The bacterial patterns suggesting the dynamic features of tick-associated microorganisms in hard ticks.

BACKGROUND: Ticks are blood-feeding significant arthropods that can harbour various microorganisms, including pathogens that pose health risks to humans and animals. Tick-symbiont microorganisms are believed to influence tick development, but the intricate interactions between these microbes and the relationships between different tick-borne microorganisms remain largely unexplored. RESULTS: Based on 111 tick pool samples presenting questing and engorged statuses including 752 questing tick and 1083 engorged tick from cattle and goats, which were collected in two types of geographic landscape (semi-desert and alpine meadow). We observed significant variations in the composition of tick-borne microorganisms across different environments and blood-engorgement statuses, with a pronounced divergence in symbionts compared to environmental bacteria. Metabolic predictions revealed over 90 differential pathways for tick-borne microorganisms in distinct environments and more than 80 metabolic variations in response to varying blood engorgement statuses. Interestingly, nine pathways were identified, particularly related to chorismate synthesis and carbohydrate metabolism. Moreover, microbial network relationships within tick-borne microorganism groups were highly distinct across different environments and blood-engorgement statuses. The microbial network relationships of symbionts involve some pathogenic and environmental microorganisms. Regression modelling highlighted positive correlations between the Coxiella symbiont and related pathogens, while some environmental bacteria showed strong negative correlations with Coxiella abundance. We also identified commensal bacteria/pathogens in bacterial cooccurrence patterns. Furthermore, we tested pathogenic microorganisms of each tick sample analysis revealed that 86.36% (1601/1855) of the tick samples carried one or more pathogenic microorganisms, The total carrier rate of bacterial pathogens was 43.77% ((812/1855). Most blood samples carried at least one pathogenic microorganism. The pathogens carried by the ticks have both genus and species diversity, and Rickettsia species are the most abundant pathogens among all pathogens. CONCLUSION: Our findings underscore that the bacterial pattern of ticks is dynamic and unstable, which is influenced by the environment factors and tick developmental characteristics.

Molecular detection and genetic characterization of Coxiella-like endosymbionts in dogs and ticks infesting dogs in Northeast India.

An epidemiological study was performed to determine the role of dogs and ticks infesting dogs in the transmission of Q fever in humans and animals from April 2019 to March 2020 in the northeastern hill states of India. In total, 245 pet and stray dogs irrespective of age or sex were sampled, without specific inclusion or exclusion criteria. In total, 478 ticks belonging to three species were detected, namely Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum anatolicum. The DNA extracted from blood and tick samples was assayed for molecular characterization of Coxiella burnetii targeting the 16S rRNA and superoxide dismutase (SOD) genes. Amplified PCR products were purified, cloned and custom sequenced. PCR assay showed 3.3% (8/245) of the dogs were positive for Coxiella-like bacteria. Coxiella-like bacterial DNA was detected in adult fully engorged females of R. sanguineus (7.7%, 13/168), R. (B.) microplus (3.3%, 4/123) and H. anatolicum (1.9%, 1/54). Coxiella-like bacterial DNA lacked in adult male or nymphal stage. The infection rate did not vary significantly between seasons, nor according to sex or age of the host. Six nucleotide sequences of 16S rRNA and SOD genes are discussed.

Molecular detection of Coxiella-like endosymbionts and absence of Coxiella burnetii in Amblyomma mixtum from Veracruz, Mexico.

Ticks are obligate ectoparasites associated with a wide range of vertebrate hosts, including domestic animals. Moreover, ticks are capable of transmitting many pathogens such as Coxiella. To date, Coxiella burnetii, the etiological agent of coxiellosis or Q fever, is the only valid species of the genera. Nevertheless, a wide range of agents denominated Coxiella-like have been detected in recent studies, mainly associated with ticks. The pathogenicity of these Coxiella-like agents is controversial as some of them can infect both birds and humans. In Mexico, knowledge about Q fever is scarce and limited to historical serological records, and there is an overall lack of molecular proof of any agent of the genus Coxiella circulating in the country. Therefore, the aim of this study was to detect the presence of Coxiella in ticks associated with cattle in all 10 regions of Veracruz, Mexico. To accomplish this objective, first, we identified ticks collected from cattle and horses in Veracruz. Then, for Coxiella detection, DNA extraction from ticks and PCR amplification of the 16S-rDNA of Coxiella was performed. Finally, we performed a phylogenetic reconstruction to determine the Coxiella lineages detected. From the 10 regions sampled we collected 888 ticks grouped in 180 pools, and only five Amblyomma mixtum from the locality of Castán, and one from Los Angeles from Tuxpan were found positive, which represents a frequency of 20% for each locality. This study represents the first attempt at molecular detection of Coxiella in ticks associated with cattle in the state of Veracruz, the major livestock producer in the country. The findings of the present study are relevant as they establish a precedent regarding the circulation of Coxiella-like agents, as well as the absence in three municipalities of the state of Veracruz of C. burnetii, an abortive agent of livestock importance.

Molecular detection of Coxiella (Gammaproteobacteria: Coxiellaceae) in Argas persicus and Alveonasus canestrinii (Acari: Argasidae) from Iran.

BACKGROUND: Coxiella burnetii and non-C. burnetii bacteria or endosymbiotic Coxiella-like were reported in various tick species. We aimed to detect C. burnetii within soft tick species, Argas persicus and Alveonasus canestrinii. METHODS: Argasid ticks were collected from different counties of Lorestan province, west of Iran. Partial fragments of 16S rRNA, IS1111 insertion sequence, com1, htpB, and icd genes related to Coxiella genus were sequenced. RESULTS: A partial 16S rRNA and com1 gene fragment as well as IS1111 was detected in four Ar. persicus and twelve Al. canestrinii pools. Moreover, partial htpB and icd gene was only detected in one pool of Ar. persicus. CONCLUSIONS: Detection of C. burnetii in tick samples was failed due to the occurrence of Coxiella-like endosymbionts and leads to misidentification. Thus, the house-keeping genes should be designated to distinguish C. burnetii within Coxiella-like endosymbionts.

Confirmation that candidatus Coxiella cheraxi from redclaw crayfish (Cherax quadricarinatus) is a close relative of Coxiella burnetii, the agent of Q-fever.

A Coxiella sp. closely related to the agent of Q-fever, Coxiella burnetii, has been associated with mortalities in redclaw crayfish, (Cherax quadricarinatus), in farms and experimental facilities for three decades. Limited sequence data including 16S rRNA have placed the rickettsial species as a new species, candidatus C. cheraxi closely related to C. burnetii. MinION sequencing was conducted on the last remaining sample from an outbreak of disease, TO-98. The accuracy of base pair reads was mostly 99·9% (error rate 1 in 1000) or better. After filtering for reads of co-isolated Citrobacter freundii, 2629 sequences remained with the longest being 12 585 base pairs (bp). The longest 21 sequences are presented with their single best hit statistics when examined by NCBI blastn (nucleotides) and the nucleotides translated into proteins NCBI blastx. All sequences hit with either C. burnetii (29/42, 69%) or Coxiella (10/42, 24%) or rickettsia (3/42, 7%) with an error rate of less than 1 in 1 million for either bp or amino acids. Sequencing in this report confirms candidatus C. cheraxi is a new species very closely related to C. burnetii. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports on the use of newer technologies on archival samples and provides significantly more data on the currently limited genome data of candidatus Coxiella cheraxi, one of the few species isolated in the genus Coxiella. Candidatus Coxiella cheraxi causes death in redclaw crayfish and has been reported as being closely related to C. burnettii, the agent of Q-fever, based on 16S rRNA sequencing. This work provides confirmation for this claim.

Characterization of microbiota diversity of field-collected Haemaphysalis longicornis (Acari: Ixodidae) with regard to sex and blood meals.

Haemaphysalis longicornis is a prominent tick species in China, and the major vector of an emerging tick-borne disease: severe fever with thrombocytopenia syndrome (SFTS). Microbiome diversity of ticks is influenced by several factors. In this study, we investigated microbiome diversity in field-collected female and male H. longicornis ticks and compared the microbial composition of fed and unfed ticks and of those feeding on different hosts using barcode sequencing of V3-V4 region of 16S RNA gene. Regardless of sex, host, and feeding status; the highest abundance among all samples was found for the genus Coxiella. The relative numbers of Coxiella sequences decreased with the length of the blood feeding, whereas the numbers of Staphylococcus and Corynebacterium increased gradually. The dominance of Coxiella across all samples indicates that it is an obligate symbiont of H. longicornis. Overall, higher microbiome richness was detected in male ticks than in female ticks. Fed ticks showed a more diverse microbe composition than unfed ticks, and ticks fed on goats exhibited the highest diversity. These findings of this study can serve as a basis for future studies of microbiota biology and interactions between the microbes and pathogens of H. longicornis.

Comparison of closely related, uncultivated Coxiella tick endosymbiont population genomes reveals clues about the mechanisms of symbiosis.

Understanding the symbiotic interaction between Coxiella-like endosymbionts (CLE) and their tick hosts is challenging due to lack of isolates and difficulties in tick functional assays. Here we sequenced the metagenome of a CLE population from wild Rhipicephalus sanguineus ticks (CRs) and compared it to the previously published genome of its close relative, CLE of R. turanicus (CRt). The tick hosts are closely related sympatric species, and their two endosymbiont genomes are highly similar with only minor differences in gene content. Both genomes encode numerous pseudogenes, consistent with an ongoing genome reduction process. In silico flux balance metabolic analysis (FBA) revealed the excess production of L-proline for both genomes, indicating a possible proline transport from Coxiella to the tick. Additionally, both CR genomes encode multiple copies of the proline/betaine transporter, proP gene. Modelling additional Coxiellaceae members including other tick CLE, did not identify proline as an excreted metabolite. Although both CRs and CRt genomes encode intact B vitamin synthesis pathway genes, which are presumed to underlay the mechanism of CLE-tick symbiosis, the FBA analysis indicated no changes for their products. Therefore, this study provides new testable hypotheses for the symbiosis mechanism and a better understanding of CLE genome evolution and diversity.

Tissue Localization and Variation of Major Symbionts in Haemaphysalis longicornis, Rhipicephalus haemaphysaloides, and Dermacentor silvarum in China.

Ticks are important disease vectors, as they transmit a variety of human and animal pathogens worldwide. Symbionts that coevolved with ticks confer crucial benefits to their host in nutrition metabolism, fecundity, and vector competence. Although over 100 tick species have been identified in China, general information on tick symbiosis is limited. Here, we visualized the tissue distribution of Coxiella sp. and Rickettsia sp. in lab-reared Haemaphysalis longicornis and Rhipicephalus haemaphysaloides by fluorescent in situ hybridization. We found that Coxiella sp. colonized exclusively the Malpighian tubules and ovaries of H. longicornis, while Rickettsia sp. additionally colonized the midgut of R. haemaphysaloides We also investigated the population structure of microbiota in Dermacentor silvarum ticks collected from Inner Mongolia, China, and found that Coxiella, Rickettsia, and Pseudomonas are the three dominant genera. No significant difference in microbiota composition was found between male and female D. silvarum ticks. We again analyzed the tissue localization of Coxiella sp. and Rickettsia sp. and found that they displayed tissue tropisms similar to those in R. haemaphysaloides, except that Rickettsia sp. colonized the nuclei of spermatids instead of ovaries in D. silvarum Altogether, our results suggest that Coxiella sp. and Rickettsia sp. are the main symbionts in the three ticks and reside primarily in midgut, Malpighian tubules, and reproductive tissues, but their tissue distribution varies in association with species and sexes.IMPORTANCE Tick-borne diseases constitute a major public health burden, as they are increasing in frequency and severity worldwide. The presence of symbionts helps ticks to metabolize nutrients, promotes fecundity, and influences pathogen infections. Increasing numbers of tick-borne pathogens have been identified in China; however, knowledge of native ticks, especially tick symbiosis, is limited. In this study, we analyze the distribution of Coxiella sp. and Rickettsia sp. in tissues of laboratory-reared Haemaphysalis longicornis and Rhipicephalus haemaphysaloides and field-collected Dermacentor silvarum We found that the localization patterns of Coxiella sp. in three Chinese tick species were similar to those of other tick species. We also found a previously undefined intracellular localization of Rickettsia sp. in tick midgut and spermatids. In addition, we demonstrate that tissue tropisms of symbionts vary between species and sexes. Our findings provide new insights into the tissue localization of symbionts in native Chinese ticks and pave the way for further understanding of their functional capabilities and symbiotic interactions with ticks.

Coxiella and Bartonella spp. in bats (Chiroptera) captured in the Brazilian Atlantic Forest biome.

BACKGROUND: The role of bats as reservoirs of zoonotic agents, especially pathogenic bacteria such as Bartonella and Coxiella, has been discussed around the world. Recent studies have identified bats as potential hosts of species from the proteobacteria phylum. In Brazil, however, the role of bats in the natural cycle of these agents is poorly investigated and generally neglected. In order to analyze the participation of bats in the epidemiology of diseases caused by Bartonella, Coxiella, Rickettsia, Anaplasma and Ehrlichia, we conducted a descriptive epidemiological study in three biogeographic regions of the Brazilian Atlantic Forest. RESULTS: Tissues of 119 bats captured in preserved areas in the states of Rio de Janeiro, Bahia and Santa Catarina from 2014 to 2015 were submitted to molecular analysis using specific primers. Bartonella spp. was detected in 22 spleen samples (18.5%, 95% CI: 11.9-26.6), whose phylogenetic analysis revealed the generation of at least two independent clusters, suggesting that these may be new unique genotypes of Bartonella species. In addition, four samples (3.4%, 95% CI: 0.9-8.3) were positive for the htpAB gene of C. burnetii [spleen (2), liver (1) and heart (1)]. Rickettsia spp., Anaplasma and Ehrlichia were not identified. This is the first study reporting C. burnetii and Bartonella spp. infections in bats from the Atlantic Forest biome. CONCLUSIONS: These findings shed light on potential host range for these bacteria, which are characterized as important zoonotic pathogens.

Determination of the microbial community features of Haemaphysalis flava in different developmental stages by high-throughput sequencing.

In this study, we analyzed the in vivo microbial community structure and diversity differences of Haemaphysalis flava (H. flava) in four developmental stages (egg, larva, nymph, and adult) to determine which bacterial genera could be propagated through transovarial transmission and transmitted by transstadial transmission during different developmental stages of H. flava. Paired-end sequencing of the V3 region of the 16S ribosomal DNA (16S rDNA) of H. flava in four developmental stage samples was conducted using the Illumina MiSeq sequencing platform. The following operational taxonomic units (OTUs) of H. flava were obtained during the four developmental stages: 89 of egg, 111 of larva, 104 of nymph, and 106 of female adult tick. Sixty-four of these OTUs had high similarity in the four developmental stages of H. flava. Eight bacterial genera had the highest abundances in all developmental stages, namely, Rickettsia, Coxiella, Pseudomonas, Ehrlichia, Escherichia, Acinetobacter, Citrobacter, and Cupriavidus. The nymph had the highest abundance of Coxiella, and the female adult tick had the highest abundance of Rickettsia. Staphylococcus and Wolbachia were detected in all developmental stages except the egg.

Candidatus Coxiella massiliensis Infection.

Bacteria genetically related to Coxiella burnetii have been found in ticks. Using molecular techniques, we detected Coxiella-like bacteria, here named Candidatus Coxiella massiliensis, in skin biopsy samples and ticks removed from patients with an eschar. This organism may be a common agent of scalp eschar and neck lymphadenopathy after tick bite.

Massive infection of seabird ticks with bacterial species related to Coxiella burnetii.

Seabird ticks are known reservoirs of bacterial pathogens of medical importance; however, ticks parasitizing tropical seabirds have received less attention than their counterparts from temperate and subpolar regions. Recently, Rickettsia africae was described to infect seabird ticks of the western Indian Ocean and New Caledonia, constituting the only available data on bacterial pathogens associated with tropical seabird tick species. Here, we combined a pyrosequencing-based approach with a classical molecular analysis targeting bacteria of potential medical importance in order to describe the bacterial community in two tropical seabird ticks, Amblyomma loculosum and Carios (Ornithodoros) capensis. We also investigated the patterns of prevalence and host specificity within the biogeographical context of the western Indian Ocean islands. The bacterial community of the two tick species was characterized by a strong dominance of Coxiella and Rickettsia. Our data support a strict Coxiella-host tick specificity, a pattern resembling the one found for Rickettsia spp. in the same two seabird tick species. Both the high prevalence and stringent host tick specificity suggest that these bacteria may be tick symbionts with probable vertical transmission. Detailed studies of the pathogenicity of these bacteria will now be required to determine whether horizontal transmission can occur and to clarify their status as potential human pathogens. More generally, our results show that the combination of next generation sequencing with targeted detection/genotyping approaches proves to be efficient in poorly investigated fields where research can be considered to be starting from scratch.

Species-level Microbiota of Biting Midges and Ticks from Poyang Lake.

Objective: The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area, namely, Qunlu Practice Base, Peach Blossom Garden, and Huangtong Animal Husbandry, and whether vectors carry any bacterial pathogens that may cause diseases to humans, to provide scientific basis for prospective pathogen discovery and disease prevention and control. Methods: Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit (OPU) analysis, we characterized the species-level microbial community structure of two important vector species, biting midges and ticks, including 33 arthropod samples comprising 3,885 individuals, collected around Poyang Lake. Results: A total of 662 OPUs were classified in biting midges, including 195 known species and 373 potentially new species, and 618 OPUs were classified in ticks, including 217 known species and 326 potentially new species. Surprisingly, OPUs with potentially pathogenicity were detected in both arthropod vectors, with 66 known species of biting midges reported to carry potential pathogens, including Asaia lannensis and Rickettsia bellii, compared to 50 in ticks, such as Acinetobacter lwoffii and Staphylococcus sciuri. We found that Proteobacteria was the most dominant group in both midges and ticks. Furthermore, the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria. Pantoea sp7 was predominant in biting midges, while Coxiella sp1 was enriched in ticks. Meanwhile, Coxiella spp., which may be essential for the survival of Haemaphysalis longicornis Neumann, were detected in all tick samples. The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors. Conclusion: Biting midges and ticks carry large numbers of known and potentially novel bacteria, and carry a wide range of potentially pathogenic bacteria, which may pose a risk of infection to humans and animals. The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

Background: Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence. Methods: We screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis. Results: Candidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study. Conclusions: The tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Coinfection of Dermacentor silvarum olenev (acari: ixodidae) by Coxiella-Like, Arsenophonus-like, and Rickettsia-like symbionts.

We report that multiple symbionts coexist in Dermacentor silvarum. Based on 16S rRNA gene sequence analyses, we prove that Coxiella-like and Arsenophonus-like symbionts, with 95.6% and 96.7% sequence similarity to symbionts in the closest taxon, respectively, are novel. Moreover, we also provide evidence that the Coxiella-like symbiont appears to be the primary symbiont.

Antimicrobial resistance gene lack in tick-borne pathogenic bacteria.

Tick-borne infections, including those of bacterial origin, are significant public health issues. Antimicrobial resistance (AMR), which is one of the most pressing health challenges of our time, is driven by specific genetic determinants, primarily by the antimicrobial resistance genes (ARGs) of bacteria. In our work, we investigated the occurrence of ARGs in the genomes of tick-borne bacterial species that can cause human infections. For this purpose, we processed short/long reads of 1550 bacterial isolates of the genera Anaplasma (n = 20), Bartonella (n = 131), Borrelia (n = 311), Coxiella (n = 73), Ehrlichia (n = 13), Francisella (n = 959) and Rickettsia (n = 43) generated by second/third generation sequencing that have been freely accessible at the NCBI SRA repository. From Francisella tularensis, 98.9% of the samples contained the FTU-1 beta-lactamase gene. However, it is part of the F. tularensis representative genome as well. Furthermore, 16.3% of them contained additional ARGs. Only 2.2% of isolates from other genera (Bartonella: 2, Coxiella: 8, Ehrlichia: 1, Rickettsia: 2) contained any ARG. We found that the odds of ARG occurrence in Coxiella samples were significantly higher in isolates related to farm animals than from other sources. Our results describe a surprising lack of ARGs in these bacteria and suggest that Coxiella species in farm animal settings could play a role in the spread of AMR.

Systemic Coxiella-like infection with myocarditis and hepatitis in an eclectus parrot (Eclectus roratus).

A multiorgan infection with a Coxiella-like organism was determined to be the cause of death of a female eclectus parrot (Eclectus roratus). The diagnosis was based on gross lesions, histopathology, Gimenez and Gram special stains, immunohistochemistry, electron microscopy, and polymerase chain reaction amplification and sequencing of a bacterial 16s rRNA gene fragment isolated from hepatic and cardiac tissue. Gross postmortem examination revealed multifocal to coalescing foci of hepatic necrosis. The most significant histologic lesions included multifocal lymphohistiocytic necrotizing hepatitis, locally extensive lymphoplasmacytic myocarditis, and myocardial degeneration and necrosis. Intralesional cytoplasmic organisms were identified in cardiomyocytes, biliary epithelium, and pancreatic exocrine cells. This is the first description of a Coxiella-like organism with wide-ranging cellular tropisms in a psittacine bird. In addition, lymphoplasmacytic neuritis, myositis, splenitis, airsacculitis, and enteritis were detected. It is also the first report of a Coxiella-like infection in an eclectus parrot.

Coxiella-like endosymbionts.

In the past two decades, many Coxiella-like bacteria have been found in hard ticks and soft ticks as well as in vertebrate hosts. It is interesting to note that many ticks harbor Coxiella-like bacteria with high prevalence. Coxiella-like bacteria and virulent Coxiella burnetii have high homology to each other; they form a monophyletic clade based on 16S rRNA sequence data and subsequent phylogenetic tree analyses. In this chapter, methods of detection, phylogeny, prevalence and density, distribution in tick organs, transmission routes, bacteria-host interactions, and putative functions of the Coxiella-like bacteria are reviewed.

Comparative Proteomics of Coxiella like Endosymbionts (CLEs) in the Symbiotic Organs of Rhipicephalus sanguineus Ticks.

Maternally transmitted obligatory endosymbionts are found in the female gonads as well as in somatic tissue and are expected to provide missing metabolite to their hosts. These deficiencies are presumably complemented through specific symbiotic microorganisms such as Coxiella-like endosymbionts (CLEs) of Rhipicephalus ticks. CLEs are localized in specialized host tissue cells within the Malpighian tubules (Mt) and the ovaries (Ov) from which they are maternally transmitted to developing oocytes. These two organs differ in function and cell types, but the role of CLEs in these tissues is unknown. To probe possible functions of CLEs, comparative proteomics was performed between Mt and Ov of R. sanguineus ticks. Altogether, a total of 580 and 614 CLE proteins were identified in Mt and Ov, respectively. Of these, 276 CLE proteins were more abundant in Mt, of which 12 were significantly differentially abundant. In Ov, 290 CLE proteins were more abundant, of which 16 were significantly differentially abundant. Gene Ontology analysis revealed that most of the proteins enriched in Mt are related to cellular metabolic functions and stress responses, whereas in Ov, the majority were related to cell proliferation suggesting CLEs function differentially and interdependently with host requirements specific to each organ. The results suggest Mt CLEs provide essential nutrients to its host and Ov CLEs promote proliferation and vertical transmission to tick progeny. IMPORTANCE Here we compare the Coxiella-like endosymbionts (CLEs) proteomes from Malpighian tubule (Mt) and the ovaries (Ov) of the brown dog tick Rhipicephalus sanguineus. Our results support the hypothesis that CLEs function interdependently with host requirements in each of the organs. The different functional specificity of CLE in the same host suggest that metabolic capabilities evolved according to the constrains imposed by the specific organ function and requirements. Our findings provide specific CLE protein targets that can be useful for future studies of CLE biology with a focus on tick population control.

Growth dynamics and tissue localization of a Coxiella-like endosymbiont in the tick Haemaphysalis longicornis.

A Coxiella-like endosymbiont (Coxiella-LE hereinafter) stably infects and influences Haemaphysalis longicornis development, indicating a mutualistic relationship of Coxiella-LE and ticks. To further elucidate the patterns of growth dynamics and tissue localization of Coxiella-LE in H. longicornis, 16S rRNA high-throughput sequencing, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) were used in this study. The density of Coxiella-LE varied among different tick life stages, and fed female ticks had the highest density, followed by unfed female and unfed larval ticks. In the four organs that were dissected from fed female ticks, the ovary carried the highest density of Coxiella-LE, which was significantly different from salivary glands, midgut and Malpighian tubules. The high abundance of Coxiella-LE in fed female ticks and in the ovaries of fed female ticks in the bacterial microbiota analyses further confirmed that Coxiella-LE rapidly proliferates in the ovary after blood feeding. The ovaries continued to develop after engorgement and oviposition began on day 5, with a significant decrease in the density of Coxiella-LE in the ovaries occurring on day 7. FISH results indicated that Coxiella-LE is mainly colonized in the cytoplasm of the oocyte and proliferates with oogenesis. Coxiella-LE was expelled from the body with the mature oocyte, ensuring its vertical transmission. In the Malpighian tubules at different days after engorgement, the white flocculent materials were increasing, and the density of Coxiella-LE raised significantly on day 7. Unlike the localization pattern in the ovary, Coxiella-LE was initially distributed in a mass and continually increased during the development of Malpighian tubules until it filled the Malpighian tubules. These findings provide new insights on the growth dynamics and tissue localization of Coxiella-LE in ticks and are useful for further investigation on the interactions of symbiont and ticks .