Autogenous reproduction by Ornithodoros turicata (Ixodida: Argasidae) females and vertical transmission of the tick-borne pathogen Borrelia turicatae (Spirochaetales: Borreliaceae)

ABSTRACT

Ornithodoros turicata is a vector of relapsing fever spirochetes in North America and transmits Borrelia turicatae to a variety of vertebrate hosts. The remarkably long lifespan of O. turicata and its ability to maintain spirochetes horizontally (between life stages) and vertically (to progeny) promote the perpetuation of B. turicatae in nature. Nevertheless, the reproductive biology of O. turicata is poorly understood. In this report, we collected ticks from a park within a neighborhood of Austin, TX. They were reared to adulthood, and male ticks were individually housed with females. We observed autogenous reproduction, which is the ability to produce eggs without the need for a blood meal, and further investigated vertical transmission of B. turicatae by quantifying filial infection rates in a cohort of progeny ticks. These results indicate that O. turicata transovarially transmits B. turicatae during autogenous reproduction and further signify the tick as a natural reservoir of the spirochetes.

IMPORTANCE

Previous research has implicated Ornithodoros ticks, including Ornithodoros turicata, as long-term reservoirs of relapsing fever (RF) spirochetes. Considering the tick’s long lifespan and their efficiency in maintaining and transferring spirochetes within the population, the infection could persist in a given enzootic focus for decades. However, little is known about the relative importance of horizontal and vertical transmission routes in the persistence and evolution of RF Borrelia. Our observations on the reproductive biology of O. turicata in the absence of vertebrate hosts indicate an additional mechanism by which Borrelia turicatae can be maintained in the environment. This work establishes the foundation for studying O. turicata reproduction and spirochete-vector interactions, which will aid in devising control measures for Ornithodoros ticks and RF spirochetes.

INTRODUCTION

The soft tick Ornithodoros turicata is a major vector of relapsing fever (RF) spirochetes in North America and is commonly found throughout the southern United States into Mexico (1). Wherever the vector occurs, enzootic foci can be formed between the pathogenic spirochete, Borrelia turicatae, and their wildlife hosts. Moreover, spillover events occur when humans or companion animals enter habitats infested with infected ticks. For example, recent work indicates that B. turicatae is an emerging threat in parks and recreational areas in highly populated cities of Texas (2 – 4). To further understand the public health impact of B. turicatae, knowledge of the tick’s biology and pathogen-vector interactions is needed.

The life cycle of O. turicata is complex and the dynamics of vertical transmission of B. turicatae are poorly understood. The life span of O. turicata is nearly 10 years (5), and adult female ticks can oviposit multiple times (6, 7). Davis reported a cohort of field-collected O. turicata females oviposited five times in a 12-month period (7). Once eggs hatch, O. turicata has upwards of five instars as nymphs (5). After molting into adults, vertically infected ticks can maintain B. turicatae for at least five generations (7). Collectively, prior work indicates that infected O. turicata can bypass the need to acquire the infection from a vertebrate host and serve as a reservoir for B. turicatae.

An interesting nuance of Ornithodoros reproduction that should be considered in combination with transovarial transmission is autogeny. This is the ability to produce eggs without the need for a blood meal. While known to occur in Ornithodoros ticks, autogeny varies between species (8). Endris and colleagues reported that during 2 years of rearing Ornithodoros puertoricensis they failed to observe autogeny (9). Alternatively, autogeny has been reported in species like Ornithodoros tholozani and Ornithodoros fonsecai (8, 10). However, we have not found a report characterizing autogeny in O. turicata.

The current study was based on a serendipitous observation while assessing O. turicata reproduction in a group of naturally infected ticks. Interestingly, as the nymphs molted into adults, we observed cases of autogeny. In one cohort of ticks, we determined rates of transovarial transmission to the F1 progeny. Our work indicates that blood feeding in the first gonotrophic cycle is not essential for O. turicata female reproduction and that transovarial transmission occurs by autogeny.

RESULTS

Collection of ticks from a park in Austin, TX

Tick collections were part of routine surveillance of Ornithodoros species in populated cities of Texas. A park was identified in a neighborhood of south Austin (Fig. 1A). The habitat consisted of limestone outcroppings and Live Oak Mesquite Savanna (Fig. 1B). As ticks were lured from outcroppings with dry ice as a source of carbon dioxide, they were collected and housed together (Fig. 1C). We collected 41 ticks. Of these, 15 were adults and 26 were nymphs. Five nymphs died between the time the ticks were collected and evaluated in our laboratory.

Fig 1.

Collection site of Ornithodoros turicata in Austin, TX, USA. Shown is an aerial view (Google Earth) of the collection site with a scale in the bottom left corner (A). The red dot represents the location where ticks were collected. A container with dry ice was placed by a limestone outcropping and used to bait ticks (B). The white arrow points to the dry ice container. Also shown is a 15 mL tube containing the ticks that were collected (C). Shown on the paper plates is the material from which the ticks were harvested (C).

Speciation of field-collected ticks

In the laboratory, we determined the species of field-collected ticks by microscopy and through a molecular approach. Morphological characterization indicated that the ticks were O. turicata. For molecular typing, we amplified a portion of the cox1 gene from two ticks, which produced a ~700 nucleotide fragment, as determined by agarose gel electrophoreses. Sequencing and BLAST analysis determined 99.71% nucleotide identity with O. turicata. The sequences were deposited to GenBank under accession numbers OR047917 and OR047918. These findings indicated an additional distribution record of O. turicata in Austin, TX.

Autogenous reproduction by O. turicata

Of the 21 live field-collected late-instar nymphs assessed, three died after blood feeding on laboratory mice, nine molted into females, and nine into males. We split the adults into individual tubes housing a male tick with a female, and within 90 days there were numerous larvae in four out of nine tubes. These findings indicated that O. turicatae females reproduced and laid eggs autogenously. We subsequently determined whether transovarial transmission occurred in one of the cohorts from a single pair of adults.

Assessment of transovarial transmission

We reared the larvae to the second-instar nymphs and transmission to susceptible animals was attempted to determine whether these ticks were infected with B. turicatae. Feeding F1 second-instar nymphs on mice indicated that the ticks were infected (Table 1). We visualized spirochetes in the blood of five of seven animals by the fifth day after feeding ticks. Spirochetes were observed in the blood of another animal on the ninth day. The mouse in which we failed to detect spirochetes by microscopy seroconverted to B. turicatae protein lysates as determined by immunoblotting (Table 1). This indicated that the infection was below the limit of detection by microscopy.

TABLE 1.

Assessment of murine infection and B. turicatae densities in autogenously produced ticks

Mouse #	Assessment of infection		Number of ticks infected/tested b	$\overline{X}$ (SD) flaB copies per infected tick b
	Microscopy (DPF) a	Seroconversion by immunoblot
M1	+(5)	ND c	3/15	2.28 × 105 (±8.2×104)
M2	−	+	7/15	2.29 × 105 (±9.49×104)
M3	+(5)	ND c	8/15	2.14 × 105 (±1.05×105)
M4	+(9)	+	5/15	2.3 × 105 (±1.05×105)
M5	+(4)	+	NA d	NA d
M6	+(4)	+	NA d	NA d
M7	+(4)	+	NA d	NA d

^a DPF: days post tick feeding.

^b As determined by qPCR.

^c ND: not determined because the animal was sacrificed for spirochete isolation.

^d NA: not applicable because the ticks were kept alive for future experiments.

Rates of filial infection in F1 nymphs

After ticks molted to the third-instar nymph, qPCR determined infection rates in 60 ticks (Table 1; Fig. S1). The remaining ticks were saved for future experiments. Out of the 60 F1 ticks, 23 were positive for B. turicatae flaB gene and filial infection rates were 38.3% (95%CI: 26.3%–51.8%). These findings determined vertical transmission rates in the offspring of ticks that autogenously reproduced.

DISCUSSION

This study arose from laboratory observations of O. turicata that were collected in a public park within a neighborhood of Austin, TX. Ticks were reared from the nymphal stage to adulthood, and we observed autogenous reproduction. While autogeny has been reported in a close relative of O. turicata, Ornithodoros parkeri (8, 11), literature on the former is absent. The reasons for this lack of data are unclear. Without designed experiments housing individual ticks and tracking their molt progress, the phenomenon may have been overlooked during colony maintenance.

In Argasidae, nuances in autogeny depend on reproductive and feeding behaviors. Several species are fully autogenous, such as Otobius spp. and Antricola spp. In the adult stage, these genera have vestigial mouthparts and never blood feed (12). Alveonasus lahorensis females are obligatory autogenous in their first gonotrophic cycle but have to secure a blood meal to produce subsequent egg batches (13). Facultative autogeny in the first gonotrophic cycle has been reported at variable rates in species currently classified in the genera Ornithodoros, Argas, and Carios (8, 10, 12, 14). Interestingly, in other arthropods like mosquitoes, autogeny varies extensively between conspecific populations across distribution ranges (15). While studies suggest that genetic and environmental factors (e.g., temperature) modulate autogeny in soft ticks (11), additional work is needed in O. turicata to identify its frequency between geographically distinct populations.

We evaluated filial infection rates and successful B. turicatae transmission to mice from the autogenously produced cohort of O. turicata. These findings indicated that after the transovarial passage through to the next generation larvae, B. turicatae remained infectious. Determining the infectivity of B. turicatae to mice was important because prior work on continuous vertical transmission through successive generations of Ornithodoros species is ambiguous. For example, ~22% of vertically infected cohorts of Ornithodoros papillipes failed to transmit Borrelia sogdiana in the eighth generation compared to ~100% in earlier generations (16). Additionally, a complete loss of transmissibility of Borrelia duttonii by tick bite was reported by the fifth generation in transovarially infected Ornithodoros moubata (17). However, Burgdorfer and Varma failed to reproduce these findings using a different strain of spirochete and population of tick (18). With B. turicatae, spirochetes remained infectious to mice after vertical maintenance by the vector for five generations (7). In these previous studies, the life stage where ticks became infected was known to the investigators. A caveat in our work was that O. turicata ticks were field collected, and it was unclear if the infected parental tick acquired spirochetes vertically or from an infectious blood meal. We have isolated and cultured the B. turicatae strain from this study and will assess vertical transmission after infecting O. turicata at different life stages.

We determined spirochete densities in individual ticks to better understand transovarial transmission rates in the offspring of autogenously reproduced ticks. Our estimates of B. turicatae loads were based on flaB copies per tick and initially appeared high. However, work with Borrelia hermsii and Borrelia burgdorferi indicates that the pathogens are polyploid having ~16 chromosomal copies per cell depending on the bacteria’s growth stage (19 – 21). With this consideration, a more accurate estimation of B. turicatae densities in the ticks was likely ~10-fold less, or ~10⁴ spirochetes per tick.

While our study began to evaluate autogeny and vertical transmission of spirochetes by O. turicata, there were limitations. For example, we fed cohorts of larvae or first-instar nymphs on mouse pups. Consequently, a portion of uninfected ticks within the cohort could have acquired B. turicatae by co-feeding with infected ticks or through hyperparasitism. Acquisition of RF Borrelia by co-feeding has not been reported for B. turicatae or other soft tick-borne RF spirochete species, presumably because the ticks are rapid feeders. Additionally, while hyperparasitism has been reported as a route of acquisition of RF spirochetes in Ornithodoros hermsi and Ornithodoros papillipes (22, 23), in O. turicata there are only occasional observations of conspecific ticks parasitizing each other (5, 24). For this study, it was impractical to feed individual larvae on mice, and we are still optimizing an approach to detect B. turicatae DNA in individual eggs and larvae.

Our findings are for one strain of B. turicatae. It will be important to know the rates of vertical transmission between B. turicatae isolates given their genomic plasticity. RF spirochetes possess complex genomes with upwards of 15 plasmids, and isolates vary significantly in their plasmid content (4). Furthermore, in this study, we evaluated one population of ticks from Texas, while O. turicata possesses a wide distribution range. More elaborate studies will identify phenotypic differences between spirochete isolates and tick populations.

It is important to define the cost-benefit ratio of autogeny for both the tick vector and pathogen. Tick eggs and larvae are less resistant to adverse environmental conditions (13, 25); however, autogenous reproduction could maximize fitness in newly established tick populations with transient hosts. This is consistent with ecological bet-hedging (26). Indeed, facultative autogeny can accelerate population growth after dispersal into new suitable habitats in patchy landscapes, as has been proposed in triatomine kissing bugs (27). Additionally, newly colonized habitats containing B. turicatae-infected larvae could facilitate a rapid expansion of enzootic foci. Studies by Davis highlighted the significance of O. turicata as a long-term reservoir of B. turicatae (7), and our findings support this notion. While more work is needed to delineate the mechanisms of vertical transmission, our work lays the foundation for studying O. turicata reproduction and spirochete-vector interactions.

MATERIALS AND METHODS

Collection and speciation of ticks

In March 2022, field efforts were implemented to collect ticks. The ticks were baited with dry ice, collected in tubes, and transferred to our lab where they were housed at 24°C ± 2°C and 85% relative humidity (28). The species of tick was confirmed by microscopy and through PCR amplification of a fragment of the mitochondrial cox1 gene using DNA from two ticks and the LCO and HCO primers (29). The amplicons were purified using the Mag-Bind Total Pure NGS beads (Omega Bio-tek, Norcross, GA, USA) and sequenced. The sequences were deposited in GenBank.

Tick feedings and detection of murine infection

To check for Borrelia infection, field-collected O. turicata were divided into pools of 8–10 ticks and fed on Institute for Cancer Research (ICR) mice, as previously described (4). Blood was collected for 10 consecutive days by tail nick, and infection was determined by visualizing spirochetes using a dark field microscope (Zeiss Axio Imager A2, Oberkochen, Baden-Württemberg, Germany). After feeding, the ticks’ life stage was determined by visualization of the genital aperture. They were sorted into pairs of males and females, individual females, or groups of nymphs (genital aperture is not apparent at this developmental stage). Ticks were housed in 50 mL TubeSpin Bioreactor tubes (MidSci, St. Louis, MO, USA).

The F1 progeny from a single female was further evaluated. The remaining larvae from the other females that autogenously reproduced were kept for other studies. For rearing O. turicata, we only used unexposed mice; the animals were never recycled to feed ticks. Larvae and first-instar nymphs were reared by feeding them on mouse pups. As second-instar nymphs, ticks were tested for infection by feeding on seven adult ICR mice (15–18 ticks per animal; n = 107). Infection was evaluated by microscopy, as stated above. Thirty days after tick feedings, all animals were euthanized and blood was collected to determine infection by evaluating seroconversion to B. turicatae protein lysates, as reported (4).

Molecular detection of B. turicatae in F1 ticks

Filial infection rates were determined on a subset of progeny using a duplex qPCR assay targeting B. turicatae flaB and the O. turicata B-actin gene, as described in reference (30). DNA was extracted from individual unfed third-instar nymphs using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). Each sample was run twice in triplicate. Spirochete loads in each sample were determined as previously described (31). The Wilson interval with continuity correction was calculated for the resulting point estimate of filial infection rates to obtain a 95% confidence interval using R (32).

ETHICS APPROVAL

Animal studies were approved by the Baylor College of Medicine (BCM) Institutional Animal Care and Use Committee (protocol AN7086). All work and animal husbandry were in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.01032-23.

Supplemental Figure 1. aem.01032-23-s0001.tif.

Calculated flaB copies per tick.

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