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. 2021 Apr 2;36:107019. doi: 10.1016/j.dib.2021.107019

Dataset of the next-generation sequencing of variable 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees kept under anthropogenic landscapes

Marek Gancarz a,b, Paul J Hurd c, Przemyslaw Latoch d,e, Andrew Polaszek f, Joanna Michalska-Madej g, Łukasz Grochowalski g, Dominik Strapagiel g, Sebastian Gnat h, Daniel Załuski i, Robert Rusinek a, Agata L Starosta e,j, Patcharin Krutmuang k,l, Raquel Martín Hernández m,n, Mariano Higes Pascual m, Aneta A Ptaszyńska c,o,1,⁎
PMCID: PMC8079459  PMID: 33937454

Abstract

Forager Apis melliefera honeybees were collected from four localities located in Europe, i.e.: London, UK; Athens, Greece; Marchamalo, Spain and Lublin, Poland. Furthermore, from Asia we have collected A. mellifera as well as A. cerana foragers form Chiang Mai in Thailand

We used next generation sequencing (NGS) to analyse the 16S rRNA bacterial gene amplicons based on the V3-V4 region and the ITS2 region from fungi and plants derived from honeybee samples. Amplicon libraries, were prepared using the 16S Metagenomic Sequencing Library Preparation, Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System (Illumina®) protocol. NGS raw data are available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA686953. Furthermore, isolated DNA was used as the template for screening pathogens: Nosema apis, N. ceranae, N. bombi, tracheal mite (Acarapis woodi), any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e., Crithidia mellificae), neogregarines including Mattesia and Apicystis spp. (i.e., Apicistis bombi).

The presented data can be used to compare the metagenomic samples from different honeybee population all over the world. A higher load of fungi, and bacteria groups such as: Firmicutes (Lactobacillus); γ-proteobacteria, Neisseriaceae, and other unidentified bacteria was observed for Nosema cearana and neogregarines infected honeybees. Healthy honeybees had a higher load of plant pollens, and bacteria groups such as: Orbales, Gilliamella, Snodgrassella, and Enterobacteriaceae. More details can be found in research article [1] Ptaszyńska et al. 2021.

Keywords: NGS, Apis cerana; Nosema sp.; Acarapis woodi; Trypanosomatida; Crithidia spp.; neogregarines; Apicystis spp.; Anthropocene

Specifications Table

Subject Biological sciences:
Entomology and insect science
Specific subject area Datasets:
Dataset 1. In the excel file are the raw original information form NGS of composition of bacteria from 16S_taxonomyReads from seasonal changes of Polish honeybee samples (collected from April to September).
Dataset 2. In the excel file are the raw original information form NGS of composition of fungi and plant pollen from ITS_taxonomyReads from seasonal changes of Polish honeybee samples (collected from April to September).
Dataset 3. In the excel file are the raw original information form NGS of composition of bacteria from 16S_taxonomyReads from UK, Greece, Spain and Thailand honeybee samples.
Dataset 4. In the excel file are the raw original information form NGS of composition of bacteria from ITS_taxonomyReads from UK, Greece, Spain and Thailand honeybee samples.
B. One-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the detected fungi and plant pollens detected on the basis of ITS NGS analysis.
Dataset 5. Table 1A shows the one-way ANOVA report from the correlation between Polish honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. In English.
Dataset 6. Table 1B shows the one-way ANOVA report from the correlation between Polish honeybees’ health status and fungi and plant pollens detected on the basis of ITS NGS analysis. In English.
Dataset 7. Table 2A shows the one-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. In English.
Dataset 8. Table 2B shows the one-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the detected fungi and plant pollens detected on the basis of ITS NGS analysis. In English.
Dataset 9. Table 1A shows the one-way ANOVA report from the correlation between Polish honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. In Polish.
Dataset 10. Table 1B shows the one-way ANOVA report from the correlation between Polish honeybees’ health status and fungi and plant pollens detected on the basis of ITS NGS analysis. In Polish.
Dataset 11. Table 2A shows the one-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. In Polish.
Dataset 12. Table 2B shows the one-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the detected fungi and plant pollens detected on the basis of ITS NGS analysis. In Polish.
Type of data Tables
Figures
How data were acquired NGS sequencing and the analysis of the 16S rRNA bacterial gene amplicon was based on the V3-V4 region and the ITS2 eukaryotic region for bee DNA samples.
PL1-PL6 samples of Apis mellifera worker honeybees were collected from an urban apiary located in Lublin city, Poland, from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September).
Other samples of Apis mellifera worker honeybees were collected in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4).
From one time and location, 3 specimens (forager honeybees) were taken, as the representative and consistent number for each group (data adequacy confirmed by the PCA analysis).
Genomic DNA was extracted from whole honeybees using QIAamp DNA Kit according to manufacturer's instructions. Before pooling samples for libraries, the concentration was measured and the final concentration of pooled libraries for sequencing was 8 pM. Prepared libraries were sequenced on an Illumina MiSeq platform, 2 × 300 sequence reading in paired ends mode. The run contained PhiX libraries (PhiX Control Kit v3, Illumina®), to serve as an internal positive quality control.
Amplicons for the 16S region and ITS2 were sequenced using the Illumina MiSeq platform. Data were trimmed and merged. For 16S analyses only full-length reads over 229 bp with medium length of all sequences at 414 bp were used [Table 1]. Sequences were assigned to taxonomy using classifier trained on SILVA 132 database with minimum similarity 90% of read matching to the reference. For ITS2 analyses only full-length reads over 269 bp with medium length of all sequences at 337 bp were used [Table 1].
Sequences were assigned to taxonomy using classifier trained on all eukaryotes UNITE database v8.2 with the minimum similarity of 90% of the read matching to the reference [2,3]. Obtained data alpha rarefaction by country is shown on Fig. 1 and Principal Component Analysis (PCA), using Jaccard 's similarity based on taxonomy composition of 16S rRNA and ITS2 on Fig. 2.
Isolated DNA was used as the template for screening pathogens: Nosema apis, Nosema ceranae, Nosema bombi, tracheal mite (Acarapis woodi), any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e., Crithidia mellificae), neogregarines including Mattesia and Apicystis spp. (i.e., Apicistis bombi). The presence of pathogens [Table 3] in collected bee samples was detected using ITS2 amplicon data and specific primers under standard PCR, according to methodology described to Nosema apis by Martín-Hernández et al. [4], Nosema ceranae by Martín-Hernández et al. [4]; Nosema bombi by Klee et al. [5] Tracheal mite (Acarapis woodi) by Yang et al. [6]; any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e. Crithidia mellificae) Meeus et al. [6]; neogregarines including Mattesia and Apicystis spp. (i.e. Apicistis bombi) Meeus et al. [6].
Data format Raw
Filtered
Parameters for data collection Forager honeybees were recognized as bees returning to the hive and captured at the hive entrance around midday. Genomic DNA was extracted from whole honeybees using QIAamp DNA Kit according to manufacturer's instructions. Isolates were sent to the Biobank, Poland for NGS analysis.
Description of data collection Forager honeybees were recognized as bees returning to the hive and captured at the hive entrance around midday. All foragers were captured individually, using tweezers.
Data source location Data source locations are presented in Table 2.
Data accessibility All raw data are available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA686953
Datasets are available at:
Ptaszynska, Aneta A (2021), “Dataset of the next-generation sequencing of variable 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees kept under anthropogenic landscapes”, Mendeley Data, V1, http://dx.doi.org/10.17632/5zrz4fmw5y.2
Related research article [1] Ptaszyńska A.A., Latoch P., Hurd P.J., Polaszek A., Michalska-Madej J., Grochowalski Ł., Strapagiel D., Gnat S., Załuski D., Gancarz M., Rusinek R., Krutmuang P., Martín Hernández R., Higes Pascual M., Starosta A.L. 2021. Amplicon sequencing of variable 16S rRNA from bacteria, and ITS2 regions from fungi and plants, reveals honeybee susceptibility to diseases results from their forage availability under anthropogenic landscapes. MDPI Pathogens. 10,3 http://dx.doi.org/10.3390/pathogens10030381
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Value of the Data

  • •

    Next-generation sequencing (NGS) has revolutionized the biological sciences and obtained data can help analysing bee biology, food preferences and susceptibility to diseases.

  • •

    Standardized data collection of honeybee microbiome derived from NGS data is crucial for proper data analysis.

  • •

    Urban beekeeping is under urgent studies due to pollinator crisis and honeybee NGS data can be useful to construct an urban ecological network.

  • •

    Correlation of honeybee microbiome from NGS data with pathogens can lead to new forms of active protection of pollinators.

  • •

    High loads of bacteria such as: Orbales, Gilliamella, Snodgrassella, Enterobacteriaceae and plant pollen can be used as honeybee well-being indicators.

1. Data Description

1.1. Datasets

NGS raw data are available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA686953.

Datasets 1-12 are available at: Ptaszynska, Aneta A (2021), “Dataset of the next-generation sequencing of variable 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees kept under anthropogenic landscapes”, Mendeley Data, V1, http://dx.doi.org/10.17632/5zrz4fmw5y.1 Table 1. describes sequences filtering statistics  Input – initial number of sequences, Filtered – number of reads after removing low-quality data, Denoised – number of reads after removing data considered as noise, Merged – number of correctly merged forward and reverse reads, Non-chimeric – number of sequences after chimera removal; final number of reads. Table 1a. describes 16S reads from Polish samples. Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland. Table 1b. describes ITS2 reads from Polish samples. Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland. Table 1c. describes 16Sreads from UK, GR, ES, TAI samples. Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4). Table 1d. describes ITS2 reads from UK, GR, ES, TAI samples. Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4). Table 2. describes localities of investigated samples.

Fig. 1.

Fig. 1

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Shows the alpha-rarefaction by country. The sampling depth was set at 14096 for 16S amplicon sequencing data and 34100 for ITS amplicon data. This parameter was selected to include all available samples in the analysis and, as can be seen in the graphs above, it is sufficient to show the full taxonomic diversity for samples from individual countries. Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4).1A.  shows the 16S alpha-rarefaction by country1B. shows ITS2 alpha rarefaction by country.

Fig. 2.

Fig. 2

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Shows Principal Component Analysis (PCA), using Jaccard 's similarity based on taxonomy composition of 16S rRNA and ITS2. Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland.

Table 1.

Sequences filtering statistics.

• Input – initial number of sequences,
• Filtered – number of reads after removing low-quality data,
• Denoised – number of reads after removing data considered as noise,
• Merged – number of correctly merged forward and reverse reads,
• Non-chimeric – number of sequences after chimera removal; final number of reads.
16S – PL.
Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland.
sample-id input filtered percentage of input passed filter denoised merged percentage of input merged non-chimeric percentage of input non-chimeric
PL1(1) 38239 20701 54,14 20637 20406 53,36 19902 52,05
PL1(2) 44576 29660 66,54 29562 29288 65,7 28528 64
PL1(3) 49083 26149 53,28 26064 25771 52,5 25092 51,12
PL2(1) 40017 24250 60,6 24216 24095 60,21 23239 58,07
PL2(2) 62869 39145 62,26 39058 38865 61,82 37217 59,2
PL3(2) 22213 14178 63,83 14140 14114 63,54 14096 63,46
PL3(3) 41999 31808 75,74 31750 31707 75,49 31521 75,05
PL4(1) 61900 41059 66,33 40969 40507 65,44 38764 62,62
PL4(2) 54334 36764 67,66 36575 36270 66,75 34761 63,98
PL4(3) 72110 49176 68,2 49052 48547 67,32 46512 64,5
PL5(1) 46551 28087 60,34 27929 27743 59,6 27473 59,02
PL5(2) 41235 25786 62,53 25673 25551 61,96 25359 61,5
PL5(3) 43505 28996 66,65 28945 28898 66,42 28820 66,25
PL6(1) 44911 24875 55,39 24748 24401 54,33 22598 50,32
PL6(2) 37642 20884 55,48 20784 20462 54,36 18948 50,34
PL6(3) 63812 34485 54,04 34327 33869 53,08 31062 48,68
ITS2 – PL.
Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland.
sample-id input filtered percentage of input passed filter denoised merged percentage of input merged non-chimeric percentage of input non-chimeric
PL1(1) 160044 51024 31,88 50799 44057 27,53 43528 27,2
PL1(2) 147968 39779 26,88 39609 34607 23,39 34223 23,13
PL1(3) 166321 45521 27,37 45319 39417 23,7 38953 23,42
PL2(1) 161433 123703 76,63 123528 122002 75,57 113293 70,18
PL2(2) 120890 90633 74,97 90481 89267 73,84 82117 67,93
PL2(3) 140416 102813 73,22 102632 102355 72,89 100746 71,75
PL3(1) 147599 106749 72,32 106540 105072 71,19 102410 69,38
PL3(2) 125356 94618 75,48 94229 92777 74,01 90241 71,99
PL3(3) 91520 64349 70,31 64054 62253 68,02 60547 66,16
PL4(1) 157768 84762 53,73 84292 82715 52,43 81679 51,77
PL4(2) 129401 79102 61,13 78655 76783 59,34 75749 58,54
PL4(3) 113704 66404 58,4 66103 64776 56,97 63820 56,13
PL5(1) 170603 128436 75,28 128035 126164 73,95 123249 72,24
PL5(2) 162549 120393 74,07 120123 118010 72,6 115383 70,98
PL5(3) 144894 98781 68,17 98569 96751 66,77 94714 65,37
PL6(1) 148006 108317 73,18 107808 106726 72,11 105435 71,24
PL6(2) 141798 100218 70,68 100005 99095 69,88 97342 68,65
PL6(3) 130099 95359 73,3 95236 94250 72,44 93123 71,58
16S –UK, GR, ES, TAI.
Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4).
sample-id input filtered percentage of input passed filter denoised merged percentage of input merged non-chimeric percentage of input non-chimeric
UK-1 125453 70721 56,37% 70261 69149 55,12% 62612 49,91%
UK-2 113210 60764 53,67% 60625 60386 53,34% 58239 51,44%
GR-1 189774 101189 53,32% 100943 98753 52,04% 98281 51,79%
GR-2 219902 124908 56,80% 124282 122054 55,50% 107053 48,68%
ES-1 165148 91760 55,56% 91535 91274 55,27% 89325 54,09%
ES-2 135182 73017 54,01% 72836 72647 53,74% 72582 53,69%
TAI-1 205607 122277 59,47% 122053 121803 59,24% 120289 58,50%
TAI-2 275928 158746 57,53% 158498 157855 57,21% 156277 56,64%
TAI-3 247489 148904 60,17% 148390 147315 59,52% 136300 55,07%
TAI-4 233312 137798 59,06% 137420 136713 58,60% 132126 56,63%
ITS2 – UK, GR, ES, TAI.
Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4).
sample-id input filtered percentage of input passed filter denoised merged percentage of input merged non-chimeric percentage of input non-chimeric
UK-1 60154 35292 58,67% 35052 34656 57,61% 34132 56,74%
UK-2 141920 76895 54,18% 76768 74365 52,40% 74196 52,28%
GR-1 192913 83547 43,31% 83462 82158 42,59% 81601 42,30%
GR-2 121281 67887 55,97% 67627 64625 53,29% 61797 50,95%
ES-1 263444 112349 42,65% 112231 105442 40,02% 105254 39,95%
ES-2 152574 108152 70,88% 108133 107913 70,73% 107913 70,73%
TAI-1 297119 148360 49,93% 148075 121757 40,98% 121337 40,84%
TAI-2 254112 138951 54,68% 138582 136814 53,84% 135843 53,46%
TAI-3 321713 160884 50,01% 160527 133804 41,59% 130792 40,65%
TAI-4 206686 123427 59,72% 123298 121189 58,63% 119821 57,97%
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Table 2.

Localities of investigated samples.

Country City Geographical coordinates Sample abbreviation Time of samplings Organisms
Poland Lublin 51°15′N 22°34′E PL1 April Apis melliefera
PL2 May Apis melliefera
PL3 June Apis melliefera
PL4 July Apis melliefera
PL5 August Apis melliefera
PL6 September Apis melliefera
UK London 51°52′N 0°03′W UK1 July Apis melliefera
51°29′N 0°10′W UK2 July Apis melliefera
Greece Athens 37°59′N 23°42′E GR1 November Apis melliefera
GR2 November Apis melliefera
Spain Marchamalo 40°68′N 3°21′W ES1 November Apis melliefera
ES2 November Apis melliefera
Thailand Chiang Mai 18°50′ 98°58″E TAI1 February Apis melliefera
TAI2 February Apis melliefera
TAI3 February Apis cerana
TAI4 February Apis cerana
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Ptaszynska, Aneta A (2021), “Dataset of the next-generation sequencing of variable 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees kept under anthropogenic landscapes”, Mendeley Data, V1, http://dx.doi.org/10.17632/5zrz4fmw5y.2.

Dataset 1. Excel 1_16S_taxonomyReads_BEES-PL. In the excel file are the row original information form NGS of composition of bacteria from 16S_taxonomyReads from seasonal changes of Polish honeybee samples (collected from April to September).

Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland.

Description of the taxonomy Excel tables

The excel tables contain the composition of 7 taxonomic level for the individual samples. The sampling depth for 16S sequencing data is 14096 and for ITS data 34100. This is the total sum of the reads at each level.

Levels denotes accordingly:

Level 1 = Kingdom,

Level 2 = Phylum,

Level 3 = Class,

Level 4 = Order,

Level 5 = Family,

Level 6 = Genus,

Level 7 = Species.

Dataset 2. Excel 2_ITS_taxonomyReads_BEES-PL. In the excel file are the row original information form NGS of composition of fungi and plant pollen from ITS_taxonomyReads from seasonal changes of Polish honeybee samples (collected from April to September).

Description of the taxonomy Excel tables

The excel tables contain the composition of 7 taxonomic level for the individual samples. The sampling depth for 16S sequencing data is 14096 and for ITS data 34100. This is the total sum of the reads at each level.

Dataset 3. Excel 3_16S_taxonomyReads_BEES-other. In the excel file are the row original information form NGS of composition of bacteria from 16S_taxonomyReads from UK, Greece, Spain and Thailand honeybee samples.

Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4).

Description of the taxonomy Excel tables

The excel tables contain the composition of 7 taxonomic level for the individual samples. The sampling depth for 16S sequencing data is 14096 and for ITS data 34100. This is the total sum of the reads at each level.

Levels denotes accordingly:

Level 1 = Kingdom,

Level 2 = Phylum,

Level 3 = Class,

Level 4 = Order,

Level 5 = Family,

Level 6 = Genus,

Level 7 = Species.

Dataset 4. Excel 4_ITS_taxonomyReads_BEES-other. In the excel file are the row original information form NGS of composition of bacteria from ITS_taxonomyReads from UK, Greece, Spain and Thailand honeybee samples.

Description of the taxonomy Excel tables

The excel tables contain the composition of 7 taxonomic level for the individual samples. The sampling depth for 16S sequencing data is 14096 and for ITS data 34100. This is the total sum of the reads at each level.

Dataset 5. The table 1A reports the sequences filtering statistics of one-way ANOVA from the correlation between Polish honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland.

Dataset 6. The table 1B reports the sequences filtering statistics of one-way ANOVA from the correlation between Polish honeybees’ health status and fungi and plant pollens detected on the basis of ITS NGS analysis. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland.

Dataset 7. The table 2A reports the sequences filtering statistics of one-way ANOVA from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4).

Dataset 8. The table 2B reports the sequences filtering statistics of one-way ANOVA from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the detected fungi and plant pollens detected on the basis of ITS NGS analysis. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4).

Dataset 9. The table 1A reports the sequences filtering statistics of one-way ANOVA report from the correlation between Polish honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland. In Polish.

Dataset 10. The table 1B reports the sequences filtering statistics of one-way ANOVA report from the correlation between Polish honeybees’ health status and fungi and plant pollens detected on the basis of ITS NGS analysis. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled from April till September: PL1 (April), PL2 (May), PL3 (June), PL4 (July), PL5 (August), PL6 (September). Honeybees were collected from an urban apiary located in Lublin city, Poland. In Polish.

Dataset 11. The table 2A reports the sequences filtering statistics of one-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the bacteria detected on the base of 16S rDNA NGS analyses. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4). In Polish.

Dataset 12. The table 2B reports the sequences filtering statistics of one-way ANOVA report from the correlation between UK, Spain, Greece and Thailand honeybees’ health status and the detected fungi and plant pollens detected on the basis of ITS NGS analysis. For the ANOVA test, the level of statistical significance was assumed to be α = 0.05 and the same level of statistical significance was used in all comparisons. The results for which p values equal to or less than 0.05 were obtained differ significantly from each other. Apis mellifera worker honeybees sampled in July in London, UK (UK1, UK2); in November in Athens, Greece (GR1, GR2); in November in Marchamalo, Spain (ES1, ES2), and Chiang Mai, Thailand (TAI1, TAI2). From Chiang Mai, Thailand were also sampled Apis cerana worker honeybees (TAI3, TAI4). In Polish.

2. Experimental Design, Materials and Methods

2.1. Materials and methods

2.1.1. Honeybee collection and DNA isolation

Forager honeybees were captured from five localities situated in urban areas of Poland, UK, Spain, Greece and Thailand (Table 2). Genomic DNA was extracted from whole honeybees using QIAamp DNA Kit according to manufacturer's instructions. Isolates were sent to the Biobank, Poland for NGS analysis.

Isolated DNA was used as the template for screening pathogens: Nosema apis, Nosema ceranae, Nosema bombi, tracheal mite (Acarapis woodi), any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e., Crithidia mellificae), neogregarines including Mattesia and Apicystis spp. (i.e., Apicistis bombi), using PCR techniques described earlier [4], [5], [6], [7]. Detected pathogens are listed in Table 3.

Table 3.

Describes the presence of pathogens in collected bee samples.

Sample abbreviation Time of samplings Presence of pathogens based on ITS2 and PCR detection* of:
  • •

    Nosema apis

  • •

    N. ceranae

  • •

    N. bombi

  • •

    tracheal mite (Acarapis woodi)

  • •

    any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e. Crithidia mellificae);

  • •

    neogregarines including Mattesia and Apicystis spp. (i.e. Apicistis bombi).
PL1 April
  • •

    Nosema ceranae
PL2 May –
PL3 June
  • •

    Betsia sp.
PL4 July
  • •

    Nosema ceranae

  • •

    neogregarines
PL5 August –
PL6 September
  • •

    Nosema ceranae

  • •

    neogregarines
UK1 July –
UK2 July
  • •

    neogregarines
GR1 November
  • •

    Cyanobacteria

  • •

    Nosema ceranae

  • •

    neogregarines
GR2 November –
ES1 November –
ES2 November
  • •

    Nosema ceranae

  • •

    neogregarines
TAI1 February
  • •

    Nosema ceranae

  • •

    neogregarines
TAI2 February –
TAI3 February –
TAI4 February
  • •

    Nosema ceranae

  • •

    Neogregarines
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Pathogens detected using ITS2 amplicon data and specific primers under standard PCR, according to methodology described to Nosema apis by Martín-Hernández et al. [4], Nosema ceranae by Martín-Hernández et al. [4]; Nosema bombi by Klee et al. [5] Tracheal mite (Acarapis woodi) by Yang et al. [6]; any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e. Crithidia mellificae) Meeus et al. [7]; neogregarines including Mattesia and Apicystis spp. (i.e. Apicistis bombi) Meeus et al. [7]; – no detected pathogens.

Ethics Statement

Although no permission is needed to administer experiments on insects, our research was planned in a way that reduced the number of honeybees to the minimum necessary for the proper conducting of these experiments.

CRediT Author Statement

Author Contributions: Marek Gancarz, and Robert Rusinek: analysed obtained data, interpreted the results, co-wrote the paper; Paul J. Hurd: analysed data, especially of metabiom and parasites, co-wrote the paper; Przemyslaw Latoch: analysed data. Patcharin Krutmuang, analysed Thai data, co-wrote the paper; Raquel Martín Hernández, and Mariano Higes Pascual: analysed UK data, co-wrote the paper; Aneta A. Ptaszyńska: co-wrote the paper; Joanna Michalska-Madej: conducted laboratory work for sequencing library preparation, sequencing, and detection of pathogens; Łukasz Grochowalski: analysed raw data from metabiom sequencing, prepared tables, co-wrote the paper; Agata L. Starosta: analysed data. Dominik Strapagiel, analysed data from metabiom sequencing, co-wrote the paper; Sebastian Gnat: performed genetic analyses; Daniel Załuski: drafted and made a correction of the manuscript; Aneta A. Ptaszyńska: (senior author), designed the experiments, analysed data, and wrote the paper.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.

Acknowledgments

The publication of the article was financed by the Polish National Agency for Academic Exchange under the Foreign Promotion Programme (NAWA), for AAP (bee-research.umcs.pl; Api Lab UMCS PPI/PZA/2019/1/00039). Honeybees were collected during the Miniatura 2 project ID 418332 founded by the NCN for AAP and the first research plans were possible thanks to EU: GB-TAF-7137 SYNTHESYS project for AAP. Work in the Hurd lab was funded by the BBSRC (BB/L023164/1) and granted to PJH.

References

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