Sampling Strategies for Experimentally Mapping Molecular Fitness Landscapes Using High-Throughput Methods.

Empirical studies of genotype-phenotype-fitness maps of proteins are fundamental to understanding the evolutionary process, in elucidating the space of possible genotypes accessible through mutations in a landscape of phenotypes and fitness effects. Yet, comprehensively mapping molecular fitness landscapes remains challenging since all possible combinations of amino acid substitutions for even a few protein sites are encoded by an enormous genotype space. High-throughput mapping of genotype space can be achieved using large-scale screening experiments known as multiplexed assays of variant effect (MAVEs). However, to accommodate such multi-mutational studies, the size of MAVEs has grown to the point where a priori determination of sampling requirements is needed. To address this problem, we propose calculations and simulation methods to approximate minimum sampling requirements for multi-mutational MAVEs, which we combine with a new library construction protocol to experimentally validate our approximation approaches. Analysis of our simulated data reveals how sampling trajectories differ between simulations of nucleotide versus amino acid variants and among mutagenesis schemes. For this, we show quantitatively that marginal gains in sampling efficiency demand increasingly greater sampling effort when sampling for nucleotide sequences over their encoded amino acid equivalents. We present a new library construction protocol that efficiently maximizes sequence variation, and demonstrate using ultradeep sequencing that the library encodes virtually all possible combinations of mutations within the experimental design. Insights learned from our analyses together with the methodological advances reported herein are immediately applicable toward pooled experimental screens of arbitrary design, enabling further assay upscaling and expanded testing of genotype space.

The genome sequence of the cinnamon sedge caddisfly, Limnephilus marmoratus (Curtis, 1834).

We present a genome assembly from an individual Limnephilus marmoratus (a caddisfly; Arthropoda; Insecta; Trichoptera; Limnephilidae). The genome sequence is 1,630 megabases in span. Most of the assembly (99.93%) is scaffolded into 30 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length.

The genome sequence of a caddisfly, Limnephilus lunatus (Curtis, 1834).

We present a genome assembly from an individual Limnephilus lunatus (a caddisfly; Arthropoda; Insecta; Trichoptera; Limnephilidae). The genome sequence is 1,270 megabases in span. Most of the assembly is scaffolded into 13 chromosomal pseudomolecules, including the assembled Z chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases long.

A sampling strategy for genome sequencing the British terrestrial arthropod fauna.

The Darwin Tree of Life (DToL) project aims to sequence and assemble high-quality genomes from all eukaryote species in Britain and Ireland, with the first phase of the project concentrating on family-level coverage plus species of particular ecological, biomedical or evolutionary interest. We summarise the processes involved in (1) assessing the UK arthropod fauna and the status of individual species on UK lists; (2) prioritising and collecting species for initial genome sequencing; (3) handling methods to ensure that high-quality genomic DNA is preserved; and (4) compiling standard operating procedures for processing specimens for genome sequencing, identification verification and voucher specimen curation. We briefly explore some lessons learned from the pilot phase of DToL and the impact of the Covid-19 pandemic.

The genome sequence of the black needle fly, Leuctra nigra (Olivier, 1811).

We present a genome assembly from an individual male Leuctra nigra (black needle fly; Arthropoda; Insecta; Plecoptera; Leuctridae). The genome sequence is 536.3 megabases in span. Most of the assembly is scaffolded into 13 chromosomal pseudomolecules , including the X sex chromosome. The mitochondrial genome has also been assembled and is 17.6 kilobases in length.

Glucose-6-phosphate dehydrogenase and 8-iso-prostaglandin F2α as potential predictors of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: Delayed cerebral ischemia (DCI) is a serious complication of aneurysmal subarachnoid hemorrhage (aSAH), which is responsible for significant death and disability. The dynamic balance between the production and elimination of reactive oxygen species (ROS) in patients with DCI is suspected be shifted to favor ROS formation. The authors assessed the relationship between F2-isoprostanes (F2-IsoPs), oxidative stress biomarkers, and glucose-6-phosphate dehydrogenase (G6PD), which are responsible for nicotinamide adenine dinucleotide phosphate (NADPH) production for glutathione system function, with post-aSAH DCI. METHODS: The authors assessed 45 aSAH patients for F2-IsoP and G6PD concentration using commercial ELISA on days 2, 4, and 6 after aSAH. The authors examined the correlation between plasma F2-IsoP and G6PD concentrations and clinical factors with DCI occurrence and aSAH outcome. RESULTS: Expectedly, the most important clinical predictors of DCI were Hunt and Hess grade and modified Fisher (mFisher) grade. Plasma F2-IsoP and G6PD concentrations were greater in aSAH patients than the control group (p < 0.01). F2-IsoP concentrations were greater and G6PD concentrations were lower in patients with DCI than those without (p < 0.01). Plasma F2-IsoP and G6PD concentrations on day 2 were correlated with DCI occurrence (p < 0.01). Plasma F2-IsoP concentrations on days 2 and 6 were correlated with outcome at 1 and 12 months (p < 0.01). CONCLUSIONS: Decreased G6PD indirectly informs the reduced antioxidant response, especially for the glutathione system. G6PD concentration was lower in patients with DCI than those without, which may explain the increased F2-IsoP concentrations. mFisher grade, plasma F2-IsoP concentration, and G6PD concentration on day 2 after aSAH, in combination, may serve as predictors of DCI. Further research is necessary to investigate the therapeutic utility of F2-IsoPs and antioxidants in clinical practice.

Orbito-cranial schwannoma-a multicentre experience.

OBJECTIVES: To describe the features, management approaches, and outcomes of orbito-cranial schwannomas. METHODS: Retrospective review of ten patients with orbito-cranial schwannomas managed in six orbital services over 22 years. Data collected included demographics, presenting features, neuroimaging characteristics, histology, management approach, complications, and outcomes. RESULTS: Mean age of the patients was 41.4 ± 19.9 years, and 6 (60%) were females. The majority presented with proptosis (90%), limited extraocular motility (80%), eyelid swelling (60%), and optic neuropathy (60%). Most lesions (80%) involved the entire anterior-posterior span of the orbit, with both intra- and extraconal involvement. All tumours involved the orbital apex, the superior orbital fissure, and extended at least to the cavernous sinus. Surgical resection was performed for all. Seven (70%) of the tumours were completely or subtotally resected combining an intracapsular approach by an orbital-neurosurgical collaboration, with no recurrence on postoperative follow-up (6-186 months). Three underwent tumour debulking. Of these, two remained stable on follow-up (6-34 months) and one showed progression of the residual tumour over 9 years (cellular schwannoma on histology) necessitating stereotactic radiotherapy (SRT) for local control. Adjuncts to the orbito-cranial resection included perioperative frozen section (n = 5), endoscopic transorbital approach (n = 2), and image-guided navigation (n = 1). Post-surgical adjuvant SRT was used in three subjects. CONCLUSIONS: These results highlight the possibility of successful surgical control in complex orbito-cranial schwannomas. A combined neurosurgical/orbital approach with consideration of an intracapsular resection is recommended. Recurrence may not occur with subtotal excision and observation may be reasonable. Adjunctive SRT for progression or residual tumour can be considered.

Comparative proteomic analysis of ventricular and cisternal cerebrospinal fluid in haemorrhagic stroke patients.

BACKGROUND: Analysis of cerebrospinal fluid (CSF) using mass spectrometry is a relatively novel analytical tool, and comparisons of ventricular and cisternal proteomes are yet to be performed. This may have implications for clinical medicine, particularly in demonstrating continuity of the ventricular system with preserved flow in the presence of ventricular blood. Other uses include the identification of novel biomarkers, including for diagnosis of subarachnoid haemorrhage and of aetiology. The primary objective was therefore to characterise and compare the proteomes of ventricular and CSF after haemorrhagic stroke. METHODS: Paired CSF samples were prospectively collected from the optico-carotid cistern and the frontal horn of the lateral ventricle at the time of craniotomy and clipping in 8 patients with haemorrhagic stroke. Six patients had an aneurysmal subarachnoid haemorrhage (aSAH) from a ruptured saccular aneurysm, one patient had an aSAH after rupture of a mycotic aneurysm and one patient had a spontaneous intracerebral haemorrhage (IPH) with an adjacent unruptured saccular aneurysm. Samples were processed and proteins identified and quantified using data-dependent liquid chromatography tandem mass spectrometry (DDA LC-MSMS). RESULTS: There was no systematic difference between the cisternal and ventricular proteomes. However, blinded principal component analysis (PCA) of the cisternal and ventricular samples separated patients according to pathophysiology. Additionally CSF D-Dimer levels were not detected in the IPH patient but were reliably measured in aSAH patients. CONCLUSIONS: Ventricular CSF is representative of cisternal CSF after aSAH. CSF proteomic PCA analysis can distinguish between haemorrhage types. CSF D-dimer levels may represent a novel diagnostic marker for aSAH. Label free DDA LC-MSMS CSF analysis may inform possible biomarkers.

The genome sequence of the White-legged damselfly, Platycnemis pennipes (Pallas, 1771).

We present a genome assembly from an individual male Platycnemis pennipes (the White-legged damselfly; Arthropoda; Insecta; Odonata; Platycnemididae). The genome sequence is 1793.3 megabases in span. Most of the assembly is scaffolded into 13 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 15.42 kilobases in length.

The genome sequence of the Common Darter, Sympetrum striolatum (Charpentier, 1840).

We present a genome assembly from an individual female Sympetrum striolatum (the Common Darter; Arthropoda; Insecta; Odonata; Libellulidae). The genome sequence is 1349.6 megabases in span. Most of the assembly is scaffolded into 12 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.16 kilobases in length.

The genome sequence of a stonefly, Nemurella pictetii Klapalek, 1900.

We present a genome assembly from an individual male Nemurella pictetii (Arthropoda; Insecta; Plecoptera; Nemouridae). The genome sequence is 257 megabases in span. The majority of the assembly (99.79%) is scaffolded into 12 chromosomal pseudomolecules, with the X sex chromosome assembled. The X chromosome was found at half coverage, but no Y chromosome was found. The mitochondrial genome was assembled, and is 16.0 kb in length.

What you sample is what you get: ecomorphological variation in Trithemis (Odonata, Libellulidae) dragonfly wings reconsidered.

BACKGROUND: The phylogenetic ecology of the Afro-Asian dragonfly genus Trithemis has been investigated previously by Damm et al. (in Mol Phylogenet Evol 54:870-882, 2010) and wing ecomorphology by Outomuro et al. (in J Evol Biol 26:1866-1874, 2013). However, the latter investigation employed a somewhat coarse sampling of forewing and hindwing outlines and reported results that were at odds in some ways with expectations given the mapping of landscape and water-body preference over the Trithemis cladogram produced by Damm et al. (in Mol Phylogenet Evol 54:870-882, 2010). To further explore the link between species-specific wing shape variation and habitat we studied a new sample of 27 Trithemis species employing a more robust statistical test for phylogenetic covariation, more comprehensive representations of Trithemis wing morphology and a wider range of morphometric data-analysis procedures. RESULTS: Contrary to the Outomuro et al. (in J Evol Biol 26:1866-1874, 2013) report, our results indicate that no statistically significant pattern of phylogenetic covariation exists in our Trithemis forewing and hindwing data and that both male and female wing datasets exhibit substantial shape differences between species that inhabit open and forested landscapes and species that hunt over temporary/standing or running water bodies. Among the morphometric analyses performed, landmark data and geometric morphometric data-analysis methods yielded the worst performance in identifying ecomorphometric shape distinctions between Trithemis habitat guilds. Direct analysis of wing images using an embedded convolution (deep learning) neural network delivered the best performance. Bootstrap and jackknife tests of group separations and discriminant-function stability confirm that our results are not artifacts of overtrained discriminant systems or the "curse of dimensionality" despite the modest size of our sample. CONCLUSION: Our results suggest that Trithemis wing morphology reflects the environment's "push" to a much greater extent than phylogeny's "pull". In addition, they indicate that close attention should be paid to the manner in which morphologies are sampled for morphometric analysis and, if no prior information is available to guide sampling strategy, the sample that most comprehensively represents the morphologies of interest should be obtained. In many cases this will be digital images (2D) or scans (3D) of the entire morphology or morphological feature rather than sparse sets of landmark/semilandmark point locations.

The genome sequence of the blue-tailed damselfly, Ischnura elegans (Vander Linden, 1820).

We present a genome assembly from an individual female Ischnura elegans (the blue-tailed damselfly; Arthropoda; Insecta; Odonata; Coenagrionidae). The genome sequence is 1,723 megabases in span. The majority of the assembly (99.55%) is scaffolded into 14 chromosomal pseudomolecules, with the X sex chromosome assembled.

Similar pattern, different paths: tracing the biogeographical history of Megaloptera (Insecta: Neuropterida) using mitochondrial phylogenomics.

The sequential breakup of the supercontinent Pangaea since the Middle Jurassic is one of the crucial factors that has driven the biogeographical patterns of terrestrial biotas. Despite decades of effort searching for concordant patterns between diversification and continental fragmentation among taxonomic groups, increasing evidence has revealed more complex and idiosyncratic scenarios resulting from a mixture of vicariance, dispersal and extinction. Aquatic insects with discreet ecological requirements, low vagility and disjunct distributions represent a valuable model for testing biogeographical hypotheses by reconstructing their distribution patterns and temporal divergences. Insects of the order Megaloptera have exclusively aquatic larvae, their adults have low vagility, and the group has a highly disjunct geographical distribution. Here we present a comprehensive phylogeny of Megaloptera based on a large-scale mitochondrial genome sequencing of 99 species representing >90% of the world genera from all major biogeographical regions. Molecular dating suggests that the deep divergence within Megaloptera pre-dates the breakup of Pangaea. Subsequently, the intergeneric divergences within Corydalinae (dobsonflies), Chauliodinae (fishflies) and Sialidae (alderflies) might have been driven by both vicariance and dispersal correlated with the shifting continent during the Cretaceous, but with strikingly different and incongruent biogeographical signals. The austral distribution of many corydalids appears to be a result of colonization from Eurasia through southward dispersal across Europe and Africa during the Cretaceous, whereas a nearly contemporaneous dispersal via northward rafting of Gondwanan landmasses may account for the colonization of extant Eurasian alderflies from the south.

Life after surgical resection of a low-grade glioma: A prospective cross-sectional study evaluating health-related quality of life.

Health related quality of life (HRQoL) has become an important consideration in LGG patients. We report the largest prospective, longitudinal, cross-sectional cohort study of HRQoL in LGG patients, aiming to identify actionable determinants of HRQoL. Post-operative LGG adults at a large tertiary center underwent HRQoL assessment using the EORTC QLQ-C30 questionnaire administered at follow-up visits and by mail. Scores at 12 month intervals were compared with those from a normative reference population. Spearman's Rho was used to evaluate correlation of subdomain and symptom scores with global HRQoL and change over time. There were 167 participants and 366 questionnaires analysed. Patients reported reduced global HRQoL at nearly every 12 month interval with significant impairments at 12, 72, 108, and 120+ months postoperative. They also reported a significant impairment in each functional subdomain at 12 months, which persisted to varying degrees over 120 months, as did significant fatigue and insomnia. Role, emotional, and social subdomains, as well as fatigue, were significantly associated with global HRQoL at the first 12 month interval. Overall, there was no significant correlation between time from surgery and global HRQoL or the subdomain functional or symptom sections of the QLQ-C30. LGG patients report considerable, sustained impairments in HRQoL after surgery, particularly in cognitive, emotional, and social function, as well as suffering significant fatigue and insomnia. These are strongly associated with global HRQoL and thus can be considered determinants of global HRQoL that with intervention, may improve HRQoL for our LGG patients. This is the largest prospective longitudinal study of HRQoL in postoperative LGG patients yet reported and is ongoing. It identifies several determinants of impaired HRQoL with available management options and interventions that have the potential to significantly improve HRQoL in these patients.

An annotated and illustrated catalogue of the Osmylidae collection (Neuroptera) at the Natural History Museum, London.

The Natural History Museum, London, houses of one of the largest insect collections in the world including several hundred specimens of the small lacewing family Osmylidae. Herein we provide the complete label information, specimen condition, locality and habitus pictures of the Osmylidae primary types of the Natural History Museum, with some historical information about the specimens.

A review of Megalogomphus sumatranus (Krüger, 1899) and its allies in Sundaland with a description of a new species from Borneo (Odonata: Anisoptera: Gomphidae).

Megalogomphus sumatranus (Krüger, 1899) and its allies in Sundaland are reviewed. The accessory genitalia of the males of this genus, hardly considered previously, are found to be taxonomically informative. The taxon from Borneo previously treated as M. sumatranus is described from both sexes as M. buddi sp. nov. (holotype ♂ Sungai Datai, Nanga Bloh, Lanjak Entimau Wildlife Sanctuary, Kapit Division, Sarawak, Malaysia, 22 viii 2013, leg. J. anak Awan M. anak Adau; deposited at the Naturalis Biodiversity Center, Leiden, the Netherlands). Megalogomphus borneensis (Laidlaw, 1914), described as a subspecies of M. icterops (Martin, 1903) and subsequently relegated to the synonymy of that species, is considered to be a distinct species. Megalogomphus icterops is however considered to be a junior synonym of M. sumatranus. A re-description of the holotype of Megalogomphus borneensis is provided as is the first description of the female. Descriptive notes with illustrations of Megalogomphus sumatranus are given.

Species-level image classification with convolutional neural network enables insect identification from habitus images.

Changes in insect biomass, abundance, and diversity are challenging to track at sufficient spatial, temporal, and taxonomic resolution. Camera traps can capture habitus images of ground-dwelling insects. However, currently sampling involves manually detecting and identifying specimens. Here, we test whether a convolutional neural network (CNN) can classify habitus images of ground beetles to species level, and estimate how correct classification relates to body size, number of species inside genera, and species identity.We created an image database of 65,841 museum specimens comprising 361 carabid beetle species from the British Isles and fine-tuned the parameters of a pretrained CNN from a training dataset. By summing up class confidence values within genus, tribe, and subfamily and setting a confidence threshold, we trade-off between classification accuracy, precision, and recall and taxonomic resolution.The CNN classified 51.9% of 19,164 test images correctly to species level and 74.9% to genus level. Average classification recall on species level was 50.7%. Applying a threshold of 0.5 increased the average classification recall to 74.6% at the expense of taxonomic resolution. Higher top value from the output layer and larger sized species were more often classified correctly, as were images of species in genera with few species.Fine-tuning enabled us to classify images with a high mean recall for the whole test dataset to species or higher taxonomic levels, however, with high variability. This indicates that some species are more difficult to identify because of properties such as their body size or the number of related species.Together, species-level image classification of arthropods from museum collections and ecological monitoring can substantially increase the amount of occurrence data that can feasibly be collected. These tools thus provide new opportunities in understanding and predicting ecological responses to environmental change.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

A Novel Automated Mass Digitisation Workflow for Natural History Microscope Slides.

The Natural History Museum, London (NHM) has embarked on an ambitious programme to digitise its collections. One aim of the programme has been to improve the workflows and infrastructure needed to support high-throughput digitisation and create comprehensive digital inventories of large scientific collections. This paper presents the workflow developed to digitise the entire Phthiraptera (parasitic lice) microscope slide collection (70,663 slides). Here we describe a novel process of semi-automated mass digitisation using both temporary and permanent barcode labels applied before and during slide imaging. By using a series of barcodes encoding information associated with each slide (i.e. unique identifier, location in the collection and taxonomic name), we can run a series of automated processes, including file renaming, image processing and bulk import into the NHM's collection management system. We provide data on the comparative efficiency of these processes, illustrating how simple activities, like automated file renaming, reduces image post-processing time, minimises human error and can be applied across multiple collection types.