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Acta Tropica 107 (2008) 90–98

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Acta Tropica

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Is Rhodnius prolixus (Triatominae) invading houses in central Brazil?

Rodrigo Gurgel-Goncalvesa,b,∗, Fernando Abad-Franchc, Jonatas B.C. Ferreiraa,b,Daniella B. Santanaa, Cesar A. Cuba Cubaa

a Laboratorio de Parasitologia Medica e Biologia de Vetores, Faculdade de Medicina, Area de Patologia, Universidade de Brasılia, Asa Norte,CEP 70910-900, Distrito Federal, Brazilb Laboratorio de Zoologia, Universidade Catolica de Brasılia, QS 07 Lote 01 EPTC Bloco M, sala 331, CEP 72030-170,Distrito Federal, Brazilc Instituto Leonidas & Maria Deane, Fiocruz Amazonia, Rua Teresina 476, Adrianapolis, CEP 69057-070, Manaus, Amazonas, Brazil

a r t i c l e i n f o

Article history:Available online 1 May 2008

Keywords:Rhodnius prolixusRhodnius neglectusGeometric morphometricsVector surveillanceChagas diseaseBrazil

a b s t r a c t

Sylvatic triatomines of the genus Rhodnius commonly fly into houses in Latin America, maintaining therisk of Chagas disease transmission in spite of control efforts. In the recent past, adult bugs collectedinside houses in central Brazil were identified as R. prolixus, a primary disease vector whose naturalgeographical range excludes this region. Three nearly sibling species (R. neglectus, R. nasutus, and R. robus-tus), secondary vectors with limited epidemiological significance, occur naturally south of the BrazilianAmazon. The specific status of Rhodnius specimens found inside houses in central Brazil is therefore anepidemiologically important (and still debated) issue. We used wing and head geometric morphometricsto investigate the taxonomic status of 230 adult specimens representing all four ‘R. prolixus group’ species(19 populations from palm trees, domiciles, and reference laboratory colonies). Discriminant analyses ofshape variation allowed for an almost perfect reclassification of individuals to their putative species. Shapepatterning revealed no consistent differences between most specimens collected inside houses in cen-tral Brazil and R. neglectus, and showed that R. robustus and R. neglectus occur sympatrically (and fly intohouses) in southern Amazonia. Furthermore, all Brazilian specimens clearly differed from our referenceR. prolixus population. Using geometric morphometrics, we confidently ascribed individual triatominesto their species within the problematic ‘R. prolixus group’, illustrating the potential value of this approachin entomological surveillance. Our results strongly support the idea that R. neglectus, and not R. prolixus,is the species invading houses in central Brazil.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The genus Rhodnius is comprised of 16 recognized species; nat-ural populations occupy arboreal ecotopes (preferentially palmtrees) in ∼28 biogeographical provinces from Central America tosouthern Brazil (Abad-Franch and Monteiro, 2007). Populations ofseveral Rhodnius species have adapted to artificial environments indifferent regions; one of them, R. prolixus, is a major domestic vec-tor of human Chagas disease. Synanthropic triatomine populationshave been targeted by successful control programs based on house-hold spraying with residual insecticides (e.g., Dias, 2007). However,the presence of sylvatic vector populations near houses limits theeffectiveness of such control interventions in many regions. Adulttriatomines recurrently invade households, maintaining the risk

∗ Corresponding author. Fax: +55 61 273 3907.E-mail address: rgurgel@ucb.br (R. Gurgel-Goncalves).

of Chagas disease transmission and occasionally originating newdomestic–peridomestic breeding colonies (Miles et al., 2003).

Such dynamics are common across Amazonia, the Orinoco basin,Central America, and the drier ecoregions of central Brazil (Barrettoet al., 1968; Silveira et al., 1984; Garcia-Zapata et al., 1985; Silvaet al., 1992, 1999; Guilherme et al., 2001; Feliciangeli et al., 2003;Romana et al., 2003; Sanchez-Martin et al., 2006; Oliveira and Silva,2007; Aguilar et al., 2007). A large fraction of these adventitiousvectors belongs to the ‘Rhodnius prolixus group’, which includesR. prolixus, R. robustus, R. nasutus, and R. neglectus (Barrett, 1991).These four species are phenotypically almost sibling, and their nat-ural distribution ranges (not always precisely defined) often overlappartially. Under these circumstances, it seems virtually unavoidablethat some field-collected specimens be misidentified (Monteiro etal., 2001; Miles et al., 2003).

R. neglectus is apparently restricted to the savanna-like cen-tral Brazilian Cerrado and the neighboring southern fringes of theAmazon, while R. nasutus occupies drier areas in the Caatinga of

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northeastern Brazil. R. robustus comprises a complex of crypticspecies that occur throughout the Amazon moist forests and theOrinoco plains. Finally, R. prolixus often infests houses in parts ofVenezuela, Colombia, and Central America; sylvatic populationshave only been confirmed to occur in palm trees of the Colombian-Venezuelan Orinoco basin (Lent and Wygodzinsky, 1979; Dujardinet al., 1998; Abad-Franch and Monteiro, 2007; Guhl, 2007). R. pro-lixus has however been also reported from Brazil after collectionsmade in areas clearly outside the currently accepted range of thespecies (Pinho et al., 1998; Schofield and Dujardin, 1999).

Until the 1950s, Rhodnius specimens collected inside houses incentral Brazil were all identified as R. prolixus. After Lent (1954)described R. neglectus, these bugs (collected from either palmtrees or houses in the states of Minas Gerais, Goias, and SaoPaulo) were reassigned to this new species. However, it was laterclaimed that R. prolixus could also be found in some areas ofthe Amazonia-Cerrado transition (Diotaiuti et al., 1984; Silveiraet al., 1984; see also Pinho et al., 1998). Dujardin et al. (1991)tackled the issue by comparing allozyme profiles of different R.prolixus and R. neglectus populations. Results revealed no dif-ferences between laboratory-reared R. prolixus and field-caughtbugs (identified as R. neglectus) from peridomestic environmentsof Mambaı, Goias. On the other hand, laboratory R. neglectusspecimens drawn from the FIOCRUZ reference colony had a dis-tinct allozyme profile. The authors concluded that a species otherthan R. neglectus was invading houses in central Brazil, leav-ing open the possibility that it was R. prolixus (Dujardin et al.,1991).

A relatively rich bibliography shows how both traditional (e.g.,Dujardin et al., 1997, 1999a; Patterson et al., 2001) and geomet-ric morphometric analyses (e.g., Matıas et al., 2001; Villegas et al.,2002; Gumiel et al., 2003; Schachter-Broide et al., 2004; Dujardinet al., 2007; Feliciangeli et al., 2007) provide valuable tools for thestudy of taxonomically problematic groups within the Triatomi-nae. In this work, we used geometric morphometric analyses forspecies-level identification of adult Rhodnius specimens collectedwithin houses in Brazil. Several field-collected (from sylvatic anddomestic environments) and laboratory bug samples were com-pared, in order to assess whether size and shape variation analyses

may provide suitable taxonomic markers for the members of theproblematic ‘R. prolixus group’.

2. Materials and methods

2.1. Bug samples

Two hundred and thirty adult Rhodnius specimens from 19 pop-ulations (five from palm trees, two from laboratory colonies, and 12from households in five Brazilian states) were analyzed (Table 1).Sylvatic samples were collected from palm trees, mainly Mauri-tia flexuosa (see Gurgel-Goncalves et al., 2003, 2004 for details),under permissions issued by the Brazilian Environmental Agency(IBAMA). Our R. neglectus reference sample was drawn from thecolony at the Centro de Pesquisas Rene Rachou (FIOCRUZ MinasGerais, Brazil), founded with specimens from the type locality.One sylvatic population from Mambaı (Goias, Brazil) was also sam-pled, allowing us to confidently reassess the results presented byDujardin et al. (1991). Our R. prolixus reference sample came froma colony (Laboratorio de Investigaciones en Parasitologıa Tropical,Universidad del Tolima, Ibague, Colombia) founded with domes-tic specimens collected in the Magdalena Valley—a trans-Andeanlocation where no other members of the ‘R. prolixus group’ occur(Abad-Franch and Monteiro, 2007). Domestic Brazilian specimenswere collected inside or around houses during routine searches byworkers of the Chagas Disease Control Program (CDCP) between2006 and 2007. All bugs were preliminarily identified after Lent andWygodzinsky (1979). Samples whose specific status was dubious(and we aimed at testing) were however treated as ‘problem spec-imens’ in our metric analyses. These samples included the Mambaıpopulation and the specimens collected within houses by CDCPagents (Rhodnius sp. in Table 1).

2.2. Morphometrics

Right hemelytra (forewings) were mounted between micro-scope slides and cover slips using a mounting resin (Entellan®),and digitally scanned (1200 dpi). Six type I (venation intersection)and one type II landmarks (Bookstein, 1991) were digitized (Fig. 1).

Table 1Origin, habitats, geographic coordinates and number of wings and heads of Rhodnius sp. populations used in geometric morphometric analyses

Populations Origine Habitats Coordinates Wings Heads

R. neglectusa Buritizal, SP, Brazil Palm trees (Mauritia flexuosa) 20◦11′S, 47◦42′W 17 30R. neglectusa Taguatinga, TO, Brazil Palm trees (M. flexuosa) 12◦23′S, 46◦24′W 23 23R. neglectusb Uberaba, MG, Brazil Laboratory colony 19◦32′S, 48◦01′W 11 8R. robustusc Maraba, PA, Brazil Palm trees (Attalea speciosa) 05◦16′S, 49◦50′W 34 45R. nasutusb Sobral, CE, Brazil Palm trees (Copernicia prunifera) 03◦40′S, 40◦13′W 30 30R. prolixusd Tolima, Ibague, Colombia Laboratory colony 02◦59′N, 74◦29′W 32 37Rhodnius sp. Mambaı, GO, Brazil Palm trees (M. flexuosa) 14◦27′S, 46◦08′W 19 21Rhodnius sp. Ituiutaba, MG, Brazil Domiciles 18◦57′S, 49◦35W 6 4Rhodnius sp. Tocantinopolis, TO, Brazil Domiciles 06◦19′S, 47◦24′W 9 8Rhodnius sp. Araguatins, TO, Brazil Domiciles 05◦38′S, 48◦05′W 3 3Rhodnius sp. Taguatinga, TO, Brazil Domiciles 12◦23′S, 46◦23′W 1 1Rhodnius sp. Quirinopolis, GO, Brazil Domiciles 18◦28′S, 50◦27′W 1 1Rhodnius sp. Cidade de Goias, GO, Brazil Domiciles 15◦54′S, 50◦08′W 4 4Rhodnius sp. Uruana, GO, Brazil Domiciles 15◦35′S, 49◦41′W 1 1Rhodnius sp. Piracanjuba, GO, Brazil Domiciles 17◦18′S, 49◦01′W 1 1Rhodnius sp. Cotriguacu, MT, Brazil Domiciles 09◦52′S, 58◦23′W 5 5Rhodnius sp. Ponte e Lacerda, MT, Brazil Domiciles 15◦13′S, 59◦21′W 1 1Rhodnius sp. Caceres, MT, Brazil Domiciles 16◦07′S, 57◦36′W 1 1Rhodnius sp. Dourados, MS, Brazil Domiciles 22◦13′S, 54◦47′W 7 6

a Sylvatic populations.b Colony from Centro de Pesquisas Rene Rachou, CPqRR-FIOCRUZ, Minas Gerais. Uberaba specimens were collected in the same locality of the holotype of R. neglectus.c F1 and F2 generations.d Colony from Laboratorio de Investigaciones en Parasitologıa Tropical, Facultad de Ciencias, Universidad del Tolima, Ibague, Colombia.e SP (Sao Paulo), TO (Tocantins), MG (Minas Gerais), PA (Para), CE (Ceara), GO (Goias), MT (Mato Grosso) and MS (Mato Grosso do Sul).

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Fig. 1. Right wing of Rhodnius neglectus with the seven landmarks used in mor-phometric analysis. Following Bookstein (1991), point 4 corresponds to a type IIlandmark, and the remain to type I landmark.

Heads were pinned on triangular cards and photographed witha digital camera (Sony® Ciber-shot 5.1 Mp) adapted to a NOVA®

stereomicroscope (25× magnification). Eight head landmarks wereused in morphometric analyses (Fig. 2). Landmark coordinates wererecorded using tpsDig 1.18 (Rohlf, 1999a).

2.3. Size and shape variation

We used “centroid size” (CS), an isometric size estimator derivedfrom coordinate data (Bookstein, 1991), to analyze wing and headsize variation. The CS value was extracted from the coordinatematrix of each individual structure using tpsRelw version 1.18(Rohlf, 1999b) and log-transformed. Shape variables were obtainedusing the Generalized Procrustes Analysis (GPA) superimposi-tion algorithm (Rohlf, 1996). First, a consensus configuration wasderived from raw coordinate data using a least-squares iterativeapproach. “Partial warps” were then computed as deformationsof each individual structure in relation to the consensus configu-ration. Both uniform and non-uniform deformation componentswere used in the analyses. Shape variables were computed andtested for variation using tpsRelw 1.18 (Rohlf, 1999b).

2.4. Statistical analyses

Size variation (wing and head CS values) among populationswas explored by means of ANOVA and Tukey tests (alpha = 0.01).The homogeneity of shape variables across groups was tested byMANOVA; the same variables (partial warps and uniform compo-nents) were used as input for multivariate discriminant functionanalysis (DFA). In a first analysis, we explored the relationshipsbetween ‘problem specimens’ and our R. prolixus and R. neglectusreference populations. We used shape variables to derive a dis-criminant model under which R. prolixus, R. neglectus (sylvatic fromTocantins and Sao Paulo and the FIOCRUZ stock), the Mambaı pop-ulation, and other ‘problem specimens’ collected in houses of fiveBrazilian states (see Table 1) could be classified. Because several‘problem specimens’ could not be confidently ascribed to any of thereference groups, we ran a second discriminant analysis including

Fig. 2. Head of Rhodnius prolixus illustrating the eight landmarks used in morpho-metric analysis. Following Bookstein (1991), points 1–2 and 5–6 correspond to typeI landmark.

field-collected Brazilian populations of R. nasutus and R. robustus,and removing R. prolixus. Factorial maps were constructed to graph-ically show the distribution of specimens and populations in theshape space defined by the two discriminant factors; to improveclarity in the graphic output, convex polygons enclosing all speci-mens within each group were overlaid on the plots and individualdots removed (except for ‘problem specimens’ whose position wewanted to examine). Reclassification of specimens to their originalputative groups was assessed by contingency table analysis andKappa statistics (Landis and Koch, 1977). Finally, we measured thecontribution of size to shape variation (allometry) using multipleregression of shape discriminant factors against CS values (wingsand heads). DFA, ANOVA, MANOVA, multiple regression and Tukeytests were computed with Statistica®. Kappa statistics were com-puted with JMP®.

3. Results

3.1. Size variation

As expected, size sexual dimorphism was consistently observed.Female wings and heads were significantly larger than those ofmales. Wing size was largest in R. robustus and smallest in R. nasu-tus. Head size was markedly smaller in R. prolixus than in any otherspecies/population analyzed here (Fig. 3). We scored statisticallysignificant differences among wings (males: ANOVA F4,61 = 41.5;p < 0.01; females: ANOVA F4,65 = 43.3; p < 0.01) and mainly heads(males: ANOVA F4,76 = 102.3; p < 0.01; females: ANOVA F4,75 = 98.6;p < 0.01) across Rhodnius species/populations. The heads and wingsof R. neglectus from Tocantins were larger on average than thosefrom Sao Paulo, but the difference was not statistically significant(p = 0.10).

3.2. Shape variation and allometry

Factorial maps of both wing and head shape variation revealedsignificant differences between ‘problem specimens’ and our ref-erence R. prolixus population (Fig. 4). ‘Problem specimens’ fromGoias (including the Mambaı population), Minas Gerais, and MatoGrosso do Sul were all indistinguishable from our R. neglectus refer-ence populations. Some specimens from Tocantins and Mato Grossowere also identified as R. neglectus, while others significantly dif-fered from both R. neglectus and R. prolixus. DFA-based correctreclassification scores were high for both head and wing shape vari-ables. All R. prolixus were correctly reclassified, while Mambaı andR. neglectus specimens often shifted groups (Table 2).

Head and wing shape differences between R. neglectus and R.prolixus were patent. R. neglectus heads were narrow and elon-gated when compared with those of R. prolixus, which had largereyes. Wing shape changes largely involved landmarks 5 (subcostalvein) and 6 (median and cubital veins) (Fig. 4). Multiple regres-sion of discriminant factors (shape variation) against CS revealedno significant allometric trend for wings (R2 = 0.004; p = 0.84), buta significant allometric content was observed in the head shapedataset (R2 = 0.45; p < 0.01).

Factorial maps derived from the second discriminant analysis(comparing R. neglectus populations with R. robustus and R. nasu-tus) showed that R. neglectus and R. robustus are sympatric in thestates of Tocantins and Mato Grosso (Fig. 5). R. neglectus was theonly species found within human dwellings in Minas Gerais, Goias,and Mato Grosso do Sul. In these analyses, correct reclassifica-tion scores were very high for both wings and heads (Table 3).Multiple regression revealed a significant allometric content inthe first discriminant factor (CV1: p < 0.01). Size predicted 18% of

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Fig. 3. Variation of wing and head centroid size (CS) among populations (males and females) of Rhodnius spp.: Rna (R. nasutus Ceara), RneSP (R. neglectus Sao Paulo), RneTO(R. neglectus Tocantins), Rp (R. prolixus) and Rr (R. robustus). Boxes show mean CS values and standard errors (S.E.); standard deviations (S.D.) are shown as lines. Populationslabeled with different letters (above boxes) were statistically different by Tukey tests (p < 0.01).

wing and 19% of head shape variation. Regarding venation confor-mational changes, the posterior apex of the forewing (landmark4) was more distant from the end of the cubital vein (landmark2) in R. robustus than in R. neglectus. The relative positions ofwing landmarks 1 and 7 were important in the discriminationof these species. R. robustus had larger eyes than R. neglectus(Fig. 5).

4. Discussion

4.1. The use of metric traits for species diagnosis within the ‘R.prolixus group’

The use of morphometric traits in systematics relies on theassumption that variation of continuous phenotypic attributes is

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Fig. 4. Factorial maps in the plane of the two canonical factors of shape variation for wing and head (CV1 and CV2) presenting the distribution of Rhodnius neglectus (fromTocantins [TO], Sao Paulo [SP], and FIOCRUZ), R. prolixus and Rhodnius Mambaı populations. The symbols represent the origin of Rhodnius specimens captured in houses. Thepercentage contribution of each component to the total shape variation is shown on the axes in parentheses. Polygons enclose each group with different patterns. Drawingsright the factorial maps are the consensus conformation of the head and wing shape (see landmarks in Figs. 1 and 2). The arrows indicate the differences in head and wingshape of R. neglectus and R. prolixus (see text for details).

the expression of underlying genetic diversity; the degree to whichphenotypic traits differ between two groups is regarded as a proxymeasure of evolutionary distance, with clear-cut differences oftensignaling mutual isolation (Sorensen and Foottit, 1992). Yet, forthese principles to hold true the two main sources of variability(genetic and environmental) must be partitioned; the standardprocedure within a strict morphometric framework entails theseparate assessment of size (largely environmental) and shape(genetic) variation (Dujardin et al., 2002; Baylac et al., 2003). Weused a geometric morphometric approach to explore the alpha sys-tematics of ‘problematic’ Rhodnius specimens collected in central

Brazil, comparing them with reference populations of known spe-cific status. Our results show that wing and head geometric traitscan be used for accurate species-level identification of ‘R. prolixusgroup’ members, and shed light on the epidemiologically signif-icant question of whether R. prolixus populations occur in Brazil(see below) (Diotaiuti et al., 1984; Silveira et al., 1984; Dujardin etal., 1991; Schofield and Dujardin, 1999).

4.1.1. Size variationCrude size disparity is often used in triatomine systemat-

ics. Species descriptions invariably refer to linear measurements,

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Table 2Reclassification scores of Rhodnius neglectus, R. prolixus, and Rhodnius sp. from Mambaı (Goias, Brazil) wings and heads after discriminant analysis of shape variables

Groups Wings Heads

R. neglectus R. prolixus Rhodnius sp.a % correct reclassification R. neglectus R. prolixus Rhodnius sp.a % correct reclassification

R. neglectus 36 0 4 90 50 0 4 92R. prolixus 0 32 0 100 0 37 0 100Rhodnius sp.a 4 0 15 79 14 0 7 33

Kappab 0.86 0.85

R. = Rhodnius.a Mambaı (Goias, Brazil) population (see text for details).b Kappa: measures the degree of agreement between observed and expected classification; it ranges from 0 to 1; a score between 0.80 and 1 is considered almost perfect

agreement (Landis and Koch, 1977).

from overall body length to the dimensions of particular struc-tures such as the head, pronotum, legs, or antennae segments(Lent and Wygodzinsky, 1979). Since Dujardin and his collabora-tors introduced modern morphometric techniques for the studyof triatomines in the 1990s (Dujardin et al., 2002), the resultsof many investigations suggest that size variation largely mirrorsenvironmental (not genetic) heterogeneity (e.g., Dujardin et al.,1997, 1998, 1999b; Schachter-Broide et al., 2004). Size-related traitsare thus prone to either convergence (when two genetically dis-tinct entities occupy similar environments) or divergence (whensubsets of a genetically homogeneous metapopulation adapt todistinct microhabitats) in triatomines (Abad-Franch et al., 2003).Consequently, size variation has to be cautiously interpreted in thecontext of species-level diagnosis (in which a declaration on thegenetic consequences of reproductive isolation is implicit), and acareful assessment of allometric trends is recommended (Dujardinet al., 2002).

Our results provide several further examples of apparentlyerratic isometric size variation. For instance, R. prolixus is expectedto be larger on average than R. neglectus, but our CS analysesrevealed the opposite for most R. neglectus specimens. Similarly,R. robustus heads and wings are generally larger than those of R.neglectus, but some of the R. neglectus specimens from Tocantinswe analyzed were similar in size to R. robustus. Our dataset alsorevealed a trend towards size-related metric divergence among R.neglectus geographical populations, a finding previously reportedby Soares et al. (1999). Habitat-related size variation has also beendetected among R. nasutus populations (Diotaiuti et al., 2005).

4.1.2. Shape variation and allometryShape patterns tend to differ significantly among geneti-

cally distinct organisms. The differences can however be subtleenough to remain undetectable to the naked eye. This isgenerally the case when cryptic species go unrecognized inqualitative systematic investigations. Geometric morphomet-ric analyses involve the explicit, quantitative assessment ofotherwise hidden shape patterning among organisms; theirpower in dealing with complex systematic problems is nowwidely recognized (see Baylac et al., 2003 and referencestherein).

Wing shape patterns are seldom used in (traditional) triatominesystematics, whereas several head characters are important tax-onomic markers. Multivariate analyses of wing metric traits mayhowever discriminate nearly sibling taxa and reveal fine-scale spa-tial structuring among populations of a single species (Matıaset al., 2001; Villegas et al., 2002; Schachter-Broide et al., 2004;Feliciangeli et al., 2007). They even helped ascribe individualtriatomine specimens to their parental lines (Dujardin et al.,2007). When applied to assign individual bugs to four nearly sib-ling Rhodnius species (R. prolixus, R. neglectus, R. nasutus, and R.robustus), our discriminant analyses of wings performed slightly

better than those of heads, but both yielded largely coherentresults.

Our first discriminant models revealed clear-cut, consistent dif-ferences between R. prolixus and R. neglectus (Fig. 4). Head variationwas in agreement with the original description of R. neglectus byLent (1954). Both wing and head shape comparisons allowed us toconfidently conclude that most of our ‘problem specimens’ (includ-ing the Mambaı population) belong to the latter species; a fewsynanthropic bugs from Tocantins and Mato Grosso did not appearto resemble any of these two taxa.

Our data also showed significant shape divergence betweenR. neglectus and R. robustus, which had more elongated headsand wings even when size was similar. The posterior apex of theforewing (landmark 4) was more distant from the end of the cubitalvein (landmark 2) in R. robustus. Villegas et al. (2002) reported sim-ilar findings in their comparison of Venezuelan populations of R.prolixus and R. robustus, underscoring the key contribution of thesetwo landmarks to wing shape changes in the ‘R. prolixus group’.Shape differences (involving the wing apex and the relative posi-tions of ocelli and eyes) were also observed between R. neglectusand R. nasutus (details not shown).

When comparing R. neglectus and R. prolixus specimens, ourwing dataset revealed no allometric content, therefore suggestingthat shape variation mainly reflects genetic variance; on the otherhand, head shape differences were under a significant influence ofsize. These findings indicate that wing shape may be less sensitiveto size changes than head shape is, but the mechanisms underlyingthis trend remain a matter of investigation. Overall, wing allometrycomponents recorded in the R. neglectus–robustus–nasutus datasetwere smaller than those described for R. prolixus and R. robustus byVillegas et al. (2002).

4.2. Which species of Rhodnius is invading houses in centralBrazil?

The ‘R. prolixus group’ is composed of four closely related, nearlysibling Rhodnius species with different epidemiological signifi-cance. R. prolixus is a major domestic vector north of the Amazon;R. neglectus often invades and colonizes artificial environmentsin the Brazilian Cerrado, while R. nasutus does so less frequentlyin the Caatinga; finally, the various lineages within the R. robus-tus complex invade (but do not colonize) households across theAmazon–Orinoco systems. For any given species within this group,presence records based on the identification of specimens afterqualitative assessment of morphological-chromatic patterns willprobably involve a certain proportion of misclassifications. Wrongrecords will in turn be used to define the species geographical range,giving rise to erroneous biogeographical inferences and mistakenestimations of epidemiological risk (Monteiro et al., 2001).

The argument over the presence of R. prolixus (an extremely effi-cient disease vector) in Brazil suitably illustrates these difficulties

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Fig. 5. Factorial maps in the plane of the two canonical factors of shape variation for wing and head (CV1 and CV2) presenting the distribution of Rhodnius neglectus (fromTocantins [TO] and Sao Paulo [SP]), R. nasutus and R. robustus populations. The symbols represent the origin of Rhodnius specimens captured in houses. The percentagecontribution of each factor to the total shape variation is shown on the axes in parentheses. Polygons enclose each group with different patterns. Drawings right the factorialmaps are the consensus conformation of the head and wing shape (see landmarks in Figs. 1 and 2). The arrows indicate the differences in head and wing shape of R. neglectusand R. robsutus (see text for details).

(e.g., Dujardin et al., 1991). While laboratory-reared R. prolixus andperidomestic R. neglectus from Mambaı shared identical allozymepatterns, both differed from a R. neglectus reference colony. It wasconsequently suggested that a species other than R. neglectus, andclosely related to R. prolixus, was invading houses in the study area(Dujardin et al., 1991).

Our results, largely based on the comparison of field-collectedmaterial, strongly suggest that R. neglectus is the species invad-

ing and occasionally colonizing artificial environments in mostof central Brazil. R. robustus and R. nasutus may also invadehouseholds in the humid Amazon and the Caatinga, respec-tively, but R. prolixus seems to be absent from the studyarea. We successfully confirmed these findings (clearly at oddswith the conclusions of Dujardin et al. (1991)) by compar-ing mitochondrial cytochrome b DNA sequences (Monteiro etal., 2003; Pavan and Monteiro, 2007) from the same spec-

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Table 3Reclassification scores of Rhodnius neglectus, R. robustus, and R. nasutus wings and heads after discriminant analysis of shape variables

Groups Wings Heads

R. neglectus R. nasutus R. robustus % correct reclassification R. neglectus R. nasutus R. robustus % correct reclassification

R. neglectus 39 1 0 97 52 1 1 96R. nasutus 30 0 0 100 0 28 2 93R. robustus 0 0 34 100 0 1 40 96

Kappaa 0.98 0.95

R. = Rhodnius.a Kappa: measures the degree of agreement between observed and expected classification; it ranges from 0 to 1; a score between 0.80 and 1 is considered almost perfect

agreement (Landis and Koch, 1977).

imens and populations; detailed results will be presentedelsewhere.

R. neglectus is a secondary vector potentially involved in humanChagas disease transmission throughout its wide distributionacross the Brazilian Cerrado and the southern fringes of the Amazon.Household infestation (with adventitious bugs occasionally estab-lishing breeding colonies) has been reported in the states of Goias,Minas Gerais, and Sao Paulo (Barretto et al., 1968; Garcia-Zapata etal., 1985; Silva et al., 1992, 1999). R. neglectus is currently the secondmost common triatomine species infesting artificial environmentsin the state of Goias. Infested households were detected in 79% ofthe municipalities in the state, and over 4500 R. neglectus speci-mens (∼1% infected with Trypanosoma cruzi) were collected over2 years (Oliveira and Silva, 2007). Extensive longitudinal surveil-lance systems capable of detecting and eliminating synanthropic R.neglectus populations are therefore needed across the range of thespecies. The development of reliable and practical methods for vec-tor species discrimination is obviously crucial in the context of suchdisease control-surveillance programs, where decision-making isoften based on taxonomic judgments (Monteiro et al., 2001).

5. Conclusions

The correct taxonomic assignment of problem organisms isnot only a cornerstone of biological research. It also has deeppractical (and ethical) implications when the lives and welfareof human beings depend on the development of suitable strate-gies for the management of a given species—and not of another,superficially similar taxon. Disease vectors obviously enter this cat-egory, together with pathogens, crop pests or endangered keystonespecies.

Most triatomine species can be confidently distinguished usingexternal morphological-chromatic characters; some groups arehowever problematic, and a few taxa are essentially isomorphic(Lent and Wygodzinsky, 1979). Several epidemiologically impor-tant species pose such difficulties; those within the ‘R. prolixusgroup’ constitute a classic example. Various alternative taxonomicmarkers have already been tested for species-level identificationof morphologically similar triatomines, but they tend to be expen-sive and laborious (Abad-Franch and Monteiro, 2005). Multivariateanalysis of metric characters is probably too complex to becomewidely used in routine, decentralized entomological surveillance.However, it is not difficult to envision a reference system wherebycontrol program managers could send ‘problem specimens’ (ordigital pictures/scans of key structures) to a supporting labora-tory where they could be compared to a reference morphometricdataset and thus ascribed to a specific taxon. We effectively mim-icked this real-life situation by treating bugs collected by CDCPworkers as ‘problem specimens’ in our analyses, and showed howthis approach can be put into practice even with paradigmaticallyproblematic triatomine species.

Acknowledgments

This study benefited from international collaboration throughthe ECLAT network. Special thanks are due to Fabio Oliveira Alvesfor fieldwork assistance. We also thank C.J. Schofield, J.P. Dujardin,M.A. Miles, and S. Catala for comments on earlier versions of themanuscript. L. Diotaiuti and F.B.S. Dias provided specimens fromthe R. neglectus FIOCRUZ colony, and the workers of the BrazilianChagas Disease Control Program supplied field-collected ‘prob-lem specimens’. Different parts of this research were funded byFAP-DF, FAPEAM and CNPq (Brazil), and by the WHO TDR SpecialProgram.

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