Fluctuating Warm and Humid Conditions Differentially Impact Immunity and Development in the Malaria Vector Anopheles stephensi

Abstract

A variety of environmental factors including temperature and humidity influence mosquito physiology and behavior. However, their direct impact on innate immunity, the core mosquito defense system against pathogen infection, remains insufficiently understood. This is particularly important as climate change is likely already altering mosquito distribution. A key limitation in most mosquito studies is the use of unrealistic static temperature and humidity settings which fail to reflect natural environmental changes. To address this, we employed a novel approach, exposing Anopheles stephensi from egg to adult to diurnal fluctuations (+4°C and +10% relative humidity above the baseline). We then investigated the impact of these elevated fluctuating conditions on innate immune gene expression, development, and adult longevity. We show that realistic elevated temperature and humidity fluctuations prime basal immune responses and accelerate pre-adult development without reducing adult lifespan. Bacterial infections under these elevated fluctuating conditions lead to a complex reprogramming of mosquito innate immunity and enhanced survival of both larvae and adults. Furthermore, fluctuating elevated temperature and humidity alter the transcriptional activity of key promoters widely used to express transgenes in genetically modified mosquitoes, highlighting the potential environmental sensitivity of these malaria control strategies. These results suggest that while elevated conditions-driven immune priming could initially decrease A. stephensi's vectorial capacity, the observed post-bacterial challenge immune suppression could enhance susceptibility to Plasmodium, with potential significant implications for vector competence and malaria transmission. Our study findings highlight the need to incorporate realistic environmental variability in mosquito research to accurately predict the impact of climate on disease transmission.

Keywords: Anopheles stephensi; bacterial infection; climate change; development & survival; diurnal fluctuation; environmental variability; innate immunity; malaria; temperature & relative humidity; vector competence.

FIGURE 1

Overview of this study's experimental design. (A) Mosquitoes from the control group were exposed to hourly variable temperatures ranging from 15°C to 22°C and hourly variable relative humidity from 75% to 86% throughout their development (egg, larva, pupa, and adult). Mosquitoes from the elevated temperature and relative humidity (ETH) group were exposed to hourly variable temperatures 4°C higher (19°C–26°C) and hourly variable relative humidity 10% higher in average than the control group (85%–94%). Hourly temperature (B) and relative humidity (C) were measured inside the control (blue line) and ETH (red line) environmental chambers for 25 consecutive days and were compared. ****p < 0.0001.

FIGURE 2

Influence of fluctuating elevated temperature and humidity on tissue‐specific immune priming in sugar‐fed and blood‐fed Anopheles stephensi. Basal transcriptional expression was assessed in the midgut and fat body‐containing carcass of sugar‐fed (3‐days post‐emergence) or blood‐fed (1‐day post‐blood feeding) A. stephensi female mosquitoes reared under ETH conditions and compared with mosquitoes reared under control conditions. Relative gene expression fold change was measured by qRT‐PCR for (A) Toll and Imd pathway key immune effectors, (B) complement‐like immune factors and promoters used to drive transgenes, and (C) melanization pathway‐related immune factors and nitric oxide synthase, and calculated using the 2^−ΔΔCT method. The line at y = 1 represents no change in gene expression under ETH conditions compared to control conditions. Bars above y = 1 indicate gene upregulation under ETH conditions; bars below y = 1 indicate gene downregulation under ETH conditions. Data represents mean and standard deviation (SD) of three to five independent biological replicates. Two‐tailed *p < 0.05; **p < 0.01.

FIGURE 3

Impact of fluctuating elevated temperature and humidity on Anopheles stephensi pre‐adult development, and adult longevity and size. (A) Hatch rates indicate the average percentage of eggs giving rise to first instar larvae, as determined by three independent biological replicates. Mean values and standard errors (SE) are indicated. No statistically significant differences were observed. (B) Larvae reared under ETH conditions developed faster than those under control conditions, demonstrated by their larger size at 3 days post‐hatch. Scale bar 1 mm. (C) Pupation in the ETH group initiated earlier than in the control group. Median time to pupation for the ETH group was 14 days (interquartile range (IQR) 1 day), compared to 25 days for the control group (IQR 2 days). (D) Pupation rate under ETH conditions was significantly higher than under control conditions. Data represents mean values and SE of three independent biological replicates. ****p < 0.0001. (E) Mosquitoes from the ETH group emerged significantly earlier than those of the control group. Data represents mean values and SE of three independent biological replicates. ****p < 0.0001. (F) The wing lengths of ETH females were significantly shorter than those of the control females. Left panel: Representative images of wings from each group; scale bar 1 mm. Right panel: Each blue dot or red triangle represents an individual wing from the control or ETH groups, respectively. Data represents mean values and SD. Two‐tailed ****p < 0.0001. (G) Lifespan did not differ significantly between ETH and control mosquitoes. Data from four replicates is presented with standard error bars.

FIGURE 4

Impact of fluctuating elevated temperature and humidity on bacterial‐infected Anopheles stephensi. Larvae and female adult mosquitoes were challenged with phosphate‐buffered saline (PBS) as control, Gram‐negative bacterium Escherichia coli (Ec), or Gram‐positive bacterium Staphylococcus aureus (Sa). (A) Larvae from the ETH group showed significantly longer lifespan than larvae from the control group, for all challenges (PBS, Ec, Sa). Data from three independent biological replicates is presented with standard error bars. *p < 0.05; ****p < 0.0001. (B) Mosquitoes from the ETH group emerged earlier than those of the control group. Data represents mean values and SE of three independent biological replicates. **p < 0.01; ***p < 0.001. (C) The lifespan of adult female mosquitoes challenged with control‐PBS or Sa was significantly longer under ETH conditions compared to control conditions. Data from three independent biological replicates is presented with standard error bars. *p < 0.05; **p < 0.01.

FIGURE 5

Reprogramming of innate immunity responses of infected Anopheles stephensi by elevated temperature and humidity. (A) Larvae and (B) female adult mosquitoes were challenged with phosphate‐buffered saline (PBS) as control, Gram‐negative bacterium Escherichia coli (Ec), or Gram‐positive bacterium Staphylococcus aureus (Sa). Gene expression was assessed at 4‐h post‐challenge in the whole body of A. stephensi larvae/adults reared under ETH conditions and compared with larvae/adults reared under control conditions. Relative gene expression fold change was measured by qRT‐PCR for Toll and Imd pathway key immune effectors, complement‐like immune factors, melanization pathway‐related immune factors and nitric oxide synthase, and calculated using the 2^−ΔΔCT method. The line at y = 1 represents no change in gene expression under ETH conditions compared to control conditions. Bars above y = 1 indicate gene upregulation under ETH conditions; bars below y = 1 indicate gene downregulation under ETH conditions. Data represents mean and SD of three to four independent biological replicates. Two‐tailed *p < 0.05; **p < 0.01.