1
1 BIOLOGICAL SCIENCES
2 EVOLUTION
3
4 A tropical bat as first mammalian model for skin carotenoid
5 metabolism
6 Short title: Mammal skin carotenoids
7
a,1 b c 8 Ismael Galván , Juan Garrido-Fernández , José J. Ríos , Antonio Pérez-
b d a 9 Gálvez , Bernal Rodríguez-Herrera , and Juan J. Negro
10
11 aDepartment of Evolutionary Ecology, Doñana Biological Station - CSIC, 41092 Sevilla,
12 Spain; bDepartment of Food Phytochemistry, Instituto de la Grasa - CSIC, 41013 Sevilla,
13 Spain; cMass Spectrometry Laboratory, Instituto de la Grasa - CSIC, 41013 Sevilla, Spain;
14 dSchool of Biology, University of Costa Rica, 2060 San José, Costa Rica.
15
16 1Correspondence: Department of Evolutionary Ecology, Doñana Biological Station - CSIC,
17 c/ Américo Vespucio s/n, 41092 Sevilla, Spain. E-mail: [email protected]. Phone:
18 +34954466700. Fax: +34954621125.
19
20 Keywords: bats, carotenoids, macular degeneration, skin colouration. 2
21 Abstract
22 Animals cannot synthesize carotenoid pigments de novo, and must take them in the diet.
23 Several mammals, including humans, are indiscriminate accumulators although
24 distribution to some body tissues like skin is inefficient. This limits the potential capacity of
25 these organisms to benefit from the antioxidant and immunostimulatory functions that
26 carotenoids fulfil. Indeed, up to date no mammal was known to have evolved physiological
27 mechanisms to incorporate and deposit carotenoids in the skin or hair, and mammals have
28 therefore been assumed to rely entirely on other pigments such as melanins to colour their
29 integument. Here, we use high-performance liquid chromatography in combination with
30 time-of-flight mass spectrometry (HPLC-TOF/MS) to show that the frugivorous Honduran
31 white bat Ectophylla alba colours its skin bright yellow with the deposition of significant
32 amounts of the xanthophyll lutein. The Honduran white bat is thus the first mammalian
33 model that may help developing strategies to understand the assimilation of carotenoids in
34 the skin of humans. This represents a change of paradigm in animal physiology showing
35 that some mammals actually have the capacity to accumulate dietary carotenoids in the
36 integument. In addition, we have also discovered that the majority of the lutein in the skin
37 of Honduran white bats is present in esterified form with fatty acids, this permitting longer-
38 lasting colouration and suggesting that bright colour traits may have an overlooked role in
39 bat visual communication.
40
41
42
43
44 3
45 Significance Statement
46 We have discovered the first mammalian species, a bat called the Honduran white bat
47 Ectophylla alba, displaying a yellow carotenoid pigment named lutein in its bare skin. Even
48 though carotenoid-based colouration has been found in birds, fish, amphibians and
49 reptiles, there are no reports of any extant mammal showing these pigments in their skin
50 or hair. The implications of this finding may be profound for human health, as carotenoids
51 are essential micronutrients and lutein in particular is involved in the preservation of the
52 macula of the eye and develop photoprotection functions in the skin. The Honduran white
53 bat, with its ability to assimilate and deposit lutein in its bare skin, may be the sought-after
54 mammalian model that is needed for enhancing studies on carotenoid function and
55 metabolism.
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65 4
66 Introduction
67 Carotenoids are photosynthetic pigments that animals use to avoid cellular oxidative
68 damage and to colour external structures (1-3). The inability of animals to synthesize de
69 novo carotenoids makes that these pigments must be obtained only from dietary sources
70 (4). Among vertebrates, the limiting nature of carotenoids is particularly marked in
71 mammalian species, which exhibit a variable, but generally low efficiency for carotenoid
72 assimilation (5,6). Here, we show for the first time that a mammal, the frugivorous
73 Honduran white bat Ectophylla alba, deposits enough amounts of carotenoids in the skin,
74 so that the colouration provided by those pigments is observed in plain sight. Indeed, up to
75 date no mammal was known to incorporate significant visible amounts of carotenoids in
76 the integument (7,8). This has prevented finding adequate animal models for carotenoid
77 assimilation in skin that would allow improving our knowledge about the bioavailability and
78 tissue distribution of carotenoids in the diet of humans (9), where these pigments are of
79 utmost importance for health by having provitamin A value and fulfilling antioxidant and
80 immunostimulatory functions (10,11). Using high-performance liquid chromatography in
81 combination with time-of-flight mass spectrometry (HPLC-TOF/MS), we found that the
82 characteristic bright yellow colouration of the bare skin of nose-leaf and ears in these bats
83 is generated by the xanthophyll lutein in both free and esterified forms. This indicates that
84 the Honduran white bat represents a change of paradigm in animal physiology showing
85 that some mammals actually have the capacity to accumulate dietary carotenoids in the
86 integument. It is thus the first mammalian model that may help developing strategies to
87 improve the assimilation of lutein in humans (12), which depend on dietary contents to
88 avoid macular degeneration (13) and skin oxidative damage (14).
89 The nocturnal Honduran white bat is a small phyllostomid distributed exclusively in
90 lowland rainforests of Mesoamerica that exhibits bright yellow colouration in the bare skin 5 91 of the nose-leaf, ears and wings (Fig. 1a,b). This distinctive colouration resembles the
92 one generated by some carotenoids, mainly lutein and zeaxanthin, in the integument of
93 other vertebrates such as bird bare integument and feathers (4,15). To our knowledge,
94 such yellow hues are not found in other mammals, which mostly rely on darker
95 endogenous melanins to colour their pelage and skin (16), with the exception of other
96 tropical bats (see below). To determine the origin of the yellow colour, we conducted in-
97 depth chemical analyses of the bare skin of ears and nose-leaf and of the liver of two male
98 Honduran white bat specimens from Costa Rica.
99 The integument of mammals represents a striking example of the difference in the
100 bioavailability of carotenoids among animals. Carotenoids are widespread pigments in the
101 integument of all vertebrates excepting mammals, as only small amounts of carotenoids
102 (30-400 pmol/g of skin) are found in the skin of humans and other mammalian species
103 (7,8) and, contrary to feathers and the scales of reptiles and fish, there seems to be a
104 complete lack of carotenoids in the mammalian hair (15). In humans, and aside from
105 pathological conditions in which carotenoids accumulate in the skin (i.e., carotenodermia),
106 these pigments only occur in those low amounts in the skin (7) in comparison with the
107 accumulation range in inner tissues, 1 mM in the human macula or 0.1-1 M in serum
108 (17), where they interact with the colour generated by keratin, melanins and blood (18).
109 Therefore, the contribution of dietary carotenoids to the perceived variation in human skin
110 colouration is negligible. To date, the role of carotenoids as the primary pigments
111 responsible for the generation of distinctive patches of bright colour in the integument, as
112 in other vertebrates (4,15), has never been reported in mammals. Beyond increasing our
113 understanding of the evolution of animal colouration and appearance, finding mammals
114 able to significantly colour their integument with carotenoids would open new possibilities
115 to obtain mammal models that help to improve our knowledge about bioavailability of
116 dietary carotenoids. Thus, once the model is found, a change in the intake pattern 6 117 providing fruit(s) with different carotenoid profile could give insights about the specificity
118 of carotenoid accumulation in the skin tissue. Also in vivo photoprotection studies could be
119 performed to span our knowledge of carotenoid physiological functions in mammals (19).
120 Mammals obtain carotenoids eating green plant products. Once ingested, these
121 pigments are released from the food matrix by the action of bile salts and pancreatic
122 lipases, which form lipid micelles that incorporate carotenoids and facilitate their transport
123 to the mucosal cells of the duodenum by passive diffusion or through facilitated transport
124 via scavenger receptor class B, type 1 (SR-B1) (20). The capacity of assembling micelles
125 with incorporated carotenoids partly determines the efficiency of carotenoid absorption,
126 which has led to the advent of functional food technologies searching for food matrices
127 that optimize the absorption of supplemented carotenoids in farmed fish (5,21) and human
128 diets (22). Once absorbed, carotenoids are transported by lipoproteins through the
129 bloodstream to the target tissues, a process in which the abundance and nature of
130 lipoproteins limit the capacity to uptake carotenoids (15). Furthermore, some animals can
131 metabolise carotenoids into other forms, and occasionally esterify them with long-chain
132 fatty acids (20). The result is that several factors, including amounts and species of
133 carotenoids ingested, molecular linkage and food matrix composition, nutrient status of the
134 host, genetic and host-related factors, and mathematical interactions (23) influence the
135 bioavailability of carotenoids, creating large differences between species in their capacity
136 to obtain these pigments from their diets and make a physiological use of them (15).
137
138 Results and Discussion
139 Our chromatographic analyses performed with extracts from ears and nose-leaf of
140 Honduran white bats clearly showed that their bright yellow colour is due to the xanthophyll
141 lutein, including trans and cis isomers and its epoxy derivative, that forms exclusively the 7 142 carotenoid profile in the extracts of the two specimens analysed, that is, both specimens
143 contains the same qualitative carotenoid profile. The identification was based on the
144 chromatographic features, UV-visible absorbance spectra and mass spectrometry
145 characteristics, including MS and MS2 spectral behaviour of the molecular ion, which
146 agree with the data available in the literature (24). Fig. 2 depicts the HPLC analyses of the
147 extracts from liver (Fig. 2a), ears and nose-leaf tissue (Fig. 2b) for both specimens, while
148 Fig. 3 shows the UV-visible absorbance spectra of the main peaks detected in the
149 analysed tissues. We compared the experimental data with those obtained from the
150 analysis of lutein standard isolated from a vegetable source, obtaining equivalent results
151 (Fig. 2c and Fig. 3). Unexpectedly, most of the lutein content in the ears and nose-leaf is
152 present as esterified forms with saturated and unsaturated fatty acids, while almost all
153 lutein content in liver is present as free form (Table 1). The probable source of dietary
154 lutein for Honduran white bats is the fig-tree Ficus colubrinae (Standley, 1917), whose ripe
155 red fruit is reported as its major food item (25) (Fig. 1c). The main carotenoids in fresh
156 cultivated figs, the congeneric species fig-tree Ficus carica, are lutein followed by
157 zeaxanthin, both concentrated in the fruit peel (26).
158 Potential applications for human health. The presence of esterified lutein and
159 epoxy-lutein in the yellow skin of Honduran white bats, but not in the liver, suggests that
160 bats possess a physiological mechanism that allows them to esterify those xanthophylls
161 that accumulate in the integument. The bioavailability of xanthophylls to humans partly
162 depends on the efficiency with which the esterified forms of these carotenoids are
163 hydrolyzed by enzymes during digestion, and in fact only free xanthophylls are normally
164 found in human serum and peripheral tissues (27). Although the nutritional use of
165 xanthophylls may require that their esters are metabolized (27), the fact that bats are able
166 to esterify them into their integument opens interesting possibilities to improve the benefits
167 of xanthophylls for human health. This is because, in plants, the esterification of 8 168 xanthophylls enhances their stability (27). Together with zeaxanthin, lutein forms the
169 macular pigment, and plays a prominent role in human vision by acting as a filter of pro-
170 oxidant short-wavelength light and as an antioxidant to prevent damage of free radicals in
171 the retina. Lutein in the human macula is not esterified (28), but if humans had the
172 mechanism to esterify this carotenoid as bats have, it may be speculated that the stability
173 of macular lutein, and then its capacity to avoid eye damage, may be improved. Almost
174 nothing is known about the physiological mechanisms by which some animals esterify
175 carotenoids (29). The Honduran white bat therefore represents the first potential
176 mammalian model that could be used to understand these mechanisms, which may help
177 to improve the stability and bioavailability of carotenoids and thus their benefits for human
178 health.
179 Evolutionary implications. The esterification of lutein that likely occurs directly at
180 the skin in Honduran white bats suggests that this process has evolved because it
181 provides some adaptive value. Given the stability that esterified xanthophylls are known to
182 confer to plants, Honduran white bats may benefit from achieving a longer-lasting skin
183 colouration, which may be related to a role in visual communication of the bats' skin as
184 suggested for the bare integument of birds (29). Honduran white bats construct tents using
185 large plant leaves under which several individuals roost together (25), and gregarious
186 behaviour is known to promote the evolution of conspicuous colour traits that facilitate
187 visual communication (30). At this stage, we can only speculate about the reasons that
188 have led to the evolution of yellow skin colouration in Honduran white bats, but we can
189 assert that at least one bat species has evolved the capacity to use dietary carotenoids to
190 generate bright skin pigmentation. However, the existence of other bat species that also
191 display vivid yellow colour in skin and hair suggests that our finding may not be limited to
192 the Honduran white bat and could have evolved in different bat lineages distributed in
193 tropical regions. It is the case of the MacConnell's bat Mesophylla macconelli, a species 9 194 sympatric with and phylogenetically close to the Honduran white bat that also feeds on
195 the fruits of fig-trees (25) and has yellow bare skin. African tropical bats such as the
196 insectivorous yellow-winged bat Lavia frons and the Sierra Leone collared fruit bat
197 Myonycteris leptodon also show integumentary yellow colouration. Although our study only
198 represents the first report of integument primarily pigmented by carotenoids in mammals,
199 recent findings indicate that a functional M/LWS opsin gene tuned to long-wavelength light
200 is functional even in species of bats that use echolocation and have a long evolutionary
201 history of nocturnality (31). Altogether, these results call into question the commonly
202 neglected role of vision in bat communication, and suggest that microbats may have
203 evolved colour traits despite constraints imposed by nocturnal habits.
204
205 Materials and Methods
206 Animals. Two adult male Honduran white bats were captured in the Tirimbina Biological
207 Reserve (10°26’ N, 83°59’ W), in the province of Heredia, on the North Eastern Caribbean
208 lowlands of Costa Rica. The specimens weighed 5 and 5.5 g, and naturally died shortly
209 after collection with no need of chemicals to sacrifice them (Honduran white bats usually
210 die after collection, probably because of stress during manipulation). They were
211 immediately flash-frozen and shipped to Spain into package with dry ice. Once the bats
212 were received at the Instituto de la Grasa - CSIC (Spain), they were left at ambient
213 temperature for 15 min. Then, the yellow ears were sliced as well as the large yellow nose-
214 leaf. After this, the body cavity was open and the viscera exposed cutting down the body
215 wall from the center of the bat until reaching the genitals. At the posterior end of the cut,
216 two lateral scissions were made to open from the center of the bat two flaps of skin. Liver
217 was located and eviscerated with the help of a clamp. 10 218 Carotenoid extraction. Extraction of carotenoids from tissues was carried out under
219 dim light and avoiding longer processing time. The method described previously (32) was
220 used with modifications as follows. The complete tissue (ears, nose-leaf or liver) was
221 hydrated with 25 ml of cold deionized water, then mixed with 50 ml of cold N,N-
222 dimethylformamide and blended with an Ultra-Turrax (T-25 IKA, Staufen, Germany) for 2
223 min. The extract was filtrated in a Buchner funnel and the solid residue returned to the
224 blender an re-extracted with 50 ml of cold N,N-dimethylformamide. The filtrates were
225 mixed and the lipid fraction extracted with 75 ml of hexane:diethyl ether (1:1) in a
226 decanting funnel. Partition of solvents was promoted by adding 50 ml of NaCl solution
227 (10% w/v). The water and organic layer were allowed to separate, and the lower aqueous
228 phase was discarded. Residual N,N-dimethylformamide was removed by washing the
229 organic phase with 50 ml portions of deionized water. After separation, the water layer was
230 drawn off and the organic phase filtered through a solid bed of Na2SO4 and solvent
231 removed in a rotary evaporator. The extracted residue was reconstituted with 30 ml of
232 chloroform, vortexed for 30 s, then diluted with 70 ml of methanol and vortexed again for
233 30 s. Samples were stored at -80 ºC until analysis.
234 Liquid chromatography - Mass Spectrometry. The carotenoid profile in extracts from
235 tissue samples was separated in a Dionex Ultimate 3000RS U-HPLC (Thermo Fisher
236 Scientific, Waltham, MA, USA) as described previously (33,34). Briefly, a C30 reversed-
237 phase YMC (YMC, Europe, Schermbeck, Germany) analytical column, 250 mm x 4.6 mm
238 (i.d.) with 3 µm particle size was used for separation. The mobile phases were mixtures of
239 methanol:tert-butyl methyl ether:water (A, 81:15:4 and B, 7:90:3), starting with 10 min
240 isocratic A (100%), then a gradient to B 50% at 40 min, B (100%) at 50 min, A (100%) at
241 55 min and isocratic A (100%) from 55 to 60 min. The flow rate was 1 ml/min and the
242 injection volume was 30 µl. UV-visible spectra were recorded from 350 to 600 nm with a
243 photodiode array spectrometer. A split post-column of 0.4 ml/min was introduced directly 11 244 on the mass spectrometer ion source. Mass spectrometry was performed in a
245 micrOTOF-QII high resolution time-of-flight mass spectrometer (UHR-TOF) with qQ-TOF
246 geometry (Bruker Daltonics, Bremen, Germany) equipped with APCI source. Instrument
247 was operated in positive ion mode using a scan range of m/z 50-1200. Mass spectra were
248 acquired through broad band Collision Induced Dissociation (bbCID) mode, providing MS
249 and MS/MS spectra, simultaneously. The instrument control was performed using Bruker
250 Daltonics Hystar 3.2. The same strategy applied to the identification of pigment catabolites
251 in plant tissues was followed for carotenoids in animal tissues (35) in the following way.
252 The in-house mass database created ex professo comprises monoisotopic masses,
253 elemental composition and, optionally, retention time and characteristic product ions for
254 360 carotenes, xanthophylls and xanthophyll esters. Data evaluation was performed with
255 Bruker Daltonics DataAnalysis 4.0. From the HPLC/TOF-MS data, an automated peak
256 detection on the extracted ion chromatograms expected for the [M+H]+ ion of each
257 compound in the database was performed with a script command editor. Identification of
258 carotenoids was performed in the basis of their chromatographic behavior, UV-vis data
259 and high-resolution mass spectrometry data. Thus, positive identification rely on mass
260 accuracy of the protonated molecule, mass error in comparison with the calculated mass
261 for the target compound, and comparison of the theoretical and experimental isotope
262 patterns for the assumed protonated molecule. This last measurement was automatically
263 calculated by the software module SigmaFit included in the data evaluation software. The
264 SigmaFit value is a measure for the goodness of fit between theoretical and experimental
265 isotopic patterns. The smaller the SigmaFit value the better the isotopic matching.
266
267 Acknowledgments. 12 268 I.G. is supported by a Ramón y Cajal Fellowship (RYC-2012-10237) from the Spanish
269 Ministry of Economy and Competitiveness. Financial support from the Spanish
270 Government (AGL2013-42757-R) is acknowledged. Sharlene Santana and Marco
271 Tschapka kindly allowed us to reproduce their Honduran white bat photographs shown in
272 Figure 1.
273
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361 Legends to figures:
362 Fig. 1. Honduran white bat images. a and b: Details of the bright yellow colouration of
363 the bare skin in ears, nose-leaf and wings of an Honduran white bat from Costa Rica. c:
364 An Honduran white bat feeding on the ripe fruit of a fig-tree Ficus colubrinae. Photographs
365 by Sharlene Santana (a and b) and Marco Tschapka (c).
366
367 Fig. 2. Chromatogram traces. The graphs correspond to the analyses of liver (a) and
368 ears+nose-leaf (b) extracts from two specimens of Honduran white bats, and to the
369 analysis of marigold Tagetes erecta extract (c). Peak identification is the same as in Table
370 1. LE means lutein esters.
371
372 Fig. 3. UV-visible spectra of the mean peaks detected in chromatographic analysis of
373 marigold, liver and ears+nose-leaf extract from Honduran white bats. Table 1
Chromatographic, UV-visible, and MS characteristics of main carotenoids from tissues of Honduran white bats E. alba
t λ Peak Carotenoid R max [M+H]+* Main product ions† (min) (nm) 420, 1 (L, 569.4335 551.4281 [M+H-18]+; Lutein 15.7 444, E+n-l)‡ (−3.1/28.8) 495.3642 472 + 418, 931.7601 [M+H-H2O] ; 2 (E+n- Dilauroyl-5,6- 949.7668 + 33.8 443, 749.5791 [M+H-FA] l) epoxy-lutein (2.6/42.3) 469 549.4184 [M+H-2FA]+ 420, 3 (E+n- Unknown¶ 35.3 444, — — l) 472 + 959.7912 [M+H-H2O] ; 418, + 4 (E+n- Lauroyl-myristoyl- 977.7970 777.6150 [M+H-FA] 36.2 443, l) 5,6-epoxy-lutein (−1.4/39.2) + 469 749.5890 [M+H-FA] ; 549.4179 [M+H-2FA]+ + 1,041.8592 [M+H-H2O] ; + 418, 831.6697 [M+H-FA] 5 (E+n- Myristoyl-oleyl- 1,059.8695 37.1 443, l) 5,6-epoxy-lutein (−2.9/45.6) 777.6196 [M+H- 469 FA]+;549.4162 [M+H- 2FA]+ + 995.8237 [M+H-H2O] ; 420, + 6 (E+n- Lauroyl-linoleoyl- 1,013.8339 813.6589 [M+H-FA] 45.5 444, l) lutein (−1.96/29.3) + 472 733.5929 [M+H-FA] ; 533.4187 [M+H-2FA]+ + 1,049.9804 [M+H-H2O] ; 420, + 7 (E+n- Palmitoyl- 1,067.8813 811.6524 [M+H-FA] 46.3 444, l) linoleoyl-lutein§ (1.74/30.2) + 472 789.6613 [M+H-FA] ; 533.4249 [M+H-2FA]+ + 1,049.9797 [M+H-H2O] ; 420, + 8 (E+n- Palmitoyl- 1,067.8859 811.6537 [M+H-FA] 47.0 444, l) linoleoyl-lutein§ (1.84/29.5) + 472 789.6595 [M+H-FA] ; 533.4262 [M+H-2FA]+ + 1,049.9992 [M+H-H2O] ; 420, + 9 (E+n- Palmitoyl- 1,067.8806 811.6529 [M+H-FA] 47.5 444, l) linoleoyl-lutein§ (1.06/32.8) + 472 789.6604 [M+H-FA] ; 533.4236 [M+H-2FA]+ + 420, 1,077.9015 [M+H-H2O] ; 10 Oleyl-linoleoyl- 1,095.9183 + 48.4 444, 813.6593 [M+H-FA] (E+n-l) lutein (1.35/19.8) 472 815.6758 [M+H- t λ Peak Carotenoid R max [M+H]+* Main product ions† (min) (nm) FA]+;533.4278 [M+H- 2FA]+ *m/z experimental value of the molecular ion. Numbers in parenthesis are mass error in ppm and SigmaFit value. †Main products ion arising from MS2-based reactions. ‡Peak present in liver extract (L) and/or in ears+nose-leaf (E+n-l) extract. ¶Peak not identified. §Trans and cis isomers.
Figure 1
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