Thermal adaptation involves higher expression levels of the lethal(2)-essential-for-life-like (l(2)efl) among the honeybee Apis mellifera L. subspecies

1 Introduction

The common honeybee Apis mellifera comprises many ecological subspecies that are highly adapted to their native habitat (Ruttner, 1988; Ilyasov et al., 2020). Yet, outside their natural distribution ranges, the survival of exotic A. mellifera subspecies is strongly impacted by extreme environmental conditions compared to the native ones (Ruttner, 1988). Under extreme desert conditions of Saudi Arabia, most colonies of the exotic A. mellifera subspecies (i.e. A. m. carnica and A. m. ligustica and their hybrids) die during the first summer season compared to the native honeybee subspecies A. m. yemenitica (Alqarni, 2006; Alattal and Alghamdi, 2015). In addition to high summer mortalities, A. mellifera winter survival is significantly influenced by previous summer temperatures “researchers refer to as “GoldiKocks” (Calovi et al., 2021; Insolia et al., 2022). In Saudi Arabia, the term over-summering describes the ability of the honeybee colonies to pass the extreme summer temperatures (Alattal and Alghamdi, 2015), a phenomenon that is restricted to regions with extreme summer temperatures. A. m. yemenitica is one of the most thermotolerant A. mellifera subspecies worldwide (Ruttner, 1988; Alqarni, 2006; Alattal and Alghamdi, 2015, Alqarni et al., 2017).

Many morphological, behavioral, and molecular aspects of A. m. yemenitica are key components of thermal adaptation. For example, smaller body size, cuticle anatomy and chemistry, efficient expression of HSPs, reduced metabolism and efficient water management are some aspects of A. m. yemenitica adaptive thermotolerance (Ruttner, 1988; Ali, 2011; Alattal et al., 2014; Alattal and Alghamdi, 2015; Porcelli et al., 2017; Alqarni et al., 2019; Alghamdi and Alattal, 2023). Additionally, as an eusocial insect, A. mellifera colony performs many cooperative functions such as fanning, migratory swarming, clustering, reduced foraging times, and hive cooling to withstand hot summer conditions (Southwick, 1983; Ruttner, 1988; Severson et al., 1990; Zhao and Jones, 2012; Zhao et al., 2021; Xinyu et al., 2023). On the other hand, beekeepers regularly employ management practices such as shading nets and providing colonies with sufficient water sources to alleviate losses during the summer months, which is considered the most critical period during the colony seasonal cycle under the desert climate of Saudi Arabia (Alghamdi and Nuru, 2013).

At the cellular level, heat shock proteins (HSP) are produced to keep protein integrity, a well-documented molecular mechanism of A. mellifera thermotolerance (Severson et al., 1990; Elekonich, 2009; Zhao and Jones, 2012; Perez and Aron, 2020). Molecular thermotolerance of A. m. yemenitica has been briefly discussed, HSP70 was reported as the main heat shock protein induced in A. m. yemenitica after exposure to heat stress with higher response thresholds compared to A. m. carnica and A. m. ligustica (Alqarni et al., 2019; Alghamdi and Alattal, 2023). A recent study reported significantly higher relative expression levels of HSP70, HSC70, HSP90, HSP83, HSP10, and HSP28 (mRNAs) in A. m. yemenitica than in A. m. carnica under the same conditions with an epigenetic layer of HSPs transcriptional regulation (Alattal and Alghamdi, 2015; Alattal and Alghamdi, 2023).

The lethal(2)-essential-for-life-like (l(2)efl) gene family encodes several cellular processes, including protein homeostasis and proteostasis (Kurzik-Dumke and Lohmann, 1995; Taylor, et al., 2014; Bakthisaran et al., 2015; Qin et al., 2019). The lethal(2)-essential-for-life-like (l(2)efl) gene family occurs in the perinuclear region of cytoplasm and is expressed in adult honeybee heads; hearts; mid-guts and in embryonic/larval muscle system (NCBI: https://www.ncbi.nlm.nih.gov). Higher levels of l(2)efl genes (724488, 724405, 724367, 724274, 724449, and 410087(a and b)) transcripts were documented in the mid-guts of A. mellifera adults exposed to 45 °C for 4 h, than in the control (Shih et al., 2021). After exposure to heat stress, A. m. carnica foragers exhibited much higher expression levels of l(2)efl mRNAs than in the control (McMenamin et al., 2020; Runtuwene et al., 2020). Yet, activation or silencing of HSP genes in A. mellifera is regulated by many factors and may require specific enzymes acting on histone remolding (Binda, 2013). Apis mellifera histone-lysine-N-methyltransferase trithorax (trx)) and Apis mellifera polycomb protein Su(z)12 are antagonistic regulators of gene transcription (Binda, 2013; Bakthisaran et al., 2015), the earlier (trx) modulates gene transcription, while the latter (Su(z)12) suppresses transcription, and their abundance is associated with the type of histone remodeling at target genes. Chromatin remolding and histone modifications are very common in A. mellifera and play a key role in many biological functions such as cast differentiation (Dickman et al., 2013; Allis and Jenuwein, 2016) and HSP expression (Alattal and Alghamdi, 2023).

In this study a day-long expression levels of three l(2)efl genes (724405; 724488; 234274), Apis mellifera histone-lysine N-methyltransferase trithorax (trx)) and Apis mellifera polycomb protein Su(z)12 (mRNAs) were measured and compared in A. m. yemenitica (thermotolerant subspecies) and A. m. carnica (thermosusceptible subspecies) under natural heat stress of desert and semiarid conditions of Saudi Arabia.

2 Materials and methods

3 Results

The results revealed highly significant differences in expression levels of the lethal(2)-essential-for-life-like (l(2)efl) genes (ID: 724405; 724488; 234274) and Apis mellifera polycomb protein Su(z)12 (ID: 409170) in A. m. yemenitica compared to A. m. carnica (Table 3). The expression of the histone-lysine N-MT (trx) (ID: 408716) was also increased in A. m. yemenitica (Table 3). The highest average fold change was reported for l(2)efl (ID:724488) in A. m. yemenitica located in Riyadh with ∼100× more expression folds compared to that of A. m. carnica in Baha (calibrator) (Fig. 1). The results also showed that absolute expression levels of l(2)efl (ID: 724405; 724488; 234274) in A. m. carnica were very modest (Fig. 1) under the study conditions.

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Fig. 1

Fold changes of (mRNA) relative expression levels for Apis mellifera lethal(2)-essential-for-life-like (l(2)efl) genes (ID: 724405; 724488; 234274); Apis mellifera histone-lysine N-MT (trx) transcript variant X4 (ID: 408716), and Apis mellifera polycomb protein Su(z)12 (ID: 409170) in A. m. yemenitica (local) and A. m. carnica (carnica) at two thermogeographical regions (Riyadh: R and Baha: B) and the three foraging times (7:00 a.m.: after ≈1 h of foraging; 12:00 p.m.; and 5:00 p.m. (≈1 h before sunset)). Δct was calculated by using actin as endogenous control, and the fold change (fold change = 2^{ct (reference-treatment)}) was calculated using the relative expression in A. m. carnica in the Baha region as reference (Calibrator). Significant fold change difference was determined based on average gene expression of each group using a three-way ANOVA analysis followed by Multiple comparisons based Bonferroni post hoc test, p < 0.05. (*) = significant variation and (ns) = non-significant variation.

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When comparing data by locations (Riyadh and Baha), the results likewise revealed significantly higher relative fold changes in expression levels for all l(2)efl genes (ID: 724488; 724405; 234274) under desert conditions (Riyadh) than in semi-arid conditions (Baha) (Table 3). The interaction between subspecies and location was significant for l(2)efl (ID: 724405; 234274) and Apis mellifera polycomb protein Su(z)12 (ID: 409170) (Table 3). Under Riyadh climatic conditions mean fold changes were significantly higher in A. m. yemenitica than in A. m. carnica for all genes under study (Fig. 1, Table 4). Under semiarid conditions of Baha, mean fold changes of genes were significantly higher in A. m yemenitica than in A. m. carnica except for Apis mellifera histone-lysine N-MT (trx) (ID: 408716) (Table 5). Although differences in expression fold changes between A. m. yemenitica and A. m. carnica within the same locality were significant, differences in fold changes between both subspecies were much higher under desert conditions (Riyadh) (Fig. 1).

Under the pertaining temperatures at the three sampling time periods (In Riyadh, temperatures were 31, 42 and 39 °C, In Baha, they were 22, 30 and 25 °C; in the morning, mid-day and evening, respectively), fold changes in expression levels were higher in A. m. yemenitica than in A. m. carnica at all time periods (Fig. 1). Relative expression levels (increase/decrease) were not consistent for either subspecies with the sampling time (Fig. 1). For two genes (ID:724405 ID: 408716) the highest expression levels occurred in the morning while for others at mid-day (ID:724488 and ID:724274) or evening (ID: 409170). Nevertheless, the maximum fold change among all time periods occurred for l(2)efl (ID:724488).

4 Discussion

The higher expression levels of Apis mellifera lethal(2)-essential-for-life-like (l(2)efl) genes (724405; 724488; 234274) in A. m yemenitica than in A. m. carnica under extreme temperature conditions (Riyadh) can be allied with the thermal adaptation of A. m. yemenitica. Above that, the daily variation in relative expression levels of l(2)efl (ID: 724405; 724488; 234274) between A. m yemenitica and the calibrator (A. m. carnica in Baha) were very high (∼100×), which demonstrate an efficient response and revealed robust transcriptional activation of these genes in A. m. yemenitica under natural thermal stress of Riyadh. Additionally, these fold changes and expression levels were as high as that of Hsp70 mRNA and other constitutively expressed Hsps reported in a previous study (done at the same locations) for both subspecies (Alqarni, et al., 2019; Alghamdi and Alattal, 2023). Consequently, these higher expression levels of l(2)efl (ID: 724405; 724488; 234274) genes can be considered key elements of survival for A. m. yemenitica under desert conditions of Riyadh.

Under semi-arid conditions of Baha, differences in expression levels between both subspecies were very modest indicating minimal stress-inducing conditions being a more suitable habitat for A. m. carnica than Riyadh. Nevertheless, significant variation in fold changes of expression levels (mRNAs) between both subspecies under Baha conditions might indicate a quicker and earlier response in A. m. yemenitica to accommodate expected changes in daily ambient temperature. Generally, the real forager body temperature is higher than the ambient temperature in many foraging insects, which is connected with foraging distance as well (Kovac et al., 2014; Davis and Moyle, 2020). The significant interaction between subspecies and location indicates that A. m. carnica had a severe locality disadvantage at Riyadh, but only minimal disadvantage at Baha, this may explain -in part- previously published data on survival rates of both subspecies under Riyadh and Baha regions, reporting that most of A. m. carnica colonies in Riyadh died within the first summer season of establishment (Alattal and Alghamdi, 2015). Consequently, keeping A. m. carnica under Baha conditions can be more reasonable than in Riyadh.

The increase/decrease of expression levels was not consistent with daytime. This was clearer in the Riyadh region which may indicate continuous adjustment of l(2)efl transcription responding to daily dynamics in ambient temperatures. Indeed, there is no data on the response threshold associated with l(2)efl gene expression under daily temperature stresses among different honeybee subspecies. Nevertheless, we cannot predict if the thermo-tolerance of A. m. yemenitica will be impacted by the global intensification of climate change including Saudi Arabia (Chen et al., 2011). Likewise, relative expression levels of A. mellifera polycomb protein Su(z)12 (ID: 409170) and Apis mellifera histone-lysine N-MT (trx) (ID: 408716) were higher in Riyadh than in Baha, which can reflect an epigenetic layer of regulation on active transcription of l(2)efls associated with heat stress. We recommend investigating expression thresholds of l(2)efl genes among different A. mellifera subspecies and try to explore how thermotolerance impacts honeybee productivity under different heat regimes. Many reports documented higher productivity but lower survival rates of exotic honeybee subspecies compared to A. m. yemenitica under hot and dry conditions in Saudi Arabia (El-kazafy and Al-kahtani, 2019), which entails the need for selection and breeding programs toward increasing productivity without impacting the thermo-adaptability of A. m. yemenitica.

5 Conclusion

In conclusion, expression of l(2)efls (mRNAs) (724405; 724488; 234274) genes can be considered a key component of A. m. yemenitica survival adaptation under extreme summer temperatures characterizing the Riyadh region. On the other hand, the semi-arid conditions of the Baha region are a more suitable habitat for A. m. carnica compared to Riyadh.

CRediT authorship contribution statement

Yehya Zaki Alattal: Conceptualization, Funding acquisition, Data curation, Writing original draft, Writing review & editing, Visualization, Investigation, Validation, Formal analysis, Methodology, Supervision, Resources, Project administration, Software. Ahmad Abdallah Alghamdi: Conceptualization, Funding acquisition, Data curation, Writing original draft, Writing review & editing, Visualization, Investigation, Validation, Formal analysis, Methodology, Supervision, Resources, Project administration, Software.