aegypti , rockefeller 
 strain , were provided by dr jos bento pereira lima [ brazilian army biology institute , 
 oswaldo cruz foundation ( fiocruz ) , state of rio de janeiro ( rj ) , brazil ] . they hatched 
 in the rearing water after about 1 h and groups of 500 newly hatched larvae were placed 
 in plastic bowls ( 25 x 15 x 7 cm ) containing 500 ml of dechlorinated water and 500 mg of 
 fish feed flakes ( tetramin tropical flakes ) . the larvae were kept in 
 incubators bod at 26  1c until third instars emerged after approximately 72 h. 
 plant material - flowers from male and fruits from female c. 
 fluminensis were collected , with the permission of the brazilian army , in a 
 rocky outcrop at forte baro do imbuhy , in the city of niteri , rj . flowers and fruits 
 were available in the summer of 2006 and autumn of 2007 , respectively . the plant 
 material was identified by dr marcelo guerra santos and a voucher specimen was deposited 
 at the herbarium of the faculty of teacher education , rio de janeiro state university 
 ( licence 9213 ) . preparation of plant extracts - fruits of c. 
 fluminensiswere dried in an oven at 40c and subsequently fragmented while 
 whole flowers were used while still partially fresh . the crude extracts were prepared by 
 macerating plant organs with analytical grade hexane as a solvent at room temperature . chemical analysis of crude extracts - gas chromatography coupled with mass 
 spectrometry ( gc - ms ) - the crude extracts obtained from flowers ofc . flumi - nensis were analysed by gc - ms performed on an agilent model 6890n gas 
 chromatograph equipped with a mass selective detector , model 5973n . a volume of 1 l of 
 the solution with a concentration 1 mg / ml was injected . the following conditions were 
 utilised : carrier gas , helium at 2.0 mlmin , split ratio 5:1 , injector 
 temperature 300c and ion source temperature 230c . the oven temperature was programmed 
 from 150 - 300c at 10c min and held at 300c for 15 min . sample injection 
 volume was 1 l . the mass spectra of the major components of c. 
 fluminensis flowers were analysed and compared with the wiley mass spectral 
 databases and the literature data . 
 nuclear magnetic resonance ( nmr ) analysis  the nmr spectra were 
 acquired using a bruker drx 400 spectrometer operating at 400 mhz for h and 
 100 mhz for c. chemical shifts were measured relatively to an internal 
 tetramethylsilane reference . spectral data of clusianone was published for our group 
 previously in silva et al . the structure of lanosterol was confirmed by ms 
 compared to the equipment s database ( wiley database ) and nmr . isolation of clusianone - the polyisoprenylated benzophenone , 
 clusianone , was isolated as a tautomeric pair from the crude hexanic extract of flowers 
 of c. flumi - nensis by countercurrent chromatography using the solvent 
 system n - hexane - acetonitrile - methanol ( 2:1.25:0.5 , v / v / v ) , as described by silva et al . ( 2012 ) . aegypti larvae were used three 
 days after hatching and were formed into groups of 10 larvae in the third instar with 
 six replicates . the groups were placed in 300 ml disposable plastic cups containing 50 
 ml of dechlorinated water . the cups were then randomly allocated to groups , with the aid 
 of the pwgen ( windows phone ) program . the dried extracts and isolates were weighed and 
 solubilised with 0.1% dimethyl sulfoxide ( dmso ) to make up to 50 l , with a final 
 concentration of 50 mg / l in the water test . these substances were thoroughly mixed in 
 the water test to minimise the possibility of adhesion of particles to the hydrophobic 
 plastic surfaces . the control groups were untreated and the solvent controls ( sc ) were 
 treated solely with 0.1% dmso used to dissolve the samples . during the tests , larvae 
 were fed with 30 mg per cup of hamster food ( alcon , brazil ) , immediately 
 after completion of the tests and three days later . all experiments were followed until the death or emergence of all insects in control 
 group . data and statistical analysis - all graphs were created with graphpad 
 prism 6.01 software ( graphpad software , usa ) . the graphics analysed the survival ( and 
 mortality ) and the developmental course of juvenile forms and adults . the 
 gehan - breslow - wilcoxon test compared the whole survival curves and larval and adult 
 development ( graphpad prism 6.01 software ) . no corrections for multiple comparisons were 
 made ( rothman 1990 ) . the barnard s test ( barnard 
 package v.1.3 ) ( erguler 2012 , r development core team 2014 ) was used to analyse 
 the development of ae . aegyptipupae , comparing the mean values only in 
 a representative day indicated by an arrow in graphs . in all experiments , aegypti were provided by dr jos 
 bento pereira lima from insects treated according to the ethical principles in animal 
 experimentation approved by the ethical committee in animal experimentation ( fiocruz ) . the protocol is from national council for the control of animal experimentation / ministry 
 of science , technology and innovation ( cobea.org.br ) , which is associated with the 
 american association for animal science , federation of european laboratory animal 
 science associations , international council for animal science and association for 
 assessment and accreditation of laboratory animal care international . the funders , from brazilian govern , had 
 no role in study design , data collection and analysis , decision to publish or 
 preparation of the paper . the bioassays with the hexanic extract of c. flumi - nensis fruit showed 
 that in the treated groups there were no effects on the survival rates or development of 
 ae . 1:effects of extracts of fruit from clusia fluminensis 
 ( fr ) on survival ( a ) and development of larvae ( b ) , pupae ( c ) and adult ( d ) of 
 aedes aegypti at different days after experimental 
 treatment ( fr ) compared with the solvent control ( sc ) that was compared with 
 untreated control ( uc ) . each point represents the mean of at least six 
 replicates with 10 insects and bars show standard error . statistical analyses ( under the graphs ) with the barnard s test were 
 used ( c ) on a representative day indicated by an arrow and the 
 gehan - breslow - wilcoxon test was used ( a , b , d ) to compare the entire curve 
 between the groups ( fr x sc and sc x c ) . significant differences ( p value < 
 0.05 ) are in bold . 
  the bioassays with the hexanic extract of c. flumi- nensis flowers ( fl ) 
 showed no significant effect on ae . aegypti survival in comparison with 
 the sc ( p = 0.1797 ) ( fig . in contrast , the 
 analyses of the effects of fl on the development of larvae to pupae and pupae to adults 
 showed significant delays in molting ( p  0.0001 ) between the fl - treated and sc groups 
 ( fig . thus , on the fifth day , 88.3% of 
 the sc group had molted to pupae , while 93.3% of the fl group were still in the larval 
 stage ( fig . furthermore , on day 7 , 81.7% of the sc had reached adults , while none of 
 the fl group had reached this stage ( fig . 2:effects of extracts of flower from clusia fluminensis 
 ( fl ) on survival ( a ) and development of larvae ( b ) , pupae ( c ) and adult ( d ) of 
 aedes aegypti at different days after experimental 
 treatment ( fl ) compared with the solvent control ( sc ) that was compared with 
 untreated control ( uc ) . the assays were executed and analysed as in the fig . 1 
 legend and materials and methods . 
  since only the flower extract affected the development of ae . the chromatogram obtained for the hexanic extract of 
 flowers of c. fluminensis showed that the major component ( retention 
 time : 18.424 min ) was the benzophenone , clusianone . this substance was identified by 
 comparison of the mass fragmentation pattern with the data provided by delle monache et al . in addition , 
 lanosterol was also detected ( retention time : 19.155 min ) in the same extract and 
 contributed to 9.74% of the sample composition ( fig . 3:chromatogram obtained for the hexanic extract of male flowers 
 ofclusia fluminensis by gas chromatography coupled with 
 mass spectrometry showing the benzophenone / clusianone and the triterpene 
 lanosterol as major components . 
  the purification protocols and chemical structures of clusianone from the flowers 
 ofc . 4a ) , so 
 that by the first day of treatment 20% of larvae had already died compared with the sc 
 group ( fig . 4a ) . in the clusianone - treated group , 
 by the last day ( 12th ) of treatment , 93.3% of insects were dead , while more than 93% 
 were still alive in the sc group ( fig . 4:effects of clusianone ( cl ) isolated from clusia 
 fluminensis on survival ( a ) and development of larvae ( b ) , pupae 
 ( c ) and adult ( d ) of aedes aegypti at different days after 
 experimental treatment ( cl ) compared with the solvent control ( sc ) that was 
 compared with untreated control ( uc ) . the assays were executed and analysed as 
 in the fig . 1 legend and materials and methods . in contrast with the effect of clusianone , no activity was detected by the isolated 
 lanosterol on the survival or development of ae . in this present study , clusianone was further identified and purified from fl , 
 following the original protocols described by silva et al . clusianone is the major component from the flowers of 
 c. fluminensis , representing almost 55% of the crude extract 
 composition . crude fruit extracts of c. fluminensis had no activity on the survival 
 or development of ae . aegypti and preliminary results showed that 
 clusianone is not present in these extracts , in which lanosterol is the main compound . in addition , the purified clusianone not only caused high larval mortality and 
 delayed development , but also completely blocked the molting of surviving 
 treated - insects . firstly , the value of 
 analysing not only mortality , but also the effect on development in the evaluation of 
 plant extracts activities against insects . secondly , that fractionated and purified 
 components may show enhanced activities in bioassays in comparison with those of crude 
 extracts . thirdly , the necessity of assaying different regions of the plant tissue for 
 activity , when testing plant extracts for insecticide properties . fourthly , they report 
 the first description of clusianone s larvicidal activity against ae . the clusianone effects , however , seem to have similar characteristics to those found 
 with synthesised benzophenone tagged indole derivatives that resulted in mortality in 
 culex quinquefasciatus larvae ( ranganatha et al . 2013 ) , as well as those reported for extracts from species 
 of hypericum against culicids ( cetin et 
 al . a fraction of h. carinatumenriched with the 
 benzophenones , cariphenone a and cariphenone b has also shown to have larvicidal 
 activity against ae . aegypti , but at a lower concentration than that 
 used in our experiments with clusianone ( da silva et al . 2013a ) . the genushypericum is often considered to be a member 
 of the family clusiaceae . benzophenyl and benzopyran compounds , although not benzophenones , have also recently 
 been reported to have similar effects to clusianone against mosquitoes . for example , 
 benzophenyl urea compounds may disrupt chitin , causing changes in development or even 
 mortality in culex and aedes mosquitoes in low doses 
 ( fontoura et al . additionally , benzopyrans , present mostly in a 
 hypericum polyanthemum lipophilic extract , showed larvicidal 
 activity against ae . aegypti in high concentrations , while in sublethal 
 levels exhibited growth - inhibiting activity to prevent pupae formation and adult 
 emergence ( da silva et al . this result is very important for the evaluation of the potential 
 use of these compounds as insecticides , because ae . aegypti treated 
 with a successful commercial analogue of jh ( methoprene ) also remained as larvae or died 
 as pupae , without emerging as adults ( braga et al . 2005a , b ) . finally , the tetranortriterpenoid , azadirachtin , purified from azadirachta 
 indica a. ( juss . ) ( meliaceae ) acts on the insect neurosecretory cells . juvenile forms after contact with this product remain irreversibly in the larval stage 
 ( garcia et al . 1986 , mulla & su 1999 ) , as similarly observed after ae . 
 aegypti treatment with clusianone isolated fromc . likewise , benzophenone-3 used in uv filters affects 
 embryogenesis by delaying hatching and mimicking the action of ecdysone , thereby 
 disrupting the insect endocrinological equilibrium ( ozez et al . 2014 ) . therefore , evidence from previous studies and for 
 clusianone indicates that many naturally - occurring plant insect control agents affect 
 the pest endocrine homeostasis to kill , delay or interrupt the development of their 
 target species . in conclusion , the present research shows , for the first time , the insecticidal activity 
 of the benzophenone , clusianone , against mosquitoes and represents a promising start for 
 the development of natural plant products for the control of insect vectors of tropical 
 diseases .
studies evaluated the effects of hexanic extracts from the fruits and flowers 
 ofclusia fluminensis and the main component of the flower 
 extract , a purified benzophenone ( clusianone ) , against aedes 
 aegypti . 
 the treatment of larvae with the crude fruit or flower extracts 
 from c. fluminensis did not affect the survival ofae . 
 aegypti ( 50 mg / l ) , however , the flower extracts significantly delayed 
 development of ae . 
 aegypti . 
 in contrast , the clusianone ( 50 mg / l ) isolate from the 
 flower extract , representing 54.85% of this sample composition , showed a highly 
 significant inhibition of survival , killing 93.3% of the larvae and completely 
 blocking development of ae . 
 aegypti . 
 the results showed , for the first time , high 
 activity of clusianone against ae . 
 aegypti that both killed and inhibited mosquito 
 development . 
 therefore , clusianone has potential for development as a biopesticide 
 for controlling insect vectors of tropical diseases . 
 future work will elucidate the 
 mode of action of clusianone isolated from c. fluminensis .