Hamilton, H.C.*; Garey, J.R.: Applying Molecular Techniques to Environmental Studies of Meiofauna

The purpose of this study is to develop sequence tags for a set of marine meiobenthic fauna (meiofauna). We have shown that DNA of meiofaunal origin can be extracted directly from sediment samples. We have also shown that primers for the 18S rRNA gene can be used to amplify the meiofaunal DNA while excluding bacterial, plant, and fungi DNA. We cloned and sequenced the amplified DNA and found that all clones belonged to meiofaunal organisms, mainly copepods and nematodes. We have identified the harpacticoid copepod species Longipedia helgolandica and a laophontid species, as well as the nematode species Metachromadora pulvinata, from sediment samples taken from Tampa Bay and extracted DNA from isolated specimens of the two harpacticoids. The 18S rRNA gene was PCR amplified from the extracted DNA and sequenced using an automated sequencer. The 18S rRNA sequence was then assembled and aligned. From the alignment, we have identified a variable loop that would be suitable for identifying the specific meiofauna species. This 20 to 25 base sequence was used to construct a sequence tag called a molecular beacon. The molecular beacon is a fluorescent molecule containing the short target sequence that can be used to identify species of meiofauna without having to sort them from the sediment and identify them by morphology. The DNA of the meiofauna can be extracted directly from sediment samples and the 18S rRNA gene PCR amplified. The molecular beacon can then be used to identify and quantify meiofauna species of interest.