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Abstracts - Dennys G. S. Ortiz

Headspace - Solid phase microextraction- Gas Chromatography- Mass Spectrometry (HS/SPME/GC-MS), method for the determination in vivo of Sex Pheromone 9-Methylgermacrene-B in sand fly Lutzomyia longipalpis

Dennys G. S. Ortiz1, Christiann D. Tosta2, Flavia B. da Rocha3, Vicente E. Machado3, Wanderson H. C. Oliveira3, Mara C. Pinto3*

1Universidade Estadual de Campinas-UNICAMP; d162706@dac.unicamp.br

2Instituto Federal de São Paulo, Campus Matão; cdtosta@ifsp.edu.br

3Universidade Estadual Paulista "Júlio de Mesquita Filho";

*Correspondence: marap@fcfar.unesp.br

Phlebotomine sand flies are important vectors of virus, bacteria and protozoan parasites. The main parasites transmitted by those vectors are Leishmania spp which causes leishmaniasis. Out of 570 sand flies identified in Americas, sexual pheromone has been detected only in Lutzomyia longipalpis, Lutzomyia cruzi and Lutzomyia cruciata, Pintomyia pessoai, Evandromyia lenti, Evandromyia carmelinoi, Lutzomyia lichyi. However, only for Lu. longipalpis the chemical structure was identified and synthesized. So far, the methodology used for sex pheromone studies in sand flies is based on classical extraction procedure with the submersion of the whole body of insects in organic solvents; methodology that ends up killing of insects and that recovers both volatile and non-volatiles compounds. We would like to use a non-lethal technique to investigate the presence of only volatile sexual pheromones in other sand fly species using the headspace - Solid phase microextraction - Gas Chromatography – mass spectrometry (HS/SPME/GC-MS) methodology. In the present study our aim is to standardized this technique using Lu. longipalpis (9-Methylgermacrene-B)1 as a positive control. Lu. longipalpis sandflies were obtained from a colony laboratory. Males and virgin females without a blood meal and with four to five days old were placed in a 2mL vial in the following conditions: a) 24 males; b) 12 females; c) 24 males and 12 females; d) one male; e) one female; f) one male and one female. Volatiles released for living insects were collected for 45 min at the surrounding environment temperature; the fiber used was 50/30µm Divinylbenzene /Carboxen/ Polydimethylsiloxane (DVB/CAR/PDMS). The conditions a - c were tested once and for the conditions d - f, six replicates were carried out for each. The mass spectra corresponding to each of the resulting peaks were compared to the NIST library and confirmed by comparison of GC retention indexes (RI) relative to n-alkanes (C8-C20). It was possible to identify a compound (m/z 218) with a similarity of 90% with germacrene-B (m/z 204) in all conditions. The peak areas of this compound varied among males. As conclusion it was possible to determine the presence of Lu. longipalpis sexual pheromone through HS-SPME-GC-MS methodology and the differences among the peaks in distinct specimens may indicate different pheromone release rates.

Refs.

  1. Hamilton, J.G.C. et al., J. Chem. Ecol.22, 1477-1491, 1996.