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Differences in saliva composition among three Brazilian populations of Panstrongylus megistus (Hemiptera, Reduviidae)
Acta Tropica 72 (1999) 91 – 98
Differences in saliva composition among three Brazilian populations of Panstrongylus megistus (Hemiptera, Reduviidae) S.E. Barbosa a,b, L. Diotaiuti b, R.P.P. Soares a,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, MG, Brazil b Centro de Pesquisas Rene´ Rachou-FIOCRUZ, A6. Augusto de Lima, 1715, Caixa Postal 1743, CEP 30.190 -002, Belo Horizonte, MG, Brazil
Received 28 January 1998; received in revised form 18 August 1998; accepted 10 September 1998
Abstract Comparisons of electrophoretic profiles of triatomines (Panstrongylus megistus) from the Brazilian states of Bahia (BA), Minas Gerais (MG) and Santa Catarina (SC) revealed differences in the composition of the saliva between the three populations. A phenogram constructed on the basis of the most representative electrophoretic banding patterns allowed the specimens tested to be divided into two groups. The BA and SC populations could be completely separated while some individuals from MG could be placed in each group. The BA population presented fewer bands than the MG and SC ones. The populations studied also differed with regard to their degree of association with human habitations (sylvatic, peridomiciliary and/or intradomiciliary) and our results suggest that the proteinaceous composition of the saliva of the three distinct populations of triatomines could be related to the hosts each is likely to encounter. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Triatomines; Saliva; Panstrongylus megistus; Interpopulation variability
* Corresponding author. Fax: + 55 31 4992970; e-mail: [email protected] 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0001-706X(98)00073-4
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1. Introduction Panstrongylus megistus is an important species in the epidemiology of Chagas’ disease in Brazil because of its high susceptibility to infection by Trypanosoma cruzi, wide geographical distribution and adaptability to a range of habitats and hosts, which allows it to colonize both domestic and peridomestic ecotopes. Nevertheless, populations of this species from different regions show behavioural variation in their capacities to colonize the domestic environment. In northeastern Brazil, P. megistus generally occurs in man-made habitats such as those encountered in the Recoˆncavo Baiano region, where repeated attempts to collect this species in sylvatic habitats have been fruitless. This was explained by Sherlock (1979) as a consequence of the destruction of its natural habitat, beginning with the colonization of Brazil and the exploitation of native timber by the settlers. This resulted in fragmentation of the Atlantic Forest, probably favoring selection of a population of triatomines with a high capacity to colonize man-made habitats. Despite the profound environmental alterations that have occurred in other regions of Brazil, in the southeast of the country P. megistus can still be encountered in sylvatic ecotopes (Barretto, 1979) as well as man-made ones (Silveira et al., 1984), the limit of these primarily sylvatic populations apparently passing through the state of Sa˜o Paulo (Araga˜o, 1961; Pessoa, 1962; Forattini, 1972; Forattini et al., 1977). The electrophoretic profile of triatomine saliva shows a complex composition of proteins, allowing the separation of species (Pereira et al., 1996a). However it is not known whether the composition of saliva varies within the species and/or populations. With this in mind, the objective of this study was to compare the electrophoretic profiles of the saliva of individuals from three Brazilian populations of P. megistus.
2. Materials and methods Triatomines were collected from the following localities: Campo Formoso, Bahia (10°30%S; 40°20%W), where the species is found exclusively in artificial ecotopes; Belo Horizonte, Minas Gerais (20°0%S; 44°0%W), where it occurs both in artificial and sylvatic ecotopes; and Floriano´polis, Santa Catarina (27°30%S; 48°30%W) where P. megistus is predominantly sylvatic and shows a low potential for the colonization of man-made habitats. Laboratory colonies were established from wild-caught specimens and the F1 (BA and MG) or F3 (SC) generations of these colonies used in analyses. The triatomines were maintained in an insectary at a temperature of 2792°C and relative humidity of 60 – 70%. Anaesthetized mice were offered to the bugs once a week as a blood meal source until adult insects were available for use in the analyses. Twenty insects from each population were analyzed 1–2 months after their final moults. Individual saliva was collected using a capillary tube (length 75 mm, internal diameter 1.1 mm; MICRON®, Sa˜o Paulo) inserted in the proboscis of each insect,
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followed by electrical stimulation of the ventral thoracic surface, which had been treated previously with an electrolytic gel (Pereira et al., 1996a). Electrophoresis (SDS-PAGE) of triatomine saliva was carried out on a 12.5% polyacrylamide slab gel (Hubbard and Lazarides, 1979) and the protein bands were stained by Coomassie blue (G-250). Protein content in the saliva was determined by the method of Bradford (1976), using bovine serum albumin as standard. Approximately 5 mg of protein were loaded in each lane. A taxonomic matrix was elaborated based on the study of banding patterns, allowing a phenogram to be drawn, using the similarity coefficient of Dice (1945). Only those bands that presented ] 5% of the total applied protein were considered in producing the matrix. Estimates of the relative amount of protein in the bands of each electrophoretic profile were made by densitometry using the Is-1000 Digital Imaging System (Alpha Innotech, San Leandro, CA).
3. Results The electrophoretic analysis of saliva of individuals of P. megistus showed a complex protein composition with molecular weights ranging from 9 to 87 kDa. The most characteristic protein band profile was observed in the region between 20 and 24 kDa. This region contains 53.8 9 10.1% (n= 60) of the total applied protein distributed in 2 – 4 bands as determined by densitometry (Figs. 1 and 2). Remarkable differences were seen between the band profiles of the Santa Catarina and Bahia populations. All SC individuals showed one 20 kDa and another 23 kDa protein band, while BA individuals did not show the 23 kDa protein band and only
Fig. 1. Electrophoretic profiles (SDS-PAGE) of Panstrongylus megistus saliva. Lanes (1 – 8): insects from Floriano´polis, Santa Catarina state, Brazil. Molecular mass markers (kDa) are indicated on the left. The arrows indicate the position of the four most characteristic protein bands of approximately 20, 21, 23 and 24 kDa.
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Fig. 2. Electrophoretic profiles (SDS-PAGE) of Panstrongylus megistus saliva. Lanes (1 – 4): insects from Campo Formoso, Bahia state, Brazil; (5– 8): insects from Belo Horizonte, Minas Gerais state, Brazil. Molecular mass markers (kDa) are indicated on the left. The arrows indicate the position of the four most characteristic protein bands of approximately 20, 21, 23 and 24 kDa.
two of them (10% of BA analyzed individuals) showed the 20 kDa band. Seventeen of the Minas Gerais individuals (85%) showed a band of 23 kDa and 14 (70%) one of 20 kDa (Figs. 1 and 2). Regarding the number of bands in this region, 19 individuals from SC population showed four bands while one individual presented three. In the BA population, 18 individuals showed two bands and only two individuals presented three. In the MG population, nine individuals showed four bands, six individuals presented three bands and five individuals showed two. No differences were observed between the electrophoretic profiles of males and females for any of the three populations studied. The phenogram constructed on the basis of these results showed the existence of two main groups, the SC population being completely separated from that of BA. The MG individuals could be distributed between the two groups, with 14 (70%) in the SC group and six (30%) in the BA one (Fig. 3). The number of bands analyzed varied from 4 to 9 per insect. With respect to this parameter, the BA individuals with 5.09 0.9 bands were different from those of SC and MG with 6.691.1 and 6.19 1.1, respectively (PB0.01). There was no significant difference in band number between the MG and SC populations (P= 0.12; Table 1).
4. Discussion This is the first time that salivary proteins have been used in the study of triatomine populations. The saliva of P. megistus obtained by electrical stimulation can be stored in a freezer at − 15°C, where it maintains its stability giving the same
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Fig. 3. Unweighted pair groups of individual adult Panstrongylus megistus males (M) and females (F) based on pairwise shared salivary protein bands. Origin: B, Bahia state; M, Minas Gerais state and S, Santa Catarina state. The similarity coefficient (horizontal scale) was derived from the Dice Index. The vertical bar represents the average level of similarity between all the pair samples analysed (phenon line).
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electrophoretic profile for up to 3 months after collection. This stability supports the finding that triatomine saliva apparently has no digestive function, no proteases, lipases or glycosidases having been described to date (Garcia, 1995). Our results show a variation in the composition of the saliva between distinct populations of P. megistus, with a clear separation between SC and BA individuals and those of MG in an intermediate position, albeit slightly closer to members of the SC population. It is interesting to note that the electrophoretic profile of the saliva appears to undergo a gradual change in populations along the south–north and/or west – east axis, also showing a tendency to concentrate more protein in a fewer number of bands. This pattern resembles the variation in the activity of the salivary vasodilator maxidilan observed among putative species of phlebotomine sand flies of the Lutzomyia longipalpis complex from Brazil, Colombia and Costa Rica, with increasing activity and a corresponding change in the pathology of visceral leishmaniasis in those further south (Warburg et al., 1994). Although the true significance of this variation in the composition of saliva of P. megistus is not known, the populations studied show different degrees of adaptation to man-made environments and it is probable that it is related to the association between the triatomines and the different hosts present in the domestic and/or sylvatic environments. According to Barretto (1979) and Sherlock (1979), P. megistus is intimately associated with opossums in sylvatic ecotopes. These animals were the source of 62.9% of blood meals in triatomines captured in man-made habitats on Santa Catarina island, based on analyses by precipitin test on one of the few collections of P. megistus made in houses (Steindel et al., 1994). By contrast studies in the southeast of Brazil on triatomines collected in peridomestic environments revealed that birds were the main source of blood meals (44.2%), other animals being represented at lower frequencies e.g. pig (10.3%), opossum (14.2%), rodent (13.9%), dog (4.9%) and man (3.2%). Nevertheless, in this area, P. megistus is able to establish colonies in the man-made habitats and represents an important vector of T. cruzi to man (Sherlock, 1979). In Bahia, 80.8% of insects captured in houses reacted positively with anti-human serum (Minter, 1975). Triatomine saliva possesses substances with pharmacological activities that assist in blood feeding, such as anticoagulants (Hellmann and Hawkins, 1964; Ribeiro et al., 1995; Pereira et al., 1996a), vasodilators (Ribeiro et al., 1990, 1993), antihistamine (Ribeiro and Table 1 Number of bands analysed of populations of Panstrongylus megistus from Bahia (BA), Minas Gerais (MG) and Santa Catarina (SC), Brazil Sex/population
Male Female Mean
Panstrongylus megistus BA
MG
SC
5.190.4 (n= 6) 4.9 91.0 (n=14) 5.09 0.9 (n=20)a
6.0 91.1 (n = 6) 6.1 9 1.1 (n = 14) 6.1 91.1 (n = 20)b
6.6 91.2 (n = 9) 6.6 9 1.1 (n =11) 6.6 91.1 (n =20)c
T-test: a/b (PB0.01); a/c (PB0.01); b/c (P= 0.12).
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Walker, 1994), anaesthetic-like (Pereira et al., 1996b) and inhibitors of platelet aggregation, the latter elicited by ADP (Smith et al., 1980; Ribeiro and Garcia, 1980), collagen (Ribeiro and Garcia, 1981; Noeske-Jungblut et al., 1994) or aracdonic acid (Ribeiro and Sarkis, 1982). Saliva also modulates the feeding success of haematophagous insects through immunogenic reactions (Lehane, 1991). Triatomine saliva can elicit host reactions varying from local (Dias, 1968; Mott et al., 1980; Costa et al., 1981) to general anaphylaxis (Teo and Cheah, 1973; Pinnas et al., 1986). Thus, variation in the composition of saliva as demonstrated here may be a consequence of selection favouring a better adaptation to local hosts. This phenomenon was observed in the Malayan pit viper Calloselasma rhodostoma, where geographical variation in the venom appeared to be linked to the composition of the local fauna, i.e. the potential prey species available (Daltry et al., 1996).
Acknowledgements Thanks to Dr John Bruce Alexander for revising the manuscript, to Dr Odair Genaro for kindly permitting use of an image analyser for densitometry and to Ana Maria Salvador Pereira for technical assistance. This study was financed by FAPEMIG, CAPES and CPqRR/FIOCRUZ.
References ´ rea de ocorreˆncia do Panstrongylus Araga˜o, M.B., 1961. Aspectos clima´ticos da doenc¸a de Chagas. II. A megistus (Burmeister, 1835). Rev. Bras. Malariol. 13, 171 – 193. Barretto, M.P., 1979. Epidemiologia. In: Brener, Z., Andrade, Z. (Eds.), Trypanosoma cruzi e doenc¸a de Chagas. Guanabara Koogan, pp. 89– 151. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Costa, C.H.N., Costa, M.T., Weber, J.N., Gilks, G.F., Castro, C., Marsden, P.D., 1981. Skin reactions to bug bites as a result of xenodiagnosis. Trans. R. Soc. Hyg. Trop. Med. 75 (3), 405 – 408. Daltry, J.C., Wu¨ster, W., Thorpe, R.S., 1996. Diet and snake venom evolution. Nature 379, 537 – 540. Dias, J.C.P., 1968. Manifestac¸o˜es cutaˆneas na pra´tica do xenodiagno´stico. Rev. Bras. Malariol. Doenc¸as Trop. 20, 247–257. Dice, L.R., 1945. Measures of the amount of ecological association between species. Ecology 26, 297–302. Forattini, O.P., 1972. Entomogeografia me´dica do Brasil. In: Lacaz, C.S., Baruzzi, R.G., Ju´nior, W.S. (Eds.), Introduc¸a˜o a` Geografia Me´dica do Brasil, Sa˜o Paulo. Edgar Blu¨cher, USP, pp. 191 – 212. Forattini, O.P., Ferreira, O.A., Rocha e Silva, E.O., Rabello, E.X., 1977. Aspectos ecolo´gicos da tripanossomı´ase americana. VIII—Domiciliac¸a˜o de Panstrongylus megistus e sua presenc¸a extradomiciliar. Rev. Sau´de Pu´bl. 11, 73– 86. Garcia, E.S., 1995. The digestion of Triatominae. In: Brenner, R., Merced Stoka, A. (Eds.), Chagas’ Disease Vectors: Anatomic and Physiological Aspects. Franklin, FL, pp. 47 – 58. Hellmann, K., Hawkins, R.I., 1964. Anticoagulant and fibrinolutic activities from Rhodnius prolixus Stall. Nature 201, 1008–1009. Hubbard, B.D., Lazarides, E., 1979. The co-purification of actin and desmin from chicken smooth muscle and their co-polymerization in 6itro to immediate filaments. J. Cell. Biol. 80, 166 – 182. Lehane, M.J., 1991. Biology of blood-sucking insects. Harper Collins, pp. 1 – 288.
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Minter, D.M., 1975. Feeding patterns of some triatomine vector species. In: New Approaches in American Trypanosomiases Research. Pan American Health Organization, Science Publication no. 318, pp. 33–47. Mott, K.E., Franc¸a, J.T., Barrett, T.B., Hoff, R., Oliveira, T.S., Sherlock, I.A., 1980. Cutaneous allergic reactions to Triatoma infestans after diagnosis. Mem. Inst. Oswaldo Cruz 75 (3-4), 3 – 10. Noeske-Jungblut, C., Kratzschmar, J., Haendler, B., Alagon, A., Possani, L., Verhallen, P., Donners, P., Schleuning, W., 1994. An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis. J. Biol. Chem. 269 (7), 5050 – 5053. Pereira, M.H., Souza, M.E.L., Vargas, A.P., Martins, M.S., Penido, C.M., Diotaiuti, L., 1996a. Anticoagulant activity of Triatoma infestans and Panstrongylus megistus saliva (Hemiptera/Triatominae). Acta Trop. 61, 255–261. Pereira, M.H., Beira˜o, P.S.L., Diotaiuti, L., 1996b. Activity of Triatoma infestans saliva on the sciatic nerve of the rat. Mem. Inst. Oswaldo Cruz 91 (Suppl. I), 143. Pessoa, S.P., 1962. Domiciliac¸a˜o de triatomı´neos e epidemiologia da doenc¸a de Chagas. Arq. Hig. Sau´de Publ. 27, 161–171. Pinnas, J.L., Lindberg, R.E., Chen, T.M.W., Meinke, G.C., 1986. Studies of kissing bug-sensitive patients: Evidence for the lack of cross-reactivity between Triatoma protracta and Triatoma rubida salivary gland extracts. J. Allergy Clin. Immunol. 77 (2), 364 – 370. Ribeiro, J.M.C., Garcia, E.S., 1980. The salivary and crop apyrase activity of Rhodnius prolixus. J. Insect Physiol. 26, 303–307. Ribeiro, J.M.C., Garcia, E.S., 1981. Platelet antiaggregating activity in the salivary secretion of the blood sucking bug Rhodnius prolixus. Experientia 37, 384 – 386. Ribeiro, J.M.C., Sarkis, J.J.F., 1982. Anti-thromboxane activity in Rhodnius prolixus salivary secretion. J. Insect Physiol. 28 (8), 655–660. 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.R., Marinotti, O., Gonzales, R., 1990. A salivary vasodilatator in the blood-sucking bug, Rhodnius prolixus. Br. J. Pharmacol. 101, 932 – 936. Ribeiro, J.M.R., Hazzard, J.M.H., Nussenzveig, R.H., Champagne, D.E., Walker, F.A., 1993. Reversible binding of nitric oxide by a salivary hem protein from a bloodsucking insect. Science 260, 539–541. Ribeiro, J.M.C., Schneider, M., Guimara˜es, J.A., 1995. Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus. Biochem. J. 308, 243–249. Sherlock, I.A., 1979. Vetores. In: Brener, Z., Andrade, Z. (Eds.), Trypanosoma cruzi e doenc¸a de Chagas. Guanabara Koogan, pp. 42–88. Silveira, A.C., Feitosa, V., Borges, R., 1984. Distribuic¸a˜o de triatomı´neos capturados no ambiente domiciliar, no perı´odo de 1975/83 no Brasil. Rev. Bras. Malar. 36, 15 – 312. Smith, J.J.B., Cornish, R.A., Wilkes, J., 1980. Properties of a calcium-dependent apyrase in the saliva of the blood-feeding bug, Rhodnius prolixus. Experientia 36, 898 – 900. Steindel, M., Toma, H.K., Carvalho Pinto, C.J., Grisard, E.C., Schlemper, B.R. Jr, 1994. Colonizac¸a˜o de eco´topos artificiais pelo Panstrongylus megistus na Ilha de Santa Catarina, Floriano´polis, Santa Catarina, Brasil. Rev. Inst. Med. Trop. Sa˜o Paulo 36 (1), 43 – 50. Teo, S.K., Cheah, J.S., 1973. Severe reaction to the bite of the triatomid bug (Triatoma rubrofasciata) in Singapore. J. Trop. Med. Hyg. 76, 161 – 162. Warburg, A.S.E., Lanzaro, G.C., Titus, R.G., Neva, F., 1994. Saliva of Lutzomyia longipalpis sibling species differs in its composition and capacity to enhance leishmaniasis. Phil. Trans. R. Soc. Lond. B, 223–230.
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Differences in saliva composition among three Brazilian populations of Panstrongylus megistus (Hemiptera, Reduviidae) S.E. Barbosa a,b, L. Diotaiuti b, R.P.P. Soares a,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, MG, Brazil b Centro de Pesquisas Rene´ Rachou-FIOCRUZ, A6. Augusto de Lima, 1715, Caixa Postal 1743, CEP 30.190 -002, Belo Horizonte, MG, Brazil
Received 28 January 1998; received in revised form 18 August 1998; accepted 10 September 1998
Abstract Comparisons of electrophoretic profiles of triatomines (Panstrongylus megistus) from the Brazilian states of Bahia (BA), Minas Gerais (MG) and Santa Catarina (SC) revealed differences in the composition of the saliva between the three populations. A phenogram constructed on the basis of the most representative electrophoretic banding patterns allowed the specimens tested to be divided into two groups. The BA and SC populations could be completely separated while some individuals from MG could be placed in each group. The BA population presented fewer bands than the MG and SC ones. The populations studied also differed with regard to their degree of association with human habitations (sylvatic, peridomiciliary and/or intradomiciliary) and our results suggest that the proteinaceous composition of the saliva of the three distinct populations of triatomines could be related to the hosts each is likely to encounter. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Triatomines; Saliva; Panstrongylus megistus; Interpopulation variability
* Corresponding author. Fax: + 55 31 4992970; e-mail: [email protected] 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0001-706X(98)00073-4
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1. Introduction Panstrongylus megistus is an important species in the epidemiology of Chagas’ disease in Brazil because of its high susceptibility to infection by Trypanosoma cruzi, wide geographical distribution and adaptability to a range of habitats and hosts, which allows it to colonize both domestic and peridomestic ecotopes. Nevertheless, populations of this species from different regions show behavioural variation in their capacities to colonize the domestic environment. In northeastern Brazil, P. megistus generally occurs in man-made habitats such as those encountered in the Recoˆncavo Baiano region, where repeated attempts to collect this species in sylvatic habitats have been fruitless. This was explained by Sherlock (1979) as a consequence of the destruction of its natural habitat, beginning with the colonization of Brazil and the exploitation of native timber by the settlers. This resulted in fragmentation of the Atlantic Forest, probably favoring selection of a population of triatomines with a high capacity to colonize man-made habitats. Despite the profound environmental alterations that have occurred in other regions of Brazil, in the southeast of the country P. megistus can still be encountered in sylvatic ecotopes (Barretto, 1979) as well as man-made ones (Silveira et al., 1984), the limit of these primarily sylvatic populations apparently passing through the state of Sa˜o Paulo (Araga˜o, 1961; Pessoa, 1962; Forattini, 1972; Forattini et al., 1977). The electrophoretic profile of triatomine saliva shows a complex composition of proteins, allowing the separation of species (Pereira et al., 1996a). However it is not known whether the composition of saliva varies within the species and/or populations. With this in mind, the objective of this study was to compare the electrophoretic profiles of the saliva of individuals from three Brazilian populations of P. megistus.
2. Materials and methods Triatomines were collected from the following localities: Campo Formoso, Bahia (10°30%S; 40°20%W), where the species is found exclusively in artificial ecotopes; Belo Horizonte, Minas Gerais (20°0%S; 44°0%W), where it occurs both in artificial and sylvatic ecotopes; and Floriano´polis, Santa Catarina (27°30%S; 48°30%W) where P. megistus is predominantly sylvatic and shows a low potential for the colonization of man-made habitats. Laboratory colonies were established from wild-caught specimens and the F1 (BA and MG) or F3 (SC) generations of these colonies used in analyses. The triatomines were maintained in an insectary at a temperature of 2792°C and relative humidity of 60 – 70%. Anaesthetized mice were offered to the bugs once a week as a blood meal source until adult insects were available for use in the analyses. Twenty insects from each population were analyzed 1–2 months after their final moults. Individual saliva was collected using a capillary tube (length 75 mm, internal diameter 1.1 mm; MICRON®, Sa˜o Paulo) inserted in the proboscis of each insect,
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followed by electrical stimulation of the ventral thoracic surface, which had been treated previously with an electrolytic gel (Pereira et al., 1996a). Electrophoresis (SDS-PAGE) of triatomine saliva was carried out on a 12.5% polyacrylamide slab gel (Hubbard and Lazarides, 1979) and the protein bands were stained by Coomassie blue (G-250). Protein content in the saliva was determined by the method of Bradford (1976), using bovine serum albumin as standard. Approximately 5 mg of protein were loaded in each lane. A taxonomic matrix was elaborated based on the study of banding patterns, allowing a phenogram to be drawn, using the similarity coefficient of Dice (1945). Only those bands that presented ] 5% of the total applied protein were considered in producing the matrix. Estimates of the relative amount of protein in the bands of each electrophoretic profile were made by densitometry using the Is-1000 Digital Imaging System (Alpha Innotech, San Leandro, CA).
3. Results The electrophoretic analysis of saliva of individuals of P. megistus showed a complex protein composition with molecular weights ranging from 9 to 87 kDa. The most characteristic protein band profile was observed in the region between 20 and 24 kDa. This region contains 53.8 9 10.1% (n= 60) of the total applied protein distributed in 2 – 4 bands as determined by densitometry (Figs. 1 and 2). Remarkable differences were seen between the band profiles of the Santa Catarina and Bahia populations. All SC individuals showed one 20 kDa and another 23 kDa protein band, while BA individuals did not show the 23 kDa protein band and only
Fig. 1. Electrophoretic profiles (SDS-PAGE) of Panstrongylus megistus saliva. Lanes (1 – 8): insects from Floriano´polis, Santa Catarina state, Brazil. Molecular mass markers (kDa) are indicated on the left. The arrows indicate the position of the four most characteristic protein bands of approximately 20, 21, 23 and 24 kDa.
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Fig. 2. Electrophoretic profiles (SDS-PAGE) of Panstrongylus megistus saliva. Lanes (1 – 4): insects from Campo Formoso, Bahia state, Brazil; (5– 8): insects from Belo Horizonte, Minas Gerais state, Brazil. Molecular mass markers (kDa) are indicated on the left. The arrows indicate the position of the four most characteristic protein bands of approximately 20, 21, 23 and 24 kDa.
two of them (10% of BA analyzed individuals) showed the 20 kDa band. Seventeen of the Minas Gerais individuals (85%) showed a band of 23 kDa and 14 (70%) one of 20 kDa (Figs. 1 and 2). Regarding the number of bands in this region, 19 individuals from SC population showed four bands while one individual presented three. In the BA population, 18 individuals showed two bands and only two individuals presented three. In the MG population, nine individuals showed four bands, six individuals presented three bands and five individuals showed two. No differences were observed between the electrophoretic profiles of males and females for any of the three populations studied. The phenogram constructed on the basis of these results showed the existence of two main groups, the SC population being completely separated from that of BA. The MG individuals could be distributed between the two groups, with 14 (70%) in the SC group and six (30%) in the BA one (Fig. 3). The number of bands analyzed varied from 4 to 9 per insect. With respect to this parameter, the BA individuals with 5.09 0.9 bands were different from those of SC and MG with 6.691.1 and 6.19 1.1, respectively (PB0.01). There was no significant difference in band number between the MG and SC populations (P= 0.12; Table 1).
4. Discussion This is the first time that salivary proteins have been used in the study of triatomine populations. The saliva of P. megistus obtained by electrical stimulation can be stored in a freezer at − 15°C, where it maintains its stability giving the same
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Fig. 3. Unweighted pair groups of individual adult Panstrongylus megistus males (M) and females (F) based on pairwise shared salivary protein bands. Origin: B, Bahia state; M, Minas Gerais state and S, Santa Catarina state. The similarity coefficient (horizontal scale) was derived from the Dice Index. The vertical bar represents the average level of similarity between all the pair samples analysed (phenon line).
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electrophoretic profile for up to 3 months after collection. This stability supports the finding that triatomine saliva apparently has no digestive function, no proteases, lipases or glycosidases having been described to date (Garcia, 1995). Our results show a variation in the composition of the saliva between distinct populations of P. megistus, with a clear separation between SC and BA individuals and those of MG in an intermediate position, albeit slightly closer to members of the SC population. It is interesting to note that the electrophoretic profile of the saliva appears to undergo a gradual change in populations along the south–north and/or west – east axis, also showing a tendency to concentrate more protein in a fewer number of bands. This pattern resembles the variation in the activity of the salivary vasodilator maxidilan observed among putative species of phlebotomine sand flies of the Lutzomyia longipalpis complex from Brazil, Colombia and Costa Rica, with increasing activity and a corresponding change in the pathology of visceral leishmaniasis in those further south (Warburg et al., 1994). Although the true significance of this variation in the composition of saliva of P. megistus is not known, the populations studied show different degrees of adaptation to man-made environments and it is probable that it is related to the association between the triatomines and the different hosts present in the domestic and/or sylvatic environments. According to Barretto (1979) and Sherlock (1979), P. megistus is intimately associated with opossums in sylvatic ecotopes. These animals were the source of 62.9% of blood meals in triatomines captured in man-made habitats on Santa Catarina island, based on analyses by precipitin test on one of the few collections of P. megistus made in houses (Steindel et al., 1994). By contrast studies in the southeast of Brazil on triatomines collected in peridomestic environments revealed that birds were the main source of blood meals (44.2%), other animals being represented at lower frequencies e.g. pig (10.3%), opossum (14.2%), rodent (13.9%), dog (4.9%) and man (3.2%). Nevertheless, in this area, P. megistus is able to establish colonies in the man-made habitats and represents an important vector of T. cruzi to man (Sherlock, 1979). In Bahia, 80.8% of insects captured in houses reacted positively with anti-human serum (Minter, 1975). Triatomine saliva possesses substances with pharmacological activities that assist in blood feeding, such as anticoagulants (Hellmann and Hawkins, 1964; Ribeiro et al., 1995; Pereira et al., 1996a), vasodilators (Ribeiro et al., 1990, 1993), antihistamine (Ribeiro and Table 1 Number of bands analysed of populations of Panstrongylus megistus from Bahia (BA), Minas Gerais (MG) and Santa Catarina (SC), Brazil Sex/population
Male Female Mean
Panstrongylus megistus BA
MG
SC
5.190.4 (n= 6) 4.9 91.0 (n=14) 5.09 0.9 (n=20)a
6.0 91.1 (n = 6) 6.1 9 1.1 (n = 14) 6.1 91.1 (n = 20)b
6.6 91.2 (n = 9) 6.6 9 1.1 (n =11) 6.6 91.1 (n =20)c
T-test: a/b (PB0.01); a/c (PB0.01); b/c (P= 0.12).
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Walker, 1994), anaesthetic-like (Pereira et al., 1996b) and inhibitors of platelet aggregation, the latter elicited by ADP (Smith et al., 1980; Ribeiro and Garcia, 1980), collagen (Ribeiro and Garcia, 1981; Noeske-Jungblut et al., 1994) or aracdonic acid (Ribeiro and Sarkis, 1982). Saliva also modulates the feeding success of haematophagous insects through immunogenic reactions (Lehane, 1991). Triatomine saliva can elicit host reactions varying from local (Dias, 1968; Mott et al., 1980; Costa et al., 1981) to general anaphylaxis (Teo and Cheah, 1973; Pinnas et al., 1986). Thus, variation in the composition of saliva as demonstrated here may be a consequence of selection favouring a better adaptation to local hosts. This phenomenon was observed in the Malayan pit viper Calloselasma rhodostoma, where geographical variation in the venom appeared to be linked to the composition of the local fauna, i.e. the potential prey species available (Daltry et al., 1996).
Acknowledgements Thanks to Dr John Bruce Alexander for revising the manuscript, to Dr Odair Genaro for kindly permitting use of an image analyser for densitometry and to Ana Maria Salvador Pereira for technical assistance. This study was financed by FAPEMIG, CAPES and CPqRR/FIOCRUZ.
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