study site - the malaria focal area of cunday - villarrica is composed of 
 the municipalities of cunday , villarrica , dolores , prado and purificacin ( 0357n 
 07436w ) ( fig . 1 ) , which are situated on the 
 western slopes of the eastern mountain chain and have an average temperature of 24c and 
 an annual rainfall of between 2,000 - 4,000 mm . this area is approximately 100 
 km and is characterised by humid forests that have an abundance of 
 epiphytes . moreover , malaria cases reported in tolima have been almost exclusively from that focal 
 area . fig . 1 : location of the study area , foci of cunday - villarica , department of 
 tolima , colombia . map realised by gegrafos del valle . 
 
 sampling strategy and adult mosquitoes - collections of biological 
 material were made between february 2009-june 2012 . adult females were captured using 
 human landing collections inside and around houses and in the forest during periods of 
 peak biting at various locations in the municipalities in this study . captured females 
 were individualised in vials and brought to the entomology laboratory of the national 
 university of colombia for preservation and taxonomic determination . additionally , adult 
 females of kerteszia , which were collected between 1981 - 1983 in the 
 same study area and belonged to the collection of the entomology laboratory of the 
 museum of entomology , university of valle , were analysed morphologically . entomological series - mosquitoes in immature stages ( larva and pupa ) 
 were captured directly from water at the base of bromeliads leaves by drawing out their 
 content with a larger pipette . larvae of anopheles were collected from 
 41 trees with 78 bromeliads and brought to the laboratory of the national university of 
 colombia for rearing to the adult stage . iv instar larvae were individualised and larval 
 and pupal skins were obtained to achieve 37 full entomological series . morphological analysis - diagnostic characters of females , iv instar 
 larvae and male genitalia were studied based on the descriptions and morphological keys 
 of zavortink ( 1973 ) and harrison et al . . 
 molecular methods - forty - four specimens were processed as belonging to 
 an . ( kerteszia ) sp . and their dna was extracted 
 using the dneasy blood & tissue kit ( qiagen , usa ) . the rdna its2 region 
 was amplified using the primers of collins and paskewitz ( 1996 ) and the polymerase chain 
 reaction ( pcr ) conditions described by linton et al . a portion ( 710 bp ) of the 
 barcoding region of coi ( mdna ) was amplified using the primers designed 
 by folmer et al . the 
 products were visualised on 1% agarose gel containing 0.5 mg / ml of ethidium bromide . the 
 pcr product was purified using exosap - it ( usb corporation , usa ) . sequencing reactions were carried out in both directions using the big dye terminator 
 kit(pe applied biosystems , england ) on an abi 3730 automated sequencer 
 ( pe applied biosystems ) . the sequences were edited with sequencher4.10.1 
 ( gene codes corporation , usa ) and aligned manually and translated to amino acids in 
 macclade v.4.06 ( maddison & maddison 2003 ) . sequence similarities were compared with 
 those available in genbank using basic local alignment search tool ( blast ) 
 ( ncbi.nlm.nih.gov/genbank/ ) and sequence statistics were calculated in mega v.5 ( tamura 
 et al . table shows the sampling locations in the study 
 area and the sampling dates . 1,596 were collected landing on humans in the period 
 between 2009 - 2012 and 141 females were sampled between 1981 - 1983 . 
 tablesampling localities of cunday - villarrica focus in the department of tolima , 
 colombiamunicipalitycoordinateslocalitysampling periodcunday044759.1nsan franciscofebruary 2009icononzo7434 50.21w  045102.1nvalencianovember 1981  7409003wcuatimbaloctober 1982  alto icononzooctober 1982villarrica035203.4npuerto llerasfebruary 1981-december 1982  743909.8wtotumaljune 1981-february 1983  guanacasjune 1982-february 1983  rio lindofebruary - november 2009  rio lindoaugust 2010  rio lindoaugust - september 2011  rio lindoapril - july 2012purificacin035158.11nvilla esperanzamay 2009  745559.83w  prado034504.15nel crucemay 2009  744955.9w 
  the first morphological analysis was conducted using the dichotomous keys of zavortink 
 ( 1973 ) . as a result of this analysis , 76% ( 1,218 ) of the collected specimens 
 corresponded to either an . pholidotus 
 and 24% ( 378 ) were in agreement with an identification of an . pholidotus is 
 restricted to the size of the scales on the female abdomen . as a result , it is difficult 
 to determine which of these two species is present in the study region using this 
 morphological key . considering this issue , a second morphological analysis was made 
 using the dichotomous key proposed by harrison et al . pholidotus females 
 according the following diagnostic features : ( i ) females with white scales on the 
 proboscis , pedicel and palpomere i ; hindtarsomeres i and 2 without apical pale band 
 ( from dorsal view ) are classified as an . lepidotus and ( ii ) females 
 without white scales on the proboscis , pedicel and palpomere i ; hindtarsomeres i and 2 
 with narrow apical pale band ( from dorsal view ) are classified as an . all females captured in tolima showed morphological characteristics that were similar to 
 those assigned to an . pholidotus exhibited numerous scales with variable widths on 
 tergites and sternites ii - vii , hindtarsal segment 2 with white bands in the apical pale 
 band between 0.1 - 0.2 as the total length of the tarsomere , mesanepimeron with large 
 curved patch of scales extending from higher bristles to below the middle of the 
 segment , scales in the proximal tergites and scales of the distal tergites forming 
 transverse apical bands ( fig . right 
 arrow shows mesepimeron with one large and curved patch of scales that extends 
 ventrally from upper setae . iv instar larvae - twenty - eight micro - preparations of iv instar larvae 
 were analysed . the larvae showed a characteristic coloration pattern in the dorsal area 
 of the thorax and abdominal segments i and v ( fig . the morphological characteristics of this instar coincided with those 
 described by zavortink ( 1973 ) for an . setae 5 - 7-c simple , 
 not plumose , moderately long , characteristics of the kerteszia 
 subgenus ; setae 6-vi moderately long , always different to 6-iii - v , setae 1-iii - vii small 
 palmate with pointed leaflets , pecten teeth of similar size all long , with marginal 
 spinules extended to the apex , setae 1-i not palmate , setae 4-c always less developed 
 than 2-c , setae 1-vii filiform , not palmate . 3 : general view that shows the dorsal coloration pattern of larvae iv instar 
 of anopheles pholidotus . 

  according to zavortink ( 1973 ) , two morphological characteristics help differentiate 
 an . pholidotus shows a setae 3-c that is moderately developed 
 and approximately 1/2 the length of 2-c , while an . lepidotus shows a 
 setae 3-c that is short and thick , fusiform and less than 1/2 the length of 2-c . the 28 
 specimens analysed in this study presented an average length of 0.19  0.01 mm for the 
 setae 2-c and of 0.11  0.004 mm for the setae 3-c , showing that in all cases the length 
 of the setae 3-c is more than half the length of the setae 2-c ( fig . the second discriminatory character is the setae 11-p , 
 which is well developed for an . all larvae tested showed the setae 11-p to 
 be well developed ( length = 0.32  0.03 mm ) , with an average of 66.7% of the length of 
 the 12-p setae ( 0.48  0.03 mm ) ( fig . 4 : larvae iv instar of anopheles pholidotus ( 400x ) . a : 
 head . lepidotus , the seta 3-c is very thick , short and usually sharply 
 pointed ; however , seta 11-p is always very short and setae vi is stout and long . as 
 mentioned above , the specimens analysed in this investigation showed characteristics 
 that do not match those described for an . male genitalia - twenty - five micro - preparations of male genitalia from 
 a series were analysed . the morphological characteristics for this structure coincided 
 with those described by zavortink ( 1973 ) and gonzlez and carrejo ( 2009 ) for an . the specific 
 characteristics were gonocoxite with long and sinusoid parabasal setae , always greater 
 than 1/3 the total length of gonocoxite ; one internal seta flattened at the apex and two 
 accessory ones located apically , an aedeagus without leaflets ( fig . 5a ) , a lateral expansion of the ventral lobe of claspette , 
 moderately spiculose except laterally and rounded ( fig . the 25 male genitalia analysed in this study showed a lateral 
 expansion of the ventral lobe of claspette corresponding to the typical shape of 
 an . pholidotus described by zavortink ( 1973 ) and gonzlez and 
 carrejo ( 2009 ) . fig . 5 : male genitalia of anopheles pholidotus . a : general view 
 ( 200x ) ( ae : aedeagus ; gc : gonocoxite ; sa : accesory setaes ; si : internal setae ; 
 sp : parabasal setae ) ; b : view of lateral expansion of ventral lobe of claspette 
 ( 400x ) ; c : ventral lobe of claspette expanded showing the characteristic form 
 of an . pholidotus ( 1,000x ) . dna sequences - ninety - five specimens were sequenced with the molecular 
 markers coi and its2 . only one haplotype was observed for each 
 marker , confirming the lack of an intraspecific variation in all of the individuals 
 analysed . the coi barcode region ( 658 bp ) consisted of a single open reading 
 frame , discarding the presence of pseudogenes . its nucleotide frequencies ( % ) were a : 
 27.7% , t : 40.9% , g : 15.5% and c : 16% ( fig . the 
 its2 fragment showed no length variability ( 546 bp ) and its nucleotide frequencies were 
 a : 26.4% , t : 19.4% , g : 26.7% and c : 27.5% ( fig . fig . 6 : cytochrome oxidase i sequence of anopheles pholidotus 
 from villarrica , department of tolima , colombia using the folmer et al . fig . 7 : internal transcribed spacer sequence of anopheles 
 pholidotus from villarrica , department of tolima , colombia using 
 the collins and paskewitz ( 1996 ) primers ( n = 10 , 546 bp ) . the sequences of its2 were submitted to blast , where high significant homology ( 100% ) 
 was found with the sequences of an . pholidotus ( from tchira , 
 venezuela ) ( accessions jn967769.1 , jn967768.1 ) published by harrison et al . coi sequences , compared with sequences in the barcode of life data 
 systems ( bold systems ) ( boldsystems.org ) , showed 100% similarity with an . ( 2012 ) , the only morphological feature that 
 had been proposed to differentiate females of an . lepidotus were , according to zavortink ( 1973 ) , the size of the 
 scales on proximal tergites . this 
 taxonomic characteristic is vague and difficult to interpret , so it would be inaccurate 
 to separate female adults of these two species based only on these characteristics . 
 therefore , it was essential to analyse other morphological structures and associated 
 stages , such as male genitalia and to use the chaetotaxy of iv instar larvae . even so , 
 genitalia analysis presents a  weak spot  or a  bottle neck  effect in the separation of 
 these two species because in zavortinks key ( 1973 ) , the presence or absence of scales 
 in vii tergite in the abdomen of adults must be verified at one point , which is an 
 external feature of the genitalia . ( 2012 ) 
 is useful for separating females of both species . as mentioned above , for several years , there was evidence of the presence of an . lepidotus in the 
 cunday - villarrica focal region ( gonzlez & carrejo 2009 ) . even just three years 
 after the report of quiones et al . 2012 ) . the limited availability of specimens collected from the area for morphological 
 analysis , the scarcity of associated stages , the poor condition of some of the samples 
 and the difficulty of accessing the area to collect new biological information most 
 likely delayed the opportunity to solve this problem by three decades . the collections of mosquitoes for this study , which occurred between february 
 2009-october 2011 , provided updated information about the species of anophelines in 
 these focal areas . according to the morphological features of females , the larvae 
 chaetotaxy , the male genitalia presented in the specimens of cunday - villarrica malaria 
 focal area in tolima and the analysis of the coi and its2 sequences , 
 an . pholidotus was the most common species found and was most likely 
 misidentified as an . lepidotus were not found by analysing 1,737 individual 
 mosquitoes captured indicates that the species that was previously reported as the 
 vector of malaria in the area since 1984 ( quiones et al . the sequences obtained in this investigation , both coi and its2 , showed 
 that the tested specimens correspond to an . sequences from three localities of tolima 
 exhibit no intraspecific variability , showing that an . pholidotus is 
 present in the study area as a single species , although this may not be true in other 
 parts of the country due to the high altitude of certain mountain ranges . ( 2001 ) and gonzlez and carrejo ( 2009 ) provided records of supposed  an . for example , we have 
 identified , by morphology , three females from valle del cauca ( pnn farallones : cali , 
 pance , vi-6 - 1984 , 1,500 m ) as an . . pholidotus has a wider 
 distribution than previously thought and that many of the specimens identified as 
 an . , it is important to determine whether this homology is generalised 
 to all countries where an . harrison et al . ( 2012 ) inadvertently forgot to include the precise location of the 
 record for an . a single female in the 
 mosquito collection located at the nmnh at the smithsonian institution was collected 
 resting on the wall of a highway tunnel . the collection data record was as follows : 
 costa rica : cartago , res . , ca . 9 km ( road nw tunnel , 9.72 n , 
 83.78 w , 8.9.vi.1988 , elv . 1,400 m , cm & os flint , molzenthal ) . one of the key aspects for optimising control measures is an appropriate taxonomic 
 determination of the species that are present and those that are responsible for 
 transmission . taxonomic studies in kerteszia species are relevant 
 because the distribution of this subgenus along the continent is still poorly known , 
 mainly due to the lack of research studies . furthermore , the association of these 
 species with areas where bromeliads are normally present means that environmental 
 changes and human activity may restrict the distribution for some species in the 
 subgenus ( marrelli et al . 2007 ) however , for other species , the same factors may 
 stimulate the proliferation of breeding sites and possibly extend their geographical 
 distribution ( solarte et al .
since 1984 , anopheles ( kerteszia ) lepidotus has been considered a 
 mosquito species that is involved in the transmission of malaria in colombia , after 
 having been incriminated as such with epidemiological evidence from a malaria 
 outbreak in cunday - villarrica , tolima . 
 subsequent morphological analyses of females 
 captured in the same place and at the time of the outbreak showed that the species 
 responsible for the transmission was not an . 
 lepidotus , but rather 
 anopheles pholidotus . however , the associated morphological 
 stages and dna sequences of an . 
 pholidotus from the foci of 
 cunday - villarrica had not been analysed . 
 using samples that were caught recently from 
 the outbreak region , the purpose of this study was to provide updated and additional 
 information by analysing the morphology of female mosquitoes , the genitalia of male 
 mosquitoes and fourth instar larvae of an . 
 pholidotus , which was 
 confirmed with dna sequences of cytochrome oxidase i and rdna internal transcribed 
 spacer . 
 a total of 1,596 adult females were collected in addition to 37 larval 
 collections in bromeliads . 
 furthermore , 141 adult females , which were captured from 
 the same area in the years 1981 - 1982 , were analysed morphologically . 
 ninety - five dna 
 sequences were analysed for this study . 
 morphological and molecular analyses showed 
 that the species present in this region corresponds to an . 
 pholidotus . 
 given the absence of an . 
 lepidotus , even in 
 recent years , we consider that the species of mosquitoes that was previously 
 incriminated as the malaria vector during the outbreak was indeed an . 
 pholidotus , thus ending the controversy .