|DOWD, S - Research And Testing Laboratories, Llc|
Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2009
Publication Date: 9/15/2009
Citation: Guerrero, F., Dowd, S.E., Djikeng, A., Wiley, G., Macmil, S., Saldivar, L., Najar, F., Roe, B.A. 2009. A database of expressed genes from the new world screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). Journal of Medical Entomology.46(5):1109-1116
Interpretive Summary: By studying gene expression patterns of an organism, one can learn how that organism responds to its environment and thereby detect vulnerabilities in its lifecycle which can be exploited to develop control technologies. We have initiated study of the genes of the New World Screwworm, Cochliomyia hominivorax, by synthesizing and sequencing DNA libraries of expressed genes from embryos, 2nd instar larvae, adult males and adult females. We utilized a novel high throughput sequencing technology called 454 pyrosequencing which produced approximately 300,000 independent sequences. Some of these sequences could be assembled into larger composite sequences (termed contigs) based on overlapping sequence identity while other sequences had no overlapping sequence identity with any other members of the database and remained as unassembled singletons. The full database consists of 6,076 assembled contigs and 58,221 unassembled singletons. Each sequence from this database was used to search public protein databases to assign putative function to as many database entries as possible. This process led to the identification of several gene coding regions with possible roles in sex determination in the developing screwworm embryo. This database will facilitate the design of experiments to study screwworm gene expression on a larger scale than previously possible. Additionally, the identification of sex determination genes in the screwworm will assist in the development of a control technology based on transgenic strains of screwworm which produce only males, a technology which would be applicable to the USDA-APHIS screwworm eradication program's production facilities.
Technical Abstract: We used an expressed sequence tag and 454 pyrosequencing approach to initiate a study of the genome of the New World Screwworm, Cochliomyia hominivorax (Coquerel). Two normalized cDNA libraries were constructed from RNA isolated from embryos and 2nd instar larvae from the Panama 95 strain. Approximately 5,400 clones from each library were sequenced from both the 5' and 3' directions. Additionally, double-stranded cDNA was prepared from random-primed polyA RNA purified from embryos, 2nd instar larvae, adult males and adult females. These 4 cDNA samples were used for 454 pyrosequencing which produced approximately 300,000 independent sequences. Sequences were assembled into a database of assembled contigs and singletons and used to search public protein databases and annotate the sequences. The full database consists of 6,076 contigs and 58,221 singletons assembled from both the traditional EST and 454 sequences. Annotation of the data led to the identification of several gene coding regions with possible roles in sex determination in the screwworm. This database will facilitate the design of microarray and other experiments to study screwworm gene expression on a larger scale than previously possible.