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First published online May 18, 2006
Journal of Experimental Biology 209, 2138-2142 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02238
Walking by Ixodes ricinus ticks: intrinsic and extrinsic factors determine the attraction of moisture or host odour
Department of Zoology, South Parks Road, Oxford OX1 3PS, UK
* Author for correspondence (e-mail: sarah.randolph{at}zoo.ox.ac.uk)
Accepted 22 March 2006
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Key words: tick, Ixodes ricinus, walking, humidity gradient, host kairomone
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Lees, 1946;
Macleod, 1935). Thus
locomotion is principally in the vertical plane, while horizontal displacement
is conventionally thought to be very limited. Recent work by Perret et al.
(Perret et al., 2003),
however, revealed that I. ricinus nymphs walk far greater distances
than was previously imagined. Continuous automated observations of ticks
constrained within vertical channels revealed walking up and down the channels
over distances of several metres, most often after periods of quiescence,
which was interpreted as the equivalent of horizontal displacement. Most
walking occurred soon after the onset of darkness, and ticks walked further
after quiescence when subjected to increasing moisture stress. In field
experiments, I. scapularis Say nymphs dispersed average distances of
2-3 m, and adults >5 m, over a period of several weeks, apparently by their
own locomotion (Carroll and Schmidtmann,
1996).
Many terrestrial invertebrates move up humidity gradients towards moisture,
for obvious reasons, and a range of Ixodid tick species respond to certain
host-produced substances (kairomones). Carbon dioxide, for example, stimulates
some tick species to move, sometimes towards the source, e.g. Amblyomma
variegatum Fabricus, A. hebraeum Koch
(Anderson et al., 1998;
Barré et al., 1997;
McMahon and Guerin, 2002;
Norval et al., 1989), although
not Ixodes spp. to any great extent
(Lavender and Oliver, 1996;
Schulze et al., 1997).
Eructions of volatile rumen metabolites from ungulates also attract
Amblyomma and Ixodes species
(Donzé et al., 2004). In
contrast, substances from glands on the legs of deer and the dorsal surface of
dogs' ears elicit an arrestant response so that ticks (Dermacentor
variabilis Say, Amblyomma americanum L. and I.
scapularis) aggregate in places associated with host presence
(Carroll, 2002;
Carroll et al., 1995;
Carroll et al., 1996).
In this study we set out to answer the following questions arising from
these observations: to what extent do I. ricinus ticks walk
spontaneously in the horizontal plane, and do they do this to seek
the two resources that are crucial for their survival, water vapour and/or
hosts? Furthermore, do they move at random, or do humidity gradients and/or
volatile host odours direct their walking? We also investigated whether ticks
behaved differently depending on the level of their energy (fat) reserves and
state of hydration, as both these factors affect the tick's critical
equilibrium humidity, the atmospheric humidity at which water efflux and
influx are balanced (Lees,
1946).
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Materials and methods |
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1°43'W,
51°33'N) by conventional blanket-dragging. In the laboratory they
were stored in 2x8 cm tubes closed with gauze, held over saturated
magnesium sulphate solution in closed vessels to maintain an atmosphere of 90%
relative humidity (RH) (Winston and Bates,
1960). Three groups of ticks were exposed to different conditions.
Group 1, high-fat, fully hydrated: ticks were kept in the dark within a
refrigerator at 4°C. Group 2, high-fat, semi-dehydrated: as group 1, but
the day before use 100 ticks were exposed to 50% RH at 24°C for 4 h,
followed by 80% RH at 4°C for 8 h overnight and finally 80% RH at 24°C
for 1 h before being introduced to the arena. Group 3, low-fat, fully
hydrated: immediately after collection, ticks were held at room temperature
(22°C), exposed to the natural light:dark regime for
May-June, over a 4-week period in order to induce a lower fat content
(Randolph and Storey, 1999),
before being returned to the refrigerator until use from July onwards.
To verify that the fat content of the high-fat and low-fat groups of ticks
did indeed differ, samples of 40 ticks from each of groups 1 and 3 were
subjected to the standard method of lipid content estimation
(Randolph and Storey, 1999).
Briefly, the ticks were dried, individually weighed, immersed in three changes
of chloroform for 24 h each, re-dried and re-weighed to the nearest 0.1
µg.
Experimental design
Four choice arenas each consisted of two transparent polystyrene boxes
(17.5 cmx12 cmx5 cm) with tight-fitting lids, joined by an acetate
tunnel (3.8 cm diameter, 28 cm long) sealed into holes cut in one side of each
box. Humidity was measured with a Squirrel data logger (Grant Instruments Ltd,
Cambridge, UK), using a probe placed within different parts of the arena.
Placing a small dish of water in one box and leaving the other box empty
produced a realistic even humidity gradient of 97-67% RH within 1 h, changing
to 100-79.5% after 24 h; At the ambient temperature within the experimental
room (20°C), this is equivalent to a saturation deficit (SD) of 5.65-3.51
mmHg at the dry end [SD=(1-RH/100)4.9463e0.0621T where RH is
percentage relative humidity and T is temperature (°C)
(Randolph and Storey, 1999)];
this is similar to midday summer conditions measured 30 cm above ground level
in woodland (S. Randolph, unpublished observations). Host odour was collected
by rubbing the back of a dog's ear thoroughly with a 4 cmx4 cm piece of
absorbent medical gauze that was then sealed in a polythene bag and chilled
until used within 24 h. The gauze was placed in one box of the arena at the
same time as the ticks were introduced, after which the apparatus was sealed
for the duration of each experiment.
For each replicate, a set of 20 nymphs was placed into a 1 cmx3 cm
clear plastic tube with both ends closed by gauze held in place by elastic
bands. A length of cotton thread was attached to each piece of gauze. These
tubes were returned to the humidity chambers within the refrigerator for
several days before use. To start an experimental run, one tick-laden tube was
slipped into the centre of each arena tunnel with the cotton threads passing
out through the closed box lids. The arena was then left undisturbed for 1 h,
after which the threads were pulled very gently to remove the gauzes from each
end of the tube. The gauzes, with any ticks on them, were left close to the
tube. Following Perret et al.'s observations
(Perret et al., 2003) that
most ticks started to walk after the onset of darkness, for the first hour
after the ticks were free to move the lights were left on, but then switched
off, leaving only a red safety light to allow observations. The positions of
the ticks (whether in the tunnel or the box) were scored at 1, 2 and 24 h
post-release. More frequent and prolonged (up to 48 h) pilot observations had
revealed that well over half the ticks that left the introduction tube had
moved to their final position within the first couple of hours; ticks were not
observed to move to and fro along the arena, and the pattern of the
distribution of ticks within each arena did not change beyond 24 h.
Each of the following conditions (a-d; not in alphabetical order, match those in Figs 1 and 2) was replicated four times simultaneously for each of the group 1 (high-fat) and group 3 (low-fat) ticks by using the four arenas; two arenas were set at right angles to the other two, and the test conditions were arranged at opposite ends within each orientation. (a) Humidity gradient (see above) with no host odour, to test the hypothesis that I. ricinus nymphs move up a humidity gradient. (c) Host odour gradient in a uniformly dry atmosphere (67% RH, 5.65 mmHg SD), and (d) host odour gradient in a uniformly wet atmosphere (100% RH, zero SD), to test the hypothesis that I. ricinus nymphs respond to host kairomones. (b) Humidity gradient plus host odour gradient, with the host odour placed at the dry end, to test whether I. ricinus nymphs move towards host odour despite the risk of desiccation or vice versa. Group 2 (semi-dehydrated high-fat) ticks were exposed only to condition a, using 25 nymphs per replicate.
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Statistical analysis
The response of each tick was treated as independent because nymphal I.
ricinus ticks are not known to interact in such a way as to influence the
onset of each other's walking activity, and ticks did not aggregate in
clusters that would indicate the operation of assembly pheromones or
con-specific arrestment stimuli (Grenacher
et al., 2001; Guerin et al.,
2000; Sonenshine,
2004). Furthermore, within each of the combinations of
environmental and physiological conditions, all four replicates could be
combined because there was no significant difference between replicates in the
distribution of ticks between each half of each arena (binary logistic
regression analysis: 0.925>P<0.078), apart from the group 1
high-fat ticks in condition c above when the percentage of nymphs counted in
the odour half varied from 25 to 83% (P=0.001).
Two distinct responses were examined by binary logistic regression, using
the GENMOP procedure in SAS, within each of two analyses. Analysis A (no
odour, condition a above): taking fat content as a categorical predictor,
response 1 - the probability that ticks moved at all, response 2 - the
probability of moving towards high humidity. Analysis B (odour at one end,
conditions b, c and d above): taking fat content and moisture conditions as
categorical predictors, response 1 - the probability that ticks moved at all,
response 2 - the probability of moving towards the odour. Where appropriate,
simple 
2 tests were also performed.
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). Fat content is positively correlated with tick size as
measured by its fat-free (reduced) dry mass. When corrected for size
(fat/reduced dry mass), the group 3 ticks did indeed have a significantly
lower mean fat content than group 1 ticks (Student's t-test=2.033,
P=0.039, d.f.=74).
Walking activity (response 1)
Between 40 and 100% of each set of 20 fully hydrated ticks (groups 1 and 3)
walked out of the introduction tube within 24 h. A higher proportion of
high-fat than low-fat ticks had walked by 24 h, and statistically this
difference was almost significant in the absence of odour
(Fig. 1A) (83.75%
vs71.25%, 2=3.51, P=0.06), and strongly
significant with the larger sample sizes in the presence of odour
(Fig. 1B-D) (74.17% vs
63.75%, 2=6.08, P=0.014). There was no effect of
moisture conditions and no interaction between moisture and fat levels. (The
data are presented in Fig. 1 as
means per replicate with standard deviations, rather than overall percentages
based on individual ticks, to show the modest degree of variation between
replicates.) Amongst the four sets of 25 high-fat semi-dehydrated ticks (group
2), on average 84% (range 72-92) walked out of the introduction tube within 24
h.
Amongst the fully hydrated ticks, the difference in activity between high-fat and low-fat groups typically (but not invariably - see Fig. 1C) started within 1 h of the ticks' release. As time passed, more ticks were counted in the boxes while the numbers within the connecting tunnel stayed more constant; by 24 h the ratio of ticks in the boxes and tunnel (2:1 for both high- and low-fat ticks) did not reflect the fourfold greater surface area of the boxes. This indicates that up to one-third of the walking ticks stopped in the tunnel before reaching the boxes and did not move up and down the arena stopping at random.
Direction of movement (response 2)
In the absence of odour, there was no significant difference in the
proportion of the fully hydrated ticks (groups 1 and 3) that moved towards the
dry or wet end of the arena, and no effect of fat content on the direction of
movement (Fig. 2A)
(2=2.03, P=0.15). There was no effect of moisture
conditions or fat levels on the proportion of ticks that walked all the way to
the boxes (59-72%) rather than stopping in the connecting tube. Of the 84
semi-dehydrated high-fat (group 2) nymphs that walked when exposed to the same
humidity gradient, a significantly greater proportion (52=62%) had moved
towards the wet end (with 45 reaching the box) than had moved towards the dry
end (32, with 26 to the box) (2=4.76, P<0.05).
The ticks' response to odour was significantly affected by moisture
conditions (Fig. 2B-D). Only in
the uniformly wet arena did a greater proportion of ticks walk towards the
odour source, and this was true of both high-fat (70%) and low-fat (73%) ticks
(Fig. 2D). As fully hydrated
ticks showed no strong preference for high humidity, or for host odour in dry
conditions, the lack of a significant directional response towards odour
despite the risk of desiccation (Fig.
2B) is not surprising [2=3.18 (not significant),
for combined high-fat and low-fat ticks]. Whatever the moisture conditions and
fat levels, a higher proportion of the ticks that walked towards the odour
went all the way to the box (70-82%), rather than stopping in the connecting
tube, than those that walked in the opposite direction (50-69%)
(2=4.20, P<0.05).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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) of long-distance walking by I. ricinus nymphs
constrained within vertical channels, walking occupied only 6.6% of the 10
days of continuous observations. Within the 24 h periods of observations of
spontaneous walking by ticks in the horizontal plane reported here, 27% of
ticks did not walk at all, consistent with observations that periods of
quiescence or questing may persist for over 20 h, even several days
(Lees and Milne, 1951;
Perret et al., 2003). Of those
that did walk, 30-50% did not walk beyond the 14 cm length of one half of the
connecting tube of the arena, although any deviation from the straight line
would have added distance. This is less than the median vertical displacement
(both up and down) of 43 cm (max 9.7 m) after periods of quiescence and 17 cm
(max 2.9 m) after questing, with the former positively correlated with SD
(Perret et al., 2003). Our
ticks were probably not fully quiescent as defined by Perret et al. (immobile
for more than 2 h within 2 cm of the wet base of the vertical tracks) when
released from the introduction tubes. Nor were they subjected to the extreme
atmospheric dryness (9.3 mmHg SD) that was associated with the longest
vertical distances. The positive geotropism shown by I. ricinus at SD
>4.4 mmHg (Macleod, 1935),
however, introduces an additional driving force in the vertical plane not
present in our horizontal arrangement. It is not known how far ticks walked
within the boxes, but prolonged walking round and round was not seen during
continuous pilot observations. Limited dispersal also applies to I.
scapularis: in a field experiment, the majority of nymphs were captured
2-3 m from a release point within 2 or 3 weeks, with a very few reaching 5-6 m
by weeks 3-4 (Carroll and Schmidtmann,
1996). Under what circumstances, therefore, do I. ricinus ticks undertake horizontal displacement, as opposed to moving simply in the vertical plane between questing and re-hydration sites in the field? The simple laboratory arena did not take account of the vegetation structure in natural field conditions, where ticks must traverse surfaces that are at many different angles (including vertical) in order to move even short horizontal distances. Nevertheless, the results presented here suggest that ticks of this ambushing species (as opposed to `hunting' species) do not walk about entirely at random, responding to stimuli thus encountered by chance, but rather are stimulated to walk, and to choose their direction, by both intrinsic and extrinsic factors. If questing ticks persistently fail to contact hosts, conserving any remaining energy is clearly an essential part of their sit-and-wait strategy, but ticks that do not move may be caught in a downward spiral towards starvation. Our results indicate that ticks with lower energy reserves were indeed less likely to walk, yet overall 66% of even the low-fat nymphs (with energy levels similar to field ticks in June, at least 4 months after the start of the questing season in southern England) did move horizontally over short distances. Intrinsic moisture conditions of ticks also exerted an influence. Whereas a mild degree of dehydration did not stimulate greater walking activity (comparing like with like, high-fat ticks in the absence of host odour), it did increase the probability of ticks moving up a humidity gradient. Biologically, this is as would be expected.
Furthermore, under conditions of uniformly high humidity both high-fat and
low-fat ticks walked towards a source of host odour. At present, we can only
speculate as to why high humidity promoted this response. The odour,
consisting of unidentified volatile compounds secreted from the back of a
dog's ear, might have been expected to saturate the whole arena over the 24 h
period of observation (Waladde and Rice,
1982). Possibly a high atmospheric vapour content impeded the
dissemination of the molecules thereby creating a stronger gradient, or
alternatively aided the detection of the molecules by the tick's olfactory
sensilla within the Haller's organ (Guerin
et al., 2000; Leonovich,
2004). This accords with recent observations that I.
ricinus is attracted to volatile rumen metabolites
(Donzé et al., 2004),
which would reach ticks within a naturally moist air stream. Alternatively,
I. ricinus might be more responsive to host odour when the moisture
stress of walking is less, which is consistent with Perret et al.'s
observation (Perret et al.,
2003) that most ticks start to walk after a period of quiescence
in moist conditions near the base of the vertical channels and with the onset
of darkness, i.e. at night, although this effect was not seen under the cruder
light conditions used here (Fig.
1). Whatever the precise cause of these humidity effects, these
results show that under certain circumstances secretions from dog skin can act
as a kairomone (a host-produced substance that stimulates tick appetance
behaviour), but do so by attracting ticks to move towards the source, rather
than merely acting as an arrestant on contact as shown for other tick species
(Carroll, 2002). The fact that
extrinsic moisture conditions had no effect on locomotory activity per
se (both the probability of walking and the probability of going as far
as the box when in the odour half of the arena) shows that the accumulation of
ticks in the odour half of the arena in high humidity was not due to increased
random walking and therefore increased chance of encountering the host odour
followed by akinesis. Odour from dog's skin appeared to be acting as an
attractant for I. ricinus, as indicated by laboratory results for
I. persulcatus and I. scapularis using a T-shaped
olfactometer (Dobrotvorsky et al., 1999). In a field experiment, when I.
scapularis adults were released centrally within a 2 m diameter circle of
upright wooden skewers, within 2 weeks they accumulated on skewers anointed
with substances rubbed from the external glands on the legs of white-tailed
deer to a significantly greater extent than on control skewers
(Carroll et al., 1996). Deer
secretions could have been acting as an attractant, but these observations do
not exclude the possibility that the adult I. scapularis, which can
walk >4 m within 2 weeks (Carroll and
Schmidtmann, 1996), ascended both types of skewers but only stayed
(i.e. were arrested) on the anointed skewers. Given the low effectiveness of
CO2 traps in attracting Ixodes spp. in the field
(Schulze et al., 1997), it is
possible that host secretions are more important than exhaled breath in
stimulating these ticks to walk.
In conclusion, the results presented here indicate that nymphal I. ricinus with higher energy reserves are more likely to walk horizontally, but only if slightly dehydrated are they more likely to walk towards fully saturated air than drier air, and only if the atmosphere is sufficiently wet are they likely to walk towards odour secreted by host skin. It seems that, under certain circumstances, ticks of this ambushing species will move short distances towards odours associated with their hosts.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Anderson, R. B., Scrimgeour, G. J. and Kaufman, W. R. (1998). Responses of the tick, Amblyomma hebraeum, to carbon dioxide. Exp. Appl. Acarol. 22, 1-15.
Barré, N., Garris, G. I. and Lorvelec, O. (1997). Field sampling of the tick Amblyomma variegatum (Acari: Ixodidae) on pastures in Guadeloupe; attraction of CO2 and/or tick pheromones and conditions of use. Exp. Appl. Acarol. 21,95 -108.[Medline]
Carroll, J. F. (2002). How specific are host-produced kairomones to host-seeking ixodid ticks? Exp. Appl. Acarol. 28,155 -161.[Medline]
Carroll, J. F. and Schmidtmann, E. T. (1996). Dispersal of blacklegged tick (Acari: Ixodidae) nymphs and adults at the woods-pasture interface. J. Med. Entomol. 33,554 -558.[Medline]
Carroll, J. F., Klun, J. A. and Schmidtmann, E. T. (1995). Evidence for kairomonal influence on selection of host-ambushing sites by adult Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 32,119 -125.[Medline]
Carroll, J. F., Mills, G. D. and Schmidtmann, E. T. (1996). Field and laboratory responses of adult Ixodes scapularis (Acari: Ixodidae) to kairomones produced by white-tailed deer. J. Med. Entomol. 33,640 -644.[Medline]
Dobrotvorsky, A. K., Tkachev, A. V., Naumov, R. L. and Carroll, J. F. (2000). Study of host odour determinants of questing behaviour in Ixodes ticks. In 3rd International Conference on Ticks and Tick-borne Pathogens: Into the 21st Century (ed. M. Kazimírová, M. Labuda and P. A. Nuttall), pp.235 -240.High Tatra Mountains, Slovakia: Slovak Academy of Sciences.
Donzé, G., McMahon, C. and Guerin, P. M.
(2004). Rumen metabolites serve ticks to exploit large mammals.
J. Exp. Biol. 207,4283
-4289.
Grenacher, S., Kröber, T., Guerin, P. M. and Vlimant, M. (2001). Behavioural and chemoreceptor cell responses of the tick, Ixodes ricinus, to its own faeces and faecal constituents. Exp. Appl. Acarol. 25,641 -660.[Medline]
Guerin, P. M., Kröber, T., McMahon, C., Guerenstein, P., Grenacher, S., Vlimant, M., Diehl, P.-A., Steullet, P. and Syed, Z. (2000). Chemosensory and behavioural adaptations of ectoparasitic arthropods. Nova Acta Leopold. 83,213 -229.
Knulle, W. and Rudolph, D. (1982). Humidity relationships and water balance of ticks. In Physiology of Ticks (ed. F. D. Obenchain and R. Galun), pp.43 -70. Oxford: Pergamon Press.
Lavender, D. R. and Oliver, J. H. J. (1996). Ticks (Acari: Ixodidae) in Bulloch County, Georgia. J. Med. Entomol. 33,224 -231.[Medline]
Lees, A. D. (1946). The water balance in Ixodes ricinus L. and certain other species of ticks. Parasitology 37,1 -20.
Lees, A. D. and Milne, A. (1951). The seasonal and diurnal activities of individual sheep ticks (Ixodes ricinus). Parasitology 41,180 -209.
Leonovich, S. A. (2004). Phenol and lactone receptors in the distal sensilla of the Haller's organ in Ixodes ricinus ticks and their possible role in host perception. Exp. Appl. Acarol. 32,89 -102.[Medline]
Macleod, J. (1935). Ixodes ricinus in relation to its physical environment. II. The factors governing survival and activity. Parasitology 27,123 -144.
McMahon, C. and Guerin, P. M. (2002). Attraction of the tropical bont tick, Amblyomma variegatum, to human breath and to the breath components acetone, NO and CO2. Naturwissenschaften 89,311 -315.[CrossRef][Medline]
Norval, R. A. I., Butler, J. F. and Yunker, C. E. (1989). Use of carbon dioxide and natural or synthetic aggregation-attachment pheromone of the bont ticks, Amblyomma hebraeum, to attract and trap unfed adults in the field. Exp. Appl. Acarol. 7,171 -180.
Perret, J.-L., Guerin, P. M., Diehl, P.-A., Vlimant, M. and
Gern, L. (2003). Darkness favours mobility and saturation
deficit limits questing duration in Ixodes ricinus, the tick vector
of Lyme disease in Europe. J. Exp. Biol.
206,1809
-1815.
Randolph, S. E. and Storey, K. (1999). Impact of microclimate on immature tick-rodent interactions (Acari: Ixodidae): implications for parasite transmission. J. Med. Entomol. 36,741 -748.[Medline]
Randolph, S. E., Green, R. M., Hoodless, A. N. and Peacey, M. F. (2002). An empirical quantitative framework for the seasonal population dynamics of the tick Ixodes ricinus. Int. J. Parasitol. 32,979 -989.[CrossRef][Medline]
Schulze, T. L., Jordan, R. A. and Hung, R. W. (1997). Biases associated with several sampling methods used to estimate abundance of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 34,615 -623.[Medline]
Sonenshine, D. E. (2004). Pheromones and other semiochemicals of ticks and their use in tick control. Parasitology 129,S405 -S425.
Waladde, S. M. and Rice, M. J. (1982). The sensory basis of tick feeding behaviour. In Physiology of Ticks (ed. F. D. Obenchain and R. Galun), pp.71 -118. Oxford: Pergamon Press.
Winston, P. W. and Bates, D. H. (1960). Saturated solutions for the control of humidity in biological research. Ecology 41,232 -237.[CrossRef]
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