Using quantitative PCR and DNA barcodes to quantify and identify marine invertebrate larvae

Information on the relationship between behavior and dispersion in marine invertebrate larvae has been hampered by the lack of suitably rapid techniques for identification and quantification from field samples. The need to understand larval dispersal dynamics of the pinto abalone (Haliotis kamtschatkana), which was recently listed as a species of concern (U.S.) and threatened (Canada), motivated the development of a high-throughput method for identification and quantification of abalone larvae in seawater samples. Seawater was filtered through a 70 mu m screen and DNA was extracted from all organisms present in sample retentate. Species-specific Cytochrome Oxidase I (COI) mitochondrial DNA sequence was used as template for Quantitative PCRs (QPCR) containing primers and a dual-labeled hydrolysis probe. Direct enumeration of larvae using light microscopy was used to verify quantity estimates derived from QPCR standard curves. To avoid bias from either underestimation of quantity or false negatives, abalone-specific reactions were multiplexed with primers, probe and exogenous template as an internal positive control (IPC) to indicate partial or full PCR inhibition. Differences between direct counts (n = 1--141) and QPCR estimates were not significant, and the average coefficient of variation was 0.56 for within-run replicates, indicative of high accuracy and precision. We estimate that 80 samples can be processed from DNA extraction through QPCR in about four hours at a cost of approximately $2/sample. Our results demonstrate that QPCR may have high utility for high throughput identification and quantification of specific marine invertebrate larvae in seawater samples via counts of mtDNA COI "barcodes" using constructed standard curves.