- Open Access
Palp ratio as a field identification tool for two members of the Anopheles gambiae complex in Ghana (A. melas and A. gambiae)
© Annan et al.; licensee BioMed Central. 2015
- Received: 13 October 2014
- Accepted: 20 May 2015
- Published: 28 May 2015
The Anopheles gambiae Giles complex is the most widely studied and the most important insect vector group. We explored the use of the palp ratio method as a field tool to identify A. melas and A. gambiae in Ghana.
Human landing catches were conducted to collect mosquitoes in the coastal area of Western Region of Ghana. Palps were removed and segments 3 and 4 + 5 measured using a compound microscope. DNA extraction and downstream PCR for species identification was carried out using the legs and wings. Known A. gambiae collected from the Ashanti Region of Ghana were used for comparison.
A total of 2120 A. gambiae were collected. Lengths of segments 3 and 4 + 5 were significantly correlated in samples from both regions. Using a palp ratio of 0.81 as the cut-off value, 14.9 % outliers (≥0.81) from our study area were confirmed by PCR as A. melas. PCR also confirmed outliers from the Ashanti Region with palp ratio < 0.81 (10.2 %) as A. gambiae.
The palp ratio method proved to be a useful tool to identify populations of salt and freshwater A. melas and A. gambiae.
- Anopheles melas
- Anopheles gambiae
- Palp ratio
The Anopheles gambiae Giles complex is the most widely studied and the most important insect vector group. It is presently made up of eight sibling species: A. gambiae Giles (formerly molecular S form), A. coluzzii Coetzee and Wilkerson (formerly molecular M form), A. arabiensis Patton, the salt water breeders A. melas Theobald in West Africa and A. merus Dönitz in East Africa, A. quadriannulatus Theobald in southern Africa, A. amharicus Hunt Wilkerson and Coetzee and A. bwambae White in Uganda . In Ghana, four species of the complex have been implicated as vectors of malaria; A. gambiae in the forest [2,3], A. melas at the coast, [4,5] and A. coluzzii and A. arabiensis in savanna areas [6,7]. A. melas and A. gambiae are vectors of lymphatic filariasis in coastal Ghana [8,9].
Identification and separation of the sibling species first became possible by using the morphology of polytene chromosomes of salivary gland cells of fourth instar larvae or the ovarian nurse cells of adult females . Later, molecular techniques such as Polymerase Chain Reaction (PCR) were developed [11,12].
As one of the morphological tools, palp ratio (length of the fourth and fifth segment divided by length of the third) has been employed to separate A. melas from A. gambiae and A. arabiensis . Bryan (1980) demonstrated that female Anopheles mosquitoes from The Gambia with palp ratios <0.81 and ≥0.81 could be identified as A. gambiae and A. melas respectively . Using this cut-off point, the number of misidentified specimens was 3.8 % for A. melas and 5.8 % for A. gambiae. Later, Palsson et al. (1998) compared the palp ratio with PCR results and demonstrated for Anopheles from Guinea Bissau that a cut off point of 0.83 correctly identified 100 % of A. melas, but erroneously identified 4 % of A. gambiae as A. melas . Akogbeto and Romano (1999) also reported the presence of A. gambiae and A. melas from coastal Benin and showed that a cut off ≤0.81 separates A. gambiae and >0.81 A. melas with an error of 3–6 % . Palsson et al. (1998) therefore suggested that the PCR was the most optimal method available to separate A. gambiae and A. melas and that the palp ratio was not sufficiently reliable .
Within the framework of a study on lymphatic filariasis and intensity of transmission in the coastal area of Nzema East (Western Region), Ghana, we investigated whether the palp ratio method can be employed as a field tool to identify A. melas. Results of palp measurements were compared with those from Anopheles populations from the forest area of the Ashanti Region, Ghana.
A. gambiae s.l. mosquitoes were collected by Human Landing Catches (HLC)  from 18:00 hours to 02:00 hours in 6 villages along the sea coast near Essiama (Nzema East, Western Region) from September 2005 to January 2006. Volunteers (adult males) gave their verbal consent before participating in the collection of mosquitoes. Malaria prophylaxis was given, and treatment (at no cost to the volunteers) was arranged with the local hospitals but none became sick during the study period.
For comparison, A. gambiae females were received from two malaria transmission projects conducted in the forest areas of the Ashanti Region in two villages near Agona , and sites near Agogo and Konongo . All mosquitoes were stored in cool boxes transferred to a field laboratory in Essiama, and dissected the following day for parity and infections with larval stages of Wuchereria bancrofti. The palps were removed, mounted in a drop of 1XPBS on a slide and segments 3 and 4 + 5 measured using a compound microscope. The legs and wings were stored in micro titre plates and transported to the laboratories of Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR). They were stored at -20 °C for later DNA extraction and downstream PCR for species identification  and identified as A. gambiae s.s. and A. melas. The molecular forms M (now A. coluzzii) and S (A. gambiae) were not separated.
Data were entered using Microsoft Excel. The same programme was used in plotting scattergrams and frequency distributions. STATISTICA for Windows 1993 (StatSoft Inc., Tulsa, OK, USA) was used for the statistical analysis of the results. Correlation coefficients were determined by Pearson Product-Moment correlation and alpha values of less than 0.05 were considered significant.
The study was part of a drug trial and treatment study  approved by the Ethical Committee of the School of Medical Sciences of the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. Study procedures were in accordance with the Helsinki Declaration of 1975 (as revised 1983 and 2000).
Comparison of PCR and palp ratio (cut-off 0.81) for identification of putative A. melas from the Western Region and A. gambiae from Ashanti Region (controls) for species identification
Palp ratio (%)
Palp ratio (%)
A. gambiae s.l.
A. gambiae s.s.
The palp ratio method proved to be a useful tool to identify populations of salt and freshwater A. melas and A. gambiae but not sufficiently reliable in identifying individual specimens. In the absence of PCR, especially in resource limited countries where students and scientists do not have access to molecular based techniques, this study recommends the use of the palp ratio to distinguish between A. gambiae and A. melas as vectors of malaria and lymphatic filariasis in the Ghana coastal area.
We are indebted to the vector collectors and the people in our study communities for their continuous cooperation. We are grateful to Drs. Kingsley Badu and Gilbert Abonuusum for providing Anopheles mosquitoes from the Ashanti Region. The staff of the Kumasi Centre for Collaborative Research (KCCR) is thanked for assistance. We are grateful to Dr. Bari Howell for proof reading the manuscript.
Rolf Garms was supported by the German Senior Expert Service in 2005 and 2006. The study was funded by the European Commission and the Bernhard Nocht Institute for Tropical Medicine. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This work is part of AAA Master of Philosophy thesis.
- Coetzee M, Hunt RH, Wilkerson R, Della Torre A, Coulibaly MB, Besansky NJ. Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. Zootaxa. 2013;3619(3):246–74.View ArticleGoogle Scholar
- Abonuusum A, Owusu-Daako K, Tannich E, May J, Garms R, Kruppa T. Malaria transmission in two rural communities in the forest zone of Ghana. Parasitol Res. 2011;108(6):1465–71.View ArticlePubMedGoogle Scholar
- Tay SC, Danuor SK, Morse A, Caminade C, Badu K, Abruquah HH. Entomological Survey of Malaria Vectors within the Kumasi Metropolitan Area-A Study of Three Communities: Emena, Atonsu and Akropong. J Environ Sci Eng. 2012;1(2):144–54.Google Scholar
- Appawu M, Baffoe-Wilmot A, Afari E, Dunyo S, Koram K, Nkrumah F. Malaria vector studies in two ecological zones in southern Ghana. Afr Entomol. 2001;9(1):59–65.Google Scholar
- Yawson AE, McCall PJ, Wilson MD, Donnelly MJ. Species abundance and insecticide resistance of Anopheles gambiae in selected areas of Ghana and Burkina Faso. Med Vet Entomol. 2004;18(4):372–7.View ArticlePubMedGoogle Scholar
- de Souza D, Kelly-Hope L, Lawson B, Wilson M, Boakye D. Environmental factors associated with the distribution of Anopheles gambiae ss in Ghana; an important vector of lymphatic filariasis and malaria. PLoS One. 2010;5(3), e9927.View ArticlePubMed CentralPubMedGoogle Scholar
- de Souza DK: Diversity in Anopheles gambiae ss and Wuchereria bancrofti, and the Distribution of Lymphatic Filariasis in Ghana. University of Science and Technology in partial fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Biological Sciences, College of Science; 2010Google Scholar
- Dunyo S, Appawu M, Nkrumah F, Baffoe-Wilmot A, Pedersen E, Simonsen P. Lymphatic filariasis on the coast of Ghana. Trans R Soc Trop Med Hyg. 1996;90(6):634–8.View ArticlePubMedGoogle Scholar
- Tuno N, Kjaerandsen J, Badu K, Kruppa T. Blood-feeding behavior of Anopheles gambiae and Anopheles melas in Ghana, western Africa. J Med Entomol. 2010;47(1):28–31.View ArticlePubMedGoogle Scholar
- Coluzzi M, Sabatini A, Della Torre A, Di Deco MA, Petrarca V. A Polytene Chromosome Analysis of the Anopheles gambiae Species Complex. Science. 2002;298(5597):1415–8.View ArticlePubMedGoogle Scholar
- Scott JA, Brogdon WG, Collins FH. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg. 1993;49(4):520–9.PubMedGoogle Scholar
- Paskewitz SM, Collins FH. Use of the Polymerase Chain Reaction to identify mosquito species of the Anopheles gambiae complex. Med Vet Entomol. 1990;4(4):367–73.View ArticlePubMedGoogle Scholar
- Coluzzi M. Morphological divergences in the Anopheles gambiae complex. Riv Malariol. 1964;43:197–232.PubMedGoogle Scholar
- Bryan JH. Use of the Palpal Ratio and the Number of Pale Bands on the Palps in Separating Anopheles gambiae Giles SS and Anopheles melas Theobald (Diptera: Culicidae). Mosq Systematics. 1967;12(1980):155.Google Scholar
- Pålsson K, Pinto J, do Rosario VE, Jaenson TGT. The palpal ratio method compared with PCR to distinguish between Anopheles gambiae s.s. and A. melas from Guinea Bissau, West Africa. Acta Trop. 1998;70(1):101–7.View ArticlePubMedGoogle Scholar
- Akogbeto M, Romano R. Anopheles melas infestation rate for Plasmodium falciparum in the coastal and lagoon area of Benin, West Africa. Bulletin de la Societe de Pathologie Exotique. 1999;92(1):57–61.PubMedGoogle Scholar
- Mboera L. Sampling techniques for adult Afrotropical malaria vectors and their reliability in the estimation of entomological inoculation rate. Tanzan J Health Res. 2006;7(3):117–24.Google Scholar
- Badu K, Brenyah R, Timmann C, Garms R, Kruppa TF: Malaria transmission intensity and dynamics of clinical malaria incidence in a mountainous forest region of Ghana. In: Malaria World Journal 2013: 4-14Google Scholar
- Hoerauf A, Mand S, Fischer K, Kruppa T, Marfo-Debrekyei Y, Debrah AY, et al. Doxycycline as a novel strategy against bancroftian filariasis—depletion of Wolbachia endosymbionts from Wuchereria bancrofti and stop of microfilaria production. Med Microbiol Immunol. 2003;192(4):211–6.View ArticlePubMedGoogle Scholar
- Afrane YA, Klinkenberg E, Drechsel P, Owusu-Daaku K, Garms R, Kruppa T. Does irrigated urban agriculture influence the transmission of malaria in the city of Kumasi, Ghana? Acta Trop. 2004;89(2):125–34.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.