Molecular Characterization of Integumental Enzymes Involved in Lipid Biosynthesis in Triatoma infestans

CALDERÓN G.M; COCCHIARARO BASTÍAS L.; PEDRINI N; JUÁREZ M.P.

La Plata

Workshop; Triatomine Genomics and Biology III; 2012

The major cuticle lipids of Triatoma infestans are hydrocarbons, fatty alcohols, waxes, and free and esterified fatty acids. These lipids are crucial to insect survival, preventing water loss and regulating chemical and biological insecticide penetration. They also participate in chemical communication events as contact pheromones. Cuticle lipids are synthesized through the concerted action of fatty acid synthases (FASs) and fatty acid elongases (ELOVLs) of the integument. We have already shown that integument FASs participate in the biosynthesis of long chain fatty acids, mostly up to 16 carbon length. Further elongation by ELOVLs will produce very long chain fatty acids, which in turn are precursors to cuticle lipids and pheromones. Materials and Methods: cDNA was synthesized by RT-PCR from total RNA purified from insect epidermis. Based on known sequences of other insects, degenerate primers were designed using the iCODEHOP (consensus-degenerate hybrid oligonucleotide primer) on-line software. These primers were used to amplify FAS and ELOVL sequences by PCR. The fragments obtained were cloned, sequenced and checked for identity by BLAST searching. The full length sequence of an ELOVL mRNA transcript was obtained by RACE. Results and Conclusions: The partial FAS sequence showed the highest identity with mammal FAS sequences (84-100%) and to a lesser extent with insect FASs (60%). The partial ELOVL sequence showed higher identity with insect ELOVL type 4 sequences (87%) than with ELOVL4 mammal sequences (60%). Further studies will be addressed in order to analyze the differential expression of these genes (i.e., developmental stage, sex, and insecticide susceptibility) as well as to identify other gene sequences.