A Predictive Analysis of Insecticide Resistance Trend on Culex quinquefasciatus (Diptera: Culicidae) Mosquito Larvae Over Generations Upon Sublethal Treatment With DDT, Malathion and Deltamethrin

A Predictive Analysis of Insecticide Resistance Trend on Culex quinquefasciatus (Diptera: Culicidae) Mosquito Larvae Over Generations Upon Sublethal Treatment With DDT, Malathion and Deltamethrin | 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 A Predictive Analysis of Insecticide Resistance Trend on Culex quinquefasciatus (Diptera: Culicidae) Mosquito Larvae Over Generations Upon Sublethal Treatment With DDT, Malathion and Deltamethrin Aditya Shankar Kataki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6425732/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 Background and objectives: Prior studies showed that, repeated exposure of insecticides during the larval stage led to increase in vector resistance. However, a gap of knowledge persisted in analysing the trend of insecticide resistance and cross resistance in mosquitoes upon sublethal treatment during its larval stage over generations with different insecticides. Therefore, the goal of the current study was to comprehend the pattern of insecticide resistance and cross resistance in Cx. quinquefasciatus larvae following four generations of sublethal deltamethrin, DDT and malathion treatment. The current study's research questions were [I] would there be an increasing trend of larval resistance observed upon repeated sublethal treatment on Cx. quinquefasciatus larvae with several insecticides in each generation, and [II] will the trend of resistance be different for each insecticide? Methods: The larvae of Cx. quinquefasciatus in their early 3 rd instar were therefore subjected to sublethal doses of deltamethrin (0.01 mg/ml), DDT (1 mg/ml), and malathion (1 mg/ml). The resilience of the larvae was noted after 24hours of exposure Statistical analyses were performed using Generalised Mixed Modelling (glmm) and Log likelihood ratio tests (LRT). Results: The results revealed a trend displaying increase in the larval resistance across the generations. Moreover, it was found that the larvae were showing more resistance against DDT followed by deltamethrin and malathion. The cross-resistance analysis demonstrated that larvae resistant to one class of insecticide exhibited an increased level of resistance to other insecticides across successive generations. Interpretation and conclusion: Thus, the study successfully evaluated a trend of increase larval resistance in Cx. quinquefasciatus upon continuous exposure with DDT followed by malathion and deltamethrin over multigeneration. The study can act as a reference for future studies especially in vector control management to develop novel vector strategies and stimulating resistance trend for different mosquito species. Entomology Mosquitoes insecticide resistance sublethal treatment deltamethrin malathion DDT multigeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Geographically extensive and frequently prolific, the Southern house mosquito, Culex quinquefasciatus (Diptera: Culicidae), is an epidemiologically significant vector for a very diverse array of infections, including filarial worms, protozoa, and several arboviruses. 1 In addition to spreading the West Nile virus (WNV), St. Louis encephalitis virus, and other arboviruses, Cx. quinquefasciatus is the main vector of Wuchereria bancrofti , the parasite that causes lymphatic filariasis, and Plasmodium relictum , which causes malaria in birds. 2 Various control measures, such as behavioural, pharmacological, and biological interventions, have been employed thus far to stop the occurrence of Culex-borne illnesses. However, the effectiveness of current control measures varies, and they face several obstacles. Global outbreaks of Culex-borne diseases are occurring in new locations due to the rising insecticide resistance in mosquitoes, inadequacy of the current conventional technique and increased contacts between Culex and people brought on by urbanization and climate change. 3 Thus, even though there are numerous integrated preventive methods for controlling mosquitoes, synthetic chemical insecticides are consistently the only method that been relied upon to reduce the densities of nuisance mosquitoes and control vector populations in disease-endemic areas. However, though insecticides are essential for reducing disease vectors, their uncontrolled and unregulated usage has led to the development of mosquito resistance to these chemicals in various parts of the world. 4 The ability of an insect to resist the harmful effects of an insecticide by developing a resistance to those effects through adaptive natural selection and mutations is known as insecticide resistance. 5 Numerous studies have demonstrated that insecticide resistance is most likely caused by a variety of intricate resistance mechanisms, including greater metabolic detoxification of insecticides and lower sensitivity of the target proteins or genes. 6 – 13 However, even though there were prior studies on effect of different insecticides on mosquitoes and evaluating their resistance status, a gap of knowledge persisted in analysing the trend of insecticide resistance in mosquitoes upon sublethal treatment during its larval stage over generations with different insecticides. Therefore, the goal of the current study was to comprehend the pattern of insecticide resistance in Cx. quinquefasciatus larvae following four generations of sublethal DDT, deltamethrin, and malathion treatment. The current study's research questions were [I] would there be a trend of increased larval resistance observed after sublethal treatment of Cx. quinquefasciatus larvae with several insecticides in each generation, [II] will the trend of resistance vary for each insecticide and [III] will there be any generational effect. Thus, the larvae of Cx. quinquefasciatus in their early 3rd instar were therefore subjected to sublethal doses of deltamethrin (0.01 µg/ml), DDT (1 µg/ml), and malathion (1µg/ml). The resilience of the larvae was noted after 90 minutes of exposure. Since the effective control of mosquito vectors is threatened by the widespread and growing resistance to insecticides, it is essential to understand their insecticide resistance trend or else the tremendous progress made in controlling West Nile virus, filariasis, malaria, dengue etc over the past ten years may be reversed if insecticide resistance is not mitigated and managed, which is likely to increased burden of disease. MATERIAL AND METHODS Experiment condition The entire experiment was carried out at temperature 25°C ± 2 and humidity 70–80% with a 12- hour day/night cycles Mosquito Larvae Collection The early 3rd instar larval samples of Cx. quinquefasciatus were collected during period (September’24 to December’24) from different locations in Guwahati Metropolitan city, Kamrup (M) district, Assam (26.1158° N, 91.7086° E). In accordance with normal procedures, mosquito larvae from various locations were gathered using scoopers and a larval collection dipper. 14 , 15 After being collected, the mosquito larvae were placed in tiny, lidded containers for identification prior to raising. The books "The Ecology of Malaria Mosquitoes" by Charlwood JD and "The Biology of Mosquitoes" by A.N. Clements were used to identify the larvae. 16 , 17 Only Cx. quinquefasciatus larvae were chosen after identification and all other larvae were thrown away. For the larval bioassay test, the larvae were moved to trays that measured 5" by 7". Pilot Experiment A pilot experiment was carried out to evaluate the sublethal dose for deltamethrin, DDT and malathion upon exposure at larval stage of Cx. quiquefasciatus that was supposed to be use later in the experiment. Serial dilations were performed according to standard protocol 18 in 1:10 to prepare different doses of the insecticides. Upon preparation, ~ 20 larvae of Cx. quiqnuefasciatus were exposed in each dose of the different insecticides along with control (distilled water) for 24 hours 15 . The alive percentage of the larvae were noted and the LC 50 dose was recorded for DDT, deltamethrin and malathion. Followed by the pilot experiment, the LC 50 dose of the insecticides was selected for exposure in the larval stage of Cx. quinquefasciatus for up to four generations to observe the insecticide resistance trend. Splitting of larvae and Larval insecticide resistance bioassay The Cx. quinquefasciatus larvae were split into two lines: control and treatment for each insecticide. According to WHO recommendations 15 , the early 3rd instar larvae of the collected Cx. quiqnuefasciatus were evaluated for susceptibility to three insecticides: DDT, malathion and deltamethrin. The insecticides were administered to 3rd instar larvae (n = ~ 500) in plastic vials containing 100 millilitres of distilled water. The sublethal concentration of the insecticides were used: DDT 0.1 µg/ml; deltamethrin 1 µg/ml and malathion 1 µg/l. The number of live and dead larvae was counted after 24 hours 15 , and a log-probit model 19 was used to estimate the LC 50 . There were three iterations of the test. Upon exposure to DDT, deltamethrin and malathion with sublethal dose on the Cx. quinquefasciatus larvae for 24 hours; the larvae were separated using fine stainer and were again released in trays containing fresh 800 ml distilled water. Mosquito food pellets were added for the larvae to grow and develop into pupae and adults for next generation to continue. Collection and separation of mosquito pupae 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. The adults were discarded in each generation upon egg laying as the experiment was only confined to the larval stage. Fecundity analysis The mosquitoes were placed on a clean slide in a similar fashion 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’ and using Generalised modelling (GLMM). Log likelihood ratio test (LRT) was used to investigate the significance of all explanatory variables on the response variables in all models. ANOVA test was performed and based on Chisq, Degrees of freedom (Df) and p-value the best model was selected and fitted. From the packages ‘ggeffects’ and ‘emmeans’; ggemmeans was used to calculate the estimates. RESULTS Upon pilot experiment it was found that LC 50 value of Deltamethrin is log − 1.4 (~ 0.1 µg/ml), Malathion is log 0.35 (~ 1µg/ml) and DDT is log 0.65 (~ 1µg/ml) as evident in [Fig. 1] . This showed that Cx. quinquefasciatus larvae were more resistant towards sublethal exposure of DDT and Malathion compared to Deltamethrin. Figure 1 Graphs representing alive percentage of Cx. quinquefasciatus larvae upon sublethal treatment with Deltamethrin, Malathion and DDT. Graph [ A ] represents that upon treatment with Deltamethrin the LC50 value is log = -1.4 (~ 0.1 µg/ml), graph [ B ] represents that upon treatment with DDT the LC50 value is log = 0.65 (~ 1µg/ml) and graph [ C ] represents that upon treatment with Malathion the LC50 value is log = 0.35 (~ 1µg/ml) Larvae Development and Survival The percentage of larvae that survive throughout the development period across four generations (f0 to f3) for each of the four strains (A, B, C, and D) under control (C) and insecticide treated (I) circumstances is shown in the graph [ Figure 2 (A) ]. The distribution of survival percentage for each strain within a generation and treatment group is displayed in each box plot. Overall, the graph shows that different strains react differently to insecticide treatment, with certain strains exhibiting improved adaptation over successive generations. The graph in [ Figure 2 (B) ] shows the number of days that larvae take to develop over the course of four generations (f0 to f3) for each of the four strains (A, B, C, and D) under two different selection conditions: control (C) and insecticide treated (I). There is no discernible difference between the control and treatment groups, nor between generations, during the 10-to-14-day development period. Figure 2 Graph [A] representing larval survival rate for larvae for treated (I) vs Control (C) and Graph [B] representing larval development period for larvae for treated (I) vs Control (C) Fecundity Analysis In [ Fig. 3(A) ], the proportion of females producing eggs is significantly lower in the insecticide-treated group compared to the control group, suggesting that exposure to insecticide reduces fecundity. The control group exhibits a higher median value and less variability, while the insecticide-treated group shows a more dispersed range, indicating a negative effect on reproductive output. Through statistical analysis it was observed that selection ( Chisq (χ 2 ) = 95.65, Df = 2, p-value = < 2.2e-16 *** ) was significant. In [ Fig. 3(B) ], the number of eggs present is analysed across four strains (A, B, C, D) and generations (F0, F1, F2, and F3). Across all generations, the control group consistently shows higher egg production compared to the insecticide-exposed group. This trend is particularly evident in strains A and B, where the reduction in egg production is most pronounced. The reduction in egg production becomes more severe in later generations, indicating a cumulative or generational effect of insecticide exposure. Strains C and D exhibit lower egg production overall, with the insecticide further exacerbating the decline. Through statistical analysis it was observed that selection and strain ( Chisq (χ 2 ) = 115.35, Df = 3, p-value = < 2.2e-16 *** ) was significant. These findings highlight that insecticide exposure has a detrimental impact on both fecundity and egg production, with potential long-term effects across generations and genetic backgrounds. Figure 3 The boxplots in [ Fig. 3 ] depict the impact of insecticide exposure on fecundity ( A ) and egg production ( B ) across different generations and strains Analysis of insecticide resistance trend upon LC 50 treatment with DDT Upon subsequent treatment of Cx. quinquefasciatus larvae with sublethal concentration of DDT (1µg/ml) across three generation from F0-F3 and performing statistical analysis, it was observed that, generation ( Chisq (χ 2 ) = 109.65, Df = 3, p-value = < 2.2e-16 *** ) and treatment ( Chisq (χ 2 ) = 192.34, Df = 1, p-value = < 2.2e-16 *** ) was significant. In addition, a trend of linear increase of larval resistance from F1 generation was observed [Fig. 4(a)] and [ Table 1 ] upon continuous exposure of DDT in each subsequent generation during the larval stage of Cx. quinquefasciatus . Table 1 Mean alive percentage of Cx. quinquefasciatus larvae upon sublethal treatment with Deltamethrin, DDT and Malathion for 24hours over four generation (F0-F3). The table clearly shows that the mean alive percentage of Cx. quinquefasciatus larvae against DDT rises across the generation when there is repeated exposure followed by malathion and deltamethrin. This indicates that larvae are developing more resistance against DDT followed by malathion. Deltamethrin is comparatively found to be more effective. Insecticide LC 50 Dose (µg/ml) Log value Generation Mean alive (%) ± S.D. Probit value Deltamethrin 0.1 -1.4 F0 52 ± 3 5.05 F1 58± 2 5.20 F2 74 ± 4 5.64 F3 80 ± 2 5.84 DDT 1 0.65 F0 53± 5 5.08 F1 68± 7 5.47 F2 85 ± 3 6.04 F3 87 ± 2 6.13 Malathion 1 0.33 F0 53± 2 5.08 F1 63± 3 5.33 F2 78± 2 5.77 F3 82± 2 5.92 Analysis of insecticide resistance trend upon LC 50 treatment with Malathion Upon subsequent treatment of Cx. quinquefasciatus larvae with sublethal concentration of Malathion (1µg/ml) across three generation from F0-F3 and performing statistical analysis, it was observed that, generation ( Chisq (χ 2 ) = 72.172, Df = 3, p-value = 1.462e-15 *** ) and treatment ( Chisq (χ 2 ) = 271.79, Df = 1, p-value = < 2.2e-16 *** ) was significant. In addition, a linear trend of increase in larval resistance from F1 generation was observed [Fig. 4(b)] and [ Table 1 ] upon continuous exposure of malathion in each subsequent generation during the larval stage of Cx. quinquefasciatus . Analysis of insecticide resistance trend upon LC 50 treatment with Deltamethrin Upon subsequent treatment of Cx. quinquefasciatus larvae with sublethal concentration of deltamethrin (0.1µg/ml) across three generation from F0-F3 and performing statistical analysis, it was observed that, generation (Chisq (χ 2 ) = 65.329, Df = 3, p-value = 4.2e-14 ***) and treatment ( Chisq (χ 2 ) = 370.27, Df = 1, p-value = < 2.2e-16 *** ) was significant. In addition, a trend of lateral increase in larval resistance was also observed [Fig. 4(c)] and [ Table 1 ] from F2 generation upon continuous exposure to deltamethrin in each subsequent generation during the larval stage of Cx. quinquefasciatus . Figure 4 Graph [ A ] showing a rising trend in insecticide resistance in Cx. quinquefasciatus larvae across generation (F0-F3) upon sublethal treatment (1 µg/ml) with DDT. Graph [ B ] showing a rising trend in insecticide resistance in Cx. quinquefasciatus larvae across generation (F0-F3) upon sublethal treatment (1 µg/ml) with Malathion. Graph [ C ] showing a rising trend in insecticide resistance in Cx. quinquefasciatus larvae across generation (F0-F3) upon sublethal treatment (0.1 µg/ml) with deltamethrin. Cross Resistance Analysis The heatmaps provided in [ Fig. 5 ] illustrate the cross-resistance investigation of resistant larvae subjected to different insecticide treatments across multiple generations (F0 to F3). The shading intensity reflects the survival rate, where darker shades indicate higher survival rates, and lighter shades represent lower survival rates. The heatmap in [ Fig. 5(A) ] focuses on resistant Deltamethrin larvae subjected to DDT and Malathion treatments across four generations. The DDT and Malathion treatments show a progressive darkening of shades from F0 to F3, indicating an increase in survival as the larvae build resistance. The Deltamethrin treatment starts with a lighter shade but shows gradual darkening, reflecting an increase in survival rates over generations. The control group remains in lighter shades, showing consistently high survival due to the lack of insecticide exposure. The heatmap in [ Fig. 5 (B) ] shows the survival rates of DDT-resistant larvae when exposed to Deltamethrin and Malathion across four generations (F0 to F3). Darker shades indicate higher survival, showing increased resistance over generations. Gradual darkening in Malathion and Deltamethrin treatments highlights the development of cross-resistance. The control group’s lighter shades reflect consistently high survival without insecticide exposure. The heatmap in [ Fig. 5(C) ] reveals the survival rates of Malathion-resistant larvae when exposed to DDT and Deltamethrin across four generations (F0 to F3). In the Malathion and Deltamethrin treatments, survival rates progressively increase, suggesting the development of cross-resistance. The DDT treatment shows a similar trend, with moderate survival in early generations and higher survival in later ones. In contrast, the control group maintains consistently high survival rates due to the absence of insecticide exposure. Thus, these patterns effectively highlight the trend of cross-resistance development in resistant larvae over successive generations. Figure 5 : The heatmaps provided illustrate the cross-resistance investigation of resistant larvae subjected to different insecticide treatments across multiple generations (F0 to F3) [(A) Resistant Deltamethrin larvae with DDT and Malathion (B) Resistant DDT larvae with Deltamethrin and Malathion (C) Resistant Malathion larvae with DDT and Deltamethrin]. The y-axis represents the treatment groups: Control, DDT, Deltamethrin, and Malathion, while the x-axis represents the generations (F0, F1, F2, and F3). The colour intensity corresponds to the survival rate, with lighter shades indicating higher survival and darker shades indicating lower survival. DISCUSSION The present study successfully evaluated and established an increasing generational larvae resistance trend in Cx. quinquefasciatus upon repeated exposure of different insecticides over multigeneration. It has been observed that the mosquito larvae of Cx. quinquefasciatus shows higher level of increase resistance against repeated exposure of DDT over multigeneration followed by malathion. The larval resistance trend against deltamethrin has been observed to be comparatively less even though it is found to be increasing too over the generation upon continuous treatment. Similar findings were observed in prior studies 20 where the populations of Cx. quinquefasciatus that were examined showed high levels of phenotypic resistance to DDT, deltamethrin, permethrin, and malathion. The results suggested a potential impact of insecticide treatment, reducing fecundity compared to the control group. Hence, it was concluded that insecticide treatment reduces fecundity. The study corresponds to the results done in previous similar studies where it showed that the resistant An. gambiae mosquitoes had a fitness disadvantage on reproductive basis in comparison to the susceptible ones suggesting the possible accumulation of deleterious effects of insecticide resistance. 21 Also, it has been suggested that the lower fecundity rate in the resistant mosquitoes could be due to lower blood digestion that may result from altered physiology of the female mosquito. The variation observed in fecundity in the resistant female mosquitoes might be because the nutrients obtained during the blood meal, were used up in other essential processes that are linked to the survival of the resistant female mosquitoes instead of egg production. 21 – 24 In other studies, it was reported that mortality rates dropped from 94.7% and 98.9% 25 to 69.9% and 80.9%, respectively, in Chiang Mai and Lampang Provinces of Thailand due to enhanced resistance to malathion. 26 In India, every insecticide tested exhibited varying degrees of reaction, which may be related to both the use of the insecticide and the varying capacities of mosquito populations. 27 Many mosquito species are known to exhibit high or moderate resistance to organochlorines, including DDT, even when it is not employed for mosquito control. 28 Despite being prohibited for use in agriculture, DDT is nonetheless used in India, particularly in rural areas, to control vectors. Numerous mosquito species in India have been seen to exhibit resistance to DDT on a regular basis. 29 Synthetic pyrethroids, which are widely employed for vector control, are typically linked to cross-resistance in mosquito species that exhibit widespread DDT resistance. 30 Both substances target the same location, the voltage-gated sodium channel, which causes cross-resistance, despite having different chemical structures. 31 Further compounding the problem of DDT resistance and raising serious concerns for the vector control effort is the discovery of residual DDT in India's numerous water resources. 32 A similar trend of increasing resistance has been observed in mosquitoes against deltamethrin. 27 Deltamethrin resistance appears to be more prevalent than resistance to other pyrethroids. It has been reported from several locations, including Tamil Nadu, Delhi, West Bengal, and Assam, that over 50% of the sites assessed against deltamethrin as a larvicide and adulticide have been classified as extremely resistant. 27 Since the 1980s, India has employed pyrethroid chemicals, such as permethrin, deltamethrin, lambda-cyhalothrin, and α-cypermethrin, to control mosquitoes. Mosquitoes are subjected to domestic insecticides, including repellents, liquidators, and coils or sprays, which are particularly employed in urban and peri-urban regions, in addition to their direct application for fogging and indoor residual spraying. Pyrethroid resistance is a serious problem because pyrethroid chemicals are used in most used mosquito control devices in homes (coils, repellents, and liquidators), including insecticide-impregnated nets used in areas where malaria is endemic. 27 , 33 Nonetheless, it is thought that the rapid emergence and spread of pyrethroid resistance in mosquito species can be explained by both overwhelming selection pressures and cross-resistance brought on by DDT. From the current study it can well understood that the steady rise in resistance to all three insecticides (DDT, deltamethrin and malathion) over the generations raises the possibility that sublethal exposure contributes significantly to the selection of resistant individuals in mosquito populations. These patterns show that to prevent the development of resistance and maintain the effectiveness of current chemical tools in vector control programs, insecticides must be used in synergistically with other organic compounds like entomopathogenic fungi or in combination and rotation. 34 – 36 The need to investigate alternate control methods is highlighted by the evidence of growing resistance. Sustainable methods to lower mosquito populations and the risks of disease transmission include the development of genetically modified mosquitoes, environmental management to decrease mosquito breeding grounds, and the use of biological control agents (such as predatory fish or bacteria like Bacillus thuringiensis Israelis ) 37 .Thus, the growing resistance to DDT, deltamethrin and malathion, which are routinely employed in public health initiatives, jeopardizes efforts to prevent mosquito-borne diseases such lymphatic filariasis and West Nile virus. If resistance continues to grow, the efficacy of these insecticides may wane, perhaps leading to increased disease transmission. Proactive efforts to combat resistance tendencies are thus critical to ensuring public health results. More research is needed to understand the biochemical and genetic pathways underlying resistance in Cx. quinquefasciatus . Studies examining gene expression changes, metabolic enzyme activity, and mutation trends in resistant populations could reveal unique resistance pathways. Advances in gene-editing technologies, like as CRISPR-Cas9, may provide new ways to combat resistance mechanisms. Furthermore, the relationship between insecticide exposure and environmental factors requires further investigation to better understand resistance mechanisms in mosquitoes. CONCLUSION Thus, the study focuses on the dynamic nature of larvae insecticide resistance in Cx. quinquefasciatus , as well as the possible consequences for vector control and public health. The identified resistance trends against DDT, deltamethrin and malathion; necessitate quick action to diversify control tactics, improve resistance monitoring, and provide novel solutions. Collaboration across the scientific, regulatory, and public health sectors will be critical in reducing the impact of insecticide resistance on mosquito control programs and disease prevention efforts worldwide. Declarations ACKNOWLEDGEMENT I 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. DATA AVAILABILITY STATEMENT All the data used to generate results in the manuscript can be found in the link provided. https://drive.google.com/drive/folders/1t0BrKoG4h_2GSOYv5Cn2QxhwJYEUyw-3?usp=share_link References Bhattacharya S, Basu P, Sajal Bhattacharya C (2016) The Southern House Mosquito, Culex quinquefasciatus: profile of a smart vector. J Entomol Zool Stud ; 4 : 73–81. 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Available from: https://books.google.com/books/about/Biology_of_Mosquitoes_Development_Nutrit.html?id=dLjwAAAAMAAJ , accessed on June 29, 2024 Serial Dilution Protocol (2024) Available from: https://bpsbioscience.com/serial-dilution-protocol , accessed on June 30 Probit RHD, Analysis. By DJ, Finney MA (1952) Sc.D., [2nd ed. Pp. xiv + 318. Cambridge University Press, 35s.]. J Inst Actuar Cambridge University Press; 1952; 78 : 388–90. Available from: https://www.cambridge.org/core/journals/journal-of-the-institute-of-actuaries/article/abs/probit-analysis-by-d-j-finney-ma-scd-2nd-ed-pp-xiv-318-cambridge-university-press-1952-35s/6A77C4EDFD67D3FD9269C796BCB74A0E , accessed on December 23, 2024 Talipouo A, Mavridis K, Nchoutpouen E, Djiappi-Tchamen B, Fotakis EA, Kopya E et al (2021) High insecticide resistance mediated by different mechanisms in Culex quinquefasciatus populations from the city of Yaoundé, Cameroon. Scientific Reports Nature Publishing Group; 2021; 11 : 1–11. Available from: https://www.nature.com/articles/s41598-021-86850-7 , accessed on December 23, 2024 Osoro JK, Machani MG, Ochomo E, Wanjala C, Omukunda E, Githeko AK et al (2022) Insecticide resistant Anopheles gambiae have enhanced longevity but reduced reproductive fitness and a longer first gonotrophic cycle. Sci Rep Nature Research; ; 12 Ma Z, Gulia-Nuss M, Zhang X, Brown MR (2013) Effects of the Botanical Insecticide, Toosendanin, on Blood Digestion and Egg Production by Female Aedes aegypti (Diptera: Culicidae): Topical Application and Ingestion. J Med Entomol Oxford Academic; ; 50 : 112–21. Available from: https://dx.doi.org/10.1603/ME12119 , accessed on July 27, 2024 Mebrahtu YB, Norem J, Taylor M (1997) Inheritance of larval resistance to permethrin in Aedes aegypti and association with sex ratio distortion and life history variation. American Journal of Tropical Medicine and Hygiene American Society of Tropical Medicine and Hygiene; ; 56 : 456–65 Sy F, Faye O, Diallo M, Dia I. Effects of insecticide resistance on the reproductive potential of two sub-strains of the malaria vector Anopheles coluzzii. J Vector Borne Dis J Vector Borne Dis; 2019; 56 : 207–11. Available from: https://pubmed.ncbi.nlm.nih.gov/32655069/, accessed on July 27, 2024 Somboon P, … LP-SAJ, 2003 undefined. Insecticide susceptibility tests of Anopheles minimus sl, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus in northern Thailand. researchgate.netP Somboon, L Prapanthadara, W SuwonkerdSoutheast Asian Journal of Tropical Medicine and Public Health, 2003•researchgate.net 2003; 34 . Available from: https://www.researchgate.net/profile/La-Aied-Prapanthadara/publication/10569078_Insecticide_susceptibility_tests_of_Anopheles_minimus_sl_Aedes_aegypti_Aedes_albopictus_and_Culex_quinquefasciatus_in_northern_Thailand/links/00b495291f3d433746000000/Insecticide-susceptibility-tests-of-Anopheles-minimus-sl-Aedes-aegypti-Aedes-albopictus-and-Culex-quinquefasciatus-in-northern-Thailand.pdf,accessed on December 23, 2024 Yanola J, Chamnanya S, Lumjuan N, Somboon P (2015) Insecticides resistance in the Culex quinquefasciatus populations from northern Thailand and possible resistance mechanisms. Acta Trop Elsevier 149:232–238 Sumitha M, Kalimuthu M, Senthil M, Paramasivan R, Kumar A, Gupta B (2023) Status of insecticide resistance in the dengue vector Aedes aegypti in India: A review. J Vector Borne Dis Wolters Kluwer Medknow Publications; ; 60 : 116–24. Available from: https://journals.lww.com/jvbd/fulltext/2023/60020/status_of_insecticide_resistance_in_the_dengue.1.aspx , accessed on December 23, 2024 Campos KB, Martins AJ, Rodovalho C, de Bellinato M, Dias DF, de Macoris L dos et al (2020) L da G,. Assessment of the susceptibility status of Aedes aegypti (Diptera: Culicidae) populations to pyriproxyfen and malathion in a nation-wide monitoring of insecticide resistance performed in Brazil from 2017 to 2018. Parasit Vectors BioMed Central Ltd; ; 13 : 1–18. Available from: https://parasitesandvectors.biomedcentral.com/articles/ 10.1186/s13071-020-04406-6 , accessed on December 23, 2024 Rahi M, Mishra AK, Chand G, Baharia RK, Hazara RK, Singh SP et al (2024) Malaria Vector Bionomics: Countrywide Surveillance Study on Implications for Malaria Elimination in India. JMIR Public Health Surveill JMIR Publications Inc.; ; 10 Chen H, Li K, Wang X, Yang X, Lin Y, Cai F et al (2016) First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid insecticides in Aedes albopictus populations from Haikou City, Hainan Island, China. Infect Dis Poverty BioMed Central Ltd.; ; 5 Hancock PA, Wiebe A, Gleave KA, Bhatt S, Cameron E, Trett A et al (2018) Associated patterns of insecticide resistance in field populations of malaria vectors across Africa. Proc Natl Acad Sci U S A Proc Natl Acad Sci U S A; ; 115 : 5938–43. Available from: https://pubmed.ncbi.nlm.nih.gov/29784773/ , accessed on December 23, 2024 Agarwal A, Prajapati R, Singh OP, Raza SK, Thakur LK (2015) Pesticide residue in water–a challenging task in India. Environ Monit Assess Environ Monit Assess; ; 187 . Available from: https://pubmed.ncbi.nlm.nih.gov/25638058/ , accessed on December 23, 2024 Pryce J, Medley N, Choi L, Sons Ltd (2022) Indoor residual spraying for preventing malaria in communities using insecticide-treated nets. Cochrane Database Syst Rev John Wiley and ; ; 2022 : CD012688. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC8763033/ , accessed on December 23, 2024 Alkhaibari AM, Maffeis T, Bull JC, Butt TM (2018) Combined use of the entomopathogenic fungus, Metarhizium brunneum, and the mosquito predator, Toxorhynchites brevipalpis, for control of mosquito larvae: Is this a risky biocontrol strategy? J Invertebr Pathol Academic Press; ; 153 : 38–50 Kataki AS, Baldini F, Naorem AS (2024) Evaluation of synergistic effect of entomopathogenic fungi Beauveria bassiana and Lecanicillium lecacii on the mosquito Culex quinquefaciatus. PLoS One Public Library of Science; ; 19 : e0308707. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0308707 , accessed on November 26, 2024 Ahmed MAI, Matsumura F (2012) Synergistic actions of formamidine insecticides on the activity of pyrethroids and neonicotinoids against aedes aegypti (Diptera: Culicidae). J Med Entomol 49:1405–1410 Huang YJS, Higgs S, Vanlandingham DL (2017) Biological Control Strategies for Mosquito Vectors of Arboviruses. Insects MDPI AG; ; 8 : 21. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC5371949/ , accessed on December 23, 2024 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-6425732","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441530817,"identity":"1405a28b-7624-4f5d-86af-8ed20fac774b","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, United Kingdom","correspondingAuthor":true,"prefix":"","firstName":"Aditya","middleName":"Shankar","lastName":"Kataki","suffix":""}],"badges":[],"createdAt":"2025-04-11 07:19:52","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6425732/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6425732/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80522451,"identity":"6a37fec2-b6f9-4bcf-8892-542c4b4c26cb","added_by":"auto","created_at":"2025-04-14 09:20:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":396846,"visible":true,"origin":"","legend":"\u003cp\u003eGraphs representing alive percentage of Cx. quinquefasciatus larvae upon sublethal treatment with Deltamethrin, Malathion and DDT. Graph [\u003cstrong\u003eA\u003c/strong\u003e] represents that upon treatment with Deltamethrin the LC50 value is log = -1.4 (~ 0.1 mg/ml), graph [\u003cstrong\u003eB\u003c/strong\u003e] represents that upon treatment with DDT the LC50 value is log = 0.65 (~1mg/ml) and graph [\u003cstrong\u003eC\u003c/strong\u003e] represents that upon treatment with Malathion the LC50 value is log = 0.35 (~ 1mg/ml)\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6425732/v1/edc139cf40076ca9f8dd7f51.jpg"},{"id":80523392,"identity":"55f74dfa-8dda-4e4c-9100-890e15646f3f","added_by":"auto","created_at":"2025-04-14 09:28:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164606,"visible":true,"origin":"","legend":"\u003cp\u003eGraph [A] representing larval survival rate for larvae for treated (I) vs Control (C) and\u003c/p\u003e\n\u003cp\u003eGraph [B] representing larval development period for larvae for treated (I) vs Control (C)\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6425732/v1/3609e963c36e9419d89979f0.png"},{"id":80522454,"identity":"afb2c5b3-3f2d-4d29-ad2a-99aa1b561045","added_by":"auto","created_at":"2025-04-14 09:20:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":153903,"visible":true,"origin":"","legend":"\u003cp\u003eThe boxplots in [\u003cstrong\u003eFig 3\u003c/strong\u003e] depict the impact of insecticide exposure on fecundity (\u003cstrong\u003eA\u003c/strong\u003e) and egg production (\u003cstrong\u003eB\u003c/strong\u003e) across different generations and strains\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6425732/v1/67c04a42616bf69bacdad57e.png"},{"id":80522458,"identity":"5b301442-0162-48a6-a3e8-56909405e1ab","added_by":"auto","created_at":"2025-04-14 09:20:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":227541,"visible":true,"origin":"","legend":"\u003cp\u003eGraph [\u003cstrong\u003eA\u003c/strong\u003e] showing a rising trend in insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae across generation (F0-F3) upon sublethal treatment (1 mg/ml) with DDT. Graph [\u003cstrong\u003eB\u003c/strong\u003e] showing a rising trend in insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae across generation (F0-F3) upon sublethal treatment (1 mg/ml) with Malathion. Graph [\u003cstrong\u003eC\u003c/strong\u003e] showing a rising trend in insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae across generation (F0-F3) upon sublethal treatment (0.1 mg/ml) with deltamethrin.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6425732/v1/5c43071b659f6b6cf423cd83.png"},{"id":80522455,"identity":"f3ba281b-3d5c-4da8-9b73-e5043757374f","added_by":"auto","created_at":"2025-04-14 09:20:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144277,"visible":true,"origin":"","legend":"\u003cp\u003eThe heatmaps provided illustrate the cross-resistance investigation of resistant larvae subjected to different insecticide treatments across multiple generations (F0 to F3) [(A) Resistant Deltamethrin larvae with DDT and Malathion (B) Resistant DDT larvae with Deltamethrin and Malathion (C) Resistant Malathion larvae with DDT and Deltamethrin]\u003cstrong\u003e.\u003c/strong\u003e The y-axis represents the treatment groups: Control, DDT, Deltamethrin, and Malathion, while the x-axis represents the generations (F0, F1, F2, and F3). The colour intensity corresponds to the survival rate, with lighter shades indicating higher survival and darker shades indicating lower survival.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6425732/v1/a3ce55d318a1d6fecf70aee0.png"},{"id":80525120,"identity":"6c0a422c-880a-4cfe-809f-77acb8942159","added_by":"auto","created_at":"2025-04-14 09:44:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1966558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6425732/v1/78d130b2-a75a-462e-8a17-a2ed7900238c.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eA Predictive Analysis of Insecticide Resistance Trend on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCulex quinquefasciatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (Diptera: Culicidae) Mosquito Larvae Over Generations Upon Sublethal Treatment With DDT, Malathion and Deltamethrin\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGeographically extensive and frequently prolific, the Southern house mosquito, \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e (Diptera: Culicidae), is an epidemiologically significant vector for a very diverse array of infections, including filarial worms, protozoa, and several arboviruses.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e In addition to spreading the West Nile virus (WNV), St. Louis encephalitis virus, and other arboviruses, \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e is the main vector of \u003cem\u003eWuchereria bancrofti\u003c/em\u003e, the parasite that causes lymphatic filariasis, and \u003cem\u003ePlasmodium relictum\u003c/em\u003e, which causes malaria in birds.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Various control measures, such as behavioural, pharmacological, and biological interventions, have been employed thus far to stop the occurrence of Culex-borne illnesses. However, the effectiveness of current control measures varies, and they face several obstacles. Global outbreaks of Culex-borne diseases are occurring in new locations due to the rising insecticide resistance in mosquitoes, inadequacy of the current conventional technique and increased contacts between Culex and people brought on by urbanization and climate change.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Thus, even though there are numerous integrated preventive methods for controlling mosquitoes, synthetic chemical insecticides are consistently the only method that been relied upon to reduce the densities of nuisance mosquitoes and control vector populations in disease-endemic areas. However, though insecticides are essential for reducing disease vectors, their uncontrolled and unregulated usage has led to the development of mosquito resistance to these chemicals in various parts of the world.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003eThe ability of an insect to resist the harmful effects of an insecticide by developing a resistance to those effects through adaptive natural selection and mutations is known as insecticide resistance.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Numerous studies have demonstrated that insecticide resistance is most likely caused by a variety of intricate resistance mechanisms, including greater metabolic detoxification of insecticides and lower sensitivity of the target proteins or genes.\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, even though there were prior studies on effect of different insecticides on mosquitoes and evaluating their resistance status, a gap of knowledge persisted in analysing the trend of insecticide resistance in mosquitoes upon sublethal treatment during its larval stage over generations with different insecticides. Therefore, the goal of the current study was to comprehend the pattern of insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae following four generations of sublethal DDT, deltamethrin, and malathion treatment. The current study's research questions were [I] would there be a trend of increased larval resistance observed after sublethal treatment of Cx. quinquefasciatus larvae with several insecticides in each generation, [II] will the trend of resistance vary for each insecticide and [III] will there be any generational effect. Thus, the larvae of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e in their early 3rd instar were therefore subjected to sublethal doses of deltamethrin (0.01 \u0026micro;g/ml), DDT (1 \u0026micro;g/ml), and malathion (1\u0026micro;g/ml). The resilience of the larvae was noted after 90 minutes of exposure.\u003c/p\u003e \u003cp\u003eSince the effective control of mosquito vectors is threatened by the widespread and growing resistance to insecticides, it is essential to understand their insecticide resistance trend or else the tremendous progress made in controlling West Nile virus, filariasis, malaria, dengue etc over the past ten years may be reversed if insecticide resistance is not mitigated and managed, which is likely to increased burden of disease.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperiment condition\u003c/h2\u003e \u003cp\u003eThe entire experiment was carried out at temperature 25\u0026deg;C \u0026plusmn; 2 and humidity 70\u0026ndash;80% with a 12- hour day/night cycles\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMosquito Larvae Collection\u003c/h3\u003e\n\u003cp\u003eThe early 3rd instar larval samples of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e were collected during period (September\u0026rsquo;24 to December\u0026rsquo;24) from different locations in Guwahati Metropolitan city, Kamrup (M) district, Assam (26.1158\u0026deg; N, 91.7086\u0026deg; E). In accordance with normal procedures, mosquito larvae from various locations were gathered using scoopers and a larval collection dipper. \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eAfter being collected, the mosquito larvae were placed in tiny, lidded containers for identification prior to raising. The books \"The Ecology of Malaria Mosquitoes\" by Charlwood JD and \"The Biology of Mosquitoes\" by A.N. Clements were used to identify the larvae. \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Only \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae were chosen after identification and all other larvae were thrown away. For the larval bioassay test, the larvae were moved to trays that measured 5\" by 7\".\u003c/p\u003e\n\u003ch3\u003ePilot Experiment\u003c/h3\u003e\n\u003cp\u003eA pilot experiment was carried out to evaluate the sublethal dose for deltamethrin, DDT and malathion upon exposure at larval stage of \u003cem\u003eCx. quiquefasciatus\u003c/em\u003e that was supposed to be use later in the experiment. Serial dilations were performed according to standard protocol\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e in 1:10 to prepare different doses of the insecticides. Upon preparation, ~\u0026thinsp;20 larvae of \u003cem\u003eCx. quiqnuefasciatus\u003c/em\u003e were exposed in each dose of the different insecticides along with control (distilled water) for 24 hours\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The alive percentage of the larvae were noted and the LC\u003csub\u003e50\u003c/sub\u003e dose was recorded for DDT, deltamethrin and malathion. Followed by the pilot experiment, the LC\u003csub\u003e50\u003c/sub\u003e dose of the insecticides was selected for exposure in the larval stage of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e for up to four generations to observe the insecticide resistance trend.\u003c/p\u003e\n\u003ch3\u003eSplitting of larvae and Larval insecticide resistance bioassay\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae were split into two lines: control and treatment for each insecticide. According to WHO recommendations \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, the early 3rd instar larvae of the collected \u003cem\u003eCx. quiqnuefasciatus\u003c/em\u003e were evaluated for susceptibility to three insecticides: DDT, malathion and deltamethrin. The insecticides were administered to 3rd instar larvae (n\u0026thinsp;=\u0026thinsp;~\u0026thinsp;500) in plastic vials containing 100 millilitres of distilled water. The sublethal concentration of the insecticides were used: DDT 0.1 \u0026micro;g/ml; deltamethrin 1 \u0026micro;g/ml and malathion 1 \u0026micro;g/l. The number of live and dead larvae was counted after 24 hours\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and a log-probit model \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e was used to estimate the LC\u003csub\u003e50\u003c/sub\u003e. There were three iterations of the test. Upon exposure to DDT, deltamethrin and malathion with sublethal dose on the \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae for 24 hours; the larvae were separated using fine stainer and were again released in trays containing fresh 800 ml distilled water. Mosquito food pellets were added for the larvae to grow and develop into pupae and adults for next generation to continue.\u003c/p\u003e\n\u003ch3\u003eCollection and separation of mosquito pupae\u003c/h3\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. The adults were discarded in each generation upon egg laying as the experiment was only confined to the larval stage.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFecundity analysis\u003c/h2\u003e \u003cp\u003eThe mosquitoes were placed on a clean slide in a similar fashion 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 \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \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; and using Generalised modelling (GLMM). Log likelihood ratio test (LRT) was used to investigate the significance of all explanatory variables on the response variables in all models. ANOVA test was performed and based on Chisq, Degrees of freedom (Df) and p-value the best model was selected and fitted. From the packages \u0026lsquo;ggeffects\u0026rsquo; and \u0026lsquo;emmeans\u0026rsquo;; ggemmeans was used to calculate the estimates.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eUpon pilot experiment it was found that LC\u003csub\u003e50\u003c/sub\u003e value of Deltamethrin is log \u0026minus;\u0026thinsp;1.4 (~\u0026thinsp;0.1 \u0026micro;g/ml), Malathion is log 0.35 (~\u0026thinsp;1\u0026micro;g/ml) and DDT is log 0.65 (~\u0026thinsp;1\u0026micro;g/ml) as evident in \u003cb\u003e[Fig.\u0026nbsp;1]\u003c/b\u003e. This showed that \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae were more resistant towards sublethal exposure of DDT and Malathion compared to Deltamethrin.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure\u0026nbsp;1\u003c/strong\u003e \u003cp\u003eGraphs representing alive percentage of Cx. quinquefasciatus larvae upon sublethal treatment with Deltamethrin, Malathion and DDT. Graph [\u003cb\u003eA\u003c/b\u003e] represents that upon treatment with Deltamethrin the LC50 value is log = -1.4 (~\u0026thinsp;0.1 \u0026micro;g/ml), graph [\u003cb\u003eB\u003c/b\u003e] represents that upon treatment with DDT the LC50 value is log\u0026thinsp;=\u0026thinsp;0.65 (~\u0026thinsp;1\u0026micro;g/ml) and graph [\u003cb\u003eC\u003c/b\u003e] represents that upon treatment with Malathion the LC50 value is log\u0026thinsp;=\u0026thinsp;0.35 (~\u0026thinsp;1\u0026micro;g/ml)\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLarvae Development and Survival\u003c/h2\u003e \u003cp\u003eThe percentage of larvae that survive throughout the development period across four generations (f0 to f3) for each of the four strains (A, B, C, and D) under control (C) and insecticide treated (I) circumstances is shown in the graph [\u003cb\u003eFigure 2 (A)\u003c/b\u003e]. The distribution of survival percentage for each strain within a generation and treatment group is displayed in each box plot. Overall, the graph shows that different strains react differently to insecticide treatment, with certain strains exhibiting improved adaptation over successive generations. The graph in [\u003cb\u003eFigure 2 (B)\u003c/b\u003e] shows the number of days that larvae take to develop over the course of four generations (f0 to f3) for each of the four strains (A, B, C, and D) under two different selection conditions: control (C) and insecticide treated (I). There is no discernible difference between the control and treatment groups, nor between generations, during the 10-to-14-day development period.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure\u0026nbsp;2\u003c/strong\u003e \u003cp\u003eGraph [A] representing larval survival rate for larvae for treated (I) vs Control (C) and\u003c/p\u003e \u003c/p\u003e \u003cp\u003eGraph [B] representing larval development period for larvae for treated (I) vs Control (C)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFecundity Analysis\u003c/h2\u003e \u003cp\u003eIn [\u003cb\u003eFig.\u0026nbsp;3(A)\u003c/b\u003e], the proportion of females producing eggs is significantly lower in the insecticide-treated group compared to the control group, suggesting that exposure to insecticide reduces fecundity. The control group exhibits a higher median value and less variability, while the insecticide-treated group shows a more dispersed range, indicating a negative effect on reproductive output. Through statistical analysis it was observed that selection (\u003cb\u003eChisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;95.65, Df\u0026thinsp;=\u0026thinsp;2, p-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16 ***\u003c/b\u003e) was significant.\u003c/p\u003e \u003cp\u003eIn [\u003cb\u003eFig.\u0026nbsp;3(B)\u003c/b\u003e], the number of eggs present is analysed across four strains (A, B, C, D) and generations (F0, F1, F2, and F3). Across all generations, the control group consistently shows higher egg production compared to the insecticide-exposed group. This trend is particularly evident in strains A and B, where the reduction in egg production is most pronounced. The reduction in egg production becomes more severe in later generations, indicating a cumulative or generational effect of insecticide exposure. Strains C and D exhibit lower egg production overall, with the insecticide further exacerbating the decline. Through statistical analysis it was observed that selection and strain (\u003cb\u003eChisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;115.35, Df\u0026thinsp;=\u0026thinsp;3, p-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16 ***\u003c/b\u003e) was significant.\u003c/p\u003e \u003cp\u003eThese findings highlight that insecticide exposure has a detrimental impact on both fecundity and egg production, with potential long-term effects across generations and genetic backgrounds.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure\u0026nbsp;3\u003c/strong\u003e \u003cp\u003eThe boxplots in [\u003cb\u003eFig.\u0026nbsp;3\u003c/b\u003e] depict the impact of insecticide exposure on fecundity (\u003cb\u003eA\u003c/b\u003e) and egg production (\u003cb\u003eB\u003c/b\u003e) across different generations and strains\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of insecticide resistance trend upon LC\u003csub\u003e50\u003c/sub\u003e treatment with DDT\u003c/h2\u003e \u003cp\u003eUpon subsequent treatment of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae with sublethal concentration of DDT (1\u0026micro;g/ml) across three generation from F0-F3 and performing statistical analysis, it was observed that, generation (\u003cb\u003eChisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;109.65, Df\u0026thinsp;=\u0026thinsp;3, p-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16 ***\u003c/b\u003e) and treatment (\u003cb\u003eChisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;192.34, Df\u0026thinsp;=\u0026thinsp;1, p-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16 ***\u003c/b\u003e) was significant. In addition, a trend of linear increase of larval resistance from F1 generation was observed \u003cb\u003e[Fig.\u0026nbsp;4(a)]\u003c/b\u003e and \u003cb\u003e[\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e]\u003c/b\u003e upon continuous exposure of DDT in each subsequent generation during the larval stage of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e.\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\u003eMean alive percentage of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae upon sublethal treatment with Deltamethrin, DDT and Malathion for 24hours over four generation (F0-F3). The table clearly shows that the mean alive percentage of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae against DDT rises across the generation when there is repeated exposure followed by malathion and deltamethrin. This indicates that larvae are developing more resistance against DDT followed by malathion. Deltamethrin is comparatively found to be more effective.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eInsecticide\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eDose\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(\u0026micro;g/ml)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLog value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eGeneration\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eMean alive (%) \u0026plusmn; S.D.\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eProbit value\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\u003eDeltamethrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e52 \u0026plusmn; 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e58\u0026plusmn; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e74 \u0026plusmn; 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e80 \u0026plusmn; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDDT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e53\u0026plusmn; 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e68\u0026plusmn; 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e85 \u0026plusmn; 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e87 \u0026plusmn; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalathion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e53\u0026plusmn; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63\u0026plusmn; 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e78\u0026plusmn; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e82\u0026plusmn; 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of insecticide resistance trend upon LC\u003csub\u003e50\u003c/sub\u003e treatment with Malathion\u003c/h2\u003e \u003cp\u003eUpon subsequent treatment of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae with sublethal concentration of Malathion (1\u0026micro;g/ml) across three generation from F0-F3 and performing statistical analysis, it was observed that, generation (\u003cb\u003eChisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;72.172, Df\u0026thinsp;=\u0026thinsp;3, p-value\u0026thinsp;=\u0026thinsp;1.462e-15 ***\u003c/b\u003e) and treatment (\u003cb\u003eChisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;271.79, Df\u0026thinsp;=\u0026thinsp;1, p-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16 ***\u003c/b\u003e) was significant. In addition, a linear trend of increase in larval resistance from F1 generation was observed \u003cb\u003e[Fig.\u0026nbsp;4(b)]\u003c/b\u003eand \u003cb\u003e[\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e] upon\u003c/b\u003e continuous exposure of malathion in each subsequent generation during the larval stage of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of insecticide resistance trend upon LC\u003csub\u003e50\u003c/sub\u003e treatment with Deltamethrin\u003c/h2\u003e \u003cp\u003eUpon subsequent treatment of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae with sublethal concentration of deltamethrin (0.1\u0026micro;g/ml) across three generation from F0-F3 and performing statistical analysis, it was observed that, generation \u003cb\u003e(Chisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;65.329, Df\u0026thinsp;=\u0026thinsp;3, p-value\u0026thinsp;=\u0026thinsp;4.2e-14 ***)\u003c/b\u003e and treatment (\u003cb\u003eChisq (χ\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u0026thinsp;=\u0026thinsp;370.27, Df\u0026thinsp;=\u0026thinsp;1, p-value\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16 ***\u003c/b\u003e) was significant. In addition, a trend of lateral increase in larval resistance was also observed \u003cb\u003e[Fig.\u0026nbsp;4(c)]\u003c/b\u003e and \u003cb\u003e[\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e]\u003c/b\u003e from F2 generation upon continuous exposure to deltamethrin in each subsequent generation during the larval stage of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure\u0026nbsp;4\u003c/strong\u003e \u003cp\u003eGraph [\u003cb\u003eA\u003c/b\u003e] showing a rising trend in insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae across generation (F0-F3) upon sublethal treatment (1 \u0026micro;g/ml) with DDT. Graph [\u003cb\u003eB\u003c/b\u003e] showing a rising trend in insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae across generation (F0-F3) upon sublethal treatment (1 \u0026micro;g/ml) with Malathion. Graph [\u003cb\u003eC\u003c/b\u003e] showing a rising trend in insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae across generation (F0-F3) upon sublethal treatment (0.1 \u0026micro;g/ml) with deltamethrin.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCross Resistance Analysis\u003c/h2\u003e \u003cp\u003eThe heatmaps provided in [\u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e] illustrate the cross-resistance investigation of resistant larvae subjected to different insecticide treatments across multiple generations (F0 to F3). The shading intensity reflects the survival rate, where darker shades indicate higher survival rates, and lighter shades represent lower survival rates.\u003c/p\u003e \u003cp\u003eThe heatmap in [\u003cb\u003eFig.\u0026nbsp;5(A)\u003c/b\u003e] focuses on resistant Deltamethrin larvae subjected to DDT and Malathion treatments across four generations. The DDT and Malathion treatments show a progressive darkening of shades from F0 to F3, indicating an increase in survival as the larvae build resistance. The Deltamethrin treatment starts with a lighter shade but shows gradual darkening, reflecting an increase in survival rates over generations. The control group remains in lighter shades, showing consistently high survival due to the lack of insecticide exposure.\u003c/p\u003e \u003cp\u003eThe heatmap in [\u003cb\u003eFig.\u0026nbsp;5 (B)\u003c/b\u003e] shows the survival rates of DDT-resistant larvae when exposed to Deltamethrin and Malathion across four generations (F0 to F3). Darker shades indicate higher survival, showing increased resistance over generations. Gradual darkening in Malathion and Deltamethrin treatments highlights the development of cross-resistance. The control group\u0026rsquo;s lighter shades reflect consistently high survival without insecticide exposure.\u003c/p\u003e \u003cp\u003eThe heatmap in [\u003cb\u003eFig.\u0026nbsp;5(C)\u003c/b\u003e] reveals the survival rates of Malathion-resistant larvae when exposed to DDT and Deltamethrin across four generations (F0 to F3). In the Malathion and Deltamethrin treatments, survival rates progressively increase, suggesting the development of cross-resistance. The DDT treatment shows a similar trend, with moderate survival in early generations and higher survival in later ones. In contrast, the control group maintains consistently high survival rates due to the absence of insecticide exposure.\u003c/p\u003e \u003cp\u003eThus, these patterns effectively highlight the trend of cross-resistance development in resistant larvae over successive generations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5\u003c/b\u003e: The heatmaps provided illustrate the cross-resistance investigation of resistant larvae subjected to different insecticide treatments across multiple generations (F0 to F3) [(A) Resistant Deltamethrin larvae with DDT and Malathion (B) Resistant DDT larvae with Deltamethrin and Malathion (C) Resistant Malathion larvae with DDT and Deltamethrin]. The y-axis represents the treatment groups: Control, DDT, Deltamethrin, and Malathion, while the x-axis represents the generations (F0, F1, F2, and F3). The colour intensity corresponds to the survival rate, with lighter shades indicating higher survival and darker shades indicating lower survival.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study successfully evaluated and established an increasing generational larvae resistance trend in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e upon repeated exposure of different insecticides over multigeneration. It has been observed that the mosquito larvae of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e shows higher level of increase resistance against repeated exposure of DDT over multigeneration followed by malathion. The larval resistance trend against deltamethrin has been observed to be comparatively less even though it is found to be increasing too over the generation upon continuous treatment. Similar findings were observed in prior studies \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e where the populations of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e that were examined showed high levels of phenotypic resistance to DDT, deltamethrin, permethrin, and malathion. The results suggested a potential impact of insecticide treatment, reducing fecundity compared to the control group. Hence, it was concluded that insecticide treatment reduces fecundity. The study corresponds to the results done in previous similar studies where it showed that the resistant \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes had a fitness disadvantage on reproductive basis in comparison to the susceptible ones suggesting the possible accumulation of deleterious effects of insecticide resistance.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Also, it has been suggested that the lower fecundity rate in the resistant mosquitoes could be due to lower blood digestion that may result from altered physiology of the female mosquito. The variation observed in fecundity in the resistant female mosquitoes might be because the nutrients obtained during the blood meal, were used up in other essential processes that are linked to the survival of the resistant female mosquitoes instead of egg production.\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn other studies, it was reported that mortality rates dropped from 94.7% and 98.9%\u003csup\u003e25\u003c/sup\u003e to 69.9% and 80.9%, respectively, in Chiang Mai and Lampang Provinces of Thailand due to enhanced resistance to malathion.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In India, every insecticide tested exhibited varying degrees of reaction, which may be related to both the use of the insecticide and the varying capacities of mosquito populations.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003eMany mosquito species are known to exhibit high or moderate resistance to organochlorines, including DDT, even when it is not employed for mosquito control.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Despite being prohibited for use in agriculture, DDT is nonetheless used in India, particularly in rural areas, to control vectors. Numerous mosquito species in India have been seen to exhibit resistance to DDT on a regular basis.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Synthetic pyrethroids, which are widely employed for vector control, are typically linked to cross-resistance in mosquito species that exhibit widespread DDT resistance.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Both substances target the same location, the voltage-gated sodium channel, which causes cross-resistance, despite having different chemical structures.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003eFurther compounding the problem of DDT resistance and raising serious concerns for the vector control effort is the discovery of residual DDT in India's numerous water resources.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e A similar trend of increasing resistance has been observed in mosquitoes against deltamethrin.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Deltamethrin resistance appears to be more prevalent than resistance to other pyrethroids. It has been reported from several locations, including Tamil Nadu, Delhi, West Bengal, and Assam, that over 50% of the sites assessed against deltamethrin as a larvicide and adulticide have been classified as extremely resistant.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Since the 1980s, India has employed pyrethroid chemicals, such as permethrin, deltamethrin, lambda-cyhalothrin, and α-cypermethrin, to control mosquitoes. Mosquitoes are subjected to domestic insecticides, including repellents, liquidators, and coils or sprays, which are particularly employed in urban and peri-urban regions, in addition to their direct application for fogging and indoor residual spraying. Pyrethroid resistance is a serious problem because pyrethroid chemicals are used in most used mosquito control devices in homes (coils, repellents, and liquidators), including insecticide-impregnated nets used in areas where malaria is endemic.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Nonetheless, it is thought that the rapid emergence and spread of pyrethroid resistance in mosquito species can be explained by both overwhelming selection pressures and cross-resistance brought on by DDT.\u003c/p\u003e \u003cp\u003eFrom the current study it can well understood that the steady rise in resistance to all three insecticides (DDT, deltamethrin and malathion) over the generations raises the possibility that sublethal exposure contributes significantly to the selection of resistant individuals in mosquito populations. These patterns show that to prevent the development of resistance and maintain the effectiveness of current chemical tools in vector control programs, insecticides must be used in synergistically with other organic compounds like entomopathogenic fungi or in combination and rotation.\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e The need to investigate alternate control methods is highlighted by the evidence of growing resistance. Sustainable methods to lower mosquito populations and the risks of disease transmission include the development of genetically modified mosquitoes, environmental management to decrease mosquito breeding grounds, and the use of biological control agents (such as predatory fish or bacteria like \u003cem\u003eBacillus thuringiensis Israelis\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.Thus, the growing resistance to DDT, deltamethrin and malathion, which are routinely employed in public health initiatives, jeopardizes efforts to prevent mosquito-borne diseases such lymphatic filariasis and West Nile virus. If resistance continues to grow, the efficacy of these insecticides may wane, perhaps leading to increased disease transmission. Proactive efforts to combat resistance tendencies are thus critical to ensuring public health results. More research is needed to understand the biochemical and genetic pathways underlying resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e. Studies examining gene expression changes, metabolic enzyme activity, and mutation trends in resistant populations could reveal unique resistance pathways. Advances in gene-editing technologies, like as CRISPR-Cas9, may provide new ways to combat resistance mechanisms. Furthermore, the relationship between insecticide exposure and environmental factors requires further investigation to better understand resistance mechanisms in mosquitoes.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThus, the study focuses on the dynamic nature of larvae insecticide resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, as well as the possible consequences for vector control and public health. The identified resistance trends against DDT, deltamethrin and malathion; necessitate quick action to diversify control tactics, improve resistance monitoring, and provide novel solutions. Collaboration across the scientific, regulatory, and public health sectors will be critical in reducing the impact of insecticide resistance on mosquito control programs and disease prevention efforts worldwide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eI 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\u003ch2\u003eDATA AVAILABILITY STATEMENT\u003c/h2\u003e \u003cp\u003eAll the data used to generate results in the manuscript can be found in the link provided. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://drive.google.com/drive/folders/1t0BrKoG4h_2GSOYv5Cn2QxhwJYEUyw-3?usp=share_link\u003c/span\u003e\u003cspan address=\"https://drive.google.com/drive/folders/1t0BrKoG4h_2GSOYv5Cn2QxhwJYEUyw-3?usp=share_link\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBhattacharya S, Basu P, Sajal Bhattacharya C (2016) The Southern House Mosquito, Culex quinquefasciatus: profile of a smart vector. \u003cem\u003eJ Entomol Zool Stud\u003c/em\u003e ; \u003cem\u003e4\u003c/em\u003e: 73\u0026ndash;81. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.researchgate.net/publication/330579096\u003c/span\u003e\u003cspan address=\"https://www.researchgate.net/publication/330579096\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on June 28, 2024\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. \u003cem\u003eScience\u003c/em\u003e American Association for the Advancement of Science; ; \u003cem\u003e330\u003c/em\u003e: 88. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pmc.ncbi.nlm.nih.gov/articles/PMC5371949/\u003c/span\u003e\u003cspan address=\"https://pmc.ncbi.nlm.nih.gov/articles/PMC5371949/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on December 23, 2024\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":"Insitute of Advanced Study in Science and Technology","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Mosquitoes, insecticide resistance, sublethal treatment, deltamethrin, malathion, DDT, multigeneration","lastPublishedDoi":"10.21203/rs.3.rs-6425732/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6425732/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and objectives:\u003c/strong\u003e Prior studies showed that, repeated exposure of insecticides during the larval stage led to increase in vector resistance. However, a gap of knowledge persisted in analysing the trend of insecticide resistance and cross resistance in mosquitoes upon sublethal treatment during its larval stage over generations with different insecticides. Therefore, the goal of the current study was to comprehend the pattern of insecticide resistance and cross resistance in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae following four generations of sublethal deltamethrin, DDT and malathion treatment. The current study's research questions were [I] would there be an increasing trend of larval resistance observed upon repeated sublethal treatment on \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e larvae with several insecticides in each generation, and [II] will the trend of resistance be different for each insecticide?\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The larvae of \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e in their early 3\u003csup\u003erd \u003c/sup\u003einstar were therefore subjected to sublethal doses of deltamethrin (0.01 mg/ml), DDT (1 mg/ml), and malathion (1 mg/ml). The resilience of the larvae was noted after 24hours of exposure Statistical analyses were performed using Generalised Mixed Modelling (glmm) and Log likelihood ratio tests (LRT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The results revealed a trend displaying increase in the larval resistance across the generations. Moreover, it was found that the larvae were showing more resistance against DDT followed by deltamethrin and malathion. The cross-resistance analysis demonstrated that larvae resistant to one class of insecticide exhibited an increased level of resistance to other insecticides across successive generations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterpretation and conclusion:\u003c/strong\u003e Thus, the study successfully evaluated a trend of increase larval resistance in \u003cem\u003eCx. quinquefasciatus \u003c/em\u003eupon continuous exposure with DDT followed by malathion and deltamethrin over multigeneration. The study can act as a reference for future studies especially in vector control management to develop novel vector strategies and stimulating resistance trend for different mosquito species.\u003c/p\u003e","manuscriptTitle":"A Predictive Analysis of Insecticide Resistance Trend on Culex quinquefasciatus (Diptera: Culicidae) Mosquito Larvae Over Generations Upon Sublethal Treatment With DDT, Malathion and Deltamethrin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-14 09:20:45","doi":"10.21203/rs.3.rs-6425732/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":"April 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47013261,"name":"Entomology"}],"tags":[],"updatedAt":"2025-04-14T09:20:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-14 09:20:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6425732","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6425732","identity":"rs-6425732","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}