Exposure to Zika and chikungunya viruses impacts aspects of the vectorial capacity of Aedes aegypti and Culex quinquefasciatus

Zika (ZIKV) and chikungunya (CHIKV) are arboviruses that cause infections in humans and can cause clinical complications, representing a worldwide public health problem. Aedes aegypti is the primary vector of these pathogens and Culex quinquefasciatus may be a potential ZIKV vector. This study aimed to evaluate fecundity, fertility, survival, longevity, and blood feeding activity in Ae. aegypti after exposure to ZIKV and CHIKV and, in Cx. quinquefasciatus exposed to ZIKV. Three colonies were evaluated: AeCamp (Ae. aegypti—field), RecL (Ae. aegypti—laboratory) and CqSLab (Cx. quinquefasciatus—laboratory). Seven to 10 days-old females from these colonies were exposed to artificial blood feeding with CHIKV or ZIKV. CHIKV caused reduction in fecundity and fertility in AeCamp and reduction in survival and fertility in RecL. ZIKV impacted survival in RecL, fertility in AeCamp and, fecundity and fertility in CqSLab. Both viruses had no effect on blood feeding activity. These results show that CHIKV produces a higher biological cost in Ae. aegypti, compared to ZIKV, and ZIKV differently alters the biological performance in colonies of Ae. aegypti and Cx. quinquefasciatus. These results provide a better understanding over the processes of virus-vector interaction and can shed light on the complexity of arbovirus transmission.

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Introduction
Complex interactions between vectors and arboviruses determine the vector competence of a species.These interactions act in association with biotic and abiotic factors; therefore, these aspects of a species' biological performance can determine its vectorial capacity [1].Among these parameters, some are related to reproductive and survival capacity [2].Studies have already demonstrated the influence of arbovirus infection on the reproductive capacity of females of different species [3][4][5][6][7][8][9].For example, the number of eggs produced in the first gonotrophic cycle indicates the total lay profile during the entire female life in mosquitoes [10].Changes in longevity and survival and changes in blood meal activity have also been associated with arbovirus exposure or infection [4,7,8].
The Zika (ZIKV) and chikungunya (CHIKV) viruses are RNA arboviruses from the Flaviviridae and Togaviridae families, respectively, which have quickly spread in recent years to various parts of the world, including Brazil [11][12][13].These pathogens are a major public health concern, as they cause infections in humans that can trigger neurological complications, such as the Guillain-Barré Syndrome [14] and the Congenital Zika Syndrome [15], as well as cardiac manifestations [16] and painful and disabling polyarthralgia [17].
Recife, a municipality located in the northeast region of Brazil, is known to have very favorable environmental conditions for mosquito reproduction and maintenance of high vector population densities.These conditions, when associated with vectorial capacity and presence of a susceptible human population, construct a scenario conducive to the rapid propagation of an arbovirus [18].Additionally, the ability of the species Ae. aegypti and Ae.albopictus to transmit ZIKV [19][20][21][22] and CHIKV [23][24][25], is an important factor for the spread of these viruses in the Americas [12,26].Also, it should be considered that other highly anthropophilic local species may be involved in arbovirus transmission as secondary vectors [27].Cx. quinquefasciatus, for example, is an abundant species in many parts of the world, including Brazil.However, most studies on its role in ZIKV transmission were only published as of 2016 [28][29][30][31][32][33][34].
In general, the information available on different aspects of the vectorial capacity of Aedes aegypti is not sufficient to explain the dynamics of arbovirus transmission, especially after pathogenic infections, considering that viruses modulate several parameters of the biological performance of vectors, and the mechanisms responsible for this modulation are also not well understood [9].Further studies addressing aspects of the vector capacity of species involved in arbovirus transmission can elucidate fundamental questions for the area, pointing out the real role of the vector in a given territory and in an epidemiological context.Thus, the present study aimed to evaluate the effect of exposure to ZIKV and CHIKV on fecundity; fertility; survival; longevity and blood meal activity in Ae. aegypti from the city of Recife (Pernambuco, Brazil) and in a laboratory colony, as well as the effect of ZIKV on Cx. quinquefasciatus, a laboratory colony, considering the same aspects, which are relevant for arbovirus transmission [4,7,[35][36][37].

Study area
The study was carried out in Recife (8º03'S 34º52'W), capital of the state of Pernambuco, Brazil, located in the northeast region.In the dry season, rainfall is scarce in the region, but it can be intense in the rainy season (from April to July).The temperature and relative air humidity range from 22 to 32ºC and from 70 to 90% at different times of the year [38].Recife is an hyperendemic area, with multiple arboviruses circulating simultaneously, and it has been considered as the epicenter of the first Zika outbreak in Brazil.

Mosquito samples
Three different mosquito populations were used in the present study: two laboratory colonies (RecL and CqLab) and one natural population (AeCamp).The laboratory colony of Ae. aegypti (RecL) has been maintained without contact with any larvicide or adulticide since 1996, when it was established from collections performed in the neighborhood of Graças, in Recife [39].The other Ae.aegypti population, called AeCamp, came from the field, and was established through egg collections performed between 2017 and 2018, in 13 neighborhoods in Recife (Santo Amaro, Várzea, Afogados, Dois Irmãos, Apipucos, Monteiro, Ipsep, Boa Viagem, Nova Descoberta, Vasco da Gama, Cidade Universitária and Mustardinha).Experiments with AeCamp were carried out with individuals of the F2 generation.As for the Culex species, we have used the Cx.quinquefasciatus laboratory colony, known as CqSLab, which was originated in the municipalities of Ipojuca, Olinda and Jaboatão dos Guararapes (Metropolitan Region of Recife), and it has been maintained since 2011 [40].
Field collections were performed using BR-OVT traps [38], as well as larvae and pupae, through shelling made directly in the breeding sites.All colonies were kept in the insectary of the Entomology Department of the Instituto Aggeu Magalhães (IAM-FIOCRUZ-PE), under controlled conditions of temperature (26 ºC ± 1 ºC), relative humidity (50 to 90%) and photoperiod (14:10 h -L/D).Larvae and pupae were kept in breeding containers with potable water and were fed cat food (friskies®).Adult mosquitoes were kept in aluminum mesh cages (50 x 40 cm) and were fed a 10% sucrose solution ad libitum on a daily basis.For females, blood feeding was offered weekly, in an artificial feeder, using defibrinated rabbit blood (Oryctolagus cuniculus).

Artificial bloodmeal for virus infection
Two to three independent experiments were performed with ZIKV and CHIKV.
For Ae. aegypti, each experiment was performed with two groups for each colony (RecL, AeCamp and CqSLab): exposed to the virus (E) and non-exposed (NE, the control group).
Two hundred adult females were used for the control groups (NE) and 300, for the test groups; they were aged between 7 and 10 days of emergence.In all groups, females were starved for 24 hours prior to oral exposure to the virus.
Oral blood-feeding was provided with an artificial feeder comprised of a Petri dish and Parafilm®, with a mixture of cultures of Vero cells inoculated with virus and defibrinated rabbit blood, in a volume of 10 mL, in a 1:1 ratio, as described in Guedes et al. [31].The stock virus dose used was 10 6 and 10 9 plaque forming units per ml (PFU/ml), for ZIKV and CHIKV, respectively.The negative control was a mixture of equal volume of virusfree cell culture and defibrinated rabbit blood.Approximately 0.5 ml of each mixture was aliquoted for further titration.Females were exposed to a blood meal for one hour.All females had three more blood meals devoid of virus, after exposure to the virus, with the objective of evaluating blood meal activity and keeping them in active gonadotropic cycles for a better evaluation of longevity.These blood meals were provided once a week for three consecutive weeks.

Assessment of the biological parameters after exposure to viruses
For the analysis of the putative biological cost, groups were defined according to the results of exposure to the viruses.Thus, for ZIKV, groups were divided into three: not exposed (NE); exposed, but not infected (E) and exposed and infected (EI).For CHIKV, groups were divided into two: not exposed (NE) and exposed and infected (EI), considering that, for this virus, infection rates were higher than ZIKV (above 90%), which made the number of exposed individuals and not infected not enough for statistical analysis (representing 4 and 8% for RecL and AeCamp, respectively).

Fecundity and fertility assessment
Approximately 24 hours after the blood meal, 50 engorged Ae. aegypti females from each experimental group were transferred to individual cages.On the 3 rd day postexposure (dpe), each cage received a container (measuring 3.14 cm 3 ) to mimic an oviposition site that contained 30% grass infusion and, in the case of the experiments with Ae. aegypti, posture supports made of cardboard, measuring approximately 4 x 2 cm.
In the Ae.aegypti assays, cardboards were changed twice a week, in three gonadotropic cycles.The collected eggs were counted through a stereomicroscope to determine the fecundity rate of each female.After 15 to 20 days of quiescence, the eggs were placed in recipients containing 2 mL of 30% grass infusion to stimulate the synchronous hatching of the larvae.These were counted to estimate the individual fertility rate of each female.Fecundity and fertility analyses were performed in the first gonadotropic cycle.
In the Cx.quinquefasciatus assays, the recipients were removed from the individual cages after each oviposition (once a week), in three gonotrophic cycles.The number of eggs present in each collected raft were determined with the aid of a magnifying glass, up to 24 hours after laying.Larvae hatching percentage was evaluated approximately 72 hours after oviposition, because the eggs of this species do not go into quiescence.

Survival and longevity assessment
Mortality notification was performed daily to assess survival and longevity.To detect viral infection, 10 females in each experimental group of Ae. aegypti, were collected at three time points: 7; 14 and 21 dpe (days post exposure).There was a total of 20 females from each group until the end of life.For these, FTA -classic card Whatman ® cards (cards), containing Manuka honey blend ® honey, were made available on the screens of the cages, from the 7th to the 14th dpe.All females contributed to survival, and longevity assessments up to the point at which they left the study.For the analysis of average lifetime (longevity), females collected on the 7th, 14th and 21st dpe were excluded.
Cx. quinquefasciatus females were only collected when death occurred throughout the experiments.Additionally, FTA classic card (Whatman® card) containing Manuka honey blend® were made available on the screens of all cages, from the 7th to the 14th dpe to ensure viral detection, through saliva collection, since females had not been collected on 7th, 14th and 21st dpe, as performed for Ae.aegypti.
Survival and longevity analysis was also performed among Ae.aegypti females in relation to viral load, represented by the RNA copy number (CN), detected by RT-qPCR.
A cut-off point was determined to define the groups (with the highest and lowest viral loads) based on the median CN values found for the infected females.

Search for blood meal
To assess the influence of ZIKV exposure on the blood meal activity of Ae. aegypti, females were fed with blood devoid of virus at 7, 14 and 21 dpe.After each feeding event, the completely engorged females were selected and counted.For Cx. quinquefasciatus exposed to ZIKV and Ae.aegypti exposed to CHIKV, evaluations were carried out exclusively with the first post-exposure blood meal (7 dpe).

RNA extraction and RT-qPCR
Females collected at 7, 14 and 21 dpe, as well as those that died during the study, were placed separately in 1.5 ml microtubes, containing 300 µl of a mosquito diluent and stored at -80 ºC until RNA extraction and RT-qPCR, described in Guedes et al. [31], with some modifications.The primers used for detection of CHIKV and ZIKV viral particles are described in Lanciotti et al. [42,43].Virus detection was performed by quantitative RT-qPCR on a QuantStudio ® 5 Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA), according to conditions described in Guedes et al. [31].Cycle threshold values (Ct) were used to estimate the amount of viral RNA, using the standard curve as a reference for each RT-qPCR assay, obtained through isolated transcripts from the ZIKV BRPE243/2015 and CHIKV PE480/2016 strains, as described in Kong et al. [44].Negative controls for the feeding experiment and RT-qPCR consisted of mosquitoes fed with virus-free blood and water, respectively.Whole mosquitoes were processed, except for Ae.aegypti collected at 7 dpe, whose abdomens and thorax were analyzed separately from the heads.To calculate the infection rate (IR), the number of positive females was divided by the total number of analyzed samples.To calculate the dissemination rates (DR), head samples or positive cards were used, divided by the total number of positive samples.Females and cards with Ct lower than 38 were considered as positive.
The FTA cards were placed in 1.5 mL tubes and stored at -80 °C until use.To prepare the inoculum, cards were cut using multipurpose scissors and placed in 1.5 mL tubes.Next, 400 µL of ultrapure water was added to each tube, following homogenization for 5 times for 10 seconds, with 5 minute-intervals.Finally, the cards were transferred to a 10 mL syringe and filtered to enable recovery of the eluate only.The prepared inoculums were stored at -80 °C until RNA extraction and RT-qPCR were performed, following the same protocol used for detection of viral RNA in mosquitoes.

Statistical analysis
A descriptive analysis was performed: the variables were presented through graphs, followed by the presentation of the confidence interval and the p-value.Normality assumptions were made by applying the Shapiro Wilk tests.To assess the differences in means for the independent variables, the T-Student test was used, when the assumptions of normality were met.Otherwise, the Mann-Whitney test was applied, and the medians were evaluated.Also, the Bartlett test was used to assess homogeneity.When the assumption of homogeneity was met, the ANOVA mean test was used with Tukey's post hoc test; if not, the median was evaluated by applying the Kruskal-Wallis' test, with the post hoc test for Fisher's test.The survival curve was determined using the Kaplan-Meier plot.The Cox test was applied to assess survival, and the proportionalities were evaluated using the Schoenfeld test.Conclusions were taken at a significance level of 5%.Results of the analysis were obtained using the R Core Team (2022).R: A language and environment for statistical computing.R Foundation for Statistical Computing, Vienna, Austria.URL https://www.R-project.org/

Infection and dissemination rates of Zika (ZIKV) and chikungunya (CHIKV) viruses
Infection and dissemination rates varied between the viruses and colonies evaluated, being higher in the experiments carried out with the chikungunya virus (IR of 96.00% for RecL and 93.00% for AeCamp).The dissemination rates of the same virus ranged between 100.00% and 94.00% for RecL and AeCamp, respectively (Table 1).
With Zika virus, these values were lower in CqSLab (22.50% IR and 57.00% DR), in relation to the two colonies of Ae. aegypti (Table 1).The percentage of positive cards varied among the colonies and viruses analyzed.
In mosquitoes infected with ZIKV, the percentage rate was 30% for RecL, and 60% for AeCamp, while for CqSLab from the first experiment, the percentage was low: 14.28%.
No evaluation was possible for the cards from the second and third experiments carried out with this colony because contamination was detected in the samples.Surprisingly, no cards were found to be positive for CHIKV in the two colonies (RecL and AeCamp), and we have no explanation for this.
The ZIKV viral load (number of RNA copies per mL -CN) was significantly higher (p = 0.019) among females from the RecL colony who underwent a second blood meal in blood free of viral particles, at 7 dpe).The mean number of RNA copies increased from 4.31E+11 among those who did not have a second meal, to 5.81E+11 among those who ingested blood at 7 dpe.As a function of time of life after infection, it was found that the CN was significantly higher among RecL females that had completed engorgement at 7 dpe (p = 0.008) and died between the 8th and 22nd dpe (Fig 1).Although it was not statistically significant, there was an increase in CN in the period between 8 and 22 days in AeCamp (Fig 1).

Biological cost of infection with Zika (ZIKV) and chikungunya (CHIKV) viruses
In general, ZIKV and CHIKV had a significant impact on the parameters of vectorial capacity of the evaluated colonies.This impact negatively altered the reproductive capacity of females exposed to artificial oral infection by these arboviruses.

Survival and Longevity
The analysis of the survival curve of the groups exposed to ZIKV, showed that the risk of death for females from RecL was about twice as high (E =

Blood Feeding Activity
The blood meal activity of RecL, AeCamp and CqSLab colonies was not altered by exposure to ZIKV and CHIKV, regarding the search for a second blood meal.Detailed numbers are shown in Table 2.

Discussion
Relevant aspects of the biological performance of vector mosquitoes can be altered by the process of pathogen infection, e.g., parameters involved in vectorial capacity.This event may lead to a consequent change in the pattern of occurrence of an epidemic in a given epidemiological context [45].Studies have shown the biological cost of arbovirus infection, namely reduced survival, longevity, reproductive capacity, blood meal activity, among other impacts in mosquitoes.The different results found between them are explained by the direct relationship between the virus lineages and vectors involved [3,[5][6][7][8]46].Significant changes in reproductive capacity, for example, can limit the number of offspring of infected females and determine the transmission dynamics of an arbovirus.In general, this dynamic also occurs as a function of the age at which the vector acquires the infection, as well as survival and longevity [5,6,45].
In this sense, the two species investigated here, suffered a negative impact on biological performance, especially on reproductive capacity, after exposure to Zika (ZIKV) or chikungunya viruses (CHIKV), with a reduction in the number of individuals for the subsequent generation.This has evolutionary implications, once any trait that may offer an adaptive advantage for the mosquito's defense against viral infection is unlikely to be selected for.However, the ability of a vector to transmit a pathogen is multifactorial and, therefore, isolated assessments in any of its parameters must be made with caution [45].
Our results showed that the natural population (AeCamp) did not suffer any impact on its survival or longevity caused by the infection of both viruses.This may suggest that infected mosquitoes can keep transmitting the virus for long periods in the environment.The interaction between vectors and viruses, genetically determined and triggered by the adaptive process in an environment, can result in a more efficient transmission dynamics, with less impact on the biological performance of Ae. aegypti [46].
On the other hand, for the Ae.aegypti colony, RecL, there was a significant reduction in survival when it was exposed and infected with both viruses.This result demonstrates a greater fragility of this colony, since the infection and dissemination rates for the two viruses were always higher when compared to the field population.It is known that the colonization process causes genetic diversity loss, which may impact on mosquito defense, metabolism, development, among other important traits for mosquito survival.
Therefore, the results found in studies using laboratory colonies should always be interpreted with caution, before being extrapolated to what actually occurs in nature.The reduction in the survival rate and number of eggs laid by Ae. aegypti was associated with the process of adaptation to CHIKV infection, in a study developed by Sirisena, Kumar and Sunil [6].The authors demonstrated that there is a negative regulation of genes involved in the egg laying pathway in infected females, through the analysis of transcript expression [6].
Previous studies have addressed cost on longevity [4] and survival [46] of Ae.
aegypti populations infected by ZIKV.Petersen et al. [4], for example, used the ZIKV strain BRPE243/2015, the same strain used in the present study, to assess the reproductive capacity and longevity of Ae. aegypti collected in Rio de Janeiro, and they found a negative impact of the infection on the longevity of females.Thus, owing to genetic background and environmental factors, the viral strain used for infection in the laboratory should be considered, because especially in Ae. aegypti, the interaction between pathogen and vector can vary even between geographically close populations [46].
Similarly, the longevity of Ae. aegypti from Palm Beach County, Florida was also unaltered using the CHIK strain LR2006-OPY1.However, the same study suggests that physiological restrictions on the evolution of CHIKV infection in Ae. albopictus result in biological cost on this species, considering that they found a significant reduction in longevity.Additionally, the body titer of female Ae.albopictus infected with CHIKV and longevity were inversely correlated [47].
The number of eggs laid in the first gonotrophic cycle was not altered by exposure and infection with ZIKV in the two colonies of Ae. aegypti.On the other hand, the fertility of the field colony, AeCamp, was significantly reduced even among females exposed to ZIKV and that did not develop the infection.According to Li et al. [48], the ovaries of mosquitoes are affected by ZIKV on the second day after infection.Despite this fact, ZIKV did not reduce the fecundity of Ae. aegypti, as also found by Padilha et al. [3], Resck et al. [5] and Silveira et al. [46].In contrast, a field population in Rio de Janeiro showed a reduction in fecundity.Interestingly, in the same study, one of the groups infected by ZIKV showed an increase in this parameter, between the first and third gonadotropic cycles [4].
The negative impact on AeCamp fertility suggested that the cost resulting from exposure to the virus was directed to the viability of the eggs, regardless of the establishment of the infection.Resck et al. [5] did not find an impact of ZIKV infection on the fertility of females from an Ae.aegypti from laboratory, corroborating the results reported in this paper for RecL.However, they differ from those found for the field colony.As demonstrated by Ciota et al. [49], the adaptive process is a determining factor for viral replication, in the case of successful infection, with subsequent increase in the immune response and consequent impact on the biological performance of the mosquito vector.This hypothesis explains the results regarding the fertility of AeCamp, which had contact with the strain used in these experiments during the Zika fever epidemic.
Parameters of reproductive capacity, especially fertility, showed a biological cost for infection with CHIKV, for both colonies, from the laboratory or field.Resck et al. [5] evaluated the reproductive capacity of Ae. aegypti after infection with CHIKV, and found a negative effect on fertility, but not on fecundity.A similar result was found with the CHIKV strain from Italy and Ae.albopictus from the same area.This alteration in fertility suggests that CHIKV can affect embryonic development and embryo survival [35] as described for other viruses [4,7].In contrast, Carvalho-Leandro et al. [50] found no change in the reproductive capacity of Ae. aegypti infected with DENV, although the ovaries showed similar titers to other organs.The relevant effect of CHIKV infection on fertility, found in this study, suggests the need for further investigation, as this seems to be a critical point in the vector/parasite interaction mechanism that may guide the development of new vector control strategies.
For Cx. quinquefasciatus (CqSLab), survival and longevity were not affected by ZIKV exposure or infection.Styer, Meola and Kramer [7] found no difference in this aspect of vectorial capacity between Cx. tarsalis females, in a laboratory colony, after exposure to West Nile virus (WNV).The survival of Cx. tarsalis, however, was altered by Western equine encephalitis virus (WEEV) infection [51].Exposure to ZIKV, but not infection, impacted the reproductive capacity of females, significantly reducing fecundity and fertility.For a laboratory colony of Cx. pipiens, pre-selected by continuous exposure to a WNV strain, fecundity was also altered; however, unlike what was found in the present study, infection, not just exposure, reduced this parameter [49].The fecundity and fertility of Cx. tarsalis infected by WNV was also reduced in the infected groups [7].
Additionally, the same authors reported that the percentage of larvae hatching was higher in the exposed group (65.8%), than in the non-exposed (55.6%) and exposed and infected groups (42.5%).
The results analyzed for post-exposure blood feeding activity in an artificial feeder suggest that there is no effect of exposure or infection on the search for subsequent blood feeding in Ae. aegypti and Cx.quinquefasciatus, for both viruses and colonies studied here, considering the criterion evaluated (percentage of females that completed the blood meal within 30 minutes of exposure to the feeder).On the other hand, when evaluating the time spent by females of Ae. aegypti to complete engorgement, Sylvestre, Gandini and Maciel-de-Freitas [8] found that infected females spent more time compared to the non-exposed group.Additionally, a higher percentage of females of Cx. tarsalis infected with WNV had a blood meal on artificial feedings after infection, compared with unexposed females and exposed females that had not been infected.However, the same authors reported no significant difference in the amount of blood ingested between the three groups evaluated [7].
In general, the findings suggest that exposure to ZIKV and CHIKV significantly impact the reproductive capacity and longevity of the colonies evaluated.CHIKV had a greater impact on Ae. aegypti, in comparison to ZIKV, considering that the former reduced both parameters of the reproductive capacity of the field mosquitoes as well as the fecundity of RecL.The results also indicate that ZIKV differently impacted reproductive capacity when comparing Ae. aegypti and Cx.quinquefasciatus exposed and/or infected.Only exposure to ZIKV, but not infection, was enough to reduce the fecundity and fertility of Cx. quinquefasciatus females, suggesting that the triggering of defense mechanisms associated with the midgut barriers generates a biological cost for the species.Although Cx. quinquefasciatus showed much lower infection and dissemination rates than Ae.aegypti, this species is much more abundant in the environment in Recife; thus, its role in ZIKV transmission is not clear yet.In Ae. aegypti, ZIKV infection reduced the fertility of field females, but not their fecundity.However, this impact may have little relevance, considering that longevity, survival and search for blood source after exposure to the virus were not affected in the field population.
In the colony of Ae. aegypti, females infected with ZIKV, which had a second blood meal at 7 dpe, had a significantly higher number of RNA copies, compared to those that did not have a second meal.Similarly, Cui et al. [52] reported a three to four-fold increase in viral load among Ae.aegypti infected with DENV-4 after blood feeding at 5 dpe.However, owing to the limited number of females analyzed for this aspect, new experiments should be carried out, as they may allow a better statistical evaluation of this relationship, involving other variables to possibly confirm this result.
In summary, despite the significant reduction in some aspects of the biological performance of Ae. aegypti, for both study viruses, and for Cx.quinquefasciatus, with respect to ZIKV, it is suggested that the vectorial capacity of these species, supported by a successful global biological performance, close relationship with the host and availability of oviposition sites, poses a serious threat to public health, when associated with the susceptibility of the human population and the successful interaction with the pathogen.These results may, above all, increase the knowledge about the biological performance of mosquitoes in endemic and epidemic situations, in order to facilitate the definition and implementation of more effective methods of control of vector populations.
Such knowledge can also trigger the development of future investigations, with a view to elucidating the several gaps that still exist on the various aspects of the vectorial capacity of mosquitoes.
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Fig 1 .
Fig 1. Viral load (number of RNA copies of ZIKV -NC, per mL, detected by RT-

Fig 4 .
Fig 4. Average number of eggs laid and average percentage of larval

Fig 5 .
Fig 5. Mean or median number of eggs and median percentage of hatching of

Table 2 .
Number and percentage of females of Aedes aegypti, from colonies RecL and AeCamp, and of Culex quinquefasciatus, from colony CqSLab, which completed the blood meal in weeks following exposure to Zika virus (ZIKV) and from colonies RecL and AeCamp exposed to chikungunya (CHIKV), in blood free of viral particles.