Effects of host age on susceptibility to infection and immune gene expression in honey bee queens (Apis mellifera) inoculated with Nosema ceranae
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Nosema ceranae is a microsporidium parasite infecting honey bees worldwide. All colony members including workers, drones, and queens can become infected. In this study, we inoculated queens of age 1, 6, and 12 days post-adult emergence, with N. ceranae spores of different doses and allowed them to age an additional 12 days. The results indicated that younger queens were indeed more susceptible to N. ceranae inoculation than older queens. Moreover, this is the first report of the effects of N. ceranae inoculation on immune gene expression in queens of different ages. Our results demonstrated that the expression of genes related to the bee immunity apidaecin, eater, and vitellogenin in the gut and the remaining abdomen was different among queens of different ages when inoculated with N. ceranae. All three ages of queens inoculated by N. ceranae showed upregulation of apidaecin in gut tissue 6 days after inoculation, but only in queens aged 1 day post-emergence were the differences significant. However, transcript levels of eater were increased in all three ages of queens when sampled on day 12, and significant differences were obtained in queens inoculated at 6 and 12 days post-emergence. We clearly show that immune responses to N. ceranae changes as queen age and this knowledge may provide clues for understanding the ability of queens to resist infection by this gut parasite.
KeywordsNosema ceranae honey bee queen Apis mellifera supersedure immunity
Two microsporidian parasites, Nosema apis and Nosema ceranae, have been reported to cause Nosema disease of honey bees. N. apis was first described in the European honey bee, Apis mellifera (Zander 1909) and N. ceranae was first known to infect the Asian cavity nesting honey bee, Apis cerana (Fries et al. 1996). However, recent studies have shown that N. ceranae is becoming the dominant species infecting A. mellifera worldwide and may be more virulent than N. apis (Fries et al. 2006; Higes et al. 2006; Chauzat et al. 2007; Cox-Foster et al. 2007; Huang et al. 2007; Klee et al. 2007; Paxton et al. 2007; Chen et al. 2008; Williams et al. 2008; Chaimanee et al. 2010). Nosema infection occurs when spores are ingested through contaminated food and water (Webster 1993). The spores can germinate and develop in the epithelial cells of the midgut of all colony members including workers, drones, and queens. Following N. ceranae infection, the epithelial ventricular cells of infected honeybees were degenerated (Higes et al. 2007). N. ceranae has been detected not only in the midgut tissue but also other tissues including Malpighian tubules, hypopharyngeal glands, salivary glands, and fat bodies using a PCR method (Chen et al. 2009). N. ceranae can change the physiology and behavior of honey bees (Dussaubat et al. 2013; Goblirsch et al. 2013). N. ceranae infection has been reported to suppress the honey bee immune response (Antúnez et al. 2009; Chaimanee et al. 2012) and to affect hormone production by increasing ethyl oleate content (Dussaubat et al. 2010). Moreover, N. ceranae induces an energetic stress (Mayack and Naug 2009; Alaux et al. 2010; Martín-Hernández et al. 2011) and oxidative stress in honey bees (Dussaubat et al. 2012) and decreases the carbohydrate level in honeybee forager hemolymph (Mayack and Naug 2010). Similar to N. apis infection, honey bees, when infected with N. ceranae, have a shorter life span (Malone et al. 1995; Goblirsch et al. 2013).
Since a honey bee queen is the primary reproductive of the colony and her pheromones and presence produce cohesion between colony members, Nosema infection in queens could cause serious problems for the colony. A honey bee queen is susceptible to N. apis infection. In commercial queen production, a queen may become infected in the mating nuc or colony via spore ingestion from infected attendants bees in shipment or upon placement in a new colony. Nosema-infected queens have smaller ovaries when compared to the non-infected queens (Farrar 1947). Eggs of infected queens show decreased hatching rates and queens are often rapidly superseded once they become infected (Farrar 1947). Similarly, N. ceranae can be transmitted horizontally from infected worker bees to queens and can affect the epithelial ventricular cells of queens (Higes et al. 2009). Low levels of N. ceranae were detected in tissues of the head, thorax, abdomen, and ovaries from naturally infected queens (Traver and Fell 2012). Recently, Alaux et al. (2011) explored the effects of N. ceranae on honey bee queen physiology. Their results demonstrated that N. ceranae infection affected the physiological functions and modified pheromone production of queens. This may explain the rapid replacement of infected queens.
In this study, to demonstrate the susceptibility of queens to N. ceranae infection, we determined the rate of N. ceranae infection in queens of different ages (queens aged for 1, 6 and 12 days post-adult emergence). We hypothesized that younger queens would be more susceptible to N. ceranae infection than older queens. The 12-day period was selected as it corresponds with the time that queens often spend in small nucleus colonies for mating before being shipped to the beekeeper. If queens are more susceptible during this initial period of adult life then infection-mitigation measures could improve queen health and queen longevity in colonies. Moreover, even though there are some published studies about the effects of N. ceranae on immune response of honey bees, little is known about molecular defense mechanisms of honey bee queens to defend themselves from pathogen infections. Hence, the immune responses of honey bee queens to N. ceranae infection were also investigated.
2 Materials and methods
2.1 Honey bee queens
The experiments were conducted in August 2010 in Beltsville, MD. Queen cells containing queens of mixed A. mellifera mellifera/A. mellifera ligustica stock were obtained from a single queen breeder in Georgia, USA. Queen cells were placed individually in new wooden Benton cages and held at 34 ± 2 °C. The queens emerged within 12 h and were held in individual new cages containing three newly emerged worker bees from a colony with no detectable Nosema. Queens aged 1, 6, and 12 days post-adult emergence were used in this study. Upon emergence, ten queens were randomly sampled and checked for the presence of microsporidian spores using light microscopy and diagnostic PCR amplification (Chen et al. 2008).
2.2 Inoculum preparation
On each day of inoculation, N. ceranae spores were isolated from a colony infected with N. ceranae obtained from an apiary at the Bee Research Laboratory, USDA-ARS in Maryland, USA. The midguts were removed and crushed in 1 ml of distilled water and N. ceranae was confirmed using PCR amplification following methods described by Chen et al. (2008). Additionally, the numbers of spores were estimated by counting spores with light microscopy after the method of Cantwell (1970). The inoculum was freshly prepared in various concentrations by mixing with 50 % sucrose solution to obtain a final concentration of 103, 104, 105, and 106 spores/ml and maintained at room temperature until used for inoculation on that day.
2.3 N. ceranae inoculation experiment
Queens of three ages: 1, 6, and 12 days post-adult emergence, were used in this study. Each age group of queens (n = 150 total) were held individually in new standard wooden queen cages with three newly emerged uninfected attendants. The top of each cage had a single hole that allowed for a 0.5-ml tube containing sucrose solution (500 μl) to serve as a food source. For each age group, 30 queens were fed with 50 % sucrose solution containing 103, 104, 105, and 106 spores/ml of N. ceranae for the first 2 days and the control group was fed with only 50 % (w/v) of sucrose solution. After exposure to N. ceranae, queens were fed ad libitum with a clean 50 % (w/v) of sucrose solution throughout the remainder of the experiment. Six queens in each group were collected at day 6 and 12 post-inoculation to be examined for N. ceranae infection. The individual queen guts were homogenized in 1 ml of distilled water and N. ceranae spores were estimated using a hemocytometer (Cantwell 1970).
2.4 Effects of N. ceranae inoculation on expression of genes related to the bee immunity
2.4.1 RNA isolation and cDNA synthesis
Total RNA was isolated from the rest of gut and remaining abdomen tissues of the individual queens (control queens and queens when the cage in which the queen was received a spore suspension with 105 and 106 spores/ml of N. ceranae) using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. DNA was removed using DNAse I incubation at 37 °C for 1 h followed by 10 min at 75 °C. First-strand cDNA was generated from approximately 2 μg total RNA using a master mix containing 50 U Superscript II (Invitrogen, Carlsbad, CA), 2 nmol dNTP mix, 2 nmol poly(dT)18, and 0.1 nmol poly(dT)(12–18). Synthesis was carried out at 42 °C for 50 min followed by 15 min at 70 °C as described in Evans (2006).
2.4.2 Quantitative PCR amplification
Oligonucleotide primers used in this study for real-time quantitative PCR (Simone et al. 2009).
Sequence 5′ to 3′
The amplification results were expressed as the threshold cycle (Ct) value, which represented the number of cycles needed to generate a fluorescent signal greater than a predefined threshold. Threshold cycle numbers for the target gene were subtracted from the reference gene (β-actin) for each sample. The amplification efficiency for target and control products was established via serial dilutions of known templates (e.g., Chen et al. 2008; Simone et al. 2009; Cornman et al. 2012).
2.5 Expression levels of genes related to the bee immunity in gut and remaining abdomen of queens at different ages
Control queens from ages 6 (n = 6), 12 (n = 12), 18 (n = 12), and 24 (n = 6) days following adult emergence were analyzed. Total RNA was isolated from gut and remaining abdomen tissues of the individual queens using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. cDNA synthesis and quantitative PCR amplification were described above (Sections 2.4.1 and 2.4.2).
2.6 Statistical analysis
Normality of data was checked using SPSS version 17.0 for Windows (SPSS, Inc.). Statistical significance was analyzed using ANOVA if they fitted a normal distribution. However, where the data were not normally distributed, the nonparametric Kruskal–Wallis H and Mann–Whitney U test were applied.
3.1 N. ceranae infection of queens aged 1, 6, and 12 days after emergence
When fed with N. ceranae spores at different doses, N. ceranae spores were quantified 6 and 12 days post-inoculation. No spores were detected in any of the control samples.
Percentage of N. ceranae infection in honey bee queen at different ages after inoculated with N. ceranae spore doses of 103–106 spores/ml.
Age of queen (days after emergence
Day of inoculation (days)
Percent of infection
Of the queens aged 6 days post-emergence and then fed N. ceranae, they became infected when receiving a spore suspension of 105 and 106 spores/ml, with an infection rate of 16.7 and 50 % at 6 days post-inoculation, respectively (Figure 1a), and a 100 % infection rate after 12 days post-inoculation (Table II). The average spore numbers were 6.64 × 106 spores/queen (SE = 3.99 × 106 spores/queen) and 1.352 × 107 spores/queen (SE = 5.87 × 106 spores/queen) at 12 days after inoculation when the cage was inoculated with 105 and 106 spores/ml, respectively (Figure 1b).
When queens were aged for 12 days post-emergence and then fed N. ceranae, no spores were observed in queens, 6 days post-inoculation (Figure 1a). By 12 days post-inoculation, the infection rates had increased to 16.7, 33.3, and 100 % when inoculated with 104, 105, and 106 spores/ml respectively, indicating decreased susceptibility with increasing queen age (Figure 1 and Table II).
3.2 Effects of N. ceranae infection on immune related gene expression in queen gut tissue
Expression of immune-related genes, apidaecin, eater, and vitellogenin of queens of different ages inoculated with N. ceranae at the spore dose of 105 and 106 spores/ml was analyzed at 6 and 12 days post-inoculation. The tissues of gut and remaining abdomen were investigated for transcript levels of these genes. We found that gene expression varies with queen age.
The expression of vitellogenin was varied in queens when inoculated with N. ceranae. In inoculated queens with 105 spores/ml, transcript levels of vitellogenin were significantly increased after 6 days of inoculation (ANOVA, P = 0.004; mean relative expression of −3.483 ± 1.086 s.e.); however, there was significantly elevated in inoculated queens with 106 spores/ml of N. ceranae at day 12 inoculation (mean relative expression of 1.912 ± 0.308 s.e.).
In queens inoculated at 6 days post-emergence, the gene expression of eater and vitellogenin in gut tissue was significantly lower in inoculated queens compared to control queens at 6 days post-inoculation (Kruskal–Wallis H, P = 0.05 and P = 0.03, respectively). Transcript levels of eater were about four times lower than control queens, with average relative expression of −14.968 ± 0.500 s.e. and −14.007 ± 1.631 s.e. for 105 and 106 spores/ml of N. ceranae-infected queens, respectively (Figure 2a). Also, vitellogenin expression in inoculated queens was about seven times lower than control queens (mean of −5.757 ± 0.265 s.e. and −4.217 ± 1.000 for 105 and 106 spores/ml of N. ceranae-inoculated queens, respectively; Figure 2c). At day 12 post-inoculation, queens inoculated with N. ceranae concentration of 106 spores/ml showed higher expression of eater (ANOVA, P = 0.036), with mean of −13.903 ± 0.306 s.e. (Figure 2d). Comparing inoculated and control queens, there were no significant differences in transcript levels of vitellogenin 12 days after inoculation (ANOVA, P > 0.05) (Figure 2f). For apidaecin, the mRNA levels were not significantly different between inoculated and control queens at 6 and 12 days after inoculation (Kruskal–Wallis H, P > 0.05 and ANOVA, P > 0.05, respectively; Figure 2b, e).
Gene expression of apidaecin and vitellogenin did not change in gut tissue of inoculated queens aged 12 days post-adult emergence after 6 and 12 days (ANOVA, P > 0.05) (Figure 2b, c, e, f). However, eater mRNA was significantly upregulated in inoculated queens with 105 spores/ml (ANOVA, P = 0.006; average of −7.271 ± 1.381 s.e.; Figure 2d).
3.3 Effects of N. ceranae infection on immune related gene expression in the remaining abdomen of queens
3.4 Expression levels in the gut and remaining abdomen of queens at different ages
In this study, we examined the effect of both queen age and spore dose on the susceptibility of queen honey bees to N. ceranae infection. The 12-day period after emergence was selected as it corresponds with the time that queens often kept in small nucleus colonies for mating before being shipped to the beekeeper. We clearly show that queens become less susceptible as they age making their time spent in the mating nuclei critical for N. ceranae infection. Our results suggest that any measures that reduce the contamination of N. ceranae spores in food sources fed to the newly emerged queens would help to prevent queens from becoming infected with N. ceranae.
The results show that queens become infected when inoculated with a high dose of either 105–106 spores/ml of N. ceranae spores, 12 days post-inoculation, except for those queens aged for 12 days, when only those queens dosed with 106 spores/ml became 100 % infected. N. ceranae fed spores at lower dosages of 103 and 104 spores/ml did not infect all queens. Younger queens are more susceptible to N. ceranae infection than older queens. This age response could be interpreted as the young queen gut cells promoting the growth, development, and germination of N. ceranae (Keeling and Fast 2002). Nevertheless, Rinderer and Elliot (1977) indicated that the protein conditions of the host are critical for the development of N. apis. It is conceivable that younger queens are in better condition than older queens that have been exposed to periods of protein depletion. If queens are more susceptible during this initial period of adult life then mitigation measures could reduce N. ceranae infection and improve queen health and longevity in colonies. When considering the queens aged for 1 day post-emergence, surprisingly the average number of spores in inoculated queens with 105 spores/ml of N. ceranae was higher than those in queens when inoculated with 106 spores/ml. As all honey bee queens originated from the same queen breeder, we suggest that the susceptibility differences likely reflect the genetic variations of host at the individual queen level. The individual immune defense can be considered one of the benefits of genetic diversity in honey bees.
Honey bee queens of all three ages (1, 6, and 12 days) inoculated by N. ceranae showed downregulation of eater in gut tissue after 6 days inoculation. A significant downregulation of eater was especially evident in queens aged 6 days post-adult emergence. Eater transcripts after 12 days post-inoculation were upregulated in all queen ages when inoculated with N. ceranae. The results suggest that queens may have to produce more eater in this period to defend against N. ceranae infection. Since N. ceranae spores can rapidly develop and multiply during day 4 to 7 of infection. Thus, eater may play important role to eliminate this pathogen. Also eater has been shown to play a role in the phagocytosis of bacterial pathogens (Ertürk-Hasdemir and Silverman 2005; Kocks et al. 2005).
For apidaecin, the upregulation of this gene was observed in the gut of inoculated queens of all ages at 6 days post-inoculation. Our results revealed that N. ceranae induces apidaecin expression in queens in early periods of inoculation. However, apidaecin expression shows an opposing response between queens and workers after inoculated with N. ceranae. N. ceranae infection suppresses immune gene response in worker bees (Autúnez et al. 2009; Chaimanee et al. 2012). Queens might be better able to defend themselves from pathogen invasion since they produce more antimicrobial peptides. Nevertheless, different immune signaling between queens and workers following N. ceranae infection cannot be excluded.
Our study demonstrates great variation in vitellogenin expression between queens of different ages. Vitellogenin transcripts were increased in inoculated queens aged 1 and 12 days post-emergence in both of gut and remaining abdomen tissues. However, queens showed decreased expression when aged 6 days old. In a recent study, Alaux et al. (2011) reported that the vitellogenin titer increased about 58 % in honey bee queens when infected with N. ceranae. In contrast, Autúnez et al. (2009) demonstrated that the mRNA levels of vitellogenin in N. ceranae infected worker bees significantly decreased after 7 days of infection. Vitellogenin is a female-specific glucolipoprotein yolk precursor synthesized in the fat body of abdomen (Byrne et al. 1989). It plays an important function in immunity and longevity of worker honey bees (Amdam et al. 2004). Therefore, it is not conclusive that vitellogenin is affected by N. ceranae infection in queens. Other factors may contribute to the expression of the vitellogenin gene.
Differences in the transcript levels of genes were clearly observed between the gut and remaining abdomen. Our results showed that the expression of eater, apidaecin, and vitellogenin in the gut was lower than that from the remaining abdomen. Not surprisingly, since the fat body distributes throughout the abdomen and it is the site of antimicrobial peptides synthesis. The results showed that eater transcripts were decreased in the gut tissue and there were elevated in the abdomen part of older queens. Contrary to eater expression, the mRNA levels of apidaecin in the gut of queens were increased in older queens. This indicates that younger queens produce lower apidaecin levels than older queens and may help explain the increased susceptibility of younger queens to N. ceranae. In this study, we showed that vitellogenin expression in the gut and abdomen did not differ between queens aged 6, 18, and 24 days old post-emergence, except in queens aged 12 days where there was a significant vitellogenin production compared to the others. In honey bee queens, vitellogenin synthesis starts 60 h before adult emergence and vitellogenin titers stays high thereafter (Barchuk et al. 2002). Moreover, Corona et al. (2007) have reported that older queens have higher vitellogenin transcript levels than do young queens. Surprisingly, queens aged 12 days old had the highest vitellogenin expression in our study. We hypothesize that high vitellogenin level may result from immunity, hormone signaling, and/or oxidative stress. Taken together, we suggest that vitellogenin expression in queens does not vary greatly between ages 6 to 24 days of age.
V.C. was supported by a grant under the program of Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program Thai Doctoral degree from the Office of the Higher Education Commission, Thailand. We thank the Thailand Research Fund (BRG 5580013), National Research University, Office of Higher Education Commission, and the USDA-ARS Bee Research Laboratory for financial support. We thank M. Hamilton, D. Lopez, V. Levi, and N. Rice for their excellent technical help with the project.
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