The draft genome of strain cCpun from biting midges establishes Cardinium as a paraphyletic group, and reveals a novel gene family expansion in a symbiont

Background: It is estimated that 13% of arthropod species carry the heritable symbiont Cardinium hertigii. 16S rRNA and gyrB sequence divides this species into three clades, with the A group infecting a range of arthropods, the B group infecting nematode worms, and the C group infecting Culicoides biting midges. To date, genome sequence has only been available for strains from clade A and B, impeding general understanding of the evolutionary history of the radiation. We present a draft genome sequence for a C group Cardinium, motivated both by the paucity of genomic information outside of the A group, and the importance of Culicoides biting midge hosts as arbovirus vectors. Methods: We reconstructed the genome of cCpun, a Cardinium strain from group C that naturally infects Culicoides punctatus, through Illumina sequencing of infected host specimens. Results: The draft genome presented has high completeness, with BUSCO scores comparable to closed group A Cardinium genomes. Phylogenomic analysis based on concatenated single copy core proteins revealed that Cardinium, as currently considered, is paraphyletic, with strains of Ca. Paenicardinium endoni from nematodes nested within the two groups infecting arthropod hosts. Analysis of the genome of cCpun revealed expansion of a variety of gene families classically considered important in symbiosis (e.g. ankyrin domain containing genes), and one set – characterized by DUF1703 domains – not previously associated with symbiotic lifestyle. This protein group encodes putative secreted nucleases, and the cCpun genome carried at least 25 widely divergent paralogs, of which 24 had a common ancestor in the C group ancestor. The genome revealed no evidence in support of B vitamin provisioning to its haematophagous host, and indeed suggests Cardinium may be a net importer of biotin. Discussion: These data indicate Cardinium, as currently conceived, to be paraphyletic. The draft genome further produces new hypotheses as to the interaction of the symbiont with the midge host, in particular the biological role of DUF1703 nuclease proteins that are predicted as being secreted by cCpun, but in contrast provides no support for a role for the symbiont in provisioning the host with B vitamins.</jats:p

Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade of heritable symbionts

A dynamic continuum exists from free-living environmental microbes to strict host-associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont Arsenophonus nasoniae, however we present multiple lines of evidence that the bee Arsenophonus deviates from a heritable model of transmission. Field sampling uncovered spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and laboratory individuals. Fluorescent in situ hybridisation showed Arsenophonus localised in the gut, and detection was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. These findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle

Cardinium symbiosis as a potential confounder of mtDNA based phylogeographic inference in Culicoides imicola (Diptera: Ceratopogonidae), a vector of veterinary viruses

BACKGROUND: Culicoides imicola (Diptera: Ceratopogonidae) is an important Afrotropical and Palearctic vector of disease, transmitting viruses of animal health and economic signifcance including African horse sickness and bluetongue viruses. Maternally inherited symbiotic bacteria (endosymbionts) of arthropods can alter the frequency of COI (cytochrome c oxidase subunit I) mitochondrial haplotypes (mitotypes) in a population, masking the true patterns of host movement and gene fow. Thus, this study aimed to assess the mtDNA structure of C. imicola in relation to infection with Candidatus Cardinum hertigii (Bacteroides), a common endosymbiont of Culicoides spp. METHODS: Using haplotype network analysis, COI Sanger sequences from Cardinium-infected and -uninfected C. imicola individuals were frst compared in a population from South Africa. The network was then extended to include mitotypes from a geographic range where Cardinium infection has previously been investigated. RESULTS: The mitotype network of the South African population demonstrated the presence of two broad mitotype groups. All Cardinium-infected specimens fell into one group (Fisher’s exact test, P=0.00071) demonstrating a linkage disequilibrium between endosymbiont and mitochondria. Furthermore, by extending this haplotype network to include other C. imicola populations from the Mediterranean basin, we revealed mitotype variation between the Eastern and Western Mediterranean basins (EMB and WMB) mirrored Cardinium-infection heterogeneity. CONCLUSIONS: These observations suggest that the linkage disequilibrium of Cardinium and mitochondria refects endosymbiont gene fow within the Mediterranean basin but may not assist in elucidating host gene fow. Subsequently, we urge caution on the single usage of the COI marker to determine population structure and movement in C. imicola and instead suggest the complementary utilisation of additional molecular markers.A BBSRC Doctoral Training Partnership studentship and a Marie Curie Individual Fellowship.https://parasitesandvectors.biomedcentral.compm2022Veterinary Tropical Disease

New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria

© 2007 Ioannidis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The definitive version was published in BMC Genomics 8 (2007): 182, doi:10.1186/1471-2164-8-182.Background: The annotated genomes of two closely related strains of the intracellular bacterium Wolbachia pipientis have been reported without the identifications of the putative origin of replication (ori). Identifying the ori of these bacteria and related alpha-Proteobacteria as well as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria. Results: Using features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus, the origin of DNA replication (ori) regions were identified in silico for Wolbachia strains and eleven other related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These features include DnaA-, CtrA- and IHF-binding sites as well as the flanking genes in C. crescentus. The Wolbachia ori boundary genes were found to be hemE and COG1253 protein (CBS domain protein). Comparisons of the putative ori region among related Wolbachia strains showed higher conservation of bases within binding sites. Conclusion: The sequences of the ori regions described here are only similar among closely related bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes, suggest that several of these obligate intracellular bacteria may have altered replication mechanisms. Based on these analyses, criteria are set forth for identifying the ori region in genome sequencing projects.PI, PS, SS, GT and KB acknowledge support of their work from intramural funding from the University of Ioannina. SB, JDH, LB and JW acknowledge support of their work from the U.S. National Science Foundation grant EF-0328363. SB also acknowledges the support from the NASA Astrobiology Institute (NNA04CC04A

Rapid molecular evolution of Spiroplasma symbionts of Drosophila

Spiroplasma is a genus of Mollicutes whose members include plant pathogens, insect pathogens and endosymbionts of animals. Spiroplasma phenotypes have been repeatedly observed to be spontaneously lost in Drosophila cultures, and several studies have documented a high genomic turnover in Spiroplasma symbionts and plant pathogens. These observations suggest that Spiroplasma evolves quickly in comparison to other insect symbionts. Here, we systematically assess evolutionary rates and patterns of Spiroplasma poulsonii , a natural symbiont of Drosophila. We analysed genomic evolution of sHy within flies, and sMel within in vitro culture over several years. We observed that S. poulsonii substitution rates are among the highest reported for any bacteria, and around two orders of magnitude higher compared with other inherited arthropod endosymbionts. The absence of mismatch repair loci mutS and mutL is conserved across Spiroplasma , and likely contributes to elevated substitution rates. Further, the closely related strains sMel and sHy (>99.5 % sequence identity in shared loci) show extensive structural genomic differences, which potentially indicates a higher degree of host adaptation in sHy, a protective symbiont of Drosophila hydei. Finally, comparison across diverse Spiroplasma lineages confirms previous reports of dynamic evolution of toxins, and identifies loci similar to the male-killing toxin Spaid in several Spiroplasma lineages and other endosymbionts. Overall, our results highlight the peculiar nature of Spiroplasma genome evolution, which may explain unusual features of its evolutionary ecology

Linking genomics and ecology to investigate the complex evolution of an invasive Drosophila pest

Drosophilid fruit flies have provided science with striking cases of behavioural adaptation and genetic innovation. A recent example is the invasive pest Drosophila suzukii, which, unlike most other Drosophila, lays eggs and feeds on undamaged, ripening fruits. This poses a serious threat for fruit cultivation, but also offers an interesting model to study evolution of behavioural innovation. We developed genome and transcriptome resources for D. suzukii. Coupling analyses of these data with field observations, we propose a hypothesis of the origin of its peculiar ecology. Using nuclear and mitochondrial phylogenetic analyses, we confirm its Asian origin, and reveal a surprising sister relationship between the eugracilis and the melanogaster subgroups. While the D. suzukii genome is comparable in size and repeat content to other Drosophila species, it has the lowest nucleotide substitution rate among the species analysed in this study. This finding is compatible with the overwintering diapause of D. suzukii, which results in a reduced number of generations per year compared to its sister species. Genome-scale relaxed clock analyses support a late Miocene origin of D. suzukii, concomitant with paleogeological and climatic conditions that suggest an adaptation to temperate montane forests, a hypothesis confirmed by field trapping. We propose a causal link between the ecological adaptations of D. suzukii in its native habitat and its invasive success in Europe and North America

Distribution, expression and molecular evolution of Wolbachia ankyrin genes

Wolbachia (α-proteobacteria, Rickettsiales) comprise a group of intracellular and maternally inherited bacteria, known to infect many species of arthropods and nematodes. Recent studies estimate that >65% of insect species (about 106 different insect species) are infected with Wolbachia making it one of the most abundant symbionts in nature. Wolbachia manipulates host reproduction in many different and unusual ways in order to insure its maternal transmission. The most common effects of Wolbachia infection includes: feminization of genetic males, thekytokous parthenogenesis, male killing and cytoplasmic incompatibility (CI) witch is the most widespread and, perhaps, the most prominent feature that Wolbachia impose on their hosts. CI results in embryonic mortality in mating between infected males with females having different Wolbachia infection status. The unique biology of Wolbachia together with its pandemic distribution has attracted considerable interest for its ecological and evolutionary effects, influencing aspects such as the adaptation to a wide variety of ecological niches and contributing to the biodiversity of the most numerous group of the animal kingdom, the group of insects. Furthermore, Wolbachia and Wolbachia-induced cytoplasmic incompatibility proposed for its potential application in pest and disease vector control. However, Wolbachia’s fastidious uncultivable nature hampers its study with the classical tools of bacterial genetics; so, little is known about the molecular mechanisms that underline the fascinating effects Wolbachia impose in their hosts. The recent sequencing of the first Wolbachia genome (wMel strain) provided novel insights into the potential molecular mechanisms used by this symbiont to manipulate its hosts. One interesting feature of wMel Wolbachia’s genome is the presence of an unusually high number of genes containing ankyrin repeat domains (23). Most commonly found in eukaryotic and viral proteins, ankyrin repeats act as scaffolds that mediate protein-protein interactions. Proteins with ankyrin repeats are known to involve in functions including: cell-cycle regulation, transcriptional regulation, cytoskeleton interactions and may act as toxins. This number of genes is in itself astonishing, as most of bacteria code for none to very few proteins with ankyrin repeats, indicating a putative role in host-Wolbachia interactions. Considering the potential importance of ankyrin repeats in mediating protein-protein interactions and their abundance in Wolbachia, we performed a comparative study to examine: a) the distribution of 23 ankyrin repeat-containing genes present in the wMel genome in various Wolbachia stains, b) the sequence variability between different ankyrin genes in various Wolbachia strains and their phylogenetic relationships and c) the transcription of ankyrin genes in different developmental stages and tissues.Η Wolbachia (α-πρωτεοβακτήρια, Rickettsiales) είναι ένα υποχρεωτικά ενδοκυττάριο και μητρι- κά κληρονομούμενο βακτήριο που απαντά σε πολλά είδη αρθροπόδων και νηματωδών. Εκτιμά- ται ότι το 65% των εντόμων (περίπου 106 είδη) είναι μολυσμένα με Wolbachia καθιστώντας την το πιο ευρέως διαδεδομένο συμβιωτικό βακτήριο που υπάρχει. Η Wolbachia επιδρά στις αναπα- ραγωγικές λειτουργίες των ξενιστών της με πολλούς και απρόσμενους τρόπους προκειμένου να εξασφαλίσει τη μητρική μεταβίβασή της. Συγκεκριμένα, η Wolbachia επάγει θηλυκοποίηση, παρθενογένεση, θανάτωση αρσενικών ατόμων και συνηθέστερα κυτταροπλασματική ασυμβατό- τητα (ΚΑ). Η ΚΑ αποτελεί ένα είδος εμβρυικής θνησιμότητας η οποία στην πιο απλή της μορφή προκύπτει όταν μολυσμένα αρσενικά άτομα γονιμοποιούν μη μολυσμένα θηλυκά. Η ΚΑ παρέχει ένα αναπαραγωγικό πλεονέκτημα στα μολυσμένα άτομα με αποτέλεσμα την εξάπλωση του βα- κτηρίου σε έναν πληθυσμό. Λόγω της ευρείας διάδοσης και των ιδιοτήτων της πιστεύεται ότι η Wolbachia έχει παίξει καταλυτικό ρόλο: (α) στην οικολογία των εντόμων, διευρύνοντας τους οικολογικούς θώκους, και (β) στην εξέλιξη, συμβάλλοντας στην αύξηση της βιοποικιλότητας, της πιο πολυπληθούς, σε αριθμό ειδών, ομάδας του ζωικού βασιλείου, των εντόμων. Επίσης, η Wolbachia και η Wolbachia-επαγόμενη κυτταροπλασματική ασυμβατότητα έχουν προταθεί, και πρόσφατα χρησιμοποιηθεί, ως ένα φιλικό προς το περιβάλλον εργαλείο πληθυσμιακού ελέγχου επιβλαβών ειδών χωρίς τη χρήση χημικών ουσιών. Παρόλο όμως το όλο και αυξανόμενο ενδιαφέρον, οι σύνθετες τροφικές ανάγκες της Wolbachia, οι οποίες υπαγορεύονται από τη συμβιωτική φύση της, έχουν παρεμποδίσει τη μελέ- τη του βακτηρίου με τα κλασικά εργαλεία της βακτηριακής γενετικής. Ωστόσο, η πρόσφατη αλ- ληλούχιση του πρώτου γονιδιώματος της Wolbachia (στέλεχος wMel) αποκαλύπτει νέα στοιχεία για τη βιολογία του βακτηρίου και τους μοριακούς μηχανισμούς αλληλεπίδρασης με τους ξενι- στές του. Για παράδειγμα, το γονιδίωμα του wMel περιέχει έναν ασυνήθιστα μεγάλο αριθμό γο- νιδίων (23) τα οποία κωδικοποιούν πρωτεΐνες με μοτίβα αγκυρινών (ankyrin domains). Τα μοτί- βα των αγκυρινών βρίσκονται κυρίως σε ευκαρυωτικές και ιικές πρωτεΐνες και είναι γνωστό ότι συμμετέχουν στις αλληλεπιδράσεις μεταξύ πρωτεϊνών και απαντούν σε κυτταροσκελετικές και μεμβρανικές πρωτεΐνες, μεταγραφικούς παράγοντες και ρυθμιστές της μεταγραφής, αναστολείς καθώς και τοξίνες. Ο αριθμός αυτός των γονιδίων είναι ιδιαίτερα εντυπωσιακός αν αναλογι- στούμε ότι τα βακτήρια κωδικοποιούν ελάχιστες πρωτεΐνες με μοτίβα αγκυρινών. Στα πλαίσια της παρούσας διατριβής, και αναλογιζόμενοι τη σημαντικότητα των αγκυρι- νών και τον ιδιαίτερα εντυπωσιακό τους αριθμό στη Wolbachia, πραγματοποιήθηκε μια συγκριτική μελέτη προκειμένου να εξετασθεί: α) η κατανομή των 23 αγκυρινών, που αρχικά εντοπί- στηκαν στο στέλεχος wMel, σε 11 διαφορετικές, φαινοτυπικά και εξελικτικά, συμβιωτικές σχέ- σεις Wolbachia-Drosophila, (β) η γενετική ποικιλομορφία καθώς και οι φυλογενετικές και εξε- λικτικές σχέσεις των γονιδίων αγκυρίνης μεταξύ των διαφορετικών στελεχών Wolbachia και (γ) η έκφραση των γονιδίων αυτών σε διαφορετικά αναπτυξιακά στάδια και ιστούς του ξενιστή και για τις 11 συμβιωτικές σχέσεις Drosophila-Wolbachia

The Tissue Tropisms and Transstadial Transmission of a <i>Rickettsia</i> Endosymbiont in the Highland Midge, <i>Culicoides impunctatus</i> (Diptera: Ceratopogonidae)

Rickettsia are a group of intracellular bacteria which can manipulate host reproduction and alter sensitivity to natural enemy attack in a diverse range of arthropods. The maintenance of Rickettsia endosymbionts in insect populations can be achieved through both vertical and horizontal transmission routes. For example, the presence of the symbiont in the follicle cells and salivary glands of Bemisia whiteflies allows Belli group Rickettsia transmission via the germline and plants, respectively. However, the transmission routes of other Rickettsia , such as those in the Torix group of the genus, remain underexplored. Through fluorescence in-situ hybridisation (FISH) and transmission electron microscopy (TEM) screening, this study describes the pattern of Torix Rickettsia tissue tropisms in the highland midge, Culicoides impunctatus (Diptera: Ceratopogonidae). Of note is high intensity of infection of the ovarian suspensory ligament, suggestive of a novel germline targeting strategy. Additionally, localisation of the symbiont in tissues of several developmental stages suggests transstadial transmission is a major route of ensuring maintenance of Rickettsia within C. impunctatus populations. Aside from providing insights into transmission strategies, Rickettsia presence in the fat body of larvae indicates potential host fitness and vector capacity impacts to be investigated in the future. Importance Statement Microbial symbionts of disease vectors have garnered recent attention due to their ability to alter vectorial capacity. Their consideration as a means of arbovirus control depends on symbiont vertical transmission which leads to spread of the bacteria through a population. Previous work has identified a Rickettsia symbiont present in several vector species of biting midges ( Culicoides spp.), however, symbiont transmission strategies and host effects remain underexplored. In this study, we describe the presence of Rickettsia in the ovarian suspensory ligament and the ovarian epithelial sheath of Culicoides impunctatus . Infection of these organs suggest the connective tissue surrounding developing eggs is important for ensuring vertical transmission of the symbiont in midges and possibly other insects. Additionally, our results indicate Rickettsia localisation in the fat body of Culicoides impunctatus . As viruses spread by midges often replicate in the fat body, this implies possible vector competence effects to be further investigated