Brendan McAndrew

Major quantitative trait loci affect resistance to infectious pancreatic necrosis in Atlantic salmon (Salmo salar).

Infectious pancreatic necrosis (IPN) is a viral disease currently presenting a major problem in the production of Atlantic salmon (Salmon salar). IPN can cause significant mortality to salmon fry within freshwater hatcheries and to smolts following transfer to seawater, although challenged populations show clear genetic variation in resistance. To determine whether this genetic variation includes loci of major effect, a genomewide quantitative trait loci (QTL) scan was performed within 10 full-sib families that had received a natural seawater IPN challenge. To utilize the large difference between Atlantic salmon male and female recombination rates, a two-stage mapping strategy was employed. Initially, a sire-based QTL analysis was used to detect linkage groups with significant effects on IPN resistance, using two to three microsatellite markers per linkage group. A dam-based analysis with additional markers was then used to confirm and position any detected QTL. Two genomewide significant QTL and one suggestive QTL were detected in the genome scan. The most significant QTL was mapped to linkage group 21 and was significant at the genomewide level in both the sire and the dam-based analyses. The identified QTL can be applied in marker-assisted selection programs to improve the resistance of salmon to IPN and reduce disease-related mortality.

Tilapia stock identification using electrophoretic markers

Abstract With the difficulties in the identification of the many tilapia species, a reliable method independent of morphological characteristics was needed to define the nature of the cultured stocks throughout the world. Starch gel electrophoresis of various body tissues and the resolution of protein loci using specific histochemical techniques has shown that a large amount of co-dominant interspecific variation exists which may be used to identify unequivocally the nine different species studied. The technique and relevant buffer systems and stains are described together with an account of the enzymes observed and their interpretation. The results are expressed as relative mobilities of the observed isozyme bands, allowing direct comparison between all the species studied. The potential of the technique is discussed regarding its possibilities in tilapia culture and in the general genetic improvement of stocks.

Masculinization of genetic female Nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation.

A series of experiments was carried out in which genetically female Nile tilapia (Oreochromis niloticus) fry were treated with Fadrozole, a nonsteroidal aromatase inhibitor (AI), in the diet during the period of sexual differentiation. Batches of tilapia fry treated with AI during the first 30 days following yolk-sac resorption (7-37 days post hatch, dph) showed a dose-dependent increase in the percentage of males from 0 to 200 mg. kg(-1). The percentage of males remained approximately constant (92.5-96.0%) from 200 to 500 mg. kg(-1). Any continuous 2- or 3-week treatment with 500 mg. kg(-1) AI in this 4-week period successfully masculinized the majority of the treated fish (>80%). Treatments of 1 week duration revealed that the most sensitive time to AI lies in the first week (between 7 and 14 dph). Progeny testing of males from AI-treated groups gave results indicating that these were XX males, as expected. These experiments strongly implicate aromatase activity as a key factor in sexual differentiation in the Nile tilapia.

The conversion of linoleic acid and linolenic acid to longer chain polyunsaturated fatty acids by Tilapia (Oreochromis) nilotica in vivo

Tilapia (Oreochromis) nilotica were fed either a commercial diet containing 2.2% (n-3) and 0.5% (n-6) polyunsaturated fatty acids (PUFA), or a diet containing 1.0% methyl linoleate as the only PUFA. The fatty acid composition of tissue lipids generally reflected that of the diet. Fish from both dietary groups were injected intraperitoneally with 14C-labelled linoleic acid, 18:2 (n-6), or linolenic acid, 18:3 (n-3), and the distribution of radioactivity in tissue lipids examined. The conversion of both 18:2 (n-6) and 18:3 (n-3) to longer chain PUFA was lower in fish fed the commercial diet than in those fed the diet containing only 18:2 (n-6). Half of the radioactivity from both substrates recovered in liver polar lipids was present in C20 and C22 PUFA with fish maintained on the experimental diet. It is concluded that T. nilotica is capable of elongating and desaturating both 18:2 (n-6) and 18:3 (n-3), but that this conversion is suppressed by dietary longer chain PUFA.

The viability of cryopreserved tilapia spermatozoa

Abstract Tilapia ( Oreochromis spp.) spermatozoa protected with 12.5% methanol (MeOH) in fish Ringers, stored in 1.5-ml cryotubes and held in a vapour phase liquid nitrogen refrigerator remained viable for at least 13 months. The results, however, showed a high degree of variation; of the 16 samples tested only 66% produced viable cells on thawing. Mean fertilization rates of eggs were independent of storage time and ranged from 38.7 to 93.4%. To minimize variability a protocol using 0.5-ml plastic straws, which were held under liquid nitrogen, was developed and tested on the ability of post-thaw spermatozoa to fertilize eggs in comparison with an unfrozen control. Choice of cryoprotectant and its concentration, cooling and dilution rates and the fertilizing capacity of 0.5 ml of extended (ca. 25 μl milt) milt were investigated. The best pre-freezing activation of spermatozoa was obtained with MeOH and maximum cell protection was achieved using 10% MeOH. Cooling spermatozoa at rates of between 5 and 50°C/min and diluting milt at rates of between 1:2 and 1:20 had no significant ( P >0.05) influence on the rates of fertilization of eggs. Mean embryo yields ranged from 86.2 to 98.6%. Milt diluted at 1:20 could satisfactorily fertilize up to 500 eggs (140×10 3 spermatozoa/egg). The growth and survival of 30-day-old fry produced from milt stored for up to 12 months were not significantly ( P >0.05) different from normally produced fry.

The susceptibility of Atlantic salmon fry to freshwater infectious pancreatic necrosis is largely explained by a major QTL

Infectious pancreatic necrosis (IPN) is a viral disease with a significant negative impact on the global aquaculture of Atlantic salmon. IPN outbreaks can occur during specific windows of both the freshwater and seawater stages of the salmon life cycle. Previous research has shown that a proportion of the variation seen in resistance to IPN is because of host genetics, and we have shown that major quantitative trait loci (QTL) affect IPN resistance at the seawater stage of production. In the current study, we completed a large freshwater IPN challenge experiment to allow us to undertake a thorough investigation of the genetic basis of resistance to IPN in salmon fry, with a focus on previously identified QTL regions. The heritability of freshwater IPN resistance was estimated to be 0.26 on the observed scale and 0.55 on the underlying scale. Our results suggest that a single QTL on linkage group 21 explains almost all the genetic variation in IPN mortality under our experimental conditions. A striking contrast in mortality is seen between fry classified as homozygous susceptible versus homozygous resistant, with QTL-resistant fish showing virtually complete resistance to IPN mortality. The findings highlight the importance of the major QTL in the genetic regulation of IPN resistance across distinct physiological lifecycle stages, environmental conditions and viral isolates. These results have clear scientific and practical implications for the control of IPN.

Relative DNA content of somatic nuclei and chromosomal studies in three genera, Tilapia, Sarotherodon, and Oreochromis of the tribe Tilapiini (Pisces, Cichlidae)

Seven Tilapiine species from three generaTilapia, Sarotherodon, andOreochromis were cytogenetically studied for chromosome number, chromosome morphology, and DNA content. The chromosome number 2n=44 was the same in all seven species. Arm number (NF) differences indicate the possible role of pericentric invasions in the karyotypic evolution of these species. C-banding of metaphase chromosomes shows that heterochromatin is localised around the centromere in all species ofOreochromis and Sarotherodon butT. zillii has more heterochromatin with six chromosomes having completely C-positive short arms. DNA values vary between 0.84 pq forO. macrochir and 1.21 pq forO. aureus. No heteromorphic sex chromosome pair could be found in any species. These findings suggest that karyotypic evolution has occurred but does not appear to be associated with speciation in this group.

Immune responses of Nile tilapia (Oreochromis niloticus L.) clones: I. Non-specific responses.

The importance of genetic variation in the non-specific immune responses of Nile tilapia (Oreochromis niloticus L.) clones was investigated. Fully inbred clones (IC) of Nile tilapia, produced using gynogenesis and sex reversal, and crosses between these lines (outbred clones) were used in this study. Non-specific immune responses were compared between the ICs, including serum lysozyme activity and phagocytosis, and significant differences were observed between the different groups. Their natural resistance to Aeromonas hydrophila infection was also assessed by bacterial challenge. A positive correlation was observed between the level of infection obtained and the non-specific immune parameters measured. Cumulative mortalities of fish obtained in the study showed that when a IC susceptible to A. hydrophila was crossed with a resistant IC, the resulting progeny exhibited intermediate levels of resistance to that of their parents.

Mapping and validation of the major sex-determining region in Nile tilapia (Oreochromis niloticus L.) Using RAD sequencing

Sex in Oreochromis niloticus (Nile tilapia) is principally determined by an XX/XY locus but other genetic and environmental factors also influence sex ratio. Restriction Associated DNA (RAD) sequencing was used in two families derived from crossing XY males with females from an isogenic clonal line, in order to identify Single Nucleotide Polymorphisms (SNPs) and map the sex-determining region(s). We constructed a linkage map with 3,802 SNPs, which corresponded to 3,280 informative markers, and identified a major sex-determining region on linkage group 1, explaining nearly 96% of the phenotypic variance. This sex-determining region was mapped in a 2 cM interval, corresponding to approximately 1.2 Mb in the O. niloticus draft genome. In order to validate this, a diverse family (4 families; 96 individuals in total) and population (40 broodstock individuals) test panel were genotyped for five of the SNPs showing the highest association with phenotypic sex. From the expanded data set, SNPs Oni23063 and Oni28137 showed the highest association, which persisted both in the case of family and population data. Across the entire dataset all females were found to be homozygous for these two SNPs. Males were heterozygous, with the exception of five individuals in the population and two in the family dataset. These fish possessed the homozygous genotype expected of females. Progeny sex ratios (over 95% females) from two of the males with the “female” genotype indicated that they were neomales (XX males). Sex reversal induced by elevated temperature during sexual differentiation also resulted in phenotypic males with the “female” genotype. This study narrows down the region containing the main sex-determining locus, and provides genetic markers tightly linked to this locus, with an association that persisted across the population. These markers will be of use in refining the production of genetically male O. niloticus for aquaculture.

Mapping the sex determination locus in the Atlantic halibut (Hippoglossus hippoglossus) using RAD sequencing

BackgroundAtlantic halibut (Hippoglossus hippoglossus) is a high-value, niche market species for cold-water marine aquaculture. Production of monosex female stocks is desirable in commercial production since females grow faster and mature later than males. Understanding the sex determination mechanism and developing sex-associated markers will shorten the time for the development of monosex female production, thus decreasing the costs of farming.ResultsHalibut juveniles were masculinised with 17 α-methyldihydrotestosterone (MDHT) and grown to maturity. Progeny groups from four treated males were reared and sexed. Two of these groups (n = 26 and 70) consisted of only females, while the other two (n = 30 and 71) contained balanced sex ratios (50% and 48% females respectively). DNA from parents and offspring from the two mixed-sex families were used as a template for Restriction-site Associated DNA (RAD) sequencing. The 648 million raw reads produced 90,105 unique RAD-tags. A linkage map was constructed based on 5703 Single Nucleotide Polymorphism (SNP) markers and 7 microsatellites consisting of 24 linkage groups, which corresponds to the number of chromosome pairs in this species. A major sex determining locus was mapped to linkage group 13 in both families. Assays for 10 SNPs with significant association with phenotypic sex were tested in both population data and in 3 additional families. Using a variety of machine-learning algorithms 97% correct classification could be obtained with the 3% of errors being phenotypic males predicted to be females.ConclusionAltogether our findings support the hypothesis that the Atlantic halibut has an XX/XY sex determination system. Assays are described for sex-associated DNA markers developed from the RAD sequencing analysis to fast track progeny testing and implement monosex female halibut production for an immediate improvement in productivity. These should also help to speed up the inclusion of neomales derived from many families to maintain a larger effective population size and ensure long-term improvement through selective breeding.