Description and characterization of the melanic morphotype of Rhodnius nasutus Stål , 1859 ( Hemiptera : Reduviidae : Triatominae )

Introduction: For the fi rst time we provide the description of the melanic (dark) morphotype of Rhodnius nasutus and determine the pattern of genetic inheritance for this characteristic. Methods: Dark morph R. nasutus specimens were crossbred with standard (typically patterned) R. nasutus. Results: We present the fi rst occurrence of the melanic morphotype in the genus Rhodnius. The crossbreeding results demonstrate that the inheritance pattern of this characteristic follows Mendel’s simple laws of segregation and an independent assortment of alleles. Conclusions: Phenotypic variation of R. nasutus reinforces the heterogeneity found in the Triatominae. Descriptions of new species in this subfamily require rigorous validation criteria.

The Rhodnius genus contains 19 species, 13 of which occur in Brazil.This genus represents one of the main genera belonging to the subfamily Triatominae, which are of epidemiologic importance for the transmission of Chagas disease.Various authors have reported the importance of palm trees as a natural ecotope for Rhodnius species [1][2][3] , particularly palm trees belonging to the genera Attalea, Acrocomia, Copernicia, Mauritia, and Syagrus 2,3 .
Rhodnius nasutus is one of the Triatominae species that is native to Brazil.It is distributed in the semi-arid region of northeastern Brazil, which is predominantly covered by scrub vegetation.This species is considered a vector of secondary importance for the transmission of Chagas disease, and is mainly associated with the carnaúba palm tree (Copernicia prunifera) [2][3][4] ; however, this species also infests other species of palm trees 2 .
The chromatic pattern of Triatominae is used as an important characteristic for species determination.However, several studies have reported intra-specifi c variation in this characteristic 2,5 .For instance, dark morphotype (dark morphs or melanic) Triatoma infestans individuals have been collected from parrot nests (Aratinga acuticaudata) in the Bolivian Chaco 6 , with intermediate coloring being recorded in the Andean Valleys and Chaco 7 .More recently, dark morph specimens of T. infestans were also found in parrot nests (Amazona aestiva) in the Chaco Province of Argentina, which is a region that is free of anthropogenic activity, with the closest domicile being located at a distance of 25km 8 .Chromatic differences were also found in Triatoma rubrovaria.Four different morphotypes of this species were identifi ed in the same geographic spot and ecotope in the State of Rio Grande do Sul, Brazil.Genetic studies based on isoenzymes confi rmed that the differences in their chromatic patterns signifi ed intra-specifi c variation only 5 .The current study aimed to present the occurrence of melanic R. nasutus and to determine the pattern of genetic inheritance for this characteristic.
Between July 8 and 11 in 2003, parental samples of R. nasutus were collected from babaçu palm trees (BA) (Attalea speciosa), in the district of Meruoca, and carnaúba palm trees (CA), in Sobral, State of Ceará.Authorization was provided by the Brazilian Institute of Environment and Natural Resources (IBAMA, Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, Authorization Nº 007/2002-COMAF; Process Nº 02001.001333/02-71).The palm trees were cut down and dissected to search triatomines, following a previously described method 2 .A total of 11 adults and 11 nymphs were collected at Sobral, while 2 adults and 23 nymphs were collected at Meruoca.The triatomines were transferred to the insectary of the Laboratório de Triatomíneos e Epidemiologia da Doença de Chagas, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz (LATEC/CPqRR/FIOCRUZ) to establish colonies.Two groups were formed, each belonging to the palm tree species from which the triatomines had been collected.The two groups were housed under semi-controlled conditions of temperature and humidity (26 ± 2°C; 70 ± 10% UR), and were fed weekly on Swiss mice anesthetized with thiopental.
The occasional appearance of adults with atypical, dark coloring (here named dark morphs) was observed in the F3 generation of the R. nasutus colony from carnaúba palm trees.

Short Communication
Dias FBS et al. -Rhodnius nasutus dark morph Dark morph (melanic) nymphs were easily identifi ed.The head and thorax of R. nasutus dark morphs are dark colored, whereas these body parts were mostly reddish or chestnut-colored in standard nymphs 2 (Figure 1).Dark morph nymphs were separated from standard nymphs to form a colony exclusively made up of this phenotype.Fifth stage nymphs were sexed 9 and separated according to head coloring (Figure 1).Then the following crosses were made, with fi ve couple being used in each cross group: I) standard male x dark morph female; II) dark morph male x standard female; III) standard male x standard female; and IV) dark morph male x dark morph female.The standard bugs were used from the carnaúba colony only.Each couple was placed together for 60 days and fed weekly on Swiss mice anesthetized with thiopental.As soon as the fi fth stage nymphs of the F1 generation were produced, all individuals were sexed following the same criteria used to obtain the F2 generation by a crossbreeding experiment.
Couples from all cross directions that exhibited the phenotype according to the standard species description produced 100% offspring with the same characteristic as the F1 generation.The couples that exhibited melanic phenotypes (male and female dark morphs) also produced 100% individuals with the same characteristic (dark morphs) in the two analyzed generations (F1 and F2).Of the fi ve couples in the crossbreeding experiment between standard females and dark morph males, one couple did not copulate, three couples produced standard triatomines in the F1 generation, and one couple produced offspring with the same proportion of both phenotypes (1:1).The couples whose progenitors were melanic females and standard males produced standard bugs only.For one couple in this cross-experiment, fewer offspring were produced in the F1 generation (n = 11) than the other couples (Table 1).This couple also produced a high number of unviable eggs, which appeared to be unfertilized (Table 1).

A B C D
The results obtained from the triatomines used in the F2 generation of the crossbreeding experiment for dark morph male x dark morph female and standard male x standard female were similar to the results obtained in the F1 generation.For the F2 generation standard female x melanic male cross, three couples with males and females that had the standard phenotype in F1, produced F2 with both phenotypes.The other two couples in this group did not copulate (Table 2).
The melanic T. infestans found in northeastern Argentina was fi rst described as a sub-species, called T. infestans melanosoma.Later, it was suggested to be a new species, which was grounded on the argument of its dark coloring 10 .However, today it is well documented that the specimens found in Argentina and the Bolivian Chaco are an intra-specifi c phenotypic variation.Among the Triatominae species, black forms have only been described for T. infestans and T. brasiliensis, being the taxonomic question of the T. brasiliensis complex recently solved 11 .However, this report is the fi rst to demonstrate this phenotypic variation for triatomine bugs belonging to the genus Rhodnius.
Despite the general chromatic pattern of the body representing an extremely useful tool identifying Triatominae species, this criterion should not be considered as absolute.In the case of Rhodnius, particularly the species of the R. prolixus complex, which are phylogenetically very close, and are still considered undecipherable by some 2,3,12 , the chromatic characteristics are very important factors for correctly identifying the species.The triatomines described here as dark morph cannot be identifi ed through the current classifi cation keys.
Inter-specifi c crossbreeding experimental studies are very useful for validating the taxonomic status of Triatominae species, even in allopatric species 13 .In the case of the R. prolixus complex, the presence of viable laboratory hybrids has been   12 .Considering that, in some geographic regions, Rhodnius species occur sympatrically, it is possible to fi nd natural hybrids with phenotypic characteristics that are distinct to the parental.R. nasutus is described as a species of the Triatominae, with a red-brownish body as its general characteristic.This coloring is very similar to the color of the fi bers and stem of the carnaúba palm tree 2,14 , which is considered as the main ecotope for R. nasutus.Thus, this coloration disguises the triatomines, protecting them from predators.A study of R. nasutus collected from fi ve species of palm trees in the south mountain region of the State of Ceará, Brazil, found chromatic variation among the collected individuals, with those from carnaúba showing typical coloring 2 .The other four morphotypes had brownish coloring, which was very similar to the stem and sheath foliage of the palm trees that they inhabited 2 .While chromatic differences were observed, melanic R. nasutus were not found at any of the sampling sites.These observations corroborate previous studies, suggesting that ecotopes the inhabited by triatomines may infl uence the coloration of these insects due to phenotypic plasticity.This association between triatomine bugs and palm trees was observed in the similar chromatic characteristics of R. pictipes, which has a darker hue, and R. robustus 15 , which has a pinker, light-brown hue, with the palm trees that they inhabit.The emergence of R. nasutus dark morphs were very rare in the colonies maintained in the insectary for four years, appearing spontaneously among standard R. nasutus collected from carnaúba.This way, we show that the existence of melanic R. nasutus is due to the genetic inheritance, and is also very rare in nature.Thus, the crossbreeding experiments between standard R. nasutus phenotypes with dark morph phenotypes confi rmed that this type of inheritance is recessive Mendelian.Only crosses of standard males x dark morph females and dark morph males x standard females were not fully successful, producing unviable eggs.The phenotypic variation shown here for R. nasutus further reinforces the intraspecifi c heterogeneity in the Triatominae subfamily, demonstrating the importance of using rigorous criteria for describing new species.

FIGURE 1 -
FIGURE 1 -A) Standard Rhodnius nasutus adult.B) Dark morph R. nasutus adult.C) Standard R. nasutus fi fth stage nymph used in the intra-specifi c cross-experiments.D) Dark morph R. nasutus fi fth stage nymph used in the intra-specifi c cross-experiments.Fifth stage nymphs were separated according to the head coloring in the intra-specifi c cross-experiments.Scale bar = 5mm.

TABLE 1 -Crossbreeding of Rhodnius nasutus showing the quantity of insects produced by each couple and the possible genotype of the parental and F1 generation.
a ♀: female; ♂: male; standard: R. nasutus of typical color; dark: melanic R. nasutus.b S: allele from standard R. nasutus; D or d: allele from dark morph R. nasutus.F1: generation one; NC: no copulation.*Many unviable eggs demonstrated