Date post: | 25-Nov-2023 |
Category: |
Documents |
Upload: | independent |
View: | 0 times |
Download: | 0 times |
The role of volatile semiochemicals in mediating hostlocation and selection by nuisance and disease-transmittingcattle flies
M. A. BIRKETT1 , N. AGELOPOULOS1 , K . -M. V . JENSEN2 ,
J . B . JESPERSEN2 , J . A . PICKETT1 , H . J . PRIJS3 , G. THOMAS3 ,
J . J . TRAPMAN3 , L . J . WADHAMS1 and C. M. WOODCOCK1
1Biological Chemistry Division, Rothamsted Research, Harpenden, U.K., 2Danish Institute of Agricultural Sciences, Danish
Pest Infestation Laboratory, Lyngby, Denmark and 3Laboratory for Behavioural Physiology, Institute for Animal Health,
Lelystad, the Netherlands
Abstract. The role of volatile semiochemicals in mediating the location and selection within
herds of Holstein-Friesian heifers by nuisance and disease-transmitting cattle flies was
investigated using coupled gas chromatography–electrophysiology (GC–EAG), coupled gas
chromatography–mass spectrometry (GC–MS), electrophysiology (EAG), laboratory beha-
viour and field studies. Using volatile extracts collected by air entrainment from heifers in the
Netherlands, a number of active peaks were located by coupled GC–EAG for Musca
autumnalis (de Geer) (Diptera: Muscidae) and Haematobia irritans (L.) (Diptera: Muscidae).
Volatile samples were also collected from two heifers in Denmark shown in previous counting
experiments to differ significantly in their fly loads. Coupled GC–EAG using Ha. irritans
antennae revealed differences in the EAG response to the samples, with additional EAG
activity in the sample collected from the heifer with the lower fly load. To identify more EAG
active compounds, volatiles were also collected from 48-h-old urine by air entrainment. In
total, 23 compounds were located and identified by coupled GC–EAG and GC–MS. Further
electrophysiological testing of these compounds with five fly species [M. autumnalis,
Ha. irritans, Hydrotaea irritans (L.) (Diptera: Muscidae), Stomoxys calcitrans (L.) (Diptera:
Musicidae) and Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae)] showed that only
some of the compounds were physiologically active across the range of flies tested. These
included 1-octen-3-ol, 6-methyl-5-hepten-2-one, (Z)-3-hexen-1-ol, naphthalene, and all EAG
active compounds identified from urine. Compounds showing significant EAG activity were
tested for behavioural activity using a wind-tunnel designed for measuring upwind flight
behaviour. At certain concentrations, 1-octen-3-ol, 6-methyl-5-hepten-2-one and 3-octanol
increased upwind flight, whereas naphthalene, propyl butanoate and linalool reduced upwind
flight. In field studies using small herds of heifers ranked according to their fly load, individual
slow-release formulations of 1-octen-3-ol and 6-methyl-5-hepten-2-one, when applied to low
and high fly loading heifers, reduced fly loads on these individuals. This study provides
evidence for the hypothesis that the natural differential attractiveness within herds of Hol-
stein-Freisian heifers, i.e. a single host species, for cattle flies is partly due to differences in
volatile semiochemicals emitted from the host. It is suggested that this phenomenon applies to
other vertebrate host species and their associated insect pests.
Key words. Haematobia irritans, Musca autumnalis, Stomoxys calcitrans,Wohlfahrtia magnifica, air entrainment, behaviour, cattle fly, electrophysiology,6-methyl-5-hepten-2-one, 1-octen-3-ol, semiochemical.
Correspondence: Professor John Pickett, Biological Chemistry Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, U.K.
Tel.: þ 44 (0)1582 763133; fax: þ 44 (0)1582 762595; e-mail: [email protected]
Medical and Veterinary Entomology (2004) 18, 313–322
# 2004 The Royal Entomological Society 313
Introduction
The behaviour of several species of nuisance, biting and
disease-transmitting cattle flies (Diptera) that settle or
feed on grazing hosts has been shown to lead to
increased disease incidence, reproductive failure and
also reduced meat and milk yields, with significant eco-
nomic losses arising as a consequence (Fraser & Broom,
1990).
Biting and blood-sucking (haematophagous) insects,
including cattle flies, use a wide variety of visual, olfac-
tory, gustatory and physical stimuli in host location and
selection. Of these, volatile semiochemicals play a major
role in mediating such behaviour (Pickett & Woodcock,
1996; Gibson & Torr, 1999). Cattle flies generally show
marked activation and attraction to carbon dioxide
(CO2), universally present in mammalian breath, but
also use other cues emanating from hosts. Numerous
studies have identified semiochemicals involved in host
location and selection for tsetse flies (Diptera: Glossini-
dae) (Hall et al., 1984; Hassanali et al., 1986; Bursell
et al., 1988; Gikonyo et al., 2002). For other cattle flies,
these cues remain largely undefined. Behavioural and elec-
trophysiological responses of Stomoxys spp., e.g. the stable
fly, S. calcitrans (L.), to host odour components have con-
centrated mainly on compounds that have been effective with
tsetse flies, with conflicting reports. In certain cases, traps
baited with 1-octen-3-ol, a component of ruminant breath,
were reported to increase catches of Stomoxys spp. (Hollo-
way & Phelps, 1991; Mihok et al., 1995), whereas in a sepa-
rate study no response was observed (Mullens et al., 1995).
Laboratory behavioural studies with S. calcitrans have
demonstrated anemotactic responses to 1-octen-3-ol and
acetone (Warnes & Finlayson, 1985; Schofield et al., 1997).
Electroantennogram recordings made from S. calcitrans
using host odour components have also been reported.
Responses to 1-octen-3-ol, 3-methylphenol and various alco-
hols, but not acetone, were observed by Schofield et al.
(1995), whereas responses to CO2, acetone and 1-octen-3-ol
were reported by Warnes & Finlayson (1986). For other
major cattle fly pests, including the sheep head fly,Hydrotaea
irritans (L.), the major transmitter for summer mastitis in
cattle, the face fly, Musca autumnalis (de Geer), the horn
fly, Haematobia irritans (L.), and the flesh fly, Wohlfahrtia
magnifica (Schiner), little or nothing is known of the role and
chemical nature of olfactory cues used in host location and
selection.
Cattle fly control is mainly obtained through the
employment of broad-spectrum toxicant insecticides, in
the form of ear-tags, pour-ons, dips, sprays, etc. Due to
concerns over the build-up of insecticide resistance, and
public concern over the possible environmental impact of
such chemicals, alternative control methods are sought. A
more benign approach is to employ insect repellents. Many
natural products derived from essential oils have been
developed, but the active compounds are extremely odifor-
ous, and represent only a weak behavioural drive away
from the potential host. An alternative approach is to
exploit the natural differentiation between members within
the host species. It has been demonstrated for several fly
species that individual animals within herds vary in their
attractiveness (Thomas et al., 1987; Brown et al., 1992;
Jensen et al., 2004). An hypothesis developed to explain
this differentiation, which applies to warm-blooded verte-
brate hosts and their associated insect pests, is that all
members of the host species naturally release volatile
attractants, but those which are less attractive release
additional compounds that interfere with the activity of
the attractants. These compounds, at higher levels, act as
true repellents but at lower, or natural physiological levels,
effectively mask the normal attractiveness of the host spe-
cies (Pickett et al., 1998).
The aim of this study was to test this hypothesis,
leading to the identification of natural repellents, and
attractants, that could form part of novel fly control stra-
tegies, such as the ‘push–pull’ strategy (Miller & Cowles,
1990). The suitability of this control strategy was con-
firmed in a preliminary study preceding this one, which
demonstrated that fly populations could be manipulated
through switching of animals between herds (Jensen et al.,
2004). The adopted approach to semiochemical identifica-
tion was to collect samples of volatile compounds emitted
by cattle that were known to differ in their attractiveness,
and to compare these by coupled gas chromatography–
electrophysiology (GC–EAG), using M. autumnalis and
Ha. irritans as the model insects. Identified EAG-active
compounds were then tested in a laboratory behavioural
bioassay, and in the field, to confirm their biological
activity.
Materials and methods
Volatile collection
Volatiles from Holstein-Friesian cattle, individually
housed in pre-washed stalls in the Netherlands and Denmark,
were collected by air entrainment. Air surrounding
the animals was drawn (1 L/min) through Pyrex glass
tubes (3.9mm o.d., 2.4mm i.d.) containing Porapak Q
(50mg per tube, 50–80 mesh, Supelco, Poole, U.K.) over
a period of several hours. The animals selected in the
Netherlands displayed significantly higher fly loads when
compared to other animals in the same herd. For volatile
collection in Denmark, animals shown in a preceding
study (Jensen et al., 2004) to have very high and low fly
loads (heifers 1257 and 1270, respectively) were selected.
The volatile collections were arranged such that relatively
free movement within the stalls was allowed, thus obviat-
ing any stress in the animals. Samples in the Netherlands
were collected by placing a single Porapak tube at a
fixed position above the animal, halfway along its length
and approximately 15–20 cm above the skin surface
when the animal was standing. In Denmark, tubes were
314 M. A. Birkett et al.
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
suspended above the neck by means of a pulley system
so that they were approximately 10–20 cm above the
animal at all times. In the Netherlands, a total of eight
entrainments, each lasting 24 h, were performed on four
individuals, i.e. two entrainments per individual. In
Denmark, two entrainments, each lasting 9 h, were per-
formed on heifer 1257, and four entrainments, each lasting
12 h, were performed on heifer 1270. Air entrainments of
clean empty stalls were carried out as controls. Porapak
tubes containing trapped volatiles were sent to the U.K.,
then eluted with freshly distilled diethyl ether (500mLper tube). To provide greater amounts of material for
subsequent identifications, samples collected in the Nether-
lands were combined prior to analysis. Samples were
concentrated to 100 mL using a gentle stream of nitrogen
and stored at �20�C in tightly capped microvials until
further use.
Volatiles were also collected from urine from a heifer
in the Netherlands. The sample was collected during
natural urination, and was left at ambient temperature
for 48 h. A portion of the aged urine (700mL) was placed
in a round-bottomed flask (1 L) and volatiles drawn
from the vessel using a purified air flow (1.5 L/min)
through glass tubes containing Porapak Q (50mg per
tube, as described previously). The trapped volatiles were
eluted with freshly distilled diethyl ether (500 mL per tube)
and stored at �20�C in tightly capped microvials until
further use.
Insects
The face fly, M. autumnalis, and the horn fly,
Ha. irritans, were reared at the Danish Pest Infestation
Laboratory (DPIL), Lyngby, Denmark, and sent to the
U.K. and the Netherlands as pupae. The stable fly, S. cal-
citrans, was obtained as pupae from the Novartis Centre
de Recherche Sante Animale, Switzerland. The flesh fly,
W. magnifica, was reared at the University of Granada,
Spain, and sent to the Netherlands as pupae (Soler Cruz
et al., 1996). The sheep head fly, Hy. irritans (L.), was
collected as adults in the field in the Netherlands. For
coupled GC–EAG studies in the U.K., M. autumnalis
and Ha. irritans were kept in controlled environment cabi-
nets at 20�C, LD16 : 8 h until required. The emerged flies
were provided with water prior to use. For EAG and
behavioural studies in the Netherlands, all fly species
were kept in cages at 21�C, 70% humidity, LD 10 : 14 h,
apart from S. calcitrans, which was kept at 24�C. All flieswere fed with sucrose solution (5% v/v) until required, and
were sexed prior to use.
Electrophysiology
For GC–EAG studies in the U.K., recordings from
female flies were made by using Ag/AgCl electrodes
filled with saline solution (composition as in Maddrell,
1969; but without the glucose). The head of the fly was
excised and the indifferent electrode positioned in the
head capsule. The recording electrode was placed over
the cut arista. The signals were passed through a high
impedance amplifier (UN-03b, Syntech, Hilversum, the
Netherlands) and displayed on a chart recorder. For
EAG studies in the Netherlands using authentic com-
pounds, recordings from excised heads of females were
made using glass electrodes filled with Ringer solution
(0.1 M KCl) and 10% PVP (polyvinylpyrrolidone). The
indifferent electrode was placed over the tip of the
antenna and the recording electrode inserted into the
back of the head. The arista of both antennae were
removed to reduce noise during recording from the sti-
mulus puff. The signals generated by the antenna were
passed through an amplifier (AM-02, Syntech) and data
storage and processing were carried out with a custom-
ized software package (Syntech). Stimuli were delivered
via standard disposable Pasteur pipettes containing a
strip of filter paper (40mm� 5mm). Solutions of test
chemicals (20 mL) were applied to filter papers, with the
solvent being allowed to evaporate for 30 s before use,
and delivered through the pipette into a continuous pur-
ified and humidified airstream (1 L/min) using a stimulus
generator (CS-27, Syntech) delivering an air puff (0.8 s,
4 L/min). The interval between stimuli was 1min. Com-
pounds were dissolved in freshly distilled diethyl ether,
and were tested in a random order. As the response of
the preparations declined with time, the mean responses
to compounds were normalized with respect to the mean
response to 1-octen-3-ol, a well-known attractant for
many haematophagous Diptera. To gain an overall pic-
ture of the EAG responses of all identified compounds,
all testing was conducted at one dose (10�2 g). For
comparison of activity, recordings were taken from
known attractants and repellents for other Diptera.
These included a tsetse lure (1-octen-3-ol/m-cresol/
p-cresol in a 1 : 4 : 8 ratio), indole, skatole (3-methylindole),
N,N-diethyltoluamide (DEET), citronellol and propionic
acid. The number of replicates for each fly species varied
according to availability of insects, but was sufficient in
each case for statistical analysis to be performed using
ANOVA.
Gas chromatography (GC)
GC analysis of air entrainment samples was carried out
on two GCs. An AI92 GC equipped with a temperature
programmable on-column injector, flame ionization
detector (FID), fitted with either a 50m� 0.32mm i.d.,
HP-1 non-polar column or a 30m� 0.3mm i.d., BP-20
polar column, and a Hewlett-Packard 5880 GC equipped
with a split-splitless injector, an FID and a 50m� 0.32mm
i.d., HP-1 non-polar column. For the AI92GC using theHP-1
column, the oven temperature was maintained at 40�C for
1min, then programmed at 5�/min to 100�C, then 10�/min
Host location and selection by flies 315
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
to 250�C. The BP-20 column was maintained at 40�C for
2min, then programmed at 10�/min to 225�C. For the 5880GC, the HP-1 column was maintained at 40�C for 2min,
then 5�/min to 150�C then 10�/min to 250�C. The carrier gasin all cases was hydrogen.
Coupled gas chromatography–electrophysiology
(GC–EAG)
The coupled GC–EAG system, in which the effluent
from the GC capillary column is delivered simultaneously
to the antennal preparation and the GC detector, has been
described elsewhere (Wadhams, 1990). Separation of air
entrainment samples was achieved on an AI93 GC
(AI, Cambridge, U.K.) equipped with a cold on-column
injector and a FID. Two columns were used. The non-
polar column (50m� 0.32mm i.d., HP-1) was maintained
at 40�C for 1min, then 5�/min to 100�C, then 10�/min to
250�C. The polar column (30m� 0.3mm i.d., BP-20) was
maintained at 40�C for 2min, then 10�/min to 225�C. Thecarrier gas was hydrogen. The outputs from the EAG
amplifier and the FID were monitored simultaneously on
a chart recorder.
Coupled gas chromatography–mass spectrometry
(GC–MS)
A Hewlett-Packard 5890 GC was connected to a VG
Autospec mass spectrometer (Fisons, Manchester, U.K.).
Ionization was by electron impact at 70 eV, 250�C, and the
GC, using the HP-1 column, was maintained at 30�C for
5min, then programmed at 5�C/min to 250�C. Compoundstentatively identified by GC–MS were confirmed by
co-injection of authentic samples on GC using non-polar
and polar columns (see above).
Chemicals
All chemicals were bought from the Aldrich Chemical
Company (Gillingham, U.K.). For peak enhancement
and internal reference standards (C9–C17 n-hydrocar-
bons), chemicals were diluted in distilled n-hexane prior
to use. For EAG studies, chemicals were diluted in
freshly distilled diethyl ether. For behavioural studies,
chemicals were dissolved in paraffin oil to provide slow
release formulations. For identified chemicals with
asymmetric carbons, racemic forms were used in
peak enhancement, electrophysiological and behavioural
studies.
Insect behaviour
A glass wind-tunnel (70 cm� 3.5 cm) was used to
monitor upwind flight behaviour of flies in response to
chemical stimuli. Musca autumnalis was selected as the
test insect, as other fly species showed little or no activ-
ity in the tunnel. The tunnel consisted of seven identical
sections of glass tubing, 10 cm long and 3.5 cm diameter.
The middle five sections were designated as the experi-
mental area, with a downwind and an upwind section
attached at either end. For each experiment, batches of
10 flies were introduced into the downwind section 1 h
prior to commencement, to allow acclimatization. The
stimulus, previously dissolved in paraffin oil, was applied
(20 mL) to a filter paper strip (4� 1 cm) and the strip
placed in the upwind section. Purified air was blown
through the tube at a speed of 0.05m/s, and recording
of the fly positions commenced 30 s after stimulus intro-
duction. Each experiment consisted of observations every
10 s for 3min. Chemicals were tested at six doses, using a
decadic series from 10�2 g to 10�7 g. The same batch of
flies was used to test a complete set of doses, each set
starting with the lowest dose. Each chemical was tested
with three batches of flies. To confirm that behaviour
was not affected by exposure to chemicals tested, and
that there was no chemical contamination of the tunnel,
a paraffin control stimulus was tested at the beginning,
middle and end of each series. Each experiment was
separated by an interval of 5min. The tunnel was
cleaned with diethyl ether and dried with purified air
for at least 30min before a new chemical was introduced.
All experiments were carried out between 08.00 hours
and 14.00 hours, as the activity of the flies tended to
drop off after this time. The response of flies to stimuli
was measured by observing the number of flies in the
upwind section of the tube. No significant differences
were observed between the first, middle and last paraffin
controls, and therefore these were pooled and averaged.
The results were analysed by comparing the difference
between the mean number of flies in the upwind section
for the stimuli and the control with Student’s t-test using
matched pairs. To test potential repellence in the tunnel,
the light and position of the tunnel was adjusted such
that flies spent more time upwind. To confirm the valid-
ity of the bioassay technique, the known fly repellent
citronellol (Bartlett, 1985) was also tested across the same
range of doses.
Field studies
Field trials were conducted in Denmark, using small
herds (n¼ 7) of Holstein-Friesian cattle that were selected
from larger herds. A single experiment comprised the use
of one compound per herd. 1-Octen-3-ol and 6-methyl-5-
hepten-2-one were released from slow release formulations
comprising polyethylene bags, protected from potential
damage caused by the animal by being placed in a dispenser
tube made of stainless steel and aluminium (4 cm length,
with 4-mm holes interspersed to allow diffusion). The ends
of the tube were closed with plates, and the tube was
316 M. A. Birkett et al.
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
hooked to a belt attached around the chest of the animal,
immediately behind the front legs. The sachets used gave
release rates of 10.5mg/day and 14mg/day for 1-octen-3-ol
and 6-methyl-5-hepten-2-one, respectively. On day 1, the
flies on each animal were identified and the number counted
using a previously established counting method (Jensen
et al., 2004) every 0.5 h, altogether four to five times in
total, and the data were used to rank the animals according
to fly load. Tube dispensers without polyethylene bags were
hung around the necks of the two least and two most fly
attractive individuals, to allow the animals to become accus-
tomed to the devices. On day 2, in the morning, the tube
dispensers on the two least attractive cattle were loaded
with a sachet containing the chemical, with the cattle
being given an hour to settle before counting was initiated.
The flies on each animal in the herd were identified and
counted every 0.5 h, four to five times in total. At the end of
day 2, the dispensers were emptied and cleaned with water
and acetone, and the sachets returned to the freezer. On day
3, the same counting procedure was carried out on the two
most attractive animals, using the same sachets. Statistical
analysis was carried out using a Kruskal–Wallis test fol-
lowed by a multiple-comparison test. Significance indicated
by different letters for each heifer indicates significant dif-
ference between medians of the ranks on a 5% level.
Results
GC analysis of the individual samples collected from
Holstein-Friesian cattle, both in the Netherlands and in
Denmark, showed that the concentration of components
was insufficient for analysis by coupled GC–MS. Samples
were therefore combined and concentrated to 100mL, toprovide separate Dutch and Danish samples, with small
volumes of each individual sample being retained. GC
analysis of the combined samples highlighted the complexity
of the odour profile of cattle (Figs 1a–c), with many compon-
ents present in very low amounts. More volatile material
Fig. 1. Gas chromatograms (GC) of volatiles collected from the headspace and urine of Holstein-Friesian cattle. Peak numbers correlate to
compounds, listed in Table 1, that were located and identified by coupled GC–electroantennography (GC–EAG) and GC–mass spectrometry
(GC–MS) and by peak enhancement with authentic samples: (a) combined and concentrated volatile sample collected from four Dutch cattle;
(b) combined and concentrated volatile sample collected from a Danish heifer (no. 1257) with known high fly load; (c) combined and
concentrated volatile sample collected from a Danish heifer (no. 1270) with known low fly load; (d) volatile sample collected from aged urine
(48 h old) of one Dutch heifer. Traces (a), (b) and (c) were obtained on a non-polar HP-1 column. Trace (d) was obtained from a polar BP-20
column. EAG-active compounds in (a) were located using M. autumnalis and Ha. irritans antennae, and in (b), (c) and (d) using Ha. irritans
antennae. Traces (b) and (c) are provided on the same scale for comparison.
Host location and selection by flies 317
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
was made available for further experiments by sampling
cattle urine that had been allowed to age (Fig. 1d).
Coupled GC–EAG using recordings from M. autumnalis
located a number of volatiles in the Dutch sample that
elicited an electrophysiological response (Fig. 1a). These
active compounds were identified tentatively by coupled
GC–MS and confirmed by peak enhancement using authen-
tic standards on both non-polar and polar GC columns
(Table 1). These compounds comprised a high proportion
of aromatic compounds, including polar aromatics such as
phenol, all three isomers of cresol (2-, 3- and 4-methylphenol)
and 4-methyl-2-nitrophenol, which were present in signifi-
cantly large amounts, together with non-polar aromatics
such as naphthalene and acenaphthene. Coupled GC–EAG
recordings using Ha. irritans showed a similar number of
active components, but with additional activity from com-
pounds including (Z)-3-hexen-1-ol and 6-methyl-5-hepten-2-
one. Coupled GC–EAG recordings from Ha. irritans,
using volatiles collected from the most and least fly attractive
cattle in ranked herds in Denmark, located a number of
volatiles that elicited an electrophysiological response (Figs
1b and c; Table 1). In the non-attractive sample, more active
peaks were located. Compounds unique to this sample were
identified as the non-polar aromatic compounds propyl-
benzene and styrene, along with camphene, 2-heptanone
and propyl butanoate. EAG active compounds for both the
attractive and non-attractive samples were identified as
phenol and m- and p-cresol. A number of EAG active com-
pounds were located forHa. irritans in the cattle urine sample
(Fig. 1d), including polar aromatics such as m- and p-cresol,
which were located in the whole animal entrainment samples,
along with 2-methoxyphenol. A range of aliphatic alcohols
were also identified, including 3-octanol, 2-nonanol,
2-decanol, 1-nonanol and linalool. Due to the complexity of
the GC traces, the stereochemistry of chiral compounds was
not assigned. Thus, racemic materials were used in further
experiments. Overall, coupled GC–MS showed that a
wide range of compounds were collected from the heifers,
Table 1. Electroantennography responses of cattle flies to volatiles collected from cattle headspace and urine (48 h old).
Haematobia Musca Hydrotaea Stomoxys Wohlfahrtia
Peak no. irritans autumnalis irritans calcitrans magnifica
Type (see Figs 1a–d) Compound (10�2 g) (n¼ 5) (n¼ 10) (n¼ 10) (n¼ 3) (n¼ 3)
Amino acid 3 Propylbenzene NS NS NS NS NS
derivative 5 Phenol NS NS NS NS NS
8 o-Cresol NS NS NS NS –
9 m-Cresol NS NS NS * –
10 p-Cresol NS NS ** ** –
12 Naphthalene *** *** *** *** ***
13 4-Methyl-2-nitrophenol * *** * * NS
14 Acenaphthene NS *** * NS *
16 Styrene NS NS NS NS NS
23 2-Methoxyphenol * *** – ** –
Fatty acid 1 (Z)-3-Hexen-1-ol NS *** ** ** NS
derivative 4 1-Octen-3-ola *** *** *** *** **
7 Decane NS NS NS NS NS
11 Undecane NS *** NS NS NS
15 2-Heptanone * NS *** *** NS
17 Propyl butanoate NS NS NS * NS
19 3-Octanola *** *** – *** ***
21 2-Decanola *** *** – *** –
22 1-Nonanol *** *** – *** –
Isoprenoid 2 a-Pinene NS NS NS NS NS
or derivative 6 6-Methyl-5-hepten-2-one *** *** *** *** **
18 Camphenea NS NS NS NS NS
20 Linaloola *** *** – *** ***
Tsetse lure *** *** – *** –
Indole NS * – NS ***
Skatole NS * – NS ***
N,N-Diethyltoluamide NS NS NS NS –
Citronellol *** *** *** *** *
Propionic acid NS NS * NS –
Compounds listed with no peak numbers are established semiochemicals or repellents for other haematophagous Diptera.aStereochemistry undefined.
*, **, ***: significantly different from control at P¼ 0.05, 0.01 and 0.001, respectively. NS, not significantly different from control at
P¼ 0.05 control¼diethyl ether, 20mL. A single dashed line indicates that the compound was not tested due to unavailability of insects.
318 M. A. Birkett et al.
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
including carboxylic acids such as isobutyric acid, but the
majority of these were not proceeded with as they were not
located by coupled GC–EAG.
To confirm the electrophysiological activity of the com-
pounds located by coupled GC–EAG and identified by
coupled GC–MS, compounds were tested at a standard
concentration (10�2 g) using five target fly species
(Ha. irritans, M. autumnalis, Hy. irritans, S. calcitrans and
W. magnifica). No significant differences were observed
between the sexes. Therefore, subsequent testing was carried
out using female flies only (Table 1). Several compounds
gave highly significant EAG responses for all or most of
the flies, including (Z)-3-hexen-1-ol, 1-octen-3-ol, 6-methyl-
5-hepten-2-one, naphthalene and linalool. When presented
individually, a number of the compounds thought to be
EAG active gave little or no response, including phenol
and the cresols (apart from p-cresol). Almost all the
compounds identified from the cattle urine (compounds
no. 19–23) gave significant EAG responses. Of the pre-
viously known semiochemicals tested, only the tsetse lure
and citronellol gave consistently significant responses. In
general, there was a good correlation between the target
species and the responses to the compounds tested.
Compounds that showed EAG activity for all fly species
were tested for behavioural activity against M. autumnalis
using a wind-tunnel bioassay (Fig. 2). Propyl butanoate was
Fig. 2. Upwind flight response of M.autumnalis in a glass wind-tunnel to semiochemicals identified from cattle headspace and urine.
Compounds were tested across a range of doses (n¼ 3). Statistical analysis was carried out with Student’s t-test using matched pairs to
compare differences between test and control data. *P< 0.05. Vertical bars represent standard errors. C¼ control response.
Host location and selection by flies 319
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
also tested as it was only found in the volatile sample of the
least attractive Danish heifer and was therefore considered
to be a good candidate for repellency. From the urine-
derived compounds, 3-octanol and linalool were selected
on the basis of their structural proximity to 1-octen-3-ol
and established roles as insect semiochemicals. 1-Octen-3-ol,
6-methyl-5-hepten-2-one and 3-octanol showed significant
attraction at certain concentrations, with 1-octen-3-ol
attracting flies at very low levels. Naphthalene and linalool
showed strong repellency at low concentrations, but at
higher concentrations, responses were not significantly dif-
ferent from the control. Propyl butanoate was repellent at
the highest concentration. Surprisingly, the known repellent
citronellol showed no statistically significant activity,
despite displaying high EAG activity.
One small field study was conducted using small, separate
herds of cattle in Denmark, and slow-release formulations
of two selected compounds, 1-octen-3-ol and 6-methyl-5-
hepten-2-one, presented individually (Table 2). On day 1,
for the experimental herd involving 1-octen-3-ol, heifers
1226 and 1170 had the lowest mean fly load, whereas heifers
1180 and 1160 had the highest mean fly load. On day 2,
when 1-octen-3-ol was applied to heifers 1226 and 1170, no
statistical significant change was obtained on either of the
heifers. On day 3, when heifer 1180 and heifer 1160 were
treated, there was a small, but not significant fall in the
percentage of flies on heifer 1180 and heifer 1160 when
compared with day 1.
When 6-methyl-5-hepten-2-one was applied to heifer
1223 and heifer 1178 on day 2, there was no significant
change in fly loads. When applied to heifer 1138 and heifer
1261 on day 3, the fly loads were reduced significantly for
heifer 1261, whereas no effect was observed for heifer 1138,
when compared with day 1.
Discussion
This study reports the use of coupled GC–EAG, GC–MS,
EAG and behavioural studies, in the laboratory and field,
to identify volatile semiochemicals used in host location and
selection by nuisance and disease-transmitting cattle flies.
A number of physiologically active components were
identified, of which 1-octen-3-ol and 6-methyl-5-hepten-2-
one were attractive, and naphthalene, linalool and propyl
butanoate were repellent, in laboratory behavioural assays.
Some of the identified compounds have already been
reported to be present in cattle volatiles and are known as
semiochemicals for dipterous pests. 1-Octen-3-ol, a known
component of ruminant breath, is an attractant for tsetse
flies (Hall et al., 1984). Phenol, m-cresol and p-cresol are
released from cattle urine through microbial degradation,
and also influence tsetse fly behaviour (Hassanali et al.,
1986; Bursell et al., 1988). Mixtures including some of
these chemicals, when released in a specific ratio, have
been used successfully in the field to trap tsetse fly popula-
tions (Vale et al., 1986, 1988; Torr et al., 1995). Surprisingly,
in this study, the phenolic compounds had little or no
impact on electrophysiological or behavioural responses of
cattle flies. 6-Methyl-5-hepten-2-one has been reported
before as a component of waterbuck odour, but was not
electrophysiologically active for tsetse flies (Gikonyo et al.,
2002). 4-Methyl-2-nitrophenol has been identified as a com-
ponent of cattle odour, and is an olfactory stimulant for
cattle tick pests (Stuellet & Guerin, 1994). No reports exist
on the presence of propyl butanoate, (Z)-3-hexen-1-ol and
naphthalene, either as components of cattle odour or as
semiochemicals for haematophagous Diptera in general.
(Z)-3-Hexen-1-ol is associated with induced defence
mechanisms in damaged plants (Agelopoulos et al., 1999),
and naphthalene is a plant-derived volatile semiochemical
mediating maize and sorghum plant interactions with stem-
borer pests (Khan et al., 1997). Aliphatic alcohols have not
been reported previously as part of the volatile profile of
cattle urine. This may be due to differences in the techniques
used in volatile collection in this study and those used in
previously published reports (Hassanali et al., 1986; Bursell
et al., 1988). In this study, the active compounds, i.e.
aliphatic alcohols and cresols, were identified by air entrain-
ment of urine followed by coupled GC–EAG. In previous
studies, urine extracts, prepared by liquid–liquid extraction
using dichloromethane, were fractionated by liquid chro-
matography prior to identification. Fractions with activity
were shown to contain only phenolic compounds, thus
excluding the need for identification of other compounds.
Where the role of volatile semiochemicals in conferring
the differential attractiveness of hosts to cattle flies has
been studied previously, these studies have focused on the
Table 2. The mean percentage of horn flies distributed on herds of
Holstein-Friesian cattle in response to the addition of slow-release
sachet formulations of 1-octen-3-ol (release rate¼ 10.5mg/day)
and 6-methyl-5-hepten-2-one (release rate¼ 14mg/day), used as
individual treatments in separate experiments.
Day of experiment
Treatment Heifer no. Day 1 Day 2 Day 3
1-Octen-3-ol 1226 5� 2ab 0� 0a 6� 2b
1170 4� 1a 2� 2a 4� 3a
1193 11� 3a 12� 8a 22� 7a
1205 24� 4a 22� 4a 18� 7a
1181 9� 7a 10� 2a 14� 4a
1180 20� 2a 12� 5b 14� 3ab
1160 28� 5ab 42� 10a 23� 4b
6-Methyl-5-hepten-2-one 1223 1� 1ab 1� 1a 8� 5b
1178 4� 2a 2� 1a 6� 4a
1225 7� 1a 7� 4a 16� 1b
1168 8� 2a 8� 6a 14� 4a
1196 24� 8a 15� 5a 20� 7a
1138 22� 17ab 44� 5a 23� 9b
1261 35� 12a 23� 14ab 13� 4b
Statistical analysis was carried out using a Kruskal–Wallis test
followed by a multiple-comparison test. Significance indicated by
different letters for each heifer indicates significant difference
between medians of the ranks on a 5% level. Bold type indicates
treated heifers.
320 M. A. Birkett et al.
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
differential attractiveness of cattle and hosts belonging to
different taxa to tsetse flies (Vale, 1974a, b; Gikonyo et al.,
2002, 2003). Recent studies have demonstrated that for horn
flies, Ha. irritans, there is natural differential attractiveness
for individuals within herds of Holstein-Friesian heifers, i.e. a
single taxa, and that these differences can be manipulated
following movement of individuals between herds (Jensen
et al., 2004). This study, together with the Jensen et al.
(2004) work, demonstrates, for the first time, the role of
volatile semiochemicals in conferring the differential attrac-
tiveness of these heifers, and the manipulation of fly popula-
tions in the field. The identification of attractants and
repellents in this study supports the hypothesis that attrac-
tiveness of individual hosts is, in part, determined by varia-
tion in the volatile profile of individuals, in particular the level
of volatiles that reduce fly loads. Those semiochemicals that
reduce fly loads may act as outright repellents, or by reducing
the sensitivity of flies to attractants, thus ‘masking’ the pres-
ence of cattle odour. In this study, a tendency towards a
repellent effect was observed when heifers with high fly
loads were treated with two compounds, 1-octen-3-ol and
6-methyl-5-hepten-2-one, which were identified in the labora-
tory as attractants. This activity may have arisen through the
use of slow-release formulations that released inappropriate
levels of the compounds. The average rate of production of
1-octen-3-ol for ox has been determined to be 0.01mg/h (Torr
et al., 1995), which is much lower than the rate used in this
study. Furthermore, higher release rates have been shown to
be repellent for tsetse flies (Vale & Hall, 1985), which may
explain the reduction of fly loads in this study. Thus, further
field-based studies are required to develop the optimum doses
and formulations for these compounds to be attractants, and
to assess the impact of the identified repellent compounds
propyl butanoate, linalool and naphthalene on reducing fly
loads on individual heifers and on whole herds.
In the plant kingdom, it has been established that herbi-
vorous insects avoid energy-wasting visits to feed on or
colonize a host on which they cannot successfully develop,
by responding to volatile components emitted by unsuitable
hosts (Pickett et al., 1998). The identification of attractants
and repellents in this study supports the hypothesis that
similar host/non-host preferences exist for haematophagous
insects in search of an appropriate vertebrate host. Differ-
ences in volatile profiles of cattle are likely to result from
differences in the physiological status of the host, i.e. diet
(Vale, 1981), age, reproductive status and general condition
of the individuals. The genetic background of cattle, i.e. the
breed, is also known to influence host susceptibility. This
has been illustrated by the strong correlation in fly load
between mothers and daughters (Thomas et al., 1987).
Clearly, the identification of naturally occurring semio-
chemicals opens up the way for the development of new
approaches to the monitoring and control of nuisance and
disease-vectoring cattle flies, where attractants and repel-
lents are utilized concurrently as part of a ‘push–
pull’ strategy (Miller & Cowles, 1990). This study also
serves as a model for the identification of new control
agents for other disease-transmitting Diptera affecting
domestic livestock and human beings, e.g. mosquitoes and
biting midges, where the natural differential attractiveness
within a host species can be exploited. The potential for
using volatile, host-derived semiochemicals in reducing
the attractiveness of human hosts to field populations of
malarial mosquitoes, Anopheles gambiae s.l. (Diptera:
Culicidae), has already been demonstrated (Costantini
et al., 2001).
Acknowledgements
The authors wish to thank Alan Todd for statistical advice.
This work was supported by the European Commission
(CEC Contract Mo. AIR3-CT 93–1445). Rothamsted
Research receives grant-aided support from the Biotechnol-
ogy and Biological Sciences Research Council (BBSRC) of
the U.K.
References
Agelopoulos, N.G., Hooper, A.M., Maniar, S.M., Pickett, J.A. &
Wadhams, L.J. (1999) A novel approach for isolation of volatile
chemicals released by individual leaves of a plant in situ. Journal
of Chemical Ecology, 25, 1411–1425.
Bartlett, C. (1985) An olfactometer for measuring the repellent
effect of chemicals on the stable fly, Stomoxys calcitrans (L.).
Pesticide Science, 16, 479–487.
Brown, A.H., Steelman, C.D., Johnson, Z.B., Rosenkrans, C.F. &
Brasuell, T.M. (1992) Estimates of repeatability and heritability
of horn fly resistance in beef cattle. Journal of Animal Science,
70, 1375–1381.
Bursell, E., Gough, A.J.E., Beevor, P.S., Cork, A., Hall, D.R. &
Vale, G.A. (1988) Identification of components of cattle urine
attractive to tsetse flies, Glossina spp. (Diptera: Glossinidae).
Bulletin of Entomological Research, 78, 281–291.
Costantini, C., Birkett, M.A., Gibson, G. et al. (2001) Electro-
antennogram and behavioural responses of the malaria vector
Anopheles gambiae to human-specific sweat components.
Medical and Veterinary Entomology, 15, 259–266.
Fraser, A.F. & Broom, D.M. (1990) Farm Animal Behaviour and
Welfare. Bailliere Tindall, London.
Gibson, G. & Torr, S.J. (1999) Visual and olfactory responses
of haematophagous Diptera to host stimuli. Medical and
Veterinary Entomology, 13, 2–23.
Gikonyo, N.K., Hassanali, A., Njagi, P.G.N., Gitu, P.M. &
Midiwo, J.O. (2002) Odor composition of preferred (Buffalo and
Ox) and nonpreferred (Waterbuck) hosts of some savanna tsetse
flies. Journal of Chemical Ecology, 28, 969–981.
Gikonyo, N.K., Hassanali, A., Njagi, P.G.N. & Saini, R.K. (2003)
Responses of Glossina mortisans mortisans to blends of electro-
physiologically active compounds in the odors of its preferred
(buffalo and ox) and nonpreferred (waterbuck) hosts. Journal of
Chemical Ecology, 29, 2331–2346.
Hall, D.R., Beevor, P.S., Cork, A., Nesbitt, B.F. & Vale, G.A.
(1984) 1-Octen-3-ol: a potent olfactory stimulant and attractant
for tsetse isolated from cattle odours. Insect Science and its
Application, 5, 335–339.
Hassanali, A., McDowell, P.G., Owaga, M.L.A. & Saini, R.A.
(1986) Identification of tsetse attractants from excretory
Host location and selection by flies 321
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322
products of a wild host animal, Synceras caffer. Insect Science
and its Application, 7, 5–9.
Holloway, M.T.P. & Phelps, R.J. (1991) The responses of
Stomoxys spp. (Diptera: Musicidae) to traps and artificial host
odours in the field. Bulletin of Entomological Research, 81,
51–55.
Jensen, K.M.V., Jespersen, J.B., Birkett, M.A., Pickett, J.A.,
Thomas, G. & Wadhams, L. (2004) Variation in the load of the
horn fly, Haematobia irritans (Diptera: Muscidae) in cattle herds
is determined by the presence or absence of individuals. Medical
and Veterinary Entomology, 18, 275–280.
Khan, Z.R., Ampong-Nyarko, K., Chiliswa, P. et al. (1997)
Intercropping increases parasitism of pests. Nature, 388,
631–632.
Maddrell, S.H.P. (1969) Secretion by the Malpighian tubules of
Rhodnius. The movement of ions and water. Journal of
Experimental Biology, 51, 71–97.
Mihok, S., Kang’ethe, E.K. & Githaiga, K.K. (1995) Trials of traps
and attractants for Stomoxys spp. (Diptera: Muscidae). Journal
of Medical Entomology, 32, 283–289.
Miller, J.R. & Cowles, R.S. (1990) Stimulo-deterrent diversion: a
concept and its possible application to onion maggot control.
Journal of Chemical Ecology, 16, 1367–1382.
Mullens, B.A., Peterson, N., Dada, C.E. & Velten, R.K. (1995)
Octenol fails to lure stable fly to insecticide. California
Agriculture, 49, 16–18.
Pickett, J.A. & Woodcock, C.M. (1996) The role of mosquito
olfaction in oviposition site location and in the avoidance of
unsuitable hosts. Olfaction in Mosquito–Host Interactions (ed. by
R. G. Bock and G. Cardew), pp. 109–123. CIBA Foundation
Symposium 200. John Wiley & Sons Ltd, Chichester, UK.
Pickett, J.A., Wadhams, L.J. & Woodcock, C.M. (1998) Insect
supersense: mate and host location by insects as model systems
for exploiting olfactory interactions. The Biochemist, August,
8–13.
Schofield, S., Cork, A. & Brady, J. (1995) Electroantennogram
responses of the stable fly, Stomoxys calcitrans, to components
of host odour. Physiological Entomology, 20, 273–280.
Schofield, S., Witty, C. & Brady, J. (1997) Effects of carbon
dioxide, acetone, and 1-octen-3-ol on the flight response of the
stable-fly, Stomoxy calcitrans, in a wind tunnel. Physiological
Entomology, 22, 380–386.
Soler Cruz, M.D., Vega Robles, M.C. & Thomas, G. (1996) In vivo
rearing and development of Wohlfartia magnifica (Diptera:
Sarcophagidae). Journal of Medical Entomology, 33, 586–591.
Stuellet, P. & Guerin, P.M. (1994) Identification of vertebrate
volatiles stimulating olfactory receptors on tarsus-I of the tick
Amblyomma-variegatum Fabricus (Ixodidae). 2. Receptors out-
side the Hallers organ capsule. Journal of Comparative
Physiology A, Sensory Neural and Behavioural Physiology, 174,
39–47.
Thomas, G., Prijs, H.J. & Trapman, J.J. (1987) Factors contribut-
ing to differential risk between heifers in contracting summer
mastitis. Summer Mastitis (ed. by G. Thomas, H. J. Over,
U. Vecht and P.Nansen), pp. 30–39.MartinusNijhof,Dordrecht.
Torr, S.J., Hall, D.R. & Smith, J.L. (1995) Responses of tsetse flies
(Diptera: Glossinidae) to natural and synthetic ox odours.
Bulletin of Entomological Research, 85, 157–166.
Vale, G.A. (1974a) The responses of tsetse flies (Diptera:
Glossinidae) to mobile and stationary baits. Bulletin of
Entomological Research, 64, 545–588.
Vale, G.A. (1974b) Direct observations on the responses of tsetse
flies (Diptera: Glossinidae) to hosts. Bulletin of Entomological
Research, 64, 589–594.
Vale, G.A. (1981) An effect of host diet on the attraction of tsetse
flies (Diptera: Glossinidae) to host odour. Bulletin of Entomo-
logical Research, 71, 259–265.
Vale, G.A. & Hall, D.R. (1985) The use of 1-octen-3-ol, acetone
and carbon dioxide to improve baits for tsetse flies, Glossina spp.
(Diptera: Glossinidae). Bulletin of Entomological Research, 75,
219–231.
Vale, G.A., Flint, S. & Hall, D.R. (1986) The field responses of
tsetse flies, Glossina spp. (Diptera, Glossinidae) to odours of
host residues. Bulletin of Entomological Research, 76, 685–693.
Vale, G.A., Flint, S. & Hall, D.R. (1988) The olfactory responses of
tsetse flies, Glossina spp. (Diptera, Glossinidae) to phenols and
urine in the field. Bulletin of Entomological Research, 78,
293–300.
Wadhams, L.J. (1990) The use of coupled gas chromatography:
electrophysiological techniques in the identification of insect
pheromones. Chromatography and Isolation of Insect Hormones
and Pheromones (ed. A. R. McCaffery and I. D. Wilson),
pp. 289–298. Plenum Press, New York.
Warnes, M.L. & Finlayson, L.H. (1985) Responses of the stable fly,
Stomoxys calcitrans (L.) (Diptera: Muscidae) to carbon dioxide
and host odours. II. Orientation. Bulletin of Entomological
Research, 75, 717–727.
Warnes, M.L. & Finlayson, L.H. (1986) Electroantennogram
responses of the stable fly, Stomoxys calcitrans, to carbon
dioxide and other odours. Physiological Entomology, 11,
469–473.
Accepted 21 September 2004
322 M. A. Birkett et al.
# 2004 The Royal Entomological Society, Medical and Veterinary Entomology, 18, 313–322