Interaction of pathogenic vibrio bacteria with the blood clot of the Pacific white shrimp, Litopenaeus vannamei

Author Posting. © Marine Biological Laboratory, 2014. This article is posted here by permission of Marine Biological Laboratory for personal use, not for redistribution. The definitive version was published in Biological Bulletin 226 (2014): 102-110.In addition to its roles in hemostasis and wound repair, the blood clot plays an underappreciated role in innate immunity, where the established clot serves as a barrier to microbial penetration into the internal milieu and where the early clot entraps and immobilizes microbes that have entered wounds to the integuments. In this report we document the behavior of the pathogenic gram-negative bacterium Vibrio harveyi that has been entrapped in the fabric of the extracellular blood clot of one of its target organisms, the Pacific white shrimp, Litopenaeus vannamei. The freshly entrapped bacteria are held tightly by the clot, losing even Brownian motility, but by 1 h post-entrapment, a fraction of the bacteria have established small domains of fibrinolysis that enlarge progressively, enabling bacteria to escape from the clot's embrace. Escape is dependent on the actions of both serine- and metallo-proteases released from the bacterial cells.This research was financially supported by a student fellowship for Vorrapon Chaikeeratisak from the Royal Golden Jubilee Ph.D. program under the Thailand Research Fund (TRF) and by grant 0344360 from the National Science Foundation (PBA)

Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

BACKGROUND: Viral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time. RESULTS: The two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR. CONCLUSIONS: This work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14-3-3 protein epsilon. The information is useful for understanding the immune system of shrimp against pathogenic bacteria

Sequence and expression analysis of HSP70 family genes in Artemia franciscana

Thus far, only one gene from the heat shock protein 70 (HSP70) family has been identified in Artemia franciscana. Here, we used the draft Artemia transcriptome database to search for other genes in the HSP70 family. Four novel HSP70 genes were identified and designated heat shock cognate 70 (HSC70), heat shock 70 kDa cognate 5 (HSC70-5), lmmunoglobulin heavy-chain binding protein (BIP), and hypoxia up-regulated protein I (HYOUI). For each of these genes, we obtained nucleotide and deduced amino acid sequences, and reconstructed a phylogenetic tree. Expression analysis revealed that in the juvenile state, the transcription of HSP70 and HSC70 was significantly (P 0.05) induction. Gene expression analysis demonstrated that not all members of the HSP70 family are involved in the response to heat stress and selection and that especially altered expression of HSC70 plays a role in a population selected for increased thermotolerance

Population structure of tropical abalone (Haliotis asinina) in coastal waters of Thailand determined using microsatellite analysis

Abstract: Three partial genomic libraries were constructed from genomic DNA of the tropical abalone (Haliotis asinina) that was digested with AluI, vortexed/sonicated, and digested with mixed enzyme (AluI, HincII, and RsaI). The libraries yielded 0.02%, 0.42%, and 1.46% positive microsatellite-containing clones, respectively. Eleven clones each of perfect, imperfect, and compound microsatellites were isolated. Ten primer pairs (CUHas1-CUHas10) were analyzed to evaluate their polymorphic level. The numbers of alleles per locus, observed heterozygosity (H 0 ), and expected heterozygosity (H e ) ranged from 3 to 26 alleles, and varied between 0.27 and 0.85 and between 0.24 and 0.93, respectively. Three microsatellite loci (CUHas2, CUHas3, and CUHas8) were further used for examination of genetic diversity and differentiation of natural H. asinina in coastal waters of Thailand. Genetic variabilities in terms of the effective number of alleles (n e ), H 0 , and H e were higher in 2 samples from the Gulf of Thailand (n e = 9.37, 7.66; H 0 = 0.62, 0.78; and H e = 0.87, 0.86) than those of one sample (n e = 6.04; H 0 = 0.58; and H e = 0.62) derived from the Andaman Sea. Assessment of genetic heterogeneity, including allele frequency comparison and pairwise F ST analysis, indicated interpopulational differentiation, between natural H. asinina from the Gulf of Thailand and that from the Andaman Sea (P < 0.0001)

The scope of the crustacean immune system for disease control

The culture or wild capture of marine and freshwater shellfish, including crustaceans, is without doubt a key source of protein for a burgeoning world population. Historically the expansion of aquaculture has, however, been accompanied by the increased incidence of economically significant diseases, most notably of viral and bacterial origin. Since the late 1970s great progress has been made in our understanding of the generalized protostome innate immune system. Distinct pathways, pathogen receptor proteins and effector molecules have since been identified that are not ancestral or homologous to those of the deuterostomes, including vertebrates. Within the past decade progress has accelerated with the rapid characterisation of new classes of recognition proteins, immune effectors and regulatory pathways. This paper provides a broad overview of our current understanding of invertebrate immunology, taking the crustacean decapod immune system as its focus. Recent developments in the field are described briefly and their implications and potential considered. These advances offer fundamental new insights in our efforts to understand disease in cultured populations and also to develop knowledge of environmental effects on host/pathogen interactions within a fishery context. Of course, challenges do remain, including the lack of an immortal cell line and the limited publically-available genomic resources. These are considered in this review as priorities for future research effort. With the continued application of more insightful technologies, coupled with associated investment, it is expected that the speed at which some of these issues are resolved will accelerate

A transcriptome analysis of mitten crab testes (Eriocheir sinensis)

The identification of expressed genes involved in sexual precocity of the mitten crab (Eriocheir sinensis) is critical for a better understanding of its reproductive development. To this end, we constructed a cDNA library from the rapid developmental stage of testis of E. sinensis and sequenced 3,388 randomly picked clones. After processing, 2,990 high-quality expressed sequence tags (ESTs) were clustered into 2,415 unigenes including 307 contigs and 2,108 singlets, which were then compared to the NCBI non-redundant (nr) protein and nucleotide (nt) database for annotation with Blastx and Blastn, respectively. After further analysis, 922 unigenes were obtained with concrete annotations and 30 unigenes were found to have functions possibly related to the process of reproduction in male crabs – six transcripts relevant to spermatogenesis (especially Cyclin K and RecA homolog DMC1), two transcripts involved in nuclear protein transformation, two heat-shock protein genes, eleven transcription factor genes (a series of zinc-finger proteins), and nine cytoskeleton protein-related genes. Our results, besides providing valuable information related to crustacean reproduction, can also serve as a base for future studies of reproductive and developmental biology

False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination

<p>Abstract</p> <p>Background</p> <p>The entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies.</p> <p>Methods</p> <p>Mosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a <it>Plasmodium </it>specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked.</p> <p>Results</p> <p>Specimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>(Pv210 and Pv247). Two new vector species were identified for the region: <it>Anopheles pampanai </it>(<it>P. vivax</it>) and <it>Anopheles barbirostris </it>(<it>Plasmodium malariae</it>). In 88% (155/176) of the mosquitoes found positive with the <it>P. falciparum </it>CSP-ELISA, the presence of <it>Plasmodium </it>sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for <it>P. vivax </it>CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of <it>P. falciparum </it>was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens.</p> <p>Conclusion</p> <p>The CSP-ELISA can considerably overestimate the EIR, particularly for <it>P. falciparum </it>and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing <it>Plasmodium </it>specific PCR followed if possible by sequencing of the amplicons for <it>Plasmodium </it>species determination.</p