Mixed-sex clusters on grass blades –breeding strategy of the ornate dog tick, Dermacentor reticulatus

doi:10.21203/rs.3.rs-3559552/v1

Download PDF

Research Article

Mixed-sex clusters on grass blades –breeding strategy of the ornate dog tick, Dermacentor reticulatus

https://doi.org/10.21203/rs.3.rs-3559552/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 09 Feb, 2024

Read the published version in Parasites & Vectors →

You are reading this latest preprint version

Introduction

Dermacentor reticulatus is second only to Ixodes ricinus in importance as a vector of infectious organisms, especially Babesia canis, the agent of canine babesiosis. The geographical range of this species, together with local densities, continue to increase in many regions of Europe. In the present study, we tested the hypothesis that D. reticulatus possesses an efficient breeding strategy allowing for a rapid increase in tick numbers and densities by formation of mixed sex clusters/aggregations, while questing in the environment.

Methods

To test this hypothesis, we carried out an observational study in spring 2023, at three sites in two regions, in Central and NE Poland, both characterised by high tick densities. At each site, a 400 metre long transect was inspected for questing ticks. All noticed ticks were collected and tick numbers and sexes per stem were recorded. Differences in tick distribution by site and sex were analysed statistically.

Results

In total 371 D. reticulatus (219 females and 152 males) were collected from 270 grass stems over a combined 1200 metres of transect. The majority of grass stems (54.2%) were occupied by just a single individual and two-tick clusters were the second most common category. Six D. reticulatus individuals were the maximum observed on only one particular grass stem.

Mixed-sex clusters were significantly more common than single-sex clusters at all three sites. With study sites combined, mixed sex clusters accounted for 17.4% (CL₉₅=13.9-21.6%) of observations, while for multiple males and multiple females the values were 2.6 % (1.4 – 4.7%) and 5.6% (3.7 – 8.3%), respectively.

Conclusion

Mixed-sex clusters of D. reticulatus ticks were significantly more common than single-sex clusters, which we hypothesise reflects an efficient, likely pheromone-mediated, breeding strategy of this expansive tick species.

behaviour

ticks

Dermacentor reticulatus

aggregations

assembly

single-sex clusters

mixed-sex clusters

Ticks and tick-borne diseases pose an increasing threat to humans and animals worldwide. Thus, there is a lot of interest in the role of ticks as vectors and their competence for transmitting multiple pathogens. Many aspects of the biology of the main tick vectors are understood in some detail [1–4], although behaviour has been less well studied. Recent studies have reported a range of different activities of ticks under laboratory conditions [5] and have identified significant differences in activity between ticks infected and uninfected with Rickettsia spp. [6], their behaviour under natural conditions is less well known [7, 8].

Dermacentor reticulatus is second only to Ixodes ricinus in importance as a vector of infectious organisms including Babesia canis (an agent of canine babesiosis), tick-borne encephalitis virus (TBEV) and Rickettsia raoulti [9–12]. This tick species is also expansive in many regions of Europe [13–16], its territorial expansion being accompanied by the emergence and spread of canine babesiosis in regions where previously dogs had been free of this disease [17–19]. Interestingly, in some areas where these two tick species occur sympatrically, D. reticulatus infestation on dogs and livestock is much more intense than infestation with I. ricinus, and mean tick density of D. reticulatus can be up to seven times higher than that of I. ricinus [20]. In a recent study, extremely high densities of adult questing D. reticulatus were recorded in South-Eastern Poland, exceeding over 340 ticks/ 100m² [21, 22]. Taking into account both their ability to expand the region of their endemicity and to reach very high densities and infestation parameters, we hypothesized that D. reticulatus has to possess an extremely efficient breeding strategy allowing for rapid increase in tick numbers and densities.

Dermacentor reticulatus belongs to the Metastriata group of ixodid ticks. The metastriate species, including Dermacentor, Amblyomma, and Rhipicephalus spp., invariably attain sexual maturity and mate solely on their hosts [1, 2, 23]. Following adult ecdysis, the germinal cells of metastriate males become arrested as late prophase primary spermatocytes. Development proceeds only when the males find a host, usually one day after the adult male ticks attach to a host [2]. In our previous study, we observed that the body shape of D. reticulatus males collected from hosts changed despite little change in weight [20]. We found indeed that these foraging males were filled with sperm containing mature spermatocytes, in contrast to questing males collected from vegetation, in which only primary spermatocytes were identified (Mierzejewska and Bębas, unpublished).

We hypothesised that for optimization of their breeding strategy, males should accompany questing females, in order to increase their chances of attachment to the same host individual, thereby enabling a blood meal for females and an opportunity for males to mature and copulate with engorged females. Thus, mixed-sex clusters of two or more ticks should be more prevalent than single sex assemblages.

To test this hypothesis, we carried out an observational study in spring 2023, at three sites in two regions: two sites in Central Poland, both characterised by high tick densities (Kury and Warsaw-Siekierki in Masovia) and one site in North-Eastern Poland (Urwitałt in the Mazury Lake District). The study was conducted between April 14th and May 5th 2023, during the spring peak of tick activity [17], when adults can be spotted easily on the tops of grass blades (Fig. 1a-h). Each of three independent researcher(s) (AB, DW, WM + WR) walked through a 400 metre long transect at one of the three sites, traversing grassy areas (two rural sites [Kury, Siekierki] and one urban site [Siekierki in Warsaw]) looking for questing ticks on the tops of grass blades. All noticed ticks were collected and tick numbers and sexes per stem were recorded.

Statistical analysis

The data recorded from each of the three study sites comprised the number of individual grass stems with just a single male tick, a single female, multiple males, multiple females or mixed sexes. For these five data subsets, the number of records was converted to a percentage of the total observations from each site and percentages are given with 95% confidence limits (CL₉₅) in the text and 95% confidence intervals illustrated on figures, and calculated in bespoke software based on the tables of Rohlf and Sokal [24].

The data were analysed using maximum likelihood techniques based on log linear analysis of contingency tables in the software package IBM SPSS (version 28). This approach is based on categorical values of factors of interest, which are used not only to fit hierarchical log-linear models to multidimensional cross-tabulations using an iterative proportional-fitting algorithm but also to detect associations between factors. We fitted a model with the following explanatory factors: site (3 levels, corresponding to Kury, Siekierki and Urwitałt), a single male tick (2 levels, present or absent), a single female tick (2 levels, present or absent), more than 1 male tick but no females (2 levels, present or absent), more than one female tick but no males (2 levels, present or absent|) and mixed sexes (2 levels, present or absent). By employing the backward selection procedure in SPSS, we simplified each model until only significant terms remained. For each level of analysis in turn, beginning with the most complex model, involving all possible main effects and interactions, those combinations that did not contribute significantly to explaining variation in the data were eliminated in a stepwise fashion beginning with the highest level interaction (backward selection procedure). A minimum sufficient model (MSM, Table 1) was then obtained, for which the likelihood ratio of Chi-square was not significant, indicating that the model was sufficient in explaining the data. The importance of each term in the final model was assessed by the probability that its exclusion would alter the model significantly and these values are given in the text, assessed by a likelihood ratio test between models with and without each term of interest.

In total 371 D. reticulatus (219 females and 152 males) were collected from 270 grass stems in the combined 1200 meters of transect. Additionally, ten I. ricinus (seven females and three males) were found accompanying D. reticulatus ticks on stems (not analysed further). Individual plants with 4, 7 and 11 ticks were observed (Figure 1 a-h).

The distribution of D. reticulatus numbers per grass stem is presented in figure 2a. The majority of grass stems were occupied by just a single individual tick (201/371= 54.2%) and two-tick clusters were the second most common category. Up to six D. reticulatus individuals were noticed on one particular grass stem (Figure 1, 2a).

Analysis of the five possible combinations of tick occupancy on grass stems (single male, single female, 2 or more females, 2 or more males, 2 or more ticks of different sexes) revealed that there was no site effect for any of the 5 combinations (for site x single male, χ²₂= 2.06, P=0.36; site x single female, χ²₂= <0.001, P= >0.99; site x multiple males, χ²₂= 4.42, P=0.110; site x multiple females, χ²₂= 3.45, P=0.178; site x mixed sexes, χ²₂= 0.061, P=0.74). The site effect in the MSM (Table 1) arose because while the number of observations from Kury and Siekierki was 100 in each case, only 70 observations were recorded from Urwitałt.

However, there were significant differences between each of the ten possible two-way comparisons of the five combinations of tick occupancy on grass stems (Table 1). The percentages for each combination by site are illustrated in Fig 2b, where it can be seen how similar the pattern was at all three sites.

Table 1. Minimum sufficient statistical model

1. m= single male; f= single female;

mm= >1 male; ff=>1 female; mf= mixed sexes

For goodness of fit of the final model to the data, χ²₇₈=12.811, P=>0.99

While occupancy by a single tick accounted for most observations, cases of occupancy by a single female were more frequently observed compared to those by a single male (Fig. 2b). Multiple occupancy was less frequently recorded, and mixed-sex clusters were significantly more common than single-sex clusters (Fig. 2b, Table 1).

With study sites combined, mixed sex-clusters accounted for 17.4% (CL₉₅=13.86-21.57%) of observations, while for multiple males and multiple females the values were 2.6 % (1.39 – 4.73%) and 5.6% (3.65 – 8.33%), respectively.

The key finding of our observational study is that mixed-sex clusters of D. reticulatus occur on grass stems more frequently than single-sex clusters, and hence are likely to result in the simultaneous transfer of both sexes on contact with a potential host. Given that mating in this species occurs only after infestation of a new host, this finding can be interpreted as reflecting a component of a successful breeding strategy of D. reticulatus ticks. Moreover, it suggests the involvement of off-host arrestment (assembly) pheromones (causing the cessation of tick kinetic activity, leading to clusters of individuals in their natural environment; [3]), partially resembling sex pheromones in their function (attractive for opposite sex; [3]).

As a breeding strategy, the more frequent occurrence of mix-sex clusters on grass stems depends on a combination of two behavioural features: formation of off-host clusters and recognition of tick sex, and requires an exchange of information between tick individuals. By definition sex pheromones are semiochemicals emitted by individuals of one sex that mediate the sexual behaviour of the opposite sex [3]. The first tick sex pheromone (2,6-dichlorophenol) was discovered in the lone star tick Amblyomma americanum over 50 years ago [25], but to the best of our knowledge, the pheromones that induce sex-related clustering of ticks off-host, have not yet been identified. Nevertheless, in recent years, an impressive amount of novel knowledge has accumulated about the chemical ‘language’ (production and release of semiochemicals, information transferring molecules) used by ticks for inter- and intra-species communication [3, 26–28].

Aggregating and sexual behaviours comprise a hierarchy of responses to different types of chemical compounds that must be recognized in a sequential order to achieve the end result [2]. In ticks these behaviours (i.e. formation of clusters, mate-finding and courtship) are regulated mostly by pheromones that induce certain behavioural responses in related organisms [2, 3, 28]. Different tick-borne semiochemicals are now known to induce (a) off-host aggregation in the environment, (b) on-host non-sexual aggregation, (c) on-host sexual aggregation, (d ) copulation, and (e) ejaculation [2]. Off-host aggregation has been attributed to arrestment (= assembly) pheromones; on-host non-sexual aggregation to attraction–aggregation–attachment (AAA) pheromones described in Amblyomma spp.; on-host sexual aggregation, copulation and ejaculation to different kinds of sex pheromones [2, 3].

The phenomenon of multi-sex aggregation on grass stems observed in the current study can be a consequence of combined arrestment behaviour (ticks clustering in the environment) and sexual aggregation (mostly mixed-sex aggregation). Arrestment/assembly pheromones are widespread in tick species and have been found in both hard (Ixodidae) and soft ticks (Argasidae). Arrestment behaviour is mostly mediated by contact with excreta from other ticks [2, 3], and although widespread among tick species, arrestment pheromones have not been reported in all tick species. For example, and in contrast to our finding, Taylor et al. [29] found no evidence of similar behaviour (off-host aggregation/assembly) in ticks of the genus Dermacentor that occur in the Nearctic region (D. variabilis and D. andersoni).

Tick clustering behaviour appears to involve a two-step process: (1) attraction to a volatile source and (2) arrestment in response to various purines. Purines have very low vapour pressure and, consequently, are not attractants [3]. Ammonia and perhaps other volatiles emanating from tick faeces gradually attract free living, unfed adults and even nymphs to the point source. Contact with the purines, especially guanine and xanthine, triggers the arrestment response and causes ticks to cease activity, forming a cluster [3]. Formation of tick clusters in the environment may actually positively affect many aspects of tick life: clusters can enhance mating and host-finding success [3, 26]; in soft ticks clustering behaviour is believed to have survival value in that the individuals forming the mass accumulate in sites favourable for avoiding stressful environmental conditions, i.e. desiccation, and where they are more likely to encounter hosts [3].

In prostriate ticks, such as I. ricinus, clusters of host-seeking ticks are often found on vegetation and this is believed to favour contact with passing hosts [3]. Clustering of I. ricinus and I. scapularis on vegetation facilitates contact between the sexes. In a pioneering study by Graf [8] up to 70% of I. ricinus females collected from vegetation were already mated. In contrast to the metastriate ticks, which invariably mate while attached to their hosts, prostriate ticks frequently mate preprandially on the ground or on vegetation. Ixodes males of nidicolous species never seek hosts and are limited to preprandial mating. However, males of the I. ricinus complex can mate once again on the surface of their hosts [2].

Perhaps the most interesting feature of our study is the repeatability of our observation of an almost identical pattern of aggregation (cluster structure) across our three study sites, which were not contiguous. Indeed the sites are isolated from one another by distances from 50 to 200 kilometres, and differ in their vegetation cover (described in detail in [20, 30]). Despite this, the distribution of questing adult ticks on grass stems was very similar in each of our three sites, and therefore supportive for our research hypothesis. It would be of interest to conduct similar studies in other D. reticulatus-endemic regions, as well as on other metastriate tick species, to assess the generality of the phenomenon.

Conclusion. Mixed-sex clusters of D. reticulatus ticks on grass stems were significantly more common than single-sex clusters, and given that this tick species mates only on newly infested hosts, such a strategy would inevitably result in the simultaneous transfer of both sexes from grass stem to host following contact of a suitable host with the supporting grass stem. The widespread occurrence of this expansive tick species and the repeatability of our observations indicate that mix-sex clusters may enhance the efficiency of mate finding and hence mating, and thereby reflect the efficient breeding strategy of D. reticulatus ticks.

Acknowledgements

Not applicable.

Author contribution

The manuscript was written by AB and JMB, observation of the ticks was carried by AB, DW, WR and WM, statistical analysis was done by JMB. All authors read and approved the manuscript.

Funding

Not applicable.

Availability of data and materials

All relevant data are included in the article.

Ethics approval and consent to participate

Not applicable.

Competing interests

AB acts as Subject Editor in Parasites and Vectors.

Consent for publication

Not applicable.

Oliver JH. Tick reproduction: sperm development and cytogenetics. In: Physiology of Ticks. Current Themes in Tropical Science 1982, Chap. 8, 245 – 75.
Kiszewski AE, Matuschka FR, Spielman A. Mating strategies and spermiogenesis in ixodid ticks. Ann Rev Entomol. 2001;46:167–82.
Sonenshine DE. Pheromones and other semiochemicals of ticks and their use in tick control. Parasitol. 2004;129:405–25.
Sonenshine D, Roe R. Ticks, people, and animals. In: Biology of Ticks 2013.
Vargová B, Pipová N, Baňas M, Majláth I, Tryjanowski P, Jankowiak Ł, Majláthová V. Behavioral repertoire on a vertical rod- an ethogram in Dermacentor reticulatus ticks. Life (Basel). 2022;12:2086.
Pipová N, Peňazziová K, Baňas M, Majláth I, Majláthová V. The behavior of Rickettsia-positive Dermacentor reticulatus ticks under laboratory conditions. Life (Basel). 2023;13:612.
Buczek A, Zając Z, Woźniak A, Kulina D, Bartosik K. Locomotor activity of adult Dermacentor reticulatus ticks (Ixodida: Ixodidae) in natural conditions. Ann Agric Environ Med. 2017;24:271–75.
Graf JF. Ecology and ethology of Ixodes ricinus L. in Switzerland (Ixodoidea: Ixodidae). III: copulation, nutrition and oviposition. Acarologia. 1974;16:636–42.
Mierzejewska EJ, Pawełczyk A, Radkowski M, Welc-Falęciak R, Bajer A. Pathogens vectored by the tick, Dermacentor reticulatus, in endemic regions and zones of expansion in Poland. Parasit Vectors. 2015;8:490.
Dwużnik-Szarek D, Mierzejewska EJ, Kiewra D, Czułowska A, Robak A, Bajer A. Update on prevalence of Babesia canis and Rickettsia spp. in adult and juvenile Dermacentor reticulatus ticks in the area of Poland (2016–2018). Sci Rep. 2022;12:5755.
Ličková M. Havlíková SF, Sláviková M, Slovák M, Drexler JF, Klempa B. Dermacentor reticulatus is a vector of tick-borne encephalitis virus. Ticks Tick Borne Dis. 2020;11:101414.
Zając Z, Obregon D, Foucault-Simonin A, Wu-Chuang A, Moutailler S, Galon C, et al. Disparate dynamics of pathogen prevalence in Ixodes ricinus and Dermacentor reticulatus ticks occurring sympatrically in diverse habitats. Sci Rep. 2023;13:10645.
Földvári G, Široký P, Szekeres S, Majoros G, Sprong H. Dermacentor reticulatus: a vector on the rise. Parasit Vectors. 2016;9:314.
Rubel F, Brugger K, Pfeffer M, Chitimia-Dobler L, Didyk YM, Leverenz S, et al. Geographical distribution of Dermacentor marginatus and Dermacentor reticulatus in Europe. Ticks Tick Borne Dis. 2016;7:224–33.
Bilbija B, Spitzweg C, Papoušek I, Fritz U, Földvári G, Mullett M, et al. Dermacentor reticulatus - a tick on its way from glacial refugia to a panmictic Eurasian population. Int J Parasitol. 2023;53:91–101.
Brugger K, Rubel F. Tick maps on the virtual globe: First results using the example of Dermacentor reticulatus. Ticks Tick Borne Dis. 2023;14:102102.
Dwużnik-Szarek D, Mierzejewska EJ, Rodo A, Goździk K, Behnke-Borowczyk J, Kiewra D, et al. Monitoring the expansion of Dermacentor reticulatus and occurrence of canine babesiosis in Poland in 2016–2018. Parasit Vectors. 2021;14:267.
Drehmann M, Springer A, Lindau A, Fachet K, Mai S, Thoma D, et al. The spatial distribution of Dermacentor ticks (Ixodidae) in Germany—Evidence of a continuing spread of Dermacentor reticulatus. Front Vet Sci. 2020;7:578220.
Daněk O, Hrazdilová K, Kozderková D, Jirků D, Modrý D. The distribution of Dermacentor reticulatus in the Czech Republic re-assessed: citizen science approach to understanding the current distribution of the Babesia canis vector. Parasit Vectors. 2022;15:132.
Mierzejewska EJ, Welc-Faleciak R, Karbowiak G, Kowalec M, Behnke JM, Bajer A. Dominance of Dermacentor reticulatus over Ixodes ricinus (Ixodidae) on livestock, companion animals and wild ruminants in eastern and central Poland. Exp Appl Acarol. 2015;66:83–101.
Zając Z. Estimated population size of Dermacentor reticulatus (Fabricius, 1794) ticks in an endemic area of the species in eastern Poland. Ann Parasitol. 2020;66:593–97.
Zając Z, Woźniak A, Kulisz J. Density of Dermacentor reticulatus ticks in eastern Poland. Int J Environ Res Public Health. 2020;17:2814.
Oliver JH, Brinton LP. Cytogenetics of ticks (Acari: Ixodoidea). 7. Spermatogenesis in the Pacific Coast tick, Dermacentor occidentalis Marx (Ixodidae). J Parasitol. 1972;58:365–79.
Rohlf FJ, Sokal RR. Statistical tables. Macmillan. 1995.
Berger RS. 2,6-dichlorophenol, sex pheromone of the lone star tick. Science. 1972;177:704–5.
Allan SA. Tick pheromones. In: Ignell R, Lazzari CR, Lorenzo MG, Hill SR. Sensory ecology of disease vectors, 2022, Chap. 15.
Sonenshine DE. Tick pheromones and their use in tick control. Ann Rev Entomol. 2006;51:557–80.
Carr A, Sonenshine DE, Strider Jr. JB, Roe M. Evidence of female sex pheromones and characterization of the cuticular lipids of unfed, adult male versus female blacklegged ticks, Ixodes scapularis. Exp Appl Acarol. 2016;68:519–38.
Taylor D, Phillips JS, Allan SA, Sonenshine DE. Absence of assembly pheromones in the hard ticks Dermacentor variabilis and Dermacentor andersoni (Acari: Ixodidae). J Med Entomol. 1987;24:628–32.
Bajer A, Rodo A, Alsarraf M, Dwużnik D, Behnke JM, Mierzejewska EJ. Abundance of the tick Dermacentor reticulatus in an ecosystem of abandoned meadows: Experimental intervention and the critical importance of mowing. Vet Parasitol. 2017;246:70–5.

Competing interest reported. Anna Bajer is Subject Editor in Parasites and Vectors

Download PDF

Journal Publication

published 09 Feb, 2024

Read the published version in Parasites & Vectors →

Editorial decision: Revision requested
26 Nov, 2023
Reviews received at journal
13 Nov, 2023
Reviewers agreed at journal
07 Nov, 2023
Reviewers invited by journal
06 Nov, 2023
Editor assigned by journal
06 Nov, 2023
Submission checks completed at journal
06 Nov, 2023
First submitted to journal
04 Nov, 2023

You are reading this latest preprint version

Mixed-sex clusters on grass blades –breeding strategy of the ornate dog tick, Dermacentor reticulatus

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Methods

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1