Development and application of LSU rRNA probes for Karenia brevis in the Gulf of Mexico, USA

Abstract

The brevetoxin producing dinoflagellate, Karenia brevis, is the target of several monitoring and research programs in the Gulf of Mexico, where it forms extensive and frequently long-lived annual blooms that can cause human intoxication and fish kills, as well as severe economic losses to coastal communities. Rapid, reliable methods for the detection and enumeration of K. brevis cells, as well as their discrimination from morphologically similar species, are valuable tools for managers and scientists alike. Our aim was to produce a species-specific molecular probe that would serve as a tool to facilitate the efficient and reliable detection of K. brevis in the Gulf of Mexico. We sequenced a fragment of the large-subunit ribosomal RNA gene (LSU rDNA) from five K. brevis cultures isolated from the Texas Gulf coast, the Florida Gulf coast, and the Atlantic coast of Florida, and detected no differences among these isolates. A consensus sequence was thus compiled and compared to a previously published sequence from Karenia mikimotoi, the closest known phylogenetic relative to K. brevis, for the purpose of identifying unique K. brevis signature sequences. Fluorescently-labeled (FITC) oligonucleotide probes targeting these regions of the K. brevis LSU rRNA were designed to include at least two base pair differences, as compared to K. mikimotoi. Among seven probes designed, one uniquely identified all K. brevis isolates to the exclusion of all other species tested (Kbprobe-7), including a Gulf of Mexico K. mikimotoi isolate (Sarasota, FL) and several additional Gymnodinium species, as well as other dinoflagellate, diatom, and raphidophyte taxa. Importantly, K. brevis cells in samples taken during a 2001 bloom, fixed with a mixture of modified saline ethanol and 10% formalin, and stored at 4 °C for 7 months were successfully labeled with Kbprobe-7. In addition, preliminary analysis of labeled cells by flow cytometry revealed that K. brevis could be distinguished from K. mikimotoi in solution, suggesting other potential applications of this probe.