Silent effect of the fungicide pyraclostrobin on the larval exposure of the non-target organism Africanized Apis mellifera and its interaction with the pathogen Nosema ceranae in adulthood

Highlights

• Larval exposure to fungicide caused cytotoxic effects in the midgut of adult bees.

• N. ceranae potentiated cytotoxic effects to bee midgut exposed to pyraclostrobin.

• Fungicide commercial formulation + pathogen intensified the damages to bee’s midgut.

• The work provides important output to be considered in higher tier risk assessments.

Abstract

The frequent exposure of bees to a wide variety of fungicides, on crops where they forage, can be considered a stressor factor for these pollinators. The organisms are exposed both to the fungicide active ingredients and to the adjuvants of commercial formulations. All these ingredients are brought to the hive by bee foragers through contaminated pollen and nectar, thus exposing also immature individuals during larval phase. This work aimed to compare the effects of larval exposure to the fungicide pyraclostrobin (active ingredient and commercial formulation) and its influence on the cytotoxicity to midguts in adults, which were inoculated with the Nosema ceranae spores in the post-emergence stage. Under laboratory conditions, Apis mellifera larvae received an artificial diet containing fungicide solution from the third to the sixth day of the feeding phase. One-day-old adult workers ingested 100,000 infectious N. ceranae spores mixed in sucrose solution. Effects on midgut were evaluated through cellular biomarkers of stress and cell death. The exposure to the fungicide (active ingredient and commercial formulation) did not affect the larval post-embryonic development and survival of adult bees. However, this exposure induced cytotoxicity in the cells of the midgut, showed by the increase in DNA fragmentation and alteration in the HSP70 immunolabeling pattern. Without the pathogen, the midgut cytotoxic effects and HSP70 immunolabeling of the organisms exposed to the commercial formulation were lower when compared to the exposure to its active ingredient. However, in the presence of the pathogen, the cytotoxic effects of the commercial formulation to the adult bees’ midgut were potentialized. The pathogen N. ceranae increased the damage to the intestinal epithelium of adult bees. Thus, realistic doses of pyraclostrobin present in beebread consumed by larvae can affect the health and induce physiological implications to the midgut functions of the adult bees.

Introduction

Non-target organisms as bees may be potentially threatened by exposure to pesticides (Pisa et al., 2014; Yoder et al., 2013). Among pesticides, fungicides are widely used to control pest and diseases in agricultural production (Cullen et al., 2019). Systemic fungicides are frequently detected in beekeeping matrices and floral resources (Long and Krupke, 2016; Raimets et al., 2020) as their active ingredient and to adjuvants of commercial preparations remain in plants (Chen and Mullin, 2013). For honeybees, these fungicide inert compounds may be more toxic than the active ingredient increasing up to 26,000 times the organisms sensitivity to pesticides (Mullin et al., 2016, 2015).

Concentrations of fungicides found in samples of pollen, nectar and beebread might vary from residual to higher concentrations than those causing lethality to 50% of honeybees, when ingested (Böhme et al., 2018). Concentrations of the fungicide strobilurin pyraclostrobin found in pollen were 8 ng/g (Vázquez et al., 2015), 145.8 ng/g (Mullin et al., 2010) and 27,000 ng/g (Pettis et al., 2013). For the beebread, the pyraclostrobin concentrations reached up to 2,170 ng/g (Yoder et al., 2013).

Fungicides have been linked to several effects in honeybees, such as: behavioral changes (Artz and Pitts-Singer, 2015; Tadei et al., 2019), reduction in longevity of adult (Fisher et al., 2017), cell alterations and microbiota changes of intestine (Anderson et al., 2011; Batista et al., 2020; Carneiro et al., 2020; DeGrandi-Hoffman et al., 2017) and changes in the immune system (Cizelj et al., 2016), facilitating their infection by pathogens (Degrandi-Hoffman et al., 2015) as the Nosema ceranae (Glavinic et al., 2019; Pettis et al., 2013). However, studies combining the exposure of bees to pathogens and fungicide are still scarce.

N. ceranae are a highly specialized fungi that exclusively parasitize the epithelial cells of the midgut of Apis mellifera (Fries et al., 2006, 1996). The intracellular parasite N. ceranae can also negatively affect in the health of bees, leading to individual energy stress (Kurze et al., 2016; Mayack and Naug, 2009) and cell lysis at the end of the pathogen cycle (Fries et al., 1996; Panek et al., 2018). When the organisms are exposed to stress conditions, they may activate mechanisms of cellular defense such as the expression of heat shock proteins, as for example the HSP70 that are ATP-dependent for their chaperone activity (Beere, 2004; King and MacRae, 2015). The expression of these proteins may inhibit cell death (Garrido et al., 2001), preventing protein aggregation and restoring the native conformation of proteins and, consequently, their functions (Bukau and Horwich, 1998; Priya et al., 2013). The HSP70 is considered a cell biomarker of exposure to xenobiotics in bees (Silva-Zacarin et al., 2006), being its level used to measure cellular stress (Kim et al., 2019; King and MacRae, 2015).

In Brazil, when assessing the Environmental Risks to bees of using pesticides in agricultural fields, only short-term experiments are normally done. These results may lead to a misunderstanding of the consequences of bee’s exposure to pesticides depending on the mode of action of the pesticide of interest. Thus, this study aimed to compare the sub-lethal effects of larval exposure to the active ingredient and the commercial formulation of the fungicide pyraclostrobin and to evaluate the influence of this exposure on the cytotoxicity to midgut of the adults’ honeybees, which were infected with the N. ceranae spores in the adult phase. Midgut effects were evaluated through cellular biomarkers of cytotoxicity and response to cell stress (HSP 70).