Loss of genetic variability in the captive stocks of tambaqui,Colossoma macropomum (Cuvier, 1818), at breeding centres in Brazil,and their divergence from wild populations

The loss of variability in farmed populations and the risks of interactions with wild populations support the need for the genetic monitoring of species farmed throughout the world. In Brazil, the tambaqui is the most widely farmed native fish species. Despite this, there are no data on the pedigree of the farmed stocks, and the potential for interactions with wild populations in the Amazon basin has raised concerns with regard to the genetic variability of these stocks. The present study analysed sequences of the mitochondrial Control Region and 12 microsatellites to characterize the genetic variability of seven historically important commercial tambaqui breeding centres located in four different regions of Brazil, and compared these sequences with those obtained from individuals collected from a wild population. High levels of genetic diversity were found in the wild population, whereas genetic diversity was reduced in both markers in most captive populations, except for the broodstock located near the Amazon River. High F_ST and D_EST indices were recorded between the wild population and most of the captive stocks analysed. The drastic reduction in genetic diversity found in most captive stocks and the difference between these stocks and the wild population may have been the result of the small size of the founding populations and the absence of breeding management. The renewal of the broodstocks and the application of breeding management techniques are recommended. In the Amazon region, in addition, the use of broodstocks that are genetically very different from local wild populations should be avoided.     

Economic effects of production scale,use of agricultural greenhouses,and integration of tropical aquaculture species when farming in a subtropical climate

Fresh and local production of tropical fish species are growing in demand in subtropical and temperate regions. However, their productions are limited by the short growing season and uncertainty related to using agricultural greenhouses. Thus, this study evaluated the economic feasibility of Amazon River prawn (Macrobrachium amazonicum) and tambaqui (Colossoma macropomum) grow-outs in monoculture and integrated multi-trophic aquaculture (IMTA) systems carried out in a subtropical region of Brazil, considering the transfer of the fish to agricultural greenhouses to complete the grow-out during the winter season. Simulations were performed of each system on small- (1 ha) and medium (5 ha)-sized properties to test the effects of production system and scale on cost return and cash flow, economic indicators, and sensitivity (risk) to productivity and market changes. Treatments were prawn monoculture (PRWN), tambaqui monoculture (FISH), IMTA of prawns and tambaqui reared free-swimming (IMTA), and IMTA of prawns reared free-swimming and tambaqui reared in net cages (CAGE). Harvested prawns were marketed for recreational fishing and the tambaqui is traded for the next grow-out phase after overwintering in greenhouses. Internal rate of return (IRR), net present value (NPV), payback period (PP), and benefit–cost ratio (BCR) showed economic feasibility for all systems, and shorter PP and greater profitability were observed with an increased size of the properties. In addition, the integrated systems showed resilience by remaining economically feasible when subjected to variations in productivity, major costs, and selling price. Further research should test the technical feasibility of producing tambaqui in greenhouses during the winter in colder climates.

     

Observations on induced breeding of the Amazonian fish tambaqui, Colossoma macropomum (Cuvier) using CPE and HCG treatments

Trials on induced breeding of the native Amazonian fish tambaqui, Colossoma macropomum (Cuvier), using CPE and HCG treatments, were carried out during 1992-93 at the Fish Culture Station in Natal, Brazil. Final maturation, ovulation and spermiation of tambaqui broodstock, reared in earthen ponds, were induced by injecting heteroplastic carp pituitary gland extracts (CPE) and human chorionic gonadotrophin (HCG) separately. The female spawners received double injections and the males received a single dose. Females were tranquillized using quinaldine and their genital openings were sutured before the resolving injection was administered. Latent period between the resolving injection and ovulation varied from 6 to 11 h according to the prevailing water temperature. Milt was obtained by extrusion. Stripping and dry fertilization of the eggs produced viable larvae. Relative fecundity was estimated at 140 000 eggs kg^-1. Mean fertilization and hatching rates varied from 50% to 70% and from 60% to 80%, respectively.     

Eugenol as an efficacious anaesthetic for tambaqui, Colossoma macropomum (Cuvier)

Anaesthetics are important in fish culture to reduce handling stress and mortality. Eugenol is a promising anaesthetic because of its low cost, efficacy, safety margin for fish and lack of toxicity to humans. The goal of this study was to establish a protocol using eugenol as a fish anaesthetic for tambaqui Colossoma macropomum (Cuvier), and provide information for regulating authorities on establishing safety dosage protocols for its use. Juvenile and sub‐adult tambaqui were first individually exposed to doses of 35, 50, 65, 85, 100 or 135 mg L^?1 eugenol for 10 min. A second experiment examined the effect of the duration of exposure to eugenol on the time required for recovery and survival of tambaqui. A eugenol dose of 65 mg L^?1 was adequate to induce fish of both sizes into a surgical anaesthetic state, and recovery time was similar for dosages up to 100 mg L^?1. Exposure to the ideal dose (65 mg L^?1) for up to 30 min did not cause fish mortality. Fish blood glucose values were similar for all the tested eugenol doses as well as with the benzocaine control. The results show that eugenol is an efficient and safe anaesthetic for tambaqui.     

DNA barcoding reveals blend of silver catfish Rhamdia species from fish farms in Southern Brazil

Rhamdia quelen is the most produced native freshwater fish in fish farms in South Brazil. Until recently, Rhamdia branneri and Rhamdia voulezi were synonyms of R. quelen, and all the species are commercialized as silver catfish (locally called jundiá or bagre sapo) by the aquaculture industry. In fact, because these species are morphologically very similar, interspecific crosses easily might occur in fish farming. We employed standard DNA barcoding to identify jundiá molecular operational taxonomic units (MOTUs) in fish cultivated and commercialized in the industry and in possible escapees in the natural environment in southern Brazil. We analysed 48 individuals from six fish farms and 48 individuals from three rivers (Uruguay, Benedito Novo and Itapocu Rivers) using the mitochondrial cytochrome oxidase subunit I (COI). Four MOTUs were identified based on the estimated optimum threshold (OT = 0.77), and these MOTUs were concordant with Bayesian Inferece (BI) and Neighbour‐Joining (NJ) trees. Our results support the existence of at least three species in our dataset: R. branneri, R. voulezi, and R. quelen 1 and R. quelen 2. The interspecific genetic divergence ranged from 1.1% to 5.1% (mean = 3.5%), and the intraspecific distance ranged from 0% to 1.4% (mean = 0.24%). The presence of cultivated fish in the Uruguay and Benedito Novo Rivers provides evidence of genetic contamination in native populations. These results show the need to regulate aquaculture activities and to characterize the species and commercial lineages of silver catfish that are cultivated in South Brazil.     

Genotyping of Streptococcus dysgalactiae strains isolated from Nile tilapia,Oreochromis niloticus (L.)

Streptococcus dysgalactiae is an emerging fish pathogen that is responsible for outbreaks of disease on fish farms around the world. Recently, this bacterium was associated with an outbreak at a Nile tilapia, Oreochromis niloticus (L.), farm in Brazil. The aim of this study was to evaluate the genetic diversity, best genotyping method and aspects of molecular epidemiology of S. dysgalactiae infections in Nile tilapia farms in Brazil. Twenty‐one isolates from four farms located in different Brazilian states were characterized genetically using pulsed‐field gel electrophoresis (PFGE), ERIC‐PCR, REP‐PCR and sodA gene sequencing. The discriminatory power of the different typing methods was compared using Simpson's index of diversity. Identical sodA gene sequences were obtained from all isolates, and ERIC‐PCR and REP‐PCR were unable to discriminate among the isolates. PFGE typing detected three different genetic patterns between the 21 strains evaluated; thus, it was the best genotyping method for use with this pathogen. The strains from Ceará State were genetically divergent from those from Alagoas State. The S. dysgalactiae isolates analysed in this study constituted a genetically diverse population with a clear association between geographical origin and genotype.     

Effect of 17β‐oestradiol on the sex ratio of tambaqui,Colossoma macropomum

The tambaqui Colossoma macropomum is the most important native fish in Brazilian aquaculture. Females are almost 20% heavier than males and therefore are more profitable. Based on this information, we tested four different concentrations (20, 40, 80, and 120 mg/kg diet) of oestradiol (E₂) offered during 6 weeks to tambaqui larvae and analysed their efficiency in producing female monosex batches of the species. The study was performed in two independent tests, using different genetic backgrounds (broodstocks). All treatments increased the percentage of females in the population, but only the highest E₂ dose was “free” of males in both tests. All treated groups contained intersex individuals (when sex inversion is not complete). Plasmatic E₂ concentration was similar between treated and non‐treated fish 60 days after the treatment in fish treated with 20, 40, and 80 mg E₂/kg diet. However, tambaqui treated with a 120 mg E₂/kg diet showed lower E₂ plasma concentration in comparison with the control group. Last, we chlorinated the water to ensure the environmental integrity of the study. Three days after chlorination, the water was completely free of any oestradiol residue (analysed by high‐performance liquid chromatography).     

Genetic diversity and relatedness estimates for captive barramundi (Lates calcarifer,Bloch) broodstock informs efforts to form a base population for selective breeding

Aquaculture of barramundi or Asian seabass (Lates calcarifer) is growing in both Australia and Southeast Asia and there is substantial interest to improve production efficiency through selective breeding. The establishment of a large and genetically diverse base population is a prerequisite for a sustainable and long‐term productive breeding program. Before selective breeding programs can begin for Australian barramundi it is important to assess the overall genetic diversity of current captive broodstock populations. To address this question, 407 captive barramundi broodstock from eight separate Australian broodstock populations were genotyped using 16 polymorphic microsatellite DNA markers. A Bayesian STRUCTURE analysis indicated that captive Australian broodstock are broadly divided into two genetic stocks. Multivariate analysis between broodstock individuals and pairwise F_ST between broodstock populations also supported the existence of two stocks. Comparisons with data obtained from natural stocks suggested that hatchery individuals were either sourced from the two stocks or represented an admixture between them. Genetic diversity was low within each broodstock population (allelic richness ranged from 2.67 to 3.42 and heterozygosity ranged from 0.453 to 0.537) and relatedness estimates within hatcheries were generally low (average r was equal to 0.141). We recommend sourcing captive individuals according to high levels of neutral genetic diversity and low levels of relatedness for the establishment of a base population. We also make recommendations about including genetically diverse wild individuals.     

Growth and production of the Amazonian tambaqui in fixed cages under different feeding regimes

Experimental culture of the native Amazonian fish tambaqui, Colossoma macropomum, in fixed cages was carried out over a period of 8 months, in Lake Urubu (Rio Grande do Norte, Brazil), to assess the viability of fixed cage culture of tambaqui and to test the influence of diet on growth rates. Nine synthetic net cages (1 m³) were each stocked with 45-day-old fish (mean weight 3 g; mean total body length 51 mm) at a density of 34 fry m^–3. During the first 2 months of culture, fish were fed a balanced formulated feed on an as-fed basis at the rate of 5% body weight day^–1. During months 3–8 this continued for fish in treatment 1 while those in treatment 2 were fed tropical regional fruits, on a wet weight basis at the rate of 5% body wt day^–1. Fish in treatment 3 were given no supplementary feed. Monthly biometric measurements were made on all fish. Fixed cage fish culture was shown to be a viable and simple technique. Survival in all treatments was 100%. With balanced supplementary feed, production was 14.4 kg m^–3, compared with 4.9 kg m^–3 and 2.1 kg m^–3, respectively, in the treatments where fish were fed with fruits and were not given any supplementary feed.     

General and specific combining ability in Serrasalmidae

This work was carried out to predict the combining abilities, both general and specific, for performance traits and bodily yields of Serrasalmidae. Ninety‐six 30‐day‐old juveniles were purchased from two commercial fish farms, 12 of each of the following eight genetic groups: pacu, pirapitinga, tambaqui, tambacu, tambatinga, patinga, paqui and piraqui. Six fish from each genetic group were grown in 500‐L fibreglass tanks (two tanks per genetic group) until they were 495 days old. At the end of the growth period fish were weighed, subjected to morphometric analysis and processed to obtain their bodily yields. Two nuclear markers and one mitochondrial marker were used to confirm the identity of the animals. Combining abilities were obtained using the method proposed by Griffing in 1956 (Australian Journal of Biological Science, 4, 463–493) adapted to a mixed models analysis, environmental effects were estimated by the empirical best linear unbiased estimator method and genetic effects (general and specific combining abilities) were estimated with the empirical best linear unbiased predictor. Predictions of the combining abilities of advanced hybrids were obtained by the mixed models mixture method with normal distributions. Tambaqui showed higher general and specific combining abilities than the other groups for most of the variables, making it the most important genetic group. General combining ability makes a greater contribution to phenotypic variance than specific combining ability for most variables, indicating a predominance of genes with an additive effect in the control of evaluated traits.     

Genotyping‐by‐sequencing for construction of a new genetic linkage map and QTL analysis of growth‐related traits in Pacific bluefin tuna

Pacific bluefin tuna (Thunnus orientalis) has high market value, but its wild populations have decreased in recent years. The broodstock of Pacific bluefin tuna that were hatched artificially and reared under aquaculture conditions is beginning to be used for production. The creation of broodstock with commercially valuable traits, such as rapid growth, is therefore of great interest. Genetic linkage map‐based identification of markers associated with quantitative trait loci (QTLs) facilitates marker‐assisted selection (MAS) breeding and allows efficient genetic improvement of broodstock. Single nucleotide polymorphism (SNP)‐based genetic linkage map construction using the genotyping‐by‐sequencing method can expand the number of mapped markers and help identify growth‐related QTLs. In this study, we constructed sex‐specific maps for 24 linkage groups consisting of 677 SNP and 651 microsatellite markers. The total lengths of 93 progenies in the mapping population followed normal distribution, with an average length of 9.4 mm. We performed composite interval mapping in the mapping population. QTL analysis revealed one significant QTL in LG10 on the female linkage map. The genetic linkage map—the second such map generated for Pacific bluefin tuna—and the growth‐related QTLs detected in this study will be useful for tuna aquaculture MAS programs.     

Effect of Fish Density During Transportation on Stress and Mortality of Juvenile Tambaqui Colossoma macropomum

The increased demand for juvenile tambaqui Colossoma macropomum for grow-out ponds and stocking programs in the Amazon state of Brazil has increased the transportation of this species. This study was designed to determine the optimum density of juvenile tambaqui during transportation in closed containers. Fish (51.9 ± 3.3 g and 14.9 ± 0.4 cm) were packed in sealed plastic bags and transported for 10 h at four densities: 78, 156, 234, and 312 kg/m³. After transportation, fish from each density were kept in separate 500-L tanks for 96 h. Mortality, 96-h cumulative mortality, water quality, and blood parameters (hematocrit, plasma cortisol, and glucose) were monitored. Fish mortality after transportation was significantly lower at densities of 78 and 156 kg/m³ than at 234 and 312 kg/m³. Cumulative mortality was significantly lower at a density of 78 kg/m³. Dissolved oxygen after 10 h of transportation remained high at a density of 78 kg/m³, but reached critically low values at all other densities. Ammonia concentration was highest at the lowest density and was lower at higher densities. Carbon dioxide concentration was lowest at the density of 78 kg/m³ but higher in the other treatments. Plasma glucose and cortisol increased significantly immediately after transportation at densities of 156, 234, and 312 kg/m³, returning to control values by 24 h. The best density for juvenile tambaqui during a 10-h transportation haul in a closed container was 78 kg/m³. At this density there was no fish mortality, water quality was kept within acceptable values, and fish were not stressed.     

Differences in fatty acid composition of egg capsules from broodstock spotted babylon, <Emphasis Type="Italic">Babylonia areolata</Emphasis>, fed a local trash fish and formulated diet under hatchery conditions

This study is the first attempt to condition broodstock Babylonia areolata using formulated diets under hatchery conditions. Samples of spotted babylon egg capsules from broodstock fed either a formulated diet or a local trash fish, carangid fish (Seleroides leptolepis) for 120 days were analyzed for proximate composition and fatty acid composition. The formulated diet contained significantly higher levels of arachidonic acid (20:4n − 6; ARA), eicosapentaenoic acid (20:5n − 3; EPA) and docosahexaenoic acid (22:6n − 3; DHA) than those of the local trash fish. The formulated diet also had significantly higher ratios of DHA/EPA and (n − 3)/(n − 6) PUFA than those of local trash fish but not for the ARA/EPA ratio. The compositions of egg capsules produced from broodstock fed formulated diet contained significantly more ARA, EPA and DHA compared to broodstock fed the local trash fish. The ARA/EPA and DHA/EPA ratios in egg capsules were significantly higher in the trash fish—fed group compared to those fed the formulated diet. However, (n − 3)/(n − 6) PUFA ratios in egg capsules produced from broodstock fed the formulated diet did not differ significantly compared to those from broodstock fed the local trash fish. The relatively low DHA/EPA, ARA/EPA and (n − 3)/(n − 6) ratios in the egg capsules produced from the formulated diet—fed broodstock B. areolata suggested that this diet is inferior, when compared to the traditional food of trash fish.     

Parentage assignment of stocked black sea bream <Emphasis Type="Italic">Acanthopagrus schlegelii</Emphasis> in Hiroshima Bay using microsatellite DNA markers

The genetic contribution of 51 broodstock, comprising 29 females and 22 males, reared at Hiroshima City Marine Products Promotion Center for the production of stocked black sea bream was monitored during two consecutive years using seven microsatellite DNA loci. The high discrimination ability of these markers was reflected in the polymorphic identification content (PIC=0.831), the exclusion probability (Q≈1), and the low probability of identity index (I=3.635⁻¹⁰). The total number of breeders contributing to the mating process was estimated at 32 (62.7%) in 2000 and 30 (58.8%) in 2001. On pedigree reconstruction, 69.3% of the offspring were successfully assigned to a single broodstock pair. Loss of alleles accounted for 16.9% during seed production; nevertheless, 90.9% of males and 69.0% of females participated in the mating process. Based on microsatellite genetic tagging, 58.9% of the fish sampled during the two months after release were identified as hatchery stock, presenting no significant differences from wild conspecifics in either fork length or body weight.     

Temporal and Spatial Variability of Limnological Characteristics in Areas under the Influence of Tilapia Cages in the Chavantes Reservoir,Paranapanema River,Brazil

The influence of tilapia cages on limnological characteristics of the Chavantes Reservoir, Paranapanema River, Brazil, was investigated in March and June 2007. Principal components analysis was used to compare the transparency; dissolved oxygen; temperature; total organic nitrogen, phosphorus, and dissolved solids; chlorophyll a and electrical conductivity of fish farm 1 (239.3 m.t.), 2 (461.5 m.t.) and areas without cages, in the lacustrine and river‐reservoir transition regions. The variables differentiated the regions and areas with and without cages. In March, in the lacustrine region, there was a thermal and chemical stratification, and in the transition region, there was a temperature and oxygen reduction gradient between the surface and bottom that was also observed in June in fish farm 1. The higher transparency confirmed the increased light penetration in the lacustrine region. In the transition region, there were higher turbidity, total dissolved solid, and conductivity values that are likely related to allochthonous material flow and transport. In fish farms, lower oxygen and pH values indicate intense decomposition and respiration. The generally higher total dissolved solid and nutrient values, turbidity, and conductivity and the lower transparency in one or both fish farms indicate their contribution to eutrophication, demonstrating the need for careful monitoring.     

The effect of fish and rapeseed oils on growth performance,egg fatty acid composition and offspring quality of sterlet sturgeon (Acipenser ruthenus)

The aim of this study was to evaluate the long‐term effects (7‐month experiment) of diets consisting of fish oil (Kilka fish) and vegetable oil (rapeseed) on the reproductive performance of sterlet sturgeon (Acipenser ruthenus) broodstock. Forty‐five broodstock (990.3 ± 20.05 g) were randomly allocated to three different diet treatments. Three experimental diets were formulated with graded levels of fish oil (100% FO), vegetable oil (100% VO), and a combination of fish and vegetable oil (50% FO + 50% VO). At the end of the 7‐month feeding trial period, the weight gain and final weight were changed significantly different between the treatments (p < 0.05). Broodstock fed the FO + VO diet had higher growth than those fed the only FO or VO diets (p < 0.05). The highest germinal vesicle migration percentage was observed in FO + VO treatment (p < 0.05). The DHA/EPA, DHA/ARA and EPA/ARA ratios in oocyte exhibited a significant difference in the different treatments (p < 0.05). This study indicates that nutrition of broodstock with diet including FO + VO (p < 0.05) can positively affect the growth performance of larvae compared with only FO or VO diets. Furthermore, the high levels of 18:1n‐9, AL and ALA contents in oocytes from broodstock fed VO and the lowest ALA content in oocytes from broodstock fed FO underlined the important role of broodstock diets in the reproductive process and embryonic and/or larval developments of sterlet.     

Forecasting the Genetic Impacts of Net Pen Failures on Gulf of Mexico Cobia Populations Using Individual‐based Model Simulations

Offshore net pen fish farming provides a cost‐efficient means for production of marine finfish, and there is great interest in development of net pen operations in domestic waters. However, there are concerns over the possible genetic and ecological impacts that escaped fish may have on wild populations. We used individual‐based simulations, with parameter values informed by life history and genetic data, to investigate the short‐term (50 yr) impacts of net pen failures on the genetic composition of cobia, Rachycentron canadum, stocks in the Gulf of Mexico. Higher net pen failure rates resulted in greater genetic impacts on the wild population. Additionally, the use of more genetically differentiated source populations led to larger influxes of non‐native alleles and greater temporal genetic change in the population as a result of net pen failure. Our results highlight the importance of considering the appropriate source population for broodstock collection in net pen aquaculture systems and help to provide a general set of best management practices for broodstock selection and maintenance in net pen aquaculture operations. A thorough understanding of the genetic diversity, stock structure, and population demography of target species is important to determine the impact escapees can have on wild populations.     

Finishing plant diet supplemented with microalgae meal increases the docosahexaenoic acid content in Colossoma macropomum flesh

The need for a sustainable aquaculture is increasing the use of plant ingredients in replacement to fishmeal and fish oil in diets for tambaqui Colossoma macropomum, which is leading to not detectable levels of docosahexaenoic acid (DHA) in its flesh. We evaluated the effect of a finishing plant diet supplemented with 5% of microalgae meal from Schizochytrium sp. (MD) on tambaqui growth, on proximal composition and fatty acid content of its flesh, comparing it to a non‐supplemented diet. One hundred and sixty‐two fish (489.67 g) were distributed into six tanks (2,000 L) and fed the experimental diets for a 90‐day period. Three fish per tank were euthanized for analyses every 15 days. The MD did not affect the growth and proximal composition of fish flesh. The MD increased the DHA content (from 14.81 to 38.60 mg/g of lipids) and the n‐3:n‐6 ratio (from 0.16 to 0.51) in the flesh of fish, beginning on the 15th day and reaching the highest DHA content on the 71st day (39.81 mg/g of lipids). We recommend C. macropomum to be fed with a finishing diet supplemented with microalgae meal for 71 days before slaughter to improve the DHA content and n‐3:n‐6 ratio in the flesh.     

Tolerance response of tambaqui Colossoma macropomum (Cuvier) to water pH

Tambaqui Colossoma macropomum (Cuvier) is a fish of primary importance in Amazon aquaculture. It has been described as an acid‐resistant species that moves seasonally between white (muddy) water and black water rivers and enters the extremely dilute acidic areas of flooded jungle to feed during the rainy season. To analyse the pH tolerance of this species, tambaqui were exposed to three water pH levels for 40 days (pH 4.0, 6.0 and 8.0). The water was acidified slowly over 3 h, allowing the fish to acclimate. A similar protocol was used to adjust water pH to 8.0. No mortality was observed during the exposure period. Several haematological parameters were significantly changed in alkaline‐exposed animals, with significant decreases in haematocrit (20%), haemoglobin concentration (8%) and red blood cells (12%). Tambaqui showed severe blood variations when exposed to alkaline pH. Fish final weight, condition factor and specific growth rate (SGR) was inversely proportional to a pH increase, and SGR were higher for fish reared in acidic water. The relative insensitivity of tambaqui to low pH confirms its acid tolerance and is in accordance with its natural occurrence in black water habitats.     

Influence of Hatchery Protocols on Mitochondrial DNA Variation in Japanese Flounder Juveniles

The Japanese flounder Paralichthys olivaceus is a commercially important fish that is stocked extensively from hatchery rearing programs in Japan. To examine the genetic variability of hatchery-raised juveniles of the Japanese flounder that are used for stocking into natural waters, we analyzed a portion of the mitochondrial genome. The mtDNA region extending from the 3'half of the cytochrome b gene to the central domain of the control region was PCR amplified and analyzed using 11 restriction endonucleases. We identified 34 polymorphic cleavage sites out of a total of 61 sites, which resulted in 67 different haplotypes in a total of 265 offspring, examined from eight hatchery stocks. Haplotype diversity of offspring at each of the eight hatcheries ranged from 0.49 ± 0.09 (SE) to 0.94 ± 0.03 (SE). Also, we observed 40 polymorphic sites out of a total of 59 sites, which resulted in a total of 50 haplotypes in 60 wild flounder. Haplotype diversity of the wild population was 0.98 ± 0.01 (SE). The use of subcultured fishes as broodstock appears to be one of the most important causes of reduced genetic diversity in hatchery-raised flounder juvenile. Our results suggest that the use of wild fish for broodstock is an effective way to maintain genetic variability in Japanese flounder offspring.