Infrequent Long-Range Dispersal and Evolution of a Top Terrestrial Arthropod Predator in the Sub-Antarctic.

AbstractThe sub-Antarctic terrestrial ecosystems survive on isolated oceanic islands in the path of circumpolar currents and winds that have raged for more than 30 million years and are shaped by climatic cycles that surpass the tolerance limits of many species. Surprisingly little is known about how these ecosystems assembled their native terrestrial fauna and how such processes have changed over time. Here, we demonstrate the patterns and timing of colonization and speciation in the largest and dominant arthropod predators in the eastern sub-Antarctic: spiders of the genus Myro. Our results indicate that this lineage originated from Australia before the Plio-Pleistocenic glacial cycles and underwent an adaptive radiation on the Crozet archipelago, from where one native species colonized multiple remote archipelagos via the Antarctic circumpolar current across thousands of kilometers. The results indicate limited natural connectivity between terrestrial macroinvertebrate faunas in the eastern sub-Antarctic and partial survival of repeated glaciations in the Plio-Pleistocene. Furthermore, our findings highlight that by integrating arthropod taxa from multiple continents, the climatically more stable volcanic Crozet archipelago played a critical role in the evolution and distribution of arthropod life in the sub-Antarctic.

Comparative anatomy of the spinneret musculature in cribellate and ecribellate spiders (Araneae).

Silk production is a prominent characteristic of spiders. The silk is extruded through spigots located on the spinnerets, which are single- to multimembered paired appendages at the end of the abdomen. Most extant spiders have three pairs of spinnerets, and in between either a cribellum (spinning plate) or a colulus (defunct vestigial organ), dividing these spiders into cribellate and ecribellate species. Previous research has shown that cribellate and ecribellate spiders differ not only in the composition of their spinning apparatus but also in the movements of their spinnerets during silk spinning. The objective of this study was to determine whether the differences in spinneret movements are solely due to variations in spinneret shape or whether they are based on differences in muscular anatomy. This was accomplished by analyzing microcomputed tomography scans of the posterior abdomen of each three cribellate and ecribellate species. It was found that the number of muscles did not generally differ between cribellate and ecribellate species, but varied considerably between the species within each of these two groups. Muscle thickness, particularly of the posterior median spinneret, varied slightly between groups, with cribellate spiders exhibiting more robust muscles, possibly to aid in the combing process during cribellar thread production. Interestingly, the vestigial colulus still possesses muscles, that can be homologized with those of the cribellum. This exploration into spinneret anatomy using microcomputed tomography data reveals that despite being small appendages, the spider spinnerets are equipped with a complex musculature that enables them to perform fine-scaled maneuvers to construct different fiber-based materials.

Facilitating taxonomy and phylogenetics: An informative and cost-effective protocol integrating long amplicon PCRs and third-generation sequencing.

Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide.

Dynamic evolution of locomotor performance independent of changes in extended phenotype use in spiders.

Many animals use self-built structures (extended phenotypes) to enhance body functions, such as thermoregulation, prey capture or defence. Yet, it is unclear whether the evolution of animal constructions supplements or substitutes body functions-with disparate feedbacks on trait evolution. Here, using brown spiders (Araneae: marronoid clade), we explored if the evolutionary loss and gain of silken webs as extended prey capture devices correlates with alterations in traits known to play an important role in predatory strikes-locomotor performance (sprint speed) and leg spination (expression of capture spines on front legs). We found that in this group high locomotor performance, with running speeds of over 100 body lengths per second, evolved repeatedly-both in web-building and cursorial spiders. There was no correlation with running speed, and leg spination only poorly correlated, relative to the use of extended phenotypes, indicating that web use does not reduce selective pressures on body functions involved in prey capture and defence per se. Consequently, extended prey capture devices serve as supplements rather than substitutions to body traits and may only be beneficial in conjunction with certain life-history traits, possibly explaining the rare evolution and repeated loss of trapping strategies in predatory animals.

Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

Geometric regularity in webs of non-orb-weaving spiders.

Geometric regularity of spider webs has been intensively studied in orb-weaving spiders, although it is not exclusive of orb weavers. Here, we document the geometrically regular, repetitive elements in the webs of the non-orb-weaving groups Leptonetidae and Telemidae for the first time. Similar to orb weavers, we found areas with regularly spaced parallel lines in the webs of Calileptoneta helferi, Sulcia sp., and cf. Pinelema sp. Furthermore, we provide a detailed account of the regular webs of Ochyrocera (Ochyroceratidae). The sections of the web with regularly disposed parallel lines are built as U-shaped modules reminiscent of orb webs. It has been suggested that the regularly spaced parallel lines in the webs of Ochyroceratidae and Psilodercidae may be produced in a single sweep of their posterior lateral spinnerets, which have regularly spaced aciniform gland spigots, perhaps involving expansion of the spinnerets. To test this hypothesis, we compared the spacing between parallel lines with the spacing between spigots, searched for expansible membranes in the spinnerets, and examined the junctions of regularly spaced lines. The distance between parallel lines was 10-20 times the distance between spigots, and we found no expansible membranes, and the intersection of parallel lines are cemented, which opposes the single sweep hypothesis. Furthermore, we found cues of viscid silk in the parallel lines of the psilodercid Althepus and broadened piriform gland spigots that may be responsible of its production. Finally, we evaluated the presence or absence of geometrically regular web elements across the spider tree of life. We found reports of regular webs in 31 spider families, including 20 families that are not orb weavers and hypothesize that the two basic aspects of regularity (parallel lines spaced at regular intervals, and radial lines spaced at regular angles) probably appeared many times in the evolution of spiders.

Prime editing in hematopoietic stem cells-From ex vivo to in vivo CRISPR-based treatment of blood disorders.

Prime editing of human hematopoietic stem cells has the potential to become a safe and efficient way of treating diseases of the blood directly in patients. By allowing site-targeted gene intervention without homology-directed repair donor templates and DNA double-stranded breaks, the invention of prime editing fuels the exploration of alternatives to conventional recombination-based ex vivo genome editing of hematopoietic stem cells. Prime editing is as close as we get today to a true genome editing drug that does not require a separate DNA donor. However, to adapt the technology to perform in vivo gene correction, key challenges remain to be solved, such as identifying effective prime editing guide RNAs for clinical targets as well as developing efficient vehicles to deliver prime editors to stem cells in vivo. In this review, we summarize the current progress in delivery of prime editors both in vitro and in vivo and discuss future challenges that need to be adressed to allow in vivo prime editing as a cure for blood disorders.

Delivering genes with human immunodeficiency virus-derived vehicles: still state-of-the-art after 25 years.

Viruses are naturally endowed with the capacity to transfer genetic material between cells. Following early skepticism, engineered viruses have been used to transfer genetic information into thousands of patients, and genetic therapies are currently attracting large investments. Despite challenges and severe adverse effects along the way, optimized technologies and improved manufacturing processes are driving gene therapy toward clinical translation. Fueled by the outbreak of AIDS in the 1980s and the accompanying focus on human immunodeficiency virus (HIV), lentiviral vectors derived from HIV have grown to become one of the most successful and widely used vector technologies. In 2022, this vector technology has been around for more than 25 years. Here, we celebrate the anniversary by portraying the vector system and its intriguing properties. We dive into the technology itself and recapitulate the use of lentiviral vectors for ex vivo gene transfer to hematopoietic stem cells and for production of CAR T-cells. Furthermore, we describe the adaptation of lentiviral vectors for in vivo gene delivery and cover the important contribution of lentiviral vectors to basic molecular research including their role as carriers of CRISPR genome editing technologies. Last, we dwell on the emerging capacity of lentiviral particles to package and transfer foreign proteins.

Fast acrobatic maneuvers enable arboreal spiders to hunt dangerous prey.

Spiders, the most specious taxon of predators, have evolved an astounding range of predatory strategies, including group hunting, specialized silk traps, pheromone-loaded bolas, and aggressive mimicry. Spiders that hunt prey defended with behavioral, mechanical, or chemical means are under additional selection pressure to avoid injury and death. Ants are considered dangerous because they can harm or kill their predators, but some groups of spiders, such as the Theridiidae, have a very high diversification of ant-hunting species and strategies [J. Liu et al., Mol. Phylogenet. Evol. 94, 658-675 (2016)]. Here, we provide detailed behavioral analyses of the highly acrobatic Australian ant-slayer spider, Euryopis umbilicata (Theridiidae), that captures much larger and defended Camponotus ants on vertical tree trunks. The hunting sequence consists of ritualized steps performed within split seconds, resulting in an exceptionally high prey capture success rate.

AnimalTraits - a curated animal trait database for body mass, metabolic rate and brain size.

Trait databases have become important resources for large-scale comparative studies in ecology and evolution. Here we introduce the AnimalTraits database, a curated database of body mass, metabolic rate and brain size, in standardised units, for terrestrial animals. The database has broad taxonomic breadth, including tetrapods, arthropods, molluscs and annelids from almost 2000 species and 1000 genera. All data recorded in the database are sourced from their original empirical publication, and the original metrics and measurements are included with each record. This allows for subsequent data transformations as required. We have included rich metadata to allow users to filter the dataset. The additional R scripts we provide will assist researchers with aggregating standardised observations into species-level trait values. Our goals are to provide this resource without restrictions, to keep the AnimalTraits database current, and to grow the number of relevant traits in the future.

Stabilized Morphological Evolution of Spiders Despite Mosaic Changes in Foraging Ecology.

A prominent question in animal research is how the evolution of morphology and ecology interacts in the generation of phenotypic diversity. Spiders are some of the most abundant arthropod predators in terrestrial ecosystems and exhibit a diversity of foraging styles. It remains unclear how spider body size and proportions relate to foraging style, and if the use of webs as prey capture devices correlates with changes in body characteristics. Here, we present the most extensive data set to date of morphometric and ecological traits in spiders. We used this data set to estimate the change in spider body sizes and shapes over deep time and to test if and how spider phenotypes are correlated with their behavioral ecology. We found that phylogenetic variation of most traits best fitted an Ornstein-Uhlenbeck model, which is a model of stabilizing selection. A prominent exception was body length, whose evolutionary dynamics were best explained with a Brownian Motion (free trait diffusion) model. This was most expressed in the araneoid clade (ecribellate orb-weaving spiders and allies) that showed bimodal trends toward either miniaturization or gigantism. Only few traits differed significantly between ecological guilds, most prominently leg length and thickness, and although a multivariate framework found general differences in traits among ecological guilds, it was not possible to unequivocally associate a set of morphometric traits with the relative ecological mode. Long, thin legs have often evolved with aerial webs and a hanging (suspended) locomotion style, but this trend is not general. Eye size and fang length did not differ between ecological guilds, rejecting the hypothesis that webs reduce the need for visual cue recognition and prey immobilization. For the inference of the ecology of species with unknown behaviors, we propose not to use morphometric traits, but rather consult (micro-)morphological characters, such as the presence of certain podal structures. These results suggest that, in contrast to insects, the evolution of body proportions in spiders is unusually stabilized and ecological adaptations are dominantly realized by behavioral traits and extended phenotypes in this group of predators. This work demonstrates the power of combining recent advances in phylogenomics with trait-based approaches to better understand global functional diversity patterns through space and time. [Animal architecture; Arachnida; Araneae; extended phenotype; functional traits; macroevolution; stabilizing selection.].

Cerotegument microstructure of whip spiders (Amblypygi: Euamblypygi Weygoldt, 1996) reveals characters for systematics from family to species level.

Like other arthropods, whip spiders of the arachnid order Amblypygi Thorell, 1883 protect themselves against external environmental influences. In this taxon, in addition to the epicuticle, the outermost layer of the exoskeleton, a cement layer (cerotegument) with superhydrophobic properties is deposited over certain body parts. Due to the high level of interspecific variation, the cerotegument structure and the morphology of its associated gland openings have been suggested to be informative for whip spider systematics. The first comparative study of the cerotegument is presented herein, based on a survey across 4 families, 16 genera, and 62 species of Euamblypygi Weygoldt, 1996, the suborder comprising all extant whip spiders except the rare monotypic family Paracharontidae Weygoldt, 1996. Results confirmed that the morphology of the colloidal particles and their assembly on cement globules differ considerably among taxa, but that the level of variation differs among lineages. Interspecific variation in cerotegument ultrastructure was highest among species of Neoamblypygi Weygoldt, 1996, making it an informative character in this clade. Evolutionary trends and intraspecific variation in the structure of the amblypygid cerotegument are briefly discussed.

Nutritionally induced nanoscale variations in spider silk structural and mechanical properties.

Spider major ampullate (MA) silk is characterized by high strength and toughness and is adaptable across environments. Experiments depriving spiders of protein have enabled researchers to examine nutritionally induced changes in gene expression, protein structures, and bulk properties of MA silk. However, it has not been elucidated if it varies in a similar way at a nanoscale. Here we used Atomic Force Microscopy (AFM) to simultaneously examine the topographic, structural, and mechanical properties of silks spun by two species of spider, Argiope keyserlingi and Latrodectus hasselti, at a nanoscale when protein fed or deprived. We found height, a measure of localized width, to substantially vary across species and treatments. We also found that Young's modulus, which may be used as an estimate of localized stiffness, decreased with protein deprivation in both species' silk. Our results suggest that nanoscale skin-core structures of A. keyserlingi's MA silk varied significantly across treatments, whereas only slight structural and functional variability was found for L. hasselti's silk. These results largely agreed with examinations of the bulk properties of each species' silk. However, we could not directly attribute the decoupling between protein structures and bulk mechanics in L. hasselti's silk to nanoscale features. Our results advance the understanding of processes inducing skin and core structural variations in spider silks at a nanoscale, which serves to enhance the prospect of developing biomimetic engineering programs.

piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac-Based DNA Transposition.

Prime editing is a novel genome editing technology that allows a wide range of tailored genomic alterations. Prime editing does not involve homologous recombination, but suffers from low efficacy. Here, we demonstrate piggyPrime, a transfected single-vector system based on piggyBac DNA transposition for genomic integration of all prime editing components in human cells allowing easy and effective transgenesis with prime editing efficacies up to 100% in cell lines.

The World Spider Trait database: a centralized global open repository for curated data on spider traits.

Spiders are a highly diversified group of arthropods and play an important role in terrestrial ecosystems as ubiquitous predators, which makes them a suitable group to test a variety of eco-evolutionary hypotheses. For this purpose, knowledge of a diverse range of species traits is required. Until now, data on spider traits have been scattered across thousands of publications produced for over two centuries and written in diverse languages. To facilitate access to such data, we developed an online database for archiving and accessing spider traits at a global scale. The database has been designed to accommodate a great variety of traits (e.g. ecological, behavioural and morphological) measured at individual, species or higher taxonomic levels. Records are accompanied by extensive metadata (e.g. location and method). The database is curated by an expert team, regularly updated and open to any user. A future goal of the growing database is to include all published and unpublished data on spider traits provided by experts worldwide and to facilitate broad cross-taxon assays in functional ecology and comparative biology. Database URL:https://spidertraits.sci.muni.cz/.

Robust substrate anchorages of silk lines with extensible nano-fibres.

Living systems are built of multiscale-composites: materials formed of components with different properties that are assembled in complex micro- and nano-structures. Such biological multiscale-composites often show outstanding physical properties that are unachieved by artificial materials. A major scientific goal is thus to understand the assembly processes and the relationship between structure and function in order to reproduce them in a new generation of biomimetic high-performance materials. Here, we tested how the assembly of spider silk nano-fibres (i.e. glue coated 0.5 µm thick fibres produced by so-called piriform glands) into different micro-structures correlates with mechanical performance by empirically and numerically exploring the mechanical behaviour of line anchors in an orb weaver, a hunting spider and two ancient web builders. We demonstrate that the anchors of orb weavers exhibit outstanding mechanical robustness with minimal material use by the indirect attachment of the silk line to the substrate through a soft domain ('bridge'). This principle can be used to design new artificial high-performance attachment systems.

Building behavior does not drive rates of phenotypic evolution in spiders.

Do animals set the course for the evolution of their lineage when manipulating their environment? This heavily disputed question is empirically unexplored but critical to interpret phenotypic diversity. Here, we tested whether the macroevolutionary rates of body morphology correlate with the use of built artifacts in a megadiverse clade comprising builders and nonbuilders-spiders. By separating the inferred building-dependent rates from background effects, we found that variation in the evolution of morphology is poorly explained by artifact use. Thus natural selection acting directly on body morphology rather than indirectly via construction behavior is the dominant driver of phenotypic diversity.

pegIT - a web-based design tool for prime editing.

Prime editing (PE) is a novel CRISPR-derived genome editing technique facilitating precision editing without double-stranded DNA breaks. PE, mediated by a Cas9-reverse transcriptase fusion protein, is based on dual-functioning prime editing guide RNAs (pegRNAs), serving both as guide molecules and as templates carrying the desired edits. Due to such diverse functions, manual pegRNA design is a subject to error and not suited for large-scale setups. Here, we present pegIT, a user-friendly web tool for rapid pegRNA design for numerous user-defined edits, including large-scale setups. pegIT is freely available at https://pegit.giehmlab.dk.

Fine structure of the epicuticular secretion coat and associated glands of Pedipalpi and Palpigradi (Arachnida).

Pedipalpi Latreille, 1810 is a poorly studied clade of arachnids comprising the whip spiders (Amblypygi Thorell, 1883), short-tailed whip scorpions (Schizomida Petrunkevitch, 1945) and whip scorpions (Thelyphonida Cambridge, 1872). It has recently been shown that whip spiders coat their exoskeleton with a solid cement layer (cerotegument) that forms elaborate microstructures and turns the cuticle into a super-hydrophobic state. The amblypygid cerotegument provides taxonomic information due to its fine structural diversity, but its presence and variation in the sister groups was previously unknown. The present contribution reports the surface structure of the cuticle in species of Palpigradi, Thelyphonida, and Schizomida to determine if these taxa possess a solid epicuticular secretion coat. Scanning electron microscopy revealed that in addition to Amblypygi only species of Thelyphonida possess solid epicuticular secretion layers. Unlike in Amblypygi, in the Thelyphonida this layer does not usually form microstructures and is less rigidly attached to the underlying cuticle. A species of Typopeltis Pocock, 1894, which exhibited globular structures analogous to the amblypygid cerotegument, was an exception. Glandular structures associated with cement secretions in Amblypygi and Thelyphonida were considered homologous due to similar structure. Solid epicuticular secretion coats were absent from Schizomida, which is interpreted as a secondary loss despite the presence of slit-like glandular openings that appear to produce such epicuticular secretions. The micro-whip scorpion order Palpigradi Thorell, 1900 exhibited markedly different cuticular surface structures and lacked solid epicuticular secretions, consistent with the hypothesis that this order is not closely related to Pedipalpi. These results enhance the knowledge of the small, enigmatic orders of Arachnida.

Locomotion and kinematics of arachnids.

A basic feature of animals is the capability to move and disperse. Arachnids are one of the oldest lineages of terrestrial animals and characterized by an octopodal locomotor apparatus with hydraulic limb extension. Their locomotion repertoire includes running, climbing, jumping, but also swimming, diving, abseiling, rolling, gliding and -passively- even flying. Studying the unique locomotor functions and movement ecology of arachnids is important for an integrative understanding of the ecology and evolution of this diverse and ubiquitous animal group. Beyond biology, arachnid locomotion is inspiring robotic engineers. The aim of this special issue is to display the state of the interdisciplinary research on arachnid locomotion, linking physiology and biomechanics with ecology, ethology and evolutionary biology. It comprises five reviews and ten original research reports covering diverse topics, ranging from the neurophysiology of arachnid movement, the allometry and sexual dimorphism of running kinematics, the effect of autotomy or heavy body parts on locomotor efficiency, and the evolution of silk-spinning choreography, to the biophysics of ballooning and ballistic webs. This closes a significant gap in the literature on animal biomechanics.