The global decline in the population of pollinator bees (Potts et al. 2010) has attracted the researchers for a comprehensive study of host microbiome community. As major contributors in pollination, honey bees are very crucial organisms in securing the agricultural produce and the maintaining ecosystem. Unlike western bees, A. florae are not reared for commercial honey or wax; rather they occur in wild functioning as major pollinators (Balachandra et al. 1999). These little dwarf bees differ from western bees in their defense against pathogens (Suwannapong et al. 2011). In the present study, Asiatic bees, A. florea were selected to study and analyze the gut microbiome. The bee symbionts are likely to play a vital role in self defense and metabolism. Apis florea, (dwarf bee) is naturally distributed in Indian subcontinent throughout south-east Asia. The most important contribution of this honeybee is its valuable pollination of many fruit plants and diversified flora in tropical ecosystems (Soman and Chawda 1996).
The defensive and metabolic capabilities of bees are highly correlated with gut microbiome interaction and to further add knowledge on role and interaction of microbial community with host, the analysis of gut bacteria of Apis florea honey bees was carried out. Healthy bees were obtained directly from single hive across Western Ghats forest area of Kodagu district, which is recognised as a global biodiversity hotspot, India. The entire thorax and abdomen was processed for analysis, thus including gut microbes and organisms attached to hemolymph or tissues. The analysis of gut bacteria was performed by culture dependent and culture-independent technique from a total of 80 worker dwarf honey bees.
In culture dependent method, the identification and characterization of culturable diversity of bee gut bacteria was done by MALDI-TOF-MS and 16S rRNA gene sequencing analysis. A total of 91 aerobic and facultative anaerobic bacteria were isolated from guts of worker A. florae bees. Based on colony characteristics, the bacterial isolates were initially subjected to MALDI-TOF-MS. Fifty six isolates were identified up to species level. The isolates with biotyper score value <2.0 could not be identified up to species level. Remaining thirty-five such isolates which were not identified up to species level from MALDI-TOF-MS were further subjected to 16S rRNA sequencing. Collectively from both MALDI-TOF-MS and 16S rRNA sequencing of culture dependent analysis, the gut bacterial isolates belonging to three bacterial phyla/classes were identified; alpha-Proteobacteria (1%), Firmicutes (19%) and Gamma-Proteobacteria (80%) (Fig.1). The percentage of isolates belonging to genera, Klebsiella, Enterobacter, Bacillus, Citrobacter, Staphylococcus and Lactobacillus are represented in Fig. 2. These isolates were found to be common in Apis clade as reported in previous work (Martinson et al. 2012; Yoshiyama and Kimura 2009; Khan et al. 2017). The presence of a diverse group of bacteria including the phyla Firmicutes, alpha and beta-Proteobacteria suggests their ecological importance (Kwong and Moran 2017). Most of these bacteria belonging to these phyla are facultative anaerobes, ferment sugars and tolerant to acidic environment. The bacterial isolates identified are common in soil, water and few of them are organisms of clinical relevance in humans. The occurrence of such bacteria in insect gut is not unusual and is described before. Staphylococcus, Enterobacter and Klbesiella found in abundance in gut of dwarf bee are typical inhabitants in human. These bacteria are considered as beneficial healthy gut microbiota in humans, insects and other animals as they are the fermenters of sugar and involved in the host defense mechanism (Anderson et al. 2011). Klbesiellaoxytoca, a prominent organism considered as probiotic, positively affects the health of host by suppressing parasite colonization (Engel et al. 2013). In termite, the role of Staphylococcus probably is involved in degradation of cellulose (Sarkar et al. 1986). Klbesiella spp (K. oxytoca) and Pantoea agglomerans were predominantly found in our analysis. Pantoea agglomerans is significantly present in desert locust, Schistocerca gregaria, and are involved in breakdown of dietary components leading to synthesis of aggregation pheromones that function in swarming behaviour of locusts (Dillon et al. 2002). It is reported that these organisms gain entry into the bee gut during foraging activity (Loncaric et al. 2009). Bacillus safensis,B. kochii and B. halotolerans were identified in the gut of dwarf bee. Bacillus safensis, Bacillus kochii and Bacillus halotolerans are reported to exist and survive in extreme environment and under stress conditions (Lateef et al. 2013, Zhang et al. 2018, Seiler et al. 2012). In two separate studies on profiling of bacterial community carried out in the gut of flesh flies and floral nectar, Bacillus safensis was identified and isolated (Gupta et al. 2014, Fridman et al. 2012). Desert locust Schistocerca gregaria, inhabits Bacillus safensis which possess high cellulolytic activity indicating the metabolic significance of this bacteria (Nelson et al. 2021). Strains of Bacillus safensis is reported to produce many industrially relevant enzymes, such as amylase, protease, lipase, inulinase and chitinase. The occurrence of Brevundimonas nasdae was prominent in our study. The pH of honey bee midgut is around 8 and this pH favors the optimal growth of Bacillus species and Brevundimonas nasdae and these may aid in degrading of carbohydrate fed by the bees. In addition to Brevundimonas nasdae (Phylum: Proteobacteria), Bacillus species and, Solibacillus silvestris (Phylum: Firmicutes) were predominant in the gut of A. florea. These bacterial species are rarely or no where reported in the available literature on the Apis gut microbiota of other species, whereas interestingly occur in Apis mellifera. The differences in the occurrence of these bacteria might be due to geographical location or existing characteristics of the environment or may be due to the feature of A.florea species itself, which requires further studies.
In culture-independent studies, a comprehensive microbial diversity analysis using high throughput sequencing approach was carried out. Bacterial community profile of honey bee gut as revealed by16S rRNA gene amplicon sequencing, yielded 15 bacterial phyla representing collective phyla in the gut of A.florea (Fig. 3a). Bacterial phyla distributions were as follows; Bacteroidetes (51.3%), Proteobacteria (45.1%), Euryarchaeota (1.3%), Actinobacteria (1.1%), Firmicutes (0.9%) and the rest (0.3%) constituted the minor phyla. The abundance of ten minor phyla (Acidobacteria, Tectomicrobia, Chloroflexi, Lentisphaerae, Verrucomicrobia, Cyanobacteria, Planctomycetes, Nitrospirae, Tenericutes and Saccharibacteria) was very less; hence the percentage of composition is not shown in Fig. 3a. At family level, OTUs with ≥0.3% abundance were filtered (Fig. 3b). The family level distribution of bacteria in the gut of A. florea were Prevotellaceae (52.1%), Enterobacteriaceae (42.7%), Halobacteriaceae (1.3%), Methylobacteriaceae (1.2%), Bifidobacteriaceae (0.9%), Orbaceae (0.4%), Lactobacillaceae (0.4%) and Halomonadaceae (0.4%). Composition percentage of bacterial family which was less than 0.9% is not shown in Fig. 3b.
At the genus level, OTUs with ≥0.3% abundance were filtered and distribution of bacterial genera in the gut of A. florea is represented as diagram in Fig. 4 and are as follows; Prevotella (59.3%), Escherichia-Shigella (33.3%), Natronomonas (1.5%), Methylobacterium (1.4%), Pantoea (1.1%), Bifidobacterium (1%), Enterobacter (0.9%), Klebsiella (0.6%), Lactobacillus (0.5%) and Nitrobacter (<0.3%). The composition percentage of bacterial genera which was less than 0.3% was not shown in Fig. 4. Amongst these, Bacteroidetes and Proteobacteria were the predominant phyla. Euryarchaeota are highly diverse and are often found in intestine, which are rarely mentioned in literature on bee gut microbiota. Along with Euryarchaeota, Tectomicrobia, Chloroflexi, Lentisphaerae and Verrucomicrobia are few of the gut symbionts, which are found in A. florea, unmentioned elsewhere in the literature on honey bee gut microbiota.
Prevotella, Escherichia-Shigella, Natronomonas, Methylobacterium, Pantoea, Bifidobacterium, Enterobacter, Klebsiella and Lactobacillus are the dominant genera found in the gut of A. florea worker bees. The bacterial genera Prevotella, Natronomonas etc. are uncultivable under typical laboratory conditions as they require strict anaerobic conditions or haloalkaliphilic conditions. Prevotella, Natronomonas and Methylobacterium are not reported in the available literature of honey bee gut microbiota, however, Methylobacterium and Prevotella are the predominant inhabitants of gut in bark beetle, (Dendroctonus rhizophagus) (Briones-Roblero et al. 2017). Species of Prevotella, are non-cellulolytic carbohydrate degrading bacteria, which bring about digestion of cell wall polysaccharides like xylan (Flint et al.2012). A relatively lower proportion of Brevundimonas, Staphylococcus, Streptococcus, Gluconobacter and Gilliamella genera was observed in our culture independent studies, whereas many of these are predominant in Apis mellifera (Kwong and Moran 2016). Functional redundancy and crosstalk among the microbes, and host has huge metabolic and physiological impact and, the significance of the presence of these bacterial communities can be untangled by further metagenomic and metatranscriptomic studies.
High throughput sequencing and quality trimming of 16S rRNA gene yielded ~0.118 million quality reads which were used for subsequent analysis. Taxonomic assignment of sequences with the reference database resulted in 589 operational taxonomic units (OTUs). Alpha diversity estimation of gut of A. florea using species richness and non-parametric Shannon index suggested higher bacterial diversity in A. florea worker bees. The alpha diversity index which is an indicator of bacterial diversity, were calculated for A. florea and is given in Table 1. Shannon index for bacterial communities was 3.121 and this observation is suggestive of richness in bacterial diversity in the gut of A. florea.
16S rRNA gene sequences of the bacterial isolates of A. florea was used to construct the phylogenetic tree showing relationship among the bacteria with reference strains of GenBank (Fig. 5). The bacterial populations in the gut were diverse among forager bees of A. florea which belonged to phyla Firmicutes, alpha and beta-Proteobacteria. A plethora of bacterial abundance in any niche suggests their significance in ecological diversity; in A.florea, Prevotella was a significant member accounting for 59% of the total gut microbe indicating the possible ecological importance.
Dominant gut inhabitants of A. mellifera belong to phyla Firmicutes, Actinobacteria and Proteobacteria (Kwong and Moran 2016; Romero et al. 2019; Ahn et al. 2012). The bee gut microbial communities in A. mellifera are specific and are dominated by nine bacterial species clusters viz., Bartonella apis, Parasaccharibacter apium, Frischella perrara, S. alvi, Gilliamella apicola, Bifidobacterium spp., Lactobacillus Firm-4, Lactobacillus Firm-5 and these are believed to impart social behavior among individuals (Kwong and Moran 2016). Snodgrassella alvi and Gilliamella apicola are ubiquitous in the gut of A. mellifera (Kwong and Moran 2012). In our study, Snodgrassella alvi was not detected whereas, Gilliamella apicola were found in traces. Acinetobacter was found in our study similar to the reports of Kim et al (2014). Acinetobacter apis spp. nov., was isolated from the intestinal tract of a honey bee, A. mellifera (Kim et al. 2014). In the metagenomic survey, the class Alpha Proteobacteria and Gamma Proteobacteria dominated the gut environment of A. mellifera (Engel and Moran 2013a). In A. florea, Bacteriodetes, Proteobacteria, Euryarcheota, Actinobacteria, Firmicutes and Acidobacteria were the predominant phyla in our study. Citrobacter spp., Providencia vermicola, Planomicrobiumo keanokoites and Exiguobacterium acetylicum were reported for the first time in the genus Apis by culture dependent 16S rRNA sequencing (Khan et al. 2017). Pyrosequecning analysis of the bacterial community structure in the midguts and hindguts of the adult honeybees of A. cerana and A. mellifera were studied. Higher frequencies of Enterobacteriaceae, Lactococcus, Bartonella, Spiroplasma, and Flavobacteriaceae-related OTUs were found in the guts of A. cerana while Bifidobacterium and Lachnospiraceae-related OTUs were more abundant in guts of A. mellifera (Ahn et al. 2012). Anjum et al. (2018) reported Firmicutes (60%), Proteobacteria (26%) and Actinobacteria (14%) in A. mellifera gut by 16S rDNA sequencing.
The dominant phyla, Proteobacteria and Firmicutes are reported in eusocial wasps (Order: Hymenoptera) inhabiting eastern and southern Asian region (Suenami et al. 2019). A variety of bacterial phyla are commonly present in insect guts, including Gammaproteobacteria, Alpha-Proteobacteria, Beta-Proteobacteria, Bacteroidetes, Firmicutes including Lactobacillus, and Bacillus species, Clostridia, Actinomycetes, Spirochetes, Verrucomicrobia, Actinobacteria, and others (Colman et al. 2012). Numerous non-culture-based studies show that dominant taxa in Drosophila melanogaster are influenced by diet and vary among laboratories however, certain taxa recur (Broderick and Lemaitre 2012). The gut inhabitants of honey bees are coevolved with bumble bees and comprise a distinctive gut community (Martinson et al. 2011). The wild flies have distinct bacterial communities and more diversification from those of reared species (Chandler et al. 2011), including bee gut bacteria (Kwong and Moran 2016; Engel et al. 2013). In major, gut flora of insects, contribute to metabolism, nutrition, immune modulation and protection against foreign entities (Engel and Moran 2013b). Understanding the bacterial network offers knowledge into bee pathology, host-microbe interaction and aid in improving honey bee health and to discover new sources of biotechnologically potential molecules and enzymes (Romero et al. 2019).