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Attractiveness of dung from ivermectin-treated cattle to Danish and afrotropical scarabaeid dung beetles
Veterinary Parasitology, 48 (1993) 159-169 Elsevier Science Publishers B.V., Amsterdam
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Attractiveness of dung from ivermectin-treated cattle to Danish and afrotropical scarabaeid dung beetles P. Holter~, C. S o m m e r
a a n d J. G r o n v o l d b
"Institute of Population Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen 0, Denmark bDepartment of Veterinary Microbiology, Section of Parasitology, Royal Veterinary and Agricultural University, Biilowsvej 13, DK-1870 Frederiksberg C, Denmark
ABSTRACT Holter, P., Sommer, C. and Gronvold, J., 1993. Attractiveness of dung from ivermectin-treated cattle to Danish and afrotropical scarabaeid dung beetles. Vet. Parasitol., 48:159-169. The effect of ivermectin treatment of cattle on the attractiveness of dung to scarabaeid dung beetles was assessed by pitfall trapping in Denmark, Tanzania and Zimbabwe. Traps were baited with dung collected at intervals after heifers were treated with ivermectin by subcutaneous injection (0.2 mg kg- i body weight). In one Danish trial, beetles preferred control dung from untreated cattle, whereas no preference was found in two other experiments. In Tanzania, the overall tendency for beetles was also to prefer control dung. In Zimbabwe, two species (Euoniticellus intermedius and Liatongus militaris) were particularly attracted to dung from treated cattle, whereas three others did not discriminate between dung types. It is concluded that at least in some cases the attractiveness of cattle dung to scarabaeid beetles is affected by avermectin therapy. Beetle discrimination between dung types is probably attributable to some unknown side effect of the treatment rather than being a direct response to the drug itself.
INTRODUCTION
Dung insects play an important role in promoting the decay of cattle dung pats in pastures. In tropical and subtropical areas this is particularly true of scarabaeid clung beetles which bury the dung as food stores for themselves and their progeny. Consequently, effects of avermectin treatment on insects may also have an impact on the general ecology of pasturelands. Negative effects of avermectin therapy on dung pat decomposition have been demonstrated in some cases (Wall and Strong, 1987; Madsen et al., 1990) but the mechanisms involved were not fully established. A priori, such effects could be brought about by ( 1 ) avoidance of pats from treated cattle by insects (or other relevant animals, e.g. earthworms) and/or (2) toxicity of avermectins, Correspondence to: P. Holter, Institute of Population Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen O, Denmark.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4017/93/$06.00
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or derived compounds, to insects once they have colonised the dung. Indeed, a variety of toxic effects of avermectins have been found in dung-living Diptera and Coleoptera; these effects have recently been reviewed by Strong (1992). Thus, although much remains to be done, the latter point has already been reasonably well documented. By contrast, the effect of avermectin therapy on the rate of colonisation seems to have been largely neglected until quite recently. In Australia, Wardhaugh and Mahon ( 1991 ) assessed the impact of avermectin treatment of cattle and sheep on the colonisation of dung by five scarabaeid dung beetle species, including two exotics. Surprisingly, it was found that all species were more attracted to dung from treated animals than to dung from untreated controls; the enhanced attraction was particularly clear with cattle dung. Wardhaugh and Mahon ( 1991 ) considered this to be a side effect of avermectin therapy and hence only indirectly attributable to the drug itself. The other possibility, i.e. direct effects of avermectins on the attractiveness of dung, seems to have been tested only once: based on very limited evidence, Strong and Wall ( 1988 ) did not find any repellent effect ofivermectin added to faeces from untreated cattle; the possibility of increased attraction was not mentioned. Obviously, we need more information about the ways in which avermectin therapy may influence the invasion of dung by insects if we are to understand how avermectins affect dung fauna and hence the ecology of pastures. The present paper contributes some evidence concerning the effects of ivermectin treatment (subcutaneous injection ) of cattle on the attractiveness of dung to beetles. The results comprise both non-burying dung beetles in a temperate country (Denmark) and afrotropical, dung-burying Scarabaeidae in Tanzania and Zimbabwe. MATERIALS AND METHODS
General In all experiments, beetles were trapped in pitfalls baited with dung collected at intervals after the treatment of cattle with ivermectin (Table 1 ). Two Danish trials included dung from untreated cattle with ivermectin added (Table 1 ). Experimental animals were heifers; ivermectin was injected subcutaneously at the recommended therapeutic dose of 0.2 mg kg- 1body weight. Controls (untreated) and treated animals were taken from the same group of heifers and were kept under exactly the same conditions. Dung from each treatment group was thoroughly mixed after collection and stored at 2-4 °C until use. Storage times did not exceed 6 clays except for the last Zimbabwean experiment in which dung was stored for up to 16 days before use (see below).
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TABLE 1 Summary of experiments and types of dung tested Experiments (location and date ) Denmark May 1990 August 1990 May 1991 Tanzania March 1990 April 1990 March-April 1990 Zimbabwe January 1991, A March 1991, B March 199 l, C
Types of dung tested
3, 10, 20, 30 5, 20; 0.30 ppm, 0.08 ppm 5, 20; 0.42 ppm, 0.015 ppm 3 6 10, 15, 21 2, 8, 16 2, 8, 16 2, 8, 16
~Number of days between treatment and dung collection, or concentration of added ivermectin (ppm, wet wt. basis).
Pitfall traps consisted of buckets (22 cm in diameter and 17 cm high in Denmark and Tanzania; slightly bigger in Zimbabwe) buried in the soil with the upper rim flush with the soil surface. The bottom was covered with 2-3 cm of detergent solution to ensure rapid submersion and drowning of captured beetles. The bait was placed centrally above the bucket opening, supported by a 15× 15 cm grid of galvanised metal (1.5 cm mesh) resting on a larger net with 5 cm mesh size. The bait was 70 g of dung in Tanzania and 300-400 g in Denmark and Zimbabwe. In the African trials, the bait was wrapped in nylon gauze (mesh size 1 m m ) to prevent the penetration of beeties; this was not necessary in Denmark where no dung-burying beetles were involved.
Details of individual experiments In Denmark, heifers (300-400 kg body weight) were stabled during the experiments. Each treatment group comprised three to four (in most cases four) animals. The groups were treated at different dates and all dung, including that of control animals, was collected on one date. Thus, the same dung served as control for all the treatment groups. In the two last experiments, powdered ivermectin was mixed into some of the dung from untreated animals at two concentrations (Table 1 ). These additions were designed to mimic field concentrations and, in the last trial, approximated to the concentrations found in dung dropped 5 (0.42 p p m ) and 20 (0.015 p p m ) days after treatment (calculated from data in Sommer et al., 1992). The remaining dung
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from untreated heifers served as control both for the dung with ivermectin added and for the dung from treated animals. The field site was a fenced part of a permanent pasture in the Strodam Nature Reserve, about 35 km north of Copenhagen. There were five traps with each type of dung, 3.5 m apart in a 5 X 5 latin square. They were baited at noon and emptied 2 days later. In the three Tanzanian experiments, there were five grazing Ayrshire heifers (200-300 kg) in each treatment and control group. The first two trials involved a single collection of dung, whereas in the last trial, cattle were injected at one date and dung collected 10, 15 and 21 days later (Table 1 ). Ten traps were placed in two rows of five traps, with rows on opposite sides of a 100-m-wide pasture at the Sokoine University of Agriculture, Morogoro, Tanzania. Traps in each row were 10 m apart, and baits with treated and control dung were placed alternately in the rows. Traps were baited in the morning (09:00 h) and harvested and rebaited at dusk (18:30 h). This was repeated so that each type of dung, in each trial, was tested on 2 successive days and nights. After the first day and night, the positions of control dung and treated dung were exchanged to eliminate possible effects of individual trap positions. In the first two Zimbabwean experiments (Experiment A, January 1991; Experiment B, March 1991; see Table 1 ), there were 12 grazing Holstein heifers (400-600 kg) in each group. Cattle were treated on different dates; dung was collected and tested on 1 day. The farm and field site was Glenara Estate, about 20 km north of Harare. The traps were placed in a pasture, 4 m apart in four rows of five traps. The four types of bait were placed at alternate positions (five traps with each) in the afternoon and the beetles were collected 24 h later. The third trial (Experiment C, March 1991 ), was performed at the Bhara Bhara-Farm, about 20 km southwest of Harare; 20 grazing Red Dane heifers (300-400 kg) were available. On Day 0, control dung was collected, and all the animals were then treated with ivermectin. Dung was collected 2, 8 and 16 days later (Table 1 ) and stored at 2 °C until Day 16, when all the dung was tested by the same procedure as at Glenara Estate. RESULTS
Denmark Table 2 shows results concerning the genus Aphodius. For each experiment and dung type, the geometric mean number of beetles per trap was calculated as antilog ( E l o g ( n + 1 ) / 5 ) - 1 (n being the number of beetles in each trap); geometric means were preferred to avoid swamping by occasional very high counts (Williams, 1937 ). In May 1990, ten species were caught, with Aphodius sticticus (Panzer), Aphodius ater (De Geer) and Aphodius prodromus (Brahm) contributing 30%, 26% and 21%, respectively, of the individuals.
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TABLE 2 Catches of the genus Aphodiusin the three Danish experiments. The dung types are characterised by the number of days between ivermectin treatment and collection of dung, or by the concentration of ivermectin (wet wt. basis) added to some of the control dung (August 1990 and May 1991 ). Beetle numbers are geometric means, with 95% confidence intervals in parentheses May 1990 Dung type
3 days 10 days 20 days 30 days Control
August 1990 Beetles per trap 13.0 (6.6-24.8) 9.5 (4.9-18.0) 15.4 (7.2-31.8) 8.8 (3.3-21.2) 26.5 (11.9-57.6)
Dung type
5 days 0.30 ppm 20 days 0.08 ppm Control
May 1991 Beetles per trap 0.8 (0-2.5) 2.1 (0-9.1) 1.3 (0.1-3.7) 1.5 (0.2-4.1) 1.8 (0.3-5.1)
Dung type
5 days 0.42 ppm 20 days 0.015 ppm Control
Beetles per trap 17.9 ( 10.1-31.1 ) 16.8 (6.3-42.2) 16.7 (5.3-48.7) 20.2 ( 10.3-38.8 ) 14.0 (8.6-22.4)
In August 1990, 90% of individuals were Aphodius rufipes (L.), but the catch was extremely small. Finally, the material from May 1991 was dominated by the same species as in May 1990 ( 12% A. ater, 42% A. sticticus and 38% A. prodromus ). In May 1990, there was a significant effect of dung type ( P < 0.01; ANOVA on In (n + 1 ) - t r a n s f o r m e d counts), with more beetles being caught in control dung than in dung from treated cattle. Catches in the four types of dung from treated cattle did not differ significantly. In the two other experiments, no significant effect of dung type could be shown. In particular, the data for 1991 (a year with reasonably high numbers of beetles) do not suggest any preference for control dung.
Tanzania The results of the five comparisons between control dung and dung collected from cattle at intervals of 3-21 days after treatment with ivermectin are summarised in Table 3. Species have been grouped according to whether they fly at night (N) or during the day (D). The combination of each dung type (days after treatment) and time of day (night/day) is represented by beetles caught in 20 trapping events (five traps with ivermectin dung and five with control dung per day for 2 consecutive days, see Methods). The species selected for analysis are those that occurred in at least ten of the 20 trapping events, or those of which at least 20 individuals were caught. For each species
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TABLE3 C~ratios ~ ~ r t h e mostnumerousscarabaeidspecies ~undduringtheTanzanianexperiments. Total beetlenumbersaregivenin parentheses. Beetleswereflyingatnight(N)orduringtheday(D) N/D
N N N N N N D D D D D
Species
Onthophagusgazella Onthophagusspfl Onthophagus variegatus Onthophaguslamelliger Onthophagus vinctus Milichus rhodesianus Onthophagus sp. 3 Onthophagus sansibaricus Caccobius schaedlei Sisyphus ocellatus Sisyphus crispatus
Days between ivermectin treatment and collection of fresh dung 3
6
10
15
21
0.8(103) 0.8(71) 3.6(21 ) 1.9(40)
1.1(47) 1.6(28) 1.4(80) 1.6(69)
1.6(19) 0.8(28) -
0.98(70) 0.7(47) 0.8(23) 1.4(49)
-
-
2.1(52) 1.7(206) 2.2*(79) 1.3(81 1.9(45
-
0.3 ( 72 )
1.4(58)
1.1(23) 1.5(58)
-
-
-
0.3" ( 97 )
-
0.7 ( 32 ) 1.2 ( 18 )
1.01(54) 0.4(15)
1.2(58) -
1.4(45 1.3(17 2.7(35 1.2(50) 1.5(41)
~C, geometric mean of beetles per trap (GMB) with control dung; L GMB with dung from cattle treated with ivermectin 3, 6, 10, 15 or 21 days earlier. 20nthophagus species, possibly identical with Onthophagusfuscivestisd'Orbigny. 3Similar to, but probably different from Onthophagus taboranus d'Orbigny (Y. Cambefort, personal communication, 1991 ). *Beetle numbers in control and ivermectin dung significantly different (P < 0.05 ).
and dung type, the geometric mean number of beetles per trap for control dung (C) and ivermectin dung (I) was calculated and the resulting 34 C/1ratios are shown in Table 3. Since the five comparisons were carried out over different 2-day periods, under varying weather conditions and with dung from three different groups of animals, a single analysis incorporating data for all species and dung types was inappropriate. Instead, separate two-way analyses of variance with treatment (control vs. ivermectin) and days (Days 1 and 2) as the two factors were performed on In (n + 1 ) -transformed counts of the selected species from each dung type. Of the 34 analyses involving 11 species (Table 3 ), only two showed a significant treatment effect. The night-flying species Onthophagus variegatus Fabricius in Day 10 dung showed a preference for control dung, whereas the day-flying Caccobius schaedlei d'Orbigny in Day 6 dung was more abundant in ivermectin dung. An alternative way of analysing such disparate data is to assume that the attractiveness of dung is unaffected by ivermectin therapy. On this basis we would expect 17 C/I ratios to be more than I and 17 C/I ratios to be less than 1. However, Table 3 shows that 24 C/I ratios were greater than 1 and ten were less than 1. This is significantly different from 1 : 1 (X 2 = 5.76; P < 0.025 ), and
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165
so there is an overall tendency for the beetles to prefer control dung. The trend seems to hold both in nocturnal ( 14 C/I ratios greater than 1, six C/I ratios less than 1 ) and in diurnal (ten C/I ratios greater than 1, four C/I ratios less than 1 ) species, although no significance can be demonstrated with these limited data. The diurnal species Euoniticellus intermedius (Reiche) is not shown in Table 3. The total number caught ( 55 beetles) was small, but there was evidence of a consistent preference for ivermectin dung with total counts in control/ ivermectin dung of 0/5 (3 days), 4/6 (6 days), 10/11 (10 days), 5/8 ( 15 days), and 2/4 (21 days). Since the difference between dung types is significant (P<0.05; sign test), this species seems to prefer ivermectin dung and thus to differ from the overall tendency seen in Table 3.
Zimbabwe Of the five species shown in Table 4, none showed an effect of dung type by ordinary one-way ANOVA (on In (n + 1 ) - transformed counts). However, an ANOVA may not be the most expedient way of analysing data when there are ordered expectations, since all information on the direction of deviations is lost (Gaines and Rice, 1990). This is a problem in the present case where effects due to ivermectin concentration in the dung are likely to decrease with time after treatment and to be absent in control dung. Accordingly, we have TABLE4 Results of the experiments in Zimbabwe. The trials were run in 1991 at the following dates and locations: 21 January, Glenara Estate (Experiment A); I I March, Glenara Estate (Experiment B); 20 March, Bhara Bhara-Farm (Experiment C). The table shows geometric mean numbers of beetles per trap (95% confidence intervals in parentheses). Dung types as in Table 2 Experiment
A A B B C C
Species
Diastellopalpus quinquedens Liatongus militaris D. quinquedens Onitis viridulus Euoniticellus intermedius Onthophagus gazella
Dung type Day 2
Day 8
Day 16
Control
10.3 (4.9-20.5) 12.0 (3.8-34.2) 14.3 (9.7-20.9) 5.1 (1.4-14.3) 8.9 (4.5-16.9) 0.9 (0-3.1)
12.5 (9.0-17.2) 12.9 (8.2-20.0) 15.3 (7.7-29.6) 2.9 (0.3-10.5) 9.9 (6.3-15.4) 3.6 (1.2-8.4)
7.3 (3.3-15.0) 6.4 (2.3-15.7) 13.3 (9.0-19.4) 4.5 (1.8-9.7) 3.1 (0.1-14.7) 1.2 (0-4.3)
12.4 (7.6-20.0) 6.2 (3.7-10.3) 17.1 (15.1-19.4) 3.5 (2.6-4.7) 5.1 (2.1-10.7) 1.3 (0.6-2.4)
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amalgamated results representing high (2 and 8 days ) and low ( 16 days and control) concentrations and compared the two groups by a t-test. In Liatongus militaris ( Castelnau ) and Euoniticellus intermedius a significant difference ( P < 0 . 0 2 5 ) between groups was found; in both cases there were more beetles in the 2 + 8 days group than in the group comprising control dung and the dung of Day 16. These two species then, both belonging to the tribe Oniticellini, seem to have preferred dung collected relatively soon after treatment and hence with a high concentration of ivermectin. No preference (i.e. no significant difference between groups) could be demonstrated in the other three species. DISCUSSION
Wardhaugh and Mahon ( 1991 ) reported a clear preference in five species of scarabaeid dung beetles for dung collected soon after avermectin treatment of cattle or sheep. Our findings confirm that avermectin treatment can indeed affect the attractiveness of cattle dung. However, the results are obviously not as simple and consistent as those of Wardhaugh and Mahon ( 1991 ). In some species (L. militaris, E. intermedius), we observed a preference for avermectin-contaminated dung, whereas no discrimination between dung types could be shown in other cases (three species in Zimbabwe and the last two Danish experiments). The third possible outcome, preference for control dung from untreated animals, was found in the first Danish and in the Tanzanian trials. As a further complication, different species did not always behave similarly within the same experiment; this is illustrated in Table 4. The mechanism behind the observed effects is still unknown, and we can only reiterate arguments already put forward by Wardhaugh and Mahon ( 1991 ). Any response to ivermectin treatment is probably not ascribable to differences in nutrition of the cattle since control animals and treated animals were kept under exactly the same conditions within any experiment. The large, non-volatile ivermectin molecules are hardly detectable at a distance, which means that responses are unlikely to be direct effects of ivermectin itself. In the two last Danish experiments,this was tested by adding ivermectin to some of the control dung. Unfortunately, the result was inconclusive in that the beetles showed no preference in these trials. Even so, some features of the Danish results indicate that a direct effect of ivermectin is not involved. First, the fact that there were different outcomes in May 1990 and 1991, with the same beetle species and with similar ivermectin concentrations (days after treatment), shows that something other than just the drug itself must have been acting. Secondly, there was no detectable discrimination between dung types, and hence ivermectin concentrations, from treated cattle even in May 1990 when the beetles clearly preferred control dung. If this preference had been governed directly by ivermectin, a
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response that was more graduated according to ivermectin concentrations would be expected. In addition, no consistent response to ivermectin concentrations (excepting the preference for control dung) is apparent in the Tanzanian material, even though - - for reasons explained in the Results section the evidence is weaker than in the Danish data. In connection with the Danish and Tanzanian experiments, one possible problem must be acknowledged. Although no exact information exists, there is probably individual variation among heifers with regard to the attractiveness of their dung. With small treatment groups (between three and five animals in these trials), such individual differences may not have been entirely eliminated in the mixed dung from the group and could provide an unknown source of variation in the comparison between dung types. However, considering that the animals used in each experiment were homogeneous for age, weight, breed and reproductive status, this may not be a serious source of error. In Zimbabwe, much larger groups were available ( 12-20 animals), and so individual animal effects were probably negated. In conclusion, it seems likely that the responses observed are attributable to some unknown side effect of the treatment. The presence or strength of this side effect is unpredictable with our present knowledge. This lack of understanding is illustrated e.g. by the unexplained difference between the outcomes of the Danish experiments and by the different behaviour of Onthophagus species in Australian (Wardhaugh and Mahon, 1991: dung with avermectin preferred ) and Tanzanian (Table 3: control dung preferred ) trials. Wardhaugh and Mahon (1991) propose that some volatile metabolite of ivermectin, or changes in the gut flora caused by ivermectin therapy, may be involved. The latter possibility in particular would seem to be in keeping with the variable response of the beetles, and we can offer no better suggestions. The findings reported here clearly have some implications concerning the possible environmental impact of avermectin treatment. Since this therapy may sometimes lead to decreased attractiveness of the dung, an impoverished beetle fauna in avermectin-contaminated dung (Wall and Strong, 1987; Madsen et al., 1990) may not only be attributable to toxic effects of the drug but also to avoidance of pats from treated cattle, i.e. a purely behavioural effect. Moreover, the varying responses of beetles (and perhaps other insects) may at least partly explain why some authors (Wall and Strong, 1987; Madsen et al, 1990) find a decreased rate of dung pat decay after ivermectin treatment, while others (e.g. McKeand et al., 1988 ) do not. Finally, it should be emphasised once more that avermectin therapy can also lead to increased attractiveness of the dung. Toxic effects of avermectins on dung beetle reproduction and survival have been shown by Ridsdill-Smith (1988), Wardhaugh and Rodriguez-Menendez ( 1988 ), Houlding et al. ( 1991 ) and Sommer and Overgaard Nielsen ( 1992 ), which means that particularly attractive dung from treated cattle may reduce the total number of dung beetles in pastureland. In -
-
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other words, increased attractiveness of dung with avermectin is clearly undesirable in terms of population effects on useful dung beetles. Therefore, we need to improve our understanding of the factors that bring about changes in the attractiveness of dung from treated cattle. The ultimate goal must be to predict, or even better to avoid, the increased attractiveness and so to minimise negative effects of avermectin therapy on dung beetle populations. ACKNOWLEDGEMENTS
Our sincere thanks are due to the following for practical help. In Denmark, staff at the farm Frihedslund, and A. Spangenberg who provided excellent technical assistance. In Tanzania, A.A. Kassuku and staff at the Sokoine University of Agriculture, Morogoro. In Zimbabwe (Harare), M. Madsen, Veterinary Research Laboratory, O.F. Barry and staffat Glenara Estates, and W. Kirk and staff at the Bhara Bhara-Farm. Y. Cambefort (Paris) kindly helped with the identification of two difficult Tanzanian Onthophagus species. Finally, we are grateful to G. Nachman (Copenhagen) for help and advice on the statistical treatment and to K. Wardhaugh (Canberra) for constructive criticism of the manuscript. The work was funded by grant 104.Dan8/464 from the Danish Council for Development Research.
REFERENCES Gaines, S.D. and Rice. W.R., 1990. Analysis of biological data when there are ordered expectations. Am. Nat., 135: 310-317. Houlding, B., Ridsdill-Smith, T.J. and Bailey, W.J., 1991. Injectable abamectin causes a delay in scarabaeine dung beetle egg-laying in cattle dung. Aust. Vet. J., 68:185-186. Madsen, M., Overgaard Nielsen, B., Holter, P., Pedersen, O.C., Brochner Jespersen, J., Vagn Jensen, K.-M., Nansen, P. and Gr~nvold, J., 1990. Treating cattle with ivermectin: effects on the fauna and decomposition of dung pats. J. Appl. Ecol., 2 7 : I - 1 5 . McKeand, J., Bairden, K. and Ibarra-Silva, A.-M., 1988. The degradation of bovine faecal pats containing ivermectin. Vet. Rec., 122: 587-588. Ridsdill-Smith, T.J., 1988. Survival and reproduction of Musca vetustissima Walker (Diptera: Muscidae) and a scarabaeine dung beetle in dung of cattle treated with avermectin B1. J. Aust. Entomol. Soc., 27: 175-178. Sommer, C. and Overgaard Nielsen, B., 1992. Larvae of the dung beetle Onthophagus gazella Fabricius (Coleoptera: Scarabaeidae) exposed to lethal and sublethal ivermectin concentrations. J. Appl. Entomol., 114: 502-509. Sommer, C., Steffansen, B., Overgaard Nielsen, B., Gr~nvold, J., Vagn Jensen, K.-M., Brochner Jespersen, J., Springborg, J. and Nansen, P., 1992. Ivermectin excreted in cattle dung after subcutaneous injection or pour-on treatment: concentrations and impact on dung fauna. Bull. Entomol. Res., 82: 257-264. Strong, L., 1992. Avermectins: a review of their impact on insects of cow pats. Bull. Entomol. Res., 82: 265-274. Strong,. L. and Wall, R., 1988. Ivermectin in cattle treatment: non-specific effects on pastureland ecology. Aspects Appl. Biol., 17:231-238.
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Wall, R. and Strong, L., 1987. Environmental consequences of treating cattle with the antiparasitic drug ivermectin. Nature, 327:418-421. Wardhaugh, K.G. and Mahon, R.J., 1991. Avermectin residues in sheep and cattle dung and their effects on dung beetle (Coleoptera: Scarabaeidae) colonization and dung burial. Bull. Entomol. Res., 81: 333-339. Wardhaugh, K.G. and Rodriguez-Menendez, H., 1988. The effects of the antiparasitic drug, ivermectin, on the development and survival of the dung-breeding fly, Orthelia cornicina (F.) and the scarabaeine dung beetles, Copris hispanus L., Bubas bubalus (Oliver) and Onitis belial F. J. Appl. Entomol., 106:381-389. Williams, C.B., 1937. The use of logarithms in the interpretation of certain entomological problems. Ann. Appl. Biol., 24: 404-414.
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Attractiveness of dung from ivermectin-treated cattle to Danish and afrotropical scarabaeid dung beetles P. Holter~, C. S o m m e r
a a n d J. G r o n v o l d b
"Institute of Population Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen 0, Denmark bDepartment of Veterinary Microbiology, Section of Parasitology, Royal Veterinary and Agricultural University, Biilowsvej 13, DK-1870 Frederiksberg C, Denmark
ABSTRACT Holter, P., Sommer, C. and Gronvold, J., 1993. Attractiveness of dung from ivermectin-treated cattle to Danish and afrotropical scarabaeid dung beetles. Vet. Parasitol., 48:159-169. The effect of ivermectin treatment of cattle on the attractiveness of dung to scarabaeid dung beetles was assessed by pitfall trapping in Denmark, Tanzania and Zimbabwe. Traps were baited with dung collected at intervals after heifers were treated with ivermectin by subcutaneous injection (0.2 mg kg- i body weight). In one Danish trial, beetles preferred control dung from untreated cattle, whereas no preference was found in two other experiments. In Tanzania, the overall tendency for beetles was also to prefer control dung. In Zimbabwe, two species (Euoniticellus intermedius and Liatongus militaris) were particularly attracted to dung from treated cattle, whereas three others did not discriminate between dung types. It is concluded that at least in some cases the attractiveness of cattle dung to scarabaeid beetles is affected by avermectin therapy. Beetle discrimination between dung types is probably attributable to some unknown side effect of the treatment rather than being a direct response to the drug itself.
INTRODUCTION
Dung insects play an important role in promoting the decay of cattle dung pats in pastures. In tropical and subtropical areas this is particularly true of scarabaeid clung beetles which bury the dung as food stores for themselves and their progeny. Consequently, effects of avermectin treatment on insects may also have an impact on the general ecology of pasturelands. Negative effects of avermectin therapy on dung pat decomposition have been demonstrated in some cases (Wall and Strong, 1987; Madsen et al., 1990) but the mechanisms involved were not fully established. A priori, such effects could be brought about by ( 1 ) avoidance of pats from treated cattle by insects (or other relevant animals, e.g. earthworms) and/or (2) toxicity of avermectins, Correspondence to: P. Holter, Institute of Population Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen O, Denmark.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4017/93/$06.00
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or derived compounds, to insects once they have colonised the dung. Indeed, a variety of toxic effects of avermectins have been found in dung-living Diptera and Coleoptera; these effects have recently been reviewed by Strong (1992). Thus, although much remains to be done, the latter point has already been reasonably well documented. By contrast, the effect of avermectin therapy on the rate of colonisation seems to have been largely neglected until quite recently. In Australia, Wardhaugh and Mahon ( 1991 ) assessed the impact of avermectin treatment of cattle and sheep on the colonisation of dung by five scarabaeid dung beetle species, including two exotics. Surprisingly, it was found that all species were more attracted to dung from treated animals than to dung from untreated controls; the enhanced attraction was particularly clear with cattle dung. Wardhaugh and Mahon ( 1991 ) considered this to be a side effect of avermectin therapy and hence only indirectly attributable to the drug itself. The other possibility, i.e. direct effects of avermectins on the attractiveness of dung, seems to have been tested only once: based on very limited evidence, Strong and Wall ( 1988 ) did not find any repellent effect ofivermectin added to faeces from untreated cattle; the possibility of increased attraction was not mentioned. Obviously, we need more information about the ways in which avermectin therapy may influence the invasion of dung by insects if we are to understand how avermectins affect dung fauna and hence the ecology of pastures. The present paper contributes some evidence concerning the effects of ivermectin treatment (subcutaneous injection ) of cattle on the attractiveness of dung to beetles. The results comprise both non-burying dung beetles in a temperate country (Denmark) and afrotropical, dung-burying Scarabaeidae in Tanzania and Zimbabwe. MATERIALS AND METHODS
General In all experiments, beetles were trapped in pitfalls baited with dung collected at intervals after the treatment of cattle with ivermectin (Table 1 ). Two Danish trials included dung from untreated cattle with ivermectin added (Table 1 ). Experimental animals were heifers; ivermectin was injected subcutaneously at the recommended therapeutic dose of 0.2 mg kg- 1body weight. Controls (untreated) and treated animals were taken from the same group of heifers and were kept under exactly the same conditions. Dung from each treatment group was thoroughly mixed after collection and stored at 2-4 °C until use. Storage times did not exceed 6 clays except for the last Zimbabwean experiment in which dung was stored for up to 16 days before use (see below).
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TABLE 1 Summary of experiments and types of dung tested Experiments (location and date ) Denmark May 1990 August 1990 May 1991 Tanzania March 1990 April 1990 March-April 1990 Zimbabwe January 1991, A March 1991, B March 199 l, C
Types of dung tested
3, 10, 20, 30 5, 20; 0.30 ppm, 0.08 ppm 5, 20; 0.42 ppm, 0.015 ppm 3 6 10, 15, 21 2, 8, 16 2, 8, 16 2, 8, 16
~Number of days between treatment and dung collection, or concentration of added ivermectin (ppm, wet wt. basis).
Pitfall traps consisted of buckets (22 cm in diameter and 17 cm high in Denmark and Tanzania; slightly bigger in Zimbabwe) buried in the soil with the upper rim flush with the soil surface. The bottom was covered with 2-3 cm of detergent solution to ensure rapid submersion and drowning of captured beetles. The bait was placed centrally above the bucket opening, supported by a 15× 15 cm grid of galvanised metal (1.5 cm mesh) resting on a larger net with 5 cm mesh size. The bait was 70 g of dung in Tanzania and 300-400 g in Denmark and Zimbabwe. In the African trials, the bait was wrapped in nylon gauze (mesh size 1 m m ) to prevent the penetration of beeties; this was not necessary in Denmark where no dung-burying beetles were involved.
Details of individual experiments In Denmark, heifers (300-400 kg body weight) were stabled during the experiments. Each treatment group comprised three to four (in most cases four) animals. The groups were treated at different dates and all dung, including that of control animals, was collected on one date. Thus, the same dung served as control for all the treatment groups. In the two last experiments, powdered ivermectin was mixed into some of the dung from untreated animals at two concentrations (Table 1 ). These additions were designed to mimic field concentrations and, in the last trial, approximated to the concentrations found in dung dropped 5 (0.42 p p m ) and 20 (0.015 p p m ) days after treatment (calculated from data in Sommer et al., 1992). The remaining dung
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from untreated heifers served as control both for the dung with ivermectin added and for the dung from treated animals. The field site was a fenced part of a permanent pasture in the Strodam Nature Reserve, about 35 km north of Copenhagen. There were five traps with each type of dung, 3.5 m apart in a 5 X 5 latin square. They were baited at noon and emptied 2 days later. In the three Tanzanian experiments, there were five grazing Ayrshire heifers (200-300 kg) in each treatment and control group. The first two trials involved a single collection of dung, whereas in the last trial, cattle were injected at one date and dung collected 10, 15 and 21 days later (Table 1 ). Ten traps were placed in two rows of five traps, with rows on opposite sides of a 100-m-wide pasture at the Sokoine University of Agriculture, Morogoro, Tanzania. Traps in each row were 10 m apart, and baits with treated and control dung were placed alternately in the rows. Traps were baited in the morning (09:00 h) and harvested and rebaited at dusk (18:30 h). This was repeated so that each type of dung, in each trial, was tested on 2 successive days and nights. After the first day and night, the positions of control dung and treated dung were exchanged to eliminate possible effects of individual trap positions. In the first two Zimbabwean experiments (Experiment A, January 1991; Experiment B, March 1991; see Table 1 ), there were 12 grazing Holstein heifers (400-600 kg) in each group. Cattle were treated on different dates; dung was collected and tested on 1 day. The farm and field site was Glenara Estate, about 20 km north of Harare. The traps were placed in a pasture, 4 m apart in four rows of five traps. The four types of bait were placed at alternate positions (five traps with each) in the afternoon and the beetles were collected 24 h later. The third trial (Experiment C, March 1991 ), was performed at the Bhara Bhara-Farm, about 20 km southwest of Harare; 20 grazing Red Dane heifers (300-400 kg) were available. On Day 0, control dung was collected, and all the animals were then treated with ivermectin. Dung was collected 2, 8 and 16 days later (Table 1 ) and stored at 2 °C until Day 16, when all the dung was tested by the same procedure as at Glenara Estate. RESULTS
Denmark Table 2 shows results concerning the genus Aphodius. For each experiment and dung type, the geometric mean number of beetles per trap was calculated as antilog ( E l o g ( n + 1 ) / 5 ) - 1 (n being the number of beetles in each trap); geometric means were preferred to avoid swamping by occasional very high counts (Williams, 1937 ). In May 1990, ten species were caught, with Aphodius sticticus (Panzer), Aphodius ater (De Geer) and Aphodius prodromus (Brahm) contributing 30%, 26% and 21%, respectively, of the individuals.
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TABLE 2 Catches of the genus Aphodiusin the three Danish experiments. The dung types are characterised by the number of days between ivermectin treatment and collection of dung, or by the concentration of ivermectin (wet wt. basis) added to some of the control dung (August 1990 and May 1991 ). Beetle numbers are geometric means, with 95% confidence intervals in parentheses May 1990 Dung type
3 days 10 days 20 days 30 days Control
August 1990 Beetles per trap 13.0 (6.6-24.8) 9.5 (4.9-18.0) 15.4 (7.2-31.8) 8.8 (3.3-21.2) 26.5 (11.9-57.6)
Dung type
5 days 0.30 ppm 20 days 0.08 ppm Control
May 1991 Beetles per trap 0.8 (0-2.5) 2.1 (0-9.1) 1.3 (0.1-3.7) 1.5 (0.2-4.1) 1.8 (0.3-5.1)
Dung type
5 days 0.42 ppm 20 days 0.015 ppm Control
Beetles per trap 17.9 ( 10.1-31.1 ) 16.8 (6.3-42.2) 16.7 (5.3-48.7) 20.2 ( 10.3-38.8 ) 14.0 (8.6-22.4)
In August 1990, 90% of individuals were Aphodius rufipes (L.), but the catch was extremely small. Finally, the material from May 1991 was dominated by the same species as in May 1990 ( 12% A. ater, 42% A. sticticus and 38% A. prodromus ). In May 1990, there was a significant effect of dung type ( P < 0.01; ANOVA on In (n + 1 ) - t r a n s f o r m e d counts), with more beetles being caught in control dung than in dung from treated cattle. Catches in the four types of dung from treated cattle did not differ significantly. In the two other experiments, no significant effect of dung type could be shown. In particular, the data for 1991 (a year with reasonably high numbers of beetles) do not suggest any preference for control dung.
Tanzania The results of the five comparisons between control dung and dung collected from cattle at intervals of 3-21 days after treatment with ivermectin are summarised in Table 3. Species have been grouped according to whether they fly at night (N) or during the day (D). The combination of each dung type (days after treatment) and time of day (night/day) is represented by beetles caught in 20 trapping events (five traps with ivermectin dung and five with control dung per day for 2 consecutive days, see Methods). The species selected for analysis are those that occurred in at least ten of the 20 trapping events, or those of which at least 20 individuals were caught. For each species
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TABLE3 C~ratios ~ ~ r t h e mostnumerousscarabaeidspecies ~undduringtheTanzanianexperiments. Total beetlenumbersaregivenin parentheses. Beetleswereflyingatnight(N)orduringtheday(D) N/D
N N N N N N D D D D D
Species
Onthophagusgazella Onthophagusspfl Onthophagus variegatus Onthophaguslamelliger Onthophagus vinctus Milichus rhodesianus Onthophagus sp. 3 Onthophagus sansibaricus Caccobius schaedlei Sisyphus ocellatus Sisyphus crispatus
Days between ivermectin treatment and collection of fresh dung 3
6
10
15
21
0.8(103) 0.8(71) 3.6(21 ) 1.9(40)
1.1(47) 1.6(28) 1.4(80) 1.6(69)
1.6(19) 0.8(28) -
0.98(70) 0.7(47) 0.8(23) 1.4(49)
-
-
2.1(52) 1.7(206) 2.2*(79) 1.3(81 1.9(45
-
0.3 ( 72 )
1.4(58)
1.1(23) 1.5(58)
-
-
-
0.3" ( 97 )
-
0.7 ( 32 ) 1.2 ( 18 )
1.01(54) 0.4(15)
1.2(58) -
1.4(45 1.3(17 2.7(35 1.2(50) 1.5(41)
~C, geometric mean of beetles per trap (GMB) with control dung; L GMB with dung from cattle treated with ivermectin 3, 6, 10, 15 or 21 days earlier. 20nthophagus species, possibly identical with Onthophagusfuscivestisd'Orbigny. 3Similar to, but probably different from Onthophagus taboranus d'Orbigny (Y. Cambefort, personal communication, 1991 ). *Beetle numbers in control and ivermectin dung significantly different (P < 0.05 ).
and dung type, the geometric mean number of beetles per trap for control dung (C) and ivermectin dung (I) was calculated and the resulting 34 C/1ratios are shown in Table 3. Since the five comparisons were carried out over different 2-day periods, under varying weather conditions and with dung from three different groups of animals, a single analysis incorporating data for all species and dung types was inappropriate. Instead, separate two-way analyses of variance with treatment (control vs. ivermectin) and days (Days 1 and 2) as the two factors were performed on In (n + 1 ) -transformed counts of the selected species from each dung type. Of the 34 analyses involving 11 species (Table 3 ), only two showed a significant treatment effect. The night-flying species Onthophagus variegatus Fabricius in Day 10 dung showed a preference for control dung, whereas the day-flying Caccobius schaedlei d'Orbigny in Day 6 dung was more abundant in ivermectin dung. An alternative way of analysing such disparate data is to assume that the attractiveness of dung is unaffected by ivermectin therapy. On this basis we would expect 17 C/I ratios to be more than I and 17 C/I ratios to be less than 1. However, Table 3 shows that 24 C/I ratios were greater than 1 and ten were less than 1. This is significantly different from 1 : 1 (X 2 = 5.76; P < 0.025 ), and
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so there is an overall tendency for the beetles to prefer control dung. The trend seems to hold both in nocturnal ( 14 C/I ratios greater than 1, six C/I ratios less than 1 ) and in diurnal (ten C/I ratios greater than 1, four C/I ratios less than 1 ) species, although no significance can be demonstrated with these limited data. The diurnal species Euoniticellus intermedius (Reiche) is not shown in Table 3. The total number caught ( 55 beetles) was small, but there was evidence of a consistent preference for ivermectin dung with total counts in control/ ivermectin dung of 0/5 (3 days), 4/6 (6 days), 10/11 (10 days), 5/8 ( 15 days), and 2/4 (21 days). Since the difference between dung types is significant (P<0.05; sign test), this species seems to prefer ivermectin dung and thus to differ from the overall tendency seen in Table 3.
Zimbabwe Of the five species shown in Table 4, none showed an effect of dung type by ordinary one-way ANOVA (on In (n + 1 ) - transformed counts). However, an ANOVA may not be the most expedient way of analysing data when there are ordered expectations, since all information on the direction of deviations is lost (Gaines and Rice, 1990). This is a problem in the present case where effects due to ivermectin concentration in the dung are likely to decrease with time after treatment and to be absent in control dung. Accordingly, we have TABLE4 Results of the experiments in Zimbabwe. The trials were run in 1991 at the following dates and locations: 21 January, Glenara Estate (Experiment A); I I March, Glenara Estate (Experiment B); 20 March, Bhara Bhara-Farm (Experiment C). The table shows geometric mean numbers of beetles per trap (95% confidence intervals in parentheses). Dung types as in Table 2 Experiment
A A B B C C
Species
Diastellopalpus quinquedens Liatongus militaris D. quinquedens Onitis viridulus Euoniticellus intermedius Onthophagus gazella
Dung type Day 2
Day 8
Day 16
Control
10.3 (4.9-20.5) 12.0 (3.8-34.2) 14.3 (9.7-20.9) 5.1 (1.4-14.3) 8.9 (4.5-16.9) 0.9 (0-3.1)
12.5 (9.0-17.2) 12.9 (8.2-20.0) 15.3 (7.7-29.6) 2.9 (0.3-10.5) 9.9 (6.3-15.4) 3.6 (1.2-8.4)
7.3 (3.3-15.0) 6.4 (2.3-15.7) 13.3 (9.0-19.4) 4.5 (1.8-9.7) 3.1 (0.1-14.7) 1.2 (0-4.3)
12.4 (7.6-20.0) 6.2 (3.7-10.3) 17.1 (15.1-19.4) 3.5 (2.6-4.7) 5.1 (2.1-10.7) 1.3 (0.6-2.4)
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amalgamated results representing high (2 and 8 days ) and low ( 16 days and control) concentrations and compared the two groups by a t-test. In Liatongus militaris ( Castelnau ) and Euoniticellus intermedius a significant difference ( P < 0 . 0 2 5 ) between groups was found; in both cases there were more beetles in the 2 + 8 days group than in the group comprising control dung and the dung of Day 16. These two species then, both belonging to the tribe Oniticellini, seem to have preferred dung collected relatively soon after treatment and hence with a high concentration of ivermectin. No preference (i.e. no significant difference between groups) could be demonstrated in the other three species. DISCUSSION
Wardhaugh and Mahon ( 1991 ) reported a clear preference in five species of scarabaeid dung beetles for dung collected soon after avermectin treatment of cattle or sheep. Our findings confirm that avermectin treatment can indeed affect the attractiveness of cattle dung. However, the results are obviously not as simple and consistent as those of Wardhaugh and Mahon ( 1991 ). In some species (L. militaris, E. intermedius), we observed a preference for avermectin-contaminated dung, whereas no discrimination between dung types could be shown in other cases (three species in Zimbabwe and the last two Danish experiments). The third possible outcome, preference for control dung from untreated animals, was found in the first Danish and in the Tanzanian trials. As a further complication, different species did not always behave similarly within the same experiment; this is illustrated in Table 4. The mechanism behind the observed effects is still unknown, and we can only reiterate arguments already put forward by Wardhaugh and Mahon ( 1991 ). Any response to ivermectin treatment is probably not ascribable to differences in nutrition of the cattle since control animals and treated animals were kept under exactly the same conditions within any experiment. The large, non-volatile ivermectin molecules are hardly detectable at a distance, which means that responses are unlikely to be direct effects of ivermectin itself. In the two last Danish experiments,this was tested by adding ivermectin to some of the control dung. Unfortunately, the result was inconclusive in that the beetles showed no preference in these trials. Even so, some features of the Danish results indicate that a direct effect of ivermectin is not involved. First, the fact that there were different outcomes in May 1990 and 1991, with the same beetle species and with similar ivermectin concentrations (days after treatment), shows that something other than just the drug itself must have been acting. Secondly, there was no detectable discrimination between dung types, and hence ivermectin concentrations, from treated cattle even in May 1990 when the beetles clearly preferred control dung. If this preference had been governed directly by ivermectin, a
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167
response that was more graduated according to ivermectin concentrations would be expected. In addition, no consistent response to ivermectin concentrations (excepting the preference for control dung) is apparent in the Tanzanian material, even though - - for reasons explained in the Results section the evidence is weaker than in the Danish data. In connection with the Danish and Tanzanian experiments, one possible problem must be acknowledged. Although no exact information exists, there is probably individual variation among heifers with regard to the attractiveness of their dung. With small treatment groups (between three and five animals in these trials), such individual differences may not have been entirely eliminated in the mixed dung from the group and could provide an unknown source of variation in the comparison between dung types. However, considering that the animals used in each experiment were homogeneous for age, weight, breed and reproductive status, this may not be a serious source of error. In Zimbabwe, much larger groups were available ( 12-20 animals), and so individual animal effects were probably negated. In conclusion, it seems likely that the responses observed are attributable to some unknown side effect of the treatment. The presence or strength of this side effect is unpredictable with our present knowledge. This lack of understanding is illustrated e.g. by the unexplained difference between the outcomes of the Danish experiments and by the different behaviour of Onthophagus species in Australian (Wardhaugh and Mahon, 1991: dung with avermectin preferred ) and Tanzanian (Table 3: control dung preferred ) trials. Wardhaugh and Mahon (1991) propose that some volatile metabolite of ivermectin, or changes in the gut flora caused by ivermectin therapy, may be involved. The latter possibility in particular would seem to be in keeping with the variable response of the beetles, and we can offer no better suggestions. The findings reported here clearly have some implications concerning the possible environmental impact of avermectin treatment. Since this therapy may sometimes lead to decreased attractiveness of the dung, an impoverished beetle fauna in avermectin-contaminated dung (Wall and Strong, 1987; Madsen et al., 1990) may not only be attributable to toxic effects of the drug but also to avoidance of pats from treated cattle, i.e. a purely behavioural effect. Moreover, the varying responses of beetles (and perhaps other insects) may at least partly explain why some authors (Wall and Strong, 1987; Madsen et al, 1990) find a decreased rate of dung pat decay after ivermectin treatment, while others (e.g. McKeand et al., 1988 ) do not. Finally, it should be emphasised once more that avermectin therapy can also lead to increased attractiveness of the dung. Toxic effects of avermectins on dung beetle reproduction and survival have been shown by Ridsdill-Smith (1988), Wardhaugh and Rodriguez-Menendez ( 1988 ), Houlding et al. ( 1991 ) and Sommer and Overgaard Nielsen ( 1992 ), which means that particularly attractive dung from treated cattle may reduce the total number of dung beetles in pastureland. In -
-
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other words, increased attractiveness of dung with avermectin is clearly undesirable in terms of population effects on useful dung beetles. Therefore, we need to improve our understanding of the factors that bring about changes in the attractiveness of dung from treated cattle. The ultimate goal must be to predict, or even better to avoid, the increased attractiveness and so to minimise negative effects of avermectin therapy on dung beetle populations. ACKNOWLEDGEMENTS
Our sincere thanks are due to the following for practical help. In Denmark, staff at the farm Frihedslund, and A. Spangenberg who provided excellent technical assistance. In Tanzania, A.A. Kassuku and staff at the Sokoine University of Agriculture, Morogoro. In Zimbabwe (Harare), M. Madsen, Veterinary Research Laboratory, O.F. Barry and staffat Glenara Estates, and W. Kirk and staff at the Bhara Bhara-Farm. Y. Cambefort (Paris) kindly helped with the identification of two difficult Tanzanian Onthophagus species. Finally, we are grateful to G. Nachman (Copenhagen) for help and advice on the statistical treatment and to K. Wardhaugh (Canberra) for constructive criticism of the manuscript. The work was funded by grant 104.Dan8/464 from the Danish Council for Development Research.
REFERENCES Gaines, S.D. and Rice. W.R., 1990. Analysis of biological data when there are ordered expectations. Am. Nat., 135: 310-317. Houlding, B., Ridsdill-Smith, T.J. and Bailey, W.J., 1991. Injectable abamectin causes a delay in scarabaeine dung beetle egg-laying in cattle dung. Aust. Vet. J., 68:185-186. Madsen, M., Overgaard Nielsen, B., Holter, P., Pedersen, O.C., Brochner Jespersen, J., Vagn Jensen, K.-M., Nansen, P. and Gr~nvold, J., 1990. Treating cattle with ivermectin: effects on the fauna and decomposition of dung pats. J. Appl. Ecol., 2 7 : I - 1 5 . McKeand, J., Bairden, K. and Ibarra-Silva, A.-M., 1988. The degradation of bovine faecal pats containing ivermectin. Vet. Rec., 122: 587-588. Ridsdill-Smith, T.J., 1988. Survival and reproduction of Musca vetustissima Walker (Diptera: Muscidae) and a scarabaeine dung beetle in dung of cattle treated with avermectin B1. J. Aust. Entomol. Soc., 27: 175-178. Sommer, C. and Overgaard Nielsen, B., 1992. Larvae of the dung beetle Onthophagus gazella Fabricius (Coleoptera: Scarabaeidae) exposed to lethal and sublethal ivermectin concentrations. J. Appl. Entomol., 114: 502-509. Sommer, C., Steffansen, B., Overgaard Nielsen, B., Gr~nvold, J., Vagn Jensen, K.-M., Brochner Jespersen, J., Springborg, J. and Nansen, P., 1992. Ivermectin excreted in cattle dung after subcutaneous injection or pour-on treatment: concentrations and impact on dung fauna. Bull. Entomol. Res., 82: 257-264. Strong, L., 1992. Avermectins: a review of their impact on insects of cow pats. Bull. Entomol. Res., 82: 265-274. Strong,. L. and Wall, R., 1988. Ivermectin in cattle treatment: non-specific effects on pastureland ecology. Aspects Appl. Biol., 17:231-238.
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Wall, R. and Strong, L., 1987. Environmental consequences of treating cattle with the antiparasitic drug ivermectin. Nature, 327:418-421. Wardhaugh, K.G. and Mahon, R.J., 1991. Avermectin residues in sheep and cattle dung and their effects on dung beetle (Coleoptera: Scarabaeidae) colonization and dung burial. Bull. Entomol. Res., 81: 333-339. Wardhaugh, K.G. and Rodriguez-Menendez, H., 1988. The effects of the antiparasitic drug, ivermectin, on the development and survival of the dung-breeding fly, Orthelia cornicina (F.) and the scarabaeine dung beetles, Copris hispanus L., Bubas bubalus (Oliver) and Onitis belial F. J. Appl. Entomol., 106:381-389. Williams, C.B., 1937. The use of logarithms in the interpretation of certain entomological problems. Ann. Appl. Biol., 24: 404-414.