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.

Detection of QTL affecting harvest traits in a commercial Atlantic salmon population.

Genetic variation in performance and quality traits measured at harvest has previously been demonstrated in Atlantic salmon aquaculture populations. To map major loci underlying this variation, we utilized data from 10 families from a commercial breeding programme. Significant QTL were detected affecting harvest weight and length traits on linkage group 1, and affecting waste weight on linkage group 5. In total, 11 of the 29 linkage groups examined showed at least suggestive evidence for a QTL. These data suggest that major loci affecting economically important harvest characteristics are segregating in commercial salmon populations.

Detection and confirmation of a major QTL affecting resistance to infectious pancreatic necrosis (IPN) in Atlantic salmon (Salmo salar).

Infectious pancreatic necrosis (IPN) is a viral disease currently presenting a major problem to the aquaculture of Atlantic salmon (Salmon salar), during both the freshwater and seawater stages of production. Genetic variation in resistance to IPN has previously been demonstrated and the purpose of this study was to determine whether this variation includes loci of major effect. The initial QTL detection methodology utilized the limited recombination seen in male salmon to detect QTL in ten large full-sib families, using a genome-wide scan of two to three markers per linkage group. QTL were then positioned by adding additional markers to the significant linkage groups in a female-based analysis. The most significant QTL was mapped to LG 21, and further confirmation of the LG 21 QTL is provided in an analysis of the QTL flanking markers in an additional nine full-sib families from the same population. The size of QTL effect is such that the QTL flanking markers can be immediately applied in marker-assisted selection programmes to improve the resistance of salmon populations to IPN, thus reducing mortality due to the disease.

Analysis of the incidence of infectious pancreatic necrosis mortality in pedigreed Atlantic salmon, Salmo salar L., populations.

A total of 77,124 Atlantic salmon post-smolts, representing 197 full-sib families produced by 149 males and 197 females, experienced a field challenge from infectious pancreatic necrosis virus (IPNV), following transfer to three separate seawater sites. The first IPN mortality was observed 45 days after transfer, and the duration of the epidemic varied between 37 and 92 days among sites. Mortalities were traced to their parental families by PIT (Passive Integrated Transpondes) tag records and DNA genotyping. Full-sib family mean incidence of mortality was calculated for each family on each site. Heritabilities were estimated based on the heterogeneity of chi-square using incidence within half-sib families and the variance in incidence among full-sib families, both on the observed and underlying liability scale. The observed correlation among families across sites was used to estimate genetic correlations. The overall mortality rate was 10.8%, with only small differences between sites, ranging from 10.3% to 11.9%. Heritabilities on the liability scale were found to be moderate to strong, and ranged between 0.24 and 0.81, with a pooled estimate of 0.43, greater than is typically associated with disease traits. Genetic correlations among sites were all substantial, between 0.71 and 0.78, and indicated that a substantial component of the genetic variation displayed within sites was common to all. The results show that field challenges can yield very good genetic information on family differences in resistance, especially when replicated over sites, which may then be developed for use in selection for breeding strains of Atlantic salmon with greater resistance to IPN.

Classification and phylogenetic relationships of African tilapiine fishes inferred from mitochondrial DNA sequences.

African cichlid fishes are composed of two major lineages, the haplochromines and the tilapiines. Whereas the phylogenetic relationships of the haplochromines have been studied extensively, primarily because of their spectacular adaptive radiations in the Great Lakes of East Africa, little is known about the relationships among the tilapiine species, despite the fact that they have become an important component of African, indeed world, aquaculture. To remedy this situation, molecular phylogenetic analysis of tilapiine fishes was undertaken. A segment of mitochondrial DNA encompassing the terminal part of the tRNA(Pro) gene and the most variable part of the control region was amplified by the polymerase chain reaction with DNA samples isolated from 42 tilapiine species, and the amplification products were subjected to heteroduplex analysis and sequencing. Phylogenetic trees based on 68 sequences revealed the existence of 11 sequence groups and 11 single-sequence branches. The groups, designated Ti1 through Ti11, were distinguished by specific combinations of diagnostic substitutions, formation of monophyletic clusters, and separation by genetic distances in excess of 0.04. Although the relationships among the groups could not be resolved, the sequences separated Oreochromis and Sarotherodon from Tilapia, as defined by Trewavas. The Oreochromis sequences clustered with the Sarotherodon sequences and thus supported the hypothesis that the mouthbrooding behavior of the tilapiine fishes evolved only once from the substrate-spawning behavior. Since on phylogenetic trees the O. alcalicus (sub)species were always separated from O. amphimelas by other Oreochromis species, it was concluded that the adaptation to life in water with a high salt concentration and high pH values evolved independently at least twice in the tilapiine fishes. The tilapiines diverged from the haplochromines more than 8 million years ago; most of the intragroup divergences among the tilapiines took place an estimated 1.1 to 6 million years ago.

Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus.

A brain aromatase gene was identified from the Nile tilapia Oreochromis niloticus. The cDNA sequence of this gene differed from that of the ovarian aromatase gene previously reported from this species. Tissue specific expression for both brain and ovarian aromatase genes was examined in the tissues of adult tilapia. Brain aromatase mRNA was expressed in the brain, kidney, eye, ovary, and testis, but not in the liver and spleen. Ovarian aromatase mRNA was expressed in the brain, spleen, ovary, and testis but not in the eye, kidney, and liver. Differential aromatase gene expression between the sexes was investigated in all-male (XY) and all-female (XX) groups of tilapia fry from fertilisation throughout the sexual differentiation period. Semi-quantitative RT-PCR analysis revealed that the initiation of expression of both aromatase genes lay between 3 and 4 dpf (days post fertilisation) in both sexes. The level of brain aromatase mRNA gradually increased throughout the period studied with little difference between the sexes. This contrasted with marked sexual dimorphism of ovarian aromatase mRNA expression. In females, the expression level was maintained or increased gradually throughout ontogeny, while the level in males was dramatically down-regulated between 15 and 27 dpf. Subsequently, the level of ovarian aromatase mRNA expression fluctuated slightly in both sexes, with the expression in females always being higher than in males. These findings clearly suggest that ovarian aromatase plays a decisive role in sexual differentiation in this species and that this is achieved by down-regulation of the expression of this gene in males. Mol. Reprod. Dev. 59: 359-370, 2001.

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.

Identification of putative sex chromosomes in the blue tilapia, Oreochromis aureus, through synaptonemal complex and FISH analysis.

Sex determination in the blue tilapia, Oreochromis aureus, is primarily a ZW female-ZZ male system. Here, by analysis of the pachytene meiotic chromosomes of O. aureus, we demonstrate the presence of two distinct regions of restricted pairing present only in heterogametic fish. The first, a subterminal region of the largest bivalent is located near to the region of unpairing found in the closely related species O. niloticus, while the second is in a small bivalent, most of which was unpaired. These results suggest that O. aureus has two separate pairs of sex chromosomes.

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.

Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.

Linkage relationships and haplotype polymorphism among cichlid Mhc class II B loci.

The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation.

A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus).

We have constructed a genetic map for a tilapia, Oreochromis niloticus, using DNA markers. The segregation of 62 microsatellite and 112 anonymous fragment length polymorphisms (AFLPs) was studied in 41 haploid embryos derived from a single female. We have identified linkages among 162 (93.1%) of these markers. 95% of the microsatellites and 92% of the AFLPs were linked in the final map. The map spans 704 Kosambi cM in 30 linkage groups covering the 22 chromosomes of this species. Twenty-four of these linkage groups contain at least one microsatellite polymorphism. From the number of markers 15 or fewer cM apart, we estimate a total map length of approximately 1000-1200 cM. High levels of interference are observed, consistent with measurements in other fish species. This map is a starting point for the mapping of single loci and quantitative traits in cichlid fishes.

Functionally significant allelic variation in myosin light chain composition in a tropical cichlid

Single fast muscle fibres in the tropical fish Oreochromis andersonii were found to contain two myosin light chains (LC1s; LC1f1* or LC1f2*). Breeding experiments confirmed that the different LC1s were of allelic origin and their inheritance patterns conformed to Mendelian expectations (1:2:1). The LC1s differed in apparent relative molecular mass by 800­900. No other differences in myosin subunits were found between the LC1 genotypes. The molar ratios of LC3:LC1(total) in the fast muscle of O. andersonii homozygous for LC1f1* or LC1f2* and heterozygous for both alleles were 2.0:1, 2.1:1 and 2.2:1, respectively, as determined by capillary electrophoresis. The maximum contraction velocity (Vmax) of single skinned muscle fibres was determined at 20 °C by the slack-test method. Vmax values (fibre lengths s-1) for fast muscle fibres from O. andersonii which were homozygous for either LC1f2* or LC1f1* were 5.3 and 3.3, respectively, compared with 3.8 when both alleles were present. Crosses between Oreochromis niloticus and O. andersonii produced F1 hybrids which were heterozygous for either LC1n/LC1f1* or LC1n/LC1f2*, where LC1n is the myosin light chain for O. niloticus. The distribution of myosin light chain genotypes in hybrid offspring was not significantly different from the expected Mendelian 1:1 ratio (47 %: 53 %). The Vmax (fibre lengths s-1) of muscle fibres containing LC1f2* from hybrid Oreochromis was 4.3 compared with 3.1 for the LC1f1* genotype. The results are consistent with a functionally significant allelic variation in myosin LC1 in fast muscle fibres from O. andersonii which is also expressed in hybrid genotypes.

Induction of diploid androgenetic and mitotic gynogenetic Nile tilapia (Oreochromis niloticus L.).

Androgenesis is a potentially valuable technique for recovering fish from gene banks composed of cryopreserved sperm, developing inbred lines, and analyzing patterns of inheritance. The procedure for producing diploid organisms whose nuclear DNA is wholly of paternal origin is dependent on: (1) the denucleation of "host" eggs, and (2) the inhibition of the first mitotic division in order to double the haploid sperm chromosome complement following fertilization of host eggs. Denucleation of tilapia (Oreochromis niloticus L.) eggs was carried out using UV irradiation. Treatment durations of 5-8 min (total dose of 450-720 J/m(2)) produced acceptable yields of viable denucleated eggs [22.9±1.6% (±SE) of controls] as estimated by the survival of haploid androgenetic tilapia to 48 h post-fertilization. Successful mitotic inhibition was accomplished using a heat-shock of 42.5 °C for 3-4 min, applied at 2.5-min intervals from 22.5 to 30 min post-fertilization (mpf). The mean survival of androgenetic diploid fish to yolk-sac absorption for treatment groups varied from 0.4% to 5.3%, relative to the controls. Differences in the suceptibility of eggs from different females to UV irradiation were a significant factor in the overall yield of androgenetic diploids. Paternal effects did not significantly influence the androgenetic yield, suggesting that individual males would not be selected against. For comparative purposes mitotic gynogenetic "mitogyne" diploids were produced from UV-irradiated sperm. Mean survival to yolk-sac absorption varied from 0.5% to 10.64%, relative to controls. Similar optima for androgenetic and gynogenetic induction were found in the period 25-27.5 mpf (minutes post-fertilization). Induction treatments would appear to be operating on the same developmental events in both these techniques, and the results suggest that the UV irradiations used do relatively little damage to the eggs beyond nuclear inactivation. The results indicate that the production of androgenetic O. niloticus is possible on a consistent basis and that the application of this technique may be useful in quantitative and conservation genetics.

Genetic variability in a family of satellite DNAs from tilapia (Pisces: Cichlidae).

We have cloned and sequenced members of a family of satellite DNAs from three genera of the tilapiine tribe of fishes: Oreochromis, Sarotherodon, and Tilapia. The satellite DNAs, visualized as intensely staining bands following electrophoretic separation of EcoRI-digested genomic DNA, consist of three size variants differentially distributed in the various tilapiine species. The sizes of the monomers are approximately 237 bp (type I), 230 bp (type II), and 209 bp (type III). Several cloned monomers were sequenced from Oreochromis niloticus (type III), Oreochromis placidus (types I and II), Sarotherodon galilaeus (type I), Tilapia zillii (type I), and Tilapia rendalli (type I). Comparison of derived consensus sequences for the monomer units of the satellite DNAs revealed sequence identities within and between species that ranged from 89 to 96%. The type II and type III size variants appear to have arisen by deletions of 9 and 29 bp, respectively, within different regions of the type I satellite. Hybridization of a cloned monomer satellite from O. niloticus (type III) to PalI digests of genomic DNA from all three genera detected polymorphic, high molecular weight restriction fragments that produced fingerprint-like patterns. The complexity of these DNA fingerprints varied from one species to another, suggesting a markedly different genomic organization for these polymorphic satellite DNAs.

Inter- and intra-specific variation in myosin light chain and troponin I composition in fast muscle fibres from two species of fish (genus Oreochromis) which have different temperature-dependent contractile properties.

The contractile properties and myofibrillar protein composition of fast muscle have been characterized in pure strains of two tropical fish Oreochromis niloticus and O. andersoni. Single fast muscle fibres were isolated from the abdominal myotomes and chemically skinned. The maximum tension-temperature relationships of fibres were similar at 25-30 degrees C, but diverged below 17 degrees C. At 10 degrees C, maximum tension was around 60% higher in O. andersoni (160 +/- 15 kN m-2) than O. niloticus (105 +/- 13 kN m-2) (mean +/- SD). The myofibrillar protein composition of fast fibres was investigated using one-dimensional and two-dimensional gel electrophoresis and peptide mapping. The two Oreochromis species differed with respect to the composition of myosin light chains, troponin I and myosin heavy chains (V8 protease and chymotrypsin peptide maps). An unexpected finding was the presence of two isoforms of myosin light chain 1 in O. andersoni, with apparent molecular masses of 27.5 kDa (LC1f1) and 26.9 kDa (LC1f2). Individuals with LC1f1 (n = 20) and LC1f1 + LC1f2 (n = 12) were represented in the population studied. The myosin light chain 3 (LC3f) content of fibres was similar in both cases. Breeding experiments established that these intra-specific variations in isoform composition were heritable. Fast muscle from O. niloticus and O. andersoni contain two isoforms of troponin I (TNIfl + TNIf2) which were both expressed in single fibres. The identity of TNI was confirmed using a stationary phase troponin-C affinity column. Of the 20 O. niloticus studied seven contained only TNIf1.(ABSTRACT TRUNCATED AT 250 WORDS)