Particle feeding in Aphodius dung beetles (Scarabaeidae): old hypotheses and new experimental evidence
1. Published studies of the highly specialized mouthparts of adult scarabaeid dung beetles have proposed that the beetles feed on (1) liquid with minute particles, squeezed out of the dung by the mandibular molars, and/or (2) dung particles that are finely ground by the same molars. These hypotheses were tested experimentally.
2. The maximum size of ingested particles was determined in six Aphodius species (Scarabaeidae, Aphodiinae) feeding on dung with latex-balls of known diameters. The probability of ingestion decreased with particle size. The diameter of balls with a 5% chance of ingestion varied between species from <5 µm to about 25 µm. This maximum size, which precludes hypothesis (1), was related to feeding habits, not to body mass.
3. Simple experiments did not indicate any comminution by the mouthparts of dung particles.
4. It is suggested that coarse (mostly indigestible) particles are rejected by filtering setae and that the remaining small particles, prior to ingestion, are concentrated on the molars by squeezing which eliminates superfluous water.
5. Since 40–50% of the dry matter in fresh dung from grazing cows, sheep and horses consisted of particles <20 µm, food competition among adult Aphodius seems unlikely in spite of their selective feeding.
Scarabaeid dung beetles have been the subject of many behavioural (e.g. Halffter & Edmonds 1982) and ecophysiological studies and have served as useful model organisms for exploring ecological processes such as decomposition, aggregation and competition. In addition, they have been the focus of extensive applied research in relation to ecotoxicology and biological control of dung and dung-breeding flies. Much of the ecological work is reviewed in Hanski & Cambefort (1991). In spite of all these studies, the feeding biology of adult dung beetles is poorly understood. These beetles colonize and feed on the dung of herbivorous mammals. However, dung is a very complex substrate, and we do not know exactly which components the beetles actually eat and digest. This lack of knowledge is a major obstacle to a rational discussion of important ecological issues such as the factors responsible for species succession in dung pats, competition (inter- as well as intraspecific) between beetles, or niche diversification in relation to different kinds of dung.
The mouthparts of scarabaeid dung beetles are highly specialized. Since only minute particles are present in the paste-like midgut contents (Madle 1934; Miller 1961), these specializations seem to ensure that ingestion is restricted to the more liquid components of the dung, including small particles. Indeed, the reproductive performance of Euoniticellus intermedius in various kinds of dung was positively, and significantly, correlated with the percentage dry matter in fluid pressed (at 2 MPa) out of the dung through gauze (Aschenborn, Loughnan & Edwards 1989; Edwards 1991). There is some experimental evidence therefore that fine particulate matter in dung liquid is the major source of nutrition for adult dung beetles.
According to the first detailed description (with numerous illustrations) of the mouthparts and feeding activity of any dung beetle (Madle 1934), Aphodius rufipes collects food via licking movements of the enlarged, hairy and padlike maxillar galeae, probably aided by the equally hairy laciniae. The material is then supposedly transferred to the mandibles, which distally are thin and leaflike without any cutting teeth, and transported to the heavily sclerotized molar areas. These are asymmetrical, convex on the right mandible and concave on the left, fitting exactly into each other. They are provided with numerous minute transverse ridges, separated by narrow ‘Filterrinnen’, i.e. filtration channels. The distal end of each ridge is dilated, and so a roof (with a very narrow central fissure) is formed by two adjoining ridges over the intervening channel. Madle suggested that the collected dung is squeezed between the molar areas, and that the resulting mixture of dung fluid and fine particulate material runs directly into the pharynx through the filtration channels. If so, the diameter of particles ingested cannot exceed the width of the ‘Filterrinnen’. Later authors (Miller 1961; Hata & Edmonds 1983), examining other scarabaeid dung beetles including Aphodius fimetarius, essentially confirmed Madle's morphological description (without mentioning it), but added one feature: the flat, dilated top of each molar ridge is subdivided into ‘tightly packed scalloped blocks’ (Hata & Edmonds 1983; like Miller 1961 this paper provides detailed illustrations of the relevant structures). These ‘tritors’ (Miller 1961) were supposed to comminute larger particles, prior to ingestion, by a grinding action of the mandibles. Several authors (Halffter & Matthews 1966; Macqueen 1975; Halffter & Edmonds 1982; Bürgis 1984; Scholtz 1989; Cambefort 1991; Mathison & Ditrich 1999) have adopted this view.
The uncertainty around the mode of action of the mouthparts extends to the exact size of particles ingested. Based on alcohol-preserved midgut content samples from four American species of scarabaeine dung beetles, Miller (1961) measured the size ranges of ‘the largest particle and most of the larger particles’. In Dichotomius (Pinotus) carolinus for example, these ranges were 6–16 µm and 6–12 µm, respectively. However, the latter size category in particular seems somewhat subjective. Besides, particles in the gut contents (of freshly killed, not preserved, beetles) can be of a highly variable and poorly defined shape (P. Holter, personal observation). Meaningful, direct measurement is therefore not easy, and a better way of characterizing ingested particle size, and hence the food of the dung beetles, seems desirable.
In this paper, a new method for determining the upper size limit of ingested particles is applied to six species of Aphodius, the dominant genus of scarabaeid dung beetles in northern Europe. In addition, the question of whether or not larger dung particles are comminuted and then ingested is addressed by a simple, experimental approach. The results are discussed in relation to the functioning of dung beetle mouthparts and to ecological characteristics of the species.
Materials and methods
Aphodius ater (Degeer) (mean fresh body mass about 15 mg), A. contaminatus (Herbst) (14 mg), A. erraticus (L.) (35 mg), A. fossor (L.) (121 mg) and A. rufipes (L.) (96 mg) beetles were collected from sheep and cattle dung in the Strødam Nature Reserve, about 35 km north-west of Copenhagen, Denmark. Since A. fimetarius (L.) (33 mg) was unusually rare in Denmark in 1997–98, it was collected (from cowpats) in pastures around Canberra, Australia, and was tested at CSIRO Entomology, Canberra. To stimulate feeding, all species were starved in moist potting soil for 2–4 days at 20–25 °C before the test. The dung used for most of the feeding experiments was fresh cattle dung from grazing cows and heifers that did not get any supplementary feed. However, since A. ater prefers sheep dung (e.g. Hirschberger 1998), fresh dung from grazing sheep was used in all experiments involving that species.
Maximum size of ingested particles
Briefly, latex balls of various known diameters were mixed into dung that was offered to the starved beetles. Balls in samples of the midgut contents were then counted by microscopy. The latex balls, which are intended for calibration of Coulter Counter® instruments (Coulter Electronics Ltd, Luton, UK), were of diameters 2, 5, 10, 14, 18 and 39 µm. Diameters between 18 and 39 µm were not available. Particles are delivered from the manufacturer in 10-ml flasks in an aqueous dispersant (plus preservative). To obtain a more concentrated dispersion, about 60% of the dispersant was gently removed by sucking while particles were settled at the bottom, and the flasks were then thoroughly shaken. Balls of two sizes (usually five or six drops of each) were mixed into about 15 g of fresh cow dung by thorough stirring. Preliminary experiments indicated that addition of more than two size categories led to a less palatable dung mixture, perhaps because too much dispersant had been added. To determine the proportions of the two particle sizes, a sample droplet of fluid was squeezed (gentle pressure with a fingertip) out of about 100 mg of mixed dung through a 100-µm sieve and placed on a microscope slide. It was then mixed with a drop of glycerol and water (1 : 1) and covered with a cover slip. All the balls in a suitable number of microscopic fields of view were counted (if possible up to at least 50 balls of the rarest size category) at 1000× magnification when 2-µm balls were involved, otherwise at 400×. The removal of coarse dung particles by the 100-µm sieve facilitated the counting. Mixing was considered satisfactory when three samples were homogeneous (P > 0·05; 2 × 3 χ2) with regard to the relative abundance of the two ball sizes.
Portions of 4–5 g of mixed dung were transferred to Petri dishes (4 cm diameter) and one (A. erraticus, A. fossor, A. rufipes) or two (other species) beetles placed in each dish. After about 45 min at room temperature in a dark, undisturbed place, the beetles were killed instantaneously in boiling water. A sample of the midgut contents was removed by dissection, placed on a slide in a droplet of glycerol/water (1 : 1), mixed with the droplet and covered by a cover slip. Since the gut content was enclosed by the peritrophic membrane, a completely ‘clean’ sample could usually be taken. Owing to the slow evaporation of glycerol, the preparation remained suitable for counting for at least two weeks at room temperature. For most combinations of species and particle sizes, samples (with >50 particles of the most abundant category) from at least three individuals were counted.
The variable number of latex balls in samples of gut contents was standardized as follows. Let the total numbers of smaller and larger balls in the three homogeneous samples of dung used as food be A and B, respectively, the corresponding numbers in a midgut sample being a and b. The expected number (e) of larger balls in the gut sample, assuming that small and large balls were present in the same proportions in gut and food, was e = (B/A) × a. A standardized abundance (β) of the larger balls in the gut was then β = (b/e) × 100 (%). In all cases where the proportion small balls to large balls did not differ significantly between dung used as food and gut content sample (P > 0·05; 2 × 2 χ2-test on ball numbers), β = 100% was recorded. This value implies that large and small particles passed the supposed filter of the mouthparts with equal ease. In practice, this means uninhibited passage of both sizes. β-values below 100%, on the other hand, indicate that entry of the larger balls into the gut was more restrained than that of the smaller ones.
Comminution of larger dung particles by the mouthparts?
Practically all well-defined larger particles in fresh dung are indigestible plant remains, consisting of cell walls. Any efficient grinding by the mandibles must lead to comminution of some of these particles. Hence, stained plant particles (initially 50–100 µm, obtained from dung) were mixed into fresh dung together with 5-µm latex balls, the latter being markers that would pass the mouthparts without difficulty when the experimental beetles are fed on this mixture. A grinding effect of the mouthparts should then reveal itself by a higher ratio of small, stained particles to latex balls in the gut contents than in the food.
Particles in the size range 50–100 µm were isolated from fresh dung by wet sieving. The particles were stained, in small nylon bags with 18 µm mesh size, with the PAS (Periodic Acid Schiff) method (e.g. Feder & O’Brien 1968) which leads to a strong and permanent magenta colouring of carbohydrates in the plant cell walls. By microscopy, stained particles stood out distinctly in mixtures with unstained material. Roughly 1 g (ww) of stained particles and four drops of 5-µm latex balls (cf. previous section) were added to about 10 g of fresh dung, followed by thorough mixing. Staining and mixing inevitably led to some fragmentation of the fragile plant remains, and so some small (≤5 µm) stained fragments were present already in the mixture eaten by the beetles. Samples of both gut contents and the original dung-latex ball mixture were taken as described previously. Five-µm balls and stained particles of 2–5 µm width were counted, usually up to at least 50 stained particles. There appeared to be rather few stained fragments <2 µm, and since their identification was difficult and somewhat subjective at 400× magnification, these were not counted. Three beetle species were tested: A. ater, A. fossor and A. rufipes.
Maximum size of ingested particles
Table 1 presents the standardized abundances, β (cf. Materials and methods) of latex balls in the midgut. Three A. erraticus (not shown in Table 1) were only tested with 5-µm balls in a preliminary experiment. Since no balls were ingested, further testing with size mixtures was omitted.
Table 1. Latex balls of known diameter in samples of midgut contents from five species of the dung beetle genus Aphodius. For each combination of latex ball sizes, the number of larger particles is expressed as a percentage (β) of the number expected based on the number of smaller particles, assuming that both sizes would pass the mouthparts with equal ease, cf. Materials and methods. Each β-value represents one tested beetle. In brackets: total number of particles (both sizes, all samples)/total number of larger particles
|2/5||31·4; 39·0; 47·8; 58·9; 100||100; 100; 100||–||–||–|
| ||(5529/137)||(2014/207)|| || || |
|5/10||4·4; 5·8; 10·8; 22·1; 22·2||30·3; 50·3; 62·8||73·5; 74·8||58·1; 80·7; 100||65·5; 75·8; 78·0; 78·3|
|5/14||0; 0; 0; 1·7; 6·3||0; 2·5; 3·0; 3·3; 3·3||18·0; 22·5; 45·2; 53·2||–||–|
| ||(678/5)||(1452/17)||(1846/391)|| || |
|5/18||–||–||1·0; 4·4; 6·5||–||27·0; 35·7; 38·1; 41·1|
| || || ||(1367/12)|| ||(1552/116)|
|10/18||–||–||–||14·2; 39·8; 44·8; 56·2||–|
| || || || ||(1278/44)|| |
|10/39||–||–||–||0; 0||0; 0; 0|
| || || || ||(504/0)|| |
Table 2 shows the probabilities of entry into the gut (minimum and maximum) for the tested particle sizes. Except for A. erraticus, 100% passage of 2-µm particles was assumed in all cases. This was supported by the β-values for 5-µm particles in A. rufipes and in A. ater. Passage for 5-µm balls of 100% was also assumed in A. contaminatus, A. fimetarius and A. fossor, based on the high values for 10 µm. In these cases, the probabilities of entry for any size were simply the percentages in Table 1 for that size in combination with 5-µm balls. Otherwise, probabilities were obtained by multiplication of the proper percentages in Table 1. For instance, the minimum probability for an 18-µm ball processed by A. contaminatus was 0·581 × 14·2% = 8·3% (Table 2).
Table 2. Probabilities, for latex balls of specified diameters, of passing the filtering mechanism of the mouthparts in six species of the dung beetle genus Aphodius. The probabilities are based on the percentages in Table 1 and assume completely unimpeded passage of 2-µm particles, cf. text
|A. rufipes||31–100|| 4–22|| 0–6·3||–||–|
|A. ater||100||30–63|| 0–3·3||–||–|
The upper size limit of ingested particles is defined arbitrarily as the largest size with a probability ≥5% of passing the filtering mechanism of the mouthparts. Obviously, the upper size limit in A. erraticus was below 5 µm, in keeping with an extremely fine-grained appearance of the gut contents by microscopy. In A. rufipes the limit was about 10 µm (or slightly lower) for some individuals, and 10–14 µm for others. The upper limit was a bit lower than 14 µm in A. ater and approximately 18 µm in A. fimetarius. The exact limit in A. fossor and A. contaminatus could not be determined since balls between 18 and 39 µm were unavailable for these experiments. However, in the other species, a small increase in ball diameter lowered probabilities considerably (Table 2), and so an upper limit of approximately 25 µm seems likely. This was confirmed by microscopy of natural gut contents, which clearly included 15–20 µm particles, but none in the 30–40 µm range.
Comminution of larger dung particles?
Comminution by the mandibles of the larger stained particles in the food should lead to a higher ratio of small (2–5 µm width), stained particles to 5-µm balls in the gut contents than in the eaten dung mixture. However, in none of the gut content samples was this ratio significantly different from that in the food (Table 3). Moreover, observations did not indicate an increased relative abundance of stained particles <2 µm in the gut contents. Thus, in none of the three species, representing a considerable variation in body size (15–121 mg) and maximum size of ingested particles (10–25 µm), was there any evidence of substantial comminution of larger plant particles.
Table 3. The ratio of stained particles (2–5 µm) to 5-µm latex balls in samples of dung and gut contents of beetles (genus Aphodius) feeding from that dung. (A) Mean ratio ±SE; (B) number of samples; (C) total number of stained particles + 5-µm balls counted in all samples. Dung 1 and 2 were different samples of sheep dung (preferred by A. ater), dung 3 and 4 were cattle dung
|Dung 1||0·53 ± 0·020||4||695|
|A. ater||0·49 ± 0·017||4||696|
|Dung 2||0·44 ± 0·019||4||775|
|A. ater||0·40 ± 0·015||4||849|
|Dung 3||0·40 ± 0·011||3||1000|
|A. fossor||0·40 ± 0·014||4||1694|
|A. rufipes||0·37 ± 0·005||3||1180|
|Dung 4||0·56 ± 0·008||3||228|
|A. rufipes||0·57 ± 0·031||5||226|
The functioning of dung beetle mouthparts
The idea (e.g. Madle 1934; Miller 1961) that scarabaeid dung beetles such as Aphodius restrict their ingestion to very small particles is supported by the present findings (Table 2). In the two species eating the largest particles (A. contaminatus and A. fossor), latex balls still had to be below about 25 µm (diameter) if their probability of passing the mouthpart filter was to be at least 5%. Moreover, in the species with the most fine-grained gut contents (A. erraticus), even 5-µm particles were too large to be ingested. These results agree with those in Bíly, Sterba & Dykova (1978) which indicate an extremely low probability of Taenia saginata eggs (30–40 µm diameter, e.g. Botero 1989) passing the mouthpart filter in A. fimetarius and A. luridus, as expected (for A. fimetarius) from Table 2. In another experiment (Bíly & Prokopic 1977), dehydrated A. fossor and A. fimetarius drank a suspension of Ascaris eggs in saline. Since a considerable number of intact eggs (70 × 50 µm) were found in at least 50% of the individuals it seems that the usual mechanism of filtering and squeezing is suspended if the animals are drinking rather than eating. However, drinking is probably rare in most Aphodius species, given their moist food and microhabitat.
Madle's (1934) assumption that the food must pass through the channels (‘Filterrinnen’) between the molar ridges on the mandibles has not been confirmed. In A. fimetarius and A. fossor, for example, the width of these channels is about 3–6 and 4–8 µm, respectively (P. Holter, unpublished observations), but 14 µm (A. fimetarius) and 18 µm (A. fossor) latex balls were always found in the gut if present in the food (Table 2). Moreover, a completely rigid filter, like the mandibular channels, would allow the passage of particles up to a certain size, and none above that size. However, the current study indicates that the probability of ingestion decreases gradually (Table 2). Hence, filtration is probably performed by some of the flexible setae on the mouthparts, as assumed by Bíly & Prokopic (1977) and (regarding the largest particles) by Hata & Edmonds (1983).
Based on interpretations of mandible morphology, Miller (1961) and Hata & Edmonds (1983) attributed the small particle size in the gut contents of various scarabaeid species to an efficient grinding and comminution of large particles by the molar areas. In addition, Miller (1961) and Miller, Chi-Rodriguez & Nichols (1961) found support in the observations (1) that charcoal dispersed in the food appeared more finely ground in the gut, and (2) that Ascaris eggs in the food were largely absent from the gut or, if found at all, were present in a ‘crushed and abraded condition’. The absence of eggs was explained by an efficient grinding so that no remnants could be recognized in the gut contents, whereas rejection of the eggs was considered unlikely (Miller et al. 1961). However, none of this evidence seems compelling. The finer charcoal particles in the gut could be ascribed to the removal of larger particles by filtration, or to squeezing between the mandibles. Squeezing could also lead to the poor condition of the few Ascaris eggs in the gut, and most of the eggs might, after all, have been rejected on the basis of size before ingestion.
At any rate, the results in Table 3 do not indicate any substantial comminution of larger dung particles prior to ingestion in Aphodius. Two additional points seem relevant here. First, the completely rigid articulation of the mandibles (e.g. Snodgrass 1935) permits outward and inward movements only. Whereas this is suitable for squeezing, it is harder to envisage an efficient grinding action (which has never been directly observed, cf. Hata & Edmonds 1983) resulting from such simple movements. Second, little could be gained by comminution and ingestion of large particles, the vast majority of which are plant remains (mainly cell walls), as these beetles lack any known specializations (such as fermentation chambers) for the digestion of cell wall components, e.g. cellulose. Easily digestible microorganisms or dead epithelial cells from the gut of the herbivore should therefore be consumed with a minimum of dilution by such intractable plant remains, and this is probably achieved by filtering setae that remove larger particles before ingestion.
In the absence of trituration, what could be the function of the elaborate ridges on the mandibular molars? The structure of the so-called pharyngeal ridges in larvae of saprophagous Cyclorrapha (Diptera) (Dowding 1967) is remarkably similar to that of the molar ridges in scarabaeid dung beetles. Moreover, the two groups feed on very similar substrates. Dowding (1967) provided convincing evidence that the larvae squeeze their food against these ridges by lowering the roof of the pharynx. Particles of 0·6 µm and above in size are retained by elaborate, lamellate substructures (not unlike the ‘tritors’) on top of the ridges, and are subsequently eaten. Superfluous fluid passes the lamellae and is eliminated through the channels between the ridges. Thus, the larvae are particle feeders that concentrate suitable particles in the rather fluid food by means of the pharyngeal ridges. As a working hypothesis it is suggested that the molar areas in scarabaeid dung beetles function in the same way. In addition, filtrating setae probably remove most of the unprofitable, large particles before the squeezing that concentrates particles to be eaten on the surface of the molar ridges. According to this hypothesis, superfluous liquid squeezed out of the dung will pass the extremely fine-meshed filter made up by ‘tritors’ and fissures between the distal parts of adjacent ridges, get into the channels between ridges and flow away from the pharynx, not into it as presumed by Madle (1934). It is tempting to suggest that this liquid may wet the fresh material coming into the mouth and hence probably facilitate the process of filtration. In other words, a recycling of liquid that would allow the beetles to filtrate even relatively dry dung if necessary. Obviously, however, this idea needs proper testing.
Feeding biology in relation to dung beetle ecology
If the beetles restrict their diet to particles below, say, 20 µm, how much of a dung pat is a potential food resource? By wet sieving, the fraction of particles >20 µm was isolated from samples of fresh dung of two cows, sheep (pooled sample from several individuals) and two horses. The dung was exclusively from grazing animals (September 1999). Each sample (about 0·5 kg) was homogenized by thorough mixing, and the wet sieving was then performed on 15-g subsamples. Particles larger than 20 µm, and therefore retained in the series of sieves, comprised 56 and 53% (cattle), 49% (sheep), and 49 and 62% (horse) of the dry matter. According to these preliminary observations, potential dung beetle food, in this example particles <20 µm, made up no less than 40–50% of the freshly voided dung. In Denmark, adult Aphodius, even in very densely populated cowpats, assimilate only 0·2–0·3% of the energy in the fresh dung (Holter 1982). Strong and/or frequent competition for food among these beetles would therefore seem unlikely.
The upper size limits of ingested particles are <5 µm (A. erraticus), 10–14 µm (A. rufipes), 12(?)–14 µm (A. ater), about 18 µm (A. fimetarius) and about 25 µm (A. contaminatus and A. fossor) (cf. Tables 1 and 2). Comparison of these values with the mean masses of the species (first section of ‘Materials and methods’) shows that there is no correlation between body masses and maximum sizes of ingested particles. This is illustrated by the fact that A. contaminatus and A. fossor, both ingesting the largest particles, are the smallest and largest species, respectively, in the assemblage.
Aphodius contaminatus is unique among the six species in that the beetles are found not only in all kinds of fresh dung but also in, for example, decaying vegetables or offal from slaughterhouses (Landin 1961; Hanski 1980a; Holter 1982 and unpublished observations). This exceptional polyphagy may require an ability to eat a wide range of particle sizes, even in a small species. Aphodius fossor and A. fimetarius are both so-called late-successional species (Hanski 1980b; Holter 1982; Gittings & Giller 1998). This means that they occur in dung of widely different ages (hence with a relatively high mean age) in which the availability and nutritional quality of any size class of particles may also differ considerably. The beetles may therefore need to be rather tolerant with regard to the size of ingested particles. Aphodius ater is mid-successional and is also intermediary as to particle size. Finally, A. erraticus and A. rufipes, eating the smallest particles, are early successional, i.e. confined to fresh dung. This could be particularly rich in easily digestible microorganisms and dead epithelial cells from the herbivore gut. In this case it may be possible and profitable to restrict the diet to very small particles, avoiding any larger, indigestible plant fragments. Thus, the mouthpart filter in these five species may be adapted to dung characteristics that are, at least to some extent, related to age. Obviously, however, valid generalizations will require an analysis of more dung beetles and of the characteristics of their food. These studies should include other taxa within the Scarabaeoidea and should represent a wider range of body sizes and preferred dung types.
I am grateful to K. Wardhaugh for generous help during my stay in Canberra and for critical reading of the manuscript. Further thanks are due to L. Bolt Jørgensen for help and advice concerning the PAS staining, and to the Strødam Nature Reserve, Frederiksborg State Forest District and L. Eberhardt for access to pastures. The work at CSIRO Entomology, Canberra, was supported by a travel grant from the Institute of Zoology, University of Copenhagen.