Bret M. Boyd

Two Bacterial Genera, Sodalis and Rickettsia, Associated with the Seal Louse Proechinophthirus fluctus (Phthiraptera: Anoplura)

ABSTRACT Roughly 10% to 15% of insect species host heritable symbiotic bacteria known as endosymbionts. The lice parasitizing mammals rely on endosymbionts to provide essential vitamins absent in their blood meals. Here, we describe two bacterial associates from a louse, Proechinophthirus fluctus, which is an obligate ectoparasite of a marine mammal. One of these is a heritable endosymbiont that is not closely related to endosymbionts of other mammalian lice. Rather, it is more closely related to endosymbionts of the genus Sodalis associated with spittlebugs and feather-chewing bird lice. Localization and vertical transmission of this endosymbiont are also more similar to those of bird lice than to those of other mammalian lice. The endosymbiont genome appears to be degrading in symbiosis; however, it is considerably larger than the genomes of other mammalian louse endosymbionts. These patterns suggest the possibility that this Sodalis endosymbiont might be recently acquired, replacing a now-extinct, ancient endosymbiont. From the same lice, we also identified an abundant bacterium belonging to the genus Rickettsia that is closely related to Rickettsia ricketsii, a human pathogen vectored by ticks. No obvious masses of the Rickettsia bacterium were observed in louse tissues, nor did we find any evidence of vertical transmission, so the nature of its association remains unclear. IMPORTANCE Many insects are host to heritable symbiotic bacteria. These heritable bacteria have been identified from numerous species of parasitic lice. It appears that novel symbioses have formed between lice and bacteria many times, with new bacterial symbionts potentially replacing existing ones. However, little was known about the symbionts of lice parasitizing marine mammals. Here, we identified a heritable bacterial symbiont in lice parasitizing northern fur seals. This bacterial symbiont appears to have been recently acquired by the lice. The findings reported here provide insights into how new symbioses form and how this lifestyle is shaping the symbiont genome.

Genome sequence of Candidatus Riesia pediculischaeffi, endosymbiont of chimpanzee lice, and genomic comparison of recently acquired endosymbionts from human and chimpanzee lice.

The obligate-heritable endosymbionts of insects possess some of the smallest known bacterial genomes. This is likely due to loss of genomic material during symbiosis. The mode and rate of this erosion may change over evolutionary time: faster in newly formed associations and slower in long-established ones. The endosymbionts of human and anthropoid primate lice present a unique opportunity to study genome erosion in newly established (or young) symbionts. This is because we have a detailed phylogenetic history of these endosymbionts with divergence dates for closely related species. This allows for genome evolution to be studied in detail and rates of change to be estimated in a phylogenetic framework. Here, we sequenced the genome of the chimpanzee louse endosymbiont (Candidatus Riesia pediculischaeffi) and compared it with the closely related genome of the human body louse endosymbiont. From this comparison, we found evidence for recent genome erosion leading to gene loss in these endosymbionts. Although gene loss was detected, it was not significantly greater than in older endosymbionts from aphids and ants. Additionally, we searched for genes associated with B-vitamin synthesis in the two louse endosymbiont genomes because these endosymbionts are believed to synthesize essential B vitamins absent in the louse’s diet. All of the expected genes were present, except those involved in thiamin synthesis. We failed to find genes encoding for proteins involved in the biosynthesis of thiamin or any complete exogenous means of salvaging thiamin, suggesting there is an undescribed mechanism for the salvage of thiamin. Finally, genes encoding for the pantothenate de novo biosynthesis pathway were located on a plasmid in both taxa along with a heat shock protein. Movement of these genes onto a plasmid may be functionally and evolutionarily significant, potentially increasing production and guarding against the deleterious effects of mutation. These data add to a growing resource of obligate endosymbiont genomes and to our understanding of the rate and mode of genome erosion in obligate animal-associated bacteria. Ultimately sequencing additional louse p-endosymbiont genomes will provide a model system for studying genome evolution in obligate host associated bacteria.

Phylogenomics from whole genome sequences using aTRAM

&NA; Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long‐term utility of the data. Currently, for organisms with moderate to small genomes (<1000 Mbp) it is feasible to sequence the entire genome at modest coverage (10‐30×). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single‐copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out‐groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close‐ to distantly related taxa at high to low levels of coverage. Both the concatenated analysis and the coalescent‐based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above 5‐10×, aTRAM was successful at assembling 80‐90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.]

Taxonomy of lice and their endosymbiotic bacteria in the post-genomic era

Recent studies of molecular and genomic data from the parasitic lice of birds and mammals, as well as their mutualistic endosymbiotic bacteria, are changing the phylogenetic relationships and taxonomy of these organisms. Phylogenetic studies of lice suggest that vertebrate parasitism arose multiple times from free-living book and bark lice. Molecular clocks show that the major families of lice arose in the late Mesozoic and radiated in the early Cenozoic, following the radiation of mammals and birds. The recent release of the human louse genome has provided new opportunities for research. The genome is being used to find new genetic markers for phylogenetics and population genetics, to understand the complex evolutionary relationships of mitochondrial genes, and to study genome evolution. Genomes are informing us not only about lice, but also about their obligate endosymbiotic bacteria. In contrast to lice and their hosts, lice and their endosymbionts do not share common evolutionary histories, suggesting that endosymbionts are either replaced over time or that there are multiple independent origins of symbiosis in lice. Molecular phylogenetics and whole genome sequencing have recently provided the first insights into the phylogenetic placement and metabolic characteristics of these distantly related bacteria. Comparative genomics between distantly related louse symbionts can provide insights into conserved metabolic functions and can help to explain how distantly related species are fulfilling their role as mutualistic symbionts. In lice and their endosymbionts, molecular data and genome sequencing are driving our understanding of evolutionary relationships and classification, and will for the foreseeable future.

Primates, Lice and Bacteria: Speciation and Genome Evolution in the Symbionts of Hominid Lice

Abstract Insects with restricted diets rely on symbiotic bacteria to provide essential metabolites missing in their diet. The blood-sucking lice are obligate, host-specific parasites of mammals and are themselves host to symbiotic bacteria. In human lice, these bacterial symbionts supply the lice with B-vitamins. Here, we sequenced the genomes of symbiotic and heritable bacterial of human, chimpanzee, gorilla, and monkey lice and used phylogenomics to investigate their evolutionary relationships. We find that these symbionts have a phylogenetic history reflecting the louse phylogeny, a finding contrary to previous reports of symbiont replacement. Examination of the highly reduced symbiont genomes (0.53–0.57 Mb) reveals much of the genomes are dedicated to vitamin synthesis. This is unchanged in the smallest symbiont genome and one that appears to have been reorganized. Specifically, symbionts from human lice, chimpanzee lice, and gorilla lice carry a small plasmid that encodes synthesis of vitamin B5, a vitamin critical to the bacteria-louse symbiosis. This plasmid is absent in an old world monkey louse symbiont, where this pathway is on its primary chromosome. This suggests the unique genomic configuration brought about by the plasmid is not essential for symbiosis, but once obtained, it has persisted for up to 25 My. We also find evidence that human, chimpanzee, and gorilla louse endosymbionts have lost a pathway for synthesis of vitamin B1, whereas the monkey louse symbiont has retained this pathway. It is unclear whether these changes are adaptive, but they may point to evolutionary responses of louse symbionts to shifts in primate biology.

Culture-Facilitated Comparative Genomics of the Facultative Symbiont Hamiltonella defensa

Abstract Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids to generate complete genomes and transcriptome data for four strains of H. defensa from the pea aphid Acyrthosiphon pisum. Chosen strains also spanned the breadth of the H. defensa phylogeny and differed in strength of protection conferred against parasitoids. Results indicated that strains shared most genes with roles in nutrient acquisition, metabolism, and essential housekeeping functions. In contrast, the inventory of mobile genetic elements varied substantially, which generated strain specific differences in gene content and genome architecture. In some cases, specific traits correlated with differences in protection against parasitoids, but in others high variation between strains obscured identification of traits with likely roles in defense. Transcriptome data generated continuous distributions to genome assemblies with some genes that were highly expressed and others that were not. Single molecule real-time sequencing further identified differences in DNA methylation patterns and restriction modification systems that provide defense against phage infection.

Phylogenomics using Target-restricted Assembly Resolves Intra-generic Relationships of Parasitic Lice (Phthiraptera: Columbicola )

Abstract.— Parasitic “wing lice” (Phthiraptera: Columbicola) and their dove and pigeon hosts are a well‐recognized model system for coevolutionary studies at the intersection of micro‐ and macroevolution. Selection on lice in microevolutionary time occurs as pigeons and doves defend themselves against lice by preening. In turn, behavioral and morphological adaptations of the lice improve their ability to evade host defense. Over macroevolutionary time wing lice tend to cospeciate with their hosts; yet, some species of Columbicola have switched to new host species. Understanding the ecological and evolutionary factors that influence coadaptation and codiversification in this system will substantially improve our understanding of coevolution in general. However, further work is hampered by the lack of a robust phylogenetic framework for Columbicola spp. and their hosts. Previous attempts to resolve the phylogeny of Columbicola based on sequences from a few genes provided limited support. Here, we apply a new approach, target restricted assembly, to assemble 977 orthologous gene sequences from whole‐genome sequence data generated from very small, ethanol‐preserved specimens, representing up to 61 species of wing lice. Both concatenation and coalescent methods were used to estimate the species tree. These two approaches yielded consistent and well‐supported trees with 90% of all relationships receiving 100% support, which is a substantial improvement over previous studies. We used this new phylogeny to show that biogeographic ranges are generally conserved within clades of Columbicola wing lice. Limited inconsistencies are probably attributable to intercontinental dispersal of hosts, and host switching by some of the lice. [aTRAM; coalescent; coevolution; concatenation; species tree.]

Both living bacteria and eukaryotes in the mosquito gut promote growth of larvae

We recently reported that larval stage Aedes aegypti and several other species of mosquitoes grow when living bacteria are present in the gut but do not grow when living bacteria are absent. We further reported that living bacteria induce a hypoxia signal in the gut, which activates hypoxia-induced transcription factors and other processes larvae require for growth. In this study we assessed whether other types of organisms induce mosquito larvae to grow and asked if the density of non-living microbes or diet larvae are fed obviate the requirement for living organisms prior results indicated are required for growth. Using culture conditions identical to our own prior studies, we determined that inoculation density of living Escherichia coli positively affected growth rates of Ae. aegypti larvae, whereas non-living E. coli had no effect on growth across the same range of inoculation densities. A living yeast, alga, and insect cell line induced axenic Ae. aegypti first instars to grow, and stimulated similar levels of midgut hypoxia, HIF-α stabilization, and neutral lipid accumulation in the fat body as E. coli. However, the same organisms had no effect on larval growth if heat-killed. In addition, no axenic larvae molted when fed two other diets, when fed diets supplemented with heat-killed microbes or lysed and heat-killed microbes. Experiments conducted with An. gambiae yielded similar findings. Taken together, our results indicate that organisms from different prokaryotic and eukaryotic groups induce mosquito larvae to grow, whereas no conditions were identified that stimulated larvae to grow in the absence of living organisms.

Simultaneous radiation of bird and mammal lice following the K-Pg boundary

The diversification of parasite groups often occurs at the same time as the diversification of their hosts. However, most studies demonstrating this concordance only examine single host–parasite groups. Multiple diverse lineages of ectoparasitic lice occur across both birds and mammals. Here, we describe the evolutionary history of lice based on analyses of 1107 single-copy orthologous genes from sequenced genomes of 46 species of lice. We identify three major diverse groups of lice: one exclusively on mammals, one almost exclusively on birds and one on both birds and mammals. Each of these groups radiated just after the Cretaceous–Paleogene (K-Pg) boundary, the time of the mass extinction event of the dinosaurs and rapid diversification of most of the modern lineages of birds and mammals.

Supplementary material from "Simultaneous radiation of bird and mammal lice following the K-Pg boundary"

The diversification of parasite groups often occurs at the same time as the diversification of their hosts. However, most studies demonstrating this concordance only examine single host–parasite groups. Multiple diverse lineages of ectoparasitic lice occur across both birds and mammals. Here, we describe the evolutionary history of lice based on analyses of 1107 single-copy orthologous genes from sequenced genomes of 46 species of lice. We identify three major diverse groups of lice: one exclusively on mammals, one almost exclusively on birds and one on both birds and mammals. Each of these groups radiated just after the Cretaceous–Paleogene (K-Pg) boundary, the time of the mass extinction event of the dinosaurs and rapid diversification of most of the modern lineages of birds and mammals.