malaria is an important public health problem in colombia with approximately 60% of the territory being suitable for disease transmission . in 2010 , 116,914 malaria cases were reported , for an annual parasite index ( api ) of 11.58/1,000 inhabitants , and in 2011 , only 62,969 cases were reported for api of 6.2/1,000 inhabitants , although underregistration of cases is presumed . the urab - bajo cauca and alto sin ( ucs ) epidemiological region , nw colombia , registers near 60% of the total malaria cases of the country and the amazonas region , se , 0.67% [ 2 , 5 ] .   in colombia , based on epidemiological , entomological or parasite detection records , three are of primary importance : anopheles ( nyssorhynchus ) albimanus wiedemann 1920 , anopheles ( nys . ) darlingi root 1926 , and anopheles ( nys . ) nuneztovari gabaldn 1940 , and six other species have relevance as secondary or local vectors [ 69 ] . however , there are species that in neighboring countries have been incriminated as local or regional vectors , but their role in transmission is not yet established for colombia . this is the case of anopheles ( nys . ) triannulatus neiva and pinto 1922 , a species widely distributed in the country . interestingly , in cimitarra municipality , ne colombia , it was the most abundant species collected and had an indoor biting behavior similar to the main vector , an . also , in santa rosa de lima , bolivar , in northern colombia , an . triannulatus was the predominant species , found in sympatry with the primary malaria vector an . furthermore , in localities of the amazon region , se colombia , it was collected in peridomestic areas showing anthropophilic activity and in sympatry with the main vectors an . triannulatus in brazil [ 13 , 14 ] , colombia , and venezuela , this species is suggested to play a role in malaria transmission as a secondary vector , especially , if it is in high densities , behaving as an opportunistic species depending on host availability and abundance . in brazil , triannulatus has been detected infected with plasmodium vivax grassi and felleti 1890 , in par state , rondnia state [ 19 , 20 ] and amazonas state , with plasmodium falciparum welch 1897 and plasmodium malariae laveran 1880 in amazonas   and amap state . triannulatus was reported as the dominant vector in eastern loreto department , per   and in bolivia , and it is suggested to be a secondary vector [ 23 , 24 ] . according to older reports , in venezuela , an . triannulatus was epidemiologically implicated as a vector during a malaria outbreak , and some years later detected with natural oocyst infection , being considered a potential vector in western venezuela where it showed a more anthropophilic behavior than an . triannulatus for plasmodium infection , using salivary glands and midgut dissection [ 28 , 29 ] or elisa and pcr [ 79 , 30 ] ; however , only recently , this species was detected infected with plasmodium vivax . anopheles triannulatus is considered a complex of at least three species , anopheles triannulatus s.s . neiva and pinto 1922 , anopheles halophylus silva - do - nascimento and loureno - de - oliveira 2002 , and anopheles triannulatus c [ 14 , 31 ] , that may differ in their vectorial capacity and involvement in parasite transmission [ 32 , 33 ] . triannulatus c have not been incriminated in malaria transmission [ 14 , 31 ] . in colombia , there is no evidence for the existence of more than one species of the triannulatus complex . triannulatus parasite infection , through the evaluation of specimens collected in six localities of two malaria endemic regions , the urab - bajo cauca and alto sin , nw , and the amazonas region , se colombia . anopheles triannulatus specimens were collected from january 2009 to march 2011 in six colombian localities ( figure 1 ) . the municipalities of leticia - let , puerto nario - pna , and tarapac - tar in amazonas department , account for 70% of the malaria cases reported in the amazonian region . antioquia and crdoba departments in the ucs region , nw , reported > 60% of total number of malaria cases of the country in 2010 [ 2 , 5 ] . of these , three municipalities in cordoba ( tierralta , montelbano , and puerto libertador ) contributed with 18% and three in antioquia ( bagre , caceres and zaragoza ) with 42% of those cases . available data for some of the localities indicate that , in 2010 bagre - bag reported 14,258 cases and san pedro de urab - spu 1,040 . indoor and outdoor collections ( within ~10  m of each house ) , from 18:00 to 24:00  h and at least one overnight ( 18:0006:00  h ) collection per field trip were performed , using human - landing catches under an informed consent agreement and collection protocol reviewed and approved by a university of antioquia institutional review board ( comit de biotica sede investigacin universitaria , cbeih - siu , udea , approval document 07 - 41 - 082 ) . in addition , in spu and puerto libertador ( plt ) , nw , mosquitoes resting on cattle were collected in nearby corrals ( within ~10 to 100  m from the house ) . at se localities specimens were provided by trained personnel by means of a contract for mosquito collections , and only peridomestic collections from 18:00 to 23:00  h were conducted . all anopheles specimens were identified based on morphological features . also , the species assignation for specimens with problematic morphology and for all an . triannulatus was confirmed by a pcr - rflp - its2 assay [ 37 , 38 ] , and megablast of an . triannulatus specimens processed by enzyme - linked immunosorbent assay ( elisa ) were tested in pools of five head - thoraces grouped by collection date and locality . detection of the circumsporozoite ( csp ) protein specific for p. falciparum and p. vivax variants vk247 and vk210 was carried out in separate plates following procedures optimized by personnel of the molecular microbiology group [ 8 , 9 ] . elisa positive specimens were those giving an od result ( wl : 405  nm ) equal or above the cut off value , set as twice the mean of seven negative controls run consecutively in each plate . positive pools were evaluated in a second elisa , performed at a later date to reduce the chance of reporting false positives [ 9 , 39 ] . amplifications using genus and species - specific primers designed to amplify the plasmodium small ribosomal subunit dna were performed using the primers and conditions previously described , with six  l of extracted dna used as template in a 25  l reaction mixture . for the genus nest-2 and the plasmodium species - specific amplifications , six l of the previous amplification product served as the dna template . plasmodium vivax dna was used as the positive control . specimens with a positive nested pcr and elisa result were further confirmed for plasmodium infection using a pcr directed to detect a mitochondrial cytochrome b ( cytb ) fragment specific for human plasmodium spp . , using two  l of extracted dna as the template . triannulatus ( np ) per number of total analyzed ( nt ) per 100 [ ir = ( np / nt )  100 ] . the confidence interval ( ci : 95% ) was calculated to indicate the reliability of the estimated value using the epidat program version 3.1 . triannulatus collected , 210 were from northwestern localities ( table 1 ) . in bag locality , 87 an . triannulatus were collected in four field trips and approximately 162  h of sampling , being the third most abundant species during the august and december 2009 collections ( 12% and 3% , resp . ) , after an . triannulatus were collected during two field trips and 72  h of sampling . here , it was also the third most abundant species ( 2% ) , after an . it is worth noting that in plt , after four collections with 168  h of sampling , an . triannulatus was only detected in the june 2010 collection , being the second most abundant species ( 28% ) after an . data on abundance and entomological parameters for other species collected in nw localities are available in .   in se , a total of 300 an . triannulatus were collected in two sites and approximately 15  h of sampling , being the most predominant species in both sites ( 63% and 80% ) ( table 2 ) . in pna , 89 an . this species was the predominant species during three samplings conducted in puerto rico site , pna ( 58% , 97% y 83% ) ( table 2 ) . in tar , 125 an . remarkably , in san sebastian , tar , it was the second most abundant species ( 13% ) after an . darlingi in the june 2010 collection and became the most predominant species ( 90% ) during the october 2010 collection . darlingi was the predominant species in the rest of the collections ( table 2 ) .   in nw , two an . triannulatus specimens were detected with plasmodium infection ( table 3 ) , one in bag infected with p. vivax vk247 , as detected by only the first elisa and the nested pcrs ( plasmodium and species - specific amplifications , ir = 1.51 , ci : 0.048.15 ) . this specimen was collected by hlc , indoors , between 18:00 to 19:00  h , during the january 2009 collection when an . triannulatus was found in low abundance ( 2% ) ( table 1 ) . in this locality , nuneztovari with p. vivax vk247 ( ir = 0.10 , ci : 0.0030.559 ) and an . triannulatus was detected infected with p. falciparum in plt , nw , as determined by the nested pcrs , plasmodium and species - specific amplifications ( figure 2(a ) ) and cytb pcr ( figure 2(b ) ) ( ir = 1.20 ; ci : 0.036.53 ) ; however , an elisa positive result was not obtained ( table 3 ) . this specimen was collected between 21:00 to 22:00  h , resting on cattle kept in corrals in proximity to the collection site . in se , two an . triannulatus collected by hlc were positive for p. falciparum , as detected by only the first elisa , result that was confirmed by the nested genus - specific pcr ( detects plasmodium ) , however , the species - specific nested pcr or the cytb pcr were negative ( table 3 ) . one of the infected an . triannulatus was collected in puerto rico site , pna ( ir = 1.20 , ci : 0.036.53 ) , during the august 2010 sampling , when an . the other infected specimen ( ir = 0.85 , ci : 0.024.63 ) was collected in ventura site , tar , in june 2010 , when an . triannulatus was the second most abundant species ( 20.3% ) ( table 2 ) . the determination of parasite infection in anopheline mosquitoes is an important component for vector incrimination . in colombia , triannulatus in transmission [ 79 , 30 ] , but , only recently , an . triannulatus nw linage   was reported infected with p. vivax vk247 . in this study , a more thorough evaluation of an . triannulatus infection in specimens collected at various localities of malaria endemic areas of the country provided further evidence of plasmodium - infected an . these ir values are similar or slightly higher than those found for the main colombian vectors , an . albimanus in the colombian pacific region , and in the same locality , bag , an . triannulatus in brazil , where it was detected infected with the same plasmodium species . for example , with p. vivax , ir was 0.5   and 1.1   in rondnia state ; with p. falciparum , ir was 0.8 in amazonas state   and 0.13 in amap state . however , in some sites of brazil , higher irs have been detected for an . triannulatus infected with p. vivax , of 8.9 in par state   and 3.7 in amazonas state .   the infected an . triannulatus from bag , nw , was collected indoors , which makes it even more important to further evaluate the epidemiological role of this species in this locality . triannulatus with anthropophilic tendency and active indoors and outdoors . however , the infected an . triannulatus collected resting on cattle in plt , nw , suggests a zoophilic tendency , as previously documented for this species in this particular area , where one of the main economic activities is livestock production and the high availability of cattle may influence this feeding behavior . the two specimens detected infected in pna and tar localities of the amazonian region , se colombia , had p. falciparum . triannulatus was the most or second most abundant species in pna and tar , constituting 97% and 20% of the total collected specimens , respectively ; notably in pna , the primary vector an . triannulatus in localities of par , and amazonas , brazil , that indicate that this species colonized altered environments and became abundant and even dominated , outnumbering the main vector an . darlingi . of particular interest , pna and tar are in close proximity to let ( se ) , a locality also included in a recent phylogeography study on an . of seven lineages proposed based on coi data , four were present in colombia ; particularly , lineage e , widely distributed in brazil , where an . [ 17 , 21 ] , is also present in let , se ; this makes it important to further investigate the role of an . triannulatus was detected infected with both p. vivax and p. falciparum ; however , only until recently this species was reported infected . among the reasons influencing the negative results could be that an . triannulatus were at low abundance   or not being participating in transmission when collections were carried out . also , technical reasons can produce negative results ; for example , in the case of low parasite infection in the mosquito , the elisa could show false negative results because of the lower sensitivity of this technique as compared to the pcr [ 43 , 48 , 49 ] . furthermore , negative results could originate from the specimen part used for the test in relation to the stage of the parasite cycle in the mosquito ; for example , at the initial stage of sporogony when the sporozoites have not reached the salivary glands [ 18 , 50 ] , the csp protein could be detected only in the midgut . in the case of the pcr , interfering material present in the reaction and a low dna concentration after extraction could produce a false negative result . likewise , false positive results of the test to detect plasmodium - infected anophelines have been reported ; specifically for the elisa , when testing zoophilic mosquitoes . therefore , this should be a consideration when analyzing elisa positive plasmodium infection results in an . triannulatus , especially in nw localities where cattle is a main mosquito - feeding source . therefore in this study , to avoid reporting false positive results , at least two positive tests of the ones performed ( two elisa reactions , plasmodium genus and species - specific nested pcrs or cytb pcr ) , served as criteria for defining plasmodium infected anopheles . in the case of the p. triannulatus collected in cattle in plt , nw , the positivity was determined by both nested and cytb pcr . for the other infected specimens , a positive result was obtained in the first elisa and confirmed by nested pcr . in accordance with our findings , variations in the results of the tests used to detect mosquito parasite infection have been reported in other studies [ 52 , 53 ] . triannulatus was detected infected with both p. vivax and p. falciparum , which suggests the importance of further evaluating the epidemiological importance of this species in malaria endemic regions of colombia . in addition , differences in results between the pcr and elisa advocate the need for assessing the degree of sensitivity and specificity of these two techniques . but even of greater priority is the evaluation or development of new , more accurate methodologies to test plasmodium infection in the anopheles mosquitoes . in this sense , the novel pcr based on parasite cytb sequences , rapid test as the vectesttm dipstick assay [ 54 , 55 ] , and real - time pcr amplification [ 56 , 57 ] deserve further evaluation . while this is done and given the limitations still found when using the available methodologies , it is recommended that at least two positive tests be used as the criterion for defining plasmodium infection in wild collected anopheles . this is of particular importance when investigating the role of presumptive vector species in disease transmission .
the role of anopheles triannulatus as a local vector has not yet been defined for malaria - endemic regions of colombia . therefore , the aim of this work was to detect an . 
 triannulatus naturally infected with plasmodium spp . 
 , as an approximation to determining its importance as malaria vector in the country . a total of 510 an . 
 triannulatus were collected in six malaria - endemic localities of nw and se colombia from january 2009 to march 2011 . in the nw , 
 two specimens were naturally infected ; one with plasmodium vivax vk247 , collected biting on humans and the other with plasmodium falciparum , collected resting on cattle . in the se , 
 two specimens were positive for p. falciparum . 
 although these results show an . triannulatus naturally infected with plasmodium , further studies are recommended to demonstrate the epidemiological importance of this species in malaria - endemic regions of colombia .