Differential Expression of Endocrine Regulatory Genes in Apis cerana and Apis mellifera (Hymenoptera: Apidae) at High Temperature
Drastic changes in temperature can interfere with the normal physiological and biochemical activities of bees. Temperature stress affects the endocrine system of bees and induces a series of stress responses. However, the changes that occur in hormones in bees that are exposed to environmental stress are not well understood. In this study, we analyzed the expression patterns of four genes by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) in Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) at different temperatures and different treatment times. The expression of juvenile hormone esterase, vitellogenin, corticotropin-releasing hormone binding protein, and adipokinetic hormone receptor genes was found to be increasingly affected by an increase in temperature and treatment time. Temperature stress affects the endocrine system of bees, and endogenous hormones in bees can respond to environmental stimuli. Our findings provide a basis for determining the mechanisms by which insect endocrine systems adapt to high temperatures.Abstract
Temperature stress greatly affects physiological and biochemical activities of insects (Hamblin et al. 2017, Daniel et al. 2020). Insect endocrine molecules and neuropeptides secreted by the nervous system control all aspects of insect life, and temperature stress is known to affect the insect endocrine system (Nässel et al. 2019). The physiological response to stress has been studied in bees (Hymenoptera: Apidae) in which a variety of hormones can respond to stress and involve biogenic amines (octopamine and dopamine) and metabolic hormones (juvenile hormone [JH], adipokinetic hormone [AKH], and Corticotropin-releasing hormone [CRH]) (Even et al. 2012). The reaction of the honeybee endocrine system to stress is an important physiological response.
JH is one of the most important endogenous hormones in insects. Its main function is to upregulate the synthesis of the preyolk protein of the adult female egg yolk precursor (Roy et al. 2018). In the highly social Hymenoptera bees, JH is an important endogenous hormone that regulates the development, metamorphosis, and reproduction of bees (Fahrbach and Robinson 1996). Studies have shown that diseases, hunger, and other stimuli can alter JH levels in worker bees (Goblirsch et al. 2013). Furthermore, bee genotypes with low JH reactivity show a reduced stress response and increased stress susceptibility (Ihle et al. 2010). These findings indicate that insect JH signaling plays an important role in stress response. JH-specific esterase (JHE) binds to JH with high affinity; it is the JHE activity that transforms JH-III of bees into the biologically inactive JH acid. Control of JH titer in the hemolymph is mainly regulated by JHE, and its activity is commonly considered to reflect that of JH (Mackert et al. 2008).
Vitellogenin (Vg) is a precursor of egg yolk protein produced by fat bodies. Vg is not only important for bee reproduction but also involved in their immune response (Harwood et al. 2018) and antioxidative stress response (Salmela et al. 2016). RNA interference experiments showed that this protein can reduce workers' JH levels and delay foraging behavior, enhance immunity, and increase antioxidant function to prolong the life of workers (Nelson et al. 2007). The regulation of the Vg gene in bees is not fully understood but is known to be positively affected by nutrients and hemolymph amino acids and negatively affected by JH and stress (Nilsen et al. 2011).
CRH is a neuroendocrine peptide expressed in brain neurons and plays a key role in the neurotransmission and neuroendocrine regulation of stress responses (Vuppaladhadiam et al. 2020). CRH Binding protein (CRH-BP) is considered an antagonist of the CRH signaling pathway, which binds CRH with high affinity to regulate the biological activity of CRH (Huising et al. 2004). Under stress stimulation, CRH binds to conserved CRH-BP. Recent experiments have shown that the CRH-BP gene is involved in the stress response of bees under various stresses. For example, the expression of the AccCRH-BP gene in the bee head was observed under ultraviolet light, heat, and cold stresses (Liu et al. 2011). Synaptic tissue in the adult bee brain is likely affected by temperature.
AKH is released from the neurosecretory cells of the heart and enters the hemolymph, accelerating catabolism in the fat body and leading to an increase in circulating carbohydrates in response to starvation or acute energy stress (Ahmad et al. 2019). It can mobilize energy reserves to counter immediate pressure problems and curb processes that are temporarily less time sensitive (Ibrahim et al. 2017). Some studies supported the activation of a non-energy-consuming pathway involving the AKH gene, which can help insects resist stress (Tang et al. 2020). Insecticides can cause oxidative stress in insects and increase AKH titers in the hemolymph, which is thought to initiate antioxidant processes (Velki et al. 2011). Because AKHs do not penetrate the cell membrane as peptide hormones, they must transmit information through the specific membrane receptor AKH receptor (AKHR) (Lu et al. 2018). This receptor is linked to the G protein and activates multiple biochemical pathways (Gäde and Auerswald 2003). Thus, its activity is typically used to evaluate AKH activity.
In recent years, numerous studies on insect endocrine and nervous systems have revealed the structure and function of hundreds of neuropeptides. Various endocrine substances have been shown to play a vital role against stress in insects. However, corresponding research on the environmental pressure on the bee endocrine system remains limited. Apis cerana F. and Apis mellifera L. are most widely reared and distributed, and they have closer kinship, but there are differences in the native ecological environment between the two. Therefore, we used A. cerana and A. mellifera as experimental materials to investigate the expression patterns of four genes, namely, JHE, Vg, CRH-BP, and AKHR, under high-temperature stress to understand the potential role of these genes in the resistance of honeybees to heat stress and the differences between the two.
Materials and Methods
Bees. We separately selected three healthy colonies of A. cerana and A. mellifera and one to two capped brood combs from each colony, which were placed in an incubator at 34 ± 0.5°C and 75 ± 5% relative humidity (RH). After emergence from the cell, the bees were marked on the back with nontoxic, odorless paint, returned to the original colony, and then sampled at the age of 20 d.
Temperature treatment. Bees can keep the temperature stable in the colony, whereas foraging is a huge challenge for bees because it usually occurs in a variable environment. Bees are more susceptible to heat stress when they go out to forage, so we chose 20-d-old workers for the study. Therefore, we chose a normal outdoor temperature of 25°C as the control temperature. Generally, when the temperature is above 40°C, bees stop going out to forage, so we set 45°C as the maximum temperature of the test. On the other hand, considering that higher humidity will inhibit the loss of water on the surface of bees and increase heat stress, we chose a RH of 30% to reduce the effect of humidity on the results.
Six hundred adults (200/hive) were collected, and each individual bee was placed into 15-ml centrifuge tubes with small pores. Bees were then divided into 5 groups (20/group/hive) and treated for 2 h at 25°C, 30°C, 35°C, 40°C, and 45°C with RH fixed at 30%. Similarly, 5 groups of bees (20/group/hive) were treated at 45°C and 30% RH for 0, 0.5, 1, 1.5, and 2 h, respectively. We randomly selected five bees as a biological replicate from each group and each hive with three replicates for each treatment. These samples were frozen in liquid nitrogen and stored at –80°C for RNA extraction.
RNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time polymerase chain reaction (PCR). Total RNA was extracted from the entire tissue using TRIzol Reagent (Ambion, Foster City, CA) according to the manufacturer's instructions. RNA degradation and contamination of each sample were monitored by separating the samples on a 1.5% agarose gel. The RNA concentration was determined using a Qubitl RNA Assay Kit and Qubitl 2.0 fluorometer (Life Technologies, Carlsbad, CA).
First-strand cDNA was synthesized from 1 µg of total RNA using the ReverTra Ace-a First Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan). Primers for quantitative reverse transcription PCR (RT-qPCR) were designed using Primer 3 software. Primers designed for each gene are listed in Table 1. The qRT-PCR thermal cycle procedure was as follows: 95°C for 30 s followed by 45 cycles of 95°C for 5 s and 60°C for 30 s. All samples were tested in triplicate. The relative quantification of PCR results was conducted using the 2–ΔΔCT method. The β-actin gene was used as an internal standard. This experiment was treated at 25°C as a control group.
Statistical analyses. The study was designed using the principle of complete randomization. The data were statistically analyzed by analysis of variance (ANOVA), and the means were compared by the least significant difference test (P < 0.05).We used ANOVA to analyze the data and performed normality tests and isovariance tests, and for data that failed these tests, we used the Kruskal-Wallis H test (P < 0.05).
Results
High temperature effect on JHE expression. Worker bees (20-d-old adults) were exposed to temperatures of 25°C, 30°C, 35°C, 40°C, and 45°C for 2 h. For A. mellifera, the expression was highest in the 40°C treatment group. The results with A. cerana showed that JHE expression was higher at 40°C and 45°C (Fig. 1).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
For different treatment times at high temperatures, longer-term high temperature stress can cause JHE expression to increase. For A. mellifera, the expression level for the 2-h treatment time was significantly higher than that for 0 h (F = 36.626; df = 4; P < 0.001). The results for A. cerana were similar to those for A. mellifera, with the highest JHE expression level at 2 h (Fig. 2).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
High temperature effect on Vg expression. High temperatures upregulated the expression of Vg compared to the expression at 25°C. For A. mellifera, the expression level at each temperature was higher than that at 25°C, and the expression level was highest in the 35°C treatment and was significantly higher than the 25°C treatment (F = 19.177; df = 4; P < 0.001). The results with A. cerana showed that Vg expression after treatment at 40°C and 45°C was significantly higher than that of the 25°C treatment (F = 66.169; df = 4; P40 = 0.038 and P45 = 0.005) (Fig. 3).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
For different treatment times at high temperatures, longer-term high temperature stress can cause Vg expression to increase. For A. mellifera, the expression levels at 2 h were higher than that at 0 h (F= 19.667; df = 4; P= 0.004). The results with A. cerana were similar to those with A. mellifera, and Vg expression levels at 2 h were significantly higher than the 0-h treatments (F = 69.878; df = 4; P = 0.044) (Fig. 4).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
High temperature effect on CRH-BP expression. The expression pattern of CRH-BP showed that increased temperatures upregulated gene expression compared to treatment at 25°C. For A. mellifera, the expression levels at 30°C, 40°C, and 45°C treatments were significantly higher than that at 25°C (F = 16.191; df = 4; P30 = 0.003, P40 = 0.038, and P45 < 0.001). The results with A. cerana showed that CRH-BP expression was upregulated with increasing temperature. The expression levels at 35°C and 40°C were significantly higher than that at 25°C (F = 43.307; df = 4; P35 < 0.001 and P40 < 0.001) (Fig. 5).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
For different treatment times at high temperatures, CRH-BP expression was upregulated after 0 h. For A. mellifera, the expression levels at 1.5 and 2 h were significantly higher than that at the 0-h treatment (F = 63.434; df = 4; P1.5 = 0.002 and P2 = 0.023). For A. cerana, the expression levels at 1.5 h were significantly higher than that at 0 h (F = 19.588; df = 4; P < 0.001) (Fig. 6).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
High temperature effect on AKHR expression. The expression pattern of AKHR showed that higher temperatures upregulated gene expression. For A. mellifera, the expression level at 30°C and 45°C was significantly higher than that at 25°C (F = 6.403; df = 4; P30 = 0.007 and P45 = 0.004). The results with A. cerana were similar to those of A. mellifera, in which the expression level at 40°C and 45°C was significantly higher than that for the 25°C treatment (F = 40.482; df = 4; P40 < 0.001 and P45 = 0.001) (Fig. 7).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
For different treatment times at high temperatures, AKHR expression was upregulated after 0 h. For A. mellifera, the expression levels at 1.5 h were significantly higher than those after other treatment times (F = 7.177; df = 4; P0 = 0.001, P0.5 = 0.035, and P1 = 0.03). For A. cerana, the expression levels at 1 h, 1.5 h, and 2 h were significantly higher than that of the 0-h treatment (F= 10.866; df = 4; P1 = 0.001, P1.5 = 0.003, and P2 = 0.005) (Fig. 8).
Citation: Journal of Entomological Science 57, 3; 10.18474/JES21-78
Discussion
When faced with stress factors, bees must regulate body metabolism to cope with environmental stress. These physiologically rapid changes are achieved by coordinated endocrine and neuroendocrine responses (Even et al. 2012). Temperature greatly influences the behavior and physiological changes of insects. We examined the expression patterns of several endocrine regulatory genes of A. cerana and A. mellifera at high temperatures. The genes examined have important effects on bee metabolism under high temperature stress. The aim of this study was to understand the role of these hormones in heat shock resistance.
JHE, which participates in JH metabolism, plays an important role in the behavioral differentiation and reproduction of bees (Diana et al. 2021), and Vg plays an important role in bee reproduction and antioxidative stress (Awde et al. 2020, Park et al. 2018). An antagonistic relationship exists between two, which together regulate the physiological metabolism of bees (Azevedo et al. 2016). Recent studies showed that JH and Vg play important roles in the stress response of bees. This is closely related to the antioxidant capacity of bee Vg. Previous studies showed that increased JH reduces tolerance to oxidative stress and shortens the lifespan of insects (Salmon et al. 2001). Our experiment also seems to confirm their results that high temperature will increase the expression of JHE and Vg. This result may be that high temperature causes oxidative stress in bees and Vg produced by bees can be used as an antioxidant to help bees resist oxidative damage (Seehuus et al. 2006). As Vg increases, JH titer decreases and, correspondingly, JHE expression increased. This suggests that these two genes are involved in resisting heat stress. Changes in Vg and JHE gene expression at high temperatures may be involved in the adaptation of bees to environmental stress during long-term evolution.
During evolution, highly conserved signaling molecules developed to respond to stress responses. As an important neurotransmitter, CRH plays an important role in coordinating various responses to stress (Vuppaladhadiam et al. 2020). In stress stimulation, CRH often coexists with conserved CRH-BP. The experimental results showed that high temperature can increase CRH-BP expression. Our results showed that high temperature affects the CRH of bees. This result is similar to those of previous studies, showing that A. cerana (AccCRH-BP) is expressed in the brain at higher levels and upregulated after application of various acute stressors, such as ultraviolet, heat, or cold stress (Liu et al. 2011). These results indicate that upregulating CRH-BP may protect against stress, although additional studies are needed to confirm this hypothesis.
The energy metabolism of bees is mainly controlled by AKH (Ahmad et al. 2019). Recent studies showed that AKH participates in stress reactions that do not include energy expenditure. Various environmental stresses can increase AKH titers in insects. Interestingly, these stressors also cause oxidative stress. Additionally, injection of exogenous AKH can activate antioxidant mechanisms to reduce damage caused by oxidative stress. This suggests that AKH is involved in activating antioxidant protection mechanisms (Andrea et al. 2015). The mode of action is unclear, but oxidative stress may lead to increased AKH. Our experimental results also demonstrate that high temperature increases the expression level of AKHR. This is probably due to the increased energy requirements of bees for metabolic regulation at high temperatures, and oxidative stress caused by high temperature can also lead to increased AKH.
Rapid physiological changes in bees are achieved through coordinated endocrine and neuroendocrine responses. Comparing the differences between the two bee species, their related gene expression trends are similar after being exposed to high temperature stress. This is consistent with the fact that the two bees are closely related. However, for Vg, CRH-BP and AKHR, the effect of high temperature on A. cerana seems to be greater. Considering that the temperature and humidity of the A. cerana native environment are relatively high, this may indicate that A. cerana may be more adaptable to high-temperature climates than A. mellifera. In our results, the expression levels of some genes did not increase linearly with the increase of temperature or time, which may be caused by the different genetic backgrounds of the three populations. In addition, combined with the previous high temperature survival experiment of bees, some bees died or vitality decreased at 45°C, indicating that the high temperature pressure has caused great harm to the body of bees at this time, which may be an important reason for the decreased expression levels of some genes at 45°C and 2 h. Considering these levels, more in-depth research is needed to further reveal how bees respond to thermal stimuli at the hormone titers and neurotransmitter levels, improving our understanding of the relevant physiological pathways.
Expression pattern of JHE at different temperatures was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different temperatures. Data are mean ± SEM.
Expression pattern of the JHE at different treatment times was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different treatment times. Data are mean ± SEM.
Expression pattern of the Vg at different temperatures was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different temperatures. Data are mean ± SEM.
Expression pattern of the Vg at different treatment times was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different treatment times. Data are mean ± SEM.
Expression pattern of the CRH-BP at different temperatures was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different temperatures. Data are mean ± SEM.
Expression pattern of the CRH-BP at different treatment times was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different treatment times. Data are mean ± SEM.
Expression pattern of the AKHR at different temperatures was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different temperatures. Data are mean ± SEM.
Expression pattern of the AKHR at different treatment times was determined by real-time qRT-PCR. (A) Apis cerana. (B) Apis mellifera. The different letters indicate significant differences at different treatment times. Data are mean ± SEM.
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