BACKGROUND
chikungunya virus  is an arbovirus  transmitted by aedes mosquito species, that usually induces an acute illness in humans characterized by fever, rash, and incapacitating arthralgia  <cit> .
the virus was first described in tanzania in  <dig>  <cit> .
in africa, it has been maintained in a sylvatic cycle involving forest mosquitoes feeding preferentially on wild non-human primates  <cit> .
the virus was later introduced into asia where it circulates within an urban cycle; only an inter-human transmission cycle is described on this continent, involving aedes aegypti and aedes albopictus as vectors  <cit> .
in early  <dig>  chikv was introduced into the indian ocean region, probably by viremic travelers returning from kenya where an outbreak had started in june  <dig>  <cit> .
later, in  <dig> and  <dig>  chikv spread across the indian ocean islands .
afterwards, chikv expanded into asia  <cit>  and africa  <cit> .
surprisingly, an autochthonous chikv outbreak was reported in europe in the ravenna region of italy during the summer of  <dig>  <cit> .
most recent chikv outbreaks were related to the mosquito ae.
albopictus whose geographical distribution has been expanding into new tropical and temperate regions over the past decades  <cit> .
in the indian ocean region, ae.
aegypti and ae.
albopictus are present together or alone; both vectors can be involved in the chikv transmission cycle.
phylogenetic analyses based on sequences of the e <dig> gene encoding an envelope glycoprotein revealed that the indian ocean isolates belonged to the eastern/central/southern african genotype  <cit> .
a switch to a new variant operated in the reunion island from the end of  <dig>  where most chikv isolated from patients presented an amino-acid substitution in the e <dig> glycoprotein, from an alanine  to a valine .
this mutation enhances transmission by ae.
albopictus  <cit> .
the new variant spread easily through the indian ocean region and has thus been found in countries where ae.
albopictus is the main aedes species.
subsequent chikv outbreaks were related to the transmission of the e1-226v variant by ae.
albopictus.
by experimental infections, we demonstrated that from day  <dig> post-infection , aedes mosquitoes could produce  <dig> -fold more viral particles than those ingested  <cit> .
although it was believed for many years that infection with an arbovirus had no deleterious effects upon its arthropod host, recent studies indicated that such viral infections may not be benign.
when compared with uninfected individuals, viral infections in mosquitoes can reduce fecundity  <cit> , the ability to get a blood-meal  <cit> , and survival  <cit> .
the effect on the survival of the mosquito vector is important in determining vector capacity.
how a vector responds to infection by a virus?
among the strategies developed to compensate for the cost of infection by a pathogen, a host could reduce the harm caused by infection by tolerance, which involves the alteration of host life-history traits.
these latter are defined as traits relevant to the allocation of resources in reproduction, survival and growth: for example the number of offspring, age of first reproduction, adult body size etc.
in this work, we have analyzed the effects of chikv infection on survival and reproduction of two mosquito species, ae.
aegypti and ae.
albopictus.
changes in survival and reproduction induced upon infection with two chikv strains, the original e1-226a strain and the new e1-226v variant were studied.
RESULTS
susceptibility of aedes species to chikv strains
batches of mosquitoes were orally exposed to serial 10-fold dilutions of chikv e1-226a and chikv e1-226v .
to determine whether disseminated infection rates correlated with blood-meal titers, surviving mosquitoes were analyzed at day  <dig> pi by ifa on head squashes.
ae.
aegypti mayotte showed similar susceptibility towards both chikv e1-226a and chikv e1-226v ; at a titer of  <dig>  pfu/ml,  <dig> % of mosquitoes showed a disseminated infection when infected with chikv e1-226v and  <dig> % did with respect to chikv e1-226a .
on the other hand, ae.
albopictus mayotte were found to be 1- <dig> fold more susceptible to chikv e1-226v than to chikv e1-226a ; at a titer of  <dig>  pfu/ml,  <dig> % of mosquitoes presented a disseminated infection when infected with chikv e1-226a, in contrast to  <dig> % with chikv e1-226v .
lastly, ae.
albopictus reunion displayed the same profile as ae.
albopictus mayotte, with significant differences between viruses at each titer point  except when the titer was high  where both viral strains gave similar disseminated infection rates .
all aedes species examined presented a similar susceptibility to chikv e1-226a at a titer of  <dig>  pfu/ml .
at this same viral titer, ae.
albopictus  presented a higher susceptibility to chikv e1-226v than did ae.
aegypti mayotte .
the plateau corresponding to 100% of disseminated infection rate was reached for blood-meal titers higher than  <dig>  pfu/ml.
survival of mosquitoes
the survival rates for each mosquito species, depending on the trial and according to the virus , are presented in figure  <dig>  the infectious blood-meals were at a titer of  <dig>  pfu/ml.
for ae.
aegypti mayotte , each trial led to the same results: the survival curves were not significantly different according to the infection .
a parametric model was used to estimate the effects on survival of the strain of the virus  and of the infection status of the mosquito , and to assess the existence of an interaction between these two factors.
for this purpose, we combined trials  <dig> and  <dig> as no significant difference was detected in ni mosquitoes , or mosquitoes exposed to chikv e1-226a  or to chikv e1-226v .
the risk of death was higher for ndinf mosquitoes compared to ni mosquitoes  :  <dig>   with chikv e1-226v and  <dig>   with chikv e1-226a).
on the other hand, no significant difference in the risk of death was observed between dinf mosquitoes and ni mosquitoes infected with chikv e1-226v :  <dig>   and with chikv e1-226a ) .
the table shows the life duration of ae.
aegypti and ae.
albopictus from mayotte and the reunion island after infection with two chikv strains: chikv e1-226a and chikv e1-226v.
n corresponds to the number of female analysed, sd to the standard deviation,  <dig> and  <dig> to respectively, trial  <dig> and trial  <dig>  dinf to females with disseminated infection, ndinf to females with non-disseminated infection, and ni to non-infected females.
for ae.
albopictus mayotte , the two trials mainly led to the same results: survival curves did not differ significantly according to the virus .
to compare the survival curves, we combined the two trials since no significant difference was found between ni mosquitoes , mosquitoes exposed to chikv e1-226a  and chikv e1-226v .
using a parametric survival model, compared to ni mosquitoes, mosquitoes exposed to chikv e1-226v were not at greater risk of dying, whether the infection status was ndinf :  <dig>  ) or dinf :  <dig>  ).
on the other hand, for mosquitoes exposed to chikv e1-226a, ndinf mosquitoes showed a higher risk of dying than did ni mosquitoes :  <dig>  ) while dinf mosquitoes had a significantly lower risk of dying :  <dig>  ) than did ni mosquitoes .
for ae.
albopictus reunion , the two trials led to the same results: a significant difference in the survival curves was detected according to the virus .
mosquitoes exposed to chikv e1-226a or chikv e1-226v had lower survival rates than ni mosquitoes.
as the trials differed significantly when ni mosquitoes , those exposed to chikv e1-226a  or to chikv e1-226v  were analyzed, trials were considered separately.
using a parametric survival model, we found that for mosquitoes exposed to chikv e1-226v, ndinf mosquitoes, as well as dinf mosquitoes, were more at risk of dying than ni mosquitoes in both trials :  <dig>   and  <dig>   in trials  <dig> and  <dig>  respectively; for dinf mosquitoes:  <dig>   and  <dig>   in trials  <dig> and  <dig>  respectively).
for mosquitoes exposed to chikv e1-226a, ndinf and dinf mosquitoes were more at risk of death than ni mosquitoes were, in both trials: for ndinf mosquitoes,  <dig>   and  <dig>   in trials  <dig> and  <dig>  respectively; and for dinf mosquitoes:  <dig>   and  <dig>   in trials  <dig> and  <dig>  respectively .
oviposition characteristics
time to first egg laying
in ae.
aegypti mayotte, the kaplan-meier estimates showed no significant difference in the time to egg laying according to the chikv strain  or the infection status of the mosquito .
combining chikv strain and infection status as a single factor, the parametric survival model showed no effect of these two factors on the time to egg laying .
in ae.
albopictus mayotte, the kaplan-meier estimates showed no significant difference in the time to egg laying when the virus  or the infection status of the mosquito  were examined.
the parametric survival model did not show any effect of the virus and infection status of mosquito on the time to egg laying .
in contrast, in ae.
albopictus reunion, the kaplan-meier estimates showed significant differences in the time to egg laying when considering the virus  and the infection status of mosquito .
the parametric survival model showed a significant effect of the virus and infection status of mosquitoes on the time to egg laying .
mosquitoes exposed to chikv e1-226v and chikv e1-226a laid eggs significantly earlier than ni mosquitoes .
± sd
± sd
±3
ae.
aegypti and ae.
albopictus from mayotte and the reunion island were infected with two chikv strains: chikv e1-226a and chikv e1-226v.
n corresponds to the number of female analysed, sd to the standard deviation,  <dig> and  <dig> to respectively, trial  <dig> and trial  <dig>  dinf to females with disseminated infection, ndinf to females with non-disseminated infection, and ni to non-infected females.
number of eggs laid per mosquito
the number of eggs laid according to the mosquito species, virus and infection status of the mosquito is described in table  <dig>  using a negative binomial regression model for each mosquito species, we did not find any evidence that the number of eggs laid differed according to the virus and infection status of the mosquito .
time between first oviposition and mosquito death
we also described the time between the first oviposition and mosquito death using kaplan-meier survival curves performed separately for each mosquito species.
no difference according to the virus and infection status of the mosquito was observed in ae.
aegypti mayotte  and ae.
albopictus mayotte .
in ae.
albopictus reunion, both trials led to the same conclusion: when compared to ni mosquitoes, the time was significantly shorter in mosquitoes infected with chikv e1-226a or chikv e1-226v irrespective of whether the infection status of the mosquito was ndinf or dinf .
hatching characteristics
proportion of mosquitoes with at least one egg hatched
when examining the proportion of females with at least one egg hatched, no significant differences were found whatever the mosquito species, the virus and the infection status of the mosquito, when results were considered for ae.
aegypti mayotte  and ae.
albopictus mayotte .
in contrast, for ae.
albopictus reunion a significant difference was found to be attribuable to ni mosquitoes  .
the table refers to the hatching characteristics of ae.
aegypti and ae.
albopictus from mayotte and the reunion island infected with two chikv strains: chikv e1-226a and chikv e1-226v.
n corresponds to the number of female analysed, sd to the standard deviation,  <dig> and  <dig> to respectively, trial  <dig> and trial  <dig>  dinf to females with disseminated infection, ndinf to females with non-disseminated infection, and ni to non-infected females.
hatching rate per mosquito
when the hatching rate per mosquito was examined, ae.
aegypti mayotte mosquitoes were found to show a slight difference according to the viral strains  and not to the infection status of the mosquito .
similarly, ae.
albopictus mayotte mosquitoes only showed a significant difference when viral strains were considered  and not the infection status of the mosquito .
finally, ae.
albopictus reunion also showed differences related to viral strains  and not to the infection status of the mosquito .
in most cases, mosquitoes infected with chikv e1-226v displayed the highest hatching rates .
eggs that issued from infected or non-infected mosquitoes maintained in a bsl- <dig> laboratory usually showed a lower capacity to hatch than eggs that were kept in an insectarium under standard conditions .
replication of chikv in mosquitoes
dead mosquitoes were collected once a day.
the number of viral rna copies present in dead mosquitoes did not vary within a period of  <dig> h after mosquito death .
for ae.
aegypti mayotte, mosquitoes had an average of  <dig>   viral rna copies per mosquito when a blood-meal containing chikv e1-226a was ingested, and an average of  <dig>   viral rna copies per mosquito that had ingested chikv e1-226v .
following the blood-meal, viral load increased to reach a maximum of  <dig>  ±  <dig>  viral rna copies at day  <dig> pi for chikv e1-226a and between day  <dig>  and day  <dig> pi  for chikv e1-226v.
later, a slight decrease was observed until day  <dig> pi .
for both viruses, many mosquitoes died between day  <dig> and day  <dig> pi, especially at day  <dig> for ae.
aegypti mayotte infected with chikv e1-226v.
for ae.
albopictus mayotte, mosquitoes ingested an average of  <dig>   viral rna copies when exposed to a blood-meal containing chikv e1-226a and  <dig>   with chikv e1-226v .
the maximum viral load was reached at day  <dig> pi after ingestion of both chikv e1-226a  and chikv e1-226v  followed by a plateau until day  <dig> pi.
at day  <dig> pi, the number of viral rna copies had decreased to  <dig>   for chikv e1-226a and  <dig>   for chikv e1-226v.
in the case of ae.
albopictus reunion, mosquitoes ingested an average of  <dig>   rna copies in the blood-meal containing chikv e1-226a and  <dig>   rna copies for chikv e1-226v .
after a slight increase in the amount of viral rnas from day  <dig> to day  <dig> pi, a plateau was observed until day  <dig> pi:  <dig>  ±  <dig>  with chikv e1-226a and  <dig>  ±  <dig>  with chikv e1-226v.
dead females exhibited two distinct profiles:  females which ensured a viral replication at a level similar to that of live females and  females which did not ensure any viral replication.
this is more clear-cut for alprov infected with chikv e1-226a.
discussion
whichever viral strain was examined , differential responses to chikv infection were found according to the mosquito species  and their geographical origin .
chikv infection affected the life-history traits of ae.
albopictus from the reunion island.
we have found that apart from the viral strains, chikv infection induced effects on survival, by reducing lifespan, and on oviposition characteristics, by shortening the time to egg laying of ae.
albopictus from the reunion island.
aedes albopictus is a mosquito species native to southeast asia that has spread beyond its natural habitat by means of intercontinental trades  <cit> .
this mosquito is able to transmit various arboviruses in laboratory conditions  <cit> .
in addition to this species, ae.
aegypti is native to africa and comprises two forms: the forest-dwelling ae.
a. formosus involved in the enzootic transmission of arboviruses, and the domestic form ae.
a. aegypti whose distribution has extended over the tropical region acting as the main vector of dengue viruses  <cit> .
both species are container-breeding mosquitoes that share the same breeding sites.
in asia, the introduced ae.
aegypti tends to displace ae.
albopictus from urban settlements  <cit> , whereas the spread of ae.
albopictus has been associated with declines of ae.
aegypti in the americas  <cit> .
in the indian ocean region, ae.
albopictus was introduced several centuries ago  <cit>  and in the reunion island, it was first recorded in  <dig>  <cit> .
on the island, this mosquito became the most common aedes species  <cit>  since the control program against malaria vectors in the 1950s that led to the confinement of ae.
aegypti in remote areas  <cit> .
on mayotte, ae.
aegypti was first recorded in  <dig>  <cit>  and ae.
albopictus was observed later, in  <dig>  <cit> .
larvae of both species could be found in artificial containers making inter-specific resource competition possible  <cit> .
ae.
albopictus predominates in domestic habitats in mayotte  <cit> .
the chikv outbreak began in kenya in  <dig>  and subsequently spread to the islands of the indian ocean  <cit> .
a new strain of chikv emerged that carried a point mutation, which changes the amino acid at position  <dig> in the e <dig> envelope glycoprotein from an alanine to a valine  <cit> .
this single mutation has increased the vector competence of ae.
albopictus for chikv  <cit> .
this increased transmissibility of chikv is one of the hypotheses evoked to explain the expansion of chikv transmission into new regions such as madagascar  <cit>  and cameroon  <cit>  in  <dig>  and india  <cit> , italy  <cit>  and gabon  <cit>  in  <dig>  prior to these recent chikv outbreaks, ae.
aegypti had been considered the primary epidemic vector of chikv and ae.
albopictus, a secondary vector.
a single e1-a226v mutation was sufficient to increase the ability of chikv to infect ae.
albopictus modifying the virus infectivity for a vector species other than the typical ae.
aegypti vector.
although the mutation confers a selective advantage in ae.
albopictus, there was no corresponding advantage in ae.
aegypti.
our results showed that ae.
aegypti mayotte was similarly susceptible to both viral strains, whereas ae.
albopictus mayotte and ae.
albopictus reunion were more susceptible to the chikv e1-226v than to the chikv e1-226a strain .
this may explain the predominance of e1-226v in countries where ae.
albopictus is considered to be the main vector species.
at a titer of  <dig>  pfu/ml, e1-226v virus was disseminated more effectively by ae.
albopictus  than by ae.
aegypti .
infection of ae.
aegypti mayotte requires that the chikv blood-meal titers are significantly higher than for infection of ae.
albopictus , which suggests that once introduced, e1-226v would be positively selected by ae.
albopictus.
as an intracellular parasite, chikv can mediate changes in host life-history traits.
two sorts of mechanisms could be proposed.
on one hand, the use of host resources to maintain viral replication can lead to modifications of resource allocation as pathogenic effects.
on the other hand, life-history modifications may be responses of the host to counterbalance the negative effects of parasitism.
by reacting to the parasite, the host reduces the effect of infection on its fitness  <cit> .
we have shown that replication of chikv in aedes mosquitoes is intensive, e.g.
<dig>  more viral particles in females from day  <dig> pi, and saliva became infectious from two days pi  <cit> .
in the present work, we have analyzed the effects of chikv replication on several life-history traits of aedes mosquitoes related to resource allocation, and only found significant modifications in ae.
albopictus from the reunion island.
thus chikv infection has a significant effect on the time until death and egg laying.
moreover, such negative effects were independent of the viral strain: chikv e1-226a and chikv e1-226v displayed similar effects.
nevertheless, aside from chikv infection,ae.
albopictus from the reunion island better survived in bsl- <dig> laboratory conditions than the two other mosquito strains.
regardless, infection by chikv has a negative impact on the survival of ae.
albopictus from the reunion island .
lifespan was shortened by 6- <dig> days when mosquitoes were infected with chikv e1-226a or chikv e1-226v.
moreover, by shortening the time from blood-feeding to laying, chikv-infected ae.
albopictus from the reunion island allocates an increased reproduction investment to laying eggs before death.
thus only limited consequences may affect vector dynamics, as other factors dictating oviposition  and hatching  characteristics were only slightly altered by infection.
such earlier reproduction has been reported in other animal-parasite systems  <cit> .
infection by a pathogen may modify resource distribution: infected hosts will allocate more resources to reproduction, subtracting them from those attributed to growth and survival  <cit> .
nevertheless, the question remains as to why this phenomenon is not species-specific.
effectively, ae.
albopictus from mayotte did not exhibit the same pattern.
one explanation could be that ae.
albopictus assumed to be introduced into the reunion island by immigrants from asia in the 17th-18th centuries  <cit>  has been subjected to major bottlenecks that cause strong genetic divergence from the asian mosquitoes  <cit> .
thus ae.
albopictus from the reunion island may be genetically different from ae.
albopictus detected in mayotte in  <dig>  <cit> .
from our results, a very provocative scenario could be envisioned concerning chikv evolution during the indian ocean outbreak.
since mayotte is located halfway between kenya, where the outbreak began, and the reunion island, where the chikv e1-a226v was first reported, this new variant could have already been present in mayotte.
in mayotte, no data pertaining to chikv sequences was available before  <dig> and chikv e1-226v was only identified after this time  <cit> .
in mayotte, both vectors co-exist  <cit>  and we clearly showed that ae.
aegypti mayotte was similarly susceptible to both viral strains while ae.
albopictus mayotte was more susceptible to the chikv e1-226v strain.
the mutation could therefore be interpreted as a positive selection phenomenon for better transmission by ae.
albopictus.
in addition, we found that infection with chikv e1-226a or chikv e1-226v did not induce significant consequences on either species from mayotte.
nevertheless, on the reunion island, the unique vector ae.
albopictus, maintains a better transmission of chikv e1-226v compared to chikv e1-226a.
however, an important decrease in the lifespan of ae.
albopictus from the reunion island was reported as a consequence of infection followed by changes in mosquito reproduction.
we propose that chikv e1-226a was maintained on mayotte by both ae.
aegypti and ae.
albopictus.
later, the introduction of chikv e1-226a onto the reunion island where ae.
aegypti is rare, would have led to the selection of the new variant by ae.
albopictus.
since then, chikv e1-226v has invaded some neighboring islands where ae.
albopictus is present.
thus, chikv e1-226v probably reached mayotte later, after having been selected in the reunion island.
in addition, the role of other mutations in the viral genome in the adaptation of chikv to aedes mosquitoes could not be completely excluded  <cit> .
furthermore, some characteristics of the ae.
albopictus bio-ecology might have favored chikv transmission in urban areas: high anthropophily and high population densities.
CONCLUSIONS
our results are in agreement with modifications in life-history traits reported for parasitized animals.
we found that the species ae.
albopictus was more susceptible to the chikv e1-226v than to the chikv e1-226a strain which assumes that once introduced, e1-226v would be positively selected by ae.
albopictus.
nevertheless, chikv infection only induced negative impacts on survival and on egg hatching characteristics of ae.
albopictus from the reunion island which led us to suggest that ae.
albopictus from the reunion island might be less adapted to chikv than mosquitoes from mayotte.
however, the physiological mechanisms underlying this pattern are unknown.
nevertheless, the outcome of the pathogen/host interaction often involves the immune system that is restricted to the innate immune function in insects.
this is an effective mechanism to provide protection against a wide variety of pathogens including viruses.
we have evaluated the number of viral rna copies in mosquitoes  and found that females that died a few days after infection did not host more viral particles than did live females.
thus we might suggest that mosquitoes did not die following an excess of viral replication but more probably when mounting an immune response.
modifications of life-history traits and/or mounting an efficient antiviral response should be among strategies developed by the vector to optimize its fitness.

 chikungunya virus is an arbovirus transmitted by aedes mosquito species, that usually induces an acute illness in humans characterized by fever, rash, and incapacitating arthralgia. the virus was first described in tanzania in. in africa, it has been maintained in a sylvatic cycle involving forest mosquitoes feeding preferentially on wild non-human primates. the virus was later introduced into asia where it circulates within an urban cycle; only an inter-human transmission cycle is described on this continent, involving aedes aegypti and aedes albopictus as vectors. in early chikv was introduced into the indian ocean region, probably by viremic travelers returning from kenya where an outbreak had started in june. later, in and chikv spread across the indian ocean islands. afterwards, chikv expanded into asia and africa. surprisingly, an autochthonous chikv outbreak was reported in europe in the ravenna region of italy during the summer of. most recent chikv outbreaks were related to the mosquito ae. albopictus whose geographical distribution has been expanding into new tropical and temperate regions over the past decades. in the indian ocean region, ae. aegypti and ae. albopictus are present together or alone; both vectors can be involved in the chikv transmission cycle. phylogenetic analyses based on sequences of the e gene encoding an envelope glycoprotein revealed that the indian ocean isolates belonged to the eastern/central/southern african genotype. a switch to a new variant operated in the reunion island from the end of where most chikv isolated from patients presented an amino-acid substitution in the e glycoprotein, from an alanine to a valine. this mutation enhances transmission by ae. albopictus. the new variant spread easily through the indian ocean region and has thus been found in countries where ae. albopictus is the main aedes species. subsequent chikv outbreaks were related to the transmission of the e1-226v variant by ae. albopictus. by experimental infections, we demonstrated that from day post-infection, aedes mosquitoes could produce -fold more viral particles than those ingested. although it was believed for many years that infection with an arbovirus had no deleterious effects upon its arthropod host, recent studies indicated that such viral infections may not be benign. when compared with uninfected individuals, viral infections in mosquitoes can reduce fecundity, the ability to get a blood-meal, and survival. the effect on the survival of the mosquito vector is important in determining vector capacity. how a vector responds to infection by a virus? among the strategies developed to compensate for the cost of infection by a pathogen, a host could reduce the harm caused by infection by tolerance, which involves the alteration of host life-history traits. these latter are defined as traits relevant to the allocation of resources in reproduction, survival and growth: for example the number of offspring, age of first reproduction, adult body size etc. in this work, we have analyzed the effects of chikv infection on survival and reproduction of two mosquito species, ae. aegypti and ae. albopictus. changes in survival and reproduction induced upon infection with two chikv strains, the original e1-226a strain and the new e1-226v variant were studied.  susceptibility of aedes species to chikv strains batches of mosquitoes were orally exposed to serial 10-fold dilutions of chikv e1-226a and chikv e1-226v. to determine whether disseminated infection rates correlated with blood-meal titers, surviving mosquitoes were analyzed at day pi by ifa on head squashes. ae. aegypti mayotte showed similar susceptibility towards both chikv e1-226a and chikv e1-226v; at a titer of pfu/ml, % of mosquitoes showed a disseminated infection when infected with chikv e1-226v and % did with respect to chikv e1-226a. on the other hand, ae. albopictus mayotte were found to be 1- fold more susceptible to chikv e1-226v than to chikv e1-226a; at a titer of pfu/ml, % of mosquitoes presented a disseminated infection when infected with chikv e1-226a, in contrast to % with chikv e1-226v. lastly, ae. albopictus reunion displayed the same profile as ae. albopictus mayotte, with significant differences between viruses at each titer point except when the titer was high where both viral strains gave similar disseminated infection rates. all aedes species examined presented a similar susceptibility to chikv e1-226a at a titer of pfu/ml. at this same viral titer, ae. albopictus presented a higher susceptibility to chikv e1-226v than did ae. aegypti mayotte. the plateau corresponding to 100% of disseminated infection rate was reached for blood-meal titers higher than pfu/ml. survival of mosquitoes the survival rates for each mosquito species, depending on the trial and according to the virus, are presented in figure the infectious blood-meals were at a titer of pfu/ml. for ae. aegypti mayotte, each trial led to the same results: the survival curves were not significantly different according to the infection. a parametric model was used to estimate the effects on survival of the strain of the virus and of the infection status of the mosquito, and to assess the existence of an interaction between these two factors. for this purpose, we combined trials and as no significant difference was detected in ni mosquitoes, or mosquitoes exposed to chikv e1-226a or to chikv e1-226v. the risk of death was higher for ndinf mosquitoes compared to ni mosquitoes: with chikv e1-226v and with chikv e1-226a). on the other hand, no significant difference in the risk of death was observed between dinf mosquitoes and ni mosquitoes infected with chikv e1-226v: and with chikv e1-226a ). the table shows the life duration of ae. aegypti and ae. albopictus from mayotte and the reunion island after infection with two chikv strains: chikv e1-226a and chikv e1-226v. n corresponds to the number of female analysed, sd to the standard deviation, and to respectively, trial and trial dinf to females with disseminated infection, ndinf to females with non-disseminated infection, and ni to non-infected females. for ae. albopictus mayotte, the two trials mainly led to the same results: survival curves did not differ significantly according to the virus. to compare the survival curves, we combined the two trials since no significant difference was found between ni mosquitoes, mosquitoes exposed to chikv e1-226a and chikv e1-226v. using a parametric survival model, compared to ni mosquitoes, mosquitoes exposed to chikv e1-226v were not at greater risk of dying, whether the infection status was ndinf: ) or dinf: ). on the other hand, for mosquitoes exposed to chikv e1-226a, ndinf mosquitoes showed a higher risk of dying than did ni mosquitoes: ) while dinf mosquitoes had a significantly lower risk of dying: ) than did ni mosquitoes. for ae. albopictus reunion, the two trials led to the same results: a significant difference in the survival curves was detected according to the virus. mosquitoes exposed to chikv e1-226a or chikv e1-226v had lower survival rates than ni mosquitoes. as the trials differed significantly when ni mosquitoes, those exposed to chikv e1-226a or to chikv e1-226v were analyzed, trials were considered separately. using a parametric survival model, we found that for mosquitoes exposed to chikv e1-226v, ndinf mosquitoes, as well as dinf mosquitoes, were more at risk of dying than ni mosquitoes in both trials: and in trials and respectively; for dinf mosquitoes: and in trials and respectively). for mosquitoes exposed to chikv e1-226a, ndinf and dinf mosquitoes were more at risk of death than ni mosquitoes were, in both trials: for ndinf mosquitoes, and in trials and respectively; and for dinf mosquitoes: and in trials and respectively. oviposition characteristics time to first egg laying in ae. aegypti mayotte, the kaplan-meier estimates showed no significant difference in the time to egg laying according to the chikv strain or the infection status of the mosquito. combining chikv strain and infection status as a single factor, the parametric survival model showed no effect of these two factors on the time to egg laying. in ae. albopictus mayotte, the kaplan-meier estimates showed no significant difference in the time to egg laying when the virus or the infection status of the mosquito were examined. the parametric survival model did not show any effect of the virus and infection status of mosquito on the time to egg laying. in contrast, in ae. albopictus reunion, the kaplan-meier estimates showed significant differences in the time to egg laying when considering the virus and the infection status of mosquito. the parametric survival model showed a significant effect of the virus and infection status of mosquitoes on the time to egg laying. mosquitoes exposed to chikv e1-226v and chikv e1-226a laid eggs significantly earlier than ni mosquitoes. ± sd ± sd ±3 ae. aegypti and ae. albopictus from mayotte and the reunion island were infected with two chikv strains: chikv e1-226a and chikv e1-226v. n corresponds to the number of female analysed, sd to the standard deviation, and to respectively, trial and trial dinf to females with disseminated infection, ndinf to females with non-disseminated infection, and ni to non-infected females. number of eggs laid per mosquito the number of eggs laid according to the mosquito species, virus and infection status of the mosquito is described in table using a negative binomial regression model for each mosquito species, we did not find any evidence that the number of eggs laid differed according to the virus and infection status of the mosquito. time between first oviposition and mosquito death we also described the time between the first oviposition and mosquito death using kaplan-meier survival curves performed separately for each mosquito species. no difference according to the virus and infection status of the mosquito was observed in ae. aegypti mayotte and ae. albopictus mayotte. in ae. albopictus reunion, both trials led to the same conclusion: when compared to ni mosquitoes, the time was significantly shorter in mosquitoes infected with chikv e1-226a or chikv e1-226v irrespective of whether the infection status of the mosquito was ndinf or dinf. hatching characteristics proportion of mosquitoes with at least one egg hatched when examining the proportion of females with at least one egg hatched, no significant differences were found whatever the mosquito species, the virus and the infection status of the mosquito, when results were considered for ae. aegypti mayotte and ae. albopictus mayotte. in contrast, for ae. albopictus reunion a significant difference was found to be attribuable to ni mosquitoes. the table refers to the hatching characteristics of ae. aegypti and ae. albopictus from mayotte and the reunion island infected with two chikv strains: chikv e1-226a and chikv e1-226v. n corresponds to the number of female analysed, sd to the standard deviation, and to respectively, trial and trial dinf to females with disseminated infection, ndinf to females with non-disseminated infection, and ni to non-infected females. hatching rate per mosquito when the hatching rate per mosquito was examined, ae. aegypti mayotte mosquitoes were found to show a slight difference according to the viral strains and not to the infection status of the mosquito. similarly, ae. albopictus mayotte mosquitoes only showed a significant difference when viral strains were considered and not the infection status of the mosquito. finally, ae. albopictus reunion also showed differences related to viral strains and not to the infection status of the mosquito. in most cases, mosquitoes infected with chikv e1-226v displayed the highest hatching rates. eggs that issued from infected or non-infected mosquitoes maintained in a bsl- laboratory usually showed a lower capacity to hatch than eggs that were kept in an insectarium under standard conditions. replication of chikv in mosquitoes dead mosquitoes were collected once a day. the number of viral rna copies present in dead mosquitoes did not vary within a period of h after mosquito death. for ae. aegypti mayotte, mosquitoes had an average of viral rna copies per mosquito when a blood-meal containing chikv e1-226a was ingested, and an average of viral rna copies per mosquito that had ingested chikv e1-226v. following the blood-meal, viral load increased to reach a maximum of ± viral rna copies at day pi for chikv e1-226a and between day and day pi for chikv e1-226v. later, a slight decrease was observed until day pi. for both viruses, many mosquitoes died between day and day pi, especially at day for ae. aegypti mayotte infected with chikv e1-226v. for ae. albopictus mayotte, mosquitoes ingested an average of viral rna copies when exposed to a blood-meal containing chikv e1-226a and with chikv e1-226v. the maximum viral load was reached at day pi after ingestion of both chikv e1-226a and chikv e1-226v followed by a plateau until day pi. at day pi, the number of viral rna copies had decreased to for chikv e1-226a and for chikv e1-226v. in the case of ae. albopictus reunion, mosquitoes ingested an average of rna copies in the blood-meal containing chikv e1-226a and rna copies for chikv e1-226v. after a slight increase in the amount of viral rnas from day to day pi, a plateau was observed until day pi: ± with chikv e1-226a and ± with chikv e1-226v. dead females exhibited two distinct profiles: females which ensured a viral replication at a level similar to that of live females and females which did not ensure any viral replication. this is more clear-cut for alprov infected with chikv e1-226a. discussion whichever viral strain was examined, differential responses to chikv infection were found according to the mosquito species and their geographical origin. chikv infection affected the life-history traits of ae. albopictus from the reunion island. we have found that apart from the viral strains, chikv infection induced effects on survival, by reducing lifespan, and on oviposition characteristics, by shortening the time to egg laying of ae. albopictus from the reunion island. aedes albopictus is a mosquito species native to southeast asia that has spread beyond its natural habitat by means of intercontinental trades. this mosquito is able to transmit various arboviruses in laboratory conditions. in addition to this species, ae. aegypti is native to africa and comprises two forms: the forest-dwelling ae. a. formosus involved in the enzootic transmission of arboviruses, and the domestic form ae. a. aegypti whose distribution has extended over the tropical region acting as the main vector of dengue viruses. both species are container-breeding mosquitoes that share the same breeding sites. in asia, the introduced ae. aegypti tends to displace ae. albopictus from urban settlements, whereas the spread of ae. albopictus has been associated with declines of ae. aegypti in the americas. in the indian ocean region, ae. albopictus was introduced several centuries ago and in the reunion island, it was first recorded in. on the island, this mosquito became the most common aedes species since the control program against malaria vectors in the 1950s that led to the confinement of ae. aegypti in remote areas. on mayotte, ae. aegypti was first recorded in and ae. albopictus was observed later, in. larvae of both species could be found in artificial containers making inter-specific resource competition possible. ae. albopictus predominates in domestic habitats in mayotte. the chikv outbreak began in kenya in and subsequently spread to the islands of the indian ocean. a new strain of chikv emerged that carried a point mutation, which changes the amino acid at position in the e envelope glycoprotein from an alanine to a valine. this single mutation has increased the vector competence of ae. albopictus for chikv. this increased transmissibility of chikv is one of the hypotheses evoked to explain the expansion of chikv transmission into new regions such as madagascar and cameroon in and india, italy and gabon in prior to these recent chikv outbreaks, ae. aegypti had been considered the primary epidemic vector of chikv and ae. albopictus, a secondary vector. a single e1-a226v mutation was sufficient to increase the ability of chikv to infect ae. albopictus modifying the virus infectivity for a vector species other than the typical ae. aegypti vector. although the mutation confers a selective advantage in ae. albopictus, there was no corresponding advantage in ae. aegypti. our results showed that ae. aegypti mayotte was similarly susceptible to both viral strains, whereas ae. albopictus mayotte and ae. albopictus reunion were more susceptible to the chikv e1-226v than to the chikv e1-226a strain. this may explain the predominance of e1-226v in countries where ae. albopictus is considered to be the main vector species. at a titer of pfu/ml, e1-226v virus was disseminated more effectively by ae. albopictus than by ae. aegypti. infection of ae. aegypti mayotte requires that the chikv blood-meal titers are significantly higher than for infection of ae. albopictus, which suggests that once introduced, e1-226v would be positively selected by ae. albopictus. as an intracellular parasite, chikv can mediate changes in host life-history traits. two sorts of mechanisms could be proposed. on one hand, the use of host resources to maintain viral replication can lead to modifications of resource allocation as pathogenic effects. on the other hand, life-history modifications may be responses of the host to counterbalance the negative effects of parasitism. by reacting to the parasite, the host reduces the effect of infection on its fitness. we have shown that replication of chikv in aedes mosquitoes is intensive, e.g. more viral particles in females from day pi, and saliva became infectious from two days pi. in the present work, we have analyzed the effects of chikv replication on several life-history traits of aedes mosquitoes related to resource allocation, and only found significant modifications in ae. albopictus from the reunion island. thus chikv infection has a significant effect on the time until death and egg laying. moreover, such negative effects were independent of the viral strain: chikv e1-226a and chikv e1-226v displayed similar effects. nevertheless, aside from chikv infection,ae. albopictus from the reunion island better survived in bsl- laboratory conditions than the two other mosquito strains. regardless, infection by chikv has a negative impact on the survival of ae. albopictus from the reunion island. lifespan was shortened by 6- days when mosquitoes were infected with chikv e1-226a or chikv e1-226v. moreover, by shortening the time from blood-feeding to laying, chikv-infected ae. albopictus from the reunion island allocates an increased reproduction investment to laying eggs before death. thus only limited consequences may affect vector dynamics, as other factors dictating oviposition and hatching characteristics were only slightly altered by infection. such earlier reproduction has been reported in other animal-parasite systems. infection by a pathogen may modify resource distribution: infected hosts will allocate more resources to reproduction, subtracting them from those attributed to growth and survival. nevertheless, the question remains as to why this phenomenon is not species-specific. effectively, ae. albopictus from mayotte did not exhibit the same pattern. one explanation could be that ae. albopictus assumed to be introduced into the reunion island by immigrants from asia in the 17th-18th centuries has been subjected to major bottlenecks that cause strong genetic divergence from the asian mosquitoes. thus ae. albopictus from the reunion island may be genetically different from ae. albopictus detected in mayotte in. from our results, a very provocative scenario could be envisioned concerning chikv evolution during the indian ocean outbreak. since mayotte is located halfway between kenya, where the outbreak began, and the reunion island, where the chikv e1-a226v was first reported, this new variant could have already been present in mayotte. in mayotte, no data pertaining to chikv sequences was available before and chikv e1-226v was only identified after this time. in mayotte, both vectors co-exist and we clearly showed that ae. aegypti mayotte was similarly susceptible to both viral strains while ae. albopictus mayotte was more susceptible to the chikv e1-226v strain. the mutation could therefore be interpreted as a positive selection phenomenon for better transmission by ae. albopictus. in addition, we found that infection with chikv e1-226a or chikv e1-226v did not induce significant consequences on either species from mayotte. nevertheless, on the reunion island, the unique vector ae. albopictus, maintains a better transmission of chikv e1-226v compared to chikv e1-226a. however, an important decrease in the lifespan of ae. albopictus from the reunion island was reported as a consequence of infection followed by changes in mosquito reproduction. we propose that chikv e1-226a was maintained on mayotte by both ae. aegypti and ae. albopictus. later, the introduction of chikv e1-226a onto the reunion island where ae. aegypti is rare, would have led to the selection of the new variant by ae. albopictus. since then, chikv e1-226v has invaded some neighboring islands where ae. albopictus is present. thus, chikv e1-226v probably reached mayotte later, after having been selected in the reunion island. in addition, the role of other mutations in the viral genome in the adaptation of chikv to aedes mosquitoes could not be completely excluded. furthermore, some characteristics of the ae. albopictus bio-ecology might have favored chikv transmission in urban areas: high anthropophily and high population densities.  our results are in agreement with modifications in life-history traits reported for parasitized animals. we found that the species ae. albopictus was more susceptible to the chikv e1-226v than to the chikv e1-226a strain which assumes that once introduced, e1-226v would be positively selected by ae. albopictus. nevertheless, chikv infection only induced negative impacts on survival and on egg hatching characteristics of ae. albopictus from the reunion island which led us to suggest that ae. albopictus from the reunion island might be less adapted to chikv than mosquitoes from mayotte. however, the physiological mechanisms underlying this pattern are unknown. nevertheless, the outcome of the pathogen/host interaction often involves the immune system that is restricted to the innate immune function in insects. this is an effective mechanism to provide protection against a wide variety of pathogens including viruses. we have evaluated the number of viral rna copies in mosquitoes and found that females that died a few days after infection did not host more viral particles than did live females. thus we might suggest that mosquitoes did not die following an excess of viral replication but more probably when mounting an immune response. modifications of life-history traits and/or mounting an efficient antiviral response should be among strategies developed by the vector to optimize its fitness. 
the two aedes species co-exist in the indian ocean region and were involved in the 2005- chikv outbreaks. we performed experimental infections using three mosquito strains of ae. aedes aegypti and aedes albopictus are both vectors of chikungunya virus. albopictus reunion and not of the other two mosquito strains, and chikv reduced the lifespan of ae. albopictus reunion were more susceptible to chikv e1-226v than to e1-226a. in terms of life-history traits measured by examining mosquito survival and reproduction, we found that: differences were observed between responses of mosquito species to the two viruses, chikv infection only affected significantly some life-history traits of ae. in the reunion island, a single mutation in the viral envelope has been selected that leads to high levels of replication in ae. albopictus from the reunion island is not reduced since other parameters characterizing oviposition and hatching were not affected. albopictus, and two chikv strains. aegypti mayotte were similarly susceptible to both viral strains, whereas ae. albopictus reunion and shortened the time before egg laying. an important question is whether this variant is associated with adverse effects impacting some mosquito life-history traits such as survival and reproduction.
 aedes aegypti and aedes albopictus are both vectors of chikungunya virus. the two aedes species co-exist in the indian ocean region and were involved in the 2005- chikv outbreaks. in the reunion island, a single mutation in the viral envelope has been selected that leads to high levels of replication in ae. albopictus, and a short extrinsic incubation period as the virus could be found in saliva as early as two days after infection. an important question is whether this variant is associated with adverse effects impacting some mosquito life-history traits such as survival and reproduction.  we performed experimental infections using three mosquito strains of ae. aegypti mayotte and ae. albopictus, and two chikv strains. ae. aegypti mayotte were similarly susceptible to both viral strains, whereas ae. albopictus mayotte and ae. albopictus reunion were more susceptible to chikv e1-226v than to e1-226a. in terms of life-history traits measured by examining mosquito survival and reproduction, we found that: differences were observed between responses of mosquito species to the two viruses, chikv infection only affected significantly some life-history traits of ae. albopictus reunion and not of the other two mosquito strains, and chikv reduced the lifespan of ae. albopictus reunion and shortened the time before egg laying.  we demonstrated that chikv only reduces the survival of ae. albopictus from the reunion island. by laying eggs just before death, reproduction of ae. albopictus from the reunion island is not reduced since other parameters characterizing oviposition and hatching were not affected. 