In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.
Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates ("fractions") of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.
Reptiles produce lipids in the epidermis, either directly from skin cells or from discrete glands that are used in social communication, such as mate assessment and tracking, territoriality, and intra- and interspecific recognition1,2,3,4. The isolation of these skin lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life2,3,4. Further, many reptiles, especially squamates (lizards, snakes), are invasive species of concern in sensitive ecosystems, and the development of pheromone-based lures to improve trapping and removal is ongoing5,6. The impermeability of reptile skin facilitates the extraction of the lipids present to obtain relatively pure extractions of a potentially robust source of chemical signals. The principle steps for quantifying reptile skin lipids in the described protocol include extraction, total lipid determination, and fractionation via column chromatography1,6,7. The methods have been used routinely as they yield bioactive isolates that explain much about mate choice and selection, especially in snakes2.
Skin lipids can be extracted from either living skin, shed skin, or dead whole reptiles, using nonpolar or polar organic solvents1,7,8,9. It should be noted that museum specimens stored in solvents such as ethanol are not optimal for the extraction of skin lipids, and only fresh or freshly frozen carcasses should be considered as possible sources for extraction. Skin lipids are inert, which makes them stable on the surface of the skin and easy to extract7. In their signaling roles in reptile ecology, skin lipid cues are often deposited in harsh environments, but because of their robust chemical properties, such cues can retain their information value over long periods of time10,11,12. The extraction process solubilizes the lipids, using a nonpolar solvent (e.g., hexane, benzene, toluene) over an hours-long soak, followed by the evaporation of the solvent, to leave a measurable mass of lipid extract7,8. Skin lipids are highly miscible in nonpolar solvents, and a wide range of hydrocarbons can be extracted from a similarly diverse array of sources.
Fractionation is more laborious than extraction but serves to separate the total lipid extract into specific fractions via column chromatography, to aid in the purification and possible identification of the compounds therein1,6,7,8. The total lipid extract is bound to a substrate-based column, and then, individual eluates ("fractions") of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture that have a common polarity6,7,8. In lipid chromatography, the fractions progress in polarity at some standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent (typically expressed as a percentage: 0%, 2%, 4% ether, etc.)6,7,8. Though methods like thin-layer chromatography (TLC) can be used to separate lipids in a mixture and are simpler, column chromatography is preferred because it uses a closed system, is easy to control, can separate more concentrated mixtures, and is compatible with multiplexing for efficiency. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. In many research projects involving the isolation of chemical cues, the ultimate goal is to effect change in the receivers exposed to those cues. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein1,2,6,7. In basic biological research, for example, bioassays using specific fractions can reveal to researchers that a purified source of pheromones has been isolated, and then, methods for the identification of the target compounds can be pursued. From a wildlife management perspective, identification may not be the goal, and instead, the active fraction could be used in the field to attract conspecifics to traps or inhibit mate tracking in the nonnative habitat13,14.
The extraction of skin lipids in reptiles can be applied to living or dead skin, in addition to shed skin, which offers versatility in the experimental application of this technique. Further, extractions of skin lipids can be done in the field, to enable a dynamic application of the method to a wide range of biologists2,13. Extraction of skin lipids is simple; therefore, it is easy to scale up extractions as needed per experiment or design, and practitioners need not have significant expertise to execute the methods. The only limiting factors for scaling up are the availability of fume hood space, an abundance of clean, sealable glassware, and solvent storage space.
Skin lipid extract fractionation can be tailored to a researcher's needs and, therefore, has similar flexibility to lipid extraction. For example, neutral lipids can be eluted and then discarded, to result in target fractions that may purify compounds of interest or simplify bioassays. Fractionation can be performed at multiple scales within and across lipid samples. For example, multiple columns can be run simultaneously to make the process more efficient. Or, only a portion of a total lipid extract can be fractionated on a small column to, thus, spare reagents and time. Fractionation is primarily limited by the mass of the total lipid extract and the precision of the equipment available to the researcher. For example, if the researcher has a balance with a 10.0 mg precision, the determination of the fraction mass and, therefore, the accuracy of the sample preparation for the GC-MS analysis is significantly, if not completely, impeded. The same is true for the glassware. If the researcher has a large-volume column for fractionation but has a small total lipid mass to separate, the elution or separation of the compounds will progress but will require a significant wastage of solvents, reagents, time, and potentially, the target compounds themselves.
It is advised to perform a quality control check before determining the elution scheme, as seen in Table 2, and decide what fractions may be discarded. To confirm the elution of the desired lipids, a column can be run where each fraction, 1 – 15, is collected individually and then analyzed using GC-MS. In Figure 3, representative gas chromatograph traces show that methyl ketones from garter snakes only elute from the column in a specific fraction. By performing this quality control step, a modified elution scheme can be developed for a given species to ensure the maximum yield of the compounds of interest. Changes in the materials, such as the supplier or lot of the alumina or the age of the diethyl ether, will absolutely result in differences in elution that should be controlled for by performing a quality control test.
The techniques described are limited chiefly by the chemical nature of the cues that can be obtained. Primarily, these methods only allow researchers to isolate and separate long-chain lipids from the skin of reptiles. Many species of reptiles use airborne and/or proteinaceous cues as chemical signals, and the described methods are incompatible with isolating said cues. Further, nonpolar solvents will not extract aqueous cues from the surfaces of reptiles or sources of cue deposition (e.g., cage water, fecal matter, aquatic substrate) that may indeed contain abundant chemical signals. Appropriate methods for capturing these types of cues are available to researchers (e.g., solid-phase microextraction [SPME] for volatile cues and high-performance liquid chromatography [HPLC] for aqueous cues), although, like the methods described above, there is a technical learning curve.
Most importantly, the utility of the final chemical mixture to the researcher should guide the methods used. For example, if a researcher wants to know if a male focal animal can distinguish between the cues produced by male vs. female conspecifics in a targeted bioassay, extraction is the only method needed2,3. If the identification of sexually dimorphic compounds is the goal, however, the extract ought to be purified, to enable greater confidence in assigning identifications to specific molecules or groups of molecules via chemical analysis1,6,9,11. However, to even conduct chromatography with a lipid source, a significant mass of starting extract is required so that measurable fraction masses can be obtained; otherwise, the pooling of samples can be pursued but is not optimal14.
Future developments of this protocol include measures to utilize and adapt the procedure for more reptilian species. Additional noninvasive methods of extracting skin lipids are also being developed.
The authors have nothing to disclose.
The development of these methods, especially shed skin lipid extraction, occurred during cooperative agreements (14-7412-1061-CA, 15-7412-1155-CA, and 16-7412-1269-CA) between James Madison University (JMU) and the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS). M.R.P. acknowledges the contributions of the following students to the development of the shed skin methods: S. Patel (Washington and Lee University [WLU]), J. Zachry (WLU), R. Flores (JMU), J. Noll (JMU), and S. Ashton (JMU).
Powder-free Chloroprene Gloves | Microflex | NEC-288 | See "1.1 Shed Extraction set-up" (step 1.1.2 "gloves") |
Ohaus Adventurer Precision Balance | Ohaus | AX622 | See "1.1 Shed Extraction set-up" (step 1.1.3 "balance") |
Freund Container Ball 16oz Mason Jar & Lid | Ball | NC9661590 | See "1.1 Shed Extraction set-up" (step 1.1.5 "mason jar") |
Hexane, Mixtures of Isomers | Sigma-Aldrich | 650544 | See "1.1 Shed Extraction set-up" (step 1.1.6 "hexane") |
Hi/Lo Write-On Temperature Tape | Electron Microscopy Sciences | 5029927 | See "1.1 Shed Extraction set-up" (step 1.1.8 "tape") |
Single-Neck Round-Bottom Flask, capacity 500 mL | Sigma-Aldrich | Z414514 | See "2. Extraction" (step 2.1.2.4 "500 mL") |
Single-Neck Round-Bottom Flask, capacity 100 mL | Sigma-Aldrich | Z414492 | See "3.2 Fractionating" (step 3.2.4 "100 mL") |
Cork Flask Support Ring | Sigma-Aldrich | Z512419 | See "2. Extraction" (step 2.1.2.1 "cork support ring") |
Accu-jet Pro Pipette Controller | Sigma-Aldrich | Z671533 | See "2. Extraction" (step 2.1.1.3 "electric pipette controller") |
Disposable Individually Wrapped Glass Serological Pipets, 10 mL | Pyrex | 13-666-7E | See "2. Extraction" (step 2.1.1.3 "10 mL pipette") |
Rotavapor R II | Buchi | 2422A0 | See "2. Extraction" (step 2.1.2.1 "rotary evaporator") |
Elliptical Bump Trap | Chemglass Life Science | 501215241 | See "2. Extraction" (step 2.1.2.1 "bump trap") |
7 mL Vials, Screw Top, Clear Glass | Supelco | 27151 | See "1.1 Shed Extraction set-up" (step 1.1.11 "7 mL vial") |
7 mL Vial Screw Cap, Solid Top with PTFE Liner | Supelco | 27152 | See "1.1 Shed Extraction set-up" (step 1.1.11 "cap") |
22 mL Vials, Screw Top, Clear Glass | Supelco | 27173 | See "1.1 Shed Extraction set-up" (step 1.1.11 "22 mL vial") |
22 mL Vial Screw Cap, Solid Top with PTFE Liner | Supelco | 27174-U | See "1.1 Shed Extraction set-up" (step 1.1.11 "cap") |
Excellence XS Analytical Balance | Mettler-Toledo | XS205DU | See "2. Extraction" (step 2.1.2.1 "balance") |
5 3/4" Disposable Glass Pipette | Fisherbrand | NC0418555 | See "1.1 Shed Extraction set-up" (step 1.1.11 "pipette") |
Chromatography column with PTFE Stopcock Assembly | Kimble-Chase | 17810-19300 | See "3.1 Preparing the column" (step 3.1.1 "small glass column") |
Cast-Iron L-Shaped Base Support Stands | Fischerbrand | 11474207 | See "3.1 Preparing the column" (step 3.1.1 "support stand") |
3-Prong Dual Adjust Nickel-Plated Zinc Clamp | Troemner | 2300203 | See "3.1 Preparing the column" (step 3.1.1 "clamps") |
Clamp Regular Holder | Fischerbrand | 05754Q | See "3.1 Preparing the column" (step 3.1.1 "clamp holder") |
Sand,Washed and Dried | Macron Fine Chemical | MK-7062-212 | See "3.1 Preparing the column" (step 3.1.5 "sand") |
Alumina, Neutral | Sorbtech | 15740-5 | See "3.1 Preparing the column" (step 3.1.7 "alumina"); only known manufacturer in the US |
Narrow-Neck Heavy-Duty Glass Erlenmeyer Flask, 1000mL | Pyrex | 4980-1L | See "3.1 Preparing the column" (step 3.1.6 "Erlenmeyer flask") |
Single-Neck Round-Bottom Flask, capacity 250 mL | Sigma-Aldrich | Z100684 | See "3.1 Preparing the column" (step 3.2.4 "250 mL round bottom flask") |
Calibrated Chromatography Column with Solvent Reservoir | Sigma-Aldrich | Z560553 | See "3.2 Preparing the column" (step 3.2.1 "large glass column") |
Ethyl Ether Anhydrous | Fisher Chemical | E138500 | See "3.2 Fractionating" (step 3.2.5 "ether") |
Alconox Detergnet | Sigma-Aldrich | 242985 | See "4. Cleaning" (step 4.2 "Alconox") |
Acetone (Certified ACS) | Fisher Chemical | A18P-4 | See "4. Cleaning" (step 4.4 "acetone") |