sampling protocol

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DMNS Technical Report 2007-6 1

Dung Beetle Sampling Protocols

Frank-T. KRELL

Curator of EntomologyDepartment of Zoology, Denver Museum of Nature and Science

2001 Colorado Boulevard, Denver, CO 80205-5798, [email protected]

Abstract. Two sampling protocols for dung beetles are presented which are suitable for differentapproaches relying on quantitative data. The protocol for ecological assemblage studies uses standard-sized dung pats put directly on the ground to simulate natural conditions. It enables us to record theactual users of a resource only by allowing tourists (temporary visitors) to leave. The other protocol isa rapid method suitable for RBAs (Rapid Biodiversity Assessments) and uses pitfall traps with sus-pended 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 allspecimensthat approach the bait in a way allowing them to fall into the trap. Many Aphodiinae speciesseem 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 suitablefor 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 (2004), and several unpublished theses and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 explo-

ration 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 (endo-

coprids; mainly Aphodiinae). Traps overestimate the portion of some groups containing many

tourists. These groups cannot be unambiguously identified among the trapped beetles. More-

over 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 recogniz-

able. 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 youshould 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 permanentmarker). This checking ofdung 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 6:00h1 (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

long2.

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 times from our experiments in northern Ivory Coast; have to be adjusted for sites in other latitudes2 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

pats3, 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.

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 endof the exposing period (usean air probe on an electronic thermometer),

soil temperature at a depth of 1 cm and 10 cm at start and endof the exposing period(use a piercing probe on the thermometer),

dung temperature just before collecting the sample (put the probe in the center of thedung pat),

soil humidity using a TDR probe (if available) (take two to three measurements; if they aresimilar, 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 dungpat)

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.)

3 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 spade4 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 stirredwith 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.

4 with very hard soil, other tools might be more suitable, e.g. local hoes (e.g. dabbas); check on localmarkets 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 thewater 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 float5.

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 solution6 (75%

5 in Ivory Coast, a substantial portion ofPedariaand dung-covered Neosisyphusremained in thesediment6 killing specimens with ethyl acetate and preservation with Scheerpeltz solution is not advisable forspecimens to be used for DNA extraction, whereas killing and preservation in pure or high-percentagealcohol as commonly used for DNA samples makes the specimens stiff and mounting/morphologicaldissections more difficult


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ethanol, 5% acetic acid, 20% water or around 80% pure alcohol and 20% white vinegar

essence7). 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 thebottle/tube.

7 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, plasticcup), better around 15 cm

filled with 2-3 cm water (in hot regions addition of chloral hydrate recom-mended)

rain cover at least 25 cm over the trap (plastic plate [party or camping equip-ment] 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 bya 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 bigheaped 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 compara-

bility of trapping results.

The pitfall traps are a modified model CSS (Cebo-Suspendido-Superficie [= dung sus-

pended 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

78 traps along a roughly 100 m transect set up in open places if any (easily accessible for flying dung beetles), that means: do

notchose 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 withsuspended dung in the savannaof Ivory Coast.

8 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 easilyattract 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 milieuurbain : cas de l'Universit d'Abobo-Adjam. Mmoire de fin de cycle de matrise, UFR Science de laNature, Universit dAbobo-Adjam, Cte d'Ivoire. 39 pp., annexe.

Freude, H., Harde, K.W. & Lohse, G.A. 1965. Die Kfer Mitteleuropas1 (Einfhrung in die Kferkunde).Goecke & Evers, Krefeld.

Kouakou, Y.C. 2002. Effet de lanthropisation sur les communauts des coloptres coprophages enzone 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 dupturage 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, Cted'Ivoire. 69 pp., annexe.

Krell, F.-T., Krell-Westerwalbesloh, S., Wei, I., Eggleton, P. & Linsenmair, K.E. 2003. Spatial separationof Afrotropical dung beetle guilds: a trade-off between competitive superiority and energeticconstraints (Coleoptera: Scarabaeidae). Ecography26: 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 TropicalBiology, Museum Koenig, Bonn. Springer, New York.

Krell-Westerwalbesloh, S. 2001. Tageszeitliche, saisonale und rumliche Sonderung der koprophagenGilden 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 dungbeetle guilds avoiding competition and neglecting resources. Journal of Natural History38: 2225-2249.

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 elestudio de las comunidades coprfagas de Scarabaeoidea (Col.). I. Caracteristicas determinantes desu capacidad de captura. Revue dcologie et de Biologie du Sol25: 77-100.

Mahiva, V.S. 2004. The impact of human land use activities and seasonal changes on the dung beetlediversity 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 dungin 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 coloptrescoprophages en zone forestires : cas de'une zone tropicale humide (Abidjan) et d'une zone detransition (Bringakro) en Cte d'Ivoire. Thse DEA, UFR Sciences de la Nature, Universit d'Abobo-Adjam, Cte d'Ivoire. iv + 66 pp., annexe.

Spector, S. 2006. Scarabaeine dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae): an invertebratefocal taxon for biodiversity research and conservation. Coleopterists Society Monographs5: 71-83.

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