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Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae)
Acta Tropica 71 (1998) 285 – 291
Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae) R.P.P. Soares a, N.F. Gontijo a, A.J. Romanha b, L. Diotaiuti b, M.H. Pereira a,* a
Departamento de Parasitologia, Uni6ersidade Federal de Minas Gerais, A6. Antoˆnio Carlos, 6627, Caixa Postal 486, CEP-31270 -901, Belo Horizonte, Minas Gerais, Brazil b Centro de Pesquisas Rene´ Rachou-FIOCRUZ, A6. Augusto de Lima, 1715, Caixa Postal 1743, CEP 30.190 -002, Belo Horizonte, Minas Gerais, Brazil Received 28 January 1998; received in revised form 1 August 1998; accepted 7 August 1998
Abstract Rhodnius prolixus interpopulation variability was studied based on a new approach using salivary heme proteins (nitrophorins) electrophoresis in starch gel. We compared salivary proteins profiles of R. prolixus from three different laboratory colonies from Honduras, Venezuela, Brazil and Rhodnius robustus from Venezuela, constructing a UPGMA. The Honduran and Venezuelan populations could not be distinguished from each other, but the Brazilian population was well separated from the others. The high similarity between Honduran and Venezuelan specimens lends support to current theories that the Central American populations of R. prolixus may have been introduced from a Venezuelan origin. The low polymorphism shown by the Honduran specimens is in agreement with a possible founder effect. This new approach also distinguished R. prolixus populations from R. robustus, species with extreme phenotypical similarity. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: R. prolixus; R. robustus; Saliva; Interpopulation variability; Triatominae; Heme proteins
* Corresponding author: Fax: + 55 31 4992970; e-mail: [email protected] 0001-706X/98/$ - see front matter © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0001-706X(98)00065-5
286
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
1. Introduction Rhodnius prolixus Stal (Hemiptera, Reduviidae) is widely distributed in Venezuela, Colombia, and parts of Central America, where it is an important domestic vector of Trypanosoma cruzi, causative agent of Chagas disease (american trypanosomiasis) (Schofield and Dujardin, 1997). It is morphologically similar to R. robustus Larrousse which frequently colonizes palm tree crowns in the same regions, and in parts of Ecuador, northern Peru and Brazil (Lent and Wygodzinsky, 1979). Both species are also similar to R. neglectus Lent from central Brazil, and R. nasutus Stal from northeastern Brazil, so that the four species are often described as the ‘prolixus group’ (Barrett, 1988). Distinction between these species of Rhodnius is of considerable importance because of their different epidemiological significance as vectors of T. cruzi (WHO, 1991). This is particularly relevant for R. prolixus and R. robustus since only prolixus is considered a major vector. Throughout its range, R. prolixus is known from domestic and peridomestic habitats but it has also been identified—by controversial morphological characters—from palm trees in Venezuela, Colombia, and parts of northern Brazil. By contrast, R. robustus is known only from palm tree crowns. However, although a range of biochemical and morphometric techniques are now being applied to clarify the distinction between these species (Schofield et al., 1996) the results remain equivocal. Iso-enzyme studies for example, clearly distinguished Rhodnius populations from domestic habitats and palm trees in Colombia (Lopez and Moreno, 1995), but not in Venezuela (Harry, 1993a; Harry et al., 1992a,b). As part of a wider study on the biosystematics of Rhodnius species, we report here a new technique based on electrophoresis of heme proteins from the salivary glands. All species of Rhodnius (and of the closely related genus Psammolestes, also within the tribe Rhodniini) have a characteristic red coloration in their salivary glands, which is not found in the salivary glands of other Triatominae. The red coloration is due to heme proteins, known as nitrophorins (Champagne et al., 1995) which release nitric oxide into the tissue of the insect’s host to elicit vasodilatation (Ribeiro et al., 1993). Four nitrophorins have been identified, and at least one of them also facilitates blood feeding by acting as an anticlotting factor (Ribeiro et al., 1995). The nitrophorins can also bind histamine and so reduce host irritation as the insect feeds (Ribeiro and Walker, 1994).
2. Materials and methods
2.1. The insects Specimens of Rhodnius were collected from houses in Venezuela and Honduras, and from palm tree crowns in Brazil (Tucurui, Para State) and Venezuela (origin unknown). These four populations were reared separately in the insectary of Centro de Pesquisas Rene´ Rachou, Belo Horizonte, Brazil. The insects were identified by Jose´ Jurberg of the National and International Reference Laboratory at the
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
287
Instituto Oswaldo Cruz, Rio de Janeiro, using the morphological criteria of Lent and Wygodzinsky (1979). By these criteria the domestic specimens from Venezuela and Honduras, and silvatic specimens from Brazil were determined as R. prolixus, while the silvatic specimens from Venezuela were determined as R. robustus. Nine adult insects from each population were used in the following analysis.
2.2. Sample preparation and electrophoresis Salivary glands from each adult insect were dissected out, washed in 0.9% NaC1 and transferred to 5 ml of running buffer on a microscope slide in a humid chamber where they were maintained until all insects had been prepared. The prepared glands from each insect were then disrupted with dissection needles, and the salivary contents applied to a starch gel electrophoresis plate. Electrophoresis was carried out at 300 V for 150 min. The running buffer was 0.15 M glycine/NaOH, pH 9.5; the gel buffer was the same but diluted 1:10. After electrophoresis, the gels were immersed for 10 min. in a staining solution of 0.3 mg/ml tetramethylbenzidine in a 1:1:1 mixture of ethanol, acetic acid and water. They were then transferred to 2% hydrogen peroxide until the bands began to develop. Because band development is transient, the gels were viewed by video camera to record the bands (Fig. 1) and the subsequent tapes transferred to a computer file.
2.3. Data analysis A taxon/character matrix was constructed on the basis of presence/absence of bands, considering only bands that were unequivocally visualized (Table 1). A phenogram based on shared bands was constructed using the Dice similarity coefficient (Dice, 1945) to produce a matrix of similarity which was then used for unweighted pair group method analysis (UPGMA) (Fig. 2).
3. Results Up to four hemeprotein bands were revealed by the above technique (Fig. 1) although tests with less alkaline gels generally showed more bands, up to a maximum of eight (data not shown) probably corresponding to the four native hemeproteins plus the same four bound to NO (see Section 4). The banding pattern shown by domestic specimens of R. prolixus from Honduras was very similar to that shown by domestic specimens from Venezuela, and the UPGMA dendrogram showed considerable overlap between these two populations (Fig. 2). However, these domestic populations were clearly distinguishable from the silvatic Brazilian specimens, and from the silvatic specimens determined as R. robustus which showed a banding pattern completely dissimilar to those shown by any of the specimens determined as R. prolixus (Fig. 1, Table 1).
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R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
4. Discussion Champagne et al. (1995) purified four hemeproteins from the saliva of R. prolixus, which were denoted as nitrophorins. During their purification process, each of these nitrophorins presented a double peak on chromatofocusing, corresponding to the molecule either bonded, or not, to nitric oxide (NO). Considering that alkaline pH favors the release of NO (Ribeiro et al., 1993; Andersen et al., 1997) we buffered our electrophoretic system to pH 9.5, in order to reveal just the unbounded native nitrophorins. The revealed banding patterns show that this technique may serve to help characterize Rhodnius species. The patterns unequivocally distinguished our R. robustus specimens from the various specimens determined as R. prolixus, and also provided a clear separation between the two domestic populations of R. prolixus and the silvatic Brazilian population. The high similarity between the Honduran and Venezuelan specimens lends support to current theories that the Central American populations of R. prolixus (which are invariably domestic) may have been introduced from a Venezuelan origin—possibly as the result of a laboratory escape (Schofield and Dujardin, 1997; Dujardin et al., 1998). Similarly, the lower level of
Fig. 1. Salivary nitrophorins profiles of the Rhodnius robustus from Venezuela (Rb1, Rb2 and Rb3) and Rhodnius prolixus from Venezuela (RpV1, RpV2 and RpV3), Honduras (RpH1, RpH2 and RpH3) and Brazil (RpT1, RpT2 and RpT3).
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
289
Table 1 Presence and absence of nitrophorin bands according to species and geographic origin Rhodnius prolixus
Rhodnius robustus
Nitrophorin profiles
Venezuela
Honduras
Brazil
Venezuela
1 2 3 4 5 6 7 8 9 10 11 12 13 14
+ − + + + + − + − − − + − −
+ − + + + + − + − − − − − −
− − + − − − − + + − + − − +
− + − − − − + − + + − − + +
polymorphism shown by our Honduran specimens accords with the idea that these populations represents the derivative of the Venezuelan population (rather than vice versa) and is consistent with a possible founder effect. Our results also endorse the specific status of R. robustus as an entity distinct from R. prolixus, which had been cast into doubt by their morphological and iso-enzymatic similarities (Harry, 1993b; Harry et al., 1992a,b). Nevertheless, further studies will be required to clarify the relationship between these species, because our results place the silvatic Brazilian specimens—determined as R. prolixus on morphological grounds—as a population distinct from both the true domestic R. prolixus populations and from the silvatic R. robustus population. It seems reasonable to expect that the electrophoretic profiles of the hemeproteins should reflect biogeographical distances between populations, and in turn reveal phylogenetic relationships between these species.
Acknowledgements This work was supported by FAPEMIG and Centro de Pesquisas Rene Rachou (FIOCRUZ), and benefited from international collaboration through the ECLAT network. We thank Jose Jurberg for morphological determination of the insects and Chris Schofield from the London School of Hygiene and Tropical Medicine for reviewing the manuscript.
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R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
Fig. 2. Unweighted pair group phenogram of individual adult Rhodnius based on pairwise shared nitrophorins bands (Rp, Rhodnius prolixus; Rb, Rhodnius robustus; Ve, Venezuela; Ho, Honduras and Tu, Brazil). The similarity coefficient (horizontal scale) was derived from the Index of Dice. The vertical bar represents the average level of similarity between all the pair samples analysed (phenon line) and indicates the point of reference for considering the insects as separated groups (Sneath and Sokal, 1962).
References Andersen, J.F., Champagne, D.E., Weichsel, A., et al., 1997. Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus. Biochemistry 36, 4423–4428. Barrett, T.B., 1988. Current Research on Amazonian Triatominae. Mem. Inst. Oswaldo Cruz 83 (Suppl. 1), 441–447. Champagne, D.E., Nussenzveig, R.H., Ribeiro, J.M.C., 1995. Purification, partial characterization and cloning of nitric oxide-carrying heme proteins (nitrophorins) from salivary glands of the blood-sucking insect Rhodnius prolixus. J. Biol. Chem. 270 (15), 8691 – 8695. Dice, L.R., 1945. Measures of the amount of ecological association between species. Ecology 26, 297–302.
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
291
Dujardin, J.P., Mun˜oz, M., Chavez, T., Ponce, C., Moreno, J., Schofield, C.J., 1998. The origin of Rhodnius prolixus in Central America. Med. Vet. Entomol. 12, 113 – 115. Harry, M., 1993a. Isozymic data question the specific status of blood-sucking bugs of the genus Rhodnius, vectors of Chagas disease. Trans. R. Soc. Trop. Med. Hyg. 87, 492. Harry, M., 1993b. Use of the median process of the pygophore in the identification of Rhodnius nasutus, R. neglectus, R. prolixus and R. robustus (Hemiptera: Reduviidae). Ann. Trop. Med. Parasitol. 87, 492. Harry, M., Moreno, G., Goyffon, M., 1992a. Genetic variability in populations of Rhodnius prolixus Vector of Chagas Disease in Venezuela. Evol. Biol. 6, 175 – 194. Harry, M., Galindez, I., Cariou, M.L., 1992b. Isozyme variability and the differentiation between Rhodnius prolixus strains, R. robustus and R. pictipes, vectors of Chagas disease in Venezuela. Med. Vet. Entomol. 6, 37–44. Lent, H., Wygodzinsky, P., 1979. Revision of the Triatominae (Hemiptera, Reduviidae), and their significance as vectors of Chagas disease. Bull. Am. Mus. Nat. Hist. 163, 125 – 520. Lopez, G., Moreno, J., 1995. Genetic variability and differentiation between populations of Rhodnius prolixus and R. pallescens, vectors of Chagas disease in Colombia. Mem. Inst. Oswaldo Cruz 90 (3), 353–357. Ribeiro, J.M.C., Hazzard, J.M.H., Nussenzveig, R.H., Champagne, D.E., Walker, F.A., 1993. Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science 260, 539–541. Ribeiro, J.M.C., Walker, F.A., 1994. High affinity histamine-binding and antihistaminic activity of the salivary NO-carrying heme protein (nitrophorin) of Rhodnius prolixus. J. Exp. Med. 180, 2251 – 2257. Ribeiro, J.M.C., Schneider, M., Guimaraes, J.A., 1995. Purification and characterization of prolixin S (nitrophorin 2) the salivary anticoagulant of the bloodsucking bug Rhodnius prolixus. Biochem. J. 308, 243–249. Schofield, C.J., Dujardin, J.P., 1997. Chagas disease vector control in Central America. Parasit. Today 13, 141–144. Schofield, C.J., Dujardin, J.P., Jurberg, J., (Eds.), 1996. Proceedings of the International Workshop on Population Biology and Control of Triatominae, Santo Domingo de los Colorados, Ecuador, p. 116. INDRE, Mexico City. Sneath, P.H.A., Sokal, A.R., 1962. Numerical taxonomy. Nature 193, 853 – 860. WHO, 1991. Control of Chagas’ disease. World Health Organization Technical Report Series. 811, 95.
.
Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae) R.P.P. Soares a, N.F. Gontijo a, A.J. Romanha b, L. Diotaiuti b, M.H. Pereira a,* a
Departamento de Parasitologia, Uni6ersidade Federal de Minas Gerais, A6. Antoˆnio Carlos, 6627, Caixa Postal 486, CEP-31270 -901, Belo Horizonte, Minas Gerais, Brazil b Centro de Pesquisas Rene´ Rachou-FIOCRUZ, A6. Augusto de Lima, 1715, Caixa Postal 1743, CEP 30.190 -002, Belo Horizonte, Minas Gerais, Brazil Received 28 January 1998; received in revised form 1 August 1998; accepted 7 August 1998
Abstract Rhodnius prolixus interpopulation variability was studied based on a new approach using salivary heme proteins (nitrophorins) electrophoresis in starch gel. We compared salivary proteins profiles of R. prolixus from three different laboratory colonies from Honduras, Venezuela, Brazil and Rhodnius robustus from Venezuela, constructing a UPGMA. The Honduran and Venezuelan populations could not be distinguished from each other, but the Brazilian population was well separated from the others. The high similarity between Honduran and Venezuelan specimens lends support to current theories that the Central American populations of R. prolixus may have been introduced from a Venezuelan origin. The low polymorphism shown by the Honduran specimens is in agreement with a possible founder effect. This new approach also distinguished R. prolixus populations from R. robustus, species with extreme phenotypical similarity. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: R. prolixus; R. robustus; Saliva; Interpopulation variability; Triatominae; Heme proteins
* Corresponding author: Fax: + 55 31 4992970; e-mail: [email protected] 0001-706X/98/$ - see front matter © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0001-706X(98)00065-5
286
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
1. Introduction Rhodnius prolixus Stal (Hemiptera, Reduviidae) is widely distributed in Venezuela, Colombia, and parts of Central America, where it is an important domestic vector of Trypanosoma cruzi, causative agent of Chagas disease (american trypanosomiasis) (Schofield and Dujardin, 1997). It is morphologically similar to R. robustus Larrousse which frequently colonizes palm tree crowns in the same regions, and in parts of Ecuador, northern Peru and Brazil (Lent and Wygodzinsky, 1979). Both species are also similar to R. neglectus Lent from central Brazil, and R. nasutus Stal from northeastern Brazil, so that the four species are often described as the ‘prolixus group’ (Barrett, 1988). Distinction between these species of Rhodnius is of considerable importance because of their different epidemiological significance as vectors of T. cruzi (WHO, 1991). This is particularly relevant for R. prolixus and R. robustus since only prolixus is considered a major vector. Throughout its range, R. prolixus is known from domestic and peridomestic habitats but it has also been identified—by controversial morphological characters—from palm trees in Venezuela, Colombia, and parts of northern Brazil. By contrast, R. robustus is known only from palm tree crowns. However, although a range of biochemical and morphometric techniques are now being applied to clarify the distinction between these species (Schofield et al., 1996) the results remain equivocal. Iso-enzyme studies for example, clearly distinguished Rhodnius populations from domestic habitats and palm trees in Colombia (Lopez and Moreno, 1995), but not in Venezuela (Harry, 1993a; Harry et al., 1992a,b). As part of a wider study on the biosystematics of Rhodnius species, we report here a new technique based on electrophoresis of heme proteins from the salivary glands. All species of Rhodnius (and of the closely related genus Psammolestes, also within the tribe Rhodniini) have a characteristic red coloration in their salivary glands, which is not found in the salivary glands of other Triatominae. The red coloration is due to heme proteins, known as nitrophorins (Champagne et al., 1995) which release nitric oxide into the tissue of the insect’s host to elicit vasodilatation (Ribeiro et al., 1993). Four nitrophorins have been identified, and at least one of them also facilitates blood feeding by acting as an anticlotting factor (Ribeiro et al., 1995). The nitrophorins can also bind histamine and so reduce host irritation as the insect feeds (Ribeiro and Walker, 1994).
2. Materials and methods
2.1. The insects Specimens of Rhodnius were collected from houses in Venezuela and Honduras, and from palm tree crowns in Brazil (Tucurui, Para State) and Venezuela (origin unknown). These four populations were reared separately in the insectary of Centro de Pesquisas Rene´ Rachou, Belo Horizonte, Brazil. The insects were identified by Jose´ Jurberg of the National and International Reference Laboratory at the
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
287
Instituto Oswaldo Cruz, Rio de Janeiro, using the morphological criteria of Lent and Wygodzinsky (1979). By these criteria the domestic specimens from Venezuela and Honduras, and silvatic specimens from Brazil were determined as R. prolixus, while the silvatic specimens from Venezuela were determined as R. robustus. Nine adult insects from each population were used in the following analysis.
2.2. Sample preparation and electrophoresis Salivary glands from each adult insect were dissected out, washed in 0.9% NaC1 and transferred to 5 ml of running buffer on a microscope slide in a humid chamber where they were maintained until all insects had been prepared. The prepared glands from each insect were then disrupted with dissection needles, and the salivary contents applied to a starch gel electrophoresis plate. Electrophoresis was carried out at 300 V for 150 min. The running buffer was 0.15 M glycine/NaOH, pH 9.5; the gel buffer was the same but diluted 1:10. After electrophoresis, the gels were immersed for 10 min. in a staining solution of 0.3 mg/ml tetramethylbenzidine in a 1:1:1 mixture of ethanol, acetic acid and water. They were then transferred to 2% hydrogen peroxide until the bands began to develop. Because band development is transient, the gels were viewed by video camera to record the bands (Fig. 1) and the subsequent tapes transferred to a computer file.
2.3. Data analysis A taxon/character matrix was constructed on the basis of presence/absence of bands, considering only bands that were unequivocally visualized (Table 1). A phenogram based on shared bands was constructed using the Dice similarity coefficient (Dice, 1945) to produce a matrix of similarity which was then used for unweighted pair group method analysis (UPGMA) (Fig. 2).
3. Results Up to four hemeprotein bands were revealed by the above technique (Fig. 1) although tests with less alkaline gels generally showed more bands, up to a maximum of eight (data not shown) probably corresponding to the four native hemeproteins plus the same four bound to NO (see Section 4). The banding pattern shown by domestic specimens of R. prolixus from Honduras was very similar to that shown by domestic specimens from Venezuela, and the UPGMA dendrogram showed considerable overlap between these two populations (Fig. 2). However, these domestic populations were clearly distinguishable from the silvatic Brazilian specimens, and from the silvatic specimens determined as R. robustus which showed a banding pattern completely dissimilar to those shown by any of the specimens determined as R. prolixus (Fig. 1, Table 1).
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R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
4. Discussion Champagne et al. (1995) purified four hemeproteins from the saliva of R. prolixus, which were denoted as nitrophorins. During their purification process, each of these nitrophorins presented a double peak on chromatofocusing, corresponding to the molecule either bonded, or not, to nitric oxide (NO). Considering that alkaline pH favors the release of NO (Ribeiro et al., 1993; Andersen et al., 1997) we buffered our electrophoretic system to pH 9.5, in order to reveal just the unbounded native nitrophorins. The revealed banding patterns show that this technique may serve to help characterize Rhodnius species. The patterns unequivocally distinguished our R. robustus specimens from the various specimens determined as R. prolixus, and also provided a clear separation between the two domestic populations of R. prolixus and the silvatic Brazilian population. The high similarity between the Honduran and Venezuelan specimens lends support to current theories that the Central American populations of R. prolixus (which are invariably domestic) may have been introduced from a Venezuelan origin—possibly as the result of a laboratory escape (Schofield and Dujardin, 1997; Dujardin et al., 1998). Similarly, the lower level of
Fig. 1. Salivary nitrophorins profiles of the Rhodnius robustus from Venezuela (Rb1, Rb2 and Rb3) and Rhodnius prolixus from Venezuela (RpV1, RpV2 and RpV3), Honduras (RpH1, RpH2 and RpH3) and Brazil (RpT1, RpT2 and RpT3).
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
289
Table 1 Presence and absence of nitrophorin bands according to species and geographic origin Rhodnius prolixus
Rhodnius robustus
Nitrophorin profiles
Venezuela
Honduras
Brazil
Venezuela
1 2 3 4 5 6 7 8 9 10 11 12 13 14
+ − + + + + − + − − − + − −
+ − + + + + − + − − − − − −
− − + − − − − + + − + − − +
− + − − − − + − + + − − + +
polymorphism shown by our Honduran specimens accords with the idea that these populations represents the derivative of the Venezuelan population (rather than vice versa) and is consistent with a possible founder effect. Our results also endorse the specific status of R. robustus as an entity distinct from R. prolixus, which had been cast into doubt by their morphological and iso-enzymatic similarities (Harry, 1993b; Harry et al., 1992a,b). Nevertheless, further studies will be required to clarify the relationship between these species, because our results place the silvatic Brazilian specimens—determined as R. prolixus on morphological grounds—as a population distinct from both the true domestic R. prolixus populations and from the silvatic R. robustus population. It seems reasonable to expect that the electrophoretic profiles of the hemeproteins should reflect biogeographical distances between populations, and in turn reveal phylogenetic relationships between these species.
Acknowledgements This work was supported by FAPEMIG and Centro de Pesquisas Rene Rachou (FIOCRUZ), and benefited from international collaboration through the ECLAT network. We thank Jose Jurberg for morphological determination of the insects and Chris Schofield from the London School of Hygiene and Tropical Medicine for reviewing the manuscript.
290
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
Fig. 2. Unweighted pair group phenogram of individual adult Rhodnius based on pairwise shared nitrophorins bands (Rp, Rhodnius prolixus; Rb, Rhodnius robustus; Ve, Venezuela; Ho, Honduras and Tu, Brazil). The similarity coefficient (horizontal scale) was derived from the Index of Dice. The vertical bar represents the average level of similarity between all the pair samples analysed (phenon line) and indicates the point of reference for considering the insects as separated groups (Sneath and Sokal, 1962).
References Andersen, J.F., Champagne, D.E., Weichsel, A., et al., 1997. Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus. Biochemistry 36, 4423–4428. Barrett, T.B., 1988. Current Research on Amazonian Triatominae. Mem. Inst. Oswaldo Cruz 83 (Suppl. 1), 441–447. Champagne, D.E., Nussenzveig, R.H., Ribeiro, J.M.C., 1995. Purification, partial characterization and cloning of nitric oxide-carrying heme proteins (nitrophorins) from salivary glands of the blood-sucking insect Rhodnius prolixus. J. Biol. Chem. 270 (15), 8691 – 8695. Dice, L.R., 1945. Measures of the amount of ecological association between species. Ecology 26, 297–302.
R.P.P. Soares et al. / Acta Tropica 71 (1998) 285–291
291
Dujardin, J.P., Mun˜oz, M., Chavez, T., Ponce, C., Moreno, J., Schofield, C.J., 1998. The origin of Rhodnius prolixus in Central America. Med. Vet. Entomol. 12, 113 – 115. Harry, M., 1993a. Isozymic data question the specific status of blood-sucking bugs of the genus Rhodnius, vectors of Chagas disease. Trans. R. Soc. Trop. Med. Hyg. 87, 492. Harry, M., 1993b. Use of the median process of the pygophore in the identification of Rhodnius nasutus, R. neglectus, R. prolixus and R. robustus (Hemiptera: Reduviidae). Ann. Trop. Med. Parasitol. 87, 492. Harry, M., Moreno, G., Goyffon, M., 1992a. Genetic variability in populations of Rhodnius prolixus Vector of Chagas Disease in Venezuela. Evol. Biol. 6, 175 – 194. Harry, M., Galindez, I., Cariou, M.L., 1992b. Isozyme variability and the differentiation between Rhodnius prolixus strains, R. robustus and R. pictipes, vectors of Chagas disease in Venezuela. Med. Vet. Entomol. 6, 37–44. Lent, H., Wygodzinsky, P., 1979. Revision of the Triatominae (Hemiptera, Reduviidae), and their significance as vectors of Chagas disease. Bull. Am. Mus. Nat. Hist. 163, 125 – 520. Lopez, G., Moreno, J., 1995. Genetic variability and differentiation between populations of Rhodnius prolixus and R. pallescens, vectors of Chagas disease in Colombia. Mem. Inst. Oswaldo Cruz 90 (3), 353–357. Ribeiro, J.M.C., Hazzard, J.M.H., Nussenzveig, R.H., Champagne, D.E., Walker, F.A., 1993. Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science 260, 539–541. Ribeiro, J.M.C., Walker, F.A., 1994. High affinity histamine-binding and antihistaminic activity of the salivary NO-carrying heme protein (nitrophorin) of Rhodnius prolixus. J. Exp. Med. 180, 2251 – 2257. Ribeiro, J.M.C., Schneider, M., Guimaraes, J.A., 1995. Purification and characterization of prolixin S (nitrophorin 2) the salivary anticoagulant of the bloodsucking bug Rhodnius prolixus. Biochem. J. 308, 243–249. Schofield, C.J., Dujardin, J.P., 1997. Chagas disease vector control in Central America. Parasit. Today 13, 141–144. Schofield, C.J., Dujardin, J.P., Jurberg, J., (Eds.), 1996. Proceedings of the International Workshop on Population Biology and Control of Triatominae, Santo Domingo de los Colorados, Ecuador, p. 116. INDRE, Mexico City. Sneath, P.H.A., Sokal, A.R., 1962. Numerical taxonomy. Nature 193, 853 – 860. WHO, 1991. Control of Chagas’ disease. World Health Organization Technical Report Series. 811, 95.
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