PHYLOGENETIC APPROACH TO THE STUDY OF TRIATOMINES (, HETEROPTERA)

TARTAROTTI, E.1, Azeredo-Oliveira, M. T. V.1 and Ceron, C. R.2 1Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas – IBILCE/UNESP, São José do Rio Preto, SP 2Departamento de Química e Ciências Ambientais, Instituto de Biociências, Letras e Ciências Exatas – IBILCE/UNESP São José do Rio Preto, SP Correspondence to: Ester Tartarotti and Carlos Roberto Ceron, Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto – IBILCE-UNESP, Rua Cristóvão Colombo, 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil, e-mail: [email protected]; [email protected] Received June 9, 2004 – Accepted October 4, 2004 – Distributed May 31, 2006

Abstract Triatomines are belonging to the order, Heteroptera suborder, family and Triatominae subfamily. All members of this subfamily are hematophagous. Triatomines evolved from Reduviidae predators and they are probably polyphyletic in origin. The combination of anatomical, physiological and ethological factors observed in this group, as well as the plesiomorphic and apomorphic characters that differentiate the five tribes and fourteen triatomine genera reinforce the polyphiletic hypotesis. However if we consider the five groups of triatomines, the Rhodniini, Cavernicolini, Bolboderini, Linshcosteini and Alberproseniini tribes constitute monophyletic groups, while the Triatomini tribe is considered polyphyletic. The New World is the center of triatomine diversity and seems to be the point of group origin. Of approximately 137 triatomine species, 105 are only found in the Americas. It is now considered that triatomines represent a polyphyletic group defined according to their convergent apomorphic hematophagous characters, which have appeared several times in Reduviidae. This study revises the phylogeny of these vectors of Chagas’ disease, covering such topics as the origin of in triatomines and ancestral proposal for the group. Keywords: Heteroptera, Triatominae, Reduviidae, phylogeny.

Resumo Abordagem filogenética emT riatomíneos (Triatominae, Heteroptera) Os triatomíneos são insetos pertencentes à ordem Hemiptera, subordem Heteroptera, família Reduviidae e subfamília Triatominae. Todos os membros desta subfamília são hematófagos. Os triatomíneos surgiram a partir de reduvídeos predadores e provavelmente têm origem polifilética. A combinação dos fatores anatômicos, fisiológicos e etológicos presentes no grupo, bem como os caracteres plésio e apomórficos que diferenciam as cinco tribos e os quatorze gêneros de triatomíneos reforçam a hipótese polifilética. As tribos Rhodniini, Cavernicolini, Bolboderini, Linshcosteini e Alberproseniini constituem grupos monofiléticos, per si, enquanto a tribo Triatomini é considerada polifilética. O Novo Mundo é claramente o centro de diversidade dos triatomíneos e possivelmente é a região de sua origem. Entre as aproximadamente 129 espécies desses insetos, 105 ocorrem somente nas Américas. Atualmente, os triatomíneos são considerados um grupo polifilético, definido com base em seus caracteres apomórficos convergentes relacionados à hematofagia. Acredita-se que este hábito alimentar tenha surgido várias vezes nos Reduviidae durante sua evolução. O presente trabalho faz uma revisão sobre a filogenia destes vetores da Doença de Chagas, aborda tópicos como a origem da hematofagia nos triatomíneos e ancestralidade proposta para o grupo. Palavras-chave: Heteroptera, Triatominae, Reduviidae, filogenia.

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Triatomines are insects belonging to the of these insects had no anesthetic properties, the Hemiptera order, Heteroptera suborder, Redu­ triatomines would have been driven to feed on viidae family and Triatominae subfamily. All newly born vertebrates, which would be attacked members of this subfamily are hematophagous, in a special form of . Later in this which is considered to be a recent characteristic phase, starting with for hemolysis, in evolutionary terms. In relation to the the hematophagous process would have begun and phylogenesis of triatomines, it is interesting (Carcavallo et al., 1999). to point out that the Hemiptera order has To avoid the predatory vertebrates in the dispersed representatives throughout the tropical nests and burrows, it was necessary to make and temperate regions. In this order more than adaptations, such as cryptic behavior and inverse 80 thousand species are described. Traditionally, activity pattern, for feeding while the vertebrate Hemiptera is divided into two suborders, Homoptera is asleep. In predators, the saliva has a proteolytic and Heteroptera. effect, a characteristic that was lost by most of Some Homoptera and most Heteroptera are the hematophagous insects to make it possible to adapted to feeding on plant sap. Some insects ingest by a painless bite. Hematophagy also of the Heteroptera suborder are predators on requires a rapid compensation of the enormous insects and on other invertebrates, sucking their amount of blood that triatomines ingest. The hemolymph, while other Heteroptera have become therefore excretes great amounts of water and hematophagous as for instance the Triatominae salts immediately to reduce its weight. Another subfamily (Schofield & Dolling, 1993). to hematophagy is the erythrocytic Reduviidae is an ancient family, with fossil rupture and hemolytic process at the beginning of remains suggesting that the initial predatory forms digestion (Carcavallo et al., 1999). might have derived from Hemipteran during the Primitive predatory behavior still occurs Permian/Triassic period. These forms preceded in many triatomines species, including the hematophagous group which probably appea­ T. rubrofasciata, which feeds on caterpillars, red together with the first and T. rubrovaria which can feed on spiders and during the Jurassic period, 50 million years ago. silkworm and T. circummaculata, which feeds Reduviidae predators are widely distributed and on vertebrates blood and cockroach hemolymph. their fossil remains have been found in amber from Young nymphs of Eratyrus mucronatus prefe­ the Oligocene and Eocene periods (25-65 million rentially feed on invertebrate , while years B.P.). In contrast, there is no evidence of nymphs in more advanced stages and adults feed fossil remains of hematophagous forms from those on vertebrate’s blood. Cannibalistic behavior can periods (Schofield, 2000). be a transitional stage between predation and It may be suggested that primitive predators hematophagy. There are reports of nymphs sucking and hematophagous Heteroptera developed from blood from other nymphs in laboratory colonies. sap sucking insects (phytophagous), the Proto- Such cleptohematophagous behavior occurs in Heteroptera, undergoing modifications in their Belminus herreri which obtains blood from species buccal and digestive apparatus. The terrestrial of recently fed Rhodnius. In short, all of these groups developed into two main branches, observations suggest that hematophagy is a recent Pentatomorpha and . These groups characteristic in triatomines and that adaptations to gave rise to predatorial forms such as , this habit are still occurring. Triatomines are little and hematophagous forms like Triatominae. different from Reduviidae predators, in habitat and Triatomines probably evolved from Redu­ forms, which also corroborates the argument that viidae predator groups. The Reduviidae early this group is a recent one. For mammals, the bite on in their possibly fed on soft forms from Reduviidae predators tends to be very painful of invertebrate animals that inhabited vertebrate and can cause death, especially by anaphylactic nests, such as caterpillars, larvae and spiders. Later shock in small animals. The same happens in they began to attempt perforating the skin of small the case of certain triatomines. For instance, the vertebrates. It is possible that, in the first phase, bite of P. geniculatus in pigs and humans in the hematophagy was optional, and, since the saliva Amazon leaves painful lesions and, in the case of

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T. rubrofasciata, there has been at least one report adaptation to different habitats. The comparison of human death (Schofield, 2000). between population and behavioral parameters, Gorla et al. (1997) consider that triatomines association with vertebrates and habitat, as well are polyphyletic in origin and they believe that as their biogeographical characteristics support hematophagy have appeared recently, associated the hypothesis that triatomines probably appeared with the evolution of vertebrate nests. The several times within the Reduviidae and that they polyphyletic hypothesis suggests that the adaptative represent species of polyphyletic origin, based steps from free life predators to hematophagous on their apomorphic character with relation to feeding might have occurred several times, not hematophagy (Schofield, 1988; Lymanet al., 1999; only among different groups of Reduviidae, but Bargues et al., 2000, Marcilla et al., 2001). also among other Hemiptera groups. The New World is clearly the center of This hypothesis may explain the close triatomine origin and diversity. Of the approxi­ relationship between genera and species of tria­ mately 137 triatomine species (Galvão, et al. tomines associated with certain vertebrates. For 2003), 105 occur in this area. Of the fourteen instance, Psammolestes associated with ’s nests, genera, twelve are found exclusively in America: Dendrocolaptidae, Cavernicola pilosa barber with Alberprosenia, Belminus, Bolbodera, Cavernicola, Chiroptera, Microtriatoma with the biocenosis of , Eratyrus, Microtriatoma, the great Bromeliads, P. geniculatus associated with Panstrongylus, Parabelmintos, Paratriatoma, the Edentates, and some species of the Triatoma Psammolestes and Rhodnius. Only two genera, protracta complex associated with the Neotoma Linshcosteus and Triatoma, occur in the Old World, genus. The polyphyletic hypothesis also helps to and the Triatoma is also found in the New World. explain most of the anatomical differences found The Linshcosteus genus, with five species, is between some tribes and their notable similarity confined to the Indian subcontinent, seven species with taxa of other Reduviidae subfamilies. For of Triatoma are present in Southeast Asia and example, Alberproseniini possesses morphological one species, T. rubrofasciata, is cosmopolitan in characteristics of the Cetherinae and Psammolestes the tropics. Its wide distribution can be explained possesses anatomical characteristics present in by marine transport from the XVII century to the the Physoderinae subfamily. Among the most early XX century. This species is also present in the convincing studies, it has been discovered that Brazilian northeast (Schofield & Dolling, 1993). there are fundamental differences in salivary T. rubrofasciata is considered to be an ancestor components between Rhodniini and Triatomini, of the other seven Triatoma species in Southeast as well as differences in sensorial patterns, Asia (T. Amicitiae, bouvieri, cavernicola, leopoldi, suggesting different origins for these two tribes. migrans, pugasi, sinica) because they share Therefore, the Triatominae subfamily should be morphological characteristics and are all included assumed, more correctly, to be a utilitarian group, in the Rubrofasciata group. Another interesting defined on the basis of their hematophagous habits characteristic that confirms the hypothesis that and adaptations associated to this diet, and not a T. rubrofasciata is an older species is related to its cladistic classification of individuals sharing a painful bite, considered a primitive characteristic common ancestry (Carcavallo et al., 1999). (Schofield, 1988). Some authors, including Usinger et al. The almost total absence of triatomines (1966) believe, however, that the triatomines in Africa, except T. rubrofasciata, probably represent a monophyletic group and that their brought to African ports by ships, suggests that hematophagy have appeared only once. Gaunt & the hematophagous evolution of Reduviidae Miles (2000) also postulate that the triatomines are in Africa was inhibited by the evolution of the of monophyletic origin, based on the appearance of hematophagous Anthocorideos, now known a salivary (anti-thrombin). as Cimicidae, which had already occupied the The monophyletic hypothesis is not only available niches. The high degree of morphological difficult to support, but it also causes problems specialization of Cimicidae suggests that they arose in the understanding of the insects’ distribution, prior to the triatomines and that the latter evolved association with animals, source of feeding and independently in America after the separation of

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the continents. This hypothesis is better than the around 85% of variable positions in the alignment view that triatomines may have appeared in Africa between the analyzed species of Rhodnius and and, subsequently, become locally extinguished Triatoma (Bargues et al., 2000). The analyses of the (Schofield, 2000). ITS-2 region of these tribes also presented The dispersion of triatomines by vertebrates significant differences in sequence lengths. Due to was studied on R. prolixus in Central America. It the high variation between the sequence alignments is believed that these insects migrated from South the tribes were considered separately (Marcilla America to Central America, transported by birds. et al., 2001). AFLP analysis of ITS-1 ribosomal Enzymatic and RAPD (Random Amplification of transcribed DNA intergenic spacer on Rhodnius, Polymorphic DNA) analyses corroborated this Triatoma and Panstrongylus species reinforced the view, the limited genetic variability denoting the idea of polyphyletic origin between Rhodniini and recent origin of populations from South America Triatomini tribes (Tartarotti & Ceron, 2005). (Dujardin et al., 1998). The monophyletic origin is well understood Similarly, the presence of in the Rhodnius genus. This group of species in Mexico is associated with the migration of possesses similar characteristics and would have vertebrates. The expansion and distribution of originated from a common ancestral species. It is T. infestans, for example, is closely related to postulated that the Rhodnius genus appeared in the human activity (Schofileld, 1988). The species Amazonian region during the Quaternary period. is endemic in Bolivia and suffered dispersion by Probably, the closest species of the Reduviidae human action, their domiciliary invasion obeying ancestry is the R. pictipes species, which inhabits an opportunist mechanism provided by the stimulus the Bromeliads and a variety of palm tree. Other of shelter and feeding (Forattini, 1980). species of Rhodnius are more specialized and In the triatomine group, the Rhodniini, can be considered to be derived from R. pictipes Cavernicolini, Bolboderini, Alberproseniini and (Schofield & Dujardin 1999). Linshcosteini tribes constitute monophyletic The similarity between Rhodnius species is groups, that is, each tribe possesses an ancestral in so great that one species is frequently mistaken common, while the Triatomini tribe is considered for another, due to their morphologic similarity. to be a polyphyletic group, with several ancestral R. prolixus, R. robustus, R. neglectus and giving origin to the tribe (Lent & Wygodzinsky, R. nasutus belong to the Prolixus complex, it 1979; Galvão et al., 2003). For example, the being difficult to differentiate R. robustus from Panstrongylus species is included in the Triatomini R. prolixus. The Peruvian R. robustus populations tribe. Phylogenetic analyses revealed that the are inter-fertile with R. prolixus populations from Triatoma and Panstrongylus genera were more Honduras, Colombia and Venezuela. Until now closely related to each other than when compared 13 Rhodnius species have been described with the Rhodnius genus (Stothard et al., 1998). (R. brethesi, R. dalessandroi, R. domesticus, Studies in nuclear rDNA ITS-2 sequences R. ecuadoriensis, R. nasutus, R. neglectus, revealed a possible polyphyly of Panstrongylus R. neivai, R. pallescens, R. paraensis, R. pictipes, species. The species P. megistus, P. geniculatus, R. prolixus, R. robustus and R. stali). These P. rufotuberculatus, P. lignarius, P. herreri and species are classified in the Rhodniini tribe P. chinai were analysed and the significant together with the Psammolestes genus. The differences in length and nucleotide composition classification of Rhodiniini, as a monophyletic suggest a relatively old divergence of Panstrongylus tribe, takes into account characteristics of the species (Marcilla et al., 2002) Rhodnius genus not shared with other triatomines, Analyses of ITS-2 region and rDNA such as, apical antenna insertion, body forms, 18S indicated that the divergence between the post-ocular callosities, male genital characteristics, Triatomini and Rhodniini tribes possibly occurred egg surface architecture and nitroforine presence in in the lower Tertiary period and that the Triatomini the salivary glands. Besides these characteristics, tribe had a polyphyletic origin. The nucleotide the Rhodnius and Psammolestes genera are differences between the ITS-2 sequences of the primarily arboreal species in contrast with the Triatomini and Rhodniini tribes are numerous, terrestrial habits of most of the other triatomines

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