Introduction

Honeybees belong to the genus Apis, which is known for its tremendous role in pollination. Unfortunately, honeybee population is recently declining with a potential risk on the agricultural service and subsequently the food supply, not only locally in Saudi but also globally¹. There is a known mutually beneficial relationship between honeybee gut microbiome and its host. The host provides the optimum environment for bacterial growth, while the bacterial community in honeybee guts aids in efficacy of nutrients absorption, optimum growth and development of the host and its ability to defend pathogens, and its adaptation to surrounding environment².Honeybee gut represents a simple model system to study the relationshipbetween gut microbiome with honeybeehosts^3,4.The bacterial community in adult honeybee workers is diverse and estimated to reach one billion bacterial cells in each worker’s gut^5,6. Such a diversity in bacterial community is dependent on the type of flower that hosts the insect, as well as many other environmental factors⁷.Gut microbiome of honeybee (Apis mellifera) workers is composed ofeight to nine core species^8,9, e.g., Bartonella apis¹⁰, Acetobacteraceae¹¹,Parasaccharibacter¹¹, Snodgrassella alvi¹², Bifidobacterium asteroids¹³, Lactobacillus sp.¹⁴, Frischella perrara¹⁵and Gilliamella apicola^12.

The two most common bee species that are widely distributed throughout the kingdom of Saudi Arabia are the indigenous Apis mellifera jementica, which is a native species, and Apis mellifera carnica, which is imported from Egypt¹⁶ as honey production of domestic bees does not meet the growing demands in Saudi Arabia. Moreover, the production cost is relatively high. Exotic bee colonies have been imported over time, reaching 200,000 bee packages annually¹⁶. It is well known for local beekeepers that the indigenous bees A.m. jementicahighly tolerates local stressful conditions when compared with exogenous races A.m. carnica, particularly during summer when the air temperature becomes extremely high. It is also noticed that at high temperatures, indigenous bees continue to forage for pollen and collect nectar, whereas imported bees will stop foraging¹⁶. Initial reports revealed that the subspecies of exotic honeybees have lower heat tolerance, shorter foraging durations and are more susceptible to Varroa mites when compared with indigenous bees^16.

In the present study, we compered the gut microbiomecomposition and diversity of the adult honeybees of Apis mellifera jementica and Apis mellifera carnica in Saudi Arabia using high-throughput 16S rRNA gene sequencing technology.

Material and Methods

Sample collection,isolation of guts microbiota and DNA extraction

Five samples each from honeybee workers of A.m. jemenitica and A.m. carnicawere collected in November 2019 from a single hive of Beekeeper Cooperative Association at Al Baha, Saudi Arabia. The collected samples were immediately stored at −80°C.Forwhole gut dissection of honeybee workers,surface disinfection was done using 1 ml aqueous ethanol (70%, v/v) for 45 sec. Dissected guts were, then,placed in a pre-frozen mortar and 700μl S1 lysis buffer (Invitrogen, Thermo Fischer Scientific, USA) were added and guts were transferred to bead tube for extraction process.DNA of gut samples was extracted by the genomic DNA extraction kit (Invitrogen, Thermo Fischer Scientific, USA), and stored at -20°C for further molecular analysis.

PCR amplification

PCR was run to amplify bacterial 16S rRNA gene of the variable regions V3-V4. The two universal primers used for PCR are 341F 5′-ACTCCTACGGGAGGCAGCAG-3′ (forward primer) and 806R 5′- GGACTACHVGGGTWTCTAAT-3′ (reveres primer). The PCR conditions were set as the following: one cycle for initial denaturation at 95°C for 5 min; 25 cycles of denaturation at 95°C for 30sec followed by annealing at 56°C for 30 sec andprimer extension at 72°C for 40 sec; and a one cycle for final extension at 72°C for 10 min. The generated PCR products were checked for quality and selected products were utilized in preparing Illumina DNA libraries. DNA sequencing was run using Illumina Miseq platform (Illumina, San Diego, CA) at Beijing Genome Institute (BGI), China to generate high-quality pair-ends of ~300 bp.

Statistical analysis

The high quality paired reads produced in fasta files as raw data were de-multiplexed, quality-filtered and trimmed by trimmomatic package (Version 0.33) through Quantitative Insights Into Microbial Ecology 2 pipeline (QIIME2, v1.80). Obtained reads were merged into single sequence files by the Fast Length Adjustment of SHort reads (FLASH, Version 1.2.11). In order to assign generated unique sequences into operational taxonomic units (OTUs), reads were tagged and clustered into OTUs with similarity cut off of 97% using the de novo OTU piking procedure. Usearch (Version 7.0.1090)¹⁹ was, then, used to remove Chimeric sequences. Taxonomies were plotted against the gut Microbiome Database (HOMD RefSeq, Version 13.2) through the RDP classifier (Version 2.2)¹⁷ and the Green-genes database (version 201305¹⁸16S rDNA database, http://qiime.org/home_static/dataFiles.html) with a cut off of 70%. Alpha diversity indeceswere measured in order to assess the intra-species variations within a given sample using Mothur (v1.31.2). Alpha diversity and rarefaction curve boxplots were constructed using software R (v3.1.1). To investigate the inter-species variations within samples, the beta diversity matrices were conducted and visualized using principal coordinate analysis (PCoA) by package ‘ade4’ of software R (v3.1.1). Also, heat maps were generated using the package ‘gplots’ of software R (v3.1.1), and, then, sequence alignments were searched against the Silva core set (Silva_108_core_aligned_seqs) by using PyNAST ‘align_seqs.py’. The obtained OTU phylogenetic tree was, then, plotted by software R (v3.1.1), and visualized through QIIME2 (v1.80).

Annotation of generated OTUs was done in order to detect the relative abundance at different taxonomical levels (phylum, genus and species). Finally, Metastats, PERMANOVA and Benjamini–Hochberg false discovery rate (FDR) correction were also used to correct for multiple hypothesis. The Linear Discriminant Analysis (LDA) Effect Size (LEfSe) was applied using software LEfSewith the online interface Galaxy (version 1.0.0; http://huttenhower.sph.harvard.edu/galaxy/root),to discriminate the two taxonomic races determining highly presented bacterial taxon within each race depending on statistical significance.

Results

Statistics of 16S rRNA Sequence data

The five gut microbiome samples of A.m. carnicawere identified asC1 to C5, while the five gut microbiome samples of A.m. jemeniticawereidentified as J1 to J5. Illumina MiSeq was used in sequencing thepartial 16S rRNA gene and statistics details are tabulated in Table 1. A total of 2,279,519 sequence reads were obtained with an average length of 297 bp across different samples ranging from 293 to 300 bp. The average clean reads per subject were 218,741 and 237,162 for A.m. carnicaand A.m. jemeniticaworkers, respectively. A total of 2,269,665 tag-linked sequences were obtained across samples from both taxonomic groups with an average read of 236,125 and 217,807 per subject, for both A.m. carnicaand A.m. jemeniticaworkers, respectively (Figure S1). Furthermore, a total of 601,485 tags were obtained across samples with an average reads of 63,313 and 56,983 per subject, in both A.m. carnicaand A.m. jemeniticaworkers,respectively (Figure S1). The tagged sequences were assigned to a total of 171 OTUsacross samples ranging from 45 (J3) to 154 (C5) OTUs (Table S1). The sum of OTUs in A.m. carnica (A.M.C) is 373 with an average number of 74 OTUs, while 241 in A.m. jemenitica(A.M.J)with an average number of 48 OTUs.

Table 1: Statistics of deep sequencing data generated for gut microbiomes of 10 adult honeybee workers from subspecies A.m.carnica(C1-C5) andA.m.jemenitica (J1-J5).

Alpha diversity and principle coordinate analyses and rarefaction curve measurement

Alpha diversity indices were used to analyse the complexity of the included species. These indices are observed species (S_obs), Chao1, Ace, Shannon and Simpson. The S_obs and Chao1 indices indicated significance differences between A.M.C and A.M.Jgroups with higher diversity in A.M.C group. The P-values determined were 0.03175 for S_ob and0.01587 for Chao1 indices (Figure 1 and Table 2). On theother hand, Shannon and Simpson indices revealed no significant difference between A.M.C and A.M.Jgroups. Chao1 and Shannon indicesreflect the species diversity in terms of richness, while Simpson index isindicative of evenness²⁰.The Simpson values in A.M.C and A.M.J groups were 0.1377 and 0.12313, respectively, while, Shannon index values were 2.4658 and 2.4769 in A.M.C and A.M.J groups, respectively(Figure 1 and Table 2).

Figure 1: Boxplots of alpha diversity indices illustrates richness and evenness at the group level of gut microbiomes of adult honeybee  workers of A.m. carnica (red) and A.m. jemenitica (blue) Click here to view figure

Table 2: Alpha diversity comparison results among groups of gut microbiomes of honeybee workers from subspeciesA.m.carnica(A.M.C) andA.m.jemenitica (A.M.J).

Principal coordinate analysis (PCoA) was used to display the diversity as well as the differences in OTU composition.Diversity of A.M.C subjects was higher towards positive and negative PCA 1 directions (PC1), whereasthat of A.M.J subjects was higher towards positive and negative PCA 2 directions (PC2). As an overall picture, the diagram shows that the mean value of A.M.C group was localized in positive portion of PC1 and negative portion of PC2, whereasA.M.J group was mainly localized in the positive portion of PC2(Figure 2). The principal coordinate analysis (PCoA) plots were created using a Bray-Curtis distance matrix and the samples were plotted to represent the microbial community compositional differences between samples. The plots are dimensionally scattered in accordance to their gut microbiome compositional relationships. The results of the present study indicate that the differences ingut microbiomes between these two groups arepossibly due to the different origins of worker honeybeesof the two subspecies.

Figure 2: PCoA based on OTU abundance of samples. Red boxes represent A.M.C (A.m.carnica) samples. Blue boxes represent A.M.J (A.m.jemenitica) samples. Each dot denotes one sample. X-axis is the first principal component and Y-axis is the second. Number in brackets denotes contributions of PCAs to differences among samples. Click here to view figure

The stacked number of OTUs and the number of observed species for different samples as rarefaction measures are shown in Figure S2. When the refraction curves inclines (Figure S2a) or stops climbing (Figure S2b), the produced data would be enough for further analysis. However, as long as the curve is still climbing, the complexity of the data in samples become higher; since more species being detected throughout sequencing analysis. The two rarefaction curve measures refer to the maximum number of sequences attained for all samples that allows to study taxonomic relative abundance and to assess eligibility of such data to represent all species of any microbial community. The findings from both rarefaction measures show that 54,000 is the maximumnumber of sequence reads that can be used further instudyingtaxonomic abundance (Figure S2).

Structure of gut microbiomes across the two honeybee workers

Two taxonomic ranks (phylum and species) were used in the comparison of gut microbiomesbetween adult honeybee workers A.M.CandA.M.J at the phylogenetic level (Figure 3). The results indicate that phylumFirmicutesharbours 24 genera,while Proteobacteria,Actinobacteria, Bacteroidetes andThermiharbour 23, 8, 6 and 2 genera, respectively (Figure 3).

Figure 3: Phylogenetic tree at genus level of gut microbiomes of adult honeybee workers of A.m.carnica and A.m.jemenitica.        Genera having the same color belong to the same phylum. Click here to view figure

Differential abundance of microbes due to different origin of worker

The observed microbial taxa along with their redundancies across different samples identified after OTU annotation are described in Table S2. The taxa refer to phylum, class, order, family, genus, and species. Eight phyla of the gut bacteria were identified according to relative abundance. They are Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Protobacteria, TM7, Tenericutes and Thermi(Figure 4). Aligning with the number of genera of each phylum shown in Figure 3, the most abundant phylum were Firmicutes (57%), Protobacteria (31%) and Actinobacteria (10%) inA.M.C group (Figure 4).

Figure 4: Metastatsmeasures for relative abundance of gut microbiomes at the phylum level forthe group levels of A.m.carnica andA.m.jemenitica. Click here to view figure

Meanwhile, Firmicutes (48%), Protobacteria (44%) and Actinobacteria (6%) were the most abundant in A.M.J group (Figure 4). The comparison at phylum level revealed a significant increase in Cyanobacteria in the A.M.C group (P-value = 0.031746), while a significant increaseof Protobacteria in the A.M.J group (P-value = 0.037724)(Table S3). Interestingly, Table S3 also indicates the existence of the three phyla TM7, Tenericutes and Thermi only in A.M.C group. The previous results align with those ofthe heat map at phylum level asFirmicutes, Protobacteria and Actinobacteria were shown to be the most abundant phyla across samples and groups (Figure S3).

In terms of species relative abundance in the gut microbiomes of two groups A.M.C and A.M.J Bacteroides_fragilis, Bacteroides_ovatus, Commensalibacter_intestini, Blautia_producta, Melissococcus_plutonius, Ruminococcus_gnavus,Saccharibacter_floricola and Snodgrassella_alviwere shown to be the most abundant (Figure 5).

Figure 5: Relative abundance of gut microbiomes of adult honeybee workers of A.m.carnica and A.m.jemeniticaamong(a and b, respectively) and across species (c) as measured by Metastats.**High significant difference between microbiomes of A.m.carnicaand A.m.jemenitica. Click here to view figure

The figure also indicates that a large proportion of the OTUs were not assigned to a certain species (93.80% for A.M.C and 86.20% for A.M.J). We have no explanation for these results except that a large number of species in workers of honeybee was not identified or classified before. The results in Table S4 indicates asignificant increase of Melissococcus_plutoniusin the gut microbiome of A.M.C (P-value = 0.034454), while Snodgrassella_alvi in theA.M.J group (P-value =0.008948). Results for the latter species Snodgrassella_alvi align with that presented in Figure 5c. TheRuminococcus_gnavusandSaccharibacter_floricolawere not existed in theA.M.J group. The heat map at species level indicates that Snodgrassella_alviharbours the highest relative abundanceacross all samples (Figures S4).

Linear discriminant analysis effect size (LEfSe) and its LDA scores (˃ 3) wereused to identify possible biomarkers in gut microbiota that refer to the origin of the host (Figure 6). The results in cladogram indicate that the possible marker in gut microbiome of A.M.C is Enterococcaceae family, while Neisseriaceaea, Neisseriales and Betaproteobacteria taxa of A.M.J (Figure 6a). Biomarkers in A.M.C based on LDA score includeEnterococcaceae, Saccharibactersp.,Saccharibacterflorcola, Firmicutes (Melissococcussp. andMelissococcusplutonius) and Cyanobacteria while Betaproteobacteria (Neisseriales, Neisseriaceae,Snodgrassella sp. andSnodgrassella_alvi)in A.M.J (Figure 6b).

Figure 6: The main different bacterial taxa (biomarkers) for gut microbiomes of adult honeybee workers of A.m. carnica (A.M.C) and A.m. jemenitica (A.M.J). a) Cladogram-based LEfSe analysis representing main different microbiota taxa between groups. b) taxa with LDA score > 3. Colour codes: Yellow (a) denotes no significant difference in taxa; Green denotes significantly different taxa (biomarkers) with their relative maximum abundance in (A.M.J); Red denotes significantly different taxa (biomarkers) with their relative maximum abundance in (A.M.C). Click here to view figure

Discussion

The gut microbiomestructure of honeybee workersis dependent upon monophyletic origin of the host⁹, social interactions⁸and the type of diet consumed, whether workers are beebread, pollen or nectar^1.In the present study, high-throughput sequencing was carried out for samples taken from the two honeybee subspecies A.m. carnicaandA.m. jemenitica and statistical analysis proved that the diversity of the bacterial community composition ofA.m. carnicaandA.m. jemenitica was statistically significant.

Four major bacterial phyla (Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes) were recognized in the guts of honeybee workers of the two subspecies A.m. carnicaand A.m. jemenitica. The dominant phylum ingut microbiomes of the two subspecies wasFirmicutes with values of 57.2 and 48.5%, respectively. This conclusion was also drawn in several previous reports^9,21,22,23.Genus Lactobacillus, gram-positive bacteria belongingto the family Lactobacillaceae (Firmicutes), was found to have a high relative abundance in adult workers of both A.m. carnicaandA.m. jemeniticawith values of 52% and 80%, respectively. It is a core gut bacterium that is dominant in the rectum of honeybee workers. Within this context, Ahnet al.²⁴ concluded thatLactobacillaceaedominates in both of A. cerana and A. mellifera species.This genus produces several compounds in honeybee gut with known antimicrobial activities such as organic acids, hydrogen peroxide, bacteriocin, reutericyclin and reuterin that mostly inhibit decaying and protects againstpathogenic bacteria, as well as some fungi^25,26.Therefore,Honeybeeslikely use lactobacilli as probiotic^27.In the present study, the dominance of Lactobacilli in both A.m. carnicaandA.m. jemeniticaadult workers is supported by the presence of low pH (3.9) of honey and nectar²⁸. This is concluded because of the ability of lactobacilli to ferment sugar in the gut of honeybee workers and, hence,to generate acidic environment²⁹, which inhibits the growth of many other bacteria. The low abundance in Lactobacillaceaewas reported to be associated with the presence of pathogenic bacteria³⁰.

GenusBifidobacterium,gram-positive bacteria belonging to the Actinobacteria phylum, was also identified in gut of both A.m. carnicaandA.m. jemenitica adult workers. Again, it is dominant in rectum, and a core gut bacteria of honeybee workers.Bifidobacterium strains carry large surface proteins, which have a role in adhesion or degradation of plant materials^7,31,32. Additionally, Bifidobacteriumcarriesgene clusters that are responsible for the production and utilization of trehalose, which is a disaccharide molecule used by insects as an energy reservoir, in comparison to glycogen, which is the energy storage form in mammals³³.

Family Neisseriaceaeand its descendentSnodgrassella_alvi(S. alvi), gram-negative bacteria belonging toBetaproteobacteria phylum, significantly increased in A.m. jemenitica.These bacteria participate in oxidation of carbohydrates. However, the pathway for the uptake and glycolytic breakdown of carbohydrates does not exist in S. alvi, thus,this bacteriumis located consistently within the periphery of the insect’s gut lumen. This area has high oxygen concentrations and this environment is preferable for S. alvidue to its dependence on aerobic respiration^34,35. Insects depend on the aerobic oxidation of carboxylates rather than breaking down carbohydrates resulting in various products such as citrate, malate, acetate and lactic acid that serve as energy sources^12,27. The steady co-exits of S. alvi with other fermentative bacterial taxa in the same gastrointestinal environment can result from utilizing separate sets of resources leading to metabolic variations suggesting a syntrophic interaction. For example, S. alvican utilize some of the substrates such as lactic acid, acetate and formate, which are produced from carbohydrate fermentation^36,37. Furthermore, S. alvi and G. apicola³⁸are enriched with genes encoding biofilm formation. The two species inhabit the host’s ileum, indicating that the biofilm can provide a protective layer against pathogens.

The bacteria of the family Acetobacteraceaeand its descendent genusCommensalibacter(also referred to as Alpha 2.1), gram-negative bacteria belonging to phylum Proteobacteria,wereidentifiedas a core member of the gut microbiota in honeybees and bumble bees^9,31. It was observed mainly in the midgut and hindgut of honeybee workers. In our study, Commensalibacterpresents in A.m. carnicaandA.m. jemenitica. However, Saccharibacterflorica (Alpha-2.2) presents only in A.m. carnica. Furthermore, Saccharibacterflorica is isolated from pollen, suggesting that this phylotype is associated with flowers^39.The role of these phylotypes (Alpha 2.1 andAlpha-2.2) is associatedwith their abilities to adaptwith fast growing metabolic processes, with two distinctive mechanisms. Alpha2.1 bacteria harvest energy througha wide range of substrates linkedand utilizedthrough a flexible oxidative and biosynthetic metabolism.Whereas, Alpha2.2 bacteria, that lack alternative oxidative pathways, determine metabolic processes through oxidative fermentation after harvesting glucose for rapid energy⁴⁰.

The bacteria of the familyEnterococccaceaeand its descendent species Melissococcusplutonius, gram-positive bacteria of phylum Firmicutes, present in low abundance (3%) in gut microbiome ofA.m. carnica honeybee workers. This conclusion was also noted in previous reports⁴¹.M. plutoniusis known to cause the European foulbrood (EFB) in earlystage of honeybee larvae, with assistance from secondary invaders (Enterococcus faecalis, Paenibacillus alvei and Bacillus pumilus). M. plutonius wasshown to have 30 different sequence types clustered under three clonal complexes (CC 3, CC12, and CC13)^42,44, where CC13is the least virulent complex^43,45. Honeybee workers transmit M. plutonius between colonies via robbing and drifting^46,47.Erban et al.⁴⁵ compared control samples from the EFB zone with samples from EFB zone without clinical symptoms,and bees from colonies from EFB zone with clinical symptoms. The study identified a 100-fold higher prevalence of M. plutonius in colonies with EFB symptoms, while it only presents in 3 of 16 control colonies that are distant from the EFB zone. This suggests that M. plutonius has lower abundance in healthy honeybee colonies, which is consistent with the results of the present study.

Conclusion

The present findings indicative that differences in gut microbiome structuresof honeybee workers of the two subspecies A.m. carnicaand A.m. jemeniticaare due to varied monophyletic origin of the host. These findings support previous results suggesting that honeybee workers have a mutual coevolving relationship with specific group of bacteria. This group of bacteria co-exists and is maintained throughout the descending generations of the host.Inclusion of more subspecies inhabited in Saudi Arabia along with ones of this study can further support our findings.

Supplementary Information

Additional file: Additional Table  S1, S3 and S4 Additional Figure S1 to S4

Additional file: Additional Table S2

Acknowledgement

This study was supported by Beekeeper Cooperative Association-Al Baha. The authors would like to thank Prof. Dr.Ahmad Al-Ghamdi, Dept. Plant Protection, College of Food and Agricultural Sciences, KSU, Riyadh, for his support and cooperation during this study.

Conflict of Interest

The authors declare no conflict of interest.

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