Vibrio areninigrae as a pathogenic bacterium in a crustacean.

The occurrence of infectious diseases poses a significant threat to the aquaculture industry worldwide. Therefore, characterization of potentially harmful pathogens is one of the most important strategies to control disease outbreaks. In the present study, we investigated for the first time the pathogenicity of two Vibrio species, Vibrio metschnikovii, a foodborne pathogen that causes fatalities in humans, and Vibrio areninigrae, a bacteria isolated from black sand in Korea, using a crustacean model, the signal crayfish Pacifastacus leniusculus. Mortality challenges indicated that injection of V. metschnikovii (108 CFU/crayfish) has a mortality percentage of 22% in crayfish. In contrast, injection of P. leniusculus with 108 or 107 CFU of V. areninigrae resulted in 100% mortality within one and two days post-injection, respectively. V. areninigrae was successfully re-isolated from hepatopancreas of infected crayfish and caused 100% mortality when reinjected into new healthy crayfish. As a consequence of this infection, histopathological analysis revealed nodule formation in crayfish hepatopancreas, heart, and gills, as well as sloughed cells inside hepatopancreatic tubules and atrophy. Moreover, extracellular crude products (ECP's) were obtained from V. areninigrae in order to investigate putative virulence factors. In vivo challenges with ECP's caused >90% mortalities within the first 24 h. In vitro challenges with ECP's of hemocytes induced cytotoxicity of hemocytes within the first hour of exposure. These findings represent the first report that V. areninigrae is a highly pathogenic bacterium that can cause disease in crustaceans. On the contrary, V. metschnikovii could not represent a threat for freshwater crayfish.

Correction: Caspase-1-Like Regulation of the proPO-System and Role of ppA and Caspase-1-Like Cleaved Peptides from proPO in Innate Immunity.

[This corrects the article DOI: 10.1371/journal.ppat.1004059.].

Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity.

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

Astakine 2--the dark knight linking melatonin to circadian regulation in crustaceans.

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.

Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus.

The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam), encodes 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV) infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.

A mammalian like interleukin-1 receptor-associated kinase 4 (IRAK-4), a TIR signaling mediator in intestinal innate immunity of black tiger shrimp (Penaeus monodon).

Interleukin-1 receptor associated kinase-4 (IRAK-4) has been identified as a central signal transduction mediator of the Toll-like receptor (TLR) and Toll/interleukin-1 receptor (TIR) pathways in vertebrate innate immunity. An IRAK-4 homologue was cloned from the black tiger shrimp (Penaeus monodon) (PmIRAK-4) and it shares domains and structures with other IRAK-4s. It was found to be mainly expressed in the hemocytes and midgut but also to a lower extent in several other tissues in shrimp. The PmIRAK-4 responded to bacterial infection in the intestine by an enhancement of its expression level. These results indicate that PmIRAK-4 may play a role at least in the intestinal innate immunity of P. monodon.

Bacterial community associated with the intestinal tract of P. monodon in commercial farms.

The potentially important roles of intestinal bacteria on immune response, disease resistance, and nutrition for the black tiger shrimp Penaeus monodon have been increasingly investigated. However, so far, little is known about the intestinal bacterial community of the shrimp in the commercial aquaculture settings. In this study, the intestinal bacterial communities of juvenile P. monodon (70 individuals) from eight commercial farms in Thailand were examined using 16S rDNA PCR-DGGE, and seven 16S rDNA clone libraries from representative DGGE profiles were constructed. Bacteria in the γ-Proteobacteria class were the only common bacteria group found in the intestinal tracts of shrimp from all farms. The dominant bacterial genera in the intestinal population of each shrimp varied among different farms, and these genera were Vibrio, Photobacterium, Aeromonas, or Propionigenium (phylum Fusobacteria). Other commonly found genera included Actinomyces, Anaerobaculum, Halospirulina, Pseudomonas, Mycoplasma, and Shewanella. Twelve phyla of bacteria including Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, Cyanobacteria, Tenericutes, Deinococcus-Thermus, Planctomycetes, Spirochaetes, Synergistetes, Thermotogae, and Verrucomicrobia were represented in the sequences. Additionally, strictly anaerobic bacteria such as Propionigenium and Fusibacter were found. These intestinal bacterial communities varied significantly among different commercial farms and were distinct from their rearing water. The results provide descriptive structures of the intestinal bacterial communities of P. monodon in commercial farms, which can further be applied to areas of research on the immunity, disease resistance, and nutrition of shrimp to improve aquaculture of the black tiger shrimp.

An ancient cytokine, astakine, mediates circadian regulation of invertebrate hematopoiesis.

Invertebrate circulating hemocytes are key players in the innate immune defense and their continuous renewal from hematopoietic tissues is tightly regulated in crustaceans by astakine, a new family of cytokines sharing a prokineticin (PROK) domain. In vertebrates, brain PROKs function as transmitters of circadian rhythms and we present evidence that hemocyte release from hematopoietic tissues in crayfish is under circadian regulation, a direct result of rhythmic expression of astakine. We demonstrate that the observed variation in astakine expression has an impact on innate immunity assessed as susceptibility to a pathogenic Pseudomonas species. These findings enlighten the importance of comparing immune responses at fixed times not to neglect circadian regulation of innate immunity. Moreover, our results entail an evolutionary conserved function for prokineticins as mediators of circadian rhythm, and for the first time show a role for this domain in circadian regulation of hematopoiesis that may have implications also in vertebrates.

A gC1qR prevents white spot syndrome virus replication in the freshwater crayfish Pacifastacus leniusculus.

The gC1qR/p32 protein is a multiple receptor for several proteins and pathogens. We cloned a gC1qR homologue in a crustacean, Pacifastacus leniusculus, and analyzed the expression of P. leniusculus C1qR (PlgC1qR) in various tissues. The gC1qR/p32 transcript was significantly enhanced by white spot syndrome virus (WSSV) infection 6 h after viral infection both in vitro in a hematopoietic tissue cell culture (Hpt) and in vivo compared to appropriate controls. Moreover, PlgC1qR silencing in both the Hpt cell culture and live crayfish enhanced the WSSV replication. In addition, by making a recombinant PlgC1qR protein we could show that if this recombinant protein was injected in a crayfish, Pacifastacus leniusculus, followed by injection of WSSV, this significantly reduced viral replication in vivo. Furthermore, if the recombinant PlgC1qR was incubated with Hpt cells and then WSSV was added, this also reduced viral replication. These experiments clearly demonstrate that recombinant PlgC1qR reduce WSSV replication both in vivo and in vitro. The results from a far-Western overlay and glutathione S-transferase pull-down assays showed that PlgC1qR could bind to VP15, VP26, and VP28. Altogether, these results demonstrate a role for PlgC1qR in antiviral activity against WSSV.

Expression of immune-related genes in one phase of embryonic development of freshwater crayfish, Pacifastacus leniusculus.

Crayfish do not have larval stage as other crustacean such as penaeid shrimp they spawn their eggs until hatching and what hatches out from the eggs are miniature crayfish known as juveniles. In order to address the question whether immune genes are initially expressed during the embryo development in the egg stage, the expression of some immune-related genes: prophenoloxidase (proPO), peroxinectin, hemocyanin, anti-lipopolysaccharide factor (ALF), plcrustin, astakine-1, 2 and transglutaminase (TGase) were determined in the middle phase of crayfish embryo development. Furthermore, immune challenge was used to determine the immune response of eggs by immersing them in a solution of the highly pathogenic bacterium Aeromonas hydrophila. Semi-quantitative RT-PCR analysis showed that all tested genes are present except proPO in this phase of crayfish embryo development and none of the genes tested changed their expression following immersion in A. hydrophila. The proPO transcript has been reported from hemocytes in crustaceans and it plays crucial roles in crustacean immune response. This may indicate that the development of immune-competent hemocytes in this stage of crayfish embryo is not completed and the egg shell as such plays an important role as a shield in protecting the embryo from bacteria and maybe also other pathogens.

Cloning and characterization of a melanization inhibition protein (PmMIP) of the black tiger shrimp, Penaeus monodon.

Melanization is an important component of the innate immune responses in invertebrates and it is essential for defense against invading microorganism. Melanin formation, which is a result of activation of the so called prophenoloxidase activating system, needs to be controlled due to the dangerous effects of quinones and melanin which are produced during the process of melanization. Here, a cDNA for a melanization inhibition protein (MIP), named PmMIP, was identified from the black tiger shrimp, Penaeus monodon by RT-PCR using degenerated oligonucleotide primers and RACE-PCR. The complete sequence significantly matched MIP of the freshwater crayfish Pacifastacus leniusculus (PlMIP). PmMIP contains an N-terminal signal peptide and a fibrinogen related domain (FReD). RT-PCR was applied to examine the expression profiles of PmMIP in various tissues of juvenile P. monodon. PmMIP was expressed in all examined tissues except hemocytes and at very low levels in hepatopancreas and ovaries. The expression of this gene was very low during the larval stages and hardly present in egg and at the nauplius stage. A time-course expression analysis of PmMIP upon Vibrio harveyi challenge at protein levels in plasma was determined. The result shows that MIP protein in plasma was induced at 6 h and disappeared at 12 and 24 h and then the protein reappeared at 48 and 72 h post injection. These results suggest that upon bacterial infection the PmMIP protein is first released from tissues into hemolymph and then degraded to allow melanization to occur for fighting against bacteria.

In vitro effects on bacterial growth of phenoloxidase reaction products.

An active phenoloxidase preparation from the freshwater crayfish Pacifastacus leniusculus exhibited a strong antibacterial effect in vitro on the bacteria Aeromonas hydrophila, Escherichia coli, Streptococcus pneumoniae whereas a weaker but still significant effect against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus. In most cases reduction of bacterial growth was stronger when dopamine was used as substrate as compared to L-dopa. The effect on bacteria was abolished if no substrate was available for the phenoloxidase or in the presence of the phenoloxidase inhibitor phenylthiourea.

Crustacean immunity.

This chapter provides are view of recent progress in the elucidation of innate immune mechanisms in crustaceans. Mainly due to the importance of crustacean aquaculture interest in this field is large and the subject for extensive research efforts. Here, we provide detailed data on the molecular characterisation of lectins, antiviral reactions, hemocyte formation and differentiation and on the regulation of innate immune pathways.

Antiviral immunity in crustaceans.

Viral diseases of shrimp have caused negative effects on the economy in several countries in Asia, South America and America, where they have numerous shrimp culture industries. The studies on the immunity of shrimp and other crustaceans have mainly focused on general aspects of immunity and as a consequence little is known about the antiviral responses in crustaceans. The aim of this review is to update recent knowledge of innate immunity against viral infections in crustaceans. Several antiviral molecules have been isolated and characterized recently from decapods. Characterization and identification of these molecules might provide a promising strategy for protection and treatment of these viral diseases. In addition dsRNA-induced antiviral immunity is also included.

A highly virulent pathogen, Aeromonas hydrophila, from the freshwater crayfish Pacifastacus leniusculus.

Aeromonas spp. are characteristic bacteria of freshwaters and many of them can be components of the bacterial flora of aquatic animals and may become pathogens on animals including humans. In this study Aeromonas hydrophila was isolated from the freshwater crayfish, Pacifastacus leniusculus, and was found to be a highly pathogenic bacterium among many isolated bacteria. Mortality reached 100% within 6h when 200 microl of 1.24 x 10(7)CFU/ml was applied by injection. Histopathological studies of moribund crayfish showed that extensive necrotic nuclei and clump-infiltrated hemocytes were found in observed tissues including gill, heart, hepatopancreas and the circulatory system. To verify how crayfish are susceptible to this bacterium, crude extracellular products (ECPs) obtained from culture supernatant of A. hydrophila was studied either in vivo or in vitro. ECPs (200 microl) were able to kill crayfish by injection. In an in vitro study, ECPs induced cytotoxicity of hemocytes as well as hematopoietic cells in a dose- and time-dependent manner after 30 min post inoculation. Two genes coding for endotoxins were also found in this isolate of A. hydrophila. This indicates that the bacterial endotoxins are the causative agents of crayfish mortality. Moreover, the effect of temperature on the infectivity of A. hydrophila to crayfish was also studied. At 4 degrees C, all crayfish survived, whereas at 20 degrees C the animals died rapidly after bacterial challenge. At this low temperature A. hydrophila did not replicate or replicated at a very low degree and hence crayfish could probably mount effective cellular reactions towards A. hydrophila.

Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacus leniusculus: characterization and expression pattern.

A 14 amino acid residues proline/arginine-rich antibacterial peptide designated as astacidin 2 was purified and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. Astacidin 2 has a broad range of antibacterial activity against both Gram-positive and Gram-negative bacteria. The primary sequence of astacidin 2 is RPRPNYRPRPIYRP with an amidated C-terminal and the molecular mass is 1838Da determined by mass spectrometry. Furthermore, the cDNA of three different crustin antibacterial homologs were isolated from a crayfish hemocyte EST library. RT-PCR was used to analyze the expression of the genes coding for astacidin 2 and P. leniusculus crustins (Plcrustin) 1-3 after bacterial challenge. The expression of Plcrustin1 was upregulated in both hemocytes and hematopoietic tissue after challenge with Gram-negative Escherichia coli or Acinetobacter ssp. non pathogenic bacteria as well as by a Gram negative crayfish pathogenic bacterium (Aeromonas hydrophila). The PlCrustin3 transcript was only upregulated after inoculation with the non-pathogenic Acinetobacter ssp. while there was no change in expression of Plcrustin2 or astacidin 2 following a bacterial challenge.

Characterization and function of a tachylectin 5-like immune molecule in Penaeus monodon.

Tachylectin5A and its homolog, tachylectin5B both contain a fibrinogen-related domain (FReD) and have been studied in horseshoe crabs, Tachypleus tridentatus and Carcinoscorpius rotundicauda and shown to be involved in host defense. Here, we demonstrate the presence of tachylectin5-like genes in shrimp, Penaeus monodon, designated as Penlectin5-1 (PL5-1) and Penlectin5-2 (PL5-2), which both contain a signal peptide and a single FReD with an acetyl group and a calcium binding sites and they are both structurally similar to horseshoe crab tachylectin/carcinolectin5. The PL5-1and PL5-2 transcript were expressed in various shrimp tissues in normal shrimp, and their expression was upregulated in tissues such as hemocytes and hindgut following challenge with pathogenic Vibrio harveyi. The PL5-2 protein was detected in various tissues as well as in cell-free hemolymph. The biological function of the PL5-2 protein is to recognize some Gram-positive and Gram-negative bacteria regardless whether they are non-pathogenic or pathogenic. They have hemagglutination activity on human erythrocyte and bacterial agglutination activity to both Gram negative and Gram positive bacteria. Possible binding sites of PL5-2 to bacteria could be at the N-acetyl moiety of the GlcNAc-MurNAc cell wall of the peptidoglycan since the binding could be inhibited by GlcNAc or GalNAC. The presence of PL5-2 protein in both circulating hemolymph and intestine, where host and microbes are usually interacting, may suggest that the physiological function of shrimp tachylectin-like proteins is to recognize and bind to invading bacteria to immobilize and entrap these microbes and subsequently clear them from circulation and the host body, and probably to control and maintain the normal flora in the intestine.

Involvement of a tachylectin-like gene and its protein in pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in the shrimp, Penaeus monodon.

A shrimp disease, the so-called acute hepatopancreatic necrosis disease (AHPND) is caused by a specific strain of Vibrio parahaemolyticus (VP) and it has resulted in significant losses to the global shrimp farming industry. In our previous study, three of tachylectin-like genes were cloned and characterized from the intestine of Penaeus monodon, designated as Penlectin5-1 (PL5-1), Penlectin5-2 (PL5-2) and Penlectin5-3 (PL5-3). These three genes all contain fibrinogen-related domain (FReD). The expression level of PL5-1, PL5-2 and PL5-3 was elevated in the stomach after oral administration with AHPND-causing V. parahaemolyticus 3HP (VP3HP). A polyclonal antibody to PL5-2 was successfully produced in a rabbit using the purified recombinant PL5-2 as an immunogen, and this because only the predominant protein PL5-2 could be successfully purified from shrimp plasma by affinity chromatography using a N-Acetyl-d-glucosamine column allowed us to perform functional studies of this lectin. The native purified PL5-2 protein had binding and agglutination activities towards VP3HP. To further understand the functions and the involvements of this lectin in response to AHPND in shrimp, RNAi-mediated knockdown of PL5-1, PL5-2 or PL5-3 was performed prior to an oral administration of VP3HP. As a result, Penlectin5-silencing in shrimp challenged with VP3HP showed higher mortality and resulted in more severe histopathological changes in the hepatopancreas with typical signs of AHPND. These results therefore suggest a role for crustacean fibrinogen-related proteins (FRePs) in innate immune response during the development of AHPND, and maybe also during other infections.

Transcriptomic Analysis of Male Black Tiger Shrimp (Penaeus monodon) After Polychaete Feeding to Enhance Testicular Maturation.

To reveal molecular mechanism of how polychaetes enhanced reproductive maturation in the male black tiger shrimp (Penaeus monodon), transcriptomic profiles of male reproductive organs (testes and vas deferens) between polychaete-fed and commercial pellet-fed male brooders were compared using cDNA microarray. The overall profiles were distinguishingly different between the two feed groups as well as between testes and vas deferens. Additionally, six of 11 differentially expressed gene identified by the microarray (HNRPUL1 and GCP4 in testes, MAT2B, CDC16, and CSN5 in vas deferens, and SLD5 in both organs) were validated by quantitative real-time PCR (qPCR) and found to exhibit significantly higher expression levels in polychaete-fed shrimp than those in commercial pellet-fed shrimp. From microarray and qPCR results, the differentially expressed transcripts in both testes and vas deferens between different feeds belonged to DNA replication and microtubule nucleation pathways. Interestingly, while the transcripts involved in nutrient uptake and nucleotide biosynthesis were increased only in testes, those involved in protein refolding and apoptosis were increased only in vas deferens. These findings suggest that polychaetes may enhance spermatogenesis by increasing spermatogonia proliferation in testes and by regulating mature spermatozoa in vas deferens.