Gorgonian host‐inducible cytochrome P450s from the predatory gastropod Cyphoma gibbosum: evolution and function

Background: Intense consumer pressure strongly affects the structural organization and function of marine ecosystems, while also having a profound effect on the phenotype of both predator and prey. Allelochemicals produced by prey often render their tissues unpalatable or toxic to a majority of potential consumers, yet some marine consumers have evolved resistance to host chemical defenses. A key challenge facing marine ecologists seeking to explain the vast differences in consumer tolerance of dietary allelochemicals is understanding the biochemical and molecular mechanisms underlying diet choice. The ability of marine consumers to tolerate toxinladen prey may involve the cooperative action of biotransformation enzymes, including the inducible cytochrome P450s (CYPs), which have received little attention in marine invertebrates despite the importance of allelochemicals in their evolution. Results: Here, we investigated the diversity, transcriptional response, and enzymatic activity of CYPs possibly involved in allelochemical detoxification in the generalist gastropod Cyphoma gibbosum, which feeds exclusively on chemically defended gorgonians. Twelve new genes in CYP family 4 were identified from the digestive gland of C. gibbosum. Laboratory-based feeding studies demonstrated a 2.7to 5.1-fold induction of Cyphoma CYP4BK and CYP4BL transcripts following dietary exposure to the gorgonian Plexaura homomalla, which contains high concentrations of anti-predatory prostaglandins. Phylogenetic analysis revealed that C. gibbosum CYP4BK and CYP4BL were most closely related to vertebrate CYP4A and CYP4F, which metabolize pathophysiologically important fatty acids, including prostaglandins. Experiments involving heterologous expression of selected allelochemically-responsive C. gibbosum CYP4s indicated a possible role of one or more CYP4BL forms in eicosanoid metabolism. Sequence analysis further demonstrated that Cyphoma CYP4BK/4BL and vertebrate CYP4A/ 4F forms share identical amino acid residues at key positions within fatty acid substrate recognition sites. Conclusions: These results demonstrate differential regulation of CYP transcripts in a marine consumer feeding on an allelochemical-rich diet, and significantly advance our understanding of both the adaptive molecular mechanisms that marine consumers use to cope with environmental chemical pressures and the evolutionary history of allelochemical-metabolizing enzymes in the CYP superfamily. Background The dominance of gorgonian corals (Cnidaria, Octocorallia, Gorgonacea) on Caribbean reefs can be attributed, in part, to their chemical defenses, which limit predation by most potential consumers [1,2]. Among the classes of natural products isolated from gorgonians are acetogenins, prostanoids, highly functionalized steroids, and a diverse suite of terpenoid derivatives (e.g., diterpenes and sesquiterpenes) [3,4], all of which contain representatives with anti-predatory activity (reviewed in [5]). Despite the unique toxicological challenge that gorgonians present to marine consumers, the ovulid gastropod Cyphoma gibbosum thrives solely on a diet of allelochemically-rich octocorals [6], implying that this obligate consumer has evolved mechanisms of allelochemical tolerance. Additionally, C. gibbosum’s tissues do not mirror the chemical * Correspondence: [email protected] Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA Full list of author information is available at the end of the article Whalen et al. BMC Ecology 2010, 10:24 http://www.biomedcentral.com/1472-6785/10/24 © 2010 Whalen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. composition of its octocoral prey [7], suggesting that this generalist consumer can biotransform dietary secondary metabolites. Elucidating the molecular mechanisms governing allelochemical resistance is crucial for understanding the genetic basis of adaptation in consumers like C. gibbosum that regularly feed on chemically defended prey. In marine systems, however, knowledge of genes conferring consumer tolerance to dietary allelochemicals is in its infancy [8] compared to that of terrestrial systems [9,10]. Metabolic resistance of insects to host plant toxins is known to involve a battery of cytochrome P450 (CYP) enzymes that oxidize a variety of hydrophobic compounds including dietary allelochemicals [11]. Much of the apparent diversity of CYP isoforms in insects is likely due to reciprocal evolutionary influences between host chemical defenses and consumer detoxification mechanisms, aptly termed a co-evolutionary “arms race” [12,13]. According to this view, multiple gene duplication and adaptive divergence of consumer CYP genes have allowed isoforms to gain new functions and increase the range of possible substrates while retaining ancestral metabolic capabilities [14]. For generalist consumers that must cope with a diversity of allelochemicals, having an assortment of catalytically versatile cytochrome P450 genes may provide a competitive advantage in dealing with the unpredictability of prey chemical defenses [15]. Consumer allelochemical tolerance is driven not only by the genetic and functional diversity of cytochromes P450, but also has been correlated with the elevated transcription of CYP genes [16]. In vertebrate systems, CYP induction following exposure to dietary xenobiotics (including allelochemicals) primarily involves genes within CYP families 1 through 4 [17], while in insects allelochemically inducible forms are found chiefly in CYP families 4, 6 and 9 [11]. The identification of molluscan CYP genes involved in allelochemical metabolism has proved difficult because many of the CYP families previously identified in such reactions are either taxonspecific (i.e., insect-specific CYP 6 and 9) [18], or lack full-length sequenced representatives from protostomes in general, including molluscs (i.e., CYP1, 2 and 3) [19,20]. In contrast, members of the CYP4 family represent a substantial portion of the CYP cDNA sequences identified to date in both arthropods and molluscs [18,21,22]. The CYP4 family is considered one of the most ancient CYP families, having evolved from the steroidsynthesizing CYPs and since diverged into an array of subfamilies and genes encoding enzymes acting on diverse substrates [23]. In vertebrates, CYP4 genes are the predominant fatty acid ω-hydroxylases, preventing lipotoxicity by hydroxylating eicosanoids, including prostaglandins [24]. Prostaglandins are potent signaling molecules, known as regulators of fever, inflammation, and pain response in human biology. In marine systems, prostaglandins function as feeding deterrents and can comprise up to 8% of the wet weight of some gorgonian species (reviewed by [25]). Yet the diversity of CYP4 substrates extends far beyond fatty acid derivatives to include anti-herbivory isoquinoline alkaloids [26] and terpene derivatives, including sesquiterpenoids [27]. The capacity of CYP4 genes to metabolize terpenoid derivatives is of particular interest due to the predominance of defensive diterpenoid and sesquiterpenoid compounds across all members of the Octocorallia [28]. This overlap between the diverse pool of CYP4 substrates and the major structural classes of gorgonian natural products make the CYP4 family an appropriate target for further investigation with respect to allelochemical detoxification in molluscan consumers. An initial investigation by Vrolijk and Targett [29] was the first to suggest that the ability of C. gibbosum to tolerate dietary allelochemicals may involve enhanced biotransformation by enzymes such as cytochromes P450. The authors measured both P450 content and activity from field-collected C. gibbosum feeding on four species of gorgonian corals (Briareum asbestinum, Gorgonian ventalina, Plexaura homomalla, and Pseudopterogorgia americana). However, P450-specific content in C. gibbosum digestive gland was low and only quantifiable in individuals collected from P. americana. Moreover, two spectrofluorometric assays (methoxyresorufin O-deethylase (MROD) and ethoxyresorufin O-deethylase (EROD)), classically used to detect CYP1A-specific activity in vertebrates exposed to anthropogenic pollutants, failed to detect the presence of CYP activity in C. gibbosum digestive gland. Since publication of that paper, advances in the field of molecular ecology warrant a re-evaluation of this hypothesis. Using a targeted molecular approach, we identified putative allelochemical-responsive C. gibbosum CYP4 cDNAs, measured their transcript expression following allelochemical exposure, and assessed their ability to metabolize diagnostic substrates. Evidence presented here emphasizes the role of CYP genes in the adaptation of marine consumers to their chemically defended prey.