- Email: [email protected]
Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana
Accepted Manuscript Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana Jun Guo, Jie Wu, Yanping Chen, Jay D. Evans, Rongguo Dai, Wenhua Luo, Jilian Li PII: DOI: Reference:
S0022-2011(15)00065-8 http://dx.doi.org/10.1016/j.jip.2015.03.010 YJIPA 6660
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
17 October 2014 15 March 2015 17 March 2015
Please cite this article as: Guo, J., Wu, J., Chen, Y., Evans, J.D., Dai, R., Luo, W., Li, J., Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana, Journal of Invertebrate Pathology (2015), doi: http://dx.doi.org/10.1016/j.jip.2015.03.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana Jun Guoa, b, Jie Wua*, Yanping Chenc, Jay D. Evansc, Rongguo Daib, Wenhua Luob, and Jilian Lia* a
Key Laboratory of Pollinating Insect Biology of the Ministry of Agriculture, Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing 100093, China
b
Institute of Economic animals, Chongqing Academy of Animal Sciences, Chongqing 402460, China
c
USDA-ARS, Bee Research Laboratory, Beltsville, MD 20705, USA Abstract: Previous surveys have shown that adult workers of the Asian honey
bee Apis cerana harbor four major gut microbes (Bifidobacterium, Snodgrassella alvi, Gilliamella apicola, and Lactobacillus). Using quantitative PCR we characterized gut bacterial communities across the life cycle of A. cerana from larvae to workers. Our results indicate that the presence and quantity of these four bacteria were low on day 1, increased rapidly after day 5, and then peaked during days 10-20. They stabilized from days 20-25 or days 25-30, then dropped to a low level at day 30. In addition, the larvae infected by Sacbrood virus or European foulbrood had significantly lower copies of 16SrRNA genes than healthy individuals.
Key words: Apis cerana, dynamics, gut microbiota
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]; [email protected].
1
Introduction China is one of the largest beekeeping countries in the world with a long history of bee culture (Chen, 1993). The Asian honey bee A. cerana was the chief pollinator of agricultural crops in China before the introduction of European honey bees Apis mellifera in the late 19th century. Currently, A. cerana and A. mellifera have overlapping habitats and are experiencing many of the same disease problems including infections by Deformed wing virus (DWV), Black queen cell virus (BQCV), Sacbrood virus (SBV), the European foulbrood (EFB) bacterium, the American foulbrood (AFB) bacterium, Nosema ceranae, and Crithidia bombi (Li et al., 2012). Gut microbiota can play an important role in honey bee physiology, nutrition, and health (Dillon and Dillon, 2004; Hamdi and Balloi et al., 2011; Evans and Schwarz, 2011; Engel et al., 2012). Furthermore, some symbionts in Hymenoptera (such as Bombus terrestris) protect against pathogenic infections (Koch and Schmid-Hempel, 2011; Kaltenpoth and Engl, 2014). This protective function has been suggested by the metagenome data of the gut microbiota of Apis mellifera (Engel et al., 2012) . In A. mellifera, cultivation independent approaches have revealed eight distinct bacterial species or phylotypes that have been identified as major constituents of honeybee bacterial communities(Martinson et al., 2011; Martinson et al., 2012; Cox-Foster et al., 2007). These species are made up of two related Firmicutes within Lactobacillus, one Bifidobacterium, one Betaproteobacterium (Snodgrassella alvi), two related Gammaproteobacteria (Gilliamella apicola and Frischella perrara) and two Alphaproteobacteria (Engel et al., 2012 ; Martinson et al., 2012). In A. mellifera, the newly emerged adult workers enclose with no or very few bacteria in their guts and within a few days have large populations of organ (ileum and hindgut) specific bacteria (Martinson et al., 2012; Powell et al., 2014). Developing larvae have a discontinuous gut (the foregut is not connected to the hindgut) before pupation (Winston, 1911). Thus, this stage of larvae is the target of many major pathogens including bacteria or virus (Bailey and Ball, 1991; Rauch et al., 2
2009; Blanchard et al., 2014). We focus on the fifth larval instar because the larvae infected by EFB or SBV begin to die from 4th to 6th instar (Liang and Chen, 2009). In addition, the fifth instar larvae harbored more diverse microbiota in A. mellifera than other instars (Mohr and Tebbe, 2006). However, there have been no reports regarding the gut bacterial diversity of microbes in the larvae of A. cerana in China. Previous research on the microbiota of A. cerana adult workers detected bacteria at low levels and found many of the same microbial taxa as in A. mellifera (Ahn et al., 2012; Disayathanoowat et al., 2012; Li et al., 2012). In these studies, the four most commonly identified bacterial species groups corresponded to Bifidobacterium, S. alvi (Neisseriaceae), G. apicola (family Orbaceae; order Orbales, previously categorized as Pasteurellaceae), and Lactobacillus (corresponding to the Firm-5 cluster found in A. mellifera) (Li et al., 2012). Understanding the microbial residents and its dynamics across the A. cerana life span is a prerequisite for comprehending the symbiotic relationship between A. cerana and its gut microbial residents. In the present study, we used culture-independent methods to characterize the dynamics of the gut microbiota at different developmental stages of health and disease of Asian honey bees, A. cerana by focusing on the four phylotypes: Bifidobacterium, S. alvi, G. apicola and Firm-5 (Lactobacillus). Furthermore, we compared the differences in the gut microbiota and spatiotemporal dynamics between A. mellifera and A. cerana. Materials and methods Sample collection A. cerana workers were collected from three colonies in Rong Chang county, ChongQing, China, during July and August 2013. In order to obtain adult workers of uniform age, two frames that contained capped brood were pulled out from bee colonies and held in a dark incubator at 34°C and 80% R.H. for 24 h to mimic hive conditions. For each colony, ten newly emerged workers were collected at day 1 and marked with enamel paint and then returned to their colony in the field. In total, there were 250 bees marked and returned back to the field colonies. Subsequently, five 3
marked bees were collected at days 5, 10, 15, 20, 25, and 30 individually for each colony. Samples were stored in 75% ethanol at −20°C for subsequent molecular analysis. In order to obtain larvae of the same age, we used the facility of laying controller which can be used to restrict the queen to lay eggs on only one frame. We selected three healthy colonies, EFB-infected colonies and SBV-infected colonies respectively for bee sampling in Nan Chuan county, Chong Qing, China, in August 2013. We put a frame without any eggs or brood into a laying controller, then caught the queen to the laying controller and put it back into each colony. After 24 h, we checked the frame in order to make sure that the eggs were enough for the research, then let the queen out of the laying controller. After three days of egg the stage and four days of the laval stage, the larvae were collected as 5th instar larvae from healthy, EFB-and SBV-infected colonies on the 5th day. We screened the EFB and SBV for the 5th instar larvae from EFB-and SBV-infected colonies using PCR method, and only EFB-and SBV-infected larvae were selected to do further examine. DNA, RNA extraction and disease detection For the worker samples, the whole guts (including crop, midgut, ileum and rectum) were dissected out from workers aseptically. The DNA was extracted from each
time
point
from
workers
with
a
Wizard® SV 96 Genomic DNA Purification System (Promega Corporation, Madison, USA) as described in Li et al. (2012). For the larval samples, we ground the individual larvae and separated it into two parts, RNA was extracted from one part using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, and the other oart was used for DNA extraction. All RNA samples were tested for the presence of seven viruses including Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Kashmir bee virus (KBV), Israeli acute bee paralysis virus (IAPV), and Sacbrood virus (SBV). Amplification of specific viral RNAs was carried out using the Access RT-PCR kit (Promega, Madison, WI) according to the manufacturer’s 4
instructions. PCR amplication conditions and primers were followed as our previous study (Li et al., 2012). All DNA samples were used to detect EFB, AFB and N.ceranae, separately. N.ceranae detection was based on Li et al. (2012). EFB and AFB bacteria screens were done following the methods in Govan et al. (1998) and Dobbelaere et al. (2001), respectively. Quantitative PCR (qPCR) for detection and quantification of the presence and abundance of four bacterial species The absolute qPCR method was used to detect and quantify the presence and abundance of bacterial species in adult workers. All the initial template DNA concentrations were normalized between samples, and a control PCR was performed using the A. cerana β-actin gene to verify that the DNA was of sufficient quality for qPCR. The primers were used from our previous study (Li, et al., 2012; Xu, et al., 2014). Quantitative PCR (qPCR) was performed in a total reaction volume of 25μL containing the following reagents: 9.5μL ddH2O and 12.5μL 2 x Power SybrGreen qPCR Master Mix (Applied Biosystems, Calif., USA); 0.5μL of each primer (10 μM), and 2μL template. qPCR reactions were performed on a ABI 7500 system (Applied Biosystems, Foster, CA, USA), using the following cycle conditions: 95℃ for 10 min, 40 cycles of 95℃ for 15 s, 60℃for 1 min with a melting curve observed at the end of each run. Standards were prepared for each of the four bacterial primers consisting of a 1:10 dilution series (adult worker samples) that was run with each qPCR to establish a standard curve. Cycle threshold (Ct) values of each sample were then compared to the standard curve to approximate the copy numbers for each bacterium in a given sample (Brucker and Bordenstein, 2012). The relative quantification method was used to assess the abundance of the bacterial species in larvae infected with EFB or SBV as in Li et al. (2012). The primers and bacterial phylotypes were the same as described above. Statistical analyses Statistical analysis was carried out in GraphPad Prism version 6.01 for Windows (GraphPad Software, California, USA). The 16S rRNA copy numbers estimated by 5
qPCR were compared across different time points using ANOVA with Tukey's multiple comparisons test. The significant difference of each bacterial load between EFB or SBV infected and non-infected larva with SYBR-Green used for detection was analysed using a one sample t-test. Results Dynamic of four characteristic gut bacteria in A. c. cerana gut The age of the A. cerana worker positively correlates with the abundance of the four bacteria. Because Day 1 workers have far fewer bacteria than older individuals, we omitted the Day 1 workers from the analyses. The results showed that the 16S rRNA gene copies of the four phylotypes increased with age, reaching the highest level at an age of 10 days (Lactobacillus), or 15 days (S. alvi and Bifidobacterium) or 20 days (G. apicola), then declining (Fig.1). Except for Lactobacillus, the 16S rRNA gene copies of the other three bacteria were very low at day 5 (Fig.1a, b, d). The 16S rRNA gene copies from G. apicola (P = 0.863 by Tukey's HSD) and Bifidobacterium (P = 0.863by Tukey's HSD) showed there was no significant difference between days 25 and 30; and the 16S rRNA gene copies of S. alvi and Lactobacillus decreased to a stable level from days 20 to day 25 (P = 0.532 and 0.939 by Tukey's HSD respectively), then it dropped to the lowest level at day 30 (Fig.1a, c). The gut bacteria of A. c. cerana in infected and uninfected larva. The relative numbers of the four bacterial taxa were compared between healthy and unhealthy larva infected by SBV and EFB, separately. The results showed that the 16S rRNA gene quantities of S. alvi (t = 19.89, p = 0.003) (Fig.2a) and Lactobacillus(t = 49.42, p = 0.0001) (Fig.2b) in larva infected by SBV were significantly lower than in the healthy ones. The difference in The Bifidobacterium (t = 2.67, p = 0.116) and G. apicola (t = 3.413, p = 0.076) between larvae infected by SBV and healthy ones was not significant. For the larva infected by EFB, their 16S rRNA gene quantities of S. alvi (t = 21.67, p = 0.002) (Fig. 2c) and Bifidobacterium (t = 16.84, p = 0.004) (Fig. 2d) were also significantly lower than in the healthy larvae. The difference in the 6
Lactobacillus(t = 2.294, p = 0.149) and G. apicola (t = 2.218, p = 0.157) between larvae infected by EFB and healthy ones was not significant. Discussion Research on A. mellifera showed that the larvae and new emerged bees had few or no bacteria in their guts (Martinson et al., 2012), which is consistent with our similar results for A. cerana. Our results suggest that quantities of the four bacteria in the A. cerana gut initially increases in adult workers from day 5 then later declines and remains stable for the rest of the time. Similar results have been reported in bumble bees (Xu et al., 2014). Koch et al.(2013) reported that the symbiotic bacteria (G. apicola and S. alvi) can be vertically transmitted from mother to daughters in bumble bees. Bees also acquire their gut bacteria through social contact within the hive, brood cell or older workers (Martinson et al., 2012; Powell et al., 2014). A. cerana workers likely also acquire the major gut bacteria through social contact or contact with the hive environment. When bees reach the adult workers age, they face an environment full of bacteria (Haydak, 1970). From ages 1-3 days, their task consists of cell cleaning, resting or grooming (Seeley, 1982). From ages 4-12 days, they serve as nurses and transfer their proteinaceous secretions to the younger and older bees in the nest (Ribbands, 1953; Seeley, 1982; Crailsheim, 1991, 1992). For A.cerana, the total levels of Lactobacillus peaked at day 10, suggesting a link between Lactobacillus and the production of larval food. From ages 12-21 days, the bees appear to spend more time on general colony maintenance such as comb building, then they transition to nectar processing and other tasks (Trumbo et al., 1997). So during ages 12-21, the most important physiological and biochemical processes of honeybees are participating in the food production, such as nectar and bread processing. Interestingly, the three bacteria peaked between ages 15-20 days corresponding to the ages of nectar and bee bread processing. Metagenome results also indicated that G. apicola, Firm (Lactobacillus) and Bifidobacterium have many functional capabilities linked to carbohydrate breakdown, and proposed that the Lactic acid bacteria (LAB) group 7
made up of Firm and Bifidobacterium was involved in nectar processing and metabolism of carbohydrates (Engel et al., 2012; Butler, 2013). Older A. mellifera workers (>20 days past eclosure) focus on foraging for pollen, nectar, propolis and water (Robinson,1992; Calderone,1998). Although these behavioral shifts might be expected to result in changes in the microbiota composition, Martinson et al.(2012) reported that the characteristic phylotypes were maintained throughout the worker's life. The difference between our results with A. cerana and Martinson et al.(2012)’s work might reflect species differences and colony differences. Recently, Powell et al.(2014) reported a more fine-scale sampling of A. mellifera workers from 2 colonies, with 9 sampling points up to Day 16, and did not find strong trends. Our study and the A. mellifera studies sampled only a few colonies, and whether composition shifts in older bees of either species needs further investigation. In this study, the number of gut bacteria began to decline after day 15 or day 20, but after 20 days of age, bacteria have a stable period, such as S. alvi and Lactobacillus in the age of 20 to 25 days, and G. apicola and Bifidobacterium in 25 to 30 days of age. And Martinson et al.(2012) reported the number of bacteria in the Apis mellifera gut at 9, 20 and 30 days of age remain stable, but they did not check the sample of the age of 15, our study showed that bacteria began to decline after the age of 15. This finding could be suggested that the loss decline of bacteria in the gut may be due to defecation of the foraging bee when it first leaves the hive after the 15th day. Many stressors, including various diseases, can contribute to the occurrence of dysbiosis in honeybees and potentially perturb gut bacteria (Crotti et al., 2013). Variations of gut microbial communities may be associated with pathogen infection (Cariveau et al., 2014). Interestingly, the gut community was less in larvae infected with EFB and SBV than in non-infected larvae, suggesting a negative interaction between these pathogens and the gut bacteria. These results have a similar discipline with Li et al. (2012) and Cariveau et al. (2014) which reported on the parasite infection research. Some symbiotic bacteria such as Beta (S. alvi) and Gamma-1 (G. apicola) phylotype of A. mellifera also have functions in protection against disease 8
(Martinson et al., 2012). Thus, these symbiotic bacteria in this study maybe participate in the role of virus or bacteria defense which lead to the decline after infection. Potentially, the gut symbionts confer defense or affect immune functioning. Alternatively, environmental stress could perturb the gut community and lead to higher susceptibility of the hosts (Engel and Moran, 2013). However, a causal relationship is not yet confirmed. Our results give an overview of the dynamics of A. cerana gut bacteria during different life stages. The mechanisms causing the bacteria to increase or decline are not yet known. Potentially, its gut microorganisms are key to bee health and resistance to biotic and abiotic stressors (Crotti et al., 2013). A.cerana is one of the most important pollinators in agricultural systems and is a significant link in the food supply in China and other Asian countries. We need further empirical work to understand the roles of gut bacteria in the health and disease of this important insect.
Acknowledgements We would like to thank Tang Hong, Ren Qin and Cao Lan for collecting honeybee samples. We thank Nancy A. Moran and J. Elijah Powell for very useful comments on the manuscript. This study was funded by China Agriculture Research System (CARS-45).
References Ahn, J.H., et al. 2012. Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea. J. Microbiol. 50 (5): 735-745. Bailey L, Ball B.V .1991. Honeybee pathology, 2nd Ed, Academic Press: San Diego USA. Blanchard, P., et al. 2014. Development and validation of a real-time two-step RT-qPCR TaqMan® assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies. J. Virol. Methods. 197: 7-13. Brucker, R.M. and Bordenstein, S.R. 2012. The roles of host evolutionary relationships (Genus: 9
Nasonia) and development in structuring microbial communities. Evolution, 66 (2): 349-362. Butler È, A.M.O.T., Proteins of novel lactic acid bacteria from Apis mellifera mellifera-an insight into the production of known extra-cellular proteins during microbial stress. BMC microbiology, 2013. 13 (1): p. 235. Calderone N. W. 1998. Proximate mechanisms of age polyethism in the honey bee, Apis mellifera L. Apidologie. 29 (1-2):127–158. Cariveau, D.P., et al. 2014. Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus). ISME J. Apr 24. doi: 10.1038/ismej.2014.68. Chen Y. C. Apicultura of China[M]. China Agriculture Press, 1993. Cox-Foster, D. L. and S. Conlan, et al. 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science. 318 (5848): 283-7. Crailsheim K. 1991. Interadult feeding of jelly in honeybee (Apis mellifera L) colonies. J. Comp. Physiol. B. 161:55–60. Crailsheim K. 1992. The flow of jelly within a honeybee colony. J. Comp. Physiol. B. 162:681–689. Crotti, E., et al. 2013. Microbial symbionts of honeybees: a promising tool to improve honeybee health. N. Biotechnol. 30 (6): 716-22. Dillon, R. J. and V. M. Dillon. 2004. The gut bacteria of insects: nonpathogenic interactions. Annu. Rev. Entomol. 49 (1): 71-92. Disayathanoowat, T., et al. 2012. T-RFLP analysis of bacterial communities in the midguts of Apis mellifera and Apis cerana honey bees in Thailand. FEMS Microbiol Ecol. 79 (2): 273-281. Dobbelaere, W. and Degraaf, D. C., et al. 2001. Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR. Apidologie 32: 363-370. Evans, J.D. and R.S. Schwarz. 2011. Bees brought to their knees: microbes affecting honey bee health. Trends Microbiol., 19 (12): 614-620. Engel, P. and N. A. Moran. 2013. The gut microbiota of insects - diversity in structure and function. FEMS Microbiol. Rev. 37 (5): 699-735. Engel, P. et al., 2012. Functional diversity within the simple gut microbiota of the honey bee. Proc. Nati. Acad. Sci. U.S.A. 109 (27): 11002-7. 10
Govan, V. A, Brözel V, Allsopp M H, et al. 1998. A PCR detection method for rapid identification of melissococcus pluton in honeybee larvae . Appl. Environ. Microbiol. 1998, 64 (5): 1983-1985. Hamdi, C. and A. Balloi, et al. 2011. Gut microbiome dysbiosis and honeybee health. J. Appl. Entomol. 135 (7): 524-533. Haydak, M. H. 1970. Honey bee nutrition. Annu. Rev. Entomol. 15 (1):143–156. Kaltenpoth, M. and T. Engl. 2014. Defensive microbial symbionts in Hymenoptera. Functional Ecology. 28 (2): 315-327. Koch, H. and P. Schmid-Hempel. 2011. Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. Proc. Natl. Acad. Sci. U. S. A. 108 (48): 19288-92. Koch, H, Abrol, D.P, Li J, et al. 2013. Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees. Mol. Ecol. 22 (7): 2028-2044. Li, J., et al. 2012. The prevalence of parasites and pathogens in Asian honeybees Apis cerana in China. PLoS One. 7 (11): e47955. Liang, Q. and Chen, D. F. 2009. Honeybee protection. 2nd Ed, China Agriculture Press: Beijing (in Chinese). Martinson, V. G., et al. 2011. A simple and distinctive microbiota associated with honey bees and bumble bees. Mol. Ecol. 20 (3): 619-28. Martinson, V. G., et al. 2012. Establishment of characteristic gut bacteria during development of the honey bee worker. App. Environ. Microbiol. 78 (8): 2830-2840. Mohr K.I., Tebbe C.C. 2006. Diversity and phylotype consistency of bacteria in the guts of three bee species (Apoidea) at an oilseed rape field. Environ Microbiol. 8: 258–272. Powell J. E., et al. 2014. Routes of acquisition of the gut microbiota of Apis mellifera. App. Environ. Microbiol. Sep 19. pii: AEM.01861-14. Rauch S, Ashiralieva A, Hedtke K, Genersch E. 2009. Negative correlation between individual-insect-level virulence and colony-level virulence of Paenibacillus larvae, the etiological agent of American foulbrood of honeybees. Appl. Environ. Microbiol.75: 3344–3347. Ribbands R. 1953. The behaviour and social life of honeybees. London Dover Publ Inc, New York. Robinson G. E. 1992. Regulation of division of labor in insect societies. Annu. Rev. Entomol. 37:637–665. 11
Seeley T. D. 1982. Adaptive significance of the age polyethism schedule in honeybee colonies. Behav. Ecol. Sociobiol. 11 (4):287–293. Trumbo S. T., Huang Z.Y., Robinson G. E. 1997. Division of labor between undertaker specialists and other middle-aged workers in honey bee colonies. Behav. Ecol. Sociobiol. 41:151–163. Winston M. L. 1991. The Biology of the Honeybee. Harvard University Press: Cambridge Mass USA. Xu L L and WU J et al. 2014. Dynamic variation of symbionts in Bumblebees during hosts growth and development. Scientia Agricultura Sinica. 47 (10):2030-2037.
Figure Legends Fig 1. Abundances of S. alvi (a), G. apicola (b), Lactobacillus (c) and Bifidobacterium (d) in different days and stages of A.cerana adult workers, measured as copies of the 16S rRNA gene. Letters above confidence intervals (1 standard deviation) represent significance levels (Tukey’s HSD).
Fig 2. Relative abundance of three bacterial groups between SBV-infected (a and b) and EFB-infected (c and d ) in the larvae of A.cerana.
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Graphical abstract
Highlights We detected the gut microbes of A.cerana during different developmental stage. Gut microbes in A. cerana tend to dynamic change and remains stable after 20 days. Gut microbes of larvae have a negative interaction with virus or bacterial diseases.
14
S0022-2011(15)00065-8 http://dx.doi.org/10.1016/j.jip.2015.03.010 YJIPA 6660
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
17 October 2014 15 March 2015 17 March 2015
Please cite this article as: Guo, J., Wu, J., Chen, Y., Evans, J.D., Dai, R., Luo, W., Li, J., Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana, Journal of Invertebrate Pathology (2015), doi: http://dx.doi.org/10.1016/j.jip.2015.03.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of gut bacteria at different developmental stages of Asian honey bees, Apis cerana Jun Guoa, b, Jie Wua*, Yanping Chenc, Jay D. Evansc, Rongguo Daib, Wenhua Luob, and Jilian Lia* a
Key Laboratory of Pollinating Insect Biology of the Ministry of Agriculture, Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing 100093, China
b
Institute of Economic animals, Chongqing Academy of Animal Sciences, Chongqing 402460, China
c
USDA-ARS, Bee Research Laboratory, Beltsville, MD 20705, USA Abstract: Previous surveys have shown that adult workers of the Asian honey
bee Apis cerana harbor four major gut microbes (Bifidobacterium, Snodgrassella alvi, Gilliamella apicola, and Lactobacillus). Using quantitative PCR we characterized gut bacterial communities across the life cycle of A. cerana from larvae to workers. Our results indicate that the presence and quantity of these four bacteria were low on day 1, increased rapidly after day 5, and then peaked during days 10-20. They stabilized from days 20-25 or days 25-30, then dropped to a low level at day 30. In addition, the larvae infected by Sacbrood virus or European foulbrood had significantly lower copies of 16SrRNA genes than healthy individuals.
Key words: Apis cerana, dynamics, gut microbiota
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]; [email protected].
1
Introduction China is one of the largest beekeeping countries in the world with a long history of bee culture (Chen, 1993). The Asian honey bee A. cerana was the chief pollinator of agricultural crops in China before the introduction of European honey bees Apis mellifera in the late 19th century. Currently, A. cerana and A. mellifera have overlapping habitats and are experiencing many of the same disease problems including infections by Deformed wing virus (DWV), Black queen cell virus (BQCV), Sacbrood virus (SBV), the European foulbrood (EFB) bacterium, the American foulbrood (AFB) bacterium, Nosema ceranae, and Crithidia bombi (Li et al., 2012). Gut microbiota can play an important role in honey bee physiology, nutrition, and health (Dillon and Dillon, 2004; Hamdi and Balloi et al., 2011; Evans and Schwarz, 2011; Engel et al., 2012). Furthermore, some symbionts in Hymenoptera (such as Bombus terrestris) protect against pathogenic infections (Koch and Schmid-Hempel, 2011; Kaltenpoth and Engl, 2014). This protective function has been suggested by the metagenome data of the gut microbiota of Apis mellifera (Engel et al., 2012) . In A. mellifera, cultivation independent approaches have revealed eight distinct bacterial species or phylotypes that have been identified as major constituents of honeybee bacterial communities(Martinson et al., 2011; Martinson et al., 2012; Cox-Foster et al., 2007). These species are made up of two related Firmicutes within Lactobacillus, one Bifidobacterium, one Betaproteobacterium (Snodgrassella alvi), two related Gammaproteobacteria (Gilliamella apicola and Frischella perrara) and two Alphaproteobacteria (Engel et al., 2012 ; Martinson et al., 2012). In A. mellifera, the newly emerged adult workers enclose with no or very few bacteria in their guts and within a few days have large populations of organ (ileum and hindgut) specific bacteria (Martinson et al., 2012; Powell et al., 2014). Developing larvae have a discontinuous gut (the foregut is not connected to the hindgut) before pupation (Winston, 1911). Thus, this stage of larvae is the target of many major pathogens including bacteria or virus (Bailey and Ball, 1991; Rauch et al., 2
2009; Blanchard et al., 2014). We focus on the fifth larval instar because the larvae infected by EFB or SBV begin to die from 4th to 6th instar (Liang and Chen, 2009). In addition, the fifth instar larvae harbored more diverse microbiota in A. mellifera than other instars (Mohr and Tebbe, 2006). However, there have been no reports regarding the gut bacterial diversity of microbes in the larvae of A. cerana in China. Previous research on the microbiota of A. cerana adult workers detected bacteria at low levels and found many of the same microbial taxa as in A. mellifera (Ahn et al., 2012; Disayathanoowat et al., 2012; Li et al., 2012). In these studies, the four most commonly identified bacterial species groups corresponded to Bifidobacterium, S. alvi (Neisseriaceae), G. apicola (family Orbaceae; order Orbales, previously categorized as Pasteurellaceae), and Lactobacillus (corresponding to the Firm-5 cluster found in A. mellifera) (Li et al., 2012). Understanding the microbial residents and its dynamics across the A. cerana life span is a prerequisite for comprehending the symbiotic relationship between A. cerana and its gut microbial residents. In the present study, we used culture-independent methods to characterize the dynamics of the gut microbiota at different developmental stages of health and disease of Asian honey bees, A. cerana by focusing on the four phylotypes: Bifidobacterium, S. alvi, G. apicola and Firm-5 (Lactobacillus). Furthermore, we compared the differences in the gut microbiota and spatiotemporal dynamics between A. mellifera and A. cerana. Materials and methods Sample collection A. cerana workers were collected from three colonies in Rong Chang county, ChongQing, China, during July and August 2013. In order to obtain adult workers of uniform age, two frames that contained capped brood were pulled out from bee colonies and held in a dark incubator at 34°C and 80% R.H. for 24 h to mimic hive conditions. For each colony, ten newly emerged workers were collected at day 1 and marked with enamel paint and then returned to their colony in the field. In total, there were 250 bees marked and returned back to the field colonies. Subsequently, five 3
marked bees were collected at days 5, 10, 15, 20, 25, and 30 individually for each colony. Samples were stored in 75% ethanol at −20°C for subsequent molecular analysis. In order to obtain larvae of the same age, we used the facility of laying controller which can be used to restrict the queen to lay eggs on only one frame. We selected three healthy colonies, EFB-infected colonies and SBV-infected colonies respectively for bee sampling in Nan Chuan county, Chong Qing, China, in August 2013. We put a frame without any eggs or brood into a laying controller, then caught the queen to the laying controller and put it back into each colony. After 24 h, we checked the frame in order to make sure that the eggs were enough for the research, then let the queen out of the laying controller. After three days of egg the stage and four days of the laval stage, the larvae were collected as 5th instar larvae from healthy, EFB-and SBV-infected colonies on the 5th day. We screened the EFB and SBV for the 5th instar larvae from EFB-and SBV-infected colonies using PCR method, and only EFB-and SBV-infected larvae were selected to do further examine. DNA, RNA extraction and disease detection For the worker samples, the whole guts (including crop, midgut, ileum and rectum) were dissected out from workers aseptically. The DNA was extracted from each
time
point
from
workers
with
a
Wizard® SV 96 Genomic DNA Purification System (Promega Corporation, Madison, USA) as described in Li et al. (2012). For the larval samples, we ground the individual larvae and separated it into two parts, RNA was extracted from one part using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, and the other oart was used for DNA extraction. All RNA samples were tested for the presence of seven viruses including Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Kashmir bee virus (KBV), Israeli acute bee paralysis virus (IAPV), and Sacbrood virus (SBV). Amplification of specific viral RNAs was carried out using the Access RT-PCR kit (Promega, Madison, WI) according to the manufacturer’s 4
instructions. PCR amplication conditions and primers were followed as our previous study (Li et al., 2012). All DNA samples were used to detect EFB, AFB and N.ceranae, separately. N.ceranae detection was based on Li et al. (2012). EFB and AFB bacteria screens were done following the methods in Govan et al. (1998) and Dobbelaere et al. (2001), respectively. Quantitative PCR (qPCR) for detection and quantification of the presence and abundance of four bacterial species The absolute qPCR method was used to detect and quantify the presence and abundance of bacterial species in adult workers. All the initial template DNA concentrations were normalized between samples, and a control PCR was performed using the A. cerana β-actin gene to verify that the DNA was of sufficient quality for qPCR. The primers were used from our previous study (Li, et al., 2012; Xu, et al., 2014). Quantitative PCR (qPCR) was performed in a total reaction volume of 25μL containing the following reagents: 9.5μL ddH2O and 12.5μL 2 x Power SybrGreen qPCR Master Mix (Applied Biosystems, Calif., USA); 0.5μL of each primer (10 μM), and 2μL template. qPCR reactions were performed on a ABI 7500 system (Applied Biosystems, Foster, CA, USA), using the following cycle conditions: 95℃ for 10 min, 40 cycles of 95℃ for 15 s, 60℃for 1 min with a melting curve observed at the end of each run. Standards were prepared for each of the four bacterial primers consisting of a 1:10 dilution series (adult worker samples) that was run with each qPCR to establish a standard curve. Cycle threshold (Ct) values of each sample were then compared to the standard curve to approximate the copy numbers for each bacterium in a given sample (Brucker and Bordenstein, 2012). The relative quantification method was used to assess the abundance of the bacterial species in larvae infected with EFB or SBV as in Li et al. (2012). The primers and bacterial phylotypes were the same as described above. Statistical analyses Statistical analysis was carried out in GraphPad Prism version 6.01 for Windows (GraphPad Software, California, USA). The 16S rRNA copy numbers estimated by 5
qPCR were compared across different time points using ANOVA with Tukey's multiple comparisons test. The significant difference of each bacterial load between EFB or SBV infected and non-infected larva with SYBR-Green used for detection was analysed using a one sample t-test. Results Dynamic of four characteristic gut bacteria in A. c. cerana gut The age of the A. cerana worker positively correlates with the abundance of the four bacteria. Because Day 1 workers have far fewer bacteria than older individuals, we omitted the Day 1 workers from the analyses. The results showed that the 16S rRNA gene copies of the four phylotypes increased with age, reaching the highest level at an age of 10 days (Lactobacillus), or 15 days (S. alvi and Bifidobacterium) or 20 days (G. apicola), then declining (Fig.1). Except for Lactobacillus, the 16S rRNA gene copies of the other three bacteria were very low at day 5 (Fig.1a, b, d). The 16S rRNA gene copies from G. apicola (P = 0.863 by Tukey's HSD) and Bifidobacterium (P = 0.863by Tukey's HSD) showed there was no significant difference between days 25 and 30; and the 16S rRNA gene copies of S. alvi and Lactobacillus decreased to a stable level from days 20 to day 25 (P = 0.532 and 0.939 by Tukey's HSD respectively), then it dropped to the lowest level at day 30 (Fig.1a, c). The gut bacteria of A. c. cerana in infected and uninfected larva. The relative numbers of the four bacterial taxa were compared between healthy and unhealthy larva infected by SBV and EFB, separately. The results showed that the 16S rRNA gene quantities of S. alvi (t = 19.89, p = 0.003) (Fig.2a) and Lactobacillus(t = 49.42, p = 0.0001) (Fig.2b) in larva infected by SBV were significantly lower than in the healthy ones. The difference in The Bifidobacterium (t = 2.67, p = 0.116) and G. apicola (t = 3.413, p = 0.076) between larvae infected by SBV and healthy ones was not significant. For the larva infected by EFB, their 16S rRNA gene quantities of S. alvi (t = 21.67, p = 0.002) (Fig. 2c) and Bifidobacterium (t = 16.84, p = 0.004) (Fig. 2d) were also significantly lower than in the healthy larvae. The difference in the 6
Lactobacillus(t = 2.294, p = 0.149) and G. apicola (t = 2.218, p = 0.157) between larvae infected by EFB and healthy ones was not significant. Discussion Research on A. mellifera showed that the larvae and new emerged bees had few or no bacteria in their guts (Martinson et al., 2012), which is consistent with our similar results for A. cerana. Our results suggest that quantities of the four bacteria in the A. cerana gut initially increases in adult workers from day 5 then later declines and remains stable for the rest of the time. Similar results have been reported in bumble bees (Xu et al., 2014). Koch et al.(2013) reported that the symbiotic bacteria (G. apicola and S. alvi) can be vertically transmitted from mother to daughters in bumble bees. Bees also acquire their gut bacteria through social contact within the hive, brood cell or older workers (Martinson et al., 2012; Powell et al., 2014). A. cerana workers likely also acquire the major gut bacteria through social contact or contact with the hive environment. When bees reach the adult workers age, they face an environment full of bacteria (Haydak, 1970). From ages 1-3 days, their task consists of cell cleaning, resting or grooming (Seeley, 1982). From ages 4-12 days, they serve as nurses and transfer their proteinaceous secretions to the younger and older bees in the nest (Ribbands, 1953; Seeley, 1982; Crailsheim, 1991, 1992). For A.cerana, the total levels of Lactobacillus peaked at day 10, suggesting a link between Lactobacillus and the production of larval food. From ages 12-21 days, the bees appear to spend more time on general colony maintenance such as comb building, then they transition to nectar processing and other tasks (Trumbo et al., 1997). So during ages 12-21, the most important physiological and biochemical processes of honeybees are participating in the food production, such as nectar and bread processing. Interestingly, the three bacteria peaked between ages 15-20 days corresponding to the ages of nectar and bee bread processing. Metagenome results also indicated that G. apicola, Firm (Lactobacillus) and Bifidobacterium have many functional capabilities linked to carbohydrate breakdown, and proposed that the Lactic acid bacteria (LAB) group 7
made up of Firm and Bifidobacterium was involved in nectar processing and metabolism of carbohydrates (Engel et al., 2012; Butler, 2013). Older A. mellifera workers (>20 days past eclosure) focus on foraging for pollen, nectar, propolis and water (Robinson,1992; Calderone,1998). Although these behavioral shifts might be expected to result in changes in the microbiota composition, Martinson et al.(2012) reported that the characteristic phylotypes were maintained throughout the worker's life. The difference between our results with A. cerana and Martinson et al.(2012)’s work might reflect species differences and colony differences. Recently, Powell et al.(2014) reported a more fine-scale sampling of A. mellifera workers from 2 colonies, with 9 sampling points up to Day 16, and did not find strong trends. Our study and the A. mellifera studies sampled only a few colonies, and whether composition shifts in older bees of either species needs further investigation. In this study, the number of gut bacteria began to decline after day 15 or day 20, but after 20 days of age, bacteria have a stable period, such as S. alvi and Lactobacillus in the age of 20 to 25 days, and G. apicola and Bifidobacterium in 25 to 30 days of age. And Martinson et al.(2012) reported the number of bacteria in the Apis mellifera gut at 9, 20 and 30 days of age remain stable, but they did not check the sample of the age of 15, our study showed that bacteria began to decline after the age of 15. This finding could be suggested that the loss decline of bacteria in the gut may be due to defecation of the foraging bee when it first leaves the hive after the 15th day. Many stressors, including various diseases, can contribute to the occurrence of dysbiosis in honeybees and potentially perturb gut bacteria (Crotti et al., 2013). Variations of gut microbial communities may be associated with pathogen infection (Cariveau et al., 2014). Interestingly, the gut community was less in larvae infected with EFB and SBV than in non-infected larvae, suggesting a negative interaction between these pathogens and the gut bacteria. These results have a similar discipline with Li et al. (2012) and Cariveau et al. (2014) which reported on the parasite infection research. Some symbiotic bacteria such as Beta (S. alvi) and Gamma-1 (G. apicola) phylotype of A. mellifera also have functions in protection against disease 8
(Martinson et al., 2012). Thus, these symbiotic bacteria in this study maybe participate in the role of virus or bacteria defense which lead to the decline after infection. Potentially, the gut symbionts confer defense or affect immune functioning. Alternatively, environmental stress could perturb the gut community and lead to higher susceptibility of the hosts (Engel and Moran, 2013). However, a causal relationship is not yet confirmed. Our results give an overview of the dynamics of A. cerana gut bacteria during different life stages. The mechanisms causing the bacteria to increase or decline are not yet known. Potentially, its gut microorganisms are key to bee health and resistance to biotic and abiotic stressors (Crotti et al., 2013). A.cerana is one of the most important pollinators in agricultural systems and is a significant link in the food supply in China and other Asian countries. We need further empirical work to understand the roles of gut bacteria in the health and disease of this important insect.
Acknowledgements We would like to thank Tang Hong, Ren Qin and Cao Lan for collecting honeybee samples. We thank Nancy A. Moran and J. Elijah Powell for very useful comments on the manuscript. This study was funded by China Agriculture Research System (CARS-45).
References Ahn, J.H., et al. 2012. Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea. J. Microbiol. 50 (5): 735-745. Bailey L, Ball B.V .1991. Honeybee pathology, 2nd Ed, Academic Press: San Diego USA. Blanchard, P., et al. 2014. Development and validation of a real-time two-step RT-qPCR TaqMan® assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies. J. Virol. Methods. 197: 7-13. Brucker, R.M. and Bordenstein, S.R. 2012. The roles of host evolutionary relationships (Genus: 9
Nasonia) and development in structuring microbial communities. Evolution, 66 (2): 349-362. Butler È, A.M.O.T., Proteins of novel lactic acid bacteria from Apis mellifera mellifera-an insight into the production of known extra-cellular proteins during microbial stress. BMC microbiology, 2013. 13 (1): p. 235. Calderone N. W. 1998. Proximate mechanisms of age polyethism in the honey bee, Apis mellifera L. Apidologie. 29 (1-2):127–158. Cariveau, D.P., et al. 2014. Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus). ISME J. Apr 24. doi: 10.1038/ismej.2014.68. Chen Y. C. Apicultura of China[M]. China Agriculture Press, 1993. Cox-Foster, D. L. and S. Conlan, et al. 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science. 318 (5848): 283-7. Crailsheim K. 1991. Interadult feeding of jelly in honeybee (Apis mellifera L) colonies. J. Comp. Physiol. B. 161:55–60. Crailsheim K. 1992. The flow of jelly within a honeybee colony. J. Comp. Physiol. B. 162:681–689. Crotti, E., et al. 2013. Microbial symbionts of honeybees: a promising tool to improve honeybee health. N. Biotechnol. 30 (6): 716-22. Dillon, R. J. and V. M. Dillon. 2004. The gut bacteria of insects: nonpathogenic interactions. Annu. Rev. Entomol. 49 (1): 71-92. Disayathanoowat, T., et al. 2012. T-RFLP analysis of bacterial communities in the midguts of Apis mellifera and Apis cerana honey bees in Thailand. FEMS Microbiol Ecol. 79 (2): 273-281. Dobbelaere, W. and Degraaf, D. C., et al. 2001. Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR. Apidologie 32: 363-370. Evans, J.D. and R.S. Schwarz. 2011. Bees brought to their knees: microbes affecting honey bee health. Trends Microbiol., 19 (12): 614-620. Engel, P. and N. A. Moran. 2013. The gut microbiota of insects - diversity in structure and function. FEMS Microbiol. Rev. 37 (5): 699-735. Engel, P. et al., 2012. Functional diversity within the simple gut microbiota of the honey bee. Proc. Nati. Acad. Sci. U.S.A. 109 (27): 11002-7. 10
Govan, V. A, Brözel V, Allsopp M H, et al. 1998. A PCR detection method for rapid identification of melissococcus pluton in honeybee larvae . Appl. Environ. Microbiol. 1998, 64 (5): 1983-1985. Hamdi, C. and A. Balloi, et al. 2011. Gut microbiome dysbiosis and honeybee health. J. Appl. Entomol. 135 (7): 524-533. Haydak, M. H. 1970. Honey bee nutrition. Annu. Rev. Entomol. 15 (1):143–156. Kaltenpoth, M. and T. Engl. 2014. Defensive microbial symbionts in Hymenoptera. Functional Ecology. 28 (2): 315-327. Koch, H. and P. Schmid-Hempel. 2011. Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. Proc. Natl. Acad. Sci. U. S. A. 108 (48): 19288-92. Koch, H, Abrol, D.P, Li J, et al. 2013. Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees. Mol. Ecol. 22 (7): 2028-2044. Li, J., et al. 2012. The prevalence of parasites and pathogens in Asian honeybees Apis cerana in China. PLoS One. 7 (11): e47955. Liang, Q. and Chen, D. F. 2009. Honeybee protection. 2nd Ed, China Agriculture Press: Beijing (in Chinese). Martinson, V. G., et al. 2011. A simple and distinctive microbiota associated with honey bees and bumble bees. Mol. Ecol. 20 (3): 619-28. Martinson, V. G., et al. 2012. Establishment of characteristic gut bacteria during development of the honey bee worker. App. Environ. Microbiol. 78 (8): 2830-2840. Mohr K.I., Tebbe C.C. 2006. Diversity and phylotype consistency of bacteria in the guts of three bee species (Apoidea) at an oilseed rape field. Environ Microbiol. 8: 258–272. Powell J. E., et al. 2014. Routes of acquisition of the gut microbiota of Apis mellifera. App. Environ. Microbiol. Sep 19. pii: AEM.01861-14. Rauch S, Ashiralieva A, Hedtke K, Genersch E. 2009. Negative correlation between individual-insect-level virulence and colony-level virulence of Paenibacillus larvae, the etiological agent of American foulbrood of honeybees. Appl. Environ. Microbiol.75: 3344–3347. Ribbands R. 1953. The behaviour and social life of honeybees. London Dover Publ Inc, New York. Robinson G. E. 1992. Regulation of division of labor in insect societies. Annu. Rev. Entomol. 37:637–665. 11
Seeley T. D. 1982. Adaptive significance of the age polyethism schedule in honeybee colonies. Behav. Ecol. Sociobiol. 11 (4):287–293. Trumbo S. T., Huang Z.Y., Robinson G. E. 1997. Division of labor between undertaker specialists and other middle-aged workers in honey bee colonies. Behav. Ecol. Sociobiol. 41:151–163. Winston M. L. 1991. The Biology of the Honeybee. Harvard University Press: Cambridge Mass USA. Xu L L and WU J et al. 2014. Dynamic variation of symbionts in Bumblebees during hosts growth and development. Scientia Agricultura Sinica. 47 (10):2030-2037.
Figure Legends Fig 1. Abundances of S. alvi (a), G. apicola (b), Lactobacillus (c) and Bifidobacterium (d) in different days and stages of A.cerana adult workers, measured as copies of the 16S rRNA gene. Letters above confidence intervals (1 standard deviation) represent significance levels (Tukey’s HSD).
Fig 2. Relative abundance of three bacterial groups between SBV-infected (a and b) and EFB-infected (c and d ) in the larvae of A.cerana.
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Graphical abstract
Highlights We detected the gut microbes of A.cerana during different developmental stage. Gut microbes in A. cerana tend to dynamic change and remains stable after 20 days. Gut microbes of larvae have a negative interaction with virus or bacterial diseases.
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