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.

Effects of atrazine on growth and sex differentiation, in juveniles of the freshwater crayfish Cherax quadricarinatus.

The effect of the herbicide atrazine was assayed in early juveniles of the redclaw crayfish Cherax quadricarinatus. Four cohorts of juveniles (a total of 280 animals) were exposed for 4 wk to each one of three atrazine concentrations (0.1, 0.5 and 2.5mg/L) or a control (0mg/L), from a commercial formulation having 90% of active principle. At the end of the exposure, no significant (p>0.05) differences in either mortality or molting were noted. However, the weight gain and the protein content of abdominal muscle decreased significantly (p<0.05) in the highest atrazine concentration as compared to control, indicating that atrazine acted as a relevant stressor, although at a concentration higher than those reported in the environment. Besides, the proportion of females increased progressively as the atrazine concentration increases, being significantly (p<0.05) higher than that of controls at the highest concentration assayed. Both macroscopic and histological analysis revealed a normal architecture of gonopores and gonads in both control and exposed animals. The obtained results strongly suggest that atrazine could be causing an endocrine disruption on the hormonal system responsible for the sexual differentiation of the studied species, increasing the proportion of female proportion without disturbing the gonad structure.

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.

Seminal fluid and spermatophore production in a western Atlantic invasive swimming crab, Charybdis hellerii, reveals a different pattern to Portunoidea.

The male reproductive system in Portunoidea is voluminous in order to produce the sperm plug, avoiding sperm competition. The portunid crab, Charybdis hellerii, is a successful invasive species, and this study describes the male reproductive system under light and electron microscopy and evaluates the gonadosomatic index and spermatophore dehiscence compared to other species that produce a sperm plug. The reproductive system is small, and its gonadosomatic index much lower than those of other Portunoidea that produce a sperm plug. The spermatophores are stored in the anterior part of the vas deferens, which is uncommon in Brachyura. Lateral outpocketings are found in the short, middle, and posterior parts of the vasa deferentia, where spermatophores are not found. The seminal fluid composition is different from the species that produce sperm plug. The usual portunid spermatophore wall is absent and the round coenospermic to even cleistospermic spermatophores are enclosed by secretions. This mucous-type spermatophore undergoes dehiscence in seawater or when passing through the gonopod. C. hellerii differ from other Portunoidea described regarding a low amount of seminal fluid production, spermatophore storage, and not show traits of producing sperm plugs. These different male reproductive features can lead to different female sperm storage in Portunoidea.

From copula to fertilization: The reproduction of primitive crab Hypoconcha parasitica (Linnaeus, 1763) (Podotremata: Dromiidae).

This study describes the mating behavior of Hypoconcha parasitica under laboratory conditions highlighting the spermathecal morphology and focusing on the seminal fluid storage and release of spermatozoa. The pairs were kept in aquaria where the mating behavior was recorded and described. The spermathecae of the female were analyzed using scanning electron microscopy, X-ray micro-CT, histology, and histochemistry. No pre- or post-copulatory mate guarding was observed in H. parasitica. The sperm transfer occurred with each pair maintaining the protection shield (bivalve shell) on their dorsum. The pair of spermathecae is covered exclusively by the cuticle, following the Podotremata pattern. Many muscle fiber bundles are attached to the cuticular wall facing toward the cephalothorax cavity, especially covering the lateral and slightly dorso-anterior region toward the apodeme of Sternite 7. The spermathecal organization indicates that the process of sperm release during fertilization occurs through muscular action exerted by the female on the wall of the chamber. Thus, the musculature distribution in Hypoconchinae distinguishes them from described for other Podotremata such as Homolidae, which shows the musculature associated with the spermathecae aperture. Like the Homolidae, the first pleopod in H. parasitica seems to take part in the transfer/ movement of spermatozoa and oocytes between the sternum and abdomen, which forms a temporary chamber where fertilization occurs. In conclusion, the spermathecal morphology and associated structures bring new insights to the mechanisms involved in the sperm storage and fertilization of primitive crabs and how the Dromiidae spermatheca perform a new pattern among the poorly studied Podotremata.

Effects of androgenic gland ablation on growth and reproductive parameters of Cherax quadricarinatus males (Parastacidae, Decapoda).

This work investigates the effects of androgenic gland (AG) ablation on the structure of the reproductive system, development of secondary sexual characters and somatic growth in Cherax quadricarinatus males. The AG ablation, which was performed at an early developmental stage (initial weight: 1.85±0.03 g), had no effect on the somatic growth parameters (specific growth rate and growth increment), but it prevented the re-formation of male gonopores and appendices masculinae. However, the red patch differentiation and chelae size were similar to those in control males. All the ablated animals developed a male reproductive system. Testis structure was macroscopically and histologically normal. The distal portion of the vas deferens (DVD) was enlarged in some animals, with histological alterations of the epithelium and the structure of the spermatophore. Results suggest that the higher growth in males than in females may be due to an indirect effect of the AG on energy investment in reproduction rather than to a direct effect of an androgen. This is the first report of a potential action of the AG on the secretory activity of the distal VD and the structural organization of the spermatophore. Although the AG may play a role in the development of male copulatory organs, its association with the red patch development deserves further research. The results obtained in the present study support and complement those from intersexes of the same species.

The influence of seasonal temperature variation on embryonic development in two species of spider crabs (Majoidea).

The aim of this study was to analyze the ecological influence of seasonal temperature variations on embryonic development in two species of Majoidea. Leurocyclus tuberculosus and Libinia spinosa. These species inhabit stable populations in the San José Gulf (42°25'41″S, 64°08'27″O, Patagonia-Argentina) in a wide depth range and seasonal temperature variations from 8 °C to 18 °C. The influence of seasonal temperatures on embryonic development was examined in ovigerous females of both species, through a bioassay in laboratory conditions at 8 °C, 14 °C, and 18 °C from gastrula to hatching. Additionally, monthly female samplings were conducted for one year, characterizing their spatial depth distribution associated with the seawater temperature in the study area and their ovigerous/non ovigerous condition. Laboratory experimental results showed that duration of incubation increased 67 % in L. tuberculosus and 55% in L. spinosa by a decline in temperature from 18 °C to 8 °C with one diapause period. The embryonic development for the 3 assayed temperatures was divided in five periods. The assayed temperatures did not affect the morphology of the embryo, and its growth during each period did not present significant differences among the different temperatures. Field analysis showed significant differences in the spatial distribution of ovigerous females which were associated to the assessed temperatures along the year. This distribution might modulate the development of embryos leading to two spawnings synchronized with the phyto- and zooplankton peaks in the San José gulf. Finally, ecological and potential impact of ocean warming in these species was also addressed.

Male reproductive strategies in two species of spider crabs, Leurocyclus tuberculosus and Libinia spinosa.

Reproductive strategies vary according to season length, individual reproductive traits, and factors associated with the social context such as density and sex ratio of the individuals. The social context predicts the intensity of sexual selection due to its effect on mate choice and intra-sexual competition. Moreover, it exacerbates the costs derived from sexual conflict and allows for the existence of alternative reproductive tactics to counteract such costs. However, these mechanisms are still underexplored in many animal taxa. Majoidea "spider crabs" show a diversity of life history, and behavioral and morphological adaptations for reproduction, which have evolved as an outcome of competition to maximize reproductive success. We aimed to analyze in this study, male reproductive strategies through mating behavior under different social contexts, density of individuals, and males size differences in two species of "spider crabs", Leurocyclus tuberculosus and Libinia spinosa. The experiments were as follows: one female and one male per aquarium (Experiment 1); a female and three males of similar sizes per aquarium (Experiment 2); and a female and three males of distinct sizes per aquarium (Experiment 3). The experiments showed that mating behavior and duration of mate guarding differ between species and among different social contexts. The presence of males with similar or different sizes showed a relevant different behavior, the female copulated with more than one male when males were similar in size (E2), and copulated only with the largest male when males were different in size (E3). In mate guarding, the shape and the use of the chelipeds had an important role: the male of Leurocyclus tuberculosus grasped the female with the chelipeds and did not fight with other males, but the male of Libinia spinosa surrounded the female with the chelipeds and fought with other males. The longest duration of mate guarding was during precopulatory guarding. Precopulatory guarding was present in all the experiments and its duration did not present significant differences among the experiments. However, in both species, the experiments differ in the duration of postcopulatory guarding until spawning. Gonopod morphology revealed important differences between species and are probably associated with sperm competition. The experiments under distinct social contexts linked to the use and shape of the chelipeds and the morphology of the gonopods, could allow to understand the functional significance of the different reproductive strategies.

Structural alterations in the male reproductive system of the freshwater crayfish, Cherax quadricarinatus (Decapoda, Parastacidae).

No diseases affecting reproductive performance have been previously reported in freshwater crayfishes. This study aims to characterise one reproductive system abnormality found in males of Cherax quadricarinatus reared in captivity. Fifteen adult males of C. quadricarinatus (70-110 g) were purchased from San Mateo S.A. farm (Entre Ríos, Argentina) each season during 2007. Macroscopic analysis showed that 26.6% of the animals sacrificed in winter presented brownish distal vasa deferentia. Histological analysis showed different levels of structural abnormality in the epithelium of the vasa deferentia and spermatophore. Granular and hyaline haemocytes were identified within the vasa deferentia but no significant differences were found in the sperm count between normal and brownish vas deferens. Histological analysis of the crayfishes sacrificed in autumn also showed these modifications in 22% of the animals, however, they did not show the brownish colour under macroscopic analysis. The similarities between the male reproductive system syndrome in shrimps and the abnormalities found in C. quadricarinatus are notable. An unspecific response to thermic stress is a possible explanation of these structural alterations.

Seminal fluid production and sperm packaging in dromiid crabs (Brachyura, Podotremata).

Reproductive anatomy, including sperm storage structures and sperm transfer, is an important feature used to analyze phylogenetic relationships among taxa. We describe the male reproductive anatomy, seminal fluid production and packaging of spermatozoa in the vas deferens of primitive crabs. In all species of Dromiidae, the testes were tubular type and the vas deferens is a tube with a simple epithelium. The spermatozoa are in a central mass immersed in type I secretion, forming a large spermatic cord. In Moreiradromia antillensis and Dromia erythropus the spermatic cord is surrounded by a more complex secretion layer composed by protein granules and polysaccharides that seem to be responsible to form the sperm plaque which was absent in Hypoconcha. The sperm plaque was found in all Dromiinae species, which may result from the mating and probably act as a barrier to subsequent copulations. These patterns of elongated coenospermic spermatophore found here are unique and different from the spermatophores of all true crabs and may be considered a plesiomorphic state to spermatophoric structure in Brachyura. Our results show a novel type of spermatozoa packaging and new insights into how sperm might be transferred in Podotremata.

Sexual system, reproductive cycle and embryonic development of the red-striped shrimp Lysmata vittata, an invader in the western Atlantic Ocean.

Several decapod crustaceans are invaders, but little is known about the biological characteristics that potentiate the success of these decapods in invaded ecosystems. Here, we evaluate and describe some aspects of the reproductive biology and development of Lysmata vittata, an invasive shrimp species in the Atlantic Ocean. In addition, we intend to provide important insights into the biology of invasion by comparing the reproductive traits of this shrimp with some of the predictions about aquatic invasive species. We used experimental and laboratory observations to evaluate the functionality of protandric simultaneous hermaphroditism (PSH), the macro and microscopic development of the ovarian portion of the ovotestes, the reproductive cycle, and the embryonic development of L. vittata. We confirm the functionality of PSH in L. vittata. This shrimp has a rapid reproductive cycle; the ovarian portion of the ovotestes develops (mean ± SD) 6.28 ± 1.61 days after spawning. Embryonic development also occurs over a short time, with a mean (± SD) of 8.37 ± 0.85 days. The larvae hatch without macroscopically visible yolk reserves. Our study provides evidence that the invasive shrimp L. vittata has reproductive and embryonic developmental characteristics (i.e., short generation time and high reproductive capacity) that may be favorable to the establishment of populations during invasive processes.

Dynamics of energy reserves and the cost of reproduction in female and male fiddler crabs.

The physiological costs of reproduction can be measured as the energy allocated to reproductive activities. In fiddler crabs, females allocate energy to vitellogenesis and brooding, whereas males perform expensive courtship behaviors. We evaluated in a large-scale study the reproduction cost of females and males of Leptuca uruguayensis in a temperate estuary, where their reproductive efforts are synchronized in a short reproductive season. The reproductive investments (vitellogenesis, spermatophore production, and male reproductive behaviors) were measured and related to the dynamics of storage and expenditure of energy reserves (glycogen, total lipids, and total protein) in the hepatopancreas, ovary, and muscle of the enlarged cheliped, throughout one annual cycle. Maximum energy storage occurred in winter, a period of low activity, whereas minimum energy storage occurred during the reproductive period, characterized by expensive activities. The glycogen reserves of the hepatopancreas decreased about 66% in females and 61% in males, suggesting high and similar physiological costs of reproduction between sexes, despite their different reproductive strategies to maximize their fitness.

Dynamics of oogenesis in ghost shrimp Callichirus major (Crustacea: Axiidea): a morphofunctional and histochemical study.

Callichirus major, popularly known as ghost shrimp, is a species of great importance in the fishing industry, because of its use as live bait. This study aimed to describe the different stages of the developing ovaries in C. major. Shrimps were collected along the Corujão beach, Piuma, Brazil (20°50'41.6"S 40°44'15.7"W), and the gonads were dissected for histological and histochemical analysis. The ovary consists of two elongated filaments covered by a connective tissue that divides the organ into lobules, where somatic and germ cells are found. It was possible to classify five types of germ cells: Oogonia (Oog), previtellogenic oocyte (Oc1), early vitellogenic oocyte (Oc2), late vitellogenic oocyte (Oc3) and mature oocyte (Oc4) based on their vitellogenic stage, cytoplasmic, nuclear and morphometric characteristics. The histochemical analysis demonstrated an intense reaction for proteins and polysaccharides in peripheral cytoplasm of Oc3 comparing to others cell types. According to size, volume, color intensity and distribution of oocyte types the gonads were classified into: immature, developing, developed and spent, being in females at this last stage, observed empty follicles and oocytes in reabsorption process. During oogenesis was observed a gradual increase in cytoplasmic acidophilia due to accumulation of yolk granules and the intense histochemical reaction in periphery of Oc3, which indicate the beginning of an extravitellogenic source of nutrients. Based on the microscopic analysis of the vitellogenesis, shrimp C. major showed the initial short phase of oocyte growth following with a fast vitellogenic cycle.

Compensatory Growth in Juveniles of Freshwater Redclaw Crayfish Cherax quadricarinatus Reared at Three Different Temperatures: Hyperphagia and Food Efficiency as Primary Mechanisms.

Feeding restriction, as a trigger for compensatory growth, might be considered an alternative viable strategy for minimizing waste as well as production costs. The study assessed whether juvenile redclaw crayfish Cherax quadricarinatus (initial weight 0.99 ± 0.03 g) was able to compensate for feeding restriction at different temperatures (23 ± 1, 27 ± 1 and 31 ± 1 ° C). Hyperphagia, food utilization efficiency, energetic reserves, and hepatopancreas structure were analyzed. Three temperatures and two feeding regimes (DF-daily fed throughout the experiment and CF- 4 days food deprivation followed by 4 days of feeding, intermittently) were tested. The restriction period was from day 1 to 45, and the recovery period was from day 45 to 90. The previously restricted crayfish held at 23, 27, and 31 ± 1 ° C displayed complete body weight catch-up through compensatory growth following the restriction period with depressed growth. The mechanisms that might explain this response were higher feed intake (hyperphagia), and increased food utilization efficiency. Hepatopancreatic lipids were used as a metabolic fuel and hepatosomatic index was reduced in the previously restricted crayfish, but recovery at the same level of unrestricted crayfish occurred after the shift to daily feeding. The highest temperature affected adversely growth, feed intake, food efficiency, and metabolism of crayfish, whereas the lowest temperature and feeding restriction induced a more efficient growth of the crayfish.

Toxicity of mercury during the embryonic development of Chasmagnathus granulatus (Brachyura, Varunidae).

Ovigerous females of the estuarine crab Chasmagnathus granulatus were exposed to mercury (0.1mg/L) during the entire, early, or late embryonic development. A delay in the egg incubation period and some morphological abnormalities were detected in larvae hatched from exposed females. Particularly, hypopigmentation of body chromatophores was the abnormality that showed the highest incidence, this incidence being greater when ovigerous females were exposed to mercury either during the totality or just the first half of the egg incubation period. In contrast, the effect of mercury on the morphology and pigmentation of eyes was greater when the exposure comprised the totality or just the second half of the incubation period. These results correlate with the timing of both body pigment synthesis and eye formation during embryonic development. Although these abnormalities have been observed in the same species with other heavy metals, such as zinc and copper, the responsiveness during the early and late embryonic development was different with mercury.

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.

Diferenciación sexual en la langosta de agua dulce Cherax quadricarinatus (Decapoda: Parastacidae) / Sexual differentiation in the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae)

Determinamos el momento del desarrollo postembrionario en que se produce la diferenciación sexual primaria en la langosta Cherax quadricarinatus. Esta es evidenciada por la presencia de las gónadas y sus respectivos conductos. También determinamos la diferenciación sexual definida por la aparición de los caracteres sexualessecundarios. Se observaron 797 machos, 506 hembras y 456 individuos intersexos de 0.02 a 89.96 g (de criadero y laboratorio). Disecamos una submuestra de 106 machos, 69 hembras y 59 individuos intersexos para la caracterización macroscópica de la estructura gonadal. La diferenciación de los gonoporos se inicia aproximadamente a los 0.10 g, en sincronía con la diferenciación del sistema reproductor en machos, hembras e intersexos. La adquisición de la forma definitiva de ovario, oviducto, testículo y vaso deferente son posteriores. El appendix masculina iniciasu diferenciación a los 0.12-0.2 g y adquiere los rasgos característicos del appendix de los adultos a partir de 1-2 g.La diferenciación de la mancha roja (red patch) ocurre a partir de los 2.3 g.

We determined the earliest stage of postembryonic development at which primary and secondary sexual differentiations occur in the freshwater (“red claw”) crayfish Cherax quadricarinatus. For this purpose,797 males, 506 females and 456 intersex specimens within a weight range of 0.02-89.96 g were observed under stereoscopic microscope to determine the presence of the genital openings at the basis of the third (females) or fifth (males) pair of pereiopods. Animals presenting both pairs of genital openings were considered as intersex. A subsample of 106 males, 69 females and 59 intersex were dissected for the macroscopic characterization of gonad morphology. The development of the genital openings began approximately at 0.10 g, simultaneously with the differentiation of the reproductive system in females, males and intersex. Although the differentiation of the reproductive system started very early in the postembryonic development, the definitive form and colour of ovaries, oviducts, testes and vasa deferentia was acquired later. The differentiation of the appendix masculina began at 0.12-0.2 g and acquired the elongated shape of the adult at 1-2 g. The soft red patch characteristic of adult males started at 2.3 g of body weight in both chelipeds.

Intersex females in the red claw crayfish, Cherax quadricarinatus (Decapoda: Parastacidae)

Cherax quadricarinatus is a large freshwater crayfish species Parastacidae) native of north-west Queensland and the Northern Territory of Australia. The species typically exhibits a gonochoristic sexual system, although in cultured populations various types of intersex individuals have been described as functional males. In the present study, the macroscopic morphology and the gonadal histology of one type of intersex are described and discussed. All intersexes having both pairs of genital openings (female and male openings) and lacking both appendix masculinae and red patches were functional females with normal ovaries and oviducts. From a histological point of view, they did not differ from normal females having previtellogenic and/or vitellogenic ovaries according to size.

Cherax quadricarinatus, es un astácido dulceacuícola de gran tamaño de la (familia Parastacidae) originario delnoroeste de Queensland y del norte de Australia. Presenta un sistema sexual gonocórico, aunque en poblaciones decultivo se han descrito varios tipos de individuos intersexos como machos funcionales. En el presente estudio se describe y discute la morfología macroscópica y la histología gonadal de un tipo de intersexos. Todos los intersexos que presentan ambos pares de aberturas genitales (femeninas y masculinas) y carecen de ambos apéndices masculinos y de la mancha roja, fueron hembras funcionales con ovarios y oviductos normales. Desde el punto de vista histológico no difieren de las hembras normales, presentando ovarios previtelogénicos y/o vitelogénicos de acuerdo a su tamaño.