fmicb-07-01920 November 28, 2016 Time: 12:6 # 1 ORIGINAL RESEARCH published: 30 November 2016 doi: 10.3389/fmicb.2016.01920 Microbial Communities of Lycaenid Butterflies Do Not Correlate with Larval Diet Melissa R. L. Whitaker1,2*, Shayla Salzman1, Jon Sanders3, Martin Kaltenpoth2,4 and Naomi E. Pierce1* 1 Department of Organismic and Evolutionary Biology, Museum of Comparative Zoology, Harvard University, Cambridge, MA, USA, 2 Insect Symbiosis Research Group, Max Planck Institute for Chemical Ecology, Jena, Germany, 3 Department of Pediatrics, University of California San Diego, La Jolla, CA, USA, 4 Department for Evolutionary Ecology, Johannes Gutenberg University, Mainz, Germany Herbivores possess many counteradaptations to plant defenses, and a growing body of research describes the role of symbiotic gut bacteria in mediating herbivorous diets among insects. However, persistent bacterial symbioses have not been found in Lepidoptera, despite the fact that perhaps 99% of the species in this order are herbivorous. We surveyed bacterial communities in the guts of larvae from 31 species of lycaenid butterflies whose caterpillars had diets ranging from obligate carnivory to strict herbivory. Contrary to our expectations, we found that the bacterial communities Edited by: of carnivorous and herbivorous caterpillars do not differ in richness, diversity, or Thomas Carl Bosch, composition. Many of the observed bacterial genera are commonly found in soil and University of Kiel, Germany plant surfaces, and we detected known homopteran endosymbionts in the guts of Reviewed by: Irene Lucile Garcia Newton, homopterophagous species, suggesting that larvae acquire gut bacteria from their food Indiana University Bloomington, USA and environment. These results indicate that lycaenid butterflies do not rely on specific Nichole A. Broderick, University of Connecticut, USA bacterial symbioses to mediate their diverse diets, and provide further evidence of *Correspondence: taxonomically depauperate bacterial communities among Lepidoptera. Melissa R. L. Whitaker Keywords: Lycaenidae, Lepidoptera, gut microbiome, horizontal gene transfer, herbivory, aphytophagy [email protected] Naomi E. Pierce [email protected] INTRODUCTION Specialty section: This article was submitted to Plants present numerous nutritional and defensive challenges to potential herbivores, and a Microbial Symbioses, large and growing body of research emphasizes the role of symbiotic microbes in ameliorating a section of the journal herbivorous diets. The argument that symbiotic bacteria can facilitate herbivory among insects is Frontiers in Microbiology not new (Berenbaum, 1988; Barbosa et al., 1991; Felton and Tumlinson, 2008; Feldhaar, 2011), Received: 19 September 2016 but with advances in high-throughput sequencing technologies, it has become increasingly feasible Accepted: 15 November 2016 to describe the bacterial communities associated with insects and identify the contributions that Published: 30 November 2016 symbiotic bacteria make to host nutrition. Citation: Bacterial genomes encode vastly more diverse metabolic capabilities than eukaryotic genomes Whitaker MRL, Salzman S, (Lapierre and Gogarten, 2009), such that symbiotic gut bacteria can profoundly expand the Sanders J, Kaltenpoth M and metabolic repertoire of their insect hosts. For example, bacteria can help insects to degrade Pierce NE (2016) Microbial indigestible plant fibers: bacterially mediated cellulose degradation has been shown in beetles Communities of Lycaenid Butterflies Do Not Correlate with Larval Diet. (Bayon and Mathelin, 1980; Vazques-Arista et al., 1997), and the guts of bees are known to harbor Front. Microbiol. 7:1920. bacteria that aid in the degradation of pectin found in pollen (Engel et al., 2012). Some bacteria doi: 10.3389/fmicb.2016.01920 are capable of detoxifying plant secondary compounds such as phenolics, glucosinolates, and some Frontiers in Microbiology | www.frontiersin.org 1 November 2016 | Volume 7 | Article 1920 fmicb-07-01920 November 28, 2016 Time: 12:6 # 2 Whitaker et al. The Lycaenid Microbiome alkaloids (Hammer and Bowers, 2015), thereby enabling their arisen many times within the butterfly family Lycaenidae, insect hosts to feed on chemically defended plants (e.g., Boone most likely as a result of the close associations between et al., 2013). Other bacteria are capable of synthesizing essential lycaenid larvae and ants (Pierce, 1995). The diversity of amino acids (Baumann, 2005) and vitamins that are unavailable feeding ecologies makes the Lycaenidae an ideal system to from plant tissues and which insects are incapable of synthesizing look into a potential microbial association with diet in the on their own (Salem et al., 2014). One of the most striking Lepidoptera. examples of insect-bacterial nutritional symbiosis is the well- Most lycaenid species associate with ants during their known association between aphids and their endosymbiont larval stages. Caterpillars produce nutritious secretions from Buchnera (Douglas, 1998), with similar obligate associations in specialized exocrine glands to reward and appease ants in other sap-feeding Hemiptera. Given that bacterial symbionts exchange for ants’ protective services. Ant-lycaenid interactions can so profoundly influence the diets of their insect hosts, it are diverse, and although the majority of these associations is not surprising that many nutritional partnerships between are considered mutualistic, a number of species have switched herbivorous insects and their gut bacteria have led to host plant to parasitic lifestyles in which caterpillars enter their host specialization and expansion (Tsuchida et al., 2004; Janson et al., ants’ nests and feed either on ant regurgitations (trophallaxis) 2008; Sudakaran et al., 2015). The gut microbiomes of diverse or directly on ant brood. Other species indirectly parasitize groups of insects are now recognized as key factors in shaping their host ants by feeding on ant-tended insects such as and promoting ecological diversification (Hammer and Bowers, aphids, mealybugs, and planthoppers (Fiedler, 1991; Pierce 2015). et al., 2002). Thus lycaenid diets range from phytophagy Persistent bacterial associations are known in several major on specialized host plants to strict aphytophagy on ants insect orders, including the Coleoptera (Campbell et al., 1992; and ant-associated insects. Whereas lycaenids comprise a Scott et al., 2008; Ceja-Navarro et al., 2015), Hemiptera (Buchner, relatively small number of lepidopteran species, they represent 1965; Douglas, 1998; McCutcheon and von Dohlen, 2011; Salem a disproportionate number of carnivorous Lepidoptera (Pierce, et al., 2012), Hymenoptera (Kaltenpoth et al., 2005; Russell et al., 1995), and herbivorous and carnivorous species are dispersed 2009b; Martinson et al., 2011; Sanders et al., 2014), Diptera across the group, with at least 13 independent origins of (Behar et al., 2005; Shin et al., 2011; Wang et al., 2013), and carnivory within otherwise herbivorous clades (Pierce et al., Blattodea (Potrikus and Breznak, 1981; Brune, 2014; Hongoh, 2002). 2014). However, they have yet to be described in detail for Given the unusual diversity of feeding ecologies among Lepidoptera. The Lepidoptera form one of the largest insect lycaenids, it follows that larvae with different diets might require orders and include many economically important species that different assemblages of bacteria in their guts to modulate act as agricultural pests, conservation targets, pollinators, and their extreme diets. Herbivorous species may harbor bacteria biological models. They are also a largely herbivorous group, with that assist with degrading plant cell walls, detoxifying plant the overwhelming majority of species feeding on plants (Strong allelochemicals, or provisioning nitrogen or other nutrients that et al., 1984). are unavailable from their host plants. Conversely, maintaining The prevalence of herbivorous diets makes Lepidoptera good associations with these same gut microbes would not be beneficial candidates for investigating potential bacterial contributions to for carnivorous species that experience a different suite of mediating the challenges of herbivory. Studies of lepidopteran gut dietary challenges. Instead, carnivorous species may require microbiomes have focused largely on pest species and laboratory a gut community that is effective in fatty acid or protein models (e.g., Broderick et al., 2004; Brinkmann et al., 2008; metabolism, or degrading the chitinous exoskeletons of their Robinson et al., 2010; Belda et al., 2011; Tang et al., 2012). These insect prey. studies have found relatively simple, often transient bacterial Earlier surveys of the heritable bacteria associated with communities, suggesting that specialized bacterial associations adult lycaenid butterflies and ants (Russell et al., 2009a, 2012) may not be as important for Lepidoptera as they are in other found that lycaenids harbor only two vertically transmitted insect groups. However, most of these studies focus on a single symbionts, Wolbachia and Spiroplasma – and Wolbachia was species reared under laboratory conditions, often on artificial by far the most dominant heritable symbiont found in media (but see Hammer et al., 2014; Staudacher et al., 2016). association with the species surveyed. Wolbachia are known While other studies have looked at bacterial shifts in response reproductive manipulators in many Lepidoptera (Hurst et al., to different host plant use (Priya et al.,