A Bacterial Filter Protects and Structures the Gut Microbiome of an Insect

The ISME Journal (2016) 10, 1866–1876 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 OPEN www.nature.com/ismej ORIGINAL ARTICLE A bacterial filter protects and structures the gut microbiome of an insect Michele Caroline Lanan1,6, Pedro Augusto Pos Rodrigues2,6, Al Agellon3, Patricia Jansma4 and Diana Esther Wheeler5 1Chair of the Natural Sciences, Deep Springs College, Big Pine, CA, USA; 2Graduate Interdisciplinary Program in Entomology and Insect Science, University of Arizona, Tucson, AZ, USA; 3School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, USA; 4Department of Neuroscience, University of Arizona, Tucson, AZ, USA and 5Department of Entomology, University of Arizona, Tucson, AZ, USA Associations with symbionts within the gut lumen of hosts are particularly prone to disruption due to the constant influx of ingested food and non-symbiotic microbes, yet we know little about how partner fidelity is maintained. Here we describe for the first time the existence of a gut morphological filter capable of protecting an animal gut microbiome from disruption. The proventriculus, a valve located between the crop and midgut of insects, functions as a micro-pore filter in the Sonoran Desert turtle ant (Cephalotes rohweri), blocking the entry of bacteria and particles ⩾ 0.2 μm into the midgut and hindgut while allowing passage of dissolved nutrients. Initial establishment of symbiotic gut bacteria occurs within the first few hours after pupation via oral–rectal trophallaxis, before the proventricular filter develops. Cephalotes ants are remarkable for having maintained a consistent core gut microbiome over evolutionary time and this partner fidelity is likely enabled by the proventricular filtering mechanism. In addition, the structure and function of the cephalotine proventriculus offers a new perspective on organismal resistance to pathogenic microbes, structuring of gut microbial communities, and development and maintenance of host–microbe fidelity both during the animal life cycle and over evolutionary time. The ISME Journal (2016) 10, 1866–1876; doi:10.1038/ismej.2015.264; published online 12 February 2016 Introduction consistent downstream flow of ingested food and microorganisms, how does the host maintain partner Nutritional mutualisms between animals and fidelity with its beneficial microbes? microbes are widespread (Backhed et al., 2005; In general, the composition of gut microbiomes is Hongoh, 2011), often taking place in the alimentary known to be structured through diet (Muegge et al., canal where microbes can have an important role in 2011), gut physiology (Kwong and Moran, 2015) and food digestion (McFall-Ngai et al., 2013). Maintenance compartmentalization (Engel and Moran, 2013), of fidelity between mutualistic partners seems avoidance of parasites through hygienic behavior straightforward for obligate endosymbionts that are (Cremer and Sixt, 2009), physical barriers (for cosseted inside special cells or organs associated with example, peritrophic matrix (Hegedus et al., 2009)) the gut (Moran et al., 2008). In contrast, microbes in and innate immune systems (Nyholm and Graf, the lumen of the one-way bilaterian gut generally face 2012). Here we report on a novel means of host a downstream flow of ingested content that may flush manipulation of gut microbiota: an anatomical filter away resident bacteria (for example, Nyholm and capable of protecting the host and their microbiota McFall-Ngai, 2004; Blum et al., 2013) or introduce from disturbance by non-symbiotic microbes and non-symbiotic and pathogenic microorganisms that likely involved in promoting high specificity can be harmful for both the host and the resident between host and symbiotic microbiota over bacterial community (for example, Nelson et al., 2012; evolutionary time. Jones et al., 2013; Cariveau et al., 2014). With this Turtle ants in the genus Cephalotes (118 species) consume a mostly herbivorous diet (Russell et al., 2009) supplemented by pollen, bird feces and Correspondence: MC Lanan, Chair of the Natural Sciences, Deep vertebrate urine (Baroni Urbani and de Andrade, Springs College, Big Pine, CA 93513, USA. 1997; Powell, 2008). Cephalotes hosts 16–20 core E-mail: [email protected] bacterial strains (Hu et al., 2014; Sanders et al., 2014) 6These two authors contributed equally to this work. Received 4 October 2015; revised 2 December 2015; accepted that are present in large numbers in the midgut and 14 December 2015; published online 12 February 2016 hindgut lumen (Roche and Wheeler, 1997) and likely A bacterial filter protects an insect’s gut microbiome MC Lanan et al 1867 have an important role in host nutrition (Russell et al., and summer of 2012 and 2013. Colonies live in 2009; Anderson et al., 2012). Microbial communities cavities bored by beetles into the branches of the tree in Cephalotes are highly similar among nestmates, Cercidium microphylla. During warm months, work- within and between species (Hu et al., 2014), and ers forage on the branches and leaves and rarely visit have codiversified with their hosts indicating a the ground. The average distance between collected history of vertical transmission at the colony level colonies was 0.5 miles and trees were spaced widely (Sanders et al., 2014). Such vertical transmission of in the habitat, ensuring that we used separate colonies. microbes is usually associated with obligate intracel- Colonies were maintained in the laboratory in nests lular symbionts (Moran et al., 2008; Engel and Moran, consisting of a wood cutout between two sheets of 2013) rather than inhabitants of the digestive tract, plexiglass inside a fluon-painted acrylic box, reared at where horizontal acquisition of microbes with ~ 25 °C, provided with water in cotton-stoppered test ingested food is frequently observed (Pernice et al., tubes, and fed a diet of freeze-killed, bisected 2014). This level of core gut microbiota stability cockroaches and 20% honey water. over evolutionary time is quite unusual, and the mechanism enabling this stability is unclear (Hu et al., 2014; Sanders et al., 2014). Imaging We investigated the function of the proventriculus, All specimens used for scanning electron microscopy a valve in the gut of Cephalotes rohweri,to were dissected from freshly killed ants and dehy- determine whether it could serve as the mechanism drated in an ethanol series followed by critical point for maintaining gut microbiota partner fidelity. drying. We then microdissected the dry specimens The proventriculus separates the crop and midgut and mounted them on stubs with conductive carbon in insects and exhibits a remarkably divergent shape adhesive tape, followed by sputter coating with in Cephalotes ants, the function of which has been platinum. All samples were imaged with a Hitachi speculated on (Baroni Urbani and de Andrade, 1997; S-4800 Type II Field Emission Scanning Electron Roche and Wheeler, 1997) but remains unknown. Microscope (Hitachi High Technologies America Inc., A symbiont sorting mechanism has been recently Pleasanton, CA, USA). The specimen in Figure 1c was described in the bean bug Riptortus pedestris—a gut treated with 10% KOH for 24 h, and then washed in constriction that is only permeable to the passage of Milli-Q (Millipore Corporation, EMD Millipore, Merck their gut symbiont, an environmentally acquired KGaA, Darmstadt, Germany) water for 24 h before bacterium in the genus Burkholderia (Ohbayashi dehydration to remove all tissue except the cuticular et al., 2015). Unlike R. pedestris, Cephalotes gut layer comprising the upper surface of the proventri- symbionts are maternally inherited and consistently culus and wall of the crop. found across different species of Cephalotes. Gut specimens for transmission electron micro- A second distinction is that while Burkholderia is scopy (TEM) imaging were dissected from ants hosted in a special, isolated portion of the gut in and fixed in 4% formaldehyde, 0.5% glutaraldehyde R. pedestris, the symbiont community of turtle ants in 0.1M phosphate buffer (pH 7.0) for 8 h at 4 °C, is found in the lumen throughout the alimentary rinsed in buffer and fixed in 2% osmium tetroxide canal and in constant exposure to ingested food. In for 30 min. Specimens were again rinsed and then host-gut symbiont systems, ingested, non-symbiotic dehydrated in an ethanol series followed by acetone, microorganisms may cause detrimental changes in infiltrated in a series of Spurr’s resin/acetone the community structure of the resident bacteria, mixtures before being embedded in Spurr’s. resulting in lower immune response and develop- Ultrathin sections were cut with a diamond blade ment of diseases in the host (Sansonetti, 2004; and ultramicrotome, placed on formvar film grids, Stecher et al., 2013). The Cephalotes proventriculus stained with uranyl acetate and lead citrate, and has previously been demonstrated to filter larger imaged with a Philips CM-12 TEM (FEI, Hillsboro, solid particles (412 μm, (Roche and Wheeler, 1997)) OR, USA). Light microscopy specimens were and we hypothesize that it may also prevent particles embedded as above for TEM, but with chlorazol as small as bacteria from transiting the gut. To test black and 1% methylene blue as stains. Images in this hypothesis, we investigated the morphology of Figures 1b and e and Figures 4a and d were colorized the proventriculus, its porosity and its association in Adobe Photoshop CS6 (Adobe Systems Incorpo- with the microbiome structure within

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