Aquaculture 220 (2003) 227 244 www.elsevier.

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Yields of cultured Pacific oysters Crassostrea gigas Thunberg improved after one generation of selection
Chris Langdon a,*, Ford Evans a, Dave Jacobson a, Michael Blouin b
a

Coastal Oregon Marine Experiment Station and Department of Fisheries and Wildlife, Hatfield Marine Science Center, Oregon State University, Newport, OR 97365, USA b Department of Zoology, Oregon State University, Corvallis, OR 97331, USA

Received 16 June 2002; received in revised form 5 November 2002; accepted 8 November 2002

Abstract Parental families (G0) in three lines of Pacific oysters were selected based on live weight and meat yields at harvest. The average live weight yield of progeny (G1) from crossing G0-selected lines in seven trials was 9.5% greater than that of nonselected control families and live weight yields were significantly greater (ANOVA, P < 0.001) in four out of seven trials. The response to selection was greater if G1 families were tested at the same site as their parents selection site rather than at a different site, although this effect was only significant for G1 families of cohort 5 ( P < 0.01) but not cohort 7 ( P>0.05). A significant genotype environment interaction affected yields in both cohort 5 and cohort 7 (ANOVA; P < 0.001). In addition, correlation between the yields of the same families planted at both intertidal and subtidal sites was positive but weak (cohort 5, r = 0.30; cohort 7, r = 0.35), indicating that selection for high yield in one environment would likely result in a low correlated response in a different environment. Nonetheless, it was possible to identify six families in cohort 5 and four families in cohort 7 that were among the top 10 families at both sites. Further evaluation of families across a wider range of environments is needed to determine if it is possible to substantially improve yields by selecting generalist families that perform well along the whole Pacific coast, or whether it will be necessary to select lines that are suited to particular sites. D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Oyster; Genetics; Selection; Yield; Crassostrea gigas

* Corresponding author. Tel.: +1-541-867-0231; fax: +1-541-867-0345. E-mail address: chris.langdon@hmsc.orst.edu (C. Langdon). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00621-X

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1. Introduction The Pacific oyster, Crassostrea gigas, is a major global aquaculture species, ranking first in terms of total weight and second in terms of value of landings among all other global aquaculture fish and shellfish species (FAO, 1999). In many parts of the world, water temperatures are sufficiently high for populations to reproduce, allowing farmers to collect seed from natural sources. In contrast, on the West Coast, USA, seawater temperatures are generally too low for successful reproduction and farmers depend on hatcheries for reliable supplies of oyster seed for planting. These hatcheries also provide an opportunity to genetically improve broodstock to increase yields of progeny. Selective breeding has been successful in improving growth and survival of several oyster species (see review by Sheridan, 1997). Reported heritability values and additive genetic effects (Lannan, 1972; Hedgecock et al., 1991) indicate that artificial selection has the potential to increase growth rate of Pacific oysters. Here we report on the results of a selective breeding program designed to improve yields of Pacific oysters on the West Coast, USA (Hedgecock et al., 1997).

2. Methods 2.1. Founder population (P0) Small effective population sizes of some hatchery stocks have been reported by Hedgecock and Sly (1990) and Gaffney et al. (1992); therefore, broodstock were collected from wild populations of Pacific oysters in Dabob and Willapa Bays, WA, USA, and Pipestem Inlet, Vancouver Island, BC, Canada (Fig. 1), to avoid the use of hatchery stocks and to enhance genetic diversity of the founder populations. Analysis of 17 allozymes by D. Hedgecock, University of California, Davis, indicated that oyster populations at the three founder sites were genetically different from each other ( Fst; Falconer and Mackay, 1996). 2.2. Hatchery and nursery culture conditions Broodstock oysters were conditioned for 6 weeks at 18 jC and fed ad libitum with mixed algal diet of Isochrysis galbana (Tahitian strain; T-ISO) and Chaetoceros gracile (Cg). At the time of spawning, a sample of the adductor muscle was taken for microsatellite analysis (Margoulas et al., 1998) to determine the genetic identity of every broodstock oyster spawned. Contaminated crosses were rapidly identified and discarded during the early larval stages and replaced with crosses from broodstock of the desired pedigree. Only about 60 full-sib families could be reared at one time due to limited available space and resources in the hatchery and nursery. Families were not replicated in the hatchery and nursery in order to maximize the number of families evaluated and genetic diversity of the breeding population. About 2 million fertilized eggs from each mating were incubated in a 20-l container at a concentration of 100 eggs ml 1. After 24-h incubation at 25 jC, larvae were collected on

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Fig. 1. Pedigrees of selected lines indicating founder population origins and evaluation sites for G0 and G1 generations.

a 25-Am mesh screen, rinsed and resuspended in 0.2-Am-filtered seawater. The concentrations of normal D-larvae were determined and added to a 100-l larval rearing tank to provide a final concentration of 10 larvae ml 1. Larvae of each family were reared in 100-l tanks filled with 0.2-Am-filtered seawater at 25 jC and 28 30 ppt salinity, according to methods described by Breese and Malouf (1975). After 14 21 days, eyed larvae retained on a 243-Am screen were treated with 2 10 4 M epinephrine to induce metamorphosis (Coon et al., 1986). This process was repeated until most of the eyed larvae were removed from each of the culture tanks. Metamorphosed larvae (spat) were placed in upwellers and fed ad libitum with an algal mixture of T-ISO and Cg at 18 20 jC and at a salinity of 28 30 ppt. Once the spat reached a shell length of >3 mm, they were transferred to 1.5-mm mesh bags (25 60 cm) and held at ambient water temperature (10 14 jC) and at 28 30 ppt salinity until planting at grow-out sites. The first individuals within each family that were transferred from upwellers to the bags were maintained at lower water temperatures and reduced rations to allow sibs in the upwellers to catch-up. Consequently, differences in spat size among and within families due to variable growth in the hatchery and nursery were minimized in order to reduce the effects of initial spat size at planting on final yields.

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A small proportion of individuals from most families either did not reach metamorphosis as larvae or did not reach minimum planting size and were discarded. All components of the hatchery and nursery were quarantined to ensure that spat were healthy and disease-free. All water entering the facility was either filtered to 0.2 Am or filtered to 1 Am and UV-sterilized (>90,000 AW s cm 2). All effluent was mixed with 2 ppm chlorine for 40 min before discharge. 2.3. Mating design Full-sib selection was considered more appropriate for the breeding program than halfsib selection for the following reasons: (1) It allowed the breeding program to start with the largest sample of the underlying genetic diversity from source populations. This was an important concern as practical constraints limited the number of families in each generation to two cohorts of approximately 60 families each. (2) It made the rate of inbreeding easier to control in later generations because a larger number of independent founders was initially included. (3) Creating full-sib families was much less complicated than making half-sib families for the hatchery, entailing less risk of cross-contamination of gametes. (4) Because only 60 families could be raised per cohort, only three or four dams per sire could have been used in a half-sib breeding design. When heritability is greater than about 0.05, the rate of response to selection among full-sibs is expected to be much greater than the rate among paternal half-sibs when such a small number of dams are used (assuming only additive effects; Osborne, 1957; Falconer and Mackay, 1996). (5) Plant breeders routinely see substantial gains under full-sib selection (Morena-Gonzalez and Cubero, 1993), so the main argument in favor of the full-sib family selection approach is that it works for a wide range of organisms. 2.4. Trait selection In 1995, a survey of Pacific oyster growers on the West Coast, USA, indicated that meat yield (weight of meat produced per number of planted seed oysters) was the most important economic trait for selection (Hedgecock et al., 1997). The standard approach for breeding livestock is to raise individuals of known pedigree, and then to select based on an index of performance for each individual, which can be a weighted combination of the individuals performance and the performance of relatives. Because oysters must be grown in groups and they compete for resources, individuals cannot be considered independent data points for this type of selection. More formally, environmental effects unique to each individual will account for a large fraction of the phenotypic variation, making individual phenotypes poor predictors of individual breeding values. This will be particularly true for low-heritability traits such as growth rate. Therefore, selective breeding of oysters has more in common with plant breeding, where individuals are grown in competition, the unit of replication is not individuals but plots, and the unit of selection is yield per plot (Hayward et al., 1993). Consequently, we grew oysters at a standard density in bags, and family performance was measured as the mean performance of replicate bags in each family. Here performance equaled total weight of live oysters or oyster meat per bag, which included components of survival and growth.

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Yields of families of each cohort were evaluated at intertidal and subtidal commercial sites on the West Coast, USA (Fig. 1). Unless stated otherwise, spat from each family were randomly added to small 1.5-mm mesh bags (25 60 cm) that were each placed inside a larger mesh bag or a compartment of a 10-tier lantern net. Spat were planted at a density of 75 100 spat per bag at intertidal sites or at a density of 35 50 spat per lantern net compartment at subtidal sites. Duplicate bags or compartments per family were placed in each of five blocks arranged to account for environmental variation due to intertidal exposure or subtidal depth, resulting in a total of 10 replicates per family per site. Once large enough, oysters were transferred from the 1.5 mm mesh bags into standard-size (41 78 cm), 6.3-mm mesh bags or 12-mm mesh lantern net compartments in order to reduce fouling and improve water flow and food supply. At harvest, total live oyster weight of each bag was determined, after removing fouling organisms and mud. In most trials (see Results), regression analysis indicated that initial planting weight had a significant effect on final harvest weights; therefore, final weights were adjusted for differences in initial weights before further statistical analysis. Fifteen families with the highest adjusted live weights per bag were identified. All live oysters in these top-performing families were then counted to estimate survival. Next, a random sample of 15 oysters from each bag was taken. Each group of 15 oysters was weighed, shucked and meat weight determined. Meat weights of the samples were used to estimate total meat yields per family, allowing top-performing families to be ranked according to mean adjusted meat yield per bag. Oysters from each selected G0 family were divided equally into three size groups (small, medium and large). Broodstock used to create the G1 generation were selected from the largest size group within each family. 2.5. Line 1 (cohorts 1 and 5) Forty-eight full-sib families were produced to create cohort 1 (C-1, Fig. 1), each by crossing a single pair of wild oysters collected from Dabob Bay. In October 1996, cohort 1 families were planted at an intertidal site ( + 0.30 m MLLW) in Tomales Bay, CA, USA, in 3-mm mesh bags. In July 1997, spat were transferred from the 3- to 6.3-mm mesh bags for growth to harvest in November 1997. Average monthly water temperature over the growout period was 15.7 jC, ranging from a low of 13.7 jC in April to a high of 18.0 jC in August. Broodstock were collected from the nine cohort 1 families with the highest meat yields at the Tomales Bay site to produce the G1 generation. Two high-yielding families that had a high frequency of individuals with curved hinges and two families that showed low survival ( < 80%), compared with other high-yielding families, were not included. Two additional families were selected based on their superior yield without adjustment for initial plant-out weight. A 9 9 half-matrix mating design, excluding reciprocal crosses, was employed to mate individuals from each of the nine selected G0 families. This design resulted in 36 outcrossed G1 families. In addition, sib sib crosses were made within each family to produce nine inbred G1 families. When possible, a single male oyster from a selected G0 family was crossed with four female oysters from another family to better represent the genetic composition of the selected families.

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Two kinds of control groups of oysters were included. Ten full-sib families were produced from crosses between single pairs of wild oysters collected from Dabob Bay, and used to estimate the performance of nonselected oyster families. Three groups of oyster seed from different commercial spawning events were obtained and planted with G1 families at grow-out sites to serve as industry controls. G1 families plus all controls made up cohort 5 (C-5, Fig. 1). Cohort 5 families were planted intertidally ( + 0.30 m MLLW) in Totten Inlet, Puget Sound, WA (August 1998), and subtidally in Yaquina Bay, OR (June 1999). An attempt to replant cohort 5 in Tomales Bay, CA, failed. In Totten Inlet, limited spat availability resulted in only three blocks being planted rather than the usual five, producing a total of six replicates per family. Families were harvested in May 2000. Average monthly water temperature for the grow-out period was 12.5 jC, ranging from a low of 7.6 jC in February to a high of 22.6 jC in July. In Yaquina Bay, cohort 5 spat were planted in 10-tier, 12-mm mesh lantern nets at a density of 35 spat per tier and allowed to grow to harvest (May 2000). Some families planted at Totten Inlet were not planted, or were planted with fewer than 10 replicates per family in Yaquina Bay due to insufficient numbers of spat per family. Average monthly water temperature over the grow-out period was 11.1 jC, ranging from a low of 9.3 jC in December to a high of 13.2 jC in April. 2.6. Line 2 (cohorts 1+2 and 7) Eleven full-sib G0 families (cohort 2) were produced by crossing wild oysters collected from Willapa Bay. These families were planted subtidally in Yaquina Bay together with 39 cohort 1 families. This combination of families was referred to as cohort 1 + 2 (C-1 + 2, Fig. 1). Hatchery, nursery and field methods were the same as those described above. Families were planted in July 1997 and harvested in October 1998. Average monthly water temperature over the grow-out period was 14.0 jC, ranging from a low of 9.4 jC in December to a high of 18.5 jC in September 1997. Broodstock were selected from the six cohort 1 + 2 families with the highest meat yields at the Yaquina Bay site, and crossed in a half-matrix 6 6 design, excluding reciprocal crosses, resulting in 15 outcrossed and 6 inbred G1 families. In addition, six selected G0 families identified in Tomales Bay were crossed in a half-matrix 6 6 design, excluding reciprocal crosses, to produce an additional 15 outcrossed and 6 inbred G1 families. No within-family selection was carried out for Tomales Bay G0 families. These two groups of families were planted in both Tomales and Yaquina bays to determine if their response to selection was affected by the parental selection site (Fig. 1). Ten full-sib families were produced from wild oysters collected from Dabob and Willapa bays, to estimate the performance of nonselected oyster families. Three groups of oyster seed from different commercial spawning events were planted with G1 families at the grow-out sites and used as industry controls. Together with G1 crosses, these families made up cohort 7 (C-7, Fig. 1). Cohort 7 was planted intertidally in Tomales Bay in September 1999 and harvested in September 2000. Average monthly water temperature over the grow-out period was 14.0 jC, ranging from a low of 9.7 jC in December to a high of 16.8 jC in September. These

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same families were planted subtidally in Yaquina Bay in September 1999 and harvested in November 2001. Water temperatures were not determined due to loss of a temperature probe. 2.7. Line 3 (cohorts 4 and 9) Forty full-sib G0 families were produced by crossing wild oysters collected at either Pipestem Inlet or Willapa Bay. These families were referred to as cohort 4 (C-4, Fig. 1). The families were planted intertidally in Sequim Bay, WA, USA, in June 1998 and harvested in September 1999. Average monthly water temperature over the grow-out period was 13.0 jC, ranging from a low of 7.3 jC in February to a high of 21.8 jC in September. Broodstock were selected from the six cohort 4 families with the highest meat yields at Sequim Bay, and crossed in a half-matrix 6 6 design, excluding reciprocals, to produce 15 outcrossed and 6 inbred G1 families. Ten full-sib families were produced using wild oysters collected from Dabob Bay to estimate the performance of nonselected oyster families. Three groups of oyster seed from different commercial spawning events were again obtained to serve as an industry control. These controls, together with the G1 crosses, made up cohort 9 (C-9, Fig. 1). Cohort 9 was planted intertidally ( + 0.30 m MLLW) at Totten Inlet in August 2000 and harvested in September 2001. Average monthly water temperature over the grow-out period was 12.6 jC, ranging from a low of 6.8 jC in February to a high of 17.9 jC in August. 2.8. Data analysis Family performance was expressed as mean live weight per bag or lantern net compartment at harvest. Each replicate bag was therefore an independent measure of family yield. Analysis of variance was carried out to determine the significance of block, family and block family effects on yield within each cohort. Factors were considered significant at P < 0.05. Contrasts were then performed to determine if average yield of G1 selected families differed significantly from nonselected control families within each line of selection (i.e. lines 1, 2 or 3; Fig. 1). Contrasts were also performed between control and inbred families to determine the significance of inbreeding depression. Intraclass correlation coefficients (t) were determined for each of the G0 cohorts (cohorts 1, 1 + 2, and 4) according to Falconer and Mackay (1996). Unequal numbers of replicate bags within families were corrected by following methods outlined in Sokal and Rohlf (1995). The within-family term used to calculate t was a measurement of the variance among replicate bags within a family and not the variance among full-sib individuals as is commonly reported in the literature. Here oyster bags were considered plots with 35 100 individuals within a plot. The broad-sense heritability was then estimated for each site as: H2 t r

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where t was the intraclass correlation as defined above and r was the coefficient of relationship among replicates (Falconer and Mackay, 1996). Each oyster bag weight (i.e. yield) was considered an estimate of offspring performance for a given set of parents. Therefore, all bags within a family were independent yield estimates of the same average genotype with an approximate coefficient of relationship of 1. When r = 1, the equation above reduces to H2 = t. Standard error of H2 and t estimates were calculated following Becker (1984). Realized heritability (h2) estimates for yield were calculated as: r h2 r R X F1 X Wild S irPWild

where R was the response to selection and S was the selection differential (Falconer and Mackay, 1996). Response to selection (R) was estimated simply as the difference between the average yield of G1 families and the average yield of nonselected control families at each site. The selection differential was determined based on the product of i, the intensity of selection, and rP(Wild), the phenotypic standard deviation of the nonselected wild-control family means (Falconer and Mackay, 1996). The number of families in wild-control groups ranged from 7 to 10 due to practical limitations in the number of families that could be tested at a site. Larger numbers of wild-control families would have been preferred in order to reduce the variance of family means and increase the accuracy of heritability estimates. In addition, the unselected control families in cohort 9 were not derived from the same source population as the founder families of cohort 4 (Fig. 1). Two estimates of S were derived, each differed in how the intensity of selection was determined. The first estimate assumed no genetic correlation between individual oyster size and average family yield, thus within-family selection for individual body weight (i.e. selecting broodstock from the top third largest oysters within a family) had no effect on the observed response to selection in average family yield. In this case, i was estimated as: i X Sel X Pop rPop

where X Sel is the mean of selected G0 families, X Pop is the total population family mean, and rPop is the population standard deviation of family means. It was understood that this approach would likely result in downward bias in the estimate of S and, therefore, an upward bias in the estimated realized heritability because any correlated response in yield due to within-family selection for individual size was not considered. A second estimate of i was determined in order to account for the possible correlated effect of within-family selection for individual oyster size on average family yield. This second estimate determined what each bag weight would have been if the average individual oyster weight was equivalent to the average weight of the top third heaviest oysters in a particular bag. Average family survival was assumed to remain unchanged. Selection intensity was then calculated as the difference between the new family bag

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weight (based on the individual weights of the top third heaviest oysters within each bag) and the site average bag weight divided by the site standard deviation of average family bag weights. Standard errors for all realized heritability estimates were determined as per Hadley et al. (1991). The significance of genotype environment interaction was determined by analysis of variance and regression analysis of average family yields at two sites. Graphical analysis was used to determine if multiple trait selection (the two traits being average family yield at one site and average family yield at a second site) could be effective in selecting for superior G1 families at both sites (generalists).

3. Results Initial plant-out weight was significantly correlated with harvest yield for all cohorts ( P < 0.01; average r2 = 0.34, range 0.11 0.52). Family effects were significant ( P < 0.05) for all cohorts. Block effects were significant in all cohorts except for cohort 5 families planted in Totten Inlet, WA, and cohort 7 families planted in Yaquina Bay, OR. In the intertidal sites, the highest yielding blocks tended to be those located lowest in the intertidal zone. No consistent pattern was found between yield and water depth in the subtidal sites. Family block interactions were never significant ( P>0.05). Live weights of oysters sampled from the 15 top-performing families at each site were always highly correlated with meat weights (r = 0.71 0.93); therefore, live weight data were considered a reliable estimate of meat weights in this study. 3.1. G0 generation Performances of G0 families are summarized in Table 1. Intraclass correlations were high for all G0 cohorts (0.40 0.61), indicating that a large fraction of the total phenotypic variation in yield could be attributed to genetic causes (Falconer and Mackay, 1996). Mean yields of G0 families chosen for broodstock ranged from 0.87 to 1.35 standard deviation units greater than the corresponding population mean. Survival among the top-yielding selected families was high at all sites, ranging from 76.3% to 96.6%. 3.2. Response to selection Performance of G1 families, inbred families, wild-control families and industrycontrol families are summarized in Table 2. Response to selection for high yield was positive in all selected lines ranging from 0.4% in cohort 7 (parents selected in subtidal Yaquina Bay and offspring evaluated at intertidal Tomales Bay) to a high of 25.5% in cohort 5 (parents selected at intertidal Tomales Bay and offspring evaluated at intertidal Totten Inlet). Contrast results indicated that offspring from selected parents were significantly higher yielding than offspring from nonselected wild parents ( P < 0.01) within cohort 5 (selection line 1) and cohort 9 (selection line 3; Table 2; Fig. 1).

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Table 1 Summary data for all G0 families and for G0 families selected as parents of the G1 generation Cohort 1 Trial sites Date planted Date harvested All families Mean family yield/bag (kg F 1 S.D.) n Range (kg) CV (%) Intraclass correlation (t F 1 S.E.) Selected G0 families Mean live weight (kg F 1 S.D.) n Range Mean meat weight (kg F 1 S.D.) Mean meat weight range (kg) Survival (% F 1 S.D.) Survival range (%) Tomales Bay (intertidal) 10/23/1996 10/13/1997 6.34 F 1.15 48 3.71 8.98 18.1 0.40 F 0.07 Cohorts 1 and 2 Yaquina Bay (subtidal) 7/10/1997 10/27/1998 2.71 F 0.43 50 1.86 3.53 15.9 0.36 F 0.06 Cohort 4 Sequim (intertidal) 6/8/1998 9/7/1999 4.52 F 0.84 40 2.67 6.10 18.5 0.61 F 0.06

7.35 F 0.83 9 6.07 8.98 1.24 F 0.08 1.14 1.41 82.8 F 3.6 76.3 87.3

3.30 F 0.13 9 3.12 3.53 0.60 F 0.03 0.53 0.65 85.4 F 5.1 78.6 92.6

5.65 F 0.26 6 5.41 6.10 1.15 F 0.13 1.00 1.39 88.1 F 2.4 78.0 96.6

Within cohort 7 (selection line 2), significantly higher yields of G1 families compared with yields of nonselected families were only observed when G1 families of parents selected from the subtidal Yaquina Bay site were planted back in Yaquina Bay ( P < 0.001; Table 2). No significant difference was observed if these offspring were planted intertidally in Tomales Bay. In cohort 5, derived from parents selected intertidally, offspring evaluated in Totten Inlet (intertidal) had a significantly higher standardized response to selection than offspring evaluated in Yaquina Bay (subtidal) ( P < 0.01). At both cohort 7 test sites (intertidal and subtidal), no significant differences were found in the responses to selection between offspring of parents selected in Tomales Bay (intertidal) and offspring of parents selected in Yaquina Bay (subtidal) ( P>0.05). Estimates of realized heritability (ignoring within-family selection) were high for cohort 5 (0.39 and 0.52) and cohort 9 (0.77), and for cohort 7 evaluated in Yaquina Bay (0.22 and 0.34). When cohort 7 was evaluated in Tomales Bay, however, realized heritabilities were not significantly different from zero (0.10 and 0.01; Table 2). Similar trends were seen among h2 estimates when within-family selection was considered, r although the increased selection intensity resulted in lower heritability estimates. Estimates of h2 for cohort 7 families derived from parents selected in Tomales Bay were an exception r because parents were collected from a group of average-size oysters and no within-family selection was applied.

Table 2 Summary of family performance based on cross type Cohort 5 (G1) G1 trial sites Date planted Date harvested G0 trial sites G1 families from selected parents Average family bag weight (kg F S.D.) Number of crosses Nonselected controls Average family bag weight (kg F S.D.) Number of crosses Inbred families Average family bag weight (kg F S.D.) Number of crosses Industry controls Average family bag weight (kg F S.D.) Number of spawning events Response to selection (% increase/generation) P value (Ho: selected = nonselected lines) h2r of ave. family bag wt. (F S.E.)a h2r of ave. family bag wt. (F S.E.)b
a b

Cohort 7 (G1) Yaquina (subtidal) 10/29/1999 11/20/2001 Yaquina Tomales Yaquina 6/22/1999 5/17/2000 Tomales 1.63 F 0.24 33 1.51 F 0.27 7 1.24 F 0.20 4 1.58 F 0.11 3 7.8 < 0.001 0.39 F 0.16 0.24 F 0.08 10/20/1999 10/31/2000 Tomales 1.72 F 0.25 9 1.66 F 0.42 8 1.46 F 0.30 9 2.09 F 0.19 3 3.8 0.0830 0.10 F 0.08 0.10 F 0.08 0.4 0.843 5.60 0.076

Cohort 9 (G1) Totten (intertidal) 8/29/2000 9/4/2001 Sequim C. Langdon et al. / Aquaculture 220 (2003) 227244

Totten (intertidal) Yaquina (subtidal) Tomales (intertidal) 8/31/1998 5/10/2000 Tomales 3.39 F 0.94 34 2.70 F 1.17 10 2.03 F 1.05 6 3.32 F 0.82 3 25.5 < 0.001 0.52 F 0.16 0.32 F 0.08

1.67 F 0.33 5.22 F 0.59 12 9 4.94 F 0.82 9 5.26 F 0.69 7 6.13 F 0.61 3

5.38 F 0.41 1.05 F 0.14 14 13 0.91 F 0.15 10 0.78 F 0.15 5 1.02 F 0.24 5 14.70 < 0.001

8.90 < 0.001

0.01 F 0.05 0.22 F 0.11 0.01 F 0.04 0.22 F 0.11

0.34 F 0.11 0.77 F 0.22 0.26 F 0.10 0.50 F 0.12

Realized heritability of average family live weight without adjusting for within family selection. See text for details. Realized heritability of average family live weight, adjusting for within family selection. See text for details.

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3.3. Genotypeenvironment interaction In cohort 5, analysis of variance indicated that a genotype environment interaction significantly affected yields of families planted at both Totten Inlet and Yaquina Bay ( P < 0.01). In addition, average family yields were not significantly correlated across site (r = 0.30, P = 0.06, n = 41). These results suggest that the relative performance of most families differed between the two environments (Fig. 2A). Although the correlation

Fig. 2. Identification of generalists from two MBP cohorts. (A) Cohort 5 families at intertidal Totten Inlet, WA, and subtidal Yaquina Bay, OR, sites. (B) Cohort 7 families at intertidal Tomales Bay, CA, and subtidal Yaquina Bay, OR, sites. Dotted lines represent point of truncation for the top 10 families at each site. Generalist families are located in the top-right quadrant.

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between family yields at the two sites was not significant, it was possible to identify six G1 families that were present among the top 10 performing families at both sites, i.e. generalist families. In cohort 7, analysis of variance indicated that a genotype environment interaction significantly affected yields of families planted at both Tomales Bay and Yaquina Bay ( P < 0.01); however, family yields of cohort 7 in Yaquina Bay and Tomales Bay were significantly and positively correlated (r = 0.346, P = 0.04, n = 35), although the correlation coefficient (r) was low (Fig. 2B).

4. Discussion The positive response to selection for live weight yields estimated in this study indicated that long-term selection should result in greater yields of Pacific oysters. Response to selection ranged from 0.4% to 25.6% improvement in yields of families from selected broodstock compared with families from wild broodstock. The average response to selection over seven trials resulted in an G1 family yield that was on average 9.5% heavier than that of nonselected controls. It is unclear why G1 families of cohort 7 evaluated in Tomales Bay showed very little improvement in yield. It is possible that environmental conditions within Tomales Bay changed sufficiently over time to render G0 selection in 1997 less effective in improving the yields of G1 families evaluated in 2000 (i.e. genotype time interaction). In addition, cohort 7 families in Tomales Bay were evaluated only after one growing season, which may not have been sufficient time for yields of control and selected lines to diverge and for a significant response to selection to be detected. Yield is an index of the combined effects of individual growth rate and survival (Blum, 1988). Response to selection for yield of bivalves has not been reported in the literature; however, Nell et al. (1996, 1999) found that growth (increase in live weight) of mass-selected Sydney rock oysters (Saccostrea commercialis) was 4% and 18% greater after one and two generations of selection, respectively, compared to that of nonselected controls. Similarly, after one generation of mass selection, Newkirk and Haley (1982) reported a 16 39% increase in growth rate of Crassostrea virginica and a 21 42% increase in growth rate of Ostrea edulis compared with that of nonselected controls. Survival, the other component of yield, has also been successfully improved through selection by Haskins and Ford (1988) (resistance to Haplosporidium nelsoni, MSX) and by Hershberger et al. (1984) (tolerance of elevated water temperatures in Puget Sound). Our most conservative estimates of realized heritabilities ranged from 0.01 to 0.50 over all trials (Table 2). Response to selection and heritability was highest when parents and offspring were evaluated in the same environment, as evident with cohort 5 and to a lesser degree with cohort 7, suggesting some extent of site-specific selection response. After adjustment for within-family selection, our estimates of heritability for yield become comparable to estimates reported in the literature for shellfish growth. Two studies have been performed to estimate heritability of individual growth rate in Pacific oysters. The first by Lannan (1972) used full-sib analysis to estimate broad-sense heritabilities for total

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weight (H2 = 0.31) and meat weight (H2 = 0.37). These estimates were biased upward due to inclusion of nonadditive genetic effects. More recently, Hedgecock et al. (1991) reported narrow-sense heritability of meat weight at harvest size to be approximately 0.2. Work with C. virginica (Davis, 2000), O. edulis (Toro and Newkirk, 1990; Newkirk and Haley, 1983) and Ostrea chilensis (Toro et al., 1994, 1995) have produced estimates of narrow-sense heritability of growth rate between 0.12 and 0.43. Significant heritability estimates of adult growth have also been reported for other bivalve species (Mallet et al., 1986; Stro mgren and Nielson, 1989; Paynter and Dimichele, 1990; Rawson and Hilbish, 1990, 1991; Hadley et al., 1991; Hilbish et al., 1993; Jarayabhand and Thavornyutikarn, 1995). Genotype environment interactions are pervasive in natural and agricultural environments, and breeding for yield stability is a common goal among terrestrial crop breeders (Ceccarelli, 1989; Blum, 1988; Baker, 1987). Generally, genotype environment interactions tend to occur wherever selective pressures vary with environment (Price and Schluter, 1991) and when a complex character (e.g. yield) is governed by two or more noninteracting causal components (Baker, 1987). The instability of relative family performance across environments observed in this study is consistent with other shellfish studies. Mallet and Haley (1983) found a significant genotype environment interaction effect on performance traits in C. virginica. Rawson and Hilbish (1991) also reported significant genotype environment interactions for hard clams (Mercenaria mercenaria), with reversals in the relative performance of families among five different sites along the East Coast, USA. They found no evidence for generalist clam families that performed well over a wide range of different sites. Commercial hatcheries for Pacific oysters on the West Coast commonly produce seed for growers farming in a wide range of sites and using a variety of culture methods. A single hatchery may provide seed for farms from Alaska to California, using subtidal or intertidal sites in brackish or marine environments. These hatcheries would, therefore, like to produce generalist families that perform well across a broad range of environments. Further experimentation is required to determine whether or not generalist top-performing families identified in this study would perform well across a broad range of culture environments and be suitable for commercial production. Although significant genotype environment interactions were detected and correlations of relative family performances were poor across sites, it was possible to identify six generalist families in cohort 5 and four generalists in cohort 7 that were among the top 10 families at both sites. Selecting for generalist families is one approach to breeding for yield stability, i.e. select only those families with acceptable performance at both sites. If multiple trait selection was adopted, whereby family yields at both sites were treated as two separate traits (Falconer and Mackay, 1996), then 10 out of 41 families at each cohort 5 site would need to be selected in order to include the top six generalist families, resulting in a selection intensity of 1.28. In contrast, if site-specific selection was adopted, then the top six of 41 families would be considered from each site, resulting in a higher selection intensity of 1.58, i.e. an intensity about 23% greater than with multiple trait selection. Similarly, if multiple trait selection was adopted in cohort 7, then 12 out of 35 families at each site need to be selected in order to include the top six generalist families, resulting in a selection

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intensity of 1.08. In contrast, if site-specific selection was adopted, then the top six of 35 families would be considered from each site, again resulting in a higher selection intensity of 1.49, i.e. an intensity about 38% greater than with multiple trait selection. Multiple trait selection of generalists will result in reduced selection intensity and yield improvement rates at any single site; however, multi-trait selection will be necessary in order to avoid selection of site-specific lines. The significant inbreeding depression found in this study (Table 2) agrees with previous work by Beattie et al. (1987), Hedgecock et al. (1995, 1996) and Bayne et al. (1999), and is consistent with significant nonadditive gene action determining performance characters in Pacific oysters. Strong nonadditive genetic effects on both larval survival (Lannan, 1980a,b) and larval growth (Hedgecock et al., 1995; McGoldrick and Hedgecock, 1997) have been reported for crosses among inbred lines of Pacific oysters. There has been considerable debate in the literature over the genetic basis of nonadditive genetic variation in bivalve mollusks, with either overdominance, dominance, associative dominance or epistasis being favored as explanations (see review by Hedgecock et al., 1996). Recent work by Launey and Hedgecock (2001) showed that inbreeding depression and the converse phenomenon of heterosis in Pacific oysters were due to a high load of deleterious recessive mutations, supporting the dominance hypothesis explaining heterosis. Improvement in broodstock quality will benefit from the identification and elimination of such recessive lethal mutations from the broodstock. In the present study, we did not determine whether yields were affected by differences in growth or survival or both. However, we observed high mortalities in some families with poor yields. Selection has been shown to improve survival of Pacific oysters that typically experienced high mortalities during summer periods in Puget Sound, WA (Beattie et al., 1978, 1987; Hershberger et al., 1984), and of Eastern oysters (C. virginica) exposed to MSX (Haskins and Ford, 1988) and juvenile oyster disease (Davis and Barber, 1999). Future attempts to increase yields of Pacific oysters should consider improving both survival and growth traits. In summary, this study showed that family selection resulted in improved yields of Pacific oysters planted at both an intertidal and subtidal grow-out sites, compared with yields of nonselected oysters. Family-based, long-term selection will likely lead to further improvement in yields. Although genotype environment interaction effects were evident, some families with high yields at both sites (generalists) were identified. Further research is required to determine if generalist families perform well over a broader range of conditions than used in this study and if substantial improvements in yield can be achieved by selecting generalist families versus families that are suited to specific environments. Oyster hatcheries should develop broodstock management programs to prevent unintentional inbreeding in order to avoid reduced yields due to inbreeding depression.

Acknowledgements The authors wish to thank Sean Matson, John Brake, Ebru Onal, and Matt Smith for technical assistance during the project. Dave Specht, U.S. Environmental Protection

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Agency, Newport, kindly provided seawater temperature data for some test sites. The study would not have been possible without the generous support of Hog Island Oyster, Taylor Oyster Farms, Oregon Oyster Farms, and the Jamestown SKlallam Tribe. The breeding program greatly benefited from input of members of the USDA Western Regional Coordinating Committee (WCC-99). The comments of two anonymous reviewers greatly improved the manuscript.

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