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Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species, and its growth and development are regulated by temperature, but the molecular mechanisms of the responses to temperature remain unclear. Herein, we identified TRPA1 from E. sinensis, a member of the TRP family of heat receptor potential channels, performed RACE cloning and bioinformatics analysis, and investigated the effect of TRPA1 on temperature responses and molting by real-time PCR and RNA interference (RNAi). The open reading frame of Es-TRPA1 is 3660 bp, and the encoded protein has a molecular weight of 136.91 kDa, and is expressed in embryos and juveniles. RNAi-mediated silencing decreased Es-TRPA1 expression in juvenile crabs, molting rate was decreased, mortality was increased, and crabs avoided cold areas (4 °C) much less than control juvenile crabs. The results suggest that Es-TRPA1 is involved in regulating temperature adaptation and molting processes in E. sinensis. The findings lay a foundation for further exploration of temperature regulation mechanisms in E. sinensis and other crustaceans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Crustacean hyperglycemic hormone (CHH) plays a crucial role in regulating carbohydrate metabolism in crustaceans. In this study, a new cDNA encoding type I CHH peptide, termed Sp-CHH3, was isolated from the mud crab Scylla paramamosain and its potential functions were investigated. The full length cDNA of Sp-CHH3 was identified as encoding a 127-aa precursor composed of a 27-aa signal peptide, a 23-aa CHH precursor-related peptide and a 75-aa mature peptide with a typical motif of CHH. Phylogenic analysis suggested that, Sp-CHH3 is a previously unreported CHH from S. paramamosain. Tissue distribution analysis showed that Sp-CHH3 was mainly expressed in the eyestalk ganglia, thoracic ganglia, stomach and the ovary. A RNA interference experiments showed that after injection of Sp-CHH3-targeted dsRNA, both the level of Sp-CHH3 expression in the eyestalk ganglia and hemolymph glucose level decreased significantly. A further short-term starvation experiments demonstrated that, the level of Sp-CHH3 detected in the eyestalk ganglia was significantly up-regulated at the 12th h of starvation, it then fell back at the 24th h of starvation and subsequently remained relative stability between the 24th to 96th h of starvation. The hemolymph glucose level decreased significantly (P < .05) at each sampling time during the 96 h starvation duration when compared to that of 0 h (prior to starvation) and the overall trend was largely correlated with the level of Sp-CHH3 expression in the eyestalk ganglia. In summary, the results suggest that Sp-CHH3 plays a functional role in regulating carbohydrate metabolism in S. paramamosain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study was aimed to characterize the full length of mRNA of oxytocin/vasopressin (OT/VP)-like mRNA in female Portunus pelagicus (PpelOT/VP-like mRNA) using a partial PpelOT/VP-like sequence obtained previously in our transcriptome analysis (Saetan, 2014) to construct the primers. The PpelOT/VP-like mRNA was 626 bp long and it encoded the preprohormones containing 158 amino acids. This preprohormone consisted of a signal peptide, an active nonapeptide (CFITNCPPG) followed by the dibasic cleavage site (GKR), and the neurophysin domain. Sequence alignment of the PpelOT/VP-like peptide with those of other animals revealed strong molecular conservation. Phylogenetic analysis of encoded proteins revealed that the PpelOT/VP-like peptide was clustered within the group of crustacean OT/VP-like peptide. Analysis by RT-PCR revealed the expression of mRNA transcripts in the eyestalk, brain, ventral nerve cord (VNC), ovary, intestine and gill. The in situ hybridization demonstrated the cellular localizations of the transcripts in the central nervous system (CNS) and ovary tissues. In the eyestalk, the mRNA expression was observed in the neuronal clusters 1-5 but not in the sinus gland complex. In the brain and the VNC, the transcripts were detected in all neuronal clusters but not in the glial cell. In the ovary, the transcripts were found in all stages of oocytes (Oc1, Oc2, Oc3, and Oc4). In addition, synthetic PpelOT/VP-like peptide could inhibit steroid release from the ovary. The knowledge gained from this study will provide more understanding on neuro-endocrinological controls in this crab species., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum. The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The purpose of this study was to investigate the effect of benzo[a]pyrene (B[a]P) on reproductive endocrine disruption and explore the preliminary mechanisms in crustaceans. In this study, sexually mature female Portunus trituberculatus were exposed to 0, 0.1, 0.5 and 2.5 μg/L B[a]P for 10 days. The following were investigated: (1) Gonadosomatic Index (GSI) and oocyte diameter, (2) steroid concentrations in ovary and hemolymph, and (3) mRNA levels of genes involved in sex steroid synthesis (3β-HSD,17β-HSD) or reproduction (estrogen receptor (ER), OUT (Ovarian tumor gene) domain containing ubiquitin aldehyde-binding protein 1 (OTUB1), vitellogenin (VTG),vasa). B[a]P exposure caused significant reductions in the GSI and oocyte diameter in the crabs. Furthermore, 17β-estradiol (E2), testosterone (T) and progesterone (P) levels were inhibited significantly while 3β-HSD and 17β-HSD mRNA expressions were also decreased in a dose-dependent manner at day 10, which suggests that B[a]P can disrupt sex steroid levels through steroid synthesis pathways. In addition, high levels of B[a]P activated transcription of OTUB1 while suppressed ER and VTG expression, which indicates that exposure to waterborne B[a]P could interfere with ubiquitin-proteasome pathway and subsequently affect ER and ER-mediated gene expression. We also observed a reduction in vasa gene expression reflecting the negative effect of B[a]P on oocyte development in the molecular level. This study is the first to demonstrate in vivo B[a]P toxicity in the reproductive endocrine system of female P. trituberculatus and provided a scientific basis of the decline in crustacean populations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The tumor suppressor gene p53 plays a critical role in safeguarding the integrity of genome in mammalian cells. It acts as a sequence-specific transcription factor. Once activated by a variety of cellular stresses, p53 transactivates downstream target genes, through which it regulates cell cycle and apoptosis. However, little is known about p53 as well as its downstream target genes in invertebrates. A full length cDNA that encodes a 453-amino-acid p53 protein (Lvp53) was characterized in the Pacific white shrimp (Litopenaeus vannamei) to explore the potential relationships between p53 and two antioxidant enzyme genes: Mn-superoxide dismutase (MnSOD) and glutathione peroxidase (GPx) in eliminating cell stresses in L. vannamei. Sequence analysis revealed a close phylogenetic relationship between Lvp53 and that of Marsupenaeus japonicus, and a high degree of conservation in critical amino acids residues is involved in DNA and zinc binding among species. Quantitative real-time PCR showed that Lvp53 was expressed with varied levels in all the 11 tissues under investigation. In response to acute pH challenge, the relative expression of Lvp53 was induced in a pH- and time-dependent manner, with the peak observed at pH 6.1 and after 24 h of treatment, in which condition, both the relative mRNA expressions and the enzymatic activities of LvMnSOD and LvGPx were increased correspondingly. In response to acute cadmium (Cd) exposure, the relative expression of Lvp53 was upregulated in a time- and concentration-dependent manner, with the maximum detected at Cd 6.6 μM and after 48 h of exposure, in which case, both the transcripts and the enzymatic activities of LvMnSOD and LvGPx were also induced. After Lvp53 transcripts were declined by double-strand RNA injection, the relative mRNA expressions of LvMnSOD and LvGPx were decreased correspondingly. Meanwhile, pH 6.1 or 6.6 μM Cd could not induce the transcripts or the enzymatic activities of LvMnSOD or LvGPx any more in Lvp53-silenced shrimp, but increased shrimp mortalities. These results indicated the involvement of Lvp53, LvMnSOD and LvGPx in mediating cell stress caused by suboptimal pH and elevated levels of Cd in L. vannamei, and that the expressions of LvMnSOD and LvGPx were positively regulated by Lvp53, which is a potential mechanism for shrimp to survive the oxidative stress that occurs during short-term exposure to Cd or challenge with acidic pH. This finding will contribute to better understanding of p53 signaling pathways and redox regulation in invertebrate organisms., (© 2013.)

Outbreak of Taura syndrome virus (TSV) is one of the major pathogens of the Pacific white shrimp Litopenaeus vannamei. Although selective breeding for improvement of TSV resistance in L. vannamei has been successfully developed and has led to a great benefit to the shrimp farming industry worldwide. The molecular mechanisms underlying the viral resistance in shrimp remain largely unknown. In the present study, we conducted the first transcriptomic profiling of host responses in hemolymph and hemocytes in order to identify the differentially expressed genes associated with resistance to TSV in L. vannamei. High-throughput RNA-Seq was employed, obtaining 193.6 and 171.2 million high-quality Illumina reads from TSV-resistant and susceptible L. vannamei lines respectively. A total of 61,937 contigs were generated with an average length of 546.26 bp. BLASTX-based gene annotation (E-value < 10(-5)) allowed the identification of 12,398 unique proteins against the NCBI non-redundant NR database. In addition, comparison of digital gene expression between resistant and susceptible strains revealed 1374 significantly differentially expressed contigs (representing 697 unigenes). Gene pathway analysis of the differentially expressed gene set highlighted several putative genes involved in the immune response activity including (1) pathogen/antigen recognition including immune regulator, adhesive protein and signal transducer; (2) coagulation; (3) proPO pathway cascade; (4) antioxidation; and (5) protease. The expression patterns of 22 differentially expressed genes involving immune response were validated by quantitative real-time RT-PCR (average correlation coefficients 0.94, p-value < 0.001). Our results provide valuable information on gene functions associated with resistance to TSV in L. vannamei., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The immune deficiency (IMD) signal pathway mediates immunity against Gram-negative bacteria in Drosophila. Recent studies show that the IMD pathway also involves in antiviral innate immune responses. The functions of the pathway in crustacean immunity are largely unknown. In this paper, two IMDs (FcIMD and PcIMD), one of the key elements of the IMD pathway, were identified from Chinese white shrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkii. Both proteins have a death domain located at the C-terminal. FcIMD was mainly expressed in the gills and stomach and PcIMD was mainly detected in the heart, hepatopancreas, and stomach. FcIMD peaked in hemocytes at 12 h after white spot syndrome virus (WSSV) challenge and it peaked in the gills at 6 h after WSSV challenge, but it was decreased at 2 h and kept the low level to 24 h in hemocytes and no obviously change in gill after Vibrio anguillarum challenge. PcIMD first decreased in hemocytes at 2 h and peaked at 12 h in hemocytes after V. anguillarum challenge. It was also upregulated in gill after bacterial challenge, peaked at 2 h, and decreased at 6 h, and then gradually increased at 12-24 h. PcIMD has no significant change in hemocytes and gill after WSSV challenge. Western blot analysis detected FcIMD protein in all tissues, and immunocytochemical analysis localized FcIMD in the cytoplasm of hemocytes. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of three kinds AMP genes, including crustins, anti-lipopolysaccharide factors and lysozymes, in shrimp and crayfish. They are Cru 1, Cru 2, ALF 1, ALF 2 and Lys 1 in crayfish, and Cru1, Cru 3, ALF 6, ALF 8, and Lys2 in shrimp. These results suggest that although IMD distribution and expression patterns have some differences, the IMD pathway may have conserved function for AMP regulation in shrimp and crayfish immunity against Gram-negative bacteria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Serine proteinases (SPs) participate in various biological processes and play vital role in immunity. In this study, we investigated the function of PmClipSP2 from shrimp Penaeus monodon in defense against bacterial infection. PmClipSP2 was identified as a clip-domain SP and its mRNA increased in response to infection with Vibrio harveyi. PmClipSP2-knockdown shrimp displayed a significantly reduced phenoloxidase (PO) activity and increased susceptibility to V. harveyi infection. Injection of LPS and/or β-1,3-glucan induced a dose-dependent mortality and a significant decrease in the number of total hemocytes, with clear morphological changes in the hemocyte surface, of the PmClipSP2-knockdown shrimp. Recombinant PmClipSP2 was shown to bind to LPS and β-1,3-glucan and to activate PO activity. These results reveal that PmClipSP2 acts as a pattern-recognition protein, binding to microbial polysaccharides and likely activating the proPO system, whilst it may play an essential role in the hemocyte homeostasis by scavenging LPS and neutralizing its toxicity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Due to the over-harvesting and deterioration of wild populations, the ever-growing crustacean market is increasingly reliant on aquaculture, driving the need for better management techniques. Since most cultured crustacean species exhibit sexually dimorphic growth patterns, the culture of monosex populations (either all-male or all-female) is a preferred approach for gaining higher yields, with the ecological benefit of reducing the risk of invasion by the cultured species. Sexual manipulations may also render sustainable solutions to the environmental problems caused by the presence of invasive crustacean species with detrimental impacts ranging from aggressive competition with native species for food and shelter, to affecting aquaculture facilities and harvests and causing structural damage to river banks. Recent discoveries of androgenic gland (AG)-specific insulin-like peptides (IAGs) in crustaceans and the ability to manipulate them and their encoding transcripts (IAGs) have raised the possibility of sexually manipulating crustacean populations. Sexual manipulation is already a part of sustainable solutions in fish aquaculture and in the bio-control of insect pest species, and attempts are also being made to implement it with crustaceans. As recently exemplified in a commercially important prawn species, IAG silencing, a temporal, non-genetically modifying and non-transmissible intervention, has enabled the production of non-breeding all-male monosex populations that are the progeny of sexually reversed males ('neo-females'). IAG manipulations-based biotechnologies therefore have the potential to radically transform the entire industry. We review here how this proof of concept could be broadened to meet both aquacultural and environmental needs. We include the major cultured decapod crustacean groups and suggest a sustainable solution for the management of invasive and pest crustacean species. We also review the key considerations for devising a biotechnological approach that specifically tailors the molecular technological abilities to the management of each target group., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Embryonic development of the Asian tadpole shrimp Triops granarius is arrested at an early stage of organogenesis under darkness, whereas exposure to light resumes its development and promotes hatching. To better understand the photoreception systems involved in the light-induced hatching of T. granarius eggs, we examined the spectral sensitivity of hatching and the expression of genes involved in photoreception (opsins) and phototransduction (arrestin 2). Hatching of eggs is induced by light of broad wavelengths, from ultraviolet (UV) (395 nm) to red (660 nm), suggesting that visual pigments are involved in photoreception. Next, we focused on opsins which are protein moieties of visual pigments and arrestin 2 which is involved in a phototransduction cascade. Transcripts of several opsin genes and the arrestin 2 gene were detected in T. granarius eggs. Thus, we present a possibility that T. granarius eggs use visual pigments for light perception in light-induced hatching., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.

A prophenoloxidase (proPO) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has a full length of 2663bp, with an open reading frame of 2019bp, a 124-bp 5'-untranslated region, and a 520-bp 3'-untranslated region containing a poly A signal. It encodes a protein of 673 amino acids with a predicted molecular weight of 77.5kDa and with an estimated pI of 5.96. It contains two putative tyrosinase copper-binding motifs with six histidine residues (copper A, 185, 189, 211, and copper B, 346, 350, 386). The proPO has thiol-ester-like motif (GCGWPQHM), which showed similar structural features of proPOs from other decapod crustaceans. It also contains five possible glycosylation sites, and a conserved C-terminal region common to all known proPOs. Sequence comparison showed that the proPO-deduced amino acid of mud crab S. serrata has an overall similarity of 78%, 57%, 56%, 51-55%, 54%, 53%, 52%, 52%, and 52% to that of Dungeness crab Cancer magister, American lobster Homarus americanus, European lobster Homarus gammarus, kuruma prawn Marsupenaeus japonicus, crayfish Pacifastacus leniusculus, white shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, green tiger shrimp Penaeus semisulcatus, and giant freshwater prawn Macrobrachium rosenbergii, respectively. The proPO was strongly expressed in haemocytes, but not in heart, eyestalk, gill, muscle, ovary, hepatopancreas, stomach, and intestine. The proPO transcript of mud crab S. serrata increased significantly in 12 and 24h post-lipopolysaccharide (LPS) injection, but returned to the original values in 72h post injection.

Analysis of 18S rDNA sequences isolated from Cyathura carinata (Crustacea: Isopoda) indicate that two different species of the Digenea (Platyhelminthes: Trematoda) parasitize this isopod species for which parasitic associations have previously been unknown. Phylogenetic analysis, based on the largest data set of digenetic 18S rDNA sequences published to date, also suggests that 18S rDNA is unsuitable for the reconstruction of phylogenetic relationships of the major digenetic lineages either due to rapid radiation of these lineages or the presence of homoplasy at this level.