Down Syndrome Cell Adhesion Molecule Triggers Membrane-to-Nucleus Signaling-Regulated Hemocyte Proliferation against Bacterial Infection in Invertebrates.

Down syndrome cell adhesion molecule (Dscam) generates tens of thousands of isoforms by alternative splicing, thereby providing crucial functions during immune responses. In this study, a novel Dscam signaling pathway was investigated in crab, which remains poorly characterized in invertebrates. Bacterial infection induced the cytoplasmic cleavage of Dscam intracellular domains (ICDs) by γ-secretase, and then the released ICDs carrying specific alternatively spliced exons could directly interact with IPO5 to facilitate nuclear translocation. Nuclear imported ICDs thus promoted hemocyte proliferation and protect the host from bacterial infection. Protein-interaction studies revealed that the ectodomain of Dscam bound to a disintegrin and metalloprotease domain 10 (ADAM10) rather than ADAM17. Inhibition or overexpression of ADAM10 impaired or accelerated Dscam shedding activity post-bacterial stimulation, respectively. Moreover, the shedding signal then mediated Dscam with an intact cytoplasmic domain to promote the cleavage of ICDs by γ-secretase. Furthermore, the transcription of ADAM10 was regulated by Dscam-induced canonical signaling, but not nuclear imported ICDs, to serve as a feedback regulation between two different Dscam pathways. Thus, membrane-to-nuclear signaling of Dscam regulated hemocyte proliferation in response to bacterial infection.

Antimicrobial activity of a novel hypervariable immunoglobulin domain-containing receptor Dscam in Cherax quadricarinatus.

Down syndrome cell adhesion molecule (Dscam) mediates innate immunity against pathogens in arthropods. Here, a novel Dscam from red claw crayfish Cherax quadricarinatus (CqDscam) was isolated. The CqDscam protein contains one signal peptide, ten immunoglobulin domains, six fibronectin type III domains, one transmembrane domain and cytoplasmic tail. CqDscam phylogenetically clustered with other invertebrate Dscams. Variable regions of CqDscam in N-terminal halves of Ig2 and Ig3 domains, complete Ig7 domain and TM domain can be reshuffled after transcription to produce a deluge of >37,620 potential alternative splice forms. CqDscam was detected in all tissues tested and abundantly expressed in immune system and nerve system. Upon lipopolysaccharides (LPS) and b-1, 3-glucans (Glu) challenged, the expression of CqDscam was up-regulated, while no response in expression occurred after injection with peptidoglycans (PG). Membrane-bound and secreted types of CqDscam were separated on the protein level, and were both extensively induced post LPS challenge. Membrane-bound CqDscam protein was not detected in the serum, but localized to the hemocyte surface by immuno-localization assay. In the antimicrobial assays, the recombinant LPS-induced isoform of CqDscam protein displayed bacterial binding and growth inhibitory activities, especially with Escherichia coli. These results suggested that CqDscam, as one of pattern-recognition receptors (PRRs), involved in innate immune recognition and defense mechanisms in C. quadricarinatus, possibly through alternative splicing.

Fatty acid binding protein FABP3 from Chinese mitten crab Eriocheir sinensis participates in antimicrobial responses.

Intracellular fatty acid-binding proteins (FABPs) are members of the lipid-binding protein superfamily. Aside from the main functions of FABPs in the uptake and transport of fatty acids, they are also critical in innate immunology. In this work, the full-length cDNA for a Chinese mitten crab Eriocheir sinensis FABP (Es-FABP3) was cloned with an open reading frame of 402 bp encoding a 133 amino acid polypeptide. Analysis using quantitative real-time PCR (qPCR) revealed that Es-FABP3 transcripts were widely distributed in gills, muscle, intestine, hepatopancreas, eyestalk, heart, stomach, brain, thoracic ganglia and hemocytes. After challenge with pathogen associated molecular pattern molecules (PAMPs), the relative mRNA expression levels of Es-FABP3 increased in hepatopancreas, gills and hemocytes. Moreover, the mature recombinant Es-FABP3 protein exhibited different binding activities to bacteria and fungus and inhibited the growth of different microbes. These collective results demonstrated the role of Es-FABP3 in the immunoreactions of E. sinensis to PAMPs.

Caspase-mediated apoptosis in crustaceans: cloning and functional characterization of EsCaspase-3-like protein from Eriocheir.

The caspase-3-like gene was cloned from Eriocheir sinensis, and its properties were characterized to identify the biological implications of this caspase in apoptosis in crab. Its deduced full-length protein sequence consists of 462 amino acid residues, including the prodomain and the large and small subunits. Moreover, several residues known to be critical in the caspase-3 catalytic center and binding pocket, as well as the active site pentapeptide motif Q(220)ACRG(224), were identically present in the deduced EsCaspase-3-like protein. Subsequently, the recombinant EsCaspase-3-like (rEsCaspase-3-like) protein was expressed from Escherichia coli and obtained via affinity purification. Results of the in vitro enzymatic activity assays indicated that the rEsCaspase-3-like protein is capable of hydrolyzing the substrate Ac-DEVD-pNA, suggesting a functional role in physiology. EsCaspase-3-like gene transcripts were found to be widely distributed in all tissues as detected by quantitative RT-PCR, being especially abundant in hemocytes and comparatively rare in muscles. Furthermore, EsCaspase-3-like, at both the mRNA and protein levels, was demonstrated to participate in the apoptotic process after stimulation by different pathogen-associated molecular patterns (PAMPs) in hemocytes. In conclusion, our findings suggest that the EsCaspase-3-like protein functions as an effector caspase and contributes to immune responses against pathogens.

Identification and characterization of Tube in the Chinese mitten crab Eriocheir sinensis.

As a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but has not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E. sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was 2247-bp with a 1539-bp open reading frame (ORF) encoding a 512-amino acid protein. The protein contained a 116-residue death domain (DD) at its N-terminus and a 272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4 proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in the ovary and testis, and moderately expressed in the thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2 (November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (ß-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.

Association of a hepatopancreas-specific C-type lectin with the antibacterial response of Eriocheir sinensis.

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate.

A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity.

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play significant roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (EsLecD) was identified from the crustacean Eriocheir sinensis. The cloning of full-length EsLecD cDNA was based on the initial expressed sequence tags (ESTs) isolated from a hepatopancreatic cDNA library. The full-length EsLecD cDNA of 686 bp with an open reading frame of 468 bp encodes a putative protein of 155 aa residues, including an N-terminal signal peptide and a single carbohydrate-recognition domain (CRD). By quantitative RT-PCR analysis, the EsLecD transcript was mainly detected in the hepatopancreas but rarely in other tissues, and it was significantly upregulated in the hepatopancreas after immune challenge with lipopolysaccharides. The recombinant EsLecD protein (rEsLecD) exhibited the ability to bind to all tested microorganisms, including bacteria and yeast. Meanwhile, calcium significantly increased the binding affinity of rEsLecD toward microorganisms, but it was not essential. The binding of rEsLecD induced the aggregation of microbial pathogens. Moreover, rEsLecD was capable of inhibiting the growth of microorganisms and even directly killing bacteria. Interestingly, rEsLecD could stimulate cellular encapsulation in vitro. In conclusion, results of this study suggest that EsLecD acts as an antibacterial PRR participating in the innate immunity of invertebrates.

Two novel Toll genes (EsToll1 and EsToll2) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosan.

Tolls/Toll-like receptors (TLRs) play an essential role in initiating innate immune responses against pathogens and are found throughout the insect kingdom but have not yet been reported in the crustacean, Eriocheir sinensis. For this purpose, we cloned two novel Toll genes from E. sinensis, EsToll1 and EsToll2. The full-length cDNA of EsToll1 was 3963 bp with a 3042-bp open reading frame (ORF) encoding a 1013-amino acid protein. The extracellular domain of this protein contains 17 leucine-rich repeats (LRRs) and a 139-residue cytoplasmic Toll/interleukin-1 receptor (TIR) domain. The cDNA full-length of EsToll2 was 4419 bp with a 2667-bp ORF encoding an 888-amino acid protein with an extracellular domain containing 10 LRRs and a 139-residue cytoplasmic TIR domain. By phylogenetic analysis, EsToll1 and EsToll2 clustered into one group together with Tolls from other crustaceans. Quantitative RT-PCR analysis demonstrated that a) both EsToll1 and EsToll2 were constitutively expressed in all tested crab tissues; b) EsToll1 and EsToll2 were differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsToll2 expression was significantly upregulated at almost all time intervals post-challenge with LPS, PG and GLU. Our study indicated that EsToll1 and EsToll2 are differentially inducibility in response to various PAMPs, suggesting their involvement in a specific innate immune recognition mechanism in E. sinensis.

A double WAP domain-containing protein Es-DWD1 from Eriocheir sinensis exhibits antimicrobial and proteinase inhibitory activities.

Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC) structure rich in cysteine residues. In this study, a double WAP domain (DWD)-containing protein, Es-DWD1, was first cloned from the Chinese mitten crab (Eriocheirsinensis). The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF) encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWD1 transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs). The mature recombinant Es-DWD1 (rEs-DWD1) protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1 could exert agglutination activities against Bacillus subtilis and Pichiapastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P. pastoris. Furthermore, rEs-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.

Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis.

Dorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was 2493 bp with a 2022-bp open reading frame (ORF) encoding a 673-amino acid protein. This protein contained a 171-residue conserved Rel homology domain (RHD) and a 102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that (a) EsDorsal had higher expression level in immune organs; (b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.

Two antibacterial C-type lectins from crustacean, Eriocheir sinensis, stimulated cellular encapsulation in vitro.

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.

Immunoglobulin superfamily protein Dscam exhibited molecular diversity by alternative splicing in hemocytes of crustacean, Eriocheir sinensis.

Be absent of adaptive immunity which have both specificity and memory, invertebrates seem to have evolved alternative adaptive immune strategies to resist various intruding pathogens. Whereas vertebrates could generate a wide range of immunological receptors with somatic rearrangement, invertebrates possibly depend on alternative splicing of pattern-recognition receptors (PRRs). Recently, it has been suggested that a member of the immunoglobulin superfamily (IgSF), Down syndrome cell adhesion molecule (Dscam), plays a crucial role in the alternative adaptive immune system of invertebrates. At present, we successfully isolated and characterized the first crab Dscam from Eriocheir sinensis. EsDscam has typical domain architecture compared with other Dscam orthologs, including one signal-peptide, 10 immunoglobulin (Ig) domains, 6 fibronectin type III domains (FNIII), one transmembrane domain (TM) and one cytoplasmic tail. We had detected four hypervariable regions of EsDscam in the N-terminal halves of Ig2 (25) and Ig3 domain (30), the complete Ig7 (18) and also the transmembrane domain (2), potentially generate 27,000 unique isoforms at least. Transcription of EsDscam were both a) detected in all tissues, especially in immune system, digestive system and nerve system; b) significantly induced in hemocytes post lipopolysaccharides (LPS), peptidoglycans (PG) and ß-1, 3-glucans (Glu) injection. Importantly, we had detected membrane-bound and secreted Dscam isoforms in E. sinensis, and showed that secreted isoforms were extensively transcribed post different PAMPs challenge respectively. Results from immuno-localization assay revealed that EsDscam evenly distributed in the cell surface of hemocytes. These findings indicated that EsDscam is a hypervariable PRR in the innate immune system of the E. sinensis.

Fatty acid binding proteins FABP9 and FABP10 participate in antibacterial responses in Chinese mitten crab, Eriocheir sinensis.

Invertebrates rely solely on the innate immune system for defense against pathogens and other stimuli. Fatty acid binding proteins (FABP), members of the lipid binding proteins superfamily, play a crucial role in fatty acid transport and lipid metabolism and are also involved in gene expression induced by fatty acids. In the vertebrate immune system, FABP is involved in inflammation regulated by fatty acids through its interaction with peroxidase proliferator activate receptors (PPARs). However, the immune functions of FABP in invertebrates are not well characterized. For this reason, we investigated the immune functionality of two fatty acid binding proteins, Es-FABP9 and Es-FABP10, following lipopolysaccharide (LPS) challenge in the Chinese mitten crab (Eriocheir sinensis). An obvious variation in the expression of Es-FABP9 and Es-FABP10 mRNA in E. sinensis was observed in hepatopancreas, gills, and hemocytes post-LPS challenge. Recombinant proteins rEs-FABP9 and rEs-FABP10 exhibited distinct bacterial binding activity and bacterial agglutination activity against Escherichia coli and Staphylococcus aureus. Furthermore, bacterial growth inhibition assays demonstrated that rEs-FABP9 responds positively to the growth inhibition of Vibrio parahaemolyticuss and S. aureus, while rEs-FABP10 responds positively to the growth inhibition of Aeromonas hydrophila and Bacillus subtilis. Coating of agarose beads with recombinant rEs-FABP9 and rEs-FABP10 dramatically enhanced encapsulation of the beads by crab hemocytes in vitro. In conclusion, the data presented here demonstrate the participation of these two lipid metabolism-related proteins in the innate immune system of E. sinensis.

Two novel short C-type lectin from Chinese mitten crab, Eriocheir sinensis, are induced in response to LPS challenged.

The basic mechanism of host fighting against pathogens is pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. For this reason, we investigated the immune functionality of two pattern recognition receptors, C-type lectin EsLecA and EsLecG, post lipopolysaccharides (LPS) challenge in Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The cloning of full-length EsLecA and EsLecG cDNA were based on the initial expressed sequence tags (EST) isolated from a hepatopancreatic cDNA library via PCR. The EsLecA cDNA contained a 480-bp open reading frame that encoded a putative 159-amino-acid protein, while EsLecG cDNA contained a 465-bp open reading frame that encoded a putative 154-amino-acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of carbohydrate recognition domains that were common among C-type lectin superfamilies. EsLecA and EsLecG mRNA expression in E. sinensis were (a) both detected in all tissues, including the hepatopancreas, gills, hemocytes, testis, accessory gland, ovary, muscle, stomach, intestine, heart, thoracic ganglia and brain, and (b) responsive in hepatopancreas, gill, hemocytes post-LPS immuno-challenge all appeared dramatically variation. Collectively, the data presented here demonstrate the successful isolation of two novel C-type lectins from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.

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.

Caspase and nm23: apoptosis genes linked to the antibacterial response of the Chinese mitten crab.

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), a commercially important and disease vulnerable aquaculture species. The capase and nm23 transcripts were detected in all examined crab tissue; furthermore, the expression is immuno-responsive and may be linked to immune functionality in crabs. 

Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab, Eriocheir sinensis.

Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin A (catA) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length catA cDNA (2200 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catA cDNA contained a 1398 bp open reading frame (ORF) that encoded a putative 465 amino acid (aa) protein. Comparisons with other reported vertebrate cathepsins sequences revealed percent identity range from 48 to 51%. CatA mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in gill and (b) responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 12 h post-injection. Collectively, data demonstrate the successful isolation of catA from the Chinese mitten crab, and its involvement 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.

Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP9) gene in the reproduction seasons of Chinese mitten crab, Eriocheir sinensis.

Fatty acid-binding proteins (FABPs), small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP9) was cloned based upon EST analysis of a testis cDNA library. The full length cDNA was 898 bp and encoded a 136 aa polypeptide that was highly homologous to related genes reported in shrimp. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP9 transcripts was widely distributed with high and detectable expression levels observed in intestine, ovary, testis and heart, while expression were comparable among hepatopancreas, hemolymphe, gills, muscle, stomach and brain. Real-time quantitative RT-PCR analysis revealed that Es-FABP9 expression in testis, hemolymphe, hepatopancreas and ovarian was dependent on the status of testis development. Evidence provided in the present report supports a role of Es-FABP9 in lipid transport during the period of rapid testis growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, testis, hemolymphe and ovarian in lipid nutrient absorption and utilization processes.

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.