about 28 million people live in areas at risk of chagas disease , 1114.5 million of whom are affected worldwide .
trypanosoma cruzi , the pathogen that causes chagas disease , is found in most south american countries , representing an important cause of heart damage among the economically active population . after a successful chemical control of triatoma infestans ( klugi , 1834 ) , the other main vectors of chagas causing agent , panstrongylus megistus burmeister , 1835 , rhodnius prolixus stal , 1859 , and triatoma brasiliensis sensu lato neiva 1911 .
triatoma brasiliensis sensu lato ( s.l . ) , found in anthropogenic habitats and considered the main vector in northeast brazil [ 2 , 3 ] , was recently found to be a species complex that includes t. b. brasiliensis , t. b. macromelasoma galvo , 1956 , t. juazeirensis costa & felix , 2007 , t. sherlocki papa , jurberg , carcavallo , cerqueira & barata , 2002 , and t. melanica costa et al .
these taxa exhibit wide phenotypic and morphological variability , displaying specific ecological requirements and chromatic patterns . in this respect ,
the systematic of triatominae species is based on morphological characters of the adult exoskeleton and male phallic structures .
however , insects captured during vector monitoring and control or received for identification and notification are often immature .
although their characteristics are similar to those of adult individuals , they are difficult to distinguish .
the morphology of triatominae species is not well described ; with studies on the immature forms performed for only 40 species , eggs and nymphs described for only 20 species , a key to identify nymphs to the species level has yet to be developed .
available keys are useful and partially applicable to other stages , but specific identification of all live forms remains unresolved .
members of the t. brasiliensis complex have been distinguished by analyzing isoenzymes , mitochondrial dna sequences , and random amplification of polymorphic dna - rapd . in the present study we analyzed the barcoding co1 sequences of nine t. brasiliensis s.l .
populations from northeastern brazil ( states of pernambuco , paraba , and rio grande do norte ) in order to identify their genetic relationships .
we also conducted a pcr - rflp assay to distinguish between t. b. macromelasoma and t. b. brasiliensis subspecies .
triatominae were collected by surveillance technicians during active inspections in anthropogenic environments ( domestic and peridomestic habitats ) .
live specimens were collected in nine localities in northeast brazil ( table 1 , figure 1 ) using tweezers , flashlights , and pirisa . housed in plastic boxes ( 7  cm diameter  8  cm high ) lined with folded filter paper
, the bugs were transported in coolers to the culicid and triatomine laboratory of the department of epidemiology , faculty of public health / usp .
adults were identified as t. b. brasiliensis according to the key by lent and wygodzinsky .
nymphs were assumed to be t. brasiliensis s.l . because , in addition to the difficulty in distinguishing immature triatominae based on chromatic and morphological characters , comparative material or a key for nymph identification to species level are not available .
after morphological identification , dna was extracted from individuals from the nine localities and sequenced the 520  bp barcode portion of the co1 gene ( table 1 ) .
specimens collected in pernambuco state ( even nymphs ) were assumed to be t. b. macromelasoma , according to costa et al .   and costa et al . .
genomic dna was extracted from the legs of 10 individual samples of each population using the qiagen dneasy blood and tissue kit ( qiagen , crawley , united kingdom ) following the manufacturer 's protocol .
the co1 barcode region was amplified from whole genomic dna using primers lcoi 1490 ( 5-ggt caa caa atc ata aag ata ttg g-3 ) and hcoi 2198 ( 5-taa act tca ggg tga cca aaa aat-3 ) .
pcr amplification was carried out in a final volume of 50  l containing pcr buffer , 0.2  mm of each dntp , 2.5  mm mgcl2 , and 1.25 units of taq polymerase .
initial pcr denaturation was at 94c for 5  min , followed by 40 cycles of denaturation ( 1  min ) at 94c , annealing ( 2  min ) at 50c , and extension ( 2  min ) at 72c .
amplicons were sequenced in both forward and reverse directions using abi prism bigdye terminator cycle sequencing ready reaction kits ( perkin elmer , foster city , ca ) on an abi prism 3100 genetic analyzer / hitachi .
one or two representative haplotypes for each population was chosen because of the low variation within populations .
phylogenetic reconstructions were performed by neighbor joining and maximum likelihood methods ( both using the kimura-3-parameter distance model k81 ) in mega 5.0 , and a divergence matrix was constructed under kimura two parameters ( k2p ) ( table 2 ) .
phylogenetic analyses included 1000 bootstrap replicates and a triatoma sordida co1 sequence ( genbank acc .
co1 sequences were also analyzed using nebcutter version 2.0   to select appropriate endonuclease enzymes .
individual co1 sequences were pcr - amplified using the above parameters and digested in a 10  l reaction with styi ( promega ) and hincii ( new england biolabs , ipswich , ma ) enzymes .
the reaction contained 1  l of 10x buffer , 4  l deionized water , 4  l of amplification product , and 1 unit of restriction enzyme .
the digestion mixture was incubated at 37c for 2  h and then resolved on 2.0% agarose gel .
phylogenetic trees derived from the neighbor joining , maximum likelihood , and parsimony methods showed similar topologies ( data shown for ml tree in figure 2 ) .
since most of co1 sequences were identical into and among the populations , the ml tree was constructed using only two samples of each one . this ml tree and
the nucleotide distance matrix ( table 2 ) indicated sequence divergence of up to 4% between the two main clades .
the basal clade , with about 4% divergence from the other populations , consisted of the salgueiro population ( 15a / b ) .
this population was considered to be t. b. macromelasoma due to its high sequence divergence compared to the other populations and because it was collected in its type locality .
the second clade , with pairwise distances up to 1% , showed that pernambuco populations are more basal , although those from serra talhada clustered with paraba populations likely because of the city 's proximity to the pernambuco - paraba border .
paraba populations formed a large cluster that also included the rio grande do norte population . based on sequence analysis
, a pcr - rflp assay was performed to differentiate between the subspecies t. b. macromelasoma and t. b. brasiliensis .
pcr fragment digestion using the styi enzyme produced two restriction fragments ( 342  bp and 192  bp ) in co1 sequences from salgueiro samples and only one fragment in samples of the other eight populations .
conversely , the hincii enzyme yielded two fragments ( 297  bp and 240  bp ) in all population samples ( n = 10 samples of each population ) except that from salgueiro ( figure 3 ) .
styi and hincii enzymes therefore produced distinct fingerprints for t. b. macromelasoma and t. b. brasiliensis , suggesting that they are different subspecies .
the molecular protocols described above are a useful tool in the study of populations and cryptic species , contributing to the identification of insect vectors .
the identification of adult triatominae based on morphological and chromatic pattern is considered relatively easy for most species ; however , this is commonly misguided owing to the wide phenotypic variability within this subfamily .
for instance , triatoma maculata erichson , 1848 and triatoma pseudomaculata corra & espnola , 1964 which were first treated as members of a same species complex due to morphological similarities   thereafter proved to be genetically distant [ 1719 ] .
panstrongylus herreri wygodzinsky , 1948 and panstrongylus lignarius walker , 1873 in turn , were considered to be separate species until marcilla et al .   and crossa et al .  
demonstrated that they are the same species , cytogenetically identical with regard to the second internal transcribed spacer .
( light brown tones ) and rhodnius nasutus stl , 1859 ( pinkish tones ) seems to be associated with the color of the palm trees they colonize , but others such as triatoma rubrovaria blanchard , 1843 , exhibit well - known 4 chromatic morphotypes .
other studies report the occurrence of natural homoploid hybrids between t. infestans and triatoma platensis neiva , 1913 , t. infestans and triatoma rubrovaria , and sympatric species of phyllosoma complex and species of the t. brasiliensis complex .
this interspecific crossing can be decisive in originating and diversifying wild species , resulting in important epidemiological consequences due to differential competence and the capacity of hybrid vectors [ 3 , 10 , 11 ] .
therefore , the characterization ( or identification ) of triatominae specimens based only on morphological and chromatic patterns , the most common identification method , is more complex than previously believed .
studies on immature stages are crucial for group systematics . however , literature reports on immature forms of certain groups are scarce , difficult to use , or nonexistent .
many species undergo changes in color , structure , and morphology during their development , hindering their identification [ 25 , 26 ] .
, molecular analyses are successfully used to characterize morphotypes of species complexes such as t. brasiliensis sp . , which exhibits wide chromatic and morphological variation [ 7 , 8 ]
. marked differences in color pattern and ecological features among species from the triatoma brasiliensis complex were detected by microsatellites , mitochondrial 12s , and cytochrome b genes , reinforcing species diagnosis [ 7 , 16 ] .
however , individuals from subspecies t. b. brasiliensis and t. b. macromelasoma might be clustered within the same co1 clade , since earlier studies have shown that some cytochrome b haplotypes of t. b. macromelasoma are similar to those of t. b. brasiliensis . moreover ,
the basal clade of the maximum likelihood tree ( figure 1 ) was identified as subspecies t. b. macromelasoma because it is highly divergent ( 6% , as shown in the nucleotide distance matrix , table 2 ) from the other populations and was collected in its type locality . on the other hand ,
however , interpopulation divergence values ( < 1% ) suggest that t. b. brasiliensis is still diversifying and/or exhibiting ongoing gene flow , probably due to human - assisted dispersal . based on wing morphometry , costa et al .   recently formulated a hypothesis that t. b. macromelasoma is the result of homoploidal hybridization between t. b. brasiliensis and t. juazeirensis in the state of pernambuco , and that this is a form of speciation in sympatric populations . in northeastern brazil ,
the epidemiological importance of triatominae bugs is mainly defined by their high rate of natural t. cruzi infection and ability to adapt to multiple ecotopes .
control measures therefore require a precise identification of which species of the t. brasiliensis complex is being targeted .
moreover , it is important to understand the ecoepidemiology of triatominae since these vectors are found in large numbers in their natural habitat . in this respect , the pcr - rflp protocol described here is suggested as rapid , relatively simple , and economical assay to distinguish triatoma b. macromelasoma from triatoma b. brasiliensis subspecies . even at small geographic scales , domestic populations are genetically structured by ecological parameters , thereby exhibiting small differences from the wild counterparts from which they are derived .
the present study highlights the effectiveness of the co1 gene in identifying subspecies of the t. brasiliensis complex and its contribution to classic taxonomy .
 
 triatoma brasiliensis sensu lato ( s.l . ) , the main vector of chagas disease in northeastern brazil , is a species complex comprising four species , one with two subspecies ( t. brasiliensis brasiliensis , t. brasiliensis macromelasoma , t. juazeirensis , t. sherlocki , and t. melanica ) , and each taxon displaying distinct ecological requirements . in order to evaluate the genetic relationships among nine t. brasiliensis s.l . 
 populations from northeastern brazil , we analyzed their mitochondrial cytochrome c oxidase subunit 1 sequences and suggested a pcr - rflp assay to distinguish between t. b. macromelasoma and t. b. brasiliensis subspecies . 
 all the specimens were morphologically identified as t. b. brasiliensis . 
 the resulting phylogenies identified two major clades that are congruent with the geographical populations studied . 
 based on collection sites and in accordance with type - location , one clade was identified as the subspecies t. b. macromelasoma . 
 the second clade grouped t. b. brasiliensis populations . 
 restriction endonuclease sites were observed in the sequences and used in pcr - rflp assays , producing distinct fingerprints for t. b. macromelasoma and t. b. brasiliensis populations . 
 the results suggest that these are different species and that gene flow occurs only among t. b. brasiliensis populations , possibly associated with human activity in the area . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion