study area - field work was conducted in a rural section of pampa del 
 indio municipality ( 2555 s 5658w ) , which for vector control purposes was divided into 
 four areas each including 300 - 400 dwellings in which similar intervention protocols were 
 performed . the current study was conducted in a 95 km rural section ( area 
 iii ) including 407 inhabited houses as of october 2008 and was mainly occupied by qom 
 communities and a creole minority ( gaspe et al . most houses had mud walls with plenty of refuges for triatomines , were 
 small - sized ( < 30 m ) and recently built and had few or no peridomestic 
 structures and little access to electricity . the last insecticide spraying campaign 
 conducted in the municipality occurred in 1997 - 1998 and selective sprays of a few houses 
 in area iii were conducted in 2000 and 2006 by local health workers . a house compound was defined as the set of human sleeping quarters ( i.e. , domestic 
 sites ) and structures included within the peridomestic area ( e.g. , kitchens , storerooms , 
 corrals , chicken coops ) as illustrated elsewhere ( gurevitz et al . the location of each house was georeferenced ( garmin 
 legend ) and the geographic distance between houses was assessed using arcgis 9.2 . study design - a longitudinal study was conducted in area iii to 
 monitor house infestation over a four - year period before and after community - wide 
 spraying with insecticides . all structures within each house compound were searched for 
 triatomine bugs by timed manual collections using a dislodjant aerosol ( 0.2% 
 tetramethrin ) ( espacial , argentina ) conducted by two skilled bug collectors from the 
 national or provincial vector control programs ( gaspe et 
 al . t. infestans was found by timed - manual searches in 
 28% of the 386 inhabited houses inspected at baseline ( october 2008 ) . house infestation 
 rose to 31.9% when all collection methods were considered . all sites within each house 
 compound were sprayed with pyrethroid insecticides immediately after the baseline survey 
 ( november 2008 ) as described elsewhere ( gurevitz et al . post - intervention annual 
 surveys aiming at full coverage were conducted at 10 months post - spray ( mps ) ( august 
 2009 ) , 18 mps ( april 2010 ) , 38 mps ( december 2011 ) and 49 mps ( november 2012 ) . only a 
 sample of the inhabited households was inspected at 38 mps and included households 
 deemed to be at a higher infestation risk . selective sprays of the re - infested houses 
 were performed immediately after completing each annual house survey in a similar 
 fashion . additional methods to assess bug infestation were performed during the 
 follow - up to improve the detectability of house infestation : ( i ) collection of bugs that 
 were spotted during insecticide spraying operations and ( ii ) householders collection of 
 any triatomine they sighted . insects - to assess the spatial structuring of baseline insect 
 populations , we selected collection sites with at least 10 adult t. 
 infestans of the same sex for wing geometric morphometry analysis . a random 
 sample of 15 insects of the same sex per site was chosen to achieve balanced sample 
 sizes in the few cases in which the total adult catch exceeded that number . in total , 
 132 males from 11 domiciles and 89 females from eight domiciles were analysed ( fig . 1a : map showing houses infested by triatoma infestans 
 before insecticide spraying which were included in wing geometric morphometry 
 analyses in area iii ( circles ) and the groups of houses identified in area i 
 ( triangles ) ( gaspe et al . 2012 ) , pampa 
 del indio , chaco , 2007 - 2008 ; b : map of area iii showing houses infested by 
 t. infestans included in the morphometric analyses by 
 survey , pampa del indio , 2008 - 2012 . circles show houses before insecticide 
 spraying ( october 2008 ) , squares correspond to the first post - spray survey [ 10 
 months post - spray ( mps ) , 2009 ] , triangles correspond to the second post - spray 
 survey ( 18 mps , 2010 ) and stars to the fourth post - spray survey ( 49 mps , 2012 ) , 
 the numbers and letters refer to the house i d . the letters correspond to the 
 name of the community ( cc : cuarta legua 14 ; cd : cuarta legua 17 ; pc : pampa 
 chica ; pg : pampa grande ; rn : el rincn ) . 
  to determine the putative origin of re - infestant insects , all adult bugs collected in 
 each post - spray survey were analysed except the only adult bug collected at 38 mps 
 ( table ) , which had its wings in poor condition 
 and therefore was excluded from the analysis . males and females collected in one 
 domicile ( pc152 ) at 10 mps were included as re - infestant insects to preserve the 
 temporal specificity of pre - spray bug populations despite the fact that this house had 
 not been inspected or sprayed at baseline . house pc152 was located at more than 1.5 km 
 from the nearest inhabited house and was surrounded by patches of dense forest . table distribution of triatoma infestans males and females 
 included in morphometric analyses and the results of the putative origin of 
 post - spray bugs according to bug collection ecotope and survey in area iii , 
 pampa del indio , chaco , argentina , 2008 - 2012surveydate of bug 
 collectionhouseecotopemalesfemalestotalbugs assigned ( % ) comparison with 
 pre - spray groups ( p)distance to the 
 nearest house ( km ) 
 baselineoctober 200811 housesdomicile13289221 - --post - spray10 mps ( 2009)pc36domicile112100 - 1.8pc152domicile45922 < 0.052.718 mps ( 2010)bc06chicken coop561155 < 0.056.5cc32domicile11250 - 1.738 mps ( 2011)pg156domicile0 
 00 
 ---49 mps ( 2012)pg38domicile and chicken coop11250 - 0.7pg42domicile3111440 < 0.051.4pg107domicile40475 - 0.8total--192544 - -- 

 total  --151114265 - -- 
 a : analysed using the one - by - one procedure that assigned 
 each individual to the previously identified pre - spray groups . the results 
 correspond to the percentage of insects assigned to the nearest house ; 
 b : the results correspond to the comparison of 
 mahalanobis distances between the post - spray house and pre - spray groups . only 
 high - density post - spray bug populations were included ; c : 
 considering pre - spray groups ; d : the only adult bug 
 collected at the 38 months post - spray ( mps ) survey had its wings in poor 
 condition and therefore was excluded from the analyses . a : analysed using the one - by - one procedure that assigned 
 each individual to the previously identified pre - spray groups . the results 
 correspond to the percentage of insects assigned to the nearest house ; 
 b : the results correspond to the comparison of 
 mahalanobis distances between the post - spray house and pre - spray groups . only 
 high - density post - spray bug populations were included ; c : 
 considering pre - spray groups ; d : the only adult bug 
 collected at the 38 months post - spray ( mps ) survey had its wings in poor 
 condition and therefore was excluded from the analyses . pre and post - spray triatomines were compared with those collected before insecticide 
 applications in area i , which included 93 males and 89 females from the two geographic 
 groups identified ( gaspe et al . 2012 ) ( fig . metric data - right wings were mounted between slides and cover slips 
 and photographed using a digital camera ( sony mvccd300 , usa ) and a stereo - microscope 
 ( zeiss sv11 , germany ) as described in schachter - broide 
 et al . we used 10 type - i landmarks ( vein intersections ) identified for 
 t. infestans wings as described elsewhere ( gaspe et al . the isometric estimator known as centroid size ( cs ) , defined as the 
 square root of the sum of the squared distances between the centre of the configuration 
 of landmarks and each individual landmark ( bookstein 
 1990 ) , was used to compare sexes , collection sites , surveys and study areas . size comparisons were performed for males and females separately using mann - whitney 
 u and kruskal - wallis tests implemented in stata 12.0 software ( stata 
 corp lp , usa ) . shape variables [ partial warps ( pw ) ] were obtained using the generalized procrustes 
 analysis superimposition algorithm ( rohlf 1996 ) . 
 to avoid having small sample sizes relative to the number of variables , we used a 
 restricted representation of shape ( i.e. , a set of principal components known as 
 relative warps ) derived from shape variables . in all cases , residual allometry was estimated by multivariate regression of 
 pw on size ( good 2000 ) . mahalanobis distances 
 were derived from the selected set of relative warps and then used to construct an 
 unweighted pair - group method with arithmetic average ( upgma ) dendrogram . the statistical 
 significance of these distances was computed by nonparametric permutation tests ( 1,000 
 runs ) adjusted by the bonferroni procedure . to analyse the occurrence of spatial structuring of baseline bug populations , 
 mahalanobis distances were used to perform a simple reclassification test for each 
 individual bug . the percentage of males and females that were correctly assigned to the 
 corresponding geographic group was assessed . the isolation - by - distance ( ibd ) model for 
 each sex was tested by regressing mahalanobis distance on geographic distance between 
 bug collection sites with a mantel test . significance levels were determined by 1,000 
 permutations using ibd ( bohonak 2002 ) . to assess the putative origin of re - infestant insects , two different analyses were 
 performed due to the small number of adult bugs collected in some sites . post - spray 
 insects were entered one by one to the discriminant analysis of pre - spray samples and 
 assigned to the closest reference group according to mahalanobis distances as described 
 elsewhere ( dujardin et al . not included in the pre - spray sample , the nearest infested house at baseline was 
 taken as the reference . additionally , the adults from high - density post - spray bug 
 colonies ( i.e. , > 10 adults collected ) were analysed together with the pre - spray 
 groups . the mahalanobis distances among post - spray collection sites and the pre - spray 
 groups were compared by nonparametric permutation tests and re - classification tests were 
 performed . field data and interviews with householders suggested that the infestations of houses 
 pg38 and pg107 in the 49 mps survey could have originated from the high - density bug 
 colony found in the neighbouring house pg42 . to test this hypothesis , we performed 
 additional analyses considering the highly infested household detected in the 49 mps 
 survey and before community - wide spraying ( pg42 ) as reference groups . to verify the quality of the re - classification analysis and to assess the confidence 
 levels of post - spray assignments , a cross - checked classification of males and females in 
 pre and post - spray reference groups each individual was removed , treated 
 as external data and then re - assigned to one of the groups . mitochondrial dna - due to the strong morphometric differentiation 
 found in insects from bc06 , two individuals were sequenced for a fragment of the 
 mitochondrial gene cytochrome oxidase i ( mtcoi ) as described in piccinali et al . the sequences were compared 
 with a database of haplotypes from 650 t. infestans individuals from 
 argentina , bolivia , uruguay and paraguay , including individuals from the neighbouring 
 area i. 
 software - tpsdig2 v.2.09 was used for landmark digitalisation , mog for 
 procrustes superimposition , generation of pw , assignment of   unknown specimens and 
 validated re - classification tests , var for nonparametric comparisons of 
 cs , cov for examination of residual allometry within shape variables 
 and common allometry model tests and pad for discriminant analyses , non parametric 
 permutation tests and re - classification tests . the modules mog , var , cov , pad developed by jp 
 dujardin , are included in the clic package available from mome-clic.com . house re - infestation by t. infestans - baseline house infestation by 
 t. infestans ( 31.9% ) dropped sharply and remained below 1% during 
 the four - year follow - up after community - wide insecticide spraying of area iii , unlike in 
 neighbouring area i ( fig . no additional house 
 was found infested by t. infestans as determined by householders bug 
 collections during the entire follow - up , whereas several adult specimens of 
 nondomiciliated species ( mainly triatoma sordida ) were collected , 
 supporting the very low prevalence of t. infestans . 2:prevalence of house infestation by triatoma infestans 
 during the follow - up in area iii ( bars ) and area i ( dots ) , pampa del indio , 
 chaco , argentina . the white arrow shows the timing of community - wide 
 insecticide spraying campaigns . after each survey numbers above bars indicate the number of houses 
 inspected for infestation in area iii ( black ) and area i ( grey ) . house infestation by t. infestans was concentrated in domiciles both 
 before and after community - wide spraying . seven of the 10 houses found re - infested after 
 initial insecticide spraying had not been found infested at baseline and none of the 
 infested houses sprayed selectively during the follow - up was found infested again . only 
 two houses had also been infested at baseline : one of which had a large bug colony 
 ( included in the baseline sample ) and the other one only had one adult bug ( not included 
 in the baseline sample ) . spatial structuring of pre - spray bug populations - female wing 
 cs [ mean = 754.8 mm ; standard deviation ( sd ) = 29.6 mm ] was 
 significantly larger than male cs ( mean = 721.9 mm ; sd = 25.8 mm ) 
 ( mann - whitney u test , p < 0.001 ) . significant differences in 
 cs were found among bugs from different collection sites both in 
 males [ kruskal - wallis u test , degree of freedom ( d.f . ) = 10 ; p < 
 0.01 ] and females ( d.f . no significant differences were found in the 
 wing cs of males and females from areas iii and i collected before 
 insecticide spraying ( mann - whitney u test , p > 0.05 ) . given the absence of significant differences in wing shape variables between males and 
 females collected at the same site , the analyses were performed considering both sexes 
 pooled by site . the analyses showed the existence of two groups that included bug 
 collection sites with no significant differences in mahalanobis distances , whereas 
 collection sites from different groups were significantly different . no residual 
 allometric effect was found , suggesting that the differences observed were not caused by 
 wing size . the two groups corresponded to geographic groups including sites located at a 
 mean distance of 1 and 3 km from one another , respectively ( fig . 3 ) , whereas sites from different groups were located at a 
 mean distance of 10 km ( range , 6 - 13 km ) . mahalanobis and geographic distances were 
 significantly correlated ( mantel test , r = 0.36 , p < 0.01 ) . both simple and 
 cross - checked re - classification scores were higher than 72% for both groups and 
 supported their validity as reference groups . 
 fig . 3:map of area iii showing the pre - spray groups of triatoma 
 infestans identified by wing geometric morphometry , pampa del 
 indio , chaco , argentina , 2008 . the letters correspond to the name of the community ( cc : cuarta legua 
 14 ; cd : cuarta legua 17 ; pc : pampa chica ; pg : pampa grande ; rn : el 
 rincn ) . significant differences in wing shape variables among these two groups and those 
 identified in area i before community - wide insecticide application were found . the upgma 
 dendrogram showed that groups within the same area were more similar than those from 
 different areas ( fig . no residual allometry 
 was found , indicating that these differences were not attributable to wing size 
 effects . 4:unweighted pair - grouped method with arithmetic average ( upgma ) dendrogram 
 derived from mahalanobis distances between male and female triatoma 
 infestans collected before community - wide insecticide spraying in 
 two neighbouring study areas ( iii and i ) and high - density post - spray collection 
 sites of area iii , pampa del indio , chaco , argentina . g1 and the remainder 
 groups correspond to high - density bug colonies in each post - spray survey [ 10 
 months post - spray ( mps ) , 18 mps and 49 mps ] . reinfestant insects - the wing cs of post - spray 
 females was signicantly larger than that of post - spray males ( mann - whitney 
 u test , p < 0.01 ) . the wing cs of males and 
 females significantly differed according to bug collection site and survey date 
 ( including pre and post - spray surveys ) ( kruskal - wallis u test , p < 
 0.01 in both sexes ) . insects collected in house pc152 ( domicile ) at 10 mps had 
 significantly smaller cs than those collected in other houses with a 
 high - density infestation detected after insecticide spraying ( including a domicile and a 
 chicken coop ) . the pre - spray frequency distribution of cs values was 
 overdispersed , probably due to the inclusion of insects collected in several dwellings 
 ( fig . 5 ) . 5:wing centroid size of male and female triatoma infestans 
 collected before community - wide insecticide spraying ( males , n = 104 ; females , 
 n = 74 ) and in each post - praying survey ( males , n = 4 , 5 , 3 ; females , n = 5 , 6 , 
 11 , respectively ) , pampa del indio , chaco , argentina , 2008 - 2012 . letters and 
 numbers correspond to the house i d . pc152 was infested at 10 months post - spray 
 ( mps ) ( 2009 ) , bc06 at 18 mps ( 2010 ) and pg42 at 49 mps ( 2012 ) . 
  for sites with high - density post - spray infestations , significant mahalanobis distances were found with respect to pre - spray groups from area iii ( t ) and area i ( p < 0.01 ) 
 ( fig . post - spray adult bugs from house bc06 
 strongly differed from all other groups in area iii ( complete line vs. broken - line 
 polygons in fig . the re - classication percentages 
 of insects in each group were 64 - 66% , whereas for house bc06 it was 90% . to test for any 
 procedural bias , insects from bc06 were digitised again , re - analysed and the initial 
 results confirmed . these re - infestant specimens were confirmed to be t. 
 infestans through mtcoi sequencing and had the same 
 haplotype as most insects from area i ( rv piccinali et al . , unpublished observations ) . this haplotype , called  c  previously , was the most frequent in the argentine chaco 
 ( piccinali et al . 6:factorial map of the discriminant analysis ( 1st vs. 2nd discriminant 
 factor ) of wing shape variables of triatoma infestans males 
 and females collected before community - wide insecticide spraying in two 
 neighbouring study areas and in high - density collection sites after spraying , 
 pampa del indio , chaco , argentina . cv1 and cv2 explained nearly 50% of the 
 total variance . g1 and g2 correspond to pre - spray bug collection sites with no 
 significant differences in shape variables within each area . the remainder 
 groups correspond to highly - abundant bug colonies in each post - spray survey [ 10 
 months post - spray ( mps ) , 18 mps , 49 mps ] . all the adult bugs collected at 10 mps in the only low - density infested site were 
 assigned to the nearest reference group . the percentage of assignment dropped to 50 - 75% 
 for insects collected at 18 and 49 mps ( table ) . half of the insects collected in houses pg38 and pg107 at 49 mps were assigned to the 
 high - density bug colony detected in house pg42 during the same survey . this study does not support our a priori hypothesis that the detected 
 re - infestations during the four - year follow - up of area iii were primarily due to 
 residual foci that survived community - wide insecticide spraying , unlike in area i ( gurevitz et al . sites with high - density bug colonies after 
 initial insecticide spraying displayed significant differences in wing shape variables 
 compared with pre - spray groups , suggesting that the former insects most likely 
 originated from external sources ( i.e. , houses from outside area iii or putative 
 sylvatic foci ) . moreover , pre and post - spray insects from area iii were significantly 
 different from those collected in area i , supporting the existence of independent 
 re - infestation processes within the same district . in addition , insects from house bc06 
 showed large , unexpected differences in wing shape variables with respect to all other 
 insects analysed . wing geometric morphometry of pre - spray bug populations identified two groups of 
 collection sites with significant differences in shape variables that corresponded to 
 geographic locations . bug collection sites grouped together were located at a mean 
 distance of 1 - 3 km whereas sites from the two groups were 10 km apart , which largely 
 exceeds the current estimates of the flight range of t. infestans 
 ( schweigmann et al . the geographic distribution 
 of t. infestans populations fitted to the ibd model , as in area i 
 ( gaspe et al . 2012 ) . this level of spatial 
 structuring in area iii was very similar to the patterns recorded in area i where no 
 official blanket insecticide spraying had occurred during the previous decade . the 
 sporadic selective sprays reported in some communities of area iii , combined with the 
 high levels of house instability and household mobility detected ( gaspe et al . 2015 ) , may have caused bottleneck events that led to 
 the observed differentiation of pre - intervention t. infestans 
 populations . the results of wing shape analyses of insects collected after blanket insecticide 
 spraying suggest that re - infestations were very unlikely to derive from residual bug 
 populations that survived the insecticide spraying campaign . this proposition is 
 consistent with the very low prevalence of house infestation recorded during the 
 four - year follow - up and the fact that most houses infested after initial spraying had 
 not been infested at baseline . of note , the municipality of pampa del indio was 
 surrounded by rural districts with variable rates of house infestation , which were under 
 sporadic vector surveillance and control operations ; passive bug transport from these 
 potential sources may have provided the post - spray insects in area iii . although much 
 less likely , internal re - infestation sources such as undetected foci within area iii or 
 sylvatic foci of t. infestans { so far not detected in pampa del indio 
 [ alvarado - otegui et al . ( 2012 ) , ym provecho , 
 unpublished observations ] } can not be completely excluded . in contrast , in area i , both 
 the space - time analysis of site - specific infestations and wing geometric morphometry 
 indicated that post - spray triatomines were mainly survivors or the offspring of 
 pre - spray bug populations ( gurevitz et al . 2013 ) . likewise , rhodnius prolixus and t. 
 infestans collected after insecticide applications elsewhere were primarily 
 survivors from pre - spray bug populations and included some insects invading from nearby 
 habitats ( feliciangeli et al . further studies using 
 additional markers are needed to identify the most likely mechanism underlying house 
 re - infestation after blanket interventions in area iii . pre - spray insects from area iii were significantly different from pre - spray bugs from 
 area i. moreover , post - spray triatomines from sites with high - density colonies in area 
 iii also differed from pre - spray insects from area i. these results suggest a low rate 
 of exchange of insects between these neighbouring rural areas and are of great relevance 
 for improved vector surveillance . bug populations involved in vector control failures in 
 area i were unlikely to represent an immediate risk for dwellings in area iii and 
 therefore vector control actions can be ( and were ) decided in a relatively independent 
 way . unexpected , large differences in wing shape variables occurred in post - spray triatomines 
 from house bc06 relative to all other bugs despite the fact they had the most common 
 mtcoi haplotype found in area i and in the chaco region ( rv 
 piccinali et al . , unpublished observations ) . this house was located more than 5 km from 
 the nearest infested house and belonged to a community that mainly included dispersed 
 houses . these results suggest that the insects from bc06 had a different origin or that 
 this specific bug population was reducing the exchange of individuals with the 
 surrounding bug populations . additional markers are needed to investigate the strong 
 differences detected between bugs from house bc06 and the rest . the wing cs of pre - spray insects showed significant differences among 
 collection sites for both sexes despite the fact that they had been collected at the 
 same type of ecotope and month ( schachter - broide et al . moreover , the wing cs of males and females collected 
 in one domicile at 10 mps ( which had not been treated with insecticides at the time of 
 community - wide interventions ) were significantly smaller than the cs of 
 insects collected in every infested house before and after interventions . these wing 
 size variations support the existence of a large degree of differentiation among the 
 study bug populations and may be associated with a limited exchange of insects among 
 some of them . consideration of additional factors known to affect wing size ( i.e. , bug 
 density and host - feeding source , among others ) may help explain the observed 
 differences . the criteria used to select pre - spray 
 samples restricted the analyses to domestic insects . the absence of abundant bug 
 colonies across the study area and some methodological restrictions hindered the 
 assignment of post - spray insects to the two reference groups identified . the few insects 
 collected after community - wide interventions during the four - year follow - up limited the 
 analyses of post - spray populations using conventional procedures . this issue was 
 addressed through one - by - one assignments and by using additional field data to contrast 
 the morphometric results . finally , the comparison of wing shape among temporally distant 
 bug populations may be influenced by the cumulative effect of new bug generations ( dujardin et al . 2009 ) and also further research on the 
 relationship between wing shape and environmental variables is needed . a major strength 
 is related to the detailed study of multiple georeferenced t. infestans 
 populations collected before community - wide insecticide spraying and during the 
 four - year follow - up , with clear a priori hypotheses that guided the research effort . our 
 preceding study in area i enabled an extended comparison between neighbouring rural 
 areas that showed contrasting re - infestation patterns . our results further support the use of wing geometric morphometry to investigate 
 population structuring and house re - infestation at a spatial scale of 100 
 km , a territory which has significant public health relevance . the 
 re - infestation patterns recorded suggest that post - spray specimens most likely 
 originated from external sources , unlike in area i. the different patterns observed 
 within the same municipality indicate the existence of apparently independent 
 re - infestation processes . these results provide useful input to vector control programs 
 in the gran chaco region and emphasise the need to expand the geographic coverage of 
 vector surveillance and control operations to the affected region .
house re - invasion by native triatomines after insecticide - based control campaigns 
 represents a major threat for chagas disease vector control . 
 we conducted a 
 longitudinal intervention study in a rural section ( area iii , 407 houses ) of pampa 
 del indio , northeastern argentina , and used wing geometric morphometry to compare 
 pre - spray and post - spray ( re - infestant bugs ) triatoma infestans 
 populations . 
 the community - wide spraying with pyrethroids reduced the prevalence of 
 house infestation by t. infestans from 31.9% to < 1% during a 
 four - year follow - up , unlike our previous studies in the neighbouring area i. two 
 groups of bug collection sites differing in wing shape variables before interventions 
 ( including 221 adults from 11 domiciles ) were used as a reference for assigning 44 
 post - spray adults . 
 wing shape variables from post - spray , high - density bug colonies 
 and pre - spray groups were significantly different , suggesting that re - infestant 
 insects had an external origin . 
 insects from one house differed strongly in wing 
 shape variables from all other specimens . 
 a further comparison between insects from 
 both areas supported the existence of independent re - infestation processes within the 
 same district . 
 these results point to local heterogeneities in house re - infestation 
 dynamics and emphasise the need to expand the geographic coverage of vector 
 surveillance and control operations to the affected region .