Investigations of internal interactions between the parasitic barnacle Loxothylacus texanus (Rhizocephala: Sacculinidae) and its host Callinectes sapidus (Brachyura: Portunidae) using PCR techniques.

摘要

We describe techniques that enable the preservation of tissues from the rhizocephalan barnacle, Loxothylacus texanus, inside the body cavities of blue crab hosts, Callinectes sapidus, in a manner that minimizes the degradative activities of hepatopancreatic enzymes. These procedures allow the extraction and amplification of both parasite and host 18S rDNA within the same sample and enable one to distinguish between parasitized and unparasitized crab tissue in as little as two weeks after infection, well before any external manifestations of the parasites. Two PCR-based approaches were taken to identify the presence of L. texanus. In the first approach, a set of primers specific for L. texanus was used to specifically amplify 18S sequence in a background of C. sapidus DNA or the DNA of other barnacle species. In the second approach, a set of general primers was used to amplify 18S sequence from C. sapidus and a variety of barnacle species. The products of this PCR were then digested with an enzyme that recognizes a restriction site present only in the L. texanus PCR product to yield a unique pattern of fragments. With these techniques, we could detect as few as five parasitic cypris larvae in water samples, as well as L. texanus in the tissue of a small crab collected from the field and in the four anterior periopods of a crab bearing the external stage of the parasite. In experiments with potential hosts of varying sizes and molt stages, we confirmed that the parasite was significantly more effective in infecting crabs less than 30 mm carapace width than larger individuals. These techniques will facilitate future investigations of ecological and physiological interactions between these important crustacean parasites and their hosts and will help to determine the economic impact of this parasite on blue crab fisheries.