The evolution of genetic mechanisms used to combat bacterial infections is critical for the survival of animals and plants, yet how these genes evolved to produce a robust defense system is poorly understood. Studies of the nematode Caenorhabditis elegans have uncovered a plethora of genetic regulators and effectors responsible for surviving pathogens. However, comparative studies utilizing other free-living nematodes and therefore providing an insight into the evolution of innate immunity have been lacking. Here, we take a systems biology approach and use whole genome microarrays to profile the transcriptional response of C. elegans and the necromenic nematode Pristionchus pacificus after exposure to the four different pathogens Serratia marcescens, Xenorhabdus nematophila, Staphylococcus aureus and Bacillus thuringiensis DB27. C. elegans is susceptible to all four pathogens whilst P. pacificus is only susceptible to S. marcescens and X. nematophila. We show an unexpected level of specificity in host responses to distinct pathogens within and across species, revealing an enormous complexity of effectors of innate immunity. Functional domains enriched in the transcriptomes on different pathogens are similar within a nematode species but different across them, suggesting differences in pathogen sensing and response networks. We find translation inhibition to be a potentially conserved response to gram-negative pathogens in both the nematodes. Further computational analysis indicates that both nematodes when fed on pathogens up-regulate genes known to be involved in other stress responses like heat shock, oxidative and osmotic stress, and genes regulated by DAF-16/FOXO and TGF-beta pathways. This study presents a platform for comparative systems analysis of two nematode model species, and a catalog of genes involved in the evolution of nematode immunity and identifies both pathogen specific and pan-pathogen responses. We discuss the potential effects of ecology on evolution of downstream effectors and upstream regulators on evolution of nematode innate immunity.

Wolbachia, an alpha-proteobacterium closely related to Rickettsia, is a maternally transmitted, intracellular symbiont of arthropods and nematodes. Aedes albopictus mosquitoes are naturally infected with Wolbachia strains wAlbA and wAlbB. Cell line Aa23 established from Ae. albopictus embryos retains only wAlbB and is a key model to study host–endosymbiont interactions. We have assembled the complete circular genome of wAlbB from the Aa23 cell line using long-read PacBio sequencing at 500× median coverage. The assembled circular chromosome is 1.48 megabases in size, an increase of more than 300 kb over the published draft wAlbB genome. The annotation of the genome identified 1,205 protein coding genes, 34 tRNA, 3 rRNA, 1 tmRNA, and 3 other ncRNA loci. The long reads enabled sequencing over complex repeat regions which are difficult to resolve with short-read sequencing. Thirteen percent of the genome comprised insertion sequence elements distributed throughout the genome, some of which cause pseudogenization. Prophage WO genes encoding some essential components of phage particle assembly are missing, while the remainder are found in five prophage regions/WO-like islands or scattered around the genome. Orthology analysis identified a core proteome of 535 orthogroups across all completed Wolbachia genomes. The majority of proteins could be annotated using Pfam and eggNOG analyses, including ankyrins and components of the Type IV secretion system. KEGG analysis revealed the absence of five genes in wAlbB which are present in other Wolbachia. The availability of a complete circular chromosome from wAlbB will enable further biochemical, molecular, and genetic analyses on this strain and related Wolbachia.

While fleas are often perceived simply as a biting nuisance and a cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat (Ctenocephalides felis) and dog (Ctenocephalides canis) fleas, as well as Ctenocephalides orientis (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by Bartonella spp.) and Rickettsia spp., including Rickettsia felis (agent of flea-borne spotted fever) and Rickettsia asembonensis , a suspected pathogen. These Rickettsia spp. are members of a phylogenetic clade known as the ‘transitional group’, which includes both human pathogens and arthropod-specific endosymbionts. The relatively depauperate flea microbiome can also contain other endosymbionts, including a diverse range of Wolbachia strains. Here, we present circularized genome assemblies for two C. orientis-derived pathogens ( Bartonella clarridgeiae and R. asembonensis ) from Malaysia, a novel Wolbachia strain (wCori), and the C. orientis mitochondrion; all were obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two Wolbachia strains from Malaysian C. felis into tick cell culture and recovered circularized genome assemblies for both, one of which (wCfeF) is newly sequenced. We demonstrate that the three Wolbachia strains are representatives of different major clades (‘supergroups’), two of which appear to be flea-specific. These Wolbachia genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors and the biotin operon of obligate intracellular microbes. The first circularized assembly for R. asembonensis includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the USA. Analysis of loci under positive selection in the transitional group revealed genes involved in host–pathogen interactions that may facilitate host switching. Finally, the first B. clarridgeiae genome from Asia exhibited large-scale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of Ctenocephalides-associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.

Background: An organism can respond to changing environmental conditions by adjusting gene regulation and by forming alternative phenotypes. In nematodes, these mechanisms are coupled because many species will form dauer larvae, a stress-resistant and non-aging developmental stage, when exposed to unfavorable environmental conditions, and execute gene expression programs that have been selected for the survival of the animal in the wild. These dauer larvae represent an environmentally induced, homologous developmental stage across many nematode species, sharing conserved morphological and physiological properties. Hence it can be expected that some core components of the associated transcriptional program would be conserved across species, while others might diverge over the course of evolution. However, transcriptional and metabolic analysis of dauer development has been largely restricted to Caenorhabditis elegans. Here, we use a transcriptomic approach to compare the dauer stage in the evolutionary model system Pristionchus pacificus with the dauer stage in C. elegans. Results: We have employed Agilent microarrays, which represent 20,446 P. pacificus and 20,143 C. elegans genes to show an unexpected divergence in the expression profiles of these two nematodes in dauer and dauer exit samples. P. pacificus and C. elegans differ in the dynamics and function of genes that are differentially expressed. We find that only a small number of orthologous gene pairs show similar expression pattern in the dauers of the two species, while the non-orthologous fraction of genes is a major contributor to the active transcriptome in dauers. Interestingly, many of the genes acquired by horizontal gene transfer and orphan genes in P. pacificus, are differentially expressed suggesting that these genes are of evolutionary and functional importance. Conclusion: Our data set provides a catalog for future functional investigations and indicates novel insight into evolutionary mechanisms. We discuss the limited conservation of core developmental and transcriptional programs as a common aspect of animal evolution.

Removal of the reproductive system of many animals including fish, flies, nematodes, mice and humans can increase lifespan through mechanisms largely unknown. The abrogation of the germline in Caenorhabditis elegans increases longevity by 60% due to a signal emitted from the somatic gonad. Apart from increased longevity, germline-less C. elegans is also resistant to other environmental stressors such as feeding on bacterial pathogens. However, the evolutionary conservation of this pathogen resistance, its genetic basis and an understanding of genes involved in producing this extraordinary survival phenotype are currently unknown. To study these evolutionary aspects we used the necromenic nematode Pristionchus pacificus, which is a genetic model system used in comparison to C. elegans. By ablation of germline precursor cells and subsequent feeding on the pathogen Serratia marcescens we discovered that P. pacificus shows remarkable resistance to bacterial pathogens and that this response is evolutionarily conserved across the Genus Pristionchus. To gain a mechanistic understanding of the increased resistance to bacterial pathogens and longevity in germline-ablated P. pacificus we used whole genome microarrays to profile the transcriptional response comparing germline ablated versus un-ablated animals when fed S. marcescens. We show that lipid metabolism, maintenance of the proteasome, insulin signaling and nuclear pore complexes are essential for germline deficient phenotypes with more than 3,300 genes being differentially expressed. In contrast, gene expression of germline-less P. pacificus on E. coli (longevity) and S. marcescens (immunity) is very similar with only 244 genes differentially expressed indicating that longevity is due to abundant gene expression also involved in immunity. By testing existing mutants of Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved function of both genes in resistance to bacterial pathogens and longevity. This is the first study to show that the influence of the reproductive system on extending lifespan and innate immunity is conserved in evolution. Removal of the germline in the nematode Caenorhabditis elegans can increase lifespan and resistance to bacterial pathogens. Currently there is no information on what genes are regulated to produce this resistance phenotype in other nematodes and whether they are the same as genes involved in lifespan regulation. We used the necromenic nematode, Pristionchus pacificus, a species that diverged from C. elegans 250–400 MYA, ablated its germline and found increased resistance to the pathogens Serratia marcescens and Xenorhabdus nematophila. In a novel manner we performed cell ablation of the germline, exposure to bacterial pathogens and used whole genome microarrays of the same animals to find that this resistance is due to expression of genes involved in insulin signaling, nuclear pore complexes, ribosomal translation and lipid production. Furthermore, we see little difference between germline ablated lifespan and immunity leading us to believe that living longer is due to an abundance of genes also being involved with immunity. We could also show that, similar to C. elegans, the transcription factor DAF-16/FOXO and nuclear hormone receptor DAF-12, are integral for this response. Our study is the first to understand how the reproductive system regulates both lifespan and innate immunity transcriptionally and offers insights into the signaling cascades involved with resisting pathogen attack.

The reproductive system regulates lifespan in insects, nematodes and vertebrates. In Caenorhabditis elegans removal of germline increases lifespan by 60% which is dependent upon insulin signaling, nuclear hormone signaling, autophagy and fat metabolism and their microRNA-regulators. Germline-deficient C. elegans are also more resistant to various bacterial pathogens but the underlying molecular mechanisms are largely unknown. Firstly, we demonstrate that previously identified genes that regulate the extended lifespan of germline-deficient C. elegans (daf-2, daf-16, daf-12, tcer-1, mir-7.1 and nhr-80) are also essential for resistance to the pathogenic bacterium Xenorhabdus nematophila. We then use a novel unbiased approach combining laser cell ablation, whole genome microarrays, RNAi screening and exposure to X. nematophila to generate a comprehensive genome-wide catalog of genes potentially required for increased lifespan and innate immunity in germline-deficient C. elegans. We find 3,440 genes to be upregulated in C. elegans germline-deficient animals in a gonad dependent manner, which are significantly enriched for genes involved in insulin signaling, fatty acid desaturation, translation elongation and proteasome complex function. Using RNAi against a subset of 150 candidate genes selected from the microarray results, we show that the upregulated genes such as transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. We also identify a novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila. From an evolutionary perspective, most of the genes differentially expressed in germline deficient C. elegans also show a conserved expression pattern in germline deficient Pristionchus pacificus, a nematode species that diverged from C. elegans 250-400 MYA.