Targeted rescue of synaptic plasticity improves cognitive decline in sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a frequent complication of severe systemic infection resulting in delirium, premature death, and long-term cognitive impairment. We closely mimicked SAE in a murine peritoneal contamination and infection (PCI) model. We found long-lasting synaptic pathology in the hippocampus including defective long-term synaptic plasticity, reduction of mature neuronal dendritic spines, and severely affected excitatory neurotransmission. Genes related to synaptic signaling, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and members of the transcription-regulatory EGR gene family, were downregulated. At the protein level, ARC expression and mitogen-activated protein kinase signaling in the brain were affected. For targeted rescue we used adeno-associated virus-mediated overexpression of ARC in the hippocampus in vivo. This recovered defective synaptic plasticity and improved memory dysfunction. Using the enriched environment paradigm as a non-invasive rescue intervention, we found improvement of defective long-term potentiation, memory, and anxiety. The beneficial effects of an enriched environment were accompanied by an increase in brain-derived neurotrophic factor (BDNF) and ARC expression in the hippocampus, suggesting that activation of the BDNF-TrkB pathway leads to restoration of the PCI-induced reduction of ARC. Collectively, our findings identify synaptic pathomechanisms underlying SAE and provide a conceptual approach to target SAE-induced synaptic dysfunction with potential therapeutic applications to patients with SAE.

First large-scale genomic prediction in the honey bee.

Genomic selection has increased genetic gain in several livestock species, but due to the complicated genetics and reproduction biology not yet in honey bees. Recently, 2970 queens were genotyped to gather a reference population. For the application of genomic selection in honey bees, this study analyzes the accuracy and bias of pedigree-based and genomic breeding values for honey yield, three workability traits, and two traits for resistance against the parasite Varroa destructor. For breeding value estimation, we use a honey bee-specific model with maternal and direct effects, to account for the contributions of the workers and the queen of a colony to the phenotypes. We conducted a validation for the last generation and a five-fold cross-validation. In the validation for the last generation, the accuracy of pedigree-based estimated breeding values was 0.12 for honey yield, and ranged from 0.42 to 0.61 for the workability traits. The inclusion of genomic marker data improved these accuracies to 0.23 for honey yield, and a range from 0.44 to 0.65 for the workability traits. The inclusion of genomic data did not improve the accuracy of the disease-related traits. Traits with high heritability for maternal effects compared to the heritability for direct effects showed the most promising results. For all traits except the Varroa resistance traits, the bias with genomic methods was on a similar level compared to the bias with pedigree-based BLUP. The results show that genomic selection can successfully be applied to honey bees.

Tool for genomic selection and breeding to evolutionary adaptation: Development of a 100K single nucleotide polymorphism array for the honey bee.

High-throughput high-density genotyping arrays continue to be a fast, accurate, and cost-effective method for genotyping thousands of polymorphisms in high numbers of individuals. Here, we have developed a new high-density SNP genotyping array (103,270 SNPs) for honey bees, one of the most ecologically and economically important pollinators worldwide. SNPs were detected by conducting whole-genome resequencing of 61 honey bee drones (haploid males) from throughout Europe. Selection of SNPs for the chip was done in multiple steps using several criteria. The majority of SNPs were selected based on their location within known candidate regions or genes underlying a range of honey bee traits, including hygienic behavior against pathogens, foraging, and subspecies. Additionally, markers from a GWAS of hygienic behavior against the major honey bee parasite Varroa destructor were brought over. The chip also includes SNPs associated with each of three major breeding objectives-honey yield, gentleness, and Varroa resistance. We validated the chip and make recommendations for its use by determining error rates in repeat genotypings, examining the genotyping performance of different tissues, and by testing how well different sample types represent the queen's genotype. The latter is a key test because it is highly beneficial to be able to determine the queen's genotype by nonlethal means. The array is now publicly available and we suggest it will be a useful tool in genomic selection and honey bee breeding, as well as for GWAS of different traits, and for population genomic, adaptation, and conservation questions.

Listeria monocytogenes Isolated from Illegally Imported Food Products into the European Union Harbor Different Virulence Factor Variants.

Unregulated international flow of foods poses a danger to human health, as it may be contaminated with pathogens. Recent studies have investigated neglected routes of pathogen transmission and reported the occurrence of Listeria monocytogenes in food illegally imported into the European Union (EU), either confiscated at four international airports or sold illegally on the Romanian black market. In this study we investigated the genotype diversity and the amino acid sequence variability of three main virulence factors of 57 L. monocytogenes isolates. These isolates were derived from 1474 food samples illegally imported into the EU and originated from 17 different countries. Multilocus sequence typing revealed 16 different sequence types (STs) indicating moderate genotype diversity. The most prevalent STs were ST2, ST9, and ST121. The pulsed-field gel electrophoresis (PFGE) analysis resulted in 34 unique pulsotypes. PFGE types assigned to the most prevalent STs (ST2, ST9, and ST121) were highly related in their genetic fingerprint. Internalin A (InlA) was present in 20 variants, including six truncated InlA variants, all harbored by isolates of ST9 and ST121. We detected eight ST-specific listeriolysin O (LLO) variants, and among them, one truncated form. The actin-assembly-inducing protein ActA was present in 15 different ST-specific variants, including four ActA variants with an internal truncation. In conclusion, this study shows that L. monocytogenes, isolated from illegally imported food, have moderate genotype diversity, but diverse virulence factors variants, mainly of InlA.

A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge.

Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates.

Prevalence of major foodborne pathogens in food confiscated from air passenger luggage.

The EU has issued several directives and regulations pertaining to the importation of animals and products of animal origin (POAO) and veterinary controls on importation. Unfortunately, little information is available concerning associated risks and no attempts have been made to collect baseline data on the actual prevalence of zoonotic agents in POAO carried by travellers. To meet these challenges the EU recently introduced and financed a research project "PROMISE". Its main objectives were to assess the risks involved when foodborne pathogens are introduced to the EU via uncontrolled imports. With special permission of the Austrian health authorities, spot-checks were made of the luggage of 61,355 passengers from 240 flights from non-EU countries arriving at the Vienna International Airport (VIE airport). Over a period of eight months (August 2012 through March 2013) 1473 POAO items were confiscated. A total of 600 samples were suitable for Salmonella spp., Campylobacter spp., verotoxigenic Escherichia coli and Listeria monocytogenes prevalence analysis. Foodborne pathogens could be detected in 5% (30/600) of all samples. The highest prevalence was attributed to L. monocytogenes, at 2.5%, followed by VTEC and Salmonella spp. at 1.3% and 1.2%, respectively. Campylobacter spp. was not present in any of the 600 samples. Multi-locus sequence typing (MLST) of L. monocytogenes revealed that current sequence types (ST) corresponded to the worldwide most present clonal complexes 1, 2, 3, 5, 9, and 121. Generally, L. monocytogenes ST9 was the predominant allelic profile, which was mainly isolated from Turkish meat products.

Vitamin supplementation by gut symbionts ensures metabolic homeostasis in an insect host.

Despite the demonstrated functional importance of gut microbes, our understanding of how animals regulate their metabolism in response to nutritionally beneficial symbionts remains limited. Here, we elucidate the functional importance of the African cotton stainer's (Dysdercus fasciatus) association with two actinobacterial gut symbionts and subsequently examine the insect's transcriptional response following symbiont elimination. In line with bioassays demonstrating the symbionts' contribution towards host fitness through the supplementation of B vitamins, comparative transcriptomic analyses of genes involved in import and processing of B vitamins revealed an upregulation of gene expression in aposymbiotic (symbiont-free) compared with symbiotic individuals; an expression pattern that is indicative of B vitamin deficiency in animals. Normal expression levels of these genes, however, can be restored by either artificial supplementation of B vitamins into the insect's diet or reinfection with the actinobacterial symbionts. Furthermore, the functional characterization of the differentially expressed thiamine transporter 2 through heterologous expression in Xenopus laevis oocytes confirms its role in cellular uptake of vitamin B1. These findings demonstrate that despite an extracellular localization, beneficial gut microbes can be integral to the host's metabolic homeostasis, reminiscent of bacteriome-localized intracellular mutualists.

Tissue-specific transcript profiling for ABC transporters in the sequestering larvae of the phytophagous leaf beetle Chrysomela populi.

BACKGROUND: Insects evolved ingenious adaptations to use extraordinary food sources. Particularly, the diet of herbivores enriched with noxious plant secondary metabolites requires detoxification mechanisms. Sequestration, which involves the uptake, transfer, and concentration of occasionally modified phytochemicals into specialized tissues or hemolymph, is one of the most successful detoxification strategies found in most insect orders. Due to the ability of ATP-binding cassette (ABC) carriers to transport a wide range of molecules including phytochemicals and xenobiotics, it is highly likely that they play a role in this sequestration process. To shed light on the role of ABC proteins in sequestration, we describe an inventory of putative ABC transporters in various tissues in the sequestering juvenile poplar leaf beetle, Chrysomela populi. RESULTS: In the transcriptome of C. populi, we predicted 65 ABC transporters. To link the proteins with a possible function, we performed comparative phylogenetic analyses with ABC transporters of other insects and of humans. While tissue-specific profiling of each ABC transporter subfamily suggests that ABCB, C and G influence the plant metabolite absorption in the gut, ABCC with 14 members is the preferred subfamily responsible for the excretion of these metabolites via Malpighian tubules. Moreover, salicin, which is sequestered from poplar plants, is translocated into the defensive glands for further deterrent production. In these glands and among all identified ABC transporters, an exceptionally high transcript level was observed only for Cpabc35 (Cpmrp). RNAi revealed the deficiency of other ABC pumps to compensate the function of CpABC35, demonstrating its key role during sequestration. CONCLUSION: We provide the first comprehensive phylogenetic study of the ABC family in a phytophagous beetle species. RNA-seq data from different larval tissues propose the importance of ABC pumps to achieve a homeostasis of plant-derived compounds and offer a basis for future analyses of their physiological function in sequestration processes.

ABC transporter functions as a pacemaker for sequestration of plant glucosides in leaf beetles.

Plant-herbivore interactions dominate the planet's terrestrial ecology. When it comes to host-plant specialization, insects are among the most versatile evolutionary innovators, able to disarm multiple chemical plant defenses. Sequestration is a widespread strategy to detoxify noxious metabolites, frequently for the insect's own benefit against predation. In this study, we describe the broad-spectrum ATP-binding cassette transporter CpMRP of the poplar leaf beetle, Chrysomela populi as the first candidate involved in the sequestration of phytochemicals in insects. CpMRP acts in the defensive glands of the larvae as a pacemaker for the irreversible shuttling of pre-selected metabolites from the hemolymph into defensive secretions. Silencing CpMRP in vivo creates a defenseless phenotype, indicating its role in the secretion process is crucial. In the defensive glands of related leaf beetle species, we identified sequences similar to CpMRP and assume therefore that exocrine gland-based defensive strategies, evolved by these insects to repel their enemies, rely on ABC transporters as a key element. DOI: http://dx.doi.org/10.7554/eLife.01096.001.

Prediction, microarray and northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida.

Bacterial small RNAs (sRNAs) are trans-encoded regulatory RNAs that typically bind mRNAs by short-sequence complementarities and change the expression of the corresponding proteins. Some of the well-characterized sRNAs serve critical steps in the regulation of important cellular processes, such as quorum sensing (Qrr), iron homeostasis (RyhB), oxidative stress (OxyS), and carbon metabolism (Spot 42). However, many sRNAs remain to be identified, and the functional roles of sRNAs are known for only a small fraction. For example, of the hundreds of candidate sRNAs from members of the bacterial family Vibrionaceae, the function is known for only 9. We have in this study significantly contributed to the discovery and verification of new sRNAs in a representative of Vibrionaceae, i.e. the Aliivibrio salmonicida, which causes severe disease in farmed Atlantic salmon and other fishes. A computational search for intergenic non-coding (nc) RNAs in the 4.6-Mb genome identified a total of 252 potential ncRNAs (including 233 putative sRNAs). Depending on the set threshold value for fluorescence signal in our microarray approach, we identified 50-80 putative ncRNAs, 12 of which were verified by Northern blot analysis. In total, we identified 9 new sRNAs.

Always being well prepared for defense: the production of deterrents by juvenile Chrysomelina beetles (Chrysomelidae).

In response to herbivores, plants produce a variety of natural compounds. Many beetle species have developed ingenious strategies to cope with these substances, including colonizing habitats not attractive for other organisms. Leaf beetle larvae of the subtribe Chrysomelina, for example, sequester plant-derived compounds and use them for their own defense against predators. Using systematically modified structural mimics of plant-derived glucosides, we demonstrated that all tested Chrysomelina larvae channel compounds from the gut lumen into the defensive glands, where they serve as intermediates in the synthesis of deterrents. Detailed studies of the sequestration process revealed a functional network of transport processes guiding phytochemicals through the larval body. The initial uptake by the larvae's intestine seems to be fairly unspecific, which contrasts sharply with the specific import of precursors into the defensive glands. The Malpighian tubules and hind-gut organs facilitate the rapid clearing of body fluid from excess or unusable compounds. The network exists in both sequestering species and species producing deterrents de novo. Transport proteins are also required for de novo synthesis to channel intermediates from the fat body to the defensive glands for further conversion. Thus, all the tools needed to exploit host plants' chemistry by more derived Chrysomelina species are already developed by iridoid-de novo producers. Early intermediates from the iridoid-de novo synthesis which also can be sequestered are able to regulate the enzyme activity in the iridoid metabolism.