94 Journal of Vector Ecology June2011

Cold season mortality and hatching behavior of Aedes aegypti L. (Diptera: Culicidae) eggs in Buenos Aires City, Argentina

SylviaFischer1,3,IrisSoledadAlem1,MaríaSolDeMajo1,RaúlErnestoCampos2,3,andNicolásSchweigmann1,3

1Grupo de Estudio de Mosquitos, Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina

2Instituto de Limnología “Dr. Raúl A. Ringuelet”, Universidad Nacional de La Plata - CONICET, Argentina3Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina

Received 27 June 2010; Accepted 26 December 2010

ABSTRACT: Intemperateregions,populationsofAedes aegyptisurvivethecoldseasonintheeggstage.Inthepresentwork,westudiedthecold-seasonmortalityofAe. aegyptieggsandtheirsubsequenthatchingpatterninBuenosAirescity.Eggswereexposedduringthewinterseason(threemonths)inthreeneighborhoodslocatedalongagradientofdistancetowardtheRíodelaPlataRiver,coincidentwithagradientofactivityofAe. aegypti. Resultsshowedmortalitieslower(30.6%)thanthosefromtropicalregionsduringthedryseason.SignificantdifferencesweredetectedamongtheeggmortalitiesofeachsitewithamaximumvalueatthesitenearesttheRíodelaPlataRiver(50%),andaminimumvalueatthemostcontinentalsite(9%).Post-experimentalhatchingresponseofeggsdifferedbetweensites,withthehighestproportionofhatchedeggsduringthefirstimmersioninthesitenearesttotheriverandthelowestproportioninthemostcontinentalsite.Thehatchingproportionalsodifferedbetweenageclasses,witholder(early-laid)eggshatchinglaterthannew(late-laid)ones.Ourresultsprovide the first information of Ae. aegypti egg mortality in temperate South America and support the hypothesis thatdifferencesineggmortalityareassociatedwithabundancepatternsofAe. aegyptiinBuenosAirescity.Journal of Vector Ecology 36 (1): 94-99. 2011.

Keyword Index:Populationdynamics,eggsurvival,hatching,winterdiapauses,temperateclimate.

INTRODUCTION

Aedes aegyptiL. (Diptera:Culicidae)hasaworldwidedistribution, ranging from tropical to temperate regions.This mosquito species is the main vector of dengue inAmerica, where the number of cases increases each year(Spiegel et al. 2005). Like other mosquito species withdrought-resistant eggs, Ae. aegypti populations surviveunfavorableperiodsintheeggstage(SotaandMogi1992).Intemperateregions,suchperiodsaredeterminedmainlyby the low winter temperatures, which may preclude thehatchingofeggsanddevelopmentoflarvae(Christophers1960).Therefore,survivaloftheeggsuntilthenextfavorableperiod is a key factor for the persistence of Ae. aegypti populations.Inspiteofthis, toourknowledge,nostudieshavebeenconductedon themortalityofAe. aegypti eggsundernaturalconditionsintemperateregions,andonlyafewhavebeenmadeintropicalconditions(e.g.,Trpis1972,Russelletal.2001).

After survival through the unfavorable season, egghatchingisthenextnecessarysteptoensurethecontinuityof the population cycle. It is known that the eggs of Ae. aegypti, like those of other species with drought-resistanteggs, do not necessarily hatch at the first stimulus(Gillett 1955, Vinogradova 2007). Although it has beendemonstrated that the differences in hatching time of Ae. aegyptieggsaredeterminedgenetically,acertainvariabilitycanbeobservedamongeggsof the sameeggbatch, even

amongthosesubjectedtosimilarenvironmentalconditions(Gillett 1955). It has been suggested for Aedes albopictus thatanincreaseintheriskofeggmortalitywouldincreasetheirsensitivitytoagivenhatchstimulus(VitekandLivdahl2006). The mortality risk may be higher when eggs areexposed to harsher environmental conditions, and undercomparableenvironmentalconditionsthisriskisexpectedtobehigherforoldereggs.

Buenos Aires is located near the southern limit ofthe worldwide distribution of Ae. aegypti. However, theabundanceofadultsofthismosquitospeciesishighduringthesummermonths(Schweigmannetal.2002).Giventhefactthatthepresenceofimmaturestagesandadultactivityare not detected during the cold season (Vezzani et al.2004),itcanbeassumedthatthepopulationsurvivesthisseasonintheeggstageandrestartsthecycleinspringwhenthetemperaturerisesaboveathresholdlevel(Schweigmannetal.2002,Vezzanietal.2004).

InBuenosAires,Ae. aegypti hasshownheterogeneouspatterns in its distribution. Ae. aegypti populationspresent in neighborhoods located towards the east (i.e.,nearer the Río de la Plata River) show lower ovipositionactivityonlyduringthesummermonths,whereasthoseinneighborhoodslocatedinnorthern,western,andsouthernareasofthecity(i.e.,theperiphery)havehigherinfestationwithovipositionactivityfromspringtofall(Carbajoetal.2006). The heterogeneous distribution of Ae. aegypti hasbeenrelatedmainlytotheurbanizationgradientinBuenos

Vol.36,no.1 Journal of Vector Ecology 95

Aires(Carbajoetal.2004,2006).Nevertheless,becauseofthe analysis scale used, it is most likely that these studieshave overlooked some differences in oviposition activitybetween sites with similar urbanization. For example,residential neighborhoods near the Río de la Plata Rivershow consistently lower oviposition activity than similarurbanized areas in the southwest side of the city. Suchdifferencescouldbeattributedtodifferentialeggmortalityduringthecoldseason,takingintoaccountthattheincreaseintheadultpopulationduringthenextreproductiveseasonisinitiatedbythesurvivingeggs.Furthermore,adifferentialhatchingpatternofeggsexposedtodifferentenvironmentalconditions could either increase or compensate for theeffectsofeggmortalityonthepopulationrecoveringduringthespringmonths.

Inthisstudy,wetestedthehypothesisthatareaswhereoviposition activity starts later in the season and attainslowermaximumvalues in thesummerexhibithighereggmortalityduringthecoldseason.Inaddition,weexploredthe hatching behavior of the eggs exposed to differentenvironmentalconditions.

MATERIALSANDMETHODS

Study areaBuenos Aires (34° 36 S and 58° 26’ W) lies on the

westernshoreoftheRíodeLaPlataRiver(Figure1).Thecityhasatemperatehumidclimatewithseasonallyvaryingtemperatures.Themeantemperatureinthecoldseasonis11.5°C,withthemeanhumidityinthewinterduringthelast ten years ranging between 74% and 79% (NationalMeteorologicalService2009).Thecitycoversanareaof200km2,hasapopulationofthreemillion,andissurroundedbyurbanandsuburbanareascovering3600km2withninemillionpeople(AtlasAmbientaldeBuenosAires2009).

Study sites were selected to represent the gradient ofovipositionactivitydescribedbyCarbajoetal.(2004).Weselected three residential neighborhoods with a similarconstruction type, predominantly single-family dwellingsof low height with backyard gardens and tree-shadedstreets. The sites were located in the northeast (site A, inthequarterofBelgrano),thewest(siteB,inthequarterofVillaDevoto),andthesouthwest(siteC,inthequarterofMataderos)ofthecity(Figure1).ApreliminarystudywithovitrapsduringthehighactivityseasonofAe. aegyptifromlatesummer(February)toearlyfall(April)confirmedtheexpecteddifferencesinovipositionactivity,with26%,42%,and51%ofinfestedovitrapsatsitesA,B,andC,respectively(unpublisheddata).

Experimental designEggs obtained from a city-wide monitoring program

withovitrapswereusedtostartacolonyofAedes aegypti.The breeding conditions in the laboratory were a 12:12photoperiod,andameantemperatureof22+1°C.Larvaewere fed with a solution of powdered baker´s yeast ad libitum, and, once pupated, transferred to a breeding boxwhere adults emerged. Once blood-fed, females were

transferred to a separate box, where approximately 40oviposition substrates (wooden paddles 9.5 x 1.8 cm,commerciallyavailableastonguedepressors)wereplacedincontainerswithwater.Thewoodenpaddleswerecollectedonce they accumulated at least 20 eggs and replaced bynew ones. This procedure (collection and replacement ofpaddles)wasrepeateduntilasufficientnumberofsubstrateswasobtained.Theweekofcollectionwasrecordedforeachpaddle,andeggsusedintheexperimentwereF1eggs,laidduring the first four consecutive weeks. Two age classeswere distinguished: eggs from paddles collected duringweeks1-2(early-laideggs)andeggsfrompaddlescollectedduringweeks3-4(late-laideggs).

Before starting the experiment, each paddle wasinspectedunderastereoscopicmicroscope,intacteggswerecounted,andalldamagedorcollapsedeggswereremoved.Allpaddleswithat least12eggs(mean31,range12-107)were used in the experiment, and paddles collected eachweekwereevenlyassigned to treatments.Theexperimentconsisted of exposing a known number of eggs in eachstudysiteforathree-monthperiod(July-September2008).Paddlescontainingthepreviouslycountedeggswereplacedin earthen pots (10 cm high x 10 cm diameter) with aplasticcoverattached0.8cmoverthetop,coveringthepotcompletelytopreventrainfromfallingintoitandeggsfromhatching(Figure2).Eachpaddlewasplacedinaseparatepotandfixedinaverticalpositionwithaclip.

Two treatments were differentiated (Figure 2). Oneconsisted of natural conditions as described above, and

Fig. 1: Location of the three study sites and two meteorological stations in Buenos Aires city, Argentina.

Fig. 2: Experimental pots; left: natural conditions, right: protected conditions.

Figure 1. Location of the three study sites and twometeorologicalstationsinBuenosAirescity,Argentina.

Figure2.Experimentalpots;left:naturalconditions,right:protectedconditions.

96 Journal of Vector Ecology June2011

one of protected conditions, where each pot was coveredwithafinemeshscreenofnylonontop(meshsizeof0.3mm)topreventaccessofmacroscopicorganisms.Eighteenreplicateswereplacedforeachtreatmentateachsiteatthebeginningofthewinterseason.Hourlytemperaturesateachsite were recorded with a HOBO® Pendant TemperatureDataLoggerplacedinapot.

All paddles were removed after 90 days (at thebeginning of spring), and transferred to the laboratory.Theeggsremainingineachpaddlewerecounted,andthenumber of completely collapsed, partially collapsed, orintact eggs recorded. All completely collapsed eggs wereremovedfromthepaddleandrecordedasdead.Tostimulatehatching,eggsweretransferredintowhitestyrofoamcupscontainingasolutionof40mgofpowderedbaker’syeastin168mldechlorinatedtapwater.Eachpaddlewasimmersedseparately,andaftertwotofourdaysthenumberofhatchedlarvae was counted. After four days, the paddles wereremoved from the water, and the immersion process wasrepeatedthefollowingweeks.Atotaloftenimmersionswerecarriedout,afterwhichthenumberofremainingunhatchedeggswasnegligible.Corionsofunhatchedeggsremainingon the paddles were bleached with a commercial 50%sodium hypochlorite solution to allow direct observationoftheembryos.Embryosthatwerecreamywhitewitheyespotsandpresentedahatchingspineanddistinctabdominalsegmentationwereconsideredviable.Yellow-brownorred-brown embryos were considered non-viable (McHaffeyand Harwood 1970). In this work, completely collapsedeggs and eggs with non-viable embryos were considereddead,whereaseggsthateitherhatchedorcontainedviableembryoswereconsideredalive.

Data analysesMean temperature and standard deviation were

calculatedbasedon thedatameasured.Temperaturedatawere compared between pairs of sites using the pairedt-test (Zar 1999). Humidity data were recorded in twometeorological stations of the National MeteorologicalServiceofArgentina:oneat the river side (station1)andtheotheratamorecontinentallocation(station2).Meanhourlypercenthumidityfrombothstationswascomparedusingthepairedt-test.

Egg mortality for each paddle was calculated bydividing thenumberofdeadeggsby the totalnumberofeggs recovered after the field exposure. Because on somepaddles either no eggs were retrieved or the number ofeggs was too low, we selected those paddles with morethan ten recovered eggs for this analysis. In order tomaintain a balanced design, randomly selected replicatesweredeletedfromthetreatmentswithexceedingpaddles,followingUnderwood(1997).Effectsofsiteandtreatmentwere analyzed by means of two-way ANOVA on arcsine-transformeddata.Toassesssignificantdifferences,post-hoccomparisonsweremadewithTukey´s(HSD)test.Averageand 95% confidence intervals were obtained for each sitefrom the transformed data. To express these in terms ofproportions, the inverse transformation was applied (Zar

1999).The hatching response of eggs was determined

analyzingtheintensityoftheresponseaftereachimmersionandthecumulativeresponsetothesuccessiveimmersions.The intensity of the hatching response was assessed bycalculatingtheconditionalproportionofhatchedeggs:thenumber of larvae observed on each occasion divided bythenumberofremaininglivingeggsbeforetherespectiveimmersion(eggswereconsideredaliveeitheriftheyhatchedafter any stimulation or if they contained viable embryosafterthebleaching).Theproportionsofhatchedeggsateachimmersionwerecomparedbetweencategoriesofexposuresiteandbetweenageclassesbymeansofachisquaretestfor independent proportions. This test is comparable tocomputingthePearsonchisquarestatisticforcontingencytables. Differences between pairs of sites were examinedby subdividing thecontingency tablesandcomputing thechisquarevalueonthepartialtables(Fleissetal.2003).Tostudy the cumulative hatching response, the accumulatedproportion of hatched eggs was calculated (sum of eggshatchedatthepreviousandpresentimmersiondividedbythe total number of living eggs). Results were plotted foreachsitebyageclasscombination.

The proportion of eggs that could not be recoveredafterthefieldexposure(missingeggs)oneachpaddlewascalculated as the difference between the number of eggsinitially present on the paddle and the number of eggsrecovered after the three-month exposure, divided by thenumber of eggs initially present on that paddle. For eachtreatmentandsite, themeannumberofmissingeggswascalculated from the proportions obtained for individualpaddles.Becausedifferenttransformationsdidnotmeetthenecessaryassumptions,anon-parametrictwo-wayANOVAwasperformedusingtheSheirer-Ray-Haremethod(Sokaland Rohlf 1995). The factors considered were: site (threelevels) and treatment (two levels). All 18 replicates wereincludedinthepresentanalysis.

RESULTS

The mean temperature values were 13° C, slightlyhigher at site A (13.08 ± 2.87° C), intermediate at siteB (13.05±4.5°C), and lowerat siteC (12.96±3.33°C).Statistical comparisons showed significant differencesbetweensiteAandsiteC(t=4.89,d.f.=1,799,p<0.001).The relative humidity was higher at the riverside station(76.73 ± 15.90%) than at the continental station (74.56 ±16.54%),anddifferencesweresignificant(t=10.54,d.f.=2,201,p<0.001).

Atotalof3,079(87.3%)outoftheinitial3,525eggswasrecoveredattheendoftheexperiment.Amongthem,943(30.6%)didnotsurvivethewinterseason.Theproportionofeggmortalityafterthreemonthswassignificantlyhigherat sites A and B than at site C (Figure 3). The statisticalcomparisondetectedsignificantdifferencesineggmortalitybetween sites (F = 13.82, d.f. = 2, p < 0.001), while nodifferencesweredetectedbetweenexposureconditions,eggage,andinteractionterms.

Vol.36,no.1 Journal of Vector Ecology 97

Of the 2,136 viable eggs, 2,127 hatched during theten immersions and only nine remained unhatched. Theoverall proportion of hatched eggs was highest duringthe first (0.79) and second (0.65) immersions, and lowestduringthefifth(0.10).Theremainingimmersionsinducedintermediatehatchingrates.

The intensity of hatching was highest at the firstimmersion and decreased progressively through the fifth,after which a new increase was observed (Figures 4a,b).Theageofeggsshowedasignificantrelationshipwiththepatternofhatching(Figure4a),withasignificantlyhigherproportionoflatelaideggshatchingduringthefirst(χ2=53.67,df=1,p<0.001),second(χ 2=13.73,df=1,p<0.001),and third (χ 2 = 12.27, df = 1, p<0.001) immersions. Asignificantly higher proportion of early laid eggs hatchedduringtheninthimmersion(χ 2=6.54,df=1,p<0.05).Theeggsexposedatthedifferentsitesalsoshowedadifferenceinthehatchingpattern(Figure4b).Differencesinhatchedeggsweresignificantbetweensitesforthefirst(χ2=31.64,df=2,p<0.001),thefifth(χ 2=6.07,df=2,p<0.05),andthe seventh immersion (χ 2 = 8.54, df = 2, p<0.05). TheproportionofeggshatchedduringthefirstimmersionwashighforeggsexposedatsiteA,intermediateforthoseatsiteB,and lowfor thoseat siteC.Theoppositewasobservedduring the fifth immersion, when eggs exposed at sitesA and B attained lower hatching proportions than thoseexposedatsiteC(Figure4b).

The comparison of the cumulative hatching curvesofthesixcombinationsofsitebyageshowedaconsistentpattern of early laid eggs hatching in later immersions(Figure 5). Hatching showed the highest heterogeneitybetween site and age combinations during the firstimmersion. The cumulative proportions of hatched eggsincreased faster for early laid eggs than for late laid eggs,with more than 97% of late laid eggs hatched after threeimmersions,andsimilarnumbersofearlylaideggshatchedaftersevenormoreimmersions.

Egglosswasrecordedinalltreatments.Approximately50% of the paddles from the treatments with protectedconditionshadmissingeggs,whilepercentagesofpaddles

withmissingeggsinthetreatmentswithnaturalconditionswere67%,72%,and79%insitesA,C,andBrespectively.Theproportionofmissingeggsshowedsignificantlylowervalues in the treatments with protected conditions (H =5.961,d.f.=1,p<0.02) (Figure6).Nosignificanteffectsofthestudysiteortheinteractionofsitevsexclusioneffectweredetected.

DISCUSSION

ColdseasoneggmortalityinBuenosAireswaslowerthanduringthedryseasonsintropicalregions.InTanzania,

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Fig. 4: Conditional pProportion of hatched eggs of Aedes aegypti after ten successive immersions for two egg classes (a) and three sites (b). Error bars indicate 95% confidence intervals. * indicate significant differences by the chi square test. Columns with dotted lines indicate proportions calculated from less than 10 remaining living eggs at the respective immersion.

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Fig. 5: Cumulative proportion of hatched eggs of Aedes aegypti after ten successive immersions for each age class by site combination.

Figure 3. Mean and confidence intervals (95%) for theproportionofeggmortalityofAedes aegypti at threesitesinBuenosAirescity.DifferentsymbolsindicatesignificantdifferencesdetectedbyTukeytest(p<0.05).

Figure5.CumulativeproportionofhatchedeggsofAedes aegyptiaftertensuccessiveimmersionsforeachageclassbysitecombination.

Figure4.ConditionalproportionofhatchedeggsofAedes aegyptiaftertensuccessiveimmersionsfortwoeggclasses(a)andthreesites(b).Errorbarsindicate95%confidenceintervals. * indicates significant differences by the chisquaretest.Columnswithdottedlinesindicateproportionscalculated from less than tenremaining livingeggsat therespectiveimmersion.

98 Journal of Vector Ecology June2011

East Africa, the mortality of eggs after three months wasfound to be 65% (Trpis 1972), while in Queensland,Australia, the mortality of eggs after the same periodreached86%(Russelletal.2001).

The most likely explanation for the lower mortalitiesobtainedinourstudyisthatclimaticand/orenvironmentalconditionsaresignificantlymore favorable forAe. aegyptieggs at the study area during the cold season than thoseobserved at tropical regions during the dry season. ThecoldseasoninBuenosAirescitytheyearofourstudywascharacterizedbyrelativelyhighhumidity(>70%)comparedto that in other studies (30%), and lower temperatures.LaboratorystudieshavedemonstratedthatthesurvivalofAe. aegyptieggsisnegativelyaffectedbylowenvironmentalhumidity (Juliano et al. 2002). On the other hand, alaboratoryexperimentundersaturatedhumidityconditionsshowedaslightdeclineinthesurvivalofeggsat10°Cascompared to eggs kept at 16° C (Meola 1964), indicatingthatcoldseason temperatures inBuenosAiresarealmostoptimalforthesurvivalofAedes aegyptieggs.

TheresultsofthepresentstudyshowaheterogeneousmortalityofAe. aegyptieggsamongsitesinBuenosAires.EggmortalitywashigheratsiteA,wherelowerovipositionactivity occurred during the warmer months. Theseresults support the hypothesis that the lower and delayedovipositionactivityinsomeareasneartheRíodelaPlataRivercouldbeaconsequenceofareducedeggbankattheend of the cold season, with fewer surviving individualsto start thepopulation increaseduring thenext favorableseason. Since only slight differences in temperature andhumidity were recorded between sites, there are probablyotherfactorsinvolvedinthemortalitydifferences.

The differences in hatching behavior related to theexposuresiteandeggageprovidedresults thatencouragefurtherstudiesinthisdirection.Thedifferencesinhatchingresponsebetweentheeggsexposedatthedifferentsiteswereconsistentwithdifferencesinmortality.SiteAwithhighermortalityshowedahigherhatchingproportionduringthefirst immersion,whereassiteCshowedtheopposite.Onepossible explanation for such results would be that eggssubjected to a higher mortality risk will hatch as soon aspossibleinordertoavoidorreducetheriskofdyingattheegg stage. Observations in this direction have been made

for different taxonomic groups such as some lizard, fish,spider, andamphibianspecies (WernerandGilliam1984,Gomez-MestreandWarkentin2007).Whetherornot thepositive relationship of both phenomena (egg mortalityand hatching rates) is related to a third one (i.e., depthof diapause as defined by Gillett (1955) deserves furtherresearch.StudiesonvariousotherAedes(Stegomyia)specieshaveshownhighersurvivalofdiapauseeggsthanofnon-diapauseeggs(SotaandMogi1992),whichmightsupportthishypothesis.

The negative association of hatching with age of theeggsisconsistentwithearlyqualitativeobservationsmadeby different authors (reviewed by Christophers 1960)whohave suggestedadelayedhatching ineggs stored forprolonged periods. Such behavior could be related to thewater loss experienced, even when stored in saturatedenvironments(Meola1964).Nevertheless,oppositeresultswere obtained for Ae. albopictus, which showed higherhatchingratesforoldereggs(VitekandLivdahl2006).VitekandLivdahl(2006)alsorelatedthesehigherhatchingratesof older eggs to a response to minimize the risk of deathwith increasing age. Since Ae. aegypti eggs exhibit highersurvivalratesthanAe. albopictuseggs(Julianoetal.2002),olderindividualsoftheformerspeciesmaynotnecessarilybeobligedtohatchatthefirststimulus.

An unexpected result of the experiment was theloss of eggs in both treatments of protected and naturalconditions. This suggests the possibility of a spontaneousdetachment of the eggs from the substrate (ranging invalues from 3% through 13%) during the cold months.Unfortunately, the experimental pots were not inspectedto recover eggs that could have fallen to the bottom, andwe do not know whether those eggs were living or dead.Whetherthedetachedeggsfinallydieorareabletohatchwithoutrainfalleventswhenfallingintothewaterrequiresfurtherresearch.Ontheotherhand,thenumberofmissingeggswasconsistentlyhigherinthetreatmentwithnaturalconditions than in that with protected conditions. Thesedifferences could be at least partially attributed to theaction of predators, although no direct observations weremade.Resultsinthisdirectionwereobtainedinstudiesofother regions (i.e., Russell et al. 2001, Focks et al. 1993),wherepredatoractivitysubstantiallyincreasedthenaturalmortalityofeggsofAe. aegypti.Sinceants(Leeetal.1994),coleopterans(Yang2006),cockroaches(Russelletal.2001),andpillbugs(Focksetal.1993)havebeenreportedtopreyoneggsofAe. aegyptiorrelatedspecies,theyconstitutethefirstcandidatestobeevaluatedinlaboratoryexperiments.

Acknowledgments

WearegratefultotheauthoritiesoftheInstitutoPasteur,BuenosAiresCityGovernmentforprovidingthefacilitiesforthemaintenanceofacolonyofAedes aegypti.Thisworkwas supported by the Agencia Nacional de PromociónCientíficayTecnológica(GrantPICT2006Nº01258),andby the Universidad de Buenos Aires (UBACyT X210 andX617).WewanttothankDr.ToddLivdahlandDr.Hernán

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Fig. 6: Mean proportions of missing eggs of Aedes aegypti under two experimental conditions (NC: natural conditions, PC: protected conditions) at three sites (A, B, C).

Figure6.MeanproportionsofmissingeggsofAedes aegyptiundertwoexperimentalconditions(NC:naturalconditions,PC:protectedconditions)atthreesites(A,B,C).

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Solari for theirhelpfulcommentson thismanuscript.Wealso are grateful to the two anonymous reviewers whosesuggestionsgreatlyimprovedourmanuscript.

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