Crayfish Hemocytes Research Articles

MicroRNA transcriptome analysis for elucidating the immune mechanism of the redclaw crayfish Cherax quadricarinatus under Decapod iridescent virus 1 infection

Redclaw crayfish (Cherax quadricarinatus) is a large, tropical freshwater crustacean species with considerable potential of commercial production. In recent years, infection with DIV1 in redclaw crayfish is being reported in aquaculture industries, causing high mortality and huge economic losses. However, many characteristics of this virus, including pathogenesis, transmission mechanism, and host immunity, remain largely unknown.MicroRNAs are known to play important roles in numerous biological processes, and many microRNAs are reported to be involved in the regulation of immune responses. In this study, nine-small RNA libraries were constructed using hemocytes of redclaw crayfish to characterize the differentially expressed miRNAs (DE-miRNAs) at 24 and 48 h postinfection (hpi). A total of 14 and 22 DE-miRNAs were identified in response to DIV1 infection at 24 and 48 hpi, respectively. Further, functional annotation of the predicted host target genes using GO and KEGG pathway enrichment analyses indicated that relevant biological processes and signal pathways underwent miRNA-mediated regulation after DIV1 infection. Our results enhanced the understanding of the mechanisms of miRNA-mediated regulation of immune responses under DIV1 infection in crustaceans.

Методика подсчета общего числа гемоцитов австралийского красноклешневого рака

The optimal formula for calculating the total number of hemocytes (HTN) of the Australian red-claw crayfish (Cherax quadricarinatus) has been established, which allows optimizing the time of counting HTN without re-ducing accuracy. Methodical recommendations on preparing a hemolymph sample, HTN calculation and differentiated calculation of hemocytes are given. It was found out that the differences in HTN are minimal when using different formulas, which is con-firmed by the analysis of variance F = 0.011, p = 0.998, the average values of HTN range from 2 384 to 2 427 cell/µl. Conducting multiple comparisons also demonstrated that the differences between the formulas are minimal: the significance levels between the formulas were p = 0.99. The optimal formula for the Australian red-claw crayfish is: HTN in 1 µl = N • 10, where N is the total number of hemocytes in 25 large squares of Gorjaev’s chamber. The counting chamber should be clean, since when even small dust particles are found on the grid, hemocyte clusters can form and impede counting, or the result of the calculation will be incorrect, because the distribution 
 of hemocytes on the grid will not be uniform. After filling the chamber with hemolymph, it is recommended to wait 
 1-1.5 minutes so that the movement of the hemolymph stops and the hemocytes stop moving. It is recommended to use an anticoagulant, which prevents the rapid decay of cells and promotes their uniform distribution on the grid of the chamber without developing clots (clusters) that affect the accuracy of counting. Working with the whole hemolymph implies the speed and accuracy of the researcher's actions, since it quickly coagulates and forms clusters of hemocytes. In order to avoid confusion when counting cells located on the border between squares, Egorov’s rule is applied. The optimal increase for counting is a 400-fold increase.

Hemocytes of the Australian Red Claw Crayfish (Cherax quadricarinatus): Morphology and Hemogram

Hemocytes of the Australian Red Claw Crayfish (Cherax quadricarinatus): Morphology and Hemogram

Crayfish hemocytes develop along the granular cell lineage

Despite the central role of hemocytes in crustacean immunity, the process of hemocyte differentiation and maturation remains unclear. In some decapods, it has been proposed that the two main types of hemocytes, granular cells (GCs) and semigranular cells (SGCs), differentiate along separate lineages. However, our current findings challenge this model. By tracking newly produced hemocytes and transplanted cells, we demonstrate that almost all the circulating hemocytes of crayfish belong to the GC lineage. SGCs and GCs may represent hemocytes of different developmental stages rather than two types of fully differentiated cells. Hemocyte precursors produced by progenitor cells differentiate in the hematopoietic tissue (HPT) for 3 ~ 4 days. Immature hemocytes are released from HPT in the form of SGCs and take 1 ~ 3 months to mature in the circulation. GCs represent the terminal stage of development. They can survive for as long as 2 months. The changes in the expression pattern of marker genes during GC differentiation support our conclusions. Further analysis of hemocyte phagocytosis indicates the existence of functionally different subpopulations. These findings may reshape our understanding of crustacean hematopoiesis and may lead to reconsideration of the roles and relationship of circulating hemocytes.

Characterization and function analysis of a Kazal-type serine proteinase inhibitor in the red claw crayfish Cherax quadricarinatus

Kazal-type serine proteinase inhibitors (KPIs) function in physiological and immunological processes requiring proteinase action. In the present study, the first Cherax quadricarinatus KPI gene (designated CqKPI) was identified and characterized. The open reading frame of CqKPI contains 405 nucleotides and encodes a protein of 134 amino acids. CqKPI has two Kazal domains comprising 44 amino acid residues with the conserved amino acid sequence C-X3-C-X7-C-X6-Y-X3-C-X6-C-X12-C. Each Kazal domain has six conserved cysteine residues, which can form a structural conformation of three pairs of disulfide bonds stabilizing the Kazal domain. CqKPI exhibited high similarity with previously identified KPIs from crayfish hemocytes. The results of tissue distribution showed that CqKPI had the highest expression level in hemocytes, and this was in agreement with phylogenic relationships. Recombinant CqKPI (rCqKPI) was heterologously expressed in Escherichia coli and purified for further study. The proteinase inhibition assays suggested that rCqKPI could potently inhibit elastase and weakly inhibit trypsin, subtilisin A, and proteinase K, but not α-chymotrypsin. It can firmly bind to Bacillus hwajinpoensis, Staphylococcus aureus, and Vibrio parahaemolyticus, with weak binding to Candida albicans. In addition, CqKPI inhibited bacterial secretory proteinase activity and inhibited the growth of B. hwajinpoensis and C. albicans. These data suggest that CqKPI might be involved in anti-bacterial immunity, acting as an inhibitor of the proteinase cascade in the resistance to invasion of pathogens.

Molecular characterization and expression analysis of dopa decarboxylase involved in the antibacterial innate immunity of the freshwater crayfish, Procambarus clarkii

Dopa decarboxylase (DDC) is responsible for the synthesis of dopamine, which acts as an important modulator in the nervous systems of vertebrates and invertebrates. Recent studies have indicated that DDC also plays crucial roles in the insect innate immune system. However, the functions of DDC in immunomodulation in crustaceans have not been thoroughly elucidated to date. In this study, a new full-length cDNA of the DDC protein was identified from red swamp crayfish, Procambarus clarkii (named Pc-ddc). The ORF of Pc-ddc encoded 474 amino acids, which possessed a 377-amino-acid domain. Pc-ddc was expressed at a relatively high level in the hemocytes and gills of crayfish. This protein was expressed at a relatively low level in the hepatopancreas and intestine. The expression level of Pc-ddc was clearly upregulated in hemocytes, hepatopancreas, gills, and intestine tissues after challenge with S. aureus or E. ictaluri. The results of the enzyme catalysis assay showed that the enzyme catalysis activity of rPc-DDC was 35 ± 2.8 ng h−1 mg−1 (n = 3). In addition, the results of the mimetic crayfish hemocytes encapsulation assay showed that the encapsulation rate of beads coated with rPc-DDC was clearly increased. The results of the bacterial binding assay showed that rPc-DDC strongly binds to S. aureus and E. ictaluri. Finally, when Pc-ddc was knocked down, the number of surviving crayfish clearly decreased after S. aureus or E. ictaluri was injected. All of these results indicate that Pc-DDC is an important immunomodulating enzyme in the neuroendocrine-immune (NEI) system of crayfish.

Cloning and Analysis of the Multiple Transcriptomes of Serine Protease Homologs in Crayfish (Procambarus clarkii)

ABSTRACTFive different serine protease homologs (SPH) transcripts presumably or possibly resulting from alternative splicing were cloned from the hemocytes of crayfish (Procambarus clarkii) in this paper. Although different deletions of cDNA of SPH-2 and SPH-4 were found in the 5ʹ untranslated regions, they shared the same open reading frame and encoded a 424 amino acids protein with a calculated molecular weight of 45.84 kDa compared with SPH-5. The predicted cutting site of the signal peptide was located between Ala22 and Glu23; a clip domain and a trypsin-like serine protease domain were located in the N-terminal and the C-terminal, respectively. Large deletions were found in the SPH-1 and SPH-3. Both of them lacked the clip domain. The 22 amino acids signal peptide existed in the SPH-1 coding protein, and a low complexity region (LCR) was formed in the N-terminal of it. The deduced protein of SPH-1 contained 358 amino acids with a molecular weight of 38.80 kDa. There was only one trypsin-like serine protease domain found in the C-terminal of the SPH-3 coding protein. The deduced protein of SPH-3 contained 250 amino acids with a molecular weight of 26.90 kDa. The amino acid Ser (S) of the catalytic triad in trypsin-like serine protease domain of the proteins analyzed in this paper was replaced by Gly (G), suggesting that the SPH-1, SPH-2, SPH-3, SPH-4, and SPH-5 were serine protease homologs.

Application of Crayfish Astacus Leptodactylus for the Analysis of Water Genotoxicity in the Lake Sevan Basin

Genotoxicity of water pollution at three sites of the Lake Sevan Basin was estimated by using crayfish Astacus leptodactylus as bioindicator. Levels of DNA damage in hemocytes of crayfish were analyzed using the comet assay. A significant rise of the level of DNA damage in terms of parameters of the percentage DNA content in the comet tail and the Olive tail moment was detected in crayfish inhabiting waters near the villages of Artanish and Tsapatakh (Armenia) as compared to the crayfish from the Masrik River Estuary. A significant positive correlation between the parameter of the Olive tail moment in crayfish and the content of Al, Fe, Cu, and Mo in water has been discovered. The obtained data indicate that crayfish Astacus leptodactylus is a sensitive bioindicator for monitoring of aquatic ecosystems.

Involvement of Serotonin in crayfish hematopoiesis

Serotonin (5-HT) is a conserved monoamine neurotransmitter that has several physiological functions both in vertebrates and invertebrates. In addition to its well-known function in the central nervous system, 5-HT also participates in peripheral system. However, in crustaceans, the knowledge about peripheral functions of 5-HT is limited. In this study, a role of 5-HT in hematopoiesis in crayfish, Pacifastacus leniusculus, was investigated. The presence of 5-HT in crayfish plasma and the effect of 5-HT injection on hemocyte number were examined. The effects of 5-HT on hematopoietic tissue (HPT) cell proliferation and secretion of the hematopoietic cytokine, astakine 1 (Ast 1) were determined in vitro. The results from this study suggest that 5-HT has no direct effect on HPT cell proliferation, but it participates in crayfish hematopoiesis through stimulating Ast 1 cytokine release from crayfish hemocytes, and thereby affects release of new hemocytes into the circulation.

ITRAQ-based quantitative proteomic analysis of Procambarus clakii hemocytes during Spiroplasma eriocheiris infection

As a new-found aquaculture pathogen, Spiroplasma eriocheiris, has resulted in inconceivable economic losses in aquaculture. In the infection of S. eriocheiris, the Procambarus clakii hemocytes have indicated to be major target cells. What was designed to examine in our study is the hemocytes' immune response at the protein levels. Before the pathogen was injected and after 192 h of post-injection, the differential proteomes of the crayfish hemocytes were analyzed immediately by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatogramphytandem mass spectrometry (LC-MS/MS). This research had identified a total of 285 differentially expressed proteins. Eighty-three and 202 proteins were up-regulated and down-regulated, respectively, caused by the S. eriocheiris infection. Up-regulated proteins included alpha-2-macroglobulin (α2M), vitellogenin, ferritin, etc. Down-regulated proteins, involved with serine protease, peroxiredoxin 6, 14-3-3-like protein, C-type lectin, cdc42 homolog precursor, etc. The prophenoloxidase-activating system, antimicrobial action involved in the immune responses of P. clarkii is considered to be damaged due to S. eriocheiris infection. The present work could lay the foundation for future research on the proteins related to the susceptibility/resistance of P. clarkii to S. eriocheiris. In addition, it is helpful for our understanding molecular mechanism of disease processes in crayfishes.

Different roles of crayfish hemocytes in the uptake of foreign particles

Crustacean hemocytes are known to remove invading pathogens by phagocytosis. In this study, we investigated how the semigranular cells (SGCs) and granular cells (GCs) of crayfish Cherax quadricarinatus participated in this process. By injecting the animals with excessive amounts of fluorescent microspheres (FMs), we showed that only a small portion of the circulating hemocytes were phagocytic cells, and they took up FMs in a size-dependent manner. The 0.2 μm FMs were internalized almost entirely by SGCs, while GCs and SGCs both contributed to the uptake of 2 μm FMs. Further analysis of the hemocytes from the animals injected with a mixture of FMs suggested that there were a subpopulation of SGCs specifically ingesting 0.2 μm FMs. The size-dependent manner was also applied to biological particles. Escherichia coli was internalized by both SGCs and GCs, whereas white spot syndrome virus (WSSV) was mostly ingested by SGCs. However, the bacterial cells were rapidly taken and cleared from the circulation by the hemocytes, while the WSSV virions were gradually internalized and remained in the cells for a relatively longer period of time. These findings provide basic information of the phagocytic hemocytes of crayfish and how they respond to different foreign particles.

PI3K signaling pathways modulated white spot syndrome virus (WSSV) replication in Procambarus clarkii

The PI3K/AKT signaling pathway is commonly exploited to regulate viral replication and affect the fate of infected cells. In the present study, a PI3K-specific inhibitor (LY294002) was employed to pretreat crayfish to evaluate the effects of PI3K/AKT signaling pathway in WSSV replication. The results showed that the WSSV copy numbers in crayfish pretreated with LY294002 were significantly lower than those in Tris-HCl pretreatment crayfish on the sixth and tenth day after WSSV infection. In semigranular cells, the apoptosis rates were up-regulated on the third day post-WSSV infection, and a significantly lower proportion of apoptosis cells were observed in LY294002-pretreatment group. The expression level of Bax, Bax inhibitor-1 and lectin mRNA in haemocytes of crayfish were increased after WSSV infection. After the secondary stimulation with Tris-HCl, the Bax expression level in LY294002-pretreatment crayfish was significantly higher than that of crayfish pretreated with Tris-HCl on the third or sixth day, but the Toll and lectin mRNA expression decreased significantly on the third, sixth and tenth day. The Bax mRNA expression levels in LY294002-WSSV group were significantly higher than those in Tris-HCl-WSSV group on the third and tenth day. The Bax inhibitor-1 mRNA expression levels in LY294002-WSSV group were significantly lower than those in Tris-HCl-WSSV crayfish on the third day. These results together indicated that the hosts PI3K/AKT signaling pathway play positive roles in WSSV replication through the balance between host cell apoptois and innate immune responses. This information is helpful to further understand the role of PI3K/AKT signaling pathway on WSSV replication in Decapoda crustaceans.

Antibacterial activity of hemocyanin from red swamp crayfish (Procambarus clarkii)

Hemocyanins (HMC): the copper-containing respiratory proteins present in invertebrate hemolymph, which plays many essential roles in the immune system. Currently, little is known about the HMC domains of Procambarus clarkii (P. clarkii) and their function in antimicrobial immune response. In this present study, we comparatively studied the expression pattern of native PcHMC with the three recombinant proteins of variable domains of crayfish hemocyanin (PcHMC-N, N-terminal domain of hemocyanin; PcHMC-T, tyrosinase domain of hemocyanin; PcHMC-C, C-terminal domain of hemocyanin). The results showed that three purified recombinant proteins had a strong binding to various bacteria and lipopolysaccharides that further highly agglutinated. The HMCs recombinant proteins showed strong antibacterial activity against V. parahaemolyticus and S. aureus by bacterial growth inhibition, phenoloxidase (PO) and phagocytosis assays. Specifically, rPcHMC1-T and rPcHMC1-C inhibited both the bacteria efficiently, rPcHMC1-T was highly upregulated the PO activity than the other recombinant proteins. Whereas, recombinant proteins pretreated crayfish hemocytes participated in phagocytosis activity, rPcHMC1-N and rPcHMC1-C proteins had a profound effect than the rPcHMC1-T on S. aureus and V. parahaemolyticus phagocytosis. The crayfish hemocyanin domains clearly exhibited antibacterial and phagocytic activities against both the bacteria, suggesting that its variable domains of hemocyanin have the different function on specific pathogen during the assault of pathogens.

BAX, a novel cell pro-apoptotic protein, involved in hemocytes early antiviral immune response in fresh water crayfish, Procambarus clarkii.

Apoptosis plays an important role in various biological processes and acts as a host defending mechanism by which infected cells are eliminated to restrict the virus propagation scale. Bax is a crucial pro-apoptotic protein, which mediates the release of cytochrome c from mitochondrion to cytosol in mammalian. However, its role in invertebrate is still obscure. Here, a novel pro-apoptotic protein gene was identified from hemocytes of red swamp crayfish. There was a Bcl-2 domain in the C-terminus of Pc-Bax, which possessed 497amino acids residues. And an important transmembrane region existed in the C-terminus of Pc-Bax, which implied that Pc-Bax located in mitochondrial membrane. Besides, Pc-Bax was expressed at a relative high level in hemocytes, and a relative low expression levels in hepatopancreas, gills, and intestine. In hemocytes, Pc-Bax transcript was rapidly up-regulated from 12h to 36h after WSSV infection. And there was the same trend for Pc-Bax protein expression level in hemocytes after WSSV infection. Results of qRT-PCR testing for VP28 gene showed WSSV replication was obviously enhanced after Pc-Bax knockdown. Meantime, hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group. Taken together, these results revealed that crayfish hemocytes apoptosis scale was enhanced to suppress WSSV replication by up-regulating Bax protein expression level after WSSV infection.

Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from red claw crayfish, Cherax quadricarinatus

Abstract A pattern recognition protein (PRP), lipopolysaccharide and β-1,3-glucan binding protein (LGBP), cDNA was cloned from the hemocytes of red claw crayfish, Cherax quadricarinatus by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1456 bp has an open reading frame of 1095 bp encoding a protein of 364 amino acids including a 15 amino acid signal peptide. The calculated molecular mass of the mature protein (349 aa) was 39.92 kDa with an estimated pI of 4.50. Sequence comparison of the deduced amino acid sequence of C. quadricarinatus LGBP (named as CqLGBP) showed a high identity of 82%, 77%, 76% and 74% with Procambarus clarkii LGBP, Homarus gammanus BGBP, Penaeus monodon BGBP and Litopenaeus stylirostlis LGBP, respectively. The CqLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. The recombinant CqLGBP were expressed in Escherichia coli BL21 with pGEX-6P-2 expression vector. The titer of rabbit anti-CqLGBP serum was above 1:12,800. Microorganism binding assay showed CqLGBP binds two kinds of crustacean pathogens Spiroplasma eriocheiris and Aeromonas hydrophila. Quantitative RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with S. eriocheiris and A. hydrophila. A significant enhancement of CqLGBP transcriptions was apparent post-injection in response to bacterial infection compared to the control group. These data should be helpful to better understand the function of CqLGBP in the crayfish immune system.

Crayfish hematopoietic tissue cells but not hemocytes are permissive for white spot syndrome virus replication

Hemocytes are the major immune cells of crustaceans which are believed to be essential for the pathogenesis of white spot syndrome virus (WSSV) infection. Crayfish hemocytes and hematopoietic tissue (HPT) cells have been found to be susceptible to WSSV infection, but the procedure of WSSV infection to both cell types has not yet been carefully investigated. In this study, we analyzed the infection and proliferation of WSSV in crayfish hemocytes as well as HPT cells in detail through transmission electronic microscopy (TEM). The results showed that WSSV could enter both hemocytes and HPT cells through endocytosis, but the production of progeny virus was only achieved in HPT cells. Further investigation demonstrated that although WSSV could transcribe its genes in both cell types, viral genome replication and structural protein expression were unsuccessful in hemocytes, which may be responsible for the failure of progeny production. Therefore, we propose that both hemocytes and HPT cells are susceptible to WSSV infection but only HPT cells are permissive to WSSV replication. These findings will extend our knowledge of the interaction between WSSV and the host immune system.

Identification of a c-Jun homolog from Litopenaeus vannamei as a downstream substrate of JNK in response to WSSV infection

c-Jun, a major substrate of c-Jun N-terminal kinase (JNK), participates in regulating gene transcription in response to various stimuli, including cytokines, stress signals, bacterial and viral infection. Results from our previous studies suggested that Litopenaeus vannamei JNK (LvJNK) could be utilized by white spot syndrome virus (WSSV) to facilitate viral replication and gene expression. In this article, a c-Jun homolog from Litopenaeus vannamei (designated as Lvc-Jun) was cloned and its role in WSSV infection was studied. Sequence analysis displayed that Lvc-Jun was a novel homolog of c-Jun family, which contained characteristic Jun and basic leucine zipper (bZIP) domains, and two conserved serine phosphorylation sites (Ser49/59). Semi-quantitative RT-PCR analysis showed that Lvc-Jun mRNAs were expressed in all examined tissues. Further investigation determined that Lvc-Jun was located in the nucleus through self-interaction and its phosphorylation levels could be reduced by JNK inhibitor, suggesting that Lvc-Jun could be regulated by LvJNK through phosphorylation and function as a transcription regulator in a homodimer. During the process of WSSV infection, the transcription levels of Lvc-Jun were up-regulated associating with the raising expression and phosphorylation levels of its protein. Moreover, TPA (12-O-tetradecanoylphorbol-13-acetate), a potent inducer of c-Jun, could remarkably promote viral immediate-early gene wsv069 transcription in crayfish hemocytes. Conclusively, our results provided experimental evidences that Lvc-Jun was engaged in WSSV infection and further implied that JNK-c-Jun signaling pathway might be important for WSSV replication and viral gene expression.

The superoxide dismutase from red claw crayfish, Cherax quadricarinatus: molecular cloning and characterization analysis.

In the present study, an extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of red claw crayfish, Cherax quadricarinatus. The open reading frame (ORF) of ecCuZnSOD is 498 bp and encodes a 166 amino acids (aa) protein, whereas the ORF of mtMnSOD is 654 bp and encodes a 218 aa protein. The amino acid sequences of C. quadricarinatus ecCuZnSOD and mtMnSOD showed high similarities with those of ecCuZnSODs and mtMnSODs of other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of C. quadricarinatus were highly expressed in hepatopancreas, hemocytes, intestine, and gill; low transcript levels were seen in other tissues (heart, muscle, and nerve). The immune responses of ecCuZnSOD and mtMnSOD were studied following inoculation with Spiroplasma eriocheiris and Aeromonas hydrophila. After S. eriocheiris or A. hydrophila challenge, mRNA transcription of ecCuZnSOD and mtMnSOD in hemocytes and gill was upregulated. mRNA transcription of ecCuZnSOD in the hepatopancreas was also upregulated after S. eriocheiris or A. hydrophila inoculation. mtMnSOD in hepatopancreas was upregulated after A. hydrophila inoculation, whereas this was down-regulated after S. eriocheiris challenge. After S. eriocheiris and A. hydrophila challenge, total SOD activity and CuZnSOD activity both increased compared to control group. The results showed that these SODs from C. quadricarinatus likely play an important role in protecting some tissues from reactive oxygen intermediates produced during challenge from S. eriocheiris and A. hydrophila.

The characterization, expression and activity analysis of superoxide dismutases (SODs) from Procambarus clarkii

The superoxide dismutases (SODs) are important antioxidant enzymes to remove excess biologically reactive oxygen intermediates, and thus avoid adverse conditions. In this study, an extracellular copper–zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of the freshwater crayfish, Procambarus clarkii. The open reading frame (ORF) of ecCuZnSOD is 612bp encoding a 204 amino acid (aa) protein with a 24 aa signal peptide, whereas the ORF of mtMnSOD is 654bp and encodes a 218 aa protein with a 20 aa mitochondrial-targeting sequence in the N-terminus. P. clarkii ecCuZnSOD and mtMnSOD proteins showed high similarity with ecCuZnSODs and mtMnSODs from other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of P. clarkii were highly expressed in hepatopancreas, hemocytes and gill; lower expression levels were seen in intestine, stomach, nerve, heart and muscle. The mRNA expressions of three SODs of P. clarkii (ecCuZnSOD, mtMnSOD and cytosolic MnSOD (cytMnSOD)) were studied after Spiroplasma eriocheiris and Aeromonas hydrophila stimulations. After S. eriocheiris challenge, the mRNAs of three SODs in hemocytes and gill were up-regulated, and ecCuZnSOD also increased in the hepatopancreas. However, the mtMnSOD and cytosolic MnSOD in the hepatopancreas were down-regulated. After A. hydrophila challenge, the mRNA expressions of three SODs in hepatopancreas and hemocytes were up-regulated and down-regulated in gill. The total SOD activity and CuZnSOD activity were increased compared to the control group with both S. eriocheiris and A. hydrophila challenges. The results may indicate that the SODs of P. clarkii have important roles in innate immune responses against S. eriocheiris and A. hydrophila.

Crustacean hyperglycemic hormone precursor transcripts in the hemocytes of the crayfish Procambarus clarkii: Novel sequence characteristics relating to gene splicing pattern and transcript stability

It was demonstrated in a previous study (Wu et al., 2012b) that crustacean hyperglycemic hormone (CHH) gene is expressed in the hemocyte of Procambarus clarkii. In the present study, 2 additional cDNAs (CHH2-L and tCHH2) from the hemocyte and a CHH gene (CHH2) from the abdominal muscle of the same species were cloned. Analyses of the cDNA and genomic sequences suggested that, similar to other previously reported CHH genes, 2 precursor transcripts (CHH2 and CHH2-L) would be derived from CHH2 gene through a process of RNA alternative splicing, and CHH2 and CHH2-L each encode a precursor containing a signal peptide, a CHH precursor-related peptide, and a mature peptide. Further, tCHH2 sequence consists of exon I, exon II, and a truncated segment of intron II of CHH2 gene, followed by a previously unknown 3′sequence. It is suggested that, because the truncation disrupts the highly conserved RNA splice acceptor site, the truncated segment is retained within tCHH2, resulting in encoding a precursor containing the typical precursor components except the mature peptide is truncated with only 40 residues. In addition, unlike 2 other previously identified transcripts (referred to as CHH1 and CHH1-L), CHH2-L, CHH2, tCHH2 contain in the 3′-UTRs 3–5 AU-rich elements (AREs). The data showed that multiple CHH genes are expressed in crayfish hemocytes. Novel sequence characteristics of the transcripts result in an RNA splicing pattern that yields a transcript (tCHH2) encoding a precursor with an atypical truncated mature peptide and possibly leads to a different expression dynamics of the precursors encoded by the ARE-containing transcripts.