Phylum-level distribution of bacterial taxa from pyrotag libraries based on the 16S rRNA gene from eggs and the guts of the first instar (L1 larvae), second instar (L2 larvae) and workers of <i>N</i>. <i>arborum</i>.

<p>For each stage, the inner pie chart corresponds to the relative abundance of reads affiliated to each phylum. The outer pie chart corresponds to the number of OTUs for each phylum as a fraction of the total number of OTUs. *The charts for L1 larvae are based on 50 sequences excluding <i>Wolbachia</i> unlike the charts for eggs, L2 larvae and workers stages which are based on subsamples of 1367 reads. “Minor phyla” correspond to the pool of all phyla with <1% of reads and ≤ 5 OTUs in well sequenced libraries (eggs, L2 larvae, workers).</p

Venn diagram of bacterial OTUs (at 97% identity) common to the pyrosequencing libraries for eggs (E), L2 larvae (L2) and workers (W) of <i>N</i>. <i>arborum</i>.

<p>Numbers in bold indicate the number of OTUs and numbers in italics the relative abundance of the corresponding OTUs in each stage where they are detected. Details of the common OTUs are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140014#pone.0140014.s003" target="_blank">S2 Table</a>.</p

Phylogenetic tree illustrating the position of Spirochaetes-related OTUs from 16S rRNA gene libraries.

<p>The tree was constructed using the Maximum Likelihood-method implemented in MEGA version 6.0 and the Tamura-Nei model (analysis, 1000 bootstraps). Only bootstrap values >50% are presented. Clones from this study are referred to as GL and GLS (from the second instar larvae) and GO (from workers) and numbers in brackets correspond to NCBI accession numbers of sequences. * indicates sequences undergoing NCBI submission. Branches in green are entirely made of sequences from termites.</p

Relative abundances of the genus-level bacterial groups in eggs (E), the first instar larvae (L1), the second instar larvae (L2) and the worker caste (W) of <i>N</i>. <i>arborum</i>.

<p>Only genera > 0.5% in at least one stage are presented. The remaining genera are included in “Others”. The detailed classification with all the taxonomic levels is provided in the interactive spreadsheet (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140014#pone.0140014.s002" target="_blank">S1 Table</a>). * The relative abundances for the L1 larval stage are based on 50 sequences excluding <i>Wolbachia</i>.</p

Sampling depth and number of taxa at OTU level (at 97% identity), at genus level and phylum level by 16S rRNA gene pyrosequencing from the various life stages of <i>N</i>. <i>arborum</i>. For eggs, L2 larvae and workers, the numbers of taxa are based on a sub-sample of 1367 reads.

<p>*For L1 larvae, they are based on 50 sequences excluding <i>Wolbachia</i>. They are given for illustration only and are not included in the total for the taxonomic composition, which refers to unique taxa.</p><p>** For the taxonomic composition, the total number of taxa at the phylum, genus and OTU levels is the sum of the individual taxa from the three pyrotag libraries, taking account of the taxa common to different stages.</p><p>Sampling depth and number of taxa at OTU level (at 97% identity), at genus level and phylum level by 16S rRNA gene pyrosequencing from the various life stages of <i>N</i>. <i>arborum</i>. For eggs, L2 larvae and workers, the numbers of taxa are based on a sub-sample of 1367 reads.</p

Co-localization of UT with γ subunits in neuron and glial components in rat cerebellum.

<p>(A, A′) Double-fluorescence staining for UT (green) and NeuN (red) showing the presence of UT in both mature (arrowhead, merge, A′) and unidentified cells (arrows, merge, A′) in the IGL. (B) Co-staining of UT and the marker of Purkinje cells, calbindin (red), in Purkinje cell soma and dendrites (arrowhead, B′). (C) Staining for UT and the marker of migrating neuroblasts doublecortin DCX (red) depicting a diffuse labeling in the ML. (C′) UT immunopositive fibers contiguous to DCX-expressing migrating granule cells (merge, yellow, arrowhead). (D, D′) Staining for UT and GFAP (red) in glial fibers (merge, yellow, arrowhead) of the ML. (E, F) Distribution of UT and the γ₁ (E) and γ₂ (F) GABA_AR subunits (red), in Purkinje cells (merge, arrowhead) and few extents of glia (merge, arrow) in the ML and IGL. Nuclei (blue) were counterstained with DAPI. Scale bars, 50 µm (A–F); 20 µm (A′–F′). EGL, external granule cell layer; IGL, internal granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. (A′–F′) images of digitally zoomed regions corresponding to the white boxes in A–F.</p

Schematic model depicting the mechanism of UT-mediated GABA_AR down-regulation.

<p>UII efficiently activates the G protein-coupled receptor UT, leading to a fast short-term decrease of the chloride current not sustained by G proteins, calcium, phosphorylation and endocytosis processes. This rapid effect involves the distal 19 C-terminal amino acids of UT and the presence of γ subunits within of the GABA_AR complex (1). During the washout period, a long-term inhibition develops <i>via</i> a dynamin-, calcium- and phosphorylation-dependent endocytic mechanisms, requiring at least in part the 351–370 sequence of UT and GABA_AR γ subunits (2). It is hypothesized that the directional cross-talk between UT and GABA_AR, and the extinction of the latter at the plasma membrane, may relay transition from quiescent to proliferant astrocytes.</p

UII-induced GABA_AR loss from the plasma membrane through the C-terminus fragment of UT in CHO.

<p>The effect of <i>h</i>UII on the proportion of GABA_AR and UT at the cell surface of CHO was assessed by ELISA. (A) CHO transiently transfected with cDNA encoding UT^c-myc and α₂β₃, or α₂β₃γ₂^HA GABA_AR subunits (left), or UT^c-myc, and α₂β₃γ₂^HA GABA_AR subunits cotransfected with the cDNA encoding UT_319–389YFP (right). Background bioluminescence (left) and fluorescence (right) were measured after anti-HA antibody and colorimetric alkaline phophatase substrate incubation, in the absence or presence of 30 min of <i>h</i>UII (10⁻⁸ M, left), or directly on a fluorescent plate reader (right). (B) CHO transiently transfected with cDNA encoding UT^c-myc and α₂β₃γ₂^HA GABA_AR subunits (left), or cotransfected with the cDNA encoding UT_319–389YFP, and immunodetected with anti-HA (left) or anti-c-myc (right) antibodies. Percentage of cell surface γ₂^HA GABA_AR subunit (left) or UT^c-myc (right) are represented as the proportion of receptor at the plasma membrane (non permeabilized cells) to the total expressed receptor (permeabilized cells). One hundred percent correspond to values in the absence of 30 min treatment with <i>h</i>UII (10⁻⁸ M, 37°C). Each bar corresponds to mean ± SEM percent obtained from 5 to 7 independent experiments, in triplicates. ns, non significant; *, <i>P</i><0.05; ***, <i>P</i><0.001.</p

UII-induced depression of GABA_AR in UT-expressing cerebellar astrocytes.

<p>(Aa, Ab) Double immunofluorescence labeling of UT (green) and the specific astrocyte marker GFAP (red, Aa), or the mature neuron marker NeuN (red, Ab) in astrocyte-neuron co-culture from P7 rat cerebellum. Astrocytes, recognized by strong GFAP staining show UT immunoreactivity (arrows), whereas few weaker UT-stained cells express NeuN (arrowheads), and were likely attributed to mature granule cells (arrowheads, Ab). Nuclei (blue) were counterstained with DAPI. Scale bars, 50 µm. (B) Phase contrast photomicrograph of astrocytes in mono-culture, or astrocytes and neurons in co-culture at 3 days <i>in vitro</i>. (C) Membrane depolarizations and currents evoked by the GABA_AR agonist isoguvacine (Iso, 10⁻⁴ M, 2 s for membrane potential and 5 s for chloride current) in astrocytes and cerebellar granule neurons before, during <i>r</i>UII (10⁻⁷ M, 40 s) application and after 2-min washout. Right, normalized amplitudes deduced by the mean Iso-evoked depolarization or current obtained before <i>r</i>UII application. (D) Concentration-response relationship of Iso-evoked currents from astrocytes yielding an EC₅₀ value of 43.6±23.7 10⁻¹² M. Data are mean ± SEM of 4 to 6 cells. *, <i>P</i><0.05; ** <i>P</i><0.01 compared with the corresponding control Iso-evoked current.</p

Receptor sequences involved in UT regulation of the GABA_AR activity.

<p>(A) Schematic diagrams mixed with sequence alignments of the HA epitope-tagged human UT, C-terminus truncated UT^HA₃₇₀, UT^HA₃₅₁, UT^HA₃₃₂, UT^HA₃₁₉ mutants, and peptidomimetics corresponding to the entire C-terminus cytosolic fragment of UT (UT^c-myc_319–389). (B and C) Traces of Iso (10^–4 M, 2 s)-evoked current before (1), during (2) a 1-min <i>h</i>UII (10⁻⁸ M) application and after 22-min washout (3). (B) Currents recorded from CHO coexpressing GABA_AR and UT^HA (Control), UT^HA₃₇₀, UT^HA₃₅₁, UT^HA₃₃₂ or UT^HA₃₁₉. Corresponding average time course of the current, in the absence or presence of UT truncated mutants. (C) Current traces recorded from CHO-UT-GABA_AR, in the absence or presence of UT^c-myc_319–389. Corresponding average time course of the Iso-evoked current, in the absence or presence of UT^c-myc_319–389. In B, significance was only annotated above the time course graph during <i>h</i>UII perfusion and after 18-min washout, for clarity. Data are mean ± SEM from 3 to 13 cells. ns, non significant; *, <i>P</i><0.05; ** <i>P</i><0.01 compared with the corresponding control Iso-evoked current.</p