Parasites

1. Quantitative Real-Time PCR as a tool for the quantification and characterization of microorganisms in caves and karst aquifers: Phytoplankton, lampenflora, bacterial communities, and fecal source tracking Rick Fowler Western Kentucky University Bowling Green, Kentucky USA

2. Beads were placed in dye-trace bags and recovered after one year in cave streams DNA test is specific and sensitive for Chlorella Quantitative Real Time PCR Melting point analysis Fungi HEX-Bac32F primer Parasites Bacteria Bacteroides 16S rDNA HEX HEX HEX HEX HEX HEX HEX HEX HEX HEX Protists vulgaris NED qRT-PCR Surface flow | Subsurface springs 1 kb Ladder NED-1492r primer 100 Cave DNA 1 kb Ladder Cave DNA Chlorella Chlorella 1000 ml 1000 ml 100 ml 1300 ml 750 ml 1300 ml 500 ml 500 ml 100 ml 250 ml 750 ml 250 ml 10 1 Dual-labeled Bacteroides 32f-798r amplicons 0.1 0.08ng 0.01 NED Limestone 0.001 0.0001 Algae GRGR NRNR BIGR HRTH ERES BSBS DFDF BSBC Blank Zooplankton NED Ca13CO3 NED NED NED NED Phylogenetic trees of cave bacteria Many cave bacterial 16SrDNA genes were sequenced A strong linear relationship exists between the concentration of DNA template and the number of cycles required to reach a fluorescence threshold Scheme for microbial source tracking Instrument is a thermocycler equipped with a fluorescent camera Organisms suitable for DNA analysis Acknowledgements Artificial substrates were beads containing limestone Chlorella DNA concentrations in Mammoth Cave National Park • Polymerase Chain Reaction (PCR) • Many copies of specific DNA sequence Lipid Analysis of artificial substrates Different technique based on analysis of membrane lipids Quantification of bacterial DNA on artificial substrates at five study sites Quantification of bacterial DNA in cave sediments Chlorella algae is a major food source for endangered mussel species in surface streams Green River main channel Turnhole Bend cave spring “Blue Hole” • Combination of compatible dyes: • SYBR Green • HEX • NED • ROX, VIC, Cy3, EB, BHQ, etc. Human Web site http://people.wku.edu/rick.fowler/mammoth Determination of Bacteroides 16S rDNA concentration Algae and phytoplankton in surface streams and cave springs Lampenflora Bacteroides 16S rDNA from different animals can be differentiated Microbial Source Tracking Identification of the origins of fecal pollution in karst aquifers Environmental DNA from karst surface stream Purified DNA from different aquatic organisms SYBR Green SYBR Green SYBR Green Chlamydomonas reinhardti vulgaris Dual-labeled Bacteroides 32f-798r amplicons Cave bacteria 0.4ng 0.000064ng 0.00032ng 0.0016ng Chlorella DNA(ppt) • Quantitative Real-Time PCR • (qRT-PCR) • Many copies of specific DNA sequence • Fluorescent dyes • Quantification and melting point analysis Chlorella vulgaris Sewage NED Decapods 1 kb Ladder Insects Cave DNA Bacteria Fungi 1 mm DNA Extracts 1000 500 250 200 bp PCR Product Chlorella is also found in lampenflora near cave tourist trails 10,000 R1 R2 R1 R2 3000 NED 1000 1000 500 PCR 200 Horse 500 Products 100 R2 R2 R1 R1