Date of Award

12-2009

Level of Access

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

Advisor

Laurie Connell

Second Committee Member

Paul Rawson

Third Committee Member

Chris Davis

Abstract

Attempts to characterize and study the population dynamics of the softshell clam Mya arenaria in relation to a mutation which confers resistance to paralytic shellfish toxins are complicated by a lack of non-lethal genotyping techniques, reliable tagging methods and an understanding of the inheritance patterns of the marker. Presented here, is a straightforward and non-lethal technique for clam genotyping, a new method for the long term tagging of clams, and the offspring genotype frequencies from a number of pair matings between clams of known genotype. Hemolymph extracted from M. arenaria was used directly in a polymerase chain reaction (PCR) to successfully amplify a DNA fragment suitable for sequencing. Tested M. arenaria showed 100% (n=10) survival after a period of four weeks. In a separate experiment, passive integrated transponder (PIT) tags were inserted between the mantle and shells of 72 clams that were monitored for tag retention and survival. Among all PIT tagged clams, there was 100% survival and 92 % tag retention. These methods provide a mechanism by which softshell clams can be genotyped and individually monitored, during field experiments. Sixteen pair matings were conducted with adult M. arenaria of known genotype. Using polymerase chain reaction (PCR), the domain II (DII) pore region of the voltage-gated Na+ channel a-subunit, in which the mutation is found, was identified in a total of 344 larvae from these crosses. The data support the hypothesis that the mutant allele can be inherited from either sex and that there are no barriers to fertilization between gametes of different genotypes. Of the ten cross offspring genotype frequencies analyzed using goodness-of-fit tests, seven adhere to Mendelian expectations for inheritance, while three significantly deviated from expected ratios. These deviations are assumed not to be an actual representation of the sampled larval populations, but rather due to a combination of small larval sample size and a conservatively high significance level. This study demonstrates that the single nucleotide polymorphism in question is most likely inherited in a Mendelian fashion.

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