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Article

Next Generation Sequencing of Bee Gut Microbiota in Urban and Rural Environments

by
Mustafa Rüstemoğlu
Plant Protection Department, Faculty of Agriculture, Şırnak University, İdil 73300, Şırnak, Türkiye
Diversity 2023, 15(9), 1016; https://doi.org/10.3390/d15091016
Submission received: 21 August 2023 / Revised: 8 September 2023 / Accepted: 12 September 2023 / Published: 14 September 2023
(This article belongs to the Special Issue Ecology and Diversity of Bees in Urban Environments)
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Abstract

:
The gut microbiota plays a vital role in the physiological and behavioral processes of organisms, thereby influencing the quality of life of their host. Investigating the microbial diversity of the gut microbiota of Apis mellifera, the ecological organizer, may provide key insights into the ecological and health-related factors affecting host populations. This study aims to investigate the comparative gut microbiota of forager A. mellifera collected from both rural and urban environments in each of the four provinces located in southeastern Türkiye by employing Next Generation Sequencing (NGS) technology, specifically amplicon metagenome sequencing targeting the V3–V4 region of the 16S rRNA gene. In general, the urban samples possessed a higher level of gut microbial diversity when compared with the rural samples. Interestingly, the rural samples, in addition to the majority of previously reported core microbiota members, contained human pathogens such as Salmonella enterica, Klebsiella pneumoniae, Escherichia coli, and Streptococcus pyogenes. Moreover, this study is the first to report the predominant existence of Enterococcus wangshanyuanii, Alkalihalobacillus halodurans, and Vitreoscilla sp. in several samples. This study contributes to the idea that urban apiculture practices can alter bacteria residing in the digestive tracts and discusses possible effects of potential infestation of A. mellifera by human-pathogenic bacteria on different aspects of life traits.

1. Introduction

The gut microbiota has a significant role in the health and well-being of animal life [1,2,3], affecting many aspects of an organism’s biology, including metabolism, immunity, and behavior [4,5,6]. For instance, it can aid in breaking down food and producing essential nutrients such as vitamins and amino acids that the host organism cannot produce by itself [3,4,7]. The gut microbiota also plays a crucial role in and the development and functioning of the immune system and protecting the host against pathogens [3,5,7]. Furthermore, recent studies suggest that the gut–brain axis influences behavior and brain function through the gut microbiota [6,7]. Various health problems have been associated with the disruption of the gut microbiota [5,8,9,10]. Pathogenic microorganisms have a negative impact on health, while commensal microorganisms can boost immunity, protect against pathogen invasion, and improve nutritional status [7,11,12,13]. Therefore, it is crucial to understand the interactions between the gut microbiota, the host, and the environment to promote the health and well-being of animals.
Honeybees are insects that are valuable both ecologically and economically due to the pollination services they provide to farmers and wildlife, as well as the beekeeping products that bee colonies produce [14,15]. The microbial community in the digestive tract of honey bees plays a significant role in their well-being by assisting in food digestion, detoxifying harmful substances (such as pesticides), providing essential nutrients, protecting against invading pathogens and parasites, regulating social behavior, and modulating the development and immunity [3,7,10,16]. Unfortunately, the health of honeybee colonies is threatened by colony losses, particularly in recent decades [17,18].
Honeybees are known to harbor a consistent and distinct core gut bacterial community [17,19,20,21,22,23,24]. Recent functional experimental studies and genomic analyses have identified various important roles of this core microbial community, including those related to nutrition and health [12,13,23,24,25,26,27,28]. Based on the analysis of the 16S rDNA in the gut of worker honeybees, this core microbiota is composed mostly of nine types of bacteria, which represent 95–98% of the community [16,19,20,29,30,31,32,33]. Among these core microbiota, Snodgrassella alvi, Gilliamella apicola, and Frischella perrara are single species within the Gram-negative bacterial phylum Proteobacteria [12,34]; Firm4, Firm5, and Bifido are closely related Gram-positive bacterial clusters composed of three species. While the Firm4 and Firm5 clusters have multiple Lactobacillus species, the Bifido cluster includes Bifidobacterium asteroides from honeybees [20,35,36]. Two other clusters of species come from distantly related groups of Alphaproteobacteria, initially named Alpha1 and Alpha2 [29]. Alpha1 is a close relative of Bartonella species and is present and frequently abundant among the sampled workers [31]. Alpha 2 consists of several strains of Acetobacteraceae, including the gut-specialized Alpha 2.1 and Alpha 2.2 (Parasaccharibacter apium) [32].
The transregional core bacterial communities are similar across population structures and regions [17,19,20,30,31,33]. Recent studies have shown that the relative abundance of some dominant members of the gut bacterial community of honeybees differs when bees are exposed to different environmental landscapes and different behavioral tasks [9,23,24,37].
In Türkiye, limited attention has been paid to the gut microbiota of honeybees [38]. Thus, this study employed 16S rRNA gene amplicon sequencing, which involves amplifying the 16S rRNA gene region using PCR primers and subsequently sequencing the amplified DNA using Next Generation Sequencing (NGS) technologies [39]. The NGS method enables a more thorough and in-depth examination of the honeybee gut microbiome [40,41].
Urban beekeeping has rapidly expanded globally [42]. The encouragement of urban beekeeping is gaining popularity due to reasons such as pollination, bee product production, community building, environmental education, and ecosystem sustainability [43,44].
The effects of urbanization on bees are complex, variable, and not well-understood [25,42,45]. Therefore, in this study, the gut microbial diversity of returning pollen, water, and nectar foragers in both rural and urban areas of the same province was investigated using Miseq amplicon assays of the 16S rRNA gene. The findings demonstrate how urban and rural beekeeping affect the microbial community of honeybees and indicate potential effects on important gut bacteria in honeybees. The study underscores the intricate interplay among the host, its gut bacteria, and the contrasting rural and urban environments.

2. Materials and Methods

2.1. Sampling and Dissection

Samples of A. mellifera foragers were collected from eight different localities in four provinces of Türkiye, with ten foragers collected from the same apiary at each location. Forager bees were collected from both rural (R) and urban (U) areas in the provinces of Şırnak (SR, SU), Diyarbakir (DR, DU), Mardin (MR, MU), and Van (VR, VU) in May (Figure 1). After collection, to avoid a potential contamination, living A. mellifera individuals were transported in small cages containing sugar powder to the laboratory of the Plant Protection Department of Şırnak University, where they were stored at −20 °C until processing. The samples were rinsed with 96 percent ethanol, and the entire alimentary canals were dissected aseptically. The alimentary canal preparation forceps had never been used before, and one pair of forceps was used to process only ten forager bees from the same apiary. The extracted alimentary canals were macerated in a clean 0.8% NaCl solution and then immediately frozen at −20 °C [46].

2.2. Isolation of Bacterial Genomic DNA from Samples

Ten forager bees’ digestive tracts were aggregated to create a single composite sample, which constituted one experimental replication. These pooled samples were collected from a single apiary. These composite samples were then subjected to dissection procedures in order to isolate DNA. The Zymo Research Quick-DNA TM Fecal/Soil Microbe Miniprep Kit (Cat. No.: D6010, Irvine, CA, USA) was used to isolate genomic DNA, and the directions were followed. To quantify the DNA, the Qubit dsDNA BR Assay Kit (Invitrogen, Carlsbad, CA, USA) was utilized, following the manufacturer’s protocols.

2.3. Amplification of 16S rRNA V3–V4 Region

The amplification of the V3–V4 regions of the 16S rRNA gene was carried out using the SimpliAmp Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) and universal primers (Alpha DNA, Montreal, QC, Canada) Pro341F (5′-CCTACGGGNBGCASCAG-3′) and Pro805R (5′-GACTACNVGGGTATCTAATCC-3′). Polymerase chain reaction (PCR) was conducted following the PCR Protocol for Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Hitchin, UK) as described [47]. The resulting amplification products were analyzed on a 2% agarose gel.

2.4. Preparation of NGS Library and Sequencing

To purify the 16S rRNA V3–V4 amplicons, the Column-Pure Gel and PCR Clean-Up Kit (Cat. No.: D509, ABM Good, Los Angeles, CA, USA) was employed. For the preparation of NGS libraries and indexing, the Illumina Nextera XT DNA Library Prep Kit (Cat. No.: FC-131-1096) and TG Nextera XT Index Kit v2 Set A (96 indices, 384 samples, Cat. No.: TG-131-2001) were utilized, respectively. Subsequently, the Illumina Miseq platform (Illumina, San Diego, CA, USA) was employed for sequencing, employing paired-end (PE) mode and generating 2 × 150 base read lengths.

2.5. Bioinformatics Analysis

The raw NGS reads (FASTQ) obtained from sequencing were subjected to quality control and, if required, trimming. Subsequently, they were classified into operational taxonomic units (OTUs) using the Kraken metagenomics system. This system accurately and efficiently assigns taxonomic labels to short DNA sequences [48].

2.6. Shannon and Simpson Diversity Index

The Shannon and Simpson diversity indices were employed to assess the species richness and evenness of bacteria within the honeybee’s alimentary tract. These indices provide quantitative measures of bacterial diversity [49]. The Shannon diversity index typically ranges from 1.5 to 3.5 and increases with greater evenness. On the other hand, the Simpson diversity index (1-D) ranges from 0 to 1, with a value of 1 indicating complete evenness.

3. Results

A total of 579,659 paired reads were produced, with an average length of 124.6 ± 3.87 bases and a quality score of Q30 > 90%. The variable regions V3 and V4 of the 16S rRNA gene in forager bees’ gut contained an average of 72,457 high-quality reads per sample, ranging from 7500 to 160,605 in urban and rural beekeeping areas (Table 1). Bacterial richness and diversity were estimated in the alimentary canal of forager bees from both rural and urban beekeeping areas. Substantial diversity was observed in both beekeeping areas (Table 1). For rural beekeeping forager bees, the Shannon and Simpson indices of bacterial diversity (Shannon diversity: 2.219–2.963, Simpson evenness: 0.8157–0.8768) and, for urban beekeeping forager bees, (Shannon diversity: 2.455–3.727; Simpson evenness: 0.7741–0.9336) showed differences in bacterial diversity. (Table 1). The VR (Van-Rural) sample had the lowest Shannon and Simpson index while the DU (Diyarbakır-Urban) sample had the highest. In comparison to Van, Şırnak, and Mardin, the average Shannon and Simpson indices of the samples collected in the Diyarbakır region (DU and DR) were the highest (Table 1).
Variations in the relative abundance of bacterial phyla were observed among all samples. The bacterial community was dominated by Proteobacteria, followed by Firmicutes, Actinobacteria, Bacteroidetes, and Tenerocutes. Except for the VU sample, proteobacteria were the most abundant phylum in all samples. Rural samples (DR, SR, VR, MR) contained a greater proportion of proteobacteria (66.78%) than urban samples (DU, VU, MU, and SU) (62.65%). Firmicutes, the second most abundant phylum, was found to be the most abundant phylum in the VU sample (Figure 2A). The relative abundance of Actinobacteria, Firmicutes, and Proteobacteria exceeds 96% of the total bacteria (Figure 2A).
The bacterial families with the highest relative abundances in rural average samples (RA) were Orbaceae (33.95%), Lactobacillaceae (25.18%), Neisseriaceae (14.81%), Acetobacteraceae (6.19%), and Bifidobacteriaceae (5.10%). In the samples collected from urban areas, the predominant bacterial families identified were Lactobacillaceae, Orbaceae, Enterobacteriaceae, Enterococcaceae, and Neisseriaceae, accounting for approximately 18.50%, 18.05%, 11.49%, 8.24%, and 7.62% of the total microbial composition, respectively (Figure 2B).
In the VR, SU, DR, and SR samples, the Orbaceae family was the most abundant (48.94%, 34.1%, 30.5%, and 30.5%, respectively), while it ranked second in the MU and DU samples (21.58% and 14.2%, respectively). Lactobacillaceae was the most prevalent bacterial family in the MU and MR samples (39.33% and 26.92%, respectively), whereas in the VU sample, Enterococcaceae was the most prevalent family (32.96%), which is another lactic acid family. Enterobacteriaceae was the most prevalent bacterial family, comprising 14.43% of the DU sample, and the second most prevalent family, including 27.16% of the VU sample (Figure 2B).
For all samples, reads were assigned to 1545 unique bacterial species, with 30 species found in at least one sample at a frequency of 0.5% or higher (Supplementary Table S1). Except for the VU sample, Gilliamella apicola was the most frequently encountered species, while Snodgrassella alvi was the second most abundant species, dominating in four out of eight samples. Only in the VU sample was E. wangshanyuanii measured as the most abundant species, and it was not detected in any of the other seven samples (Supplementary Table S1). The second-most prevalent bacterial species in one sample were those listed below: Lactobacillus apis, Frischella perrara, Bombilactobacillus bombi, and S. enterica (Figure 2C).
In Figure 2C, the objective was to pinpoint the bacterial taxa exhibiting a notable difference in relative abundance between urban and rural landscape categories. A. raozihei, Entomomonas moraniae, C. perfringens, C. taklimakanense, and Pseudomonos sp. C27 (2019) were detected only in the rural samples, while G. apicola, S. alvi, F. perrara, L. kullabergensis, B. bombi, B. apis, Vitreoscilla sp. C1, and Morganella. morganii bacterial taxa were more prevalent in rural areas. L. apis, Commensalibacter sp. AM001, B. asteroides, L. helsingborgensis, E. coli, S. enterica, K. pneumoniae, S. pyogenes, Ahb. halodurans, B. indicum, C. acnes, and R. leguminosarum were more abundant in urban samples. E. wangshanyuanii, S. thermophilus, Lacrimispora saccharolytica, and P. stewartii were found only in urban samples (Figure 2C).
Heatmap clustering and PCoA analyses were conducted to determine the impact of urban and rural beekeeping landscapes on metagenomic diversity and abundance. As depicted in Figure 3, the geographical distance and the landscape of the beekeeping region had an effect on the diversity of bacterial species and the similarity of species composition among samples. The PCoA results led to the identification of three primary clusters in the metagenomic analysis, with discernible geographical distinctions among these clusters, as demonstrated in Figure 4.

4. Discussion

This study employed 16S metabarcoding analysis to examine the bacterial compositions of the digestive tract of A. mellifera in both rural and urban beekeeping areas located in southeastern Türkiye.
In terms of the density of the most common human pathogenic microorganisms, there is a similarity between the urban samples. However, Figure 3 and Figure 4 show that although the samples collected from different locations have similarities in terms of carrying the same human pathogenic microorganisms, geographical similarities are more influential in terms of both the most dominant species and total species density. Furthermore, the samples collected from urban beekeeping areas demonstrated a slightly higher level of Shannon and Simpson diversity compared to those from rural areas. Specifically, Diyarbakır and Van, which experience a greater degree of human interaction compared to Mardin and Şırnak, exhibited even greater diversity (Table 1). This suggests that in urban beekeeping areas, there is an additional presence of pathogenic microorganisms originating from metropolitan environments alongside the gut microbiota of honeybees.
Previous metagenome studies on honeybee gut showed characteristic communities of bacteria that contain nine main species clusters, also known as the core microbiota of the honeybee [20,22,32,50,51,52] These include Alpha-1 (Bartonella apis), Alpha-2.1 (Commensalibacter_sp_AMU001), Alpha-2.2 (Parasaccharibacter apium), Bifido (Bifidobacterium asteroides and Bifidobacterium indicum), Gilliamella apicola, Snodgrassella alvi, Lactobacillus Firm-4 (including L. mellis and L. mellifer), Lactobacillus Firm-5 (including L. apis, L. helsingborgensis, L. kullabergensis, L. kimbladii and L. melliventris), and Frischella perrara [12,32,35,36,52,53]. These nine phylotypes make up 95% of bacterial 16S rRNA sequences in the honeybee gut, despite environmental, geographic, and subspecies differences among hosts [20,30,31,32]. In this study, these nine distinctive phylotypes accounted for 61% and 86%, respectively, of bacterial 16S amplicon sequences in urban and rural areas. In the urban samples, the relative abundance of core microbiota was 56.43% in Diyarbakır (DU), 11.32% in Van (VU), 87.30% in Mardin (MR), and 86.92% in Şırnak (SU). The human population densities around the localities where the Diyarbakır (DU) and Van (VU) samples were collected are much higher than those in Mardin (MU) and Şırnak (SU), from which other urban samples were taken (see Figure 1). Among the ten most abundant species in rural areas, the captured forager bees contained core gut microbiota members of honeybees. However, in urban areas, the captured forager bees contained some well-known pathogenic or potentially pathogenic bacteria species, such as E. coli, S. enterica, K. pneumoniae, and S. pyogenes in addition to members of the core microbiota (see Figure 3). These bacteria are known to cause various diseases in humans, including diarrhea, nausea, vomiting, and fever [54,55,56,57].
Recent studies have shed light on the presence of pathogenic or potentially pathogenic bacteria species in the gut of honeybees. Some of the well-known bacteria species identified include Hafnia alvei, Serratia marcescens, Shigella sonnei, S. enterica, Melissococcus, Paenibacillus, and Spiroplasma [57,58,59]. Research has shown that when bacteria such as S. enterica, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, and Serratia marcescens are injected into the hemocoel of A. mellifera, it can lead to bee mortality. In fact, S. aureus-infected bees can serve as vectors for other hive mates, potentially causing a 50% decline in colony size within 24 h [60].
The studies have indicated that human pathogenic bacteria can also be found in other insects [8,61]. For instance, cockroaches in government hospital environments have been found to carry K. pneumoniae [62], and Diptera flies have been discovered harboring S. enterica [63]. In certain urban areas with high human activity, honey bees collected from locations such as botanical gardens have been found to carry strains of Salmonella [58]. Another study isolated two Klebsiella species, namely K. pneumoniae and K. oxytoca, from the digestive tracts of honeybees. The prevalence of K. pneumoniae was 26.7%, and that of K. oxytoca was 23.3% [64]. Furthermore, K. pneumoniae is abundant in dwarf bees, which are often found in close proximity to humans.
Another bacterium of interest is E. coli, which is commonly present in the honeybee gut along with other bacterial species. However, certain strains of E. coli, such as LF82 associated with inflammatory bowel disease, have been shown to harm the gut and cognitive functions of honeybees. In a study, honeybees exposed to LF82 exhibited increased gut permeability, impaired learning and memory, and reduced lifespan compared to bees exposed to the non-pathogenic E. coli strain MG1655 [65].
Considering these findings, urban beekeeping areas may pose increased risks to both bee and human health due to the potential presence of a greater variety of human pathogenic bacteria. The ability of certain bacteria to thrive in the honeybee digestive system and their association with detrimental effects on bee health raise concerns about the impacts of urban environments on bee populations. It is important to note that the prevalence and impact of pathogenic bacteria in honeybees may vary across different regions and environments. Further research is needed to better understand the dynamics of bee–microbe interactions in urban areas and to develop strategies to mitigate potential risks to both bees and human health. Overall, the presence of human pathogenic bacteria in the gut of honeybees, as indicated by various studies, highlights the need for increased awareness and appropriate management practices in urban beekeeping to minimize potential health hazards and promote the well-being of both bees and humans.
In summary, while human pathogenic bacteria can affect honeybees, they are just one of many factors that can lead to colony collapse. Further research is needed to fully understand the complex interactions between pathogens, parasites, pesticides, and other stressors that affect honeybee health. In addition to the core microbiota of the honeybee and human pathogenic bacteria, the most prevalent 10 species in the samples include E. wangshanyuani, M. morganii, B. bombi, E. moraniae, Ahb. halodurans, L. saccharolytica, and Vitreoscilla sp. strain C1 types were detected at least in one sample.
Recently, E. wangshanyuani was isolated and described from yak (Bos grunniens) feces in China, and it was proposed as a new species in 2017 based on its distinct genetic and phenotypic characteristics [66]. It was detected only in the Van-Urban (VU) sample, in which it was determined to be the most prevalent species. Enterococcus is a genus of lactic acid bacteria, Gram-positive cocci, typically found in pairs or short chains, and they are facultative anaerobic organisms, meaning they can respire in both oxygen-rich and oxygen-poor environments [66,67]. E. wangshanyuani is not a well-known or studied bacterium. However, diverse Enterococcus species were isolated from the digestive tracts of honeybees and Apis nigricans [67,68,69]. Enterococcus spp. (E. faecium) act as probiotics and food preservatives [67,69].
M. morganii is a type of bacteria that has been found in honeybees. It was identified as one of the operational taxonomic units (OTUs) in the honeybee-associated bacterial community affected by American foulbrood, a bacterial disease that affects honeybee larvae [70]. In addition, M. morganii has been studied for its in vitro antagonistic potential against Paenibacillus larvae, a major bacterial pathogen of honeybee broods [71,72,73]. It is a component of the gut microflora of bees that could function as an antibiotic-resistant microorganism [74] and has probiotic effects [72]. In this study, M. morganii was found in all samples, but it was only one of the ten most abundant species in the DR sample.
Recent research in South Korea discovered B. apium, a novel species of bacteria, in the gut of Asian honeybee (Apis cerana). Even though this species is closely related to the Firm-4 species B. mellis and B. bombi, it is distinct enough to be considered a new species [75]. Multiple studies [13,75,76,77,78,79] have discovered Bombilactobacillus species in the guts of A. mellifera and bumblebees. According to studies [13,79], Bombilactobacillus species members of the gut microbiota of bees plays an important role in bee health, including pathogen defense and behavior regulation. B. bombi is one of the top ten species in this study found in all rural samples (DR, VR, MR, and SR), as well as in SU, the smallest urban sample city in the study. It was initially discovered in the digestive tract of A. cerana, where the genus Apis first appeared [75]. For this study, it was also discovered in the A. mellifera gut.
E. moraniae was detected in the DR, MR, and MU samples, but only in the DR sample was it one of the ten most common bacterial species. E. moraniae was first isolated from the digestive tract of the Asian honeybee (A. cerana) collected from Pingwu County, Sichuan Province, PR China, has a highly reduced genome, and was classified as a new species in a new genus [80]. In the metagenome studies conducted on the gut of Xylocopa species (X. micans, X. mexicanorum, X. tabaniformis parkinsoniae), blast analysis revealed sequences with a 98.4% similarity to E. moraniae [81]. This study also revealed its presence in A. mellifera’s digestive system.
Ahb. halodurans was detected in all samples with the exception of VR and is one of the ten most prevalent species in SR and SU samples. Ahb. halodurans DSM 497T and Ahb. okuhidensis DSM 13666T are the same species based on digital DNA–DNA hybridization (dDDH) and ANI value. Based on this, Ahb. okuhidensis is proposed as a synonym of Ahb. halodurans [82]. Some Ahb. halodurans strains have the greatest potential for biosurfactant production, lipase, and protease [83,84,85]. Moreover, acetic acid bacteria (AAB), which belong to the Gram-negative bacteria group within the proteobacteria clade, are commonly detected in the microbiome of honeybees [86]. While there is no specific information available on the role of Ahb. halodurans in the honeybee gut, it is possible that it may also play a role in protecting honeybees from pathogenic bacteria or improving their health. Further research is needed to understand the specific role of this bacteria in the honeybee gut.
L. saccharolytica was previously classified as Clostridium saccharolyticum. In 2019, it was reclassified as L. saccharolytica [87] and in this study, it was only found in the VU sample. Due to its extensive saccharolytic activity, this mesophilic anaerobe gained the name Clostridium saccharolyticum and fermented a wide range of carbohydrate sources [88]. Its strains have previously been isolated from the gut of termites, and their closest relatives [89].
Vitreoscilla sp. strain C1 is historically significant as the source of the first identified prokaryotic hemoglobin. In addition to hemoglobin, Vitreoscilla sp. strain C1 also produces dihydrodipicolinate reductase and has been found to be a source of antibiotic resistance genes [90]. While there is no information about Vitreoscilla sp. strain C1 specifically in honeybee guts in the literature, in this study, it is ranked ninth on the list of the most common species found in the SR sample.
This study suggests that the gut microbiota of honeybees is influenced by the environment in which they are kept. Specifically, the study found that the core microbiota of honeybees differed depending on whether they were kept in urban or rural areas.
Evidence indicates that factors such as seasonal fluctuations, caste variations, and diverse climatic conditions can influence the composition of the bee microbiota [91,92,93]. Within the scope of this study, beekeeping in environments with a notable presence of human pathogenic microorganisms consistently resulted in the incorporation of these pathogens into the bee microbiota. Notably, urban beekeeping areas, characterized by heightened human activity and potentially elevated contamination sources, exhibited a heightened prevalence of these harmful microorganisms within the honeybee microbiota. This observation underscores the potential role of human activity in urban settings in contributing to modifications in the microbiota of honeybees.
Another factor that appeared to influence the honeybee microbiota was the number of species of gut flora found in honeybee relatives in rural beekeeping areas. This suggests that the microbiota of honeybees may be influenced by the broader ecological context in which they are found, and that the microbiota of related species may play a role in shaping the microbiota of honeybees.
Overall, these findings suggest that the microbiota of honeybees is a complex and dynamic system that is influenced by a range of environmental factors. Understanding how these factors interact to shape the honeybee microbiota could have important implications for managing honeybee health and supporting pollination services.
The presence of human pathogenic bacteria in the honeybee gut raises concerns about both bee health and public health. The invasion of opportunistic pathogens into the core bacterial community of bees in urban areas indicates the potential transmission of pathogens from humans to bees. This highlights the need for further research to understand the mechanisms and routes through which these pathogens enter the bee gut. It is crucial to investigate the sources and transmission pathways of these human pathogens into the bee gut, including their presence in nutrition or water sources. Further research is needed to explore the reservoirs and transmission routes of human pathogens in honeybees and to develop strategies for preventing and managing such infections. This knowledge will contribute to the protection of honeybees, the maintenance of their essential role in pollination, and the safeguarding of public health.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d15091016/s1, Table S1: species table.

Funding

This research was funded by Şırnak University Scientific Research Projects Unit, grant number 2019.FNAP.13.02.01.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

Acknowledgments

I extend my appreciation to Esengül Özdemir and Adil Tonga for their valuable contributions and support.

Conflicts of Interest

The author declares no conflict of interest.

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Diversity 15 01016 g001
Figure 1. Sampling locations rural and urban samples are represented by black and white dots, respectively.
Figure 1. Sampling locations rural and urban samples are represented by black and white dots, respectively.
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Figure 2. The distribution of bacterial communities in rural and urban locations. Panel (A) shows the phylum level of eight samples: Diyarbakır-Urban (DU), Van-Urban (VU), Mardin-Urban (MU), Şırnak Urban (SU), Diyarbakır-Rural (DR), Van-Rural (VR), Mardin-Rural (MR), Şırnak-Rural (SR), as well as the urban sample average (UA) and rural sample average (RA). Panel (B) represents the family level and panel (C) shows the species level for UA and RA (only including species whose relative abundance exceeds %0.5).
Figure 2. The distribution of bacterial communities in rural and urban locations. Panel (A) shows the phylum level of eight samples: Diyarbakır-Urban (DU), Van-Urban (VU), Mardin-Urban (MU), Şırnak Urban (SU), Diyarbakır-Rural (DR), Van-Rural (VR), Mardin-Rural (MR), Şırnak-Rural (SR), as well as the urban sample average (UA) and rural sample average (RA). Panel (B) represents the family level and panel (C) shows the species level for UA and RA (only including species whose relative abundance exceeds %0.5).
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Figure 3. Heatmap of the variability in the relative abundance of the thirty most abundant bacterial species in the honeybee alimentary tract of NYC. The yellow color palette is used to indicate the degree of abundance, and the heatmap represents bacterial species that have at least 0.5% abundance in at least one sample. (Diyarbakır-Urban (DU), Van-Urban (VU), Mardin-Urban (MU), Şırnak Urban (SU), Diyarbakır-Rural (DR), Van-Rural (VR), Mardin-Rural (MR), Şırnak-Rural (SR), as well as the urban sample average (UA) and rural sample average (RA)).
Figure 3. Heatmap of the variability in the relative abundance of the thirty most abundant bacterial species in the honeybee alimentary tract of NYC. The yellow color palette is used to indicate the degree of abundance, and the heatmap represents bacterial species that have at least 0.5% abundance in at least one sample. (Diyarbakır-Urban (DU), Van-Urban (VU), Mardin-Urban (MU), Şırnak Urban (SU), Diyarbakır-Rural (DR), Van-Rural (VR), Mardin-Rural (MR), Şırnak-Rural (SR), as well as the urban sample average (UA) and rural sample average (RA)).
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Figure 4. The results of a Principal Coordinates Analysis (PCoA) performed on honeybee gut samples from eight different locations: Diyarbakır-Urban (DU), Van-Urban (VU), Mardin-Urban (MU), Şırnak Urban (SU), Diyarbakır-Rural (DR), Van-Rural (VR), Mardin-Rural (MR), and Şırnak-Rural (SR).
Figure 4. The results of a Principal Coordinates Analysis (PCoA) performed on honeybee gut samples from eight different locations: Diyarbakır-Urban (DU), Van-Urban (VU), Mardin-Urban (MU), Şırnak Urban (SU), Diyarbakır-Rural (DR), Van-Rural (VR), Mardin-Rural (MR), and Şırnak-Rural (SR).
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Table 1. The sequencing statistics, Shannon index, and Simpson index, representing species-level diversity within the alimentary tract of forager Apis mellifera.
Table 1. The sequencing statistics, Shannon index, and Simpson index, representing species-level diversity within the alimentary tract of forager Apis mellifera.
Samples of Honeybee *Number of ReadsAverage Reading Length (Base)Shannon Index
H = − ∑ i = 1 S P İ ln ⁡ P İ 		Simpson Index
(1-D)
DU7500130.03.727/0.63240.9336
VU157396113.583.114/0.49040.7741
MU97876125.72.611/0.41770.8534
SU23840131.52.455/0.42920.8137
DR9012132.42.735/0.50170.8573
VR160605117.242.219/0.36660.8157
MR79914117.52.963/0.44580.8768
SR43516129.52.489/0.41180.8246
*: DU: Diyarbakır Urban, VU: Van Urban, MU: Mardin Urban, SU: Şırnak Urban, DR: Diyarbakır Rural, VR: Van Rural, MR: Mardin Rural, SR: Şırnak Rural, H: Shannon diversity index, s: number of species, pi: abundance of each species.
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Rüstemoğlu, M. Next Generation Sequencing of Bee Gut Microbiota in Urban and Rural Environments. Diversity 2023, 15, 1016. https://doi.org/10.3390/d15091016

AMA Style

Rüstemoğlu M. Next Generation Sequencing of Bee Gut Microbiota in Urban and Rural Environments. Diversity. 2023; 15(9):1016. https://doi.org/10.3390/d15091016

Chicago/Turabian Style

Rüstemoğlu, Mustafa. 2023. "Next Generation Sequencing of Bee Gut Microbiota in Urban and Rural Environments" Diversity 15, no. 9: 1016. https://doi.org/10.3390/d15091016

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