study area - the study was conducted in the community of nuevo 
 chorrillo , located approximately 17 km west of panama city , at 85710.97n 
 794150.57w , 71 m altitude ( fig . nuevo 
 chorrillo belongs to the township of nuevo emperador , which is a sub - division of the 
 arraijan district , panama province . this area experienced a large dengue outbreak in 
 2009 , with 1,340 cases reported ( population in the arraijan district was estimated at 
 220,779 inhabitants in a 2010 national census ) 
 ( arraijan.municipios.gob.pa/index.php/sobre-el-distrito/poblacion ) . subsequently , the 
 number of reported dengue cases in the district for 2010 and 2011 was 37 and 174 , 
 respectively ( l diez , nuevo chorrillo community health centre , unpublished 
 observations ) . inset map shows the 
 approximate location of nuevo chorrillo in relation to panama s capital , panama 
 city . dashed square shows the limits of the southwestern section , where the 
 study was conducted ; b : location of release points ( white circles ) and 
 bg - sentinel traps ( white crosses ) in the study area . original figure under the creative commons attribution - sharealike 3.0 unported 
 license ( creativecommons.org/licenses/by-sa/3.0/deed.en ) . satellite images in 
 panel a and b : @digitalglobe 2012 , used with permission . the population of nuevo chorrillo is estimated at 4,722 inhabitants living in 1,042 
 houses ( l diez , nuevo chorrillo community health centre , unpublished observations ) , 
 which gives an average density of ~4.5 persons per house . the study area encompassed a 
 section of 0.1 km ( 10 ha ) located towards the southwestern end of the town 
 ( fig . 1 ) and contained 249 houses , for an 
 estimated population of 1,121 persons , which in turn is equivalent to ~11,000 
 persons / km ( ~112 persons / ha ) . temperature in the area fluctuates between 
 26 - 28c throughout the year . rainfall is highest between may - december ( average 228 
 mm / month ) and lowest between january - april ( average 39 mm / month ) ( the panama canal port 
 authority , unpublished observations ) . mosquito strain , rearing and marking - the study was performed using a 
 panamanian strain of ae . aegypti originally collected in march 2010 at 
 the 24 de diciembre community ( municipality of panama ) . the strain had been continuously 
 maintained at the gorgas institute s insectaries until used for this trial , undergoing 
 re - hatch cycles approximately every four months . mosquito rearing was performed in dedicated insectaries located at the gorgas 
 institute s campus in panama city . eggs were hatched en masse by 
 placing them in water and under vacuum for 1 h. first instar larvae were aliquoted in 
 plastic trays ( 29 x 23 x 6 cm ) and reared under standard insectary conditions 
 ( temperature : 28c  2c ; humidity : 80%  10% ) and a photoperiod of 12 h light:12 h 
 darkness . larvae were fed ground tetramin flakes ( tetra gmbh , germany ) 
 daily . upon pupation , male pupae were recovered and a sample of 1,620 of these 
 specimens was used to verify the quality of the mechanised sexing procedure by 
 individually sexing each pupa under a dissection microscope . only one female pupa was 
 found in the sample ( 0.06% of the total ) , implying a sorting efficiency of 99.94% [ exact 
 binomial 95% confidence interval ( ci ) : 100 - 99.66% ] . male pupae were immediately aliquoted ( on average 540 pupae per container ) in 100 ml 
 plastic weight boats ( ramboldi ltd , cyprus ) placed inside 1.8-l clear plastic 
 cylindrical containers ( produtos prafesta , brazil ) which had their inner 
 walls previously coated with dayglo  red rocket fluorescent powder 
 ( day - glo color corporation , usa ) . a round hole was cut - off the lid of each container and 
 covered with a sheet of fine mesh , to allow for air flow and provide a surface for sugar 
 feeding . a sheet of damp cotton wool was placed on top of each container to avoid 
 desiccation . after emerging , adult mosquitoes moved around and came in contact with the fluorescent 
 powder in the walls of the container , which stuck to the surface of their bodies and 
 resulted in clear and even marking of the specimens . this procedure efficiently marked 
 all mosquitoes with no obvious adverse effects ( a mckemey , unpublished observations ) . a 
 10% sucrose solution was provided ad libitum to adult males until their 
 release . water was drained from 
 the plastic containers through a small incision and adult males were held for a further 
 48 h to allow them to reach sexual maturity prior to release . 
 mosquito release and recapture - on the day of release , plastic 
 containers holding adult mosquitoes were loaded in a large cooler box ( to avoid 
 overheating ) and transported to the field site in the back of a pick - up truck . the 
 release took place between 10:00 am-11:00 am . at each one of the 27 pre - selected release 
 spots ( fig . 1 ) , one container was opened and 
 mosquitoes were allowed to fly away . following release , a sample of eight containers 
 were re - fitted with their respective lids and taken back to the laboratory , where the 
 numbers of dead and non - released specimens were counted and used to estimate the number 
 of released males . to recapture released mosquitoes , 30 bg - sentinel traps baited with bg - lures ( biogents , 
 germany ) and powered by rechargeable 9 ah/12-volt batteries ( model cp-1290 , vision 
 group , china ) were distributed around the study area ( fig . traps were thereafter visited every 24 h. during each visit , batteries 
 were replaced with fully charged units and captured mosquitoes were collected and taken 
 to the laboratories at the gorgas institute , where they were sorted and screened . aegypti specimens were checked for the presence of fluorescent 
 powder by examining them under a uvr9000 hand - held ultraviolet uv light source ( bayco , 
 usa ) . wing measurement - for each selected specimen , one wing was carefully 
 removed and mounted on a microscope slide . digital micrographs of mounted wings were 
 obtained using a leica mz 125 stereoscope fitted with a leica dfc 500 digital camera 
 ( leica microsystems , germany ) . wing length was defined as the linear 
 distance from the axillary incision to the apical margin , excluding the fringe ( harbach & knight 1980 , maciel - de - freitas et al . 2007 ) . data analysis - statistical analyses were performed using r software , 
 an open source language and environment for statistical computing ( r core team , 
 austria ) . daily survival probability ( dsp ) of released males in the field was estimated 
 via a non - linear method ( nonlinear least square function ) ( buonaccorsi et al . 2003 ) ; life expectancy ( le ) was derived from the 
 estimated survival using the formula -1/loge(dsp ) ( niebylski & craig jr 
 1994 ) . population size was estimated using the petersen - lincoln test ( petersen 1896 , lincoln 1930 ) . the number of pupae per person was determined from the wild 
 mosquito population estimates following the calculations described by focks et al . 95% ci for survival and 
 population estimates were calculated by bootstrap analysis ( 1,000 repeats ) . wing length groups were tested for normality by shapiro - wilk test and then differences 
 between groups were tested using student t test ( normal distribution ) 
 or kolmogorov - smirnov test ( non - normal distribution ) . linear relation between day of 
 recapture and size ethics - since this research work did not involve any human or other 
 vertebrate subjects , and upon consultation with panamanian health authorities ( gorgas 
 memorial institute for health studies ) , it was established that no ethical review was 
 required for the study to proceed . release and recapture numbers - an estimated 14,388 adult male marked 
 mosquitoes were released on 11 november 2011 ( average of 533 males / release point ) . trapping started the day of release and continued until 24 november 2011 , which marked 
 the end of three consecutive days without recaptures . over the 13-day trapping period , 
 4.43% ( 637/14,388 ) of released males aegypti were caught in the traps during the study , 
 equivalent to a 0.08 sex ratio in the area , given an assumption of similar 
 attractiveness of the traps to both sexes ( krckel et 
 al . a total of 
 305 culex specimens were also caught , but were not identified to 
 species level . 
 longevity - the dsp was 0.14 ( ci : 0.11 - 0.18 ) , equivalent to an le of 
 0.5 days ( ci : 0.46 - 0.58 ) . however , the data did not respect the assumption of normal 
 distribution ( shapiro - wilk normality test : p = 0.003 ) which invalidates the dsp 
 estimate ; this is likely the result of the number of recaptures on day 1 , which was much 
 higher than during the other days combined ( 503 vs. 134 ) . consequently , calculations 
 were run again after removing day 1 recaptures , resulting in a dsp of 0.65 ( ci : 
 0.57 - 0.70 ; shapiro - wilk normality test : p = 0.24 ) , equivalent to an le of 2.3 days ( ci : 
 1.78 ; 2.86 ) . wild population estimation - the wild population was calculated for 
 days 1 , 2 and 3 , as well as their average ( table ) . to estimate the number of released males remaining in the area on 
 different days the 
 population estimations were 111 , 773 and 328 males for days 1 , 2 and 3 respectively , 
 with an average of 404 males , i.e. , 43 males / ha or 0.36 males / person . table daily and average wild population and pupae / person estimationdaywild population males 
 ( ci)pupae / person 0.7:1 
 sex ratio ( ci)1111 ( 36.8 - 206.2)0.13 ( 0.04 - 0.24)2773 ( 238 - 1772.8)0.9 ( 0.28 - 2.1)3328 ( 0 - 714.6)0.38 ( 0 - 0.83)average4040.47average ( 2 and 3 combined)5500.64the wild population density was estimated from day 1 , 2 and 3 recaptures 
 separately and combined for day 2 and 3 ( petersen 1896 , , lincoln 1930 ) . the 
 confidence interval ( ci ) calculated by bootstrap using 1,000 repeats is also 
 presented . the equivalent pupae / person estimates shown use a male : female sex 
 ratio of 0.7:1 in the calculations ( focks et al . 2000 ) . the wild population density was estimated from day 1 , 2 and 3 recaptures 
 separately and combined for day 2 and 3 ( petersen 1896 , , lincoln 1930 ) . the 
 confidence interval ( ci ) calculated by bootstrap using 1,000 repeats is also 
 presented . the equivalent pupae / person estimates shown use a male : female sex 
 ratio of 0.7:1 in the calculations ( focks et al . 2000 ) . the male : female sex ratio observed among captured specimens in this study ( 0.08 ) is 
 extremely low in comparison to other reports , which usually place this value between 
 0.5 - 1 when bg - traps are used [ maciel - de - freitas et al . ( 2012 ) and our unpublished observations ] and has been reported to reach values 
 as high as 1.4 ( williams et al . the reasons behind 
 the unusually low figure obtained in the present study remain to be determined . for 
 calculations , we will consider a more realistic sex ratio of 0.7 [ representing an 
 average of male : female ratios reported by krckel et al . ( 2012 ) ] . assuming similar recapture of males and 
 females , the density of females in the area can be estimated at 83 females / ha or 0.7 
 females / person . the pupal development time chosen was two 
 days , the proportion of pupae successfully emerging as adults 0.83 and the dsp of 
 wild - type males 0.8 ( focks et al . 2000 ) . on 
 average the pupae / person ratio was 0.64 ; taking each day separately , the two estimates 
 were 0.38 ( ci : 0 - 0.83 ) and 0.9 ( ci : 0.28 - 2.10 ) for day 2 data . 
 wing length - wing size , an indicator of body size ( nasci 1990 , ponlawat 
 & harrington 2007 ) , was measured for a sample of 50 marked male mosquitoes 
 randomly selected before release , 190 recaptured ( marked ) male mosquitoes and 10 wild 
 ( non - marked ) captured male mosquitoes ( fig . 2 ) 
 ( the 9 remaining wild mosquitoes were too damaged to use for this purpose ) . the average 
 (  standard error of the mean ) wing lengths of the analysed groups were as follows : 
 released males = 2.86 mm (  0.019 ) , recaptured males = 2.87 mm (  0.006 ) , wild males = 
 2.61 mm (  0.069 ) . the average wing length of marked recaptured males was not 
 significantly different from that of released males ( p = 0.96 ) , indicating that size did 
 not affect the probability of recapture . both released and recaptured males were 
 significantly larger than wild males ( p = 0.0002 and p = 0.0006 , respectively ) . there 
 was no linear relation between day of recapture and size of the males ( linear 
 regression : p = 0.92 , data not shown ) . 2 : size of the released , wild - type and recaptured males . the size of the 
 released males ( n = 50 ) , recaptured males ( n = 190 ) and wild type males ( n = 
 10 ) was assessed by measuring their wing length ( mm ) . wild - type males were 
 significantly smaller than both released males ( kolmogorov - smirnov test : p = 
 0.0006 ) and recaptured males ( kolmogorov - smirnov test : p = 0.0002 ) . there was 
 no difference between released and recaptured males ( student t 
 test : p = 0.96 ) . this study adds to the relatively sparse information available on survival and wild 
 population of male ae . although various population - monitoring tools can 
 provide indirect measures of the size of specific mosquito populations or their relative 
 change over time , estimating the absolute size of a wild mosquito population is best 
 accomplished using pupal surveys which are expensive , time consuming and dependent on 
 personnel s ability ( focks & chadee 1997 ) or 
 mrr experiments . though many mathematical methods have been developed to support these field studies , 
 there is unfortunately no consensus as to what the most appropriate methodology is for 
 population size estimation . deterministic population estimation models are based on a 
 dilution effect observed from mrr experiments and involve single release / single capture 
 [ petersen - lincoln method ( petersen 1896 , lincoln 1930 ) ] , single release / multiple captures 
 [ yamamura method ( yamamura et al . 1992 ) , jackson 
 positive method ( jackson 1939 ) ] , multiple 
 releases / single captures [ jackson negative method ( jackson 1939 ) ] or multiple releases / multiple captures [ fisher and ford s 
 method ( fisher & ford 1947 ) , bailey s stochastic models involving multiple releases / multiple 
 captures have also been developed ( jolly 1965 , 
 seber 1965 , manly & parr 1968 ) . recent studies have used the shift in the sex ratio 
 of captures after releases of wolbacchia infected ae . this study was designed to include a single 
 multi - point massive release from laboratory reared males without background monitoring . consequently , the petersen - lincoln index was used as an estimator of mosquito population 
 and the estimated survival rate for the calculation of the number of survivors on each 
 day ( similar to the yamamura method ) . 2005 ) which are not always respected or explicitly assessed : ( i ) 
 closed population , ( ii ) all individuals have the same probability of capture , ( iii ) 
 marking individuals does not affect their capabilities and behaviour , ( iv ) animals do 
 not lose their marking and ( v ) all marked individuals are reported . fluorescent powder 
 marking is widely used in mosquito mrr and is considered not to interfere with 
 assumptions ii , iv and v when applied correctly ( hagler 
 & jackson 2001 ) ; preliminary tests were performed to ensure that the 
 marking did not affect mortality of the mosquitoes . due to the limited range of dispersal typical of male ae . aegypti 
 ( muir & kay 1998 , maciel - de - freitas et al . 2007 , lacroix et al . 2012 ) and the virtual absence of human dwellings 
 immediately to the north , south and west of the study area ( fig . 1 ) , it was assumed that immigration and emigration were limited 
 to the 150 m to the east of the study area and should balance each other , as should the 
 proportions of marked and wild males emigrating . as the study was conducted at the end 
 of the rainy season , the population should have been stable - emergence and mortality of 
 adults were assumed to be equivalent . estimations of dsp make the assumption of equal probability of recapture over time . 
 similarly , mrr statistics assume equal probability of recapture of marked / released and 
 unmarked ( wild ) mosquitoes . however , behaviour associated with release ( altered flight 
 activity / search for mates , resting sites etc . ) could affect probability of recapture on 
 the release day compared to wild mosquitoes and to subsequent recapture days . it was 
 considered that after the first 24 h , subsequent activity and behaviour of released 
 males resulting in contact with traps and probability of recapture were comparable to 
 that of wild males ; thus only estimates not including recapture data for day 1 will be 
 discussed further . overall , the male population was estimated at 58 males / ha ( range 12 - 81 males / ha ) , which 
 falls in the low range of the estimates from a study in kenya , where 37 - 107 males / ha 
 were reported ( trpis & husermann 1986 ) . in a 
 different study by focks et al . ( 1981 ) the local 
 male population of a study area in new orleans ( united states of america ) was estimated 
 by pupae survey to be between 264 - 558 males , depending on dsp ( focks et al . although the area surveyed in that study is not 
 stated clearly in the report , we estimated around 5 ha ( using the cars visible in the 
 report s aerial photo as a scale ) , which would correspond to a density of 53 - 111 
 males / ha . much higher male population estimates ( > 1,700 males / ha ) have been reported 
 from tyre dumps in delhi , india ( conway et al . 
 1974 ) . however we 
 anticipated that the released males would show different behaviour right after release 
 that would likely affect their survival . supporting this hypothesis , we found that 
 incorporating data from day 1 in the dsp calculation led to an estimated dsp of 0.14 
 ( 95% ci : 0.11 - 0.18 ) , equivalent to 0.51 days le . this low value would imply that all the 
 released males would have died by day 5 , yet we were able to recapture marked specimens 
 until day 10 ( fig . daily recaptures are presented on a 
 logarithmic scale for the 13 day trapping period of the study . the full recapture data did not conform to the normality distribution necessary to the 
 estimation of the dsp , however , as dsp is assumed constant it can also be estimated if 
 one day is excluded from the data . in this study , day 1 recaptures were much higher than 
 other days and likely biased the dsp estimates . using data only from day 2 onwards gave 
 a calculated dsp of 0.65 ( 95% ci : 0.57 - 0.71 ) ( equivalent to 2.3 days le ) where , additionally , the model s 
 sensitivity to the number of males released is low [ dsp estimate is still 0.65 if only 
 6,000 males ( < 50% of total released ) are assumed to remain on day 2 ] . this dsp value 
 is similar to those reported previously in studies that used either laboratory or 
 recently colonised males , where dsp values range from 0.48 - 0.85 ( sheppard et al . 1969 , trpis & 
 husermann 1986 , muir & kay 1998 , 
 valerio et al . focks and chadee ( 1997 ) proposed that the 
 estimation of populations by performing pupal surveys and relating them to the local 
 human population ( effectively measuring the number of pupae per person in the area of 
 interest ) is a more accurate method than traditional aedes indexes . 
 during a multi - country pupae survey project that applied this concept in the americas , 
 caribbean , africa and asia , the reported mosquito densities ranged from 0.02 - 7.63 
 pupae / person , with most values falling between 0.5 - 1.5 ( focks & alexander 2006 ) . in our study , the estimated population is 
 equivalent to 0.64 pupae / person on average , a value that falls in the typical range 
 reported by focks and alexander ( 2006 ) . many 
 mosquito populations undergo seasonal fluctuation ; our study was performed at the end of 
 the rainy season , when the population might be expected to be at or near its peak . the recapture rate in our study ( 4.4% ; 0.93% without day 1 ) was lower than the 7.4% and 
 12.3% reported in brazil using bg - sentinel traps ( maciel - de - freitas et al . 2007 ) and in the low range of values reported by 
 studies that used other trapping tools : 6.6% and 11.5% ( reuben et al . 1973 ) , 23% ( trpis & 
 husermann 1986 ) , 4.6% and 14.8% ( muir & 
 kay 1998 ) , 17% ( harrington et al . recapture 
 rates for bg - sentinel traps vary between areas depending , at least in part , on the 
 density of human populations that compete with the traps to attract mosquitoes . indeed , 
 the recapture rates of marked males in an uninhabited area ( 17.2% and 50.2% ) were in the 
 highest range of the literature ( lacroix et al . therefore , the relatively high human density found in our study site 
 ( ~112 persons / ha ) might explain , at least in part , the low recapture rates observed . the average wing lengths of the released and recaptured males were not significantly 
 different , so there was no indication of a size effect in recapture probability of 
 individuals . additionally , although bigger males have been reported to survive longer 
 ( maciel - de - freitas et al . 2007 ) , our study 
 showed no significant correlation between wing size and day of recapture . however , it is 
 possible that this lack of correlation is an artefact caused by the small numbers of 
 specimens recaptured during the later days and differences could be revealed with larger 
 samples or multiple studies . insectary - reared released males were significantly larger than their wild counterparts , 
 an effect that has been observed in previous studies ( johnson et al . this may reflect the different larval environments of each group : while 
 insectary - reared specimens are maintained in relatively uniform conditions intended to 
 be reasonably nurturing , the harsher breeding environment faced by wild specimens ( i.e. 
 variations in food availability , temperature and specimen density ) may explain the 
 relatively small size of wild specimens . it is worth mentioning that although the strain used for release was originally 
 collected within the municipality of panama , it had been reared for approximately 20 
 months under insectary conditions prior to its use in the current experiment ( roughly 
 equivalent to 5 generations , since the strain was re - hatched every 4 months ) . although 
 the strain did not undergo any kind of purposeful selection during this time , it is 
 nonetheless possible that released specimens presented slight genetic variations from 
 their wild ancestors due to the effect of either genetic drift or selective pressures 
 unintendedly imposed on the strain during insectary rearing . however , considering the 
 limited time the strain was maintained in the laboratory prior to its release , we 
 believe that any phenotypic variation caused by this process is likely to be limited and 
 therefore should not significantly impact the results of the current work . furthermore , in spite of the high efficiency of the sexing process - estimated at 99.94% 
 - it is impossible to rule out the unintended release of a minimal number of female 
 individuals during the experiment . however , considering that ( i ) the species released is 
 endemic to the study area , ( ii ) the strain released is of panamanian origin and ( iii ) 
 the number of released females is likely to be extremely low ( 14,388 specimens released , 
 with 99.94% sexing accuracy , corresponds to ~9 females released in total ) , it was 
 estimated that the potential risks for the population and environment in the study area 
 were negligible . as previously stated , detailed knowledge of male mosquito biology is a 
 crucial element for the design of genetic population control strategies which rely on 
 the release of adult male transgenic individuals ( thomas 
 et al . 2007 , alphey et al . 2008 , 2010 , harris et al . 2011 , 2012 ) . even considering that the data generated by 
 this study would have to be locally validated for transgenic strains of ae . aegypti , they give encouraging insights for potential genetic control 
 programs in the area : mean survival of two to three days would imply the need to perform 
 releases of 10,000 males two - three times a week to maintain a 10:1 standing crop ratio 
 of sterile to wild males in the environment . however , it should be considered that field 
 studies in the cayman islands and malaysia report a lower dsp for ox513a males ( mean dsp 
 = 0.33 , equivalent to ~0.9 days ) ( lacroix et al . taking 
 this lower dsp reported for ox513a , a release of 25,000 males three times a week ( for a 
 total of 75,000 males / week ) would be required to achieve a 10:1 ratio of ox513a : wild 
 males in the study area . furthermore , the larger size of insectary - reared specimens may 
 prove advantageous since larger males have been reported to be more successful at mating 
 with females , as well as carrying more spermatozoids ( ponlawat & harrington 2009 ) .
traditional mosquito control strategies rely heavily on the use of chemical 
 insecticides . 
 however , concerns about the efficiency of traditional control methods , 
 environmental impact and emerging pesticide resistance have highlighted the necessity 
 for developing innovative tools for mosquito control . 
 some novel strategies , 
 including release of insects carrying a dominant lethal gene ( ridl ) , rely 
 on the sustained release of modified male mosquitoes and therefore benefit from a 
 thorough understanding of the biology of the male of the species . in this report 
 we 
 present the results of a mark - release - recapture study aimed at : ( i ) establishing the 
 survival in the field of laboratory - reared , wild - type male aedes 
 aegypti and ( b ) estimating the size of the local adult ae . 
 aegypti population . 
 the study took place in panama , a country where 
 recent increases in the incidence and severity of dengue cases have prompted health 
 authorities to evaluate alternative strategies for vector control . 
 results suggest a 
 life expectancy of 2.3 days for released male mosquitoes ( confidence interval : 
 1.78 - 2.86 ) . 
 overall , the male mosquito population was estimated at 58 males / ha ( range 
 12 - 81 males / ha ) , which can be extrapolated to an average of 0.64 pupae / person for the 
 study area . 
 the practical implications of these results are discussed .