Assortative mating in mixed swarms of the mosquito Anopheles gambiae s.s. M and S molecular forms, in Burkina Faso, West Africa.

The molecular form composition of Anopheles gambiae Giles s.s. (Diptera: Culicidae) mating swarms and the associated mating pairs (copulae) were investigated during two rainy seasons (July to October, 2005 and July to November, 2006) in the villages of Soumousso and Vallée du Kou (VK7). Although the habitats of these villages differ markedly, sympatric populations of M and S molecular forms of An. gambiae s.s. occur in both places periodically. The main aim was to assess the degree to which these molecular forms mate assortatively. In Soumousso, a wooded savannah habitat, the majority of swarm samples consisted of only S-form males (21/28), although a few M-form males were found in mixed M- and S-form swarms. In VK7, a rice growing area, the majority of swarm samples consisted of only M-form males (38/62), until October and November 2006, when there were nearly as many mixed-form as single-form swarms. Overall, ∼60% of M- and S-form swarms were temporally or spatially segregated; the two forms were effectively prevented from encountering each other. Of the remaining 40% of swarms, however, only about half were single-form and the rest were mixed-form. Of the 33 copulae collected from mixed-form swarms, only four were mixed-form pairs, significantly fewer than expected by random pairing between forms (χ(2) = 10.34, d.f. = 2, P < 0.01). Finally, all specimens of inseminated females were of the same form as the sperm contained within their spermatheca (n = 91), even for the four mixed-form copulae. These findings indicate that assortative mating occurs within mixed-form swarms, mediated most probably by close-range mate recognition cues.

Contribution of Anopheles funestus, An. gambiae and An. nili (Diptera: Culicidae) to the perennial malaria transmission in the southern and western forest areas of Côte d'Ivoire.

The involvement of members of the Anopheles gambiae complex Giles and An. funestus Giles and An. nili Theobald groups in the transmission of Plasmodium falciparum was recently investigated in the villages of Gbatta and Kpéhiri, which lie, respectively, in forest areas in the west and south of Côte d'Ivoire. Adult female mosquitoes were collected, using human landing catches, inside and outside dwellings. After identification and dissection, the heads and thoraces of all the anopheline mosquitoes were tested, in an ELISA, for circumsporozoite protein (CSP). All the female anopheline mosquitoes collected and identified to species using PCR were found to be An. gambiae s.s., An. nili s.s. or An. funestus s.s., with An. gambiae s.s. and An. funestus s.s. predominant in Gbatta but An. nili s.s. the most common species in Kpéhiri. In Gbatta, 3·1% of the female An. gambiae collected, 5·0% of the female An. funestus and 1·8% of the female An. nili were found CSP-positive. The corresponding values in Kpéhiri were even higher, at 5·9%, 6·2% and 2·4%, respectively. The estimated entomological inoculation rates (EIR) were very high: 302 infected bites (139 from An. gambiae, 146 from An. funestus and 17 from An. nili)/person-year in Gbatta and 484 infected bites (204 from An. gambiae, 70 from An. funestus and 210 from An. nili)/person-year in Kpéhiri. In Gbatta, An. gambiae s.s. was responsible for most of the rainy-season transmission while An. funestus became the main malaria vector in the dry seasons. In Kpéhiri, however, An. nili appeared to be the main vector throughout the year, with An. gambiae of secondary importance and An. funestus only becoming a significant vector during the rainy season. Although, in both study sites, intense transmission was therefore occurring and the same three species of anopheline mosquito were present, the relative importance of each mosquito species in the epidemiology of the human malaria at each site differed markedly.

Distribution of insensitive acetylcholinesterase (ace-1R) in Anopheles gambiae s.l. populations from Burkina Faso (West Africa).

OBJECTIVE: To investigate through countrywide sampling at 20 localities across the three different agro-climatic zones of Burkina Faso, the distribution of the acetylcholinesterase insensitive mutation ace-1(R), which confers resistance to organophosphates (OP) and carbamates (CM) insecticides in An. gambiae s.l. METHODS: Adult mosquitoes were collected by indoor aerosol spraying from August to October 2006. Specimens were identified to species by polymerase chain reaction (PCR) assay and characterized for the ace-1(R) mutation using a PCR-restriction fragment length polymorphism diagnostic. RESULTS: Collected mosquitoes were a mixture of An. gambiae s.s. and An. arabiensis across the Sudan (98.3%vs. 1.7%), Sudan-sahelian (78.6%vs. 21.4%) and the Sahel (91.5%vs. 8.5%) ecotypes. The An. gambiae S-form predominated in the Sudan sites from the West (69%vs. 31% for the M form) but was not found in the Sahel (100% M form). The ace-1(R) mutation was dispersed throughout the Sudan and Sudan-sahelian localities at moderate frequency (<50%) but was absent in the Sahel. It was far more prevalent in S form than M form mosquitoes (0.32 for the S form vs. 0.036 for the M form). No An. arabiensis was detected carrying the mutation. The geographic distribution of ace-1(R) in the Sudan and Sudan-sahelian correlated with the cotton growing areas dispersed throughout the two climatic zones. CONCLUSIONS: These results have special significance as OP and CM insecticides have been proposed as alternatives or additions to pyrethroids which are currently used exclusively in many vector control programmes.

[Comparison of PCR, ELISA-CSP and direct microscopic observation methods for the detection of Plasmodium falciparum sporozoites in Anopheles gambiae M in Senegal]. / Comparaison des méthodes de la PCR, d'ELISA-CSP et d'observation microscopique directe pour la détection des sporozoïtes de Plasmodium falciparum chez Anopheles gambiae M au Sénégal.

A comparative study between the Enzyme-Linked Immuno Sorbent Assay (ELISA-CSP) for circumsporozoitic antigen detection method, the direct observation after dissection and the polymerase chain reaction (PCR) technique used to identify Plasmodium falciparum genomic DNA markers was carried out. This to evaluate the sensibility and the specificity of the PCR, for the determination of both sporozoitic index (ICSP) and the entomological inoculation rate (EIR). The study is conducted in laboratory on eighty six specimens of Anopheles gambiae M infected after being fed with the blood of a gametocytes carrier from Dielmo (Senegal). Salivary glands of forty-eight specimens randomly selected (test A) among the infected eighty six are microscopically observed after manual dissection for the sporozoites detection. The content of these salivary glands and the crushed head/thorax of the remaining 38 specimens (test B) are tested in ELISA-CSP and PCR. The positive and negative results obtained were recorded and summarized for each method. A pair-comparison of the results obtained with each method generally revealed a good sensibility and an excellent specificity The kappa coefficient (K) of test A indicated a "moderate" to "excellent" concordance between the three different methods performed. By using the crushed head/thorax sample, generally used to determine the transmission parameters (ICSP and EIR), the PCR/ELISA-CSP concordance was excellent. In the light of the values of sensibility and specificity obtained, this PCR is comparable to the other methods for the assessment of sporozoitic index and entomological inoculation rate.

Molecular evidence for similarity between Anopheles hyrcanus (Diptera: Culicidae) and Anopheles pseudopictus (Diptera: Culicidae), sympatric potential vectors of malaria in France.

Malaria was a former public health problem in the Camargue, southeastern France, where members of the Hyrcanus group were recently described as the main malaria potential vectors. However, the systematic status in this group, which includes at least two sympatric sibling species, Anopheles hyrcanus (Pallas) and Anopheles pseudopictus Grassi as well as a morphologically intermediate form in the Camargue, is unclear. Indeed, both species have been alternatively considered as separated or synonymous species. We examined sequence variation of the internal transcribed spacer (ITS) 2 and domain-3 (D3) of 28S ribosomal DNA and the cytochrome oxidase subunit I and II (COI and COII) genes of mitochondrial DNA of the Hyrcanus group mosquitoes from the Camargue and Turkey to infer the taxonomic status of the members of this group. DNA sequence analysis of ITS2 and D3 showed no difference between either species or geographical origin (mean pairwise genetic distances d = 0.000-0.003). The COI and COII sequences between French specimens also were nearly identical (d = 0.001-0.002), whereas French and Turkish Anopheles were genetically distinct (d = 0.009-0.014). The distinction between populations of the two areas, supported, respectively, by four and five fixed mutations, attested the differentiation by the distance. Finally, the high degree of genetic similarity, despite morphological differences between An. hyrcanus, An. pseudopictus, and an intermediate form, suggests that these three taxa may belong to a single species in the Camargue.

[Vectorial transmission of malaria in shrubby Savannah area at Ganse, Ivory Coast]. / Transmission vectorielle du paludisme en savane arborée a Ganse en Côte d'Ivoire.

A longitudinal entomologic study was carried out in the village of Ganse located in the Northeastern Ivory Coast from July 2000 to July 2001. The threefold purpose of the study was to index Plasmodium-carrying Anopheles species by capturing mosquitoes on human volunteers, collecting larvae in different dwelling types, and evaluating the involvement each species in the malaria transmission. A total of 4 species belonging to the Anopheles genus were collected in the village. Identification of circumsporozoite protein using the ELISA technique demonstrated that three species were plasmodium vectors. These species belonged to the An. gambiae complex (An. gambiae s.s. 100%), to the An. funestus group (An. funestus s.s. 95.6%) and to the An. nill group (An. nili s.s. 100%). The estimated mean sporozoite index was 5.9% for An. gambiae s.l., 4.3% for the An. funestus group and 2.6% for the An. nili group. The main larva breeding sites were standing water such as puddles for An. gambiae s.l., streams with tall plants for the An. funestus group and the Comoe River for An. nili group. Because peak breeding of these three species occurs at three successive times; i.e., in May, September and July respectively, transmission of P. falciparum is continuous throughout the year. The transmission rate is high since we recorded up to up to 410 infected bites per person per year. In addition to showing the presence of An. rivulorum-like, our findings in the area demonstrates the important role of An. nili s.s. in the transmission and the complexity of the vectorial system.

[Malaria vectors: from the field to genetics. Research in Africa]. / Vecteurs de paludisme: du terrain à la génétique moléculaire. Recherches en Afrique.

Only about 60 Anopheline species transmit malaria among more than 3,000 mosquito species recorded in the world. In Africa, the major vectors are Anopheles gambiae,An. arabiensis, An. funestus, An. nili and An. moucheti. They all belong to species complexes or groups of closely related species that are very difficult to set apart on morphological grounds, but which may have highly variable behaviours and vectorial capacities. Understanding this complexity is of major importance in vector control programs or for implementing any public health intervention program such as drugs or vaccine trials. Among the seven species of the complex,Anopheles gambiaes.s. shows a huge chromosomal polymorphism related to adaptation to specific natural or anthropic environments, from equatorial forested Africa to dry sahelian areas. Recent studies conducted in West and Central Africa suggest an incipient speciation into 2 molecular forms provisionally called M and S. A similar evolutionary phenomenon is observed in An. funestus, in which sympatric populations carrying specific chromosomal paracentric inversions showed restricted gene flow. Distribution of species from An. nili group and An. moucheti complex is restricted to more humid regions of Africa. However in some areas these species play the major role in malaria transmission. Comprehensive knowledge of transmission cycles and of behavioural and underlying genetic heterogeneities that exist within and among natural vector populations will thus benefit the whole area of malaria control and epidemiology. Molecular and genetic studies, as well as in depth monitoring of vector biology, have been recently facilitated by advances in functional and comparative genomics, including recent publication of the nearly complete genome sequence of An. gambiae. Challenge for the next years is to answer to the very simple question: why is an insect a vector?

Natural swarming behaviour of the molecular M form of Anopheles gambiae.

In Anopheles gambiae, as in most species of mosquitoes, mating is initiated in flight. The males aggregate in aerial swarms and conspecific females individually fly to these swarms where they mate with males. In this study, we investigated the swarming behaviour of A. gambiae and conducted 2 surveys in the rice field area of the Vallée du Kou in Burkina Faso in 1999 and 2002. A high number of anopheline mosquitoes were observed in this area and both molecular M and S forms of A. gambiae were found in sympatry. Swarms formed a few minutes after sunset in different places and no obvious markers were associated with their occurrence. However, swarms occurred close to cow herds generally in open flat areas, 2-3 m above the ground. Overall, 2829 anopheline mosquitoes were collected from 21 swarms composed primarily of males. A few specimens of Culex quinquefasciatus were collected from 3 swarms. Although both molecular M and S forms were found in sympatry in the village, swarms were composed almost exclusively of the molecular M form. This suggests that there are alternative swarming habits for both molecular M and S forms of A. gambiae in nature.

Description and bionomics of Anopheles (Cellia) ovengensis (Diptera: Culicidae), a new malaria vector species of the Anopheles nili group from south Cameroon.

Mosquito species of the Anopheles nili group (Diptera: Culicidae) transmit malaria to humans along rivers in Africa. To date, the An. nili group includes the species Anopheles nili s.s. and its pale-winged variant known as the "Congo form," Anopheles somalicus and Anopheles carnevalei. Larval and adult mosquito collections in the forest region of Campo, in southern Cameroon, uncovered an additional morphological variant provisionally called "Oveng form" that was subsequently found to be genetically distinct from the other members of the An. nili group. In this study, we provide further biological data that characterizes this new taxon and justifies elevation to specific rank. We propose calling this new species Anopheles ovengensis, after its geographical origin. We present a morphological description of the adult female and fourth instars and original data on the biology, ecology, and role as a human malaria vector of this new species in its type location. We provide dichotomous keys for identification of adult females and fourth instars that can be used at least in tropical areas of west and central Africa.

Malaria: use of restriction endonuclease digestion and mutation-specific PCR for antifolate resistance isolate detection.

Antifolate resistance isolates of Plasmodium falciparum in the blood of 56 patients was investigated by using PCR technology. DNA was extracted with three different methods from parasite lysate by phenol-chloroform, or from whole blood and from blood collected onto dry filter paper, by chelex-100. The expected 727-bp PCR product was obtained in all samples extracted by chelex-100, while three samples prepared by phenol-chloroform failed to show any amplified product. The crucial point mutation within the dhfr gene leading to pyrimethamine and cycloguanil resistance is localised in an Alul recognition site. Thus, the 727-bp PCR product was submitted to endonuclease digestion. Fifty out of the 56 blood samples analysed yielded the two expected restriction fragments and an undigested 727-bp band. These 50 samples likely represent mixed infection as also confirmed the specific mutation PCR. The six undigested samples amplify a 339-bp fragment using a nested PCR-specific for pyrimethamine resistance mutation. Our results show that, the rapid DNA extraction from blood using chelex-100 and the PCR endonuclease assay can be efficiently used for accurate chemosensitivity analysis in the field.

[Development of a PCR (polymerase chain reaction) specific for complex Anopheles nili (Theobald) 1904 species in Cameroon ]. / Développement d'une PCR (polymerase chain reaction) spécifique d'espèces du complexe Anopheles nili (Théobald) 1904 au Cameroun.

The objective of this study was to develop new molecular tools for the identification of members of An. nili group, a malaria vector in Africa. Our strategy was based on the sequence analysis of portions of the rDNA. The ITS2 fragment of An. nili collected in Cameroon was sequenced and compared. The analysis of these sequences has revealed a great variability of ITS2 sequence. Three molecular forms: An. nili typical form, An. nili Oveng form and An. carnevalei were observed within the six morphological types. Specific primers were selected on ITS2 sequence to develop an allele-specific PCR giving 3 size bands. 169 specimens of An. nili collected in Cameroon were successfully tested. This method has been validated on specimens collected in others localities of tropical Africa. The multiplex PCR developed was very sensitive practical and applicable on large scale.

[Anopheline population genetics]. / Génétique des populations anophéliennes.

Population genetic studies of vectors are essential for (i) the determination of their taxonomic status and consequently the definition of their vectorial role in the transmission of pathogenic agents; (ii) the evaluation of the species genetic variability and the estimation of their capacities of adaptation to selection pressure; (iii) an estimation of gene flow among populations in order to evaluate their degree of isolation and gene circulation, especially resistance genes. Among the malaria vectors taken as examples on three continents, Africa, South-East Asia and Latin America, the large majority of the species showed an important polymorphism. The Gambiae Complex, which is by far the most studied one, includes at present 7 species with the recent description of An. quadriannulatus A and B from Ethiopia. An. gambiae s.s. includes itself 5 chromosomal forms. One of them, the Mopti form, should be considered as a species unto itself. For An. arabiensis, a strong differentiation has been observed among the populations from Senegal and the Indian Ocean Islands. The kdr mutation, which confers resistance to pyrethroid knockdown effect, has never been found either in the Mopti form, or An. arabiensis, indicating a restricted gene flow between these latter two and An. gambiae s.s. The speciation process of the Gambiae Complex seems to be a recent phenomenon due to environmental selection pressure. Species of the Funestus Group are distinguishable by morphological characters. The genetic study of An. funestus s.s. did not show the presence of a complex, in spite of high polymorphism and population structure. Anophelines from eastern areas present an important biodiversity. The Minimus Complex includes two species, A and C, which are widely distributed in South-East Asia. Species A is strongly endophilic, on the contrary species C is at once more exophilic and zoophilic. The latter species might have been selected by DDT indoor house spraying. After numerous taxonomic investigations, the Dirus Complex includes now 7 species. In Latin America, An. pseudopunctipennis clustered into three geographic populations which are under a speciation process. One covers North America and Guatemala, the other South America and Belize, whilst the last one is restricted to Grenada Island. On the contrary, An. darlingi showed little morphologic and genetic variability throughout the species geographic range suggesting the existence of a single species. The main objective of these studies is to implement a more selective approach of vector control programs in relation to the incriminated species, their bioecology and their role in malaria transmission. The improvement of efficiency and selectivity of vector control is becoming a major goal in order to make the best out of the available tools and control the impact of interventions on the environment.

[Systematics and biology of Anopheles vectors of Plasmodium in Africa, recent data]. / Systématique et biologie des anophèles vecteurs de Plasmodium en Afrique, données récentes.

Renewed interest in research on Plasmodium vectors in Africa and development of genetic and molecular biology techniques has been spearheaded by the WHO and the PAL+ program of the French research ministry. New findings have led to a better understanding of the systematics and biology of the main vector groups. The purpose of this article is to describe the newest data on the Anopheles gambiae complex and the M and S forms of An. gambiae s.s., on species in the An. funestus group and genetic polymorphism of An. funestus, on the two probable species in the An. moucheti complex, and on An. mascarenesis.

Swarming behaviour in natural populations of Anopheles gambiae and An. coluzzii: review of 4 years survey in rural areas of sympatry, Burkina Faso (West Africa).

The swarming behaviour of natural populations of Anopheles gambiae and An. coluzzii (formerly known as An. gambiae S and M forms, respectively) were investigated through longitudinal surveys conducted between July 2006 and October 2009 in two rural areas of south-western Burkina Faso where these forms are sympatric. In both sites, the majority of swarms were recorded above visual markers localised among houses. In Soumousso, a wooded area of savannah, 108 pairs caught in copula from 205 swarms were sampled; in VK7, a rice growing area, 491 couples from 250 swarms were sampled. If segregated swarms were the norm in both sites, many visual markers were shared by the two forms of An. gambiae. Furthermore, mixed swarms were collected annually in frequencies varying from one site to another, though no mixed inseminations were recorded, corroborating the low hybrid rate previously reported in the field. The occurrence of inter-specific mate-recognition mechanisms, which allow individuals to avoid hybridisation, is discussed.

Distribution of pyrethroid and DDT resistance and the L1014F kdr mutation in Anopheles gambiae s.l. from Burkina Faso (West Africa).

This study reports on the distribution of pyrethroid and DDT resistance and the L1014F knockdown resistance (kdr) mutation in Anopheles gambiae s.l. populations from 21 localities in three different climatic zones of Burkina Faso from August to October 2006. The susceptibility of these populations was assessed by bioassay using DDT (4%), permethrin (1%) and deltamethrin (0.05%). Anophelesgambiae were resistant to both permethrin and DDT in the Sudanian regions but were susceptible in the central and sahelian areas and susceptible to deltamethrin at all sites except Orodara, although mortality values in some populations were close to the resistance threshold. The kdr frequency varied from 0.4 to 0.97 in populations from the Sudanian region and was lower in populations from the Sudano-sahelian and sahelian areas (0.047 to 0.54). Compared to the last survey of kdr in An. gambiae populations conducted in 2000, the kdr frequency did not differ in the S form but had increased in the M form (0.6), with an extended distribution into the Sudano-sahelian region. The frequency of kdr was also found to have increased in An. arabiensis populations (0.28), where it was formerly reported in only a single specimen. These results have practical significance for malaria vector control programs.

Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism.

Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.

Trichomonas vaginalis: repeated DNA target for highly sensitive and specific polymerase chain reaction diagnosis.

Trichomoniasis is recognised as a major sexually transmitted disease (STD) in the world and may act as an acquired immunodeficiency syndromes (AIDS) co-factor by enhancing the transmission of human immunodeficiency virus (HIV). Diagnosis of Trichomonas vaginalis can be achieved by several methods, but sensitive detection means are still lacking. In this study a 2000-bp repeated DNA fragment of T. vaginalis was cloned. Part of a conserved region of this insert was sequenced, two primers (TVK3 and TVK4) were chosen and a highly sensitive detection by polymerase chain reaction (PCR) was then developed for T. vaginalis. All strains of T. vaginalis analysed with these primers gave the expected 350-bp fragment and a 450-bp additional fragment. Sequence analysis of these PCR amplification products revealed that the 450-bp fragment contained the 350-bp with a 100-bp insertion characterised by a TGG microsatellite. A second primer set, namely TVK3 and TVK7 (determined at the border of the insertion), yielded PCR products of expected sizes. After amplification we were able to detect a single parasite. We also detected T. vaginalis in vaginal fluids of patients with STD. There was no reaction with human DNA or other infectious agents. It appears that the two set primers are highly specific of T. vaginalis and provide a useful tool for PCR diagnosis in asymptomatic and symptomatic patients especially among the HIV at risk individuals.

Molecular identification of the Anopheles nili group of African malaria vectors.

Distinction between members of the Anopheles nili group of mosquitoes (Diptera: Culicidae), including major malaria vectors in riverside villages of tropical Africa, has been based mainly on doubtful morphological characters. Sequence variations of the ribosomal DNA second internal transcribed spacer (ITS2) and D3 28S region between morphological forms revealed four genetic patterns corresponding to typical An. nili (Theobald), An. carnevalei Brunhes et al., An. somalicus Rivola & Holstein and the newly identified variant provisionally named Oveng form. Primers were designed based on ITS2 fixed nucleotide differences between haplotypes to develop a multiplex PCR for rapid and specific identification of each species or molecular form. Specimens of the An. nili group from Cameroon, Burkina Faso, Ivory Coast and Senegal were successfully identified to species, demonstrating the general applicability of this technique based on criteria described in this paper.

A multiplex PCR-based method derived from random amplified polymorphic DNA (RAPD) markers for the identification of species of the Anopheles minimus group in Southeast Asia.

Effective control of Anopheles minimus s.l., an important malaria vector in Southeast Asia, is based on the accurate identification of species within An. minimus complex, which cannot be distinguished using morphological characters. Derived from individual random amplified polymorphic DNA markers, sequence characterized amplified regions were analysed for the design of species-specific paired-primers. Combination of these primers resulted in the development of a simple, robust multiplex PCR able to identify both species An. minimus A and C belonging to the complex, hybrids AC, and three sympatric and closely related species, An. aconitus, An. pampanai and An. varuna. Hybrids AC do not possess alleles of both parents but exhibit novel adaptive potentials resulting from recombination among parental genes leading to hybrizyme.

SCAR markers and multiplex PCR-based identification of isomorphic species in the Anopheles dirus complex in Southeast Asia.

The Anopheles dirus Peyton & Harrison complex of mosquitoes (Diptera: Culicidae) comprises seven known species, including important malaria vectors in Southeast Asia. Specific identification of each species of the complex, which cannot be distinguished using morphological characters, is crucial for understanding vector ecology and implementing effective control measures. Derived from individual random amplified polymorphic DNA (RAPD) markers, sequence characterized amplified regions (SCAR) were analysed for the design of specific paired-primers. Combination of six SCAR primers resulted in the development of a simple, robust, single multiplex PCR able to identify three important malaria vectors among the four most common species (A, B, C, D) of the complex: species A from several Southeast Asian countries, species B from Perlis, Malaysia, and species C and D from Thailand.