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Am. J. Trop. Med. Hyg., 78(1), 2008, pp. 169-175
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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Distribution and Chromosomal Characterization of the Anopheles gambiae Complex in Angola

Maria Calzetta, Federica Santolamazza, Gian Carlo Carrara, Pedro J. Cani, Filomeno Fortes, Maria Angela Di Deco, Alessandra della Torre*, AND Vincenzo Petrarca
Dipartimento di Scienze di Sanità Pubblica, Università degli Studi di Roma "La Sapienza"; Ministry of Health, National Program of Malaria Control, Luanda, Angola; Istituto Pasteur-Fondazione Cenci-Bolognetti; Dipartimento di Genetica e Biologia Molecolare, Università degli Studi di Roma "La Sapienza," Rome, Italy


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Mosquitoes of the Anopheles gambiae complex (N = 1,336) were sampled (2001–2005) across Angola to identify taxa, study inversion polymorphisms, and detect the circumsporozoite protein of Plasmodium falciparum. Anopheles gambiae s.s. was found in all sites; it was characterized as M-form in localities of the tropical dry and semi-desertic belts, whereas the S-form was predominant in comparatively more humid and less anthropized sites. Both forms were characterized by low degrees of chromosomal polymorphism based solely on the 2La inversion, a pattern usually associated with An. gambiae populations from forested, humid, and derived savanna areas. Unexpectedly, this pattern was also observed in M-form populations collected in dry/pre-desertic areas, where this form largely predominates over An. arabiensis, which was also detected in central/inland sites. Anopheles melas was found in northern coastal sites. Three of 534 An. gambiae s.s. were positive for P. falciparum CS-protein, whereas none of the 105 An. melas were positive.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Information on malaria vectors in Angola and in the whole central part of the African continent is limited, especially concerning the member species of the Anopheles gambiae complex. In particular, the distribution and the chromosomal inversion patterns of An. gambiae molecular forms in West Africa has only been extensively analyzed in northern and southern Guinea savanna areas, where the M-form and S-form show high frequencies of paracentric inversion polymorphisms, and in forested and humid areas north of the Equator, where they are characterized by a low degree of polymorphism, mainly based on the 2La inversion, or even monomorphism.1,2

Based on chromosomal data, Coluzzi (1982) proposed that co-adapted inversions may act as selective forces leading to the formation of new species within An. gambiae s.s.3,4 If inversions have had a crucial role as selective forces, the presence of uninverted homokaryotypic M and S in forested and humid areas of west-central Africa could be interpreted as a convergence due to the role of the standard arrangement on chromosome-2 in the ecotypic adaptation to these environments. In this case, the core of the speciation process would have been in west African savanna areas, and the southern humid areas would have been re-colonized afterward.1 However, the scarceness of data on the distribution and the absence of data on chromosomal inversion polymorphisms of molecular forms in west-African dry areas south of the Equator, do not allow further speculations on the speciation process within An. gambiae s.s. and on the role of the inversions in the ecotypic adaptation to these areas.

The few existing papers on the Angolan mosquito fauna were published mainly by Portuguese scientists before the beginning of the war events (November 1975), which came to an end in 2002. The major pre-war article was by Ribeiro and Ramos,5 who sampled species of the genus Anopheles at 147 sites, covering predominantly the western and central areas of the Country. Anopheles gambiae s.l. was sampled at many of those sites, but no attempt was made to methodically differentiate the member species of the complex, except to discriminate between generic "fresh-water species" and An. melas, and to report in the Cacuaco area a few records as "Anopheles gambiae species A" (= An. gambiae s.s.), through the crossing experiment technique. However, based on eco-climatic speculations, Ribeiro and Ramos stated that "species A will prove to be more common in the West Africa biomes in Angola, while species B (= An. arabiensis) is expected to be more associated with the Rhodesian Highland and SW Arid Zone." Moreover, in the same article Ribeiro and Ramos (citing Ribeiro and others, unpublished reports) reported Plasmodium sporozoite indexes of fresh-water An. gambiae (presumably An. gambiae s.s.) ranging from 0–8%.

Very recently, Cuamba and others reported the results of the PCR–RFLP-based species identification of about 400 An. gambiae s.l., collected in 2001 in Angola at three central and western localities, and at Luanda, the capital city.6 Cuamba and others identified An. gambiae s.s. M molecular form (94%), S-form (6%), a single An. arabiensis larval specimen in the site of Samba (Luanda), where An. melas was also found. No data on the chromosomal inversion patterns of the collected samples were reported. Cuamba and others also assessed, through an ELISA-based method, the Plasmodium falciparum infection rates of 580 An. gambiae s.s., reporting an overall infection rate of less than 2%.

The end of wartime in 2002 opened up the perspective of updating entomological data in the region. Here we present the results of sampling activities (2001–2005) aimed at collecting entomological baseline information, especially in and around urban coastal sites, mainly to study the distribution and chromosomal characterization of the taxonomic units of the An. gambiae complex, with particular reference to the M and S molecular forms of An. gambiae s.s.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Study area and climate. The sampling activity was carried out in peri-urban and rural sites belonging to 11 Provinces of Angola (Table 1Go, Figure 1Go), which are situated in four main different eco-climatic domains (sampling sites between parentheses):


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TABLE 1
Relative frequencies of Anopheles gambiae species and molecular forms sampled in sites in Angola listed north to south
 

Figure 1
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  		    FIGURE 1. Relative frequencies of indoor-resting Anopheles gambiae M-form, An. gambiae S-form, An. Melas, and An. arabiensis at sites across Angola. Smaller cyclograms refer to collections with a sample size < 10. * The Luanda cyclogram refers to the sum of the Nazaré and Bairro Pinto samples.

 
  1. The Provinces of Cabinda (Cabinda), Zaire (Soyo District: Kikudo, Matajor), Cuanza Norte (N’dalatando), Malanje (Malanje), and Lunda Sul (Saurimo) are located in the northern tropical humid zone, characterized by areas ranging from broadleaf evergreen forest to savanna. The northern Cabinda coastal enclave and the Soyo District are located in an oil well area; the other localities are in rural zones.
  2. The Provinces of Luanda (Samba, Nazarè, Bairro Pinto, Mateba, Cazenga, Viana, Mussulo) and Bengo (Cabungo) are located in the northern-central part of the coast, characterized by tropical dry climate and mangrove swamps along the sea-shore, and grassland and woodland further inland. The collection sites were mainly located in peri-urban areas, except for Cabungo, a rural zone, and for the peninsular strip of Mussulo, a tourist area of Luanda.
  3. The Provinces of Benguela (Cavaco) and Namibe (Namibe) are located on the southern coastal area, characterized by an even drier climate, which degrades into desert in the extreme south; the collecting sites were in peri-urban rural areas.
  4. The Provinces of Huambo (Cossango, Camussamba) and Huila (Lubango) are located on an inland plateau (Bié plateau) that extends as far as 2/3 of the country (average altitude of 1200–1600 m above sea-level), characterized by evergreen tropical forest in the north, humid savanna in the center and southeast, and desert steppe in the southwest.

Annual rainfall ranges from 1750–1250 mm in northern and central areas, to 600–400 mm in the extreme southeast, up to 250–100 mm in the southwest and less than 100 mm in the extreme southwest. The rainy season is from November to March in the extreme southwest, from September to May in the northeast, and from November to April on the inland plateau, where the dry and cool season is from May to October. In Luanda the rainy season is from October to April/May, and the average rainfall (about 600 mm per year) is lower than surrounding areas.

Mosquito sampling and identification. Collections of indoor-resting adult An. gambiae s.l. were carried out from June 2001 to March 2005: date details are given for each collecting site in Table 1Go. Sampling was performed by hand-operated aspirators on domestic walls (IR-HC) or inside bed-nets (IR-NET), or by pyrethrum spray collections (IR-PSC) (Table 1Go). The living mosquitoes were kept in moistened cool-boxes until they reached the half-gravid gonotrophic stage, suitable for polytene chromosome analysis. Either whole female mosquitoes or their dissected ovaries were dropped in Carnoy’s fixative (one part of glacial acetic acid in three parts of absolute ethanol). Specimens were stored at –20°C until processing. The non-half–gravid mosquitoes were kept in vials with desiccant. Larval collections were carried out at Samba (Luanda): larvae were bred to adults and then processed as previously outlined. Anophelines were identified using the morphologic identification keys of Gillies and de Meillon7 and Gillies and Coetzee.8 Anopheles gambiae s.l. specimens were identified by species and molecular forms following the PCR-RFLP protocol by Fanello and others.9

Cytogenetic analysis. The half-gravid Carnoy’s fixed An. gambiae s.l. females and ovaries were processed for ovarian polytene chromosome analysis following della Torre.10 Paracentric inversion karyotypes were scored according to the nomenclature of Coluzzi and others11 and Petrarca and others.12 Observed karyotype frequencies were tested against Hardy-Weinberg expectations by {chi}2 test.

Plasmodium infection rates. Plasmodium falciparum circumsporozoite protein (Pf-CSP) detection was carried out following Beier and others,13 on a subsample of An. gambiae s.l. collected in 2001–2005 in sites of the Provinces Zaire, Luanda, Bengo, Lunda Sul, Benguela, Huila, and Namibe.

Blood-meal identification. A sub-sample of blood-fed An. gambiae s.l. females collected in the Luanda Province in 2002 was analyzed for host preference: blood from the mosquito stomach was absorbed and dried on filter paper and the blood-meal source was determined following Beier and others,14 using human, bovine, swine, and canid antisera. Forty An. gambiae s.s. from Cavaco (Benguela) were tested with human and rat antisera.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Distribution of Anopheles gambiae species and molecular forms. A total of 1,336 An. gambiae s.l. specimens from 22 sites of Angola were identified by species/form: 3 were An. arabiensis, 214 An. melas, 904 An. gambiae M-form, and 215 An. gambiae S-form. No hybrids between these species/forms were found. The relative frequencies of the species and molecular forms at the collection sites are reported in Table 1Go and summarized in Figure 1Go.

Two of the three An. arabiensis specimens were sampled in highland sites (Provinces of Huambo and Huila); the third specimen was found in the arid area of Namibe, where An. gambiae M-form largely prevailed (99.5%).

Anopheles melas was found in most coastal sites where mangrove brackish swamps were present. More in detail, this species was found in sympatry with the S-form of An. gambiae s.s. in the Zaire Province sites (30.7%) and with the M-form in most of the sampling sites in the Luanda area with frequencies ranging from 19–94%.

Anopheles gambiae s.s. was found in all the localities sampled; the two molecular forms have been found in sympatry in a single sampling site (N’dalatando, Cuanza Norte Province). The M-form was mainly recorded in coastal localities of Luanda, Benguela, and Namibe Provinces, whereas the S-form was found in northern coastal sites (i.e., Cabinda and Zaire Provinces) and inland localities.

Chromosomal characterization. The single karyotyped An. arabiensis was inverted homozygote for the inversion 2Rb (Figure 2AGo); all the rest of the complement were standard (i.e., uninverted for any polymorphic inversions).


Figure 2
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  		    FIGURE 2. Schematic representation of the paracentric inversions recorded in samples of Anopheles arabiensis (A), An. melas (B), and An. gambiae s.s. (C) in Angola. The homozygote 2Rb/b is shown for An. arabiensis. For An. melas, the polymorphic inversions 2Rm1 and 2Rm1#9 are drawn on the species-specific 2Rm/m arrangement. For An. gambiae s.s., the polymorphic inversion 2La is drawn on the standard (i.e., uninverted) 2L arrangement

 
The 50 karyotyped specimens of Anopheles melas were polymorphic for the inversion 2Rm1 (Figure 2BGo), with a mean frequency of the inverted arrangement of 33%; the three 2Rm1 karyotypes were in Hardy-Weinberg equilibrium [{chi}2 = 0.85, degrees-of-freedom (df) = 1, P = 0.36], even when lumping together all the samples. A single specimen from Kikudo (Soyo District, Province of Zaire) was heterozygous for an inversion provisionally named 2Rm1#9, a short inversion based on the inverted arrangement 2Rm1.

We karyotyped 259 An. gambiae s.s., 236 of which were subsequently discriminated as M-form (191 from Luanda area, 39 from Cavaco, and 6 from Namibe) and 12 as S-form (9 from Kikudo and 3 from Cabinda); 11 have not been molecularly identified, mostly due to unavailability of the carcasses. All of them were standard homozygotes for chromosomal arms 3R, 3L, and 2R (i.e., uninverted for any of the known polymorphic inversions), whereas all samples showed the common 2La/+ polymorphism (Figure 2CGo), with a mean frequency of the inverted arrangement of 13.9%. No significant differences were recorded among the 2La frequencies of the M-populations from Luanda area, Cavaco and Namibe (2La mean frequency = 13.6%). The M-form populations were in Hardy-Weinberg equilibrium. The small S-form sample had a 2La frequency of 20.8%, not significantly greater than that of the M-form (Fisher exact probability test P = 0.23).

Plasmodium infection rates. We analyzed for the presence of Pf-CSP a total of 712 An. gambiae s.l. specimens. All 105 An. melas and the single An. arabiensis tested were Pf-CSP negative. Three of 606 An. gambiae s.s. specimens were positive: 1 of 355 M-form from Luanda area (0.28%; standard error [SE] = 0.28), 1 of 130 M-form from Namibe (0.77%; SE = 0.77), and 1 of 27 S-form from Zaire Province (3.70%; SE = 3.63). The Pf-CSP index was not significantly different between the M- and S-form samples (0.41%, SE = 0.29 and 3.70%, SE = 3.63, respectively; Fisher exact probability test, P = 0.15).

Host preferences. Blood-meal identification of 141 An. gambiae M-form from the Luanda area and 40 from Cavaco (Benguela Province), showed that 61% and 70%, respectively, of the indoor-resting females had fed on humans, whereas the rest had bitten animals other than bovines, swine, canids (at Luanda) and rats (at Cavaco); the difference was not significant. Fifteen of 25 (60%) indoor-resting An. melas of the coastal area of Luanda had fed on humans.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Anopheles arabiensis. Our data show that An. arabiensis is largely outnumbered by the M-form of An. gambiae s.s. in the drier coastal sites, in spite of the eco-climatological conditions apparently very suitable for arabiensis, which could suggest better fitness and possibly a process of competitive exclusion of the M-form over An. arabiensis, as suggested by Touré and others.15 The single An. arabiensis from Namibe was among the southernmost records of this species in west Africa (see Coetzee and others, for references)16: the Namibe area is characterized by a nearly desert climate (annual rainfall: about 100 mm), which is known to be tolerable for this species, particularly in East Africa (see Petrarca and others, for references).12 The other two specimens were found at highland sites (> 1.000 m asl; annual rainfall > 1000 mm), in agreement with other findings at mid/high altitude areas of Ethiopia,17 Madagascar,18 and Mozambique.19 Cuamba and others did not report the presence of An. arabiensis in the highland zone (i.e., Huambo), while reporting a single specimen of this species from the Luanda area.6 Apart from obvious sample-size biases, these differences could possibly be due to the different sampling periods (e.g., late versus early rainy season), which could reflect differentiated seasonal prevalence for this species. An additional, reasonable hypothesis is that the minimal presence of An. arabiensis in indoor samples could also reflect a marked exophilic behavior (as it was observed on the Madagascar Plateau),18 which seems to be supported by the fact that the single An. arabiensis found by Cuamba and others was a larva.6

Anopheles melas. As expected, An. melas was found in the coastal sites characterized by the presence of brackish swampy zones with mangroves.7 In more detail, this species was found in sympatry with An. gambiae M-form in most of the coastal sampling sites of the Luanda area, as observed also by Cuamba and others,6 and by Wondji and others on the Atlantic shores of southern Cameroon.2 The indoor relative frequencies of Luandan An. melas varied from zero to more than 90%, even on limited temporal and/or spatial scales (i.e., Mateba, Mussulo and peri-urban sites of Luanda city; see Table 1Go and Figure 1Go), not an uncommon finding when dealing with a species that is known to have non-uniform population dynamics (see references in Bryan and others.20) In the sites of Zaire Province, An. melas was sympatric with the S-form of An. gambiae; to our knowledge, this is the first time such an event is reported south of Ghana coasts: in fact, Yawson and others21 reported the presence of a single An. melas specimen together with 133 M- and S-forms at a site of coastal Ghana.

The paracentric inversion set-up of Angolan An. melas has shown remarkable similarities with coastal populations sampled at the Congo River mouth in 19903 (Petrarca & Coene, unpublished data). In fact, although separated by more than 10 years in time and about 100 km in space, the Congolese and Angolan populations share the 2Rm1 and 2Rm1#9 floating inversions, which have thus proven to make stable local polymorphisms. They have not been recorded elsewhere in west Africa20,22–25 (Petrarca and others, unpublished data).

Although no Pf-CSP positive An. melas individuals have been found in the sub-sample analyzed, the high indoor relative frequency reached by this species in some sites, associated with a human blood index around 60%, suggests a potential role of this species in local malaria transmission.7,8,20

Anopheles gambiae s.s. Anopheles gambiae s.s. was present at all sampled sites; both molecular forms were found, with M-form being the most abundant taxon. The two molecular forms apparently showed a complementary distribution. In fact, we found the M-form only in the sites in the Luanda area (with the exception of a single S-specimen collected in Cabungo, Bengo Province), Benguela, and Namibe Provinces. Although Cuamba and others reported the presence of both forms in the Benguela Province,6 the prevalence of M-form in coastal sites in the Luanda area and southward is a consistent finding, which could be attributed to a better adaptation of this form to the anthropized environment where the collections have been carried out. Conversely, the S-form was the only one observed in our samples from northern coastal sites (i.e., Cabinda and Zaire) and in the small samples from humid inland sites (i.e., at Malanjie, Lunda Sul, and Huambo Provinces). This may possibly reflect a better adaptation of the S-form to more humid and rural environments, although the co-occurrence of both forms in the inland areas is supported by our finding of one M-specimen in Cuanza Norte and by the report of several M-specimens in Huambo by Cuamba and others.6 Although these results are probably affected by collection and/or season biases, the observed distribution of the two molecular forms is likely to reflect a definite prevalence of one form over the other in different eco-climatic conditions, which is consistent with previous observations in other west-African areas, where M-form predominates in urban or peri-urban settings, whereas the S-form is usually more abundant in rural environments.1,2,26

Both forms had Pf–CSP-positives, with non-significant differences between M- and S-form. The overall Pf–CSP-positivity (0.59%, SE =0.34) was non-significantly different from that reported by Cuamba and others (1.90%, SE =0.57),6 and within the range of the sporozoite rates reported by Ribeiro & Ramos for "fresh-water An. gambiae" (presumably An. gambiae s.s.).5

The cytogenetic analysis of the Angolan An. gambiae s.s. populations showed that both molecular forms are characterized by the same low degree of chromosomal polymorphism based on the floating inversion 2La only, and also share similar frequencies of its inverted arrangement. Populations of An. gambiae s.s. very similar to the Angolan ones for the same inversion set-up have been recorded in sites generally characterized by a humid tropical climate, like in some West African sites (southern Ghana,27 southern Cameroon,2 and Kinshasa area, Democratic Republic of Congo [Petrarca & Coene, unpublished data]), and especially in extensive areas of East Africa, namely eastern Mozambique,19 coastal Madagascar,28 and Comoro archipelago.29 Thus, the inversion set-up of the Angolan samples is strictly comparable to that of the An. gambiae s.s. populations from rain forest areas and humid and derived savannas.1,30 However, it must be noted that, although the central and northern areas of western Angola actually lie in the humid tropical belt (where the S-form predominates), the coastal sites (Luanda and Cavaco) and particularly the southernmost collection locality (Namibe) lie in comparatively more arid or even pre-desertic areas, where the potential larval sites are almost exclusively associated with, or even provided by, human activities, where the M-form thrives. In fact, the ability of the M-form to exploit man-made larval breeding sites in dry areas has already been shown in Mali and Burkina Faso,15,31,32 where Sudan-savanna or Sahelian rice fields are almost exclusively colonized by this form. In these regions the ability and the highly successful adaptation of the M-form to arid habitats have been associated with 2R inversion polymorphisms typical of this form, such as 2Rbc/+ and 2Ru/+. On the contrary, the very low degree of inversion polymorphism characterizing the M-populations from the comparatively drier zones of Angola suggests that the M-form local adaptation to man-made larval sites and arid conditions could be completely independent from the 2R inversions. Moreover, these M-populations also show unexpected relatively low frequencies of the inverted arrangement of the catholic 2La/+a polymorphism, which is known to be associated with an adaptation to aridity.11,33,34

If these preliminary observations were confirmed on a larger scale, it would open a new debate on the role of chromosomal inversions in the ecotypic adaptation to the ecological conditions in south dry areas and on the speciation process within An. gambiae s.s.


CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In conclusion, our results provide new information on the Anopheles gambiae complex in Angola, highlighting regional peculiarities, such as the unexpected predominance of an almost chromosomally monomorphic M-form over An. arabiensis in dry/pre-desertic areas. However, knowledge of the bionomics of the Angolan member species of the An. gambiae complex and their genetic make-up still requires a significant strengthening. In particular, it would be of great interest: i) to clarify how the M-form of An. gambiae is capable of exploiting dry and pre-desertic areas without making use of paracentric inversions as tools for ecotypic adaptation; ii) to extend the temporal/spatial sampling activity, particularly toward the inland and highland zones, to study the temporal/ spatial distribution boundaries of the species/forms1; iii) to carry out a genetic characterization and comparison of the S-populations of An. gambiae and of An. arabiensis from Angola with those from East Africa, with which they share similarities (e.g., a generalized low degree of chromosomal polymorphism; presence of An. arabiensis on highlands); and iv) to study with much higher detail the trophic behavior and the abilities of the species/forms in transmitting Plasmodium.

Received March 12, 2007. Accepted for publication July 2, 2007.

Acknowledgments: Collections have been done in a framework of collaboration among University of Rome "La Sapienza," Italy, and representatives of the Italian Ministry of Foreign Affairs and Angolan Ministry of Health. We thank André Francisco Sebastião, Mpova Zambote, Alberto Bunga, and Manuel Alfredo Paulo (Instituto Nacional da Saúde Publica-Luanda) for technical assistance during the field collections. We are especially grateful to Stefano Ferroni, Project Manager of the "Programma di Cooperazione Socio-Sanitaria AID 5810." We thank Joao Pinto for commenting on the manuscript. We thank Mr. Gianni Petrangeli and Mrs. Graziella Croce for laboratory support and Mario Coluzzi for advice and support.

Work was funded by Istituto Pasteur-Fondazione Cenci-Bolognetti, Italian Ministry for University and Research (funds MIUR/PRIN), University of Rome "La Sapienza" (Faculty funds), and by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR).

* Address correspondence to Alessandra della Torre, Piazzale Aldo Moro 5, Rome, Italy 00185. E-mail: alessandra.dellatorre{at}uniromal.it Back

Authors’ addresses: Maria Calzetta, Federica Santolamazza, Gian Carlo Carrara, Maria Angela Di Deco, Alessandra della Torre, and Vincenzo Petrarca, Dipartimento di Scienze di Sanità Pubblica, Sezione di Parassitologia, Università "La Sapienza," Piazzale Aldo Moro, 5, 00185 Roma, Italy, Telephone: +39-06-4455780, Fax: +39-06-49914653, E-mails: Maria.Calzetta{at}uniroma1.it, Federica.Santolamazza{at}uniroma1.it, carbriga{at}alice.it, Mariaangela.Dideco{at}uniroma1.it, Alessandra.dellatorre{at}uniroma1.it, and Vincenzo.Petrarca{at}uniroma1.it. Filomeno Fortes and Pedro J. Cani, Ministério da Saúde–Programa Nacional de Controle da Malária, Luanda, Angola.

Reprint requests: Alessandra della Torre, Dipartimento di Scienze di Sanità Pubblica, Sezione di Parassitologia, Università "La Sapienza", Rome, Italy. E-mail: alessandra.dellatorre{at}uniroma1.it.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

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