Papers by Saengchan Senapin
Developmental & Comparative Immunology, 2015
Pacifastin is a recently classified family of serine proteinase inhibitors that play essential ro... more Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.
The Toll pathway is an evolutionarily conserved signaling pathway which exists in both vertebrate... more The Toll pathway is an evolutionarily conserved signaling pathway which exists in both vertebrates and invertebrates, implying an essential role in innate immunity. Additionally, proteins involved in the Toll pathway have been reported to be a target of viruses. Recently, the whiteleg shrimp Penaeus vannamei (Pv) Toll pathway has been shown to be targeted by white spot syndrome virus (WSSV). MyD88, an essential component of the Toll pathway also has been shown to be targeted by vaccinia virus and Hepatitis C virus in mammalian cells. Therefore, P. vannamei MyD88 might also be targeted by WSSV. To identify the potential interacting partners, a yeast two-hybrid screening was performed by screening PvMyD88 with WSSV library. After preliminary screening, results showed that PvMyD88 was able to interact with four WSSV proteins, WSSV164, WSSV189, WSSV454 and WSSV458. The results presented here provides preliminary data of the interactions between WSSV and the shrimp Toll pathway which ope...
Shrimp aquaculture is one of the most rapid growing businesses and is practiced worldwide, includ... more Shrimp aquaculture is one of the most rapid growing businesses and is practiced worldwide, including in Thailand. According to the statistics of the Food and Agriculture Organization (FAO) of the United Nations, marine and brackish water shrimp culture production expanded from less than 10,000 metric tons in 1970 to more than 3,000,000 metric tons in 2011. Among the cultivated shrimps, the most popular farmed shrimp is the Whiteleg shrimp (Penaeus vannamei) which accounts for 67% of the whole shrimp production. Each year, enormous losses come from disease outbreaks which are mainly from viruses. One of the most fatal shrimp diseases is the White spot syndrome virus (WSSV) which causes 90-100% mortality within 3-10 days. In order to dissect the functions of viral and host cell proteins involved in pathogenesis and defense mechanisms, protein interaction analysis was used. From the previous study of a genome-wide analysis of White spot syndrome virus showed that WSSV449 was similar to...
Developmental & Comparative Immunology, 2015
The viral responsive protein, PmHHAP, plays an important role in the control of hemocyte homeosta... more The viral responsive protein, PmHHAP, plays an important role in the control of hemocyte homeostasis in shrimps during viral infection. In this study, we further investigate the role of PmHHAP in the regulation of hemocyte apoptosis. RNA interference (RNAi) mediated gene silencing was used to suppress the PmHHAP expression and the change in hemocyte apoptosis was determined in the knockdown shrimp. Within circulating hemocytes, PmHHAP knockdown increased the number of annexin V-positive apoptotic cells and the combined caspase-3/-7 activity and induced the characteristic apoptotic DNA ladder. Furthermore, PmHHAP down-regulation was accompanied by significantly altered expression of apoptosis-related proteins including the effector caspases, PmCaspase and PmCasp. Yeast two-hybrid and co-immunoprecipitation assays showed that PmHHAP binds to the p20 domain of PmCasp. Moreover, the recombinant PmHHAP protein was able to reduce the caspase activity in the actinomycin D-treated hemocyte cells and rPmCasp-treated hemocyte cells. Taken together, our data indicate that PmHHAP regulates hemocyte homeostasis by inhibits apoptotic cell death through caspase activation.
The Journal of parasitology, 2014
Many species of fish in the world are infected with digenean trematodes belonging to the genera C... more Many species of fish in the world are infected with digenean trematodes belonging to the genera Clinostomum and Euclinostomum. In this study, metacercariae, identified as Euclinostomum sp. on the basis of morphological characteristics and molecular data, were taken from 3 osphronemid fish- Trichopsis vittata , Trichopsis schalleri, and Betta imbellis, in Thailand. Phylogenetic analysis based on a mitochondrial gene (cytochrome c oxidase subunit I) and 2 nuclear genes (18S rDNA and ITS-internal transcribed spacer) of these Euclinostomum parasites indicated a clear distinction from those belonging to the Clinostomum genus. These are the first records of partial mitochondrial and nuclear DNA sequences of an Euclinostomum sp.
PLOS ONE, 2015
Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune respons... more Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gramnegative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system.
Meta Gene, 2015
Air-breathing fish species of the genus Trichopsis have been reported in Cambodia, Lao PDR, Indon... more Air-breathing fish species of the genus Trichopsis have been reported in Cambodia, Lao PDR, Indonesia, Malaysia, Singapore, Thailand and Vietnam. It is only in Thailand that all three recognized species (Trichopsis vittata, Trichopsis schalleri and Trichopsis pumila), as judged by distinct external features, are found. Cambodia and Lao PDR harbor two species each. The present work involves first-time DNA sequencing and analysis based on mitochondrial (COI) and nuclear (RAG1) DNA of numerous specimens of these species and specimens of a controversial Phetchaburi (Thailand) fish population with a mixed outward appearance. In addition to confirming the morphologically clear-cut taxonomic division of the three fish species, our DNA results show that whereas the T. pumila populations form one single species, there are cryptic species in the T. vittata and T. schalleri populations and possibly a new one in the latter. Members of the putative Phetchaburi fish population have been proven to be hybrids between T. pumila and T. vittata. In addition, a new the phylogenetic tree indicating ancestral relationships is also
Developmental & Comparative Immunology, 2015
Here we show that knockdown of laminin receptor (Lamr) with PvLamr dsRNA in the whiteleg shrimp P... more Here we show that knockdown of laminin receptor (Lamr) with PvLamr dsRNA in the whiteleg shrimp Penaeus (Litopenaeus) vannamei (Pv) caused a dramatic reduction specifically in hyaline hemocytes prior to death. Since apoptosis was not detected in hemocytes or hematopoietic cells, other possible causes of hemocyte loss were investigated. Reports that suppression of crustacean hematopoietic factor (CHF)-like protein or hemocyte homeostasis-associated protein (HHAP) also reduced shrimp hemocyte counts led us to carry out yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays to test for interactions between Lamr and Pv homologues to these proteins (PvCHF-like and PvHHAP). The assays revealed that Lamr bound to both these homologues, but that the homologues did not bind to each other. Subsequent RT-PCR assays confirmed that PvLamr dsRNA injection significantly reduced expression levels for both PvCHF-like and PvHHAP genes. Further work is needed to determine how interaction among these three proteins can regulate shrimp hemocyte homeostasis.
Meta Gene, 2014
In Thailand, there are currently five recognized species members of the bubble-nesting Betta genu... more In Thailand, there are currently five recognized species members of the bubble-nesting Betta genus, namely Betta splendens, B. smaragdina, B. imbellis, B. mahachaiensis and B. siamorientalis. In 2010, we indicated the possibility, based on COI barcoding evidence, that there might be two additional species, albeit cryptic, related to the type-locality B. smaragdina in some provinces in the northeast of Thailand. In the present study, after a more extensive survey of the northeast, and phylogenetic analyses based on COI and ITS1 sequences, the B. smaragdina group may be composed of at least 3 cryptic species members. The phylogenetic positions of these B. smaragdina group members in the bubble-nesting bettas' tree together with those of their congeners have been consolidated by better DNA sequence quality and phylogenetic analyses. With a better supported tree, the species statuses of B. siamorientalis and the Cambodian B. smaragdina-like fish, B. stiktos, are also confirmed.
Virus Research, 2006
Viruses are a major cause of production losses in the world shrimp-farming industry. Despite this... more Viruses are a major cause of production losses in the world shrimp-farming industry. Despite this, little is known about viral-host interactions in shrimp due in part to the lack of continuous shrimp cell lines. Here, the yeast two-hybrid assay system was employed to study interactions between three Taura syndrome viral capsid proteins (VP1-VP3) and proteins from a cDNA library of the black tiger shrimp Penaeus monodon. VP1 interacted with -actin, elongation factor 1␣ (EF1␣), lysozyme (Lys) and laminin receptor/ribosomal protein p40 (Lamr/p40) containing a putative palindromic laminin binding region LMWWML. VP2 interacted with -actin and EF1␣, while VP3 bound to the same proteins as VP1 except for Lamr/p40. In vitro pull-down assays confirmed these interactions. The most interesting interaction was specific binding between VP1 and Lamr/p40 since Lamr/p40 has been identified as the mammalian cell receptor for several arthropod-borne viruses (arboviruses). A search of mosquito vector and Drosophila sequences at available databases revealed the presence of putative Lamr/p40 proteins with high homology to the Lamr/p40 from shrimp.
The Journal of General and Applied Microbiology, 1999
Previously established PCR amplification and Southern hybridization procedures were developed for... more Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.
The Journal of General and Applied Microbiology, 1998
Aquaculture Research, 2015
Protein Expression and Purification, 2013
HtrA2 is an apoptosis-activating protein to enhance the apoptotic process by preventing the forma... more HtrA2 is an apoptosis-activating protein to enhance the apoptotic process by preventing the formation of the IAP-caspase complex, thus freeing caspase to trigger the apoptosis pathway. Here, we presented the full-length sequence of HtrA2 from the black tiger shrimp (PmHtrA2). The full-length PmHtrA2 transcript was 1403 bp with a 1338 bp open reading frame encoding 445 amino acids and contains 5 conserved domains, namely, a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a serine protease domain, and a PDZ domain normally found in HtrA2 proteins of other organisms. The mature form of PmHtrA2 was cloned into the pET28b(+) and pET15bThio vectors, and the expression of the protein was compared in Escherichia coli BL21 DE3 and BL21 RIL (CodonPlus) strains. Greater quantities of stable and soluble PmHtrA2 were expressed as a thioredoxin fusion protein in E. coli BL21 RIL (CodonPlus) cells with the recombinant pET15bThio-PmHtrA2 vector. To investigate the expression of PmHtrA2 in shrimp, the crude proteins from several shrimp tissues were imaged by Western blot using the polyclonal antibody specific to the recombinant PmHtrA2. The expression of the 47-kDa immature PmHtrA2 protein could be detected in shrimp lysates from the gills and the muscles. This study is the first to report the full-length PmHtrA2 gene, which is functional in black tiger shrimp and will lead to more focused studies on the function of PmHtrA2 in apoptosis regulation during immune responses to viral infection in shrimp.
Fish & Shellfish Immunology, 2015
Apoptosis is an essential immune response to protect invertebrates from virus infected cells. In ... more Apoptosis is an essential immune response to protect invertebrates from virus infected cells. In shrimp, virus infection has been reported to induce apoptosis. Macrobrachium rosenbergii (Mr) was considered to be a disease-resistant host when compared to penaeid shrimps. Caspase-3 was classified as an executioner caspase which played a key role in virus-induced apoptosis. In this study, an effector caspase gene of M. rosenbergii (Mrcasp) was cloned and characterized. The open reading frame (ORF) of Mrcasp was 957 nucleotide encoding 318 amino acid with a deduced molecular mass of 35.87 kDa. RT-PCR analysis showed the presence of Mrcasp in all examined tissues. The phylogenetic tree indicated that Mrcasp was closely related with caspase 3 of shrimp. The functions of the Mrcasp, B2 and capsid proteins of M. rosenbergii nodavirus (MrNV) were assayed in Sf-9 cells. The results showed that Mrcasp induce apoptotic morphology cells; however, capsid protein of MrNV could inhibit apoptotic cells whereas B2 could neither induce nor inhibit apoptotic cells by DAPI staining. The protein interaction between Mrcasp and viral MrNV structure revealed that Mrcasp did not bind to B2 or capsid protein whereas B2 and capsid proteins could bind directly to each other. This study reported a novel sequence of a full-length Mrcasp and its functional studies indicated that Mrcasp could induce apoptotic cells. Our data is the first report demonstrating the direct proteineprotein interaction between capsid protein and B2 protein of MrNV.
Molecular and Cellular Probes, 2010
In this work, a probe-free, multiplex RT-PCR was combined with high resolution melt (HRM) analysi... more In this work, a probe-free, multiplex RT-PCR was combined with high resolution melt (HRM) analysis for the simultaneous detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) infection in the freshwater prawn Macrobrachium rosenbergii. This first application of HRM multiplex RT-PCR in shrimp reveals a new potential for rapid and sensitive detection of multiple pathogens. In addition, sequence variation in XSV could be observed from the high resolution melt peaks, as confirmed by sequence analysis. In 19 field samples of the freshwater prawn M. rosenbergii the technique revealed samples negative for both viruses, positive for both viruses or positive for MrNV alone. No sample was found positive for XSV alone. Comparison of these results to those obtained using the same samples in analysis by traditional nested RT-PCR combined with gel electrophoresis revealed that HRM multiplex RT-PCR was more sensitive. Thus, the latter technique allows for rapid and sensitive, simultaneous detection of MrNV and XSV and also has the potential to be adapted for simultaneous detection of other mixed viral infections in shrimp.
Antiviral Research, 2014
In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathog... more In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24h and 48h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter's neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins.
Journal of Negative Results in BioMedicine, 2011
Background: Infectious myonecrosis virus (IMNV) disease outbreaks in cultivated whiteleg shrimp P... more Background: Infectious myonecrosis virus (IMNV) disease outbreaks in cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei are characterized by gross signs of whitened abdominal muscles and by slow mortality reaching up to 70%. In 2006 the first disease outbreaks caused by IMNV in Asia occurred in Indonesia. Since then rumours have periodically circulated about IMNV disease outbreaks in other Asian countries. Our findings indicate that these are false rumours. Findings: Our continual testing by nested RT-PCR of shrimp samples suspected of IMNV infection from various Asian countries since 2006 has yielded negative results, except for samples from Indonesia. Our results are supported by the lack of official reports of IMNV outbreaks since January 2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndrome that also commonly causes muscle whitening in stressed whiteleg shrimp. Thus, we suspect that most of the false rumours in Asia about IMNV outside of Indonesia have resulted because of muscle cramp syndrome. Conclusions: Results from continual testing of suspected IMNV outbreaks in Asian countries other than Indonesia since 2006 and the lack of official country reports of IMNV outbreaks since January 2007, indicate that rumours of IMNV outbreaks in Asian countries outside of Indonesia are false. We suspect that confusion has arisen because muscle cramp syndrome causes similar signs of whitened tail muscles in whiteleg shrimp.
Virus Research, 2010
Field specimens of post-larvae of the giant freshwater prawn (Macrobrachium rosenbergii) from Tha... more Field specimens of post-larvae of the giant freshwater prawn (Macrobrachium rosenbergii) from Thailand showed hepatopancreatic tubule epithelial cells that contained central, eosinophilic inclusions within enlarged nuclei and marginated chromatin. These inclusions resembled those produced by some baculoviruses prior to formation of occlusion bodies that enclose virions in a polyhedrin protein matrix. By electron microscopy, the intranuclear inclusions contained bacilliform, enveloped virions (approximately 327 ± 29 nm × 87 ± 12 nm) with evenly dense, linear nucleocapsids surrounded by trilaminar envelopes with lateral pockets containing nucleoproteinic filaments. In some cases, these were accompanied by moderately electron dense, spherical particles of approximately 20 nm diameter resembling polyhedrin subunits of occlusion bodies (OB) of a bacilliform virus of the black tiger shrimp Penaeus monodon, previously reported from Thailand and called monodon baculovirus (MBV). It is currently listed by the International Committee on Taxonomy of viruses as Penaeus monodon nucleopolyhedrovirus (PemoNPV). Two polymerase chain reaction (PCR) assays for MBV gave positive results with DNA extracts prepared from M. rosenbergii samples using the hot phenol technique. One of these assays targeted the polyhedrin gene of MBV to which the resulting amplicon showed 100% sequence identity. Presence of the Penaeus monodon virus polyhedrin gene was confirmed by in situ hybridization assays and by positive immunohistochemical reactions in one sample batch. The data revealed that MBV can be found but may rarely produce polyhedrin occlusion bodies in M. rosenbergii.
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Papers by Saengchan Senapin