Comparison of morphology and genetic diversity between broodstock and hybrid offspring of oriental river prawn, Macrobrachium nipponense based on morphological analysis and SNP markers.

Hybridization is an effective method for the genetic improvement of farmed species. In this study, three broodstock populations (Changjiang, CJ, Dongting, DT, and Dianshan, DS) of oriental river prawn, Macrobrachium nipponense were used, and DS was used as the female broodstock. Through three-line hybridization, two hybrid populations were finally obtained. The F3 generation of the broodstock population and the F1 generation of the hybrid population were cultured indoors for 3 months. Through morphological analysis (cluster analysis, discriminant analysis and path analysis) it was found that the hybrid population and the broodstock had some differences, but not enough to reach the subspecies level, and the dominant traits exhibited differentiation and reorganization. This study identified SNP genetic markers, carried out systematic evolution analysis and genetic diversity analysis and found that the nucleotide diversity π and heterozygosity Het of the hybrid population were higher than those of the broodstock. Among broodstocks, the differentiation index (Fst ) of SCD and SDC was smallest (0.055). This research provides some valuable reference for genetic breeding.

Effects of reclamation methods and habitats on macrobenthic communities and ecological health in estuarine coastal wetlands.

This study compared the community structure of macrobenthos in bare tidal areas and vegetated regions under two reclamation patterns, fully enclosed and opened reclamation, in Yangtze wetland, China. Compared with fully enclosed reclamation areas, opened reclamation areas had higher water salinity, higher number of species and abundance, and less disturbed habitat health status. Compared with bare tidal areas, vegetated regions were more suitable for the survival of macrobenthos and its habitat health status was more stable. Salinity, pH and larger soil particle diameter were positively associated with macrobenthic diversity, while total nitrogen and total phosphorus were negatively associated. Overall, compared with fully enclosed reclamation and vegetated regions, opened reclamation and bare tidal areas had less negative effects upon macrobenthic community and habitat health. Opened reclamation is preferred for wetland reclamation, and planting indigenous plants and releasing species adapted to fresh water is beneficial to the restoration of macrobenthic communities.

Two genes related to reproductive development in the juvenile prawn, Macrobrachium nipponense: Molecular characterization and transcriptional response to nanoplastic exposure.

Plastic pollution in the form of nanoplastics poses a global threat to aquatic ecosystems and the organisms inhabiting them. However, few studies have been conducted on the effects of nanoplastic exposure on reproductive development in crustaceans. In order to address this issue, juvenile oriental river prawns (Macrobrachium nipponense) were exposed to different concentrations of 75-nm polystyrene nanoplastics (0, 5, 10, 20, 40 mg/L) for 28 days. In order to study the regulation of reproduction-related genes in the presence of nanoplastics, the Wee1 protein kinase gene (Wee1) and OTU domain ubiquitin aldehyde binding protein gene (OTUB) were selected. In this study, for the first time, the full-length cDNA of Mn-Wee1 and Mn-OTUB were cloned from M. nipponense. Homologous alignments revealed that Mn-Wee1 had a highly conserved function-critical sequence, and that Mn-OTUB was more closely related to OTUB1 than OTUB2. With increasing concentration of nanoplastics, the expression of both genes increased initially, then decreased. The inhibition of expression of Wee1 and OTUB occurred in 40 mg/L group, respectively. Analysis of the data also indicated that nanoplastic exposure might have differing effects on gene expression in M. nipponense male and female reproductive organs.

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.

Effects of nanoplastics on energy metabolism in the oriental river prawn (Macrobrachium nipponense).

Nanoplastics are common pollutants in aquatic environments and have attracted widespread research attention. However, few studies focus on the effects of nanoplastic exposure on energy metabolism in crustaceans. Accordingly, we exposed juvenile oriental river prawns (Macrobrachium nipponense) to different concentrations of 75-nm polystyrene nanoplastics (0, 5, 10, 20, and 40 mg/L) for 7, 14, 21, or 28 days. Thereafter, the effects of nanoplastic exposure on metabolite content, energy metabolism-related enzyme activity, and gene expression were evaluated. Our results showed that (1) with increasing nanoplastic concentration and exposure time, the survival rate decreased, while weight gain rate and molting number increased and then decreased; glycogen, triglyceride, and total cholesterol content all declined while lactic acid content increased with higher exposure to nanoplastic concentrations; (2) the activities of acetyl-CoA carboxylase (ACC), hexokinase (HK), carnitine palmitoyl transferase-1, pyruvate kinase (PK), lipase, and fatty acid synthase tended to decrease, while the activity of lactate dehydrogenase (LDH) increased. In particular, the activity of 6-phosphofructokinase exposed to 5 mg/L nanoplastics increased significantly (P < 0.05). (3) Expression of the metabolism-related genes 6-phosphate glucokinase (G-6-Pase), HK, PK, ACC, Acetyl-CoA-binding protein (ACBP), CPT-1, and fatty-acid-binding protein 10 (FABP 10) increased and then decreased, while expression of the LDH gene showed an upward trend. These results indicate that nanoplastics affect growth, enzyme activity, and the gene expression of energy metabolism in M. nipponense, and that high concentrations of nanoplastics have a negative impact on energy metabolism.

Cold stress regulates lipid metabolism via AMPK signalling in Cherax quadricarinatus.

Lipids play an important role in protecting poikilotherms from cold stress, but relatively little is known about the regulation of lipid metabolism under cold stress, especially in crustaceans. In the present study, red-clawed crayfish Cherax quadricarinatus was employed as a model organism. Animals were divided into four temperature groups (25, 20, 15 and 9 °C) and treated for 4 weeks, with the 25 °C group serving as a control. The total lipid content in the hepatopancreas as well as the triglyceride, cholesterol and free fatty acid levels in the hemolymph were determined. Lipids stored in the hepatopancreas and hemolymph decreased with decreasing temperature, with changes in the 9 °C group most pronounced, indicating that lipids are the main energy source for crayfish at low temperatures. Furthermore, enzyme activity of lipase, fatty acid synthase, acetyl-CoA carboxylase, and lipoprotein esterase, and gene expression analysis of fatty acid synthase gene, acetyl-CoA carboxylase gene and carnitine palmitoyltransferase gene showed that the digestion, synthesis and oxidation of lipids in the hepatopancreas were inhibited under low temperature stress, but expression of sphingolipid delta-4 desaturase (DEGS) was increased, indicating an increase in the demand for highly unsaturated fatty acids at low temperatures. Analysis of the expression of genes related to the AMP-activated protein kinase (AMPK) signalling pathway revealed that the adiponectin receptor gene was rapidly upregulated at low temperatures, which may in turn activate the expression of the downstream AMPKα gene, thereby inhibiting lipid anabolism.

Characterization and expression of arginine kinase 2 from Macrobrachium nipponense in response to salinity stress.

Salinity is a fundamental environmental factor in aquaculture, and arginine kinase (AK) plays imperative roles in innate immune feedback and stress resistance in invertebrates. In the current study, we cloned a full-length cDNA of arginine kinase 2 (MnAK2, GenBank number, MN149533) in Macrobrachium nipponense and analyzed its function through a salinity challenge using bioinformatic approaches. MnAK2 was expressed at the highest levels in hepatopancreas and muscle. Changes in the expression levels of MnAK2, enzymes involved in innate immunity, antioxidant enzymes, and antioxidant enzyme-related genes, and the glutathione and malondialdehyde contents were investigated after 6-week salinity treatment. The expression of MnAK2 gradually increased as salinity increased, and western blotting showed that MnAK2 was significantly upregulated in the 14 and 22 ppt salinity-treatment groups relative to the control group. The findings indicate that high salinity produces oxidative stress and that salinity below isotonic salinity might improve the antioxidant response in M. nipponense. MnAK2 may play a crucial role in the response to salinity stress in M. nipponense.

Effects of nanoplastics on antioxidant and immune enzyme activities and related gene expression in juvenile Macrobrachium nipponense.

Nanoplastics are widely distributed in aquatic environments, and nanoplastic pollution has become a global concern. However, few studies have evaluated the toxicity of nanoplastics to freshwater crustaceans. In this study, by adding different concentrations of nanoplastics to water, we explored the effects of nanoplastics on the survival, antioxidant activity, immune enzyme activity, and related gene expression levels in juvenile Macrobrachium nipponense. The results showed that the 96 -h half-lethal concentration of nanoplastics to juvenile shrimp was 396.391 mg/L. As the concentration of nanoplastics increased, the activities of antioxidant enzymes generally decreased, while the contents of hydrogen peroxide and lipid peroxidation products increased. The activities of non-specific immune enzymes first increased and then decreased with increasing nanoplastic concentration. The trends in the expressions of antioxidant-related genes were generally consistent with those in the activities of antioxidant enzymes. As the nanoplastic concentration increased, the expressions of immune-related genes generally increased at first and then decreased. These results indicate that low concentrations of nanoplastics (5 mg/L) may enhance the viability of juvenile shrimp, whereas high concentrations (10,20, 40 mg/L) have inhibitory and/or toxic effects. The findings provide basic information on the toxic effects of nanoplastics in juvenile shrimp.

Polystyrene nanoplastic induces ROS production and affects the MAPK-HIF-1/NFkB-mediated antioxidant system in Daphnia pulex.

Recently, research on the biological effects of nanoplastics has grown exponentially. However, studies on the effects of nanoplastics on freshwater organisms and the mechanisms of the biological effects of nanoplastics are limited. In this study, the content of reactive oxygen species (ROS), gene and protein expression in the MAPK-HIF-1/NFkB pathway, and antioxidant gene expressions and enzyme activities were measured in Daphnia pulex exposed to polystyrene nanoplastic. In addition, the full-length extracellular signal-regulated kinases (ERK) gene, which plays an important role in the MAPK pathway, was cloned in D. pulex, and the amino acid sequence, function domain, and phylogenetic tree were analyzed. The results show that nanoplastic caused the overproduction of ROS along with other dose-dependent effects. Low nanoplastic concentrations (0.1 and/or 0.5 mg/L) significantly increased the expressions of genes of the MAPK pathway (ERK; p38 mitogen-activated protein kinases, p38; c-Jun amino-terminal kinases, JNK; and protein kinase B, AKT), HIF-1 pathway (prolyl hydroxylasedomain, PHD; vascular endothelial growth factor, VEGF; glucose transporter, GLUT; pyruvate kinase M, PKM; hypoxia-inducible factor 1, HIF1), and CuZn superoxide dismutase (SOD) along with the activity of glutathione-S-transferase. As the nanoplastic concentration increased, these indicators were significantly suppressed. The protein expression ratio of ERK, JNK, AKT, HIF1α, and NFkBp65 (nuclear transcription factor-kB p65) as well as the phosphorylation of ERK and NFkBp65 were increased in a dose-dependent manner. The activities of other antioxidant enzymes (catalase, total SOD, and CuZn SOD) were significantly decreased upon exposure to nanoplastic. Combined with our previous work, these results suggest that polystyrene nanoplastic causes the overproduction of ROS and activates the downstream pathway, resulting in inhibited growth, development, and reproduction. The present study fosters a better understanding of the biological effects of nanoplastics on zooplankton.

Effects and transcriptional responses in the hepatopancreas of red claw crayfish Cherax quadricarinatus under cold stress.

The red claw crayfish, Cherax quadricarinatus, is an economically important freshwater crustacean that cannot tolerate low temperature, which diminishes survival via unknown mechanisms. Herein, physiological regulation of C. quadricarinatus was investigated following exposure to low temperature stress at 9 ±â€¯2 °C for 4 weeks. Hepatopancreas tissue was tested for nonspecific enzyme activity, histological structure, and transcriptome sequencing analyses. The results showed that the activities of nonspecific enzymes were inhibited following low temperature stress. Ultrastructural observation revealed that the hepatopancreas structure was oxidatively damaged at low temperature, with numerous autophagic vesicles visible. Apoptosis in the hepatopancreas was significantly increased in the cold stress group, indicating diminished function. Transcriptome sequencing identified 2615 differentially expressed genes (DEGs) following low temperature stress, of which 1147 and 1468 were up- and down-regulated, respectively. Functional analysis of DEGs indicated involvement in substance metabolism, antioxidant defences, signal transduction, and immune responses. Therefore, chronic cold stress can suppress metabolism and cause oxidative damage and immune deficiency in crayfish. The findings provide fundamental molecular information for further study of the regulatory mechanisms of cold tolerance in red claw crayfish.

Assessing the ecological health of the Chongming Dongtan Nature Reserve, China, using different benthic biotic indices.

To assess the ecological health of the Chongming Dongtan Nature Reserve (CDNR), univariate and multimetric indices-AZTI's Marine Biotic Index (AMBI), multivariate AMBI (M-AMBI), abundance-biomass comparison curves, taxonomic diversity, Shannon-Wiener index (H') (log2), and Benthic Opportunistic Polychaetes Amphipods index (BOPA) - were used to translate the macrobenthic community into classifications of ecological health in 2016 and 2017. Based on the results of the various indicators, the overall habitat status of the CDNR ranged from good to high status. Sites 4 and 5, which fell in the remaining tidal flats following reclamation activities, were classified as disturbed, whereas the degree of disturbance at site 3 was low. The results indicate that the CDNR is under some form of human disturbance, which includes reclamation and animal (mainly cattle) grazing. H' (log2) and M-AMBI index may be more suitable for assessing ecological quality in intertidal zones, including the CDNR.

Changes in ultrastructure of gonads and external morphology during aging in the parthenogenetic cladoceran Daphnia pulex.

The water flea Daphnia pulex, a small crustacean that lives in freshwater ponds, undergoes parthenogenesis and gamogenesis according to environmental conditions. In D. pulex, different morphological characteristics can be observed using a microscope during aging. In this study, we recorded the growth, reproduction, and survival indicators of parthenogenetic D. pulex females to analyze the morphological changes that occur during senescence. We observed the growth and development of the parthenogenetic females on the 7th, 14th, 21st, and 28th days by using scanning electron microscopy and ultrastructure of the gonads on the 7th, 14th and 21st days with transmission electron microscopy. We found that body length and reproductive capacity in the parthenogenetic individuals first increased and then decreased with aging, and the survival rate decreased gradually. The depression of the brood pouch on the back of the parthenogenetic females implied a decline in gonadal function and gradual aging. During senescence, the number of lipid droplets in the gonads of the parthenogenetic females decreased, and the integrity of the mitochondria and peroxisome was destroyed. The results of our study revealed the morphological characteristics of senescence in D. pulex.

Cloning and characterisation of Na+/K+-ATPase and carbonic anhydrase from oriental river prawn Macrobrachium nipponense.

Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) are ion transporters that play important roles in osmotic pressure balance in crustaceans. Oriental river prawn (Macrobrachium nipponense) is one of the most economically important shrimps in China. This species can live in freshwater and low-salt water, but the molecular mechanism of salinity regulation is unclear. In this study, full-length cDNAs of M. nipponense α-NKA and CA were obtained by rapid amplification of cDNA ends (RACE)-PCR (GenBank accession numbers MH378774 and MH827971, respectively), and characterised. Full-length cDNAs of Mnα-NKA (3778bp) and MnCA (1407bp) contain open reading frames of 3030bp and 930bp, respectively, encoding polypeptides of 1009 and 930 amino acids. Bioinformatic analysis showed that Mnα-NKA and MnCA harbour the representative features of NKAs and CAs, and share high homology with orthologs in other crustaceans. Quantitative real-time PCR revealed that Mnα-NKA and MnCA were expressed in various tissues, with highest expression in posterior gills and hepatopancreas. Both Mnα-NKA and MnCA were up-regulated in response to salinity acclimation, but expression patterns differed. Our results provide a theoretical basis for the mechanism regulating salinity acclimation in M. nipponense.

Polystyrene nanoplastic exposure induces immobilization, reproduction, and stress defense in the freshwater cladoceran Daphnia pulex.

The widespread occurrence and accumulation of plastic waste have been globally recognized as a critical issue. However, there is limited information on the adverse effects of nanoplastics on freshwater invertebrates. In this study, the effects of a polystyrene nanoplastic on physiological changes (e.g., survival, growth, and reproduction) and expression levels of stress defense genes (oxidative stress-mediated and heat shock proteins) in the freshwater flea Daphnia pulex were measured. The results showed that the digestive organs of D. pulex were strongly fluorescent after exposure to the nanoplastic particles, and the 48-h median lethal concentration (LC 50) of the nanoplastic was determined to be 76.69 mg/L. In the 21-day chronic toxicity test, dose- and time-dependent relationships were observed for body length, and the time to first eggs was significantly prolonged in the 0.5 and 1 mg/L groups. The time to clutch was delayed, and total offspring per female and number of clutches were decreased in all the treatment groups. In addition, the offspring per clutch were significantly decreased in the 0.1 mg/L group. As the nanoplastic concentration increased, expression of stress defense genes (SOD, GST, GPx, and CAT) was first induced and then inhibited. The gene expressions of heat shock proteins (HSP70 and HSP90) were induced in all the treatment groups. Our results suggest that nanoplastics can be ingested by the freshwater cladoceran D. pulex and affect its growth and reproduction as well as induce stress defense.

Macrobenthic community characteristics and ecological health of a constructed intertidal oyster reef in the Yangtze Estuary, China.

Development of substrate organisms (oysters, barnacles) and the health of a monitored oyster reef were investigated in the Yangtze Estuary. Very low salinity suppressed oyster survival. Nevertheless, middle- to high-salinity significantly increased the abundance and biomass of substrate organisms, and macrobenthos species and diversity. Long-term variation in substrate organisms was steady after a major fluctuation, yet the macrobenthic community structure lagged behind that of oysters. Overall, the oyster reef was in a healthy state. The M-AMBI results showed that its ecological status under high-salinity was better than medium-salinity conditions. Redundancy analysis indicated these results were associated with changes in water salinity and substrate factors. Taken together, our results suggest this constructed intertidal oyster reef has had a positive effect on the community and health status of macrobenthos in the Yangtze Estuary. Further, these ecological benefits increased going from medium- to high-salinity waters, but were generally absent under low salinity.

Molecular cloning, tissue expression and regulation of nutrition and temperature on Δ6 fatty acyl desaturase-like gene in the red claw crayfish (Cherax quadricarinatus).

Desaturase enzymes play an important role in the synthesis of unsaturated fatty acids. In this study, a complete cDNA sequence of a Δ6 desaturase-like gene was cloned from the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. The full-length 1885 bp sequence comprises a 5' UTR of 254 bp, 3' UTR of 234 bp, and an open reading frame (ORF) of 1377 bp encoding a 458 amino acid polypeptide (GenBank accession no. MF497442). Bioinformatics analysis revealed three conserved histidine-rich regions, a cytochrome b5 domain at the N-terminus, and a haem binding site (HPGG) in the cytochrome b5 domain, all of which are typical of Δ6 desaturases. Quantitative real-time PCR demonstrated significantly higher expression in the hepatopancreas than other tissues. After feeding crayfish four formulated diets in which fish oil was replaced by 0, 33, 67, or 100% highly unsaturated soybean oil for 8 weeks, Δ6 desaturase-like mRNA expression and enzyme activity were higher than in the fish oil only group. Additionally, a 4-week low temperature treatment at 25, 20, 15, or 9 °C increased Δ6 desaturase mRNA expression and enzyme activity with decreasing water temperature. These results may help us better understand the biosynthesis of unsaturated fatty acids in C. quadricarinatus.

Different effects of reclamation methods on macrobenthos community structure in the Yangtze Estuary, China.

The effects of enclosed, semi-closed, and opened reclamation methods on the macrobenthos community structure were investigated. Compared with their paired controls, water salinity decreased sharply in the enclosed reclamation region with no apparent change in the opened reclamation region. Declining species and biodiversity was observed in the reclamation regions, but the extent of this declining trend was weaker in the semi-closed and opened reclamations than in the enclosed reclamation region. The ABC curve indicated that the enclosed reclamation was disturbed, whereas the semi-closed and opened reclamations were undisturbed. Taken together, these results suggest that reclamation may have a negative effect on the community and health status of macrobenthos in the intertidal wetlands of the Yangtze Estuary. Semi-closed and opened reclamation methods may mitigate the problem of a salinity decrease caused by enclosed reclamation, while also having a relatively weaker negative effect on community structure and wetland habitat.

Effects of cadmium on lipid metabolism in female estuarine crab, Chiromantes dehaani.

Due to the nature of their habitat, which contains a high level of pollutants, estuarine crabs are at great risk of exposure to contaminants such as cadmium. Thus, in this study, the effects of cadmium on lipid metabolism were investigated in estuarine crab Chiromantes dehaani. Adult female estuarine crabs were randomly exposed to 0.05, 0.1, 0.5, and 1mg/L of CdCl2 for 7, 14 and 21days, after which the lipid contents of the hepatopancreas and ovary were measured. Also, the substance contents and the activities of the enzyme in lipid digestion, lipid synthesis and lipid transport metabolism were analyzed. The results showed that the lipid contents in the hepatopancreas and ovary of the exposed crabs decreased after prolonged exposure to cadmium compared to the control. The lipase activity decreased while the activities of fatty acid synthase and acetyl coenzyme A in the hepatopancreas increased on day 7 but decreased on days 14 and 21. Moreover, the change in non-esterified fatty acid level was similar to fatty acid synthase. The level of low-density lipoprotein increased in the exposed crabs compared to the control group while the level of high-density lipoprotein and the activity of lipoprotein lipase decreased at a higher concentration of cadmium and longer exposure time. These observations suggest that cadmium decreases the lipid content by weakening the ability of digestion, transportation and synthase of lipid, thus affecting hepatopancreas and ovary indices.

Loss and self-restoration of macrobenthic diversity in reclamation habitats of estuarine islands in Yangtze Estuary, China.

In this study, macrobenthic diversity data were collected from intertidal habitats of island wetlands in Yangtze Estuary before and after reclamation. Three survey regions based on habitat features were investigated: protected region, normal region, and self-restored region. The pattern of diversity variation showed a sharp decrease in reclamation sites and an obvious increase in vegetated sites of the self-restored region before and after reclamation. A declining trend in habitat health was observed in reclamation sites, but the degree of perturbation was relatively weaker in protected region than in normal region. The vegetated site showed a better self-restoration of biodiversity than the bald site. These results suggest that reclamation may have a negative influence on biodiversity and habitat health status in the intertidal wetland. Also, there is a possibility of self-restoration in tidal flats disturbed by reclamation and the resistance effect in nature reserve may reduce the disturbances resulting from reclamation.

Application of macrobenthic diversity to estimate ecological health of artificial oyster reef in Yangtze Estuary, China.

In this study, several macrobenthic diversity investigations were performed in Yangtze Estuary Oyster Reef, the largest artificial oyster reef in China, from 2012 to 2014. The sampling sites of the south branch showed considerably higher diversity than those of the north branch. The richness measures exhibited a significant increasing trend from low- to high-salinity zone; however, the evenness measures were typically high in the middle-salinity zone. During the past decade, the results were combined with historical data to detect the changes in macrobenthos. The variation in substrate organisms and macrobenthic diversity followed a steady trend after a major fluctuation. Redundancy analysis indicated that the water salinity and substrate factors were the main indicators that influence macrobenthic distribution. All sampling sites in the south branch were protected by a nature reserve. However, the N2 and N6 sites in the north branch were subjected to severe and mild human interventions, respectively.