The Black Fly Simulium vittatum Trypsin Gene: Characterization of the 5′-Upstream Region and Induction by the Blood Meal

The events leading to blood digestion in hematophagous insects are nor well understood at the molecular level. Here we report on the characterization of a trypsin gene from the black fly Simulium vittatum. Southern blot analysis indicated that the S. vittatum genome contains at least two trypsin genes. Primer extension experiments identified two closely spaced transcription initiation sites, 3 bp apart. A genomic clone containing the trypsin gene was restriction mapped and the nucleotide sequence of the 5′-upstream region was determined. Several sequence elements were identified, including a consensus TATA box 35 nucleotides upstream from the transcription initiation sire, a consensus arthropod initiator site at position -7, and two conserved sequence elements, 5′GGATTAA (position -77) and 5′TGTTTCCT (position -148). The latter two elements are present at comparable distances from the transcription initiation sites in black fly and mosquito trypsin genes. Significant amounts of trypsin mRNA were detected in guts of sugar-fed flies. Upon blood feeding, trypsin mRNA levels gradually increased to reach twice the initial abundance at 8 hr after blood ingestion. In contrast to other hematophagous insects, the induced mRNA level persists for a prolonged period of time (at least 48 hr) after blood ingestion. The results of this study provide information that may be useful for the expression of transgenes in insects of medical importance.