Bacterial Community and PHB-Accumulating Bacteria Associated with the Wall and Specialized Niches of the Hindgut of the Forest Cockchafer (Melolontha Hippocastani)
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fmicb-08-00291 February 25, 2017 Time: 15:46 # 1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Frontiers - Publisher Connector ORIGINAL RESEARCH published: 28 February 2017 doi: 10.3389/fmicb.2017.00291 Bacterial Community and PHB-Accumulating Bacteria Associated with the Wall and Specialized Niches of the Hindgut of the Forest Cockchafer (Melolontha hippocastani) Pol Alonso-Pernas1*†, Erika Arias-Cordero1†, Alexey Novoselov1, Christina Ebert2,3, Jürgen Rybak4, Martin Kaltenpoth5, Martin Westermann6, Ute Neugebauer2,3 and Edited by: Wilhelm Boland1* Martin Grube, University of Graz, Austria 1 Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Jena, Germany, 2 Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany, 3 Leibniz Institute of Photonic Technology, Jena, Germany, Reviewed by: 4 Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany, 5 Department of Armin Erlacher, Evolutionary Ecology, Institute of Zoology, Johannes Gutenberg University Mainz, Mainz, Germany, 6 Electron Microscopy Graz University of Technology, Austria Center, Jena University Hospital, Jena, Germany Tomislav Cernava, Austrian Centre of Industrial Biotechnology, Austria A characterization of the bacterial community of the hindgut wall of two larval and *Correspondence: the adult stages of the forest cockchafer (Melolontha hippocastani) was carried Pol Alonso-Pernas [email protected] out using amplicon sequencing of the 16S rRNA gene fragment. We found that, Wilhelm Boland in second-instar larvae, Caulobacteraceae and Pseudomonadaceae showed the [email protected] highest relative abundances, while in third-instar larvae, the dominant families were †These authors have contributed equally to this work. Porphyromonadaceae and Bacteroidales-related. In adults, an increase of the relative abundance of Bacteroidetes, Proteobacteria (g- and d- classes) and the family Specialty section: Enterococcaceae (Firmicutes) was observed. This suggests that the composition of the This article was submitted to Microbial Symbioses, hindgut wall community may depend on the insect’s life stage. Additionally, specialized a section of the journal bacterial niches hitherto very poorly described in the literature were spotted at both Frontiers in Microbiology sides of the distal part of the hindgut chamber. We named these structures “pockets.” Received: 01 May 2016 Amplicon sequencing of the 16S rRNA gene fragment revealed that the pockets Accepted: 13 February 2017 Published: 28 February 2017 contained a different bacterial community than the surrounding hindgut wall, dominated Citation: by Alcaligenaceae and Micrococcaceae-related families. Poly-b-hydroxybutyrate (PHB) Alonso-Pernas P, Arias-Cordero E, accumulation in the pocket was suggested in isolated Achromobacter sp. by Nile Novoselov A, Ebert C, Rybak J, Kaltenpoth M, Westermann M, Blue staining, and confirmed by gas chromatography–mass spectrometry analysis (GC- Neugebauer U and Boland W (2017) MS) on cultured bacterial mass and whole pocket tissue. Raman micro-spectroscopy Bacterial Community allowed to visualize the spatial distribution of PHB accumulating bacteria within the and PHB-Accumulating Bacteria Associated with the Wall pocket tissue. The presence of this polymer might play a role in the colonization of and Specialized Niches of the Hindgut these specialized niches. of the Forest Cockchafer (Melolontha hippocastani). Front. Microbiol. 8:291. Keywords: hindgut, Melolontha hippocastani, gut bacteria, poly-b-hydroxybutyrate, PHB, Achromobacter, Raman doi: 10.3389/fmicb.2017.00291 microscopy Frontiers in Microbiology | www.frontiersin.org 1 February 2017 | Volume 8 | Article 291 fmicb-08-00291 February 25, 2017 Time: 15:46 # 2 Alonso-Pernas et al. Hindgut Bacterial Community M. hippocastani INTRODUCTION In view of this unexpected stability of the gut microbial community, we considered appropiate to characterize the Bacteria not only thrive as free-living organisms in the bacterial communities inhabiting the hindgut wall of both environment, they also engage in complex symbiotic below- and above-ground stages of the forest cockchafer, relationships with higher organisms (Wells and Varel, 2011). thus complementing the above-mentioned midgut-based study Insects, in particular, are associated with a large diversity (Arias-Cordero et al., 2012). We put our focus on the hindgut of microorganisms that play important roles for their host’s wall itself, and also on particular bacterial niches attached physiology, ecology, and evolution. The insect gut is colonized and connected to it, at both sides of the distal part of the by a wide range of bacterial phylotypes that interact with the larval hindgut. These small structures, called from now on host and allow it to subsist on nutritionally imbalanced diets. “pockets,”have been hitherto only once described in the literature The recycling of nitrogen, the provisioning of essential amino (Wildbolz, 1954). They consist of several tubular poles connected acids and cofactors, and the digesting of recalcitrant polymers to the hindgut chamber, which contain bacterial phylotypes that in the host’s diet are among the functions for which symbiotic are minor or not detected in the hindgut wall. We detected the microorganisms play an integral role (Potrikus and Breznak, presence of PHB within the pockets, and Achromobacter sp., one 1981; Douglas, 2009; Watanabe and Tokuda, 2010), increasing of the major pocket bacterial species, is able to accumulate PHB the overall fitness of the insect host. in pure culture. This suggests that some of the pocket symbionts Typically, the insect gut is divided into three regions, may be horizontally transmitted, as previous studies found this i.e., foregut, midgut and hindgut. The symbiotic bacteria are type of inclusions in symbiotic Burkholderia of environmental either attached to the gut wall or colonize the gut as free- origin harbored in the midgut crypts of the midgut of Riptortus living organisms, usually mostly in the mid- and hindgut pedestris (Kim et al., 2013). The question of whether PHB plays a regions. The structure of these communities differs among role in host nutrition remains unknown. insect species, influenced by the host’s diet and taxon (Egert et al., 2003, 2005; Colman et al., 2012; Jones et al., 2013). In the Scarabaeidae family, the hindgut region is of special MATERIALS AND METHODS importance. It is anatomically modified to serve as fermentation chamber. This chamber, in addition to its original function, Sample Collection and DNA Extraction namely, absorbing water and salts from the gut content, is Second-instar (L2) and third-instar (L3) larvae of M. hippocastani also devoted to aiding digestion, probably with the help of and actively flying adults were collected in forests of red oak ◦ 0 00 ◦ 0 00 the fermentative bacteria that colonize it (Egert et al., 2005; in Mannheim (49 29 20 N 8 28 9 E), and Graben-Neudorf ◦ 0 00 ◦ 0 00 Huang et al., 2010; Arias-Cordero et al., 2012; Engel and (49 9 55 N 8 29 21 E), respectively, between December 2010 Moran, 2013). These microbial associates are transmitted either and May 2014. Beetles were collected at the same sites. The vertically, directly from mother to offspring, or horizontally, insects were transported alive in boxes with soil or tree leaves. ◦ that is, being taken anew from the environment by each Before dissection, the insects were kept at −20 C for 20 min host generation (Bright and Bulgheresi, 2010). In horizontally to kill them, and then rinsed three times alternately with sterile transmitted symbiosis, the host usually ingests the symbiont distilled water and 70% ethanol. Dissection was performed on along with unwanted microbes that may compete for the ice in a phosphate-buffered saline (PBS) solution. Hindguts, as colonization of the gut. The selection of the right symbiont shown between dotted lines in Figure 1D (top for larva and may depend on its phenotypic traits. Kim et al. (2013) bottom for adult), were excised, cut open, and carefully washed showed that poly-b-hydroxybutyrate (PHB) accumulation by three times with sterile PBS in order to remove any unattached the symbiont is crucial for the maintenance of host–microbe bacteria. The pockets were separated from the hindgut wall, and relationship. as much of the surrounding epithelium was removed as possible. ◦ In this study, we investigate the forest cockchafer (Melolontha Samples were stored at −20 C before DNA extraction. The day hippocastani). This scarabaeid constitutes an interesting model of the extraction, frozen samples were thawed on ice and dried at ◦ due to its particular life cycle, consisting in two well-differentiated 45 C for 90 min in a Speedvac (Concentrator 5301, Eppendorf), stages: the rhizophagous larvae spend up to 4 years underground, then crushed in a 1.5 ml tube with a sterile pestle. For 454- while the adults, after pupation, emerge from the soil and shift pyrosequencing, DNA extractions of the tissue were carried out to a diet based exclusively on foliage. To date, there is a lack using the PowerSoilTM DNA Isolation Kit (MO BIO Laboratories of comparative studies on the variation of the gut bacterial Inc., Carlsbad, CA, USA) according to the protocol provided by community associated with the transition from larva to adult. the manufacturer. Final DNA concentrations were determined Only one study addressed this question, a study conducted using a Nanovue