A DNA barcoding framework for taxonomic verification in the Darwin Tree of Life Project.

Biodiversity genomics research requires reliable organismal identification, which can be difficult based on morphology alone. DNA-based identification using DNA barcoding can provide confirmation of species identity and resolve taxonomic issues but is rarely used in studies generating reference genomes. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We present a standardised framework for DNA barcode sequencing and data interpretation that is then adapted for diverse organismal groups. DNA barcoding data from over 12,000 DToL specimens has identified up to 20% of samples requiring additional verification, with 2% of seed plants and 3.5% of animal specimens subsequently having their names changed. We also make recommendations for future developments using new sequencing approaches and streamlined bioinformatic approaches.

Identifying species based solely on their morphology can be difficult. DNA-based identification using DNA barcoding can aid species identification, but can be challenging to implement in biodiversity projects sampling diverse organismal groups. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We discuss how a standardised approach has been adapted by each partner to suit different organismal groups, show the efficacy of this approach for confirming species identities and resolving taxonomic issues, and make recommendations for future developments.

Temporal variability in host availability alters the outcome of competition between two parasitoid species.

Variability in the availability of resources through time is a common attribute in trophic interactions, but its effects on the fitness of different consumer species and on interspecific competition between them are not clearly understood. To investigate this, we allowed two parasitoid species, Trichopria drosophilae and Pachycrepoideus vindemiae, to exploit Drosophila host pupae under different temporal variability treatments, either on their own or simultaneously. When tested individually (in the absence of interspecific competition), both parasitoid species had lower fitness when hosts were exposed for a short duration at high density than when exposed for a long duration at low density. When both parasitoid species exploited hosts simultaneously, interspecific competition significantly decreased the number of offspring for both parasitoid species. The outcome of this interspecific competition depended on host temporal variability, with T. drosophilae or P. vindemiae dominating in short and long host exposure treatments, respectively. These results can be explained by the combination of host availability and egg load of female adult parasitoids. When abundant hosts are provided for a short period, the ample mature eggs of the proovigenic T. drosophilae enable them to exploit hosts more efficiently than P. vindemiae, which is synovigenic. However, P. vindemiae is an intrinsically superior competitor and dominates when multiparasitism occurs. Multiparasitism is more frequent when hosts are at low levels relative to the egg load of the parasitoids. Our results clearly demonstrate that resource temporal availability can alter the outcome of competition between consumers with different reproductive traits.

The genome sequence of the Mottled Pug, Eupithecia exiguata (Hübner, 1813).

We present a genome assembly from an individual male Eupithecia exiguata (the Mottled Pug; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 372.9 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.39 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,194 protein coding genes.

The genome sequence of the Marbled White Spot, Protodeltote pygarga (Hufnagel, 1766).

We present a genome assembly from an individual male Protodeltote pygarga (the Marbled White Spot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 421.1 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.48 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,784 protein coding genes.

A Model of Gastric Mucosal pH Regulation: Extending Sensitivity Analysis Using Sobol' Indices to Understand Higher Moments.

Several recent theoretical studies have indicated that a relatively simple secretion control mechanism in the epithelial cells lining the stomach may be responsible for maintaining a neutral (healthy) pH adjacent to the stomach wall, even in the face of enormous electrodiffusive acid transport from the interior of the stomach. Subsequent work used Sobol' Indices (SIs) to quantify the degree to which this secretion mechanism is "self-regulating" i.e. the degree to which the wall pH is held neutral as mathematical parameters vary. However, questions remain regarding the nature of the control that specific parameters exert over the maintenance of a healthy stomach wall pH. Studying the sensitivity of higher moments of the statistical distribution of a model output can provide useful information, for example, how one parameter may skew the distribution towards or away from a physiologically advantageous regime. In this work, we prove a relationship between SIs and the higher moments and show how it can potentially reduce the cost of computing sensitivity of said moments. We define γ -indices to quantify sensitivity of variance, skewness, and kurtosis to the choice of value of a parameter, and we propose an efficient strategy that uses both SIs and γ -indices for a more comprehensive sensitivity analysis. Our analysis uncovers a control parameter which governs the "tightness of control" that the secretion mechanism exerts on wall pH. Finally, we discuss how uncertainty in this parameter can be reduced using expert information about higher moments, and speculate about the physiological advantage conferred by this control mechanism.

Research priorities in regional anaesthesia: an international Delphi study.

BACKGROUND: Regional anaesthesia use is growing worldwide, and there is an increasing emphasis on research in regional anaesthesia to improve patient outcomes. However, priorities for future study remain unclear. We therefore conducted an international research prioritisation exercise, setting the agenda for future investigators and funding bodies. METHODS: We invited members of specialist regional anaesthesia societies from six continents to propose research questions that they felt were unanswered. These were consolidated into representative indicative questions, and a literature review was undertaken to determine if any indicative questions were already answered by published work. Unanswered indicative questions entered a three-round modified Delphi process, whereby 29 experts in regional anaesthesia (representing all participating specialist societies) rated each indicative question for inclusion on a final high priority shortlist. If ≥75% of participants rated an indicative question as 'definitely' include in any round, it was accepted. Indicative questions rated as 'definitely' or 'probably' by <50% of participants in any round were excluded. Retained indicative questions were further ranked based on the rating score in the final Delphi round. The final research priorities were ratified by the Delphi expert group. RESULTS: There were 1318 responses from 516 people in the initial survey, from which 71 indicative questions were formed, of which 68 entered the modified Delphi process. Eleven 'highest priority' research questions were short listed, covering themes of pain management; training and assessment; clinical practice and efficacy; technology and equipment. CONCLUSIONS: We prioritised unanswered research questions in regional anaesthesia. These will inform a coordinated global research strategy for regional anaesthesia and direct investigators to address high-priority areas.

Opsin Gene Duplication in Lepidoptera: Retrotransposition, Sex Linkage, and Gene Expression.

Color vision in insects is determined by signaling cascades, central to which are opsin proteins, resulting in sensitivity to light at different wavelengths. In certain insect groups, lineage-specific evolution of opsin genes, in terms of copy number, shifts in expression patterns, and functional amino acid substitutions, has resulted in changes in color vision with subsequent behavioral and niche adaptations. Lepidoptera are a fascinating model to address whether evolutionary change in opsin content and sequence evolution are associated with changes in vision phenotype. Until recently, the lack of high-quality genome data representing broad sampling across the lepidopteran phylogeny has greatly limited our ability to accurately address this question. Here, we annotate opsin genes in 219 lepidopteran genomes representing 33 families, reconstruct their evolutionary history, and analyze shifts in selective pressures and expression between genes and species. We discover 44 duplication events in opsin genes across ∼300 million years of lepidopteran evolution. While many duplication events are species or family specific, we find retention of an ancient long-wavelength-sensitive (LW) opsin duplication derived by retrotransposition within the speciose superfamily Noctuoidea (in the families Nolidae, Erebidae, and Noctuidae). This conserved LW retrogene shows life stage-specific expression suggesting visual sensitivities or other sensory functions specific to the early larval stage. This study provides a comprehensive order-wide view of opsin evolution across Lepidoptera, showcasing high rates of opsin duplications and changes in expression patterns.

Experimental heatwaves facilitate invasion and alter species interactions and composition in a tropical host-parasitoid community.

As mean temperatures increase and heatwaves become more frequent, species are expanding their distributions to colonise new habitats. The resulting novel species interactions will simultaneously shape the temperature-driven reorganization of resident communities. The interactive effects of climate change and climate change-facilitated invasion have rarely been studied in multi-trophic communities, and are likely to differ depending on the nature of the climatic driver (i.e., climate extremes or constant warming). We re-created under laboratory conditions a host-parasitoid community typical of high-elevation rainforest sites in Queensland, Australia, comprising four Drosophila species and two associated parasitoid species. We subjected these communities to an equivalent increase in average temperature in the form of periodic heatwaves or constant warming, in combination with an invasion treatment involving a novel host species from lower-elevation habitats. The two parasitoid species were sensitive to both warming and heatwaves, while the demographic responses of Drosophila species were highly idiosyncratic, reflecting the combined effects of thermal tolerance, parasitism, competition, and facilitation. After multiple generations, our heatwave treatment promoted the establishment of low-elevation species in upland communities. Invasion of the low-elevation species correlated negatively with the abundance of one of the parasitoid species, leading to cascading effects on its hosts and their competitors. Our study, therefore, reveals differing, sometimes contrasting, impacts of extreme temperatures and constant warming on community composition. It also highlights how the scale and direction of climate impacts could be further modified by invading species within a bi-trophic community network.

The genome sequence of the Dark Spectacle, Abrostola triplasia (Linnaeus, 1758).

We present a genome assembly from an individual male Abrostola triplasia (the Dark Spectacle; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 362.7 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.34 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,532 protein coding genes.

The genome sequence of the Broken-barred Carpet, Electrophaes corylata (Thunberg, 1792).

We present a genome assembly from an individual male Electrophaes corylata (the Broken-barred Carpet; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 347.5 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.36 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,031 protein coding genes.

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.

Latitudinal and anthropogenic effects on the structuring of networks linking blood-feeding flies and their vertebrate hosts.

Biting flies (Diptera) transmit pathogens that cause many important diseases in humans as well as domestic and wild animals. The networks of feeding interactions linking these insects to their hosts, and how they vary geographically and in response to human land-use, are currently poorly documented but are relevant to understanding cross-species disease transmission. We compiled a database of biting Diptera-host interactions from the literature to investigate how key interaction network metrics vary latitudinally and with human land-use. Interaction evenness and H2' (a measure of the degree of network specificity) did not vary significantly with latitude. Compared to near-natural habitats, interaction evenness was significantly lower in agricultural habitats, where networks were dominated by relatively few species pairs, but there was no evidence that the presence of humans and their domesticated animals within networks led to systematic shifts in network structure. We discuss the epidemiological relevance of these results and the implications for predicting and mitigating future spill-over events.

Microbiome Structure of a Wild Drosophila Community along Tropical Elevational Gradients and Comparison to Laboratory Lines.

Variation along environmental gradients in host-associated microbial communities is not well understood compared to free-living microbial communities. Because elevational gradients may serve as natural proxies for climate change, understanding patterns along these gradients can inform our understanding of the threats hosts and their symbiotic microbes face in a warming world. In this study, we analyzed bacterial microbiomes from pupae and adults of four Drosophila species native to Australian tropical rainforests. We sampled wild individuals at high and low elevations along two mountain gradients to determine natural diversity patterns. Further, we sampled laboratory-reared individuals from isofemale lines established from the same localities to see if any natural patterns are retained in the lab. In both environments, we controlled for diet to help elucidate other deterministic patterns of microbiome composition. We found small but significant differences in Drosophila bacterial community composition across elevation, with some notable taxonomic differences between different Drosophila species and sites. Further, we found that field-collected fly pupae had significantly richer microbiomes than laboratory-reared pupae. We also found similar microbiome composition in both types of provided diet, suggesting that the significant differences found among Drosophila microbiomes are the products of surrounding environments with different bacterial species pools, possibly bound to elevational differences in temperature. Our results suggest that comparative studies between lab and field specimens help reveal the true variability in microbiome communities that can exist within a single species. IMPORTANCE Bacteria form microbial communities inside most higher-level organisms, but we know little about how the microbiome varies along environmental gradients and between natural host populations and laboratory colonies. To explore such effects on insect-associated microbiomes, we studied the gut microbiome in four Drosophila species over two mountain gradients in tropical Australia. We also compared these data to individuals kept in the laboratory to understand how different settings changed microbiome communities. We found that field-sampled individuals had significantly higher microbiome diversity than those from the lab. In wild Drosophila populations, elevation explains a small but significant amount of the variation in their microbial communities. Our study highlights the importance of environmental bacterial sources for Drosophila microbiome composition across elevational gradients and shows how comparative studies help reveal the true flexibility in microbiome communities that can exist within a species.

Effects of storage conditions and digestion time on DNA amplification of biting midge (Culicoides) blood meals.

BACKGROUND: Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. METHOD: We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a - 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. RESULTS: We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. CONCLUSIONS: These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera.

Diversity, duplication, and genomic organization of homeobox genes in Lepidoptera.

Homeobox genes encode transcription factors with essential roles in patterning and cell fate in developing animal embryos. Many homeobox genes, including Hox and NK genes, are arranged in gene clusters, a feature likely related to transcriptional control. Sparse taxon sampling and fragmentary genome assemblies mean that little is known about the dynamics of homeobox gene evolution across Lepidoptera or about how changes in homeobox gene number and organization relate to diversity in this large order of insects. Here we analyze an extensive data set of high-quality genomes to characterize the number and organization of all homeobox genes in 123 species of Lepidoptera from 23 taxonomic families. We find most Lepidoptera have around 100 homeobox loci, including an unusual Hox gene cluster in which the lab gene is repositioned and the ro gene is next to pb A topologically associating domain spans much of the gene cluster, suggesting deep regulatory conservation of the Hox cluster arrangement in this insect order. Most Lepidoptera have four Shx genes, divergent zen-derived loci, but these loci underwent dramatic duplication in several lineages, with some moths having over 165 homeobox loci in the Hox gene cluster; this expansion is associated with local LINE element density. In contrast, the NK gene cluster content is more stable, although there are differences in organization compared with other insects, as well as major rearrangements within butterflies. Our analysis represents the first description of homeobox gene content across the order Lepidoptera, exemplifying the potential of newly generated genome assemblies for understanding genome and gene family evolution.

The genome sequence of the Lilac Beauty, Apeira syringaria (Linnaeus, 1758).

We present a genome assembly from an individual female Apeira syringaria (the Lilac Beauty; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 544.4 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.5 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,426 protein coding genes.

The genome sequence of the Pebble Hook-tip, Drepana falcataria (Linnaeus, 1758).

We present a genome assembly from an individual male Drepana falcataria (the Pebble Hook-tip; Arthropoda; Insecta; Lepidoptera; Drepanidae). The genome sequence is 326.7 megabases in span. The whole assembly is scaffolded into 31 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 the Small Angle Shades, Euplexia lucipara (Linnaeus, 1758).

We present a genome assembly from an individual male Euplexia lucipara (the Small Angle Shades; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 661.8 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.37 kilobases in length. Gene annotation of this assembly on Ensembl identified 20,395 protein coding genes.

The genome sequence of the Ruby Tiger, Phragmatobia fuliginosa (Linnaeus, 1758).

We present a genome assembly from an individual male Phragmatobia fuliginosa (the Ruby Tiger; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 629.4 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,338 protein coding genes.

The genome sequence of the Orange Footman, Eilema sororcula (Hufnagel, 1766).

We present a genome assembly from an individual male Eilema sororcula (the Orange Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 729.4 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.46 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,093 protein coding genes.