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

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1 48 Journal of Vector Ecology June 2016 Vector capacity of members of Triatoma brasiliensis species complex: The need to extend Chagas disease surveillance to Triatoma melanica Elaine Folly-Ramos 1*, L. Lynnette Dornak 2, Guilherme Orsolon 3, Teresa Cristina Monte Gonçalves 4, Mauricio Lilioso 1, Jane Costa 5, and Carlos Eduardo Almeida 1 1 Laboratório Ecologia Animal, Programa de Pós-Graduação em Ecologia e Monitoramento Ambiental - PPGEMA, Universidade Federal da Paraíba-UFPB, elafolly@yahoo.com.br 2 Department of Geography and Geology, University of Wisconsin-Platteville, WI, U.S.A. 3 Centro Federal de Educação Tecnológica Celso Suckow da Fonseca - CEFET/RJ, Campus Valença 4 Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera, Instituto Oswaldo Cruz, IOC/ FIOCRUZ, Rio de Janeiro, RJ, Brazil 5 Laboratório 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 insects 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 = vs 6.63) and survived for a shorter period (mean =330.8 days) than T. melanica (mean = days), probably due to the cost of reproduction (all significant at P<0.05). These data support the idea that T. 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 of T. melanica. Regarding features analyzed related to vector capacity, insects of all life stages of both species were considered competent to transmit Trypanosoma cruzi because they needed many feedings (mean = ) 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): Keyword Index: Vector potential, Triatominae, comparative bionomics, eco-epidemiology, behavior. INTRODUCTION Chagas disease is caused by a flagellated protozoan parasite, Trypanosoma cruzi, and is transmitted to humans mainly by bloodsucking true bugs, the triatomines. These insects are the primary vectors from which humans contract the parasite, and thus, it is plausible that triatomine elimination from domiciles may interrupt the epidemiological chain of Chagas disease transmission and reduce overall infection rates. More than eight million people in Latin America, most of whom are from rural populations, are still at risk of contracting T. cruzi, placing this illness among the most serious parasitic diseases in the hemisphere. Considerable advances have been made to avoid vector transmission in Brazil, including use of insecticide treatments for domiciles. However, Abad-Franch et al. (2013) noted that the elimination of the non-native species Triatoma infestans from Brazil by the late 1990s led health authorities to mistakenly assume a total interruption of vector transmission. As a result of the near-elimination of T. infestans, however, native triatomines, previously considered exclusively sylvatic, have been found in domiciles and are emerging as potential risks in several Brazilian regions (Almeida et al. 2000, Coutinho et al. 2014). Members of T. brasiliensis species complex are the most important vectors in the semi-arid, northeastern region of Brazil. This group comprises four species, one of which includes two distinct subspecies: T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis, T. melanica and T. sherlocki (Costa et al. 2013). Taxa within this group are distinctively important epidemiologically, morphological characteristics, natural history traits, ecological requirements, genetic characteristics, and dispersion abilities (Almeida et al. 2009, 2012, Costa et al., 2002, Monteiro et al. 2004). Recently, Gardim et al. (2014) highlighted the nonmonophyly for the T. brasiliensis subcomplex defined by Schofield and Galvão (2009) and, additionally, Alevi et al. (2014) suggested that T. lenti may be the newest member of this complex, although its position has not yet been evaluated fully save for comparisons of morphology and cytogenetics (Panzera et al Alevi et al. 2012). The focus of this study is on two allopatric members of this species complex, Triatoma b. brasiliensis (hereafter referred to as T. brasiliensis) and T. melanica. Triatoma brasiliensis is unquestionably the best-studied member of the complex, primarily because of its remarkable capacity to colonize domiciles (Costa et al. 2003) compared to the lesser known T. melanica, a species that does not colonize domiciles (Costa et al. 2013). These species also differ greatly in geographic range and in the ecoregion in which they are distributed: the extent of occurrence of T. brasiliensis is approximately km 2 across five states (Maranhão, Piauí, Ceará, Rio Grande do Norte, and Paraíba) and within the Caatinga biome. Triatoma melanica is confined to an

2 Vol. 41, no. 1 Journal of Vector Ecology 49 area of roughly km 2 in the northwestern states of Minas Gerais and Bahia and within the cerrado biome (Costa et al. 2014). Several bionomic characteristics can be related to vector capacity. Those most commonly evaluated in lab conditions are (i) time required to reach adulthood, as the shorter the life cycle, the more insects that can be produced and thus the greater the potential for home infestation; (ii) the more feedings, the greater the chance to become infected as well as to transmit T. cruzi; (iii) the shorter the elapsed time between feeding and defecation, the greater the chance to defecate on the animal and thus to transmit T. cruzi if the vector is infected (Almeida et al. 2003). These bionomic features have been traditionally analyzed for isolated species (Almeida et al. 2005, Luitgards-Moura et al. 2005) but not with an experimental comparison among closelyrelated taxa, such as members of the same species complex. For Mexican species, a set of similar studies was conducted on Meccus phyllosomus complex (Martínez-Ibarra et al. 2007, 2012). Few efforts have attempted to explain the interspecific variability on bionomic parameters for closely related Brazilian triatomines. Hence, we chose T. brasiliensis and T. melanica for this comparative analysis on bionomic characteristics regarding vector capacity because these species exhibit considerable ecological and genetic variation (Costa et al. 1997, 2002, Monteiro et al. 2004, Gardim et al. 2014). MATERIALS AND METHODS Triatomines used for this study came from colonies kept in the Entomological Collection of Oswaldo Cruz Institute. Colonies were founded by 73 and 75 samples collected in the municipality of Caicó-RN (6 27ʹ30 S, 37 05ʹ52 W) and Espinosa- MG (14 55ʹ34 S; 42 49ʹ09 W) for T. brasiliensis and T. melanica, respectively. Of the colonies, 40 nymphs of each species, which hatched from the eggs on the same day, were randomly separated. Specimens used in these experiments were the first generation from colonies kept under the same environmental conditions and food source (anesthetized Swiss mice, according to Bioethics License for Laboratory Animal Care and Use - LW-18/11). Immediately post-hatch, nymphs of T. brasiliensis and T. melanica were transferred to individual plastic containers (3x4x9 cm), each with a perforated lid. All containers had pieces of folded filter paper inside to increase the interior surface area and to reduce humidity generated from excrement. The experiment was conducted in the Centro Universitário de Barra Mansa (UBM), Rio de Janeiro, Brazil under laboratory conditions: relative humidity (mean 77.6%; range 53 to 96%), and temperature at 9 h (mean 26.2 C; range 23 to 29 C) and at 15h (mean 28.2 C; range 24 to 30 C). Bugs were observed daily and blood meals (Mus musculus) were offered individually twice a week during the whole life cycle, during which all insects were maintained under identical conditions. The bionomic parameters analyzed were adapted from previous studies (Almeida et al. 2003): (1) intermolting period (i.e., the elapsed time in days between hatching/moulting and the next moult); (2) maturity is the developmental time (i.e., period in days from hatching until the imaginal molt); (3) longevity (i.e., the period from hatching until death); (4) number of feedings; (5) duration of blood meal in hours: minutes: seconds (h:m:s) for each instar; (6) voluntary starvation post-ecdysis/hatching (i.e., the period in days that bugs take to accept the first blood meal); (7) voluntary starvation pre-ecdysis (i.e., the time taken in days between the feeding and the next molting); and (8) elapsed time (in h:m:s) for defecation after the first blood meal. For this last parameter, the end of the blood meal was recorded when the rostrum was retracted and all defecation during the blood meal was considered as immediate, as well as defecations within 30 min. Following recommendations of Almeida et al. (2003) we fragmented information regarding elapsed time for defecation after the blood meals in four time frames: i) immediately to 30 s, ii) from 31 s to 1 min, iii) from 1 min and 1 s to 1 min and 30 s, and iv) over 1 min and 31 s. Features were calculated per evolutionary instar and the first to the fifth nymph stages were named as N1- N5. Adults were referred to by their gender. Means of each parameter were compared between species with Student s t-test, using the software Systat version To check whether each pattern followed a normal distribution, we used the Shapiro-Wilk normality test. Differences were considered statistically significant at P<0.05, and Bonferoni adjustments were made to account for multiple comparisons. RESULTS In the fifth nymphal stage, the intermolt period was significantly higher for T. melanica than for T. brasiliensis (58.2 vs 40.4 days, P<0.001). Intermolt periods were clearly prolonged by the diapause of a few specimens of a given nymphal stage. It can be illustrated by N5 of T. melanica, of which only three (not shown) of the 38 samples spent more than 110 days in this stage. There was no significant difference in the period since hatching until the imaginal molt between T. melanica (175 days) and T. brasiliensis (153.6 days), which may allow both species to produce more than two generations per year. The mean observed for female longevity was significantly higher for T. melanica, which survived 35 more days, than for T. brasiliensis (Table 1). For males, however, there was no significant difference between species, which survived for periods between for T. melanica and for T. brasiliensis (five days of difference). Males spent the shortest mean time (mean=00:19:05) feeding, whereas some nymph stages (N4 and N5) spent more than an hour feeding. Except for T. brasiliensis N1, an ascendant curve was observed for nymph stages from N1 to N5, as expected due to an increasing of body mass. There were no significant differences in feeding period between species in any of the evolutionary stages. There was no significant variation in the mean number of feedings between T. melanica (Tm) and T. brasiliensis (Tb) in N1 (Tm= 2.08 and Tb =2.13), N2 (Tm= 1.50 and Tb=1.85), N4 (Tm= 2.05 and Tb=2.25), and males (Tm= 5.95 and Tb=6.18). However, N3 of T. brasiliensis exhibited 2.46 times (mean= 3.82) more feedings than T. melanica (mean=1.55, P<0.001). An inverse relationship was observed for N5, in which T. melanica fed more often (mean= 3.47) than T. brasiliensis (2.32, P<0.001). Females of T. brasiliensis engaged almost twice as often in feeding (mean=12.92) than T. melanica (mean=6.63, P<0.001; Table 2). Meal acceptance after ecdysis (post-ecdysis fasting) of N3 of T. brasiliensis was significantly (P=0.03) more rapid (mean= 4.69 days) than acceptance by N3 individuals of T. melanica (mean=

3 50 Journal of Vector Ecology June 2016 Table 1. Intermolt period, maturity, and longevity in each life stage of Triatoma melanica (Tm) and T. brasiliensis (Tb). Significant values are in bold. Intermolting period (days) Maturity (days) Longevity (days) N1 N2 N3 N4 N5 Male Female Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb n Mean SD ±9.5 ±5 ±5 ±13 ±3 ±3 ±2 ±4 ±18 ±11 ±22 ±17.8 ±86 ±37.8 ±19. ±35.1 Var Max Min T df Two-sample t-test P < By nymphal stage Final % mortality n = number of specimens; N = nymphal stages; SD = standard deviation; Var = variance; Max = maximum; Min = minimum; df = degree of freedom; p= significance. Maturity is the developmental time (i.e., period in days from hatching until the imaginal moult) and longevity refers to the period from hatching until death days). On the contrary, N4 of T. melanica accepted meals more promptly (mean=2.76 days) than N4 of T. brasiliensis (4.06 days, P=0.02). Longer fasting periods before molting (voluntary fasting pre-ecdysis) were observed for N2 (mean=18.05 vs days), N3 (mean=17.82 vs days), and N5 (mean=28.92 vs 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, results showed that high proportions (all 39%) of bugs for both species and sexes defecate within a time to allow them to transmit T. cruzi. Among nymph stages, N3 of T. melanica had the highest proportion (67%) of immediate defecation, as the first nymphal 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 could be applied to test groups for this feature (Figure 1). DISCUSSION Bionomic parameters of Chagas disease vectors have provided valuable information regarding vector capacity to transmit T. cruzi (Zeledón et al. 1977). Due to the epidemiological importance of T. brasiliensis, these features have been extensively studied for this isolated species. Because T. brasiliensis and T. pseudomaculata overlap in geographic distribution and epidemiological importance, comparative studies between them have already been conducted (Guarneri et al. 2000, Soares et al. 2000). However they are not genetically related (Gardim et al. 2014), belonging to distinct species complexes. Within the T. brasiliensis species complex, Costa and Marchon-Silva (1998) and Almeida et al. (2012) have demonstrated distinctions among species as regards 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 parameters related to vector capacity such as those explored in this study. Overall, the results of this study highlighted some bionomic distinctions between T. brasiliensis and T. melanica, including those related to vector capacity. The findings support previous studies that showed pronounced differentiation among two members of the T. brasiliensis species complex, T. brasiliensis and T. melanica (Costa et al. 1997, Monteiro et al. 2004). Regarding life span, both species are able to produce more than two annual lab-generations, similar to other triatomines recognized for the high capacity to infest homes, such as T. infestans (Perlowagora-Szumlewicz 1969). It is known that triatomines are able to molt with a single feeding and sometimes even without feeding (Lent and Wygodzinsky 1979, Perlowagora- Szumlewicz 1969). Results for both species indicated that they need a minimum average of 1.5 feedings to molt, reaching a maximum average of 3.82 feedings, both for N3 of T. melanica and T. brasiliensis, respectively. Total number of feedings (all 11) to reach adulthood for both species was similar to values 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 period, these results might reflect the cost of reproduction, and consequently the capacity to infest domiciles, although this feature

4 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. Number of blood meals N1 N2 N3 N4 N5 Male Female Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb n Mean SD± Var Max Min Twosample t-test T df P < < <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. Fasting postecdysis period (days) Fasting preecdysis period (days) N1 N2 N3 N4 N5 Tm Tb Tm Tb Tm Tb Tm Tb Tm Tb N Mean SD± Var Max Min T Two-sample t-test df P Mean SD± Var Max Min T Two-sample df t-test P <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.

5 52 Journal of Vector Ecology June 2016 % of insects that defecated after feeding 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 productive colonies of T. brasiliensis under lab conditions than T. melanica (J. Costa and C.E. Almeida, unpublished data.). Usually, colonies of this last species begin to decline after the fourth lab generation. It may also be inferred in an epidemiologic context that T. brasiliensis could present higher plasticity, easily adapting to an artificial environment, such in the lab and domiciles (Costa et al. 2003). According to Menu et al. (2010), a general characteristic of triatomines is that a portion of a given population will have longer intermolt periods, which serves as an adaptive strategy for species maintenance to avoid synchronization of intermolt periods when environmental conditions become unfavorable (Seger and Brockmann 1987). This diapause may have been responsible for variation between N-stages and within each species. For example, a few specimens of T. brasiliensis N5 took a long period (more than 110 days) to molt. This may have also influenced results for the number of feedings, because prolonged stagnation in a given stage consequently results in more feedings. Some features we evaluated are not directly related to vector capacity but contribute to basic knowledge of the biological system. The voluntary time taken to accept the first blood meal (post-hatching/ecdysis) in each stage could be related to voracity and also to the adaptability to laboratory conditions. Indeed, this characteristic did not differ significantly between the species. Conversely, the time taken before molting was significantly greater for T. melanica nymph stages (N2, N3, and N5) than T. brasiliensis. The effect of this parameter has yet to be fully explained (Martínez-Ibarra et al. 2012). However, it might be inferred that the previous blood meals taken by T. melanica might have been sufficient to allow for longer periods without feeding between moults, providing evidence for bionomic distinctions between these two species. Some triatomine species do not defecate within a time that allows them to transmit T. cruzi, as is with T. vitticeps, and most may take more than 10 min to defecate after feedings (Santos et al. 2006). Almeida et al. (2003, 2005) stated that in the 30 s after feeding ceases, insects might not be in contact with the host anymore, because they may have retreated to shelter. Because high proportions of insects for both species in the current study defecated within this post-feeding period, we conclude that each is competent to transmit T. cruzi regarding this feature. Following Almeida et al. (2003, 2005) recommendations, we fragmented the elapsed time for defecation after blood meals in four time frames. This method was suitable for the members of species complex in this study, which based on Soares et al. (2000) were expected to be able to defecate rapidly after feedings. However, statistical comparisons were not possible. Indeed, this comparison would be unnecessary because the main question in the epidemiological context was answered: both were considered competent to transmit T. cruzi regarding this feature for all life stages. Among the bionomic features, the time taken to defecate after feeding is considered one of the most important factors regarding vector capacity (Crocco and Catalá 1996). The transitory nature of epidemiological importance in triatomines can be illustrated by the case of T. sherlocki, a member of T. brasiliensis species complex originally considered exclusively sylvatic. This species was found infesting human domiciles in a recently colonized mining community (Almeida et al. 2009), highlighting the rapid changes that can occur in triatomine ecology in the face of anthropogenic environmental changes. Despite the fact that T. melanica is still considered exclusively

6 Vol. 41, no. 1 Journal of Vector Ecology 53 sylvatic, and because it was also considered competent to transmit T. cruzi based on features explored herein, we highlight the need to extend surveillance to this vector. Finally, we reinforce previous findings (Costa and Lorenzo 2009, Rebaudo et al. 2014) that T. brasiliensis complex provides a suitable model to evaluate a series of phenomenon on speciation and species differentiation. Acknowledgments We are grateful to the vector control and surveillance staff of the municipalities of Caicó, RN and Espinosa, MG, Brazil for the technician support for collecting insects. This study is in honor of Prof. Élio Gouvea (in memoriam), who idealized and created the Museu de Ciências-UBM. This study was funded by the following programs: the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for DCR fellowships, PPGEMA, Universidade Federal da Paraíba-UFPB supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process numbers 2010/ and 2011/ ), and the Centro Universitário de Barra Mansa, RJ, Brazil. REFERENCES CITED Abad-Franch, F., L. Diotaiuti, R. Gurgel-Gonçalves, and R.E. Gurtler Certifying the interruption of Chagas disease transmission by native vectors: cui bono? Mem. Inst. Oswaldo Cruz 108: Alevi, K.C., P. P. Mendonça, N. P. Pereira, J. A. Rosa, and M. T. Oliveira Karyotype of Triatoma melanocephala Neiva and Pinto (1923). Does this species fit in the Brasiliensis subcomplex? Infect. Genet. Evol. 12: Alevi, K.C., J.A. Rosa, and M.T. 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