Artigo Comparative biology of the two sister species of Triatominae ( Hemiptera : Reduviidae ) Biologia comparativa de duas espécies irmãs de Triatominae ( Hemiptera : Reduviidae )

1. Laboratory, Leishmaniasis Transmitters, Medical and Forensic Entomology. Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil. 2. Laboratory of Triatominae and Epidemiology of the Chagas Disease. Rene Rachou Institute. Oswaldo Cruz Foundation, Belo Horizonte, MG, Brazil. 3. National and International Reference Laboratory in Taxonomy of Triatominae (LNIRTT). Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil. 4. Institut de Recherche pour le Développement (IRD), Montpellier, France.

Autochthonous species of Triatominae originally restricted to the wild environment are increasingly reported invading houses and peridomestic structures where they may act as vectors of Trypanosoma cruzi (Chagas, 1909), the agent of Chagas disease.Among these, Triatoma pseudomaculata (Corrêa & Espínola, 1964), an arboricolous species in sylvatic environment, is considered a vector of T. cruzi in peridomestic structures.Nevertheless, it does not display significant ability for colonizing human dwelling, as verified by the scarcity of domestic colonies notified indoors 1,2,3 .Recently, one case of T. cruzi transmission by T. pseudomaculata to a woman was reported in Ceará State 4 .Therefore, the role of this species as an effective vector needs to be accurately investigated.A second autochthonous species is Triatoma wygodzinskyi Lent, 1951 (morphologically very similar to T. pseudomaculata).This rupicolous species was described among a small number of specimens collected in southern Minas Gerais State 5,6 .Recently, genetic and morphometric comparisons suggest that both species belong to the same evolutionary lineage; consequently, they should be classified together in the same group 7,8 .
The development cycle of triatomines is related to the species and environmental conditions and is deeply influenced by the accessibility of adequate blood sources 9 .The hypothesis of this work is that sister species, such as T. pseudomaculata and T. wygodzinskyi, share biological traits.In order to test this assumption, comparative studies of the development cycle and blood ingestion of both species were conducted under the same laboratory conditions.

Development cycle
Eggs laid by five field females of each species were randomly selected and individually placed in plastic containers (12 cm high and 3 cm diameter), with filter paper inside to provide the developing insects with access to the food source.Each container was numbered and examined daily until egg hatching.The nymphs were observed daily and fed on mice weekly (Protocol CEuA: P0100-01) and the time required to pass through the different stages to adulthood recorded in days (d).The development times (total and by stage) were expressed in median values.

Blood ingestion
Throughout the different development stages, the triatomines were weighed individually on an analytical balance (DAHuS ® ) before and after feeding and the number of grams of blood ingested (mg) were calculated.

Mortality
The mortality rate according to each nymphal stage was calculated.

Statistical methods
For data interpretation concerning the development cycle and blood meal ingestion, normality and homogeneity of variances were assessed using the Levene and Bartlett tests, respectively.The median variables were analyzed using the Mann-Whitney test.For statistical tests, we used JMP v.6.0.0 (SAS Institute Inc., 2005).For the Chi square test on mortality range, we used GraphPad InStat v.3.05 (GraphPad Software, San Diego, CA, uSA).

Development cycle
The total life cycle was slightly longer in T. pseudomaculata than T. wygodzinskyi (232 and 205 days, respectively, p = 0.0014).Nevertheless, interspecies differences were only observed for egg and N5 duration, all the other stages showed similar durations (Table 1).Moreover, the differences were exclusively observed among female nymphs (data not shown).

Blood ingestion
T. pseudomaculata and T. wygodzinskyi needed a similar number of feeding periods (10 and 11, respectively) to reach the adult stage.No significant difference was observed in the number of blood meals between both species (Table 2); however, the fourth and fifth nymphal instars of T. pseudomaculata ingested more blood than the corresponding stages in T. wygodzinskyi (Table 3).

Mortality
The mortality rates for each nymphal instar are shown in   the adult stage (global mortality rate of 58.1%); a similar rate was determined for T. pseudomaculata (56.3%).Mortality for each stage varied from 7.3 to 25.7% for T. wygodzinskyi and from 3.9 to 24.4% for T. pseudomaculata, the highest mortality rate was registered in N1 (25.67%) for the former species and in N2 (24.4%) for the latter.The interspecies comparison for each stage did not reveal any significant difference between the two species.
Several papers involve studies related to the biology of T. pseudomaculata, a triatomine species with an epidemiological role 10,11,12,13,14 .In contrast, no readily available information exists concerning the biology of T. wygodzinskyi, which is an exclusively sylvatic vector.In fact, the study conducted by Juarez (1970)  15 on the biological cycle of T. arthurneivai actually involves misidentified populations that are currently known as T. wygodzinskyi 7,16 .
In most cases, the disparities between the conditions maintained in individual insectaries, the methodologies and statistical analyses complicate the comparison of different studies relating to the same species 11 .For example, the egg to adult life cycle showed an average duration of 255 days in this work, whereas it varied from 212 to 349 days according to other authors 11,14 .The differences observed are partly due to the influence of the blood meal source 13 .
The comparison of biological traits of two species under the same laboratory conditions is more informative.In the present study, the study began with eggs laid by field-collected females in order to limit the endogamy that occurs after a few generations in the laboratory.Concerning the life cycle, the differences observed between the two species are minor and limited to the development time of the egg and N5 stage in female insects.Regarding the blood meal parameters, the number of feeding periods showed no significant differences between the two species.Only the late nymphal instars of T. pseudomaculata ingest more blood than T. wygodzinskyi.This factor is epidemiologically important and suggests an increased risk of T. cruzi transmission in T. pseudomaculata.Finally, the interspecies comparison of the mortality rate showed no significant difference.
For a long time, T. maculata and T. pseudomaculata were considered closely related species 17,18,19 .Recently, this hypothesis was undermined by genetic and morphological studies 7,8,20 .When comparing the life cycles between these two species, it is clear that the life cycle of T. maculata is shorter than that of T. pseudomaculata.According to different authors, the N1-adult life cycle does not exceed 144 days for T. maculata, a much shorter duration than that observed for T. pseudomaculata (Table 5).The coherence of our results with the genetic and morphological studies suggests that biological traits are important criteria to determine the relationship between species.Regarding T. pseudomaculata and T. wygodzinskyi, sister species derived from a common ancestor, they have maintained similar biological traits even though they show a different ecology; the former is arboricolous and the latter is rupicolous.

TABLE I -
Time of development (in days) according to the stage in T. wygodzinskyi and T. pseudomaculata.

TABLE 2 -
Number of blood meals according to the nymphal stage in T. wygodzinskyi and T. pseudomaculata.

TABLE 3 -
Blood meal ingested (in mg) according to nymphal stage in T. wygodzinskyi and T. pseudomaculata.

TABLE 5 -
Comparison between T. maculata and T. pseudomaculata life cycles according to the feeding source.