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.     

海湾扇贝南部亚种遗传多样性和选择特征的基因组学解析

为了探究海湾扇贝南部亚种(Argopecten irradians concentricus)的遗传多样性特征,通过全基因组重测序分析,系统解析了其群体遗传结构以及其与海湾扇贝北部亚种(Argopecten irradians irradians)间的遗传差异,共筛查出15059961个高质量单核苷酸多态性(single nucleotide polymorphisms,SNPs)位点。基于全基因组SNP的群体祖先成分推断、主成分分析和系统发生重构皆表明,南北亚种之间存在较为显著的遗传分化。其中,海湾扇贝南部亚种的近交水平和连锁不平衡程度更高。通过选择性清除分析,在海湾扇贝南部亚种中共鉴定出349个正选择基因,其中GHSR、HSF1、HABP2和Dna J等基因可能与其生长速度较快、耐热性较强以及免疫功能较强等环境适应性特征相关。该研究揭示了海湾扇贝亚种间遗传分化和选择特征,为扇贝的基因组研究和分子育种提供了数据支持。     

Crosses between two subspecies of bay scallop Argopecten irradians and heterosis for yield traits at harvest

In order to improve the yield of bay scallop Argopecten irradians by exploiting geographic subspecies heterosis, crosses between and within two subspecies, the northern A. i. irradians (Lamarck) and the southern A. i. concentricus (Say), were created and the offspring were cultured under the same environmental conditions. The two parental populations were collected from Bohai Bay in northern China and Beibu Bay in southern China, which were distinct in particular shell colours. The results indicated that the shell colour of bay scallop was inheritable and could be used as a morphological mark to distinguish hybrid and purebred individuals. At harvest, hybrid offspring always produced a significantly higher yield than purebred offspring. Positive heterosis was achieved for shell length, shell height, shell width, total weight and adductor weight, which was 5.02, 5.22, 3.88, 7.53 and 9.47 respectively. Hybrid gain was 10.86% for shell length, 10.48% for shell height, 9.77% for shell width, 34.90% for total weight and 41.69% for adductor weight, respectively, and all were significantly >5%. The present study reveals that it is effective for improving yield by hybridization between different geographic subspecies in the bay scallop.     

Evaluating spawning performance among captive Florida pompano Trachinotus carolinus broodstock using microsatellite‐based parentage assignment

Florida pompano has been identified as a promising candidate for commercial‐scale aquaculture production, but to date, little information is available regarding captive broodstock spawning characteristics. Genetic markers were tested for their power in monitoring mating outcomes and potential in analysing heritability of rapid growth trait in Trachinotus carolinus. A total of 20 unrelated adults (10 females and 10 males) were chosen for a hormone‐induced mass spawning event. The 515 fastest growing and 485 slowest growing fish of the total 4852 offspring were considered a selected progeny stock, and fish were collected at 45 days post hatch based on their growth traits. Parentage analyses based on the 20 breeders and 1,000 selected progeny were performed using a total of nine microsatellite markers, a 100% assignment rate was achieved, and a four‐marker set was the minimum number for the parentage assignment. The effective breeding number for the selected progeny was 11 (six females and five males), among which three females and two males were predominant contributors with the total contribution of 95.8% and 94.7% respectively. The proportion of fast‐growing offspring from broodfish and each mating cross (sire/dam) was used for detecting whether variation in growth of the offspring was related to parental stocks. Results showed that three adults and their mated combination exhibited the greatest fast‐growing offspring proportion (69.73% and 55.95%). This research provided new information regarding spawning performance and parental contribution during mass spawning events; both important first steps towards developing improved management strategies for captive Florida pompano broodstock.     

Sustained, Natural Spawning of Southern Flounder Paralichthys lethostigma Under an Extended Photothermal Regime

Hormone‐induced spawning of southern flounder Paralichthys lethostigma has produced substantial numbers of viable eggs, but wide variations in fertilization and hatch rates have been reported. Recently, sustained natural spawning of southern flounder broodstock, without hormone induction, has been achieved in our laboratory. Adults (average weight = 1.12 kg; N= 25), including 6 captured as juveniles in 1993 and 19 captured as adults during September 1998, were stocked in two 4.8‐m³ controlled‐environment tanks in October 1998 and held under natural photothermal conditions until January 1999, when an artificial winter photo‐period of 10 L:14 D was initiated and then maintained through April 1999. Sex ratio was approximately 13 females:8 males:7 unknown. Natural spawning was observed in early December 1998 and increased in frequency to a peak in March 1999, before declining in late April. Water temperature ranged from 13.9 to 24.5 C during the spawning period. Natural spawnings over 142 d produced a total of 18.3 × 10⁶ eggs, with a mean fertilization rate of 28.0% (range = 0–100%), yielding 4.94 × 10⁶ fertilized eggs. The mean percentage of eggs that remained buoyant in full‐strength seawater (34 ppt) was 41.3% (0–98%), while hatching rate of buoyant eggs was 37.3% (0–99%) and survival of yolksac larvae to the first‐feeding stage was 30.2% (0–100%). Gonadal biopsies in late April identified six females from both tanks as probable spawners. A preliminary comparison suggests that natural spawning produced much larger numbers of viable eggs per female, with higher egg quality (i.e., fertilization and hatching success) than hormone‐induced spawning. In contrast to natural spawning, hormone‐induced strip‐spawning enabled timing of spawnings to be more precisely controlled. These results suggest that a combination of both natural and hormone‐induced spawning of photothermally conditioned fish will help produce the large numbers of eggs required to support commercial production.     

Connectivity of the bay scallop (Argopecten irradians) in Buzzards Bay,Massachusetts, U.S.A.

The harvest of bay scallops (Argopecten irradians) from Buzzards Bay, Massachusetts, U.S.A. undergoes large interannual fluctuations, varying by more than an order of magnitude in successive years. To investigate the extent to which these fluctuations may be due to yearly variations in the transport of scallop larvae from spawning areas to suitable juvenile habitat (settlement zones), a high‐resolution hydrodynamic model was used to drive an individual‐based model of scallop larval transport. Model results revealed that scallop spawning in Buzzards Bay occurs during a time when nearshore bay currents were principally directed up‐bay in response to a persistent southwesterly sea breeze. This nearshore flow results in the substantial transport of larvae from lower‐bay spawning areas to settlement zones further up‐bay. Averaged over the entire bay, the spawning‐to‐settlement zone connectivity exhibits little interannual variation. However, connectivities between individual spawning and settlement zones vary by up to an order of magnitude. The model results identified spawning areas that have the greatest probability of transporting larvae to juvenile habitat. Because managers may aim to increase scallop populations either locally or broadly, the high‐connectivity spawning areas were divided into: (i) high larval retention and relatively little larval transport to adjoining settlement areas, (ii) both significant larval retention and transport to more distant settlement areas, and (iii) little larval retention but significant transport to distant settlement areas.     

Early assessment of gilthead sea bream (Sparus aurata) spawning dynamics by mini‐broodstocks

The implementation of any sustainable breeding program requires a maximization of effective size (Ne) to maintain inbreeding rate at minimum levels. However, the control of Ne remains a major challenge for those species whose reproduction in captivity is based on a spawning strategy where there is no control over the established matings. The present study aims to assess the spawning dynamic of gilthead sea bream (Sparus aurata) at different dates from two mini‐broodstocks (4 ♀ and 4 ♂). Here we report the use of a simple protocol based on the genotyping of fertilized eggs from a reduced set of microsatellite loci which provides a quick evaluation of daily spawning in terms of family structuring (number of active breeders and number and family size). According to our results, the daily spawning dynamics is characterized by a fluctuation in the number of active breeders, reaching maximum values (until 100%) during peak production phase. However, the proportion of possible mating (families) in a single daily spawning never exceeded more than 81%. Unbalanced parental contributions were highly frequent, with extreme cases involving the assignment of 50%–90% of the offspring to a single family. Therefore, the use of mini‐broodstocks and the grouping of spawning harvested at different dates are proposed as strategies to minimize the inbreeding risk by providing a more optimal pattern of family structuring (increase in the number of families obtained and a more balanced parental contributions) that entails a more conservative Ne/N ratio.     

SNPs of myostatin (MSTN) gene and their association with growth traits in three bay scallop (Argopecten irradians) populations

Genetic diversity and correlation analysis between single nucleotide polymorphism (SNP) of myostatin (MSTN) gene and the growth traits were performed in Argopecten irradians. Around 279 individuals from three A. irradians populations were screened for the polymorphic sites of the MSTN gene. Two SNPs were found: T910G and G1162A. The observed heterozygosity of the two SNPs ranged from 0.1310 to 0.3299, whereas the expected heterozygosity ranged from 0.1436 to 0.2973. The values of D′ and r² of linkage disequilibrium (LD) ranged from 0.475 to 1.0 and 0.052 to 0.174 respectively. Association analysis shows that individuals with genotype TT and TG of locus W4 had significantly higher adductor muscle mass than that with genotype GG in population CA (P < 0.05). Individuals with genotype AA of locus W5 had significantly higher body mass, soft‐tissue mass, adductor muscle mass, shell width and shell mass than those of other genotypes in population RZ (P < 0.05). The two SNPs found in our study can be used for molecule marker‐assisted breeding of A. irradians.     

Effects of Varying Dietary Protein Levels and Feeding Frequencies on Condition and Reproductive Performance of Channel Catfish to Produce Hybrid Catfish

The interspecific hybridization of channel catfish, Ictalurus punctatus, females with blue catfish, I. furcatus, males has been identified as a method to further improve production; however, lack of spawning success has affected its commercial application. To facilitate our understanding of the interaction of brood stock nutrition and reproductive performance, we evaluated the interaction of feed quality and feeding frequency. Channel catfish females were classified into two genetic groups, namely, high and low spawning. The treatments were offered during the spring season 70–90 d prior to the start of the spawning season. Induced reproduction was performed using luteinizing hormone releasing hormone analog. Condition of the fish as well as reproductive performance using spawning success, egg production, egg size, and fertilization at 48 h were determined. Changing protein level of the diet from 32 to 42% did not influence spawning, fecundity, or fertilization, but affected egg size and biochemical composition of the eggs. Increasing the feeding frequency from three to six times per week negatively affected spawning in one of the two genetics groups, did not affect egg production and egg fertilization, but had a significant effect on egg size. Older fish performed better than younger fish in terms of spawning success and egg production.     

Utility of high-density plantings in bay scallop, Argopecten irradians irradians, restoration

Two different methods of establishing high-density spawner sanctuaries for bay scallop (Argopecten irradians irradians) restoration were evaluated over 2 years at a site in Northwest Harbor, East Hampton, New York, USA. Hatchery-reared scallops, which had been overwintered at nearby sites, were free-planted directly to the bottom in late March/early April at an initial target density of 94?C128 scallops/m². In addition, scallops were stocked in off-bottom culture units consisting of three vertically stacked 15-mm mesh ADPI^® bags at densities of 50, 100, or 200 scallops/bag (=117, 234, or 468 scallops/m²), respectively. Survival of scallops differed significantly by year, planting method, and scallop source. Survival of free-planted scallops was generally lower than caged scallops. Better survival of free-planted scallops in 2005 versus 2006 likely reflected the presence of luxuriant eelgrass beds in 2005, which were absent in 2006. Survival of scallops in ADPI bags was not appreciably related to stocking density. Shell growth was highest for free-planted scallops; in cages, growth was somewhat better at 50 versus 200 scallops/bag. Wet weights of epibionts were significantly higher in caged versus free-planted scallops. Reproductive condition of scallops stocked at 50/bag was usually higher than at 200/bag. Both free-planting and off-bottom systems yielded high densities of adult bay scallops at the time of spawning, which ensures a higher probability of successful fertilization of spawned eggs and thus a greater potential for success of restoration efforts.     

Microsatellites markers to depict the reproductive and genetic patterns of farmed gilthead seabream (Sparus aurata): illustration by a case study on mass spawning

In the absence of breeding strategy, natural spawning constitutes the breeding ground for fish farmers to empirically manage their commercial broodstock. In this context, we used six microsatellite markers to characterize the genetic pattern of six commercial seabream broodfish tanks having a common history spanning four generations. The progeny of one tank single‐day mass‐spawning event, reared in two different environments, was used to estimate the genetic parameters for body weight. Limited genetic differentiation was observed among broodfish groups. A panel of nine loci allowed us to unambiguously assign 95.4% of the offspring (1692) and identify 37 parents (65% of the total broodfish). The limited effective population size (N_e = 15.3) was due to the elevated variance of parental contributions and to broodfish failing to contribute to the progeny. The fluctuation of the allele frequency highlighted the risks of genetic drift and reduction in the heterozygosity in the next generations. Heritability for body weight was moderate at commercial size (0.40 ± 0.10) and the high genetic correlation at later stages laid the groundwork for precocious selection criteria for growth. The discussion opens on the opportunity to use mass spawning for selective breeding.     

Multiple paternity,reproductive skew and correlates of male reproductive success in a wild population of the Trinidadian guppy

Abstract – In most species, males have a higher reproductive potential than females, leading to skewed reproductive success, particularly in mating systems where pre‐ or postcopulatory sexual selection reinforces inequality in male mating success. We investigated multiple paternity, reproductive skew and correlates of male reproductive success in a wild population of the Trinidadian guppy (Poecilia reticulata). We used nine microsatellite loci to assess the frequency of multiple paternity, number of sires per brood and reproductive skew. Across broods, the frequency of multiple paternity was high with 94% of broods having multiple sires (range: 1–5), resulting in a reproductive skew of 0.14. Variation in male reproductive success was high (range: 0–14 offspring per male), suggesting that there is considerable opportunity for sexual selection. Next, we examined correlations between male reproductive success and sexual coloration, sperm velocity and gonopodium length. Relative area of orange, black, iridescent and total coloration, and sperm velocity were not correlated with reproductive success. However, gonopodium length explained 14% of the variation in reproductive success, suggesting that gonopodium length is likely a sexually selected trait. We discuss these findings in the light of other studies that genetically dissect joint‐sex parentage and examine correlates of male reproductive success in wild populations.     

Assessing the impact of population decline on mating system in the overexploited Mediterranean red coral

1. Understanding the interactions among demographic parameters, mating system and population dynamics is key to predict the response of populations to global change. The Mediterranean red coral is a precious octocoral suffering from population decline due to overfishing and warming‐driven mass mortality events.
2. While the demographic consequences of these two pressures are well characterized, little is known regarding their impact on population dynamics and evolution of red coral populations. The main objective of this study was to fill this gap focusing more particularly on mating pattern and genetic drift.
3. Combining sibship and progeny arrays analyses, a genetic characterization of the red coral mating system was conducted. In addition, a synchronic approach was developed comparing mating patterns in two populations with contrasting demographic patterns: a pristine‐like population and a declining population.
4. The results show that polyandry is likely to be the norm in red coral. The similar patterns of genetic diversity between adults and larvae combined with the lack of differential reproductive success among putative fathers did not support significant sweepstakes effects during larval production. While instantaneous biparental inbreeding was detected, no long‐term inbreeding was observed even in the declining population. Mating patterns and effective population sizes in the two populations were not statistically different. Nevertheless, a trend towards a slightly higher inbreeding and a lower number of breeders was observed in the declining population.
5. Accordingly, we hypothesized that an increase in male gamete dispersal may buffer the increase of genetic drift expected in the declining population. This feedback between demographic decline and reproductive pattern may potentially take part in the long‐term persistence of red coral populations. However, the negative trend reported in the declining population unambiguously supports the need to maintain high densities of reproductive colonies to the functioning of red coral populations.

     

Evaluation of egg quality in broodstock cobia Rachycentron canadum L.

Twenty egg batches spawned naturally from 17 different females over two spawning seasons were used to evaluate the egg quality of cobia Rachycentron canadum. A reduction in egg size was observed towards the end of the spawning season for both years. The proportion of floating eggs demonstrated a positive linear relationship with both yolk‐sac larval survival (r²=0.91, P<0.05) and batch larval production (r²=0.80, P<0.01). Viable egg batches (i.e. fertilization success >50%) were of higher batch fecundity, had larger eggs and a higher proportion of floating eggs than non‐viable batches (i.e. 0% fertilization success). Also, biochemical analyses revealed that these viable eggs had significantly higher protein and amino acid contents. A multiple regression model based on the proportion of floating eggs, batch fecundity and fertilization success provided the most accurate predictions of batch larval production (r²=0.95, P<0.001). Similarly, using the egg content of arginine/glycine and methionine significantly increased the correlation coefficient in the multiple regression model predicting larval deformity (r²=0.92, P=0.002). This study reveals that accurate determination of egg quality in cobia can be improved using a combination of several variables rather than a single variable.     

Estimating number of spawning white sturgeon adults from embryo relatedness

The goal for using genetic information derived from embryo samples is to gain insight into the reproductive biology of adult white sturgeon, Acipenser transmontanus Richardson. The relationship between egg development stages on the extraction of quality DNA was determined, and subsequent genotype data were used to infer contributing spawner numbers. White sturgeon DNA was present in extractions from all stages; however, pre‐gastrulation stage embryos generally failed to amplify sufficient genetic loci for analysis. Using experimentally created families, multivariate ANOVA and maximum‐likelihood sibship methods estimated the presence of 10 contributing parents from 11 actual adult spawners. It was observed that unidentified half‐sibling relationships (offspring sharing one parent) caused error for inferring parent numbers. Genetic methods estimated 13–40 parents potentially contributed to a collection of wild‐caught embryos, with variable numbers of parents inferred from separate spawning events (varying from 8 to 24). Egg source material provides a means to enhance the accuracy of annual spawning population size estimates, which is important for informing habitat restoration, harvest and water management decisions and recovery planning.     

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.     

Artificial Spawning of Tilapia Eggs1

A stripspawning methodology was evaluated for tilapia (oreochromid) species. This technique achieved an average hatching success of 68.6 ± 3.6% (N= 31). Female fecundity and spawning frequency were dependant on both genetical and husbandry factors. Egg yields for Oreochromis niloticus, O. mossambicus, and O. niloticus±O. mossambicus hybrids averaged 4.54, 10.86 and 10.36 eggs/g female/spawn, respectively. Female broodstock that were adapted to an intensive spawning regime exhibited a significant increase in fecundity. Additionally, egg survival was not affected by hydration for up to 15 minutes prior to fertilization. Results suggest that the strip spawning of tilapia species may be an efficient method of providing viable gametes for hatchery purposes.     

Effect of parental stock size on F1 genetic structure in the bay scallop, Argopecten irradians (Lamarck, 1819)

Three F₁ families of the bay scallop, Argopecten irradians, were produced from one, two and 10 individuals. The genetic changes in these populations, which suffered recent and different levels of bottleneck, were analysed using amplified fragment length polymorphism (AFLP) techniques. In the parental stock, a total of 330 bands were detected using seven AFLP primer pairs, and 70% of the loci were polymorphic. All F₁ groups had a significantly lower proportion of polymorphic loci when compared with the initial stock, and loss of the rare loci and reduction in heterozygosity both occurred. The progeny of the larger population (i.e., N=10) exhibited a lesser amount of genetic differentiation compared with the progeny from N=2, which showed lesser differentiation than progeny from N=1. The effective population sizes (N_e) in N=1, 2 and 10 were estimated as 1.50, 1.61 and 2.49. Based on regression analysis, we recommend that at least 340 individuals be used in hatchery populations to maintain genetic variation.     

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.     

Extensive dispersal of Roanoke logperch (Percina rex) inferred from genetic marker data

The dispersal ecology of most stream fishes is poorly characterised, complicating conservation efforts for these species. We used microsatellite DNA marker data to characterise dispersal patterns and effective population size (N_e) for a population of Roanoke logperch Percina rex, an endangered darter (Percidae). Juveniles and candidate parents were sampled for 2 years at sites throughout the Roanoke River watershed. Dispersal was inferred via genetic assignment tests (ATs), pedigree reconstruction (PR) and estimation of lifetime dispersal distance under a genetic isolation‐by‐distance model. Estimates of N_e varied from 105 to 1218 individuals, depending on the estimation method. Based on PR, polygamy was frequent in parents of both sexes, with individuals spawning with an average of 2.4 mates. The sample contained 61 half‐sibling pairs, but only one parent–offspring pair and no full‐sib pairs, which limited our ability to discriminate natal dispersal of juveniles from breeding dispersal of their parents between spawning events. Nonetheless, all methods indicated extensive dispersal. The AT indicated unrestricted dispersal among sites ≤15 km apart, while siblings inferred by the PR were captured an average of 14 km and up to 55 km apart. Model‐based estimates of median lifetime dispersal distance (6–24 km, depending on assumptions) bracketed AT and PR estimates, indicating that widely dispersed individuals do, on average, contribute to gene flow. Extensive dispersal of P. rex suggests that darters and other small benthic stream fishes may be unexpectedly mobile. Monitoring and management activities for such populations should encompass entire watersheds to fully capture population dynamics.