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Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location.

Hroncova Z, Havlik J, Killer J, Doskocil I, Tyl J, Kamler M, Titera D, Hakl J, Mrazek J, Bunesova V, Rada V - PLoS ONE (2015)

Bottom Line: However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence.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.We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

No MeSH data available.


Heatmap summarising the relative density of dominant denaturing electrophoresis bands of the 16S rRNA amplicon profiles of the total gastrointestinal tract contents of several honey bee ontogenetic stages.1-, 3- and 6-day old larvae (L1, L3 and L6, respectively), white and black pupae (PW, PB), young bees, drones and flying bees (BY, DR and BF, respectively) collected in 4 different locations (Dol, Postrizin, Ustrasice, Hostice). Samples are sorted by ontogenetic stage (A) and location (B). The colours refer to relative band strength according to the colour key.
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pone.0118707.g001: Heatmap summarising the relative density of dominant denaturing electrophoresis bands of the 16S rRNA amplicon profiles of the total gastrointestinal tract contents of several honey bee ontogenetic stages.1-, 3- and 6-day old larvae (L1, L3 and L6, respectively), white and black pupae (PW, PB), young bees, drones and flying bees (BY, DR and BF, respectively) collected in 4 different locations (Dol, Postrizin, Ustrasice, Hostice). Samples are sorted by ontogenetic stage (A) and location (B). The colours refer to relative band strength according to the colour key.

Mentions: Samples of honey bees in various ontogenetic stages were acquired from 4 apiary sites within 10–100 km distance of each other (S4 Fig.), and 3 colonies from each apiary site were investigated to determine bacterial diversity. This experiment is referred to as EXP1. DGGE fingerprinting profiles of 200-base pair (bp) amplicons of the 16S rRNA V3 region showed between 20–30 bands. Of these, the 17 abundant or well aligning bands (S5 Fig.) were matched manually through multi-gel alignment of all 96 samples. Selected major band intensities were displayed in a heatmap (Fig. 1). This visualisation revealed that all three G. apicola (Gamma1) strains occurred rather randomly and at very low intensities in 1st instar larvae (L1) and at even lower intensities in 4th larval instar (L3), but became abundant bands in 5th instar larvae prior capping and defecation (L6). In later stages, they were mostly absent in both pupal stages (white pupa, PW, and black pupa, PB) and began occurring randomly in samples of young bees (BY) in some apiary sites. These strains were very abundant in foraging bees (BF) and drones (DR) with similar distribution profiles. At each apiary site, there were colonies with absent or highly abundant Gilliamella sp. (Gil) strains. Gil 1 (100% similarity to wkB1T) and Gil 2 was generally present as a more intense band than Gil 3. Frischella perrara (Fri) (Gamma-2) showed a very similar pattern to Gilliamella sp. strains (Fig. 1).


Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location.

Hroncova Z, Havlik J, Killer J, Doskocil I, Tyl J, Kamler M, Titera D, Hakl J, Mrazek J, Bunesova V, Rada V - PLoS ONE (2015)

Heatmap summarising the relative density of dominant denaturing electrophoresis bands of the 16S rRNA amplicon profiles of the total gastrointestinal tract contents of several honey bee ontogenetic stages.1-, 3- and 6-day old larvae (L1, L3 and L6, respectively), white and black pupae (PW, PB), young bees, drones and flying bees (BY, DR and BF, respectively) collected in 4 different locations (Dol, Postrizin, Ustrasice, Hostice). Samples are sorted by ontogenetic stage (A) and location (B). The colours refer to relative band strength according to the colour key.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358834&req=5

pone.0118707.g001: Heatmap summarising the relative density of dominant denaturing electrophoresis bands of the 16S rRNA amplicon profiles of the total gastrointestinal tract contents of several honey bee ontogenetic stages.1-, 3- and 6-day old larvae (L1, L3 and L6, respectively), white and black pupae (PW, PB), young bees, drones and flying bees (BY, DR and BF, respectively) collected in 4 different locations (Dol, Postrizin, Ustrasice, Hostice). Samples are sorted by ontogenetic stage (A) and location (B). The colours refer to relative band strength according to the colour key.
Mentions: Samples of honey bees in various ontogenetic stages were acquired from 4 apiary sites within 10–100 km distance of each other (S4 Fig.), and 3 colonies from each apiary site were investigated to determine bacterial diversity. This experiment is referred to as EXP1. DGGE fingerprinting profiles of 200-base pair (bp) amplicons of the 16S rRNA V3 region showed between 20–30 bands. Of these, the 17 abundant or well aligning bands (S5 Fig.) were matched manually through multi-gel alignment of all 96 samples. Selected major band intensities were displayed in a heatmap (Fig. 1). This visualisation revealed that all three G. apicola (Gamma1) strains occurred rather randomly and at very low intensities in 1st instar larvae (L1) and at even lower intensities in 4th larval instar (L3), but became abundant bands in 5th instar larvae prior capping and defecation (L6). In later stages, they were mostly absent in both pupal stages (white pupa, PW, and black pupa, PB) and began occurring randomly in samples of young bees (BY) in some apiary sites. These strains were very abundant in foraging bees (BF) and drones (DR) with similar distribution profiles. At each apiary site, there were colonies with absent or highly abundant Gilliamella sp. (Gil) strains. Gil 1 (100% similarity to wkB1T) and Gil 2 was generally present as a more intense band than Gil 3. Frischella perrara (Fri) (Gamma-2) showed a very similar pattern to Gilliamella sp. strains (Fig. 1).

Bottom Line: However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence.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.We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

No MeSH data available.