Variability of the salivary proteins of 20 Brazilian populations of Panstrongylus megistus (Hemiptera: Reduviidae: Triatominae)

Acta Tropica 92 (2004) 25–33

Variability of the salivary proteins of 20 Brazilian populations of Panstrongylus megistus (Hemiptera: Reduviidae: Triatominae) S.E. Barbosa a,b , L. Diotaiuti a , E.M. Braga b , M.H. Pereira b,∗ a

b

Centro de Pesquisas René Rachou-FIOCRUZ, Av. Augusto de Lima 1715, Caixa Postal 1743, CEP 30.190-002, Belo Horizonte, MG, Brazil Departamento de Parasitologia, Universidade Federal de Minas Gerais, Av. Antˆonio Carlos 6627, Caixa Postal 486, CEP-31270-901, Belo Horizonte, MG, Brazil Received 15 August 2003; received in revised form 3 March 2004; accepted 27 May 2004 Available online 21 July 2004

Abstract The objective of the present study was to study variability in the salivary proteins of 20 Panstrongylus megistus populations from different ecotopes and verify whether this variability influenced the intensity of the response to specific anti-saliva antibodies. Electrophoretic analysis of P. megistus saliva showed a complex protein composition and great interpopulation variability. A higher concentration of bands was observed in the 17–29 kDa region. The phenogram constructed from the electrophoretic profiles of the P. megistus study populations revealed the existence of two main groups. However, there was no evident relationship between these groups and geographical regions, ecotopes or hosts. Saliva inoculated by P. megistus during feeding elicited production of low level of anti-saliva antibodies in rabbit. The homologous and heterologous salivary proteins were recognised by serum of rabbit sensitised with saliva from only one population. Qualitative and quantitative differences were observed for recognised bands in the saliva of all eight populations studied by Western blot analysis. The most recognised bands were those of greatest molecular weight (68.0–97.4 kDa). Published by Elsevier B.V. Keywords: Triatomines; Saliva; Panstrongylus megistus; Immune recognition

1. Introduction The triatomine Panstrongylus megistus is important in the epidemiology of Chagas’ disease in Brazil. Currently thought to be the principal vector of Trypanosoma cruzi in Central, East and Southeastern ∗ Corresponding author. Tel.: +55 31 3499 2867; fax: +55 31 3499 2970. E-mail address: [email protected] (M.H. Pereira).

0001-706X/$ – see front matter Published by Elsevier B.V. doi:10.1016/j.actatropica.2004.05.012

Brazil, its geographical distribution is restricted to eastern South America (Correa et al., 1963). Its centre of dispersal is Brazil (Forattini, 1980, 1985), where it occurs in a wide range of habitats and ecotopes from State of Pará until Santa Catarina, including Atlantic forest, cerrado and caatinga (dry and mesic forests of the interior), occupying silvatic, domestic and peridomestic environments. This species exhibits behavioural variations in its capacity to colonise and adapt to human habitations,

26

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33

its epidemiological importance varying among different geographical regions. The presence or absence of domiciliation is delimited geographically by a hypothetical line that crosses the north–northeast of São Paulo State. To the north of this line, P. megistus occupies both silvatic and artificial ecotopes and assumes epidemiological importance as a vector of T. cruzi, as in the States of Minas Gerais and Bahia. To the south of this limit it is encountered only in natural biotopes, rarely being found inside houses and therefore not playing a role in the transmission of Chagas’ disease (Pessoa, 1962; Forattini et al., 1970; Forattini, 1980). Several biological and biochemical studies have been published in recent years in an attempt to clarify whether behavioural differences among populations of this species are due to genetic factors and/or to distinct climatic conditions of their respective areas of distribution (Dórea et al., 1982; Barbosa et al., 1998, 1999, 2001, 2003). A comparative study of three geographically distinct populations of P. megistus revealed differences in the protein compositions of their saliva (Barbosa et al., 1999). This could reflect adaptation to feeding on different hosts present in their areas of origin. The objective of the present study was to follow up these findings by studying variability in the salivary proteins of 20 P. megistus populations from different ecotopes and verify whether this variability influenced the intensity of their response to specific anti-saliva antibodies.

1998) and stored at −20 ◦ C until used. Pooled saliva obtained from 15 adult insects (±10 ␮l) was used to evaluate its stability and compare electrophoretic profiles. Stability of the saliva was evaluated by incubating it at 37 ◦ C in 400 ␮l PBS (pH 7.2) for up to 5 h. Salivary glands (D1 and D2) were extracted in 0.9% saline solution containing 5 ␮g/ml Pepstatin A. After dissection, the glands were broken prior to use with an ultrasonic cleaner (Branson) for 10 s and the tube centrifuged (10000 g, 5 min). The supernatant was used for ELISAs and immunoblotting assays. The concentration of soluble proteins was determined by the method of Bradford (1976), using BSA as a standard. 2.3. Electrophoresis (SDS–PAGE) Salivary profiles (5 ␮g) were obtained on 12.5% polyacrylamide gels (Laemmli, 1970) stained with Coomassie blue. The relative quantity of protein in each electrophoretic profile was estimated with a densitometer using the Digital Images IS-1000 system. Only bands that constituted >5% of the total protein were used in the analyses (Barbosa et al., 1999). A matrix character/taxon was elaborated based on the presence or absence of bands, from which Dice’s coefficient of similarity (Dice, 1945) was calculated and used to construct a phenogram (UPGMA). 2.4. Rabbit immunisation

2. Material and methods

Specimens of P. megistus were obtained from different ecotopes and geographical regions of Brazil, as shown in Table 1. Adult insects of the first filial generation (F1) were used and two populations maintained for more than 15 years in the laboratory, both reared under the same conditions (27 ◦ C ± 2 ◦ C; 60% ± 10% RH). The insects were fed every two weeks on chickens and were starved for 7–15 days.

An adult rabbit (1.5 kg) was exposed to successive bites of P. megistus adults (population 16PMJ-Jacu´ı/MG). The rabbit was immobilised and each ear exposed to the bites of 10 insects for 1 h over an immunisation period of approximately one month, the rabbit being submitted to bites at one-week intervals (on days 3, 10, 17, 23, 27 and 34). Five collections of blood were made, on days 0, 14, 20, 30 and 37. Blood was obtained by puncturing the ear of the rabbit and the serum obtained stored at −20 ◦ C.

2.2. Collection of saliva

2.5. ELISA

Saliva was collected from adult insects by mechanical stimulation using capillary tubes (Amino et al.,

Salivary gland extracts from nine P. megistus populations were coated onto NUNC MAXISORP

2.1. Insects

Table 1 Origin, capture site ecotopes, codes used in different studies and different morphoclimatic dominions that characterise the region of origin of the different P. megistus populations studied State

Locality

Ecotope

Code

Coordinates

Morphoclimatic dominion

Palmeira dos Indios Paulo Jacinto Palmeira dos Indios

I (bed, wall) P (pile of roofing tiles) P (henhouse)

10PMAL 11PMAL∗ 1PMAL

9◦ 24 14S/36◦ 26 24W 9◦ 27 41S/36◦ 25 28W

Atlantic forest Atlantic forest Atlantic forest

Cear´a

Alcˆantaras Meruoca

P P (henhouse)

13PMCE∗ 12PMCE

3◦ 36S/40◦ 28W 3◦ 32 08S/40◦ 29 47W

Atlantic forest remnant Atlantic forest remnant

Bahia

Castro Alves Campo Formoso

L I (bed, wall)

19PMBA∗ 21PMBAL

12◦ 46S/39◦ 33W 10◦ 26 10S/40◦ 16 74W

Atlantic forest Cerrado

Minas Gera´ıs

Olhos d’agua Olhos d’agua Carmo do Parana´ıba Carmo do Parana´ıba Santana do Riacho Bambu´ı Jacu´ı

P (henhouse) P (henhouse) P (henhouse/pile of roofing tiles) I (woodpile on veranda) P (henhouse) P P (henhouse, rat’s nest)

3PMO∗ 24PMG∗ 1PMMG 6PMI 4PMS 2PMB 16PMJ∗

17◦ 30 80S/43◦ 34 24W 17◦ 28 41S/43◦ 33 48W 18◦ 54 55S/46◦ 15 54W 19◦ 00 14S/46◦ 09 79W 19◦ 20 124S/43◦ 37 868W 20◦ 01S/45◦ 59W 21◦ 04 16S/46◦ 47 05W

Cerrado Cerrado Cerrado Cerrado Cerrado Cerrado Atlantic forest

Goi´as

Corumb´a de Goi´as

P (paiol)

14PMGO∗

15◦ 54 22S/48◦ 29 17W

Cerrado

São Paulo

Juqui´a Caconde

L I

9PMSP 20PMSP∗

– 21◦ 34 66S/46◦ 39 07W

Atlantic forest Atlantic forest

Paran´a

Arapongas

P

18PMPR∗

15◦ 54 22S/48◦ 29 17W

Cerrado

15PMSC 22PMSC

27◦ 43S/48◦ 30W

Atlantic forest Atlantic forest

Santa Catarina

Lagoa da Conceiçˇao Costeira do Pirajuba´e

S S

27◦ 43S/48◦ 30W

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33

Alagoas

9◦ 29 32S/36◦ 23 30W

The letters I, P, S and L refer to intradomicile, peridomicile, silvatic and laboratory, respectively. ∗ Populations used in ELISAs and Western Blot experiments.

27

28

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33

(Dynatech) microlitre plates in 0.1 M NaHCO3 by overnight incubation (2.0 ␮g/well). After blocking the free binding sites using 5% non-fat milk, serum from immunised rabbit was added in duplicate at a dilution of 1:10 in 0.05% Tween 20/phosphate-buffered saline (PBS). The serum was allowed to react for 3 h with the antigen, followed by six washings with 0.3% Tween 20/PBS. Mouse anti-rabbit IgG conjugated to peroxidase (Sigma) was used at a dilution of 1:500 for the detection of bound antibodies. After 90 min, the plates were washed as above and residual peroxidase activity was revealed by adding ortho-phenylenodiamine (Sigma) and interrupted by 30 ␮l of 4N H2 SO4 per well. Absorbance was read at 490 nm on a BioRad 3550 microplate reader. 2.6. Western blot Salivary gland extracts from eight distinct P. megistus populations (50 ␮g) were separated by 12.5% sodium dodecyl sulfact–polyacrilamide gel electrophoresis and transferred onto nitrocellulose membranes (Immobilon-NC; Millipore) according to Mini Trans-Blot manual® (Bio-Rad). Serum from immunised rabbit (day 37) was diluted 1:10 and incubated for 90 min. After incubation the membrane was probed with 1:500 monoclonal anti-rabbit immunoglobulin-peroxidase conjugated from mouse (Sigma) and then developed using a diaminobenzidine

peroxidase substrate kit (SK-4100, Vector Laboratories, Inc.).

3. Results 3.1. Electrophoresis (SDS–PAGE) Electrophoretic analysis of P. megistus saliva showed a complex protein composition and great interpopulation variability. A higher concentration of bands was observed in the 17–29 kDa region, representing a mean of 90.40 ± 4.02% of the total protein applied. A total of 3–8 bands was found in this region. The 16PMJ population presented fewest bands (three) while 19PMBA presented the greatest number i.e., eight (Fig. 1). The electrophoretic profile of salivary gland extract was similar to the profile observed for saliva. In the salivary gland extract, stronger bands of low molecular weight (below 14.3 kDa) could be observed (Fig 2A). Saliva remained stable even after 5 h incubation in PBS (pH 7.2) at 37 ◦ C, as can be observed from the electrophoretic profile (Fig. 2B), particularly in the 17–29 kDa region, where the bands could be used to separate the study populations. The phenogram constructed from the electrophoretic profiles of the P. megistus study populations (Fig. 3) revealed the existence of two main groups. However, there was no evident relationship

Fig. 1. Electrophoretic profile (SDS–PAGE) of saliva (pool) of the 20 populations of Panstrongylus megistus. Values of molecular weights are shown on the left. (A) Lanes: (1) molecular weight standard; (2) 24PMG; (3) 5PMG; (4) 16PMJ; (5) 9PMSP; (6) 18PMPR; (7) 19PMBA; (8) 11PMAL; (9) 1PMALP; (10) 12PMCE; (11) 15PMSC. B. Lanes: (1) pattern of molecular weight; (2) 22PMSC; (3) 13PMCE; (4) 10PMAL; (5) 21PMBAL; (6) 14PMGO; (7) 4PMS; (8) 3PMO; (9) 2PMB; (10) 6PMI; (11) 1PMMG.

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33

29

Fig. 2. (A) Electrophoretic profile (SDS–PAGE) of population 9PMSP (pool) of Panstrongylus megistus. Lanes: (1) molecular weight standard; (2 and 3) saliva; (4 and 5) salivary gland extract. (B) Electrophoretic profile (SDS–PAGE) of population 15PMSC after incubation of saliva with PBS 37 ◦ C (test of stability of proteins present in Panstrongylus megistus saliva). Lanes: (1) molecular weight standard; (2–7) time 0, 1, 2, 3, 4 and 5 h incubation, respectively.

between these groups and geographical region, ecotopes or hosts. 3.2. ELISA Repeated exposure to the bites of P. megistus led to the production of specific antibodies against

homologous salivary proteins (16PMJ). This effect was particularly marked after the third sensitisation, the levels being maintained until after the sixth sensitisation (Fig. 4). Different optical densities were obtained when heterologous antigens were used, the salivary extracts of insects of the populations 11PMAL, 20PMSP, 24PMG, 13PMCE

Fig. 3. Phenogram constructed by UPGMA method from electrophoretic profiles (pool) of salivary proteins of 20 populations of Panstrongylus megistus. The vertical bar represents the mean level of similarity among all pairs analysed (phenon line). The horizontal scale represents the similarity index derived from the Dice index. The numbers and letters on the right indicate the codes for geographical origin of the study populations.

30

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33

Fig. 4. IgG antibodies against salivary proteins from different populations of Panstrongylus megistus studied at different times of immunisation. The arrows indicate the days of sensitisation of the rabbit.

presenting higher values than the homologous population. Recognition by antibodies of the remaining four populations (18PMPR, 3PMO, 14PMGO and 19PMBA) showed low absorbance values (<0.3). 3.3. Western blotting Serum from rabbit immunised by bites of P. megistus (population 16PMJ) was able to recognise proteins with molecular weights of 17–100 kDa, present in the saliva of 16PMJ and the other seven distinct populations (Fig. 5). Nevertheless, the most recognised bands were those of greatest molecular weight (68.0–97.4 kDa). Qualitative and quantitative differences were observed for this band in the saliva of all eight populations studied.

4. Discussion In general, the electrophoretic profile (SDS–PAGE) of salivary proteins in P. megistus was similar to that observed in other Triatominae, showing a complex protein composition in which most prominent bands

Fig. 5. Immunoblot analysis of different populations of Panstrongylus megistus salivary proteins with IgG antibodies from one rabbit immunized. Lanes: (1) 19PMBA; (2) 14PMGO; (3) 20PMSP; (4) 3PMO; (5) 18PMPR; (6) 24PMG; (7) 11PMAL and (8) 16PMJ.

had molecular weights of less than 45 kDa (Chapman et al., 1986; Volf et al., 1993; Pereira et al., 1996; Barbosa et al., 1999). Comparison of the principal bands obtained by SDS–PAGE (≥5% of total protein) of the saliva of 20 P. megistus populations did not permit them to be grouped based on their respective geographical regions, ecotopes, hosts or morphoclimatic

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33

dominions. On the other hand, the great variability observed in the crude saliva (SDS–PAGE) or salivary haemoproteins of triatomines did allow separation at the species level (Pereira et al., 1996; Soares et al., 2000). It was recently demonstrated that both the crude saliva of Triatoma brasiliensis and the salivary haemoproteins of Rhodnius prolixus varied during the development of these triatomines (Guarneri et al., 2003; Moreira et al., 2003). The great variability found in P. megistus profiles during the present study suggests that saliva could be used as a population marker, or in detecting re-infestation of houses following insecticidal control measures. A previous study using the same methodology and three co-specific populations that presented different degrees of association with human habitation allowed individuals of two strains (SC and BA) to be separated completely. Although this was linked to interaction of these populations with their local hosts (Barbosa et al., 1999), this was not confirmed in the present study. Recognition by specific anti-saliva antibodies may be considered a good parameter to differentiate P. megistus populations, given the great variability of protein patterns from the salivary gland extracts. This hypothesis was tested by ELISA and immunoblotting. Antibody levels increased from the third sensitisation by bite onwards. In addition, homologous and heterologous proteins were recognised by serum of rabbit sensitised with only one population. It appears from our results that the antibodies present in the sera of rabbit exposed to P. megistus bites reacted more strongly to proteinaceous components with high molecular weight (68–97.4 kDa), although the most prominent salivary proteins (17–29 kDa) were also recognised, albeit with less intensity. The pattern obtained was similar to that observed for Triatoma infestans, although low molecular weight proteins have not been shown to be immunogenic for this species (Volf et al., 1993). On the other hand, in the North American species Triatoma protracta, the most immunogenic salivary proteins have low molecular weights (17–25 kDa). The principal immunogenic protein of T. protracta was recently purified and characterised as a 20 kDa lipocalin known as procalin (Paddock et al., 2001). It is interesting to note that much of the biological activity of triatomine saliva is due to lipocalins which present great structural similarity to each other (Fuentes-Prior et al., 1997;

31

Andersen et al., 1998), although severe reactions such as those observed in response to the bite of T. protactra are rare in South American species. A salivary protein very similar to procalin was recently described in T. brasiliensis, a species whose saliva does not induce strong reactions (Sant’Anna et al., 2002). In is interesting to note that in phlebotomine sandflies, distinct electrophoretic profiles of salivary proteins can also be observed, either between different species or distinct populations of different geographical origins (Volf et al., 2000). A high variability was also found in the amino acid sequence of the salivary peptide maxadilan in the Lutzomyia longipalpis complex (Lanzaro et al., 1999). However, unlike in sandflies where the bite induces production of high levels of antibodies (Volf and Rohousova, 2001), we observed a low humoral immune response elicited by P. megistus bites. In the present study we were only able to detect the presence of antibodies by ELISA of rabbit serum at a dilution of 1:10. At higher dilutions (1:20, 1:40, 1:50 and 1:100) the responses were very low (data not shown). Originally an Atlantic forest species (Forattini, 1980), P. megistus may have begun to invade the domestic environment during the post-colonial period. Following the destruction of its natural habitat and consequent agricultural developments this species presumably invaded and became adapted to the houses (Litvoc et al., 1990). A series of population adaptations could thus be expected associated with the transition from silvatic habitats to domestic ones (Schofield, 1988). The variability observed in the present study as well as previous studies of P. megistus populations (Barbosa et al., 1998, 1999, 2001, 2003) could be due to the founder effect, relatively few individuals giving rise to a new populations in different habitats and geographical areas (Schofield, 1985; Pires et al., 1998).

Acknowledgements Thanks to Dr. Bruce Alexander for revising the manuscript and to Dr. Evaldo Nascimento for kindly permitting use of an image analyser for densitometry. We would like to thank FUNASA, Dr. Vera Rodrigues (SUCEN/SP), Dr. Mário Steindel (UFSC) and Dr. Rogério Luiz Koop (UFPR) for the support

32

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33

during the capture of the insects. This study was financed by CAPES, FAPEMIG, TDR/WHO, ECLAT and CPqRR/FIOCRUZ.

References Amino, R., Porto, R.M., Chammas, R., Egami, M.I., Schenkman, S., 1998. Identification and characterization of a sialidase released by the salivary gland of the hematophagous insect Triatoma infestans. J. Biol. Chem. 273 (38), 24575–24582. Andersen, J.F., Weichsel, A., Balfour, C.A., Champagne, D.E., Montfort, W.R., 1998. The crystal structure of nitrophorin 4 at 1.5 Å resolution: transport of nitric oxide by lipocalin-based heme protein. Structure 10, 1315–1327. Barbosa, S.E., Soares, R.P.P., Pires, H.H.R., Melo, M.D., Pimenta, P.F.P., Margonari, C., Dujardin, J.P., Catalá, S.S., Panzera, F., Romanha, A.J., Pereira, M.H., Diotaiuti, L., 1998. Biossistemática de Panstrongylus megistus (Burmeister, 1835). Rev. Soc. Brasil. Med. Trop. 31 (Suppl. III), 29–31. Barbosa, S.E., Diotaiuti, L., Soares, R.P.P., Pereira, M.H., 1999. Differences in saliva composition among three Brazilian populations of Panstrongylus megistus (Hemiptera, Reduviidae). Acta Trop. 72, 91–98. Barbosa, S.E., Soares, R.P.P., Pires, H.H.R., Diotaiuti, L., 2001. Experimental evidence for a demographic cline in Panstrongylus megistus populations. Mem. Inst. Oswaldo Cruz 96 (6), 773–775. Barbosa, S.E., Dujardin, J.P., Soares, R.P.P., Pires, H.H.R., Margonari, C.M., Romanha, A.J., Panzera, F., Linardi, P.M., Duque-de-Melo, M., Pimenta, P.F.P., Pereira, M.H., Diotaiuti, L., 2003. Interpopulation variability among Panstrongylus megistus (Hemiptera: Reduviidae) from Brazil. J. Med. Entomol. 40 (4), 411–420. 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. Chapman, M.D., Marshall, N.A., Saxon, A., 1986. Identification and partial purification of species-specific allergens from Triatoma protacta (Heteroptera, Reduviidae). J. Allergy Clin. Immunol. 78, 436–443. Correa, R.R., Silva, E.O.R., Schiavi, A., 1963. Observações sobre o Panstrongylus megistus, transmissor da moléstia de Chagas (Hemiptera, Triatominae). Arq. Hig Saúde Públ. 28, 165–174. Dórea, R.C.C., Póvoa, M.M., Miles, M.A., Souza, A.A.A., Barata, J.M., 1982. Eletroforese de isoenzimas para estudo de triatom´ıneos com referˆencia especial a subpopulações de P. megistus. Rev. Bras. Biol. 42 (3), 521–526. Dice, L.R., 1945. Measures of the amount of ecological association between species. Ecology 26, 297–302. Forattini, O.P., 1980. Biogeografia, origem e distribuição da domiciliação de triatom´ıneos no Brasil. Rev. Saúde Públ. 14, 265–299. Forattini, O.P., 1985. Panstrongylus megistus (Burmeister). In: Carcavallo, R.U., Rabinovich, J.E., Tonn R.J (Eds.), Factores Biologicos y Ecologicos en la Enfermedad de Chagas. Tomo I.

Epidemiologia-Vectores. Ministerio de Salud y Accion Social, Imprenta Central “Prof. Dr. Ramón Carrillo”, Buenos Aires, pp. 219–223. Forattini, O.P., Rabello, E.X., Castanho, M.L.S., Patolli, D.G.B., 1970. Aspectos ecológicos da tripanossom´ıase americana. I. Observações sobre o Panstrongylus megistus e suas relações com focos naturais da infecção em área urbana na cidade de São Paulo. Rev. Saúde Publ. 4, 19–30. Fuentes-Prior, P., Noeske-Jungblut, C., Donner, P., Schleuning, W., Huber, R., Bode, W., 1997. Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug. Proc. Natl. Acad. Sci U.S.A. 94, 11845. Guarneri, A.A., Diotaiuti, L., Gontijo, N.F., Gontijo, A.F., Pereira, M.H., 2003. Blood-feeding performance of nymphs and adults of Triatoma brasiliensis on human hosts. Acta Trop. 87 (3), 361–370. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lanzaro, G.C., Lopes, A.H.C.S., Ribeiro, J.M.C., Shoemaker, C.B., Warburg, A., Soares, M., Titus, R.G., 1999. Variation in the salivary peptide, maxadilan, from species in the Lutzomyia longipalpis complex. Insect Mol. Biol. 8 (2), 267–275. Litvoc, J., Goldbaum, M., da Silva, G.R., 1990. Determinants of the process of domiciliary infestation by Panstrongylus megistus: the role of housing and deforesting. Rev. Inst. Med. Trop. Sao Paulo 32 (6), 443–449. Moreira, M.F., Coelho, H.S.L., Zingali, R.B., Oliveira, P.L., Masuda, H., 2003. Changes in salivary nitrophorin profile during the life cycle of the blood-sucking bug Rhodnius prolixus. Insect Bichem. Mol. Biol. 33, 23–28. Paddock, C.D., McKerrow, J.H., Foreman, K.W., Hsich, I., Marshall, N., 2001. Identification, cloning, and recombinant expression of Procalin, a major Triatomine allergen. J. Immunol. 167, 2694–2699. Pereira, M.H., Souza, M.E.L., Vargas, A.P., Martins, M.S., Penido, C.M., Diotaiuti, L., 1996. Anticoagulant activity of Triatoma infestans and Panstrongylus megistus saliva (Hemiptera/Triatominae). Acta Trop. 61, 255–261. Pessoa, S.P., 1962. Domiciliação de triatom´ıneos e epidemiologia da Doença de Chagas. Arq. Hig. Saúde Publ. 27, 161–171. Pires, H.H.R., Barbosa, S.E., Margonari, C., Jurberg, J., Diotaiuti, L., 1998. Variations of the external male genitalia in three populations of Triatoma infestans Klug, 1834. Mem. Inst. Oswaldo Cruz 93 (4), 479–483. Sant’Anna, M.R.V., Araújo, J.G.V.C., Pereira, M.H., Pesquero, J.L., Diotaiuti, L., Lehane, S.M., Lehane, M.J., 2002. Molecular cloning and sequencing of salivary gland-specific cDNAs of the blood-sucking bug Triatoma brasiliensis (Hemiptera: Reduviidae). Insect Mol. Biol. 11 (6), 585–593. Schofield, C.J., 1985. Population dynamics and control of Triatoma infestans. Ann. Soc. Belge Med. Trop. 65, 149–164. Schofield, C.J., 1988. The biosystematics of Triatominae. In: MW Service, Biosystematic of Haematophagous Insects. Systematics Association, Special Volume 37. Clarenden Press, Oxford, pp. 284–312. Soares, R.P.P., Sant’anna, M.R.V., Gontijo, N.F., Romanha, A.J., Diotaiuti, L., Pereira, M.H., 2000. Identification

S.E. Barbosa et al. / Acta Tropica 92 (2004) 25–33 or morphologically similar Rhodnius species (Hemiptera: Reduviidae: Triatominae) by electrophoresis of salivary heme proteins. Am. J. Hyg. 62 (1), 157–161. Volf, P., Grubhoffer, L., Hosek, P., 1993. Characterization of salivary gland antigens of Triatoma infestans and antigen-specific serum antibody response in mice exposed to bites of T. infestans. Vet. Parasitol. 47, 327–337.

33

Volf, P., Rohousova, I., 2001. Species-specific antigens in salivary glands of phlebotominae sandflies. Parasitology 1, 37– 41. Volf, P., Tesarova, P., Nohynkova, E.N., 2000. Salivary proteins and glycoproteins in phlebotomine sandflies of various species, sex and age. Med. Vet. Entomol. 14 (3), 251– 256.