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.     

Use of Microsatellite DNA Profiling to Identify Japanese Flounder,Paralichthys olivaceus of Hatchery Origin

Japanese flounder, Paralichthys olivaceus, is an economically important marine fish species in Asia. A suite of 18 microsatellite markers chosen from published genetic linkage maps was used to carry out parentage assignments of 188 hatchery‐reared juveniles from a small number of breeders. The probabilities of exclusion for the 18 microsatellite markers were 0.604–0.913, and the effectiveness of combined probability of exclusion reached 100% when using the eight microsatellite markers with higher Excl 1 probabilities. The cultured and wild stocks (WSs) were differentiated in a release‐recapture population based on these markers. Of the 321 recaptured offspring, 28.34% were assigned to their parental pairs in our broodstock, whereas the remaining offspring could not be traced back to a possible sire or dam. Significant reduction in genetic diversity of the cultured stock (CS) had not been found compared with that of the WS. The results suggest that CSs released into the wild will not adversely affect the genetic structure of natural populations. Our results demonstrate that these markers provide an efficient tool for parentage assignments and genetic analysis of Japanese flounder.     

Loss of genetic diversity due to hatchery culture practices in barramundi (Lates calcarifer)

Many aquaculture hatchery practices are detrimental to the long-term viability of restocking and selective improvement programs. Small effective broodstock population sizes, differential broodstock contribution, differential larval/juvenile survival during metamorphosis and size-based grading, all have the potential to drastically reduce the level of genetic variation remaining in hatchery populations. Monitoring levels of genetic variation and maintaining detailed pedigrees on progeny is the key to circumventing these problems. In this study we used microsatellites, coupled with DNA parentage analyses, to track the loss of genetic diversity in two independent commercial barramundi (Lates calcarifer) hatcheries over three mass spawning events, where up to two females and seven males had the opportunity to participate. Initial broodstock contributions were observed to be highly skewed, with significant differences observed in both the level of contribution by females to each mass spawning, as well as in the number of males participating and subsequently contributing to the genetic composition of cohorts. Effective population sizes were around half that of census sizes. We then examined whether differential family survival through metamorphosis (27 days post-hatch) and/or first size grading further influenced the retention of genetic diversity levels initially sampled during spawning. Parentage analyses indicated that some families that had been initially represented in cohorts had been lost, or that the contribution by particular broodstock had changed. In one cohort, as many as 55% of progeny were found to be sired by a single male individual. Size grading was also found to potentially impact on genetic diversity, with data suggesting that family representation in each of the grades was non-uniform and that some families were on average faster or slower growing than others. These results illustrate that hatchery management practices have the potential to significantly impact on the retention of genetic diversity in this species.     

Identification of differential broodstock contribution affecting genetic variability in hatchery stocks of Atlantic salmon (Salmo salar)

Supportive breeding of Atlantic salmon (Salmo salar) is commonly employed to maintain numbers of fish where the species has become locally endangered. Increasingly, one of the main aims of population management is the preservation of natural genetic diversity. If the stocks employed in supportive breeding exhibit reduced variation they can alter the natural pattern of genetic variation observed in wild populations. In northern Spain, wild adult salmon are caught every year from local rivers and artificially crossed in order to create supportive stocks. The offspring are hatchery reared until the juvenile stage, then released into the same river where their parents were caught. In the current study, our findings demonstrate that although adult broodstock exhibit a pattern of variation similar to the wild populations, variability at microsatellite loci was drastically reduced in the juveniles released into one of three rivers analyzed. The contribution of broodstock to this juvenile stock was examined by pedigree analysis. A restricted number of females contributing to the hatchery stock was identified as the main cause of loss in genetic variation, possibly due to overmaturity of some multi-sea-winter females. We suggest that better monitoring and control of parental contribution will help in solving the problem of loss of genetic diversity in hatchery populations.     

AFLP and microsatellites as genetic tags to identify cultured gilthead seabream escapees: data from a simulated floating cage breaking event

Genetic discrimination using DNA fingerprinting is rapidly developing for cultured stock and wild fish populations. Microsatellites and AFLPs are being widely used in aquaculture to assign fish or processed fish products, to their claimed origin, paternity or strain. In the present study, 147 AFLP and 4 microsatellite markers were used as genetic tags in gilthead seabream, Sparus auratus. Specimens from two different hatchery broodstocks (one of Atlantic and one of Mediterranean origin) and wild fishes from a natural population were fingerprinted. Putative offspring from these broodstocks were computer-generated, and the confidence in the parentage assignment of their genetic profiles to the hatchery broodstock assessed. The virtual offspring were then mixed with specimens from a natural population to simulate an accidental escape from a floating cage. The risk of false paternity inclusion was evaluated to test the ability to identify either Atlantic or Mediterranean hatchery offspring among wild fish. The method proved to be reliable, and could therefore be used to forecast the impact of fish farm escapees.     

Pedigree analysis of recaptured fish in the stock enhancement program of spotted halibut Verasper variegatus

ABSTRACT:   Spotted halibut Verasper variegatus hatchery juveniles produced in 2002 were genotyped using three microsatellite DNA markers (msDNA) and then released into natural waters. Subsequently, recaptured individuals were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, from the genetic point of view, a pairwise F _ST test was implemented to estimate the genetic divergence between the wild captive broodstock, the hatchery offspring and the recaptured samples. The analysis showed significant differentiation between the broodstock and recaptured samples. Pedigree determination using msDNA was used to calculate the effective population size of the recaptured stock, which was found to be very low ( N _e ≈ 8). Equal family survivability was observed between the two recaptured stocks, but not between the released and recaptured stock. The number of identified families was higher and more equalized in the hatchery offspring compared to the recaptured samples, where the number of families declined. This fact was caused by an unequal family survivability just before or just after release. Separately, the number of contributing parents to the hatchery offspring was lower than the broodstock census number. Consequently, these two facts caused the genetic divergence of the recaptured stock from the broodstock.     

Monitoring of the genetic variability of the hatchery and recaptured fish in the stock enhancement program of the rare species barfin flounder <Emphasis Type="Italic">Verasper moseri</Emphasis>

ABSTRACT:   In relation to the stock enhancement program for barfin flounder, hatchery juveniles produced in 2001 were genotyped using microsatellite DNA markers (msDNA) and then released to natural waters. Subsequently, recaptured individuals, designated as 'tentative recaptured', were examined using msDNA. In order to evaluate the effectiveness of the stock enhancement program, pairwise F _ST and genic differentiation tests were used to estimate the genetic divergence between the wild samples, the hatchery broodstock, and the tentative recaptured samples. Analysis showed significant differentiation among these three groups. Pedigree determination by msDNA was used to establish the origin of the tentative recaptured individuals, in order to elucidate whether they were hatchery produced, 'real recaptured', or wild specimens. Wild individuals were not found. The effective population size of the real recaptured stock was very low ( N _e = 16.6). Equal family survivability was observed between the released and real recaptured stocks, indicating that the genetic variability of the released stock was maintained in the natural environment. Future broodstock management, breeding designs, and family contribution equalization of the offspring to be released will be required in this rare species to avoid unintended genetic differentiation between the wild population and the hatchery broodstock.     

Parentage assignment in hatchery population of brown sole <Emphasis Type="Italic">Pleuronectes herzensteini</Emphasis> by microsatellite DNA markers

ABSTRACT:   Five loci ( Phz2 , Phz6 , Phz7 , Phz12 , and Phz14 ) of microsatellite DNA markers developed in a previous study for parentage assignment in the hatchery population generated by mating among 61 broodstock fish (35 females and 26 males) in a spawning tank, were selected. After natural spawning in the same tank, larvae collected at three different times were categorized into early phase (EP), middle phase (MP), and late phase (LP) groups. In the parental broodstock, the mean number of alleles per locus was 21.8 and expected heterozygosity ( H _E ) was 0.813. In the progeny, the mean number of alleles per locus decreased to 11.6 (EP), 14.4 (MP), and 6.4 (LP) and H _E to 0.796 (EP), 0.833 (MP), and 0.681 (LP). Parental assignment determined eight dams and six sires as major parents for the EP group. In the MP group, 13 dams and ten sires genetically contributed to spawning, but only three dams and two sires were involved in LP group progeny. In the hatchery population produced from a limited number of parental fish such as the LP group, genetic variability was apparently decreased.     

Genetic structure and genetic relatedness of a hatchery stock of Senegal sole (Solea senegalensis) inferred by microsatellites

Solea senegalensis is a fish species in an early stage of domestication. A commercial hatchery in southern Spain experienced a dramatic failure in spawning and embryo survival, after incorporation into the broodstock of fish reared in their hatchery. To assess the impact of management on the stock quality, a population genetics study was made of adult broodstock present at this hatchery in 2000. The broodstock was composed of a group of fish of wild origin and two more groups from F₁ progeny. A set of eight microsatellite loci was used to compare genetic variability among the three groups and to establish the relationships between pairs of individuals within each group.Individuals from F₁ origin showed a substantial reduction in genetic variability when compared to those from the wild. There was a greater than 50% decrease in the number of alleles per locus corrected by sample size and 16% and 26% reductions in H_e values in the two F₁ groups, respectively. The degree of genetic relationship between individuals, evaluated by the relatedness estimator, rxy, and by graphical approaches, revealed a high proportion, almost 75%, of full-sibs and half-sibs in both F₁ groups, which might account for the reduction in overall variability. The high proportion of siblings also suggests a reproductive pattern in this stock in which few individuals breed successfully. This mating behaviour, along with factors inherent to selection and management systems, might be responsible for the negative effects on the reproductive capacity of the stock.     

Forecasting the genetic influences of red drum stock enhancement in South Carolina

The South Carolina Department of Natural Resources (SCDNR) began stocking red drum, Sciaenops ocellatus (Linnaeus), in 1989 to augment the abundance of juveniles available for recreational harvest in South Carolina estuaries. While stock enhancement can help supplement wild populations under high fishing pressure, releasing hatchery‐raised fish into the wild also presents the risk of decreased genetic diversity. An individual‐based model (IBM) was developed to forecast the genetic influences of stocking on the wild spawning population to inform responsible stocking strategies. Model results indicated the SCDNR red drum stock enhancement programme should maintain mean contributions of stocked fish no greater than 30% per year class over a 45‐year stocking period, coupled with at least 10 effective breeders in the hatchery replaced annually, to maintain current levels of genetic diversity estimated in the wild population. The IBM is a useful tool for hatchery managers to guide responsible stock enhancement.     

Application of Polymorphic Microsatellite Loci in a Channel Catfish Ictalurus punctatus Breeding Program

Abstract.— In order to monitor spawning success in earthen ponds, individual channel catfish Ictalurus punctatus broodfish were identified prior to stocking by genotype analysis with polymorphic microsatellite DNA markers. Individual spawns were collected and reared in a hatchery, and eight sac-fry per spawn were sampled for genotype analysis. Mendelian inheritance of microsatellite alleles was used to identify parents of each spawn. The probability of detecting a falsely matched offspring and spawning pair was >99%. Average spawning success was 44% (17–80%). Multiple spawning by males was found in seven of the eight ponds sampled. In these ponds, 47% of the males fertilized 2–6 egg masses over 1–8 wk, although most were 2–3 wk apart. Four different spawns were identified as two full-sib families and were probably due to interrupted spawning. Parent/offspring genotype analysis will be useful for production of half-sib families for estimation of genetic components of variation, estimation of effective breeding population, and selection of broodstock for multiple reproductive traits under natural spawning conditions.     

Application of DNA markers to the management of Atlantic halibut (Hippoglossus hippoglossus) broodstock

For many aquaculture finfish species, the current broodstock have been collected from the wild or have undergone only a few generations of domestication. The Atlantic halibut (Hippoglossus hippoglossus) aquaculture industry in Atlantic Canada has retained F₁ juveniles (n=145) from the 1996 spawning of wild adults for candidate broodstock. Through the development and use of a five-microsatellite DNA marker multiplex, we determined the parentage of these 1996 F1 individuals, which are being reared at one government and two industry hatcheries, and evaluated the change in genetic variation between the wild and the 1996 F₁ stock. In the three groups of F₁ fish, single parental pairs were assigned to 98%, 96% and 100% of individuals. Large full- and half-sibling groups were identified within and across F₁ groups and, overall, only 36% of attempted crosses were represented in the retained fish. Effective population size in the parental group decreased from 27 to 13 when variance in family size was accounted for and to 12.5 when changes in gene diversity (compared to the combined F₁ stocks) were considered. Statistically significant differences in measures of genetic variation were not widely observed between groups (original wild sample, parental group, three F₁ groups and combined F₁). However, the F₁ population shows a 26% decrease in total allele numbers compared to the wild sample. These observations demonstrate the utility of genetic tools in the evaluation of genetic diversity and determination of pedigree during the establishment of new broodstock. They also emphasize the necessity for closely monitoring future matings among these fish and suggest the need to introduce additional genetic variation into this group of Atlantic halibut broodstock.     

Impacts of hatchery release on genetic structure of rock carp <Emphasis Type="Italic">Procypris rabaudi</Emphasis> in the upper Yangtze River,China

Rock carp Procypris rabaudi is a vulnerable endemic fish in the upper Yangtze River. Hatchery release has been carried out as a major stock enhancement strategy for this species. Ten microsatellite loci were chosen to compare genetic variation between one wild population and two hatchery groups to evaluate the potential impacts of hatchery release on the genetic structure of the wild population. Two different models indicated strong evidence of recent bottlenecks in all groups. The hatchery groups were lower in the mean number of alleles per locus, allelic richness, and allelic diversity compared with the wild population. The 80% membership coefficient indicated that 14% of the wild fish could be assigned as hybrids of wild and hatchery fish. Our results suggested that hatchery release will further reduce the natural genetic diversity in the wild population, change the genetic structure of the rock carp population, and may not benefit restoration of this vulnerable fish species.     

Parentage Determination and Effective Population Size Estimation in Mass Spawning Pacific Oyster,Crassostrea gigas,Based on Microsatellite Analysis

Seven high polymorphic microsatellite loci were used to determine the pedigrees in a mass spawning of Pacific oyster, Crassostrea gigas, and to estimate the genetic variability between broodstock and offspring. Parental assignment was performed on a total of 155 individuals, including 141 offspring, 8 candidate mothers, and 6 candidate fathers. The assignment results of real offspring were generally in agreement with simulation with a success rate over 99% using only six of these loci. The allelic diversity and observed heterozygosity (H_o) exhibited similarity between parents and offspring populations, but the expected heterozygosity (H_e) had a significant decrease in offspring. Although all the males and females contributed to the next generation, the variances of reproductive success and unequal sex ratio resulted in a decline in effective population size (N_e = 11.42). The inbreeding rate of this small‐scale, mass spawning population was estimated at approximately 16.5% per generation. This gave us an insight that when designing breeding programs based on mass spawning for future oyster cultivation generations, the higher inbreeding and lower effective population size should be considered.     

Microsatellite pedigree analysis reveals high variance in reproductive success and reduced genetic diversity in hatchery-spawned northern abalone

The northern (or pinto) abalone, Haliotis kamtschatkana, is a broadcast-spawning marine gastropod that was recently listed as endangered in Canada. To aid in species recovery, a captive-breeding and supplementation program is underway in Barkley Sound, British Columbia. We genotyped first generation progeny for five microsatellite loci and used a pedigree reconstruction program (Pedigree 2.2) to identify their genealogical relationships in the absence of information on parental genotypes. We analyzed progeny from three separate group-spawning events and inferred considerable variation in the number of offspring produced by each parent; in the most severe case a single male sired all the progeny produced during one spawning event. After only one generation of captive-breeding we found a 55–60% reduction in allelic richness and a 17–18% reduction in heterozygosity relative to the diverse wild source population. This study illustrates the difficulty of managing genetic diversity in hatchery populations of a broadcast-spawning species, even when gametes are collected separately from each individual broodstock.     

Decreased genetic diversity and a reduced effective population size in cultured silver-lipped pearl oysters (Pinctada maxima)

Before selection programmes for aquaculture species can commence, a fundamental knowledge of how the culture process affects genetic diversity is requisite. “South Sea” pearl production, through the culture of the silver-lipped pearl oyster, Pinctada maxima, stands to gain substantially from genetic selection. Aquaculture techniques for P. maxima have progressed vastly, to a point where the majority of current operations are based on hatchery-propagated stock; however, there has been no investigation on how the culture process is affecting the genetic diversity of this species. To address this deficiency, we investigated the genetic diversity and effective population sizes (N_e) in three wild and five hatchery-produced P. maxima populations. Using six microsatellite DNA markers, cultured populations exhibited low N_e (3.5–9.2) and reduced allelic richness (29–44%) compared with their wild progenitors. Observed heterozygosities were not affected greatly, however, an increased mean pairwise genetic relatedness was observed in all cultured populations. The practice of mass spawning was identified as a major factor in the reduction of diversity, although diversity was not necessarily maintained when a more controlled spawning approach was utilised. Pedigree reconstructions revealed genetic diversity and N_e is affected by large skews in full-sib family representations (up to 40% from a single family, in a cohort using 28 broodstock), and could be due to differential survival rates amongst communally reared families. In order to maximise future selective breeding efforts, further research should be directed towards identifying critical stages of the culture process in which genetic diversity is lost.     

Estimating reproductive success of brooders and heritability of growth traits in Asian sea bass (Lates calcarifer) using microsatellites

Asian sea bass (Lates calcarifer) is one of the most important marine food fish species in Asia and Australia. To estimate the reproductive success of broodstock and heritabilities of growth‐related traits, two independent full‐factorial crosses (PI and PII) were created by crossing 10 males and 10 females. At 90 days post hatch (dph), the body weight (BW) and total length (TL) of 804 individuals from PI and 900 individuals from PII were measured and tissues samples of each fish were collected. Parents and offspring were genotyped with nine polymorphic microsatellites. Out of 1704 offspring from the two crosses, 98.7% were assigned to their parents. In PI, 19 of 20 parents produced offspring, while in PII, only five parents contributed to offspring. Low contribution of parents to offspring could lead to reduced genetic variation in the next generation. Heritabilities for growth‐related traits were estimated using the pedigrees reconstructed using microsatellite genotypes. The estimates of heritability were 0.22±0.16 and 0.25±0.18 for BW, 0.31±0.14 and 0.24±0.21 for TL and 0.22±0.22 and 0.15±0.09 for Fulton's condition factor in the two crosses respectively. Body weights at 90 dph and at harvest (289 dph) were significantly correlated (r=0.601, P<0.01). Therefore, growth‐related traits could be improved by exploiting additive genetic effects through selective breeding, and broodstock candidates could be selected early in the production cycle.     

放流三疣梭子蟹遗传多样性和贡献率初步研究

近年来我国沿海三疣梭子蟹自然资源明显下降,为恢复现有资源,辽宁省自2012年开始,连续实施了4年大规模人工放流工作。在目前的增殖放流中,为检测参与繁殖的三疣梭子蟹亲本和即将放流的子代间的遗传差异以及亲本对其子代的繁殖贡献率水平,利用10对具有丰富遗传多态性的微卫星分子标记,分别对9只三疣梭子蟹雌性亲本和179只即将放流的子代进行了遗传多样性分析及亲子鉴定。结果发现,子代的平均观测杂合度、平均期望杂合度和平均多态性信息含量数值均低于参与繁殖的雌性亲本,亲本和放流子代之间在观测杂合度、多态性信息含量参数方面并无显著差异(P0.05),但期望杂合度遗传参数存在显著差异(P0.05),子代的遗传多样性较雌性亲本呈下降的趋势。使用4个微卫星分子标记时,累积排除率≥0.998,亲子鉴定的准确率为56.98%;微卫星分子标记为6个时,累积排除率≥0.9999,准确率达到97.21%;当使用8个微卫星分子标记鉴定时,准确率达100%。同时发现,9只雌性亲本对子代均有贡献,最高为29.61%,最低为3.35%,不同亲本之间的贡献率存在显著差异(P0.05)。研究表明,微卫星可以作为有效的标记手段用于三疣梭子蟹增殖放流遗传评估中。上述试验结果将为我国三疣梭子蟹增殖放流的科学发展提供基础资料。     

Genetic Variability in Eight Cultured and Two Wild Populations of Japanese Flounder,Paralichthys olivaceus,Based on the Mitochondrial DNA Control Region

To examine the present population genetic diversity and variability of Japanese flounder, a 394‐bp hypervariable fragment of mtDNA control region was sequenced. A total of 215 individuals from two wild and eight cultured populations were analyzed. The 91 variable sites defined 61 haplotypes and 12 of them were shared. Six single base pair insertion/deletions were detected. The haplotype diversity (h), the nucleotide diversity (π), and mean number of pairwise differences (k) in cultured populations (h = 0.443–0.844; π = 0.010–0.030; k = 3.745–11.838) were obviously lower than those in the wild populations (h = 0.987–0.988; π = 0.032; k = 12.443–12.718). Fixation index (F_st) and analysis of molecular variance (AMOVA) revealed that significant genetic differentiation mainly existed among cultured populations. The results of the exact test of population differentiation (nondifferentiation exact P values) rejected a panmictic mtDNA gene pool in all cultured populations. The results of this study indicated that genetic diversity of cultured Japanese flounder populations in China had significantly declined due to farm propagation and an increase in broodstock number should increase genetic diversity in cultured Japanese flounder base on the genetic theory.     

Completion of the Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel) life cycle

Tuna aquaculture is currently dependent on the wild capture of juveniles for production. The development of hatchery technology for bluefin and other tunas would be a major step forward in improving sustainability of their aquaculture. The present study overviews the technology in the life cycle completion of the Pacific bluefin tuna (PBT) Thunnus orientalis (Temminck et Schlegel) under aquaculture conditions in Kinki University, and the problems to be solved for the establishment of tuna hatchery technology. On 23 June 2002, broodstock of PBT that were artificially hatched and reared spontaneously spawned in captivity. The resulting eggs hatched and were subsequently reared to the juvenile stage. The spawning fish were the result of a research project started in 1987 to rear wild‐caught juvenile PBT that were several months old. Fertilized eggs were obtained from these fish in 1995 and 1996. Resulting juveniles (the artificially hatched first generation) were reared to maturity and spawned in 2002. Over the summer of 2002, 1.63 million eggs from these fish were used for a mass rearing experiment, and 17 307 juveniles were produced and transferred to an open sea net cage. Of these artificially hatched second‐generation PBT, 1100 grew to approximately 95 cm total length and 14 kg body weight in 22 months. This procedure means the completion of PBT life cycle under aquaculture conditions, which was first attained among large tuna species. The problems awaiting solution in PBT hatchery production are their unpredictable spawning in captivity, to improve survival during the first 10 days post hatch, to reduce cannibalism in larval and juvenile stages, and to solve collision problem causing high mortality during the juvenile stage.