**ABSTRACT
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2 1066 Bogue Street,
Room 420, Department of Horticulture, Michigan State University, East Lansing,
MI 48824, nairm@msu.edu, 517 353
0406
3 1066 Bogue Street, Room 426, Department of
Horticulture, Michigan State University, East Lansing, MI 48824, amila@msu.edu, 517 353 0409
4 332
Natural Resources, Department of Fisheries and Wildlife, Michigan State
University, East Lansing, MI 48824, mwagner@msu.edu,
517 353 5485
December 2018
ABSTRACT:
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.