Rumen metabolites serve ticks to exploit large mammals
Institute of Zoology, University of Neuchâtel, Rue Emile-Argand 11, 2007 Neuchatel, Switzerland
* Author for correspondence (e-mail: patrick.guerin{at}unine.ch)
Accepted 14 August 2004
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Summary |
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Key words: tick, ectoparasite, rumen, behaviour, neurophysiology, rumen metabolite, Amblyomma, Ixodes
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Introduction |
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In contrast to soft ticks (Acari: Argasidae), which have successfully
exploited the nidicolous habitat, hard ticks predominate in open range
(Klompen et al., 1996). Hard
ticks typically spend most of their life cycle on the ground, where they must
survive prevailing environmental conditions until host encounter. For this
reason, detection of a host at a distance is paramount. Host-emitted odours
readily arouse ticks that ambush from questing sites, such as Ixodes
adults and immature stages of all hard tick species, or provoke orientation in
ticks that walk towards their prey, i.e. Amblyomma adults. Rumen
fluid odour is the product of a stable bioreactor whose major chemical
constituents do not vary greatly between ruminant species
(Garcia et al., 1994
). Given
the affiliation of hard ticks for large wandering ungulates such as ruminants,
we hypothesised that chemicals eructed from the rumen to the exterior might be
significant in mediating their recruitment to suitable hosts.
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Materials and methods |
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Odour delivery
Rumen fluid collected from freshly slaughtered cattle was conserved under
N2 at 5°C (<2 weeks) for behavioural tests. This fluid
(0.41 ml) was applied to a filter paper disk in a borosilicate 500 ml
gas-wash flask. Vapours from the flask were evacuated into an air stream (0.18
m s1; dilution 25x) flowing over an adult tick (male
or female) walking on the locomotion compensator
(McMahon and Guerin, 2002). No
change in the CO2 concentration in the air stream was detected
after presentation of rumen fluid odour using an infrared gas analyser
(detection limit ±2 p.p.m.; BINOS1; Leybold-Heraeus, Hanau, Germany).
Behavioural responses were recorded for A. variegatum or I.
scapularis to entrained rumen odour, acetic, propionic, butanoic and
isobutanoic acid, 3-methylindole and 4-methylphenol, all diluted in
dichloromethane and applied to filter paper. After evaporation of the solvent,
the filter papers were placed in 500 ml gas-wash flasks from which the test
odours are evacuated into an air stream (as above). A. variegatum
adults were also presented with methane from a pressurised gas cylinder (2.5%
v/v in synthetic air) injected into the air stream. The response of each tick
was tested only once.
Statistics
Paired tests eliminated most of the biological variation among the
behaviours of the individual ticks tested. The significance of the difference
between test and control for a given treatment in terms of attraction and %
change in speed (not normally distributed) was analysed using the Wilcoxon
signed rank test (two-tailed). Differences between treatments were compared
with the WilcoxonMannWhitney test (two-tailed). Comparisons of
local search behaviours made by A. variegatum in response to removal
of different treatments from the air stream were made with the Fisher exact
test (two-tailed). These statistical tests were run on S-Plus (v.3.3 release
1, 1995).
Collection of rumen volatiles
The odour of rumen fluid was entrained immediately after collection from a
steer (Bos taurus) or a roe deer (Capreolus capreolus) on a
porous polymer (500 mg of Soxhlet-extracted Porapak Q®; Waters, Milford,
MA, USA; 5080 mesh, packed into a 6 cmx6.23 mm-diameter glass
tube and stoppered with glass wool plugs). Before use, the cartridge was
ventilated for 1.5 h with pure N2 at 180°C. Pure N2
(150 ml min1) was bubbled through the cud to the porous
polymer cartridge for 25 h. The entrained volatiles were eluted with
dichloromethane, and the first 35 drops (4060 µl) were
transferred to a glass ampoule and flame sealed for conservation at
20°C until use.
Gas chromatography linked single sensillum recordings
In ticks, odours are perceived via 20 multiporous olfactory
sensilla borne dorsally on each foreleg tarsus, with each sensillum housing
between 4 and 12 receptor cells (Hess and
Vlimant, 1986
). Specific volatiles present in rumen fluid odour
that excite olfactory receptors in A. variegatum and I.
ricinus were identified by coupling recordings of action potentials from
tick olfactory receptor cells to components of the entrained rumen odour
eluting in succession from a high-resolution capillary column in a gas
chromatograph (Steullet and Guerin,
1994
). 2 µl aliquots of the porous polymer-extracted volatiles
were injected on-column on a 30 m DB-WAX column (J&W Scientific, Folsom,
CA, USA) in a Carlo-Erba 5160 gas chromatograph (Carlo Erba Instruments,
Milan, Italy) for the single chemosensillum electrophysiology recordings
coupled with flame ionisation detection (FID). The column was held at 40°C
for 5 min and then programmed at 5 deg. min1 to 250°C
with hydrogen as carrier gas. The column effluent was split (glass Y-splitter)
so that 60% was directed to the FID (280°C) and 40% was directed from a
heated transfer line (240°C) in the wall of the chromatograph to the
electrophysiological preparation in such a way that the column effluent was
simultaneously monitored by the FID and the single sensillum preparation. To
facilitate electrical contact, the tip of the sensillum was cut under the
microscope by prising its tip between two blades held on holders mounted on
micromanipulators.
Gas chromatographymass spectrometry (GCMS)
The Carbowax column (see above) was installed in a Hewlett-Packard 5890 gas
chromatograph (Hewlett-Packard, Meyrin, Geneva, Switzerland) with helium as
carrier gas and connected via 1 m deactivated fused-silica capillary
(0.25 mm i.d.) to the mass selective detector (Hewlett-Packard 5971,
ionisation chamber temperature 160°C; ionisation energy 70 eV). The mass
selective detector (EI mode) scanned for masses of 20 to 300. The eluting
volatiles were identified by comparing their mass spectra with those in a
library of the Hewlett Packard Chemstation software and by matching retention
times and mass spectra of synthetic analogues.
Quantification of rumen carboxylic acids and aromatics
Rumen supernatant was first brought to pH 14 by the addition of 2 mol
l1 NaOH, after which amines plus neutral molecules were
extracted into ether. The aqueous layer was brought to pH 1 with 5 mol
l1 HCl, and the remaining organic compounds extracted into
ether (fraction F1). This ether fraction was then washed with saturated
NaHCO3 solution so that phenols were retained in the organic phase
(fraction F2) and carboxylic acids were retained in the aqueous phase. These
acids were subsequently extracted into ether after acidification as above
(fraction F3). Fractions F2 and F3 were washed with saturated NaCl and
desiccated using Na2SO4 before analysis by GCMS.
The ratio of butanoic acid:isobutanoic acid:4-methylphenol:3-methylindole
recovered was 138:12:4:1.
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Results |
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Tick sensory responses to rumen metabolites
Olfactory receptor cells of both I. ricinus and A.
variegatum were stimulated by the same fractions of rumen fluid odour
(Fig. 2). These compounds are
from four sources: (1) straight-chain carboxylic acids derived from plant
carbohydrates and aliphatic amino acids, i.e. acetic, propionic, butanoic,
pentanoic and hexanoic acids; (2) a branched carboxylic acid derived from the
branched aliphatic amino acids leucine or valine, i.e. isopentanoic acid; (3)
phenols derived from tyrosine, i.e. phenol, 2-nitrophenol,
4-methyl-2-nitrophenol-2- and 4-methylphenol and (4) indoles derived from
tryptophan, i.e. indole and 3-methylindole
(Fig. 3). This complements a
previous study where olfactory receptor cells responding to another branched
amino acid catabolite present in the rumen, isobutanoic acid, were identified
in A. variegatum (Steullet and
Guerin, 1994). These four classes of rumen volatiles for which
receptors have been identified in ticks arise, not as intermediates, but as
anticipated stable end-products of different pathways of ruminal fermentation.
Short-chain carboxylic acids such as butanoic and isobutanoic acid are
absorbed by the host and enter amphibolic pathways whereas toxic aromatic
catabolites such as 4-methylphenol and 3-methylindole are excreted in urine
(Martin, 1982
;
Yang and Carlson, 1972
).
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Tick behavioural responses to rumen metabolites
We then proceeded to measure the behavioural responses of A.
variegatum to the rumen volatiles that excite its receptor cells. The
compounds were chosen from the four classes of chemostimulants. Butanoic and
isobutanoic acid, 4-methylphenol and 3-methylindole each attracted A.
variegatum on their own (Table
2). However, a mixture of these products where butanoic acid,
isobutanoic acid, 4-methylphenol and 3-methylindole were presented together at
an identical source dose (1:1:1:1) failed to induce attraction in A.
variegatum (median increase=2%, P>0.4, N=20;
Table 2). As straight-chain
carboxylic acids predominate among the organic compounds of rumen fluid
(Clarke and Bauchop, 1977), we
increased the dose of butanoic acid to 100 times the dose of the other three
constituents, i.e. at 100:1:1:1, but this mixture also failed to attract
(median increase=3%, P>0.5, N=11;
Table 2). The relative amounts
of each component of the mixture were then adjusted according to their
approximate proportions (g ml1) in rumen fluid to give a
100:10:1:1 mixture of butanoic acid:isobutanoic
acid:4-methylphenol:3-methylindole. This four-component mixture induced
attraction at approximately half that recorded to rumen fluid odour (median
increase=25%, P<0.001, N=18;
Table 2). This same synthetic
mixture (at a 10x higher dose) also induced attraction in I.
scapularis, similarly at half that induced by the natural odour (median
increase=12%, P<0.01, N=16). These results indicate that
both the nature and proportions at which rumen volatiles are present in an
odour are important to ticks. The discrepancy recorded here between the
behavioural responses to the synthetic mixtures and the natural rumen odour
may be in part due to the absence in our test mixtures of gaseous rumen fluid
components (NH3, H2S) for which ticks have receptor
cells (Steullet and Guerin,
1992
,
1994
). It is also the case
that ticks respond to other constituents of natural rumen odour absent in the
test mixtures such as acetic and propionic acid
(Table 2). Methane, a major
end-product of rumen metabolism (Clarke and
Bauchop, 1977
), failed to induce any behavioural response
(Table 2).
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Discussion |
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The attraction of ticks to volatile rumen end-products explains how ticks
track their ruminant hosts. The rumen fermenter generates a great variety of
partially oxidized volatiles in which short-chain carboxylic acids (acetic,
propionic, butanoic and isobutanoic) predominate
(Garcia et al., 1994).
Ruminants eruct at regular intervals (once every 23 min), voiding half
the rumen gaseous contents into breath in 1 h
(Waghorn and Stafford, 1993
).
Breath itself is a fundamental host cue for both ticks
(McMahon and Guerin, 2002
) and
other haematophagous arthropods (Guerin et
al., 2000
; Stange,
1996
). Nevertheless, anaerobic environments are not exclusive to
the rumen, and hard ticks feed on a wide range of both mono- and digastric
vertebrates (Cumming, 1999
).
Moreover, no compound formed in the rumen is specific to the foregut, and all
occur to a lesser extent in the hindgut, particularly in the large bioreactors
in the caecum and large intestine of other ungulates, although it is difficult
to quantify volatiles released from such regions. Similar products may also be
released from protected areas of the host pelage where oxygen may be limiting,
such as the axillar and genital areas, preferred tick predilection sites. It
is also interesting that these same classes of compounds feature prominently
elsewhere in the biology of these ticks: 2-nitrophenol, derived from the
anaerobic degradation of tyrosine and released from cattle
(Steullet and Guerin, 1994
),
forms the major component of the aggregationattachment pheromone
secreted from dermal glands in at least two Amblyomma spp.
(Diehl et al., 1991
;
Guerin et al., 2000
), and
isobutanoic acid, derived from valine or leucine, is a component of the
aggregationattachment pheromone of A. hebraeum
(Apps et al., 1988
). Such
scents are secreted by feeding males to enhance the recruitment of
conspecifics to the same host (Norval et
al., 1989
). Similarly, other phenols (2-, 3- as well as
4-methylphenol) derived from tyrosine are important attractants for tsetse
flies (Saini and Hassanali,
1994
). 3-Methylindole, derived from tryptophan, is a known
oviposition stimulus for mosquitoes
(Beehler et al., 1994
), and the
rumen products indole, acetic acid and propionic acid are also olfactory
stimulants for mosquitoes (Meijerink et
al., 2000
). Recent investigations show that aliphatic acids
present on human skin induce mosquito attraction
(Bosch et al., 2000
). The
importance of products released from an anaerobic milieu may represent a
general motif in the sensory ecology of haematophagous arthropods and,
particularly, in their resource-tracking habit on vertebrates.
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Acknowledgments |
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