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Am. J. Trop. Med. Hyg., 78(6), 2008, pp. 872-877
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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Malaria Vectors in Areas of Plasmodium falciparum Epidemic Transmission in the Amazon Region, Brazil

José Almir M. da Rocha, Sabrina B. de Oliveira, Marinete M. Póvoa, Luciano A. Moreira, AND Antoniana Ursine Krettli*
Universidade do Estado do Pará, Belém, PA, Brazil; Instituto Evandro Chagas, Secção de Parasitologia, SVS/MS, Belém, PA, Brazil; Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, MG, Brazil


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main vectors of malaria in Brazil are Anopheles darlingi, An. aquasalis, and some species of the An. albitarsis complex, whereas others have questionable importance with regard to the disease transmission. To identify these vectors in the State of Pará, Brazil, in a high-prevalence P. falciparum area, 565 anophelines were captured and identified while the seasonal variation and daily biting activity were determined. Of the seven anopheline species (An. strodei, An. albitarsis s.l., An. rondoni, An. darlingi, An. triannulatus, An. oswaldoi, and An. nuneztovari), the plasmodia circumsporozoite protein (CSP) was detected in three of them, with a total infection rate of 6.2%. An. darlingi was the most prevalent species (22.4%), followed by An. albitarsis (5.2%) and An. rondoni (3.6%). An. rondoni was found to be infected for the first time, which was also confirmed through PCR. This result possibly represents a new malaria vector based on its highest frequency, biting and seasonal activities in the peak of malaria transmission.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the Amazon region of Brazil, malaria remains endemic, with up to 600,000 new human cases detected yearly, based on examination of thick blood smears by technical officers of the Brazilian Ministry of Health (Fundação Nacional de Saúde). The State of Pará accounted for about 45% of the cases (278,204 diagnosed and treated cases)1 in 2000 when this study started. Of those, 13,000 cases (8,397 of Plasmodium vivax, 4,324 of Plasmodium falciparum, and 239 mixed infections) were recorded in Marabá, the locality chosen for the present study. Plasmodium malariae is also prevalent in the area (< 1%), but is considered to be underestimated due to diagnosis using thick blood smears.

Anopheles darlingi Root, 1926 is the primary malaria vector in the Brazilian Amazon region,2,3 followed by species of the An. albitarsis Lynch–Arribalzaga, 1878 complex in some areas.2,4–6 Description of the Albitarsis Complex was first based on distinct chromosomes,7 and more recently by PCR analysis, allowing the inclusion of An. albitarsis E species.8–10 Species such as An. deaneorum Rosa-Freitas, 1989, An. oswaldoi Peryassú, 1922, An. albitarsis E and An. braziliensis Chagas, 1908 are considered potential vectors.6,8,11–15

In the present work, we aimed to identify possible Anopheles species involved in malaria transmission in a region of P. falciparum and P. vivax epidemics. We carried this out in Marabá, southeastern Pará, where we captured and identified mosquitoes during the peak of disease transmission. Seasonal variations and daily biting activity of the most abundant mosquito species were studied followed by ELISA (enzyme-linked immunosorbent assay) using monoclonal antibodies against the circumsporozoite protein (CSP) of Plasmodium parasites. We detected An. darlingi, An. albitarsis sensu lato (s.l.), and An. rondoni Neiva and Pinto, 1922, positive for the malarial CSP of P. falciparum and P. vivax. These data were further confirmed by PCR to check for the presence of plasmodia in mosquito pools, when An. rondoni mosquitoes were shown to be positive, for the first time, for Plasmodium spp. This turns the species into a newly recognized potential vector of malaria in the state of Pará, Brazil, although further studies with dissections of salivary glands would be ideal to confirm the presence of sporozoites.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study area. Mosquitoes were captured in the periphery of Marabá, southeastern Pará (05° 22' 07' ' S, 49° 07' 04' ' W), approximately 438 km from the capital, Belém, at the confluence of the Tocantins and Itacaiunas rivers. This area comprises 15,092 km2 and had approximately 168,020 inhabitants in the year of the study (IBGE, Instituto Brasileiro de Geografia e Estatística, 2000. Available at: www.ibge.com.br). Marabá was chosen because at that time an epidemic of P. falciparum malaria, co-transmitted with P. vivax, was reported.

Ethical approval. Oral consent to capture mosquitoes indoors on sleeping adult volunteers was obtained from each individual in a protocol previously approved by the Ethics Committees of the University of Pará (UFPA) and the Instituto Evandro Chagas, in Belém.

Mosquito collections and processing for malaria tests. Mosquitoes were collected outdoors, monthly from June to September 2001, being captured hourly on one of us (JAMR) and on inhabitants of the village with their prior consent, most of them while they slept in hammocks. Mosquitoes were captured on the legs and arms with a hand-held aspirator, between 6 and 12 pm, and also using CDC light traps (Centers for Disease Control and Prevention, Atlanta, GA) placed outdoors overnight. The number of mosquitoes captured per hour was recorded and biting activity time determined. Identification was based on available taxonomic keys.16–18 Identified mosquitoes had their thoraces and abdomens carefully separated and these parts were kept in tubes containing silica gel for further use. Each dissected thorax was individually triturated in 50 µL of blocking buffer solution containing 0.5% NP-40 in a small tube and immediately used to search for Plasmodium CSP by ELISA, or frozen for further studies. Individual or groups of three to five pooled abdomens were kept frozen until used in PCR reactions.

ELISA. Detection of CSP was performed through ELISA as previously described19,20 using material from individual mosquito thoraces in plates previously coated with monoclonal antibodies anti-CSP specific for P. falciparum, P. vivax VK210, P. vivax VK247, or P. malariae; positive controls for each Plasmodium species were used as well as a negative control antigen, represented by material from uninfected mosquitoes (Anopheles reared in the laboratory). The monoclonal antibodies were obtained from the National Center for Infectious Disease (CDC), and produced by Kirkegaard and Perry Laboratories (Atlanta, GA). The same batches of capture monoclonal, peroxidase-labeled monoclonal, and positive control antibodies were used in all tests. The absorbance of solutions at 405 to 414 nm was determined 30 min after adding the substrate to an ELISA plate reader. The negative cutoff was calculated as twice the mean values of the negative controls.

PCR analysis. Nested PCR analysis was performed as previously described21 to confirm the presence of Plasmodium DNA. Because the thoracic material had been used for ELISA and was no longer available, DNA was extracted from abdomens, either from individual mosquitoes (in the case of An. darlingi and An. albitarsis s.l.) or from pools of three to five abdomens, following a previously described protocol.22 Briefly, mosquito abdomens were homogenized in grinding buffer (0.1M NaCl; 0.2M sucrose; 0.1M Tris-HCl pH 9.1; 0.05M EDTA and 0.05% SDS) following incubation at 65°C for 30 min, and precipitated by adding potassium acetate (8M) and placing on ice for 60 min. After centrifugation, DNA pellet was washed with 70% ethanol, air dried, and suspended in 50 µL of H2O. PCR reactions were performed as follows: for the first reaction, 333 nM of each primer (rPLU6 and rPLU5), 1x PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.4, 1.5 mM MgCl2, and 0.1 mg/mL gelatin), 200 µM dNTP, 1 U Taq DNA polymerase (Invitrogen, San Diego, CA), and 6 µL of each DNA sample (or 2 µL plus 4 µL H2O of the Plasmodium spp.-positive control DNA) in a total of 15 µL reaction; in the second reaction, 2 µL of the first reaction product were used as template with P. vivax- and P. falciparum-specific primers.21 Cycling parameters for both reactions were: 95°C for 5 min, 30 cycles (reaction 1) and 25 cycles (reaction 2), 58°C for 2 min; 72°C for 2 min, 94°C for 1 min, then one cycle of each of 58°C for 2 min and 72°C for 5 min.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have found 7 anopheline species (An. strodei, An. albitarsis s.l., An. rondoni, An. darlingi, An. triannulatus Neiva and Pinto, 1922, An. oswaldoi and An. nuneztovari Galbadon, 1942) in a total of 565 mosquitoes (Table 1Go). All mosquitoes were captured through human landing catches. Only 9 specimens were captured using the CDC trap; these were not included in the results. The mosquitoes were identified and kept individually for further analysis for the presence of plasmodial parasites. The largest number of adults was obtained in August, when An. rondoni was the predominant species. Mosquito biting activity was evaluated during the first 6 h of capture (between 6 and 12 pm). An. strodei and An. darlingi were found throughout this period with no evident peaks, whereas An. albitarsis s.l. and An. rondoni showed peaks of activity between 6 and 8 pm and between 7 and 8 pm, respectively (data not shown).


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TABLE 1
Total number of mosquitoes, percentage of Anopheles species collected, and their seasonal variation at the peak of transmission in Marabá, State of Pará, Brazil, in 2001
 
After identification, each specimen was processed and tested by ELISA for the presence of CSP of human malaria parasites. This was detected in 35 of the 565 mosquitoes examined, with an overall natural infection rate of 6.2% (Table 2Go); only 3 species of mosquitoes were CSP-positive: An. darlingi, which had the highest natural infection rate (22.4%), followed by An. albitarsis s.l. (5.2%) and An. rondoni (3.6%). Overall, P. vivax was the most prevalent malaria species (54.3% in both variants), followed by P. falciparum (37.1%) and P. malariae (8.6%). No mixed infections were found in the samples tested by ELISA; An. darlingi was found to be positive for P. vivax VK247, P. falciparum, and P. malariae; An. albitarsis s.l. was positive for P. vivax VK210 and P. vivax VK247; An. rondoni was found to be positive for P. vivax VK210 and P. falciparum (Table 2Go).


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TABLE 2
Numbers of Anopheles mosquitoes positive for the circumsporozoite protein of Plasmodium vivax (variants VK247 and VK210), P. falciparum (Pf), and P. malariae (Pm) among specimens collected in Marabá, State of Pará, Brazil, in 2001, detected by ELISA
 
Because this is the first finding of An. rondoni harboring human malarial parasites (P. falciparum and P. vivax VK210), we also tested the available specimens by PCR for further confirmation of the presence of Plasmodium DNA. Because all mosquitoes thoraces had been used in ELISA, PCR was carried out with frozen abdomens kept in pools of 3 or 5 mosquitoes each. We used primers that span the ssurRNA gene, an abundant sequence in Plasmodium genomic DNA,21 making PCR a sensitive technique to detect Plasmodium spp. sequences in mosquitoes (Table 3Go; Figures 1Go and 2Go). The expected product sizes were 205 bp for P. falciparum and 120 bp for P. vivax as illustrated in the figures; this shows An. rondoni infected with P. falciparum (4 pools), and with both P. vivax and P. falciparum (1 pool).


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TABLE 3
Presence of Plasmodium spp. ssurRNA in different Anopheles mosquitoes captured in Marabá, State of Pará, as detected by PCR in individual abdomens (An. darlingi, An. albitarsis) or in pools for the other species
 

Figure 1
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  		    FIGURE 1. Nested-PCR analysis using primers for Plasmodium spp. ssurRNA.20 Agarose gels show the results of the second nested-PCR reaction (see Materials and Methods) of 7 Anopheles rondoni mosquito pools. (A) Nested primers specific to Plasmodium falciparum ssurRNA gene; negative (–) (H2O instead of DNA) and positive (+) (DNA from malaria-infected blood) control, respectively; M, DNA marker (100 bp DNA ladder; New England Biolabs); (B) The same mosquito pools DNAs using primers specific to P. vivax; – and +, negative and positive controls as above. Note that mosquito pool 271 is positive for both P. falciparum and P. vivax.

 

Figure 2
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  		    FIGURE 2. Nested-PCR analysis using primers for Plasmodium spp. ssurRNA confirm the double positivity with P. vivax and P. falciparum parasites on mosquito pool 265. M, DNA marker (50 bp DNA ladder; New England Biolabs); Neg, negative control; falc and viv, positive controls (DNA from malaria-infected blood).

 
We also searched for Plasmodium ssurRNA sequences in the other mosquito species using mosquito pools. For the primary vectors—An. darlingi and An. albitarsis s.l.2–5—individual mosquitoes were used for PCR. The number of reactions positive for Plasmodium was higher by PCR (up to 68%) than ELISA (up to 22.4%) and most of the mosquitoes positive by ELISA were also positive by PCR regardless of the use of pools or of individual abdomens for PCR (Table 4Go).


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TABLE 4
Comparison between PCR and ELISA positive for Plasmodium spp. in different Anopheles species collected in Marabá, State of Pará, Brazil, in the year 2001
 
The overall presence of mosquitoes harboring plasmodial DNA as measured by PCR, compared with ELISA in the same groups of mosquitoes (Tables 3Go and 4Go), shows that: (i) PCR was far more sensitive, as expected; (ii) in the An. rondoni mosquitoes, PCR was particularly positive (68%) because only 4 of 110 mosquitoes were positive by ELISA (3.6%); (iii) An. triannulatus were the least positive by PCR (14%); (iv) in An. nuneztovari and An. strodei about 40% of the samples were PCR positive, whereas the respective samples were negative by ELISA.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Among the Anopheles collected in Marabá, southeastern Pará, an area of epidemic malaria in the Brazilian Amazon region, An. strodei (23.9%) and An. albitarsis s.l. (23.7%) were more abundant, followed by An. rondoni (19.5%) and An. darlingi (18.9%). Although An. darlingi was not the most frequently captured species in Marabá, it seems to have been the primary vector at that time because it presented the highest infection rate (22.4%) by ELISA tests, which detected CSP of Plasmodium parasites in the mosquito thoraces. This was followed by An. albitarsis s.l. (5.2%), which has been recognized as the main vector of malaria transmission in several areas in the east Amazon region.2,4,5 It was not surprising to discover that most of these infected mosquitoes (54%) harbored CSP of P. vivax. In the past 10 years, P. vivax has accounted for 80% of human malaria cases diagnosed in the Brazilian Amazon.1 An. darlingi and An. albitarsis s.l. also had high rates of parasite infection, which further confirms that they are the primary malaria vectors in the area.2–5

Only some species of the An. albitarsis complex here referred to as An. albitarsis sensu lato (s.l.) are incriminated as primary malaria vectors (namely An. marajoara, Galvão and Damasceno, 1942 and An. albitarsis E). The mosquito identification used here was based on morphological criteria alone, although we are aware that PCR would be necessary to differentiate between species within this complex.9,10 Similarly, identification of the An. rondoni was based on morphological criteria (i.e., the prescutellar area of the scutum), which has a large, subtriangular, bare dark brown to black spot and hindtarsomere 3, with a dark band in basal 0.20 to 0.3.16–18 These typical details make the possibility of misidentification unlikely.

Although An. rondoni has previously been identified in other tropical areas, including some areas of Brazil,23,24 we present here the first report of An. rondoni bearing malaria parasites, although positivity was rather low by ELISA (4 of 110 mosquitoes infected). This may explain why there are no previous reports on infections by this species. By PCR, of 19 mosquito pools tested, 13 were positive for plasmodia. Amplified products of representative samples, illustrated in the figures, reveal mosquito pools positive for P. vivax and for P. falciparum, as well as mixed infections.

Our results confirm previous investigations showing a higher sensitivity of the PCR technique to detect plasmodia compared with other methods.21,25–27 It was not surprising to find a higher number of positive abdomens (by PCR) compared with mosquito thoraces (by ELISA). Firstly, due to innate immunity, oocyst with sporozoites may develop in the mosquito midgut, with no further sporozoite migration to the salivary glands in some species28; secondly, during the initial stages of sporogony (i.e., in mosquitoes with early infections before oocysts have reached maturation), only the midguts would have been found positive for the CSP protein, as shown in experimental models.29 However, the most suitable method to confirm the mosquito vectorial capacity is to find sporozoites in their salivary glands; this task is difficult when performing surveys, as in the present study where both ELISA and PCR revealed plasmodia in Anopheles species. Although PCR was significantly more sensitive it is still an expensive technique for most countries where malaria is endemic.

In further studies in the Amazon (data not shown), we found no An. rondoni in 2 distinct areas of epidemic transmission of P. vivax malaria, both in Prainha, northwest Pará: (i) in Franca, we captured 310 specimens of An. albitarsis s.l. (3 positive for Pv-210 by ELISA) and 46 An. darlingi (all CSP-negative); (ii) in Santa Maria, a nearby area, we captured 410 An. albitarsis s.l. (1 positive for Pv-210) and 58 An. darlingi (one positive for Pv 210). Hence, it appears that in P. vivax areas fewer species of Anopheles are involved in the transmission when comparing to areas where P. falciparum epidemics occur. Nevertheless, only ELISA was used in the search of malaria parasites in these groups of mosquitoes.

An. rondoni—reported here for the first time to be infected with human plasmodia—may be considered a potential vector of human malaria because: (i) it was the third most frequently captured mosquito species in an area of epidemic malaria; (ii) its natural infection ratio was 3.6% with the highest seasonal activity in August—a month when An. rondoni was the most frequent species captured; (iii) its peak occurred at the highest rate of malaria transmission in the area (August, according to reports of the Brazilian Ministry of Health, www.funasa.gov.br). Thorough investigations are required to further clarify whether this species is a malaria vector and to take measures to improve mosquito control, especially in areas where P. falciparum is transmitted. Our results showing the participation of several anophelines species involved in the transmission of P. falciparum could improve the planning and implementation of malaria control activities in regions of intense transmission in the Brazilian Amazon. Despite being more expensive, PCR should be included in future surveys in the search of malaria infections in possible mosquito vectors.

Received June 13, 2005. Accepted for publication January 21, 2008.

Acknowledgments: The authors thank FAPEMIG for financial support, the Fundação Nacional de Sa úde (FUNASA) for technical assistance during mosquito field collections, Maria Anice M. Sallum and Simon Luke Elliot for a critical review of the manuscript, and Marino R. F. Barbosa for technical help with the PCR analysis. We are grateful to the community of Maraba for volunteer participation.

* Address correspondence to Antoniana Ursine Krettli, Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima, 1715, Belo Horizonte, MG, Brasil, 30190-002. E-mail: akrettli{at}cpqrr.fiocruz.br Back

Authors’ addresses: José Almir M. daRocha, Laboratory of Parasitology, Universidade Federal do Pará, Av. Augusto Correa, 01, CEP 66075-000, Belém, PA, Brazil, E-mail: jrocha{at}ufpa.br; and Laboratório de Malária, Centro de Pesquisas René Rachou, FIOCRUZ. Av. Augusto de Lima 1715, CEP 30190-002, Belo Horizonte, MG, Brazil. Marinete M. Póvoa, Instituto Evandro Chagas, Secção de Parasitologia, SVS/MS, Belém, PA, Brazil. Sabrina B. de Oliveira, Laboratório de Malária, Centro de Pesquisas René Rachou, FIOCRUZ. Av. Augusto de Lima 1715, CEP 30190-002, Belo Horizonte, MG, Brazil. Luciano A. Moreira, Laboratório de Malária, Centro de Pesquisas René Rachou, FIOCRUZ. Av. Augusto de Lima 1715, CEP 30190-002, Belo Horizonte, MG, Brazil. Antoniana U. Krettli, Laboratório de Malária, Centro de Pesquisas René Rachou, FIOCRUZ. Av. Augusto de Lima 1715, CEP 30190-002, Belo Horizonte, MG, Brazil, E-mail: akrettli{at}cpqrr.fiocruz.br.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ministry of Health. Available at: www.funasa.gov.br; http://portal.saude.gov.br/portal/arquivos/pdf/painel_%20indicadores_do_SUS.pdf. Accessed December 10, 2007.
  2. Arruda M, Carvalho MB, Nussenzweig RS, Maracic M, Ferreira AW, Cochrane AH, 1986. Potential vectors of malaria and their different susceptibility to Plasmodium falciparum and Plasmodium vivax in Northern Brazil identified by immunoassay. Am J Trop Med Hyg 35: 873–881.[Abstract/Free Full Text]
  3. Deane LM, 1986. Malaria vectors in Brazil. Mem Inst Oswaldo Cruz 81 (Suppl II): 5–14.
  4. Oliveira-Ferreira J, Oliveira LR, Teva A, Deane LM, Daniel-Ribeiro CT, 1990. Natural malaria infections in anophelines in Rondônia state, Brazilian Amazon. Am J Trop Med Hyg 43: 6–10.[Abstract/Free Full Text]
  5. Póvoa MM, Wirtz RA, Lacerda RNL, Miles MA, Warhurst DC, 2001. Malaria vectors in the municipality of Serra do Navio, State of Amapá, Amazon Region, Brazil. Mem Inst Oswaldo Cruz 96: 179–184.[Web of Science][Medline]
  6. Rosa-Freitas MG, Deane L, Momen H, 1990. A morphological, isoenzymatic and behavioural study of ten populations of Anopheles albitarsis Lynch-Arribalzaga, 1878 (Diptera: Culicidae) including from the type-locality Baradero, Argentina. Mem Inst Oswaldo Cruz 85: 275–289.[Web of Science]
  7. Narang SK, Klein TA, Perera OP, Lima JB, Tang AT, 1993. Genetic evidence for the existence of cryptic species in the Anopheles albitarsis complex in Brazil: allozymes and mitochondrial DNA restriction fragment length polymorphisms. Biochem Genet 31: 97–112.[Web of Science][Medline]
  8. Povoa MM, de Souza RT, Lacerda RN, Rosa ES, Galiza D, de Souza JR, Wietz RA, Schilichting CD, Conn JE, 2006. The importance of Anopheles albitarsis E and An. darlingi in human malaria transmission in Boa Vista, state of Roraima, Brazil. Mem Isnt Oswaldo Cruz 101: 163–168.
  9. Wilkerson RC, Gaffigan TV, Lima JB, 1995. Identification of species related to Anopheles (Nyssorhynchus) albitarsis by random amplified polymorphic DNA-polymerase chain reaction (Diptera: Culicidae). Mem Inst Oswaldo Cruz 90: 721–732.[Web of Science][Medline]
  10. Wilkerson RC, Foster PG, Li C, Sallum MAM, 2005. Molecular phylogeny of the neotropical Anopheles (Nyssorhynchus) albitarsis species complex (Diptera: Culicidae). Ann Entomol Soc Am 98: 918–925.[Web of Science][Medline]
  11. Klein TA, Lima JB, Tada MS, 1991. Comparative susceptibility of anopheline mosquitoes to Plasmodium falciparum in Rondonia, Brazil. Am J Trop Med Hyg 44: 598–603.[Abstract/Free Full Text]
  12. Branquinho MS, Lagos CB, Rocha RM, Natal D, Barata JM, Cochrane AH, Nardin E, Nussenzweig RS, Kloetzel JK, 1993. Anophelines in the state of Acre, Brazil, infected with Plasmodium falciparum, P. vivax, the variant P. vivax VK247 and P. malariae. Trans R Soc Trop Med Hyg 87: 391–394.[Web of Science][Medline]
  13. Branquinho MS, Araújo MS, Natal D, Marrelli MT, Rocha RM, Taveira FA, Kloetzel JK, 1996. Anopheles oswaldoi a potential malaria vector in Acre, Brazil. Trans R Soc Trop Med Hyg 90: 233.[Web of Science][Medline]
  14. Silva-Vasconcelos A, Kato MYN, Mourão EN, Souza RTL, Lacerda RNL, Sibajev A, Tsouris P, Póvoa MM, Momen H, Rosa-Freitas MG, 2002. Biting indices, host-seeking and natural infection rates of anopheline species in Boa Vista, Roraima, Brazil from 1996 to 1998. Mem Inst Oswaldo Cruz 97: 151–161.[Web of Science][Medline]
  15. Conn JE, Wilkerson RC, Segura MN, de Souza RT, Schlichting CD, Wirtz RA, Povoa MM, 2002. Emergence of a new neo-tropical malaria vector facilitated by human migration and changes in land use. Am J Trop Med Hyg 66: 18–22.[Abstract]
  16. Forattini OP, 1962. Entomologia Médica. v.1. São Paulo: Facul-dade de Higiene e Saúde Pública. Universidade de São Paulo.
  17. Consoli RA, Lourençode-Oliveira R, 1994. Principais Mosquitos de Importância Sanitária no Brasil. Rio de Janeiro: FIOCRUZ.
  18. Faran ME, 1980. Mosquito studies (Diptera: Culicidae) XXXIV. A revision of the albimanus section of the subgenus Nyssorhynchus of Anopheles. Contrib Amer Entomol Inst 15: 1–215.
  19. Wirtz RA, Burkot TR, Graves PM, 1987. Field evaluation of enzyme-linked immunosorbent assays for Plasmodium falciparum and Plasmodium vivax sporozoites in mosquitoes (Diptera: Culicidae) from Papua New Guinea. J Med Entomol 24: 433–437.[Web of Science][Medline]
  20. Wirtz RA, Zavala F, Charoenvit Y, Campbell GH, Burkot TR, Schneider I, Esser KM, Beaudoin RL, André RG, 1987. Comparative testing of monoconal antibodies against Plasmodium falciparum sporozoites for ELISA development. Bull World Health Organ 65: 39–45.[Web of Science][Medline]
  21. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, doRosario VE, Thaithong S, Brown KN, 1993. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 61: 315–320.[Web of Science][Medline]
  22. Black WC 4th, Munstermann LE, 1996. Molecular taxonomy and systematics of arthropod vectors. BJ Beaty and WC Marquardt, eds. The Biology of Disease Vectors, 1st ed. Denver: Colorado University Press.
  23. Wilkerson RC, Hribar LJ, Milstrey EG, Calderon GF, 1995. The identification of Anopheles (Nyssorhynchus) rondoni (Diptera: Culicidae) in Mato Grosso, Brazil: an analysis of key character variability, 1995. Mem Inst Oswaldo Cruz 90: 575–582.[Web of Science]
  24. Guimarães AE, Mello RP, Lopes CM, Alencar J, Gentile C, 1997. Prevalencia de anofelinos (Diptera: Culicidae) no crepusculo vespertino em areas da Usina Hidreletrica de Itaipu, no Municipio de Guaira, Estado do Parana, Brasil. Mem Inst Oswaldo Cruz 92: 745–754.[Web of Science][Medline]
  25. Povoa MM, Machado RL, Segura MN, Vianna GM, Vasconcelos AS, Conn JE, 2000. Infectivity of malaria vector mosquitoes: correlation of positivity between ELISA and PCR-ELISA tests. Trans R Soc Trop Med Hyg 94: 106–107.[Web of Science][Medline]
  26. Vythilingam I, Nitiavathy K, Yi P, Bakotee B, Hugo B, Singh B, Wirtz RA, Palmer K, 1999. A highly sensitive, nested polymerase chain reaction based method using simple DNA extraction to detect malaria sporozoites in mosquitoes. Southeast Asian J Trop Med Public Health 30: 631–635.[Medline]
  27. Santos-Ciminera PD, Achee NL, Quinnan GV Jr, Roberts DR, 2004. Use of polymerase chain reaction technique to confirm VecTest screening results in Plasmodium falciparum and Plasmodium vivax VK 210 laboratory-infected Anopheles stephensi mosquitoes. J Am Mosq Control Assoc 20: 265–271.[Web of Science][Medline]
  28. Meister S, Koutsos AC, Christophides GK, 2004. The Plasmodium parasite—a new ‘ ’ challenge for insect innate immunity. Int J Parasitol 34: 1473–1482.[Web of Science][Medline]
  29. Boulanger N, Charoenvit Y, Krettli A, Betschart B, 1995. Developmental changes in the circumsporozoite proteins of Plasmodium berghei and P. gallinaceum in their mosquito vectors. Parasitol Res 81: 58–65.[Web of Science][Medline]




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