title: Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology.
abstract: BACKGROUND
  The malaria vector Anopheles culicifacies (sensu lato) is an important malaria vector in Southeast Asia which comprises of five sibling species namely A, B, C, D and E. However, only a few forms have been identified as malaria vectors in various endemic countries. Currently, for the first time egg morphometry and morphology has been used to differentiate the three known vector sibling species of Anopheles culicifacies collected from malaria endemic Madhya Pradesh state of central India.
  
  
  METHODS
  The adult An. culicifacies (s.l.) was collected from five districts using standard mosquito collection methods. Adult female mosquitoes were allowed to lay eggs individually. The emerged mosquitoes were identified using allele specific polymerase chain reaction (AS-PCR) to sibling species. Eggs of sibling species A, D and E were studied using scanning electron microscopy (SEM) for morphometric and morphological characteristics.
  
  
  RESULTS
  Currently AS-PCR identified four known sibling species (B, C, D and E) of An. culicifacies in the study area. The surface morphology and morphometric attributes of the sibling species A, D and E eggs considerably differed from each other. An. culicifacies E had a narrow deck as compared to A and D, while An. culicifacies A had a bigger micropyle with 6-7 sectors as compared to D and E that had 6 sectors. An. culicifacies D had the smallest float (the structure present on sides of the egg surface in which air is filled that help in floating) and the number of ribs was also fewer than for An. culicifacies A and E.
  
  
  CONCLUSIONS
  The present study provides the first evidence that in addition to PCR assay, sibling species of An. culicifacies can also be differentiated using morphological and morphometric characteristics of the egg stage. The results also advocate that the sibling species of An. culicifacies are morphologically dissimilar and can be resolved using advanced microscopy.
authors.affiliation: Vector Management Division, Defence R&D Establishment, Jhansi Road, Gwalior, 474002, Madhya Pradesh, India. varundrl@rediffmail.com.|Medical Entomology Division, Defence Research Laboratory, Tezpur, 784001, Assam, India. varundrl@rediffmail.com.
authors.forename: Varun
authors.initials: V
authors.lastname: Tyagi
authors.affiliation: Vector Management Division, Defence R&D Establishment, Jhansi Road, Gwalior, 474002, Madhya Pradesh, India.
authors.forename: A K
authors.initials: AK
authors.lastname: Sharma
authors.affiliation: Medical Entomology Division, Defence Research Laboratory, Tezpur, 784001, Assam, India.
authors.forename: Sunil
authors.initials: S
authors.lastname: Dhiman
authors.affiliation: Vector Management Division, Defence R&D Establishment, Jhansi Road, Gwalior, 474002, Madhya Pradesh, India.
authors.forename: A R
authors.initials: AR
authors.lastname: Srivastava
authors.affiliation: Vector Management Division, Defence R&D Establishment, Jhansi Road, Gwalior, 474002, Madhya Pradesh, India.
authors.forename: Ruchi
authors.initials: R
authors.lastname: Yadav
authors.affiliation: Vector Management Division, Defence R&D Establishment, Jhansi Road, Gwalior, 474002, Madhya Pradesh, India.
authors.forename: D
authors.initials: D
authors.lastname: Sukumaran
authors.affiliation: School of Studies in Zoology, Jiwaji University, Gwalior, 474002, Madhya Pradesh, India.
authors.forename: O P
authors.initials: OP
authors.lastname: Agrawal
authors.affiliation: Medical Entomology Division, Defence Research Laboratory, Tezpur, 784001, Assam, India.
authors.forename: Vijay
authors.initials: V
authors.lastname: Veer
mesh_terms: D000818:Animals; D000852:Anopheles / Q000145:classification* / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D005260:Female; D006801:Humans; D007194:India / Q000453:epidemiology; D007303:Insect Vectors / Q000145:classification* / Q000235:genetics / Q000648:ultrastructure; D008288:Malaria / Q000453:epidemiology / Q000635:transmission; D008297:Male; D008855:Microscopy, Electron, Scanning; D010063:Ovum / Q000145:classification / Q000648:ultrastructure
keywords: Anopheles culicifacies; Malaria; Morphometry; PCR; SEM; Sibling species