isoenzyme variability of five principal triatomine vector species of chagas disease in mexico

Infection, Genetics and Evolution 1 (2001) 21–28

Isoenzyme variability of five principal triatomine vectorspecies of Chagas disease in Mexico

Angelica Flores a, Ezequiel Magallón Gastélum b, Marie-France Bosseno c, Rosalinda Ordoñez d,Felipe Lozano Kasten b, Bertha Espinoza a, Janine Ramsey d, Simone Frédérique Brenière c,∗

a UNAM, Instituto de Investigaciones Biomédicas, AP 70228, CP 04510, México D.F., Mexicob Universidad de Guadalajara, Depto. de Salud Pública del Centro Universitario de Ciencias de la Salud, AP 4-119, Guadalajara, Jalisco, Mexico

c Institut de Recherche pour le Développement (IRD), UR 008, Pathogénie des Trypanosomatidés,911 Av. Agropolis, BP 5045, 34032 Montpellier Cedex 1, France

d Center for Infectious Disease Research, National Institute for Public Health, Cuernavaca, Morelos 62508, Mexico

Received 3 November 2000; received in revised form 15 February 2001; accepted 19 February 2001

Abstract

Triatoma barberi, T. dimidiata, T. longipennis, T. pallidipennis and T. picturata, all key Chagas disease vectors in Mexico, were analysedby multilocus enzyme electrophoresis (MLEE) at 17 putative loci. The majority of insect specimens studied were collected from domesticand peridomestic structures from multiple geographic locations while others were collected from sylvatic areas. T. barberi was the leastpolymorphic species (P (0.95) = 0.18), with polymorphism rates of the other species ranging from 0.29 to 0.50. T. barberi, a member ofthe protracta complex, clustered apart from the other studied species by Nei’s genetic distance with >1.36, and at least eight loci were foundto be diagnostic for this species. T. dimidiata was more related to T. longipennis, T. pallidipennis and T. picturata (phyllosoma complex)than to T. barberi, with a genetic distance averaging 0.36 with the phyllosoma complex species. In contrast, the genetic distances betweenthe three phyllosoma complex species were not significantly different from zero, and there were no species-specific loci differentiatingamong them. The results strongly support the grouping of these three species in one complex, separate from the two other species studied.© 2001 Elsevier Science B.V. All rights reserved.

Keywords: Triatoma barberi; Triatoma dimidiata; Triatoma longipennis; Triatoma pallidipennis; Triatoma picturata; Mexico; Multilocus enzymeelectrophoresis (MLEE); Taxonomy

1. Introduction

The majority of the 18 Trypanosoma cruzi-infectedspecies of Triatominae (Hemiptera: Reduviidae) found inMexico have been collected from human dwellings andperidomestic sites, as well as from sylvatic areas (Zárateand Zárate, 1985; Velasco-Castrejón and Guzmán-Bracho,1986). All 129 species classified within the subfamily, ofwhich 28 are known to occur in Mexico, are currentlyidentified on the basis of morphological (including malegenitalia), as well as behavioural and ecological character-istics (Lent and Wygodzinsky, 1979). Many species showconsiderable overlap in many key morphological character-istics, providing the need for species complex groupings.Species or subspecies assignment for complex membersremains controversial, however, since at least in some

∗ Corresponding author. Fax: +33-467-547800.E-mail address: [email protected] (S.F. Breniere).

complex groups, viable hybrid crosses between species havebeen obtained (Mazzotti and Osorio, 1940; Ryckman, 1962).

Triatoma barberi, one of Mexico’s most important vectorspecies, is currently classified within the protracta complex(Ryckman, 1962). Although differentiation among adultspecimens from different species within the complex is fea-sible based on few morphological characteristics, differen-tiation between nymphs from the different species is not. Inmuch the same way, the phyllosoma complex is composedof multiple species including several of epidemiological im-portance in Mexico: Triatoma longipennis (Usinger, 1939),Triatoma mazzottii (Usinger, 1941), Triatoma pallidipennis(Stal, 1872), Triatoma phyllosoma (Burmeister, 1835) andTriatoma picturata (Usinger, 1939). Both Triatoma mex-icana (Herrich–Schaeffer, 1848) and Triatoma dimidiata(Latreille, 1811) were tentatively assigned to the phyllo-soma complex by Lent and Wygodzinsky (1979), this latterspecies representing one of the most ubiquitous membersdue to its range from Mexico to Peru. Recent descrip-tion of other Mexican species with similar morphological

1567-1348/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S1 5 6 7 -1 3 48 (01 )00005 -3

22 A. Flores et al. / Infection, Genetics and Evolution 1 (2001) 21–28

characteristics have motivated Dujardin et al. (1999) tosuggest the additional inclusion of Triatoma bolivari(Carcavallo, Martınez and Peláez, 1987), Triatomabrailovskyi (Martinez et al., 1984), Triatoma hegneri(Mazzotti and Osorio, 1940), and Triatoma ryckmani (Car-cavallo, Martınez and Peláez, 1987) to the phyllosomacomplex based uniquely on morphological characteristics.

The phylogenetic and taxonomic structure of the protractaand phyllosoma complexes have received little attentionin recent decades, and no comparative molecular studieshave been conducted to address relationships among epi-demiologically relevant Mexican species. Since isoenzymepattern analyses have been successfully used for phyloge-netic reconstruction with other Triatominae (Harry et al.,1992b; Chavez et al., 1999; Pereira et al., 1996; Noireauet al., 1998), the present study has used isoenzyme markersidentified through multilocus electrophoresis to evaluatetaxonomic relationships among Mexico’s five principalvector species: T. barberi, T. dimidiata, T. pallidipennis,T. longipennis and T. picturata.

2. Materials and methods

Triatominae: all triatomines were collected followingactive daytime searches in or around dwellings and perido-

Table 1Origin of the insectsa

Species State Municipality Community N Instar Colection sites

T. barberi Oaxaca San Felipe Tejalapa Jalapa del Valle 5 Adults (1) and nymphs (4) DSan Bartolo de Coyotepec San Bartolo de Coyotepec 28 Adults (19) and nymph (9) D (1), P (24)

Jalisco San Martin de Hidalgo Cruzero de Santa Maria 2 Adults (1) and nymph (1) P

T. dimidiata Oaxaca San Juan Comaltepec San Juan 4 Adults D (0), PChinantequilla 1

San Luis Potosı San Antonio Tanchahuil 13 Adults (6) and nymphs (7) D

T. longipennis Nayarit Compostela Carrillo Puerto 6 Adults D (1), P (4), S (1)Jalisco San Martin de Hidalgo Ipazoltic 1 Adult P

Cruzero Santa Maria 2 Adults PTepehuaje 7 Adults PSan Martin de Hidalgo 14 Adults P

Zacatecas Moyagua Cuxpala 7 Adults D (4), P (3)Palo verde 2 Adults P

T. pallidipennis Colima Comala Nogueras 8 Adults (7) and nymph (1) DMorelos Cuernavaca Cuernavaca 4 adults D (2), P (2)

E. Zapata E. Zapata 5 Adults DJantetelco Chacaltzingo 2 Adults PJiutepec Jiutepec 4 Adults D (3), P (1)

Ecological reservation El Texcal 2 Adults STemixco Temixco 3 Adults D

Acatlipa 1 Adult PXochitepec Xochitepec 1 Adult DYautepec Yautepec 1 Adult D

T. picturata Nayarit Compostela Carrillo Puerto 25 Adults D (1), P (18), S (6)Platanito 2 Adults D (1), P (1)

Jalisco San Martin de Hidalgo Cruzero de Santa Maria 1 Adult P

a D: dwelling, P: peridomestic, S: sylvatic.

mestic structures, or from sylvatic areas, from differentMexican states between 1997 and 1998. Collection sites foreach population studied are classified by village and state(Table 1).

T. barberi: specimens were collected from the states ofOaxaca and Jalisco, representing the southern and northernextensions of this species’ longitude distribution, respec-tively. Those specimens studied from Oaxaca were collectedin two communities within the Central Valleys, approxi-mately 15 km distant from each other. T. barberi is the onlytriatomine species present in this region. In the communityof the state of Jalisco 7.2% of triatomines (adults+nymphs)were T. barberi. Sympatric triatomine species in this lattercommunity were T. longipennis and T. picturata, from thephyllosoma complex.

T. dimidiata: the insects were collected by Health localAuthorities in three different localities.

T. pallidipennis: specimens were collected from Colimaand Morelos states, representing areas from the northern andsouthern regions of this species’ distribution, respectively.Samples from Morelos were collected from the metropolitanarea of Cuernavaca (six counties) and a village in the east-ern region of the state, Chalcatzingo, approximately 85 kmdistant from the former.

T. longipennis: in Zacatecas, T. longipennis was the onlyspecies encountered inside and surrounding dwellings. In

A. Flores et al. / Infection, Genetics and Evolution 1 (2001) 21–28 23

Jalisco, T. longipennis was the principal species encounteredfrom four communities, which were within 10 km of eachother. All samples from these villages represent approxi-mately 93% of all adult triatomines collected (sympatricwith T. barberi and T. picturata). In Nayarit, T. longipen-nis represents only 10% of the adult triatomine population(sympatric with T. picturata). Specimens from Nayarit werecollected from peridomestic and sylvatic sites within andsurrounding Carrillo Puerto.

T. picturata: specimens collected in domestic, peridomes-tic and sylvatic sites in Carrillo Puerto were sympatric withT. longipennis species. Triatomines collected from Platan-ito, located on the ocean 50 km from Carrillo Puerto, werecollected inside a single house. One specimen was collectedin Jalisco state where T. longipennis is the primary species.

Isoenzyme electrophoresis: thoracic muscle from eachinsect was dissected, frozen in liquid nitrogen, groundin a microtube, and then suspended in 60–80 �l of en-zyme stabiliser (diothiothreitol, E-aminocaproic acid andEDTA, at 2 mM each). Extracts were stored at −70◦C un-til used. Multilocus enzyme electrophoresis (MLEE) wasperformed on cellulose acetate plates (Helena Laborato-ries, Beaumont, TX). Twelve different enzyme systemswere surveyed (17 enzymatic loci): diaphorase (DIA, EC1.6.2.2), glutamate oxaloacetate transaminase (GOT, EC2.6.1.1), glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49), glucose phosphate isomerase (GPI, EC 5.3.1.9),isocitrate dehydrogenase (IDH, EC 1.1.1.42), malate dehy-drogenase (MDH, EC 1.1.1.37), malic enzyme (ME, EC1.1.1.40), peptidases (substrates: l-leucyl–leucine–leucineand l-leucyl–l-alanine) (PEP1 and PEP2, EC 3.4.11 or13∗), 6-phospho-gluconate dehydrogenase (6PGDH, EC1.1.1.44), and phospho-glucomutase (PGM, EC 2.7.5.1).Conditions for electrophoresis and enzyme staining werecarried out as described previously by Noireau et al. (1998).

Statistical methods: genetic interpretation was based onprevious descriptions to identify homozygotes and hetero-zygotes (Pasteur et al., 1987). Each specimen was char-acterised by its multilocus genotype, represented by thedifferent allelic combinations found at all identified putativeloci. Genetic variability was estimated by the percentageof polymorphic loci (P) (0.95 criteria), mean number ofallele per locus (A), observed heterozygosity (Ho) and unbi-aised expected heterozygosity (He) (also known as geneticdiversity) and their respective standard deviations (sHe)including the monomorphic loci. Each index was calcu-lated using the GENETIX package version 4.0 by Belkhir(1999).

Genetic similarity between species was estimated with theunbiased genetic distance of Nei (1978) calculated for pairsof species (D). A permutation test, which consists of ran-dom permutation of individuals between two species, testedthe null hypothesis D = 0 between each pair of species,and therefore, genetic homogeneity between two species.These statistical procedures were also performed using theGENETIX package.

The unweighted pair group method with arithmeticaverage (UPGMA) (Sokal and Sneath, 1963) was used tocluster the species according to Nei’s distance matrix. Thedendrogram was constructed using Mac Dendro and MacGraph programs (Thioulouse, 1989).

3. Results

3.1. Isoenzyme polymorphism

All 12 enzyme systems tested exhibit activity in the fivespecies studied. Five of these systems (IDH, MDH, ME,PEP1 and PGM) had two activity zones, which have been in-terpreted, as separate loci, and consequently, the 12 enzymesystems represented a total of 17 gene loci. The scored pat-terns were in all cases either one or two bands, correspondingto homozygote or heterozygote specimens, respectively. Themost characteristic band patterns observed for all speciesare represented in Fig. 1: three loci (Gpi, Idh2 and Pep1b)appear to be monomorphic within each species, with fixedalleles differentiating T. barberi from the other four species.The allele frequencies, mean number of alleles, propor-tions of polymorphic loci (P), observed and expected meanheterozygosity (Ho and He) for all species are presented inTable 2.

Triatoma barberi: 13 loci were monomorphic for bothadults and nymphs: 6Pgdh, Got, Gpi, Idh1, Idh2, Lap, Me1,Me2, Pep1, Pep2, Pep3, Pgm1 and Pgm2. All polymorphicloci segregated for two alleles, and there was a lower pro-portion of polymorphic loci for T. barberi as compared withother species P (0.95) = 0.18. No heterozygotic specimenswere identified for this species.

Triatoma dimidiata: polymorphism within specimens ofthis species was high P (0.95) = 0.50 (data are missingfor PEP2 enzyme system), segregating for two alleles, withno heterozygote scored. Eight loci were monomorphic: Dia,Gpi, Idh2, Lap, Mdh1, Mdh2, Me2, Pep1a.

Triatoma longipennis: seven gene loci were monomor-phic in this species: Got, Gpi, Idh2, Mdh1, Mdh2, Me2 andPep1a. Among the polymorphic loci, the Lap locus had twoalleles, with allele one present at a frequency less than 0.05,(corresponding to one triatomine). Other polymorphic locisegregated for two to four alleles, and among them, twodisplayed heterozygotic electrophoretic patterns (G6pd, andDia). The observed heterozygosity for the Dia locus washigh: Ho = 0.27. The average number of alleles per locuswas the highest in this species, A = 1.88.

Triatoma pallidipennis: nine loci were monomorphic inthis species, 6Pgdh, Got, Gpi, Idh1, Idh2, Mdh1, Mdh2, Me2,and Pep1b. Among the variable loci, three (Lap, Me1 andPep2) had allelic frequencies less than 0.05, correspondingto specimens from Morelos. The polymorphism rate fromspecimens collected from Colima was P (0.95) = 0.18,as compared with those from Morelos P (0.95) = 0.29.Heterozygotes were observed for Dia and Pep2.


Fig. 1. Scanned record of isoenzyme profiles on acetate gel electrophoresis of Triatoma individuals of the three enzyme loci (Gpi, Idh2 and Pep1b)differentiating T. barberi species (a) from the others studied species (T. dimidiata (b); T. longipennis (c); T. pallidipennis (d); T. picturata (e)). Weakband (· · · ).

Table 2Allelic frequencies at polymorphic loci among five Mexican species of triatomine bugs

Locus and alleles T. barberi T. dimidiata T. longipennis T. pallidipennis T. picturata

6Pgd 32 17 26 22 231 1.00 0.00 0.00 0.00 0.002 0.00 0.00 0.04 0.00 0.043 0.00 0.41 0.92 1.00 0.964 0.00 0.59 0.04 0.00 0.00

Dia 26 13 38 30 281 0.96 0.00 0.00 0.00 0.002 0.00 0.00 0.16 0.07 0.093 0.04 1.00 0.84 0.93 0.91

G6pd 19 14 34 20 241 0.00 0.36 0.00 0.00 0.002 0.05 0.64 0.84 0.82 0.623 0.95 0.00 0.09 0.05 0.214 0.00 0.00 0.04 0.00 0.045 0.00 0.00 0.03 0.13 0.13

Got 9 12 34 21 221 0.00 0.33 0.00 0.00 0.002 0.00 0.67 1.00 1.00 1.003 1.00 0.00 0.00 0.00 0.00

Gpi 30 17 35 27 241 1.00 0.00 0.00 0.00 0.002 0.00 1.00 1.00 1.00 1.00

Idh1 32 17 35 27 261 1.00 0.00 0.00 0.00 0.002 0.00 0.41 0.91 1.00 1.003 0.00 0.00 0.09 0.00 0.004 0.00 0.59 0.00 0.00 0.00

Idh2 32 17 35 27 261 0.00 1.00 1.00 1.00 1.002 1.00 0.00 0.00 0.00 0.00


Table 2 (Continued)

Locus and alleles T. barberi T. dimidiata T. longipennis T. pallidipennis T. picturata

Lap 28 17 38 30 281 0.00 0.00 0.03 0.03 0.002 0.00 0.00 0.97 0.97 1.003 1.00 1.00 0.00 0.00 0.00

Mdh1 22 17 29 29 281 0.14 1.00 1.00 1.00 1.002 0.86 0.00 0.00 0.00 0.00

Mdh2 24 13 30 28 281 0.87 0.00 0.00 0.00 0.002 0.13 1.00 1.00 1.00 1.00

Me1 23 16 38 27 281 0.00 0.13 0.00 0.00 0.002 1.00 0.87 0.00 0.00 0.003 0.00 0.00 0.92 0.96 0.754 0.00 0.00 0.08 0.04 0.25

Me2 20 12 37 23 281 0.00 1.00 1.00 1.00 1.002 1.00 0.00 0.00 0.00 0.00

Pep1 22 13 36 23 251 1.00 0.46 0.19 0.17 0.122 0.00 0.54 0.81 0.83 0.88

Pep1b 25 13 38 25 261 0.00 1.00 1.00 1.00 1.002 1.00 0.00 0.00 0.00 0.00

Pep2 8 0 32 18 211 0.00 nd 0.03 0.00 0.002 1.00 nd 0.91 0.97 0.863 0.00 nd 0.03 0.03 0.144 0.00 nd 0.03 0.00 0.00

Pgm1 30 15 37 28 261 1.00 0.93 0.00 0.03 0.002 0.00 0.07 0.92 0.93 0.813 0.00 0.00 0.08 0.04 0.19

Pgm2 31 10 38 30 281 0.00 0.90 0.00 0.00 0.002 0.00 0.00 0.08 0.05 0.073 0.00 0.10 0.92 0.91 0.934 1.00 0.00 0.00 0.04 0.00

P (0.95) 0.18 0.50 0.53 0.29 0.41A 1.23 1.50 1.88 1.65 1.59Ho 0.000 0.000 0.017 0.018 0.016He 0.038 0.187 0.109 0.072 0.124sHe 0.079 0.223 0.112 0.103 0.170

Triatoma picturata: seven loci were monomorphic for thisspecies: Got, Gpi, Idh1, Idh2, Lap, Mdh1, Mdh2, Me2, Pep1b.Among the variable loci, one had an allele frequency lessthan 0.05 (6Pgdh, allele two). In Tepehuaje (Jal.) and Pla-tanitos (Nay.) only one and two specimens are analysed andthe genotypes, except for G6pg and Pgm2, correspond to themost frequent allele in Carrillo Puerto. Heterozygote geno-types were observed in some specimens from Carrillo Puertofor Dia and Pep2.

T. barberi was the least variable species, with T. longipen-nis, T. pallidipennis and T. picturata having similar variabil-ity indices. Only four alleles were common to the five species

studied, whereas 15 common alleles (32%) were observedfor T. dimidiata, T. longipennis, T. pallidipennis and T. pic-turata. In contrast, 22 (47%) common alleles were sharedamong the three closest species of the phyllosoma complex(T. longipennis, T. pallidipennis and T. picturata).

3.2. Between species diagnostic loci

The locus with absence of common allele between thecompared species was considered as a diagnostic locus. T.barberi can be distinguished from the other four species by8–11 loci (Table 3), while only two loci can differentiate


Table 3Isoenzyme diagnosis loci between five species of triatomine of epidemiological relevance in Mexicoa

T. barberi T. dimidiata T. longipennis T. pallidipennis

T. dimidiata 6Pgd, Got, Gpi, Idh1, Idh2, Me2, Pep1b, Pgm2 (50%)T. longipennis 6Pgd, Got, Gpi, Idh1, Idh2, Lap, Me1, Me2, Pep1b, Pgm1, Pgm2 (65%) Lap, Me1 (12.5%)T. pallidipennis 6Pgd, Got, Gpi, Idh1, Idh2, Lap, Me1, Me2, Pep1b (53%) Lap, Me1 (12.5%) NoneT. picturata 6Pgd, Got, Gpi, Idh1, Idh2, Lap, Me1, Me2, Pep1b, Pgm1, Pgm2 (65%) Lap, Me1 (12.5%) None None

a Pep2 locus is not considered for T. dimidiata comparisons (missing data).

Table 4Nei’s unbiaised genetic distances among five Triatoma species from Mexico (above the diagonal) and P-values (below the diagonal)

Species T. barberi T. dimidiata T. longipennis T. pallidipennis T. picturata

T. barberi 1.366 2.336 2.303 2.340T. dimidiata <10−4 0.362 0.362 0.360T. longipennis <10−4 <10−4 <10−4 0.005T. pallidipennis <10−4 <10−4 0.76 0.005T. picturata <10−4 <10−4 0.09 0.09

Fig. 2. An UPGMA tree derived from Nei’s standard unbiased genetic distances between five Triatoma Mexican species.

T. dimidiata from the three other species of the phyllosomacomplex (Lap and Me1). No diagnostic loci were identifiedfor differentiation among T. longipennis, T. pallidipennis andT. picturata, although some alleles (six) were present in onespecies and absent in the other two. These latter alleles were,however, present in very low frequencies, 0.03–0.09.

3.3. Phylogenetic clustering

The unbiased genetic distances estimated among the fivespecies indicate significant difference from zero only forT. barberi and T. dimidiata (Table 4). T. barberi clustersseparately from the other four species, as illustrated by thedendrogram based on Nei’s distances (Fig. 2). The geneticdistances among T. longipennis, T. pallidipennis and T. pic-turata ranged from <10−4 to 0.005 (Table 4).

4. Discussion

Twelve enzyme systems were studied among five of themost important Chagas disease vector species in Mexico.The majority of specimens used in this study were collectedfrom domestic habitats representative of individual species’distribution ranges, although all species can be collectedfrom domestic as well as sylvatic sites (Zárate and Zárate,1985; Ramsey et al., 2000).

Enzyme polymorphism ranged from the lowest of 0.18 forT. barberi to the highest of 0.53 for T. longipennis (P (0.95)).These rates are greater than those for Triatoma infestanspopulations, a species that has little polymorphism (Dujardinand Tibayrenc, 1985; Brenière et al., 1998; Garcıa et al.,1995), and whose primarily domestic and minor sylvatic (inlimited areas of Bolivia) populations are strictly separated,


as demonstrated by morphometric studies (Dujardin et al.,1997). T. barberi has similar polymorphism to that of thegenus Rhodnius (Harry et al., 1992a), while T. dimidiata,T. longipennis, T. pallidipennis and T. picturata have higherrates than those described previously for other triatomines(Pereira et al., 1996; Costa et al., 1997; Noireau et al., 1998).The vast geographic distribution and diversity of Mexicantriatomine ecosystems may partially explain this variability,along with the relatively recent domestication of most of itsautochtonous species.

T. barberi is classified within the protracta group ofthe genus Triatoma, in the protracta complex. This genusgroup is different from that of the other four species stud-ied herein, which are classified within the rubrofasciatagroup, rubrofasciata subgroup, phyllosoma complex (Lentand Wygodzinsky, 1979). The present study has identifiedat least eight diagnostic loci differentiating T. barberi fromthe other four species, in agreement with current genusgroup taxonomy. T. dimidiata differs from other species ofthe phyllosoma complex by only two loci, although the ge-netic distance between T. dimidiata and all other complexmembers is higher than that among complex members stud-ied herein. Despite recent studies which suggest a similardistance between T. dimidiata and other phyllosoma com-plex members (Lyman et al., 1999; Bargues et al., 2000),other species of this complex will need to be studied inorder to define the range of genetic distances separatingcore members of the complex. Independent of this distanceand its taxonomic value, two enzyme systems can be usedhenceforth to distinguish T. dimidiata nymphs from thoseof the other phyllosoma complex species.

The absence of diagnostic loci to distinguish amongT. longipennis, T. pallidipennis and T. picturata supportsthe current taxonomic phyllosoma complex cohesion. Sur-prisingly, the genetic distances among the three species arenot significantly different from zero, even though analy-ses included distances calculated from isoenzyme data andcomparison of geographically isolated populations from in-dividual species. Despite the lack of enzyme differentiationmarkers for these species, which strongly suggests relativelyrecent divergence from a common ancestor, morphologicalcharacteristics effectively differentiate adults through theuse of taxonomic keys, even when these species are sym-patric, as in the case of T. longipennis and T. picturata inNayarit. Hence, adult morphological characteristics appearto be stable parameters for individual species and for dif-ferentiation between species, even when species’ sympatryprovides the potential for hybrid individuals, and viablecrosses are feasible, as demonstrated by in-laboratory stud-ies (Mazzotti and Osorio, 1940; Zárate unpublished; Ryck-man unpublished). Although Mazzotti and Osorio notedthe production of F1 individuals with certain characteristicsof both parents, as well as individuals with characteris-tics strictly the same as either of the parents, Ryckman(unpublished) obtained complete sexual segregation of allmorphological characteristics by viable F1 hybrids from

certain phyllosoma complex species’ crosses (Ryckman, un-published data). The segregation of phenotype may explainthe absence of morphological hybrids during this and otherstudies, as well as from bugs collected from infested villagesby vector control personnel throughout Mexico. However,the study of molecular genetic markers other than enzymeswould better elucidate taxonomic relationships among suchsimilar species as those from the phyllosoma complex.

Acknowledgements

This work was supported by the “Institut de Recherchepour le Développement (IRD)”, the WHO special pro-gramme (TDR) ID no. 970943, DGAPA IN 224 798 UNAMand Conacyt 27951M.

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