Analysis of gene expression in endosulfan exposed Homarus americanus larvae using an oligonucleotide microarray

Potato farming and lobster fishing are two important industries on Prince Edward Island. However for both industries to be sustainable, they must be managed responsibly. Recently, agricultural runoff has been suspected to have caused numerous fish kills in rivers throughout the island. Although a pesticide research and monitoring program was implemented for freshwater areas in Canada by Environment Canada, there is insufficient information about pesticide levels and impacts in estuarine and marine environments. One agricultural pesticide of concern is the organochlorine endosulfan that is used to combat the Colorado potato beetle. Endosulfan is a potent neurotoxin and moult inhibitor that can have serious effects on non-target organisms such as the larvae of the American lobster, Homarus americanus.;The lobster life cycle consists of a larval pelagic phase followed by the migration of postlarvae to a benthic habitat. For the lobster to undergo normal development, they must moult their hardened exoskeleton to allow for growth and tissue expansion. One specific moult event is linked to the crucial developmental stage of metamorphosis, wherein the lobster transitions from a larva to a postlarva. Metamorphosis is a sensitive developmental period during which the lobster experiences significant morphological, physiological, biochemical, behavioural and ecological changes. Since this is a critical stage of development, exposure to endosulfan could have deleterious effects on development and survival of lobster larvae.;The focus of this study was to determine the effects of environmentally relevant concentrations of endosulfan on gene expression during metamorphosis of lobster larvae. Endosulfan causes serious developmental delays and deformities in an array of species, however very little is known about the regulation of gene expression during pesticide exposure. The use of a custom made high throughput lobster, H. americanus, microarray allowed for monitoring of 14,592 genes based on unique lobster expressed sequence tags (EST). A pooled reference design was used to identify changes in gene expression between 5 endosulfan concentrations and a control. Genes with >1.5 fold change and identified as being significant at p < 0.05 using one-way ANOVA were selected for further analysis. There were 707 genes identified as being significantly differentiated. However with only ∼40% annotation of the array, the majority of these genes were unknown. Annotated genes were involved in many processes: development, metabolism, immune and oxidative stress response and gene regulation.;Nine genes of interest (GOI) were selected for reverse transcription quantitative PCR (RT-qPCR) analysis to validate the microarray results. For optimal RT-qPCR normalization, 5 housekeeping genes were identified and validated using geNorm. Although the RT-qPCR detected a similar expression pattern as the microarray, the microarray results were often greatly under expressed. Due to discrepancies between expression levels in the microarray compared to the RT-qPCR method, the correlation values between the two were low.;Endosulfan had a serious effect on survival, development and gene expression during metamorphosis. The long term objective of this research will be to use microarray gene expression profiles as screening tools for identifying what contaminants are present in the environment.