Function analysis and molecular characterization of cyclin A in ovary development of oriental river prawn, Macrobrachium nipponense.

Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA, with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycAmRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense.

Developmental and reproductive effects in grass shrimp (Palaemon pugio) following acute larval exposure to a thin oil sheen and ultraviolet light.

Many early stages of estuarine species congregate at the surface or in the upper mixing layer making them prone to UV light exposure and oil sheens. Laboratory testing was used to assess UV-oil sheen interactions with grass shrimp (Palaemon pugio). Newly hatched grass shrimp larvae were exposed to a 1-µm thick oil sheen for 24 h with or without an 8-h pulse of UV light. Grass shrimp were then transferred to clean seawater and non-UV conditions to measure development, growth, and reproductive fitness. Minimal toxicity was observed after the initial exposure but larval development was significantly delayed in shrimp exposed to the UV enhanced sheen. After reaching sexual maturity, shrimp were paired to evaluate effects on reproduction. Shrimp initially exposed to the UV enhanced sheen as larvae had a significant reduction in fecundity compared to controls. This demonstrates the importance of examining interactions between UV light and oil since negative effects to aquatic organisms may be underestimated if based on standard laboratory fluorescent lighting. Acute exposures of early life stages to thin oil sheens and UV light may lead to long-term impacts to individuals and ultimately to grass shrimp populations.

Ultraviolet-B radiation induces transcriptional modulation of components associated with the extracellular matrix in embryos of decapod Macrobrachium olfersii.

The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.

Kinetic characterization of the gill (Na+, K+)-ATPase in a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from a hololimnetic population of the diadromous Amazon River shrimp Macrobrachium amazonicum. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)ATP-ase activity, but also containing other microsomal ATPases. Only a single immune-reactive (Na+, K+)-ATPase with Mr of ≈110 kDa is present that hydrolyzes ATP with VM = 130.3 ±â€¯4.8 nmol Pi min-1 mg protein-1 and K0.5 = 0.065 ±â€¯0.00162 mmol L-1, exhibiting site-site interactions. Stimulation by Na+ (VM = 127.5 ±â€¯5.3 nmol Pi min-1 mg protein-1, K0.5 = 5.3 ±â€¯0.42 mmol L-1), Mg2+ (VM = 130.6 ±â€¯6.8 nmol Pi min-1 mg protein-1, K0.5 = 0.33 ±â€¯0.042 mmol L-1), K+ (VM = 126.7 ±â€¯7.7 nmol Pi min-1 mg protein-1, K0.5 = 0.65 ±â€¯0.0079 mmol L-1) and NH4+ (VM = 134.5 ±â€¯8.6 nmol Pi min-1 mg protein-1, K0.5 = 1.28 ±â€¯0.44 mmol L-1) also obeys cooperative kinetics. Ouabain (KI = 0.18 ±â€¯0.058 mmol L-1) inhibits total ATPase activity by ≈70%. This study reveals considerable differences in the kinetic characteristics of the gill (Na+, K+)-ATPase in a hololimnetic population that appear to result from the adaptation of diadromous Macrobrachium amazonicum populations to different limnic habitats.

Growth, antioxidant capacity, intestinal morphology, and metabolomic responses of juvenile Oriental river prawn (Macrobrachium nipponense) to chronic lead exposure.

Understanding the mechanisms of metal toxicity to organisms farmed for food may suggest mitigation strategies. We determined the 24-, 48-, 72-, and 96-h median lethal concentrations of lead in juvenile oriental river prawn (Macrobrachium nipponense). The prawns were then exposed to sub-lethal concentrations (13.13 and 26.26 µg/L) of lead for 60 days and growth, antioxidant enzyme activity, intestinal morphology, and metabolite profiles were assessed. Prawns exposed to 26.26 µg/L but not to 13.13 µg/L lead exhibited lower weight gain than controls. The lead burden in muscle was 0.067 and 0.25 µg/g of dry weight exposed to 13.13 and 26.26 µg/L, respectively. Levels of glutamic oxaloacetic transaminase and glutamic-pyruvic transaminase were not altered following exposure. Exposure increased malondialdehyde activity in the hepatopancreas and decreased superoxide dismutase and glutathione peroxidase activities. Catalase activity first increased and then decreased as lead concentrations increased. Some intestinal epithelial cells disassociated from the basement membrane in prawns exposed to 13.13 µg/L lead. Intestinal epithelial cells in prawns exposed to 26.26 µg/L lead separated completely from the basement membrane. Gas chromatography-mass spectrometry metabolomics assays showed the 13.13-µg/L exposure did not elicit significant metabolic alterations. Exposure to 26.26 µg/L lead differentially up-regulated 58 metabolites and down-regulated 21 metabolites. The metabolites identified were involved in galactose, purine, glutathione, and carbon metabolism, biosynthesis of amino acids and steroids, and neuroactive ligand-receptor interaction. These data indicate that chronic lead exposure can adversely affect growth, increase accumulation in muscle, impair intestinal morphology, and induce oxidant stress or neurotoxicity-related effects in M. nipponense.

Toxicokinetics of cadmium in Palaemon varians postlarvae under waterborne and/or dietary exposure.

The present study assessed cadmium uptake and depuration rates in the euryhaline estuarine shrimp Palaemon varians under different exposure routes. Postlarval shrimp were exposed for 4 d under different exposure routes: contaminated water, contaminated diet, and a 2-way exposure scenario where both contaminated water and diet were used. After exposure, postlarval shrimp were transferred to a clean medium and fed a noncontaminated diet for 96 h. Bioaccumulation via the different exposure routes was modeled with a standard first-order, one-compartment toxicokinetics model and one with an additional parameter reflecting an inert fraction or storage compartment. The simultaneous 2-way exposure (through water and diet) resulted in accumulation being almost twice as high as the sum of the individual exposure routes, thus indicating that accumulation from multiple routes may be more than additive. Cadmium uptake from water was faster than uptake from food maintained for 48 h at that same cadmium concentration. Shrimp were unable to eliminate cadmium from their body, showing no depuration during 96 h after exposure via different routes, thus suggesting that a longer depuration period is needed. Model comparisons did not provide a significantly better fit when the model included the presence of an inert fraction. The present study highlights the importance of assessing accumulation using multiple exposure routes compared with individual routes because the latter may underestimate bioaccumulation. Environ Toxicol Chem 2018;37:1614-1622. © 2018 SETAC.

Abundance and spatial-temporal distribution of Macrobrachium surinamicum Holthuis, 1948 (Palaemonidae) in the Amazon estuary, north of Brazil / Abundância e distribuição espaço-temporal de Macrobrachium surinamicum Holthuis, 1948 (Palaemonidae) em um estuário Amazônico, norte do Brasil

Abstract Macrobrachium surinamicum is a small shrimp that inhabits rivers of low salinity. It is mainly caught as bycatch in Amazon shrimp Macrobrachium amazonicum fisheries, which is widely exploited by artisanal fisheries for food and economic needs of the riverside population. This study aimed to characterize the spatial and temporal distribution of the freshwater shrimp M. surinamicum in the Guajará Bay and on Mosqueiro Island, correlating the abundance of this species with abiotic factors (temperature and salinity). Samples were taken from May 2006 to April 2007 in six locations: Mosqueiro Island (Furo das Marinhas and Porto do Pelé); Icoaraci district; Arapiranga Island, edge of the city of Belém; and Combu Island, using traps named 'matapis'. A total of 361 shrimps were caught. The abundance was higher in December and lower in July 2006. The biggest catch occurred on Arapiranga Island and the lowest on Mosqueiro Island. The abundance differed significantly in December 2006 and no variable studied had significant influence on M. surinamicum abundance. In Guajará Bay, particularly the more sheltered places, as Arapiranga and Combu islands, favor the development of M. surinamicum, indicating that this species has preference for less disturbed areas.

Resumo Macrobrachium surinamicum é um camarão de pequeno porte que habita a foz de rios e águas de baixa salinidade. Sua captura na Amazônia ocorre principalmente como fauna associada à pesca do camarão-da-Amazônia Macrobrachium amazonicum que é largamente explorado pela pesca artesanal atendendo as necessidades alimentícias e econômicas da comunidade ribeirinha. O presente estudo teve como objetivo caracterizar a distribuição espaço-temporal do camarão dulcícola M. surinamicum na Baía do Guajará e Ilha de Mosqueiro, correlacionando a abundância desta espécie com fatores abióticos (temperatura e salinidade). Os exemplares foram capturados no período de maio/06 a abril/07 em seis locais: Ilha de Mosqueiro (Furo das Marinhas e Porto do Pelé), Distrito de Icoaraci, Ilha do Arapiranga, orla de Belém e Ilha do Combu com armadilhas (matapis). Um total de 361 camarões foi capturado, sendo a maior abundância em dezembro e a menor em julho de 2006. A maior captura foi na Ilha de Arapiranga e a menor na Ilha de Mosqueiro. A abundância diferiu significativamente em dezembro/06 e nenhuma variável estudada teve influência significativa na abundância de M. surinamicum. A Baía do Guajará, especialmente os locais mais abrigados como a Ilha de Arapiranga e do Combu, propiciam o desenvolvimento de M. surinamicum, o que indica que esta espécie tenha preferência para áreas menos antropizadas.

An eclosion hormone-like gene participates in the molting process of Palaemonid shrimp Exopalaemon carinicauda.

Molting behavior is an important physiological process related to metamorphosis, growth, and reproduction in crustaceans. Previous studies indicated that the molting process was controlled by 20-hydroxyecdysone (20E) and upstream hormones, peptides, and environmental factors, which regulate 20E function. Eclosion hormone (EH) in insect is a kind of neuropeptide that is regulated by 20E and triggers ecdysis behavior at the end of molting process. However, the function of eclosion hormone gene during the molting process in crustaceans is still largely unknown. In the present study, an eclosion hormone-like gene EcEHL was identified from Exopalaemon carinicauda. The deduced amino acid sequence of EcEHL contained a signal peptide, a typical eclosion domain, and six conserved cysteine residues forming three putative disulfide bonds. EcEHL was predominantly expressed in the epidermis, gill, and eyestalk of shrimp. In situ hybridization analysis showed that EcEHL transcripts were localized in gill cells and in medulla externa X-organ, medulla terminalis X-organ, sinus gland, and lamina ganglionaris of eyestalks. During the molting process of shrimp, EcEHL showed the highest expression level in shrimp at the premolt stage. The expression level of EcEHL in shrimp at mid premolt stage was up-regulated by injection of exogenous 20E. Silencing of EcEHL using double-stranded RNA delayed both the molting process and ecdysis rate of E. carinicauda. Furthermore, injection of exogenous 20E to shrimp at mid premolt stage (D2) could remarkably speed up the molting process and also raise the ecdysis rate of E. carinicauda. The results revealed that EcEHL might participate in the molting process of shrimp and its expression was regulated by 20E. These data will help us to understand the molecular mechanism of molting in crustacean.

Dietary Supplementation of Magnesium Oxide (MgO) Nanoparticles for Better Survival and Growth of the Freshwater Prawn Macrobrachium rosenbergii Post-larvae.

This study was performed to assess the growth-promoting potential of dietary magnesium oxide nanoparticles (MgO NPs) in Macrobrachium rosenbergii post-larvae (PL). MgO NPs were supplemented at 0, 100, 200, 300, 400 and 500 mg kg-1 with the basal diet (containing 0.95 g Mg kg-1); the concentrations of Mg in MgO NP-supplemented diets were increased correspondingly (1.07, 1.15, 1.24, 1.37 and 1.46 g Mg kg-1 respectively). MgO NP-supplemented diets were fed to M. rosenbergii PL (initial weight 0.11 ± 0.04 g) for a period of 90 days. In the carcasses of experimental prawns, the content of Mg was found to be elevated significantly with respect to the individual diet (102.14, 183.29, 205.46, 221.03, 237.10 and 254.36 µg Mg g-1 respectively) when compared with that of the control. The contents of Cu, Zn, Fe, Ca, Na and K levels were also found to be elevated in the carcasses of experimental prawns. Significant (P < 0.05) improvements were observed in nutritional indices [survival rate (SR), weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER)], activities of digestive enzymes (protease, amylase and lipase), concentrations of basic biochemical constituents (total protein, amino acid, carbohydrate, lipid, profiles of amino acids and fatty acids) and population of haemocytes [total and differential (hyalinocytes, semigranulocytes and granulocytes)] in all the test PL. Maximum performance was recorded in 500 mg kg-1 MgO NP-supplemented-feed-fed PL. There were no significant elevations recorded in activities of antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)], lipid peroxidation (LPO) and metabolic enzymes [glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)] recorded in any of the MgO NP-supplemented-feed-fed PL when compared with the control. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed increases in the staining intensity of polypeptide bands resolved in 500 mg kg-1 MgO NP-supplemented-feed-fed PL when compared with the control. Based on the gradual improvement in attaining survival, growth, FCR, biochemical constituents and haemocyte population, this study recommends MgO NP supplementation of 500 mg kg-1 for sustainable maintenance of M. rosenbergii PL. As the studied highest concentration of MgO NPs showed the best performance, it is necessary to study with beyond 500 mg kg-1 of MgO NPs to optimize the actual concentration.

Development and assays estradiol equivalent concentration from prawn (p-EEQ) in river prawn, Macrobrachium nipponense, in Taiwan.

The present study established a fast and convenient bioassay method for aqueous ecosystems using the prawn estradiol equivalent concentration (p-EEQ) of male Macrobrachium nipponense, which produce vitellogenin (VTG) after exposure to xeno-estrogens. This method was then used to determine the concentrations of xeno-estrogen pollutants in the rivers of Taiwan. To establish the calibration curve for the concentrations based on the p-EEQ, the induced VTG content was determined using the alkali-labile phosphate method after male M. nipponense were exposed to 0, 10, 100, 1,000 and 10,000ng/L of 17ß-estradiol for 1, 3, 5, 7, 10 and 14 days, respectively. The results of the experiments showed that the induced VTG content in all of the experimental groups stabilized after 10 days, except for the 10,000ng/L experimental group, in which the induced VTG content decreased after 10 days. A 17ß-estradiol-VTG10day response curve was then established based on the induced VTG content in the 0, 10, 100 and 1000ng/L experimental groups at day 10. After establishing the curve, male M. nipponense were captured from the upper, middle and lower reaches of the Chuo-shui River, the Beigang River, the Jishui River, the Agongdian River and the Sichong River in Taiwan, and the VTG content in these prawns was determined. In addition, the p-EEQ in the waters was determined based on the VTG content, and the estradiol equivalent concentration (EEQ) in the waters was also measured immediately after sampling using the solid-phase extraction-enzyme-linked immunosorbent assay (SPE-ELISA) method. The results showed that the p-EEQ in the middle and lower reaches of the rivers in certain parts of Taiwan ranged from 38 to 400ng/L, and the detection rate was 100%. Moreover, the EEQ ranged from 7.9 to 92.9ng/L, and the detection rate was 42.9%, indicating that most of the middle and lower reaches of the rivers in Taiwan were polluted by xeno-estrogens. The 17ß-estradiol concentrations determined based on the p-EEQ were all higher than those based on the EEQ (SPE-ELISA method). The results of the present study showed that the use of M. nipponense to determine the p-EEQ in environmental waters provided advantages that included a high detection rate, high sensitivity and convenience. However, the p-EEQ cannot be used in waters that do not contain M. nipponense.

Risk assessment of pesticides used in rice-prawn concurrent systems in Bangladesh.

The objectives of the current study were to determine the occupational health hazards posed by the application of pesticides in rice-prawn concurrent systems of south-west Bangladesh and to assess their potential risks for the aquatic ecosystems that support the culture of freshwater prawns (Macrobrachium rosenbergii). Information on pesticide use in rice-prawn farming was collected through structured interviews with 38 farm owners held between January and May of 2012. The risks of the pesticide use to human health were assessed through structured interviews. The TOXSWA model was used to calculate pesticide exposure (peak and time-weighted average concentrations) in surface waters of rice-prawn systems for different spray drift scenarios and a simple first tier risk assessment based on threshold concentrations derived from single species toxicity tests were used to assess the ecological risk in the form of risk quotients. The PERPEST model was used to refine the ecological risks when the first tier assessment indicated a possible risk. Eleven synthetic insecticides and one fungicide (sulphur) were recorded as part of this investigation. The most commonly reported pesticide was sulphur (used by 29% of the interviewed farmers), followed by thiamethoxam, chlorantraniliprole, and phenthoate (21%). A large portion of the interviewed farmers described negative health symptoms after pesticide applications, including vomiting (51%), headache (18%) and eye irritation (12%). The results of the first tier risk assessment indicated that chlorpyrifos, cypermethrin, alpha-cypermethrin, and malathion may pose a high to moderate acute and chronic risks for invertebrates and fish in all evaluated spray drift scenarios. The higher tier assessment using the PERPEST model confirmed the high risk of cypermethrin, alpha-cypermethrin, and chlorpyrifos for insects and macro- and micro-crustaceans thus indicating that these pesticides may have severe adverse consequences for the prawn production yields.

Developmental effects of exposure to ultraviolet B radiation on the freshwater prawn Macrobrachium olfersi: Mitochondria as a target of environmental UVB radiation.

In South America, increased UVB radiation has become an important environmental issue that is potentially threatening aquatic ecosystems. Considering that species exhibit different degrees of sensitivity to UVB radiation and that embryos are more sensitive than organisms at later life stages, the aim of this study was to characterize the effects of UVB radiation on subcellular compartments of embryos of the freshwater prawn Macrobrachium olfersi. This species lives and reproduces in clear and shallow waters, where UV radiation can fully penetrates. Embryos were irradiated with a UVB 6W lamp for 30min and examined after 1h, 12h, 24h and 48h of exposure. The irradiance of the UVB used simulates the UV radiation that embryos receive in the natural environment. The subcellular compartment most affected by the UVB radiation was the mitochondria, which exhibited a circular shape, a decrease in mitochondrial cristae, rupture of membranes and a morphology compatible with fission. These impairments were observed simultaneously with increased ROS production, just after 1h of UVB exposure. Thus, we investigated proteins related to mitochondrial fission (Drp-1) and fusion (Mfn-1), which are essential to cell maintenance. We found a significant increase in Drp-1 expression at all analyzed time-points and a significant decrease in Mfn-1 expression only after 24h of UVB exposure. Additionally, a decrease in embryonic cell viability was verified via the mitochondrial integrity assay. To conclude, we observed important mitochondrial dysfunctions against the environmental stress caused by UVB radiation. Moreover, the cellular responses found are critical and should not be disregarded, because they impact embryos that can potentially compromise the aquatic ecosystems.

Osmoregulation in larvae and juveniles of two recently separated Macrobrachium species: Expression patterns of ion transporter genes.

In this comparative study, osmoregulatory mechanisms were analyzed in two closely related species of palaemonid shrimp from Brazil, Macrobrachium pantanalense and Macrobrachium amazonicum. A previous investigation showed that all postembryonic stages of M. pantanalense from inland waters of the Pantanal are able to hyper-osmoregulate in fresh water, while this species was not able to hypo-osmoregulate at high salinities. In M. amazonicum originating from the Amazon estuary, in contrast, all stages are able to hypo-osmoregulate, but only first-stage larvae, late juveniles and adults are able to hyper-osmoregulate in fresh water. The underlying molecular mechanisms of these physiological differences have not been known. We therefore investigated the expression patterns of three ion transporters (NKA α-subunit, VHA B-subunit and NHE3) following differential salinity acclimation in different ontogenetic stages (stage-V larvae, juveniles) of both species. Larval NKAα expression was at both salinities significantly higher in M. pantanalense than in M. amazonicum, whereas no difference was noted in juveniles. VHA was also more expressed in larvae of M. pantanalense than in those of M. amazonicum. When NHE3 expression is compared between the larvae of the two species, further salinity-related differences were observed, with generally higher expression in the inland species. Overall, a high expression of ion pumps in M. pantanalense suggests an evolutionary key role of these transporters in freshwater invasion.

Characterization of the male-specific lethal 3 gene in the oriental river prawn, Macrobrachium nipponense.

In this study, male-specific lethal 3 homolog (Mnmsl3) was cloned and characterized from the freshwater prawn Macrobrachium nipponense (Crustacea: Decapoda: Palaemonidae) by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnmsl3 showed high-sequence homology to the insect Msl3 and contained a conserved chromatin organization modifier domain and an MORF4-related gene domain. Real-time quantitative reverse transcription-polymerase chain reaction showed that the Mnmsl3 gene was expressed in all the investigated tissues, with the highest level of expression in the testis. The expression level of Mnmsl3 between males and females was different in the gonad (testis or ovary), abdominal ganglion, and heart. The results revealed that the Mnmsl3 gene might play roles in regulating chromatin and in dosage compensation of M. nipponense. Real-time quantitative reverse transcription-polymerase chain reaction also revealed that Mnmsl3 mRNA expression was significantly increased in both 5 and 20 days post-larvae after metamorphosis, suggesting that Mnmsl3 plays complex and important roles in the early embryonic development and sex differentiation of M. nipponense.

Effect of salinity on the metabolism and osmoregulation of selected ontogenetic stages of an amazon population of Macrobrachium amazonicum shrimp (Decapoda, Palaemonidae) / Efeito da salinidade sobre o metabolismo e a osmorregulação de estágios ontogenéticos selecionados de uma população amazônica do camarão Macrobrachium amazonicum (Decapoda, Palaemonidade)

Probably as a function of their wide geographical distribution, the different population of Macrobrachium amazonicum shrimp may present distinct physiological, biochemical, reproductive, behavioral, and ecological patterns. These differences are so accentuated that the existence of allopatric speciation has been suggested, although initial studies indicate that the genetic variability of populations happen at an intraspecific level. Among the biological responses described for M. amazonicum populations, those regarding osmoregulation and metabolism play a key role for being related to the occupation of diverse habitats. To this effect, we investigated osmoregulation through the role of free amino acids in cell volume control and metabolism, through oxygen consumption in larvae (zoeae I, II, V and IX) and/or post-larvae of a M. amazonicum population from Amazon, kept in aquaculture fish hatcheries in the state of São Paulo. The results add information regarding the existence of distinct physiological responses among M. amazonicum populations and suggest that possible adjustments to metabolism and to the use of free amino acids as osmolytes of the regulation of the larvae and post-larvae cell volume depend on the appearance of structures responsible for hemolymph osmoregulation like, for example, the gills. In this respect, we verified that zoeae I do not alter their metabolism due to the exposition to fresh or brackish water, but they reduce intracellular concentration of free amino acids when exposed to fresh water, what may suggest the inexistence or inefficient performance of the structures responsible for volume regulation and hemolymph composition. On the other hand, in zoeae II and V exposed to fresh and brackish water, metabolism alterations were not followed by changes in free amino acids concentration. Thus it is possible, as the structures responsible for osmoregulation and ionic regulation become functional, that the role of free amino acids gets diminished and oxygen consumption elevated, probably due to greater energy expenditure with the active transportation of salts through epithelial membranes. Osmotic challenges also seem to alter throughout development, given that in zoeae II oxygen consumption is elevated on brackish water of 18, but in zoeae V it happens in fresh water. After M. amazonicum metamorphosis, free amino acids begin to play an important role as intracellular osmolytes, because we verified an increase of up to 40% in post-larvae exposed to brackish water of 18. The main free amino acids involved in cell volume regulation of ontogenetic stages evaluated were the non essential ones: glutamic acid, glycine, alanine, arginine, and proline. Interestingly, larvae from estuarine population studied here survived until the zoeae V stage in fresh water, but in some populations far from the sea, zoeae die right after eclosion in fresh water or they do not reach zoeae III stage. In addition, given that in favorable conditions caridean shrimp larvae shorten their development, we may infer that the cultivation environment, in which larvae developed in the present work, was appropriate, because almost all zoeae VIII kept on brackish water underwent metamorphosis directly to post-larvae and did not go through zoeae IX stage.

Provavelmente como função da sua ampla distribuição geográfica, as diferentes populações do camarão Macrobrachium amazonicum podem apresentar distintos padrões fisiológicos, bioquímicos, reprodutivos, comportamentais e ecológicos. Essas diferenças são tão acentuadas que tem sido sugerido a existência de especiação alopátrica embora estudos iniciais indiquem que a variabilidade genética das populações ocorre ao nível intraespecífico. Dentre as respostas biológicas descritas para as populações de M. amazonicum, aquelas relacionadas à osmorregulação e metabolismo têm papel central por estarem relacionadas à ocupação dos diversos habitats. Nesse sentido, investigou-se a osmorregulação, por meio do papel dos aminoácidos livres no controle do volume celular e o metabolismo, por meio do consumo de oxigênio, em larvas (zoeas I, II, V e IX) e/ou pós-larvas de uma população de M. amazonicum oriunda da Amazônia e mantida em viveiros de aquicultura no estado de São Paulo. Os resultados adicionam informações a respeito da existência de respostas fisiológicas distintas entre as populações de M. amazonicum e sugerem que possíveis ajustes no metabolismo e no uso de aminoácidos livres como osmólitos da regulação do volume celular das larvas e pós-larvas dependem do surgimento de estruturas responsáveis pela osmorregulação da hemolinfa como, por exemplo, as brânquias. Nesse sentido, verificou-se que as zoeas I não alteram seu metabolismo em função da exposição à água doce ou salobra, mas reduzem a concentração intracelular de aminoácidos livres quando expostas à água doce, o que pode sugerir a inexistência ou um desempenho ineficiente das estruturas responsáveis pela regulação do volume e composição da hemolinfa. Por outro lado, nas zoeas II e V expostas à água doce ou salobra alterações no metabolismo não foram acompanhadas por mudanças na concentração dos aminoácidos livres. Assim é possível que à medida que estruturas responsáveis pela osmo e ionorregulação tornam-se funcionais, o papel dos aminoácidos livres se torne reduzido e o consumo de oxigênio elevado, provavelmente em função do maior gasto energético com o transporte ativo de sais através das membranas epiteliais. Os desafios osmóticos também parecem se alterar ao longo do desenvolvimento visto que em zoeas II o consumo de oxigênio é elevado em água salobra de 18 mas em zoeas V essa resposta ocorre em água doce. Após a metamorfose de M. amazonicum, os aminoácidos livres passam a ter papel importante como osmólitos intracelulares, pois se verificou um aumento de até 40% nas pós-larvas expostas à água salobra de 18. Os principais aminoácidos livres envolvidos na regulação do volume celular dos estágios ontogenéticos avaliados foram os não essenciais ácido glutâmico, glicina, alanina, arginina e prolina. Interessantemente, as larvas da população estuarina aqui estudada sobrevivem até o estágio de zoea V em água doce mas em algumas populações distantes do mar as zoeas morrem logo após a eclosão em água doce ou não chegam ao estágio de zoea III. Adicionalmente, visto que em condições favoráveis as larvas de camarões carídeos abreviam o seu desenvolvimento pode ser inferido que o meio de cultivo em que as larvas se desenvolveram no presente trabalho foi adequado, pois quase todas as zoeas VIII mantidas em água salobra sofreram diretamente a metamorfose para pós-larvas e não passaram pelo estágio de zoeas IX.

Integrated multi-trophic culture of Nile tilapia (Oreochromis niloticus) and Amazon river prawn (Macrobrachium amazonicum) in brackish water / Cultivo multi-trófico integrado de tilápia-do-nilo (Oreochromis niloticus) e camarão da Amazônia (Macrobrachium amazonicum) em água salobra

The aim of this study was to assess the feasibility of integrated multi-trophic culture of Nile tilapia (Oreochromis niloticus) and Amazon River prawn (Macrobrachium amazonicum) in brackish water by evaluating its limnological characteristics and economic performance. The experiment was completely randomized with four treatments and four repetitions: control treatment with Nile tilapia only, stocked with 2 tilapias/m² (P2C0) and three integrated multi-trophic culture treatments stocked with 2 tilapias/m² and prawns at densities of 4, 8 and 16 prawns/m² (P2C04, P2C08 and P2C16, respectively). The limnological variables of temperature, pH, dissolved oxygen, turbidity, ammonia, orthophosphate and chlorophyll "a" were evaluated and throughout the experiment remained within the limits recommended for culture. The experiment lasted 150 days with monthly animal sampling. No significant differences were observed for total fish biomass or for fish and prawn total survival rates. However, prawn individual weight decreased as stocking density increased. Gross revenue was not significantly different between treatments, as well as profitability. The profitability was 40.1% (P2C0), 36.7% (P2C04), 41.2% (P2C08) and 50.1% (P2C16). It is concluded that although feasible from the view point of husbandry, the integrated multi-tropic culture of M. amazonicum and O. niloticus did not influence significantly profitability compared to the monoculture system.

O objetivo deste estudo foi verificar a viabilidade do cultivo multitrófico integrado da tilápia-do-nilo (Oreochromis niloticus) e do camarão-da-amazônia (Macrobrachium amazonicum) em água salobra, mediante a avaliação de suas características limnológicas e de seu desempenho econômico. O experimento foi inteiramente ao acaso, com quatro tratamentos e quatro repetições: tratamento controle somente com tilápia-do-nilo, estocado com duas tilápias/m² (P2C0) e três tratamentos de cultivo multitrófico integrado, estocados com duas tilapias/m² e camarões nas densidades de quatro, oito e 16 camarões/m² (P2C04, P2C08 e P2C16, respectivamente). As variáveis limnológicas temperatura, pH, oxigênio dissolvido, turbidez, amônia, ortofosfato e clorofila "a" foram avaliadas e, durante todo o experimento, mantiveram-se dentro dos limites recomendados para o cultivo. O experimento durou 150 dias, com amostragem mensal dos animais. Diferenças significativas não foram observadas para a biomassa total de peixe nem para a taxa de sobrevivência total em peixes e camarões. Entretanto, o peso individual do camarão diminuiu à medida que a densidade de estocagem aumentou. A receita bruta não foi significativamente diferente entre os tratamentos, assim como a rentabilidade. A rentabilidade foi 40,1% (P2C0), 36,7% (P2C04), 41,2% (P2C08) e 50,1% (P2C16). Conclui-se que, apesar de viável do ponto de vista da larvicultura, o cultivo multitrófico integrado de M. amazonicum e O. niloticus não influenciou significativamente a rentabilidade quando comparado com o sistema de monocultura.

[Effects of salinity on spawning and larval development of Exopalaemon carinicauda].

Female Exopalaemon carinicauda at ovarian maturation stage II rearing by artificial propagation in the laboratory were chosen as test material. The shrimps were gradually acclimated to the experimental salinity levels of 2, 5, 10, 15, 20, 25 and 30 to determine the effects of salinity on spawning, embryonic development and larval growth of E. carinicauda. The results showed that the ovaries of female E. carinicauda could mature at all test salinity levels. However, it failed to spawn at salinity 2. Salinity levels from 10 to 20 were the most favorable for female E. carinicauda spawning. Although larval development was most successful in eggs incubated at salinity levels of 5 to 30, embryonic development were significantly affected by salinity, and the egg incubation period was shorter at salinities of 15, 20 and 25 than at the other salinities. There were no significant effects of salinity on the rates of larval metamorphosis and survival, but the dry mass of individuals was significantly affected by salinity. The dry mass of shrimps reared at salinities of 15 and 20 were significantly higher than at the other salinities. The growth of 20-day old shrimps was significantly affected by salinity. The specific growth rate increased with the increasing salinity level from 5 to 20, and then decreased at the salinity above 20. The mRNA level of gill Na(+)-K(+)-ATPase was high at high and low salinities, and the lowest at the theoretical salinity 17.5, which might be equal to the hemolymph osmotic pressure of E. carinicauda. It was implied that female parent E. carinicauda could reproduce in a wide range of salinities, while 20-day old shrimps presented higher growth rates at salinities near its theoretical isosmotic point.

Reproductive aspects of Macrobrachium amazonicum (Decapoda: Palaemonidae) in the State of Amapá, Amazon River mouth / Aspectos reprodutivos do camarão Macrobrachium amazonicum (Decapoda: Palaemonidae) no estado do Amapá, foz do rio Amazonas

Macrobrachium amazonicum is an indigenous prawn vastly distributed in basins of South America, widely exploited by artisanal fisheries in northern and northeastern Brazil and, with great potential for aquaculture. This study aimed to investigate general aspects of population structure and reproductive characteristics (size at first maturity, fecundity and reproductive output) of M. amazonicum from two important areas to artisanal prawn fishing located at the mouth of the Amazon River, State of Amapá. The specimens were captured using 20 handcrafted traps called "matapi". A number of 5,179 prawns were captured, 2,975 females and 2,195 males resulting in 1.35:1 female to male ratio. Santana Island and Mazagão Velho showed females predominated in the population. A reproductive peak period was observed from January to April/2009 and in December/2010, coinciding with the period of higher rainfall. The recruitment peak occurred in June and July/2009. Egg-bearing females ranged in size (carapace length) from 11.10 to 29.6 mm. Fecundity increased with female size and reached up to 7,417 eggs. This amount of eggs is considered low if compared with other Macrobrachium estuarine species. Mean egg volume increased gradually from 0.121 to 0.24 mm³ during embryogenesis, representing 68.5% of overall increase from Stage I to Stage III. Eggs of M. amazonicum are small; this is typical for Macrobrachium species, which depends on brackish water to complete the larval development. Irrespective of female size, reproductive output of M. amazonicum varied between 4.8 and 21.85% of their body weight into eggs production.

Macrobrachium amazonicum é uma espécie nativa vastamente distribuída nas bacias da America do Sul, sendo largamente explorada por pescadores artesanais no Norte e Nordeste do Brasil e com grande potencial para aquicultura. O objetivo deste trabalho foi abordar aspectos gerais da estrutura populacional e biologia reprodutiva (tamanho de primeira maturação, fecundidade e investimento reprodutivo) de M. amazonicum de duas importantes áreas de pesca artesanal de camarão localizada na foz do rio Amazonas, no Estado do Amapá. Os exemplares foram capturados usando 20 armadilhas artesanais, conhecidas localmente como matapi. Um total de 5.179 camarões foi capturado, dos quais 2.975 eram fêmeas e 2.195 eram machos, dando uma razão sexual geral de 1,35: 1 em favor das fêmeas. As proporções sexuais nas áreas de Ilha de Santana e Mazagão mostraram fêmeas predominando na população. Os picos reprodutivos nas duas áreas ocorreram de Janeiro a Abril/2009 e em Dezembro/2010, coincidindo com o período mais chuvoso. Os picos de recrutamento foram observados em junho e julho de 2009. O tamanho das fêmeas ovígeras variou de 11,10 mm a 29,6 mm de comprimento de carapaça. A fecundidade aumentou com o tamanho das fêmeas, chegando até 7.417 ovos, podendo ser considerada baixa comparada a outras espécies estuarinas de Macrobrachium. Volume médio dos ovos aumentou gradualmente durante a embriogênese saindo de 0,112 para 0,220 mm³, representando um aumento global de 68,5% do I ao III estágio embrionário. Os ovos de M. amazonicum podem ser considerados pequenos, típicos de espécies de Macrobrachium que dependem de água salgada para completar seu desenvolvimento larval. Independente do tamanho da fêmea, o investimento reprodutivo de M. amazonicum variou entre 4,8 e 21,85% do seu peso corporal para a produção de ovos.

Epidermal growth factor receptor in the prawn Macrobrachium rosenbergii: function and putative signaling cascade.

Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase receptor superfamily found in metazoans and plants. In arthropods, EGFRs are vital for the proper development of embryos and of adult limbs, gonads, and eyes as well as affecting body size. In searching for genes involved in the growth and development of our model organism, the decapod crustacean (Macrobrachium rosenbergii), a comprehensive transcript library was established using next-generation sequencing. Using this library, the expression of several genes assigned to the signal transduction pathways mediated by EGFRs was observed, including a transcript encoding M. rosenbergii EGFR (Mr-EGFR), several potential ligands upstream to the receptor, and most of the putative downstream signal transducer genes. The deduced protein encoded by Mr-EGFR, representing the first such receptor reported thus far in crustaceans, shows sequence similarity to other arthropod EGFRs. The M. rosenbergii gene is expressed in most tested tissues. The role of Mr-EGFR was revealed by temporarily silencing the transcript through weekly injections of double-stranded Mr-EGFR RNA. Such treatment resulted in a significant reduction in growth and a delay in the appearance of a male secondary sexual characteristic, namely the appendix masculina. An additional function of Mr-EGFR was revealed with respect to eye development. Although the optic ganglion appeared to have retained its normal morphology, Mr-EGFR-silenced individuals developed abnormal eyes that presented irregular organization of the ommatidia, reflected by unorganized receptor cells occupying large areas of the dioptric portion and by a shortened crystalline tract layer.

The apparent quorum-sensing inhibitory activity of pyrogallol is a side effect of peroxide production.

There currently is more and more interest in the use of natural products, such as tea polyphenols, as therapeutic agents. The polyphenol compound pyrogallol has been reported before to inhibit quorum-sensing-regulated bioluminescence in Vibrio harveyi. Here, we report that the addition of 10 mg · liter(-1) pyrogallol protects both brine shrimp (Artemia franciscana) and giant river prawn (Macrobrachium rosenbergii) larvae from pathogenic Vibrio harveyi, whereas the compound showed relatively low toxicity (therapeutic index of 10). We further demonstrate that the apparent quorum-sensing-disrupting activity is a side effect of the peroxide-producing activity of this compound rather than true quorum-sensing inhibition. Our results emphasize that verification of minor toxic effects by using sensitive methods and the use of appropriate controls are essential when characterizing compounds as being able to disrupt quorum sensing.