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Background color is a crucial factor influencing the growth performance and feed utilization in crustaceans. However, little is known about its impacts on the culture performance of Chinese mitten crab (Eriocheir sinensis). In this study, we examined the impacts of six different background colors (white-W, black-BK, green-G, red-R, yellow-Y, and blue-BL) on growth, inter-molt period, and coloration and total carotenoids content of carapace of juvenile E. sinensis. The culture experiment comprised two trials: a 68-day individual culture, and a 30-day group culture. Individual culture results: No significant effects of background color were found on weight gain rate (WGR), specific growth rate (SGR), molting interval, carapace length and width, and survival rate (SR) of the juvenile crabs (P > 0.05). However, during the second molting cycle, the BL group exhibited the highest WGR, and the G group showed the lowest hepatopancreas index (HSI). Meanwhile, markedly lower feed conversion ratio (FCR) was obtained for the W group compared to the G and BK groups. Group culture results: Significantly higher SR was recorded for the BK group, and the highest limb loss rate was detected for Y group indicating the significant effects of background color on survival and aggressiveness of juvenile E. sinensis. Moreover, background color impacted the carapace coloration indices where the W, BK and Y groups showed the highest brightness (L*), redness (a*), and yellowness (b*) values, respectively. Furthermore, correlation analysis revealed a positive linear relationship between the L* value and background color (y =0.1638x+27.961, R2=0.613). In conclusion, our results demonstrated that background color significantly affects culture performance and carapace color. Black background color was shown to enhance the survival rate and reduce the limb amputation rate under group culture condition. These results provide new insights on the optimization of farming technology for juvenile E. sinensis.

Abstract This study aimed to investigate the effects of adding different levels (0, 50, 100, 200 and 300 mg/kg) of dietary β‐carotene on the color and flavor quality of adult female E. sinensis ovaries. Adding moderate amount of β‐carotene to the feed could regulate the distribution of carotenoids, fatty acids, and free amino acids in the ovaries of E. sinensis, as well as increase the content of total carotenoids, total fatty acids, sweet and umami amino acids in the ovaries. The dietary β‐carotene have limited coloring effects and a significant impact on the flavor of the ovaries. During thermal process, the addition of 100 mg/kg β‐carotene could effectively promote formation of aroma precursors and generate more characteristic aroma components (2‐methylbutanal, 3‐methylbutanal, hexanal, benzaldehyde, methylpyrazine). However, excessive β‐carotene supplementation (300 mg/kg) led to adverse effects on physiological metabolism and flavor of E. sinensis ovaries. Considering both feed cost and quality improvement, β‐carotene supplementation at 100 mg/kg can be considered the optimal feed supplementation for adult female E. sinensis within a 70‐day rearing period to improve edible quality of ovaries.

Seawater contains a variety of mineral ions, such as Na+, Mg2+, Ca2+, and K+. Although previous studies have shown that brackish water can promote ovarian development of the Chinese mitten crab (Eriocheir sinensis), it remains unclear whether these ions directly affect the ovarian development and vitellogenesis of E. sinensis. Therefore, this study was designed, using in vitro tissue incubation mediums with different concentrations of Na+ (280, 320, 360, and 400 mmol/L), Mg2+ (5, 10, and 15 mmol/L), Ca2+ (5, 10, 15, and 20 mmol/L), and K+ (5.5, 9, and 13 mmol/L), to investigate their effects on Vg/Vn contents and the expression of ovarian development-related genes in the hepatopancreas and ovaries of E. sinensis. The results showed that: (1) Different Na+ concentrations had no significant impact on vitellogenesis and the expression of ovarian development-related genes in both tissues, except for an enhancement of the esEcR2-mRNA levels in the hepatopancreas. (2) The Mg2+ (15 mmol/L) significantly increased the Vg/Vn content in hepatopancreas and ovary. However, the 10 mmol/L Mg2+ group decreased esVgR-mRNA levels and esEcR2-mRNA levels in the ovary compared to the control group. (3) Ca2+ (10 ∼ 15 mmol/L) significantly promoted Vg/Vn content and esVg-mRNA levels in both tissues, which also upregulated the expression of ovarian development-related genes (esERR, esEcR2, and esRXR). (4) Increasing K+ content significantly decreased the Vg/Vn content in hepatopancreas and ovaries. Furthermore, the K+ significantly reduced esRXR-mRNA levels in the ovaries but significantly increased esERR-mRNA levels in both tissues. In summary, under in vitro conditions, Na+ concentration had no significant effect on the vitellogenesis, while Ca2+ (10 ∼ 15 mmol/L) and Mg2+ (15 mmol/L) significantly promoted the vitellogenesis in the hepatopancreas and ovaries of E. sinensis. Additionally, Ca2+ significantly enhanced the expression of genes related to ovarian development, while K+ had a negative impact on the vitellogenesis.

β-carotene has been shown to be beneficial for the color formulation, survival, antioxidation, immunity and stress resistance of many crustaceans. This study was conducted to investigate the effects of dietary supplementation of synthetic β-carotene on growth, antioxidant status, high pH resistance, color parameters, and carotenoids composition of pre-adult Chinese mitten crab Eriocheir sinensis. Three diets were formulated to contain 3.06, 52.40 and 103.59 mg/kg dry diets of synthetic β-carotene (defined as Diet 1 - 3). The results showed that: (1) There were no marked differences in body weights or feed utilization, suggesting that β-carotene was not particularly efficient in enhancing the growth of pre-adult E. sinensis. (2) Indicators of antioxidant capacity (T-AOC, MDA) and immunity (AKP, ACP) both got improved in the hepatopancreas and hemolymph with the supplementation of 50–100 mg/kg β-carotene in the diets. (3) The stress test with high pH indicated Diet 1 treatment had the highest mortality among all treatments, and the lowest mortality was found in Diet 3 treatment. (4) For color enhancement, the redness of the carapace and hepatopancreas increased significantly with the increase of dietary β-carotene supplementation at Day 30. Combined, the inclusion of β-carotene in the diet could enhance the antioxidative capability, non-specific immunity, stress resistance to high pH and tissue carotenoid contents, and the optimal level of dietary synthetic β-carotene supplementation was suggested to be around 50 mg/kg for pre-adult male E. sinensis.

The Chinese mitten crab (Eriocheir sinensis) is an important economic crab in China. Across most of China, juvenile Chinese mitten crabs are cultured in earthen ponds. Juvenile crab cultured in rice fields is mainly restricted to the northeastern region. Rice-crab coculture is a new mode of sustainable development, which has developed over the past 30 years in China with progress in field engineering, culture density of the Chinese mitten crab, and ecological benefits. However, there are no reports on appropriate feeds in rice-crab coculture. Presently, crabs reared in an earthen pond consume a traditional diet (including soybean meal, corn, bran, and wheat) or formulated feed. Due to the numerous advantages of formulated feed (including comprehensive nutrition, stable quality, and ease of feeding) the development of practical and cheap formulated feed for Chinese mitten crab is the latest focus for promoting the sustainable development of the Chinese mitten crab industry. Although researchers have developed a series of formulated feed for Chinese mitten crab, traditional feed is more commonly used in the culture of Chinese mitten crab due to farmers feeding habits and the low price of traditional products. However, numerous studies have shown that feeding Chinese mitten crabs formulated diets has many advantages, for example: it improves the survival rate, reduces the early maturity rate, improves non-specific immunity and disease resistance of juvenile crabs, improves the quality and culture performance of crab species, and economic benefits, etc. However, research were usually conducted under indoor or pond culture conditions. The culture environment of rice fields and ponds are quite different to each other. The feed sources and environmental stress on juvenile crabs may also be quite different between the culture environments. Therefore, it is important to explore the feeding mode of juvenile crabs cultured in rice fields. However, the optimum feed and the effect of different diets on the growth and physiological metabolism of Chinese mitten crab reared in rice fields remains unclear. It is important to optimize the rice-crab breeding mode to achieve a high-quality Chinese mitten crab industry. Formulated diets and traditional diets (soybean meal, bran, corn, and wheat) were fed to juvenile E. sinensis cultured in a rice field for 152 days. Each feeding group included three replicates. This study was conducted to compare formulated diets with traditional diets using culture performance, protein metabolism, digestive enzymes, and the antioxidant and immune indexes of juvenile crabs. The results showed that: (1) the body weights of female and male crabs from the formulated diet group were higher than those in the traditional diet group, but the differences were not significant (P > 0.05) (Fig.1–3); (2) in terms of culture performance, the average body weight and yield of juvenile crabs, 1-year-old precocious crabs, and the overall total yield and survival rate of juvenile crabs in the formulated diet group were slightly higher than those of the traditional diet group. The feed conversion ratio of the formulated diet group was much lower than that of the traditional diet group (P < 0.05); (3) in terms of protein metabolism, the total protein content in the hemolymph of both female and male crabs fed the formulated diet was significantly higher than that in the traditional diet group (P < 0.05). Conversely, the contents of urea nitrogen in the hemolymph of male crabs and the activity of glutamic oxaloacetic transaminase in the hepatopancreas of the traditional diet group were significantly higher than those of the formulated diet group (P < 0.05); (4) lipase in the hepatopancreas of female and male crabs in the formulated feed group was significantly higher than that in the traditional diet group (P < 0.05). Conversely, the α-amylase activity of the male crabs in the formulated diet group was significantly lower than that of the traditional diet group; (5) the total antioxidant capacity, alkaline phosphatase, and acid phosphatase activities in the hemolymph of female crabs and the acid phosphatase activity in the hepatopancreas of the formulated diet group were significantly higher than those of the traditional feed group (P < 0.05). In conclusion, feeding a formulated diet can improve the culture performance, promote protein deposition and lipid absorption, and enhance the antioxidant and immune capacity of juvenile E. sinensis reared in a rice field. These factors result in improved crab quality. This study provides a basis for optimizing the culture technology of Chinese mitten crab and the development of a formulated diet for improved rice-crab coculture.

Pond culture is the primary method for cultivating Chinese mitten crab (Eriocheir sinensis), with phytoplankton significantly influencing their growth. Green algae benefit crab growth by serving as supplementary food, while cyanobacteria, particularly during blooms, hinder it and pose health risks. Environmental changes in nutrient levels, temperature, and light significantly affect phytoplankton communities in ponds, impacting both ecosystem stability and crab growth. However, there is a limited understanding regarding the patterns of phytoplankton changes within adult Chinese mitten crab culture ponds. This study conducted monthly collection and analysis of phytoplankton throughout the culture cycle in typical adult Chinese mitten crab culture ponds, concurrently measuring physical and chemical water parameters to explore the correlation between phytoplankton changes and environmental factors. The results revealed distinct seasonal variations in phytoplankton composition. Chlorophyta and Bacillariophyta, such as Chlorella, Pediastrum, and Cocconeis, predominated in spring, while Chlorophyta and cyanobacteria, such as Volvox, Anabaena, and Microcystis, dominated in summer, and cyanobacteria and Bacillariophyta, such as Microcystis, Dolichospermum, and Cocconeis, prevailed in autumn. Total phytoplankton density consistently increased throughout the culture period. Microcystis constituted the majority of cyanobacteria biomass throughout most months. Although the total phytoplankton biomass fluctuated, cyanobacteria biomass consistently rose each month, reaching a peak of 61.7 mg/L in October. Water temperature and pH emerged as the primary environmental drivers influencing changes in phytoplankton community structure. Cyanobacteria density reached its peak between 18 and 26 °C and at a pH range of 7.5–8.5. These findings highlight the need for environmental regulation and cyanobacteria control in Chinese mitten crab culture ponds, thus promoting the health and sustainability of the Chinese mitten crab culture.

Carotenoid cleavage oxygenases can cleave carotenoids into a range of biologically important products. Carotenoid isomerooxygenase (NinaB) and β, β-carotene 15, 15′-monooxygenase (BCO1) are two important oxygenases. In order to understand the roles that both oxygenases exert in crustaceans, we first investigated NinaB-like (EsNinaBl) and BCO1-like (EsBCO1l) within the genome of Chinese mitten crab (Eriocheir sinensis). Their functions were then deciphered through an analysis of their expression patterns, an in vitro β-carotene degradation assay, and RNA interference. The results showed that both EsNinaBl and EsBCO1l contain an RPE65 domain and exhibit high levels of expression in the hepatopancreas. During the molting stage, EsNinaBl exhibited significant upregulation in stage C, whereas EsBCO1l showed significantly higher expression levels at stage AB. Moreover, dietary supplementation with β-carotene resulted in a notable increase in the expression of EsNinaBl and EsBCO1l in the hepatopancreas. Further functional assays showed that the EsNinaBl expressed in E. coli underwent significant changes in its color, from orange to light; in addition, its β-carotene cleavage was higher than that of EsBCO1l. After the knockdown of EsNinaBl or EsBCO1l in juvenile E. sinensis, the expression levels of both genes were significantly decreased in the hepatopancreas, accompanied by a notable increase in the redness (a*) values. Furthermore, a significant increase in the β-carotene content was observed in the hepatopancreas when EsNinaBl-mRNA was suppressed, which suggests that EsNinaBl plays an important role in carotenoid cleavage, specifically β-carotene. In conclusion, our findings suggest that EsNinaBl and EsBCO1l may exhibit functional co-expression and play a crucial role in carotenoid cleavage in crabs.

Astaxanthin is an important pigment for the rainbow trout Oncorhynchus mykiss. This study was conducted to investigate the effects of different sources of dietary astaxanthin on the growth, coloration, and antioxidant capacity of the commercial-sized O. mykiss during long-term feeding. Haematococcus pluvialis (HP), yeast Phaffia rhodozyma (PR), and synthetic astaxanthin (SA) were added to the basic feed (no astaxanthin, NA) to prepare the isonitrogenous and isolipidic experimental diets; the actual astaxanthin content values in the diets were 31.25, 32.96, and 31.50 mg/kg, respectively. Eighteen hundred O. mykiss, averaging 670 ± 20 g, were randomly divided into four groups and then fed with the experimental diet for four months. Dietary supplementation of P. rhodozyma and synthetic astaxanthin had no significant effects on the growth and tissue indexes of O. mykiss. In contrast, dietary supplementation with astaxanthin from H. pluvialis significantly increased the weight gain rate after four months of feeding. The fillet lightness of O. mykiss in the PR and SA was statistically lower than that in the NA and HP; the redness and astaxanthin content of fillet in the HP, PR, and SA groups were statistically higher than those in the NA. The total antioxidant capacity of the liver and serum in the HP was statistically higher than that in other diet groups, and a higher liver total superoxide dismutase activity was detected in the HP compared with the PR. Dietary supplementation of astaxanthin significantly increased the glutathione peroxidase activity in the liver and serum, and the highest serum glutathione peroxidase activity was detected in the HP, while dietary astaxanthin significantly decreased the malondialdehyde content in the liver and serum. Dietary supplementation of PR significantly increased the fillet ash content, while the highest fillet total lipid content was detected in the HP. Dietary astaxanthin significantly improved fillet redness and antioxidant capacity, among which H. pluvialis astaxanthin has greater effects on improving weight gain, antioxidant capacity, and fillet total lipid content.

Haematococcus pluvialis is an important source of natural astaxanthin, which plays a key role in the regulation of color, antioxidant capacity, and immunity in aquatic animals. However, it is unclear that the effects of long-term H. pluvialis astaxanthin feeding on the growth performance, coloration, and antioxidant capacity of commercial-sized rainbow trout Oncorhynchus mykiss. The commercial feed of O. mykiss was used as the basic feed, and 15, 30 mg/kg H. pluvialis astaxanthin were added to prepare isonitrogenous and isolipidic experimental diets, defined as HPA0, HPA15, and HPA30, respectively. Juvenile O. mykiss with an initial weight of 0.60 kg were fed with three experimental diets for 4 months, which was conducted to investigate the effects of long-term H. pluvialis astaxanthin feeding on the growth performance, coloration, antioxidant capacity, and biochemical composition. The results showed that the weight gain rate and specific growth rate of the HPA30 from 0 to 4 months were significantly higher than those of the other two diet groups. In the 4th month, the viscerosomatic index of O. mykiss significantly increased with increasing dietary astaxanthin levels. The redness (a*), DSM SalmoFan™ scores, and astaxanthin content of fillets significantly increased with increasing dietary astaxanthin levels during the whole feeding trial. The activities of total antioxidant capacity and glutathione peroxidase in the serum and liver, and serum immunoglobulin (IgM) content significantly increased with increasing dietary astaxanthin levels, while the levels of total antioxidant capacity, and malondialdehyde in the serum and liver showed a decreasing trend. The contents of total lipids in the fillet of O. mykiss increased significantly with increasing dietary astaxanthin levels, while the percentages of total saturated fatty acid (∑SFA), C18:3n3, and n-3/n-6 PUFA ratio showed a decreasing trend; the percentages of C18:2n6 and total n-6 polyunsaturated fatty acids (∑n-6PUFA) in the HPA15 and HPA30 were significantly higher than those of the HPA0. In conclusion, four months of feeding with 30 mg/kg H. pluvialis astaxanthin diet could improve the growth performance, fillet coloration, antioxidant capacity, and fillet total lipids content of commercial-sized O. mykiss.

Molting is one of the most important biological processes of crustacean species, and a number of molecular mechanisms facilitate this complex procedure. However, the understanding of the immune mechanisms underlying crustacean molting cycle remains very limited. This study performed transcriptome sequencing in hemolymph and hepatopancreas of the swimming crab (Portunus trituberculatus) during the four molting stages: post-molt (AB), inter-molt (C), pre-molt (D), and ecdysis (E). The results showed that there were 78,572 unigenes that were obtained in the hemolymph and hepatopancreas of P. trituberculatus. Further analysis showed that 98 DEGs were involved in immunity response of hemolymph and hepatopancreas, and most of the DEGs participated in the process of signal transduction, pattern recognition proteins/receptors, and antioxidative enzymes system. Specifically, the key genes and pathway involved in signal transduction including the GPCR126, beta-integrin, integrin, three genes in mitogen-activated protein kinase (MAPK) signaling cascade (MAPKKK10, MAPKK4, and p38 MAPK), and four genes in Toll pathway (Toll-like receptor, cactus, pelle-like kinase, and NFIL3). For the pattern recognition proteins/receptors, the lowest expression level of 11 genes was found in the E stage, including C-type lectin receptor, C-type lectin domain family 6 member A and SRB3/C in the hemolymph, and hepatopancreatic lectin 4, C-type lectin, SRB, Down syndrome cell adhesion molecule homolog, Down syndrome cell adhesion molecule isoform, and A2M. Moreover, the expression level of copper/zinc superoxide dismutase isoform 4, glutathione peroxidase, glutathione S-transferase, peroxiredoxin, peroxiredoxin 6, and dual oxidase 2 in stage C or stage D significantly higher than that of stage E or stage AB. These results fill in the gap of the continuous transcriptional changes that are evident during the molting cycle of crab and further provided valuable information for elucidating the molecular mechanisms of immune regulation during the molting cycle of crab.

The Pacific white shrimp Litopenaeus vannamei is an economically important penaeid species worldwide. Under farming conditions, the full ovarian maturation of L. vannamei generally depends on the combination of artificial ablation of unilateral eyestalk and feeding high-quality natural diets, suggesting that nutrient accumulation is important to ovarian maturation. In this study, we aimed to investigate the gene expression related to nutritional accumulation in L. vannamei during ovarian development by transcriptomic analysis. A total of 52.45 Gb of high-quality transcriptome data were obtained from 8 samples from the hepatopancreas and ovaries from shrimp in gonadal developmental stages I-IV. A total of 23,149 expressed genes were detected, of which 19,852 were known genes and 3,297 were novel genes. Our study found that genes related to amino acid, carbohydrate and lipid metabolism were expressed at higher levels in the hepatopancreas than in the ovary. The genes for most lipoproteins and their receptors were predominantly expressed in the hepatopancreas, while vitellogenin receptor (VgR) was specifically expressed in the ovary. Moreover, two vitellogenin (Vg) genes were identified, in which one was specifically expressed in the hepatopancreas and defined as Hp-Vg, and the other was specifically expressed in the ovary and defined as Ov-Vg. In addition, genes related to carotenoid metabolism were enriched in the hepatopancreas. This study provides solid evidence that the hepatopancreas is the major exogenous vitellogenesis site for ovarian maturation of L. vannamei. Two distinct Vg genes perform exogenous and endogenous vitellogenesis in the hepatopancreas and ovary in L. vannamei, respectively. The results of this study also presented some new insights for understanding the nutritional dynamics from the hepatopancreas to ovary during ovarian maturation in penaeids.

Ovarian development is a key physiological process that holds great significance in the reproduction of the Chinese mitten crab (Eriocheir sinensis), which is an economically important crab species for aquaculture. However, there is limited knowledge for the regulatory mechanisms of ovarian development. To study the molecular mechanisms of its ovarian development, transcriptome analysis was performed in the ovary and hepatopancreas of E. sinensis during ovarian stages I (oogonium proliferation), II (endogenous vitellogenesis), and III (exogenous vitellogenesis). The results showed that 5,520 and 226 genes were differentially expressed in the ovary and hepatopancreas, respectively. For KEGG enrichment analysis, the differentially expressed genes in the ovary were significantly clustered in phototransduction-fly, phagosome, and ECM-receptor interaction. Significantly enriched pathways in the hepatopancreas included fatty acid biosynthesis, fatty acid metabolism, and riboflavin metabolism. Further analysis showed that 25 genes and several pathways were mainly involved in oogenesis, including the ubiquitin-proteasome pathway, cyclic AMP-protein kinase A signaling pathway, and mitogen-activated protein kinase signaling pathway. Twenty-five candidate genes involved in vitellogenesis and endocrine regulation were identified, such as vitellogenin, vitellogenin receptor, estrogen sulfotransferase, ecdysone receptor, prostaglandin reductase 1, hematopoietic prostaglandin D synthase and juvenile hormone acid O-methyltransferase. Fifty-six genes related to nutritional metabolism were identified, such as fatty acid synthase, long-chain-fatty-acid-CoA ligase 4, 1-acyl-sn-glycerol-3-phosphate acyltransferase 4, fatty acid-binding protein, and glycerol-3-phosphate acyltransferase 1. These results highlight the genes involved in ovarian development and nutrition deposition, which enhance our understanding of the regulatory pathways and physiological processes of crustacean ovarian development.

Abstract Fish culture in paddy fields is a traditional aquaculture mode, which has a long history in East Asia. Large-scale loach (Paramisgurnus dabryanus) fast growth is suitable for paddy fields aquaculture in China. The objective of this study was to identify differential expression genes (DEGs) in the brain, liver and muscle tissues between large (LG, top 5% of maximum total length) and small (SG, top 5% of minimum total length) groups using RNA-seq. In total, 150 fish were collected each week and 450 fish were collected at twelfth week from three paddy fields for all the experimental. Histological observation found that the muscle fibre diameter of LG loaches was greater than that of SG loaches. Transcriptome results revealed that the high expression genes (HEGs) in LG loaches (fold change ≥ 2, p

This study analyzed the tissue indices, proximate composition, fatty acid and amino acid composition of edible tissues of male E. sinensis under three feeding modes (formulated diets (FD), natural diets (ND), and traditional diets (TD)). The results showed that different feeding modes had no impacts on the overall gonadal development, as no significant difference was found in gonadsomatic index among three treatments. For edible yield, three modes all had similar levels of the meat yield, hepatosomatic index, and total edible yield. Although different feeding modes did not affect the individual edible yield and gonadal development, significant differences were detected in the nutritional composition of males from three modes. For proximate composition, significant differences were found in the total lipids of gonads and the carbohydrate of hepatopancreas and meat. For fatty acids, crabs from ND had a higher proportion of monounsaturated fatty acids. In gonads and hepatopancreas, FD had a higher proportion of long-chain polyunsaturated fatty acids. For amino acids, FD had higher amino acid contents, while ND had the lowest. Except tryptophan, the essential amino acids scores of the meat were all greater than 100. As there was no significant difference on meat quality and meat yields between three feeding modes, this study suggests that the formulated diet could replace the natural diet and traditional diet towards sustainable crab farming in future.

In this study, the mating behavior of Portunus trituberculatus under indoor conditions and the role of shelters in its mating process were systematically studied by using behavioral observation methods. The results showed that the mating behavior of P. trituberculatus was divided into four stages: courtship and pre-copulatory guarding, reproductive moulting, copulation, and post-copulatory guarding. P. trituberculatus in the no shelter group (NSG) usually preferred to gather at the edges or corners of the cement tank, while P. trituberculatus in the shelter group (SG) preferred to hide in the shelters. During the whole experiment, the shelter occupancy rate of P. trituberculatus during the day (57.40%) was significantly higher than that at night (38.08%) (P

Abstract The effect of heating medium (with water and steam) and initial temperature of heating medium (cold and boiling) on the taste quality and sensory properties of Portunus trituberculatus was investigated. Nonvolatile taste active components in the meat and gonad of crabs cooked under four different methods were detected and compared. Sensory evaluation and electronic tongue assessment were also conducted. Sensory evaluation and electronic tongue indicated that taste quality was different under four cooking methods. The results showed that steaming cooking preserved more water‐soluble substances as compared with boiling cooking, especially cooking began with boiling water. It is noteworthy that the umami intensity in gonad cooked from boiling water was stronger, either steaming or boiling. Therefore, this study can provide a theoretical basis and suggest the dynamic chemical changes of different organs during heating process need to be further studied.

To investigate the effect of steaming time (0, 5, 10, 15, 20, and 25 min) on the protein degradation and non-volatile flavor substances of swimming crab (Portunus trituberculatus), the moisture content, total nitrogen (TN), non-protein nitrogen (NPN), free amino acids (FAAs), flavor nucleotides, electronic tongue analysis, and sensory evaluation were determined. The results showed that the contents of NPN and total FAAs were the highest after crabs steamed for 10 min. Meanwhile, the AMP (adenosine monophosphate) content reached the maximum value (332.83 mg/100 g) and the taste active value (TAV) reached 6.67, which indicated that AMP contributes the most to the taste of steamed crab at 10 min. The electronic tongue distinguished the taste difference well, and the sensory score was the highest at 15 min. Combined with equivalent umami concentration (EUC) and TAV value, swimming crab (weight = 200 ± 20 g) steamed for 10–15 min tasted best.

Prostaglandins (PGs) play many essential roles in the development, immunity, metabolism, and reproduction of animals. In vertebrates, arachidonic acid (ARA) is generally converted to prostaglandin G2 (PGG2) and H2 (PGH2) by cyclooxygenase (COX); then, various biologically active PGs are produced through different downstream prostaglandin synthases (PGSs), while PGs are inactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH). However, there is very limited knowledge of the PG biochemical pathways in invertebrates, particularly for crustaceans. In this study, nine genes involved in the prostaglandin pathway, including a COX, seven PGSs (PGES, PGES2, PGDS1/2, PGFS, AKR1C3, and TXA2S), and a PGDH were identified based on the Pacific white shrimp (Litopenaeus vannamei) genome, indicating a more complete PG pathway from synthesis to inactivation in crustaceans than in insects and mollusks. The homologous genes are conserved in amino acid sequences and structural domains, similar to those of related species. The expression patterns of these genes were further analyzed in a variety of tissues and developmental processes by RNA sequencing and quantitative real-time PCR. The mRNA expression of PGES was relatively stable in various tissues, while other genes were specifically expressed in distant tissues. During embryo development to post-larvae, COX, PGDS1, GDS2, and AKR1C3 expressions increased significantly, and increasing trends were also observed on PGES, PGDS2, and AKR1C3 at the post-molting stage. During the ovarian maturation, decreasing trends were found on PGES1, PGDS2, and PGDH in the hepatopancreas, but all gene expressions remained relatively stable in ovaries. In conclusion, this study provides basic knowledge for the synthesis and inactivation pathway of PG in crustaceans, which may contribute to the understanding of their regulatory mechanism in ontogenetic development and reproduction.

Oocyte maturation and ovarian development are sequentially coordinated events critical to reproduction. In the ovaries of adult oviparous animals such as birds, bony fish, insects, and crustaceans, vitellogenin receptor (VgR) is a plasma membrane receptor that specifically mediates vitellogenin (Vg) transport into oocytes. Accumulation of Vg drives sexual maturation of the female crustaceans by acting as a pivotal regulator of nutritional accumulation within oocytes, a process known as vitellogenesis. However, the mechanisms by which VgR mediates vitellogenesis are still not fully understood. In this study, we first identified a unique VgR (Lv-VgR) and characterized its genomic organization and protein structural domains in Litopenaeus vannamei, a predominant cultured shrimp species worldwide. This newly identified Lv-VgR phylogenetically forms a group with VgRs from other crustacean species within the arthropod cluster. Duplicated LBD/EGFD regions are found exclusively among arthropod VgRs but not in paralogs from vertebrates and nematodes. In terms of expression patterns, Lv-VgR transcripts are specifically expressed in ovaries of female shrimps, which increases progressively during ovarian development, and rapidly declines toward embryonic development. The cellular and subcellular locations were For analyzed by in situ hybridization and immunofluorescence, respectively. The Lv-VgR mRNA was found to be expressed in the oocytes of ovaries, and Lv-VgR protein was found to localize in the cell membrane of maturing oocytes while accumulation of the ligand Vg protein assumed an even cytoplasmic distribution. Silencing of VgR transcript expression by RNAi was effective for stunting ovarian development. This present study has thus provided new insights into the regulatory roles of VgR in crustacean ovarian development.

As an important freshwater aquaculture species in China, the Chinese mitten crab (Eriocheir japonica sinensis) has high economic and nutritional value. However, limited genomic information is currently available for studying its basic development and genetic diversity. Here, we performed whole-genome sequencing on Oxford Nanopore Technologies Limited's platform using promethION. The assembled size of E. j.sinensis genome was approximately 1.27 Gb, which is close to the estimated size (1.19 Gb). Furthermore, based on assessment using Benchmarking Universal Single-Copy Orthologs (BUSCO) (Simao et al., 2015), 94.00% of the expected eukaryotic genes were completely present in the genome assembly. In addition, repetitive sequences accounted for ~61.42% of the assembled genome, and 22,619 protein-coding genes were annotated. Comparative genomics analysis demonstrated that the Chinese mitten crab diverged from Penaeus vannamei ~373.6 million years ago, with a faster evolution rate than shrimp. We anticipate that the annotated high-quality genome of E. j. sinensis will promote research on its basic development and evolution and make substantial contributions to comparative genomic analyses of crustaceans.

The Chinese mitten crab (Eriocheir sinensis) possesses an open circulatory system, in which hemolymph moves through interconnected sinuses or spaces surrounding organs called hemocoels. The hemocytes are classified into hyalinocytes, semigranulocytes, and granulocytes; and limited transcriptomics research is available. In this study, the transcriptome of the crab E. sinensis hemocytes was characterized. A total of 14,380,229 clean reads representing a total of 4.31 Gb nucleotides dataset were produced. A total of 67,047 contigs were obtained and 12.49% (8375) and 9.74% (6533) of the contigs were matched to data publicly available from the GenBank nr nucleotide and Uniprot databases, respectively. Among these contigs, 4344 contigs belong to three categories of Gene Ontology, 126 contigs to 21 subcategories of KEGG, and 4962 contigs to 25 categories of the COG database. A total of 508,379 and 345 transcripts of the E. sinensis showed >40% sequence similarity with transcripts expressed in the vertebrate blood cells from tilapia (Oreochromis niloticus), Chinese softshell turtle (Trionyx sinensis) and chicken (Gallus gallus) respectively. A total of 53,077 single nucleotide polymorphisms were identified and 9912 SSRs for microsatellite mining were found. The most frequent repeat motifs detected were dinucleotide repeats, accounting for 37.52% of the total repeats. Our data provides an important gene resource for the exploitation of molecular marker-assisted breeding, immune mechanisms and conservation of germplasm in the crab E. sinensis. Keywords: Eriocheir sinensis, Hemocytes, Transcriptome

The Chinese mitten crab (Eriocheir sinensis) is an euryhaline crustacean, whose adults migrate downstream to estuaries for reproduction. Lipids are believed to be involved in salinity adaptation during migration. This study investigated the effects of different salinities (0, 6, 12, and 18‰) on the total lipids, neutral lipids, and polar lipids contents, and fatty acid profiles in the gonads, hepatopancreas, and muscles of adult E. sinensis after 40 days of salinity adaptation. The results showed that the males and females from 12‰ treatment had the highest contents of total lipids and neutral lipids in their hepatopancreas and total lipids in the muscles. Notably, salinity had a greater effect on the fatty acid profiles in the hepatopancreas compared to that in the gonads and muscles. The male hepatopancreas treated with 18‰ salinity had the highest percentage of total n-6 polyunsaturated fatty acid (∑n-6PUFA) in both neutral lipids and polar lipids, while the percentage of total n-3 polyunsaturated fatty acid (∑n-3PUFA) in neutral lipids and polar lipids decreased significantly with increasing salinity in males. In females, the 0‰ treatment had the highest percentages of total saturated fatty acids in neutral lipids and polar lipids in the hepatopancreas, while the highest ∑n-3PUFA and ∑n-6PUFA in neutral lipids and polar lipids were detected in the 12‰ treatment group. In conclusion, brackish water could promote the accumulations of total lipids and neutral lipids in the hepatopancreas and change the fatty acid profiles of adult E. sinensis, particularly in the hepatopancreas after long-term salinity adaptation.

Estradiol is an important sex steroid hormone that is involved in the regulation of crustacean ovarian development. However, the molecular regulatory mechanisms of estradiol on ovarian development are largely unknown. This study performed transcriptome sequencing of ovary, hepatopancreas, brain ganglion, eyestalk, and mandibular organ of crabs after estradiol treatment (0.1μg g-1 crab weight). A total of 23, 806 genes were annotated, and 316, 1300, 669, 142, 383 genes were expressed differently in ovary, hepatopancreas, brain ganglion, eyestalk, and mandibular organ respectively. Differentially expressed gene enrichment analysis revealed several crucial pathways including protein digestion and absorption, pancreatic secretion, insect hormone biosynthesis, drug metabolism-cytochrome P450 and signal transduction pathway. Through this study, some key genes in correlation with the ovarian development and nutrition metabolism were significantly affected by estradiol, such as vitelline membrane outer layer 1-like protein, heat shock protein 70, Wnt5, JHE-like carboxylesterase 1, cytochrome P302a1, crustacean hyperglycemic hormone, neuropeptide F2, trypsin, carboxypeptidase B, pancreatic triacylglycerol lipase-like, and lipid storage droplet protein. Moreover, RT-qPCR validation demonstrated that expression of transcripts related to ovarian development (vitelline membrane outer layer 1-like protein and cytochrome P302a1) and nutrition metabolism (trypsin, glucose dehydrogenase and lipid storage droplet protein) were significantly affected by estradiol treatment. This study not only has identified relevant genes and several pathways that are involved in estradiol regulation on ovarian development of P. trituberculatus, but also provided new insight into the understanding of the molecular function mechanisms of estradiol in crustacean.

Hypoxia has important effects on biological activity in crustaceans, and modulation of energy metabolism is a crucial aspect of crustaceans’ ability to respond to hypoxia. The adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) enzyme is very important in cellular energy homeostasis; however, little information is known about the role of AMPK in the response of prawns to acute hypoxia. In the present study, three subunits of AMPK were cloned from the oriental river prawn, Macrobrachium nipponense. The full-length cDNAs of the α, β, and γ AMPK subunits were 1,837, 3,174, and 3,773 bp long, with open reading frames of 529, 289, and 961 amino acids, respectively. Primary amino acid sequence alignment of these three subunits revealed conserved similarity between the functional domains of the M. nipponense AMPK protein with AMPK proteins of other animals. The expression of the three AMPK subunits was higher in muscle tissue than in other tissues. Furthermore, the mRNA expression of AMPKα, AMPKβ, and AMPKγ were significantly up-regulated in M. nipponense muscle tissue after acute hypoxia. Probing with a phospho-AMPKα antibody revealed that AMPK is phosphorylated following hypoxia; this phosphorylation event was found to be essential for AMPK activation. Levels of glucose and lactic acid in hemolymph and muscle tissue were significantly changed over the course of hypoxia and recovery, indicating dynamic changes in energy metabolism in response to hypoxic stress. The activation of AMPK by hypoxic stress in M. nipponense was compared to levels of muscular AMP, ADP, and ATP, as determined by HPLC; it was found that activation of AMPK may not completely correlate with AMP:ATP ratios in prawns under hypoxic conditions. These findings confirm that the α, β, and γ subunits of the prawn AMPK protein are regulated at the transcriptional and protein levels during hypoxic stress to facilitate maintenance of energy homeostasis.

The carnitine palmitoyltransferase (CPT) family includes CPT 1 and CPT 2 that transport long-chain fatty acids into the mitochondrial compartment for β-oxidation. In this study, three isoforms (CPT 1α, CPT 1β and CPT 2) of the CPT family were cloned from Chinese mitten crab (Eriocheir sinensis) and their complete coding sequences (CDS) were obtained. Sequence analysis revealed deduced amino acid sequences of 915, 775 and 683 amino acids, respectively. Gene expression analysis revealed a broad tissue distribution for all three isoforms, with high CPT 1α and CPT 2 mRNA levels in the hepatopancreas of males and females. In males, CPT 1β was highly expressed in gill, heart, brain ganglia and muscle, while in females, CPT 1β-mRNA levels were relatively high in muscle, hepatopancreas and ovary tissue. The effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development were investigated using five experimental diets formulated with replacement of 0, 25, 50, 75 and 100% fish oil by 1:1 rapeseed oil: soybean oil. The results showed that Diets 2# and 5# yielded higher CPT 1α and CPT 2 mRNA expression in males (P < 0.05), while in females, expression of all three CPT isoforms increased then declined in the hepatopancreas with increasing dietary fish oil replacement. The observed changes in CPT gene expression varied in different isoforms and gender, suggesting the three CPT genes might play different roles in fatty acid β-oxidation in E. sinensis.

Salinity plays a key role affecting ovarian development, osmoregulation and metabolism of female Chinese mitten crab, Eriocheir sinensis during reproductive migration. In this study, female E. sinensis after their puberty molt were subjected to four salinities of 0, 6, 12, and 18‰ for 40 days to investigate the salinity effects on their ovarian development as well as a range of important physiological parameters. Elevated salinity accelerated the ovarian development with ovigerous crabs found at salinity treatments of 12 and 18‰ despite no copulation had occurred. Meanwhile the survival rate of female crabs showed a decreasing trend with increasing salinity. Higher salinity also led to increased hemolymph Na+, K+, Ca2+, Cl−, and Mg2+ concentrations. The 6‰ treatment had the highest contents of hemolymph total and major free amino acids while the Na+/K+ -ATPase activity in the posterior gills was the lowest among treatments. Total n-3 polyunsaturated fatty acids (∑n-3PUFA) and n-3/n-6 PUFA ratio in the anterior gills showed a decreasing trend with salinity while 18‰ had the highest ∑PUFA and ∑n-6PUFA. The ∑n-3PUFA content and n-3/n-6 PUFA ratio of the posterior gills showed a fluctuating pattern and the highest value was detected at 0‰, while an increasing trend was found for the ∑n-6PUFA with increasing salinity. The hemolymph glucose showed a decreasing trend with increasing salinity and the highest total cholesterol in hemolymph was detected at 12‰. The 18‰ treatment had the highest levels of hemolymph γ-glutamyltransferase, alkaline phosphatase and acid phosphatase, as well as glucose, urea and acid phosphatase in hepatopancreas while the highest hemolymph superoxide dismutase and malondialdehyde were detected at 0‰. Overall, the results showed that salinity increase from freshwater to brackish conditions led to lower metabolism, accelerated ovarian development, and the appearance of ovigerous crabs without copulation in female E. sinensis post puberty molt.

Taxonomic classification of Eriocheir hepuensis was ambiguous, and it has long been controversial. In this study, the whole mitochondrial genome of E. hepuensis was determined to be 16,397 bp, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region. A total of 20 intergenic gaps were detected, and the AT content of whole mitochondrial genome was 71.78%. Phylogenetic analysis confirmed that the evolutionary relationship of E. hepuensis, E. sinensis, and E. japonica are most likely to be three species with the same taxonomic status. The whole mitogenome of this species will be useful for the future animal evolutionary, phylogenetic relationship, phylogeny and genomic studies in the genus Eriocheir.

As a catadromous species, salinity is a key parameter that affects gonadal development of Chinese mitten crab Eriocheir sinensis during reproductive migration. It is however unclear the effects of salinity on the gonadal development of male E. sinensis as well as their physiological responses to salinity during reproductive migration. This study investigated the effects of four salinities (0 ‰, 6 ‰, 12 ‰ and 18 ‰) on gonadal development, osmoregulation and metabolism of adult male E. sinensis over a 40-day period. The results showed that elevating salinity promote gonadal development, increase hemolymph osmolality and K+ and Mg2+ concentrations (P < 0.05). The 12 ‰ salinity resulted in the highest contents of taurine and arginine in the hemolymph while the highest contents of threonine, phenylalanine, lysine, ß-alanine, tryptophan, ornithine and total free amino acids were found for 0 ‰ treatment (P < 0.05). A decreasing trend was detected for the Na+/K+-ATPase activity and its mRNA expression level in the posterior gills with salinity (P < 0.05). Total saturated fatty acids in the anterior gills decreased with increasing salinity (P < 0.05); the 0 ‰ treatment had the highest total polyunsaturated fatty acids in the posterior gills while total n-6 polyunsaturated fatty acids increased with salinity (P < 0.05). The hemolymph glucose and uric acid showed a decreasing trend as salinity while an increasing trend was found for the hemolymph triglyceride and high-density lipoprotein cholesterol (P < 0.05). The 12 ‰ treatment had the highest levels of hemolymph malonaldehyde and hepatopancreatic γ-glutamyltranspeptidase (P < 0.05). In conclusion, these results suggested that the brackish water promote gonadal development of male E. sinensis, and increase osmolality and ionic concentrations in hemolymph while reduced the activity of Na+ /K+- ATPase and its mRNA expression in the posterior gills as well as metabolism.

The present study was conducted to investigate the size distribution and seasonality variation of ovarian development for females of the blue swimmer crab, Portunus pelagicus, from Beibu Gulf, China. The results show that, based on pubertal moult, gonadosomatic index (GSI), morphology and histological configurations, the ovarian development cycle of P. pelagicus can be divided into five stages; Stage I, dominated by oogonia with some previtellogenic oocytes and endogenous vitellogeic oocytes; Stage II, dominated by endogenous vitellogenic oocytes with some previtellogeic oocytes and oogonia; Stage III, dominated by exogenous vitellogeic oocytes; Stage IV, of nearly mature oocytes; and Stage V, of mature oocytes with fully ripened ovary. GSI increased significantly during the ovarian development (P < 0.05), while the hepatosomatic index (HSI) appeared to decrease. Thus, a significant negative correlation was found between GSI and HSI (P < 0.01). The smallest carapace width of a mature female and that at which 50% of females reach maturity were 79.2 mm and 108.2 mm, respectively. The carapace width and body weight of the dominant size class ranged between 86 and 115 mm and 40 and 120 g, respectively. Furthermore, the highest GSI of female P. pelagicus was found in March, while the peak of ovigerous females was found in April.

Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn (Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.

Metabolic adaption to hypoxic stress in crustaceans implies a shift from aerobic to anaerobic metabolism. Lactate dehydrogenase (LDH) is a key enzyme in glycolysis in prawns. However, very little is known about the role of LDH in hypoxia inducible factor (HIF) pathways of prawns. In this study, full-length cDNA of LDH (MnLDH) was obtained from the oriental river prawn Macrobrachium nipponense, and was characterized. The full-length cDNA is 2267-bp with an open reading frame of 999 bp coding for a protein of 333 amino acids with conserved domains important for function and regulation. Phylogenetic analysis showed that MnLDH is close to LDHs from other invertebrates. Quantitative real-time PCR revealed that MnLDH is expressed in various tissues with the highest expression level in muscle. MnLDH mRNA transcript and protein abundance in muscle, but not in hepatopancreas, were induced by hypoxia. Silencing of hypoxia-inducible factor 1 (HIF-1) α or HIF-1β subunits blocked the hypoxia-dependent increase of LDH expression and enzyme activity in muscle. A series of MnLDH promoter sequences, especially the full-length promoter, generated an increase in luciferase expression relative to promoterless vector; furthermore, the expression of luciferase was induced by hypoxia. These results demonstrate that MnLDH is probably involved a HIF-1-dependent pathway during hypoxia in the highly active metabolism of muscle.

The crustacean hepatopancreas has different functions including absorption, storage of nutrients and vitellogenesis during growth, and ovarian development. However, genetic information on the biological functions of the crustacean hepatopancreas during such processes is limited. The swimming crab, Portunus trituberculatus, is a commercially important species for both aquaculture and fisheries in the Asia-Pacific region. This study compared the transcriptome in the hepatopancreas of female P. trituberculatus during the growth and ovarian maturation stages by 454 high-throughput pyrosequencing and bioinformatics. The goal was to discover genes in the hepatopancreas involved in food digestion, nutrition metabolism and ovarian development, and to identify patterns of gene expression during growth and ovarian maturation. Our transcriptome produced 303,450 reads with an average length of 351 bp, and the high quality reads were assembled into 21,635 contigs and 31,844 singlets. Based on BLASTP searches of the deduced protein sequences, there were 7,762 contigs and 4,098 singlets with functional annotation. Further analysis revealed 33,427 unigenes with ORFs, including 17,388 contigs and 16,039 singlets in the hepatopancreas, while only 7,954 unigenes (5,691 contigs and 2,263 singlets) with the predicted protein sequences were annotated with biological functions. The deduced protein sequences were assigned to 3,734 GO terms, 25 COG categories and 294 specific pathways. Furthermore, there were 14, 534, and 22 identified unigenes involved in food digestion, nutrition metabolism and ovarian development, respectively. 212 differentially expressed genes (DEGs) were found between the growth and endogenous stage of the hepatopancreas, while there were 382 DEGs between the endogenous and exogenous stage hepatopancreas. Our results not only enhance the understanding of crustacean hepatopancreatic functions during growth and ovarian development, but also represent a basis for further research on new genes and functional genomics of P. trituberculatus or closely related species.