Mixtures of Two Bile Alcohol Sulfates Function as a Proximity Pheromone in Sea Lamprey

RESEARCH ARTICLE Mixtures of Two Bile Alcohol Sulfates Function as a Proximity Pheromone in Sea Lamprey Cory O. Brant, Mar Huertas, Ke Li, Weiming Li* Department of Fisheries and Wildlife, Room 13 Natural Resources Building, 480 Wilson Road, Michigan State University, East Lansing, MI, 48824, United States of America * Abstract OPEN ACCESS Citation: Brant CO, Huertas M, Li K, Li W (2016) Mixtures of Two Bile Alcohol Sulfates Function as a Proximity Pheromone in Sea Lamprey. PLoS ONE 11 (2): e0149508. doi:10.1371/journal.pone.0149508 Editor: Michel Renou, INRA-UPMC, FRANCE Received: July 29, 2015 Unique mixtures of pheromone components are commonly identified in insects, and have been shown to increase attractiveness towards conspecifics when reconstructed at the natural ratio released by the signaler. In previous field studies of pheromones that attract female sea lamprey (Petromyzon marinus, L.), putative components of the male-released mating pheromone included the newly described bile alcohol 3,12-diketo-4,6-petromyzonene-24-sulfate (DkPES) and the well characterized 3-keto petromyzonol sulfate (3kPZS). Here, we show chemical evidence that unequivocally confirms the elucidated structure of DkPES, electrophysiological evidence that each component is independently detected by the olfactory epithelium, and behavioral evidence that mature female sea lamprey prefer artificial nests activated with a mixture that reconstructs the male-released component ratio of 30:1 (3kPZS:DkPES, molar:molar). In addition, we characterize search behavior (sinuosity of swim paths) of females approaching ratio treatment sources. These results suggest unique pheromone ratios may underlie reproductive isolating mechanisms in vertebrates, as well as provide utility in pheromone-integrated control of invasive sea lamprey in the Great Lakes. Accepted: February 1, 2016 Published: February 17, 2016 Copyright: © 2016 Brant et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files (see S1 Data). Funding: This work was funded by the Great Lakes Fishery Commission, Ann Arbor, MI, USA. Competing Interests: The authors have declared that no competing interests exist. Introduction A common source of conspecific information used in orientation strategies and mate location across the animal kingdom is provided by pheromones, or unique chemical signatures that are released by animals and influence behavior or development of members of the same species [1, 2]. Together, the sending and receiving of pheromones result in movement patterns that reduce the distance between conspecifics across their odor landscape (attraction) and/or maintain individuals in place (arrestant) to gain advantage in mating or feeding [2, 3]. In insects, pheromones that function in sex and aggregation are often comprised of multiple components at specific ratios [4–6]. For pheromones of unique ratios to function as species-specific attractants, a level of discrimination against individual components must occur at the sensory and behavioral level. In fishes, olfactory systems have been shown, via electrophysiological cross- PLOS ONE | DOI:10.1371/journal.pone.0149508 February 17, 2016 1 / 14 Mixture of Bile Alcohols Function as Proximity Pheromone adaptation experiments, to detect and discriminate between compounds with separate receptors [7–9]. Detection and discrimination of individual compounds in phylogenetically similar groups of fishes has been proposed as an adaptation involved in speciation [8]. However, behavioral evidence is rarely presented to evaluate the specificity of pheromone ratios in vertebrates [2]. The olfactory epithelium of the sea lamprey (Petromyzon marinus, L.) has been shown to detect and discriminate multiple compounds that are structurally similar [10–12]. Using pheromones for key aspects of their life history [13–16], sea lamprey present a useful vertebrate model for studies regarding pheromone ratio specificity. Sea lamprey begin their single reproductive season by migrating into freshwater tributaries to the Atlantic Ocean (native range), or the Laurentian Great Lakes (invasive range), that are activated with compounds released by stream-residing conspecific larvae [13, 16]. Males often move upstream in greater numbers earlier in the migratory season through April–May, and establish nests in suitable spawning habitat in early June [17, 18]. Upon reaching sexual maturation in streams, males release a pheromone across gill epithelia that contains a main component, 3-keto petromyzonol sulfate (3kPZS).[19, 20]. Synthesized 3kPZS alone draws significant numbers of mature females upstream towards the source [14, 21]. However, 3kPZS as a single component is often less attractive than the whole male odor [14]. Upon analyzing whole male odor (termed spermiated male washings, or SMW, herein), the chemical structure of a new sulfate-conjugated compound, 3,12-diketo-4,6-petromyzonene-24-sulfate (DkPES) was elucidated [22]. Mature females were shown to increase their preference towards mixtures of 3kPZS and DkPES compared to 3kPZS alone. However, many critical issues have not been addressed regarding identity and function of DkPES [22], including chemical synthesis of DkPES and confirmation of the elucidated structure, discrimination of DkPES from 3kPZS by the olfactory epithelia, the orientation mechanisms used by sea lamprey to locate a mixture of these two compounds, and the effective range of ratios between the two pheromone components required for attraction. This study reports the next step in identifying the function of DkPES as a pheromone component in sea lamprey. Here, we confirm the structure elucidated for DkPES with its synthesized copy, show the olfactory epithelium of sea lamprey discriminates DkPES from 3kPZS, demonstrate that females are attracted to a reconstructed ratio similar to that seen in extracts from mature male sea lamprey [22], and further define the orientation strategies of mature females to the mixture of DkPES and 3kPZS. Materials and Methods Test Subjects All surgical and behavioral procedures using sea lamprey presented in this manuscript were approved by the Michigan State University Institutional Animal Care and Use Committee prior to any experimentation (AUF# 05/09-088-00 and 03/11-053-00). Adult sea lamprey used for electro-olfactogram (EOG) recordings were collected in spring 2012 by commercial fishing companies in Lake Huron and transported to United States Geological Survey and Great Lakes Science Center—Hammond Bay Biological Station (HBBS). Sea lamprey were later shipped to Michigan State University, East Lansing, Michigan for EOGs. Migrating adult sea lamprey were captured by (...truncated)