Disinfectant dodecyl dimethyl benzyl ammonium chloride (DDBAC) disrupts gut microbiota, phospholipids, and calcium signaling in honeybees (Apis mellifera) at an environmentally relevant level

Abstract

One of the impacts of the Coronavirus disease 2019 (COVID-19) pandemic has been a profound increase in the application amounts of disinfectants. Dodecyl dimethyl benzyl ammonium chloride (DDBAC) is a widely used disinfectant, yet its hazards to non-target species remain largely unknown. We are unaware of any studies assessing DDBAC's impacts on honeybee, a pollinator species that is a useful indicator of environmental pollution essential for many forms of agricultural production. Here, we assessed the potentially negative effects of DDBAC on honeybees. After conducting a formal toxicity evaluation of DDBAC on honeybee mortality, we detected an accumulation of DDBAC in the honeybee midgut. We subsequently studied the midgut tissues of honeybees exposed to sub-lethal concentrations of DDBAC: histopathological examination revealed damage to midgut tissue upon DDBAC exposure, microbiome analysis showed a decreased abundance of beneficial midgut microbiota, lipidomics analysis revealed a significant reduction in cell membrane phospholipids with known functions in signal transduction, and a transcriptome analysis detected altered expression of genes involved in calcium signaling pathways (that variously function in calcium absorption, muscle contraction, and neurotransmission). Thus, our study establishes that DDBAC impacts honeybee midgut functions at multiple levels. Our study represents an early warning about the hazards of DDBAC and appeals for the proper stewardship of DDBAC to ensure the protection of our ecological environment.

Keywords: Calcium signaling; Dodecyl dimethyl benzyl ammonium chloride; Gut microbiota; Honeybee midgut; Phospholipids.

Graphical abstract

Fig. 1

The toxic effects of DDBAC on adult honeybees. (A) Acute toxicity test showing mortality of adult honeybees after DDBAC exposure at the indicated concentrations at 24 h and 48 h. (B) Survival of adult honeybees exposed to the indicated sublethal concentrations of DDBAC during a 2-week exposure window. (C) In situ distribution of DDBAC molecule in honeybee midgut after DDBAC exposure at the indicated concentrations for 14 days, assessed by MALDI-MS imaging analysis. (D) Profile signals of MALDI-MS imaging analysis showing the accumulation of DDBAC in honeybee midgut after DDBAC exposure at the indicated concentrations for 14 days. (E) Midgut pathology results for honeybees after DDBAC exposure at the indicated concentrations for 14 days; midgut cells were stained by hematoxylin & eosin.

Fig. 2

The hazards of DDBAC exposure on the microbial composition of honeybee midgut. (A) The changes in microbial alpha diversity of honeybee midgut under the indicated sublethal concentrations of DDBAC exposure for 14 days based on Shannon and Simpson index analysis. (B) The changes in microbial beta diversity of honeybee midgut under the indicated sublethal concentrations of DDBAC exposure for 14 days based on partial least squares discriminant analysis (PLS-DA). (C) Taxonomic cladogram showing the bacterial taxa meeting an LDA-significant threshold > 4.0. The taxa shaded in different colors are with significant abundance in those corresponding groups. The abundance of those highlighted taxa in the control, 1 mg/L, and 100 mg/L DDBAC groups is significantly higher than that in the 10 mg/L DDBAC group. (D) Differences in the abundance of Lactobacillus and Bifidobacterium among different sublethal concentrations of DDBAC exposures by Kruskal-Wallis H test.

Fig. 3

Effects of 10 mg/L DDBAC exposure for 6 and 18 days on lipid composition of the honeybee midgut. (A) Principal component analysis (PCA) showing the different lipidomic profiles in the honeybee midgut with/without DDBAC exposure for short (6 days) and long (18 days) duration. (B) Volcano plot showing the distinct lipid compositions in the honeybee midgut for comparison of 6- and 18-day (after eclosion) DDBAC-exposed samples relative to their time-matched controls. Blue points represent the significantly down-regulated compounds (Fold change > 2 and P value < 0.05) and red points represent the significantly up-regulated compounds (Fold change > 2 and p-value < 0.05). Four kinds of phospholipids are labeled in the panel, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and Phosphatidylinositol (PI). (C) Heat map showing the effects of 6- and 18-day DDBAC exposure on the abundance of four major cell membrane constituent phospholipids (PC, PE, PS, and PI). D6, adult honeybees exposed to 10 mg/L DDBAC for 6 days after eclosion; D18, adult honeybees exposed to 10 mg/L DDBAC for 18 days after eclosion; CK6, adult honeybees without DDBAC exposure and sampled at 6-day after eclosion; CK18, adult honeybee without DDBAC exposure and sampled at 18-day after eclosion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Transcriptional alterations in the honeybee midgut responding to 10 mg/L DDBAC exposure for 6 or 18 days. (A) Venn diagram presenting differentially expressed genes (DEGs) from comparisons of the DDBAC exposure groups to the time-matched vehicle controls. D6, adult honeybees exposed to 10 mg/L DDBAC for 6 days after eclosion; D18, adult honeybees exposed to 10 mg/L DDBAC for 18 days after eclosion; CK6, adult honeybees without DDBAC exposure and sampled at 6-day after eclosion; CK18, adult honeybee without DDBAC exposure and sampled at 18-day after eclosion. (B) Volcano plot presenting the DEGs between the DDBAC exposure group and time-matched vehicle control groups. Red dots indicate upregulated genes; blue dots indicate downregulated genes. (C) Bubble chart for GO enrichment analysis of DEGs. P value indicates adjusted P value. (D) KEGG pathways analysis of the DEGs between the DDBAC exposure group and time-matched vehicle control groups. The horizontal coordinate of the bar chart indicates the -log₁₀(P value), while the horizontal coordinate of the line chart indicates the number of genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Effects of 10 mg/L DDBAC exposure on the expression of genes involved in calcium signaling pathways. (A) DDBAC exposure altered the expression of genes involved in calcium signaling pathways that variously function in calcium absorption, muscle contraction, and neurotransmission. The purple box presents DEGs responsive to 10 mg/L DDBAC for 6 or 18 days. The pathway components presented according to resources of the KEGG database: specifically, the “calcium signaling pathway” (map04020), “endocrine and other factor-regulated calcium reabsorption” (map04961), “vascular smooth muscle contraction” (map04270), “dopaminergic synapse” (map04728), and “dilated cardiomyopathy” (map05414). The full names of the components are shown in the abbreviation table. (B) Heat map presenting the expression levels (mean) of differentially expressed genes involved in calcium-related signaling pathways shown in (A). D6, adult honeybees exposed to 10 mg/L DDBAC for 6 days after eclosion; D18, adult honeybees exposed to 10 mg/L DDBAC for 18 days after eclosion; CK6, adult honeybees without DDBAC exposure and sampled at 6-day after eclosion; CK18, adult honeybee without DDBAC exposure and sampled at 18-day after eclosion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)