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.

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.

Specifications Table

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.

Data Description

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.

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%

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

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.

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.

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.

Experimental Design, Materials and Methods

Materials and methods

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

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.

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	TablesFigures
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	RawFiltered
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/PRJNA686953Datasets 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