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Am. J. Trop. Med. Hyg., 75(4), 2006, pp. 656-658
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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MULTILOCUS ENZYME ELECTROPHORESIS SUPPORTS SPECIATION WITHIN THE ANOPHELES NILI GROUP OF MALARIA VECTORS IN CAMEROON

HERMAN P. AWONO-AMBENE*, FRÉDÉRIC SIMARD, CHRISTOPHE ANTONIO-NKONDJIO, ANNA COHUET, PIERRE KENGNE, AND DIDIER FONTENILLE
Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), Yaoundé, Cameroon; Institute of Medical Research and studies of Medicinal Plants (IMPM), Ministry of Scientific Research and Innovation, Yaoundé, Cameroon; Institut de Recherche pour le Développement (IRD), Research Unit 016, Montpellier, France


ABSTRACT
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Multilocus enzyme analysis of the genetic variability and population structure was conducted among three malaria vector species of the Anopheles nili group in Cameroon: An. nili, An. carnevalei, and the recently described An. ovengensis. We detected species-specific alleles and large differences in shared allele frequencies at six of nine loci (e.g., PGM, GOT1, IDH1, IDH2, PGI, and {alpha}-GPD). This non-random distribution of alleles leads to high and significant values of differentiation indexes (0.569 < Fst < 0.874, P < 10–4). These results fully agree with standard morphologic descriptions, and therefore provide further support for recent taxonomic classification within the An. nili group.

The bionomics of Anopheles nili mosquitoes was recently updated after the description of a new malaria vector species from central African regions, namely Anopheles ovengensis.1 The An. nili group includes 4 recognized species that can be identified through slight morphologic diagnostic characters observable at the larval and/or adult stages: An. nili, An. somalicus, An. carnevalei, and An. ovengensis. Mosquitoes of this group are recognized as major human malaria vectors in tropical Africa, especially along streams and rivers that represent typical larval development sites.2,3 However, in spite of its epidemiologic importance for malaria transmission, very few studies have yet targeted this species group. Recent analysis of DNA sequence variation in the ribosomal DNA region revealed 4 different clusters corresponding each to one species.4 This genetic polymorphism provided the basis for the development of a PCR-based molecular identification assay. However, no study was undertaken to explore the level of intra-specific genetic variation or genetic relationship between these taxonomic units. Here, we used multilocus enzyme electrophoresis as a preliminary tool for assessing genetic variability and differentiation within the An. nili group of malaria vectors.

Adult females An. nili were captured from March to September 2001 in 4 villages within the equatorial forest domain, south of Cameroon (Figure 1Go): Mbébé (4°10'N; 11°04'E), Afan Essokié (2°23'N; 10°00'E), Oveng (2°24'N; 10°21'E), and Simbock (3°51'N; 11°30'E). Geographic distance between locations ranged from 50 km (Afan Essokié-Oveng) to 250 km (Mbébé-Afan Essokié). Species within the An. nili group were determined morphologically according to dichotomous keys and recent descriptions.1,5 Each individual mosquito was stored in liquid nitrogen until processed in the laboratory for isoenzyme analysis. Seven enzyme systems were studied: mannose-6-phosphate-isomerase (MPI, E.C 5.3.1.8.); phosphoglucomutase (PGM, E.C 2.7.5.1); glutamate-oxaloacetate transaminase (GOT, E.C 2.6.1.1); hexokinase (HK, E.C 2.7.1.1); isocitrate dehydrogenase (IDH, E.C 1.1.1.42); phosphogluco-isomérase (PGI, E.C 5.3.1.9); alpha glycerophosphate dehydrogenase ({alpha}-GPD, E.C 1.1.1.8). Migration patterns were analyzed at 9 interpretable neutral loci, namely MPI, PGM, GOT1, HK1, HK2, IDH1, IDH2, PGI, and {alpha}-GPD, using horizontal starch gel electrophoresis.6 For each locus, conformity with Hardy-Weinberg (HW) expectations was estimated for each species and overall. Statistical significance and linkage disequilibrium between pairs of loci were assessed using the exact probability tests available in GENEPOP 3.2.7 Genetic differentiation between cryptic species of the An. nili group was examined by Wright’s F statistics.8,9 Significance of Fst was assessed using the G-based exact test for genotypic differentiation.10


Figure 1
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  		    FIGURE 1. Map of Cameroon showing the main hydrographic network, the capital city, and the 4 mosquito collection sites.

 
We analyzed 127 specimens including 66 An. nili (from Mbébé and Simbock), 45 An. ovengensis (from Oveng), and 16 An. carnevalei (from Afan-Essokié). Samples from Mbébé and Simbock were merged together because all specimens were morphologically identified as An. nili. Indeed, as shown in Table 1Go, no deficit in heterozygote could be indicative of a mixing of different gene pools (Whalund effect). The percentage of polymorphic loci (with more than 1 allele) was 55.6% for An. nili and 66.7% for both An. ovengensis and An. carnevalei. The average number of alleles (± standard error) per locus ranged from 1.78 (± 0.63) to 2.00 (± 0.82), and the observed heterozygosity from 0.030 (± 0.015) in An. nili to 0.437 (± 0.022) in An. carnevalei. These estimates fall within the range of values reported for other anophelines species.11–13 When we considered all samples as a single gene pool, significant heterozygote deficits inducing a deviation from HW equilibrium was observed at PGM, GOT1, IDH1, IDH2, and {alpha}-GPD loci (P < 0.05 after correction for multiple tests). In agreement with this genetic heterogeneity, significant linkage disequilibrium was detected for 18 of 36 pairs of loci (P < 0.05; single test level). In contrast, HW expectations were respected at all loci when we analyzed each species as a different taxonomic unit, with the exception of locus MPI that showed marginally significant heterozygote deficit in An. ovengensis. High values of genetic differentiation indexes (Fst) across all loci were observed between An. nili and An. ovengensis (Fst = 0.874, P < 10–4), between An. nili and An. carnevalei (Fst = 0.791, P < 10–4), and between An. ovengensis and An. carnevalei (Fst = 0.569, P < 10–4). These findings are consistent with the occurrence of non-overlapping species-diagnostic alleles and large differences in shared allele frequencies at PGM, GOT1, IDH1, PGI, and {alpha}-GPD loci. An.nili displayed a diagnostic private allele at IDH2, differing from the allele shared by both An. ovengensis and An. carnevalei at the same locus. No heterozygote was observed at this locus. Moreover, An. carnevalei and An. ovengensis revealed different fixed alleles for the locus GOT. As shown in Table 2Go, the combination of such species-diagnostic loci can be used for species identification within the An. nili group.


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TABLE 1
Genetic variability and goodness of fit to Hardy-Weinberg equilibrium in An. nili, An. ovengensis, and An. carnevalei populations from Cameroon
 

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TABLE 2
Loci showing species diagnostic alleles (*) or significant differences in shared allele frequencies among the An. nili group of malaria vectors
 
The amount of fixed and/or large differences in allele profiles and differentiation indexes across all loci we observed in this study further supports speciation between An. nili, An. carnevalei, and An. ovengensis, and is in agreement with current classification based on specific morphologic traits and segregating sequences in the ribosomal DNA.1,4 As shown in this study, the combination of multiple polymorphic enzyme loci can be useful to explore the extent and distribution of genetic polymorphism within the An. nili group, as was formerly demonstrated in other anopheline11–16 and culicine groups.17 Multiple enzyme electrophoresis provided preliminary data on the population genetic structure of members of the An. nili group in Cameroon. Recent availability of microsatellite DNA markers should allow more refined assessment of the genetic diversity and population structure of these neglected mosquito vector groups.11,18

Received March 7, 2006. Accepted for publication May 31, 2006.

Acknowledgments: The authors thank Maïte Marquine, Maurice Adja Akré, Vohahirana Rajaonarivelo, and Jean Claude Toto for technical assistance during mosquito collections and laboratory processing.

Financial support: This work received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR-REG N° 990831), the Pal+ French Ministry of Research, and the French Institut de Recherche pour le Développement (IRD).

* Address correspondence to Herman P. Awono-Ambene, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), P.O. Box 288, Yaoundé, Cameroon. E-mail: hpaawono{at}yahoo.fr Back

Authors’ addresses: Herman Parfait Awono-Ambene, Frédéric Simard, and Christophe Antonio-Nkondjio, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), P.O. Box 288, Yaoundé, Cameroon, Telephone: (+237) 223-2232, Fax: (+237) 223-0061. Anna Cohuet, Pierre Kengne, and Didier Fontenille, Institut de Recherche pour le Développement (IRD), Research Unit 016, Laboratoire de Lutte contre les Insectes Nuisibles (LIN), 911 av. Agropolis, BP 64501, 34 394 Montpellier Cedex 5, France, Telephone: (+33)4-6704-1924, Fax: (+33)4-6754-2044.

Reprint requests: Herman P. Awono-Ambene, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), P.O. Box 288, Yaoundé, Cameroon, Telephone: (+237) 223-2232, Fax: (+237) 223-0061, E-mail: hpaawono{at}yahoo.fr


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