Fine-scale temporal analysis of genotype-dependent mortality at settlement in the Pacific oyster Crassostrea gigas

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

Contents lists available at ScienceDirect

Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe

Fine-scale temporal analysis of genotype-dependent mortality at settlement in the Pacific oyster Crassostrea gigas

T

Louis V. Plough1 University of Southern California, AHF 130, 3616 Trousdale Pkway, Los Angeles, CA 90089, United States

A R T I C L E I N F O

A B S T R A C T

Keywords: Recruitment Larval Metamorphosis Life history Genetic load

Settlement and metamorphosis mark a critical transition in the life cycle of marine invertebrates, during which substantial mortality occurs in both field and laboratory settings. Previous pair-crossing experiments with the Pacific oyster Crassostrea gigas have revealed significant selective or genotype-dependent mortality around the metamorphic transition, but the fine-scale nature and timing of this mortality is not known, particularly whether it occurs before, during or after metamorphosis. In this laboratory study, microsatellite marker segregation ratios were followed daily throughout the settlement and metamorphosis of an F2 cross of the Pacific oyster to examine the fine-scale patterns of genotype dependent mortality at this transition and whether settlement timing (early vs. late) might be under genetic control and affect inference of genotype dependent mortality. Settlement occurred over nine days (day 18 to day 27 post-fertilization) with 68% of individuals settling either early (day 19) or late (day 24). Tracking the survival of spat for 40 days after initial settlement revealed almost no mortality and thus no appreciable genetic mortality. Temporal genetic analysis revealed that 3/11 loci exhibited genotype dependent mortality around the metamorphic transition, one of which (Cg205) was followed throughout settlement and metamorphosis. Alternative temporal patterns of strong selection against each homozygous genotype at Cg205 revealed possible defects in both the competency pathway (inability to initiate metamorphosis) and the morphogenesis pathway (mortality during the metamorphic transition). Quantitative trait locus (QTL) mapping of settlement timing identified three individual and one epistatic QTL with significant genetic effects on this trait (29% of the variance explained in total); however, two of these loci were linked to markers exhibiting selective mortality at metamorphosis, potentially confounding their apparent association with settlement timing. Overall, the results of this study highlight the complex nature of mortality and behavior during settlement and metamorphosis in oysters and suggest that endogenous sources of mortality at settlement may play an important role in the recruitment dynamics of oysters and possibly other broadcast spawning marine invertebrates.

1. Introduction The period of settlement and metamorphosis is critical in the life cycle of many marine invertebrates (Gaines and Roughgarden, 1985; Hunt and Scheibling, 1997; Roughgarden et al., 1988; Rodriguez et al., 1993). It is the final developmental hurdle to successful recruitment, wherein larvae go through the dramatic ecological and biological transformation from a free-swimming planktonic larval form to a sessile benthic juvenile. In marine mollusks, metamorphosis is well described, characterized by the complete rearrangement of the body plan, coinciding with the loss of larval features, such as the velum, and the emergence of juvenile or adult characteristics, such as the gills (which first begin to form in the larval stages; e.g. Cole, 1938; Hickman and Gruffydd, 1971; Bonar, 1976; Cannuel and Beninger, 2006; Cannuel et al., 2009). The process of metamorphosis comprises two distinct

1

phases: 1) the attainment of “competency”, the developmental capacity to respond to appropriate settlement cues and to exhibit settlement behavior and 2) the morphogenetic transformation that occurs when larvae attach to the substratum and complete metamorphosis (e.g. Pawlik, 1992; Degnan and Morse, 1995). A suite of genes control an anticipatory, ‘competency’ pathway, which begins to form juvenile structures prior to metamorphosis (e.g. digestive and shell formation pathways; e.g. Jackson et al., 2007), and then the morphogenetic transformation is dictated by the up-regulation of genes related to apoptosis, cell cycling, and calcium flux pathways, among others (the ‘morphogenetic’ pathway; Jackson et al., 2005; Jackson and Degnan, 2006; Jackson et al., 2007; Williams et al., 2009). Though there appear to be clear patterns of gene classes expressed across invertebrate taxa during competency and metamorphosis (e.g. Heylund and Moroz, 2006), most studies have focused on only a few marine gastropods (e.g.

E-mail address: [email protected]. Permanent address: University of Maryland Center for Environmental Science, Horn Point Laboratory, 2020 Horns Pt. Rd., Cambridge MD 21613, United States.

https://doi.org/10.1016/j.jembe.2018.01.006 Received 8 May 2017; Received in revised form 28 December 2017; Accepted 19 January 2018 Available online 08 February 2018 0022-0981/ © 2018 Elsevier B.V. All rights reserved.

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

shown that many of the deleterious alleles observed in inbred crosses have significant dominance (fitness effects on heterozygotes; Plough and Hedgecock, 2011; Plough, 2012) and thus would likely have fitness effects in wild or outbred crosses (Plough et al., 2016; Plough, 2016). Three hypotheses are proposed to explain how selection and mortality might proceed during settlement based on the differential timing of GDM observed in spat and larvae: 1) larvae are able to advance through metamorphosis and settle, but die soon after, which is reflected as high mortality of, and genotypic shifts in, juveniles after settlement, 2) larvae begin metamorphosis but die sometime during the transition, which is reflected by genotype deficiencies of both larvae and spat during metamorphosis, and 3) larvae with particular genotypes are delayed or fail to initiate metamorphosis, which is reflected by the deficiency of certain genotypes in spat that remain and perhaps accumulate in the larval population. Finally, the effect of genotype on settlement timing (larval duration) was analyzed in a quantitative trait locus (QTL) mapping framework to determine if settlement behavior could potentially affect the inference of loci causing GDM at metamorphosis. Overall, the novel sampling and genotyping strategy carried out in this controlled experiment sheds light on how genotype can influence settlement behavior and mortality at metamorphosis in a family of oysters, setting the stage for future genetic studies of genotype dependent mortality and settlement variation in a broader, population context.

Aplysia and Haliotus spp.) and ascidians (e.g. Degnan et al., 1997; Eri et al., 1999; Jackson et al., 2002; Kawashima et al., 2005; Jacobs et al., 2006), all of which have lecithotrophic larvae. Less is known about genetic processes at metamorphosis in broadcast spawning marine invertebrates, such as marine bivalves with planktotrophic larvae. Metamorphosis in marine invertebrates, and marine bivalves in particular, is accompanied by substantial mortality. High rates of mortality in newly settled juveniles have been observed for a wide variety of marine invertebrates, and, generally, survival curves of new settlers are type III: mortality is initially high but decreases rapidly after the first few days or weeks and then levels off (e.g. Rodriguez et al., 1993; Gosselin and Qian, 1996' reviewed by Gosselin and Qian, 1997; Hunt and Scheibling, 1997). Early mortality in invertebrates has primarily been estimated in field studies that examine rates and patterns of mortality after settlement; however, many of these studies are only able to measure “recruitment”, i.e. the survival of a population to a certain time point after settlement, because monitoring mortality during settlement is difficult if not impossible for many species (e.g. Keough and Downes, 1982). Few studies accurately measure larval supply, metamorphosis, and early and late post-settlement periods within the same experiment, thus, a comprehensive understanding of when mortality occurs during settlement and metamorphosis is lacking. Experimental laboratory studies allow for more detailed analysis of mortality during settlement in marine invertebrates, but few such studies exist. Jones and Jones (1983) found for the flat oyster (Ostrea edulis) that only a small fraction (10–30%) of late-stage larvae successfully completed metamorphosis under laboratory conditions. Haws et al. (1993) also noted that substantial mortality occurred during the metamorphic transition in their detailed examination of biochemical and physiological changes during metamorphosis of the Pacific oyster Crassostrea gigas. Culture experiments have shown significantly greater post-metamorphic survival associated with improved diet during the larval stages, suggesting that endogenous processes related to energy acquisition prior to metamorphosis are important (e.g. Gallager et al., 1986; Gallager and Mann, 1986; Helm et al., 1991; Coutteau et al., 1994; Pernet and Tremblay, 2004). Recent experimental genetic studies of offspring from inbred crosses of the Pacific oyster have confirmed high mortality at metamorphosis and revealed a large load of deleterious recessive mutations that were ‘expressed’ (acted) during metamorphosis, which are detected by their negative fitness effects on neutral marker genotypes (microsatellites) carrying alleles that are physically linked to these deleterious loci (i.e. genotype-dependent mortality or GDM; Launey and Hedgecock, 2001; Plough and Hedgecock, 2011). Plough and Hedgecock (2011) determined the stagespecific timing of GDM in two inbred crosses by following temporal changes in genotype frequencies relative to their Mendelian expectation (i.e. tests for distortion of Mendelian segregation ratios; Hedrick and Muona, 1990; Launey and Hedgecock, 2001; Plough and Hedgecock, 2011; Plough, 2012) in daily larval samples (day 1–18), a post settlement sample (day 30), and an adult sample (> 1 yr). The finding of substantial GDM during settlement highlights the potential important physiological and developmental changes associated with this transition, however, there were no temporal genetic data collected during settlement and metamorphosis in Plough and Hedgecock (2011), and thus the fine-scale temporal patterns of selection remain unknown. In the current study, an inbred, F2 (second generation bi-parental) family was allowed to set on adult shell in the laboratory, and daily samples of spat (recently settled juvenile oysters) and larvae were taken throughout the settlement period to determine the timing of endogenous (i.e. non-environmental) genotype-dependent mortality (GDM) during metamorphosis. While the F2, laboratory-based design limits somewhat the application of results to understand the causes of oyster larval mortality in nature, a laboratory approach is required to eliminate or minimize environmental sources of mortality (e.g. predation, food limitation, etc.), which are difficult to distinguish from the endogenous sources (the focus of this study). Recent work has also

2. Materials and methods 2.1. Crosses and culturing Inbred lines 51 and 35 were derived from a naturalized population of C. gigas in Dabob Bay, WA, with initial families made from pair crosses of wild individuals in 1996 (Hedgecock and Davis, 2007). These lines were inbred (full-sib mating) for four generations leading up to the F1 hybrid cross that was made in 2007. In 2009, the experimental F2 family was created by mating a pair of male and female full-siblings from the 2007 51 × 35 F1 hybrid cross. Crosses were performed at the University of Southern California (USC) Wrigley Marine Science Center (WMSC) on Catalina Island, CA. Pedigrees of parents were verified with microsatellite DNA markers (e.g. Hedgecock and Davis, 2007). An inbred cross was used here because deleterious recessive alleles affecting offspring survival (viability selection) can be identified from their characteristic fitness effects on linked alleles in homozygous marker genotypes (homozygote deficiencies; e.g. Launey and Hedgecock, 2001; Plough and Hedgecock, 2011; Plough, 2012). The cross was performed by stripping ripe gametes from a single male and a single female and combining ~ 1,000,000 eggs and an appropriate amount of sperm in a two-liter beaker of fresh seawater for fertilization (Breese and Malouf, 1975; Hedgecock and Davis, 2007). Observations of fertilizations in 100 μl sub-samples indicated that fertilization success was ~80%, so the record of initial expected stocking density was adjusted to 800,000 embryos. After a one-hour incubation, 800,000 zygotes were stocked in a 200-l vessel (4 embryos·ml−1) with fresh seawater. Starting at 24 h post-fertilization, larvae were fed a diet of exponential phase Isochrysis galbana (Tahitian Isolate) every two days, at a starting concentration of 30,000 cells·ml−1, which was increased up to 120,000 cells·ml−1 as larvae grew, following typical larval rearing protocols for the Pacific oyster (Breese and Malouf, 1975; Launey and Hedgecock, 2001; Plough and Hedgecock, 2011). Mean density of larvae ( ± SEM) was calculated every 2–4 days after fertilization, from replicated (n = 4) volumetric counts of 20–100 μl aliquots taken from a concentrated culture (~1 l volume) containing all larvae. These estimates were converted to mean survival from egg (embryo) or the first shelled, prodissoconch I stage, also described as the ‘d-hinge’ stage.

91

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

of 1× PCR buffer (Promega, Madison,Wisconsin), 0.2 mM EDTA, 1.0 μg/μl proteinase K (Shelton Scientific, Connecticut) and purified H2O. Plates were frozen at −80 °C until extraction (Plough and Hedgecock, 2011; Plough et al., 2014). Larvae were extracted in 40 μl volumes in a 96-well thermalcycler (BioRad Tetrad, Hercules, CA) with a three-hour incubation at 56 °C followed by a 15 min boil at 95 °C. Parent tissue was stored in 70% ethanol at 4 °C until extraction from 10 to 25 mg of tissue using the Qiagen DNeasy blood and tissue kit, following the manufacturer's protocols. Eighty-four microsatellite markers cloned from the Pacific oyster were screened for polymorphism (i.e. at least two alleles segregating in the offspring) for use in this study (Magoulas et al., 1998; McGoldrick et al., 2000; Huvet et al., 2000; Li et al., 2003; Sekino et al., 2003; Yamtich et al., 2005; Yu and Li, 2007; Wang et al., 2008). Genotyping of microsatellites was carried out as described previously (Plough and Hedgecock, 2011; Plough, 2012) and 46 markers were found to be polymorphic or informative for the cross, all of which are on published linkage maps (Hubert and Hedgecock, 2004; Hubert et al., 2009; Plough and Hedgecock, 2011). From these 46 markers, a subset of 24 was chosen for genotyping, with the aim of having at least two markers on each of the 10 linkage groups, thereby maximizing genomic coverage with the fewest markers.

2.2. Deployment of shell and sampling protocol At day 18, when a substantial proportion of the larvae were displaying characteristic settlement behavior (e.g. Bonar, 1976; Kennedy et al., 1996), cured adult shell that had been soaked in FSW for 12–24 h was deployed on a cylindrical harness with a bottom of Vexar® mesh, as a substrate for natural settlement. Adult shell was placed with the nacreous side facing both up and down and with shells overlapping to form random 3-D structures, in order to give larvae a number of different substrates and angles to settle on regardless of their orientation to light or gravity, which can affect settlement behavior (Kennedy et al., 1996; Baker, 1997; Baker and Mann, 1998). Adult shell was used because it provides a substrate that would be found in the natural environment and oysters are known to settle gregariously and preferentially on conspecific shells (e.g Vasquez et al., 2013 and see references therein). Shells were also soaked prior to deployment in order to stimulate biofilm production that would provide natural settlement cues. It is noted however, that the experimental setup likely does not completely represent the dynamic physical and chemical environment of a natural oyster reef and thus observed settlement patterns may differ between lab experiments and the field environment. Starting in the afternoon on day 18 post-fertilization, and at about the same time each day thereafter, the harness was carefully pulled up, and the surface of shells were sprayed with seawater from a small squirt bottle to return any probing or non-cemented larvae back to the 200-l vessel and the larval pool. Larvae were filtered out of the 200-l vessel and concentrated for survival estimates each day during settlement. Survival was calculated by counting the number of larvae in four to six random sub-samples of known volume (50–100 μl) from homogeneously mixed, concentrated cultures (500–1000 ml). These counts were then multiplied by the inverse of the fraction of the subsample volume to the total volume (count ×

2.4. Temporal genetic analysis of mortality during settlement To identify markers exhibiting GDM at settlement, goodness of fit chi-square tests for segregation distortion (relative to Mendelian expectation) were carried out for the 24 selected markers as in Plough and Hedgecock (2011) and Plough (2012). The observation of significant segregation distortion (relative to Mendelian expectation) reflects the differential viability of genotypes (genotype dependent mortality, GDM), and thus, is a measure of viability selection (selection against particular genotypes) in the controlled hatchery or culture environment (e.g. Launey and Hedgecock, 2001; Plough and Hedgecock, 2011). Tests for segregation distortion were first performed on the end-point sample of 60-day-old juveniles (spat) that had set on day 19 or 24 (combined n = 192), representing the bulk of successful settlement (68% of all successful spat settled on day 19 or day 24; see Fig. 1). Day 60 was chosen as the end point to allow recent settlers some grow-out time to track potential endogenous, post-settlement mortality, and to provide sufficient amounts of tissue for robust DNA extractions of the spat. Observation of segregation distortion at the day 60 time point identifies markers that exhibited viability selection at some point during larval development and metamorphosis. Markers showing significant segregation distortion at day 60 were then genotyped in day 18 larval samples to identify markers that were already distorted before metamorphosis and thus were not candidates of genotype-dependent mortality during metamorphosis (Plough and Hedgecock, 2011). Markers demonstrating Mendelian segregation (no selection) at day 18 (but not at day 60, post-metamorphosis) were then genotyped in larvae and spat each day throughout settlement and metamorphosis to track the temporal onset of genotype-dependent mortality in this cross.

total ml ⎞ ⎟, subsample ml

and averaged across ⎠ replicates to estimate the mean number of larvae remaining (surviving) each day. For each daily estimate of settlement, spat were counted by eye from each shell under a dissecting microscope at 8–48× magnification until no more spat could be found. Fresh shells that had been incubating for 24 h in filtered seawater were then placed on the harness, and lowered back into the 200-l tank, which had been rinsed and refilled with fresh seawater and algae. Finally, the counted larvae were placed back into the tank. This process was repeated each day until no larvae remained in the water column. From the counts of larvae, L, at time i (ti) and time i + 1 (ti+1), and the count of settlers, S, at ti+1, larval mortality (M; the number larvae that died) between two adjacent days was calculated as: M =Li − (Li+1 + Si+1). Shells that had spat settling on a particular day were then collected and placed in Vexar® mesh bags in the upwelling nursery system and grown out until day 60 to increase the amount of tissue for DNA extraction. Spat were fed live Isochrysis galbana and Shellfish Diet 1800 (Reed Mariculture, Campbell, CA) every six to 12 h during this grow out period. To determine if mortality occurred post-settlement (after the day on which individuals were recorded as settling), a subset of spat from day 19, 21, and 24, were followed to day 60 to determine survival through the early juvenile stage. This was done by marking an area around 50 (live) spat on two shells from each of the three settlement days, and then counting the number of live spat in these marked shell areas at day 60.

2.5. QTL analysis of settlement timing Spat settling on day 19 and day 24 (Fig. 1) were used to examine possible genetic differences between early and late settling individuals (i.e. differences in larval duration). For an initial, single-marker analysis, 96 spat from both the early and the late settlement samples (combined n = 192) were genotyped at 24 loci and tested for heterogeneity between the two settlement samples with contingency chisquare tests using the program ‘Chirxc’ (Zaykin and Pudovkin, 1993; See table S1). For a more integrated, genome-wide approach to examine genetic variation in settlement timing, a quantitative trait locus (QTL) mapping approach was taken. First, linkage maps were constructed with day 19 and day 24 spat samples (n = 192), under the CP (cross

2.3. DNA extraction and genotyping At 60 days, individual spat were scraped off shells and placed directly in Qiagen tissue lysis buffer (ATL buffer; Qiagen, Valencia CA) in individual tubes and stored at −80 °C until extraction using the Qiagen DNeasy blood and tissue kit. Live larvae were pipetted individually into 96-well PCR plates containing a homemade extraction buffer consisting 92

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

Fig. 1. Settlement and larval mortality during metamorphosis. Percent settlement (relative to the total or cumulative count of settled spat) and percent remaining larvae (relative to day 18, which is set at 100%) for each day are plotted over the period of settlement and metamorphosis. Larval mortality is calculated as the percent difference in the abundance of larvae between days, accounting for the number of new settlers (spat). Error bars on the larval abundance estimates represent one standard error of the mean from volumetric counts, converted to percent.

these were not sampled. On the initial day of shell deployment, the number of surviving larvae was 132,000 ± 7500 (from a starting stock of ~800,000 embryos), which represents 16.5% survival from embryo to the end of the larval stage, or ~30% survival from d-hinge to the end of larval stage. Settlement occurred over nine days and appeared to follow a bimodal distribution; settlement peaks were observed at day 19 (1084 counted spat) and day 24 (1484 spat) and these days account for ~68% of all observed settlement. In total, 3776 settlers were counted, which represents 0.45% survival to the spat stage over the 60 days of the experiment (or just under 1% from d-hinge to day 60). Larval abundance appeared to drop sharply on the days when a substantial number of settlers were recorded (e.g. day 19 and day 24) and a corresponding spike in larval mortality was observed (Fig. 1). After day 24, the abundance of larvae steadily declined despite few additional spat setting. Mortality of new settlers followed for approximately 40 days after initial settlement (examined at day 60) was very low, with estimates of 2%, 0% and 2% for day 19, 21, and 24 settlers, respectively. Because negligible post-settlement mortality was observed in these samples, no further analysis of segregation ratios was performed.

pollinator) population type in JoinMap 3.0 (Van Ooijen and Voorrips, 2001) and using the Kosambi mapping function, with a minimum likelihood of the odds (LOD) score of 2.0 (most linkage groups had LOD scores well above 4.0). Phase was determined by JoinMap 3.0 from the frequencies of parental and recombinant types. Deviations from Mendelian segregation ratios may affect linkage mapping (e.g. Zhu and Zhang, 2007), so marker order and distances were compared with previously published linkage maps, some of which were constructed using larval samples that showed little segregation distortion (e.g. Hubert and Hedgecock, 2004). If markers that should have mapped failed to map, or were in very different locations compared with previously published maps, locations were taken from published maps (Hubert and Hedgecock, 2004; Hubert et al., 2009; Plough and Hedgecock, 2011). Genomic coverage was estimated with the equation GC = 1 − e−2∗dn/L, where d = mean inter-marker distance and n = total number of markers assigned to linkage groups, and L is the estimated map length (Bishop et al., 1983). QTL analysis of time to settlement was performed in R/qtl (v.1.1; Broman et al., 2003), under a binary trait model, (early vs. late settlement, day 19 vs. day 24), using the outcross settings. A one-dimensional scan for single QTL was first performed (‘scan-one’ module) and 5% significance thresholds at the genome-wide and chromosome-wide level were calculated with 1000 permutations. Next, genetic interaction was tested at every position in the genome, using a two-QTL model (‘scan-two’ module; significance adjusted with 1000 permutations). Once significant QTL were identified, they were fit into an overall multiple regression ANOVA model, using a backwards selection procedure to find the best model with lowest model comparison criterion.

3.2. Temporal genetic analysis of mortality during settlement Significantly distorted segregation ratios at the α = 0.05 level were found at 13 of 21 (62%) markers (none of the three informative markers on LG 2 could be scored in the spat, so these markers and this linkage group were not included in the analyses; Table S1). These distortions were mainly caused by deficiencies of homozygous genotypes or heterozygote carriers of identical-by-descent deleterious alleles (e.g. Plough and Hedgecock, 2011). Markers with distorted segregation ratios were found on eight out of the nine linkage groups analyzed. Genotyping all but two (Cg162 and Cg156) post-settlement distorted markers in the day-18 larval sample identified three markers exhibiting metamorphosis-related selection; Cg109, Cg205 and Cg175 from LG 4, LG 6, and LG 8, respectively (three of 11 tested; Table S1). Difficulties in genotyping larval samples (inconsistent amplification from low DNA yields of larvae) only allowed complete temporal analysis of marker Cg205 after day 19, and not for every daily time point (Fig. 2). Significant deficiencies of both homozygous genotypes (aa and bb from cross type ab × ab) were evident for Cg205 in the pooled, endpoint spat sample (post-metamorphosis “cumulative” sample; Fig. 2).

3. Results 3.1. Patterns of observed settlement and mortality The cross showed typical survival (55%) to the first shelled, prodissoconch-I (d-hinge) stage (day 2 post-fertilization) and larvae developed typically though the late pediveliger stage, exhibiting the characteristic signs of early settlement behavior (e.g. eyespots and probing foot) on day 17 post-fertilization. Adult shells were deployed starting on day 18 but around 30–50 larvae had already settled, sticking to the bottom of the poly‑carbonate tank in the absence of adult shell; 93

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

Fig. 2. Cg205 genotype proportions in larvae and spat during settlement. Asterisks denote the level of significance for the goodness-of-fit chi-square test (cf. Mendelian expectations of 2:1:1, AB:AA:BB) of genotype proportions at Cg205 (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001); ns is non-significant. Days post fertilization are marked along the x-axis, with ‘Expected’ being the expected Mendelian genotype proportions for Cg205 with no selection, and ‘Cumulative’ denoting the pooled sample of all spat collected during settlement (i.e. d19, d24, and d26 together).

dominant genotype remaining in the water column late in the larval period but never setting in appreciable numbers even in the last days of settlement. The differing temporal patterns of mortality and segregation distortion between the two homozygous genotypes indicate that Cg205 is linked to two different deleterious recessive mutations in repulsion phase, each with very different patterns of selection (Plough and Hedgecock, 2011).

Starting with the inspection of genotypic data for day-19 spat, observed segregation ratios were highly distorted with substantial deficiencies of the two homozygous genotypes compared to their Mendelian expectation (Fig. 2). Segregation ratios in day-19 larvae did not deviate from their Mendelian expectation however, indicating that genotypes deficient in the spat sample were still present among larvae in the water column. At the next larval sample, day 21, segregation ratios were significantly distorted but only the AA genotype was deficient, while the BB genotype did not deviate from its expected 1:2 ratio, relative to the frequency of AB. At day 24, segregation ratios in the larval sample were dramatically different from the previous time point (day 21). There was a much larger proportion of BB genotypes than expected (60% compared with the expected 25%) and significantly fewer AB genotypes, while the number (and relative frequency) of AA genotypes remained highly deficient (only five spat with AA genotypes were observed in both day 21 and day 24 larval samples; Fig. 2). In the day-24 spat sample, both homozygous genotypes were again observed to be deficient and segregation ratios were dramatically different from the day 19 sample, owing to a greater frequency of BB individuals settling (P < 0.001, r × c contingency chi-square test; Zaykin and Pudovkin, 1993). At the final larval time point (day 26) the BB genotype was in even greater excess (79% compared to the expected 25%) while the AA genotype was completely absent and the AB genotype was deficient at this time point as well (Fig. 2). The day-26 spat sample exhibited a slight increase in BB genotypes but both the BB and AA genotypes were significantly deficient relative to Mendelian expectation, a result consistent with segregation data for the “cumulative” spat sample (days 19, 24, and 26 combined). In summary, individuals with the AA genotype suffered relatively swift mortality early in settlement (day 19 to day 21) with few larvae surviving long in the water column or setting successfully after this point. In contrast, while BB individuals were also deficient in the spat samples relatively early in settlement (day 19) they were present in the larval pool throughout settlement, accumulating to become the

3.3. QTL analysis of settlement timing Twenty-one markers were assigned to 8 of 10 linkage groups, with assignments as expected from previous crosses and studies (Hubert and Hedgecock, 2004; Hubert et al., 2009; Plough and Hedgecock, 2011). Linkage group two (LG 2) lacked informative markers, and map distances for the two markers on LG 7 were obtained from previously published maps because marker positions were estimated to be > 40 cM. Marker phase was successfully estimated for each linkage group. Total map length was 369 cM, calculated by summing all inter-marker distances, with a mean inter-marker distance of 17.65 cM (maximum 42 cM; LG 7). Despite low marker density, estimated genomic coverage for this map was high at 86% (Bishop et al., 1983). Single-marker analysis revealed that four of the 21 markers, Cg205, Cg162, Cg140 and Cg109, had significant heterogeneity in genotype proportions between days 19 and 24, indicating that these markers might be associated with differential settlement timing or larval duration (Table S2). Associations between markers and settlement timing were further assessed in a genome-wide context with quantitative trait locus (QTL) mapping analyses, in which the phenotype, time to settlement, was coded as a binary trait (early or late). The one-dimensional scan identified three significant QTL on linkage groups 4, 6, and 10 (QTL 1–3; Table 1). The most significant QTL was well above the genome-wide threshold LOD score of 2.99 (QTL 3, LOD = 3.794; Table 1) and was located on linkage group 10, closely linked to marker Cg140. The significance of the other two QTL, one on LG 4 near Cg109 (QTL 1, 94

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

settlement in the marine environment could be related to a number of factors, including variation in reproductive condition and timing of spawning or oceanographic features, such as currents or food availability, none of which are relevant in this laboratory experiment. Instead, variation in settlement timing here, particularly the early and late peaks of settlement, may have a genetic basis (see below). Alternatively, the clustering of settlement around these two days could simply reflect a pattern of gregarious or aggregative settlement, wherein settlement is stimulated by conspecific larvae (e.g. Cole and Knight-Jones, 1939; Hidu et al., 1978; Burke, 1986). Only one family was examined in the current study, thus, it is not known if the observed settlement patterns (the bimodal distribution in particular) are unique to this family. Survival over the duration of the experiment (embryos to day 60 spat) was low, (< 1%) which is consistent with previous reports of substantial early mortality in the Pacific oyster (e.g. Bucklin, 2003; Ernande et al., 2003; Helm et al., 2004; Plough and Hedgecock, 2011; Plough, 2012; Plough et al., 2016). Accurate estimation of survival during the larval period is difficult in general due to the high variability in volumetric counts, but it was particularly challenging in post-metamorphosis counts of settled spat on dark, rugose, irregularly-shaped adult shells. Thus, it is possible that some spat were missed in our visual counts and that metamorphic mortality was overestimated. However, other laboratory studies of metamorphosis in Pacific oysters report similarly low survival during this period (5–30%; e.g. Jones and Jones, 1983; Haws et al., 1993; Ernande et al., 2003). Oyster larval mortality can vary substantially across experiments, and a single study reported a much lower rate of larval mortality for the Pacific oyster (Rico-Villa et al., 2008); however, it did not include the entire life cycle (egg to juvenile/adult) and used a different methodology to estimate larval mortality (enumerating dead shells). In a well-replicated study of the Pacific oyster, Ernande et al. (2003) reported 51% survival of fertilized eggs at the D-hinge stage, 6.1% by day 24, and 0.9% survival at the spat stage. Mortality is generally quite high in the early stages of oyster culture, and our study is in accord with what has been observed previously. Analysis of spat survival 40 days after initial settlement showed conclusively that very little endogenous (genotype-dependent) mortality occurred post-settlement. Essentially, once larvae had completed metamorphosis and appeared as spat on adult shell (time to census was 24 h or less), individuals survived at a rate of ~99%. These findings support previous observations of low genotype-dependent morality after metamorphosis (Bucklin, 2003; Plough and Hedgecock, 2011). Studies of recruitment in natural populations do, of course, show substantial post-settlement mortality in a variety of marine invertebrates due to predation and other sources of mortality in the environment (e.g. Hunt and Scheibling, 1997; Gosselin and Qian, 1997; Pineda et al., 2006) and for oysters in particular (e.g. Kennedy et al., 1996; Newell et al., 2000). No external sources of mortality, such as predation or environmental stressors were present in this experiment, thus, these results suggest only that mortality from endogenous sources (the expression of genetic load and genotype dependent mortality) is minimal once metamorphosis is complete, at least under the laboratory conditions used in this study and for the family examined.

Table 1 QTL and ANOVA model results for settlement timing. QTL

LGa

Position (cM)b

Markerd

LODe

% Var.f

P-value (χ2)

1 2 3

4 6 10 5 8 5:8c

0.0 1.0 1.0 10.0g 40g 10:40c

Cg109 Cg205 Cg140 – – –

2.567 2.714 3.794 – – 5.642

3.84 7.085 8.7 6.66 5.18 6.97 29.9

0.030 0.001 < 0.001 0.210 0.079 0.052 < 0.00001

4 Full model a

Linkage group number. Position along linkage group, in centimorgans (cM). The colon represents interaction between the two positions for QTL 4. d Markers closely linked to QTL, if any (within 15 cM). e LOD represents the log of the odds (LOD) score for each identified QTL. f Percent variance explained by the individual QTL or the full model. g These genomic positions were not significant QTL in the 1-dimensional scan and, thus, were not assigned numbers. Their effects are reported, however, because a significant interaction between them was detected in the two-dimensional scan and R/qtl includes them in the hierarchical ANOVA model by convention. b c

LOD = 2.567) and one on LG 6 near Cg205 (QTL 2, LOD = 2.714), were slightly below the genome-wide threshold but well above the chromosome-wide thresholds of 2.36 and 1.60 for LG 4 and LG 6 respectively, and are therefore considered suggestive QTL (Table 1). The two-dimensional scan for interacting QTL identified one significant interaction between genomic regions on LG 5 and LG 8 (QTL 4; LG 5 at 10 cM, and LG 8 at 40 cM, LOD = 5.642), which was above the α = 0.05 genome-wide interaction threshold LOD score of 5.46. Fitting a model for the four identified QTL (from the single QTL scan and the 2-locus scan), the ‘drop-one’ analysis of variance (ANOVA) method was used to examine the fit of the model with each QTL included and then dropped in sequential order. In the drop-one analysis, all QTL remained significant except for the interaction QTL, which was marginally significant (P = 0.057). Results of the ANOVA with four QTL indicated that the model was highly significant (P < 0.00001), with the four QTL explaining almost 29% of the variance in settlement time. The percent variation explained by the four QTL can be considered a rough estimate of broad-sense heritability for this trait. Overall, the strongest effect was at QTL3 (Cg140), which alone explained 8.7% of the variance in settlement timing. Examining the gene effects of the single QTL at the nearest markers, Cg140 (QTL 3) demonstrated a qualitatively additive pattern; the binomial probability of settlement on day 24 (late settlement) was 0.83, 0.53, and 0.33 for individuals with genotypes AA, AB, and BB, respectively (Fig. 3). Patterns of gene action at Cg205 and Cg109 were less straightforward but appeared to be qualitatively nonadditive (Supplemental Fig. S1). 4. Discussion 4.1. Patterns of observed mortality and settlement Tracking near daily changes in the larval and spat populations over the course of the settlement produced striking fine-scale patterns of settlement and mortality in the oyster family examined. High rates of mortality in the larval population appeared to coincide with the peaks of settlement (Fig. 1), suggesting that larval mortality was associated with some aspect of the metamorphic transition: larvae were “trying and dying” (see next section). Another striking result was the bimodal distribution in settlement timing: pulses of settlement on days 19 and 24 accounted for 68% of all settlement in this family over the nine-day period. Field-based recruitment studies have shown that settlement over the course of a season or reproductive period is often non-random, with specific peaks of settlement and highly variable survival over time (e.g. Raimondi, 1990; Pineda, 1994; Balch and Scheibling, 2000; Broitman et al., 2008; Pineda et al., 2006). Temporal variation in larval

4.2. Temporal genetic analysis of mortality during settlement At the outset of this study, three hypotheses were proposed to explain the previously observed patterns of substantial genotype-dependent mortality at metamorphosis in inbred crosses of the Pacific oyster (Plough and Hedgecock, 2011): 1) mortality occurred post-settlement owing to genetic or developmental abnormalities affecting fitness after metamorphosis, 2) mortality occurred during the process of metamorphosis owing to the expression of deleterious mutations in genes critical for morphogenesis and, 3) mortality occurred prior to metamorphosis due to the expression of mutations affecting the development of 95

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

Fig. 3. Plot of genotype effects on settlement timing for Cg140. Values (0 or 1, corresponding to early and late settlement, respectively) are jittered and represented by shaded circles. Large red markers (filled circles with dash) represent mean probability p, the binomial probability of late settlement for individuals of each genotype, AA, AB, and BB, which shows an additive pattern of gene effects at this marker. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Similar studies of the fine-scale timing of selective mortality at metamorphosis in other marine invertebrates are lacking, so it is not known how important this type of mortality might be for the recruitment dynamics of marine animals generally. A recent review of the literature on genetic load and genotype dependent mortality in marine animals suggested that oysters and other marine bivalves (e.g. scallops and mussels) may have higher genetic loads relative to other marine invertebrates and marine fish, which manifests as substantial early life history mortality and segregation distortion (selective mortality) in experimental crosses of these species (reviewed by Plough, 2016). However, a lack of data for most marine invertebrate groups makes it difficult to draw any major conclusions at this point – much of the available experimental data on segregation distortion across marine animals synthesized in Plough (2016) come from aquaculture species, thus, many major marine invertebrate groups were missed. Experiments similar to the current study will need to be performed across a number of less-represented marine invertebrate phyla to begin understand how universal this phenomenon might be. Nevertheless, the results here support the idea that selective mortality at settlement and metamorphosis can represent a major bottleneck in the recruitment of Pacific oysters, and more insight into the non-environmental, endogenous sources of selective mortality at metamorphosis may prove important for understanding the recruitment dynamics of other marine invertebrates as well. Still, caution must be exercised in extrapolating results of this labbased experiment to settlement processes in the natural environment, as only a single cross was examined, and the myriad physical forces and chemical cues present on a natural oyster reef were not likely replicated. Efforts were taken during the design of the study to minimize these effects (e.g. natural shell substrate, 3-D structure, inoculated for biofilms), but the small scale of the experiment must be acknowledged and additional work needs to be done, perhaps in a more natural setting and with additional families or crosses, to validate the findings.

competency or the delay of metamorphosis initiation. The observation of very minimal mortality after settlement suggests that the expression of genetic load does not greatly affect viability post settlement and thus, hypothesis one is falsified, at least based on data from the current experiment. However, given the alternative temporal patterns of selection against the two homozygous genotypes (AA and BB) at marker Cg205 during settlement (which implies selection against two marker alleles linked to two different deleterious loci in repulsion phase; Plough and Hedgecock, 2011) there is support for the hypotheses that selection is acting during and possibly just preceding metamorphosis (hypotheses two and three). The early and rapid mortality (deficiencies) of AA individuals during metamorphosis in both the larval and spat samples implies the expression of a linked, deleterious recessive mutation possibly affecting the morphogenetic transition (hypothesis 2; Fig. 2). In contrast, selection against individuals possessing the BB genotype appeared to be delayed in the larval samples, which suggests that the B allele may be linked to a different mutation causing either a delay in metamorphosis or the inability to develop competence (hypothesis 3). As microsatellite markers are typically neutral, the affected or implicated alleles at Cg205 are most likely physically linked to protein coding genes or regulatory variants that are the actual targets of selection, thus it is unknown what genes or physiological functions are associated with the observed selective mortality. Because it is not possible to directly link the observed temporal patterns of selection to specific defects in the morphogenetic or competency pathways, the evaluation of the developmental processes affected is necessarily somewhat qualitative at this point. However, the two different temporal patterns of selection at Cg205 align well with what might be expected for individuals that fail to initiate metamorphosis or that die during the metamorphic transition. Because only a single marker was followed throughout metamorphosis, it is unknown whether mortality related to pre-metamorphic processes (competency or initiation of metamorphosis) or defects to the morphogenetic transition are more frequent. Clearly, more genetic markers need to be included across a large number of families to make any conclusive statements about the patterns of metamorphic mortality in Pacific oysters. Nevertheless, the single marker evaluated here provided significant information on the fine-scale selective changes during this period, showing effects on both metamorphic mortality and the delay of metamorphic initiation.

4.3. QTL analysis of settlement timing The QTL mapping analyses showed that settlement timing has a strong genetic basis (29% variance explained by four QTL), which agrees with previous studies of marine invertebrates that showed 96

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

processes may be not always be distinguishable. Ultimately, the results of the study suggests that underlying genetic variation can have a substantial effect on success or failure at metamorphosis, and more work needs to be done to identify the function of these variants and whether or not they are in common among different oyster families (across other genetic backgrounds).

significant genetic (paternal) effects on competency and early settlement (Levin et al., 1991; Jackson et al., 2005). That a developmental larval trait is under partial genetic control is not particularly surprising, but the current study is one of the first to associate specific genetic markers or genomic regions with a larval trait in the oyster, even with relatively sparse genomic coverage (21 markers). With only a single F2 family examined and the relatively large haplotype blocks present (high linkage disequilibrium), it is not known whether these QTL will be associated with settlement variation across families or if they are specific only to the F2 family examined. While significant QTL were identified for time (days) to settlement, it is possible that other correlated larval traits may contribute to or even drive the genetic differences observed between early and late settling larvae. Time to settlement is a composite larval trait that can essentially be described as larval duration, integrating growth rate at multiple developmental stages as well as the attainment of competency. Genetic variance in any of these developmental traits may be reflected in the association detected between genotype and time to settlement. Indeed, previous studies of the Pacific oyster have shown that hatchery selection for larval growth rate resulted in a simultaneous decrease in the average time to settlement (Taris et al., 2006). Additional evidence from quantitative genetic studies indicates that strong genetic correlations exist between larval growth rate and time to settlement (e.g. Ernande et al., 2003). Larval growth rate was not independently measured in this study, so a direct comparison of genetic variance for growth vs. settlement timing (e.g. via QTL mapping) is not possible. Nevertheless, the identification of specific genetic markers that are associated with variance in larval duration is a significant result, and future QTL-mapping or genome-wide marker studies employing greater marker densities and measurements of multiple larval traits will be better able to characterize genetic variation underlying the larval developmental program. Finally, it is important to discuss the possibility that the association between specific genetic markers and time to settlement could confound our interpretation of genotype-dependent mortality (at those markers) during settlement or vice-versa, genotype-dependent mortality could confound settlement timing QTL. For example, the observed delay (and almost complete failure) in metamorphosis associated with one of the linked deleterious alleles at locus Cg205 (the B allele) in the temporal analysis, may also generate the association between the marker allele and delayed settlement timing (larval duration) in the QTL analysis. Indeed, two of the three identified QTL for settlement timing were closely linked to markers showing significant GDM during metamorphosis (Cg205 and Cg109). In these cases, it is difficult to distinguish between the effects of linked deleterious alleles causing GDM at settlement and alleles that are not necessarily deleterious, but are associated with the settlement timing phenotype, altering growth rate or settlement behavior and ultimately the timing of settlement. On the other hand, the most significant settlement timing QTL identified appears to be independent of genotype-dependent mortality during metamorphosis. Cg140 (closely linked to QTL 3; Table 1) did not show significant genotype-dependent mortality (chi-square goodness-of-fit test in the final, pooled, day 60 spat sample, P = 0.088; Table S1), and while there is another marker on the linkage group that did (goodnessof-fit chi-square test for Cg129 in d18 larvae, P < 0.0001; Table S1), it was thirty centiMorgans (cM) away and was likely independent, acting prior to metamorphosis. The epistatic QTL identified in this study (QTL 4) is also not likely to be associated with genotype-dependent mortality at metamorphosis, because interactions among loci linked to deleterious mutations were virtually absent in all previous crosses examined (Bucklin, 2003; Plough and Hedgecock, 2011; Plough, 2012; Plough et al., 2016). Thus, loci involved with GDM at metamorphosis and loci affecting settlement timing QTL may be distinct, at least in some cases. In other cases, genotypes associated with variation in settlement behavior (e.g. delay) may lead to metamorphic failure (and observed genotype-dependent mortality at metamorphosis), indicating the two

5. Conclusions In this study, genetic marker data and a novel temporal sampling scheme were employed to uncover the fine-scale nature of genotype dependent mortality and settlement behavior in a pair-crossed F2 family of the Pacific oyster. Near-daily sampling and genotyping of oyster larvae and spat during settlement revealed significant genotype-dependent mortality possibly acting just prior to, and during the metamorphic transition, but not after. Quantitative trait locus (QTL) mapping also revealed at least three regions of the genome with significant effects on time to settlement, though genotype-dependent mortality at metamorphosis may confound the interpretation of some (but not all) settlement timing QTL. Overall, endogenous mortality caused by the expression of deleterious allelic variants (genetic load) appears to be substantial during settlement and metamorphosis of Pacific oysters, and results from this study highlight the complex temporal patterns of selection that can occur during this important life history transition. However, the results presented here come from a single cross, and additional studies of selective mortality at metamorphosis are needed in other oyster families and other marine invertebrate species so that this work can be put into a broader context. Because mortality at settlement and metamorphosis represents a major bottleneck in the recruitment of oysters and many other marine invertebrates, experimental insight into the endogenous or selective sources of mortality during this critical period can significantly advance our understanding of the recruitment dynamics of oysters and possibly other marine invertebrates. Acknowledgements The author wishes to thank D. Hedgecock and two anonymous reviewers for helpful comments and suggestions on previous versions of the manuscript. Funding This work was funded in part by an endowed graduate fellowship (Oakley Fellowship) and a Rose Hills foundation fellowship to L. Plough at U.S.C., The Deerbrook Cheritable Trust (DCT 15-30), and the University of Maryland Center for Environmental Science. Data accessibility Microsatellite genotype data from this study are deposited in the Dryad digital repository, https://doi.org/10.5061/dryad.6fm12. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jembe.2018.01.006. References Baker, P., 1997. Settlement site selection by oyster larvae, Crassostrea virginica: evidence for geotaxis. J. Shellfish Res. 16, 125–128. Baker, P., Mann, R., 1998. Response of settling oyster larvae, Crassostrea virginica, to specific portions of the visible light spectrum. J. Shellfish Res. 17, 1081–1083. Balch, T., Scheibling, R.E., 2000. Temporal and spatial variability in settlement and recruitment of echinoderms in kelp beds and barrens in Nova Scotia. Mar. Ecol. Prog. Ser. 205, 139–154. Bishop, D.T., Cannings, C., Skolnick, M., Williamson, J.A., 1983. The number of polymorphic DNA clones required to map the human genome. In: Weir, B.S. (Ed.),

97

Journal of Experimental Marine Biology and Ecology 501 (2018) 90–98

L.V. Plough

abalone Haliotis asinine. Mar. Biol. 147, 681–697. Jackson, D.J., Worheide, G., Degnan, B.M., 2007. Dynamic expression of ancient and novel molluscan shell genes during ecological transitions. BMC Evol. Biol. 7, 1–17. Jacobs, M.W., Degnan, S.M., Woods, R., Williams, E., Roper, K., Green, K., Degnan, B.M., 2006. The effect of larval age on morphology and gene expression during ascidian metamorphosis. Integr. Comp. Biol. 46, 60–776. Jones, G., Jones, B., 1983. Methods for Setting Hatchery Produced Oyster Larvae. Marine Resources Branch, Ministry of Environment, BC, Canada (Information report No. 4., 94pp). Kawashima, T., Satou, Y., Murakami, S.D., Satoh, N., 2005. Dynamic changes in developmental gene expression in the basal chordate Ciona intestinalis. Develop. Growth Differ. 47, 187–199. Kennedy, V.S., Newell, R.I.E., Eble, A.F., 1996. The Eastern Oyster: Crassostrea virginica. Maryland Sea Grant press. Keough, M.J., Downes, B.J., 1982. Recruitment of marine invertebrates: the role of active larval choices and early mortality. Oecologia (Berl) 54, 348–352. Launey, S., Hedgecock, D., 2001. High genetic load in the Pacific oyster Crassostrea gigas. Genetics 159, 255–265. Levin, L.A., Zhu, J., Creed, E., 1991. The genetic basis of life history characters in a polychaete exhibiting planktotrophy and lecithotrophy. Evolution 45, 380–397. Li, G., Hubert, S., Bucklin, K., Ribes, V., Hedgecock, D., 2003. Characterization of 79 microsatellite DNA markers in the Pacific oyster Crassostrea gigas. Mol. Ecol. Notes 3, 228–232. Magoulas, A., Gjetvag, B., Tersoglou, V., Zouros, E., 1998. Three polymorphic microsatellites in the Japanese oyster, Crassostrea gigas (Thunberg). Anim. Genet. 29, 69–70. McGoldrick, D.J., Hedgecock, D., English, L.J., Baoprasertkul, P., Ward, R.D., 2000. The transmission of microsatellite alleles in Australian and North American stocks of the Pacific oyster (Crassostrea gigas): selection and null alleles. J. Shellfish Res. 19, 779–788. Newell, R.I.E., Alspach, G.S., Kennedy, V.S., Jacobs, D., 2000. Mortality of newly metamorphosed eastern oysters (Crassostrea virginica) in mesohaline Chesapeake Bay. Mar. Biol. 136, 665–676. Pawlik, J.R., 1992. Induction of marine invertebrate larval settlement: evidence for chemical cues. In: Paul, V.J. (Ed.), Ecological Roles of Marine Natural Products. Cornell University Series in Chemical Ecology. Cornell University Press, pp. 189–236. Pernet, F., Tremblay, R., 2004. Effect of varying levels of dietary essential fatty acid during early ontogeny of the sea scallop Placopecten magellanicus. J. Exp. Mar. Biol. Ecol. 310, 73–86. Pineda, J., 1994. Spatial and temporal patterns in barnacle settlement rate along a southern California rocky shore. Mar. Ecol. Prog. Ser. 107, 125–138. Pineda, J., Starczak, V., Stueckle, T.A., 2006. Timing of successful settlement: demonstration of a recruitment window in the barnacle Semibalanus balanoides. Mar. Ecol. Prog. Ser. 320, 233–237. Plough, L.V., 2012. Environmental stress increases selection against and dominance of deleterious mutations in inbred families of the Pacific oyster, Crassostrea gigas. Mol. Ecol. 21, 3974–3987. Plough, L.V., 2016. Genetic load in marine animals: a review. Curr. Zool. 62, 567–579. Plough, L.V., Hedgecock, D., 2011. QTL analysis of stage-specific inbreeding depression in the Pacific oyster Crassostrea gigas. Genetics 189, 1473–1486. Plough, L.V., Moran, A., Marko, P., 2014. Density drives polyandry and relatedness influences paternal success in the gooseneck barnacle, Pollicipes elegans. BMC Evol. Biol. 14, 81. Plough, L.V., Shin, G., Hedgecock, D., 2016. Genetic inviability is a major driver of type III survivorship in experimental families of a highly fecund marine bivalve. Mol. Ecol. 25, 895–910. Raimondi, P.T., 1990. Patterns, mechanisms, and consequences of variability in settlement and recruitment in an intertidal barnacle. Ecol. Monogr. 60, 283–309. Rico-Villa, B., Woerther, P., Mingant, C., Lepiver, D., Pouvreau, S., Hamon, M., Robert, R., 2008. A flow-through rearing system for ecophysiological studies of Pacific oyster Crassostrea gigas larvae. Aquaculture 282, 54–60. Rodriguez, S.R., Patricio-Ojedal, F., Inestrosa, N.C., 1993. Settlement of benthic marine invertebrates. Mar. Ecol. Prog. Ser. 97, 193–207. Roughgarden, J., Gaines, S., Possingham, H., 1988. Recruitment dynamics in complex life cycles. Science 241, 1460–1466. Sekino, M., Hamaguchi, M., Aranishi, F., Okoshi, K., 2003. Development of novel microsatellite DNA markers from the Pacific oyster, Crassostrea gigas. Mar. Biotechnol. 5, 227–233. Taris, N., Ernande, B., McCombie, H., Boudry, P., 2006. Phenotypic and genetic consequences of size selection at the larval stage in the Pacific oyster (Crassostrea gigas). J. Exp. Mar. Biol. Ecol. 333, 147–158. Van Ooijen, J.W., Voorrips, R.E., 2001. JoinMap 3.0, Software for the Calculation of Genetic Linkage Maps. Plant Research International, Wageningen, The Netherlands. Vasquez, H.E., Hashimoto, K., Yoshida, A., Hara, K., Imai, C.C., Kitamura, H., Satuito, C.G., 2013. A glycoprotein in shells of conspecifics induces larval settlement of the Pacific oyster Crassostrea gigas. PLoS One 8, e82358. Wang, Y., Ren, R., Yu, Z., 2008. Bioinformatic mining of EST-SSR loci in the Pacific oyster, Crassostrea gigas. Anim. Genet. 39, 287–289. Williams, E.A., Degnan, B.M., Gunter, H., Jackson, D.J., Woodcroft, B.J., Degnan, S.M., 2009. Widespread transcriptional changes pre-empt the critical pelagic–benthic transition in the vetigastropod Haliotis asinine. Mol. Ecol. 18, 1006–1025. Yamtich, J., Voigt, M.L., Li, G., Hedgecock, D., 2005. Eight microsatellite loci for the Pacific oyster Crassostrea gigas. Anim. Genet. 36, 524–526. Yu, H., Li, Q., 2007. EST-SSR markers from the Pacific oyster, Crassostrea gigas. Mol. Ecol. Notes 7, 860–862. Zaykin, D.V., Pudovkin, A.I., 1993. Two programs to estimate significance of Chi-square values using pseudo-probability test. J. Hered. 84, 152. Zhu, C., Zhang, Y.-M., 2007. An EM algorithm for mapping segregation distortion loci. BMC Genet. 8, 82.

Statistical Analysis of DNA Sequence Data. Marcel Dekker, New York, pp. 181–200. Bonar, D.B., 1976. Molluscan metamorphosis: a study in tissue transformation. Am. Zool. 16, 573–591. Breese, W.P., Malouf, R.E., 1975. Hatchery manual for the Pacific Oyster, Crassostrea gigas (Gould). In: Oregon Agricultural Experiment Station Special Report No, 443, and Oregon State University Sea Grant College Program Publ. No. ORESU-H-75-002, (22 pp). Broitman, B.R., Blachette, C.A., Menge, B.A., Lubchenko, J., Krenz, C., Foley, M., Raimondi, P.T., Lohse, D., 2008. Spatial and temporal patterns of invertebrate recruitment along the west coast of the United States. Ecol. Monogr. 78 (3), 403–421. Broman, K.W., Wu, H., Sen, S., Churchill, G.A., 2003. R/qtl: QTL mapping in experimental crosses. Bioinformatics 19, 889–890. Bucklin, K.A., 2003. Analysis of the Genetic Basis of Inbreeding Depression in the Pacific Oyster Crassostrea gigas. University of California at Davis (Ph.D. Dissertation). Burke, R.D., 1986. Pheremones and the gregarious settlement of marine invertebrate larvae. Biol. Bull. 39, 323–331. Cannuel, R., Beninger, P.G., 2006. Gill development, functional and evolutionary implications in the Pacific oyster Crassostrea gigas (Bivalvia: Ostreidae). Mar. Biol. 149, 547–563. Cannuel, R., Beninger, P.G., McCombie, H., Boudry, P., 2009. Gill development and its functional and evolutionary implications in the blue mussel Mytilus edulis (Bivalvia: Mytilidae). Biol. Bull. 217, 173–188. Cole, H.A., 1938. The fate of the larval organs in the metamorphosis of Ostrea edulis. J. Mar. Biol. Assoc. UK 23, 469–484. Cole, H.A., Knight-Jones, E.W., 1939. Some observations and experiments in the setting behavior of larvae of Ostrea edulis. J. Cons. Cons. Int. Explor. Mer. 14, 86–105. Coutteau, P., Caers, M., Mallet, A., Moore, W., Manzi, J.J., Soorgeloos, P., 1994. Effect of lipid supplementation on growth, survival, and fatty acid composition of bivalve larvae. In: Kestemont, P., Muir, J., Williot, P. (Eds.), Measures for Success Proceedings of Bordeaux Aquaculture, 1994, Mar. 23–25, (Bordeaux, France). Degnan, B.M., Morse, D.E., 1995. Developmental and morphogenetic gene regulation in Haliotis rufescens larvae at metamorphosis. Am. Zool. 35, 391–398. Degnan, B.M., Souter, D., Degnan, S.M., Long, S.C., 1997. Induction of metamorphosis with potassium ions requires develop- ment of competence and an anterior signalling centre in the ascidian Herdmania momus. Dev. Genes Evol. 206, 370–376. Eri, R., Arnold, J.M., Hinman, V.F., Green, K.M., Jones, M.K., Degnan, B.M., Lavin, M.F., 1999. Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis. Development 126, 5809–5818. Ernande, B., Clobert, J., McCombie, H., Boudry, P., 2003. Genetic polymorphism and trade-offs in the early life-history strategy of the Pacific oyster, Crassostrea gigas (Thunberg, 1795): a quantitative genetic study. J. Evol. Biol. 16, 399–414. Gaines, S., Roughgarden, J., 1985. Larval settlement rate: a leading determinant of structure in an ecological community of the marine intertidal zone. Proc. Natl. Acad. Sci. U. S. A. 82, 3707–3711. Gallager, S.M., Mann, R., 1986. Growth and survival of larvae of mercfenaria-mercenaria (L) and Crassostrea virginica (Gmelin) relative to broodstock conditioning and lipid content of eggs. Aquaculture 56, 105–12.1. Gallager, S.M., Mann, R., Sasaki, G.C., 1986. Lipid as an index of growth and viability in 3 species of bivalve larvae. Aquaculture 56, 81–103. Gosselin, L.A., Qian, P.Y., 1996. Early post-settlement mortality of an intertidal barnacle: a critical period for survival. Mar. Ecol. Prog. Ser. 135, 69–75. Gosselin, L.A., Qian, P.Y., 1997. Juvenile mortality in benthic marine invertebrates. Mar. Ecol. Prog. Ser. 146, 265–282. Haws, M.C., DiMichele, L., Hand, S.C., 1993. Biochemical changes and mortality during metamorphosis of the Eastern oyster, Crassostrea virginica, and the Pacific oyster, Crassostrea gigas. Mol. Mar. Biol. Biotechnol. 2, 207–217. Hedgecock, D., Davis, J.P., 2007. Heterosis for yield and crossbreeding of the Pacific oyster Crassostrea gigas. Aquaculture 272S, S17–S29. Hedrick, P.W., Muona, O., 1990. Linkage of viability genes to marker loci in selfing organisms. Heredity 64, 67–72. Helm, M.M., Holland, D.L., Utting, S.D., East, J., 1991. Fatty acid composition of early non-feeding larvae of the European flat oyster, Ostrea edulis. J. Mar. Biol. Assoc. UK 71, 691–705. Helm, M.M., Bourne, N., Lovatelli, A., 2004. Hatchery Culture of Bivalves — A Practical Manual. FAO Fisheries Technical Paper, FAO, Rome. Heylund, A., Moroz, L.L., 2006. Signaling mechanisms underlying metamorphic transitions in animals. Integr. Comp. Biol. 46, 743–759. Hickman, R.W., Gruffydd, L.L.D., 1971. The Histology of the Larva of Ostrea edulis during Metamorphosis. In: Crisp, E.J. (Ed.), Fourth European Marine Biology Symposium. University Press, Cambridge, pp. 281–294. Hidu, H., William, G.V., Veitch, F.P., 1978. Gregarious setting in European and American oysters-response to surface chemistry versus waterborne pheromones. Proc. Natl. Shellfish. Assoc. 68, 11–16. Hubert, S., Hedgecock, D., 2004. Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas. Genetics 168, 351–362. Hubert, S., Cognard, E., Hedgecock, D., 2009. Centromere-mapping in triploid families of the Pacific oyster Crassostrea gigas (Thunberg). Aquaculture 288, 172–183. Hunt, H.L., Scheibling, R.E., 1997. Role of early post settlement mortality in recruitment of benthic marine invertebrates. Mar. Ecol. Prog. Ser. 155, 269–301. Huvet, A., Boudry, P., Ohresser, M., Delsert, C., Bonhomme, F., 2000. Variable microsatellites in the Pacific oyster Crassostrea gigas and other cupped oyster species. Anim. Genet. 31, 71–72. Jackson, D.J., Degnan, B.M., 2006. Expressed sequence tage analysis of genes expressed during development of the tropical abalone Haliotis asinine. J. Shellfish Res. 25, 225–231. Jackson, D.J., Leys, S.P., Hinman, V.F., Woods, R., Lavin, M.F., Degnan, B.M., 2002. Ecological regulation of development: induction of marine invertebrate metamorphosis. Int. J. Dev. Biol. 46, 679–686. Jackson, D.J., Ellemor, N.E., Degnan, B.M., 2005. Correlating gene expression with larval competence, and the effect of age and parentage on metamorphosis in the tropical

98