.If
6116 OlKOS 34: 54-58. Copenlaagea 1980
Insect effects on bacteria and fungi in cattle dung
John Lussenbop, Rabinder Kumar, D. T. Wick10w and J. E. Lloyd
Lussenhop, J., Kumar. R.• Wicklow. D. T. and Uoyd. J. E. 1980. Insect effects on booteria and fungi in cattle dung. - Oikos 34: 54-58.
Insect-microbial interactions were studied during the first month of dung decomposi tion in Wyoming and Michigan. At both sites. screen cones placed over fresh dung were used to exclude insect colonists and confine normal field densities of Aphodius beetle adults or enough sarcophagid adults to produce normal larval densities. The effect of these insects on bacterial and hyphal densities as well as on fungal species numbers was assayed after the dung had been in the field 3 to 4 wk. Presence of maggots and Aphodius beetles increased bacterial and decreased hyphal density in Wyoming but not in Michigan. We hypothesize that these effects are due to insect mixing of the substrate. giving bacteria a competitive advantage over fungi. Normal insect colonization increased the number of fungal species per pat in Michigan and Wyoming. although the toW number of fungal species was not affected by treat· ments.
J. LUS$~nhop, D~pt of Biological Scienc~$, Uni". of Illinois at Chicago Ci~l~. Box 4348, Chicago, IL 60680, USA. R. Kumar and J. E. L./oyd, Entomology S«tion. Uni" of Wyoming, Box 3354, LartmriIl, Wy 82071, USA. D. T. Wicklow, Northern RegioNli RUHl'Ch C~ntD', F~rkraJ. Research, Science and EdlIcation Administration. USDA, Peorl&, IL 6J604, USA.
~ HaCe!CCMilC K ~~ B 'n!!'IeHHe nep BClE'O foeClIIUIll ~ lIl!IJIC3& B B!lAI::MlHre K MINHrltHe. B 0Clc:lKX MeC'l'l'Xl&t '1'aHHIIX l1CI8ePX e::ae:.ro lIl!IJIC3& r1l:M'!laIlH ~ KCI\'YOlI, ~ rrpe .rs>124*llllllM ~ HaC!!!KCMiIlC H ~ Iq:MI1IIMfYIO IlCJIeIIYlO l"l11O'" 'ntClC'l'It llI.YICC8 AphadilUJ JlHClO ~ It1I:71"HlX'no B3pc)C1HC ~ nc, ~ ItCl\:'M&f1IJHYIO It1I:71"HlX'no JllNHHCllHOA nonymIUHH. It3Y'UUlR B1IIDIHIIe 3'1HlC IfollICI!ICCMlI H& I'UlI:lTHOC'lh ~ H rpHOHbIX l'14lSI K H& IIMJI:)808 pa.1tCQC5paaHe rr;::tIClClII I1CC118 '1'OE"O, Kale HaBC3 HlIlCIQI:lWICII B naneBIIIC yc1I:l8IIIIX 3-4 1Il!!lmtIlH. ~ MYX HI1H lIY1
Accepted IS March 1979 C OIKOS 0030.12991801010054-05 $02.5010
54 OIKOS 34:1 (19MII) was placed around the bases of the cones to exclude Introdudion arthropods. • The effect of invertebrate activity on microbial The five treatments were: (I) presence of maggots metabolism in decomposing substrates is believed to belonging to carly colonizing fly genera (in Wyoming. enhance decomposition of all but the "softest" tissues only Ravil/ia species were used; in Michigan half or (Edwards et a1. 1970). However. the basis for the more of the individuals were Ravinia, the remainder. stimulus. the specific effects of invertebrates on par species of Orthel/ia and Mwca), (2) presence of early ticular microbial groups. are poorly known (Satchell beetle colonists of approximately equal size (Aphodiw 1974). When is grazing and when is substrate mixing the vittalw Say and A. scabriceps LeConte were used in important effect? Do invertebrates which stimulate Wyoming, A. haemorrhoidalis L. in Michigan). (3) bacterial metabolism also stimulate fungal metabolism? presence of maggots and beetles, (4) no insects. (5) How are fungal species numbers affected by inverteb normal insect colonization. Each treatment was repli rates? These questions motivated this study of in cated five times in Wyoming and four times in sect-microbial interactions during the first month of de Michigan. Wire screen cones were placed over dung composition of cattle dung in an arid short-grass range subjected to each treatment; treatments were effected in Wyoming and a mesic pasture in Michigan. At both by adding unsexed adults as indicated in Tab. 1. In the sites we contrasted dung with and without insects in case of normal insect colonization. treatment 5. open order to show how their activity affected bacterial and topped cones were raised about 15 em above the ground fungal densities as well as fungal species composition. on wooden blocks. Two dung pats were placed under' We chose cattle dung for an examination of in cones to which adult insects were added. and the repro sect-microbial interactions because rapid development ductive success ofadded adults was assayed by assuming of bacterial. fungal. and arthropod populations might be that the number of arthropods extracted from one of the expected to result in strong interactions. Among the pats, under each cone was a reasonable estimate of ar first colonists are insects whose mouthparts allow then thropods in the other. to concentrate fine particles from moist dung. Cyclor Comparisons were made with arthropod densities in rhaphous fly larvae. such as species in the sarcophagid cohorts of dung pats put out in the same pastures on I genus Ravinia used in the present study. sieve particles August 1977 in Wyoming and Michigan; these are 0.6 J.lm or more from dung between pharyngeal ridges termed unmanipulated in contrast to dung manipulated (Dowding 1967). Adult Aphodiw beetles concentrate with use of cones. particles by pressing dung between labial and labraJ Dung was exposed in the field for 28 d in, Wyoming pads of setae (Madle 1934. Landin 1961). At the same and 21 d in Michigan. These intervals were chusen to time insects arrive. spores of coprophilous fungi. stimu allow for sporulation of the earlier appearing cop lated to germinate by gut passage through cattle. are rophilous fungi during field exposure at either site. believed to begin development of hyphal networks. Within 30 min of collection. manipulated dung pats were divided and returned to the laboratory: arthropods were extracted from one half in Berlese funnels; one quarter was frozen for later microbial biomass esti Materials and methods mates; one quarter was frozen until each could be incu Dung was obtained from pastured cattle so that the bated for 40 d to allow sporulation of fungi which in the fungal innoculum they consumed would be fresh and field might have sporulated during the next period of representative of the site and season. The experiments suitable moisture and temperature. Percent muisture were conducted near Laramie. Wyoming (Univ. of was determined from frozen samples. Water potential Wyoming Experimental Farm) from 29 July to 26 Au of dung was estimated on the basis of a pF- % moisture gust. and in southeastern Michigan (Kellogg Biological relationship determined using filter paper of known Station. Michigan State Univ.) from 13 August to 3 matric potential (Fawcett and Collis-George 1967). September 1977. Adult Herefords were used in Microbial biomass was determined in the 3-4 0101 Wyoming; 12 to 16 month old Guernseys were used in thick crust as well as in the center of each dung pal. Michigan. Dung was collected in the field of Wyoming, Fungal biomass was determined by counting hyphae in while in Michigan dung was collected from cows tem dung-agar suspensions. and bacterial biomass by porarily confined in a bam. and was frozen three days counting bacteria stained with fluorescein isothiocyan until use. We will not make between-site comparisons ate (FITC). Frozen 2 g dung samples were dispersed for of microbial densities and species numbers because of 2 min in a Waring blender with enough !;terile distilled the freezing. However. microbial densities in frozen and water to make a 10-1 dilution. Preparations for fungal unfrozen dung incubated in the field for 3 wk in JUly counts were prepared by pipetting (pipette tip Ld. = 5 1977 and June 1978 did not show a response to freez mm) 20 011 of the dispersed solution from the center of ing. At both sites 1 kg pats were immediately placed the column of the 10-1 suspension and mixing with 30 inside wire scre'en cones. The screen cones (O.n mesh ml of 2% purified agar solution. This mixture was used 1 1 0101- ) were lined with muslin (3 mesh 0101- ) and sand to prepare agar films in haemocytometer wells as de-
OIKOS 34:1 (l91l0) 55 ..
scribed by Jones and Mollison (1948), and Parkinson et Michigan. Aplwdius larvae are sedentary; we argue that al. (1971). Then, using phase contrast microscopy, the they had much less effect on dung microorganisms than number of hyphal intersections with an occular grid . adult beetles (P > O. 10 for the correlations between were counted and converted to a length estimate using density of Aplwdius larvae and fungal or hacterial de the formula of Olson (1950). Preparations for bacterial nsity in each of the 10 Wyoming pats with beetles). counts were made by pipetting (pipette tip i.d. = 2.5 The initial percent moisture of dung used was similar mm) 0.01 ml of the 10-1 solution onto microscope (81, and 80 in Michigan and Wyoming, respectively). slides, heat fixing, and following the staining procedure Averaged over treatments, the % moisture ofdung pats of Babiuk and Paul (1970). The me-stained cells when collected in Michigan was 80 in contrast to 37 in were then counted with dark-field illumination; globu Wyoming. The high percent moisture in Michigan was lar cells above 2 microns in diameter were considered partly due to 28 mm rainfall during the two days prior to yeasts. Fungal species numbers were determined by collection. In Wyoming only the dung crust dried suUi identifying species which sporulated during a 40 d ciently to limit microbial growth (a pF estimate of 4.5). laboratory incubation at 24"C with optimal moisture. Arthropods had no effect on final moisture content in We counted bacteria in 20 fields, and hyphae in 80 Michigan. In Wyoming, however, dung with lowest ar microscope fields per replicate. The F-max test indi thropod densities (the no-arthropod, and normal col cated that statistical analysis should be performed on onization treatments) contained 33% moisture after 28 the square root of hyphal density, and on untrans d, significantly lower (p < 0.05; t-test) than the 4(}% formed averages of bacterial numbers per field. moisture in dung to which arthropods were added. In spite of slightly higher water content associated with arthropod activity, rapid evaporation from Wyoming dung concentrates biological activity in a smaller vol Results ume. Independent field drying experiments in which dry The goal of our treatments was production of compara and wet center dung was weighed separately suggest ble numbers of both adult AphodiU3 and maggots in the that after 3 d. at the end of maggot activity. S%. and dung at both sites. Tab. 1 shows that this was ac after 7 d. at the end of adult AphodiU3 activity. 20% of complished. although after a week there was much the volume of wet center dung has dried. The lower higher AphodiU3 reproduction in Wyoming than arthropod density in un manipulated Wyoming pats re lative to those in Michigan (Tab. 1) may be caused by this loss of volume. Tab. I. Arthropod density in manipulated and unmanipuJated The field site and crust-eenter contrasts were respon I kg dung pats. sible for larger microbial density differences than treat Treatment Micbigan Wyoming ments effected (Fig. 2). The high densities of bacteria and yeast in Michigan dung may be due to higher mois (1) Number of adult ture levels. Different moisture levels at the two sites flies added •.. •.. . .. •.•. .. 10 10 interacted with the microbial response to arthropod (2) Number of adult treatments, because the treatment effects were consis Aphodius added .... 4Q-.45 30 tently strongest in the crust of Michigan dung and in the (3) Number of adult flies and AphodUu added...... 10+40-45 10+30 center of Wyoming dung. F-ratios of the 12 one-way analyses-of-variance performed on the data were al LlUVaJ d~nsilin in ways higher for crust in Michigan and for center in Dung Wyoming; this pattern is evident in Fig. 1. ( 1) Maggots after 3 d 20.4 38.0 Arthropods affected microbial density more strongly (2) Aphudius after 7 d ...•.. 19.0 123.0 in Wyoming than in Michigan (Tab. 2). and their effects (3) Maggots after 3 din maggot + bccUe were opposite at the two sites. In Wyoming. microbial treatment . 24.0 26.6 density in dung crusts was unaffected by presence or (5) Aphodius larvae 14 d absence of arthropods. In the center of Wyoming dung after natural pats. presence of Aphodius alone increased bacterial colonization . 4.0 1.0 density by 53% and maggots increased density by 43% Densities and spec:i~s relative to dung without insect... (Fig. 1). But in the "umb~1'$ (par~ntht!Sis) presence of both Aphodius and maggots. bacterial in unmanipuiat~d dung numbers only increased 27%. Wyoming Aphodius Maggots after 3 d .. 9 31 Adult Aphodius after 3 d . 9(2) 8(1.75) treatments also show a similar. though statistically non Larval Aphodius after 14 d .. 15 12 significant, trend in increased yeast density. Hyphal de Larval Aphodius after 21 d .. 14 25 nsity, however. was decreased 40% by Aphodius. Other anhropods after 3 d ... 143(17) 68(8.5) In Michigan, the opposite microbial responses to ar Other anhmpods after 14 d .. 250(16) 131(8.5) Other arthropods after 21 d .. 129(12) 63(6.25) thropods were observed. Arthropods had no effect on microbial biomass in the center of Michigan dung. In
56 OIIi.OS 14'1 (lflKIII WYO"' •• MiCHl .... caul' Fig. I. Microbial rcspullse to arthropod addition to cow dung incubated in the field • under cOlles. Treatments arc: - (nu arthropuUs). r (maggots). B (ApllOdius beetles). B+F (magguts + Aphudius). C (cunes raised to allow colonization). Lellers indicate statistical differences based on one-sided ~% confidence intervals (Dunnell 19~ ~) for no-arthropod vs. treatment contrasts.
IACTr;Il'. ' •••T HYPMAe
Tab. 2. The average number of fungai species sporulating on laboratory incubation. the rate of sporulation was the 250 g ponions ofcattie dung in the field and during subsequent same for aU treatments and sites. 40 d laboratory incubation. Norma! colonization significantly Arthropods were important in dispersing some cop increased species numbers in both Michigan and Wyoming (P OIKOS 34:1 (1980) 57 'j sheep dung (Breymeyer et al. 1975). Breymeyer and trated by Dickinson and Underhay (1977) who re- . her colleagues added ten times the insect densities of corded densities similar to those observed in the present the present experiment, and quantified microbial abun study. By using two field sites, we have shown that al dance using agar plate cultures. They found, as in the though fungal reproduction is not affected. hyphal de Wyoming dung of the present study. that ApllOditlS nsity may be reduced by insects. Over a range of mois adults decreased numbers of fungal propagules, while ture conditions, insect activity may result in bacterial both maggots and Aphodius adults increased bacterial and fungal activity occurring sequentially under dry density. conditions, or simultaneously under moist conditions. Why was no microbial response observed in Michigan dung? An answer cannot be given because both climate and dung properties differ between sites. We speculate Ackllowledgt'mt'nu. We thank Drs T. L Poulson and D. B. Mertz for reading the manuscript. and D. Nyberg for helpful that more rapid drying of dung in Wyoming concen discussion. J. Knieser performed the laboratory work on which trates arthropod activity. Further, forage plants and this study is based. S. Carta and B. Martin helped in the field. thus dung texture differ resulting in flatter pats with a Research supported by NSF Grants DEB75-09443. greater surface area in Wyoming; this might also con DEB77-Q9443. centrate arthropod activity. Finally, freezing the Michigan dung may have increased initial microbial growth. Nevertheless, existence of a different response References at each site is important, because it demonstrates that Babiuk. L.A. and Paul E. A. 1970. The use of fluorescein very similar insect congeners may have contrasting ef isothiocyanate in the determination of the bacterial fect on microorganisms in different habitats. biomass of grassland soil. - Can. J. Microbial. 16: 57-62. Why did bacterial and hyphal density respond differ Breymeyer. A., Jakubc:zvk, H.• and Olechowiez, E. 1975. In e ently to insects in Wyoming dung? One hypothesis is fluence of coprophilous arthropods on microorganisms in that adult Aphodius feeding destroys bacterivorous sheep feces - laboratory investigations. - Bull. Acad. Pol. Sci. 23: 257-262. nematodes and their eggs. This does not explain the Dickinson, C. H., and Underhay. V. H. S. 1977. Growth of bacterial increase in response to maggots, which be fungi in cattle dung. - Trans. Br. mycol. Soc. 69: 473-477. cause of differently constructed mouthparts are unlikely Dowding, V. M. 1967. The function andecologicalsignificance to affect nematodes. A more consistent hypothesis is of the pharyngeal ridges occurring in the larvae of some cyc:lorraphous Diptera. - Parasitology 57: 371-3R8. that in the presence of insects the competitive advan Dunnett, C. W. 1955. A muJtiple comparison procedure for tage of hyphal growth form is lost to fungi. With ar comparing !leveral treatments with a control. -J. Am. Stat. thropod mixing, bacteria are .continually exposed to Assn. 50: 1096-1 t 21. fresh substrate, they do not deplete resources within Edwards, C. A.. Reichle, D. and Cros.'ilay, D. A. Jr. 1970. The role of soil invertebrates in turnover of organic matter and diffusion distance of their cells and stop growing. In nutrients. - In: Reichle, D. E. (ed.), Analysis of Tempe addition, nutrients in arthropod feces, and possibly low erature Forest Ecosystems. Springer, New York, pp. ered antibacterial effects of fungi may contribute to the 147-172. effect. With arthropod mixing of dung, the ability to Fawcett, R. G. and Collis-George. G. 1967. A filter paper method for determining the moi.'iture characteristic of soil. extend a hyphal network into fresh substrate is less ad Austr. J. Exp. Age. Animal Hush. 7. 162-167. vantageous to fungi because their growth rates are gen Fenchel. T. 1970. Studies on the decomposition of organic: erally lower than those of bacteria. detritus derived from the turtle grass Thalassi4 testlU.linum. Aquatic amphipods appear to increase bacterial pro - Limnol. Oceanogr. 15: 14-211. Hargrave. B. T. 19711. The effect of deposit feeding amphipods ductivity by mixing similarly to dung insects (Fenchel on the metabolism of benthic micmnora. - Limnol. 1970, Harrison 1977). Hargrave's (1970) study of Oceano~r. 15: 21-30. HyaJella aueca Saussure feeding on sediment cores Harrison, P. G. 1977. Decomposition of macrophyte detritus from the benthos of lakes is especially pertinent because in seawater: effects of grazing by amphipods. - Oikas 28: 165-169. substrate disturbance by HyaJella azteca increased bac Jones. P. C. T. and Mollison. J. E. 1948. A technique for the terial productivity, but. at high densities, decreased quantitative extimation of soil micro-organisms. J. gen. algal productivity. Microbiol 2: 54-{)9. The present study suggests that insects may reduce Landin. B. O. 1961. Ecological studies on dung beetles. Opuscula Entomologica Suppl. 19. microenvironmental extremes in dung. In wet, possibly Mad1e. H. 1934. Zur Kenntnis der Morphologie. Okologie und anaerobic Michigan dung. insects may aerate dung. In physiologie von Aphodiw rufifHs Lin. und einigen ver Wyoming. where insects were associated with higher % wandten Arten. - Zool. Jb. Abt. Anat. 58: 3113-39h. moisture, separation of a crust from moist dung under Olson, F. C. W. 195U. Quantitative estimates of filamentous algae. - Trans. Am. micro Soc. 59: 272-279. neath by arthropod activity may reduce capillary water Parkinson. D.. Gray, T. R. G.. and Williams, S. T. 1971. movement between the wet dung center and the sur Methods for studying the ecology ofsoil microorganisms. face. Blackwell. Oxford. As dung ages and dries, insects emigrate or pupate, Satchell. J. E. 1974. Litter-interface of animatelinanimate matter. - In: Dickinson. C. H. and Pugh. G.. lEd.). and hyphal density increases. The expansion of hyphal Biology of Plant Litter Decomposition. Vol. I. Academic networks into the center of drying dung has been iIIus- Press. London. pp. xii-xliv. 58 OIKOS )4, I \ 19KtI)