Molecular Ecology (2008) 17, 1156–1162 doi: 10.1111/j.1365-294X.2007.03627.x EBlackwell Puvblishing Ltod lution of ecological specialization and venom of a predatory marine gastropod E. A. REMIGIO* and T. F. DUDA JR*† *Department of Ecology and Evolutionary Biology/Museum of Zoology, University of Michigan, 1109 Geddes Avenue, Ann Arbor, MI 48109, USA, †Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancón, Panama Abstract Understanding the evolution of ecological specialization is important for making inferences about the origins of biodiversity. Members of the predatory, marine gastropod genus Conus exhibit a variety of diets and the ability to capture prey is linked to a venom comprised of peptide neurotoxins, termed conotoxins. We identified conotoxin transcripts from Conus leopardus, a species of Conus that uniquely preys exclusively on hemichordates, and compared its venom duct transcriptome to that of four other Conus species to determine whether a shift to a specialized diet is associated with changes in the venom composition of this species. We also examined the secondary structure of predicted amino acid sequences of conotoxin transcripts of C. leopardus to identify substitutions that may be linked to specialization on hemichordates. We identified seven distinct conotoxin sequences from C. leopardus that appear to represent transcripts of seven distinct loci. Expression levels and the diversity of conotoxins expressed by C. leopardus are considerably less than those of other Conus. Moreover, gene products of two transcripts exhibited unique secondary structures that have not been previously observed from other Conus. These results suggest that transition to a specialist diet is associated with reduction in the number of components expressed in venoms of Conus and that diverse venoms of Conus are maintained in species with a broad dietary width. Keywords: conotoxins, Conus, ecological specialization, gene expression Received 15 September 2007; revision accepted 26 October 2007 Species of Conus prey on fishes, other marine snails, Introduction errant and sedentary polychaetes, and hemichordates Much of evolution involves the origin of adaptations (Kohn 1959). Based on phylogenetic reconstructions, errant that enable organisms to utilize new resources or to use polychaete-eating is the ancestral diet and the derived resources in new or more efficient ways. However, these feeding habits evolved only a few times during the initial adaptations and their contribution to the origins of major diversification of Conus species (Duda et al. 2001; biodiversity cannot be fully realized until their genetic Duda & Palumbi 2004). The particular diets of vermivorous foundation is identified. Members of the predatory, tropical species also differ, with species-specific preferences for marine gastropod genus Conus exhibit a number of feeding particular worm taxa (Kohn 1959, 1966, 1980; Marsh 1971; specializations and their ability to capture prey is linked Kohn & Nybakken 1975; Leviten 1980; Reichelt & Kohn to a venom comprised of peptide neurotoxins. Studies of 1985; Kohn & Almasi 1993). Conus utilize an assortment of the molecular evolution of genes that are directly potent peptide neurotoxins, termed conotoxins, in their associated with species’ ecologies, such as those expressed venom to capture prey. Conotoxins are small peptides in the venoms of Conus, will illuminate key issues in consisting of 10–35 amino acids with highly conserved evolution, particularly the patterns of evolution of ecolo- framework of cysteine (Cys) residues and are expressed gically relevant genes and their association with ecological in the venom ducts of Conus. These peptides target specific adaptations. ion channels and receptors of prey (Olivera 1997; McIntosh et al. 1999; Terlau & Olivera 2004). The active components Correspondence: T. F. Duda Jr, Fax: 734-763-4080; E-mail: of cone snail venoms are remarkably diverse; venom of a [email protected] single species may contain up to 200 different toxin peptides © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd EVOLUTION OF ECOLOGICAL SPECIALIZATION AND VENOM 1157 (Olivera et al. 1990, 1991). These toxins vary extensively abilities, foraging behaviours and search strategies, phy- within and between species, and across the snails’ range siological processes associated with digestion and waste of dietary preferences (e.g. Olivera et al. 1991; Walker et al. removal, and other features involved with prey capture, 1999; Conticello et al. 2001; Olivera 2002; Duda & Palumbi with some traits being more labile than others. Among 2004; Corpuz et al. 2005). predatory taxa that use venom to subdue prey, the evolu- Among cone snails, Conus leopardus is unique in that it is tion of a specialized diet is likely to be strongly linked to the only Conus species known to prey exclusively on the the evolution of venoms, many of which are comprised of hemichordate Ptychodera flava (Kohn 1959, 1980; Pickens direct gene products such as the conotoxins of Conus. Thus, 1973; Kohn & Nybakken 1975; Röckel et al. 1995). While although the evolution of many of these characters may be other vermivorous Conus include P. flava in their diets (e.g. difficult to trace for a species that has evolved a narrow C. lividus; Kohn 1959), no other Conus are known to feed diet, the evolution of venoms can be inferred from analyses solely on this species and C. leopardus is perhaps the only of expression of genes encoding venom components. How Conus to exhibit such a narrow dietary breadth. Conus has the venom of C. leopardus evolved with the origin of leopardus is most closely related to other Conus that pre- the specialized diet of this species? As noted above, Conus dominantly prey on sedentary polychaetes, errant and venoms are typically comprised of a plethora of conotoxins. sedentary polychaetes, sedentary polychaetes and hemi- Complex venoms may be associated with broad diets of chordates, or fishes (Duda et al. 2001; see also Duda & Kohn species. Are transitions to more specialized feeding modes 2005). Although reconstructed phylogenies are not well then accompanied by the evolution of a more streamlined resolved with regards to the history of divergence of C. venom such that fewer venom components are needed to leopardus and its closest relatives, the diet of this species capture a narrower range of prey? The answers to questions reflects the evolution of a unique feeding ecology and a such as these can give broad insight into the evolution shift to a narrow diet. and maintenance of the diverse venom compositions of Because the venoms of Conus are used primarily to Conus and illuminate the genetic changes associated with subdue prey, the evolution of the unique feeding mode shifts in feeding ecology and the origins of specialization. exhibited by C. leopardus is likely to be associated with the To determine whether the dietary transition of C. leopardus evolution of its venom. Dietary shifts and venom evolution is related to the evolution of its venom, we generated a were examined previously in sea snakes (Li et al. 2005a, b). cDNA library from mRNA recovered from the venom duct Phospholipase A2 toxins of Aipysurus eydouxii, a sea snake of a single individual of this species. We sequenced 200 that feeds on fish eggs and does not utilize venom to obtain transcripts obtained from this library to identify putative food, are less diverse and exhibit slower rates of evolution conotoxin transcripts. Because C. leopardus shows no close than these toxins from sea snakes that use venom to cap- affinity to other Conus and venom duct transcriptomes ture prey (Li et al. 2005a). Moreover, a gene encoding the have so far only been described from four other Conus species, sole three-finger toxin expressed by A. eydouxii possesses a we are limited in our ability to compare the venom com- two-base deletion that significantly alters the function of position of C. leopardus to that of these four species: C. arenatus, this peptide (Li et al. 2005b). Clearly, the loss of a venomous a species that preys on capitellid, eunicid, maldanid and feeding mode in this sea snake is associated with the neried polychaetes (Kohn 1968; Kohn & Nybakken 1975; reduced effectiveness of its toxins and decelerated evo- Reichelt & Kohn 1985; Röckel et al. 1995); C. striatus, a lution of genes that encode venom components (Li et al. piscivore (Röckel et al. 1995); and C. pennaceus and C. textile, 2005a, b). Shifts in diet from venomous to nonvenomous two molluscivorous species (Röckel et al. 1995). Based on feeding modes may have predictable effects on the evolu- phylogenies derived from mitochondrial and nuclear tion of venoms, but the association of venom evolution sequence data, C. arenatus represents the closest relative and dietary shifts among venomous taxa remains largely of C. leopardus among the above four species (Duda et al. unexplored despite the potential utility of these systems 2001; Duda & Kohn 2005) and the lineages that gave rise to examine the molecular basis of ecological adaptations. to C. arenatus and C. leopardus likely diverged about 21 The evolution of ecological specialization is of funda- million years ago (Duda & Kohn 2005). Because of the mental concern in biology because it is likely to foster hindrance of not being able to make direct comparisons adaptive radiations and the origins of biodiversity (Schluter among a very close relative of C. leopardus, we were not 2000). The process of specialization typically includes the able to specifically examine the evolution of conotoxin loci loss or reduction of characters that result in the origin of of C. leopardus or identify changes in expression of these a narrow niche breadth (Futuyma & Moreno 1988). For genes. Nonetheless, we did compare the venom duct tran- predators, transitions from a generalist to specialist diet scriptome of C. leopardus to that of the four other Conus listed should be accompanied by the modification of characters above to determine whether the origin of a unique feeding that permit greater exploitation of a narrower breadth mode in C.