The sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) is the principal vector of Leishmania infantum (Protozoa: Kinetoplastida) in the Americas, a protozoan parasite responsible for American visceral leishmaniasis (AVL) [1–3]. L. longipalpis uses male-produced pheromones for mate recognition. Females perceive and respond to a specific chemical signal from distances of a few meters [4, 5]. Additionally, there is evidence that the same pheromone also produces male aggregation . These attractants are synthesized by specialized gland cells located beneath the cuticle in abdominal segments of adult insects . Recently, a study with L. longipalpis obtained from Lapinha Cave, Minas Gerais, Brazil reported that pheromone biosynthesis started around 12 hours after emergence and increased for 3 days from then on, stabilizing thereafter . Once the pheromone is produced, it is disseminated to the environment through papular structures located in pale abdominal spots . In L. longipalpis populations these pale-spots phenotypes can be found as a single pair (1S) on the fourth abdominal segment, or as two pairs (2S), on both the third and fourth abdominal segments. The lighter-colored appearance of these segments, exposing the pheromone disseminating structure, is generally attributed to the absence of macrotrichias, widely distributed over the rest of the abdominal surface of the insect .
Lutzomyia longipalpis is apparently a complex of sibling species (, reviewed in ), although the number and distribution of potential component species remains to be established [13–15]. Early studies with different L. longipalpis populations identified the main chemical components of the pheromone blends belong to the terpene class of compounds [16–18]. Four distinct terpenes have been identified, two of which have been completely characterized to have 16 carbons (homosesquiterpenes) and two compounds having 20 carbons (diterpenes) of unknown stereochemistry. The homosesquiterpenes of the L. longipalpis sex phermone are (1S,3S,7R)-3-methyl-a-himachalene and (S)-9-methylgermacrene-B. In Brazil, these pheromones are produced by insects found in Jacobina, state of Bahia, and Lapinha Cave, state of Minas Gerais, respectively [17, 19]. Other populations, such as L. longipalpis from Sobral, state of Ceará and Jaíba, state of Minas Gerais, produce two diterpene isomeres: cembrene1 and cembrene2, respectively . A fifth pheromone-producing L. longipalpis population (chemotype) found in Sobral, state of Ceará, is distinguished for its varying amounts of terpenes, relative to the populations previously mentioned, as well as other morphological differences . These diverse populations make the L. longipalis complex an interesting model for research on genetic regulation, enzymatic components, and pathways of pheromone production.
In addition to the Lutozmyia genus, there has been progress in the chemical communication research of other phlebotomine genera such as Sergentomyia and Phlebotomus. Although terpenes similar to those of the L. longipalpis species complex have been found to be produced in species such as Sergentomyia minuta and Sergentomyia fallax, their role in pheromone communication or other biological functions remains unclear. On the other hand, studies have presented behavioral evidence for the presence of sex pheromones in Phlebotomus papatasi, without identifying their pheromone structures [23, 24].
Terpene compounds are one of the largest groups of natural products that have significant roles as repellents or attractants to many organisms . They are derived from a five-carbon precursor isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) . These compounds are produced by a cellular metabolic pathway, the mevalonate route or mevalonate dependent pathway (MAD) , which is present in higher eukaryotes and many bacteria. Alternatively, IPP and DMAPP can also be produced through a different pathway, the methylerithritol phosphate pathway (MEP), also known as the deoxysylulose phosphate pathway (DOXP), which operates in many bacteria, plant chloroplast, and some eukaryotic parasites .
The MAD route involves a series of enzymatic reactions whose rate-limiting step is the reduction of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to a mevalonic acid catalyzed by the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R) . Therefore HMG-R is considered the rate-limiting enzyme of this route. In insects, several studies of the mavalonate pathway have been motivated by its role in providing important bioactive molecules such as juvenile hormones  and aggregation pheromones (reviewed in ).
Following the mevalonate pathway, the two C5 isomeric products (IPP and DMAPP) are condensed into the monoterpe precursor (C10), geranyl diphosphate (GPP), catalyzed by the geranyl diphosphate synthase (GPPS). Further condensation of the C5 units with the GPP molecule convert into farnesyl diphosphate (FPP) (C15) and geranylgeranyl pyrophosphate (GGPP) (C20), the precursors of sesqui- and diterpene compounds among others (reviewed in ). These reactions are catalyzed by farnesyl diphosphate synthase (FPPS) and geranyl geranyl diphosphate synthase (GPPS), respectively, also known as short-chain prenyltransferases . By applying molecular methods, these enzymes have been sequenced in several insects [34–38], some of which were directly involved in pheromone production [39, 40]. Based on this, it is conceivable to hypothesize that pheromone biosynthesis in L. longipalpis involves the mevalonate pathway as well as prenyltransferase activity.
Molecular technologies allow the exploration of biosynthetic pathways by recognizing the expression of their enzymatic machineries . This method has been successfully employed in recent studies that have analyzed enzymes in pheromone production of moths [42, 43] and diterpene secretion in termites . Additionally, the construction and sequencing of cDNA libraries for L. longipalpis has been used to obtain genetic profiles from whole insects  as well as from separate tissues such as salivary glands , midgut [47, 48] and more recently, the male reproductive organ .
Besides the chemical identity of pheromones, there is no information about the molecular basis of pheromone production in L. longipalpis. In this respect, the main objective of this study was to identify the genes expressed in the pheromone gland and flanking tissues of one L. longipalpis population and consequently generate the first sequence catalogue for a specialized tissue where the pheromone (S)-9-methylgermacrene-B is produced. For this purpose, we constructed a cDNA library from three abdominal segments, the 3rd, 4th (containing the pheromone gland) and 5th tergites. Analyses of the expressed sequence tags (ESTs) from these segments allowed us to compare their genetic profiles and expand the spectrum of molecules possibly associated with the terpenoid pheromone. In addition, this source can also serve as a basis for future molecular research on pheromone biosynthesis and contribute to the understanding of other aspects of this insect’s biology.