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Keywords:

  • An. gambiae;
  • malaria vector;
  • distribution;
  • Central Highlands;
  • Madagascar

ABSTRACT:

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

In Madagascar, Anopheles gambiae has been found below altitudes of 1,000 m. We sampled An. gambiae sensu lato (sl) between 2008 and 2010 in the Central Highlands of Madagascar at altitudes over 1,200 m. The study site consists of rainforest, rainforest edge, and an open savanna biotope. Anopheles gambiae and An. arabiensis, as well as molecular forms of An. gambiae, were identified molecularly. An. gambiae accounted for 26.7% at the edge of the rainforest and 2.3% in the open savanna biotope. One specimen of this species was caught in the forest. An. arabiensis accounted for 66.3% at the edge of the rainforest and 97.7 % in the open savanna biotope. All An. gambiae adults tested belonged to the S molecular form. An. gambiae is present at high altitudes in Madagascar, with a high prevalence at the rainforest edge. Several factors, including the appearance of new favorable biotopes, recolonization after a reduction of indoor vector control, and climate change, may contribute to its distribution. The changing distribution of An. gambiae may have consequences for the distribution and incidence of malaria in the Malagasy Highlands.


INTRODUCTION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Anopheles gambiae sensu lato is a complex of species, including two freshwater species (Anopheles arabiensis and An. gambiae) and a brackish water species (An. merus) in Madagascar (Chauvet 1969, Léong Pock Tsy et al. 2003). The presence of two freshwater species was first suspected by observing their both zoophilic and exophilic behavior on the Central Highlands (Chauvet et al. 1964). Members of the complex present zoophilic, anthropophilic, exophilic, and endophilic behaviors in other regions of Madagascar (Chauvet et al. 1964, Grjebine 1966). The existence of different species was confirmed on the basis of chaetotaxy on the larval stage (Chauvet et al. 1969) and recently by molecular analyses, including polymerase chain reaction (PCR) (Léong Pock Tsy et al. 2003).

In addition to the differences in behavior, species of the An. gambiae complex are distributed differently in the four bioclimatic domains of Madagascar. An. merus is present on the coast, An. arabiensis in four bioclimatic domains, and An. gambiae is generally limited to areas below 1,000 m, especially in the wet zone in the east, with the exception of some localities on the margins of the Malagasy Highlands. Indeed, on the margins of the Central Highlands, An. gambiae has been caught, albeit in small numbers: two of 121 An. gambiae sl caught in Ankazobe, six of 33 An. gambiae sl in Anjeva, and one of eight An. gambiae sl at Betatao, respectively, at 1,300 m, 1,280 m, and 1,350 m elevations (Chauvet 1969, Léong Pock Tsy et al. 2003). The absence of this species from the capital, Antananarivo, with an altitude of 1,200 m, has been reported (Ravoahangimalala et al. 2008).

Although An. gambiae has not been rigorously described at Ankazobe, its presence there may be because Anjeva and Betatao, located on the eastern margin of the Central Highlands, have characteristics similar to the eastern slope where it is abundant (Chauvet 1969). Understanding factors influencing the presence of An. gambiae in the Central Highlands would be epidemiologically informative relative to the transmission of Plasmodium parasites. Indeed, temperature and rainfall have direct effects on the density of these vectors by acting both on larval development and on the sporogonic cycle (Bodeker et al. 2003). Similarly, the distribution of these species depends strongly on moisture and much less on altitude (Minakawa et al. 2002).

The factors favoring the presence of An. gambiae, and in particular those in the Central Highlands, are poorly understood. We studied An. gambiae in the municipality of Ambongamarina located on the eastern margin of the Central Highlands, between Betatao and Anjeva, where An. gambiae has been reported to be present (Chauvet 1969). The study site included three different habitats: forest, forest edge, and an open savanna biotope, ideal for estimating the effect of the forest on the presence of An. gambiae at high altitude.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Study sites

The study site in Ambongamarina is 30 km northeast of the city of Anjozorobe and is composed of rainforest, rainforest edge, and an open savanna biotope with several villages. The rainforest biotope is in the rainforest of Anorana (18°18′44″ S, 048°00′98″ E), at an altitude above 1,150 m, with the highest peak at 1,345 m. The rainforest of Anorana is part of the rainforest corridor of Anjozorobe-Angavo. In the rainforest edge biotope, the village of Anorana (18°18′19″ S, 048°00′21″ E) is at an altitude 1,285 m and is surrounded by interspersed relics of forest, swamps, and rice fields. The village consists of 39 houses with 204 inhabitants. The main activity is agriculture, with two crops of rice annually: one in mid-March (vary vakiambiaty) and the second between December and January (vary aloha). In the open savanna biotope, the village of Antanifotsy (18°19′04″ S, 047°58′32″ E) is 4.7 km southwest of the rainforest at an altitude of 1,295 m. The landscape consists of narrow strips of lowland rice and food crops, sisal hedges and banana plantations, and 120 houses with 553 inhabitants in the village. There are two crops of rice annually.

During the rainy season, the climate of Ambongamarina is characterized by a high rainfall with peaks in January and March. The night time temperature varies from 14° to 20° C in the primary forest and 10° to 22° C in the two villages. The relative humidity at night is 94–99% in the rainforest and its edge, and 73–99% in the village of Antanifotsy. During the dry season, there is little rainfall, although drizzle is not uncommon and night time temperatures varies from 9° to 15° C in the rainforest and between 11° and 15° C in both villages. In the forest and forest-edge, the relative humidity at night is between 82–99%, and in the village of Antanifotsy it is between 78–97%.

The entomological investigations in the rainforest were conducted between November, 2008, and July, 2010, with sampling every month during the rainy season from November to March, and once in June or July for the dry season. For the open biotope and rainforest edge, the same sampling frequency was used, but the study started in January and February, 2009, respectively. Several methods were used: butterfly nets and a backpack were used for daytime catches of imagoes in outdoor and indoor shelters and in Muirhead Thomson (1958) pit traps. This latter is a rectangular pit dug about 150–180 cm deep, 120–150 cm long, and 90–120 cm wide. In each of the four vertical sides of the pit, a little cavity is dug about 30 cm deep with a horizontal roof. CDC-light traps and net traps baited with animals or humans were used for night catches in only the two villages. Mosquitoes were identified in the field, then frozen in liquid nitrogen and the identification subsequently confirmed in more detail morphologically in the laboratory on a chill table. After identification, mosquitoes were sorted according to species, sex, and female status (fed or unfed) and then pooled and stored at –20° C for molecular studies.

The dynamics of An. gambiae complex was studied with ten CDC-light traps used together between November, 2009 and July, 2010 at the rainforest edge. There were three nights of capture at each site and traps were placed near swamps, bushes, gardens, outside houses, and near the poultry houses and sheep and cattle parks in both villages.

Mosquito processing

PCR assays were used to identify mosquitoes within the An. gambiae sl complex according to the method of Scott et al. (1993). Molecular forms were identified according to the protocol of Favia et al. (2001). The origin of the blood meal of fed mosquitoes was determined according the method of Beier et al. (1988). Eight antibodies corresponding to human, rat, cow, pig, sheep, goat, poultry, and dog were tested by ELISA. Fed mosquitoes were collected using a backpack aspirator, from indoor and outdoor resting places, and particularly Muirhead Thomson's pit traps, dug in and around both villages.

RESULTS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

A total of 235 adults of the Anopheles gambiae complex was caught during the two years of study. PCR analysis gave exploitable results for 226 specimens and both An. arabiensis and An. gambiae were present (Table 1). Nine specimens were PCR negative despite three independent PCR runs.

Table 1.  Number of anopheline and culicine mosquitoes species present in the municipality of Ambongamarina.
SpeciesForestEdge of forestVillage AntanifotsyTotal
Anopheles coustani 124,8162,8407,668
Anopheles fuscicolor 0973100
Anopheles arbiensis 067128195
Anopheles gambiae 127331
Anopheles mascarensis 587163255
Anopheles squamosus/cydippis 07,03521,58528,620
Culex annulioris 5911070
Culex antennatus 211,9071,4333,361
Culex argenteopunctatus 604489661
Culex decens 1,369139361,544
Culex giganteus 8997372701,906
Culex pipiens 5,7083266396,673
Culex univittatus 42,1631,4183,585
Lutzia tigripes 71210
Total8,68917,46128,52954,679

An. arabiensis was the most abundant species: 71.3% (67/94) and 97.7% (128/131) of identified specimens of the Anopheles gambiae complex from the edge of the rainforest and from the village of Antanifotsy, respectively. No specimen of this species was captured in the forest of Anorana.

In total, 13.2% (31/235) of all specimens identified by PCR were An. gambiae. This species was captured in the three biotopes with a high prevalence of 28.7% (27/94) in the village at the edge of the forest. One specimen of An. gambiae was captured in the forest and three of the 131 PCR-identified specimens in the village of Antanifotsy were An. gambiae. Twenty-six An. gambiae were studied for molecular form identification. Five specimens were PCR negative and 21 belonged to the molecular form S. None belonged to the molecular form M.

Different types of traps captured these two species; light traps were the most productive, yielding 68.5% (155/226) of the capture. Both species were collected in nets traps baited with domestic animals (zebu, poultry, and sheep) (8%, 18/226), but none were collected in net traps baited with humans. Muirhead Thomson's wells (Figure 1) and catches in outdoor shelters provided 18% (40/226) and 5% (12/226) of all captures, respectively. None were caught in indoor shelters. All 31 blood meals tested by ELISA were taken from cattle (Table 3). An. gambiae sl was abundant during the rainy season, with an abundance peak in December for An. gambiae and in January for An. arabiensis (Figure 2).

image

Figure 1. Map of the distribution of Malagasy rainforest; the places and periods where An. gambiae has been reported in the Central Highlands and the three study sites in the municipality of Ambongamarina, in the district of Anjozorobe.

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Table 3.  Sex and engorgement status of An. gambiae complex collected in the municipality of Ambongamarina.
 MalesFemales
  unfedfed
  1. *31 fed mosquitoes were tested by ELISA. Nine were caught from outdoor resting places and 22 were collected in Muirhead Thomson's pit traps.

An. gambiae 1273
An. arabiensis 1914135*
       Total2016838
image

Figure 2. Monthly densities of An. gambiae (dark) and An. arabiensis (gray) in Anorana village (rainforest edge).

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DISCUSSION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Anopheles gambiae appears to be well established in this area (above 1,200 m), particularly at the rainforest's edge where its prevalence is high (≥28%) (Table 2). This species was found in our study site during several periods, albeit at a very low proportion of all captures in the rainforest (one specimen) and the open biotopes (2%). Consistent with previous observations in Madagascar (LeGoff et al. 2006), all specimens belonged to the S molecular form. An. gambiae was known to be present only at altitudes below 1,000 m, with the exception of small numbers observed on the northern and eastern margins of the Central Highlands (Chauvet 1969, Léong Pock Tsy et al. 2003). Recent studies confirm the absence of this species from the capital Antananarivo, which is at 1,200 m altitude (Ravoahangimalala et al. 2008). The possible presence in Madagascar of a fourth unidentified species of the An. gambiae complex has been suggested (Léong Pock Tsy et al. 2003) and may correspond to the PCR–negative mosquitoes we captured. However, these negative results may have also been the consequence of poor DNA preparations.

Table 2.  Annual catch of species of the Anopheles gambiae complex in each biotope at the municipality of Ambongamarina.
 ForestEdge of ForestVillage Antanifotsy
 Nov 08–Jun 09Nov 09–Jul 10Feb 09–Jun 09Nov 09–Jul 10Jan 09–Jun 09Nov 09–Jul 10
An. gambiae 1072012
An. arabiensis 0026515113
PCR negative206100

In this study, the highest proportion of An. gambiae was observed in the village at the rainforest edge. The higher humidity in the forest-edge biotope compared to the open biotope of Antanifotsy may favor the development of An. gambiae. This species prefers wetter areas than An. arabiensis (Coetzee et al. 2000) and aridity is one of the factors limiting the spread of An. gambiae, which is very abundant in the wetlands of eastern Madagascar (Léong Pock Tsy et al. 2003). Anjeva and Betatao are close to the eastern slope of the highlands, 40 and 10 km, respectively, from the edge of the eastern cliff. Both sites present characteristics similar to the eastern slope where this species present in abundance (Chauvet 1969). In any case, An. gambiae is less abundant than An. arabiensis in the deforested area in the eastern slope of Madagascar (Chauvet 1969). Leong Pock Tsy et al. (2003) suggest that the humidity that favors An. gambiae may explain the presence of some individuals during the period of maximum rainfall in the northern and western margins of the Malagasy Central Highlands (Léong Pock Tsy et al. 2003). However, some populations of An. gambiae, established from the outset in an environment with large seasonal variation, may become dynamically adapted to this environment (Chauvet 1969). In view of the absence of An. gambiae from the capital Antananarivo (Ravoahangimalala et al. 2008), our results are consistent with the Kenyan findings of Minakawa et al. (2002) that An. gambiae is the predominant species in saturated environments, whereas An. arabiensis is more tolerant of dry weather. The distribution of An. gambiae and An. arabiensis is influenced more by moisture than by altitude in Kenya (Minakawa et al. 2002). Moreover, higher indoor humidity observed in the forested area and lower indoor humidity in the deforested area may have negatively affected the survival of An. gambiae in the Kenyan Highland, (Afrane et al. 2006). These observations suggest that rainforest corridors could provide a favorable biotope for An. gambiae by maintaining the indoor and outdoor humidity in the margins of the Central Highlands of Madagascar.

An. gambiae would still be present at high altitudes in the eastern part of the Central Highlands, but insecticide pressure during indoor vector control from 1998 to 2010 may explain the low proportion of An. gambiae complex in this study. Indeed, before the 1949 start date of indoor vector control, both An. arabiensis and An. gambiae were present on the Malagasy Highlands (Lumaret 1963), as shown in the map of distribution of An. gambiae complex (http://www.cartographie.ird.fr/). They were anthropo-zoophilic and anthropophilic, respectively (Jouncour 1956, Chauvet 1973). However, during the subsequent extensive DDT spraying campaigns that targeted houses, more exophilic and zoophilic vector populations may have escaped, while endophilic and anthropophilic vector populations were more likely to have been eliminated in the Central Highlands (Chauvet and Rajaonarivelo 1973), except in Anjozorobe, Ankazobe, and Lack Itasy, where the indoor treatment of the 1950s had less effect on the endophilic and antropophilic vector populations (Lumaret 1963). Interestingly, our observations concerning the behavior of the An. gambiae complex indicate that both An. arabiensis and An. gambiae were zoophilic and exophilic at the sites studied. Our results are thus consistent with the observation that at the time of the eradication campaign and after the recent indoor anti-vector interventions of 1998 to 2010, surviving populations of An. gambiae sl were more exophilic and zoophilic in the municipality of Ambongamarina. Indeed, such trends have been observed in other parts of Madagascar (Fontenille and Campbell 1992), in Equatorial Guinea (Reddy et al. 2011), and in Tanzania (Russell et al. 2011). The low proportion of An. gambiae population we report in this study may also be the result of indoor vector control pressure. This case was observed in Anjeva and Betatao 43 years ago (Chauvet 1969). This would be the case of Ankazobe, where An. gambiae was collected in low proportions (Léong Pock Tsy et al. 2003). Further studies are needed to understand this issue.

Lastly, climate change may also be having an effect on the distribution of An. gambiae. In Madagascar, the maximum temperature has increased by 2° C between 1980 and 2010. A high ambient temperature, at least with certain limits, is likely to accelerate the development from egg to adult and may contribute to changes in geographic distribution (Chastel 2006).

The changing distribution of An. gambiae may have epidemiological consequences, as An. gambiae is thought to be a better malaria vector than An. arabiensis. The climatic and environmental conditions at this site may allow the coexistence of the two species, which were caught at high abundance by CDC light traps during the rainy season (Figure 2). This method was used not only as a substitute for the human-landing and daytime indoor resting collections (Sadanandane et al. 2004, Mathenge et al. 2004) but it can provide means to study the trend in monthly density of Anopheles species (Faraj et al. 2009).

The presence of An. gambiae in different biotopes demonstrates its ecological plasticity, allowing it to adapt to the different environments of the Central Highlands. If the changes we report in its distribution become widespread, the malaria status of the Central Highlands may change, with a malaria epidemic followed by endemic persistence of the disease, as already described in the Mascarene Islands (Julvez et al. 1998). Insufficient mosquito control may be a significant factor allowing continued recolonization of the Central Highlands by this species, especially in areas close to the Anjozorobe-Angavo forest corridor. These areas are not far from eastern lower-altitude areas where both An. gambiae and An. arabiensis exhibit both zoo-anthropophilic behavior and endo-exophilic behavior. Continued monitoring of mosquito biology and distribution is an essential part of the fight against malaria.

Acknowledgments

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

We thank Dr. Sandra Telfer, Pr. Christophe Rogier, and Gilbert LeGoff for critical reading of the manuscript. This study was conducted as part of the research project (RIFT-OI) on emerging infectious diseases transmitted by arthropod vectors in the geographical area of the Indian Ocean, financed by the Institut Pasteur de Madagascar (IPM) and the Centre de Recherche et de Veille sur les maladies émergentes dans l'Océan Indien (CRVOI).

REFERENCES CITED

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
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