Title

A molecular approach to the study of saxitoxin resistance in copepods

Date of Completion

January 2010

Keywords

Biology, Molecular|Biology, Neurobiology

Degree

Ph.D.

Abstract

Previous work has demonstrated adaptation (resistance) of the copepod Acartia hudsonica to saxitoxin (STX), a neurotoxin that blocks sodium channels and interrupts nerve transmission signals. STX resistance in other organisms has been linked to mutations in the outside pore of the sodium channel, which dramatically reduce toxin binding affinity. To study the mechanism of STX resistance in A. hudsonica, I isolated and sequenced the full-length cDNAs of its sodium channel α subunit. Two types of channels were obtained, with identical amino acid (aa) sequences except for a three-aa insertion and one-aa substitution in the inner pore of the channel, near the inactivation gate, in one of the sequences. The sequence with the 3-aa-insertion/1-aa-substitution is considered the mutant type. ^ The role of the mutation on sodium channel function was examined by introducing the insertion into a rat sodium channel cDNA, expressing it in frog oocytes, and measuring sodium currents in patch clamp experiments. Unlike other mutations conferring resistance to STX, this mutation has no apparent effects on STX binding affinity. Instead, the main feature of the mutation is that it results in a leaky channel; i.e., a channel with residual currents when the channel is inactivated. ^ To test my hypothesis that a leaky channel accounts for copepod resistance to STX, egg production rate of individuals was measured in the presence and absence of STX, and the channel genotype of those same individuals was determined. The assays showed that for heterozygous copepods (those having both the mutant and wild type channel), egg production rate was higher when they were fed a toxic diet compared to a non-toxic diet. This finding partly supports the hypothesis and suggests an entirely new mechanism of toxin adaptation. ^ In natural populations, heterozygotes represent the most abundant genotypes and wild homozygotes are the rarest. The highest frequency of heterozygotes occurs in areas where blooms are most common and frequent. The suggested novel mechanism of adaptation and the observed heterozygote advantage have important implications for understanding the dynamics of toxic algal blooms and the nature of grazer adaptability to neurotoxins. ^