Dedication: In memory of the late Lothar Jaenicke
Reptiles possess a number of macroscopic integumentary glands, many of which are unique to a particular order or suborder (see  for recent bibliography). Snakes (order Squamata, suborder Serpentes) possess a paired exocrine organ, called the scent gland, situated in the base of the tail and opening through two ducts at the margin of the cloacal orifice. Foul-smelling fluids typically are released from this gland when snakes are molested, thus inspiring the frequent suggestion that these secretions repel predators. Other proposed functions for the scent glands include the production by females of courtship deterrents against unpreferred suitors , .
Chemical analyses reveal that scent gland secretions consist chiefly of proteins , , . Lipids and other low-molecular components, including volatiles that impart the characteristic secretion odors of some species, are also present. Cholesterol , , , , , , which is a common tetrapod skin lipid, and carboxylic acids , , , , , ,  are widely documented in scent gland secretions. Nitrogen-containing compounds, including piperidone , amines , , and amides , , also have been indicated in some taxa. 1-O-Monoalkylglycerols have been reported in the secretions of the western diamondback rattlesnake (Crotalus atrox) .
The Middle American burrowing python (Loxocemus bicolor Cope) occurs in moist to dry forests from southwestern Mexico through Guatemala, Honduras, El Salvador, Nicaragua, and into Costa Rica (Figure 1). This fossorial snake feeds predominantly on small vertebrates and reptile eggs , . Loxocemus bicolor is the sole member of its genus and family, the Loxocemidae. Molecular studies indicate that this snake is a basal alethinophidean, closely allied with or properly placed within the Pythonidae , . We chose to investigate the scent gland secretions of L. bicolor because of its unique phylogenetic position in a clade of primitive constricting snakes. This secretion proved to be particularly diverse, consisting of more than 300 lipid components.
The analysis of the scent gland secretions revealed the presence of many peaks in the total ion chromatograms. The bad peak shapes leading to largely overlapping peaks suggested the presence of a high percentage of compounds with active hydrogens, such as alcohols and acids (Figure 2). Therefore, the samples were analyzed again after derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA, Figure 3). This procedure led to better peak separation and allowed the identification of the major compound classes. Over 300 components were observed. Cholesterol was a major constituent of all samples. The other components belonged to the following compound classes, in order of abundance: fatty acids, glyceryl monoalkyl ethers, and alcohols.
2.1 Fatty acids
The acids comprise most of the compounds in the secretions and showed the highest structural diversity. For GC/MS analysis of these acids, the crude extracts were treated with diazomethane to yield the corresponding methyl esters, which improved gas chromatographic behavior (Figure 3). Mass spectra of methyl esters (3) allow the detection of several structural features such as methyl groups near the ester group  and the number of double bonds in the chain. The retention index I can be used to identify methyl groups near the alkyl end of the chain . Nevertheless, internal methyl groups or double-bond positions cannot be determined, and other derivatives must be used to locate such groups. The best results are usually obtained by converting of the acids into 3-pyridiylmethyl esters 4 , but other methods such as the formation of dimethyloxazolines 5  or pyrrolidides 6  are also used.
A major problem in the analysis of the snake acids proved to be the large number of compounds present. This led to extensive coelution of compounds, making proper identification difficult. All three derivatization procedures mentioned were performed. It turned out that the analysis of both 3-pyridiylmethyl esters and pyrrolidides were necessary for proper identification, while dimethyloxazolines did not add additional information. To complement this approach, dimethyl disulfide derivatization was used to assign the position of double bonds in addition to 3-pyridiylmethyl esters.
As an example, the identification of a major methyl-branched acid, 14-methyloctadecanoic acid, and a major unsaturated acid, 11-icosenoic acid will be discussed in detail. The various spectra of their derivatives are shown in Figure 4 as examples. These spectra allow the localization of the methyl branch and the location of the double bond in the two compounds. While the spectrum of methyl 14-methyloctadecanoate (Figure 4A) is difficult to interpret a priori , the respective pyrrolidide (Figure 4C) lacks ion m/z 280 of the ion series [C4H8NCOCnH2n ]+. This ion series is formed by sequential alkyl cleavage along the chain. Similarly, the respective 3-pyridiylmethyl ester (Figure 4E) lacks ion m/z 318 of the ion series [C6H7NOCOCnH2n ]+. The intensities of the neighboring ions in these homologous series (m/z 266/294 and m/z 304/332, respectively) are enhanced. The gaps in the ion series indicate a methyl branch in the chain that can be located by the ions mentioned at C-14.
The localization of the double bond is more difficult but can also be achieved. The methyl ester (Figure 4B) mass spectrum does not give any hints on the localization of the double bond. The mass spectrum of the pyrrolidide shows 14 amu gaps between the methylene groups, but only 12 amu between m/z 252 and 264 (Figure 4D). Obviously, the double bond is near C-11 but cannot exactly be located. Somewhat better results are obtained with the 3-pyridiylmethyl esters (Figure 4E). The strong allylic cleavage ion at m/z 330 and the 12 amu gap between m/z 290 and 302 locates the double bond at C-11. Nevertheless, a good and clean spectrum is required for reliable identification. This was not always the case for the other acids of the secretion. The most reliable results were obtained by the mass spectra of dimethyl disulfide (DMDS) adducts (10) of the unsaturated methyl esters , . 11-Icosenoic acid was identified because the mass spectrum of the DMDS adduct of its methyl ester furnished prominent ions at m/z 245 [CH3SC10H20CO2CH3+], 213 [CH3SC10H20CO-CH3OH+] and 173 [C9H20SCH3+] . Two isomers, 12-icosenoic acid, characterized by m/z 259, 227, and 159, as well as 13-icosenoic acid, characterized by m/z 273, 241, and 145, were also present in the secretion.
The individual acids were identified by interpretation of the mass spectra of the derivatives as just discussed, according to well established rules , , , , , , , and by correlation with gas chromatographic retention indices I , . Over 100 acids were identified. The acids of L. bicolor chiefly consist of long-chain, unbranched, saturated, and unsaturated acids, as well as mono-, di-, and trimethyl-branched acids (Table 1). Major constituents are 13-docosenoic acid, docosanoic acid, 15-tricosenoic acid, 15-tetracosenoic acid, and 14-methylhexadecanoic acid. Methyl branching occurs predominately at C-4, C-10, C-12, and C-14. Double bonds prevail at the ω-9 position, a typical location for fatty acids of animal origin. A second double bond can occur in very long tetracosadienoic acids. The two acids F99 and F101 (Table 1) represent the two acid types present, those with remote double bonds and those with bishomoconjugated double bonds. Structures of minor amounts of similar acids could not be fully assigned.
Acids with both methyl branches and double bonds occur as well but in minor amounts. Even-numbered carbon chains are dominating, although odd-numbered acids also occur. 2,3-Dihydrofarnesoic acid is the only acid likely originating from the terpene biosynthetic pathway. No attempt was made to elucidate the configuration of the double bonds, but the common (Z)-configuration seems most likely. Structures of some of the acids are shown in Figure 5.
A general biosynthetic model explaining the diversity of the acids is shown in Figure 6. Fatty acids are biosynthesized starting from acetyl-coenzyme A (12) that is elongated by malonate (15) until a typical chain length is reached, e.g. stearic acid (13). Dehydrogenation by a ∆9-desaturase leads to oleic acid, (Z)-9-octadecenoic acid (16), a ω-9 unsaturated acid (Figure 6). Several variations of this pathway obviously occur during biosynthesis of the scent gland acids. Replacement of one malonyl-unit with methylmalonate (14) during chain formation leads to methyl groups in the chain. An additional ∆11-desaturase furnishes ω-7 unsaturated acids. The chain can be elongated by additional malonate units or biosynthesis can stop earlier, leading to shorter acids than C18. After elongation, a second desaturation by a ∆7-desaturase leads to dienoic acids. Finally, odd-numbered acids can be formed by using propionate-CoA (11) instead of 12 as chain starter, or alternatively, acids can be shortened by α-oxidation by one carbon. Combining all these elements of chain-length diversity from C12 to C24 including odd-numbered acids, desaturation on a few positions, and methyl groups near the acid group and in the middle/end of the chain leads to the extraordinary diversity in acid structures.
Although variation in the relative proportions of individual compounds occur, neither the acids nor other compounds appeared to be sex specific; the secretion looks remarkably similar for males and females (Figure 7). Furthermore, the composition of the gland does not vary greatly; many compounds were present in all samples analyzed, despite their different origin. It seems, therefore, that the composition of the secretion is relatively stable.
Glyceryl alkyl ethers in the scent gland secretions were identified by their mass spectra and the trimethylsilyl (TMS) derivatives 8. The mass spectrum of 1-O-tetradecylglyceride and the respective TMS-ether is shown in Figure 8. The mass spectrum of the latter features a base peak at m/z 205 [(CH3)3SiOCH2CHOSi(CH3)3+], the ion m/z 147 indicating the presence of two TMS groups , and a small M-15 ion , . The ion m/z 205 clearly indicates that the terminal alcohol is used in an ether linkage, as do the ions m/z 61 and 227 in the original spectrum. Over 40 ethers were present in the secretion in which a homologous series of unbranched, saturated ethers from C13 to C16 as well as 1-O-14-methylhexadecylglycerol dominated. They were accompanied by minor amounts of methyl-branched or unsaturated ethers with longer chains (Table 2 and Figures 8 and 9). The locations of branching points or double-bond positions were not determined; however, iso- and anteiso-branched compounds were identified based on established rules for calculating their gas chromatographic retention indices (I) in long-chain compounds , .
A homologous series of 1-alkanols were identified after derivatization with MSTFA. The chain length ranged from C13 to C24. Even-numbered alcohols were present in higher amounts than the odd-numbered alcohols, with increasing concentration according to chain length. No branched alcohols could be found; tetracosen-1-ol was identified as the only unsaturated alcohol.
Carboxylic acids evidently are ubiquitous scent gland products of snakes, having been documented in colubrids , boids , , , pythonids , elapids , viperids , , and leptotyphlopids . These compounds typically possess 2–26 carbon atoms and chiefly feature saturated or monounsaturated straight chains. Lower molecular weight acids (C4 and C5) with a single methyl branch , , hydroxypropanoic acid , , methylbenzoic acid , and phenylacetic as well as phenylpropanoic acids , ,  also have been documented in some species. The fatty acids of Loxocemus bicolor include some of the straight-chain compounds reported in other snakes, but distinctly contain more than 65 mono-, di-, and trimethyl-branched compounds, and many compounds with one, two, or three double bonds. The significance of this structural diversity is open to speculation. The Texas blindsnake (Leptotyphlops dulcis), another fossorial snake, discharges repellent cloacal fluids, including scent gland secretions, when attacked by ants . Blum et al.  suggested that fatty acids in the secretions of L. dulcis, which they identified as 13 straight-chain C12 to C20 compounds, act against ants as insecticides or by exploiting out-of-context semiochemical responses. Fatty acids of L. bicolor should be tested for similar allomonal properties against ants and other offending leaf-litter arthropods.
Glyceryl monoethers possessing n-alkyl residues ranging in carbon-chain length from 12 to 20, chiefly C14, C16, and C18 chains, were reported in the scent gland secretions of male and female western diamondback rattlesnakes (Crotalus atrox) . Young et al. ., however, failed to observe bands corresponding to this compound class in thin-layer chromatograms of the secretions of two pitvipers (Crotalinae), the eastern diamondback rattlesnake (C. adamanteus) and the Florida cottonmouth (Agkistrodon piscivorus conanti). Our analysis of L. bicolor affirms 43 glyeryl alkyl monoethers as scent gland products, revealing C13 to C24 straight-chain or methyl-branched alkyl chains. Glyceryl ethers, mostly showing saturated C15 to C22 n-alkyl chains, have also been observed in the femoral gland secretions of a lacertid lizard (Acanthodactylus boskianus) . These compounds may occur widely among squamate reptiles.
We observed straight chain alcohols in the scent gland secretions of L. bicolor ranging in carbon-chain length from 13 to 24. This compound class has not previously been described from snake scent glands. However, alcohols possessing chains of more than 30 carbons have been observed in extracts of the shed or intact epidermis of colubrid snakes , . Such long-chain compounds are among the nonpolar lipids that may contribute to the transepidermal water barrier of the epidermis. The significance of alcohols in the scent gland secretions is unclear.
4 Experimental part
Scent gland secretions were collected from three male and seven female snakes (total lengths=61–127 cm) maintained on rodents at the Memphis Zoo (TN, USA) and Vida Preciosa International, Inc. (Boerne, TX, USA). Most specimens were captured in Central America, possibly Honduras; two females were reared in captivity from these wild-caught individuals. Snakes were restrained while manual pressure was applied to the base of the tail. The emerging stream of scent gland fluids was directed into glass vials to which several milliliters of dichloromethane was added. Samples were kept frozen until analysis.
4.2 GC/MS analyses
GC/MS analyses were performed on an HP7890A GC connected to an HP5975C mass selective detector fitted with an HP-5ms fused silica capillary column (30 m, 0.22 mm i.d., 0.25 μm film, Agilent Technologies, USA). Helium served as carrier gas. Conditions were as follows: injection volume 1 μL, transfer line 300 °C, injector 250 °C, electron energy 70 eV. Linear retention indices were determined from a homologous series of n-alkanes (C8–C32).
N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (50 μL) was added to 100 μL of an extract, followed by heating for 1 h at 60 °C. Excess MSTFA and solvent were removed under a gentle stream of nitrogen to a volume of 5 μL. Finally, dichloromethane was added (100 μL) and the derivatized extract was analysed by GC/MS.
N-methyl-N-nitroso-p-toluenesulfonamide (0.41 M in 1:1 diethyl ether/diethylethylenglykol monoethyl ether, 1 eq.) was added to a 5-mL vial equipped with a Teflon-lined septum cap carrying a small teflon tube as gas outlet. The tubing was connected to a second vial serving as gas washer. This vial was empty and carried a second Teflon tube as gas outlet. This tube was connected to a third vial, carrying diethyl ether. The latter vial was cooled with ice. KOH (1.1 eq, 0.37 M solution in 1:1 methanol/water) was added to the first tube by a syringe. The produced yellow was trapped in the diethyl ether vial. Subsequently, the yellow solution was added to 100 μL of an extract until the gas formation ceased. The derivatized extract was analyzed by GC/MS. Caution: Pure diazomethane is explosive.
4.3.3 3-Methylpyridyl esters
About 100 μL dichloromethane extract was treated with 20 μL oxalyl chloride for 1 day in a 2-mL vial. Excess oxalyl chloride was removed by evaporation with a stream of nitrogen. One drop 3-pyridinemethanol and 100 μL dichloromethane were added. The mixture was heated for 1 h in a heating block at 60 °C, followed by GC/MS analysis.
About 100 μL dichloromethane extract was treated with 50 μL of a 9:1 mixture of pyrrolidine and pyridine in a 2-mL vial. After 1 h at room temperature, the 100 μL saturated NaHCO3 solution was added. The organic phase was removed, dried with molecular sieve, and analyzed by GC/MS.
4.3.5 Dimethyl disulfide derivatives
Dimethyl disulfide (DMDS) adducts were obtained by stirring equal amounts of freshly distilled DMDS and 100 μL of a natural extract with 5 μL of a 5% I2-solution in diethyl ether at 60 °C overnight. Then excess I2 was removed with saturated aqueous Na2S2O3. The organic phase was separated and the aqueous phase was extracted twice with 100 μL pentane. The combined organic phases were dried with NaCl and reduced to a volume of 50 μL. The derivatized extract was then analyzed by GC/MS.
S. Reichling (Memphis Zoo, TN, USA) and D. and T. Barker (Vida Preciosa International, Inc., Boerne, TX, USA) made specimens available for our study.
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Published Online: 2017-03-15
Published in Print: 2017-07-14