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Since the discovery of natural malaria vector populations infected by the endosymbiont bacteriumWolbachia, a renewed interest has arisen for using this bacterium as an alternative for malaria control. Among naturally infected mosquitoes,Anopheles moucheti, a major malaria mosquito in Central Africa, exhibits one of the highest prevalences ofWolbachiainfection. To better understand whether this maternally inherited bacterium could be used for malaria control, we investigatedWolbachiainfluence inAn. mouchetipopulations naturally infected by the malaria parasitePlasmodium falciparum. To this end, we collected mosquitoes in a village from Cameroon, Central Africa, where this mosquito is the main malaria vector. We found that the prevalence ofWolbachiabacterium was almost fixed in the studied mosquito population, and was higher than previously recorded. We also quantifiedWolbachiain whole mosquitoes and dissected abdomens, confirming that the bacterium is also elsewhere than in the abdomen, but at lower density. Finally, we analyzed the impact ofWolbachiapresence and density onP. falciparuminfection.Wolbachiadensity was slightly higher in mosquitoes infected with the malaria parasite than in uninfected mosquitoes. However, we observed no correlation between theP. falciparumandWolbachiadensities. In conclusion, our study indicates that naturally occurringWolbachiainfection does not affectP. falciparumdevelopment withinAn. mouchetimosquitoes.

Babesiasp. YLG has been recently described in Yellow-legged gull (Larus michahellis) chicks and belongs to the Peircei clade in the new classification of Piroplasms. Here, we studiedBabesiasp. YLG vectorial transmission by ticks in the simplified environment of a single seabird breeding colony where the Yellow-legged gull is the sole vertebrate host,Ornithodoros maritimus(syn.Alectorobius maritimus, Mans et al. 2021) the sole tick species, andBabesiasp. YLG is the unique blood parasite species previously detected in gull chicks in the colony. We collected ticks over four years, maintained certain individuals through moulting or oviposition, and dissected fresh ticks to isolate different organs and test for the presence of the parasite using molecular assays. We report the first strong evidence of a Piroplasmidae transmitted by a soft tick. Indeed,Babesiasp. YLG DNA was detected in the salivary glands of nymphs, females and males, a necessary organ to infect for transmission to a new vertebrate host. Parasite DNA was also found in tick ovaries, which could indicate possible transovarial transmission. Our detection ofBabesiasp. YLG DNA in several male testes and in endospermatophores, and notably in a parasite-free female (uninfected ovaries and salivary glands), raise the interesting possibility of sexual transmission from infected males to uninfected females. Future work in this system will now need to focus on the degree to which the parasite can be maintained locally by ticks and the epidemiological consequences of infection for bothO. maritimusand the avian host.

The Coxiellaceae family is composed of five genera showing lifestyles ranging from free-living to symbiosis. Among them,Coxiella burnetiiis a well-known pathogen causing Q fever in humans. This bacterium presents both intracellular (parasitic) and environmental (resistant) forms. Recently, several environmentalCoxiellagenomes have been reported, among which several have come from intracellular mutualistic symbionts of ticks, termedCoxiella-like endosymbionts. We sequenced two newCoxiella-LE genomes fromDermacentor marginatus(CLEDm) andOrnithodoros maritimus(CLEOmar) ticks, the latter belonging to theC. burnetiilineage. Using these newly sequencedCoxiella-LEs and 43 Coxiellaceae genomes, we conducted comparative genomic and phylogenomic analyses to increase our knowledge ofC. burnetiipathogenicity and the emergence ofCoxiella-LEs. Results highlight the probably parasitic nature of the common ancestor of the Coxiellaceae. Indeed, the virulence factor Dot/Icm T4 Secretion System is present in most, but not all, Coxiellaceae. Whereas it is part of a putative pathogenic island inC. burnetii, it has been entirely lost or inactivated inCoxiella-LEs, suggesting its importance in pathogenesis. Additionally, we found that a Sha/Mrp antiporter was laterally acquired in theC. burnetiilineage. This antiporter might be involved in alkali resistance and the development of the resistant form that is able to persist in the environment for long periods of time. The Sha operon is eroded or absent inCoxiella-LEs. Finally, we found that allCoxiellarepresentatives produce B vitamins and co-factors indicating a pre-adaptation ofCoxiellato mutualism with hematophagous arthropods. Accordingly, the ancestor ofC. burnetiiandCoxiella-LEs was likely a parasitic bacterium able to manipulate its host cell and to produce vitamins and co-factors for its own use.

Symbiosis with vitamin-provisioning microbes is essential for the nutrition of animals with specialized feeding habits. While coevolution stabilizes the interactions between symbiotic partners, their associations are not necessarily permanent: Recently acquired symbionts can replace ancestral symbionts. In this study, we demonstrate successful replacement dynamics of Francisella-Like Endosymbionts (-LE), a group of invasive B-vitamin-provisioning endosymbionts, across tick communities driven by a complex web of horizontal transfers. Using a broad collection of Francisella-LE-infected tick species, we determined the diversity of Francisella-LE haplotypes through a multi-locus strain typing approach, and further characterized their phylogenetic relationships and their association with biological traits of their tick hosts. The patterns observed showed that Francisella-LE commonly transfer through similar ecological networks and geographic distributions shared among different tick species, and, in certain cases, through preferential shuffling across congeneric tick species. Altogether, these findings reveal the importance of both routes in shaping the invasive pattern in which new nutritional symbioses are initiated.

The symbiont-associated (SA) environmental package is a new extension to the minimum information about any (x) sequence (MIxS) standards, established by the Parasite Microbiome Project (PMP) consortium, in collaboration with the Genomics Standard Consortium. The SA was built upon the host-associated MIxS standard, but reflects the nestedness of symbiont-associated microbiota within and across host-symbiont-microbe interactions. This package is designed to facilitate the collection and reporting of a broad range of metadata information that apply to symbionts such as life history traits, association with one or multiple host organisms, or the nature of host-symbiont interactions along the mutualism-parasitism continuum. To better reflect the inherent nestedness of all biological systems, we present a novel feature that allows users to co-localize samples, to nest a package within another package, and to identify replicates. Adoption of the MIxS-SA and of the new terms will facilitate reports of complex sampling design from a myriad of environments.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions: Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

International audience; Mutualistic interactions with microbes have facilitated the adaptation of major eukaryotic lineages to restricted diet niches. Hence, ticks with their strictly blood‐feeding lifestyle are associated with intracellular bacterial symbionts through an essential B vitamin supplementation. In this study, examination of bacterial diversity in 25 tick species of the genus Amblyomma showed that three intracellular bacteria, Coxiella‐like endosymbionts (LE), Francisella‐LE and Rickettsia, are remarkably common. No other bacterium is as uniformly present in Amblyomma ticks. Almost all Amblyomma species were found to harbour a nutritive obligate symbiont, Coxiella‐LE or Francisella‐LE, that is able to synthesize B vitamins. However, despite the co‐evolved and obligate nature of these mutualistic interactions, the structure of microbiomes does not mirror the Amblyomma phylogeny, with a clear exclusion pattern between Coxiella‐LE and Francisella‐LE across tick species. Coxiella‐LE, but not Francisella‐LE, form evolutionarily stable associations with ticks, commonly leading to co‐cladogenesis. We further found evidence for symbiont replacements during the radiation of Amblyomma, with recent, and probably ongoing, invasions by Francisella‐LE and subsequent replacements of ancestral Coxiella‐LE through transient co‐infections. Nutritional symbiosis in Amblyomma ticks is thus not a stable evolutionary state, but instead arises from conflicting origins between unrelated but competing symbionts with similar metabolic capabilities.

Rickettsia are obligate intracellular bacteria often associated with ticks and best known for causing human diseases (rickettsiosis), including typhus fever and sporadic cases of serious infection. In this study, we conducted a large survey of ticks in French Guiana to understand the overall diversity of Rickettsia in this remote area largely covered by dense rainforests. Out of 819 individuals (22 tick species in six genera), 252 (30.8%) samples were positive for Rickettsia infection. Multilocus typing and phylogenetic analysis identified 19 Rickettsia genotypes, but none was 100% identical to already known Rickettsia species or strains. Among these 19 genotypes, we identified two validated Rickettsia species, Rickettsia amblyommatis (spotted fever group) and Rickettsia bellii (bellii group), and characterized a novel and divergent Rickettsia phylogenetic group, the guiana group. While some tick hosts of these Rickettsia genotypes are among the most common ticks to bite humans in French Guiana, their potential pathogenicity remains entirely unknown. However, we found a strong association between Rickettsia genotypes and their host tick species, suggesting that most of these Rickettsia genotypes may be nonpathogenic forms maintained through transovarial transmission.

International audience; Ticks are commonly infected by Coxiella-like endosymbionts (Coxiella-LE) which are thought to supply missing B vitamin nutrients required for blood digestion.While this nutritional symbiosis is essential for the survival and reproduction of infected tick species, our knowledge of where Coxiella-LE is localized in tick tissues is partial at best since previous studies have focused on a limited number of Asian or American tick species. To fill this gap, we investigated the tissue localization of Coxiella-LE in three European tick species, Ornithodoros maritimus, Dermacentor marginatus and Ixodes hexagonus, using a diagnostic fluorescence in situ hybridization (FISH) assay, combined with PCR-based detection. Specific fluorescent foci were observed in several tick tissues. We visualized a pronounced tissue tropism of Coxiella-LE for tick ovaries and Malpighian tubules, a pattern suggestive of a high degree of lifestyle specialization toward mutualism: infection of the ovaries is indicative of transovarial transmission, whereas infection of the Malpighian tubules suggests a nutritional function. We postulate that Malpighian tubules are key organs for the nutritional symbiosis, notably the synthesis of B vitamins by Coxiella-LE, whereas the infection of the ovaries ensures vertical transmission of the symbionts to future generations. We also detected occasional infections in other organs, such as salivary glands and the midgut. Finally, we discuss the potential significance of the different tissue tropism for tick biology.

Q fever is a widespread zoonotic disease caused by the intracellular bacterium Coxiella burnetii. While transmission is primarily but not exclusively airborne, ticks are usually thought to act as vectors on the basis of early microscopy studies. However, recent observations revealed that endosymbionts of ticks have been commonly misidentified as C. burnetii, calling the importance of tick-borne transmission into question. In this study, we re-evaluated the vector competence of the African soft tick Ornithodoros moubata for an avirulent strain of C. burnetii. To this end, we used an artificial feeding system to initiate infection of ticks, specific molecular tools to monitor further infections, and culture assays in axenic and cell media to check for the viability of C. burnetii excreted by ticks. We observed typical traits associated with vector competence: The exposure to an infected blood meal resulted in viable and persistent infections in ticks, trans-stadial transmissions of infection from nymphs to adults and the ability of adult ticks to transmit infectious C. burnetii. However, in contrast to early studies, we found that infection differed substantially between tick organs. In addition, while adult female ticks were infected, we did not observe C. burnetii in eggs, suggesting that transovarial transmission is not effective. Finally, we detected only a sporadic presence of C. burnetii DNA in tick faeces, but no living bacterium was further isolated in culture assays, suggesting that excretion in faeces is not a common mode of transmission in O. moubata., Author summary The intracellular bacterium Coxiella burnetii is the agent of Q fever, a widespread zoonotic disease. Some early detection reports and microscopy studies identified ticks as vectors of Q fever but more recent studies and molecular analyses revealed that endosymbionts of ticks have been commonly misidentified as C. burnetii: It raises questions of whether ticks play an important role in Q fever transmission. In our study, we therefore experimentally re-evaluate the vector competence of the African soft tick Ornithodoros moubata for C. burnetii. We found that O. moubata can be infected by C. burnetii after the exposure to an infected blood meal. It resulted in viable and persistent infections in ticks, a trans-stadial transmission and the ability of adult ticks to transmit infection when feeding. Infection was however not transmitted transovarially or by faeces as early reported. Overall, we conclude that O. moubata may act as a driver of the transmission and of the spatial dispersal of Q fever among vertebrates where this tick is present in Africa.

Lyme disease (LD) and relapsing fevers (RF) are vector-borne diseases caused by bacteria of the Borrelia genus. Here, we report on the widespread infection by a non-described Borrelia species in passerine-associated ticks in tropical rainforests of French Guiana, South America. This novel Borrelia species is common in two tick species, Amblyomma longirostre and A. geayi, which feed on a broad variety of neotropical mammal and bird species, including migratory species moving to North America. The novel Borrelia species is divergent from the LD and RF species, and is more closely related to the reptile- and echidna-associated Borrelia group that was recently described. Genome sequencing showed that this novel Borrelia sp. has a relatively small genome consisting of a 0.9-Mb-large chromosome and an additional 0.3 Mb dispersed on plasmids. It harbors an RF-like genomic organization but with a unique mixture of LD- and RF-specific genes, including genes used by RF Borrelia for the multiphasic antigen-switching system and a number of immune-reactive protein genes used for the diagnosis of LD. Overall, our data indicate that this novel Borrelia is an intermediate taxon between the LD and RF species that may impact a large host spectrum, including American mammals. The designation “Candidatus Borrelia mahuryensis” is proposed for this species.

Assays used to evaluate the transmission-blocking activity of antimalarial drugs are largely focused on their potential to inhibit or reduce the infectivity of gametocytes, the blood stages of the parasite that are responsible for the onward transmission to the mosquito vector. For this purpose, the drug is administered concomitantly with the gametocyte-infected blood, and the results are evaluated as the % reduction in the number of oocysts in the mosquito midgut.We report the results of a series of experiments that explore the transmission blocking potential of two key antimalarial drugs, artesunate (AS) and sulfadoxine-pyrimethamine (SP), when administered to mosquitoes already infected from a previous blood meal. For this purpose, uninfected mosquitoes and mosquitoes carrying a 6-day old Plasmodium relictum infection (early oocyst stages) are allowed to feed either on a drug-treated or an untreated host in a fully factorial experiment. This protocol allows us to bypass the gametocyte stages and establish whether the drugs are able to arrest the ongoing development of oocysts and sporozoites, as would be the case when a mosquito takes a post-infection treated blood meal. In a separate experiment, we also explore whether a drug-treated blood meal impacts key life history traits of the mosquito relevant for transmission, and if this depends on their infection status.Our results show that feeding on an AS- or SP-treated host has no epidemiologically relevant effects on the fitness of infected or uninfected mosquitoes. In contrast, when infected mosquitoes feed on an SP-treated host, we observe both a significant increase in the number of oocysts in the midgut, and a drastic decrease in both sporozoite prevalence (−30%) and burden (−80%) compared to the untreated controls. We discuss the potential mechanisms underlying these seemingly contradictory results and contend that, provided the results are translatable to human malaria, the potential epidemiological and evolutionary consequences of the current preventive use of SP in malaria-endemic countries could be substantial.

International audience

International audience; Rickettsia are obligate intracellular bacteria often reported from hard ticks but more rarely from soft ticks. In this study, we detected in Northern Africa two putatively novel Rickettsia species in soft tick species of the Ornithodoros erraticus complex: Ornithodoros occidentalis from Morocco, Ornithodoros erraticus from Algeria and Ornithodoros normandi from Tunisia. We characterized these two novel Rickettsia species on the basis of comparative DNA sequence analyses and phylogenetics of four genes (gltA, 16S rRNA, coxA and ompB). These Rickettsia, provisionally named 'Candidatus Rickettsia africaseptentrionalis' and 'Candidatus Rickettsia maur-etanica', differed in nucleotide sequence from those of other Rickettsia species by 0.38-21.43 % depending on the gene examined. Phylogenetics further showed that the two novel Rickettsia species are closely related to each other and represent sister taxa to R. hoogstraalii, R. felis and R. asembonensis within the transitional Rickettsia group. While Ornithodoros host species of 'Candidatus Rickettsia africaseptentrionalis' and 'Candidatus Rickettsia mauretanica' are among the most common soft ticks to bite humans, their pathogenicity remains to be investigated .

Summary Mutualistic interactions with microbes have facilitated the radiation of major eukaryotic lineages [ 1 , 2 ]. Microbes can notably provide biochemical abilities, allowing eukaryotes to adapt to novel habitats or to specialize on particular feeding niches [ 2 , 3 , 4 ]. To investigate the importance of mutualisms for the exclusive blood feeding habits of ticks, we focused on a bacterial genus of medical interest, Francisella, which is known to include both virulent intracellular pathogens of vertebrates [ 5 , 6 ] and maternally inherited symbionts of ticks [ 7 , 8 , 9 ]. Through a series of physiological experiments, we identified a Francisella type, F-Om, as an obligate nutritional mutualist in the life cycle of the African soft tick Ornithodoros moubata. Francisella F-Om mutualism synthesizes B vitamins that are deficient in the blood meal of ticks. Indeed, experimental elimination of Francisella F-Om resulted in alteration of tick life history traits and physical abnormalities, deficiencies which were fully restored with an oral supplement of B vitamins. We also show that Francisella F-Om is maternally transmitted to all maturing tick oocytes, suggesting that this heritable symbiont is an essential adaptive element in the life cycle of O. moubata. The Francisella F-Om genome further revealed a recent origin from a Francisella pathogenic life style, as observed in other Francisella symbionts [ 6 , 7 , 10 ]. Though half of its protein-coding sequences are now pseudogenized or lost, Francisella F-Om has kept several B vitamin synthesis pathways intact, confirming the importance of these genes in evolution of its nutritional mutualism with ticks.

Creatine concentration in red blood cells was determined after ammoniumsulfate precipitation on a clear hemoglobin-free filtrate with a new enzymatic assay making use of bacterial creatinase. The method described is more specific than Griffiths' method and can easily be mechanised and adapted for use in a routine laboratory using classical automated equipment. By contrast with Griffiths' method no significant interferences of amino acids and creatine-like molecules were found. Reference values for this method were 0.379 +/- 0.076 mmol/l. In patients with high turnover of erythrocytes, e.g. haemodialysis patients (0.529 +/- 0.122 mmol/l), and renal insufficiency patients (0.565 +/- 0.145 mmol/l), significantly increased creatine concentration in erythrocytes were observed. Low erythrocyte creatine concentrations were found in chronic ambulatory dialysis patients (0.311 +/- 0.042 mmol/l).