Expression analysis and phylogeny studies based on de novo assembled transcript libraries of leaf beetle larvae

Chrysomelina larvae possess a sophisticated strategy in terms of chemical defense adapted to their natural habitat. In case of a predatory attack, the deterrent secretions are presented from dorsal glands. Three different ways to produce the repellent glucosides are proposed. Irrespective of the deterrent production, all larvae show common principles: the glucosidically bound precursors are transferred via the hemolymph into the glandular reservoir. Until recently, almost no genomic and transcriptomic data were available for leaf beetles. In order to comprehensively study the defense metabolism with respect to transport proteins in leaf beetle species, the transcriptomes of Phaedon cochleariae as well as Chrysomela populi were sequenced by applying next-generation sequencing technologies. The transcript sequences of these leaf beetles were assembled de novo to provide essential transcript catalogues. These sequences were then annotated and putative functions assigned. Thereafter, in addition to phylogenetic studies, RNA interference (RNAi) methodology was applied to characterize particular enzymes and proteins. To circumvent the knock-down of non-target transcripts, an off-target prediction method was implemented. Combining RNA-seq with RNAi, and subsequently studying differential expression, promoted the observation of enzymes involved in the sequestration process. Two enzymes namely a salicyl alcohol oxidase in C. populi and a juvenile hormone-binding protein of P. cochleariae were observed, RNAi targeting the most abundant sugar transporters in P. cochleariae revealed a counter-regulation which ensures the defense ability, and RNAi targeting the most abundant glandular ABC transporter in C. populi validated its significant function. In conclusion, sequencing of transcriptomes and RNA-seq after triggering RNAi followed by the analysis of differential gene expression holds great opportunities for the study of genes of interest in non-model organisms

AmpliSeq screening of genes encoding the C-Type lectin receptors and their signaling components reveals a common variant in MASP1 associated with pulmonary tuberculosis in an Indian population

Tuberculosis (TB) is a multifactorial disease governed by bacterial, host and environmental factors. On the host side, growing evidence shows the crucial role that genetic variants play in the susceptibility to Mycobacterium tuberculosis (Mtb) infection. Such polymorphisms have been described in genes encoding for different cytokines and pattern recognition receptors (PRR), including numerous Toll-like receptors (TLRs). In recent years, several members of the C-type lectin receptors (CTLRs) have been identified as key PRRs in TB pathogenesis. Nevertheless, studies to date have only addressed particular genetic polymorphisms in these receptors or their related pathways in relation with TB. In the present study, we screened the main CTLR gene clusters as well as CTLR pathway-related genes for genetic variation associated with pulmonary tuberculosis (PTB). This case-control study comprised 144 newly diagnosed pulmonary TB patients and 181 healthy controls recruited at the Bhagwan Mahavir Medical Research Center (BMMRC), Hyderabad, India. A two-stage study was employed in which an explorative AmpliSeq-based screening was followed by a validation phase using iPLEX MassARRAY. Our results revealed one SNP (rs3774275) in MASP1 significantly associated with PTB in our population (joint analysis p = 0.0028). Furthermore, serum levels of MASP1 were significantly elevated in TB patients when compared to healthy controls. Moreover, in the present study we could observe an impact of increased MASP1 levels on the lectin pathway complement activity in vitro. In conclusion, our results demonstrate a significant association of MASP1 polymorphism rs3774275 and MASP1 serum levels with the development of pulmonary TB. The present work contributes to our understanding of host-Mtb interaction and reinforces the critical significance of mannose-binding lectin and the lectin-complement pathway in Mtb pathogenesis. Moreover, it proposes a MASP1 polymorphism as a potential genetic marker for TB resistance

Independently recruited oxidases from the glucose-methanol-choline oxidoreductase family enabled chemical defences in leaf beetle larvae (subtribe Chrysomelina) to evolve

Larvae of the leaf beetle subtribe Chrysomelina sensu stricto repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors (e.g. salicylaldehyde). The autonomous production of iridoids, as in Phaedon cochleariae, is the ancestral chrysomeline chemical defence and predates the evolution of salicylaldehyde-based defence. Both biosynthesis strategies include an oxidative step of an alcohol intermediate. In salicylaldehyde-producing species, this step is catalysed by salicyl alcohol oxidases (SAOs) of the glucose-methanol-choline (GMC) oxidoreductase superfamily, but the enzyme oxidizing the iridoid precursor is unknown. Here, we show by in vitro as well as in vivo experiments that P. cochleariae also uses an oxidase from the GMC superfamily for defensive purposes. However, our phylogenetic analysis of chrysomeline GMC oxidoreductases revealed that the oxidase of the iridoid pathway originated from a GMC clade different from that of the SAOs. Thus, the evolution of a host-independent chemical defence followed by a shift to a host-dependent chemical defence in chrysomeline beetles coincided with the utilization of genes from different GMC subfamilies. These findings illustrate the importance of the GMC multi-gene family for adaptive processes in plant–insect interactions

Bacterial colonization dynamics and antibiotic resistance gene dissemination in the hospital environment after first patient occupancy: a longitudinal metagenetic study

Background!#!Humans spend the bulk of their time in indoor environments. This space is shared with an indoor ecosystem of microorganisms, which are in continuous exchange with the human inhabitants. In the particular case of hospitals, the environmental microorganisms may influence patient recovery and outcome. An understanding of the bacterial community structure in the hospital environment is pivotal for the prevention of hospital-acquired infections and the dissemination of antibiotic resistance genes. In this study, we performed a longitudinal metagenetic approach in a newly opened ward at the Charité Hospital (Berlin) to characterize the dynamics of the bacterial colonization process in the hospital environment after first patient occupancy.!##!Results!#!The sequencing data showed a site-specific taxonomic succession, which led to stable community structures after only a few weeks. This data was further supported by network analysis and beta-diversity metrics. Furthermore, the fast colonization process was characterized by a significant increase of the bacterial biomass and its alpha-diversity. The compositional dynamics could be linked to the exchange with the patient microbiota. Over a time course of 30 weeks, we did not detect a rise of pathogenic bacteria in the hospital environment, but a significant increase of antibiotic resistance determinants on the hospital floor.!##!Conclusions!#!The results presented in this study provide new insights into different aspects of the environmental microbiome in the clinical setting, and will help to adopt infection control strategies in hospitals and health care-related buildings. Video Abstract

Putative Sugar Transporters of the Mustard Leaf Beetle <i>Phaedon cochleariae</i>: Their Phylogeny and Role for Nutrient Supply in Larval Defensive Glands

<div><p>Background</p><p>Phytophagous insects have emerged successfully on the planet also because of the development of diverse and often astonishing defensive strategies against their enemies. The larvae of the mustard leaf beetle <i>Phaedon cochleariae</i>, for example, secrete deterrents from specialized defensive glands on their back. The secretion process involves ATP-binding cassette transporters. Therefore, sugar as one of the major energy sources to fuel the ATP synthesis for the cellular metabolism and transport processes, has to be present in the defensive glands. However, the role of sugar transporters for the production of defensive secretions was not addressed until now.</p><p>Results</p><p>To identify sugar transporters in <i>P. cochleariae</i>, a transcript catalogue was created by Illumina sequencing of cDNA libraries. A total of 68,667 transcripts were identified and 68 proteins were annotated as either members of the solute carrier 2 (SLC2) family or trehalose transporters. Phylogenetic analyses revealed an extension of the mammalian GLUT6/8 class in insects as well as one group of transporters exhibiting distinctive conserved motifs only present in the insect order Coleoptera. RNA-seq data of samples derived from the defensive glands revealed six transcripts encoding sugar transporters with more than 3,000 counts. Two of them are exclusively expressed in the glandular tissue. Reduction in secretions production was accomplished by silencing two of four selected transporters. RNA-seq experiments of transporter-silenced larvae showed the down-regulation of the silenced transporter but concurrently the up-regulation of other SLC2 transporters suggesting an adaptive system to maintain sugar homeostasis in the defensive glands.</p><p>Conclusion</p><p>We provide the first comprehensive phylogenetic study of the SLC2 family in a phytophagous beetle species. RNAi and RNA-seq experiments underline the importance of SLC2 transporters in defensive glands to achieve a chemical defense for successful competitive interaction in natural ecosystems.</p></div

The phylogenetic tree of the nine sequences derived from <i>P. cochleariae</i> belonging to the purple branch (see <b>Figure 3</b> and <b>4</b>, Figure S4) and homologous sequences derived from the whole tree of life, especially from <i>Dendroctonus ponderosae</i> (Dc) as well as from <i>Tribolium castaneum</i> (Tc), was calculated using RaxML.

<p>Indicated in purple, it can be seen that the beetles’ sequences build up a separate branch.</p

Heatmap of the variance stabilization transformed data (vsd) of ds<i>Pcsut2</i>-injected <i>versus</i> ds<i>gfp</i>-injected samples.

<p>Samples derived from glandular tissue. For further explanation see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084461#pone-0084461-g008" target="_blank">Figure 8</a>.</p

Heatmap of the variance stabilization transformed data (vsd) of ds<i>Pcsut6</i>-injected <i>vs</i>. ds<i>gfp</i>-injected samples.

<p>Samples derived from glandular tissue. For further explanation see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084461#pone-0084461-g008" target="_blank">Figure 8</a>.</p

Number of assembled transcripts and average length after assembly and reassembly showing the usefulness of reassembling.

<p>Number of assembled transcripts and average length after assembly and reassembly showing the usefulness of reassembling.</p

Heatmap of the variance stabilization transformed data (vsd) of ds<i>Pcsut1</i>-injected <i>vs</i>. ds<i>gfp</i>-injected samples.

<p>Samples derived from glandular tissue. For this, the transcript counts of the sugar transporters of each sample after dsRNA-injection have been normalized to the effective library size and the variance over all samples has been stabilized by applying the DESeq package. For each heatmap, the 30 most abundant sugar transporter transcripts are shown. Ds<i>gfp</i>-injected samples are the same in each heatmap.</p