Jason W. Kempton

Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond

Abstract A dinoflagellate bloom was found associated with a fish kill event in a South Carolina brackish water retention pond. A multi-analytical approach was used to confirm the identity of the bloom dinoflagellate and evaluate its potential toxicity. Karlodinium micrum was confirmed through light microscopy, pigment profile comparisons, species-specific PCR, and gene sequence data. Necropsy findings on several fish were suggestive of an acute kill event. Toxicity of filtrate from bloom samples was tested by a hemolytic assay using rainbow trout ( Oncorhynchus mykis ) erythrocytes and an ichthyotoxicity assay using larval zebrafish ( Danio rerio ). Hemolytic activity was measurably high (>80% hemolysis) in both whole filtrate and fractionated filtrate (from the 80% MeOH C 18 column elution). This fraction also demonstrated high ichthyotoxic activity as exposed fish experienced rapid death. These results implicate toxic K. micrum as a causative factor in fish death in a non-aquaculture brackish pond associated with a housing development, and extend recent findings linking this species to fish kills in aquaculture ponds.

Potential Indicators of Stress Response Identified by Expressed Sequence Tag Analysis of Hemocytes and Embryos from the American Oyster, Crassostrea virginica

Abstract: A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS.

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.

Harmful algal blooms in South Carolina residential and golf course ponds

The South Carolina coastal zone is among the fastest growing areas in the U.S., and population epicenters are marked by dense brackish water pond (lagoon) coverage associated with housing complexes and golf courses. Surveillance efforts in 2001–2002 documented the widespread occurrence of several types of potentially or measurably toxic harmful algal blooms (HABs) in these ponds. These man-made retention ponds were constructed in order to serve as a buffer between developed areas and open estuaries or for aesthetic reasons. However, the combination of restricted tidal flow and nutrient and/or contaminant deposition creates a stimulatory environment for potential HAB formation. These discoveries introduce the need to consider mitigation measures to existing ponds and HAB preventive strategies for future pond construction.

Kryptoperidinium foliaceum blooms in South Carolina: a multi–analytical approach to identification

Abstract Observations following the discovery of Kryptoperidinium foliaceum blooms in South Carolina (SC), USA, suggest that a multi-analytical approach, using a standard, minimal set of criteria, should be adopted for determining dinoflagellate species identity and taxonomic placement. A combination of morphological, molecular, and biochemical analyses were used to determine the identity of this “red tide” dinoflagellate, first documented in SC waters in the spring of 1998. Results from thecal plate tabulations (based on scanning electron and epifluorescence microscopy), gene sequence data, species-specific PCR probe assays, and microalgal pigment profiles were analyzed and compared to reference cultures of K. foliaceum . Comparative data showed marked inconsistencies among the K. foliaceum reference culture isolates. In addition, the SC bloom isolate was shown to be mononucleate, contrary to previous reports for K. foliaceum , suggesting a more transient endosymbiotic association than previously considered.