Lipids (2014) 49:1143–1150 DOI 10.1007/s11745-014-3951-2
ORIGINAL ARTICLE
Fatty Acid Methyl Ester Profiles of Bat Wing Surface Lipids Evan L. Pannkuk • Nathan W. Fuller • Patrick R. Moore • David F. Gilmore • Brett J. Savary • Thomas S. Risch
Received: 7 April 2014 / Accepted: 3 September 2014 / Published online: 17 September 2014 Ó AOCS 2014
Abstract Sebocytes are specialized epithelial cells that rupture to secrete sebaceous lipids (sebum) across the mammalian integument. Sebum protects the integument from UV radiation, and maintains host microbial communities among other functions. Native glandular sebum is composed primarily of triacylglycerides (TAG) and wax esters (WE). Upon secretion (mature sebum), these lipids combine with minor cellular membrane components comprising total surface lipids. TAG and WE are further cleaved to smaller molecules through oxidation or host enzymatic digestion, resulting in a complex mixture of glycerolipids (e.g., TAG), sterols, unesterified fatty acids (FFA), WE, cholesteryl esters, and squalene comprising surface lipid. We are interested if fatty acid methyl ester (FAME) profiling of bat surface lipid could predict species specificity to the cutaneous fungal disease, white nose syndrome (WNS). We collected sebaceous secretions from 13 bat spp. using SebutapeÒ and converted them to FAME with an acid catalyzed transesterification. We found that
E. L. Pannkuk (&) Graduate Program of Environmental Science, Arkansas State University, P.O. Box 847, Jonesboro, AR 72467, USA e-mail: [email protected] N. W. Fuller Department of Biology, Center for Ecology and Conservation Biology, Boston University, 5 Cummington Mall, Boston, MA 02215, USA P. R. Moore D. F. Gilmore T. S. Risch Department of Biological Sciences, Arkansas State University, P.O. Box 599, Jonesboro, AR 72467, USA B. J. Savary Arkansas Biosciences Institute, Arkansas State University, P.O. Box 639, Jonesboro, AR 72467, USA
SebutapeÒ adhesive patches removed *69 more total lipid than SebutapeÒ indicator strips. Juvenile eastern red bats (Lasiurus borealis) had significantly higher 18:1 than adults, but 14:0, 16:1, and 20:0 were higher in adults. FAME profiles among several bat species were similar. We concluded that bat surface lipid FAME profiling does not provide a robust model predicting species susceptibility to WNS. However, these results provide baseline data that can be used for lipid roles in future ecological studies, such as life history, diet, or migration. Keywords White nose syndrome Sebaceous lipids Bat integument Fatty acid methyl esters Gas chromatography Mass spectrometry Abbreviations C Cholesterol CE Cholesteryl ester C:M Chloroform/methanol DAG Diacylglyceride(s) FA Fatty acid(s) FAME Fatty acid methyl ester(s) FFA Unesterified fatty acid(s) GC Gas chromatography IT Ion trap LC Liquid chromatography LOQ Limit of quantification MAG Monoacylglyceride(s) MS Mass spectrometry PL Phospholipid(s) PUFA Polyunsaturated fatty acid(s) SL Sphingolipid(s) TAG Triacylglyceride(s) WE Wax ester(s) WNS White nose syndrome
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Introduction Pseudogymnoascus destructans is a psychrophilic fungus responsible for unprecedented mortality in North American cave dwelling bats [1–4]. Infection by P. destructans, dubbed white nose syndrome (WNS), is responsible for an estimated death of over five million North American cave bats and may result in $52B/year in agricultural damages [5, 6]. The little brown bat (Myotis lucifugus), a once common species, may face local extinctions [7]. P. destructans invades the connective tissue of wings leading to visible necrosis, salt imbalance and decreased glucose levels, [8–12]. Bat species are not equally susceptible to WNS. Currently species confirmed with WNS include M. lucifugus, northern long-eared bat (M. septentrionalis), Indiana bat (M. sodalis), eastern small-footed bat (M. leibii), gray bat (M. grisescens), big brown bat (Eptesicus fuscus), and tricolored bat (Perimyotis subflavus); while the silver-haired bat (Lasionycteris noctivagans), Virginia bigeared bat (Corynorhinus townsendii), and southeastern myotis (M. austroriparius) have tested PCR positive for P. destructans. Among species that exhibit clinical signs of WNS, mortality events are not the same. An explanation for species specificity of WNS susceptibility would be invaluable in WNS management. Species susceptibility is likely due to myriad interactions including group size and sociality [13], immune function [14], body mass [15], life history, location and cave microclimates [16], and sebaceous secretions [17, 18]. Sebaceous lipids play important roles in the interactions of pathogenic microbes with skin and provide molecular fingerprints of disease states in mammals [19–22]. Integumentary secretions in bats are predominantly cholesterol (C) and unesterified fatty acids (FFA), with lower amounts of glycerolipids (TAG, DAG, MAG), squalene, wax esters (WE), cholesteryl esters (CE), phospholipids (PL), and sphingolipids (SL) [17, 18]. The chemical composition of secreted lipid is different from that of other layers of bat integument [23]. Lipid profiling may obtain evidence of species molecular specific sebaceous secretions aiding in vitro studies on response to fungal growth substrate. Changes in sebum molecular profiles may be clinical signs of disease, thus detecting these changes could aid in biomarker discoveries and therapeutic advancements. To date, there has not been a thorough analysis on the molecular composition of bat sebum across multiple bat species with differing susceptibilities to WNS. We hypothesize that sebaceous secretions vary among bat species. Differences in sebaceous chemical composition may play a role in species specificity of WNS. The purpose of the present study is to investigate bat species specific fatty acid methyl esters (FAME). We collected
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sebaceous secretions from multiple bat species using SebutapeÒ and converted to FAME with an acid catalyzed transesterification. We sampled sebaceous secretions from 13 bat species representing seven different genera and 155 individuals [Eptesicus (E. fuscus), Lasiurus (L. cinereus and L. borealis), Nycticeius (N. humeralis), Myotis (M. lucifugus, M. austroriparius, M. septentrionalis, M. leibii, and M. grisescens), Lasionycteris (L. noctivagans), Perimyotis (P. subflavus), and Corynorhinus (C. rafinesquii and C. townsendii ingens)]. Additionally, we sampled juvenile and adult L. borealis to compare FAME content by age. Isolated lipids were derivatized as FAME and analyzed by ion trap gas chromatography/mass spectrometry (GC/MS). Quantitative ratios were compared between bat species.
Materials and Methods Lipid Sampling Bats sampled include sites from Middlesex and Worcester counties in Massachusetts (E. fuscus and M. lucifugus; MA SCI #107.13SCM), Acadia National Park (M. leibii; IACUC #NER-ACAD-Divoll-Bats-2013A3; NPS #ACAD2013-SCI-0042), Ozark Plateau National Wildlife Refuge (C. townsendii ingens), and the remaining from Earl Buss Bayou DeView WMA or Ozark National Forest Sylamore Ranger District (USFWS #TE075913-3; ASU IBC #135349-1). Lipids were collected from 13 bat species by pressing SebutapeÒ Adhesive Patches (28.6 9 19 mm, cat #S100) (tapes) onto the dorsal portion of the plagiopatagium for 1 min [24–26]. For the first 12 samples, both SebutapeÒ indicator strips (15.9 9 15.9 mm, cat #S232) and SebutapeÒ adhesive patches were compared for their effectiveness at collecting 16:0 and 18:0 FA from plagiopatagium (CuDerm Corporation, Dallas, TX, USA). Adhesive patches alone (hereafter referred to as tape) were used for the remainder of the study. Tape samples were placed into 2.0 ml of 3:2 chloroform:methanol (C:M) with 50 mg/l of butylated hydroxytoluene and a TeflonÒ lined cap [27]. Samples were placed on dry ice and shipped overnight to the Arkansas Biosciences Institute (Jonesboro, AR, USA) and stored at -20 °C until analysis. Samples were stored in a freezer within 8 h for a period no longer than 4 weeks. Tapes were removed from the tubes and the C:M was evaporated under a stream of N2. FAME were prepared with an acid-catalyzed esterification and transesterification by adding 0.2 ml toluene, 1.5 ml MeOH, and 0.3 ml MeOH–35 % HCl (used within 2 months of preparation). The solvents were mixed and samples were heated at 45 °C
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overnight. After transesterification 1.0 ml ultrapure water and 1.0 ml hexane were added to the tubes and vortexed for 1 min. The top hexane portion containing FAME was removed and evaporated under a stream of N2. The remaining lipid residue was reconstituted in 50.0 ll hexane and placed in a GC vial with a 0.25 ml conical glass insert (Agilent cat #5183-2085). All solvents were of HPLC grade (Fisher Scientific, USA). Gas Chromatography/Mass Spectrometry FAME were analyzed using a Varian (Santa Clara, CA, USA) 450-GC unit equipped with Agilent Durabond HP-88 column (60 m 9 0.25 mm, with a 0.20 lm film thickness; Agilent application note #5990-8429EN) and a Varian CP8400 autosampler coupled to an ion trap (IT) Varian 240-MS/4000 MS. Injector and transfer line temperatures were 140 and 240 °C, respectively. Oven temperature was programed at 100 °C for 1 min, then 100–200 °C at 5 °C/ min; 200–250 °C at 20 °C/min; and held at 250 °C for 1 min. The injection conditions were 1.0 ll sample volume, splitless injection, carrier gas helium, 0.7 ml/min flow rate, 24.0 psi initial pressure, 8.0 min solvent delay. EI mass spectra were recorded as normal full scan mode. Operating conditions for EI-MS, manifold, transfer-line, and trap temperatures were performed as previously described [18].
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Results Standards Analysis and Comparison of SebutapeÒ Products Total lipid removed was low, so to obtain a response within a reasonable limit of quantification (LOQ) we took total lipid collected per bat, transesterified total lipid content, and used GC vial inserts with 50 ll sample per insert, and injected lipid splitless into the GC/MS. Concentrations of FAME from experimental samples fell within the ranges of the appropriate standard for the major FAME collected, and the standard curves for these FAME showed high linearity (16:0, 18:0, 18:1, 18:2 R2 = 0.99). The 18:0 and 16:0 fatty acyls (FA) were chosen to determine quantitative amounts of total lipid removed because they accounted for dominant peak area of GC chromatograms (67–83 %). SebutapeÒ adhesive patches (tapes, 28.6 9 19 mm) removed significantly higher amounts of 16:0 (22.4 vs 3.5 mM) and 18:0 (20.5 vs
Table 1 Total 16:0 and 18:0 fatty acid methyl ester collected by SebutapeÒ adhesive patches or skin indicators FAME
N
Adhesive patches
Skin indicators
z value
P value
16:0 (mM)
12
22.4 ± 2.7
3.5 ± 0.4
-6.84
\0.001
Statistical Analysis and Quality Control
18:0 (mM)
12
20.5 ± 2.4
3.2 ± 0.4
-7.19
\0.001
Total 16:0 and 18:0 removed by SebutapeÒ type was arcsin transformed compared between 24 Myotis spp. with a Mann–Whitney U test. A blank (29) and external standard (200 lg/ml) were run every five samples to ensure no carryover between samples. A standard curve (7–500 lg/ml) was performed to test for linearity within the ranges acquired. Data acquisition/processing were performed with Varian Workstation Software (version 6; Walnut Creek, CA, USA). Target peaks were identified by reference to a standard where available (Sigma GLC 20 & 80) and by EI spectral matching to the NIST/EPA/NIH mass spectral library (NIST 11) and the NIST mass spectral search program (Version 2.0f) (Gaithersburg, MD, USA). FAME results are expressed as a percentage of total FAME peak area. Values were arcsin transformed and means were compared between bat species with a Kruskal–Wallis test, using a Bonferroni corrected P value of 0.008, and post hoc Duncan test. Juvenile and adult L. borealis were compared with a Mann–Whitney U test (SAS version 9.2; SAS Institute Inc., Cary, North Carolina, USA).
Mean ± SE
Fig. 1 Proportion of fatty acid methyl esters in juvenile (n = 10) or adult (n = 10) Lasiurus borealis sebum (* denotes significance)
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Fig. 2 Proportion of fatty acid methyl esters in sebum of 13 species of North American bats
3.2 mM) FA than indicator strips (15.9 9 15.9 mm), and were used for all subsequent analysis (Table 1). SebutapeÒ adhesive patches were also more efficient at lipid
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adsorption, as they remove 78.9 nM/mm2 (1.12 lg/mm2) of the dominant FA while indicator strips only removed 19.0 nM/mm2 (0.38 lg/mm2).
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Comparison of Adult and Juvenile Eastern Red Bats Differences between juvenile and adult L. borealis included adults having significantly higher 14:0 (z = 6.80, P = 0.009), 16:1n-7 (z = 5.71, P = 0.017), and 20:0 (z = 11.10, P = 0.001) than juveniles (Fig. 1). Juveniles had higher amounts of 18:1n-9 (z = 4.11, P = 0.043) than adults and non-significantly higher amounts of 18:0 and 16:0.
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significant differences in FAME proportion except for 16:0 (Table 2). The 18:0 FAME was the dominant peak detected followed by 16:0, except in N. humeralis and C. rafinesquii were 16:0 was dominant over 18:0.
Discussion
The majority of the FAME detected from the surface lipid of bat wings are 16:0, 18:0, and 18:1n9 with lower amounts of 12:0, 14:0, 16:1n-7, 18:2n-6, 20:0, 20:1, 21:0, 21:1, 22:0, and 24:0 (Figs. 2, 3, 4; Table 2). FAME present \1.0 % were excluded from statistical analysis. Surprisingly, 18:3 was rarely detected and may be due to elevated secreted lipid oxidation rates and increased hydrolysis leading to shorter FA chains. Spectra of N. humeralis FAME revealed the absence of 20:0. N. humeralis total lipid has higher levels of unsaturation (Fig. 2). We found
We compared SebutapeÒ brand efficiency to remove sufficient lipid for downstream GC/MS analysis, compared sebaceous FAME between juvenile and adult L. borealis, and quantified 13 bat spp. sebaceous wing lipid FAME. We found SebutapeÒ adhesive patches (tapes) removed *69 the amount of 16:0 and 18:0 than indicator strips. Indicator strips can be utilized to estimate sebaceous gland density, but tapes are more efficient to collect sufficient lipid for downstream analytical analysis. Perhaps of importance is that we applied tapes for 1 min (to minimize distress to the animal) that to the best of our knowledge is the shortest application time mentioned in the literature. Reported application times on humans have been C30 min [25, 26]. Indicator strips may be efficient for collecting sufficient
Fig. 3 Total ion current obtained for Myotis septentrionalis sebaceous lipid and mass spectrum for 18:0 fatty acid methyl ester
Fig. 4 Total ion current obtained for Nycticeius humeralis sebaceous lipid and mass spectrum for 16:0 fatty acid methyl ester
Comparison Between Species
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41.44 21.58 30.35 50.71 45.13 55.84 \0.001 0.04 0.003 \0.001 \0.001 \0.001 10 3.5 ± 0.5b,c,d,e 39.7 ± 1.5a 1.2 ± 0.3b,c 44.2 ± 2.0a,b,c 9.6 ± 1.0c,d 4.4 ± 0.5d 16 2.4 ± 0.2d,e 34.8 ± 1.2a,b 1.1 ± 0.1c 46.0 ± 1.5a,b 11.4 ± 0.6b,c,d 6.7 ± 0.3c,d 11 5.5 ± 1.0a,b 30.0 ± 2.8b 1.6 ± 0.2b,c 37.3 ± 3.5c,d,e 20.0 ± 4.6a 7.7 ± 1.1b,c,d Mean ± SE a, b, c, d, e Statistically different groups
5 2.6 ± 0.2d,e 32.1 ± 0.4a,b 3.9 ± 0.8a 48.1 ± 1.0a 7.1 ± 0.3d 6.2 ± 0.2c,d N 14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6
11 4.8 ± 0.8a,b,c 32.4 ± 1.5a,b 1.4 ± 0.3b,c 41.4 ± 1.9b,c,d 12.0 ± 1.0b,c,d 9.9 ± 1.1a,b,c
H value P value M. grisescens M. leibii M. septentrionalis M. austroriparius Myotis lucifugus Species
12 3.1 ± 0.7c,d,e 33.9 ± 0.7a,b 2.0 ± 0.4a,b,c 38.7 ± 1.1b,c,d,e 16.4 ± 1.6a,b 7.7 ± 0.6b,c,d 17 3.7 ± 0.3b,c,d,e 36.5 ± 2.6a,b 2.9 ± 0.3a,b,c 31.3 ± 1.6e 15.8 ± 1.2a,b,c 11.7 ± 1.0a 6 1.9 ± 0.4e 35.3 ± 1.2a,b 1.5 ± 0.5b,c 46.1 ± 3.0a,b 13.1 ± 1.5b,c,d 4.7 ± 0.7d 8 2.8 ± 0.3d,e 34.3 ± 1.0a,b 2.8 ± 0.6a,b,c 39.6 ± 1.9a,b,c 17.7 ± 1.4a,b 6.8 ± 0.6c,d 6 4.3 ± 1.1a,b,c,d 37.4 ± 4.6a,b 3.7 ± 1.7a 38.3 ± 6.1b,c,d,e 10.4 ± 0.8b,c,d 8.2 ± 1.7a,b,c,d 11 5.9 ± 0.6a 37.7 ± 2.9a,b 3.1 ± 0.9a,b 33.6 ± 2.2d,e 12.0 ± 0.9b,c,d 9.5 ± 1.7a,b,c N 14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6
5 2.4 ± 0.2d,e 36.7 ± 1.3a,b 2.2 ± 0.5a,b,c 45.0 ± 2.5a,b,c 11.0 ± 1.1b,c,d 5.2 ± 0.7d
Eptesicus fuscus Nycticeius humeralis L. cinereus Lasiurus borealis Lasionycteris noctivagans C. townsendii ingens Corynorhinus rafinesquii Species
Table 2 Fatty acid methyl ester percentages of North American bat sebum
9 3.7 ± 0.7b,c,d,e 30.6 ± 1.6b 1.4 ± 0.2b,c 42.4 ± 1.4a,b,c 13.0 ± 0.9b,c,d 10.7 ± 1.8a,b
Lipids (2014) 49:1143–1150 Perimyotis subflavus
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total lipid for downstream analysis when used for longer application times. Furthermore, SebutapeÒ is non-invasive and less harmful for lipid biomarker collection from wild mammals than other techniques, such as solvent soaked cotton ball scrubs [28] or inverting solvent vials on integumentary surfaces (CAMAG application note #A-89.1). We determined differences in FAME between adults and juveniles of a ubiquitous bat species, L. borealis. Sebaceous output and chemical composition change during aging or are different due to ethnic background [29, 30]. Female African Americans had higher proportions of 14:0, 16:1, and 18:1 FAME than Caucasian or northern Asian females [30]. Some reports suggest that sebum is stable through most adulthood, declining after menopause or lower testosterone production in males [29]. Contrasting reports suggest sebum production gradually declines through adulthood, with higher decline rates in females than in males [31]. In humans, 16:1 WE FAME increase during infancy to the 20’s and begins to decline, with the reverse pattern for 16:1 isobranched FAME [32]. WE 18:1 FAME decreases from infancy to the 20’s and shows no change throughout adulthood. Similar results were found for L. borealis, where 18:1 was significantly higher in juveniles than adults but 14:0, 16:1, and 20:0 were higher in adults. We found significant differences in FAME proportions except for 16:0. However, the hypothesis that bat sebaceous FAME may provide a model predicting species susceptibility to WNS was not supported by the present analysis. FAME profiling partially differentiated species, but it does not predict species according to known susceptibilities to WNS or current taxonomic status. While FAME profiling is used extensively to characterize microbial communities, mammalian FAME profiles may be too similar for taxonomic purposes [33]. Dominant FAME include 16:0, 18:0, and 18:1 as previously reported [18]. FAME profiles were similar among all species tested; however, 20:0 is absent in evening bats. Evening bats have higher PUFA percentage than other bats [11.7 %; however, not significantly higher than C. rafinesquii (9.5 %), L. noctivagans (8.2 %), P. subflavus (10.7 %), and M. austroriparius (9.9 %)], which may contribute to their oily texture. The 16:1 and 18:2 FAME was in lower percentage in most species susceptible to contracting WNS [i.e., 16:1, P. subflavus (1.4 %), M. septentrionalis (1.6 %), M. leibii (1.1 %), and M. grisescens (1.2 %); 18:2, M. lucifugus (6.2 %), M. leibii (6.7 %), and M. grisescens (4.4 %)]. Sapienic acid 16:1n-10 is present in high abundance on human skin and observed to be highly antimicrobial and defends against colonization of Staphylococcus aureus [19, 34]. Antifungal properties for 18:2 FFA have also been observed in the growth inhibition of P. destructans (Pannkuk data not shown). However, M. lucifugus (the species
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most heavily affected by WNS) had higher proportions of 16:1 (3.9 %) than other bats species. L. cinereus (not affected by WNS) had lower proportions of 16:1 (1.5 %). For 18:2, two bat species affected by WNS [P. subflavus (10.7 %); M. austroriparius (9.9 %)] had higher amounts than other species and Lasiurine bats had lower amounts (L. borealis and L. cinereus; 6.8 and 4.7 % respectively). Knowledge on the bat integumentary lipidome has increased greatly. Bat wing/sebaceous gland morphology [35, 36], broad lipid class of sebaceous secretions [17], individual lipid molecule variation among body site [30, 37], and stratum corneum lipid composition [23] have been described. To proceed with lipid biomarker identification utilized in assessing WNS progression and disease state, high throughput liquid chromatography (LC) tandem MS methods should be developed for more sensitive and broad scale lipid analysis of bat integument [37] and differentiated from fungal lipids [38]. Also, biases from lipid types collected by SebutapeÒ should be determined if this method is to be applied to broad scale ecological studies. To achieve these objectives, lipid types detected on the mammalian host vs. P. destructans must be determined. These baseline data on fungal and bat lipids will provide a framework to facilitate further investigations determining lipid biomarkers and explaining interactions in a complex host/pathogen system. Acknowledgments This project was funded by the Arkansas State Wildlife Grant, the National Speleological Society, the graduate program of environmental science at Arkansas State University (ASU), Bat Conservation International, and the Center for North American Bat Research and Conservation at Indiana State University. Laboratory assistance was provided by K. Arter and H. Southe (Arkansas State University). Samples were collected by C. Gerdes (Missouri State University), T. Divoll (Biodiversity Research Institute), and P. Jordan (Arkansas State University). We thank the ecotoxicology research facility (Arkansas State University; J. Bouldin and T. Woodruff) for assistance with GC/MS (NSF Grant #1040466).
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Lipids (2014) 49:1143–1150 31. Jacobsen E, Billings JK, Frantz RA, Kinney CK, Stewart ME, Downing DT (1985) Age-related changes in sebaceous wax ester secretion rates in men and women. J Invest Dermat 85:483–485 32. Yamamoto A, Serizawa S, Ito M, Sato Y (1987) Effect of aging on sebaceous gland activity and on the fatty acid composition of wax esters. Soc Invest Dermatol 89:507–512 33. Zelle L (1999) Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Bio Fer Soil 29:111–129 34. Takigawa H, Nakagawa H, Kuzukawa M, Mori H, Imokawa G (2005) Deficient production of hexadecenoic acid in the skin is associated in part with the vulnerability of atopic dermatitis patients to colonisation by Staphylococcus aureus. Dermat 211:240–248 35. Cortese TA, Nicoll PA (1970) In vivo observations of skin appendages in the bat wing. J Invest Dermatol 54:1–11 36. Nassar JM, Salazar MV, Quintero A, Stoner KE, Go´mez M, Cabrera A, Jaffe K (2008) Seasonal sebaceous patch in the nectar-feeding bats Leptonycteris curasoae and L. yerbabuenae (Phyllostomidae: Glossophaginae): Phenological, histological, and preliminary chemical characterization. Zoology 111:363–376 37. Pannkuk EL, McGuire LP, Gilmore DF, Savary BJ, Risch TS (2014) Glycerophospholipid analysis of Eastern red bat (Lasiurus borealis) hair by electrospray ionization tandem mass spectrometry. J Chem Ecol 40:227–235 38. Pannkuk EL, Blair HB, Fischer AE, Gerdes CL, Gilmore DF, Savary BJ, Risch TS (2014) Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures. Fungal Biol 118:792–799
ORIGINAL ARTICLE
Fatty Acid Methyl Ester Profiles of Bat Wing Surface Lipids Evan L. Pannkuk • Nathan W. Fuller • Patrick R. Moore • David F. Gilmore • Brett J. Savary • Thomas S. Risch
Received: 7 April 2014 / Accepted: 3 September 2014 / Published online: 17 September 2014 Ó AOCS 2014
Abstract Sebocytes are specialized epithelial cells that rupture to secrete sebaceous lipids (sebum) across the mammalian integument. Sebum protects the integument from UV radiation, and maintains host microbial communities among other functions. Native glandular sebum is composed primarily of triacylglycerides (TAG) and wax esters (WE). Upon secretion (mature sebum), these lipids combine with minor cellular membrane components comprising total surface lipids. TAG and WE are further cleaved to smaller molecules through oxidation or host enzymatic digestion, resulting in a complex mixture of glycerolipids (e.g., TAG), sterols, unesterified fatty acids (FFA), WE, cholesteryl esters, and squalene comprising surface lipid. We are interested if fatty acid methyl ester (FAME) profiling of bat surface lipid could predict species specificity to the cutaneous fungal disease, white nose syndrome (WNS). We collected sebaceous secretions from 13 bat spp. using SebutapeÒ and converted them to FAME with an acid catalyzed transesterification. We found that
E. L. Pannkuk (&) Graduate Program of Environmental Science, Arkansas State University, P.O. Box 847, Jonesboro, AR 72467, USA e-mail: [email protected] N. W. Fuller Department of Biology, Center for Ecology and Conservation Biology, Boston University, 5 Cummington Mall, Boston, MA 02215, USA P. R. Moore D. F. Gilmore T. S. Risch Department of Biological Sciences, Arkansas State University, P.O. Box 599, Jonesboro, AR 72467, USA B. J. Savary Arkansas Biosciences Institute, Arkansas State University, P.O. Box 639, Jonesboro, AR 72467, USA
SebutapeÒ adhesive patches removed *69 more total lipid than SebutapeÒ indicator strips. Juvenile eastern red bats (Lasiurus borealis) had significantly higher 18:1 than adults, but 14:0, 16:1, and 20:0 were higher in adults. FAME profiles among several bat species were similar. We concluded that bat surface lipid FAME profiling does not provide a robust model predicting species susceptibility to WNS. However, these results provide baseline data that can be used for lipid roles in future ecological studies, such as life history, diet, or migration. Keywords White nose syndrome Sebaceous lipids Bat integument Fatty acid methyl esters Gas chromatography Mass spectrometry Abbreviations C Cholesterol CE Cholesteryl ester C:M Chloroform/methanol DAG Diacylglyceride(s) FA Fatty acid(s) FAME Fatty acid methyl ester(s) FFA Unesterified fatty acid(s) GC Gas chromatography IT Ion trap LC Liquid chromatography LOQ Limit of quantification MAG Monoacylglyceride(s) MS Mass spectrometry PL Phospholipid(s) PUFA Polyunsaturated fatty acid(s) SL Sphingolipid(s) TAG Triacylglyceride(s) WE Wax ester(s) WNS White nose syndrome
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Introduction Pseudogymnoascus destructans is a psychrophilic fungus responsible for unprecedented mortality in North American cave dwelling bats [1–4]. Infection by P. destructans, dubbed white nose syndrome (WNS), is responsible for an estimated death of over five million North American cave bats and may result in $52B/year in agricultural damages [5, 6]. The little brown bat (Myotis lucifugus), a once common species, may face local extinctions [7]. P. destructans invades the connective tissue of wings leading to visible necrosis, salt imbalance and decreased glucose levels, [8–12]. Bat species are not equally susceptible to WNS. Currently species confirmed with WNS include M. lucifugus, northern long-eared bat (M. septentrionalis), Indiana bat (M. sodalis), eastern small-footed bat (M. leibii), gray bat (M. grisescens), big brown bat (Eptesicus fuscus), and tricolored bat (Perimyotis subflavus); while the silver-haired bat (Lasionycteris noctivagans), Virginia bigeared bat (Corynorhinus townsendii), and southeastern myotis (M. austroriparius) have tested PCR positive for P. destructans. Among species that exhibit clinical signs of WNS, mortality events are not the same. An explanation for species specificity of WNS susceptibility would be invaluable in WNS management. Species susceptibility is likely due to myriad interactions including group size and sociality [13], immune function [14], body mass [15], life history, location and cave microclimates [16], and sebaceous secretions [17, 18]. Sebaceous lipids play important roles in the interactions of pathogenic microbes with skin and provide molecular fingerprints of disease states in mammals [19–22]. Integumentary secretions in bats are predominantly cholesterol (C) and unesterified fatty acids (FFA), with lower amounts of glycerolipids (TAG, DAG, MAG), squalene, wax esters (WE), cholesteryl esters (CE), phospholipids (PL), and sphingolipids (SL) [17, 18]. The chemical composition of secreted lipid is different from that of other layers of bat integument [23]. Lipid profiling may obtain evidence of species molecular specific sebaceous secretions aiding in vitro studies on response to fungal growth substrate. Changes in sebum molecular profiles may be clinical signs of disease, thus detecting these changes could aid in biomarker discoveries and therapeutic advancements. To date, there has not been a thorough analysis on the molecular composition of bat sebum across multiple bat species with differing susceptibilities to WNS. We hypothesize that sebaceous secretions vary among bat species. Differences in sebaceous chemical composition may play a role in species specificity of WNS. The purpose of the present study is to investigate bat species specific fatty acid methyl esters (FAME). We collected
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sebaceous secretions from multiple bat species using SebutapeÒ and converted to FAME with an acid catalyzed transesterification. We sampled sebaceous secretions from 13 bat species representing seven different genera and 155 individuals [Eptesicus (E. fuscus), Lasiurus (L. cinereus and L. borealis), Nycticeius (N. humeralis), Myotis (M. lucifugus, M. austroriparius, M. septentrionalis, M. leibii, and M. grisescens), Lasionycteris (L. noctivagans), Perimyotis (P. subflavus), and Corynorhinus (C. rafinesquii and C. townsendii ingens)]. Additionally, we sampled juvenile and adult L. borealis to compare FAME content by age. Isolated lipids were derivatized as FAME and analyzed by ion trap gas chromatography/mass spectrometry (GC/MS). Quantitative ratios were compared between bat species.
Materials and Methods Lipid Sampling Bats sampled include sites from Middlesex and Worcester counties in Massachusetts (E. fuscus and M. lucifugus; MA SCI #107.13SCM), Acadia National Park (M. leibii; IACUC #NER-ACAD-Divoll-Bats-2013A3; NPS #ACAD2013-SCI-0042), Ozark Plateau National Wildlife Refuge (C. townsendii ingens), and the remaining from Earl Buss Bayou DeView WMA or Ozark National Forest Sylamore Ranger District (USFWS #TE075913-3; ASU IBC #135349-1). Lipids were collected from 13 bat species by pressing SebutapeÒ Adhesive Patches (28.6 9 19 mm, cat #S100) (tapes) onto the dorsal portion of the plagiopatagium for 1 min [24–26]. For the first 12 samples, both SebutapeÒ indicator strips (15.9 9 15.9 mm, cat #S232) and SebutapeÒ adhesive patches were compared for their effectiveness at collecting 16:0 and 18:0 FA from plagiopatagium (CuDerm Corporation, Dallas, TX, USA). Adhesive patches alone (hereafter referred to as tape) were used for the remainder of the study. Tape samples were placed into 2.0 ml of 3:2 chloroform:methanol (C:M) with 50 mg/l of butylated hydroxytoluene and a TeflonÒ lined cap [27]. Samples were placed on dry ice and shipped overnight to the Arkansas Biosciences Institute (Jonesboro, AR, USA) and stored at -20 °C until analysis. Samples were stored in a freezer within 8 h for a period no longer than 4 weeks. Tapes were removed from the tubes and the C:M was evaporated under a stream of N2. FAME were prepared with an acid-catalyzed esterification and transesterification by adding 0.2 ml toluene, 1.5 ml MeOH, and 0.3 ml MeOH–35 % HCl (used within 2 months of preparation). The solvents were mixed and samples were heated at 45 °C
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overnight. After transesterification 1.0 ml ultrapure water and 1.0 ml hexane were added to the tubes and vortexed for 1 min. The top hexane portion containing FAME was removed and evaporated under a stream of N2. The remaining lipid residue was reconstituted in 50.0 ll hexane and placed in a GC vial with a 0.25 ml conical glass insert (Agilent cat #5183-2085). All solvents were of HPLC grade (Fisher Scientific, USA). Gas Chromatography/Mass Spectrometry FAME were analyzed using a Varian (Santa Clara, CA, USA) 450-GC unit equipped with Agilent Durabond HP-88 column (60 m 9 0.25 mm, with a 0.20 lm film thickness; Agilent application note #5990-8429EN) and a Varian CP8400 autosampler coupled to an ion trap (IT) Varian 240-MS/4000 MS. Injector and transfer line temperatures were 140 and 240 °C, respectively. Oven temperature was programed at 100 °C for 1 min, then 100–200 °C at 5 °C/ min; 200–250 °C at 20 °C/min; and held at 250 °C for 1 min. The injection conditions were 1.0 ll sample volume, splitless injection, carrier gas helium, 0.7 ml/min flow rate, 24.0 psi initial pressure, 8.0 min solvent delay. EI mass spectra were recorded as normal full scan mode. Operating conditions for EI-MS, manifold, transfer-line, and trap temperatures were performed as previously described [18].
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Results Standards Analysis and Comparison of SebutapeÒ Products Total lipid removed was low, so to obtain a response within a reasonable limit of quantification (LOQ) we took total lipid collected per bat, transesterified total lipid content, and used GC vial inserts with 50 ll sample per insert, and injected lipid splitless into the GC/MS. Concentrations of FAME from experimental samples fell within the ranges of the appropriate standard for the major FAME collected, and the standard curves for these FAME showed high linearity (16:0, 18:0, 18:1, 18:2 R2 = 0.99). The 18:0 and 16:0 fatty acyls (FA) were chosen to determine quantitative amounts of total lipid removed because they accounted for dominant peak area of GC chromatograms (67–83 %). SebutapeÒ adhesive patches (tapes, 28.6 9 19 mm) removed significantly higher amounts of 16:0 (22.4 vs 3.5 mM) and 18:0 (20.5 vs
Table 1 Total 16:0 and 18:0 fatty acid methyl ester collected by SebutapeÒ adhesive patches or skin indicators FAME
N
Adhesive patches
Skin indicators
z value
P value
16:0 (mM)
12
22.4 ± 2.7
3.5 ± 0.4
-6.84
\0.001
Statistical Analysis and Quality Control
18:0 (mM)
12
20.5 ± 2.4
3.2 ± 0.4
-7.19
\0.001
Total 16:0 and 18:0 removed by SebutapeÒ type was arcsin transformed compared between 24 Myotis spp. with a Mann–Whitney U test. A blank (29) and external standard (200 lg/ml) were run every five samples to ensure no carryover between samples. A standard curve (7–500 lg/ml) was performed to test for linearity within the ranges acquired. Data acquisition/processing were performed with Varian Workstation Software (version 6; Walnut Creek, CA, USA). Target peaks were identified by reference to a standard where available (Sigma GLC 20 & 80) and by EI spectral matching to the NIST/EPA/NIH mass spectral library (NIST 11) and the NIST mass spectral search program (Version 2.0f) (Gaithersburg, MD, USA). FAME results are expressed as a percentage of total FAME peak area. Values were arcsin transformed and means were compared between bat species with a Kruskal–Wallis test, using a Bonferroni corrected P value of 0.008, and post hoc Duncan test. Juvenile and adult L. borealis were compared with a Mann–Whitney U test (SAS version 9.2; SAS Institute Inc., Cary, North Carolina, USA).
Mean ± SE
Fig. 1 Proportion of fatty acid methyl esters in juvenile (n = 10) or adult (n = 10) Lasiurus borealis sebum (* denotes significance)
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Fig. 2 Proportion of fatty acid methyl esters in sebum of 13 species of North American bats
3.2 mM) FA than indicator strips (15.9 9 15.9 mm), and were used for all subsequent analysis (Table 1). SebutapeÒ adhesive patches were also more efficient at lipid
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adsorption, as they remove 78.9 nM/mm2 (1.12 lg/mm2) of the dominant FA while indicator strips only removed 19.0 nM/mm2 (0.38 lg/mm2).
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Comparison of Adult and Juvenile Eastern Red Bats Differences between juvenile and adult L. borealis included adults having significantly higher 14:0 (z = 6.80, P = 0.009), 16:1n-7 (z = 5.71, P = 0.017), and 20:0 (z = 11.10, P = 0.001) than juveniles (Fig. 1). Juveniles had higher amounts of 18:1n-9 (z = 4.11, P = 0.043) than adults and non-significantly higher amounts of 18:0 and 16:0.
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significant differences in FAME proportion except for 16:0 (Table 2). The 18:0 FAME was the dominant peak detected followed by 16:0, except in N. humeralis and C. rafinesquii were 16:0 was dominant over 18:0.
Discussion
The majority of the FAME detected from the surface lipid of bat wings are 16:0, 18:0, and 18:1n9 with lower amounts of 12:0, 14:0, 16:1n-7, 18:2n-6, 20:0, 20:1, 21:0, 21:1, 22:0, and 24:0 (Figs. 2, 3, 4; Table 2). FAME present \1.0 % were excluded from statistical analysis. Surprisingly, 18:3 was rarely detected and may be due to elevated secreted lipid oxidation rates and increased hydrolysis leading to shorter FA chains. Spectra of N. humeralis FAME revealed the absence of 20:0. N. humeralis total lipid has higher levels of unsaturation (Fig. 2). We found
We compared SebutapeÒ brand efficiency to remove sufficient lipid for downstream GC/MS analysis, compared sebaceous FAME between juvenile and adult L. borealis, and quantified 13 bat spp. sebaceous wing lipid FAME. We found SebutapeÒ adhesive patches (tapes) removed *69 the amount of 16:0 and 18:0 than indicator strips. Indicator strips can be utilized to estimate sebaceous gland density, but tapes are more efficient to collect sufficient lipid for downstream analytical analysis. Perhaps of importance is that we applied tapes for 1 min (to minimize distress to the animal) that to the best of our knowledge is the shortest application time mentioned in the literature. Reported application times on humans have been C30 min [25, 26]. Indicator strips may be efficient for collecting sufficient
Fig. 3 Total ion current obtained for Myotis septentrionalis sebaceous lipid and mass spectrum for 18:0 fatty acid methyl ester
Fig. 4 Total ion current obtained for Nycticeius humeralis sebaceous lipid and mass spectrum for 16:0 fatty acid methyl ester
Comparison Between Species
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41.44 21.58 30.35 50.71 45.13 55.84 \0.001 0.04 0.003 \0.001 \0.001 \0.001 10 3.5 ± 0.5b,c,d,e 39.7 ± 1.5a 1.2 ± 0.3b,c 44.2 ± 2.0a,b,c 9.6 ± 1.0c,d 4.4 ± 0.5d 16 2.4 ± 0.2d,e 34.8 ± 1.2a,b 1.1 ± 0.1c 46.0 ± 1.5a,b 11.4 ± 0.6b,c,d 6.7 ± 0.3c,d 11 5.5 ± 1.0a,b 30.0 ± 2.8b 1.6 ± 0.2b,c 37.3 ± 3.5c,d,e 20.0 ± 4.6a 7.7 ± 1.1b,c,d Mean ± SE a, b, c, d, e Statistically different groups
5 2.6 ± 0.2d,e 32.1 ± 0.4a,b 3.9 ± 0.8a 48.1 ± 1.0a 7.1 ± 0.3d 6.2 ± 0.2c,d N 14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6
11 4.8 ± 0.8a,b,c 32.4 ± 1.5a,b 1.4 ± 0.3b,c 41.4 ± 1.9b,c,d 12.0 ± 1.0b,c,d 9.9 ± 1.1a,b,c
H value P value M. grisescens M. leibii M. septentrionalis M. austroriparius Myotis lucifugus Species
12 3.1 ± 0.7c,d,e 33.9 ± 0.7a,b 2.0 ± 0.4a,b,c 38.7 ± 1.1b,c,d,e 16.4 ± 1.6a,b 7.7 ± 0.6b,c,d 17 3.7 ± 0.3b,c,d,e 36.5 ± 2.6a,b 2.9 ± 0.3a,b,c 31.3 ± 1.6e 15.8 ± 1.2a,b,c 11.7 ± 1.0a 6 1.9 ± 0.4e 35.3 ± 1.2a,b 1.5 ± 0.5b,c 46.1 ± 3.0a,b 13.1 ± 1.5b,c,d 4.7 ± 0.7d 8 2.8 ± 0.3d,e 34.3 ± 1.0a,b 2.8 ± 0.6a,b,c 39.6 ± 1.9a,b,c 17.7 ± 1.4a,b 6.8 ± 0.6c,d 6 4.3 ± 1.1a,b,c,d 37.4 ± 4.6a,b 3.7 ± 1.7a 38.3 ± 6.1b,c,d,e 10.4 ± 0.8b,c,d 8.2 ± 1.7a,b,c,d 11 5.9 ± 0.6a 37.7 ± 2.9a,b 3.1 ± 0.9a,b 33.6 ± 2.2d,e 12.0 ± 0.9b,c,d 9.5 ± 1.7a,b,c N 14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6
5 2.4 ± 0.2d,e 36.7 ± 1.3a,b 2.2 ± 0.5a,b,c 45.0 ± 2.5a,b,c 11.0 ± 1.1b,c,d 5.2 ± 0.7d
Eptesicus fuscus Nycticeius humeralis L. cinereus Lasiurus borealis Lasionycteris noctivagans C. townsendii ingens Corynorhinus rafinesquii Species
Table 2 Fatty acid methyl ester percentages of North American bat sebum
9 3.7 ± 0.7b,c,d,e 30.6 ± 1.6b 1.4 ± 0.2b,c 42.4 ± 1.4a,b,c 13.0 ± 0.9b,c,d 10.7 ± 1.8a,b
Lipids (2014) 49:1143–1150 Perimyotis subflavus
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total lipid for downstream analysis when used for longer application times. Furthermore, SebutapeÒ is non-invasive and less harmful for lipid biomarker collection from wild mammals than other techniques, such as solvent soaked cotton ball scrubs [28] or inverting solvent vials on integumentary surfaces (CAMAG application note #A-89.1). We determined differences in FAME between adults and juveniles of a ubiquitous bat species, L. borealis. Sebaceous output and chemical composition change during aging or are different due to ethnic background [29, 30]. Female African Americans had higher proportions of 14:0, 16:1, and 18:1 FAME than Caucasian or northern Asian females [30]. Some reports suggest that sebum is stable through most adulthood, declining after menopause or lower testosterone production in males [29]. Contrasting reports suggest sebum production gradually declines through adulthood, with higher decline rates in females than in males [31]. In humans, 16:1 WE FAME increase during infancy to the 20’s and begins to decline, with the reverse pattern for 16:1 isobranched FAME [32]. WE 18:1 FAME decreases from infancy to the 20’s and shows no change throughout adulthood. Similar results were found for L. borealis, where 18:1 was significantly higher in juveniles than adults but 14:0, 16:1, and 20:0 were higher in adults. We found significant differences in FAME proportions except for 16:0. However, the hypothesis that bat sebaceous FAME may provide a model predicting species susceptibility to WNS was not supported by the present analysis. FAME profiling partially differentiated species, but it does not predict species according to known susceptibilities to WNS or current taxonomic status. While FAME profiling is used extensively to characterize microbial communities, mammalian FAME profiles may be too similar for taxonomic purposes [33]. Dominant FAME include 16:0, 18:0, and 18:1 as previously reported [18]. FAME profiles were similar among all species tested; however, 20:0 is absent in evening bats. Evening bats have higher PUFA percentage than other bats [11.7 %; however, not significantly higher than C. rafinesquii (9.5 %), L. noctivagans (8.2 %), P. subflavus (10.7 %), and M. austroriparius (9.9 %)], which may contribute to their oily texture. The 16:1 and 18:2 FAME was in lower percentage in most species susceptible to contracting WNS [i.e., 16:1, P. subflavus (1.4 %), M. septentrionalis (1.6 %), M. leibii (1.1 %), and M. grisescens (1.2 %); 18:2, M. lucifugus (6.2 %), M. leibii (6.7 %), and M. grisescens (4.4 %)]. Sapienic acid 16:1n-10 is present in high abundance on human skin and observed to be highly antimicrobial and defends against colonization of Staphylococcus aureus [19, 34]. Antifungal properties for 18:2 FFA have also been observed in the growth inhibition of P. destructans (Pannkuk data not shown). However, M. lucifugus (the species
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most heavily affected by WNS) had higher proportions of 16:1 (3.9 %) than other bats species. L. cinereus (not affected by WNS) had lower proportions of 16:1 (1.5 %). For 18:2, two bat species affected by WNS [P. subflavus (10.7 %); M. austroriparius (9.9 %)] had higher amounts than other species and Lasiurine bats had lower amounts (L. borealis and L. cinereus; 6.8 and 4.7 % respectively). Knowledge on the bat integumentary lipidome has increased greatly. Bat wing/sebaceous gland morphology [35, 36], broad lipid class of sebaceous secretions [17], individual lipid molecule variation among body site [30, 37], and stratum corneum lipid composition [23] have been described. To proceed with lipid biomarker identification utilized in assessing WNS progression and disease state, high throughput liquid chromatography (LC) tandem MS methods should be developed for more sensitive and broad scale lipid analysis of bat integument [37] and differentiated from fungal lipids [38]. Also, biases from lipid types collected by SebutapeÒ should be determined if this method is to be applied to broad scale ecological studies. To achieve these objectives, lipid types detected on the mammalian host vs. P. destructans must be determined. These baseline data on fungal and bat lipids will provide a framework to facilitate further investigations determining lipid biomarkers and explaining interactions in a complex host/pathogen system. Acknowledgments This project was funded by the Arkansas State Wildlife Grant, the National Speleological Society, the graduate program of environmental science at Arkansas State University (ASU), Bat Conservation International, and the Center for North American Bat Research and Conservation at Indiana State University. Laboratory assistance was provided by K. Arter and H. Southe (Arkansas State University). Samples were collected by C. Gerdes (Missouri State University), T. Divoll (Biodiversity Research Institute), and P. Jordan (Arkansas State University). We thank the ecotoxicology research facility (Arkansas State University; J. Bouldin and T. Woodruff) for assistance with GC/MS (NSF Grant #1040466).
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