48 Journal of Vector Ecology June 2016

Vector capacity of members of brasiliensis species complex: The need to extend Chagas disease surveillance to Triatoma melanica

Elaine Folly-Ramos1*, L. Lynnette Dornak2, Guilherme Orsolon3, Teresa Cristina Monte Gonçalves4, Mauricio Lilioso1, Jane Costa5, and Carlos Eduardo Almeida1

1Laboratório Ecologia , Programa de Pós-Graduação em Ecologia e Monitoramento Ambiental - PPGEMA, Universidade Federal da Paraíba-UFPB, [email protected] 2Department of Geography and Geology, University of Wisconsin-Platteville, WI, U.S.A. 3Centro Federal de Educação Tecnológica Celso Suckow da Fonseca - CEFET/RJ, Campus Valença 4Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e , Instituto Oswaldo Cruz, IOC/ FIOCRUZ, Rio de Janeiro, RJ, 5Laboratório de Biodiversidade Entomológica, IOC/FIOCRUZ-RJ

Received 18 August 2015; Accepted 10 October 2015

ABSTRACT: We conducted a lab-based comparative study on vector capacity features of two species of triatomines: Triatoma brasiliensis and T. melanica. Both are members of the T. brasiliensis species complex. The former is the most important Chagas disease vector in the northeastern region of Brazil. To date, no transmission via T. melanica has been recorded. Immature exhibited distinct intermoult periods without a direct relationship to a given species. Females of T. brasiliensis consumed an average of 1.9 times more meals (mean = 12.92 vs 6.63) and survived for a shorter period (mean =330.8 days) than T. melanica (mean = 365.2 days), probably due to the cost of reproduction (all significant at P<0.05). These data support the idea thatT. brasiliensis is more adapted to lab conditions and is more able to infest domiciles than T. melanica. We also found significant distinctions in other features between these species, such as the elapsed time without eating before molting, which was higher for the second, third, and fifth nymph stages ofT. melanica. Regarding features analyzed related to vector capacity, insects of all life stages of both species were considered competent to transmit because they needed many feedings (mean =1.5-3.82) to moult and because a high proportion (>39%) of insects defecated rapidly (<30 s) after feeding. Overall, results highlight the need to extend vector surveillance to T. melanica. Journal of Vector Ecology 41 (1): 48-54. 2016.

Keyword Index: Vector potential, , comparative bionomics, eco-epidemiology, behavior.

INTRODUCTION distinct subspecies: T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis, T. melanica and T. sherlocki (Costa et al. 2013). Taxa Chagas disease is caused by a flagellated protozoan parasite, within this group are distinctively important epidemiologically, Trypanosoma cruzi, and is transmitted to humans mainly by blood- morphological characteristics, natural history traits, ecological sucking true bugs, the triatomines. These insects are the primary requirements, genetic characteristics, and dispersion abilities vectors from which humans contract the parasite, and thus, it is (Almeida et al. 2009, 2012, Costa et al., 2002, Monteiro et al. plausible that triatomine elimination from domiciles may interrupt 2004). Recently, Gardim et al. (2014) highlighted the non- the epidemiological chain of Chagas disease transmission and monophyly for the T. brasiliensis subcomplex defined by Schofield reduce overall infection rates. More than eight million people in and Galvão (2009) and, additionally, Alevi et al. (2014) suggested Latin America, most of whom are from rural populations, are still that T. lenti may be the newest member of this complex, although at risk of contracting T. cruzi, placing this illness among the most its position has not yet been evaluated fully save for comparisons serious parasitic diseases in the hemisphere. of morphology and cytogenetics (Panzera et al. 2000 Alevi et al. Considerable advances have been made to avoid vector 2012). transmission in Brazil, including use of insecticide treatments The focus of this study is on two allopatric members of for domiciles. However, Abad-Franch et al. (2013) noted that this species complex, Triatoma b. brasiliensis (hereafter referred the elimination of the non-native species Triatoma infestans to as T. brasiliensis) and T. melanica. Triatoma brasiliensis from Brazil by the late 1990s led health authorities to mistakenly is unquestionably the best-studied member of the complex, assume a total interruption of vector transmission. As a result of primarily because of its remarkable capacity to colonize domiciles the near-elimination of T. infestans, however, native triatomines, (Costa et al. 2003) compared to the lesser known T. melanica, previously considered exclusively sylvatic, have been found in a species that does not colonize domiciles (Costa et al. 2013). domiciles and are emerging as potential risks in several Brazilian These species also differ greatly in geographic range and in the regions (Almeida et al. 2000, Coutinho et al. 2014). ecoregion in which they are distributed: the extent of occurrence Members of T. brasiliensis species complex are the most of T. brasiliensis is approximately 1.6 × 106 km2 across five states important vectors in the semi-arid, northeastern region of Brazil. (Maranhão, Piauí, Ceará, , and Paraíba) and This group comprises four species, one of which includes two within the Caatinga biome. Triatoma melanica is confined to an Vol. 41, no. 1 Journal of Vector Ecology 49 area of roughly 1.1 × 106 km2 in the northwestern states of Minas for each instar; (6) voluntary starvation post-ecdysis/hatching Gerais and Bahia and within the cerrado biome (Costa et al. 2014). (i.e., the period in days that bugs take to accept the first blood Several bionomic characteristics can be related to vector meal); (7) voluntary starvation pre-ecdysis (i.e., the time taken in capacity. Those most commonly evaluated in lab conditions are days between the feeding and the next molting); and (8) elapsed (i) time required to reach adulthood, as the shorter the life cycle, time (in h:m:s) for defecation after the first blood meal. For this the more insects that can be produced and thus the greater the last parameter, the end of the blood meal was recorded when the potential for home infestation; (ii) the more feedings, the greater rostrum was retracted and all defecation during the blood meal the chance to become infected as well as to transmit T. cruzi; (iii) was considered as immediate, as well as defecations within 30 the shorter the elapsed time between feeding and defecation, the min. Following recommendations of Almeida et al. (2003) we greater the chance to defecate on the animal and thus to transmit fragmented information regarding elapsed time for defecation T. cruzi if the vector is infected (Almeida et al. 2003). after the blood meals in four time frames: i) immediately to 30 s, These bionomic features have been traditionally analyzed ii) from 31 s to 1 min, iii) from 1 min and 1 s to 1 min and 30 s, and for isolated species (Almeida et al. 2005, Luitgards-Moura et al. iv) over 1 min and 31 s. Features were calculated per evolutionary 2005) but not with an experimental comparison among closely- instar and the first to the fifth nymph stages were named as N1- related taxa, such as members of the same species complex. For N5. Adults were referred to by their gender. Mexican species, a set of similar studies was conducted on Meccus Means of each parameter were compared between species phyllosomus complex (Martínez-Ibarra et al. 2007, 2012). Few with Student’s t-test, using the software Systat® version 11.0. To efforts have attempted to explain the interspecific variability on check whether each pattern followed a normal distribution, we bionomic parameters for closely related Brazilian triatomines. used the Shapiro-Wilk normality test. Differences were considered Hence, we chose T. brasiliensis and T. melanica for this comparative statistically significant at P<0.05, and Bonferoni adjustments were analysis on bionomic characteristics regarding vector capacity made to account for multiple comparisons. because these species exhibit considerable ecological and genetic variation (Costa et al. 1997, 2002, Monteiro et al. 2004, Gardim et RESULTS al. 2014). In the fifth nymphal stage, the intermolt period was MATERIALS AND METHODS significantly higher for T. melanica than for T. brasiliensis (58.2 vs 40.4 days, P<0.001). Intermolt periods were clearly prolonged by Triatomines used for this study came from colonies kept the diapause of a few specimens of a given nymphal stage. It can be in the Entomological Collection of Oswaldo Cruz Institute. illustrated by N5 of T. melanica, of which only three (not shown) Colonies were founded by 73 and 75 samples collected in the of the 38 samples spent more than 110 days in this stage. There municipality of Caicó-RN (6°27ʹ30˝S, 37°05ʹ52˝W) and Espinosa- was no significant difference in the period since hatching until the MG (14°55ʹ34˝S; 42°49ʹ09˝W) for T. brasiliensis and T. melanica, imaginal molt between T. melanica (175 days) and T. brasiliensis respectively. Of the colonies, 40 nymphs of each species, which (153.6 days), which may allow both species to produce more than hatched from the eggs on the same day, were randomly separated. two generations per year. The mean observed for female longevity Specimens used in these experiments were the first generation was significantly higher for T. melanica, which survived 35 more from colonies kept under the same environmental conditions days, than for T. brasiliensis (Table 1). For males, however, there and food source (anesthetized Swiss mice, according to Bioethics was no significant difference between species, which survived for License for Laboratory Animal Care and Use - LW-18/11). periods between 338.4 for T. melanica and 343.5 for T. brasiliensis Immediately post-hatch, nymphs of T. brasiliensis and T. (five days of difference). melanica were transferred to individual plastic containers (3x4x9 Males spent the shortest mean time (mean=00:19:05) feeding, cm), each with a perforated lid. All containers had pieces of folded whereas some nymph stages (N4 and N5) spent more than an filter paper inside to increase the interior surface area and to hour feeding. Except for T. brasiliensis N1, an ascendant curve was reduce humidity generated from excrement. The experiment was observed for nymph stages from N1 to N5, as expected due to an conducted in the Centro Universitário de Barra Mansa (UBM), increasing of body mass. There were no significant differences in Rio de Janeiro, Brazil under laboratory conditions: relative feeding period between species in any of the evolutionary stages. humidity (mean 77.6%; range 53 to 96%), and temperature at 9 There was no significant variation in the mean number of h (mean 26.2° C; range 23° to 29° C) and at 15h (mean 28.2° C; feedings between T. melanica (Tm) and T. brasiliensis (Tb) in range 24° to 30° C). Bugs were observed daily and blood meals N1 (Tm= 2.08 and Tb =2.13), N2 (Tm= 1.50 and Tb=1.85), N4 (Mus musculus) were offered individually twice a week during the (Tm= 2.05 and Tb=2.25), and males (Tm= 5.95 and Tb=6.18). whole life cycle, during which all insects were maintained under However, N3 of T. brasiliensis exhibited 2.46 times (mean= identical conditions. 3.82) more feedings than T. melanica (mean=1.55, P<0.001). An The bionomic parameters analyzed were adapted from inverse relationship was observed for N5, in which T. melanica previous studies (Almeida et al. 2003): (1) intermolting period fed more often (mean= 3.47) than T. brasiliensis (2.32, P<0.001). (i.e., the elapsed time in days between hatching/moulting and the Females of T. brasiliensis engaged almost twice as often in feeding next moult); (2) maturity is the developmental time (i.e., period (mean=12.92) than T. melanica (mean=6.63, P<0.001; Table 2). in days from hatching until the imaginal molt); (3) longevity (i.e., Meal acceptance after ecdysis (post-ecdysis fasting) of N3 of the period from hatching until death); (4) number of feedings; T. brasiliensis was significantly (P=0.03) more rapid (mean= 4.69 (5) duration of blood meal in hours: minutes: seconds (h:m:s) days) than acceptance by N3 individuals of T. melanica (mean= 50 Journal of Vector Ecology June 2016

7.05 days). On the contrary, N4 of T. melanica accepted meals more promptly (mean=2.76 days) than N4 of T. brasiliensis (4.06 11 Tb 365 281 ±35.1 12356

330.82 days, P=0.02). Longer fasting periods before molting (voluntary fasting pre-ecdysis) were observed for N2 (mean=18.05 vs 15.79 14.8 -2.93 0.011

Female days), N3 (mean=17.82 vs 15.66 days), and N5 (mean=28.92 vs 15 Tm 365 391 393 313 ±19. 21.83 days) of T. melanica than individuals of the same stages in T. brasiliensis (for all comparisons P≤0.02, Table 3). By considering immediate defecation as t<30 s, overall, 19 Tb

390 251 results showed that high proportions (all ≥ 39%) of bugs for both 1433 343.5 ±37.8 Longevity (days) species and sexes defecate within a time to allow them to transmit 41.1 Male 0.872 T. cruzi. Among nymph stages, N3 of T. melanica had the highest -0.0161 23 40 proportion (67%) of immediate defecation, as the first nymphal Tm 393 ±86 7404 338.4 stages were the ones lowest for both species (39-41%). Higher proportions (Tm=65% and Tb=68%) of females for both species defecated faster than males (both 50%). No statistical treatment 29 Tb 319 224 131 27.5 153.6 ±17.8 could be applied to test groups for this feature (Figure 1). 62.2 0.174 Final -1.375 DISCUSSION 38 5.0 Tm 175 480 230 148 ±22 Maturity (days) Maturity Bionomic parameters of Chagas disease vectors have provided valuable information regarding vector capacity to transmit T. cruzi 30 90 29 Tb 139 ±11 40.4 25.0 (Zeledón et al. 1977). Due to the epidemiological importance of T. brasiliensis, these features have been extensively studied for this N5 65.1 -3.773 <0.001 isolated species. Because T. brasiliensis and T. pseudomaculata 38 37 5.0 Tm 336 114 ±18 58.2 overlap in geographic distribution and epidemiological importance, comparative studies between them have already been conducted (Guarneri et al. 2000, Soares et al. 2000). However ). Significant values are in bold. are values ( Tb ). Significant brasiliensis T. ) and 32 45 21 ±4 Tb they are not genetically related (Gardim et al. 2014), belonging 26.8 20.0 22.4 to distinct species complexes. Within the T. brasiliensis species ( Tm N4 67.1 0.875 0.386 complex, Costa and Marchon-Silva (1998) and Almeida et al. (2012) have demonstrated distinctions among species as regards 38 32 21 ±2 5.0 7.3 Tm 27.1 bionomic and behavior factors, despite the capacity to breed in lab-conditions (Correia et al. 2013). Indeed, Costa and Marchon- Silva (1998) and Almeida et al. (2012) were not focused on 32 35 20 ±3 Tb 9.3 26.3 20.0 parameters related to vector capacity such as those explored

N3 in this study. Overall, the results of this study highlighted some 67.1 -0.84 0.409 bionomic distinctions between T. brasiliensis and T. melanica, 38 35 15 ±3 5.0 Tm 27.3 14.7 including those related to vector capacity. The findings support By nymphal stage nymphal By previous studies that showed pronounced differentiation among

Intermolting period (days) Intermolting two members of the T. brasiliensis species complex, T. brasiliensis 34 96 17 Tb 160 ±13 31.2 15.0 and T. melanica (Costa et al. 1997, Monteiro et al. 2004). Regarding life span, both species are able to produce more N2 47.7 0.583 0.563 than two annual lab-generations, similar to other triatomines 38 20 40 21 ±5 5.0 Tm recognized for the high capacity to infest homes, such as T. 29.5 infestans (Perlowagora-Szumlewicz 1969). It is known that triatomines are able to molt with a single feeding and sometimes 38 46 28 even without feeding (Lent and Wygodzinsky 1979, Perlowagora- ±5 Tb 5.0 32.3 27.6 Szumlewicz 1969). Results for both species indicated that they N1

55.1 need a minimum average of 1.5 feedings to molt, reaching a 0.080 2.952 maximum average of 3.82 feedings, both for N3 of T. melanica 39 34 60 26 2.5 Tm 89.5 ±9.5 and T. brasiliensis, respectively. Total number of feedings (all ≥11) to reach adulthood for both species was similar to values P T df obtained for T. pseudomaculata and T. rubrovaria, both of which were considered competent to transmit T. cruzi by Almeida et al. (2005). Because females of T. brasiliensis fed at a rate 1.5 times

higher than T. melanica but survived for a significantly shorter n SD Var Min Max t-test Mean period, these results might reflect the cost of reproduction, and % mortality Two-sample Two-sample consequently the capacity to infest domiciles, although this feature Triatoma melanica Triatoma of stage in each life longevity and period, maturity, 1. Intermolt Table Maturity p= significance. freedom; of df = degree = minimum; Min = maximum; Max = variance; Var deviation; = standard SD stages; N = nymphal specimens; of n = number death. until the period to hatching from refers longevity and the moult) imaginal until hatching period from time (i.e., in days the developmental is Vol. 41, no. 1 Journal of Vector Ecology 51

Table 2. Number of feedings in each life stage of Triatoma melanica (Tm) and T. brasiliensis (Tb). Significant values are in bold.

N1 N2 N3 N4 N5 Male Female Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb n 39 38 38 34 38 32 38 32 38 30 23 18 15 11 Mean 2.08 2.13 1.50 1.85 1.55 3.82 2.05 2.25 3.47 2.32 5.95 6.18 6.63 12.92 Number SD± 0.42 0.41 0.56 0.74 0.65 0.63 0.61 1.22 1.11 0.83 2.94 1.24 1.89 1.62 of blood Var 0.17 0.17 0.31 0.55 0.42 0.39 0.38 1.48 1.23 0.69 8.62 1.53 3.58 2.63 meals Max 4 3 3 5 3 5 3 8 7 5 12 8 10 15 Min 1 1 1 1 1 2 1 1 2 1 1 4 4 10 Two- T 0.573 1.499 18.851 0.832 -4.923 0.32 9.453 sample df 77 60.3 36.9 43.9 66.7 29.7 25.5 t-test P 0.569 0.139 <0.001 0.410 <0.001 0.751 <0.001 n = number of specimens; N = nymphal stages; SD = standard deviation; Var = variance; Max = maximum; Min = minimum; df = degree of freedom; p= level of significance.

Table 3. Post- and pre-ecdysis/hatching voluntary starvation in each life stage of Triatoma melanica (Tm) and T. brasiliensis (Tb). Significant values are in bold.

N1 N2 N3 N4 N5 Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb N 39 38 38 34 38 32 38 32 38 30 Mean 9.41 9.58 7.97 8.29 7.05 4.69 2.76 4.06 3.89 5.20 SD± 4.78 4.43 4.06 4.31 5.52 3.16 2.22 2.32 2.45 3.32 Var 22.88 19.66 16.46 18.58 30.44 10.01 4.94 5.40 5.99 10.99 Fasting post- Max 20 21 16 21 32 14 9 10 12 14 ecdysis period Min 6 7 1 2 1 2 1 2 2 1 (days) T 0.161 0.324 -2.188 2.36 1.8 Two-sample t-test df 74.8 68 59.2 63 52.1 P 0.873 0.747 0.033 0.021 0.078 Mean 14.82 13.53 18.05 15.79 17.82 15.66 16.42 15.34 28.92 21.83 SD± 4.44 2.75 4.33 4.00 4.19 3.20 3.38 3.41 8.62 6.91 Var 19.68 7.55 18.75 15.99 17.56 10.23 11.44 11.65 74.29 47.80 Fasting pre- Max 40 18 28 21 34 22 21 22 58 39 ecdysis period (days) Min 12 4 12 4 12 11 9 10 13 4 T -1.543 -2.301 -2442 -1.321 -3.763 Two-sample df 63.7 69.9 67.4 65.8 66 t-test P 0.128 0.024 0.017 0.191 <0.001 n = number of specimens; N = nymphal stages; SD = standard deviation; Var = variance; Max = maximum; Min = minimum; df = degree of freedom; p= level of significance. 52 Journal of Vector Ecology June 2016 % of insects that defecated after feeding after defecated % that of insects

Figure 1. Distribution of percentage of insects that defecated in four elapsed frame-periods after the first feeding: 1- immediately to 30 s, 2- from 31 s to 1 min, 3- from 1 min and 01 s to 1 min and 30 s, and 4- over 1 min and 31 s Tm = Triatoma melanica and Tb = T. brasiliensis. was not been fully explored in this study. It is clearly easier to keep between moults, providing evidence for bionomic distinctions productive colonies of T. brasiliensis under lab conditions than T. between these two species. melanica (J. Costa and C.E. Almeida, unpublished data.). Usually, Some triatomine species do not defecate within a time that colonies of this last species begin to decline after the fourth lab allows them to transmit T. cruzi, as is with T. vitticeps, and most generation. It may also be inferred in an epidemiologic context may take more than 10 min to defecate after feedings (Santos et that T. brasiliensis could present higher plasticity, easily adapting al. 2006). Almeida et al. (2003, 2005) stated that in the 30 s after to an artificial environment, such in the lab and domiciles (Costa feeding ceases, insects might not be in contact with the host et al. 2003). anymore, because they may have retreated to shelter. Because According to Menu et al. (2010), a general characteristic of high proportions of insects for both species in the current study triatomines is that a portion of a given population will have longer defecated within this post-feeding period, we conclude that each intermolt periods, which serves as an adaptive strategy for species is competent to transmit T. cruzi regarding this feature. Following maintenance to avoid synchronization of intermolt periods Almeida et al. (2003, 2005) recommendations, we fragmented the when environmental conditions become unfavorable (Seger and elapsed time for defecation after blood meals in four time frames. Brockmann 1987). This diapause may have been responsible for This method was suitable for the members of species complex variation between N-stages and within each species. For example, in this study, which based on Soares et al. (2000) were expected a few specimens of T. brasiliensis N5 took a long period (more to be able to defecate rapidly after feedings. However, statistical than 110 days) to molt. This may have also influenced results for comparisons were not possible. Indeed, this comparison would the number of feedings, because prolonged stagnation in a given be unnecessary because the main question in the epidemiological stage consequently results in more feedings. context was answered: both were considered competent to Some features we evaluated are not directly related to vector transmit T. cruzi regarding this feature for all life stages. Among capacity but contribute to basic knowledge of the biological the bionomic features, the time taken to defecate after feeding is system. The voluntary time taken to accept the first blood meal considered one of the most important factors regarding vector (post-hatching/ecdysis) in each stage could be related to voracity capacity (Crocco and Catalá 1996). and also to the adaptability to laboratory conditions. Indeed, this The transitory nature of epidemiological importance in characteristic did not differ significantly between the species. triatomines can be illustrated by the case of T. sherlocki, a member Conversely, the time taken before molting was significantly of T. brasiliensis species complex originally considered exclusively greater for T. melanica nymph stages (N2, N3, and N5) than sylvatic. This species was found infesting human domiciles in a T. brasiliensis. The effect of this parameter has yet to be fully recently colonized mining community (Almeida et al. 2009), explained (Martínez-Ibarra et al. 2012). However, it might be highlighting the rapid changes that can occur in triatomine inferred that the previous blood meals taken by T. melanica might ecology in the face of anthropogenic environmental changes. have been sufficient to allow for longer periods without feeding Despite the fact that T. melanica is still considered exclusively Vol. 41, no. 1 Journal of Vector Ecology 53 sylvatic, and because it was also considered competent to transmit Rosa, and J. Costa. 2013. Cross-mating experiments detect T. cruzi based on features explored herein, we highlight the need reproductive compatibility between Triatoma sherlocki and to extend surveillance to this vector. Finally, we reinforce previous other members of the Triatoma brasiliensis species complex. findings (Costa and Lorenzo 2009, Rebaudo et al. 2014) that T. Acta Trop. 128: 162-167. brasiliensis complex provides a suitable model to evaluate a series Costa, J. and M. Lorenzo. 2009. Biology, diversity and strategies of phenomenon on speciation and species differentiation. for the monitoring and control of triatomines-Chagas disease vectors. Mem. Inst. Oswaldo Cruz 1: 46-51. Acknowledgments Costa, J. and V. Marchon-Silva. 1998. Período de Intermuda e Resistência ao jejum de Diferentes PopulaçÕes de We are grateful to the vector control and surveillance staff of Triatoma brasiliensis Neiva, 1911 (Hemiptera, , the municipalities of Caicó, RN and Espinosa, MG, Brazil for the Triatominae). Entomol. Vect. 5: 23-34. technician support for collecting insects. This study is in honor Costa, J., C. Almeida, E. Dotson, A. Lins, M. Vinhaes, A. Silveira, of Prof. Élio Gouvea (in memoriam), who idealized and created and C. Beard. 2003. The epidemiologic importance of the Museu de Ciências-UBM. This study was funded by the Triatoma brasiliensis as a Chagas disease vector in Brazil: a following programs: the Conselho Nacional de Desenvolvimento revision of domiciliary captures during 1993-1999. Mem. Científico e Tecnológico (CNPq) for DCR fellowships, Inst. Oswaldo Cruz 98: 443-449. PPGEMA, Universidade Federal da Paraíba-UFPB supported by Costa, J., N.C. Correia, V.L. Neiva, T.C. Gonçalves, and M. Felix. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 2013. Revalidation and redescription of Triatoma brasiliensis (Capes), Fundação de Amparo à Pesquisa do Estado de São Paulo macromelasoma Galvão, 1956 and an identification key for (FAPESP, process numbers 2010/17027-0 and 2011/22378-0), and the Triatoma brasiliensis complex (Hemiptera: Reduviidae: the Centro Universitário de Barra Mansa, RJ, Brazil. Triatominae). Mem. Inst. Oswaldo Cruz 108: 785-789. Costa, J., L. Dornak, C. Almeida, and A. Peterson. 2014. 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