Parental and stock effects on larval growth and survival to metamorphosis in winter flounder (Pseudopleuronectes americanus)

Aquaculture 269 (2007) 339 – 348 www.elsevier.com/locate/aqua-online

Parental and stock effects on larval growth and survival to metamorphosis in winter flounder (Pseudopleuronectes americanus) Ian A.E. Butts ⁎, Matthew K. Litvak Department of Biology and Centre for Coastal Studies and Aquaculture, University of New Brunswick (Saint John), Ganong Hall, P.O. Box 5050, Saint John, New Brunswick, Canada E2L 4L5 Received 9 June 2006; received in revised form 4 April 2007; accepted 7 April 2007

Abstract Geographically separated winter flounder (Pseudopleuronectes americanus) populations in the northwest Atlantic Ocean are both phenotypically and genetically distinct from one another. This has important implications for winter flounder aquaculture with respect to broodstock selection; however, few studies have investigated the effect of population on larval growth and survival in a hatchery setting. In this study, eggs from Passamaquoddy Bay females were fertilized with sperm from Georges Bank and Passamaquoddy Bay males. Larvae were reared in common environmental conditions to evaluate population and parental contributions to variations in growth, and survival during early life history. Mixed-model nested ANOVAs revealed that larvae sired by Georges Bank males were significantly larger with respect to standard length, eye diameter, head depth, and jaw length during certain stages in larval development. Maternal, paternal, and parental interactions all contributed to morphological variation in developing larvae. Survival was strongly influenced by the paternal variance component. These results have two major implications: 1) they provide further supporting evidence that Georges Bank winter flounder are genetically selected for faster growth than larvae from inshore stocks, and 2) they suggest that aquaculture operations should also account for paternal variation so that the best broodstock can be selected for production. © 2007 Elsevier B.V. All rights reserved. Keywords: Winter flounder; Stock; Parental; Georges Bank

1. Introduction Winter flounder (Pseudopleuronectes americanus) has been identified as a species with potential for aquaculture in Atlantic Canada and the Northeast United States (Howell and Litvak, 2000; Litvak, 1994, 1996, 1999). These fish possess several desirable characteristics for aquaculture. They are euryhaline (McCracken, 1963), and eurythermal (Pearcy, 1961, 1962; Duman and DeVries, 1974), they ⁎ Corresponding author. Tel.: +1 506 529 5873; fax: +1 506 529 5862. E-mail address: [email protected] (I.A.E. Butts). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.012

respond well to stripping of gametes (Rideout et al., 2003), can be photo-manipulated to produce good quality eggs seven months of the year (Butts, unpublished data), their sperm can be cryopreserved (Rideout et al., 2003), and they process antifreeze proteins that allow then to survive temperatures less than −1 °C (Pearcy 1961; Duman and DeVries, 1974). Overall winter flounder are an extremely hardy fish; however, a major developmental constraint for winter flounder aquaculture is the lack of knowledge of larval growth and survivorship to the juvenile stage (Litvak, 1999). Larval growth and survival are the most important traits during hatchery production and define the success

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of an aquaculture venture in the early production stage (Litvak, 1999). Several factors may contribute to larval growth and survival, including genetic effects (at both the parental and population level), and non-genetic effects (such as maternal non-genetic contributions and environmental conditions). Most research aimed at improving growth and survival of winter flounder during early life history (ELH) has concentrated on non-genetic factors such as the effect of diet (King and Howell, 1997; Ben Khemis et al., 2000; Mercier et al., 2004), appropriate weaning procedures (Lee and Litvak, 1996; Ben Khemis et al., 2003), and optimal environmental conditions (Williams, 1975; Bejda et al., 1992; Keller and Klein-MacPhee, 2000). The next step in developing winter flounder as an aquaculture species is to determine genetic effects on larval growth and survival at the parental, and population levels. This will allow for the selection of the most suitable broodstock for aquaculture production. Several studies have reported different growth rates between winter flounder populations in the wild (Lux, 1973; Buckley et al., 1991). Winter flounder from different locales have been found to be phenotypically (Kendall, 1912; Lux et al., 1970) and genetically distinct (Crivello et al., 2004; McClelland et al., 2005). Recent work has demonstrated Georges Bank winter flounder yolk-sac larvae have higher growth rates than Passamaquoddy Bay flounder when individuals of the two populations are held under identical conditions (Butts and Litvak, 2007). Further comparisons among the different stocks (populations) are important to understanding which individuals will produce larvae with the highest growth and survival rates. Maternal effects tend to have large impacts on larval production (reviews in Hempel, 1979; Kamler, 1992; Chambers and Leggett, 1996; Brooks et al., 1997; Solemdal, 1997); however, research on paternal effects has been under-represented in the literature (Kirpichnikov, 1981; Trippel, 2003; Rideout et al., 2004). Two studies have examined paternal contributions to winter flounder growth (Chambers and Leggett, 1992; Butts and Litvak, 2007). Butts and Litvak (2007) found that during certain points in embryo and early larval development, maternal, paternal, and the parental interactions all contributed to differences in morphological variation and performance. Chambers and Leggett (1992) examined parental effects present at metamorphosis. Although a significant maternal effect for length at metamorphosis was found, no significant paternal effect was evident. It was noted that differences caused by parentage may have occurred during an earlier stage in the ELH of the fish and disappeared by metamorphosis, and that the degree of each parental contribution is likely to be trait specific (Chambers and

Leggett, 1992). Therefore, it may be more beneficial to study paternal effects on ELH success at earlier larval stages, examine various larval morphological traits, and/or survival during larval development to metamorphosis rather than just size at metamorphosis (Rideout et al., 2004). To fully understand all contributing factors to larval growth and survival it is important to evaluate the magnitude of stock and parental effects as larvae develop to metamorphosis. A half-sibling breeding design was used to: 1) determine if larvae sired by Georges Bank males were larger at age than larvae sired by Passamaquoddy Bay males; and 2) examine the maternal, paternal, and family contributions to larval morphological development, and survival during ELH. 2. Materials and methods 2.1. Broodstock collection and management Broodstock were collected from Georges Bank (40– 42°N) between Feb. 5–17 2004, and from Passamaquoddy Bay, N.B., Canada (44–45°N), on Nov. 3 2003 and Apr. 23 2004 using bottom trawls. The fish were held at the University of New Brunswick, Saint John campus (UNBSJ), in a 2700 l closed recirculation system. Temperature and salinity were maintained at 2– 4 °C, 28–30 ppt, respectively. 2.2. Gamete collection and egg incubation Sperm was obtained from 6 males: 3 males from Passamaquoddy Bay and 3 males from Georges Bank. In order to synchronize the spawning of several males and to provide consistency in sperm availability thought-out the entire sampling period, sperm collected from each male was cryopreserved following Rideout et al. (2003). Eggs were collected from three Passamaquoddy Bay females. Females produced eggs either naturally or after luteinizing hormone-releasing hormone (LHRH, 10 μg/ kg; Syndel International) injections. Results from previous research have shown that hormone injections have no effect on egg quality, fertilization rates or larval ELH traits (Smigielski, 1975; Harmin and Crim, 1992; Shangguan and Crim, 1999). Expelled eggs were collected into dry 100 ml polyethylene beakers. Using a 1 ml syringe, 0.2 ml of eggs (approximately 900) was placed into petri dishes (150 mm × 15 mm). Sperm was immediately added to the eggs in each petri dish (referred to as a “cross”). Eggs from each female were crossed with the sperm from each male producing six half-sibling families per female. Three

I.A.E. Butts, M.K. Litvak / Aquaculture 269 (2007) 339–348 Table 1 Summary of fixed effects statistics (DFN = numerator degrees of freedom, DFD = denominator degrees of freedom, f = f value, p = p value) obtained from the nested mixed-model repeated measures ANOVAs used to examine each of the winter flounder larval morphological traits (SL = standard length, MH = myotome height, ED = eye diameter, JL = jaw length, HD = head depth, BA = body area) Morphological trait

Effect

DFN DFD

f

p

SL

Population Day Population × day Population Day Population × day Population Day population × day Population Day Population × day Population Day Population × day Population Day Population × day

1 4 4 1 4 4 1 4 4 1 4 4 1 4 4 1 4 4

11.70 163.15 2.20 7.37 467.83 2.31 12.90 160.57 4.88 49.37 482.73 4.37 15.11 257.29 3.96 9.78 132.94 5.05

0.026 b0.001 n.s 0.067 b0.001 n.s 0.019 b0.001 0.008 b0.001 b0.001 0.004 0.017 b0.001 0.021 0.034 b0.001 0.008

MH

ED

JL

HD

BA

4.08 11.50 16.40 3.23 11.60 16.50 4.52 9.33 17.00 14.40 8.79 47.80 4.04 10.80 15.70 4.08 11.90 15.40

Note that when a non-significant first-order interaction was detected (P N 0.05; n.s.), the model was re-run with the interaction effect removed.

replicate crosses were completed for each male–female pairing. Once the milt was added to the petri dish, the eggs and milt were quickly swirled together and 100 ml of 28 ppt sterilized seawater (8 °C) containing 13 mg/l of penicillin G, and 13 mg/l of streptomycin sulfate was added to the dish. The sperm–egg–seawater combination sat for 20 s and then milt was rinsed away (Rideout et al., 2003). Petri dishes were immediately refilled. The fertilized eggs were kept in petri dishes. Embryos were incubated in 24 h darkness, in a temperature controlled room at 8 °C. Seventy-five percent of the sterilized seawater in each petri dish was replaced every second day.

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2.3. Larval rearing Cohorts of hatched larvae were removed daily. When larvae from modal hatch had reached 3 and 4 day post hatch, 100 larvae (50 larvae from each daily cohort) were counted and placed into 1.3 l black polyethylene containers (12 cm diameter; 12 cm height). This stocking day represents experimental day 0. Larvae were kept under continuous overhead lights (Litvak, 1999) for the duration of their development, at an intensity of ∼1100 lx. Temperature, which has been shown to affect larval growth and body size (Blaxter, 1992), was recorded daily in each experimental tank. No significant differences in temperature (mean ± SD of 14.3 °C ± 0.61) existed between treatments (all P N 0.05, nested analysis of variance (ANOVA) model using SAS PROC GLM procedure; SAS Institute, 2003). Salinity was kept constant at 28 ppt. A pipette attached to an air source was placed at the bottom of each tank to prevent prey from settling out, and to maintain oxygen levels. Half the water in each tank was changed every second day, at which time mortalities were counted and removed. Larvae were fed Instant Algae® (Pavlova sp; 40,000 cells/ml of culture water; Reed Mariculture Inc., U.S.A.) for the duration of the experiment. From days 0–21, larvae were fed rotifers (Brachionus plicatilis; 5 rotifers/ml). A mixture of rotifers (3 rotifers/ml) and Artemia sp. (2 Artemia/ml) were fed to the larvae from days 21–28. Rotifers and Artemia were enriched with Microfeast® MB-30 (Aquaculture supply, USA) and a combination of algae (Isochrysis sp., Tahitian strain (TISO) and Pavlova lutheri). Larvae were fed twice daily at 900 h and 1800 h. 2.4. Data collection Images of eggs, embryos and larvae were captured using a digital camera (Coolpix Nikon 990) attached to a dissecting microscope (Olympus SZ60 with SZ-CTV

Table 2 Summary of fixed effects statistics (DFN = numerator degrees of freedom, DFD = denominator degrees of freedom, f = f value) obtained from the nested mixed-model ANOVAs used to examine each of the winter flounder larval morphological traits (ED = eye diameter, HD = head depth, JL = jaw length, BA = body area) Trait

Day 0 DFN

ED(mm) HD (mm) JL (mm) BA (mm2) ⁎ P b 0.05.

1 1 1 1

Day 7 DFD 3.76 2.3 2.11 2.27

F 3.05 2.13 0.61 2.04

DFN 1 1 1 1

Day 14 DFD

F

3.56 1.91 2.02 1.89

8.67 ⁎ 5.58 20.85 ⁎ 6.44

DFN 1 1 1 1

Day 21

Day 28

DFD

F

DFN

DFD

F

DFN

DFD

F

4.11 3.42 4.13 2.76

12.93 ⁎ 5.42 17.61 ⁎ 2.37

1 1 1 1

1.88 4.33 3.58 4.32

10.78 8.22 ⁎ 9.02 ⁎ 5.85

1 1 1 1

6.49 3.72 4.64 4.3

0.4 9.6 ⁎ 13.23 ⁎ 5.89

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Fig. 1. Results of mixed-model nested ANOVAs for stock effects on winter flounder larval morphological traits. Separate ANOVAs were run for days 0, 7, 14, 21, and 28 because the nested repeated measures ANOVA revealed significant first-order interactions. Populations with different letters are significantly different (P b 0.05, least square means, ANOVA). Error bars represent least square means standard error (Proc Mixed; SAS Institute, 2003).

attachment). Image J analysis software (Version 1.32j) was used to analyze the digital images. 2.4.1. Size of eggs Fifty eggs were randomly sampled from each female to analyze egg diameter (mm). 2.4.2. Larval growth and survival For the analysis of larval growth four larvae were randomly sampled from each tank at day 0, day 7, day 14, day 21, and day 28. Larvae were anesthetized using MS-222 (0.1–0.5 mg/ml), and then digitally imaged. Measurements of standard length (SL; distance from tip of snout to tip of notochord (days 0–14), and to hypural plate (day 21 and day 28)), myotome height (MH; immediately posterior to anus), eye diameter (ED; maximum eye diameter), jaw length (JL; length of the lower jaw), head depth (HD; total depth of the head at the midpoint of the eye) and body area (BA; excluding finfold and yolk-sac) were obtained from each larva. The number of surviving larvae at day 28 was recorded. 2.5. Statistical analysis All data were analyzed using SAS statistical analysis software (v.9.1; SAS Institute Inc., Cary, NC, USA).

Prior to analyses, data were tested for normality (Shapiro–Wilk test; PROC UNIVARIATE; SAS Institute, 2003) and homogeneity of variance (plot of residuals vs. predicted values; PROC GPLOT; SAS Institute, 2003). Data were transformed to meet the assumptions of normality and homoscedasticity when necessary. Alpha was set at 0.05. 2.5.1. Size of eggs Differences in mean egg diameter (mm) between females were analyzed using a single-factor ANOVA (PROC GLM; SAS Institute, 2003). Tukeys a-posteriori analysis was used to compare treatment means. 2.5.2. Larval growth and survival All morphological characters were analyzed using mixed-model nested repeated measures ANCOVAs based on mean values per tank (PROC MIXED; SAS Institute, 2003): P
Yijkmn ¼ l þ b Xijkmn  X þ Di þ Pj þ Sð PÞk ð jÞ þDPij þ DSð PÞik ð jÞ þTm þ TDmi þ TPmj þ TSð PÞmk ð jÞ þTDPmij þ TDSð PÞmik ð jÞ þenðijkmÞ

ðModel1Þ

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Fig. 2. Restricted maximum likelihood (REML) variance components expressed as a percentage for growth of winter flounder larvae. Separate analyses were run for experimental days 0, 7, 14, 21, and 28 data because of significant first-order fixed interactions with sampling time. To test for significant variability among variance components that were greater than zero, likelihood ratio statistics were generated (Littell et al., 1996; Messina and Fry, 2003; Fry, 2004). Variance components with an asterisk are significant (P b 0.05).

Fig. 3. Mean standard length and myotome height for stock (1 and 3) and day (2 and 4) effects generated from the mixed-model repeated measures nested ANOVA. Treatments significantly different from one another (P b 0.05, determined from a Scheffé multiple comparisons procedure; Proc Mixed; SAS Institute 2003) are indicated by different letters (A–E). Error bars represent least square means standard error (Proc Mixed; SAS Institute, 2003).

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Table 3 Restricted maximum likelihood (REML) variance components expressed as a percentage for larval standard length (SL) and myotome height (MH) Variance component

SL

MH

Maternal Maternal × population Day × maternal Paternal (population) Maternal × paternal (population) Day × paternal (population) Day × maternal × paternal (population) Residual

0.0 0.0 29.2 ⁎⁎ 6.1 0.0 10.8 ⁎ 10.4 ⁎ 43.5

0.0 23.7 ⁎⁎ 0.1 4.8 0.8 9.1 24.2 ⁎⁎ 37.3

Variance components were obtained from repeated measures nested ANOVAs. To test for significant variability among variance components that were greater than zero, likelihood ratio statistics were generated (Littell et al., 1996; Messina and Fry, 2003; Fry, 2004). ⁎ P b 0.05. ⁎⁎ P b 0.001.

where μ is the true mean; β is the combined regression coefficient between X and Y within each group; X¯ is the mean value of the covariate; Di is the maternal effect (where i = 1–3); Pj is the population effect (where j = 1, 2); S(P)k( j ) is the paternal effect (k = 1–3) nested within each population; DPij is the maternal × population interaction; DS(P)ik( j ) is the maternal × paternal nested within population interaction; Tm is the day main effect (where m = 1–5); TDmi is the day × maternal interaction; TPmj is the day × population interaction; TS(P)mk( j ) is the day × paternal nested within population interaction; TDPmij is the day × maternal × population interaction; TDS(P)mik( j ) is day × maternal × paternal nested within population interaction; and εn(ijkm) is the residual error. Population and day effects were considered fixed while maternal and paternal effects were considered random. In addition to testing for normality and homogeneity of variances, sphericity and homogeneity of slopes were tested to ensure that the data followed all the assumptions of the model (Quinn and Keough, 2002; SAS Institute, 2003).The Kenwardroger (Kenward and Roger, 1997) procedure was used to approximate the denominator degrees of freedom for all F-tests (Spilke et al., 2005). When a non-significant first-order fixed interaction was detected, the model was re-run with the interaction effect removed. In the case of a significant first-order fixed interaction, the model was decomposed into individual nested mixed-model ANCOVAs at days 0, 7, 14, 21, and 28 using the following model: P
Yijkm ¼ l þ b Xijkmn  X þ Di þ Pj þ S ð PÞk ð jÞ þDPij þ DSð PÞik ð jÞ þemðijk Þ

ðModel2Þ

where μ is the true mean; β is the combined regression coefficient between X and Y within each group; X¯ is the mean value of the covariate; Di is the maternal effect (where i = 1–3); Pj is the population effect (where j = 1, 2); S(P)k( j ) is the paternal effect (k = 1–3) nested within each population; DPij is the maternal × population interaction; DS(P)ik( j ) is the maternal × paternal nested within population interaction; and εm(ijk) is the residual error. Egg diameter was used as the covariate in the models to partition out maternal non-genetic effects of egg size. The ANCOVA models were first run as saturated models, and then when appropriate were re-run with non-significant covariates removed. Percent survival of larvae was calculated from day 0 to day 8, day 16, and day 28. Survival data were analyzed using Model 2. Treatment means for fixed effects were contrasted using the least squares means method (LSMEANS / CL adjust= SCHEFFE, PROC MIXED; SAS Institute, 2003). A-posteriori analyses were not performed on random effects. Instead, variance components were constructed using the REML method in SAS PROC Mixed, and expressed as a percentage. To test for significant variability among variance components that were greater than zero, likelihood ratio statistics were generated based on the difference in −2 Res[tricted] log-likelihood between the full model and a model with a given variance component (VC) constrained to zero (PROC MIXED; SAS Institute, 2003). As the resulting chi-square statistic is one-tailed, the probabilities were halved to obtain significance levels for each VC (Littell et al., 1996; Messina and Fry, 2003; Fry, 2004). 3. Results 3.1. Size of eggs The overall mean egg diameter (±SD) was 0.65 mm (± 0.04) with egg sizes for the females ranging from Table 4 Restricted maximum likelihood (REML) variance components expressed as a percentage for larval survival to experimental days 8, 16, and 28 Variance component

Day 8

Day 16

Day 28

Maternal Paternal(population) Maternal × population Maternal × paternal(population) Residual

7.4 ⁎ 58.1 ⁎⁎ 0.0 0.5 34.0

0.0 75.7 ⁎⁎ 0.0 10.3 ⁎⁎ 14.0

0.5 79.3 ⁎⁎ 0.0 9.5 ⁎ 10.7

To test for significant variability among variance components that were greater than zero, likelihood ratio statistics were generated (Littell et al., 1996; Messina and Fry, 2003; Fry, 2004). ⁎ P b 0.05. ⁎⁎ P b 0.001.

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Table 5 Review of fish studies (larval and juvenile) that have shown significant paternal effects Species

Common name

Significant paternal effect

Authors

Salmo salar

Atlantic salmon

Thorpe and Morgan (1978)

S. trutta

Brown trout

Oncorhynchus mykiss O. tshawytscha O. masou Clupea harengus

Rainbow trout

• Growth rate after 171–174 days feeding • Mortality rate • Larval life span after hatching (without external food) • Larval growth rate (measured as the increase in length from hatching until death) • Larval growth and survival (1 year old progeny) • Growth performance of progeny • Survival of progeny • Otolith size in swim-up fry • Larval size at hatch

Chinook salmon Masu salmon Herring

Dicentrarchus labrax European sea bass • Proportion of females among progeny Melanogrammus Haddock • Standard length, yolk area, eye diameter, myotome height, aeglefinus and growth rate during larval development (0–5 days post hatch) • Jaw length, myotome height, yolk area, and standard length during larval development (0–10 days post hatch) Pseudopleuronectes Winter flounder • Larval jaw length and standard length at 8 and 12 days post americanus hatch, respectively Amphiprion Clownfish • Larval growth and survival to metamorphosis melanopus Poecilia reticulate Trinidadian guppy • Growth rate, fecundity, and offspring weight

0.55–0.77 mm. The mean (± SD) egg diameter for Females 1, 2 and 3 was 0.64 mm ± 0.04, 0.64 mm ± 0.04, and 0.66 mm ± 0.03, respectively. A significant difference between mean egg diameter was detected (F2,147 = 8.83, P b 0.001, single-factor ANOVA), and eggs produced by Female 3 were significantly larger than eggs produced by Female 1 or 2. 3.2. Larval growth and survival Containers were not stocked with Passamaquoddy Bay Male 3 for Female 1 due to an insufficient amount of hatched larvae. As a result, unbalanced ANCOVA models were used to analyze the data. The results of the ANCOVA models showed non-significant effects for the covariate egg size for all analyses performed (all Pvalues N 0.05). Therefore all models were re-run with the covariates removed. The nested repeated measures mixed-model ANOVA's revealed that there were significant first-order population × day interaction effects for ED, HD, JL, and BA (P ≤ 0.021; Table 1). Therefore the models were broken down into five separate nested mixed-model ANOVAs for days 0, 7, 14, 21, and 28 (for each morphological characteristic). There were no significant population effects for any of the morphological traits on day 0 (Table 2, Fig. 1). There were significant population effects for ED on day 7, and day 14; HD on day 21, and day 28; and JL on day 7, day 14, day 21 and day 28 (Table 2, Fig. 1). BA showed no significant stock effects on any of the sampling days

Vøllestad and Lillehammer (2000)

Herbinger et al. (1995) Rinchard et al. (2003) Unwin et al. (2003) Yamamoto and Reinhardt (2003) Panagiotaki and Geffen (1992) Evans and Geffen (1998) Gorshkov et al. (2003) Probst et al. (2006) Rideout et al. (2004) Butts and Litvak (2007) Green and McCormick (2005) Reynolds and Gross (1992)

(Table 2, Fig. 1). Maternal variance components were significant on at least one of the sampling days for ED, HD, and BA (Fig. 2). Paternal variance components were significant for ED at day 0 (VC = 4.85%, P = 0.050, nested mixed-model ANOVA), and day 28 (VC = 36.10%, P = 0.006, nested mixed-model ANOVA), and HD at day 28 (VC = 13.35%, P = 0.035, nested mixed-model ANOVA). After day 7, the paternal VC was larger than the maternal VC on at least one sampling day for all morphological traits (Fig. 2). There was a significant maternal × paternal (population) interaction for ED, HD, JL, and BA on at least one of the days (P ≤ 0.035, nested mixed-model ANOVAs; Fig. 2). The maternal × population VC was not significant for any of the sampling days (Fig. 2). SL and MH had non-significant first-order interactions of population × day for the repeated measures nested mixed-model ANOVAs (Table 1). The model was therefore re-run with the interaction effect removed for these two morphological traits. Significant effects of stock and day were detected for SL (Table 1, Fig. 3). Mean SL of Georges Bank larvae were larger than the Passamaquoddy Bay larvae (Fig. 3). The day × maternal VC was significant for SL and had the largest VC in the model (29.16%; Table 3). The day × paternal (population) and the day × maternal × paternal (population) variance components were also significant for SL (Table 3). No significant stock effect was detected for MH; however, the effect of day was significant (Table 1, Fig. 3). The

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maternal × population and day × maternal × paternal (population) variance components were significant for MH (Table 3). The mean (±SD) percent survival at days 8, 16, and 28 was 70.90% (±11.6), 41.9% (±19.32), and 38.35% (±20.89), respectively. There were no significant stock effects for survival at day 8 (F1,4.04 = 0.06, P = 0.8245, nested mixed-model ANOVA), day 16 (F1,4.02 = 0.09, P = 0.7759, nested mixed-model ANOVA), and day 28 ( F 1,4.01 = 0.07, P = 0.8031, nested mixed-model ANOVA). The paternal (population) variance components were significant for percent survival at all sampling days and had the largest VC in the models (P ≤ 0.03, nested mixed-model ANOVAs; Table 4). The maternal effect was only significant at day 8 (P = 0.05, nested mixed-model ANOVA), and had a VC of 7.40% (Table 4). With the exception of survival at day 8, the maternal × paternal (population) VC were all significant (all Pvalues ≤ 0.005, nested mixed-model ANOVAs; Table 4). 4. Discussion Winter flounder caught on Georges Bank have different pigmentation (Kendall, 1912), have more fin rays (Lux et al., 1970), are reported to be larger (Bigelow and Schroeder, 1953; McClelland et al., 2005), grow faster (Lux, 1973), and are genetically distinct compared to winter flounder from other locales (McClelland et al., 2005). Winter flounder generally grow to 30.5–38.5 cm in length (Bigelow and Schroeder, 1953), although fish as large as 66–70 m have been caught on Georges Bank (Lux, 1973). Butts and Litvak (2007) used sperm from Passamaquoddy Bay and Georges Bank winter flounder males to fertilize eggs from Passamaquoddy Bay females. They found that during early larval development, larvae sired by Georges Bank males grew significantly faster than larvae sired by males from Passamaquoddy Bay. In this study, larvae sired by Georges Bank males had a significantly larger mean SL, ED, HD, and JL than larvae sired by Passamaquoddy Bay males. Until now we did not know if these differences between the populations existed during the exogenous feeding stage. Results from both these studies provide further supporting evidence that Georges Bank winter flounder are genetically predisposed for faster growth under those experimental conditions tested. Besides growth rates, traits such as survival, disease resistance, and appearance are some of the most important traits to select for in an aquaculture setting (Newkirk, 1980; Knibb, 2000). Although survival was not significantly influenced by stock effects in the present study, survival has been shown to vary among laboratory-reared

populations of winter flounder (Buckley et al., 1991). Further stock comparisons are needed that examine survival during weaning to artificial diets (where considerable mortality has been shown to occur; Walford et al., 1991; Person Le Ruyet et al., 1993), and up to market size under different environmental regimes. Survival was significantly influenced by paternal VC. It has been demonstrated in a limited number of fish studies that paternal genetic effects contribute to variations in survival (Table 5). In most r-selected marine species, such as winter flounder, the greatest mortality occurs during ELH in both natural and aquaculture conditions. Buckley et al. (1991) reported mean survival of laboratory-reared winter flounder to be 3% after 28 days. Thus, attempts centered towards improving survival of winter flounder during ELH will result in a more cost-effective aquaculture venture. Results of the present study suggest that males may be selected to overcome such survival bottlenecks during ELH. Maternal, paternal, and the parental interactions affected growth rates from larval development to metamorphosis. Egg diameter also varied between individual females. In the present study egg size was found to not to be a significant covariate associated with the larval variables examined. This suggests that the maternal effects found in the present study were not related to egg size. However, there are other maternal non-genetic effects (i.e. yolk and lipid quality, hormones, immunoglobulin, mRNAs) that are not easily partitioned from maternal genetic contributions (Lam, 1994; Santacruz and Vris, 1996; Takemura and Takano, 1997; Kronnie et al., 2000). Significant differences in maternal non-genetic factors may mask paternal effects during early larval stages since the male contributes only 50% of the nuclear genomic DNA to the initial egg, while the female contributes 50% nuclear genomic DNA plus mitochondrial DNA, and a non-genetic environmental component (yolk and lipid reserve) (Rideout et al., 2004). Thus, the strong maternal effects found for several morphological traits at the beginning of this experiment (which coincides with the larval yolk-sac stage) were expected. Once maternal extra-nuclear contributions are used, it is possible that maternal and paternal nuclear-genetic effects may either equally contribute to larval variation (since the genomic framework of the offspring would be 50% maternal and 50% paternal), or vary according to the specific trait and time through ontogeny it was examined. Studies showing paternal effects contributing to larval growth and performance during ELH, however, are limited (Table 5) partly due to the fact that researchers pool milt when fertilizing eggs (Trippel, 2003; Rideout et al., 2004). Regardless of underlying patterns, the driving force behind

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each parental contribution is likely to be strongly modified by the sum of genetic effects, environmental effects, the genotype and environmental interaction, and the covariance between genetic and environmental effects (Conover, 1998). Therefore, caution must be exercised when interpreting data that measure genetic and environmental contributions to phenotypic variation. The present study found that maternal and paternal effects equally contribute to larval size at day 14 and day 21 (although they were not significant effects). Significant parental effects were evident at metamorphosis (day 28). The degree of parental influence during winter flounder development also appears to be trait and age specific. Chambers and Leggett (1992) examined maternal and paternal effects present at metamorphosis in winter flounder. Their results found that size at metamorphosis was under stronger parental influence than was age at metamorphosis. Results of the present study, and those reported by Butts and Litvak (2007) provide additional evidence that the degree of parental influence is determined by the parameter and what time through ontogeny it is measured. Acknowledgements We would like to thank E. Trippel, H. Stone, L. Van Eeckhaute, F. Purton, E. Carter, and the crew of the Wilfred Templeman for helping to collect broodstock. Special thanks to G. Guptill (Bayshore Lobster Ltd.) and S. Neil for housing the broodstock. Thanks to M. Horne, S. Leadbeater, and K. Howes for assisting in live food production and larval culture. I would also like to thank H. Moors, T. Benfey, B. MacDonald, and D. Methven for reviewing this manuscript. Funding for this study was provided by the Natural Sciences and Engineering Research Council of Canada (Matthew K. Litvak — NSERC Discovery, NSERC strategic and CRD grants) and AquaNet (AP20: Litvak, Lall, Hammell and Trippel). All winter flounder used in this study were handled according to CCAC guidelines. References Bejda, A.J., Phelan, B.A., Studholme, A.L., 1992. The effect of dissolved oxygen on the growth of young-of-the-year winter flounder, Pseudopleuronectes americanus. Environ. Biol. Fisches 34, 321–327. Ben Khemis, I., de la Noűe, J., Audet, C., 2000. Feeding larvae of winter flounder Pseudopleuronectes americanus (Walbaum) with live prey or microencapsulated diet: linear growth and protein, RNA and DNA content. Aquac. Res. 31, 377–386. Ben Khemis, I., Audet, C., Fournier, R., de la Noűe, J., 2003. Early weaning of winter flounder (Pseudopleuronectes americanus

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