Acta Tropica 102 (2007)

Transcription

1 Acta Tropica 102 (2007) Identification of four members of the Anopheles funestus (Diptera: Culicidae) group and their role in Plasmodium falciparum transmission in Bagamoyo coastal Tanzania E.A. Temu a,, J.N. Minjas b,n.tuno a,h.kawada a, M. Takagi a a Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan b Department of Parasitology and Medical Entomology, Muhimbili College of Health Sciences, Dar es Salaam, Tanzania Received 20 November 2006; received in revised form 21 February 2007; accepted 17 April 2007 Available online 22 April 2007 Abstract The role of Anopheles funestus group in malaria transmission was investigated in Bagamoyo coastal Tanzania, in the process of characterizing the area as a malaria vaccine testing site. Mosquitoes were sampled inside houses and multiplex PCR was used to identify 649 specimens. The following species were found: A. funestus s.s. (84.3%), A. leesoni (13.6%), A. rivulorum (1.5%) and A. parensis (0.6%). Multiplex PCR of 147 blood-fed specimens showed that over half (57.1%) of the identifiable blood meals were taken from human hosts, and human blood index in A. funestus and A. leesoni was 55% and 82% respectively. Plasmodium falciparum infection rate determined by nested PCR was 11% in A. funestus s.s. Although the abundance was low, 26 specimens of A. leesoni, twoofa. rivolurum and one of A. parensis were found positive for P. falciparum. The presence of four A. funestus species in Tanzania emphasizes the relevance to define precisely their spatial and temporal distribution, specific behaviour, ecology and exact role in malaria transmission Elsevier B.V. All rights reserved. Keywords: Malaria; Anopheles funestus; Anopheles rivulorum; Anopheles leesoni; Anopheles parensis; PCR; Tanzania 1. Introduction The Anopheles funestus Giles group consists of 9 species that are widely distributed throughout subtropical Africa (Gillies and Coetzee, 1987; Gillies and De Meillon, 1968). Members of the group are difficult to distinguish morphologically: A. funestus Giles, A. vaneedeni Gillies and Coetzee, A. leesoni Evans, A. parensis Gillies, A. rivulorum Leeson, A. fuscivenosus Leeson, A. Corresponding author. Tel.: ; fax: address: emmanuel.temu@gmail.com (E.A. Temu). brucei Service, A. aruni Sobti and A. confusus Evans and Leeson (Gillies and Coetzee, 1987; Gillies and De Meillon, 1968). Despite the morphological similarities that exist between them, the biology and the vector competence of these species are very different. With exception of A. funestus, these species are mostly zoophilic. Due to its close association with humans and their habitations, A. funestus is an important malaria vector in Africa sometimes rivaling or exceeding the role of other vectors in malaria transmission. In Burkina Faso, at the end of the rainy season, A. funestus follows in peak abundance after the vectors of the A. gambiae complex, thus extending malaria transmission into the dry period X/$ see front matter 2007 Elsevier B.V. All rights reserved. doi: /j.actatropica

2 120 E.A. Temu et al. / Acta Tropica 102 (2007) (Costantini et al., 1999). In A. funestus, parasite rates of 22% (De Meillon, 1933) and 27% (Swellengrebel et al., 1931) determined by salivary gland dissections have been recorded in South Africa, and of late 11% determined by ELISA in Tanzania (Temu et al., 1998) toas much as 50% (n = 56) determined by ELISA in Burkina Faso (Costantini et al., 1999). Anopheles rivulorum has been implicated as a minor vector of malaria in Tanzania (Wilkes et al., 1996) and yet its human feeding preference is as high as 40% (42/106) in southern Nigeria (Awolola et al., 2005). Although A. vaneedeni feeds readily on humans outdoors and was experimentally infected with P. falciparum in the laboratory, this species has never been implicated in malaria transmission in nature (De Meillon et al., 1977). The report of large numbers of A. parensis found inside human dwellings in Kenya coupled with low human blood index raises the question of whether it is a vector as well (Kamau et al., 2003a). Nevertheless, earlier studies in Africa have found no A. parensis infected with sporozoites (Awolola et al., 2005; Gillies and De Meillon, 1968; Hargreaves et al., 2000; Kamau et al., 2003a). In most parts of Africa, A. funestus is sympatric with other members of the group, such as A. rivulorum, A leesoni and A. parensis (Awolola et al., 2005; Gillies and Coetzee, 1987; Kamau et al., 2003a). Historical evidence suggests that in order to conduct an efficient vector control program, precise identification of species is necessary to separate vector from non-vector species. Control measures leading to a reduction of A. funestus, an anthropophilic and endophilic vector, can lead to upsurge of other members of the A. funestus group that are more exophilic (Gillies and Smith, 1960). For example, in South Africa (De Meillon et al., 1977; Hargreaves et al., 2000), Kenya (Gillies and Furlong, 1964) and Tanzania (Gillies and Smith, 1960 and Matola et al., 1990), indoor spraying used to eliminate A. funestus was followed by an upsurge of funestus look-alike specimens suggesting failure of the control program. However, careful identification revealed that these mosquitoes were in fact A. vaneedeni, A. parensis, A. rivolurum or A. leesoni, which only occasionally or rarely transmit human malaria. In this situation, zoophilic and exophilic habits probably reduced exposure to insecticides. In another situation in Zambia A. longipalpis (Theobald) a predominant zoophilic non-vector mosquito found indoors was initially misidentified morphologically and molecularly as A. funestus s.l. Although these specimens were collected during human landing catches, around 89% of A. longipalpis were found feeding on cattle. This case highlights how crucial correct identification of A. funestus s.l. is to malaria control programmes in areas where A. funestus s.l. and A. longipalpis co-exist (Kent et al., 2006). Identification of members of the A. funestus group has, until recently, depended on the use of morphological characters (Gillies and Coetzee, 1987; Gillies and De Meillon, 1968) and polytene chromosomes (Green and Hunt, 1980). Morphological and cytogenetics methods of identification are time consuming, cumbersome and require a great deal of expertise. For example, morphological identification requires the use of rearing of field collected specimens, due to similarity or overlapping characteristics of some members of the group in immature stages (Gillies and Coetzee, 1987). Cytogenetics, although more rapid than morphological examination, can only be used on half-gravid specimens for species identification, and therefore limit the sample size. Recently, a reliable PCR based molecular technique has been developed to identify five of the most commonly found members of the group (A. rivulorum, A. funestus, A. vaneedeni, A. parensis and A. leesoni), making identification easier and requiring limited amount of DNA (e.g. a mosquito wing or leg) from any life stages of the mosquito (Koekemoer et al., 2002). Reliable species identification is indeed important to assess the relative role played by each species in the transmission of Plasmodium and improve our ability to evaluate efficacy of vector control measures implemented in areas where several species of the A. funestus group are present. As part of the on going project to establish baseline data on demography and malaria burden for future malaria vaccine trials in Bagamoyo coastal Tanzania, this paper investigates the composition of species of A. funestus group to determine their biology and epidemiologic importance in the area. Anopheles gambiae complex constitute other species of malaria vectors which have been previously described in the study area (Temu et al., 1998). 2. Materials and methods 2.1. Study area The study was carried out in the Bagamoyo district located 70 km north of Dar es Salaam in coastal Tanzania. The two villages, Kongo and Matimbwa, used in this study are situated south of the town of Bagamoyo ( S, E; elevation 50 m) bordered on the western edge by Ruvu River, in the Yombo administrative division with a population of about 21,000. The coastal climate is tropical, hot and has high relative humidity with little variation in annual temperature. The

3 E.A. Temu et al. / Acta Tropica 102 (2007) pattern of rainfall is bimodal with a long period of rain between April May and a shorter period of rain in October or November. Economic activities of the majority of people in Bagamoyo villages include subsistence farming, cultivation of cassava, maize, rice, cashew nuts, small coconut plantations, and rice irrigation farming that is both small and large-scale. Cattle goat, dogs and chickens are common domesticated animals kept around dwellings inhabited by people. Houses are mostly traditional (mud walls) with a few western style (cement bricks and corrugated iron roofs). Both A. gambiae and A. funestus are important vectors in Bagamoyo (Temu et al., 1998) and their density fluctuates following rainfall pattern. As a consequence, malaria transmission is high and occurs throughout the year (Shiff et al., 1995). Following extensive public campaigns on insecticide treated nets (ITN), people in these villages are quite aware of the role of mosquitoes in malaria transmission Ethical consideration Verbal informed consent was obtained from each household before the field team accessed their houses Mosquito collection and processing Adult anophelines were sampled, twice every second and the last week of the month of collection, at five households from March to June in 2002, and ten households from March to December in All collections were done inside houses, one house in each of the household, using un-baited CDC miniature light trap from 19:00 to 06:00 h, as described previously (Temu et al., 1998). This was supplemented by use of an early dawn pyrethrum spray catch method and indoor searches by tube aspirator, in rooms that were not used by light trapping or neighboring houses close to a sentinel house. Light trapping was always collected in the same sentinel house, but rooms or houses around sentinel sites used for PSC and indoor catches varied. In the laboratory, Anopheles mosquitoes were identified using the morphological keys (Gillies and Coetzee, 1987; Gillies and De Meillon, 1968) and only A. funestus group mosquitoes were included in the final analysis. The mosquito abdomen was separated from the head and thorax, placed in separate tubes with matching identification numbers. Genomic DNA from the head and thorax was processed for PCR identification of species of the A. funestus group and presence of P. falciparum. Likewise, genomic DNA from engorged abdomens was used to determine the source of the host s blood meal. Since loss of PCR amplification efficiency is likely to result from inhibitors present in the mosquito tissues (Arez et al., 2000), we used IsoQuick DNA isolation kit (ORCA Research Inc.), a silica/quanidium based template preparation method (Chanteau et al., 1994) that efficiently removes PCR inhibitors (Arez et al., 2000) for parasite detection from infected mosquitoes PCR identification of Anopheles funestus Mosquito specimens belonging to the A. funestus group were analysed by a multiplex PCR assay (Koekemoer et al., 2002). In each case, DNA extracted from the head and thorax were amplified and the PCR product of unknown specimens together with positive controls (A. funestus, A. rivulorum and A. leesoni) were separated on a 2.5% agarose gel stained with ethidium bromide and visualized on a UV transilluminator PCR identification of Plasmodium falciparum DNA extracted from the head and thorax of mosquito specimens belonging to A. funestus group were analysed by a nested PCR assay for identification of P. falciparum (Snounou et al., 1993). The amplified PCR product was separated on a 2.5% agarose (1:2 ratios of SaeKem and Nusieve) gel stained with ethidium bromide and visualized on a UV transilluminator. Positive and negative controls constituting of P. falciparum strain K1 and master mix without template DNA was used for each run PCR identification of blood meal source DNA extracted from the abdomen of blood-engorged mosquito were analysed by a multiplex PCR assay for identification of blood meal source (Kent and Norris, 2005). This assays targets a mammalian cytochrome B and primers specific for human, cattle, dog and goat, the common mammals found in the study areas, were employed. Standard specimens of genomic DNA extracted from human, cattle, goat and dog blood were used as positive controls. The amplified PCR product was separated on a 2.5% agarose gel stained with ethidium bromide and visualized on a UV transilluminator Statistical analysis The mosquito populations from each of the households were pooled by year of collection and analysed together. Infection rates were determined as the percentage of mosquito species tested positive for P. falciparum over total number of specimens tested. Differences

4 122 E.A. Temu et al. / Acta Tropica 102 (2007) Table 1 The distribution of species of Anopheles funestus group and specimens infected with P. falciparum collected in Bagamoyo coastal Tanzania Period of collections Number of species caught (% positive for P. falciparum) Year Month funestus leesoni rivulorum parensis 2002 March 31(0%) April 60(20%) 6(16.6%) 1(0%) May 32(15.6%) 24(37.5%) June 16(6.25%) 1(0%) Total 139(12.9%) 32(31.2%) 1(0%) 2004 March 49(0%) July 119(17.6%) 14(14.2%) 5(20%) 3(33.3%) August 26(3.8%) 8(12.5%) September 19(0%) 1(0%) October 2(0%) November 31(6.4%) 2(0%) 2(50%) December 162(11.1%) 32(40.6%) 2(0%) Total 408(10.2%) 56(28.5%) 9(22.2%) 4(25%) Fig. 1. A representative of results of PCR identification of species of Anopheles funestus group found in the Bagamoyo villages, coastal Tanzania. Lanes 1, 2 and 4 are positive controls for A. rivulorum, A. funestus and A. leesoni respectively; lanes 3 and 25 is 100bp DNA ladder, lanes 5 17, are A. leesoni, lane 18 and 24 are A. rivulorum and lane 23 is A. parensis. between year of collection and mosquito species collected were analysed using the 2 test. 3. Results 3.1. Species composition of Anopheles funestus group in the study area A total of 657 A. funestus group were collected inside human dwellings in the Kongo/Matimbwa village of the Bagamoyo district, costal Tanzania. The number of mosquitoes collected from any one house ranged from 0 to 87 with an overall mean of A. funestus group of mosquitoes per house. Of the 649 specimens that were positively identified, four were A. parensis, ten A. rivulorum, 88A. leesoni, 547 were A. funestus (Table 1 and Fig. 1) and there was no PCR amplification in eight samples Infectivity rate and blood meal sources Among the 147 specimens tested by multiplex PCR to determine the source of host blood meal, 57.2% had fed Fig. 2. A representative of results of Anopheles funestus species found positive for P. falciparum in the Bagamoyo villages, coastal Tanzania. Lanes 1, 26 and 46 are 100kb DNA ladder, lanes 4 (funestus), 8(leesoni), 10 (leesoni), 13 (rivulorum), 15 (funestus), 19 (leesoni, weak signal), 24 (leesoni), 27 (funestus), 34 (leesoni), 37 (leesoni), 42 (funestus) are positive for P. falciparum. Lanes 25 and 47 are positive controls for P. falciparum (K1 strains). on human, 14.3% on goat, 8.8% were multiple feeding positive for both human and goat. None of the sample had fed on cattle (Table 2). At least 19.7% (n = 29) samples were scored as negative indicating amplification failure. A total of 90 specimens were positive for P. falciparum (Table 3). Although the infection rate of A. funestus appeared higher in the year 2002 (12.9%) compared to 2004 (10.3%), the difference was not statistically significant ( 2 = 1.4, d =2, p>0.05). Out of ten A. rivulorum caught indoors, two were positive for P. falciparum. Unexpectedly, one out of four A. parensis and remarkably 29.5% (26 out of 88) A. leesoni caught tested positive for P. falciparum (Table 3, Fig. 2). Taking the specimen blood meal status into consideration, at least 37% (range 17.8% 50.0%) of infection rates were found in specimens that tested positive for human Table 2 Number of blood fed Anopheles funestus group mosquitoes caught indoors found to contain blood from human, cattle and goat hosts in Bagamoyo villages, coastal Tanzania Anopheles Species No Tested Cattle only n(%) Goat only n(%) Human only n(%) Human + goat n(%) Negative for all n(%) A. funestus (15.8%) 66(55.0%) 11(9.2%) 24(20%) A. leesoni (81.8%) 2(9.1%) 2(11.1%) A. rivulorum 5 0 2(40%) 0 0 3(60%) A. parensis

5 E.A. Temu et al. / Acta Tropica 102 (2007) Table 3 Species composition of Anopheles funestus group and Plasmodium falciparum infection determined by nested PCR assay in mosquitoes sampled indoors, year 2002 and 2004, from Bagamoyo villages, coastal Tanzania Anopheles species Blood meal status Total number caught indoors Number (%) positive A. funestus A. leesoni Overall Overall Unfed + nonhuman (6.8%) 32(9.1%) 40(8.5%) Human fed a (47.6%) 10(17.8%) 20(25.9%) All (12.9%) 42(10.3%) 60(11.0%) Unfed + nonhuman (22.2) 14(28.0%) 18(26.5%) Human fed a (50.0%) 2(33.3%) 8(40.0%) All (34.3%) 16(28.6%) 26(29.5%) A. rivulorum Unfed + nonhuman (22.2%) 2(20%) A. parensis (25%) 1(25%) a All abdomen samples tested positive for human blood or a mixture of human with other blood source. blood compared with 14.2% (range 6.8% 28%) infection rates among unfed or specimens fed on non-human host (Table 3). Since the dominant malaria parasite at Bagamoyo is P. falciparum (Premji et al., 1995), other parasites such as P. malariae or P. ovale were not tested. 4. Discussion and conclusion Malaria vector control programs in most of sub- Saharan Africa have to deal with different Anopheles species involved in transmission, however, most information available on malaria vector bionomics involves the A. gambiae complex. Due to the increasing recognition of A. funestus as an important vector, tools have been developed for quick identification of species in A. funestus group. This report on the molecular analysis of the A. funestus group has established the presence of A. funestus, A. rivulorum, A. leesoni and A. parensis in coastal Tanzania. About 1.2% of the specimens morphologically recorded as A. funestus could not be identified by PCR. This could be due to misidentification of some of the specimens, problems associated with handling and molecular processing, or the presence of other members of the A. funestus group which could not be identified for one reason or another. The presence of the same four members of A. funestus group has also been reported in western and coastal Kenya (Kamau et al., 2003a), suggesting that sympatric occurrence of these species is common in Africa (Awolola et al., 2005; Hargreaves et al., 2000). Collections performed inside human dwellings reported here show that the abundance of A. funestus and A. leesoni is significantly higher than both A. parensis and A. rivulorum (p < ). Low numbers of A. rivulorum observed indoors at Bagamoyo is indicative of exophilic/exophaghic behaviors that have been demonstrated elsewhere (Hargreaves et al., 2000; Wilkes et al., 1996). Indeed, A. funestus was found almost exclusively inside human dwellings while A. rivulorum was found almost exclusively outdoors in Kenya and Nigeria (Awolola et al., 2005; Kamau et al., 2003b). In this study, A. parensis was the least abundant species of A. funestus group, but it was the most frequent and common species found inside human dwelling in Kenya (Kamau et al., 2003a). These field observations suggest that the abundance of species belonging to A. funestus group fluctuates throughout the year and varies from one area to another, highlighting the need to systematically investigate seasonal dynamics in relation to malaria transmission. Separating complex species of malaria carrying mosquitoes is important for effective malaria vector control programs. The results of the multiplexed PCR assays of blood meal sources indicate that A. funestus is predominantly anthropophagic (55%), whereas A. leesoni shows both strong and weak feeding preference to human (>80%) and goat (9%) respectively. Although A. rivulorum had fed exclusively on goat, this observation should be considered with caution due to the small number of specimens tested. The high degree of anthropophagic found among A. leesoni contradicts an earlier report indicating that this species feed mainly on cattle even in the presence of human hosts (Gillies and De Meillon, 1968). While all specimens were collected indoors, the presence of goat fed A. funestus, A. leesoni and A. rivu-

6 124 E.A. Temu et al. / Acta Tropica 102 (2007) lorum demonstrates exophagic and endophilic behavior for these species. Likewise, A. leesoni collected inside human dwellings in Zambia had taken blood from either cattle or goat, which suggests exophagic and endophilic behaviors (Kent and Norris, 2005). Although 29 samples were negative for the presence of either human, cattle or goat blood, this finding does not rule out the possibility that members of A. funestus group could be feeding on other hosts. Besides, negative reaction could also be a result of PCR failure due to degradation of DNA in the blood (Kent and Norris, 2005), or the blood source being from other animals that were not tested (e.g. bird) or it was truly negative. In some parts of Kenya, A. parensis is the most abundant species found inside human dwellings and has shown a tendency to feed on humans (Kamau et al., 2003a). Likewise, the reports of A. leesoni found inside human dwellings in Kenya (Kamau et al., 2003b) and West Africa (Awolola et al., 2005) demonstrate endophilic and anthropophagic tendencies. In our study, A. leesoni was the second most abundant member of the A. funestus group found inside human dwellings and the majority of specimens had fed primarily on human and secondarily on goats, suggestive of strong anthropophagic, weak exophaghic and endophilic tendencies for A. leesoni in coastal Tanzania. Unexpectedly, specimens of A. leesoni and A. parensis were positive for P. falciparum and closer scrutiny revealed several important observations. First, the intensity of PCR band signal in some of positive samples was weak, suggesting the integrity of parasite DNA was compromised. Second, taking the source of blood meal into consideration, infection rates in samples that tested positive for human blood was much higher than unfed samples, or samples with blood sources other than human. Despite the fact that the abdomen was separated from the head/thorax and given the high sensitive of PCR used, it is possible that parasite found in thoracic hemocele or parasite residual of human blood found in the head/thorax were amplified and this could explain higher infection rates among individuals that had fed on human. However, several unfed specimens of A. leesoni tested positive, even though the intensity of PCR signals in most of these samples were comparatively low compared to A. funestus that tested positive for parasites. Such huge infection rates in non-vectors should be interpreted with caution because none of the previous studies have reported any sample of A. parensis or A. leesoni infected with the Plasmodium parasite, either by salivary gland dissections or ELISA detection methods (Awolola et al., 2005; Kamau et al., 2003a,b; Gillies and De Meillon, 1968; Hargreaves et al., 2000). While one cannot rule out the possibility that the PCR is detecting sporozoites in the salivary glands themselves, it is likely that the method is also picking up parasite in the thoracic hemocele. PCR, therefore, unlike salivary gland dissections, does not guarantee that the mosquito was infective unless it was carried out on the salivary glands alone. Further studies are required involving detection of parasite by PCR performed on salivary glands dissected from wild members of the A. funestus group. Overall, 11% of A. funestus were infected with P. falciparum in Bagamoyo, concurring with earlier reports indicating the major role played by this species in malaria transmission in Tanzania (Davis et al., 1995; Temu et al., 1998), West Africa (Awolola et al., 2005) and Southern Africa (De Meillon, 1933; Hargreaves et al., 2000; Swellengrebel et al., 1931). Malaria transmission in coastal Tanzania is intense, occurs throughout different seasons and a child can be expected to have at least five malarial episodes a year (Premji et al., 1995). This situation is reflected by high infection rates among vectors found in the area (Temu et al., 1998). Although the abundance was low, the presence of infected individuals of A. rivulorum in Bagamoyo concurs with a report from Tanga (Wilkes et al., 1996), confirming this species to be a vector playing a minor role in malaria transmission. During the course of this study, a number of other anophelines were encountered, including A. gambiae s.s., A. arabiensis, A. merus, A. coustani and A. squamosus. The first three species are important malaria vectors in the area (Davis et al., 1995; Temu et al., 1998). More than one species within the A. funestus group was found infected with P. falciparum, therefore substantiating the need for correct identification of malaria vectors that belong to species groups or complexes in order to establish areas of sympatric existence and to assess the role played by each species in malaria transmission. This information will improve our ability to evaluate efficacy and strategic planning of vector control measures. In conclusion, since mosquito abundance displays temporal and spatial fluctuation and since more than one species within the A. funestus group was found infected with P. falciparum, it will be relevant to define precisely the distribution of the A. rivulorum, A. leesoni and A. parensis, to determine their relationship with other populations of A. funestus, in areas of differing levels of malaria endemicity, as well as to determine their specific behavior, ecology, and to assess their relative role in malaria transmission. Among members of the A. funestus group, relative species composition is likely to differ from one locality to another; therefore local characterization of species diversity is required for effective management of vector control.

7 E.A. Temu et al. / Acta Tropica 102 (2007) Conflicts of Interest The authors have no conflicts of interest concerning the work reported in this paper. Acknowledgements Our sincere thanks go to the villagers of Matimbwa and Kongo in Bagamoyo for their cooperation and field staffs for their skilful assistance during the field work, Dr M Alifrangis, Centre for Medical Parasitology, Copenhagen Denmark for kindly providing the P. falciparum strain K1 used as positive control, and for the financial support received from the Japanese Society for the Promotion of Science (JSPS) to EAT through its Centre of Excellence (COE). References Arez, A.P., Lopes, D., Pinto, J., Franco, A.S., Snounou, G., do Rosario, V.E., Plasmodium sp: optimal protocols for PCR detection of low parasite numbers from mosquito (Anopheles sp.) samples. Exp. 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