Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey

From Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey by Sorenson, et al. (1)

Pheromones

Pheromones are most commonly thought of as the chemicals that attract a person of the opposite gender.  However, they are also used to elicit different social responses, which can be seen in the activity of many aquatic vertebrates.  Many aquatic vertebrates must migrate in order to find suitable spawning locations, and this migration occurs due to an innate attraction to species-specific pheromones.

The study of pheromones and their detection by animals is important for several reasons.  It will provide a better understanding of some of the methods by which animals interact socially with one another, locate food, and avoid predators (2).  It will also provide insight into biological processes regulating the human body.  For example, some pheromones have been found to have an effect on puberty, female cycles, hormone regulation, and attraction between males and females (3).

The sea lamprey is an ideal model organism for the study of pheromones.

Sea Lamprey (Petromyzon marinus)

The sea lamprey, also known as Petromyon marinus, is a jawless vertebrate found in the Atlantic Ocean, the Mediterranean Sea, and the Great Lakes.  The sea lamprey preys on fish by suctioning its mouth onto the skin of a fish and sucking out its tissue and blood. They are aggressive predators that invaded the Great Lakes about 100 years ago and have been detrimental to fisheries there due to their preying and parasitic activity (4).  In fact, current research is being performed into the possibility of using certain pheromones to repel sea lampreys away from these areas (5).

Sea Lamprey Panic Response.

Sea lampreys are ideal model organisms for the study of pheromone-signaling because the life history of the organism is very well-understood.  First, it is in the larval phase in freshwater for 3-20 years.  Then it undergoes metamorphosis into the parasitic phase, which lives in the sea for one year.  It then enters into a sexually mature phase that lives only a few weeks in freshwater to spawn (6).  The sea lamprey also has a very intricate olfactory system, which can be studied in order to gain a better understanding of the chemical basis of the attraction to pheromones.  This is because the olfactory system contains an organ called the vomeronasal organ (VNO) that detects pheromones via a G-protein coupled receptor (7).

Sea lampreys and pheromones

Pheromones have been studied as a means of controlling sea lamprey population in places such as the Great Lakes, where they have become destructive towards other fish populations.  Pheromones are potent, specific, and inexpensive, making them ideal subjects for research in this area.

Data from the trapping of lampreys has shown that there is a strong correlation between the amount of larval lampreys living in a stream and the migration of adult lampreys towards that stream.  This indicates that lampreys might rely on a pheromone released by larval lamprey to direct them towards streams that are good habitats for spawning.  This makes sense because the presence of larval lamprey would indicate a suitable environment for growth.

Search for the migratory pheromone

It was found that adult lampreys were attracted to larval holding water, which is water taken from tanks containing sea lamprey larvae 12 hours after feeding. Sorensen, et al. sought to identify the principal compounds found in the migratory pheromone that attracted them to areas of high larval density.  They found that larvae released petromyzonol sulfate (PS) along with its precursor, allocholic acid (ACA) (8).  However, they also found that migratory adults were more attracted to larval holding water than water that just contained PS and ACA, indicating the presence of additional components in the migratory pheromone. Larval water was collected 12 hours after feeding because that is when the release of PS is greatest.

Larval lampreys release pheromones that attract adult lampreys

Isolation of the principal components of the migratory pheromone

The Sorenson, et al. group fractionated larval holding water using high-pressure liquid chromatography (HPLC), and they screened 20 fractions for olfactory activity using electro-olfactogram (EOG) recording.  In order to obtain EOG fractions, the olfactory epithelium of freshly captured adult sea lamprey were exposed to odorous stimuli.  Responses were captured using electrodes.  Five fractions had very significant olfactory activity, which can be seen in the figure below.

This figure shows the attraction of adult sea lamprey to fractions of larval holding water, with control water being tank water without lampreys

They then tested the five most active fractions using a two-choice maze, and they found that most pheromonal activity occurred in fractions 9-11.  The two-choice maze contained natural river water in which test odors were pumped down one side of the maze, while the other side was used as the control.  Sea lampreys were released in the maze, and the level of pheromonal activity was determined by the amount of time the lampreys spent in each side of the maze.

Responses of adult lamprey between larval sea lamprey extract or fractions of it versus control water.

They sub fractionated fractions 9-11 using electrospray ionization mass spectrometry (ESI-MS), which revealed three compounds with strong EOG activity.  The three compounds were then isolated so that their structures and activity could be studied.

Petromyzonamine disulfate (PADS)

Structure and biological activity of PADS, the most active component in the migratory pheromone.

PADS was the most potent of the three compounds, having behavioral and physiological activity at concentrations as low as 10^-13 M.  Mass spectrometry (MS) and proton NMR were used to deduce the structure.  It is a steroid containing two sulfates and at least one basic amine.  The structure was confirmed by comparing the proton NMR, C13 NMR, MS, and behavioral activity of the natural compound to a synthesized PADS molecule.  The proton NMR for its C20-C27 side chain was found to be almost identical to squalamine, which is a compound with strong antiangiogenic and antimicrobial activity produced by the dogfish shark.  It is an aminosterol like PADS, and the dogfish shark is an ancient organism like the sea lamprey, which indicates a possible biological connection between the two organisms.

Petromyzosterol disulfate (PSDS)

Structure and biological activity of PSDS, the second most active component in the migratory pheromone.

PSDS is the second most active component of the migratory pheromone, with a physiological response at 10^-13 M and a behavioral response at 10^-11 M.  It is a steroid containing two sulfates and an additional hydroxyl group. The structure was determined using an approach similar to the structural determination of PADS.

Petromyzonol sulfate (PS)

Structure and biological activity of PS, the least active component of the three in the migratory pheromone.

PS is the least abundant compound of the three in the migratory pheromone, with physiological activity at 10^-12 M and moderate behavioral activity at 10^-11 M.  It was discovered based on molecular mass, HPLC retention time, and proton NMR data.  There is a possible biosynthetic link between PS and 3-keto-petromyzonol sulfate, which is the male sea lamprey sex pheromone.

Application in freshwater bodies

The migratory pheromone works as a mixture, which was verified because PADS alone has less behavioral activity than the complete larval water extract.

Behavioral preferences of sea lampreys given the choice of whole larval extract or varying amounts of purified PADS

Each compound is detected by a different olfactory receptor.  It is likely that PADS and PSDS are present in higher concentrations than 10^-12 M in stream water, which is why adult lampreys are still attracted to stream water that has been found to be very dilute.  This type of dilution is found at the entrances to the Great Lakes, which explains the natural migration of sea lampreys to that area.

There is promising potential for the three compounds to be put to use in controlling the parasitic sea lamprey.  It has been shown that only a small amount of the compound would be required; therefore, trapping efforts could be enhanced at a low cost.  Since the pheromone PADS is also common to other lamprey species, it could be used to lead endangered lamprey species along the Atlantic and Pacific seaboards to locations where they could thrive.

Future research

These findings lead to several areas of potential future research, which could focus on the following areas:

  • Improvement of the efficiency of synthesizing PADS in order to allow for industrial production so that it could be used towards a lamprey control effort.
  • Confirmatory synthesis studies of PSDS.
  • Exploration of the possible biosynthetic link between PS and 3-keto-petromyzonol sulfate.
  • Exploration of the biosynthetic link between PADS and squalamine.
  • Tests to verify whether PADS and PSDS are actually present in higher concentrations than 10^-12 M in sea water.

Paper Critiques

  • The Sorenson, et al. group did not test the activity of PADS at 10^-11 M as they did with PSDS and PS.
  • Figure 1b is not explained well, making it hard to understand the attraction of the sea lampreys to the fractions over the control water.

References

  1. Sorensen, Peter W., et al. “Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey.” Nature Chemical Biology 1.6 (2005): 324-328.
  2. Wyatt, Tristram D. Pheromones and animal behaviour: communication by smell and taste. Cambridge University Press, 2003.
  3. Wysocki, Charles J., and George Preti. “Facts, fallacies, fears, and frustrations with human pheromones.” The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology 281.1 (2004): 1201-1211.
  4. “Sea Lamprey – Animals Town.” Sea Lamprey – Animals Town. Animals Town, n.d. Web. 03 Apr. 2013.
  5. Imre, István, et al. “Use of chemosensory cues as repellents for sea lamprey: potential directions for population management.” Journal of Great Lakes Research 36.4 (2010): 790-793.
  6. Nonindigenous Aquatic Species Factsheet: Petromyzon marinus U.S. Geological Survey (USGS), Nonindigenous Aquatic Species Program (NAS). Retrieved on 2007-08-04.
  7. Dulac C, Axel R. A novel family of genes encoding putative pheromone receptors in mammals. Cell. 1995 Oct 20;83(2):195-206.
  8. Li, W., Sorensen, P.W. & Gallaher, D. The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae. J. Gen. Physiol.105, 569–589 (1995).

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