Study areas
This study took place along two mountain slopes in Norway during 2017 and 2018 (Fig 1). The first study area was a southern facing mountain slope on the Lifjell massif (N59°26.495’ E9°0.603’), north of Bø i Telemark. It is characterized by a continental climate, located within the boreonemoral to southern boreal zone. Below the tree line, which is situated between 800 and 900 m.a.s.l., the vegetation is a blend of deciduous and coniferous forests with birch (Betula pubescens) and spruce (Picea abies) as the dominant tree species, and blueberries (Vaccinium myrtillus) as the dominant species at ground layer. Below the tree line the vegetation is mostly homogeneous. Above the tree line the vegetation is primarily dominated by common heather (Calluna vulgaris) and blueberries. Boulder fields occur frequently throughout the gradient, and the highest peak found on the plateau is 1288 m.a.s.l. Most of the data collection points were located on topographically open hillside. Temperature and precipitation normals for the study areas can be found in Appendix 3. The second study area was located in the Erdal valley (N61°05.817’ E7°24.688’) near Lærdalsøyri (hereafter referred to as Lærdal). It is a northern facing mountain slope close to the innermost part of the Sognefjorden fjord, approximately 150 km east of the western coastline. Due to its proximity to the fjord, the climate is more maritime than Lifjell, characterized by cooler summers and milder winters (Appendix 3). Sampling points were mostly located within the valley formed by the Erdal river. The tree line is here situated between 900 and 1000 ma.s.l., and below the tree line the vegetation consists primarily of homogeneous deciduous forests with birch and alder (Alnus glutinosa) as the dominant tree species. At ground layer the vegetation is dominated by blueberries, and different species of ferns and tall perennial herbs. Above the tree line common heather, dwarf birch (Betula nana), common juniper (Juniperus communis) and crowberry (Empetrum nigrum) are the dominant species. Surrounding the study area are several mountain peaks exceeding 1500 m.a.s.l.
Study species
I. ricinus is the most common and the most studied tick species in Europe [18]. It ranges latitudinally from North Africa to Scandinavia, and longitudinally from Ireland to Russia [40]. It is a three-host tick feeding on a wide range of mammals, birds and reptiles [41]. Immature life stages (larvae and nymphs) parasitize small mammals in larger proportions [42], whereas adults tend to feed on larger mammals [41]. It is common to deciduous, and to some extent, coniferous forests, and is dependent on sufficient temperature and humidity to be able to quest (actively seeking a host). It is sensitive to desiccation and temperature extremes [43].
I. trianguliceps occurs throughout Europe, ranging latitudinally from Italy up to well above the Arctic circle [44]. Contrary to I. ricinus, I. trianguliceps specializes primarily on rodents and other small mammals during all life stages [45, 46]. It is endophilic (nest-dwelling), spending its off-host time within the burrows of its host to molt and quest [47]. It occurs in widely different habitats, ranging from meadows, peat bogs to dark-coniferous forests, mixed and deciduous forests, as well as high altitude treeless zones [42, 45]. It is generally considered to be one of the most cold resistant ticks of the genus Ixodes in the Palearctic region [45]. Since it rarely infests humans or livestock due to its host seeking behaviour [48], cases of tick-borne infection are considered exceptional [15], but it does contribute to maintaining the infection cycle of several pathogens between I. ricinus and their hosts such as Borrelia burgdorferi [49] and Anaplasma phagocytophilum [50].
The bank vole is a commonly found rodent throughout Europe and occurs virtually everywhere in Fennoscandia [51]. It is the most common rodent species in both study areas. Other small mammal species present are the field vole (Microtus agrestis), tundra vole (M. oeconomus), grey red-backed vole (Myodes rufocanus), wood mouse (Apodemus sylvaticus), yellow-necked mouse (A. flavicollis) and house mouse (Mus musculus). The common shrew (Sorex araneus), pigmy shrew (S. minutus) and water shrew (Neomys fodiens) are also found in these areas. The bank vole is a reservoir host for several tick-borne pathogens such as B. burgdorferi [52], Babesia microti [53], Candidatus Neoehrlichia mikurensis [54], and A. phagocytophylum [55]. It is possibly the most important host for all life stages of I. trianguliceps [42], and is heavily infested by the immature stages of I. ricinus [56, 57]. Because it was common at both study areas and at all altitudes, we chose to base our analysis on this species.
Host trapping
Ticks are commonly collected by two methods: cloth dragging for questing (unfed) ticks and host examination for feeding ticks. The cloth dragging method is only applicable for the collection of exophilic species, and only when the vegetation is dry. During most days of data collection the vegetation was either partly wet or weather conditions did not allow for cloth dragging. Additionally, as we aimed to include the infestations of I. trianguliceps in this study, we focused solely on the capturing of hosts. However, examining tick burdens accurately from live small mammals can be difficult [58], therefore we opted for a combination of lethal traps and euthanized live captures. In both study areas, 10 trapping stations were set up along a vertical gradient ranging from 100 to 1000 m.a.s.l. at every 100 m altitude interval. Bank voles have relatively small home ranges [59], and altitude stations were located several hundred meters from each other, hence we can be reasonably confident that ticks collected from host at a certain altitude were also acquired in the immediate vicininy. At each trapping station, two plots of 20 traps each were deployed, one with live traps (Ugglan Special Nr. 2, Grahnab AB, Sweden, www.grahnab.se), the other with lethal traps (Rapp2 Mousetrap, www.rappfellene.no). The traps in both plots were arranged in a 4 by 5 grid, with 10 meters spacing between each trap. Live traps were baited with a slice of apple for hydration and whole oats for caloric value, and a lining of sawdust was provided on the trap floor as insulation. Lethal traps were baited with peanut butter for practical reasons as it is easily applied to the inside of the trap body. Trap type does not influence tick burden size on the captured animals [60]. At each altitude, the live and lethal plots were placed at approximately 100 m distance from each other, but in locations with similar vegetation structure and habitat characteristics. As humidity and temperature have a direct influence on tick activity, they are important drivers of phenological patterns and host-seeking behavior [61]. For this reason, a datalogger (TinyTag Plus 2 – TGP 4017, housed in a DataMate instrument cover ACS-5050) was placed in between the two plots at each trapping station, approximately 50 cm above ground level measuring air temperature and relative air humidity at a 1-hour interval for the duration of the trapping period. The height of 50 cm was chosen to capture the general variation in environmental conditions at each altitude. Trapping took place during the spring (May 20th – 30th), summer (July 20th-30th) and autumn (September 20th-30th) seasons of the years 2017 and 2018. As an exception, during the spring season of 2017, capturing took place from June 1st until June 7th, and only up to 700 m.a.s.l. in both areas, as there was too much snow to allow for the operation of traps earlier and above this altitude. During each trapping period, traps were checked every 24 hours, and the collection of trapped animals started at 8h30. When checking the trapping grids, triggered lethal traps were rebaited and reset. As examining live small mammals for ticks can be stressful and cause injury or death [62], all animals captured in live traps were euthanized by cervical dislocation of the head upon collection, and each individual was kept separately in a sealed and coded plastic bags. Activated live traps were emptied of the remaining contents, and new insulation and food was provided before resetting the traps. At the end of every collection day, all animals were place in a freezer at -20°C.
Laboratory processing
At the end of every trapping season, captured bank voles were examined for ticks in the laboratory, as a full body post-mortem examination provides the highest degree of sensitivity [63] The day before the examination, the voles were removed from the freezer and left to thaw overnight at 10°C. The voles were examined one by one and taken out of the plastic bags individually. The empty bags were checked for ticks that might have dropped off. It was our observation that a number of ticks would drop off the host when the animals were placed in the freezer, possibly in an attempt to escape the extreme temperatures. Animals that were wet were dried with a hairdryer before examination. The hosts were checked for ticks starting with the head, ears and snout, followed by the neck and throat, back and abdomen, legs, feet and tail. Attached or detached ticks were removed from the host using tweezers. Collected ticks were removed and placed in a 1.5 mL plastic Eppendorf tube containing a 70 % ethanol solution (1 vial per host). Finally, a lice comb was brushed through the fur of the animal from tail to head (against the hair orientation), and the vole was shaken by the tail above a white plastic tray to collect any ticks that might have been missed during the examination. The hosts were then weighed to the nearest 10th of a gram, and the sex was determined. The minimum amount of time needed to process one animal was 20 minutes. After the examination the animals were bagged in new plastic bags and refrozen at -20°C.
Ticks were determined for life stage and species under a Zeiss Discovery V20 stereomicroscope, using an established publication key as reference [64]. Because more than 94 % and 75 % of all I. ricinus and I. trianguliceps collected were larvae, only the larval stage was included in the analysis.
Data analysis
The statistical analyses were performed using the software package R version 3.5.3 [65]. The analysis of I. ricinus larvae and I. trianguliceps larvae was performed separately. As is usually the case with tick presence on small mammals, neither species was evenly distributed on the hosts [66], and 13.8 and 82.0 % of the hosts had no I. ricinus and I. trianguliceps larvae present, respectively. We therefore chose to use the presence or absence of larvae as the response variable and applied generalized linear modeling with a binomial distribution, i.e. logistic regression. The probability of encountering a tick is defined as prevalence. As predictor variables we considered altitude (ranging from 100 to 1000 ma.s.l. – as a continuous variable), study area (Lifjell and Lærdal), collection year (2017 and 2018), season (spring, summer and autumn) and humidity (%). Because altitude and temperature were negatively correlated (Pearson correlation test, t=-8.1578, df=1323, p<0.001, r=-0.219), temperature per se was not used as a predictor variable to avoid introducing collinearity into the model. Two-way interactions between season and altitude, study area and altitude, year and altitude, study area and year, as well as year and season were included in the starting model. We also considered individual bank vole body mass (in grams) and sex (male or female) as intrinsic co-variates. Starting with a full starting model containing all variables and the two-way interactions listed above, we used a backward step model selection process to progressively remove non-significant predictors - by comparing the residual deviance and degrees of freedom of nested models using a Chi-square test - until an optimal model, containing only significant predictor variables (α=0.05), was found. To visually represent the infestation probabilities of both species across study areas, collection years and seasons, we created multiple regression line plots using the ggplot2 package in R [67].
Ethics statement
This study was carried out in strict accordance with regulations issued by the Norwegian Environment Agency, and a permit was provided prior to the start of the sampling (Miljødirektoratet, reference number: 2017/4651) for the duration of the trapping period. The trapping protocol for animal capture was approved by the Animal Ethics Committee of the Department of Nature, Health and Environment (University of South-Eastern Norway). All efforts were made to minimize animal suffering.