Biological Aspects of Rhodnius montenegrensis (Hemiptera,Reduviidae,Triatominae) Under Laboratory Conditions

Introduction

Triatomines are hematophagous insects that feed on vertebrate hosts, including humans (Abad-Franch et al. 2013). They have epidemiological importance because they are vectors of the parasitic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease, and other flagellated protozoa such as Trypanosoma rangeli (Abad-Franch et al. 2013). Chagas disease affects 10 million people in Latin American countries with an increase of the reported cases in nonendemic regions (Grijalva et al. 2014).

Initially, Chagas disease had an enzootic cycle maintained among triatomines and wild animals. The transmission to human beings occurred when they invaded the wild ecotypes or when the triatomines invaded human dwellings in rural and urban areas (Coura 2015). This process leads to the colonization of domestic and peridomestic areas, which contributes to increase in the transmission to humans (Coura 2015).

In recent years, the increasing number of Chagas disease cases by oral transmission occurring due to ingestion of contaminated fruits with T. cruzi such as açaí, bacaba, and jaci was reported in the Brazilian Amazon (Dias et al. 2016). Between 2000 and 2011, ∼1200 acute cases of Chagas disease were reported in Brazil, most of them occurring in the Amazon region. Besides the acute cases, Dias et al. (2016) also report the occurrence of 112 outbreaks involving 35 municipalities between the years 2005 and 2013. In the state of Acre, Brazilian Western Amazon, from 2015 to 2016, the number of cases of Chagas disease increased >300%, which is mostly attributed to oral transmission (Oliveira et al. 2018).

It has been observed that home invasion by triatomines in the Amazon region is related to anthropogenic environmental changes and occurs mostly by species of the genus Rhodnius, commonly associated with palm trees (Abad-Franch et al. 2015, Bilheiro et al. 2018, Oliveira et al. 2018). Previous studies have shown that Rhodnius montenegrensis is found constantly colonizing palms. Meneguetti et al. (2013) reported the occurrence of this species in Attalea speciosa in the municipality of Buritis, also in the state of Rondônia. On account of this, there is a need for a better biological characterization of the species R. montenegrensis. The proximity of rural domiciles to palm trees colonized with triatomines and the presence of mammal hosts are considered risk factors for the occurrence of outbreaks of Chagas disease in the form of both vector and oral infection (Abad-Franch et al. 2010, Coura and Junqueira 2012). The proximity of palm trees was already associated with home invasion by R. montenegrensis in the state of Acre in previous studies (Meneguetti et al. 2014). The specimens were found inside rural domiciles in the proximity of a forest fragment with several specimens of palm trees of the genus Attalea located close to the dwelling.

Among the genus Rhodnius, the species R. montenegrensis, first described in the state of Rondônia, Brazil, by Rosa et al. (2012), was already identified as infected by T. rangeli (Meneguetti et al. 2014) and by T. cruzi (Bilheiro et al. 2018).

Knowing the biological characteristics of secondary vectors that have the potential to generate domiciliary populations, such as the sylvatic species R. montenegrensis, is essential to design vector control strategies (Cardozo-de-Almeida et al. 2014). Since the laboratory populations can be considered models for domiciliary populations of triatomines, they can help to predict population sizes and any reinfestation process (Dujardin et al. 1999, Costa and Lourenzo 2009). The study of the biological parameters of this newly described triatomine can shed light on its life cycle and, therefore, contribute to improving the understanding of the epidemiological role of those vectors, guiding surveillance actions against Chagas disease (Barreto-Santana et al. 2011).

Materials and Methods

For the biological analysis, insects from colonies maintained in the Biomedical Sciences Institute of University of São Paulo (ICB5-USP), in Monte Negro, Rondônia, Brazil, were observed in the laboratory for 4 months. The colonies originated from eggs of R. montenegrensis captured on Attalea sp specimens (babaçu trees) in the municipality of Monte Negro (permanent authorization of IBAMA, Nr. 52260-1). The specimens of R. montenegrensis were identified by morphological and morphometric parameters as well as by the morphology of the internal male genital. Both analyses were made using the differential morphological characters proposed by Rosa et al. (2012).

Initially, 5th-instar nymphs of R. montenegrensis were selected randomly from the forest to obtain adult specimens. After ecdysis, adult specimens were separated into three groups of six female and two male specimens for obtaining eggs. The groups were maintained in plastic recipients covered with cotton cloth and lined with paper filter. In this step, the insects were fed weekly ad libitum in Balb/C mice anesthetized with ketamine 100 mg/kg applied to the peritoneal region (CEUA-Fiocruz N. 2014/15). One hundred and eighty-four eggs were obtained, then they were individualized in plastic containers, numbered, and observed daily until emergence. After emergence, the 1st-instar nymphs were weighted and maintained in plastic containers lined with filter paper and covered by a thin cotton fabric. The nymphs were also fed in anesthetized Balb/C mice weekly ad libitum until they were engorged to repletion. For those not engorged to repletion, the feeding process was repeated. The insects were maintained at a temperature of 27–29°C and 55–75% of humidity recorded daily in a thermohygrometer. A 12-h period of photophase was used intercalated with a 12-h period of scotophase. The insects were observed daily to verify the time required for emergence, the mortality rate per instar, and the time required for the nymphs to reach the last instar. The weight gain in R. montenegrensis was obtained by measuring the weight before and after the bloodmeal in each development stage, the after-meal weighting occurring after the postmeal diuresis.

The software Statistical Package for Social Sciences (SPSS) 17.0 (SSP, 2008) was used for statistical analysis. Kolmogorov–Smirnov test and Shapiro–Wilk test were used to test the normality of the distribution in all analyzed variables. Since the variables had a non-normal distribution, the parameters were evaluated using median, mean, and standard deviation (SD). The heterogeneity chi-square test was used to calculate the egg hatching rate and sex ratio. Statistical significance was considered if p < 0.05.

Results

The emergence rate for R. montenegrensis eggs was 63%. No statistical difference between male and female rate was noted using the heterogeneity chi-square test (p > 0.05). The mean time required for emergence was 13.9 ± 1.7 days. The biological cycle from egg to adult stage was 105.2 ± 9.2 days. The data, according to the nymphal stages both in mean and median deviation, are given in Table 1.

The number of bloodmeals required for each nymphal stage to reach the next stage, expressed in terms of median, mean, and SD, is given in Table 2.

The bloodmeal volume and weight gain in each stage are given in Table 3. The highest weight gains registered were 10.6 times for 1st-instar nymphs, 6.6 times for 2nd-instar nymphs, 5.4 times for 3rd-instar nymphs, 5.9 times for 4th-instar nymphs, and 3.9 times for 5th-instar nymphs. The lowest weight gain was observed in the adult phase: 2.2 times in male specimens and 2.3 times in female specimens.

Out of 81 adult specimens of R. montenegrensis, 44 (54.3%) were female and 37 (45.7%) were male. No statistical significance was observed using the chi-square test (p > 0.05).

Discussion

The egg hatching rate in R. montenegrensis was 63.0%. This result was higher than those obtained by Guarneri et al. (1998) in Rhodnius domesticus (57%) and by Soares et al. (1995) in Rhodnius nasutus (53.3%). However, it was lower than those described by Aldana et al. (2005) in Rhodnius robustus, where the emergence rate was 78.3%. Peixoto and Jurberg (2014) observed an emergence rate of 83.3% in Rhodnius stali and 86.7% in Rhodnius pictipes. Previous studies demonstrated that egg hatching rates in Rhodnius species can usually vary (Guarneri et al. 1998). These rates are related to the fertility in female specimens, which can decline with aging (Guarneri et al. 1998).

The incubation period observed for eggs of R. montenegrensis was 13.9 ± 1.7 days. This result was shorter than those observed by Rocha et al. (2001) in R. robustus specimens (16.3 ± 2.9 days), under the same conditions used in the present study. In R. domesticus, Guarneri et al. (1998) observed a longer incubation period (15.6 ± 1.0 days) than in R. montenegrensis, also under the same conditions used in this study. According to previous studies, the incubation period varies with temperature, usually declining with higher temperatures (Rocha et al. 2001).

The development cycle of triatomines varies according to the different species, environmental conditions, and availability of bloodmeal sources (Guarneri et al. 1998). In this study, the time required for R. montenegrensis to complete the biological cycle from egg to adult phase was 105.2 ± 9.2 days. This result was higher than those observed in R. robustus (70 days) by Aldana et al. (2005). However, the duration of the biological cycle in R. montenegrensis was lower than that observed by Lent and Valderrama (1977) for R. pictipes (190.7 days) and for Rhodnius neivai (181.2 days). Parameters such as egg hatching rates, the time required for the nymphal development, and mortality rates are related to the population size of the species in the natural environment, as a shorter development cycle can generate larger populations. Considering that, it could be expected that the population size may reflect the dispute for bloodmeal sources in the natural environment and possibly leads to dispersion of these vectors (Barreto-Santana et al. 2011, Cardozo-de-Almeida et al. 2014).

Regarding the increase in weight in R. montenegrensis after bloodmeal, the higher weight gain was observed in the 1st-instar specimens (10.6 times), a decrease being noticed in the next stages of development. Lower weight gain was observed in the adult specimens (2.3 times in female specimens and 2.2 times in male specimens). This result was lower than those observed in R. robustus by Aldana et al. (2005), which varied between 14.7 and 8.8 times. These variables are of great importance in vector competence. The number of bloodmeals and its volume are related to the risk to become infected with trypanosomatids and to the number and viability of eggs as well. Besides that, this variable is also related to the ability of dispersion by flight (Friend et al. 1965, Buxton 1930, Regis 1979).

Conclusion

For the first time, biological aspects of this newly described species are registered. Their importance lies in the fact that they can guide more effective vector control strategies. This information is of great relevance mainly in regions where household invasions by triatomines commonly occur and there is a noticeable rise in acute Chagas disease cases, as in the Amazon region.