Apidologie (2016) 47:389–411 Review article * INRA, DIB and Springer-Verlag France, 2015 DOI: 10.1007/s13592-015-0400-z

Pharmacophagy and pharmacophory: mechanisms of self-medication and disease prevention in the honeybee colony (Apis mellifera )

1 1,2,3 Silvio ERLER , Robin F. A. MORITZ

1Institut für Biologie, Molekulare Ökologie, Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 4, 06099, Halle (Saale), Germany 2Department of Zoology and Entomology, University of Pretoria, 0002, Pretoria, South Africa 3German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103, Leipzig, Germany

Received 20 May 2015 – Revised 3 August 2015 – Accepted 8 October 2015

Abstract – Apitherapy promises cures for diseases in human folk medicine, but the effects of honeybee produced and foraged compounds on bee health are less known. Yet, hive products should chiefly facilitate medication and sanitation of the honeybees themselves rather than other organisms. We here review the impact of both self-produced gland secretions and foraged hive products (pharmacognosy) on colony health. Although foraged plant-derived compounds vary highly in antibiotic activity depending on the floral and regional origins, secondary plant metabolites in honey, pollen and propolis are important for the antibiotic activity against pathogens and parasites. However, specific bee health-enhancing activities of bee products should clearly be distinguished from the effects of an intact nutrition ensuring the basic immune competence of bees. Further unravelling the interactions among groups of active substances or individual compounds used in concert with specific behavioural adaptations will deepen our understanding of the natural potential of honeybees to maintain colony health. honey / propolis / pollen / bee bread / royal jelly / antimicrobial activity / self-medication / host-parasite interaction

1. INTRODUCTION to infectious viruses (Mutinelli 2011; Schmid- Hempel 1998). Hence, it is not surprising to see a The colony-based life history and social organi- suite of bacterial, viral, fungal and protozoan infec- zation of honeybees (Apis mellifera )makethem tions as well as various parasitic arthropod infesta- ideal targets for any parasite or pathogen infection tions that cause serious damage to both individual (Schmid-Hempel 1998). The high density of closely bees and the colony as a whole (Bailey and Ball related individuals, the constant environmental con- 1991; Morse and Flottum 1997; Schmid-Hempel ditions, and the rich stores of proteins and carbohy- 1998). Indeed, some of these agents (e.g. Varroa drates in the nest provide ideal conditions for ene- destructor , Nosema sp. and viruses) are currently mies, ranging from large vertebrate predators down considered to be involved in recently discussed global colony losses (Cornman et al. 2012; Genersch et al. 2010; vanEngelsdorp et al. 2009). Yet, in spite of these dramatic reports, honey- Corresponding author: R. Moritz, bees are by no means defenceless against diseases [email protected] and pests neither at the individual nor at the col- Handling Editor: Yves Le Conte ony level (Evans and Spivak 2010). External 390 S. Erler and R.F.A. Moritz individual defence mechanisms (e.g. the insect there are several recent reviews available on hy- cuticle) and also social defence mechanisms (e.g. gienic behaviour and the importance of grooming seclusion of the nest cavity) provide efficient lines for social insect colony defence, we will not of defence against parasites and pathogens. The address this issue in any deeper detail and would release of the honeybee genome gave a valuable like to direct the reader to the excellent reviews of insight into the genomic background of individual Evans and Spivak (2010) and Wilson-Rich et al. immune defence (Honeybee Genome Sequencing (2009) and references therein. Consortium 2006). We now know that the four We will focus this review on those behavioural major innate immune pathways (Toll, Imd, JNK, defence mechanisms that result from the bees’ JAK/STAT) are involved in the innate immune exceptional capacity as generalist foragers of plant response of honeybees upon infection and products. The co-evolution of floral plants and wounding. A broad molecular toolbox is used pollinators has driven plants to provide nectar for most effective defence mechanisms including and pollen in exchange for the pollination service. proteasome-dependent degradation, apoptosis, In order to be attractive to bees, the nectar provid- melanisation and antimicrobial effector proteins ed in a flower should not be fermented nor should which reduce or eliminate pathogens. Yet, com- pollen be contaminated with fungal pathogens. paring the honeybee with other insects shows that Hence, plants need to provide pathogen-free food the Apis innate immune system is based on a for the bees if co-evolution is going to be a success severely reduced number of immunity-related story. It is therefore not that surprising to see genes (Evans et al. 2006). This came as a surprise, plants adding antibiotic secondary metabolites to given the excellent intra-colonial conditions for the nectar that prevent bacterial fermentation. In pathogens and the apparent need for control. general, plants themselves need protection against However, colony immunity is governed not just bacterial and fungal pathogens and suites of high- by individual immune competence but also by ly active antimicrobial and antifungal compounds external immune defence (Otti et al. 2014). are known and used in many medical treatments Mechanisms at the colony level have been shown in human medicine. When bee pollinators (both to be equally important, and the reduction of social and solitary) collect pollen and nectar from immune genes may be more than compensated plants, they will therefore not just forage for pro- for by well-developed behavioural defence mech- tein and carbohydrate; they will inevitably also anisms, ‘social’ or ‘collective immunity’ (Cotter forage associated secondary plant metabolites that and Kilner 2010;Cremeretal.2007). will have immediate impact on the individual bee Social immunity summarizes all antiparasitic and the colony, enhancing colony health. By for- colony-level mechanisms, not just reducing any aging to satisfy their basic nutritional demand, parasite intake into the colony but also reducing honeybees inevitably gather compounds as part the spread, and transmission between individuals of the nectar or pollen that may become relevant and colonies (Cremer et al. 2007). This disease to fight pests and pathogens. However, there is a resistance resulting from social behaviour has concise difference between the nutritional func- been known since the ground-breaking work of tion and the health function. The nutritional value Walter Rothenbuhler on hygienic behaviour of the food relates to the amount of carbohydrates, (Rothenbuhler 1964; Rothenbuhler and proteins, and other essential compounds ensuring Thompson 1956). Workers uncap cells with larvae basic physiological functioning and can always be infected with Paenibacillus larvae and remove needed in the colony irrespective of its health the infected individuals from the colony. status. Health-relevant compounds may not be Hygienic behaviour has therefore successfully needed immediately, and this generates a problem. been used in selective breeding in the control of Specific foraging for health may not be possible various brood diseases (e.g. American foulbrood, because plants producing a specific compound chalk brood and Varroa mites) (Harbo and Harris may not be flowering when needed to fight spe- 1999;Palacioetal.2010; Rothenbuhler and cific diseases. So storage of these compounds Thompson 1956; Spivak and Gilliam 1998). As becomes an issue, and it is here where the Bee products for self-medication 391 honeybee excels. The huge difference between pheromones and other secretions typically com- eusocial honeybees with perennial colonies and posed of various volatile and nonvolatile com- all other bee pollinators is the exceptional capacity pounds (Mizrahi and Lensky 1997). The secre- of the honeybee colony to store foraged plant tions of the hypopharyngeal, mandibular, venom products over extended periods of time. It is the and wax glands have been identified to be most ability to store the huge variety of foraged antimi- important regarding self-medication by self- crobial substances that lends the honeybee colony produced bee products. Substances secreted by an enormous advantage not just within the bee these exocrine glands do not just comprise the pollinators but also over many other social insects whole range of nutritive primary elements, includ- that require animal protein in their diet. The ca- ing carbohydrates, proteins and fatty acids, but pacity to store food provides the honeybees with also compounds with antimicrobial activity. an opportunity to selectively choose among the variety of stored products in an adaptive way dependent on their own or the colony’s health 2.1. Cuticular hydrocarbons status. Given the huge field of apitherapy and the Cuticular hydrocarbons covering the entire arguably not always convincing use of bee prod- body of the honeybee primarily function as a ucts in human medicine, it is clearly overdue to barrier to prevent water loss (Figure 1). In addi- study the effects of antibiotic plant secondary tion, they also affect the absorption of chemicals metabolites more widely in the context of honey- from the environment, can serve as pheromones, bee health rather than human health (the latter and most important for honeybee health, form a reviewed in some older reviews by Crane 1975 most efficient obstacle against the attachment to or and Ghisalberti 1979). penetration of the insect cuticle by microorgan- Bees use native or processed hive products in isms (Blomquist and Jackson 1979; Lockey two alternative pharmacological ways defined as 1988). They form an oily layer on the cuticle, pharmacophagy and pharmacophory. which is continuously renewed by glandular se- Pharmacophagy relates to all defence mechanisms cretions and prevents fungi and bacteria from resulting from the direct consumption (e.g. honey, entering the bees’ body (Blomquist et al. 1980; pollen, royal jelly) to decrease the disease or in- Koidsumi 1957;Gołębiowski et al. 2013). The crease honeybee health whereas pharmacophory cuticular hydrocarbon composition varies during refers to the nonedible hive products (e.g. propolis, individual development and is dependent on both resin) (König 1988). In addition to foraged com- the environment and the individual health status pounds, we will also highlight those honeybee- (Blomquist et al. 1980). The honeybees’ cuticular produced compounds that are not related to the wax layer mainly consists of hydrocarbons, innate immune system but nevertheless are highly mono- and polyester, free fatty acids and other effective against pests and pathogens. This will polar substances (Blomquist et al. 1980). These particularly include the compounds secreted in cuticular hydrocarbons serve as a kind of ‘body the various glandular systems of honeybees. lotion’ washing off any potentially intruding path- Hence, we will address the self-produced and for- ogen and are therefore most essential for individ- aged compounds in honeybee colonies and discuss ual bee health. In addition, compounds that are the health-promoting activity of these products. easily dissolved in the oily hydrocarbons can be transported across the body surface. These com- 2. ANTIBIOTIC AND BEE HEALTH- pounds include esters and fatty acids, many of ENHANCING ACTIVITIES OF SELF- which are known to have antibiotic activity. PRODUCED HIVE AND BEE Hence, although cuticular hydrocarbons them- PRODUCTS selves have no direct antimicrobial or antifungal function, they are crucial for self-sanitation and The honeybees’ glands and glandular tissues may serve as a carrier substance for antibiotic produce a broad variety of well-identified compounds to be spread on the body surface, 392 S. Erler and R.F.A. Moritz

Figure 1. In-hive repertoire of foraged and self-produced agents used by honeybees not only for self- and nest sanitation but also for antibiotic feeding of brood and other nestmates. In the field: Secondary plant metabolites with antibiotic potential are inevitably foraged together with pollen and nectar. Propolis with highly antiviral and antibiotic compounds is collected by specific foragers for nest cavity sanitation. In the hive: Stored honey and bee bread can be selectively used for feeding diseased and healthy larvae and other nest members. On the bee: Glandular secretions with antibiotic activity can be used for individual and colony health.

which prevents adhesion of microorganisms or beeswax extracts against bee parasites and may be toxic to them. pathogens, but the natural comb has never been tested in situ in a biological setting. 2.2. Beeswax Wax extracts in acetone, ethanol and metha- nol repeatedly proved to have antimicrobial The physiochemical and biological proper- activity (Table I). In vitro assays verified the ties of beeswax have been comprehensively antibacterial activity against honeybee patho- described in two monographs by the late gens including Paenibacillus alvei and Randall Hepburn (Hepburn 1986; Hepburn P. l arvae (bacteria associated with American et al. 2014). Beeswax is secreted by the and European foulbrood) (Lavie 1960a), but wax glands and is composed of a complex also fungicide activity against Aspergillus mix of alkanes, alkenes, hydrocarbons flavus , Aspergillus fumigatus and (14 %), free fatty acids (12 %), monoesters Aspergillus niger —fungi associated with (35 %), diesters (14 %), hydroxymonoesters stone brood disease (Kacániová et al. 2012). and some minor constituents (fatty alcohols Both assays used extracts of small pieces of and hydroxydiesters) (Hepburn et al. 2014; comb heated (80 °C) or boiled for 1 h in the Mizrahi and Lensky 1997). The beeswax an- respective solvent. Any potential compounds tibiotic activity is known for the fatty acids in these extracts that might have caused the rather than the esters (Koidsumi 1957; inhibitory activity had not been identified. Gołębiowski et al. 2013). Only very few Moreover, the wax had been extracted from studies address the biological activity of old combs rather than wax from newly Bee products for self-medication 393

Table I. Effects of glandular secretions on bee parasites, pathogens and predators.

Product Application Parasite/ Observation Technique/ Reference pathogen/ experiment predator

Wax Acetone extract P. al ve i , P. Bacteria In vitro assay Lavie 1960a larvae growth inhibition Ethanol and methanol A. flavus , A. Fungi growth In vitro assay Kacániová et al. 2012 extracts fumigatus , inhibition A. niger Royal Pure material E. faecalis , P. Bacteria In vitro assay Hornitzky 1998; jelly larvae growth Sauerwald et al. inhibition 1998 Acidic extract P. larvae Bacteria In vitro assay Bachanová et al. growth 2002 inhibition Aqueous-ethanol extract P. larvae Bacteria In vitro assay Crailsheim and growth Riessberger-Gallé inhibition 2001 Ether extract P. al ve i , P. Bacteria In vitro assay Lavie 1960b larvae growth inhibition/ delay Major royal jelly protein P. larvae Bacteria In vitro assay Bachanová et al. 2, defensin-1 growth 2002;Bílikováetal. inhibition 2001, 2009 Pure material A. apis , A. Fungi growth In vitro assay Chu et al. 1992; niger inhibition/ Sauerwald et al. weakening 1998 Crude extract and V. destructor Deterrent Arena Calderone et al. 2002; fractions activity, experiment Drijfhout et al. 2005 (dichloromethane- repellent methanol) effect Octanoic acid V. destructor Repellent Arena Nazzi et al. 2009 effect experiment, bee colony Worker Aqueous-ethanol extract P. larvae Bacteria In vitro assay Crailsheim and jelly growth Riessberger-Gallé inhibition 2001 Pure material P. larvae Bacteria In vitro assay Rose and Briggs growth 1969 inhibition Crude extract V. destructor Arrestment Arena Calderone and Lin (dichloromethane- response experiment 2001 methanol) Drone Crude extract and V. destructor Arrestment Arena Calderone and Lin jelly fractions response experiment 2001 (dichloromethane- methanol) constructed virgin combs or freshly secreted wax combs may also have contributed to the observed scales. Hence, compounds transferred from stored antibiotic effects, and not just wax itself. honey, pollen, resin or bee larvae reared in these Nevertheless, given the antibiotic potential of 394 S. Erler and R.F.A. Moritz many compounds in wax, it seems most likely that varies significantly among honeybee colonies both the individual bees producing wax and the (Rose and Briggs 1969). Royal jelly from developing larvae may profit from the antibiotic P. larvae -resistant colonies had much stronger potential in wax. antimicrobial activity than that from susceptible colonies (Rose and Briggs 1969). Honeybees may 2.3. Food jelly be able to adjust the quality of royal jelly in response to pathogen infections. Workers of The nurse worker bees feed the larvae with a P. larvae -infected colonies produce royal jelly protein-rich secretion of the hypopharyngeal with much higher amounts of antibacterial pep- glands, termed royal jelly if fed to queen larvae, tides (Bachanová et al. 2002). Most likely, the worker jelly if fed to worker larvae and drone jelly combination of high-quality proteinous food, en- if fed to drone larvae. This food jelly is the exclu- hancing bee health, with increasing antimicrobial sive diet of the adult queen but is also fed to all activity offers a simple and effective method for other adult members of the colony albeit as sup- reducing or eliminating intra-colony P. larvae ti- plementary food. Food jelly is composed of water tres. However, given the variability of royal jelly (60–70 %), proteins (12–15 %), sugar (10–16 %), quality even within the same colony, there is lipids (<10 %) and traces of vitamins, salts and certainly a wide open research field looking at free amino acids. The majority of the protein the effects of the interactions between nutrition, fraction comprises the so-called major royal jelly pathogen and royal jelly quality. Nothing is proteins (for review, see Buttstedt et al. 2014). known about seasonal, colony-specific or biogeo- Pure and even water-diluted royal and worker graphic effects relating to the numerous subspe- jelly has been shown to have antibiotic activity cies of Apis mellifera or the other Asian Apis against bacteria (Enterococcus faecalis ) and fungi species. (Ascosphaera apis and Aspergillus niger ) In addition to the antimicrobial and antifungal (Table I). In addition, the antimicrobial effects of effects, royal jelly also interferes with parasites. the various compounds in royal and worker jelly Crude extracts (dichloromethane-methanol) and have been tested in vitro using acidic, aqueous- fractions of royal, worker and drone jelly used in ethanol and ether extracts on P. alvei and arena experiments with V. de st ru ct or mites had P. larvae cultures (Table I). In general, royal jelly repellent or arresting effects (Table I). extracts had higher inhibitory effects than those of Experiments using food jelly and V. destructor worker jelly (Crailsheim and Riessberger-Gallé were conducted to understand why these mites 2001). Whereas the vegetative P. l arv ae cells are prefer drone cells over queen cells to complete killed after 5 min of treatment with water-diluted their reproductive cycle. A mix of 15 polar semio- royal jelly, the spores showed no reduced survival chemicals, originally characterized in native royal (Hornitzky 1998). jelly fractions, showed to have a comparable de- In vivo larval infection studies with the fungal terrent activity against V. destructor (Drijfhout pathogen Aspergillus fumigatus show a reduction et al. 2005). Octanoic acid, as single repellent of larval mortality with increasing royal jelly con- substance, is the first volatile substance being centration (Foley et al. 2012). Whether this is due active against the mite under lab and field condi- to malnutrition resulting from a lack of proteins in tions (Nazzi et al. 2009). This fatty acid is as the diet or due to antifungal compounds in royal repellent as royal jelly itself and might be involved jelly needs to be determined. in the repellency of queen cells (Nazzi et al. 2009). Two isolated proteins (major royal jelly protein A single experiment has been conducted to 2 and defensin-1) and the fatty acid 10-hydroxy-2- unravel the self-medication potential of royal jelly decenoic acid are discussed as candidates of the by feeding different types of royal jelly antimicrobial activity of royal jelly (Bachanová (heterospecific feeding) to Chinese sacbrood virus et al. 2002;Bílikováetal.2001, 2009; Chu et al. (CSBV)-infected bee larvae. Heterospecific feed- 1992;Hornitzky1998). The antimicrobial activity ing, the feeding of bee larvae with royal jelly of of royal jelly is not just dose dependent but also non-species-specific origin, showed that feeding Bee products for self-medication 395

Apis cerana larvae with Apis mellifera royal jelly worker specialization govern the allocation reduces mortality rates and therefore enhances of foragers to the various tasks (Page 2013). resistance to Chinese sacbrood virus in compari- In-hive workers further process the collected son to feeding A. cerana larvae with A. cerana pollen and nectar to bee bread and honey. royal jelly. This effect was not observed for Apis Both products can be stored for extended mellifera larvae (Zhang et al. 2014). periods of time to bridge phases of dearth Heterospecific feeding might induce antiviral pro- including winters and drought. However, pol- tein expression or royal jelly molecules can direct- len and nectar do not just comprise water, ly inhibit Chinese sacbrood viral replication in bee sugars and proteins. Trace elements, vitamins larvae (Zhang et al. 2014). Subsequent differential and particularly various secondary plant me- protein expression analysis found that proteins tabolites are included, which may have im- involved in stress response, phagocytosis, antiox- portant effects on bee health and bee diseases idation and energy metabolism are candidates (Brodschneider and Crailsheim 2010; Vaudo explaining the mortality-reducing activity of et al. 2015). heterospecific feeding (Zhang et al. 2014). In addition, there are workers that specialize in the collection of plant resins. These resins are 2.4. Bee venom mixed with wax and other bee-derived substances to produce propolis (see Section 3.1)thatisused Honeybee venom is a complex mix of proteins to seal and protect potentially exposed regions of (phospholipase A, hyaluronidase), peptides the nest cavity. (melittin, apamin), physiological active amines (histamine, dopamine), sugars, phospholipids 3.1. Resin and propolis and some volatile compounds (Mizrahi and Lensky 1997). The most active compound, with Resins are tree bud-, leaf- and wound- up to 50 % of dry weight, is melittin. Its high excreted sticky substances, which are foraged antibiotic and biological activity has been studied by the bees and termed propolis once used in for many decades in human apitherapy (Mizrahi the hive and mixed with pollen and wax by and Lensky 1997; Ratcliffe et al. 2011). Only few honeybees. The use of resins to improve and studies analysed honeybee health-enhancing fea- reshape the nest cavity is not unique to Apis tures of bee venom. Female honeybees smear mellifera . Stingless bees (Meliponini) also venom proteins (melittin and apamin) on their process resin either as cerumen (mixed with body surface and on comb wax for self- and nest wax) and batumen or geopropolis (mixed sanitation (Baracchi and Turillazzi 2010; Baracchi with wax, mud, seeds, wood or vertebrate et al. 2011). However, given the high antibiotic faeces) (Roubik 2006). The chemical compo- potential, there is a surprising paucity of studies sition (with more than 300 chemical com- on the antibiotic activity of bee venom or venom pounds in complex and varied mixtures), flo- proteins including potential interactions with oth- ral origin and verification, folk medicine us- er cuticular compounds. age and general characteristics of propolis are comprehensivelysummarizedinvariousre- 3. ANTIBIOTIC AND BEE HEALTH- views (Bankova et al. 2000, 2014; Burdock ENHANCING ACTIVITIES 1998; Ghisalberti 1979; Greenaway et al. OF FORAGED HIVE PRODUCTS 1990; Huang et al. 2014; Marcucci 1995; Mizrahi and Lensky 1997; Simone-Finstrom Honeybees are vegetarians in a strictly vegan and Spivak 2010; Walker and Crane 1987). sense: nectar and pollen are the only protein and The composition even from the same plant carbohydrate food sources. Clearly, the primary species can vary markedly even depending aim of foraging workers is to collect pollen, nectar on the sex in sexually dimorphic plants with and water for feeding the colony members. It is two-times higher activity of female plant res- well understood how division of labour and in (Lokvam and Braddock 1999). 396 S. Erler and R.F.A. Moritz

Healthy worker bees treated with such ex- P. larvae spore loads in honey were reduced tracts (ethanol and methanol) reduce their after propolis extract treatment (Antúnez et al. individual innate immunity and upregulate 2008). These colony-level examples suggest that detoxification genes (Johnson et al. 2012; propolis compounds either directly inhibit the Simone et al. 2009). In addition, much lower replication of vegetative P. l arv ae cells in the microbe levels were detected in healthy col- larval gut or stimulate the honeybee’simmune onies treated with different ethanolic propolis system (Antúnez et al. 2008). Flavones/flavonols extracts (Simone et al. 2009). This effect was and flavanones/dihydroflavonols, the two major explained by inhibition due to direct contact groups of propolis compounds, are most likely of extracts and bacteria or by the volatile candidates to trigger the proposed mechanisms compounds released in the colony (Simone (Mihai et al. 2012). et al. 2009). Both studies were the first A recent study tested several propolis sam- claiming the self-medication potential of for- ples, from 12 climatically diverse regions, aged hive products. against P. larvae and A. apis (chalk brood Nearly all studies characterizing the antibiotic pathogen) and found that some samples did activity of resin and its products used extracts deviate from the generally high antibiotic made with a huge variety of various solvents activity observed against both pathogens. (acetone, acetonitrile, dimethylsulfoxide, ethanol, Four of the 12 samples were poor inhibitors ethylacetate, hexane, methanol, petrol ether) or of P. larvae growth, but good inhibitors of simply water (Table II). In vitro antibacterial A. apis growth, suggesting that resin avail- activity is in a dose-dependent manner well con- ability in these climatic regions was deficient firmed for E. faecalis , P. al vei and P. l arv ae in terms of providing broad-spectrum antibi- (associated with American and European foul- otic activity (Wilson et al. 2015). Thus, re- brood) using honeybee propolis (Table II). gional differences in floral vegetation (resin Climate, local environment and plant origin are donors) can very strongly determine antibiot- the major drivers of varying antibiotic activity, ic activity with highly variable therapeutic with wet-tropical rainforest-type climate propolis potential. showing the strongest activity (Seidel et al. Propolis extracts inhibit the growth of not only 2008). The antibiotic activity seems to be very A. apis but also other fungal pathogens including stable and the antibiotic effects of propolis A. flavus , Aspergillus fumigatus and Aspergillus showed no decline over time (Schmidt et al. niger (Table II). Enhanced resin foraging was 2014). Metabolomic studies on cottonwood and observed in chalk brood-infected and balsam poplar resin detected neither significant V. destructor -infested colonies (Simone- seasonal nor regional changes in major com- Finstrom and Spivak 2012;Popovaetal.2014). pound composition, but the different resin donor Increased amounts of resin reduced chalk brood species varied in inhibition of P. l arv ae (Wilson infection intensities, however, without any knowl- et al. 2013). They showed that foraging honey- edge of the exact causal mechanisms (Simone- bees make discrete choices among many closely Finstrom and Spivak 2012). related resinous plant species, depending on me- Resins have also been suggested to have tabolite content and antimicrobial activity. Some an impact on Nosema infections. In cage studies showed that spraying or feeding experiments with Apis florea , ethanolic ex- ethanolic and aqueous propolis extracts can also tracts of stingless bee cerumen (Trigona act at the colony level. Treated colonies repeat- apicalis ) reduced Nosema ceranae infection edly have been shown to partially recover from rates (Suwannapong et al. 2011). However, the P. larvae infection. Typically treated colo- this study did not use A. florea -collected nies showed disease suppression and a reduction propolis, so the results on honeybees infected in diseased larvae compared to untreated control with N. ceranae have to be treated with colonies (Kamel et al. 2013; Lindenfelser 1968; caution in the context of self-medication of Mlagan and Sulimanovic 1982). Also the a honeybee colony. Bee products for self-medication 397

Table II. Effects of propolis on bee parasites, pathogens and predators.

Application Parasite/ Observation Technique/ Reference pathogen/ experiment predator

Acetone extract E. faecalis , P. Bacteria growth In vitro Lokvam and Braddock 1999 alvei , P. inhibition assay larvae Dimethylsulfoxide E. faecalis Bacteria growth In vitro Netíková et al. 2013 extract inhibition assay Ethanol extract E. faecalis Bacteria growth In vitro Derevici et al. 1964; Kartal et al. inhibition assay 2003; Koo et al. 2000;Kouidhiet al. 2010; Mavri et al. 2012; Nieva Moreno et al. 1999; Schmidt et al. 2014;Seideletal.2008; Stepanović et al. 2003;Uzeletal. 2005 Methanol extract E. faecalis , P. Bacteria growth In vitro Boonsai et al. 2014; Erkmen and larvae inhibition assay Özcan 2008; Vardar-Ünlü et al. 2008 Aqueous extract P. al ve i Bacteria growth In vitro Lavie 1960a inhibition assay Acetonitrile P. l arv ae Bacteria growth In vitro Wilson et al. 2013 extract inhibition assay Ethanol extract P. l arv ae Spore load Bee colony Antunez et al. 2008 reduction in honey Ethanol extract P. l arv ae Bacteria growth In vitro Bastos et al. 2008; Bíliková et al. inhibition assay 2013; Lindenfelser 1967;Mihai et al. 2012;Özkırımetal.2014; Wilson et al. 2015 Ethanol extract, P. l arv ae Bacteria growth In vitro Lavie 1960a; Mlagan and aqueous extract inhibition assay Sulimanovic 1982 Petrol ether, P. l arv ae Bacteria growth In vitro Bíliková et al. 2013 ethylacetate inhibition assay fraction Ethanol extract A. apis , A. Fungi growth In vitro Falcão et al. 2014; Garedew et al. flavus , A. inhibition assay 2004b; Ghaly et al. 1998; fumigatus , A. Sahinler and Kurt 2004; niger Samšiňáková et al. 1977; Wilson et al. 2015 Ethanol and A. flavus , A. Fungi growth In vitro Kacániová et al. 2012 methanol fumigatus , A. inhibition assay extracts niger Dimethylsulfoxide A. fumigatus Fungi growth In vitro Netíková et al. 2013 extract inhibition assay Ethanol extract G. mellonella Reduced larval In vivo assay Eischen and Dietz 1987;Garedew growth and et al. 2004a; Johnson et al. 1994 survival, metabolism regulation Ethanol extract V. destructor Narcotic and In vivo assay Damiani et al. 2010; Garedew et al. lethal effects, 2002; Garedew et al. 2003a metabolism regulation 398 S. Erler and R.F.A. Moritz

Table II (continued)

Application Parasite/ Observation Technique/ Reference pathogen/ experiment predator

Ethanol extract V. destructor Reduced mature Bee colony Simone-Finstrom and Spivak 2010 female production Pure material A. tumida Beetle Bee colony Ellis et al. 2003;Neumann encapsulation et al. 2001 Pure material O. smaragdina Repellent effect Open arena Duangphakdee experiment et al. 2009 Pure material Ants, hunting Sticky barrier Bee colony Seeley et al. 1982 predators Pure material Dead Encapsulation Bee colony Visscher 1980 mammals, large insects

Ethanolic extracts seem to act not only on insects within the colony (reviewed in pathogenic and parasitic microorganisms but Visscher 1980). also on larger enemies (Table II). Feeding Raw honeybee propolis is further used to propolis ethanolic extracts to wax moth exclude honey- or brood-robbing ants, and (Galleria mellonella )larvaeininvivoassays other hunting predators from the nest reduced wax moth larval growth, survival (Table I). Hive entrances are glued with a and heat production with increasing propolis propolis layer as a sticky barrier against concentration (Eischen and Dietz 1987; small, crawling insects to protect bee colo- Garedew et al. 2004a; Johnson et al. 1994). nies (Seeley et al. 1982). Such sticky barrier Topical application of propolis extracts on defence may also be a combination of me- V. destructor in vivo in the honeybee colony chanical and chemical defence. Open arena resulted in narcotic and lethal effects on the experiments showed a repellent effect of mite, and again interfered negatively with the propolis against the weaver ant Oecophylla larval heat production (Damiani et al. 2010; smaragdina . That effect was explained by Garedew et al. 2002, 2003a). Indeed, the adhesive and viscous characteristics of Simone-Finstrom and Spivak (2010) sug- the plant resins (Duangphakdee et al. 2009). gested a propolis treatment to reduce mature female mite production at the colony level. 3.2. Pollen and bee bread Few studies show how native propolis is used in the context of colony health Pollen as collected by the foragers has on av- (Table II). Invading adults of the small hive erage about 35 % proteinaceous content including beetle Aethina tumida were encapsulated in about 50 % free amino acids. The amounts of ‘propolis prisons’ to prevent establishment carbohydrate are highly variable, the lipid content and spreading (Ellis et al. 2003;Neumann is below 10 % and vitamins, minerals and trace et al. 2001). Both studies highlighted signif- elements are present in quantities <10 % (Campos icant differences in the observed encapsula- et al. 2008; Mizrahi and Lensky 1997). Protein- tion behaviour and effectiveness against the carbohydrate ratios and chemical composition de- beetle, comparing European and South pend not only on the floral origin but also on African cape honeybees. This mechanism is climatic and environmental conditions, plant age also used to isolate dead mammals or large and nutrient status during pollen development and Bee products for self-medication 399 the water content of the pollen grain (Campos origin is correlated to low infection intensities et al. 2008). The pollen foragers deposit the col- with acute bee paralysis virus (ABPV), black lected pollen pellets directly into the storage cell. queen cell virus (BQCV), DWV and sacbrood The stored pollen is then transformed to bee bread virus (SBV) (Antúnez et al. 2015). The observed by in-hive bees through lactic acid fermentation of antiviral activity of pollen was thought to be due pollen mixed with regurgitated nectar, honey and to the presence of quercetin and other phenolic glandular secretions (Brodschneider and compounds (Antúnez et al. 2015). But again, the Crailsheim 2010). The nutritive value and physi- results can be explained by either malnutrition or a cochemical characteristics of fresh and processed potential antiviral activity of specific pollen types. pollen are not different from each other, suggest- For more details on Varroa - and virus-infection- ing that both are equally important for bee nutri- driven immunity changes and trade-offs with tion (Herbert and Shimanuki 1978). The pollen (mal-)nutrition, please see the current review of diet is not only important for the growing larvae, DeGrandi-Hoffmann and Chen (2015). but also for the queen to ensure egg laying and the Antibacterial and antifungal activities of pollen nurse bees to produce food jelly. Although other were assessed by testing the effects of solvent adult bees typically have a respiratory quotient extracts in ethanol, methanol or water and water- close to 1 and primarily use carbohydrates to ethanol, on honeybee pathogenic bacteria satisfy energy metabolism, they also need protein (P. alvei , P. larvae )andfungi(A. flavus , in their diet. This is of particular importance when Aspergillus fumigatus , Aspergillus niger ) it comes to immunity and fighting diseases. Alaux (Table III). Smith et al. (1949) were the first to et al. (2010) studied the nutrigenomics and show that aqueous pollen extracts inhibited immuno-competence of caged honeybee workers P. larvae sporulation in a concentration- and found increased glucose oxidase and dependent manner. In a second experiment, they phenoloxidase (PO) activity in bees fed with showed that this effect was linked to fractions polyfloral compared to monofloral pollen. The more soluble in ether than in water, but did not pollen diet also increased vitellogenin and identify specific antibiotic compounds in the ex- spätzle gene expression and the upregulation of tracts (Smith et al. 1949). Crailsheim and TOR pathway and immunity genes, and enhanced Riessberger-Gallé (2001) compared the antibacte- antioxidative enzymes in honeybees compared to rial activity against P. larvae of pollen collected those kept on no-pollen diets (Alaux et al. 2011). directly from flowers, pollen pellets collected Both studies highlight that pollen nutrition affects from honeybee corbiculae and bee bread (water- baseline immuno-competence and the diversity of ethanol extracts). They consistently found floral resources have direct impact on bee health. concentration-dependent growth inhibition. Hence, constraints in nutrient diversity can result Furthermore, the pollen-mediated growth inhibi- in honeybees that cannot establish an efficient tory effect greatly increased the more the bees had immune defence and thus are a higher infection processed the pollen. This was attributed to two risk for the colony. nonexclusive mechanisms: (1) an enhanced activ- DeGrandi-Hoffmann et al. (2010) tested the ity resulting from substances added by the bees by impact of pollen nutrition on deformed wing virus regurgitating liquids from food glands or the hon- (DWV) infection in cage experiments. They fed ey stomach or (2) fermentation of pollen progres- fresh pollen mixed with granulated sugar to hon- sively releasing substances that were already in eybees infected with DWV. Pollen feeding re- the pollen (Crailsheim and Riessberger-Gallé duced titres of DWV compared to controls (no 2001). protein at all). Whether this resulted from a direct Feeding P. larvae -infected colonies with antiviral potential of the pollen (DeGrandi- pollen-water mixes reduced worker larvae mortal- Hoffmann et al. 2010) or indirectly by enhancing ity (Rinderer et al. 1974). More recent cage ex- the bees’ immune system is not known. A recent periments showed a weak upregulation of antimi- colony-based experiment showed that feeding crobial effectors at the protein level in infected honeybee colonies on pollen of diverse botanical bees fed with a diet of polyfloral pollen and 400 S. Erler and R.F.A. Moritz

Table III. Effects of pollen and bee bread on bee parasites, pathogens and predators.

Product Application Parasite/ Observation Technique/ Reference pathogen/ experiment predator

Pollen Pure material DWV Reduced virus Cage DeGrandi-Hoffman mixed with concentration experiment et al. 2010 sugar Pure material ABPV, Reduced virus Bee colony Antúnez et al. 2015 BQCV, concentration DWV, SBV Ethanol extract P. al ve i , Bacteria growth In vitro assay Lavie 1960b P. l arv ae inhibition Aqueous P. l arv ae Concentration- In vitro assay Smith et al. 1949 extract dependent sporulation increase and inhibition Aqueous- P. l arv ae Bacteria growth In vitro assay Crailsheim and ethanol inhibition Riessberger-Gallé extract 2001 Ethanol and A. flavus , Fungi growth In vitro assay Kacániová methanol A. fumigatus , inhibition et al. 2012 extracts A. niger Pure material N. apis Increased spore Cage Beutler and Opfinger mixed with development experiment, 1950; Rinderer and syrup caged Elliot 1977 colonies Pure material N. ceranae Increased spore Bee colony Invernizzi et al. 2011 development, with lower pollen diversity Bee Aqueous- P. l arv ae Bacteria growth In vitro assay Crailsheim and bread ethanol inhibition Riessberger-Gallé extract 2001 Pure material, N. apis Increased spore Cage Beutler and Opfinger and mixed development experiment 1950; Porrini et al. with syrup 2011 Pure material N. ceranae Increased spore Cage Basualdo et al. 2014 development experiment

honeydew honey (Höcherl et al. 2012). Again, cannot be inhibited by feeding raw pollen or either pollen could have facilitated an immune bee bread mixed with syrup (Table III). On system upregulation in P. l a rv ae -infected the contrary, nutritional enrichment by individuals or specific antibacterial compo- proteinfeedingseemstobelinkedwithan nents may be released by the partial digestion expansion of the bees’ midgut, facilitating an of pollen (Rinderer et al. 1974). Polyfloral increase in Nosema spore production pollen further increases survival rates for (Beutler and Opfinger 1950;Rindererand stone brood-infected bees, as shown with Elliot 1977). However, colonies fed with in vivo experiments using bee larvae, in pollen from diverse botanical origins had comparison to monofloral pollen (Foley lower levels of N. ceranae spores com- et al. 2012). pared to colonies fed only with mono- With the exception of a single study, spore floral pollen (Invernizzi et al. 2011). Again, development of Nosema apis and N. ceranae this result remains inconclusive regarding a Bee products for self-medication 401 differentiation between potential malnutrition Consequently, Varroa parasitism prevented bees and true self-medication. from accessing the beneficial effects of pollen Increased survival rates and therefore higher (Alaux et al. 2011). The colony’s nutritional status longevity may be a general effect of (polyfloral- affected not only the transcriptomic changes but ) pollen feeding in micro-parasite infection also behavioural adaptations in individual bees. studies. Both N. apis and N. ceranae infec- For example, Varroa jacobsoni -infested colonies tion cage experiments showed comparable increased the removal of infested brood when they results on longevi-ty for pollen feeding per had higher pollen stores (Janmaat and Winston se and in particular for polyfloral pollen 2000). (Table III). Mostly, polyfloral pollen achieved Honeybee larvae are typically fed pollen in the better results in comparison with monofloral form of bee bread rather than of corbicular pollen pollen. (Brodschneider and Crailsheim 2010). Up to now, Protein and amino acid quality and quantity bee bread-induced beneficial bee health effects are the main criteria regarding the health- were only studied on Nosema -infected caged promoting activity of pollen. For example, a honeybees (Beutler and Opfinger 1950; recent study showed that polyfloral pollen is Basualdo et al. 2014; Porrini et al. 2011). not necessarily better than a monofloral pollen Feeding N. apis -andN. ceranae -infected worker diet of high protein content (Di Pasquale et al. bees bee bread mixed with sugar syrup or as raw 2013). The nurse bees’ physiology is expected material leads to increased longevity as already to be affected by a qualitatively and quantita- known for pollen. On the physiological level, tively enhanced amino acid consumption. As a N. ceranae -infected bees increase their result, they might be able to produce a better haemolymph protein titre if fed with bee bread quality food jelly that in turn might counteract which may help them to tolerate the parasite in- the lifespan-reducing effect of Nosema infec- fection (Basualdo et al. 2014). Regarding pollen tions in other bees. However, experi-ments in as a foraged and stored item with a potential bee colonies and observation hives found exactly importance for colony health, the interpretation the opposite effects comparing both rearing of the empirical data remains ambiguous because methods. Supplementary pollen feeding increased of the obvious role of pollen as a food source. bee longevity in observation hives independent of Unless it is possible to separate food quality from infection status, whereas no lifespan effect was direct medicinal effects of specific compounds, it observed for supplementary-fed infected bee will be difficult at best to dissect the effects of colonies (Mattila and Otis 2006). When it comes potential malnutrition and impaired immunity to studying bee longevity, Nosema infection and from those of a lack of pollen-derived antimicro- pollen feeding, results from cage, observation bial compounds. hive and bee colony experiments showed oppos- ing results and need to be evaluated with caution. 3.3. Honey Varroa mite infestation studies using pollen and no-pollen feeding yielded similar gene ex- Honey is primarily composed of glucose and pression patterns (increasing vitellogenin and im- fructose as the main sugar compounds and water munity gene expression, upregulation of proteol- (~20 %). In addition, it also comprises, at a much ysis, peptidase activity and carbohydrates metab- lower level, di- and polysaccharides, minerals, olism) for bees with access to pollen independent amino acids and even some proteins (Crane of the infestation status. Comparing Varroa - 1975;Doner1977). The high sugar concentration infested and not infested bees, mite infestation results in a strong osmotic pressure lethal for any led to much lower gene expression levels and microbe (except for symbiotic lactic acid bacteria, mostly downregulation of target genes (Alaux Olofsson and Vásquez 2008). This is the primary et al. 2011). The pollen feeding could not reverse reason why honeybees can prevent honey from the Varroa -induced negative effects on bee me- fermenting. So why should we bother about any tabolism and immunity (Alaux et al. 2011). other antimicrobial effects of honey if the high 402 S. Erler and R.F.A. Moritz sugar concentration does it all? Indeed there is Secondary plant metabolites including alka- everyreasontoalsolookatdilutedhoney. loids, phenolic acids (e.g. caffeic, p -coumaric, Although honey is stored with high sugar concen- ellagic and gallic acid) and flavonoids (e.g. trations, it becomes diluted as soon as it is added chrysin, galangin, pinocembrin, quercetin, to the larval food and hence it would be highly kaempferol and tectochrysin) are prime candi- adaptive if it also had antimicrobial effects at dates for causing the observed interaction spec- lower concentrations where osmotic pressure ificity (Erler et al. 2014; Cushnie and Lamb alone is insufficient as an antimicrobial factor. 2005). In vitro assays using pure honey Various antimicrobial compounds and chemical (Table IV) and distillate fractions (Obaseiki- properties other than the high sugar concentration Ebor et al. 1983) showed that these com- have been identified (Molan 1992a, b) including pounds also have antifungal activity against hydrogen peroxide produced by glucose oxidase, the stone brood pathogens Aspergillus flavus , low pH (acidity), methylglyoxal, the antimicrobial and Aspergillus niger. peptide bee defensin-1, major royal jelly protein 1 Several studies focusing on Nosema spp. and various phenolic compounds which are im- (Table IV) addressed the antibiotic effect of honey portant for the antimicrobial potential of honey by counting dormant spores (Gherman et al. 2014; (Brudzynski and Sjaarda 2015; Dustmann 1979; Gregorc 1993; Pohorecka and Skubida 2004). Kwakman and Zaat 2012). In addition, many Both, cage and colony experiments showed that highly plant-specific secondary metabolites have such spores are still infectious after storage in been identified in honey. honey (but see White 1919). The honeys’ antifun- Most studies used natural (unprocessed) honey gal activity, as direct measurement of spore load that makes it often difficult to differentiate be- reduction, lead to the assumption that honey may tween the effects of osmosis and antibiotic com- inhibit at some point the life cycle of N. apis and pounds (Table IV). Two studies used solvent ex- N. ceranae (Table IV). Nonetheless, honey con- tract fractions (methanol-water and acetone) to sumption does not always improve bee health study the antibacterial activity of honey on hon- even when impairing parasite development. For eybee pathogens. This seems like an odd approach example, honeydew honey fed to wintering hon- as clearly in the colony, compounds will only eybees did inhibit N. apis development; neverthe- operate in a water solvent condition (Table IV). less, it also resulted in increased bee mortality Except for the historical French studies of the (Gregorc 1993; Pohorecka and Skubida 2004). 1950s–1960s testing natural honey against The nonfloral origin of honeydew honey may P. al vei and P. larvae (Gonnet and Lavie 1960; have lacked the health-enhancing substances Lavie 1960b; Verge 1951), mainly the European present in floral nectar. foulbrood-associated bacterial strain E. faecalis The pharmacophagic effect of stored hon- has been tested (Table IV). More recent studies ey was tested on N. apis and N. ceranae addressed how the interactions among the various infection experiments. Malone et al. (2001) antimicrobial substances affect the antibacterial compared the effect of two different honeys activity of honey, against American and (manuka and thyme honey) and found European foulbrood bacteria. There were pro- that this decreased the individual bees’ nounced honey type-dependent and honey N. apis spore load. However, the results pathogen-specific interaction effects (Bobiş et al. were less conclusive since sugar syrup-fed 2011, 2013; Erler et al. 2014). For instance, black bees had the highest longevity (Malone et al. locust honey inhibited the growth of European 2001). foulbrood-specific and European foulbrood- Also, Gherman et al. (2014) showed honey associated bacteria much more effectively than type-specific spore load reduction for sunflower honey, whereas sunflower honey N. ceranae . They suggested multiple nonex- inhibited the growth of P. larvae strains more clusive mechanisms that might be important: strongly than black locust honey (Erler et al. The active compounds may (1) kill Nosema 2014). spores, (2) increase the activity of the Bee products for self-medication 403

Table IV. Effects of honey on bee parasites, pathogens and predators.

Application Parasite/pathogen/ Observation Technique/ Reference predator experiment

Pure material B. pumilus , Bacteria In vitro Bobiş et al. 2011, 2013; Erler et al. B. laterosporus , growth assay 2014; Gonnet and Lavie 1960; M. plutonius , inhibition Lavie 1960b P. al ve i , P. l arv ae Pure material E. faecalis Bacteria In vitro Chan-Rodríguez et al. 2012;daCruz growth assay et al. 2014;Efemetal.1992;Erler inhibition et al. 2014; Gallardo-Chacón et al. 2008; Islaa et al. 2011;Temaruetal. 2007; Tenore et al. 2012; Ulusoy et al. 2010 Methanol-water E. faecalis Bacteria In vitro Escuredo et al. 2012 extract growth assay inhibition Acetone extract P. l arv ae Bacteria In vitro Lavie 1960b growth assay delay Pure material A. flavus , A. niger Fungi In vitro Boukraâ et al. 2008; Chanchao growth assay 2009a;Efemetal.1992; Fahim inhibition et al. 2014; Gulfraz et al. 2010; Radwan et al. 1984;Tenoreetal. 2012; Wellford et al. 1978 Distillate fractions A. niger Fungi In vitro Obaseiki-Ebor et al. 1983 growth assay inhibition Pure material N. apis Reduced Cage Malone et al. 2001 infectivity experiment Pure material N. apis Spore load Bee colony Gregorc 1993; Pohorecka and reduction Skubida 2004 Pure material N. ceranae Spore load Cage Gherman et al. 2014 reduction experiment

honeybee immune system to fight against result, genes related to detoxification processes microsporidian infections, or (3) inhibit the and innate immunity (antimicrobial peptide replication of vegetative forms of Nosema genes) were upregulated in groups of bees fed spp. Whatever the actual mechanisms, those with these extracts. A single substance seemed specific honey types that resulted in a reduc- to modulate the measured transcriptional changes. tion of a N. ceranae spore load had also The phenolic acid p -coumaric acid, the monomer been selected in a choice assay by workers of sporopollenin (the major plant spore and pollen infected with N. ceranae but not by healthy outer wall compound), was isolated from these bees, suggesting a self-medication potential extracts and shown to actively detoxify pesticides of honey at the level of the individual bee and to activate innate immune system gene ex- (Gherman et al. 2014). pression (Mao et al. 2011, 2013). p -Coumaric Nutrigenomic studies used methanol and acid can be detected in honey, pollen, beebread ethylacetate extracts of honey to measure tran- and propolis, but not in nectar. The combination scriptional changes in healthy bees (Johnson of its nutraceutical and antibiotic activity raises et al. 2012; Mao et al. 2011, 2013). As a common the possibility that p -coumaric acid together with 404 S. Erler and R.F.A. Moritz other honey compounds (nectar-derived flavo- the supposed pharmacophagic potential of food noids: pinobanksin, pinobanksin 5-methyl ether, jelly. We can only speculate on the pharmacolog- pinocembrin) may well interact with additive or ical potential of beeswax and bee venom as their synergistic consequences for innate immunity and antibiotic activity for honeybee pathogens re- detoxification (Mao et al. 2013). mains largely unknown. The antibiotic origin of A second group of plant-derived products en- gland secretions is even more complex than ven- hancing bee health are phytohormones. Abscisic om proteins and peptides. Solitary bees use vola- acid, a phytohormone regulating physiological tile acyclic terpenoids, lipids and fatty acid deriv- functions in plants, can be detected in nectar, atives, released from the mandibular gland, for honey and honeybees at all stages. This phytohor- nest cell sanitation and as predator repellent mone enhances the immune response (cellular and (Cane et al. 1983;Cane1986). These fungistatic humoral) and wound healing of individual worker and bacteriostatic substances are applied against honeybees and the colony (Negri et al. 2015). Aspergillus niger and other microorganisms, and Phytohormones are a new group of candidate may also be distributed during self-grooming. The substances that may be used by honeybees for mixture of proteinous substances, lipids and acids various medicinal purposes. Currently, we are has potentially additive antibiotic effects or acts only at the beginning of understanding the poten- selectively on species-specific diseases. tial function of these phytohormones for honey- In stark contrast, the results of hundreds of bees and more studies are needed to evaluate their studies deal with the pharmacological potential antibiotic potential. of foraged hive products. The in-hive pharmacy provides three major types of natural medicine 4. CONCLUSION (honey, pollen/bee bread and propolis) for self- medication usage. Propolis, not consumable by Gland-produced and foraged products have a bees, can only be seen as indirect hive medicine, highly diverse specificity and efficacy on bee but nonetheless harbours a very high parasites and pathogens. Scientists have tried over pharmacophoric activity. Plant resins are widely decades to unravel the molecular mechanisms used in bee societies as honeybees and other bees behind the antibiotic effects of these products share a common spectrum of diseases and preda- against viruses, bacteria and fungi. However, with tors. Stingless bees’ resinous products have increasing availability of high-resolution analyti- known antifungal effects (Aspergillus niger ) cal tools, the puzzle gets more and more compli- (Garedew et al. 2003b;Mulietal.2008) and are cated rather than disentangled. Hundreds to thou- highly effective when used to mummify sands of substances can be identified with ever- A. tumida beetles by deposing resin on the body decreasing detection limits. How do these com- of the intruder (Greco et al. 2010; Halcroft et al. pounds interact? Which of these compounds are 2011). Comparative studies (single type resin vs. the ones used by the honeybees when choosing mixtures) revealed that single resins may have between two types of honey? These remain diffi- different effects, and mixtures are more effective cult questions to answer in spite of modern ana- indicating functional complementarity for repel- lytical instrumentation. lent effects against predatory ants and A. tumida Pharmacophagy and pharmacophory are wide- (Drescher et al. 2014). Resins of different plant ly known for self-produced gland products. In species not only target different organisms; they particular, the antimicrobial substances of royal also act synergistically (Drescher et al. 2014). jelly (10-hydroxy-2-decenoic acid, major royal Stingless bees combine the repellent effect against jelly protein 2 and defensin-1) help the bees in ants and the advantage of the sticky barrier against fighting against brood diseases. Self-medication predatory insects (Duangphakdee et al. 2009; per se has not been proven for them as yet. Studies Schwarz 1948, reviewed in Roubik 2006). analysing seasonal and between-hive antibiotic During an ant attack, they increase their resin variance in combination with disease-associated intake to build barriers of resin droplets, effective- feeding behaviour are needed to fully substantiate ly entangling ants (Leonhardt and Blüthgen Bee products for self-medication 405

2009), and emit ‘glue’,presumablyresin,onthe against a gut disease (Crithidia bombi )ofbum- invading ants (Lehmberg et al. 2008). These ex- blebees (Baracchi et al. 2015; Manson et al. 2010; amples show that honeybees and stingless bees Richardson et al. 2015). Studies on secondary use resinous hive products in a very broad but plant metabolites in nectar should be extended to similar pharmacophoric way. all groups of bee diseases to better understand the Plant secondary metabolites, including the pharmacophagic potential of highly diverse forag- groups of flavones/flavonols and flavanones/ ing sites shared by honey and bumblebees dihydroflavonols among others, are the prime collecting nectar and pollen. candidates for the overall observed very high bac- Whereas a general intact nutrition will be es- tericide and fungicide activities. Most of the stud- sential to operate the honeybee’s innate immune ies describing the antibiotic activity of resin and and detoxification system, the specific glandular propolis use organic solvent extracts; bees never and foraged compounds may be of particular im- will use or have access to these, as they only portance to prevent and fight specific infections. collect water. Consequently, the results obtained Future studies should not just single out specific by studying propolis extracts, but also extracts of substances for their antibiotic activity and effects other bee and hive products, have to be viewed on the bees’ health. If we want to comprehensive- with caution as providing causal explanations for ly understand self-medication in the honeybee, we their activity. must understand how the individual worker bee Pollen and honey were shown to have the with its excellent recognition senses combines its highest variability in their pharmacophagic activ- behaviour with the potentially available com- ity for several Apis (Tables III and IV) and non- pounds from its rich glandular system and those Apis bee species (Boorn et al. 2010; Chanchao available in the hive and its environment to max- 2009b; Chan-Rodríguez et al. 2012;daCruzetal. imize its own health and that of the colony. 2014; Kimoto-Nira and Amano 2008; Logan et al. 2005;Temaruetal.2007; Vandenberg 1994). An ACKNOWLEDGMENTS outstanding nonpharmacophagic but defensive usage of stored honey was observed for the sting- We would like to thank Alexis Beaurepaire for the less bee Hypotrigona braunsi . Field observations help with translations of the French literature and Robin described invasion of Hypotrigona colonies by M. Crewe and two reviewers for comments on the the honey robber Lestrimellita cubiceps .Asa manuscript. Financial support was granted by the Fed- defensive strategy, worker bees deposit honey in eral Ministry of Food, Agriculture and Consumer Pro- the colony entrance (Michener 1959; de Portugal- tection (Germany): FIT-BEE project (grant no. 511- Araújo 1958). This blockage prevented honey 06.01-28-1-71.007-10 to R.F.A.M.) and the DFG (pri- ority programme SPP 1399 to R.F.A.M., and ER 786/1- robbing and shows that pharmacophory not only 1toS.E.). exists for propolis but also for honey. At least for honey, many factors are known to be related with its antibiotic activity (hydrogen peroxide, low pH, methylglyoxal, etc.). Again, it Pharmacophagie et pharmacophore : mécanismes d 'auto -médication et de prévention des maladies dans is the secondary plant metabolites that are gaining la colonie d 'abeilles (Apis mellifera ) more and more importance in studying pharmacophagy and self-medication for individu- miel / propolis / pollen / pain d 'abeille / gelée royale / al bees and the brood. Alkaloids, phenolic acids activité antimicrobienne / interactions hôte -parasite (e.g. caffeic, p -coumaric, ellagic and gallic acid) and flavonoids (e.g. chrysin, galangin, Pharmakophagie und Pharmakophore : Mechanismen pinocembrin, quercetin, kaempferol and der Selbstmedikation und Krankheitsvorbeugung in Bienenvölkern (Apis mellifera ) tectochrysin) are linked with decreased parasite and pathogen loads in infected individuals. Honig / Propolis / Pollen / Bienenbrot / Recent studies report the antimicrobial potential Königinnenfuttersaft / antimikrobielle Aktivität / of alkaloids, terpenoids and iridoid glycosides Selbstmedikation / Parasit -Wirt -Interaktion 406 S. Erler and R.F.A. Moritz

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