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Larval size and metamorphosis are significantly reduced in second generation of inbred sea urchins Strongylocentrotus intermedius
Aquaculture 452 (2016) 402–406
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Larval size and metamorphosis are significantly reduced in second generation of inbred sea urchins Strongylocentrotus intermedius Chong Zhao 1, Ping Sun 1, Jing Wei, Lisheng Zhang, Weijie Zhang, Jian Song, Yaqing Chang ⁎ Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China
a r t i c l e
i n f o
Article history: Received 6 August 2015 Received in revised form 3 November 2015 Accepted 16 November 2015 Available online 17 November 2015 Keywords: Sea urchin Strongylocentrotus intermedius Inbreeding Larval size Metamorphosis
a b s t r a c t Little experimental evidence of multiple generations of inbreeding depression is available for marine invertebrates. Here, we report that two generations of inbreeding significantly reduced larval growth and metamorphosis, but did not significantly impact fertilization and hatching in the sea urchin Strongylocentrotus intermedius. A higher inbreeding coefficient had significantly more negative impacts on some traits of S. intermedius larvae (for example, larval length) but not all. Maternal effects of inbreeding depression probably appear in the eight-arm stage and metamorphosis of S. intermedius. The present study provides convincing evidence of inbreeding depression on larval growth and metamorphosis in S. intermedius and highlights the importance of avoiding multiple generations of inbreeding in sea urchin aquaculture. Statement of relevance: Multiple inbreeding dramatically impacts aquaculture of marine species. Here, we investigated effects of second generation of inbreeding on fertilization, hatching, larval growth and metamorphosis in sea urchins. The present study provides the convincing evidence on the inbreeding depression of larval growth and metamorphosis in sea urchins and highlights the importance of avoiding multiple generation of inbreeding in sea urchin aquaculture. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inbreeding depression refers to a general decline in fitness traits with increasing homozygosity in the offspring of biologically related individuals during their life history (Lynch and Walsh, 1998). This phenomenon is especially common in cultured marine invertebrates because of their high fecundity and limited effective population size (Rhode et al., 2014). In addition to impacted commercially important traits and consequent reduced marketing values (Ma et al., 2005), another important concern in aquaculture about decreasing effective population size and increasing inbreeding levels is the potentially decline in hatchery success, larval growth and metamorphosis (Moss et al., 2008; Luo et al., 2014). For example, significant inbreeding depression was found in the larval size in only one generation of inbred sea urchins Strongylocentrotus purpuratus, clearly suggesting a large load of recessive deleterious mutations (Anderson and Hedgecock, 2010). Further, even random mating of multiple generations significantly decreased the effective population size and increased the inbreeding rate of cultured abalones Haliotis midae (Rhode et al., 2014). Thus, inbreeding depression probably worsens in the second or third generation of inbreeding even if one generation of inbreeding does not induce a significant depression in fitness traits (Oosterhout et al., ⁎ Corresponding author. E-mail address: [email protected] (Y. Chang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.aquaculture.2015.11.024 0044-8486/© 2015 Elsevier B.V. All rights reserved.
2003). This raises an interesting question about the consequence of inbreeding marine invertebrates for multiple generations on embryos and larvae. In addition, inbreeding depression probably worsens on the embryo and larvae of invaded (or introduced) marine invertebrates considering the relatively small population size of founders and consequent limited genetic diversity (Zheng et al., 2012). Thus, it is especially essential to investigate the potential inbreeding depression of multiple generations on fertilization, hatching, larval growth and metamorphosis of introduced marine invertebrates of commercial importance. However, we know of little information on this issue in spite of its importance to aquaculture (Zheng et al., 2012). The sea urchin Strongylocentrotus intermedius, which is endemic to intertidal and subtidal bottoms in northern Pacific coastal waters of Hokkaido of Japan, Korea and Far East Russia (Agatsuma, 2013), was introduced into China from Japan in 1989 for its commercial value (Chang et al., 2004). Increasing market demand encourages the development of aquaculture in both Japan and China. The annual production of sea urchins from fisheries and aquaculture in China was 6791 t 2014 (Zhao, 2015). We previously studied the effects of one generation of inbreeding on fertilization, hatchability and larval development of S. intermedius, which lays a solid foundation for further studies on multiple generations of inbreeding (Zhou et al., 2013). Thus, S. intermedius is a good research model to study the effects of multiple generations of inbreeding. The purposes of the present study are to investigate 1) whether fertilization rate, hatching rate, larval growth and metamorphosis rate are significantly impacted by two generations
C. Zhao et al. / Aquaculture 452 (2016) 402–406
403
of inbreeding in S. intermedius; 2) whether a higher inbreeding coefficient shows significantly more negative impacts on all experimental traits of S. intermedius; and 3) whether maternal effects of inbreeding depression appear in all experimental traits of S. intermedius.
was used to evaluate hatching rates. Fertilization rate and hatching rate were calculated as follows:
2. Materials and methods
where F = fertilization rate, a = number of embryo in cleavage, and b = total number of embryo and eggs.
2.1. Experimental design Breeding and culture methods of full-sib families used for brother– sister mating in the present study were fully described in Chang et al. (2012) and Zhang et al. (2013). As S. intermedius is a non-native species in China, 43, 58 and 24 individuals were collected for breeding from three cultured populations in Rongcheng (122.59°E, 37.14°N), Lingshui (121.56°E, 38.88°N) and Lvshun (121.19°E, 38.72°N) in 2007, respectively (Chang et al., 2012). From these initial populations, 190 full-sib families were established at the end of October 2007 (Chang et al., 2012). From the sea urchins produced in October 2007, ninety seven full-sib families were established in May 2009 (Zhang et al., 2013). Inbreeding was strictly avoided in the breeding programs (Chang et al., 2012; Zhang et al., 2013). The establishing methods of these families had no influence on further inbreeding studies. Based on the families haphazardly collected from the ninety seven full-sib families produced in May 2009, 27 families of one generation inbred and crossbred S. intermedius were produced in 2010 at three inbreeding coefficients of 0.25, 0.125 and 0 (designated F, H and C, 9 families for each group), presuming no genetic exchange had occurred among the ancestors of the basic populations collected in 2007. Two F families and two C families were haphazardly collected from the one generation inbred and crossbred families for the establishment of the second generation inbred/crossbred families. Four second generation inbred families (designated FF) were produced by mating males and females from the same F families. In order to investigate maternal effects of inbreeding, individuals from F and C families were crossbred to produce second generation crossbred families with a genetic load (designated FC and CF, respectively). FC refers to F as the male parent and CF refers to C as the male parent. Four fully crossbred families (designated CC) were produced by mating males and females from different C families. The inbreeding coefficients of FF, FC, CF and CC were estimated as 0.375, 0.219, 0.219 and 0, according to the possibility of sharing a common ancestor.
a 100 b
Fð%Þ ¼
Hð%Þ ¼
c 100 d
where H = hatching rate, a = number of prism larvae, and b = total number of larvae and embryo. 2.4. Measurements of larval size Four-arm, six-arm and eight-arm larvae were sampled in all experimental groups at 3 days, 14 days and 24 days after artificial fertilization, respectively. All samples were placed into tubes for 10 min to allow larvae to settle on the bottom. Larvae were then transferred to clear plastic tubes and placed on slides for further measurements under a microscope (Fig. 1). The larvae were photographed with a known size scale. One hundred larvae were examined each time for each individual sea urchin (the only exception was that two hundred larvae were used at the four-arm larval stage). The stomach is the dominant organ of sea urchins for storing nutrients. Thus, stomach size directly determines the ability of larvae to store nutrients. Larval length, larval width, stomach length and stomach width were calculated as follows: Y¼X
m n
where Y = the actual size of the target trait, X = the measured size of the target trait in the photograph, m = the actual length of the scale, and n = the measured size of the scale in the photograph. 2.5. Metamorphosis Two hundred larvae were haphazardly collected and placed into a 2 L beaker 28 days after artificial fertilization to measure metamorphosis in each group. Two polyethylene plates with benthic diatoms (8 cm × 8 cm and 10 cm × 14 cm) were placed into each beaker to
2.2. Culture of inbred and crossbred sea urchins Spawning of S. intermedius was induced by injecting 1.5 mL KCl (0.5 mol.L−1). The gametes were collected using individual containers to avoid potential mixture. All fertilized eggs were placed into labeled 70 L tanks for hatchability studies after careful washing to remove excess sperm. All tanks were randomly distributed in the laboratory to balance non-experimental environmental factors. The conditions of hatchability were 18–19 °C seawater temperature, 30 ‰ salinity, 0–300 lx natural light density and no aeration. The hatching density was adjusted to 0.5–1.0 ind ∙ mL− 1 in all tanks 30 days after fertilization. The larvae were equally fed the microalga Chaetoceros gracilis in all tanks four times a day in weakly aerated seawater at concentrations from 2 × 10 4 to 5 × 10 4 cells ∙ mL − 1 . Sea urchins in all experimental groups were cultured using the same methods and in similar conditions. 2.3. Fertilization and hatchability Fertilization was observed using a microscope in 3 h after artificial fertilization. An embryo with cleaving cells indicates development of fertilized eggs. Hatchability of S. intermedius was observed using a microscope 30 h after artificial fertilization. The number of prism larvae
Fig. 1. Measurements of larval length (ll), larval width (lw), stomach length (sl), and stomach width (sw) of Strongylocentrotus intermedius.
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encourage settlement of larvae. The number of settled S. intermedius was recorded 20 days after placing the polyethylene plates. 2.6. Statistical analysis The data were first tested for normal distribution and homogeneity of variance before statistical analysis. One-way ANOVA was performed to detect differences in fertilization rate, hatching rate, larval development and metamorphosis rate. Duncan's multiple comparisons were then performed when significant differences were found with the ANOVA analysis. All analyses were done with SPSS 16.0 statistical software. A probability level of P b 0.05 was considered statistically significant. 3. Results 3.1. Fertilization and hatchability Fertilization rates of S. intermedius were not significantly different among all the experimental groups (P N 0.05), ranging from 94.4% to 95.4%. Hatching rates showed no significant difference among all the experimental groups (P N 0.05), although it was slightly less in the FF group. 3.2. Larval growth The FF group showed an obvious high rate of abnormal four-arm larvae, although the P value was not significant (P N 0.05). Larval length of inbred sea urchins was significantly less than that of crossbred individuals in four-arm larva, six-arm larva and eight-arm larva stages, respectively (P b 0.05, Fig. 2). The length of eight-arm larvae was significantly shorter in the FF group than in the FC and CF groups (P b 0.05). Larval length in the FC group was significant shorter than in the CF group in the six-arm larva stage (P b 0.05) and significantly longer than in the eight-arm larva stage (P b 0.05). The width of six-arm and eight-arm larvae was significantly greater in the CC group than in the inbred groups (P b 0.05, Fig. 2). The width of four-arm larvae, however, was significantly less in the CC group than in the CF and FF groups (P b 0.05). The width of eight-arm larvae was not significantly different among the experimental inbreeding groups (P N 0.05). The larval width of the CF group was significantly greater
than that of the FC group at the four-arm larva stage (P b 0.05), but showed no significant difference at the six-arm larva and eight-arm larva stages (P N 0.05). Stomach length in the CC group was significantly greatest at the four-arm, six-arm and eight-arm larva stages, respectively (P b 0.05, Fig. 3). The FF group had a significantly less stomach length at the sixarm larva stage than in the FC and CF groups (P b 0.05) and a longer stomach length at the eight-arm larva stage (P b 0.05). The CF group showed significantly longer stomach length than the FC group at the four-arm larva stage (P b 0.05). The CC group showed significantly wider stomach only in the four-arm larva stage (P b 0.05, Fig. 3). The stomach width of eight-arm larvae in the FF group was significantly greater than in the other groups (P b 0.05). There was no significant difference of stomach width between the CF and FC groups at the four-arm larva stage (P N 0.05). However, the stomach width in the FC groups was significantly larger than in the CF group at both the six-arm and eight-arm stages (P b 0.05). 3.3. Metamorphosis Significantly more larvae in the CC group settled in 20 days after placing polyethylene plates into the beakers than in the inbred groups (P b 0.05, Fig. 4). However, there was no significant difference of settlement among the CF, FC and FF groups (P N 0.05). 4. Discussion This study increases evidence of the effect of multiple generations of inbreeding depression in marine invertebrates (Zheng et al., 2012; Luo et al., 2014), although depression after one generation of inbreeding has been well documented in larvae of scallops (Ibarra et al., 1995; Zheng et al., 2012), Pacific oysters (Taris et al., 2007), and sea urchins (Anderson and Hedgecock, 2010, Zhou et al., 2013). In the present study, second generation of inbreeding did not significantly affect fertilization and hatchability of S. intermedius, which agrees to our previous study on effects of one generation of inbreeding on fertilization and hatchability of S. intermedius (Zhou et al., 2013). This result indicates of the robustness of fertilization and hatchability in S. intermedius with an inbreeding coefficient of 0.375, but does not ensure that in higher inbreeding coefficient in other species (Zheng et al., 2012).
Fig. 2. Larval length and width of Strongylocentrotus intermedius in the four-arm, six-arm and eight-arm stages in different experimental groups (N = 200 for four-arm larvae and N = 100 for the others, mean ± SD). F refers to one generation full-sibling inbred families, C refers to the families without inbreeding, FF refers to second generation fully inbred families by mating males and females from the same F families, FC refers to F as the male parent, while CF refers to C as the male parent, CC refers to the families produced by mating males and females from different C families.
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Fig. 3. Stomach length and width of Strongylocentrotus intermedius in the four-arm, six-arm and eight-arm stages in different experimental groups (N = 200 for four-arm larvae and N = 100 for the others, mean ± SD). See Fig. 2 for abbreviation for groups.
In the present study, we found significant depression of larval size (especially in larval length) and metamorphosis in second generation of inbred S. intermedius. This result agrees with the finding that two successive generations of self inbreeding significantly reduced larval survival and juvenile growth in the scallop Argopecten irradians irradians (Zheng et al., 2012). Compared to our previous finding that one generation of inbreeding did not significantly affect larval growth of S. intermedius (Zhou et al., 2013), the present result clearly indicates of a larger load of recessive deleterious mutation in the second generation of inbred S. intermedius and highlights the potential risks of multiple generations of inbreeding on aquaculture of sea urchins. This agrees with a behavioral study with the guppy Poecilia reticulata (Oosterhout et al., 2003), in which male following behavior was not affected by one generation of inbreeding but was significantly depressed in the second and third generation of inbreeding. Together, the present results indicate that inbreeding depression of sea urchins is highly trait dependent and that larval growth and metamorphosis are more susceptible to inbreeding than fertilization and hatchability. The larva is one of the most fragile stages in the life history of sea urchins (Metaxas, 2013), which would greatly affect metamorphosis and consequently the whole breeding success in aquaculture (Chang
Fig. 4. Number of settled sea urchins of Strongylocentrotus intermedius in different experimental groups 20 days after placing polyethylene plates with benthic diatoms into the beakers (N = 3, mean ± SD). See Fig. 2 for abbreviation for groups.
et al., 2004). In the present study, we found the larval length was significantly less in second generation inbred sea urchins than in the crossbred individuals. This result is consistent with the finding of Anderson and Hedgecock (2010) that one generation inbred larvae were significantly smaller in S. purpuratus. Larval length, especially post-oral arms, is important for feeding and consequent fitness in sea urchins (Adams et al., 2011). Thus, we hypothesize that inbreeding depression in larval length probably affects feeding ability and consequently metamorphosis in sea urchins. Metamorphosis is the life history stage in which larval features are lost and the larva is irreversibly transformed into a juvenile in invertebrates (Hadfield, 2000). This stage is fundamentally important for the successful recruitment of sea urchins (Swanson et al., 2012). Consequently it would determine breeding success in aquaculture. It is well known that metamorphosis of sea urchins can be induced or affected by various physical and chemical factors (Swanson et al., 2012; Metaxas, 2013). However, little information is available on the genetic basis of metamorphosis in sea urchins. For the first time, we report that second generation of full-sib mating significantly decreased metamorphosis of S. intermedius. This clearly indicates that second generation of full-sib mating S. intermedius carried a large load of recessive detrimental mutation in genes, which significantly impacted fitness related traits, including larval growth and consequent metamorphosis. A negative linear relationship has been generally accepted for fitness traits and the inbreeding coefficient in both plants and animals (Lynch and Walsh, 1998). In the present study, we found that this phenomenon appeared in some traits (for example, larval length), but not all. This result is consistent with a recent report that different levels of multiple generation inbreeding significantly affected body weight, but did not significantly impact the WSSV resistance of the Chinese shrimp, Fenneropenaeus chinensis (Luo et al., 2014). In addition, larval growth of S. intermedius at the three inbreeding levels differed greatly among different developmental stages. A reasonable explanation is that larval development of sea urchins is a well-ordered process with highly spatial and temporal expression. Thus, different levels of loci homozygosis probably have different effects on sea urchin larvae at different developmental stages. A number of studies have verified a significant maternal inbreeding effect on progeny performance (Lynch and Walsh, 1998). In general, we found that larval length of S. intermedius in the CF group was significantly larger than in the FC group in the six-arm stage, while significantly shorter in the eight-arm stages. Deng et al. (2005) reported a similar maternal effect of inbreeding depression on metamorphosis and larval
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size in the Pacific abalone Haliotis discus hannai. This clearly indicates that maternal effects of inbreeding depression probably happen in the late developmental larval stage and metamorphosis of sea urchins. Our result increases the evidence of maternal effect of inbreeding depression in marine invertebrates. However, it remains unknown of whether inbreeding depression in maternal traits or offspring traits contributed to the maternal inbreeding effects, because we did not measure the maternal traits of parent sea urchins (for example, egg size and egg nutrient components). Further studies should be carried out to reveal the contributions of the maternal inbreeding effects of sea urchins. The present study provides convincing evidence of significant inbreeding depression in second generation inbred S. intermedius. Notably, inbreeding depression can be further aggravated by the interaction with environmental factors (Franke and Fischer, 2013). Thus, we suggest that sea urchin aquaculture should involve a large effective population size to avoid inbreeding, especially multi-generation inbreeding, to ensure the whole successful hatchery in aquaculture. Further, we found that the impact of inbreeding is variable among traits. This is well consistent with the finding of Nakadate et al. (2003) that inbreeding significantly impacted some of the experimental traits of the guppy Poecilia reticulate, but not all. In conclusion, two generations of inbreeding significantly affected larval growth and metamorphosis, but not fertilization and hatchability in S. intermedius. Higher inbreeding coefficient showed significantly more negative impacts on some traits of S. intermedius larvae (for example, larval length), but not all. Maternal effects of inbreeding depression on larval length probably appeared in the eight-arm stage and metamorphosis of S. intermedius. The present study provides convincing evidence of inbreeding depression of larval growth and metamorphosis in S. intermedius and highlights the importance of avoiding multiple generation of inbreeding in sea urchin aquaculture. Acknowledgments This work was supported by the Chinese National 863 Project (2012AA10A412), a research project granted by Liaoning Department of Education (L2015087) and a public welfare project granted by the State Oceanic Administration of China (201305027). We are grateful to Prof. John Lawrence for academic and editorial suggestions and to Jinyong Zhang for his assistance. All authors do not have any conflict of interest. References Adams, D.K., Sewell, M.A., Angerer, R.C., Angerer, L.M., 2011. Rapid adaptation to food availability by a dopamine-mediated morphogenetic response. Nat. Commun. 2, 592.
Agatsuma, Y., 2013. Strongylocentrotus intermedius. In: Lawrence, J.M. (Ed.), Edible Sea Urchins: Biology and Ecology. Elsevier, Amsterdam, pp. 437–447. Anderson, D., Hedgecock, D., 2010. Inbreeding depression and growth heterosis in larvae of the purple sea urchin Stronglyocentrotus purpuratus (Stimpson). J. Exp. Mar. Biol. Ecol. 384, 68–75. Chang, Y., Ding, J., Song, J., Yang, W., 2004. Biology and Aquaculture of Sea Cucumbers and Sea Urchins. Ocean Press, Beijing, China (in Chinese). Chang, Y., Zhang, W., Zhao, C., Song, J., 2012. Estimates of heritabilities and genetic correlations for growth and gonad traits in the sea urchin Strongylocentrotus intermedius. Aquac. Res. 43 (2), 271–280. Deng, Y., Liu, X., Zhang, G., Guo, X., 2005. Inbreeding depression and maternal effects on early performance of Pacific abalone. N. Am. J. Aquac. 67, 231–236. Franke, K., Fischer, K., 2013. Effects of inbreeding and temperature stress on life history and immune function in a butterfly. J. Evol. Biol. 26 (3), 517–528. Hadfield, M.G., 2000. Why and how marine-invertebrate larvae metamorphose so fast. Semin. Cell Dev. Biol. 11, 437–443. Ibarra, A.M., Cruz, P., Romero, B.A., 1995. Effects of inbreeding on growth and survival of self-fertilized Catarina scallop larvae, Argopecten circularis. Aquaculture 134, 37–47. Luo, K., Kong, J., Luan, S., Meng, X., Zhang, T., Wang, Q., 2014. Effect of inbreeding on survival, WSSV tolerance and growth at the postlarval stage of experimental full-sibling inbred populations of the Chinese shrimp Fenneropenaeus chinensis. Aquaculture 420–421, 32–37. Lynch, M., Walsh, B., 1998. Genetics and Analysis of Quantitative Traits. Sinauer Associates, Inc., Publishers, USA, pp. 251–291. Ma, D.Y., Hu, H.L., Kong, J., 2005. Inbreeding and its impact on aquaculture. J. Fish. China 29 (6), 849–856 (in Chinese). Metaxas, A., 2013. Larval ecology in echinoids. In: Lawrence, J.M. (Ed.), Edible Sea Urchins: Biology and Ecology. Elsevier, Amsterdam, pp. 69–81. Moss, D.R., Arce, S.M., Otoshi, C.A., Moss, S.M., 2008. Inbreeding effects on hatchery and growout performance of Pacific white shrimp, Penaeus (Litopenaeus) vannamei. J. World Aquacult. Soc. 39 (4), 467–476. Nakadate, M., Shikano, T., Taniguchi, N., 2003. Inbreeding depression and heterosis in various quantitative traits of the guppy, Poecilia reticulate. Aquaculture 220, 219–226. Oosterhout, C.V., Trigg, R.E., Carvalho, G.R., Magurran, A.E., Hauser, L., Shaw, P.W., 2003. Inbreeding depression and genetic load of sexually selected traits: how the guppy lost its spots. J. Evol. Biol. 16, 273–281. Rhode, C., Maduna, S.N., Roodt-Wilding, R., Bester-van der Merwe, A.E., 2014. Comparison of population genetic estimates amongst wild, F1 and F2 cultured abalone (Haliotis midae). Anim. Genet. 45 (3), 456–459. Swanson, R.L., Byrne, M., Prowse, T.A.A., Mos, B., Dworjanyn, S.A., Steinberg, P.D., 2012. Dissolved histamine: a potential habitat marker promoting settlement and metamorphosis in sea urchin larvae. Mar. Biol. 159, 915–925. Taris, N., Batista, F.M., Boudry, P., 2007. Evidence of response to unintentional selection for faster development and inbreeding depression in Crassostrea gigas larvae. Aquaculture 272 (1), 69–79. Zhang, W., Zhao, C., Chen, M., Chang, Y., Song, J., Luo, S., 2013. Family growth response to different laboratory culture environments shows genotype by environment interaction in the sea urchin Strongylocentrotus intermedius. Aquac. Res. 44 (11), 1706–1714. Zhao, X. (Ed.), 2015. China Fishery Statistical Yearbook 2014. China Agriculture Press, Beijing (in Chinese). Zheng, H.P., Li, L., Zhang, G.F., 2012. Inbreeding depression for fitness-related traits and purging the genetic load in the hermaphroditic bay scallop Argopecten irradians irradians (Mollusca: Bivalvia). Aquaculture 366–367, 27–33. Zhou, H., Luo, S., Zhang, W., Gao, L., Zhao, C., Chang, Y., 2013. Effect of inbreeding on fertility rate, hatchability and larva development of sea urchin Strongylocentrotus intermedius. J. Dalian Ocean Univ. 28 (5), 445–449 (in Chinese).
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Larval size and metamorphosis are significantly reduced in second generation of inbred sea urchins Strongylocentrotus intermedius Chong Zhao 1, Ping Sun 1, Jing Wei, Lisheng Zhang, Weijie Zhang, Jian Song, Yaqing Chang ⁎ Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China
a r t i c l e
i n f o
Article history: Received 6 August 2015 Received in revised form 3 November 2015 Accepted 16 November 2015 Available online 17 November 2015 Keywords: Sea urchin Strongylocentrotus intermedius Inbreeding Larval size Metamorphosis
a b s t r a c t Little experimental evidence of multiple generations of inbreeding depression is available for marine invertebrates. Here, we report that two generations of inbreeding significantly reduced larval growth and metamorphosis, but did not significantly impact fertilization and hatching in the sea urchin Strongylocentrotus intermedius. A higher inbreeding coefficient had significantly more negative impacts on some traits of S. intermedius larvae (for example, larval length) but not all. Maternal effects of inbreeding depression probably appear in the eight-arm stage and metamorphosis of S. intermedius. The present study provides convincing evidence of inbreeding depression on larval growth and metamorphosis in S. intermedius and highlights the importance of avoiding multiple generations of inbreeding in sea urchin aquaculture. Statement of relevance: Multiple inbreeding dramatically impacts aquaculture of marine species. Here, we investigated effects of second generation of inbreeding on fertilization, hatching, larval growth and metamorphosis in sea urchins. The present study provides the convincing evidence on the inbreeding depression of larval growth and metamorphosis in sea urchins and highlights the importance of avoiding multiple generation of inbreeding in sea urchin aquaculture. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inbreeding depression refers to a general decline in fitness traits with increasing homozygosity in the offspring of biologically related individuals during their life history (Lynch and Walsh, 1998). This phenomenon is especially common in cultured marine invertebrates because of their high fecundity and limited effective population size (Rhode et al., 2014). In addition to impacted commercially important traits and consequent reduced marketing values (Ma et al., 2005), another important concern in aquaculture about decreasing effective population size and increasing inbreeding levels is the potentially decline in hatchery success, larval growth and metamorphosis (Moss et al., 2008; Luo et al., 2014). For example, significant inbreeding depression was found in the larval size in only one generation of inbred sea urchins Strongylocentrotus purpuratus, clearly suggesting a large load of recessive deleterious mutations (Anderson and Hedgecock, 2010). Further, even random mating of multiple generations significantly decreased the effective population size and increased the inbreeding rate of cultured abalones Haliotis midae (Rhode et al., 2014). Thus, inbreeding depression probably worsens in the second or third generation of inbreeding even if one generation of inbreeding does not induce a significant depression in fitness traits (Oosterhout et al., ⁎ Corresponding author. E-mail address: [email protected] (Y. Chang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.aquaculture.2015.11.024 0044-8486/© 2015 Elsevier B.V. All rights reserved.
2003). This raises an interesting question about the consequence of inbreeding marine invertebrates for multiple generations on embryos and larvae. In addition, inbreeding depression probably worsens on the embryo and larvae of invaded (or introduced) marine invertebrates considering the relatively small population size of founders and consequent limited genetic diversity (Zheng et al., 2012). Thus, it is especially essential to investigate the potential inbreeding depression of multiple generations on fertilization, hatching, larval growth and metamorphosis of introduced marine invertebrates of commercial importance. However, we know of little information on this issue in spite of its importance to aquaculture (Zheng et al., 2012). The sea urchin Strongylocentrotus intermedius, which is endemic to intertidal and subtidal bottoms in northern Pacific coastal waters of Hokkaido of Japan, Korea and Far East Russia (Agatsuma, 2013), was introduced into China from Japan in 1989 for its commercial value (Chang et al., 2004). Increasing market demand encourages the development of aquaculture in both Japan and China. The annual production of sea urchins from fisheries and aquaculture in China was 6791 t 2014 (Zhao, 2015). We previously studied the effects of one generation of inbreeding on fertilization, hatchability and larval development of S. intermedius, which lays a solid foundation for further studies on multiple generations of inbreeding (Zhou et al., 2013). Thus, S. intermedius is a good research model to study the effects of multiple generations of inbreeding. The purposes of the present study are to investigate 1) whether fertilization rate, hatching rate, larval growth and metamorphosis rate are significantly impacted by two generations
C. Zhao et al. / Aquaculture 452 (2016) 402–406
403
of inbreeding in S. intermedius; 2) whether a higher inbreeding coefficient shows significantly more negative impacts on all experimental traits of S. intermedius; and 3) whether maternal effects of inbreeding depression appear in all experimental traits of S. intermedius.
was used to evaluate hatching rates. Fertilization rate and hatching rate were calculated as follows:
2. Materials and methods
where F = fertilization rate, a = number of embryo in cleavage, and b = total number of embryo and eggs.
2.1. Experimental design Breeding and culture methods of full-sib families used for brother– sister mating in the present study were fully described in Chang et al. (2012) and Zhang et al. (2013). As S. intermedius is a non-native species in China, 43, 58 and 24 individuals were collected for breeding from three cultured populations in Rongcheng (122.59°E, 37.14°N), Lingshui (121.56°E, 38.88°N) and Lvshun (121.19°E, 38.72°N) in 2007, respectively (Chang et al., 2012). From these initial populations, 190 full-sib families were established at the end of October 2007 (Chang et al., 2012). From the sea urchins produced in October 2007, ninety seven full-sib families were established in May 2009 (Zhang et al., 2013). Inbreeding was strictly avoided in the breeding programs (Chang et al., 2012; Zhang et al., 2013). The establishing methods of these families had no influence on further inbreeding studies. Based on the families haphazardly collected from the ninety seven full-sib families produced in May 2009, 27 families of one generation inbred and crossbred S. intermedius were produced in 2010 at three inbreeding coefficients of 0.25, 0.125 and 0 (designated F, H and C, 9 families for each group), presuming no genetic exchange had occurred among the ancestors of the basic populations collected in 2007. Two F families and two C families were haphazardly collected from the one generation inbred and crossbred families for the establishment of the second generation inbred/crossbred families. Four second generation inbred families (designated FF) were produced by mating males and females from the same F families. In order to investigate maternal effects of inbreeding, individuals from F and C families were crossbred to produce second generation crossbred families with a genetic load (designated FC and CF, respectively). FC refers to F as the male parent and CF refers to C as the male parent. Four fully crossbred families (designated CC) were produced by mating males and females from different C families. The inbreeding coefficients of FF, FC, CF and CC were estimated as 0.375, 0.219, 0.219 and 0, according to the possibility of sharing a common ancestor.
a 100 b
Fð%Þ ¼
Hð%Þ ¼
c 100 d
where H = hatching rate, a = number of prism larvae, and b = total number of larvae and embryo. 2.4. Measurements of larval size Four-arm, six-arm and eight-arm larvae were sampled in all experimental groups at 3 days, 14 days and 24 days after artificial fertilization, respectively. All samples were placed into tubes for 10 min to allow larvae to settle on the bottom. Larvae were then transferred to clear plastic tubes and placed on slides for further measurements under a microscope (Fig. 1). The larvae were photographed with a known size scale. One hundred larvae were examined each time for each individual sea urchin (the only exception was that two hundred larvae were used at the four-arm larval stage). The stomach is the dominant organ of sea urchins for storing nutrients. Thus, stomach size directly determines the ability of larvae to store nutrients. Larval length, larval width, stomach length and stomach width were calculated as follows: Y¼X
m n
where Y = the actual size of the target trait, X = the measured size of the target trait in the photograph, m = the actual length of the scale, and n = the measured size of the scale in the photograph. 2.5. Metamorphosis Two hundred larvae were haphazardly collected and placed into a 2 L beaker 28 days after artificial fertilization to measure metamorphosis in each group. Two polyethylene plates with benthic diatoms (8 cm × 8 cm and 10 cm × 14 cm) were placed into each beaker to
2.2. Culture of inbred and crossbred sea urchins Spawning of S. intermedius was induced by injecting 1.5 mL KCl (0.5 mol.L−1). The gametes were collected using individual containers to avoid potential mixture. All fertilized eggs were placed into labeled 70 L tanks for hatchability studies after careful washing to remove excess sperm. All tanks were randomly distributed in the laboratory to balance non-experimental environmental factors. The conditions of hatchability were 18–19 °C seawater temperature, 30 ‰ salinity, 0–300 lx natural light density and no aeration. The hatching density was adjusted to 0.5–1.0 ind ∙ mL− 1 in all tanks 30 days after fertilization. The larvae were equally fed the microalga Chaetoceros gracilis in all tanks four times a day in weakly aerated seawater at concentrations from 2 × 10 4 to 5 × 10 4 cells ∙ mL − 1 . Sea urchins in all experimental groups were cultured using the same methods and in similar conditions. 2.3. Fertilization and hatchability Fertilization was observed using a microscope in 3 h after artificial fertilization. An embryo with cleaving cells indicates development of fertilized eggs. Hatchability of S. intermedius was observed using a microscope 30 h after artificial fertilization. The number of prism larvae
Fig. 1. Measurements of larval length (ll), larval width (lw), stomach length (sl), and stomach width (sw) of Strongylocentrotus intermedius.
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encourage settlement of larvae. The number of settled S. intermedius was recorded 20 days after placing the polyethylene plates. 2.6. Statistical analysis The data were first tested for normal distribution and homogeneity of variance before statistical analysis. One-way ANOVA was performed to detect differences in fertilization rate, hatching rate, larval development and metamorphosis rate. Duncan's multiple comparisons were then performed when significant differences were found with the ANOVA analysis. All analyses were done with SPSS 16.0 statistical software. A probability level of P b 0.05 was considered statistically significant. 3. Results 3.1. Fertilization and hatchability Fertilization rates of S. intermedius were not significantly different among all the experimental groups (P N 0.05), ranging from 94.4% to 95.4%. Hatching rates showed no significant difference among all the experimental groups (P N 0.05), although it was slightly less in the FF group. 3.2. Larval growth The FF group showed an obvious high rate of abnormal four-arm larvae, although the P value was not significant (P N 0.05). Larval length of inbred sea urchins was significantly less than that of crossbred individuals in four-arm larva, six-arm larva and eight-arm larva stages, respectively (P b 0.05, Fig. 2). The length of eight-arm larvae was significantly shorter in the FF group than in the FC and CF groups (P b 0.05). Larval length in the FC group was significant shorter than in the CF group in the six-arm larva stage (P b 0.05) and significantly longer than in the eight-arm larva stage (P b 0.05). The width of six-arm and eight-arm larvae was significantly greater in the CC group than in the inbred groups (P b 0.05, Fig. 2). The width of four-arm larvae, however, was significantly less in the CC group than in the CF and FF groups (P b 0.05). The width of eight-arm larvae was not significantly different among the experimental inbreeding groups (P N 0.05). The larval width of the CF group was significantly greater
than that of the FC group at the four-arm larva stage (P b 0.05), but showed no significant difference at the six-arm larva and eight-arm larva stages (P N 0.05). Stomach length in the CC group was significantly greatest at the four-arm, six-arm and eight-arm larva stages, respectively (P b 0.05, Fig. 3). The FF group had a significantly less stomach length at the sixarm larva stage than in the FC and CF groups (P b 0.05) and a longer stomach length at the eight-arm larva stage (P b 0.05). The CF group showed significantly longer stomach length than the FC group at the four-arm larva stage (P b 0.05). The CC group showed significantly wider stomach only in the four-arm larva stage (P b 0.05, Fig. 3). The stomach width of eight-arm larvae in the FF group was significantly greater than in the other groups (P b 0.05). There was no significant difference of stomach width between the CF and FC groups at the four-arm larva stage (P N 0.05). However, the stomach width in the FC groups was significantly larger than in the CF group at both the six-arm and eight-arm stages (P b 0.05). 3.3. Metamorphosis Significantly more larvae in the CC group settled in 20 days after placing polyethylene plates into the beakers than in the inbred groups (P b 0.05, Fig. 4). However, there was no significant difference of settlement among the CF, FC and FF groups (P N 0.05). 4. Discussion This study increases evidence of the effect of multiple generations of inbreeding depression in marine invertebrates (Zheng et al., 2012; Luo et al., 2014), although depression after one generation of inbreeding has been well documented in larvae of scallops (Ibarra et al., 1995; Zheng et al., 2012), Pacific oysters (Taris et al., 2007), and sea urchins (Anderson and Hedgecock, 2010, Zhou et al., 2013). In the present study, second generation of inbreeding did not significantly affect fertilization and hatchability of S. intermedius, which agrees to our previous study on effects of one generation of inbreeding on fertilization and hatchability of S. intermedius (Zhou et al., 2013). This result indicates of the robustness of fertilization and hatchability in S. intermedius with an inbreeding coefficient of 0.375, but does not ensure that in higher inbreeding coefficient in other species (Zheng et al., 2012).
Fig. 2. Larval length and width of Strongylocentrotus intermedius in the four-arm, six-arm and eight-arm stages in different experimental groups (N = 200 for four-arm larvae and N = 100 for the others, mean ± SD). F refers to one generation full-sibling inbred families, C refers to the families without inbreeding, FF refers to second generation fully inbred families by mating males and females from the same F families, FC refers to F as the male parent, while CF refers to C as the male parent, CC refers to the families produced by mating males and females from different C families.
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405
Fig. 3. Stomach length and width of Strongylocentrotus intermedius in the four-arm, six-arm and eight-arm stages in different experimental groups (N = 200 for four-arm larvae and N = 100 for the others, mean ± SD). See Fig. 2 for abbreviation for groups.
In the present study, we found significant depression of larval size (especially in larval length) and metamorphosis in second generation of inbred S. intermedius. This result agrees with the finding that two successive generations of self inbreeding significantly reduced larval survival and juvenile growth in the scallop Argopecten irradians irradians (Zheng et al., 2012). Compared to our previous finding that one generation of inbreeding did not significantly affect larval growth of S. intermedius (Zhou et al., 2013), the present result clearly indicates of a larger load of recessive deleterious mutation in the second generation of inbred S. intermedius and highlights the potential risks of multiple generations of inbreeding on aquaculture of sea urchins. This agrees with a behavioral study with the guppy Poecilia reticulata (Oosterhout et al., 2003), in which male following behavior was not affected by one generation of inbreeding but was significantly depressed in the second and third generation of inbreeding. Together, the present results indicate that inbreeding depression of sea urchins is highly trait dependent and that larval growth and metamorphosis are more susceptible to inbreeding than fertilization and hatchability. The larva is one of the most fragile stages in the life history of sea urchins (Metaxas, 2013), which would greatly affect metamorphosis and consequently the whole breeding success in aquaculture (Chang
Fig. 4. Number of settled sea urchins of Strongylocentrotus intermedius in different experimental groups 20 days after placing polyethylene plates with benthic diatoms into the beakers (N = 3, mean ± SD). See Fig. 2 for abbreviation for groups.
et al., 2004). In the present study, we found the larval length was significantly less in second generation inbred sea urchins than in the crossbred individuals. This result is consistent with the finding of Anderson and Hedgecock (2010) that one generation inbred larvae were significantly smaller in S. purpuratus. Larval length, especially post-oral arms, is important for feeding and consequent fitness in sea urchins (Adams et al., 2011). Thus, we hypothesize that inbreeding depression in larval length probably affects feeding ability and consequently metamorphosis in sea urchins. Metamorphosis is the life history stage in which larval features are lost and the larva is irreversibly transformed into a juvenile in invertebrates (Hadfield, 2000). This stage is fundamentally important for the successful recruitment of sea urchins (Swanson et al., 2012). Consequently it would determine breeding success in aquaculture. It is well known that metamorphosis of sea urchins can be induced or affected by various physical and chemical factors (Swanson et al., 2012; Metaxas, 2013). However, little information is available on the genetic basis of metamorphosis in sea urchins. For the first time, we report that second generation of full-sib mating significantly decreased metamorphosis of S. intermedius. This clearly indicates that second generation of full-sib mating S. intermedius carried a large load of recessive detrimental mutation in genes, which significantly impacted fitness related traits, including larval growth and consequent metamorphosis. A negative linear relationship has been generally accepted for fitness traits and the inbreeding coefficient in both plants and animals (Lynch and Walsh, 1998). In the present study, we found that this phenomenon appeared in some traits (for example, larval length), but not all. This result is consistent with a recent report that different levels of multiple generation inbreeding significantly affected body weight, but did not significantly impact the WSSV resistance of the Chinese shrimp, Fenneropenaeus chinensis (Luo et al., 2014). In addition, larval growth of S. intermedius at the three inbreeding levels differed greatly among different developmental stages. A reasonable explanation is that larval development of sea urchins is a well-ordered process with highly spatial and temporal expression. Thus, different levels of loci homozygosis probably have different effects on sea urchin larvae at different developmental stages. A number of studies have verified a significant maternal inbreeding effect on progeny performance (Lynch and Walsh, 1998). In general, we found that larval length of S. intermedius in the CF group was significantly larger than in the FC group in the six-arm stage, while significantly shorter in the eight-arm stages. Deng et al. (2005) reported a similar maternal effect of inbreeding depression on metamorphosis and larval
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size in the Pacific abalone Haliotis discus hannai. This clearly indicates that maternal effects of inbreeding depression probably happen in the late developmental larval stage and metamorphosis of sea urchins. Our result increases the evidence of maternal effect of inbreeding depression in marine invertebrates. However, it remains unknown of whether inbreeding depression in maternal traits or offspring traits contributed to the maternal inbreeding effects, because we did not measure the maternal traits of parent sea urchins (for example, egg size and egg nutrient components). Further studies should be carried out to reveal the contributions of the maternal inbreeding effects of sea urchins. The present study provides convincing evidence of significant inbreeding depression in second generation inbred S. intermedius. Notably, inbreeding depression can be further aggravated by the interaction with environmental factors (Franke and Fischer, 2013). Thus, we suggest that sea urchin aquaculture should involve a large effective population size to avoid inbreeding, especially multi-generation inbreeding, to ensure the whole successful hatchery in aquaculture. Further, we found that the impact of inbreeding is variable among traits. This is well consistent with the finding of Nakadate et al. (2003) that inbreeding significantly impacted some of the experimental traits of the guppy Poecilia reticulate, but not all. In conclusion, two generations of inbreeding significantly affected larval growth and metamorphosis, but not fertilization and hatchability in S. intermedius. Higher inbreeding coefficient showed significantly more negative impacts on some traits of S. intermedius larvae (for example, larval length), but not all. Maternal effects of inbreeding depression on larval length probably appeared in the eight-arm stage and metamorphosis of S. intermedius. The present study provides convincing evidence of inbreeding depression of larval growth and metamorphosis in S. intermedius and highlights the importance of avoiding multiple generation of inbreeding in sea urchin aquaculture. Acknowledgments This work was supported by the Chinese National 863 Project (2012AA10A412), a research project granted by Liaoning Department of Education (L2015087) and a public welfare project granted by the State Oceanic Administration of China (201305027). We are grateful to Prof. John Lawrence for academic and editorial suggestions and to Jinyong Zhang for his assistance. All authors do not have any conflict of interest. References Adams, D.K., Sewell, M.A., Angerer, R.C., Angerer, L.M., 2011. Rapid adaptation to food availability by a dopamine-mediated morphogenetic response. Nat. Commun. 2, 592.
Agatsuma, Y., 2013. Strongylocentrotus intermedius. In: Lawrence, J.M. (Ed.), Edible Sea Urchins: Biology and Ecology. Elsevier, Amsterdam, pp. 437–447. Anderson, D., Hedgecock, D., 2010. Inbreeding depression and growth heterosis in larvae of the purple sea urchin Stronglyocentrotus purpuratus (Stimpson). J. Exp. Mar. Biol. Ecol. 384, 68–75. Chang, Y., Ding, J., Song, J., Yang, W., 2004. Biology and Aquaculture of Sea Cucumbers and Sea Urchins. Ocean Press, Beijing, China (in Chinese). Chang, Y., Zhang, W., Zhao, C., Song, J., 2012. Estimates of heritabilities and genetic correlations for growth and gonad traits in the sea urchin Strongylocentrotus intermedius. Aquac. Res. 43 (2), 271–280. Deng, Y., Liu, X., Zhang, G., Guo, X., 2005. Inbreeding depression and maternal effects on early performance of Pacific abalone. N. Am. J. Aquac. 67, 231–236. Franke, K., Fischer, K., 2013. Effects of inbreeding and temperature stress on life history and immune function in a butterfly. J. Evol. Biol. 26 (3), 517–528. Hadfield, M.G., 2000. Why and how marine-invertebrate larvae metamorphose so fast. Semin. Cell Dev. Biol. 11, 437–443. Ibarra, A.M., Cruz, P., Romero, B.A., 1995. Effects of inbreeding on growth and survival of self-fertilized Catarina scallop larvae, Argopecten circularis. Aquaculture 134, 37–47. Luo, K., Kong, J., Luan, S., Meng, X., Zhang, T., Wang, Q., 2014. Effect of inbreeding on survival, WSSV tolerance and growth at the postlarval stage of experimental full-sibling inbred populations of the Chinese shrimp Fenneropenaeus chinensis. Aquaculture 420–421, 32–37. Lynch, M., Walsh, B., 1998. Genetics and Analysis of Quantitative Traits. Sinauer Associates, Inc., Publishers, USA, pp. 251–291. Ma, D.Y., Hu, H.L., Kong, J., 2005. Inbreeding and its impact on aquaculture. J. Fish. China 29 (6), 849–856 (in Chinese). Metaxas, A., 2013. Larval ecology in echinoids. In: Lawrence, J.M. (Ed.), Edible Sea Urchins: Biology and Ecology. Elsevier, Amsterdam, pp. 69–81. Moss, D.R., Arce, S.M., Otoshi, C.A., Moss, S.M., 2008. Inbreeding effects on hatchery and growout performance of Pacific white shrimp, Penaeus (Litopenaeus) vannamei. J. World Aquacult. Soc. 39 (4), 467–476. Nakadate, M., Shikano, T., Taniguchi, N., 2003. Inbreeding depression and heterosis in various quantitative traits of the guppy, Poecilia reticulate. Aquaculture 220, 219–226. Oosterhout, C.V., Trigg, R.E., Carvalho, G.R., Magurran, A.E., Hauser, L., Shaw, P.W., 2003. Inbreeding depression and genetic load of sexually selected traits: how the guppy lost its spots. J. Evol. Biol. 16, 273–281. Rhode, C., Maduna, S.N., Roodt-Wilding, R., Bester-van der Merwe, A.E., 2014. Comparison of population genetic estimates amongst wild, F1 and F2 cultured abalone (Haliotis midae). Anim. Genet. 45 (3), 456–459. Swanson, R.L., Byrne, M., Prowse, T.A.A., Mos, B., Dworjanyn, S.A., Steinberg, P.D., 2012. Dissolved histamine: a potential habitat marker promoting settlement and metamorphosis in sea urchin larvae. Mar. Biol. 159, 915–925. Taris, N., Batista, F.M., Boudry, P., 2007. Evidence of response to unintentional selection for faster development and inbreeding depression in Crassostrea gigas larvae. Aquaculture 272 (1), 69–79. Zhang, W., Zhao, C., Chen, M., Chang, Y., Song, J., Luo, S., 2013. Family growth response to different laboratory culture environments shows genotype by environment interaction in the sea urchin Strongylocentrotus intermedius. Aquac. Res. 44 (11), 1706–1714. Zhao, X. (Ed.), 2015. China Fishery Statistical Yearbook 2014. China Agriculture Press, Beijing (in Chinese). Zheng, H.P., Li, L., Zhang, G.F., 2012. Inbreeding depression for fitness-related traits and purging the genetic load in the hermaphroditic bay scallop Argopecten irradians irradians (Mollusca: Bivalvia). Aquaculture 366–367, 27–33. Zhou, H., Luo, S., Zhang, W., Gao, L., Zhao, C., Chang, Y., 2013. Effect of inbreeding on fertility rate, hatchability and larva development of sea urchin Strongylocentrotus intermedius. J. Dalian Ocean Univ. 28 (5), 445–449 (in Chinese).