Bile Salts as Semiochemicals in Fish

Chem. Senses 39: 647 654, 2014 doi:10.1093/chemse/bju039 Advance Access publication August 23, 2014 Bile Salts as Semiochemicals in Fish Tyler J. Buchinger 1, Weiming Li 1 and Nicholas S. Johnson 2 1 Department of Fisheries and Wildlife, Room 13 Natural Resources Building, Michigan State University, East Lansing, MI 48824, USA and 2 United States Geological Survey, Great Lakes Science Center, Hammond Bay Biological Station, 11188 Ray Road, Millersburg, MI 49759, USA Correspondence to be sent to: Nicholas S. Johnson, United States Geological Survey, Great Lakes Science Center, Hammond Bay Biological Station, 11188 Ray Road, Millersburg, MI 49759, USA. e-mail: njohnson@usgs.gov Accepted July 7, 2014 Abstract Bile salts are potent olfactory stimuli in fishes; however the biological functions driving such sensitivity remain poorly understood. We provide an integrative review of bile salts as semiochemicals in fish. First, we present characteristics of bile salt structure, metabolism, and function that are particularly relevant to chemical communication. Bile salts display a systematic pattern of structural variation across taxa, are efficiently synthesized, and are stable in the environment. Bile salts are released into the water via the intestine, urinary tract, or gills, and are highly water soluble. Second, we consider the potential role of bile salts as semiochemicals in the contexts of detecting nearby fish, foraging, assessing risk, migrating, and spawning. Lastly, we suggest future studies on bile salts as semiochemicals further characterize release into the environment, behavioral responses by receivers, and directly test the biological contexts underlying olfactory sensitivity. Key words: chemical cue, communication, electro-olfactogram, olfaction, pheromone Introduction Bile salts are functionally diverse metabolites of cholesterol in vertebrates (Hofmann 1999). The primary function of bile salts is the solubilization of dietary fats in the intestine. However, additional functions include cholesterol homeostasis, antimicrobial effects, and endocrine signaling (Houten et al. 2006; Hofmann and Hagey 2008). Interest in bile salts spans a diverse range of disciplines, including medicine (Hofmann and Hagey 2008), evolution (Hagey et al. 2010), physiology (Cai et al. 2012; Yeh et al. 2012), and environmental chemistry (Li et al. 2011). Hypotheses pertaining to the role of bile salts as olfactory cues have garnered the interest of chemical ecologists (Døving et al. 1980). Bile salts are potent olfactory stimuli in fish (Table 1). Døving et al. (1980) first proposed that bile salts excreted by stream resident conspecifics guide migrating Arctic char (Salvelinus alpinus) to spawning streams. A widespread sensitivity of fish to bile salts has now become apparent. Olfactory detection of bile salts appears throughout the phylogeny of fish, including early vertebrates (Li et al. 1995) and more recently diverged fishes (Michel and Lubomudrov 1995). Species residing in marine (Velez et al. 2009) and freshwater (Zhang et al. 2001) habitats, and those migrating between the 2 (Sola and Tosi 1993; Li et al. 1995; Baker et al. 2006) detect bile salts with high sensitivity. While olfactory detection of bile salts is well-supported by electrophysiological evidence (Table 1), the biological function of fish sensitivity to bile salts is understood only in the sea lamprey (Petromyzon marinus; Li et al. 1995, 2002; Bjerselius et al. 2000). Here, we review evidence for the role of bile salts as semiochemicals (molecules which carry information to individuals) in fishes. We begin by highlighting characteristics of bile salt chemistry and physiology that are particularly relevant to olfaction. Second, we consider the information fishes receive when bile salts are detected in several contexts. We conclude by suggesting research to further characterize bile salts as semiochemicals. Bile salt chemistry and physiology Structure Bile salts display many minor structural variations (Hofmann et al. 2010) and have high potential to function as specific olfactory cues. Minor structural differences between bile Published by Oxford University Press on behalf of US Government 2014.

648 T.J. Buchinger et al. salts can have major consequences for their affinity for a given olfactory receptor (Li et al. 1995; Siefkes and Li 2004; Johnson et al. 2012). The steroid nucleus of bile salts varies in ring stereochemistry, hydroxyl and oxo groups, and conjugation, and the side chain varies in length, hydroxyl and carboxyl groups, saturation, stereochemistry, and conjugation (Figure 1; Hofmann et al. 2010). Variation in bile salt structure follows a systematic pattern (Hofmann et al. 2010) and may contribute to taxon-specific odor profiles (Huertas et al. 2010). Hofmann et al. (2010) proposed a progression from C 27 bile alcohols to C 27 bile acid intermediates to C 24 bile acids throughout the evolution of vertebrates (Figure 1). The pattern generally holds in fishes; hagfish primarily produce 5α C 27 bile alcohols, lampreys use 5α C 27 and C 24 bile alcohols, cartilaginous fishes use 5β C 27 bile alcohols, and ray-finned fish use 5β C 24 bile acids (Figure 2; Hagey et al. 2010). Lobe-finned Table 1 Evidence for bile salt olfactory cues in fish, including physiological detection as determined by electroolfactograms (EOG), release of bile salts into the water, and behavioral responses in the laboratory and in the field Species Common name Detection Release Response Reference laboratory field Petromyzon marinus Sea lamprey x x x x Li et al. (1995, 2002); Polkinghorne et al. (2001); Yun et al. (2003a) Ichthyomyzon unicuspis Silver lamprey x x x x Fine et al. (2004); Buchinger et al. (2013) Entosphenus tridentatus Pacific lamprey x x Yun et al. (2003b, 2011); Robinson et al. (2009); Fine et al. (2004) Petromyzontiforme sp. Various lampreys x Fine et al. (2004); Stewart et al. (2011) Sphyrna tiburo Bonnethead shark x Meredith et al. (2012) Dasyatis Sabina Atlantic stingray x Meredith et al. (2012) Anguilla Anguilla European eel x x Sola and Tosi (1993); Huertas et al. (2007) Danio rerio Zebrafish x Michel and Lubomudrov (1995); Friedrich and Korsching (1997) Carassius auratus Goldfish x Sorensen et al. (1987); Huertas et al. (2010) Catostomus catostomus Longnose sucker x Cardwell et al. (1992) Catostomus commersoni White sucker x Cardwell et al. (1992) Ictalurus punctatus Channel catfish x Erickson and Caprio (1984) Oncorhynchus mykiss Rainbow trout x x Hara et al. (1984); Giaquinto and Hara (2008); Vermeirssen and Scott (2001) Salvelinus namaycush Lake trout x x Zhang et al. (2001); Zhang and Hara (2009) Salvelinus alpinus Arctic char x x x Døving et al. (1980); Selset and Døving (1980) Solea senegalensis Senegalese sole x x Velez et al. (2009) Sparus auratus Gilthead seabream x Hubbard et al. (2003) Galaxias fasciatus Banded kokopu x x Baker et al. (2006) Oreochromis mossambicus Mozambique tilapia x Frade et al. (2002); Huertas et al. (2010) Gadus morhua Atlantic Cod x Hellstrøm and Døving (1986) Figure 1 Generic structures of (A) C 27 bile acids, (B) C 27 bile alcohols, and (C) C 24 bile acids. Common sites of hydroxylation are represented by R. See Hofmann et al. (2010) for additional details on structural variants. Modified from Hofmann et al. (2010).

Bile Salts as Semiochemicals in Fish 649 Figure 2 Phylogeny of fishes overlaid with patterns of bile salt evolution (Hagey et al. 2010; Hofmann et al. 2010). (A) C 27 bile alcohol of hagfishes, myxinol disulfate, (B) C 24 bile alcohol of lampreys, (C) C 27 bile alcohol of cartilaginous fishes, and (D) C 24 bile acid of ray-finned fishes. fishes use unusual 25-hydroxylated C 26, C 28, C 29, and C 30 bile alcohols (Hagey et al. 2010). Variation in the ratios and primary constituents of bile salt profiles occur down to the species level (Hagey et al. 2010). Although the importance of odorant ratios has yet to be determined in fishes, research on insect pheromones suggests that organisms can distinguish between ratios of the same odor blend (Wyatt 2014). A higher structural stability increases the time over which bile salts can function as signals (Wyatt 2014). For example, extremely stable odorants eventually provide false information in the context of mate search, but are needed to retain information over the long temporal scale of migration. Bile salts are generally stable as they must be resistant to digestive enzymes within an organism (Hofmann and Mysels 1987). Intestinal bacteria produce secondary bile salts by modifying those synthesized in the liver (Philipp 2011). Degradation of the steroid skeleton occurs quite slowly (Shimada et al. 1969; Aries and Hill 1970; Ternes et al. 1999), however transformations such as deconjugation and dehydroxylations are likely to occur sooner (Philipp 2011). Putative migratory pheromones of the sea lamprey have half-lives 2 3 times longer than mating pheromones, which makes sense ecologically because mating pheromones are more likely to be used over relatively short distances (Polkinghorne et al. 2001; Fine and Sorensen 2010; Wang et al. 2013). Metabolism Vertebrates synthesize bile salts in the large quantities (500 mg/day in humans; Russell 2003) required to be detected in high-volume environments. The active space of a semiochemical depends upon the olfactory sensitivity, volume of environment, and the release rate. Approximately 90% of cholesterol, accumulated through dietary intake or through biosynthesis from acetyl CoA (Liscum 2008), is catabolized into bile salts in the hepatocyte (in humans; Figure 3; Hofmann and Hagey 2008), while only 10% is converted into sex steroids (Russell 2003). Metabolic differences between bile salts and reproductive hormones (sex steroids and prostaglandins) translate into release rates, as known bile salt release rates are several orders of magnitude higher than those of reproductive hormones (Zhang et al. 2001; Li et al. 2002; Baker et al. 2006; Scott and Ellis 2007; Velez et al. 2009). The synthesis of large quantities of bile salts may lead to bile salt mating pheromones being used by species reproducing in high-volume environments, such as the sea lamprey (Li et al. 2002; Li 2005). Release Fish excrete bile salts through the intestine (Polkinghorne et al. 2001; Zhang et al. 2001), urinary tract (Sato and Suzuki 2001; Cai et al. 2012), and gills (Li et al. 2002; Siefkes et al. 2003; Brant et al. 2013). Intestinal release of bile salts via feces is the result of foraging and digestion. Marine teleost fishes excrete an intestinal fluid containing bile salts as a result of salt regulation (Hubbard et al. 2003; Huertas et al. 2007). Renal excretion has been reported in rainbow trout (Oncorhynchus mykiss; Sato and Suzuki 2001) and sea lamprey (Cai et al. 2012), and may be used to alleviate the toxic effects of bile salts (Cai et al. 2012) or signal to mates (Sato and Suzuki 2001). Excreted bile salts are often conjugated with a sulfate (bile alcohols), glycine, taurine, or a taurine derivative (bile acids) and thus highly water soluble and accessible to the olfactory organ of fish. Solubility is as critical for olfaction in aquatic environments as volatility is for terrestrial environments (Wyatt 2014). Bile salts excreted into the environment are

650 T.J. Buchinger et al. Figure 3 Schematic of bile acid metabolism and excretion. Bile salts are synthesized in the liver, stored in the gallbladder, secreted via the bile duct into the intestine, and excreted via feces. often sulfated; sulfation decreases intestinal permeability, increases solubility in urine, and may be an adaptive mechanism for excretion (De Witt and Lack 1980). In several cases, the olfactory epithelium is more sensitive to sulfateconjugated bile salts (Michel and Lubomudrov 1995; Siefkes and Li 2004; Venkatachalam 2005). The bile alcohol mating pheromone of the sea lamprey does not function without the sulfate group (Johnson et al. 2012). Release of bile salts is not restricted to fish that are actively feeding (Polkinghorne et al. 2001; Zhang et al. 2001; Fine and Sorensen 2010) or of a specific life stage (Zhang et al. 1996; Buchinger et al. 2013). Consistent release of bile salts may allow fish to gather information from the odor of bile across many ecological contexts. Reproductive hormones, in contrast, are primarily excreted by individuals of a specific reproductive status (Stacey et al. 2003), and thus only suited to function in reproductive contexts. Detection Bile salts elicit strong responses in the fish olfactory epithelium (Table 1). The detergent properties of bile salts have been hypothesized to irritate the olfactory epithelium (Døving et al. 1980). For example, Erickson and Caprio (1984) found a portion of the electrical response of channel catfish (Ictalurus punctatus) to bile salts could not be attributed to the olfactory neural activity. Non-specific irritation of the olfactory epithelium may increase conspicuity of bile salts as chemical signals (Endler and Basolo 1998). However, several fish detect bile salts with highly specific olfactory receptors (Li and Sorensen 1997; Zhang and Hara 2009). Kittredge and Takahashi (1972) hypothesized that receptors required for pheromone signaling evolved from receptors required for endocrine signaling. Bile salt receptors involved in feedback regulation of cholesterol catabolism are present throughout the biliary system (Houten et al. 2006), thus the transition from intra to inter-individual signaling required only the mutation externalizing receptors from within the organism to the olfactory epithelium. Behavioral context of bile salt olfactory cues Fishes evaluate their local environment using conspecific and heterospecific olfactory cues (Chung-Davidson et al. 2011). The sensitivity of the olfactory epithelium to bile salts suggests a role in the chemical evaluation of a fish s surroundings. In this section, we discuss the information gathered when fishes smell bile salts in the contexts of detecting nearby fish, foraging, assessing risk, migrating, and spawning. Interspecific awareness The detection of many bile salts is not species-specific, and may reflect a mechanism for detecting nearby individuals regardless of species (Huertas et al. 2010). Taurocholic acid is detected with acute sensitivity by all species investigated, including char (Salvelinus sp.; Døving et al. 1980; Zhang et al. 2001), eels (Anguilla anguilla; Huertas et al. 2010), goldfish (Carassius auratus; Sorensen et al. 1986), and sea lamprey (Li et al. 1995). The release of taurocholic acid is also common across many fish species (Zhang et al. 2001; Baker et al. 2006; Velez et al. 2009). The general detection of bile salts may even extend to those not produced by conspecifics (Huertas et al. 2010). Non-specific detection of bile salts may provide fish with a mechanism to detect nearby individuals in an environment where other sensory modalities can be unreliable. Foraging Detection of conspecific bile salts may confer information regarding foraging opportunities as their release coincides with digestion. While amino acids suggest food may be present (Hara 1994, 2006), bile salts, involved in digestion and influenced by diet (Polkinghorne et al. 2001), could indicate that successful feeding is occurring. Bile salts display a pattern of systematic structural variation (Hofmann et al. 2010) and therefore, hold specific information regarding conspecific foraging success. Specific foraging information may be particularly important to fishes

Bile Salts as Semiochemicals in Fish 651 undergoing extensive foraging migrations. For example, glass eels (A. rostrata and A. anguilla) have been hypothesized to cue onto conspecific bile salts during their migration from sea to streams to forage (Sorensen 1986; Sola and Tosi 1993), and are attracted to several bile salts in laboratory behavioral assays (Sola and Tosi 1993). Given the great investment of long-distance migrations, individuals likely face a substantial decline in fitness with incorrectly choosing a system lacking suitable foraging opportunity. Amino acids and bile salts are likely both important foraging cues, as amino acids will present more immediate information while bile salts provide delayed information after successful foraging has occurred. Risk While the identity of alarm cues remains generally elusive (Ferrari et al. 2010, but see Mathuru et al. 2012), a role of bile salts in anti-predator behaviors is conceivable (Huertas et al. 2010). Alarm responses of some fish are elicited specifically by predators which have consumed conspecifics (Mathis and Smith 1993), and replicated by feces of the predator (Brown et al. 1995). Although the synthesis of primary bile salts is unlikely to be influenced by diet (Hofmann 1999), the secretion of bile salts into the intestine and excretion into the water are effected by diet (Polkinghorne et al. 2001; Fine and Sorensen 2010). Bacteria ingested may also change with diet, which results in different secondary bile salts. Some insects use bacteria-modified compounds as chemical cues (Dillon et al. 2000), and the hypothesis that secondary bile salts modified by bacteria living within certain prey species may also function as alarm cues seems plausible. Reproductive migrations Bile salts emanating from stream-resident conspecifics have been hypothesized to guide fishes undertaking reproductive migrations (Døving et al. 1980; Li et al. 1995). The systematic diversity (Hofmann et al. 2010), structural stability (Wang et al. 2013), and relation to offspring success (Polkinghorne et al. 2001) makes bile salts well-suited to direct high-investment reproductive migrations. Arctic char are acutely sensitive to bile salts (Døving et al. 1980), show innate behavioral preference for conspecific bile salts (Jones and Hara 1985), and return to streams containing conspecific odors (Nordeng 1977). Similarly, sea lampreys are sensitively tuned to conspecific bile salts (Li et al. 1995; Siefkes and Li 2004), display behavioral preferences for conspecific odors (Teeter 1980) and bile salts (Bjerselius et al. 2000), and return to streams tributaries emanating larval odor (Wagner et al. 2009). In contrast, several migratory species appear to learn the odor of their natal stream (Hasler and Wisby 1951), specifically recognizing amino acids mixtures (Shoji et al. 2000, 2003). Whether the 2 mechanisms of natal homing are mutually exclusive is unknown, however the role of amino acids verses bile salts in natal homing remains debated (Quinn 1990). Spawning Bile salts function as mating pheromones in the sea lamprey (Li et al. 2002, 2013) and have been hypothesized as mating pheromones in several additional species (Zhang et al. 1996; Vermeirssen and Scott 2001; Huertas et al. 2007). Many freshwater teleost fishes are hypothesized to use sex steroids and prostaglandins as mating pheromones, which is unsurprising given their role in endocrine control of reproduction (Stacey et al. 2003) and release via urine (Appelt and Sorensen 2007). Freshwater teleost fishes constantly produce large amounts of dilute urine while marine fishes sporadically excrete small amounts of highly concentrated urine (Karnaky 1998). Hubbard et al. (2003) hypothesized that the little urine released by marine fishes favors the use intestinal fluids and bile salts as mating pheromones. Reproductive pheromones of marine fish are poorly understood, but the few studies completed support the hypothesis (Huertas et al. 2006, 2007, 2010; Velez et al. 2009). Changes in bile salt release stemming from diet changes (Polkinghorne et al. 2001; Fine and Sorensen 2010) during reproduction or osmoregulatory changes associated with a saltwater to freshwater transition during spawning (Hubbard et al. 2003) may provide information regarding the reproductive status of individuals. However, the information is likely quite vague compared with reproductive hormones, and is unlikely to explain preferences for bile salts in a spawning context. In the sea lamprey, female preference for a bile salt mating pheromone may have evolved outside the context of spawning, whereas females use juvenile-released bile salts to locate suitable rearing habitat (Li et al. 1995; Bjerselius et al. 2000) and males take advantage of the existing preference for bile salts to lure females to nests (Buchinger et al. 2013). Current state of the olfactory hypothesis Although olfactory detection of bile salts by fish is well documented (Table 1), the biological function remains poorly understood. Two lines of evidence are generally lacking: 1) release of bile salts into the water, and 2) behavioral responses to bile salts. To our knowledge, the sea lamprey mating pheromone is the only bile salt pheromone for which the release, detection, and behavioral response in natural environments is understood (Li et al. 2002, 2013; Siefkes et al. 2005; Johnson et al. 2009). Although we present indirect evidence that bile salts may function as semiochemicals in several contexts, direct evidence provided through an integration of electrophysiology, chemistry, laboratory assays, and field testing is critical (Johnson and Li 2010). For example, the hypothesis that bile salts guide migratory fishes is supported by observations that Arctic char are acutely sensitive to bile salts (Døving et al. 1980), show laboratory preference

652 T.J. Buchinger et al. for conspecific bile salts (Jones and Hara 1985), and return to the stream containing conspecific odors (Nordeng 1977), but specific bile salts have not been identified nor tested in laboratory and field assays. Likewise, a role of bile salts in the migratory behavior of sea lamprey is supported by chemical, electrophysiological, and laboratory evidence (Li et al. 1995; Bjerselius et al. 2000; Polkinghorne et al. 2001; Sorensen et al. 2005), but field assays suggest additional, unknown, components are critical (Meckley et al. 2012, 2014). Conclusion The behavioral function underlying widespread olfactory sensitivity to bile salts remains unclear. Here, we review the chemistry and physiology of bile salts relevant to olfaction and consider possible roles of bile salts in the chemical evaluation of nearby individuals, foraging opportunities, predatory threats, rearing habitat, and suitable mates. The widespread olfactory detection and range of possible behavioral influences of bile salts offers a useful system to evaluate inter- and intra-specific interactions between fish across ecological contexts. Continued studies on bile salt pheromones may shed light on pest management (Johnson et al. 2009), restoration of native fishes (Zhang et al. 2001), and evolution of chemical signals (Buchinger et al. 2013). Funding This work was supported by the Great Lakes Fisheries Commission. Acknowledgments Dr. Mar Huertas, Trevor Meckley, Carrie Kozel, and 3 anonymous reviewers provided valuable critique of early drafts. Thanks to Dr. Ke Li and Cory Brant for assistance with figures. This manuscript is contribution number 1855 of the Great Lakes Science Center. References Appelt CW, Sorensen PW. 2007. Female goldfish signal spawning readiness by altering when and where they release a urinary pheromone. Anim Behav. 74:1329 1338. Aries V, Hill MJ. 1970. 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