In times of climate change, organisms need to adjust to new climatic conditions. Short-term adjustments can be achieved by phenotypic plasticity, i.e. the ability to adjust phenotypic traits to local environmental conditions within their lifetime, contributing significantly to their survival and fitness. Phenotypic plasticity can affect morphological traits in response to predator pressure(Tollrian, 1990) but it also concerns responses to climatic conditions, such as certain temperatures or humidity levels (Angilletta, 2009; Seebacher et al., 2015). Phenotypic plasticity stands in contrast to genetic (evolutionary) adaptation, which takes place over generations with respect to prevailing environmental conditions to maximize fitness. Thus, the expression of a particular phenotype is primarily influenced by a mixture of the genomic makeup of an organism and its environment (Pigliucci, 2001). Acclimation, i.e. the physiological adjustment of an organism to current environmental conditions, may increase tolerance and performance under different conditions(Angilletta, 2009; Chown et al., 2011) and thus lead to higher resilience to environmental variation (Colinet et al., 2015; Rohr et al., 2018; Seebacher et al., 2014).
Especially ectothermic organisms such as insect, must be able to flexibly adjust to different temperature regimes, and insects must respond quickly to changes in humidity to prevent desiccation. Temperature directly affects desiccation risk because firstly, higher temperature increases vapour pressure deficit, and secondly because, the cuticular hydrocarbon layer, which protects against water loss, melts and becomes more permeable for water (Menzel et al., 2019). Acclimation usually results in enhanced survival, as has been shown for drought resistance (Baumgart et al., 2022; Chown et al., 2011), enhanced heat shock response and heat tolerance (Hoffmann & Watson, 1993; Menzel et al., 2018), and/or changes in life history traits (Franke et al. 2019), but it matters whether organisms acclimate to fluctuating or constant regimes. Fluctuating conditions often led to better performance concerning temperature tolerance (Peng et al., 2014; Salachan & Sørensen, 2022), egg survival (Tippelt et al., 2020), or drought resistance (Baumgart et al 2022). Thus, plastic changes can increase the resistance of ectothermic animals to climate change (Seebacher et al., 2015).
Acclimatory processes include a wide range of traits, such as the expression of heat shock proteins, changes in metabolism, the synthesis of protective osmolytes, and modulation of potassium conductance in neural cells (Chown et al., 2011; Chown & Terblanche, 2006). A further acclimatory response comprises changes in the composition of lipid membranes and cuticular hydrocarbon (CHC) layers (van Dooremalen et al., 2011). Cuticular hydrocarbons cover the body surface of virtually all insects, serving as the most important desiccation barrier (Beament, 1945; Ramsay, 1935), but also as communication signals between sexes and in social insects among castes (Blomquist & Bagnères, 2010). Acclimatory changes of lipid membranes and CHC are probably homeostatic, i.e., they are used to maintain physiological properties such as viscosity, and thus ensure continued waterproofing. Indeed, acclimatory CHC changes to warm or dry conditions are linked to increase in heat and/or drought survival (Baumgart et al., 2022; Menzel et al., 2018). In lipid membranes of arthropods, molluscs and fishes, warm conditions generally favour the expression of saturated fatty acids and/or lower amounts of cholesterol compared to colder conditions (Harwood & Takata, 1965; Koštál et al., 2011; Pernet et al., 2007). Similarly, CHC layers of cold-acclimated individuals are enriched in unsaturated hydrocarbons (alkenes and alkadienes) or dimethyl alkanes, and depleted in compounds with higher melting points like n-alkanes. Thus, in both membranes and CHC layers, cold-acclimation favours less viscous substances, while warm conditions promote the expression of more viscous or solid components (Hadley, 1977; Sprenger & Menzel, 2020). However, CHC layers can include more than 100 different compounds, and their composition is highly species-specific (Sprenger & Menzel, 2020). Hence, the precise CHC changes in response to climate conditions are species-specific as well, and likely adapted to their longer-term environmental conditions, including microhabitat-specific climatic fluctuations. Therefore, acclimatory changes may differ even among closely related species, depending on the species’ ecological niches; i.e. the climatic conditions they encounter and are adapted to.
The genomic basis of CHC biosynthesis is still not fully resolved. CHC synthesis is mediated by an assembly pipeline of CHC synthesis genes, including fatty acid synthases, elongases, desaturases, reductases, and decarbonylases (Holze et al., 2021). Many of those genes are involved in fatty-acid synthases and only few studies have functionally annotated CHC synthesis genes, e.g., in Drosophila (Dembeck et al., 2015), the German cockroach (Pei et al., 2019), the migratory locust (Yang et al., 2020) and the mason wasp (Moris et al., 2023). The number of elongase and desaturase genes expanded in many eusocial Hymenoptera (Hartke et al., 2019). Only few studies have investigated the underlying gene expression patterns associated with differences in CHC profiles between species (Li et al., 2021; Sprenger et al., 2021), between species and life stages (Falcon et al., 2019), and desiccation resistance between populations (Etges et al., 2017; Ostwald et al., 2023).
Studying the transcriptome of differently acclimated individuals reveals underlying molecular changes during acclimation. By assigning transcripts to GO terms, we can assess which physiological processes are up- or downregulated during acclimation. Furthermore, the extent of gene expression changes between acclimatory regimes allows to assess an organism’s acclimatory plasticity, i.e. the degree to which its gene expression can respond to environmental changes. This is particularly important in times of climate change, where a species’ ability to acclimate may be vital for its future survival.
The present study investigates gene expression changes during acclimation in three congeneric ant species. While many life-history traits such as colony size and life cycle are relatively similar (Seifert 2018), the three species inhabit different microhabitats. Lasius niger nests in mesic to dry, sun-exposed meadows and thus should be adapted to cope with high temperatures. Its sister species, Lasius platythorax, was only recognised as a separate species 30 years ago (Seifert, 1991) and inhabits dead logs or tree stumps on moist forest floors and in bogs. Since open habitats generally possess higher temperatures, higher temperature fluctuations, and lower humidity compared to forest habitats in similar locations (Renaud et al., 2011), L. niger should be more able to cope with hot temperatures and fluctuations compared to L. platythorax. The third species, Lasius brunneus, nests in the wood of trunks or the canopy of living trees and has a largely arboreal lifestyle. It is phylogenetically more distant from the other two (Maruyama et al., 2008).
A previous study, which the current one is based on, showed that acclimation to either constant 20°C, constant 28°C, or fluctuating (20°C-28°C) temperatures resulted in species-specific CHC changes in these three species (Baumgart et al., 2022). Warm (28°C)-acclimated L. niger and L. platythorax produced more monomethyl alkanes and had less dimethyl (L. niger) or trimethyl alkanes (L. platythorax) compared to 20°C-acclimated ones. CHCs of ants from the fluctuating treatment were in between the two constant regimes. The acclimation was beneficial, enhancing drought survival in warm- and fluctuating-acclimated ants. Lasius brunneus differed in that, firstly, its CHC profile is dominated by unsaturated CHCs, i.e. n-alkenes, alkadienes and methyl-branched alkenes, while that of L. niger and L. platythorax consists mainly of methyl-branched alkanes but has hardly any unsaturated compounds. Secondly, acclimation did not alter drought survival in L. brunneus and it had the worst desiccation tolerance of all three species (Baumgart et al., 2022).
Using conditions currently experienced by the three species, we analysed 1) whether acclimatory responses differ between species in terms of (i) the number and identity of differentially expressed genes, (ii) their functions, and (iii) gene co-expression networks. We tested whether the three species apply the same acclimatory strategies, i.e. they activate the same pathways when they adjust to new conditions, or whether each species has a specific strategy that might be linked to its ecological niche. Secondly, 2) we studied how the expression of CHC biosynthesis genes differed among acclimation treatments and species, and whether this fits observed CHC changes. In particular, we were interested in whether CHC-candidate genes are co-expressed and associated with other genes linked to acclimation, and link these to the desiccation resistance results of Baumgart et al. (2022) as a fitness measure.
We predicted that
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Gene expression differences should be highest between the two constant temperature regimes, with the fluctuating regime in-between.
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The three species adapted to different niches, differ in their response to the different acclimation regimes. We expect the meadow-dwelling L. niger to show a lower stress response in terms of upregulation of metabolism and heat shock genes at higher temperatures.
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We expected CHC-candidate genes to be co-expressed together with genes associated to temperature treatments.
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Since the species differ in their CHC-profiles and niche adaptation, we do not expect gene-coexpression modules associated with temperature to be preserved between species.
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L. brunneus is the only species with unsaturated CHC-compounds, and we thus expect to find desaturase expression only in this species.