Carbendazim exposure during the larval stage suppresses major royal jelly protein expression in nurse bees (Apis mellifera)

Highlights

• A carbendazim-related proteins library of honeybee head is constructed.

• Carbendazim reduces the expression of major royal jelly proteins in honeybee head.

• Proteins related to immune loss, olfactory and visual deficiencies are downregulated.

Abstract

Studying the sublethal effects of agrochemical pesticides on nontarget honeybees (Apis mellifera) is important for agricultural development. Carbendazim is a widely used broad-spectrum fungicide that inhibits mitotic microtubule formation and cell division. However, the impact of carbendazim on bee health and development has not been fully elucidated. Here, using proteomics approaches, we assessed in vitro the changes in the expression of functional proteins in the head of newly emerged adults following treatment with field concentration of carbendazim during the larval stage. Treatment with carbendazim severely altered 266 protein expression patterns in the heads of adults and 218 of them showed downregulation after carbendazim exposure. Notably, major royal jelly proteins, a crucial multifunctional protein family with irreplaceable function in sustaining the development of colonies, were significantly suppressed in carbendazim-treated bees. This result was verified in both head and hypopharyngeal gland of nurse bees. Moreover, visual and olfactory loss, immune functions, muscular activity, social behavior, neural and brain development, protein synthesis and modification, and metabolism-related proteins were likely inhibited by carbendazim treatment. Together, these results suggest that carbendazim is an environmental risk factor that likely weakens bee colonies, partially due to reduced expression of major royal jelly proteins, which may be potential causes of colony collapse disorder.

Introduction

In the past few decades, combined stress from pesticides, parasites, and lack of flowers have negatively affected the species richness of pollinators (Goulson et al., 2015). Honeybees (Apis mellifera) are one of the most important pollinating insects in natural ecosystems and make significant economic and ecological contributions by increasing agricultural production and maintaining ecosystem diversity. The intensification of agriculture has resulted in a boom in pesticide industries. Owing to their foraging activities, honeybees are more likely to be exposed chronically to pesticide-contaminated environments. This has become a global concern for environmental pollution and honeybee conservation.

There are increasing reports on the unexplained decline of honeybee colonies (Potts et al., 2010; Goulson et al., 2015), which is ascribed to colony collapse disorder (CCD) and is likely associated with pesticides(Goulson et al., 2015). Genome sequence revealed honeybees lack detoxification genes compared to other insects (Weinstock et al., 2006; Berenbaum and Johnson, 2015). In fact, deficiency of immunity- and detoxification-related genes contributes to the extreme sensitivity of bees to pesticides, although a “social detoxification system” typical to bees, could be considered as a complementary stress resistance mechanism (Berenbaum and Johnson, 2015). Many studies have demonstrated the sublethal effects of pesticides on physiological functions, such as nervous and hormone system functions, development, sense of direction, immunity, and longevity (Christopher Cutler and Scott-Dupree, 2007; Juraske et al., 2007; Schmehl et al., 2014; Wu et al., 2017; Wang et al., 2018) and behavior, such as mobility, feeding, learning floral traits, navigation, and foraging (Gill et al., 2012; Whitehorn et al., 2012; Fischer et al., 2014; Henry et al., 2012; Kessler et al., 2015). Moreover, aside from the effects of pesticides on on the honeybee itself, dysbiosis of gut microbes resulting from antibiotic or glyphosate exposure, also indirectly affects the health of honeybee, due to increased susceptibility to ubiquitous opportunistic pathogens (Raymann et al., 2017; Motta et al., 2018). It is worth noting that besides their direct action on individual bees, the side effects of pesticides on the overall colony is significant. For example, imidacloprid exposure can inhibit the expression of major royal jelly proteins (MRJPs) in honeybees (Wu et al., 2017; Li et al., 2019; Heylen et al., 2011; Hatjina et al., 2013). MRJPs are involved in differential development of queen larva and worker larvae, thus establishing division of labor in the bee colony (Buttstedt et al., 2014). Thus, the suppression of MRJPs is of great concern because it likely leads to colony collapse.

Carbendazim, a broad-spectrum fungicide, act by inhibiting fungal mitotic microtubule formation and cell division (Akbarsha et al., 2001; Zhou et al., 2018). with remarkable control effects on fungal diseases in honey plants including camellia and rapeseed, thereby allowing residues of carbendazim to accumulate in nectar and pollen (Calatayud-Vernich et al., 2018; Li et al., 2017). Shi et al. (2018) reported that carbendazim at field concentration (0.0456–4.56 ppb) was not acutely lethal to worker bees but significantly inhibited the expression of antimicrobial peptides and detoxifying enzymes. In addition, carbendazim has been found to possess properties, and delays larvae development (Wang et al., 2018) in honeybees. Zhou et al. (2006) have reported that carbendazim (detection rate: 77.1%, maximum value: 4516 ng g⁻¹) presented the highest detection ratio from 48 bee pollen sampled from eight provinces in mainland China. Owing to the migratory beekeeping method adopted by most beekeepers in China, honey bees have to travel across large numbers of Intricate fields or farms to forage, which increases the likelihood of pesticide exposure. Considering the detection of high residue ratios of carbendazim (Zhou et al., 2016), investigation on the effects of this fungicide on A. mellifera becomes relevant, particularly on its chronic sublethal impacts.

The “omic” technologies have become increasingly important in evaluating the side effects of pesticides on bees (Christen et al., 2018; Roat et al., 2020). Therefore, in the present study, we evaluated the molecular mechanisms associated with carbendazim response in honeybees using proteomic approaches. The elucidation of differentially expressed proteins (DEPs) after carbendazim exposure aids our understanding of the biological processes in the head of honeybees and provides insights into the molecular mechanisms underlying the action of this fungicide.