
 mosquitoes - the mosquitoes used in this study were from a laboratory 
 colony of ae .
aegypti ( rockefeller strain ) maintained at institute of 
 biology of the brazilian army , state of rio de janeiro , brazil .
for all experiments , 
 they were pre - synchronised from pupa stage to 12 h of light and 12 h of dark ( ld 12:12 ) 
 at 25c  0.5c in a precision scientific incubator mod .
818 and newly emerged females 
 were separated from males while still virgins and maintained at the same conditions . for experiments with blood - fed females , four - five - days
old females were fed on 
 anesthetised guinea pig ( ketamine : xylazine 80 - 120:10 - 16
mg / kg ) for 1 h , according to 
 institutional procedures , oriented by the national guideline the brazilian legal 
 framework on the scientific use of animals ( filipecki 
 et al .
samples for blood - feeding and insemination experiments - virgin females 
 were initially separated in two groups of 200 individuals . to one group
we added 300 
 males for insemination for two days ; the other group remained virgin .
all mosquitoes 
 were provided with 10% glucose solution and placed under the same conditions used to 
 synchronise the pupae .
the inseminated females were separated from the males and split 
 into two groups of 80 females .
virgin females were also split in two groups of 80 
 individuals . on the same day ,
5 h after lights - on or at zeitgeber - time ( zt ) 5 , one cage 
 of virgin females and one cage of inseminated females were provided with a source of 
 blood meal ( guinea pig ) for 1 h. after this procedure , there were four distinct groups 
 of ae .
aegypti females : ( i ) virgins / unfed , ( ii ) inseminated / unfed , 
 ( iii ) virgins / blood - fed and ( iv ) inseminated / blood - fed . to allow one day entrainment 
 after blood meal , we collected the females at zt11 of the following day ( 11 h after 
 lights - on and ~30 h after the blood meal ) , a time point of the average expression of 
 most clock genes ( gentile et al .
all 
 mosquitoes were frozen in liquid nitrogen and each of the four groups was separated into 
 five samples of 10 individuals each . in the case of the two blood - fed groups ,
only 
 females with visual evidence of blood intake ( colour and volume of the abdomen ) were 
 considered .
we beheaded the mosquitoes on a metal plate placed on dry ice to ensure rna 
 integrity .
therefore , the five replicas of each group of females generated two 
 sub - samples : ( i ) 10 heads and ( ii ) 10 beheaded bodies .
rna interference ( rnai ) experiments - we used the rnai technique to 
 achieve the knockdown of tim expression and evaluate its effects on the 
 activity of ae .
2006 ) were amplified and cloned into   pgem -t easy 
 vector ( promega ) according to the manufacturer s protocol .
the plasmid containing the 
 cloned tim sequence fragment was used in a polymerase chain reaction 
 ( pcr ) with oligonucleotides complementary to tim and containing the 
 sequence of t7 promoter ( underlined ) in their 5 end : 5 
 -taatacgactcactataggg agaagcggagaaggatggaatg-3 ( forward 
 primer ) and 5 -taatacgactcactataggg agatggaaggaagtgttatgattgg-3 
 ( reverse primer ) , generating an amplicon of about 590 bp .
the product of the reaction 
 was purified with microspin s-400 hr columns ( ge healthcare life sciences ) and used in 
 the synthesis of double - stranded rna ( dsrna ) with megascript ( life technologies ) , 
 according to the manufacturer s protocol .
the dsrna was purified with megaclear ( life 
 technologies ) and quantified in a spectrophotometer .
the same procedure was conducted to 
 generate a dsrna of lacz gene , used as a control , using the primers 5 
 -taatacgactcactataggg agaatccgacgggttgttact-3 ( forward ) and 
 5 -taatacgactcactataggg agacaccacgctcatcgataattt-3 
 ( reverse ) .
the dsrna was injected at a concentration of 3 g/l into two - three days old virgin 
 ae .
aegypti   females using femtojet micro - injector ( eppendorf ) , 
 adjusted to 200 hectopascal and for 0.5 sec .
the mosquitoes were temporarily paralysed 
 by cooling , placed on a metal plate ( on ice ) and injected in the mesothorax with the 
 dsrna solution ( garver & dimopoulos 2007 ) . 

four hundred virgin females were injected , half with dsrna for tim ( 
 dstim ) and half with dsrna for lacz   ( 
 dslacz ) .
after the injection , the mosquitoes were transferred to 
 cages provided with 10% glucose solution and placed in the precision scientific 
 incubator ( mod .
after a couple of hours of 
 recovery , 32 females from each of the four groups were prepared for the behavioural 
 assay , while the remaining mosquitoes were kept in the incubator under the same 
 conditions used for the behavioural assay and later collected for gene expression 
 analysis .
two independent experiments ( called  ld  and  dd  ) were performed , differing on the 
 light regime in which the females were kept after the injections . in the ld experiment , 

the same regime of the pre - synchronization of ld 12:12 for seven days was used ; in the 
 dd experiment they were kept under ld 12:12 for two days followed by constant darkness 
 for five days .
the samples for gene expression analysis were collected in five replicas 
 with four mosquitoes each on the fourth day after the injection , on the zt11 in the ld 
 experiment ( 11th h after lights - on ) and on the circadian time 11 ( corresponding to the 
 11th h of the  subjective  day ) in the dd experiment . as before , mosquitoes were 
 beheaded on a metal plate and placed on dry ice to ensure rna integrity , generating 
 independent sub - samples of heads and beheaded bodies .
gene quantification was performed 
 with quantitative real - time pcr ( qrt - pcr ) . 

rna extractions and cdna synthesis - total rna was extracted with 1 ml 
 trizol ( life technologies ) , purified with lithium chloride 2.5 m and resuspended in 
 rnase - free water .
cdna was synthesised with taqman reverse transcription reagents ( life 
 technologies ) using oligo - dt in a final volume of 20 l , according to the manufacturer s 
 protocol and temperature conditions of 25c for 10 min , 48c for 1 h and 95c for 5 min . 

the cdna used for qpcr assays was diluted in mili - q water in a 1:9 proportion . 

qrt - pcr - relative quantification of gene expression was conducted 
 using qrt - pcr , performed in abi prism 7000 sequence detection systems ( applied 
 biosystems ) and using power sybr green pcr master mix ( life technologies ) , 
 oligonucleotides at a final concentration of 0.5 m and 4.0 l of cdna , in a final 
 reaction volume of 20 l .
reactions were performed in four replicas for each sample and 
 independently for the clock genes per , tim , 
 cyc and clk plus the endogenous control 
 rp49 , using the same oligonucleotides designed for a previous work 
 ( gentile et al .
the relative 
 quantification of the gene expression was determined using the compara- tive cycle 
 threshold ( ct ) method , also known as the ct or 2 ( pfaffl 2001 ) .
analysis of the data was carried out 
 using microsoft excel software . for more details
aegypti were recorded automatically using a larger version of 
 the drosophila activity monitoring system ( trikinetics ) as previously described ( gentile et al .
two independent behavioural experiments were conducted : one under ld 12:12 for 
 seven days ( ld experiment ) and
the other under ld 12:12 for two days followed by 
 constant darkness for five days ( dd experiment ) .
we used actogramj to analise the actograms and the  
 periodograms ( schmid et al .
statistical analysis - the student t test and anova 
 analysis were performed using spss v.17 ( ibm 2008 , chicago , usa ) .

 effect of blood - feeding and insemination on clock gene expression - we 
 tested the effect of blood - feeding and insemination on the expression of the clock genes 
 per , tim , cyc and 
 clk , both in the head and body of ae .
aegypti 
 females , comparing four groups of mosquitoes in distinct physiological states : ( i ) 
 virgin / unfed , ( ii ) inseminated / unfed , ( iii ) virgin / blood - fed and ( iv ) 
 inseminated / blood - fed .
analysis of variance of two factors ( 2-way anova ) was performed 
 to determine the significance of the effect of insemination , blood - feeding and the 
 interaction of these two factors on the expression of the clock genes investigated .
the 
 results demonstrate blood - feeding alone is the only factor to cause consistent and 
 significant impact on the gene expression .
therefore , for the graph representation , we 
 joined the virgins and inseminated females and plotted only the difference between 
 blood - fed and unfed status ( fig . 1
its effect on the body was significant 
 for all four genes and significant for per   and cyc 
 and borderline for tim in the head ( table i ) .
1 shows that in all 
 cases a reduction in clock gene expression was observed following blood - feeding . 

fig . 1 : expression of clock genes period , timeless , 
 cycle   and   clock relative to the constitutive 
 gene rp49 in the head and body of aedes aegypti 
  females either fed or unfed on blood .
asterisks indicate when rna expression is significantly different between 
 groups of blood - fed females and control ( unfed ) .
since the statistics 
 demonstrate blood - feeding is the only relevant factor in the analysis , for the 
 graph representation , we joined the virgins and inseminated females and plotted 
 only the difference between blood - fed and unfed status . for details on 
 statistic results
see table i. 
 
 table ianalysis of variance ( 2-way anova ) of the expression of the clock genes 
 period , timeless , cycle 
 and clock , concerning the effect of insemination and 
 blood - feeding either separately or interacting  factors 
 period 

 timeless 

 cycle 

 clock 
 bodyinsemination 
 f 
 1,15 = 1.554p = 0.232 
 f 
 1,16 = 1.162p = 0.297 
 f 
 1,15 = 0.253p = 0.623 
 f 
 1,16 = 1.352p = 0.262  blood - feeding 
 f 

= 9.364 

 p = 0.007 
 insemination and blood - feeding 
 ( interaction ) 
 f 
 1,15 = 2.076p < 0.170 
 f 
 1,16 = 0.213p = 0.650 
 f 
 1,15 = 0.205p = 0.657 
 f 
 1,16 = 0.085p = 0.775headinsemination 
 f 
 1,14 = 3.140p = 0.098 

f 
 1,15 = 1.289p = 0.274 
 f 
 1,16 = 1.173p = 0.295 
 f 
 1,15 = 0.802p = 0.385  blood - feeding 
 f 
 1,14 
 = 15.637 

 p = 0.001 

 f 
 1,15 = 3.901p = 0.067 
 f 
 1,16 
 = 6.917 

 p = 0.018 

 f 
 1,15 = 3.159p = 0.096  insemination and blood - feeding 
 ( interaction ) 
 f 
 1,14 = 0.020p = 0.888 
 f 
 1,15 = 1.622p = 0.222 
 f 
 1,16 = 2.896p = 0.108 
 f 
 1,15 = 0.115p = 0.739heads and beheaded bodies were analysed independently .
rnai knockdown of tim expression and its effects on activity behaviour 
 - we conducted injections of in vitro synthesised tim dsrna in adult 
 ae .
aegypti   females and monitored the effect of this intervention 
 on the pattern of the mosquitoes activity / rest behaviour under two different conditions , 
 ld and dd .
the efficacy of the rnai in reducing tim expression was 
 evaluated in head and body of mosquitoes collected on the fourth day after the dsrna 
 injection using qrt - pcr .
a significant reduction in tim expression was 
 detected in both heads and bodies in the two experiments ( fig .
2 : expression of timeless relative to the constitutive gene 
 rp49 in the head and body of aedes aegypti 
  females injected with either timeless ( 
 tim ) or lac z double - stranded rna 
 ( dsrna ) . the highest value between these
a t 
  test was used to compare dstim and dslacz 
  in each condition and all presented significant differences ( p 
  < 0.05 ) , marked by asterisks .
statistics indicates that 
 concerning gene expression results , there are no differences between the two 
 independent experiments ( dd : constant darkness ; ld : light / dark cycles ) .
it also 
 indicates only the fourth day after the injection with the dsrna has 
 significant change in gene expression on the group injected with 
 dstim relative to the control group injected with dsrna 
 lacz ( anova , f 
 1.58 = 9.708 ; p < 0.003 )
. therefore , this figure depicts only the 
 statistically significant result of the fourth day after injections of both 
 experiments .
the greater reduction in tim expression observed 
 in the head relative to the body specifically on the fourth day after injection 
 had also been proven significant ( anova , f 
 1.58 = 8.226 ; p < 0.006 ) .
error bars show standard error . 
  the graph of locomotor activity depicted in fig .
3 
 shows similar patterns between dstim and dslacz , but 
 with different levels of activity during seven days in ld . under ld cycles
, we expect an 
 activity pattern with 24 h , which represents a proper entrainment of mosquitoes to the 
 ld regime . however , we were unable to observe any significant differences in the 
 proportion of rhythmic individuals between dslacz and 
 dstim groups .
the power of the rhythm is 
 usually defined as the amplitude from the top of the peak to the confidence level in the 
 2 periodogram ( liu et al .
1991 ) and it is used 
 as a parameter to measure the strength of the rhythm in a given environmental cycle . 

thus we can assume that the power reduction in dstim mosquitoes is a 
 consequence of a deficient light entrainment in this group . 

3 : activity / rest behaviour of two groups of aedes aegypti 
 female after the injection of double - stranded rna ( dsrna ) for seven days in 
 light - dark cycles ( ld ) .
one group was injected with timeless 
 dsrna ( black line ) and the other with lacz dsrna ( grey area ) , 
 used as control .
white horizontal bars indicate lights - on and black horizontal 
 bars indicate light - off .
readings on the 30 min after the transitions ld 
 and dark - light were interpolated to smooth startle responses . 
 

table iilocomotor activity rhythms of aedes aegypti in constant 
 darkness injected with dslacz and dstim 
 mosquitoes injected withtotal ( n)rhythmic mosquitoes ( % ) period ( mean  sd)/hpower ( mean  sd ) 
 ld 
 dslacz 
 2379.324.23  0.11116.80  17.58  dstim279024.11  0.2381.68  9.93 
 dd 
 dslacz 
 3033.322.5  0.837.72  6.10  dstim2733.325.25  1.0 
 30.46  5.81 
 a : the power of the rhythm was defined as the amplitude 
 from the peak to the cut - off line (  = 0.05 ) in the  
 periodogram ( liu et al .
1991 ) ; 
 b :   t test , borderline statistical 
 significance ( p = 0.07 ) ; c :   t test , p 
 < 0.05 ; dd : constant darkness ; dslacz : double - stranded 
 rna ( dsrna ) for lacz ; dstim : dsrna for 
 timeless ; ld : light / dark cycles ; sd : standard 
 deviation . 
 

a : the power of the rhythm was defined as the amplitude 
 from the peak to the cut - off line (  = 0.05 ) in the  
 periodogram ( liu et al .
1991 ) ; 
 b :   t test , borderline statistical 
 significance ( p = 0.07 ) ; c :   t test , p 
 < 0.05 ; dd : constant darkness ; dslacz : double - stranded 
 rna ( dsrna ) for lacz ; dstim : dsrna for 
 timeless ; ld : light / dark cycles ; sd : standard 
 deviation . in another experiment , after two days in ld the mosquitoes were submitted to five days 
 in dd ( fig .
, the number of rhythmic 
 individuals diminishes equally between the two groups ( table ii ) .
nevertheless , we observed a dramatic decrease in the activity 
 levels in the dstim group in the second dd day , which corresponds to 
 the fourth day after injection .
after that , there is a gradual increase in the activity 
 levels , but with a delayed phase shift as compared to the dslacz group . 
 also , there is a significant difference in the free running period between the groups ( 
 table ii ) , probably as a result of a great 
 variety between individuals from the dstim group , which is not observed 
 in the dslacz mosquitoes ( fig . 

aegypti female after the injection of double - stranded rna ( dsrna ) 
 for two days in light - dark cycles ( ld ) followed by five days in constant 
 darkness ( dd ) .
a : double - plotted actograms of representative mosquitoes 
 injected with dslacz and dstim .
the data was 
 smoothed ( gaussian smoothing ) using actogramj to reduce the noise and improve 
 the visualisation of actograms .
the shaded dark grey areas indicate dark phase 
 and light grey areas indicate the subjective days in dd .
the arrow shows a 
 reduction of activity in dstim mosquitoes on the fourth day 
 after the injection ; b : mean  standard error of the means ( lines ) and 
 individual mosquito ( dslacz , circles ; dstim 
 , squares ) free running period length ( hours )
. statistical analysis by 
 t test show a difference between dslacz 
 and dstim ( t = 2.158 ; p < 0.05 ) .
there is a wide interest in the behaviour of blood - sucking insects , due not only to the 
 distress they cause to humans and other animals , but predominantly due to their role in 
 the transmission of disease , with mosquitoes one of the most important groups in this 
 regard .
a particularly interesting aspect of the behaviour of haematophagous insects is 
 the change observed in females according to their physiological state .
for example , it 
 has long been known that insemination , blood - feeding and the interaction of these two 
 factors cause changes in the pattern of flight activity and rest behaviour in female 
 mosquitoes ( jones & gubbins 1977 , 1978 , jones 
 1981 , rowland 1989 , clements 1999 )
. the explanation could rely on the 
 ecological needs of each species . in nature
, anopheles mosquitoes 
 generally mate in swarms and then females go chasing a blood meal .
stephensi that 
 after mating , females change the profile of the flight activity and rest behaviour such 
 that they no longer perfectly overlap with the males pattern , as is the case for virgin 
 females .
instead , they switch to a mode of persistent activity throughout the night that 
 would favour their encounter with a source of blood meal . when inseminated and 
 blood - fed
, the female does nt need to do much else but develop its eggs and shows almost 
 no activity at all for two days .
at which time it becomes active again to proceed with 
 egg laying and the next search for a blood meal ( jones 
 & gubbins 1978 , rowland 1989 , 
 hawkes et al .
however , the 
 blood - feeding alone does not seem to alter the flight activity pattern of virgin 
 females , which continues to overlap with the males activity , favouring the encounter 
 for mating ( rowland 1989 , clements 1999 ) . on the other hand , ae .
they mate 
 close to where the source of blood meal can be found ( jones 1981 , clements 1999 ) .
after 
 mating , females do not need to be active for long periods in order to find a source of 
 blood meal and in fact , they show a strong reduction in the overall activity after 
 insemination . when mated and blood - fed , the activity of ae .
aegypti 
 females is drastically suppressed , as seen in anopheles , while 
 blood - feeding alone causes only mild effects in the activity pattern ( jones 1981 , clements 
 1999 , tn lima - camara , unpublished observations ) . despite an abundance of information on the behavioural and ecological observations ,
not 
 much is known about the molecular mechanism of the circadian clock in mosquitoes . the 
 expression pattern of the main clock genes in ae .
2006 , 
 2009 ) and shown to present a robust cycling 
 expression , similar to drosophila ,   with the exception of cyc 
  expression . even less is known about the molecular changes in clock genes 
 triggered by insemination or blood - feeding .
one of our aims in this study was to 
 investigate the impact of the blood meal on the expression of clock genes .
blood - feeding 
 behaviour has been shown to be under circadian clock control ( das & dimopoulos 2008 ) and it is possible that this behaviour is 
 not only regulated by , but also feeds back into , the circadian clock .
this question has 
 been addressed before in a study on the sandfly vector of leishmaniasis , lu . 

the authors show ~40% reduction in the overall activity as a 
 result of blood intake ( meireles - filho et al . 

2006 ) and discuss that this reduction could be driven by the central 
 pacemaker .
it is known that two genes acting in haeme metabolism , -aminolevulinate 
 synthase and haemeoxygenase cycle in anti - phase in drosophila   heads ( 
 ceriani et al . 2002 ) .
so it is possible that 
 the oxidative stress caused by the blood meal could directly interfere with the 
 regulation of the clock genes , causing an impairment on clk s ability to bind dna and 
 driving the observed reduction in the expression of per and 
 tim after blood intake .
aegypti reinforce this idea , since we show here that blood - feeding , and not 
 insemination , is capable of causing a down regulation in the expression of the canonical 
 clock genes per , tim , cyc and 
 clk in the head and body of the females of this mosquito species ( 
 fig . 1 , table 
 i ) .
in addition , cyc   regulation is under clk control ( 
 gentile et al .
2009 ) and it is also known 
 that haeme selectively regulates the rev - erb targets in mammals , including bmal1 , the 
 drosophila cyc   orthologue gene ( raghuram et al .
the rev - erb orthologue e75 
  is a nuclear receptor essential for haeme functions in the fat body ( cruz et al .
these findings together suggest 
 that two pathways may be acting together to reduce cyc   expression .
the central control of the circadian clock lies in a group of brain neurons called the 
 clock neurons . while head extracts will not exclusively contain mrna from clock neurons , 
 they will closely reflect the overall expression pattern of clock genes in the central 
 clock and for this reason are typically used as a proxy for the central clock molecular 
 profile ( hardin 2011 ) .
our results show that 
 the blood meal is responsible for a significant reduction in the expression of 
 per and cyc in the head of females and a borderline 
 reduction for tim and clk ( fig . 1
one 
 explanation for the borderline result with the latter two clock genes could lie in the 
 fact that these genes have the lowest amplitude in their circadian fluctuation in the 
 head of ae .
aegypti ( gentile et al . 
 2009 ) , making it harder to detect a statistically significant reduction in 
 their expression levels . in the case of the clock genes down - regulation in the body ( fig . 1
it is not possible to infer if it is caused 
 by the down - regulation of the central clock or by a general mechanism responsible for 
 the broader gene down - regulation observed after a blood meal ( ribeiro 2003 ) or by both .
irrespective , the effect on the 
 peripheral clock may be relevant to the observed output behaviour downstream from the 
 central clock .
peripheral clocks have already been shown to be important and sometimes even sufficient 
 for maintaining some circadian aspects of behaviour and/or physiology ( hall 2005 ) .
the fat body is an insect analogue of the vertebrate 
 liver and adipose tissue that also functions in other physiological processes such as 
 innate immunity , detoxification and reproduction ( leclerc & reichhart 2004 , lazareva et 
 al .
a drosophila   microarray analysis on fat bodies 
 along a circadian day identified several rhythmically - expressed genes that are 
 controlled by this organ , including five cytochrome p450 genes and eleven oxidoreductase 
 genes ( xu et al .
so , it is possible to speculate that blood - feeding affects the insects 
 peripheral clock via fat bodies . on the other hand , the reduction observed in the body 
 could relate more to the non - clock related functions these genes have in peripheral 
 tissues .
as discussed previously , mating seems to be the most relevant event in terms of 
 the modulation of behaviour in ae .
aegypti females are usually inseminated only once by males 
 because accessory gland substances transferred from males make mated females refractory 
 to subsequent inseminations ( craig jr 1967 , 
 gillott 2003 ) .
the evidence from drosophila studies reinforces mating as the crucial 
 event that modulates behaviour in females through changes at the molecular level 
 [ reviewed in greenspan and ferveur ( 2000 ) ] . in 
 mosquitoes ,
the molecular mechanism could be similar and some groups had been trying to 
 investigate this .
gambiae that 
 male accessory glands express genes that could potentially work as sex peptide - like 
 modulators ( dottorini et al .
2007 ) and that 
 mating triggers a wide range of gene expression changes ( rogers et al . 2008 ) .
however , we could not detect any significant 
 change in clock gene expression in ae .
we can not discard the 
 possibility that insemination alone is able to affect the clock gene expression in a 
 subset of clock neurons or peripheral clocks .
if the alteration in the gene expression 
 is restricted , it could be undetectable with the strategy we adopted , using rna extract 
 from whole heads or beheaded bodies .
alternatively , the molecular changes following 
 mating might not involve the circadian clock at all , but other behavioural controlling 
 genes downstream from the clock .
this hypothesis could only be tested when more 
 information about the genes controlling output behaviour is available .
understanding in detail the behaviour of female mosquitoes that are vectors of disease 
 is crucial for the epidemiology of the parasites / virus they transmit ( clements 1999 ) .
it has been known for a long time , 
 both in the field and in the laboratory , that females in different physiologic states 
 will show different patterns of circadian activity ( jones & gubbins 1977 , 1978 , 
 jones 1981 , lima - camara et al .
we know , for example , that dengue virus infected 
 females show an increase in their locomotor activity ( lima - camara et al .
2013 ) . in a simplified mathematical model , 
 if we assume an increase in the biting rate of infected ae .
aegypti , 
 the number of primary and secondary infections also increases ( luz et al .
we aimed in this paper to investigate the consequences 
 of different physiologic states of female ae .
aegypti on the expression 
 of the clock genes , fundamental in the control of the circadian rhythms , but further 
 experiments are still needed to address this question .
another aim of this work was to test the assumption of cause and effect in relation to 
 clock gene expression and circadian behaviour .
this assumption is based on what is known 
 from drosophila ( hall 2005 , 
 hardin 2011 ) , but testing this in ae 
 aegypti presents challenges due to limitations in the available genetic 
 tools .
we therefore chose to perform rnai assays to knockdown tim and 
 correlate the reduced expression with any circadian behavioural changes .
the choice of 
 tim was based on the sensitivity of the clock to a decrease in tim 
 protein concentrations caused by light - triggered degradation , resulting in a resetting 
 of the whole system ( hall 2005 , hardin 2011 ) .
results from das and dimopoulos ( 2008 ) , who used a similar dsrna injection 
 technique , found a link between tim expression and blood - feeding 
 behaviour .
aegypti   ( danbara et al . 2010 , kamae & tomioka 2012 ) indicates that although 
 the functional domains a reasonably similar , tim may play distinct roles on the 
 circadian clock of each species .
recent studies on the firebrat thermobia 
 domestica showed that tdtim knockdowns ( using dsrna ) 
 abolished the locomotor rhythm in most of the treated firebrats for more than 30 days . 

in contrast , experiments using rnai in the orthoptera gryllus bimaculatus 
  ( cricket ) showed that gbtim dsrna was able to reduce 
 tim levels significantly , but was not sufficient to disrupt the 
 circadian locomotor activity in dd . despite not losing rhythmicity , the
free running 
 period is significantly shorter than control crickets injected with 
 dsred2 ( danbara et al .
our results show in independent experiments that tim   gene silencing 
 was significant on the fourth day after injection ( fig . 
 2 ) .
this effect seems to influence the levels of locomotor activity in dd , 
 four days after the injection ( fig .
this 
 reduction of tim could also promote long - term behaviour effects in both 
 regimes .
in fact , tim   rnai introduces changes that resemble the 
 effects observed in the tim 
  mutant in drosophila ( sehgal et 
 al . 1994 ) , since these mutants have rhythmic patterns of activity in ld , but 
 are arrhythmic in dd . in ld , the increased activity of dstim in 
 ae .
aegypti suggests changes in the masking effect by light , 
 possibly due to the mosquitoes becoming less sensitive to light than the control group . 

also , the lower levels of power in dstim ( table ii ) imply a small impairment in their entrainment to light . 

together these results suggest that in mosquitoes tim plays a role in 
 light masking and entrainment , as similarly observed with drosophila 
 [ reviewed in hardin ( 2011 ) ] .
in addition , under 
 dd , we observed an endogenous period longer than 24 h for dstim ( table ii ) , but this likely reflects a substantial 
 individual variation in the free - running periods as detected by the periodogram analysis 
 ( fig .
it has 
 been observed under constant conditions ( constant light or constant darkness ) that one 
 peak of activity often splits into two or more components that free run with different 
 phases ( short and long periods ) ( saunders 2002 
 ) .
thus , it is possible that the reduction of tim favours the 
 occurrence of splitting patterns of activity .
the average endogenous period observed for 
 dstim mosquitoes might be the mean of individuals in dd with both 
 components free running simultaneously , but only one of these components can be detected 
 by a periodogram analysis ( fig .
4b ) . interestingly , dstim injection into adult gryllus 
 bimaculatus has also generated increased activity in ld and a decrease in 
 amplitude in dd ( danbara et al .
thus , although it is still premature 
 to say , tim may have similar roles in the clock of these two species , 
 playing an important role in both the light entrainment and modulation masking in ld and 
 in dd , as well as the proper coupling of oscillators , the fine - tuning of the endogenous 
 period and the amplitude control of locomotor activity . finally
, our results on the 
 silencing of tim show that it is possible to achieve gene silencing in 
 the head of the mosquito by thorax injection of dsrna , however we could only show mild 
 effect on the behaviour . as a consequence
, we did not pursue with the silencing of the 
 other individual clock genes .
 mosquitoes are the culprits of some of the most important vector borne diseases . 
 a 
 species potential as a vector is directly dependent on their pattern of behaviour , 
 which is known to change according to the female s physiological status such as 
 whether the female is virgin / mated and unfed / blood - fed . 
 however , the molecular 
 mechanism triggered by and/or responsible for such modulations in behaviour is poorly 
 understood . 
 clock genes are known to be responsible for the control of circadian 
 behaviour in several species . here 
 we investigate the impact mating and blood - feeding 
 have upon the expression of these genes in the mosquito aedes 
 aegypti . 
 we show that blood intake , but not insemination , is responsible 
 for the down - regulation of clock genes . using rna interference , we observe a slight 
 reduction in the evening activity peak in the fourth day after dstim 
 injection . 
 these data suggest that , as in drosophila , clock gene 
 expression , circadian behaviour and environmental light regimens are interconnected 
 in ae . aegypti . 
MATERIALS AND METHODS
RESULTS
DISCUSSION