Identifying low salinity adaptation gene expression in the anterior and posterior gills of the mud crab (Scylla paramamosain) by transcriptomic analysis.

In the present study, BGISEQ-500 RNA-Seq technology was adopted to investigate how Scylla paramamosain adapts to salinity tolerance at the molecular level and explores changes in gene expression linked to salinity adaptation following exposure to both low salinity (5 ‰) and standard salinity (23 ‰) conditions. A total of 1100 and 520 differentially expressed genes (DEGs) were identified in the anterior and posterior gills, respectively, and their corresponding expression patterns were visualized in volcano plots and a heatmap. Further analysis highlighted significant enrichment of well-established gene functional categories and signaling pathways, including those what associated with cellular stress response, ion transport, energy metabolism, amino acid metabolism, H2O transport, and physiological stress compensation. We also selected key DEGs within the anterior and posterior gills that encode pivotal stress adaptation and tolerance modulators, including AQP, ABCA1, HSP 10, A35, CAg, NKA, VPA, CAc, and SPS. Interestingly, A35 in the gills might regulate osmolality by binding CHH in response to low salinity stress or serve as a mechanism for energy compensation. Taken together, our findings elucidated the intricate molecular mechanism employed by S. paramamosain for salinity adaptation, which involved distinct gene expression patterns in the anterior and posterior gills. These findings provide the foothold for subsequent investigations into salinity-responsive candidate genes and contribute to a deeper understanding of S. paramamosain's adaptation mechanisms in low-salinity surroundings, which is crucial for the development of low-salinity species cultivation and the establishment of a robust culture model.

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain.

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

Identifying sex-differential gene expression in the antennal gland of the swimming crab by transcriptomic analysis.

The antennal glands (AnGs) are recognized as an important organ that functions in ion regulation and excretion in decapods. Previously, many studies had explored this organ at the biochemical, physiological, and ultrastructural levels but had few molecular resources. In this study, the transcriptomes of the male and female AnGs of Portunus trituberculatus were sequenced using RNA sequencing (RNA-Seq) technology. Genes involved in osmoregulation and organic/inorganic solute transport were identified. This suggests that AnGs might be involved in these physiological functions as versatile organs. A total of 469 differentially expressed genes (DEGs) were further identified between male and female transcriptomes and found to be male-biased. Enrichment analysis showed that females were enriched in amino acid metabolism and males were enriched in nucleic acid metabolism. These results suggested differences in possible metabolic patterns between males and females. Furthermore, two transcription factors related to reproduction, namely AF4/FMR2 family members Lilli (Lilli) and Virilizer (Vir), were identified in DEGs. Lilli was found to be specifically expressed in the male AnGs, whereas Vir showed high expression levels in the female AnGs. The expression of up-regulated metabolism and sexual development-related genes in three males and six females was verified by qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our results suggest that although the AnG is a unified somatic tissue composed of individual cells, it still demonstrates distinct sex-specific expression patterns. These results provide foundational knowledge of the function and differences between male and female AnGs in P. trituberculatus.

Identification and characterization of expression profiles of neuropeptides and their GPCRs in the swimming crab, Portunus trituberculatus.

Neuropeptides and their G protein-coupled receptors (GPCRs) regulate multiple physiological processes. Currently, little is known about the identity of native neuropeptides and their receptors in Portunus trituberculatus. This study employed RNA-sequencing and reverse transcription-polymerase chain reaction (RT-PCR) techniques to identify neuropeptides and their receptors that might be involved in regulation of reproductive processes of P. trituberculatus. In the central nervous system transcriptome data, 47 neuropeptide transcripts were identified. In further analyses, the tissue expression profile of 32 putative neuropeptide-encoding transcripts was estimated. Results showed that the 32 transcripts were expressed in the central nervous system and 23 of them were expressed in the ovary. A total of 47 GPCR-encoding transcripts belonging to two classes were identified, including 39 encoding GPCR-A family and eight encoding GPCR-B family. In addition, we assessed the tissue expression profile of 33 GPCRs (27 GPCR-As and six GPCR-Bs) transcripts. These GPCRs were found to be widely expressed in different tissues. Similar to the expression profiles of neuropeptides, 20 of these putative GPCR-encoding transcripts were also detected in the ovary. This is the first study to establish the identify of neuropeptides and their GPCRs in P. trituberculatus, and provide information for further investigations into the effect of neuropeptides on the physiology and behavior of decapod crustaceans.

Identification of differentially abundant mRNA transcripts and autocrine/paracrine factors in oocytes and follicle cells of mud crabs.

The late vitellogenic stage of the mud crab is characterized by large and obvious follicle cells as well as an enlarged oocyte nucleus and a prominent germinal vesicle (GV). The aim of this study was evaluation of functions of oocytes and follicle cells during meiosis as well as at identifying associated ovarian autocrine/paracrine factors using comparative transcriptomics. The results from the KEGG pathway analysis indicated DNA replication, nucleotide excision repair, spliceosome and the ribosome pathways were highly associated with oocyte maturation across both transcriptomes. In addition, there was a larger abundance of mRNA transcripts for cell cycle-related genes in the oocyte, as well as cyclin A, cyclin B and CKS1B in the GV than at the time of germinal vesicle breakdown (GVBD). These findings indicate these cell cycle-related genes might be involved in GVBD induction. Results when there was localization of ligands and the respective receptors of VEGF, TGFß propeptide and BMP9/10 indicated these proteins might be autocrine/paracrine factors. Results from functional analysis of VEGF, TGFß propeptide and BMP9/10 in oocyte maturation using RNA interference revealed that these proteins might be involved in oocyte maturation by regulating cyclin abundance. This is the first study on the functions of VEGF in oocyte maturation in invertebrates.

The Chinese mitten crab genome provides insights into adaptive plasticity and developmental regulation.

The infraorder Brachyura (true or short-tailed crabs) represents a successful group of marine invertebrates yet with limited genomic resources. Here we report a chromosome-anchored reference genome and transcriptomes of the Chinese mitten crab Eriocheir sinensis, a catadromous crab and invasive species with wide environmental tolerance, strong osmoregulatory capacity and high fertility. We show the expansion of specific gene families in the crab, including F-ATPase, which enhances our knowledge on the adaptive plasticity of this successful invasive species. Our analysis of spatio-temporal transcriptomes and the genome of E. sinensis and other decapods shows that brachyurization development is associated with down-regulation of Hox genes at the megalopa stage when tail shortening occurs. A better understanding of the molecular mechanism regulating sexual development is achieved by integrated analysis of multiple omics. These genomic resources significantly expand the gene repertoire of Brachyura, and provide insights into the biology of this group, and Crustacea in general.

Large-scale lysine crotonylation analysis reveals its potential role in spermiogenesis in the Chinese mitten crab Eriocheir sinensis.

Lysine crotonylation (Kcr) is a recently-discovered type of post-translational modification. Although Kcr has been reported in many species, little is known about this process in crustaceans. In this study, pan anti-lysine crotonylation antibody enrichment and high-resolution liquid chromatogram-mass spectrometry analysis were employed to characterize Kcr in testis of the Chinese mitten crab Eriocheir sinensis testis. Overall, 2799 Kcr sites were identified on 908 proteins with 14 conserved motifs. Bioinformatics analysis showed that Kcr was predominant on proteins found in cytoplasm, mitochondria and nucleus, and those involved in ribosome, proteasome, carbon metabolism and protein processing in endoplasmic reticulum. In total, 83 up-regulated and 12 down-regulated non-histone crotonylated sites were identified during spermiogenesis. These differentially expressed proteins were enriched in protein processing in endoplasmic reticulum pathway during formation of acrosome. In contrast, histone Kcr associated with mammalian spermatogenesis. These results provide foundational knowledge on the role of non-histone Kcr in spermiogenesis of E. sinensis. SIGNIFICANCE: Lysine crotonylation (Kcr) is a recently-identified post-translational modification, and histone Kcr was found to associate with mammalian spermatogenesis. However, crotonylation of non-histone proteins has not been reported in spermatogenesis regulation. Further, there is no information on crotonylation in crustaceans. This study was the first large-scale Kcr proteome characterization in crustaceans. A total of 2799 Kcr sites on 908 proteins with 14 conserved motifs were identified from Eriocheir sinensis testis. Of which, 83 up-regulated and 12 down-regulated non-histone crotonylated sites were identified during spermiogenesis. Our results provide the basic information for further functional validation of Kcr proteins and revealed new roles of Kcr in spermiogenesis of E. sinensis.

Identification of Peptides and Their GPCRs in the Peppermint Shrimp Lysmata vittata, a Protandric Simultaneous Hermaphrodite Species.

Peptide hormones commonly binding with G-protein coupled receptors (GPCRs) achieve their function in reproduction. The peppermint shrimp Lysmata vittata popular in marine ornamental trade and is known to display protandric simultaneous hermaphrodite (PSH). Knowledge on reproductive biology of this commercial species is critical for resources management and aquaculture. This study employed Illumina sequencing and bioinformatics analysis to identify peptides and their candidate GPCRs from male phase (MP) and euhermaphrodite phase (EP) of L. vittata. A total of 61 peptide and 40 peptide GPCR transcripts derive from 44 peptide families and 13 peptide GPCR families were identified, respectively. Among them, insulin-like androgenic gland hormone and crustacean female sex hormone have two unique mature peptides, respectively, and their transcripts showed higher expression levels in MP than EP, which suggest that these sex differentiation hormones might be involved in sexual characters than spermatogenesis or vitellogenesis. Overall, the first study on identification of peptides and their GPCRs in the genus Lysmata extends our knowledge of peptidergic signaling in PSH species, and provides an important basis for development of aquaculture strategies.

Serotonergic Mechanisms of Oocyte Germinal Vesicle Breakdown in the Mud Crab, Scylla paramamosain.

The mechanism of serotonin (5-HT)-induced oocyte germinal vesicle breakdown (GVBD) in the mud crab, Scylla paramamosain, was investigated in this study. Histological staining showed that there were two meiotic arrests in oocyte, appearing at prophase I and metaphase I. This result indicated that meiosis I arrest at prophase I in S. paramamosain was similar to that of vertebrates, but meiosis II arrest at metaphase I was different from that of vertebrates. Resumption of oocytes arrest at meiosis prophase I could be induced by 5-HT rapidly within 5 min in S. paramamosain. We obtained the sequence of the 5-HT receptor type 1A (5-HTR1A ) from the NCBI database, and found that 5-HTR1A was expressed in oocytes and follicle cells. In addition, we found that an agonist 8-OH-DPAT which binds 5-HTR1A induced GVBD and an antagonist WAY100635 which inhibited 5-HT induced GVBD in S. paramamosain. This result showed that 5-HTR1A mediated the regulation of oocyte GVBD by 5-HT. To explore the functional mechanism of 5-HT in inducing oocyte GVBD, forskolin, a cAMP agonist was used. Results showed that, forskolin significantly blocked 5-HT-induced GVBD, and there was a negative correlation between GVBD rate and cAMP level. Our data indicate that there are two meiotic arrests in S. paramamosain, and the resumption of prophase I arrest can be induced by 5-HT, which binds to 5-HTR1A , and this process is mediated by cAMP, which acts as negative regulator via cAMP signaling pathway.

Identifying neuropeptide GPCRs in the mud crab, Scylla paramamosain, by combinatorial bioinformatics analysis.

Neuropeptides, ubiquitous signaling molecules, commonly achieve their signaling function via interaction with cell membrane-spanning G-protein coupled receptors (GPCRs). In recent years, in the midst of the rapid development of next-generation sequencing technology, the amount of available information on encoded neuropeptides and their GPCRs sequences have increased dramatically. The repertoire of neuropeptides has been determined in many crustaceans, including the commercially important mud crab, Scylla paramamosain; however, determination of GPCRs is known to be more difficult and usually requires in vitro binding tests. In this study, we adopted a combinatorial bioinformatics analysis to identify S. paramamosain neuropeptide GPCRs. A total of 65 assembled GPCR sequences were collected from the transcriptome database. Subsequently these GPCRs were identified by comparison to known neuropeptide GPCRs based on the sequence-similarity-based clustering and phylogenetic analysis, which showed that many of them are closely related to insect GPCR families. Of these GPCRs, most of them were detected in various tissues of the mud crab and some of them showed differential expression by gender, suggesting they are involved in different physiological processes, such as sex differentiation. By employing ligand-receptor binding tests, we demonstrated that the predicted crustacean cardioactive peptide (CCAP) receptor was activated by CCAP peptide in a dose-dependent manner. This is the first CCAP receptor that has been functionally defined in crustaceans. In summary, the present study shortlists candidate neuropeptide GPCRs for ligand-receptor binding tests, and provides information for subsequent future research on the neuropeptide/GPCR signaling pathway in S. paramamosain.

Inhibitory Role of the Mud Crab Short Neuropeptide F in Vitellogenesis and Oocyte Maturation via Autocrine/Paracrine Signaling.

Neuropeptides, in addition to their classical role in the nervous system, act on intraovarian factors to regulate reproductive functions in vertebrates. However, this function of neuropeptides has not been characterized in crustaceans. Short neuropeptide F (sNPF), a highly conserved invertebrate neuropeptide, has been reported to be involved in feeding, metabolism, and in differentiation processes including reproduction. Although sNPF and its receptor (sNPFR) have been detected in the ovary in different species, ovarian colocalization of sNPF/sNPFR has not been investigated. In this study, we identified Scylla paramamosain (mud crab) sNPF (Sp-sNPF) as an endogenous ligand for the S. paramamosain orphan G protein-coupled receptor NPY2R in mammalian cell line HEK293T. We designated this receptor as Sp-sNPFR. RNA in situ hybridization in pre-vitellogenic ovary and reverse transcription-PCR on isolated denuded oocytes and follicle layers showed that Sp-sNPF was exclusively localized to the follicle cells, whereas Sp-sNPFR was detected in both follicle cells and oocytes. We also found that Sp-sNPF partly suppressed spontaneous maturation of denuded oocytes and caused intracellular cAMP accumulation and Ca2+ mobilization. Moreover, injection of synthetic Sp-sNPF peptides inhibited the expression of vitellogenin and vitellogenin receptor genes in vivo. These combined results suggest for the first time that Sp-sNPF may have inhibitory functions in vitellogenesis and oocyte maturation possibly via the autocrine/paracrine pathway.

Voltage-dependent calcium channels in the neurosecretory cells of cerebral ganglia of the mud crab, Scylla paramamosain.

Voltage-dependent calcium channels (VDCCs) play a critical role in stimulus-secretion coupling in neurosecretory cells (NSCs). The crustacean cerebral ganglion plays a crucial role in neuromodulation and controls neuropeptide release. The present study used patch-clamp and Illumina sequencing techniques to investigate the potential features of VDCC in the cerebral ganglia of the mud crab (Scylla paramamosain). The electrophysiological characteristics of VDCC were analyzed in three types of NSCs with a patch clamp. The thresholds for activation of Ca channel current recorded from all the three types of NSCs were all above -40 mV, with peak amplitudes occurring around 0 mV. Therefore, it was concluded that the currents recorded in NSCs were mediated by high-voltage-activated Ca channels. Ca channel current densities in I type NSCs were significantly lower than those in II and III type NSCs. Four VDCC subunits derived from three transcripts were predicted from a transcriptome database of the cerebral ganglia. Among these transcripts, Cavα1, Cavß, and Cavα2/δ were predicted to encode 1674, 554, and 776 amino acids, respectively, and they shared conservative domains with VDCC subunits in other species. Overall, these findings provide an important basis for further studies on the neuroendocrine mechanisms in crustaceans.

Transcriptome profiling of claw muscle of the mud crab (Scylla paramamosain) at different fattening stages.

In crustaceans, muscle growth and development is complicated, and to date substantial knowledge gaps exist. In this study, the claw muscle, hepatopancreas and nervous tissue of the mud crab (Scylla paramamosain) were collected at three fattening stages for sequence by the Illumina sequencing. A total of 127.87 Gb clean data with no less than 3.94 Gb generated for each sample and the cycleQ30 percentages were more than 86.13% for all samples. De Bruijn assembly of these clean data produced 94,853 unigenes, thereinto, 50,059 unigenes were found in claw muscle. A total of 121 differentially expressed genes (DEGs) were revealed in claw muscle from the three fattening stages with a Padj value < 0.01, including 63 genes with annotation. Functional annotation and enrichment analysis showed that the DEGs clusters represented the predominant gene catalog with roles in biochemical processes (glycolysis, phosphorylation and regulation of transcription), molecular function (ATP binding, 6-phosphofructokinase activity, and sequence-specific DNA binding) and cellular component (6-phosphofructokinase complex, plasma membrane, and integral component of membrane). qRT-PCR was employed to further validate certain DEGs. Single nucleotide polymorphism (SNP) analysis obtained 159,322, 125,963 and 166,279 potential SNPs from the muscle transcriptome at stage B, stage C and stage D, respectively. In addition, there were sixteen neuropeptide transcripts being predicted in the claw muscle. The present study provides a comprehensive transcriptome of claw muscle of S. paramamosain during fattening, providing a basis for screening the functional genes that may affect muscle growth of S. paramamosain.

Vitellogenin2: spermatozoon specificity and immunoprotection in mud crabs.

As the precursor of vitellin (Vn), vitellogenin (Vg) has initially been considered as a female-specific protein involved in vitellogenesis, while it was also present in males induced by hormones or organs manipulation. Distinct from vtg1 we previously found in female mud crab Scylla paramamosain, vtg2 was intriguingly detected in male testis under normal physiological conditions in this study. Sequence analysis showed that vtg2 and vtg1 were actually two isoforms of Vg caused by different types of alternative splicing. PCR and in situ hybridization analysis revealed that vtg2 was localized only in the testicular spermatozoa, while Vn was detected in both the spermatozoa of the testis and seminal vesicle. Therefore, we speculated that Vn was initially translated in testicular spermatozoa, then migrated with spermatozoa, and finally stored in the seminal vesicle, where spermatozoa gradually accomplished maturation. We presumed that vtg2/Vn might act as an immune-relevant molecule in the male reproduction system. In the subsequent experiment, the expression of vtg2/Vn in testis was significantly induced in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) injection at both transcriptional and translational levels. In the light of the results presented above, we deemed that vtg2/Vn is a novel candidate of immune-relevant molecules involved in immunoprotection during the spermatozoon maturation, and this research helps to open a new avenue for further exploring the role of Vg.

The retinoid X receptor from mud crab: new insights into its roles in ovarian development and related signaling pathway.

In arthropods, retinoid X receptor (RXR) is a highly conserved nuclear hormone receptor. By forming a heterodimeric complex with the ecdysone receptor (EcR), RXR is known to be vital importance for various physiological processes. However, in comparison to EcR, the RXR signaling pathway and its roles in crustacean reproduction are poorly understood. In the present study, the RXR mRNA was detected in the ovarian follicular cells of mud crab Scylla paramamosain (SpRXR) and during ovarian maturation, its expression level was found to increase significantly. In vitro experiment showed that both SpRXR and vitellogenin (SpVg) mRNA in the ovarian explants were significantly induced by 20-hydroxyecdysone (20E) but not methyl farnesoate (MF). However, differing from the in vitro experiment, injection of MF in in vivo experiment significantly stimulated the expressions of SpRXR and SpVg in female crabs at early vitellogenic stage, but the ecdysone and insect juvenile hormone (JH) signaling pathway genes were not induced. The results together suggest that both MF and SpRXR play significant roles in regulating the expression of SpVg and ovarian development of S. paramamosain through their own specific signaling pathway rather than sharing with the ecdysone or the insect JH.

The mechanism of regulation of ovarian maturation by red pigment concentrating hormone in the mud crab Scylla paramamosain.

In this study a full-length cDNA (Sp-RPCH) was cloned from the eyestalk ganglia of the mud crab Scylla paramamosain. Sp-RPCH is 660 base pairs in length and its open reading frame encodes a precursor that is predicted to be processed into a 25-residue signal peptide, a mature red pigment concentrating hormone (RPCH, an octapeptide), and a 75-residue precursor-related peptide. Phylogenetic analysis indicates that it clusters with other crustacean RPCHs and belongs to the adipokinetic hormone/RPCH peptide superfamily. Sp-RPCH gene expression was detected, using an end-point polymerase chain reaction (PCR), not only in the eyestalk ganglia but also in the brain and thoracic ganglia. Quantified using a real-time PCR, Sp-RPCH gene expression levels in the three tissues fluctuated along a cycle of ovarian maturation, with the levels progressively increased from stages I to IV, after which the expression levels decreased (although they remained significantly higher than stage I levels) when the ovary reached the mature stage (stage V). It was demonstrated using a patch clamp analysis that synthetic RPCH was able to evoke a Ca(2+) current in dissociated brain neurons and synthetic RPCH significantly increased the mean oocyte diameter of the ovarian tissues co-cultured with the eyestalk ganglia, brain, or thoracic ganglia; the stimulatory effect of RPCH was absent when the nervous tissues were not included in the ovarian incubation. Animals administrated with RPCH had significantly higher levels of gonad-somatic index, hepatopancreas-somatic index, and vitellogenin gene expression, when compared to control animals receiving a saline injection. The combined results clearly show that RPCH is involved in ovarian maturation in the mud crab; the stimulatory effects of RPCH are likely mediated by its actions on the release from the nervous tissues of factor(s) that directly regulate vitellogenesis in the ovary and hepatopancreas.

Neuropeptides in the cerebral ganglia of the mud crab, Scylla paramamosain: transcriptomic analysis and expression profiles during vitellogenesis.

Neuropeptides play a critical role in regulating animal reproduction. In vertebrates, GnRH, GnIH and kisspeptin are the key neuropeptide hormones of the reproductive axis, however, the reproductive axis for invertebrates is vague. Knowledge on ovarian development of the mud crab, Scylla paramamosain, is critical for aquaculture and resources management of the commercially important species. This study employed Illumina sequencing, reverse transcription-polymerase chain reaction and quantitative real-time PCR techniques to identify neuropeptides that may be involved in ovarian development of S. paramamosain. A total of 32 neuropeptide transcripts from two dozen neuropeptide families, 100 distinct mature peptides were predicted from the transcriptome data of female S. paramamosain cerebral ganglia. Among them, two families, i.e. GSEFLamide and WXXXRamide, were first identified from the cerebral ganglia of crustaceans. Of these neuropeptides, 21 transcripts of interest were selected for further confirmation and all of them were detected in the cerebral ganglia, as well as in other nervous tissues and the ovary. Most of them also had differential expression in the cerebral ganglia during various vitellogenic stages, suggesting their likely involvement in regulating vitellogenesis and ovarian maturation. Overall, these findings provide an important basis for subsequent studies on peptide function in reproduction of S. paramamosain.

Immune responses of prophenoloxidase in the mud crab Scylla paramamosain against Vibrio alginolyticus infection: in vivo and in vitro gene silencing evidence.

Phenoloxidase (PO) plays an important role in arthropod melanization. In the present study, a proPO gene was obtained from the mud crab Scylla paramamosain, then we localized the proPO mRNA in hemocytes and detected the expression of proPO after bacterial challenge. In vivo and in vitro gene silencing mediated by dsRNA was also used to investigate the function of proPO in innate immune. The full-length of the proPO cDNA was 2600 bp and the predicted ORF encoded a protein of 673 amino acids with a predicted molecular mass of 77.3 kDa. The deduced amino acid and the main functional domain of proPO shared a high similarity to the mud crab Scylla serrata. In situ hybridization assay showed that the proPO mRNA was localized in the granular and semi-granular cells. The expression level of proPO in hemocytes showed a clear time-dependent pattern during the 96 h course after stimulated by Vibrio alginolyticus. In this study, high expression levels were observed at 3, 12, 24 and 48 h, respectively and the highest expression level was observed at 12 h, and this suggested that proPO was induced by bacteria and involved in immune response. In vivo proPO and GFP dsRNA treatment experiments showed that, proPO mRNA transcript was reduced to 39%, but the PO activity showed no significant difference (P > 0.05). Results indicated that the expression of proPO could be inhibited by dsRNA, and the enzyme activity may be influenced by incomplete knockdown of proPO, or hemocyanin, and other proPO isoforms as well. In vitro proPO-silenced experiments showed that the levels of proPO were decreased by 36%, 64% and 77% at 8, 16 and 32 h, respectively. Meanwhile, the quantity of bacteria was significantly larger in proPO dsRNA treatment than that in control at 3 h, calculated by 4,6-diamino-2-phenylindole staining (P < 0.01). These data demonstrated that the proPO gene plays an important role in the control of systemic bacterial infections and could help us to elucidate the defense role of the proPO-activating system in crabs. In addition, in vitro gene silencing operation mediated by dsRNA was expected to be a new tool for investigating the function of genes in crustaceans in the case of lacking cell line.