title: An effective method for the identification and separation of Anopheles minimus, the primary malaria vector in Thailand, and its sister species Anopheles harrisoni, with a comparison of their mating behaviors.
abstract: Species of the Anopheles minimus complex are considered to be the primary vectors of malaria in South and Southeast Asia. Two species of the complex, Anopheles minimus and Anopheles harrisoni, occur in Thailand. They are sympatric and difficult to accurately distinguish based on morphological characters. The aim of this study was to investigate the potential of antennal sensory organs to distinguish these two species. Additionally, we investigated their ability to mate in cages of different sizes, as well as the possible mechanism(s) that evokes stenogamous behavior.
  
  
  
  Large sensilla coeloconica present on the antennae of females of An. minimus and An. harrisoni were counted under a conventional light microscope and various types of antennal sensilla were examined under a scanning electron microscope (SEM). Determinations of mating ability were carried out in 20 and 30 cm3cages with a density resting surface (DRS) of 7.2. The insemination rate, frequency of clasper (gonocoxopodite) movement of the male genitalia during induced copulation and duration of mating of the two species were compared.
  
  
  
  The mean numbers of large sensilla coeloconica on antennal flagellomeres 1-8 and the mean number of large sensilla coeloconica on each flagellum in An. minimus (26.25) and An. harrisoni (31.98) were significantly different. Females of both species bear five types of antennal sensilla: chaetica, trichodea, basiconica, coeloconica and ampullacea. Marked differences in the structure of the large sensilla coeloconica were observed between the two species. Furthermore, only An. minimus could copulate naturally in the small cages. The frequency of clasper movement in the stenogamous An. minimus was significantly higher than in An. harrisoni, but there was no difference in the duration of mating.
  
  
  
  To our knowledge, this study is the first to examine and discover the usefulness of large sensilla coeloconica on the antennae of females and the frequency of clasper movement in males for distinguishing the sibling species An. minimus and An. harrisoni. The discovery provides an effective and relatively inexpensive method for their identification. Additionally, the greater frequency of clasper movement of An. minimus might influence its ability to mate in small spaces.
authors.affiliation: Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand.
authors.forename: Kritsana
authors.initials: K
authors.lastname: Taai
authors.affiliation: Department of Life Sciences, Natural History Museum, Cromwell Road, London, SW7 5BD, UK.
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Harbach
authors.affiliation: Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand.
authors.forename: Kittipat
authors.initials: K
authors.lastname: Aupalee
authors.affiliation: Entomology Section, Queen Sirikit Botanic Garden, P.O. Box 7, Chiang Mai, 50180, Thailand.
authors.forename: Wichai
authors.initials: W
authors.lastname: Srisuka
authors.affiliation: Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand.
authors.forename: Thippawan
authors.initials: T
authors.lastname: Yasanga
authors.affiliation: Research Center for the Pacific Islands, Kagoshima University, Kagoshima, 890-8580, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Otsuka
authors.affiliation: Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand. atisaeung.noi@gmail.com.
authors.forename: Atiporn
authors.initials: A
authors.lastname: Saeung
mesh_terms: D000818:Animals; D000852:Anopheles / Q000033:anatomy & histology / Q000145:classification* / Q000502:physiology*; D057506:Arthropod Antennae / Q000033:anatomy & histology*; D001699:Biometry; D005260:Female; D008297:Male; D008853:Microscopy; D000072138:Mosquito Vectors; D058618:Sensilla / Q000033:anatomy & histology; D012726:Sexual Behavior, Animal; D013785:Thailand
keywords: Anopheles harrisoni; Anopheles minimus; Antennal sensilla; SEM; Stenogamous behavior