Washboarding: Are Man-Managed Honeybees Performing a Vestigial Activity Fostered by Cryptogams?

Cavity-nesting in a tree or rock crevices is how the honeybee Apis mellifera naturally sets up a colony. This is well documented, even from the rock paintings of prehistoric peoples (Crane, 2001). Oleksa et al. (2013) first assessed free-living honeybees in tree cavities in Europe and they recorded the frequency of feral colonies of honeybee inhabiting rural avenues in Poland. Density of feral colonies was lower than that of managed populations. Certainly, most colonies are man-managed, even if free-living ones are commonly reported; however, wild colonies (which never inhabited a beehive) or feral ones (possibly swarmed from a nearby apiary and settled free) are poorly investigated.

Following Oleksa et al. (2013), other authors focused on wild/feral colonies, carrying out systematic studies on free-living colonies. Kohl & Rutschmann (2018) surveyed a beech forest in Germany estimating an average colony density of 0.13 colonies per square kilometre. Requier & Leonhardt (2020, and citations herein) mentioned trees as primary nesting sites of wild colonies, and Kohl et al. (2022) found that colonies were not confined to the forest edges (as commonly expected) but were also recorded deep inside the forests. Other monitoring also found colonies in anthropogenic environments. Browne et al. (2020) detected the largest number of free-living colonies (almost 70%) settled in cavities in buildings, and Rutschmann et al. (2022) found colonies adopting electric power poles as nesting sites. Monitoring free-living colonies is often performed thanks to the collaboration of beekeepers or citizens (Browne et al., 2020; Bila Dubaić et al., 2021).

Life-history traits connected to free-living colonies may help to disentangle washboarding, an activity frequently recorded and reported by beekeepers. While performing this behaviour, individual bees can be observed with their heads down, scraping the substrate with their mandibles while rocking back and forth in an almost synchronous way. Inferences among scientist and beekeepers date back to the end of 19^th century. Alfonsus (1932) nicely summed up all available hypotheses (Tab. 1), describing the behaviour in great details and as a mechanical cleaning. The behaviour is frequently observed on the front of the hive, seldomly also on the inner walls. Seeley & Morse (1976) inferred that areas of smoothed bark, up to 30 cm around the entrance holes of natural nests in hollow trees, could be the result of washboarding to facilitate nest defence and traffic flow. Walton & Toth (2016) employed washboarding as a behaviour to estimate individual worker variability, finding a paired correlation with another behaviour, the application and manipulation of wax. The authors also confirmed washboarding as possibly related to nest maintenance tasks but underlined the purpose still to be a mystery. Despite the diversity of reasons hypothesized for this behaviour, washboarding still lacks solid evidence for any particular explanation. This could be linked to the fact that so far washboarding has mostly been observed on box hives, structures made by man to facilitate bee manipulation, favouring hypotheses linked with artificial conditions. The sole reports on washboarding in natural conditions were by Taulman (2017), who directly observed and videorecorded washboarding at two different natural forest hives. He hypothesised that the behaviour could be related to the application of tarsal pheromone, an oily substance which may be rubbed on the surface.

After surveying all hypotheses, we detected that no author mentioned washboarding as a vestigial behaviour, originally put in place in such natural surfaces as tree barks and rocky surfaces. Bees may act in response to potentially self-restoring cues, which elicit the inconsistently observed response. This hypothesis does not fit with the mechanical polishing or with the application of pheromones. If the aim is smoothing the texture, once polished the work is done, no need to repeat it; if the aim is the application of a scent, that should be constantly resumed. Among the possible cues for a preliminary investigation, we decided to address cryptogams, and lichens in particular. Lichens are found on any surface, including bark, rock crevices and man-made surfaces of almost all textures. Moreover, lichens release undetachable spores, propagules and Volatile Organic Compounds (VOCs), still poorly studied, which may be the cue to which honeybees respond.

Lichens produce a great variety of secondary metabolites, mostly depsides and depsidones. Numerous secondary metabolites growth regulators and lichen acids are produced by the symbiotic microorganisms (Pankratov et al., 2017). Lichen secondary metabolites represent from 0.1–5.0% up to 25% of thallus dry weight (Fahselt, 1994; Romagni et al., 2004) and are mainly deposited on the surface of the hyphae. Out of over one-thousand secondary metabolites known to occur in lichens, only approximately 7% have also been found in nonlichenized fungi or in higher plants (Hauck et al., 2009; Beckett & Minibayeva, 2013). Secondary compounds have various biological activities with antibiotic, antimycobacterial, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative and cytotoxic effects (e.g. Triggiani et al., 2009; Bačkorová et al., 2012; Munzi et al., 2014; Goga et al., 2017). While many animals feed on lichens, including primates (Wang et al., 2019), lepidoptera (Hesbacher et al., 1995; Karunaratne et al., 2008), orbit mites (Syed & Seaward, 1984) and terrestrial gastropods (Baur et al., 1992), bees have never been mentioned in relation to lichens, but the interaction could either be positively (employing some compounds in bee products) or negatively (removing lichens as a response to mechanical disturbance or toxic volatiles) correlated.

We ran an exploratory study to 1) investigate the frequency and distribution of the behaviour through data mining, internet search and a tailored-designed questionnaire circulated among researchers and beekeepers; 2) verify the presence of lichens on hives; 3) test the responses to transplants of bark strips covered with lichens placed on the hives. Because the test was a simplified preliminary approach to verify if any kind of reaction could be detected, some weaknesses should be expected. For example, we decided to allow beekeepers themselves to assess behavioural changes in their hives, and we did not run a simultaneous control, not being able to constantly register all hives at once. However, in the present study a possible bees-lichens relationship is investigated for the first time and a citizen science approach is combined with experimental manipulations to validate the interest of stakeholders and the feasibility of further analyses.

The main aim of this study was to aggregate existing knowledge on washboarding. Descriptions originate from various sources, but this behaviour was rarely investigated scientifically. We found it first mentioned in 1860, and later washboarding was described and addressed only occasionally up to 2017. No experiments were performed, apart from those mentioned in the abstract of a conference. Washboarding, however, stimulate beekeepers’ curiosity and hypotheses on reasons behind it. Many reports were found among beekeepers’ blogs, associations’ newsletters and videos uploaded to the YouTube platform. In agreement with our analyses (Fig. 3; Tab. S1 and S2), and with the responses to our questionnaire, the interest of this important group of stakeholders emerged and should be undertaken by the scientific community. As a preliminary step and hoping to promote interest, we performed an experimental manipulation of hives with the help of volunteer beekeepers. Our working hypothesis was that this behaviour, now performed on the clean surfaces of man-made hives, could be a vestigial trait linked to the presence of cryptogams on surfaces of natural nesting sites (tree trunks or rock crevices).

The absence of lichen colonization on the hives is not surprising, since hives are periodically subjected to maintenance and lichens are slow-growing organisms requiring years to reach relevant sizes. Washboarding has been observed most frequently outside but in some case on the inner surfaces of the hive. Although light conditions are not suitable for lichen colonization in hive interiors, we cannot exclude that lichen propagules (like spores and soredia) reach the inside walls. Most observations occurring on the outside surfaces match with the possibility of an organism, eliciting the behaviour, that needs light to grow. Moreover, we could also expect VOCs to penetrate the hives and elicit the behaviour: both lichens and/or microorganisms hosted by lichens could be responsible for their production.

The lichen stripes underwent a physical and chemical processing by honeybees. Foliose lichens were removed from the bark, possibly since they are anchored to the substrate through rhizines or hapteria (root-like structures). Occasional detachment and fragmentation of the thallus could have been caused by bees just walking on their surface. However, the total and precise removal of lichens from the substrate observed on some bark stripes is hardly compatible with accidental removal due to bees’ passage or environmental conditions. When dead, lichens equally remain attached to the substrate for a long time in absence of a direct effect on the thalli, and no extreme meteorological events occurred during the transplant period. Crustose lichens did not undergo the same process, but their tight attachment to the substrate makes their mechanical removal impossible. Our experiment confirmed that washboarding causes lichens’ manipulation in the proximity of beehives. However, what must be solved is whether lichens are removed for being unwanted guests or if they are collected as a nutrition source.

The two hypotheses also match with recent findings of lichens and their relationship with animals, insects included. Several papers recently investigated the relationship between invertebrates and lichens, used as hiding places, shelter or food (Karunaratne et al., 2008 and references therein). Numerous butterflies and moths showed different feeding habits and a certain specificity for lichen species (Pringle et al., 1994; Pöykkö, 2006). In the lichen association, the fungal symbiont is responsible for the production of a large range of metabolites, often unique to lichens. The presence of lichen compounds recorded in lichen-feeding populations of butterflies (Karunaratne et al., 2002, 2008) suggested that invertebrates obtain protection against predators by assimilating lichen secondary compounds and using them as a chemical defence. Possibly, lichen substances may be included in the propolis, similar to many different substances assimilated from plants (Burdock, 1998; Anjum et al., 2019). Otherwise, such lichen compounds as usnic acid, vulpinic acid, and stictic acid, and lichen extracts were studied for toxicity and antifeedant activity against larvae of the polyphagous insect herbivore Spodoptera littoralis (Emmerich et al., 1993), adults of wheat weevil, Sitophilus granarius (Emsen et al., 2015) and other insects (Sachin et al., 2018). Toxicity may be the reason for our observations of propolis distributed on top of all lichen stripes which we had placed on hives.

To our knowledge no investigation linking lichens and bees exists. The above speculations are aimed at considering a newly emerging opportunity to investigate the disregarded relationship of bees and lichens and the intriguing washboarding behaviour.