Vector Control Programmes

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PROJECT

European Community. Specific Support ActivitiesAmerican Trypanosomiasis Update. INCO-CT 2004-515942

Biological and environmental causes of the spatialstructuration in Triatoma infestans and theimplications for vector control programmes

2005-2006


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PARTICIPANTS (Group heads underlined)

Project coordinator. Silvia Catal, CRILAR , Anillaco, La Rioja, Argentina

Antennal PhenotypesSilvia Catal, Luciana Abrahan, Maria Laura Hernandez. CRILAR

BiogeographyDavid Gorla, Ximena Porcasi, Mariana Moreno. CRILAR.

CitogeneticsFrancisco Panzera, Ruben Prez, Claudia Lucero, Ines Ferrandis, Luca Calleros,Mara Jos Ferreiro URU, Lourdes Cardozo IICS.

Cuticular hydrocarbonsPatricia Jurez, Gustavo Caldern, Juan Girotti, Sergio Mijailovski. INIBIOLP

Field Collection and samples coord ination activitiesSilvia Catal, Mariana Moreno y Ximena Porcasi, Luciana Abrahan CRILAR,BlancaHerrera DNCH, Alberto Gentile DEPI, Abrahan Gemio PNCH, Francisco Panzera URU,Ruben Cardozo UNSA. Pilar Alderete PPCH, Patricio Diosque, UNSA, Elsa Lopes,Pedro Recalde, Cesar Zelaya y Victoria Bogado IICS.

Susceptibility to insecticidesMaria Ines Picollo, Eduardo Zerba, Claudia Vassena CIPEIN

Sylvatic fociMolecular analysis: Franois Noireau, Wilfrid Richer, Pierre Kengne, Marie MathildePerrineau, Mirko Rojas Cortez*, Anna Cohuet and Didier Fontenille, IRD.Field works: Mirko Rojas Cortez, Abraham Gemio, Alberto Llanos PNCH, RubnCardoso y Alejandro Uncos, UNSA, Gustavo Caldern INIBIOLP.Elsa Lopes, PedroRecalde, Cesar Zelaya y Victoria Bogado IICS, Gustavo Calderon INIBIOLP.

Wing MorphometryJean Pierre Dujardin IRD. Silvia Catal, CRILAR. Judith Schater Broide y RicardoGurtler. UBA

INSTITUTIONS

CIPEIN. Centro de Investigaciones en Plagas e insecticidas. Buenos Aires, Argentina.CRILAR.Centro Regional de Investigacion Cientifica de La Rioja. Anillaco, Argentina.DEPI. Direccion de Epidemiologia. Salta. Argentina.DNCH. Delegacion Catamarca, Argentina.IICS.Instituto de Investigaciones en Ciencias de la Salud, Asucncion, ParaguayINIBIOLP.Instituto de Investigaciones Bioqumicas de La Plata, ArgentinaIRD. Institut de Recherche pour le Dveloppement, UR016, Montpellier, FrancePNCH. Programa Nacional de Control de Chagas, BoliviaPPCH. Programa provincial de Chagas, Santiago del Estero, Argentina.UBA. Universidad de Buenos Aires, Facultad de Cs Exactas. Argentina.

UNSA Universidad Nacional de Salta, ArgentinaURU. Universidad de la Republica, Fac. de Ciencias, Genetica Evolutiva, Montevideo,Uruguay.


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INTRODUCTIONTriatoma infestansis the main vector of Trypanosoma cruzi,the causal agent of

Chagas disease in the southern cone of South America. The geographical distributionof the vector is being substantially reduced because of the activities of the vectorcontrol programme of the countries of the region, especially after the Southern ConeInitiative started by 1991. The distribution of T. infestansby the 1960s was much morewidespread, including most parts of Argentina, Bolivia, Paraguay, southeastern Brazil,northern Uruguay and Chile and southern Peru. At present, Uruguay, Chile, most ofBrazil and some areas of Argentina had certified the interruption of the vectorialtransmission of the disease by T infestans, with continued vigilance, especiallybecause of the occurrence of the species in neighboring countries. T infestans stillrepresents a serious public health problem in the endemic areas of Argentina, Bolivia,Paraguay and south of Peru.

The interruption of vector control activities by 2000 due to the economic crisis in

Argentina has lead to a recrudescence of the problem in the historically endemicregion. Northwestern areas of Argentina show the increasing occurrence of acutecases, with small areas of La Rioja, Chaco, Formosa and Santa Fe showingseropositive rates in children below 15 years as high as 45% (Biancardi et al 2003,Galvn et al. 2003, Diosque et al. 2004, Provincial Programs Reports La Rioja andSanta F).

In Paraguay, by 2002, Departments of Paraguari and Cordillera showedseroprevalence in children below 6 years of 5.6% and 4.2%, respectively. In a nationalsample in children below 5 years, seroprevalence was 0.48%, whereas in blood banksit was about 4 to 5%. The Paraguayan Chaco, although less populated than theoriental region, shows higher house infestation rate and high abundance of T. infestanspopulations (Incosur, 2003).

In Bolivia, Chagas disease is identified as the most important public healthproblem, with an overall seroprevalence of 40% (total population), reaching to 70% insome areas. Vector control shows a highly significant reduction of house infestationafter a nation-wide insecticide spraying programme, down to 4.1% house infestationrate at the national level (Jemio 2004).

Among the Triatominae, T.infestans is the best adapted to the domesticenvironment, living almost exclusively in houses and peridomestic structures. However,the presence of the species in sylvatic ecotopes has been confirmed in several andeanvalleys of Cochabamba and Sucre in Bolivia (Dujardin et al. 1987, Noireau et al. 2005)and in the bolivian chaco, where a chromatic variant called dark morph was found(Noireau et al. 2002). These findings, historical reconstructions (Schofield 1988) andgenetic analysis (Dujardin et al. 1998) suggest central Bolivia as the center of originand dispersion of T. Infestans towards the rest of the Southern Cone of South America.Another view, (Gorla 2001) based on environmental variables and potential dispersionroutes, analyses the possibility that a Chaco area around central Argentina couldconstitute the most probable dispersion center of the species. Additional efforts toelucidate the migratory routes of the species and its adaptation to domestic ecotopesare of importance in the epidemiology of Chagas disease.

Another important issue for the vector control programmes is the origin of theinsects that reinfest rural houses after insecticide application. Residual populations(surviving to the effect of the insecticide within the house) and populations moving fromthe peridomestic structures are the two candidate sources identified to play a role in thedomestic reinfestation, with positive evidences for both sources (Dujardin et al 1996,

1997, 1999 and Schachter-Broide et al 2004). Studies of Noireau et al (2005) reportedsylvatic foci near human houses, arising an important issue in the ecology of T.infestansand a warning for the vector control programmes.


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Recently, domestic populations of T. infestansin localities of northern Argentinaand southwest Bolivia showed resistance to pyrethroid insecticides. The issue forced

the use of an organophosphate (OP) to eliminate the domestic infestation (successfullyattained), with the consequent complications of a much better supervision needed forthe OP manipulation.

Panzera et al (2004) identified two chromosomic allopatric groups clearlydifferentiated in T. infestans populations, identified as "andean" (andean samples fromBolivia and Peru) and "non andean" (samples from central Argentina, Paraguay, Braziland the Bolivian Chaco). The "andean" group has C bands in almost all 22chromosomes, whereas the "non andean" group has only 5 to 8 banded chromosomes.This difference in the heterochromatin content is the main cause of the high variation inthe DNA content between the andean and non andean groups, according to laser fluxcytometry (about 35% more heterochromatin in the andean group). Panzera and co-workers suggest that the variations in the heterochromatin and DNA are reflecting

adaptive genomical changes, contributing to the ability of T. infestans to survive andreproduce in environments with different altitudes. Because of the high variabilityobserved in northern Argentina, it has been suggested that this region was thedispersion center of the non-andean populations. The existence of geographical clinesin T. infestanspopulations, is also supported by morphometrics (Dujardin et al. 1996,1997, 1999; Noireau et al. 2000, Catal et al. 2000, Catal and Dujardin, 2001, Cataland Torres 2001).

The general objective of this project was the study of the diversity of T.infestanspopulations in the Gran Chaco and mesothermic andean valleys of Bolivia,where the resilience of the vector populations and the reinfestation of domestic andperidomestic structures has shown to be widespread, in spite of the efforts of the vectorcontrol programmes in the region. The observed biological and environmental features

suggest there might be particular factors impeding the elimination of the vectorpopulation. Finding out what these factors are and how they operate, might help designcontrol strategies to produce an increase the impact of the vector control activities inthe region.

Within the context of the known spatial structuration of T. infestans populationsseveral questions were defined: Is the variability of the T. infestans population acontinuous or discrete phenomenon? Is the spatial structuration associated with theexistence of the "andean" - "non andean" genetic groups, and a consequence of itsdispersion to the south?. The occurrence of resistant and susceptible populations topyrethroids is linked to these two genotypes?. Is the limitation imposed byenvironmental variables associated with the spatial structuration of populations?. Aresylvatic populations unable to colonize domestic structures?

MAIN OBJECTIVES1. To study the diversity (ecological, morphological, biochemical, genetic) of T.

infestans populations of the Gran Chaco and mesothermic andean valleys ofBolivia, and the factors that determine this diversity

2. To evaluate the existence of new T. infestans silvatic population throughoutBolivia, Northern Argentina and Paraguay, in order to assess their geographicdistribution.

3. To discuss and propose migratory routes used by T. infestans to colonize thecentral and southern areas of the Southern Cone

4. To produce recommendations for the vector control programme, based on the

findings on the diversity of T. infestanspopulations.

Specific objectives are included with the specific methodologies.


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GENERAL METHODOLOGYBased on current knowledge of Triatoma infestanspopulations from studies on

cuticular hydrocarbons, morphometry, genetics, ecology, cytogenetics, resistance toinsecticides, sylvatic foci and geographic distribution, specific sampling areas wereselected from Argentina, Paraguay and Bolivia. The approach aimed at optimizing theuse of resources in terms of full coverage of the environmental heterogeneity withinthe Gran Chaco and the andean valleys of Bolivia. The depicted region included areaswhere T. infestans is still very abundant. The vector populations occupy theperidomestic structures constituted by shelters of domestic animals and otherstructures that rural people uses to store grain and goods, frequently occupied also, bywild mammals.

A total of 23 field sites were visited to collect T. infestans specimens. Thegeographic area covered by the sampling area spans from 6745 to 5948W and from1836 to 2813S. Elevations of the field sites are in the range 129 to 3095 meters over

sea level (mosl) (Table 1).

Table 1. Collecting sites of T. infestansspecimens.

CoordinatesCountry Province(Dept) Locality Altitude(masl)

Longi tude W Latitude S

ARGENTINA Chaco Pampa Avila 107 613044 265950

Chaco Tres estacas 122 614023 265430

S. del Estero Silipica 138 64078 280612

S. del Estero Huachana 165 640713 281325

Salta La Union 169 630815 234538

Salta San Carlos 2088 655753 254833

Salta Salvador Mazza 834 634121 220124

Catamarca Palo Blanco 1828 674535 272026

BOLIVIA Tarija San Antonio 429 632847 211539

Tarija Estacin Sunchal 558 632201 214117

Tarija San Fco del Inti 610 633635 214941

Tarija Palmar Chico 610 633645 215350

Tarija Yaguacua 650 633600 214200

Tarija Suarurito 853 635633 211656

Tarija Tacuarandy 958 635957 212303

Tarija Tarupayo 974 635717 212014

Cochabamba Bandorniyoc 1853 651104 183003

Cochabamba Mataral 1892 650919 183640

Chuquisaca Carapari 1892 651030 183718

Cochabamba Cotapachi 2618 661725 172555

Potosi La Deseada 2921 654134 213119

Potosi Urulica 3143 654954 213523PARAGUAY Boqueron Jerico 115 594841 223546


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More than 1000 adults and nymphs (V stage) of T. infestans were collected from 13

sampling sites and sent to CRILAR in order to be classified and processed. A sampleof 450 adults (males and females) were individually numbered and dissected. Specificbody parts were distributed to groups:

Wings (dried) were wrapped individually in aluminum foil and stored at roomtemperature prior to cuticular hydrocarbons analysis (INIBIOLP).

And then, the wings were sent to CRILAR for morphometric analysis (togetherwith UBA and IRD Fr),

Legs, fixed in ethanol 70 %, were sent to IRD Fr, for microsatellite DNAanalysis.

Heads were fixed in alcohol 70 % for the study of the antennal phenotype, atCRILAR.

Gonads were fixed in 3:1 ethanol-acetic acid, and sent to cytogenetics lab in

UdeRU.

A sample of live insects (eggs and/or nymphs and/or adults) was sent toCIPEIN in order to check susceptibility to insecticides on each locality. All no-usedinsects were killed and preserved in ethanol 70 %, as part of the SSA project collection(whole insects and legs, wings gonads and antennae of dissected insects).

GPS data collected during trips were mailed to CRILAR in order to carry out theenvironmental study.

1. Environmental features of SSA sampling sites

The environmental characteristics of the sites where Triatoma infestansspecimens were collected were studied using remotely sensed imagery and digital-format data.

The analysis included information about 6 environmental variables: elevation(DEM), air (AT) and land surface (LST) temperature, medium infrared radiation (MIR),vegetation index (NDVI) and vapour pressure deficit (VPD). Data on elevation wereextracted from the digital elevation model with 90 mts horizontal spatial resolution and16 mts vertical precision, produced by the NASA Shuttle Radar Topographic Mission

(SRTM) and downloaded from the CGIAR Consortium of Spatial Information (CGIAR-CSI, www.srtm.csi.cgiar.org). Data from the rest of the environmental variables were

extracted from a temporal time series of monthly satellite images for the period 1981-2000, produced by the sensor AVHRR onboard of the NOAA satellites, with a spatialresolution of 8x8 kms. The time series was analysed through a temporal Fourieranalysis, that produced a set of 14 descriptive statistics for each variable: average (a0),minimum (mn), maximum (mx), variance (vr), amplitude (ax), phase (px) and variabilityassociated to the annual, 2-annual or 3-annual cycles (x=1,2,3), and the variability ofthe 3 cyles (da). Spatial resolution of the data on terrain elevation was degraded tomatch the 8x8 km pixel size of the other variables. The set 14 descriptive statistics ofthe 5 environmental time-series, plus the DEM gives a total of 71 descriptor variablesfor each sampling site (Gorla, 2002).

Collection sites were spread over an area covering approximately 1.1 millionsquare kilometres that includes phytogeographical provinces of the Yungas, Chaco,

Pre-puna, Puna and Alto-Andina (Cabrera & Willink 1980).


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Fig. 1. Geographic location of the sampling sites. Black lines show internacionalboundaries, white lines are provincial boundaries (lowercase underlined). Colour

palette represents elevation: green is lowest, white is highest. Localities: 1: Cotapachi, 2:Bandorniyoc, 3: Mataral; 4: Carapari; 5: La Deseada; 6: Urulica; 7: Suarurito; 8: Tarupayo; 9:Tacuarandy; 10: San Antonio; 11: Estacin Sunchal; 12: Yaguacua; 13: San Francisco del Inti;14: Palmar Chico; 15: Salvador Mazza; 16: Jeric; 17: La Unin; 18: San Carlos; 19: PaloBlanco; 20: Pampa Avila; 21: Tres Estacas; 22: Huachana; 23: Silpica.

100


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2. Susceptibility to pyrethroid insecticides in Triatoma infestans populations

According to the previous results, the purpose of this sub-project was thecharacterization of the pyrethroid resistance of T.infestans from the areas included inthe general project, in order to establish the geographical distribution of thephenomenon, and explore a possible pyrethroid resistance route.

Insects: Samples of T. infestans eggs, nymphs or adults were received in thelaboratory and kept at 28 1C, 50% RH, 12: 12 h (L: D) photoperiod. A total of 49samples collected in Argentina (Catamarca, Chaco and Salta), Paraguay and Bolivia,were received. The insects were reared until the eclosion of the field eggs or theappearance of first instars of the further generations of the field insects. CIPEIN is aninsecticide susceptible strain maintained in our laboratory since 1975 without anyexposure to insecticides (Picollo et al. 1976). From all colonies, 3 day old first instars,starved since eclosion (mean weight 1.2 0.2 mg), were selected for toxicity tests

according to the WHO protocol (1994).Chemicals: Technical grade deltamethrin (97%, Bayer, Argentina), and

analytical grade acetone (Merck, Buenos Aires, Argentina) were used for thetoxicological tests.

Topical application bioassays: For determining resistance, discriminative dose(DD) of deltamethrin (2 ng/i) was delivered in 0.2 l acetone to the dorsal abdomen offirst instars (WHO 1994). At least 10 nymphs per replicate were treated for eachsample and for the susceptible strain. Control groups received acetone. Fordetermining lethal dose 50 (LD50), a minimum of three doses giving >0% and


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The C-banding pattern for each specimen was determined by analyzing at least10 cells. In males, both mitotic (spermatogonial prometaphase) and meiotic (first and

second metaphases) plates were observed. For females, only oogonial prometaphaseswere studied because no meiotic stages can be detected. The identification of eachchromosomal pair was based on size differences and on the analysis of the meioticconfigurations. Three autosomal morphs, denoted A, B, and C, were recognized on thebasis of previous reports (Panzera et al. 1992; 1998): A morph (a subterminal C-heterochromatic block is present at one chromosomal end; the other end iseuchromatic or has a very small C-band); B morph (C-heterochromatic blocks areclearly present at both chromosomal ends); and C morph (the chromosome is totallyeuchromatic or has a very small C-band). This nomenclature was used to describe thecharacteristic of the chromosome complement of each individual. In this description wealso include additional information about the presence of interstitial bands andchromosome fragments or satellites.

In order to quantify the autosomal C-heterochromatin, we estimated the relativeamount of C-heterochromatin presented in the autosomal complement. For eachspecimen, at least 3-5 images of gonial metaphases plates were analyzed by means ofappropriate software (IPP plus, Media Cybernetics, Carlsbad, CA).

4. Cuticular hydrocarbons of T. infestans

In this project we propose to help improve the knowledge on Triatoma infestanspopulation structure along most of its geographical distribution, using the cuticularhydrocarbon fingerprint as phenetic marker. The specific objectives were: To establishphenotype diversity based on comparative analyses of the cuticular hydrocarbons as afunction of sex, habitat and geographic distribution and, to evaluate the role of

hydrocarbons on insecticide-cuticle interaction based on comparative analyses ofinsecticide resistant and susceptible populations.

Hydrocarbon analysis: Wings from each specimen were washed with redistilledwater, dried out, then transferred to a glass vial with Teflon-lined caps, and submergedin redistilled hexane (6 ml/g) overnight, to extract total lipids. After solvent reduction involume under nitrogen, hydrocarbons were separated from other components byadsorption chromatography on a mini-column of activated Biosil A (10 mm x 5 mmI.D.), eluting with redistilled hexane (6 ml/mg hydrocarbon). The extract then wasevaporated to an appropriate volume for gas chromatography. Capillary gaschromatographic (CGC) analysis was performed using a Hewlett-Packard (HP) 6890gas chromatograph equipped with a split/splitless injection port, fitted with a non-polarfused silica (0.25 m) HP-5 capillary column (30 m x 0.32 mm I.D.). The oventemperature was programmed from 50 C (hold time 2 min) to 180 C at 20 C/min,then 180 C to 310 C at 3 C/min (hold 10 min). Chain length was estimated bycalculating the Kovats Indices (KI) according to Kovats (1965). The KI number providesinformation on the total carbon number and the branching pattern; i.e. KI 2900corresponds to a hydrocarbon of 29 carbon atoms in a straight chain whereas last twodigits different from zero indicate the presence of methyl groups inserted in the straightchain and/or unsaturation. The methyl-branching pattern of T. infestanswas previouslydescribed in Jurez & Blomquist 1993.

In order to evaluate the role of hydrocarbons on insecticide-cuticle interactionbased on comparative analyzes of insecticide resistant and susceptible populations,cuticular lipids were extracted from resistant specimens (S. Mazza, Salta, AR) and

compared to susceptible lab-reared ones by thin layer chromatography (TLC) andCGC analyzes.


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logistic reasons, investigations were carried out only in Paraguay for the Chacoecosystem, and in Bolivia and Northern Argentina for the Andean valleys. Local

residents will be asked about the knowledge of wild bugs and the insects will be searchusing the trapping system described by Noireau et al. (1999).

6 b. Genetic structure of wild and domestic bug populationsMicrosatellite DNA markers, which are useful tools for determining population

structure, were recently isolated and characterized in T. infestans(Garca et al., 2004;Marcet et al., 2006). To assess the dispersion of wild T. infestans in an area of theBolivian Andes, we studied genetic variability at 9 microsatellite loci in vectorpopulations from different collecting sites.

Study area - Cotapachi (2,750 m asl.; 17 26 S, 66 17 W) is a rural arealocated in the Eastern Andean Cordillera, near the city of Cochabamba. It is a smallcirque-valley open to the east and encircled by three hills (northern, western and

southern hills) that overhang a lagoon and culminate at 60 m above it. Somedwellings and cultivated areas (market-gardening, maize) are located in the valley,between hills and lagoon. Two separate rocky outcrops made of huge blocks occur inthe valley. They are named Inca wall (for its similitude with a man-building) andperidomestic rocks (for their location near the houses). The zone located on the footand slopes of hills is more homogeneous. It is covered by rocky outcrops of differentdimensions and displays a low diversity of vegetation dominated by shrubs ofMimosoideae. The climate of the Cochabamba valley is dry and hot during thesummer, temperate in the winter (mean annual temperature 16.3C, mean annualrainfall 362 mm). The vegetation of the study area is damaged, on account of theantiquity of human occupation. It is characterized by the dominance of thickets withscarce arboreal components (principally shrubs). The inhabitants belong to the

Qeshua ethnic group. Their dwellings are mainly of wattle and daub construction androofed with tile.

Collection of triatomines - Five sylvatic sites were investigated in June 2005:southern, eastern and northern hills, Inca wall and peridomestic rocks. T. infestanswassearched in its natural habitat consisting of cracks between rocks and shelters of smallmammals located under the rocks (Table 2). Baited traps as described by Noireau etal. (1999) were used to capture the insects. Collections were also made inside twodwellings of Cotapachi. Legs from triatomines were stored individually at -20C inethanol 70%. Analyzed samples are given in Table 1. As outgroup, we used 13domestic T. infestans collected at Mataral, a locality situated 200 km southwest ofCotapachi.

DNA extraction - Genomic DNA was extracted from legs of each individualtriatomine following a slightly modified version of the protocol of Cornel et al.(1996).

Microsatellite amplification, genotyping and analysis - Ten microsatelliteloci were selected from published T. infestans sequence data and amplified: TiA02,TiC02, TiC08, TiC09, TiD09, TiE02, TiE12, TiF03, TiF11 et TiG03 (Garca et al., 2004).Two reaction mixtures were utilized. For TiA02, TiC02, TiD09, TiE02, TiE12, TiF03,TiF11 et TiG03, PCR amplification was carried out in 25 l reaction volume, from 1 lof template DNA; reaction mixture contained 1X PCR buffer with 0.2 mM dNTP, 1.5mM MgCl2, 10 m of each primer and 0.5 U Taq Polymerase (QIAgen, France). ForTiC08 et TiC09, the reaction mixture contained 1X PCR buffer with 0.2 mM dNTP, 2.5mM MgCl2, 10 m of each primer and 0.75 U Taq Polymerase. Amplification wasperformed under the following conditions: an initial denaturation step at 94C for 3 min

followed by 35 cycles of 30 sec. at 94C, annealing at 49C for 30 seconds, extensionat 72C for 30 min followed by a 72C final extension for 30 min, and 10C indefinitely.PCR products were separated and visualized on 2% agarose gel in TBE buffer. PCR


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products were diluted at 1:30 (A02, C09, E02, F11 and G03 loci) or 1:60 (other loci).Pools consisted of 3.5 l aliquots of PCR reactions, 0.4 l of size standard (Applied

Biosystems) and 16.1 l dHi-Di formamide (Applied Biosystems). Alleles were resolvedon an Applied Biosystems 3130xl Genetic and sized GeneMapper 3.7 software. Foreach microsatellite locus, deviation from Hardy-Weinberg expectation was tested ineach location and the pooled population. Statistical significance for linkagedisequilibrium between pairs of microsatellites loci in the pooled population and withineach population was computed using Genepop 3.4. Genetic differentiation betweengeographical populations was examined by F statistics according to Weir & Cockerham(1984) using Fstat 2.9.3.2 software.

Table 2. Samples analyzed according to the collecting site at Cotapachi.*This population was considered as sylvatic because: i) its habitat is natural and not artificial; ii)complementary studies have shown that it feed on small wild mammals.

Site of collectionSort of

triatomine populationNo. of insects analyzed

Northern hill (NH) sylvatic 6

Western hill (WH) sylvatic 20

Southern hill (SH) sylvatic 12

Inca wall (IW) sylvatic 32

Peridomestic rocks (PR) sylvatic* 32

Dwelling 1 (DW1) domestic 3

Dwelling 2 (DW2) domestic 7

Total 113


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RESULTS

1. Environmental analysis

The analysis proceeded in four steps. In the first step, the values of theenvironmental variables were extracted from the available digital imagery containingdata on air and land surface temperature (AT and LST), vegetation index (NDVI),medium infrared radiation (MIR), vapour pressure deficit (VPD) and elevation. Eachlocality had an associated set of 71 values of environmental variables: 14 statisticsderived from the Fourier-processed time series for each of the the five mentionedvariables (AT, LST, NDVI, MIR, VPD), plus the elevation data.

In the second step, an environmental similarity analysis of the localities wascarried out. This analysis grouped the localities according to their environmentalsimilarities.

In the third step, a discriminant analysis of the locality groups obtained in step 2was carried out, to obtain linear models with the ability to describe the membership tothe locality groups as a function of the environmental variables.

In the fourth step, the linear models obtained by the discriminant analysis wereused to produce thematic maps to predict areas with similar environmental propertiesto the groups identified in step 2.

The environmental similarity analysis of the localities was carried out through anUPGMA cluster analysis over the Mahalanobis distances using the environmentalvariables and the time series statistics of the 27 sampling sites. Because of the spatialresolution of the resampled AVHRR images (8x8 kms), several sites were included inthe same pixel. This resolution issue reduced the number of sampling sites from 27 to23.

The locality groups identified in the cluster analysis were used as the input for astepwise linear discriminant analysis using the set of 71 environmental variables. This

analysis allowed for the identification of the variables that have the highest weight todifferentiate the sample site groups and to produce a model that identifies the areaswith similar environmental properties to the locality groups identified in the clusteranalysis (Rogers 2000, Gorla 2002). The most important environmental variables in thediscrimination of locality groups were used to make a descriptive analysis of theenvironmental properties of the sampling sites.

The similarity analysis showed that the environmental profile could beaggregated into 2 main groups (Fig. 4). A first group includes 12 localities of the GranChaco of Argentina, Bolivia and Paraguay (labelled as GC). A second group includes15 localities of the Andean valleys and high Andes (HA). This second group showsmore variability, including high Andes (Urulica and La Deseada, labelled as HA group),high valleys (Cotapachi, Palo Blanco and San Carlos, labelled as HV), intermediate

valleys (Bandorniyoc and Mataral, labelled as IV) and low valleys (including the last 8localities within this group, labelled as LV). High Andes and intermediate valleys (HA-IV) are more similar than high and low valleys subgroup ( HV-LV).


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Figure 4. Similarity of sampling sites according to a cluster analysis (UPGMA withEuclidean distances) of 71 independent environmental variables.

At euclidean distances of 2400, 2100 and 1600, 2, 3 or 5 groups of localitiescan be identified (Fig 4). A separate analysis of grouping relationships was carried outconsidering either 2, 3 or 5 groups. A stepwise linear discriminant analysis was carriedout using the data on elevation and the variables of the Fourier time series statistics toidentify the environmental variables that best describe the sampling site groups.

In the analysis that considered 5 groups of localities, a model with 2 variables(DEM and MIRP1) was able to discriminate the locality groups with 3.7% error(classifying incorrectly a locality belonging to GC as LV). The first discriminant functionexplained 96.7% of the total variation; elevation (in meters above sea level masl,labelled as DEM) and the annual phase of the medium infra red (MIRP1) show asimilar weight in the linear model. According to the annual phase of the medium

infrared (MIRP1), the MIR peaks between November and December in the Chaco andhigh valleys and high Andes, but peaks around October in the low and intermediatevalleys (Table 3).

When 3 groups of localities (according to the similarity analysis ofenvironmental variables, Fig. 4) were considered, a linear model with two variableswere able to discriminate the localities without error. The variables are elevation andthe average of the vapour pressure deficit (VPDA0). VPD shows more weight in themodel than the DEM. The high Andes and intermediate valleys (non-Chaco areas)were discriminated by a higher elevation and lower humidity of the first. Within thiscomparison, the Gran Chaco area showed as the lower elevation and lower humidity(=higher pressure deficit) (Table 3).

When only two groups of localities were considered (chaco and non-chaco), amodel with two variables was able to discriminate the localities with a 3.7% error (onechaco locality classified as non-chaco). The variables are the variability of the vapourpressure deficit (VPDVR) and the minimum of the land surface temperature (LSTMN).VPDVR has more weight in the model than the LSTMN. Chaco localities show higher

GC

HA

IV

LV

HV


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minimum temperature and variability of the vapour deficit pressure than the non-chacolocalities (Table 3).

In general, the discriminant analysis shows that irrespective the number ofenvironmental groups considered, the Gran Chaco area always appears as a wellidentified group, with low elevation, lower and more variable humidity (as measured bythe vapour pressure deficit) and higher minimum land surface temperature. Areasoutside the Gran Chaco show high heterogeneity, specially because of the east-westelevation gradient imposed by the Andes.

Table 3. Mean (standard deviations) of the variables identified as the best todiscriminate the environmental properties of locality groups identified by the similarityanalysis (Fig. 4). GC: Gran Chaco; LV low valleys, HV: high valleys; IV: intermediatevalleys, HA: high Andes.

Locality Group

Number oflocality groups Discriminatingvariables GC LV HV IV HA

Elevation 193.5 790 2178 1873 3032

(masl) (144.2) (148) (403) (27.6) (157)

Annual phase 11.45 10.7 11.5 9.8 11.7

5 groupserror = 3.7%

of MIR (0.25) (0.21) (0.72) (0.00) (0.14)

GC LV-HV IV-HA

VPD Average 3333 (299.6) 2209 (229) 2963 (131)3 groupserror = 0% Elevation

(masl) 193.5 (144.2) 1169 (684) 2452 (676)

GC No-GC

VPD Variante 351.6 (69.6) 164 (44.5)2 groupserror = 3.7% LST minimum 25.11 (1.96) 24.1 (2.79)

The three linear models were used to calculate temathic maps of areas with similarenvironmental properties as those ones that defined the groups by the similarityanalysis. Figure 5 shows the 3 resulting maps with 2, 3 and 5 area groups. The resultshows that as more groups are considered, more discrimination is obtained over theAndean region. The map including only two groups shows the Gran Chaco region andthe Andean region; the map including 3 groups shows the Gran Chaco unchanged but

the Andean region splitted in two (a higher and lower region); when 5 groups wereconsidered, the lower Andean region was again splitted in 3 subregions, showing low,intermediate and high valleys (Figure 5).

Air temperature shows the known negative relationship with altitude, althoughthe relation is not linear over all the observed range of altitude. There is a lineardecrease of temperature with altitude up to 1000 meters (adjusted R2= 0.80, n=20).Above 1000 meters the variation of temperature with altitude shows higher variability,especially due to temperature values of Bandorniyoc, Mataral, Palo Blanco and SanCarlos, within the elevation range of 1500 2500 meters (Fig. 6). A logarithmic functionfits the whole data set with an R2= 0.66.


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Figure 5. Thematic map showing areas with similar environmental properties to thelocality groups (2, 3 or 5 groups, see text for explanation) identified in the similarityanalysis, according to discriminant analysis. Map on the left (two groups) identifieschaco (green) and non-chaco (red) areas; discriminating variables are variability of thevapour pressure deficit and the minimum of the land surface temperature. Middle mapidentifies chaco (green), non-chaco (red) and an intermediate region (yellow);discriminating variables are elevation and the average of the vapour pressure deficit.Map on the right (five groups) is calculated using data on elevation and the annualphase of the middle infrared red.

Figure 6. Average of air temperature (C) and altitude (in meters above sea level) foreach sample site

The annual phase of the infrared radiation (MIRP1) shows a non linear relation withaltitude, with a minimum at 1800 masl (Palo Blanco and San Carlos are out of trend)(Fig. 7).

15

20

25

30

35

40

0 500 1000 1500 2000 2500 3000 3500

Al ti tude (mos l)

AverageofAirTemperature(C)


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9

9.5

10

10.5

11

11.5

12

0 500 1000 1500 2000 2500 3000 3500

Elevation (masl)

Annualphas

eMIR

Figure 7. Relationship between altitude and the annual phase of the medium infrared

radiation (MIRP1) of the sampling sites. The two points out of the curvilinear trend arePalo Blanco and San Carlos.

2. Susceptibilit y to pyrethroid insectic ides

Toxic effect of deltamethrin was studied in field samples of T. infestans from thenorth of Argentina, Paraguay and Bolivia. Mortality data of T. infestansfirst instars afterapplication of DD of deltamethrin are shown in Table 4. These values were used toassess resistance associated to field control failures, according to our previous results,which demonstrated that mortalities higher than 40 (%M >40) measured by topical

application in the laboratory, indicate that field populations are being successfullycontrolled, and mortalities equal or lower than 20% is a signal of potential controlfailures in the field. The determination of resistance based on the survival to thedeltamethrin discriminative dose could be done on thirty (30) samples. Some sampleswere discarded because the insects were dead or there was not enough biologicalmaterial.

Table 4. Mortality data of T. infestans first instars after application of DD ofdeltamethrin. S:susceptible insects, R: resistant insects

COUNTRY, PROVINCE/STATE LOCALITYTested

samples%

MortalitySusceptibility

Argentina, Catamarca Palo Blanco 1 95 4,5 S

Argentina, Chaco Pampa Avila 3 100 S

Tres Estacas 1 100 S

Argentina, Salta La Union 1 76 7 S

Salvador Mazza 1 13,7 9,6 R

San Carlos 1 20 5 R

Bolivia, Potosi (Tupiza) La Deseada 1 0 R

Bolivia, Chuquisaca (Sucre) Carapari 2 0 R

Bolivia, Tarija (Entre Rios) Suarurito 1 0 R

Bolivia, Tarija (Entre Rios) Tacuarandy 3 0 R

Bolivia, Tarija (Villa Montes) San Antonio 1 0 R


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COUNTRY, PROVINCE/STATE LOCALITYTested

samples%

MortalitySusceptibility

Bolivia, Tarija (Yacuiba) S Franc. del Inti 2 0 R

Bolivia, Tarija (Yacuiba) Quebrada Buzuy 3 0 R

Bolivia, Tarija (Yacuiba) Palmar Chico 4 17 8 RBolivia, Tarija (Yacuiba) Estacin Sunchal 2 0 R

Bolivia, Tarija (Yacuiba) El Barrial 1 0 R

Paraguay, (Boqueron) Jerico 1 90 0 S

Lethal dose 50 (LD50) and resistance ratio to deltamethrin were assessed in firstnymphs from samples with enough number of insects (8 samples). These data forsusceptible and resistant samples and CIPEIN strain are shown in Table 5.

The laboratory toxicity tests have demonstrated pyrethroid resistance at aenough level to cause control failures in field T. infestans populations from Bolivia(Potos, Chuquisaca, Cochabamba y Tarija) and Salta (Salvador Mazza and SanCarlos). Otherwise, no resistance to topically applied deltamethrin existed in fieldinsects from Paraguay (Boqueron), Catamarca (Palo Blanco), Chaco (P.Avila y TresEstacas), and Salta (La Unin ).

The toxicity data estimated for field populations demonstrated high resistancelevel in collected insects from Salta- Salvador Mazza, Salta- San Carlos, Bolivia-Chuquisaca-Carapari and Bolivia-Tarija-Yacuiba. In these localities, ineffective fieldcontrol is expected after pyrethroid treatment. Otherwise the low resistant estimated forCatamarca-Palo Blanco (4.48x), Chaco-P. Avila and Tres Estacas (2.97x), Salta-LaUnion (5x) and Paraguay-Boqueron (3.7x), indicate probable field control withpyrethroids. These results demonstrated similar toxicological profile in field populationsfrom Bolivia and Salta north and west. Otherwise, populations from Salta east are

toxicologically similar to those from Paraguay, Chaco and Santiago del Estero.The resistance to fenitrothion, bendiocarb and fipronil, and the determination ofenzyme activity, could not be studied because the number of descendent nymphs wasnot large enough.

Table 5: Bioassay statistics and resistance ratios for deltamethrin in T. infestansfieldpopulations. *DL50: Lethal dose of deltamethrin nedeed to eliminate 50% of exposedinsects, *GR: Resistance ratio.

Sample n Slope SDDL50(ng/i)*(95% LC)

GR**(95% LC)

CIPEIN (Lab. Strain) 145 1,76 0,250,10

(0,07 0,14)

-

Palo Blanco (Catamarca,Argentina) 220 2.72 0.09

0.58(0.07- 0.69)

4.48(2.47-8.13)

P. Avila andT.Estacas (Chaco, Argentina..)

49 1.32 0.100.39

(0.07-1.52)2.97

(2.18-4.05)

La Unin (Salta, Argentina)30 1. 29 0.11

0.65(0.1-1.79)

5.01(4.06-6.19

Salvador Mazza (Salta,Argentina)210 1.59 0.08

61.43( 36.2-149.4)

380.70(316.88-457.38

San Carlos (Salta, Argentina)30 1.71 0.20 8.31

64.16(49.34-83.43)

Carapari (Chuquisaca,Bolivia) 80 1.15 0.09

30.12

(20.7-42.6)

233.0

(184.6- 294.1)

S. Francisco Inti (Tarija, Bolivia) 70 1.39 0.0983.89

(39.40-309.0)645.24

(518.72-802.63

Jerico (Boqueron, Paraguay) 90 1.57 0.100.48

(0.01-1.59)3.68

(2.99-4.52)


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3. Chromosomal analysisDiploid Chromosome number and chromosomal fragments

All T. infestans specimens had the same diploid chromosome number (2n =22), constituted by 20 autosomes and two sex chromosomes (XY in the males and XXin the females). However, some individuals from specific populations presentedchromosomal fragments or supernumerary chromosomes (also called B or accessorychromosomes) (Table 6). The same individual could have one or two chromosomalfragments. Within an individual, these fragments appeared in several, but not all mitoticprometaphases. Only in a few individuals, they were also observed in meiotic plates. Inall cases, these B chromosomes are conspicuously smaller than the smallestautosomes but their size was variable. In general, these fragments appearedeuchromatic but some of them were C-heterochromatic.

C-banding techniqueC-heterochromatic blocks were usually located in terminal and subterminalpositions. The existence of interstitial C-bands was variable according the populationanalyzed. Each specimen exhibited a specific C-banding pattern, without intraindividualvariation. Table 6 and Figures 8 A and B summarize the variability observed in the C-banding karyotype of T. infestans from different localities. All populations showedvariation in the number and/or the position of C-bands, allowing us to differentiate threeclearly distinct groups:

Group 1 or Andean Group.It comprised insects from Andean Bolivia. The number ofautosomes with C-blocks varied from 12 to 16. Both sex chromosomes (X and Y)had always C-bands but with different sizes. Within group 1, the similar size and

shape of the 10 chromosomal pairs made it very difficult to identify each pair.The C-heterochromatin content varied from 46% to 56% of the autosomalcomplement because of the heterochromatin polymorphism already mentioned.

Group 2 or Non-Andean.It included specimens from several non-Andean populationsin Argentina and Paraguay (see Table 6). The number of autosomes with C-bands varied from 4 to 7 chromosomes (Table 6), but most insects presented 6C-heterochromatic autosomes. In this group, the three first autosomal C-heterochromatic pairs were identified based on size differences and meioticconfigurations. The karyotype described in previous reports, BB BB AA, was byfar the most frequent. The Y chromosome always exhibited C-blocks, whereasthe X chromosome did not show any C-banding. C-heterochromatin content

ranges from 24% to 30% of the total autosomal length.

Group 3 or Intermediate. It comprised individuals from Salvador Mazza in SaltaProvince (Argentina, neighboring with Tarija, Bolivia) and several localities ofTarija Province (Bolivia). This group had an intermediate number ofheterochromatic chromosomes (7 to 11) (Figure 8), compared with the groups 1and 2 previously described. The Y chromosome always exhibited C-blocks,whereas the X chromosome did not show C-banding. In only 2 specimens a verysmall C band were observed in the X chromosome.


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Table 6. Analyzed material of Triatoma infestans classified by procedence, number ofspecimens analyzed (N) with C-banding, number of autosomes with C-blocks (meanand standard deviation), total number of C-block, presence of chromosomal fragmentsor autosomes with satellite, and presence of X chromosome with C-block. M= males;F=females.

Geographic or igin N(M,F)

Number ofautosomes withC-blocks (mean

and SD)

Total numberof C-blocks(mean and

SD)

Presence ofchromosomalfragments orautosomes

with satellite

Xchromosome withC-blocks

Group 1. AndeanLa Deseada, Potos, Bolivia 5M 12.60 + 0.89 20.20 + 1.30 No Yes

Cochabamba & Sucre,Bolivia.

7M 13.71 + 2.29 18.43 + 2.76 No Yes

Group 2. Non-AndeanSan Carlos, Salta, Arg. 2M, 1F 6.33 + 0.58 10.67 + 1.53 Yes NoLa Unin, Salta, Arg. 2M 6.00 + 0.00 10.00 + 0.00 Yes NoPalo Blanco, Catamarca, Arg. 6M 6.00 + 0.00 9.33 + 0.82 Yes NoTres Estacas, Chaco, Arg. 6M, 2F 6.00 + 0.00 10.00 + 0.00 Yes NoPampa Avila, Chaco, Arg. 18M,

5F5.33 + 0.64 8.63 + 0.58 Yes No

Huachana, Stgo. Estero, Arg. 3M 5.00 + 0.00 8.67 + 0.58 No NoSilipica, Stgo. Estero, Arg. 3M 5.00 + 0.00 8.67 + 0.58 No NoJeric, Boquern, Paraguay. 9M 6.00 + 0.00 11.00 + 0.00 No No

Group 3. Intermediate.Yacuiba, Tarija, Bolivia 14M,

8F8.82 + 2.08 13.27 + 2.59 Yes No

Salvador Mazza, Salta,Argentina.

33M,20F

8.25 + 1.21 13.30 + 1.58 Yes No

Figure 8. Mitotic prometaphases of Triatoma infestans (2n= 22 chromosomes) oftwo specimens from Salvador Mazza (Salta, Argentina) showing chromosomalfragments (arrows)Figure A: Eleven autosomes with C-banding are observed, two of them with interstitialC-bands. Figure B: Nine heterochromatic chromosomes are observed, two of them withinterstitial C-bands.


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4. Cuticular hydrocarbons

T. infestanshydrocarbons comprise a mixture of straight chains with prevalenceof 27 to 33 carbons (KIs 2700 to 3300) together with a variety of methyl-branchedchains of more than 40 carbons (KIs from 2970 to 4040). The relative amount of thehydrocarbon components was analyzed in order to evaluate intraspecific variability.Cuticular hydrocarbon fingerprints showed high quantitative variability, although noqualitative differentiation was detected. A total number of 328 pair of wings obtainedfrom the collection sites detailed in Table 7, were used for hydrocarbon analyses.

Table 7. Triatoma infestans collection sites in Argentina, Bolivia and Paraguay.

Country State Locality Numberofinsects

Catamarca Palo Blanco 38Pampa Avila 38Chaco

Tres Estacas 11La Unin 8Morillo 6Salv. Mazza 7

Salta

San Carlos 7Capital 7Huachana 7

Argentina

Sgo. DelEstero

Silpica 14

Bandorniyoc 13CochabambaMataral 14

Potos La Deseada 13Urulica 1

Chuquisaca Carapari 20

El Barrial 4Est. Sunchal 8Palmar Chico 7Qda. Buzuy 8San Francisco 8Suarurito 15San Antonio 10Tacuarandy 5Tarupayo 5

Bolivia

Tarija

Yaguacua 4

Paraguay Boquern Jeric 40

Typical examples of the hydrocarbon pattern are shown in Figure 9.


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Figure 10. Dendrogram showing the population structure of Triatoma infestans inArgentina, Bolivia and Paraguay, based on squared Mahalanobis distances fromdiscriminant analysis.

Comparative analyses of cuticular hydrocarbons in insecticide resistant and susceptiblepopulations of T.infestans

Thin layer chromatography (TLC) from resistant (S. Mazza, Salta, AR) andsusceptible lab-reared specimens, showed a significant increase (> 2-fold) in the total

hydrocarbon amount of the resistant insects (Fig 11), also confirmed by CGC analyses(data not shown).

Figure 11. Epicuticular lipids of resistant (A) and susceptible (B) T.infestans.Upper dark bands correspond to the hydrocarbon fraction;the other spots represent the wax ester, acylglycerol, fatty acid, sterolsand fatty alcohol components.

5. Cuantitative morphologya. The antennal phenotype of Triatoma infestans

The antennae showed a great variability in the number of trichoidea sensilla,either thin (TH) or thick walled (TK), over all Triatoma infestansgeographical range. Onthe other hand, mechanoreceptors (BR) and basiconica (BA) showed null or low

variability. Sexual differences account for all populations, mainly on the number of THon the pedicel, more abundant in males than females. The main changes in theantenna occur on the first segment of the flagel and pedicel of females but, in bothsegments of the flagel in males.

Samples from Pampa Avila and Yacuiba, which included specimens from


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domiciles and goat corrals were used to study the habitat effect. Results showed theoccurrence of minor variations in the number of TK sensilla on flagel 1 and 2, with alower number of these sensilla in insects collected in the intradomestic ecotopes. Thesylvatic population of Cotapachi (Bolivia, Cochabamba) showed a significant decreaseon the TK of flagel and TH from pedicel when compared with domestic populations ofthe same region.

Variability of geographical populations of T.infestans based on their antennalphenotypes.

The data from 184 individuals collected in 11 geographic populations studied bymultivariate discriminant analysis (with 10 variables), allowed to produce a dendrogramderived from the Mahalanobis distances (Fig. 12) Two main groups were identified.One group comprised the Andean populations of Bolivia (Carapari, Aiquile, LaDeseada and the sylvatic population of Cotapachi). The second group includedpopulations from Gran Chaco localities of Argentina, Paraguay and Bolivia. Theexception is the population of Palo Blanco located in the Sierras Pampeanas,Catamarca, Argentina (1900 mosl). Females showed better definition of the antennal

morphological markers than males.The argentinean populations of San Carlos (Salta, south-west) and La Union(Salta, north-east) were incorporated afterwards into the discriminant analysis asexternal groups, because they had low number of individuals. The analysis classifiedthe individuals of both localities within the Gran Chaco group.

The populations coming from the humid oriental Chaco: Jerico (Paraguay) andPampa Avila (Argentina) were clearly separated from populations of the occidental andmore dry, Chaco (Fig 12).

Figure 12. Phenetic distances between T.infestanspopulations. Dendrogram for11 geographic populations derived from Mahalanobis distances.

Distance

La Deseada

Carapari

A iq ui le

Cotapachi

P.Av ila

Jer ico

Yacuiba

E.Rios

P.Blanco

Huachana

S.Mazza

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Bolivia, Chuq uisac

Bolivia, Coch abamb

Bolivia, Potos i

Arg en tina , Ca tama rc a

Gran Chaco

Andes

Bolivia, Tarija

Paraguay, Boquero

Arg en tina , Ch ac o

Arg en tina , Santia go

Arg en tina , Salta


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Phenotipic variability was apparently high within some populations,suggesting heterogeneous composition. To test this hypothesis, 99 females wereanalyzed using k-means clustering. Following the structure showed in fig 13, theconformation of 3 cluster was proposed for the analysis. The two most significantvariables of the phenotype PTH (F=84.81, p


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The phenotipic expression of T.infestans genotypes

When all the geographic populations were identified by its chromosomalcharacteristics, we searched for their relationship with the antennal phenotypes. Table11 shows the means for the number of each sensillum type. The three genotypes couldbe recognized by trichoidea sensilla (Th and TK), less abundant in Andean T.infestans.The bristles (BR) from the 3 segments and basiconica (BA) on flagel, did not showedsignificant differences among groups. Sexual differences were evident for allgenotypes.

Table 11. Mean number of antennal sensilla (10 types) according to the sex andgenotype of individuals.

PEDICEL FLAGEL 1 FLAGEL 2Genotype

BR TH BR TH TK BA BR TH TK BA

Andean 126 60 13 68 239 37 7 33 200 29

Inter-mediate

124 74 12 61 309 29 7 27 223 28

FEMALES

NoAndean 124 121 12 87 387 35 8 32 236 30

Andean 124 134 13 85 245 38 7 35 209 32

Inter-mediate

123 138 12 78 299 28 7 32 240 26MALES No

Andean

120 160 12 95 362 37 7 35 241 30

A discriminant analysis, allowed the significant characterization of the 3 genotypes(data not shown).

5.b .Population analysis based on wings morphometry

No relationship was found between wing size (as Centroid Size, CS) and thealtitude on which the insects were captured (data not shown). Figure 13 shows the

variation of CS in 13 geographic populations, both sex included. Sexual dimorphism isevident in wings size for all populations, except S. Mazza (Argentina).The largest wings were found on both sex from Cochabamba and Carapari

(Bolivia), as well as in males from Palo Blanco (Argentina). Males and females from LaDeseada (Bolivia) showed the smallest wings.


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Figure 13. Triatoma infestans wing size (as CS) on different populations. Circle:males,star: females. Arrow: no-sexual dimorphism.

Symmetry of size.Eight populations were checked for asymmetry, in wing size and conformation.

All of them exhibited directional asymmetry of wing conformation, at significant levels.Three populations (P.Avila, La Union and Yacuiba peridomestic) showed low

levels of fluctuating asymmetry (FA) and no significant directional asymmetry (DA) ofwing size. The other five populations demonstrated variable levels of directionalasymmetry of wing size with highest values in Palo Blanco (Catamarca) and Yacuiba

(domestic). No relationship was found among directional assimetry and the degree ofsusceptibility to pyrethroids, the genotype, the wing size, the altitude or thegeographic area (Fig.14).

Figure 14. Directional (DA) assymetry for wing size in individuals from 7 localities.Blue:Andean genotype, Pink: intermediate, Yellow: No-Andean.

P.Avila La Union P. Blanco S.Mazza Yacuibaperi

Yacuibadom

LaDe-

Carapari

05

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

Directionalassymetry

Jerico P.Avila La

Union

S.Estero S.Mazza P.Blan

co

S.Carlos Yacuiba E.Rios Villam. Cbba Carapari De-

seada

350

400

450

Locality or Province

CS


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Analysis of wing conformationWhen wings conformation was analyzed by sex, the UPGMA dendrograms

derived from Mahalanobis distances, showed lack of coincidence between females andmales in several localities, but Mantel test for covariation of both matrices, indicate agood correlationship (r=0,61). Figure 15 shows the dendrogram produced whenfemales and males were analyzed together.

Figure 15. Dendrogram derived from Mahalanobis distances obtained by discriminantanalysis of wing conformation variables.

6. Triatoma infestans sylvatic populations.

In the Paraguayan Gran Chaco, triatomines was searched in a variety ofecotopes such as hollow trees and mammal shelters. In sylvatic environment nearbythe Municipality of Loma Plata, Province of Boqueron), seventy traps were placed andonly some T. guasayana specimens were caught. In Bolivia, forty traps were usednearby the community of Urulica (Tupiza Province, Department of Potosi) and 40others nearby the community of Carapari, Sucre rural, Department of Chuquisaca.Investigated ecotopes were stony walls, hollow trees and shelters. At Urulica, wild T.infestanswas collected in a stony wall 300 m from the first houses. Four traps (10%)

were positive and 5 triatomine bugs were caught (1 adult and 4 nymphal instars). AtCarapari, only T. guasayanaspecimens were collected in the sylvatic environment. InArgentina, we have searched wild T. infestans in sylvatic environment nearby thecommunity of La Rinconada, Murillo Municipality, Salta Province. Sixty traps wereplaced in hollow trees and mammal shelters. Only Triatoma guasayanaand Triatomasordidawere collected (Table 12).

Euclidean Distances

Villamontes

Sucre

Cochabamba

Jerico

S.Estero

S.Mazza

Yacuiba

E.Rios

La Union

La Deseada

P.Avila

S.Carlos

P.Blanco

0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 0.022


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Table 12. Sites investigated for the searching of sylvatic Triatoma infestans

Country Locality investigated GPS point Altitude Wild triatominescollected

Argentina La Rinconada (Murillo,

Salta)

S 23.61296

O 62.89844

206 T. guasayana

T. sordidaParaguay Loma Plata (Boqueron) S 22.31240

W 59.73916133 T. guasayana

Bolivia Urulica(Tupiza, Potosi)

S 21 35' 384"W 65 49' 900''

3000 T. infestans

Bolivia Carapari(Sucre, Chuquisaca)

S 18 37' 306'' W65 10' 497''

2900 T. guasayana

Genotypes at nine microsatellite loci were determined. The locus TiC08 wasrejected due to the absence of amplification product. Except for the locus TiF11, all lociwere highly polymorphic (Table 13). The mean number of alleles per locus was 10.4(range 2-17) and expected heterozygosity ranged from 0.29 to 0.85. Similar levels ofvariability were observed in all populations.

Table 13. Genetic variability within T. infestanspopulations from the different collectingsites at Cotapachi (Nsp: number of specimens; Nall: number of alleles; He: expectedheterozigosity; Ho: observed heterozygosity) *Triatomines collected from both dwellingswere pooled.

Locus Sylvatic populations Domestic Outgroup Total

NH WH SH IW PR population* (Mataral)

TiA02 Nsp 6 20 12 31 32 10 13 124

Nall 4 6 4 4 4 4 6 11

He/Ho 0.68/0.60TiC02 Nsp 6 20 12 29 26 10 0 103

Nall 4 7 8 7 6 5 0 12

He/Ho 0.56/0.57

TiC09 Nsp 6 20 12 31 32 10 13 124

Nall 3 4 5 5 5 3 5 8

He/Ho 0.60/0.51

TiD09 Nsp 6 20 12 31 32 10 13 124

Nall 6 8 6 8 6 7 10 19

He/Ho 0.80/0.84

TiE02 Nsp 6 20 12 31 32 10 13 124

Nall 5 3 5 5 5 4 6 7

He/Ho 0.68/0.71

TiE12 Nsp 6 20 12 31 32 10 13 124

Nall 4 7 6 6 7 6 5 9

He/Ho 0.72/0.64

TiF03 Nsp 6 20 12 31 32 10 13 124

Nall 5 10 9 9 7 9 9 15

He/Ho 0.85/0/78

TiF11 Nsp 6 20 12 31 32 10 13 124

Nall 2 2 2 2 2 2 1 2

He/Ho 0.29/0.13TiG03 Nsp 6 20 12 31 32 10 13 124

Nall 4 5 4 4 4 4 8 11

He/Ho 0.67/0.64


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When all specimens were considered as belonging to one single population,

departure from Hardy-Weinberg equilibrium was found in 6 of the 9 loci (5 afterapplying the Bonferroni correction) and always with positive Fisvalues (heterozygosisdeficit), suggesting a Wahlund effect (Table 14). At the population level, only slightdeviations from Hardy-Weinberg expectations occurred. They were never significant

when the Bonferroni procedure was applied. Hardy-Weinberg desequilibrium across allpopulations remained significant only in one locus (TiF11; P= 0.0061 after Bonferronicorrection). This locus specific deviation from Hardy-Weinberg expectations could bedue to null alleles and the locus therefore was discarded for further analyses.

Linkage disequilibrium measures the nonrandom association of alleles atdifferent gene loci in a population. Among 196 pairwise tests (28 pairwise comparisonwithin each population) 12 were significant (P< 0.05) and only 2 remained significantafter applying the Bonferroni correction suggesting the absence of statistical linkagebetween loci.

Table 14. Fiscoefficient according to the locus and collecting site

1All specimens were considered as belonging to one single panmictic population

2Outgroup population

* P < 0.05 assessed through Fisher exact test; ** P< 0.05 after Bonferroni correction

Pairwise Fstestimates between populations and across all 8 loci are shown inTable 15. Fstvalues between the six Cotapachi populations and outgroup (Mataral)were high and highly significant, suggesting restricted gene flow between T. infestanspopulations collected 200 km apart. At the level of the sylvatic area at Cotapachi, the

PR population showed significant genetic differentiation with all other populations (Fstestimates ranging from 0.0503 to 0.1101). Another significant genetic differentiationwas detected between IW and WH. On the other hand, no microsatellite differentiationwas detected between T. infestans populations from the hills (P=0.1383). Finally,restricted or absent gene flows were suggested between the domestic population (DW;populations of both dwellings pooled) and all sylvatic populations.

Locus Sylvatic populations Domestic populations Total

1

NH WH SH IW PR Cotapachi Mataral2

TiA02 0.231 0.076 0.149 0.025 0.027 -0.068 0.406 0.137**

TiC02 -0.351 -0.090 0.246* 0.195 0.167 -0.220 - 0.108

TiC09 0.750* 0.231 -0.215 0.176 0.071 -0.516 0.459* 0.271**

TiD09 -0.200 -0.022 0.141 -0.022 0.050 -0.125 -0.095 0.045**

TiE02 -0.042 -0.249 0.089 0.041 -0.029 -0.417 0.185 0.009*

TiE12 0.184 -0.010 0.218 -0.003 0.193 0.007 0.256 0.121

TiF03 -0.064 0.205* 0.083 0.120 -0.007 -0.013 0.226 0.131**

TiF11 0.706 0.782* 0.585 0.077 0.000 0.757* - 0.467**

TiG03 -0.190 0.052* 0.054 0.042 0.065 0.045 0.246 0.155

Acrossloci

0.304 0.039 0.096 -0.143 0.065 0.065 0.226** 0.137**


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Table 15. Microsatellite differentiation (Fst) between T. infestanspopulations from thedifferent collecting sites at Cotapachi

NH WH SH IW PR DW

NH -

WH 0.0227

SH 0.0051 0.0086

IW 0.0134 0.0108 0.0019

PR 0.0539 0.1101 0.0503 0.1048

DW 0.0434 0.0720 0.0260 0.0475 0.0695

Outgroup 0.1613 0.1635 0.1276 0.1509 0.1634 0.1422

Statistical significance of Fstwas assessed through Fisher exact test (Fstwith 0.01


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sylvatic populations, that appear as separate reproductive units, even within shortdistances (in the order of hundred meters). There was no genetic flow betweendomestic and the sylvatic sites. This observation only applies to this type of highaltitude populations and cannot be generalized to other regions of the soutrhern cone,in particular to the chaco populations.

The spatial structuration of T. infestans populations is associated with theexistence of the andean, non andean and intermediate groups. However, the datacollected during this project do not allow concluding about the continuity ordiscretedness of the T. infestansvariability. To answer it, data on the phylogeographyof the species should be collected from specimens captured along an Andes-Chacotransect, increasing the number of localities studied with mtDNA. Phenotypic featuresas the antennal phenotypes could also be useful, as they express genetic andenvironmental factors.

Results of this project do not allow shedding a different light to the previouslyproposed dispersion route of T. infestans. According to the most common opinion, theoriginal population of T. infestansappeared in the bolivian Andes and dispersed south.Arguments that support this opinion are the existence of sylvatic andean populations,

human migrations and the enzimatic characterization that establishes the center oforigin in the mesothermic valleys of Bolivia (Cochabamba and Chuquisaca). However,Andean citotypes of T.infestans were not found in the Chaco region, even at highaltitudes. Another alternative opinion suggests that the center of origin would be thechaco, and it based on the existence of sylvatic populations in the chaco and theoccurrence of other species members of the infestans sub-complex in this chacoregion.

This project also show that the populations ofT. infestanshave a very importantphenotypic plasticity. Markers to differentiate T. infestans populations, used extensivelywithin this project, are available to the vector control programmes interested in theidentification of the different populations of the main vector of Chagas disease in theSouthern Cone of South America.


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Type (from citogeneticcriteria) (1)

Antennal phenotypesCuticular

hydrocarbonsmtDNA

AndeanMainly AP 1

Some individuals AP 2Andean Andean Hp

Intermediate(localities 7-15)

Mainly AP 2Some individuals AP 1

and AP 3

Non andeanMainly AP 3

Some individuals AP 2

Non Andeanincluding theintermediate

Non andean Hincluding theintermediate

(1) Andean: 14 or more heterochromatic chromosomes; intermediate: 7 to 11 heterochromatic chromoso

heterochromatic chromosomes

Table 16. Triatoma infestanstypes (from citogenetic criteria) and their correlationships with Antennal phenotmtDNA and Susceptibility to pirethroids.


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Anexo: Informe del trabajo:

CRUZAMIENTOS EXPERIMENTALES PARAANALIZAR LA TRANSMISION DEL CARCTER

DE RESISTENCIA A INSECTICIDASPIRETROIDES EN Triatoma infestans

Cardozo Rubn, Segura Maria, Diosque Patricio, Perez Ruben,

Panzera Francisco y Miguel Basombro


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INTRODUCCIN

Triatoma infestanses el principal vector transmisor de Trypanosoma cruzi, el

agente causal de la Enfermedad de Chagas. Para el control de este insecto

vector se han usado, intensiva y extensivamente, insecticidas piretroides desde

hace ms de 20 aos (Zerba et al 1999). Este hecho trae aparejado el riesgo

de desarrollar fenmenos preadaptativos de resistencia.

En la Argentina el monitoreo de la resistencia se ha iniciado en 1997. En

principio se detectaron cambios en la susceptibilidad y niveles de resistencia

bajos (Grado de Resistencia: GR) en algunos departamentos de solo cinco

provincias de las estudiadas. En 1999 las estimaciones realizadas indicaron

que el rango del GR oscilaba entre 2, para una colonia de San Luis, y 7,9 parauna colonia de Salvador Mazza, Salta. A pesar de los valores observados no se

registraron problemas de control qumico en las poblaciones de campo (Vasena

C, Picollo MI 2003)

En el ao 2002 el Servicio Nacional de Chagas informo sobre la poca eficiencia

de la deltametrina y otros insecticidas piretroides en el tratamiento de casas

infestadas en parajes rurales aledaos a Salvador Mazza. Se detectaron focos

con altos niveles de resistencia, (el GR estimado para distintas poblaciones deesta zona vario desde 50,5 a 133,1) mucho ms elevados que los estimados

previamente en este mismo paraje en 1999 (GR = 7,9) (Picollo et al 2005). Por

otro lado en reas aledaas en Bolivia se registran actualmente serios

problemas en cuanto al control vectorial debido al grado de resistencia a la

deltametrina encontrado.

El gran incremento en los niveles de resistencia observado en una poblacin de

campo de un rea restringida en un corto periodo de tiempo, sugiere que

existen fuertes mecanismos genticos involucrados en la transmisin del

carcter de la resistencia a la deltametrina y que el o los genes involucrados

podran introducirse en poblaciones susceptibles a estos insecticidas

generando descendientes con ciertos niveles de resistencia.

En el marco de lo expuesto, y con el objeto de contribuir al proyecto "Biological

and environmental causes of the spatial structuration in Triatoma infestansand

the implications for vector control programmes incluido en el proyecto ATU-


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parajes rurales aledaos a Salvador Mazza, departamento San Martn provincia

de Salta (22 01 22,9S, 63 41 24,5W). La segunda muestra esta

constituida por insectos atrapados en ecotopos domiciliarios de diversos

parajes rurales del departamento Chacabuco en la provincia de Chaco (26 55

11 S, 61 37 42W). Ambas poblaciones se encuentran separadas

geogrficamente por 600 Km aproximadamente. (Figura 1). Mediciones previas

realizadas sobre insectos de ambas poblaciones confirmaron la presencia de

altos niveles de resistencia (GR = 120) y completa susceptibilidad (GR < 1).

Figura 1: Mapa del noreste argentino mostrando los sitios donde fueron colectados (crculos rojos) losinsectos utilizados en este estudio

De cada poblacin se utilizaron ninfas pertenecientes al quinto estadio. Se las

mantuvo en el insectario del Instituto de Patologa Experimental de la

Universidad Nacional de Salta en condiciones controladas de temperatura,

humedad y fotoperodo (27 +/- 2 C, 60 +/- 5 % HR, 12:12 hs de luz/oscuridad).

Se alimentaron con sangre de ratn una vez por semana, e inmediatamente

emergidos los imagos, se separaron machos de hembras. Posteriormente se

formaron las parejas experimentales de acuerdo al diseo experimental que se

plantea a continuacin.


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Cruzamientos experimentales y anlis is de fertil idad.

Se formaron parejas de T. infestans de acuerdo a las siguientes combinaciones

de cruzamientos:

Un macho resistente (R) con una hembra resistente (R).

Un individuo resistente (R) con un individuo no resistente (no R): este

cruzamiento se realiz en ambas direcciones (macho de una poblacin

por hembra de la otra y viceversa) para analizar la posibilidad de algn

componente ligado al sexo. Por esta razn cada direccin de

cruzamiento se considero como un como un cruzamiento diferente.

Un macho no resistente (no R) con una hembra no resistente (no R).

El siguiente cuadro resume las distintas combinaciones de cruzamientos

realizados.

Cruzamientos Parejas

C1 Macho R x Hembra R

C2 Macho R x Hembra no R

C3 Macho no R x Hembra R

C4 Macho no R x Hembra no R

Se utilizaron 5 parejas por cada grupo de cruzamientos, se observo la

oviposicin de cada hembra semanalmente y se registro el nmero de huevos

colocados. Los huevos de cada pareja se mantuvieron aislados para analizar

su fertilidad. A las 12 semanas posteriores a la formacin de cada pareja se

calculo el porcentaje de eclosin de cada grupo, como parmetro de viabilidad

de los huevos.

Evaluacin del efecto insecticida

Una vez obtenida la descendencia (F1) de cada pareja, las ninfas del primer

estadio (ayunadas desde la eclosin) fueron sometidas a pruebas de

resistencia a la deltametrina.

Para ello se aplic una serie de diluciones seriales de deltametrina de grado

tcnico (97%, Bayer, Buenos Aires, Argentina), usando acetona como solvente.


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Se aplicaron por los menos 3 diluciones, con el criterio de que dieran mas 0% y

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