Honey bees are social insects of great ecological and economic importance, but they have experienced substantial losses over the last years due to the action of several interacting biotic and abiotic stressors. These include invasive alien predators, parasites and pathogens, pesticides, and climate change [reviewed by Zhao et al. (2021), Even et al. (2012)]. One of the main impacts of climate change is the intensification of extreme weather events, such as heavy rainfall, prolonged droughts, and heat waves (IPCC-Intergovernmental Panel on Climate Change, 2007), which may severely impact honey bees’ wellbeing. On one hand, these events can indirectly affect honey bee nutrition by disrupting the availability of floral resources and reducing the quantity and quality of nectar and pollen [reviewed by Obeso and Herrera (2018)]. On the other hand, they can directly cause heat stress on honey bees, impairing their foraging efficiency and metabolic functions [reviewed by Zhao et al. (2021)]. Whether honey bees are able to adapt and survive changes in temperature, humidity, and ultraviolet (UV) radiation, and to a reduction in the quantity and diversity of food resources, is a question of utmost importance. However, the vulnerability of bees to climatic vagancies is not necessarily the same worldwide. Even in the same environment, different bee species and subspecies may suffer different stress levels and exhibit different behaviours, weaknesses and lifespans [reviewed by Zhao et al. (2021)]. This would mean that bee species and subspecies may differ in their ability to adapt, resist and be resilient to environmental changes.

Genetic variability is key to resistance and adaptation in stressful environments. Several candidate Single Nucleotide Polymorphisms (SNPs) mapped to genes involved in reproduction, immunity, olfaction, circadian rhythm, lipids biosynthesis and storage were linked to local adaptation in the western honey bee Apis mellifera (Chávez-Galarza et al., 2013; Wallberg et al., 2014; Chen et al., 2016; Henriques et al., 2018). However, beyond genetic determination, complex interactions between genetic and epigenetic factors are known to shape the diversity of organismal phenotypes (West-Eberhard, 2003). A phenotype can change in response to environmental signals without altering its genotype, resulting in modifications to an organism’s physiology and behaviour. This ability is called “phenotypic plasticity” and is remarkably developed in A. mellifera, which can respond to environmental cues to generate dramatically distinct phenotypes, such as queens or workers, through nutritional stimuli such as royal jelly (Winston, 1991; Corona et al., 2016). Interestingly, the A. mellifera genome has been described as structured with respect to plasticity, where stress-related genes are organised into clusters that show coordinated gene expression in response to environmental changes (Duncan et al., 2020). In addition to epigenetics, RNA processing mechanisms are also involved in phenotypic plasticity; splicing is the process by which introns are removed from a gene’s primary transcript, resulting either in a single functional protein (constitutive splicing) or in various structurally and functionally isoforms (alternative splicing). Alternative splicing (AS) can be triggered by physiological needs and environmental stimuli, often representing a primary source of phenotypic diversity within the proteome of eukaryotic cells [reviewed by Ast (2004), Blencowe (2006), Maniatis and Tasic (2002)]. It also plays a key role in cellular stress tolerance [reviewed by Biamonti and Caceres (2009)].

Genetic studies are a valuable tool to understand the effects of stressors on different molecular mechanisms. This review proposes genetic markers for Apis mellifera focusing mainly on climate change stressors such as temperature, humidity, UV exposure and food scarcity. These markers belong to different molecular mechanisms summarized in Figure 1, ranging from genetics to epigenetics, and affecting an organism´s phenotype. Thus, it can serve as a basis for designing new studies that consider the relationship with the stressor(s), as well as the type of gene expression (constitutive or inducible), splicing-related processes (key in PCR designs based on RNA) and epigenetic factors that might affect gene expression. In addition, the knowledge of the role of each marker in the corresponding cellular pathways will help future studies to select accurate molecular markers and correctly interpret the results.

This review was based on a published bibliography of original articles, reviews, book chapters and web pages obtained from scientific sources such as PubMed (PubMed, 2025), Scopus (Scopus, 2025), Google Scholar (Google Scholar, 2025), and Web of Science (Web Of Science, 2025). Candidate search terms are shown in Supplementary Material (Supplementary Table S1).

Gene descriptions and nomenclature follow the conventions established by NCBI. Given that the Apis mellifera genome has been characterized as organized in clusters associated with phenotypic plasticity (Duncan et al., 2020), the genetic markers referenced throughout this study were visualized as loci mapped onto their respective chromosomes (Figure 2). This was accomplished using the R package chromoMap v0.4.1 (Anand and Rodriguez Lopez, 2022). Such spatial representation of genomic features may prove instrumental for future research, particularly when selecting genetic markers based on their chromosomal location.

Although the term “stress” is commonly understood to have a negative connotation, stress is a natural state pervasive in all known biological systems. These systems maintain a complex dynamic equilibrium called homeostasis that is vulnerable to destabilization in the presence of stressors. After many years of controversy, stress is defined as a “state of homeostasis being challenged,” and stressors as “the factors with the potential to directly challenge homeostasis” [reviewed by Lu et al. (2021)]. An optimal stress level plays a key role in the health and adaptability of organisms. Depending on the level of exposure to the stressor(s), stress can be classified as: (i) distress (or bad stress), which occurs when the state of homeostasis is severely challenged by high levels of stress, inducing a severe response that impairs homeostasis and threatens health; (ii) sustress (or inadequate stress), which is the consequence of inappropriate stressor effects that do not challenge homeostasis but undermine its capacity, and threaten health, and (iii) eustress (or good stress), in which the system is mildly challenged by moderate levels of stressors, inducing a mild response, enhancing the buffering capacity of homeostasis and benefiting health [reviewed by Lu et al. (2021)]. Previous terminology defined “physiological” stress as encompassing environmental stress, intrinsic developmental stress and ageing [reviewed by Kagias et al. (2012)], initially understood as eustress. However, if an organism is unable to cope with one or more of these stressors, the level of stress increases (distress) and becomes pathological.

Regardless of its type or intensity, stress has various consequences at different levels. In honey bees, these consequences can be observed in their behavioral, physiological, and cellular responses. The first consists of the term “fight-or-flight.” For example, sting extension has been used to evaluate sensitivity to stressors in honey bees. This behaviour is widely considered to be indicative of stress as well as an aggressive response. Physiological responses include hormone and neurotransmitter levels. Finally, cellular responses consist of the activation of several molecular mechanisms to control the stressful state and restore the damage. Perhaps the best known and most studied cellular response is the production of stress-related proteins, such as Heat Shock Proteins (HSPs) and antioxidant enzymes, known as stress biomarkers (review by Even et al. (2012)]. At all these levels, cells and systems use different strategies to respond and adapt to environmental changes. Suppose these strategies are effective in coping with the stressful situation. In this case, the organism would undergo a process of adaptation, enabling it to survive and reproduce under new conditions.

Rising temperature is one of the main effects of climate change (IPCC-Intergovernmental Panel on Climate Change, 2007) and is of concern because temperature is one of the most stressful abiotic stresses on living organisms. But in addition to the effects of temperature on individual organisms, the consequences for an entire biocenosis depend on how each organism changes to adapt. For example, changes in temperature are affecting the hibernation cycle of honey bees (which would affect pollination, and hence plant reproduction), but also the timing and amount of flowering (which changes the availability of food resources for bees) (Obeso and Herrera, 2018). This example is very important because nutritional stress is an important indirect stressor to honey bees from rising temperatures [reviewed by Quinlan and Grozinger (2023)] and is discussed in section 7 of this review.

Focusing only on honey bees, the effect of temperature must be analysed from two perspectives: as a super-organism (colony) and at the individual level (single bee). In both cases, honey bees have developed mechanisms to resist stress and restore homeostasis. As a super-organism, they can regulate the temperature inside the colony, maintaining it constant at approximately 35 °C. In a cold environment, all the bees cluster around the queen and brood to generate and maintain heat (Southwick, 1985; Southwick and Heldmaier, 1987). In a warm environment, the colony employs a combination of ventilation, achieved through coordinated wing fanning by honey bees at hive entrance (at a rate of up to 60 L/min), and evaporative cooling, achieved through the use of water from the bee´s bodies to create droplets that cool down the nest (Southwick and Heldmaier, 1987; Jones and Oldroyd, 2006). All these actions at the community level require also actions at the individual level. Here, cellular mechanisms are key to coping with temperature changes.

Heat stress negatively impacts key biological processes in honey bees, including physiological and behavioral development and immunocompetence (Bordier et al., 2017; Medina et al., 2018; Alqarni, 2020; Greenop et al., 2020). It is worth mentioning that the impact of heat stress on honey bees and their defence mechanisms vary across species and even subspecies [reviewed by Abou-Shaara et al. (2017), Zhao et al. (2021)]. In Apis mellifera, two of the 31 currently recognized subspecies (Ruttner, 1988; De la Rúa et al., 2009), A. mellifera ligustica (native to Italy) and A.m. carnica (native to the Balkan region), have been the focus of several comparative studies due to their widespread commercial use beyond their native ranges. A study on thermal limits and metabolic rates showed that A .m. ligustica foragers are more tolerant to high temperatures than A. m. carnica (Kovac et al., 2007), suggesting that the Italian honey bee is better fit for warmer climates. In Saudi Arabia, several studies have compared the two commercial subspecies with the native A. m. jemenitica, which evolved in semi-arid and desert environments. Interestingly, all the studies showed that A. m. ligustica and A. m. carnica had lower heat tolerance and survival rates than A. m. jemenitica (Abou-Shaara et al., 2012; Alattal et al., 2015; Alqarni et al., 2019). In another study comparing the native subspecies of Algeria, A. m. sahariensis and A. m. intermissa, a differential response to heat stress was also found. Interestingly, A.m. sahariensis, the subspecies adapted to the desert environment with extreme temperatures, reacted better to heat than A.m. intermissa, the subspecies native to the southern shore of the Mediterranean, where the average temperatures are comparatively milder (Khedidji et al., 2024). As for interspecific variability, a study in China showed a higher temperature adaptability in Apis cerana than in A. mellifera. However, the contrary was observed on survival assays; under constant heat and humidity, A. mellifera showed higher survival rates than A. cerana (Li et al., 2019). These findings suggest that variations within the genus Apis must be studied to understand the different thermotolerances and metabolic responses to heat stress, as well as the expected differential transcriptional regulation. However, there are still many honey bee species and subspecies for which no information is available.

While temperature has been the main focus of climate change studies, changes in precipitation patterns are also key, not only because of water availability but also because of fluctuations in relative humidity (RH) (IPCC-Intergovernmental Panel on Climate Change, 2007). In A. mellifera colonies, a suitable RH of up to 75% is required for egg hatching (Ellis et al., 2008), and changes in RH can significantly affect larval development during incubation (Human et al., 2006) [reviewed by Abou-Shaara et al. (2017)]. In fact, when RH falls below 50%, there is a significant reduction in the number of normally hatched eggs (Li et al., 2016). Comparative studies of A. mellifera subspecies revealed that A.m. jemenitica eggs exhibit higher hatching rates than those of A.m. carnica at 50%–75% RH, while no eggs from either subspecies hatched at 30% RH (Al-Ghamdi et al., 2014).

The bees can regulate humidity within the colony by evaporating water from nectar and regurgitating liquid droplets, helping to restore favourable RH conditions while also aiding in thermoregulation (Heinrich, 1980; Human et al., 2006; Kovac et al., 2007). As for external conditions, no clear direct impact of RH in honey bees, including foraging activity, has been reported (Joshi and Joshi, 2010).

High humidity alone appears to have a limited impact on bees compared to high temperatures (Ma et al., 2019). However, rather than acting as an isolated stressor, humidity most relevant effect likely occurs in combination with temperature. These two variables are closely interconnected and, when combined, act as a new independent stressor. For example, low RH levels combined with high temperature can exacerbate heat stress, whereas high RH can reduce its severity. The highest rate of body water loss in A. mellifera occurs at 35 °C/0% RH, and the lowest occurs at 25 °C/75% RH and 30 °C/100% (Atmowidjojo et al., 1997). Studies on A. m. jemenitica and A. m. carnica showed that body water loss rates increased with higher temperature and lower RH, while higher RH generally improved workers’ survival (Abou-Shaara et al., 2012). In A. mellifera and A. cerana, high RH was shown to have a protective effect on bee survival under high temperatures, which may be due to the inhibition of body water loss (Li et al., 2019). Therefore, although the effect of RH on bees was previously considered negligible (Joshi and Joshi, 2010), it appears to become an important stress factor when it drops below 50% in warm environments, conditions increasingly common in arid regions under a climate change scenario.

The protective effect of high RH under high temperatures is also evident at the molecular level. Ma et al. (2019) demonstrated this in their study on A. mellifera when examining its responses to various combinations of temperatures and RH. The authors reported 434 differentially expressed genes (DEGs) under high-temperature treatments, 86 under high-humidity treatments, and 266 in combined high-temperature and humidity treatments. This suggests that high humidity reduces the expression of heat response genes by nearly half in a heat-stress environment. However, when focusing solely on molecular responses to humidity stress under optimal temperature conditions, the expression of genes such as Pla2 (phospholipase A2) and Afp (antifreeze protein) increases with increasing humidity (Table 1), starting at RH levels above 50% (Ma et al., 2019). Pla2 encodes a cell membrane enzyme (PLA2) that cleaves fatty acids. When its activity increases, the hydrolysis reaction is enhanced, producing a variety of fatty acids, disturbing cell membrane metabolism and nerve signal transmission (Mahalka et al., 2011). It is important to note that the PLA2 protein is involved in immunity through the arachidonic acid release in the eicosanoid pathway (McMenamin et al., 2018). There are 10 genes in the genome of A. mellifera referred to Pla2 (Supplementary Table S4). Afp encodes AFP, a proteinaceous compound with enhanced antifreeze properties, enabling it to bind to small ice crystals and inhibiting their growth and recrystallization. This mechanism helps minimize the damage caused by frozen water/ice to living organisms (Jia and Davies, 2002). There is one gene in the genome of A. mellifera referred to Afp (Supplementary Table S4).

Climate change has been linked to stratospheric ozone depletion, which increases the amount of ultraviolet-B (UV-B) radiation that reaches the Earth’s surface (IPCC-Intergovernmental Panel on Climate Change, 2007). This affects how organisms and ecosystems respond to it (Bernhard et al., 2020). UV radiation is well-known for its detrimental effect on organisms. Yet, it is the universal source of non-ionizing radiation and is essential for life and its development on Earth. According to CIE (Commission Internationale de l’Eclairage, International Commission on Illumination), UV radiation can be divided into three ranges: UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280 nm). Earth’s ozone layer blocks the majority of UVC, and a significant portion of UV-B, so the light near the Earth’s surface is enriched by UV-A, which has also been linked to oxidative types of mutation due to oxidative stress following irradiation [reviewed by Cockell and Knowland (1999)].

UV promotes photochemical reactions of ROS formation. In fact, one of the most susceptible biological targets of UV radiation is the DNA. When UV radiation enters a cell, it is absorbed by the aromatic rings of nucleotides and amino acids, leading to DNA and protein damage, respectively [reviewed by Cockell and Knowland (1999)]. However, it is important to note that repair mechanisms are not 100% efficient, so the lower the exposure to UV-radiation, the lower the damage and the greater the benefit to the organism [reviewed by Cockell and Knowland (1999)]. Hence, UV protection is key, particularly in a scenario of climate change with a prospective increase of UV radiation exposure.

Cells use a wide array of biological macromolecules to protect themselves against ROS by quenching oxygen free radicals. In insects, melanins are good examples of such macromolecules. Melanins can be classified into two groups: brown to black pigments termed eumelanin, and alkali-soluble yellow to reddish-brown pigments termed pheomelanin (Ito and Wakamatsu, 2008). They enhance the protective properties of the cuticle, acting as important barriers against environmental stressors such as UV radiation (Charlier et al., 2020). Melanins are produced by the enzymatic oxidation of tyrosine by tyrosinase followed by the conversion of dopa to 5,6-dihydroxy-indole. This is a phenolic and indolic compound and the basic building block of the eumelanin polymeric structure. This structure acts as the UV-absorbing chromophore [reviewed by Cockell and Knowland (1999)]. Recently, Charlier et al. (2020) used the electron paramagnetic resonance imaging (EPRI) technique to detect melanin in A. mellifera. They identified free radicals almost exclusively in the cuticle of the bee periphery of A. mellifera consistent with a eumelanin signal. This finding suggests that melanin–chitin complexes in the honey bee cuticle play a key role in UV defence (Charlier et al., 2020). Interestingly, these authors detected the presence of other free radicals in the centre of the honey bee head, suggesting the possible presence of neuromelanin in its brain, as in Drosophila (Barek et al., 2018).

At the molecular level, melanization is an immunological process that results from the combination of humoral and cellular processes that occur during encapsulation and healing to cope with non-pathogen-mediated and pathogen-mediated injuries. In the honey bee, and insects in general, melanization acts as an important cellular defence mechanism responsible for eliminating a large number of bacterial cells, parasites and xenobiotics (Eleftherianos et al., 2009; McMenamin et al., 2018). Simultaneously with the formation of melanin and its polymerization together with other proteins to encapsulate the invading agent, reactive oxygen and nitrogen intermediates are released, which collaborate with the destruction of the agent (Negri et al., 2016). Melanization is mediated by a humoral protein, prophenoloxidase (PPO) (González-Santoyo and Córdoba-Aguilar, 2012; Negri et al., 2019) and A. mellifera possesses only one PPO gene (Supplementary Table S5).

Several markers involved in temperature stress (see “Temperature” section) are also involved in UV stress (Table 1). For example, in A. cerana, the expression of DnaJA1, DnaJB12 and DnaJC8 are upregulated under exposure to both climatic stressors (Li et al., 2018a). Furthermore, as for temperature, sHSPs can develop the protection function under UV stress conditions not only in honey bees but also in other animals and even plants (Dasgupta et al., 1992; Waters et al., 2008). Although very few studies have focused on CRH-BP response to stress in invertebrates, it has been shown that A. cerana cerana subjected to UV radiation exhibited increased CRH-BP mRNA in the head in a time-dependent manner (Liu et al., 2011). On the other hand, it is worth noting that UV promotes photochemical reactions leading to ROS formation, so antioxidant enzymes are also involved in UV stress. Finally, the serine protease AccSp1 gene, which is directly involved in ROS metabolism, seems to play different roles in resistance to UV radiation in A. cerana (Gao et al., 2019).

Although the interactions between plants and their insect pollinators are often the result of a long history of co-evolution, climate change can quickly disrupt the timing of their life cycles by altering their phenology and distribution (Hughes, 2000; Scaven and Rafferty, 2013; Gérard et al., 2020). Changes in plant physiology under realistic climate change scenarios may alter flowering patterns and the duration and intensity of blooming (Scaven and Rafferty, 2013; Inouye, 2020). This disruption could lead to food shortages for pollinators and, in some cases, even contribute to their extinction (Memmott et al., 2007; Dalton et al., 2023).

The abundance and diversity of floral resources play a crucial role in honey bee health, as they significantly influence immune response (Alaux et al., 2010; Di Pasquale et al., 2013; Martelli et al., 2022; Corona et al., 2023). The survival of bee colonies relies heavily on the availability of pollen, which provides proteins, lipids, and micronutrients, and nectar, which supplies carbohydrates (Brodschneider and Crailsheim, 2010). In temperate climates, bees typically experience nutritional stress as winter approaches due to the diminishing availability of food resources (Mattila and Otis, 2007; Knoll et al., 2024). This issue has gained particular attention because many bees face low dietary diversity due to climate change (Goulson et al., 2015).

At the molecular level, bees that consume a protein-rich diet exhibit signs of anabolism, lipid metabolism, and an increased expression of genes encoding nursing-related proteins, such as vitellogenin and major royal jelly protein 1 (Bitondi and Simoes, 1996; Corona et al., 2007; 2023; Ament et al., 2008; Azzouz-Olden et al., 2018). In contrast, bees subjected to suboptimal nutrition, typically characterized by the intake of diets lacking proteins, display a reduction in nitrogen and lipid metabolism, reduction in antioxidant response, and different gene expression patterns (Martelli et al., 2022). These include altered expression in cuticle maturation genes, the over-expression of genes involved in the modulation of circadian rhythm and other genes related to the foraging behavioral state, such as those involved in the insulin/insulin-like growth signaling (IIS), juvenile hormone (JH) and Target of Rapamycin (TOR) (Figure 4) (Supplementary Table S6) (Corona et al., 2007; 2023; Ament et al., 2008; Azzouz-Olden et al., 2018; Martelli et al., 2022). These nutrient-sensing pathways include the Forkhead box O subfamily of gene (FOXO) [reviewed by Murtaza et al. (2017)], a transcription factor involved in multiple biological processes including the regulation of aging, nutrient levels response, and stress response (Hwangbo et al., 2004; Semaniuk et al., 2021a; Semaniuk et al., 2021b). FOXO proteins are known to regulate translation of environment-induced stimuli into gene expression and their antioxidant role [reviewed by Murtaza et al. (2017)]. In A. mellifera genome there is only one FOXO gene (Supplementary Table S6).

The connection between honey bee nutrition and behavioral development is further highlighted by the fact that nutritional stress is associated to precocious foraging (Fewell and Winston, 1992; Schulz et al., 1998), changes in colony demographics, and, ultimately, colony collapse (Perry et al., 2015). Research indicates that similarly to nutritional stress, thermal stress reduces the expression levels of nurse-associated genes (vg and mrjp1) and increases the expression of foraging-associated genes (e.g., juvenile hormone esterase) (Bordier et al., 2017; Corona et al., 2023) suggesting that thermal stress can induce the physiological changes linked to precocious foraging, potentially affecting the overall fitness of the colony. In support of this idea, it has been shown that bees raised under high temperatures exhibited an increased probability of dancing and foraging earlier in life (Becher et al., 2009).

In addition to the pathways that are directly or indirectly involved in stress management, cells have several general mechanisms that are activated in stressful situations, such as changes in RNA processing and epigenetic factors. They all act independently of the organism’s genetic background and directly influence its phenotypic plasticity (Figure 1), and, therefore, its resistance and adaptability to new environments. This section reviews the major ones in A. mellifera and their role in stress.

Immunity is one of the main physiological mechanisms that regulate the overall survival of an organism, and, therefore, immune stress requires a substantial nutritional and energetic cost [reviewed by Lochmiller and Deerenberg (2000)]. The immune system of honey bees consists of a complex set of individual immune mechanisms and a special type of behavioural adaptations that protect against biotic and abiotic stress factors. As social insects, at the colony level, honey bees exhibit a collective defence mechanism known as “social immunity,” while at the individual level, they rely on cellular and humoral immune reactions. Within humoral immunity, antimicrobial peptides (AMPs) play a key role (Danihlík et al., 2015). At the molecular level, several immune pathways have been described in A. mellifera. These are mainly Jak/STAT (Janus Kinase/Signal Transducer and Activator of Transcription), RNAi (RNA interference), Toll via NF-ĸΒ (Nuclear Factor ĸΒ/Dorsal), Imd (immune deficiency) via NF-ĸΒ/Relish, JNK (c-Jun N-terminal kinase), MAPK (Mitogen-Activated Protein Kinases), autophagy, eicosanoid biosynthesis, endocytosis, melanization, and Prophenoloxidase (PPO) (McMenamin et al., 2018) (Figure 7).

Most of the stressors described in this review, directly or indirectly can modify the expression of genes related to at least one of these pathways (Table 1). For example, the first step when pathogens enter honey bee is the host recognition pathogen-associated molecular patterns from the invading microorganisms (PAMP recognition), which rapidly promote the activation of an SP cascade (Figure 7) [reviewed by McMenamin et al. (2018)]. Serine proteases, which have been described related to heat stress (see section 4), are also directly involved in immunity, as they are key in melanisation, wound healing, and phagocytosis stimulation by participating in the prophenoloxidase (PPO) activation pathway, RNAi, and SP proteolytic cascade in the Toll signalling [reviewed by Lu et al. (2014), McMenamin et al. (2018)] (Figure 3). Three genes of SP putative substrates, PPO, spätzle-1, and spätzle-2, are described in the A. mellifera genome (Zou et al., 2006). However, Phenoloxidase has been also linked to heat stress, existing different levels of enzymatic activity in workers, queens and drones exposed to the same stressful situation during development (Medina et al., 2020).

Regarding heat, there is a close relationship between the iRNAs and HSPs in honey bees. The RNAi pathway is initiated by Dicer-2 cleavage of viral dsRNA into 21–22 bp siRNAs, which are then loaded into Argonaute-2 (Ago2), the catalytic component of the RNA Induced Silencing Complex (RISC). Within this route, a putative serine/threonine cyclin-dependent kinase (MF116383) acts in non-specific dsRNA-mediated antiviral responses [reviewed by McMenamin et al. (2018)] (Figure 7). McMenamin et al. (2020) conducted an in-depth study of A. mellifera, comparing the transcriptomic response to heat shock and viral infections. The mf116383 gene was the only immune-related gene of this pathway consistently induced by heat treatment alone and it has been suggested to serve as a point of cross-talk between the generalized antiviral immune response and the HSR in honey bees. On the other hand, the expression of Hsc70-3, Hsc70-4, and Hsp90 was found to be positively correlated with Dicer-like (Dcr-like) and Argonaute2 (Ago2), suggesting co-regulation of these genes (McMenamin et al., 2020). Cognate forms of Hsp70 are involved in regulating the life cycle of various viruses, such as mediating attachment and endocytosis (Iša et al., 2004), penetration and uncoating, transcription and replication (Du et al., 2011), assembly and budding (Prange et al., 1999), and modulating autophagy (Beere, 2004). HSC70-4 is an important chaperone for the assembly of the RISC in Drosophila S2 cells and other flies (Dorner et al., 2006; Iwasaki et al., 2010). While further studies are needed to determine the mechanisms leading to the co-regulation of immune genes and HSP-encoding genes, it may be beneficial to co-regulate HSPs and HSP client proteins (Iwasaki et al., 2010). Meanwhile, heat stress in honey bee colonies has been linked to a reduction in virus and parasite infections [reviewed by Zhao et al. (2021)]. Finally, RNAi is not only key to antiviral defence and closely related to HSR, but may also act as epigenetic factors. The RNAi pathways is constituted by piRNAs, miRNAs and siRNAs, and is involved in regulating gene expression in most organisms [reviewed by Ding (2010)] (see “epigenetics” subsection).

Within the Toll pathway (Figure 7), NF-ĸB transcriptional factors are crucial in immunity, inflammatory response, cellular adhesion, differentiation, proliferation, autophagy, senescence, and apoptosis (Bonizzi and Karin, 2004). In A. mellifera, NF-ĸB factors Dorsal are encoded by two dorsal homologues genes, Dorsal-1 and Dorsal-2 (Evans et al., 2006). Dorsal-1 produces two isoforms through alternative splicing (DORSAL-1A and DORSAL-1B) (Fan, 2001; Evans et al., 2006), and arguably this allows for a refinement of Toll-induced immune function. At this point, there is an important relationship between immunity and oxidative stress, consisting of both ROS influencing the activation of NF-κB pathway, and NF-κB pathway influencing the ROS levels by increasing the expression of antioxidant genes (Zhang et al., 2016). On the other hand, in adult bees, Dorsal genes are directly involved in the regulation of AMP genes together with Imd pathway (McMenamin et al., 2018), as Defensine1 (Lourenço et al., 2018) and Apidaecin (Prisco et al., 2016). The activation of both Toll and Imd pathways also governs the expression of Abaecin (McMenamin et al., 2018). Defensin genes have been described as predictive markers of honey bee colony health linked to viral infections (Barroso-Arévalo et al., 2019). Two Defensin genes are described in honey bees: Defensin1 and Defensin2. Sequences of these two genes revealed their different structure in a phylogenetic analysis, with Defensin1 forming a clade with two other hymenopteran defensins, whereas Defensin2 grouped with coleopteran defensins (Klaudiny et al., 2005). In contrast to other invertebrates, heat shock in A. mellifera represses the expression of certain antimicrobial peptide genes such as Hymenoptaecin, Defensin1 and Abaecin (McKinstry et al., 2017). This may be due to an energetic trade-off in the honeybee between cellular mechanisms for maintaining proteostasis and the immune response, as both are energetically costly (Powers and Balch, 2013).

The link between nutrition and immunity is well known. In fact, “nutritional immunology” is a discipline that studies the composition of the diet to optimise the immune response (Ponton et al., 2011). The interaction between nutritional and immune pathways is as wide as complex. For example, miRNAs have been described as acting on the insulin and TOR pathways (Shi et al., 2015), while PiP3, AKT and TOR inhibit autophagy (Figure 4) (McMenamin et al., 2018). On the other hand, inadequate nutrition stimulates adenylate kinase (Ak6) (involved in stress-induced pathways), NF-ĸB, and genotoxic/non-genotoxic stress (Hou et al., 2008; Barrio et al., 2014; Hasygar and Hietakangas, 2014; Corona et al., 2023). Furthermore, VG is involved in immunity in honey bees (Amdam et al., 2004). Recently, Azzouz-Olden et al. (2018) conducted an in-depth study comparing well- and poorly-fed honey bees between two groups comprising infected and non-infected individuals with the microsporidium Nosema apis. The authors showed that poorly fed and infected individuals underwent a significant upregulation of some genes related to the Toll pathway, such as Sph, serpine-1 (Nec), Tub, Cactus, PGRP-S3 and AMP genes (like Apisimin). On the other hand, during melanisation (see section 6), melanins are produced by the enzymatic oxidation of tyrosine by tyrosinase, which is the enzyme’s main activating system (PPO). Tyrosinase depends upon tyrosine, which derives from phenylalanine, an essential amino acid that can only be obtained from ingested food. In addition, the defensive compounds produced by the bees are rich in nitrogen, requiring a significant investment of their resources. Hence, the PO response involves a high energetic cost for each bee (González-Santoyo and Córdoba-Aguilar, 2012; Negri et al., 2019).

It is important to note that the PLA2 protein, involved in stress induced by humidity, is involved in immunity through the arachidonic acid release in the eicosanoid pathway (McMenamin et al., 2018).

Predicting how climate change will impact honey bees is complex and challenging. It will not only directly affect individual bees and colonies but also compound existing stressors, alter the ecosystems they inhabit, and introduce new and additional pressures. Higher temperatures, changes in humidity, and increased exposure to UV radiation will not only affect bee biology but also contribute to colony stress, alter plant flowering (and thus bee food resources), and influence disease dynamics. These factors can both benefit the pathogens under the new conditions and weaken the host organisms. In this review, we aimed to summarise a guide of molecular targets potentially linked to climate change stress, along with the cellular pathways directly involved or related. Genetic studies are a valuable tool for addressing these issues, and understanding the related mechanisms and pathways is essential for correctly interpreting molecular insights. Knowing which genes are involved in a specific pathway, what they are like, what their transcripts are like, how they express under stress, and how they affect other pathways, provides a basis for future studies aimed at measuring not only the degree of stress in honey bees but also its consequences for their cell biology.