Chemical Characterization of Lipophilic Constituents in the Skin of Migratory Adult Sea Lamprey from the Great Lakes Region

The sea lamprey (Petromzons marinus) is an invasive ectoparasite of large-bodied fishes that adversely affects the fishing industry and ecology of the Laurentian Great Lakes. Lipid content in the whole sea lamprey and muscles, liver and kidney of metamorphosing larval stages has been reported. Similarly, the fatty acid profile of the rope tissues of sexually-mature male sea lampreys has also been reported. The average body weight of a sub-adult migratory sea lamprey is 250 g, which includes 14.4% skin (36 g). Our preliminary extraction data of an adult sea lamprey skin revealed that it contained approximately 8.5% of lipophilic compounds. Lamprey skin is home to a naturally aversive compound (an alarm cue) that is being developed into a repellent for use in pest management. As part of an ongoing investigation to identify the chemical structure of the sea lamprey alarm cue, we extracted the skin with water and methanol, respectively. The methanolic extract (1.55%) contained exclusively lipophilic compounds and did not include the alarm cue. We chemically characterized all compounds present in the methanolic extract as cholesterol esters (CE), tri- and di-glycerides (TG and DG), cholesterol, free fatty acids (FFA) and minor amounts of plasticizers. The free fatty acids fraction was composed of saturated (41.8%), monounsaturated (40.7%) and polyunsaturated (17.4%) fatty acids, respectively. The plasticizers characterized were phthalate and benzoate and found to be 0.95 mg and 2.54 mg, respectively, per adult sea lamprey skin. This is the first report of the chemical characterization of all the lipophilic constituents in the skin of sub-adult migratory sea lamprey. The CEs isolated and characterized from sea lamprey skin are also for the first time.


Introduction
Lampreys, along with hagfishes, represent the two extant groups of basal vertebrates. These most primitive cartilaginous fishes evolved from jawless vertebrates more than 400 million years ago and include several parasitic species [1,2]. Among these the sea lamprey (Petromzons marinus) has become a critically important model species for the study of behavior, developmental biology, skin that may prove important in the bioenergetics of migration and responses to climate change induced warming in its native and introduced ranges.

Materials and Methods
General procedures for chromatographic purification and spectroscopic analyses ACS reagent grade solvents were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) and used for all isolation and purification steps. Merck silica gel (60 mesh size, 35−70 μm) with particle size of 60 μm was used for preparative medium-pressure liquid chromatography (MPLC). Silica gel plates (250 μm; Analtech, Inc., Newark, DE, USA) were used for preparative thin-layer chromatography (TLC). After developing, TLC plates were observed under UV light at 254 and 366 nm in a Spectroline CX-20 ultraviolet fluorescence analysis cabinet (Spectroline Corp., Westbury, NY, USA) and sprayed with 10% sulfuric acid solution. NMR spectra were recorded on 500 MHz (Varian Unity ±500, 1 H NMR) and 125 MHz (Varian Unity ±500, 13 C NMR) VRX instruments. ESIMS spectra were recorded on a Waters Xevo G2-S Q-TOF LC mass spectrometer (Waters Corporation, Milford, MA, USA) and GCMS on Thermo DSQ-II GC/single quadrupole mass spectrometer (Thermo Electron Corporation, Austin, TX USA).

Collection of adult migratory sea lamprey and its skin samples
We obtained 429 live, actively migrating male and female sea lampreys from the U.S. Fish and Wildlife Service (USFWS) in May and June 2015. The lampreys were captured in traps placed near dams in two tributaries to Lake Huron (the Cheboygan and Ocqueoc Rivers) and one tributary to Lake Michigan (Manistique River), in Michigan, USA. Following capture, the subjects were transported to the Hammond Bay Biological Station (Millersburg, Michigan, USA) in aerated live-wells and placed into 1000 L holding tanks that received a continuous flow of fresh Lake Huron water (100% exchange every 2 h). Prior to removal of the skin, each subject was euthanized via cervical dislocation with a scalpel. A single incision was made around the circumference of the body immediately anterior to the posterior gill opening. The skin was peeled from anterior to posterior in a single piece, cleaned of any muscle tissue, and rinsed for several minutes in distilled water. The skins were placed into plastic bags and frozen at -20˚C until use in the extraction protocol. All procedures for lamprey maintenance, euthanasia, and processing were approved by the Michigan State University Institutional Animal Care and Use Committee (permit # AUF 01/14-007-00).

Hydrolysis and methylation of triglycerides and GCMS analyses of resulting fatty acid methyl esters
An aliquot of (1 mg) sample was stirred with KOH in MeOH (3M, 3 h), acidified with HCl and evaporated under vacuum. The resulting fatty acids were then methylated with CH 2 N 2 separately to afford fatty acid methyl esters, according to the reported procedure [22]. The methyl esters thus obtained from samples were analyzed separately by GC using a capillary column, Agilent J&W VF-5ms GC Column, 30 m x 0.25 mm, 0.25 μm film thickness attached to a 10 m EZ-Guard column with a 7-inch cage. The conditions for the analyses were 1 μL sample dissolved in chloroform, helium carrier gas at a flow rate of 1.5 mL/min and temperature gradient with an injector port temperature at 40˚C, held for 1 min, raised to 160˚C at a rate of 40˚C and then raised to 210˚C at a rate of 3˚C followed by raising the temperature up to 250˚C at a rate of 40˚C. Triplicate GC analyses were preformed and the results expressed in GC area percent as mean ± SD. Chromatographic peaks were identified by comparing retention times with a standard mixture containing 37 fatty acid methyl esters (Supelco 1 37 Component FAME Mix, 47885-U, Bellefonte, PA, USA).

Methylation of FFA and GCMS analyses of resulting fatty acid methyl esters
Free fatty acids were methylated with CH 2 N 2 to afford corresponding fatty acid methyl esters, according to the reported procedure [22].

Extraction of whole sea lamprey and skin
Frozen sea lamprey skins 1.88 kg (180 skins) were cut in to small pieces and sequentially extracted with water (2 L, 3x) and methanol (2 L, 3x). Water extract was lyophilized to yield a water-soluble powder (32 g). Evaporation of methanol under vacuum afforded an oily extract (29.2 g). Similarly, a whole sea lamprey (frozen, 292 g) was sliced and sequentially extracted with water (500 mL × 3, 5 h) and MeOH (500 mL × 3, 5 h). Water extract was lyophilized to yield a water-soluble powder (12 g). Methanolic extract was partitioned with hexane and evaporation of hexane under vacuum afforded the total lipids (21.4 g). Also, whole frozen sea lampreys (5.09 Kg) were cut to small pieces and lyophilized (1.37 Kg). An aliquot of the lyophilized whole sea lamprey (399 g) was sequentially extracted with hexane, ethyl acetate, methanol and water. Removal of solvent afforded 118.9, 4.85, 45.6 and 10.2 g of solvent-free extracts, respectively. The hexane and ethyl acetate extracts contained the total lipids.
Lipid components in the hexane extract of the whole sea lamprey were identified by 1 H NMR spectral data and showed it as a mixture of lipids. It contained mixtures of glycerides and FFAs that were identical to the lipid components characterized in the methanolic extract of sea lamprey skin ( Figures A-B in S3 File). Characterization of fatty acid composition was achieved by GCMS analyses of the methylated products from its hydrolysis [22]. GCMS Data revealed that fatty acid profiles were identical to the lipids obtained from whole sea lamprey and its skin separately.

Results
The chemical identity of all pure compounds isolated from sea lamprey skin were determined by 1 H-and 13 C-NMR and MS analyses. Identity of FFA was achieved by its methylation using   [31]. In addition, the NMR spectra of compound 1 showed proton and carbon signals with chemical shifts that were characteristic of long chain fatty acid esters. The fatty acid ester was assigned at C-3 of the cholesterol moiety on the basis of HMBC correlations observed between C3 (δ C 73.6) to C1 0 (δ C 173.3) (Figure D in S1 File). Compound 1 was hydrolyzed and methylation of the resulting product afforded the corresponding fatty acid methyl ester [22]. The molecular ions  at m/z 270 (methyl palmitate) in its HRESIMS further confirmed the identity of the fatty acid moiety in 1 and its proposed structure (Fig 1) Fig 1) (Figure K in S1 File). In addition to signals for a cholesteryl moiety, the NMR spectra of compound 2 showed proton and carbon signals with chemical shifts that were characteristic of long chain fatty acid esters with one unsaturation (δ C 129.7, 129.8 and δH 5.32-5.36) (Figures G-J in S1 File). The fatty acid ester was assigned at C-3 of the cholesterol moiety on the basis of HMBC correlations observed between C3 (δ C 73.7) to C1 0 (δ C 173.3) (Figure J in S1 File). Compound 2 was hydrolyzed and methylation of the resulting product afforded the corresponding fatty acid methyl ester [22]. GCMS and HRESIMS analyses of the resulting fatty acid ester gave molecular ions at m/z 270 (methyl oleate) and further confirmed the proposed structure of 2 (Fig 1) [24].
As in the case of compounds 1 and 2, compound 3 was also a colorless oil. It showed the molecular formula of C 45 Fig 1) (Figure Q in S1 File). In addition to signals for a cholesterol moiety, NMR spectra of compound 3 showed proton and carbon signals with chemical shifts that were characteristic of long chain fatty acid esters with four degrees of unsaturation (δ C 127.5-129 and δH 5.28-5.42) (Figures M-O in S1 File). The fatty acid ester moiety was assigned at C-3 of the cholesterol moiety on the basis of HMBC correlations observed between C3 (δ C 73.7) to C1 0 (δ C 173) (Figure P in S1 File). Compound 3 was hydrolyzed and methylation of the resulting product afforded the corresponding fatty acid methyl esters [22]. GCMS and HRESIMS analyses of the resulting fatty acid ester gave molecular ions at m/z 318 (methyl arachidonate) and further confirmed the proposed structure of 3 (Fig 1) [25].
Compound 4 was also a colorless oil and showed the molecular formula of C 45 Fig 1) (Figure W in S1 File). In addition to signals for a cholesterol moiety, NMR spectra of compound 4 showed proton and carbon signals with chemical shifts that were characteristic of long chain fatty acid ester with five degrees of unsaturation (δ C 126.9-129.1 and δH 5.24-5.40) (Figures S-U in S1 File). This fatty acid ester moiety was assigned at C-3 of the cholesterol moiety on the basis of HMBC correlations observed between C3 (δ C 73.9) to C1 0 (δ C 173) (Figure V in S1 File). Compound 4 was hydrolyzed and methylation of the resulting product afforded the corresponding fatty acid methyl esters [22]. GCMS and HRESIMS analyses of the resulting fatty acid ester gave a molecular ion at m/z 316 (methyl eicosapentaenoate) and further confirmed proposed structure of 4 (Fig 1) [26].
Compound 8 was identified as 1,3 di-substituted glycerol based on the characteristic signals observed for a tertiary hydroxyl group (C-2 δ C 68.4, δ H 4.09) in its H-NMR spectrum [27,28]. The chemical shift of the carbonyl group in its 13 C-NMR at δ C 173.9 revealed that the glycerol moiety was substituted with identical fatty acids at 1 and 3 ( Figures R-S in S2 File). Hydrolyses and methylation [22] of the resulting products from compound 8 afforded a single fatty acid methyl ester. The GCMS analysis of this fatty acid ester gave molecular ion at m/z 296 and confirmed it as methyl oleate and further confirmed the identity of compound 8. As in the case of compound 8, based on NMR and GCMS analyses, compound 9 was identified as 1,2 di-substituted glycerol. Its NMR spectra showed C3-position of the glycerol moiety at δ H 3.72 and δ C 61.5 and carbonyl group at C-1 0 (δ C 173.4). This conformed that the glycerol was substituted with identical fatty acids at 1 and 2 [27,28] (Figures T-U in S2 File). Hydrolyses and methylation of compound 9 afforded only one fatty acid methyl ester [22]. GCMS analysis of this fatty acid ester (R t = 11.56 min) gave a molecular ion at m/z 296 and confirmed it as methyl oleate. Compound 10 was isolated as a white powder and identified as cholesterol [31] (Figure V in S2 File) (Fig 2).

Characterization of free fatty acids (FFA) in sea lamprey skin
Components of the fraction D were identified by the 1 H NMR spectral data as a mixture of FFAs ( Figure D in S3 File). The chemical characterization of the FFA was carried out by GCMS analyses. The observed peaks in the GC profile at R t = 5.35, 6.57, 6.90, 8.44, 8.81, 11.06, 11.43, 11.55, 12.26, 16.45, 17. 3). The relative abundance of these fatty acids in fraction D was calculated from the GC profile ( Table 2) (Figures E-N in S3 File).

Discussion
Liver and intestinal tissues from metamorphosing larvae of sea lamprey in the Great Lakes region have been reported to contain cholesterol ester and lipids, identified by GC analysis of  Fatty Acids and Esters in Migratory Adult Sea Lamprey Skin the tissue extract [32]. In this study, we report the isolation of pure cholesterol esters from adult sea lamprey skin extract and its characterization by NMR and GCMS methods ( Table 1). The acid moiety of the four cholesterol esters (CE) characterized in the extract from adult sea lamprey skin were palmitic (C16:0), oleic (C18:1), arachidonic (C20:4) and eicosapentaenoic (C20:5) acids. These CEs contributed to 2.53% of the total lipophilic constituents of adult migratory sea lamprey skin. Kaoa et al, reported an increase in monounsaturated CEs while decreasing the saturated CEs in liver and intestine during the larval metamorphosis of sea lamprey [32]. Our results on adult sea lamprey skin showed high amounts of monounsaturated cholesterol esters relative to saturated CEs, which is in support of the turnover of saturated CEs in larvae during metamorphosis to adult. In addition, our results also confirmed significantly high amounts polyunsaturated cholesterol esters (ω-6 C20:4, and ω-3 C20:5) compared to metamorphosing larvae, where trace amounts or absence of polyunsaturated cholesterols reported from liver and intestine [32]. During metamorphosis, larvae of sea lamprey begin to store significant amounts of triglycerides in kidney, liver, fat column, subcutaneous tissues and myosepta as energy reserves for migration, which requires maximum energy input [33,34]. Our lipid analysis of sea lamprey skin showed 30.3% of triglycerides in the total lipophilic compounds isolated. Among this total triglyceride mixture, 1,3-di(cis-9-hexadecenoyl)-2-hexadecanoyl-glycerol (compound 5) was found to be the major component (Fig 2). Apart from the major triglyceride, compound 5, other triglycerides characterized from the skin extract were glycerol esters of SFAs (C14:0 and C16:0), MUFAs (C16:1 and C18:1), and polyunsaturated fatty acids (PUFAs) (ω-6 C20:4, ω-3 C20:5, and ω-3 C22:6) (Fig 2, supplemental data for spectroscopic and chromatographic data). Significant amounts of free cholesterol and diglycerides of C18:1 ω-9 acids, 20.1 mg and 5.6 mg per animal respectively, have also been isolated and characterized along with TGs from the migratory sea lamprey skin (Fig 2). Hydrolysis of CEs and TGs could result in the production of these compounds.
Like teleost fishes, triglyceride composition in sea lamprey depends on the lipid metabolism, nutritional status, environmental (thermal), and physiological (hormonal) factors [35]. Changes in triglyceride composition have been reported on several parasitic sea lamprey species such as Mordacia mordax [36], Geotria australis [37] and landlocked parasitic sea lamprey Petromzons marinus [38]. Therefore, composition of the triglycerides in sea lamprey depends on the migratory state and the specific organ of the animal. Nevertheless, the adult migratory sea lamprey skin contain high amounts of triglycerides of PUFAs (ω-6 C20:4, ω-3 C20:5, and ω-3 C22:6), which are essential nutrients. As reported in the case of larvae [33,34], these triglycerides of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) may serve as essential energy pool via lipid metabolism during the migration for the adult sea lamprey.
Two synthetic compounds that are lipid soluble and isolated along with TGs from sea lamprey skin were a phthalate and a benzoate. Recently, parabene derivatives, widely used as consumer product preservatives and pharmaceuticals [39,40], have been reported to accumulate in fish, birds and bears in United States [41]. The phthalate, bis-(2-ethylhexyl) terephthalate (compound 6) [29] and benzoate, diethylene glycol dibenzoate (compound 7) [30] isolated from the adult migratory sea lamprey skin (Fig 2), 0.95 mg and 2.54 mg per animal respectively, are common plasticizers. Phthalate has been rated as priority pollutant by USA Environmental Protection Agency [42,43]. Its use has been restricted in European Union as well. Benzoate plasticizers are introduced as an alternative to phthalate plasticizers due their low toxicity and high rate of biodegradations [43,44]. However, recent reports indicate that diethylene glycol dibenzoates (such as compound 7) metabolizes to monobenzoates, which exhibit higher toxicity than dibenzoates [45][46][47][48] and accumulate in tissues. Bioaccumulation of these plasticizers in sea lamprey could be attributed to its infaunal larval stage, or the parasitic feeding stage where it targets larger, older individuals that are enriched in environmental pollutants [49]. Nevertheless, accumulation of these plasticizers in the sea lamprey skin provides good indication of the pollution levels in the Great Lakes and surrounding regions. Importantly, these pollutants will be deposited back into rivers at the conclusion of the reproductive migration, shunting the chemicals from the top of the lake food web to the base of the river food web.
The FAs in glycerides and FFAs in the total lipid extract of the whole sea lampreys were identical to FAs and FFAs characterized from the sea lamprey skin. Also, our findings revealed that frozen and lyophilized adult sea lamprey afforded 7.34% and 8.55% of total lipids, respectively. The skin from the frozen sea lamprey gave 1.55% of total lipids. The difference in total lipid yields from fresh and lyophilized sea lamprey was due to the extraction protocols. That is, lipids can be extracted from the lyophilized sample much better than from a fresh or frozen sample. Also, total lipid content in adult sea lampreys could vary and depends on the maturity of the animals, nutritional status, and environmental factors [35].
The fatty acid composition in fish oil from common fish species with high economical values such as mackerel (Rastrilliger kanagurta), [58] salmon (Salmo salar L), [59] sardines (Sardinella Brasiliensis), [60] Atlantic cod (Gadus morhua L) [61] and anchovy (Engraulis encrasicolus L) [62] have been reported. Although data on total lipids from whole fish is not available, salmon (Salmo salar L) [59] fillet reportedly contain about 7% of total lipids to its body weight. The relative amount of fatty acids in the fish oil from these fishes was comprised of SFAs, MUFAs and PUSAs at 23.2%, 45.6% and 26.2%, respectively, and identical to the lipids characterized in sea lamprey. MUFAs content reported in salmon fillet was similar to our finding of MUFAs in sea lamprey. The FA composition in fish was depended on the FA content of the feed and environmental factors. Studies on total fatty acid content in wild and farm raised salmon [59] indicated that fish diet can alter the fatty acid composition significantly. The total lipids in the farm raised salmon was found to be 12% of its body weight while wild salmon contained only 6% of total lipids. This significant increase in total lipid content in the farm raised salmons was attributed to the high vegetable oil content in its diet [59]. Sea lampreys feed only on blood of its host fish. Therefore, a comparison of its total lipid to the total lipid in fish species that feed on diets other than blood may not be relevant.
In summary, adult migratory sea lamprey skin contains significant amounts of triglycerides, cholesterol and free fatty acids relative to its skin mass. These lipid-soluble compounds are important sources of energy that may support migration, sexual maturation and spawning. In addition, we believe the large amount of TGs in sea lamprey skin may reduce frictional energy loss during swimming by increasing the water-repellency of the epidermis (an "easy to glide" benefit) that would also inhibit microbial attachment to the animal's surface, perhaps establishing a strong selective pressure for accumulation of these water-repellent triglycerides in its skin. The presence of plasticizer pollutants in sea lamprey skin is an indication of the accumulation of contaminants in the Great Lakes regional waterways. Therefore, further studies are warranted to determine the bioaccumulation and biotransfer pathways of these pollutants, as well as any human health concerns since these compounds are likely accumulated in salmon, trout and other fame fish in the Great Lakes basin.