Evidence for resting cyst production in the cosmopolitan toxic dinoflagellate Karlodinium veneficum and the cyst distribution in the China seas

• Published in: Harmful algae 2020-03, Vol.93, p.101788, Article 101788
• Main Authors: Liu, Yuyang, Hu, Zhangxi, Deng, Yunyan, Tang, Ying Zhong
• Format: Article
• Language: English
• Subjects: China; Cyst mapping; Dinoflagellida - genetics; Distribution; Fluorescence in situ hybridization (FISH); In Situ Hybridization, Fluorescence; Karlodinium veneficum; Oceans and Seas; Resting cyst
• ISSN: 1568-9883; 1878-1470
• DOI: 10.1016/j.hal.2020.101788

•Karlodinium veneficum produces sexual resting cysts homothallically in clonal cultures.•Presence of resting cysts in sediments was confirmed via LM-FISH and single-cell PCR sequencing.•Karlodinium veneficum cysts widely distribute in Chinese coasts as mapped using real-time PCR and FISH. The naked dinoflagellate Karlodinium veneficum is a cosmopolitan and toxic species that frequently forms harmful algal blooms (HABs) in coastal waters. This species has been intensively studied from multiple aspects including toxicology, toxins, nutrition mode (e.g., mixotrophy, phagotrophy, etc.), blooming dynamics, allelopathy, and behavior, while the mechanisms accounting for its global distribution and possible invasion to new regions have not been investigated. Since the first report of a bloom of this species from the South China Sea in 2003, K. veneficum has been frequently detected in coastal waters of China. While resting cyst has been well documented to play vital roles in the initiation and decline of HABs and in facilitating geographical expansion of HABs species, whether or not K. veneficum forms resting cyst remains an open question. Here, we provide proofs for the resting cyst formation in K. veneficum based on both the observations on the life history of clonal cultures and cyst detections from field sediment. We microscopically observed the mating gametes, gametes in fusion, planozygotes (judged from the two longitudinal flagella and cell morphology such as a larger size), dark brown, thick-walled cysts with smooth surface, and cyst germination. The resting cyst was produced homothallically (i.e. from single clonal culture). We also determined the diploidity of cysts via measuring the copy numbers of the large subunit (LSU) rRNA gene in resting cysts and vegetative cells. The presence of K. veneficum cysts in field sediments was detected via fluorescence in situ hybridization (FISH) using species-specific probes, and further confirmed by single-cell PCR sequencing for the FISH-detected cysts. The distribution and abundance of K. veneficum cysts in the China Seas (Bohai Sea, Yellow Sea, East China Sea, and South China Sea) were mapped using a combined approach of real-time PCR and FISH, and quantified after measuring and taking into account the copy numbers of LSU rRNA gene in vegetative cells and cysts. We found a wide distribution of resting cysts of this organism in the seas of China, but generally with a low abundance in most of the samples (0 to 15 cy