Mathew T. Cook

Exploring RNAi as a therapeutic strategy for controlling disease in aquaculture.

Aquatic animal diseases are one of the most significant constraints to the development and management of aquaculture worldwide. As a result, measures to combat diseases of fish and shellfish have assumed a high priority in many aquaculture-producing countries. RNA interference (RNAi), a natural mechanism for post-transcriptional silencing of homologous genes by double-stranded RNA (dsRNA), has emerged as a powerful tool not only to investigate the function of specific genes, but also to suppress infection or replication of many pathogens that cause severe economic losses in aquaculture. However, despite the enormous potential as a novel therapeutical approach, many obstacles must still be overcome before RNAi therapy finds practical application in aquaculture, largely due to the potential for off-target effects and the difficulties in providing safe and effective delivery of RNAi molecules in vivo. In the present review, we discuss the current knowledge of RNAi as an experimental tool, as well as the concerns and challenges ahead for the application of such technology to combat infectious disease of farmed aquatic animals.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways.

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways.

Exploiting genomic data to identify proteins involved in abalone reproduction

UNLABELLED 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.

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.

Pseudocyst formation in the marine parasitic amoeba Neoparamoeba perurans: a short-term survival strategy to abrupt salinity variation

Amoebic gill disease (AGD), caused by the freeliving marine amoeba Neoparamoeba perurans, has long been recognized as the major health concern affecting the Atlantic salmon aquaculture industry in Australia (Munday et al. 1993) and is now emerging as a significant threat worldwide (Mitchell & Rodger 2011). Bathing affected fish in freshwater is the current treatment strategy employed by the Australian industry (Parsons et al. 2001). However, despite the effectiveness of the treatment, the number of gill-associated amoebae has been shown to return to prebath levels within 10 days (Clark, Powell & Nowak 2003), in part attributed to a small percentage of amoebae survival following bathing (Adams & Nowak 2004). Although exposure of individual amoebae to freshwater may vary with localized gill structure and mucus cover, an adaptation mechanism to survive sudden freshwater immersion for an extended period of time should also be expected for N. perurans. The occurrence of round cyst-like cells has been reported for the Acanthamoeba T4 genotype in response to organic solvents exposure (Kliescikova, Kulda & Nohynkova 2011). The spherical structures were classified by the authors as pseudocysts as, unlike double-walled true cysts, these cells were coated by a single-layered envelope and survived for shorter periods of time when exposed to the stress factor. Interestingly, pseudocysts were eventually able to revert to viable trophozoites once returned back to optimum culture conditions. The development of non-motile spherical cells has been also described for N. perurans when submitted to in vitro freshwater incubation (Powell & Clark 2003; Nowak, Crosbie & Adams 2010). In both studies, rounded amoebas were counted as dead cells based on either trypan blue or neutral red viability assays which were performed at the end of each in vitro challenge. Therefore, given the frequent reports of amoeba survival following freshwater bathing, the possibility of N. perurans recovering from the apparently dead spherical stage should be accounted for. To investigate this hypothesis, in vitro cultured trophozoites were submitted to freshwater incubation and the dynamics of rounded cells closely monitored by microscopy, once returned to optimum culture conditions. Briefly, N. perurans trophozoites were freshly isolated from gill swabs taken from AGD-affected fish and maintained on seawater agar plates (Crosbie et al. 2012) until reaching 50% confluence. Prior to the freshwater challenge, the cells were evenly spread into 24-well culture plates to a final concentration of 1000 amoebae/well and allowed to adhere at 16 °C overnight (Lima et al. 2014). At this stage, an aliquot of cells was used to Correspondence P C Lima, Integrated Sustainable Aquaculture Production, CSIRO Agriculture and Food, BIRC, 144 North Street, Woorim, QLD 4507, Australia (e-mail: paula.lima@csiro.au)

Evaluation of Non-destructive Molecular Diagnostics for the Detection of Neoparamoeba perurans

Amoebic gill disease (AGD) caused by Neoparamoeba perurans, has emerged in Europe as a significant problem for the Atlantic salmon farming industry. Gross gill score is the most widely used and practical method for determining AGD severity on farms and informing management decisions on disease mitigation strategies. As molecular diagnosis of AGD remains a high priority for much of the international salmon farming industry, there is a need to evaluate the suitability of currently available molecular assays in conjunction with the most appropriate non-destructive sampling methodology. The aims of this study were to assess a non-destructive sampling methodology (gill swabs) and to compare a range of currently available real-time PCR assays for the detection of N. perurans. Furthermore a comparison of the non-destructive molecular diagnostics with traditional screening methods of gill scoring and histopathology was also undertaken. The study found that all molecular protocols assessed performed well in cases of clinical AGD with high gill scores. A TaqMan based assay (protocol 1) was the optimal assay based on a range of parameters including % positive samples from a field trial performed on fish with gill scores ranging from 0 to 5. A higher proportion of gill swab samples tested positive by all protocols than gill filament biopsies and there was a strong correlation between gill swabs tested by protocol 1 and gross gill score and histology scores. Screening for N. perurans using protocol 1 in conjunction with non-destructive gill swab samples was shown to give the best results.

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.