Ultrastructure Expansion Microscopy applied to C. elegans embryos.

Visualization of organelles using expansion microscopy has been previously applied to Caenorhadbitis elegans adult gonads or worms. However, its application to embryos has remained a challenge due to the protective eggshell barrier. Here, by combining freeze-cracking and ultrastructure expansion microscopy (U-ExM), we demonstrate a four-time isotropic expansion of C. elegans embryos. As an example structure, we chose the nuclear pore and demonstrate that we achieve sufficient resolution to distinguish them individually. Our work provides proof of principle for U-ExM in C. elegans embryos, which will be applicable for imaging a wide range of cellular structures in this model system.

Cytokines as fast indicator of infectious virus titer during process development.

Measuring infectious titer is the most time-consuming method during the production and process development of live viruses. Conventionally, it is done by measuring the tissue culture infectious dose (TCID50) or plaque forming units (pfu) in cell-based assays. Such assays require a time span of more than a week to the readout and significantly slow down process development. In this study, we utilized the pro-inflammatory cytokine response of a Vero production cell line to a recombinant measles vaccine virus (MVV) as model system for rapidly determining infectious virus titer within several hours after infection instead of one week. Cytokines are immunostimulatory proteins contributing to the first line of defence against virus infection. The probed cytokines in this study were MCP-1 and RANTES, which are secreted in a virus dose as well as time dependent manner and correlate to TCID50 over a concentration range of several logarithmic levels with R2 = 0.86 and R2 = 0.83, respectively. Furthermore, the pro-inflammatory cytokine response of the cells was specific for infectious virus particles and not evoked with filtered virus seed. We also discovered that individual cytokine candidates may be more suitable for off- or at-line analysis, depending on the secretion profile as well as their sensitivity towards changing process conditions. Furthermore, the method can be applied to follow a purification procedure and is therefore suited for process development and control.

Foraging valor linked with aggression: selection against completely abandoning aggression in the high-elevation ant Tetramorium alpestre?

Aggression has multiple benefits and is often coupled with other behaviors ("behavioral syndromes"). The level of aggressiveness is influenced by an adaptive benefit-cost ratio suggesting that benefits should outweigh the costs of aggression. Here, we assess if several behaviors are coupled in two behaviorally different populations (aggressive, peaceful) of the high-elevation ant Tetramorium alpestre. For three weeks, we collected colony fragments and analyzed boldness, exploring, foraging, and risk-taking behaviors. We hypothesized that the aggressive population is bolder, more explorative and risk-prone, and forages more food than the peaceful population. To test whether (a) the combination of experiments and parameters used yields a good setup, (b) populations differ behaviorally, and (c) populations display behavioral syndromes, we assessed (a) the frequency of repeatable behaviors of each experiment, (b) the behavioral means among populations, and (c) the behavioral repeatability, respectively. We found that (a) boldness and exploring were most repeatable and represent a good experimental setup, (b) the aggressive population was bolder and more explorative and risk-prone than the peaceful population, (c) boldness and exploring behaviors were highly repeatable in both populations, thus corroborating our hypothesis. The results suggest that boldness, exploring, and risk-taking but not foraging are presumably coupled with aggression and indicate the presence of behavioral syndromes in this ant. Under specific ecological conditions, aggression may be coupled with other behaviors and important for finding food. Aggression is probably adaptive in T. alpestre, possibly indicating that selection favors aggression at least partially, which may counteract the complete loss of intraspecific aggression.

Evolutionary history of an Alpine Archaeognath (Machilis pallida): Insights from different variant.

Reconstruction of species histories is a central aspect of evolutionary biology. Patterns of genetic variation within and among populations can be leveraged to elucidate evolutionary processes and demographic histories. However, interpreting genetic signatures and unraveling the contributing processes can be challenging, in particular for non-model organisms with complex reproductive modes and genome organization. One way forward is the combined consideration of patterns revealed by different molecular markers (nuclear vs. mitochondrial) and types of variants (common vs. rare) that differ in their age, mode, and rate of evolution. Here, we applied this approach to RNAseq data generated for Machilis pallida (Archaeognatha), an Alpine jumping bristletail considered parthenogenetic and triploid. We generated de novo transcriptome and mitochondrial assemblies to obtain high-density data to investigate patterns of mitochondrial and common and rare nuclear variation in 17 M. pallida individuals sampled from all known populations. We find that the different variant types capture distinct aspects of the evolutionary history and discuss the observed patterns in the context of parthenogenesis, polyploidy, and survival during glaciation. This study highlights the potential of different variant types to gain insights into evolutionary scenarios even from challenging but often available data and the suitability of M. pallida and the genus Machilis as a study system for the evolution of sexual strategies and polyploidization during environmental change. We also emphasize the need for further research which will be stimulated and facilitated by these newly generated resources and insights.

Short-time development of among-colony behaviour in a high-elevation ant.

Standardised assays are often used to characterise aggression in animals. In ants, such assays can be applied at several organisational levels (e.g., colony, population) and at specific times during the season. However, whether the behaviour differs at these levels and changes over a few weeks remains largely unexplored. Here, six colonies from the high-elevation ant Tetramorium alpestre were collected weekly for five weeks from two behaviourally-different populations (aggressive and peaceful in intraspecific encounters). We conducted one-on-one worker encounters at the colony and population levels. When analysing the colony combinations separately, the behaviour was peaceful and remained so within the peaceful population; initial aggression became partially peaceful within the aggressive population; and initial aggression decreased occasionally and increased in one combination but remained constant for most across-population combinations. When analysing all colony combinations together, within-population behaviour remained similar, but across-population behaviour became peaceful. The observed behavioural differences among organisational levels emphasise the relevance of assessing both levels. Moreover, the effect of decreasing aggression is discernible already over a few weeks. Compression of the vegetation period at high elevations may compress such behavioural changes. Addressing both organisational levels and seasonality is important, particularly in studies of behavioural complexity such as in this ant.

Wolbachia endosymbiotic bacteria alter the gut microbiome in the fly Drosophila nigrosparsa.

Wolbachia are known to cause reproductive manipulations and in some arthropod species, Wolbachia were reported to cause changes in gut microbiome. However, the effects of Wolbachia bacteria on the microbiomes of their hosts, including Drosophila flies, have not been fully accessed. Here, we checked the bacterial microbiome in guts of Wolbachia-uninfected and of Wolbachia-infected Drosophila nigrosparsa, both separated into a bleach-only (embryos bleached) and a gnotobiotic (embryos bleached and inoculated with bacteria) treatment. We observed a clear separation between the Wolbachia-infected and the Wolbachia-uninfected samples, and the infected samples had higher variation in alpha diversity than the uninfected ones. There were reductions in the abundances of Proteobacteria (Pseudomonadota), especially Acetobacter, in the infected samples of both treatments. These findings highlight that Wolbachia change the gut microbiome in D. nigrosparsa as well as that the interactions between Wolbachia and bacteria like Acetobacter need to be investigated.

Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy.

3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques with fluorescence imaging. Here, we achieve 3D super-resolution by combining the 2D localization of pMINFLUX with the axial information of graphene energy transfer (GET) and the single-molecule switching by DNA-PAINT. We demonstrate <2 nm localization precision in all 3 dimension with axial precision reaching below 0.3 nm. In 3D DNA-PAINT measurements, structural features, i.e., individual docking strands at distances of 3 nm, are directly resolved on DNA origami structures. pMINFLUX and GET represent a particular synergetic combination for super-resolution imaging near the surface such as for cell adhesion and membrane complexes as the information of each photon is used for both 2D and axial localization information. Furthermore, we introduce local PAINT (L-PAINT), in which DNA-PAINT imager strands are equipped with an additional binding sequence for local upconcentration improving signal-to-background ratio and imaging speed of local clusters. L-PAINT is demonstrated by imaging a triangular structure with 6 nm side lengths within seconds.

Phylogenomic inference and demographic model selection suggest peripatric separation of the cryptic steppe ant species Plagiolepis pyrenaica stat. rev.

The ant Plagiolepis taurica Santschi, 1920 (Hymenoptera, Formicidae) is a typical species of the Eurasian steppes, a large grassland dominated biome that stretches continuously from Central Asia to Eastern Europe and is represented by disjunct outposts also in Central and Western Europe. The extent of this biome has been influenced by the Pleistocene climate, and steppes expanded recurrently during cold stages and contracted in warm stages. Consequently, stenotopic steppe species such as P. taurica repeatedly went through periods of demographic expansion and severe isolation. Here, we explore the impact of these dynamics on the genetic diversification within P. taurica. Delimitation of P. taurica from other Plagiolepis species has been unclear since its initial description, which raised questions on both its classification and its spatiotemporal diversification early on. We re-evaluate species limits and explore underlying mechanisms driving speciation by using an integrative approach based on genomic and morphometric data. We found large intraspecific divergence within P. taurica and resolved geographically coherent western and eastern genetic groups, which likewise differed morphologically. A morphometric survey of type material showed that Plagiolepis from the western group were more similar to P. barbara pyrenaica Emery, 1921 than to P. taurica; we thus lift the former from synonymy and establish it as separate species, P. pyrenaica stat. rev. Explicit evolutionary model testing based on genomic data supported a peripatric speciation for the species pair, probably as a consequence of steppe contraction and isolation during the mid-Pleistocene. We speculate that this scenario could be exemplary for many stenotopic steppe species, given the emphasized dynamics of Eurasian steppes.

Global change may make hostile - Higher ambient temperature and nitrogen availability increase ant aggression.

Behaviour is a response of organisms to internal and external stimuli and comprises various activities such as searching for food. Aggression is important in such activities, for example, improving the chances of winning competition for food, but animals differ in their level of aggression. This behavioural plasticity allows individuals to respond to environmental changes and is important for the survival of animals. It may be an important asset in facing global changes, which affect all organisms, for example, via rising temperature and eutrophication. The latter have steadily increased since 1900, especially in high elevations. Their effects may first become visible in stationary organisms such as ants because their nests are strictly associated with the conditions on site. Here, we analysed eight populations of the high-elevation ant Tetramorium alpestre along several elevations spanning the European Alps. We conducted a correlative approach and analysed several genetic and environmental proxies, namely within- and across-colony genetic relatedness, cuticular hydrocarbons, body size, across-colony geographic distance, air temperature, and worker nitrogen values additionally to within-population aggressive behaviour. We hypothesised that a) these proxies and aggressive behaviour differ among populations and that b) one or more of these proxies influence aggression. We found that a) some environmental proxies and aggression differed among populations but not the genetic proxies and that b) air temperature and worker nitrogen-isotope values correlated positively with worker aggression. The results indicate an environmental but not social-structural influence on this ant's aggressive behaviour, even though social structure varied among populations (single- and multiple-queened colonies). We infer that global change affects aggression in our study system and propose five mutually non-exclusive scenarios to explain the behavioural change mechanistically. Using the space-for-time principle, we speculate that aggression may increase due to future increases in temperature and nitrogen availability in this ant and other species living in high elevations.

Distribution of soil macrofauna across different habitats in the Eastern European Alps.

Macro-invertebrates are important components of soil ecosystems as they provide a wide range of crucial functions and ecosystem services. Knowledge on their distribution in mountain soils is scarce despite the importance of such soils for people living in mountain regions as well as downstream. The present dataset contains records on soil macro-invertebrates belonging to nineteen taxa listed at class or order level and earthworms listed at species level from 22 different habitat types characteristic for the Eastern European Alps. Data were collected over a period of more than 30 years (1987-2020) following a standard protocol. We compiled 1572 single records from 241 unique sampling sites, providing default site parameters (GPS coordinates, habitat type, type of management, elevation, exposition, inclination, bedrock, soil type following WRB classification). Such data are important to analyse global trends and macroecological patterns and to set a basis for tracking long-term changes in macrofauna composition. In addition, this dataset will add to the still sparse knowledge on the occurrence and abundance of alpine soil fauna taxa.

Recurrent but Short-Lived Duplications of Centromeric Proteins in Holocentric Caenorhabditis Species.

Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis species, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) were previously identified in C. elegans and C. remanei. Using phylogenomic analyses in 32 Caenorhabditis species, we find strict retention of the ancestral hcp-3 gene and 10 independent duplications. Most hcp-3L (hcp-3-like) paralogs are only found in 1-2 species, are expressed in both males and females/hermaphrodites, and encode histone fold domains with 69-100% identity to ancestral hcp-3. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential subfunctionalization or functional loss following duplication. An N-terminal extension in the hcp-3L gene of C. afra revealed an unprecedented protein fusion, where hcp-3L fused to duplicated segments from hcp-4 (nematode CENP-C). By extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, which appear to have been retained independent of hcp-3 duplications. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid "revolving door" of kinetochore protein paralogs.

Reply to Letter to the Editor: Huang, M., 2022: Comments on "Persistent, bioaccumulative, and toxic chemicals in insects: Current state of research and where to from here?". Sci Total Environ. 838, 155624, and Corrigendum.

In our recently published study in Science of the Total Environment, we used a systematic literature search to investigate the current state of research of persistent, bioaccumulative, and toxic chemicals (PBTs) and insects. We found a distinct increase of human, animal, and vertebrate publications related to PBTs in the early 1990s but did not identify a conclusive cause for this. In her Letter to the Editor, Huang (2022) offered an explanation for our result, and we have used her initiative to repeat our analysis with refined methodology. We present a corrected version of our original Fig. 1; importantly, though, our main finding, the general underrepresentation of insects in the research of PBTs, has remained the same. We conclude by addressing difficulties such as the reproducibility in literature searches and by discussing consequences of unequal resource distributions in science.

Analyses of locomotion, wing morphology, and microbiome in Drosophila nigrosparsa after recovery from antibiotics.

Antibiotics, such as tetracycline, have been frequently used to cure arthropods of Wolbachia endosymbionts. After the symbionts have been removed, the hosts must recover for some generations from the side effects of the antibiotics. However, most studies do not assess the direct and indirect longer-term effects of antibiotics used to remove Wolbachia, which may question the exact contribution of this endosymbiont to the effects observed. Here, we used the fly Drosophila nigrosparsa treated or not with tetracycline for three generations followed by two generations of recovery to investigate the effects of this antibiotic on the fly locomotion, wing morphology, and the gut microbiome. We found that antibiotic treatment did not affect fly locomotion two generations after being treated with the antibiotic. In addition, gut-microbiome restoration was tested as a more efficient solution to reduce the potential side effects of tetracycline on the microbiome. There was no significant difference in alpha diversity between gut restoration and other treatments, but the abundance of some bacterial taxa differed significantly between the gut-restoration treatment and the control. We conclude that in D. nigrosparsa the recovery period of two generations after being treated with the antibiotic is sufficient for locomotion, and suggest a general assessment of direct and indirect effects of antibiotics after a particular recovery time.

Congruent evolutionary responses of European steppe biota to late Quaternary climate change.

Quaternary climatic oscillations had a large impact on European biogeography. Alternation of cold and warm stages caused recurrent glaciations, massive vegetation shifts, and large-scale range alterations in many species. The Eurasian steppe biome and its grasslands are a noteworthy example; they underwent climate-driven, large-scale contractions during warm stages and expansions during cold stages. Here, we evaluate the impact of these range alterations on the late Quaternary demography of several phylogenetically distant plant and insect species, typical of the Eurasian steppes. We compare three explicit demographic hypotheses by applying an approach combining convolutional neural networks with approximate Bayesian computation. We identified congruent demographic responses of cold stage expansion and warm stage contraction across all species, but also species-specific effects. The demographic history of the Eurasian steppe biota reflects major paleoecological turning points in the late Quaternary and emphasizes the role of climate as a driving force underlying patterns of genetic variance on the biome level.

Persistent, bioaccumulative, and toxic chemicals in insects: Current state of research and where to from here?

The ongoing decline in the biomass, abundance, and species number of insects is an established fact. Persistent, bioaccumulative, and toxic chemicals (PBTs) - persistent organic pollutants (POPs) and, in the case of our study, mercury (Hg) - play an important role, but their effect on insect populations is insufficiently investigated. Here, the current state of research on PBTs related to insects is examined with a systematic literature study using Web of Science™. We investigate time trends of research intensity compared with other organisms, insect orders and chemicals analyzed, chemicals' effects on insects, and geographical aspects. We show that research intensity increased in the early 1990s, but studies on PBTs in insects are still underrepresented compared with other organisms. The taxonomic focus lies strongly on dipterans. The predominance of studies on DDT suggests its relevance in the context of disease-vector management. Phenotypic and acute effects on insects were more often investigated than genotypic and chronic effects. Laboratory-bred insects and wild-bred insects were examined equally often, pollutant exposure and analysis were conducted predominantly in the laboratory. Mostly habitats with a medium or high human impact were studied, and natural and near-natural habitats are understudied. The sources of the substances are often unknown. Most studies were carried out in economically rich continents, including North America, Europe, and Australia. The numbers of publications dealing with Asia, South America, and Africa are comparatively low, although the control of vector-borne diseases with POPs is still intensively practiced there. We identify gaps in the research - among others, refined analytical methods for biomarkers and for the examination of chronic effects, combinations of field and laboratory experiments to analyze the same problem, and a global approach for the monitoring of PBTs will be needed for accelerating the dearly needed progress in the research of PBTs in insects.

Persistent, Bioaccumulative, and Toxic Chemicals in Wild Alpine Insects: A Methodological Case Study.

With their high persistence in the environment and their potential for long-range atmospheric transport, persistent, bioaccumulative, and toxic chemicals (PBTs) may be among the numerous anthropogenic threats to insect populations worldwide. The effects of PBTs on insects have been investigated in the laboratory, but topical field studies are scarce. A reason might be the multiple challenges faced by PBT-related field studies on wild insects. We studied two species of bumblebees (Bombus spp.) and of ants (Formica spp.) in two high-elevation locations in the Austrian and German Alps to tackle two of these challenges. First, PBTs occur in minuscule concentrations compared with other substances in the environment. Therefore, the practicability of body burden data from pooled individuals was tested. Second, fitness proxies like fecundity, which typically are endpoints for chemical toxicity, are difficult to quantify in the field. Hence, fluctuating asymmetry of bumblebee wings and ant heads was tested as an alternative endpoint. To exclude the possibility that fluctuating asymmetry was caused by genetic stressors, inbreeding levels were estimated using population-genetic markers, and their relationships to fluctuating asymmetry in the same individuals were assessed. We successfully quantified polychlorinated biphenyls and Hg as PBTs using the pooled samples and found PBT data from pooled individuals useful, in that significant correlations to fluctuating asymmetry were identified in bumblebees and ants. This finding confirmed the potential of fluctuating asymmetry to indicate PBT effects in wild insects. Inbreeding did not interfere with PBT links to fluctuating asymmetry in any instance. Our findings contribute to the development of a quantitative methodological framework for investigating the effects of persistent environmental chemicals on wild insects. Environ Toxicol Chem 2022;41:1215-1227. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Transmission of chromatin states across generations in C. elegans.

Epigenetic inheritance refers to the transmission of phenotypes across generations without affecting the genomic DNA sequence. Even though it has been documented in many species in fungi, animals and plants, the mechanisms underlying epigenetic inheritance are not fully uncovered. Epialleles, the heritable units of epigenetic information, can take the form of several biomolecules, including histones and their post-translational modifications (PTMs). Here, we review the recent advances in the understanding of the transmission of histone variants and histone PTM patterns across generations in C. elegans. We provide a general overview of the intergenerational and transgenerational inheritance of histone PTMs and their modifiers and discuss the interplay among different histone PTMs. We also evaluate soma-germ line communication and its impact on the inheritance of epigenetic traits.

Hybridization Dynamics and Extensive Introgression in the Daphnia longispina Species Complex: New Insights from a High-Quality Daphnia galeata Reference Genome.

Hybridization and introgression are recognized as an important source of variation that influence adaptive processes; both phenomena are frequent in the genus Daphnia, a keystone zooplankton taxon in freshwater ecosystems that comprises several species complexes. To investigate genome-wide consequences of introgression between species, we provide here the first high-quality genome assembly for a member of the Daphnia longispina species complex, Daphnia galeata. We further resequenced 49 whole genomes of three species of the complex and their interspecific hybrids both from genotypes sampled in the water column and from single resting eggs extracted from sediment cores. Populations from habitats with diverse ecological conditions offered an opportunity to study the dynamics of hybridization linked to ecological changes and revealed a high prevalence of hybrids. Using phylogenetic and population genomic approaches, we provide first insights into the intra- and interspecific genome-wide variability in this species complex and identify regions of high divergence. Finally, we assess the length of ancestry tracts in hybrids to characterize introgression patterns across the genome. Our analyses uncover a complex history of hybridization and introgression reflecting multiple generations of hybridization and backcrossing in the Daphnia longispina species complex. Overall, this study and the new resources presented here pave the way for a better understanding of ancient and contemporary gene flow in the species complex and facilitate future studies on resting egg banks accumulating in lake sediment.

Mitotic chromosome condensation requires phosphorylation of the centromeric protein KNL-2 in C. elegans.

Centromeres are chromosomal regions that serve as sites for kinetochore formation and microtubule attachment, processes that are essential for chromosome segregation during mitosis. Centromeres are almost universally defined by the histone variant CENP-A. In the holocentric nematode C. elegans, CENP-A deposition depends on the loading factor KNL-2. Depletion of either CENP-A or KNL-2 results in defects in centromere maintenance, chromosome condensation and kinetochore formation, leading to chromosome segregation failure. Here, we show that KNL-2 is phosphorylated by CDK-1 in vitro, and that mutation of three C-terminal phosphorylation sites causes chromosome segregation defects and an increase in embryonic lethality. In strains expressing phosphodeficient KNL-2, CENP-A and kinetochore proteins are properly localised, indicating that the role of KNL-2 in centromere maintenance is not affected. Instead, the mutant embryos exhibit reduced mitotic levels of condensin II on chromosomes and significant chromosome condensation impairment. Our findings separate the functions of KNL-2 in CENP-A loading and chromosome condensation, and demonstrate that KNL-2 phosphorylation regulates the cooperation between centromeric regions and the condensation machinery in C. elegans. This article has an associated First Person interview with the first author of the paper.

How Blinking Affects Photon Correlations in Multichromophoric Nanoparticles.

A single chromophore can only emit a maximum of one single photon per excitation cycle. This limitation results in a phenomenon commonly referred to as photon antibunching (pAB). When multiple chromophores contribute to the fluorescence measured, the degree of pAB has been used as a metric to "count" the number of chromophores. But the fact that chromophores can switch randomly between bright and dark states also impacts pAB and can lead to incorrect chromophore numbers being determined from pAB measurements. By both simulations and experiment, we demonstrate how pAB is affected by independent and collective chromophore blinking, enabling us to formulate universal guidelines for correct interpretation of pAB measurements. We use DNA-origami nanostructures to design multichromophoric model systems that exhibit either independent or collective chromophore blinking. Two approaches are presented that can distinguish experimentally between these two blinking mechanisms. The first one utilizes the different excitation intensity dependence on the blinking mechanisms. The second approach exploits the fact that collective blinking implies energy transfer to a quenching moiety, which is a time-dependent process. In pulsed-excitation experiments, the degree of collective blinking can therefore be altered by time gating the fluorescence photon stream, enabling us to extract the energy-transfer rate to a quencher. The ability to distinguish between different blinking mechanisms is valuable in materials science, such as for multichromophoric nanoparticles like conjugated-polymer chains as well as in biophysics, for example, for quantitative analysis of protein assemblies by counting chromophores.