Darkness induces mobility, and saturation deficit limits questing duration, in the tick Ixodes ricinus
Institute of Zoology, University of Neuchâtel, Rue Emile-Argand 11, 2007 Neuchâtel, Switzerland
* Author for correspondence (e-mail: patrick.guerin{at}unine.ch)
Accepted 28 February 2003
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Summary |
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Key words: questing, quiescence, tick, Ixodes ricinus, dark, desiccation, saturation deficit, photoreceptor, behaviour.
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Introduction |
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The life span of free-living stages of ticks (larvae, nymphs and adults) is
limited by the fixed energy reserves they possess at emergence. To find a
blood meal, I. ricinus first climbs onto low vegetation to quest for
a passing vertebrate host. During `questing', ticks lose water
(Lees, 1946), which they
normally regain by descending at intervals to the litter zone
(Lees, 1946
;
Milne, 1950
;
Lees and Milne, 1951
) where
they actively reabsorb water vapour from the atmosphere
(Rudolph and Knülle,
1979
; Gaede and Knülle,
1997
; Kahl and Alidousti,
1997
) during a period called `quiescence'
(Lees and Milne, 1951
). In
addition to water sorption, movements up and down the vegetation also require
energy. These ectoparasites would therefore be expected to display behavioural
and physiological adaptations that minimize energy use to facilitate prolonged
questing activities.
Most studies on I. ricinus behaviour have focused on field observations and have been limited to observing questing ticks. To gain more insight into hitherto unobserved tick behaviours, we developed a computerized video-tracking system for continuous recording of tick behaviours under controlled climatic conditions and in the absence of any host stimuli. We studied factors governing the alternation of questing and quiescence and the duration of these states by continuously following the behaviours of individual I. ricinus nymphs.
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Materials and methods |
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Constant infrared illumination (950 nm) enabled video observations independent of lighting conditions. Images were acquired every 3 s using up to four Philips ToUCam PRO webcams with their built-in infrared-block filter replaced with a Kodak 87 infrared-pass filter. The position of ticks in the vertical channels was determined on the acquired images by a homemade tracking software (`camtud') running on a Linux computer, resulting in time-coded tracks. This set-up could follow the position of up to 40 nymphs simultaneously with a spatial resolution of 0.5 mm, independent of light conditions (the error rate in position detection was inferior to 1/10 000, as determined by following a mechanically driven moving object).
Time-coded tracks were further analysed by splitting them into three event types: (1) questing (ticks immobile for more than 2 h at a position higher than 2 cm above the wet cotton wick; i.e. outside the humid zone of the wet cotton, as measured with a Vaisala Humitter 50Y humidity sensor), (2) quiescence (ticks immobile for more than 2 h within 2 cm of the wet cotton) and (3) walking (ticks that moved more than 3 cm or changed their position by more than 1.5 cm within the channels). For each event, we analysed the start time, end time and duration. A few events for which the start or the end time could not be precisely determined were discarded. Actographs of tick movements recorded every 3 s were also generated from the time-coded tracks.
Experimental conditions
Tick behaviour was observed under three climatic conditions: 25°C, 60%
relative humidity (RH); 25°C, 85% RH; and 15°C, 85% RH; corresponding
to saturation deficits of 9.3 mmHg, 3.5 mmHg and 1.9 mmHg (1 mmHg=133.3 Pa),
respectively, as calculated by Randolph and Storey
(1999). Saturation deficit
integrates temperature and relative humidity to derive a measure of the drying
power of the atmosphere. Ticks were observed for 10 days under a light cycle
of 14 h:10 h L:D at the three climatic conditions. In addition, the ticks that
were observed at 9.3 mmHg saturation deficit under the 14 h:10 h L:D cycle
were then left for 5 days in complete darkness and subsequently observed under
a 8 h:4 h L:D cycle for 10 days. During the 14 h:10 h L:D cycle, light
increased from 08.00 h to 10.00 h to reach a maximum of 1400 lux and decreased
from 20.00 h to 22.00 h to reach a minimum of 0 lux. During the 8 h:4 h L:D
cycle, light increased from 08.00 h to 10.00 h and from 20.00 h to 22.00 h and
decreased from 14.00 h to 16.00 h and from 02.00 h to 04.00 h.
Statistics
Questing and quiescence event durations were not normally distributed, so
we used the nonparametric Jonckheere test
(Siegel and Castellan, 1988)
to evaluate the null hypothesis of independence against the alternative
hypothesis of a quantitative relationship between saturation deficit and event
duration. The Rayleigh test (Batschelet,
1981
) for cyclic data was used to evaluate the null hypothesis of
a uniform distribution for start and end of behavioural events over the day.
Calculations were made using R for Linux
(Ihaka and Gentleman,
1996
).
Light and electron microscopy
Laboratory reared I. ricinus were examined for the presence of
photosensitive cells by light and electron microscopy. Six larvae, four
nymphs, two adult females and three adult males were cut longitudinally in
half and immediately fixed for 4 h at 4°C in paraformaldehyde and
glutaraldehyde, prepared according to Karnovsky
(1965), using a 0.1 mol
l1 cacodylate buffer (pH 7.4) with 4% sucrose added.
Specimens were washed four times in the same buffer and postfixed for 1 h at
room temperature in 1% buffered OsO4. After three washes with the
cacodylate buffer, specimens were dehydrated for 10 min in three solutions of
increasing ethanol concentration (3070%). For intermediate block
contrasting, specimens were held for 2 h in darkness in 2% uranyl acetate
diluted in 75% ethanol (Philis and
Cromroy, 1977
). Dehydration was terminated in 90% ethanol, 90%
acetone and then in three successive baths of absolute acetone. Finally, the
samples were embedded in Spurr's resin and polymerised for 24 h at 60°C.
Semi-thin sections (500 nm) and thin serial sections (100150 nm) were
obtained on a Reichert Ultracut S microtome. Semi-thin sections were stained
with toluidine blue and observed by light microscopy at a magnification of
400x. Thin sections were mounted on copper grids, poststained with
uranyl acetate and lead citrate and observed with a Philips CM 100
transmission electron microscope at 60 kV.
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Results |
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During these experiments, tick movements recorded in the actographs were not uniformly distributed during the day (Rayleigh test, P<0.001; Fig. 1A) but occurred preferentially during darkness, with only 6% of movements occurring during the period of maximum light intensity. It was thus hypothesised that dropping light intensity could trigger tick mobility. To test this, we changed the photoperiod from a 14 h:10 h L:D cycle to a 8 h:4 h L:D cycle; temperature and relative humidity remained unchanged (25°C and 60% RH; saturation deficit, 9.3 mmHg). Tick walking still occurred predominantly during darkness, i.e. twice in 24 h (Fig. 1B). Only 1% of movements occurred during the period of maximum light intensity (Rayleigh test, P<0.001).
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Since ticks lose water during questing, the duration of questing bouts should be related to desiccating conditions. To test this hypothesis, we followed different groups of ticks under three different saturation deficit conditions: at 9.3 mmHg (N=58 ticks), 3.5 mmHg (N=50 ticks) and 1.9 mmHg (N=27 ticks) under a 14 h:10 h L:D cycle. In these experiments, the duration of questing events was inversely related to saturation deficit: means of 19.4 h, 25.9 h and 39.8 h at 9.3 mmHg, 3.5 mmHg and 1.9 mmHg, respectively (Jonckheere test, P<0.05; N=33, N=44 and N=66, respectively; Fig. 2; 1 mmHg=133.3 Pa). A negative relationship between questing duration and saturation deficit was also observed when another group of ticks was successively exposed to saturation deficits of 9.3 mmHg and 3.5 mmHg (data not shown). By contrast, the duration of quiescence was not related to saturation deficit (means of 28.6 h, 17.2 h and 20.6 h at 9.3 mmHg, 3.5 mmHg and 1.9 mmHg, respectively; Jonckheere test, P=0.93, N=148, N=106 and N=68, respectively; Fig. 3). The durations of quiescence events were not normally distributed but strongly skewed in favour of events of short duration (83% of events were below the mean at 9.3 mmHg).
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In these experiments, we also examined walking to ascertain whether ticks walked preferentially during darkness under the different climatic conditions. Indeed, walks associated with the start and end of quiescence and questing events occurred mainly during darkness under all three climatic conditions (Fig. 4; Rayleigh test, P<0.005 for the start and end times of all event types under all conditions). The distance walked after questing interruption was not related to saturation deficit (Jonckheere test, P=0.28) and reached a median of 13 cm and a maximum of 2.92 m. By contrast, the distance walked after quiescence interruption was positively related to saturation deficit (Jonckheere test, P<106; median distance at 1.9 mmHg=20.5 cm, at 3.5 mmHg=26.9 cm and at 9.3 mmHg=42.6 cm) and reached a maximum of 9.65 m at 9.3 mmHg. This means that the dryer the conditions, the further nymphs walked after quiescence. By contrast, independent of saturation deficit, nymphs interrupting questing walked similar distances before entering quiescence.
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The mean walking speed of nymphs during walking events was influenced by temperature. At 25°C, this speed reached a median of 0.97 cm min1 (50% of observations between 0.56 cm min1 and 1.53 cm min1), either at 60% or at 85% RH (Wilcoxon test, P=0.35), and was similar after quiescence and questing (Wilcoxon test, P=0.38 at 60% RH and P=0.14 at 85% RH). At 15°C, which is closer to the temperature encountered by ticks at night, the mean speed was reduced compared with that at 25°C, either after questing (Wilcoxon test, P<105) or after quiescence (Wilcoxon test, P<1015). However, at 15°C, nymphs walked faster after questing (median 0.67 cm min1; 50% of observations between 0.48 cm min1 and 0.84 cm min1) than after quiescence (median 0.43 cm min1; 50% of observations between 0.31 cm min1 and 0.56 cm min1; Wilcoxon test, P<106). This shows that the walking speed of nymphs is reduced at lower temperature, but this reduction is dependent on whether nymphs are interrupting questing or quiescence.
Since I. ricinus movements occurred preferentially during
darkness, we suspected the presence of photoreceptors in this species, as
previously suggested for other ticks
(Binnington, 1972). Examination
of semi-thin sections of larvae, nymphs and adults by light microscopy
revealed two rows of 2021 photosensitive cells located dorsolaterally
in a pearl-string fashion in all three life stages of I. ricinus
(Fig. 5A). The morphology of
these cells was examined in thin sections by electron microscopy
(Fig. 5B), which revealed cells
containing a major rhabdomere, attached to the hypodermis
(Fig. 5C). Axons from the
photoreceptor cells join to form the optic nerves that pass antero-dorsally to
reach the synganglion rostrally, parallel to the cheliceral nerves.
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Discussion |
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Questing was almost always followed by quiescence in the humid zone of our
observation channels. Furthermore, the walking speed to reach the humid zone
was higher than the walking speed to leave it at 15°C, and the distance
walked to reach the humid zone was shorter than the distance walked after
leaving it. This demonstrates that ticks need to hydrate after questing.
Indeed, I. ricinus is very susceptible to desiccation and loses water
during questing on vegetation (Lees,
1946). In the humid zone, ticks restore their water content by
practicing water sorption (Rudolph and
Knülle, 1979
; Gaede and
Knülle, 1997
; Kahl and
Alidousti, 1997
). Recent studies have suggested an influence of
saturation deficit on the questing duration of I. ricinus in both
quasi-natural arenas (Randolph and Storey,
1999
) and the field (Perret et
al., 2000
; Randolph et al.,
2002
). Here, we demonstrate that the duration of questing is
indeed limited by saturation deficit. When conditions are less desiccating,
ticks will quest for longer periods. The proportion of questing individuals in
the tick population is influenced by the prevailing saturation deficit. Longer
periods of questing may increase the host-finding probability and thus
influence tick population dynamics
(Randolph et al., 2002
).
Abrupt declines in the density of questing ticks have been shown to coincide
with abrupt increases in saturation deficit at field sites in both Switzerland
(Perret et al., 2000
) and the
UK (Randolph et al., 2002
).
This demonstrates the important role of saturation deficit on tick populations
in different ecozones occupied by I. ricinus.
Although desiccation may dictate when ticks should interrupt questing to
move down the vegetation to rehydrate (quiescence), the factors driving
interruption of quiescence remain largely unknown. We did not investigate
this, but we discovered that quiescence was often interrupted by walking
events that did not necessarily lead to questing. We suggest that some of
these movements represent activities that enable ticks to find a favourable
questing site in nature. Good questing sites are those where the probability
of host encounter is high during the questing bout permitted by the prevailing
climatic conditions. It has been shown that another Ixodes tick
species, I. scapularis, chooses questing sites where chemostimuli
have been left by hosts (Carroll et al.,
1998). In the present study, we observed that the distance walked
after quiescence increased with saturation deficit. I. ricinus nymphs
were ready to repeatedly walk long distances (up to 9.65 m) during the night.
Although the walks recorded in our experimental set-up occurred in vertical
channels, we assume that at least some of these movements could represent
horizontal walks if the ticks were not confined to the vertical channels. This
suggests that I. ricinus nymphs can undertake extensive displacement
in search of a questing site with appropriate microclimatic conditions.
Most tick walking occurred during darkness under all our climatic
conditions. Preference for questing interruption during darkness was described
in the field by Lees and Milne
(1951), who suggested it to be
due to an immediate response to temperature drops at night. Our data show that
a drop in light intensity is sufficient to trigger mobility in I.
ricinus and need not necessarily be accompanied by shifts in temperature
or RH. Similarly, Carroll et al.
(1998
) observed that I.
scapularis moves preferentially during darkness. In our experiments, we
show that even when the L:D cycle was changed to a 12 h period, the ticks
continued to move during darkness. In addition, we show that, irrespective of
the climatic conditions we applied, not only interruption of questing but also
most walking occurs during darkness.
When active, respiration and thus spiracle opening increases in ticks
(Lighton et al., 1993),
resulting in increased water loss (Rudolph
and Knülle, 1979
;
Knülle and Rudolph,
1982
). In addition, the time needed to search for suitable
questing and quiescence loci is unpredictable for a tick in nature. By
undertaking movements during less desiccating conditions, I. ricinus
minimizes water loss and the energy costs to reabsorb it. Since darkness
generally coincides with less severe desiccating conditions in the field,
I. ricinus uses darkness to trigger mobility when desiccation risk is
lowest. As a wide range of tick hosts show peak activity just after dark
(Hausser, 1995
), tick mobility
during this period may increase the probability of finding a host either by
direct encounter or by orientation to host stimuli by mobile ticks
(Carroll et al., 1998
;
McMahon and Guerin, 2002
).
Unlike other ticks (Philis and Cromroy,
1977; Kaltenrieder et al.,
1989
), I. ricinus does not have eyes with corneas. So,
how can I. ricinus perceive the changes in light intensity that
trigger its movements after dark? Although the existence of photosensitive
cells in a variety of tick species, including Ixodes holocyclus, was
suspected by Binnington (1972
),
none was described for I. ricinus. In the present study, we found
2021 cells containing a rhabdomere, located dorsolaterally behind coxa
2 on each side of larvae, nymphs and adult I. ricinus. These cells
most probably play a role in the perception of shifts in light intensity that
trigger tick movements during darkness. They may also be implicated in the
perception of changes in photoperiod that trigger morphogenetic diapause
(Belozerov, 1982
). Since
photosensitive cells were found in all three life stages of I.
ricinus, we expect nocturnal preference for movements in larvae and
adults as well. Beyond avoidance of water loss, synchronised movements during
darkness would carry a further advantage for adult ticks; since mating in
I. ricinus occurs mostly off the host
(Graf, 1978
), synchronised
mobility of adults with sundown could serve to increase encounters between
sexes.
I. ricinus has a very wide geographical distribution, occurring throughout Europe and extending to North Africa. The climatic, photoperiod and vegetational conditions experienced by this tick species, as well as its host range, are extremely heterogeneous, which has diverse effects on tick populations. Therefore, individual I. ricinus populations may have developed particular physiological and behavioural adaptations to optimize energy use in a particular ecozone. The video-tracking system developed for this study might help to estimate such local adaptations in populations of I. ricinus from diverse climatic and photoperiod conditions.
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Acknowledgments |
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