Heredity 77 (1996) 47—54 Received 11 September 1995 Enzymatic variability and phylogenetic relatedness among Triatoma infestans, T platensis, T delpontei and T rubrovaria J. PEREIRA1, J. P. DUJARDIN*, A. SALVATELLA & M. TIBAYRENC UMR CNRS-ORS TOM 9926 'Gdnétique Mo/ecu/a/re des Parasites et des Vecteurs BP 5045, 34032, Montpelller cedex 1, France and Departamento de Paras/to/ogla, Instituto de Higiene, Universidad de Ia RepUb/ica, BP 11600, Montevideo, Uruguay. Fourcongeneric species of triato mine bugs, Triatoma platensis, T delpontei, T rubrovaria and T infestans, were examined by multilocus enzyme electrophoresis. These four species are distrib- uted sympatrically throughout Argentina, Paraguay and Uruguay, and were found to be closely related according to their known ethological, ecological and morphological traits. In order to evaluate previous hypotheses concerning the phylogenetic branching within this group, isoen- zyme patterns were submitted to classical phenetic and genetic clustering analysis. Results are discussed in the light of occasional natural gene flow between T infestans and T platensis, and indicate that ecology is the main factor explaining both their present status and their morpho- logical differentiation. Keywords:Chagasdisease, genetic distance, isoenzymes, Triatominae, Uruguay. transmission of human Chagas disease (Salvatella, Introduction 1986a,b, 1987); T platensis commonly infests the Intheir major review of the biosystematics of Tria- woven stick nests of furnariid weaver birds (Anum- tominae, Usinger et a!. (1966) grouped together the bius spp.) in northern Argentina, Paraguay, Uruguay four species considered in the present work, on the and southern Brazil, but has also been found in basis of their morphological, behavioural and peridomestic habitats such as chicken coops. Tria- ecological similarities. The four species share many toma delpontei seems more ecologically restricted, common features, particularly T platensis and T found almost exclusively in woven stick nests of delpontei (Lent & Wygodzinsky, 1979), although they colonial monk parrots (Myiopsitta monarcha), but are not difficult to distinguish by overt morpholog- occupying a similar geographical range to T ical characteristics such as connexival colour platensis. patterns and degree of pilosity (Lent & Wygod- The fourth member of the group, T rubrovaria, is zinsky, 1979). Triatoma infestans is an important usually found amongst limestone outcrops in vector of American trypanosomiasis (Chagas Uruguay and southern Brazil, often forming abun- disease) and is almost exclusively associated with dant colonies feeding on a wide range of rupicoline humans and domestic animals. It is widely distrib- vertebrates (Salvatella, 1988). However, it is increas- uted in the southern part of Latin America (Argen- ingly reported in domestic and peridomestic habitats tina, Bolivia, Brazil, Paraguay, Peru and Uruguay) in parts of Uruguay where T infestans has been and typically found in the cracks and crevices of eliminated by Chagas vector control activities (Salva- poor quality rural houses, emerging at night to feed tella et a!., 1991b; Salvatella, 1993). on the people and domestic animals sleeping there. Considering a large number of morphological, In contrast, T platensis and T delpontei are ornitho- physiological and behavioural characteristics, philic arboreal species, without relevance for the Usinger et al. (1966) concluded that T platensis and T delpontei were more closely related to each other *Correspondence than to T infestans. They suggested that T platensis tPresent address: Sección Genética Evolutiva, Facultad de Ciencias, Universidad de Ia Repüblica, BP 11200, Montevideo, and T delpontei were 'most probably derived one Uruguay. from the other or from a direct common ancestor'. 1996 The Genetical Society of Great Britain. 47 48 J.PER8RAETAL. Nevertheless, epidemiological considerations have led other authors to suggest that T infestans may Enzyme electrophoresis have arisen as a domesticated form of T platensis Analysiswas performed by electrophoretic separa- (Carcavallo et a!., 1986). Since then, no more data tion of isoenzymes on cellulose acetate plates have been added to improve our knowledge about (Helena Laboratories, Beaumont, TX). Seventeen this closely linked species group, except for a recent enzyme systems were assayed, as follows: aconitase study relating T. infestans with T. platensis on the (aconitate hydratase, EC 4.2.1.3, ACON); aspartate basis of their chromosome C-banding (Panzera et al., aminotransferase (also known as glutamate-oxalo- 1995). Isoenzyme markers, although they are consid- acetate transaminase) (EC 2.6.1.1, GOT); diaphor- ered to be more suitable for phylogenetic recon- ase (EC 1.8.1.4, DIA); glyceraldehyde-3-phosphate struction than phenotypic traits (Richardson et at., dehydrogenase (EC 1.2.1.12, GAPD); glucose- 1986), were not applied. 6-phosphate dehydrogenase (EC 1.1.1.49, G6PDH); The goal of the present work was to clarify the glycerol-3-phosphate dehydrogenase (EC 1.1.1.8, relationships within this group of species by using GPD); glucose-6-phosphate isomerase (EC 5.3.1.9, multilocus enzyme electrophoresis, and to see if the GPI); hexokinase (EC 2.7.1.1, HK); isocitrate enzyme patterns could be related to ecological dehydrogenase (EC 1.1.1.42, IDH); leucine amino- associations of the four species. peptidase (cytosol aminopeptidase) (EC 3.4.11.1, LAP); malate dehydrogenase (EC 1.1.1.37, MDH); Materials and methods malate dehydrogenase (malic enzyme) (EC 1.1.1.40, Insects ME); peptidase 1 (EC 3.4.11, PEP-i; leucyl-leucyl- leucine substrate); peptidase 2 (EC 3.4.11, PEP-2; The origins of the 1044 insects used in this study are leucyl-L-alanine substrate); peptidase 3 (EC 3.4.11, as follows. PEP-3; L-leucine-L-tyrosine substrate); phosphoglu- comutase [EC 5.4.2.2 (formerly EC 2.7.5.1), PGMJ; Triatoma infestans One hundred and thirty-one and 6-phosphogluconatedehydrogenase (EC specimens from Uruguay were collected from 1.1.1.44, 6-PGDH). domestic and peridomestic habitats in the Depart- Starved fifth instar nymphs and adults (both males ments of Soriano (south of Uruguay) and Rivera and females) were used. Head and thorax samples (North of Uruguay). The results obtained from these were ground in liquid nitrogen with a glass mortar in specimens were then plotted together with data an Eppendorf tube. Stabilizer was added (Godfrey previously obtained by Dujardin (1990) on 777 Boli- & Kilgour, 1976) and the rest of the material was vian T infestans using similar techniques. cut with entomological scissors until all the pieces has been crushed. Other conditions for sample Triatoma rubrovaria Thirty-one specimens were preparation, electrophoresis and enzyme staining collected from peridomestic habitats in the Depart- were as previously described by Dujardin & Tibay- ment of Artigas, Uruguay. Most were collected by renc (1985) except for GOT, HK, GAPD and DIA, the National Chagas Control Programme between which were processed following the techniques of 1989 and 1990. Richardson et at. (1986). Triatoma platensis Ninety-two specimens were studied: 82 were collected from nests of Anumbius Statisticalmethods annumbi in the Palmar de Quebracho region, Wecharacterized each specimen by its multilocus Department of Paysandü, Uruguay); the remaining genotype (MLG), represented by the different allelic 10 were collected from similar nests in the Barra do combinations (genotypes) found at all identified QuaraI region, State of RIo Grande do Sul, Brazil putative loci. For the whole sample, we could retain (Salvatella et al., 1991a). 53 different MLGs (Table 1) which were used for statistical analysis. Triatoma delpontei Thirteenspecimens were Our cladistic approach (Fig. 1) was based on the studied, of which 10 were laboratory-reared adults maximum parsimony method ('Wagner tree') of representing the fourth generation of material Farris (1970), and was computed using the software originally collected in Santiago del Estero, Argen- package PHYLIP 3.4 (Felsenstein, 1985). The 'inde- tina. Three further specimens were captured in a pendent alleles' coding of electrophoretic data monk parrot nest in the Department of Artigas, (Agnese, 1989) was used, whereby each allele is Uruguay. considered as a character and the character-states The Genetical Society of Great Britain, Heredity, 77, 47—54. ISOZYMIC COMPARISON BETWEEN TRJATOMTNAE 49 Table1 The isoenzyme patterns of the 53 different multilocus genotypes (MLGs) found among the four Triatoma species studied Locus MLG 1 2 3 5 6 7 9 10 11 12 13 14 16 18 19 21 22 23 24 n 1* 11 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 22 11 65 2* 33 22 22 22 00 11 11 11 11 33 11 22 ii 22 22 22 22 22 11 107 3 33 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 12 53 4 22 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 12 1 5 33 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 11 18 6 11 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 11 3 7 33 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 12 1 8 333322220011 11 1111 33 1122 112222222223 22 1 9 33 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 22 108 10 33 12 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 22 1 11 33 23 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 22 1 12 33 22 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 23 22 6 13 33 23 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 22 11 1 14 33 11 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 22 12 1 15 33 12 22 22 00 11 11 11 11 33 11 22 11 22 22 22 22 22 12 10