Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay

Abstract

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm ؊3 ) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species. 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 (1-10). Resting cysts, therefore, play an important role in the ecology of harmful algal blooms (HABs) caused by dinoflagellates (8, 11) and have been considered a fundamental attribute of dinoflagellate life cycles (12). About 200 marine and freshwater dinoflagellates are known to produce resting cysts, a small number relative to the ϳ2,300 extant dinoflagellate species (5, 11, 13). More than 20 of these cyst-producing dinoflagellates are known to cause harmful algal blooms (HABs) (5), including Cochlodinium polykrikoides Margalef (10).Cochlodinium polykrikoides Margalef is an unarmored dinoflagellate that has caused fish-killing HABs in locations across much of Asia and North America (14-17). The initiation and development of C. polykrikoides blooms have been shown to be related to multiple factors, including stimulation by nutrients such as nitrogen and vitamins (18, 19), mixotrophy (20), the production of extracellular toxins lethal to grazers (21-23), bacterial mutualism (24), and allelopathic effects on competing phy...

Mapping distribution of cysts of recent dinoflagellate and Cochlodinium polykrikoides using next-generation sequencing and morphological approaches in South Sea, Korea

“…According to the hypothesis of Hattenrath-Lehmann et al . 28 , vegetative cells of C. polykrikoides were present during 2015, as evident from observation of the sediments in Tongyeong area in 2014. However, vegetative cells of C. polykrikoides were not detected during the summers of 2015 and 2017 (Supplementary Fig.…”

“…Hattenrath-Lehmann et al . 28 reported that low cyst densities after high-density blooms may be affected by several physical, chemical, and biological factors, which suggest that a small portion of the vegetative population is involved in sexual reproduction 22 . Moreover, the other factors affecting density of cysts include grazing of cysts in sediments 29 , bacterial degradation of cysts 8 , and/or the dispersal of cysts/vegetative cells out of the bloom region via currents 30 , followed by deposition of these cysts elsewhere 31 .…”

“…Hattenrath-Lehmann et al . 28 suggested that the modest densities of cysts are important as ‘seed banks for initiating blooms’. However, the present results indicate some distinct patterns of C. polykrikoides cysts, as reported by Hattenrath-Lehmann et al .…”

Evidence for Production of Sexual Resting Cysts by the Toxic Dinoflagellate Karenia mikimotoi in Clonal Cultures and Marine Sediments

“…Because of the occasional non‐specific fluorescence in non‐target cells or particles (e.g., pollen grains) in FISH samples as reported in previous work (Hattenrath‐Lehmann et al. ), the fluorescing cells observed in the samples that were subject to FISH using both Cy3‐ and FITC‐labeled probes were carefully observed under bright field microscopy for the cyst morphology (i.e. to compare with that observed in laboratory cultures) and were then selectively micropipetted for single‐cell PCR and sequencing (see below).…”

Heterogeneous distribution in sediments and dispersal in waters of Alexandrium minutum in a semi-enclosed coastal ecosystem

“…Real-time PCR amplification to quantify DNA genes from sediments has been shown to be a good proxy for cyst abundances, including some Alexandrium species (Kamikawa et al, 2007, Erdner et al, 2010, Park and Park, 2010. Lastly, cyst species have been mapped using the fluorescence in situ hybridization (FISH) technique (Hattenrath-Lehmann et al, 2016). Genetic techniques can therefore be used to provide reliable information on accumulation spots of cysts and to infer ecological patterns.…”