USE OF MICROARRAY HYBRIDIZATION TO IDENTIFY BRUGIA GENES INVOLVED IN MOSQUITO INFECTIVITY
Griffiths, Kathryn Greer
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Filarial nematodes are a family of insect-borne parasitic worms that cause a number of diseases in man and animals. The most widespread filarial disease of humans is lymphatic filariasis, caused by worms in the genera Wuchereria and Brugia. Lymphatic filariasis is an economic and social burden in endemic countries and affects approximately 119 million people worldwide. Brugia malayi and B. pahangi microfilariae (mf) require a maturation period of at least five days in the mammalian host before they can infect mosquito vectors. This maturation process correlates with changes in the surface composition of mf that likely are associated with changes in gene expression. To test this hypothesis, we verified the differential infectivity of immature (<3 day) and mature (>30 day) Brugia mf for black-eyed Liverpool strain of Aedes aegypti (LVP), and then assessed transcriptome changes associated with microfilarial maturation by competitively hybridizing microfilarial cDNAs to the B. malayi oligonucleotide microarray. We identified transcripts that were more abundant in immature (94 in B. pahangi and 29 in B. malayi) and mature (64 in B. pahangi and 14 in B. malayi) mf. In each case, >40% of Brugia transcripts shared no similarity to known genes, or were similar to genes with unknown function; the remaining transcripts were categorized by putative function based on sequence similarity to known genes/proteins. Microfilarial maturation was not associated with demonstrable changes in the abundance of transmembrane or secreted proteins; however, differences in transcript abundance were observed for many that have predicted functions. For example, immature mf were enriched for transcripts putatively associated with immune modulation, neurotransmission, transcription and cellular cytoskeleton elements. In mature mf, there was an increase in transcripts potentially encoding hypodermal/muscle and surface molecules, i.e., cuticular collagens and sheath components. These finding lend support to the underlying hypothesis that changes in microfilarial gene expression drive surface modifications that influence the parasite to begin development in compatible vectors. Brugia malayi genes corresponding to differentially abundant transcripts were identified, and transcript abundance validated by quantitative polymerase chain reaction. These studies serve as a starting point towards gaining a better understanding of the parasite side of the intricate parasite/mosquito relationship.
Nematoda Genetic aspects
Filarial worms Molecular genetics
Mosquitoes as carriers of diseases
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science-Biology Microbiology