Effects of temperature and nutrition stress on Anopheles stephensi, an Indian urban malaria vector

doi:10.21203/rs.3.rs-5214107/v1

Background: Changing climate and complexity in ecological landscape can potentially expand the geographic distribution of mosquitoes (Diptera: Culicidae) to adapt and transmit various vector-borne diseases like malaria globally. Anopheles stephensi is a potential urban malaria vector in the Indian subcontinent. Temperature and nutrients are the important environmental stressors, which influence the life cycle and vector competence of mosquitoes.

Methods: Three experimental setups were designed in the laboratory by exposing An. stephensi to low-temperature (LT, 4°Cand 18±1°C), high-temperature (HT, 35.5±1°C) and nutrition-deficient (NT, 33 mg) conditions. Stress was induced to both juvenile and adult stages for eight consecutive generations. Data of life-stage parameters were recorded. Additionally, LT, HT and NT strains were infected with in vitro culture of Plasmodium falciparum to compare their vectorial competence. Cytological analysis using the ovarian polytene chromosomes of the three stressed strains was carried out to check the structural differences in chromosomal arms due to extreme stress. Through predictive mathematical modelling using linear regression analysis, we estimated the number of generations required for the stressed lines to become comparable to the control lines.

Results: Fecundity, egg–wing sizes, egg-to-adult developmental time, pupal weight, male-to-female ratio, and longevity of adults were decreased in HT and NT lines, whereas these parameters were increased in LT lines. The HT and NT lines developed faster from egg to adult emergence (7.5 and 11 days), whereas the duration was longer (25 days) in the LT line. The oocyst infection rates in in-vitro Plasmodium falciparum were higher in HT, LT, and NT lines than the control line. The NT line revealed paracentric inversions on the 3L polytene chromosome arm in 2 locations. Further, the predictive model suggested faster adaptation of the LT line than HT and NT lines.

Conclusion: Being an invasive species and responsible for urban malaria transmission, the present multi-transgenerational laboratory-based study provides novel insights into the effects of stress on the life cycle, vectorial competence, cytology and rate of adaptation of An. stephensi. The findings aid in assessing disease risk and developing suitable intervention measures.

stress

Anopheles stephensi

Plasmodium falciparum

vectorial competence

Linear model

polytene chromosomal inversion

Climate change, rapid globalization, and natural disasters are matters of concern as they might amplify the abundance and dispersal of insect vectors, significantly affecting disease transmissions [1–3]. The World Meteorological Organization (WMO), in its Global Annual to Decadal Climate Update (GADCU), observed a 98% probability that the 5-year mean environmental temperature for 2023–2027 will be higher than the previous 5 years (2018–2022) [4]. Temperature fluctuations due to climate change have a greater impact on the survivability of vector mosquitoes. Vector-borne diseases (VBDs) have increased globally. Since 2014, major outbreaks of VBDs transmitted by mosquitoes like malaria, dengue, chikungunya, yellow fever and Zika have caused troubles to human populations, claimed lives and challenged public health [5, 6].

According to World Malaria Report 2023, climate change is a major threat that drives new malaria cases worldwide. Approximately 249 million cases were recorded globally, resulting in more than 608000 deaths annually [7]. Malaria is a protozoan disease transmitted by Anopheline mosquitoes. Anopheles stephensi is one of the important urban malaria vectors in the Indian subcontinent and Middle East [8]. Recently, it has emerged as an invasive vector in Africa competent in transmitting both Plasmodium falciparum and P. vivax parasites. The range of An. stephensi is expanding in 8 countries of the African continent – Djibouti in 2012, Ethiopia and Sudan (2016), Somalia (2019), Nigeria (2020), Ghana, Kenya and Eritrea (2022) [9]. Studies revealed that environmental stressors such as nutrient and temperature are the most important factors affecting mosquitoes by altering their life cycles, habits, behaviour, biting habits, and pathogen transmission [10–13].

Previous experiments showed that under nutritional stress, larval growth and development have carry-over effects on adult life-history traits and affect the human-to-mosquito transmission cycle in An. coluzzii–P. falciparum trajectory [10]. Few studies explored that the suboptimal temperatures, crowding, and starvation reduced the length of the gonotrophic cycle, fecundity, larval growth, adult mosquito size, longevity, and ultimately affected vectorial capacity [14, 15]. Similarly, the larvae of Culex pipiens, Aedes aegypti and Aedes albopictus reared under insufficient nutrition and extreme temperature conditions have observed reduced number of eggs (fecundity), smaller egg size and decreased adult size [16–18]. Studies revealed that various pathogens affected differently regulated by larval diets [19, 20]. Effects of temperature on Corythucha ciliata (Say) life stages have a significant role in the duration of the egg-to-adult stages, adult body size, reproductive capability, and vectorial capacity [21]. One of the most significant cytogenetic mechanisms is chromosomal inversions that have been frequently involved in local adaptation in many insects. It allows them to survive and reproduce better in its environment [22].

Mathematical models for predicting the effects of climate change on vector survivability and VBD transmissions are of great use not just in the field of epidemiology, but also in developing effective vector management strategies. Various studies have shown the impact of temperature on the distribution and dynamics of disease in view of climate change [23]. Laboratory-based assays on temperature dependencies of life stages are being increasingly studied to make mathematical models to predict the impact of climatic and environmental factors on vector populations and vector-borne disease dynamics [24, 25].

Although there have been specific studies on the impact of stress on mosquitoes, they are not comprehensive and transgenerational. The evolutionary changes and adaptations of the vectors to stress are imperative in understanding the disease epidemiology and forecasting the vulnerability of the new geographical areas to mosquito-borne diseases. Moreover, the pathogen–host interactions are also directly related to epigenetic factors and climate change directly impacts them. The present study investigated how temperature and nutritional deficiency influence the transgenerational traits, life cycle, vectorial competence, chromosomal inversions and adaptation rate of An. stephensi.

In the present study, 3 independent experiments were designed to expose mosquito life stages to different environmental stressors such as low temperature, high temperature, and nutritional deficiency over 8 consecutive generations. Data were generated and recorded for various parameters: (a) morphometrics of eggs and (b) wings, (c) pupal body mass (weight), (d) life-cycle stages, i.e., fecundity, egg hatchability, duration time of eggs to larvae, number of males and females, and adult longevity; (e) vectorial competence by estimating oocyst rates of P. falciparum; (f) chromosomal inversions; and (g) a linear model using the transitional life-cycle data.

Colonization of An. stephensi

Two wild strains of An. stephensi (T₂ andT₆) were used for this study and are maintained in TIGS insectary.

T₂ Strain – The mosquito was initially collected from Anna Nagar, Chennai, Tamil Nadu, India (13.018410°N, 80.223068°E). Approximately 50–60 gravid females were collected from human dwellings with the help of aspirators in the morning between 6 and 8 a.m. during the summer season. The strain was brought and established in the insectary. This strain was used for high- and low-temperature stress experiments along with their corresponding control lines.

T₆ Strain – The mosquito was collected from Sriramanahalli, Bangalore semi-urban area, Karnataka, India (12.972442°N, 77.580643°E). Around 250 larvae were collected at 2^nd to 4^th instar stages from freshwater of a cemented tank. The strain was brought and established in the insectary. This strain was used for nutritional stress experiments along with their corresponding control lines.

The rearing of mosquitoes in the insectary was followed as per our previous published protocols [26, 27]. Adults were maintained in the insectary at 28±1°C, with a relative humidity (RH) of 75±5% and a photoperiod of 12h:12h light and dark cycles. Adult foods were provided with a mixture of sugar (8%), glucose (2%), and multivitamin soln. (5%). On day 6 or 7, the adults were fed human blood through artificial Hemotek membrane feeding systems. Ovicups or individual vials containing RO water were lined with filter paper strips and placed inside the adult cages for oviposition. Hatched-out larvae were reared in white trays provided with larval food (1 part of yeast: 3 parts of dog biscuits). The temperature of the larval room was maintained at 28±1°C. Pupae were bleached with 1% sodium hypochlorite before being kept inside adult cages. Emerged males and females were allowed to inbreed within the filial generation and blood-fed on day 5 or 6 to get eggs for maintaining the next progeny [26].

Morphometric analyses

The size of the experimental (LT, HT and NT) eggs and wings and control lines (T₂ and T₆) were measured and compared to determine the patterns and trends between variables.

Egg morphometrics – Eggs from 5 individual females of both experimental and control lines were counted following our earlier published protocols [28]. The measurement was performed for 10 consecutive generations. Eggs were placed on wet filter paper on a glass slide. Then, the eggs were measured under the microscope using a stage and ocular micrometre (Unilab GE-34, Binocular Research Microscope, India). Different egg parameters, i.e., ridge number of the egg float, length and width of both eggs and egg floats, were measured, and data were recorded for 10 generations.

Wing morphometrics – Male and female mosquitoes (6–7-day old) were randomly selected from LT, HT, NT and control lines in 8 consecutive generations and measured for their wing sizes. A total of 10 wing samples from each experimental strain were selected for measurement. The mosquitoes were anesthetized on CO₂ pads, and the right wing of each mosquito was dissected under a microscope (Olympus, SZX2-ILLK, Germany). The length of wings was measured from the distal end of the alula to the utmost extreme tip between R₁ and R₂ veins, except the fringe scale [29]. The measurement data were recorded using Leica Application Suite X (LAS X) software for 8 generations.

Pupal body mass

Pupa samples of LT, HT, NT, and their corresponding control lines were sorted out for weight measurement. The measurement was performed for 8 consecutive generations. Around 30 female pupae were selected based on their larger size, and their sex was confirmed under a microscope. Pupae from each batch were taken in a strainer, dried properly soaking with the filter paper and weighed to record the body mass [13].

Thermal and nutritional stress on the life cycle of An. stephensi

To fix the optimal level of stress 3 experimental setups were designed independently in the laboratory by exposing them to low (LT) and high temperatures (HT) and nutritional deficient (NT) conditions along with their corresponding control sets (Figure 1).

For the control sets of LT and HT lines, the egg, larvae and pupae were reared at 29±1°C, whereas the adults were reared at 28±1°C and RH at 75±5% with a photoperiod of 12h:12h light and dark cycles. The fecundity, hatchability, duration of eggs to adults, male-to-female ratios, and adult longevity of the experiment and control lines were recorded up to 8^th generation.

LT stress – In the baseline assay, 3 egg cups were kept at 4°C and each cup contained approximately 250–300 eggs. Each egg cup was removed from 4°C for 3 different time durations (24, 48 and 72 h). The percent egg hatching was counted and recorded.

Experimental:

In the G₀generation, around 600–700 eggs were collected from 5 gravid females and divided into 2 batches: one for the control line and the other for the experimental. The experimental egg cups were kept at 4°C for 48 h. After removal from 4°C the eggs were allowed to hatch. The larvae, pupae and adults were reared at 18°C and 64% humidity.
In experimental sets, approximately 100 larvae were reared in 750 ml of RO water, with 230 mg of larval food added on alternate days. The mortality of larvae, pupae and adults were checked and recorded. The adults were provided with 10% sugar solution and then a blood meal on day 5to obtain eggs.

HT stress – In the baseline assay, 5 sets were made to conduct the test and approximately 100 larvae were taken in each set. The larvae were reared at variable temperatures, i.e., 30.5, 32.5, 35.5, 36.5, and 40°C. Larval mortalities were recorded from all the sets.

Experimental:

In the G₀generation, around 500–600 eggs were collected from 3 gravid females and divided into 2 batches: one for the control line and the other for the experimental. Based on the HT baseline assay, all the developmental stages, i.e., eggs, larvae, pupae, and adults, were reared inside the incubator at 35.5±1°C. The humidity was maintained at 54%.
In every set, about 100 larvae were kept in 750 ml of RO water and 230 mg of larval food was added for every alternate day. The adults were provided with sugar soln. Water change was made 2 times. Larval to adult mortality was checked and recorded. The emerged adults were given sugar solution and blood meal on day 5 or 6 to obtain eggs.

NT stress – Both larvae and adults of T₆ strains were exposed to nutritional stress.

Larval stress – In the baseline assay, 6 groups of food amount were considered – (a) 75 mg, (b) 46 mg, (c) 33 mg, (c) 23 mg, (d) 15 mg and (e) 10 mg – to determine the optimal amount of food required for larvae to develop and survive. In these 6 sets, the water volume (750 ml) and the number of larvae (~100 no.) were kept constant. For comparison, the experimental sets were considered in 2 replicates, along with the control sets. The percentage of larval mortality and survivability was recorded for each set.

Experimental:

In the G₁ generation, the eggs from 3 gravid females were collected, and the emerged larvae were divided into 2 groups: one control and one experimental set.
In the experimental set, for every set about 100 larvae were reared in 750 ml of RO water in the rearing trays.
The amount of food was measured depending on the larval sizes: 1^st to 2^nd instar larvae were provided with 15 mg larval food, and at 3^rd and 4^th instar larval stages with 33 mg larval food. Food was provided every alternate day for 4 days until the pupation and at the same time as scheduled.
Water was changed twice, i.e., on day 4 (2^nd instar larval stage) and day 6 (4^th instar larval stage), to ensure the complete removal of extra larval food. The number of larvae was counted daily, and mortality was recorded.

Control – In the control set, about 100 larvae were reared in 750 ml of RO water and were provided with sufficient larval food. Approximately 150 mg of food was added for 1^st and 2^nd instar larvae and 230 mg for 3^rd and 4^th instar larvae. Water change was also made two times like the experimental set. The number of larvae was counted daily, and mortality was recorded. Temperature was maintained at 29±1°C in the larval room. The adults were reared at 28±1°C and RH at 75±5% with a photoperiod of 12h:12h light and dark cycles. The fecundity, hatchability, duration of eggs to adults, male-to-female ratios, and adult longevity of the experiment and control lines were recorded up to 8^th generation.

Adult stress

In the experimental set, the adults that emerged from stressed pupae were provided with plain water without sugar solution. On day 5, alive females were given blood meal to obtain eggs and continue to the next progenies.

In the control set, the adults emerged from normal (unstressed) pupae were provided with sugar solution and blood meal on day 5 following the standard protocol [30].

The adults of both experimental and control sets were maintained at 28±1°C and relative humidity at 75±5% with a photoperiod of 12:12h light and dark cycles.
The number of adults, sex ratio, and adult longevity (in days) were recorded in every generation (up to 8^th generation).

Control – For NT stress lines, the adults were reared at 28±1°C and RH at 75±5% with a photoperiod of 12h:12h light and dark cycles. The fecundity, hatchability, duration of eggs to adults, male-to-female ratios, and adult longevity of the experiment and control lines were recorded up to 8^th generation.

Vectorial competence

Vectorial competence was performed among LT, HT and NT stress lines along with the control strain (An. stephensi) at the 8^th generation, and in-vitro infection was estimated using P. falciparum parasites.

Parasites – P. falciparum (PfNF54 Line E) was procured from BEI resources (MRA-1000). The in-vitro culture was initiated by seeding 0.15–0.2% asexual parasitaemia and 5% haematocrit in 10 ml complete medium based on the inhouse standard protocol published in Ghosh et al [27]. The culture was maintained in an atmosphere of 5% O₂, 5% CO₂ and 90% N₂ for 16–18 days with daily media change. For the mosquito feeding experiment, staggered cultures (14- and 17-day old) were selected based on their stage-V gametocytaemia and exfagellation activities (above 10 exflagellating centres per 40× field) and pooled [31].

Standard membrane feeding assay (SMFA) – One day before SMFA, 5–6-day-old non-blood-fed adult female mosquitoes were collected from the cages of 3 experimental (LT, HT and NT) and one control line and kept in 8 paper containers. Approximately 40 mosquitoes were kept in each container, and 2 replicates. Mosquitoes were given 5% D-glucose overnight in dark conditions until feeding. Sugar cotton was removed 4 hours prior to SMFA [32]. They were fed with mature gametocytes (stage V) of P. falciparum NF54 simultaneously using an artificial glass feeder through parafilm. A sample of the infective feed was examined under a light microscope (Nikon Eclipse Ni-U upright microscope, Japan), and exflagellation was recorded. After 30 min of blood-feeding, all the cups containing fully engorged mosquitoes were placed inside an environmental chamber maintaining temperature at 26°C and 75% RH for 8–9 days (Percival I-30VL insect chamber, Perry, IA, USA). Mosquitoes were fed with fresh wet cotton balls soaked in 10% D-glucose and 0.05% para-aminobenzoic acid (PABA) solution daily. The midguts of infected mosquitoes were dissected on day 9 post-feeding for the presence of oocysts and stained with 0.1% mercurochrome soln. Oocyst numbers per midgut were recorded under a light microscope under 10X magnification.

Chromosomal inversion

Ovarian polytene chromosomes from the semi-gravid females were prepared from LT, HT, and NT stressed and control lines at the 8^th generation. The semi-gravid females were dissected, fixed in modified Carnoy’s solution (3 parts of methanol: 1 part of glacial acetic acid), and stained in LAO (Lacto Aceto Orcein) as per the protocol published by Ghosh and Shetty [33] Gentle pressure was applied for squashing and sealed with nail polish around the coverslip. Using a microscope, the slides were checked for chromosomal inversions under 40X and 60X (Nikon Eclipse, Model SC600). The inversion photomap was followed according to the earlier methods [34–36].

Linear model

Linear model is used to describe the linear relationship between the response variable (number of eggs, pupae, larvae, and adults) and the predictor variable (the number of generations). The impact of varied stress conditions (elevated temperature, low temperature, and nutrition stress) on the response variables compared to their respective control groups was studied. Linear model is given by the equation: , where is the number of eggs/pupae/ larvae/adults; is the number of generations ( 1 to 8), is the intercept (the value of Y when X=0), is the slope (the change in Y for a one-unit change in X) and is the error term. The study identifies the generations required for optimal adaptation under each stress condition by comparing the equations derived with their control groups.

Statistical analyses

The study identifies the generations required for optimal adaptation under each stress condition by comparing the equations derived with their control groups of An. stephensi over multiple generations. Exploratory analysis and predictive modelling have been carried out in the present study. Chi-square (χ²) test, F-test and independent sample t-tests were carried out to determine the mean and variance and the association between the control and experimental lines. The egg and wing measurements were calculated following t-test analysis for independent or correlated samples using Vassar Stat software (http://vassarstats.net/ ). The Mean, Standard Error of the Mean (SEM), Standard Deviation (SD), p-value and F value were compared between stressed mosquito samples and with their corresponding control lines. The non-parametric Mann–Whitney t-test was performed on pooled oocyst counts per midgut from infected mosquitoes of experimental and control sets using GraphPad Prism 10.0 (ignoring uninfected mosquitoes). Linear models were then developed for each scenario; Root Mean Square Error and R2 were calculated to assess model accuracy. Karl Pearson’s correlation coefficient was computed to explore the relationship between stress and control conditions at each developmental stage. The developed linear models used the number of generations necessary to adapt to the control conditions (R software, version 4.1.3). P-value >0.05 was considered statistically non-significant (NS).

The results of our investigation on the impact of temperature and nutrition on An. stephensi are presented under 7 distinctive groups: (a) morphometrics of eggs, (b) wing, (c) pupal body mass, (d) transgenerational life-cycle parameters, (e) vector competence, (f) variations in polytene chromosomes, and (g) Linear model. Each of these headings has subheadings that specifically demonstrate the impact of higher temperature stress (HT), lower temperature stress (LT) and nutritional stress (NT).

Egg morphometrics

Morphometric analyses of eggs were carried out from LT, HTand NT lines along with their control lines (see Methods section) to ascertain any significant changes due to stress in the egg length (EL), egg width (EW), egg-float length (EFL), and egg-float width (EFW). The eggs were black, boat-shaped, and blunt at both anterior and posterior ends. The results of LT, HT, NT and their control lines are presented in Tables S1–S3. Their comparative significance data were generated from 10 consecutive generations and are presented in Tables 1 and S4.

LT stress – The results showed that the EL, ER, EFL and EFR of LT strains were significantly larger compared to their corresponding control (unstressed) strains. In LT line, the minimum and maximum EL of LT were 440 and 480 µm (470±4.47 µm), respectively and were significantly larger (p=0.0035) compared to their control (unstressed) lines (450–460 µm, 455±1.66 µm). The range of EW of LT and the control were 150–170 µm (164±2.21 µm) and 130–170 µm (152±4.16 µm), respectively, with a significant difference (p=0.036). The range of EFL of LT and their corresponding control were 220–250 µm (240±3.94 µm) and 210–230 µm (220±2.10 µm), respectively (p=0.038). No significant difference was observed in EFW (p=0.460, NS>0.05). See Tables 1, S1 and S4.

HT stress – The results showed that the EL, EW and EFL of HT lines were significantly smaller than the control (unstressed) lines. The range of minimum and maximum EL of HT and control lines were 380–430 µm (407±5.26 µm) and 450–470 µm (458±2.49 µm), respectively, showing significant differences (p=0.045). The range of EW of HT and control lines were 130–150 µm (140±1.88 µm) and 130–170 µm (152±4.16 µm) with a significance value of p=0.027. The range of EFL of HT and control lines were 200–210 µm (206±1.82 µm) and 220–240 µm (230±2.98 µm) with a significance value of p=0.049 (Tables 1, S2 and S4).

Significant differences were observed in the EL (p=0.0032), EW (p<0.0001) and EFL (p=0.0074) while compared between LT and HT stress lines (Tables 1, S2 and S4).

NT stress – The results indicate that the EL, EW and EFL of NT lines were significantly smaller compared to their control (unstressed) lines. The minimum and maximum range of EL of NT and its corresponding control line were 360 and 390 µm (370±2.981 µm) and 390 and 460 µm (417±6.675 µm), respectively, and showed a significant difference (p=0.012) compared to the control lines. The EW range of NT was 100 and 130 µm (112±3.266 µm) and control lines was 120 and 140 µm (131±1.795 µm), respectively, and showed a significant difference (p=0.044) between them. The EFL of NT and the control lines were 130–160 (145±4.01 µm) and 190–210 µm (206±2.21 µm), respectively, with a significant difference (p=0.045). The ranges of EFW of NT and control were 30–40 and 70–90 µm, respectively, showing a significant difference compared to control lines (p=0.038) (Tables 1, S3 and S4).

Wing morphometrics

The results of wing measurements of LT, HT, and NT, along with their control lines, are presented in Tables 2 and S5–S8.

LT stress – The mosquitoes exposed to lower temperature showed comparatively smaller wing length in males (p=0.038) and wing width in females (p=0.008) than their control (unstressed) lines. The ranges of wing length and width in LT males were 2.701–2.933 mm (2.804±0.032 mm) and 0.602–0.824 (0.719±0.028 mm), respectively. In LT females, ranges of wing length and width were 2.992–3.655 mm (3.325±0.08 mm) and 0.791–0.953 mm (0.879±0.02 mm), respectively (Tables 2, S5 and S8).

HT stress – The mosquitoes subjected to high temperature showed significantly smaller in wing size than their control (unstressed) counterparts. In HT males, the wing length and width ranges were 2.391–3.021 mm (2.665±0.0787 mm) and 0.501–0.617 mm (0.546±0.016 mm), respectively. In HT females, the range of wing length and width were 2.413–3.135 mm (2.827±0.082 mm) and 0.612–0.712 mm (0.657±0.016 mm), respectively. Differences were also observed in the length and width of LT-stressed males (p=0.012 and 0.018) and females (p=0.011 and 0.031), respectively, compared to their control lines (Tables 2, S6 and S8).

When compared between male and female wings of HT and LT lines, significant difference was observed in the wing length of males (p=0.0149) (see Table 2).

NT stress – The mosquitoes reared with low nutrition had significantly smaller wing sizes compared to control mosquitoes. In NT males, the range of wing length is 2.401–2.501 mm (2.4226±0.011 mm) and width 0.406–0.567 mm (0.445±0.017 mm), whereas in NT females, the range of wing length is 2.225–2.595 mm (2.431±0.044 mm) and width 0.507–0.616 mm (0.557±0.016 mm), respectively. Compared with the control males and females, the wing length of NT males was significantly smaller (p=0.0001), and in NT females, the wing length and width were observed to be smaller (p=0.04 and 0.049) (Tables 2, S7 and S8).

Pupal body mass

The average body mass of female pupae of LT, HT, and NT stress lines was analysed (see Materials and methods) and the results are presented in Tables 3 and S9 (see Figure 2A–C). The pupal body mass of LT, HT and NT stress lines were 57.45±0.32, 52.03±0.22 and 50.59±1.17 mg, respectively. Significant differences were observed while compared the values between LT and NT lines with their corresponding control (unstressed) lines (p=0.025 and 0.0004). However, significant differences were also observed in the pupal body mass when compared between LT and HT lines (p<0.0001) (see Table 3).

LT, HT and NT stress on transgenerational life-cycle parameters

Baseline assays – The results of baseline assays of LT, HT and NT stress lines are presented in Figure 3A–C and Tables S10–S12. Based on baseline results the following optimal levels were chosen for 3 sets of stress experiments. (i) 48 h at 4°C was considered as optimal duration time of eggs hatching for LT stress experiment, (ii) 35.5°C as optimal temperature for HT stress experiment, and (iii) 33 mg as optimal amount of larval diet for NT stress experiment, respectively (see the Methods section). The results of baseline data of LT, HT and NT are presented in Tables S10–S12.

During the stress experiments, the parameters like fecundity, percent egg hatchability, number of larvae and pupae, sex ratio (male: female), and longevity of adults (lifespan) were recorded for 8 generations along with their control lines. The comparative life-cycle data of LT, HT and NT stress lines of the G₀ generation is presented in Table S13. Results of life-cycle data (for both experimental and control) of LT lines are presented in Tables S14 and S15, HT lines in Tables S16 and S17 and NT lines in Tables S18 and S19. Significant values of LT, HT and NT stress lines are presented in Table S20.

LT stress – Based on the baseline result, all the life stages (i.e., eggs to adults) were exposed to lower temperature (at 4°C and 18±1°C) in the G₁ generation (see the Materials section) and the experiment was continued up to 8 generations. The results of LT stress and control lines are presented in Figure 3A and Tables S14 and S15. The LT-stressed lines took a longer time from egg to adult emergence (~25 days). The lowest number of eggs (fecundity) was 213 observed in the 1^st generation, and the highest was 327 in the 7^th generation (M=262.625±16.299). The lowest and highest numbers of larvae were 172 observed in the 1^st generation and 266 in the 7^th generation (M=213.375±13.901). The lowest and highest numbers of pupae were 130 and 243 (M=176.125±17.279), and adults were 90 and 203 in the 1^st and 7^th generations (M=140.75±17.092), respectively. The adult survived for ~30 days (for control, 40 days).

Significant differences were observed in the number of eggs (M=306.063, p=0.0001, F=4.34), larvae (M=269, p<0.0001, F=5.09), pupae (M=249.125, p<0.0001, F=7.41) and adults (M=230.375, p<0.0001, F=7.53) when compared with the mean values of their corresponding control lines over 8 generations (p>0.05 indicates NS). See Table S20.

Further, in gonotrophic cycles of LT adult females, oviposition time (egg maturation) was prolonged (4 days) and the frequency of blood meal uptake was 3 times with decreasing temperature compared to their control strains (3 days and 4 times). The developmental time (duration) of egg-to-adult stages of LT and control lines using mean over 8 generations is shown in Figure 4A. The longevity of LT adults (in days) is shown in Figure 5A. The male-to-female ratio of LT was 1.54:1 (vs. control, 1.4:1; see Tables S14 and S15).

HT stress – Based on the baseline assay, all the life stages, i.e., eggs to adults were exposed to higher temperature (35.5°C) and continued up to 8 generations (see the Methods section). The results of HT-stressed and control lines are presented in Figure 3B and Tables S16 and S17. The HT-stressed lines developed faster from egg to adult emergence (~7.5 days). The lowest and the highest fecundity were 192 and 240 (M=218.25±6.23) observed in the 2^nd and 7^th generations, larvae were 141 and 210 (M=164.75±9.07) in the 5^th and 8^th generations, pupae were 129 and 195 (M=153.25±8.51) in the 2^nd and 8^th generations and adults were 95 and 159 (M=128.125±7.825) in the 2^nd and 6^th generations, respectively. The adult survived for ~25 d (vs. 40 days for control).

Significant differences were observed in eggs (p=0.0001, F=5.08, M=282.312), larvae (p<0.0001, F=3.8, M=242.5), pupae (p<0.0001, F=3.14, M=234.87) and adults (p<0.0001, F=2.77, M=221.37) of HT lines compared to the control lines (Table S20).

In the gonotrophic cycle of the HT line, the oviposition time (egg maturation) was shortened (1.5 days) and the frequency of blood meal was 2 times with increasing temperature compared to their control lines (3 days and 4 times). The developmental time (duration) from eggs to adults of HT lines using mean value over 8 generations are shown in Figure 4B. The longevity of HT adults (in days) is shown in Figure 5B. The male-to-female ratio of HT was 1.44:1 (vs. control, 1.49:1; see Tables S16 and S17).

Significant differences were also observed in eggs, larvae, pupae and adults while compared between HT and LT stress lines and the results are presented in Table 4.

NT stress –Based on the baseline assay, all the life stages, i.e., larvae and adults were exposed to nutrient-deficient condition (33 mg larval food) and continued up to 8 generations (see the Methods section).

The life-cycle data of NT and control lines of 8 generations are presented in Figure 3C and Tables S18 and S19. The NT-stressed lines developed faster from egg to adult emergence (~11 days). The adult survived for ~5 days (vs. 40 days for the control). The lowest and the highest numbers of NT eggs (fecundity) were observed at 79 and 129 in the 1^st and 6^th generations, respectively (Table S18). The mean number of fecundities of NT was 105.125±6.807 (SD=19.253). For control lines, fecundity was 329.38±3.25 (SD=9.101) (Table S19). The lowest and the highest numbers of NT larvae were 52 and 85 in the 2^nd and 6^th generations, respectively [M=69.5±4.238 (SD=11.988)]. For control lines, the number of larvae was 310.5±3.789 (SD=10.72). Similarly, the lowest and the highest numbers of pupae were 45 and 74 in the 1^st and 7^th generations, respectively (M=60.875±3.856; SD=10.907), and for the control lines, it was M=308.63±3.6593 (SD=10.35). The lowest and highest numbers for NT adults were 39 and 62 in the 1^st and 7^th generations, respectively (M=50±3.251; SD=9.196). For control adults, it was 307.13±3.6618 (SD=10.357) (Tables S18 and S19). Moreover, in the gonotrophic cycle of NT female adults, the oviposition time was shortened (~1.5 days) and the frequency of blood meal was only for 2 times under NT-stressed conditions compared to their control strains (3 days and 4 times). The developmental time (duration) of eggs, larvae, pupae, and adults of NT lines using mean over 8 generations is shown in Figure 4C, and the longevity (in days) is given in Figure 5C. The male-to-female ratio of NT was 1.04:1 (vs. control, 1.42:1). See Tables S18 and S19.

Significant differences were observed in eggs (M=217.25, F=4.47, p<0.0001), larvae (M=190, F=1.25, p<0.0001), pupae (M=184.75, F=1.11, p<0.0001) and adults (M=178.563, F=1.27, p<0.0001) of NT lines compared to their corresponding control lines (Table S20).

In NT-stressed lines, the sex ratio (M:F), number of male and female adults, number of survived adults till day 5, and the frequency of blood meal taken by adult females are presented in Table 5.

Vectorial competence

The results of 3 experimental lines, i.e., LT, HT, NT, along with the control line of An. stephensi fed with P. falciparum-infected blood (see the Methods section) are presented in Figure 6 and Table 6. The positive midguts and percent oocysts rate were calculated from 3 stressed and control lines. It was observed that the oocyst rate was significantly higher in HT lines (Median=16; SEM=18.32±2.67), LT lines (Median=5; SEM=26.31±3.3) and NT lines (Median=4; SEM=28.44±3.35) compared to the control line (Median=1; SEM=19.81±1.94), respectively. The non-parametric Mann–Whitney test between the HT and the control lines showed a significant difference (p<0.0001). However, differences were non-significant in LT (p<0.06) and NT (p<0.08) lines compared to the control line. However, the variations in the prevalence of infection were observed among the stress lines of LT (71.4%), HT (85.1%) and NT (63.9%) compared to the control line (55.8%)(Figure 6).

Chromosomal inversion

At the 8^th generation, the inversion study of polytene chromosome reveals that 11 out of 40 gravid females from the NT stress line showed chromosomal inversions. Inversion break points were observed on the 3L arm at 2 locations, i.e., 39A–39C and 42A–44C (Figures 7 and S1). Approximately 15 similar type double inversions were observed in the same NT line and the inversion rate was ~27.5%. No inversions were observed in the polytene chromosomes of LT and HT lines (Table 7).

Linear model

In this study, 2 sets of linear models were developed from the 4 variables (number of eggs, pupae, larvae, and adult) under different conditions (high temperature, low temperature, and nutrition stress). The first set corresponds to the specific conditions, while the second represents the respective control groups. Further, the equations are compared with their control groups to identify the generations required for optimal adaptation in the corresponding stress condition.

The values obtained from this comparison of linear models reveal the number of generations required to adapt to the given stressors. Considering the adult stage, it will take 21 generations for LT, 144 generations for HT and 4354 generations for NT lines, respectively to gain the normal stage (Table 8). A strong correlation was observed in LT stress followed by HT stress compared to control conditions. Also, a weak correlation between low nutrition and control lines was found in stressed individuals. An interesting observation is that the number of generations required to adapt in LT stress conditions is lower than in the case of HT-stressed conditions (Figures 8 and S2). This suggests that the LT-stressed mosquitoes exhibit a higher adaptability rate, and the NT-stressed mosquitoes exhibit a larger number of generations required to adapt, indicating the lowest adaptability rate (Table 8).

Correlation analysis demonstrated a notable positive correlation under LT stress, suggesting a high adaptability potential. Equating models from specific stress conditions with their respective controls illuminated the generations required for optimal adaptation, providing insights into mosquito resilience to environmental stressors.

Climate is a significant factor influencing density, distribution, and dispersal of mosquito populations [6]. Climatological variables include rise or fall of temperature, humidity, rainfall, wind flow and direction, duration of photoperiods, etc., which may modify the complex set of demographics and environmental scenarios that influence the dynamics of transmission of mosquito-borne diseases. The differences in the life-cycle stages of the mosquito population are known to be associated with the variation of temperature of the environment and food availability in the habitat, which has long been a matter of interest.

Temperature is one of the most important extrinsic factors affecting the growth and developmental rates of mosquito in the environment. Life-stage parameters are efficient tools for understanding and analysing the effect that an external factor has on the growth of development, growth rate, reproduction, and survival of a mosquito population. Although several studies have examined the effect of temperature and nutrition on mosquito and parasite traits that determine the transmission potential of human malaria, our transgenerational study over 8 generations specifically emphasizes the impact of temperature and nutrition on life-stage parameters, egg and wing morphometrics, duration of life stages, longevity of adults, vectorial competence, chromosomal inversion and prediction model of An. stephensi. We have observed that the length and width of eggs and the length of egg float in LT-stressed lines were larger at lower temperatures (see the Results section). In contrast, the length and width of eggs and egg floats in HT and NT stressed lines are shorter compared to their control lines. To our knowledge, For the first time, this study reveals the effect of temperature and nutrition on the size of mosquito eggs.

The pupa is the best stage for estimating body mass weight as it is a non-feeding stage. The environment of the larval mosquitoes has a significant effect on pupal body mass due to diverse larval rearing conditions like high and low temperatures and availability of nutrients. In our study, the average pupal body masses in females were significantly lower in LT (8%), HT (16%), and NT (19%) stressed lines compared to their corresponding control lines due to high-stress conditions. Armbruster and Hutchinson estimated the pupal body mass and wing length as significant estimators to accurately predict the fecundity in Aedes albopictus and Aedes geniculatus [37]. Price et al showed that the average body mass of female mosquitoes reared under uncrowded conditions is larger than the mosquito (smaller) size reared under crowded and less nutrition conditions [13].

Many experiments used wing shape and size of Drosophila melanogaster due to seasonal variation [38]. The wing sizes varied significantly in LT, HT and NT lines of An. stephensi due to stress. The wing length of LT males and females was larger, and the width was smaller at lower temperatures. The wing sizes of HT and NT males and females were smaller at higher temperature and nutritional stress conditions. Based on our findings, the morphometric results indicate that the variation in egg and wing sizes due to temperature variations and food deficiency is an outcome of rapid adaptation in response to environmental changes. Our study supports the previous reports where the similar results were observed. Yeap et al tested the wing shape, size and wing–thorax ratio to ascertain field fitness of Wolbachia-infected Ae. aegypti [39]. Kolliker-Ott et al correlated the wing size, shape and asymmetry to field fitness of Trichogramma egg parasitoids used in biological control [40]. Hoffmann et al have demonstrated the effect of temperature-related factors and food availability due to seasonal changes that generate variations in wing size and shape in Drosophila melanogaster [41].

In the present study, transgenerational data shows a distinct progressive trend toward adaptation from 1^st or 2^nd generation to 7^th or 8^th generations in LT, HT and NT stressed lines. This indicates that the mosquito population can adapt quickly to various temperature fluctuations and nutritional deficiencies, which is quite normal in natural (wild) conditions. It was observed that the average percent hatchability rates of LT, HT and NT were decreased by 13, 19 and 29% compared to their corresponding control (unstressed) lines. A study showed that food restriction caused a 50% reduction in clutch size and a 25% reduction in fecundity in Lepidoptera, specifically in butterflies [42]. Bauerfeind et al experimented to assess the direct and transgenerational effects of food deprivation in the Glanville fritillary butterfly, Bicyclus anynana [43]. To our knowledge, this is the first time such a transgenerational study in An. stephensi was conducted to understand the effect of environmental stressors on mosquitoes in the laboratory.

In the LT, HT and NT-stressed lines, the male-to-female ratio was approximately consistent within 8 generations. However, among the starved mosquitoes, the male adults survived only 3–4 days, whereas the females survived a maximum of 5 days. The maximum number of blood meal uptake was 2 times in their lifetimes, with reduced adult longevity in NT-stressed mosquitoes than control (unstressed) ones. This indicates that the nutrients stored in the pupal stage were utilized for the energy required for adult emergence and early development.

The developmental rate of egg-to-adult in LT lines was slower, and the duration was prolonged at lower temperatures (~25 days). In comparison, the developmental rate in HT lines was faster, and the duration period was shorter (~7.5 days). However, in NT-stressed lines, the development time from egg to adult was ~11 days. The mean of adult longevity was 30, 25 and 5 days for LT, HT and NT lines, respectively. Similar results were observed in the study of Couret et al, where temperature, larval density and diet directly affected the development rate and survival of Aedes aegypti [44, 45]. Behrman et al demonstrated seasonal heterogenicity in adaptive phenotypic traits like thermal and starvation tolerance and innate immunity in Drosophila melanogaster populations [46–48]. They suggested that many loci respond to seasonal selection.

Niitepõld demonstrated the effect of 2 stress factors, like increased flight and dietary restriction, on bioenergetics and the life history of the Glanville fritillary butterfly (Melitaea cinxia). The study reported that larval food restriction significantly affects the life cycle, specifically in reproduction. However, the study also showed insufficient adult feeding cannot reach the maximum reproductive potential [49].

However, we still have limited knowledge of these responses, mainly when multiple stress factors exist simultaneously, as it is often found in nature. We intended to determine the relative effect of temperature and diet on developmental processes and evaluate their vectorial competence under laboratory conditions.

Climate change influences malaria transmission, correlated mainly with temperature and malaria parasite development inside the mosquito host [50]. More studies are needed to understand and enhance our knowledge to clarify this relationship. The sensitivities of Anopheles mosquitoes to temperature vary among different species and even among the same species, with various responses. Experimentally stressed LT, HT and NT lines of An. stephensi were used to determine the vector competence status infected with in-vitro culture of P. falciparum [51].

Plasmodium is very sensitive parasite in the mosquito host. Oocyst formation undergoes 2 important stages – the development of ookinetes from micro and macrogametes in the midgut lumen, followed by oocyst development in the midgut epithelium [13–15]. Various extrinsic factors, including temperature and nutritional status, regulate vectorial competence. Previous studies showed that food stress during larval development may cause variations in adult mosquito immunity that can increase their susceptibility to pathogens, such as An. quadrimaculatus and An. albimanus to Plasmodium falciparum [52], Ae. aegypti to Sindbis virus [53] and Zika virus [54], An. stephensi to Plasmodium yoelii nigeriensis [15]. The food-stressed larvae might utilize their stored energy mostly in development and survival instead of immunity and defence [55, 56]; thus, the adults become more susceptible to infections. This concept might also apply to temperature-stressed lines. Susceptibility with 2 isofemale strains of An. stephensi against P. berghei ANKA and P. falciparum was reported earlier [27].

Our result of vectorial competence supports the above observations. Susceptibility of LT, HT and NT stressed lines of An. stephensi to P. falciparum infection was observed more than the corresponding control line (Table 7). Life-cycle stages reared under stress conditions might be more susceptible when exposed to malaria parasite infection. Distinct variations were noticed in the P. falciparum oocyst infection rates among LT, HT and NT lines of An. stephensi. The stressed lines showed medium-to-high oocyst infection rates, e.g. 85.1% for HT, 71.4% for LT and 63.9% for NT lines, respectively, compared to the control strain (55.8%). HT showed a significant difference (p<0.0001) in the oocyst infection rate compared to the control strain.

Furthermore, in the chromosomal study, 2 break points (inversions) were observed from NT lines in 2 locations (39A–39C and 42A–44C) on the 3L arm with an inversion frequency of 27.5%. One of the inversions (42A–44C) is already reported on 3L arm in the earlier studies [57, 58]. However, in the present study an additional inversion at breakpoint 39A–39C has been observed. Such finding might have a great significance in the adaptation process under stress conditions. Chromosomal inversions were also reported earlier by several authors involving desiccation resistance (2La, 2Rs), indoor/outdoor behaviour (2Rd, 2La), infectivity to Plasmodium (2La), geographical location (2R, 3R, 23L), resting behaviour (2Rb), etc. in Anopheles sp. [22] and insecticide resistance (DDT, Dieldrin, Propoxur, Temephos, Neem oil, etc.) in An. stephensi [59]. Likewise, in experiments on Drosophila pseudoobscura, Dobzhansky suggested that the fitness of alleles present in inversions involves local adaptation and varies with environmental conditions [60]. Earlier studies on Drosophila reported that inversion polymorphisms have a great role in local adaptation [61, 62]. Resistance to desiccation and thermal stresses was observed in carriers and non-carriers of 2La inversion in Anopheles gambiae, and inversions on chromosomes exhibited greater resistance to both stresses [63, 64].

Recent studies increasingly focus on laboratory-derived temperature dependencies of various life stages of vectors to develop mathematical models that predict how climatic and environmental factors influence the dynamics of vector-borne diseases. These models assess the impact on the global abundance, distribution, and invasion of vector populations [65–68]. Transgenerational data was used to analyse and identify specific patterns and relationships concerning the effects of temperature and nutrition on the developmental and survival rates of An. stephensi. A predictive model using life-cycle data from 8 generations of the LT, HT, and NT stress lines enables us to understand how quickly the mosquitoes would adapt to these stressors. The model indicates that the LT stress lines adapt more quickly, requiring approximately 21 generations, compared to the HT lines that take about 144 generations. In contrast, the NT lines adapt very slowly, taking nearly 4354 generations to return to normal conditions.

This comprehensive laboratory study would help understand the relationship between environmental stressors and mosquito biology and improve understanding of the epidemiology of vector-borne diseases. Vector-borne diseases including malaria are climate-sensitive tropical diseases because of global climate change and local land use practices [69]. Global health community is concerned in finding more connections between climate change and diseases risk and trying for appropriate interventions. Prediction of the effect of temperature on both the biology and distribution of ectotherms requires a deep understanding of the relation between temperature and performance traits [70].

Malaria has caused suffering to humanity for centuries and continues to create a severe threat to public health globally. India bears a significant burden of mosquito-borne diseases where, despite concerted efforts, barriers to effective prevention and treatment persist, sustaining the transmission cycle. Climate changes due to rising temperatures, increased humidity, draughts and water stagnation, excess rainfall and flooding create favourable conditions for the mosquito, in general, leading to extended transmission windows. Our near-real study under laboratory conditions provides a picture of the possible effects of climate change on nature.

In summary, this is a comprehensive multi-transgenerational study that reveals novel insights into the effect of stress on the life cycle, vectorial competence, cytology, and a predictive model of An. stephensi carried out in the laboratory under temperature and food stress conditions. The present study provides an experimental framework for further research to understand better the biology of An. stephensi and its vector competence in the context of climatic and environmental changes, which in turn would help design more effective vector control strategies.

VBD, vector-borne disease; HT, high temperature; LT, low temperature; NT, nutritional stress; EL, egg length; EW, egg width; EFL, egg-float length; EFW, egg-float width.

Data availability

All study data are included in the article and/or Supplementary materials.

Ethics statement

Biosafety approval for mosquito maintenance in the insectary facility (Approval Ref. No. TIGS 3rd IBSC March 2018), for malaria infection study (approval ref. no. – inStem/IEC-12-001/27E, dated 26/12/2022) and for blood feeding of mosquitoes (approval ref. no. – inStem/IEC-12-002/27E, dated 26/12/2022).

Authors’ contributions

CG - conceptualized, designed research; performed egg morphometrics, transgenerational life cycle, and polytene chromosome studies; curated and analysed data; maintained all experimental and control lines of An. stephensi; and drafted the manuscript. NK - performed in vitro P. falciparum culture. SM - performed wing morphometric study and maintained An. stephensi experimental lines. CKR and SJ performed wing morphometric study and maintained insectary populations. SL and PC performed linear modelling. SK and SS reviewed the manuscript. All authors reviewed the manuscript.

Acknowledgements

Sincere gratitude to Dr. Rakesh Mishra, Director, Tata Institute for Genetics and Society (TIGS), Bangalore, India and Dr. Suresh Subramani, UCSD, San Diego for their support, insightful inputs and quick review of the manuscript. Their guidance from time to time have been instrumental in the completion of this research work. The authors are thankful to Dr. Susanta Kumar Ghosh, ICMR-National Institute of Malaria Research, Bangalore, India for his guidance in the establishment of TIGS insectary. We are thankful to Mr. Joydeep Roy from TIGS-insectary for his technical support.

Funding:

No fund was received for this study.

Conflict of interest statement

The authors have declared that no competing interests exist.

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Table 1. Comparative significance of different egg parameters among NT, HT and LT eggs with control lines of An. stephensi (LT, low temperature; HT, high temperature; NT, nutritional stress; EL, egg length; EW, egg width; EFL, egg-float length; EFW, egg-float width; p>0.05, NS).

An. stephensi	LT vs. control				HT vs. control				NT vs. control				LT vs. HT
	EL	EW	EFL	EFW	EL	EW	EFL	EFW	EL	EW	EFL	EFW	EL	EW	EFL	EFW
Mean (µm)	462.5	158	230	63	431.5	146	217.5	52.5	393.5	121.5	175.5	57	430	144.7	223	48
F value	7.2	3.55	3.5	1	3.3	3.9	3.2	2.67	5.01	3.31	3.3	3.5	7.4	37.64	5.83	1.95
P value	0.0035	0.036	0.038	0.5	0.045	0.027	0.049	0.079	0.012	0.044	0.045	0.038	0.00321	<0.0001	0.0074	0.167

Table 2. Comparative significance of length and width of male and female wings among LT, HT, and NT lines with their control lines of An. stephensi [LT, low temperature stress; HT, high temperature stress; NT, nutritional stress; non-significant (p>0.05)].

An. stephensi	Male		Female
	Length	Width	Length	Width
LT vs. control
P	0.038	0.119	0.433	0.008
F	4.2	2.55	1.14	7.45
Mean (mm)	2.9908	0.679	3.411	0.831
HT vs. control
P	0.012	0.018	0.011	0.031
F	6.54	5.64	6.73	4.57
Mean (mm)	2.8325	0.5929	3.058	0.7203
NT vs. control
P	0.0001	0.069	0.04	0.049
F	29.35	3.29	4.12	3.8
Mean (mm)	2.673	0.547	2.768	0.657
LT vs. HT
P	0.0149	0.095	0.475	0.267
F	6.05	2.84	1.05	1.63
Mean (mm)	2.7346	0.632	3.076	0.768

Table 3. Comparative significance of pupal body masses among LT, HT, and NT lines compared to their control lines of An. stephensi [LT, low temperature; HT, high temperature; NT, nutritional stress; non-significant (p>0.05)].

	LT vs. control	HT vs. control	NT vs. control	LT vs. HT
Number	30	30	30	30
Generation	8	8	8	8
Mean (mg)	59.73	57.237	56.65	51.52
F	4.94	1.78	19.78	883.06
P	0.025	0.232	0.0004	<0.0001

Table 4. Significant differences in life-cycle stages between HT and LT lines over 8 generations (HT, high temperature; LT, low temperature).

HT vs. LT	Eggs	Larvae	Pupae	Adults
N	16 (8+8)	16 (8+8)	16 (8+8)	16 (8+8)
Mean	240.437	189.062	164.687	134.437
F	6.84	2.35	4.12	4.77
P	0.0106	0.14109	0.0407	0.0281

Table 5. Number of male and female adults of NT lines and their survivability over 8 generations (NT, nutritional stress).

NT-line	Generations	No. of adults	No. of males	No. of females	M:F ratio	No. of alive adults (male+female) post-eclosion			Blood meal
						3rd day	4th day	5th day
T₆	1	39	25	14	1.7	14 (10M+4F)	20 (15M+5F)	5F (0M+5F)	2
T₆	2	37	11	26	0.4	12(6M+6F)	18 (5M+13F)	7F (0M+7F)	2
T₆	3	49	15	34	0.4	16 (2M+14F)	30 (13M+17F)	3F (0M+3F)	1
T₆	4	50	29	21	1.3	15 (5M+10F)	31 (24M+7F)	4F (0M+4F)	2
T₆	5	62	35	27	1.2	16 (8M+8F)	42 (27M+15F)	4F (1M+3F)	1
T₆	6	53	31	22	1.4	14 (6M+8F)	34 (25M+9F)	5F (0M+5F)	2
T₆	7	62	33	29	1.1	26 (13M+13F)	30 (20M+10F)	6F (0M+6F)	2
T₆	8	48	25	23	1.0	19 (11M+8F)	22 (14M+8F)	7F (0M+7F)	2

Table 6. Comparative oocyst infectivity among LT, HT and NT stress lines of An. stephensi with P. falciparum (LT, low temperature; HT, high temperature; NT, nutritional stress).

Parameters	Control	Experimental
		LT	HT	NT
No. of mosquito exposed	104	63	47	72
Positive midguts	58	45	40	46
Oocyst rate	55.8%	71.4%	85.1%	63.9%
Range of oocysts	0–111	0–150	0–68	0–121
Mean	10.86	14.86	18.83	18.15
Median	1	5	16	4
SEM	19.81±1.94	26.31±3.3	18.32±2.67	28.44±3.35
P-value	–	<0.06	<0.0001	<0.08

Table 7. Chromosomal inversions in NT lines of An. stephensi (LT, low temperature; HT, high temperature; NT, nutritional stress).

Expt. Lines	Total no. of females dissected	Percent inversion (%)	No. of positive females	Chromosomal arm	Inversion break points on the 3L arm in 2 locations
NT	40	27.5	11	3L arm	39A–39C and 42A–44C
HT	30	0	–	–	–
LT	30	0	–	–	–

Table 8. Comparative account of stress on different development stages of An. stephensi and the number of generations required for adaptation.

Stress Type	Stage	Control condition	Stress condition	Correlation r (control, stress)	Generations required recovery (~)
HT	Egg	= 338.179+1.821 RMSE = 6.01 =0.3257	= 189.857+6.31 RMSE = 7.92 =0.7693	0.413	33
	Larvae	= 301.71+ 4.119 RMSE = 7.897031 =0.5882	= 125.107+ 8.810 RMSE = 12.99 =0.7071	0.467	38
	Pupae	= 296.89+ 4.357 RMSE = 7.863228 = 0.6171	= 115.536+ 8.381 RMSE = 11.75481 = 0.7274	0.537	45
	Adults	= 295.61+ 4.226 RMSE = 7.808058 = 0.606	= 103.107+ 5.560 RMSE = 16.31995 = 0.3786	0.656	144
LT	Egg	= 314.89+7.690 RMSE = 10.87414 = 0.7242	= 189.607+16.226 RMSE = 21.84527 = 0.7434	0.935	15
	Larvae	= 296.50+6.250 RMSE = 7.804246 = 0.771	= 152.143+13.607 RMSE = 19.51087 = 0.7186	0.905	20
	Pupae	= 293.79+ 6.298 RMSE = 8.581628 = 0.7387	= 99.893+ 16.940 RMSE = 24.14986 = 0.7209	0.899	18
	Adults	= 292.04+ 6.214 RMSE = 8.307161 = 0.7461	= 64.786+16.881 RMSE = 23.42783 = 0.7316	0.902	21
NT	Egg	= + 1.82 RMSE = 7.420712 = 0.2403	RMSE = 9.67054 = 0.7117	0.288	51
	Larvae	2.57298.93 RMSE = 8.110839 = 0.3454	RMSE = 5.793058 = 0.7331	0.264	153
	Pupae	297.1 RMSE = 7.703296 = 0.3669	45.29 RMSE = 6.411137 = 0.6052	0.146	279
	Adults	RMSE = 7.66961 = 0.3733	RMSE = 6.368636 = 0.4519	0.146	4354

No competing interests reported.

ChaitaliSIFig1.jpg
S1 Figure. Photomap of the polytene chromosome from An. stephensi ovarian nurse cells. Chromosomal inversions show breakpoints at two locations, i.e., 39A–39C and 42A–44C, on the 3L arm of NT-stressed lines (NT, nutritional stress).
ChaitaliSIFig2.jpg
S2 Figure. Plots show the transgenerational changes in the larval and pupal stages of An. stephensiunder stress and control conditions. (A, D) low-temperature (LT) stress, (B, E) high-temperature (HT) stress and (C, F) nutritional (NT) stress.
ChaitaliStressSupportingTables.docx
Supplementary Table legends: S1 Table. Egg measurement (in µm) of LT experimental and control lines for ten generations of An. stephensi (LT, low temperature; ER, egg-float ridge; EL, length of egg; EW, width of egg; EFL, length of egg-float; EFW, width of egg-float). S2 Table. Egg measurement (in µm) of HT experimental and control lines for ten generations of An. stephensi (HT, high temperature; ER, egg-float ridge; EL, length of egg; EW, width of egg; EFL, length of egg-float; EFW, width of egg-float). S3 Table. Egg measurement (in µm) of NT experimental and control lines for ten generations of An. stephensi (NT, nutritional stress; ER, ridge number of egg-float; EL, egg length; EW, egg width; EFL, length of egg-float; EFW, width of egg-float). S4 Table. Comparative statistics of egg measurements (in µm) of NT, HT and LT experimental lines of An. stephensi (LT, low temperature; HT, high temperature; NT, nutritional stress; ER, ridge number of egg-float; EL, egg length; EW, egg width; EFL, length of egg-float; EFW, width of egg-float). S5 Table. Wing measurement (in mm) of male and female adults of LT experimental and control strains for eight generations of An. stephensi (LT, low temperature). S6 Table. Wing measurement (in mm) of male and female adults of HT experimental and control lines for eight generations of An. stephensi (HT, high temperature). S7 Table. Wing measurement (in mm) of male and female adults of NT experimental and control lines of An. stephensi for eight generations (NT, nutritional stress). S8 Table. Wing measurements (in mm) of NT, HT and LT lines of An. stephensi (NT, nutritional stress; HT, high temperature; LT, low temperature). S9 Table. Pupal body mass (in mg) of LT, HT and NT-stressed and control lines for eight generations of An. stephensi (LT, low temperature; HT, high temperature, and NT, nutritional stress). S10 Table. Baseline assay of eggs of LT lines for fixing the optimal lower temperature of An. stephensi (LT, low temperature). S11 Table. Baseline assay for fixing the optimal higher temperature for HT lines of An. stephensi (HT, high temperature). S12 Table. Baseline study for fixing the optimal amount of larval food for NT lines of An. stephensi (NT, nutritional stress). S13 Table. LT, HT and NT stress data at G₀generation of An. stephensi (LT, low temperature; HT, high temperature; NT, nutritional stress). S14 Table. Life-cycle data of LT stress lines of An. stephensi for eight generations (LT, low temperature). S15 Table. Life-cycle data of control lines for corresponding LT lines for eight generations of An. stephensi. (LT, low temperature). S16 Table. Life-cycle data of HT stress lines for eight generations of An. stephensi (HT, high temperature). S17 Table. Life-cycle data of control strains for corresponding HT strains of all the life stages for eight generations of An. stephensi (HT, high temperature). S18 Table. Life-cycle data of NT stress lines for eight generations of An. stephensi (NT, nutrition stress). S19 Table. Life-cycle data of control strains for corresponding NT strains for eight generations of An. stephensi (NT, nutritional stress). S20 Table. Comparative p values of eggs, larvae, pupae and adults among LT, HT, and NT strains with the control strains of An. stephensi [LT, low-temperature stress; HT, high-temperature stress; NT, nutritional stress; non-significant (p>0.05)].

Effects of temperature and nutrition stress on Anopheles stephensi, an Indian urban malaria vector

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Abstract

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