Review of the Minimus Complex of Anopheles, main malaria vector in Southeast Asia: from taxonomic issues to vector control strategies.

BACKGROUND: The Minimus Complex of Anopheles subgenus Cellia is composed of two sibling species, A and C, on the Southeast Asian mainland, and a third allopatric species E that occurs in the Ryukyu Archipelago (Japan), a malaria-free region. Anopheles minimus s.l. is considered to be one of the main malaria vector in the hilly forested regions of Southeast Asia. Despite a large number of studies over its range of distribution, it is difficult to have a global view of the ecological and bionomical characteristics of the individual species as different identification methods were used, generally without specific identification of the sibling species. OBJECTIVES: (1) To review the main malaria studies on An. minimus s.l.; (2) to discuss recently published data on the biology and ecology of each sibling species; and (3) to identify gaps in our understanding of the Minimus Complex. REVIEW RESULTS: Major biological and ecological trends are addressed, such as the high plasticity of trophic behaviour and the sympatry of species A and C over the Southeast Asian mainland. Despite the availability of rapid molecular identification methods, we still lack important information concerning the biological characteristics of each sibling species. These gaps must be filled in the future because An. minimus species A and C may exhibit different abilities to transmit malaria. CONCLUSION: We expect that entomological surveys will employ molecular methods to clearly identify these two species, and thus elucidate the biological characteristics of each species. As a consequence, current vector control strategies will be improved by targeting the most efficient vectors.

Malaria transmission and major malaria vectors in different geographical areas of Southeast Asia.

During the last decade, major progress in malaria control has been achieved in Vietnam, Laos and Cambodia. However, malaria is still a potentially fatal disease in some hilly-forested areas and continues to be endemic in a few coastal foci. To estimate the risk that stems from the major vectors after a decade of intensive malaria control, an entomological study based on human landing collections was conducted between April 1998 and November 2000 in six study villages (four in Vietnam, one in Cambodia and one in Laos) located in different physio-geographical areas. Five villages were selected in places where new cases of malaria still occurred. In the sixth village, in the northern hilly area of Vietnam, no case of malaria was detected during the past 3 years. In three study villages of the hilly forested areas of Cambodia and central Vietnam, Anopheles dirus A still played an important role in malaria transmission and maintain perennial transmission inside the villages despite its low density. Anopheles minimus A was found in all study villages except in the southern coastal village of Vietnam. Its role in malaria transmission, however, varied between localities and surveys. In one study village of central Vietnam it was almost absent (one specimen collected over 480 man nights), and in another village sporozoite positive specimens (2.8%) were only observed during the first two surveys whereas this species disappeared from the collections from November 1998 onwards (six surveys: 360 man nights). In the northern study site An. minimus A and C were found in all collections, but no local malaria transmission occurred. However, the constant presence of these two species associated with a high longevity (parous rate up around 80% and 65%, respectively), suggests that transmission can occur at almost any time if parasite reservoirs are reintroduced in the area. The proper management of malaria cases and population movement is, therefore, important to prevent outbreaks and the reintroduction of malaria in northern Vietnam. In the study site of the Mekong delta, An. sundaicus occurred at high densities (up to 190 bites/man/night). The recent changes in land use from rice cultivation to shrimp farming probably explains the increase of this brackish water breeding species during the study period. However, none of the 11,002 specimens was positive for Plasmodium circumsporozoite protein (CSP). The relative low survival rate as estimated by the parous rate (around 47%) may reflect its low vectorial status that could explain the very low malaria incidence (1.9 case/100 persons/year) in this study site. A calculated sporozoite rate of maximum 1/300,000 is enough to explain this low malaria incidence. Despite the successes in malaria control, the vector An. dirus A continues to play an important role in malaria transmission, whereas An. minimus A showed temporal and spatial variation in its role as vector. The role of An. sundaicus as vector could not be confirmed because of the low incidence in the coastal study village. Other Anopheles species may be locally involved, but in the five study villages where malaria is still present they probably do not contribute significantly to malaria transmission. The study also points towards the fact that in Southeast Asia it will become increasingly difficult to incriminate Anopheles species in malaria transmission while the risk for malaria transmission still persist.

Population genetic structure of the malaria vector Anopheles minimus A in Vietnam.

Anopheles minimus A, a major malaria vector in Southeast Asia, is the main target of vector control in this area. The impact of these control measures can be influenced by the population structure of the target species. In rural areas, An. minimus breeds along the banks of small clear-water streams, yet in the suburbs of Hanoi, northern Vietnam, there is an An. minimus population whose immature stages develop in water tanks. This study uses allozyme data (1) to assess the population structure of An. minimus A and (2) to evaluate the taxonomic status of the urban An. minimus population from Hanoi. The population from the suburbs of Hanoi was identified as An. minimus A. Although significant genetic differentiation was observed between rural and urban An. minimus A populations, they have not differentiated substantially by genetic drift. Limited macrogeographical differentiation was observed between two rural populations at distances of more than 1000 km. Consequently, geographical distance is not the primary factor in differentiating An. minimus A populations having the typical breeding ecology. The estimated effective population size is consistent with the moderate macrogeographical differentiation. Furthermore, no genetic structuring was observed between adult mosquitoes having different behaviour. The macrogeographical population structure indicates that genes may spread over large areas, whereas the presence of an 'urban' An. minimus A population shows the ability of this species to adapt to anthropogenic environmental changes.

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.

Confirmation of Anopheles varuna in vietnam, previously misidentified and mistargeted as the malaria vector Anopheles minimus.

Malaria control programs in Southeast Asia are faced with several questions concerning vector behavior and species identification, which need to be answered to consolidate and further improve the results of control practices. The vector system in Southeast Asia is complex because of the number of species potentially involved in malaria transmission. Additionally, the follow-up and evaluation of preventive control measures are hampered by the misidentification of vectors due to overlapping morphological characters of the female mosquitoes. In central Vietnam, control practices are aimed at 2 main species, Anopheles dirus s.l. and Anopheles minimus s.l. These reputed vectors were studied in an area of Binh Thuan Province of south-central Vietnam. Different collection methods were used to capture mosquitoes quarterly during a 1-year period. Mosquitoes were identified in the field and later subjected to detailed morphological examination and polymerase chain reaction-restriction fragment length polymorphism analysis. What was thought to be an unusual morphotype of An. minimus was shown to be Anopheles varuna, and most specimens identified as the former species in the field proved to be the latter species. Very few An. minimus individuals were found during the study period. The population of An. varuna was found to be highly zoophilic, and based on this behavior, it cannot be considered a vector in Vietnam. Because this species was previously being misidentified as An. minimus, a nonvector was mistargeted as a malaria vector in Binh Thuan Province. Anopheles dirus, which was found positive for Plasmodium falciparum circumsporozoite via enzyme-linked immunosorbent assay, is clearly the main vector in this area. Despite the fact that several potential secondary vectors were found during the study, the primary target for vector control in the region should be An. dirus.

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.

Molecular identification of Anopheles minimus s.l. beyond distinguishing the members of the species complex.

Correct species identification is the starting point for studying the epidemiological role of vectors. Identification is mostly achieved using morphological criteria, but this fails when sibling species and species with overlapping morphological characters are involved. The problem with the identification of Anopheles minimus s. l., one of the most widespread malaria vectors in South-East Asia, is twofold: it is a complex of at least two isomorphic species, and based on morphology, members of the complex are difficult to distinguish from closely related species. An identification method was developed for An. minimus species A and C, and four related species, An. aconitus, An. pampanai, An. varuna and An. jeyporiensis. PCR-amplified internal transcribed spacer 2 (ITS2) ribonuclear DNA (rDNA) fragments were digested with restriction endonuclease BsiZI. Clear diagnostic banding patterns for the six species were obtained on agarose gels. Testing field-collected specimens from different regions in South-East Asia indicated that the technique will be applicable over a wide geographical area. From this it is clear that molecular identification has to focus not only on the species of complexes, but also on related species if they hamper the morphological identification of the 'sensu lato species'.

Identification of two species within the Anopheles minimus complex in northern Vietnam and their behavioural divergences.

Elucidating the complex taxonomic status of the major malaria vector taxa and characterising the individual species within each complex is important for understanding the complexity of the vector system in the south-east Asian region and will allow to estimate the impact of vector control measures. This applies to countries such as Laos, Cambodia and Vietnam that spend about 60% of their malaria control budget on implementing vector control activities. We used isozyme electrophoresis to clarify the Anopheles minimus s.l. species composition in northern Vietnam and identify behavioural divergences of individual species. Using different collection methods, adult mosquitoes were caught at monthly intervals from June to November 1995 in four villages. An. minimus s.l. could be distinguished from closely related species, An. aconitus and An. jeyporiensis, at the Octanol dehydrogenase (Odh) enzyme locus. Significant positive Fis values gave clear evidence of nonrandom mating within the An. minimus s.l. population. The highest heterozygote deficiency was observed at locus Odh, which was diagnostic for 2 sympatric An. minimus species in Vietnam similar to the An. minimus A and C species known from Thailand. We found no evidence for restricted gene flow between monthly samples, villages, or collection methods in either of the two An. minimus species. They occurred in sympatry, but in different proportions depending on the collection site, and had dissimilar resting and biting behaviours. Thus a vector control strategy will have a nonuniform effect on the various components of this diverse vector system.