The relative prevalence and distribution limits of the two main species An. gambiae and An. arabiensis have remained extremely stable since the first available data , more than 35 years ago. Attentive examination did not reveal notable differences. However, huge environmental modifications have occurred, mainly due to demographic increase and human activity resulting in dramatic deforestation and an increase in the areas given over to rice-fields. This did not obviously affect the distribution of the members of the An. gambiae complex.
Because An. arabiensis is present in all regions, the question of understanding the limiting factors of distribution is only of concern in the case of An. gambiae. Bioclimatic domain and altitude appear to be two key variables. An. gambiae is limited in its habitat in Madagascar by only two factors: aridity in the south and south-west and altitude > 1000 m. This picture is perfectly concordant with the findings of this study, except at site No.1 Ampasimajina), perhaps due to the small sample size, near a place in which Chauvet  had identified 3 An. gambiae s.l. in December 1966. The moisture that favours An. gambiae may explain why some individuals have been observed at low prevalence (always <5%) during the period of maximum rainfall in the northern and western margins of the central highlands (site N°32 Ankazobe, and also N°25 Fenoarivo with 1 An. gambiae caught in March 1997). A recent study performed in Kenya  presents two interesting findings: as in Madagascar the moisture index was the most important variable explaining the distribution of An. gambiae, but unlike Madagascar An. arabiensis was not observed in areas above 1400 m in western Kenya.
An. merus was collected from 3 different areas: in the extreme south and a site near Mahajanga in these studies and had been previously collected by Chauvet in a salt swamp near Toliara . It is possible that all the western and southern coasts have suitable habitats but the observation that An. merus is not closely associated with mangroves (in line with Chauvet's findings ) does not support this assumption. Why is this species not observed on the west coast? This is surprising in view of the climatic conditions that prevail, which resemble those observed on the east coast of Kenya where An. merus is found . The different nature of the soils between west and east coast in Madagascar may explain this: mainly sedimentary rocks (alluvial and lake deposits, unconsolidated sands) in the west and metamorphic rocks (basement rocks, lava) in the east .
It is likely that the collection methods influenced the observed distributions, but this influence is not considered to lead to a major bias. The ratio gambiae/gambiae+arabiensis indoor- versus outdoor-landing collections was significantly different, with a higher exophily for An. arabiensis as classically observed in continental Africa, but the percentages were rather similar (41.2% indoor and 35.6% outdoor). Furthermore, each of the 3 species was collected using indoor-landing, outdoor-landing and pyrethrum spray collections. Considering the large variations in zoophilic behaviour of populations of the An. gambiae complex only tens of kilometres apart , an ideal sampling would involve at least 3 different collection methods in each prospected site, which was only achieved in 10 of the 38 sites sampled.
Another point that merits discussion is that of the 45 "PCR negative" mosquitoes. It is conceivable that PCR sometimes fails: this is supported by the decreasing number of "PCR negative" mosquitoes after second then third attempt. Poor DNA is also a possibility. The eventuality of a fourth and unknown species of the An. gambiae complex present in Madagascar, is an unlikely hypothesis but one which cannot be ruled out. Overall, the PCR multiplex method gave reliable results for 98% of tested mosquitoes, which is considered an acceptable result.
An. merus is confirmed as having a role as a malaria vector in Madagascar, which had not previously been recorded. These observations are compatible with those performed in Tanzania where, in places, it is the main vector . A fifth species can now be added to the list of anopheline vectors of human malaria in Madagascar: An. gambiae, An. arabiensis, An. funestus, An. mascarensis and now An. merus.