Antimicrobial activity of indoleacetic, gibberellic and coumaric acids against Paenibacillus larvae and its toxicity against Apis mellifera

Aim of study: To explore three isolated phytomolecules: indoleacetic acid (IAA), gibberellic acid (GA), and the secondary me-tabolite p-coumaric acid (CUM): (1) evaluating their toxicity against Apis mellifera larvae and adults under controlled conditions in the laboratory; (2) searching for antimicrobial activity against Paenibacillus larvae . Area of study : Honey bee larvae and adults were collected from the experimental apiary of the “Centro de Investigación en Abejas Sociales (CIAS)” (-37.9348798, -57.682817), Institute of the National University of Mar del Plata (UNMdP), Argentina. Material and methods : Paenibacillus larvae strains were isolated from beehives from different provinces of Argentina (Buenos Aires, Córdoba and Entre Ríos) showing clinical symptoms of the American foulbrood. All strains (S1, S2, S3, S4) were genotypically identified using PL5 and PL4 primers and characterized as genotype ERIC1. Then standard essays were performed to determined toxicity of phytomolecules in honey bees and antimicrobial activity through the broth microdilution method. Main results : The diet with GA, IAA and CUM did not present toxic effects in larvae or adult bees, and only CUM showed antimicrobial activity against P. larvae. In this study, we obtained in vitro values of MNIC (minimum non-inhibitory concentration) of 500 μg mL -1 and a MIC (minimum inhibitory concentration) of 650 μg mL -1 for CUM. Research highlights: The obtained results remark its potential as a natural alternative for the control of P. larvae , avoiding the problems generated by the use of synthetic antibiotics such as the resistance phenomena and the contamination of hive’s products.

2 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.

Biological material
Honey bee larvae and adults were collected from the experimental apiary of the "Centro de Investigación en Abejas Sociales (CIAS)" (-37.9348798, -57.682817), Institute of the National University of Mar del Plata (UNMdP), Argentina.

Introduction
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 agrochemicals 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 3 Short communication: Phytochemicals CUM, GA and IAA toxicity in honey bees and control against P. larvae of CO 2 , 37°C, 48 hs). Afterwards, vegetative cells of P. larvae (previously cultivated) were suspended in sterile peptone water (peptone 0.1 % (w/v) and sodium chloride 0.85 % (w/v)) to a final optical density at 600 nm of 0.1 using a UV-VIS spectrophotometer Spectrum SP-1103 (Spectrum Instr. Co. Ltd., Shanghai, China). Brain-heart infusion (3.7 %, w/v) was used as growth media during the broth microdilution assay. Paenibacillus larvae growth was detected using resazurin sodium salt. We evaluated in a range of concentrations between 15.6 to 1000 μg mL -1 against P. larvae strains and determined two threshold concentration for each phytomolecule: the minimum inhibitory concentration (MIC) and the minimum non-inhibitory concentration (MNIC) of in vitro bacterial growth (De Graaf et al., 2013). Positive and negative controls (P. larvae strains viability and water respectively) were used.

Statistical analyses
Kaplan-Meier survival analyses (Stalpers & Kaplan, 2018) were performed in order to compare survival curves (number of living bees vs time) for each treatment. The non-parametric Log-rank test was performed to determine differences between survival curves. This method builds up curves of chi-square values by comparing the observed and expected number of deaths (GraphPad Prism 5.0).

Results and discussion
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, χ 2 =14.14; df =10; p=0.1668) (Fig. 1A) or bee larvae survival, until 8 days in vitro (Long-rank test, χ 2 =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 purity) was used to prepare the stock. The stock solutions concentrations were 10 mM GA, 50 mM IAA and 25 mM CUM.

Toxicity of phytomolecules in honey bees
In vitro experiments were conducted in the CIAS laboratory at the UNMdP. For CUM, GA and IAA toxicity bioassays of adult honey bees, we followed the methodology described in Porrini et al. (2010). For this, combs-sealed brood from healthy colonies were carried to the laboratory within insulated containers and placed into an incubator (30 ± 0.79 °C, 60 ± 3.3% HR). Newly emerged bees were removed from the combs. Each treatment consisted of 30 adult bees randomly confined within acrylic boxes of 8 cm × 15 cm, using a total of three replica (N=90 individuals per treatment). The phytochemicals were administered ad libitum through a solution made of powdered sugar and glucose (candy), which was replaced daily. Mortality was recorded daily for 5 days (120 h). Adult bees were kept under incubator conditions during the experiment of toxicity. For honey bee larvae, the in vitro breeding trials were carried out according to the methodology proposed in Aupinel et al. (2005). We used 30 bee larvae per treatment in each of the three replica, involving a total of 90 (N=90) individuals per treatment. The bee larvae were incubated at 34 ± 0.5 °C and 90% RH. The phytochemicals were administered in individual doses diluted in the food during the whole feeding stage. Mortality was recorded daily for 8 days. The treatments for both growing stages of bees (adults and larvae) were grouped as follows: (i) Control (only candy or larvae diet respectively); (ii) control diet supplemented with the solvent (ethanol) used to do the stock solutions for the molecules tested (C Et); (iii) CUM 300/600/1200 μM; (iv) IAA 100/200/400 μM; and (v) GA 2.5/25/250 μM.

Assays of antimicrobial activity
The antimicrobial activity of the IAA, GA and CUM, were determined by the broth microdilution method on four P. larvae strains (S1, S2, S3, S4) within the same day (in triplicate for each antimicrobial agent and strain) and with triplicate essays (experimental replicas) (Cugnata et al., 2017). First, the bacterial strains were grown and maintained on Mueller-Hinton broth, yeast extract, glucose, and sodium pyruvate (MYPGP) (Dingman & Stahly, 1983) agar supplemented with 9 mg mL -1 of nalidixic acid to inhibit Paenibacillus alvei growth, and incubated under microaerobic conditions (5-10% 4 pounds 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.
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 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 com- Figure 1. Kaplan-Meier plot for honey bee survival. The diet with GA (gibberelic acid), IAA (indoleacetic acid) and CUM (pcoumaric acid) did not present toxic effects in larvae and adult bees. A: Survival of adult bees (N=90 per treatment) fed ad libitum during 5 days (Longrank test, p=0.1668). B: Survival of bee larvae (N=90 per treatment) reared in vitro during 8 days (Longrank test, p=0.9583). Controls involved adult and larvae bees fed only by candy or larvae diet respectively and control diet supplemented with the solvent (ethanol) used to do the stock solutions for the molecules tested (C Et). Table 1. Antimicrobial activity of three phytomolecules (CUM: p-coumaric acid; GA: gibberellic acid; IAA: indole acetic acid) against Paenibacillus larvae. The bactericidal activity of each molecule was evaluated in a range of concentrations between 15.6 to 1000 μg mL -1 . The results were the same for the four P. larvae strains used (S1, S2, S3, S4) 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.