Populations, hybrids and the systematic concepts of species and subspecies in Chagas disease triatomine vectors inferred from nuclear ribosomal and mitochondrial DNA.

In Chagas disease, triatomine vectors are the main target for control measures because of the absence of effective drugs. The broad usefulness of nuclear rDNA and mtDNA sequences explains why triatomine studies using these markers have increased so pronouncedly in recent years. This indicates the appropriateness of an updated review about these molecular markers, concentrating on aspects useful for research on Chagas disease vectors. A comparative analysis is presented on the efficiency, weight of their different characteristics, limitations and problems of each of the different DNA markers in the light of the results obtained in studies on populations, hybrids, subspecies and species of the subfamily Triatominae. The use of a standardized composite haplotype code nomenclature for both nuclear rDNA and mtDNA markers is strongly encouraged to avoid difficulties in comparative studies. Triatomine aspects related to concerted evolution, microsatellites, minisatellites and insertions/deletions in nuclear rDNA and silent/non-silent mutations, pseudogenes and weaknesses of partial sequences in mtDNA are analysed. Introgression and hybrids, nuclear and mitochondrial DNA strengths, and compared evolutionary rates of nuclear rDNA and mtDNA in triatomines are discussed. Many conclusions are obtained thanks to the availability, for the first time in triatomines, of a complete sequence of a protein-coding mtDNA gene as ND1 from very numerous triatomine species covering from different populations of a species up to members belonging to different tribes. The evolutionary rates of each nuclear rDNA marker and mtDNA marker are analysed by comparison at subspecies level (intrapopulational, interpopulational, between morphs, and between subspecies) and species level (close and distant species of the same genus, species of different genera, and species of different tribes). Weaknesses of mtDNA for systematic-taxonomic purposes detected recently and newly in insects and triatomines, respectively, are discussed in detail. Emphasis is given to taxonomic units and biological entities presenting well-known problematics, both from the systematic-taxonomic and/or epidemiological-control points of view, as well as to molecular situations which can give rise to erroneous conclusions. All these aspects constitute the background on which the key question about the systematic concepts of species and subspecies in triatomines is focused. The global purpose is to facilitate future work on triatomines by highlighting present gaps, how better choice the appropriate markers, and marker aspects which should be taken into account. Key characteristics as alpha, CI and transformation rate matrices ought to be obtained and noted to get appropriate results and allow correct interpretations. The main aim is to offer a baseline for future fundamental research on triatomines and applied research on transmission, epidemiology and control measures related to Chagas disease vectors.

Mas-Coma S ，Bargues MD 《-》

Nuclear rDNA pseudogenes in Chagas disease vectors: evolutionary implications of a new 5.8S+ITS-2 paralogous sequence marker in triatomines of North, Central and northern South America.

A pseudogene, paralogous to rDNA 5.8S and ITS-2, is described in Meccus dimidiata dimidiata, M. d. capitata, M. d. maculippenis, M. d. hegneri, M. sp. aff. dimidiata, M. p. phyllosoma, M. p. longipennis, M. p. pallidipennis, M. p. picturata, M. p. mazzottii, Triatoma mexicana, Triatoma nitida and Triatoma sanguisuga, covering North America, Central America and northern South America. Such a nuclear rDNA pseudogene is very rare. In the 5.8S gene, criteria for pseudogene identification included length variability, lower GC content, mutations regarding the functional uniform sequence, and relatively high base substitutions in evolutionary conserved sites. At ITS-2 level, criteria were the shorter sequence and large proportion of insertions and deletions (indels). Pseudogenic 5.8S and ITS-2 secondary structures were different from the functional foldings, different one another, showing less negative values for minimum free energy (mfe) and centroid predictions, and lower fit between mfe, partition function, and centroid structures. A complete characterization indicated a processed pseudogenic unit of the ghost type, escaping from rDNA concerted evolution and with functionality subject to constraints instead of evolving free by neutral drift. Despite a high indel number, low mutation number and an evolutionary rate similar to the functional ITS-2, that pseudogene distinguishes different taxa and furnishes coherent phylogenetic topologies with resolution similar to the functional ITS-2. The discovery of a pseudogene in many phylogenetically related species is unique in animals and allowed for an estimation of its palaeobiogeographical origin based on molecular clock data, inheritance pathways, evolutionary rate and pattern, and geographical spread. Additional to the technical risk to be considered henceforth, this relict pseudogene, designated as "ps(5.8S+ITS-2)", proves to be a valuable marker for specimen classification, phylogenetic analyses, and systematic/taxonomic studies. It opens a new research field, Chagas disease epidemiology and control included, given its potential relationships with triatomine fitness, behaviour and adaptability.

Bargues MD ，Zuriaga MA ，Mas-Coma S 《-》

The ITS-2 of the nuclear rDNA as a molecular marker for populations, species, and phylogenetic relationships in Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease.

:The nucleotide sequences of the rDNA second internal transcribed spacer (ITS-2) of 31 populations of 12 and 3 species of the two main Triatominae tribes Triatomini and Rhodniini, including the most important Chagas disease vectors, were obtained. Sequence comparisons and parsimony, distance, and maximum-likelihood analyses indicate that ITS-2 is a useful marker for resolving supraspecific, specific, subspecific, and even sometimes population-level relationships in Triatominae. Results were markedly different between species of Triatomini and Rhodniini, suggesting polyphyly. Phylogenetic trees support an old divergence between South American and North-Central American Triatomini and query the validity of some genera (Dipetalogaster, Psammolestes). The very low sequence variation between species of the phyllosoma complex suggests that subspecific ranking would be more appropriate. Triatoma dimidiata proves to be a clearly differentiated species, with several populations evidencing a clinal variation along a north-south axis and a population from Yucatan showing differences consistent with specific status.

Marcilla A ，Bargues MD ，Ramsey JM ，Magallon-Gastelum E ，Salazar-Schettino PM ，Abad-Franch F ，Dujardin JP ，Schofield CJ ，Mas-Coma S ... -  《MOLECULAR PHYLOGENETICS AND EVOLUTION》

Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size.

For about half of all Chagas disease cases T. infestans has been the responsible vector. Contributing to its genetic knowledge will increase our understanding of the capacity of geographic expansion and domiciliation of triatomines. Populations of all infestans subcomplex species, T. infestans, T. delpontei, T. platensis and T. melanosoma and the so-called T. infestans "dark morph", from many South American countries were studied. A total of 10 and 7 different ITS-2 and ITS-1 haplotypes, respectively, were found. The total intraspecific ITS-2 nucleotide variability detected in T. infestans is the highest hitherto known in triatomines. ITS-1 minisatellites, detected for the first time in triatomines, proved to be homologous and thus become useful markers. Calculations show that ITS-1 evolves 1.12-2.60 times faster than ITS-2. Despite all species analyzed presenting the same n=22 chromosome number, a large variation of the haploid DNA content was found, including a strikingly high DNA content difference between Andean and non-Andean specimens of T. infestans (mean reduction of 30%, with a maximum of up to 40%) and a correlation between presence/absence of minisatellites and larger/smaller genome size. Population genetics analysis of the eight composite haplotypes of T. infestans and net differences corroborate that there are clear differences between western and eastern populations (60%), and little genetic variation among populations (1.3%) and within populations (40%) within these two groups with migration rates larger than one individual per generation corresponding only to pairs of populations one from each of these groups. These values are indicative either of a large enough gene flow to prevent population differentiation by drift within each geographic area or a very recent spread, the latter hypothesis fitting available data better. Phylogenetic trees support a common ancestor for T. infestans and T. platensis, an origin of T. infestans in Bolivian highlands and two different dispersal lines, one throughout Andean regions of Bolivia and Peru and another in non-Andean lowlands of Chile, Paraguay, Argentina, Uruguay and Brazil.

Bargues MD ，Klisiowicz DR ，Panzera F ，Noireau F ，Marcilla A ，Perez R ，Rojas MG ，O'Connor JE ，Gonzalez-Candelas F ，Galvão C ，Jurberg J ，Carcavallo RU ，Dujardin JP ，Mas-Coma S ... -  《INFECTION GENETICS AND EVOLUTION》

Ribosomal DNA ITS-1 intergenic spacer polymorphism in triatomines (Triatominae, Heteroptera).

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.

Tartarotti E ，Ceron CR 《BIOCHEMICAL GENETICS》