Differential Exoproteome and Biochemical Characterisation of Neoparamoeba perurans.

Infection with the protozoan ectoparasite Neoparamoeba perurans, the causative agent of AGD, remains a global threat to salmonid farming. This study aimed to analyse the exoproteome of both an attenuated and virulent N. perurans isolate using proteomics and cytotoxicity testing. A disproportionate presence of proteins from the co-cultured microbiota of N. perurans was revealed on searching an amalgamated database of bacterial, N. perurans and Amoebozoa proteins. LC-MS/MS identified 33 differentially expressed proteins, the majority of which were upregulated in the attenuated exoproteome. Proteins of putative interest found in both exoproteomes were maltoporin, ferrichrome-iron receptor, and putative ferric enterobactin receptor. Protease activity remained significantly elevated in the attenuated exoproteome compared with the virulent exoproteome. Similarly, the attenuated exoproteome had a significantly higher cytotoxic effect on rainbow trout gill cell line (RTgill W1) cells compared with the virulent exoproteome. The presence of a phosphatase and serine protease in the virulent exoproteome may facilitate AGD infection but do not appear to be key players in causing cytotoxicity. Altogether, this study reveals prolonged culture of N. perurans affects the exoproteome composition in favour of nutritional acquisition, and that the current culturing protocol for virulent N. perurans does not facilitate the secretion of virulence factors.

Host-Parasite Interaction of Atlantic salmon (Salmo salar) and the Ectoparasite Neoparamoeba perurans in Amoebic Gill Disease.

Marine farmed Atlantic salmon (Salmo salar) are susceptible to recurrent amoebic gill disease (AGD) caused by the ectoparasite Neoparamoeba perurans over the growout production cycle. The parasite elicits a highly localized response within the gill epithelium resulting in multifocal mucoid patches at the site of parasite attachment. This host-parasite response drives a complex immune reaction, which remains poorly understood. To generate a model for host-parasite interaction during pathogenesis of AGD in Atlantic salmon the local (gill) and systemic transcriptomic response in the host, and the parasite during AGD pathogenesis was explored. A dual RNA-seq approach together with differential gene expression and system-wide statistical analyses of gene and transcription factor networks was employed. A multi-tissue transcriptomic data set was generated from the gill (including both lesioned and non-lesioned tissue), head kidney and spleen tissues naïve and AGD-affected Atlantic salmon sourced from an in vivo AGD challenge trial. Differential gene expression of the salmon host indicates local and systemic upregulation of defense and immune responses. Two transcription factors, znfOZF-like and znf70-like, and their associated gene networks significantly altered with disease state. The majority of genes in these networks are candidates for mediators of the immune response, cellular proliferation and invasion. These include Aurora kinase B-like, rho guanine nucleotide exchange factor 25-like and protein NDNF-like inhibited. Analysis of the N. perurans transcriptome during AGD pathology compared to in vitro cultured N. perurans trophozoites, as a proxy for wild type trophozoites, identified multiple gene candidates for virulence and indicates a potential master regulatory gene system analogous to the two-component PhoP/Q system. Candidate genes identified are associated with invasion of host tissue, evasion of host defense mechanisms and formation of the mucoid lesion. We generated a novel model for host-parasite interaction during AGD pathogenesis through integration of host and parasite functional profiles. Collectively, this dual transcriptomic study provides novel molecular insights into the pathology of AGD and provides alternative theories for future research in a step towards improved management of AGD.

Comparative proteomic profiling of newly acquired, virulent and attenuated Neoparamoeba perurans proteins associated with amoebic gill disease.

The causative agent of amoebic gill disease, Neoparamoeba perurans is reported to lose virulence during prolonged in vitro maintenance. In this study, the impact of prolonged culture on N. perurans virulence and its proteome was investigated. Two isolates, attenuated and virulent, had their virulence assessed in an experimental trial using Atlantic salmon smolts and their bacterial community composition was evaluated by 16S rRNA Illumina MiSeq sequencing. Soluble proteins were isolated from three isolates: a newly acquired, virulent and attenuated N. perurans culture. Proteins were analysed using two-dimensional electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The challenge trial using naïve smolts confirmed a loss in virulence in the attenuated N. perurans culture. A greater diversity of bacterial communities was found in the microbiome of the virulent isolate in contrast to a reduction in microbial community richness in the attenuated microbiome. A collated proteome database of N. perurans, Amoebozoa and four bacterial genera resulted in 24 proteins differentially expressed between the three cultures. The present LC-MS/MS results indicate protein synthesis, oxidative stress and immunomodulation are upregulated in a newly acquired N. perurans culture and future studies may exploit these protein identifications for therapeutic purposes in infected farmed fish.

Immersion challenge of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. did not increase the severity of Neoparamoeba perurans-induced amoebic gill disease (AGD).

Amoebic gill disease (AGD) is one of the main health issues impacting farmed Atlantic salmon. Neoparamoeba perurans causes AGD; however, a diversity of other amoeba species colonizes the gills and there is little understanding of whether they are commensal or potentially involved in different stages of gill disease development. Here, we conduct in vivo challenges of naïve Atlantic salmon with cultured Nolandella sp. and Pseudoparamoeba sp. to investigate their pathogenicity to Atlantic salmon gills. Additionally, we assessed whether the presence of Nolandella sp. and Pseudoparamoeba sp. influences the onset and/or severity of N. perurans-induced AGD. All three strains attached and multiplied on the gills according to qPCR analysis. Furthermore, minor gross gill lesions and histological changes were observed post-exposure. While N. perurans was found associated with classical AGD lesions, Nolandella sp. and Pseudoparamoeba sp. were not found associated with lesion sites and these lesions did not meet the expected composite of histopathological changes for AGD. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of AGD. This trial provides evidence that cultured Nolandella sp. and Pseudoparamoeba sp. do not induce AGD and do not influence the severity of AGD during the early stages of development.

Gill Mucus and Gill Mucin O-glycosylation in Healthy and Amebic Gill Disease-Affected Atlantic Salmon.

Amoebic gill disease (AGD) causes poor performance and death in salmonids. Mucins are mainly comprised by carbohydrates and are main components of the mucus covering the gill. Since glycans regulate pathogen binding and growth, glycosylation changes may affect susceptibility to primary and secondary infections. We investigated gill mucin O-glycosylation from Atlantic salmon with and without AGD using liquid chromatography-mass spectrometry. Gill mucin glycans were larger and more complex, diverse and fucosylated than skin mucins. Confocal microscopy revealed that fucosylated mucus coated sialylated mucus strands in ex vivo gill mucus. Terminal HexNAcs were more abundant among O-glycans from AGD-affected Atlantic salmon, whereas core 1 structures and structures with acidic moieties such as N-acetylneuraminic acid (NeuAc) and sulfate groups were less abundant compared to non-infected fish. The fucosylated and NeuAc-containing O-glycans were inversely proportional, with infected fish on the lower scale of NeuAc abundance and high on fucosylated structures. The fucosylated epitopes were of three types: Fuc-HexNAc-R, Gal-[Fuc-]HexNAc-R and HexNAc-[Fuc-]HexNAc-R. These blood group-like structures could be an avenue to diversify the glycan repertoire to limit infection in the exposed gills. Furthermore, care must be taken when using skin mucus as proxy for gill mucus, as gill mucins are distinctly different from skin mucins.

A High Throughput Viability Screening Method for the Marine Ectoparasite Neoparamoeba perurans.

The marine protozoan parasite Neoparamoeba perurans has been established as the causative agent for amoebic gill disease (AGD) in Atlantic salmon (Salmo salar). Freshwater bathing is the only routinely used treatment for AGD in Australia while hydrogen peroxide (H2O2) is used in countries with cooler water temperatures. The identification of new treatments that do not rely on either freshwater or H2O2 bathing is highly sought. However, in vitro based methods for high throughput screening of antiparasitic compounds have not been established for this parasite. To this end the present study evaluated two in vitro bioassays based on metabolic energy production and cellular membrane integrity to distinguish between amoebistatic (crenated or pseudocyst forms with recovery possible) and amoebicidal (death) activity. Amoebae were subject to either freshwater, H2O2 or chloramine-T for 4h treatment and assessed 24h after recovery. Visualization by microscopy and bioassay assessment 24h post-treatment confirmed that H2O2 and freshwater are 95% amoebicidal albeit due to different mechanisms of action. These data are consistent with other studies where amoebae have been observed to recover following exposure to these compounds and provide evidence for the inclusion of a recovery component to differentiate between the mechanism of action of amoebicidal and amoebistatic treatments. Together these bioassays are a critical tool for high throughput screening of novel and more effective treatments against AGD.

Genetic diversity among geographically distant isolates of Neoparamoeba perurans.

The present study explored the use of 2 common genetic markers, the mitochondrial cytochrome oxidase I (COI) and the ribosomal internal transcribed spacer (ITS) to infer the relationship between geographically distant isolates of the protozoan gill parasite Neoparamoeba perurans, the agent responsible for amoebic gill disease in farmed Atlantic salmon worldwide. Present data confirmed that the ITS marker is suitable for Neoparamoeba species discrimination; however, it is not recommended as a population marker due to the presence of multiple copies of ITS within both N. perurans clonal and polycultures. On the other hand, in the partial COI gene analysed here, a low variability was observed, with 8 haplotypes recovered from N. perurans samples collected from Europe (Ireland, Norway, Scotland) and Tasmania (Australia). In Europe, the COI haplotypes which have more recently been detected in aquaculture are different to the haplotypes associated with the original gill disease emergence in Ireland in 1997 and Norway in 2006. The presence of unique COI haplotypes in different continents suggests the presence of multiple distinct reservoirs of the pathogen in both Europe and Tasmania. Isolates from additional geographical locations are required to fully understand the origins and routes for the spread of N. perurans worldwide.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning.

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian 'greenlip' abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone 'omics website, http://abalonedb.org.

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD).

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.

Characterization of lipid metabolism genes and the influence of fatty acid supplementation in the hepatic lipid metabolism of dusky grouper (Epinephelus marginatus).

Dusky grouper is an important commercial fish species in many countries, but some factors such as overfishing has significantly reduced their natural stocks. Aquaculture emerges as a unique way to conserve this species, but very little biological information is available, limiting the production of this endangered species. To understand and generate more knowledge about this species, liver transcriptome sequencing and de novo assembly was performed for E. marginatus by Next Generation Sequencing (NGS). Sequences obtained were used as a tool to validate the presence of key genes relevant to lipid metabolism, and their expression was quantified by qPCR. Moreover, we investigated the influence of supplementing different dietary fatty acids on hepatic lipid metabolism. The results showed that the different fatty acids added to the diet dramatically changed the gene expression of some key enzymes associated with lipid metabolism as well as hepatic fatty acid profiles. Elongase 5 gene expression was shown to influence intermediate hepatic fatty acid elongation in all experimental groups. Hepatic triglycerides reflected the diet composition more than hepatic phospholipids, and were characterized mainly by the high percentage of 18:3n3 in animals fed with a linseed oil rich diet. Results for the saturated and monounsaturated fatty acids suggest a self-regulatory potential for retention and oxidation processes in liver, since in general the tissues did not directly reflect these fatty acid diet compositions. These results indicated that genes involved in lipid metabolism pathways might be potential biomarkers to assess lipid requirements in the formulated diet for this species.

Genome Sequencing of Blacklip and Greenlip Abalone for Development and Validation of a SNP Based Genotyping Tool.

Abalone breeding in southern Australia often involves the production of interspecies hybrids through crossing blacklip (Haliotos rubra) and greenlip (H. laevigata) parental populations. To assist applied breeding and investigate genetic divergence, this study applied genome sequencing and variant detection to develop and validate a SNP genotyping tool. Skim short read Illumina sequencing was performed using 24 individuals from each of the two parental species and a hybrid population. Raw reads were assembled into three population specific pools (each 12-15 fold coverage), before mapping was performed against a draft greenlip abalone reference genome. Variant detection identified 22.4 M raw variants across the three populations (SNP and indels), suggesting they are highly heterozygous. First stage filtering defined a high quality SNP collection of 2.2 M variants independently called in each of the three populations. Second stage filtering identified a much smaller set of variants for assay design and genotyping using a validation set of 191 abalone of known population and pedigree. Comparison of allele frequency data revealed a high proportion of SNP (43%) had divergent allele frequency (< 0.2) between the two parental populations, suggesting they should have utility for parentage assignment. A maximum likelihood approach was used to successfully assign 105 of 105 progeny to their known true parent amongst a set of 86 candidate parents, confirming the genotyping tool has utility for applied breeding. Analysis of pairwise allele sharing successfully discriminated animals into populations, and PCA of genetic distance grouped the hybrid animals with intermediate values between the two parental populations. The findings present a library of DNA polymorphism of utility to breeding and ecological application, and begins to characterize the divergence separating two economically important aquaculture species.

Identification of differentially expressed reproductive and metabolic proteins in the female abalone (Haliotis laevigata) gonad following artificial induction of spawning.

Inefficient control of temperate abalone spawning prevents pair-wise breeding and production of abalone with highly marketable traits. Traditionally, abalone farmers have used a combination of UV irradiation and application of temperature gradients to the tank water to artificially induce spawning. Proteins are known to regulate crucial processes such as respiration, muscle contraction, feeding, growth and reproduction. Spawning as a pre-requisite of abalone reproduction is likely to be regulated, in part, by endogenous proteins. A first step in elucidating the mechanisms that regulate spawning is to identify which proteins are directly involved during spawning. The present study examined protein expression following traditional spawning induction in the Haliotis laevigata female. Gonads were collected from abalone in the following physiological states: (1) spawning; (2) post-spawning; and (3) failed-to-spawn. Differential protein abundance was initially assessed using two-dimensional difference in-gel electrophoresis coupled with mass spectrometry for protein identification. A number of reproductive proteins such as vitellogenin, vitelline envelope zona pellucida domain 29 and prohibitin, and metabolic proteins such as thioredoxin peroxidase, superoxide dismutase and heat shock proteins were identified. Differences in protein abundance levels between physiological states were further assessed using scheduled multiple reaction monitoring mass spectrometry. Positive associations were observed between the abundance of specific proteins, such as heat shock cognate 70 and peroxiredoxin 6, and the propensity or failure to spawn in abalone. These findings have contributed to better understand both the effects of oxidative and heat stress over abalone physiology and their influence on abalone spawning.

Expression analysis of sex-determining pathway genes during development in male and female Atlantic salmon (Salmo salar).

We studied the expression of 28 genes that are involved in vertebrate sex-determination or sex-differentiation pathways, in male and female Atlantic salmon (Salmo salar) in 11 stages of development from fertilization to after first feeding. Gene expression was measured in half-sibs that shared the same dam. The sire of family 1 was a sex-reversed female (i.e., genetically female but phenotypically male), and so the progeny of this family are all female. The sire of family 2 was a true male, and so the offspring were 50% male and 50% female. Gene expression levels were compared among three groups: 20 female offspring of the cross between a regular female and the sex-reversed female (family 1, first group), ∼ 10 females from the cross between a regular female and a regular male (family 2, second group) and ∼ 10 males from this same family (family 2, third group). Statistically significant differences in expression levels between males and the two groups of females were observed for two genes, gsdf and amh/mis, in the last four developmental stages examined. SdY, the sex-determining gene in rainbow trout, appeared to be expressed in males from 58 days postfertilization (dpf). Starting at 83 dpf, ovarian aromatase, cyp19a, expression appeared to be greater in both groups of females compared with males, but this difference was not statistically significant. The time course of expression suggests that sdY may be involved in the upregulation of gsdf and amh/mis and the subsequent repression of cyp19a in males via the effect of amh/mis.

Exploiting genomic data to identify proteins involved in abalone reproduction.

Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. BIOLOGICAL SIGNIFICANCE: A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases.

Development of an in vitro model system for studying bacterially expressed dsRNA-mediated knockdown in Neoparamoeba genus.

RNA interference (RNAi) has been extensively used to study gene function in non-model organisms and has the potential to identify parasite target molecules in order to develop alternative treatment strategies. This technology could assist in further development of preventive methods against amoebic gill disease (AGD), the main health problem affecting the Atlantic salmon aquaculture industry in Tasmania (Australia) and now a significant emerging issue in Europe. Using ß-actin and EF1-α as candidate genes, we investigated the feasibility of gene knockdown by double-stranded RNA (dsRNA) in Neoparamoeba pemaquidensis, the non-infective strain closely related to the causative agent of AGD, Neoparamoeba perurans. Bacterially expressed dsRNA targeting the selected target genes was administered by soaking (2, 20 and 50 µg/mL) and a time course sampling regime performed. Quantitative real-time PCR analysis showed that candidate genes were successfully downregulated with silencing efficiency and duration both target and dose-dependent. Additionally, ß-actin deficient trophozoites unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. An effective RNAi model system for N. pemaquidensis was validated in the current study. Such findings will greatly facilitate further application of RNAi in the aetiological agent of AGD. To our knowledge, this is the first time that RNAi-mediated technology has been successfully employed in a member of the Neoparamoeba genus.

Control of gibberellin levels and gene expression during de-etiolation in pea.

Gibberellin A(1) (GA(1)) levels drop significantly in wild-type pea (Pisum sativum) plants within 4 h of exposure to red, blue, or far-red light. This response is controlled by phytochrome A (phyA) (and not phyB) and a blue light receptor. GA(8) levels are increased in response to 4 h of red light, whereas the levels of GA(19), GA(20), and GA(29) do not vary substantially. Red light appears to control GA(1) levels by down-regulating the expression of Mendel's LE (PsGA3ox1) gene that controls the conversion of GA(20) to GA(1), and by up-regulating PsGA2ox2, which codes for a GA 2-oxidase that converts GA(1) to GA(8). This occurs within 0.5 to 1 h of exposure to red light. Similar responses occur in blue light. The major GA 20-oxidase gene expressed in shoots, PsGA20ox1, does not show substantial light regulation, but does show up-regulation after 4 h of red light, probably as a result of feedback regulation. Expression of PsGA3ox1 shows a similar feedback response, whereas PsGA2ox2 shows a feed-forward response. These results add to our understanding of how light reduces shoot elongation during de-etiolation.