First report of Anaplasma spp., Ehrlichia spp., and Rickettsia spp. in Amblyomma gervaisi ticks infesting monitor lizards (Varanus begalensis) of Pakistan.

Ticks pose significant health risks to both wildlife and humans due to their role as vectors for various pathogens. In this study, we investigated tick infestation patterns, tick-associated pathogens, and genetic relationships within the tick species Amblyomma gervaisi, focusing on its prevalence in monitor lizards (Varanus bengalensis) across different districts in Pakistan. We examined 85 monitor lizards and identified an overall mean intensity of 19.59 ticks per infested lizard and an overall mean abundance of 11.98 ticks per examined lizard. All collected ticks (n = 1019) were morphologically identified as A. gervaisi, including 387 males, 258 females, 353 nymphs, and 21 larvae. The highest tick prevalence was observed in the Buner district, followed by Torghar and Shangla, with the lowest prevalence in Chitral. Lizard captures primarily occurred from May to October, correlating with the period of higher tick infestations. Molecular analysis was conducted on tick DNA, revealing genetic similarities among A. gervaisi ticks based on 16S rDNA and ITS2 sequences. Notably, we found the absence of A. gervaisi ITS2 sequences in the NCBI GenBank, highlighting a gap in existing genetic data. Moreover, our study identified the presence of pathogenic microorganisms, including Ehrlichia sp., Candidatus Ehrlichia dumleri, Anaplasma sp., Francisella sp., Rickettsia sp., and Coxiella sp., in these ticks. BLAST analysis revealed significant similarities between these pathogenic sequences and known strains, emphasizing the potential role of these ticks as vectors for zoonotic diseases. Phylogenetic analyses based on nuclear ITS2 and mitochondrial 16S rDNA genes illustrated the genetic relationships of A. gervaisi ticks from Pakistan with other Amblyomma species, providing insights into their evolutionary history. These findings contribute to our understanding of tick infestation patterns, and tick-borne pathogens in monitor lizards, which has implications for wildlife health, zoonotic disease transmission, and future conservation efforts. Further research in this area is crucial for a comprehensive assessment of the risks associated with tick-borne diseases in both wildlife and humans.

Tick-borne microorganisms in Amblyomma tigrinum (Acari: Ixodidae) from the Patagonian region of Argentina.

This study presents the results of the molecular detection of tick-borne microorganisms in Amblyomma tigrinum Koch collected near the city of Viedma, Río Negro, Argentina. Ticks were collected in their non-parasitic stage, on pet dogs and on Lycalopex gymnocercus (Pampa fox). Also, six tick samples from humans were analyzed. All ticks were morphologically identified to species level and genomic DNA was extracted. The DNA samples were examined by end point PCR assays to amplified DNA of Anaplasma sp., Babesia sp., Ehrlichia sp., Rickettsia sp. and Theileria sp. Although all tested DNA samples from the collected ticks resulted negative to the detection of Piroplasmida and Rickettsia spp., 16 samples (16.5%, including all hosts) were positive in the 16S rDNA gene PCR that detects bacteria from the Anaplasmataceae family. Phylogenetic analysis of seven obtained partial sequences resulted in the identification of three bacteria: two Ehrlichia spp. (related to Ehrlichia sp. strain Iberá and strain Viedma) and Candidatus Anaplasma boleense. The latter finding represents the first detection of this novel Candidatus species in A. tigrinum. Based on the results of this study, it must be assumed that the diversity of bacteria of the Anaplasmataceae family in Argentina is greater than previously thought, and that these bacteria can infect a wide range of domestic and wild animals.

Identification, structural modeling, gene expression analysis and RNAi effect of putative phospholipase A2 in the lone star tick Amblyomma americanum.

Amblyomma americanum, also known as the lone star tick, is a small arachnid that feeds on blood and can spread disease to humans and other animals. Despite the overlapped ecological niche, geographic distribution, and host selection, there is no proof that A. americanum transmits the pathogen Borrelia burgdorferi that causes Lyme disease. Studies have shown that phospholipase A2 (PLA2) may act as a tool to eliminate B. burgdorferi, but particular PLA2 genes in A. americanum have not been identified and functionally characterized. Using the de novo sequencing method, we identified 42 putative A. americanum PLA2 (pAaPLA2) homologs in the present study, of which three pAaPLA2 had calcium binding sites and canonical histidine catalytic sites. Then, we determined phylogenetic relationships, sequence alignments, and conserved protein motifs of these pAaPLA2s. Protein structural analysis demonstrated that pAaPLA2s primarily consisted of α-helices, ß-sheets, and random coils. These genes were predicted to be engaged in the phospholipid metabolic process, arachidonic acid secretion, and PLA2 activity by functional annotation analysis. A transcriptional factor (Bgb) was discovered that interacted with pAaPLA2 proteins that may have unrecognized roles in regulating neuronal development. Based on the RNA-seq data, we surveyed expression profiles of key pAaPLA2-related genes to reveal putative modulatory networks of these genes. RNAi knockdown of pAaPLA2_1, a dominant isoform in A. americanum, led to decreased bacterial inhibition ability, suggesting pAaPLA2 may play an important role in mediating immune responses. Collectively, this study provides essential evidence of the identification, gene structure, phylogeny, and expression analysis of pAaPLA2 genes in A. americanum, and offers a deeper understanding of the putative borreliacidal roles in the lone star tick.

Description of Amblyomma monteiroae n. sp. (Acari: Ixodidae), a parasite of the great horned owl (Strigiformes: Strigidae) in southern Brazil.

In 2020, adult hard ticks (males and females) were collected from great horned owls [Bubo virginianus (Gmelin, 1788)] in the coastal region in southern Brazil. The engorged females were allowed to oviposit in the laboratory and hatched larvae could be obtained. Analyses of the external morphology of the adult ticks revealed that they represent a new species, which was named Amblyomma monteiroae n. sp. Partial sequences of the mitochondrial 16S rRNA gene and the nuclear second internal transcribed spacer (ITS2) were generated from a male and a female. Their 16S rRNA haplotypes were identical to each other and closest (96% identity) to corresponding sequences of Amblyomma parvitarsum Neumann, 1901, and 90% identical to Amblyomma neumanni Ribaga, 1902. Their ITS2 haplotypes were 95.8 to 96.0 identical to the single ITS-2 partial sequence of A. parvitarsum available in GenBank. In the phylogenetic trees inferred by both 16S rRNA and ITS2 partial sequences, A. monteiroae n. sp. formed a clade with A. parvitarsum, with A. neumanni branching sister to this clade. Amblyomma monteiroae n. sp. is genetically and morphologically related to A. parvitarsum. Both tick species are unique in combining the following morphological characters: scutum extensively ornate; eyes rounded and bulging; coxa I with two moderate pointed spurs, the external longer than the internal; a single triangular short spur on coxae II-III; presence of two spines on the tibia of legs II-IV; hypostomal dentition 3/3, trochanters without spurs. However, the males of the two species can be separated by specific features in palps and festoons, whereas the females differ in specific features of the coxal spurs. The larva of A. monteiroae n. sp. can be morphologically distinguished from A. parvitarsum only by morphometry, with the former species being slightly smaller. Currently, A. monteiroae n. sp. is restricted to southern Brazil, and the only known host is B. virginianus (Strigiformes: Strigidae). The present study increases the Amblyomma Brazilian fauna to 34 species.

Detection of Bartonella DNA in Yellow Flies, Lone Star Ticks, and a Human Patient with Concurrent Evidence of Borrelia burgdorferi Infection in Northeast Florida, USA.

Background: Bartonella species and Borrelia burgdorferi sensu lato (Bbsl) are emerging zoonotic pathogens. The vectors and frequency of infections with both pathogen groups in the southern United States is understudied. This study describes an investigation of Bartonella and Bbsl in yellow flies collected at a residence in northeast Florida, USA, that led to subsequent discoveries of both organisms in lone star ticks (Amblyomma americanum) and a human patient. Materials and Methods: DNA samples from flies, ticks, and human patient blood specimens were tested via polymerase chain reaction assays for Bartonella or Bbsl species. DNA sequences were compared to reference strains for identification and characterization. Results: An exploratory investigation of arthropod-borne pathogens in yellow flies collected at a residence in northeast Florida revealed the presence of uncharacterized Bartonella species DNA sequences similar to ones previously detected in two lone star ticks from Virginia. Subsequent testing of several lone star ticks from the area detected similar sequences of Bartonella in three ticks. Testing of stored blood samples from a resident of the site, who had experienced chronic relapsing and remitting symptoms for over a decade, identified nearly identical Bartonella DNA sequences in multiple samples collected over a 10-year period. Two lone star ticks and several samples from the same patient and time period also tested positive for Bo. burgdorferi DNA, suggesting possible long-term coinfection of the patient with both organisms. Conclusion: This investigation identified highly similar Bartonella DNA sequences in yellow flies, lone star ticks, and a human patient in northeast Florida. Similarly, Bo. burgdorferi DNA was detected in two lone star ticks and multiple specimens from the patient. Positive PCR results from archived patient blood samples documented the presence of both organisms at multiple time points over more than a decade. More studies on human patients with chronic undefined illness and on the presence of Bartonella and Bbsl in hematophagous arthropods and animal hosts in the southeastern United States are needed.

Anaplasmataceae presence in Amblyomma calcaratum associated with anteaters (Tamandua tetradactyla) in the rainforest ecoregion, Argentina.

Bacteria of the sister genera Ehrlichia and Anaplasma (Anaplasmataceae) are obligate intracellular Alphaproteobacteria that are transmitted mostly through arthropod vectors. These agents can infect different vertebrate cells, depending on the species involved, and can cause diseases in animals and humans. In this study, we evaluated the presence of Anaplasmataceae bacteria in Amblyomma calcaratum ticks collected from a road-killed Tamandua tetradactyla in the Rainforest ecoregion in Argentina. All samples were screened for Anaplasmataceae DNA using a real-time PCR assay targeting the 16S rRNA gene. Evidence of Anaplasmataceae DNA was detected in three out of thirty-nine Am. calcaratum ticks. Phylogenetic analysis of a portion of 16S rRNA gene positioned one sample (Ehrlichia sp. strain Ac124) with Ehrlichia sequences and the other two samples with Anaplasma sequences; Anaplasma sp. strain Ac145 close to Anaplasma odocoilei and Anaplasma sp. strain Ac152 in an ancestral position to most Anaplasma species. The groEL sequence obtained showed that Ehrlichia sp. strain Ac124 was phylogenetically related to Ehrlichia sp. strain Iberá reported infecting Amblyomma tigrinum from Iberá wetlands in Argentina. Phylogenetic analysis using the rpoB sequence positioned Anaplasma sp. strain Ac145 close to the canine pathogen Anaplasma platys, while Anaplasma sp. strain Ac152 was positioned close to the bovine pathogen Anaplasma marginale. In this study, three Anaplasmataceae agents were detected in adults of Am. calcaratum associated with a T. tetradactyla. These results suggest that the number of Anaplasmataceae species, as well as their distribution, is largely unknown.

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (Acari: Ixodidae).

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens.

Detection of Rickettsia tamurae-like and other spotted fever group rickettsiae in ticks (Acari: Ixodidae) associated with wild birds in the Western Amazon, Brazil.

Ticks are vectors for several pathogens, including bacteria belonging to the Rickettsia genus, such as Rickettsia rickettsii and Rickettsia parkeri, the causative agents of spotted fever. The aim of the present study was to investigate the tick species richness and rickettsial agents associated with wild birds captured in the Humaita Forest Reserve, Acre, in the Western Amazon region. Wild birds were captured with ornithological nets for visual inspection with the purpose of collecting ticks, which were identified through morphological analyses and molecular tests for several genes (12S rDNA, 16S rDNA, gltA, ompA, and sca4). A total of 607 wild birds were captured, 12% of which were parasitized by 268 ticks of the Amblyomma genus, with new host-parasite associations reported for Amblyomma calcaratum, Amblyomma geayi, Amblyomma longirostre, Amblyomma naponense, Amblyomma nodosum, and Amblyomma varium. Of the total ticks collected, 113 were tested for the presence of rickettsial DNA fragments, with 19 testing positive for R. parkeri in A. geayi, Rickettsia tamurae-like in Amblyomma sp., and Rickettsia amblyommatis in A. geayi, A. longirostre, and Amblyomma sp. We detected R. tamurae-like in Amblyomma larvae for the first time in the Western Brazilian Amazon biome, and registered spotted fever group rickettsiae, although the relevance of the detected species in a public health context should be further explored in South America, as well as new host-parasite interactions in this underexplored region.

First molecular evidence of Wolbachia occurrence in Amblyomma sculptum (Acari: Ixodidae).

As the main vector for the bacterium Rickettsia rickettsii in Brazil, the tick Amblyomma sculptum is a parasite of great public health importance in this country. Wolbachia is an endosymbiont bacterium highly widespread among invertebrates and because of its impact on its hosts' biology, form a powerful alternative for pests and disease control. The aim of this study was to investigate the occurrence of this bacterium in A. sculptum. For this, 187 adult ticks collected in two municipalities in the interior of the state of São Paulo, Brazil, were analyzed using molecular techniques and bioinformatics tools. A total of 15 ticks were positive for the presence of Wolbachia. Phylogenetic analysis on the 16S rRNA gene indicated that the Wolbachia DNA sequences obtained in this investigation belonged to different clades, probably in supergroups B and F. This was the first study to report the occurrence of Wolbachia in A. sculptum and it enriches knowledge about the susceptibility of ticks to this bacterium. Now that we know that Wolbachia can be found in A. sculptum, the objective for a next study must be to investigate Wolbachia's possible origin in this tick.

Phylogenetic relationships of the Amblyomma cajennense complex (Acari: Ixodidae) at mitogenomic resolution.

The genus Amblyomma is the third most diverse in the number of species within the Ixodidae, with practically half of its species distributed in the Americas, though there are also species occurring in Africa, Asia, and Australia. Within the genus, there are several species complexes with veterinary and public health importance. The Amblyomma cajennense complex, in the Americas, is represented by six species with a wide distribution, from Texas to northern Argentina. We combined two sequencing techniques to generate complete mitogenomes of species belonging to the Amblyomma cajennense complex: genome skimming and long-range PCRs sequencing methods. Thus, we generated seven new mitochondrial genomes for all species of the Amblyomma cajennense complex, except for Amblyomma interandinum. Genetic distances between the mitogenomes corroborate the clear differentiation between the five species of the Amblyomma cajennense complex. The phylogenetic relationships of these species had previously been evaluated by combining partial nuclear and mitochondrial genes and here these relationships are corroborated with a more robust framework of data, which demonstrates that the conjunction of mitochondrial and nuclear partial genes can resolve close relationships when entire genes or genomes are unavailable. The gene order, structure, composition, and length are stable across these mitogenomes, and they share the general characteristics of Metastriata. Future studies should increase the number of available mitogenomes for this genus, especially for those species from the Indo-Pacific region and Africa, by means of a better understanding of their relationships and evolutionary process.

A draft of the genome of the Gulf Coast tick, Amblyomma maculatum.

The Gulf Coast tick, Amblyomma maculatum, inhabits the Southeastern states of the USA bordering the Gulf of Mexico, Mexico, and other Central and South American countries. More recently, its U.S. range has extended West to Arizona and Northeast to New York state and Connecticut. It is a vector of Rickettsia parkeri and Hepatozoon americanum. This tick species has become a model to study tick/Rickettsia interactions. To increase our knowledge of the basic biology of A. maculatum we report here a draft genome of this tick and an extensive functional classification of its proteome. The DNA from a single male tick was used as a genomic source, and a 10X genomics protocol determined 28,460 scaffolds having equal or more than 10 Kb, totaling 1.98 Gb. The N50 scaffold size was 19,849 Kb. The BRAKER pipeline was used to find the protein-coding gene boundaries on the assembled A. maculatum genome, discovering 237,921 CDS. After trimming and classifying the transposable elements, bacterial contaminants, and truncated genes, a set of 25,702 were annotated and classified as the core gene products. A BUSCO analysis revealed 83.4% complete BUSCOs. A hyperlinked spreadsheet is provided, allowing browsing of the individual gene products and their matches to several databases.

Seventy-eight entire mitochondrial genomes and nuclear rRNA genes provide insight into the phylogeny of the hard ticks, particularly the Haemaphysalis species, Africaniella transversale and Robertsicus elaphensis.

Hoogstraal and Kim (1985) proposed from morphology, three groups of Haemaphysalis subgenera: (i) the "structurally advanced"; (ii) the "structurally intermediate"; and (iii) the "structurally primitive" subgenera. Nuclear gene phylogenies, however, did not indicate monophyly of these morphological groups but alas, only two mitochondrial (mt) genomes from the "structurally intermediate" subgenera had been sequenced. The phylogeny of Haemaphysalis has not yet been resolved. We aimed to resolve the phylogeny of the genus Haemaphysalis, with respect to the subgenus Alloceraea. We presented 15 newly sequenced and annotated mt genomes from 15 species of ticks, five species of which have not been sequenced before, and four new 18S rRNA and 28S rRNA nuclear gene sequences. Our datasets were constructed from 10 mt protein-coding genes, cox1, and the 18S and 28S nuclear rRNA genes. We found a 132-bp insertion between tRNA-Glu (E) gene and the nad1 gene in the mt genome of Haemaphysalis (Alloceraea) inermis that resembles insertions in H. (Alloceraea) kitaokai and Rhipicephalus (Boophilus) geigyi. Our mt phylogenies had the three species of Amblyomma (Aponomma) we sequenced embedded in the main clade of Amblyomma: Am. (Aponomma) fimbriatum, Am. (Aponomma) gervaisi and Am. (Aponomma) latum. This is further support for the hypothesis that the evolution of eyes appears to have occurred in the most-recent-common-ancestor of Amblyocephalus (i.e. Amblyomminae plus Rhipicephalinae) and that eyes were subsequently lost in the most-recent-common-ancestor of the subgenus Am. (Aponomma). Either Africaniella transversale or Robertsicus elaphensis, or perhaps Af. transversale plus Ro. elaphensis, appear to be the sister-group to the rest of the metastriate Ixodida. Our cox1 phylogenies did not indicate monophyly of the "structurally primitive", "structurally intermediate" nor the "structurally advanced" groups of Haemaphysalis subgenera. Indeed, the subgenus Alloceraea may be the only monophyletic subgenus of the genus Haemaphysalis sequenced thus far. All of our mt genome and cox1 phylogenies had the subgenus Alloceraea in a clade that was separate from the rest of the Haemaphysalis ticks. If Alloceraea is indeed the sister to the rest of the Haemaphysalis subgenera this would resonate with the argument of Hoogstraal and Kim (1985), that Alloceraea was a subgenus of "primitive" Haemaphysalis. Alectorobius capensis from Japan had a higher genetic-identity to A. sawaii, which was also from Japan, than to the A. capensis from South Africa. This indicates that A. capensis from Japan may be a cryptic species with respect to the A. capensis from South Africa.

Evidence of the presence of Borrelia burgdorferi in dogs and associated ticks in Egypt.

BACKGROUND: Borrelia burgdorferi is the spirochete that causes Lyme Borreliosis (LB), which is a zoonotic tick-borne disease of humans and domestic animals. Hard ticks are obligate haematophagous ectoparasites that serve as vectors of Borrelia burgdorferi. Studies on the presence of Lyme borreliosis in Egyptian animals and associated ticks are scarce. METHODS: This study was conducted to detect B. burgdorferi in different tick vectors and animal hosts. Three hundred animals (dogs=100, cattle=100, and camels=100) were inspected for tick infestation. Blood samples from 160 tick-infested animals and their associated ticks (n=1025) were collected and examined for the infection with B. burgdorferi by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene. The identified tick species were characterized molecularly by PCR and sequencing of the ITS2 region. RESULTS: The overall tick infestation rate among examined animals was 78.33% (235/300). The rate of infestation was significantly higher in camels (90%), followed by cattle (76%) and dogs (69%); (P = 0.001). Rhipicephalus sanguineus, Rhipicephalus (Boophilus) annulatus, and both Hyalomma dromedarii and Amblyomma variegatum, were morphologically identified from infested dogs, cattle, and camels; respectively. Molecular characterization of ticks using the ITS2 region confirmed the morphological identification, as well as displayed high similarities of R. sanguineus, H. dromedarii, and A. Variegatu with ticks identified in Egypt and various continents worldwide. Just one dog (1.67%) and its associated tick pool of R. sanguineus were positive for B. burgdorferi infection. The 16S rRNA gene sequence for B. burgdorferi in dog and R. sanguineus tick pool showed a 100% homology. CONCLUSION: Analyzed data revealed a relatively low rate of B. burgdorferi infection, but a significantly high prevalence of tick infestation among domesticated animals in Egypt, which possesses a potential animal and public health risk. Additionally, molecular characterization of ticks using the ITS2 region was a reliable tool to discriminate species of ticks and confirmed the morphological identification.

Phylogenies from mitochondrial genomes of 120 species of ticks: Insights into the evolution of the families of ticks and of the genus Amblyomma.

The evolution and phylogenetic relationships of the ticks at both the family and genus levels are contested. The genus Amblyomma and its subgenera are in a state of flux; moreover, the relationships among the three tick families are controversial due to conflicting phylogenetic support for different arrangements of the three families of living ticks. With 18 newly sequenced mitochondrial (mt) genomes of ticks included, we executed the largest mt genome phylogenetic study of ticks so far. Phylogenetic trees were inferred from one sea spider mt genome, one horseshoe crab, five mite mt genomes and 146 tick mt genomes from 120 species: 153 mt genomes in total. Sixteen phylogenetic trees were inferred from 10 datasets using both maximum likelihood and Bayesian inference methods. We describe the first novel mt gene-arrangement for the metastriate Ixodidae in Amblyomma (Africaniella) transversale. Also, three unusual partial 16S rRNA gene inserts were found in the mt genome of Haemaphysalis (Alloceraea) kitaokai: we consider the possible role of past genome translocation events in the formation of these inserts. Our phylogenies revealed evidence that: (i) the genus Amblyomma is polyphyletic with respect to Amblyomma (Africaniella) transversale; (ii) the subgenus Aponomma is apparently embedded in the genus Amblyomma; (iii) Haemaphysalis (Segalia) parva and Haemaphysalis (Alloceraea) kitaokai form a clade to the exclusion of other Haemaphysalis species; and (iv) the phylogenetic position of the family Nuttalliellidae is unstable among phylogenies from different datasets.

First detection of Amblyomma variegatum and molecular finding of Rickettsia africae in Sardinia, Italy.

Here we present the first detection of a male Amblyomma variegatum tick infesting a sheep on the island of Sardinia, as well as the detection of a pathogen, Rickettsia africae, in DNA extracted from this tick. The tick, the second individual of this species reported in Italy (the first one was reported in Sicily by Albanese in 1971) was collected in August 2018 from the inguinal region of an adult female sheep in a farm located near Sassari (North-West Sardinia). The tick was identified as an adult A. variegatum male under a stereomicroscope using morphological keys. A phylogenetic analysis showed that the 12S sequence clustered with that of African A. variegatum individuals and was embedded within the previously identified West African group. We tested the tick for the presence of microorganisms of the genera Ehrlichia, Rickettsia, Anaplasma, Theileria and Babesia, using published PCR protocols. The tick was found positive to Rickettsia and the obtained sequence matched at 100 % identity with R. africae. The area where the tick was detected was inspected on multiple occasions, looking for other specimens of A. variegatum, without any results. In the same period another male specimen of A. variegatum was found in Haute Corse in 2019. The authors' hypothesis is that the presence of the A. variegatum specimen is an occasional finding, probably linked to the migrating birds that cross Sardinia and Corsica from Africa during summer. Although this may have been an incidental finding, it must be considered that global warming could increase the risk of establishment of colonies of these ticks, that show a strong spreading capability. It is also important to emphasize that this tick species is a proven vector and reservoir of R. africae, an uncommon zoonotic pathogen in Italy, thus additional monitoring must be performed as the establishment of a stable population in Sardinia could represent a serious veterinary and public health issue.

The sialotranscriptome of the gopher-tortoise tick, Amblyomma tuberculatum.

The gopher tortoise tick, Amblyomma tuberculatum, is known to parasitize keystone ectotherm reptile species. The biological success of ticks requires precise mechanisms to evade host hemostatic and immune responses. Acquisition of a full blood meal requires attachment, establishment of the blood pool, and engorgement of the tick. Tick saliva contains molecules which counter the host responses to allow uninterrupted feeding on the host. RNASeq of the salivary glands of Amblyomma tuberculatum ticks were sequenced resulting in 138,030 pyrosequencing reads which were assembled into 29,991 contigs. A total of 1875 coding sequences were deduced from the transcriptome assembly, including 602 putative secretory and 982 putative housekeeping proteins. The annotated data sets are available as a hyperlinked spreadsheet. The sialotranscriptome assembled for this tick species made available a valuable resource for mining novel pharmacological activities and comparative analysis.

Sequence of a Coxiella Endosymbiont of the Tick Amblyomma nuttalli Suggests a Pattern of Convergent Genome Reduction in the Coxiella Genus.

Ticks require bacterial symbionts for the provision of necessary compounds that are absent in their hematophagous diet. Such symbionts are frequently vertically transmitted and, most commonly, belong to the Coxiella genus, which also includes the human pathogen Coxiella burnetii. This genus can be divided in four main clades, presenting partial but incomplete cocladogenesis with the tick hosts. Here, we report the genome sequence of a novel Coxiella, endosymbiont of the African tick Amblyomma nuttalli, and the ensuing comparative analyses. Its size (∼1 Mb) is intermediate between symbionts of Rhipicephalus species and other Amblyomma species. Phylogenetic analyses show that the novel sequence is the first genome of the B clade, the only one for which no genomes were previously available. Accordingly, it allows to draw an enhanced scenario of the evolution of the genus, one of parallel genome reduction of different endosymbiont lineages, which are now at different stages of reduction from a more versatile ancestor. Gene content comparison allows to infer that the ancestor could be reminiscent of C. burnetii. Interestingly, the convergent loss of mismatch repair could have been a major driver of such reductive evolution. Predicted metabolic profiles are rather homogenous among Coxiella endosymbionts, in particular vitamin biosynthesis, consistently with a host-supportive role. Concurrently, similarities among Coxiella endosymbionts according to host genus and despite phylogenetic unrelatedness hint at possible host-dependent effects.

Comparative population genetics of Amblyomma maculatum and Amblyomma americanum in the mid-Atlantic United States.

The Gulf Coast tick, Amblyomma maculatum, is undergoing a northward expansion along the United States East Coast, most recently establishing populations in Virginia, Maryland, and Delaware. This expansion has human health implications, as A. maculatum is the primary natural vector of the bacterium Rickettsia parkeri, which causes a spotted fever-type rickettsiosis. Newly established populations of A. maculatum in Virginia tend to have high prevalence of R. parkeri, compared to lower infection rates in the historical range. The factors contributing to high R. parkeri prevalence in Virginia are not known. Investigating connectivity between sites colonized with A. maculatum can help determine whether sites with higher prevalence are isolated or well-connected through migration, thus serving as a source of infected individuals. We characterized 16S rRNA haplotypes of A. maculatum and, for comparison, the congeneric Amblyomma americanum collected from sites where these species co-occur. We then explored connectivity and genetic structure among Virginia populations using pairwise ΦST and AMOVA analyses. Our study identified one recently restored native grassland site with low A. maculatum haplotype diversity and strong evidence of a founder effect, whereas most sites are haplotypically diverse but with no clear genetic structure or connectivity between sites. These findings contrast with high connectivity and a slight mainland/island structure among A. americanum populations. Our results suggest that A. maculatum populations occasionally arise following long-distance drop-offs of few individual ticks in suitable habitat, but no clear migration patterns were observed. The distinct population genetic patterns between species might result from differences in host utilization.

Morphological and molecular characterization supporting Amblyomma mixtum presence in Cuba.

Amblyomma cajennense Fabricius, 1787 (Acari: Ixodidae) is a widely distributed tick taxon. Recent studies have reassessed this taxon as a complex of six species. Amblyomma mixtum Koch, 1844 has been suggested by some authors as the only species of this complex that is present in Cuba. Other authors have pointed a niche overlapping for A. mixtum and A. cajennense s.s. in the country. Detailed taxonomic studies on the Cuban species belonging to this complex are needed in order to evaluate their current distribution according to the recent classification. This study aimed to characterize Cuban populations from the A. cajennense complex by using tick samples obtained from 3 occidental provinces and 1 central province of the country. Morphological identification and measurements of the main relevant taxonomic structures were conducted by using Scanning Electron Microscopy. Phylogenetic analyzes were carried out with 16S ribosomal RNA, internal transcribed spacer 2 and the subunit I of mitochondrial cytochrome c oxidase gene sequences. The results of these studies demonstrated that all samples belonged to the species A. mixtum (Koch, 1844). This study constitutes the first molecular characterization of this Amblyomma species in Cuba. Further studies will be necessary in order to corroborate if A. cajennense s.s. is also present in the island.

Molecular phylogeny of Amblyomma exornatum and Amblyomma transversale, with reinstatement of the genus Africaniella (Acari: Ixodidae) for the latter.

The phylogeny of the hard tick genus Amblyomma Koch, 1844 deserves special attention, because several poorly studied tick species associated with reptiles still bear the name of this genus, although they may not belong to it. This study focuses on the phylogeny of two such species with uncertain taxonomic status, i.e., Amblyomma transversale (Lucas, 1845) and Amblyomma exornatum Koch, 1844, analyzing two mitochondrial (cytochrome c oxidase subunit 1 and 16S rRNA) and two nuclear (18S and 28S rRNA) genes. In the cox1 phylogenetic analysis, both Am. transversale and Am. exornatum were part of a sister group to all other Metastriata, whereas in the 16S rRNA gene analysis, Am. transversale belonged to a sister group to three subfamilies (Amblyomminae Neumann, 1911; Haemaphysalinae Hoogstraal and Aeschlimann, 1982; Bothriocrotoninae Klompen, Dobson and Baker, 2002), and Am. exornatum formed a sister group to other Amblyomminae. However, based on the 18S and 28S rRNA genes, Am. transversale belonged to a sister group of either Bothriocrotoninae alone or of both Bothriocrotoninae and Haemaphysalinae, respectively. In the latter two phylogenetic analyses Am. exornatum always clustered within Amblyomminae. Morphological comparisons revealed that Am. transversale has at least four unique characters and shares a high number of traits with the genera Robertsicus Barker and Burger, 2018 and Archaeocroton Barker and Burger, 2018, as well as with the subgenus Alloceraea Schulze, 1918 (represented by Haemaphysalis inermis Birula, 1895). These results justify that the genus Africaniella Travassos Dias, 1974 should be reinstated, and the species name of Am. transversale should be used as Africaniella transversale (Lucas, 1845).