Microbiota dysbiosis in honeybee (Apis mellifera L .) larvae infected with brood diseases and foraging bees exposed to agrochemicals

Abstract

American foulbrood (AFB) disease and chalkbrood disease (CBD) are important bacterial and fungal diseases, respectively, that affect honeybee broods. Exposure to agrochemicals is an abiotic stressor that potentially weakens honeybee colonies. Gut microflora alterations in adult honeybees associated with these biotic and abiotic factors have been investigated. However, microbial compositions in AFB- and CBD-infected larvae and the profile of whole-body microbiota in foraging bees exposed to agrochemicals have not been fully studied. In this study, bacterial and fungal communities in healthy and diseased (AFB/CBD) honeybee larvae were characterized by amplicon sequencing of bacterial 16S rRNA gene and fungal internal transcribed spacer1 region, respectively. The bacterial and fungal communities in disordered foraging bees poisoned by agrochemicals were analysed. Our results revealed that healthy larvae were significantly enriched in bacterial genera Lactobacillus and Stenotrophomonas and the fungal genera Alternaria and Aspergillus. The enrichment of these microorganisms, which had antagonistic activities against the etiologic agents for AFB and CBD, respectively, may protect larvae from potential infection. In disordered foraging bees, the relative abundance of bacterial genus Gilliamella and fungal species Cystofilobasidium macerans were significantly reduced, which may compromise hosts' capacities in nutrient absorption and immune defence against pathogens. Significantly higher frequency of environmentally derived fungi was observed in disordered foraging bees, which reflected the perturbed microbiota communities of hosts. Results from PICRUSt and FUNGuild analyses revealed significant differences in gene clusters of bacterial communities and fungal function profiles. Overall, results of this study provide references for the composition and function of microbial communities in AFB- and CBD-infected honeybee larvae and foraging bees exposed to agrochemicals.

Keywords: American foulbrood disease; agrochemical exposure; bacterial and fungal compositions; chalkbrood disease; honeybee.

Figure 1.

Relative abundance of the top 10 taxa at the genus level calculated for each group. (a,b) Bacterial microbiota. (c,d) Fungal microbiota.

Figure 2.

Plot of mean alpha-diversity for different groups. Community richness and diversity is characterized by the observed species and the Shannon index, respectively. (a,b) Bacterial communities. (c,d) Fungal communities. Asterisks indicate a significant difference between groups from the same apiary. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Significantly different taxa between groups from the same apiary identified by LEfSe. (a, c, e and g) LefSe analysis (the threshold of the LDA score was 4.0). (b, d, f and h) Taxonomic cladogram based on the results of LEfSe analysis. The size of the circles was based on relative abundance. Yellow represents no significant difference in taxa; green and red represent significantly different taxa in the correspondingly colour-labelled groups. Only taxa with greater than 0.1% proportional abundance are visualized. (a, b, c, d), based on 16S rRNA sequencing data. (e, f, g, h), based on ITS sequencing data.

Figure 3.

Significantly different taxa between groups from the same apiary identified by LEfSe. (a, c, e and g) LefSe analysis (the threshold of the LDA score was 4.0). (b, d, f and h) Taxonomic cladogram based on the results of LEfSe analysis. The size of the circles was based on relative abundance. Yellow represents no significant difference in taxa; green and red represent significantly different taxa in the correspondingly colour-labelled groups. Only taxa with greater than 0.1% proportional abundance are visualized. (a, b, c, d), based on 16S rRNA sequencing data. (e, f, g, h), based on ITS sequencing data.

Figure 4.

Non-metric multidimensional scaling (NMDS) plots showing the bacterial (a,b) and fungal (c,d) community diversity between groups from the same apiary.

Figure 5.

(a) Heatmap of microbial function pathways in different groups. (b) Fungal function classification by FUNGuild tool. Asterisks represent significant differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001.