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Transcriptome exploration of the sex pheromone gland of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae)

Natalia González-Caballero1, Jesus G Valenzuela2, José MC Ribeiro2, Patricia Cuervo3 and Reginaldo P Brazil1*

Author Affiliations

1 Laboratório de Bioquímica e Fisiologia de Insetos, IOC, FIOCRUZ, Av. Brasil 4365, Manguinhos, Pav. Leônidas Deane, Sala 213, Rio de Janeiro, RJ, CEP: 21040-360, Brasil

2 Laboratory of Malaria and Vector Research, NIAID, NIH, Rockville, MD, USA

3 Laboratório de Pesquisa em Leishmaniose, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brasil

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Parasites & Vectors 2013, 6:56  doi:10.1186/1756-3305-6-56

The electronic version of this article is the complete one and can be found online at: http://www.parasitesandvectors.com/content/6/1/56


Received:6 December 2012
Accepted:19 February 2013
Published:7 March 2013

© 2013 González-Caballero et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Molecules involved in pheromone biosynthesis may represent alternative targets for insect population control. This may be particularly useful in managing the reproduction of Lutzomyia longipalpis, the main vector of the protozoan parasite Leishmania infantum in Latin America. Besides the chemical identity of the major components of the L. longipalpis sex pheromone, there is no information regarding the molecular biology behind its production. To understand this process, obtaining information on which genes are expressed in the pheromone gland is essential.

Methods

In this study we used a transcriptomic approach to explore the pheromone gland and adjacent abdominal tergites in order to obtain substantial general sequence information. We used a laboratory-reared L. longipalpis (one spot, 9-Methyl GermacreneB) population, captured in Lapinha Cave, state of Minas Gerais, Brazil for this analysis.

Results

From a total of 3,547 cDNA clones, 2,502 high quality sequences from the pheromone gland and adjacent tissues were obtained and assembled into 1,387 contigs. Through blast searches of public databases, a group of transcripts encoding proteins potentially involved in the production of terpenoid precursors were identified in the 4th abdominal tergite, the segment containing the pheromone gland. Among them, protein-coding transcripts for four enzymes of the mevalonate pathway such as 3-hydroxyl-3-methyl glutaryl CoA reductase, phosphomevalonate kinase, diphosphomevalonate descarboxylase, and isopentenyl pyrophosphate isomerase were identified. Moreover, transcripts coding for farnesyl diphosphate synthase and NADP+ dependent farnesol dehydrogenase were also found in the same tergite. Additionally, genes potentially involved in pheromone transportation were identified from the three abdominal tergites analyzed.

Conclusion

This study constitutes the first transcriptomic analysis exploring the repertoire of genes expressed in the tissue containing the L. longipalpis pheromone gland as well as the flanking tissues. Using a comparative approach, a set of molecules potentially present in the mevalonate pathway emerge as interesting subjects for further study regarding their association to pheromone biosynthesis. The sequences presented here may be used as a reference set for future research on pheromone production or other characteristics of pheromone communication in this insect. Moreover, some matches for transcripts of unknown function may provide fertile ground of an in-depth study of pheromone-gland specific molecules.

Keywords:
Lutzomyia longipalpis; Male pheromone gland; Transcriptome; Mevalonate pathway

Background

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 [6]. These attractants are synthesized by specialized gland cells located beneath the cuticle in abdominal segments of adult insects [7]. 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 [8]. Once the pheromone is produced, it is disseminated to the environment through papular structures located in pale abdominal spots [9]. 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 [10].

Lutzomyia longipalpis is apparently a complex of sibling species ([11], reviewed in [12]), 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 [20]. 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 [16]. 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[21]. 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[22], 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 [25]. They are derived from a five-carbon precursor isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) [26]. These compounds are produced by a cellular metabolic pathway, the mevalonate route or mevalonate dependent pathway (MAD) [27], 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 [28].

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) [29]. 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 [30] and aggregation pheromones (reviewed in [31]).

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 [32]). These reactions are catalyzed by farnesyl diphosphate synthase (FPPS) and geranyl geranyl diphosphate synthase (GPPS), respectively, also known as short-chain prenyltransferases [33]. 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 [41]. 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 [44]. Additionally, the construction and sequencing of cDNA libraries for L. longipalpis has been used to obtain genetic profiles from whole insects [45] as well as from separate tissues such as salivary glands [46], midgut [47,48] and more recently, the male reproductive organ [49].

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.

Methods

Insects

A L. longipalpis (Diptera: Psychodidae) one-spot population was collected from Gruta da Lapinha, Municipality of Lagoa Santa Minas Gerais, Brazil (19° 38S; 43° 53 W). This population was colonized according to [50] at a temperature of 26°C, 80% relative humidity, and a 12:12 LD photoperiod. We used sand flies from the 1st to 7th generations after checking for their (S)-9-methylgermacrene-B production according to the procedures indicated in [17]. This population was maintained at the Laboratorio de Bioquímica e Fisiologia de Insetos at the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. A permit for sand fly collection was obtained from the Brazilian Ministry of Environment (SISBIO#26066-1). Tissues were extracted from male sand flies fed on a 70% sucrose solution for up to 7 days post-emergence.

Dissections and RNA preparations

Three different tissues were carefully dissected, the 4th abdominal segment (LL-phg) containing the pheromone gland, as well as the 3rd (LL3-seg) and 5th (LL5-seg) abdominal tergites (Figure 1). After removing the midgut, the dissected segments were thoroughly washed in sterilized 0.9% saline solution in order to minimize any risk of contamination. One hundred units of the 3rd abdominal segment were pooled together and preserved in an RNA preservation solution (RNAlater/Ambion) to obtain 200 ng of mRNA, the mRNA quantity used in [51]. The same procedure was repeated for each of the tissues analyzed. Messenger RNA from each tissue was purified with the Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, CA, USA) and kept at −80°C.

thumbnailFigure 1. Tissues of L. longipalpis male included in this study. The stereoscope image of a L. longipalpis male illustrates the three abdominal segments (3rd, 4th and 5th) analyzed. Characteristics of these segments are shown by scanning electron microscopy. Pheromone disseminating structures, which are present in the 4th abdominal segment are indicated by the arrowhead. Macrotrichias present in the 3rd and 5th segment are indicated by the asterisks, and microtrichias, which are present in all abdominal segments, are indicated by the star.

Library construction and DNA sequencing

Following [47,51,52], a full-length, enriched, directionally cloned cDNA library was generated for each abdominal segment using the SMART (SwitchingMechanismAt 5end ofRNATranscript) cDNA library construction kit (Clontech Laboratories, Montana View, CA). This technique uses oligoribonucleotide (SMART IV) to attach an identical sequence at the 5end of each reverse-transcribed cDNA strand. This sequence is then utilized in subsequent PCR reactions and restriction digests.

First-strand cDNA was synthesized using PowerScript reverse transcriptase at 42°C for 1 h in the presence of SMART IV and CDS III (3) primers. Second-strand synthesis was performed by a long-distance PCR-based protocol using Advantage Taq polymerase (Clontech) mix in the presence of the 5 PCR prime and the CDS III (3) primer. These two primers create SfiI A and B restriction enzyme sites at the ends of the nascent cDNA. Double-strand cDNA synthesis was carried out on a Perkin Elmer 9700 Thermal cycler (Perkin Elmer Corp., Foster City, CA, USA) in the following conditions: 95°C for 1 min, 19 cycles of 95°C for 10 s, and 68°C for 6 min. Double-stranded cDNA was immediately treated with proteinase K (0.8 μg/ml) at 45°C for 20 min, and the enzyme was removed by ultrafiltration though a Microcon YM-100 centrifugal filter device (Amicon Inc., Beverly, California, USA). The cleaned, double-stranded cDNA was then digested with SfiI at 50°C for 2 h, followed by size fractionation on a ChromaSpin-400 column (Clontech Laboratories, Mountain View,CA) into small (S), medium (M), and large (L) transcripts. The profile of the fractions was checked on a 1.1% agarose gel, and fractions containing cDNAs of more than 400 bp were pooled and concentrated using a Microcon YM-100.

The cDNA was ligated into the λ TripIEx2 vector (Clontech), and the resulting ligation reaction was packed using the Gigapack® Gold III Plus packaging extract (Stratagene, La Jolla, California, USA) according to the manufacturer’s instructions. The package library was plated by infecting log-phase XL1-Blue Escherichia coli cells (Contech). The percentage of recombinant clones was determined by blue-white selection screening on LB plates containing X-gal/IPTG. Recombinant plaques were selected and picked using a sterilized wooden stick and placed into 75 μL of ultrapure water in a 96-well v-bottom plate. The plates were covered and placed on a gyrating shaker for 30 min at room temperature. The phage suspension was either immediately used for PCR or stored at 4°C for future use. The inserts were amplified using specific library vector primers, PT2F1 (5-AAGTACTCTAGCAATTGTGAGC-3) and PT2R1 (5-CTCTTCGCTACGCCAGCTG-3), flanking the inserted cDNA. The PCR conditions were: 1 cycle of 75°C for 3 min; 1 cycle of 94°C for 3 min; 33 cycles of 94°C for 1 min, 49°C for 1 min, 72°C for 2 min, and 72°C for 7 min. We analyzed the quality of the PCR products by running 5 μL of the sample on 1.1% agarose gel and revealing them with SYBR Green.

In order to clean off salts, dNTPs, and primers, the PCR products were washed with ultrapure water three times using ExcelaPure 96-well UF PCR purification plates (Edge Biosystems, Gaithersburg, MD). Approximately 200–250 ng of each PCR product was then transferred to Thermo-Fast 96-well PCR plates (ABgene Corp, Epson, Surrey, UK) and shipped for sequencing to the Rocky Mountain Laboratories Genomics Unit as described in [52] along with the PT2F3 (5-TCTCGGGAAGCGCGCCATTGT-3) primer. Sequencing reactions were prepared according to the manufacturer’s protocol (BigDye®Terminator, Applied Biosystems, Inc.) by adding 1 μL ABI BigDye® Terminator ready reaction mix v3.1 (P/N 4336921), 1.5 μL 5x ABI sequencing buffer (P/N 4336699), and 3.5 μL of water for a final volume of 10 μL. Cycle sequencing was performed at 96°C for 10 sec, 50°C for 5 sec, 60°C for 4 min for 27 cycles on either a Bio-Rad Tetrad 2 (Bio-Rad Laboratories, Hercules, CA) or ABI 9700 thermal cycler (Applied Biosystems, Inc.). Fluorescently labeled extension products were purified following Applied Biosystems’ BigDye® XTerminator™ purification protocol and subsequently processed on an ABI 3730xL DNA Analyzer (Applied Biosystems, Inc.). Fluorescently labeled extension products were purified following the Applied Biosystems BigDye® XTerminator™ purification protocol and subsequently processed on an ABI 3730xL DNA Analyzer (Applied Biosystems, Inc.).

Bioinformatics analyses

Primers or vector sequences were removed from the expressed sequence tags (EST) and then assembled into contigs using a CAP assembly program [53]. As previously described, BLAST packages and ClustalW software were used to compare and align sequences, respectively [54]. The BLASTtx algorithms were performed for functional annotation of each transcript searching against the NCBInr protein and Gene Ontology databases [55]. Conserved protein domains were predicted using reverse position specific BLAST (rpsBLAST), which searches for matches in the conserved domain database CDD [56] as well as the Pfam [57], SMART [58], and KOG [59] databases. Subsets of several organism proteomes available in the NCBI, ENSEMBL, or VectorBase were also searched. Phylogenetic analysis and statistical neighbor-joining bootstrap tests were performed using MEGA package [60]. The presence of a signal peptide was investigated using the SignalP server. Beginning with methionine found in the first 300 predicted amino acids, expressed sequence tag (EST) segments were examined every three-frame translations. Protein O-glycosylation sites were predicted using the NetOGlyc 26 software. Statistical significance in the number of transcripts per contigs, between tissues, was analyzed using a Chi-square test.

Results and discussion

Sequences overview

In total 3,547 phage plaques were randomly selected and sequenced, of which approximately 1,200 came from each of the three abdominal segments and were sequenced separately. After Sanger sequencing, a total of 2,502 high quality sequences were obtained. From these sequences, 1,387 clusters or contiguous sequences were assembled, of which 1,273 were represented by a single EST or singleton (Table 1). We use the term “cluster” or “contig” to refer to consensus sequences and singletons from this point on. A putative function was assigned to each contig based on the sequence homology to the databases mentioned in the methods section.

Table 1. Summary of the sequence results from L. longipalpis pheromone gland and adjacent tissues

Sequences of most of the contigs (751) had a significant BLAST match (E-val < 10E-5) according to all reference databases, whereas 636 contigs, mostly singletons, presented low or no homology to the consulted databases. A large portion of the low homology transcripts may represent untranslated regions of genes or novel L. longipalpis-specific gene products.

Each contig was assigned a possible function and grouped into functional categories according to the sequence homology to molecules identified by the BLAST results (E-value of less than 10E-5) from the NCBI non-redundant protein, the Gene Ontology and the conserved domain databases. Figure 2 shows the sequence abundance compared by tissue of origin across each functional category. This comparison was made after summing the contig sequences within each functional category for each tissue.

thumbnailFigure 2. Distribution of the number of ESTs with homology to previously described proteins grouped into functional classes from the three tissues: LL4SEG (4th segment containing the pheromone gland), LL3SEG (3rd abdominal segment) and LL5SEG (5th abdominal segment). Functional classification of the transcripts was based on their sequence similarity to the proteins in the reference databases. A Chi-square test was performed for each EST tissue group across the functional categories to examine the differences between tissues. The double-asterisks represents functional classes where both differences in the number of ESTs were statistically significant (4th and 3rd; 4th and 5th) and the single asterisk represents functional classes where only one difference in the number of ESTs was statistically significant.

Although this histogram only shows the molecules identified by the sequence homology, it may approximately describe the biochemical characteristics of each tissue. We found more ESTs on the 4th abdominal segment than the 3rd and 5th segments in some functional categories. For example, in the protein synthesis functional class we observed an increase of 35% in the number of ESTs on the 4th segment relative to the 3rd, and an increase of 31% on the same segment relative to the 5th. These differences were statistically significant (4th and 3rd segments, χ2== 7.8, p-value = 5.E-03; 4th and 5th segments, χ2 = 3.13, p-value = 8.E-02). Similarly, in the energy metabolism functional class the greater number of ESTs found on the 4th abdominal segment relative to the 3rd and 5th segments were also statistically significant (4th seg and 3rd seg χ2 = 7.88 and p = 5E-03; 4thseg and 5thseg χ2 = 12.37 and p = 4E-04). This is interesting considering greater production of proteins in the 4th abdominal segment may be related to the pheromone biosynthetic machinery and the consequent energy cost. We also observed the same pattern of a greater number of ESTs on the 4th abdominal segment for the lipid metabolism functional class, though the difference was statistically significant between the 4th and 5th segments only (4th seg and 3rd seg, χ2 = 0,17, p = 6,8E-01; 4th seg and 5th seg, χ2 = 20,83 p = 5.E-06). Similarly, we also observed more ESTs of the detoxification/oxidation functional class on the 4th abdominal segment than the 3rd or 5th segments where the difference was statistically significant with the 5th segment only (4th seg and 3rd seg, χ2 = 2,12, p = 1E-01; 4th seg and 5th seg, χ2 = 23,68 p = 1.E-06). Figure 2 highlights these functional classes and differences. These are not surprising findings considering the lipid metabolism and detoxification/oxidation machinery functional classes may include genes potentially associated to pheromone biosynthesis. The assembled contigs and BLAST results described in this paper are available in the hyperlinked Additional file 1.

Additional file 1. Excel File with assembled contigs and BLAST results.

Format: XLSX Size: 705KB Download fileOpen Data

Transcripts possibly representing enzymes of the classical isoprenoid pathway

In looking further into the group of sequences of the lipid metabolism and detoxification/oxidation functional class, we identified molecules grouped into 10 contigs potentially representing four of the seven enzymes of the mevalonate pathway. Additionally, we also found sequences similar to farnesyl diphosphate synthase (intermediate metabolism functional class) and NADP + dependent farnesol dehydrogenase of the isoprenoid pathway (Table 2). All these potentially isoprenoid-pathway related sequences were found on the 4th abdominal segment only. These were not such surprising findings considering, as observed in other insects, the site where terpenoid production occurs activates the mevalonate pathway genes [44,61,62]. Figure 3 shows the classical isoprenoid biosynthetic pathway adapted from the 00900 interactive map of the Kyoto Encyclopedia of genes and genomes KEGG [63]. Enzymes for which transcripts have been identified are highlighted in the red boxes of the same figure.

Table 2. Putative L. longipalpis pheromone gland-associated proteins; best matched results with corresponding E values from BLAST inquiries of a GenBank derived non-redundant protein database (NR) and Swiss protein database

thumbnailFigure 3. Scheme of the classical isoprenoid pathway exhibiting enzymes for which transcripts have been identified. Enzymes for which transcripts have been identified in the L. longipalpis pheromone gland only are highlighted in red.

3-hydroxy-3-methyl-glutaryl-CoA reductase – HMG-R (EC 1.1.1.34)

We identified sequences grouped in two clusters (contigs 215 and 400) potentially encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). This enzyme has two known forms, Class I and Class II, which are present in eukaryotes and prokaryotes catalyzing the synthesis of mevalonate [64]. Although the sequences found in our library seem to be incomplete, they presented high homology to the HMG-Rs described in other insects such as Culex quinquefasciatus and Aedes aegypti (See Additional file 1 for BLAST results). They putatively encode part of the C-terminal portion of the enzyme, which is a highly conserved region containing the catalytic domain [65]. The alignment of the HMG-R C-terminal sequence of the longest transcript (contig 215) and the homologous sequences of other insects is shown in Figure 4. The conserved motifs common to HMG-R class I and II [64] are also displayed in the same figure. These motifs include the ENVIG sequence, which has a role in the HMG-CoA binding, and the DAMGXN motif, which has a role in the NADP(H) binding. The LLphg-contig_400 sequence was not used in this analysis due to its incompleteness. The differentiated expression for the gene encoding HMG-R and its links to terpenoid pheromone production have been reported in various coleopteran species such as Anthonomus grandis[66], Ips paraconfusus[67], Ips pini[68] and Dendroctonus jeffreyi[69,70].

thumbnailFigure 4. 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-R) sequence analysis. Sequence alignment of Culex quinquefasciatus (Cq), Aedes aegypti (Aa), and L. longipalpis (LLphg-contig_215) HMG-R. Identical residues are highlighted in black and similar residues highlighted in gray (>70%). The illustrated sequences represent a subset of this alignment and include residues that extend from positions four hundred to seven hundred. The predicted HMG-CoA binding motif (ENVIG) is shown in the blue box and the predicted NADP(H) binding motif (DAMGXN) is shown in the green box. The accession numbers of the sequences used are between parentheses.

Phosphomevalonate kinase - PMK (EC 2.7.4.2)

A unique phosphomevalonate kinase transcript (contig 437) was identified with a predicted peptide sequence of 187 amino acids. The role of this enzyme is to catalyze the reversible reaction of mevalonate-5-phosphate and ATP to produce mevalonate-5-diphosphate as well as ADP. Sequence searches against the consulted databases presented the highest similarity score to the PMK of Aedes aegypti (XP_001664006), Culex quinquefasciatus (XP_001866014) and to the PMK found in Bombix mori (BAF62110). Figure 5A also exhibits the comparison of our sequences to the PMK sequence found in the specialized tissues for pheromone production of Dendroctonus pondesorase[61]. Protein domains directly involved in mevalonate 5-phosphate phosphorylation [71,72] and ATP binding [73] were recognized in the predicted amino acid sequence of PMK (Figure 5B).

thumbnailFigure 5. Phosphomevalonate kinase (PMK) sequence analysis. (A) Neighbor-joining tree of putative PMK from the L. longipalpis pheromone gland (LLphg-contig_437) Aedes aegypti (Aa), Culex quinquefasciatus (Cq), Dendroctonus pondersae (Dp) and Bombix mori (Bm). The accession numbers of the sequences used are between parentheses and node support is indicated by the bootstrap values. (B) Sequence alignment of PMK sequences. Identical residues are highlighted in black and similar residues highlighted in gray (>70%). Portions of the N-terminal region of these putative proteins with the phosphate binding loop is shown in the blue box and the putative mevalonate 5-phosphate binding site in the green boxes.

Diphosphomevalonate decarboxylase – DMD (EC 4.1.1.33)

From contig 397 we identified a short cDNA fragment having the putative domain of diphosphomevalonate decarboxylase according to the KOG database. Despite its incompleteness, we have included this sequence in our analysis considering it may be used in future studies. This enzyme, also known as mevalonate diphosphate decarboxylase (MDD) or mevalonate pyrophosphate decarboxylase (MPD) performs the crucial step in isoprenoids biosynthesis [74] catalyzing the ATP dependent decarboxylation of the mevalonate 5-diphosphate (MDP) to yield isopentenyl diphosphate (IPP) [75]. The transcript for this enzyme was also described in the midgut of male Ips pini, where monoterpenoid pheromone production occurs [76].

Isopentenyl diphosphate delta isomerase – IDI (EC 5.3.3.2)

This enzyme catalyzes an essential activation in the isoprenoid biosynthetic pathway by converting isopentenyl diphosphate isomerase (IPP) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). IPP and DMAPP are the assembly blocks of isoprenoid compounds. We identified thirteen sequences grouped into six contigs possibly representing IDI. Contrary to the singletons (contig 56 and 118), the remaining contigs were assembled using more than one sequence. Contigs number 53, 54, and 117 were assembled with three sequences each, while contig 55 resulted from two sequences. The longest transcripts accessed from our library were compared to isopentenyl diphosphate delta isomerase found in the genome of Aedes aegypti (XP_001657533), Culex quinquefasciatus (XP_001868438) and expressed in the corpora allata of Bombix mori (NP_001040323.2). We also compared our transcripts to IDI sequences identified in coleopteran insects such as Dendroctonus jeffreyi (AAX78437) and Dendroctunus ponderosae (AFI45054) (Figure 6). Our phylogenetic analysis separated the L. longipalpis IDI-like sequences into two groups (Figure 6B), which may indicate that two forms of IDI are probably occurring in this species. The catalytic residues (reviewed in [77]) are shown in Figure 6A. The same figure exhibits the high level of similarity and identity among the sequences.

thumbnailFigure 6. Isopenteyl diphosphate isomerase (IDI) sequence analysis. (A) Sequence alignment of IDIs from L. longipalpis (contigs 53, 54, 55 and 117) to reference sequences. Identical residues are highlighted in black and similar residues highlighted in gray (>70%). (B) Neighbor-joining tree of IDI full transcripts of L. longipalpis pheromone gland (contigs 53, 54, 55 and 117), Dendroctonus jeffreyi (Dj), Dendroctonus ponderosae (Dp), Bombix mori (Bm), Culex Aedes aegypti (Aa) and quinquefasciatus (Cq). The accession numbers of the sequences used are between parentheses and the node support indicated by the bootstrap values.

Farnesyl diphosphate synthase - FPPS (EC 2.5.1.1./2.5.1.10)

In the group of sequences classified as part of the intermediate metabolism functional class we identify molecules from three contigs (93, 94 and 257) potentially encoding farnesyl diphosphate synthase, FPPS. Enzymes catalyzing the consecutive condensation of IPP with DMAPP are known as prenyltransferases or isoprenyl diphosphate synthase [33]. Geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), and geranylgeranyl diphosphate synthase (GGPPS) are classified as short chain prenyltransferases and are named according to their final product: geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) (reviewed in [78]). In this study we identified FPPS and no GPPS, which may indicate that FPPS is possibly involved in the synthesis of both GPP and FPP. This dual enzyme activity has been proposed and supported in independent studies [40,79-81]. Since the first characterization of FPPS in the moth Agrotis ipsilin[82], searches for this enzyme have been conducted in other species [35,83,84] and extended to other orders such as Coleoptera [79], Hemiptera [40,81,85], as well as Diptera [36]. Although FPPS characterizations have been traditionally associated to juvenile hormone (JH) biosynthesis (reviewed in [30]), representations of this enzyme have also been related to terpenoid-pheromone production in some insects [40,79,86]. A comparison of the L. longipalpis FPPS from contigs 93 and 94 to homologous sequences from other insects is shown in Figure 7. This figure also exhibits the characteristic aspartate-rich motifs, which play an essential role in substrate binding and catalysis [87,88].

thumbnailFigure 7. Farnesyl diphosphate synthase (FPPS) sequence analysis. (A) Subset of the sequence alignment of FPPS including residues extending from positions one hundred to four hundred forty two. Sequence alignment of the largest predicted FPPS sequences of our library (contig 94 and 93) and Aedes aegypti (Aa), Drosophila melanogaster (Dm), Glossina morsitans morsitans (Gm), Dendroctonous ponderosae (Dp), and Dendroctonus jeffreyi (Dj) sequences. Identical residues are highlighted in black and similar residues highlighted in gray. The aspartate-rich motifs are shown in a blue square. (B) Neighbor-joining tree of FPPS sequences of L. longipalpis pheromone gland (contigs 93 and94), Aedes aegypti (Aa), Drosophila melanogaster (Dm), Glossina morsitans morsitans (Gm), Dendroctonus jeffreyi (Dj), Dendroctonus ponderosae (Dp). The accession numbers of the sequences used are between parentheses and the node support indicated by the bootstrap values.

Farnesol dehydrogenase - FDH

From contigs 314 and 333 we accessed two full-length cDNAs having 771 bp ORFs encoding predicted peptides of 247-amino acids each. The predicted protein sequences were associated to the short chain dehydrogenase/reductase (SDR) family as indicated by the SwissProt match. Additionally, by searching the NR database of the NCBI, our sequences presented a high degree of similarity to the NADP + −dependent farnesol dehydrogenase 2 (71-72%) and NADP + −dependent farnesol dehydrogenase 1 (69-70%) of Aedes aegypti (see Additional file 1 for blast results). This type of oxidoreductase has been previously characterized as being responsible for farnesol oxidation [89]. Figure 8 compares putative L. longipalpis FDH sequences with the previously described enzymes and also exhibits amino acid motifs of the enzymes’ active site [90].

thumbnailFigure 8. Farnesol dehydrogenase FDH sequence analysis. (A) Neighbor-joining tree and (B) sequence alignment of two Lutzomyia longipalpis farnesol dehydrogenases sequences and previously described enzymes from Culex quinquefasciatus (Cq) and Aedes aegypti (Aa). Identical residues are highlighted in black and similar residues in gray.

This is a first study describing transcripts potentially related to enzymes making up the mevalonate pathway and prenyltransferase in L. longipalpis. Considering we identified these transcripts from a specialized tissue where terpenoid pheromone production occurs, these genes may be the subject of future research addressing their relationship with pheromone production.

Transcripts associated with secreted proteins

From the sequences obtained from all three tissues containing peptide signals, an indication of secretion (Table 3), we identified molecules with predicted protein domains of odorant/pheromone binding proteins and juvenile hormone binding proteins (JHBP).

Table 3. Putative L. longipalpis proteins; best matched results and corresponding E values from BLAST inquiries of a PFAM database

3.1 Transcripts related to insect odorant binding protein (OBP)

Members of this family are ubiquitous in insects and are specialized in carrying small hydrophobic ligands through aqueous media [91]. According to PFAM database searches, sequences grouped in seven contigs (78, 79, 80, 81, 1059, 954 and 1236) presented conserved domains of pheromone/odor binding proteins. Additionally, sequences from contig 954 and 1236 presented similarity to the 99a OBP sequence described in C. quinquefasciatus (EDS45783) deposited in the NR database. Another group of sequences reunited in contigs 95, 96, and 98 also presented a high degree of similarity to proteins referred as OS-D proteins, chemosensory proteins (CSPs) or sensory appendage proteins (SAPs), based on their association with insect sensory organs [92]. Given these molecules are highly conserved across species [92], our finding led us to consider the possibility that they may also facilitate pheromone transportation between the pheromone gland and the external environment, as in moths [93-95].

Juvenile hormone binding protein (JHBP)

Ten molecules were identified as probable haemolymph juvenile hormone binding proteins (JHBP). It is known that JHBPs transport the highly hydrophobic insect juvenile hormone (JH) from the most important site of its synthesis (the corpora allata) to target tissues [96,97]. By analogy to the role of JHs in the regulation of pheromone production in beetles, cockroaches, and moths [98-101] the possible expression of JHBP in the tissues analyzed here may represent trace evidence of a similar function in L. longipalpis. However, it is also possible that due to the chemical similarity between the JHs and the L. longipalpis sesquiterpene pheromone, the JHBP gene products might bind both JH and L. longipalpis pheromones.

Transcripts related to detoxification/oxidation machinery

Cytochrome P450

Another interesting group of transcripts were those encoding putative cytochrome P450 enzymes. These sequences were grouped into thirteen contigs obtained from all three tissues used in this analysis (Table 4). Cytochrome P450 (CYP) comprises a large family of heme-thiolate enzymes that metabolize a wide range of endogenous and exogenous compounds. In insects, these enzymes are found in virtually all tissues [102] and catalyze the metabolism of exogenous substances and participate in the biosynthesis and degradation of juvenile hormones, ecdysteroids, and pheromones [103-105]. In our work, two P450 protein subfamilies were identified: CYP4/CYP19/CYP26 and CYP3/CYP5/CYP6/CYP9, according to the eukaryotic orthologous group database (KOG). The CYP4/CYP19/CYP26 subfamily was the most abundant and it was represented in contigs 39, 40, 41, 42, 43, 615, and 792. The occurrence of a higher abundance of transcripts for CYP4 and CYP3 P450 protein families was also recently described in a transcriptome study of different Dendroctonus ponderosae tissues, including the terpenoid pheromone production site [61].

Table 4. Putative L. longipalpis proteins; best matched results and corresponding E values from BLAST inquiries of a GenBank derived non-redundant protein database (NR), KOG and Swiss prot databases

Glutathione S-transferase

Another noticeable gene involved in biotransformation is the glutatione S-transferase. We identified sequences grouped into six contigs (156, 157, 158, 774, 789, and 1066) with high similarity to glutation S-transfererase previously described in L. longipalpis (Table 4 and Additional file 1). Besides its important role for detoxification, especially in phytophagous insects [106], these molecules have also been described as odor degradation enzymes in some insects [107].

Juvenile hormone epoxide hydrolase - JHEH

After consulting the SWISSP database two transcripts presented high sequence similarity to juvenile hormone epoxide hydrolase (JHEH), one obtained from the 4th abdominal segment library containing the pheromone gland (contig 367), and the other from the 3rd abdominal segment library (contig 868). The JHEHs together with the juvenile hormone esterase (JHE) are known as enzymes having a role in JH degradation [108].

Conclusion

This study has confidently generated a list of transcripts expressed in the pheromone gland and adjacent tissues. Molecules associated to enzymes of the mevalonate pathway were found only in the 4th abdominal segment (containing the pheromone gland). The identification of these enzymes was facilitated by the conservation of the mevalonate pathway between insect species and vertebrates [30]. However, this conservation is not present in the later steps of isoprenoid compound production, which involves several paths leading to different final products. With the exception of juvenile hormones [30], there is little available information on the biosynthesis of cyclic sesquiterpene or homosesquiterpene compounds in insects. Such advances in this field could contribute to the identification of the specialized enzymatic machinery through a classical homology inference. In this context, the unmatched groups of sequences we obtained from the 4th abdominal segment constitute a useful resource for future explorations of the specialized enzymatic components of (S)-9-methylgermacrene-B production.

Furthermore, in this work we also present a set of interesting genes potentially related to the insect OBP protein family, including molecules similar to chemosensory proteins. This finding also provides an interesting subject for future studies considering its potential implication for pheromone transportation [93] or other functions [109]. This transcriptome sequence resource may also provide new molecular tools for future studies of other terpene-producing L. longipalpis populations. Finally, our results may also serve to identify potential targets for genetic manipulation techniques for population monitoring and control of L. longipalpis, considering this insect carries the parasite causing an important and neglected tropical disease, visceral leishmaniasis.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Conceived and designed the experiments: NGC, PC and RPB. Performed the experiments: NGC and JGV. Analyzed the data: NGC, JGV and JMR. Contributed reagents/materials/analysis tools: RPB, JGV and JMR. Wrote the paper: NGC, PC and RPB. All authors read and approved the final version of the manuscript.

Acknowledgements

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Division of Intramural Research, the National Institute of Allergy and Infectious Disease. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We are thankful to the Research Technologies Section, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA, for DNA sequencing. We are also grateful to Dr. Jacenir Reis Santos-Mallet, Dr. Susana Corte-Real, and Rozana Corte-Real for their collaboration, and the multiuser platform for electron microscopy at the Instituto Oswaldo Cruz (FIOCRUZ) for the use of its scanning electron microscope. We would also like to acknowledge Francisco de Assis Marra for technical assistance, Mariel Figueroa, Caroline da Silva Moraes, and Andressa Fuzari Rodrigues for their valuable assistance in experimental work, and Nathalia Pinho de Souza for her help with the figures.

References

  1. Deane LM, Deane MP: Visceral leishmaniasis in Brazil: geographical distribution and transmission.

    Rev Inst Med Trop Sao Paulo 1962, 4:149-212. PubMed Abstract OpenURL

  2. Lainson R, Rangel EF: Lutzomyia longipalpis and the eco-epidemiology of American visceral leishmaniasis, with particular reference to Brazil: a review.

    Mem Inst Oswaldo Cruz 2005, 100(8):811-827. PubMed Abstract | Publisher Full Text OpenURL

  3. Palatnik-de-Sousa CB, Day MJ: One Health: the global challenge of epidemic and endemic leishmaniasis.

    Parasit Vectors 2011, 4(1):1-10. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  4. Bray DP, Alves GB, Dorval ME, Brazil RP, Hamilton JG: Synthetic sex pheromone attracts the leishmaniasis vector Lutzomyia longipalpis to experimental chicken sheds treated with insecticide.

    Parasit Vectors 2010, 3:16. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  5. Morton IE, Ward RD: Laboratory response of female Lutzomyia longipalpis sandflies to a host and male pheromone source over distance.

    Med Vet Entomol 1989, 3(3):219-223. PubMed Abstract | Publisher Full Text OpenURL

  6. Quinnell RJ, Dye C: An experimental study of the peridomestic distribution of Lutzomyia longipalpis (Diptera: Psychodidae).

    Bull Entomol Res 1994, 84(3):379-382. Publisher Full Text OpenURL

  7. Lane R, Souza Bernardes D: Histology and ultrastructure of pheromone secreting glands in males of the phlebotomine sandfly Lutzomyia longipalpis.

    Ann Trop Med Parasitol 1990, 84(1):53-61. PubMed Abstract OpenURL

  8. Spiegel CN, Batista-Pereira LG, Bretas JAC, Eiras ÁE, Hooper AM, Pelxoto AA, Soares MJ: Pheromone Gland Development and Pheromone Production in Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae).

    J Med Entomol 2011, 48(3):489-495. PubMed Abstract | Publisher Full Text OpenURL

  9. Spiegel CN, Brazil RP, Soares MJ: Ultrastructure of male sex pheromone glands in abdominal tergites of five Lutzomyia sandfly species (Diptera: Psychodidae).

    Arthropod Struct Dev 2002, 30(3):219-227. PubMed Abstract | Publisher Full Text OpenURL

  10. Ward R, Hamilton J, Dougherty M, Falcão A, Feliciangeli M, Perez J, Veltkamp C: Pheromone disseminating structures in tergites of male phlebotomines (Diptera: Psychodidae).

    Bull Entomol Res 1993, 83:437-445. Publisher Full Text OpenURL

  11. Lanzaro GC, Ostrovska K, Herrero MV, Lawyer PG, Warburg A: Lutzomyia longipalpis is a species complex: genetic divergence and interspecific hybrid sterility among three populations.

    AmJTrop Med Hyg 1993, 48(6):839-847. OpenURL

  12. Uribe S: The status of the Lutzomyia longipalpis species complex and possible implications for Leishmania transmission.

    Mem Inst Oswaldo Cruz 1999, 94:729-734. PubMed Abstract OpenURL

  13. Arrivillaga J, Mutebi JP, Piñango H, Norris D, Alexander B, Feliciangeli MD, Lanzaro GC: The taxonomic status of genetically divergent populations of Lutzomyia longipalpis (Diptera: Psychodidae) based on the distribution of mitochondrial and isozyme variation.

    J Med Entomol 2003, 40(5):615-627. PubMed Abstract | Publisher Full Text OpenURL

  14. Bauzer LGSR, Souza NA, Maingon RDC, Peixoto AA: Lutzomyia longipalpis in Brazil: a complex or a single species? A mini-review.

    Mem Inst Oswaldo Cruz 2007, 102(1):1-12. PubMed Abstract | Publisher Full Text OpenURL

  15. Maingon RDC, Ward RD, Hamilton JGC, Bauzer LGSR, Peixoto AA: The Lutzomyia longipalpis species complex: does population sub-structure matter to Leishmania transmission?

    Trends Parasitol 2008, 24(1):12-17. PubMed Abstract | Publisher Full Text OpenURL

  16. Hamilton JG, Maingon RD, Alexander B, Ward RD, Brazil RP: Analysis of the sex pheromone extract of individual male Lutzomyia longipalpis sandflies from six regions in Brazil.

    Med Vet Entomol 2005, 19(4):480-488. PubMed Abstract | Publisher Full Text OpenURL

  17. Hamilton JGC, Ibbotson HC, Hooper AM, Pickett JA: 9-Methylgermacrene-B is confirmed as the sex pheromone of the sandfly Lutzomyia longipalpis from Lapinha, Brazil, and the absolute stereochemistry defined as S.

    Chem Commun 1999, 23:2335-2336. OpenURL

  18. Hamilton JG: Sandfly pheromones. Their biology and potential for use in control programs.

    Parasite (Paris, France) 2008, 15(3):252-256. Publisher Full Text OpenURL

  19. Hamilton JGC, Dawson GW, Pickett JA: 3-Methyl-α-himachalene: Proposed structure for novel homosesquiterpene sex pheromone of Lutzomyia longipalpis (diptera: Psychodidae) from Jacobina, Brazil.

    J Chem Ecol 1996, 22(12):2331-2340. Publisher Full Text OpenURL

  20. Hamilton JG, Brazil RP, Maingon R: A fourth chemotype of Lutzomyia longipalpis (Diptera: Psychodidae) from Jaibas, Minas Gerais State, Brazil.

    J Med Entomol 2004, 41(6):1021-1026. PubMed Abstract | Publisher Full Text OpenURL

  21. Bray D, Ward R, Hamilton J: The chemical ecology of sandflies. In Olfaction in vector-host interactions. Volume 2. Edited by Takken W, Knols B. The Netherland: Wageningen Academic Publishers; 2010:203-215. OpenURL

  22. Ward RD, Hamilton JG, Dougherty M, Falcao AL: Pheromones in Old and New World sandflies (Diptera: Psychodidae).

    Ann Trop Med Parasitol 1991, 85(6):667-668. PubMed Abstract OpenURL

  23. Chelbi I, Bray DP, Hamilton JG: Courtship behaviour of Phlebotomus papatasi the sand fly vector of cutaneous leishmaniasis.

    Parasit Vectors 2012, 5:179. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  24. Chelbi I, Zhioua E, Hamilton JG: Behavioral evidence for the presence of a sex pheromone in male Phlebotomus papatasi scopoli (Diptera: Psychodidae).

    J Med Entomol 2011, 48(3):518-525. PubMed Abstract | Publisher Full Text OpenURL

  25. Langenheim JH: Higher plant terpenoids: a phytocentric overview of their ecological roles.

    J Chem Ecol 1994, 20(6):1223-1280. Publisher Full Text OpenURL

  26. Cane DE: Isoprenoid Biosynthesis: Overview. In Comprehensive Natural Products Chemistry Volume 2. Oxford: Pergamon Press; 1999:1-13. OpenURL

  27. Goldstein JL, Brown MS: Regulation of the mevalonate pathway.

    Nature 1990, 343(6257):425. PubMed Abstract | Publisher Full Text OpenURL

  28. Eisenreich W, Bacher A, Arigoni D, Rohdich F: Biosynthesis of isoprenoids via the non-mevalonate pathway.

    Cell Mol Life Sci 2004, 61(12):1401-1426. PubMed Abstract | Publisher Full Text OpenURL

  29. Bochar DA, Freisen J, Stauffacher CV, Rodwell VW, Otto M-C, Sir Derek B, Koji N: Biosynthesis of Mevalonic Acid from Acetyl-CoA. In Comprehensive Natural Products Chemistry. Oxford: Pergamon; 1999:15-44. OpenURL

  30. Bellés X, Martin D, Piulachs MD: The mevalonate pathway and the synthesis of juvenile hormone in insects.

    Annu Rev Entomol 2005, 50:181-199. PubMed Abstract | Publisher Full Text OpenURL

  31. Blomquist GJ, Figueroa-Teran R, Aw M, Song M, Gorzalski A, Abbott NL, Chang E, Tittiger C: Pheromone production in bark beetles.

    Insect Biochem Mol Biol 2010, 40(10):699-712. PubMed Abstract | Publisher Full Text OpenURL

  32. Nürenberg G, Volmer DA: The analytical determination of isoprenoid intermediates from the mevalonate pathway.

    Anal Bioanal Chem 2012, 402(2):671-685. PubMed Abstract | Publisher Full Text OpenURL

  33. Ogura K, Koyama T: Enzymatic aspects of isoprenoid chain elongation.

    Chem Rev 1998, 98(4):1263-1276. PubMed Abstract | Publisher Full Text OpenURL

  34. Sen SE, Cusson M, Trobaugh C, Beliveau C, Richard T, Graham W, Mimms A, Roberts G: Purification, properties and heteromeric association of type-1 and type-2 lepidopteran farnesyl diphosphate synthases.

    Insect Biochem Mol Biol 2007, 37(8):819-828. PubMed Abstract | Publisher Full Text OpenURL

  35. Kinjoh T, Kaneko Y, Itoyama K, Mita K, Hiruma K, Shinoda T: Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata.

    Insect Biochem Mol Biol 2007, 37(8):808-818. PubMed Abstract | Publisher Full Text OpenURL

  36. Sen SE, Trobaugh C, Béliveau C, Richard T, Cusson M: Cloning, expression and characterization of a dipteran farnesyl diphosphate synthase.

    Insect Biochem Mol Biol 2007, 37(11):1198-1206. PubMed Abstract | Publisher Full Text OpenURL

  37. Vandermoten S, Charloteaux B, Santini S, Sen SE, Béliveau C, Vandenbol M, Francis F, Brasseur R, Cusson M, Haubruge É: Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity.

    FEBS Lett 2008, 582(13):1928-1934. PubMed Abstract | Publisher Full Text OpenURL

  38. Zhang YL, Li ZX: Two different farnesyl diphosphate synthase genes exist in the genome of the green peach aphid, Myzus persicae.

    Genome 2008, 51(7):501-510. PubMed Abstract | Publisher Full Text OpenURL

  39. Gilg AB, Bearfield JC, Tittiger C, Welch WH, Blomquist GJ: Isolation and functional expression of an animal geranyl diphosphate synthase and its role in bark beetle pheromone biosynthesis.

    Proc Natl Acad Sci USA 2005, 102(28):9760-9765. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  40. Lewis MJ, Prosser IM, Mohib A, Field LM: Cloning and characterisation of a prenyltransferase from the aphid Myzus persicae with potential involvement in alarm pheromone biosynthesis.

    Insect Mol Biol 2008, 17(4):437-443. PubMed Abstract | Publisher Full Text OpenURL

  41. Morgan D: Biosynthesis in Insects. Great Britain: Royal Society of Chemistry; 2010. OpenURL

  42. Strandh M, Johansson T, Ahrén D, Löfstedt C: Transcriptional analysis of the pheromone gland of the turnip moth, Agrotis segetum (Noctuidae), reveals candidate genes involved in pheromone production.

    Insect Mol Biol 2008, 17(1):73-85. PubMed Abstract | Publisher Full Text OpenURL

  43. Vogel H, Heidel AJ, Heckel DG, Groot AT: Transcriptome analysis of the sex pheromone gland of the noctuid moth Heliothis virescens.

    BMC Genomics 2010, 11:29. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  44. Hojo M, Maekawa K, Saitoh S, Shigenobu S, Miura T, Hayashi Y, Tokuda G, Maekawa H: Exploration and characterization of genes involved in the synthesis of diterpene defence secretion in nasute termite soldiers.

    Insect Mol Biol 2012, 21(6):545-557. PubMed Abstract | Publisher Full Text OpenURL

  45. Dillon RJ, Ivens AC, Churcher C, Holroyd N, Quail MA, Rogers ME, Soares MB, Bonaldo MF, Casavant TL, Lehane MJ: Analysis of ESTs from Lutzomyia longipalpis sand flies and their contribution toward understanding the insect-parasite relationship.

    Genomics 2006, 88(6):831-840. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  46. Anderson JM, Oliveira F, Kamhawi S, Mans BJ, Reynoso D, Seitz AE, Lawyer P, Garfield M, Pham MV, Valenzuela JG: Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis.

    BMC Genomics 2006, 7(1):52. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  47. Jochim R, Teixeira C, Laughinghouse A, Mu J, Oliveira F, Gomes R, Elnaiem DE, Valenzuela J: The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies.

    BMC Genomics 2008, 9(1):15. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  48. Pitaluga AN, Beteille V, Lobo AR, Ortigao-Farias JR, Davila AM, Souza AA, Ramalho-Ortigao JM, Traub-Cseko YM: EST sequencing of blood-fed and Leishmania-infected midgut of Lutzomyia longipalpis, the principal visceral leishmaniasis vector in the Americas.

    Mol Genet Genomics 2009, 282(3):307-317. PubMed Abstract | Publisher Full Text OpenURL

  49. Azevedo RV, Dias DB, Bretas JA, Mazzoni CJ, Souza NA, Albano RM, Wagner G, Davila AM, Peixoto AA: The Transcriptome of Lutzomyia longipalpis (Diptera: Psychodidae) Male Reproductive Organs.

    PLoS One 2012, 7(3):e34495. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  50. Brazil RP, Brazil BG: Biologia de Flebotomíneos Tropicais. In Flebotomíneos do Brasil. Edited by Rangel E, Lainson R. Rio de Janeiro: Editora Fiocruz; 2003:257-274. OpenURL

  51. Valenzuela JG, Pham VM, Garfield MK, Francischetti IMB, Ribeiro JMC: Toward a description of the sialome of the adult female mosquito Aedes aegypti.

    Insect Biochem Mol Biol 2002, 32(9):1101-1122. PubMed Abstract | Publisher Full Text OpenURL

  52. Francischetti I, Anderson JM, Manoukis N, Pham VM, Ribeiro J: An insight into the sialotranscriptome and proteome of the coarse bontlegged tick, Hyalomma marginatum rufipes.

    J Proteomics 2011, 74(12):2892-2908. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  53. Huang X, Madan A: CAP3: A DNA sequence assembly program.

    Genome Res 1999, 9(9):868-877. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  54. Valenzuela JG, Francischetti I, Pham VM, Garfield MK, Ribeiro J: Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito.

    Insect Biochem Mol Biol 2003, 33(7):717-732. PubMed Abstract | Publisher Full Text OpenURL

  55. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT: Gene Ontology: tool for the unification of biology.

    Nat Genet 2000, 25(1):25. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  56. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Church DM, DiCuccio M, Edgar R, Federhen S, Helmberg W: Database resources of the national center for biotechnology information.

    Nucleic Acids Res 2005, 33(suppl 1):D39-D45. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  57. Bateman A, Birney E, Durbin R, Eddy SR, Howe KL, Sonnhammer ELL: The Pfam protein families database.

    Nucleic Acids Res 2000, 28(1):263-266. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  58. Schultz J, Copley RR, Doerks T, Ponting CP, Bork P: SMART: a web-based tool for the study of genetically mobile domains.

    Nucleic Acids Res 2000, 28(1):231-234. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  59. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN: The COG database: an updated version includes eukaryotes.

    BMC Bioinforma 2003, 4(1):41. BioMed Central Full Text OpenURL

  60. Kumar S, Tamura K, Nei M: MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment.

    Brief Bioinform 2004, 5(2):150-163. PubMed Abstract | Publisher Full Text OpenURL

  61. Keeling CI, Henderson H, Li M, Yuen M, Clark EL, Fraser JD, Huber DPW, Liao NY, Docking TR, Birol I: Transcriptome and full-length cDNA resources for the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major insect pest of pine forests.

    Insect Biochem Mol Biol 2012, 42(8):525-536. PubMed Abstract | Publisher Full Text OpenURL

  62. Keeling C, Blomquist G, Tittiger C: Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae).

    Naturwissenschaften 2004, 91(7):324-328. PubMed Abstract | Publisher Full Text OpenURL

  63. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M: KEGG for integration and interpretation of large-scale molecular data sets.

    Nucleic Acids Res 2012, 40(Database issue):D109-D114.

    http://www.genome.jp/kegg/pathway/map/map00900.html webcite

    PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  64. Bochar DA, Stauffacher CV, Rodwell VW: Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

    Mol Genet Metab 1999, 66(2):122-127. PubMed Abstract | Publisher Full Text OpenURL

  65. Hampton R, Dimster-Denk D, Rine J: The biology of HMG-CoA reductase: the pros of contra-regulation.

    Trends Biochem Sci 1996, 21(4):140. PubMed Abstract | Publisher Full Text OpenURL

  66. Taban AH, Fu J, Blake J, Awano A, Tittiger C, Blomquist GJ: Site of pheromone biosynthesis and isolation of HMG-CoA reductase cDNA in the cotton boll weevil, Anthonomus grandis.

    Arch Insect Biochem Physiol 2006, 62(4):153-163. PubMed Abstract | Publisher Full Text OpenURL

  67. Ivarsson P, Tittiger C, Blomquist C, Borgeson CE, Seybold SJ, Blomquist GJ, Högberg HE: Pheromone precursor synthesis is localized in the metathorax of Ips paraconfusus Lanier (Coleoptera: Scolytidae).

    Naturwissenschaften 1998, 85(10):507-511. Publisher Full Text OpenURL

  68. Hall GM, Tittiger C, Andrews GL, Mastick GS, Kuenzli M, Luo X, Seybold SJ, Blomquist GJ: Midgut tissue of male pine engraver, Ips pini, synthesizes monoterpenoid pheromone component ipsdienol de novo.

    Die Naturwissenschaften 2002, 89(2):79-83. PubMed Abstract | Publisher Full Text OpenURL

  69. Hall GM, Tittiger C, Blomquist GJ, Andrews GL, Mastick GS, Barkawi LS, Bengoa C, Seybold SJ: Male jeffrey pine beetle, Dendroctonus jeffreyi, synthesizes the pheromone component frontalin in anterior midgut tissue.

    Insect Biochem Mol Biol 2002, 32(11):1525-1532. PubMed Abstract | Publisher Full Text OpenURL

  70. Nardi J, Young AG, Ujhelyi E, Tittiger C, Lehane M, Blomquist G: Specialization of midgut cells for synthesis of male isoprenoid pheromone components in two scolytid beetles, Dendroctonus jeffreyi and Ips pini.

    Tissue Cell 2002, 34(4):221-231. PubMed Abstract | Publisher Full Text OpenURL

  71. Herdendorf TJ, Miziorko HM: Phosphomevalonate kinase: functional investigation of the recombinant human enzyme.

    Biochemistry 2006, 45(10):3235-3242. PubMed Abstract | Publisher Full Text OpenURL

  72. Miziorko HM: Enzymes of the mevalonate pathway of isoprenoid biosynthesis.

    Arch Biochem Biophys 2011, 505(2):131-143. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  73. Herdendorf TJ, Miziorko HM: Functional evaluation of conserved basic residues in human phosphomevalonate kinase.

    Biochemistry 2007, 46(42):11780-11788. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  74. Berges T, Guyonnet D, Karst F: The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity.

    J Bacteriol 1997, 179(15):4664-4670. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  75. Bloch K, Chaykin S, Phillips AH, De Waard A: Mevalonic acid pyrophosphate and isopentenylpyrophosphate.

    J Biol Chem 1959, 234(10):2595-2604. PubMed Abstract | Publisher Full Text OpenURL

  76. Eigenheer AL, Keeling CI, Young S, Tittiger C: Comparison of gene representation in midguts from two phytophagous insects, Bombyx mori and Ips pini, using expressed sequence tags.

    Gene 2003, 316:127-136. PubMed Abstract | Publisher Full Text OpenURL

  77. Berthelot K, Estevez Y, Deffieux A, Peruch F: Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis.

    Biochimie 2012, 94(8):1621-1634. PubMed Abstract | Publisher Full Text OpenURL

  78. Vandermoten S, Haubruge E, Cusson M: New insights into short-chain prenyltransferases: structural features, evolutionary history and potential for selective inhibition.

    Cell Mol Life Sci 2009, 66(23):3685-3695. PubMed Abstract | Publisher Full Text OpenURL

  79. Taban AH, Tittiger C, Blomquist GJ, Welch WH: Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil, Anthonomus grandis.

    Arch Insect Biochem Physiol 2009, 71(2):88-104. PubMed Abstract | Publisher Full Text OpenURL

  80. Vandermoten S, Santini S, Haubruge E, Heuze F, Francis F, Brasseur R, Cusson M, Charloteaux B: Structural features conferring dual geranyl/farnesyl diphosphate synthase activity to an aphid prenyltransferase.

    Insect Biochem Mol Biol 2009, 39(10):707-716. PubMed Abstract | Publisher Full Text OpenURL

  81. Ma GY, Sun XF, Zhang YL, Li ZX, Shen ZR: Molecular cloning and characterization of a prenyltransferase from the cotton aphid, Aphis gossypii.

    Insect Biochem Mol Biol 2010, 40(7):552-561. PubMed Abstract | Publisher Full Text OpenURL

  82. Castillo Gracia M, Couillaud F: Molecular cloning and tissue expression of an insect farnesyl diphosphate synthase.

    Eur J Biochem 1999, 262(2):365-370. PubMed Abstract | Publisher Full Text OpenURL

  83. Kikuchi K, Hirai M, Shiotsuki T: Molecular cloning and tissue distribution of farnesyl pyrophosphate synthase from the silkworm Bombyx mori.

    J Insect Biotechnol Sericol 2001, 70(3):167-172. OpenURL

  84. Cusson M, Béliveau C, Sen SE, Vandermoten S, Rutledge RG, Stewart D, Francis F, Haubruge É, Rehse P, Huggins DJ: Characterization and tissue-specific expression of two lepidopteran farnesyl diphosphate synthase homologs: Implications for the biosynthesis of ethyl-substituted juvenile hormones.

    Proteins 2006, 65(3):742-758. PubMed Abstract | Publisher Full Text OpenURL

  85. Zhang Y-L, Li Z-X: Functional analysis and molecular docking identify two active short-chain prenyltransferases in the green peach aphid, Myzus persicae.

    Arch Insect Biochem Physiol 2012, 81(2):63-76. PubMed Abstract | Publisher Full Text OpenURL

  86. Keeling CI, Blomquist GJ, Tittiger C: Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae).

    Die Naturwissenschaften 2004, 91(7):324-328. PubMed Abstract | Publisher Full Text OpenURL

  87. Ashby MN, Edwards PA: Elucidation of the deficiency in two yeast coenzyme Q mutants.

    Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase. J Biol Chem 1990, 265(22):13157-13164. OpenURL

  88. Ohnuma S, Hirooka K, Ohto C, Nishino T: Conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase.

    J Biol Chem 1997, 272(8):5192-5198. PubMed Abstract | Publisher Full Text OpenURL

  89. Mayoral JG, Nouzova M, Navare A, Noriega FG: NADP + −dependent farnesol dehydrogenase, a corpora allata enzyme involved in juvenile hormone synthesis.

    Proc Natl Acad Sci 2009, 106(50):21091-21096. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  90. Perssom B, Krook M, Jörvall H: Characteristics of short‐chain alcohol dehydrogenases and related enzymes.

    Eur J Biochem 2005, 200(2):537-543. OpenURL

  91. Hekmat-Scafe DS, Dorit RL, Carlson JR: Molecular evolution of odorant-binding protein genes OS-E and OS-F in Drosophila.

    Genetics 2000, 155(1):117-127. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  92. Wanner KW, Willis LG, Theilmann DA, Isman MB, Feng Q, Plettner E: Analysis of the insect os-d-like gene family.

    J Chem Ecol 2004, 30(5):889-911. PubMed Abstract OpenURL

  93. Dani FR, Michelucci E, Francese S, Mastrobuoni G, Cappellozza S, La Marca G, Niccolini A, Felicioli A, Moneti G, Pelosi P: Odorant-binding proteins and chemosensory proteins in pheromone detection and release in the silkmoth Bombyx mori.

    Chem Senses 2011, 36(4):335-344. PubMed Abstract | Publisher Full Text OpenURL

  94. Jacquin-Joly E, Vogt RG, Francois MC, Nagnan-Le Meillour P: Functional and expression pattern analysis of chemosensory proteins expressed in antennae and pheromonal gland of Mamestra brassicae.

    Chem Senses 2001, 26(7):833-844. PubMed Abstract | Publisher Full Text OpenURL

  95. Zhou JJ, Kan Y, Antoniw J, Pickett JA, Field LM: Genome and EST analyses and expression of a gene family with putative functions in insect chemoreception.

    Chem Senses 2006, 31(5):453-465. PubMed Abstract | Publisher Full Text OpenURL

  96. Goodman W, Gilbert L: Hemolymph protein binding of juvenile hormone in Manduca sexta.

    Am Zool 1974, 14:1289. OpenURL

  97. Hidayat P, Goodman WG: Juvenile hormone and hemolymph juvenile hormone binding protein titers and their interaction in the hemolymph of fourth stadium Manduca sexta.

    Insect Biochem Mol Biol 1994, 24(7):709-715. Publisher Full Text OpenURL

  98. Tillman JA, Holbrook GL, Dallara PL, Schal C, Wood DL, Blomquist GJ, Seybold SJ: Endocrine regulation of de novo aggregation pheromone biosynthesis in the pine engraver, Ips pini (Say)(Coleoptera: Scolytidae).

    Insect Biochem Mol Biol 1998, 28(9):705-715. Publisher Full Text OpenURL

  99. Tillman JA, Seybold SJ, Jurenka RA, Blomquist GJ: Insect pheromones–an overview of biosynthesis and endocrine regulation.

    Insect Biochem Mol Biol 1999, 29(6):481-514. PubMed Abstract | Publisher Full Text OpenURL

  100. Rafaeli A, Jurenka RA: PBAN regulation of pheromone biosynthesis in female moths. In Insect Pheromone Biochemistry and Molecular Biology. Edited by Gary B, Richard V. San Diego: Academic Press; 2003:107-136. OpenURL

  101. Jurenka R: Insect Pheromone Biosynthesis. In The Chemistry of Pheromones and Other Semiochemicals I. 239th edition. Edited by Schulz S. Berlin: Springer Berlin Heidelberg; 2004:97-132. OpenURL

  102. Feyereisen R: Insect P450 enzymes.

    Annu Rev Entomol 1999, 44(1):507-533. PubMed Abstract | Publisher Full Text OpenURL

  103. Berenbaum MR: Postgenomic chemical ecology: from genetic code to ecological interactions.

    J Chem Ecol 2002, 28(5):873-896. PubMed Abstract | Publisher Full Text OpenURL

  104. Schuler MA: The role of cytochrome P450 monooxygenases in plant-insect interactions.

    Plant Physiol 1996, 112(4):1411. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  105. Wen Z, Pan L, Berenbaum MR, Schuler MA: Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase.

    Insect Biochem Mol Biol 2003, 33(9):937-947. PubMed Abstract | Publisher Full Text OpenURL

  106. Wadleigh RW, Yu SJ: Glutathione transferase activity of fall armyworm larvae toward α, β-unsaturated carbonyl allelochemicals and its induction by allelochemicals.

    Insect Biochem 1987, 17(5):759-764. Publisher Full Text OpenURL

  107. Robertson H, Martos R, Sears C, Todres E, Walden K, Nardi J: Diversity of odourant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae.

    Insect Mol Biol 1999, 8(4):501-518. PubMed Abstract | Publisher Full Text OpenURL

  108. De Kort CAD, Granger NA: Regulation of JH titers: the relevance of degradative enzymes and binding proteins.

    Arch Insect Biochem Physiol 1996, 33(1):1-26. Publisher Full Text OpenURL

  109. Picimbon JF: Biochemistry and evolution of OSD and OBP proteins. In Insect Pheromone Biochemistry and Molecular Biology. New edition. Edited by Blomquist G, Vogt RG. USA: NY Academic Press; 2003:539-566. OpenURL