Marine actinomycetes Nocardiopsis alba MCCB 110 has immunomodulatory property in the tiger shrimp Penaeus monodon.

Shrimp farming constitutes an important source of revenue and employment in many developing countries. However, the shrimp industry has always been plagued with infectious diseases having varied aetiologies. Dominated by non - specific immune mechanism, preventive health care strategy is the most appropriate approach to protect the crop. The present study evaluated the efficacy of an actinomycete, Nocardiopsis alba MCCB 110 in eliciting non - specific immune mechanism in Penaeus monodon having Vibrio harveyi as the challenge organism. Haemocyte count, total protein, phenoloxidase, reactive oxygen intermediates, acid and alkaline phosphatase as well as the gene expression of proPO, peroxinectin, transglutaminase, alpha 2-macroglobulin, astakine, crustin, and penaeidin-3 were evaluated. The results demonstrated that the phenoloxidase, respiratory burst, total protein, acid and alkaline phosphatases were higher in the haemolymph of shrimps fed with Nocardiopsis alba MCCB 110 incorporated feed before and after challenge with Vibrio harveyi, compared to those of placebo fed animals. Up-regulation of six immune genes (alpha 2 macroglobulin, penaeidin -3, transglutaminase, proPO, crustin and peroxinectin) during the post-challenge were recorded. Survival of shrimp among the Nocardiopsis alba administered ones was 83% while it was 50% in placebo fed group. The elevated levels of nonspecific immune gene transcripts and concurrent increase in non specific immunity besides the higher survival rate in the Nocardiopsis alba administered group demonstrated the immunomodulatory property of the marine actinomycete Nocardiopsis alba MCCB 110 in the tiger shrimp Penaeus monodon, and on administering it through diet shrimp could be protected from vibriosis especially of V. harveyi.

Identification, ontogeny and expression analysis of a novel laboratory of genetics and physiology 2 (LGP2) transcript in Asian seabass, Lates calcarifer.

LGP2 (laboratory of genetics and physiology 2) is an important member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which plays a significant role in antiviral innate immunity. In this study, we have cloned the full-length cDNA sequence of LGP2 from Asian seabass, Lates calcarifer (AsLGP2). The complete AsLGP2 cDNA sequence consisted of 2586 nucleotides encoding a putative protein of 681 amino acids with a molecular mass of 77.6 kDa. From the AsLGP2 protein, four different conserved domains were predicted: a DExDc (DEAD/DEAH box helicase domain), a bacterial type III restriction enzyme domain (RES III), a HELICc (Helicase superfamily c-terminal domain and a RIG-I_C-RD (RIG-I C-terminal regulatory domain). The transcript of AsLGP2 could be detected in all the 11 tissues tested in healthy animals with high expression noticed in tissues facing external environment such as gill, hindgut and skin. The ontogenic expression profile of AsLGP2 implies a possible maternal transfer of this gene as it has been detected in all early embryonic developmental stages along with unfertilized eggs. Viral analogue, poly I:C, injection resulted in rapid up-regulated expression in different tissues with the highest modulation of expression observed in kidney followed by liver and gill. A rapid response of AsLGP2 expression was also observed in the different tissues of Vibrio alginolyticus-injected L. calcarifer, while significant change in expression was noticed following Staphylococcus aureus infection. Similarly, exposure to different pathogen-mimicking microbial analogues such as poly I:C, LPS and PGN resulted in enhanced expression of AsLGP2 in SISK cell-line. Taking together, these observations suggest that AsLGP2 can act as both antiviral and antibacterial cytosolic receptor and may play a significant role in embryonic and larval development in marine euryhaline teleosts like Asian seabass.

Identification of Nod like receptor C3 (NLRC3) in Asian seabass, Lates calcarifer: Characterisation, ontogeny and expression analysis after experimental infection and ligand stimulation.

Nod like receptors (NLRs) are a large group of cytoplasmic PRRs believed to play an important role in bacterial recognition in higher vertebrates. In this study, a novel Nod like receptor C3 (AsNLRC3) has been identified, cloned and characterised from Asian seabass, Lates calcarifer. The full-length AsNLRC3 transcript composed of a 4142 bp nucleic acid sequence encode for a protein of 1134 deduced amino acids. Three signature domains identified are conserved NACHT-domain, C-terminal LLR domain and N-terminal CARD effector domain. From the domain architecture and phylogenetic analysis, it was quite evident that AsNLRC3 is different from the NLR subfamily C of other teleosts. AsNLRC3 expressed in all the 11 tissues tested but highly expressed in tissues facing external environment such as gill, hindgut and midgut. The ontogenic expression profile of this receptor showed constitutive expression throughout the embryonic and larval developmental stages, which could be an innate immune strategy against different marine pathogens for larval survival. Infection with Vibrio alginolyticus and poly I:C induction showed an alteration of expression pattern in different tissues but did not show significant alteration in expression with Staphylococcus aureus infection. In vitro study in Asian seabass kidney cell line (SISK) stimulated with different ligands such as LPS, PGN and poly I:C showed considerable up-regulation at some of the time-points tested. These results suggest that AsNLRC3 can be a pivotal cytosolic innate immune receptor for recognizing wide array of pathogens in a euryhaline teleost model like Asian seabass in diverse environmental conditions.

Molecular cloning, characterisation and expression analysis of melanoma differentiation associated gene 5 (MDA5) of green chromide, Etroplus suratensis.

Innate immune system recognises pathogen-associated molecular patterns (PAMPs) by limited number of germline encoded and non-clonally developed pathogen recognition receptors (PRRs). Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are important cytosolic PRRs for sensing viral RNAs. The receptor encoded by melanoma differentiation associated gene 5 (MDA5), an RLR, recognises viral RNA and enhances antiviral response in host cells. The full-length MDA5 cDNA in Etroplus suratensis was cloned and found to have 3673 nucleotides encoding a polypeptide of 978 amino acids. The deduced amino acid sequence contains four main structural domains: two CARD domains in the N-terminal region, a DExDc (DEAH/DEAD box helicase domain), HELICc (C-terminal helicase) domain and a C-terminal regulatory domain (RD). Phylogenetic analysis revealed a close relationship of E. suratensis MDA5 (EsMDA5) with MDA5 of Neolamprologus brichardi and Oreochromis niloticus, both belonging to Cichlidae family. EsMDA5 transcripts were ubiquitously expressed in all the 12 tissues tested in healthy fish. Although, transcript level was found to be the highest in muscle, high expression was also detected in the spleen, head kidney and hindgut. In poly I:C-injected fish, EsMDA5 transcripts showed peak expression in the spleen, intestine and heart at 12h post-injection (hpi). However, in gill and kidney tissues, maximum up-regulation of EsMDA5 was observed at 6 and 48 hpi, respectively. Further, liver tissue showed an increasing trend in expression profile from 6 to 48 hpi. Interferon promoter stimulator-1 (IPS-1) gene, an adaptor triggering RIG-I- and MDA5-mediated type I interferon induction, also showed up-regulated expression at initial time-points in poly I:C-injected E. suratensis. The constitutive expression and up-regulation of EsMDA5 and the IPS-1 genes in different tissues indicate that EsMDA5 may play an important role in sensing viral PAMPs in conjunction with IPS-1.

Toll-pathway in tiger shrimp (Penaeus monodon) responds to white spot syndrome virus infection: evidence through molecular characterisation and expression profiles of MyD88, TRAF6 and TLR genes.

The Toll-pathway plays key roles in regulating the innate immune response in invertebrates. Myeloid differentiation factor 88 (MyD88) and Tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) are key molecules in this signalling pathway. To investigate the role of Toll-pathway in innate immune response of shrimp, Penaeus monodon, MyD88 (PmMyD88) and TRAF6 (PmTRAF6) were identified and characterised. PmMyD88 cDNA is 1716 bp long with an open reading frame (ORF) of 1449 bp encoding a putative protein of 482 amino acids, with a death domain, a TIR domain and C-terminal extension domain. PmTRAF6 cDNA is 2563 bp long with an ORF of 1785 bp (594 amino acids) with an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled region and a MATH domain. In healthy shrimp, PmMyD88, PmTRAF6 and PmToll were detected in 15 tissues with the highest expression in midgut, eyestalk and lymphoid organ, respectively. Responses of these genes to WSSV in experimentally-infected P. monodon as well as in cultured haemocytes and also effect of poly I:C on the gene expression in vitro was investigated at six time-points in seven tissues. PmToll showed significant up-regulation at all time-points of infection in six tissues and until 24 h post-infection in vitro. However, poly I:C-induced haemocytes showed up-regulation of the gene until 48 h post-exposure. WSSV caused significant up-regulation of PmMyD88 in most of the tissues tested. The virus challenge as well as poly I:C induction in vitro also resulted in significant up-regulation of the gene. Up-regulated expression of PmTRAF6 was detected in haemocytes and lymphoid organ at late stage of infection. In vitro virus challenge showed significant up-regulation of PmTRAF6 at almost all time-points whereas no significant change in the expression was observed on poly I:C induction. The responses of these key genes, observed in the present study, suggest that Toll-pathway as a whole may play a crucial role in the immune response against viruses in shrimp.

Toll-like receptor of mud crab, Scylla serrata: molecular characterisation, ontogeny and functional expression analysis following ligand exposure, and bacterial and viral infections.

Toll-like receptors are sentinels of innate immune system, which recognise pathogen-associated molecular patterns, and subsequently activate production of antimicrobial peptides to contain the infection. In the present study, we cloned and characterised a Toll gene from Scylla serrata (SsToll) encoding 1005 amino acids with typical Toll-like receptor domain topology. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family showing 100 % identity with Scylla paramamosain (SpToll). The expression pattern of mRNA in different tissues indicated that SsToll is constitutively expressed in all the tissues examined, with varying expression levels. The expression was also detected in all the life-stages (egg, zoea stages 1-5, megalopa and crab instar) with the highest level observed in zoea 2. In-vitro studies using crab haemocyte culture demonstrated that SsToll transcripts are distinctly modulated in response to ligands such as peptidoglycan and lipopolysaccharide at all time-points. A significant change in SsToll expression was also noticed in haemocytes exposed to poly I:C (3-9 h). On the contrary, the transcription level of SsToll in response to white spot syndrome virus (WSSV) challenge was noticeably different. The change in expression in vitro was not significant at early time-points until 3 h; the transcripts showed a significant up-regulation commencing from 6 h, but not beyond 12 h. However, in vivo expression was unaffected at early time-points of WSSV challenge (until 12 h) and a gradual up-regulation was detected at 24 h. In-vivo challenge with Vibrio parahaemolyticus resulted in delayed up-regulation of the gene. The results obtained in the present study suggest that SsToll might be involved in the innate immunity of mud crab.

Isolation and characterization of virulent Aeromonas veronii from ascitic fluid of oscar Astronotus ocellatus showing signs of infectious dropsy.

The cichlid oscar Astronotus ocellatus has worldwide commercial value in the pet fish industry because of its early maturation, relatively high fecundity, ability to identify its caretaker and also to alter colouration amongst conspecifics. Pathogenic strains of Aeromonas veronii resistant to multiple antibiotics were isolated from A. ocellatus individuals showing signs of infectious abdominal dropsy. The moribund fish showed haemorrhage in all internal organs, and pure cultures could be obtained from the abdominal fluid. The isolates recovered were biochemically identified as A. veronii biovar sobria and genetically confirmed as A. veronii based on 16S rRNA gene sequence analysis (GenBank accession no. FJ573179). The RAPD profile using 3 primers (OPA-3, OPA-4 and OPD-20) generated similar banding patterns for all isolates. They displayed cytotoxic and haemolytic activity and produced several exoenzymes which were responsible for the pathogenic potential of the isolates. In the representative isolate MCCB 137, virulence genes such as enterotoxin act, haemolytic toxin aerA, type 3 secretion genes such as aexT, ascVand ascF-ascG, and gcat (glycerophospholipid-cholesterol acyltransferase) could be amplified. MCCB 137 exhibited a 50% lethal dose (LD50) of 10(5.071) colony-forming units ml(-1) in goldfish and could be subsequently recovered from lesions as well as from the internal organs. This is the first description of a virulent A. veronii from oscar.

Identification of components of male-produced pheromone of coffee white stemborer, Xylotrechus quadripes.

The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is the foremost pest of arabica coffee in India, Sri Lanka, China, Vietnam, and Thailand. Previous work showed that female beetles were attracted to traps baited with male beetles. Analyses of volatiles from male X. quadripes of Indian origin by gas chromatography (GC) linked to electroantennographic (EAG) recording from a female beetle antenna showed three male-specific components comprising more than 90% of the volatiles, two of which elicited EAG responses. The major EAG-active component was produced at up to 2 microg hr(-1) insect(-1) and was identified as (S)-2-hydroxy-3-decanone (I) by comparison of GC data, and mass (MS), infrared, and nuclear magnetic resonance (NMR) spectra with those of synthetic standards. The second component was identified as 3-hydroxy-2-decanone (II) produced in part by isomerization of I under the conditions of the GC analysis, although the NMR spectrum suggested it is naturally produced at up to 7% of I. The minor component that elicited an EAG response, present at 7% of the amount of I, was identified as (S,S)-2,3-dihydroxyoctane (III) from GC and MS data. 2-Hydroxy-3-octanone (0.2-0.5% of I), 2,3-decanedione (2% of I), 2-phenylethanol (3% of I), and octanoic acid (4% of I) were also identified in volatiles from male beetles. A general, stereospecific synthetic route to the enantiomers of 2-hydroxy-3-alkanones from the enantiomers of ethyl lactate was developed. The enantiomers of III were synthesized from (E)-2-octene by Sharpless asymmetric dihydroxylation. (S)-(I) was attractive to male X. quadripes in laboratory bioassays, but addition of (S,RS)-(III) at 10% of I reduced attractiveness. In field trials carried out in India with sticky, cross-vane traps, (S)- and (RS)-(I) attracted male X. quadripes and addition of (S,S)-(III) at 10% of I reduced attractiveness. Significant numbers of female Demonax balyi Pascoe (Coleoptera: Cerambycidae) were sometimes caught in traps baited with (S)-(I) alone.