aegypti was maintained in the laboratory of chemical ecology of 
 vector insects of the federal university of minas gerais from 2007 . the breeding room was kept at a temperature of 27  1c , 75 - 80% relative humidity ( rh ) 
 and a 12l:12d photoperiod ( eiras & jepson 
 1991 ) . the larvae were kept in plastic tubs containing water ( approximately 5 
 cm deep ) and fed reptile food ( reptolife , alcon , brazil ) until they reached 
 the pupa stage . the pupae were collected daily and transferred to adult screened cages 
 ( bugdorm-1 , mega view science education services , taiwan ) ( 30  30  30 cm ) and fed 10% 
 sucrose solution . a selection cage was used to separate the gravid females that were 
 used in the assays . blood meal was prepared using chicken blood ( gallus 
 domesticus ) , which was obtained from a slaughterhouse , including the 
 anticoagulant heparin ( gomes et al . . aegyptifemales ( f6 and f7 ) of five-10 days of chronological age 
 and three days after blood meal ( gomes et al . experimental area - the tests were performed in laboratory and 
 semi - field conditions . in laboratory conditions , four transparent acrylic boxes ( 150  
 50  41 cm ) were kept in a room with monitored temperature and humidity conditions ( 27  
 3c , 60 - 90% rh ) and a photoperiod of 12l:12d ( eiras 
 & jepson 1991 ) . the semi - field tests were performed in an experimental 
 area of 14.0  7.0  3.5 m , where eight cages ( 2.5  2.5  2.0 m each ) were installed 
 ( roque & eiras 2008 ) . the temperature , rh 
 and the photoperiod ranged according to the external environment and were monitored with 
 a thermo - hygrometer . the tests were considered valid only when the temperature reached a 
 minimum of 25c ( roque & eiras 2008 ) . 
 bioassays - the tests evaluating the pattern of distribution of eggs by 
 ae . the females 
 were evaluated in laboratory conditions in experiment 1 and in semi - field conditions in 
 experiment 2 . experiment 1 ( laboratory ) was performed in the four acrylic boxes in which four 
 different densities of breeding sites ( 2 , 4 , 8 and 16 ) were simulated . in each box , a 
 single gravid female of ae . aegypti was released and allowed to remain 
 for 96 h in the interior of the box . the breeding sites were ovitraps that were set with 
 300 ml of tap water and a rectangular wooden paddle ( 12 cm  3 cm ) ( fay & perry 1965 , fay & eliason 1966 ) . for each density level , 12 repetitions of 
 the experiment were performed . 1 ) . sugar solution ( 10% ) absorbed into 
 cotton was provided as food to the insects during the experimental processes . 1:representation of the distribution of the different densities ( 2 , 4 , 8 
 and 16 breeding sites ) and the positions of the breeding sites in the interior 
 of the acrylic boxes . in the centre , after 96 h , the paddle and the water that were present in each trap were collected , 
 labelled and taken to the laboratory . to verify the distribution of eggs at each 
 breeding site , the number of eggs that were present in the water and in the paddle of 
 each ovitrap was counted using a manual counter and a stereoscopic microscope ( 20 
  ) . experiment 2 ( semi - field ) was conducted in the interior of the four fabric cages ( 2.5  
 2.5  2.0 m ) ( roque & eiras 2008 ) . the same 
 densities ( 2 , 4 , 8 and 16 ovitraps ) that were evaluated in the laboratory conditions 
 were evaluated in this environment . for each density the positions of the breeding sites were kept constant and the same 
 methodology as used in experiment 1 was followed . however , to avoid visual effects of 
 the treatment itself , each cage received all 16 of the ovitraps , but only the test traps 
 ( densities 2 , 4 , 8 or 16 ) received water ( fig . 
 2 ) . 
 fig . 2:diagram of the distribution of the ovitrap with and without water of each 
 treatment / density of breeding sites . 
 
 statistical analyses - for statistical analyses , the specific locations 
 of egg deposition ( water and paddle ) were called habitats and the local of performance 
 of the experiment ( laboratory or semi - field ) was called environment . to compare the number of eggs that were deposited in each environment / density , data were 
 submitted to a normality test at a significance level of 95% . when the distributions were not in accordance with 
 the criteria of normality , medians were compared using the kruskal - wallis test . the proportions of eggs that were deposited on water by each single female ( n eggs on 
 the water / n total eggs ) at the different densities of breeding sites both in laboratory 
 and semi - field conditions were compared by two - factors anova . before analyses , the data 
 were transformed in arcsin(sqrt ) to normalise the distribution and stabilise the 
 variances . the number of colonised breeding sites in relation to the amount of available ones , both 
 in laboratory and semi - field conditions , was compared by the kruskal - wallis test . by investigating the female groups above and below the regression lines , we obtained two 
 groups : those that colonise various breeding sites and those that colonise few sites . we 
 compared these two groups by the mann - whitney u test to determine 
 whether there was any difference between the groups with higher and lower frequencies of 
 colonised breeding sites and the proportion of eggs that were deposited in the 
 preferential breeding site . the r program ( available from : r-project.org ) environment ( laboratory or semi - field ) of the experiment and egg deposition 
 - the oviposition behaviour of ae . aegypti was maintained 
 in both environments . under laboratory conditions , the mean number [  standard deviation 
 ( sd ) ] of eggs that were laid by one ae . aegyptiper repetition was 73.9 
 (  25.64 ) , ranging between 40 - 142 eggs . similarly , under semi - field conditions , the mean 
 number (  sd ) of eggs that were laid by oneae . aegypti per repetition 
 was 72.2 (  20.57 ) , ranging between 41 - 123 eggs . on average , females in both the experimental environments distributed the same number of 
 eggs and colonised the same number of breeding sites ( anova p > 0.05 ) . therefore , 
 these oviposition behaviours do not seem to be influenced by changes in the tested 
 environments . oviposition on the water - the tested females exhibited a strong 
 tendency to deposit their eggs on the water of the ovitrap . the mean proportion of eggs 
 that were laid on the water surface tended to be higher than that on the paddle when the 
 number of available breeding sites was greater ( fig . habitat received significantly more eggs in 
 semi - field than in laboratory conditions ( anova p < 0.05 ) ( fig . 3:box plot of the proportion of eggs on the water in different density of 
 oviposition breeding sites . 4:box plot of the number of eggs laid on water and paddle in semi - field and 
 laboratory conditions . breeding sites colonised in relation to available ones - the 
 availability of breeding sites directly influenced the dispersion of eggs by females . there was a significant difference ( p < 0.05 ) between the densities 2 * 4 , 2 * 8 , 2 * 16 
 and 4 * 16 both in laboratory and semi - field conditions ( fig . 5 ) . one female dispersed 
 the eggs among 11 breeding sites , the highest number observed in the study . skip 
 oviposition behaviour , although widely used , may not occur , as observed in eight ( 7.4% ) 
 females . 5:mean and standard deviation of breeding sites colonised on the basis of 
 available ones . different letters mean statistical difference ( kruskal - wallis p 
 < 0.05 ) . one - by counting the number of eggs at each breeding site , we noticed that 
 one of the sites usually received most of the eggs ( 40% or more of the total deposited 
 eggs ) . although all the breeding sites were identical and in semi - field and were not 
 statistically different from each other , it one site generally received more eggs than 
 the others . the amount of eggs that were deposited at the  favourite  site was similar in both 
 laboratory and semi - field conditions . the average percentage of eggs that were deposited 
 in the favourite site was significantly higher with a density of two breeding sites and 
 did not vary among the other densities ( fig . 6 ) , 
 showing that females tend to aggregate more eggs when there are only two available 
 breeding sites . this number can be reduced by an increase in the dispersion among 
 several breeding sites . there was a negative relationship between the number of 
 colonised breeding sites and the percentages of eggs that were laid at the favourite 
 breeding site ( simple linear regression y = -0.0682x + 0.9256 , f1,106 = 65.9 , 
 r = 0.38 , p < 0.001 ) ( fig . 7 ) . 
 however , even those females that used many breeding sites seemed to deposit at least 40% 
 of their eggs at the favourite breeding site , as shown by the dashed line ( fig . 7 ) . nevertheless , comparing the percentages of 
 eggs that were laid by the ae . aegypti females of the two groups ( above 
 and below the regression lines , fig . 8) , a 
 significant difference was observed in both semi - field ( t = -6.62 , p < 0.001 ) and 
 laboratory ( t = 2.87 , p = 0.001 ) ( fig . 7:percentage of eggs laid on favourite breeding site in relation to the 
 number of colonised breeding sites under laboratory and semi - field 
 conditions . 8:percentage of eggs laid in the favourite breeding site in laboratory by 
 aedes aegypti female s who had higher frequency of breeding 
 site colonisation ( a ) and smaller frequency ( b ) . percentage of eggs laid in the 
 favourite breeding site in semi - field by ae . aegypti female s 
 who had higher frequency of breeding site colonisation ( a ) and smaller 
 frequency ( b ) . 
  in other words , both environments ( laboratory and semi - field ) showed higher percentages 
 of eggs laid at favourite breeding sites by females those that distributed their eggs in 
 small numbers of containers . although all the breeding sites were identical and in 
 semi - field they were not statistically different from each other , one site generally 
 received more eggs than the others . the results that were obtained under laboratory and semi - field conditions were similar , 
 showing that the general aspects of the oviposition behaviour were maintained despite 
 the individual variations that were observed between the tested females ( wong et al . the variation in the number of 
 eggs per female is common in experiments that assess a single gravidae . these 
 variations are probably due to the blood - feeding efficiency ( xue et al . 2008 ) and the size of the adults , which may interfere 
 with the reproductive capacity of males and females ( blackmore & lord 2000 , ponlawat & 
 harrington 2009 ) . the average number of eggs was similar to that found by other authors ( christophers 1960 , madeira et al . as expected , the average number of eggs that were laid did not 
 significantly differ between densities ( 2 , 4 , 8 and 16 breeding sites ) or between 
 environments ( laboratory or semi - field ) . this result is probably due to the duration of 
 the tests , which were performed between the third - seventh day after the consumption of 
 blood meal , during which the females laid almost all of the eggs that were produced 
 ( gomes 2006 , reiter 2007 , chadee 2010 ) . it was demonstrated for the first time that the behavioural response of females depended 
 on the resources ( number of breeding sites ) that were provided to them . in an experiment 
 with eight available containers , chadee ( 2010 ) noted that 87% of females used one to four breeding sites , with a maximum of seven . in 
 our study , the number of containers that were used by the females increased following 
 the increased availability of breeding sites under both laboratory and semi - field 
 conditions . however , the number of breeding sites that were colonised seemed to 
 stabilise at around five , even when there were 16 breeding sites available and reached a 
 maximum of 11 under semi - field conditions . this result has epidemiological importance , 
 as the search for breeding sites seems to be a crucial factor in the dispersal of the 
 females and hence the diseases that they transmit ( edman 
 et al . the determination of the average and maximum 
 number of breeding sites that were used by each female can aid in the development of 
 methodologies for monitoring and controlling the vector . therefore , further studies 
 should be conducted to verify this behaviour in other environments and with different 
 densities of breeding sites , given that few females colonised more than eight 
 ovitraps . the results of the present study support the existence of the  skip oviposition aegypti females , as previously observed ( christophers 1960 , fay & perry 1965 , corbet & chadee 
 1993 , apostol et al . 2003 , reiter 2007 , 
 chadee 2010 ) . the ability of ae . aegypti females to distinguish potential breeding sites that will sustain 
 the survival of their offspring during their development is a crucial factor in the life 
 cycle of mosquitoes ( zahiri & rau 1998 ) . the 
 selective pressure in favour of the females that make choices that may maximise the 
 survival of their offspring ( reiter 2007 , harrington et al . nevertheless , this behaviour does not occur sometimes , as 
 observed in a few females in this study ( 7.4% of females ) and by other authors ( harrington & edman 2001 , chadee 2010 ) . the large proportion of eggs that were laid on water contradicts the findings of most 
 authors , who reported that the number of eggs that were deposited on water is negligible 
 compared to that deposited on the walls , filter paper or paddles of the breeding site 
 ( chadee & corbet 1987,chadee et al . however , a study in brazil demonstrated a large number of 
 eggs that were deposited on water by females of two populations and at different 
 humidities . the obtained figures were 42.9% and 57.3% ( 80% rh ) and 61.4% and 63.2% ( 51% rh ) for populations l and b , respectively ( madeira et 
 al . 2002 ) . these findings were similar to the observations of the present work 
 and almost 10 times higher than those reported by other authors ( chadee & cobert 1987 , chadee et 
 al . aegypti because females of the same population distributed their 
 eggs in different available proportions in oviposition substrates , as observed in our 
 study . considering the behavioural plasticity of ae . aegypti , it is possible 
 that the oviposition in both environments ( water or paddle / side walls of the breeding 
 site ) can be advantageous depending on the circumstance . in anthropic environments , females can lay eggs in a large range of ephemeral containers 
 that are very susceptible to disturbance ( reiter 
 2007 ) . this behaviour can make the choice of laying most of their eggs out of 
 water relevant in places that can only be achieved by increasing the level of the water 
 and can offer more chances of the larvae hatching and reaching adulthood . furthermore , 
 deposition on the walls of the breeding sites may be an example of  germ banking  for 
 the future of the ae . aegyptipopulation ( tsunoda et al . 2010 ) . in unfavourable conditions , the maintenance of 
  germ banking until the return of favourable periods may be more advantageous to the 
 offspring than the immediate eclosion of the larvae . on the other hand , the behaviour of laying eggs directly on the water is also relevant 
 to the vector from an epidemiological perspective , as it helps to maintain their 
 populations when there is no rainwater to supply breeding sites , thus allowing the 
 existence of floating populations of mosquitoes during the dry season ( gomes et al . the deposition of eggs on water can also prevent 
 predation by ants and cockroaches and the reduction of viability due to delays in 
 hatching ( madeira et al . this result could 
 perhaps explain why the number of eggs on the water was higher in semi - field conditions , 
 where the eggs would be more exposed to these attacks . the increase in the number of 
 eggs on water as observed in this study could also be explained by the abundance of 
 resources ( breeding sites ) . the quick hatching of the larvae would increase the chances 
 of the offspring finding the same available resources . however , to be considered a viable strategy , the eggs that were laid on the water 
 surface must have hatching rates similar to those that were laid outside of the water . the literature is controversial 
 on this issue because some authors have reported an extremely low hatching rate ( 2% ) 
 ( silva et al . 2003 ) , while others have found 
 higher rates , such as from 47 - 53% ( madeira et al . 2002 ) and from 73 - 80% ( rey & oconnell 
 2014 ) . therefore , we believe that the deposition of eggs on the water may be , 
 at least in certain cases , a good choice for the survival of ae . 
 aegypti . our results are also relevant in the context of the strategies of monitoring vector 
 populations . in brazil , ovitraps can be used to obtain the indices of infestation 
 ( ovitrap positivity index = number of ovitraps positive  100/number of ovitraps 
 inspected and egg density index = number of eggs / number of positive ovitraps ) by 
 counting the number of eggs on the paddle of the installed ovitraps ( gomes 1998 , ms / svs 
 2009 ) . in these surveys , the liquids ( water or infusion grass ) that are 
 present in the ovitraps are discarded without performing any type of analysis . however , 
 madeira et al . ( 2002 ) and the present study , 
 both conducted in brazil , found high amounts of eggs in the ovitrap water . additional studies should be 
 performed to determine if this trend is maintained in other populations of the country 
 and under field conditions . the present work demonstrated for the first time the existence of a favourite breeding 
 site and showed that egg distribution was not equal . the term favourite breeding site 
 was used to denote the breeding site that received the highest percentage of eggs . this 
 site was observed in semi - field and in laboratory conditions and with different amounts 
 of available breeding sites . the ae . aegypti females usually choose the most productive containers 
 to deposit most of their eggs . these containers are usually large , dark and unmanaged 
 ( maciel - de - freitas & loureno - de - oliveira 
 2011 , wong et al . 2012 ) . in the present 
 study , this behaviour was maintained even when all of the breeding sites were identical . either the females were able to 
 recognise clues that were insignificant to us or this was such an intrinsic behaviour 
 that it was maintained even when there was no advantage among the breeding sites . it is not known if the females lay their eggs in a single visit to the breeding site or 
 if the breeding site receives a portion of the eggs and then the female returns to the 
 same place and deposits eggs again . the observations on the proportion of eggs demonstrated the existence of two distinct 
 patterns : ( i ) females lay many eggs at one favourite breeding site ( 40% or more ) and 
 spread the remainder at other breeding sites and ( ii ) females lay few eggs ( less than 
 40% ) at the favourite breeding site and display a greater potential for spreading the 
 eggs remaining over other breeding sites . ( 2012 ) , who observed an increase 
 in the distribution of eggs in semi - field conditions when the highly productive 
 containers were removed . the first pattern was more frequently observed in our study , 
 although these two strategies can be important , depending on the condition of the 
 breeding site and the environment in which the mosquitoes live . the behaviour of females laying most of their eggs at the favourite breeding site may 
 favour the species in periods during which temporary breeding sites are scarce . this 
 behaviour can also be advantageous when a female finds high - productivity breeding sites 
 containing ideal conditions , such as large volumes and diameters , dark colouring and the 
 presence of co - specific larvae . this behaviour would indicate the capacity to support 
 large quantities of immature forms ( harrington et al . 2008 , maciel - de - freitas & 
 loureno - de - oliveira 2011 , wong et al . females that choose other strategies , spreading the higher proportion of their eggs and 
 depositing some at the favourite breeding site or spreading their eggs without 
 considering the favourite breeding site , can favour their offspring during the rainy 
 season , when there is a wide variety of breeding sites that are constantly supplied by 
 rain water . thus , the plasticity of behaviour that was observed in this study and in previous 
 studies ( madeira et al . the choice 
 of a favourite breeding site and the deposition of large percentage of eggs in water 
 were remarkable behaviours in the assessed population . aegypti ( madeira et al . 2002 , paduan et al . 
 2006 , hiragi et al . 2009 ) because they 
 inhabit a variety of environmental conditions and present short life cycles . aegyptimosquitoes contain populations of individuals of various origins that 
 have experienced different selective pressures . mosquito populations show great 
 adaptability , primarily because their larval stages develop in different seasons and 
 under different environmental conditions ( becker 
 1989 ) and because different selective pressures of the environment can lead to 
 populations presenting distinct characteristics and great genetic and behavioural 
 plasticity ( begon et al . 2011 ) . to determine whether the oviposition behaviour is the result of phenotypic plasticity or 
 intraspecific differences between different populations , studies are required . however , 
 regardless of the origin of behavioural plasticity in the oviposition ofae . . therefore , studies that elucidate the vector behaviour are important to the definition 
 of control measures and despite efforts , little is known about the oviposition 
 behaviour . thereby , it is extremely important that further studies investigate these 
 aspects , especially under field and different conditions . finally , it is also important 
 to understand the behaviour of mosquitoes that are used in the most modern and promising 
 control techniques , such as those infected with the bacteria wolbachia 
 and genetically modified mosquitoes .
despite the importance of the mosquito aedes aegypti in the 
 transmission of arboviruses , such as yellow fever , chikungunya fever and dengue 
 fever , some aspects of their behaviour remain unknown . in the present study , the 
 oviposition behaviour of ae . 
 aegypti   females that were exposed to 
 different densities of breeding sites ( 2 , 4 , 8 and 16 ) was evaluated in laboratory 
 and semi - field conditions . 
 the number of breeding sites that were used was 
 proportional to the number available , but tended towards stabilisation . 
 females used 
 four - six breeding sites on average , with a maximum of 11 . 
 a high percentage of eggs 
 was observed in the water , along with the presence of a breeding site termed 
  favourite  , which received at least 40% of the eggs . 
 the results are discussed in 
 ecological , evolutionary and epidemiological approaches .