Triatomines (Hemiptera, Triatominae) are hematophagous insects of great epidemiological importance because they are vectors of the protozoan Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae), the etiological agent of Chagas disease—a neglected disease that affects about eight million people, resulting in approximately 10,000 deaths per year from clinical manifestations (such as terminal heart failure, thromboembolic complications, refractory ventricular arrhythmias, and sudden death). 1,2 Although all of triatomine species, of both sexes, are considered as potential vectors of Chagas disease, the Triatoma (Laporte, 1832), Panstrongylus (Berg, 1879), and Rhodnius (Stål, 1859) genera are the most important from the epidemiological point of view. 3
The genus Rhodnius is a paraphyletic group composed of 23 species grouped into three large groups (pallescens, pictipes, and prolixus) (Table 1). The genus paraphilia is related to the fact that the species of the prolixus group present greater phylogenetic proximity with the Psammolestes (Bergroth, 1911 genus) than with other groups of Rhodnius spp. 4,5 In an attempt to solve this issue, Hypsa et al. 6 suggested changing the generic status from Psammolestes to Rhodnius. However, because of morphological and habitat divergences, the genus Psammolestes was maintained by the scientific community. 7
Cytogenetic characteristics of Rhodnius spp.
| C-banding | CMA3/DAPI | |||||||
|---|---|---|---|---|---|---|---|---|
| Rhodnius genus | Karyotype | A | X | Y | A | X | Y | |
| prolixus group | ||||||||
| Rhodnius barretti | – | – | – | – | – | – | – | |
| Rhodnius dalessandroi | – | – | – | – | – | – | – | |
| Rhodnius domesticus | 2n = 22 (20A + XY)* | Yes† | No† | Yes† | CMA3 + | CMA3 + | DAPI+ | |
| Rhodnius milesi | 2n = 22 (20A + XY)‡ | No | No | Yes | – | CMA3 + | DAPI+ | |
| Rhodnius marabaensis | 2n = 22 (20A + XY) | No | No | Yes | – | CMA3 + | DAPI+ | |
| Rhodnius montenegrensis | 2n = 22 (20A + XY)† | No | No | Yes | – | CMA3 + | DAPI+ | |
| Rhodnius nasutus | 2n = 22 (20A + XY)§ | Yes‖ | No‖ | Yes‖ | CMA3 + | CMA3 + | DAPI+ | |
| Rhodnius neglectus | 2n = 22 (20A + XY)¶ | No¶ | No¶ | Yes¶ | – | CMA3 + | DAPI+ | |
| Rhodnius neivai | 2n = 22 (20A + XY)# | No | No | Yes | – | CMA3 + | DAPI+ | |
| Rhodnius prolixus | 2n = 22 (20A + XY)** | No‖ | No‖ | Yes‖ | – | CMA3 + | DAPI+ | |
| Rhodnius robustus | 2n = 22 (20A + XY)†† | No‖ | No‖ | Yes‖ | – | CMA3 + | DAPI+ | |
| pictipes group | ||||||||
| Rhodnius amazonicus | – | – | – | – | – | – | – | |
| Rhodnius brethesi | 2n = 22 (20A + XY)‖ | No‖ | No‖ | Yes‖ | – | CMA3 + | DAPI+ | |
| Rhodnius paraensis | – | – | – | – | – | – | – | |
| Rhodnius pictipes | 2n = 22 (20A + XY)†† | Yes§ | No§ | Yes§ | CMA3 + | CMA3 + | DAPI+ | |
| Rhodnius stali | 2n = 22 (20A + XY)* | No | No | Yes | – | CMA3 + | DAPI+ | |
| Rhodnius zeledoni | – | – | – | – | – | – | – | |
| pallescens group | ||||||||
| Rhodnius colombiensis | 2n = 22 (20A + XY)* | Yes‡‡ | No‡‡ | Yes‡‡ | CMA3 + | CMA3 + | DAPI+ | |
| Rhodnius ecuadoriensis | 2n = 22 (20A + XY)‖ | No‖ | No‖ | Yes‖ | – | CMA3 + | DAPI+ | |
| Rhodnius pallescens | 2n = 22 (20A + XY)§§ | Yes‖ | No‖ | Yes‖ | CMA3 + | CMA3 + | DAPI+ | |
The species of the genus Rhodnius show little interspecific chromosomal variation because all species analyzed so far have karyotype 2n = 22 (20A + XY) (Table 1) and 45S rDNA clusters restricted to sex chromosomes. 8 Besides, most species have heterochromatin restricted to the Y sex chromosome (Table 1). However, the knowledge of the genomic composition of heterochromatin base pairs (AT and CG) is limited to Rhodnius pallescens (Barber, 1932), 9 and Rhodnius prolixus (Stål, 1859), 10 highlighting the necessity for further studies on the molecular cytogenetics of these vectors. Based on the data presented earlier, we analyzed the distribution of AT- and CG-rich DNA in the chromatin and chromosomes of the genus Rhodnius, and discuss the chromosome evolution of these vectors.
At least five adult males from each species (10 R. prolixus, 10 Rhodnius robustus (Larrousse, 1927), 10 Rhodnius neglectus (Lent, 1954), five Rhodnius nasutus (Stål, 1859), five Rhodnius domesticus (Neiva and Pinto, 1923), 10 Rhodnius montenegrensis (Rosa et al. [2012]), and five Rhodnius marabaensis (Souza et al. [2016])) were used. They had been assigned by the Triatominae Insectarium within the Department of Biological Sciences, in the College of Pharmaceutical Sciences, at Sao Paulo State University’s Araraquara campus (FCFAR/UNESP), São Paulo, Brazil. The seminiferous tubules of the specimens were squashed and fixed to a coverslip. Then, they underwent the cytogenetic technique of CMA3/4′,6-diamidino-2-phenylindole (DAPI) 9 and C banding. 11 The biological material was analyzed using a Jenaval light microscope (Zeiss) attached to a digital camera, with the AxioVision LE 4.8 image analyzer (Copyright 2006–2009 Carl Zeiss Imaging Solutions GmbH), and using a fluorescence microscope Zeiss-Axioskop and Olympus BX-FLA.
All species had the same number of chromosomes, 2n = 22 (20A + XY) (Table 1), confirming the hypothesis that all species of the Rhodniini tribe have 22 chromosomes. 23 Taking into account that the ancestral karyotype is 2n = 22, 23,24 Alevi et al. 23 highlight that during the diversification of the pallescens, pictipes, and prolixus groups, there were no evolutionary events (as agmatoploidy and simploidy) that resulted in changes in the number of chromosomes.
Except for R. domesticus, R. nasutus, Rhodnius pictipes (Stål, 1872), Rhodnius colombiensis (Mejia, Galvão and Jurberg, 1999), and R. pallescens, all Rhodnius species have euchromatic autosomes with the absence of AT- and CG-rich blocks (Figure 1A and B) (Table 1). Curiously, the same species that have heterochromatin blocks in the autosomes also have CMA3 + blocks dispersed in the prophasic nucleus (Figure 1D) (Table 1), emphasizing that the heterochromatin present in the chromosomes of R. domesticus, R. nasutus, R. pictipes, R. colombiensis, and R. pallescens is rich in CG regions. Besides, all species had the euchromatic X sex chromosome and CMA3 + (Figure 1B and D), and the heterochromatic Y chromosome and DAPI+ (Figure 1A and C) (Table 1).
Composition of the heterochromatin base pairs in prophases of Rhodnius marabaensis (A and B) and Rhodnius domesticus (C and D). (A and C) Y sex chromosome rich in AT. (B) X sex chromosome rich in CG. (D) X sex chromosome and autosomes rich in GC. Bar: 10 μm.
Citation: The American Journal of Tropical Medicine and Hygiene 104, 2; 10.4269/ajtmh.20-0875
Taking into account that Rhodnius species whose autosomes present heterochromatic blocks and CMA3 + belong to different groups (prolixus group: R. domesticus and R. nasutus; pictipes group: R. pictipes; and pallescens group: R. colombiensis and R. pallescens), we suggest that the gain/loss of CG-rich heterochromatin was an event that took place independently in the three groups of Rhodnius and can be related to different evolutionary factors. Alevi et al., 25 for example, suggest that the different patterns of heterochromatin losses observed in the pallescens group can be associated with adaptation to different environments occupied by species.
The divergences in the composition of the autosomes can also be used as taxonomic tools to differentiate some species of Rhodnius because events of phenotypic plasticity and cryptic speciation have been reported for this genus of medical importance. 26 Within the prolixus group, for example, R. domesticus and R. nasutus can be distinguished from all other species with euchromatic autosomes; within the pictipes group, R. pictipes can be distinguished from all other species by cytotaxonomy; besides, R. colombiensis and R. pallescens can be differentiated from R. ecuadoriensis (Lent and León, 1958) (euchromatic autosomes). Still, R. colombiensis and R. pallescens can also be differentiated from each other by the amount of heterochromatin in the autosomes. 25
Recently, the heterochromatin pattern and the composition of AT and CG was one of the tools used to describe R. taquarussuensis Rosa et al. 27 However, molecular analyzes and experimental crosses showed that R. taquarussuensis was R. neglectus with chromosomal polymorphisms. 28 Based on this, it is evident that although cytogenetic analyses are of great taxonomic importance, 29,30 the confirmation that different cytotypes represent valid taxa must be performed through integrative taxonomy. 31
Thus, we characterize the AT- and CG-rich DNA pattern for the genus Rhodnius, and we suggest that the pattern of CG-rich heterochromatin in the autosomes of these vectors evolved independently in pallescens, pictipes, and prolixus groups.
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