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.

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.

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.

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.

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.

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.

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 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.

Trait-mediated competition drives an ant invasion and alters functional diversity.

The assumption that differences in species' traits reflect their different niches has long influenced how ecologists infer processes from assemblage patterns. For instance, many assess the importance of environmental filtering versus classical limiting-similarity competition in driving biological invasions by examining whether invaders' traits are similar or dissimilar to those of residents, respectively. However, mounting evidence suggests that hierarchical differences between species' trait values can distinguish their competitive abilities (e.g. for the same resource) instead of their niches. Whether such trait-mediated hierarchical competition explains invasions and structures assemblages is less explored. We integrate morphological, dietary, physiological and behavioural trait analyses to test whether environmental filtering, limiting-similarity competition or hierarchical competition explain invasions by fire ants on ant assemblages. We detect both competition mechanisms; invasion success is not only explained by limiting similarity in body size and thermal tolerance (presumably allowing the invader to exploit different niches from residents), but also by the invader's superior position in trait hierarchies reflecting competition for common trophic resources. We find that the two mechanisms generate complex assemblage-level functional diversity patterns-overdispersion in some traits, clustering in others-suggesting their effects are likely missed by analyses restricted to a few traits and composite trait diversity measures.

Recommendations for environmental risk assessment of gene drive applications for malaria vector control.

Building on an exercise that identified potential harms from simulated investigational releases of a population suppression gene drive for malaria vector control, a series of online workshops identified nine recommendations to advance future environmental risk assessment of gene drive applications.

Effects of distance from semi-natural habitat on fall armyworm (Spodoptera frugiperda, J. E. Smith) and its potential natural enemies in Ghana.

Insect crop pests are a major threat to food security in sub-Saharan Africa. Configuration of semi-natural habitat within agricultural landscapes has the potential to enhance biological pest control, helping to maintain yields whilst minimising the negative effects of pesticide use. Fall armyworm (Spodoptera frugiperda, J. E. Smith) is an increasingly important pest of maize in sub-Saharan Africa, with reports of yield loss between 12 and 45%. We investigated the patterns of fall armyworm leaf damage in maize crops in Ghana, and used pitfall traps and dummy caterpillars to assess the spatial distribution of potential fall armyworm predators. Crop damage from fall armyworm at our study sites increased significantly with distance from the field edge, by up to 4% per m. We found evidence that Araneae activity, richness and diversity correspondingly decreased with distance from semi-natural habitat, although Hymenoptera richness and diversity increased. Our preliminary findings suggest that modifying field configuration to increase the proximity of maize to semi-natural habitat may reduce fall armyworm damage and increase natural enemy activity within crops. Further research is required to determine the level of fall armyworm suppression achievable through natural enemies, and how effectively this could safeguard yields.

Altered structure of bat-prey interaction networks in logged tropical forests revealed by metabarcoding.

Habitat degradation is pervasive across the tropics and is particularly acute in Southeast Asia, with major implications for biodiversity. Much research has addressed the impact of degradation on species diversity; however, little is known about how ecological interactions are altered, including those that constitute important ecosystem functions such as consumption of herbivores. To examine how rainforest degradation alters trophic interaction networks, we applied DNA metabarcoding to construct interaction networks linking forest-dwelling insectivorous bat species and their prey, comparing old-growth forest and forest degraded by logging in Sabah, Borneo. Individual bats in logged rainforest consumed a lower richness of prey than those in old-growth forest. As a result, interaction networks in logged forests had a less nested structure. These network structures were associated with reduced network redundancy and thus increased vulnerability to perturbations in logged forests. Our results show how ecological interactions change between old-growth and logged forests, with potentially negative implications for ecosystem function and network stability.

Increased mortality of tropical tree seedlings during the extreme 2015-16 El Niño.

As extreme climate events are predicted to become more frequent because of global climate change, understanding their impacts on natural systems is crucial. Tropical forests are vulnerable to droughts associated with extreme El Niño events. However, little is known about how tropical seedling communities respond to El Niño-related droughts, even though patterns of seedling survival shape future forest structure and diversity. Using long-term data from eight tropical moist forests spanning a rainfall gradient in central Panama, we show that community-wide seedling mortality increased by 11% during the extreme 2015-16 El Niño, with mortality increasing most in drought-sensitive species and in wetter forests. These results indicate that severe El Niño-related droughts influence understory dynamics in tropical forests, with effects varying both within and across sites. Our findings suggest that predicted increases in the frequency of extreme El Niño events will alter tropical plant communities through their effects on early life stages.

Natural enemies have inconsistent impacts on the coexistence of competing species.

The role of natural enemies in promoting coexistence of competing species has generated substantial debate. Modern coexistence theory provides a detailed framework to investigate this topic, but there have been remarkably few empirical applications to the impact of natural enemies. We tested experimentally the capacity for a generalist enemy to promote coexistence of competing insect species, and the extent to which any impact can be predicted by trade-offs between reproductive rate and susceptibility to natural enemies. We used experimental mesocosms to conduct a fully factorial pairwise competition experiment for six rainforest Drosophila species, with and without a generalist pupal parasitoid. We then parameterised models of competition and examined the coexistence of each pair of Drosophila species within the framework of modern coexistence theory. We found idiosyncratic impacts of parasitism on pairwise coexistence, mediated through changes in fitness differences, not niche differences. There was no evidence of an overall reproductive rate-susceptibility trade-off. Pairwise reproductive rate-susceptibility relationships were not useful shortcuts for predicting the impact of parasitism on coexistence. Our results exemplify the value of modern coexistence theory in multi-trophic contexts and the importance of contextualising the impact of generalist natural enemies to determine their impact. In the set of species investigated, competition was affected by the higher trophic level, but the overall impact on coexistence cannot be easily predicted just from knowledge of relative susceptibility. Methodologically, our Bayesian approach highlights issues with the separability of model parameters within modern coexistence theory and shows how using the full posterior parameter distribution improves inferences. This method should be widely applicable for understanding species coexistence in a range of systems.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Experimental warming influences species abundances in a Drosophila host community through direct effects on species performance rather than altered competition and parasitism.

Global warming is expected to have direct effects on species through their sensitivity to temperature, and also via their biotic interactions, with cascading indirect effects on species, communities, and entire ecosystems. To predict the community-level consequences of global climate change we need to understand the relative roles of both the direct and indirect effects of warming. We used a laboratory experiment to investigate how warming affects a tropical community of three species of Drosophila hosts interacting with two species of parasitoids over a single generation. Our experimental design allowed us to distinguish between the direct effects of temperature on host species performance, and indirect effects through altered biotic interactions (competition among hosts and parasitism by parasitoid wasps). Although experimental warming significantly decreased parasitism for all host-parasitoid pairs, the effects of parasitism and competition on host abundances and host frequencies did not vary across temperatures. Instead, effects on host relative abundances were species-specific, with one host species dominating the community at warmer temperatures, irrespective of parasitism and competition treatments. Our results show that temperature shaped a Drosophila host community directly through differences in species' thermal performance, and not via its influences on biotic interactions.