CBS/CSE mediated H2S production induced AMPs expression through Toll pathway in crabs with black gill syndrome.

The occurrence of black gill syndrome (BGS) is a serious threat to the healthy culture of Eriocheir sinensis. Studying the innate immune ability of E. sinensis with BGS can help develop new strategies for disease prevention and treatment. Antimicrobial peptides (AMPs) have crucial roles in crustacean humoral immunity. In this study, we found that the expression levels of two antilipopolysaccharide factor (EsALF7 and EsALF-L), one Toll receptor 3 (EsToll3), and one Pelle kinase (EsPelle) were upregulated in E. sinensis with BGS. Moreover, ALFs expressions in E. sinensis with BGS were positively regulated by EsToll3 and EsPelle. The content of hydrogen sulfide (H2S) in the gills of E. sinensis with BGS was increased. Further studies showed that the expressions of cystathionine ß-synthase (EsCBS) and cystathionine γ-lyase (EsCSE) in the gills of E. sinensis with BGS were upregulated, which positively regulate the production of H2S. Whether there was a correlation between the upregulation of ALFs expression and changes in H2S content? Further studies showed that 1) the expressions of EsToll3, EsPelle, EsALF7, and EsALF-L in the gills of E. sinensis were upregulated under H2S exposure and 2) the knockdown of EsCBS and EsCSE in E. sinensis reduced the transcriptions of EsToll3, EsPelle, EsALF7, and EsALF-L. To sum up, these findings suggest that upregulation of H2S content induced by CBS/CSE promotes the expression of ALFs through the Toll pathway in E. sinensis suffering from BGS.

Characterization of the molecular, cellular, and behavioral changes caused by exposure to a saline-alkali environment in the Chinese mitten crab, Eriocheir sinensis.

In the context of global warming, the accelerated evaporation of seawater will lead to a continuous expansion of saline-alkali land area. As an important economic freshwater crustacean, investigation on the mechanism of damage to Eriocheir sinensis (E. sinensis) under saline-alkali environment will provide a valuable precedent for understanding the detrimental effect of climate change on crustaceans. In this study, histopathological analysis and integrative omics analysis were employed to explore the injury mechanism on the cerebral nervous system of E. sinensis exposure to saline-alkali stress. Our findings revealed that under this stress E. sinensis exhibited behavioral disorders and damage to cerebral neurosecretory cells and key organelles. Additionally, several pathways related to detoxification metabolism, neurotransmitter synthesis, and antioxidant defense were significantly down-regulated. Collectively, these results show, for the first time, that saline-alkali stress can induce neurodegenerative disease-like symptoms in E. sinensis, and provide critical information for understanding the harmful effects of saline-alkali environments.

Molecular insights into information processing and developmental and immune regulation of Eriocheir sinensis megalopa under hyposaline stress.

Eriocheir sinensis is an important euryhaline catadromous crustacean of the Yangtze River and an important commercial species for breeding in China. However, wild E. sinensis have suffered serious damage attributed to overfishing, climate change, etc. The Ministry of Agriculture of China issued a notice banning the commercial fishing of wild E. sinensis. E. sinensis megalopa migrates upriver into fresh water for growth and fattening, which creates optimal conditions to experimentally explore its hyposaline osmoregulation mechanism. We performed comparative transcriptome analyses of E. sinensis megalopae under hyposaline stress. The results suggest that KEGG pathways and genes related to genetic information processing, developmental regulation, immune and anti-stress responses were differentially expressed. The present study reveals the most significantly enriched pathways and functional gene groups, and explores the hyposaline osmoregulation mode of E. sinensis megalopae. This study lays a theoretical foundation for further studies on the osmoregulation and developmental mechanisms of E. sinensis.

Exploration of Synergistic Regulation Mechanisms of Cerebral Ganglion and Muscle in Eriocheir sinensis Activated in Response to Alkalinity Stress.

The cerebral ganglion and muscle are important regulatory tissues in Eriocheir sinensis. Therefore, it is of great significance to explore their synergistic roles in this organism's anti-stress response. In this study, proteomics, metabolomics, and combination analyses of the cerebral ganglion and muscle of E. sinensis under alkalinity stress were performed. The cerebral ganglion and muscle played a significant synergistic regulatory role in alkalinity adaptation. The key regulatory pathways involved were amino acid metabolism, energy metabolism, signal transduction, and the organismal system. They also played a modulatory role in the TCA cycle, nerve signal transduction, immune response, homeostasis maintenance, and ion channel function. In conclusion, the present study provides a theoretical reference for further research on the mechanisms regulating the growth and development of E. sinensis in saline-alkaline environments. In addition, it provides theoretical guidelines for promoting the vigorous development of the E. sinensis breeding industry in saline-alkaline environments in the future.

The crosstalk between Toll and AMPK signaling pathways mediates growth inhibition of Eriocheir sinensis under deltamethrin stress.

Hepatopancreatic necrosis disease (HPND) broke out in 2015 in the Eriocheir sinensis aquaculture region of Xinghua, Jiangsu Province; however, the specific cause of HPND remains unclear. A correlation was found between HPND outbreak and the use of deltamethrin by farmers. In this study, E. sinensis specimens developed the clinical symptoms of HPND after 93 days of deltamethrin stress. The growth of E. sinensis with HPND was inhibited. Adenosine monophosphate-activated protein kinase (AMPK) is a central regulator of energy homeostasis, and its expression was up-regulated in the intestine of E. sinensis with HPND. Growth inhibitory genes (EsCabut, Es4E-BP, and EsCG6770) were also up-regulated in the intestine of E. sinensis with HPND. The expression levels of EsCabut, Es4E-BP, and EsCG6770 decreased after EsAMPK knockdown. Therefore, AMPK mediated the growth inhibition of E. sinensis with HPND. Further analysis indicated the presence of a crosstalk between the Toll and AMPK signaling pathways in E. sinensis with HPND. Multiple genes in the Toll signaling pathway were upregulated in E. sinensis under 93 days of deltamethrin stress. EsAMPK and its regulated growth inhibition genes were down-regulated after the knockdown of genes in the Toll pathway. In summary, the crosstalk between the Toll and AMPK signaling pathways mediates the growth inhibition of E. sinensis under deltamethrin stress.

Top-Down Effect of Arthropod Predator Chinese Mitten Crab on Freshwater Nutrient Cycling.

Aquatic litter decomposition is highly dependent on contributions and interactions at different trophic levels. The invasion of alien aquatic organisms like the channeled apple snail (Pomacea canaliculata) might lead to changes in the decomposition process through new species interactions in the invaded wetland. However, it is not clear how aquatic macroinvertebrate predators like the Chinese mitten crab (Eriocheir sinensis) will affect the nutrient cycle in freshwater ecosystems in the face of new benthic invasion. We used the litter bag method to explore the top-down effect of crabs on the freshwater nutrient cycle with the help of soil zymography (a technology previously used in terrestrial ecosystems). The results showed significant feeding effects of crabs and snails on lotus leaf litter and cotton strips. Crabs significantly inhibited the intake of lotus litter and cotton strips and the ability to transform the environment of snails by predation. Crabs promoted the decomposition of various litter substrates by affecting the microbial community structure in the sediment. These results suggest that arthropod predators increase the complexity of detrital food webs through direct and indirect interactions, and consequently have an important impact on the material cycle and stability of freshwater ecosystems. This top-down effect makes macrobenthos play a key role in the biological control and engineering construction of freshwater ecosystems.

Glutathione S-transferase gene diversity and their regulation by Nrf2 in Chinese mitten crab (Eriocheir sinensis) during nitrite stress.

Eriocheir sinensis is one of the most important economic aquatic products in China. However, nitrite pollution has become a serious threat to the healthy culture of E. sinensis. Glutathione S-transferase (GST) is an important phase II detoxification enzyme, which plays a leading role in the cellular detoxification of exogenous substances. In this study, we obtained 15 GST genes (designated as EsGST1-15) from E. sinensis, and their expression and regulation in E. sinensis under nitrite stress were studied. EsGST1-15 belonged to different GST subclasses. EsGST1, EsGST2, EsGST3, EsGST4, and EsGST5 belonged to Delta-class GSTs; EsGST6 and EsGST7 are Theta-class GSTs; EsGST8 is a mGST-3-class GST; EsGST9 belonged to mGST-1-class GSTs; EsGST10 and EsGST11 belonged to Sigma-class GSTs; EsGST12, EsGST13, and EsGST14 are Mu-class GSTs; EsGST15 is a Kappa-class GST. Tissue distribution experiments showed that EsGSTs were widely distributed in all detected tissues. The expression level of EsGST1-15 was significantly increased in the hepatopancreas under nitrite stress, indicating that EsGSTs were involved in the detoxification of E. sinensis under nitrite stress. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a transcription factor that can activate the expression of detoxification enzyme. We detected the expression of EsGST1-15 after interfering with EsNrf2 in the hepatopancreas of E. sinensis with or without nitrite stress. Results showed that EsGST1-15 were all regulated by EsNrf2 with or without nitrite stress. Our study provides new information about the diversity, expression, and regulation of GSTs in E. sinensis under nitrite stress.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress.

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential "nitrite stress-Duox activation-ROS production" pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for "nitrite stress-Duox activation-ROS production-Dorsal activation-AMP synthesis" that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

Isolation of yellow catfish ß-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein.

Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of ß-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own ß-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

Bioinformatic identification and validation of conservative microRNAs in Ictalurus punctatus.

Catfish (Ictalurus spp.) is an important aquaculture species around the world, accounting for over 60 % of the domestic aquaculture output in the United States. However, little information is available about I. punctatus miRNAs which play an important role in the regulation of almost every biological process. In the present studies, we applied a bioinformatic strategy to identify 16 miRNAs which represent 12 miRNA families in I. punctatus by searching both expressed sequence tags and genome survey sequences databases. The A + U contents of the candidate pre-miRNA sequence range from 51 to 63 %, and the pre-miRNA sequences vary from 55 to 63 bp in length. To verify the predicted miRNAs, real-time PCR was used to profile the expression of 16 miRNAs with different tissues of I. punctatus. All the miRNA candidates were detectable in five tissues except for ipu-miR-9-3p. Based on sequence complementarity between miRNAs and their mRNA targets, potential targets for I. punctatus miRNAs were predicted. Due to the limited information for the I. punctatus transcripts, only one sequence targeted by ipu-miR-135 was identified to be an I. punctatus EB1 mRNA. Bioinformatic analyses indicated that the 3' untranslated region (3'-UTR) of EB1 mRNA contains an ipu-miR-135 target site, which are perfectly complementary to the seed region (positions 2-8) of the mature ipu-miR-135. I. punctatus miRNAs characterized in this study may provide useful information for the miRNAs research in I. punctatus and other aquaculture species.

[Determination of free amino acids in Eriocheir sinensis by ultra-high performance liquid chromatography-high resolution mass spectrometry].

Eriocheir sinensis is a unique freshwater crab found in China, which is well known for its rich nutrition and sweet and delicious taste. Free amino acids in Eriocheir sinensis are not only important nutrients but also are closely related to their unique taste and aroma. Therefore, the determination of the free amino acid contents in Eriocheir sinensis is of great significance for product quality evaluation, flavor research, authenticity, and origin identification. Herein we proposed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based method for the determination of 17 free amino acids in Eriocheir sinensis. First, 5 g of the Eriocheir sinensis sample was weighed into a 50-mL polypropylene centrifuge tube. Then, 10 mL of extraction solvents was added to the centrifuge tube, and the resultant solution was mixed well using a vortex mixer. We compared a variety of solvents and finally selected 5%(v/v) perchloric acid aqueous solution as the optimum extraction solvent. The supernatant was transferred to another polypropylene centrifuge tube after centrifuging at 8000 r/min for 5 min. The extraction procedure was repeated according to the above-mentioned steps, and the extraction solution was combined with the supernatant. The extracts were then adjusted to pH 6.5 with 1 mol/L potassium hydroxide solution, and were diluted to 50 mL with water. After filtering by both qualitative filter paper and a 0.45-µm polyether sulfone syringe filter, the extracts were determined by UHPLC-HRMS. We compared three types of mobile phases and chose 0.1%(v/v) formic acid aqueous solution mixed with acetonitrile as the optimum one. Precise parent ion and ion source parameters were also optimized. The 17 analytes, viz. aspartic acid, threonine, serine, glutamic acid, proline, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, glycine, alanine, and histidine, were separated on an XDB-C18 column (100 mm×4.6 mm, 1.7 µm) with gradient elution. The amino acids were then detected by HRMS in electrospray ionization and selected ion monitoring modes, and the analytes were quantified using external standards. The instrumental detection limit (IDL) and the instrumental quantification limit (IQL) were 0.3 mg/L and 1.0 mg/L, respectively. The linear correlation coefficients were all above 0.9990 in the concentration range of 10.0-200.0 mg/kg. Three levels of free amino acid standards were spiked into the edible parts of Eriocheir sinensis. The recoveries of the amino acids were between 78.4% and 105.3%. The intra-sample, intra-day, and inter-day repeatabilities were below 4.2%, 5.2%, and 11.4%, respectively, which were within reasonable ranges. Twenty samples of Eriocheir sinensis were tested using the proposed method. Thus, in this study, we developed an alternative method for the determination of free amino acids in Eriocheir sinensis with simple pretreatment, good selectivity, and high accuracy.

Transcriptome Analysis of Hepatopancreas Provides Insights into Differential Metabolic Mechanisms of Eriocheir sinensis Feeding on Trash Fish and Formulated Feed.

BACKGROUND: The Chinese mitten crab, Eriocheir sinensis (E. sinensis), is a popular crab species in both domestic and foreign markets. Trash fish are essential for E. sinensis breeding, but have caused serious water pollution. The municipal party committee for the main production areas of E. sinensis implemented a ban on feeding on trash fish since 2020. METHODS: In this study, we performed a culture experiment of E. sinensis feeding on trash fish and formulated feed, with comparative transcriptome analysis on hepatopancreas of E. sinensis. RESULTS: The results indicate that formulated feed causes no significant difference in growth, survival rate or content of amino acids in the muscles of adult E. sinensis. Formulated feed caused a slight downregulation of pathways involved in amino acid metabolism, development, energy metabolism and homeostasis maintenance. CONCLUSIONS: On the whole, formulated feed can serve as an undifferentiated substitution for trash fish. This study provides a theoretical foundation for optimizing research on E. sinensis feed.

Exploration of the regulatory mechanisms of regeneration, anti-oxidation, anti-aging and the immune response at the post-molt stage of Eriocheir sinensis.

Eriocheir sinensis is widely appreciated by the surrounding population due to its culinary delicacy and rich nutrients. The E. sinensis breeding industry is very prosperous and molting is one of the important growth characteristics. Research on the regulation of molting in E. sinensis is still in the initial stages. There is currently no relevant information on the regulatory mechanisms of heart development following molting. Comparative transcriptome analysis was used to study developmental regulation mechanisms in the heart of E. sinensis at the post-molt and inter-molt stages. The results indicated that many regulatory pathways and genes involved in regeneration, anti-oxidation, anti-aging and the immune response were significantly upregulated after molting in E. sinensis. Aside from cardiac development, the differentially expressed genes (DEGs) were relevant to myocardial movement and neuronal signal transduction. DEGs were also related to the regulation of glutathione homeostasis and biological rhythms in regard to anti-oxidation and anti-aging, and to the regulation of immune cell development and the immune response. This study provides a theoretical framework for understanding the regulation of molting in E. sinensis and in other economically important crustaceans.

Reproductive performance and semen characteristics of pond-reared and wild-caught large-sized male broodstock of the Chinese mitten crab Eriocheir sinensis.

There has been a recent emphasis on production of large-sized Eriocheir sinensis broodstock. In China, aquaculturists generally prefer wild-caught (WC) crabs from the Yangtze River as broodstock because offspring performance is superior to that of pond-reared (PR) broodstock. Currently, however, there is a ban on fishing in the Yangtze River, and effects on E. sinensis breeding have not been ascertained. There was comparison in the present study of reproductive performance and semen characteristics of male broodstock of PR and WC groups. After copulation, sperm quantity in the vas deferens of crabs in specimens of both groups was large, although there was a consistent decrease in vaso-somatic index. Although sperm density of PR crabs was less, that of WC specimens remained relatively constant. Specimens of neither group, however, had changes in the hepatopancreas index or condition factor, and sperm survival was close to 100%. Although the acrosome reaction was detected in response to cold-temperature induction, there were differences in extent of reaction to cold temperatures. Importantly, in as many as 98% of sperm from female spermathecae, the reaction was completed, which was considerably greater than 15% for sperm of males post-mating. It is concluded there was no difference between PR and WC crabs with respect to reproductive performance or semen characteristics, and, notably, sperm from PR crabs were of sufficient quality for use in E. sinensis aquaculture enterprises. Accordingly, it is predicted the Yangtze River fishing ban would only have a limited effect on supply of male E. sinensis broodstock.

A novel JNK induces innate immune response by activating the expression of antimicrobial peptides in Chinese mitten crab Eriocheir sinensis.

c-Jun NH2-terminal kinase (JNK) is a member of mitogen-activated protein kinases (MAPKs) that participates in the regulation of various physiological and pathological processes. In this study, we identified a novel JNK (EsJNK) and determined the cDNA sequence of its isoform (EsJNK-a) from the Chinese mitten crab Eriocheir sinensis. The open reading frame (ORF) of EsJNK was predicted to encode 421 peptides with a serine/threonine protein kinase, a catalytic (S_TKc) domain, and a low complexity region. The ORF of EsJNK-a was 1380 bp encoding a protein with 459 amino acids, which was 38 amino acids more than that of EsJNK. The predicted tertiary structure of EsJNK was conserved and contained 15 α-helices and 10 ß-sheets. Phylogenetic tree analysis revealed that EsJNK was clustered with the JNK homologs of other crustaceans. Quantitative real-time PCR assays showed that EsJNK was expressed in all the tissues examined, but it was relatively higher in hemocytes, muscles, and intestines. The expression of EsJNK mRNA in the hemocytes was upregulated by lipopolysaccharides and peptidoglycans, as well as by Staphylococcus aureus or Vibrio parahaemolyticus challenge. Functionally, after silencing EsJNK by siRNA in crabs, the expression levels of two antimicrobial peptides (AMPs), namely, anti-lipopolysaccharide factor and crustin, were significantly inhibited. The purified recombinant EsJNK protein with His-tag accelerated the elimination of the aforementioned bacteria in vivo. However, knockdown of EsJNK had an opposite effect. These findings suggested that EsJNK might be involved in the antibacterial immune defense of crabs by regulating the transcription of AMPs.

Alterations of amino acid metabolism and intestinal microbiota in Chinese mitten crab (Eriocheir sinensis) fed on formulated diet and iced trash fish.

Formulated diet (FD) and iced trash fish (ITF) are common diets during E. sinensis farming. However, whether FD can completely replace ITF during long-term E. sinensis farming is still unclear. Thus this study was conducted to compare the differences in amino acid metabolism and intestinal microbiota of the E. sinensis fed on different diets. The crabs were randomly divided into three groups fed on FD, ITF and mixed diet (MD, FD: ITF = 1:1), respectively. The results showed that there were no significant differences in amino acid composition among FD, MD and ITF groups. The activities of AST and ALT, and the mRNA levels of amino acid metabolism-related genes were significantly up-regulated in FD or/and MD groups compared with ITF group. The diversity of intestinal bacterial community was similar between the FD and ITF groups, but the relative abundance of dominant taxa showed marked differences between the two groups. At the phylum level, the relative abundance of Firmicutes was significantly higher, but the relative abundance of Proteobacteria was significantly lower in the FD group than that in ITF group. Meanwhile, at genus level, the relative abundance of Candidatus_Hepatoplasma in FD was higher than that in ITF group. Data related to functional prediction demonstrated that the significantly differenced pathways between the two groups were observed in metabolism (Pyrimidine metabolism, Glycolysis/Gluconeogenesis and Citrate cycle) and environmental information processing (transporters). The overall results indicated that replacement of ITF by FD did not affect amino acid composition, but altered amino acid metabolism and the relative abundance of intestinal microbiota. Our data provided a valuable reference for FD application replacing ITF during long-term E. sinensis farming.

Risk to humans of consuming metals in anchovy (Coilia sp.) from the Yangtze River Delta.

Concentrations of metals were determined in four species of anchovy (Coilia sp.) from the Yangtze River, Taihu Lake, and Hongze Lake in Jiangsu Province, China. Concentrations of Cr in anchovy fish muscle ranged from 2.6 × 10(-2) to 5.0 mg/kg ww, and Coilia nasus taihuensis in Jiaoshan, Taihu Lake contained the highest concentrations of Cr, which was almost 111-fold higher than the mean value at other locations. Concentrations of Pb ranged from 1.5 × 10(-2) to 1.3 × 10(-1) mg/kg ww. Comparisons of concentrations of lead (Pb) among the four species indicated that anadromous species contained higher concentrations of Pb than did freshwater species. However, concentrations of Pb in C. nasus from the Nanjing and Haimen locations in the Yangtze River were not significant higher than those of two freshwater species: C. nasus taihuensis from Taihu Lake and C. brachygnathus from Hongze Lake (Duncan's test, α = 0.05). While concentrations of Cd and Zn ranged from 7.0 × 10(-4) to 3.6 × 10(-3) mg/kg ww and 3.4 to 4.8 mg/kg ww, respectively, there were no significant differences in concentrations among the eight locations. The only concentration of the metals studied that exceeded the Chinese National Standard was Cr in Coilia from Jiaoshan, Taihu Lake, which was 2.5-fold higher than the standard. These results indicate that people who consume the genus Coilia are not at risk due to concentrations of metals, except Cr in C. nasus taihuensis from Jiaoshan in Taihu Lake. Concentrations of all of the metals studied except for Cr were similar to or less than those of metals in most other areas in the world.

Concentrations of pentachlorophenol (PCP) in fish and shrimp in Jiangsu Province, China.

Pentachlorophenol (PCP) concentrations were determined in 55 samples of fish, shrimp, crabs, frogs and turtles collected in Jiangsu Province and 55 freshwater shrimp samples collected from the Huiming Fish Market, Nanjing, China between 2003 and 2004. PCP concentrations ranged from less than the method detection limit (MDL: 0.5 microg/kg ww) to 61 microg/kg ww, with a mean of 5.2 microg/kg ww in 55 samples collected across Jiangsu Province and a mean of 0.5 microg/kg ww for freshwater shrimp collected from the Huiming Fish Market. Concentrations of PCP in common carp (Carassius auratus) were significantly greater than concentrations of PCP in other species from all of the areas studied. Grass carp (Ctenopharyngodon idella) contained the second greatest concentrations and crustacean species contained the least concentrations. Concentrations of PCP were greater in samples collected from Southern Jiangsu than those collected from other areas of Jiangsu Province. Concentrations of PCP in the freshwater shrimp (Macrobrachium nipponense) from the Huiming Fish Market were greater in March and September than those collected in June and December. Based on the allowable daily intake (ADI) set by the US National Academy of Science, current concentrations of PCP measured in aquatic products from Jiangsu Province presented minimus health risks to humans.

Molecular cloning, characterization and expression analysis of two juvenile hormone esterase-like carboxylesterase cDNAs in Chinese mitten crab, Eriocheir sinensis.

Precise regulation of methyl farnesoate (MF) titer is of prime importance throughout the crustacean life-cycle. Although the synthetic pathway of MF is well-documented, little is known about its degradation and recycling in crustaceans. Juvenile hormone esterase-like (JHE-like) carboxylesterase (CXE) is a key enzyme in MF degradation, thus playing a significant role in regulating the MF titer. We identified and characterized two cDNAs, Es-CXE1 and Es-CXE2, encoding JHE-like CXEs in Chinese mitten crab. Full-length cDNAs of Es-CXE1 and Es-CXE2 encode proteins composed of 584 and 597 amino acids, respectively, both of which contain a typical carboxylesterase domain. Alignment and phylogenetic analyses revealed that the Es-CXEs are highly similar to those of other crustaceans. To further validate their functions, we evaluated the mRNA expression patterns of the Es-CXEs in various tissues and in different physiological conditions. Tissue-specific expression analysis showed that the two Es-CXEs were predominantly expressed in the hepatopancreas and ovaries, which are the major tissues for MF metabolism. Es-CXE2 expression levels in the hepatopancreas and ovaries were about 100 and 25-fold higher, than the respective Es-CXE1 expressions. During ovarian rapid development stage, the global expressions of Es-CXEs were up-regulated in the hepatopancreas and down-regulated in the ovaries. After eyestalk ablation (ESA), the mRNA expressions of the two Es-CXEs were up-regulated in the hepatopancreas, further indicating their potential in degrading MF. Taken together, our results suggest that Es-CXEs, the key component of the juvenile hormone degradation pathway, may play vital roles in the development and reproduction of the Chinese mitten crab.

Transcriptome profiling of the eyestalk of precocious juvenile Chinese mitten crab reveals putative neuropeptides and differentially expressed genes.

Chinese mitten crabs that reach maturity 1 year earlier than normal crabs are known as precocious juvenile crabs. The molecular mechanisms underlying the precocity of the Chinese mitten crab are poorly understood. To identify the genes that may be involved in the control of precocity in Chinese mitten crab, we measured the expression profile of eyestalk genes in precocious and normally developed juvenile crabs using high-throughput sequencing on an Illumina HiSeq 2500 platform. We obtained 56,446,284 raw reads from the precocious crabs and 58,029,476 raw reads from the normally developed juvenile crabs. Reads from the two libraries were combined into a single data set. De novo assembly of the combined read set yielded 78,777 unigenes with an average length of 1563 bp. A total of 41,405 unigenes with predicted ORFs were selected for functional annotation. Among these genes, we identified three neuropeptide genes belonging to the crustacean hyperglycemic hormone family and two neuropeptide genes encoding the chromatophorotropic hormones. Transcriptome comparison between the two libraries revealed 42 genes that exhibited significant differential expression, of which 29 genes were up-regulated and 13 genes were down-regulated in the precocious crabs. To confirm the sequencing data, six differentially expressed genes with functional annotations were selected and validated by qRT-PCR. In conclusion, we obtained the comprehensive transcriptome of the eyestalk tissues of precocious juvenile crabs. The sequencing results may provide new insights into the biomolecular basis of precocity in the Chinese mitten crab.