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)2–4; however, this species also infests other species of palm trees2.
The chromatic pattern of Triatominae is used as an important characteristic for species determination. However, several studies have reported intra-specific variation in this characteristic2,5. For instance, dark morphotype (dark morphs or melanic) Triatoma infestans individuals have been collected from parrot nests (Aratinga acuticaudata) in the Bolivian Chaco6, with intermediate coloring being recorded in the Andean Valleys and Chaco7. 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 25km8. Chromatic differences were also found in Triatoma rubrovaria. Four different morphotypes of this species were identified in the same geographic spot and ecotope in the State of Rio Grande do Sul, Brazil. Genetic studies based on isoenzymes confirmed that the differences in their chromatic patterns signified intra-specific variation only5. 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 N° 007/2002-COMAF; Process N° 02001.001333/02-71). The palm trees were cut down and dissected to search triatomines, following a previously described method2. 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. Dark morph (melanic) nymphs were easily identified. The head and thorax of R. nasutus dark morphs are dark colored, whereas these body parts were mostly reddish or chestnut-colored in standard nymphs2 (Figure 1). Dark morph nymphs were separated from standard nymphs to form a colony exclusively made up of this phenotype. Fifth stage nymphs were sexed9 and separated according to head coloring (Figure 1). Then the following crosses were made, with five 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 fifth 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.
![](http://www.scielo.br/img/revistas/rsbmt/v47n5//0037-8682-rsbmt-47-05-637-gf01.jpg)
FIGURE 1- A) Standard Rhodnius nasutus adult. B) Dark morph R. nasutus adult. C) Standard R. nasutus fifth stage nymph used in the intra-specific cross-experiments. D) Dark morph R. nasutus fifth stage nymph used in the intra-specific cross-experiments. Fifth stage nymphs were separated according to the head coloring in the intra-specific cross-experiments. Scale bar = 5mm.
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 five 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).
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.
1. ♀ standard x ♂ darka | Dark (%) | Standard (%) | Genotype (parental and F1)b | Probable F1 |
---|---|---|---|---|
Couple 1 | zero | 73 (100.0) | dd x SS = 100% Sd | 100% standard |
Couple 2 | zero | 75 (100.0) | dd x SS = 100% Sd | 100% standard |
Couple 3 | NC | NC | NC | NC |
Couple 4 | zero | 65 (100.0) | dd x SS = 100% Sd | 100% standard |
Couple 5 | 44 (50.0) | 44 (50.0) | dd x Sd = 50% Sd + 50% dd | 50% standard and 50% dark |
Total | 44 (14.6) | 257 (85.4) | ||
2. ♀ dark x ♂ standard | Dark (%) | Standard (%) | Genotype (parental and F1) | Probable F1 |
Couple 1 | zero | 39 (100.0) | dd x SS = 100% Dd | 100% standard |
Couple 2 | zero | 11 (100.0)* | dd x SS = 100% Dd | 100% standard |
Couple 3 | zero | 63 (100.0) | dd x SS = 100% Dd | 100% standard |
Couple 4 | zero | 56 (100.0) | dd x SS = 100% Dd | 100% standard |
Couple 5 | zero | 39 (100.0) | dd x SS = 100% Dd | 100% standard |
Total | zero | 208 (100.0) | ||
3. ♀ standard x ♂ standard | Dark (%) | Standard (%) | Genotype (parental and F1) | Probable F1 |
Couple 1 | NC | NC | NC | NC |
Couple 2 | zero | 72 (100.0) | SS x SS = 100% SS | 100% standard |
Couple 3 | zero | 65 (100.0) | SS x SS = 100% SS | 100% standard |
Couple 4 | zero | 55 (100.0) | SS x SS = 100% SS | 100% standard |
Couple 5 | zero | 83 (100.0) | SS x SS = 100% SS | 100% standard |
Total | zero | 275 (100.0) | ||
4. ♀ dark x ♂ dark | Dark (%) | Standard (%) | Genotype (parental and F1) | Probable F1 |
Couple 1 | 83 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Couple 2 | 83 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Couple 3 | 69 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Couple 4 | 87 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Couple 5 | 117 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Total | 439 (100.0) |
a♀: female; ♂: male; standard: R. nasutus of typical color; dark: melanic R. nasutus.
bS: allele from standard R. nasutus; D or d: allele from dark morph R. nasutus. F1: generation one; NC:no copulation.
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).
TABLE 2- Crossbreeding experiment of Rhodnius nasutus showing the quantity of insects produced by each couple and the possible genotype of the F1 and F2 generations.
1. ♀ standard x ♂ darka | Dark (%) | Standard (%) | Genotype (F1 and F2)b | Probable F2 |
---|---|---|---|---|
Couple 1 | 28 (36.8) | 48 (63.2) | Sd x Sd = SS, 2Sd, dd | 75% standard and 25% dark |
Couple 2 | 15 (27.8) | 39 (72.2) | Sd x Sd = SS, 2Sd, dd | 75% standard and 25% dark |
Couple 3 | NC | NC | NC | NC |
Couple 4 | NC | NC | NC | NC |
Couple 5 | 28 (36.8) | 48 (63.2) | Sd x dd = 50% Sd + 50% dd | 50% standard and 50% dark |
Total | 71 (34.5) | 135 (65.5) | ||
2. ♀ dark x ♂ standard | Dark (%) | Standard (%) | Genotype (F1 and F2) | Probable F2 |
Couple 1 | 25 (24.8) | 76 (75.2) | Sd x Sd = SS, 2Sd, dd | 75% standard and 25% dark |
Couple 2 | 14 (28.0) | 36 (72.0)* | Sd x Sd = NN, 2Sd, dd | 75% standard and 25% dark |
Couple 3 | NC | NC | NC | NC |
Couple 4 | 14 (20.6) | 54 (79.4) | Sd x Sd = SS, 2Sd, dd | 75% standard and 25% dark |
Couple 5 | 38 (37.3) | 94 (62.7) | Sd x Sd = SS, 2Sd, dd | 50% standard and 50% dark |
Total | 91 (25.9) | 260 (74.1) | ||
3. ♀ standard x ♂ standard | Dark (%) | Standard (%) | Genotype (F1 and F2) | Probable F2 |
Couple 1 | – | – | – | – |
Couple 2 | Zero | 57 (100.0) | SS x SS = 100% SS | 100% standard |
Couple 3 | Zero | 32 (100.0)** | SS x SS = 100% SS | 100% standard |
Couple 4 | Zero | 67 (100.0) | SS x SS = 100% SS | 100% standard |
Couple 5 | Zero | 94 (100.0) | SS x SS = 100% SS | 100% standard |
Total | Zero | 250 (100.0) | ||
4. ♀ dark x ♂ dark | Dark (%) | Standard (%) | Genotype (F1 and F2) | Probable F2 |
Couple 1 | – | – | – | – |
Couple 2 | 63 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Couple 3 | 91 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Couple 4 | 24 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Couple 5 | 37 (100.0) | Zero | dd x dd = 100% dd | 100% dark |
Total | 215 (100.0) | Zero |
a♀: female; ♂: male; standard: R. nasutus of typical color; dark: melanic R. nasutus.
bS: allele from standard R. nasutus; d: allele from dark morph R. nasutus. F1: generation one; F2:generation two; NC: no copulation.
The melanic T. infestans found in northeastern Argentina was first 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 coloring10. However, today it is well documented that the specimens found in Argentina and the Bolivian Chaco are an intra-specific 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. brasiliensiscomplex recently solved11. However, this report is the first 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 some2,3,12, the chromatic characteristics are very important factors for correctly identifying the species. The triatomines described here as dark morph cannot be identified through the current classification keys.
Inter-specific crossbreeding experimental studies are very useful for validating the taxonomic status of Triatominae species, even in allopatric species13. In the case of the R. prolixus complex, the presence of viable laboratory hybrids has been demonstrated12. Considering that, in some geographic regions, Rhodnius species occur sympatrically, it is possible to find 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 fibers and stem of the carnaúba palm tree2,14, which is considered as the main ecotope for R. nasutus. Thus, this coloration disguises the triatomines, protecting them from predators. A study of R. nasutuscollected from five 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 coloring2. The other four morphotypes had brownish coloring, which was very similar to the stem and sheath foliage of the palm trees that they inhabited2. 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 influence 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. robustus15, 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 morphphenotypes confirmed that this type of inheritance is recessive Mendelian. Only crosses of standard males x dark morphfemales 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 intraspecific heterogeneity in the Triatominae subfamily, demonstrating the importance of using rigorous criteria for describing new species.