Survival effects of antibiotic exposure during the larval and adult stages in the West Nile virus vector Culex pipiens

The ability of mosquitoes to transmit a pathogen is affected, among other factors, by their survival rate, which is partly modulated by their microbiota. Mosquito microbiota is acquired during the larval phase and modified during their development and adult feeding behavior, being highly dependent on environmental factors. Pharmaceutical residues including antibiotics are widespread pollutants potentially being present in mosquito breeding waters likely affecting their microbiota. Here, we used Culex pipiens mosquitoes to assess the impact of antibiotic exposure during the larval and adult stages on the survival rate of adult mosquitoes. Wild‐collected larvae were randomly assigned to two treatments: larvae maintained in water supplemented with antibiotics and control larvae. Emerged adults were subsequently assigned to each of two treatments, fed with sugar solution with antibiotics and fed only with sugar solution (controls). Larval exposure to antibiotics significantly increased the survival rate of adult females that received a control diet. In addition, the effect of adult exposure to antibiotics on the survival rate of both male and female mosquitoes depended on the number of days that larvae fed ad libitum in the laboratory before emergence. In particular, shorter larval ad libitum feeding periods reduced the survival rate of antibiotic‐treated adult mosquitoes compared with those that emerged after a longer larval feeding period. These differences were not found in control adult mosquitoes. Our results extend the current understanding of the impact of antibiotic exposure of mosquitoes on a key component of vectorial capacity, that is the vector survival rate.


Introduction
Vector-borne pathogens are currently a major concern due to their impact on public health (WHO, 2020), livestock production (Ndiva Mongoh et al., 2008;Pendell et al., 2016), and wildlife (Folly et al., 2020).Among temperature, humidity and larval nutrition (Lefèvre et al., 2013;Carvajal-Lago et al., 2021), among others.Additionally, recent studies identified the mosquito microbiota, which is the microbial community that lives in contact with the mosquito epithelia, as a major factor affecting different components of the vectorial capacity, through their effects on the mosquito lifespan and pathogen development (Caragata et al., 2013;Martínezde la Puente et al., 2018;Cansado-Utrilla et al., 2021).Mosquito larvae partially obtain the microbiota from their breeding sites and, therefore, it is highly dependent on the environment (Coon et al., 2014).After emergence, adult mosquitoes maintain part of the larval microbial community (Lindh et al., 2008) but also acquire new symbionts from their diet (Muturi et al., 2019;Sarma et al., 2022).
Antibiotics and other pharmaceutical residues are widespread pollutants in freshwater worldwide (Wilkinson et al., 2022), with mosquitoes and other aquatic organisms being commonly exposed to them (Endersby-Harshman et al., 2019).In addition, adult mosquitoes feeding on animals treated with antibiotics, including humans, may be also exposed to these pollutants (Gendrin et al., 2015).Antibiotic exposure may largely affect the composition of the mosquito microbiota and, consequently, affect their lifespan and their competence for the transmission of vector-borne pathogens (e.g., Dong et al., 2009;Gendrin et al., 2015;Martínez-de la Puente et al., 2021).In addition, antibiotic treatments may also affect mosquito fecundity (Ha et al., 2021), blood digestion and egg production (Gaio et al., 2011), and the larval development period (Chouaia et al., 2012).Despite the importance of antibiotics in the epidemiology of mosquito-borne pathogens, studies so far have mainly focused on female mosquitoes belonging to the genera Anopheles (Dos Santos et al., 2022) and Aedes (Gómez-Govea et al., 2022), with very limited information available for other major mosquito vector groups.This is especially the case of the carryover effects of antibiotic exposure during the larval and adult stages on adult survival that, to our knowledge, have not been investigated in detail.
Here we assessed the synergistic impact of antibiotic exposure during the larval and adult stages on the survival rate of both male and female adult mosquitoes.We used wild-collected Culex pipiens mosquitoes due to: (i) the abundance of this species in human habitats in the temperate northern hemisphere (Haba & McBride, 2022) and (ii) their role as major vectors of pathogens such as West Nile virus and avian malaria parasites (Santiago-Alarcón et al., 2012;Engler et al., 2013;Gutiérrez-López et al., 2020).We predict that exposing Cx. pipiens mosquitoes to antibiotics solely during the adult stage will enhance survival rates of mosquitoes, as suggested by prior research (Gendrin et al., 2015;Martínez-de la Puente et al., 2021;Santos et al., 2022).However, we hypothesize that exposure to antibiotics during both larval and adult stages will negatively affect adult survival rates, as this treatment may lead to a more profound alteration of the microbiome, potentially removing crucial bacterial taxa.In our analyses, we control for the potential effect of the development time of mosquitoes feeding ad libitum as larva, because this variable may determine the nutrition and size of adults with potential consequences on the survival rate of mosquitoes (Carvajal-Lago et al., 2021).

Mosquito collection and rearing conditions
We collected Cx. pipiens larvae in September 2022 from two sampling points in a rural area in Granada, southern Spain.The larvae were then placed in separate sterilized plastic trays containing 1.5 L of dechlorinated water according to treatment (see below), collection date and sampling point.Each tray was kept in a distinct incubation tent and fed ad libitum with 40 mg of shredded fish food (JBL Propond All Seasons S ® ) per liter of dechlorinated water.Food was provided to larvae every day.Emerged adults were isolated in insectaries according to emergence date, sampling point, sex and treatment as larvae.From emergence until assignment to the different experimental groups, mosquitoes were provided ad libitum with sterilized, dechlorinated 10% sugar solution.Larvae and adult mosquitoes were maintained in a climatic chamber under controlled conditions at a mean temperature of 26.7 °C (Range: 25.2−27.7 °C), with a mean relative humidity of 58% (range: 52%−67%) and a 16 : 8 light : dark photoperiod cycle.

Experimental design and antibiotic treatments
We used a paired two-way factorial design to investigate how antibiotic exposure during the larval and/or adult stages affected the survival rate of female and male adult mosquitoes.Larvae from each sampling point and collection date were separated into two experimental groups, each placed in separate trays and incubation tents: (i) larvae in dechlorinated water (larvae control group), and (ii) larvae in dechlorinated water with antibiotics (larvae antibiotic-treated group).For the antibiotictreated group, we added a single dose of antibiotics to the dechlorinated water at the beginning of the experiment to a final concentration of 0.6 µg of gentamicin sulfate (Sigma-Aldrich, Stockholm, Sweden), and 1.2 units/1.2µg of penicillin-streptomycin (Gibco TM , Grand Island, NY, USA) per liter of water solution.These concentrations are in line with those reported in treated wastewater (Mutuku et al., 2022).
Adults that emerged during the first day after capture were discarded in order to include in the experiment only mosquitoes that were exposed to antibiotics for at least 24 h.The rest of mosquitoes were extracted from each tent every 1−3 d, anesthetized with ether, sexed and identified to the species level following Schaffner et al. (2001).Culex pipiens mosquitoes of each sex and experimental origin (control and antibiotic-treated larvae) were divided into two experimental groups: (i) adults fed with sterilized 10% sugar solution (adult control group), and (ii) adults fed with sterilized 10% sugar solution and antibiotics (adult antibiotic-treated group).The adult antibiotic treatment included 15 µg gentamicin sulfate and 10 units/10 µg of penicillin-streptomycin per milliliter of water solution (Dong et al., 2009;Martínez-de la Puente et al., 2021).The sugar solution with or without antibiotics was replaced every day to avoid product degradation.To avoid density being a confounding factor, we used a paired experimental design, assigning the same number of individuals (±1; average = 22.32, SD = 5.43) to each replicate of control and antibiotic treatments for males and females emerged at the same date (Table 1).We monitored the daily mosquito survival rate of adult mosquitoes for 68 d postemergence.

Statistical analysis
To test the effect of antibiotic treatment on mosquito survival rate, we generated survival curves using Kaplan-Meier estimates, and employed a Cox proportionalhazards model to fit the data (estimated as the probability of a mosquito of surviving 24 h).We included three independent variables in the model: treatment of larvae (control or antibiotic-treated larvae), treatment of adults (control and antibiotic-treated adults), and the number of days that the larvae remain in the trays feeding ad libitum until their emergence.We performed a backward stepwise model selection beginning with the model including each independent variable and all the interactions among them.Then, we simplified the models by removing the least significant variable or interaction at each step until the coefficient of all variables and interactions were significant (P ≤ 0.05).We used Bonferroni correction for multiple-comparisons among the different levels of the interactions included in the final model.Separate models were constructed for female and male mosquitoes.Analyses were conducted in R (R Core Team, 2022), using the survival (v3.3-1;Therneau, 2022) and survminer (v0.4.9;Kassambara et al., 2021) packages.

Results
We monitored the daily survival rate of 2723 adult mosquitoes corresponding to 122 replicates (14−17 insectaries per sex and larvae-adult treatment; Table 1).Results of the models for both females and males are summarized in Table 2.
For female mosquitoes, the final model included the interactions between larval and adult treatments and between adult treatment and larval feeding period (Table 2).Post hoc analyses revealed that the only significant differences were found in control adult females with respect to the antibiotic treatment received during the larval stage.In particular, control females treated with antibiotics during the larval stage showed a higher survival than those treated as controls (Fig. 1; Z = 3.07, P = 0.01).Nonsignificant differences between the treatments during the larval stage were found in female mosquitoes treated with antibiotics as adults (Fig. 1; Z = 0.25, P = 1), or between the different adult treatments for larvae treated as controls (Fig. 1; Z = 1.14, P = 1), and for those treated with antibiotics (Fig. 1; Z = −1.68,P = 0.55).Regarding the interaction between adult treatment and larval ad libitum feeding period, a shorter larval ad libitum feeding period reduced the survival rate of mosquitoes when they were exposed to antibiotics as adults, while adult females fed with the control diet showed a similar mortality rate regardless of the larval ad libitum feeding period (Fig. 2; Table 2).
For male mosquitoes, the final model included the interaction between adult treatment and the larval ad libitum feeding period (Table 2, Z = −3.95,P < 0.0001) showing a similar pattern to that found in females.Shorter larval feeding periods rapidly reduced the survival rate of adults when they were subsequently exposed to antibiotics, but as larval feeding period increased, the Table 2 Summary of the adjusted Cox models for female and male mosquitoes showing the coefficient (Coef.)and its standard variation (Coef.Std.Dev.), the hazard ratio (HR), Z statistic (Z), and adjusted P value (P) corresponding to each variable or their interactions included in the final model: larval treatment (LT), adult treatment (AT), and larval ad libitum feeding period (LFP).For the larval and adult treatments, the reference level was control and, therefore, the coefficient was computed for the antibiotic-treated level (A).

Variable
Coef Fig. 1 Kaplan-Meier curve showing the survival rate of Culex pipiens female mosquitoes according to their treatments during the larval and adult stages.Blueish curves correspond to control larvae and reddish curves to antibiotic-treated larvae; darker curves correspond to control adults and lighter curves to antibiotic-treated adults.Significant differences were found only between control adults that were treated and those that were not treated (control) with antibiotics at the larval stage.

Discussion
The habitat conditions during the larval stage, such as food availability and the presence of pollutants, can affect different mosquito traits including survival rate and vector competence (Lefèvre et al., 2013;Carvajal-Lago et al., 2021;Neff & Dharmarajan, 2021).However, despite the widespread distribution of antibiotics in nature, particularly in freshwater (Maghsodian et al., 2022), little is known about their effects on mosquito life history traits.Here, we exposed larvae and adults of Cx. pipiens to antibiotics including penicillin, gentamycin and streptomycin.We found significant effects on the survival Fig. 2 Survival rate of Culex pipiens mosquitoes through the experiment duration (x-axis) according to the duration of the larval ad libitum feeding period in days (y-axis).Figures correspond to each adult treatment group (control and antibiotic-treated mosquitoes) for females (upper) and males (lower).The interaction between the adult treatment and the larvae ad libitum feeding period was significant for both males and females.
rates of adult males and females.Our findings indicate that larvae exposed to antibiotics showed increased survival rates in control adult female mosquitoes, and the duration of ad libitum feeding period during the larval stage determined the impact of antibiotic treatment during the adult stage on the survival rate of both female and male mosquitoes.Disruption of larvae microbiota composition by antibiotics may result in a modification of adult microbiota (Qing et al., 2020;Tikhe & Dimopoulos, 2022), which is known to modulate mosquito traits such as survival rate or reproduction (Caragata et al., 2013).However, few studies have assessed the effect of larval microbiota disruption in adult survival, showing contradictory results.Using Aedes aegypti females, Dickson et al. (2017) did not find any significant difference in the adult lifespan among different groups of mosquitoes emerged from monoaxenic larvae with different bacterial symbionts.Giraud et al. (2022) found that several larval monoaxenic treatments resulted in differences in the adult lifespan.On the other hand, Carlson et al. (2020) exposed larvae to two different bacteria species and found no differences in the survival rate of adult mosquitoes.Here, we used an antibiotic cocktail to modify the microbiota composition of Cx. pipiens larvae and observed an increase in the survival rate of adult females, but only when they received a control diet.Taken together, these results suggest that, in mosquitoes, the effects of the larval environment on adult fitness, here measured as survival rate, may be due in part to differences in the microbial community present in their breeding habitat, although the underlying mechanisms are still unknown.One potential reason explaining this pattern could be that antibiotics may remove or reduce the presence of bacteria that are detrimental to mosquito survival, including pathogenic bacteria (Ramirez et al., 2014;Contreras et al., 2019).Additionally, mosquito microbiota triggers mosquito immune responses (Gabrieli et al., 2021), which can induce important costs for mosquitoes (Ahmed et al., 2002), affecting their survival.Thus, a simplified microbiota community due to antibiotic exposure during the larval period is expected to result in lower immune responses and, consequently, reduced survival cost for mosquitoes.
Larval ad libitum feeding period modulates the negative effect of antibiotics on adult survival rate, showing a similar pattern in both female and male mosquitoes.Microorganism-based detritus that larvae consume in the field usually contain lower amounts of macronutrients than laboratory diets (Merritt et al., 1992;Souza et al., 2019), and field-collected adult mosquitoes contain fewer nutritional reserves than those reared under laboratory conditions (Day & Van Handel, 1986).In addition, the associated microbiota plays a key role in mosquito nutrition being a direct source of macronutrients (Steyn et al., 2016), providing essential nutrients like vitamins (Danchin & Braham, 2017;Guégan et al., 2020;Wang et al., 2021), and participating in functions such as fructose assimilation (Guégan et al., 2020), and metabolism of sugar, protein, and nitrogen (Samaddar et al., 2011;Chabanol et al., 2020;Guégan et al., 2020).Chabanol et al. (2020) exposed Anopheles coluzzii mosquitoes to the same antibiotic cocktail that we used here, which affected the tricarboxylic acid (TCA) cycle with subsequent loss of amino acids and other nitrogen-containing metabolites.In this context, the pattern found here with a lower survival rate of mosquitoes exposed to antibiotics as adults and fed during fewer days ad libitum as larvae suggests that microbiota disruption of adults may have higher deleterious effects in individuals with lower nutritional reserves.These results may have important implications for pathogen transmission as the effects of antibiotic exposure of adult mosquitoes (e.g., feeding on blood of antibiotic-treated individuals) may have different effects on mosquito survival depending on the development time and diet during the larval period, a factor that has not been considered in previous studies (e.g., Martínezde la Puente et al., 2021).Unfortunately, information on the exact instar of the larvae included in our experiment is lacking, and therefore we cannot separate the effect of the development time from that of the larval diet.Further studies are necessary to (1) control for the larval instar when studying the interaction between the larval diet and antibiotic treatment in adult mosquitoes and (2) identify the role of antibiotic treatments of humans and livestock on the survival rate of mosquitoes and their impacts on parasite epidemiology.
The conclusions of our study are limited by the fact that we do not know the identity and richness of microorganisms affected by the treatment.However, the antibiotics used were previously employed to disrupt the microbiota of mosquitoes, showing significant effects on survival and pathogen transmission (Dong et al., 2009;Martínez-de la Puente et al., 2021;Santos et al., 2022).In addition, using field-collected larvae, we observed significant effects resulting from exposure to antibiotics at concentrations commonly found in environmental freshwater (Mutuku et al., 2022).Our findings underscore the importance of considering the impact of pharmaceutical pollutants on vectorial capacity, affecting the survival rate of mosquito vectors of pathogens causing, among others, West Nile fever and avian malaria.A mosquito with a longer lifespan might have more opportunities to bite different hosts and, therefore, more chances of becoming infected and transmitting pathogens (Cansado-Utrilla et al., 2021).Further research must be done to identify the mechanisms underlying these results and the consequences of antibiotic exposure on the epidemiology of mosquito-borne pathogens.

Disclosure
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
This study was financed by MCIN/AEI/10.13039/501100011033 [grant number PID2020-118205GB-I00], the Spanish Ministry of Science and Innovation [grant numbers PRE2021-098544 and FJC2021-048057-I], and the Spanish Ministry of Universities [Margarita Salas and María Zambrano programs].Two anonymous reviewers provided valuable comments on a previous version of the manuscript.Funding for open access charge: Universidad de Granada / CBUA.

Table 1
Number of replicates and total number of mosquitoes (in brackets) included in this study according to the larval and adult treatments.