Humidity modulates the effects of high temperature on the thermal tolerance of 4 aphid species

Abstract

Accurate prediction of aphid population trends depends on understanding the characteristics of aphids’ responses to high temperature and humidity. Currently, most studies on the interactive effects of high temperature and humidity on aphids focus on a single species, and whether multiple species exhibit similar response characteristics warrants investigation. This study examined the effects of combinations of temperatures (38 °C, 42 °C) and relative humidities (20%, 60%, and 90%) on the mortality and heat tolerance (characterized by LT₅₀) of Myzus persicae (Sulzer, 1776), Acyrthosiphon pisum (Harris, 1776), Sitobion avenae (Fabricius, 1775), and Aphis craccivora Koch, 1854. Results showed that low relative humidity (20%) generally enhanced heat tolerance in aphids, although in some species, temperature combinations, the effect of humidity was not statistically significant. In contrast, high relative humidity exacerbated mortality under high-temperature conditions. Additionally, different aphid species exhibited species-specific responses to changes in temperature and humidity. A. craccivora showed the highest heat tolerance among the species tested. This study identified the temperature–humidity synergistic effect as a key factor regulating aphid survival and provided ecological insight for predicting aphid populations and formulating adaptive management strategies under climate change.

Introduction

Global warming has drawn increasing attention for its profound effects on agricultural pests (Ma et al. 2021). Rising temperatures significantly influence the population dynamics and geographic distribution of insect pests (Cammell and Knight 1992, Bale et al. 2002, Deutsch et al. 2018). Recent projections suggest that elevated temperatures may intensify outbreaks of piercing—sucking pests such as aphids, whiteflies, and thrips (Crossley et al. 2024, Wang et al. 2024). Moreover, the interaction between high temperature and environmental humidity is considered a key determinant in predicting insect population abundance and habitat range.

Most previous studies have focused on the effects of temperature on individual aphid species, often overlooking the combined influence of temperature and relative humidity. Significant interspecific differences exist in the heat tolerance of aphids. The upper developmental threshold of Myzus persicae (Sulzer, 1776) is approximately 37.3 °C (Davis et al. 2006); Acyrthosiphon pisum (Harris, 1776) can still develop successfully at 39 °C (Mastoi et al. 2023); Aphis craccivora Koch, 1854 is capable of surviving at temperatures as high as 42 °C (Kataria and Kumar 2017); and the critical thermal maximum (CT_max) of Sitobion avenae (Fabricius, 1775) is reported to be 36.79 ± 0.46 °C (Majeed et al. 2022). Although these studies have provided insights into temperature limits of individual species, the combined influence of temperature and humidity has received limited attention. Humidity can profoundly affect insect water balance and cuticular permeability, thereby modulating thermal tolerance. Yet, how multiple aphid species respond to the joint effects of heat and humidity remains poorly understood.

In this study, we investigated the interactive effects of temperature (38 °C and 42 °C) and relative humidity (20%, 60%, and 90%) on the mortality and thermal tolerance (LT₅₀) of 4 aphid species: M. persicae, A. pisum, S. avenae, and A. craccivora. We hypothesized that low humidity would enhance aphid heat tolerance, whereas high humidity would intensify heat-induced mortality. Understanding how temperature–humidity interactions affect insect survival provides crucial insights into the physiological limits of aphids and their adaptive potential under climate stress, helping improve predictions of pest dynamics in a warming and increasingly variable climate.

Materials and Methods

Biological Materials

The populations of M. persicae, S. avenae, A. craccivora, and A. pisum were collected in 2024 from the alfalfa, wheat, and pepper fields in Changyi City, Shandong Province, China. These aphids had been continuously reared for >1 yr in the laboratory on their preferred hosts (M. persicae on pepper, S. avenae on wheat, and A. craccivora and A. pisum on pea) under controlled conditions of 25 ± 1 °C, 60 ± 5% relative humidity (RH), and a 16:8 h (L:D) photoperiod.

Seeds of pea, wheat, and pepper were purchased from a commercial supplier and germinated in the laboratory under 26 ± 1 °C, 60 ± 10% RH, and a 16:8 h (L:D) photoperiod. Plants used for these tests were at the 2-true leaf growth stage, and plant leaves were placed in plastic basins (20 × 10 × 6 cm) for testing.

Temperature and Humidity Settings

To examine the tolerance of high temperature in the 4 aphid species, we set 2 constant temperatures (38 °C and 42 °C), with a photoperiod of 16:8 h (L:D). Humidity control followed the method described by Fisher et al. (2021), using saturated salt solutions to maintain specific relative humidity levels (Fig. 1).

Fig. 1.

Humidity chamber used to maintain controlled temperature and humidity conditions for aphid experiments. The chamber consisted of a sealed plastic container with a wooden rack placed above a saturated salt solution. Petri dishes containing aphids and a GSP-6 temperature-humidity data logger were placed on the rack to monitor conditions. Relative humidity was controlled using saturated salt solutions for low (∼20%, potassium acetate) and high (∼90%, zinc sulfate) humidity levels, while the intermediate level (∼60%) was achieved by adjusting the climate chamber settings.

For low humidity conditions, a 5-liter plastic container with a saturated potassium acetate (CH₃COOK) solution was used to maintain a relative humidity of 21.61 ± 0.53%. For high humidity, a saturated zinc sulfate (ZnSO₄·7H₂O) solution was placed inside another container and, together with the climate chamber settings, maintained a relative humidity of 90 ± 2%. The climate chamber was further adjusted to achieve an intermediate humidity of 60 ± 2%.

A wooden rack was positioned above the salt solution. Petri dishes containing aphids, along with a GSP-6 temperature and humidity data logger, were arranged on the rack to monitor internal conditions. Temperature and humidity records during the 120 min exposure period showed that temperature variation was greater under the 20% RH treatment than under the other humidity levels, as indicated by the higher standard deviations (Table 1; Fig. 2).

Table 1.

The mean and standard error of environmental temperature fluctuations under three relative humidity levels (20%, 60%, and 90%) were statistically analyzed

Humidity (%)	Temperature (°C)	Mean value	Standard deviation
20	38	37.4	0.39
	42	42.8	0.44
60	38	37.6	0
	42	41.6	0.02
90	38	37.8	0.26
	42	41.6	0.23

Fig. 2.

Temperature and humidity records during 120 min. Dashed lines represent temperature, and solid lines represent humidity. Panels show variations at A) 38 °C, and B) 42 °C. For both temperatures, 3 humidity levels (20%, 60%, and 90%) were set and recorded. Temperature at the 20% humidity level showed greater variation compared with the other levels.

Leaf-Disc Preparation and Treatment

Leaf discs were prepared by placing fresh host plant leaves on an agar medium within Petri dishes. A small circular hole was cut in the center of each dish lid and covered with fine mesh to allow air exchange with the external environment. Each leaf disc was infested with 10 4-d-old aphids. For each aphid species, 10 leaf discs were prepared under each temperature and humidity combination, totaling 100 aphids per treatment.

Mortality Observation

After the relative humidity within the chamber had stabilized at 20%, 60%, or 90% for at least 24 h, aphids were introduced for observation. Aphid mortality was recorded every 10 min during the first 2 h of exposure, and every 30 min thereafter until all individuals had died. Mortality rate was calculated from cumulative deaths. Aphids were gently touched with a soft brush; individuals showing no leg or antenna movement were removed and held at room temperature for 5 min. If no signs of life were observed after this period, the aphid was recorded as dead.

Data Analysis

Survival curves were constructed using the Kaplan–Meier method based on groups of 10 aphids per leaf disc, which were treated as independent experimental units. Differences among treatments were assessed using log-rank tests. The median lethal time (LT₅₀), defined as the time required for 50% mortality, was calculated for each species under different temperature and humidity conditions. Graphs and figures were generated using Origin software. Linear regression between mortality and exposure duration was fitted using the least squares method in SPSS, with the slope of the fitted line representing the strength of the mortality trend.

Results

Survival analysis showed that death time decreased as both temperature and humidity increased, indicating reduced heat tolerance under harsher conditions (Fig. 3). Survival curves differed significantly among the 4 aphid species, such as 38 °C with RH 20% (log-rank test: χ² = 50.56, P < 0.001), and the separation of the curves confirmed clear interspecific variation in heat tolerance. LT₅₀ derived from the Kaplan–Meier estimates further supported these differences, with LT₅₀ values ranging from 49.8 to 70.5 min across species under the same stress conditions (Table 2).

Fig. 3.

Kaplan–Meier survival curves for 4 aphid species, Myzus persicae, Acyrthosiphon pisum, Sitobion avenae, and Aphis craccivora, under different temperature and humidity combinations. Low humidity appeared to increase aphid tolerance to high temperature.

Table 2.

Linear regression parameters and survival-based LT₅₀ estimates for 4 aphid species (Myzus persicae, Acyrthosiphon pisum, Sitobion avenae, and Aphis craccivora) exposed to different combinations of temperature and relative humidity

			Linear regression		Survival curve
Aphid	Temperature (°C)	Relative humidity (%)	Slope (β1)	R2	LT50	95% LCL	95% UCL
M. persicae	38	20	8.9	0.942	570	480	659.4
	38	60	17.8	0.833	303	298.8	307.2
	38	90	24.85	0.781	241.2	238.2	244.8
	42	20	1.18	0.814	87.2	86.1	88.4
	42	60	1.32	0.679	63.4	62.5	64.3
	42	90	1.73	0.869	58.9	57.8	60.0
A. pisum	38	20	7.64	0.795	630	528	731.4
	38	60	21.45	0.803	232.2	228.6	235.8
	38	90	33.57	0.821	156.6	154.2	159.6
	42	20	0.55	0.940	174.9	171.4	178.4
	42	60	1.58	0.932	59.7	58.7	60.8
	42	90	1.85	0.903	49.8	48.6	51.0
S. avenae	38	20	5.07	0.796	870	757.2	982.2
	38	60	8.57	0.901	644.4	634.8	653.4
	38	90	18.6	0.815	286.2	282.6	290.4
	42	20	0.87	0.780	98.9	97.7	100.1
	42	60	1.1	0.825	80.4	79.3	81.5
	42	90	1.69	0.926	58.4	57.3	59.5
A. craccivora	38	20	1.98	0.823	2340	2017.8	2661.6
	38	60	2.4	0.809	2370	2343.6	2396.4
	38	90	3.13	0.860	1831.8	1806.6	1856.4
	42	20	0.52	0.789	172.9	170.5	175.2
	42	60	0.94	0.884	105.0	103.0	106.9
	42	90	1.26	0.809	70.5	69.5	71.6

Linear regression showed strong mortality–time relationships across all treatments (R² > 0.78, P < 0.001). Slopes increased with temperature and humidity, indicating faster mortality rates under harsher conditions (Table 2).

Among the 4 species, A. craccivora exhibited the strongest heat tolerance, while M. persicae and A. pisum were more sensitive to elevated temperature and humidity (Fig. 3). Overall, the results demonstrate that low humidity enhances aphid heat tolerance, while high humidity intensifies heat-induced mortality.

Discussion

Our study showed that the 4 aphid species generally exhibited higher resistance to high temperatures under low-humidity conditions, with A. craccivora displaying comparatively higher heat tolerance than the other species. Across species, LT₅₀ values at 90% relative humidity were reduced by approximately 30% to 70% compared with those at 20% relative humidity, indicating that high humidity can substantially exacerbate heat-induced mortality. One possible explanation is that aphids exposed to low humidity may develop enhanced desiccation resistance by reducing cuticular water loss (Yang et al. 2020). This physiological adjustment helps maintain cellular water balance, which can indirectly improve tolerance to heat stress (Zhang et al. 2019).

The comparatively higher heat tolerance observed in A. craccivora may be related to its ecological background. Previous studies have statistically characterized the temperature and relative humidity ranges associated with the survival of different aphid species, indicating that A. craccivora occupies a broader and warmer climatic niche (10 to 42.7 °C; RH: 31% to 99%) (Kataria and Kumar 2017) than M. persicae (14.61 to 34.69 °C; RH: 40.57% to 98.85%) (Pathipati et al. 2020), A. pisum (12 to 36 °C; RH: 52% to 73%) (Hassan et al. 2023), and S. avenae (3.4 to 33.7 °C; RH: 69% to 95%) (Mir 2013). This comparative ecological context suggests that A. craccivora is more frequently exposed to high-temperature and relatively low-humidity conditions, which is consistent with its higher tolerance to heat stress observed in the present study, although this association does not imply a direct causal relationship.

Another factor that may contribute to species differences is the presence of facultative symbionts. These microbial communities are known to shape how aphids respond to thermal stress by modulating host physiological processes and facilitating acclimation under unfavorable temperature (Hudson et al. 2024). For example, certain symbiotic bacterium of A. pisum, can enhance reproduction under heat stress (Heyworth and Ferrari 2015), and symbionts in S. avenae have also been linked to improved heat tolerance (Majeed et al. 2022). We did not examine the symbiont communities in the 4 species tested, and this is an important limitation. Because symbiont composition can vary widely even within a single aphid species, it likely contributes to hidden variation in thermal performance. Including symbiont screening, symbiont-removal approaches, or controlled reinfection experiments in future work would help determine how much of the heat tolerance we observed is due to the aphids’ own physiology versus the assistance of their microbial partners.

Host plants further influence thermal tolerance. Different pea varieties affect the lifespan of A. pisum under temperature stress (Mastoi et al. 2023), R. padi avoids plants experiencing severe water stress (Kansman et al. 2020), and plant hormones can indirectly enhance aphid tolerance by altering host physiology (Grover et al. 2022). In our study, however, aphids were tested on isolated leaf discs rather than intact plants. This approach allowed us to control temperature and humidity precisely, but it also excluded the broader influence of plant tolerance, plant-mediated stress responses, or plant–aphid interactions. As a result, any potential buffering or amplifying effects from the host plant were not captured here. Future studies should consider testing aphids on whole plants or incorporating plant physiological responses to better understand how plant–aphid–environment interactions jointly shape heat tolerance.

Overall, the enhanced heat tolerance of aphids under low humidity conditions likely reflects an integrated physiological response involving water conservation and metabolic regulation. These findings highlight the importance of considering combined temperature–humidity interactions when predicting aphid population dynamics under future climate scenarios.

Author Contributions

Qingsen Yuan (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Project administration [equal]), Zhaoke Dong (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Funding acquisition [equal], Investigation [equal], Methodology [equal], Project administration [equal]), and Zhaozhi Lu (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Funding acquisition [equal], Investigation [equal], Methodology [equal], Project administration [equal])

Conflicts of Interest

Authors declare no conflicts of interest.