The western honeybee, Apis mellifera
, is one of the most important pollinators in the world [1
]. They contribute to the production of many different fruits, vegetables, crops, and aromatic seeds. However, honeybees are weak at resisting their stressors, which can cause direct/indirect injuries and affect their development, thus influencing the structure and functionality of plant-pollination networks. In recent years, the incidence rate of colony collapse disorder (CCD) [3
], characterized by hives missing their worker bees, has increased significantly [5
]; this has contributed to significant losses in the produce economy. Several factors have been suggested as contributing to this phenomenon, including poor nutrition, pesticides, pathogens, parasites, and interactions between these two factors resulting in a stressful environment for the colony [6
Colonies suffering from CCD often exhibit signs of viral infections caused by Israel acute paralysis virus (IAPV), acute bee paralysis virus (ABPV), and deformed wing virus (DWV) [9
]. DWV has been isolated in over 90% of colonies with CCD, making it the most prevalent bee virus. This RNA virus infection, spread by Varroa destructor
mites, can cause the abnormal development of wings and memory loss since the highest titer of the virus is found in the head after infection [12
]. Furthermore, DWV has a negative impact on the immune system of the honeybee [15
]: the virus tends to negatively regulate the transcription of some genes such as dorsal 1A, a transcription factor in the family NF-κB [17
], and the Toll pathway, which are known to target viruses [19
]. As a result of this transcription dysregulation, transcription of antimicrobial peptides, clotting and melanization are reduced in infected honeybees [23
Plants and pollinators benefit each other since plants provide nectar as a source of food and, in return, their pollen grains are disseminated by the visiting insect. In nectars of plants in the genera Citrus
, low concentrations of caffeine (1,3,7-trimethylxanthine) are found, which acts as an antiherbivory compound [24
]. Previous studies have indicated that the ingestion of caffeine as nectar ingredient could increase the foraging efficiency of honeybees and thus promote pollination [26
]. Furthermore, it is believed that caffeine could enhance both the learning behavior and long-term memory of forager bees [29
]. In other insects, it has been found that adequate caffeine consumption influences locomotion and enhances memory; this includes studies involving hornets [Vespa orientalis
]], honeybees, the green scale insect [Coccus viridis
]], and flour beetles [Tribolium castaneum
and Tribolium confusum
]]. In addition, one study of European honeybees indicated that caffeine can enhance the tolerance to infection with the fungus parasite Nosema ceranae
as well as extend their lifespan [34
]. However, the beneficial effects of caffeine on the immune response against other pathogens, including viruses, are unknown.
Based on the above information, we hypothesized that caffeine could help the honeybee resist viral infection. We compared bees supplied with/without caffeine and assessed their immune responses against DWV infection. Furthermore, we analyzed the expression of a set of immune-related genes to determine how they are influenced by caffeine. Our results showed that the expression of immune-related genes was up-regulated after the infected honeybees were fed with caffeine, and that the replication of DWV was inhibited. These results provide evidence that caffeine is beneficial to the honeybee in fighting against pathogens in the environment. Furthermore, it provides us with a greater understanding of why bees prefer to harvest nectar from caffeine-synthesizing plants.
2. Materials and Methods
2.1. Bee Rearing
Western honeybees (Apis mellifera
) were purchased in Hsinchu county, Taiwan, and kept at the National Taiwan University. About 50~100 bees for the experiment were collected directly from one hive as a mixed group. Then, the bees were caged in different Bugdorms (18 × 18 × 18 cm, MegaView Science, Taichung, Taiwan) at 37 °C. Prior to the experiments, bees were fed with 0.7 M sucrose water with/without caffeine (0.1 mM, Sigma-Aldrich, St. Louis, MO, USA) for one week [29
]. One frame of the hive was taken back to the lab and incubated in 30 °C, waiting for newly emerged bees to come out. The newly emerged bees were first fed with 0.7 M sucrose water and pollen as protein source for one week to stabilize their physiological condition, then caffeine (0.1 mM) was added to the diet for another week. All food for the bees were changed every three days, and the dead bees were removed. After this feeding stage, all bees were collected for gene analysis or bioassays.
2.2. Deformed Wing Virus (DWV) Purification
About 80~100 bees were directly collected from their hive, and then put into cages, incubated in 30 °C. DWVs were mixed with sugar solution (0.7 M) as a regular treatment for one week. After treatment, bees were frozen in −80 °C, and then homogenized with 10 mL phosphate-buffered saline (PBS). The liquid was collected through a nylon filter to keep out residue, then we centrifuged it (16,000× g). Supernatant was moved to another tube and centrifuge again, then use Minisart® Syringe Filters (0.45 µm) to filtrate the supernatant.
2.3. DWV Infection and Caffeine Treatment
Four treatment groups were prepared: control (0.7 M sucrose water only), caffeine only (0.1 mM) [29
], DWV only (106
virus copies), and both (caffeine/DWV). DWV was diluted with phosphate-buffered saline (PBS) to prepare a working virus solution with 5 × 106
copies/µL. The sucrose water was replaced with water only one day prior to experiment and the following day the bees were forced to take 4 µL of solution mixed with 2 µL of virus and 2 µL of sucrose. After 48 h, the bees were frozen at −80 °C for total RNA extraction and gene expression analysis. After one week stabilizing the physiological condition, the bees were separated into two groups, and caffeine was then added to the diet of one of the groups for another week as the caffeine-only treatment group. Then, bees were randomly chosen from each group and forced to take 4 µL of solution mixed with 2 µL of virus and 2 µL of sucrose as virus only and both treatment group. After this feeding stage, all bees were collected for gene analysis or bioassays.
2.4. Total RNA Extraction
After the treatments, total RNA was extracted using a TRIzol™ reagent kit (Thermo Fisher Scientific). Whole bodies of two bees were pooled together for homogenization. RNA quantification was determined using a NanoDrop 2000 (Thermo Fisher Scientific).
2.5. cDNA Synthesis
cDNA synthesis of each treatment group was performed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For each sample, 2 µg of total RNA was used. The reaction was incubated in a FlexCycler 2 PCR thermal cycler (Analytic Jena AG) for 10 min at 25 °C, 120 min at 37 °C, 5 min at 85 °C, and then stopped at 4 °C. The final products were used for quantitative real-time polymerase chain reaction (RT-PCR) analysis or stored at −20 °C for later analysis.
2.6. Real-Time Polymerase Chain Reaction (PCR) and Data Analysis
Real-time PCR was performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using SensiFAST™ SYBR Hi-ROX Kit (BIOLINE, London, UK). The result (fold change) was calculated following the relative quantification theory [36
]. For quantitative PCR, honeybee-specific primers for including immune, viral and carbohydrate metabolism genes (Table 1
) were used as described in previous studies [35
]. All samples were amplified simultaneously, and three independent experiments were performed. PCR-array images were analyzed with the software R (Version XX). Fold changes were calculated using the relative quantification method (2−△△Ct
]. Each group of tested genes was normalized to a reference gene (18s rRNA), and fold changes in the control group were used as a calibrator.
2.7. DWV Titer Calculation
A plasmid containing a DNA fragment of DWV was transformed to Escherichia coli (DH5) and then grown in LB broth (ARROWTEC) containing ampicillin as a selection marker at 37 °C for 16 h. Plasmid extraction was conducted using a Presto™ Mini Plasmid Kit (Geneaid) and the concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific). A DNA Copy Number and Dilution Calculator (Thermo Fisher scientific website) were used to determine the amount of DNA sample equivalent to 1010 plasmid copies. Serial dilution was performed to prepare 1010 to 101 plasmid copies. Real-time PCR was conducted using serially diluted plasmid samples. A standard curve was plotted using data obtained from the real-time PCR results on serially-diluted plasmid samples; regression analysis was used to calculate the virus copy numbers in the virus-treated bees.
2.8. Other Latent Infecting Viruses in Taiwan
Based on a previous study in Taiwan [35
], we choose the also latent-infecting virus species to see if caffeine can aid the bees to resist them. The virus we use including Vorroa destructor
virus-1 (VDV), Kashmir bee virus (KBV), Kakugo virus (KV), Israeli acute bee paralysis virus (IAPV), Sacbrood Virus (SBV), black queen cell virus (BQCV), and Chronic bee paralysis virus (CBPV) to see whether caffeine can also suppress their replication or not. After treated with caffeine, honeybees were freeze-killed and the mRNA extraction, complimentary DNA (cDNA) reverse transcription, and RT-PCR was performed to compare the amount of viral infection of honeybees treated with/without caffeine.
2.9. Statistical Analysis
Gene expression level results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for significance using Statistica software (Version 8). The virus number was analyzed using one-way Student’s t-test. A p < 0.05 indicated a statistically significant result.
In this study, the influence of caffeine on the immune system of honeybees was evaluated. Gene expression levels were measured to demonstrate the interaction between caffeine and DWV. Although the effect of caffeine on mammals is well known [41
], particularly its effects on anti-oxidation and neural activation, there are only a limited number of similar studies on insects. The results of the present study provide a basic yet valuable insight into the effect of caffeine on gene regulation in insects.
The effect of caffeine on the immune system of honeybees is almost unknown. In humans, a high concentration of caffeine can mitigate the damage caused by inflammation [42
]. This is due to the inhibition on phosphodiesterase (PDE) activity, leading to an increased level of intracellular cAMP and the activation of the PKA pathway [43
]. This may explain the changes in gene expression found in the present study, since the PKA pathway is also involved in regulating the immune system [44
]. Analysis of the expression level of immune genes showed that they were significantly up-regulated after caffeine treatment in DWV-infected bees (Figure 1
A–C), namely for parseph
in the Toll pathway, PGRPLC
in the Imd pathway, and lysozyme-1 (Lys-1
), prophenoloxidase-activating enzyme (PPOact
), and the AMP gene amPPO
D). Both the Toll and Imd pathways have been previously demonstrated to be involved in fighting against viral invasion in insects [45
]. Our results also showed that caffeine treatment prior to DWV infection could sufficiently inhibit DWV infection (Figure 2
). This indicates that caffeine can boost the immune system by up-regulating the expression of genes involved in pathways known to influence immune responses (such as Toll and Imd) and protect honeybees from the external stress caused by viral infections. In the results, after DWV infection, the gene expression level of the immune system is up-regulated. Although it is indicated in previous study that DWV can suppress the host’s immune system, this is the consequence of long-term and latent infection of Vorroa destructor
and DWV [16
]. In our experiment, the virus was fed to the healthy bees, so this situation is different from the previous study. This acute infection induced the spike in the immune response of honeybees against the viral invasion [47
Until now, the effect of caffeine on gene expression in 16-day-old bees has remained unexplored. Caffeine is a natural compound present in the nectar of certain plants and, therefore, honeybees can easily consume caffeine from the environment [25
]. Bees that are 16 days old, however, were not exposed to caffeine (and other compounds) as the foragers because they were kept in lab. The effect of a stressor and caffeine treatment was, therefore, worth investigating for these honeybees with “clean” background. Interestingly, the results are contrary to those obtained in the mixed group: caffeine does not stimulate the immune system of 16-day-old bees as it does to the mixed group in the presence of DWV infection and the expression of most genes involved in the Toll and Imd pathways and AMPs are down-regulated (Figure 4
One previous study indicated that older honeybees, such as foragers, have a high basal gene expression level related to detoxification and immune pathways for dealing with more environmental stressors [48
]. This may explain different outcomes under the same experimental conditions from mixed nursing bees/forager bees and 16-day-old bees. Nevertheless, the expression of PPOact
were also significantly enhanced in DWV-infected bees, similar to in the caffeine/DWV group (Figure 4
), suggesting that caffeine has a marginal boosting effect on the immune system of 16-day-old bees. The differential influences of caffeine on the immune system during pathogen infection in 16-day-old bees and older nursing bees/forager bees thus require further study.