Association of clonal diversity and population growth in the small-type rotifer Brachionus koreanus during hatchery mass production Eitaro Sawayama a , Wilma Moka b , Daiki Noguchi c , Motohiro Takagi d, ⁎ a R&D Division, Marua Suisan Co., Ltd., 4472 Iwagi, Kamijima, Ehime 794-2410, Japan b The United Graduate School of Agricultural Sciences, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-5866, Japan c Nippon Total Science, Inc., 456-2 Minomi cho, Fukuyama, Hiroshima 720-0832, Japan d South Ehime Fisheries Research Center, Tarumi Branch, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan abstract article info Article history: Received 10 February 2016 Received in revised form 1 September 2016 Accepted 11 September 2016 Available online 12 September 2016 In this study, we identify clones of S-type rotifers Brachionus koreanus based on haplotypes of mitochondrial gene cytochrome c oxidase subunit I (mtCOI) and microsatellite DNA genotypes. We then study how clonal diversity affects the population growth of rotifers in a mass culture at a hatchery using a lot of B. koreanus rotifers for anal- ysis. Microsatellite DNA markers were also developed to identify clones of B. koreanus. Clones of B. koreanus were identified based on haplotypes of mtCOI, microsatellite genotypes, as well as a combination of these haplotypes and genotypes. Three haplotypes of mtCOI, three clones based on microsatellites, and six clones based on a com- bination of mtCOI haplotypes and microsatellite genotypes were identified. The population growth rate of mass- cultured rotifers was monitored for a month, and the correlation between population growth rate and these clon- al diversities was analyzed. Observed was a significant positive correlation between the population growth rate and haplotype diversity (r = 0.695, P = 0.004), however no correlations were found between the population growth rate and clonal diversity based on either microsatellite genotypes (r = 0.320, P = 0.245) or a combination of mtCOI haplotype and microsatellite genotypes (r = 0.435, P = 0.105). Some clones shared mtCOI haplotypes and microsatellite genotypes suggesting sexual reproduction occurred in the hatchery stock of B. koreanus. Statement of relevance: This study showed the correlation between population growth rate and clonal diversities based on mtCOI haplotypes and microsatellite DNA genotypes in Brachionus koreanus. There was a significant correlation between the population growth rate and haplotype diversity and suggested genetic factor is one of the possible causes affecting population growth rate. Our results will be useful in mass-production and mainte- nance of Brachionus koreanus at hatcheries. © 2016 Elsevier B.V. All rights reserved. Keywords: Rotifer Brachionus koreanus Clone Haplotype Microsatellite Multi-locus genotype Multi-locus lineage 1. Introduction Small-morphotype (S-type) rotifers belonging to the Brachionus species have been used as larval food in the larviculture of many marine fish species (Hagiwara et al., 2001). Rotifers are used as an initial food in larval rearing for 10–30 days after mouth opening (Lubzens et al., 1989), before feeding with Artemia and formula diets. S-type rotifers are com- monly used in hatcheries worldwide because this morphotype of roti- fers is easily adopted and grown in hatchery environments (Papakostas et al., 2006). Several studies based on DNA barcoding anal- ysis revealed that hatchery-used S-type rotifers in Europe and Japan be- long to B. koreanus, formally called as B. sp. “Cayman” (Papakostas et al., 2006; Baer et al., 2008; Hwang et al., 2013; Moka et al., 2016; Mills et al., 2016). Rotifers can suddenly die in very large numbers with a subsequent decrease in culture density, usually called a “Crash”. Such Crashes have been one major obstacle to rotifer production since rotifer culture started. Once rotifer production has Crashed, sufficient numbers of roti- fers for larval rearing are not produced in a hatchery, therefore, seed production is not able to continue. Crashes are thought to be caused by environmental factors such as: food quality and the absence of vita- min B12 (Scott, 1981; Hirayama, 1987), or competition with other or- ganisms such as viruses, bacteria, and ciliates (Hagiwara et al., 1995; De Araujo et al., 2000). However, based on data, a few genetic factors has been considered a cause of such Crashes (Papakostas et al., 2007). We previously identified three haplotypes of mitochondrial gene cyto- chrome c oxidase subunit I (mtCOI) in the S-type rotifer B. koreanus of hatchery strains in Japan, and usually several haplotypes were mixed in a mass culture (Moka et al., 2016). This finding suggests that the ge- netic diversity of S-type rotifers changes during mass culturing and it may be one cause of the Crashes. Billions of rotifers are cultured in mass-production tanks every day. Although sexual recombination may occur in continuous and batch culture systems (Declerck et al., 2015), standing genetic variation may be a key factor affecting population growth in mass culture conditions. Therefore, genetic diversity using Aquaculture 465 (2016) 296–302 ⁎ Corresponding author. E-mail address: takagi@agr.ehime-u.ac.jp (M. Takagi). http://dx.doi.org/10.1016/j.aquaculture.2016.09.020 0044-8486/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aquaculture