Single-cell PCR of the luciferase conserved catalytic domain reveals a unique cluster in the toxic bioluminescent dinoflagellate Pyrodinium bahamense

Abstract

Pyrodinium bahamense is a toxic, bioluminescent dinoflagellate with a record of intense bloom formation in both the Atlantic-Caribbean and Indo-Pacific regions. To date, limited genetic information exists for P. bahamense in comparison to other closely related harmful algal bloom taxa such as Alexandrium, or other bioluminescent taxa such as Pyrocystis. This study utilized single-cell PCR to explore the molecular diversity of P. bahamense within the Indian River Lagoon (IRL), Florida, USA, and a bioluminescent bay in Puerto Rico. Pyrodinium-specific primers targeting a ca.1.2-kb region of the 18S rRNA gene and degenerate primers targeting the conserved catalytic domain of the luciferase gene (lcf ) were applied to single cells isolated from both geographic regions as well as single cells of clonal isolates from the IRL. Phylogenetic analysis revealed that while P. bahamense is more closely related to Alexandrium spp. at the 18S rRNA gene level, its lcf sequences are more closely related to Pyrocystis spp. than Alexandrium spp. Pyrodinium bahamense lcf sequences from the Western Atlantic formed 2 distinct clusters. These clusters were defined by a set of core amino acid substitutions, and the extent of variation was greater than that recorded between the established variants of Pyrocystis lcf. lcf sequences from an Indo-Pacific strain formed a third distinct cluster. Based on these results, the potential of lcf for use in tracking sub-populations of P. bahamense is discussed.

sxtA4+ and sxtA4- Genotypes Occur Together within Natural Pyrodinium bahamense Sub-Populations from the Western Atlantic

“…Large subunit rRNA gene sequence analysis indicated the existence of Indo-Pacific and Atlantic-Caribbean ribotypes, leading to the suggestions that P. bahamense is a species complex. A previous study showed 18S rRNA gene sequences of P. bahamense from multiple locations in the IRL, Puerto Rico, and the Indo- Pacific to be nearly identical and forming a single cluster [ 48 ]. Comparison of 18S rRNA gene sequences from sxtA4+ and sxtA4- genotype cells in this study were compared to existing 18S rRNA gene sequences from the Indo-Pacific and Atlantic-Caribbean.…”

“…Single cells were isolated for lysis and subsequent multiplex PCR using a slight modification of a protocol described previously [ 48 ]. Briefly, an aliquot (ca.…”

“…Multiplex PCR was performed on single cells using Pyrodinium -specific primers targeting the 18S rRNA gene developed previously [ 48 ] and the sxtA4F1/stxA4680R primers. The single-cell multiplex PCR was first optimized on single cells from lab cultures of the toxin-producing P. bahamense isolate from the IRL using the same methods for cell isolation and washing, lysis, and PCR as for environmental samples.…”

Paralytic Toxin Producing Dinoflagellates in Latin America: Ecology and Physiology

“…than Alexandrium spp. suggesting a much greater variation than that seen in bioluminescent species with known gene variants (Cusick et al, 2016).…”

“…Its distribution in the Pacific coast extends from southern GOLCA to Colombia (Martínez-López et al, 2007;Rodríguez-Salvador and Meave del Castillo, 2007;Morquecho, 2008;Gárate-Lizárraga and González-Armas, 2011;Usup et al, 2012). In the Atlantic coast it has been reported from the Gulf of Mexico to Uruguay Limoges et al, 2015;Mertens et al, 2015;Poot-Delgado et al, 2015;Cusick et al, 2016). HABs have been linked in several occasions to human poisoning (Rosales-Loessener, 1989;Rodríguez et al, 1990;Saldate-Castañeda et al, 1991;Núñez-Vázquez et al, 2011;Gárate-Lizárraga et al, 2012;Callejas et al, 2015) as well as to epizootic events (fish and sea turtles) Amaya et al, 2018) ( Table 1).…”