One strain may hide another: Cryptic male-killing Wolbachia.

While heritable symbionts are common in insects, strains that act as male-killers are considered rare. A new study in PLOS Biology identifies a novel male-killer hidden by coinfection and host resistance, highlighting the complexity of host-microbial interactions in natural systems.

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.

Sex determination systems as the interface between male-killing bacteria and their hosts.

Arthropods host a range of sex-ratio-distorting selfish elements, including diverse maternally inherited endosymbionts that solely kill infected males. Male-killing heritable microbes are common, reach high frequency, but until recently have been poorly understood in terms of the host-microbe interaction. Additionally, while male killing should generate strong selection for host resistance, evidence of this has been scant. The interface of the microbe with host sex determination is integral to the understanding of how death is sex limited and how hosts can evolve evasion of male killing. We first review current knowledge of the mechanisms diverse endosymbionts use to induce male-specific death. We then examine recent evidence that these agents do produce intense selection for host nuclear suppressor elements. We argue, from our understanding of male-killing mechanisms, that suppression will commonly involve evolution of the host sex determination pathways and that the host's response to male-killing microbes thus represents an unrecognized driver of the diversity of arthropod sex determination. Further work is required to identify the genes and mechanisms responsible for male-killing suppression, which will both determine the components of sex determination (or other) systems associated with suppressor evolution, and allow insight into the mechanism of male killing itself.

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

The small cabbage white butterfly, Pieris rapae, 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 P. rapae, 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 P. rapae introductions. We provide molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies of P. rapae (∼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 P. rapae 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.

Investigating the genomic basis of discrete phenotypes using a Pool-Seq-only approach: New insights into the genetics underlying colour variation in diverse taxa.

While large-scale genomic approaches are increasingly revealing the genetic basis of polymorphic phenotypes such as colour morphs, such approaches are almost exclusively conducted in species with high-quality genomes and annotations. Here, we use Pool-Seq data for both genome assembly and SNP frequency estimation, followed by scanning for FST outliers to identify divergent genomic regions. Using paired-end, short-read sequencing data from two groups of individuals expressing divergent phenotypes, we generate a de novo rough-draft genome, identify SNPs and calculate genomewide FST differences between phenotypic groups. As genomes generated by Pool-Seq data are highly fragmented, we also present an approach for super-scaffolding contigs using existing protein-coding data sets. Using this approach, we reanalysed genomic data from two recent studies of birds and butterflies investigating colour pattern variation and replicated their core findings, demonstrating the accuracy and power of a Pool-Seq-only approach. Additionally, we discovered new regions of high divergence and new annotations that together suggest novel parallels between birds and butterflies in the origins of their colour pattern variation.

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

Symbionts 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.

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Integrative analyses unveil speciation linked to host plant shift in Spialia butterflies.

Discovering cryptic species in well-studied areas and taxonomic groups can have profound implications in understanding eco-evolutionary processes and in nature conservation because such groups often involve research models and act as flagship taxa for nature management. In this study, we use an array of techniques to study the butterflies in the Spialia sertorius species group (Lepidoptera, Hesperiidae). The integration of genetic, chemical, cytogenetic, morphological, ecological and microbiological data indicates that the sertorius species complex includes at least five species that differentiated during the last three million years. As a result, we propose the restitution of the species status for two taxa often treated as subspecies, Spialia ali (Oberthür, 1881) stat. rest. and Spialia therapne (Rambur, 1832) stat. rest., and describe a new cryptic species Spialia rosae Hernández-Roldán, Dapporto, Dinca, Vicente & Vila sp. nov. Spialia sertorius (Hoffmannsegg, 1804) and S. rosae are sympatric and synmorphic, but show constant differences in mitochondrial DNA, chemical profiles and ecology, suggesting that S. rosae represents a case of ecological speciation involving larval host plant and altitudinal shift, and apparently associated with Wolbachia infection. This study exemplifies how a multidisciplinary approach can reveal elusive cases of hidden diversity.

Interkingdom interactions shape the fungal microbiome of mosquitoes.

BACKGROUND: The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). RESULTS: Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. CONCLUSION: Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.

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.

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.

Microbial interactions in the mosquito gut determine Serratia colonization and blood-feeding propensity.

How microbe-microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host-microbe interactions and provides evidence that microbial interactions influence mosquito behavior.

Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes.

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti, and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria.

Male-killing bacteria trigger a cycle of increasing male fatigue and female promiscuity.

Sex-ratio distorters are found in numerous species and can reach high frequencies within populations. Here, we address the compelling, but poorly tested, hypothesis that the sex ratio bias caused by such elements profoundly alters their host's mating system. We compare aspects of female and male reproductive biology between island populations of the butterfly Hypolimnas bolina that show varying degrees of female bias, because of a male-killing Wolbachia infection. Contrary to expectation, female bias leads to an increase in female mating frequency, up to a point where male mating capacity becomes limiting. We show that increased female mating frequency can be explained as a facultative response to the depleted male mating resources in female biased populations. In other words, this system is one where male-killing bacteria trigger a vicious circle of increasing male fatigue and female promiscuity.

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.

Competing selfish genetic elements in the butterfly Hypolimnas bolina.

Maternally inherited selfish genetic elements are common in animals . Whereas host genetics and ecology are recognized as factors that may limit the incidence of these parasites , theory suggests one further factor-interference with other selfish elements-that could affect their prevalence . In this paper, we show that spatial heterogeneity in the occurrence of the male-killing Wolbachia wBol1 in the tropical butterfly Hypolimnas bolina is caused by a second infection that can exclude the male-killer. We first provide evidence of a second Wolbachia strain, wBol2, present in most populations that do not carry the male-killer but rare or absent when the male-killer is present. Crossing data indicate that wBol2 in males induces cytoplasmic incompatibility to both uninfected and wBol1-infected females. The wBol2 infection can therefore not only spread through uninfected populations but also resist invasion by wBol1. Thus, we provide empirical support for the hypothesis that the incidence of particular selfish genetic elements can limit the presence of competing types.

Evolution of male-killer suppression in a natural population.

Male-killing bacteria are widespread in arthropods, and can profoundly alter the reproductive biology of their host species. Here we detail the first case of complete suppression of a male killer. The nymphalid butterfly Hypolimnas bolina is infected with a strain of the bacterium Wolbachia, wBol1, which kills male host embryos in Polynesian populations, but does not do so in many areas of Southeast Asia, where both males and female adults are naturally infected, and wBol1-infected females produce a 1:1 sex ratio. We demonstrate that absence of male killing by wBol1 is associated with dominant zygotic suppression of the action of the male killer. Simulations demonstrate host suppressors of male-killer action can spread very rapidly, and historical data indicating the presence of male killing in Southeast Asia in the very recent past suggests suppressor spread has been a very recent occurrence. Thus, male killer/host interactions are much more dynamic than previously recognised, with rapid and dramatic loss of the phenotype. Our results also indicate that suppression can render male killers completely quiescent, leading to the conclusion that some species that do not currently express a male killer may have done so in the past, and thus that more species have had their biology affected by these parasites than previously believed.

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.

Unprecedented reorganization of holocentric chromosomes provides insights into the enigma of lepidopteran chromosome evolution.

Chromosome evolution presents an enigma in the mega-diverse Lepidoptera. Most species exhibit constrained chromosome evolution with nearly identical haploid chromosome counts and chromosome-level gene collinearity among species more than 140 million years divergent. However, a few species possess radically inflated chromosomal counts due to extensive fission and fusion events. To address this enigma of constraint in the face of an exceptional ability to change, we investigated an unprecedented reorganization of the standard lepidopteran chromosome structure in the green-veined white butterfly (Pieris napi). We find that gene content in P. napi has been extensively rearranged in large collinear blocks, which until now have been masked by a haploid chromosome number close to the lepidopteran average. We observe that ancient chromosome ends have been maintained and collinear blocks are enriched for functionally related genes suggesting both a mechanism and a possible role for selection in determining the boundaries of these genome-wide rearrangements.

You can't keep a good parasite down: evolution of a male-killer suppressor uncovers cytoplasmic incompatibility.

Maternally inherited parasites are known to impose a wide variety of reproductive manipulations upon their host. These often produce strong selection on the host to suppress the parasite, resulting in a reduction in the frequency of the parasite. However, in the butterfly Hypolimnas bolina, infected with a Wolbachia bacterium, field data demonstrate that suppression of the male-killing phenotype does not depress parasite frequency. Here we test and verify one hypothesis to explain this apparent paradox-Wolbachia induces a second phenotype, Cytoplasmic Incompatibility (CI), in populations where host suppression has evolved. We further demonstrate that the capacity to induce CI has not evolved de novo, but instead is instantaneously expressed upon the survival of infected males. The significance of these results is threefold: (1) multiple phenotypes can provide Wolbachia with the means to maintain itself in a host following suppression of a single manipulative phenotype; (2) the ability to induce CI can remain hidden in systems in which male-killing is observed, just as the ability to induce male-killing may be obscured in strains exhibiting CI; (3) the evolutionary maintenance of CI in a system in which it is not expressed suggests a functional link with male-killing or other traits under selection.