Sex-Specific Survival and Morphological Adaptations in Culex quinquefasciatus (Diptera: Culidae) Mosquitoes Under Multi-Generational Insecticide Selection in North-East Guwahati, Assam, India

Sex-Specific Survival and Morphological Adaptations in Culex quinquefasciatus (Diptera: Culidae) Mosquitoes Under Multi-Generational Insecticide Selection in North-East Guwahati, Assam, India | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sex-Specific Survival and Morphological Adaptations in Culex quinquefasciatus (Diptera: Culidae) Mosquitoes Under Multi-Generational Insecticide Selection in North-East Guwahati, Assam, India Aditya Shankar Kataki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7201858/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The growing threat of insecticide resistance in the southern house mosquito, Culex quinquefasciatus , presents a significant obstacle to effective vector control efforts worldwide. While previous research has largely focused on elucidating the genetic and biochemical mechanisms of resistance, there remains a critical gap in understanding how prolonged insecticide exposure influences mosquito physiology across generations—particularly in a sex-specific manner. In this study, we investigated the effects of multigenerational insecticide exposure on key life history traits, including adult survival, body size, and reproductive output in Cx. quinquefasciatus . We hypothesized that sustained exposure would (1) increase resistance, (2) influence survival and tolerance, and (3) impact physiological parameters such as body size and fecundity across generations. Our results demonstrate that female mosquitoes subjected to repeated insecticide exposure exhibit increased survival, larger body size, and modified fecundity patterns over successive generations, while males showed comparatively limited changes. These findings suggest that physiological adaptations are emerging under selection pressure, potentially enhancing the vectorial capacity of resistant mosquito populations. This study provides novel insights into the evolutionary consequences of insecticide pressure on mosquito biology and underscores the urgent need for integrated, long-term vector management strategies tailored to resistance dynamics in endemic regions. Entomology Culex quinquefasciatus body size fecundity insecticide resistance survival multi-generation Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION The prevention, management, and eradication of vector-borne illnesses have been greatly aided by different classes of insecticides over the years. However, the gradual increase in insecticide resistance in mosquitoes and other vectors causing diseases, questions the effectiveness of current vector control methods (Bhattacharya et al. 2016). Previous studies have shown that, the malaria mosquitoes are gradually becoming resistant to different classes of insecticides all around the globe. The malaria vectors have been found to be most resistant against pyrethroids. In addition, resistance to carbamates, organochlorines, and organophosphates has also been observed to increase (Bartholomay et al. 2010; Rai et al. 2019; Madhav et al. 2024 ). Research and studies have shown that mosquitoes exhibit insecticide resistance through the genes encoding for target proteins that interact with insecticides and results in structural point mutations, developing target site resistance. In addition, when cytochrome P450s, esterase, and glutathione S-transferases are transcriptionally upregulated in mosquitoes, it results in metabolic resistance and elevated protein synthesis and enzyme activity(Hemingway et al.1998a ; Hemingway et al. 2002b). The ace-1 mutation in the acetylcholinesterase gene G119S, which results in resistance to organophosphates and carbamates, and the voltage-gated sodium channel (Vgsc) mutations L1014F or L1014S are common target site resistance mechanisms in malaria vectors.(Liu 2015) Current research highlighted that, the primary mechanism of pyrethroid resistance in malaria vectors is knockdown resistance (kdr) mutations involving the substitution of leucine by serine (L1014S) or phenylalanine (L1014F) at amino acid position 101(Liu et al. 2005).Moreover, the use of insecticides has also been linked to several negative consequences on the life history features of the insect population, including survivorship, fecundity, and biting rate (Liu et al. 2011).The worrisome rise of insecticide resistance, particularly to pyrethroids, has made it clear that a more thorough and advanced approach to monitoring pesticide resistance is required.(Liu et al. 2011)However, though there are studies showing how insecticide treatment influences the physiological processes of mosquito vectors which includes the biting frequency, adult survivorship, blood-feeding and reproductive fitness of malaria vectors (Ranson et al. 2002; Xu et al. 2005; Ma et al 2013; Talipouo 2021; Osoro et al. 2022); details on how insecticide treatment over generation influences the survival rate of larvae and adult mosquitoes, body length and fecundity rate is not clear and needs further research. Survivorship and fecundity are two important criteria of the population growth dynamics of mosquitoes(Ma et al. 2013; Alout et al.2013; Tchouakui et al.2018; Barreaux et al.2018; Tchakounte et al. 2019; Derua et al.2019; Osoro et al. 2022).The natural survival rate of adult mosquitoes is one of the fundamental factors that influences vectorial capacity and a change in it due the fitness cost can have a profound effect on the disease transmission risks and distribution of malaria (Xu et al 2005; Barreaux et al. 2018). Fecundity is the number of offspring an individual female mosquito can produce and is considered as an important trait as it is a major fitness component and plays a role in determining the reproductive rate and intrinsic growth rate of a population.(Xu et al. 2005; Derua et al. 2019; Batume et al. 2022; Antonio et al.2022) Thus, the fact how these physiological life history traits influence the insecticide resistance over the generations in Anopheles gambiae is not clearly understood and a gap of knowledge is existing. This research was an attempt to bridge up all these gaps of knowledge that were existing. The research questions of the study were: [1] How does insecticide treatment affect the natural survival of the adult mosquitoes over generations? [2] Does the natural survival of the mosquitoes upon treatment with insecticide over generations is influenced by sex? [3] Does insecticide treatment over generations influences change in body length of mosquitoes and consequently increase their resistance? MATERIAL AND METHODOLOGY Sample Collection Area Mosquito larvae were collected from eight georeferenced sites across Guwahati, Assam, India representing a range of urban aquatic habitats such as drains, stagnant pools, and peri-urban water bodies. The sites (Site 1–8) were strategically selected to ensure broad spatial coverage. Larvae were sampled using standard dippers during early mornings and transported to the lab for rearing and identification. The main map highlights the specific collection points within the city, while the inset locates Guwahati within India, supporting spatial context for vector surveillance efforts. Insectary Condition The entire experiment was carried out in the insectary having temperature 25°C to 33°C and humidity 70–80% with a 12- hour day/night cycles. Mosquito pupae to adults When the pupal stage of development arrived, the pupae were collected and separated into conical cups and were placed inside mosquito cages to let them develop into adults. Fresh cotton pads soaked in 5% glucose solution were placed on top of the cage. Upon becoming adults, they were blood fed and then fresh batch of eggs were collected for the experiment to continue. Adult Insecticide Bioassay Test The insecticide susceptibility/ resistance bioassay tests were carried out using standard protocols of World Health Organisation (WHO) and susceptibility test kits. For testing the tolerance of adults to insecticide, CDC filters papers were prepared initially. Eight filter papers were prepared in total from which four were impregnated with distilled water (control) and four were impregnated with insecticide (deltamethrin). The insecticide treatment was a composition of 0.66 µl silicon + 2ml acetone + 1.32 µl deltamethrin which all together made up 0.02% of the standard dose of deltamethrin. The filter papers were allowed to impregnate for 24 hrs and then were wrapped in aluminium foil for later use in the experiment. Once the mosquitoes reached the adult stage of their life cycle, ~ 40 female mosquitoes were separated out from each of the four cages for CDC bottle bioassay test. The filter papers for control and treatment were applied accordingly in the CDC bottles. The exposure was carried out by exposing mosquitoes in two equal batches for control and treatment respectively. The female mosquitoes were exposed in the CDC bottles for 60 mins (WHO Standard protocol). During the exposure, the number of mosquitoes knocked down was recorded after 60 mins. Upon exposure they were transferred to the opposite holding end of the bottle and were fed on glucose solution via a cotton pad soaked in 5% glucose solution placed on the mesh ends of the holding tube. Upon 24 hrs, the mortality of the adults was noted and recorded. An adult mosquito was considered alive if it was able to fly. Mosquitoes were classified as knocked down or dead if they were immobile or unable to move and fly. This was done for each generation to evaluate the resistance status of the mosquitoes. On completion of the susceptibility/resistant test, the mosquitoes were transferred to clearly labelled tubes with a lid for airtight locking for preservation and further body length analysis. Adult Natural Survival After the CDC bioassay test was conducted, the remaining mosquitoes in the cage were blood fed cautiously. The most recent blood in lab was used for feeding and it was done for 25–30 mins. Upon feeding, fresh cotton pads soaked in 5% glucose solution were added on a regular basis each day on top of the cages for their energy, survival and reproduction cycle. In addition, the natural survival period for both male and female were also observed and noted down for the adult natural survival assessment upon insecticide treatment. Body Length Analysis The mosquitoes were collected from the labelled tubes and were placed on a clean slide under stereo microscope. The specimen was fixed with a drop of 5% PBS (Phosphate Buffer Solution). With the help of fine forceps, the wings of the mosquitoes were separated. Upon dissection, wing measurements were noted down with the help of the software Dino eye and microscope at magnification 1X. Fecundity Analysis The separated female mosquitoes (~ 40) from each cage that were collected upon death were placed on a clean slide and were fixed with a drop of 5% PBS solution. The abdomen of the mosquitoes was dissected using fine forceps. After dissection, the presence or absence of eggs in mosquitoes was observed and if present the total number of eggs were counted and recorded for both control and treated mosquitoes. Statistical Analysis Statistical analysis was performed in RStudio software (version 2024.04.2 + 764) with packages ‘dplyr’ ‘ggplot2’ ‘ggeffects’, ‘emmeans’ ‘lme4’ ‘lmerTest’ ‘Matrix’ ‘splines’ ‘survival’ ‘survminer’ and ‘ggfortify’ and using linear mixed models (LMM) and generalised linear mixed-effects model (GLMM) to investigate the significance of several explanatory variables on the response variables. The models used were binary distribution and gaussian distribution. For linear mixed models, any significant explanatory variables and significant interactions were determined using log likelihood ratios and for generalised linear mixed effects model, ANOVA test was performed and based on Chisq, Df and p-value the best model was selected and fitted. The map was created in R using the tidyverse, sf, osmdata, ggspatial, ggrepel, rnaturalearth, and cowplotpackages. RESULTS AND DISCUSSION Figure 2 explores how mosquito survival (Panel A) and body length (Panel B) vary across strains, selection lines, and generations. For body length, selection and generation were statistically significant (Table 1 ). For adult insectide tolerance, selection and generation were significant (Table 2 ). Panel A shows the proportion of mosquitoes alive across four generations (F0–F3) under control (C) and insecticide-selected (I) conditions for strains A–D. In the initial generation (F0), survival rates are similar across selection lines, but by F1 and F2, selected lines often show improved survival, particularly in some strains. However, by F3, the survival advantage in selected lines diminishes or becomes more variable, suggesting strain-specific and possibly transient benefits of insecticide selection. Panel B illustrates predicted body length across generations. Strains A and B consistently exhibit larger body sizes than strains C and D. While body size increases slightly over generations in strains A and B, it remains lower and more stable in strains C and D. This indicates that genetic background strongly influences morphological traits, with selection and generational change having varied effects. Overall, the data highlight that both survival and body size are shaped by interactions between strain, selection pressure, and generational dynamics. These results underscore the importance of genetic variability in determining how mosquito populations respond to environmental and selective pressures. Table 1 Result of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for body length in Cx. quinquefasciatus. Factor df χ2 p Selection 1 0.71 0.652 Generation 1 8.17 0.0035 Strain 1 90.52 < 0.0001 Generation x selection 1 0.69 0.3245 Generation x strain 1 0.55 0.4567 Selection x strain 1 0.47 0.3452 Generation X strain X selection 1 0.52 0.4532 Table 2 Result of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for adult insecticide tolerance in Cx. quinquefasciatus. Factor df χ2 p Selection 1 102.33 < 0.0001 Generation 1 9.14 0.0024 Strain 1 0.91 0.78 Generation x selection 1 2.38 0.128 Generation x strain 1 0.76 0.36 Selection x strain 1 2.38 0.15 Generation X strain X selection 1 0.63 0.457 Figure 3 illustrate the impact of insecticide selection on the reproductive performance of female mosquitoes. For fecundity, selection, generation and strain was found to be statstically significant (Table 3 ). Panel A shows that a significantly higher proportion of females from the control group produced eggs compared to those from the insecticide-selected group. The median proportion of egg-laying females in the control was around 0.75, whereas it dropped to approximately 0.5 in the insecticide-selected population, indicating a potential reproductive cost associated with insecticide resistance. Panel B further explores this trend across four generations (F0–F3), showing the number of eggs present in dissected females from four different strains (A–D) under both selection regimes. In the F0 generation, strains A and D had the highest egg counts, particularly in the control group. As generations progressed, variation in egg production became more pronounced. Notably, in F2 and F3, strains B and C in the insecticide-selected lines showed reduced egg numbers compared to their control counterparts. This generational decline in fecundity among selected mosquitoes suggests that insecticide selection may negatively affect reproductive capacity over time. These findings support the hypothesis that while resistance may enhance survival, it could simultaneously impose physiological trade-offs affecting reproduction. Table 3 Result of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for fecundity in Cx. quinquefasciatus. Factor df χ2 p Selection 1 98.40 < 0.0001 Generation 1 9.17 0.0028 Strain 1 7.19 0.0073 Generation x selection 1 0.10 0.7575 Generation x strain 1 2.98 0.0876 Selection x strain 1 0.25 0.6234 Generation X strain X selection 1 0.67 0.4545 Table 4 Result of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for adult natural survival in Cx. quinquefasciatus. Factor df χ2 p Selection 1 117.89 < 0.0001 Generation 1 0.11 0.0011 Sex 1 271.79 < 0.0001 Strain 1 0.75 0.456 Generation x selection 1 0.48 0.575 Generation x strain 1 0.12 0.763 Selection x strain 1 0.91 0.642 Generation X Sex 1 0.14 0.786 Generation X strain X selection X sex 1 0.47 0.4545 Figure 4 depicts the survival dynamics of adult mosquitoes post blood-feeding with stratification by generation, selection line, sex, and strain. In the adult natural survival, selection, generation and sex was statistically significant (Table 4 ). Panel A shows the Kaplan–Meier survival curves across six generations (F0–F5). A significant decline in survival is observed across generations, with F0 showing higher survival probabilities and F5 demonstrating the lowest. This trend may suggest cumulative effects of selection pressure or physiological adaptation over time, with each successive generation exhibiting reduced longevity. Panel B compares survival between two selection lines: control (C) and insecticide-selected (I). A marked difference is evident, where the insecticide-selected line demonstrates significantly greater survival over the 30-day period. The vertical dashed lines indicate median survival times, further highlighting the extended longevity in the selected line. This suggests that selection for insecticide resistance may confer a survival advantage post blood-feeding, possibly due to enhanced physiological robustness or stress tolerance mechanisms. Panel C displays sex-specific survival probabilities. Females consistently show higher survival than males throughout the observation period. This trend is statistically significant and biologically meaningful, as females must survive longer to complete gonotrophic cycles and ensure successful reproduction. The survival advantage in females may reflect evolutionary adaptations favoring longevity for reproductive success. Panel D illustrates strain-specific survival curves. Among the four strains (A–D), strain A has the lowest survival, while strain D exhibits the highest, with B and C showing intermediate patterns. These differences may arise from inherent genetic variation, differential resistance mechanisms, or variability in physiological responses to blood-feeding stress. Overall, the data emphasize that both intrinsic (strain, sex) and extrinsic (selection, generational change) factors strongly influence mosquito survival. The observed patterns have important implications for vector competence, insecticide resistance evolution, and long-term vector control strategies. Table 5 Cox proportional hazards ratios (HRs) for mosquito survival across generations F0–F5 by selection line, strain, and sex. Hazard ratios (HRs) represent the relative risk of mortality compared to the reference group over time. Values less than 1 indicate a lower risk (greater survival), while values close to or above 1 indicate higher or similar mortality risk. The insecticide-selected mosquitoes, particularly strain C in both sexes, exhibited the lowest HRs, suggesting enhanced survival under selection pressure Cox Proportion Hazard Ratio [F0-F5 Generation] Selection Strain Sex Mean HR exp (coef) CONTROL A F 0.76 CONTROL B F 0.69 INSECTICIDE C F 0.50 INSECTICIDE D F 0.81 CONTROL A M 0.96 CONTROL B M 0.91 INSECTICIDE C M 0.63 INSECTICIDE D M 0.81 DISCUSSION This study demonstrates that adult survival following blood feeding and insecticide exposure in Cx. quinquefasciatus is significantly influenced by generation, sex, and the interaction of selection across different mosquito strains. Notable generational differences and survival patterns were observed, with insecticide-selected groups consistently exhibiting higher survival probabilities compared to controls—indicating enhanced resistance and longevity. This trend likely reflects the progressive development of resistance mechanisms under sustained insecticide pressure, which contribute to increased lifespan across generations (Xiu et al 2005; Vantaux et al. 2016; Takken et al. 2013; Gimnig et al. 2022). While survival probabilities declined with time for both sexes, females consistently displayed higher survival rates than males. Given that mosquito longevity is a critical determinant of vectorial capacity (Hien et al 2017; Fodjo et al. 2018), the extended lifespan observed in resistant females may have significant implications for malaria transmission dynamics (Fodjo et al. 2018). These findings suggest that long-term insecticide exposure could inadvertently favor traits that enhance vectorial capacity, thereby complicating control efforts. Similar trends have been observed in F1-resistant An. funestus carrying the 119F-GSTe2 allele, which exhibited both increased longevity and transmission potential (Keitta et al. 2022). Although survival among treated mosquitoes fluctuated across generations, the consistently high and stable survival observed in control groups suggests possible cycles of adaptation and vulnerability. These dynamics likely reflect the evolving nature of resistance mechanisms driven by sustained insecticide selection pressure, particularly from widespread pyrethroid use in both vector control and agriculture (Mebrahtu et al. 1997; Ma et al. 2013). Morphometric analyses revealed physiological adaptations associated with resistance, as insecticide-treated mosquitoes consistently showed increased body size alongside enhanced survival. These traits support the hypothesis that resistance is linked to overall fitness gains in certain contexts. This study successfully demonstrates that multigenerational insecticide exposure significantly alters key life-history traits in Cx. quinquefasciatus . Traits such as survival probability, body size, and reproductive output were progressively affected over generations, indicating the development of physiological resistance. These changes were particularly pronounced in females, suggesting sex-specific adaptations that may enhance vectorial capacity. The observed trends underscore the importance of considering long-term ecological and evolutionary effects of insecticide use in malaria control programs. The findings provide a foundation for future investigations into resistance mechanisms and can inform the development of more sustainable vector control strategies. Integrating physiological and ecological data into resistance management frameworks will be essential to mitigate the spread of resistance and reduce malaria transmission in endemic areas. Declarations Competing Interests No Cometing Interest. FINANCIAL SUPPORT No financial support was received. ACKNOWLEDGEMENTS The author would like to show my sincere gratitude to the Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam, India for their support and guidance. References Alout H, Ndam NT, Sandeu MM, Djégbe I, Chandre F, Dabiré RK, et al.(2013) Insecticide resistance alleles affect vector competence of Anopheles gambiae s.s. for Plasmodium falciparum field isolates. PLoS One 8(5). Antonio-Nkondjio C, Nkahe DL, Kopya E, Djiappi-Tchamen B, Toussile W, Sonhafouo-Chiana N, et al.(2020) Fitness cost of insecticide resistance on the life-traits of a Anopheles coluzzii population from the city of Yaoundé, Cameroon. Wellcome Open Res 5. Bartholomay LC, Waterhouse RM, Mayhew GF, Campbell CL, Michel K, Zou Z, et al. (2010) Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens. Science American Association for the Advancement of Science 330: 88. 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Scientific Reports 11:1 Takken W, Smallegange RC, Vigneau AJ, Johnston V, Brown M, Mordue-Luntz AJ, et al.(2013) Larval nutrition differentially affects adult fitness and Plasmodium development in the malaria vectors Anopheles gambiae and Anopheles stephensi. Parasit Vectors 6(1):1–10. Tchouakui M, Riveron JM, Djonabaye D, Tchapga W, Irving H, Takam PS, et al.(2018) Fitness Costs of the Glutathione S-Transferase Epsilon 2 (L119F-GSTe2) Mediated Metabolic Resistance to Insecticides in the Major African Malaria Vector Anopheles Funestus. Genes (Basel) 9(12). Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7201858","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":490097899,"identity":"770b15e8-9df6-42c8-ae1d-b726a6a5a080","order_by":0,"name":"Aditya Shankar Kataki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYFCCxAYgkQDEbAwHPlTYABmMjQcIaGlsAGnhAWo5OONMGkhLAwEtCYxwLcy8bYfBYni18Lcntz/mqUmTt2c/lniY58x5u7Xth4G21NhE49IiceZhYzPPsRzDHp60AwfnVNxO3nYmEajlWFpuAy49NxKBWtgqGHsY0hsOvDlzO9nsAFALY8NhnFrkwVr+Vdj38D9vOMDbdi7Z7PxD/FoMQFp423ISeySADuNtO2BndoOALYZAv8yc25eW3HPjWQIwkJMTzG4AbUnA4xe54+kPPrz5lmzb3p9m/OFDhZ292fn0hw8+1Njg9j4QMPEgccCJAZwa8AHGH0gcewKKR8EoGAWjYAQCAKohchtXd6gfAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0005-2864-6916","institution":"University of Glasgow","correspondingAuthor":true,"prefix":"","firstName":"Aditya","middleName":"Shankar","lastName":"Kataki","suffix":""}],"badges":[],"createdAt":"2025-07-24 05:56:11","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7201858/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7201858/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87544627,"identity":"9497b76d-144f-447e-84c7-1a4a098d3bb7","added_by":"auto","created_at":"2025-07-25 04:33:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":552864,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing eight mosquito larval collection sites (Site 1–8) across Guwahati, Assam, India. Sampling locations were selected to represent diverse aquatic habitats across the city. The inset map highlights Guwahati’s geographic location within India.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7201858/v1/6b342e7d2a086924e6e7c87e.png"},{"id":87544456,"identity":"58725f11-7543-4aba-a159-37b8ba22c65c","added_by":"auto","created_at":"2025-07-25 04:25:36","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInsecticide tolerance and body size variation across mosquito strains, selection lines, and generations. \u003c/strong\u003e[A] Proportion of adult mosquitoes surviving 24 hours post-insecticide exposure in generations F0 to F3, separated by selection line (C: control, I: insecticide-selected). Colored points represent individual strains (A–D), while boxplots summarize group-level variation. \u003cstrong\u003e[\u003c/strong\u003eB] Predicted adult body length (mm) of female mosquitoes across generations 1 to 4, stratified by strain (A–D). Each point denotes the mean prediction per group with boxplots showing distribution. Strain A and B consistently exhibit greater body size than C and D across generations.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7201858/v1/151335ca174ee3de6fc152b8.jpeg"},{"id":87544457,"identity":"a7597c49-87be-4ad6-a3c8-85d9a7b4db28","added_by":"auto","created_at":"2025-07-25 04:25:36","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61021,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of insecticide selection on female mosquito reproductive traits. \u003cstrong\u003e[A] \u003c/strong\u003eProportion of blood-fed females producing eggs in control and insecticide-selected lines. Females from the insecticide-selected population show a lower reproductive output compared to controls.\u003cstrong\u003e[B] \u003c/strong\u003eNumber of eggs present in dissected females across four generations (F0 to F3), stratified by strain (A–D) and selection line. Egg production varied by strain and was generally reduced in insecticide-selected lines compared to controls.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7201858/v1/b125b9e63e19dbb2a6eabefc.jpeg"},{"id":87544462,"identity":"55cf43ae-728b-420d-8325-67db155bc400","added_by":"auto","created_at":"2025-07-25 04:25:36","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":396707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan–Meier survival curves of adult mosquitoes post blood feeding. [A]\u003c/strong\u003e Survival probability across six consecutive generations (F0–F5). [\u003cstrong\u003eB]\u003c/strong\u003e Comparison between two selection lines: control (C) and insecticide-selected (I) mosquitoes, with vertical dashed lines indicating median survival times. [\u003cstrong\u003eC]\u003c/strong\u003e Sex-specific survival showing differences between females (F) and males (M).[\u003cstrong\u003eD]\u003c/strong\u003e Survival among mosquitoes from four different genetic strains (A, B, C, and D). Risk tables below each plot indicate the number of individuals at risk at different time points. All comparisons were statistically significant ( \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7201858/v1/3f7e8e9bcb6a7d31081efb10.jpeg"},{"id":87545421,"identity":"74b93d5d-272f-42a0-aefb-14a52d952c69","added_by":"auto","created_at":"2025-07-25 04:49:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1665508,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7201858/v1/b7e48d05-56c3-4759-84e6-dbcf2c75315c.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eSex-Specific Survival and Morphological Adaptations in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCulex quinquefasciatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (Diptera: Culidae) Mosquitoes Under Multi-Generational Insecticide Selection in North-East Guwahati, Assam, India\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe prevention, management, and eradication of vector-borne illnesses have been greatly aided by different classes of insecticides over the years. However, the gradual increase in insecticide resistance in mosquitoes and other vectors causing diseases, questions the effectiveness of current vector control methods (Bhattacharya et al. 2016). Previous studies have shown that, the malaria mosquitoes are gradually becoming resistant to different classes of insecticides all around the globe. The malaria vectors have been found to be most resistant against pyrethroids. In addition, resistance to carbamates, organochlorines, and organophosphates has also been observed to increase (Bartholomay et al. 2010; Rai et al. 2019; Madhav et al. 2024 ). Research and studies have shown that mosquitoes exhibit insecticide resistance through the genes encoding for target proteins that interact with insecticides and results in structural point mutations, developing target site resistance. In addition, when cytochrome P450s, esterase, and glutathione S-transferases are transcriptionally upregulated in mosquitoes, it results in metabolic resistance and elevated protein synthesis and enzyme activity(Hemingway et al.1998a ; Hemingway et al. 2002b). The ace-1 mutation in the acetylcholinesterase gene G119S, which results in resistance to organophosphates and carbamates, and the voltage-gated sodium channel (Vgsc) mutations L1014F or L1014S are common target site resistance mechanisms in malaria vectors.(Liu 2015) Current research highlighted that, the primary mechanism of pyrethroid resistance in malaria vectors is knockdown resistance (kdr) mutations involving the substitution of leucine by serine (L1014S) or phenylalanine (L1014F) at amino acid position 101(Liu et al. 2005).Moreover, the use of insecticides has also been linked to several negative consequences on the life history features of the insect population, including survivorship, fecundity, and biting rate (Liu et al. 2011).The worrisome rise of insecticide resistance, particularly to pyrethroids, has made it clear that a more thorough and advanced approach to monitoring pesticide resistance is required.(Liu et al. 2011)However, though there are studies showing how insecticide treatment influences the physiological processes of mosquito vectors which includes the biting frequency, adult survivorship, blood-feeding and reproductive fitness of malaria vectors (Ranson et al. 2002; Xu et al. 2005; Ma et al 2013; Talipouo 2021; Osoro et al. 2022); details on how insecticide treatment over generation influences the survival rate of larvae and adult mosquitoes, body length and fecundity rate is not clear and needs further research. Survivorship and fecundity are two important criteria of the population growth dynamics of mosquitoes(Ma et al. 2013; Alout et al.2013; Tchouakui et al.2018; Barreaux et al.2018; Tchakounte et al. 2019; Derua et al.2019; Osoro et al. 2022).The natural survival rate of adult mosquitoes is one of the fundamental factors that influences vectorial capacity and a change in it due the fitness cost can have a profound effect on the disease transmission risks and distribution of malaria (Xu et al 2005; Barreaux et al. 2018). Fecundity is the number of offspring an individual female mosquito can produce and is considered as an important trait as it is a major fitness component and plays a role in determining the reproductive rate and intrinsic growth rate of a population.(Xu et al. 2005; Derua et al. 2019; Batume et al. 2022; Antonio et al.2022) Thus, the fact how these physiological life history traits influence the insecticide resistance over the generations in \u003cem\u003eAnopheles gambiae\u003c/em\u003e is not clearly understood and a gap of knowledge is existing. This research was an attempt to bridge up all these gaps of knowledge that were existing.\u003c/p\u003e\u003cp\u003eThe research questions of the study were: [1] How does insecticide treatment affect the natural survival of the adult mosquitoes over generations? [2] Does the natural survival of the mosquitoes upon treatment with insecticide over generations is influenced by sex? [3] Does insecticide treatment over generations influences change in body length of mosquitoes and consequently increase their resistance?\u003c/p\u003e"},{"header":"MATERIAL AND METHODOLOGY","content":"\u003cp\u003e\u003cstrong\u003eSample Collection Area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMosquito larvae were collected from eight georeferenced sites across Guwahati, Assam, India representing a range of urban aquatic habitats such as drains, stagnant pools, and peri-urban water bodies. The sites (Site 1\u0026ndash;8) were strategically selected to ensure broad spatial coverage. Larvae were sampled using standard dippers during early mornings and transported to the lab for rearing and identification. The main map highlights the specific collection points within the city, while the inset locates Guwahati within India, supporting spatial context for vector surveillance efforts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsectary Condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe entire experiment was carried out in the insectary having temperature 25\u0026deg;C to 33\u0026deg;C and humidity 70\u0026ndash;80% with a 12- hour day/night cycles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMosquito pupae to adults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the pupal stage of development arrived, the pupae were collected and separated into conical cups and were placed inside mosquito cages to let them develop into adults. Fresh cotton pads soaked in 5% glucose solution were placed on top of the cage. Upon becoming adults, they were blood fed and then fresh batch of eggs were collected for the experiment to continue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdult Insecticide Bioassay Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe insecticide susceptibility/ resistance bioassay tests were carried out using standard protocols of World Health Organisation (WHO) and susceptibility test kits. For testing the tolerance of adults to insecticide, CDC filters papers were prepared initially. Eight filter papers were prepared in total from which four were impregnated with distilled water (control) and four were impregnated with insecticide (deltamethrin). The insecticide treatment was a composition of 0.66 \u0026micro;l silicon\u0026thinsp;+\u0026thinsp;2ml acetone\u0026thinsp;+\u0026thinsp;1.32 \u0026micro;l deltamethrin which all together made up 0.02% of the standard dose of deltamethrin. The filter papers were allowed to impregnate for 24 hrs and then were wrapped in aluminium foil for later use in the experiment. Once the mosquitoes reached the adult stage of their life cycle, ~ 40 female mosquitoes were separated out from each of the four cages for CDC bottle bioassay test. The filter papers for control and treatment were applied accordingly in the CDC bottles. The exposure was carried out by exposing mosquitoes in two equal batches for control and treatment respectively. The female mosquitoes were exposed in the CDC bottles for 60 mins (WHO Standard protocol). During the exposure, the number of mosquitoes knocked down was recorded after 60 mins. Upon exposure they were transferred to the opposite holding end of the bottle and were fed on glucose solution via a cotton pad soaked in 5% glucose solution placed on the mesh ends of the holding tube. Upon 24 hrs, the mortality of the adults was noted and recorded. An adult mosquito was considered alive if it was able to fly. Mosquitoes were classified as knocked down or dead if they were immobile or unable to move and fly. This was done for each generation to evaluate the resistance status of the mosquitoes. On completion of the susceptibility/resistant test, the mosquitoes were transferred to clearly labelled tubes with a lid for airtight locking for preservation and further body length analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdult Natural Survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the CDC bioassay test was conducted, the remaining mosquitoes in the cage were blood fed cautiously. The most recent blood in lab was used for feeding and it was done for 25\u0026ndash;30 mins. Upon feeding, fresh cotton pads soaked in 5% glucose solution were added on a regular basis each day on top of the cages for their energy, survival and reproduction cycle. In addition, the natural survival period for both male and female were also observed and noted down for the adult natural survival assessment upon insecticide treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBody Length Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mosquitoes were collected from the labelled tubes and were placed on a clean slide under stereo microscope. The specimen was fixed with a drop of 5% PBS (Phosphate Buffer Solution). With the help of fine forceps, the wings of the mosquitoes were separated. Upon dissection, wing measurements were noted down with the help of the software Dino eye and microscope at magnification 1X.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFecundity Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe separated female mosquitoes (~\u0026thinsp;40) from each cage that were collected upon death were placed on a clean slide and were fixed with a drop of 5% PBS solution. The abdomen of the mosquitoes was dissected using fine forceps. After dissection, the presence or absence of eggs in mosquitoes was observed and if present the total number of eggs were counted and recorded for both control and treated mosquitoes.\u003c/p\u003e\n\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analysis was performed in RStudio software (version 2024.04.2\u0026thinsp;+\u0026thinsp;764) with packages \u0026lsquo;dplyr\u0026rsquo; \u0026lsquo;ggplot2\u0026rsquo; \u0026lsquo;ggeffects\u0026rsquo;, \u0026lsquo;emmeans\u0026rsquo; \u0026lsquo;lme4\u0026rsquo; \u0026lsquo;lmerTest\u0026rsquo; \u0026lsquo;Matrix\u0026rsquo; \u0026lsquo;splines\u0026rsquo; \u0026lsquo;survival\u0026rsquo; \u0026lsquo;survminer\u0026rsquo; and \u0026lsquo;ggfortify\u0026rsquo; and using linear mixed models (LMM) and generalised linear mixed-effects model (GLMM) to investigate the significance of several explanatory variables on the response variables. The models used were binary distribution and gaussian distribution. For linear mixed models, any significant explanatory variables and significant interactions were determined using log likelihood ratios and for generalised linear mixed effects model, ANOVA test was performed and based on Chisq, Df and p-value the best model was selected and fitted. The map was created in R using the tidyverse, sf, osmdata, ggspatial, ggrepel, rnaturalearth, and cowplotpackages.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e explores how mosquito survival (Panel A) and body length (Panel B) vary across strains, selection lines, and generations. For body length, selection and generation were statistically significant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For adult insectide tolerance, selection and generation were significant (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePanel A shows the proportion of mosquitoes alive across four generations (F0\u0026ndash;F3) under control (C) and insecticide-selected (I) conditions for strains A\u0026ndash;D. In the initial generation (F0), survival rates are similar across selection lines, but by F1 and F2, selected lines often show improved survival, particularly in some strains. However, by F3, the survival advantage in selected lines diminishes or becomes more variable, suggesting strain-specific and possibly transient benefits of insecticide selection. Panel B illustrates predicted body length across generations. Strains A and B consistently exhibit larger body sizes than strains C and D. While body size increases slightly over generations in strains A and B, it remains lower and more stable in strains C and D. This indicates that genetic background strongly influences morphological traits, with selection and generational change having varied effects. Overall, the data highlight that both survival and body size are shaped by interactions between strain, selection pressure, and generational dynamics. These results underscore the importance of genetic variability in determining how mosquito populations respond to environmental and selective pressures.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResult of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for body length in \u003cem\u003eCx. quinquefasciatus.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFactor\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003edf\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eχ2\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.652\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0035\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x selection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.3245\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.4567\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.3452\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration X strain X selection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.4532\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResult of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for adult insecticide tolerance in \u003cem\u003eCx. quinquefasciatus.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFactor\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003edf\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eχ2\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e102.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0024\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x selection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.128\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration X strain X selection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.457\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrate the impact of insecticide selection on the reproductive performance of female mosquitoes. For fecundity, selection, generation and strain was found to be statstically significant (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Panel A shows that a significantly higher proportion of females from the control group produced eggs compared to those from the insecticide-selected group. The median proportion of egg-laying females in the control was around 0.75, whereas it dropped to approximately 0.5 in the insecticide-selected population, indicating a potential reproductive cost associated with insecticide resistance. Panel B further explores this trend across four generations (F0\u0026ndash;F3), showing the number of eggs present in dissected females from four different strains (A\u0026ndash;D) under both selection regimes. In the F0 generation, strains A and D had the highest egg counts, particularly in the control group. As generations progressed, variation in egg production became more pronounced. Notably, in F2 and F3, strains B and C in the insecticide-selected lines showed reduced egg numbers compared to their control counterparts. This generational decline in fecundity among selected mosquitoes suggests that insecticide selection may negatively affect reproductive capacity over time. These findings support the hypothesis that while resistance may enhance survival, it could simultaneously impose physiological trade-offs affecting reproduction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResult of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for fecundity in \u003cem\u003eCx. quinquefasciatus.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFactor\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003edf\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eχ2\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e98.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0028\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0073\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x selection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7575\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0876\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.6234\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration X strain X selection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.4545\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResult of statistical analysis for generation (F0-F5), sex (M and F) strain (A,B,C,D,) and selection (C and I ) for adult natural survival in \u003cem\u003eCx. quinquefasciatus.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFactor\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003edf\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eχ2\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e117.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0011\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e271.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.456\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x selection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.575\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.763\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelection x strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.642\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration X Sex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.786\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeneration X strain X selection X sex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.4545\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depicts the survival dynamics of adult mosquitoes post blood-feeding with stratification by generation, selection line, sex, and strain. In the adult natural survival, selection, generation and sex was statistically significant (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePanel A shows the Kaplan\u0026ndash;Meier survival curves across six generations (F0\u0026ndash;F5). A significant decline in survival is observed across generations, with F0 showing higher survival probabilities and F5 demonstrating the lowest. This trend may suggest cumulative effects of selection pressure or physiological adaptation over time, with each successive generation exhibiting reduced longevity. Panel B compares survival between two selection lines: control (C) and insecticide-selected (I). A marked difference is evident, where the insecticide-selected line demonstrates significantly greater survival over the 30-day period. The vertical dashed lines indicate median survival times, further highlighting the extended longevity in the selected line. This suggests that selection for insecticide resistance may confer a survival advantage post blood-feeding, possibly due to enhanced physiological robustness or stress tolerance mechanisms. Panel C displays sex-specific survival probabilities. Females consistently show higher survival than males throughout the observation period. This trend is statistically significant and biologically meaningful, as females must survive longer to complete gonotrophic cycles and ensure successful reproduction. The survival advantage in females may reflect evolutionary adaptations favoring longevity for reproductive success. Panel D illustrates strain-specific survival curves. Among the four strains (A\u0026ndash;D), strain A has the lowest survival, while strain D exhibits the highest, with B and C showing intermediate patterns. These differences may arise from inherent genetic variation, differential resistance mechanisms, or variability in physiological responses to blood-feeding stress. Overall, the data emphasize that both intrinsic (strain, sex) and extrinsic (selection, generational change) factors strongly influence mosquito survival. The observed patterns have important implications for vector competence, insecticide resistance evolution, and long-term vector control strategies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCox proportional hazards ratios (HRs) for mosquito survival across generations F0\u0026ndash;F5 by selection line, strain, and sex. Hazard ratios (HRs) represent the relative risk of mortality compared to the reference group over time. Values less than 1 indicate a lower risk (greater survival), while values close to or above 1 indicate higher or similar mortality risk. The insecticide-selected mosquitoes, particularly strain C in both sexes, exhibited the lowest HRs, suggesting enhanced survival under selection pressure\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eCox Proportion Hazard Ratio [F0-F5 Generation]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSelection\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eStrain\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eSex\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eMean HR exp (coef)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eINSECTICIDE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eINSECTICIDE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCONTROL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eINSECTICIDE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eINSECTICIDE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study demonstrates that adult survival following blood feeding and insecticide exposure in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e is significantly influenced by generation, sex, and the interaction of selection across different mosquito strains. Notable generational differences and survival patterns were observed, with insecticide-selected groups consistently exhibiting higher survival probabilities compared to controls\u0026mdash;indicating enhanced resistance and longevity. This trend likely reflects the progressive development of resistance mechanisms under sustained insecticide pressure, which contribute to increased lifespan across generations (Xiu et al 2005; Vantaux et al. 2016; Takken et al. 2013; Gimnig et al. 2022).\u003c/p\u003e\u003cp\u003eWhile survival probabilities declined with time for both sexes, females consistently displayed higher survival rates than males. Given that mosquito longevity is a critical determinant of vectorial capacity (Hien et al 2017; Fodjo et al. 2018), the extended lifespan observed in resistant females may have significant implications for malaria transmission dynamics (Fodjo et al. 2018). These findings suggest that long-term insecticide exposure could inadvertently favor traits that enhance vectorial capacity, thereby complicating control efforts. Similar trends have been observed in F1-resistant An. funestus carrying the 119F-GSTe2 allele, which exhibited both increased longevity and transmission potential (Keitta et al. 2022). Although survival among treated mosquitoes fluctuated across generations, the consistently high and stable survival observed in control groups suggests possible cycles of adaptation and vulnerability. These dynamics likely reflect the evolving nature of resistance mechanisms driven by sustained insecticide selection pressure, particularly from widespread pyrethroid use in both vector control and agriculture (Mebrahtu et al. 1997; Ma et al. 2013). Morphometric analyses revealed physiological adaptations associated with resistance, as insecticide-treated mosquitoes consistently showed increased body size alongside enhanced survival. These traits support the hypothesis that resistance is linked to overall fitness gains in certain contexts.\u003c/p\u003e\u003cp\u003eThis study successfully demonstrates that multigenerational insecticide exposure significantly alters key life-history traits in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e. Traits such as survival probability, body size, and reproductive output were progressively affected over generations, indicating the development of physiological resistance. These changes were particularly pronounced in females, suggesting sex-specific adaptations that may enhance vectorial capacity. The observed trends underscore the importance of considering long-term ecological and evolutionary effects of insecticide use in malaria control programs. The findings provide a foundation for future investigations into resistance mechanisms and can inform the development of more sustainable vector control strategies. Integrating physiological and ecological data into resistance management frameworks will be essential to mitigate the spread of resistance and reduce malaria transmission in endemic areas.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eNo Cometing Interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eFINANCIAL SUPPORT\u003c/h2\u003e\u003cp\u003eNo financial support was received.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eThe author would like to show my sincere gratitude to the Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam, India for their support and guidance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlout H, Ndam NT, Sandeu MM, Dj\u0026eacute;gbe I, Chandre F, Dabir\u0026eacute; RK, et al.(2013) Insecticide resistance alleles affect vector competence of Anopheles gambiae s.s. for Plasmodium falciparum field isolates. PLoS One 8(5).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAntonio-Nkondjio C, Nkahe DL, Kopya E, Djiappi-Tchamen B, Toussile W, Sonhafouo-Chiana N, et al.(2020) Fitness cost of insecticide resistance on the life-traits of a Anopheles coluzzii population from the city of Yaound\u0026eacute;, Cameroon. Wellcome Open Res 5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBartholomay LC, Waterhouse RM, Mayhew GF, Campbell CL, Michel K, Zou Z, et al. (2010) Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens. Science American Association for the Advancement of Science 330: 88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarreaux AMG, Stone CM, Barreaux P, Koella JC.(2018) The relationship between size and longevity of the malaria vector Anopheles gambiae (s.s.) depends on the larval environment. Parasit Vectors 11(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBatume C, Akol AM, Mukwaya LG, Birungi J, Kayondo JK. (2022) Life-history attributes of juvenile Anopheles gambiae s.s. in central Uganda; implications for malaria control interventions. Med Vet Entomol 36(2):212.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhattacharya S, Basu P, Sajal Bhattacharya C (2016) The Southern House Mosquito, Culex quinquefasciatus: profile of a smart vector. J Entomol Zool Stud 4 : 73\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDerua YA, Kweka EJ, Kisinza WN, Githeko AK, Mosha FW.(2019) Bacterial larvicides used for malaria vector control in sub-Saharan Africa: review of their effectiveness and operational feasibility. 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Med Vet Entomol Med Vet Entomol 12: 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHemingway J, Field L, Science JV- (2002) An overview of insecticide resistance. science. orgJ Hemingway, L Field, J VontasScience 298: 96\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHien AS, Soma DD, Hema O, Bayili B, Namountougou M, Gnankin\u0026eacute; O, et al. (2017) Evidence that agricultural use of pesticides selects pyrethroid resistance within Anopheles gambiae s.l. populations from cotton growing areas in Burkina Faso, West Africa. PLoS One12(3).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKe\u0026iuml;ta M, Sogoba N, Kan\u0026eacute; F, Traor\u0026eacute; B, Zeukeng F, Coulibaly B, et al. (2021) Multiple Resistance Mechanisms to Pyrethroids Insecticides in Anopheles gambiae sensu lato Population from Mali, West Africa. 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Genes (Basel) 9(12).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Institute of Advanced Study in Science and Technology","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Culex quinquefasciatus, body size, fecundity, insecticide, resistance, survival, multi-generation","lastPublishedDoi":"10.21203/rs.3.rs-7201858/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7201858/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe growing threat of insecticide resistance in the southern house mosquito, \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e, presents a significant obstacle to effective vector control efforts worldwide. While previous research has largely focused on elucidating the genetic and biochemical mechanisms of resistance, there remains a critical gap in understanding how prolonged insecticide exposure influences mosquito physiology across generations\u0026mdash;particularly in a sex-specific manner. In this study, we investigated the effects of multigenerational insecticide exposure on key life history traits, including adult survival, body size, and reproductive output in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e. We hypothesized that sustained exposure would (1) increase resistance, (2) influence survival and tolerance, and (3) impact physiological parameters such as body size and fecundity across generations. Our results demonstrate that female mosquitoes subjected to repeated insecticide exposure exhibit increased survival, larger body size, and modified fecundity patterns over successive generations, while males showed comparatively limited changes. These findings suggest that physiological adaptations are emerging under selection pressure, potentially enhancing the vectorial capacity of resistant mosquito populations. This study provides novel insights into the evolutionary consequences of insecticide pressure on mosquito biology and underscores the urgent need for integrated, long-term vector management strategies tailored to resistance dynamics in endemic regions.\u003c/p\u003e","manuscriptTitle":"Sex-Specific Survival and Morphological Adaptations in Culex quinquefasciatus (Diptera: Culidae) Mosquitoes Under Multi-Generational Insecticide Selection in North-East Guwahati, Assam, India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 04:25:32","doi":"10.21203/rs.3.rs-7201858/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"47c4bd42-2cf5-4b60-b528-6944f3d1d31d","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52035202,"name":"Entomology"}],"tags":[],"updatedAt":"2025-07-25T04:25:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-25 04:25:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7201858","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7201858","identity":"rs-7201858","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}