Evaluation of commercially available DNA extraction kits for the analysis of the broiler chicken cecal microbiota.

16S rRNA gene amplicon sequencing is a state of the art technology to analyze bacterial communities via microbiome profiling. Choosing an appropriate DNA extraction protocol is crucial for characterizing the microbial community and can be challenging, especially when preliminary knowledge about the sample matrix is scarce. The aim of the present study was to evaluate seven commercial DNA extraction kits suitable for 16S rRNA gene amplicon sequencing of the bacterial community of the chicken cecum, taking into account different criteria such as high technical reproducibility, high bacterial diversity and easy handling. The DNA extraction kits differed strongly with respect to extractable DNA quantity, DNA quality, technical reproducibility and bacterial diversity determined after 16S rRNA gene amplicon sequencing and subsequent bioinformatic and biostatistical data processing. While some of the DNA extraction protocols under-represented specific bacterial community members, the removal of PCR inhibitors supported technical reproducibility and subsequently enhanced the recovered bacterial diversity from the chicken cecum community. In conclusion, the removal of PCR inhibitors from the sample matrix seemed to be one of the main drivers for a consistent representation of the bacterial community even of low abundant taxa in chicken cecum samples.

A Synbiotic Formulation Comprising Bacillus subtilis DSM 32315 and L-Alanyl-L-Glutamine Improves Intestinal Butyrate Levels and Lipid Metabolism in Healthy Humans.

The gut microbiota is a crucial modulator of health effects elicited by food components, with SCFA (short chain fatty acids), especially butyrate, acting as important mediators thereof. We therefore developed a nutritional synbiotic composition targeted at shifting microbiome composition and activity towards butyrate production. An intestinal screening model was applied to identify probiotic Bacillus strains plus various amino acids and peptides with suitable effects on microbial butyrate producers and levels. A pilot study was performed to test if the synbiotic formulation could improve fecal butyrate levels in healthy humans. A combination of Bacillus subtilis DSM (Number of German Collection of Microorganisms and Cell Cultures) 32315 plus L-alanyl-L-glutamine resulted in distinctly increased levels of butyrate and butyrate-producing taxa (Clostridium group XIVa, e.g., Faecalibacterium prausnitzii), both in vitro and in humans. Moreover, circulating lipid parameters (LDL-, and total cholesterol and LDL/HDL cholesterol ratio) were significantly decreased and further metabolic effects such as glucose-modulation were observed. Fasting levels of PYY (Peptide YY) and GLP-1 (Glucagon-like Peptide 1) were significantly reduced. In conclusion, our study indicates that this synbiotic composition may provide an effective and safe tool for stimulation of intestinal butyrate production with effects on e.g., lipid and glucose homeostasis. Further investigations in larger cohorts are warranted to confirm and expand these findings.

Family matters: skin microbiome reflects the social group and spatial proximity in wild zebra finches.

BACKGROUND: So far, large numbers of studies investigating the microbiome have focused on gut microbiota and less have addressed the microbiome of the skin. Especially in avian taxa our understanding of the ecology and function of these bacteria remains incomplete. The involvement of skin bacteria in intra-specific communication has recently received attention, and has highlighted the need to understand what information is potentially being encoded in bacterial communities. Using next generation sequencing techniques, we characterised the skin microbiome of wild zebra finches, aiming to understand the impact of sex, age and group composition on skin bacteria communities. For this purpose, we sampled skin swabs from both sexes and two age classes (adults and nestlings) of 12 different zebra finch families and analysed the bacterial communities. RESULTS: Using 16S rRNA sequencing we found no effect of age, sex and family on bacterial diversity (alpha diversity). However, when comparing the composition (beta diversity), we found that animals of social groups (families) harbour highly similar bacterial communities on their skin with respect to community composition. Within families, closely related individuals shared significantly more bacterial taxa than non-related animals. In addition, we found that age (adults vs. nestlings) affected bacterial composition. Finally, we found that spatial proximity of nest sites, and therefore individuals, correlated with the skin microbiota similarity. CONCLUSIONS: Birds harbour very diverse and complex bacterial assemblages on their skin. These bacterial communities are distinguishable and characteristic for intraspecific social groups. Our findings are indicative for a family-specific skin microbiome in wild zebra finches. Genetics and the (social) environment seem to be the influential factors shaping the complex bacterial communities. Bacterial communities associated with the skin have a potential to emit volatiles and therefore these communities may play a role in intraspecific social communication, e.g. via signalling social group membership.

Impact of the dual defence system of Plantago lanceolata (Plantaginaceae) on performance, nutrient utilisation and feeding choice behaviour of Amata mogadorensis larvae (Lepidoptera, Erebidae).

Iridoid glycosides are plant defence compounds with potentially detrimental effects on non-adapted herbivores. Some plant species possess ß-glucosidases that hydrolyse iridoid glycosides and thereby release protein-denaturing aglycones. To test the hypothesis that iridoid glycosides and plant ß-glucosidases form a dual defence system, we used Plantago lanceolata and a polyphagous caterpillar species. To analyse the impact of leaf-age dependent differences in iridoid glycoside concentrations and ß-glucosidase activities on insect performance, old or young leaves were freeze-dried and incorporated into artificial diets or were provided freshly to the larvae. We determined larval consumption rates and the amounts of assimilated nitrogen. Furthermore, we quantified ß-glucosidase activities in artificial diets and fresh leaves and the amount of iridoid glycosides that larvae feeding on fresh leaves ingested and excreted. Compared to fresh leaves, caterpillars grew faster on artificial diets, on which larval weight gain correlated positively to the absorbed amount of nitrogen. When feeding fresh young leaves, larvae even lost weight and excreted only minute proportions of the ingested iridoid glycosides intact with the faeces, indicating that the hydrolysis of these compounds might have interfered with nitrogen assimilation and impaired larval growth. To disentangle physiological effects from deterrent effects of iridoid glycosides, we performed dual choice feeding assays. Young leaves, their methanolic extracts and pure catalpol reduced larval feeding in comparison to the respective controls, while aucubin had no effect on larval consumption. We conclude that the dual defence system of P. lanceolata consisting of iridoid glycosides and ß-glucosidases interferes with the nutrient utilisation via the hydrolysis of iridoid glycosides and also mediates larval feeding behaviour in a concentration- and substance-specific manner.

The effects of mineral nitrogen limitation, competition, arbuscular mycorrhiza, and their respective interactions, on morphological and chemical plant traits of Plantago lanceolata.

Plants are sessile organisms that suffer from a multitude of challenges such as abiotic stress or the interactions with competitors, antagonists and symbionts, which influence their performance as well as their eco-physiological and biochemical responses in complex ways. In particular, the combination of different stressors and their impact on plant biomass production and the plant's ability to metabolically adjust to these challenges are less well understood. To study the effects of mineral nitrogen (N) availability, interspecific competition and the association with arbuscular mycorrhizal fungi (AMF) on biomass production, biomass allocation patterns (root/shoot ratio, specific leaf area) and metabolic responses, we chose the model organism Plantago lanceolata L. (Plantaginaceae). Plants were grown in a full factorial experiment. Biomass production and its allocation patterns were assessed at harvest, and the influence of the different treatments and their interactions on the plant metabolome were analysed using a metabolic fingerprinting approach with ultra-high performance liquid chromatography coupled with time-of-flight-mass spectrometry. Limited supply of mineral N caused the most pronounced changes with respect to plant biomass and biomass allocation patterns, and altered the concentrations of more than one third of the polar plant metabolome. Competition also impaired plant biomass production, yet affected the plant metabolome to a much lesser extent than limited mineral N supply. The interaction of competition and limited mineral N supply often caused additive changes on several traits. The association with AMF did not enhance biomass production, but altered biomass allocation patterns such as the root/shoot ratio and the specific leaf area. Interestingly, we did not find significant changes in the plant metabolome caused by AMF. A targeted analysis revealed that only limited mineral N supply reduced the concentrations of one of the main target defence compounds of P. lanceolata, the iridoid glycoside catalpol. In general, the interaction of competition and limited mineral N supply led to additive changes, while the association with AMF in any case alleviated the observed stress responses. Our results show that the joint analysis of biomass/allocation patterns and metabolic traits allows a more comprehensive interpretation of plant responses to different biotic and abiotic challenges; specifically, when multiple stresses interact.

Impact of defoliation on the regrowth capacity and the shoot metabolite profile of Plantago lanceolata L.

After defoliation challenges, plants should invest their resources in a way that maximizes their fitness, which may lead to trade-offs in investment in physiological versus chemical traits. Thereby, the regrowth capacity of plants may highly depend on the type and the severity of defoliation. In the present study, we investigated the phenotypic plasticity of Plantago lanceolata L. in response to different defoliation events in a comprehensive way, measuring various traits. This herbaceous species grows on ruderal sites and typically suffers from repeated substantial losses of shoot biomass due to mowing and/or herbivory during the growing season. To study treatment-specific effects of defoliation on resource allocation and induction of defence responses, plants were exposed either to (repeated) mechanical clipping or to herbivory by the generalist Grammia incorrupta (Lepidoptera). Next to regrowth and physiological leaf traits such as the water content, the specific leaf area and C/N ratios of these plants, primary and secondary metabolites in leaves were analyzed with a metabolite profiling approach using gas chromatography coupled with mass spectrometry. The different defoliation treatments strongly affected the regrowth capacity of clipped and herbivore-treated plants, leading to additive changes in physiological leaf traits. As a response to the defoliation treatments, clipped and herbivore-treated plants reduced mainly carbon-based primary metabolites such as sorbitol, and glucose, suggesting that the different defoliation challenges similarly limited the ability of carbon fixation. Yet, only in response to herbivory but not to clipping, plants induced defence compounds, which indicates the importance of treatment-specific responses despite severe resource limitations. Overall, the different responses to defoliation by clipping and herbivory may reflect allocation constraints and/or adaptive plasticity.

Role of plant ß-glucosidases in the dual defense system of iridoid glycosides and their hydrolyzing enzymes in Plantago lanceolata and Plantago major.

The typical defense compounds of Plantaginaceae are the iridoid glycosides, which retard growth and/or enhance mortality of non-adapted herbivores. In plants, glycosidic defense compounds and hydrolytic enzymes often form a dual defense system, in which the glycosides are activated by the enzymes to exert biological effects. Yet, little is known about the activating enzymes in iridoid glycoside-containing plants. To examine the role of plant-derived ß-glucosidases in the dual defense system of two common plantain species, Plantago lanceolata and Plantago major, we determined the concentration of iridoid glycosides as well as the ß-glucosidase activity in leaves of different age. To investigate the presence of other leaf metabolites potentially involved in plant defense, we used a metabolic fingerprinting approach with ultra-high performance liquid chromatography coupled with time-of-flight-mass spectrometry. According to the optimal defense hypothesis, more valuable parts such as young leaves should be better protected than less valuable parts. Therefore, we expected that both, the concentrations of defense compounds as well as the ß-glucosidase activity, should be highest in younger leaves and decrease with increasing leaf age. Both species possessed ß-glucosidase activity, which hydrolyzed aucubin, one of the two most abundant iridoid glycosides in both plant species, with high activity. In line with the optimal defense hypothesis, the ß-glucosidase activity in both Plantago species as well as the concentration of defense-related metabolites such as iridoid glycosides correlated negatively to leaf age. When leaf extracts were incubated with bovine serum albumin and aucubin, SDS-PAGE revealed a protein-denaturing effect of the leaf extracts of both plantain species, suggesting that iridoid glycosides and plant ß-glucosidase interact in a dual defense system.

The interplay between toxin-releasing ß-glucosidase and plant iridoid glycosides impairs larval development in a generalist caterpillar, Grammia incorrupta (Arctiidae).

Herbivores with polyphagous feeding habits must cope with a diet that varies in quality. One of the most important sources of this variation in host plant suitability is plant secondary chemistry. We examined how feeding on plants containing one such group of compounds, the iridoid glycosides, might affect the growth and enzymatic activity in a polyphagous caterpillar that feeds on over 80 plant species in 50 different families. Larvae of the polyphagous arctiid, Grammia incorrupta, were reared exclusively on one of two plant species, one of which contains iridoid glycosides (Plantago lanceolata, Plantaginaceae) while the other does not (Taraxacum officinale, Asteraceae). Larval weight was measured on the two host plants, and midgut homogenates of last instar larvae were then assayed for activity and kinetic properties of ß-glucosidases, using both a standard substrate, 4-nitrophenyl-ß-D-glucose (NPßGlc), and the iridoid glycoside aucubin, one of the two main iridoid glycosides in P. lanceolata. Larvae feeding on P. lanceolata weighed significantly less and developed more slowly compared to larvae on T. officinale. While the larval midgut ß-glucosidase activity determined with NPßGlc was significantly decreased when fed on P. lanceolata, aucubin was substantially hydrolyzed and the larval ß-glucosidase activity towards both substrates correlated negatively with larval weight. Our results demonstrate that host plants containing high concentrations of iridoid glycosides have a negative impact on larval development of this generalist insect herbivore. This is most likely due to the hydrolysis of plant glycosides in the larval midgut which results in the release of toxic aglycones. Linking the reduced larval weight to the toxin-releasing action of an iridoid glycoside cleaving ß-glucosidase, our results thus support the detoxification limitation hypothesis, suggesting fitness costs for the larvae feeding solely on P. lanceolata. Thus, in addition to the adaptive regulation of midgut ß-glucosidase activity, host plant switching as a behavioral adaptation might be a prerequisite for generalist herbivores that allows them to circumvent the negative effects of plant secondary compounds.

Coping with toxic plant compounds--the insect's perspective on iridoid glycosides and cardenolides.

Specializing on host plants with toxic secondary compounds enforces specific adaptation in insect herbivores. In this review, we focus on two compound classes, iridoid glycosides and cardenolides, which can be found in the food plants of a large number of insect species that display various degrees of adaptation to them. These secondary compounds have very different modes of action: Iridoid glycosides are usually activated in the gut of the herbivores by ß-glucosidases that may either stem from the food plant or be present in the gut as standard digestive enzymes. Upon cleaving, the unstable aglycone is released that unspecifically acts by crosslinking proteins and inhibiting enzymes. Cardenolides, on the other hand, are highly specific inhibitors of an essential ion carrier, the sodium pump. In insects exposed to both kinds of toxins, carriers either enabling the safe storage of the compounds away from the activating enzymes or excluding the toxins from sensitive tissues, play an important role that deserves further analysis. To avoid toxicity of iridoid glycosides, repression of activating enzymes emerges as a possible alternative strategy. Cardenolides, on the other hand, may lose their toxicity if their target site is modified and this strategy has evolved multiple times independently in cardenolide-adapted insects.

Influence of iridoid glycoside containing host plants on midgut ß-glucosidase activity in a polyphagous caterpillar, Spilosoma virginica Fabricius (Arctiidae).

Iridoid glycosides are secondary plant compounds that have deterrent, growth reducing or even toxic effects on non-adapted herbivorous insects. To investigate the effects of iridoid glycoside containing plants on the digestive metabolism of a generalist herbivore, larvae of Spilosoma virginica (Lepidoptera: Arctiidae) were reared on three plant species that differ in their secondary plant chemistry: Taraxacum officinale (no iridoid glycosides), Plantago major (low iridoid glycoside content), and P. lanceolata (high iridoid glycoside content). Midguts of fifth instar larvae were assayed for the activity and kinetic properties of ß-glucosidase using different substrates. Compared to the larvae on T. officinale, the ß-glucosidase activity of larvae feeding on P. lanceolata was significantly lower measured with 4-nitrophenyl-ß-d-glucopyranoside. Using the iridoid glycoside aucubin as a substrate, we did not find differences in the ß-glucosidase activity of the larvae reared on the three plants. Heat inactivation experiments revealed the existence of a heat-labile and a more heat-stable ß-glucosidase with similar Michaelis constants for 4-nitrophenyl-ß-d-glucopyranoside. We discuss possible mechanisms leading to the observed decrease of ß-glucosidase activity for larvae reared on P. lanceolata and its relevance for generalist herbivores in adapting to iridoid glycoside containing plant species and their use as potential host plants.