Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology.

Varun Tyagi,Vijay Veer,O P Agrawal,D Sukumaran,A R Srivastava,Sunil Dhiman,A K Sharma,Ruchi Yadav

doi:10.1186/s13071-016-1478-5

Abstract

BackgroundThe 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.MethodsThe 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.ResultsCurrently 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.ConclusionsThe 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.

Highlights

The malaria vector Anopheles culicifacies is an important malaria vector in Southeast Asia which comprises of five sibling species namely A, B, C, D and E
At least twenty specimens from each egg batch were used for allele specific polymerase chain reaction (AS-PCR) assay for the identification of An. culicifacies sibling species [2, 33, 34]
In all 23 taxa of anopheline sibling species have been recognized across the world bearing distinct gene pools and Previous studies have attempted to find out the morphological markers for the members of An. culicifacies species complex and reported variation in the spermatheca of species A and B [39], but could not successfully differentiate all the sibling species

Summary

Introduction

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. 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. Anopheles culicifacies (sensu lato) is one of the most important malaria vectors in Southeast Asia and has been recorded in the majority of the malaria affected countries [1, 2]. Previous studies conducted have demonstrated that identification of sibling species using adult mosquitoes is. Molecular methods using PCR assays have been effectively able to identify the sibling species in An. culicifacies mosquitoes [2, 17]

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