title: Clocks do not tick in unison: isolation of Clock and vrille shed new light on the clockwork model of the sand fly Lutzomyia longipalpis.
abstract: BACKGROUND
  Behavior rhythms of insect vectors directly interfere with the dynamics of pathogen transmission to humans. The sand fly Lutzomyia longipalpis is the main vector of visceral leishmaniasis in America and concentrates its activity around dusk. Despite the accumulation of behavioral data, very little is known about the molecular bases of the clock mechanism in this species. This study aims to characterize, within an evolutionary perspective, two important circadian clock genes, Clock and vrille.
  
  
  RESULTS
  We have cloned and isolated the coding sequence of L. longipalpis' genes Clock and vrille. The former is structured in eight exons and encodes a protein of 696 amino acids, and the latter comprises three exons and translates to a protein of 469 amino acids. When compared to other insects' orthologues, L. longipalpis CLOCK shows a high degree of conservation in the functional domains bHLH and PAS, but a much shorter glutamine-rich (poly-Q) C-terminal region. As for L. longipalpis VRILLE, a high degree of conservation was found in the bZIP domain. To support these observations and provide an elegant view of the evolution of both genes in insects, phylogenetic analyses based on maximum-likelihood and Bayesian inferences were performed, corroborating the previously known insect systematics.
  
  
  CONCLUSIONS
  The isolation and phylogenetic analyses of Clock and vrille orthologues in L. longipalpis bring novel and important data to characterize this species' circadian clock. Interestingly, the poly-Q shortening observed in CLOCK suggests that its transcription activity might be impaired and we speculate if this effect could be compensated by other clock factors such as CYCLE.
authors.affiliation: Laboratório de Biologia Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. joaogesto@gmail.com.|Mosquitos Vetores: Endossimbiontes e Interação Patógeno Vetor, Centro de Pesquisas René Rachou-Fiocruz, Belo Horizonte, Minas Gerais, Brazil. joaogesto@gmail.com.
authors.forename: João Silveira Moledo
authors.initials: JS
authors.lastname: Gesto
authors.affiliation: Laboratório de Biologia Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. gbsrivas@gmail.com.
authors.forename: Gustavo Bueno da Silva
authors.initials: GB
authors.lastname: Rivas
authors.affiliation: Laboratório de Biologia Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. marciopavan@gmail.com.|Laboratório de Epidemiologia e Sistemática Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. marciopavan@gmail.com.
authors.forename: Marcio Galvão
authors.initials: MG
authors.lastname: Pavan
authors.affiliation: Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. antonio.meirelesfilho@epfl.ch.
authors.forename: Antonio Carlos Alves
authors.initials: AC
authors.lastname: Meireles-Filho
authors.affiliation: Laboratório de Biologia Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. pauloamoretty@yahoo.com.br.
authors.forename: Paulo Roberto de
authors.initials: PR
authors.lastname: Amoretty
authors.affiliation: Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. souzana@ioc.fiocruz.br.
authors.forename: Nataly Araújo de
authors.initials: NA
authors.lastname: Souza
authors.affiliation: Laboratório de Biologia Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. rafaelav@ioc.fiocruz.br.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM)/CNPq, Rio de Janeiro, Brazil. rafaelav@ioc.fiocruz.br.
authors.forename: Rafaela Vieira
authors.initials: RV
authors.lastname: Bruno
authors.affiliation: Laboratório de Biologia Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil.|Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM)/CNPq, Rio de Janeiro, Brazil.
authors.forename: Alexandre Afranio
authors.initials: AA
authors.lastname: Peixoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001522:Behavior, Animal / Q000502:physiology*; D056926:CLOCK Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011576:Psychodidae / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D019476:Insect Proteins; D014157:Transcription Factors; D056926:CLOCK Proteins