Apis mellifera colonies are threatened by different biotic and abiotic factors which compromise their fitness causing depopulation or entire colony losses (Steinhauer et al., 2018). Due to phenology and climate, there are times of the year where the bees’ food resources are scarce (De Grandi-Hoffman & Chen, 2015). This phenomenon is enhanced by the beekeepers’ management, who harvest almost all the colony’s stored honey, leaving those bees with a nutritional challenge. The depletion of food reserves induces a stress in honey bee colonies, negatively affecting their health and increasing their susceptibility to agro-chemicals and different diseases (Nazzi et al, 2012; De Grandi-Hoffman & Chen, 2015; Sánchez-Bayo et al, 2016).

One of the most important pathogens that affect bee health is the sporulated bacterium (gram positive) Paenibacillus larvae, the causative agent of the American foulbrood (AFB) (Hansen & Brødsgaard, 1999). For its control, the most effective treatments are based on the use of a broad spectrum of antibiotics, such as sulfathiazole and oxytetracycline hydrochloride (OTC). Those molecules are capable of inhibiting the growth of P. larvae, but in most cases, they have been wrongly used in its quantity and frequency of application, leading to the appearance of resistant strains and residues which contaminate the commercial products of the hive (Wilson, 1974; Hansen & Brodsgaard, 1999). Consequently, the use of antibiotics for AFB treatment and prevention is forbidden in several countries (Mutinelli, 2003), leading to an increasing need for natural alternatives for its control. In this venue, there are reports of a wide variety of natural control of P. larvae tested through in vitro assays, such as the use of essential oils, plant extracts, propolis, among others (Alonso-Salces et al, 2016).

Plants contain an enormous variety of chemical compounds that are present in nectar, pollen and/or resins and seems to play an important role in honey bee health (Mao et al., 2013; Couvillon et al., 2015; Negri et al., 2015; Richardson et al., 2015; Erler & Moritz, 2016). Indeed, plant-derived compounds are involved in bees’ “self-medication” a phenomenon defined as an individual responding to infection by ingesting (“pharmacophagy”: e.g. honey, pollen, royal jelly) or to the nonedible hive products (pharmacophory: e.g. propolis, resins) (Erler & Moritz, 2016).

Mao et al. (2013) identified that p-coumaric acid (CUM), a phytochemical found in pollen and honey, up-regulates different detoxification and antimicrobial genes in A. mellifera. Accordingly, Liao et al. (2017) performed dietary trials with CUM (500 μgL^-1) and two pyrethroids insecticides (which are known to reduce the lifespan of bees), observing that this acid enhanced tolerance of both pyrethroids. Isidorov et al. (2017) carried out a study in vitro proving the antimicrobial activity of European propolis against P. larvae, where the GC-MS analysis of those extracts reveals the presence of some flavonoids and also phenolics components including p-coumaric acid. Nevertheless, there is a lack of evidence regarding the antimicrobial activity of CUM against P. larvae.

From a sanitary point of view, phytomolecules found in nectars or in pollen need to be continuously explored regarding its potential effects on bee health. Gibberelic acid (GA) and indoleacetic acid (IAA), are involved in the regulation of plants’ nectar production and other functions (Aloni et al., 2006; Wiesen et al., 2015). These phytomolecules are regulators of growth, development and pathogens resistances in plants, acting through transduction pathways (Richards et al., 2001; Denancé et al., 2013). In addition, these phytohormones are present in honey (Wang et al., 2017), but there are no reports of potential effects on bee health. Here, we aim to assess the potential bactericide effect of three isolated phytomolecules against P. larvae. For this purpose, we evaluated two main aspects: a) the toxicity of CUM, GA and IAA in adults and larvae of A. mellifera; and b) their antimicrobial activity against P. larvae through the broth microdilution method.

In this study, we explored the beneficial properties of p-coumaric acid (CUM) and other previously unexplored phytomolecules, the phytohormones indole acetic (IAA) and gibberellic (GA) acids, on bee health. First, we determined the toxicity of these molecules in larvae and adult bees, in a range of concentrations that include those found naturally in plants (Aloni et al., 2006; Wiesen et al., 2015) and honey (Wang et al., 2017). Our results indicated that these molecules are not toxic to adult bees feed ad libitum for 5 days (Long-rank test, χ²=14.14; df =10; p=0.1668) (Fig. 1A) or bee larvae survival, until 8 days in vitro (Long-rank test, χ²=3.741; df=10; p=0.9583) (Fig. 1B). This is the first condition in the development of anti-parasite treatments to be used in beekeeping (e.g. Maggi et al., 2013).

Secondly, our search of antimicrobial activity in IAA, GA and CUM by the broth microdilution method on four P. larvae strains (S1, S2, S3, S4) suggested that IAA and GA are not suitable antimicrobial molecules in the range from 15.6 to 1000 μg mL^-1 to be used against P. larvae. Only CUM showed antimicrobial activity against P. larvae, obtaining a MIC equal to 650 μg mL^-1 and MNIC to 500 μg mL^-1 (for all P. larvae isolates) (Table 1).

Similar to Tunçel & Nergiz (1993) results, CUM showed antibacterial activity resembling different hydroxycinnamic acids respect their effect against gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli and Salmonella typhimurium) showing similar MIC values (400 to 600 μg mL^-1). In the study of Isidorov et al. (2017), all propolis extracts tested inhibited the growth of P. larvae, with a MIC of 7.8 to 62.4 μg mL^-1. But this antimicrobial activity was associated with a very complex mixture of compounds present in the diethyl ether extracts of propolis (on the chromatograms of nine samples of propolis), where 278 organic components were recorded, among them, monoglycerides and diglycerides of CUM.

Similar MICs values were found between our MIC results of CUM (500 μg mL^-1) and other organic compounds (all assessed by broth microdilution method). For instance, MIC value for essential oils of Artemisia absinthium was 416 μg mL^-1; for Aloysia polystachia was 700-800 μg mL^-1 (Fuselli et al, 2008). Also, individual propolis compounds have been tested such as benzyl ferulate and pentenyl ferulate, with MIC values of 500 μg mL^-1 (Biliková et al., 2013). However, there are other organic compounds with MIC values against P. larvae better and closer to the synthetic antibiotic oxytetracycline hydrochloride (0.5-5 μg mL^-1; Gende et al, 2010), such as cinnamon (Cinnamomum zeylanicum) essential oil (CEO): 41.67 ± 19.17 μg mL^-1 (Gende et al, 2010a), or the individual propolis compound Pinocembrin: 62.5 μg mL^-1 (Biliková et al, 2013).

In our study, we obtained in vitro values of MNIC for CUM (500 μg mL^-1) that remarks its potential as a natural alternative for the control of the American foulbrood, avoiding the problems generated by the use of synthetic antibiotics (resistance phenomena and bee product contamination). In addition to our results, previous reports also demonstrated that in the presence of pesticides, the CUM somewhat enhanced different mechanism of detoxification in honey bees (Mao et al., 2013; Liao et al., 2017). Thus, we found evidence suggesting that CUM is a promising molecule, which could perform either as a pharmacophagy-related compound and/or as a pharmacophory-like substance. Future studies should test CUM effects on A. mellifera colonies in order to improve current knowledge about their integrated management.