crANKing up the infection: ankyrin domains in Rickettsiales and their role in host manipulation.

Intracellular bacteria use secreted effector proteins to modify host biology and facilitate infection. For many of these microbes, a particular eukaryotic domain-the ankyrin repeat (ANK)-plays a central role in specifying the host proteins and pathways targeted by the microbe. While we understand much of how some ANKs function in model organisms like Legionella and Coxiella, the understudied Rickettsiales species harbor many proteins with ANKs, some of which play critical roles during infection. This minireview is meant to organize and summarize the research progress made in understanding some of these Rickettsiales ANKs as well as document some of the techniques that have driven much of this progress.

Occurrence and molecular prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African wildlife: A systematic review and meta-analysis.

INTRODUCTION: Tick-borne pathogens (TBPs) constitute an emerging threat to public and animal health especially in the African continent, where land-use change, and wildlife loss are creating new opportunities for disease transmission. A review of TBPs with a focus on ticks determined the epidemiology of Rhipicephalus ticks in heartwater and the affinity of each Rickettsia species for different tick genera. We conducted a systematic review and meta-analysis to collect, map and estimate the molecular prevalence of Anaplasmataceae, Rickettsiaceae and Coxiellaceae in African wildlife. MATERIALS AND METHODS: Relevant scientific articles were retrieved from five databases: PubMed, ScienceDirect, Scopus, Ovid and OAIster. Publications were selected according to pre-determined exclusion criteria and evaluated for risk of bias using the appraisal tool for cross-sectional studies (AXIS). We conducted an initial descriptive analysis followed by a meta-analysis to estimate the molecular prevalence of each pathogen. Subgroup analysis and meta-regression models were employed to unravel associations with disease determinants. Finally, the quality of evidence of every estimate was finally assessed. RESULTS: Out of 577 retrieved papers, a total of 41 papers were included in the qualitative analysis and 27 in the meta-analysis. We retrieved 21 Anaplasmataceae species, six Rickettsiaceae species and Coxiella burnetii. Meta-analysis was performed for a total of 11 target pathogens. Anaplasma marginale, Ehrlichia ruminantium and Anaplasma centrale were the most prevalent in African bovids (13.9 %, CI: 0-52.4 %; 20.9 %, CI: 4.1-46.2 %; 13.9 %, CI: 0-68.7 %, respectively). Estimated TBPs prevalences were further stratified per animal order, family, species and sampling country. DISCUSSION: We discussed the presence of a sylvatic cycle for A. marginale and E. ruminantium in wild African bovids, the need to investigate A. phagocytophilum in African rodents and non-human primates as well as E. canis in the tissues of wild carnivores, and a lack of data and characterization of Rickettsia species and C. burnetii. CONCLUSION: Given the lack of epidemiological data on wildlife diseases, the current work can serve as a starting point for future epidemiological and/or experimental studies.

Natural infection of free-ranging capybaras (Hydrochoerus hydrochaeris) with Anaplasmataecea and Rickettsiaceae bacteria in the Iberá wetlands ecoregion, Argentina.

The current work assessed the infection with Ehrlichia and Anaplasma species, and exposure to Rickettsia spp. in free-ranging capybaras in the Iberá wetlands ecoregion in Argentina. By indirect immunofluorescence assay, 37 out of 51 (73%) capybara sera were seropositive to Rickettsia spp., with 23.5% and 4% samples considered homologous to Rickettsia parkeri and Rickettsia bellii, respectively (or very closely related serotypes). Anaplasmataceae DNA was found to be highly prevalent in capybaras, with 33 out of 62 samples positive for Anaplasma sp. with Ct values of 28.64 ± 0.35 (average ± standard error), and 12 samples positive for Ehrlichia sp. with Ct values of 31.74 ± 0.87. Anaplasma sp. from capybaras was closely related to Anaplasma sp. reported to infect Amblyomma dubitatum in Iberá wetlands and to Anaplasma odocoilei, while the detected Ehrlichia sp. was closely related to "Candidatus Ehrlichia hydrochoerus" previously reported to infect capybaras in Brazil and A. dubitatum in Iberá wetlands. Structures compatible with Anaplasma morulae were observed in the cytoplasm of platelets from Anaplasma-positive capybaras. Our findings show that capybaras from the Iberá wetlands were exposed to Rickettsia species related to R. bellii and to the pathogen R. parkeri, and were infected with "Ca. Ehrlichia hydrochoerus" and a novel Anaplasma species, herein named "Candidatus Anaplasma capybara".

Rickettsia tillamookensis (Rickettsiales: Rickettsiaceae) in Ixodes scapularis (Acari: Ixodidae) in Oklahoma.

Some of the most prevalent arthropod-borne pathogens impacting humans in the United States are transmitted by Ixodes ticks. However, little is known regarding the Rickettsia species that inhabit Ixodes scapularis in the United States. The aim of this study was to screen adult I. scapularis collected in central Oklahoma over an 8-yr period for the presence of tick-borne rickettsial pathogens or potential pathogens. During 2014-2021, 112 adult specimens of I. scapularis were collected from central Oklahoma. Amplicons for Rickettsia spp. were amplified from 53 (47.3%) of the samples. Of the positive ticks, 42 (79.2%) amplicon-positive Rickettsia samples were 100% identical to Rickettsia buchneri, 10 (18.9%) were 100% identical to R. tillamookensis strain Tillamook 23, and 1 (1.9%) specimen showed high identity for Rickettsia amblyommatis. This study highlights the importance of considering Rickettsia-specific assays when assessing Ixodes species ticks for potential pathogens.

Optimization of artificial membrane feeding system for lone star ticks, Amblyomma americanum (Acari: Ixodidae), and experimental infection with Rickettsia amblyommatis (Rickettsiales: Rickettsiaceae).

With the introduction of siliconized artificial membranes, various artificial feeding systems (AFS) for hard ticks (Ixodidae) have been developed over the last decades. Most AFS utilize similar core components but employ diverse approaches, materials, and experimental conditions. Published work describes different combinations of the core components without experimental optimizations for the artificial feeding of different tick species. Amblyomma americanum L., (Acari: Ixodidae) (lone star tick) is a known vector and reservoir for diverse tick-borne pathogens, such as Rickettsia amblyommatis and Ehrlichia chaffeensis. Ongoing environmental changes have supported the expansion of A. americanum into new habitats, contributing to increased tick-borne diseases in endemic areas. However, a significant knowledge gap exists in understanding the underlying mechanisms involved in A. americanum interactions with tick-borne pathogens. Here, we performed a systematic analysis and developed an optimized AFS for nymphal lone star ticks. Our results demonstrate that Goldbeater's membranes, rabbit hair, hair extract, and adult lone star ticks significantly improved the attachment rate of nymphal ticks, whereas tick frass and frass extract did not. With the optimized conditions, we achieved an attachment rate of 46 ±â€…3% and a success rate of 100% (i.e., one or more attached ticks) in each feeding experiment for nymphal lone star ticks. When fed on sheep blood spiked with R. amblyommatis, both nymphal and adult lone star ticks acquired and maintained R. amblyommatis, demonstrating the feasibility of studying A. americanum-pathogen interactions using AFS. Our study can serve as a roadmap to optimize and improve AFS for other medically relevant tick species.

Acquisition of Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Haemaphysalis longicornis (Acari: Ixodidae) through co-feeding with infected Dermacentor variabilis (Acari: Ixodidae) in the laboratory.

Haemaphysalis longicornis (Neumann) is an invasive ixodid tick originating from eastern Asia which recently has become established in the United States. In its native range, this tick can transmit several pathogens to animals and humans, but little is known about its ability to acquire and transmit pathogens native to the United States. Geographic overlap with ticks such as Dermacentor variabilis (Say), a known vector of Rickettsia rickettsii, makes investigation into the interactions between H. longicornis and D. variabilis of interest to the public health community. Previous studies have shown that H. longicornis can serve as a competent vector of R. rickettsii under laboratory settings, but there is little information on its ability to acquire this pathogen via other biologically relevant routes, such as co-feeding. Here, we assess the ability of H. longicornis nymphs to acquire R. rickettsii through co-feeding with infected D. variabilis adults on a vertebrate animal model under laboratory conditions. The median infection prevalence in engorged H. longicornis nymphs across 8 cohorts was 0% with an interquartile range (IQR) of 4.13%. Following transstadial transmission, the median infection prevalence in flat females was 0.7% (IQR = 2.4%). Our results show that co-feeding transmission occurs at low levels in the laboratory between these 2 species. However, based on the relatively low transmission rates, this may not be a likely mechanism of R. rickettsii introduction to H. longicornis.

'Candidatus Tisiphia' is a widespread Rickettsiaceae symbiont in the mosquito Anopheles plumbeus (Diptera: Culicidae).

Symbiotic bacteria can alter host biology by providing protection from natural enemies, or alter reproduction or vectoral competence. Symbiont-linked control of vector-borne disease in Anopheles has been hampered by a lack of symbioses that can establish stable vertical transmission in the host. Previous screening found the symbiont 'Candidatus Tisiphia' in Anopheles plumbeus, an aggressive biter and potential secondary vector of malaria parasites and West Nile virus. We screened samples collected over 10-years across Germany and used climate databases to assess environmental influence on incidence. We observed a 95% infection rate, and that the frequency of infection did not fluctuate with broad environmental factors. Maternal inheritance is indicated by presence in the ovaries through FISH microscopy. Finally, we assembled a high-quality 1.6 Mbp draft genome of 'Ca. Tisiphia' to explore its phylogeny and potential metabolic competence. The infection is closely related to strains found in Culicoides biting midges and shows similar patterns of metabolism, providing no evidence of the capacity to synthesize B-vitamins. This infection offers avenues for onward research in anopheline mosquito symbioses. Additionally, it provides future opportunity to study the impact of 'Ca. Tisiphia' on natural and transinfected hosts, especially in relation to reproductive fitness and vectorial competence and capacity.

Newer Surveillance Data Extends our Understanding of the Niche of Rickettsia montanensis (Rickettsiales: Rickettsiaceae) Infection of the American Dog Tick (Acari: Ixodidae) in the United States.

Background: Understanding the geographic distribution of Rickettsia montanensis infections in Dermacentor variabilis is important for tick-borne disease management in the United States, as both a tick-borne agent of interest and a potential confounder in surveillance of other rickettsial diseases. Two previous studies modeled niche suitability for D. variabilis with and without R. montanensis, from 2002 to 2012, indicating that the D. variabilis niche overestimates the infected niche. This study updates these, adding data since 2012. Methods: Newer surveillance and testing data were used to update Species Distribution Models (SDMs) of D. variabilis, and R. montanensis-infected D. variabilis, in the United States. Using random forest models, found to perform best in previous work, we updated the SDMs and compared them with prior results. Warren's I niche overlap metric was used to compare between predicted suitability for all ticks and "R. montanensis-positive niche" models across datasets. Results: Warren's I indicated <2% change in predicted niche, and there was no change in order of importance of environmental predictors, for D. variabilis or R. montanensis-positive niche. The updated D. variabilis niche model overpredicted suitability compared with the updated R. montanensis-positive niche in key peripheral parts of the range, but slightly underpredicted through the northern and midwestern parts of the range. This reinforces previous findings of a more constrained R. montanensis-positive niche than predicted by D. variabilis records alone. Conclusions: The consistency of predicted niche suitability for D. variabilis in the United States, with the addition of nearly a decade of new data, corroborates this is a species with generalist habitat requirements. Yet a slight shift in updated niche distribution, even of low suitability, included more southern areas, pointing to a need for continued and extended monitoring and surveillance. This further underscores the importance of revisiting vector and vector-borne disease distribution maps.

Detection and Isolation of Rickettsia tillamookensis (Rickettsiales: Rickettsiaceae) From Ixodes pacificus (Acari: Ixodidae) From Multiple Regions of California.

The western black-legged tick (Ixodes pacificus) is the most frequently identified human-biting tick species in the western United States and the principal vector of at least three recognized bacterial pathogens of humans. A potentially pathogenic Rickettsia species, first described in 1978 and recently characterized as a novel transitional group agent designated as Rickettsia tillamookensis, also exists among populations of I. pacificus, although the distribution and frequency of this agent are poorly known. We evaluated DNA extracts from 348 host-seeking I. pacificus nymphs collected from 9 locations in five California counties, and from 916 I. pacificus adults collected from 24 locations in 13 counties, by using a real-time PCR designed specifically to detect DNA of R. tillamookensis. DNA of R. tillamookensis was detected in 10 (2.9%) nymphs (95% CI: 1.6-5.2%) and 17 (1.9%) adults (95% CI: 1.2-3.0%) from 11 counties of northern California. Although site-specific infection rates varied greatly, frequencies of infection remained consistently low when aggregated by stage, sex, habitat type, or geographical region. Four novel isolates of R. tillamookensis were cultivated in Vero E6 cells from individual adult ticks collected from Alameda, Nevada, and Yolo counties. Four historical isolates, serotyped previously as 'Tillamook-like' strains over 40 yr ago, were revived from long-term storage in liquid nitrogen and confirmed subsequently by molecular methods as isolates of R. tillamookensis. The potential public health impact of R. tillamookensis requires further investigation.

Abiotic factors affecting the prevalence of Wolbachia (Rickettsiaceae) in immature Aedes albopictus (Skuse) (Culicidae).

Wolbachia is a genus of gram-negative endosymbiotic bacterium of maternal transmission, located mainly in the gonads of arthropods, including mosquitoes such as Aedes albopictus. The current distribution of Ae. albopictus in Argentina is restricted to the subtropical northeastern region of the country. Here, we studied the seasonal prevalence of Wolbachia detected in Ae. albopictus larvae and the relationship between the abiotic factors of the larval microhabitat and the infection status, in Eldorado city, Misiones province, subtropical region. The prevalence of Wolbachia infection found was 76.89% (n = 312). From the total samples examined, 52.80% (n = 214) showed double infection with the wAlbA/wAlbB strains, 23.84% (n = 97) infection only with wAlbB, and 0.25% (n = 1) only with wAlbA. The prevalence of double infection did not present statistically significant differences between the sites studied. For single infection, the lowest prevalence value of the wAlbB strain (13.33%) was found in the natural park, whereas the highest was found in the family dwellings and cemeteries. Tire repair shops showed an intermediate value. The wAlbA single infection was identified once. Our results also showed an association between temperature and slightly turbid waters with exposure to the sun in the larval habitats and the probability of infection by Wolbachia.

Rickettsiaceae in two reptile-associated tick species, Amblyomma exornatum and Africaniella transversale: First evidence of Occidentia massiliensis in hard ticks (Acari: Ixodidae).

All species of hard ticks associated with reptiles as hosts throughout their life cycle, are currently assigned to genera including Amblyomma and Africaniella. Among these species, based on literature data, Africaniella transversale has never been investigated for the presence of tick-borne pathogens. In this study, seven DNA extracts (two from A. transversale and five from Amblyomma exornatum) were screened for the presence of important tick-borne protozoa (piroplasms) and bacteria (Anaplasmataceae and Rickettsiaceae) with conventional PCRs and sequencing. A new heat shock protein chaperonin (groEL) gene-specific PCR was also developed to identify Occidentia spp. in these samples. In A. transversale, Occidentia massiliensis (previously detected in rodent-associated soft ticks) and Rickettsia hoogstraalii were present. While the latter was molecularly identical with formerly reported sequences of this rickettsia, the genotype of O. massiliensis was new based on sequence and phylogenetic analyses of its groEL gene. In A. exornatum, a Rickettsia genotype closely related to R. tamurae and R. monacensis, was detected. The ompA sequence of this genotype was identical to that of Rickettsia sp. Ae-8 reported from A. exornatum in a reptile breeding facility in the USA. These results show that A. transversale might carry O. massiliensis which (unless having a symbiotic nature in ticks) may originate either from the reptile host of this hard tick species or the rodent prey of reptiles. This is also the first detection of the reptile tick-associated Rickettsia sp. Ae-8 (phylogenetically aligning with R. tamurae, R. monacensis) in Africa, i.e. within the original geographical range of A. exornatum.

Discovery of a Diverse Set of Bacteria That Build Their Cell Walls without the Canonical Peptidoglycan Polymerase aPBP.

Peptidoglycan (PG) is a highly cross-linked peptide-glycan mesh that confers structural rigidity and shape to most bacterial cells. Polymerization of new PG is usually achieved by the concerted activity of two membrane-bound machineries, class-A penicillin binding proteins (aPBPs) and class-B penicillin binding proteins (bPBPs) in complex with shape, elongation, division, and sporulation (SEDS) proteins. Here, we have identified four phylogenetically distinct groups of bacteria that lack any identifiable aPBPs. We performed experiments on a panel of species within one of these groups, the Rickettsiales, and found that bacteria lacking aPBPs build a PG-like cell wall with minimal abundance and rigidity relative to cell walls of aPBP-containing bacteria. This reduced cell wall may have evolved to minimize the activation of host responses to pathogens and endosymbionts while retaining the minimal PG-biosynthesis machinery required for cell elongation and division. We term these "peptidoglycan-intermediate" bacteria, a cohort of host-associated species that includes some human pathogens. IMPORTANCE Peptidoglycan (PG) is a large, cross-linked polymer that forms the cell wall of most bacterial species and confers shape, rigidity, and protection from osmotic shock. It is also a potent stimulator of the immune response in animals. PG is normally polymerized by two groups of enzymes, aPBPs and bPBPs working together with shape, elongation, division, and sporulation (SEDS) proteins. We have identified a diverse set of host-associated bacteria that have selectively lost aPBP genes while retaining bPBP/SEDS and show that some of these build a minimal PG-like structure. It is expected that these minimal cell walls built in the absence of aPBPs improve the evolutionary fitness of host-associated bacteria, potentially through evasion of PG-recognition by the host immune system.

The neglected challenge: Vaccination against rickettsiae.

Over the last decades, rickettsioses are emerging worldwide. These diseases are caused by intracellular bacteria. Although rickettsioses can be treated with antibiotics, a vaccine against rickettsiae is highly desired for several reasons. Rickettsioses are highly prevalent, especially in poor countries, and there are indications of the development of antibiotic resistance. In addition, some rickettsiae can persist and cause recurrent disease. The development of a vaccine requires the understanding of the immune mechanisms that are involved in protection as well as in immunopathology. Knowledge about these immune responses is accumulating, and efforts have been undertaken to identify antigenic components of rickettsiae that may be useful as a vaccine. This review provides an overview on current knowledge of adaptive immunity against rickettsiae, which is essential for defense, rickettsial antigens that have been identified so far, and on vaccination strategies that have been used in animal models of rickettsial infections.

Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae.

Attributed to the tropism for host microvascular endothelium lining the blood vessels, vascular inflammation and dysfunction represent salient features of rickettsial pathogenesis, yet the details of fundamentally important pathogen interactions with host endothelial cells (ECs) as the primary targets of infection remain poorly appreciated. Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase of the phosphatidylinositol kinase-related kinase family, assembles into two functionally distinct complexes, namely mTORC1 (Raptor) and mTORC2 (Rictor), implicated in the determination of innate immune responses to intracellular pathogens via transcriptional regulation. In the present study, we investigated activation status of mTOR and its potential contributions to host EC responses during Rickettsia rickettsii and R. conorii infection. Protein lysates from infected ECs were analyzed for threonine 421/serine 424 phosphorylation of p70 S6 kinase (p70 S6K) and that of serine 2448 on mTOR itself as established markers of mTORC1 activation. For mTORC2, we assessed phosphorylation of protein kinase B (PKB or Akt) and protein kinase C (PKC), respectively, on serine 473 and serine 657. The results suggest increased phosphorylation of p70 S6K and mTOR during Rickettsia infection of ECs as early as 3 h and persisting for up to 24 h post-infection. The steady-state levels of phospho-Akt and phospho-PKC were also increased. Infection with pathogenic rickettsiae also resulted in the formation of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) puncta and increased lipidation of LC3-II, a response significantly inhibited by introduction of siRNA targeting mTORC1 into ECs. These findings thus yield first evidence for the activation of both mTORC1 and mTORC2 during EC infection in vitro with Rickettsia species and suggest that early induction of autophagy in response to intracellular infection might be regulated by this important pathway known to function as a central integrator of cellular immunity and inflammation.

Trends in clinical diagnoses of typhus group rickettsioses among a large U.S. insurance claims database.

Typhus group rickettsioses (TGRs) are vector-borne diseases that include murine typhus (Rickettsia typhi) and epidemic typhus (R. prowazekii). Twentieth-century public health interventions led to dramatic decreases in incidence; little is known about the contemporary TGR prevalence because neither disease is nationally notifiable. We summarized administrative claims data in a commercially insured population to examine trends in TGR medical encounters. We analysed data from 2003 to 2016 IBM® MarketScan® Commercial Databases to identify persons with inpatient or outpatient visits with an International Classification of Diseases, Ninth or Tenth Revision, Clinical Modification TGR-specific code. We summarized epidemiologic characteristics associated with incident diagnosis. We identified 1,799 patients diagnosed with a TGR. Patients resided in 46 states, and most were female (n = 1,019/1,799; 56.6%); the median age was 42 years (range: 0-64 years). Epidemic typhus (n = 931/1,799; 51.8%) was the most common TGRs, followed by murine typhus (n = 722/1,799; 40.1%). The majority of TGR patients were diagnosed in an outpatient setting (n = 1,725/1,799; 95.9%); among hospitalized patients, the majority received a murine typhus diagnosis (n = 67/74; 90.5%). TGRs are rarely diagnosed diseases. More patients were diagnosed with epidemic than murine typhus, even though R. prowazekii transmission requires body louse or flying squirrel exposure. Patients from all geographic regions were diagnosed with murine and epidemic typhus, despite historically recognized ranges for these diseases. The epidemiologic misalignment of insurance claims data versus historic TGRs data highlights the challenges of finding appropriate alternative data sources to serve as a proxy when national surveillance data do not exist.

Highly Reduced Genomes of Protist Endosymbionts Show Evolutionary Convergence.

Genome evolution in bacterial endosymbionts is notoriously extreme: the combined effects of strong genetic drift and unique selective pressures result in highly reduced genomes with distinctive adaptations to hosts [1-4]. These processes are mostly known from animal endosymbionts, where nutritional endosymbioses represent the best-studied systems. However, eukaryotic microbes, or protists, also harbor diverse bacterial endosymbionts, but their genome reduction and functional relationships with their hosts are largely unexplored [5-7]. We sequenced the genomes of four bacterial endosymbionts from three species of diplonemids, poorly studied but abundant and diverse heterotrophic protists [8-12]. The endosymbionts come from two bacterial families, Rickettsiaceae and Holosporaceae, that have invaded two families of diplonemids, and their genomes have converged on an extremely small size (605-632 kilobase pairs [kbp]), similar gene content (e.g., metabolite transporters and secretion systems), and reduced metabolic potential (e.g., loss of energy metabolism). These characteristics are generally found in both families, but the diplonemid endosymbionts have evolved greater extremes in parallel. They possess modified type VI secretion systems that could function in manipulating host metabolism or other intracellular interactions. Finally, modified cellular machinery like the ATP synthase without oxidative phosphorylation, and the reduced flagellar apparatus present in some diplonemid endosymbionts and nutritional animal endosymbionts, indicates that intracellular mechanisms have converged in bacterial endosymbionts with various functions and from different eukaryotic hosts across the tree of life.

Pilot survey of mosquitoes (Diptera: Culicidae) from southeastern Georgia, USA for Wolbachia and Rickettsia felis (Rickettsiales: Rickettsiaceae).

BACKGROUND & OBJECTIVES: Mosquito surveillance is one of the critical functions of local health departments, particularly in the context of outbreaks of severe mosquito-borne viral infections. Unfortunately, some viral and parasitic infections transmitted by mosquitoes, manifests non-specific clinical symptoms which may actually be of rickettsial etiology, including Rickettsia felis infections. This study tested the hypothesis that mosquitoes from southeastern Georgia, USA may be infected with Rickettsia felis and Wolbachia, an endosymbiotic bacterium of the order Rickettsiales. METHODS: Specimens of the five most common mosquito species occurring in the region were collected using gravid and light-traps and identified using morphological keys. Mosquitoes were then pooled by species, sex, trap and collection site and their DNA was extracted. Molecular methods were used to confirm mosquito identification, and presence of Wolbachia and R. felis. RESULTS: Wolbachia DNA was detected in 90.8% of the mosquito pools tested, which included 98% pools of Cx. quinquefasciatus Say (Diptera: Culicidae), 95% pools of Ae. albopictus Skuse (Diptera: Culicidae), and 66.7% of pools of Cx. pipiens complex. Samples of An. punctipennis Say (Diptera: Culicidae) and An. crucians Wiedemann (Diptera: Culicidae) were tested negative for Wolbachia DNA. Three genotypes of Wolbachia sp. belonging to Group A (1 type) and Group B (2 types) were identified. DNA of R. felis was not found in any pool of mosquitoes tested. INTERPRETATION & CONCLUSIONS: This study provides a pilot data on the high presence of Wolbachia in Cx. quinque-fasciatus and Ae. albopictus mosquitoes prevalent in the study region. Whether the high prevalence of Wolbachia and its genetic diversity in mosquitoes affects the mosquitoes' susceptibility to R. felis infection in Georgia will need further evaluation.

Diversity and environmental distribution of the cosmopolitan endosymbiont "Candidatus Megaira".

Members of the order Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont "Candidatus Megaira", which is phylogenetically closely related to the pathogen Rickettsia. "Candidatus Megaira" was first described as an intracellular bacterium in several ciliate species. Since then it has been found in association with diverse evolutionary distantly-related hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species "Candidatus Megaira polyxenophila", and the multidisciplinary description of a novel species, "Candidatus Megaira venefica", presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different separate species-level clades of "Candidatus Megaira", and we designed a set of specific probes allowing easy recognition of the four major clades of the genus.

Plastid Genomes and Proteins Illuminate the Evolution of Eustigmatophyte Algae and Their Bacterial Endosymbionts.

Eustigmatophytes, a class of stramenopile algae (ochrophytes), include not only the extensively studied biotechnologically important genus Nannochloropsis but also a rapidly expanding diversity of lineages with much less well characterized biology. Recent discoveries have led to exciting additions to our knowledge about eustigmatophytes. Some proved to harbor bacterial endosymbionts representing a novel genus, Candidatus Phycorickettsia, and an operon of unclear function (ebo) obtained by horizontal gene transfer from the endosymbiont lineage was found in the plastid genomes of still other eustigmatophytes. To shed more light on the latter event, as well as to generally improve our understanding of the eustigmatophyte evolutionary history, we sequenced plastid genomes of seven phylogenetically diverse representatives (including new isolates representing undescribed taxa). A phylogenomic analysis of plastid genome-encoded proteins resolved the phylogenetic relationships among the main eustigmatophyte lineages and provided a framework for the interpretation of plastid gene gains and losses in the group. The ebo operon gain was inferred to have probably occurred within the order Eustigmatales, after the divergence of the two basalmost lineages (a newly discovered hitherto undescribed strain and the Pseudellipsoidion group). When looking for nuclear genes potentially compensating for plastid gene losses, we noticed a gene for a plastid-targeted acyl carrier protein that was apparently acquired by horizontal gene transfer from Phycorickettsia. The presence of this gene in all eustigmatophytes studied, including representatives of both principal clades (Eustigmatales and Goniochloridales), is a genetic footprint indicating that the eustigmatophyte-Phycorickettsia partnership started no later than in the last eustigmatophyte common ancestor.