Medical and Veterinary Entomology (1992) 6, 103- 109

Responses of female New World screwworm flies, Cochliom yia hominivorax, to co I ou red targets in the laboratory C . H . G R E E N and M . L . W A R N E S Tsetse Research Laboratory, ODAIUniversity of Bristol, Langford House, Bristol

Abstract. The responses of unmated female New World screwworm flies, Cochliomyia horninivorux Coquerel, to visual targets were studied in a wind- tunnel. Both activity and frequency of contacts with targets increased greatly when the screwworm attractant mixture swormlure-4 was added to the airstream. Target-orientated responses depended on target colour, with red and black targets being preferred over blue, white and yellow ones; this preference was much greater in the presence of odour than in its absence. No preference was detected for different shapes and orientations of red targets, all of equivalent surface area. Omitting different components from swormlure-4 generally resulted in a large reduction in activation and target contacts. Attempts to substitute 1- octen-3-01 for the butanol fraction were unsuccessful, but skatole may substitute to some extent for indole; the two isomers of butanol normally present in swormlure-4 may substitute partly or completely for each other. This type of measurement forms a suitable bioassay in the development of attractive targets for monitoring and control of wild adult screwworm populations.

Key words. New World screwworm fly, Cochliomyia hominivorux, targets, colour, attraction, swormlure.

Introduction

The highly successful control campaigns against the New World screwworm, Cochliomyia hominivorax Coquerel, in the southern U.S.A. and Mexico have been based on the sterile insect technique (SIT), in conjunction with veterinary surveillance and cure of myiases (Krafsur el al., 1987). The need for an effective adult control system, capable of rapid deployment to contain an outbreak until SIT can be put into place, has been underlined by the recent outbreak of C.hominivorax in Libya (El-Azazy, 1989). A possible technique already available, the screw- worm adult suppression system (SWASS) (Coppedge et al., 1978) was ruled unacceptable on environmental grounds, and no effective alternatives were available. As part of a joint research effort (involving the Tsetse Research Laboratory, the British Natural History Museum, the Natural Resources Institute, and the Joint Food and Agriculture Organization/International Atomic Energy

Correspondence: Dr C. H. Green, Tsetse Research Laboratory, Bristol University, Langford House, Langford, Bristol BS18 7DU.

Agency Division) it was decided to explore a technique previously used against the tsetse fly, Gfossiria spp., known as the odour-baited target (reviewed by Warnes, 1991). In this technique, flies are lured with the help of a synthetic host odour mixture to an insecticide-impregnated target (consisting of cloth, or cloth and mosquito netting). This has proved highly effective against tsetse flies, and has a low environmental impact (Vale et al., 1988). A highly effective synthetic attractant has already been developed for Chominivorax, known as ‘swormlure’ (Jones et ul. , 1976; Coppedge et al., 1977). A n improved mixture. swormlure-4, was developed by Mackley & Brown (1984).

Laboratory studies formed an integral part of the devel- opment of baits used against tsetse flies (Bursell ct a/.. 1988), as they permit rapid evaluation of different elements of the system under controlled conditions. Warnes & Green (1992) demonstrated activation and changes in flight behaviour in female Chominivorax in a wind-tunnel when exposed to swormlure-4. This study reports on orientation of female C.hominivorax to targets in the presence of swormlure-4 and derived mixtures, in an attempt to develop a bioassay for the development of a target-based control system for this species.

Materials and Methods

Flir,\. Flies were rcceivecl as sterilized pupae from the USDA ARS Screwwornm Research Laboratory, Tuxtla Gutierrez, Mexico, each week. and were matured as described by Warnes & Green (1992) with access t o sugar and water. Unmated female flies were used when between 4 and 0 days post-emergence.

Ohwr1utioti chiir?ihi,r arid recorditig techiiiqiies. Behav-. iour of groups of thirty-five flies at a time was recorded using video apparatus (Cohu h712-20OCl CCD camera. Panasonic AG-6702 S-VHS time lapse video recorder and For.,\ VTG-22 tinicr) in an observation chamber. The chanibix was similar to that described by Bursell (1990). except that the ceiling and floor were made from acrylic sheet ('Pcrspex', 1CI; OX02 grade, 5 mni) covered with paper (Benchkote, Whatman International Ltd), and the walls were covered with grey cardboard (Daler-Rowney no. 51) (Fig. 1 ) . Illumination was provided by four 20W tungsten halogen bulbs, positioned 0.5m above the chanihcr: the Bcnchkote on the ceiling acted as a light diffuser-. Thc chamber had a flow of clean air (passed through a charcoal filter) at 0.2-0.5 mis. The chamber,

odours and camera were sited in a windowless room maintained at 2XT, with the video recorder and observer in an adjacent room.

Turgars. Two targets were present in all experiments, in the positions indicated in Fig. 1; unless otherwise stated, they were 240mm high squares, one covered with red cloth and the other yellow cloth. Black, red, white and yellow cloths were similar to those described by Wall et ul. (1992); the blue was a 'phthailogen' blue, similar to sample [40] in Green (1989).

For the experiment on target shape, flies had a choice between the standard target (a red upright square), and one o f five different targets: square (standard), diamond (square rotated through 45"), upright oblong, oblong on its side, and circle (Fig. 4). each covered in red cloth.

Odoim. Odours were supplied to the chamber by passing a stream of clean air through a U-tube containing the mixture to be tested, and then out through pipes in the upwind section of the chamber (Fig. 1; NB: flies are separated from this section by a netting screen). For earlier experiments with standard sworrn\ure-4, outlets were three silicone rubber tubes terminating within 30 mm of each other, and the air flow rate was I l/min, giving a

I+ - - SIDE

c *

1

1 -

I - I /- TOP

t

c *

SCALE *------.------f 1 metre

Fig. 1. D i a p r n 01 flight space of observation chamber, and view o f chamber through video apparatus (c=video camera. r=odour dclivery i-oscttc. c-=targct; dotted lines indicate netting. dashed lines the baffle plate).

Coloirr responses of New World .screwworm 105

release rate (measured by weight loss in the U-tube) of 15mg/min. For experiments on target colour and shape, fly behaviour was recorded over 15 min; odour was supplied over minutes 6- 10. A further 15 rnin was left before the next treatment.

For experiments testing different odour mixtures, a number of measures were introduced to combat both habituation and cross-contamination, both shown t o be problems in early trials. The air flow rate was reduced to 0.2 llmin (giving a variable odour release rate depending on the odour substances). Odour release time was reduced to 4 min. Outlet tubes were changed to PTFE plastic, and each odour was given an individual tube, and U-tube. Between experiments, PTFE tubes were cleaned with a sequence of dichloromethane, acetone, water and air; other tubing, U-tubes and plastic connectors were dis- carded. Contamination of upstream equipment (pump, tubing, etc.) was prevented by occluding upstream pipes between odour pulses.

Experimettial design. Both the experiment with colours, and that with shapes, made use of a 5 x 5 Latin square design, in which the five treatments were presented during each of 5 days in varying sequences. In the odour exper- iments, a replicate consisted of a run with the control odour (swormlure-4), followed by the other treatments in random order, finishing up with a further control run.

20 5 15 r z 10

5 0

2 0 r ( b ) 4

n

Landing C]Brief contact m ,

Results

Effect of swormlure-4 on different aspects of fly behaviour

Flights were generally slow and meandering, with rela- tively little activity in the absence of odour, as has been described by Warnes & Green (1992). Flies at rest in the field of view of the camera at different positions in the chamber were scored every 30s, and changes monitored over a 12 min period, including 6 min when swormlure-4 odour was blown into the chamber. Relative numbers at different positions in the chamber did not change signifi- cantly over this period, and so were pooled into the cat- egory of total at rest; these are shown in Fig. 2(a). Flies at rest declined significantly during minutes 4-9 in the presence of odour. Totals in flight were also scored every 3Os, and these increased greatly during minutes 4-9 (Fig. 2b).

A continuous record of contacts of flies with the two targets was made. Those longer than 0.3s were scored separately and are termed 'landings'. Totals during each 30s period are shown in Fig. 2(c) for the red target and Fig. 2(d) for the yellow target. The frequency of both landings and briefer contacts on the red target increased greatly during minutes 4-9. Relatively few contacts were observed on the yellow target.

The totals of flies at rest and those in flight give the total numbers in the field of view at 30s intervals. Only upwind flies were visible to the camera and since the number of flies in the chamber did not alter, changes in the number of flies visible allow estimates of the downwind or upwind movement of flies. In the first minute there was a small

1 = 5 4 + 0 , , , , , , ~ , , , ~ ~ s , , , n , ~ , , , 9 ,

I I 2 2 3 3 4 4 5 5 6 6 7 7 8 899101011111212 Minute

Fig. 2. Behaviour in a group of thirty-five female C.horninivorax during a single 12min observation period. (a) Flies at rest in field of view at end of each 30s period; (b) flies in flight at end of caeh 30s period; ( c ) total landingdbrief contacts with red targct during each 30s period; (d) total landingslbrief contacts with yellow target during each 30s period.

increase in flies in view, and therefore moving upwind, but this was not significant. In the first 30s after odour switch-off (minute 10) the number of flies in the field of view dropped from thirty to sixteen, implying a rapid movement of flies downwind.

The effects of odour on the different aspects of behaviour measured appears to be subject to some time-lag, especially in the target-orientated responses; the frequencies of these behaviours did not reach a maximum until several minutes after odour-on, and did not decline immediately on odour-off.

Effect of target colour

Five differently coloured targets were tested, each being paired with a yellow control target. Total contacts with the test targets over a 15min session are shown in Fig. 3, with odour on for minutes 6-10. Black and red targets received many more contacts than the other colours. Red appears

30 t Blue

3 cr f 10 LL

0 I 2 3 4 5 6 7 8 9101112131415

Minute

30 t- Yel low Mlnu te

U

LL 10

0 I 2 3 4 5 6 7 8 9101112131415

M i n u t e

Whi te

: 20

I 2 3 4 5 6 7 8 9 1011 12131415 Minute Minute

Fig. 3. I_,tridinga (Iiorizontal hatching) aiid brief contacts (stipple:) of fcmalc C'./~orniniiwro~ over a 15 inin period, with odour for minutcs h- I0 (m,ii-kcd h! arrows). on targets of five different colours: totals from fivc replicatcs.

to be more attractive than black, but this could he an artefact. as the flies were difficult to see against a black background on video. and some flies may have been missed. Once again there appears to be a time-lag in the starting and stopping of odour-mediated activity relative t o the stimulus.

Statistical analysis shows overall effects of colour and odour on the frequencies of total contacts and landings. I t also shows a significant colour X odour interaction ( P < O.(n)l for ;ill main effects and interactions). This reflects the much greater effect of target colour on behav- iour whcn odour is present compared to when i t is absent.

Contacts with the control targets were also analysed. 'These were generally similar to contact frequencies on the blue. white and yellow treatments. They also increased in the presence of odour .

( ontdcts m d landings on the test targets during odour itiniuLition (minutes 9 and 10 only), along with diagrams of the h p e \ tr\ted. dre given in Fig. 4 There was no .ignifiLmt effect of shape on either overall contacts, o r I,rncling~ eithei during odour ytimulation (as shown on the bdr-chart), o r during other minutes.

lmportuirce of diSfrrriit components of swormlure-4

A series of tests was conducted to check the importance of the various sworrnlure-4 components in mediating specifically target-orientated responscs. The response of flies to red targets (described above) was chosen as a suitable assay. A yellow target was placed opposite the red in all tests, in case of an odour-mediated change in

Londing O B r i e f conioci

8o r x 60 C 0

2 40

t 20

0

El

Shape

Fig. 4. Summary of landings/bricf contacts of female C ~ . hornini- rlorax made with red targets of diffcrcnt shapes. but equal surface area. during 2 mill with odour stimulation; totals from fivc replicates.

Colour responses of New World .scriwworrn 107

colour preferences, but this was never observed. Both overall contacts and landings for the different mixtures tested are given in Table 1.

Standard swormlured is a mixture of dimethyl disul- phide; sec and is0 butanols; acetic, butyric, valeric and benzoic acids; phenol and 4-methyl phenol; and indole. In the first two experiments these different groups of compounds were omitted in turn, and all were shown to be necessary to give a full response, as the fractions all produced significantly lower levels of contacts with the red target than did complete swormlure-4.

In experiment 3 a simple substitution was attempted by omitting each isomer of butanol, whilst increasing the other to compensate. The results suggested that the two butanols might indeed be able to substitute for each other (although see butanol did give a response that was just significantly lower than the standard mixture).

Experiments 4 and 5 investigated two compounds which produce strong electroantennogram (EAG) responses, 1-

octen-3-01 (octenol) and 4-methyl indole (skatole) (A. Cork, pers. comm.). Octenol was tested in place of the butanols, to which it shows the greatest chemical similarity. None of the three concentrations tested gave any indication of increasing the target-orientated responses above the level of swormlure-4 without both butanols. Skatole was tested in place of indole at two concentrations, one similar to the standard indole concentration, and one at 0.1 times this concentration. Skatole does appear to substitute for indole to a great extent, although target contact frequency was still significantly less with skatole than with either indole (standard swormlure-4), or with swormlure-4 with a reduced indole concentration.

Among the partial swormlure-4 mixtures, some gave a much higher level of response than others. Apart from the butanols (where it may be possible to substitute them for each other without reducing the response), the fraction giving the highest response was that omitting dimethyl disulphide, which gave over 50% of the control response.

Table 1. Contacts of female C.hominivorux with a red target when stimu- lated by swormlurc-4, and by swormlure-4 with different components dclctcd or substituted (mean contactslmin for minutes 2-4 of 4 min observation periods, replicated as indicated).

Mixture (S4 = swormlure-4) Contacts (Landings) % control

Experiment 1 (3 replicates) s 4 S4-dimethyl disulphide S4- bu tanols S4- acids S4-indole and phenols

Experiment 2 (4 replicates) s 4 S4-indole and phenols S4- indole S4- phenols

Experiment 3 (3 replicates) s 4 S4- butanols s4-iso butanolt S4- sec butanolt

Experiment 4 (1 replicate) s4 S4- butanols S4-butanols + octenol ( lYo) S4-butanols + octenol (5%) S4- butanols + octenol (10%)

Experiment 5 (4 replicates) s 4 S4 (indolc at low dosei) S4- indole S4-indole + skatole S4-indole + skatole (low dose)'

30.1 15.4 1.2 2.0 3.3

18.8 3.6

10.2 4.8

28.2 2.3

25.6 22.7

28.7 1.3 4 0.7 0.7

23.1 22.7 6.3

17.5 16.3

(100.0) 51.3*** 4.1*** 6.6***

11.1***

(100.0) 19.1*** 54.1*** 25.7***

(100.0) 8.3***

90.6 80.3*

(100.0) 4.h***

14.0*** 2.3*** 2.3***

(100.0) 98.0 27.3*** 75.7** 70.3***

Difference from control ( x ' ) : * P < 0.05, **P< 0.01, ***P< 0.001, ' When only one butanol was omitted, amounts of the other were doubled. ' 'Low-dose' was 0.1 of normal amount.

Omitting indole alone gave results o f 54% and 27% of control in different experiments. Omitting larger groups of chemicals gave the greatest reductions in target-orientated responses.

On occasions where the same fractions were re-tested, the responses showed a reasonable degree of stability (for example, swormlurc-4 less butanols gave 4.1%, 8.3% arid 4.6% of control in separate tests). even though compai-i- sons were based on relatively few repetitions. Activity level5 wcre not measured. but generally correspondcd closely with levels o f target-orientated activity.

Landing frequencies followed the frequencies of overall contacts. making up about one-third of the total contacts.

Discussion

.I'hc most striking changes observed in female C.honiitii- w r u . ~ when stimulated by swormlure-4 were an increase in flight activity. a n d an increase in target-orientated behav- iour. ,A high Iwel of fly contacts was observed on red and black targets in the presence of swormlure-4 odour: rela- tivcl! tcw contacts wcre observed in the absence of odour, or o n bluc. uhite and yellow targets. The importance of target colour indicates that orientation to visual stimuli was involved. and not just the incidental results of increased activit?. Whether the response is truly to colour (in the wnsc of differcntial reflectivity at different wavelengths) is doubtful. Red wavelengths are generally poorly perceived by Diptera (hlciirel, 10791, and C.I?orriirzitmax- may per- ceiw red objects as dark. like black. There was no indi- cation of colour preferences among blue. yellow, or white, a s i \ often found among other Diptera (e.g. Green & Flint, 1986: Kirk. 1984). The response observed here may be to an object showing dark-contrast with a background. It is not known what natural stimuli swornilure-4 imitates (it was dcveloped simply as a replacement for the rotting liver previously employed as an attractant; Jones et ul. , 1Y76), but i t swornilure-4 imitates wound odours, the visual responsc might he part of the wound-locating strategy, ;IS

wounds might appear dark, or reddish, against a background of fur or fleece.

N o evidence was found o f preference for some shapes o f targets over others. This contrasts with similar studies o n (;ios\ijiu (Doku & Brady, lY89), in which a marked preference for vertically-orientated shapes was demon- strated. This may indicate a different priority for visud and olfactory stimuli in host-location between tsetse and scren\vorni flies.

The increaw i n flight activity observed in the presence o f sworrnlure-4 odour was probably due both to activation of individual flies. and an increase in the time spent by individuals in flight (Warnes & Green, 1992). The latter study also described a tendency to upwind flight in the presence of otlour, and downwind flight when odour was turned of f , as was observed here.

Odour experiments demonstrated that this experimental protocol is suitable for the rapid assessment of candidate attractant rniutures. Although not exhaustive, these

expcriments did show that most if not all of the components in swormlure-4 are necessary to produce maximum re- sponse to targets. Swormlure-4, in common with earlier versions of swormlure, was developed in the field using traps, but these experiments show that it is probably suitable in its present form for usc with targets, also. 'The literature on swormlure development does not allow much assessment of the contributions of individual com- ponents to overall swormlure attraction. An exception to this is the study of Coppedge rt al. (1977), in which dimethyl disulphide was added to an earlier version of the swormlure mixture, leading to a two-fold increase in catches of screwworni flies (sex unspecified). It is interesting that in the present experiments a halving of target con- tacts was produced by omitting this compound from swornilure-4.

The strong visual element in the response, demonstrated by the present study, is encouraging for the use of target technology in controlling C.hominivorux; similar principles might also be used for a population monitoring system, for example with sticky targets. Most contacts observed here were brief (over 60% less than 0.3s), and it remains to be seen whether insecticide formulations can be devel- oped giving a satisfactory kill rate for insecticidal devices (although contacts may be repeated many times, giving much longer cumulative contacts). It may be possible to develop further olfactory or gustatory stimuli to keep flies in contact with targets for longer, as contacts of female flies with wounds are much more prolonged (Guillot ef d., 1977). Improved attractants based on extracts of wound fluid or blood (Hammack & Holt, 1083) may also be developed for use with targets, and the present assay would seem highly suitable for this type of bait development.

Acknowledgments

We thank the USDA ARS Screwworm Research Labora- tory, Tuxtla Gutierrez, Mexico, for supplying screwworm pupae: Dr P. A. Langley for arranging their importation and providing rearing facilities; and Mrs K. Stafford for help in fly maintenance and harvesting. Drs D. Hall and A. Cork of the Natural Resources Institute (NRI) advised on the odour testing aspects of this work. Drs A . M. Jordan, M. J. K. Hall and Mr R. Allsopp are gratefully acknowledged for helpful comments on the manuscript. This work was funded by the Overseas Development Administration of the U.K. Government through thc Livestock Protection Programme of the NRI . Additional support was provided by the Food and Agriculture Organiz- zation, Rome, and Joint FAO/IAEA Division, Vienna.

References

Burscll, E. (1990) The effect of host odour on thc landing rc- sponses of tsctsc flies (Glossina morsitans morsitans) in a wind tunnel with and without visual targcts. Physidogicuf En'rrtomo- log.v, 15, 369-376.

Colour responses of New World ,screwworm 109

Burscll, E., Gough, A.J.E., Beevor, P.S., Cork, A., Hall, D.R. & Vale, G.A. (1988) Identification of components of cattle urinc attractive to tsetse flies, Clossina spp. (Diptera: Gloss- inidae). Bulletin of Entomological Research, 78, 281-291.

Coppedgc, J.R., Ahrens, E., Goodcnough, J.L., Guillot, F.S. & Snow. J.W. (1977) Field comparisons of liver and a new chcmical mixture as attractants for the screwworm fly. Environ- mental Entomology, 6, 66-68.

Coppedgc, J.R., Broce, A.B., Tannahill, F.H., Goodenough, J.L., Snow, J.W. & Crystal, M.M. (1978) Development of a bait system for suppression of adult screwworms. Journal of Economic Entomology, 71, 483-486

Doku, C. & Brady, J. (1989) Landing site preferences of Glossina morsitans morsitans Westwood (Diptera: Glossinidae) in the laboratory: avoidance of horizontal features? Bulletin of Entomological Research, 79, 52 1-528

El-Azazy, O.M.E. (1989) Wound myiasis caused by Cochliomyia hominivorax in Libya. Veterinary Record, 124, 103.

Grccn, C.H. (1989) The use of two-coloured screens for catching Glossina palpalis palpalis (Robineau-Desvoidy) (Diptera: Glossinidae). Bulletin of Entomological Research, 79, 81-93.

Green, C.H. & Flint, S. (1986) An analysis of colour effects in the performance of the F2 trap against Glossina pallidipes Austen and G.morsitans morsitans Westwood (Diptera: Gloss- inidae). Bulletin of Entomological Research, 76, 409-418.

Guillot, F.S., Coppedge, J.R., Goodenough, J.L., Adam, T.S. & Ahrcns, E. (1977) Behaviour and reproductive status of native female screwworm flies attracted to a host. Journal of Economic Entomology, 70, 588-590.

Hammack, L. & Holt, G.G. (1983) Responses of gravid screwworm flies, Cochliomyia hominivorax, to whole wounds, wound fluid, and a standard blood attractant in olfactometer tests. Journal

of Chemical Ecology, 9, 913-922. Jones, C.M., Oehlcr, D.D., Snow, J.W. & Grabbc, R.R. (1976)

A chemical attractant for screwworm flies. Journal of Economic Entomology, 69, 389-391.

Kirk, W.D.J. (1984) Ecologically selective colourcd traps. Eco- logical Entomology, 9, 35-41.

Krafsur, E.S., Whitten, C.J. & Novy, J.E. (1987) Scrcwworm eradication in North and Central America. Parasitology Today,

Mackley, J.W. & Brown, H.E. (1984) Swormlurc-4: a ncw formu- lation of the swormlure-2 mixture as an attractant for adult screwwonns, Cochliomyia hominivorax (Diptera: Calliphoridac). Journal of Economic Entomology, 77, 1264-1268.

Menzel, R. (1979) Spectral sensitivity and color vision in invcrt- ebrates. Handbook of Sensory Physiology, Vol. 7 , part 6A (ed. by A. H. Autrum), pp. 503-580. Springer, New York.

Vale, G.A., Lovemore, D.F., Flint, S. & Cockbill, G.F. (1988) Odour-baited targets to control tsetse flies. Clossina spp. (Diptera: Glossinidae), in Zimbabwe. Bulletin of Entomological Research, 78, 31-49.

Wall, R., Green, C.H., French, N. & Morgan. K.L. (1992) Development of an attractant target for the sheep blowfly Lucilia sericata. Medical and Veterinary Entomology (in press).

Warnes, M.L. (1991) The control of savanna species of tsetse flies using odour baited traps and targets. Pesticide Outlook, 2.

Warnes, M.L. & Green, C.H. (1992) Responses of fcmalc New World screwworm flies, Cochliomyia hominivora.u. to sworrnlure-4 in the laboratory. Medical and Veterinary Enlo- mology, 6, 98- 102.

3, 131-137.

32-35.

Accepted 30 September 1991