Chang and Duda BMC Evolutionary Biology (2016) 16:27 DOI 10.1186/s12862-016-0592-5 RESEARCH ARTICLE Open Access Age-related association of venom gene expression and diet of predatory gastropods Dan Chang1,2,4* and Thomas F. Duda Jr1,3 Abstract Background: Venomous organisms serve as wonderful systems to study the evolution and expression of genes that are directly associated with prey capture. To evaluate the relationship between venom gene expression and prey utilization, we examined these features among individuals of different ages of the venomous, worm-eating marine snail Conus ebraeus. We determined expression levels of six genes that encode venom components, used a DNA-based approach to evaluate the identity of prey items, and compared patterns of venom gene expression and dietary specialization. Results: C. ebraeus exhibits two major shifts in diet with age—an initial transition from a relatively broad dietary breadth to a narrower one and then a return to a broader diet. Venom gene expression patterns also change with growth. All six venom genes are up-regulated in small individuals, down-regulated in medium-sized individuals, and then either up-regulated or continued to be down-regulated in members of the largest size class. Venom gene expression is not significantly different among individuals consuming different types of prey, but instead is coupled and slightly delayed with shifts in prey diversity. Conclusion: These results imply that changes in gene expression contribute to intraspecific variation of venom composition and that gene expression patterns respond to changes in the diversity of food resources during different growth stages. Keywords: Conus, Conotoxin, Developmental plasticity, Predator–prey interactions Background fed with the same food items as those consumed by Phenotypes for resource acquisition may evolve in re- human and chimp, levels of differential expression in liver sponse to changes in resource availability or utility. Gill tissues of these mice are comparable to levels of differen- rakers of alewives [1], drilling behavior of marine snails tial expression between liver tissues of human and chimp [2], venoms of snakes [3–5] and beaks of Darwin’sfinches [9]. This indicates that dietary changes exert more influ- [6] all exhibit specific phenotypes that correspond to ence on gene expression than the inherent regulating particular resources. But the genetic mechanisms under- mechanisms among species. To understand the genetic lying these phenotypic changes are mostly unknown. In mechanisms underlying the dynamics of predator–prey addition to non-synonymous mutations, gene regulation interactions, it is essential to evaluate the effect of diets on also influences phenotypic changes. Expression of genes expression of genes that are directly involved in resource that contribute to the ability to consume particular utilization. resources is often regulated by characteristics of the The developmental process, accompanied by drastic resources [7, 8]. For example, for groups of mice that are changes of phenotypes with age, represents an ideal case to explore the connection between gene expression and * Correspondence: [email protected] environmental cues [10]. Predatory marine snails of the 1Department of Ecology and Evolutionary Biology and Museum of Zoology, family Conidae (‘cone snails’) exhibit particular dietary University of Michigan, Ann Arbor, Michigan, USA 2Department of Statistics, University of Michigan, Ann Arbor, Michigan, USA changes that are associated with increase in body size Full list of author information is available at the end of the article [11, 12], and many venom genes used for predation have © 2016 Chang and Duda. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chang and Duda BMC Evolutionary Biology (2016) 16:27 Page 2 of 12 already been well characterized [13, 14]. Leviten [11] After collecting feces we returned most samples back to suggested that diets of vermivorous Conus species shift their original collecting location at Pago Bay, and dis- from being trophic specialists as juveniles, to generalists sected 60 samples following the permit of Guam Depart- as subadults, and then to specialists as adults. Characters ment of Agriculture. We determined sexual maturity associated with predation also exhibit vast changes dur- and sex of each specimen based on the presence/absence ing development. For example, radular teeth of Conus of a penis. We preserved venom ducts in RNAlater magus that are used to inject venom into prey, are mor- (Ambion, Inc.) and stored them at −20 °C prior to prep- phologically distinct in juvenile and adult stages that aration of cDNA. specialize on polychaetes and fish respectively [15–17]. Cone snails use venom, a cocktail of numerous com- Identification of prey items pounds including conotoxins, to capture prey and, for We examined feces from 243 individuals with microscopy some species, to defend against predators [13, 18]. Con- to determine tentative identifications of prey items. Then otoxins genes undergo extensive gene duplication and we used a DNA-based approach described by Duda et al. rapid evolution [19, 20], and their expression patterns [24] to further evaluate identifications. In brief, we ob- are highly divergent among species [21–23]. Similar to tained sequences of a region of the mitochondrial 16S the changes of radula teeth through development, the ribosomal RNA gene from DNA extracts of feces, and quantity and diversity of conotoxins may change with aligned these with 16S rRNA sequences of polychaetes growth, but no prior study has tested this hypothesis. downloaded from GenBank (accession numbers shown in To investigate patterns of changes of conotoxin gene Fig. 1) in Se-Al 2.0 [29]. We obtained the relative positions expression and diet among individuals of different shell of fecal sequences in neighbor-joining trees and from sizes, we chose Conus ebraeus, a vermivorous species, as these results assigned fecal sequences to major taxonomic our study organism. This species is abundant at numer- groups of Polychaeta (e.g., Eunicida, Nereididae and Sylli- ous shallow water sites in the Indo-West Pacific and its dae). We selected the best substitution models with the diet has been studied previously [11, 12, 24]. We also Bayesian Information Criterion in jModelTest v0.1.1 [30] have sequence alignments of several conotoxin genes for alignments of 16S gene sequences for each of the taxo- from previous population genetic and molecular evolu- nomic groups, and built Bayesian consensus phylogenies tion studies of this species [22, 24–27], all of which for each group separately with these models (10,000,000 facilitate the experimental design of this study. generations, two runs, four chains, 25 % burn-in) in We specifically addressed the following questions. Do MrBayes v3.1.2 [31]. We ultimately determined the iden- conotoxin gene expression patterns differ among individ- tity of prey species based on the sequence similarity and uals of different ages? If so, how does expression change phylogenetic positions of fecal sequences with sequences through time? Are some genes uniquely expressed only in of known or pre-defined polychaete species in these particular stages? Do shifts in conotoxin gene expression estimated species phylogenies. patterns correspond with shifts in diet? If so, are the diet- ary shifts associated with changes in the types of prey or Analysis of dietary data diversities of prey items? To answer these questions, we Shell lengths provide an approximate estimate of ages of collected individuals from a single population at Guam, Conus individuals [11, 12]. To determine if individuals of determined the identity of prey species based on micro- different sizes show differences in prey selection, we scopic examination of feces and a DNA-based approach, performed one-way Analysis of Variance (ANOVA) of quantified conotoxin gene expression levels among indi- shell lengths of individuals consuming different prey viduals of different shell sizes, and evaluated the relation- species and higher taxonomic levels with the function ship between shifts in conotoxin gene expression and diet. lm in R v2.15.0 [32]. To identify patterns of transition in dietary composition, we built a heatmap of percentages Methods of each prey species captured by individuals of a specific Specimens shell length bin with the heatmap.2 function in the We collected specimens of C. ebraeus at Pago Bay, gplots package [33]. We used Shannon-Weiner (H’) [34] Guam in May 2010. We measured shell lengths of each and Gini-Simpson’s(S) [35] indices and average genetic specimen upon collection. We placed individual speci- distances (GD) to quantify levels of prey diversity in each mens in separate cups that contained enough seawater size bin. The two parameters H’ and S were estimated to cover the animal, collected feces upon defecation, and with the function diversity in the package vegan [36]. To preserved feces in the 95 % ethanol. Members of this calculate GD, we estimated pairwise genetic distances of species typically consume only one prey item every other the mitochondrial 16S rRNA sequences of prey species night [28], and therefore each of our fecal samples usu- with the Tamura-Nei [37] + G distance model and complete ally contains the remains of a single prey individual [24]. deletion of gaps in MEGA 5.05 [38], and computed the Chang and Duda BMC Evolutionary Biology (2016) 16:27 Page 3 of 12 Fig. 1 Phylogenies of 16S rRNA sequences of fecal samples of C. ebraeus and known polychaete species. Bayesian posterior probabilities are labeled at nodes of major clades.