study was aimed at evaluating the effects of the commercial formulation Roundup Ultramax® on sperm mortality and viability, in terms of the capacity of spermatozoids (spz) to trigger the acrosome reaction (AR), using the estuarine crab Neohelice granulata as a model. To this, an in vivo assay comprising 100 days duration was carried out, on a control group and two groups exposed to the formulation (0.01 mg/L and 0.2 mg/L of glyphosate) under controlled conditions of photoperiod, feeding, and temperature. At the end of the assay, the right vas deferens (VD, proximal and middle portion) was dissected, and after homogenizing it in calcium-free saline solution, the acrosome reaction was induced in the phase containing the spz. In each treatment, the percentage of spz with total and partial AR was calculated, as well as that of dead spz. Compared to the control, crabs exposed to the herbicide showed a significant decrease in spz with full AR, together with an increase in the percentage of spz with partial AR. Furthermore, spz mortality was significantly higher in both glyphosate concentrations compared to the control, in a concentration-dependent manner. On the other hand, abnormal spermatophores, showing expanded walls and coalescence, were observed in a significant percentage in the left VD of the groups treated with the herbicide. The results obtained are compared with those from other studies on several invertebrate and vertebrate species that found inhibition of the AR and abnormal sperm, together with inhibition of spermatogenesis, endocrine disruption, and reduced sperm motility by effect of pure glyphosate and/or different glyphosate formulations. In summary, the available evidence highlights the possible impact of glyphosate on sperm quality, in a wide variety of species.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle.

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

Effects of the Herbicide Atrazine on Crustacean Reproduction. Mini-Review.

Atrazine, one of the most intensively applied herbicides worldwide, is commonly found in several water bodies, affecting the associated fauna. Autochthon crustacean species have been relatively less studied, compared to vertebrate species, particularly concerning reproductive success. In this mini-review, we summarize the relevant information about the effects of atrazine exposure on the main reproductive aspects of crustaceans. One of these effects is related to the inhibition of ovarian growth. In this respect, a diminished vitellogenin content was found in the ovary of crabs exposed to atrazine during the entire period of ovarian growth, in correlation with a reduced oocyte size and a delay of ovarian maturation. Similar results were observed in crayfish. Atrazine was also able to affect the reproductive process, acting as an endocrine disruptor. In this sense, this herbicide was suspected to affect the secretion of some neurohormones involved in the gonadal growth, as well as to alter the circulating levels of steroid hormones which promote the synthesis of vitellogenin for ovarian growth. Moreover, atrazine induced sexual differentiation in juvenile crayfish toward a higher proportion of females, while it produced an increment of males in daphnids. Another aspect affected by this herbicide was the reduction of offspring production, as well as several embryonic abnormalities; genotoxic effects have been also reported in crayfish. Finally, some metabolic imbalances, such as reduction in energy reserves, have been observed in some species, together with oxidative stress and histopathological effects.

The Effect of Glyphosate on the Reproduction of Estuarine Crabs: Neohelice granulata as a Study Model.

This review summarizes the bulk of evidence about the effect of glyphosate, both technical and formulated, on the ovarian maturation of Neohelice granulata female crabs, as well as the effects of glyphosate on sperm production in males of the same species. After long-term in vivo assays, made during the 3-month pre-reproductive period of this species, both formulated and technical glyphosate were able to produce a significant incidence of oocyte reabsorption in the ovary, together with a concomitant decreased of vitellogenin content, at concentrations ranging from 0.2 to 1 mg/L. Despite this, after 32-day in vivo assays, glyphosate stimulated oocyte growth, in terms of a higher percentage of vitellogenic oocytes, suggesting that glyphosate could be acting as an endocrine disruptor. In vitro assays made with isolated ovarian pieces showed a decrease of vitellogenin content, in correlation with lower protein synthesis, although some advance in maturation was observed in the histological analysis. In male crabs exposed in vivo to both technical and formulated glyphosate at 1 mg/L, several reproductive imbalances were noted, such as a significant decrease of the sperm count, abnormal spermatophores, and possible disrupting effects of glyphosate on the androgenic gland.

Interference of an atrazine commercial formulation with the endocrine control of ovarian growth exerted by the eyestalks.

Atrazine is currently one of the most used herbicides worldwide. We tested the possible effect of the widely used herbicide atrazine on the endocrine control of ovarian growth exerted by the neurohormones secreted at the eyestalk of the estuarine crab Neohelice granulata. For this, both in vivo and in vitro assays were carried out. The in vivo assay comprised the exposure for 1 month to 3 mg/L of a commercial formulation containing 90% of atrazine as active ingredient (Gesaprim 90 WDG®, Syngenta) on three categories of females: intact, ablated of one eyestalk, and ablated of both eyestalks. At the end of the assay, only the intact females showed a significant (p < 0.05) decrease in both content of vitellogenic ovarian proteins and proportion of vitellogenic oocytes, compared to a concurrent control. The results of the in vitro incubation of ovarian pieces with the eventual addition to the incubation medium of eyestalk tissue and/or atrazine at 3 mg/L showed a significant (p < 0.05) decrease in the proportion of vitellogenic oocytes only when atrazine and eyestalk tissue were added. Taken together, these results strongly suggest that the assayed atrazine formulation may act as an endocrine disruptor at the eyestalk level, by altering the normal secretion of some eyestalk hormone, therefore inhibiting ovarian growth.

Ovarian growth impairment after chronic exposure to Roundup Ultramax® in the estuarine crab Neohelice granulata.

Adult females of the estuarine crab Neohelice granulata were exposed to the glyphosate formulation Roundup Ultramax® during the entire 3-month pre-reproductive period. At the end of the assay, a significant higher increment of glycemia was noted at both glyphosate concentrations assayed (0.01 and 0.2 mg/L, acid equivalent). Although no differences were observed in the gonadosomatic index, a significantly higher proportion of reabsorbed vitellogenic oocyte was observed at the highest glyphosate concentration, together with a significant decrease of vitellogenin content in the ovary. In addition, some in vitro assays were carried out by co-incubating small pieces of ovary with or without the addition of Roundup; at both concentrations tested (same as those used in vivo), a decrease in the ovarian vitellogenin content was observed, whereas the ovarian protein synthesis was significantly inhibited by glyphosate at 0.2 mg/L in the Roundup formulation used.

Involvement of the crustacean hyperglycemic hormone (CHH) in the physiological compensation of the freshwater crayfish Cherax quadricarinatus to low temperature and high salinity stress.

This study was aimed at determining the role of the crustacean hyperglycemic hormone (CHH) in the physiological compensation to both saline and thermal stress, in the freshwater crayfish Cherax quadricarinatus. By determining the expression of the CHH gene in the eyestalk of juvenile crayfish, we found that maximal induction of CHH was induced at high salinity (10 g/L) and low temperature (20 °C). In order to investigate the role of CHH in the physiological compensation to such stressful conditions, recombinant CHH was supplied to stressed animals. CHH-injected crayfish showed increased hemolymphatic levels of glucose, in accordance with a significant utilization of glycogen reserves from the hepatopancreas. Furthermore, CHH administration allowed stressed animals to regulate hemolymphatic sodium and potassium at more constant levels than controls. Taken together, these results suggest a relevant role of CHH in increasing the energy available intended for processes involved in the physiological compensation of C. quadricarinatus to both saline and thermal stress.

Endocrine disruption in crustaceans due to pollutants: a review.

The main endocrine-regulated processes of crustaceans have been reviewed in relation to the effects of endocrine-disrupting compounds (EDCs). Molting has been shown to be inhibited by several organic pollutants, such as xenoestrogens and related compounds, as well as by some pesticides. Most of these disrupters are thought to interfere with ecdysone at target tissues, although only for a few has this action been demonstrated in vitro. The heavy metal cadmium appears to inhibit some ecdysone secretion. Juvenoid compounds have also been shown to inhibit molting, likely by interfering with the stimulatory effect of methyl farnesoate. A molt-promoting effect of emamectin benzoate, a pesticide, has also been reported. As for reproduction, a variety of organic compounds, including xenoestrogens, juvenoids and ecdysteroids, has produced abnormal development of male and female secondary sexual characters, as well as alteration of the sex ratio. Cadmium and copper have been shown to interfere with hormones that stimulate reproduction, such as methyl farnesoate, as well as with secretion of the gonad inhibiting hormone, therefore affecting, for example, ovarian growth. Several heavy metals were able to produce hyperglycemia in crustaceans during short times of exposure; while a hypoglycemic response was noted after longer exposures, due to inhibition of secretion of the crustacean hyperglycemic hormone. The ecological relevance of EDCs on crustaceans is discussed, mainly in relation to the identification of useful biomarkers and sentinel species. New experimental approaches are also proposed.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

La langosta de agua dulce Cherax quadricarinatus es una especie tropical de gran interés para la acuicultura. Se midió la concentración de vitelogenina (Vg) en hemolinfa, ovario y hepatopáncreas de hembras adultas de esta especie, por medio de ELISA. Las mediciones fueron hechas en los cuatro períodos del ciclo reproductivo: pre-reproductivo, reproductivo medio, reproductivo tardío y post-reproductivo. Se detectaron variaciones significativas en los niveles de Vg tanto en hemolinfa como en hepatopáncreas, se observó el mayor valor durante el período reproductivo medio. Además, tales variaciones se correlacionaron positivamente con el índice hepatosomático. Se observó además una correlación positiva de los niveles de Vg entre hepatopáncreas y ovario. Estos resultados apoyan evidencias previas sobre el papel central del hepatopáncreas como sitio de síntesis de Vg, en esta especie, y también enfatizan la importancia de la hemolinfa para el transporte de la Vg del hepatopáncreas al ovario. Para propósitos de acuicultura, la medición de Vg en hemolinfa podría ser utilizada como un método no lesivo, con el fin de constatar la actividad reproductiva de hembras de C. quadricarinatus.

Effect of methyl farnesoate, alone and in combination with other hormones, on ovarian growth of the red swamp crayfish, Procambarus clarkii, during vitellogenesis.

The effect of methyl farnesoate (MF) on the ovaries of female red swamp crayfish, Procambarus clarkii, undergoing vitellogenesis was determined both in vivo and in vitro. The in vivo assay showed a positive effect of MF on oocyte growth when injected alone and in combination with 17 beta-estradiol, but not in combination with JHIII or 17 alpha-hydroxyprogesterone. A higher level of incorporation of labeled leucine was induced by MF on isolated pieces of ovary. The same effect was seen when ovary and mandibular organ (MO) were coincubated. These results suggest that MF stimulated the synthesis of vitellin in the ovary of crayfish. In vitro, 17 alpha-hydroxyprogesterone completely suppressed the stimulatory action of the MO on the ovary, suggesting a competitive inhibition between 17 alpha-hydroxyprogesterone and MF on the ovary and/or a negative feedback by that steroid on the MO.

Effects of some steroids and other compounds on ovarian growth of the red swamp crayfish, Procambarus clarkii, during early vitellogenesis.

The effects of spiperone, a dopaminergic receptor blocker, the terpenoid JHIII, and the steroids 17alpha-hydroxyprogesterone and 17beta-estradiol (10(-7) mol/crayfish) were assayed in vivo on females of the red swamp crayfish, Procambarus clarkii, during early vitellogenesis. All these compounds were injected twice a week for 3 weeks. The rate of molting in response to these compounds was also determined. At the end of the experiment the ovaries were dissected out, in order to determine the gonadosomatic indexes and oocyte diameters. Spiperone, 17alpha-hydroxyprogesterone, and 17beta-estradiol produced a significant increase in the gonadosomatic index, while only the first two compounds were able to significantly increase the oocyte diameter; therefore, the effect of 17beta-estradiol the on gonadosomatic index could be merely due to increased water uptake by the ovary. Spiperone could be causing a decrease in secretion of the gonad and/or mandibular inhibiting hormones from the eyestalks, and 17alpha-hydroxyprogesterone could be having a direct stimulatory effect on vitellogenin production. Concerning molting, of the compound tested, only JHIII had a significant (P < 0.05) stimulatory effect, producing a 100% molting rate versus 66.7% among the controls.