Desiccation tolerance and possible starvation trade-offs in larvicide resistant Culex and Aedes mosquitoes

Summary

Larvicides are widely used for mosquito control, but resistance development complicates efforts. We investigated the interplay between insecticide resistance and environmental stress in Aedes albopictus, Culex pipiens pipiens, and Culex pipiens molestus. Mosquitoes were selected for resistance to diflubenzuron and Bacillus thuringiensis subsp. israelensis, acclimated at four temperatures, and subjected to desiccation and starvation assays. Desiccation and starvation survival were affected by species, sex, and thermal acclimation, but not by larvicide resistance. Larvicide resistance affected starvation survival in Ae. albopictus, which also exhibited higher thermal plasticity under both stressors. Females outlived males across all species and conditions. These findings highlight ecological consequences of larvicide resistance, as it does not compromise stress resistance and thermal plasticity, complicating mosquito control amid climate change. Trade-offs between starvation and desiccation in Ae. albopictus suggests potential mechanistic links between these traits and larvicide mode-of-action. Species-, context- and trait-specific larvicide resistance responses complicates mosquito control efforts.

Graphical abstract

Highlights

• •Larvicide resistant Culex pipiens adults suffer no-cost under desiccation and starvation
• •Larvicide resistant Ae. albopictus suffer survival cost under starvation
• •Larvicide resistance does not affect thermal plasticity of Cx. pipens and Ae. albopictus
• •Species, context and trait-specific larvicide resistance responses complicate mosquito control

Biological sciences; Entomology

Introduction

Environmental factors such as temperature and humidity play a crucial role in regulating mosquito population dynamics, survival, and vector competence. Furthermore, the emergence of larvicide resistance in several mosquito species has raised concerns about the effectiveness of current control strategies. While much is known about the thermal biology and insecticide resistance in mosquitoes, the relationship between larvicide resistance and their responses to other forms of environmental stressors, such as desiccation and starvation, remain underexplored.

Aedes and Culex (Culicidae) mosquitoes pose significant public health risks. In Europe, Aedes albopictus and Culex pipiens biotypes are among the most prevalent and extensively studied species. High invasion rates of Ae. albopictus due to plastic and adaptive responses to environmental stress, allowing it to thrive in novel and often harsh environments have increased the burden on mosquito control efforts.^1,2 Furthermore, the two forms of the Cx. pipiens complex, Culex pipiens pipiens (aboveground, ornithophilic) and Culex pipiens molestus (underground, mammophilic), coexist in Europe, exploiting distinct habitats and expressing different host preferences.³ The behavior of various hybrids between the two Cx. pipiens forms further amplifies public health risks by driving unpredictable patterns of animal-to-human pathogen transmission.^4,5

Among biotic and abiotic factors affecting mosquito development and survival, temperature is considered the most crucial. Despite Ae. albopictus capacity to sustain survival and population growth in a wide range of temperatures, the thermal environment can substantially affect various aspects of its biology, including developmental duration of immature stages, adult longevity, but also vector competence and hence disease transmission patterns.^6,7,8 Culex pipiens embryonation is prolonged in cooler temperatures, while larval and pupal development is accelerated in warmer conditions. Regarding adults, female mosquitoes live longer in cooler temperatures.^9,10,11 Additionally, in Cx. p. pipiens temperature and daylength are key stimuli for inducing and terminating females’ facultative reproductive diapause.¹² Aside from temperature, among other environmental factors affecting the species, humidity should also be considered.

Humidity, associated with water regulation, directly affects adult and egg survival and indirectly the dispersion of breeding sites and hence the performance and survival of aquatic larvae. Relative humidity may affect various biological traits of Cx. pipiens populations, such as survival, fecundity, fertility, dispersal capacity, and distribution, overall regulating invasion success.¹³ Furthermore, humidity can indirectly impact Ae. albopictus populations by affecting persistence and abundance of ephemeral breeding sites (e.g., natural and artificial water cavities like pots, tanks, leaves, tree hollows). Lower humidity can accelerate evaporation and thus water volume in these sites, leading to increased larval crowding and suppressing population growth causing developmental stress, decreased size in emerged adults and differential vectorial capacity of mosquitoes.¹⁴ Increased water loss due to low humidity can lead to desiccation stress affecting physiological function in adults, leading to death.¹⁵ Hence, desiccation resistance is a key ecological trait supporting invasion success and dispersion dynamics. In general, dormant insects are more tolerant of desiccation stress since they use different physiological mechanisms and behavioral responses, that may restrict water loss by regulating water intake and select a proper hibernation site, respectively.¹⁶ In Ae. albopictus desiccation resistance plays a pivotal role in overwintering success, as following a photoperiodic stimulus, these mosquitoes produce desiccation-resistant eggs that can survive extended transport and establish populations in new habitats.^14,17 Similarly, while dry conditions may impact adult survival and reproduction, diapausing overwintering females of Cx. p. pipiens exhibit greater desiccation resistance compared to non-dormant individuals, further enhancing their survival and adaptability in challenging conditions.¹²

Among other environmental factors described above, adult mosquitoes’ access to nutrients also affects various parameters of their performance, like survival and disease transmission. Carbohydrate deficiency for example has been shown to increase mosquito vulnerability in parasite infections, by altering immunity related genetic factors like resistant polymorphisms or susceptibility alleles.^18,19 Furthermore, starvation can affect mosquitoes survival time in high temperatures, by accelerating their metabolic rates.^20,21

All three species described above are of high importance for public health and effective control approaches are required. Application of insecticides, especially treatment of breeding sites with larvicides, still remains the main control method against mosquitoes. Diflubenzuron (DFB) and Bacillus thuringiensis subsp. israelensis (Bti) are among the most widely used larvicides. DFB, a benzoylurea compound belongs in the chitin synthesis inhibitors pesticides, distorts the normal formulation of chitin structures in mosquito larvae, resulting in molting failures and death during adult emergence.²² Bti is a gram-positive bacterium with toxic action in Diptera species.²³ Bti interferes with the production of toxins (Cry and Cyt families, crystal proteins) that cause cell failure in midgut membrane, during sporulation.²⁴ Despite the high effectiveness and low environmental impact of both insecticides, concerns regarding resistance development from target species are rising. Recent reports of gene mutations in Cx. pipiens mosquitoes, that survived higher than recommended DFB field doses, question the future efficiency of this insecticide.²⁵ Also, high resistant rates of Cx. pipiens mosquitoes to DFB have been confirmed in different laboratory studies.^26,27 Recently, Ioannou et al.²⁸ has shown that selection in the laboratory for a few generations can dramatically increase the resistance levels of Ae. albopictus to DFB but not to Bti. It seems that the Bti toxins synergistic action has prevented resistance development. Other mosquito killing Bacillus species have shown considerable levels of resistance in mosquitoes.²⁴

Some rather limited recent efforts explore the tradeoffs between larvicide resistance and some biological traits.^26,27,29 Ioannou et al.^28,30 following meticulously designed and laboriously executed experimental protocols, created DFB and Bti resistant selected populations of Cx. p. pipiens, Cx. p. molestus, and Ae. albopictus, demonstrating that pesticide-resistant populations suffer high fitness costs in terms of egg viability and winter longevity, but not regarding their responses to thermal stress or thermal plasticity.³¹ Nonetheless, the response of resistant populations to stress induced by desiccation and starvation has yet to be explored. In addition, to the best of our knowledge there are no comparative studies exploring the desiccation and starvation resistance of the above three species.

The research cited above leads to the hypothesis that resistance to two widely used insecticides will not affect mosquitoes’ thermal plasticity but is expected to decrease their survival under environmental stressors. The present paper focuses on responses of larvicide resistant populations (DFB and Bti) of three mosquito species to desiccation and starvation, exploring possible trade-offs between larvicide resistance, stress tolerance and thermal plasticity. The current knowledge, regarding mosquito resistance to widely used insecticides and its potential impacts on populations fitness under environmental stress, is rather poor. Furthermore, plastic responses among resistant species during stress remain unexplored. This study aims to deepen our understanding about these interactions, while our results are expected to contribute to mosquito control strategies and address future challenges in vector management.

Results

Response to desiccation stress

The full factorial model of Cox-Regression analysis revealed significant variation in survival rate among species and thermal acclimation regimes (Table S1). Acclimation∗species was the only significant interaction highlighting the deferential plastic responses among the mosquito species (Table S1). Due to full model complication non-significant interactions were removed, to better understand the source of variation, and presented as a minimum adequate model (Table 1). In the minimum adequate model species, sex and thermal acclimation affected survival under desiccation stress. The acclimation∗species and sex∗species interactions were significant. Non-significant interactions have been removed. Selection to insecticides did not affect mosquitoes’ performance. Separate analysis per species revealed that sex significantly affected survival in all three species, while selection treatment was not a significant predictor (Tables S2–S4). Females outlived males across all species and treatments. Aedes albopictus and Cx. p. molestus showed longer survival when acclimated in higher temperatures, with stronger effects in females and no influence from selection treatments (Figures 1C and 1B). In Cx. p. pipiens, no significant differences were observed, though females showed a similar survival pattern to the other species (Figure 1A).

Table 1.

Minimum adequate model of the Cox regression analysis, where adult survival under desiccation stress (time in h) is the dependent variable; species, sex, and treatment are the main factors; and acclimation the covariate

Minimum adequate model
Factor	Wald	df	P
Species	55.1769	2	<0.001
Sex	135.052	1	<0.001
Acclimation	96.562	1	<0.001
Treatment	0.404	2	0.817
Acclimation∗Species	36.2	2	<0.001
Sex∗Species	32.967	2	<0.001

Figure 1.

Survival (l_x) of adult mosquitoes under desiccation stress

(A) Culex pipiens pipiens. Different colors represent different thermal acclimations prior to stress assays (blue, yellow, orange and red for 15, 20, 25, and 30°C respectively). Different insecticide selection treatments are presented in each row (no selection/control in the first line, selection to DFB in the second line and selection to Bti in the third line). Different sexes are presented in different columns (females in the left column and males in the right one for each species).

(B) Culex pipiens molestus. Different colors represent different thermal acclimations prior to stress assays (blue, yellow, orange and red for 15, 20, 25, and 30°C respectively). Different insecticide selection treatments are presented in each row (no selection/control in the first line, selection to DFB in the second line and selection to Bti in the third line). Different sexes are presented in different columns (females in the left column and males in the right one for each species).

(C) Aedes albopictus. Different colors represent different thermal acclimations prior to stress assays (blue, yellow, orange and red for 15, 20, 25, and 30°C respectively). Different insecticide selection treatments are presented in each row (no selection/control in the first line, selection to DFB in the second line and selection to Bti in the third line). Different sexes are presented in different columns (females in the left column and males in the right one for each species).

To better understand the response of different factors during desiccation stress, in association with thermal acclimation prior to assays, we estimated the reaction norms between species, treatment and sex (Figure 2). Reaction norms indicate effects of a single covariate in the different genotypes included in a group leading to different survival responses.³² In our case different species presented different responses to thermal acclimation with Ae. albopictus revealing a strong plastic response to thermal adaptation. Culex p. molestus showed a similar pattern with milder plasticity while Cx. p. pipiens had no plastic response (Figure 2A). Thermal acclimation affected mosquito survival in almost identical ways after selection to the two larvicides, while non-selected individuals presented a milder plastic response (Figure 2B). Between sexes females revealed stronger plastic response following thermal acclimation compared to males (Figure 2C).

Figure 2.

Effects of thermal acclimation on desiccation resistance of different mosquito genotypes

(A) Different mosquito species (green for Culex pipiens pipiens, purple for Culex pipiens molestus and orange for Aedes albopictus). Error bars are presented with vertical lines.

(B) Selection to larvicides (blue for non-selected individuals, red for selection to DFB and green for selection to Bti). Error bars are presented with vertical lines.

(C) Sex (pink for females and green for males). Error bars are presented with vertical lines.

Response to starvation stress

Full factorial model resulted from Cox-Regression analysis, revealed significant variation in survival rate among species, sex and thermal acclimation. The sex∗treatment interaction was the only significant factor affecting survival. Among three-way interactions sex∗species∗treatment and acclimation∗sex∗species were significant as well (Table S6). Due to full model complication non-significant interactions were removed, to better understand the source of variation (Table 2). In the minimum adequate model species, insecticide selection (treatment), sex and thermal acclimation significantly affected survival under starvation stress. Species∗treatment and sex∗species were significant two-way interactions. Non-significant interactions have been removed. Among three-way interactions sex∗species∗treatment and acclimation∗sex∗species were significant (Table 2). Due to three-way significant interactions, we conducted a separated analysis per factor.

Table 2.

Minimum adequate model of the Cox regression analysis, where adult survival under desiccation stress (time in h) is the dependent variable; species, sex, and treatment are the main factors; and acclimation the covariate

Minimum adequate model
Factor	Wald	df	P
Species	37.706	2	<0.001
Treatment	25.692	2	<0.001
Sex	823.669	1	<0.001
Acclimation	87.916	1	<0.001
Species∗Treatment	9.267	4	0.055
Sex∗Species	28.791	2	<0.001
Sex∗Species∗Treatment	28.325	4	<0.001
Acclimation∗Sex∗Species	12.433	2	0.002

To better understand the higher-order interactions and investigate effects of insecticide resistance in the three populations, our dataset was split by species. Cox regression analysis revealed significant effects of sex in all three species. Larvicide treatment significantly affected Ae. albopictus survival under starvation stress and had no significant effects in Cx. pipiens. Interactions among factors were significant in both Ae. albopictus and Cx. p. pipiens (Table 3). Females lived longer than males across species and treatments. Aedes albopictus and Cx. p. pipiens acclimated in higher temperatures performed better under starvation stress (Figures 3C and 3A). Culex p. pipiens, females selected to Bti had shorter lifespans compared to control and DFB-selected individuals (Figure 3A). Culex p. molestus adults without insecticide selection lived longer after higher thermal acclimation, while Ae. albopictus selected to larvicides had shorter lifespans than non-selected individuals (Figures 3B and 3C).

Table 3.

Results of Cox regression analysis, within each species, where survival (h) under starvation is the dependent variable, treatment and sex the main factors

Culex pipiens pipiens
Factor	Wald	df	P
Sex	171.59	1	<0.001
Treatment	2.330	2	0.31
Sex∗Treatment	20.61	2	<0.001
Culex pipiens molestus
Factor	Wald	df	P
Sex	68.64	1	<0.001
Treatment	2.66	2	0.26
Sex∗Treatment	3.42	2	0.18
Aedes albopictus
Factor	Wald	df	P
Sex	233.81	1	<0.001
Treatment	27.79	2	<0.001
Sex∗Treatment	7.40	2	0.025

Figure 3.

Survival (l_x) of adult mosquitoes under starvation stress

(A) Culex pipiens pipiens. Different colors represent different thermal acclimations prior to stress assays (blue, yellow, orange and red for 15, 20, 25, and 30°C respectively). Different insecticide selection treatments are presented in each row (no selection/control in the first line, selection to DFB in the second line and selection to Bti in the third line). Different sexes are presented in different columns (females in the left column and males in the right one for each species).

(B) Culex pipiens molestus. Different colors represent different thermal acclimations prior to stress assays (blue, yellow, orange and red for 15, 20, 25, and 30°C respectively). Different insecticide selection treatments are presented in each row (no selection/control in the first line, selection to DFB in the second line and selection to Bti in the third line). Different sexes are presented in different columns (females in the left column and males in the right one for each species).

(C) Aedes albopictus. Different colors represent different thermal acclimations prior to stress assays (blue, yellow, orange and red for 15, 20, 25, and 30°C respectively). Different insecticide selection treatments are presented in each row (no selection/control in the first line, selection to DFB in the second line and selection to Bti in the third line). Different sexes are presented in different columns (females in the left column and males in the right one for each species).

Complex interactions between acclimation and main tested factors can easier be understood by employing a reaction norm approach (Figure 4). Species differed in their capacity to withstand starvation stress following thermal acclimation. Aedes albopictus revealed a strong plastic response compared to both Cx. pipiens forms (Figure 4A). DFB selected mosquitoes showed weak plastic effects while mosquitoes selected to Bti had almost zero plastic response (Figure 4B). Between sexes, females expressed stronger plastic response following thermal acclimation compared to males (Figure 4C).

Figure 4.

Effects of thermal acclimation on starvation resistance of different mosquito genotypes

(A) Different mosquito species (green for Culex pipiens pipiens, purple for Culex pipiens molestus and orange for Aedes albopictus). Error bars are presented with vertical lines.

(B) Selection to larvicides (blue for non-selected individuals, red for selection to DFB and green for selection to Bti). Error bars are presented with vertical lines.

(C) Sex (pink for females and green for males). Error bars are presented with vertical lines.

Discussion

Our results revealed no correlation between larvicide resistance and survival under desiccation stress. On the other hand, selection to insecticides significantly affected the survival of Ae. albopictus under starvation, while in Cx. pipiens mosquitoes no survival cost was observed. Females presented a longer lifespan compared to males regardless of species or larvicide treatment. Mosquito survival during desiccation was affected by thermal acclimation in both Ae. albopictus and Cx. p. molestus, but not Cx. p. pipiens, while under starvation stress only Ae. albopictus survival was affected. Notably, plastic responses varied among species, with Ae. albopictus showing the strongest plasticity in response to environmental stressors.

Desiccation stress

Selection for resistance to both DFB and Bti did not affect mosquito performance during desiccation stress, in all three species tested. These findings align with previous studies on DFB resistance, which have identified certain mechanisms that may explain this outcome. Specifically, DFB-resistant mosquitoes bear a thicker cuticle and increased chitin content, which reduce water loss and thereby delay mortality.^16,33

Interplay between thermal stress, water balance, and desiccation resistance seems crucial to interpret our results. While previous experiments have found no correlation between thermal stress and larvicide resistance,³¹ broader studies indicate that water balance is a critical factor influencing both thermal tolerance and cold hardiness. For instance, insects pose various techniques to avoid or tolerate dehydration, including suppression of metabolic and thermoregulatory activities, which helps conserve water under thermal or cold stress.¹⁶ However, at higher temperatures metabolic rates increase, accelerating respiratory and cuticular water loss, making it hard to separate death from extreme heat from dehydration, also due to closely related physiological and omic markers.³⁴ Similarly, complex relationships link cold hardiness and dehydration. Freeze-tolerant insects, for example, gain resistance to extremely low temperatures, under extreme cellular dehydration, as supercooling mechanisms can be used in both stresses. Additionally, many insects decrease water content prior to cold periods to increase their lipid stores and hemolymph concentration, decreasing supercooling point and mitigating freeze damage.^16,34

In this context, the ability of DFB- and Bti-resistant mosquitoes to survive desiccation stress as effectively as their non-resistant counterparts may reflect a broader physiological resilience linked to shared mechanisms of water conservation.

Regarding different sex responses in desiccation stress, female mosquitoes of all three species tested lived longer compared to males. Dehydration tolerance can vary within the same species depending on sex.³⁴ Water loss rate depends mostly on fat and cuticle reserves in insects’ bodies. Females normally have more lipid reserves due to egg development, while due to their bigger body size they have smaller surface area to volume ratio delaying water loss along cuticle surface.³⁵

Starvation stress

Our results support that larvicide selection treatment to both DFB and Bti significantly affected the survival of Ae. albopictus mosquitoes under starvation, but not that of Cx. pipiens. DFB is an insect growth regulator (IGRs) and its effects on mosquito control are well documented. DFB disrupts hormonal control related to insect growth and development, primarily inhibiting chitin synthesis leading to molding failure prior to adult emergence.³⁶ However, its impact extends beyond affecting chitin synthesis. Juvenile hormone (JH), a key regulator for insect growth and development, remains at high levels during first larval stages and gradually decrease, by activating JH esterase, and disappears right before pupation. Despite DFB considered as chitin inhibitor its role on the maintenance of high JH levels during later larval stages has been proven.³⁷ Furthermore, in Aedes mosquitoes, resistance to DFB has been linked to significant physiological trade-offs, including decreased lipid reserves and disrupted JH synthesis. These interruptions suggest that resistance might involve mutations in JH regulation pathways, that under starvation gets more disrupted leading to higher mortality.³⁸ These mechanisms might explain why Ae. albopictus, which exhibited nearly double the DFB resistance ratio compared to Cx. pipiens, was more negatively impacted under starvation stress in our study.

On the other hand, resistance in Bti was relatively low in all three species.³¹ Previously, resistance mechanisms to Bti toxins have been found to be based on detoxification enzymes present in larval midgut and result in important fitness costs in Aedes mosquitoes, since an important energetic cost results from enzymes production.³⁹

By integrating these findings with prior research, our study underscores the importance of species-specific physiological and biochemical responses to larvicide resistance. The differential effects observed in Ae. albopictus and Cx. pipiens highlight how resistance mechanisms, particularly those involving hormonal regulation and energy metabolism, influence survival under environmental stressors like starvation. This knowledge can be complementary in mosquito control supporting resistance monitoring, and integrated pest management (IPM) combining chemical and non-chemical methods that can enhance control effectiveness while slowing resistance evolution. Addressing ecological factors, such as breeding site reduction and food availability, can impose additional stress on mosquito populations, complementing chemical interventions. Developing synergists to inhibit resistance mechanisms can also extend larvicide efficacy, promoting sustainable and environmentally friendly mosquito control practices.

Females showed higher survival under starvation stress compared to males, in all three species tested. During starvation, survival mechanisms dealing with lipid metabolism are activated and stored lipids are used as an energy source following complex metabolic paths.⁴⁰ Female mosquitoes have greater lipid reserves due to their bigger body size. Furthermore, the protein yolk produced during vitellogenesis can support molecular and biochemical regulations during starvation. This protein is normally produced by the insect’s fat body, but under starvation stress this tissue is reduced and secondary sources like midgut (exogenous) and oocyte (endogenous), can also produce it, supporting longer survival in females.⁴¹ The enhanced survival of female mosquitoes under starvation stress poses challenges for vector control programs, as females are the primary disease vectors. This resilience may reduce the effectiveness of larvicides, challenge the efficacy of sterile insect techniques (SITs), and accelerate resistance development due to shared physiological mechanisms, while may increase mosquito invasion rates due to climate change.⁴² Aedes species developing under stressful conditions potentially exhibit higher pathogen infection and dissemination rates a fact that is associated with alteration in disease transmission patterns that might impact public health.⁴³ Effective control strategies should integrate larval and adult-targeted measures, resistance monitoring, and environmental modeling to address these challenges and improve the efficacy of vector management efforts.

Thermal acclimation responses among species and stresses

All three species tested presented different responses to thermal acclimation. Survival during desiccation stress was affected by thermal acclimation in both Ae. albopictus and Cx. p. molestus, but not Cx. p. pipiens, while under starvation stress only Ae. albopictus survival was affected. Strong, mild and no thermal plasticity was revealed in Ae. albopictus, Cx. p. molestus, and Cx. p. pipiens respectively, under desiccation stress. Under starvation stress Ae. albopictus revealed a strong plastic response compared to both Cx. pipiens forms. Environmentally driven plastic responses, seem to highly impact population dynamics and disease transmission patterns, as vector transmitting ability can be associated with different thermal backgrounds, resulted from geographically distant regions, in mosquito species.⁴⁴ Aedes albopictus is well known for its quick adaptability and thermal plasticity as it originates from tropical Southeast Asia. Furthermore, inheritance of thermal-related traits, leading to evolutionary adaptation is reasonable in Aedes mosquitoes due to species quick biological circle, large population dynamics and close relation of their activity and physiology with thermal regimes.⁴⁵ Culex pipiens dispersed through thermally distant habitats in North America, contributing a variability of gene flow between populations originating from different thermal backgrounds.⁴⁶ Differences in thermal plasticity among the two Cx. pipiens forms might be related to co-species different origin and dispersion, but also winter biology.^3,47,48

Selection to the two larvicides did not affect mosquitoes’ thermal plasticity under desiccation and starvation stresses. We have previously suggested that in mosquitoes, genes inducing resistance to larvicides are distant from those responsible for thermal plasticity. The lack of trade-offs between thermal response and insecticide resistance raises critical implications for vector control strategies in the context of climate change. Populations selected for larvicide resistance may experience increased survival in limited resources or extreme thermal environments, thereby enabling their persistence and potential northward expansion as global temperatures rise. This phenomenon could exacerbate the spread of vector-borne diseases in previously unaffected regions, requiring extensive control measures.³¹

Between sexes females showed stronger plastic response following thermal acclimation compared to males in both assays. Larger body mass, in female ectotherms has already been associated with greater thermal tolerance, due to bigger energy reserves and lower mass specific metabolic rates.⁴⁹ Our results come in alignment with Lyberger et al.⁵⁰ research, revealing that female mosquitoes have presented a greater response across temperatures and higher thermal plasticity compared to males.

Conclusions

Insecticide-resistant mosquitoes can maintain their thermal plasticity and resistance to environmental stress, challenging mosquito control efforts. This is particularly worrying since larvicide-resistant populations are able to perform and survive effectively in the environment. Additionally, preservation of their plasticity raises concerns about their possible expansion into northern regions fronting climate change. However, our evidence suggests that Ae. albopictus may experience trade-offs between larvicide resistance and starvation survival mechanisms. This finding offers valuable insights into the mode of action of larvicides and could support mosquito control efforts by helping prevention of resistance development.

Limitations of the study

Although DFB resistance has been well-documented in both laboratory and natural settings, resistance to Bti under field conditions remains largely unexplored. Field validation of laboratory findings is crucial to ensure their ecological relevance. Understanding how environmental variables interact with resistance and plasticity, along with modeling mosquito range expansion in response to climate change, could provide valuable predictive insights and inform effective mosquito control strategies. Additionally, resistance mechanisms must be further investigated using both physiological and molecular techniques to better understand trade-offs between larvicide resistance and survival under stress, particularly in Ae. albopictus. Comparative studies across mosquito species are also essential to identify broader patterns and species-specific differences in resistance and plasticity, enhancing the development of targeted and sustainable control measures.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Nikos T. Papadopoulos (nikopap@uth.gr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •Data: All data reported in this paper will be shared by the lead contact upon request.
• •Code: This paper does not report original code.
• •All other requests: Any additional information required to reanalyze the data reported will be shared by the lead contact upon request.

Acknowledgments

This research was supported by the University of Thessaly and the Hellenic National Public Health Organization (grant on Enhanced Entomological Surveillance of Major Mosquito Vectors in Greece, 2024).

Author contributions

E.C.S.: Investigation, data curation, writing–Original draft preparation, formal analysis, visualization, writing–review and editing C.S.I.: Conceptualization, methodology, validation, writing–review and editing, investigation. L.A.: Investigation, data curation, writing–review and editing. J.S.T.: Methodology, writing–original draft, formal analysis, writing–review and editing. N.T.P.: Conceptualization, methodology, formal analysis, writing–original draft, supervision, funding acquisition, writing–reviewing and editing.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Bacillus thuringiensis subsp. israelensis (Bti)	Valent BioSciences Corporation, Libertyville, IL, USA	Vectobac® 12AS, 11.61% w/w Bti serotype H-14, strain AM65-52, 1200 ITU/mg
Chemicals, peptides, and recombinant proteins
Diflubenzuron (DFB)	Sigma-Aldrich, Taufkirchen, Germany	Purity ≥ 99.8%, Pestanal®
Experimental models: Organisms/strains
Culex pipiens pipiens	Greece, Volos
Culex pipiens molestus	Greece, Volos
Aedes albopictus	Greece, Volos
Software and algorithms
Statistical analysis	IBM SPSS Statistics 29

Experimental model and study participant details

Populations and rearing methods

All three mosquito populations considered in the current study originated from Volos, Greece and reared in the laboratory of Entomology and Agricultural Zoology at the University of Thessaly, since 2017. Aedes albopictus, Cx. pipiens pipiens and Cx. pipiens molestus reared inside walk-in chambers under constant environmental conditions (25 ± 1°C, 65 ± 5% RH and 14L:10D). Detailed rearing methods for all populations are given in Ioannou et al.^28,30,31

Larvicide selected populations

Larvicide resistant populations were developed for all three species, following selection in both DFB and Bti, for 12 successive generations. Detailed methodologies on the selection processes as well as the resulting Resistance Ratios (RR), for each population and selection treatment are detailed in Ioannou et al.^28,30 and Ioannou et al.³¹ respectively. Populations not subjected to larvicide selection process were used as control.

Method details

Thermal acclimation

Upon emergence, twenty adults per species, sex and selection treatment, were placed, in groups of five, inside small plastic cages.⁵¹ On the sixth day of age, the cages were transferred from 25°C at either 15, 20, 30 or maintained at 25°C for a week until being subjected to desiccation and starvation assessments.

Response to desiccation stress

Following the thermal acclimation treatment, adult mosquitoes were placed individually inside 1.5ml Eppendorf tubes, which were pierced with a distilled needle 12 times. To achieve low relative humidity (below 10%), vials containing mosquitoes were placed inside glass desiccators containing 300g of silica gel. Twenty replicates per species, treatment and acclimation were used. For each species vials separated in 4 desiccators, each of them hosting 60 vials. To achieve homogeneity among desiccators 5 mosquitoes per treatment and acclimation were placed inside each desiccator. Every three hours adult survival was recorded by inspecting the desiccators for dead individuals. Dead mosquitoes were removed as quickly as possible, to avoid condition sift inside the desiccators.

Response to starvation stress

The same methodology described above was used to estimate the effect of starvation on adult mosquito survival. The Pyrex desiccators used contained 300ml of distilled water to assure a saturated hydric environment (RH>98%).

Quantification and statistical analysis

The Cox regression analysis was selected for analyzing time to event data,⁵² while proportional hazard test was used to assess model’s fit.⁵³ Adult mosquito survival variation among, species, thermal acclimation conditions, sexes and insecticide selection lines, was tested. Pairwise comparisons were estimated using the log-rank test with the Kaplan–Meier method. Due to significant 3- and 4-way interactions, the data were split and re-analyzed by species to better understand the sources of variation. The statistical software IBM SPSS Statistics for Windows, Version 26.0 was used for data analysis. Both results from the full factorial (Supplementary Materials) and minimum adequate model analyses are provided. In order to better understand the source of variance among factors, reaction norms were created with two-way Anova using pairwise estimated marginal means to separate, statistically heterogeneous groups. All data were tested for normality and transformed for normal distribution prior to Anova tests.

Published: April 23, 2025