Genomic signatures of climate adaptation in bank voles.

Evidence for divergent selection and adaptive variation across the landscape can provide insight into a species' ability to adapt to different environments. However, despite recent advances in genomics, it remains difficult to detect the footprints of climate-mediated selection in natural populations. Here, we analysed ddRAD sequencing data (21,892 SNPs) in conjunction with geographic climate variation to search for signatures of adaptive differentiation in twelve populations of the bank vole (Clethrionomys glareolus) distributed across Europe. To identify the loci subject to selection associated with climate variation, we applied multiple genotype-environment association methods, two univariate and one multivariate, and controlled for the effect of population structure. In total, we identified 213 candidate loci for adaptation, 74 of which were located within genes. In particular, we identified signatures of selection in candidate genes with functions related to lipid metabolism and the immune system. Using the results of redundancy analysis, we demonstrated that population history and climate have joint effects on the genetic variation in the pan-European metapopulation. Furthermore, by examining only candidate loci, we found that annual mean temperature is an important factor shaping adaptive genetic variation in the bank vole. By combining landscape genomic approaches, our study sheds light on genome-wide adaptive differentiation and the spatial distribution of variants underlying adaptive variation influenced by local climate in bank voles.

Molecular-phylogenetic analyses of Ixodes species from South Africa suggest an African origin of bird-associated exophilic ticks (subgenus Trichotoixodes).

BACKGROUND: Among hard ticks (Acari: Ixodidae), the genus Ixodes comprises the highest number of species, which in turn are most numerous in the Afrotropical zoogeographic region. In South Africa extensive morphological studies have been performed on Ixodes species but only few reports included molecular analyses. METHODS: In this study, 58 Ixodes spp. ticks, collected from ten mammalian and eight avian host species in South Africa, were molecularly and phylogenetically analyzed. In addition, a newly collected sample of the Palearctic Ixodes trianguliceps was included in the analyses. RESULTS: Among the ticks from South Africa, 11 species were identified morphologically. The majority of ticks from mammals represented the Ixodes pilosus group with two species (n = 20), followed by ticks resembling Ixodes rubicundus (n = 18) and Ixodes alluaudi (n = 3). In addition, single specimens of Ixodes rhabdomysae, Ixodes ugandanus, Ixodes nairobiensis and Ixodes simplex were also found. Considering bird-infesting ticks, Ixodes theilerae (n = 7), Ixodes uriae (n = 4) and ticks most similar to Ixodes daveyi (provisionally named I. cf. daveyi, n = 2) were identified. Molecular analyses confirmed two species in the I. pilosus group and a new species (I. cf. rubicundus) closely related to I. rubicundus sensu stricto. Phylogenetic trees based on concatenated mitochondrial or mitochondrial and nuclear gene sequences indicated that the subgenus Afrixodes forms a monophyletic clade with bird-associated exophilic ticks (subgenus Trichotoixodes). Ixodes trianguliceps clustered separately whereas I. alluaudi with their morphologically assigned subgenus, Exopalpiger. CONCLUSIONS: Phylogenetic analyses shed new lights on the relationships of Ixodes subgenera when including multiple sequences from subgenus Afrixodes and African as well as Palearctic species of subgenera Trichotoixodes and Exopalpiger. Subgenera Afrixodes and bird-associated Trichotoixodes share common ancestry, suggesting that the latter might have also originated in Africa. Regarding the subgenus Exopalpiger, I. alluaudi is properly assigned as it clusters among different Australian Ixodes, whereas I. trianguliceps should be excluded.

Three-dimensional images reveal the impact of the endosymbiont Midichloria mitochondrii on the host mitochondria.

The hard tick, Ixodes ricinus, a main Lyme disease vector, harbors an intracellular bacterial endosymbiont. Midichloria mitochondrii is maternally inherited and resides in the mitochondria of I. ricinus oocytes, but the consequences of this endosymbiosis are not well understood. Here, we provide 3D images of wild-type and aposymbiotic I. ricinus oocytes generated with focused ion beam-scanning electron microscopy. Quantitative image analyses of endosymbionts and oocyte mitochondria at different maturation stages show that the populations of both mitochondrion-associated bacteria and bacterium-hosting mitochondria increase upon vitellogenisation, and that mitochondria can host multiple bacteria in later stages. Three-dimensional reconstructions show symbiosis-dependent morphologies of mitochondria and demonstrate complete M. mitochondrii inclusion inside a mitochondrion. Cytoplasmic endosymbiont located close to mitochondria are not oriented towards the mitochondria, suggesting that bacterial recolonization is unlikely. We further demonstrate individual globular-shaped mitochondria in the wild type oocytes, while aposymbiotic oocytes only contain a mitochondrial network. In summary, our study suggests that M. mitochondrii modulates mitochondrial fragmentation in oogenesis possibly affecting organelle function and ensuring its presence over generations.

The evolution of intramitochondriality in Midichloria bacteria.

Midichloria spp. are intracellular bacterial symbionts of ticks. Representatives of this genus colonise mitochondria in the cells of their hosts. To shed light on this unique interaction we evaluated the presence of an intramitochondrial localization for three Midichloria in the respective tick host species and generated eight high-quality draft genomes and one closed genome, showing that this trait is non-monophyletic, either due to losses or multiple acquisitions. Comparative genomics supports the first hypothesis, as the genomes of non-mitochondrial symbionts are reduced subsets of those capable of colonising the organelles. We detect genomic signatures of mitochondrial tropism, including the differential presence of type IV secretion system and flagellum, which could allow the secretion of unique effectors and/or direct interaction with mitochondria. Other genes, including adhesion molecules, proteins involved in actin polymerisation, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, to fuse with the organelles or manipulate the mitochondrial network.

Phylogenetic analyses of Ixodes rugicollis with notes on its morphology in comparison with Ixodes cornutus.

BACKGROUND: The subgenus Pholeoixodes contains Ixodes species typically associated with birds that nest in cavities or with carnivorous mammals that are burrow-dwelling. Among ticks infesting the latter, Ixodes rugicollis is regarded as the rarest species in the western Palearctic. Despite the unique morphology of this species, its identification (especially of subadult stages) is difficult, and molecular-phylogenetic data to offer other diagnostic methods and a better understanding of its taxonomy are not available. METHODS: In this study, a female and a male of I. rugicollis were collected in Romania. The female was compared morphologically to another female of this species collected in France and to the lectotype of Ixodes cornutus (from Tajikistan), which has similar morphology and host association. Following DNA extraction, two mitochondrial (cytochrome c oxidase subunit I: cox1 and the 16S rRNA gene) and two nuclear genetic markers (18S and 28S rRNA genes) of I. rugicollis were amplified and analyzed in a phylogenetic context. RESULTS: Females of I. rugicollis and I. cornutus differed in the shape of their palps, scutum and areae porosae and the size of peritremes, but they were similar in palpal setal length, dental formula and arrangement of anal setae. Measurements of two I. rugicollis females examined were not less different from each other than from I. cornutus. Phylogenetically, I. rugicollis clustered with other members of its subgenus. The topology of all trees showed the position of bat-associated tick species of the subgenus Eschatocephalus among Pholeoixodes species. CONCLUSIONS: For the first time to our knowledge, this study provides high-resolution digital pictures of male and female I. rugicollis as well as corresponding molecular data. Morphological comparison of this species with I. cornutus could not resolve uncertainties in the validity of the latter species, which can only be accomplished after collecting new specimens of I. cornutus and consequent molecular comparisons. This study includes the first comprehensive molecular-phylogenetic analysis of western Palearctic Pholeoixodes species based on both nuclear and mitochondrial genetic markers and including I. rugicollis. The results of these confirm the phylogenetic position of subgenus Eschatocephalus within Pholeoixodes, justifying the need to merge them to comply with the taxonomic criterion of monophyly.

Phylogenetic evidence for a clade of tick-associated trypanosomes.

BACKGROUND: Trypanosomes are protozoan parasites of vertebrates that are of medical and veterinary concern. A variety of blood-feeding invertebrates have been identified as vectors, but the role of ticks in trypanosome transmission remains unclear. METHODS: In this study, we undertook extensive molecular screening for the presence and genetic diversity of trypanosomes in field ticks. RESULTS: Examination of 1089 specimens belonging to 28 tick species from Europe and South America led to the identification of two new trypanosome strains. The prevalence may be as high as 4% in tick species such as the castor bean tick Ixodes ricinus, but we found no evidence of transovarial transmission. Further phylogenetic analyses based on 18S rRNA, EF1-α, hsp60 and hsp85 gene sequences revealed that different tick species, originating from different continents, often harbour phylogenetically related trypanosome strains and species. Most tick-associated trypanosomes cluster in a monophyletic clade, the Trypanosoma pestanai clade, distinct from clades of trypanosomes associated with transmission by other blood-feeding invertebrates. CONCLUSIONS: These observations suggest that ticks may be specific arthropod hosts for trypanosomes of the T. pestanai clade. Phylogenetic analyses provide further evidence that ticks may transmit these trypanosomes to a diversity of mammal species (including placental and marsupial species) on most continents.

Effect of the combination of DEET and flupyradifurone on the tick Ixodes ricinus: Repellency bioassay and pharmacological characterization using microtransplantation of synganglion membranes.

Ticks are vectors of many human and animal pathogens, and represent a major threat to public health. In recent years, an increase in tick-borne diseases has been observed, and new strategies are therefore needed in order to control tick numbers and reduce human tick bites. In the present study, we adapted the previous tick repellency bioassay based on the exploration behavior of the tick, using the ToxTrac software and video-tracking, to compare the repellent effect of two compounds on the tick Ixodes ricinus: N,N-diethyl-methyl-m-toluamide (DEET), and butenolide, flupyradifurone (FLU). We found that when applied alone, 10% DEET or FLU have no/or low repellency effect. But, the combination of both 10% DEET and FLU demonstrated a significant repellency effect against I. ricinus, similar to the repellency of 20% DEET. Using membrane microtransplantation, we evaluated the effect of DEET and FLU on native acetylcholine receptors expressed on the tick synganglion. We found that DEET has no effect on acetylcholine-evoked currents, but significantly reduced nicotine-induced current amplitudes. FLU induced an ionic current but was not able to reduce acetylcholine or nicotine evoked currents. The combination of both DEET and FLU strongly reduced nicotine-evoked currents. Finally, we demonstrated that our recording device for repellency, as well as the use of membrane microtransplantation, could be used as methods to study the mode of action of active compounds on ticks.

Transcriptome of the synganglion in the tick Ixodes ricinus and evolution of the cys-loop ligand-gated ion channel family in ticks.

BACKGROUND: Ticks represent a major health issue for humans and domesticated animals. Exploring the expression landscape of the tick's central nervous system (CNS), known as the synganglion, would be an important step in understanding tick physiology and in managing tick-borne diseases, but studies on that topic are still relatively scarce. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs, or cysLGICs) are important pharmacological targets of acaricides. To date their sequence have not been well catalogued for ticks, and their phylogeny has not been fully studied. RESULTS: We carried out the sequencing of transcriptomes of the I. ricinus synganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). The de novo assembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were associated with neurotransmission and/or localized in neurons or the synaptic membrane. As the first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homolog, and functionally reconstituted the first GABA-gated receptor of Ixodes ricinus. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and other cys-loop LGICs respectively, revealing tick-specific expansions of some types of receptors (especially for Histamine-like subunits and GluCls). CONCLUSIONS: We established a large catalogue of genes preferentially expressed in the tick CNS, including the cysLGICs. We discovered tick-specific gene family expansion of some types of cysLGIC receptors, and a case of intragenic duplication, suggesting a complex pattern of gene expression among different copies or different alternative transcripts of tick neuro-receptors.

Meteorological and climatic variables predict the phenology of Ixodes ricinus nymph activity in France, accounting for habitat heterogeneity.

Ixodes ricinus ticks (Acari: Ixodidae) are the most important vector for Lyme borreliosis in Europe. As climate change might affect their distributions and activities, this study aimed to determine the effects of environmental factors, i.e., meteorological, bioclimatic, and habitat characteristics on host-seeking (questing) activity of I. ricinus nymphs, an important stage in disease transmissions, across diverse climatic types in France over 8 years. Questing activity was observed using a repeated removal sampling with a cloth-dragging technique in 11 sampling sites from 7 tick observatories from 2014 to 2021 at approximately 1-month intervals, involving 631 sampling campaigns. Three phenological patterns were observed, potentially following a climatic gradient. The mixed-effects negative binomial regression revealed that observed nymph counts were driven by different interval-average meteorological variables, including 1-month moving average temperature, previous 3-to-6-month moving average temperature, and 6-month moving average minimum relative humidity. The interaction effects indicated that the phenology in colder climates peaked differently from that of warmer climates. Also, land cover characteristics that support the highest baseline abundance were moderate forest fragmentation with transition borders with agricultural areas. Finally, our model could potentially be used to predict seasonal human-tick exposure risks in France that could contribute to mitigating Lyme borreliosis risk.

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

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

Borrelia afzelii Infection in the Rodent Host Has Dramatic Effects on the Bacterial Microbiome of Ixodes ricinus Ticks.

The microbiome of blood-sucking arthropods can shape their competence to acquire and maintain infections with vector-borne pathogens. We used a controlled study to investigate the interactions between Borrelia afzelii, which causes Lyme borreliosis in Europe, and the bacterial microbiome of Ixodes ricinus, its primary tick vector. We applied a surface sterilization treatment to I. ricinus eggs to produce dysbiosed tick larvae that had a low bacterial abundance and a changed bacterial microbiome compared to those of the control larvae. Dysbiosed and control larvae fed on B. afzelii-infected mice and uninfected control mice, and the engorged larvae were left to molt into nymphs. The nymphs were tested for B. afzelii infection, and their bacterial microbiome underwent 16S rRNA amplicon sequencing. Surprisingly, larval dysbiosis had no effect on the vector competence of I. ricinus for B. afzelii, as the nymphal infection prevalence and the nymphal spirochete load were the same between the dysbiosed group and the control group. The strong effect of egg surface sterilization on the tick bacterial microbiome largely disappeared once the larvae molted into nymphs. The most important determinant of the bacterial microbiome of I. ricinus nymphs was the B. afzelii infection status of the mouse on which the nymphs had fed as larvae. Nymphs that had taken their larval blood meal from an infected mouse had a less abundant but more diverse bacterial microbiome than the control nymphs. Our study demonstrates that vector-borne infections in the vertebrate host shape the microbiome of the arthropod vector. IMPORTANCE Many blood-sucking arthropods transmit pathogens that cause infectious disease. For example, Ixodes ricinus ticks transmit the bacterium Borrelia afzelii, which causes Lyme disease in humans. Ticks also have a microbiome, which can influence their ability to acquire and transmit tick-borne pathogens such as B. afzelii. We sterilized I. ricinus eggs with bleach, and the tick larvae that hatched from these eggs had a dramatically reduced and changed bacterial microbiome compared to that of control larvae. These larvae fed on B. afzelii-infected mice, and the resultant nymphs were tested for B. afzelii and for their bacterial microbiome. We found that our manipulation of the bacterial microbiome had no effect on the ability of the tick larvae to acquire and maintain populations of B. afzelii. In contrast, we found that B. afzelii infection had dramatic effects on the bacterial microbiome of I. ricinus nymphs. Our study demonstrates that infections in the vertebrate host can shape the tick microbiome.

Infection with Borrelia afzelii and manipulation of the egg surface microbiota have no effect on the fitness of immature Ixodes ricinus ticks.

Arthropod vectors carry vector-borne pathogens that cause infectious disease in vertebrate hosts, and arthropod-associated microbiota, which consists of non-pathogenic microorganisms. Vector-borne pathogens and the microbiota can both influence the fitness of their arthropod vectors, and hence the epidemiology of vector-borne diseases. The bacterium Borrelia afzelii, which causes Lyme borreliosis in Europe, is transmitted among vertebrate reservoir hosts by Ixodes ricinus ticks, which also harbour a diverse microbiota of non-pathogenic bacteria. The purpose of this controlled study was to test whether B. afzelii and the tick-associated microbiota influence the fitness of I. ricinus. Eggs obtained from field-collected adult female ticks were surface sterilized (with bleach and ethanol), which reduced the abundance of the bacterial microbiota in the hatched I. ricinus larvae by 28-fold compared to larvae that hatched from control eggs washed with water. The dysbiosed and control larvae were subsequently fed on B. afzelii-infected or uninfected control mice, and the engorged larvae were left to moult into nymphs under laboratory conditions. I. ricinus larvae that fed on B. afzelii-infected mice had a significantly faster larva-to-nymph moulting time compared to larvae that fed on uninfected control mice, but the effect was small (2.4% reduction) and unlikely to be biologically significant. We found no evidence that B. afzelii infection or reduction of the larval microbiota influenced the four other life history traits of the immature I. ricinus ticks, which included engorged larval weight, unfed nymphal weight, larva-to-nymph moulting success, and immature tick survival. A retrospective power analysis found that our sampling effort had sufficient power (> 80%) to detect small effects (differences of 5% to 10%) of our treatments. Under the environmental conditions of this study, we conclude that B. afzelii and the egg surface microbiota had no meaningful effects on tick fitness and hence on the R0 of Lyme borreliosis.

Sequence diversity and evolution of a group of iflaviruses associated with ticks.

We studied a group of tick-associated viruses with characteristics of members of the family Iflaviridae, a family of viruses frequently found in arthropods. Our aim was to gain insight into the evolutionary dynamics of this group of viruses, which may be linked to the biology of ticks. We explored assembled RNA-Seq data sets for different species of ticks. We identified members of five different iflavirus species, four of them novel, and discovered nine new genome sequences, including variants. Five variants represented a virus species associated with Ixodes ricinus. Unexpectedly, a sequence found in the Ixodes scapularis cell line ISE6 was nearly identical to the sequences of I. ricinus variants, suggesting a contamination of this cell line by I. ricinus material. Analysing patterns of substitutions between these variants, we detected a strong excess of synonymous mutations, suggesting evolution under strong positive selection. The phylogenies of the viruses and of their tick hosts were not congruent, suggesting recurrent host changes across tick genera during their evolution. Overall, our work constitutes a step in the understanding of the interactions between this family of viruses and ticks.

Symbiont dynamics during the blood meal of Ixodes ricinus nymphs differ according to their sex.

Ticks harbour rich and diverse microbiota and, among the microorganisms associated with them, endosymbionts are the subject of a growing interest due to their crucial role in the biology of their arthropod host. Midichloria mitochondrii is the main endosymbiont of the European tick Ixodes ricinus and is found in abundance in all I. ricinus females, while at a much lower density in males, where it is even absent in 56 % of the individuals. This endosymbiont is also known to increase in numbers after the blood meal of larvae, nymphs or females. Because of this difference in the prevalence of M. mitochondrii between the two sexes, surveying the density of these bacteria in nymphs that will become either females or males could help to understand the behaviour of Midichloria in its arthropod host. To this aim, we have set up an experimental design by building 3 groups of unfed nymphs based on their scutum and hypostome lengths. After engorgement, weighing and moulting of a subset of the nymphs, a significant difference in sex-ratio among the 3 groups was observed. In parallel, Midichloria load in individual nymphs was quantified by qPCR both before and after engorgement. No difference in either body mass or Midichloria load was observed at the unfed stage, but following engorgement, both features were significantly different between each size group. Our results demonstrate that symbiont dynamics during nymphal engorgement is different between the two sexes, resulting in a significantly higher Midichloria load in nymphs that will become females. The consequences of those findings on our understanding of the interplay between the endosymbiont and its arthropod host are discussed.

Morphology of Pholeoixodes species associated with carnivores in the western Palearctic: Pictorial key based on molecularly identified Ixodes (Ph.) canisuga, I. (Ph.) hexagonus and I. (Ph.) kaiseri males, nymphs and larvae.

Three Palearctic members of the subgenus Pholeoixodes, i.e., Ixodes canisuga, Ixodes hexagonus and Ixodes kaiseri are frequently collected from dogs, cats, red foxes, badgers and other carnivorous/insectivorous hosts in Europe. While a pictorial identification key has been reported for female Pholeoixodes ticks, a similar work has not been done on their male, nymphal and larval specimens. This study was initiated in order to clarify and re-examine those morphological characters of these three tick species, which can be used relatively easily to identify/distinguish them. In the case of larvae the aims included finding alternatives to chaetotaxy, which is difficult to observe and its usefulness is also affected by uncertainties in literature data. For this, 609 Pholeoixodes ticks (males, nymphs and larvae) were collected from carnivores, hedgehogs and their environment in six European countries (representing Western, Central and Southeastern Europe), followed by detailed morphological examination and/or molecular analyses to confirm the identity of their species. Based on the morphology of 84 molecularly analyzed specimens and a new identification key compiled accordingly, altogether 116 I. canisuga, 277 I. hexagonus and 216 I. kaiseri males, nymphs and larvae were identified. Ixodes kaiseri was not found in Western Europe, where I. canisuga predominated. In Central Europe, all three Pholeoixodes species were collected, the largest number of specimens represented by I. hexagonus. On the other hand, in Southeastern Europe I. kaiseri had the highest abundance. In conclusion, the morphology of internal spur on the first coxae (as the traditionally used character to distinguish I. hexagonus from other Pholeoixodes species) is trustworthy to recognize males but is less informative in the case of nymphs and larvae. The latter can be identified more properly by observing the morphology of basis capituli. In particular, nymphs and larvae of I. canisuga have anteriorly flattened basis capituli, forming a plateau that surrounds the base of the hypostome. On the other hand, nymphs and larvae of I. hexagonus and I. kaiseri lack a similar plateau, but (unlike I. canisuga) have cornuae, which are either posterolaterally or caudally directed, respectively.

Where to find questing Ixodes frontalis ticks? Under bamboo bushes!

Tick-borne diseases have a complex epidemiology that depends on different ecological communities, associating several species of vertebrate hosts, vectors and pathogens. While most studies in Europe are focused on Ixodes ricinus, other Ixodes species may also be involved in the transmission or maintenance of pathogens. This is the case of Ixodes frontalis, a poorly known species associated with different bird species such as blackbirds, thrushes and robins, with a wide distribution covering most European countries. In a previous study, high densities of questing I. frontalis larvae were found during autumn-winter at a site close to Nantes (western France) where a long-term survey focused on I. ricinus was conducted. These I. frontalis were mostly observed under bamboo bushes. In the present study, we investigated the presence of I. frontalis under bamboo bushes at various locations. With that aim in mind, a systematic search for questing I. frontalis was undertaken by the flagging method in public urban parks and private gardens presenting bamboo bushes (32 sites). This survey was carried out during autumn-winter to maximize the probability of finding the most abundant stage, i.e. larvae. We searched for I. frontalis first in the area of Nantes (10 sites), then in other regions of France (21 sites) and at one site in northern Italy. A single visit to each site revealed the presence of I. frontalis at 29 out of 32 sites: larvae were always present, nymphs were frequent (59 % of the positive sites), while adults were found at only 14 % of the sites. Questing stages of this understudied species are thus easy to find, by dragging or flagging under bamboo bushes in autumn or winter. We make the assumption that bamboo offers a favourable place for birds to roost overnight outside their breeding period (i.e. spring), sheltered from both predators and wind. This would explain higher densities of I. frontalis under bamboo, relative to other biotopes. As I. frontalis is known to harbour zoonotic pathogens, the consequences of this discovery on the epidemiology of tick-borne diseases are discussed.

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.

Nicotinic acetylcholine receptors in the synganglion of the tick Ixodes ricinus: Functional characterization using membrane microtransplantation.

Nicotinic acetylcholine receptors are an important class of excitatory receptors in the central nervous system of arthropods. In the ticks Ixodes ricinus, the functional and pharmacological properties of nicotinic receptors located in their neurons are still unknown. The objective of this study was to characterize the pharmacological properties of tick nicotinic receptors using membrane microtransplantation in Xenopus laevis oocytes and two-electrodes voltage clamp method. The membranes microtransplanted were extracted from the tick synganglion. We found that oocytes microtransplanted with tick synganglion membranes expressed nicotinic acetylcholine receptor subtypes which were activated by acetylcholine (1 mM) and nicotine (1 mM). Currents induced by pressure application of acetylcholine and nicotine were diminished by 10 nM α-bungarotoxin and methyllycaconitine, suggesting that they expressed two subtypes of nicotinic receptors, α-bungarotoxin-sensitive and -insensitive, respectively. In addition, we found that nicotine receptors expressed in the synganglion membranes were poorly sensitive to the neonicotinoid insecticides clothianidin (CLT), imidacloprid (IMI), acetamiprid (ACE) and thiamethoxam (TMX), in agreement with their lack of activity as acaricides. Interestingly, current amplitudes were strongly potentialized in the presence of 1 µM PNU-120596. CLT was more active as an agonist than IMI, TMX and ACE. Finally, we demonstrated that microtransplantation of purified membrane from the tick synganglion can be a valuable tool for the development and screening of compounds targeting tick nicotinic acetylcholine receptor subtypes.