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MicroRNAs are regulatory molecules that can be packaged into exosomes to modulate cellular response of recipients. While the role of exosomes during viral infection is beginning to be appreciated, the involvement of exosomal miRNAs in immunoregulation in invertebrates has not been addressed. Here, we observed that exosomes released from WSSV-injected mud crabs could suppress viral replication by inducing apoptosis of hemocytes. Besides, miR-137 and miR-7847 were found to be less packaged in mud crab exosomes during viral infection, with both miR-137 and miR-7847 shown to negatively regulate apoptosis by targeting the apoptosis-inducing factor (AIF). Our data also revealed that AIF translocated to the nucleus to induce DNA fragmentation, and could competitively bind to HSP70 to disintegrate the HSP70-Bax (Bcl-2-associated X protein) complex, thereby activating the mitochondria apoptosis pathway by freeing Bax. The present finding therefore provides a novel mechanism that underlies the crosstalk between exosomal miRNAs and apoptosis pathway in innate immune response in invertebrates., Competing Interests: The authors declare no conflicts of interest.

Matrix-assisted laser desorption/ionization (MALDI)-MS imaging has been utilized to image a variety of biomolecules, including neuropeptides. Washing a tissue section is an effective way to eliminate interfering background and improve detection of low concentration target analyte molecules; however, many previous methods have not been tested for neuropeptide analysis via MALDI-MS imaging. Using crustaceans as a neurological model organism, we developed a new, simple washing procedure and applied this method to characterize neuropeptide changes due to hypoxia stress. With a 10 s 50:50 EtOH:H 2 O wash, neuropeptide coverage was improved by 1.15-fold, while normalized signal intensities were increased by 5.28-fold. Specifically, hypoxia and hypercapnia stress conditions were investigated due to their environmental relevance to marine invertebrates. Many neuropeptides, including RFamides, pyrokinin, and cardioactive peptides, showed distinct up- and down-regulation for specific neuropeptide isoforms. Since crustacean neuropeptides are homologous to those found in humans, results from these studies can be applied to understand potential roles of neuropeptides involved in medical hypoxia and hypercapnia.

The metal binding nature of heterologously expressed metallothionein of Sinopotamon henanense (ShMT) had been demonstrated previously. In this study, we analysed the stoichiometry of ShMT yielded in vivo and exchange reactions of the Zn-ShMT with Cd 2+ , Pb 2+ and Cu 2+ in vitro via electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), and isothermal titration calorimetry (ITC). The results of ESI-TOF-MS analyses showed that metal-ShMT synthesized in vivo had three major forms, namely Zn 15 -, Cd 9 -, and Pb 5 -ShMT. The ITC analyses of exchange reactions demonstrated that Zn-ShMT exhibited up to 6, 6, and 7 binding sites for Cd 2+ , Pb 2+ and Cu 2+ . By the analyses of the UV and CD spectra in the substitution experiments showed that the geometric structural stability of metal-ShMT could be influenced when excess of over 6, 6, or 7 equivalents of Cd 2+ , Pb 2+ , or Cu 2+ were added into Zn-ShMT. Although both the reconstructed apo-ShMT and substituted Zn-ShMT with three metal ions fitted the same M 6 Ⅱ- and M 7 Ⅰ-ShMT binding models for divalent and monovalent metals, the differences in their thermodynamic data suggested that discrepancies exit in their physiological functions. These results suggested that ShMT yielded in vivo had a higher storage capability for Zn 2+ and a uptake ability for Cd 2+ , and Zn-ShMT was more easy to release Zn 2+ as well as to uptake Cd 2+ , Cu 2+ , or Pb 2+ . The results presented in this work will be very valuable to understand the function(s) of ShMT not only in a normal physiological condition but also in the presence of non-essential metals in crabs., (Copyright © 2019. Published by Elsevier Ltd.)

The development of Portunus trituberculatus egg cells is directly related to the nutritional status of the fertilized egg, which affects the key production stages of offspring hatching. Vitellogenin plays a key role in the nutrient supply required for the development of the egg cells. The c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase (MAPK) superfamily and plays an important role in cell proliferation, transformation, differentiation, and apoptosis. At present, there are no reports on the involvement of the JNK signaling pathway in the reproductive regulation of P. trituberculatus. In this study, rapid amplification of complementary DNA ends amplification technology was used to clone the full length of JNK complementary DNA, which has a length of 2094 bp, including an open reading frame (ORF) of 1266 bp encoding a 421-amino acid protein. The protein includes the S&#95;TKC conserved domain with a TPY phosphorylation site, which is a typical feature of the JNK gene family. Observing tissue sections found the oocytes in the inhibitor group developed slowly, while the oocytes in the activated group showed accelerated development. Meanwhile, Portunus trituberculatus JNK and vitellogenin (Vg) genes exhibited the same trend in the hepatopancreas and ovaries, and the expression of the SP600125 group was downregulated (P < 0.05), while the anisomycin group was upregulated (P < 0.05). In addition, JNK enzyme activity and vitellin (Vn) content in the ovarian tissue showed that the JNK activity of the SP600125 group decreased, while activity increased in the anisomycin group. The accumulation of Vn content in the SP600125 group decreased, and that in the anisomycin group increased. In summary, after injection with inhibitor or activator, the JNK signaling pathway of P. trituberculatus was inhibited or activated, the accumulation of Vn in the ovary was reduced or increased, and ovarian development was inhibited or accelerated, respectively. These results indicated that the JNK signaling pathway is involved in the regulation of Vg synthesis and ovarian development in P. trituberculatus. The results of this study further add to the knowledge of the breeding biology of P. trituberculatus and provide a theoretical reference for the optimization of breeding techniques in aquaculture production systems.

This study investigated the properties of Scy p 9 in mud crab (Scylla paramamosain). The gene sequence of filamin C obtained from crabs, which was denoted as Scy p 9, contains a 2544 bp open reading frame and encodes 848 amino acid residues. Recombinant Scy p 9 (rScy p 9) is expressed in Escherichia coli, which exhibits tertiary structure changes, and the IgE binding activity of rScy p 9 is higher than that of native Scy p 9 (nScy p 9). Moreover, this study explored the possibility of the presence and cross-reactivity of filamin C in 8 shellfish. IgE-specific binding to nScy p 9 and rScy p 9 in patients allergic to shellfish revealed that rScy p 9 was more sensitive than nScy p 9. The gene sequence of filamin C fills in the blank in shellfish. This study contributes to the understanding of the properties of Scy p 9, and the results indicate that rScy p 9 can be used as a candidate for component-resolved diagnosis in shellfish.

Matrix metalloproteinases (MMPs) are a cluster of enzymes that degrade the extracellular matrix (ECM) and some intracellular proteins; as such, they play an important role in tissue regeneration, infant growth, animal reproduction, and immunity. Most research into MMPs focuses mainly on their effects on the mammalian immune system. However, it is not clear how MMPs affect immune processes in crustaceans. Here, we cloned the open reading frame (ORF) of Eriocheir sinensis (Chinese mitten crab) MMP-14 (EsMMP-14) to explore the role of MMPs in crustacean innate immune responses. RT-PCR results showed that stimulation of crab with LPS and poly I:C upregulated expression of EsMMP-14 markedly. Besides, following the stimulation of 20-Hydroxyecdysone, the expression level of EsMMP-14 increased robustly, suggesting that EsMMP-14 involved in the molt process of E. sinensis. Hematoxylin and eosin staining of hepatopancreas and intestine revealed that knocking down EsMMP-14 maintained morphology following infection by Bacillus thuringiensis. Moreover, downregulated expression of EsMMP-14 increased the survival rate of infected E. sinensis. These results show that EsMMP-14 plays a role in innate immune responses of E. sinensis and fills a gap in our knowledge about the function of MMPs in crustaceans., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In mammals, casitas B-lineage lymphoma (CBL) family proteins, a RING-type E3 ubiquitin ligase, are involved in many signal transduction pathways. However, the functions of CBL in invertebrates are not well elucidated. In this study, Sp-CBL containing CBL-N, CBL-2, CBL-3 and RING domains was identified in mud crab Scylla paramamosain. Sp-CBL was widely expressed in all tissues tested and found to be significantly up-regulated in the hemocytes of mud crab challenged by white spot syndrome virus (WSSV). The RNA interference of Sp-CBL increased the copy number of WSSV and declined the apoptosis rate of hemocytes. In addition, Sp-CBL could affect the activities of caspase 3 and the mitochondrial membrane potential. Taken together, the results of this study revealed that Sp-CBL could restrict WSSV proliferation through enhancing the apoptosis of the hemocytes, which would provide a novel insight into the anti-viral response in the innate immunity system of mud crab., (Copyright © 2019. Published by Elsevier Ltd.)

Elongation of very long-chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of long-chain and very long-chain polyunsaturated fatty acids (LC-PUFA and VLC-PUFA). In the present study, an elovl4 cDNA was cloned from the swimming crab Portunus trituberculatus by PCR techniques and functionally characterized using recombinant expression in yeast Saccharomyces cerevisiae. The elovl4 cDNA sequence contained an open reading frame of 1038 base pairs, encoding a protein of 346 amino acids. The elovl4 has typical Elovl structures, with transmembrane domains (6) and a histidine box. The elovl4 was expressed in various tissues analyzed, with the highest expression found in intestine and hepatopancreas, followed by stomach and eyestalk. The functional characterization of Elovl4 yeast showed that the P. trituberculatus Elovl4 can elongate C 18 - 22 polyunsaturated fatty acids (PUFA), reaching in some cases products of C 24 and C 26 . Along its ability to elongate PUFA, the P. trituberculatus Elovl4 was also efficient in the elongation of saturated fatty acids, with 28:0 and 30:0 being prominent elongation products. These results provide insight into the LC-PUFA biosynthetic capability of commercially important species of crustaceans., Competing Interests: Declaration of Competing Interest The authors declared that there were no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The environmental quality of a Ramsar wetland site located at the Cananéia-Iguape-Peruíbe Protected Area (CIP-PA), in São Paulo, Brazil, was assessed by geochemical analyses and biomarker assessments (GPx, GST, GSH, GST, MT, LPO, DNA damage) performed in swimming crab Callinectes danae Smith, 1869 organs (posterior and anterior gills and hepatopancreas) to estimate sediment contaminant bioavailability. The results indicated that two sampling stations, PT and PM, exhibited the worst environmental conditions, as sediments collected at both points contained metal contamination, while crabs exhibited significant responses for GPx, GST, and LPO (mostly during winter). Sediment contamination tended to be associated to fine sediments (both seasons) and organic matter (winter). During the summer survey, Pb concentrations in sediments of station PT exceeded the Brazilian Sediment Quality Guidelines (SQGs) and the Canadian Interim Marine Sediment Quality Guidelines. Metal concentrations in sediments sampled in winter were higher compared with summer, with Co, Ni, and Pb exceeding SQGs levels at PT, whereas Co, Ni, Hg, Zn, and Pb exceeded SQGs at PM. Biomarker induction during summer appeared to be caused by natural variables (water salinity and temperature, and molting cycle), whereas oxidative stress and tissue damage during winter appeared to be more clearly linked to metal contamination. Anterior gills presented the clearest signs of seasonal variability, being more responsive to sediment contamination. The results suggest that metals originated from the upper Ribeira de Iguape River are transported toward the estuarine system, causing effects on C. danae individuals. Additionally, seasonality is a strong factor concerning CIP-PA toxicity, since the rainfall regime significantly modifies the freshwater flow and, consequently, estuarine water salinity, suspended particle and metal inputs, as well as the location of depositional areas. Thus, efforts to mitigate CIP-PA contamination should be based on the control of upstream pollution sources.

Oxytocin (OT)/vasopressin (VP) signaling system is important to the regulation of metabolism, osmoregulation, social behaviours, learning, and memory, while the regulatory mechanism on ovarian development is still unclear in invertebrates. In this study, Sp ot/vp-like and its receptor ( Sp ot/vpr-like) were identified in the mud crab Scylla paramamosain. Sp ot/vp-like transcripts were mainly expressed in the nervous tissues, midgut, gill, hepatopancreas, and ovary, while Sp ot/vpr-like were widespread in various tissues including the hepatopancreas, ovary, and hemocytes. In situ hybridisation revealed that Sp ot/vp-like mRNA was mainly detected in 6-9 th clusters in the cerebral ganglion, and oocytes and follicular cells in the ovary, while Sp ot/vpr-like was found to localise in F-cells in the hepatopancreas and oocytes in the ovary. In vitro experiment showed that the mRNA expression level of Sp vg in the hepatopancreas, Sp vgr in the ovary, and 17β-estradiol (E 2 ) content in culture medium were significantly declined with the administration of synthetic Sp OT/VP-like peptide. Besides, after the injection of Sp OT/VP-like peptide, it led to the significantly reduced expression of Sp vg in the hepatopancreas and subduced E 2 content in the haemolymph in the crabs. In brief, Sp OT/VP signaling system might inhibit vitellogenesis through neuroendocrine and autocrine/paracrine modes, which may be realised by inhibiting the release of E 2 ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Crustaceans have a more persistent starvation tolerance than mammals, birds, reptiles, and even fish. This study is aimed at assessing the survival strategy and regulatory mechanism of crustaceans in response to starvation through an animal model using Eriocheir sinensis . In the 42-day starvation experiment, the hepatopancreas was found to become the target organ, which was characterized by atrophy of the thin wall in the hepatic tubules and expansion of the lumen. During short-term starvation, E. sinensis activates lipid and glycogen metabolism in the hepatopancreas with lipid metabolism dominating. In lipid metabolism, there was a significant decline in triglyceride, whereas cholesterol did not change significantly. Meanwhile, the fatty acid metabolism pathway was inhibited, but autophagy increased in the hepatopancreas, which may be the selective pathway for the decomposition of intracellular substances. However, under long-term starvation, the stored energy in the hepatopancreas was depleted, and E. sinensis selects to consume hepatopancreatic cells and maintain energy metabolism through apoptosis, which was triggered by both the death receptor pathway and the mitochondrial pathway. In addition, cell proliferation was blocked to reduce unnecessary energy consumption., Competing Interests: The authors declare no competing financial interests., (Copyright © 2020 Xiaoli Huang et al.)

Troponin C (TnC) is one member of the EF-hand superfamily. In many species, this gene had been identified and related functions had been elucidated. The TnC gene was still blank in the Scylla paramamosain. We obtained the TnC gene for the first time in the S. paramamosain. And we systematically analyzed the possible role of this gene in the innate immunity of S. paramamosain while infected with white spot syndrome virus (WSSV) or Vibrio alginolyticus. The full-length 1427 bp sequence of TnC contains a 453 bp open reading frame (ORF) for encoding a 151 amino acid protein. Detection of tissue specificity of gene expression showed that the TnC was primarily expressed in muscle tissue. The expression of TnC was successfully inhibited by RNA interference technology, and several immune genes were affected. The activity of phenoloxidase and superoxide dismutase increased, and the total hemocytes counts increased after RNAi of TnC. It was found that after infection with V. alginolyticus and WSSV, the expression of TnC in hemocytes decreased. Infected with V. alginolyticus and WSSV, the cumulative mortality and apoptotic rate of hemocytes increased after silencing the TnC gene. Our results indicate that TnC takes participate in the innate immunity of S. paramamosain and may plays a different role in the antiviral and antibacterial immune response., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Toll is essential in innate immune system which is important for defense against bacterial, fungal and viral infections in invertebrates. Our previous study showed that cadmium (Cd) could change the expression pattern of ShToll3 in the epithelium (gills and midgut from the freshwater crab Sinopotamon henanense) infected by Aeromonas hydrophila. To investigate the diverse innate immune roles of crustacean homolog Tolls, in this study, we cloned Shtoll1 from S. henanense. The full-length cDNA of Shtoll1 was 4746 bp, with an ORF of 3033 bp encoding a putative protein of 111 amino acids, a 5'-untranslated region of 255 bp and a 3'-untranslated region of 1713 bp. Phylogenetic analysis showed that ShToll1 was clustered into the group of DmToll1, DmToll 4 and DmToll 5. In addition, the tissue distribution results showed that Shtoll1 was expressed widely in different tissues, with the highest expression in heamocytes. Besides, Shtoll1 expressions were upregulated in heamocytes and hepatopancreas after A. hydrophila infection. At the same time, the increase of Shtoll1 expressions were examined in heamocytes in response to Cd exposure and A. hydrophila infection in combination. Through western blotting and immunohistochemical analysis, the ShToll1 expressions in heamocytes were increased in response to A. hydrophila and Cd independently as well as in combination. Moreover, the mRNA level of three antimicrobial peptides (AMPs) alf5, alf6, and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll1, were analyzed. Thus, we conclude that Cd expand the susceptibility of ShToll1 to A. hydrophila infection in heamocytes. This suggest that ShToll1 may contribute to the innate immune defense of S. henanense against A. hydrophila and Cd in heamocytes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Glutathione peroxidases (GPx) are parts of the enzymatic antioxidant system that can eliminate the peroxides produced as effect of reactions of molecules with reactive oxygen species (ROS). In this study, a selenium-dependent glutathione peroxidase 3 cDNAs (designated as SpGPx3) was obtained from the mud crab Scylla paramamosain. The open reading frame (ORF) of SpGPx3 was 639 bp, which encoded a putative protein of 212 amino acids. SpGPx3 protein contained a characteristic GPx signature motif, and an active site motif. Mud crabs were exposed to 20 mg L - 1 nitrite for 72 h. Quantitative real-time PCR analysis revealed that the SpGPx3 mRNA was distributed abundantly in mud crab. The transcript levels of antioxidant enzyme genes (SpGPx3, SpSOD and SpCAT) were obviously induced after acute nitrite exposure. After knockdown of the SpGPx3 level, the mortality of mud crabs and malondialdehyde (MDA) content significantly increased under nitrite stress. These results suggested that SpGPx3 played an important role in protecting organisms against oxidative stress., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Juvenile hormone binding proteins (JHBPs) serve as the carriers that transport juvenile hormone (JH) to target tissues to activate JH signals. In this study, seven JHBPs from mud crab Scylla paramamosain were obtained. All Sp-JHBPs contained one JHBP domain, and Sp-JHBP4, 5, 6 also contained one Grp7&#95;allergen domain. The predicted secondary and 3D structures are conserved among the seven Sp-JHBP domains, but the complete 3D structure of Sp-JHBP4, 5, 6 were more similar to lipopolysaccharide binding protein. Additionally, the potential ligands for Sp-JHBP1, 2, 3, 7 were ubiquinone 8 (UQ8) and JH II or JH III, and the predicted binding location and sites for JHs were similar to the structured known BmJHBP, suggesting that MF is a potential ligand for these Sp-JHBPs. Furthermore, the expression analysis showed that Sp-JHBP1 expression is relatively low in the 14 detected tissues, and Sp-JHBP2 has the highest expression in the ovary, while the other five JHBPs are highly expressed in the hepatopancreas. During larval development, Sp-JHBP2 is highly expressed during the Z1, M and C1 stages, which are three post-metamorphosis stages, while Sp-JHBP3 and 4 expression levels were significantly lower during the Z5 and M stages, which are two pre-metamorphosis stages. Moreover, the in vivo and in vitro study revealed that Sp-JHBP2 expression was reduced by MF, while the in vitro studies showed that Sp-JHBP3, 6, 7 are induced by MF in a concentration-independent manner. Taken together, we hypothesized Sp-JHBP1, 2, 3, 7 has the potential to bind MF and Sp-JHBP2, 3, 7 play a more important roles in MF signal, while Sp-JHBP4, 5, 6 were more likely to participate in immune response., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chinese mitten crab Eriocheir sinensis is probably the most important freshwater cultured crab in China. A tiny minority of brownish-orange individuals have been discovered in the long period of artificial breeding history of E. sinensiss. Those mutants are usually accompanied with slow growth rate, low molting frequency and poor survival rate, which may be the results of growth defects and immunodeficiency. To better understand the relationship between body color determination and the immune system as well as the related genes expression in E. sinensiss, we performed the whole-body transcriptome analysis in different color of first stage zoea (ZI) larvae using next-generation sequencing (NGS) technology. We randomly assembled 175.40 and 177.52million clean reads from the wild and mutant ZIs, respectively. Finally, we identified 7153 differentially expressed genes (DEGs) (p < 0.05), with 5194 up-regulated and 1959 down-regulated. A total of 13 KEGG pathways related to immune system were detected among 248 pathways. Except the first whole-body RNA sequencing of color-specific transcriptomes for E. sinensis, this study will offer a better understanding of the underlying molecular mechanisms of interaction between color determination and the immune system., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hepatopancreatic necrosis disease (HPND) is a newly emerging disease in the Chinese mitten crab, Eriocheir sinensis, which has resulted in large economic losses. However, the underlying cause of this disease remains unclear. To better understand the pathogenesis and pathogenic mechanism of HPND, we compared the transcriptome differences of the hepatopancreas of E. sinensis with and without HPND. The analysis yielded > 30 million reads for each sample of three test (with HPND) and three control groups (without HPND). We observed 978 downregulated genes and 644 upregulated genes. Among the gene ontology categories "biological process," "cellular component," and "molecular function", the subcategories cellular process, single-organism process, biological regulation, metabolic process, cell part, organelle, organelle part, binding, and catalytic were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that "metabolism of xenobiotics by cytochrome P450," "drug metabolism-cytochrome P450," "chemical carcinogenesis," and "material metabolism" were the "five" most significantly enriched pathways in the hepatopancreas of E. sinensis with HPND. The results revealed that material metabolic abnormalities and drug effects from the external environment might be associated with HPND in the Chinese mitten crab. Considering the wide use of pyrethroids for pond cleaning in Xinghua city, we speculated that pyrethroids might cause HPND in the Chinese mitten crab. Our study provided useful information about the cause and pathogenetic mechanisms of HPND and could help to prevent this disease in production practice., Competing Interests: The authors have declared that no competing interests exist.

The environmental health of Buenaventura Bay, a highly impacted tropical estuary, is influenced by numerous human activities, including mining upstream. Large- and small-scale fishing plays an important role in the local economy, so we investigated the dynamic processes of bioaccumulation of mercury at basal trophic levels. Four samples were taken at each of the four locations in Buenaventura Bay during each of the four seasons of 2015. We measured the total mercury content (T-Hg, dry weight) in sediments and in muscle tissue across 17 macroinvertebrate species. The most abundant were the blue crab (C. arcuatus) and the mantis shrimp (S. aculeata aculeata). Blue crab showed an average muscle T-Hg value ​​exceeding the limit of 0.2 g·g -1 , which is the maximum T-Hg level suggested for food consumption by vulnerable humans and populations: pregnant women, children, and the community that feeds from this source of protein on a daily basis. It was found that, 6.22% of individuals exceeded the 0.5 g·g -1 level, which is the maximum T-Hg level suggested for food consumption by the general population: the population that consumes it sporadically. Significantly high values ​​of T-Hg in blue crab and mantis shrimp occurred during low salinity conditions in the estuary, suggesting that Hg mainly originates from river runoff during the rainy season. Nevertheless, the biota-sediment accumulation factor (BSAF) was favored in high salinity, which could mean greater availability of Hg for higher levels of the estuarine food web in the dry season and in marine waters. In general, the T-Hg levels in some samples exceeded 0.2 g·g -1 . Therefore this pollutant must be monitored due to its biomagnification potential and as a threat to human health, especially that for the local population of fishermen and their families.

The mud crab, Scylla paramamosain, is an economically-important crab in China. Air exposure is an important environmental stressor during mud crab culture and transportation. Adaptive mechanisms responding to air exposure in mud crabs are still poorly understood. In this study, mud crabs were exposed to air for 120 h. Air exposure decreased total hemocyte counts, led to cytological damage, and caused high mortality. Transcriptomic analysis was conducted at 0, 6 and 96 h after air exposure. A total of 3530 differentially expressed genes (DEGs) were identified. DEGs were mainly involved in the oxidative stress response, metabolism, cellular processes, signal transduction, and immune functions. Transcriptomic analysis also revealed that genes of glycolysis and of the tricarboxylic acid cycle were key factors in regulating the mud crab adaptation to air exposure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. In this study, we cloned the PHB gene from the testis of the swimming crab Portunus trituberculatus (PtPHB) and analyzed the deduced amino acid sequence. The expression level of phb mRNA in larvae was analyzed using qRT-PCR. The expression level of phb mRNA and PHB protein in different tissues were analyzed using qRT-PCR and Western blot respectively. Enzyme-linked immunosorbent assay analyses of the PHB protein were conducted with the testis and ovaries from P. trituberculatus specimens at different developmental stages. PHB was localized with mitochondria and ubiquitin in the testis and ovaries. The PtPHB gene was found to contain an open reading frame of 825 bp, encoding a predicted peptide with 275 amino acids, sharing between 65.9% and 96.7% similarity with that of other species. The qRT-PCR and Western blot results showed that the phb gene and PHB protein both expressed less in the testis and ovary than in other tissues, and the phb gene presented the lowest expression in the Z1 stage. Furthermore, the phb gene and PHB protein expression were different in the testis and ovaries at different developmental stages. PHB was mainly found to be co-localized with mitochondria and ubiquitin in cytoplasm and acrosome complex during spermatogenesis and in follicular cells during oogenesis. Interestingly, PHB-mitochondria signals and ubiquitin signal were also found in oocytes. These results indicated that PHB might play important roles during spermatogenesis and oogenesis by regulating mitochondrial activities., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In this study, the Chinese mitten crabs, Eriocheir sinensis were exposed to avermectin at 0.03, 0.06, 0.12, 0.24, and 0.48 mg/L respectively for 96 hours. The results showed that levels of superoxide dismutase, catalase, and glutathione peroxidase in hepatopancreas were slightly induced at concentration of 0.03 and 0.06 mg/L, but significantly (P < .05) decreased at higher concentrations, meanwhile similar trend of the activities of acid phosphatase, alkaline phosphatase and lysozyme were observed. Significant induction of HSP70 and HSP90 mRNA expression was detected at 24 hours whereas no significant change was found in HSP60. In addition, levels of reactive oxygen species in hepatocytes increased in dose- and time- dependent manners, and cell viabilities of hepatocytes and haemocytes decreased. These results indicated that sublethal concentration exposure of avermectin had a prominent oxidative stress effect on E. sinensis based on the antioxidative and immunological activity inhibition, and HSP60, 70, and 90 perform a protective response during the exposure., (© 2019 Wiley Periodicals, Inc.)

It is known that abamectin (ABM) inflicts oxidative damage on aquatic animals; however, knowledge about the immune response under pesticide-induced oxidative stress is incomplete. In the present study, several cellular and humoral immune parameters, including total haemocyte counts (THC), lysosomal membrane stability (LMS), activities of acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LZM) were investigated to reveal the effects of ABM exposure on the immune defence mechanisms of the important freshwater crab, Erocheir sinensis. According to the results, a significant increase of THC was found in low concentration groups (0.03 and 0.06 mg/L), while dramatic decreases occurred in high concentration groups (0.12 and 0.24 mg/L) after 96 h of exposure. We also detected significant increases of reactive oxygen species (ROS) in haemocytes at 0.12 and 0.24 mg/L, and there was a dose- and time-dependent decrease of lysosomal membrane stability. These results suggest that the excessive generation of ROS induced by ABM may be leading the massive collapse of lysosomal membrane, which in turn may be causing the sharp drop of haemocyte counts in E. sinensis. The increase of hydrolytic enzymes ACP and AKP at low concentrations and the decrease at high concentrations also indicate an immune response associated with haemocytes status under stress. However, activities of LZM decreased significantly. After injection of Aeromonas hydrophil, mortalities increased under exposure to ABM and were positively related to ABM concentration. These results confirm that ABM exposure has the ability to impair immune defence and result in the host's susceptibility to pathogens., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The regulation of lipogenesis and lipolysis mechanisms related to consumption of lipid has not been studied in swimming crab. The aims of the present study were to evaluate the effects of dietary lipid levels on growth, enzymes activities and expression of genes of lipid metabolism in hepatopancreas of juvenile swimming crab. Three isonitrogenous diets were formulated to contain crude lipid levels at 5·8, 9·9 and 15·1 %. Crabs fed the diet containing 15·1 % lipid had significantly lower growth performance and feed utilisation than those fed the 5·8 and 9·9 % lipid diets. Crabs fed 5·8 % lipid had lower malondialdehyde concentrations in the haemolymph and hepatopancreas than those fed the other diets. Highest glutathione peroxidase in haemolymph and superoxide dismutase in hepatopancreas were observed in crabs fed 5·8 % lipid. The lowest fatty acid synthase and glucose 6-phosphate dehydrogenase activities in hepatopancreas were observed in crabs fed 15·1 % lipid, whereas crabs fed 5·8 % lipid had lower carnitine palmitoyltransferase-1 activity than those fed the other diets. Crabs fed 15·1 % lipid showed lower hepatopancreas expression of genes involved in long-chain-PUFA biosynthesis, lipoprotein clearance, fatty acid uptake, fatty acid oxidation, lipid anabolism and lipid catabolism than those fed the other diets, whereas expression of some genes of lipoprotein assembly and fatty acid oxidation was up-regulated compared with crabs fed 5·8 % lipid. Overall, high dietary lipid level can inhibit growth, reduce antioxidant enzyme activities and influence lipid metabolic pathways to regulate lipid deposition in crab.

Neuroparsin (NP) is an important neuropeptide in invertebrates. It is well-known that NP displays multiple biological activities, including antidiuretic and inhibition of vitellogenesis in insects. However, the information about its effect in crustaceans is scarce. In this study, the sequence of Sp-NP1 was selected from the transcriptome database from the mud crab, Scylla paramamosain. Sequence analyses indicate that the Sp-NP1 amino acid (AA) sequences consist of a 27 AA signal peptide and a 74 AA mature peptide, which contains 12 cysteine residues. qRT-PCR analysis has revealed that the expressions of Sp-NP1 gene are high in the nervous tissues and extremely low in the ovary and hepatopancreas. In situ hybridization has shown that the positive signals are localized in cell cluster 6 of protocerebrum and cell clusters 10 and 11 of deutocerebrum. The presence of Sp-NP1 in the haemolymph has been detected in S. paramamosain through western blot, which indicates that Sp-NP1 serves as an endocrine factor in the regulation of physiological activities. In vitro experiments have further shown that the mRNA level of vitellogenin in the hepatopancreas notably decreases following administration of recombinant Sp-NP1, while the mRNA level of vitellogenin receptor and cyclin B in the ovary shows no significant differences. Collectively, Sp-NP1 possibly can inhibit the production of vitellogenin in the hepatopancreas and has no direct effect on the ovary in S. paramamosain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

PIWI-interacting RNAs (piRNAs) are abundantly found in germ cells and involved in gametogenesis and gonadal development. Information on the regulatory roles of piRNAs in crustacean reproduction, however, is scarce. Thus, we identified gonadal piRNAs of mud crab Scylla paramamosain. Of the 115,491 novel piRNAs, 596 were differentially expressed. Subsequently, 389,887 potential piRNA-target genes were predicted. The expression of 4 piRNAs and 9 genes with high piRNA interactions were validated with the inclusion of additional immature specimens, including LRP2 that is involved in growth and reproduction, MDN1 in ribosome biogenesis pathway and gametogenesis, and PRKDC, a DNA repair gene involved in gonadal differentiation and maturation. KEGG analysis further revealed the involvement of predicted piRNA target genes in gametogenesis- and reproduction-related pathways. Our findings provide baseline information of mud crab piRNAs and their differential expression between testes and ovaries suggests that piRNAs play an essential role in regulating gametogenesis and gonadal development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Copper (Cu) is an essential metal capable to alter many metabolic and physiological processes in animal species, depending on the environmental concentration and salinity. The present study evaluated the effects of Cu exposure on the metabolism of the blue crab Callinectes sapidus under different osmotic situations. Crabs were acclimated at two different salinities conditions (30 and 2). Subsequently, they were exposed to Cu during 96 h at each salinity and under hypo-osmotic shock. Results demonstrated that Cu exposure increased whole-body oxygen consumption. In addition, the activity of LDH decreased while citrate synthase increased in anterior gills from animals submitted to hypo-osmotic shock. This scenario indicates extra stress caused by sudden environmental osmotic changes, as commonly observed in estuarine environments, when combined with copper exposure. Therefore, the activity of LDH and citrate synthase enzymes might be sensitive indicators for aquatic toxicology studies approaching Cu contamination in estuarine environments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Serotonin (5-HT) plays pivotal roles in many physiological processes including reproduction of crustaceans, which are mediated 5-HT receptors. The distributions of 5-HT and its receptor have never been explored in Portunus pelagicus. To validate the targets which indirectly indicate the roles of 5-HT in this crab, we have investigated the distribution of 5-HT in the central nervous system (CNS) and ovary using immunohistochemistry and tissue expression of its receptor by RT-PCR. In the brain, 5-HT immunoreactivity (-ir) was detected in clusters 6, 7, 8, 11, 14, 15 and the fibers. In the ventral nerve cord (VNC), 5-HT-ir was detected in pairs of neurons and the fibers connected to the neurons. In the ovary, 5-HT-ir was intense in the oocyte step 1 (Oc1) and Oc2, and its intensity was slightly decreased in Oc3 and Oc4. The 5-HT receptor was molecularly characterized to be type 7, and it was strongly expressed in the eyestalk, brain, VNC, mature ovary and muscle. Due to the presence of 5-HT receptor we suggest that 5-HT acts primarily at the CNS and ovary, thus implicating its role in reproduction especially in the development of oocytes though its exact function in this crab needed to be explored further., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background and Objective: The accumulation of lipofuscin (LF) is an alternative technique to identify age of crustacean species. However, the exact sites and the level of the LF concentration were unknown especially for different sexes of blue swimming crab, Portunus pelagicus. Thus, the present study was aimed to identify which part of the eyestalk of P. pelagicus contains more LF levels in order to establish a specific target part of samples., Materials and Methods: Thus, crab samples for this study were sampled from the wild habitat at Setiu wetlands, Terengganu, Malaysia. A total of 100 samples of with the same size (80±5 mm carapace width) were sampled and the eyestalk dissected for LF extraction. The determination of LF sites and levels in the eyestalks organ was taken from the area between the distal tangential layer (DTL) and proximal tangential layer (PTL). The lower part of the eyestalk was taken from the PTL until the end of the eyestalk., Results: The upper part of the crab's eyestalk was higher in the males crabs compared to the females crabs. LF index also shown that the upper part of crab's eyestalk have higher concentration compared to the lower part., Conclusion: The left crab's eyestalk had the higher LF index especially in males compared to females but the total concentration was higher in female crabs. Knowing which part has highly dense accumulation of LF helps in LF detection of the tissue and further helps for age determination for this species.

The transforming growth factor-β (TGF-β) signaling pathway is conserved across animals, and knowledge of its roles during the molt cycle in crustaceans is presently very limited. This study investigates the roles of the TGF-β receptor in molting-related muscle growth in Eriocheir sinensis. Using the RT-PCR and RACE techniques, we obtained a 1722 bp cDNA sequence encoding a transforming growth factor-β type I receptor in Eriocheir sinensis, designated EsTGFBRI, which contains a 124 bp 5'-untranslated region, a 20 bp partial 3'-untranslated region and a 1578 bp open reading frame encoding 525 amino acids. The deduced EsTGFBRI contains an N-terminal 24 amino acid signal peptide, an activin type I and II receptor domain, a transmembrane helix region, a glycine-serine-rich motif, and a conserved serine/threonine kinase catalytic domain including an activation loop. The qRT-PCR results showed that EsTGFBRI gene was highly expressed in the intermolt testis and ovary in mature crabs. In juvenile crabs, the mRNA levels of EsTGFBRI in claw and abdominal muscles in the later premolt D 3-4 stage were significantly higher than those in the intermolt C and postmolt A-B stages. There was no significant change in EsTGFBRI mRNA levels in walking leg muscles during the molt cycle. The results suggest that EsTGFBRI is probably play roles in molting-related muscle growth in E. sinensis. This study provides a necessary basis for elucidating the functions of TGF-β-like signaling mediated by TGFBRI in molting-related muscle growth in crustaceans.

Background and Objective: Fundamental information on how environmental conditions affect lipofuscin (LF) levels could be useful to those furthering the development of an effective management plan to control certain populations in certain geographical areas. Thus, the effects of salinity upon LF levels were examined using 7 salinities in the range 10-40% (increasing in steps of 5%)., Materials and Methods: The impacts of freezing preservation were also examined for 60 crab samples with carapace widths of 7-8 cm randomly allocated for either immediate or post-freezing (-80°C) analysis., Results: The result showed that 25% salinity was optimum for LF accumulation in both males and females crabs. The mean value of LF production was higher for males compared to females. Higher and lower salinity of 10 and 40% were the most stressed conditions for LF accumulation. The LF index (LI) of P. pelagicus decreased in salinities below and above the typical range., Conclusion: For both sexes, the frozen samples exhibited notably higher LI values than the fresh samples (p<0.05). These results will be applicable in numerous respects, for example in the selection of farm locations and for optimising productivity by controlling salinity levels.

The Chinese mitten crab (Eriocheir sinensis), an economically valuable crustacean that is popular for its flavor, exhibits catadromous spawning migration. Overfishing and environmental pollution have inflicted serious damage on wild E. sinensis populations, and the Chinese government has banned the commercial fishing of this species in the Yangtze River. Studies have examined the sexual dimorphism in the body size and morphology of crabs, but there are few reports on the molecular regulatory mechanisms that occur during the reproduction of E. sinensis. In this study, we performed the first comparative transcriptome analyses of the cerebral ganglion and hepatopancreas of E. sinensis during reproduction. The results indicate that E. sinensis has significant sexual dimorphism in signal transduction, metabolism, substance transportation, and cellular protection. This study aims to provide information that can be used as a basis for further research on the molecular mechanisms that underlie sexual dimorphism in E. sinensis during reproduction. Furthermore, the results can be used to support the development of the E. sinensis breeding industry and the restoration of wild E. sinensis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Inhibitor of apoptosis proteins (IAPs) maintain the balance between cell proliferation and cell death by inhibiting caspase activities and mediating immune responses. In the present study, a homolog of IAP (designated as EsIAP1) was identified from Chinese mitten crab Eriocheir sinensis. EsIAP1 consisted of 451 amino acids containing two baculoviral IAP repeat (BIR) domains with the conserved Cx2 Cx6 Wx3 Dx5 Hx6 C motifs. EsIAP1 mRNA was expressed in various tissues and its expression level in hemocytes increased significantly (p < 0.01) at 12-48 h after lipopolysaccharide stimulation. In the hemocytes, EsIAP1 protein was mainly distributed in the cytoplasm. The hydrolytic activity of recombinant EsCaspase-3/7-1 against the substrate Ac-DEVD-pNA decreased after incubation with rEsIAP1. Moreover, rEsIAP1 could directly combine with rEsCaspase-3/7-1 in vitro. After EsIAP1 was interfered by dsRNA, the mRNA expression and the hydrolytic activity of EsCaspase-3/7-1 increased significantly, which was 2.26-fold (p < 0.05) and 1.71-fold (p < 0.05) compared to that in the dsGFP group, respectively. These results collectively demonstrated that EsIAP1 might play an important role in apoptosis pathway by regulating the activity of EsCaspase-3/7-1 in E. sinensis.

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., Competing Interests: The authors have declared that no competing interests exist.

NFIL3 is a transcriptional activator of the IL-3 promoter in T cells. In vertebrates, it has been characterized as an essential regulator of several cellular processes such as immunity response, apoptosis and NK cells maturation. However, the identification and functional characterization of NFIL3 still remains unclear in arthropods. In this study, the NFIL3 homologue was firstly cloned and characterized in mud crab Scylla paramamosain. The full-length of SpNFIL3 was 2, 041 bp in length with an open reading frame of 1, 509 bp, containing a conserved basic region of leucin zipper domain. The qRT-PCR analysis indicated that SpNFIL3 was significantly highly expressed in hepatopancreas and in hemocytes. Moreover, the SpNFIL3 transcription could be up-regulated after the challenge of Vibrio alginolyticus or virus-analog Poly (I:C). The dual-luciferase reporter assays revealed that SpNFIL3 could activate NF-κB pathway. The immunofluorescence assay indicated SpNFIL3 was located in nucleus. After NFIL3 was interfered in vivo and in vitro, the expressions of two NF-κB members (SpRelish and SpDorsal), six antimicrobial peptide genes (SpCrustin and SpALF2-6) and pro-inflammatory cytokine SpIL-16 were suppressed, and the bacteria clearance capacity of crabs was also markedly impaired in NFIL3 silenced crabs. These results indicated that SpNFIL3 played crucial role in the innate immunity of S. paramamosain and it also brought new insight into the origin and evolution of NFIL3 in arthropods and even in invertebrates., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Avermectin is commonly used in aquaculture systems for pest control in recent decades in China. However, no information is provided for the toxic effect to the important commercial species, Chinese mitten crab, Eriocheir sinensis. To investigate the aquatic toxicity of avermectin, an acute toxic test was performed in this study. The results showed that the 48 h- and 96 h- LC50 were 1.663 and 0.954 mg/L, respectively. For further research, crabs were exposed to sublethal concentrations of 0.03, 0.06, 0.12, 0.24 and 0.48 mg/L. Levels of antioxidants, including superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) were significantly (P<0.05) decreased with dose- and time- dependent responses, meanwhile the oxidative products including malondialdehyde (MDA), hydrogen peroxide (H2O2) and protein carbonyl in serum increased significantly (P<0.05) at concentrations of 0.24 and 0.48 mg/L throughout the experiment. A significant (P<0.05) increase of intracellular ROS and decrease of phagocytic activity was observed in high concentration groups, with dose- and time- dependent manners during the exposure. In addition, serious genetic damage was detected, for the significant increase (P<0.05) of both comet ratio and %DNA in tail at each concentration, and micronucleus (MN) frequency at concentrations of 0.12, 0.24 and 0.48 mg/L at 96 h. These results indicated that sublethal concentration exposure of avermectin had a prominent toxic effect on E. sinensis based on the oxidative stress induced by generated ROS, immunological activity inhibition and genotoxicity., Competing Interests: The authors have declared that no competing interests exist.

Background: Cadmium (Cd) is a ubiquitous environmental toxicant for aquatic animals. The freshwater crab, Sinopotamon henanense ( S. henanense ), is a useful model for monitoring Cd exposure since it is widely distributed in sediments whereby it tends to accumulate several toxicants, including Cd. In the recent years, the toxic effects of Cd in the hepatopancreas of S. henanense have been demonstrated by a series of biochemical analysis and ultrastructural observations as well as the deep sequencing approaches and gene expression profile analysis. However, the post-transcriptional regulatory network underlying Cd toxicity in S.henanense is still largely unknown., Results: The miRNA transcriptional profile of the hepatopancreas of S. henanense was used to investigate the expression levels of miRNAs in response to Cd toxicity. In total, 464 known miRNAs and 191 novel miRNAs were identified. Among these 656 miRNAs, 126 known miRNAs could be matched with the miRNAs of Portunus trituberculatus , Eriocheir sinensis and Scylla paramamosain . Furthermore, a total of 24 conserved miRNAs were detected in these four crab species. Fifty-one differentially expressed miRNAs were identified in the Cd-exposed group, with 31 up-regulated and 20 down-regulated. Eight of the differentially expressed miRNAs were randomly selected and verified by the quantitative real-time PCR (qRT-PCR), and there was a general consistency (87.25%) between the qRT-PCR and miRNA transcriptome data. A total of 5258 target genes were screened by bioinformatics prediction. GO term analysis showed that, 17 GO terms were significantly enriched, which were mainly related to the regulation of oxidoreductase activity. KEGG pathway analysis showed that 18 pathways were significantly enriched, which were mainly associated with the biosynthesis, modification and degradation of proteins., Conclusion: In response to Cd toxicity, in the hepatopancreas of S. henanense , the expressions of significant amount of miRNAs were altered, which may be an adaptation to resist the oxidative stress induced by Cd. These results provide a basis for further studies of miRNA-mediated functional adaptation of the animal to combat Cd toxicity., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Mercury is a potent neurotoxin. Its main source of entry into the human body is the consumption of fish and seafood. Within the past few decades a new species of crab has appeared in the food web of the southern Baltic: Rhithropanopeus harrisii. The aim of the present study was to estimate the level of total and labile mercury concentrations in this species collected from the southern Baltic Sea. Analyses were performed using the thermodesorption method. The share of organic mercury fraction, as well as the Hgtot, increased with the size class of the crabs. Due to the increasingly common occurrence of R. harrisii and the low Hg TOT concentration in its body, and assuming that it is bound to become a more and more common component of the diet of fish, it can be supposed that the load of toxic mercury entering the food web is likely to decrease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We characterized the metal tolerance of recombinant strains harboring metallothionein from the freshwater crab Sinopotamon henanense (ShMT) in vivo and metal binding properties of ShMT purified in vitro. The recombinant strains harboring ShMT were exposed to 0.1 mM Cd 2+ , 0.3 mM Cu 2+ , 0.5 mM Pb 2+ , and 0.8 mM Zn 2+ . The growth curves and spot assays of recombinant strains and the contents of heavy metal ions were analysed in the media supplemented with above metal ions provided to recombinant E. coli synthesis. The structural characteristics of the Cd-, Cu-, Pb-, and Zn-ShMT were determined through ultraviolet spectroscopy (UV-vis), circular dichroism (CD), and isothermal titration calorimetry (ITC). The in vivo results showed that, compared to control strains, recombinant strains tolerated Cd 2+ , Cu 2+ , Pb 2+ , and Zn 2+ . Furthermore, the contents of Cd 2+ and Pb 2+ in media decreased substantially. In vitro and the Cd-ShMT had a higher degree of folding compactness in solution. 5,5'-Dithiobis-(2-nitrobenzoic) acid (DTNB) reaction and ITC results demonstrated that ShMT yielded Cd 6 -, Cu 7 -, and Pb 6 -ShMT. The binding stability order was Cu-ShMT > Cd-ShMT > Pb-ShMT > Zn-ShMT. Overall, ShMT is a canonical crustacean MT and is defined as a Cd-specific MT isoform that functions mainly in a detoxifying Cd 2+ and Pb 2+ and in regulating Zn 2+ homeostasis in S. henanense. This research on the metal binding properties of ShMT provides a better understanding of the physiological function of ShMT reducing heavy metal bioavailability and by regulating essential trace metals., (Copyright © 2019. Published by Elsevier Ltd.)

Five different diets with different ratio of fish oil to vegetable oil were prepared. The biological index and proximate composition of Eriocheir sinensis fed with different diets were compared, and then sensory analysis, electronic nose (E-nose) and headspace-solid phase micro-extraction combined with gas chromatography-mass spectrometer (HS-SPME-GC-MS) analysis were applied to determine the odor profile of E. sinensis. The results showed that partial replacement (50%-75%) of fish oil by vegetable oil (FO/VO) was beneficial to the weight increment, nutrition accumulation, and odor-active compounds (OACs) formation of E. sinensis. A total of 7 and 11 OACs were detected in the hepatopancreas and gonad, respectively, these OACs contributed greatly to the overall odor profiles of E. sinensis when the dietary replacement levels were at 50% and 75%, respectively. The results could provide the guide for dietary fish oil replacement as well as improving the odor quality of E. sinensis. Practical application The objective of this research is to compare the effects of dietary replacement of fish oil by vegetable oil on proximate composition and odor profiles of E.sinensis. The results obtained from this study would not only chose an optimal dietary replacement level and serve as a useful database for the odor of female and crabs, but also provide some guide for the improvement of Chinese mitten crab aquaculture., (© 2018 Wiley Periodicals, Inc.)

The SIFamides are a broadly conserved arthropod peptide family characterized by the C-terminal motif -SIFamide. In decapod crustaceans, two isoforms of SIFamide are known, GYRKPPFNGSIFamide (Gly 1 -SIFamide), which is nearly ubiquitously conserved in the order, and VYRKPPFNGSIFamide (Val 1 -SIFamide), known only from members of the astacidean genus Homarus. While much work has focused on the identification of SIFamide isoforms in decapods, there are few direct demonstrations of physiological function for members of the peptide family in this taxon. Here, we assessed the effects of Gly 1 - and Val 1 -SIFamide on the cardiac neuromuscular system of two closely related species of Cancer crab, Cancer borealis and Cancer irroratus. In each species, both peptides were cardioactive, with identical, dose-dependent effects elicited by both isoforms in a given species. Threshold concentrations for bioactivity are in the range typically associated with hormonal delivery, i.e., 10 -9 to 10 -8  M. Interestingly, and quite surprisingly, while the predicted effects of SIFamide on cardiac output are similar in both C. borealis and C. irroratus, frequency effects predominate in C. borealis, while amplitude effects predominate in C. irroratus. These findings suggest that, while SIFamide is likely to increase cardiac output in both crabs, the mechanism through which this is achieved is different in the two species. Immunohistochemical/mass spectrometric data suggest that SIFamide is delivered to the heart hormonally rather than locally, with the source of hormonal release being midgut epithelial endocrine cells in both Cancer species. If so, midgut-derived SIFamide may function as a regulator of cardiac output during the process of digestion., (Copyright © 2019. Published by Elsevier Inc.)

To investigate the effects of arginine (Arg) on the growth, antioxidant capacity, immunity and disease resistance of juvenile Chinese mitten crab, three diets containing Arg levels at 1.72% (control), 2.73% and 3.72% were formulated and fed to Chinese mitten crab (0.22 ± 0.03 g) for eight weeks. The weight gain, ecdysterone and growth hormone in the serum, relative expression of insulin-like growth factor 2 in the hepatopancreas significantly increased in crabs fed the 2.73% and 3.72% Arg diets. The protein and lipid contents significantly increased in crabs fed the 3.72% Arg diet. The feed conversion ratios in crabs fed the diets with Arg additions were lower than in the control. Arg supplementation also enhanced the antioxidative capacity by increasing the activities of superoxide dismutase, catalase and the relative expression of Kelch-like ECH-associated protein 1 gene in the hepatopancreas, which subsequently decreased malondialdehyde content in the hepatopancreas. Besides, Arg also decreased nitric oxide content in the serum and the activity of nitric oxide synthetase in the hepatopancreas. The relative mRNA levels of crustin, relish, lysozyme and cryptocyanin genes were significantly upregulated by Arg supplementation. The activities of acid phosphatase and alkaline phosphatase in the serum significantly increased in crabs fed the 3.72% Arg diet than those in the control. Similarly, the relative mRNA levels of crustin, cryptocyanin and proPO genes were significantly upregulated in crabs fed the 2.73% Arg diet after lipopolysaccharide challenge, and in crabs fed the 3.72% Arg diet after the Poly (I:C) challenge. The crabs fed the 2.73% and 3.72% Arg diets had higher survival rate after bacterial infection than those fed the control diet. This study indicates that the addition of Arg to the diet at 2.7-3.7% can improve the growth, survival, antioxidant capacity, immunity and disease resistance in juvenile Chinese mitten crab., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study evaluates the effect of dietary supplementation of immunostimulants on the Chinese mitten crab (Eriocheir sinensis) with a single administration of mannan oligosaccharide (MOS), or its combination with either β-glucan or with inulin for 8 weeks. Four diets included an untreated control diet (C), MOS alone (3 g kg -1 , M), MOS with β-glucan (3 g kg -1 MOS + 1.5 g kg -1 β-glucan, MB), and MOS with inulin (3 g kg -1 MOS + 10 g kg -1 inulin, MI). The weight gain and specific growth rate of the crabs fed M, MB, and MI diets were improved by lowing feed conversion ratio. The growth and feed utilization of the crabs fed the MB diet were improved compared with the other three groups. The crabs fed the M, MB and MI diets showed a higher intestinal trypsin activity than that in the M and control groups. The highest trypsin activity in the hepatopancreas was observed in the MB group. Crabs fed M, MB and MI diets increased antioxidant system-related enzyme activities, but reduced malondialdehyde. The highest activities of alkaline phosphatase, acid phosphatase, lysozyme and phenol oxidase in the gut and the respiratory burst of the crabs were found in the MB group. The MB diet promoted the mRNA expression of E. sinensis immune genes (ES-PT, ES-Relish, ES-LITAF, p38MAPK and Crustin) compared with the control. After 3 days of infection with Aeromonas hydrophila, the highest survival of crabs was also found in the MB group. This study indicates that the combination of MOS with β-glucan or with inulin can improve growth, antioxidant capacity, non-specific immunity and disease resistance in E. sinensis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Estradiol is an important sex steroid hormone that involved in regulation of animal lipid metabolism. However, the effect of estradiol on lipid metabolism in swimming crab (Portunus trituberculatus) is unclear. The present study investigated the effect of four concentrations of exogenous estradiol (0, 0.01, 0.1 and 1 μg g -1 crab weight) on the expression levels of lipid metabolism-related genes, lipid composition and histology of hepatopancreas in the P. trituberculatus. The results showed that the mRNA levels of carnitine palmitoyltransferase I and II (CPT-I and CPT-II) increased significantly at the low concentrations (0.01 μg g -1 and 0.1 μg g -1 ), while decreased significantly in the highest concentration (1 μg g -1 ). The mRNA levels of acyl-CoA oxidase (ACOX), fatty acid transport protein (FATP), fatty acid-binding protein (FABP), diacylglycerol acyltransferase 1 (DGAT1) and acetyl-CoA carboxylase (ACC) were significantly down-regulated. The transcripts of fatty acid synthase (FAS) and fatty acyl desaturase (FAD) decreased significantly only in 1 μg g -1 treatment. All estradiol treatments (0.01, 0.1 and 1 μg g -1 ) had significantly higher percentages of 20:4n6, 20:5n3 and 22:6n3, but lower percentages of total monounsaturated fatty acids and polar lipids than the control treatment (0 μg g -1 ). Histological observations indicated the size of B cell became larger under estradiol treatment. The results indicated that estradiol promoted lipid catabolism in the hepatopancreas of P. trituberculatus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Copper is essential, but can be toxic to aquatic organisms when present in high concentrations. In freshwater crustaceans, copper inhibits enzymes related to ionic and osmoregulation and to the ammonia efflux, that leads to Na + imbalance and inhibition of ammonia excretion. In the animals inhabiting estuarine or seawater, mechanisms of copper toxicity is not clear, but had been described as disruption of ionregulation and metabolism. To clarify the mechanism of copper toxicity in crustaceans inhabiting variable salinity, this work investigated whether copper affects ammonia excretion and enzymes used for ammonia balance and osmoregulation in the blue crab Callintectes sapidus acclimated to salinity 2 and 30 ppt. To achieve this, juveniles of the blue crab were exposed to 63.5 µg/L of copper at both salinities for 96 h. This is an environmentally realistic copper concentration. Results of ammonia efflux, free amino acids and Na + concentrations in hemolymph, Na + /K + -ATPase, H + -ATPase and, carbonic anhydrase (CA) activities in gills were consistent with the osmoregulatory pattern adopted by the blue crab, which hyperosmoregulates at salinity 2 ppt and osmoconforms at 30 ppt. At 30 ppt copper reduced free amino acid in hemolymph of crabs, suggesting an effect of the metal on osmotic performance. At 2 ppt, copper significantly increased the H + -ATPase activity involved in ammonia excretion. This may be a compensatory response of crabs to maintain low levels of ammonia in their hemolymph; which can be increased by copper exposure. Results presented here are useful for the improvement of the Biotic Ligand Model (BLM) to predict copper toxicity for saltwater environments.

The importance of trace elements in ecotoxicological investigations is a well-known issue when monitoring polluted areas such as commercial harbors. Copper represents one of the most common metal contaminants, often detected in these areas as it is widely employed in various fields and has many sources of inflow in the marine environment. Pachygrapsus marmoratus is a widespread intertidal crab species that has been extensively studied in ecology, ethology and population genetics. Ecotoxicological studies have also been performed, exclusively on the adult stage. In the present study we investigated the mortality and biochemical (oxidative stress and neurotoxicity) responses of P. marmoratus larvae exposure to environmental relevant concentration of copper. Results showed dose-dependent responses in terms of larval mortality, with a calculated LC 50 value of 0.5 mg/L of Cu 2+ . The LC 50 concentration was used as the starting point for subsequent biochemical response evaluation. Results also demonstrated dose-dependent activation of antioxidant systems assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. Moreover, a significant enhancement of neurotransmitter activities was observed, assuming a possible direct interaction of copper with the enzymes or an increase of free copper ion aliquot into the cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The land crabs, Chiromantes haematocheir (Akate-gani) and Chiromantes dehaani (Kurobenkei-gani) inhabit seaside forests in Japan. The crabs mainly consume plant material and its detritus. Therefore, they are expected to possess the ability to degrade the major components of biomass, cellulose and lignin in order to digest plant materials. In this study, we analyzed biomass-degrading activities of the land crabs, especially guaiacol oxidation activity, which seems to be related to lignin degradation. Cellulase activity was detected from almost all gut samples including the stomach, midgut gland and intestine of all dissected crabs. Conversely, high guaiacol oxidation activity was detected in the midgut gland of all C. dehaani and several female C. haematocheir crabs. This is consistent with a previous study showing that female crabs were more herbivorous than male crabs were and observation that C. dehaani crabs are more herbivorous than C. haematocheir. Guaiacol oxidation activity might play an important role in the herbivorous behavior of land crabs., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Global trends in pesticide use can increase aquatic pollution and affect resident fisheries. Crabs exposed to organophosphate pesticides, such as chlorpyrifos, may increase production of reactive oxygen species (ROS), affecting the pro-oxidant/antioxidant balance. Zichiopsis collastinensis crabs were exposed to environmentally relevant concentrations of chlorpyrifos (0.1 and 0.5 µg L -1 ). Effects on the oxidative stress enzymes catalase, superoxide dismutase, glutathione S-transferases, glutathione reductase, and on thiobarbituric acid reactive substances and hydrogen peroxide concentrations were evaluated at four intervals during 96 h exposures. Exposures caused decreased GST activity and increased H 2 O 2 levels in gills. There were modifications of GST, CAT and SOD activities in the hepatopancreas after 12 h of exposure, and an increase of H 2 O 2 levels at every exposure interval observed. The present study proved that chlorpyrifos lead to oxidative stress in Z. collastinensis. However other enzymatic/non-enzymatic responses should be further investigated in order to be included as part of a battery of biomarkers, together with H 2 O 2 levels, which is a parameter highly recommended to be taken into account.

The functions of Wnt signalling in crab limb regeneration are poorly understood. Herein, we isolated and characterised the full-length cDNA of WNT4 from Portunus trituberculatus, designated PtWNT4. The 4831 bp cDNA encodes 323 amino acid polypeptide. Protein structure prediction showed that PtWNT4 has a conserved WNT domain. The PtWNT4 gene was expressed in all regenerative limb stages, and was upregulated from stage I, with highest expression in stage III, and expression then declined in stages IV and V. Immunohistochemistry revealed strong localisation at loose connective tissue during limb regeneration, but PtWNT4 protein levels decreased upon formation of muscle fibers. Injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels from 36 h to 5 days after injection, indicating successful gene silencing. RNA interference knockdown of PtWNT4 expression greatly retarded limb regeneration compared with the control group. Blastema emergence phenotype analysis revealed limb regeneration rates of 0, 31.5% and 40.5% for the dsWNT4 group at 36 h, 3 days and 5 days, respectively, compared with 29.95%, 83.0% and 92.5% for the saline-injected control groups (p <0.05). Expression analysis on the WNT4 using RNAi provide important information for understanding its functional mechanism during limb regeneration in P. trituberculatus. The results also contribute to our understanding of the role of Wnt signalling during limb regeneration in crustaceans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The objective of the present study was to test whether the Atlantic ghost crab Ocypode quadrata is a reliable biomonitor of microplastic (MP) pollution of beach sediments. To test the hypothesis (H1) that the sediment is the main source of MP ingestion, the proportion of MP types (hard plastic, microfibers, pellet, soft plastic, and extruded polystyrene foam) in the gut content was compared with that on the strandline. The types of MPs in the gut content and sediment had similar proportions; black (~49%) and blue (~45%) microfibers were responsible for this similarity (55%), hence confirming H1. However, the second hypothesis (H2) that prevalence of MP in the gut content is related to its density on beach with distinct urbanization degree was not accepted. These results indicate that high trophic plasticity of the ghost crab and, consequently, multiple-sources of contamination may interfere with its use as a biomonitor of MP pollution., (Copyright © 2019. Published by Elsevier Ltd.)

Microplastic (MP) has become ubiquitous in the marine environment. Its threat to marine organisms has been demonstrated under laboratory conditions, yet studies on wild populations still face methodological difficulties. We reviewed the methods used to separate MP from soft animal tissues and highlighted a lack of standardised methodologies, particularly critical for synthetic microfibres. We further compared enzymatic and a potassium hydroxide (KOH)-based alkaline digestion protocols on wild crabs (Carcinus aestuarii) collected from three coastal lagoons in the north Adriatic Sea and on laboratory-prepared synthetic polyester (PES) of different colour and polypropylene (PP). We compared the cost-effectiveness of the two methods, together with the potential for adverse quantitative or qualitative effects on MP that could alter the capability of the polymers to be recognised via microscopic or spectroscopic techniques. Only 5.5% of the 180 examined crabs contained MP in their gastrointestinal tracts, with a notably high quantitative variability between individuals (from 1 to 117 particles per individual). All MP found was exclusively microfibres, mainly PES, with a mean length (±SE) of 0.5 ± 0.03 mm. The two digestion methods provided comparable estimates on wild crabs and did not cause any visible physical or chemical alterations on laboratory-prepared microfibres treated for up to 4 days. KOH solution was faster and cheaper compared to the enzymatic extraction, involving fewer procedural steps and therefore reducing the risk of airborne contamination. With digestion times longer than 4 days, KOH caused morphological alterations of some of the PES microfibres, which did not occur with the enzymatic digestion. This suggests that KOH is effective for the digestion of small marine invertebrates or biological samples for which shorter digestion time is required, while enzymatic extraction should be considered as alternative for larger organisms or sample sizes requiring longer digestion times., (Copyright © 2019 Elsevier Ltd. All rights reserved.)