Abstract
Background Gut microbiota plays an essential role in bee’s health. To elucidate the effect of food and Nosema ceranae infection on the gut microbiota of honeybee Apis cerana, we used 16S rRNA sequencing to survey the gut microbiota of honeybee workers fed with sugar water or beebread and inoculated with or without N. ceranae.
Results The gut microbiota of A. cerana is dominated by Serratia, Snodgrassella, and Lactobacillus genera. The overall gut microbiota diversity was significantly differential by food type. The N. ceranae infection significantly affects the gut microbiota only at bees fed with sugar water. Higher abundance of Lactobacillus, Gluconacetobacter and Snodgrassella and lower abundance of Serratia were found in bees fed with beebread than with sugar water. N. ceranae infection led to higher abundance of Snodgrassella and lower abundance of Serratia in sugar-fed bees. Imputed bacterial KEGG pathways showed the significant metagenomics functional differences by feeding and N. ceranae infections. Furthermore, A. cerana workers fed with sugar water showed lower N. ceranae spore loads but higher mortality than those fed with beebread. The cumulative mortality was strongly positive correlated (rho=0.61) with the changes of overall microbiota dissimilarities by N. ceranae infection.
Conclusions Both food and N. ceranae infection significantly affect the gut microbiota in A. cerana workers. Beebread feeding not only provide better nutrition but also help establish a more stabled gut microbiota therefore protect bee in response to N. ceranae infection.
Abstract Importance Gut microbiota plays an essential role in bee’s health. Scientific evidence suggests the diet and infection can affect the gut microbiota and modulate the gut health, however the interplay between those two factors and bee gut microbiota is not well known. In this study, we used high-throughput sequencing method to monitor the changes of gut microbiota by both food intake and the Nosema ceranae infection. Our result showed that the gut microbiota composition and diversity of Asia Honeybee was significantly associated with both food intake and the N. ceranae infection. More interestingly, bees fed with beebread showed higher microbiota stability and less mortality than those fed with sugar water when infected by N. ceranae. Those data suggest the potential role of beebread, not only providing better nutrition but also helping establish a more stabled gut microbiota to protect bee against N. ceranae infection.
Background
European honey bees (Apis mellifera) and Asian honey bees (A. cerana) are two truly domesticated bee species that play a vital role in agriculture and ecosystem by providing pollination service to food crops and natural plants. However, both bee species are confronted with many biotic and abiotic stressors including diseases caused by pathogens and parasites, acute and sublethal toxicity of pesticides, malnutrition due to loss of foraging habitat, and etc that act separately or synergistically to cause the significant decline of bee health and population worldwide[1–3]. As a result, the health of managed honey bees has drawn much attention worldwide in recent years. There has been growing evidence that gut bacteria play very important roles in animal health by maintaining homeostasis, modulating immunity, regulating nutrition metabolism, and supporting host development, and reproduction[4–6]. Although most insect guts harbor relatively few microbiota species as compared to mammalian guts, insect bacteria have been shown to be vital in regulating various aspects of their host biology [7–9]. Over the past decade, progress has been made in understanding the composition and functional capacity of microbes living in honey bee guts [10–12]. Honey bee gut microbiota is established gradually through trophallaxis, food consuming, and interacting with the hive environment[13]. Many factors, like genetics, age, diet, geography, and medication can affect the gut microbiota composition[14,15]. Several types of bacteria have been identified in the guts of A. mellifera including the genera of Bacillus, Lactobacilli and Staphylococcus from Firmicutes phylum, Coliforms from Enterobacteriaceae family of Proteobacteria phylum [16–18].A previous study reported that species within the Apis genus share rather simple and similar gut bacterial microbiota. At phylum level, among proteobacteria, Gammaproteobacteria class was the most abundant, while other proteobacteria including Betaproteobacteria and Alphaproteobacteria classes, Firmicutes and Actinobacteria were less frequent but widespread organisms. Less than ten members formed a core species, including Lactobacillus, Bifidobacterium, Neisseria, Pasteurella, Gluconobacter and newly named species: Snodgrassella and Gilliamella [19–21]. However, most the studies about the microbiota in Apis were conducted in European honey bees, A. mellifera. The food influence on the microbiota of A. cerana has barely been investigated.
Nosema ceranae is an intracellular parasite that disrupts a bee’s digestive system. It was first discovered in the A. cerana but has recently jumped host from A. cerana to A. mellifera[22,23], N. ceranae can seriously shorten the life expectancy of adults, decrease the productivity of the colony, and cause severe colony lost especially during wintering in the temperate area[24,25]. Furthermore stresses caused by Nosema would be more severe when mixed infection happened with other parasites or pathogens, such as Varroa mites, and viruses [26–30]. Now Nosema is one of the major threats to the honey bee populations and has been often implemented in honey bee colony losses worldwide[31,32]. The survey of microbial communities from the digestive tracts of A. cerana workers showed that N. ceranae infection might have detrimental effects on the gut microbiota[1]. However, the relationship between N. ceranae and microbiota in A. cerana is largely unknown. In this study, we challenged A. cerana workers with N. ceranae, and then fed them with either beebread or sugar water. The intent of the current study was to evaluate the effects of N. ceranae infection and food types on the gut microbiota.
Methods
Honey bees
Three A. cerana colonies without identified diseases were chosen for sample collection, which located at the campus of College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China. Capped brood-combs with pupae near emergence were taken out of the colonies and then kept in the incubator with 35±1°C and 55%-65%RH. Workers emerged within 24h were collected for the study.
Purification of Nosema ceranae spore
Because the prevalence and spore loads of N. ceranae in A. cerana are less than A. mellifera[33,34], we purified N. ceranae spores from A. mellifera foragers. First, adult workers were captured at entrances of A. mellifera colonies and immobilized in the refrigerator for few minutes, and then the guts of the bees were dissected, pooled, and ground in a mortar. Afterward, the spores were purified by differential centrifugation to exclude most of the debris, finally, the suspension was loaded on Percoll (Sigma-Aldrich, St.Louis, USA) and centrifuged to eliminate unsaturated spores [33]. The purity and maturity of spore were confirmed under phase contrast microscopy. The Nosema species was confirmed by PCR method [35].
Treatments and sampling
The newly emerged workers (<24h) were randomly distributed into 18 laboratory rearing cages. 30 bees were transferred to each cage The experimental cages were divided into two groups: 1) group supplied with only 50% (W/V) sugar water in modified syringe feeder [36], and 2) group supplied with both 50% (W/V) sugar water and beebread freshly collected from the A. cerana colonies (thereafter call beebread). For each group, three subgroups were set up one without spore inoculation which was used as a negative control, one inoculated with N. ceranae 5000 spores per bee, and one inoculated with 50000 spores per bee (Figure 1). Each subgroup consisted of three cages as replicates. Cages were kept in an incubator with 30±1°C and 55%-65% RH. About eight workers were collected at day 5, 10, and 15 post treatment (dpi) from each subgroup. The gut tissue was collected from each bee at 5-day, 10-day, and 15-day post infection and then stored into −80°C freezer until the subsequent microbial composition analysis Foods were changed each other day; dead bees were counted and removed every day.
DNA extraction from gut tissue samples
Sample bees were taken out of the refrigerator and rinsed with 7% benzalkonium bromide for 2min and then rinsed four times with sterilized water to minimize the bacterial contamination from the body surface. The intestine tissues were collected with tweezers clamping the end of the abdomen and each gut tissue was further separated and transferred into a labeled 1.5ml tube on ice. The entire procedure was conducted under the aseptic condition and all tools used were sterilized. The total DNA of the gut tissue samples was extracted using Insect DNA Extraction Kit II (Beijing Demeter Biotech Ltd, Beijing, China) following the manufacturer’s instruction. The quality and yield of DNA samples were assessed using a Quawell Q5000 UV-Vis spectrophotometer (Quawell, San Jose, CA, USA).
Gut Nosema ceranae spore counting
After caged bees were sampled at day 5, 10, and 15 post treatment, the quantity of the spores in the gut specimen was counted as previously described with slight modification[33]. Briefly, the sediments of gut were resuspended in 100μl ddH2O, then vortexed evenly. The suspension was loaded onto the hemocytometer for N. ceranae spore inspection and counting under a microscope. We conducted three to four repeated measurements for each sample.
Bacterial 16S ribosomal RNA gene PCR amplification
The phylogenetically informative V3-V4 region of 16S ribosomal RNA (rRNA) gene was amplified using universal primer 347F/803R [37]. The dual-barcoding approach as previously described[38] was applied to label the 16S rRNA gene amplicons of each sample. Briefly, the 6-mer barcodes were attached on the 5’ends of both forward and reverse PCR primers so that 16S rRNA gene PCR amplicons from each sample contained a unique dual barcode combination. The PCR Primers were synthesized by Sangon Biotech, Shanghai, China, and the primer sequences are shown in Supplementary Table 1. The 25-μL PCR reaction mixes contain 300ng of sample DNA as PCR template, 1μL of 10μM forward and reverse 16S primers, and 12.5μL of 2×HotMaster Taq DNA mix (Tiangen Biotech, Beijing, China). The PCR reaction was performed on Applied Biosystem 2720 thermal cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 94°C for 3 minutes, then 94°C 30 seconds, 58°C 30 seconds, and 72°C 20sec for 30cycles, and 72°C for 4 min. The integrity of the PCR products was verified by agarose gel electrophoresis. After purified with gel purification kit (Promega, Madison, WI, USA), the 16S PCR amplicons were pooled at equal molarity, freeze-dried, and submitted to New York Genome Center for sequencing.
16S rRNA gene sequencing and microbiota profiling
The 16S rRNA gene PCR amplicons were sequenced on the Illumina HiSeq platform using 2×250 paired-end fast-run mode. In total, we generated 21 million high-quality 16S reads obtained by NGS sequencing on pooled barcoded PCR amplicons from 86 samples. After splitting by barcodes, ~ 2.5×105 reads per sample were obtained. After the merge, the sequencing reads with length >400 and the quality score >Q30 at more than 99% of bases were further split by barcode and trimmed of primer regions using CLC Genomic workbench 6 (Qiagen Bioinformatics, Redwood City, CA, USA). The filtered and trimmed high-quality reads were further processed by QIIME 1.9.0[39]. We used the command pick_open_reference_otus.py with the defaulted cutoff =97% to a cluster of nearly-identical sequencing reads as an Operational Taxonomic Unit (OTU) using Uclust[40]. Representative sequences for each OTU were aligned using PyNAST. Finally, the program built a biom-formatted OTU table with assigned taxonomical information for each OTUs. Using Chimera Slayer[41], chimera sequences arising from the PCR amplification were detected and excluded from the aligned representative sequences and the OTU table.
Statistical Analysis
The mortality data of different groups were transformed by square root and degrees and Asin, and then compared by using two-way ANOVA of the SPSS program. The overall microbiota dissimilarities among all samples were accessed using the Bray-Curtis distance matrices[42] generated at the genus level. The PERMANOVA (Permutational Multivariate Analysis of Variance) procedure [43,44] using the [Adonis] function of the R package vegan 2.0-5), with the maximum number of permutations = 999, was performed to test the significance of the overall microbiota differences between the gut microbiota grouped by feeding types and N. ceranae infections. The diversity within each microbial community, so-called alpha-diversity was calculated using the Shannon Index as metric and represented the measure of the diversity at genus level [45]. Using the linear discriminant analysis (LDA) effect size (LEfSe) method[46], we further selected the microbiota features significantly associated with feeding types and N. ceranae infections. The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States)[47] was used to predict the metagenome functional content based on our 16S rRNA gene sequencing data. Briefly, a close reference-based OTU table was generated using the QIIME pipeline and input into PICRUSt to bin individual bacterial genes into Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, thereby predicting their function.
Dataset
16S rRNA gene sequencing information has been deposited in the European Nucleotide Archive with study accession number: PRJEB21090.
Results
1. Simple core bacterial clusters in the gut of Apis cerana
As illustrated in Figure 1, the A. cerana adult workers were grouped by foods and the level of N. ceranae infection. The microbial composition analysis of gut tissue collected at 5-day, 10-day and 15-day post infection for each subgroup following the method described previously [18,49] showed that the gut microbiota of A. cerana is rather simple and mainly contain three phyla, Proteobacteria, Firmicutes, and Bacteroidetes, counting for over 97% of the total microbiota composition (Figure 1, Figure 2). At the genus level, less than 6 taxa from Proteobacteria and Firmicutes are dominant in the A. cerana gut bacterial community. In details, they were the genera Snodgrassella, Acetobacteraceae, Serratia, Gilliamella, Lactobacillus and unclassified genus from Bacteroidetes, of which Serratia was not in the core species clusters of A. mellifera[50].
2. Foods and N. ceranae infection changed the relative abundance of microbes in the gut
The overall microbiota dissimilarity in samples grouped by food or Nosema infection was visualized in NMDS plots (Figure2). The overall gut microbiota is significant different between bees fed with beebread fed and sugar (p=0.018 with N. ceranae infection, and p=0.001 without infection, PERMANOVA test using Bray-Curtis distance). In sugar fed bees, we found N. ceranae infection significantly altered the microbiota (p=0.001). However, N. ceranae infection caused no significant alteration in gut microbitota in bees fed with beebread (p=0.23). LEfSe method was applied to select the microbiota taxa which are significantly associated with either food types or N. ceranae infections. In subgroups without N. ceranae infection, the bees fed with beebread showed more abundant Lactobacillus, Snodgrassella, Weeksellaceae, and less abundant Serratia genus than bees fed with sugar. However, in the subgroups with N. ceranae infection, the bees fed with beebread showed more abundant OTUs of Lactobacillus and less abundant Serratia and Acetobacteraceae than bees fed with sugar (Figure2). Among bees fed with sugar solution, N. ceranae infection caused major changes in microbiota and was associated to increased OTUs of Weeksellaceae, Snodgrassella and Gluconacetobacter and decreased Proteobacteria phyla, in particular, Telluria, Serratia, and Acinetobacter. Among bees fed with beebread, N. ceranae infection had a minor effect on microbiota, with merely decreased the abundance of Massilia, Aggregatibacter and Gluconacetobacter genera.
3. Differential metagenome features predicted by PICRUSt and their association with food and N. ceranae infection status
We performed PICRUSt analysis to predict the full metagenomic content of microbial communities using 16S gene surveys33 and compared the predicted metagenomic pathways by food and N. ceranae infection status (FigureS1). The Nearest Sequenced Taxon Index (NSTI), which quantifies the uncertainty of the prediction (lower values mean a better prediction), ranged from 0.027 to 0.11 with mean value=0.067, indicating fair reliability and accuracy in the metagenome reconstruction. The heat map (FigureS1) with clustering analysis showed the overall changes in predicted KEGG pathways. Among those significantly differential pathways, we found the food type could affect the bacterial Glycolysis/Gluconeogenesis, Fructose and mannose metabolism, metabolism of several amino acids and etc. N. ceranae infection could affect biosynthesis of several amino acids, the signal transduction mechanism, and the lipopolysaccharide biosynthesis and phosphotransferase system (PTS).
4. The cumulative mortality of caged bees with different feeding type and infection status
When inoculated with N. ceranae spores, the average cumulative mortality of caged bees increased gradually during the experimental observation, our results showed that N. ceranae infection significantly shortened the longevity of workers fed with only sugar water than those fed with beebread (Figure 3A). Interestingly, the spore load in the gut fed with beebread were significantly higher than those with sugar water (p-value=0.01 and 0.007 for low N. ceranae and high N. ceranae, respectively) at 15 days after inoculation (Figure 3B). This was consistent with the earlier report by Zheng et al[51]. There is not interaction between food type and spore dosage on the mortality (p-value=0.868, F=0.029). There was no significant difference in gut N. ceranae spore counts between low and high dosage N. ceranae inoculations. This may be due to the late sampling time that the spore load in the gut has reached the plateau.
5. The richness of the gut microbiota of caged bees with different feeding type and infection status
We used Shannon index, a commonly used metrics, for richness assessment within the given community[45]. Without N. ceranae infection, the richness of the microbiota in bees fed with sugar solution was significantly lower than those fed with beebread (Figure 3C, p-values<0.05 at 5, 10 and 15days). Furthermore, the richness of the microbiota decreased by time in bees fed with sugar, but not in bees fed with beebread. In subgroups with N. ceranae infection, the microbiota of bees fed with sugar showed increased richness at all time points with a slightly higher mean but no significant differences to that of bee fed with beebread.
6. The stability of the gut microbiota is significantly correlated with the cumulative mortality
The stability of the microbiota in response to the N. ceranae infection in groups with different of feeding conditions showed that the bee fed with beebread showed relatively stable microbiota. The mean dissimilarity was not significantly different from either sampling time post infection or N. ceranae doses. However, among bees fed with sugar solution, the microbiota dissimilarities significantly increased by time, with the most dissimilarities and higher consistency at 15 days with the high N. ceranae infection (Figure 3D), suggesting the most diverged microbiota within this group. Further, the mean microbiota dissimilarities were significantly correlated with the cumulative mortality rate (r=0.61, spearman correlations, p-value=0.035).
Discussion
Our result demonstrated that the gut microbiota of the A. cerana adult workers are composed of three major phyla, Proteobacteria, Firmicutes, and Bacteriodetes. This result is consistent with the previous reports [21] except that the most abundant taxa in our study was Proteobacteria, which was the second in Ahn’s study [21]. At the genus level, we found that the gut microbiota of Asian honey bees is dominated by a few core bacterial species, including Lactobacillus, Snodgrassella and Gilliamella, among the major genera found in both our study and previous studies[52].
Food constituents can influence the gut microbiota composition. Our results confirmed that food type significantly shapes the bees’ gut microbiota composition (Figure 1 and 2). Beebread contains high protein and comprehensive nutrients, which may favor those proteolytic species. In addition, beebread provides additional microbiota [53,54] inoculations especially lactic acid bacteria, and may benefit the gut microflora too.
Our study also showed that Lactobacillus and Snodgrassella genera were much more abundant in those bees fed on beebread (Fig 2B). The genera, Lactobacillus, Bifidobacterium and the family Pasteurelaceae, were also found in beebread from colonies of A. mellifera [51]. Lactobacillus had been found in flora and hive environment, including honey, royal jelly, beebread, and honey sac. Lactobacillus was also found in honey bee crop and showed inhibition effect on Paenibacillus larvae in vitro [55]. Therefore, it is plausible to speculate that the Lactobacillus found in gut of adult workers fed with were obtained through food trophallaxis. In contrast, bees fed with sugar only showed more abundant Enterobacteraceae. Overgrowth of Enterobacteraceae has been linked to gut inflammation in many studies.
Our data showed that higher proportion of Serratia harbored in the gut of 10-day old bees fed with sugar. Serratia was further confirmed as S. marcescens by sequencing near full-length 16S rRNA gene(data no showed). S. marcescens is commonly found in adult A. mellifera, A. cerana, and bumble bee gut. It is generally harmless to honey bee, and commonly used to explore the host immune reaction to microbes[56], There were two cases, that Serratia had detrimental effects on A. mellifera survivorship after host microbiota was erased by antibiotics. In our study, N. ceranae infection broke the balance of Serratia in the microflora, and shortened host lifespan. Future investigations are necessary to further explore complex interactions among N. ceranae, host, and gut microbiotas.
Nosema resides in the gut of the bee and the infection by N. ceranae can profoundly change honey bees physiology [57], and change the host-microbiota relationship in the gut. Investigation conducted by Li et al. showed that four common bacterial clusters, Bifidobacterium, Neisseriaceae, Pasteurellaceae, and Lactobacillus in N. ceranae infected adult A. cerana workers were less abundant compared to non-infected ones[58]. However, we found minor changes in gut microbiota by N. ceranae infection in beebread fed bees. When sugar water is the only food supplied, N. ceranae infection showed a stronger effect on the overall gut microbiota with more abundant Neisseriaceae/Snodgrassella, Weeksellaceae, Gluconacetobacter and less abundant Serratia, Telluria and Enterobacteriaceae. Further, the lower stability of gut microbiota in bee fed with sugar could lead to increased susceptibility to Nosema infections in bees.
Our data showed that the N. ceranae infection caused much higher cumulative mortality in bees fed with sugar than bees fed with beebread. Interestingly, the changes in microbiota dissimilarity were highly correlated to bee’s mortality. N. ceranae infection caused significant increases in both the microbiota richness and the dissimilarity in sugar fed bees, but not beebread fed bees. We speculated that N. ceranae infection in sugar fed bees resulted in a more diverged microbiota, among which many are not considered as probiotic in bees. The gut microbiota in bees fed with beebread was stable with N. ceranae infection. This stability of gut microbiota could play a protective role and result in less mortality.
Having a biological measure of the effect of N. ceranae infection might help us further understand the controversy of honey bee health and N. ceranae infection, which bees with pollen feeding resulted in higher spore load but less mortality compared to those with sugar water[59].
Although the 16S sequencing based taxonomy analysis is sufficient in current technology development, it only identified bacterial taxa to genus level. It is difficult to identify a specific species or strain that is strongly correlated to either the food feeding or N. ceranae infection.
In summary, the gut microbiota of A. cerana workers is significantly differentiated by both food types and N. ceranae infection. The higher stability of the gut microbiota in the bees fed with plays a role in bees ability to defend N. ceranae infection and warrants further exploration
Conflict of Interest
The authors declare that they have no competing interests
Authors’ contributions
SKH and JZH conceived an designed the study; KTY and SKH performed the bee experiment, KTY, BHY, XS, and LHL counted spore loads; JZH and XLB performed the microbiota analysis; WFH, JHL and YPC analyzed the phenotype data; SKH, JZH and JLL contributed reagents/materials/analysis tools; SKH, JZH wrote the paper; WFH and YPC revised the paper. All authors read and approved the final manuscript.
Acknowledgement
We thank the genomic center of NYU to perform the NGS. This project was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (K4115005A); The Ministry of Agriculture 948 project (2015-Z9).
Footnotes
Kun T. Ye, Email:905797485{at}qq.com;, Wei F. Huang, Email: wfhuang{at}fafu.edu.cn, Bi H. Ying, Email: bihuaying{at}qq.com;, Xin Su, Email: 903469055{at}qq.com;, Li H. Lin, Email: 891140246{at}qq.com, Jiang H. Li, Email: <496939124{at}qq.com>, Yan P. Chen, Email: judy.Chen{at}ARS.USDA.GOV, Ji L. Li, Email:bumblebeeljl{at}126.com, Xiu L. Bao, Email: xiuliangbao{at}gmail.com