Flight-induced transgenerational maternal effects influence butterfly offspring performance during times of drought.

Maternal condition can generate resource-related maternal effects through differential egg provisioning that can negatively affect offspring performance especially when offspring growth occurs in stressful or sub-optimal environments. Using the Speckled Wood butterfly, Pararge aegeria (L.) we tested the hypothesis that repeated periods of intensive flight during female oviposition affects egg provisioning and reduces offspring performance when larval development occurs under stressful conditions on drought stressed host plants. We investigated whether (after controlling for egg size) maternal age and flight treatment resulted in changes in egg provisioning and whether this contributed to variation in offspring traits across life stages. Age-related changes in maternal condition were found to generate resource-related maternal effects that influenced offspring traits across all life stages. Flight-induced changes in maternal egg provisioning were found to have direct consequences for offspring development in the egg and larval stages. There were significant interactive effects between maternal age and flight on larval development and growth. Compared to offspring from forced flight mothers, offspring from control (no forced flight) mothers that hatched from eggs laid early in the oviposition period (i.e. by younger mothers) had shorter larval development times and heavier pupal masses, suggesting that offspring from mothers in relatively good condition may be able to buffer some of the costs associated with growth on drought stressed host plants. Our multi-factor study demonstrates the importance of considering the various, and often interacting, mechanisms by which maternal effects may influence offspring performance in stressful environments.

Vertically transmitted rhabdoviruses are found across three insect families and have dynamic interactions with their hosts.

A small number of free-living viruses have been found to be obligately vertically transmitted, but it remains uncertain how widespread vertically transmitted viruses are and how quickly they can spread through host populations. Recent metagenomic studies have found several insects to be infected with sigma viruses (Rhabdoviridae). Here, we report that sigma viruses that infect Mediterranean fruit flies (Ceratitis capitata), Drosophila immigrans, and speckled wood butterflies (Pararge aegeria) are all vertically transmitted. We find patterns of vertical transmission that are consistent with those seen in Drosophila sigma viruses, with high rates of maternal transmission, and lower rates of paternal transmission. This mode of transmission allows them to spread rapidly in populations, and using viral sequence data we found the viruses in D. immigrans and C. capitata had both recently swept through host populations. The viruses were common in nature, with mean prevalences of 12% in C. capitata, 38% in D. immigrans and 74% in P. aegeria We conclude that vertically transmitted rhabdoviruses may be widespread in a broad range of insect taxa, and that these viruses can have dynamic interactions with their hosts.

Sub-lethal viral exposure and growth on drought stressed host plants changes resource allocation patterns and life history costs in the Speckled Wood butterfly, Pararge aegeria.

This study investigated the interactive effects of growth on drought stressed host plants and pathogen challenge with the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) on survival and fitness-related traits using the Speckled Wood butterfly, Pararge aegeria (L.). Exposure to AcMNPV significantly reduced survival to pupation. For surviving larvae, sub-lethal infection significantly decreased daily mass acquisition rates and pupal mass. Growth on drought stressed plants increased daily mass acquisition rates resulting in heavier pupae, and increased resource allocation to adult reproduction. The interaction between host plant drought and viral exposure resulted in different resource allocation strategies, and thus different growth trajectories, between larvae. This in turn resulted in significantly different allometric relationships between larval mass (at inoculation) and both development time and investment in flight muscles. For larvae with relatively lighter masses there was a cost of resisting infection when growth occurred on drought stressed host plants, both within the larval stage (i.e. longer larval development times) and in the adult stage (i.e. lower investment in flight muscle mass). This multi-factor study highlights several potential mechanisms by which the complex interplay between low host plant nutritional quality due to drought, and pathogen exposure, may differentially influence the performance of P. aegeria individuals across multiple life stages.

Ancient expansion of the hox cluster in lepidoptera generated four homeobox genes implicated in extra-embryonic tissue formation.

Gene duplications within the conserved Hox cluster are rare in animal evolution, but in Lepidoptera an array of divergent Hox-related genes (Shx genes) has been reported between pb and zen. Here, we use genome sequencing of five lepidopteran species (Polygonia c-album, Pararge aegeria, Callimorpha dominula, Cameraria ohridella, Hepialus sylvina) plus a caddisfly outgroup (Glyphotaelius pellucidus) to trace the evolution of the lepidopteran Shx genes. We demonstrate that Shx genes originated by tandem duplication of zen early in the evolution of large clade Ditrysia; Shx are not found in a caddisfly and a member of the basally diverging Hepialidae (swift moths). Four distinct Shx genes were generated early in ditrysian evolution, and were stably retained in all descendent Lepidoptera except the silkmoth which has additional duplications. Despite extensive sequence divergence, molecular modelling indicates that all four Shx genes have the potential to encode stable homeodomains. The four Shx genes have distinct spatiotemporal expression patterns in early development of the Speckled Wood butterfly (Pararge aegeria), with ShxC demarcating the future sites of extraembryonic tissue formation via strikingly localised maternal RNA in the oocyte. All four genes are also expressed in presumptive serosal cells, prior to the onset of zen expression. Lepidopteran Shx genes represent an unusual example of Hox cluster expansion and integration of novel genes into ancient developmental regulatory networks.

The genome sequence of the Brown Oak Tortrix, Archips crataeganus (Hübner, 1796).

We present a genome assembly from an individual female Archips crataeganus (the Brown Oak Tortrix; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 626.9 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 16.64 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,596 protein coding genes.

Unscrambling butterfly oogenesis.

BACKGROUND: Butterflies are popular model organisms to study physiological mechanisms underlying variability in oogenesis and egg provisioning in response to environmental conditions. Nothing is known, however, about; the developmental mechanisms governing butterfly oogenesis, how polarity in the oocyte is established, or which particular maternal effect genes regulate early embryogenesis. To gain insights into these developmental mechanisms and to identify the conserved and divergent aspects of butterfly oogenesis, we analysed a de novo ovarian transcriptome of the Speckled Wood butterfly Pararge aegeria (L.), and compared the results with known model organisms such as Drosophila melanogaster and Bombyx mori. RESULTS: A total of 17306 contigs were annotated, with 30% possibly novel or highly divergent sequences observed. Pararge aegeria females expressed 74.5% of the genes that are known to be essential for D. melanogaster oogenesis. We discuss the genes involved in all aspects of oogenesis, including vitellogenesis and choriogenesis, plus those implicated in hormonal control of oogenesis and transgenerational hormonal effects in great detail. Compared to other insects, a number of significant differences were observed in; the genes involved in stem cell maintenance and differentiation in the germarium, establishment of oocyte polarity, and in several aspects of maternal regulation of zygotic development. CONCLUSIONS: This study provides valuable resources to investigate a number of divergent aspects of butterfly oogenesis requiring further research. In order to fully unscramble butterfly oogenesis, we also now also have the resources to investigate expression patterns of oogenesis genes under a range of environmental conditions, and to establish their function.

The genome sequence of the Variegated Golden Tortrix, Archips xylosteana (Linnaeus, 1758).

We present a genome assembly from an individual female Archips xylosteana (the Variegated Golden Tortrix; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 650.6 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 16.39 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,861 protein coding genes.

Exploring sub-lethal effects of exposure to a nucleopolyhedrovirus in the speckled wood (Pararge aegeria) butterfly.

This study investigated the sub-lethal effects of larval exposure to baculovirus on host life history and wing morphological traits using a model system, the speckled wood butterfly Pararge aegeria (L.) and the virus Autographa californica nucleopolyhedrovirus. Males and females showed similar responses to the viral infection. Infection significantly reduced larval growth rate, whilst an increase in development time allowed the critical mass for pupation to be attained. There was no direct effect of viral infection on the wing morphological traits examined. There was, however, an indirect effect of resisting infection; larvae that took longer to develop had reduced resource investment in adult flight muscle mass.

The genome sequence of the clay, Mythimna ferrago (Fabricius, 1787).

We present a genome assembly from an individual female Mythimna ferrago (the clay; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 861 megabases in span. The majority of the assembly (99.98%) is scaffolded into 32 chromosomal pseudomolecules, with the W and Z chromosomes assembled. The complete mitochondrial genome was also assembled and is 15.3 kilobases in length. Gene annotation of this assembly on Ensembl has identified 14,075 protein coding genes.

The influence of chalk grasslands on butterfly phenology and ecology.

The influence of large-scale variables such as climate change on phenology has received a great deal of research attention. However, local environmental factors also play a key role in determining the timing of species life cycles. Using the meadow brown butterfly Maniola jurtina as an example, we investigate how a specific habitat type, lowland calcareous grassland, can affect the timing of flight dates. Although protracted flight periods have previously been reported in populations on chalk grassland sites in the south of England, no attempt has yet been made to quantify this at a national level, or to assess links with population genetics and drought tolerance. Using data from 539 sites across the UK, these differences in phenology are quantified, and M. jurtina phenology is found to be strongly associated with both site geology and topography, independent of levels of abundance. Further investigation into aspects of M. jurtina ecology at a subset of sites finds no genetic structuring or drought tolerance associated with these same site conditions.

Maternal effects, flight versus fecundity trade-offs, and offspring immune defence in the speckled wood butterfly, Pararge aegeria.

BACKGROUND: Maternal condition can generate resource-related maternal effects through differential egg provisioning, and can greatly affect offspring performance. In the present study, the speckled wood butterfly Pararge aegeria (L.) was used to investigate whether (after controlling for egg size) maternal age, and increased flight during the oviposition period, resulted in changes in egg provisioning and whether this contributed to variation in offspring performance, i) early in development (egg stage and early post-hatching development), and ii) later in larval development after being exposed to the model viral pathogen system; the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). RESULTS: Age-related changes in maternal egg provisioning were observed to influence egg stage development only. Flight-induced changes in maternal egg provisioning had direct consequences for offspring growth and survival across each life stage from egg to adulthood; offspring from forced flight mothers had lower larval masses and longer development times. Offspring with lower larval masses also had reduced survival after exposure to the viral pathogen. CONCLUSION: The present study demonstrates that a change in maternal provisioning as a result of increased flight during the oviposition period has the potential to exert non-genetic cross-generational fitness effects in P. aegeria. This could have important consequences for population dynamics, particularly in fragmented anthropogenic landscapes.

Organisms on the move: ecology and evolution of dispersal.

The symposium and workshop 'Organisms on the move: ecology and evolution of dispersal', held in Ghent (Belgium), 14-18 September 2009, brought together a wide range of researchers using empirical and modelling approaches to examine the dispersal process. This meeting provided an opportunity to assess how much cross-fertilization there has been between empiricists and theoreticians, to present novel insights on dispersal patterns in plants, animals and micro-organisms and to measure the progress made in examining the causes and consequences of dispersal.

Butterfly flight activity affects reproductive performance and longevity relative to landscape structure.

Due to an overlap in the resources used by the flight muscles with the resources used during egg production, it has been hypothesised that an increased dispersal in fragmented landscapes may result in a physiological trade-off between flight and reproduction. In a common garden experiment, we investigated the effects of increased flight on the reproductive output of female speckled wood butterflies (Pararge aegeria, L.) from closed continuous woodland populations versus open highly fragmented agricultural landscapes in central France. Our flight treatment significantly affected resource allocation to egg size, but had no effect on mean daily fecundity. This treatment effect was similar for females from the two landscapes of origin, and suggests that energetic costs associated with increased flight result in a decrease in resource allocation to egg provisioning. There was a landscape-specific effect of flight on longevity: flight reduced longevity by 21% in woodland females, but had no affect on agricultural females. This result suggests that woodland landscape females further compensate for excessive flight by redirecting resources away from somatic maintenance, resulting in reduced life spans. Our results strongly indicate that increased flight caused by changes in landscape structure may impact on key life history traits such as reproductive success and longevity.

Individual tree traits shape insect and disease damage on oak in a climate-matching tree diversity experiment.

Diversifying planted forests by increasing genetic and species diversity is often promoted as a method to improve forest resilience to climate change and reduce pest and pathogen damage. In this study, we used a young tree diversity experiment replicated at two sites in the UK to study the impacts of tree diversity and tree provenance (geographic origin) on the oak (Quercus robur) insect herbivore community and a specialist biotrophic pathogen, oak powdery mildew. Local UK, French, and Italian provenances were planted in monocultures, provenance mixtures, and species mixes, allowing us to test whether: (a) local and nonlocal provenances differ in their insect herbivore and pathogen communities, and (b) admixing trees leads to associational effects on insect herbivore and pathogen damage. Tree diversity had variable impacts on foliar organisms across sites and years, suggesting that diversity effects can be highly dependent on environmental context. Provenance identity impacted upon both herbivores and powdery mildew, but we did not find consistent support for the local adaptation hypothesis for any group of organisms studied. Independent of provenance, we found tree vigor traits (shoot length, tree height) and tree apparency (the height of focal trees relative to their surroundings) were consistent positive predictors of powdery mildew and insect herbivory. Synthesis. Our results have implications for understanding the complex interplay between tree identity and diversity in determining pest damage, and show that tree traits, partially influenced by tree genotype, can be important drivers of tree pest and pathogen loads.

Viral exposure effects on life-history, flight-related traits, and wing melanisation in the Glanville fritillary butterfly.

Infections represent a constant threat for organisms and can lead to substantial fitness losses. Understanding how individuals, especially from natural populations, respond towards infections is thus of great importance. Little is known about immunity in the Glanville fritillary butterfly (Melitaea cinxia). As the larvae live gregariously in family groups, vertical and horizontal transmission of infections could have tremendous effects on individuals and consequently impact population dynamics in nature. We used the Alphabaculovirus type strain Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and demonstrated that positive concentration-dependent baculovirus exposure leads to prolonged developmental time and decreased survival during larval and pupal development, with no sex specific differences. Viral exposure did not influence relative thorax mass or wing morphometric traits often related to flight ability, yet melanisation of the wings increased with viral exposure, potentially influencing disease resistance or flight capacity via thermal regulation. Further research is needed to explore effects under sub-optimal conditions, determine effects on fitness-related traits, and investigate a potential adaptive response of increased melanisation in the wings due to baculovirus exposure.

The effects of insecticides on butterflies - A review.

Pesticides, in particular insecticides, can be very beneficial but have also been found to have harmful side effects on non-target insects. Butterflies play an important role in ecosystems, are well monitored and are recognised as good indicators of environmental health. The amount of information already known about butterfly ecology and the increased availability of genomes make them a very valuable model for the study of non-target effects of pesticide usage. The effects of pesticides are not simply linear, but complex through their interactions with a large variety of biotic and abiotic factors. Furthermore, these effects manifest themselves at a variety of levels, from the molecular to metapopulation level. Research should therefore aim to dissect these complex effects at a number of levels, but as we discuss in this review, this is seldom if ever done in butterflies. We suggest that in order dissect the complex effects of pesticides on butterflies we need to integrate detailed molecular studies, including characterising sequence variability of relevant target genes, with more classical evolutionary ecology; from direct toxicity tests on individual larvae in the laboratory to field studies that consider the potentiation of pesticides by ecologically relevant environmental biotic and abiotic stressors. Such integration would better inform population-level responses across broad geographical scales and provide more in-depth information about the non-target impacts of pesticides.

The sleep phenotype of Borderline Personality Disorder: A systematic review and meta-analysis.

AIM: To delineate the sleep profile of Borderline Personality Disorder (BPD). METHOD: A meta-analysis to synthesise findings on the objective and subjective sleep characteristics of BPD. RESULTS: We identified 32 studies published between 1980 and December 2015. Meta-analysis indicated significant differences between BPD and healthy control groups across objective sleep continuity (sleep onset latency, total sleep time, sleep efficiency) and architecture (rapid eye movement latency/density, slow wave sleep) measures, and self-reported sleep problems (nightmares, sleep quality). Findings were independent of depression (in clinical and community populations), and concomitant psychotropic medication use. There were few significant differences between BPD and clinical (majority depressed) control groups. CONCLUSION: BPD is associated with comparable sleep disturbances to those observed in depression. These disturbances are not solely attributable to comorbid depression. Given growing evidence that sleep disturbance may exacerbate emotional dysregulation and suicide risk, treatments for BPD should explicitly address sleep problems. Future studies should utilise prospective designs to ascertain whether (and in which circumstances) sleep problems predate or follow the onset of the disorder.

Multiple host-plant use may arise from gender-specific fitness effects.

Ovipositing females are predicted to select host-plants that will maximise offspring survival and fitness. Yet hosts often differ in the component of larval fitness affected so host-selection often involves a trade-off between short development times and large size and high fecundity of offspring. If host-species can directly affect development rates and body size, and if there are gender differences in resource allocation during development, there can be different sex-specific selection pressures associated with different hosts. Using a Madeiran population of the speckled wood butterfly Pararge aegeria (L.) as the model species gender differences in larval development and size were examined in response to the hosts Brachypodium sylvaticum, Holcus lanatus and Poa annua. It was observed that male and female P. aegeria larvae differed, with their responses dependent on the host species. These results would suggest that oviposition behavior is a complex process, and use of multiple hosts may have evolved to balance the conflicting needs of male and female larvae. Co-evolution of host selection and oviposition behaviors may help to balance the differing performance needs of offspring.

Studying Oogenesis in a Non-model Organism Using Transcriptomics: Assembling, Annotating, and Analyzing Your Data.

This chapter provides a guide to processing and analyzing RNA-Seq data in a non-model organism. This approach was implemented for studying oogenesis in the Speckled Wood Butterfly Pararge aegeria. We focus in particular on how to perform a more informative primary annotation of your non-model organism by implementing our multi-BLAST annotation strategy. We also provide a general guide to other essential steps in the next-generation sequencing analysis workflow. Before undertaking these methods, we recommend you familiarize yourself with command line usage and fundamental concepts of database handling. Most of the operations in the primary annotation pipeline can be performed in Galaxy (or equivalent standalone versions of the tools) and through the use of common database operations (e.g. to remove duplicates) but other equivalent programs and/or custom scripts can be implemented for further automation.

Divergent RNA Localisation Patterns of Maternal Genes Regulating Embryonic Patterning in the Butterfly Pararge aegeria.

The maternal effect genes responsible for patterning the embryo along the antero-posterior (AP) axis are broadly conserved in insects. The precise function of these maternal effect genes is the result of the localisation of their mRNA in the oocyte. The main developmental mechanisms involved have been elucidated in Drosophila melanogaster, but recent studies have shown that other insect orders often diverge in RNA localisation patterns. A recent study has shown that in the butterfly Pararge aegeria the distinction between blastodermal embryonic (i.e. germ band) and extra-embryonic tissue (i.e. serosa) is already specified in the oocyte during oogenesis in the ovariole, long before blastoderm cellularisation. To examine the extent by which a female butterfly specifies and patterns the AP axis within the region fated to be the germ band, and whether she specifies a germ plasm, we performed in situ hybridisation experiments on oocytes in P. aegeria ovarioles and on early embryos. RNA localisation of the following key maternal effect genes were investigated: caudal (cad), orthodenticle (otd), hunchback (hb) and four nanos (nos) paralogs, as well as TDRD7 a gene containing a key functional domain (OST-HTH/LOTUS) shared with oskar. TDRD7 was mainly confined to the follicle cells, whilst hb was exclusively zygotically transcribed. RNA of some of the nos paralogs, otd and cad revealed complex localisation patterns within the cortical region prefiguring the germ band (i.e. germ cortex). Rather interestingly, otd was localised within and outside the anterior of the germ cortex. Transcripts of nos-O formed a distinct granular ring in the middle of the germ cortex possibly prefiguring the region where germline stem cells form. These butterfly RNA localisation patterns are highly divergent with respect to other insects, highlighting the diverse ways in which different insect orders maternally regulate early embryogenesis of their offspring.