Presence of sodefrin precursor-like factor pheromone candidates in mental and dorsal tail base glands in the plethodontid salamander, Karsenia koreana

PLOS ONE RESEARCH ARTICLE Presence of sodefrin precursor-like factor pheromone candidates in mental and dorsal tail base glands in the plethodontid salamander, Karsenia koreana Jared H. DeBruin ID1*, Damien B. Wilburn2,3, Richard C. Feldhoff4, Nancy L. Staub1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Biology, Gonzaga University, Spokane, Washington, United States of America, 2 Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America, 3 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, United States of America, 4 Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, Kentucky, United States of America * Abstract OPEN ACCESS Citation: DeBruin JH, Wilburn DB, Feldhoff RC, Staub NL (2023) Presence of sodefrin precursorlike factor pheromone candidates in mental and dorsal tail base glands in the plethodontid salamander, Karsenia koreana. PLoS ONE 18(8): e0289296. https://doi.org/10.1371/journal. pone.0289296 Editor: M. Caitlin Fisher-Reid, Bridgewater State University, UNITED STATES Received: October 31, 2022 Accepted: July 16, 2023 Published: August 1, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0289296 Copyright: © 2023 DeBruin 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. Plethodontid salamanders are well known for their distinct courtship rituals and the associated pheromonal signaling. However, little is known about pheromones produced in the lone Asian plethodontid species Karsenia koreana. Here, we examined the localization patterns of proteins of the sodefrin precursor-like factor (SPF) pheromone system in K. koreana. Using an antibody generated against SPF proteins from another plethodontid, Desmognathus ocoee, we tested three types of skin glands in K. koreana males via immunohistochemistry: the mental gland and two types of dorsal tail base glands–caudal courtship glands and dorsal granular glands. SPF immunoreactivity was detected in the known courtship gland, the mental gland, as well as granular glands, but not in caudal courtship glands. Due to immunoreaction specificity, we hypothesize the proteins of the SPF system in K. koreana and D. ocoee are structurally and functionally related and are used as courtship pheromones in K. koreana. Also, we hypothesize that K. koreana males transmit SPF to the female during the tail-straddling walk via dorsal granular glands. Finally, K. koreana male caudal courtship glands may be producing SPF proteins that are not recognized by our SPF antibody or these glands may play a different role in courtship than anticipated. Introduction Pheromones, chemical signals that produce a response in members of the same species [1], are used throughout the tree of life. Identifying which signals elicit which behavioral or physiological responses is of great interest [2–4]. To confirm that a substance is a pheromone, however, can be a challenge. Since pheromones are often released as a part of chemical mixtures, isolating the signal, its receptor, and measuring the effects of a signal, can be difficult [5–8]. As a result, only a few pheromone-receptor pairs have been described [9]. Some examples of PLOS ONE | https://doi.org/10.1371/journal.pone.0289296 August 1, 2023 1/9 PLOS ONE Funding: The funding received for this study is outlined as follows: NLS, no grant number (internal funding), Gonzaga Science Research Program Fund, https://www.gonzaga.edu/academics/ centers-institutes/center-for-undergraduateresearch-and-creative-inquiry/gonzaga-scienceresearch-program DBW, NICHD R00-HD090201, National Institute of Child Health and Human Development, https://www.nichd.nih.gov/grantscontracts The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. SPF pheromone-candidates in salamander skin glands pheromone-receptor pairs can be found in insects, where pheromones are used for mate selection, food localization, predator warning signals, and more [10–12]. Because of the difficulty in identifying pheromones, chemical signals that scientists hypothesize to be pheromones are aptly named “pheromone-candidates.” One example in frogs is the sodefrin precursor-like factor (SPF) family of proteins [13]. These signals were described as pheromone candidates in frogs because they are known to be pheromones in salamandrids and plethodontids [14–16], but their function in frogs is, as of yet, unknown [13]. Behavioral evidence indicates that SPF proteins are involved in courtship in salamandrids and plethodontids [14]. Courtship is defined as behaviors that maintain reproductive actions between mating partners; it does not refer to initial mate attraction [17]. The courtship pheromone SPF was identified in Desmognathus ocoee, a species of plethodontid salamander [14]. During courtship, a male D. ocoee will scratch the female’s dorsal skin with hypertrophied teeth and rub over these scratches with his submandibular region [14, 18, 19]. In D. ocoee and in other plethodontids, the submandibular region of the male contains a group of exocrine glands called the mental gland [14, 20]. Within the individual secretory glands, cells secrete substances into the gland lumen for the eventual release from the gland’s secretory duct [15, 21]. SPF proteins are present in the mental gland tissue of D. ocoee [14]. When a proteinaceous extract, that is primarily SPF proteins, is applied to the dorsal skin of female D. ocoee, female receptivity of courtship behavior increases [14]. Receptivity refers to the female’s “acceptance” of the courtship behavior, quantified as a decrease in courtship duration [14]. The target organ of this transdermal-based method of pheromone delivery is unknown. However, the proteinaceous extract ellicited a distinct behavioral response in females, which resulted in classifying the major components of the fraction, SPF proteins, as pheromones [14, 22]. Interestingly, SPF proteins are linked to multiple courtship behaviors in plethodontids. For example, in addition to being associated with courtship behaviors involving the mental gland in D. ocoee, recent studies have detected SPF mRNA in dorsal tailbase glands in other plethodontids [23]. These glands, named caudal courtship glands aft (...truncated)