Functional Study of the Role of the Methyl Farnesoate Epoxidase Gene in the Ovarian Development of Macrobrachium nipponense.

Methyl farnesoate epoxidase (MFE) is a gene encoding an enzyme related to the last step of juvenile hormone biosynthesis. Mn-MFE cDNA has a total length of 1695 bp and an open reading frame (ORF) length of 1482 bp, encoding 493 amino acids. Sequence analysis showed that its amino acid sequence has a PPGP hinge, an FGCG structural domain, and other structural domains specific to the P450 family of enzymes. Mn-MFE was most highly expressed in the hepatopancreas, followed by the ovary and gill, weakly expressed in heart and muscle tissue, and barely expressed in the eyestalk and cranial ganglion. Mn-MFE expression remained stable during the larval period, during which it mainly played a critical role in gonadal differentiation. Expression in the ovary was positively correlated and expression in the hepatopancreas was negatively correlated with ovarian development. In situ hybridization (ISH) showed that the signal was expressed in the oocyte, nucleus, cell membrane and follicular cells, and the intensity of expression was strongest at stage O-IV. The knockdown of Mn-MFE resulted in a significantly lower gonadosomatic index and percentage of ovaries past stage O-III compared to the control group. However, no differences were found in the cumulative frequency of molting between the experimental and control groups. Moreover, the analysis of ovarian tissue sections at the end of the experiment showed differences between groups in development speed but not in subcellular structure. These results demonstrate that Mn-MFE promotes the ovarian development of Macrobrachium nipponense adults but has no effect on molting.

Studies on the Relationships between Growth and Gonad Development during First Sexual Maturation of Macrobrachium nipponense and Associated SNPs Screening.

In this study, we used full-sib families to investigate the association between growth and gonad development during first sexual maturation of M. nipponense. We found that male GSI was significantly negatively correlated with growth traits (p < 0.01) and there were no significant correlations between female GSI (Gonadosomatic index) and growth traits (p > 0.05). HSI (Hepatopancreas index) in both males and females showed no significant correlations with growth traits (p > 0.05). We furthermore investigated the association between the specific allele of Mn-CTS L1 polymorphism and gonad development and growth traits. In total, 35 mutation loci were screened and 16 high-quality single-nucleotide polymorphisms (SNPs) loci were obtained after validation. Four and two SNPs proved to be strongly associated with all growth traits in female and male M. nipponense separately, among which A+118T might be a candidate SNP positively associated with large growth traits. Two and one SNPs were screened, respectively, in males and females to associate with GSI, while three SNPs were detected to associate with female HSI, among which A+1379C may be applied as a potential molecular marker for gene-assisted selection to improve both reproduction speed and growth traits in M. nipponense.

NPC Intracellular Cholesterol Transporter 1 Regulates Ovarian Maturation and Molting in Female Macrobrachium nipponense.

NPC intracellular cholesterol transporter 1 (NPC1) plays an important role in sterol metabolism and transport processes and has been studied in many vertebrates and some insects, but rarely in crustaceans. In this study, we characterized NPC1 from Macrobrachium nipponense (Mn-NPC1) and evaluated its functions. Its total cDNA length was 4283 bp, encoding for 1344 amino acids. It contained three conserved domains typical of the NPC family (NPC1_N, SSD, and PTC). In contrast to its role in insects, Mn-NPC1 was mainly expressed in the adult female hepatopancreas, with moderate expression in the ovary and heart. No expression was found in the embryo (stages CS-ZS) and only weak expression in the larval stages from hatching to the post-larval stage (L1-PL15). Mn-NPC1 expression was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. A 25-day RNA interference experiment was employed to illustrate the Mn-NPC1 function in ovary maturation. Experimental knockdown of Mn-NPC1 using dsRNA resulted in a marked reduction in the gonadosomatic index and ecdysone content of M. nipponense females. The experimental group showed a significant delay in ovarian maturation and a reduction in the frequency of molting. These results expand our understanding of NPC1 in crustaceans and of the regulatory mechanism of ovarian maturation in M. nipponense.

Regulation and Response Mechanism of Acute Low-Salinity Stress during Larval Stages in Macrobrachium rosenbergii Based on Multi-Omics Analysis.

Macrobrachium rosenbergii is an essential species for freshwater economic aquaculture in China, but in the larval process, their salinity requirement is high, which leads to salinity stress in the water. In order to elucidate the mechanisms regulating the response of M. rosenbergii to acute low-salinity exposure, we conducted a comprehensive study of the response of M. rosenbergii exposed to different salinities' (0‱, 6‱, and 12‱) data for 120 h. The activities of catalase, superoxide dismutase, and glutathione peroxidase were found to be significantly inhibited in the hepatopancreas and muscle following low-salinity exposure, resulting in oxidative damage and immune deficits in M. rosenbergii. Differential gene enrichment in transcriptomics indicated that low-salinity stress induced metabolic differences and immune and inflammatory dysfunction in M. rosenbergii. The differential expressions of MIH, JHEH, and EcR genes indicated the inhibition of growth, development, and molting ability of M. rosenbergii. At the proteomic level, low salinity induced metabolic differences and affected biological and cellular regulation, as well as the immune response. Tyramine, trans-1,2-Cyclohexanediol, sorbitol, acetylcholine chloride, and chloroquine were screened by metabolomics as differential metabolic markers. In addition, combined multi-omics analysis revealed that metabolite chloroquine was highly correlated with low-salt stress.

Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense.

Dmrt (doublesex and mab-3 related transcription factor) is a protein family of transcription factors implicated in sexual regulation. Dmrt proteins are widely conserved and known for their involvement in sex determination and differentiation across species, from invertebrates to humans. In this study, we identified a novel gene with a DM (doublesex/Mab-3)-domain gene in the river prawn, Macrobrachium nipponense, which we named MniDmrt1B due to its similarities and close phylogenetic relationship with Dmrt1B in Macrobrachium rosenbergii. Through amino acid alignments and structural predictions, we observed conservation and identified putative active sites within the DM domain. qRT-PCR analysis revealed that MniDmrt1B exhibited high expression levels in the testis, with consistently higher expression in males compared to females during development. Additionally, similar to other sex-regulated genes, the MniDmrt1B gene exhibited high expression levels during the sex differentiation-sensitive periods in M. nipponense. These results strongly indicated that MniDmrt1B probably plays an important role in testis development and sex differentiation in M. nipponense.

RNA interference shows that Spook, the precursor gene of 20-hydroxyecdysone (20E), regulates the molting of Macrobrachium nipponense.

The aim of this study was to explore the function of the Mn-Spook gene, which was found in the ovary transcriptome of the Oriental river prawn (Macrobrachium nipponense). The Spook gene, which is the precursor gene of 20-hydroxyecdysone (20E), plays an important role in the process of molting in many arthropods, but its function in M. nipponense is unclear. We cloned the full-length Mn-Spook gene from the ovary of M. nipponense and found that it had the same conserved domains as the P450 gene of the Halloween family of genes. The Mn-Spook gene was highly expressed in ovary and gill tissue during the breeding period. During ovarian development, Mn-spook gene expression was highest at the nearly-ripe stage, and it also was highly expressed in the zoea developmental stage. Cellular localization analysis showed that Mn-Spook signals accumulated in the cytoplasmic membrane and nucleus of oocytes. Finally, we used RNA interference to evaluate the function of the Mn-Spook gene. Compared with the control group, in vivo injection of Mn-Spook dsRNA effectively downregulated the expression of Mn-Spook and the content of 20E. The molting frequency of M. nipponense in the experimental group also was significantly inhibited. These results demonstrated that the Mn-Spook gene played an important role in the molting process of M. nipponense.

Impact of different dietary lutein levels on growth performance, biochemical and immuno-physiological parameters of oriental river prawn (Macrobrachium nipponense).

A 56-day trial was conducted to evaluate the effects of dietary lutein pigment on growth, biochemical, and immuno-physiological parameters of the oriental river prawn. Prawns were fed five formulated diets containing different lutein levels, 0 (control), 50, 100, 150, and 200 mg/kg. Growth performance, except hepatosomatic index, was affected by different lutein levels, and biochemical parameters (urea, uric acid, glucose, creatinine, and triglycerides) decreased. However, high-density and low-density lipoprotein elevated significantly compared to the control treatment. Furthermore, calcium, phosphorus, and cholesterol did not show a significant difference. Hemato-immunological parameters (albumin, total protein, cortisol, lysozyme, phenoloxidase, total hemocyte count, granular cells, semi-granular cells, hyaline cells, alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase), and hepatopancreatic antioxidant statuses (total antioxidant capacity, superoxide dismutase, catalase, and malondialdehyde), were significantly affected; however, alkaline phosphatase and glutathione peroxidase were not affected by lutein treatments. By increasing dietary lutein levels, digestive enzyme activities, total bacteria count, total carotenoid content, significantly increased. Conversely, lactic acid bacteria were not affected. Overall, the research results demonstrated that adding 200 mg/kg of lutein to the diet improved growth performance, biochemical and immuno-physiological parameters of the oriental river prawn.

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.

Comparison of growth performance and biochemical components between parent and hybrid offspring in the oriental river prawn, Macrobrachium nipponense.

Macrobrachium nipponense, as one of the large-yield farmed shrimp, is facing germplasm degradation. Genetic improvement through hybridization is one of the effective methods to solve this problem. In this study, using a three-line hybrid strategy, two-hybrid F1 populations were obtained using three local populations of M. nipponense as parents for crossbreeding. Five populations were then cultured for 3 months. Growth rate performance was measured by the hepatosomatic index, weight gain, body length growth rate and special growth rate. Biochemical components were also assessed. The results showed that the survival rate and growth performance of the hybrid progeny were better than those of the parents. The levels of triglycerides, total cholesterol, glycogen and lactic acid of the hybrid population were higher than those of the parents. This was consistent with variation in the activity of four digestive enzymes. Compared with the results of the fatty acid and amino acid analysis, it was found that the contents of polyunsaturated fatty acids such as eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid and eight essential amino acids in the hybrid populations were significantly higher than those of their parents, and the contents of flavor amino acids were higher. The expression level of molting genes related to the growth of the parent populations was lower than that of the hybrids. These results show that crossbreeding is effective for the genetic improvement of M. nipponense germplasm. Hybrids showed advantages in growth and nutrition and multigenerational breeding will be required to form a stable germplasm.

Identification of a novel germ cell marker MnTdrd from the oriental river prawn Macrobrachium nipponense.

Germ cell-specific genes play an important role in establishing the reproductive system in sexual organisms and have been used as valuable markers for studying gametogenesis and sex differentiation. Previously, we isolated a vasa transcript as a germ cell marker to trace the origin and migration of germ cells in the oriental river prawn Macrobrachium nipponense. Here, we identified a new germ cell-specific marker MnTdrd RNA and assessed its temporal and spatial expression during oogenesis and embryogenesis. MnTdrd transcripts were expressed in high abundance in unfertilized eggs and embryos at cleavage stage and then dropped significantly during late embryogenesis, suggesting that MnTdrd mRNA is maternally inherited. In situ hybridization of ovarian tissue showed that MnTdrd mRNA was initially present in the cytoplasm of previtellogenic oocyte and localized to the perinuclear region as the accumulation of yolk in vitellogenic oocyte. Whole-mount in situ hybridization of embryos showed that MnTdrd-positive signals were only localized in one blastomere until 16-cell stage. In the blastula, there were approximately 16 MnTdrd-positive blastomeres. During embryonized-zoea stage, the MnTdrd-positive cells aggregated as a cluster and migrated to the genital rudiment which would develop into primordial germ cells (PGCs). The localized expression pattern of MnTdrd transcripts resembled that of the previously identified germ cell marker vasa, supporting the preformation mode of germ cell specification. Therefore, we concluded that MnTdrd, together with vasa, is a component of the germ plasm and might have critical roles in germ cell formation and differentiation in the prawn. Thus, MnTdrd can be used as a novel germ cell marker to trace the origin and migration of germ cells.

Hepatopancreatic metabolomics shedding light on the mechanism underlying unsynchronized growth in giant freshwater prawn, Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii (M. rosenbergii) as an important freshwater aquaculture species with high commercial value, exhibited unsynchronized growth. However, the potentially metabolic mechanism remains unclear. In this study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the hepatopancreatic metabolic profiles of twenty giant freshwater prawns between the fast-growing group and slow-growing group. In the metabolomics assay, we isolated 8,293 peaks in positive and negative iron mode. Subsequently, 44 significantly differential metabolites were identified. Functional pathway analysis revealed that these metabolites were significantly enriched in three key metabolic pathways. Further integrated analysis indicated that glycerophospholipid metabolism and aminoacyl-tRNA biosynthesis have significant impact on growth performance in M.rosenbergii. Our findings presented here demonstrated the critical metabolites and metabolic pathways involved in growth performance, moreover provided strong evidence for elucidating the potentially metabolic mechanism of the unsynchronized growth in M. rosenbergii.

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.

Effects of ZnO nanoparticles on the Giant freshwater prawn (Macrobrachium rosenbergii, de Man, 1879): Reproductive performance, larvae development, CHH concentrations and anti-oxidative enzymes activity.

The Giant freshwater prawn (Macrobrachium rosenbergii) breeds when in captive conditions. The eggs of a clutch are attached to the abdomen of berried females. Zinc oxide (ZnO) is one of the most important metal oxide-nanoparticles (NPs) that is widely used in various industries and is released into aquatic environments from wastewater management facilities. The present study was conducted to evaluate effects of ZnO on values for the reproductive variables: larvae development, crustacean hyperglycemic hormone (CHH) release from the X-organ into the hemolymph and anti-oxidative enzymes activity of M. rosenbergii. There were five groups including a group not treated (control), and groups treated with10, 20, 50, 100 mg/L ZnO in triplicate during a 90-day period. Results indicated that ZnO-NPs have marked effects on reproductive performance, offspring development, CHH release from the X-organ into the hemolymph and anti-oxidant enzymes activities with there being no spawning of brood-stock in the 100 mg/L ZnO group and in the prawns treated with 50 mg/L there was spawning but there was larvae mortality immediately subsequent to hatching. Also, values for viability rate of eggs, dry weight of eggs, brood-stock inter-spawn period and egg clutch somatic index (ESI) reproductive variables were affected by the NP. This NP was found to have a dose-dependent effect on CHH release from the X-organ into the hemolymph and also superoxide dismutase (SOD) and catalase activities in M. rosenbergii. The results indicate that M. rosenbergii, a freshwater decapod crustacean, is an appropriate species to study nano-material effects on reproduction in freshwater ecosystems.

Investigation of growth retardation in Macrobrachium rosenbergii based on genetic/epigenetic variation and molt performance.

Giant freshwater prawn, Macrobrachium rosenbergii is an important freshwater aquaculture species worldwide, and China contributes the most to its global production. However, in recent years in China, many prawns have shown serious growth retardation, which is referred to as "iron prawn." To explore the mechanism behind this phenomenon, we compared the difference between these "iron prawns" and normal prawns in three aspects-changes in genetic diversity, DNA methylation, and transcriptomes-as well as comparing differences in their molt performance. The results are as follows: first, compared with normal prawns, "iron prawns" showed no significant decrease in genetic diversity, but they did show obvious genetic differentiation, and different DNA methylation levels were observed. The genetic and epigenetic variations that existed between "iron prawn" and normal prawn indicated the influence of germplasm on growth performance. Second, transcriptome analysis revealed 1813 differentially expressed genes (DEGs) between the "iron prawn" and normal prawn, and the DEGs mainly enriched the glucose metabolism- and immune-related pathways, such as in glycolysis/gluconeogenesis metabolism, insulin secretion, glucagon signaling pathway, antigen processing and presentation, as well as in complement and coagulation cascades. Enrichment analysis indicated the importance of the glucose level and pathogen attacks to growth performance in the "iron prawn." Finally, a comparison of the molt performance showed that the length of the molt cycle in the "iron prawn" was comparable to normal prawns with the same size, but the specific growth was much lower in the "iron prawn." This result suggested that lower body weight gain per molt cycle should be responsible for growth retardation in the "iron prawn," but not in the longer molt cycle. The results in this study provided fundamental information about the mechanism behind growth retardation in M. rosenbergii.

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.

The composition of the microbial community associated with Macrobrachium rosenbergii zoeae varies throughout larval development.

The giant river prawn, Macrobrachium rosenbergii, is an economically important freshwater prawn. The cultivation of zoea larvae is crucial for the success of the M. rosenbergii industry. In this study, we surveyed the microbial community diversity and structure associated with M. rosenbergii zoeae at different stages of larval development. Samples of zoea larvae from different developmental stages were collected and subjected to high-throughput DNA sequencing. Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria were the dominant phyla in all six sample groups. At the genus level, the relative abundance of Bacillus decreased, and that of Enterobacter increased with the growth of the zoeae. This may have been related to the intestinal development of the zoea larvae. The microbial diversity of M. rosenbergii zoea larvae decreased significantly with development. The beta diversity analysis showed that the closer the developmental stage of M. rosenbergii, the more similar the structure of the associated bacterial communities.

Comparative metabolomics analysis of ovarian developmental stages in Macrobrachium nipponense.

Rapid sexual maturity of female Macrobrachium nipponense is a severe problem for the aquaculture industry. To date, there have been only transcriptome studies investigating ovarian development, and studies using other tools, such as metabolomics are lacking. Metabolomics reveals changes in the level of metabolites in tissues in relation to current physiological characteristics, and can yield valuable insight into the growth and development of organisms. In this study, we systematically analyzed 15 samples from five different ovarian developmental stages in M. nipponense to learn more about how metabolites change over reproduction. Gas chromatography/time-of-flight mass spectroscopy revealed an array of different compounds and 83-162 pathways depending on the stage. Furthermore, 89 metabolites and 14 pathways were significantly different across stages. It is hypothesized that N-acetyl-N-formyl-5-methoxykynurenamine, ascorbate, fructose-2,6-bisphosphate, cortexolone and other metabolites that significantly differed by stage are regulated by hormones and are closely related to ovarian development. However, for other metabolites that changed with development, such as cytidine and xanthine, an association with ovarian development has yet to be revealed. Quantitative polymerase chain reaction was used to correlate gene changes to metabolites in the pathway for biosynthesis of plant secondary metabolites. We found that the TCA cycle rate may be the cause of female miniaturization during the reproductive period, and that the control of fatty acid content via aquaculture nutrition may be an exogenous tool for regulatory control of maturation. This study provides a systematic and comprehensive metabolomics analysis of ovarian development in M. nipponense and lays a foundation for addressing the problem of rapid sexual maturity.

Validation of growth-related quantitative trait loci markers in different Exopalaemon carinicauda families for marker-assisted selection.

We detected growth-related QTL and associated markers from the backcross population of Exopalaemon carinicauda in the previous study. Based on our previous study, the 47 SNP markers associated with candidate growth trait QTL were selected to analyze the association between these markers and body weight (BW), body length and abdominal segment length traits in four different populations including wild population, a full-sib family, a half-sib family and a backcross population for evaluating their potential application of marker-assisted selection in E. carinicauda. The general linear model (GLM) and mixed linear model were applied and the associations between SNP loci and three growth-related traits verified. The results showed that the Marker79268 and Marker100644 were significantly associated with the BW trait in more than three populations by the GLM method. The Marker100644 was significantly associated with BW in the full-sib family, half-sib family and backcross populations by the GLM and mixed linear model methods. Our findings will provide useful SNP markers to go forward to improve growth performance through more refined marker-assisted selection in E. carinicauda.

Effects of pH and nitrites on the toxicity of a cypermetrin-based pesticide to shrimps.

The Pantanal (Brazil) is a wetland region characterized by seasonal flooding. Hydrological cycles influence the water physicochemical parameters, causing seasonal variations in pH and nitrites. Thus, the objective of this study was to evaluate the effects of varying pH and nitrite concentrations on the toxicity of the cypermethrin-based pesticide Barrage®, considering both lethal (mortality) and sublethal endpoints (growth and development). Larvae of the endemic shrimp Macrobrachium pantanalense and of the estuarine Amazonian congener Macrobrachium amazonicum were exposed to cypermethrin (through Barrage®) under several pH levels (6.5, 7.5 and 8.5) or nitrite concentrations (0.1, 0.2 and 0.4 mg/L). The pH had direct effects on all the tested endpoints for both species. For M. pantanalense, the lethal effects of the cypermethrin formulation were more pronounced at low pH (96-h LC50 = 0.004 µg/L at pH 6.5, and 0.146 µg/L at pH 8.5). For M. amazonicum, an opposite response was observed, with increased toxicity of the formulation at high pH (96-h LC50 = 0.110 µg/L at pH 6.5 and 0.044 µg/L at pH 8.5). Variations in pH also seemed to modify the sublethal effects of the formulation on larval growth and development of M. pantanalense. Nitrite concentrations affected larval growth of both species, modifying also the effects of the cypermethrin formulation on the larval development of M. amazonicum. This work shows the importance of considering abiotic factors for risk assessment either due to possible direct effects on the physiology of organisms and/or due to interactions with other stressors, particularly in fragile biomes such as Pantanal.

Effects of ammonia-N exposure on the growth, metabolizing enzymes, and metabolome of Macrobrachium rosenbergii.

Ammonia nitrogen elevated is one of the commonest problem in the aquatic system, which caused a great threat to the survival and growth of prawn. However, little is know about the ammonia metabolism and detoxification strategy of prawn. In this study, the effects of ammonia-N (0, 0.108, 0.216, 0.324, or 0.54 mg L-1) on growth and metabolizing enzymes in hepatopancreas of Macrobrachium rosenbergii, including glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GDH), were investigated. The metabolome of its muscle was also analyzed after exposure to ammonia-N (0, 0.108, 0.324, or 0.54 mg L-1) for 20 days. The survival rate of M. rosenbergii decreased significantly after treatment with 0.54 mg L-1 ammonia-N compared with that in the other groups. However, ammonia-N had no significant effect on the growth of the river prawn after exposure for 20 days. GS activity increased significantly after exposure to 0.108 mg L-1 ammonia-N compared with the control and other ammonia-N-treated groups. Hepatopancreatic GDH activity was lower in the prawns treated with 0.216, 0.324, or 0.54 mg L-1 ammonia-N than in the control by 34.70%, 38.80%, or 41.94%, respectively. Ammonia-N had no significant effect on hepatopancreatic AST or ALT activity. Urea nitrogen was higher in the prawns treated with 0.216 mg L-1 ammonia-N than in the control or those treated with 0.54 mg L-1 ammonia-N. Ammonia-N had significant effects on the lipid, carbohydrate. and protein metabolism of M. rosenbergii, including purine metabolism, amino sugar and nucleotide sugar metabolism, α-linolenic acid metabolism, arginine and proline metabolism, glutathione metabolism, and phosphonate and phosphate metabolism, and on the terpenoid biosynthesis, lysine degradation, and lysine biosynthesis pathways. High concentrations of ammonia-N stress increased the content of glutamate and arginine, which may participate in the urea cycle, which synthesizes glutamine or urea to eliminate ammonia toxicity.