The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts

Abstract

While harmful algal blooms (HABs) caused by the toxic dinoflagellate Cochlodinium polykrikoides have been known to science for more than a century, the past two decades have witnessed an extraordinary expansion of these events across Asia, North America, and even Europe. Although the production of resting cysts and subsequent transport via ships’ ballast water or/and the transfer of shellfish stocks could facilitate this expansion, confirmative evidence for cyst production by C. polykrikoides is not available. Here, we provide visual confirmation of the production of resting cysts by C. polykrikoides in laboratory cultures isolated from North America. Evidence includes sexually mating cell pairs, planozygotes with two longitudinal flagella, formation of both pellicular (temporary) cysts and resting cysts, and a time series of the cyst germination process. Resting cyst germination occurred up to 1 month after cyst formation and 2–40% of resting cysts were successfully germinated in cultures maintained at 18–21 °C. Pellicular cysts with hyaline membranes were generally larger than resting cysts, displayed discernable cingulum and/or sulcus, and reverted to vegetative cells within 24 h to ∼1 week of formation. A putative armored stage of C. polykrikoides was not observed during any life cycle stage in this study. This definitive evidence of resting cyst production by C. polykrikoides provides a mechanism to account for the recurrence of annual blooms in given locales as well as the global expansion of C. polykrikoides blooms during the past two decades.

Introduction

Resting cysts of dinoflagellates can be associated with genetic recombination, maintenance of blooms, termination of blooms, recurrence of annual blooms, resistance against unfavorable environmental conditions, protection from viruses, grazers or parasite attacks, and geographical expansion of populations (Anderson and Wall, 1978, Anderson and Morel, 1979, Anderson, 1989, Hallegraeff and Bolch, 1991, Nehring, 1993, Matsuoka and Fukuyo, 2002, Zingone et al., 2002, Figueroa et al., 2010, Anglès et al., 2012). Resting cysts, therefore, play an important role in the ecology of harmful algal blooms (HABs) of dinoflagellates (Matsuoka and Fukuyo, 2003, Figueroa et al., 2010) and have been considered a fundamental attribute of dinoflagellate life cycles (Elbrăchter, 2003). About 100 marine and freshwater dinoflagellates have been shown to produce resting cysts, a small number relative to ∼2000 extant species of dinoflagellates (Nehring, 1993, Matsuoka and Fukuyo, 2003). More than 20 of these cyst-producing dinoflagellates are known to cause HABs (Nehring, 1993).

Cochlodinium polykrikoides Margalef is an unarmored dinoflagellate that has caused catastrophic HABs in the Caribbean Sea, eastern and western Pacific Ocean, the western Atlantic Ocean, Indian Ocean, Mediterranean Sea, and the Arabian Gulf (Margalef, 1961, Matsuoka et al., 2008, Richlen et al., 2010, Kudela and Gobler, 2012). The initiation and development of C. polykrikoides blooms have been shown to be related to diurnal migration behavior (Park et al., 2001, Kim et al., 2010), mixotrophy (Jeong et al., 2004), light quantity and quality (Oh et al., 2006), the production of toxins lethal to grazers (Jiang et al., 2009, Tang and Gobler, 2009a, Tang and Gobler, 2009b), allelopathic effects on competing phytoplankton (Tang and Gobler, 2010), transport by large-scale currents (Onitsuka et al., 2010), and stimulation by nutrients such as nitrogen and vitamins (Tang et al., 2010, Gobler et al., 2012). None of these factors, however, can account for the rapid and large geographic expansion of the species across Asia, North America, and even Europe during the past two decades. While the production of resting cysts could contribute toward such an expansion, whether C. polykrikoides produces resting cysts is still an open question (Fukuyo, 1982, Matsuoka, 1985, Matsuoka, 1987, Kim et al., 2002, Kim et al., 2007, Richlen et al., 2010).

Prior studies have reported the identification of resting cysts of C. polykrikoides or Cochlodinium sp. from sediments (Rosales-Loessener et al., 1996, Matsuoka and Fukuyo, 2000, Matsuoka and Fukuyo, 2002, Orlova et al., 2004, Seaborn and Marshall, 2008, Rubino et al., 2010, Mohamed and Al-Shehri, 2011), but the identity of the observed cysts was not fully confirmed in these studies. For example, Orlova et al. (2004) identified cysts in sediments from the East coast of Russia as C. cf. polykrikoides based on micrographs in Fukuyo (1982, as Cochlodinium sp. 1) and Matsuoka and Fukuyo (2000; as Cochlodinium sp. 1, or C. cf. polykrikoides). Seaborn and Marshall (2008) identified C. polykrikoides cysts from sediments in a US east coast estuary but micrographs were not provided, germination experiments were not performed, and a detailed description and identification of cysts was not included. Rubino et al. (2010) and Mohamed and Al-Shehri (2011) reported the identification and germination of C. polykrikoides cysts from the Red Sea and the Mediterranean, respectively, but, again, the cysts and germlings were not identified unambiguously. Park and Park (2010) detected C. polykrikoides by PCR in sediment samples, suggesting the presence of C. polykrikoides in sediments, but no morphological information was provided and hence the detection of vegetative cells or DNA residues of vegetative cells could not be excluded.

C.-H. Kim et al. (2002) reported on the production of hyaline cysts from cultivated C. polykrikoides bloom water while C.-J. Kim et al. (2007) and Tomas and Smayda (2008) reported similar hyaline cysts in laboratory cultures and field samples, respectively. These studies all described temporary cysts (or more appropriately called ‘pellicles’, see Bravo et al., 2010) with hyaline membranes formed by modification of the vegetative cells without sexual mating (Kim et al., 2002, Kim et al., 2007, Tomas and Smayda, 2008). Interestingly, C.-J. Kim et al. (2007) described a life cycle of C. polykrikoides comprising two different morphological stages—an armored and an unarmored vegetative swimming stage, with the latter forming long chains of cells that are commonly observed in this taxon and a hyaline cyst as in C.-H. Kim et al. (2002). Still, C.-J. Kim et al. (2007), by their own admission, did not convincingly document the production of resting cysts by C. polykrikoides.

Here, we provide clear visual evidence of the production of resting cysts by C. polykrikoides from laboratory cultures isolated from the estuaries of Long Island, NY, USA, Cotuit Bay, MA, USA, and Bahia de La Paz, Mexico. Evidence presented includes sexual mating cell pairs, planozygotes with two longitudinal flagella, and time series micrographs of the cyst formation and cyst germination processes. We believe this information provides a mechanism that may account for the recurrence of annual blooms in some locations and the global expansion of C. polykrikoides blooms during the past two decades.