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 causeclinical complications, representing a worldwide public health problem. Aedes aegypti is the primary vector of these pathogens and Culex quinquefasciatusmay 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. quinquefasciatusexposed 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 fertilityinthe natural population, 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.

For the analysis of the putative biological cost, groups were defined according to 135 the results of exposure to the viruses. Thus, for ZIKV, groups were divided into three: 136 not exposed (NE); exposed, but not infected (E) and exposed and infected (EI). For 137 CHIKV, groups were divided into two: not exposed (NE) and exposed and infected 138 (EI), considering that, for this virus, infection rates were higher than ZIKV (above 139 90%), which made the number of exposed individuals and not infected not enough for 140 statistical analysis (representing 4 and 8% for RecL and AeCamp, respectively).  To assess the influence of ZIKV exposureon the blood meal activity of Ae. 182 aegypti, females were fed with blood devoid of virus at 7, 14 and 21 dpe. After each 183 feeding event, the completely engorged females were selected and counted. For Cx. 184 quinquefasciatusexposed to ZIKV and Ae. aegypti exposed to CHIKV, evaluations were 185 carried out exclusively with the first post-exposure blood meal (7 dpe).

187
Females collected at 7, 14 and 21 dpe, as well as those that died during the 188 study, were placed separately in 1.5 ml microtubes, containing 300 µl of a mosquito with Ct lower than 38 were considered as positive.

205
The FTA cards were placed in 1.5 mL tubes and stored at -80 °C until use. To 206 prepare the inoculum, cards were cut using multipurpose scissors and placed in 1.5 mL 207 tubes. Next, 400 µL of ultrapure water was added to each tube, following 208 homogenization for 5 times for 10 seconds, with 5 minute-intervals. Finally, the cards 209 were transferred to a 10 mL syringe and filtered to enable recovery of the eluate only.

210
The prepared inoculums were stored at -80°C until RNA extraction and RT-qPCR were 211 performed, following the same protocol used for detection of viral RNA in mosquitoes.

213
A descriptive analysis was performed: the variables were presented through 214 graphs, followed by the presentation of the confidence interval and the p-value. 215 Normality assumptions were made by applying the Shapiro Wilk tests. To assess the 216 differences in means for the independent variables, the T-Student test was used, when 217 the assumptions of normality were met. Otherwise, the Mann-Whitney test was applied, 218 and the medians were evaluated.Also, the Bartlett test was used to assess homogeneity.

219
When the assumption of homogeneity was met, the ANOVA mean test was used with 220 samples. Surprisingly, no cards were found to be positive for CHIKV in the two 248 colonies (RecL and AeCamp), and we have no explanation for this.

249
The ZIKV viral load (number of RNA copies per mL -CN) was significantly 250 higher (p = 0.019) among females from the RecL colony who underwent a second blood 251 meal in blood free of viral particles, at7 dpe). The mean number of RNA copies 252 increased from 4.31E+11 among those who did not have a second meal, to 5.81E+11 253 among those who ingested blood at 7 dpe. As a function of time of life after infection, it 254 was found that the CN was significantly higher among RecL females that had 255 completed engorgement at 7 dpe(p = 0.008)and died between the 8th and 22nd dpe (Fig   256   1). Although it was not statistically significant, there was an increase in CNin the period 257 between 8 and 22 days in AeCamp (Fig 1).  The analysis of the survival curve of the groups exposed to ZIKV, showed that 280 the risk of death for females from RecL was about twice as high (E = 1.845: p = 0.014 281 and EI = 2.014: p = 0.003), compared to the control group (non-exposed -NE) (Fig 2A   282 and S1-S3 Tables). Mean lifespans for the three groups did not differ significantly: there was no significant difference in survival between the three groups analyzed, as the 285 risk of death was 1,289 for group E and 1,212 for EI (Fig 2B and S1 NE, E and EI, respectively) were not altered among females exposed to ZIKV ( Fig 2C   290 and S1-S3 Tables).  showed an impact of infection on survival, with a higher risk of death (3.963) for the EI 299 group (Fig3A and S1-S3 Tables); however, this difference appeared only between day 300 zero and the 20th day of observation (p = 0.001), i.e., it was not found after the 21st day 301 (Fig 3B). The longevity of RecL females was also reduced by CHIKV infection:38 and 302 17 days for NE and E, respectively (p = 0.002). On the other hand, in AeCamp, the 303 survival curves showed no statistical difference between the two study groups (Fig 3C   304 and S1-S3 Tables). Longevity was 37.5 days for the control group (non-exposed -NE) 305 and 32 days for the EI group.  Note: NE -Control (non-exposed); E -exposed; EI -exposed and infected. A: RecL 333 colony; B: AeCamp colony; C:CqSLab colony. significantly altered by the infection with CHIKV in the first gonadotropic cycle. 336 However, the infection impacted fertility, with a reduction in the median percentage of 337 hatching from 63.48 % in group NE to 40.67 % in group EI (Fig 5). Differently, in 338 AeCamp, CHIKV had an impact on fecundity, reducing the median number of eggs 339 from 48 in NE to 38 in group EI. The fertility of this colony was also altered by the 340 infection, with a reduction in the median percentage from 57.50 to 37.50, between 341 groups NE and EI, respectively (Fig 5). Note: NE -Control (non-exposed); EI -exposed and infected. A: RecL colony; B: 348 AeCamp colony.

349
The blood meal activity of RecL, AeCamp and CqSLab colonies was not altered 351 by exposure to ZIKV and CHIKV, regarding the search for a second blood meal.

352
Detailed numbers are shown inTable 2.  capacity. This event may lead to a consequent change in the pattern of occurrence of an 362 epidemic in a given epidemiological context [45]. Studies   unlikely to be selected for. However, the ability of a vector to transmit a pathogen is 376 multifactorial and, therefore, isolated assessments in any of its parameters must be made 377 with caution [45].

378
Our results showed that the natural population (AeCamp) did not suffer any  The fecundity and fertility of Cx. tarsalis infected by WNV was also reduced in the 456 infected groups [7]. Additionally, the same authors reported that the percentage of 457 larvae hatching was higher in the exposed group (65.8%), than in the non-exposed 458 (55.6%) and exposed and infected groups (42.5%).

459
The results analyzed for post-exposure blood feeding activity in an artificial 460 feeder suggest that there is no effect of exposure or infection on the search for