A deleterious effect associated with UNH159 is attenuated in twin embryos of an inbred line of blue tilapia Oreochromis aureus.

Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12-fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed-atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub-sets of twins and normal full-sibs. Consistent with previous reports, the normal embryo sub-set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2-8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA-binding motif protein, X-linked (rbmx), SRY-box containing gene 3 (sox3) and alpha-thalassemia/mental retardation syndrome X-linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X-chromosome inactivation.

Linkage and Physical Mapping of Sex Region on LG23 of Nile Tilapia (Oreochromis niloticus).

Evidence supports that sex determination (SD) in tilapia is controlled by major genetic factors that may interact with minor genetic as well as environmental factors, thus implying that SD should be analyzed as a quantitative trait. Quantitative trait loci (QTL) for SD in Oreochromis niloticus were previously detected on linkage groups (LG) 1 and 23. Twenty-one short single repeats (SSR) of >12 TGs and one single nucleotide polymorphism were identified using the unpublished tilapia genome sequence on LG23. All markers showed two segregating alleles in a mapping family that was obtained by a cross between O. niloticus male (XY) and sex-reversed female (ΔXY) yielding 29 females (XX) and 61 males (XY and YY). Interval mapping analysis mapped the QTL peak between SSR markers ARO172 and ARO177 with a maximum F value of 78.7 (P < 7.6 × 10(-14)). Twelve adjacent markers found in this region were homozygous in females and either homozygous for the alternative allele or heterozygous in males. This segment was defined as the sex region (SR). The SR encompasses 1.5 Mbp on a single tilapia scaffold (no. 101) harboring 51 annotated genes. Among 10 candidate genes for SD that were tested for gene expression, anti-Müllerian hormone (Amh), which is located in the center of the SR, showed the highest overexpression in male vs. female embryos at 3 to 7 days postfertilization.

Fine-mapping of a locus on linkage group 23 for sex determination in Nile tilapia (Oreochromis niloticus).

Genetic markers in tilapia species associated with loci affecting sex determination (SD), sex-specific mortality or both were mapped to linkage groups (LG) 1, 2, 3, 6 and 23. The objective of this study was to use these markers to fine-map the locus with the greatest effect on SD in Oreochromis niloticus. Our parental stock, full-sibs of Nile tilapia (Swansea origin), were divided into three groups: (i) untreated, (ii) feminized by diethylstilbestrol and (iii) masculinized by 17α-methyltestosterone. We analysed the first group for association of microsatellite markers representing these five LGs. The strongest association with gender was found on LG23 for marker UNH898 (χ(2) ; P=8.6×10(-5) ). Allele 276 was found almost exclusively in males, and we hypothesized that this allele is a male-associated allele (MAA). Sex-reversed individuals were used for mating experiments with and without the segregating MAA. Mating of individuals lacking the MAA resulted in all-female progeny. Mating of two heterozygotes for MAA gave rise to 81 males and 30 females. Analysis of association between gender and genotypes identified the MAA in 98.6% of males as opposed to 8.0% of females (χ(2) ; P=2.5×10(-18) ). Eight markers that flank UNH898 were genotyped to map the locus on LG23 within a confidence interval of 16-21 cM. Mating of homozygous individuals for MAA is underway for production of all-male populations.

Copy number variation of lipocalin family genes for male-specific proteins in tilapia and its association with gender.

Lipocalins are involved in the binding of small molecules like sex steroids. We show here that the previously reported tilapia male-specific protein (MSP) is a lipocalin encoded by a variety of paralogous and homologous genes in different tilapia species. Exon-intron boundaries of MSP genes were typical of the six-exon genomic structure of lipocalins, and the transcripts were capable of encoding 200 amino-acid polypeptides that consisted of a putative signal peptide and a lipocalin domain. Cysteine residues are conserved in positions analogous to those forming the three disulfide bonds characteristic of the ligand pocket. The calculated molecular mass of the secreted MSP (20.4 kDa) was less than half of that observed, suggesting that it is highly glycosylated like its homologue tributyltin-binding protein. Analysis of sequence variations revealed three types of paralogs MSPA, MSPB and MSPC. Expression of both MSPA and MSPB was detected in testis. In haploid Oreochromis niloticus embryos, each of these types consisted of two closely related paralogs, and asymmetry between MSP copy numbers on the maternal (six copies) and the paternal (three copies) chromosomes was observed. Using this polymorphism we mapped MSPA and MSPC to linkage group 12 of an F(2) mapping family derived from a cross between O. niloticus and Oreochromis aureus. Females with high MSP copy number were more frequent by more than twofold than males. Gender-MSPC combinations showed significant deviation from expected Mendelian segregation (P=0.009) suggesting elimination of males with MSPC copies. We discuss different hypotheses to explain this elimination, including possibility for allelic conflict resulted by the hybridization.

Genetics of sex determination in tilapiine species.

We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongae and Tilapia mariae the sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticus and T. zillii the sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. Sex-specific recombination suppression was found in the female heterogametic sex. Sequence analysis showed the accumulation of repetitive elements in this region. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in this clade. This variation in sex determination mechanisms among closely related species makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates.

Genome-scan analysis for quantitative trait loci in an F2 tilapia hybrid.

We searched for genetic linkage between DNA markers and quantitative trait loci (QTLs) for innate immunity, response to stress, biochemical parameters of blood, and fish size in an F2 population derived from an interspecific tilapia hybrid (Oreochromis mossambicusx O. aureus). A family of 114 fish was scanned for 40 polymorphic microsatellite DNA markers and two polymorphic genes, covering approximately 80% of the tilapia genome. These fish had previously been phenotyped for seven immune-response traits and six blood parameters. Critical values for significance were P <0.05 with the false discovery rate (FDR) controlled at 40%. The genome-scan analysis resulted in 35 significant marker-trait associations, involving 26 markers in 16 linkage groups. In a second experiment, nine markers were re-sampled in a second family of 79 fish of the same species hybrid. Seven markers (GM180, GM553, MHC-I, UNH848, UNH868, UNH898 and UNH925) in five linkage groups (LG 1, 3, 4, 22 and 23) were associated with stress response traits. An additional six markers (GM47, GM552, UNH208, UNH881, UNH952, UNH998) in five linkage groups (LG 4, 16, 19, 20 and 23) were verified for their associations with immune response traits, by linkage to several different traits. The portion of variance explained by each QTL was 11% on average, with a maximum of 29%. The average additive effect of QTLs was 0.2 standard deviation units of stress response traits and fish size, with a maximum of 0.33. In three linkage groups (LG 1, 3 and 23) markers were associated with stress response, body weight and sex determination, confirming the location of QTLs reported by several other studies.

Two unlinked loci controlling the sex of blue tilapia (Oreochromis aureus).

Sex determination in the blue tilapia (Oreochromis aureus) is thought to be a WZ-ZZ (female heterogametic) system controlled by a major gene. We searched for DNA markers linked to this major gene using the technique of bulked segregant analysis. We identified 11 microsatellite markers on linkage group 3 which were linked to phenotypic sex. The putative W chromosome haplotype correctly predicts the sex of 97% of male and 85% of female individuals. Our results suggest the W locus lies within a few centimorgans of markers GM354, UNH168, GM271 and UNH131. Markers on LG1 also showed a strong association with sex, and indicate the segregation of a male-determining allele in this region. Analysis of epistatic interactions among the loci suggests the action of a dominant male repressor (the W haplotype on LG 3) and a dominant male determiner (the Y haplotype on LG1). These markers have immediate utility for studying the strength of different sex chromosome alleles, and for identifying broodstock carrying copies of the W haplotype.

Sex determination in crayfish: are intersex Cherax quadricarinatus (Decapoda, Parastacidae) genetically females?

In the Australian red-claw crayfish Cherax quadricarinatus (von Martens) (Decapoda, Parastacidae), a gonochoristic species, seven different combinations of intersex individuals (with both male and female genital openings) have been described. However, to date, the genetic basis for this phenomenon has not been investigated. This study was designed to test a simple chromosome-based sex-determination model for C. quadricarinatus that assumes the male to be the homogametic (ZZ) sex. According to our model, intersex individuals that are functionally males are genetically females (WZ). Individual crosses were performed between intersex and female crayfish, with control crosses being performed between normal males and females. The control crosses yielded, in most cases, the expected 1:1 sex ratio in the F1 progeny. Crosses between intersex individuals and females yielded a 1:3 (male:female) sex ratio in most crosses. According to our hypothesis, one-third of the females produced in a cross of a female with an intersex animal should be WW females. The hypothesis was tested by crossing normal males with F1 females, which were progeny of intersex fathers. These crosses yielded almost 100% females, a finding that conforms to the above-suggested sex determination model for C. quadricarinatus and the female WZ genotype of intersex individuals.

Association between loci with deleterious alleles and distorted sex ratios in an inbred line of tilapia (Oreochromis aureus).

Three microsatellite markers (UNH159, UNH231, and UNH216) were examined for association with both deleterious genes and sex-ratio distortions in a full-sib family of 222 progeny from the fourth generation of a meiogynogenetic tilapia line (Oreochromis aureus). The three markers were mapped previously to different linkage groups and were shown to be associated with genes with deleterious alleles in this line. A restricted maximum likelihood model was used for analysis of major effects and their interactions on sex ratio and viability. This model was based on selective mortality of genders, ignoring effects of possible sex-determining genes. The results showed that deleterious genes linked to UNH216 and UNH231 exert higher lethality in females than in males (P < .0005 and P < .05, respectively). UNH159 was not associated directly with sex ratio distortion, but acts strongly as a modifier of sex ratio in combination with UNH216 and UNH231. Each of the three loci was found to have a significant effect on viability (P < .05) in the maximum likelihood analysis. The deleterious single-locus effects act strongly against females, while most of the epistatic interactions exert higher lethality in males. This contradiction results in a close to 1:1 sex ratio at maturity. The genetic mechanism and significance of such a balance between genders are still unknown. A detailed analysis of sex-specific lethality may be applied by screening in appropriate series of matings and fine mapping with additional markers. Our data suggest that UNH216 and UNH231 are linked to sex ratio distortion genes and that UNH159 may be linked to a modifier of these genes.

Genetic manipulations in aquaculture: a review of stock improvement by classical and modern technologies.

The aim of this review was to highlight the extent to which the genetic technologies are implemented by the aquaculture industry. The review shows that some of the modern genetic technologies are already extensively applied by the diverse aquaculture industries, though not to the same extent for all important aquacultured species (according to FAO 1998 figures). Some species (common carp, Atlantic salmon, rainbow trout, channel catfish, Nile tilapia, and the Pacific oyster) received concentrated breeding efforts, while other major cultured species (Chinese and Indian carps and the giant tiger shrimp) received, so far, relatively limited attention, and a few species (Yesso scallop, blue mussel, white Amur bream, and milkfish) have, apparently, not been genetically improved at all. Most of the genetically improved strains reaching the aquaculture industry were developed through traditional selective breeding (selection, crossbreeding, and hybridization). Emerging, more modern technologies for genetic manipulation seem to take 10-20 years from being established experimentally until applications affect the industry. Thus, chromosome-set and sex manipulations started to affect the industry during the 1980's and 1990's. DNA marker technology and gene manipulations have yet hardly affected the industry. The former have not matured yet, but hold much promise. The latter could have affected the industry already had it not been restricted by public concern.

Intersex Red Claw Crayfish, Cherax quadricarinatus (von Martens): Functional Males with Pre-vitellogenic Ovaries.

Intersex individuals, possessing both male and female genital openings, were assessed in two groups-7 and 19 months old-of Australian red claw crayfish (Cherax quadricarinatus). All intersex individuals investigated were functional males, as suggested by their malelike morphology and the presence of testes, sperm ducts, androgenic glands, and viable spermatozoa. When an ovary was present in an intersex individual from either group, the gonadosomatic index, the diameter of the oocytes, and the ovarian cytosolic polypeptide profile were similar to those of immature, pre-vitellogenic females. We conclude that intersexuality in C. quadricarinatus does not indicate a case of protandric sequential hermaphroditism, as previously suggested. The case of intersexuality described here presents a unique model for the study of the role of the androgenic gland in the regulation of sex differentiation in crustaceans.

Induced diploid gynogenesis and polyploidy in the ornamental (koi) carp, Cyprinus carpio L. : 4. Comparative study on the effects of high- and low-temperature shocks.

The effects of high- and low-temperature shock treatments, applied at different phases of the 2nd meiotic division within the limits of 0.05-0.60 τ0 (τ0 = relative unit of embryological age) in order to induce gynogenesis in the common carp, were studied. A remarkable difference in the effect of two temperature treatments applied at the same biological age after insemination (expressed in τ0) was revealed. The curves of embryo survival and diploid gynogenetic larva output showed a bimodal response in cold-shocked gynogenetic progenies, with the highest level of diploid larva output at the periods 0.05-0.10 τ0 and 0.30-0.40 τ0 (after insemination), separated by a period of high sensitivity to cold shock (0.15-0.25 τ0). In contrast to this, the curves of embryo survival and diploid gynogenetic larva output showed a single, narrow, peak corresponding to 0.15-0.25 τ0 in heat-shocked gynogenetic progenies. The results obtained are in general accord with those of previous experiments on induced gynogenesis and triploidy in common carp, in which either cold- or heat-shock was used.

Induced diploid gynogenesis and polyploidy in the ornamental (koi) carp Cyprinus carpio L. : 3. Optimization of heat-shock timing during the 2nd meiotic division and the 1st cleavage.

The results of a series of experiments conducted in our laboratory on the ornamental common carp (koi), aimed at optimizing heat-shock chromosome-set manipulation procedures, are described. The timing of heat-shock initiation was expressed in the relative unit of embryological age (τ0) in order to standardize this parameter, the absolute time for heat-shock initiation being calculated from duration of one τ0 at two different pre-treatment water temperatures. Heat shocks were applied within the periods of 0.05-0.60 τ0 and 1.20-2.20 τ0 which, respectively, cover the successive phases of the 2nd meiotic division and the 1st cleavage. The highest production of diploid gynogenetic offspring was observed when heat shocks were initiated at 0.15-0.25 τ0 and at 1.5 τ0, after insemination, corresponding to anaphase of meiosis-II, and metaphase of the 1st cleavage, respectively. Similar results were obtained irrespective of the different pre-treatment water temperatures, thus confirming the possibility of standardizing heat-shock timing by τ0.

Growth, Size Rank, and Maturation of the Freshwater Prawn, Macrobrachium rosenbergii: Analysis of Marked Prawns in an Experimental Population.

For four months we marked and followed through female maturation and adult male mophotypic differentiation, the growth of all 150 individuals in an experimental population of Malaysian giant freshwater prawns (Macrobrachium rosenbergii). Small immature female prawns had high growth rates. Growth of female prawns nearly ceased after maturation. This compensatory growth process produces adult females having a unimodal, symmetrical size distribution with a mean above the size threshold for maturation (about 18-26 g). The small male morphotype has a low growth rate, while the orange claw male morphotype has a high growth rate. As the orange claw males transform to the blue claw morphotype, growth ceases. Examination of changes in size rank during the maturation process supported the leapfrog phenomenon. The fastest growing, largest orange claw male is the first to metamorphose to the blue claw morphotype (at a size of 35 g). As other orange claw males exceed this size, they transform in a sequential process so that the most recent blue claw male is generally the largest blue claw male in the population. Thus, growth of males is depensatory throughout the process of morphotypic differentiation, leading to a wide size range of orange and blue claw males. The leapfrog phenomenon is discussed in terms of the reproductive success of the blue claw males and compared with related growth processes in male poeciliid fishes. Implications of this growth process for aquacultural productivity includes the stimulatory effect on the remaining prawns of selectively harvesting the largest blue claw and orange claw prawns and suggests that the inclusion of a small proportion of large "target" blue claw males might stimulate the rapid growth of orange claw males in a population of smaller prawns.

Genetic differences between the Chinese and European races of common carp : 5. Differential adaptation to manure and artificial feeds.

Common carp of the Chinese and European races and their cross were tested in different environments. The test groups were either stocked together into the same pond, or each group was stocked separately. Mean growth, taken as a measure of the quality of the environment, varied widely between treatments. Genotype-environment interactions were estimated by the regression of growth of different genetic groups on this measure of environment. Proportional growth differences between the European and European X Chinese crossbreds, were several times higher in manured ponds than in ponds with artificial feed. The Chinese fish showed the fastest relative growth in poor conditions, with manure as the major nutrient input, while the European fish showed the fastest relative growth under improved conditions and irrespective of its source of food. The Chinese X European crossbred is heterotic over a range of intermediate conditions with manure as the principal nutrient.

Genetic improvement of wild fish populations.

A plan for the genetic improvement of commercially exploited wild animals is presented. It consists of crossing wild with domesticated breeds to produce heterotic hybrids and to upgrade the wild stocks. Empirical evidence is presented from experiments with the carp. Procedures for monitoring the manipulated populations are outlined. The suggested plan is ecologically reasonable and would counteract the negative genetic changes caused by excessive commercial exploitation of many species.