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 both moths and butterflies. We also reveal that the vast majority of research undertaken of Lepidopteran endosymbionts concerns Wolbachia. While this highly prevalent bacteria 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.Peer reviewe

Quantitative RNA-Seq analysis in non-model species: assessing transcriptome assemblies as a scaffold and the utility of evolutionary divergent genomic reference species

Background How well does RNA-Seq data perform for quantitative whole gene expression analysis in the absence of a genome? This is one unanswered question facing the rapidly growing number of researchers studying non-model species. Using Homo sapiens data and resources, we compared the direct mapping of sequencing reads to predicted genes from the genome with mapping to de novo transcriptomes assembled from RNA-Seq data. Gene coverage and expression analysis was further investigated in the non-model context by using increasingly divergent genomic reference species to group assembled contigs by unique genes. Results Eight transcriptome sets, composed of varying amounts of Illumina and 454 data, were assembled and assessed. Hybrid 454/Illumina assemblies had the highest transcriptome and individual gene coverage. Quantitative whole gene expression levels were highly similar between using a de novo hybrid assembly and the predicted genes as a scaffold, although mapping to the de novo transcriptome assembly provided data on fewer genes. Using non-target species as reference scaffolds does result in some loss of sequence and expression data, and bias and error increase with evolutionary distance. However, within a 100 million year window these effect sizes are relatively small. Conclusions Predicted gene sets from sequenced genomes of related species can provide a powerful method for grouping RNA-Seq reads and annotating contigs. Gene expression results can be produced that are similar to results obtained using gene models derived from a high quality genome, though biased towards conserved genes. Our results demonstrate the power and limitations of conducting RNA-Seq in non-model species.Peer reviewe

Male-killer symbiont screening reveals novel associations in Adalia ladybirds

While male-killing bacteria are known to infect across arthropods, ladybird beetles represent a hotspot for these symbioses. In some host species, there are multiple different symbionts that vary in presence and frequency between populations. To further our understanding of spatial and frequency variation, we tested for the presence of three male-killing bacteria: Wolbachia , Rickettsia and Spiroplasma , in two Adalia ladybird species from a previously unexplored UK population. The two-spot ladybird, A. bipunctata, is known to harbour all three male-killers, and we identified Spiroplasma infection in the Merseyside population for the first time. However, in contrast to previous studies on two-spot ladybirds from continental Europe, evidence from egg-hatch rates indicates the Spiroplasma strain present in the Merseyside population does not cause embryonic male-killing. In the related ten-spot ladybird, A. decempunctata, there is only one previous record of a male-killing symbiont, a Rickettsia , which we did not detect in the Merseyside sample. However, PCR assays indicated the presence of a Spiroplasma in a single A. decempunctata specimen. Marker sequence indicated that this Spiroplasma was divergent from that found in sympatric A. bipunctata. Genome sequencing of the Spiroplasma -infected A. decempunctata additionally revealed the presence of cobionts in the form of a Centistes parasitoid wasp and the parasitic fungi Beauveria. Further study of A. decempunctata from this population is needed to resolve whether it is the ladybird or wasp cobiont that harbours Spiroplasma , and to establish the phenotype of this strain. These data indicate first that microbial symbiont phenotype should not be assumed from past studies conducted in different locations, and second that cobiont presence may confound screening studies aimed to detect the frequency of a symbiont in field collected material from a focal host species

The joint evolutionary histories of Wolbachia and mitochondria in Hypolimnas bolina

Background. The interaction between the Blue Moon butterfly, Hypolimnas bolina, and Wolbachia has attracted interest because of the high prevalence of male-killing achieved within the species, the ecological consequences of this high prevalence, the intensity of selection on the host to suppress the infection, and the presence of multiple Wolbachia infections inducing different phenotypes. We examined diversity in the co-inherited marker, mtDNA, and the partitioning of this between individuals of different infection status, as a means to investigate the population biology and evolutionary history of the Wolbachia infections. Results. Part of the mitochondrial COI gene was sequenced from 298 individuals of known infection status revealing ten different haplotypes. Despite very strong biological evidence that the sample represents a single species, the ten haplotypes did not fall within a monophyletic clade within the Hypolimnas genus, with one haplotype differing by 5% from the other nine. There were strong associations between infection status and mtDNA haplotype. The presence of wBol1 infection in association with strongly divergent haplotypes prompted closer examination of wBol1 genetic variation. This revealed the existence of two cryptic subtypes, wBol1a and wBol1b. The wBol1a infection, by far the most common, was in strict association with the single divergent mtDNA haplotype. The wBol1b infection was found with two haplotypes that were also observed in uninfected specimens. Finally, the wBol2 infection was associated with a large diversity of mtDNA haplotypes, most often shared with uninfected sympatric butterflies. Conclusion. This data overall supports the hypothesis that high prevalence of male-killing Wolbachia (wBol1) in H. bolina is associated with very high transmission efficiency rather than regular horizontal transmission. It also suggests this infection has undergone a recent selective sweep and was introduced in this species through introgression. In contrast, the sharing of haplotypes between wBol2-infected and uninfected individuals indicates that this strain is not perfectly transmitted and/or shows a significant level of horizontal transmission

The evolution of sex ratio distorter suppression affects a 25 cM genomic region in the butterfly Hypolimnas bolina

Open Access ArticleSymbionts that distort their host's sex ratio by favouring the production and survival of females are common in arthropods. Their presence produces intense Fisherian selection to return the sex ratio to parity, typified by the rapid spread of host 'suppressor' loci that restore male survival/development. In this study, we investigated the genomic impact of a selective event of this kind in the butterfly Hypolimnas bolina. Through linkage mapping, we first identified a genomic region that was necessary for males to survive Wolbachia-induced male-killing. We then investigated the genomic impact of the rapid spread of suppression, which converted the Samoan population of this butterfly from a 100:1 female-biased sex ratio in 2001 to a 1:1 sex ratio by 2006. Models of this process revealed the potential for a chromosome-wide effect. To measure the impact of this episode of selection directly, the pattern of genetic variation before and after the spread of suppression was compared. Changes in allele frequencies were observed over a 25 cM region surrounding the suppressor locus, with a reduction in overall diversity observed at loci that co-segregate with the suppressor. These changes exceeded those expected from drift and occurred alongside the generation of linkage disequilibrium. The presence of novel allelic variants in 2006 suggests that the suppressor was likely to have been introduced via immigration rather than through de novo mutation. In addition, further sampling in 2010 indicated that many of the introduced variants were lost or had declined in frequency since 2006. We hypothesize that this loss may have resulted from a period of purifying selection, removing deleterious material that introgressed during the initial sweep. Our observations of the impact of suppression of sex ratio distorting activity reveal a very wide genomic imprint, reflecting its status as one of the strongest selective forces in nature.Natural Environment Research Council (NERC

Mosquitoes reared in distinct insectaries within an institution in close spatial proximity possess significantly divergent microbiomes

The microbiome affects important aspects of mosquito biology and differences in microbial composition can affect the outcomes of laboratory studies. To determine how the biotic and abiotic conditions in an insectary affect the composition of the bacterial microbiome of mosquitoes we reared mosquitoes from a single cohort of eggs from one genetically homogeneous inbred Aedes aegypti colony, which were split into three batches, and transferred to each of three different insectaries located within the Liverpool School of Tropical Medicine. Using three replicate trays per insectary, we assessed and compared the bacterial microbiome composition as mosquitoes developed from these eggs. We also characterised the microbiome of the mosquitoes' food sources, measured environmental conditions over time in each climate-controlled insectary, and recorded development and survival of mosquitoes. While mosquito development was overall similar between all three insectaries, we saw differences in microbiome composition between mosquitoes from each insectary. Furthermore, bacterial input via food sources, potentially followed by selective pressure of temperature stability and range, did affect the microbiome composition. At both adult and larval stages, specific members of the mosquito microbiome were associated with particular insectaries; and the insectary with less stable and cooler conditions resulted in slower pupation rate and higher diversity of the larval microbiome. Tray and cage effects were also seen in all insectaries, with different bacterial taxa implicated between insectaries. These results highlight the necessity of considering the variability and effects of different microbiome composition even in experiments carried out in a laboratory environment starting with eggs from one batch; and highlights the impact of even minor inconsistencies in rearing conditions due to variation of temperature and humidity.</p

Suppression of Wolbachia-mediated male-killing in the butterfly Hypolimnas bolina involves a single genomic region.

BACKGROUND: Sex ratio distorting agents (maternally inherited symbionts and meiotically-driving sex chromosomes) are common in insects. When these agents rise to high frequencies they create strong population sex ratio bias and selection then favours mutations that act to restore the rare sex. Despite this strong selection pressure, the evolution of mutations that suppress sex ratio distorting elements appears to be constrained in many cases, where sex-biased populations persist for many generations. This scenario has been observed in the butterfly Hypolimnas bolina, where Wolbachia-mediated male killing endured for 800-1,000 generations across multiple populations before the evolution of suppression. Here we test the hypothesis that this evolutionary lag is the result of suppression being a multilocus trait requiring multiple mutations. METHODS: We developed genetic markers, based on conservation of synteny, for each H. bolina chromosome and verified coverage using recombinational mapping. We then used a Wolbachia-infected mapping family to assess each chromosome for the presence of loci required for male survival, as determined by the presence of markers in all surviving sons. RESULTS: Informative markers were obtained for each of the 31 chromosomes in H. bolina. The only marker that cosegregated with suppression was located on chromosome 25. A genomic region necessary for suppression has previously been located on this chromosome. We therefore conclude that a single genomic region of the H. bolina genome is necessary for male-killing suppression. DISCUSSION: The evolutionary lag observed in our system is not caused by a need for changes at multiple genomic locations. The findings favour hypotheses in which either multiple mutations are required within a single genomic region, or the suppressor mutation is a singularly rare event

Optix and cortex/ivory/mir-193 again: the repeated use of two mimicry hotspot loci

The extent to which evolution is repeatable has been a debated topic among evolutionary biologists. Although rewinding the tape of life perhaps would not lead to the same outcome every time, repeated evolution of analogous genes for similar functions has been extensively reported. Wing phenotypes of butterflies and moths have provided a wealth of examples of gene re-use, with certain ‘hotspot loci’ controlling wing patterns across diverse taxa. Here, we present an example of convergent evolution in the molecular genetic basis of Batesian wing mimicry in two Hypolimnas butterfly species. We show that mimicry is controlled by variation near cortex/ivory/mir-193, a known butterfly hotspot locus. By dissecting the genetic architecture of mimicry in Hypolimnas misippus and Hypolimnas bolina, we present evidence that distinct non-coding regions control the development of white pattern elements in the forewing and hindwing of the two species, suggesting independent evolution, and that no structural variation is found at the locus. Finally, we also show that orange coloration in H. bolina is associated with optix, a well-known patterning gene. Overall, our study once again implicates variation near the hotspot loci cortex/ivory/mir-193 and optix in butterfly wing mimicry and thereby highlights the repeatability of adaptive evolution

Mosquitoes reared in distinct insectaries within an institution in close spatial proximity possess significantly divergent microbiomes

The microbiome affects important aspects of mosquito biology and differences in microbial composition can affect the outcomes of laboratory studies. To determine how the biotic and abiotic conditions in an insectary affect the composition of the bacterial microbiome of mosquitoes we reared mosquitoes from a single cohort of eggs from one genetically homogeneous inbred colony, which were split into three batches, and transferred to each of three different insectaries located within the Liverpool School of Tropical Medicine. Using three replicate trays per insectary, we assessed and compared the bacterial microbiome composition as mosquitoes developed from these eggs. We also characterised the microbiome of the mosquitoes' food sources, measured environmental conditions over time in each climate-controlled insectary, and recorded development and survival of mosquitoes. While mosquito development was overall similar between all three insectaries, we saw differences in microbiome composition between mosquitoes from each insectary. Furthermore, bacterial input via food sources, potentially followed by selective pressure of temperature stability and range, did affect the microbiome composition. At both adult and larval stages, specific members of the mosquito microbiome were associated with particular insectaries; and the insectary with less stable and cooler conditions resulted in slower pupation rate and higher diversity of the larval microbiome. Tray and cage effects were also seen in all insectaries, with different bacterial taxa implicated between insectaries. These results highlight the necessity of considering the variability and effects of different microbiome composition even in experiments carried out in a laboratory environment starting with eggs from one batch; and highlights the impact of even minor inconsistencies in rearing conditions due to variation of temperature and humidity

Global invasion history of the agricultural pest butterfly revealed with genomics and citizen science.

The small cabbage white butterfly, , is a major agricultural pest of cruciferous crops and has been introduced to every continent except South America and Antarctica as a result of human activities. In an effort to reconstruct the near-global invasion history of , we developed a citizen science project, the "Pieris Project," and successfully amassed thousands of specimens from 32 countries worldwide. We then generated and analyzed nuclear (double-digest restriction site-associated DNA fragment procedure [ddRAD]) and mitochondrial DNA sequence data for these samples to reconstruct and compare different global invasion history scenarios. Our results bolster historical accounts of the global spread and timing of introductions. We provide molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies of (∼1,200 y B.P.) coincides with the diversification of brassicaceous crops and the development of human trade routes such as the Silk Route (Silk Road). The further spread of over the last ∼160 y was facilitated by human movement and trade, resulting in an almost linear series of at least 4 founding events, with each introduced population going through a severe bottleneck and serving as the source for the next introduction. Management efforts of this agricultural pest may need to consider the current existence of multiple genetically distinct populations. Finally, the international success of the Pieris Project demonstrates the power of the public to aid scientists in collections-based research addressing important questions in invasion biology, and in ecology and evolutionary biology more broadly