Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis

Apoptosis is a central regulatory feature of the immune system, and the most common form of death among immunological cells. However, the function of apoptosis, within the innate immune system of invertebrates, remains largely unknown. For this reason, we investigated the immune functionality of two apoptosis genes, caspase and nm23, in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The entire length caspase and nm23 cDNA genes were cloned using PCR, based on an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The caspase cDNA contained an 1119 bp open reading frame that encoded a putative 372 amino acid protein, while nm23 cDNA contained a 456 bp open reading frame that encoded a putative 151 amino acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of conserved enzyme active sites that were common among caspase and nm23 superfamilies. In brief, caspase and nm23 mRNA expression in E. sinensis were (a) both detected in all tissues, including the hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain and eyestalk, and (b) responsive in hemocytes, gill and hepatopancreas to a Vibrio anguillarum immuno-challenge all appeared sharp increase. Collectively, the data presented here demonstrate the successful isolation of caspase and nm23 apoptosis genes from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.     

Molecular cloning, characterization and expression analysis of macrophage migration inhibitory protein (MIF) in Chinese mitten crab, Eriocheir sinensis

Macrophage migration inhibitory factor (MIF) as a multi-functional cytokine mediating both innate and adaptive immune responses, however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of MIF in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full-length MIF cDNA (704 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a E. sinensis cDNA library. The MIF cDNA contained a 363 bp open reading frame (ORF) that encoded a putative 120 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate MIF sequences revealed conserved enzyme active sites. MIF mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, and (b) responsive in hemocytes, hepatopancreas and gill to a Vibrio anguillarum challenge, with peak exposure observed 8 h, 12 h and 12 h post-injection, respectively. Collectively, data demonstrate the successful isolation of MIF from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.     

Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from mixed tissues of E. sinensis challenged with LPS. Eight genes involved in immune response were identified from 319 single colonies. Among them, two different C-type lectin-like domain (CTLD)-containing proteins were firstly identified in Chinese mitten crab. The full-length cDNA sequences of two C-type lectin-like domain (CTLD)-containing proteins named EsCTLDcp-1 and EsCTLDcp-2 were cloned by 5' RACE. The deduced amino acid sequences of EsCTLDcp-1 and EsCTLDcp-2 possessed several conserved features of C-type lectin subfamily. The tissue distribution of EsCTLDcp-1 and EsCTLDcp-2 was examined by Real-time PCR. In the normal Chinese mitten crab, the expression of EsCTLDcp-2 was detected in all tested tissues such as haemolymph, muscle, intestine, gill, heart, gonad and hepatopancreas, whereas in muscle, intestine, gill, heart and hepatopancreas for EsCTLDcp-1. The highest expressions of EsCTLDcp-1 and EsCTLDcp-2 were both observed in hepatopancreas. LPS significantly induced the expression of EsCTLDcp-1 and EsCTLDcp-2 in the hepatopancreas at the different time points. The induced fold change of EsCTLDcp-1 and EsCTLDcp-2 increased significantly from 2 h for EsCTLDcp-1 and 4 h for EsCTLDcp-2, and reached a maximum at 12 h, then dropped at 24 h. A differential pattern was found in Chinese mitten crab challenged with Chinese mitten crab pathogen Aeromonas hydrophila. The expression of EsCTLDcp-1 increased significantly at 2 h post-challenge crabs with A. hydrophila, then decreased at 4 h and 8 h, after that increased at 12 h and 24 h. The expression of EsCTLDcp-2 was decreased at the all time points. All these data suggest a differential role of EsCTLDcp-1 and EsCTLDcp-2 in the crab innate immune response to bacterial infection.     

Reproductive function of Selenoprotein M in Chinese mitten crabs (Eriocheir sinesis)

Selenoproteins are present in all major forms of life, including eukaryotes, bacteria and archaea. In eukaryotic animals, selenoproteins often function as antioxidants, but rare or absent in other phyla, such as plants and fungi (except for the green alga Chlamydomonas). Selenoprotein M (SelM) is a selenocysteine containing protein with redox activity, which is involved in the antioxidant response. However, information remains limited about SelM physiology and function in marine invertebrates, particularly in crustaceans. Hence, we investigated the reproductive functionality of SelM in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important yet disease vulnerable aquaculture species. The full-length SelM cDNA (928bp) strand was cloned by using PCR, based on an initial expressed sequence tag (EST) that was isolated from a hepatopancreatic cDNA library. The SelM cDNA contained a 390bp open reading frame (ORF) that encoded a putative 129 amino acid (aa) protein. SelM mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in the hepatopancreas, testis, ovaries and intestines. Based on this information, we then detected the different stages of tissue expression for SelM in the testis, ovary, and male crab hepatopancreas and hemolymph, and the enzyme activity of SelM in the testis. Overall, SelM was isolated successfully from the Chinese mitten crab, and its involvement in the regulation of reproduction during the period of rapid development in E. sinensis was confirmed.     

叉角厉蝽触角转录组及嗅觉相关基因分析

叉角厉蝽Eocanthecona furcellata是一种在生物防治方面有重要作用和潜力的捕食性天敌昆虫,触角是昆虫进行信息交换的重要器官,而嗅觉相关基因则是调控天敌昆虫捕食行为的重要分子基础。为获得叉角厉蝽触角转录组数据库,挖掘叉角厉蝽嗅觉相关基因,本研究利用Illumina高通量测序平台对叉角厉蝽雌雄成虫与5龄若虫触角进行转录组测序。成功构建了叉角厉蝽触角转录组,获得了67 843条unigenes, N50长度为2 300 bp。与七大公共数据库比对注释到27 686条unigenes,其中NR数据库注释最多(33.33%),且与茶翅蝽Halyomorpha halys相似度最高(64.20%)。14 258条注释到GO数据库中,分为生物过程、细胞组分和分子功能3个大类42个亚类;KEGG代谢途径分析表明,7 703条形成282条代谢通路,其中被注释在信号传导通路中的unigenes最多(11.50%)。进一步基因注释分析,鉴定得到134个候选嗅觉相关基因,32个化学感应蛋白基因(Chemosensory protein genes, CSP),10个气味结合蛋白基因(Odor...     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

Multiple isoforms of immune-related genes from hemocytes and eyestalk cDNA libraries of swimming crab Portunus trituberculatus

Expressed sequence tags (ESTs) analysis has been shown to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. Two full-length enriched cDNA libraries were constructed from hemocytes and eyestalk of Portunus trituberculatus, respectively, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. A total of 99 unigenes including 64 unigenes (6.00% of 1066 unigenes) in hemocytes library and 35 unigens (6.86% of 510 unigenes) in eyestalk library are identified to be immune genes. These genes are categorized into six classes, viz. antimicrobial peptides, redox proteins, melanization related proteins, chaperone proteins, clottable proteins and other immune factors. The content and category of immune genes in eyestalk library indicate eyestalk might have unrecognized role in crab immunity. Five immune genes containing multiple protein isoforms are identified and characterized, including anti-lipopolysaccharide factor (PtALF1-7), crustin (PtCrustin1-3), thioredoxin (PtTrx1-2), clip domain serine proteinase (PtcSP1-5) and kazal-type proteinase inhibitor (PtKPI1-4). Sequence alignment and phylogenetic analysis reveal PtALF1-7 contain two conserved cysteine residues and might be encoded by multiple genomic loci. PtCrustin1-3 share the consensus cysteine motif and are considered as Type I crustins. PtTrx1 possesses the critical structural cysteine residue C?3 of Trx-1, while PtTrx2 has the N-terminal mitochondrial translocation signal of Trx-2. Sequence analysis shows PtcSP1-5 contain one clip domain and one partial SP catalytic triad domain. PtKPI1-4 present one typical Kazal domain consisting of six conserved cysteine residues. Some protein isoforms are tissue-specific, which might suggest they have different origins and perform diverse functions. Except PtALF1-3 and PtCrustin1, the other isoformes in this study are firstly identified from P. trituberculatus. Especially, PtTrx2 are firstly identified from crustaceans. Our research will provide useful genomic information of P. trituberculatus and be helpful in understanding the molecular mechanisms of crab immunity.