Effect of Thermal Stress on the Cuticular Chemical Composition of the Amazonian Social Wasp Polybia rejecta (Fabricius, 1798)

Abstract

Insects are facing challenges with climate change, especially in tropical regions where small variations in temperature can affect their survival and behavior. The insect cuticle is a barrier against water loss and a source of signals for chemical communication triggered mainly by cuticular hydrocarbons. Knowing that tolerance in social wasps to temperature variations mainly depends on changes in the chemical composition of the cuticle, the objective was to evaluate how high temperatures affect the cuticular hydrocarbon composition of the social wasp Polybia rejecta. The wasps were exposed to a temperature of 40 °C for 1 h, 3 h, and 6 h following analyses of the cuticular hydrocarbons by GC-MS. The results revealed five long-chain hydrocarbons and one fatty alcohol. The relative percentages to each class of compounds indicated alkanes as the principal component in all samples. Tricosane was only identified after the third hour of exposure, increasing in the sixth hour, suggesting a possible chemical communication mechanism to alert critical situations between individuals. These results open up new avenues of research into insect communication in response to environmental stress.

1. Introduction

Ectothermic animals depend on the environmental temperature to regulate their body temperature. These animals constitute the majority of terrestrial biodiversity and may be vulnerable to climate change because their physiological functions, such as locomotion, growth, and reproduction, are affected by temperature [1].

Climate change has contributed to a decrease in insect populations, especially in tropical regions, where they live close to the optimal physiological state with a low thermal safety margin and are therefore susceptible to small variations in temperature [2], resulting in changes in phenology, ecology, migration, and geographic distribution [3].

The body of insects is covered by a cuticle with different layers. The outermost layer, epicuticle, is composed of alcohols, alkyl esters, sterols, aldehydes, and mainly cuticular hydrocarbons (CHCs) that consist of complex mixtures of long-chain hydrocarbons, particularly linear alkanes, branched alkanes, and alkenes [4]. The water retention capacity of insects is influenced by the wax layer covering the cuticle [5]. Heating this layer at critical temperatures reduces the viscosity of the lipids, resulting in higher permeability and water losses. Therefore, melting temperatures of the lipids that constitute the insect wax layer play a fundamental role in the adaptation of insects to the environment, influencing their relationship with water availability [6,7]. In addition to their waterproofing function, these compounds are important for chemical communication in social insects [4,8,9].

Linear alkanes are primarily responsible for waterproofing [7,10,11], while branched alkanes and alkenes are responsible for chemical signaling, or communication, between individuals [12,13,14]. However, compounds responsible for chemical communication can potentially interact with those that prevent water loss [15].

In the social insects, the tolerance to temperature variations depends on changes in the chemical composition of the cuticle [16]. Adaptations to different temperatures in the ants Crematogaster and Camponotus are related to changes in the CHC composition [17].

As CHCs also play a role in communication, changes in the cuticular chemical composition may affect this feature. Nestmate social wasps showed increased aggression at high temperatures due to lack of individual recognition [16].

The 2023 Intergovernmental Panel on Climate Change (IPCC) [18] estimates that global warming will exceed 1.5 °C by 2040 and may reach to 4.4 °C between 2081–2100. Furthermore, anthropogenic activities are affecting climate and meteorological extremes worldwide. In fact, the 2021 IPCC [19] indicated that the Amazon biome is close to the point of no return, beyond which the biome loses its basic characteristics and degrades. Therefore, studying the chemical compounds in the body cuticle is fundamental to understanding the ecology and behavior of social insects and their interactions with the environment, especially in tropical regions. The objective was to evaluate how high temperature affects the cuticular chemical composition of the social wasp Polybia rejecta (Fabricius, 1798).

2. Materials and Methods

Polybia rejecta (Figure 1) was selected given its abundance and representation in the region. Females were collected in September 2023 in the early hours of the morning, in the urban area of Manaus (3°6″ S, 60°1″ W), state of Amazonas, Brazil, with an entomological net for the bioassays.

Immediately after collection, five individuals (control I—CI) were killed by freezing temperatures. Another five individuals were stored in a 250 mL plastic container and kept at room temperature throughout the experiment period to exclude the stress variable (control II—CII). Then, 15 females were caged in three 250 mL plastic containers with five individuals each and kept in an incubator at 40 °C for 1 h, 3 h, and 6 h, respectively. After each of these periods, the individuals were killed by freezing and then each individual was immersed for two minutes [20] in 2 mL of hexane (Nuclear^®, São Paulo, Brazil, Code 311782) in glass microtubes to extract the CHCs. After removing the wasps from the microtubes, the extracts were left in a fume hood to evaporate the hexane.

The samples were resolubilized in 150 µL of HPLC grade hexane and the chemical constitution of the extracts was determined by Gas Chromatography-Mass Spectrometry (CG-MS) analysis using a Shimadzu GCMS-QP5050A apparatus (Shimadzu Europe, Duisburg, Germany) under the following operational conditions: capillary column DB-5 (30 m × 0.25 mm, 0.25 µm); carrier gas (He) flow 1.6 mL min⁻¹; splitless; injector and detector temperature 290 °C; sample injection volume 1.0 μL; electron impact method (70 eV); scan mode m/z 35.00 to 700.00; oven temperature 40 °C for 4 min, heating to 300 °C at a gradient of 20 °C min⁻¹ and keeping the final temperature for 30 min. Compounds were identified through a comparative analysis of their experimentally obtained mass spectra against data from the equipment libraries (Wiley 7 and NIST). Additionally, calculated retention indices were compared with values documented in the literature [21,22,23,24]. Only those compounds exhibiting a minimum of 90% similarity in mass spectra with the libraries data were classified as identified.

The GC–MS instrument was calibrated prior to analysis using perfluorotributylamine (PFTBA) in automatic tune mode to ensure proper mass axis calibration and ion abundance ratios within the manufacturer’s specifications. Column bleed was evaluated by holding the oven at 300 °C for 10 min with a blank injection; no significant background rise or interfering peaks were observed, confirming column stability under the analytical conditions.

Hydrocarbon concentrations below detection limits were recorded as zero, and the data were analyzed separately for each compound. A generalized linear model (GLM) with time (1, 3, and 6 h) as a continuous predictor and concentration as the response was fitted, and the time coefficient and its p-value were extracted to assess temporal trends. Point comparisons were performed using CI and CII controls as references, testing the mean differences after the 6 h of exposure. p-values were adjusted for multiple testing using the false discovery rate (Benjamini–Hochberg), and model assumptions (normality and homoscedasticity of residuals) were assessed by visual inspection.

3. Results

The GC-MS analysis (Figure 2) revealed five long-chain hydrocarbons and one fatty alcohol (

Table 1. Chemical constituents of hexane extracts of Polybia rejecta obtained at increasing time intervals of exposure at 40 °C identified by GC-MS. RI, Retention Index; ^a calculated; ^b Literature [reference], ^c means of three replicates; CI and CII, control groups; nd, not detected.

Peak	Compound	MF	MW	RI		Relative Percentage (%) c
				Calc a	Lit b	CI	CII	1 h	3 h	6 h
1	Tricosane	C23H48	324.6	2303	2300 [21]	nd	nd	nd	1.6	9.6
2	Pentacosane	C25H52	352.7	2502	2500 [21]	27	34	23.6	28.3	42.2
3	Heptacosane	C27H56	380.7	2702	2700 [22]	37.8	20.8	18.1	19.4	12.5
4	Nonacos-1-ene	C29H58	406.8	2888	2888 [23]	nd	nd	3.5	3.5	1.0
5	Nonacosane	C29H60	408.8	2901	2900 [22]	35.2	34.9	25.7	22.6	28.8
6	Octacosan-1-ol	C28H58O	410.8	3091	3110 [24]	nd	10.2	10.9	5.2	1.0

). The relative percentages to each class of compounds indicated alkanes as the principal component in all samples. GLM analysis revealed distinct variations in cuticular hydrocarbons over time. After 6 h of exposure there was a decrease in Nonacos-1-ene (β = –0.839; p = 0.001) and octacosan-1-ol (β = –1.70; p = 0.002), and an increase in Tricosane (β = 1.99; p < 0.001) and Pentacosane (β = 3.79; p = 0.008). In contrast, Heptacosane (β = –1.22; p = 0.203) and Nonacosane (β = 0.684; p = 0.385) did not show significant changes, indicating no temporal effect.

4. Discussion

It was possible to observe that in the laboratory, social wasps do not survive for long at a constant temperature of 40 °C, indicating that these insects mitigate heat exposure through different behavioral strategies in the natural environment, such as remaining in the nest during the hottest periods or using substrates with low heat absorption for nest building [25], or nesting without direct sunlight exposure (TTM pers. obs.). Furthermore, social wasps have different mechanisms to regulate the temperature of their nests, such as ventilation, which involves individuals flapping their wings to expel hot air from the nest, and evaporative cooling through depositing water droplets on the surface of the nest [26].

Regarding the composition of cuticular hydrocarbons, all linear hydrocarbons found are main compounds involved in waterproofing in social insects [7,10,11] and commonly reported in other social wasp species, showing a typical cuticular chemical profile consistent with literature. Point comparisons with controls C1 (wasps immediately collected in the field) and C2 (control group) showed that some cuticular hydrocarbons responded significantly after 6 h of exposure to 40 °C. Heptacosane showed a significant decrease in the sixth hour compared to C1 (t = –39.8; p < 0.001) and C2 (t = –13.0; p = 0.006), also demonstrating a relationship with stress. Tricosane and nonacos-1-ene were not detected in the control samples (CI and CII), indicating a direct relationship with the increase in temperature.

Tricosane was only identified after the third hour of exposure, increasing in the sixth hour (β = 1.99; p < 0.001), in addition to its waterproofing function, this compound is also a sexual pheromone, which indicates that under heat stress, wasps also use Tricosane to communicate/alarm. If confirmed, this could open new directions for research into insect communication in response to environmental stress.

In adverse environments, such as those with high temperatures, communication between insects becomes even more crucial for survival. CHCs, which act in communication between individuals, function as chemical signals to alert other members of the colony about dangerous conditions/situations [7,27,28,29]. When exposed to environmental stress, such as extreme heat, insects release these compounds, as we observed, to inform their companions about the overheating alert and the thermal limit tolerated by them. Thus, the CHCs recorded may be acting as communication that regulates specific behaviors, such as the recruitment of individuals to perform cooling behaviors, as mentioned above, promoting a coordinated response that improves the survival of the colony against high temperatures [28].

In conclusion, the social wasp P. rejecta demonstrates an adaptive response to high temperatures through alterations in the composition and proportion of cuticular hydrocarbons (CHCs). These findings provide fundamental data that contributes to how climate change may impact insect biodiversity and thermal resilience. While this study advances our understanding of insect adaptation, significant gaps remain regarding the key factors that underpin their success under thermal stress. Moreover, ongoing research continues to uncover novel roles of CHCs, indicating that these compounds may be far more integral to insect ecology and behavior than previously assumed. Unraveling these complexities is essential to fully grasp how insects persist and thrive in increasingly challenging environments.