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☆
Graphical abstract
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).
Section snippets
Exposure to fungicides
Three bioassays were conducted in order to assess the effects of the fungicide pyraclostrobin (active ingredient and commercial formulation) on the development of the honeybees’ larvae (oral exposure) and the emerging adults. For each bioassay, which followed the OECD no. 239 guideline (OECD, 2016), 576 larvae of the bee Apis mellifera were used, being 144 per experimental group.
First instar larvae from three healthy colonies of Africanized Apis mellifera were individually transferred to
Exposure to pesticides
There was no difference in larval mortality between controls and the fungicide-exposed groups (Table S2, p = 0.08). Pupation and emergence rates did not differ among the different experimental groups (Table S2, p = 0.41 and 0.20, respectively). The larvae exposed to dimethoate showed a mean mortality of 91.67%, validating the bioassays, according to OECD protocol no. 239. The fungicide, isolated or in combination with the pathogen N. ceranae, did not affect the survival of adult honeybees (
Discussion
Larval exposure to pyraclostrobin fungicide, both as active ingredient and commercial formulation, did not affect the development or survival of adult honeybees. The absence of effects on survival was also observed in larval exposure to the dose of the pyraclostrobin fungicide (Tadei et al., 2019) and other fungicides such as iprodione (Carneiro et al., 2020), propiconazole and pyraclostrobin + boscalide (Wade et al., 2019).
In the adult phase, sublethal effects from the larval exposure to
Conclusion
Exposure to the fungicide pyraclostrobin in the larval phase of Apis mellifera affected the morphology of the midgut of emerging bees. The combination of stressors (exposure to the fungicide and pathogen Nosema ceranae) increased the damages to the bees’ midgut epithelium. However, these effects did not affect bee survival at any stage of development and post-emergence, although it may potentially compromise their fitness. The cytotoxic effects of the commercial formulation, in the absence of
Author contributions
Rafaela Tadei, Conceptualization, Formal analysis, Investigation, Methodology, Writing. Vanessa B. Menezes-Oliveira, Investigation, Methodology, Writing. Elaine C.M. Silva-Zacarin, Conceptualization, Methodology, Project administration, Resources, Supervision, Writing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
We would like to acknowledge the support from São Paulo Research Foundation (FAPESP no.2017/21097–3) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) by master’s degree scholarship for first author and post-doctoral scholarship for the second author. We thank also Monique S. Souza and Caio E. C. Domingues for their technical help during bioassays, to the beekeeper Edson Sampaio (COAPIS) for providing bee colonies for experiments, and Profa. Dra. Letícia Silva Souto from
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This paper has been recommended for acceptance by Sarah Harmon.