
 dengue epidemiology in rio de janeiro in 2008 - dengue transmission in 
 rio de janeiro is strongly associated with summer ( december - april ) , when higher 
 temperatures and intense rainfall are frequent ( honrio 
 et al .
, the city of rio de janeiro suffered one of its most 
 severe dengue outbreaks when denv-2 and denv-3 co - circulated . between january - april 
 2008 ,
more than 155,000 cases and 110 deaths were reported and an incidence of 
 2,453.7/100,000 inhabitants was observed ( teixeira et 
 al .
the epidemic reached its peak in march and april , with 86,036 and 
 90,945 human cases reported , respectively , and had ended by june ( malho et al .
aegypti survival in the laboratory - 
 to estimate the effect of dengue infection on ae .
aegypti longevity and 
 how that effect may vary with mosquito size , we reanalysed data reported by sylvestre et al .
( 2013 ) , who gave a full description 
 of experimental conditions and methods .
briefly , we orally challenged females with 
 denv-2 at a viral titre of 2 x 10 tcid50 .
aegypti populations : paea ( a population maintained as laboratory 
 colony at oswaldo cruz institute , rio de janeiro , for almost 13 years ) ( sylvestre et al . 2013 ) and a field population from 
 rio de janeiro .
2013 ) reported 
 that 93.3% of females challenged in this way had detectable dengue in the head , 
 suggesting disseminated infection .
after being orally infected , females were maintained 
 individually in plastic vials ( 6.5 cm height , 2.5 cm diameter ) with cotton soaked in a 
 10% sucrose solution ad libitum and an oviposition substrate at the 
 bottom for egg laying . during the longevity study one blood meal on an anesthetised 
 mouse
[ oswaldo cruz foundation ethical committee for animal use ( ceua l-0007/09 ) ] was 
 offered to mosquitoes once a week .
after 
 death , wing length ( axillary incision to wing tip , excluding fringe ) ( alto et al .
aegypti field collections - mosquito collections started in march 
 2008 during the dengue outbreak and continued until june 2009 .
aegypti were collected in 23 districts that encompassed three municipalities 
 in metropolitan region of rio de janeiro ( supplementary 
 data ) .
district selection was done together with the health municipal 
 secretary , which directed collectors to neighbourhoods in which dengue transmission had 
 recently become intense . at each field - site
, we randomly choose 10 - 20 houses per day for 
 mosquito collection using backpack aspirators or bg - sentinel traps ( clark et al .
mosquito collections began after explaining 
 the objectives of this research project to householder and receiving their oral consent 
 to use an aspirator or a mosquito trap to collect mosquitoes .
aspirating was performed 
 for 15 - 20 min per house , depending on the premise area , and was also done in the 
 peridomestic environment .
bg - sentinel traps were installed inside houses and remained in 
 the same house for a 24-h trapping period . 

mosquito preparation - all mosquitoes collected in the field ( 1,586 
 males and 1,213 females ) were brought to the laboratory for identification using 
 taxonomic keys ( consoli & loureno - de - oliveira 
 1994 ) .
aegypti females had their wing length measured 
 from axillary incision to wing tip excluding the fringe ( alto et al . 2008b ) . each adult
adult females from all 23 sites were individually macerated 
 in 1 ml of leibovitz l-15 medium ( sigma ) plus antibiotics ( penicillin - streptomycin , 
 10,000 units - invitrogen ) and centrifuged ( 6,000 rpm at 4c for 30 min ) .
supernatant 
 was transferred to an eppendorf tube containing 100 ml of streptomycin / fungizone and 
 penicillin , kept in an ice bath for 1 h and centrifuged ( 3,000 rpm at 4c for 15 min ) . 

supernatant was transferred to an eppendorf tube containing 0.3 ml of foetal calf serum 
 ( fcs ) ( invitrogen ) and frozen ( -70c ) ( de castro et al . 

virus isolation - virus isolation was performed by inoculation into 
 monolayers of c6/36 ae .
albopictus cells ( igarashi 1978 ) in leibovitz l-15 medium ( sigma ) supplemented with 2% 
 fcs ( invitrogen ) and 0.2 mm of nonessential amino acids ( invitrogen ) .
cells were 
 incubated at 28c for five - seven days and observed for cytopathic effects ( de castro et al .
isolates were identified by 
 indirect fluorescent antibody test ( ifat ) using dengue serotype - specific monoclonal 
 antibodies ( gubler et al .
1984 ) and infected 
 supernatant was clarified by centrifugation and virus stocks stored in 1-ml aliquots at 
 -70c . 

rna extraction and reverse transcriptase polymerase chain reaction 
 ( rt - pcr ) - viral rna was extracted using qiaamp viral rna mini kit ( qiagen ) 
 following the manufacturer s instructions .
rt - pcr for detecting denv was performed as 
 described previously ( lanciotti et al .
1992 ) . 
 statistical analysis - laboratory - for the laboratory longevity study , 
 time to death was analysed by a proportional hazards model with classification effects 
 of population ( field , lab ) and dengue infection ( yes , no ) and continuous effects of wing 
 length ( mm ) and wing length(mm ) ( proc phreg ) ( sas 2011 ) .
we 
 initially tested a model with all interactions of the class variables and wing length 
 and wing length .
all interactions were far from significant ( p > 0.15 ) 
 and this full model yielded a poorer information theoretic index [ akaike s information 
 criterion with a correction for small sample sizes ( aicc ) ] ( burnham et al .
similarly , effects of population on longevity were also far from significant ( p > 
 0.15 ) and a model with population yielded a poorer aicc ; hence our final analysis was a 
 reduced model including only dengue infection , wing length and wing length . 

this same model also was the endpoint of using the stepwise option in proc phreg ( sas 2011 ) . 

aegypti were 
 collected from march to early june 2008 , corresponding to epidemic period reported by 
 the ministry of health ( malho et al .
aegypti field collected during this 
 epidemic period , testing effects of date of collection ( expressed as days post 1 march 
 2008 ) and ( date of collection ) and the random effect of site for 545 
 individuals from all 23 sites .
we used a mixed model anova ( sas proc mixed ) ( sas 2011 ) .
aegypti females and we 
 analysed only these sites for frequency of dengue infection because we could not be sure 
 dengue was active at a site unless we detected it .
we analysed the relationship between 
 frequency of dengue infection and wing length ( mm ) and ( wing length ) across 
 these 13 sites .
all models tested included date of collection ( days post 1 march 2008 ) 
 ( date of collection ) and the random effect of site for 492 individuals from 
 these 13 positive sites .
we used a mixed effects generalised linear model ( proc glimmix ) 
 ( sas 2011 ) , employing an information theoretic 
 approach to model comparison ( anderson 2008 ) using 
 aicc .
a major advantage of this approach is quantification of weight of evidence 
 ( wi ) for different models ( anderson 
 2008 , burnham et al .
we tested 
 three models that included wing length as both linear and quadratic terms , wing length 
 as a linear term only and no wing length term .
we used a binary distribution of the 
 dependent variable dengue ( yes , no ) , with a logit link function ( sas 2011 ) .
we used multimodel estimation ( anderson 2008 ) to estimate weighted average consensus predicted 
 probabilities of infection and their weighted standard errors ( se ) , as a function of 
 wing length and date of collection based on all the models evaluated and weighting model 
 contributions by wi ( anderson 
 2008 ) .
for all 23 sites , including those with no dengue - positive females , we determined mean 
 female size ( wing length ) and proportion of females infected .
we then tested for a 
 relationship across sites of proportion dengue - infected and mean female size per site 
 using pearson s correlation ( proc corr ) ( sas 
 2011 ) .

 laboratory - sizes of the 134 females used in the laboratory study 
 ranged from 2.10 - 3.20 mm wing length , with a mean  se of 2.89  0.01 mm .
infection with 
 dengue significantly shortened the life of females [ = 7.71 , degrees of 
 freedom ( df ) = 1 , p = 0.0055 ] , with a hazard ratio of control / infected of 0.560 , 
 indicating that controls had nearly half the hazard of death as infected individuals . 

both wing length ( = 4.06 , df = 1 , p = 0.0439 ) and ( wing 
 length ) ( = 4.81 , df = 1 , p = 0.0283 ) were significantly 
 related to the hazard of death .
the linear term (  se ) was negative ( -20.81  10.30 ) , 
 indicating that hazard of death initially declines with adult size .
the quadratic term 
 was positive ( 4.09  1.90 ) , indicating that hazard of death increases as larger sizes 
 are reached .
thus this analysis describes a hazard function of size that is 
 significantly concave upward . predicted survival curves for infected and uninfected 
 females of wing lengths = 2.10 , 2.70 and 3.30 mm ( fig . 

1 ) indicated that longevity peaked at 2.70 mm and was actually least at 3.30 
 mm . 

fig . 1 : predicted survivorship curves for adult female aedes 
 aegypti in the laboratory that are not infected with dengue ( a ) or 
 infected with dengue ( b ) .
statistical analysis reported in the results section . 
 plotted wing lengths were chosen to span the range of sizes of females included 
 in the experiment . 
 

field - sizes of 545 field collected females ranged from 1.60 - 3.95 mm 
 wing length , with a mean  se of 2.69  0.01 mm .
wing length varied significantly among 
 sites ( z = 2.24 , p = 0.0124 ) , but was unrelated to date of collection , 
 as a model omitting date of collection , and only including site yielded the best 
 aicc .
frequency of dengue infection was best predicted by a model with wing length as a linear 
 effect ( table i ) and this linear effect was 
 positive ( slope  se = 1.24  0.56 , t 
 476 = 2.20 , p = 0.0285 ) .
wi indicated that the model with linear + 
 quadratic effects of wing length also had some support , given this data set , based on 
 aicc [ and , indeed , any of the other information theoretic indices reported by sas institute ( 2011 ) ] .
for the quadratic model , the 
 linear effect of wing length was again positive ( linear coefficient  se = 9.33  7.33 , 
 t 
 475 = 1.27 , p = 0.2034 ) , whereas the quadratic effect was negative ( quadratic 
 coefficient  se = -1.46  1.32 , t 
 475 = 1.11 , p = 0.2688 ) .
the model omitting wing length entirely was 
 substantially less likely as a model generating these data ( table i ) . predicted frequencies of dengue infection for these two 
 most likely models , holding the random site effect constant at the value for the 
 valqueire collection ( supplementary data , fig .
2a , 
 b , table 
 ii ) show that in both cases predicted frequency of dengue infection increases 
 from wing lengths of 2.0 mm to about 3.5 mm . at wing lengths from 3.5 - 4.0 mm
the 
 quadratic model predicts frequency of dengue infection declines , though never to the low 
 levels observed for the smallest females ( fig . 

we expected frequency of dengue infection to be related to day of collection 
 and the relationship proved to be quadratic , with frequency first increasing to a peak 
 in the middle of the epidemic , then declining ( fig . 

table imixed effect generalised linear models for the relationship of wing length 
 to frequency of dengue infectionmodel 
 effectsaiccaiccexp[-0.5(aicc)]wi 
 wing , day , day , site301.87010.5608wing , wing , day , day , 
 site302.470.600.74080.4155day , day , site308.206.330.04220.0237sum--1.7830-all considered models included linear and quadratic effects of date of 
 collection and the random effect of site .
weight of evidence ( wi ) 
 expresses exp[-0.5(aicc ) ] as a proportion of the sum of that column and 
 indicates the evidence for the model ( i.e. , the probability that the model 
 is the correct one ) .
aicc : akaike s information criterion with a correction 
 for small sample sizes . 
 
all considered models included linear and quadratic effects of date of 
 collection and the random effect of site .
weight of evidence ( wi ) 
 expresses exp[-0.5(aicc ) ] as a proportion of the sum of that column and 
 indicates the evidence for the model ( i.e. , the probability that the model 
 is the correct one ) .
aicc : akaike s information criterion with a correction 
 for small sample sizes . 

2 : predicted frequencies of dengue infection among field collected female 
 aedes aegypti from rio de janeiro , brazil .
all models included a 
 random effect for collection site ( n = 13 dengue positive sites ) .
the predicted 
 relationships are shown holding site effect constant at the value for 
 collections from valqueire ( supplementary data , table ii ) .
statistical 
 assessment of support for linear ( a ) and quadratic ( b ) models of frequency vs. 
 wing length ( mm ) is given in table i. uncertainty of predicted frequencies 
 given in table ii . 
 

table iiestimates of standard error ( se ) of prediction for models of the 
 relationship of frequency of dengue infection to wing lengthwing 
 ( mm)modelmodel 
 ( wi)predicted frequency 
 ( dengue)conditional se 
 predictedmodel selection 
 uncertaintyaverage predicted 
 frequency ( dengue)unconditional se 
 predicted2.0wing , wing 
 0.41550.0780.0620.0010.1120.335wing0.56080.1450.0860.011 - 0.02370.2750.1160.0553.0wing , wing 
 0.41550.3990.1470.0740.2990.547wing0.56080.3680.1410.059 - 0.02370.2750.1160.0224.0wing , wing 
 0.41550.2200.3520.0040.3480.590wing0.56080.6670.2080.260 - 0.02370.2750.1160.014all models include date and date . for prediction ,
the random 
 effect of site was held constant at the value for valqueire ( supplementary 
 data ) , which had an intermediate frequency of dengue - positive females 
 ( 0.1719 ) and date was held constant at 21 april ( middle of the epidemic ) . 

unconditional 
 se includes that uncertainty plus model selection uncertainty weighted by 
 evidence ( wi ) .
for prediction , the random 
 effect of site was held constant at the value for valqueire ( supplementary 
 data ) , which had an intermediate frequency of dengue - positive females 
 ( 0.1719 ) and date was held constant at 21 april ( middle of the epidemic ) . 

unconditional 
 se includes that uncertainty plus model selection uncertainty weighted by 
 evidence ( wi ) . for details
multimodel consensus prediction of frequency of dengue infection , based on the weighted 
 averages of the three models ( table ii ) indicated 
 that frequency of dengue infection likely increased monotonically across the range of 
 wing lengths from 2.0 - 4.0 mm .
multimodel predictions , particularly at the largest wing 
 lengths , had high uncertainty ( table ii ) due to 
 both inherent error of prediction and model selection error ( anderson 2008 ) .
site - wide frequency of dengue infection was not significantly correlated with mean wing 
 length for a site ( fig .
3 ) , but highly 
 variable and unlikely to be linear . 
 fig . 3 : relationship of frequency of female aedes aegypti 
 infected with dengue [  standard error ( se ) , binomial formula ] and mean female 
 body size (  se ) for 23 sites in rio de janeiro , brazil , and vicinity during 
 the dengue epidemic of 2008 .
our data support the competition - longevity hypothesis and suggest that the 
 competition - susceptibility hypothesis does not account for patterns of dengue infection 
 of these vectors in nature .
however , a central point of this paper is that the frequency 
 of dengue infection in the field may have a complex relationship to vector body size . 

thus , it is important to determine that empirical relationship and to understand the 
 ecological processes that cause heterogeneity in frequency of infection of vectors . 

though our data refute the competition - susceptibility hypothesis , we do not reject the 
 physiological mechanism underlying that hypothesis ( i.e. , physiological effects of 
 competition and body size on probability of dengue infection given an infectious blood 
 meal ) .
that mechanism has been demonstrated in laboratory studies of infection 
 of aedes with dengue ( alto et al .
our data indicate , however , that the demographic 
 effects of competition ( longevity of adults ) are more important in 
 nature than these physiological effects of competition ( susceptibility to 
 infection ) . in the epidemic in rio
aegypti indicates 
 that longevity is not linearly related to size , but rather shows a quadratic 
 relationship , with maximal predicted longevity at intermediate sizes ( wing lengths of 
 2.7 mm ) and lower longevity at smaller or larger sizes .
typically mosquito ecologists 
 assume , based on some data ( e.g. , hawley 1985 , 
 reiskind & lounibos 2009 ) , that adult 
 aedes longevity is positively related to body size .
some field 
 studies have failed to find a relationship ( e.g. , scott 
 et al . 2000 , maciel - de - freitas et al . 
 2007 ) and others find evidence for non - monotonic relationships ( e.g. , hawley 1985 ) .
our laboratory study of longevity had 
 a relatively limited range of female wing lengths represented ( 2.1 - 3.2 mm ) compared to 
 our field collected females ( 1.6 - 4.0 mm ) ; nevertheless this range was sufficient to show 
 the nonlinear relationship of size to longevity . in the field ,
frequency of dengue infection increases with female size and the linear 
 model was the best description of our field data .
even if we accepted the quadratic 
 model of frequency of dengue , it yields a peak predicted frequency of dengue for females 
 between 3.0 - 3.5 mm wing length ( relatively large females ) ( fig .
wing lengths  3.5 mm represented less than 1% of all 
 females collected for this study , which indicates that for the vast majority of females 
 in the field in rio de janeiro during the epidemic , frequency of dengue infection 
 increases with size for both the linear and quadratic models ( fig .
if the decline in frequency of dengue among this 
 largest 1% of females is real , its cause remains unknown .
that decline may result from 
 low crowding of larvae during development , yielding very large , less physiologically 
 susceptible adults , as observed by alto et al .
( 2008a , b ) . alternatively , low crowding 
 may yield very large , but short - lived adults , as observed in our laboratory 
 analysis . reduced longevity of small ae .
aegypti adults from crowded larval 
 conditions was evident at adult body sizes at or below 2.6 mm mean wing length ( reiskind & lounibos 2009 ) , which suggests that 
 the very small females < 2.5 mm wing length , which constituted 25% of our field 
 sample , may be expected to have significantly reduced longevity relative to larger 
 females .
in contrast , based on logistic regressions , estimated physiological 
 susceptibility to dengue infection and disseminated infection in the laboratory decline 
 rapidly with increasing size for female ae .
aegypti across the range of 
 wing lengths from 2.0 - 3.8 mm ( alto et al .
this wing length range encompasses 
 about 99% of the field collected females in our study .
as we observe increased frequency 
 of dengue infection across virtually all of that size range , we conclude that the field 
 patterns we observe are not consistent with the competition - susceptibility hypothesis 
 and that the demographic mechanisms postulated in the competition - longevity hypothesis 
 are most important in producing patterns of dengue infection with body size in the 
 field .
aegypti clearly vary considerably in size in 
 nature and this variation is related to probability of infection at the 
 individual level .
we found little evidence that inter - site variation in size , which was 
 significant , was related to local frequency of dengue infection at the site level .
thus , 
 individual variation does not obviously relate to larger - scale site - level patterns of 
 risk of dengue . whether spatial variation in dengue risk is associated with female body 
 size and the ecological conditions that produce that size variation at smaller spatial 
 scales
( e.g. , the urban block or the house level ) remains to be tested it is also well 
 established that susceptibility to dengue infection has a genetic component ( black et al .
aegypti varies 
 due to environmental factors beyond larval density and competition [ e.g. , temperature 
 ( muturi & alto 2011 ) ] .
thus , for 
 genetically competent populations , we expect larval density and other environmental 
 factors to foster heterogeneity in susceptibility to dengue infection among adult 
 females .
whether genotypic variation in dengue susceptibility interacts with 
 environmental effects remains to be investigated . beyond the present study ,
our results indicate that there are important patterns of 
 frequency of dengue infection among ae .
larger females appear to be the most dangerous 
 dengue vectors during the outbreak in rio de janeiro .
this suggests that larval 
 conditions that yield large , robust adult females are those that are most likely to 
 produce the most dangerous individual vectors of dengue .
uncrowded aquatic habitats for 
 larvae , with ample food , thus seem likely to be sources of vectors that pose the 
 greatest threat .
this indicates an important future direction for research in 
 vector - borne disease : quantifying factors creating heterogeneity of disease vectors .
not 
 all individual vectors are alike in their likelihood of infection and disease 
 transmission and approaches to limit vector - borne disease need to consider the 
 ecological conditions , particularly during larval development for vectors with complex 
 life cycles that produce the most dangerous individuals .
a similar concept is already 
 enshrined in general ecology of host - pathogen interactions ( lloyd - smith et al .
2012 
 ) where some individual hosts are considered  superspreaders  because of 
 their disproportionately high tendency to spread the infection to other hosts .
similar 
 effects are likely for insect vectors of human and animal disease , even though those 
 effects may be more difficult to detect among the large numbers of individuals in vector 
 populations . even more important than understanding the lack of homogeneity of vectors 
 ( or hosts ) for contributions to disease transmission
is the identification of measurable 
 traits , such as body size , that can be used to identify those individuals that are the 
 most likely to transmit pathogens .
this can enable us to identify the ecological 
 conditions that are likely to produce those most dangerous vectors and thus may aid in 
 limiting the transmission of vector - borne disease .
 two hypotheses for how conditions for larval mosquitoes affect vectorial capacity 
 make opposite predictions about the relationship of adult size and frequency of 
 infection with vector - borne pathogens . 
 competition among larvae produces small adult 
 females . 
 the competition - susceptibility hypothesis postulates that small females are 
 more susceptible to infection and predicts frequency of infection should decrease 
 with size . 
 the competition - longevity hypothesis postulates that small females have 
 lower longevity and lower probability of becoming competent to transmit the pathogen 
 and thus predicts frequency of infection should increase with size . 
 we tested these 
 hypotheses for aedes aegypti in rio de janeiro , brazil , during a 
 dengue outbreak . in the laboratory , longevity increases with size , then decreases at 
 the largest sizes . for field - collected females , 
 generalised linear mixed model 
 comparisons showed that a model with a linear increase of frequency of dengue with 
 size produced the best akaike s information criterion with a correction for small 
 sample sizes ( aicc ) . 
 consensus prediction of three competing models indicated that 
 frequency of infection increases monotonically with female size , consistent with the 
 competition - longevity hypothesis . 
 site frequency of infection was not significantly 
 related to site mean size of females . 
 thus , our data indicate that uncrowded , low 
 competition conditions for larvae produce the females that are most likely to be 
 important vectors of dengue . 
 more generally , ecological conditions , particularly 
 crowding and intraspecific competition among larvae , are likely to affect 
 vector - borne pathogen transmission in nature , in this case via 
 effects on longevity of resulting adults . 
 heterogeneity among individual vectors in 
 likelihood of infection is a generally important outcome of ecological conditions 
 impacting vectors as larvae . 
MATERIALS AND METHODS
RESULTS
DISCUSSION