Anopheles species of the mount Cameroon region: biting habits, feeding behaviour and entomological inoculation rates

Authors


Authors
Caroline Ajonina, Tendongfor Nicholas and Theodore Tanke, University of Buea, Department of Life Sciences, P.O. Box 63, Buea, Cameroon. E-mail: ntendongfor@yahoo.com
Sali N. Atanga, Research Foundation in Tropical Diseases and Environment, P.O. Box 474, Buea, Cameroon. E-mail: atangandi@yahoo.com
Dr Didier Fontenille, Laboratoire IRD-ORSTOM, OCEAC, BP. 288, Yaoundé, Cameroon. E-mail: didier.fontenille@mpl.ird.fr
Dr. Samuel Wanji, Research Foundation in Tropical Diseases and Environment, P.O. Box 474, Buea-Cameroon. E-mail: swanji@yahoo.fr or refotde@yahoo.fr (corresponding author).

Summary

There is a lack of data on the Anopheles fauna, its biology and the roles played by different vector species in the transmission of malaria in the mount Cameroon region. The biting habits, feeding behaviour and entomological inoculation rates of different Anopheles species during the dry and rainy season were investigated. A total of 2165 Anopheles was collected, 805 in the rainy season and 1360 in the dry season. Five Anopheles species were identified: Anopheles gambiae s.l., An. funestus, An. hancocki, An. moucheti and An. nili. An. gambiae, An. funestus and An. hancocki, recorded during both seasons, were the main vectors of malaria in the region. An. gambiae s.s. was the only member of the An. gambiae (Giles) complex. These three species had their peak activity between 1 and 2 am. A human blood index (HBI) of 98.29% was recorded for fed Anopheles. The sporozoite rate, for all vectors together, was significantly higher in the rainy season (9.4%) than in the dry season (4.2%) with all the species infected by Plasmodium falciparum. The average inoculation rate was 0.44 infective bites per man per night, which adds up to 161 infective bites per year in this study area. Analyses of relative abundance and infection rate of malaria vectors at different sites situated along a transect of 20 km during the dry season showed high heterogeneity in biting and sporozoite rates. No malaria vector was caught at 1200 m a.s.l. The mount Cameroon region should be considered an area of high malaria transmission intensity.

Introduction

Although tremendous progress has been made in understanding and fighting malaria over the last century, the disease remains one of the biggest public health problems of mankind. The World Health Organization estimates that malaria affects some 300–500 million people, killing 1.5–2.5 million every year. More than 90% of these cases are registered in sub-Saharan Africa with >70% of inhabitants in certain areas of this region being chronically infected by Plasmodium falciparum. Fontenille and Lochouarn (1999) have discussed the complexity of malaria vectorial systems in Africa, with different species involved in transmission in different bio-geographical zones.

The principal vectors of malaria in Cameroon are Anopheles gambiae s.s. and An. funestus, which are widespread. An. arabiensis is restricted to the northern part of the country and An. melas to the coastal zones of the southern part. An. hancocki, An. nili and An. moucheti play a secondary role in the transmission of malaria in the forest block, and their relative importance may vary from one bio-ecological zone to another (Carnevale et al. 1992; Njan et al. 1993; Fontenille et al. 2000; Nkondjio et al. 2002).

The mount Cameroon region can be considered as an area of hyperendemic malaria. Parasitological and clinical studies recently revealed a spleen rate of 62% (65 of 105) and a parasite rate of 71% (75 of 105) in children of 2–9 years of age (unpublished data). Despite this high incidence of malaria in the region, no research has been carried out on malaria transmission, and on the roles played by different vectors at different seasons of the year. Cameroon is characterized by several bio-geographical zones and the national programme for malaria control under the roll-back malaria initiative requires information on the malaria transmission from all the bio-ecological zones of the country. This paper reports on work carried out in the mount Cameroon region on the biting habits, feeding behaviours and entomological inoculation rates (EIR) of malaria vectors, during two seasons of the year.

Materials and methods

Study area

Mount Cameroon, the highest mountain in West Africa and an active volcano, rises from the Atlantic Ocean at the Golf of Guinea and culminates at 4100 m above sea level in Buea. The mountain is formed of a continuous pile of terraces from the base to the summit; at an altitude of 50 m from the coast, is a sedimentary plain that extends from Limbe to Mutengene and Tiko. From Mutengene, the terrain gradually elevates to an altitude of 800–1200 m in Buea town. Hydrologically, some 20 streams are of prime importance and empty into the Atlantic Ocean. Two of these, Ombe and Onge, flow southeast and northwest, respectively.

In this forested area of southern Cameroon, the equatorial climate has been modified by the double influence of the ocean and the mountain. The temperatures are lower than in the other areas of the southern part of the country: the mean values of the minimum temperatures are 20 °C in December and 18 °C in August, the mean values of the maximum temperatures are 35 °C in August and 30 °C in March. Rainfall is also important. Debundsha, located at the western flanks of mount Cameroon registers up to 11 000 mm of rainfall, making this area one of the wettest in the world. The mount Cameroon area has a long rainy season that starts in March and ends in late October with maximum rainfall in August and September. The dry season starts in late October and ends in February.

More than 400 000 people live around mount Cameroon in the towns of Tiko, Limbe, Mutengene, Buea and Muyuka. The population is composed of indigenous Bakweri people, Creoles (from Liberia and Sierra Leone) and immigrants from other parts of Cameroon, especially the North West Province.

Mosquito collection

The study was conducted during the rainy months of June, July, August and September 1998 and the dry months of November and December 1999, January and February 2000. Four stations representing the major settlements along a gradient of altitude (transect) in the mount Cameroon region were selected for the study: Mutengene (4°05′ N; 9°18′ E, 100 m a.s.l.), Molyko (4°08′ N; 9°17′ E (600 m a.s.l.), Likoko (4°13′ N; 9°14′ E, 800 m a.s.l.) and Vasingi (4°17′ N; 9°15′ E, 1200 m a.s.l.). Mosquitoes were collected by the same workforce at all stations during the two seasons through standardized house-resting collections and human-biting collections.

Two human-landing collections were carried out at each month of the 8 months of collection in the four stations, using two teams each with four trained collectors. The activities of the collectors were monitored throughout the night by a supervisor. The first team worked from 6 pm to 12 am, and the second from 12 am to 6 am. Two homes were selected per station with one collector being indoors (generally in the bedroom) and the other outdoors. Collections took place over eight man-nights per station per month with different houses used for subsequent collections. Tubes were labelled hourly and mosquitoes that came to feed on collectors were captured using aspirators and then transferred into the tubes. This gave a total of 256 man-nights during the entire period of collection.

Pyrethrum spray collections as described by Service (1977) were carried out in all four stations every month of the 8 months of collection. Fifteen bedrooms were selected randomly from each locality, with at most three bedrooms from the same house; 480 rooms were sprayed during the entire period of the study. The mosquitoes collected in test tubes were kept in cold boxes and taken to the laboratory for processing.

Collections were made using the same team of collectors who expressed consent to a research protocol approved by the Ethical Committee of the University of Buea and the Research Foundation in Tropical Diseases and Environment. All volunteers were given prophylactic treatment for malaria.

Identification of mosquitoes

Two approaches were used for the identification of mosquitoes: the morphological identification of different species of Anopheles and the molecular identification of members of the An. gambiae s.l. All mosquitoes collected were identified using the morphological key of Gillies and De Meillon (1968) and Gillies and Coetze (1987) and ‘The IRD Software for the Identification of Anopheles of the Afro-Tropical Region’ by Hervy et al. (1998).

Anopheles gambiae s.l. mosquitoes collected in the dry season were further identified into the sibling species using polymerase chain reaction (PCR) techniques demonstrated by Paskewitz and Collins (1990), with minor modifications. Two methods were used: the direct method where a single leg or a wing of mosquito was placed directly into PCR reaction mixture and amplified; and in situations where this method gave negative results, probably because of significant DNA degradation, the extraction method was used.

Estimation of infective rate of Anopheles

Two methods were used for the estimation of sporozoite rates. Initially, microscopy was used for Anopheles collected in the rainy season and later on the enzyme-linked immunosorbent assay (CSP-ELISA) was used for dry season mosquitoes as soon as this assay system was established in the laboratory (Fontenille et al. 2001). The head and thorax of Anopheles caught in the dry season were separated from the abdomen and tested for the presence of P. falciparum, P. ovale and P. malariae CSP by the method described by Burkot et al. (1984) and modified by Wirtz et al. (1987).

Determination of source of blood meal of Anopheles

Fed Anopheles from the house-resting collection were dissected and blood smeared on Whatman filter paper. The technique used for the ELISA was that described by Beier et al. (1988). Antibodies used for the test were of human, goat, chicken and bovine origin. Only Anopheles collected during the dry season were tested using this method.

Estimation of physiological age

Physiological age was calculated using the method of Detinova (1962) where Anopheles mosquitoes collected during the rainy season were dissected and their ovaries observed for tracheolar appearance.

Expression of results

The following entomological indices of malaria transmission were determined: the sporozoite rate – the proportion of Anopheles with positive salivary gland after dissection or positive by CSP-ELISA; the biting rate – the number of Anopheles bites experienced by a collector during an entire night of activity (the biting cycle is the hourly variation of the biting rate and it is also referred to as the aggressiveness of the species); the infective biting rate – the product of the biting rate and the sporozoite rate estimated from human-landing catches; the coefficient of endophagy – the ratio of the number of Anopheles caught biting indoors to that caught biting outdoors; the human blood index (HBI) – the proportion of Anopheles that fed on humans; and the parity rate – the proportion of Anopheles that were found parous.

Simple chi-square analysis was performed to compare different proportions and the chi-square analysis of trend was performed to find out if there is any relationship between the variation in the altitude and the Anopheles infectivity. All the tests were performed at the 5% significance level.

Results

Anopheles species: biting densities, feeding behaviour and infection rates

Between June 1998 and February 2000, a total of 2165 Anopheles were collected: 805 (37.2%) in the rainy season (Table 1a) and 1360 (62.8%) in the dry season (Table 1b). Five Anopheles species were identified: An. gambiae, An. funestus, An. hancocki, An. nili and An. moucheti. An. nili and An. moucheti were rare and found only during the dry season. PCR analysis of the sibling species of An. gambiae revealed that all the 125 specimens tested were An. gambiae s.s. The major species of Anopheles collected from the mount Cameroon region were therefore An. gambiae s.s., An. funestus and An. hancocki.

Table 1.  Species and sporozoite rates of malaria vectors found in the Mount Cameroon region
Anopheles speciesNumber capturedNumber dissectedNumber positiveSporozoite rate (95% CI)
(a) Rainy season
An. gambiae4571572214.01% (8.5–19.4)
An. funestus3101278 6.29% (2.0–10.5)
An. hancocki38380 0
Anopheles speciesNumber capturedNumber tested (ELISA)Number positiveSporozoite rate (95%CI)
(b) Dry season
An. gambiae95655125 4.53% (2.8–6.26)
An. funestus3142458 3.26% (1.04–5.48)
An. hancocki83794 5.06% (0.23–9.89)
An. moucheti550 0
An. nili220 0

The three major species of Anopheles showed different biting rates at the rainy and dry seasons (Table 2a,b). An. gambiae was the most aggressive species at both seasons with three bites per man per night in the rainy season and 5.53 bites per man per night in the dry season. The hourly variations of the aggressiveness of the three major species of Anopheles are shown in Figure 1. All three species display the same biting cycle during the night with a minor peak of activity between 8 and 11 pm and a major peak of activity between 1 and 4 am.

Table 2.  Entomological inoculation rates of malaria vectors in the Mount Cameroon region (1998–2000)
Anopheles speciesNumber captured bitingMan-nightsNumber dissectedSporozoite rate (95% CI)Biting rate (bites/man/night)Infective biting rate (infective bites/man/night)
(a) Rainy season
An. gambiae38412810515% (8.1–21.8)30.45
An. funestus23812864 6.0% (0.2–11.8)1.860.11
An. hancocki3512835 00.270
Anopheles speciesNumber captured bitingMan-nightsNumber tested by ELISASporozoite rate (95% CI)Biting rate (bites/man/night)Infective biting rate (infective bites/man/night)
(b) Dry season
An. gambiae7061283672.17% (0.7–3.6)5.530.12
An. funestus96128954.2% (0.17–8.2)0.750.03
An. hancocki79128765.3% (0.27–10.37)0.620.03
Figure 1.

Night biting cycle of the malaria vectors in the Mount Cameroon region (1998–2000).

The three main species of Anopheles varied in their feeding behaviour during the two seasons (Figure 2); An. funestus and An. hancocki were more exophagous during the dry season while An. gambiae was both endo- and exophagus in equal measure during both seasons. The three main species also displayed heterogeneity in their sporozoite rates and EIR for the two seasons. An. gambiae was more infected during the rainy season (14%; 22 of 157) while An. hancocki was more infected during the dry season (5%; four of 79). An. hancocki was found infected only during the dry season. An. funestus maintains relatively stable infection rates in both seasons: 6.3% (eight of 127) during the rainy season and 3.26% (eight of 245) during the dry season. The EIR was estimated based on Anopheles collected from landing catches (Table 2a,b). The highest infective biting rate was recorded with An. gambiae during the rainy season (0.45 infective bite per man per night).

Figure 2.

Proportions of Anopheles biting outdoors (exophagy) during the rainy and dry seasons.

Overall infective biting rates of 0.61 and 0.2 were obtained for the three species of Anopheles for the rainy and dry seasons, respectively. Therefore, an individual may receive an infective bite every other night during the rainy season and once every five nights during the dry season.

Species infecting Anopheles sp. in the study area

Of 882 Anopheles collected during the dry season and tested for P. falciparum, P. malariae, and P. ovale, all 37 infected specimens had P. falciparum.

Source of blood meal of Anopheles

Of 235 fed Anopheles collected during the dry season and tested for sources of blood meal, 156 An. gambiae, 72 An. funestus and three An. hancocki had fed on humans. One An. funestus had fed both on humans and goats. Four specimens were negative, suggesting that they may have taken blood from sources different from those with available antibodies.

Parous proportions and longevity of Anopheles

The parous proportions for the three major species of Anopheles of the mount Cameroon region were as follows: An. gambiae 80.6 ± 5.14%;An. funestus 78.0 ± 6.32%; An. hancocki 80% ± 6.76%. Assuming an interval of 3 days between blood meals, the probability of surviving for 1 day was very high (0.92–0.93) for all three species of Anopheles and their life expectation was 12–14 days.

Relative abundance and circumsporozoite rates of malaria vectors at different points of a transect of altitude during the dry season

For vector density analyses, 888 Anopheles were used (all species considered) collected during the dry season (Table 3). There was great heterogeneity in the biting rates at different altitudes taken together (χ2 = 16.89, df = 3, P < 10−3). The highest biting rate was recorded at Molyko (600 m a.s.l.), the lowest at Vasingi (1200 m a.s.l.), where no mosquito was captured during the 32 man-nights of collection.

Table 3.  Human biting rates and entomological inoculation rates of malaria vectors at different altitudes of the mount Cameroon region during the dry season (1999–2000)
Station (altitude)Number collectedBiting rateNumber testedNumber positiveCSP rate (95% CI)Infective biting rate
Mutengene (100 m)692.156469.3% (2.48–16.9)0.20
Molyko (600 m)64520.1530941.3% (0.04–2.54)0.26
Likoko (800 m)1745.4317263.4% (0.75–6.21)0.19
Vasingi (1200 m)0

As for CSP rates and infective biting rate, 545 Anopheles (all species taken together) collected from different points of the gradient of altitude and analysed for circumsporozoite protein also displayed heterogeneity in their infection rates (χ2 = 12.42, df = 3, P < 10−2). The highest infection rate was recorded at Mutengene (9.37%), and the lowest at Molyko (1.3%). There was no linear trend between sporozoite rates and altitudes. The EIR did not vary significantly between Mutengene, Molyko and Likoko (χ2 = 3.2, df = 3, P > 0.10).

Discussion

The main species of Anopheles in the mount Cameroon region are An. gambiae s.s., An. funestus and An. hancocki. These results confirm the confinement of the sibling species An. gambiae s.s. to the forested areas of Africa (Coetzee et al. 2000). An. gambiae s.s. and An. funestus are the major vectors of malaria in this study area as they were found infected during the dry and rainy seasons. These two species are well known as efficient vectors of malaria in other areas of Africa and Madagascar (Meyus et al. 1962; Manga et al. 1997; Shililu et al. 1998; Lindblade et al. 1999; Jambou et al. 2001). The presence of An. hancocki in the mount Cameroon region and its implication in malaria transmission is noteworthy; An. hancocki has only rarely been found infected with Plasmodium species (Vaucel & Campourcy 1943; Fontenille et al. 2000). In the mount Cameroon region it was found that An. hancocki had a relatively high sporozoite rate (5%) during the dry season, topping the malaria transmission; hence it should be considered an important malaria vector there.

Anopheles species of the mount Cameroon area were highly anthropophagous (almost 100% of specimens tested fed on human beings despite the presence of other blood sources such as goats, dogs, chickens and pigs that are commonly reared by the inhabitants of this area). These findings are in agreement with other reports from equatorial Africa regions (Nkondjio et al. 2002) and are in contrast with the anthropophagous rate of Anopheles in west Africa and savannah regions where the blood meal preference is determined by the availability of potential hosts (Lemasson et al. 1997; Killeen et al. 2001).

Anopheles funestus and An. hancocki displayed variations in their biting behaviour according to the time of year, being more endophagous during the rainy season and more exophagous during the dry season. This change may be related to the weather conditions in the study area. During the wet season, the rainfall is very heavy and the temperature low. These environmental factors may probably influence these two Anopheles species to be more endophagous than exophagous during the rainy season. An. gambiae seems to be more adapted to the weather conditions of this area as its biting behaviour was similar in the two seasons. This seasonal shift in the feeding behaviour of An. funestus and An. hancocki would have important implications for the vector control in the mount Cameroon region during the rainy season, as insecticide treated nets (ITN) or residual spraying could be very effective in preventing these two Anopheles species from biting, or reducing their vectorial capacity.

By employing the same workforce at different points of a transect of altitude from 100–1200 m a.s.l., it was found that the malaria vectors were heterogeneously distributed along the transect. At Vasingi, a station at 1200 m a.s.l, no mosquito was collected during the research period, probably because of weather conditions: temperatures at this altitude are usually <15 °C. The absence of mosquitoes could also be due to the lack of appropriate breeding sites. The slope at this altitude is abrupt and the streams flow very fast.

The relative abundance of vectors at altitudes where they were found varied greatly, with fewer Anopheles collected at low-altitude Mutengene than high-altitude Molyko and Likoko. The relatively low abundance of malaria vectors at Mutengene is paradoxical as this station is characterized by high temperature (30–36 °C) and a high relative humidity throughout the year. These results could be explained by the fact that the locality is more urbanized than the other two stations and that relatively lower hygienic conditions lead to the contamination of breeding places with polluted materials, making them unsuitable for the development of Anopheles larvae (Bruce-Chwatt 1985). Culex spp. and Mansonia sp. (data not shown) were also collected at this station during the surveys. Mutengene nevertheless seems to be very suitable for malaria transmission as the highest infectivity rate was recorded there.

In contrast to Mutengene, Molyko displayed high malaria density. Molyko is a newly urbanized area, which is experiencing a rapid increase of population since the creation of the University of Buea in 1993. The increase in population at this station is associated to the environmental modifications for the infrastructure required for accommodating and supporting the new population and to the creation of more man-made breeding places for Anopheles (Trape & Zoulani 1987).

Plasmodium falciparum was the only parasite found in the vectors using ELISA-CSP, but this does not mean that it is the only species of malaria parasite in this area. In a recent study (unpublished data) conducted in 105 children in the area (71% prevalence), the specific prevalence of P. malariae was 4%. No P. ovale was found. Therefore it is more likely that P. falciparum is the dominant species in this hilly area.

This study shows that mount Cameroon region is an area of high malaria transmission intensity, inhabited by the major vectors of malaria in Africa. Every other night during the rainy season, and every five nights during the dry season, an inhabitant of this region may receive an infective bite from Anopheles. The abundance of Anopheles species, their highly anthropophagous behaviour and their longevity, coupled with high EIRs, indicate the necessity of introducing the vector control measures in the region as one of the strategies of fighting malaria. Such vector control needs to focus on providing an effective personal protection for the most susceptible age groups against vector contact rather than aiming at reducing the potential for transmission at the regional level. The most appropriate vector control option in this area could be the use of ITN as these tools are currently the most effective and practical vector control option in areas where malaria is highly transmitted (Diallo et al. 1999; Guillet 2001).

Acknowledgements

This study received financial support from the Vihpal programme ‘Anophèles d'Afrique’ of the French Ministry of Research and from the Research Foundation in Tropical Diseases and Environment, Cameroon. We are indebted to Mr Djouaka Rousseau and Ivo Nchendia for their technical assistance. We are also especially grateful to the volunteers who participated in mosquito collection.

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