Cloning and characterisation of NMDA receptors in the Pacific oyster, Crassostrea gigas (Thunberg, 1793) in relation to metamorphosis and catecholamine synthesis.

Bivalve metamorphosis is a developmental transition from a free-living larva to a benthic juvenile (spat), regulated by a complex interaction of neurotransmitters and neurohormones such as L-DOPA and epinephrine (catecholamine). We recently suggested an N-Methyl-D-aspartate (NMDA) receptor pathway as an additional and previously unknown regulator of bivalve metamorphosis. To explore this theory further, we successfully induced metamorphosis in the Pacific oyster, Crassostrea gigas, by exposing competent larvae to L-DOPA, epinephrine, MK-801 and ifenprodil. Subsequently, we cloned three NMDA receptor subunits CgNR1, CgNR2A and CgNR2B, with sequence analysis suggesting successful assembly of functional NMDA receptor complexes and binding to natural occurring agonists and the channel blocker MK-801. NMDA receptor subunits are expressed in competent larvae, during metamorphosis and in spat, but this expression is neither self-regulated nor regulated by catecholamines. In-situ hybridisation of CgNR1 in competent larvae identified NMDA receptor presence in the apical organ/cerebral ganglia area with a potential sensory function, and in the nervous network of the foot indicating an additional putative muscle regulatory function. Furthermore, phylogenetic analyses identified molluscan-specific gene expansions of key enzymes involved in catecholamine biosynthesis. However, exposure to MK-801 did not alter the expression of selected key enzymes, suggesting that NMDA receptors do not regulate the biosynthesis of catecholamines via gene expression.

Bivalves are NO different: nitric oxide as negative regulator of metamorphosis in the Pacific oyster, Crassostrea gigas.

BACKGROUND: Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish. RESULTS: In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot's smooth muscle relaxation. CONCLUSION: Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

Gene expression comparison of resistant and susceptible Atlantic salmon fry challenged with Infectious Pancreatic Necrosis virus reveals a marked contrast in immune response.

BACKGROUND: Infectious Pancreatic Necrosis (IPN) is a highly contagious birnavirus disease of farmed salmonid fish, which often causes high levels of morbidity and mortality. A large host genetic component to resistance has been previously described for Atlantic salmon (Salmo salar L.), which mediates high mortality rates in some families and zero mortality in others. However, the molecular and immunological basis for this resistance is not yet fully known. This manuscript describes a global comparison of the gene expression profiles of resistant and susceptible Atlantic salmon fry following challenge with the IPN virus. RESULTS: Salmon fry from two IPNV-resistant and two IPNV-susceptible full sibling families were challenged with the virus and sampled at 1 day, 7 days and 20 days post-challenge. Significant viral titre was observed in both resistant and susceptible fish at all timepoints, although generally at higher levels in susceptible fish. Gene expression profiles combined with gene ontology and pathway analyses demonstrated that while a clear immune response was observed in both resistant and susceptible fish, there were striking differences between the two phenotypes. The susceptible fish showed marked up-regulation of genes related to cytokine activity and inflammatory response that evidently failed to protect against the virus. In contrast, the resistant fish demonstrated a less pronounced immune response including up-regulation of genes relating to the M2 macrophage system. CONCLUSIONS: While only the susceptible phenotype shows appreciable mortality levels, both resistant and susceptible fish can become infected with IPNV. Susceptible fish are characterized by a much larger, yet ineffective, immune response, largely related to cytokine and inflammatory systems. Resistant fish demonstrate a more moderate, putative macrophage-mediated inflammatory response, which may contribute to their survival.

Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression.

BACKGROUND: Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). RESULTS: Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. CONCLUSIONS: Avermectins are believed to exert their toxicity to invertebrates through interaction with glutamate-gated and GABA-gated chloride channels. Further potential drug targets include other Cys-loop ion channels such as nAChR. The present study demonstrates decreased transcript abundances of GABA-Cl and nAChR subunits in EMB-resistant salmon lice, suggesting their involvement in avermectin toxicity in caligids.

Transcriptomic analysis of the host response to early stage salmonid alphavirus (SAV-1) infection in Atlantic salmon Salmo salar L.

Salmon pancreas disease, caused by salmonid alphavirus (SAV) of the family Togaviridae, is an economically important disease affecting farmed Atlantic salmon (Salmo salar L.) in Scotland, Norway, and Ireland. The virus causes characteristic lesions in the pancreas, heart, kidney and skeletal muscle of infected fish. The mechanisms responsible for the pathology and the immune responses elicited in infected Atlantic salmon are not fully understood. A microarray-based study was therefore performed to evaluate the host transcriptomic response during the early stages of an experimentally-induced SAV-1 infection. Atlantic salmon parr were injected intra-peritoneally with viral cell culture supernatant or cell culture supernatant without virus. RNA, extracted from head kidney sampled from infected and control fish at 1, 3 and 5 days post-injection (d.p.i.), was interrogated with the 17 k TRAITS/SGP cDNA microarray. The greatest number of significantly differentially expressed genes was recorded at 3 d.p.i., mainly associated with immune and defence mechanisms, including genes involved in interferon I pathways and Major Histocompatibility Complex Class I and II responses. Genes associated with apoptosis and cellular stress were also found to be differentially expressed between infected and uninfected individuals, as were genes involved in inhibiting viral attachment and replication. The microarray results were validated by follow-on analysis of eight genes by real-time PCR. The findings of the study reflect mechanisms used by the host to protect itself during the early stages of SAV-1 infection. In particular, there was evidence of rapid induction of interferon-mediated responses similar to those seen during mammalian alphavirus infections, and also early involvement of an adaptive immune response. This study provides essential knowledge to assist in the development of effective control and management strategies for SAV-1 infection.

Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification.

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA).

Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.