High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae).

The subfamily Triatominae (Hemiptera-Reduviidae) includes more than 150 blood-sucking species, potential vectors of the protozoan Trypanosoma cruzi, causative agent of Chagas disease. A distinctive cytogenetic characteristic of this group is the presence of extremely stable chromosome numbers. Unexpectedly, the analyses of the chromosomal location of ribosomal gene clusters and other repetitive sequences place Triatominae as a significantly diverse hemipteran subfamily. Here, we advance the understanding of Triatominae chromosomal evolution through the analysis of the 45S rDNA cluster chromosomal location in 92 Triatominae species. We found the 45S rDNA clusters in one to four loci per haploid genome with different chromosomal patterns: On one or two autosomes, on one, two or three sex chromosomes, on the X chromosome plus one to three autosomes. The movement of 45S rDNA clusters is discussed in an evolutionary context. Our results illustrate that rDNA mobility has been relatively common in the past and in recent evolutionary history of the group. The high frequency of rDNA patterns involving autosomes and sex chromosomes among closely related species could affect genetic recombination and the viability of hybrid populations, which suggests that the mobility of rDNA clusters could be a driver of species diversification.

High dynamics of rDNA cluster location in kissing bug holocentric chromosomes (Triatominae, Heteroptera).

In this paper, we determine by fluorescent in situ hybridization the variability in the chromosomal location of 45S rDNA clusters in 38 species belonging to 7 genera of the Triatominae subfamily, using a triatomine-specific 18S rDNA probe. Our results show a striking variability at the inter- and intraspecific level, never reported so far in holocentric chromosomes, revealing the extraordinary genomic dynamics that occurred during the evolution in this group of insects. Our results also demonstrate that the chromosomal position of rDNA clusters is an important marker to disclose chromosomal differentiation in species karyotypically homogenous in their chromosome number.

Inheritance of resistance to pyrethroids in Triatoma infestans, the main Chagas disease vector in South America.

An outbreak of pyrethroid resistance was recently detected in Triatoma infestans from northern Argentina. To analyze the inheritance of the resistant phenotype, we carried out experimental crosses between resistant (R) and susceptible (S) strains captured in Argentina during 2005. The R strain was collected from sprayed houses in the north of the province of Salta while the S strain was collected in the province of Chaco. Both strains were bred in the laboratory for reciprocal crosses (F1), intercrosses (F2) and backcrosses (BC). The descendents were tested by a standard insecticide resistance bioassay. Resistance ratios were 1 for S strain, 103.36 for R strain and 18.34 for F1. The regression lines of F1 generations (R×S and S×R) showed no significant differences and were closer to that of the R parents, indicating that inheritance of deltamethrin resistance in T. infestans is autosomal and incompletely dominant (D=0.20). Chi-square analysis from responses of intercross and backcross progenies rejected the hypothesis of a single gene being responsible for resistance. The minimum number of independent segregation genes was three, as calculated with Lande's method. The genetic basis here described for the resistant phenotype indicate that, under pyrethroid selective pressure, the resistant genotypes could be easily spread to susceptible insects from resistant individuals, posing a major threat to vectorial control of Chagas disease.

Systematics of Mepraia (Hemiptera-Reduviidae): cytogenetic and molecular variation.

The haematophagous insects of the subfamily Triatominae (Hemiptera-Reduviidae) have great epidemiological importance as vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Mepraia was originally described as a monotypic genus comprised of Mepraia spinolai, distributed along coastal areas of northern Chile (from Region I to the Metropolitan Region). Recently, some M. spinolai populations have been ranked as a new species named Mepraia gajardoi. Several populations along the distribution range of the genus were sampled, and genetic differentiation was studied based upon the analysis of three molecular markers: cytogenetics (karyotype and chromosome behaviour during meiosis using the C-banding technique), mitochondrial DNA (a cytochrome oxidase I gene fragment), and nuclear ribosomal DNA (intergenic region including the two internal transcribed spacers ITS-1 and ITS-2 and the 5.8S rRNA gene). The data here presented indicate that populations within the Mepraia genus (excluding Region II specimens) can be divided into two separate lineages. One lineage is comprised of specimens from the northernmost Region I and represents M. gajardoi. The other includes samples from the southern III, IV and the Metropolitan Regions, and represents M. spinolai. Region II individuals deserve particular attention as their relationship to the two identified lineages is not clear-cut. While they appear to belong to M. spinolai based on cytogenetics and rDNA markers, COI results indicate a closer relationship to M. gajardoi. This disagreement can be due to mitochondrial DNA introgression or the retention of ancestral polymorphisms.

Genetic variability of Triatoma rubrovaria (Reduviidae: Triatominae) from Brazil, Argentina and Uruguay as revealed by two different molecular markers.

Randomly amplified polymorphic DNA (RAPD) and nuclear ribosomal DNA sequence analyses were used to assess the genetic population structure of the South American triatomine species Triatomo rubrovario throughout its geographical distribution. To investigate the genetic variability at both intraspecific and intrapopulational levels the RAPD profiles and the nucleotide sequences of the rDNA intergenic spacers, ITS-1 and ITS-2, were analysed and compared. The phenetic analysis based on RAPD profiles show three distinct clusters diverging by similarity coefficients ranging from 0.62 to 0.96. The ITS-1 and ITS-2 sequence variability detected may be considered very high, suggesting reproductive isolation between populations. A total of seven composite haplotypes (CH) were found, among which three are specific for Brazil, other three for Uruguay, and the last one common for the three countries studied. The population studied in Argentina does not represent an independent CH. Sequence analyses proved that the five populations studied are easily differentiable and that there is heterogeneity within each one. True mutations and indels are the responsible of sequence differences between haplotypes and populations, suggesting that divergence processes may presently go on within this species. The large intraspecific variability detected may underlie the known plasticity of T. rubrovaria, making it a potential intradomiciliary invader and consequently an appropriate vector for Chagas disease transmission. Therefore, this triatomine species must be continuously monitored throughout.

Evolutionary relationships based on genetic and phenetic characters between Triatoma maculata, Triatoma pseudomaculata and morphologically related species (Reduviidae: Triatominae).

The maculata group currently comprises two species of Triatominae, Triatoma maculata and Triatoma pseudomaculata, which share morphologic and chromatic characteristics. In order to clarify the systematic status of these two vectors of Trypanosoma cruzi and to infer their evolutionary relationships, we performed an enzymatic, morphometric and cytogenetic comparison of them, also taking into account two sister species not included in the group (T. arthurneivai and T. wygodzinskyi). According to our results, T. maculata and T. pseudomaculata belong to distinct evolutionary lineages. Similarly, T. arthurneivai topotypes from Minas Gerais form an independent isolated group by morphometrics. Our results also support the specific status of the Triatoma population from São Paulo State (formerly referred to T. arthurneivai), and suggest the possibility that it is T. wygodzinskyi. Finally, we suggest that only the arboricolous T. pseudomaculata from northeast Brazil and the rupicolous sister species originated from São Paulo State should be classified together in the same group.

Chromosomal variation and genome size support existence of cryptic species of Triatoma dimidiata with different epidemiological importance as Chagas disease vectors.

The wide geographical distribution of Triatoma dimidiata, one of the three major vectors of Chagas disease, ranges from Mexico to northern Peru. Since this species occupies a great diversity of artificial and natural ecotopes, its eradication is extremely difficult. In order to assist control efforts, we used chromosome analyses and DNA amount as taxonomic markers to study genetic variability in populations of T. dimidiata from Mexico, Guatemala, El Salvador and Colombia. We differentiated three groups or cytotypes defined by characteristic chromosome C-banding patterns and genome size measured by flow cytometry. The three cytotypes are restricted to different geographic locations. Cytotype 1 occurs in Mexico (excluding Yucatán), Guatemala (excluding Petén), El Salvador and Colombia. Cytotype 2 occurs in Yucatán and cytotype 3 occurs in Petén. Cytotype 1, commonly associated with domestic and peridomestic environments but also inhabiting sylvatic ecotopes, is the most widespread and with major epidemiological significance. In contrast, the Yucatán cytotype inhabits wild ecotopes but increasingly enters houses, while the Petén cytotype appears exclusively sylvatic. We suggest that these cytotypes represent cryptic species of T. dimidiata with different epidemiological relevance as Chagas disease vectors. Poor ability to colonize human dwellings, together with their restricted geographic distribution, indicate that the Yucatán and Petén putative species probably have much less epidemiological significance than cytotype 1. Thus, the genetic markers we describe are powerful tools to differentiate cryptic species in T. dimidiata with different epidemiological significance, contributing to planning the most effective control measures.

Chromosome homogeneity in populations of Triatoma brasiliensis Neiva 1911 (Hemiptera - reduviidae - triatominae).

Triatoma brasiliensis is the most important vector of Chagas disease in the semiarid zone of the Northeast of Brazil. Several authors have reported the occurrence of four chromatic patterns with morphological, ecological, and genetic differences. In order to determine the existence of cytogenetic differentiation between these chromatic forms, we analyzed their karyotypes and the chromosome behavior during the male meiotic process. Triatoma brasiliensis shows distinct and specific chromosome characteristics, which differ from those observed in all other triatomine species. However, no cytogenetic differences were observed between the four chromatic forms of T. brasiliensis. The lack of chromosome differentiation among them could indicate that the populations of this species are in a process of differentiation that does not involve their chromosomal organization.

Meiosis in holocentric chromosomes: orientation and segregation of an autosome and sex chromosomes in Triatoma infestans (Heteroptera).

The meiotic behaviour of the X chromosome and one autosomal pair of the heteropteran Triatoma infestans was analysed by means of C-banding plus DAPI staining. At first metaphase, the X univalent is oriented with its long axis parallel to the equatorial plate, which suggests a holocentric interaction with the spindle fibres. After this initial orientation, kinetic activity is restricted to one of both chromatid ends. The election of the active chromatid end is random and it is independent of the end selected in the sister chromatid. At second metaphase, the X and Y chromatids associate side by side forming a pseudobivalent. After that, the kinetic activity is again restricted to either of both chromosomal ends in a random fashion. At first metaphase, the fourth autosomal bivalent shows two alternative random orientations depending on the chromosome end showing kinetic activity (DAPI positive or opposite). At second metaphase, half bivalents are oriented with their long axis parallel to the equatorial plate. Three different segregation patterns are observed. The kinetic activity can be localised: (i) in the end with the DAPI signal (46.9%), (ii) in the opposite end (44.6%) or (iii) in one DAPI-positive end in one chromatid and in the opposite end in the other one (8.5%). The existence of the last pattern indicates that the same end can show kinetic activity during both meiotic divisions. Our results provide new information on the comparative meiotic behaviour of autosomes and sex chromosomes in holocentric systems.

Sylvatic population of Triatoma infestans from the Bolivian Chaco: from field collection to characterization.

A sylvatic Triatoma infestans DM (dark morph) population detected in the Bolivian Chaco was characterized and compared with various domestic ones. The degree of differentiation of DM was clearly within the T. infestans intra-specific level. Nevertheless marked chromatic and morphometric differences as well as differences in antennal pattern, chromosome banding and randomly amplified polymorphic DNA support the hypothesis of a distinct population. Continuous exchange of insects between wild and domestic habitats seems unlikely in the Chaco.

Mitochondrial DNA variation of Triatoma infestans populations and its implication on the specific status of T. melanosoma.

DNA sequence comparison of 412 base-pairs fragments of the mitochondrial cytochrome B gene was used to infer the genetic structure of nine geographical Triatoma infestans populations and their phylogenetic relationship with T. melanosoma and T. brasiliensis. T. infestans and T. melanosoma were compared by morphometry, allozyme and cytogenetic analyses, as well as subjected to reciprocal crosses, in order to clarify the taxonomic status of the latter. No differences were found to distinguish the two species and the crosses between them yielded progeny. T. infestans populations presented four haplotypes that could be separated in two clusters: one formed by the samples from Bolivia (Andes and Chaco) and the other formed by samples from Argentina and Brazil. Silvatic and domestic T. infestans populations from Bolivia (Andes) were genetically identical.

Alary polymorphism in Triatoma spinolai and its possible relationship with demographic strategy.

Among collections of Triatoma spinolai from various sites in northern Chile, adults from coastal populations are invariably wingless, whereas inland populations show balanced alary polymorphism between wingless females and males that are either winged or wingless. Laboratory crosses showed that male offspring from normal-winged parents were always winged (88% long-winged) and those from long-winged male parents were all long-winged. The male offspring from wingless males always included winged males: 11/33 = 33%, of which 8/11 = 73% were long-winged. An X-linked mutation is proposed to inhibit wing development. Field studies of population demography indicate that male alary polymorphism is advantageous in the desert environment of northern Chile.

Genetic variability and geographic differentiation among three species of Triatomine bugs (Hemiptera-Reduviidae).

Three species of triatomine bugs, Triatoma sordida, T. guasayana, and T. patagonica, were examined by cytogenetic (C-banded karyotypes and male meiotic process) and isoenzymatic studies. These three species, with different importance as Chagas' disease vectors, were found to be closely related according to their known ethologic, ecologic, and morphologic traits. Although they have the same diploid chromosome number (2n = 22 constituted by 20 autosomes and an XY male/XX female sex mechanism), each species has a distinct chromosomal behavior during male meiosis and a specific amount and localization of C-heterochromatic blocks. Moreover, these chromosome characteristics allowed us to differentiate two T. sordida populations. Isoenzymatic data confirmed the taxonomic status of the three species and together with our cytogenetic results questioned the species homogeneity of T. sordida.

Chromosome numbers in the Triatominae (Hemiptera-Reduviidae): a review.

The chromosome numbers of 46 out of the 122 currently recognized species of Triatominae (Hemiptera, Reduviidae) are summarized. We present the number of autosomes, the sex mechanism and the first reference for each karyotype.

Karyotype evolution in holocentric chromosomes of three related species of triatomines (Hemiptera-Reduviidae).

C-banded karyotypes, DNA content and the male meiiotic process of Triatoma platensis and Triatoma delpontei are compared with those of Triatoma infestans, the main vector of Chagas disease in South America. These three species present the same diploid chromosome number 2n = 22 (20 autosomes + XX male/XY female). They also have several cytogenetic traits that differ from all other triatomines: large autosomes, C-heterochromatic blocks and meiotic heteropycnotic chromocenters formed by autosomes and sex chromosomes. In spite of these similarities, each species presents different chromosomal behavior during male meiosis, distinct DNA content and a specific amount and localization of the C-heterochromatin. The differences in DNA content are mainly due to the variation in C-heterochromatin amount, which may be interpreted as loss and/or gain of C-regions. This interpretation is supported by the presence of meiotic and mitotic chromocenters that facilitate the transference of C-positive material. The cytogenetic data presented in this work suggest that T. infestans and T. platensis are more closely related to each other than to T. delpontei. It can also be inferred that the differences in distribution and amount of heterochromatin do not play a direct role in speciation in this group.

Cytogenetics as a tool for triatomine species distinction (Hemiptera-Reduviidae).

Several cytogenetic traits were tested as species diagnostic characters on five triatomine species: Rhodnius pictipes, R. nasutus, R. robustus, Triatoma matogrossensis and T. pseudomaculata. Four of them are described for the first time. The detailed analysis of the meiotic process and the application of C-banding allowed us to identify seven cytogenetic characters which result useful to characterize and differentiate triatomine species.