A Worked Example of Screening Genomic Material for the Presence of Wolbachia Infection.

This chapter gives a brief overview of how to screen existing host genomic data for the presence of endosymbionts, such as Wolbachia. The various programs used provide test examples, and the corresponding manuals and discussion boards provide invaluable information. Please do consult these resources.

Morphological changes in female reproductive organs in the African monarch butterfly, host to a male-killing Spiroplasma.

Background: Sexual selection and conflicts within and between sexes promote morphological diversity of reproductive traits within species. Variation in the morphology of diagnostic reproductive characters within species offer an excellent opportunity to study these evolutionary processes as drivers of species diversification. The African monarch, Danaus chrysippus (Linnaeus, 1758), is widespread across Africa. The species is polytypic, with the respective geographical ranges of the four colour morphs only overlapping in East Africa. Furthermore, some of the populations host an endosymbiotic bacterium, Spiroplasma, which induces son-killing and distorts the local host population sex-ratio, creating sexual conflicts between the females seeking to optimize their fecundity and the limited mating capacity of the rare males. Methods: We dissected females from Kenya, Rwanda and South Africa, where Spiroplasma vary in presence and prevalence (high, variable and absent, respectively), and conducted microscopy imaging of their reproductive organs. We then characterized the effect of population, female body size, and female mating status, on the size and shape of different genitalia characters of the D. chrysippus female butterflies. Results: We showed that although the general morphology of the organs is conserved in D. chrysippus, female genitalia vary in size and shape between and within populations. The virgin females have smaller organs, while the same organs were expanded in mated females. Females from highly female-biased populations, where the male-killing Spiroplasma is prevalent, also have a larger area of their corpus bursae covered with signa structures. However, this pattern occurs because a larger proportion of the females remains virgin in the female-biased populations rather than because of male depletion due to the symbiont, as males from sex-ratio distorted populations did not produce significantly smaller nutritious spermatophores.

One's trash is someone else's treasure: sequence read archives from Lepidoptera genomes provide material for genome reconstruction of their endosymbionts.

BACKGROUND: Maternally inherited bacterial symbionts are extremely widespread in insects. They owe their success to their ability to promote their own transmission through various manipulations of their hosts' life-histories. Many symbionts however very often go undetected. Consequently, we have only a restricted idea of the true symbiont diversity in insects, which may hinder our understanding of even bigger questions in the field such as the evolution or establishment of symbiosis. RESULTS: In this study, we screened publicly available Lepidoptera genomic material for two of the most common insect endosymbionts, namely Wolbachia and Spiroplasma, in 1904 entries, encompassing 106 distinct species. We compared the performance of two screening software, Kraken2 and MetaPhlAn2, to identify the bacterial infections and using a baiting approach we reconstruct endosymbiont genome assemblies. Of the 106 species screened, 20 (19%) and nine (8.5%) were found to be infected with either Wolbachia or Spiroplasma, respectively. Construction of partial symbiotic genomes and phylogenetic analyses suggested the Wolbachia strains from the supergroup B were the most prevalent type of symbionts, while Spiroplasma infections were scarce in the Lepidoptera species screened here. CONCLUSIONS: Our results indicate that many of the host-symbiont associations remain largely unexplored, with the majority of associations we identify never being recorded before. This highlights the usefulness of public databases to explore the hidden diversity of symbiotic entities, allowing the development of hypotheses regarding host-symbiont associations. The ever-expanding genomic databases provide a diverse databank from which one can characterize and explore the true diversity of symbiotic entities.

Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map.

BACKGROUND: The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. RESULTS: The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92-94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. CONCLUSIONS: The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.

Exploring bycatch diversity of organisms in whole genome sequencing of Erebidae moths (Lepidoptera).

Models estimate that up to 80% of all butterfly and moth species host vertically transmitted endosymbiotic microorganisms, which can affect the host fitness, metabolism, reproduction, population dynamics, and genetic diversity, among others. The supporting empirical data are however currently highly biased towards the generally more colourful butterflies, and include less information about moths. Additionally, studies of symbiotic partners of Lepidoptera predominantly focus on the common bacterium Wolbachia pipientis, while infections by other inherited microbial partners have more rarely been investigated. Here, we mine the whole genome sequence data of 47 species of Erebidae moths, with the aims to both inform on the diversity of symbionts potentially associated with this Lepidoptera group, and discuss the potential of metagenomic approaches to inform on host associated microbiome diversity. Based on the result of Kraken2 and MetaPhlAn2 analyses, we found clear evidence of the presence of Wolbachia in four species. Our result also suggests the presence of three other bacterial symbionts (Burkholderia spp., Sodalis spp. and Arsenophonus spp.) in three other moth species. Additionally, we recovered genomic material from bracovirus in about half of our samples. The detection of the latter, usually found in mutualistic association to braconid parasitoid wasps, may inform on host-parasite interactions that take place in the natural habitat of the Erebidae moths, suggesting either contamination with material from species of the host community network, or horizontal transfer of members of the microbiome between interacting species.

Wolbachia-driven selective sweep in a range expanding insect species.

BACKGROUND: Evolutionary processes can cause strong spatial genetic signatures, such as local loss of genetic diversity, or conflicting histories from mitochondrial versus nuclear markers. Investigating these genetic patterns is important, as they may reveal obscured processes and players. The maternally inherited bacterium Wolbachia is among the most widespread symbionts in insects. Wolbachia typically spreads within host species by conferring direct fitness benefits, and/or by manipulating its host reproduction to favour infected over uninfected females. Under sufficient selective advantage, the mitochondrial haplotype associated with the favoured maternally-inherited symbiotic strains will spread (i.e. hitchhike), resulting in low mitochondrial genetic variation across the host species range. METHOD: The common bluetail damselfly (Ischnura elegans: van der Linden, 1820) has recently emerged as a model organism for genetics and genomic signatures of range expansion during climate change. Although there is accumulating data on the consequences of such expansion on the genetics of I. elegans, no study has screened for Wolbachia in the damselfly genus Ischnura. Here, we present the biogeographic variation in Wolbachia prevalence and penetrance across Europe and Japan (including samples from 17 populations), and from close relatives in the Mediterranean area (i.e. I. genei: Rambur, 1842; and I. saharensis: Aguesse, 1958). RESULTS: Our data reveal (a) multiple Wolbachia-strains, (b) potential transfer of the symbiont through hybridization, (c) higher infection rates at higher latitudes, and (d) reduced mitochondrial diversity in the north-west populations, indicative of hitchhiking associated with the selective sweep of the most common strain. We found low mitochondrial haplotype diversity in the Wolbachia-infected north-western European populations (Sweden, Scotland, the Netherlands, Belgium, France and Italy) of I. elegans, and, conversely, higher mitochondrial diversity in populations with low penetrance of Wolbachia (Ukraine, Greece, Montenegro and Cyprus). The timing of the selective sweep associated with infected lineages was estimated between 20,000 and 44,000 years before present, which is consistent with the end of the last glacial period about 20,000 years. CONCLUSIONS: Our findings provide an example of how endosymbiont infections can shape spatial variation in their host evolutionary genetics during postglacial expansion. These results also challenge population genetic studies that do not consider the prevalence of symbionts in many insects, which we show can impact geographic patterns of mitochondrial genetic diversity.

Long-term spatiotemporal genetic structure of an accidental parasitoid introduction, and local changes in prevalence of its associated Wolbachia symbiont.

Population bottlenecks associated with founder events strongly impact the establishment and genetic makeup of populations. In addition to their genotype, founding individuals also bring along parasites, as well as symbionts that can manipulate the phenotype of their host, affecting the host population establishment, dynamics and evolution. Thus, to understand introduction, invasion, and spread, we should identify the roles played by accompanying symbionts. In 1991, the parasitoid wasp, Hyposoter horticola, and its associated hyperparasitoid were accidentally introduced from the main Åland islands, Finland, to an isolated island in the archipelago, along with their host, the Glanville fritillary butterfly. Though the receiving island was unoccupied, the butterfly was present on some of the small islands in the vicinity. The three introduced species have persisted locally ever since. A strain of the endosymbiotic bacterium Wolbachia has an intermediate prevalence in the parasitoid H. horticola across the main Åland population. The infection increases its susceptibility of to hyperparasitism. We investigated the establishment and spread of the parasitoid, along with patterns of prevalence of its symbiont using 323 specimens collected between 1992 and 2013, from five localities across Åland, including the source and introduced populations. Using 14 microsatellites and one mitochondrial marker, we suggest that the relatively diverse founding population and occasional migration between islands might have facilitated the persistence of all isolated populations, despite multiple local population crashes. We also show that where the hyperparasitoid is absent, and thus selection against infected wasp genotypes is relaxed, there is near-fixation of Wolbachia.

Promoting inclusive metrics of success and impact to dismantle a discriminatory reward system in science.

Success and impact metrics in science are based on a system that perpetuates sexist and racist "rewards" by prioritizing citations and impact factors. These metrics are flawed and biased against already marginalized groups and fail to accurately capture the breadth of individuals' meaningful scientific impacts. We advocate shifting this outdated value system to advance science through principles of justice, equity, diversity, and inclusion. We outline pathways for a paradigm shift in scientific values based on multidimensional mentorship and promoting mentee well-being. These actions will require collective efforts supported by academic leaders and administrators to drive essential systemic change.

Wolbachia impairs post-eclosion host preference in a parasitoid wasp.

Host preference behavior can result in adaptive advantages with important consequences for the fitness of individuals. Hopkin's host-selection principle (HHSP) suggests that organisms at higher trophic levels demonstrate a preference for the host species on which they developed during their own larval stage. Although investigated in many herbivorous and predatory insects, the HHSP has, to our knowledge, never been tested in the context of insects hosting selfish endosymbiotic passengers. Here, we investigated the effect of infection with the facultative bacterial symbiont Wolbachia on post-eclosion host preference in the parasitoid wasp Trichogramma brassicae (Hymenoptera: Trichogrammatidae). We compared host preference in Wolbachia-infected individuals and uninfected adult female parasitoids after rearing them on two different Lepidopteran hosts, namely the flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) or the grain moth Sitotroga cerealella (Lepidoptera: Gelechiidae) in choice and no choice experimental design (n = 120 wasps per each choice/no choice experiments). We showed that in T. brassicae, Wolbachia affects the post-eclosion host preference of female wasps. Wolbachia-infected wasps did not show any host preference and more frequently switched hosts in the laboratory, while uninfected wasps significantly preferred to lay eggs on the host species they developed on. Additionally, Wolbachia significantly improved the emergence rate of infected wasps when reared on new hosts. Altogether, our results revealed that the wasp's infection with Wolbachia may lead to impairment of post-eclosion host preference and facilitates growing up on different host species. The impairment of host preference by Wolbachia may allow T. brassicae to shift between hosts, a behavior that might have important evolutionary consequences for the wasp and its symbiont.

Towards unravelling Wolbachia global exchange: a contribution from the Bicyclus and Mylothris butterflies in the Afrotropics.

BACKGROUND: Phylogenetically closely related strains of maternally inherited endosymbiotic bacteria are often found in phylogenetically divergent, and geographically distant insect host species. The interspecies transfer of the symbiont Wolbachia has been thought to have occurred repeatedly, facilitating its observed global pandemic. Few ecological interactions have been proposed as potential routes for the horizontal transfer of Wolbachia within natural insect communities. These routes are however likely to act only at the local scale, but how they may support the global distribution of some Wolbachia strains remains unclear. RESULTS: Here, we characterize the Wolbachia diversity in butterflies from the tropical forest regions of central Africa to discuss transfer at both local and global scales. We show that numerous species from both the Mylothris (family Pieridae) and Bicyclus (family Nymphalidae) butterfly genera are infected with similar Wolbachia strains, despite only minor interclade contacts across the life cycles of the species within their partially overlapping ecological niches. The phylogenetic distance and differences in resource use between these genera rule out the role of ancestry, hybridization, and shared host-plants in the interspecies transfer of the symbiont. Furthermore, we could not identify any shared ecological factors to explain the presence of the strains in other arthropod species from other habitats, or even ecoregions. CONCLUSION: Only the systematic surveys of the Wolbachia strains from entire species communities may offer the material currently lacking for understanding how Wolbachia may transfer between highly different and unrelated hosts, as well as across environmental scales.

The gut bacterial community affects immunity but not metabolism in a specialist herbivorous butterfly.

Plant tissues often lack essential nutritive elements and may contain a range of secondary toxic compounds. As nutritional imbalance in food intake may affect the performances of herbivores, the latter have evolved a variety of physiological mechanisms to cope with the challenges of digesting their plant-based diet. Some of these strategies involve living in association with symbiotic microbes that promote the digestion and detoxification of plant compounds or supply their host with essential nutrients missing from the plant diet. In Lepidoptera, a growing body of evidence has, however, recently challenged the idea that herbivores are nutritionally dependent on their gut microbial community. It is suggested that many of the herbivorous Lepidopteran species may not host a resident microbial community, but rather a transient one, acquired from their environment and diet. Studies directly testing these hypotheses are however scarce and come from an even more limited number of species.By coupling comparative metabarcoding, immune gene expression, and metabolomics analyses with experimental manipulation of the gut microbial community of prediapause larvae of the Glanville fritillary butterfly (Melitaea cinxia, L.), we tested whether the gut microbial community supports early larval growth and survival, or modulates metabolism or immunity during early stages of development.We successfully altered this microbiota through antibiotic treatments and consecutively restored it through fecal transplants from conspecifics. Our study suggests that although the microbiota is involved in the up-regulation of an antimicrobial peptide, it did not affect the life history traits or the metabolism of early instars larvae.This study confirms the poor impact of the microbiota on diverse life history traits of yet another Lepidoptera species. However, it also suggests that potential eco-evolutionary host-symbiont strategies that take place in the gut of herbivorous butterfly hosts might have been disregarded, particularly how the microbiota may affect the host immune system homeostasis.

Uncovering the hidden players in Lepidoptera biology: the heritable microbial endosymbionts.

The Lepidoptera is one of the most widespread and recognisable insect orders. Due to their remarkable diversity, economic and ecological importance, moths and butterflies have been studied extensively over the last 200 years. More recently, the relationship between Lepidoptera and their heritable microbial endosymbionts has received increasing attention. Heritable endosymbionts reside within the host's body and are often, but not exclusively, inherited through the female line. Advancements in molecular genetics have revealed that host-associated microbes are both extremely prevalent among arthropods and highly diverse. Furthermore, heritable endosymbionts have been repeatedly demonstrated to play an integral role in many aspects of host biology, particularly host reproduction. Here, we review the major findings of research of heritable microbial endosymbionts of butterflies and moths. We promote the Lepidoptera as important models in the study of reproductive manipulations employed by heritable endosymbionts, with the mechanisms underlying male-killing and feminisation currently being elucidated in moths and butterflies. We also reveal that the vast majority of research undertaken of Lepidopteran endosymbionts concerns Wolbachia. While this highly prevalent bacterium is undoubtedly important, studies should move towards investigating the presence of other, and interacting endosymbionts, and we discuss the merits of examining the microbiome of Lepidoptera to this end. We finally consider the importance of understanding the influence of endosymbionts under global environmental change and when planning conservation management of endangered Lepidoptera species.

Silk properties and overwinter survival in gregarious butterfly larvae.

All organisms are challenged by encounters with parasites, which strongly select for efficient escape strategies in the host. The threat is especially high for gregarious species entering immobile periods, such as diapause. Larvae of the Glanville fritillary butterfly, Melitaea cinxia, spend the winter in diapause in groups of conspecifics each sheltered in a silk nest. Despite intensive monitoring of the population, we have little understanding of the ecological factors influencing larval survival over the winter in the field. We tested whether qualitative and quantitative properties of the silk nest contribute to larval survival over diapause. We used comparative proteomics, metabarcoding analyses, microscopic imaging, and in vitro experiments to compare protein composition of the silk, community composition of the silk-associated microbiota, and silk density from both wild-collected and laboratory-reared families, which survived or died in the field. Although most traits assessed varied across families, only silk density was correlated with overwinter survival in the field. The silk nest spun by gregarious larvae before the winter acts as an efficient breathable physical shield that positively affects larval survival during diapause. Such benefit may explain how this costly trait is conserved across populations of this butterfly species and potentially across other silk-spinning insect species.

Impact of male condition on his spermatophore and consequences for female reproductive performance in the Glanville fritillary butterfly.

In butterflies, male reproductive success is highly related to the quality and the size of the spermatophore transferred to the female. The spermatophore is a capsule produced by the male during copulation, which in many species contains sperm in addition to a nuptial gift, and which is digested by the female after copulation. The nuptial gift may contribute to egg production and offspring quality, and in some cases also to female body maintenance. The production of the spermatophore, however, represents a cost for the male and, in polyandrous species, ejaculates are sometimes allocated adaptively across matings. Nonetheless, although the ecological factors affecting the reproductive success of female butterflies have been the topic of numerous studies, little information exists on the factors affecting males' contribution to reproduction, and the indirect impacts on female fecundity and fitness. We used the Glanville fritillary butterfly, Melitaea cinxia (Linnaeus, 1758) (Nymphalidae), in order to assess variation in male allocation to matings. In this species, smaller males produce smaller spermatophores, but variation in spermatophore size is not correlated with female reproductive success. We show that spermatophore size increases with male age at first mating, decreases with mating frequency and adult food-deprivation, and is not influenced by developmental food-limitation. The length of copulation period does not influence the spermatophore size nor influences the polyandrous mating behavior in this species. Male contribution to his spermatophore size is clearly influenced by his condition and adult-resource at the time of mating. Despite this variation, spermatophore size does not seem to have a direct impact on female reproductive output or mating behavior.

Wolbachia in the Genus Bicyclus: a Forgotten Player.

Bicyclus butterflies are key species for studies of wing pattern development, phenotypic plasticity, speciation and the genetics of Lepidoptera. One of the key endosymbionts in butterflies, the alpha-Proteobacterium Wolbachia pipientis, is affecting many of these biological processes; however, Bicyclus butterflies have not been investigated systematically as hosts to Wolbachia. In this study, we screen for Wolbachia infection in several Bicyclus species from natural populations across Africa as well as two laboratory populations. Out of the 24 species tested, 19 were found to be infected, and no double infection was found, but both A- and B-supergroup strains colonise this butterfly group. We also show that many of the Wolbachia strains identified in Bicyclus butterflies belong to the ST19 clonal complex. We discuss the importance of our results in regard to routinely screening for Wolbachia when using Bicyclus butterflies as the study organism of research in eco-evolutionary biology.

Genetic effects on life-history traits in the Glanville fritillary butterfly.

BACKGROUND: Adaptation to local habitat conditions may lead to the natural divergence of populations in life-history traits such as body size, time of reproduction, mate signaling or dispersal capacity. Given enough time and strong enough selection pressures, populations may experience local genetic differentiation. The genetic basis of many life-history traits, and their evolution according to different environmental conditions remain however poorly understood. METHODS: We conducted an association study on the Glanville fritillary butterfly, using material from five populations along a latitudinal gradient within the Baltic Sea region, which show different degrees of habitat fragmentation. We investigated variation in 10 principal components, cofounding in total 21 life-history traits, according to two environmental types, and 33 genetic SNP markers from 15 candidate genes. RESULTS: We found that nine SNPs from five genes showed strong trend for trait associations (p-values under 0.001 before correction). These associations, yet non-significant after multiple test corrections, with a total number of 1,086 tests, were consistent across the study populations. Additionally, these nine genes also showed an allele frequency difference between the populations from the northern fragmented versus the southern continuous landscape. DISCUSSION: Our study provides further support for previously described trait associations within the Glanville fritillary butterfly species across different spatial scales. Although our results alone are inconclusive, they are concordant with previous studies that identified these associations to be related to climatic changes or habitat fragmentation within the Åland population.

Wolbachia increases the susceptibility of a parasitoid wasp to hyperparasitism.

The success of maternally transmitted endosymbiotic bacteria, such as Wolbachia, is directly linked to their host reproduction but in direct conflict with other parasites that kill the host before it reaches reproductive maturity. Therefore, symbionts that have evolved strategies to increase their host's ability to evade lethal parasites may have high penetrance, while detrimental symbionts would be selected against, leading to lower penetrance or extinction from the host population. In a natural population of the parasitoid wasp Hyposoter horticola in the Åland Islands (Finland), the Wolbachia strain wHho persists at an intermediate prevalence (∼50%). Additionally, there is a negative correlation between the prevalence of Wolbachia and a hyperparasitoid wasp, Mesochorus cf. stigmaticus, in the landscape. Using a manipulative field experiment, we addressed the persistence of Wolbachia at this intermediate level, and tested whether the observed negative correlation could be due to Wolbachia inducing either susceptibility or resistance to parasitism. We show that infection with Wolbachia does not influence the ability of the wasp to parasitize its butterfly host, Melitaea cinxia, but that hyperparasitism of the wasp increases in the presence of wHho. Consequently, the symbiont is detrimental, and in order to persist in the host population, must also have a positive effect on fitness that outweighs the costly burden of susceptibility to widespread parasitism.

Wolbachia Infection in a Natural Parasitoid Wasp Population.

The maternally transmitted bacterium Wolbachia pipientis is well known for spreading and persisting in insect populations through manipulation of the fitness of its host. Here, we identify three new Wolbachia pipientis strains, wHho, wHho2 and wHho3, infecting Hyposoter horticola, a specialist wasp parasitoid of the Glanville fritillary butterfly. The wHho strain (ST435) infects about 50% of the individuals in the Åland islands in Finland, with a different infection rate in the two mitochondrial (COI) haplotypes of the wasp. The vertical transmission rate of Wolbachia is imperfect, and lower in the haplotype with lower infection rate, suggesting a fitness trade-off. We found no association of the wHho infection with fecundity, longevity or dispersal ability of the parasitoid host. However, preliminary results convey spatial associations between Wolbachia infection, host mitochondrial haplotype and parasitism of H. horticola by its hyperparasitoid, Mesochorus cf. stigmaticus. We discuss the possibility that Wolbachia infection protects H. horticola against hyperparasitism.

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera.

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.