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We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.
The original version of this Article contained errors where Table S5 and Table S6 were incorrectly cited. As the result, in the Methods section, under the subheading ‘Germline transformation, crossing setups and insertion junction sequencing’, “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S4).” now reads: “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S5).” And, under the subheading ‘Light sheet-based fluorescence microscopy’, “Metadata for the three datasets are provided in Table S5.” now reads: “Metadata for the three datasets are provided in Table S6.” In Data availability section, “Microscopy data can be accessed as described in Table S5.” now reads: “Microscopy data can be accessed as described in Table S6.” Additionally, in the Supplementary Information 8 file, the “Data Access” row was omitted in Table S6. The “Data Access” row now reads: Dataset (DS) DS0001 DS0002 DS0003 Dataset Access DOI: 10.5281/zenodo.4892363 DOI: 10.5281/zenodo.4892373 DOI: 10.5281/zenodo.4892381 The original Supplementary Information 8 file is provided below. Finally, the Supplementary Information 1 and 5 files published with this Article contained tracked changes, these have now been removed. The original Article and accompanying Supplementary Information files have been corrected.
Nomadic movements are often a consequence of unpredictable resource dynamics. However, how nomadic ungulates select dynamic resources is still understudied. Here we examined resource selection of nomadic Mongolian gazelles (Procapra gutturosa) in the Eastern Steppe of Mongolia. We used daily GPS locations of 33 gazelles tracked up to 3.5 years. We examined selection for forage during the growing season using the Normalized Difference Vegetation Index (NDVI). In winter we examined selection for snow cover which mediates access to forage and drinking water. We studied selection at the population level using resource selection functions (RSFs) as well as on the individual level using step-selection functions (SSFs) at varying spatio-temporal scales from 1 to 10 days. Results from the population and the individual level analyses differed. At the population level we found selection for higher than average NDVI during the growing season. This may indicate selection for areas with more forage cover within the arid steppe landscape. In winter, gazelles selected for intermediate snow cover, which may indicate preference for areas which offer some snow for hydration but not so much as to hinder movement. At the individual level, in both seasons and across scales, we were not able to detect selection in the majority of individuals, but selection was similar to that seen in the RSFs for those individuals showing selection. Difficulty in finding selection with SSFs may indicate that Mongolian gazelles are using a random search strategy to find forage in a landscape with large, homogeneous areas of vegetation. The combination of random searches and landscape characteristics could therefore obscure results at the fine scale of SSFs. The significant results on the broader scale used for the population level RSF highlight that, although individuals show uncoordinated movement trajectories, they ultimately select for similar vegetation and snow cover.
Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.
The combined behaviours of individuals within insect societies determine the survival and development of the colony. For the western honey bee (Apis mellifera), individual behaviours include nest building, foraging, storing and ripening food, nursing the brood, temperature regulation, hygiene and defence. However, the various behaviours inside the colony, especially within the cells, are hidden from sight, and until recently, were primarily described through texts and line drawings, which lack the dynamics of moving images. In this study, we provide a comprehensive source of online video material that offers a view of honey bee behaviour within comb cells, thereby providing a new mode of observation for the scientific community and the general public. We analysed long-term video recordings from longitudinally truncated cells, which allowed us to see sideways into the cells in the middle of a colony. Our qualitative study provides insight into worker behaviours, including the use of wax scales and existing nest material to remodel combs, storing pollen and nectar in cells, brood care and thermoregulation, and hygienic practices, such as cannibalism, grooming and surface cleaning. We reveal unique processes that have not been previously published, such as the rare mouth-to-mouth feeding by nurses to larvae as well as thermoregulation within cells containing the developing brood. With our unique video method, we are able to bring the processes of a fully functioning social insect colony into classrooms and homes, facilitating ecological awareness in modern times. We provide new details and images that will help scientists test their hypotheses on social behaviours. In addition, we encourage the non-commercial use of our material to educate beekeepers, the media and the public and, in turn, call attention to the general decline of insect biomass and diversity.
Premise: Both universal and family-specific targeted sequencing probe kits are becoming widely used for reconstruction of phylogenetic relationships in angiosperms. Within the pantropical Ochnaceae, we show that with careful data filtering, universal kits are equally as capable in resolving intergeneric relationships as custom probe kits. Furthermore, we show the strength in combining data from both kits to mitigate bias and provide a more robust result to resolve evolutionary relationships.
Methods: We sampled 23 Ochnaceae genera and used targeted sequencing with two probe kits, the universal Angiosperms353 kit and a family-specific kit. We used maximum likelihood inference with a concatenated matrix of loci and multispecies-coalescence approaches to infer relationships in the family. We explored phylogenetic informativeness and the impact of missing data on resolution and tree support.
Results: For the Angiosperms353 data set, the concatenation approach provided results more congruent with those of the Ochnaceae-specific data set. Filtering missing data was most impactful on the Angiosperms353 data set, with a relaxed threshold being the optimum scenario. The Ochnaceae-specific data set resolved consistent topologies using both inference methods, and no major improvements were obtained after data filtering. Merging of data obtained with the two kits resulted in a well-supported phylogenetic tree.
Conclusions: The Angiosperms353 data set improved upon data filtering, and missing data played an important role in phylogenetic reconstruction. The Angiosperms353 data set resolved the phylogenetic backbone of Ochnaceae as equally well as the family specific data set. All analyses indicated that both Sauvagesia L. and Campylospermum Tiegh. as currently circumscribed are polyphyletic and require revised delimitation.
The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene-for-gene interactions have been identified in the maize–U. maydis pathosystem. Thus, maize resistance to U. maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how U. maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA sequencing (RNA-Seq) analysis of six U. maydis-infected maize lines of highly distinct resistance levels. The different maize lines showed specific responses of diverse cellular processes to U. maydis infection. For U. maydis, our analysis identified 406 genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated U. maydis CRISPR/Cas9 knock-out mutants for selected candidate effector sets. After infections of different maize lines with the fungal mutants, RNA-Seq analysis identified effectors with quantitative, maize line-specific virulence functions, and revealed auxin-related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing insights into the molecular mechanisms underlying QDR in the maize–U. maydis interaction.
Background: Efficient transfer of chemical signals is important for successful mating in many animal species. Multiple evolutionary lineages of animals evolved direct sex pheromone transmission during traumatic mating—the wounding of the partner with specialized devices—which helps to avoid signal loss to the environment. Although such direct transmission modes of so-called allohormone pheromones are well-documented in invertebrates, they are considered rare in vertebrates. Males of several species of the frog genus Plectrohyla (Hylidae, Anura) have elongated teeth and develop swollen lips during the breeding season. Here we investigated the possibility that these structures are used to scratch the females’ skin and apply allohormone pheromones during traumatic mating in several Plectrohyla species.
Results: Our behavioural observations revealed that males press their upper jaw onto the females’ dorsum during amplexus, leaving small skin scratches with their teeth. Histological examinations of the males’ lips identified specialized mucus glands, resembling known amphibian pheromone glands. Whole-transcriptome sequencing of these breeding glands showed high expression of sodefrin precursor-like factor (SPF) proteins, which are known to have a pheromone function in multiple amphibian species.
Conclusions: Our study suggests SPF delivery via traumatic mating in several anuran species: the males have specialized breeding glands in the lips for production and secretion and use their elongated teeth as wounding devices for application. We hypothesize that these SPF proteins end up in the females’ circulatory system, where understanding their exact function will require further molecular, physiological and behavioural testing.
Translation is an important step in gene expression. Initiation of translation is rate-limiting, and it is phylogenetically more diverse than elongation or termination. Bacteria contain only three initiation factors. In stark contrast, eukaryotes contain more than 10 (subunits of) initiation factors (eIFs). The genomes of archaea contain many genes that are annotated to encode archaeal homologs of eukaryotic initiation factors (aIFs). However, experimental characterization of aIFs is scarce and mostly restricted to very few species. To broaden the view, the protein–protein interaction network of aIFs in the halophilic archaeon Haloferax volcanii has been characterized. To this end, tagged versions of 14 aIFs were overproduced, affinity isolated, and the co-isolated binding partners were identified by peptide mass fingerprinting and MS/MS analyses. The aIF–aIF interaction network was resolved, and it was found to contain two interaction hubs, (1) the universally conserved factor aIF5B, and (2) a protein that has been annotated as the enzyme ribose-1,5-bisphosphate isomerase, which we propose to rename to aIF2Bα. Affinity isolation of aIFs also led to the co-isolation of many ribosomal proteins, but also transcription factors and subunits of the RNA polymerase (Rpo). To analyze a possible coupling of transcription and translation, seven tagged Rpo subunits were overproduced, affinity isolated, and co-isolated proteins were identified. The Rpo interaction network contained many transcription factors, but also many ribosomal proteins as well as the initiation factors aIF5B and aIF2Bα. These results showed that transcription and translation are coupled in haloarchaea, like in Escherichia coli. It seems that aIF5B and aIF2Bα are not only interaction hubs in the translation initiation network, but also key players in the transcription-translation coupling.
Animals living in human care for several generations face the risk of losing natural behaviors, which can lead to reduced animal welfare. The goal of this study is to demonstrate that meerkats (Suricata suricatta) living in zoos can assess potential danger and respond naturally based on acoustic signals only. This includes that the graded information of urgency in alarm calls as well as a response to those alarm calls is retained in captivity. To test the response to acoustic signals with different threat potential, meerkats were played calls of various animals differing in size and threat (e.g., robin, raven, buzzard, jackal) while their behavior was observed. The emitted alarm calls were recorded and examined for their graded structure on the one hand and played back to them on the other hand by means of a playback experiment to see whether the animals react to their own alarm calls even in the absence of danger. A fuzzy clustering algorithm was used to analyze and classify the alarm calls. Subsequently, the features that best described the graded structure were isolated using the LASSO algorithm and compared to features already known from wild meerkats. The results show that the graded structure is maintained in captivity and can be described by features such as noise and duration. The animals respond to new threats and can distinguish animal calls that are dangerous to them from those that are not, indicating the preservation of natural cooperative behavior. In addition, the playback experiments show that the meerkats respond to their own alarm calls with vigilance and escape behavior. The findings can be used to draw conclusions about the intensity of alertness in captive meerkats and to adapt husbandry conditions to appropriate welfare.
An increasing number of voices highlight the need for science itself to transform and to engage in the co-production of knowledge and action, in order to enable the fundamental transformations needed to advance towards sustainable futures. But how can global sustainability-oriented research networks engage in co-production of knowledge and action? The present article introduces a strategic tool called the ‘network compass’ which highlights four generic, interrelated fields of action through which networks can strive to foster co-production. It is based on the networks’ particular functions and how these can be engaged for co-production processes. This tool aims to foster self-reflection and learning within and between networks in the process of (re)developing strategies and activity plans and effectively contributing to sustainability transformations.
Background: Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in biological sciences and in biodiversity-related applied fields such as environmental management and natural product research. Advances in long-read DNA sequencing make it feasible to generate high-quality genomes for many non–genetic model species. However, long-read sequencing today relies on sizable quantities of high-quality, high molecular weight DNA, which is mostly obtained from fresh tissues. This is a challenge for biodiversity genomics of most metazoan species, which are tiny and need to be preserved immediately after collection. Here we present de novo genomes of 2 species of submillimeter Collembola. For each, we prepared the sequencing library from high molecular weight DNA extracted from a single specimen and using a novel ultra-low input protocol from Pacific Biosciences. This protocol requires a DNA input of only 5 ng, permitted by a whole-genome amplification step.
Results: The 2 assembled genomes have N50 values >5.5 and 8.5 Mb, respectively, and both contain ∼96% of BUSCO genes. Thus, they are highly contiguous and complete. The genomes are supported by an integrative taxonomy approach including placement in a genome-based phylogeny of Collembola and designation of a neotype for 1 of the species. Higher heterozygosity values are recorded in the more mobile species. Both species are devoid of the biosynthetic pathway for β-lactam antibiotics known in several Collembola, confirming the tight correlation of antibiotic synthesis with the species way of life.
Conclusions: It is now possible to generate high-quality genomes from single specimens of minute, field-preserved metazoans, exceeding the minimum contig N50 (1 Mb) required by the Earth BioGenome Project.
Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding.
The development of photosynthesis was a highlight in the progression of bacteria. In addition to the photosystems with their structural proteins, the photosynthesis apparatus consists of different cofactors including essential carotenoids. Thus, the evolution of the carotenoid pathways in relation to the functionality of the resulting structures in photosynthesis is the focus of this review. Analysis of carotenoid pathway genes indicates early evolutionary roots in prokaryotes. The pathway complexity leading to a multitude of structures is a result of gene acquisition, including their functional modifications, emergence of novel genes and gene exchange between species. Along with the progression of photosynthesis, carotenoid pathways coevolved with photosynthesis according to their advancing functionality. Cyanobacteria, with their oxygenic photosynthesis, became a landmark for evolutionary events including carotenogenesis. Concurrent with endosymbiosis, the cyanobacterial carotenoid pathways were inherited into algal plastids. In the lineage leading to Chlorophyta and plants, carotenoids evolved to their prominent role in protection and regulation of light energy input as constituents of a highly efficient light-harvesting complex.
Identification and regulation of tomato Serine/Arginine-rich proteins under high temperatures
(2021)
Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.
Acetogenic bacteria are a polyphyletic group of organisms that fix carbon dioxide under anaerobic, non-phototrophic conditions by reduction of two mol of CO2 to acetyl-CoA via the Wood–Ljungdahl pathway. This pathway also allows for lithotrophic growth with H2 as electron donor and this pathway is considered to be one of the oldest, if not the oldest metabolic pathway on Earth for CO2 reduction, since it is coupled to the synthesis of ATP. How ATP is synthesized has been an enigma for decades, but in the last decade two ferredoxin-dependent respiratory chains were discovered. Those respiratory chains comprise of a cytochrome-free, ferredoxin-dependent respiratory enzyme complex, which is either the Rnf or Ech complex. However, it was discovered already 50 years ago that some acetogens contain cytochromes and quinones, but their role had only a shadowy existence. Here, we review the literature on the characterization of cytochromes and quinones in acetogens and present a hypothesis that they may function in electron transport chains in addition to Rnf and Ech.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Toxicogenomic differentiation of functional responses to fipronil and imidacloprid in Daphnia magna
(2021)
Active substances of pesticides, biocides or pharmaceuticals can induce adverse side effects in the aquatic ecosystem, necessitating environmental hazard and risk assessment prior to substance registration. The freshwater crustacean Daphnia magna is a model organism for acute and chronic toxicity assessment representing aquatic invertebrates. However, standardized tests involving daphnia are restricted to the endpoints immobility and reproduction and thus provide only limited insights into the underlying modes-of-action. Here, we applied transcriptome profiling to a modified D. magna Acute Immobilization test to analyze and compare gene expression profiles induced by the GABA-gated chloride channel blocker fipronil and the nicotinic acetylcholine receptor (nAChR) agonist imidacloprid. Daphnids were expose to two low effect concentrations of each substance followed by RNA sequencing and functional classification of affected gene ontologies and pathways. For both insecticides, we observed a concentration-dependent increase in the number of differentially expressed genes, whose expression changes were highly significantly positively correlated when comparing both test concentrations. These gene expression fingerprints showed virtually no overlap between the test substances and they related well to previous data of diazepam and carbaryl, two substances targeting similar molecular key events. While, based on our results, fipronil predominantly interfered with molecular functions involved in ATPase-coupled transmembrane transport and transcription regulation, imidacloprid primarily affected oxidase and oxidoreductase activity. These findings provide evidence that systems biology approaches can be utilized to identify and differentiate modes-of-action of chemical stressors in D. magna as an invertebrate aquatic non-target organism. The mechanistic knowledge extracted from such data will in future contribute to the development of Adverse Outcome Pathways (AOPs) for read-across and prediction of population effects.
Research on Podospora anserina unraveled a network of molecular pathways affecting biological aging. In particular, a number of pathways active in the control of mitochondria were identified on different levels. A long-known key process active during aging of P. anserina is the age- related reorganization of the mitochondrial DNA (mtDNA). Mechanisms involved in the stabilization of the mtDNA lead to lifespan extension. Another critical issue is to balance mitochondrial levels of reactive oxygen species (ROS). This is important because ROS are essential signaling molecules, but at increased levels cause molecular damage. At a higher level of the network, mechanisms are active in the repair of damaged compounds. However, if damage passes critical limits, the corresponding pathways are overwhelmed and impaired molecules as well as those present in excess are degraded by specific enzymes or via different forms of autophagy. Subsequently, degraded units need to be replaced by novel functional ones. The corresponding processes are dependent on the availability of intact genetic information. Although a number of different pathways involved in the control of cellular homeostasis were uncovered in the past, certainly many more exist. In addition, the signaling pathways involved in the control and coordination of the underlying pathways are only initially understood. In some cases, like the induction of autophagy, ROS are active. Additionally, sensing and signaling the energetic status of the organism plays a key role. The precise mechanisms involved are elusive and remain to be elucidated.
In the past two decades, an increasing body of studies has been published on the intersex phenomenon in separate-sexed crustaceans from marine and freshwater ecosystems. Various causes are being considered that could have an influence on the occurrence of intersex. Besides genetic factors, environmental conditions such as photoperiodicity, temperature, salinity and parasitism, but also environmental pollution with endocrine disrupting chemicals (EDCs) are discussed. As part of a long-term monitoring (2012 – 2020) in north-west Brittany, we recorded the occurrence of intersex in the marine amphipod Echinogammarus marinus. We quantified the intersex incidence at marine and estuarine sites and analyzed the incidence in relation to the endocrine potential of the sediments. Intersex occurred with mean frequencies between 0.87% and 12%. It was striking that the incidence of intersex increased with increasing distance from the sea. Since the highest incidence was observed at the range boundary of this stenohaline species, we assume that intersex is triggered by endocrine potential and increasing stress due to increasing freshwater content − and thus an interplay of different environmental factors.