Expression of the mevalonate pathway enzymes in the Lutzomyia longipalpis (Diptera: Psychodidae) sex pheromone gland demonstrated by an integrated proteomic approach

Abstract

In Latin America, Lutzomyia longipalpis is the main vector of the protozoan parasite Leishmania infantum, which is the causal agent of American Visceral Leishmaniasis. This insect uses male-produced pheromones for mate recognition. Elucidation of pheromone biogenesis or its regulation may enable molecular strategies for mating disruption and, consequently, the vector's population management. Motivated by our recent results of the transcriptomic characterization of the L. longipalpis pheromone gland, we performed a proteomic analysis of this tissue combining SDS-PAGE, and mass spectrometry followed by an integrative data analysis. Considering that annotated genome sequences of this sand fly are not available, we designed an alternative workflow searching MS/MS data against two customized databases using three search engines: Mascot, OMSSA and ProLuCID. A total of 542 proteins were confidently characterized, 445 of them using a Uniref100-insect protein database, and 97 using a transcript translated database. In addition, use of PEAKS for de novo peptide sequencing of MS/MS data confirmed ∼90% identifications made with the combination of the three search engines. Our results include the identification of six of the seven enzymes of the mevalonate-pathway, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. Biological significance L. longipalpis is the main vector of the protozoan parasite L. infantum, which is the causal agent of American Visceral Leishmaniasis. One of the control measures of such disease is focused on vector population control. As this insect uses male-produced pheromones for mate recognition, the elucidation of pheromone biogenesis or its regulating process may enable molecular strategies for mating disruption and, consequently, this vector's population management. On this regard, in this manuscript we report expression evidence, at the protein level, of several molecules potentially involved in the pheromone production of L. longipalpis. Our results include the identification of the mevalonate-pathway enzymes, plus the enzymes involved in sesquiterpenoid biosynthesis, all of which are proposed to be involved in pheromone production in L. longipalpis. In addition, considering that the annotated genome sequences of this sand fly are not yet available, we designed an alternative workflow searching MS/MS data against proteomic and transcript translated customized databases, using three search engines: Mascot, OMSSA, and ProLuCID. In addition, a de novo peptide sequencing software (PEAKS) was used to further analyze the MS/MS data. This approach made it possible to identify and annotate 542 proteins for the pheromone gland of L. longipalpis. Importantly, all annotated protein sequences and raw data are available for the research community in protein repositories that provide free access to the data.

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus

“…Three gels from three independent midgut suspensions were performed. Proteins were enzymatically digested following procedures previously described [ 35 ] with some modifications. Briefly, fine slices from each protein lane were manually excised and de-stained three times in 400 μL of 50 % acetonitrile, 25 mM NH 4 HCO 3 pH 8.0 for 15 min.…”

The Lutzomyia longipalpis complex: a brief natural history of aggregation-sex pheromone communication

“…The costs of production are rarely demonstrated in insects (but see Johansson et al [94]), however these can be inferred indirectly in species where signal production commences after adult eclosion (as is the case for L. longipalpis [83]). Recent transcriptomics and proteomics studies have revealed much about the biosynthetic pathway for glandular secretions in Lutzomyia including the specific enzyme pathways involved in sesquiterpenoid biosynthesis [95, 96]. It was observed in L. longipalpis that if the diet is changed, e.g.…”

“…In L. longipalpis it is conceivable that an analogous process is occurring. González-Caballero [95] and colleagues used transcriptome and proteome data to explore the enzymatic cascade from the sex pheromone gland. Molecules which are associated with enzymes of the mevalonate pathway were found in the pheromone gland.…”

Expression of active trypsin-like serine peptidases in the midgut of sugar-feeding female Anopheles aquasalis

“…The proteins were re-suspended in buffer containing 0.5 M Hepes and 7 M urea, and the protein concentration was determined using Qubit. The proteins were enzymatically digested in a solution following previously established protocols [ 37 ] with some modifications. Briefly, the proteins were reduced in 65 mM DTT for 30 min at 56 °C and then alkylated with 200 mM iodoacetamide at 25 °C in the dark for 30 min.…”