Identification and expression analysis of meiosis-related genes in the harmful alga Heterosigma akashiwo (Raphidophyceae)

Highlights

• Seven meiosis-related genes were identified from Heterosigma akashiwo.

• H. akashiwo has the genetic potential for sexual reproduction.

• All detected meiosis-related genes may regulate cell division cycle in H. akashiwo.

Abstract

Sexual reproduction (SR), hallmarked by meiosis, is widespread in eukaryotes. In phytoplankton, SR has been observed in many lineages, but molecular information on SR or meiosis of harmful algal bloom (HAB) species is scarce. The raphidophyte Heterosigma akashiwo is a globally distributed and devastating HAB species, but molecular evidence of its SR or meiosis is lacking. Here, to address the gap of knowledge, the presence of meiotic genes in H. akashiwo were examined. Interestingly, seven meiosis-specific or related genes (SPO11, MND, RAD21, RAD51, MSH2, MSH6 and MEI2) were identified from H. akashiwo transcriptomes. Furthermore, expression patterns of these genes except MSH6 (excluded due to primer failure) were investigated using quantitative reverse-transcription PCR. Results showed that the examined genes exhibited similar diel rhythms, typically, highest in early dark period and then gradually decreasing until mid-day. In addition, the expression of these six genes was not higher in the stationary growth stage than in the exponential stage, as would be expected if meiosis was to form cysts, and their elevated expression in response to colchicine treatment (arresting cells in the G2/M transition) indicated a potential role of these genes in cell division and population growth in H. akashiwo. Consistent with this, we also found a morning to afternoon shift in the expression of these genes during the bloom of H. akashiwo. This study documents a part of the typical SR gene repertoire and its potential role in regulating cell division in H. akashiwo, offering candidates for population growth markers for bloom monitoring although its linkage to meiosis and SR remain to be studied further in the future.

Introduction

In the marine ecosystem, phytoplankton not only serve as the base of the food chain, but also play an important role in biogeochemical cycle (Falkowski et al., 1998). However, some of species form exceptional blooms, known as harmful algal blooms (HABs), which cause significant damages to local economies, public health and marine ecosystems around the world (Anderson et al., 2012; Grattan et al., 2016; Wells et al., 2015). To form a bloom, the HABs species must increase their population faster than the other species among phytoplankton community (Jeong et al., 2015). As typical in phytoplankton, HABs species usually reproduce by asexual fission, or vegetative reproduction (Steidinger and Garccés, 2006). Sexual reproduction (SR) is known to occur only under stress such as nutrient limitation, or unusual cellular status in which cell size reduces to below the threshold in diatoms, and to result in the formation of resting cysts or spores (Grigorszky et al., 2006).

SR is widespread in eukaryotes and evolved very early in eukaryotic evolution (Speijer et al., 2015; Vesteg and Krajčovič, 2011). Sex is an important vehicle of chromosome recombination and source of genetic diversity, and hence is essential for evolutionary success. In phytoplankton, SR has been observed in diatoms, chlorophytes, rhodophytes, and many other lineages, some of which are not known to form resting cysts or equivalent forms of dormant cells (Deng et al., 2017; Grimsley et al., 2009; Vesteg and Krajčovič, 2011). Indeed, some earlier and recent studies have suggested an alternative role of SR not necessarily forming a cyst, but instead, as a way of vegetative cell division (Figueroa and Bravo, 2005). For instance, Noctiluca scintillans often forms a massive bloom in a very short time period, and is able to produce 256–1024 gametes within 24 h (Fukuda and Endoh, 2006). Furthermore, SR in Alexandrium minutum was found to be capable of producing vegetative cells. In this case, a zygote produces four daughter cells through a round of meiosis and a round of mitosis, and resting cyst formation is the fate of only a small fraction of the diploid mobile zygotes formed (Figueroa et al., 2015).

The raphidophyceae Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara is one of flagellates species that cause HABs, and distribute widely in the world’s oceans (Honjo, 1993; Ki and Han, 2007). Blooms of H. akashiwo are notorious for their dramatic destruction of fish, especially in aquaculture facilities (Rensel et al., 2010). In order to survive under dark and low temperature conditions, H. akashiwo lives in sediments as three different cell types, cysts, resting cells or benthic stage cells (Kim et al., 2015; Shikata et al., 2007). Among these three cell types, resting cells have been induced in the laboratory (Han et al., 2002). In the field, only cysts have been identified from sediment (Kim et al., 2015). In addition, many results showed that light, temperature and dissolved oxygen concentration could affect the germination of H. akashiwo cysts (Portune et al., 2009; Shikata et al., 2007). Despite the many physiological studies, the molecular mechanism of H. akashiwo reproduction is still unknown and underexplored. Especially, it is unclear whether H. akashiwo has the molecular machinery needed for SR (meiosis), and whether SR in this species occurs during cyst germination or active cell proliferation. Evaluating genetic consequence of sex and meiotic recombination is a common molecular method to detect SR (Normark et al., 2003; Schurko and Logsdon, 2008). In addition, with the availability of phytoplankton genome and transcriptome data, identification of genes involved in meiosis has become more effective than before. Currently, there are 14 transcriptomes of H. akashiwo generated by Marine Microbial Eukaryotic Transcriptome Sequencing Program (MMETSP) (Keeling et al., 2014) and a set of bloom metatranscriptomes of this species (Ji et al., 2018), allowing us to uncover meiosis-related genes in this species. Moreover, the “meiosis detection toolkit”, a set of meiotic genes that represent the best markers for the presence of meiosis exists for referencing; the presence of all, or most, of these genes in a genome indicate that the ability of sexual reproduction has been maintained (Schurko and Logsdon, 2008).

In this study, several meiosis-related genes were identified from H. akashiwo. The expression patterns of six of these genes under different growth conditions were then investigated. The results showed that all of the detected genes displayed a clear diel rhythm under diel cycle, and their high expression levels might be related to certain cell cycle stages. The possible roles played by these genes in H. akashiwo were also discussed.