DMNS Technical Report 2007-6

Dung Beetle Sampling Protocols

Frank-T. KRELL
Curator of Entomology Department of Zoology, Denver Museum of Nature and Science 2001 Colorado Boulevard, Denver, CO 80205-5798, USA frank.krell@dmns.org

Abstract. Two sampling protocols for dung beetles are presented which are suitable for different approaches relying on quantitative data. The protocol for ecological assemblage studies uses standardsized dung pats put directly on the ground to simulate natural conditions. It enables us to record the actual users of a resource only by allowing tourists (temporary visitors) to leave. The other protocol is a rapid method suitable for RBAs (Rapid Biodiversity Assessments) and uses pitfall traps with suspended bait.

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Dung beetles are becoming an increasingly important focal taxon for ecological and conservation studies (Spector 2006). Taking advantage of the easy attraction of dung beetles by suitable baits (faeces, carrion, other rotting matter), baited pitfall traps are commonly used for experiments with a variety of scopes, disregarding the fact that traps catch all specimens that approach the bait in a way allowing them to fall into the trap. Many Aphodiinae species seem to avoid traps by not landing in the trap whereas Scarabaeinae generally tumble into traps. Specimens of both groups might approach a resource but not use it (tourists). Trapped in the trap, tourists cannot be separated from actual users. Here two different protocols are proposed for two different approaches, written for novices to the field. Protocol 1 is suitable for dung beetle assemblage studies, focusing on recording the actual users of a resource. Protocol 2 is suitable for assessing the local species pool and species abundances of a study site (RBA Rapid Biodiversity Assessment).

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Protocol 1: Comparing Dung Beetle Assemblages without traps

Although the following protocol has been developed for Afrotropical conditions (Ivory Coast, Kenya), it is likely to work in other regions as well. It has been followed by Krell et al. (2003, 2005), Krell-Westerwalbesloh et al. (2004), and several unpublished theses and dissertations (Dosso 2002; Kouakou 2002, 2004; Krell-Westerwalbesloh 2001; Mahiva 2004; Newman 2005; N'goran 2002, 2004). Traps are commonly used for quantitative sampling of dung beetle assemblages, and they are valuable in constructing inventories of local dung beetle assemblages (see Protocol 2). However, if we want to record established assemblages after a fixed period of exposure of the resource, and not all of the beetles visiting the resource during that period of time, traps are inappropriate. Some beetles may approach the dung pat but fly away after a short exploration period (Landin 1961: 207; personal observations) or when the pat is already highly populated. With this protocol we register only the actual users of a resource whereas traps collect both users and tourists. In a comparative study, Lobo et al. (1988) demonstrated that baited pitfall traps contain a much larger number of individuals than the same amount of dung exposed on the soil surface. Our own experiences and experiments indicate that small dung beetles avoid traps, leading to a strong bias against the functional group of dwellers (endocoprids; mainly Aphodiinae). Traps overestimate the portion of some groups containing many tourists. These groups cannot be unambiguously identified among the trapped beetles. Moreover with the condensed succession that we find in tropical ecosystems, the condition and, hence, the attractive effect of a bait in a trap develops differently from a bait exposed on soil, even in a short period of time. However, with our method we do underestimate the proportion of rollers in the dung invertebrate assemblages since many of them have already left the pat before we collect the sample. Although we obviously get a biased pattern, we are able to identify the bias, whereas in pitfall trap sampling the overestimated groups are not recognizable. Hence, the data obtained by our method result in an accurate picture on assemblage structures and differences. A method to estimate the portion of rollers missed is currently under development. Dung Collecting and Preparing 1. Collect fresh cattle dung in a bucket with lid. The dung should be very fresh, so that it is not populated by dung beetles already. It should not have a crust, because in a crust, small beetles may easily be hiding. Collect the dung best in the early morning when less beetles are active. Always transport the dung in a bucket with a closed lid to avoid dung beetles flying into the dung. The lid might need to be taped during transport.

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2. The dung we expose has to be free of any beetles. So you have to scrutinize the collected dung for beetles before you start the experiments. For that you need a second bucket in which to put the sorted dung. Only take a spoonful of dung and look through it carefully using a pair of forceps. Remove all beetles you find, put them in a collecting jar and put the dung in the second bucket. (You may preserve the extracted beetles or not, but you should not release them at this stage to prevent them from flying back into the dung buckets.) Always write on the bucket what it contains (with a permanent marker). This checking of dung is very time-consuming. Under certain conditions, e.g. if the dung has been produced in a cold night and collected early in the morning, or if you have seen the dung falling and collected it instantly, checking before the experiment is not necessary (take random samples to make sure). Experiments: Exposing the Dung 3. The period of exposing the dung for day samples is from 6:00h 1 (i.e., just after dawn) to 16:00h (not much later, but not earlier than 15:00), and for night samples from 18:00h (i.e., just before dusk) to 6:00h (just after dawn, before sunrise). Since dusk is the main activity period for many nocturnal dung beetles, we must include dusk in the night sample period, but exclude it from the day sample period. 4. Always expose around 1 kg (900 ml) dung per sample. Weigh out 1 kg of dung, fill it in a plastic potty (Fig. 1) or similar plastic pot (best with handle) and mark the surface level with a permanent marker pen. You can then use this jar for all experiments without weighing dung again. Fill the jar with sorted dung up to the marked line. 5. The dung has to be exposed on bare ground, not on grass tussocks and not on leaf litter, because that would make collecting the dung and extracting the beetles more difficult. You should push aside the leaf litter in the forest and choose a bare area between grass tussocks in grassland. Avoid big roots under the dung pat. 6. The odor of the dung should be freely dispersible. Avoid exposing the dung directly at the bottom of a big tree or of any kind of wall. In open areas (grassland) the dung should be exposed to the sun for at least some time. Avoid places which are presumably shady all day long 2 . 7. Expose the dung by filling it into the pot (Fig. 1) up to the marked line, then turn the pot upside down and bang it onto the ground. If a considerable amount of dung remains in the pot, remove it and add it to the dung pat. The pat should be in one piece and nearly round. Leave the pat in the field as indicated in 3.
1 2

times from our experiments in northern Ivory Coast; have to be adjusted for sites in other latitudes not applicable for experiments on the influence of solar radiation or shade

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8. In case of rain during the exposure period of the dung, you have to abandon the pats , because many beetles leave the dung during strong rain or wont fly during a long moderate rain. If there was only a slight rain, you can collect the dung.
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Fig. 1. Preparing a 1 kg sample of fresh dung.

Measurements 9. To characterize the microclimatic properties of the sites and microhabitats we measure air temperature at about 1 m above ground at start and end of the exposing period (use an air probe on an electronic thermometer), soil temperature at a depth of 1 cm and 10 cm at start and end of the exposing period (use a piercing probe on the thermometer), dung temperature just before collecting the sample (put the probe in the center of the dung pat), soil humidity using a TDR probe (if available) (take two to three measurements; if they are similar, take the arithmetic means; if one is much lower, dismiss it and take the other(s) because this lower measurement is likely to be caused by a hollow in the ground), penetration resistance (if device is available) (take three measurements around the dung pat) shear strength (if device is available) (take three measurements around the dung pat) (dimensions (length, width, and height) of the dung pat just before collecting. The height can most easily be measured by sticking a stick or a twig vertically into the pat and measure how deep it goes. If there are soil ejections, take them as a part of the dung. This measurement is useful only if the dung beetle abundance is very high so that dung beetle activity influences the dimensions of the pat.)
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not applicable for experiments on the influence of rain

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10. When you do the measurements, try not to disturb the beetles on the pat. Avoid casting a shadow on the pat; otherwise some rollers will flee instantly. If you see rollers or other beetles leaving the pat during your measurements, collect them in a collecting tube and put it into the bucket provided for collecting the pat. 11. Note any rainfall in the week before the experiment and during the experimental period, because rainfall events trigger the emergence and flight activity of many dung beetle species. Collecting the Samples 12. After the exposure period and after having done all the measurements, you collect the dung and the soil beneath (that is populated by dung beetles) into a bucket with a lid. Use a spade 4 to dig out the dung pat and the soil beneath at best as a single cube (at least 10 cm deep) and put it quickly into the bucket. If you see soil mounds (like small molehills on the side of the dung pat), tunnelling dung beetles have made their nests beyond them. You should extent the soil cube in the direction of the soil hill to get the nests with the beetles. After you have collected the soil/dung cube, you should inspect the hole that you made for further dung beetles. If the soil in the hole is still populated, you have to dig deeper and collect all soil into the bucket till no beetle remains in the hole. You should particularly inspect the area where molehills were present so that you dont miss the big tunnelers that made the hills. They make big tunnels (sometimes 2 cm in diameter or more) which you can feel with your fingers. Extracting the Beetles 13. Bring the bucket to a place where fresh water is available. Process the sample as soon as possible. Two days storage is the absolute maximum because there are always predators in the sample which may prey on a good portion on the dung beetles. 14. If the bucket is full of soil and dung, fill another bucket with water and put portions of soil/dung into the water. Most of the beetles will float (Fig. 2) and can be collected with a sieve or a strainer from where they can be picked with a pair of forceps and put into a collecting bottle (labelled with a permanent marker: for the bottle see 16). After you collected the beetles from the water surface, the sediment in the water-bucket must be stirred with a strong stick time and again, because some beetles stick in the mud and wont float without mechanical help. Use a strainer with a small mesh width, because some beetles are very small.
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with very hard soil, other tools might be more suitable, e.g. local hoes (e.g. dabbas); check on local markets what farmers use

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15. If there is only a little soil and dung in the bucket, you may pour the water directly into the collecting bucket.

Fig. 2. Beetles floating on the water surface in a bucket.

16. After all the soil and dung is floated, inspect the sediment, best with a big spoon/ladle and a pair of forceps. Sometimes particular groups of dung beetles do not float 5 . According to our experiences, this inspecting was most necessary with dung pats exposed during the day in grassland. 17. Use big plastic bottles for collecting the beetles (250ml-1l). Put toilet paper into the bottle first. The beetles may clean themselves by digging through the toilet paper. If it is warm, dung beetles are very agile. It may then be necessary to put some drops of ethyl acetate onto the toilet paper in the bottle. 18. When all beetles are in the bottle, put some more ethyl acetate into the bottle. If many wet beetles are in the bottle, it is necessary to add some more toilet paper. After half an hour all the beetles should be dead. (An alternative method to kill the beetles is putting them into a bottle of water with detergent to kill them, or directly into alcohol, but this is more expensive and takes equally long). Preserving the Beetles 19. The dead beetles are to be transferred to the smallest possible bottles/tubes for conservation. Put some toilet paper into the bottle, soak it with Scheerpeltz solution 6 (75%
in Ivory Coast, a substantial portion of Pedaria and dung-covered Neosisyphus remained in the sediment 6 killing specimens with ethyl acetate and preservation with Scheerpeltz solution is not advisable for specimens to be used for DNA extraction, whereas killing and preservation in pure or high-percentage alcohol as commonly used for DNA samples makes the specimens stiff and mounting/morphological dissections more difficult
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ethanol, 5% acetic acid, 20% water or around 80% pure alcohol and 20% white vinegar essence 7 ). Put the beetles in. Pour some Scheerpeltz solution onto the beetles. Fill up the bottle with Scheerpeltz-soaked toilet paper and close it tightly (if liquid is visible from outside, pour it out; the material should be stored in moist, not liquid condition). Label the bottle with a marker pen and by scratching the code with a diamond pen or a knife into the plastic of the bottle/tube.

Scheerpeltz originally proposed a lower alcoholic percentage for use as a soaking agent (Freude et al. 1965: 130); for temporary conservation 70-80% alcohol should be used.

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Protocol 2: RBA Using Traps with Human Dung

Traps (Figs 3-4)
(minimum requirements bold)

pitfall trap of more than 10 cm in diameter and depth (small bucket, plastic cup), better around 15 cm filled with 2-3 cm water (in hot regions addition of chloral hydrate recommended) rain cover at least 25 cm over the trap (plastic plate [party or camping equipment] on the top of four wooden sticks) closed cage for bait, highly pervious to air (two tea strainers fixed together by tape; or sacs made from mosquito net or fly screen material or from net curtains) bait suspended not lower than soil surface level; sack or streamers with bait fixed by a rope on the rain cover ca. 40 g of bait (fresh human faeces; max. 2 days old, in cool environment 3 days since a human produces at least 80-120 g faeces per day, 2 persons can provide the bait for 7 traps within two days, 1 person within 3 days; 40 g is a big heaped spoonful of dung, and a spoon is the best device to handle it) The trap design may be changed due to the locally available components, but should be around the given size, since a standardized amount of dung is necessary to allow comparability of trapping results. The pitfall traps are a modified model CSS (Cebo-Suspendido-Superficie [= dung suspended at surface level], Lobo et al. 1988) because this is the most effective design among the trap types with limited access to the bait. In tropical environments with a high abundance of coprophagous insects, we have to avoid them accessing the dung, because a small amount of dung may be degraded within less than one hour (pers. obs. in Cte dIvoire). During the trapping time of two days, a dung bait of a small size (40-60 g) is easily completely degraded by maggots. Advice for sampling carrion feeders: Since carrion, especially fish, is generally attractive for necrophagous mammals, pitfall traps with this kind of bait are regularly destroyed in the field in due course. As a preventive measure, the number of carrion baited traps should be higher than needed. However, omnivorous dung like human faeces attracts generally not only coprophagous dung beetles but to a certain extent necrophagous species as well.

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Trapping

7 8 traps along a roughly 100 m transect set up in open places if any (easily accessible for flying dung beetles), that means: do not chose places randomly 44-48 hours continuous trapping period; we mostly started around noon (avoid samples from days with long rain during the main activity periods of dung beetles [10-16h, dusk and dawn]) distance between traps at least 3 m

Killing and preserving the beetles follows Protocol 1. Avoid having too much water and too many flies in the preserved material.

Figs 3-4. Pitfall traps with suspended dung in the savanna of Ivory Coast.

The minimum number of traps for cost-efficient sampling and for completeness is currently evaluated. The number of 7 might be unnecessarily high in Afrotropical environments where one trap may easily attract 1500 specimens.

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Acknowledgments. The sampling protocols were developed and applied in projects Ivory Coast and Kenya funded by the German Research Council (Deutsche Forschungsgemeinschaft; Li150-18,1-3; Prof. K.E. Linsenmair) and German Federal Ministry of Education and Research (BIOTA 09; Prof. C.M. Naumann). I thank my students Clstin Kouakou, Alexandre Koffi, Vincent Mahiva, Dorothy Newman, Paul N'goran, Uta Schneider, Ingo Wei and Sylvia Westerwalbesloh for their efforts in the field having helped to refine the method.

References
Dosso, K. 2002. Effets de l'anthropisation sur les communauts de coloptres coprophages en milieu urbain : cas de l'Universit d'Abobo-Adjam. Mmoire de fin de cycle de matrise, UFR Science de la Nature, Universit dAbobo-Adjam, Cte d'Ivoire. 39 pp., annexe. Freude, H., Harde, K.W. & Lohse, G.A. 1965. Die Kfer Mitteleuropas 1 (Einfhrung in die Kferkunde). Goecke & Evers, Krefeld. Kouakou, Y.C. 2002. Effet de lanthropisation sur les communauts des coloptres coprophages en zone urbaine : Cas dAbidjan-Vridi. Mmoire de fin de cycle de matrise, UFR Science de la Nature, Universit dAbobo-Adjam, Cte d'Ivoire. 34 pp., annexe. Kouakou, Y.C. 2004. Influence des perturbations anthropiques sur les coloptres coprophages: cas du d'Ivoire. 69 pp., annexe.

pturage et de l'urbanisation respectivement en rgions de savane (Toumodi) et de fort (Abidjan) de la Cte d'Ivoire. Thse DEA, UFR Sciences de la Nature, Universit d'Abobo-Adjam, Cte

Krell, F.-T., Krell-Westerwalbesloh, S., Wei, I., Eggleton, P. & Linsenmair, K.E. 2003. Spatial separation of Afrotropical dung beetle guilds: a trade-off between competitive superiority and energetic constraints (Coleoptera: Scarabaeidae). Ecography 26: 210-222. Krell, F.-T., Mahiva, V.S., Kouakou, C., Ngoran, P., Krell-Westerwalbesloh, S., Newman, D.H., Wei, I. & Doumbia, M. 2005. Human influence on the dung fauna in Afrotropical grasslands (Insecta: Coleoptera). Pp. 133-139 in: Huber, B.A., Sinclair, B.J. & Lampe, K.-H. (eds): African Biodiversity:

Molecules, Organisms, Ecosystems. Proceedings of the 5th International Symposium on Tropical Biology, Museum Koenig, Bonn. Springer, New York.

Krell-Westerwalbesloh, S. 2001. Tageszeitliche, saisonale und rumliche Sonderung der koprophagen

Gilden bei der Besiedelung von Bffeldung im Como-Nationalpark, Elfenbeinkste Diversittserhaltene Mechanismen? Diplomarbeit, Universitt Wrzburg, Germany. 123 pp., Anhang.

Krell-Westerwalbesloh, S., Krell, F.-T. & Linsenmair, K.E. 2004. Diel separation of Afrotropical dung beetle guilds avoiding competition and neglecting resources. Journal of Natural History 38: 22252249. Landin, B.-O. 1961. Ecological studies on dung-beetles. Opuscula Entomologica, Suppl. 19: 228 pp. Lobo, J.M., Martn-Piera, F. & Veiga, C.M. 1988. Las trampas pitfall con cebo, sus posibilidades en el estudio de las comunidades coprfagas de Scarabaeoidea (Col.). I. Caracteristicas determinantes de su capacidad de captura. Revue dcologie et de Biologie du Sol 25: 77-100. Mahiva, V.S. 2004. The impact of human land use activities and seasonal changes on the dung beetle diversity of Kakamega Forest ecosystem. MSc Thesis, University of Nairobi, Kenya. Xix + 90 pp.

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Newman, D.H. 2005. Anthropogenic effects on the structure of dung beetle assemblages in cattle dung in Ivory Coast. PhD Thesis, University of Reading, UK. Ngoran, K.P. 2002. Effet de lanthropisation sur les communauts des coloptres coprophages (bousiers) en zone urbaine: Commune de Yopougon (Abidjan). Mmoire de fin de cycle de matrise, UFR Science de la Nature, Universit dAbobo-Adjam. Abidjan. 29 pp., annexe. N'goran, K.P. 2004. Influence des exploitations agricoles sur les communautes de coloptres Adjam, Cte d'Ivoire. iv + 66 pp., annexe.

coprophages en zone forestires : cas de'une zone tropicale humide (Abidjan) et d'une zone de transition (Bringakro) en Cte d'Ivoire. Thse DEA, UFR Sciences de la Nature, Universit d'Abobo-

Spector, S. 2006. Scarabaeine dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae): an invertebrate focal taxon for biodiversity research and conservation. Coleopterists Society Monographs 5: 71-83.

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