title: Age and prior blood feeding of Anopheles gambiae influences their susceptibility and gene expression patterns to ivermectin-containing blood meals.
abstract: BACKGROUND
  Ivermectin has been proposed as a novel malaria transmission control tool based on its insecticidal properties and unique route of acquisition through human blood. To maximize ivermectin's effect and identify potential resistance/tolerance mechanisms, it is important to understand its effect on mosquito physiology and potential to shift mosquito population age-structure. We therefore investigated ivermectin susceptibility and gene expression changes in several age groups of female Anopheles gambiae mosquitoes.
  
  
  METHODS
  The effect of aging on ivermectin susceptibility was analyzed in three age groups (2, 6, and 14-days) of colonized female Anopheles gambiaemosquitoes using standard survivorship assays. Gene expression patterns were then analyzed by transcriptome sequencing on an Illumina HiSeq 2500 platform. RT-qPCR was used to validate transcriptional changes and also to examine expression in a different, colonized strain and in wild mosquitoes, both of which blood fed naturally on an ivermectin-treated person.
  
  
  RESULTS
  Mosquitoes of different ages and blood meal history died at different frequencies after ingesting ivermectin. Mortality was lowest in 2-day old mosquitoes exposed on their first blood meal and highest in 6-day old mosquitoes exposed on their second blood meal. Twenty-four hours following ivermectin ingestion, 101 and 187 genes were differentially-expressed relative to control blood-fed, in 2 and 6-day groups, respectively. Transcription patterns of select genes were similar in membrane-fed, colonized, and naturally-fed wild vectors. Transcripts from several unexpected functional classes were highly up-regulated, including Niemann-Pick Type C (NPC) genes, peritrophic matrix-associated genes, and immune-response genes, and these exhibited different transcription patterns between age groups, which may explain the observed susceptibility differences. Niemann-Pick Type 2 genes were the most highly up-regulated transcripts after ivermectin ingestion (up to 160 fold) and comparing phylogeny to transcriptional patterns revealed that NPCs have rapidly evolved and separate members respond to either blood meals or to ivermectin.
  
  
  CONCLUSIONS
  We present evidence of increased ivermectin susceptibility in older An. gambiae mosquitoes that had previously bloodfed. Differential expression analysis suggests complex midgut interactions resulting from ivermectin ingestion that likely involve blood meal digestion physiological responses, midgut microflora, and innate immune responses. Thus, the transcription of certain gene families is consistently affected by ivermectin ingestion, and may provide important clues to ivermectin's broad effects on malaria vectors. These findings contribute to the growing understanding of ivermectin's potential as a transmission control tool.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. jon.a.seaman@gmail.com.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Seaman
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. haoues.alout@gmail.com.
authors.forename: Haoues
authors.initials: H
authors.lastname: Alout
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. jacob.i.meyers@gmail.com.
authors.forename: Jacob I
authors.initials: JI
authors.lastname: Meyers
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. mark.stenglein@colostate.edu.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Stenglein
authors.affiliation: Institute de Recherche en Sciences de la Santé (IRSS)/Centre Muraz, Direction Régionale de l'Ouest, 399 Ave de la Liberté, Bobo Dioulasso, Houet, 10400-000, Burkina Faso. dabire_roch@hotmail.com.
authors.forename: Roch K
authors.initials: RK
authors.lastname: Dabiré
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. lozano.saul@gmail.com.
authors.forename: Saul
authors.initials: S
authors.lastname: Lozano-Fuentes
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. timaburton@gmail.com.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Burton
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. wojtek94@yahoo.com.
authors.forename: Wojtek S
authors.initials: WS
authors.lastname: Kuklinski
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. william.black@colostate.edu.
authors.forename: William C
authors.initials: WC
authors.lastname: Black
authors.affiliation: Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University 1692 Campus Delivery, Fort Collins, CO, 80525, USA. brian.foy@colostate.edu.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Foy
mesh_terms: D000818:Animals; D000852:Anopheles / Q000187:drug effects / Q000235:genetics* / Q000469:parasitology; D001769:Blood / Q000187:drug effects*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007559:Ivermectin / Q000494:pharmacology*; D008288:Malaria / Q000097:blood / Q000469:parasitology / Q000517:prevention & control* / Q000635:transmission
chemical_list: D007559:Ivermectin