ARTÍCULO ORIGINAL

doi: http://dx.doi.org/10.7705/biomedica.v35i1.2213

Author´s contributions:

Daniella Barreto-Santana: experimental design, data collection in the laboratory, data analysis and the writing of this manuscript

Liliane Santos-Schuenker and Aline Rosa da Fonseca: data collection in the laboratory

Rodrigo Gurgel-Gonçalves and Cesar Augusto Cuba-Cuba: contribution of data bases for each registry, experimental study design, participation in the discussion, drafting and reviewing of this manuscript

Recibido: 02/12/13; aceptado: 05/11/14

Introduction: Specific host-parasite a ssociations have been detected experimentally and suggest that triatomines of the genus Rhodnius act as biological filters in the transmission of Trypanosoma rangeli .

Objective: To analyze the susceptibility of four Rhodnius species ( Rhodnius robustus , Rhodnius neglectus , Rhodnius nasutus and Rhodnius pictipes ) to a Brazilian strain of T. rangeli (SC-58/KP1-).

Materials and methods: We selected t hirty nymphs of each species, which were fed on blood infected with T. rangeli . Periodically, samples of feces and hemolymph were analyzed. Triatomines with T. rangeli in their hemolymph were fed on mice to check for transmission by bites. Later, the triatomines were dissected to confirm salivary gland infection.

Results: Specimens of R. pictipes showed higher rates of intestinal infection compared to the other three species. Epimastigotes and trypomastigotes were detected in hemolymph of four species; however, parasitism was lower in the species of the R. robustus lineage. Rhodnius robustus and R. neglectus specimens did not transmit T. rangeli by bite; after dissection, their glands were not infected. Only one specimen of R. nasutus and two of R. pictipes transmitted the parasite by bite. The rate of salivary gland infection was 16% for R. pictipes and 4% for R. nasutus .

Conclusions: Both infectivity (intestinal, hemolymphatic and glandular) and transmission of T. rangeli (SC58/KP1-) were greater and more efficient in R. pictipes. These results reinforce the hypothesis that these triatomines may act as biological filters in the transmission of T. rangeli .

Key words: Trypanosoma rangeli , Rhodnius, host-parasite interactions.

doi: http://dx.doi.org/10.7705/biomedica.v35i1.2213

Sensibilidad de diferentes especies de Rhodnius (Hemiptera, Reduviidae, Triatominae) a una cepa brasileña de Trypanosoma rangeli (SC58/KP1-)

Introducción. Se han detectado asociaciones biológicas huésped-parásito específicas que sugieren que los triatominos del género Rhodnius podrían actuar como filtros biológicos en la transmisión de Trypanosoma rangeli .

Objetivo. Estudiar la sensibilidad de cuatro especies de Rhodnius ( Rhodnius robustus , Rhodnius neglectus , Rhodnius nasutus y Rhodnius p ictipes ) frente a la cepa de T. rangeli (SC-58/KP1-).

Materiales y métodos. Se seleccionaron treinta ninfas de cada especie después de xenodiagnóstico artificial en sangre infectada con T. rangeli. Se examinaron periódicamente m uestras de heces y hemolinfa. Los insectos con hemolinfas infectadas fueron alimentados en ratones a fin de comprobar la transmisión por picadura y posteriormente disecados para confirmar la infección de las glándulas salivales .

Resultados . En Rhodnius pictipes se encontró un mayor porcentaje de infección intestinal que en las otras especies . Se detectaron epimastigotes y tripomastigotes en la hemolinfa de las cuatro especies , y se encontró que el parasitismo fue menor en las especies del linaje R. robustus . Rhodnius robustus y R. neglectus no transmitían T. rangeli a ratones por picadura: después de la disección , sus glándulas no estaban infectadas. Solo un espécimen de R. nasutus y dos de R. pictipes transmitieron el parásito por la picadura . La tasa de infección glandular fue de 16 % para R. pictipes y de 4 % para R. nasutus .

Conclusiones . La capacidad infecciosa ( hemolinfática, intestinal y glandular ) y la transmisión de T. rangeli (SC-58/KP1-) fueron mayores y más eficientes en R. pictipes . Estos resultados refuerzan la hipótesis de que estos triatominos actúan como filtros biológicos en la transmisión de T. rangeli .

Palabras clave: Trypanosoma rangeli , Rhodnius, interacciones huésped-parásitos .

doi: http://dx.doi.org/10.7705/biomedica.v35i1.2213

Trypanosoma rangeli is a protozoan parasite that has been found in different species of mammals and triatomines throughout South and Central America. Its geographic distribution often overlaps with that of Trypanosoma cruzi , the etiologic agent of Chagas disease. Mixed infections with both protozoan species have been detected in different host species and humans, which causes difficulties in the diagnosis of Chagas disease. The most notable biological characteristic of T. rangeli in its invertebrate hosts is the passage of parasites from the intestine into the hemolymph and then to the insect salivary glands, where the protozoan develops infective metacyclic trypomastigotes. Then, they become infective to the vertebrate host through the bite of the insect vector (1-3). Rhodnius specimens are particularly susceptible to infection with T. rangeli; 13 of 19 Rhodnius species were reported to be infected with this parasite (4,5).

Biological, biochemical and molecular parameters have demonstrated polymorphism between strains of T. rangeli isolated from different triatomines and vertebrate hosts in different geographic regions (6-9). Vallejo, et al. (10), characterized two major lineages of T. rangeli in Latin America based on the presence or absence of a minicircle of kDNA (KP1+ and KP1-, respectively). T. rangeli presents an ex tensive genetic variability demonstrated by kDNA and nuclear DNA analysis (7,9,11,12). Moreover, analysis of the karyotype profiles permitted the division of the T. rangeli strains into two groups coinciding with the KP1+ and KP1- genotypes (13). Subsequent studies clearly showed host-parasite specific associations between T. rangeli - Rhodnius spp. and a hypothesis that these triatomines act as biological filters in the transmission of genetically distinct populations of T. rangeli (11,14,15).

Abad-Franch and Monteiro (16) and Abad-Franch, et al. (17), hypothesized that there are two major evolutionary lineages within the genus Rhodnius : the R. pictipes lineage, composed by Andean and Amazonian species (such as R. pallescens , R. pictipes, R. colombiensis and R. ecuadoriensis ), and the R. robustus lineage, composed by Amazonian species ( R. robustus sensu lato and R. prolixus ) and species that occur in other ecoregions, such as the caatinga ( R. nasutus ), cerrado ( R. neglectus ), and Atlantic forest ( R. domesticus ). It has been demonstrated that T. rangeli strains isolated from species of the R. pictipes lineage (KP1-), are genetically divergent to those isolated from the R. robustus lineage (KP1+), indicating possible differences in the susceptibility of vectors to different genotypes of T. rangeli (11). Such association agrees with the report of a trypanolytic protein present in R. prolixus hemolymph, which selectively lyses (KP1-) strains isolated from R. pallescens , R. colombiensis and R. ecuadoriensis , but not (KP1+) strains (18). However, the susceptibility of the species that give the name to these evolutionary lineages ( R. pictipes and R. robustus ) has not yet been evaluated.

Arthropod-borne parasites have possibly co-evolved with their biological vectors, especially those that are hematophagous (19). This process is complex and poorly explored in trypanosomatids. Our work offers some insights into this matter. Based on the current hypothesis regarding T. rangeli and Rhodnius spp. co-evolution (11,14 ,15), we propose that infectivity (intestinal, hemolymphatic and glandular) and the transmission of the T. rangeli strain SC-58 (charac terized as KP1-) would be greater and more efficient in R. pictipes . Therefore, the goal of the present study was to analyze the susceptibility of four Rhodnius species ( R. robustus , R. neglectus , R. nasutus and R. pictipes ) to a Brazilian strain of T. rangeli (SC-58/KP1-).

Materials and methods

Insects and T. rangeli strain

Triatomines were obtained from colonies maintained at the Laboratório de Parasitologia Médica e Biologia de Vetores, Faculdade de Medicina, Universidade de Brasília. Specimens of R. neglectus were originally collected in Ituiutaba , in the state of Minas Gerais, R. nasutus in Sobral, state of Ceará, whereas the specimens of R. robustus were collected in Marabá, state of Pará (20). Rhodnius pictipes specimens were obtained from colonies maintained at the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Fiocruz, Rio de Janeiro, and originated from Barcarena, in the state of Pará.

Males and females of each species ( R. robustus , R. nasutus , R. neglectus and R. pictipes ) were housed together in order to obtain eggs (21). After oviposition, 50 eggs of each species were separated. The temperature and relative humidity in the insectary were monitored weekly.

Triatomines were fed by using 30-to-40-day-old Swiss albino mice weighing approximately 30 to 35 g. Mice were obtained from the Bioterium at the Universidade de Brasília. They were anesthetized intraperitoneally with ketamine 80 mg/kg + xylazine 10 mg/kg. Environmental conditions, management and animal care followed standards recommended by the Guide for the Care and Use of Laboratory Animals, and the ethical principles in animal experi ments suggested by the Ethics Committee on Animal Use (CEUA), Universidade de Brasília – Instituto de Ciências Biológicas (UnBDoC: 42003/2010).

The Brazilian T. rangeli strain SC-58 was used in the study. This strain was isolated by Steindel, et al. (22), from a wild rodent, Echimys dasythrix , in the state of Santa Catarina. The strain was maintained in culture medium blood agar at 25-28°C and with successive passages made every two weeks. According to Vallejo, et al. (23), this strain is characterized molecularly as KP1-.

Experimental infection

Forty specimens of R. robustus , R. pictipes , R. nasutus and R. neglectus were fed on blood infected with T. rangeli . For that purpose, 3 ml of a SC-58 culture (2.8 x 10 7 , mainly epimastigotes) were added to 5 ml of human blood in tubes with heparin, using an artificial feeder (24). At the end of the procedure, 30 nymphs at stages IV and V that engorged with blood were selected, placed individually in a suitable container and labeled.

After seven days of infection, the feces of triatomines were examined microscopically. The stools were obtained by gentle pressure on the last abdominal segments, and the material was diluted in saline solution and freshly examined at 400X magnifi cation. Negative bugs were examined again at 5 to 7 day intervals.

Hemolymphatic samples of triatomines were collected to determine the approximate period of hemocoel invasion by T. rangeli . Samples were obtained after a tarsal section of a single leg and they were freshly examined at 400X magnification (25).

Triatomines with positive hemolymph were placed to feed on albino mice 1 , 3, 5, 7 and 9 days after first diagnosis to determine the approximate period of salivary gland invasion by T. rangeli and transmission by bite (26). Fresh blood samples were obtained from tails of anesthetized mice. Samples were examined at 400X magnification. At the end of the experiment, mice were euthanized in a CO2 chamber with a concentration above 70%.

Forty-two days after the infective feeding, the salivary glands of the surviving triatomines were extracted and immediately examined to identify the presence of parasites (2). Then, the insects were isolated and subsequently dissected. Finally, their glands were stained with Giemsa after acid hydrolysis (27). The effects of parasitism (rejection of feeding, molting deformations, mortality) were recorded and quantified daily throughout the experiment.

To study the morphogenesis of T. rangeli in the intestine, hemolymph and salivary glands, each slide was stained with Giemsa 10% for 45 minutes. During the microscopic observation of the material, the flagellates that most represented the T. rangeli life cycle were photographed (Canon: PowerShot SD 700 IS).

Statistical analysis

Chi-square tests were performed to verify differ ences in the proportion of triatomines infected and not infected by T. rangeli among the four Rhodnius species studied, considering p <0 .05 as statistically significant. Proportions and confidence intervals ( lower and upper) of infected specimens per spe cies were estimated after 7, 21 and 35 days of experimental infection with T. rangeli . The propor tions were calculated using the method of Agresti and Coull (28). Separate analyses were conducted for intestinal, glandular and hemolymphatic infection. Moreover, the survival curves of Rhodnius species experimental groups were compared using the Kaplan-Meier method. Statistical significance of any differences observed was then assessed by the log rank test. Statistical analysis was performed using SPSS ® , v. 20.0 (IBM Corp., Armonk, NY, USA).

Results

Intestinal infection

After 7 days, 15 (50%) specimens of R. pictipes had a large number of epimastigotes in the feces (table 1). After 35 days, the proportion of infected specimens of R. pictipes differed from the proportions of the other species of Rhodnius that were analyzed ( p <0.05). R. pictipes showed a higher percentage of infected triatomines in a shorter period of time, as well as a gradual increase when compared with the three other species of Rhodnius (figure 1).

Hemolymphatic infection

All analyzed Rhodnius species showed parasites (epimastigotes and trypomastigotes) in their hemolymph. The low number of insects with hemolymph infection did not allow us to test for differences between the species. Parasitism was less frequent in the species of the R. robustus lineage (figure 1). Seven days after infection, R. nasutus and R. neglectus presented only one specimen each (3.3%) with flagellates in the hemolymph; and after 28 days, they presented a total of four (13.3%) positive specimens. Three specimens of R. robustus showed epimastigotes of T. rangeli in the hemolymph 42 days after infection (table 1, figure 2).

R. pictipes showed the highest rate of hemo lymphatic infection, with four specimens (13.3%) infected after 14 days, totaling seven specimens (23.3%) with infected hemolymph after 28 days (table 1, figure 1, figure 2).

Salivary gland infection and transmission by bite

In total, 25 transmission trials by bite were made with hemolymph-infected specimens. R. neglectus and R. robustus did not transmit T. rangeli after 11 trials. In contrast, two specimens of R. pictipes and one of R. nasutus succeeded in transmitting T. rangeli via biting after 2-3 days of hemolymphatic infection (table 2, figure 2).

Forty-two days after infection, we extracted the glands of six survivors; only one specimen of R. nasutus and two of R. pictipes showed glandular infection (table 2).

Mortality rates

High mortality rates were observed during the experiment (table 1). After 28 days, 90% of R. pictipes were dead, the highest rate among the four species. A Kaplan-Meier plot of survival is shown in figure 3. There was a significant difference in survival for the Rhodnius species analyzed (Log rank = 48.5, p <0.01 ); R. pictipes showed the lowest survival rate.

Discussion

The four Rhodnius species studied here were susceptible to infection with strain SC-58/KP1- of T. rangeli at different levels of outcomes following exposure. Clearly, the genetic profile of both the insect and the parasite must influence the final outcome of host-parasite interactions. R. pictipes showed higher rates of intestine, hemolymph and salivary gland infections. These observations play an important role in the transmission of T. rangeli . R. pictipes appears to offer a compatible biochemical environment for the successful development and reproduction of T. rangeli KP1-. Our data on intestinal infection of R. pictipes (76.7% in 14 days) were compared with those obtained by Cuba-Cuba (26), who, using a Peruvian strain of T. rangeli , obtained 83% of intestinal infections in R. ecuadoriensis 15 days after experimental infection.

Machado, et al. , (2), using the same strain as in the present paper (SC-58/KP1-), observed glandular infection in R. nasutus and R. neglectus , but to a lesser extent compared to the other strain (Choach í/ KP1 +), which developed much better in these species of the R. robustus lineage. We observed the same behavior when comparing the development of strain SC-58. The specimens of the R. robustus lineage had a lower percentage of infections in the hemolymph than those of R. pictipes . A plausible explanation for this is the immune response of the vector to the parasite. A strong response can reduce or restrict the vectorial capacity of some hosts. The cellular and or humoral components in the hemolymph, for instance, elicited an immune response that can effectively destroy the parasite. In contrast, a weakened response can enhance the ability to support parasite development (29).

The morphogenesis studied followed the pattern expected for Rhodnius spp. infections by T. rangeli (27,30), with the presence of intestinal epimastigotes, intracellularly dividing flagellates in hemocytes, extracellular epimastigotes and meta-cyclic trypomastigotes in the salivary glands, in the case of R. pictipes infection. The importance of epimastigote forms is well established in the lite rature suggesting distinct infectivity and metabolic pathways for two parasite morphotypes (long and short) within triatomine bugs. Infections with short epimastigotes of T. rangeli are able to mobilize the proteases stored in the fat body, whereas long epimastigotes in some way inhibit protease activities in the fat body (31). Short epimastigotes were more resistant to lysis and stimulated greater superoxide and prophenoloxidase responses than long form epimastigotes of T. rangeli (32,33). Moreover, the ecto-phosphatase activity of short forms was more sensitive than that found in the long form (34,35). Finally, long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels (36). In the present study, we did not characterize the epimastigote forms inoculated in Rhodnius specimens. Future studies selecting the forms inoculated during experimental infection should clarify the role of long/short epimastigotes on infectivity for Rhodnius specimens.

Isolates of T. rangeli from different geographical origins show variable behavior in different Rhodnius species; moreover, transmission by biting, as described, is virtually restricted to the local vector (3,37,38). The difference in susceptibility to the strains of T. rangeli among the species of Rhodnius reinforces the existence of a complex relationship between vector and parasite, and shows that the ability of T. rangeli to reach the hemolymph and the salivary glands of the insect is dependent on both the strain used and the triatomine species (2). This study shows for the first time that R. pictipes is susceptible to T. rangeli strain SC-58/KP1-, which was expected, considering previous hypotheses of parasite-vector coevolution (11,15).

According to Urrea, et al . (15), the fact that identical genotypes of T. rangeli were isolated in vectors of the same evolutionary lineage supports a possible coevolutionary association between T. rangeli and its vectors, which most likely means that these genotypes have the same biological, biochemical and molecular characteristics determining their association with the Rhodnius lineages.

Despite the presence of T. rangeli in the hemolymph of all four Rhodnius species in this study, only R. pictipes and R. nasutus showed glandular infection. Both species have been reported to be infected naturally. It appears that some triatomine species become infected with T. rangeli , which reaches the hemolymph, but never invade the salivary glands. These triatomine species would be permissive to the parasite but they are not able to transmit T. rangeli via biting as showed for Panstrongylus herreri (39). Evidence of glandular infection of R. nasutus by T. rangeli (SC-58/KP1-) had already been found by Machado, et al. (2), but with a smaller proportion compared to infection with the Choachí strain (KP1+).

With regard to establishing the approximate period in which the SC-58/KP1- strain of T. rangeli led to invasion of the salivary glands, our observations are quite similar to those described by Castaño, et al. (40), who studied the development of the Choachí strain of T. rangeli (KP1+) in R. prolixus , observing flagellates in the salivary glands starting only on the 10th day after infection; this infection pattern was also observed by Cuba-Cuba (26) in R. ecuadoriensis.

Pulido, et al. (18), demonstrated the existence of a trypanolytic protein, which acts against T. rangeli (KP1-) isolated from R. colombiensis , but not against T. rangeli (KP1+) isolated from R. prolixus . According to Vallejo, et al. (11), the lytic factor in R. prolixus hemolymph appears to act as a biological barrier. The occurrence of this protein in other species of the R. robustus lineage ( R. robustus, R. neglectus, R. nasutus ) may prevent the glandular development and transmission of T. rangeli (KP1-). This suggests that in their natural habitat, some Rhodnius species are biological filters for certain populations of parasites , confirming a close coevolutionary association between the two subpopulations of T. rangeli and the two main Rhodnius lineages. In addition, the interactions between T. rangeli and the triatomine immune system or between T. rangeli and symbionts in triatomine intestines may be determinant factors for parasite survival in a particular vector (11).

T. rangeli is considered to be pathogenic to its invertebrate hosts, being responsible for triato mine death (1,6,41,42 ). It is known that several aspects of the physiology of the triatomines are altered during infection by T. rangeli , including reduced immune responses, anti-hemostatic activity of the salivary glands, and behavioral changes such as molting delays, changes in the movements of the insects, decreased reproductive performance and increased mortality (29,43,44). However, the mortality rates in our study were much higher than those observed in other studies analyzing T. rangeli experimental infection (2,25). This may be due to the manipulation of the insects during the experiment by their tarsal sections to obtain hemolymphatic samples. Machado, et al. (2), also showed that the manipulation of insects in experiments can be harmful to the triatomines, and therefore have an influence on the observed mortality rates.

The infectivity of strain SC-58 of T. rangeli (KP1-) was higher in R. pictipes compared with the species of the R. robustus lineage analyzed ( R. robustus, R. neglectus and R. nasutus ), confirming existing hypothesis of coevolution between T. rangeli and Rhodnius spp. (11,14,15,45). We may assume from these studies that R. pictipes is a biological vector of proven competence and vector capacity. Future experimental infections comparing the susceptibility of these species of Rhodnius to the population of T. rangeli KP1 + may further clarify the role of these triatomines in the transmission of these protozoans in nature.

Acknowledgements

Our thanks to Dr. Cleber Galvão of the National Reference Laboratory on Taxonomy of Triatominae, at Fiocruz, in Rio de Janeiro, for sending the R. pictipes ; to technicians Shigueru Ofugi and Walcymar P. Santiago, at the Chagas Disease Laboratory at the University of Brasilia (NMT-UnB), for their support for utilization of the artificial feeding apparatus, and to Dr. Fernando Abad-Franch and Dr. Gustavo A. Vallejo for insightful comments on the manuscript.

Conflicts of interest

The authors have no conflicts of interest to declare.

Financing

The research was partially funded by the   National Council for Scientific and Technological Develop ment of Brazil (CNPq).

Corresponding author: Daniella Barreto-Santana, Laboratório de Parasitologia Médica e Biologia de Vetores, Faculdade de Medicina, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, 70910-900 Brasília, DF, Brasil Telephone: (5561) 3107 1786; fax: (5561) 3273 3907 daniellabaag@hotmail.com

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