Abstract
Queen honey bees (Apis mellifera) have a much longer lifespan than worker bees; however, the longevity-promoting mechanisms of queen bees are still unclear. Assaying cellular activities can explore the longevity-promoting mechanisms of queen bees because the longevity of individuals is based on the longevity of their cells. In this study, NAD+ levels, NAD+/NADH ratio, ATP levels, AMPK activity, lysosome activity, and ribosomal protein S6 mRNA levels were assayed to evaluate whether queen bees have higher cellular activities than worker bees. The results showed that the trophocytes and oenocytes of queen bees have higher cellular activities than that of worker bees. To explore which signaling pathway increases these cellular activities, the cAMP concentration and the mRNA levels of AC, PKA, RAS, MEK, and RSK were assayed. The results showed that the trophocytes and oenocytes of queen bees exhibit higher cAMP concentration and mRNA levels of AC, PKA, RAS, MEK, and RSK than that of worker bees. Combining these findings suggested that the cAMP/PKA and RAS/MAPK signaling pathways may increase cellular activities leading to the longevity of queen bees.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
1 Introduction
Queen and worker honey bees (Apis mellifera) are females and share the same genome; however, the lifespan of queen bees is much longer than worker bees. Queen bees live in the hive except during mating flights and colony fission and lay 1000–2000 eggs per day throughout their lives (Bodenheimer 1937). Worker bees spend only the first two weeks of life in the hive and perform tasks such as comb-building, cell-cleaning, and brood-nursing before transitioning to the foraging stage. In the foraging stage, worker bees collect nectar and pollen for the hive (Neukirch 1982). The difference in lifespan between queen and worker bees is mainly related to food intake. Queen bees feed on royal jelly (RJ) throughout their lives, whereas worker bees consume honey and pollen (Winston 1987).
The aging and longevity of individuals are based on the aging and longevity of their cells. In other words, studying the causes of aging and longevity of cells can clarify the aging and longevity of the individual. Trophocytes, which are large and irregularly shaped, and oenocytes, which are small and spherical, attach to form a single layer of cells around each segment of the honey bee abdomen. They are so tight that they cannot be separated. The biological functions of trophocytes and oenocytes are analogous to the white adipose tissue or liver of vertebrates (Law and Wells 1989). Trophocytes and oenocytes have been used for studying the mechanisms of aging and longevity due to the ease of isolation from the abdomen, convenient manipulation, and lack of cell division during adulthood (Hsieh and Hsu 2011a, b).
The cellular activities of trophocytes and oenocytes of worker or queen bees have been assayed, respectively, but those between worker and queen bees are unknown. The cellular activities include mitochondrial energy utilization activity, cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity (Chuang and Hsu 2013; Hsu and Lu 2015; Hsu and Chuang 2014; Hsu and Hu 2014; Hsu et al. 2014, 2016). In this study, the cellular activities in the trophocytes and oenocytes of young and old worker bees and young and old queen bees were assayed to reveal whether queen bees have higher cellular activities than worker bees. Furthermore, which signaling pathway triggers the cellular activities in the trophocytes and oenocytes of queen bees were determined.
2 Materials and methods
2.1 Honey bees (Apis mellifera)
The brood combs containing pupae and a few emerged worker bees from the different colonies were transferred to an incubator (34 °C, 75% relative humidity). Seventy newly emerged worker bees were collected in a cage (15 × 10 × 12 cm), put into a 34 °C thermostat (NK system, Nippon, Japan), and daily fed honey and fresh pollen grains mixed with honey (3:1). Young (5 days old) and old (30 days old) worker bees were used for the following studies; thus, the difference between them was only age (Hsu and Chan 2013). Young (2-month-old) and old (16-month-old) queen bees were collected from different hives for the following experiments. The young and old queen bees were mated with drones and were able to lay eggs. Young and old worker bees and young and old queen bees were collected on the same dates for subsequent experiments.
2.2 NAD+ and NADH concentration
NAD+ and NADH levels were measured using a NAD/NADH quantification kit (K337-100; BioVision, Mountain View, CA, USA) (Chuang and Hsu 2013; Hsu and Lu 2015). Bees were anesthetized on ice. The abdomens were collected and dissected with scissors. Their digestive tracts, trachea system, and nerve system on the abdomen were carefully removed. The cuticle of the abdomen was washed with phosphate-buffered saline. Trophocytes and oenocytes were detached from the cuticle of the abdomen using a knife. Trophocytes and oenocytes from two young or old worker bees or one young or old queen bee were extracted with NADH/NAD extraction buffer. The extracted samples of queen bees were diluted to obtain a similar protein concentration as worker bees because the cells of queen bees had higher protein concentration than that of worker bees and the protein concentration interfered with the measurement using the spectrometer. The dilution factor was used for the value calculation. The extracted samples were heated for NADH measurement. After background luminescence was recorded, the extracted samples and the extracted and heated samples were measured. The resultant luminescence was normalized to the background. Dilutions of an NADH standard solution (0, 20, 40, 60, 80, and 100 pmol l−1) were processed concurrently to generate a standard curve, which was used to obtain NAD+ and NADH values. The NAD+ and NADH concentrations were expressed as nanomoles mg−1 of protein and were used to determine the NAD+/NADH ratio. This experiment was performed with five biological replicates simultaneously using a total of ten young and ten old worker bees and five young and five old queen bees.
2.3 ATP concentration
ATP concentration was determined using an ATP determination kit (A22066; Invitrogen, Carlsbad, CA, USA) (Chuang and Hsu 2013; Hsu and Lu 2015). The manipulation of trophocytes and oenocytes was as described above. Trophocytes and oenocytes from two young or old worker bees or one young or old queen bee were homogenized in phosphate buffer containing protease inhibitors and centrifuged to obtain the resulting supernatant. The resulting supernatants of queen bees were diluted to obtain a similar protein concentration as worker bees. The dilution factor was used for the value calculation. After recording background luminescence, diluted ATP standard solution (1, 10, 100, 1000, and 5000 nM) or supernatant was added to the standard reaction solution to measure values. The ATP concentration was expressed as nanomoles per milligram of protein. This experiment was performed with five biological replicates simultaneously using a total of ten young and ten old worker bees and five young and five old queen bees.
2.4 AMPK activity
AMPK activity was evaluated using the CycLex AMPK kinase assay kit (CY-1182, CycLex, Nagano, Japan) (Hsu and Chuang 2014; Hsu and Hu 2014). The manipulation of trophocytes and oenocytes was as described above. Briefly, trophocytes and oenocytes were isolated from two young or old worker bees or one young or old queen bee, homogenized with a blue pestle and sonicator in 50 μl of cell lysis buffer containing protease inhibitors (11,697,498,001; Roche Applied Science, Indianapolis, IN, USA) and centrifuged at 5000 g for 10 min at 4 °C to obtain the supernatant. The supernatants of queen bees were diluted to obtain a similar protein concentration as worker bees. The dilution factor was used for the value calculation. The supernatant (10 μl) and kinase reaction buffer (90 μl) were applied to each well of a 96-well plate and incubated for 30 min at 30 °C. After washing, 100 μl of the anti-phospho-mouse IRS-1 S789 monoclonal antibody was added and incubated for 30 min at room temperature. After washing, 100 μl of HRP-conjugated anti-mouse IgG was added and incubated for 30 min at room temperature. After washing, 100 μl of substrate reagent was added and incubated for 15 min at room temperature. Finally, 100 μl of stop solution was added to stop the reaction. The absorption was measured at 450 nm using an ELISA plate reader (Synergy HT, BioTek, VT, USA). The AMPK activity was expressed as the absorbance at 450 nm mg−1 of protein. This experiment was performed with five biological replicates simultaneously using a total of ten young and ten old worker bees and five young and five old queen bees.
2.5 Lysosomal activity
The acid phosphatase activity was used to evaluate the lysosomal activity because acid phosphatase is one of the acid hydrolases in the lysosome. The acid phosphatase activities in the trophocytes and oenocytes of two young or old worker bees or one young or old queen bee were determined using the acid phosphatase assay kit (CS0740; Sigma, Saint Louis, MO, USA) (Hsu et al. 2014, 2016). Fifty microliters of the supernatants (described in AMPK activity) (replaced with citrate buffer for the blank samples) and 50 μl of the substrate solution were mixed and incubated for 10 min at 37 °C. The reactions were stopped by adding 0.2 ml of stop solution. The absorption was measured at 405 nm using an ELISA plate reader, and the specific activity was expressed as milliunits per milliliter. One unit of acid phosphatase hydrolyzed 1 μmol of 4-nitrophenyl phosphate per minute at pH 4.8 and 37 °C. This experiment was performed with five biological replicates simultaneously using a total of ten young and ten old worker bees and five young and five old queen bees.
2.6 Quantitative real-time polymerase chain reaction (qPCR) analysis
Total RNA was extracted from the trophocytes and oenocytes of two young or old worker bees or one young or old queen bee using TRIzol® Reagent (15,596,018; Invitrogen) (Hsu et al. 2014, 2016). RNA concentration and quality were determined using a Synergy™ HT multi-mode microplate reader (7,091,000; BioTek). The complementary DNA (cDNA) synthesis was performed using an iScript™ cDNA synthesis kit (170–8891; Bio-Rad Laboratories, CA, USA). Each reaction contained 1 μg of total RNA in a 20-µl reaction volume. The qPCR was performed using a CFX connect RT-PCR detection system (Bio-Rad Laboratories) and each reaction contained 0.5 µl of 10 µM of each primer, 12.5 µl of SYBR Green (170–8882; Bio-Rad Laboratories), 1 µl of diluted cDNA, and 10.5 µl of ddH2O in a final volume of 25 μl. The β-actin gene was used as a reference gene (Hsu et al. 2014). Primers were designed based on GenBank nucleotide sequences (Table I). The PCR program was at 95 °C for 3 min, followed by 39 cycles of denaturation at 95 °C for 10 s and annealing at 60 °C for 30 s (Hsu et al. 2014). The cycle threshold (Ct) values were normalized to β-actin (ΔCt) and the gene expression was expressed as 2−ΔΔCt (Livak and Schmittgen 2001). This experiment was performed with five biological replicates simultaneously using a total of ten young and ten old worker bees and five young and five old queen bees.
2.7 cAMP concentration
The cAMP concentration was assayed using the cAMP direct immunoassay kit (ab65355, Abcam, MA, USA) (Hsu and Chuang 2014; Hsu and Hu 2014). The manipulation of trophocytes and oenocytes is as described above. Briefly, trophocytes and oenocytes from two young or old worker bees or one young or old queen bee were treated with 110 µl of 0.1 M HCl, incubated for 20 min at room temperature, and centrifuged at 10,000 g for 10 min at 4 °C to obtain the supernatant. The supernatants of queen bees were diluted to obtain a similar protein concentration as worker bees. The dilution factor was used in the value calculation. The supernatant was used directly in the assay. After recording the background luminescence, 100 µl of the diluted cAMP standard solution (0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, and 10 pmol/µl) or 100 µl of the supernatant was added to the standard reaction solution and measured spectrophotometrically at 450 nm at room temperature using an ELISA plate reader (Synergy HT, BIO-TEK, VT, USA). The cAMP values, expressed as picomoles per nanogram of protein, were obtained from the luminescence measurements (standardized to background) by reference to a cAMP standard curve that was generated concurrently. This experiment was performed with five biological replicates simultaneously using a total of ten young and ten old worker bees and five young and five old queen bees.
2.8 Statistical analysis
Differences in the mean values among the four groups were determined by the Kruskal–Wallis test and followed by Mann–Whitney U-test for pairwise comparisons. A P-value of less than 0.01 was considered statistically significant (Lu et al. 2017).
3 Results
3.1 NAD+ concentration, NAD+/NADH ratio, and ATP concentration
To determine the mitochondrial energy utilization activity and cellular energy metabolism activity in the trophocytes and oenocytes of worker and queen bees, we assayed NAD+ levels, NAD+/NADH ratios, and ATP levels in the trophocytes and oenocytes of young and old worker bees, and young and old queen bees. The fold changes in the mean NAD+ levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.25 ± 0.05, 12.25 ± 2.27, and 11.51 ± 1.22, respectively. The NAD+ levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 1A). However, there was no significant difference between young and old queen bees. The fold changes in the mean NAD+/NADH ratios in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.25 ± 0.03, 5.49 ± 0.77, and 5.25 ± 1.11, respectively. The NAD+/NADH ratios significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 1B). However, there was no significant difference between young and old queen bees. The fold changes in the mean ATP levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.82 ± 0.01, 3.25 ± 0.33, and 2.94 ± 0.33, respectively. The ATP levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 1C). However, there was no significant difference between young and old queen bees. These findings indicated that the trophocytes and oenocytes of queen bees had better mitochondrial energy utilization activity and cellular energy metabolism activity than worker bees.
3.2 AMPK activity, lysosome activity, and ribosomal protein S6 mRNA level
To determine the cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity in trophocytes and oenocytes of worker and queen bees, we assayed AMPK activities, lysosome activities, and ribosomal protein S6 mRNA levels in the trophocytes and oenocytes of young and old worker and queen bees. The fold changes in the mean AMPK activities in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.56 ± 0.08, 18.31 ± 2.67, and 16.06 ± 2.33, respectively. The AMPK activities significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 2A). However, there was no significant difference between young and old queen bees. The fold changes in the mean lysosome activities in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.31 ± 0.07, 2.43 ± 0.17, and 2.20 ± 0.19, respectively. Lysosome activities significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 2B). However, there was no significant difference between young and old queen bees. The fold changes in the mean ribosomal protein S6 mRNA levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.49 ± 0.07, 2.48 ± 0.36, and 2.37 ± 0.21, respectively. Ribosomal protein S6 mRNA levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 2C). However, there was no significant difference between young and old queen bees. These results further revealed that the trophocytes and oenocytes of queen bees had better cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity than worker bees.
3.3 AC mRNA level, cAMP concentration, and PKA mRNA level
To explore the signaling pathways that increased cellular activities in the trophocytes and oenocytes of queen bees, we analyzed AC mRNA levels, cAMP concentrations, and PKA mRNA levels in the trophocytes and oenocytes of worker and queen bees. The fold changes in the mean AC mRNA levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.31 ± 0.05, 7.36 ± 0.72, and 6.48 ± 0.44, respectively. The AC mRNA levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 3A). However, there was no significant difference between young and old queen bees. The fold changes in the mean cAMP levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.67 ± 0.07, 3.27 ± 0.15, and 3.16 ± 0.30, respectively. The cAMP levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 3B). However, there was no significant difference between young and old queen bees. The fold changes in the mean PKA mRNA levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.40 ± 0.07, 2.57 ± 0.29, and 2.35 ± 0.28, respectively. The PKA mRNA levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 3C). However, there was no significant difference between young and old queen bees. These results indicated that the trophocytes and oenocytes of queen bees had higher signaling of the cAMP/PKA pathway than worker bees.
3.4 RAS, MEK, and RSK mRNA levels
To further investigate the signaling pathways that increased cellular activities in the trophocytes and oenocytes of queen bees, we examined the mRNA expression of RAS, MEK, and RSK in the trophocytes and oenocytes of young and old worker and queen bees. The fold changes in the mean RAS mRNA levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.62 ± 0.07, 2.87 ± 0.31, and 2.53 ± 0.22, respectively. The RAS mRNA levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 4A). However, there was no significant difference between young and old queen bees. The fold changes in the mean MEK mRNA levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.56 ± 0.05, 3.24 ± 0.20, and 3.55 ± 0.11, respectively. The MEK mRNA levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 4B). However, there was no significant difference between young and old queen bees. The fold changes in the mean MEK mRNA levels in old worker bees, young queen bees, and old queen bees relative to young worker bees were 0.50 ± 0.08, 5.34 ± 0.79, and 5.04 ± 0.54, respectively. The MEK mRNA levels significantly differed between young and old worker bees, between young worker bees and young and old queen bees, and between old worker bees and young and old queen bees (n = 5, P < 0.01; Figure 4C). However, there was no significant difference between young and old queen bees. These results indicated that the trophocytes and oenocytes of queen bees had higher signaling of the RAS/MAPK pathway than worker bees.
4 Discussion
Cellular activities including mitochondrial energy utilization activity, cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity in the trophocytes and oenocytes of queen bees are higher than that of worker bees based on the results of NAD+ levels, NAD+/ NADH ratio, ATP levels, AMPK activities, lysosome activities, and ribosomal protein S6 mRNA levels. The molecules of cAMP/PKA and RAS/MAPK signaling pathways in the trophocytes and oenocytes of queen bees are higher than that of worker bees. These findings suggested that the cAMP/PKA and RAS/MAPK signaling pathways may increase cellular activities leading to the longevity of queen bees.
4.1 NAD+ concentration, NAD+/ NADH ratio, and ATP concentration
NADH can be synthesized in the tricarboxylic acid cycle through glycolysis and β-oxidation and converted to NAD+ and H+ by NADH dehydrogenase. H+ increases mitochondrial membrane potential and promotes ATP synthesis by ATP synthase. Therefore, NAD+ level, NAD+/NADH ratio, and ATP level have been used to evaluate mitochondrial energy utilization activity and cellular energy metabolism activity in honey bees (Chuang and Hsu 2013; Hsu and Hu 2014; Lu et al. 2017, 2018).
The NAD+ levels and NAD+/NADH ratios decrease with advancing age in pancreatic β cells and neurons of mice (Imai 2009), the livers, hearts, kidneys, and lungs of rats (Braidy et al. 2011), and the trophocytes and oenocytes of worker honey bees (Chuang and Hsu 2013). Additionally, caloric restriction, which extends the lifespan of organisms, is associated with high NAD+ levels (Moroz et al. 2014). High NAD+ levels also extend the lifespan of Caenorhabditis elegans (Hashimoto et al. 2010). The ATP levels decrease with advancing age in the brains of mice (Joo et al. 1999) and the hearts of rats (Guerrieri et al. 1996). Mitochondrial respiration and electron transport decrease with advancing age in the flight muscle of Drosophila melanogaster (Ferguson et al. 2005). The daf-2 C. elegans mutant (e1370), which has an extended lifespan, has a higher ATP concentration than the wild-type nematode (Brys et al. 2010). Ambient temperature reduction, which can extend the lifespan of organisms, is associated with an increase in ATP concentration (Hsu and Chiu 2009). These studies indicated that young or long-lived cells have higher mitochondrial energy utilization activity and cellular energy metabolism activity than old or short-lived cells.
In this study, the trophocytes and oenocytes of young and old queen bees exhibit higher NAD+ levels, NAD+/NADH ratios, and ATP levels than that of young and old worker bees indicating that the trophocytes and oenocytes of queen bees have higher mitochondrial energy utilization activity and cellular energy metabolism activity than that of worker bees.
4.2 AMPK and lysosome activity and ribosomal protein S6 mRNA level
AMPK is a metabolic energy gauge and regulates cellular metabolism including the activation of glycolysis, fatty acid oxidation, mitochondrial biogenesis, and autophagy (Hardie et al. 2012; Reznick et al. 2007). Lysosomes are single-membrane organelles that contain acid hydrolase enzymes to degrade cellular macromolecules and organelles. Ribosomal protein S6 induces protein synthesis, cell growth, proliferation, and glucose homeostasis (Ruvinsky et al. 2005). Therefore, AMPK activity, lysosome activity, and ribosomal protein S6 have been used to evaluate cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity in honey bees (Hsu and Chuang 2014; Hsu and Hu 2014; Lu et al. 2017, 2018).
AMPK activity decreases with advancing age in rat muscles (Reznick et al. 2007) and mouse left ventricular tissues (Turdi et al. 2010). Caloric restriction, which extends the lifespan of organisms, is associated with an increase in AMPK activity through AMPK-α phosphorylation (Salminen et al. 2016). The lifespan extension of D. melanogaster is mediated by activating AMPK (Su et al. 2019). The lysosomal activity decreased with aging in the liver of rats (Cuervo and Dice 2000) and C. elegans (Sun et al. 2020). Caloric restriction, which extends the lifespan of organisms, decreases lipofuscin accumulation in the lysosome of the mouse brain (Moore et al. 1995). Mitochondrial protein synthesis decreased with age in the heart of Wistar rats (Hudson et al. 1998), the skeletal muscle of humans (Rooyackers et al. 1996), and D. melanogaster (Yang et al. 2019). Lipoprotein lipase synthesis decreased with age in the postural skeletal muscle of Fischer 344 rats (Bey et al. 2001). These studies indicated that young or long-lived cells have higher cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity than old or short-lived cells.
In this study, the trophocytes and oenocytes of young and old queen bees exhibit higher AMPK activities, lysosome activities, and ribosomal protein S6 mRNA levels than that of young and old worker bees indicating that the trophocytes and oenocytes of queen bees have higher cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity than that of worker bees.
4.3 AC mRNA level, cAMP concentration, and PKA mRNA level
The cAMP is transformed from ATP by AC and as a second messenger in signaling transduction for triggering many cell processes. The cAMP can trigger calcium signaling and activate PKA which phosphorylates liver kinase B1 to activate AMPK (Park et al. 2012; Collins et al. 2000; Perisse et al. 2009).
AC mRNA expression decrease with age in mice hippocampus (Mons et al. 2004) and AC activity decline with age in rat myocardium (O’Connor et al. 1981). The cAMP levels decline with age in rat isolated aorta (Schoeffter and Stoclet 1990). PKA activity declines with age in the Ceratitis capitata brain (Laviada et al. 1997). These studies indicated that young cells have higher signaling of the AC/cAMP/PKA pathway than old cells.
In this study, the trophocytes and oenocytes of young and old queen bees exhibit higher AC mRNA levels, cAMP levels, and PKA mRNA levels than that of young and old worker bees indicating that the trophocytes and oenocytes of queen bees have higher signaling of AC/cAMP/PKA pathway than that of worker bees.
The studies of cAMP and PKA in this study are consistent with previous studies indicating that caffeine increase cAMP and calcium levels in the brain of honey bees (Scheiner et al. 2006), that dopamine and homovanillyl alcohol increase the cAMP level in the brain of honey bees (Beggs et al. 2007), that tyramine increase cAMP level via tyramine receptor 2 in honey bees (Rein et al. 2017), and that PKA RNAi decreased PKA activity and impaired long-term memory formation of honey bees (Fiala et al. 1999).
4.4 RAS, MEK, and RSK mRNA levels
The RAS/RAF/MEK/ERK, also called RAS/MAPK, signaling pathway is important in regulating cell survival and proliferation and in inhibiting cell apoptosis (Chung and Kondo et al. 2011). This signaling pathway phosphorylates RSK to activate ribosomal protein S6 for protein synthesis (Sawicka et al. 2016). The RAS/RAF/MEK/ERK signaling declines with age in rat hepatocytes (Hutter et al. 2000) and rat brains (Zhen et al. 1999). These studies indicated that young cells have higher signaling of the RAS/RAF/MEK/ERK pathway than old cells.
In this study, the trophocytes and oenocytes of young and old queen bees exhibit higher RAS, MEK, and RSK mRNA levels than that of young and old worker bees indicating that the trophocytes and oenocytes of queen bees have higher signaling of the RAS/MAPK pathway than that of worker bees. The study of the RAS/MAPK signaling pathway in this study is consistent with a previous study indicating that Varroa-parasitized honey bees with visibly deformed wing virus clinical signs activate the RAS pathway (Erban et al. 2019).
Parallel increases in AC mRNA level, cAMP concentration, PKA mRNA level, AMPK activity, RAS mRNA levels, MEK mRNA levels, RSK mRNA levels, ribosomal protein S6 mRNA levels, and cellular functions of mitochondrial energy utilization activity, cellular energy regulation activity, cellular degradation activity, and cellular energy metabolism activity indicate that AC/cAMP/PKA and RAS/MAPK signaling pathways may be related to the increase of cellular activities. AC may increase the production of cAMP which activates AMPK activity through PKA to prompt cellular activities. This inference is supported by a previous study indicating that the activated receptor by biogenic amines stimulates AC to increase the concentration of cAMP which activates PKA in the brain to modulate the behavior of honey bees (Scheiner et al. 2006). The RAS pathway may activate RSK to activate ribosomal protein S6 for protein synthesis and cellular activities. This inference is consistent with a previous study indicating that RSK activates the ribosomal protein S6 of the brain in the mouse (Sawicka et al. 2016).
In addition, these results are consistent with our previously published transcriptional data showing that the mRNA expression of adenylyl cyclase, PKA, calcium/calmodulin-dependent protein kinase, RAS, RAF, and mitogen-activated protein kinase phosphatase 3 of queen bees are significantly higher than that of worker bees (Lu et al. 2021). Furthermore, the lifespan of queen and worker bees depends on their food intake. Queen bees eat RJ throughout their lives, and worker bees consume honey and pollen (Winston 1987). It infers that RJ might promote cellular activity through cAMP/PKA and RAS/MAPK signaling to increase NAD+ and ATP concentration, AMPK and lysosome activities, and protein synthesis in the cells of queen bees.
Taken together, a parallel increase in cellular activities and signaling of cAMP/PKA and RAS/MAPK pathways in the trophocytes and oenocytes of queen bees suggests that the cAMP/PKA and RAS/MAPK signaling pathways may increase cellular activities leading to the longevity of queen bees.
Data availability
The raw data of the study are available from the corresponding author upon reasonable request.
Code availability
Not applicable.
Abbreviations
- NAD+ :
-
Nicotinamide adenine dinucleotide oxidized form
- NADH:
-
Nicotinamide adenine dinucleotide reduced form
- ATP:
-
Adenosine triphosphate
- AMP:
-
Adenosine monophosphate
- AMPK :
-
AMP-activated protein kinase
- cAMP :
-
Cyclic AMP
- AC:
-
Adenylyl cyclase
- PKA :
-
Protein kinase A
- MAPK :
-
Mitogen-activated protein kinase
- RAS:
-
Ras GTPase
- RAF:
-
MAPK kinase kinase
- MEK :
-
MAPK kinase
- RSK :
-
Ribosomal protein S6 kinase
References
Beggs KT, Glendining KA, Marechal NM, Vergoz V, Nakamura I, Slessor KN, Mercer AR (2007) Queen pheromone modulates brain dopamine function in worker honey bees. Proc Natl Acad Sci USA 104:2460–2464. https://doi.org/10.1073/pnas.0608224104
Bey L, Areiqat E, Sano A, Hamilton MT (2001) Reduced lipoprotein lipase activity in postural skeletal muscle during aging. J Appl Physiol 91:687–692. https://doi.org/10.1152/jappl.2001.91.2.687
Bodenheimer FS (1937) Studies in animal populations. II. Seasonal population-trends of the honey-bee. Q Rev Biol 12:406–425
Braidy N, Guillemin GJ, Mansour H, Chan-Ling T, Poljak A, Grant R (2011) Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in Wistar rats. PLoS ONE 6:e19194. https://doi.org/10.1371/journal.pone.0019194
Brys K, Castelein N, Matthijssens F, Vanfleteren JR, Braeckman BP (2010) Disruption of insulin signaling preserves bioenergetic competence of mitochondria in ageing Caenorhabditis elegans. BMC Biol 8:91. https://doi.org/10.1186/1741-7007-8-91
Chuang YL, Hsu CY (2013) Changes in mitochondrial energy utilization in young and old worker honeybees (Apis mellifera). Age 35:1867–1879. https://doi.org/10.1007/s11357-012-9490-y
Chung E, Kondo M (2011) Role of Ras/Raf/MEK/ERK signaling in physiological hematopoiesis and leukemia development. Immunol Res 49:248–268. https://doi.org/10.1007/s12026-010-8187-5
Collins SP, Reoma JL, Gamm DM, Uhler MD (2000) LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo. Biochem J 345:673–680
Cuervo AM, Dice JF (2000) Age-related decline in chaperone-mediated autophagy. J Biol Chem 275:31505–31513. https://doi.org/10.1074/jbc.M002102200
Erban T, Sopko B, Kadlikova K, Talacko P, Harant K (2019) Varroa destructor parasitism has a greater effect on proteome changes than the deformed wing virus and activates TGF-β signaling pathways. Sci Rep 9:9400. https://doi.org/10.1038/s41598-019-45764-1
Ferguson M, Mockett RJ, Shen Y, Orr WC, Sohal RS (2005) Age-associated decline in mitochondrial respiration and electron transport in Drosophila melanogaster. Biochem J 390:501–511. https://doi.org/10.1042/BJ20042130
Fiala A, Muller U, Menzel R (1999) Reversible downregulation of protein kinase A during olfactory learning using antisense technique impairs long-term memory formation in the honeybee, Apis mellifera. J Neurosci 19:10125–10134. https://doi.org/10.1523/JNEUROSCI.19-22-10125.1999
Guerrieri F, Vendemiale G, Turturro N, Fratello A, Furio A, Muolo L, Grattagliano I, Papa S (1996) Alteration of mitochondrial F0F1 ATP synthase during aging. Possible involvement of oxygen free radicals. Ann N Y Acad Sci 786:62–71. https://doi.org/10.1111/j.1749-6632.1996.tb39052.x
Hardie DG, Ross FA, Hawley SA (2012) AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol 13:251–262. https://doi.org/10.1038/nrm3311
Hashimoto T, Horikawa M, Nomura T, Sakamoto K (2010) Nicotinamide adenine dinucleotide extends the lifespan of Caenorhabditis elegans mediated by sir-2.1 and daf-16. Biogerontology 11:31. https://doi.org/10.1007/s10522-009-9225-3
Hsieh YS, Hsu CY (2011a) Honeybee trophocytes and fat cells as target cells for cellular senescence studies. Exp Gerontol 46:233–240. https://doi.org/10.1089/rej.2013.1420
Hsieh YS, Hsu CY (2011b) The changes of age-related molecules in the trophocytes and fat cells of queen honeybees (Apis mellifera). Apidologie 42:728–739. https://doi.org/10.1007/s13592-011-0085-x
Hsu CY, Chan YP (2013) The use of honeybees reared in a thermostatic chamber for aging studies. Age 35:149–158. https://doi.org/10.1007/s11357-011-9344-z
Hsu CY, Chiu YC (2009) Ambient temperature influences aging in an annual fish (Nothobranchius rachovii). Aging Cell 8:726–737. https://doi.org/10.1111/j.1474-9726.2009.00525.x
Hsu CY, Chuang Y, Chan YP (2014) Changes in cellular degradation activity in young and old worker honeybees (Apis mellifera). Exp Gerontol 50:128–136. https://doi.org/10.1016/j.exger.2013.12.003
Hsu CY, Chuang YL (2014) Changes in energy-regulated molecules in the trophocytes and fat cells of young and old worker honeybees (Apis mellifera). J Gerontol A Biol Sci Med Sci 69:955–964. https://doi.org/10.1093/gerona/glt163
Hsu CY, Hu TH (2014) Energy-regulated molecules maintain young status in the trophocytes and fat cells of old queen honeybees. Biogerontology 15:389–400. https://doi.org/10.1007/s10522-014-9509-0
Hsu CY, Lu CY (2015) Mitochondrial energy utilization maintains young status in the trophocytes and oenocytes of old queen honeybees. Apidologie 6:583–594. https://doi.org/10.1007/s13592-015-0348-z
Hsu CY, Qiu JT, Chan YP (2016) Cellular degradation activity is maintained during aging in long-lived queen bees. Biogerontology 17:829–840. https://doi.org/10.1007/s10522-016-9652-x
Hudson EK, Tsuchiya N, Hansford RG (1998) Age-associated changes in mitochondrial mRNA expression and translation in the Wistar rat heart. Mech Ageing Dev 103:179–193. https://doi.org/10.1016/s0047-6374(98)00043-8
Hutter D, Yo Y, Chen W, Liu P, Holbrook NJ, Roth GS, Liu Y (2000) Age-related decline in Ras/ERK mitogen-activated protein kinase cascade is linked to a reduced association between Shc and EGF receptor. J Gerontol A Biol Sci Med Sci 55:B125–B134. https://doi.org/10.1093/gerona/55.3.b125
Imai S (2009) The NAD world: a new systemic regulatory network for metabolism and aging-Sirt1, systemic NAD biosynthesis, and their importance. Cell Biochem Biophys 53:65–74. https://doi.org/10.1007/s12013-008-9041-4
Joo HJ, Ma JY, Choo YG, Choi BK, Jung KY (1999) Age-related alteration of intracellular ATP maintenance in the cell suspensions of mice cerebral cortex. Mech Aging Dev 110:1–12. https://doi.org/10.1016/s0047-6374(99)00027-5
Laviada ID, Galve-Roperh I, Malpartida JM, Haro A (1997) cAMP signaling mechanisms with aging in the Ceratitis capitata brain. Mech Ageing Dev 97:45–53. https://doi.org/10.1016/s0047-6374(97)01899-x
Law JH, Wells MA (1989) Insects as biochemical-models. J Biol Chem 264:16335–16338
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lu CY, Chuang YL, Hsu CY (2017) Aging results in a decline in cellular energy metabolism in the trophocytes and oenocytes of worker honeybees (Apis mellifera). Apidologie 48:761–775. https://doi.org/10.1007/s13592-017-0521-7
Lu CY, Qiu JT, Hsu CY (2018) Cellular energy metabolism is maintained during aging in long-living queen bees. Arch Insect Biochem Physiol 2018:e21468. https://doi.org/10.1002/arch.21468
Lu CY, Weng YT, Tan B, Hsu CY (2021) The trophocytes and oenocytes of worker and queen honeybees (Apis mellifera) exhibit distinct age-associated transcriptome profiles. Geroscience 43:1863–1875. https://doi.org/10.1007/s11357-021-00360-y
Mons N, Segu L, Nogues X, Buhot MC (2004) Effect of age and spatial learning on adenylyl cyclase mRNA expression in the mouse hippocampus. Neurobiol Aging 25:1095–1060. https://doi.org/10.1016/j.neurobiolaging.2003.10.014
Moore WA, Davey VA, Weindruch R, Walford R, Ivy GO (1995) The effect of caloric restriction on lipofuscin accumulation in mouse brain with age. Gerontology 2:173–185. https://doi.org/10.1159/000213741
Moroz N, Carmona JJ, Anderson E, Hart AC, Sinclair DA, Blackwell TK (2014) Dietary restriction involves NAD+-dependent mechanisms and a shift toward oxidative metabolism. Aging Cell 13:1075–1085. https://doi.org/10.1111/acel.12273
Neukirch A (1982) Dependence of the lifespan of the honeybee (Apis mellifera) upon flight performance and energy consumption. J Comp Physiol 146:35–40. https://doi.org/10.1007/BF00688714
O’Connor SW, Scarpace PJ, Abrass IB (1981) Age-associated decrease of adenylate cyclase activity in rat myocardium. Mech Ageing Dev 16:91–95. https://doi.org/10.1016/0047-6374(81)90036-1
Park SJ, Ahmad F, Philp A, Barr K, Williams T, Luo H, Ke H, Rehmann H, Taussig R, Brown AL, Kim MK, Beaven MA, Burgin AB, Manganiello V, Chung JH (2012) Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell 148:421–433. https://doi.org/10.1016/j.cell.2012.01.017
Perisse E, Raymond-Delpech V, Neant I, Matsumoto Y, Leclerc C, Moreau M, Sandoz JC (2009) Early calcium increase triggers the formation of olfactory long-term memory in honeybees. BMC Biol 7:30. https://doi.org/10.1186/1741-7007-7-30
Rein T, Balfanz S, Baumann A, Blenau W, Thamm M, Scheiner R (2017) AmTAR2: functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity. Insect Biochem Mol Biol 80:91–100. https://doi.org/10.1016/j.ibmb.2016.12.004
Reznick RM, Zong H, Li J, Morino K, Moore IK, Yu HJ, Liu ZX, Dong J, Mustard KJ, Hawley SA, Befroy D, Pypaert M, Hardie DG, Young LH, Shulman GI (2007) Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis. Cell Metab 5:151–156. https://doi.org/10.1016/j.cmet.2007.01.008
Rooyackers OE, Adey DB, Ades PA, Nair KS (1996) Effect of age on in vivo rates of mitochondrial protein in human skeletal muscle. Proc Natl Acad Sci USA 93:15364–15369. https://doi.org/10.1073/pnas.93.26.15364
Ruvinsky I, Sharon N, Lerer T, Cohen H, Stolovich-Rain M, Nir T, Dor Y, Zisman P, Meyuhas O (2005) Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev 19: 2199–2211. https://doi.org/10.1101/gad.351605
Salminen A, Kaarniranta K, Kauppinen A (2016) Age-related changes in AMPK activation: role for AMPK phosphatases and inhibitory phosphorylation by upstream signaling pathways. Ageing Res Rev 28:15–26. https://doi.org/10.1016/j.arr.2016.04.003
Sawicka K, Pyronneau A, Chao M, Bennett MVL, Zukin RS (2016) Elevated ERK/p90 ribosomal S6 kinase activity underlies audiogenic seizure susceptibility in fragile X mice. Proc Natl Acad Sci USA 113, E6290–E6297. https://doi.org/10.1073/pnas.1610812113
Scheiner R, Baumann A, Blenau W (2006) Aminergic control and modulation of honeybee behaviour. Cur Neuropharmacol 4:259–276. https://doi.org/10.2174/157015906778520791
Schoeffter P, Stoclet JC (1990) Age-related differences in cyclic AMP metabolism and their consequences on relaxation induced by isoproterenol and phosphodiesterase inhibitors in rat isolated aorta. Mech Ageing Dev 54:197–205. https://doi.org/10.1016/0047-6374(90)90050-p
Su Y, Wang T, Wu N, Li D, Fan X, Xu Z, Mishra SK, Yang M (2019) Alpha-ketoglutarate extends Drosophila lifespan by inhibiting mTOR and activating AMPK. Aging 11:4183–4197. https://doi.org/10.18632/aging.102045
Sun Y, Li M, Zhao D, Li X, Yang C, Wang X (2020) Lysosome activity is modulated by multiple longevity pathways and is important for lifespan extension in C. elegans. eLife 9, e55745. https://doi.org/10.7554/eLife.55745
Turdi S, Fan X, Li J, Zhao J, Huff AF, Du M, Ren J (2010) AMP-activated protein kinase deficiency exacerbates aging-induced myocardial contractile dysfunction. Aging Cell 9:592–606. https://doi.org/10.1111/j.1474-9726.2010.00586.x
Winston ML (1987) The biology of the honey bee. Harvard University Press, Cambridge
Yang Lu, Cao Y, Zhao J, Fang Y, Liu N, Zhang Y (2019) Multidimensional proteomics identifies declines in protein homeostasis and mitochondria as early signals for normal aging and age-associated disease in Drosophila. Mol Cell Proteomics 18:2078–2088. https://doi.org/10.1074/mcp.RA119.001621
Zhen X, Uryu K, Cai G, Johnson GP, Friedman E (1999) Age-associated impairment in brain MAPK signal pathways and the effect of caloric restriction in Fischer 344 rats. J Gerontol A Biol Sci Med Sci 54:B539–B548. https://doi.org/10.1093/gerona/54.12.b539
Funding
This work was supported by grants (CMRP1K0481 and CMRP1M0481) from the Chang Gung Memorial Hospital, Linkou, Taiwan, and a grant (MOST 108–2320-B-182–037-MY3) from the Ministry of Science and Technology, Taiwan.
Author information
Authors and Affiliations
Contributions
CH designed the research; CH and YW performed research; CH, YW, and CC analyzed data; CH and CC wrote the paper. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Conflict of interest
The authors declare no competing interests.
Additional information
Manuscript editor: Klaus Hartfelder
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Hsu, CY., Weng, YT. & Chen, CH. The longevity of queen honey bees (Apis mellifera) is associated with the increase of cellular activities through the cAMP/PKA and RAS/MAPK signaling pathways. Apidologie 53, 65 (2022). https://doi.org/10.1007/s13592-022-00976-9
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13592-022-00976-9