Ternary Mixture of Azoxystrobin, Boscalid and Pyraclostrobin Disrupts the Gut Microbiota and Metabolic Balance of Honeybees (Apis cerana cerana)

Abstract

There is a growing risk of pollinators being exposed to multiple fungicides due to the widespread use of fungicides for plant protection. A safety assessment of honeybees exposed to multiple commonly used fungicides is urgently required. Therefore, the acute oral toxicity of the ternary mixed fungicide of ABP (azoxystrobin: boscalid: pyraclostrobin = 1:1:1, m/m/m) was tested on honeybees (Apis cerana cerana), and its sublethal effect on foragers' guts was evaluated. The results showed that the acute oral median lethal concentration (LD₅₀) of ABP for foragers was 12.6 μg a.i./bee. ABP caused disorder of the morphological structure of midgut tissue and affected the intestinal metabolism; the composition and structure of the intestinal microbial community was perturbed, which altered its function. Moreover, the transcripts of genes involved in detoxification and immunity were strongly upregulated with ABP treatment. The study implies that exposure to a fungicide mixture of ABP can cause a series of negative effects on the health of foragers. This work provides a comprehensive understanding of the comprehensive effects of common fungicides on non-target pollinators in the context of ecological risk assessment and the future use of fungicides in agriculture.

Keywords: Apis cerana; azoxystrobin; boscalid; gut microbiota; metabolome; pyraclostrobin.

Figure 1

Toxicity regression curve of ternary mixed fungicide of ABP (azoxystrobin: boscalid: pyraclostrobin = 1:1:1, m/m/m) on Apis cerana by acute oral toxicity test.

Figure 2

Light micrographs of the midgut of honeybees after 10 d of oral continuous exposure to ABP. (A) CTL—acetone syrup control; (B) ABP—0.063 mg/mL of fungicide mixture (azoxystrobin: pyraclostrobin: boscalid = 1:1:1, m/m/m). Legend: mu = muscle, n = nucleus, v = vacuole, cc = columnar cells, lu = lumen, mi = microvilli, pm = peritrophic membrane, rc = regenerative cells.

Figure 3

(A) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot between ABP-treated and control honeybees. (B) The heatmap visualization of the significant different metabolites between the ABP and CTL groups. Relative abundance is represented as color range from low level (−2, blue) to high level (2, red). (C) Fold change analysis describing alteration trend in computed means of related metabolites in ABP (purple bars) compared against the CTL (red bars). (D) Summary diagram of the pathway analysis with MetPA.

Figure 4

Effects of ternary mixed fungicide of ABP (azoxystrobin: boscalid: pyraclostrobin = 1:1:1, m/m/m) on gut microbial communities and function of A. cerana cerana. Comparison of the (A) gene richness and (B) species richness between fungicide exposure and CTL group. Each group includes three experimental repeats. (C) Principal coordinate analysis (PCoA) shows beta diversity among gut microbiota of fungicide exposure and CTL group based on Bray–Curtis distance. (D) Taxon differences in the gut microbiota between ABP and control group. Sizes of each dot is proportional to taxon abundance. Red-shaded areas indicate ABP-enriched taxa; green-shaded areas indicate the CTL-enriched taxa. Only the taxa that meet a significant LDA threshold value of >3 are shown. (E) Stacked bar plot of relative abundances of main bacteria at the genus level for each sample. (F) Hierarchical clustering heatmap of KEGG functional pathways from all samples. An increasing relative abundance is indicated by a color gradient from blue to red.

Figure 5

Relative expression of the (A) antimicrobial peptide family and (B) cytochrome P450-dependent monooxygenase genes of A. cerana foragers fed on ternary mixed fungicide (ABP) and 50% sucrose as control (CTL) for 10 days. Asterisks indicate differences between treatments via the t-test (** p < 0.01).