title: Investigating associations between biting time in the malaria vector Anopheles arabiensis Patton and single nucleotide polymorphisms in circadian clock genes: support for sub-structure among An. arabiensis in the Kilombero valley of Tanzania.
abstract: BACKGROUND
  There is growing evidence that the widespread use of Long-Lasting Insecticidal Nets (LLINs) is prompting malaria vectors to shift their biting towards times and places where people are not protected, such as earlier in the evening and/or outdoors. It is uncertain whether these behavioural shifts are due to phenotypic plasticity and/or ecological changes within vector communities that favour more exophilic species, or involve genetic factors within vector species to limit their contact with LLINs. Possibly variation in the time and location of mosquito biting has a genetic basis, but as yet this phenomenon has received little investigation. Here we used a candidate gene approach to investigate whether polymorphisms in selected circadian clock genes could explain variation in the time and location of feeding (indoors versus outside) within a natural population of the major African malaria vector Anopheles arabiensis.
  
  
  METHODS
  Host-seeking An. arabiensis were collected from two villages (Lupiro and Sagamaganga) in Tanzania by Human Landing Catch (HLC) technique. Mosquitoes were classified into phenotypes of "early" (7 pm-10 pm) or "late" biting (4 am -7 am), and host-seeking indoors or outdoors. In these samples we genotyped 34 coding SNPs in 8 clock genes (PER, TIM, CLK, CYC, PDP1, VRI, CRY1, and CRY2), and tested for associations between these SNPs and biting phenotypes. SNPs in 8 mitochondrial genes (ATP6, ATP8, COX1, COX2, COX3, ND3, ND5 and CYTB) were also genotyped to test population subdivision within An. arabiensis.
  
  
  RESULTS
  The candidate clock genes exhibited polymorphism within An. arabiensis, but it was unrelated to variation in the timing and location of their biting activity. However, there was evidence of strong genetic structure within An. arabiensis populations in association with the TIM, which was unrelated to geographic distance. Substructure within An. arabiensis was also detected using mitochondrial markers.
  
  
  CONCLUSIONS
  The variable timing and location of biting in An. arabiensis could not be linked to candidate clock genes that are known to influence behaviour in other Diptera. This finding does not rule out the possibility of a genetic basis to biting behaviour in this malaria vector, but suggests these are complex phenotypes that require more intensive ecological, neuronal and genomic analyses to understand.
authors.affiliation: Environmental Health and Ecological Sciences Thematic Group, Ifakara Health Institute, Ifakara, Morogoro, Tanzania. dmaliti@ihi.or.tz.|Nelson Mandela African Institute of Science and Technology Tanzania, School of Life Sciences, Arusha, Tanzania. dmaliti@ihi.or.tz.|Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, Lancashire, UK. dmaliti@ihi.or.tz.
authors.forename: Deodatus Vincent
authors.initials: DV
authors.lastname: Maliti
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, CA, USA. cmarsdenresearch@gmail.com.
authors.forename: C D
authors.initials: CD
authors.lastname: Marsden
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, CA, USA. bmain@usc.edu.
authors.forename: B J
authors.initials: BJ
authors.lastname: Main
authors.affiliation: Environmental Health and Ecological Sciences Thematic Group, Ifakara Health Institute, Ifakara, Morogoro, Tanzania. govella@ihi.or.tz.
authors.forename: N J
authors.initials: NJ
authors.lastname: Govella
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, CA, USA. ykyamasaki@ucdavis.edu.
authors.forename: Y
authors.initials: Y
authors.lastname: Yamasaki
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, CA, USA. travcollier@gmail.com.
authors.forename: T C
authors.initials: TC
authors.lastname: Collier
authors.affiliation: Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, Lancashire, UK. kkreppel@ihi.or.tz.
authors.forename: K
authors.initials: K
authors.lastname: Kreppel
authors.affiliation: Department of Entomology and Nematology, University of California Davis, Davis, CA, USA. jcchiu@ucdavis.edu.
authors.forename: J C
authors.initials: JC
authors.lastname: Chiu
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, CA, USA. gclanzaro@gmail.com.
authors.forename: G C
authors.initials: GC
authors.lastname: Lanzaro
authors.affiliation: Department of Entomology and Nematology, University of California Davis, Davis, CA, USA. Heather.Ferguson@glasgow.ac.uk.
authors.forename: H M
authors.initials: HM
authors.lastname: Ferguson
authors.affiliation: School of Veterinary Medicine, University of California Davis, Davis, CA, USA. yoosook.lee@gmail.com.
authors.forename: Y
authors.initials: Y
authors.lastname: Lee
mesh_terms: D000818:Animals; D000852:Anopheles / Q000235:genetics* / Q000502:physiology*; D057906:Circadian Clocks / Q000235:genetics*; D005247:Feeding Behavior; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D007303:Insect Vectors; D020641:Polymorphism, Single Nucleotide; D013636:Tanzania; D013997:Time Factors