Cosmetic colouring by Bearded Vultures Gypaetus barbatus: still no evidence for an antibacterial function

Feather analyses

We collected feathers of Bearded Vultures found near an ochre bath and at adjacent sites (Margalida & Pelayo, 1998) for analysis using a scanning electronic microscope (SEM). Seven feathers coloured with iron oxides from three different territories were mounted on a stub and coated with carbon to ensure a conductive surface. The SEM morphological and analytical feather images were produced using a Jeol JSM 840, which allowed imaging and a semi-quantitative microanalysis of mineral elements on the feathers. In addition to the morphology, it was possible to observe the density contrast of the different metallic surfaces based on discrimination of the backscattered elements.

Feather-degrading bacteria and iron oxides (hypothesis 2)

The degradation of feathers from selected species containing different natural pigments has been described previously by Grande, Negro & Torres (2004). It has been hypothesized that iron oxides applied externally would protect Bearded Vulture feathers from feather-degrading bacteria (Arlettaz et al., 2002; Tributsch, 2016) in addition to any protection normally afforded by pigments naturally present in feathers. The effects of Bacillus licheniformis applied in vitro were tested on Bearded Vulture covert and body feathers of different original colours covered or not by iron oxides, as explained in Table 1. The methods used for exposing these feathers to feather-degrading Bacilli, and to assess the level of degradation with time, were described in Grande, Negro & Torres (2004), as the vulture feathers were in fact part of the same experimental setting. The data corresponding to the bearded vultures were never published because their samples were collected in the wild as shed feathers, and might have been exposed to feather-degrading agents. The material from the remaining species was obtained from museum specimens. One of us (JJN) participated in the aforementioned study and was one of the co-authors. Given that the results reported in Grande, Negro & Torres (2004) were obtained using the same bacterial strains (Bacillus licheniformis CMP L5, isolated from soil in California, USA), as well as same culture media and culture conditions , we will discuss our results in relation to those previously reported for feathers of other species containing different pigments.

Antimicrobial activity of ochre and iron oxides: Medium and culture conditions (hypothesis 2)

The antimicrobial activities of iron salts, and of ochre, collected from Bearded Vulture bathing sites, were investigated under light and dark conditions using Escherichia coli XL1-Blue MRF‘ (Gram-negative) and Kocuria rhizophila DSM 11926^T(Gram-positive). In addition, the keratinolytic Bacillus licheniformis DSM 13^T was used to assess the effects of ochre on feather-degrading microorganisms. This strain of Bacillus licheniformis was different to the one used for the degradation experiment on feathers described above (Grande, Negro & Torres, 2004). All bacteria were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), except for E. coli which was purchased from Stratagene (San Diego, CA, USA). All bacteria were maintained on Müller-Hinton Agar (MHA) at optimum temperatures, and fresh cultures were used for each test.

Suspension tests (hypothesis 2)

Suspension tests were conducted to evaluate the antimicrobial potential of ochre and iron oxides when exposed to visible light (16 W, 880 lumen, 140 mA, 2,700 K) compared with dark conditions. The ochre sample was sterilized using dry heat (180 °C for 2 h) and adjusted to 10 mg/mL (w/v) in Müller-Hinton Broth (MHB). Iron(II) oxide and iron(III) oxide (Sigma Aldrich, Steinheim, Germany) were combined at a concentration of 5 mg/mL each in MHB and tested for antimicrobial effects in parallel with the ochre sample.

For the suspension tests, the cell numbers of the indicator strains were adjusted to comparable values using a densitometer (Biosan DEN-1, Biosan Laboratories, Warren, MI, USA). The bacteria were added to the samples to create a final cell density of approximately 5 ×10⁵ cfu/mL. The samples were incubated in liquid media under agitation at 150 rpm at 37 °C. A desk lamp (LM-106B, BAHAG AG, Mannheim, Germany) equipped with a 16 W bulb (Müller-Licht GmbH, Lilienthal, Germany) was used to provide visible light, while the samples to be incubated in the dark were wrapped in aluminium foil during incubation.

Antimicrobial effects were determined by means of viable cell counts as follows: 10 µL aliquots of the samples were taken at the beginning of the incubation and after 18 h. The diluted bacterial suspensions were transferred to MHA and incubated overnight. A 2.5 mg/mL solution of MTT (Sigma Aldrich, Steinheim, Germany) was used to facilitate the visualization of the bacterial colonies which were subsequently counted. Cells counts were plotted on a logarithmic scale using GraphPad Prism 5.01 (GraphPad Software Inc., La Jolla, CA, USA).

All dilutions were done in duplicate per experiment and all tests were conducted independently three times. Ampicillin (32 µg/mL, AppliChem, Darmstadt, Germany) served as a positive control, while MHB without the addition of samples served as negative controls. Non-inoculated samples were used as sterility controls.

Effects of iron oxides on breeding success (hypothesis 3)

To assess whether iron oxides have any influence on the reproductive success of Bearded Vultures (Arlettaz et al., 2002), we randomly selected five breeding pairs with very pale feather colours and six pairs with very intense colours, and compared their breeding success (the total number of chicks fledged divided by the number of pairs with hatched clutches), obtaining data from 122 breeding attempts (from 1990–2006). Using telescopes, we categorised the observed neck and ventral plumage colour of individual breeding Bearded Vultures as either pale or orange. The observations were made by A.M. during a long-term breeding season monitoring programme. Pairs with paler colourations came mainly from territories located in the outer mountain ranges (average: 1,170 m, range: 900–1,450 m asl), whereas the most intensely coloured ones inhabited the axial zone of the Pyrenees (average: 1,533 m, range: 1,150–2,100 m asl). Although females are more intensely coloured than males (Negro et al., 1999) in all the territories selected, both sexes presented similar colouration to be assigned as paler or intensely coloured. The absence, or limited presence, of sources of ferruginous material in the Outer Sierras (in contrast to their much greater abundance in the axial zone, A Margalida, pers. obs., 2005) results in the paler appearance of breeding individuals there as a result of the scarcity of cosmetic soils (ochre).

A priori, food availability and human disturbance, two factors known to influence breeding success (Donázar, Hiraldo & Bustamante, 1993; Margalida et al., 2003; Arroyo & Razin, 2006) could be confounding factors affecting the pairs under investigation. However, recent studies quantifying the food available suggest that, in the study area, the amount of available natural food is 10-times greater than the energetic requirements needed for the breeding population (Margalida, Pérez-García & Moreno-Opo, 2017). Thus, food availability could be discarded as a factor potentially affecting breeding success. With respect to human disturbance, this factor should be more present in the Outer Sierras, affecting more intensely to territories occupied by paler individuals.

Breeding experience and the mating system (trio or pair) could also influence breeding success (Margalida et al., 2003; Carrete et al., 2006; Bertran, Margalida & Arroyo, 2009). To avoid biases related to the age of a breeding individual or its mating system (birds breeding as trios have lower breeding success than pairs; (Carrete et al., 2006), the breeding pairs selected had attempted breeding at least seven times previously, and only territories occupied by breeding pairs which had completed the incubation period were included (52–56 days; Margalida et al., 2004). To avoid pseudoreplication, we estimated the average breeding success individually in each territory and then calculated the group mean of the two categories of territory studied.

Chemical analysis of ochre

Chemical analysis was performed on red-coloured soil (ochre) from a water bath in the Pyrenean Mountains (Fig. 2) known to be frequented by Bearded Vultures (Table 2). More than 40% of the dry matter was from iron oxides, explaining the reddish colour of the ochre soil and bath water. Other minerals were of minor occurrence.

Feather analyses: chemical composition

The results of the X-ray microanalyses performed on red-coloured Bearded Vulture feathers revealed the chemical compositions shown in Table 3. Iron was present in the feathers as goethite FeOOH-a, lepidocrocite FeOOH-g and ferrihydrite Fe(OH)₃. Although sulphur (S) and iron (Fe) are the dominant elements, silicon (Si) and calcium (Ca) are also clearly present. The microphotograph (Fig. 3) indicates a thin homogeneous coating throughout the distal part of the down, due to normally hydrated sulfates that balance their charges with metal cations. The X-ray microanalyses at the different points of the spine, middle and distal part of the down show that while Fe, Si and S are present in minor amounts, Ca, copper (Cu), nickel (Ni), titanium (Ti), aluminium (Al) and sodium (Na) occur as the dominant elements.

The morphology of the feather from territory 14 shows that the coating is practically identical to that of the feather from territory 13. The feather from territory 16 had higher quantities of Fe and S, and lower quantities of Si, Ca and Cu.

Effects of iron oxides on feather-degrading bacteria (hypothesis 2)

Feather degradation caused by Bacillus licheniformis in vitro was monitored daily, and no variation was detected among Bearded Vulture feathers of different basal colouration, with or without a cover of iron oxides. The feathers were fully degraded by day 7 after the inoculation of the bacilli (Table 1), as were those of eight other bird species of different colouration (including unpigmented white feathers) similarly tested by Grande, Negro & Torres (2004). Feathers from two species (black feathers from white storks, Ciconia ciconia, and ravens, Corvus corax) were fully degraded one day earlier (day 6), while those that resisted bacterial damage longest (day 8) were from the blue-crowned parakeet, T hectocercus acuticaudata, which contained a psittacofulvin. Therefore, the iron oxides naturally coating Bearded Vulture feathers did not significantly retard feather degradation by the particular strain of Bacillus licheniformis used by Grande, Negro & Torres (2004), at least in vitro.

General antimicrobial effects of ochre (hypothesis 2)

We investigated the potential antibacterial activities of ochre and iron oxides, both after incubation in the dark and in the light, against Bacillus licheniformis (a gram-positive bacterium), Kocuria rhizophila (a gram-positive bacterium from soil) and Escherichia coli (a common gram-negative gut bacterium of vertebrates). The growth of all three species of bacteria was unaffected either by ochre (10 mg/ mL, Fig. 4) or by iron oxides (10 mg/mL). Light conditions did not affect antibacterial activity.

Influence of red colouration on breeding success (hypothesis 3)

The five territories held by pale individuals (n = 64 breeding attempts) had a mean breeding success of 0.88 chicks/pair (range 0.7–1), excluding clutches where no eggs hatched, whereas the six territories inhabited by orange individuals (n = 58 breeding attempts) produced a mean of 0.76 chicks/pair (range 0.56–0.91, n = 6), excluding clutches where no eggs hatched (Mann–Whitney U-test, z = 1.67, P = 0.095, Fig. 5). Therefore, there were no significant differences in breeding success between the two colour categories (pale vs. orange).

Chemical analysis of ochre and feathers

The orange colouration of the plumage is mostly mediated by iron oxide which is accompanied by Cu, Ni and Ti. Unlike the red soils studied in the Alps (Houston, Hall & Frey, 1993), the ochre used by Bearded Vultures in the Pyrenees is not a product of altered dolomitic limestones but of metamorphic rocks. The ferruginous waters of our study come from rocks from the Palaeozoic era. In Pyrenean ferruginous waters, recent cores of goethite coexist with other ancient minerals that have already changed to a more stable form, such as ferrihydrite. Ferruginous waters are not ubiquitous in the landscape, so that that potential Bearded Vulture bathing sites are geographically restricted (see Houston, Hall & Frey, 1993; Negro et al., 1999), and probably only some individual vultures have ready access to cosmetic soils.

Re-evaluating the antibacterial hypothesis (hypothesis 2)

Recently, Tributsch (2016) proposed that the habit of Bearded Vultures bathing in iron-oxides might have a sanitary function (hypothesis 2). This author suggested that under visible light iron oxides kill viruses and bacteria, and convert smelly organic substances into neutral, volatile carbon dioxide gas. Ochre would therefore act as a sterilizing agent in sunlight, and provide a strategy against feather wear (Tributsch, 2016). It is noteworthy that cave-dwelling hominins occupying the same distributional ranges, and possibly the same rocky outcrops, as Bearded Vultures, both in Africa (Homo sapiens) and Europe (H. neanderthalensis and H. sapiens), applied ochre to themselves for at least 140,000 years (Wolf 2018). Anthropologists favour the idea that ochre application in humans was related to ritual activities, but a photoprotective action has recently been demonstrated (Rifkin et al., 2015) and other functions, such as a mosquito repellent (Rifkin, 2015) or use as a hide tanning agent have also been proposed (reviewed in Wolf et al., 2018).

The in vitro exposure of bearded vulture feathers to feather-degrading bacteria showed no effect of iron oxides. Degradation was not retarded in iron-coated feathers, and all tested feathers, irrespective of their basal color prior to staining degraded at exactly the same rate as the feathers of other birds. When iron oxides were applied directly to bacterial cultures, there was no perceptible inhibition of bacterial growth. Taken together, these two lines of evidence weaken the antibacterial hypothesis for ochre use in bearded vultures.

Could iron oxides improve breeding success? (hypothesis 3)

The functional hypotheses in humans are in line with Arlettaz et al. (2002), who hypothesized that Bearded Vultures probably use iron oxides because they have pro-oxidative effects. These authors suggested that iron oxides reduce the negative effects of bacteria on egg and nestling mortality. Some experiments suggested that melanised feathers are more resistant to feather degrading bacteria than unmelanised feathers (Goldstein et al., 2004; Gunderson et al., 2008; but see Grande, Negro & Torres, 2004). However, our experiments were unable to show any antibacterial properties of ochre and iron oxides (hypothesis 2). The in vitro experiment with feather degrading bacteria demonstrated that iron oxides naturally coating Bearded Vulture feathers did not retard feather degradation by at least one strain of Bacillus licheniformis. In addition, the potential general properties of ochre and iron oxides against three bacterium species, showed no influence of ochre (10 mg/ mL) or iron oxides (10 mg/mL) on bacterial growth, after either incubation in the dark or in the light. In particular, light did not enhance any potential antibacterial activity (hypothesis 2). Natural sunlight could not be used experimentally, but we believe that the artificial light used in our experiments was a realistic substitute.

The pro-oxidation hypothesis should directly relate to the impact of egg and chick mortality caused by bacteria, and the natural absence of iron oxides should be expected to result in reduced hatching and fledging success (Arlettaz et al., 2002) (hypothesis 3). According to hypothesis 3, breeding success under similar conditions (i.e., human disturbance and food availability) should be higher in orange vulture territories. The majority (51%) of bearded vulture breeding failures take place during the hatching period (Margalida et al., 2003). Thus, the pro-oxidative effects of iron oxides should increase hatchability by reducing the negative effects of bacteria on eggs and improving nestling survival. However, our results show instead that breeding success did not differ significantly between territories occupied by orange individuals vs. pale ones, with, if anything a reverse tendency, with slightly higher average breeding success in the territories with pale individuals (with a lower frequency of visits to ferruginous water sources or a lower tendency to bathe in them) compared with orange ones. In fact, better habitat quality with respect feeding resources and lower human disturbance is present in the axial zone (Margalida, Colomer & Sanuy, 2011) with higher frequency or intensely coloured individuals with respect Outer Sierras. Thus, taking into account hypothesis 3 it should be expected that the intensely coloured individuals (with more access to ferruginous sources) inhabiting the axial area could transfer oxide particles to the eggs during the incubation process improving breeding success. Our findings do not support this hypothesis suggesting that probably breeding success can be regulated by other factors (Margalida et al., 2003; Margalida, Colomer & Oro, 2014). Therefore, the antibacterial activity hypothesis, which implies indirect effects of iron oxide on breeding success, was not supported.

The function of orange colouration in a behavioural context (hypothesis 1)

As already mentioned, Bearded Vultures might use the orange colouration in a behavioural context (hypothesis 1; Negro et al., 1999). There are descriptions of birds exhibiting secretive behaviour when visiting water baths—bathing captive birds are extremely wary and stop bathing if they are disturbed (Houston, Hall & Frey, 1993; Frey & Roth-Callies, 1994). Anecdotal observations of wild individuals also confirm this secretive behaviour. This may be due to its behavioural function as a conspecific status signal because ochre baths are a scarce resource that would be defended against conspecifics and not revealed to them (Negro et al., 1999; Duchateau & Tellechea, 2019). The signal function of plumage colour may operate by communicating the ability of an individual to obtain limited resources (Hill, 2002; Searcy & Nowicki, 2005). In the case of Bearded Vultures, the costs involved in acquiring a bright colouration (i.e., the significant investment involved in finding the rare cosmetic soils), would allow the more orange adults to express their superiority over conspecifics. This visual signal would also indicate a good knowledge of their own territory (Negro et al., 1999). The status-signalling hypothesis (Rohwer, 1975; Searcy & Nowicki, 2005) posits that dominants benefit from their ‘badges of status’ because they reduce the number of aggressive contests in which they are involved, and subordinates benefit by avoiding interactions with superior individuals (Rohwer & Rohwer, 1978; Senar & Camerino, 1998). Accordingly, more intensively coloured individuals may be those of higher quality. However, our results do not support that more intensely coloured individuals are better breeders. This is because other confounding factors can operate on the results of reproduction (i.e., breeding experience, human disturbance, weather), being independent the higher quality status from the breeding performance. In the case of Egyptian Vultures, Van Overveld, de la Riva & Donázar (2017) suggested that the primary function of feather painting may serve pair formation and bonding and/or that it is used to show off during sexual conflicts. In fact, in bearded vultures, females are more intensely coloured than males, and in the case of polyandrous trios the subordinate male (beta male), who performed least copulations, was always the less intensively coloured individual. If feather protection was the only function of cosmetic colour, no such sex- and age-related colour differences would be expected (see Negro et al., 1999). We believe that strongly coloured individuals may be preferred as mates by individuals of the opposite sex, but that other potential explanations deserve equal consideration. In any case, as Delhey, Peters & Kempenaers (2007) pointed out, even if a protective function for the iron oxides is eventually found, “such additional functions would merely add informational content to the signal without invalidating a potential status-signalling function (Negro et al., 2002)”.

Concluding remarks

Our findings do not support an antibacterial function for ochre application to the white feathers of Bearded Vultures (hypothesis 2), nor that incubating birds pass any protective effect of iron oxides on to their developing embryos or growing chicks (hypothesis 3). In fact, the selective application of pigments to white feather tracts—Bearded Vultures do not stain their entire plumage by rolling in the ferruginous baths—provides additional support to the hypothesis that colouring of the plumage acts as a territorial-status signal (Negro et al., 1999), although it may also benefit other behaviours including pair formation and pair-bonding, as suggested for Egyptian Vultures (Van Overveld, de la Riva & Donázar, 2017). From our findings, we conclude that the primary function of applied colouration in adult Bearded Vultures is that of status signalling at breeding sites, with a secondary function in enhancing their competitive ability to find a mate as have been proposed for greater flamingos (Phoenicopterus roseus) (see Amat & Rendón, 2017). Monitoring ferruginous sources using video-cameras could provide extra useful information regarding their frequency of use with respect to breeding phenology, as well as the interactions occurring there between Bearded Vultures of various age and sex classes (https://www.hbw.com/ibc/news/bearded-vulture-gypaetus-barbatus-bathing-iron-rich-mountain-springs).