Pestic. Sci. 1985, 16, 479-487

An Olfactometer for Measuring the Repellent Effect of Chemicals on the Stable Fly Stomoxys calcitrans (L.)"

Christopher Bartlett

Coopers Animal Health Ltd, Berkhamsted Hill, Berkhamsted, Hertfordshire HP4 2QE

(Manuscript received 12 December 1984)

An olfactometer is described that permits the evaluation of chemicals acting in the vapour phase against the stable fly Stomoxys calcitrans (L.). Hungry flies, held in a metal frame cage covered with soft gauze, are attracted to a target held adjacent to one side of the cage, and emitting an attractive airflow. Flies landing on the target are counted at intervals of 15s. Flies starved for 24h were tested in the olfactometer when 2, 3 and 4 days old. With the exception of the first exposure of the 4-day-old flies, all the flies of the three ages showed greater than 80% attraction to the olfactometer target after exposure for 2 min, for each of four successive exposures at 10-min intervals. For all ages there was a significant increase in attraction at the second exposure but no further increase with subsequent exposures. It is likely that the sensillae, stimulated by the first exposure, subsequently responded faster. Eight chemicals were incorporated into the airflow behind the target to test their effect on flies. Permethrin (a low vapour pressure pyrethroid) and crotoxyphos (a low vapour pressure organophosphate) did not act in the vapour phase. Empenthrin (a high vapour pressure pyrethroid) and dichlorvos (a high vapour pressure organophosphate) both exhibited the toxic effect of knock-down on the flies without repellency. Oil of citronella and citronellol, two known fly repellents, gave a general reduction of attraction that was dose dependent; fewer flies found the target and those that did stayed a shorter length of time. Natural pyrethrum gave a similar effect to these two at lower concentrations, but at higher concentrations, it also showed a repellent effect. N , N-Diethyl-m-toluamide gave a low reduction in attraction. The olfactometer shows potential for use with other flying insects, both to observe the effects of an insecticide and also to study the behavioural responses in the absence of insecticides.

1. Introduction

The stable fly Stomoxys calcitrans (L.) is an important blood-sucking pest of cattle, horses and dogs throughout the world. Both sexes are haematophagous and when large numbers of flies are present, milk yield'X2 and weight gain3 in cattle may be adversely affected. In addition, the fly has been implicated in the spread of animal diseases such as trypanomyiasis, equine infectious anaemia, Dermatophila congolensis, anaplasmosis4 and bovine Herpes mammalitis.'

Chemicals that have been used successfully to control flies on cattle and other domestic stock include organophosphates, such as chlorfenvinphos and crotoxyphos, natural pyrethrins and synthetic pyrethroids, notably permethrin. However, these chemicals must contact the fly on the body surface before any effect is possible, and therefore treated animals may still be worried by the flies.

"This paper was presented, in part, at the symposium Control of ectoparasites of veterinury importance on 26 November 1984, organised by the Pesticides Group, Society of Chemical Industry.

479

C. Bartlett 480

A chemical giving a vapour-repellent effect and thus preventing flies landing on the animal is likely to give more complete freedom from fly worry. The olfactometer described in this paper was designed to select chemicals with such an effect.

2. Experimental methods

2.1. Description of the olfactometer The olfactometer attracts starved Stomoxys culcitruns to a target by generating conditions known to be important in attracting the fly to its mammalian host. The requisite factors are carbon dioxide and olfactory stimuli, which are important in long-range host location, and a local increase in warmth and humidity, which induces the flies to settle and probe.6

A diagram of the olfactometer is shown in Figure 1. Compressed air enters at (a) and is first cleaned of odours by passing through an activated charcoal filter (b) of volume 176cm3. The air then passes through a needle valve (c) and flowmeter (d) that enable the flow-rate to be accurately adjusted. Cattle odour and humidity are then imparted to the air by bubbling it through the olfactory flask (e). The cattle odour is provided by freshly cut cattle hair in water (f); the water provides an increase in humidity. The air then flows out into the target (h). Flask (9 ) prevents water accidentally bubbling out of flask (e) and entering the target. The required amount of carbon dioxide can be added to the airflow by adjusting the flow of carbon dioxide gas entering at (i) with the needle valve (c).

The target (h) is situated at one end of the box. The outer jacket (k) consists of a stainless steel tube of internal diameter 62 mm within another stainless steel tube of external diameter 92 mm. Both tubes are 75mm long and have a 92-mm diameter circle of stainless steel welded to each end, to cover the area between the tubes. The space between the inner and outer tube walls is insulated with expanded polystyrene fragments. The jacket is positioned securely at one end of the olfactometer.

A stainless steel funnel is glued to the inner end of the jacket and air enters the target from a tube inserted tightly into a rubber bung pushed into the narrow end of this funnel. A 45-W bulb ( I ) , painted matt black, is mounted inside the jacket. This bulb warms an adjacent copper gauze circle (m) (62-mm diameter; mesh size 1mm2; 64 holes cm-’), and in this way distributes heat evenly over the air flow and raises the temperature of the target face to about 37°C. The target face consists of a brass ring, 19mm wide with an external diameter of 12.5mm, with a circle of copper gauze (mesh size as for the circle m) welded to one side. This target ring (n) fits securely into another brass ring 25mm wide (o) , which pushes over the end of the target jacket.

A moist cattle hair pad (p) is positioned behind the target face to provide a humid, tactile, surface. The hair pad is formed by placing 3g of cattle hair evenly in a 14-cm Buchner funnel over a Whatman No. 1 filter paper. Approximately 150ml of water is added and by gentle tamping of the hair, with a circular piece of gauze while the water is removed under suction, a pad 4 5 mm thick is formed.

A test chemical is incorporated into the target as follows. The target ring (n) has four equally-spaced metal rods attached which extend 8mm from the inside of the ring, opposite to the gauze face. The test chemical is dissolved in a suitable volatile solvent, such as acetone, and applied evenly to a circle of nylon gauze (9) (150-mm diameter; mesh size 2.5mm2; 27 holes cm-*). When dry, the gauze is pushed firmly over the four metal rods and hence in position over the area of the ring. When ring n is pushed into ring 0 , and ring o is pushed on to the target jacket, the gauze circle is held tightly over the area of the target 19mm behind the face. The airflow can then pick up chemical from the treated gauze, but the test insects have no contact with the treated gauze.

The flies on test are held in a cage (r) which consists of a square brass frame with sides 230 mm long. The cage is covered with tubular ‘Stockinette‘ (Kershaw and Co. Ltd., Manchester) stretched tightly over the frame and secured at both ends with a rubber band. The cage stands on a brass stand (s), which holds the cage with one side adjacent to the target, and the top of the target level with the top of the cage. If the cage extends higher than the top of the target, flies

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482 C. Bartlett

attracted by ascending emanations from the target may be found above the target and therefore not included in the test, although they have responded.

The target face and fly cage and stand are contained in a fume cupboard (v) which removes air at a rate of 3m3 min-’. This ensures that there is no build-up of chemical vapour in the target area. Gauze circles are treated in a fume cupboard which is isolated from the test area to ensure no contamination of the atmosphere with chemical vapour.

2.2. Evaluation of the olfactometer 2.2.1. Test insects and procedure A culture of Stomoxys culcitrans, originating from flies collected from the field in the UK over 20 years ago and since maintained in culture at the Wellcome Research Laboratories, provided the flies for all the tests. The insects were maintained at 27 (k1)OC and 55 (+l)% relative humidity, with a photoperiod of 12h light, 12h dark. Larvae were reared on an artificial medium comprising bran, granmeal, vermiculite, malt, yeast and water. Adult flies were fed citrated bovine blood.

Flies starved for 24 h before the test were used throughout. It has been shown that the activity of S. calcitrans peaks after 21-22 h of starvation with an active period spanning 6 h;’ optimal attraction to the host was observed when flies starved for 24 h, were released into a large flight chamber containing stalled cattle.8 No attempt was made to separate the sexes for any of the experiments; observations of the fly culture had shown a male: female ratio of 52:48.

The olfactometer was prepared by placing freshly-cut cattle hair in water in the olfactory flask and a moist hair pad in the target. The target bulb was turned on, and the air and carbon dioxide flows were adjusted to the desired rates. These rates were not critical but air and carbon dioxide flow rates of 13.4 and 0.26litre min-’ respectively, gave good results. The apparatus was then allowed to run for 5 rnin, by which time the target face had been heated to 3TC, approximately that of the skin temperature of the flies’ host, and shown to be near optimal for fly response.’ The apparatus was then used for one day’s testing before the hair in the olfactory flask was replaced.

Before the start of a test exposure, 25 flies were placed in a gauze-covered cage and left for 30 min. During the test, the cage was placed with one side adjacent to the target face, and the number of flies present on the target area was counted every 15 s for 2 min.

2.2.2. Fly age tests Flies, 2, 3 and 4-days-old, were tested in the olfactometer in the absence of chemicals. Prior to starvation, the flies were offered blood for a period of 2 h at 24-h intervals. For each age of fly, four cages of flies were tested. Each cage was exposed four times to test the effect of repeated exposures on the flies’ response. One hair pad was used throughout and was remoistened after every four exposures.

2.2.3. Test with chemicals Eight chemicals were tested in the olfactometer against 4-day-old flies that had been offered free access to blood until they were starved. The chemicals are listed in Table 1, together with the concentrations tested, and the vapour pressures where known. Each chemical was tested twice, the two tests being on separate days. On the first day, the tests were carried out in ascending order of concentration, and on the second day, the order was reversed. This eliminated any effect due to the flies getting ‘hungrier’ over the day.

For each day, 20 cages of flies were used. These were each exposed twice in the absence of a chemical. The first exposures were ignored; the second exposures served as controls for the third exposures (see section 3.1. for the explanation). The 20 cages were then sorted into four groups of five cages, and each group used for one of the four dose levels of the chemical.

The chemicals were dissolved in Analar grade acetone to the required concentration, and 1.6ml of the solution was applied evenly to a gauze circle laid on ‘Benchkote’. The gauze was

An olfactometer for measuring repellent effect 483

Table 1. Chemicals tested in the olfactometer against 4-day-old Sfomoxys culcirruns that had been starved for 24 h

Chemical Vapour pressure Application

(mPd) rates (gm-')

Permethrin" Empenthrinb Oil of citronella Natural pyrethrum (25% pale extract) Dichlorvos' Crotoxyphosd Citronellol N , N-diethyl-m-toluamide'

0.045 at 25°C 200 at 25°C

4.0, 2.0, 1.0 and 0.5 0.2, 0.1, 0.05 and 0.025 4.0, 2.0, 1.0 and 0.5 16.0, 8.0, 4.0 and 2.0 0.5, 0.1, 0.05 and 0.01 16.0, 8.0, 4.0 and 2.0 16.0, 8.0, 4.0 and 2.0 16.0, 8.0, 4.0 and 2.0

1600 at 20°C 1.8 at 20°C

"3-Phenoxybenzyl (1RS)-cis,tru~-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate. b(E)-(RS)-l-Ethynyl-2-methylpent-2-enyl (lRS)-cis,truns-2,2-dimethyl-3-(2-methylprop-l-enyl)-

'2,2-Dichlorovinyl dimethyl phosphate. dDimethyl (E)-l-methyl-2-(l-phenylethoxycarbonyl)vinyl phosphate. "Deet'.

cyclopropanecarboxylate (S-2852 Forte), ('Vaporthrin', Sumito Chemical Co.).

then left for 1 min before being placed in the target. After a 30-s equilibration period the exposure was made. A freshly treated gauze was used for each exposure.

During control tests, one hair pad was used throughout and this was remoistened after every four exposures. When chemicals were used, the hair pad was changed, and the olfactometer target was rinsed with acetone between each chemical dose, to avoid contamination.

2.2.4. Analysis of data The 15-s counts from the fly age tests were converted by the arc-sin transformation to stabilise the data over the full response range, and the means and standard errors were calculated for the

I00

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Time (rnin)

Figure 2. Attraction of: (a) 2-day-old flies; (b) 3-day-old flies; (c) 4-day-old flies to the olfactometer. Cages of the flies were tested four times, with 10-min intervals between the tests; four replicates were tested and the results pooled; (m) first test; (U) second test; (0) third test; (0) fourth test. Standard error (arcsin units) for control flies were 0.037 for 2-day-old flies, 0.028 for 3-day-old flies, and 0.030 for 4-day-old flies.

484 C. Bartlett

Table 2. Values of P from tests of significant difference on pairs of overall mean percentage attraction for repeated tests 1-4

Attraction compared for the following

Value of P for age of fly indicated -

pairs of tests 2-day-old 3-day-old 4-day-old

1 and 2 1 and 3 I and 4 2 and 3 2 and 4 3 and 4

0.1 0.026 0.001 0.034 0.024 0.001 0.004 0.001 <O.OoI

NS NS NS NS NS NS NS NS NS

~ ~~ ~

NS=not significant.

four replicates of each consecutive exposure for each age of fly (Table 2; Figure 2). The overall mean percentage attractions for the four consecutive exposures, for each age of fly, were tested for significant differences (Table 2 ) .

The data from the chemical tests were also converted by the arc-sin transformation and averaged over the replicates (Figures 3 and 4). There were ten replicates for each chemical dose, and 40 replicates for the controls for each chemical. Standard errors for the means were also calculated. For each chemical, the overall mean percentage attraction for the control tests and the overall mean percentage attraction for the treated tests were compared for significant differences (Table 3). For these comparisons, the mean percentage attraction for the controls was calculated from only ten tests. Thus the mean percentage attraction for a chemical dose was compared with the mean control percentage attraction for the same ten cages.

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Figure 3. Attraction of 4-day-old flies to the olfactometer with four dose levels of: (a) permethrin; (b) crotoxyphos, (c) dichlorvos, and (d) empenthrin. Each point on the control curve (0) is the pooled result of 40 cages of flies; each time point on the remaining curves is the pooled result of ten cages of flies. For permethrin: (0) 0.5g permethrin m-’; ( A ) l .0gm-2;(0) 2.0gm-’; (0) 4.0gm-’ was placed in the airflow; standard errors (arcsin units) for treated flies=O.O35, for control flies=0.015. For crotoxyphos: (0) 2.0g crotoxyphos m-2; (A) 4.0gm-’; (0) 8.0gm-’; (V) 16.0g11-~ was placed in the airflow; standard errors (arcsin units) for treated flies=O.O68, for control flies=O.O30. For dichlorvos: (0) 0.01 g dichlorvos m-’; ( A ) O.OSgm-’; (0) O.lgm-’; (0 ) 0.5grf2 was placed in the airflow; standard errors (arcsin units) for treated flies=O.O87, for control flies=O.O39. For empenthrin: (0) 0.025g empenthrin m-’; ( A ) 0.05gm-’; (0) 0.1 gm-’; (0) 0.2gm-’ was placed in the airflow; standard errors (arcsin units) for treated flies=O.O43, for control flies=O.O22.

An olfactometer for measuring repellent effect 485

I 1 I I I I I 1 I I I I I I I I 0 0.5 1-0 1.5 2.0 0 0.5 I .o 1.5 2 0

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Figure 4. Attraction of 4-day-old flies to the olfactometer with four dose levels of (a) oil of citronella, (b) citronellol, (c) pyrethrum, and (d) N,N-diethyl-m-toluamide. Each point on the control curve (0) is the pooled result of 40 cages of flies; each time point on the remaining curves is the pooled result of ten cages of flies. For oil of citronella: (0) 0.5g oil of citronella m-’; ( A ) l.Ogm-’; (0) 2.0gm-’; (0) 4.0gm-’ was placed in the airflow; standard errors (arcsin units) for treated flies=0.055, for control flies=O.O25. For citronellol: (0) 2.Og citronellol m-’; ( A ) 4.0gm-’; (0) 8.0gm-’; (V) 16.0gm-’ was placed in the airflow; standard errors (arcsin units) for treated flies=0.130. for control flies=0.057. For pyrethrum: (0) 2.Og pyrethrum m-’; (a) 4.0gm-*; (U) S.Ogm-’; (V) 16.0gm-’ was placed in the airflow; standard errors (arcsin units) for treated flies=O.O86, for control flies=0.041. For N,N-diethyl-m-toluamide: (0) 2.Og N,N-diethyl-m- toluamide m-’; (A) 4.0gm-*; (0) 8.0gm-’; (0) 16.0gm-’ was placed in the airflow; standard errors (arcsin units) for treated flies=O.lOO, for control flies=O.O34.

3. Results and discussion 3.1. Fly age experiments All three ages of fly tested, 2, 3 and 4-day-old, responded satisfactorily in the olfactometer; the mean percentage attraction to the target after 2 min was greater than 80%, except in the case of exposure 1 for 4-day-old flies, for which it was a little over 60% (Figure 2). The flies differed,

Table 3. Values of P from tests of significant difference on pairs of overall mean percentage attraction for control and chemical tests 1-4

Value of P for indicated chemical Pairs of

treatments Oil of N , N-Diethyl- compared Pennethrin Pyrethrum Empenthrin Crotoxyphos Dichlorvos citronella Citronellol -m-toluamide

C and 1 C and 2 C and 3 C and 4 1 and 2 1 and 3 1 and 4 2 and 3 2 and 4 3 and 4

NS NS NS NS NS NS NS NS NS NS

NS 0.05 0.01 0.001 NS NS

0.001 NS 0.001 0.01

0.001 0.001 0.001 0.001 NS NS NS NS NS NS

NS NS NS NS NS NS 0.05 NS NS 0.05

NS 0.05 0.05 0.001 NS

0.01 0.001 0.01 0.001 0.001

0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.5 0.001 0.5

0.01 NS 0.001 NS 0.001 0.01 0.001 0.01

NS NS NS 0.01 NS 0.01 NS NS NS NS NS NS

C=control. NS=not significant.

32

486 C. Bartlett

however, in their response in repeated exposures. When comparing the mean percentage attraction over the 2-min exposure period, for all three ages of fly, the response for the first exposure was always significantly lower than those for the second, third and fourth exposures, which did not differ from one another (Table 2 ) .

Two-day-old flies showed the highest level of variation between replicate exposures, with a standard error of 0.037. This was significantly different at the 5% level to the standard error of 0.028 for 3-day-old flies. The standard error for 4-day-old flies (0.030) was not significantly different to the standard errors for either the 2 or 4-day-old flies, at the 5% level.

The reason for the enhanced attraction of flies after one exposure may be because the sensillae on the antennae, once stimulated by the first exposure, respond faster in later exposures. Further work would be required to show how long this enhancement lasts, as the length of time between successive exposures in the fly age experiments was only 10 min.

When considering the design of the experiments with chemicals, the problem of the different responses for the first and subsequent exposures was resolved by exposing a cage of flies twice in the absence of the chemical, and then once with the chemical. The first exposure was discarded and the second taken as the control for the third (treated) exposure. The choice of age of fly for the chemical tests lay between 3 and 4-day-old flies because these flies showed similar attraction curves for the second and third exposures (important for comparing control and test attractions) and a lower level of variation in response than 2-day-old flies. As 4-day-old flies were used routinely in other tests in the laboratory, these were chosen.

3.2. Tests with chemicals Two chemicals, crotoxyphos and permethrin, showed no significant differences between control attraction and attraction at any chemical dose (Figures 3a and 3b; Table 3). Previous work in this laboratory has shown both these chemicals to be effective as rapid contact toxicants and both are sold as treatments for controlling flies on animals. The present tests showed that neither chemical acts in the vapour phase, and their action when controlling flies on cattle is likely to be contact toxicity. Both chemicals have low vapour pressures (Table 1).

The remaining six chemicals showed at least some effect on the flies with attraction on at least one chemical dose being significantly lower than the control attraction (Table 3). In addition, the shapes on the attraction curves suggested the possible type of action of the chemical.

Dichlorvos and empenthrin were similar in effect as shown by the shape of their attraction curves (Figures 3c and 3d). For both chemicals, overall attraction at all dose levels was significantly lower than the control attraction (Table 3). The flies were attracted normally for the first 1530s , after which the flies on the target began to be knocked down by the chemical. Flies arriving subsequently were similarly affected. Both chemicals were clearly acting in the vapour phase but were not affecting the flies’ ability to locate the host, nor were they repelling the flies; the effect appeared to be simply one of toxicity. Both chemicals have high vapour pressures (Table 1).

Oil of citronella and citronellol (a major fraction of oil of citronella) both strongly affected the flies but in a different way to the two preceding chemicals. The attraction curves for the chemical tests were similar in shape to the control curves but at a lower level of attraction (Figures 4a and 4b). Fly attraction appeared to be suppressed from the start of a test, and there was no evidence of knockdown or toxicity effects. Compared to the controls, fewer flies in treated exposures found the target and they remained there for a shorter time. Thus the chemicals, acting in the vapour phase, may have been both inhibiting location of the host, and acting as repellents at the target.

Natural pyrethrum appeared to exert both toxic and repellent effects on the flies, depending on the dose. At 2, 4 and 8grnp2, the effect was similar to empenthrin and dichlorvos (as judged by the shape of the attraction curve; Figure 4c); the flies responded normally at first and then were affected on the target after approximately 1 min. At these concentrations, there appeared to be no inhibition of attraction. However, at 16gm-’, as well as the toxic effect, the number of

An olfactometer for measuring repellent effect 487

flies finding the target initially was also reduced, suggesting that the chemical also acted as a repellent at the highest concentration. Both effects were clearly operating in the vapour phase.

N,N-Diethyl-m-toluamide showed a low level of effect on the flies, attraction at the two highest dose levels being significantly lower than the control attraction at P=O.Ol (Table 3). This effect appeared to be similar to the effect seen with oil of citronella and citronellol, as indicated by the shape of the attraction curve (Figure 4d) and was likely to be due either to inhibition of attraction or to repellency of the flies from the target. No toxic effect was observed.

The chemical tests show that the apparatus identifies only the chemicals that act in the vapour phase; these tend to be those with a high vapour pressure. Thus some chemicals, for example, permethrin which is highly active on contact, may show no effect in the olfactometer because of their low volatility.

With this apparatus, the shape of the attraction curve over time gives an indication of the type of effect of a chemical. A chemical may act with a toxic effect only and give a curve of similar shape to that found with dichlorvos (Figure 3c), or it may act as a repellent or inhibit attraction of the flies and give a curve of similar shape to that found with citronellol (Figure 4b).

In the field, a chemical would be required to work for, ideally, at least 24 h. The residual life of a chemical may be tested using the olfactometer by testing treated gauze circles at various time intervals after treatment.

Besides the above applications, the apparatus has other potential uses. By changing the constituents of the airflow, other species, such as blowflies or other biting flies, could be tested; the airflow could either simulate a host or an oviposition site. Preliminary work with mosquitoes (Anopheles and Aedes spp.) has shown promising results. Crawling insects could be tested by modifying the design of the holding cage. Finally, in the absence of insecticide, the apparatus could be used for investigating behavioural responses of flying insects.

Acknowledgements The author would like to thank all those colleagues who provided advice and assistance during the course of this study, especially Mr I. S. MacPherson and Mr M. I. Burch who provided the statistical analyses, and Mr M. D. Matthewson who critically reviewed the manuscript.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Bruce, W. M.; Decker, G. C. J . Econ. Entomol. 1947, 40, 530. Morgan, D. W. T.; Bailie, H. D. Vet. Rec. 1980, 106, 121. Campbell, J. B.; White, R. G.; Wright, J. E.; Crookshank, R.; Clanton, D. C. J . Econ. Entomol. 1977, 70, 592. Greenburg, B. Flies and Diseuse Vol. 11, Biology and Disease Transmission Princeton University Press, Princeton, 1971. Gibbs, E. P. J . ; Johnston, R. H.; Gatehouse, A. G. Res. Ver. Sci. 1973, 14, 145. Gatehouse, A. G. ; Lewis, C. T. Entomol. Exp. Appl. 1973, 16,275. Bidgood, H. M . , PhD Thesis, University of Birmingham, 1980. Martin, S . J . S . Proc. Br. hec t i c . Fungic. Conf. 8th 1975, 2, 539. Gatehouse, A. G. J . Insect. Physiol. 1970, 16, 61.