The microbiome at the interface between environmental stress and animal health: an example from the most threatened vertebrate group.

Nitrate pollution and global warming are ubiquitous stressors likely to interact and affect the health and survival of wildlife, particularly aquatic ectotherms. Animal health is largely influenced by its microbiome (commensal/symbiotic microorganisms), which responds to such stressors. We used a crossed experimental design including three nitrate levels and five temperature regimes to investigate their interactive and individual effects on an aquatic ectotherm, the European common frog. We associated health biomarkers in larvae with changes in gut bacteria diversity and composition. Larvae experienced higher stress levels and lower body condition under high temperatures and nitrate exposure. Developmental rate increased with temperature but decreased with nitrate pollution. Alterations in bacteria composition but not diversity are likely to correlate with the observed outcomes in larvae health. Leucine degradation decreased at higher temperatures corroborating accelerated development, nitrate degradation increased with nitrate level corroborating reduced body condition and an increase in lysine biosynthesis may have helped larvae deal with the combined effects of both stressors. These results reinforce the importance of associating traditional health biomarkers with underlying microbiome changes. Therefore, we urge studies to investigate the effects of environmental stressors on microbiome composition and consequences for host health in a world threatened by biodiversity loss.

High throughput screening identifies repurposable drugs for modulation of innate and acquired immune responses.

A balanced immune system is essential to maintain adequate host defense and effective self-tolerance. While an immune system that fails to generate appropriate response will permit infections to develop, uncontrolled activation may lead to autoinflammatory or autoimmune diseases. To identify drug candidates capable of modulating immune cell functions, we screened 1200 small molecules from the Prestwick Chemical Library for their property to inhibit innate or adaptive immune responses. Our studies focused specifically on drug interactions with T cells, B cells, and polymorphonuclear leukocytes (PMNs). Candidate drugs that were validated in vitro were examined in preclinical models to determine their immunomodulatory impact in chronic inflammatory diseases, here investigated in chronic inflammatory skin diseases. Using this approach, we identified several candidate drugs that were highly effective in preclinical models of chronic inflammatory disease. For example, we found that administration of pyrvinium pamoate, an FDA-approved over-the-counter anthelmintic drug, suppressed B cell activation in vitro and halted the progression of B cell-dependent experimental pemphigoid by reducing numbers of autoantigen-specific B cell responses. In addition, in studies performed in gene-deleted mouse strains provided additional insight into the mechanisms underlying these effects, for example, the receptor-dependent actions of tamoxifen that inhibit immune-complex-mediated activation of PMNs. Collectively, our methods and findings provide a vast resource that can be used to identify drugs that may be repurposed and used to promote or inhibit cellular immune responses.

Phylotranscriptomic relationships of the Oophila clade of green algae associated to amphibian egg masses.

Green algae usually assigned to the genus Oophila are known to colonize egg capsules of amphibian egg masses across the Nearctic and Palearctic regions. We study the phylogenetic relationships of these algae using a phylotranscriptomic data set of 76 protein-coding single-copy nuclear genes. Our data set includes novel RNAseq data for six amphibian-associated and five free-living green algae, and draft genomes of two of the latter. Within the Oophila clade (nested within Moewusinia), we find samples from two European frogs (Rana dalmatina and R. temporaria) closely related to those of the North American frog R. aurora (Oophila subclade III). An isolate from the North American R. sylvatica (subclade IV) appears to be sister to the Japanese isolate from the salamander Hynobius nigrescens (subclade J1), and subclade I algae from Ambystoma maculatum are sister to all other lineages in the Oophila clade. Two free-living algae (Chlamydomonas nasuta and Cd. pseudogloeogama) are nested within the Oophila clade, and a strain of the type species of Chlorococcum (Cc. infusionum) is related to this assemblage. Our phylotranscriptomic tree suggests that recognition of different species within the Oophila clade ("clade B" of earlier studies) is warranted, and calls for a comprehensive taxonomic revision of Moewusinia.

Variation in the Spectrum of New Mutations among Inbred Strains of Mice.

The mouse serves as a mammalian model for understanding the nature of variation from new mutations, a question that has both evolutionary and medical significance. Previous studies suggest that the rate of single-nucleotide mutations (SNMs) in mice is ∼50% of that in humans. However, information largely comes from studies involving the C57BL/6 strain, and there is little information from other mouse strains. Here, we study the mutations that accumulated in 59 mouse lines derived from four inbred strains that are commonly used in genetics and clinical research (BALB/cAnNRj, C57BL/6JRj, C3H/HeNRj, and FVB/NRj), maintained for eight to nine generations by brother-sister mating. By analyzing Illumina whole-genome sequencing data, we estimate that the average rate of new SNMs in mice is ∼µ = 6.7 × 10-9. However, there is substantial variation in the spectrum of SNMs among strains, so the burden from new mutations also varies among strains. For example, the FVB strain has a spectrum that is markedly skewed toward C→A transversions and is likely to experience a higher deleterious load than other strains, due to an increased frequency of nonsense mutations in glutamic acid codons. Finally, we observe substantial variation in the rate of new SNMs among DNA sequence contexts, CpG sites, and their adjacent nucleotides playing an important role.

Physiological responses to a changing winter climate in an early spring-breeding amphibian.

Climate change is swiftly altering environmental winter conditions, leading to significant ecological impacts such as phenological shifts in many species. As a result, animals might face physiological mismatches due to longer or earlier activity periods and are at risk of being exposed to late spring freezes. Our study points for the first time to the complex physiological challenges that amphibians face as a result of changing thermal conditions due to winter climate change. We investigated the physiological responses to a period of warmer winter days and sudden spring freeze in the common toad (Bufo bufo) by acclimating them to 4°C or 8°C for 48 h or exposing them to 4°C or -2°C for 6 h, respectively. We assessed the daily energy demands, determined body condition and cold tolerance, explored the molecular responses to freezing through hepatic tissue transcriptome analysis, and measured blood glucose levels. Toads acclimated to higher temperatures showed a higher daily energy expenditure and a reduced cold tolerance suggesting faster depletion of energy stores and the loss of winter acclimation during warmer winters. Blood sugar levels were higher in frozen toads indicating the mobilization of cryoprotective glucose with freezing which was further supported by changed patterns in proteins related to glucose metabolism. Overall, our results emphasize that increased thermal variability incurs physiological costs that may reduce energy reserves and thus affect amphibian health and survival. This might pose a serious threat to breeding adults and may have subsequent effects at the population level.

Mitochondrial DNA variants and microbiota: An experimental strategy to identify novel therapeutic potential in chronic inflammatory diseases.

We previously demonstrated that mice carrying natural mtDNA variants of the FVB/NJ strain (m.7778 G>T in the mt-Atp8 gene in mitochondrial complex V), namely C57BL/6 J-mtFVB/NJ (B6-mtFVB), exhibited (i) partial protection from experimental skin inflammatory diseases in an anti-murine type VII collagen antibody-induced skin inflammation model and psoriasiform dermatitis model; (ii) significantly altered metabolites, including short-chain fatty acids, according to targeted metabolomics of liver, skin and lymph node samples; and (iii) a differential composition of the gut microbiota according to bacterial 16 S rRNA gene sequencing of stool samples compared to wild-type C57BL/6 J (B6) mice. To further dissect these disease-contributing factors, we induced an experimental antibody-induced skin inflammatory disease in gnotobiotic mice. We performed shotgun metagenomic sequencing of caecum contents and untargeted metabolomics of liver, CD4+ T cell, and caecum content samples from conventional B6-mtFVB and B6 mice. We identified D-glucosamine as a candidate mediator that ameliorated disease severity in experimental antibody-induced skin inflammation by modulating immune cell function in T cells, neutrophils and macrophages. Because mice carrying mtDNA variants of the FVB/NJ strain show differential disease susceptibility to a wide range of experimental diseases, including diet-induced atherosclerosis in low-density lipoprotein receptor knockout mice and collagen antibody-induced arthritis in DBA/1 J mice, this experimental approach is valuable for identifying novel therapeutic options for skin inflammatory conditions and other chronic inflammatory diseases to which mice carrying specific mtDNA variants show differential susceptibility.

High parasite diversity maintained after an alga-virus coevolutionary arms race.

Arms race dynamics are a common outcome of host-parasite coevolution. While they can theoretically be maintained indefinitely, realistic arms races are expected to be finite. Once an arms race has ended, for example due to the evolution of a generalist-resistant host, the system may transition into coevolutionary dynamics that favour long-term diversity. In microbial experiments, host-parasite arms races often transition into a stable coexistence of generalist-resistant hosts, (semi-)susceptible hosts, and parasites. While long-term host diversity is implicit in these cases, parasite diversity is usually overlooked. In this study, we examined parasite diversity after the end of an experimental arms race between a unicellular alga (Chlorella variabilis) and its lytic virus (PBCV-1). First, we isolated virus genotypes from multiple time points from two replicate microcosms. A time-shift experiment confirmed that the virus isolates had escalating host ranges, i.e., that arms races had occurred. We then examined the phenotypic and genetic diversity of virus isolates from the post-arms race phase. Post-arms race virus isolates had diverse host ranges, survival probabilities, and growth rates; they also clustered into distinct genetic groups. Importantly, host range diversity was maintained throughout the post-arms race phase, and the frequency of host range phenotypes fluctuated over time. We hypothesize that this dynamic polymorphism was maintained by a combination of fluctuating selection and demographic stochasticity. Together with previous work in prokaryotic systems, our results link experimental observations of arms races to natural observations of long-term host and parasite diversity.

Gut-associated functions are favored during microbiome assembly across a major part of C. elegans life.

The microbiome expresses a variety of functions that influence host biology. The range of functions depends on the microbiome's composition, which can change during the host's lifetime due to neutral assembly processes, host-mediated selection, and environmental conditions. To date, the exact dynamics of microbiome assembly, the underlying determinants, and the effects on host-associated functions remain poorly understood. Here, we used the nematode Caenorhabditis elegans and a defined community of fully sequenced, naturally associated bacteria to study microbiome dynamics and functions across a major part of the worm's lifetime of hosts under controlled experimental conditions. Bacterial community composition initially shows strongly declining levels of stochasticity, which increases during later time points, suggesting selective effects in younger animals as opposed to more random processes in older animals. The adult microbiome is enriched in genera Ochrobactrum and Enterobacter compared to the direct substrate and a host-free control environment. Using pathway analysis, metabolic, and ecological modeling, we further find that the lifetime assembly dynamics increase competitive strategies and gut-associated functions in the host-associated microbiome, indicating that the colonizing bacteria benefit the worm. Overall, our study introduces a framework for studying microbiome assembly dynamics based on stochastic, ecological, and metabolic models, yielding new insights into the processes that determine host-associated microbiome composition and function. IMPORTANCE: The microbiome plays a crucial role in host biology. Its functions depend on the microbiome composition that can change during a host's lifetime. To date, the dynamics of microbiome assembly and the resulting functions still need to be better understood. This study introduces a new approach to characterize the functional consequences of microbiome assembly by modeling both the relevance of stochastic processes and metabolic characteristics of microbial community changes. The approach was applied to experimental time-series data obtained for the microbiome of the nematode Caenorhabditis elegans across the major part of its lifetime. Stochastic processes played a minor role, whereas beneficial bacteria as well as gut-associated functions enriched in hosts. This indicates that the host might actively shape the composition of its microbiome. Overall, this study provides a framework for studying microbiome assembly dynamics and yields new insights into C. elegans microbiome functions.

Template-specific optimization of NGS genotyping pipelines reveals allele-specific variation in MHC gene expression.

Using high-throughput sequencing for precise genotyping of multi-locus gene families, such as the major histocompatibility complex (MHC), remains challenging, due to the complexity of the data and difficulties in distinguishing genuine from erroneous variants. Several dedicated genotyping pipelines for data from high-throughput sequencing, such as next-generation sequencing (NGS), have been developed to tackle the ensuing risk of artificially inflated diversity. Here, we thoroughly assess three such multi-locus genotyping pipelines for NGS data, the DOC method, AmpliSAS and ACACIA, using MHC class IIß data sets of three-spined stickleback gDNA, cDNA and "artificial" plasmid samples with known allelic diversity. We show that genotyping of gDNA and plasmid samples at optimal pipeline parameters was highly accurate and reproducible across methods. However, for cDNA data, the gDNA-optimal parameter configuration yielded decreased overall genotyping precision and consistency between pipelines. Further adjustments of key clustering parameters were required tο account for higher error rates and larger variation in sequencing depth per allele, highlighting the importance of template-specific pipeline optimization for reliable genotyping of multi-locus gene families. Through accurate paired gDNA-cDNA typing and MHC-II haplotype inference, we show that MHC-II allele-specific expression levels correlate negatively with allele number across haplotypes. Lastly, sibship-assisted cDNA-typing of MHC-I revealed novel variants linked in haplotype blocks, and a higher-than-previously-reported individual MHC-I allelic diversity. In conclusion, we provide novel genotyping protocols for the three-spined stickleback MHC-I and -II genes, and evaluate the performance of popular NGS-genotyping pipelines. We also show that fine-tuned genotyping of paired gDNA-cDNA samples facilitates amplification bias-corrected MHC allele expression analysis.

Phylogenetic insights into Central European Chorthippus and Pseudochorthippus (Orthoptera: Acrididae) species using ddRADseq data.

The evolution of several orthopteran groups, especially within the grasshopper family Acrididae, remains poorly understood. This is particularly true for the subfamily Gomphocerinae, which comprises cryptic sympatric and syntopic species. Previous mitochondrial studies have highlighted major discrepancies between taxonomic and phylogenetic hypotheses, thereby emphasizing the necessity of genome-wide approaches. In this study, we employ double-digest restriction site-associated DNA sequencing (ddRADseq) to reconstruct the evolution of Central European Chorthippus and Pseudochorthippus species, especially C.smardai, P.tatrae and the C.biguttulus group. Our phylogenomic analyses recovered deep discordance with mitochondrial DNA barcoding, emphasizing its unreliability in Gomphocerinae grasshoppers. Specifically, our data robustly distinguished the C.biguttulus group and confirmed the distinctiveness of C.eisentrauti, also shedding light on its presence in the Berchtesgaden Alps. Moreover, our results support the reclassification of C.smardai to the genus Pseudochorthippus and of P.tatrae to the genus Chorthippus. Our study demonstrates the efficiency of high-throughput genomic methods such as RADseq without prior optimization to elucidate the complex evolution of grasshopper radiations with direct taxonomic implications. While RADseq has predominantly been utilized for population genomics and within-genus phylogenomics, its application extends to resolve relationships between deeply-diverged clades representative of distinct genera.

Plant-derived environmental DNA complements diversity estimates from traditional arthropod monitoring methods but outperforms them detecting plant-arthropod interactions.

Our limited knowledge about the ecological drivers of global arthropod decline highlights the urgent need for more effective biodiversity monitoring approaches. Monitoring of arthropods is commonly performed using passive trapping devices, which reliably recover diverse communities, but provide little ecological information on the sampled taxa. Especially the manifold interactions of arthropods with plants are barely understood. A promising strategy to overcome this shortfall is environmental DNA (eDNA) metabarcoding from plant material on which arthropods leave DNA traces through direct or indirect interactions. However, the accuracy of this approach has not been sufficiently tested. In four experiments, we exhaustively test the comparative performance of plant-derived eDNA from surface washes of plants and homogenized plant material against traditional monitoring approaches. We show that the recovered communities of plant-derived eDNA and traditional approaches only partly overlap, with eDNA recovering various additional taxa. This suggests eDNA as a useful complementary tool to traditional monitoring. Despite the differences in recovered taxa, estimates of community α- and ß-diversity between both approaches are well correlated, highlighting the utility of eDNA as a broad scale tool for community monitoring. Last, eDNA outperforms traditional approaches in the recovery of plant-specific arthropod communities. Unlike traditional monitoring, eDNA revealed fine-scale community differentiation between individual plants and even within plant compartments. Especially specialized herbivores are better recovered with eDNA. Our results highlight the value of plant-derived eDNA analysis for large-scale biodiversity assessments that include information about community-level interactions.

Evolution of E. coli in a mouse model of inflammatory bowel disease leads to a disease-specific bacterial genotype and trade-offs with clinical relevance.

Inflammatory bowel disease (IBD) is a persistent inflammatory condition that affects the gastrointestinal tract and presents significant challenges in its management and treatment. Despite the knowledge that within-host bacterial evolution occurs in the intestine, the disease has rarely been studied from an evolutionary perspective. In this study, we aimed to investigate the evolution of resident bacteria during intestinal inflammation and whether- and how disease-related bacterial genetic changes may present trade-offs with potential therapeutic importance. Here, we perform an in vivo evolution experiment of E. coli in a gnotobiotic mouse model of IBD, followed by multiomic analyses to identify disease-specific genetic and phenotypic changes in bacteria that evolved in an inflamed versus a non-inflamed control environment. Our results demonstrate distinct evolutionary changes in E. coli specific to inflammation, including a single nucleotide variant that independently reached high frequency in all inflamed mice. Using ex vivo fitness assays, we find that these changes are associated with a higher fitness in an inflamed environment compared to isolates derived from non-inflamed mice. Further, using large-scale phenotypic assays, we show that bacterial adaptation to inflammation results in clinically relevant phenotypes, which intriguingly include collateral sensitivity to antibiotics. Bacterial evolution in an inflamed gut yields specific genetic and phenotypic signatures. These results may serve as a basis for developing novel evolution-informed treatment approaches for patients with intestinal inflammation.

Time-calibrated phylogeny and full mitogenome sequence of the Galapagos sea lion (Zalophus wollebaeki) from scat DNA.

Background: The Galapagos sea lion, Zalophus wollebaeki, is an endemic and endangered otariid, which is considered as a sentinel species of ecosystem dynamics in the Galapagos archipelago. Mitochondrial DNA is an important tool in phylogenetic and population genetic inference. In this work we use Illumina sequencing to complement the mitogenomic resources for Zalophus genus-the other two species employed Sanger sequencing-by a complete mitochondrial genome and a molecular clock of this species, which is not present in any case. Materials and Methods: We used DNA obtained from a fresh scat sample of a Galapagos sea lion and shotgun-sequenced it on the Illumina NextSeq platform. The obtained raw reads were processed using the GetOrganelle software to filter the mitochondrial Zalophus DNA reads (∼16% survive the filtration), assemble them, and set up a molecular clock. Results: From the obtained 3,511,116 raw reads, we were able to assemble a full mitogenome of a length of 16,676 bp, consisting of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNA), and two ribosomal RNAs (rRNA). A time-calibrated phylogeny confirmed the phylogenetic position of Z. wollebaeki in a clade with Z. californianus, and Z. japonicus, and sister to Z. californianus; as well as establishing the divergence time for Z. wollebaeki 0.65 million years ago. Our study illustrates the possibility of seamlessly sequencing full mitochondrial genomes from fresh scat samples of marine mammals.

Forward genetics and functional analysis highlight Itga11 as a modulator of murine psoriasiform dermatitis.

Psoriasis is a chronic inflammatory skin condition. Repeated epicutaneous application of Aldara® (imiquimod) cream results in psoriasiform dermatitis in mice. The Aldara®-induced psoriasiform dermatitis (AIPD) mouse model has been used to examine the pathogenesis of psoriasis. Here, we used a forward genetics approach in which we compared AIPD that developed in 13 different inbred mouse strains to identify genes and pathways that modulated disease severity. Among our primary results, we found that the severity of AIPD differed substantially between different strains of inbred mice and that these variations were associated with polymorphisms in Itga11. The Itga11 gene encodes the integrin α11 subunit that heterodimerizes with the integrin ß1 subunit to form integrin α11ß1. Less information is available about the function of ITGA11 in skin inflammation; however, a role in the regulation of cutaneous wound healing, specifically the development of dermal fibrosis, has been described. Experiments performed with Itga11 gene-deleted (Itga11-/- ) mice revealed that the integrin α11 subunit contributes substantially to the clinical phenotype as well as the histopathological and molecular findings associated with skin inflammation characteristic of AIPD. Although the skin transcriptomes of Itga11-/- and WT mice do not differ from one another under physiological conditions, distinct transcriptomes emerge in these strains in response to the induction of AIPD. Most of the differentially expressed genes contributed to extracellular matrix organization, immune system, and metabolism of lipids pathways. Consistent with these findings, we detected a reduced number of fibroblasts and inflammatory cells, including macrophages, T cells, and tissue-resident memory T cells in skin samples from Itga11-/- mice in response to AIPD induction. Collectively, our results reveal that Itga11 plays a critical role in promoting skin inflammation in AIPD and thus might be targeted for the development of novel therapeutics for psoriasiform skin conditions. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Adult and Larval Tracheal Systems Exhibit Different Molecular Architectures in Drosophila.

Knowing the molecular makeup of an organ system is required for its in-depth understanding. We analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance our knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless (btl)-Gal4 is not able to target all parts of the adult tracheal system. Here, we have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system.

Non-native hosts of an invasive seaweed holobiont have more stable microbial communities compared to native hosts in response to thermal stress.

Seaweeds are colonized by a microbial community, which can be directly linked to their performance. This community is shaped by an interplay of stochastic and deterministic processes, including mechanisms which the holobiont host deploys to manipulate its associated microbiota. The Anna Karenina principle predicts that when a holobiont is exposed to suboptimal or stressful conditions, these host mechanisms may be compromised. This leads to a relative increase of stochastic processes that may potentially result in the succession of a microbial community harmful to the host. Based on this principle, we used the variability in microbial communities (i.e., beta diversity) as a proxy for stability within the invasive holobiont Gracilaria vermiculophylla during a simulated invasion in a common garden experiment. Independent of host range, host performance declined at elevated temperature (22°C) and disease incidence and beta diversity increased. Under thermally stressful conditions, beta diversity increased more in epibiota from native populations, suggesting that epibiota from non-native holobionts are thermally more stable. This pattern reflects an increase in deterministic processes acting on epibiota associated with non-native hosts, which in the setting of a common garden can be assumed to originate from the host itself. Therefore, these experimental data suggest that the invasion process may have selected for hosts better able to maintain stable microbiota during stress. Future studies are needed to identify the underlying host mechanisms.

Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers.

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring. However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.

Hemocytes and fat body cells, the only professional immune cell types in Drosophila, show strikingly different responses to systemic infections.

The fruit fly Drosophila is an excellent model to study the response of different immunocompetent organs during systemic infection. In the present study, we intended to test the hypothesis that the only professional immune organs of the fly, the fat body and hemocytes, show substantial similarities in their responses to systemic infection. However, comprehensive transcriptome analysis of isolated organs revealed highly divergent transcript signatures, with the few commonly regulated genes encoding mainly classical immune effectors from the antimicrobial peptide family. The fat body and the hemocytes each have specific reactions that are not present in the other organ. Fat body-specific responses comprised those enabling an improved peptide synthesis and export. This reaction is accompanied by transcriptomic shifts enabling the use of the energy resources of the fat body more efficiently. Hemocytes, on the other hand, showed enhanced signatures related to phagocytosis. Comparing immune-induced signatures of both cell types with those of whole-body responses showed only a minimal correspondence, mostly restricted again to antimicrobial peptide genes. In summary, the two major immunocompetent cell types of Drosophila show highly specific responses to infection, which are closely linked to the primary function of the respective organ in the landscape of the systemic immune response.

Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities.

A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of α-diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere α-diversity loss, but can be driven by ß-diversity decay across space and time.

Insects are a barometer of environmental health. Ecosystems around the world are being subjected to unprecedented man-made stresses, ranging from climate change to pollution and intensive land use. These stresses have been associated with several recent, dramatic declines in insect populations, particularly in areas with heavily industrialised farming practices. Despite this, the links between insect decline, environmental stress, and ecosystem health are still poorly-understood. A decline in one area might look catastrophic, but could simply be part of normal, longer-term variations. Often, we do not know whether insect decline is a local phenomenon or reflects wider environmental trends. Additionally, most studies do not go far back enough in time or cover a wide enough geographical range to make these distinctions. To understand and combat insect decline, we therefore need reliable methods to monitor insect populations over long periods of time. To solve this problem, Krehenwinkel, Weber et al. gathered data on insect communities from a new source: tree leaves. Originally, these samples were collected to study air pollution, but they also happen to contain the DNA of insects that interacted with them before they were collected ­ for example, DNA deposited in chew marks where the insects had nibbled on the leaves. This is called environmental DNA, or eDNA for short. To survey the insect communities that lived in these trees, Krehenwinkel, Weber et al. first extracted eDNA from the leaves and sequenced it. Analysis of the different DNA sequences from the leaf samples revealed not only the number of insect species, but also the abundance (or rarity) of each species within each community. Importantly, the leaves had been collected and stored in stable conditions over several decades, allowing changes in these insect populations to be tracked over time. eDNA analysis revealed subtle changes in the make-up of forest insect communities. In the forests where the leaves were collected, the total number of insect species remained much the same over time. However, many individual species still declined, only to be replaced by newcomer species. These 'colonisers' are also widespread, which will likely lead to an overall pattern of fewer species that are more widely distributed ­ in other words, more homogeneity. The approach of Krehenwinkel, Weber et al. provides a reliable method to study insect populations in detail, over multiple decades, using archived samples from environmental studies. The information gained from this has real-world significance for environmental issues with enormous social impact, ranging from conservation, to agriculture and even public health.

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803.

The cyanobacterial bidirectional [NiFe]-hydrogenase is a pentameric enzyme. Apart from the small and large hydrogenase subunits (HoxYH) it contains a diaphorase module (HoxEFU) that interacts with NAD(P)+ and ferredoxin. HoxEFU shows strong similarity to the outermost subunits (NuoEFG) of canonical respiratory complexes I. Photosynthetic complex I (NDH-1) lacks these three subunits. This led to the idea that HoxEFU might interact with NDH-1 instead. HoxEFUYH utilizes excited electrons from PSI for photohydrogen production and it catalyzes the reverse reaction and feeds electrons into the photosynthetic electron transport. We analyzed hydrogenase activity, photohydrogen evolution and hydrogen uptake, the respiration and photosynthetic electron transport of ΔhoxEFUYH, and a knock-out strain with dysfunctional NDH-1 (ΔndhD1/ΔndhD2) of the cyanobacterium Synechocystis sp. PCC 6803. Photohydrogen production was prolonged in ΔndhD1/ΔndhD2 due to diminished hydrogen uptake. Electrons from hydrogen oxidation must follow a different route into the photosynthetic electron transport in this mutant compared to wild type cells. Furthermore, respiration was reduced in ΔhoxEFUYH and the ΔndhD1/ΔndhD2 localization of the hydrogenase to the membrane was impaired. These data indicate that electron transfer from the hydrogenase to the NDH-1 complex is either direct, by the binding of the hydrogenase to the complex, or indirect, via an additional mediator.