title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratório de Bioquímica e Fisiologia de Insetos, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Natalia
authors.initials: N
authors.lastname: González-Caballero
authors.affiliation: Laboratório de Pesquisa em Leishmaniose, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Andrés
authors.initials: A
authors.lastname: Rodríguez-Vega
authors.affiliation: Pós-graduação Biologia Parasitaria, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Geovane
authors.initials: G
authors.lastname: Dias-Lopes
authors.affiliation: Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institutes of Health Rockville, MD, USA.
authors.forename: Jesus G
authors.initials: JG
authors.lastname: Valenzuela
authors.affiliation: Vector Biology Section, Laboratory of Malaria and Vector Research, National Institutes of Health Rockville, MD, USA.
authors.forename: Jose M C
authors.initials: JM
authors.lastname: Ribeiro
authors.affiliation: Laboratório de Proteômica e Engenharia de Proteínas, Instituto Carlos Chagas, FIOCRUZ, Curitiba, Brazil.
authors.forename: Paulo Costa
authors.initials: PC
authors.lastname: Carvalho
authors.affiliation: Laboratório de Toxinologia, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Valente
authors.affiliation: Laboratório de Bioquímica e Fisiologia de Insetos, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
authors.forename: Reginaldo P
authors.initials: RP
authors.lastname: Brazil
authors.affiliation: Laboratório de Pesquisa em Leishmaniose, IOC, FIOCRUZ, Rio de Janeiro, RJ, Brazil. Electronic address: pcuervo@fiocruz.br.
authors.forename: Patricia
authors.initials: P
authors.lastname: Cuervo
mesh_terms: D000818:Animals; D004175:Diptera / Q000201:enzymology* / Q000235:genetics / Q000469:parasitology; D005088:Exocrine Glands / Q000201:enzymology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D019476:Insect Proteins / Q000096:biosynthesis* / Q000235:genetics; D007303:Insect Vectors / Q000201:enzymology* / Q000235:genetics / Q000469:parasitology; D018314:Leishmania infantum; D007898:Leishmaniasis, Visceral; D008297:Male; D013058:Mass Spectrometry; D008798:Mevalonic Acid / Q000378:metabolism*; D012724:Sex Attractants / Q000235:genetics / Q000378:metabolism*
chemical_list: D019476:Insect Proteins; D012724:Sex Attractants; D008798:Mevalonic Acid
keywords: Lutzomyia longipalpis; Male pheromone gland; Mevalonate pathway; Proteome; Proteomics