Maximum likelihood phylogenetic trees of defensin (A) and hemiptericin (B) transcripts.

<p>Sequences recovered in this study are marked with a grey diamond, sequences used as an outgroup are marked in grey font. Bootstrap values above 50% are given at the nodes (based on 500 replicates).</p

Awareness:Tactility and Experience as Transformational Stategy

<div><p>The acquisition and vertical transmission of bacterial symbionts plays an important role in insect evolution and ecology. However, the molecular mechanisms underlying the stable maintenance and control of mutualistic bacteria remain poorly understood. The cotton stainer <i>Dysdercus fasciatus</i> harbours the actinobacterial symbionts <i>Coriobacterium glomerans</i> and <i>Gordonibacter</i> sp. in its midgut. The symbionts supplement limiting B vitamins and thereby significantly contribute to the host’s fitness. In this study, we experimentally disrupted the symbionts’ vertical transmission route and performed comparative transcriptomic analyses of genes expressed in the gut of aposymbiotic (symbiont-free) and control individuals to study the host immune response in presence and absence of the mutualists. Annotation of assembled cDNA reads identified a considerable number of genes involved in the innate immune system, including different protein isoforms of several immune effector proteins (specifically i-type lysozyme, defensin, hemiptericin, and pyrrhocoricin), suggesting the possibility for a highly differentiated response towards the complex resident microbial community. Gene expression analyses revealed a constitutive expression of transcripts involved in signal transduction of the main insect immune pathways, but differential expression of certain antimicrobial peptide genes. Specifically, qPCRs confirmed the significant down-regulation of c-type lysozyme and up-regulation of hemiptericin in aposymbiotic individuals. The high expression of c-type lysozyme in symbiont-containing bugs may serve to lyse symbiont cells and thereby harvest B-vitamins that are necessary for subsistence on the deficient diet of Malvales seeds. Our findings suggest a sophisticated host response to perturbation of the symbiotic gut microbiota, indicating that the innate immune system not only plays an important role in combating pathogens, but also serves as a communication interface between host and symbionts.</p></div

Annotation and normalized expression values (RPKM) of transcripts putatively involved in recognition and signalling of the innate immune response.

<p>Differentially expressed genes (>2 fold) are marked with asterisks.</p><p>Annotation and normalized expression values (RPKM) of transcripts putatively involved in recognition and signalling of the innate immune response.</p

Relative expression of antimicrobial peptides in symbiotic and aposymbiotic bugs based on qPCR experiments.

<p>Expression values were calculated as the dCT based on the expression of a housekeeping gene (60S ribosomal protein L13a). Grey boxes represent expression values of symbiotic bugs, open boxes expression values of aposymbionts. Significant differential expression is indicated with an asterisk (Wilcoxon signed rank tests, p<0.05).</p

Supplemental_data2_Transcript_annotation

Transcript annotatio

Supplemental_data1_Transcript_contigs

Supplemental_data1_Transcript_contig

Transcription of selected long asRNAs (lasRNAs):

<p>(A) Internalin protein; (B) Internalin protein (note the different scales of x-axis); (C) a novel long antisense transcript with more than 2,400–3,800 nt; (D) predicted SAM-dependent methyltransferase; (E) a rRNA methylase homolog; (F) similar to a methylated DNA protein cystein methyltransferase (note the different scales of x-axis). The upper half of each transcription profile represents the plus strand and the lower one the minus strand. Number of displayed reads is limited to 20. Dark purple – detected ncRNA candidates; lightgreen – NCBI annotation; darkgreen – BacProt annotation; black – reads of the extracellular library; dark blue – reads of the intracellular library; violet – locally stable secondary structure (analyzed with RNALfold); blue – conserved region among other <i>L. monocytogenes</i> serotypes (analyzed with POMAGO); cyan blue – potential new ncRNAs predicted by RNAz; pink – annotated ncRNAs. A better resolution of the figure can be found in the supplement.</p

Scoring system.

<p>For evaluation of the ncRNA candidates, a scoring system retrieved from known ncRNAs (Rfam, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-ToledoArana1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Wurtzel1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Hain4" target="_blank">[26]</a>, see supplemental material) was developed. For increasing length, number of reads and GC content, scores are summed up along the column; for example, an ncRNA candidate of length 100 nt receives a score of +1. The higher the score of a candidate, the higher its probability to be an ncRNA.</p><p>Scoring system.</p

Comparative analysis of ncRNA transcriptome data:

<p>Comparison of our ncRNA candidates with results of previous studies performed by Toledo-Arana <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-ToledoArana1" target="_blank">[13]</a>, Mraheil <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Mraheil1" target="_blank">[11]</a> and Wurtzel <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108639#pone.0108639-Wurtzel1" target="_blank">[15]</a>. Note that whenever an ncRNA prediction of this study overlaps with multiple previously described candidates, it is a single hit in the diagram. Altogether, including previous literature, Rfam and this work, now 741 putative ncRNAs are described. In this work we defined 611 to be putative ncRNAs, of which 474 ncRNAs are not part of previous literature, 33 of them known ncRNAs from Rfam.</p

Overview of RNA-seq libraries.

<p>Libraries were retrieved by next generation semiconductor sequencing technology. Number of reads before and after clipping and their mean length.</p><p>Overview of RNA-seq libraries.</p