Applying genetic technologies to combat infectious diseases in aquaculture.

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies-sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon.

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.

Surrogate broodstock to enhance biotechnology research and applications in aquaculture.

Aquaculture is playing an increasingly important role in meeting global demands for seafood, particularly in low and middle income countries. Genetic improvement of aquaculture species has major untapped potential to help achieve this, with selective breeding and genome editing offering exciting avenues to expedite this process. However, limitations to these breeding and editing approaches include long generation intervals of many fish species, alongside both technical and regulatory barriers to the application of genome editing in commercial production. Surrogate broodstock technology facilitates the production of donor-derived gametes in surrogate parents, and comprises transplantation of germ cells of donors into sterilised recipients. There are many successful examples of intra- and inter-species germ cell transfer and production of viable offspring in finfish, and this leads to new opportunities to address the aforementioned limitations. Firstly, surrogate broodstock technology raises the opportunity to improve genome editing via the use of cultured germ cells, to reduce mosaicism and potentially enable in vivo CRISPR screens in the progeny of surrogate parents. Secondly, the technology has pertinent applications in preservation of aquatic genetic resources, and in facilitating breeding of high-value species which are otherwise difficult to rear in captivity. Thirdly, it holds potential to drastically reduce the effective generation interval in aquaculture breeding programmes, expediting the rate of genetic gain. Finally, it provides new opportunities for dissemination of tailored, potentially genome edited, production animals of high genetic merit for farming. This review focuses on the state-of-the-art of surrogate broodstock technology, and discusses the next steps for its applications in research and production. The integration and synergy of genomics, genome editing, and reproductive technologies have exceptional potential to expedite genetic gain in aquaculture species in the coming decades.

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes.

Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system.

The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0.

Harnessing genomics to fast-track genetic improvement in aquaculture.

Aquaculture is the fastest-growing farmed food sector and will soon become the primary source of fish and shellfish for human diets. In contrast to crop and livestock production, aquaculture production is derived from numerous, exceptionally diverse species that are typically in the early stages of domestication. Genetic improvement of production traits via well-designed, managed breeding programmes has great potential to help meet the rising seafood demand driven by human population growth. Supported by continuous advances in sequencing and bioinformatics, genomics is increasingly being applied across the broad range of aquaculture species and at all stages of the domestication process to optimize selective breeding. In the future, combining genomic selection with biotechnological innovations, such as genome editing and surrogate broodstock technologies, may further expedite genetic improvement in aquaculture.

Temperature-induced testicular germ cell loss and recovery in Nile tilapia Oreochromis niloticus.

Water temperature is a critical external factor influencing gonadal development in fish. This research aimed to study the impact of elevated temperature on testicular germ cell survival and reproductive capacity of Nile tilapia. Male Nile tilapia were exposed to high temperatures of either 36 (HT1) or 37 °C (HT2) for 3000 degree-days (DD) and thereafter returned to the control temperature of 27 °C (CT) for 2200 DD. The deleterious effects on testicular germ and somatic cells were observed histologically, characterised by vacuolisation, atrophy and the loss of spermatogenic cells in testes with a more severe impact of HT2 compared to HT1. Interestingly, serum 11-ketotestosterone (11-KT) and testosterone (T) levels tended to be higher during the heat treatments than CT. Expression levels of germline-specific genes piwil1, piwil2 and nanos2 and Bcl-2 family genes, bcl-xLb and baxa were significantly reduced during the heat treatment compared to CT, more so in the HT2, while the levels of nanos3 and gfra1 transcripts were only significantly reduced in HT2, implying a significant loss of spermatogonial stem cell (SSC) and spermatogonia in HT2. The effect of HT2 is further evidenced by the significantly reduced sperm density and fertilisation rate compared to CT and HT1 at the end of the recovery period but complete sterility was not induced by HT2. Overall, the present study showed significant effects of HT2 on germ cell survival with histological changes in testes, reduced milt quality, increased 11-KT, and decreased expression of germline-specific genes, SSC marker genes and Bcl-2 family genes in testes which could therefore be potential target genes for sterilisation by genome editing.

Kisspeptin2 stimulates the HPG axis in immature Nile tilapia (Oreochromis niloticus).

It has been suggested that kisspeptin influences reproduction and onset of puberty in fishes. Unlike mammals, which produce only one kisspeptin (Kiss1), some teleosts have two, Kiss1 and Kiss2, both thought to be involved in the stimulation of gonadotropin (GTH) secretion. In Nile tilapia (Oreochromis niloticus), however, only Kiss2 has been identified so far. The effect of Kiss2 on GTH release varies significantly depending on species and reproductive stage. Furthermore, its physiological function in this species is not clearly defined. In this study, kiss2 gene expression profiles were examined using quantitative real-time PCR (qRT-PCR) in the brain, pituitary, and gonads of Nile tilapia at different reproductive stages (male: immature, pre-spermiation, post-spermiation; female: immature, pre-spawning, post-spawning). The kiss2 mRNA expression profiles of the brain, pituitary, and gonads of both sexes shared a similar pattern their expression was significantly higher at the immature stage than at the mature or post-spawning stages, implying it is involved in early gonadal maturation in this species. To investigate the effect of kisspeptin on the hypothalamus-pituitary-gonad (HPG) axis in vivo, synthetic kisspeptin2 (FNYNPLSLRF) was injected into immature male and female tilapia intraperitoneally (i.p.) at a dose of 200pmol/g body weight. The results showed that synthetic Kiss2 administration increased the expression of GnRH I, fshß and lhß mRNA in the brain and increased 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) levels in the plasma. These results suggest that Kiss2 stimulates the expression of GnRH and GTH genes in immature Nile tilapia.

Neurokinin B-related Peptide Suppresses the Expression of GnRH I, Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus.

Neurokinin B (NKB) and neurokinin B related peptide (NKBRP) belong to tachykinin peptide family. Theyact as a neurotransmitter and/or neuromodulator. Mutation of NKB and/or its cognate receptor, NK3R resulted in hypogonadotropic hypogonadism in mammals, implying a strong involvement of NKB/NK3R system in controlling mammalian reproduction. Teleosts possess NKBRP as well as NKB, but their roles in fish reproduction need to be clarified. In this study, NKB and NKBRP coding gene (tac3) was cloned from Nile tilapia and sequenced. Based on the sequence, Nile tilapia NKB and NKBRP peptide were synthesized and their biological potencies were tested in vitro pituitary culture. The synthetic NKBRP showed direct inhibitory effect on the expression of GTH subunits at the pituitary level. This inhibitory effect was confirmed in vivo by means of intraperitoneal (ip) injection of synthetic NKB and NKBRP to mature female tilapia (20 pmol/g body weight [BW]). Both NKB and NKBRP had no effect on the plasma level of sex steroids, E2 and 11-KT. However, NKBRP caused declines of expression level of GnRH I, Kiss2 and tac3 mRNAs in the brain while NKB seemed to have no distinct effect. These results indicate some inhibitory roles of NKBRP in reproduction of mature female Nile tilapia, although their exact functions are not clear at the moment.

The Expression Pattern of Melatonin Receptor 1a Gene during Early Life Stages in the Nile tilapia (Oreochromis niloticus).

The action of melatonin within the body of animals is known to be mediated by melatonin receptors. Three different types of melatonin receptors have been identified so far in fish. However, which of these are specifically involved in puberty onset is not known in fish. We cloned and analyzed the sequence of melatonin receptor 1a (mel 1a) gene in Nile tilapia Oreochromis niloticus. In addition, we examined the tissue distribution of gene expressions for three types of receptors, mel 1a, 1b and lc and investigated which of them is involved in the onset of puberty by comparing their expression with that of gonadotropin-releasing hormone receptor I (GnRHr I) gene using quantitative real-time PCR from 1 week post hatch (wph) to 24 wph. The mel 1a gene of Nile tilapia consisted of two exons and one bulky intron between them. Mel 1a gene was found to be highly conserved gene showing high homology with the corresponding genes from different teleost. All three types of melatonin receptor genes were expressed in the brain, eyes and ovary in common. Expression of mel 1a gene was the most abundant and ubiquitous among 3 receptors in the brain, liver, gill, ovary, muscle, eye, heart, intestine, spleen and kidney. Mel 1b and mel 1c genes were, however, expressed in fewer tissues at low level. During the development post hatch, expressions of both mel 1a and GnRHr I genes significantly increased at 13 wph which was close to the putative timing of puberty onset in this species. These results suggest that among three types of receptors mel 1a is most likely associated with the action of melatonin in the onset of puberty in Nile tilapia.