Molecular detection of Cercopithifilaria, Cruorifilaria and Dipetalonema-like filarial nematodes in ticks of French Guiana

Filarial nematodes of the Dipetalonema lineage are widespread parasites and include some species that are transmitted by ticks. In this study, we conducted a large molecular survey of ticks in French Guiana, South America, to understand the overall diversity of tick-borne filarioids in this remote region largely covered by dense tropical forests. Out of 682 ticks belonging to 22 species and 6 genera, 21 ticks (3.1%) of the species Amblyomma cajennense, A. oblongoguttatum, A. romitii, Ixodes luciae and Rhipicephalus sanguineus sensu lato were positive for infection by filarioids. Molecular typing and phylogenetic analysis identified all these filarioids as members of the Dipetalonema lineage. While the filarioid of R. sanguineus sensu lato is a previously described species, the canine worm Cercopithifilaria bainae Almeida & Vicente, 1984, all other filarioids detected in this study are related but distinct to already known species in the genera Cercopithifilaria, Cruorifilaria and Dipetalonema. Their vertebrate host range may include a wide variety of mammals present in French Guiana, but dogs, capybaras, and opossums are the best candidate hosts for some of these filarioids. Although the detection of members of the Dipetalonema lineage in ticks of significant medical or veterinary interest is of concern, the risk of contracting a tick-borne filarial infection is still largely unknown. The pathogenicity of these filarioids, their epidemiology, developmental cycles, and mechanisms of transmission by South American tick species now require further study.


Introduction
Ticks are important vectors of viruses, bacteria, and protozoan parasites of medical and veterinary importance [31,44].
Ticks can also harbor filarial nematodes of the family Onchocercidae Leiper, 1911, commonly known as filariae or filarioids, but their presence is not often the focus of research on tick-borne pathogens [10,13,45,46,56]. Members of the Onchocercidae family have evolved specialized parasitic life cycles in terrestrial vertebrates and in a variety of blood feeding dipterans (mosquitoes, biting midges, sand flies, horse flies, black flies, etc.) which are the main vectors for most species [29,46,64,67]. In vertebrate hosts, mature filarioids live in the circulatory system or in particular tissues and release microfilariae into the bloodstream. The microfilariae are next taken up by blood-feeding arthropod vectors in which they develop into infective larvae that can be transmitted to a new vertebrate host through further biting. Some filarioids are causative agents of major human (lymphatic filariasis, onchocerciasis, loiasis) and animal diseases (canine heartworm disease) [29, 64,67].
Experimental infection assays have confirmed the vector competence of ticks for filarioids of the genera Acanthocheilonema, Monanema, Yatesia and Cercopithifilaria. Baltazard et al. [11] first experimentally showed that the soft tick O. tartakovskyi can be infected by Acanthocheilonema viteae (Krepkogorskaya, 1933; formerly Dipetalonema viteae) after feeding on infected field vertebrates and that this tick species can further transmitted viable infective larvae to domestic rodents through biting. This tick-borne infection cycle has been conducted for several decades in the laboratory using O. tartakovskyi as an exclusive vector [5,6,48], or O. moubata as an alternative vector [63]. Further experimental infection assays showed similar vector competence in pairing of hard ticks (belonging to the genera Ixodes, Rhipicephalus, Amblyomma, Haemaphysalis and Hyalomma) and some filarioids of the Dipetalonema lineage: ticks feeding on infected vertebrates ingest microfilariae, which can develop up to the viable infective third stage in a few weeks and are further excreted with saliva during biting [4, 6, 9, 14-16, 24, 42, 52, 53, 58, 66, 73, 74]. Filarioids of the Dipetalonema lineage also survive transstadially in such ticks since the development from microfilariae to infective third-stage larvae occurs only while the tick is off-host, that is, during ecdysis from tick larva to nymph or from nymph to adult [53,66]. While all these observations converge to confirm that ticks are effective vectors for these filarioids, members of the Dipetalonema lineage are currently neglected tick-borne parasites [10,46,56].
In this study, we conducted a molecular survey of filarioids in ticks in French Guiana. This remote territory is a vast equatorial land located on the north-east coast of South America, mostly covered by dense rainforests and with a low density of human population [68]. We know little on the presence and diversity of tick-borne diseases in French Guiana, but recent surveys led to the description of novel tick-borne pathogens [17,21,32,43]. Here, we surveyed 682 field ticks belonging to 22 tick species of the 33 known from French Guiana [19]. We further used cytochrome c oxidase subunit I (cox1) mitochondrial gene sequences, and phylogenetics for the description of filarioid infections in these ticks. Lastly, we examined and discussed their genetic proximity with already known species of filarioids associated with ticks.

Tick collection
Field ticks (n = 682; belonging to 22 tick species) were collected in French Guiana either on vegetation by the dragflag method (for questing ticks) or directly from their vertebrate hosts or seabird nests (for engorged or questing ticks) (Tables 1 and S1). Samples included five major tick genera belonging to the family Ixodidae (hard ticks): Amblyomma (16 species), Rhipicephalus (2 species), Ixodes (1 species), Dermacentor (1 species) and Haemaphysalis (1 species). Samples include one species of the Ornithodoros genus, belonging to the Argasidae family (soft ticks). All ticks were stored in 75% ethanol until examination. Ticks were further examined under a Leica Z16 APO A macroscope and categorized by species, stage and sex in previous studies [18][19][20]. Use of genetic resources was approved by the French Ministry of the Environment under reference #TREL19028117S/156, in compliance with the Access and Benefit Sharing procedure implemented by the Loi pour la Reconquête de la Biodiversité.

Molecular detection and typing of filarioids in ticks
DNA was extracted from individual ticks using a DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions. Each individual extract was then tested by a polymerase chain reaction (PCR) assay for filarioid infection by amplifying a fragment of the cox1 mitochondrial gene (689 bp) using the CO1intF (5 0 -TGA TTG GTG GTT TTG GTA A-3 0 ) and CO1intR (5 0 -ATA AGT ACG AGT ATC AAT ATC-3 0 ) primers [26]. PCRs were performed in a total volume of 25 lL containing 10-50 ng of genomic DNA, 8 mM of each dNTP (Thermo Fisher Scientific, Waltham, MA, USA), 10 mM of MgCl 2 (Thermo Fisher Scientific), 7.5 lM of each of the internal primers, 2.5 lL of 10Â PCR buffer (Thermo Fisher Scientific), and 1.25 U of Taq DNA polymerase (Thermo Fisher Scientific). All PCR amplifications were performed as follows: initial denaturation at 93°C for 3 min, 35 cycles of denaturation (93°C, 30 s), annealing (50°C, 30 s), extension (72°C, 1 min), and a final extension (72°C, 5 min). Positive (DNA of the filarioid Loa loa) and negative (water) controls were included in each PCR assay. Following visualization via electrophoresis in 1.5% agarose gel, positive PCR products were sequenced by Eurofins. Sequence chromatograms were cleaned with Chromas Lite (http://www. technelysium.com.au/chromas_lite.html), and alignments were performed using ClustalW, implemented in the MEGA software package (https://www.megasoftware.net/). New sequences obtained in this study were deposited in GenBank under accession numbers OR030075-OR030095.

Molecular phylogenetic analyses
Phylogenetic analyses were based on sequence alignments of the filarioid cox1 gene sequences obtained in this study. Sequences of other filarioids obtained from GenBank, including representative members of the Dipetalonema lineage (Acanthocheilonema, Yatesia, Cercopithifilaria, Cruorifilaria, Litomosoides and Dipetalonema) and of other filarial nematodes were also included in the phylogenetic analyses. The Basic Local Alignment Search Tool (BLAST; https://blast. ncbi.nlm.nih.gov/blast/Blast.cgi) was used to find additional sequences available on GenBank and showing the highest nucleotide similarities with the filarioid gene sequences we characterized in this study. The Gblocks program with default parameters was used to obtain non-ambiguous sequence alignments [27]. Tree-based phylogenetic analyses were performed using maximum-likelihood (ML) analyses using the MEGA software package (https://www.megasoftware.net/). The evolutionary models that best fit the sequence data were determined using the Akaike information criterion. Clade robustness was assessed by bootstrap analysis using 1,000 replicates.

Statistical analyses
All statistical analyses were carried out using the R statistical package (https://www.r-project.org/).

Molecular typing of filarioids in ticks
On the basis of cox1 gene sequences, we characterized six genetically distinct filarioids (pairwise nucleotide identity: 84.1-99.8%) in A. cajennense, A. oblongoguttatum, A. romitii, I. luciae and R. sanguineus s.l. Two genetically distinct filarioids were detected in A. cajennense (pairwise nucleotide identity: 87.1%), two in I. luciae (pairwise nucleotide identity: 99.5%), and one in each of the other infected tick species (A. oblongoguttatum, A. romitii and R. sanguineus s.l.). Of the six genetically distinct filarioids, each was specific to its respective tick species, expect one which was shared by A. cajennense and I. luciae.
The filarioid cox1 gene sequence we detected in R. sanguineus s.l. was identical to those of Cercopithifilaria bainae Almeida [62].

Phylogeny of filarioids in ticks
A ML analysis based on cox1 nucleotide sequences was further conducted to examine the phylogenetic proximity between the filarioids of A. cajennense, A. oblongoguttatum, A. romitii, I. luciae and R. sanguineus s.l. and with other filarioids. Phylogenetic reconstruction revealed a clustering of the genera Cercopithifilaria, Cruorifilaria, Litomosoides, Yatesia, Acanthocheilonema and Dipetalonema in a unique monophyletic clade, the Dipetalonema lineage, distinct to other members of the family Onchocercidae (Fig. 1). Phylogenetic reconstruction further showed within the Dipetalonema lineage that: 1. The filarioids of A. oblongoguttatum and R. sanguineus s.l.
formed a robust subclade, supported by a 91% bootstrap value, together with all species of the genus Cercopithifilaria (Fig. 1). This suggested that the filarioids of A. oblongoguttatum and R. sanguineus s.l. belong to the genus Cercopithifilaria. While the filarioid of R. sanguineus s.l. was probably Cercopithifilaria bainae (as suggested by their 100% pairwise nucleotide identity), the filarioid of A. oblongoguttatum was distinct from already known Cercopithifilaria species. 2. The filarioid of A. romitii and one of A. cajennense delineated a robust subclade (95% bootstrap value) together with Cruorifilaria tuberocauda (Fig. 1), suggesting that all belong to the genus Cruorifilaria. The filarioid of A. romitii and one of A. cajennense were distinct to already known Cruorifilaria species. 3. The other filarioid of A. cajennense and those of I. luciae clustered in a unique robust subclade (provisionally named Dipetalonema-like hereafter; 99% bootstrap value) substantially divergent from all other genera of the Dipetalonema lineage (Fig. 1). The phylogenetic proximity of this filarioid of A. cajennense to those of I. luciae, and their 99.8-100% pairwise nucleotide identities, suggested that they probably belong to the same species. The closest relative of Dipetalonema-like is the genus Dipetalonema, while other genera are more distantly related. This suggests that these Dipetalonemalike filarioids may belong to a potential novel genus which could be a sister genus of Dipetalonema.

Discussion
We show here that substantial diversity of filarioids of the Dipetalonema lineage is present in ticks of French Guiana. We conducted extensive molecular screening of 22 tick species, and identified filarioids in five tick species, A. cajennense, A. oblongoguttatum, A. romitii, I. luciae and R. sanguineus s.l. No filarioid was detected in the 17 other tick species. While pairwise nucleotide identities and phylogenetics assigned the filarioid of R. sanguineus s.l. to an already described species, Cercopithifilaria bainae, all other filarioids detected in this study are distinct to already known species of Cercopithifilaria, Cruorifilaria and Dipetalonema on the basis of their cox1 gene sequences. To our knowledge, this is also the first observation of Cruorifilaria in ticks (and, more broadly, in blood-feeding arthropods), suggesting that A. cajennense and A. romitii are vectors of this filarioid genus.
The presence of filarioid DNA in A. cajennense, A. oblongoguttatum, A. romitii, I. luciae and R. sanguineus s.l. suggests that these tick species may serve as vectors for filarial nematodes. Obviously, these observations are not conclusive evidence of vector competence: ticks feeding on parasitemic vertebrates can be positive for filarioid DNA due to the presence of infected blood in the tick digestive tract, but may not be competent to transmit infectious filarioids during their next blood meal. However, experimental infection assays have directly confirmed the vector competence of Ixodes, Amblyomma and Rhipicephalus ticks for filarioids of the Dipetalonema lineage, including the brown dog tick R. sanguineus s.l. for Cercopithifilaria bainae [4,6,24,42,52,53,58,66,73,74]. In the field, the detection of viable microfilariae and infective filarial larvae in the hindgut, haemocoele, fat cells or ducts of salivary glands of ticks also support these observations [5,7,12]. In addition, the accumulation of microfilariae in the dermis of infected vertebrates at the sites of attachment of ticks suggest a specific chemotactic response of filarioids to tick saliva [42,50]. Furthermore, our detection of filarioids from questing (unfed) A. cajennense, A. oblongoguttatum, and R. sanguineus s.l. ticks that have already digested their previous blood meals, have further moulted, and are seeking vertebrates for their next blood meal suggests that these tick species can acquire and maintain filarioids of the Dipetalonema lineage, a pattern of transstadial transmission also observed for other tick species [53,66]. Finally, the observation that all tick-associated filarioids cluster in a monophyletic clade, the Dipetalonema lineage, distinct from clades of filarioids specifically associated with transmission by blood-feeding dipterans, supports the conclusion that ticks are vectors of these filarioids.
Tick-borne filarioids have been detected from most continents, suggesting worldwide distribution of the Dipetalonema lineage [10,46]. Some species may have global distribution such as Cercopithifilaria bainae, specifically associated with dogs and R. sanguineus s.l., which was first formally identified in Brazil [3], and further in Europe [41,56,65], Asia [13,45], Iran [61], North America [23,47], and now in French Guiana (this study). The other filarioids we characterized in French Guiana have not been detected anywhere and their transmission cycles remain unknown. In fact, the vertebrate hosts of the Cercopithifilaria sp. of A. oblongoguttatum remain unidentified since this tick is a generalist species feeding on a wide range of mammals [19].
For the Cruorifilaria spp. of A. romitii and A. cajennense, two biological features suggest that capybara could be their vertebrate host. First, the single described Cruorifilaria species, Cruorifilaria tuberocauda, has been reported as a parasite of capybara in other South American regions [25,33], and, second, A. romitii is a tick species highly specific to capybara, while A. cajennense is a generalist tick species often found on these animals [19]. For the Dipetalonema-like filarioids, opossums could be vertebrate hosts since one of the infected tick species, I. luciae, is a specialized tick species that feeds primarily on opossums [19]. Past studies have morphologically identified four filarial species, all of the Dipetalonema lineage, in South American opossums: Acanthocheilonema pricei (Vaz et Pereira, 1934), Cercopithifilaria didelphis (Esslinger et Smith, 1979), Skrjabinofilaria skrjabini Travassos, 1925, and Cherylia guyanensis Bain et al., 1985 [9, 35]. No molecular data are currently available for these four species, which prevents any definitive conclusions.
The detection of members of the Dipetalonema lineage in ticks of significant medical or veterinary interest is of concern, but the risk of acquiring a tick-borne filarial infection is unknown. While A. cajennense and A. oblongoguttatum are the tick species most commonly biting humans in South America [36,39], in the present study, we found that 3-6.6% of field specimens were infected with filarioids of the Dipetalonema lineage. Humans are therefore exposed to bites of filarioid-infected ticks without the risk of infection being documented to date. In North America, a survey of humans with a tick-bite history for the presence of antibodies against the I. scapularis-associated filarioid found, however, no evidence of infection [69]. In animals, filarioids of the Dipetalonema lineage are often considered innocuous, but some studies and evidence suggest that they are not. In dogs, recent surveys showed that Cercopithifilaria bainae can induce erythematous and pruritic dermatitis, subcutaneous nodules, ulcerative skin lesions and chronic polyarthritis [23,38,54]. Infections of dogs by other members of the Dipetalonema lineage, Acanthocheilonema spp., were also associated with clinical symptoms, such as anemia and high levels of plasma proteins [22,34]. In capybaras, adults of Cruorifilaria tuberocauda form tangled masses in the kidneys, and less frequently in the pulmonary arteries, where they cause extensive tissue damage with severe endarteritis, leading to obstruction of vessels and infarction of adjacent tissues [33].
In conclusion, we have identified a wide range of Cercopithifilaria, Cruorifilaria and Dipetalonema-like filarial nematodes in French Guiana. The repeated detection of the Dipetalonema lineage in major tick genera on most continents confirms that these are widespread but neglected tick-borne filarioids of largely undocumented veterinary (and perhaps medical) concern. Their pathogenicity, epidemiology, developmental cycles and transmission mechanisms by ticks must be further investigated.