Omics Tools Applied to the Study of Chagas Disease Vectors: Cytogenomics and Genomics

Cytogenomics.

The application of cytogenomics in the study of triatomines began in 2005 with the study of the physical mapping of chromosomes, with emphasis on the number and arrangement of the 45S ribosomal DNA (rDNA) probe (which characterizes nucleolus organizing regions [NORs]) by the fluorescence in situ hybridization (FISH) technique.⁷ Currently, the distribution of NORs is known for more than 50 species of triatomines that allow to clarify systematic, taxonomic, and evolutionary issues (Table 1).^5,7–14 The NORs have been described in the X chromosome, in the X and Y chromosomes, in one autosomal pair, two autosomal pairs, in the X chromosome plus one autosomal pair, in two autosomal pairs plus X chromosome, or two autosomal pairs (with one heterozygote) plus X chromosome (Table 1).

In the Rhodniini tribe, the location of the rDNA clusters was restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes).^5,12 The cytogenomic characterization of the Rhodniini tribe species made it possible to suggest that three evolutionary events may have happened during the chromosomal evolution of this tribe, namely, ectopic recombination and/or rDNA transposition (if the ancestor is marked only on sex X chromosomes) or loss of rDNA loci on the Y chromosome (if the ancestor is marked on both sex chromosomes).¹²

The Triatomini tribe has the most diversity in the location of the rDNA clusters (Table 1). The species that make up the genera Dipetalogaster Usinger, 1939, Eratyrus Stål, 1859, Panstrongylus, Mepraia Mazza Gajardo and Jörg, 1940, Nesotriatoma Usinger, 1944, and Meccus Stål, 1859 have the same location for the rDNA clusters (Table 1). On the other hand, species of the genus Triatoma present all the possibilities of NORs described for Triatominae (Table 1).

Based on this, it was observed that, in general, evolutionarily related species (grouped into complexes and subcomplexes by Schofield and Galvão¹⁵—and reorganized by Pita et al.¹³ and Alevi et al.¹⁶) present the same FISH markings: a large autosomal pair for Triatoma dimidiata subcomplex species, one autosomal pair for Triatoma protracta and Triatoma lecticularia complexes, X chromosome for Triatoma dispar complex, one autosomal pair for the Triatoma rubrovaria subcomplex, one (X) or two sex chromosomes (XY) for the Triatoma sordida subcomplex, sex chromosomes (XY) for the Triatoma maculata subcomplex, one autosomal pair for the Triatoma pseudomaculata subcomplex, the largest autosomal pair for the Triatoma brasiliensis subcomplex, and X sex chromosomes for the Triatoma vitticeps subcomplex. The same evolutionary phenomena suggested for Rhodniini¹² possibly are related to the great diversification of NORs in Triatoma; as for triatomines, heterologous associations among heterosomes (Xs/Y sex chromosomes), as well as nonhomologous autosomes, are very common phenomena and could favor the observed rDNA variation.⁵

In addition to the application of cytogenomics in taxonomy, systematics, and evolution (as discussed earlier), this tool has also been used in studies associated with population genetics: Panzera et al.^5,11 analyzed the karyotype, heterochromatin distribution, and 45s rDNA gene mapping of Triatoma infestans (Klug, 1834), from seven different countries using the techniques of C-banding and FISH. The authors observed that all individuals had 20 autosome chromosomes and two sex chromosomes (2n = 22). However, through the analyses with FISH, the authors observed different intraspecific chromosomal patterns between the Andean and non-Andean groups (Table 1), as well as a high diversity of rDNA in the Andean group compared with the non-Andean group, so they suggested that the Andean group is ancestral in T. infestans, with the non-Andean being a derived group.^5,11 Taking into account the two most common patterns of the Andean group (autosomal pair and autosomal pair and X chromosome), Panzera et al.¹¹ conclude that a single event of autosomal loss is the most accepted hypothesis to explain the pattern of the non-Andean group (X chromosome). Thus, they support the hypothesis that T. infestans originated in the Andean valleys of Bolivia, as a wild species with polymorphic populations in terms of high amounts of heterochromatic autosomes and variant positions of the ribosomal cluster.¹¹

Besides that, a third group of chromosomes (intermediate) for T. infestans was identified by Panzera et al.¹¹ This group had three chromosomal characteristics: 1) the number of heterochromatic autosomes is less in the Andean group, but more in the non-Andean group; 2) this group includes individuals with and without heterochromatic X chromosome, as observed in Andean and non-Andean individuals; and 3) about 50% of individuals have an exclusive rDNA pattern. Thus, the authors suggested two hypotheses to explain the origin of this intermediate group. In the first hypothesis, called “ancestral origin,” the authors suppose that the intermediate group represents an intermediate stage of the gradual variation of heterochromatin that occurred during the initial divergence between the Andean and non-Andean groups. In the second hypothesis, called “secondary contact,” they assume that the intermediate group is a very recent formation and not a legacy of the old process of divergence between Andean and non-Andean groups.¹¹ The authors suggest that the intermediate group has recently originated from crossing between Andean and non-Andean individuals, which were previously isolated.¹¹

The FISH technique was also used by Pita et al.¹³ to analyze whether telomeric regions were present in triatomines, because there was a chance of loss of this region in insects of the Cimicomorpha order.¹⁷ For this analysis, the TTAGG telomeric probe was used in four triatomine species, namely, T. infestans, T. dimidiata (Latreille, 1811), Dipetalogaster maxima (Uhler, 1894), and R. prolixus. All species showed evident telomeres at the end of the chromosomes, contradicting the currently accepted hypothesis¹⁷ that evolutionary recent heteropterans lack this ancestral insect telomeric sequence.

Pita et al.,¹⁸ using in situ hybridization by genomic probes DNA of Triatoma delpontei (Romaña and Abalos 1947) and T. infestans (genomic in situ hybridization [GISH]), showed that the repetitive sequences are shared in closely related species. Two different hybridization patterns were demonstrated by the probes: 1) strong signals accumulated in some regions along the chromatin and 2) strong signals scattered throughout the chromosome.

The Triatomini tribe species analyzed (T. delpontei, Andean T. infestans, non-Andean T. infestans, Triatoma platensis Neiva, 1913, T. dimidiata, Triatoma carrioni Larrousse, 1926 and T. protracta (Uhler, 1894), Mepraia spinolai (Porter, 1934), D. maxima, Eratyrus mucronatus Stål, 1859, and Panstrongylus geniculatus) presented the Y chromosome with highly repeated sequences. These results show that the genomes of Triatomini species share scattered sequences of repetitive DNA: T. infestans, T. delpontei, T. platensis, and M. spinolai share accumulated repetitive sequences in the same regions, whereas other species exhibit these sequences only on the heterochromatic Y chromosome. Among Triatomini species and R. prolixus (Rhodniini tribe), only repeated and scattered DNA sequences are shared. All species studied exhibit a strong hybridization pattern in the autosome chromosome and/or sex chromosome regions, except for R. prolixus which showed a scattered hybridization pattern without strong hybridization regions. These scattered signals in chromatin in all species show that transposition elements were an important component in genomes.¹⁸

Pita et al.¹⁹ using genomic probes of T. dimidiata and Triatoma rubrofasciata (De Geer, 1773) observed Y chromosome–restricted GUISH markings for T. dimidiata, T. lecticularia (Stål, 1859), and Triatoma nitida (Usinger, 1939), whereas M. spinolai and Triatoma barberi (Usinger, 1939) showed Y-marking and one of the X chromosomes. Triatoma infestans was already in the Y mark and three autosomes (using T. dimidiata probe), and in the Y and five autosomes (using T. rubrofasciata probe). Similarly, using genomic probes of T. infestans, Pita et al.¹⁹ observed markings restricted to the sex chromosomes of T. dimidiata and T. rubrofasciata.

Pita et al.^18,19 also suggested that X and Y sex chromosomes’ triatomines present differences in DNA composition because the authors confirmed that the Y chromosome of the North and South American Triatoma share repeated DNA sequences and that this Y heterochromatic chromosome conservation may be an ancestral characteristic of the Triatomini tribe. Although the X chromosome is euchromatic, it has no fluorescent staining, but in some species (T. barberi, Andean T. infestans, T. platensis, and T. delpontei), X chromosomes have heterochromatic regions similar to those observed in Y chromosomes, may be due to transfer of sequences from Y to X chromosome. Transfer of repeated sequences between sex chromosomes without recombination or pairing events may occur because of transposition elements, as it was already suggested by Pita et al.¹²

Pita el al.²⁰ performed analyses with three new satellite DNAs (satDNAs) in T. infestans using the FISH technique and revealed that these satDNAs are located in the euchromatic regions of the autosomes and the X chromosome. The authors also showed that only four satDNA families of the 42 families that were isolated of the T. infestans genome had some similarity to the R. prolixus genome. The data show that the differentiation between the species of triatomines was accompanied by major changes in their repetitive DNAs. One of the most striking differences between the hybridization patterns in these species is the uneven distribution of the four satDNA families on the Y chromosome. The role of satDNA in speciation processes has been a topic of discussion for a long time; several mechanisms have been proposed in which satDNA could interrupt chromosomal pairing in hybrids, acting as a reproductive barrier.^20–22

Genomics.

The first use of genomic techniques for triatomines was in 1998, where mitochondrial DNA sequences from the 12S, 16S genes, and the genes encoding cytochrome oxidase I were used to establish phylogenetic relationships between Triatoma species.²³ In 1999, Lyman et al.²⁴ used fragments of the mitochondrial large subunit ribosomal RNA (mtlsurRNA) and cytochrome B (mtCytB) to differentiate triatomine species. The following year, Monteiro et al.²⁵ used the same gene fragments (mtlsurRNA and mtCytB) as well as the 28S nuclear RNA variable region D2 to establish phylogenetic analyses among 11 Rhodnius species. However, it was not until 2001 that the first complete mitochondrial genome of a triatomine species (T. dimidiata) was published and the analysis of the mitochondrial genome supported the theory that crustaceans and insects are more related to each other than to other arthropods, providing more information on the phylogenetic position of arthropods.²⁶

In 2015, the complete genome of a triatomine species (R. prolixus) was sequenced.⁶ According to the analyses performed on its genome, transposable elements (TEs) (5.6%) were identified; immune-mediated immune responses (IMD) and toll effectors (immune pathway) and IMD were found to control the intestinal microbiota of the insect, but do not affect the development of T. cruzi. Furthermore, the work showed that R. prolixus lost selenoprotein-related genes and has only two proteins related to glutathione peroxidase. Many lineage-specific gene expansions and absence of genes have been observed in these triatomines through protein comparison (these expansions are from genes that are related to chemoreception, feeding, and digestion, and may have contributed to the evolution of lifestyle of these insects).⁶

Castro et al.²⁷ analyzed the genome of Rhodnius montenegrensis Rosa et al. (2012) and Rhodnius marabaensis Souza et al. (2016) and reanalyzed the totality of repetitive genome sequences (repeatome) of R. prolixus. The analyses revealed that the total amount of TEs present in Rhodnius genomes (19–23.5%) is three to four times greater than that expected, based on the quantifications already performed for R. prolixus by Mesquita et al.⁶ The three species showed similar proportions of TEs in their genomes: 19% in R. prolixus, 21.2% in R. marabaensis, and 23.5% in R. montenegrensis. Of the TEs listed, the most abundant were classified as DNA transposons (class II), with 61.4% of the total TEs analyzed being correspondent to the genome of R. prolixus, 63% to that of R. montenegrensis, and 66.3% to that of R. marabaensis.²⁷ Long terminal repeat retrotransposons (class I) were the least represented class of TEs, with a minimum value of 0.8% in R. prolixus and R. marabaensis and a maximum of 1.1% in R. montenegrensis.²⁷

Comparative analyses between the three Rhodnius genomes based on the presence/absence pattern showed that a high percentage of TE families is shared between the pairs of genomes (74% for R. prolixus–R. montenegrensis; 76% for R. prolixus–R. marabaensis; and 80% for R. montenegrensis–R. marabaensis).²⁷ The three genomes compared revealed three characteristics necessary to understand the evolutionary dynamics of their repeatome: 1) the size of the TE families shared between the genomes is highly correlated, both in high and low abundance; 2) several identical families are present in very different proportions between pairs of genomes; and 3) some families seem unique to a species, but this event is observed only for families with very low proportion estimates.²⁷ The comparable proportions of TEs noted in the three species suggest that stochastic events and/or different selective pressures acting during speciation did not affect the general evolutionary routes of TEs. Alternatively, they could have provided the molecular basis that led to the radiation of the R. prolixus–Rhodnius robustus species complex.²⁷

The mitochondrial genome of five other species has already been sequenced: Triatoma migrans Breddin, 1903, Panstrongylus rufotuberculatus (Champion, 1899), Rhodnius pictipes Stål, 1872, T. infestans, and T. rubrofasciata.^28–30 For T. infestans, the number, order, and orientation of mitochondrial genes are the same as T. dimidiata; the main differences are found in the control region and in the intergenic spacer between the nd1 and tRNA-Ser genes.²⁸ The T. rubrofasciata species was characterized through genomic analysis and the phylogenetic tree with the genes that encode T. rubrofasciata proteins, and 13 other related species of hemiptera established and demonstrated the phylogenetic relationships.²⁹ For T. migrans, P. rufotuberculatus, and R. pictipes, it can be observed that mitochondrial genomes shared a similar pattern (nucleotide composition, gene order, and control region structure), a high conservation of genome organization, that different genes protein coders have different rates of molecular evolution and some genes had higher evolutionary rates.³⁰ Furthermore, genome and molecular phylogeny analysis using all available genomes of Reduviidae species allowed the authors to confirm a close relationship between Triatominae and Stenopodainae.^28–30

Recently, Liu et al.³¹ analyzed a chromosomal-level genome assembly of the T. rubrofasciata. The application of a combination of techniques allowed researchers to assemble the genome with higher quality than the other sequenced and assembled genomes. Protein sequences of the single copy genes were aligned along with molecular clock data and divergence time analysis, phylogenetic relationships were constructed to conclude that T. rubrofasciata is closely related to R. prolixus, and the two species diverged from their common ancestor about 60–95 million years ago.³¹ In addition, the work of Liu et al.³¹ resulted in a successfully reconstructed entire genome assembly (680-Mb) that covers 90% of the nuclear genome (757 Mb) and also reconstructed complete female chromosomes using 13 unique chromosomes.

Henriques et al.³² showed that the number and diversity of peptidases found in R. prolixus are within the normal values found in arthropods. These proteins are essential for various functions in animal metabolism (like digestion, blood clotting, and immune system), which explain the high diversity of this type of molecule among different arthropods.³² When the authors compared the frequencies of the R. prolixus peptidase families with the other genomes of arthropods, only eight families had higher gene frequencies, suggesting that, during the evolution of hematophagy in this group, gene duplication for new physiological functions occurred only on a limited set of proteases.³² Genetic expansion was observed in three families with a large number of peptidases in the R. prolixus genome, namely, pepsin A, calpain-2, and leucyl aminopeptidase, which suggests that these families may be involved in the process of adapting to blood feeding in triatomines.³²

This study suggested that there was a horizontal transfer of six genes belonging to three different peptidase families associated with bacteria or viruses (M74, S24, and S29) to the R. prolixus genome, one these families are not common in any eukaryotic organism, but most of them have been found in genomic regions that are surrounded by genes associated with insect metabolism.³² By amplifying the genomic DNA of R. prolixus, the presence of these proteins was confirmed and they were expressed in the intestine of adults fed with blood. It is not clear how the genes can be transferred to the insect's genome from vertebrate viruses, but the authors suggest that the infection may occur during blood meal in a contaminated vertebrate host.³² The RPRC003168 gene was identified in R. prolixus genome as a peptidase of the M74 family, and this peptidase is closely related to genes of Gram-negative bacteria, such as Dickeya spp. and Yersinia spp., that may be associated with hematophagous arthropods.³² The similarity reinforces the hypothesis of horizontal gene transfer from the microorganism to the R. prolixus genome because this type of bacteria had previously been implicated in horizontal gene transfer in arthropods.³³