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Experimental & Applied Acarology, 21 (1997) 747–754

Questing behaviour of Ixodes ricinus ticks (Acari: Ixodidae) H. A. Mejlona and T. G. T. Jaensonb* a

Department of Zoology, Uppsala University, Villavägen 9, S-752 36 Uppsala, Sweden b Medical Entomology, Department of Entomology, Swedish University of Agricultural Sciences and Zoological Museum, Uppsala University, Villavägen 9, S-752 36 Uppsala, Sweden (Received 19 April 1997; accepted 22 June 1997) ABSTRACT The vertical distribution in the vegetation of questing Ixodes ricinus ticks was investigated in two different vegetation types (‘high’ and ‘low’ vegetation) at two localities in south–central Sweden during 1992–1993 (Torö) and 1995 (Bogesund). Significant correlations were found between the vertical distribution of immature ticks and the height of the vegetation. The greatest mean availabilities of the larvae and nymphs in low vegetation were in the intervals 0–9 and 30–39 cm, respectively. The larval numbers were greatest close to the ground (0–29 cm) in both high and low vegetation. The larval : nymphal ratio, at ground level at localities free of ground vegetation, varied between 8 : 1 and 32 : 1. In high vegetation, the greatest mean numbers of nymphal and adult ticks were at height intervals of 50–59 and 60–79 cm, respectively. These ranges are within the estimated height interval (40–100 cm) of the main part of the body surface of their ‘preferred’ host, the roe deer (Capreolus capreolus). The presence of most questing I. ricinus larvae at ground level would favour the transmission of Borrelia burgdorferi s.l., since this is where the highly reservoir-competent rodents and shrews usually occur. Key words: Ixodes ricinus, Borrelia burgdorferi, vertical distribution, host-seeking activity, questing behaviour, host. INTRODUCTION

The vertical distribution of questing Ixodes ricinus is influenced by many factors including the height and other physical properties of the vegetation. For instance, investigations by Lees and Milne (1951) on the vertical distribution of questing I. ricinus ticks in natural vegetation, showed that the majority of ticks were questing close to the tips of the vegetational parts. The gravity, humidity and temperature will also influence the movement and vertical distribution of questing ticks (Lees, 1948; Belozerov, 1982). The desiccation tolerance generally increases with tick age (stage). Therefore, the larvae tend to quest lower in the vegetation than the nymphs or adults (Gigon, 1985). However, such a distribution pattern could also be due to a ‘preference’ of particular stages of I. ricinus to quest for hosts of certain sizes. Tälleklint and Jaenson (1994) recorded that near Stockholm approximately 70% of all larval engorgements took place on small mammals such as shrews (Sorex spp.) *To whom correspondence should be addressed at: Fax: +46 18 559888; e-mail: [email protected]

0168–8162 © 1997 Chapman & Hall

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H. A. MEJLON AND T. G. T. JAENSON

and rodents (Apodemus spp., Clethrionomys glareolus and Microtus agrestis), whereas the majority of nymphal and female ticks fed on larger mammals such as roe deer (Capreolus capreolus) and hares (Lepus spp.). Thus, in order to maximize the likelihood of encountering the main part of their ‘preferred’ hosts, nymphs and adults should be questing higher above ground than larvae. The aim of this study was to investigate whether questing I. ricinus ticks tend to occur in relatively distinct, tick stage-associated vertical zones in the vegetation and if such a potential distribution pattern may be related to one or more of the following factors: (1) meteorological variables, (2) vegetation structure and (3) size of the ‘preferred’ mammalian hosts. MATERIALS AND METHODS

Study localities The main field investigations were carried out at Torö (58°50'N, 17°51'E), an island situated 57 km south of Stockholm (June–August 1992 and 1993) and at Bogesund (59°25'N, 18°10'E), 10 km north of Stockholm (June and September 1995). Ticks were collected once monthly. Additional studies were performed at three localities ¨ in south-western Sweden: Dagsås (57°04'N, 12°30'E) and Anggårdsbergen (57°41'N, 11°57'E) in July 1995 and Hallands Väderö (56°26'N, 12°34'E) in August 1995. The study area at Torö was located in a mixed forest clearing made for electrical power lines. The vegetation was dense and consisted mainly of young alder bushes (Alnus glutinosa) approximately 2 m high and grasses and ferns up to approximately 1 m high. At Bogesund, two different vegetation types were studied: a meadow at a forest edge and a herbaceous pine forest. The herbal layer in the pine forest at Bogesund was considered to represent the ‘low’ vegetation type (i.e. generally 0–50 cm high but occasionally reaching 80 cm). The vegetations studied at Torö and the open site at Bogesund were considered to represent the ‘high’ vegetation type (i.e. 0–150 cm and occasionally higher). The study sites in broad¨ leaf forests at Dagsås, Anggårdsbergen and Hallands Väderö were, in general, almost devoid of any ground vegetation but covered with dead beech (Fagus sylvatica) leaves. Environmental variables and tick abundance For both the high and low vegetation types, the vertical distribution of the vegetation, i.e. the potential tick questing sites, was estimated by visual quantification for each 10 cm increment. This measure, i.e. the distribution of the vegetational apices, is henceforth referred to as the height of vegetation. The air temperature and relative humidity (RH) were measured at Torö and Bogesund. At Torö, these variables were measured 10 cm above the ground. At Bogesund, the temperature was measured at 0, 10, 50 and 100 cm above the ground and the RH at 10 and 50 cm. Sampling was not conducted during or shortly after rainfall. To sample the ticks, a dress of white cotton flannel cloth was used. The dress totally covered the collector between neck and ankles and was marked at every

QUESTING BEHAVIOUR OF IXODES RICINUS

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10 cm from 10 to 140 cm above the ground. The collector walked slowly with his arms raised through the vegetation. Ticks that attached to the cloth were collected at approximately every 15 m. The stage of each tick and its location on the dress was recorded. The ticks were subsequently released. In each biotope, eight to 18 stops to collect ticks were made. At Torö and Bogesund it was, in general, not possible to record ticks at the 0–10 cm level. However, in the pine forest at Bogesund, where the ground cover was relatively sparse, the tick availability between 0 and 10 cm was estimated using a small flag (10 3 20 cm). To estimate the ratio between the abundance (availability) of the different active tick stages, blanket-dragging with a 1 3 1 m white flannel cloth was performed at ¨ Dagsås, Anggårdsbergen and Hallands Väderö in the forested study areas where the ground cover was very sparse or absent. The tick availability is defined here as the number of questing ticks that attached to the cloth per unit of walking distance. To calculate the effective area sampled on each occasion and at each of the 13 different height intervals, we estimated the collector’s horizontal body coverage to be 30 cm, i.e. approximately the hip width of the collector. The total area covered per height interval per sampling occasion thus ranged from 42 to 88 m2. The surface areas of the different potential mammalian host species were estimated from drawings of museum specimens. A height range of 0–10 cm represents the surface areas of Sorex minutus, Sorex araneus, M. agrestis, C. glareolus, Apodemus flavicollis and Apodemus sylvaticus, a range of 10–30 cm Lepus timidus and a range of 40–100 cm roe deer. To calculate the likelihood of questing ticks to encounter hosts at Bogesund and Torö, we used previously published data on the density of the main tick hosts at Bogesund (Tälleklint and Jaenson, 1994). We considered the vertical distribution of the host surface areas as approximately the same in all biotopes investigated. This approximation was deemed acceptable since, in general, small mammals tend to be more abundant than medium-sized or large hosts in most mainland biotopes in southern and central Sweden, even if the species composition of the potential hosts for I. ricinus may vary among biotopes. Statistical methods Each tick collected was treated as a separate case and assigned a value corresponding to the height interval where the tick was collected. Comparisons of the height locations between the tick stages were made using the Kruskal–Wallis test (Sokal and Rohlf, 1981). Spearman rank order correlations were used to compare the frequency of the host surface area and height of the vegetation with the mean tick availability. RESULTS

Vertical distribution of ticks in high vegetation All stages of I. ricinus were found in all 13 different height intervals from 10 to 140 cm above ground level, except adult ticks, which were absent from one interval (Table 1). The intervals with the greatest availability were 10–19 cm for the larvae,


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50–59 cm for the nymphs and 60–79 cm for the adults (Table 1). In the height range 10–140 cm the mean heights (6 S.D.) of occurrence of the larvae, nymphs and adults were 50.2 6 28.9, 58.8 6 29.9 and 66.0 6 31.3 cm, respectively. The questing heights recorded in high vegetation differed significantly among the larvae, nymphs and adults (Kruskal–Wallis test, H(2,1381) 5 33.1 and p , 0.0001). This difference was mainly due to the larvae questing significantly lower than nymphs (Kruskal–Wallis test, H(1,1346) 5 27.4 and p 5 0.0001). There was no difference in questing heights between the nymphs and adults. Vertical distribution of ticks in low vegetation Ixodes ricinus subadults were absent from at least two different height intervals (Table 1). The greatest mean availabilities of the larvae and nymphs were in the intervals 0–9 and 30–39 cm, respectively. No adult ticks were found here. The mean questing heights for the larvae and nymphs were 17.6 6 10.0 and 27.0 6 16.4 cm (only five ticks), respectively. These means are not significantly different. Vertical distribution of ticks in relation to vegetation and mammalian hosts The vertical distributions of the I. ricinus larvae, nymphs and adults in relation to the height of the vegetation and host surface areas are shown in Fig. 1(a) and (b). TABLE 1 Estimated proportion of host target area (% host; 100% 5 7740 cm2/hectare), vegetational heights (% veg) and estimated density (mean numbers) of I. ricinus larvae, nymphs and adults per 100 m2 in different vertical zones between 0 and 140 cm above ground level High vegetation

Low vegetation

Height interval (cm)

% host % vegetation Larvae Nymphs Adults

% vegetation Larvae Nymphs Adults

0–9 10–19 20–29 30–39 40–49 50–59 60–69 70–79 80–89 90–99 100–109 110–119 120–129 130–139

41.5 5.4 5.4 1.1 9.9 11.0 11.0 11.0 1.7 1.7 0.3 NH NH NH

15.0 25.0 25.0 13.0 7.0 7.0 4.0 4.0 NV NV NV NV NV NV

11.0 14.0 14.0 13.0 12.0 9.4 5.7 4.9 3.5 2.6 2.2 1.6 1.1 1.1

– 20.0 19.3 14.6 12.7 15.7 13.0 9.7 7.5 5.7 7.3 2.2 1.4 0.4

– 6.3 9.7 10.2 10.4 11.5 10.1 8.1 6.2 5.2 4.5 1.8 3.6 1.0

– 0.5 0.5 0.2 0.5 0.3 1.0 1.0 0.3 0.6 0.5 0.2 0.0 0.2

61.9 35.7 50.3 4.0 1.3 1.3 0.0 0.0 NV NV NV NV NV NV

2.4 1.3 0.0 2.6 1.3 0.0 0.0 0.0 NV NV NV NV NV NV

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NV NV NV NV NV NV

–, no data; NH, no hosts; NV, no vegetation Two different vegetational types were sampled: high (semi-open bush/meadow) and low (herbaceous pine forest). Ticks were collected at Torö (June–August 1992–1993) and Bogesund (June and September 1995) near Stockholm, Sweden.


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Fig. 1. Estimated proportion of main hosts’ surface areas (shaded bars) and vegetation heights (open bars) in (a) high and (b) low vegetation types. The lines represent the density, i.e. the mean numbers per 100 m2, of questing I. ricinus larvae (dotted line), nymphs (broken line) and adults (solid line) at the vertical range 0–140 cm. The ticks were sampled near Stockholm on eight occasions in 1992 (Torö) and 1995 (Bogesund).


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In high vegetation, within the interval 10–140 cm, the larval and nymphal distribution patterns were similar to that of the height of the vegetation. They were clearly dissimilar from the distribution pattern of the host surface areas. In addition, in low vegetation the larval tick distribution data fitted the distribution of the height of the vegetation. The nymphal data were considered inadequate for statistical evaluation. Correlations between the tick numbers and the estimated proportions of the height of the vegetation gave significant positive coefficients in three out of five possible combinations, whereas the correlations between the tick numbers and the host surface area variable were non-significant in all combinations (Table 2). The temperature and RH varied only slightly between the different levels (0, 10, 50 and 100 cm) and were not significantly associated with tick vertical distribution. Proportion of questing larvae to questing nymphs In each biotope, the larval to nymphal availability ratio was estimated from the total numbers of each stage recorded for the whole range of vegetation sampled, i.e. 10–140 cm (high vegetation) or 10–80 cm (low vegetation). For comparison, the following data from the blanket drag samplings were used: (1) areas free of ground ¨ vegetation but covered with dead beech leaves (Anggårdsbergen, Dagsås and Hallands Väderö) in 1995 and (2) low vegetation biotopes at Bogesund and high vegetation biotopes at Torö in 1991–1992. By dress sampling, the larval : nymphal ratios were 1.4 : 1 (high vegetation, 620 m2 sampled) and 19 : 1 (low vegetation, 80 m2 sampled). The larval : nymphal ratios based on previous blanket dragging (H. A. Mejlon unpublished data) in these areas were 7.5 : 1 (1800 m2), and 25 : 1 (500 m2), respectively. Finally, the areas free of ground vegetation yielded larval : nymphal ratios of 18 : 1 (Hallands ¨ 100 m2) and 32 : 1 (Dagsås, 100 m2). Väderö, 40 m2), 8.3 : 1 (Anggårdsbergen, TABLE 2 Spearman rank correlations between tick availability, i.e. the numbers of questing I. ricinus collected and the estimated vertical proportion in 10 cm increments of host surface area or vegetation heights Host surface area

Vegetation height

Tick stage

Vegetation

Number of ticks

rs

p value

n

rs

p value

n

Larva Nymph Adult Larva Nymph Adult

High High High Low Low Low

796 550 35 96 5 0

0.37 0.60 0.47 –0.14 –0.17 No ticks

NS NS NS NS NS observed

10 10 10 8 8

0.96 0.80 0.34 0.92 0.45

0.0001 0.001 NS 0.01 NS

13 13 13 8 8

The ticks were sampled in the interval 0–140 cm above ground level in high and low vegetation types at two localities (Torö and Bogesund) near Stockholm. rs, Spearman rank correlation coefficient. n, number of vertical increments. NS, not significant at the 0.05 level.


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Thus, in high vegetation, the larval : nymphal ratio based on dress sampling (1.4 : 1) was considerably less than that of both blanket dragging (7.5 : 1) and vegetation-free ‘control’ areas (8.3 : 1–32 : 1). In low vegetation, the larval : nymphal ratios seemed less variable between the dress sampling (19 : 1), blanket dragging (25 : 1) and control areas (8 : 1 –32 : 1). DISCUSSION

Not surprisingly, in high, partly open vegetation, the distribution of larval and nymphal ticks appeared strongly associated with the vegetation structure, i.e. the height of the vegetation (Table 2). Nevertheless, both larvae and nymphs were recorded at all height intervals between 10 and 140 cm. However, the mean height at which the larvae were present was significantly lower than those of nymphs or adults. In low vegetation, only the larval vertical distribution appeared to be correlated with the vegetation structure. The vertical distribution of the adult ticks appeared to be less dependent on the vegetation structure, although larger samples are needed to confirm this. The vegetation structure affects the microclimate in which the ticks live and will therefore influence their water balance. According to Lees and Milne (1951), adult I. ricinus spend only approximately 30% of their time questing above ground. The remainder is spent on the ground where the humidity is usually high, whereby the water balance can be restored. The same should apply to immature ticks, but since nymphs and in particular larvae are more sensitive to desiccation than adults, these stages are likely to quest lower and to spend less time above ground level. The work by Gigon (1985) on Swiss I. ricinus populations in artificial arenas showed that subadults generally quest at lower heights (7–11 cm) than adults (10–50 cm) and that this pattern of tick vertical distribution is related to habitat type. Because I. ricinus larvae mainly infest small mammals while nymphs and adults usually feed on medium-sized and large mammals (Tälleklint and Jaenson, 1994), a lower mean height of distribution of questing larvae compared to that of questing nymphs and adults conforms to their partly different host associations. This is supported by the fact that the larval density in both high and low vegetation was greatest at the lowest intervals sampled (10–19 and 0–9 cm, respectively). The mean questing heights for larvae in high (50.2 cm) and low (17.6 cm) vegetation types indicate that the host size (small mammals) as well as the vegetation structure and the relatively low degree of desiccation tolerance of the larvae may influence their vertical distribution in the habitat. Since they presumably spend most of their time at ground level, they would actually have greater opportunities to contact small mammals here. The mean questing heights recorded at Torö and Bogesund are biased since ticks questing at 0–10 cm could not be recorded. It is reasonable to assume that the majority of questing tick larvae occurred below 10 cm. Therefore, we estimated the larval and nymphal availabilities below 10 cm by blanket dragging in vegetation-free areas. It should be emphasized that the ratio of questing larvae to questing nymphs at a particular locality is likely to vary during the season because of their varying seasonal

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densities. As expected, at ground level, the larval : nymphal ratios were 8.3 : 1–32 : 1 by blanket dragging compared to 1.4 : 1 by dress sampling in vegetation of .10 cm. Approximately 80% of the feeding nymphs are expected to take their meals from hares and cervids (Tälleklint and Jaenson, 1994). This means that an optimal questing site for these nymphs would be located approximately 10–100 cm above ground. Our data conform to this and show that the greatest mean abundance of the nymphs was in the height range of 30–39 cm in low vegetation and at 50–59 cm in high vegetation (Table 1 and Fig. 1). Approximately 45% of feeding adult I. ricinus females will take their blood meal from roe deer (Tälleklint and Jaenson, 1994). The proportion of adult I. ricinus questing in the ‘roe deer zone’ (40–100 cm) was 63%. Thus, for the adult ticks our data also conform to the ‘host size-dependent questing strategy hypothesis’. Other investigations on the host-seeking behaviour of ticks include that of Loye and Lane (1988). They recorded that adult Ixodes pacificus were mainly questing close to the tips of 25 and 50 cm wooden dowels, while 75 cm dowels were used less often. Cervids and lagomorphs are major hosts of adult I. pacificus. The body sizes of these hosts correspond well to the mean questing height of the adult ticks. ACKNOWLEDGEMENTS

We are very grateful to Jeremy Gray and Lars Tälleklint for valuable comments on an earlier version of this paper. This work was supported by grants from the Swedish Natural Science Research Council to T. G. T. Jaenson. REFERENCES Belozerov, V.N. 1982. Diapause and biological rhythms in ticks. In Physiology of ticks, F.D. Obenchain and R. Galun (eds), pp. 469–500. Pergamon Press, New York. Gigon, F. 1985. Biologie d’Ixodes ricinus L. sur le Plateau Suisse – une contribution a` l’écologie de ce vecteur. Doctoral thesis, Faculty of Sciences, University of Neuchâtel. Lees, A.D. 1948. The sensory physiology of the sheep tick Ixodes ricinus L. J. Exp. Biol. 25: 145–207. Lees, A.D. and Milne, A. 1951. The seasonal and diurnal activities of individual sheep ticks (Ixodes ricinus L.). Parasitology 41: 189–208. Loye, J.E. and Lane, R.S. 1988. Questing behavior of Ixodes pacificus (Acari: Ixodidae) in relation to meteorological and seasonal factors. J. Med. Entomol. 25: 391–398. Sokal, R.R. and Rohlf, F.J. 1981. Biometry, 2 edn. W.H. Freeman and Co, New York. Tälleklint, L. and Jaenson, T.G.T. 1994. Transmission of Borrelia burgdorferi s.l. from mammal reservoirs to the primary vector of Lyme borreliosis, Ixodes ricinus (Acari: Ixodidae), in Sweden. J. Med. Entomol. 31: 880–886.