Description: The dinoflagellate genus Prorocentrum is globally represented by a wide variety of species found upon benthic and/or epiphytic substrates. Many epibenthic Prorocentrum species produce lipophilic polyether toxins, some of which act as potent protein phosphatase inhibitors and tumor-promoters associated with Diarrheic Shellfish Poisoning (DSP). Most members of the Prorocentrum lima species complex (PLSC) commonly found in the tropics and sub-tropics are toxigenic. Epiphytic and planktonic bacteria co-occur with toxigenic Prorocentrum but reciprocal allelochemical interactions are under-investigated. The aim of the present study was to identify the culturable bacteria collected together with isolates of the PLSC from seagrass (Thalassia testudinum) and macroalgae along tropical Atlantic coasts of Mexico, and to explore potential species interactions with selected isolates. Twenty-one bacterial genera belonging to Proteobacteria, Actinobacteria, and Bacteroidetes were identified by amplification of the 16S rRNA gene marker from nine clonal Prorocentrum cultures, with g-proteobacteria comprising the dominant class. A positive correlation was found between the bacterial genera associated with two Prorocentrum clones and the esterified toxin analog DTX1a-D8, but there was no apparent correlation between the other PLSC clones and their associated bacteria with the other five DSP toxins detected. No bacteriostatic or allelochemical response was found for cell- and culture medium extracts of five Prorocentrum isolates assayed for bioactivity against Staphylococcus sp. DMBS2 and Vibrio sp. HEL66. Bulk cell-washing of Prorocentrum PA1, followed by growth with antibiotics, was only effective in reducing bacterial load in the initial growth stages, but did not yield axenic cultures or lower bacterial cell densities throughout the culture cycle. Antibiotic treatment did not impair growth or survival of the dinoflagellate, or apparently affect DSP toxin production. There was no significant correlation between Prorocentrum cell volume, growth rate, bacterial cell counts, or cellular toxin concentration over the entire time-series culture cycle. Benthic Prorocentrum and associated bacterial communities comprise highly diverse and characteristic microbiomes upon substrates, and among compartments in culture, but this study provides little evidence that allelochemical interactions among Prorocentrum cells and associated bacteria originating from epibenthic substrates play a definable role in growth and toxigenicity.

Description: The marine dinoflagellate genus Prorocentrum Ehrenberg comprises many species occupying primarily benthic or epiphytic habitats, particularly in tropical and sub-tropical waters. Despite concerted efforts to establish phylogenetic associations, there remain unresolved issues in defining morphospecies and membership in species complexes. The study described herein addressed the inter- and infraspecific relationships of members of the Prorocentrum lima and Prorocentrum hoffmannianum species complexes (PLSC and PHSC, respectively) by applying multivariate approaches in morphotaxonomy, molecular phylogenetics and chemodiversity to establish affinities among multiple clonal isolates. Morphotaxonomic analysis showed consistency with classical morphospecies descriptors, and high variability in cell size and dimensions, but did not challenge current species complex concepts. Phylogenetic analysis of ITS/5.8S rDNA sequences from isolates from the Gulf of California, Caribbean Sea, and Gulf of Mexico coasts compared with archived global GenBank sequences served to define five consistent clades with separation of the PLSC and PHSC. Secondary structure modeling of ITS2 rRNA variation based on compensatory base changes (CBC) was effective in resolving details of the respective species complexes and even indicated putative incipient or cryptic speciation due to potential hybridization barriers. This study represents the largest (n = 67 isolates) chemodiversity analysis of polyketide-derived toxins associated with diarrheic shellfish poisoning (DSP) from a benthic dinoflagellate genus. Relative composition of some analogs (OA, OA-D8, DTX1, DTX1a, and DTX1a-D8), including two new undescribed isomers, distinguished P. lima from P. hoffmannianum sensu lato, but without clear associations with substrate type or geographical origin. Although all P. lima and most (one exception) P. hoffmannianum were toxigenic, the total cell toxin content could not be linked at the species level. This research demonstrates that clonal chemodiversity in toxin composition cannot yet be effectively applied to define ecological niches or species interactions within local assemblages. Phylogenetic analysis of the ITS/5.8 rDNA, particularly when combined with secondary structure modeling, rather than only a comparison of LSU rDNA sequences, is a more powerful approach to identify cryptic speciation and to resolve species complexes within benthic dinoflagellate groups.