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3 1066 Bogue Street, Room 426, Department of Horticulture, Michigan State University, East Lansing, MI 48824, firstname.lastname@example.org, 517 353 0409
4 332 Natural Resources, Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, email@example.com, 517 353 5485
The sea lamprey (Petromzons marinus) is an invasive ectoparasite of large-bodied fishes that adversely affect the fishing industry and ecology of the Laurentian Great Lakes. Lamprey tissue, in particular the skin, contains naturally aversive compounds that constitute a conspecific alarm cue. The goal of this project was to identify and characterize potential repellent compounds for potential use in the management of sea lampreys in the Great Lakes region. We conducted bioassay-directed isolation and purification of compounds from the aqueous ethanolic extract of sea lamprey skin; the material that has proven effective in several previous laboratory and field examinations of the alarm cue. The average body weight of a sub-adult migratory sea lamprey is 250 g, which includes 36 g of skin (14.4%). We executed a sequential extraction of the lyophilized whole sea lamprey with hexane, ethyl acetate, methanol and water afforded extracts containing compounds of varying polarity. Laboratory bioassays revealed that only methanolic and water extracts showed sea lamprey deterrent activity. Based on the assay results and several other extraction methods, we simplified the extraction procedure to soxhlet extraction of sea lamprey skin with aqueous ethanol (80:20 ethnaol:water). The lipophilic extracts showed limited deterrent activity in the laboratory. Because lipids are sometimes important carriers for semiochemicals, we completely characterized the lipophilic compounds in the whole sea lamprey extract and showed that sea lamprey contained approximately 8.5% of its total body weight as lipophilic compounds. The lipid compounds were characterized as cholesterol esters, tri- and di-glycerides, cholesterol, free fatty acids and minor amounts of plasticizers. The free fatty acids 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 individual sea lamprey skin. Publication of these results in PLOS ONE (Dissanayake et al. 2016) was the first report of the characterization of all lipophilic constituents in sea lamprey including the cholesterol esters. In order to isolate and characterize alarm cue constituents, we focused on the extracts of sea lamprey skin prepared by soxhlet extraction using aqueous ethanol (80:20 v/v) that showed the highest behavioral reactivity in laboratory tests. The resulting crude extract, dissolved in water and partitioned with chloroform, yielded about equal amounts of chloroform-soluble and water-soluble fractions. Chromatographic analyses revealed that the chloroform-soluble fraction was identical to the components in the lipid soluble fraction as published in Dissanayake et al. 2016. Based on activity profiling, the repellent activity was maximal in the water-soluble fraction. Therefore, this modified extraction and fractionation method successfully partitioned the active sea lamprey deterrent compound(s) into a single fraction that is free from lipid-soluble compounds. The behaviorally active water-soluble fraction was subjected to repeated chromatographic fractionation and purifications to yield numerous sub-fractions. These fractions were assayed in the laboratory for deterrent activity and results indicated varying degrees of sea lamprey deterrence by a number of sub-fractions. The active sub-fractions, after repeated HPLC purifications, yielded 20 pure compounds to date (several minor components, by mass, remain to be structurally elucidated). Spectroscopic (NMR and MS) experiments helped to chemically identify these pure isolates. The major components in the active water-soluble fraction identified to date are 14 amino acids (arginine, valine, leucine, isoleucine, tyrosine, histidine, glutamic acid, phenylalanine, tryptophan, threonine, asparagine, methionine, cysteine, and glycine) and 6 nitrogenous compounds (creatine, hypoxanthine, inosine, adenine, xanthine, and adenosine). Results indicated that the active water-soluble fraction consisted of primarily creatine (700 mg/g), heterocyclic nitrogen compounds (43 mg/g) and free amino acids (184 mg/g), respectively. Among the free amino acids characterized in our study, essential amino acids constituted 130 mg/g of the water-soluble fraction. Free amino acids isolated from the water-soluble fraction composed of arginine, phenylalanine, threonine, and asparagine 39, 27, 26 and 24 mg/g, respectively. This is the first report of the chemical characterization of nitrogenous constituents in the skin of a migratory-phase sea lamprey. Several pure isolates (12 compounds) from the active water-soluble fraction were tested in the laboratory raceway assay and showed varying degrees of deterrent activity. Among these tested pure compounds, the amino acids isoleucine and tyrosine exhibited good activity while phenylalanine showed moderate repellent activity against the migratory sea lampreys. Similarly, nitrogenous compound, hypoxanthine also exhibited good repellent activity against the migratory-phase sea lampreys. It is important to point out that there are additional active compounds yet to be isolated from several of the active sub-fractions from the water-soluble fraction of the sea lamprey skin extract.