State and federal regulatory agencies increasingly call upon NCDOT biologists to determine whether the site for a proposed construction project is HOME to an endangered or threatened species of freshwater mussel. Biologists conducting surveys currently depend on species identification based primarily on examination of diagnostic shell characteristics, which are difficult to discern and may be unreliable, even in the hands of experts. Genetic identification offers a powerful alternative, but creates new challenges that this proposal aims to help solve. First, we will overcome the need to sacrifice animals or to obtain tissue biopsies, neither of which is a viable approach to sampling a threatened population. The methods we develop for obtaining, storing, and processing hemolymph for DNA extraction will allow even the rarest species to be sampled with little or no risk of mortality. Secondly, genetic methods based on DNA sequence analysis require equipment and expertise not generally available to NCDOT and other state agencies. However, the end result of our work will be standard operating procedures that will involve DNA extraction, Polymerase Chain Reaction, restriction digestion, and agarose gel electrophoresis that will greatly simplify genetic analysis as well as establish a standard set of procedures that will ensure consistency across the state. Standardization of identification procedures will streamline the process, and each additional analysis, if added to a common database, will further our ability to quickly evaluate the genetic distinctiveness of mussel populations. Such methodologies could substantially facilitate the evaluation process of potential impacts from proposed NCDOT construction projects.
The objectives of this research project are:
- To obtain hemolymph samples from several freshwater mussel individuals from several species collected at multiple sites in Lake Waccamaw.To monitor the survivorship of sampled individuals for several weeks to evaluate the effects of hemolymph withdrawal on survivorship under field conditions.To develop methods for hemolymph transport, storage and processing to allow extraction of PCR-amplifiable DNA from this tissue.To PCR amplify and sequence the DNA of 2 mtDNA regions and 1 nuclear DNA region from a subset of the individuals sampled.To analyze the DNA sequences phylogenetically to define monophyletic groups (i.e. species and subspecies), and to use these analyses to design genus- and species-specific restriction endonuclease digestion assays.To apply methods developed in (5) to new locations, we will sample and sequence the DNA, then perform diagnostic restriction digests on selected animals from adjacent and more distant collection sites in North Carolina.
