Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession.

Bee Research Institute at Dol Libcice nad Vltavou Czech Republic

Bee Research Institute at Dol Libcice nad Vltavou Czech Republic; Department of Zoology and Fisheries Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Prague Czech Republic

Department of Forage Crops and Grassland Management Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Prague Czech Republic

Department of Microbiology Nutrition and Dietetics Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Prague Czech Republic

Department of Microbiology Nutrition and Dietetics Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Prague Czech Republic; Institute of Animal Physiology and Genetics v v i Academy of Sciences of the Czech Republic Prague Czech Republic

Institute of Animal Physiology and Genetics v v i Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Klein AM, Vaissiere BE, Cane JH, Steffan-Dewenter I, Cunningham SA, et al. Importance of pollinators in changing landscapes for world crops. Proc Biol Sci. 2007;274: 303–313. PubMed PMC

Genersch E. Honey bee pathology: current threats to honey bees and beekeeping. Appl Microbiol Biotechnol. 2010;87: 87–97. 10.1007/s00253-010-2573-8 PubMed DOI

Forsgren E, Olofsson TC, Vásquez A, Fries I. Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie. 2010;41: 99–108.

Evans JD, Lopez DL. Bacterial Probiotics induce an immune response in the honey bee (Hymenoptera: Apidae). J Econ Entomol. 2004;97: 752–756. PubMed

Martinson VG, Moy J, Moran NA. Establishment of characteristic gut bacteria during development of the honeybee worker. Appl Environ Microbiol. 2012;78: 2830–2840. 10.1128/AEM.07810-11 PubMed DOI PMC

Jeyaprakash A, Hoy MA, Allsopp MH. Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences. J Invertebr Pathol. 2003;84: 96–103. PubMed

Engel P, Moran NA. Functional and evolutionary insights into the simple yet specific gut microbiota of the honey bee from metagenomic analysis. Gut Microbes. 2013;4: 60–65. 10.4161/gmic.22517 PubMed DOI PMC

Mattila HR, Rios D, Walker-Sperling VE, Roeselers G, Newton ILG. Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse. PloS One. 2012;7: e32962 10.1371/journal.pone.0032962 PubMed DOI PMC

Mrazek J, Strosova L, Fliegerova K, Kott T, Kopecny J. Diversity of insect intestinal microflora. Folia Microbiol (Praha). 2008;53: 229–233. 10.1007/s12223-008-0032-z PubMed DOI

Yoshiyama M, Kimura K. Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood. J Invertebr Pathol. 2009;102: 91–96. 10.1016/j.jip.2009.07.005 PubMed DOI

Mohr KI, Tebbe CC. Diversity and phylotype consistency of bacteria in the guts of three bee species (Apoidea) at an oilseed rape field. Environ Microbiol. 2006;8: 258–272. PubMed

Corby-Harris V, Maes P, Anderson KE. The Bacterial Communities Associated with Honey Bee (Apis mellifera) Foragers. PloS One. 2014;9: e95056 10.1371/journal.pone.0095056 PubMed DOI PMC

Ludvigsen J. Seasonal trends in the midgut microbiota of honeybees. PhD Thesis, Norwegian University of Life Sciences. 2013. Available: http://brage.bibsys.no/xmlui/handle/11250/186492. Accessed 17 June 2014.

Gilliam M, Prest DB. Microbiology of feces of the larval honey-bee, Apis-mellifera . J Invertebr Pathol. 1987;49: 70–75.

Kwong WK, Moran NA. Cultivation and characterization of the gut symbionts of honey bees and bumble bees: description of Snodgrassella alvi gen. nov., sp nov., a member of the family Neisseriaceae of the Betaproteobacteria, and Gilliamella apicola gen. nov., sp nov., a member of Orbaceae fam. nov., Orbales ord. nov., a sister taxon to the order 'Enterobacteriales' of the Gammaproteobacteria. Int J Syst Evol Microbiol. 2013;63: 2008–2018. 10.1099/ijs.0.044875-0 PubMed DOI

Moran NA, Hansen AK, Powell JE, Sabree ZL. Distinctive gut microbiota of honey bees assessed using deep sampling from individual worker bees. PloS One. 2012;7: e36393 10.1371/journal.pone.0036393 PubMed DOI PMC

Koch H, Abrol DP, Li JL, Schmid-Hempel P. Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees. Mol Ecol. 2013;22: 2028–2044. 10.1111/mec.12209 PubMed DOI

Engel P, Stepanauskas R, Moran NA. Hidden diversity in honey bee gut symbionts detected by single-cell genomics. PLoS Genet. 2014;10: e1004596 10.1371/journal.pgen.1004596 PubMed DOI PMC

Corby-Harris V, Snyder LA, Schwan MR, Maes P, McFrederick QS, et al. Origin and effect of Acetobacteraceae Alpha 2.2 in honey bee larvae and description of Parasaccharibacter apium, gen. nov., sp. nov. Appl Environ Microbiol. 2014;80: 7460–7472. 10.1128/AEM.02043-14 PubMed DOI PMC

Ahn J-H, Hong I-P, Bok J-I, Kim B-Y, Song J, et al. Pyrosequencing analysis of the bacteria communities in the guts of honey bees Apis cerana and Apis mellifera in Korea. Internet J Microbiol. 2012;50: 735–745. PubMed

Olofsson TC, Vasquez A. Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apis mellifera . Curr Microbiol. 2008;57: 356–363. 10.1007/s00284-008-9202-0 PubMed DOI

Patruica S, Mot D. The effect of using prebiotic and probiotic products on intestinal micro-flora of the honeybee (Apis mellifera carpatica). Bull Entomol Res. 2012;102: 619–623. 10.1017/S0007485312000144 PubMed DOI

Olofsson TC, Alsterfjord M, Nilson B, Butler É, Vásquez A. Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov., and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera . Int J Syst Evol Microbiol. 2014;64: 3109–3119. 10.1099/ijs.0.059600-0 PubMed DOI PMC

Killer J, Dubna S, Sedlacek I, Svec P. Lactobacillus apis sp. nov., from the stomach of honeybees (Apis mellifera) having in vitro inhibitory effect on causative agents of American and European Foulbrood. Int J Syst Evol Microbiol. 2013;64: 152–157. 10.1099/ijs.0.053033-0 PubMed DOI

Killer J, Kopecny J, Mrazek J, Rada V, Benada O, et al. Bifidobacterium bombi sp nov., from the bumblebee digestive tract. Int J Syst Evol Microbiol. 2009;59: 2020–2024. 10.1099/ijs.0.002915-0 PubMed DOI

Killer J, Kopecny J, Mrazek J, Havlik J, Koppova I, et al. Bombiscardovia coagulans gen. nov., sp nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees. Syst Appl Microbiol. 2010;33: 359–366. 10.1016/j.syapm.2010.08.002 PubMed DOI

Killer J, Kopecny J, Mrazek J, Koppova I, Havlik J, et al. Bifidobacterium actinocoloniiforme sp nov and Bifidobacterium bohemicum sp nov., from the bumblebee digestive tract. Int J Syst Evol Microbiol. 2011;61: 1315–1321. 10.1099/ijs.0.022525-0 PubMed DOI

Engel P, Kwong WK, Moran NA. Frischella perrara gen. nov., sp. nov., a gammaproteobacterium isolated from the gut of the honeybee, Apis mellifera . Int J Syst Evol Microbiol. 2013;63: 3646–3651. 10.1099/ijs.0.049569-0 PubMed DOI

Engel P, Martinson VG, Moran NA. Functional diversity within the simple gut microbiota of the honey bee. Proc Natl Acad Sci U S A. 2012;109: 11002–11007. 10.1073/pnas.1202970109 PubMed DOI PMC

Yun Ji-Hyun, et al. Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host. Appl Environ Microbiol. 2014;8017: 5254–5264 10.1128/AEM.01226-14 PubMed DOI PMC

de F Michelette ER, Soares AEE. Characterization of preimaginal developmental stages in Africanized honey bee workers (Apis mellifera L). Apidologie. 1993;24: 431–440.

Rembold H, Kremer J-P, Ulrich GM. Characterization of postembryonic developmental stages. Apidologie. 1980;11: 29–38.

Rettedal EA, Clay S, Brözel VS. GC‐clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles. FEMS Microbiol Lett. 2010;312: 55–62. 10.1111/j.1574-6968.2010.02097.x PubMed DOI

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 2010;28: 2731–2739. PubMed PMC

Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000;17: 540–552. PubMed

De Gregoris TB, Aldred N, Clare AS, Burgess JG. Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa. J Microbiol Methods. 2011;86: 351–356. 10.1016/j.mimet.2011.06.010 PubMed DOI

Braak CJFt, Šmilauer P. CANOCO reference manual and CanoDraw for Windows user's guide: software for canonical community ordination (version 4.5). Section on Permutation Methods Microcomputer Power, Ithaca, New York; 2002.

Huang Z-Y, Robinson GE. Regulation of honey bee division of labor by colony age demography. Behav Ecol Sociobiol. 1996;39: 147–158.

Vasquez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, et al. Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees. Plos One. 2012;7: e33188 10.1371/journal.pone.0033188 PubMed DOI PMC

Vojvodic S, Rehan SM, Anderson KE. Microbial Gut diversity of Africanized and European honey Bee larval instars. PloS One. 2013;8: e72106 10.1371/journal.pone.0072106 PubMed DOI PMC

Gilliam M. Microbial sterility of the intestinal content of the immature honey bee, Apis mellifera . Ann Entomol Soc Am. 1971;64: 315–316.

Kikuchi Y, Hosokawa T, Fukatsu T. Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation. Appl Environ Microbiol. 2007;73: 4308–4316. PubMed PMC

Powell JE, Martinson VG, Urban-Mead K, Moran NA. Routes of acquisition of the gut microbiota of Apis mellifera . Appl Environ Microbiol. 2014;80: 7378–7387. PubMed PMC

Ahn J-H, Hong I-P, Bok J-I, Kim B-Y, Song J, et al. Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea. Internet J Microbiol. 2012;50: 735–745. PubMed

Killer J, Votavova A, Valterova I, Vlkova E, Rada V, et al. Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris). Int J Syst Evol Microbiol. 2014;64: 2611–2617. 10.1099/ijs.0.063602-0 PubMed DOI

Killer J, Mrázek J, Bunešová V, Havlík J, Koppová I, et al. Pseudoscardovia suis gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa). Syst Appl Microbiol. 2013;36: 11–16. 10.1016/j.syapm.2012.09.001 PubMed DOI

In-hive variation of the gut microbial composition of honey bee larvae and pupae from the same oviposition time