APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1982, p. 000-0000099-2240/82/080001-00$02.00/0

Vol. 44, No. 2

Role of Aerobic Microbial Populations in Cellulose Digestionby Desert Millipedes

ELSA C. TAYLORDepartment of Biology, University ofNew Mexico, Albuquerque, New Mexico 87131

Received 15 January 1982/Accepted 22 April 1982

I examined the role of aerobic microbial populations in cellulose digestion bytwo sympatric species of desert millipedes, Orthoporus ornatus and Comanchelussp. High numbers of bacteria able to grow on media containing cellulose,carboxymethyl cellulose, or cellobiose as the substrate were found in thealimentary tracts of the millipedes. Enzyme assays indicated that most celluloseand hemicellulose degradation occurred in the midgut, whereas the hindgut wasan important site for pectin degradation. Hemicellulase and 0-glucosidase in bothspecies and possibly Cx-cellulase and pectinase in 0. ornatus were of possiblemicrobial origin. Degradation of [14C]cellulose by millipedes whose gut floraswere reduced by antibiotic treatment and starvation demonstrated a reduction in14Co2 release and 14C assimilation and an increase in 14C excretion over valuesfor controls. It appears that the millipede-bacterium association is mutualistic andmakes available to millipedes an otherwise mostly unutilizable substrate. Such anassociation may be an important pathway for decomposition in desert ecosys-tems.

In desert ecosystems, rates of decompositionare limited by available water, nitrogen, andcarbon (see literature cited in reference 20).Decomposition and nutrient cycling are there-fore key processes affecting primary productionin these arid regions. Cellulose decomposition, acomplex process mediated by a series of en-zymes, is carried out by a wide variety oforganisms. In soils, decomposition can be ac-complished directly through the activities offungi (21) or aerobic and anaerobic bacteriacapable of degrading cellulose to glucose and amixture of acids (25). Indirect degradation isbelieved to be effected by the production ofenzymes by microorganisms in invertebrate ani-mal alimentary tracts. Evidence of this is oftenconflicting and inconclusive, owing to difficul-ties in culturing bacteria and distinguishing en-zymes of microbial origin from those of inverte-brate origin. Nevertheless, some cellulases havebeen fairly conclusively shown to originate ininvertebrate animals (26; see references 23, 24,and 38 for more conclusive evidence) and bacte-ria (11, 30; see references 14, 16, 17, 36, 37, and41 for definite evidence). Studies of some milli-pede species have indicated that cellulose isdigested during passage through the intestinaltract (7, 34); however, the origin of cellulolyticenzymes is unknown. In another millipede spe-cies, ingestion of a cellulose diet has been shownto result in midgut bacterial population develop-ment (2).

Cellulose decomposition is effected by threeclasses of enzymes: C1-enzymes (active uponcrystalline cellulose), Cx-erizymes (active uponnoncrystalline cellulose and soluble derivativesor degradation products of cellulose), and ,B-glucosidases or cellobiases (active upon cellu-biose) (28). Because any of these enzymes maybe produced by microflora or invertebrates, it isimportant in quantifying cellulolytic activity totrace the origin of these enzymes.The present study compares the levels of

activity, origin, and ultimate function of cellulo-lytic enzymes found in two sympatric species ofdesert millipedes. Orthoporus ornatus (Spiro-streptidae) is a large, desiccation-resistant milli-pede (13) that forages over a broad area. Coman-chelus sp. (Atopetholidae) is smaller, lessdesiccation-resistant, and more restricted in for-aging area and habitat. The digestive tract of 0.ornatus is composed of a small foregut (FG),larger midgut (MG), and very large, soil-filledhindgut (HG) which usually averages a littleover half the length of the animal (Fig. 1). InComanchelus sp., the MG is large and soil filled,whereas the HG is much smaller, although it isalso usually packed with soil.The following questions are posed in this

paper. (i) Are bacteria in the guts of either orboth species capable of utilizing cellulose? (ii) Ifcellulolytic enzymes are present in detectableamounts in the guts of these millipedes, in whatpart of the gut is such activity found? (iii) Is the

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FIG. 1. Digestive tracts of 0. ornatus and Comanchelus sp. Top, Comanchelus sp. FG. 1.9 to 2.1 cm; MG,2.1 to 4.2 cm; HG, 4.2 to 5.2 cm. Bottom, 0. ornatus FG, 0 to 0.9 cm; MG, 0.9 to 2.8 cm; HG, 2.8 to 6.8 cm.

activity probably of microbial origin, or is itassociated with intestinal tissue, indicating thatthese enzymes have a millipede origin? (iv) Dobacteria, through cellulose degradation, makeavailable to millipedes otherwise unutilizablesubstrates, or are bacteria and millipedes com-peting for the same food sources?

MATERIALS AND METHODS

Study site. Millipedes were collected from a site atthe base of the volcanic escarpment immediatelynorthwest of Albuquerque, N.M. This Chihuahuandesert site is covered with large, dark, basalt bouldersand a mixture of shrubs, mainly Gutierrezia sarothrae,Atriplex canescens, and Rhus trilobata (see reference35 for a more complete description of habitat). Beforeeach experiment, both species of millipedes werecollected from the same habitat. Each species wasthen maintained separately in the laboratory on habitatsoil and detritus food until used.

Bacterial isolation. Selective media were used toisolate bacteria capable of producing any of the threecellulases. Agar plates contained Skinner cellulosemedium B (33) with Hoagland trace element solutionand either 1% Whatman powdered cellulose with 1.5%agar and cycloheximide (0.1 mg ml-') to inhibit fungi(12) or 0.5% carboxymethyl cellulose (CM-cellulose)with 1% agar (22). Whatman cellulose was purified bymethod of Leedle and Hespell (27). Because cellulosesettled during solidification in the petri dishes, thehardened agar disks were inverted before inoculation,which gave bacteria a higher concentration of cellu-lose. Medium for estimates of most probable numberscontained Skinner cellulose medium B, Hoaglandtrace element solution, 1% cellobiose, and phenyl redindicator.Medium containing Skinner cellulose medium B,

Hoagland trace element solution, and 1.5% agar but nocellulose, CM-cellulose, or cellobiose served as acontrol. In addition, I isolated numerically dominantmorphotypes from cellulose and CM-cellulose plates,purified them by three subculturings, and then testedthem for the presumptive ability to breakdown CM-cellulose by flooding 9-day old cultures on CM-cellu-

lose plates with 1% hexadecyltrimethyl ammoniumbromide (22). Zones of clearing around or undercolonies (which were scraped from the plates beforeflooding) constituted a positive result, and those mor-photypes which showed clearing were further charac-terized morphologically (5). (Results are based upongrowth on CM-cellulose or cellulose medium, sincesome cultures would not grow on nutrient agar.)The midsegment width (diameter of the middle of

the millipede as measured with calipers) of each milli-pede was measured, and then the FG, MG, and HGwere extracted under sterile conditions and trituratedseparately in crucibles with 10 ml of sterile distilledwater. Initial dilutions were shaken for 40 min on awrist action shaker (time predetermined from thestandard curve of number of colonies versus timeshaken). Serial dilutions of each initial dilution werespread on three replicate plates, and 1-ml aliquotswere added to five tubes for estimates of most proba-ble numbers. All inoculated media were incubatedaerobically at 30°C (mean daily field temperature dur-ing feeding season) and 60% relative humidity for 14days (cellulose), 4 days (CM-cellulose), or 3 days(cellobiose). To facilitate counting, I air dried celluloseplates for 2 h and then flooded them with dilute (1:10[vol/vol]) Safranin for 5 min, which yielded dark-orange colonies on a pale-orange background. Directmicroscopic counts were made in a Petroff-Haussercounting chamber.To standardize data for millipedes of different sizes,

I measured the midsegment width of five individuals ofeach species. The FG, MG, and HG were extractedand then dried at 60°C under vacuum for 48 h. A linearregression of the midsegment width with the weight ofdried gut tissue plus contents gave standard curveswith high correlations.Enzyme assays. To assess the presence in millipedes

of cellulolytic enzymes and other enzymes capable ofdegrading plant polymers, I maintained both speciesseparately in the laboratory for 2 weeks before assayand gave them normal field detritus. Four 0. ornatusand five Comanchelus sp. were pooled to make eachsample. FGs of millipedes in each sample were re-moved and triturated in a crucible with 0.001 Mphosphate buffer, pH 7.0. For MG and HG samples,

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gut parts were slit open, and the contents were flushedout with buffer and then triturated for assay of en-zymes. (Hereafter, MC will refer to MG contents andHC to HG contents.) Tissue was flushed with twoadditional 10-ml aliquots of buffer (subsequently dis-carded) and then triturated in a 10-ml aliquot of freshbuffer. (Hereafter, MT will refer to MG tissue and HTto HG tissue.) All homogenates were centrifuged(10,000 x g; 4°C; 20 min), and the supernatant fluidwas then passed through columns of PD-10 Sephadex(5-cm bed height) with 0.001 M phosphate as theelution buffer. The protein-containing eluate fractionswere then pooled for use.

Substrates were suspended in 0.1 M buffers thatreflected the mean pH of the gut part being assayed:for FG, phosphate buffer (pH 6.0); for MT and MC,sodium acetate buffer (pH 5.5); and for HT and HC,Tris buffer (pH 8.5) (31). Assay procedures were thoseof Martin and Martin (29). To determine the presenceof Cx-cellulase, hemicellulase, and pectinase, I made1% suspensions of CM-cellulose, locust bean gum,and citrus pectin in appropriate buffers. A 0.3-mlportion of substrate was incubated with 0.3 ml ofenzyme for S to 60 min at 35°C. I terminated incuba-tion by adding 0.6 ml of 3,5-dinitrosalicylic acid rea-gent (Bernfield reagent; 6) and heating the mixture in aboiling water bath for 5 min. A 0.9-ml portion of waterwas then added, and the optical density at 540 nm ofBernfield reagent-reducing sugar complex was deter-mined. Controls were run with enzyme denatured byheating in a boiling water bath for 15 min.

I determined the presence of Cl-cellulase by incu-bating the enzyme with microcrystalline cellulose inbuffer (50 mg ml-'). A drop of toluene, to inhibitbacterial growth, was added before incubation for 24to 27 h with shaking at 35°C. Incubation was terminat-ed by rapid filtration through Celite; the assay condi-tions were those described above.A 3.32 mM solution of p-nitrophenyl-3-D-glucoside

was used to determine the presence of aryl-f-glucosi-dase. A 0.5-ml portion of the enzyme was incubatedwith 0.5 ml of substrate for 5 to 60 min at 35°C.Incubation was terminated by the addition of 1 ml of 1M NH40H-NH4Cl buffer (pH 9.8), and the opticaldensity at 420 nm of liberated nitrophenol was deter-mined. Controls were run with enzyme denatured asdescribed above.For the determination of the amount of protein

present in each sample, the Bradford protein assay(10) was used to run enzyme extracts and proteinstandards containing bovine albumin.

Radioisotope assay. Each species of millipede wasdivided into two groups: insects with reduced flora andcontrol insects. Gut floras were reduced by a combina-tion of starvation and antibiotic treatment. Individualsthat were newly emerged from dormancy in the soiland that had just started to eat were collected andstarved in the laboratory for 7 days (0. ornatus) or 5days (Comanchelus sp.). Preliminary tests indicatedthat these lengths of time caused a gut flora reductionin each species without imposing undue physiologicalstress. During the last 36 h, individuals consumeddrops of 10%o dextrose-water (0. ornatus) or 10%fructose-water (Comanchelus sp.) (sugars were cho-sen on the basis of species preference) with 0.044 mgeach of tetracycline and chlortetracycline per g ofmillipede weight.

To determine the probable level of decrease in flora,I dissected the guts of four additional millipedes ofeach species that had received the starvation-antibiot-ic treatment and determined bacterial levels by thetechniques described above.

Uniformly labeled, powdered ['4C]cellulose (specif-ic activity, 7.7 ,uCi mg-'; dose, 1.4 mg per six milli-pedes) was triturated with a glass rod in a small (75 by12 mm) test tube; 0.15 ml of molten sterile agar wasthen added (potato dextrose for 0. ornatus and treha-lose-fructose for Comanchelus sp. [final concentra-tions: sugars, 0.5% each; agar, 1.5%]). Preliminarypreference tests with various prepared media andmixtures of sugar and agar in distilled water indicatedthat 0. ornatus readily ingested potato dextrose agar,whereas Comanchelus sp. preferred a combination oftrehalose and fructose (unpublished data). Cellulosewas dispersed into the agar by mixing on a Vortexmixer; drops were then quickly placed on tin foil witha pipette. Each millipede (individuals were in separateTupperware containers) was then offered a drop to eat.The cellulose could not be powdered finely enough tobe evenly dispersed throughout the agar, so the drops(which also differed in size) did not contain a uniformquantity of cellulose. In addition, individual millipedesdid not always ingest all of the agar drop offered. As aresult of these problems, each millipede ingested adifferent and undetermined quantity of cellulose.As soon as a millipede had eaten, it was placed in an

experimental flask in a train in which CO2 could betrapped. The flask in which a millipede with reducedflora was placed contained a moist soil-detritus layerwhich had been autoclaved (20 lb/in2, 220°C, 1 h) andcooled before the millipede (surface sterilized by beingdipped in 2% Lysol for 20 s) was added. This flask waspreceded in the train by an air filter (15) to preventbacteria from entering as the air was bubbled throughthe train and into the cocktail at the approximate rateof 30 ml s-1. "CO2 was trapped for 6-h periods in acocktail containing 55% toluene, 39o ethylene glycolmonomethyl ether, 5.5% ethanolamine, and 0.5% PPO(2,5-diphenyloxazole). Every 12 h, all fecal pellets and5 ,ul of hemolymph were taken. Hemolymph wasmixed in a 99.5% toluene-0.5% PPO cocktail. Fecalpellets were air dried, triturated, and suspended in aCab-o-sil cocktail (99.5% toluene, 0.5% PPO, 4% cab-o-sil). The experiment was terminated after 54 h, whenanimals were dissected and two portions of fat bodyper millipede were removed, weighed, solubilized inNCS solubilizer, and mixed with a 99% toluene-0.5%PPO cocktail. Radioactivity was determined with amodel LS230 liquid scintillation counter (BeckmanInstruments, Inc.). Corrections were made for back-ground radiation levels and for counting efficiency andquenching among cocktails and among vials of eachcocktail type (40).

Control animals were treated in a similar mannerexcept that starvation of these millipedes, which hadbeen eating in the field for 2 weeks before collection,was for 36 h. (Preliminary tests indicated that thislength of time rendered millipedes hungry enough toeat readily without affecting population levels of gutflora.) Antibiotics were omitted from the sugar-waterdrops, millipedes were dipped in distilled water, andthe experiment was run under unsterile conditions.

Statistics. Bacterial and enzyme data for differencesbetween both species and within each species were

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A B A B A BFIG. 2. Number of bacteria from millipede guts cultured on three media. Values are for each milligram of gut

part tissue plus contents. A, 0. ornatus; B Comanchelus sp. Each vertical bar represents one standard error.Results for 10 of each millipede species are shown.

analyzed by the Friedman nonparametric analysis ofvariance. Where differences were found, the Newman-Kuels multiple range test was employed. Radioisotopedata and degree of flora reduction effected by antibiot-ic-starvation treatment were analyzed by the Wilcox-on rank sums test (46).

Chemicals. All chemicals were purchased from Sig-ma Chemical Co., with the following exceptions: theagar was from BBL Microbiology Systems; the potatodextrose agar was from Difco Laboratories, PD-10Sephadex columns were from Pharmacia Fine Chemi-cals, Inc., microcrystalline cellulose was from Poly-science, [14C]cellulose and NCS solubilizer were fromAmersham-Searle, Cab-o-sil and ethanolamine werefrom Eastman Kodak Co., and toluene was fromFisher Scientific Co.

RESULTSBacteria. All data were standardized as de-

scribed above so that results could be expressedas the number of bacteria per milligram of guttissue plus contents. Both millipede species con-tained large numbers of bacteria capable ofgrowing on media containing cellulose, CM-cellulose, or cellobiose as the substrate (Fig. 2).Control plates without added cellulose sourcessupported low numbers of colonies (0 to 9 perplate) at dilutions which yielded 30 to 300 colo-

nies on plates with cellulose sources, indicatingthat most of the colonies on plates with celluloseor CM-cellulose were probably using these car-bon sources. There was no significant differencebetween millipede species in overall numbers ofbacteria counted by direct microscopic countsor cultivated (Fig. 3A).

In pooling results for 0. ornatus organismsisolated from all media, I found that the HG hadthe highest number of bacteria per milligram,followed by the MG and then the FG (all signifi-cantly different at P < 0.001; Fig. 2). In poolingdata for gut parts and testing for density differ-ences among media, I found that cellobiosesupported larger numbers of bacteria (P < 0.05)than did cellulose or CM-cellulose, whichranked equally. Each substrate supported thegrowth of the same number of FG bacteria(Table 1). Cellobiose supported significantlyhigher numbers ofMG and HG bacteria than didcellulose or CM-cellulose (probability: <0.025for MG and <0.01 and <0.005 for cellulose andCM-cellulose, respectively, in HG).Each substrate supported the growth of the

same number of Comanchelus MG and HGbacteria, whereas the FG housed significantlyless bacteria (P < 0.001; Fig. 2). Overall, the

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CELLULOSE DIGESTION BY DESERT MILLIPEDES_|°x-W. g -O R OO0;0 -3 X 8z U,RHOOU

42~ ~ ~ ~ ~ ~ ~ ~~&CMANCQHLLUh2--

FIG. 3. Direct microscopic counts of total numbers of bacteria from millipede guts. Values are for eachmilligram of gut part tissue plus contents. A, Gounts of normal flora. Results for 10 of each millipede species areshown. B, Gounts after gut flora reduction by starvation and antibiotics. Results for four of each millipedespecies are shown. Each vertical bar represents one standard error.

highest numbers of bacteria in Comanchelus sp.grew in cellobiose media, followed by celluloseand then CM-cellulose media (all significant at P< 0.05). Equal numbers of FG bacteria grew inall three substrates (Table 1). The number ofMGbacteria growing in cellobiose media was greaterthan the numbers growing in cellulose and CM-cellulose media (P < 0.025; P < 0.001, respec-tively), whereas HG bacteria that grew in cello-biose outnumbered those that grew in CM-cellulose only (P < 0.005).

Direct microscopic counts showed that foreach millipede species (Fig. 3A), there was lessbacteria in the FG than in the MG and HG,which had equal numbers (P < 0.05).Many types of bacteria were able to establish

zones of clearing on CM-cellulose (Table 2).Many of these were, in addition, able to grow onmedia with cellulose, although no attempt wasmade to determine whether cellulose was uti-lized, owing to difficulties in seeing clearing onsuch thick plates and to the drying of the platesduring the long incubation period required. As

TABLE 1. Differences among bacteria from eachgut part in ability to grow on three types of mediaa

Gut p Growth of Growth of0. ornatus on: Comanchelus sp. on:

FG CB, CEL, CMC CEL, CMC, CBMG CB,CELCMC CB, CEL, CMCHG CB, CEL, CMC CB, CEL, CMCa Underlined variables are not significantly different

at P < 0.05. Relative values are given in the text. CB,Cellobiose; CEL, cellulose; CMC, CM-cellulose.

could be expected given its more restrictedforaging area, Comanchelus sp. had a far smallerdiversity of bacteria than did 0. ornatus.Enzymes. Results of total enzyme activity

assays indicated that both millipede species hadthe capacity to degrade cellulose and other plantpolymers and that the MG was the main site forcellulose and hemicellulose (but not necessarilypectin) degradation in both millipede species(Table 3). The amount of pectin degradation inthe 0. ornatus HG was surprisingly high. CG-cellulase activity was higher in 0. ornatus thanin Comanchelus (P < 0.0001); otherwise, totalenzyme activity levels for the entire alimentarytract were the same for both species. Assayresults for separated gut tissue and contents areexpressed as micromoles of equivalent reducingsugar liberated per microgram of protein inenzyme homogenate per unit of time so that theresults could be standardized with the leastamount of bias: the millipedes used were ofdifferent sizes, and expressing results on a per-milligram-of-gut-weight basis (versus per micro-gram of protein) would have strongly biasedresults toward the FG, MT, and HT, owing totheir low weight, compared with the high weightof soil present in the MC and HC (Table 4).

C1-cellulase in 0. ornatus was significantlyhigher in MC than in HT (P < 0.001), FG (P <0.05), or MT and HC (P < 0.05) (Table 5). (Forclarity of discussion gut contents will be consid-ered as a separate gut part.) Activity of Cx-cellulase in MC was higher than those in HT,MT, FG, and HC (probability: <0.001, <0.005,<0.01, and <0.025, respectively). HC, FG, and

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TABLE 2. Morphology of bacteria able to clear media containing CM-cellulose

Isolate Colony morphology Cell morphology

no. Color Configura- Marg b Eleva- Gram Type and shape Spore Motilitytion' ri tion' stain

3d White 1 1 3 - Streptococci in sheaths4d White 1 1 1 - Large bacilli, round ends, single or chains of2 _-7d White 6 3 2 - Long, thin bacilli, rounded ends, some curved +8d White 6 4 7 - Bacilli, single or chains of 2 -

gd White 1 3 2 - Coccobacilli, single - +13e White 2 2 7 - Coccobacilli, irregular form - +14e White 2 2 5 - Coccobacilli, single or clumped - +16e White 1 1 5 +/- Coccobacilli, single - +25e White 1 1 3 _ Bacilli, short, clumped -

2d Yellow 6 4 7 + Bacilli, single or branching -

6d Yellow 5 2 6 - Bacilli, single or clumped - +18d Yellow 1 4 7 + Cocci, single or short chains -

21d Yellow 1 3 5 + Cocci, chains of .2 -

23d Yellow 6 4 8 - Bacilli, long, thin, flexible - +ise Yellow 1 1 2 - Bacilli, long, thin, curved, single - +17e Yellow 1 1 3 + Coccobacilli, cornyiform, single or rows - +ld Pink 2 2 3 + Coccobacilli, single, rows, V-form -

24d Orange 1 1 3 + Bacilli, V-form -

20d Orange 2 2 3 + Cocci, chains of -2 -

5d Orange 1 1 3 - Coccobacilli, single or clumped - +12 Orange 1 1 7 - Bacilli, single or clumped -

19" Orange 1 1 3 + Coryniform, club-shaped rods in clumps - +lld Beige 2 2 3 - Coccobacilli, single

a 1, Round; 2, round with scalloped margin; 5, concentric; 6, irregular and spreading.b 1, Smooth; 2, wavy; 3, lobate; 4, irregular.c 1, Flat; 2, raised; 3, convex; 5, umbonate; 6, hilly; 7, ingrowing into medium; 8, crateriform.d Isolated from 0. ornatus.e Isolated from Comanchelus sp.

MT all showed significantly higher activity thanHT (probability: <0.001, <0.005, and <0.01,respectively). P-Glucosidase activity was high-est in MC (P < 0.001 for HT, MT, FG; P < 0.005for HC). FG and HC showed higher activity thandid HT and MT (P < 0.001). HC showed higherpectinase activity than did MT, HT, or FG (P <0.0001). Hemicellulase activity was highest inMC (P < 0.0001); FG ranked next (P < 0.001 forHT and MT; P < 0.025 for HC), followed by HC(P < 0.005 higher than HT).

In Comanchelus sp., there was no significantdifference among gut parts in C1-cellulase activi-ty (Table 5). Cx-cellulase was highest in MC (P< 0.001 higher than HC, HT, and FG; P < 0.01higher than MT), whereas MT showed signifi-cantly greater activity than HT and HC (P <0.05). ,B-Glucosidase activity in MC was higherthan that in other gut parts (P < 0.001); FG andHC differed from HT and MT (P < 0.001), andMT differed from HT (P < 0.05). Pectinaseactivity in HC was higher than those in MT andHT (probability: <0.005 and <0.025, respective-ly). Hemicellulase was higher in MC (P < 0.001for MT, HT, and HC; P < 0.01 for FG), followedby FG (P < 0.001 higher than HT, HC, and MT),and then MT (P < 0.005 higher than HT).The above results, indicating that ,-glucosi-

dase and hemicellulase were present in extreme-ly low levels in tissue (although total-gut-partlevels were high [Table 3]), suggest that theseenzymes may not be made by either millipedespecies but rather by microbes present in theMG. For 0. ornatus, Cx-cellulase found in theMG, as well as pectinase found in the HG, mayalso partly be of microbial origin.

Radioisotope assay. Direct microscopic countsindicated that, with the exception of the 0.ornatus FG, the gut floras of both millipedespecies were significantly (P < 0.006) reducedby antibiotic treatment-starvation: 0. ornatusMG by 97.67% and HG by 97.64%; and Coman-chelus sp. FG by 98.59%, MG by 98.87%, andHG by 96.90% (Fig. 3B). The numbers of orga-nisms capable of growing on cellulose mediawere also reduced by various amounts in eachmillipede species (Table 6). Reduction of gutflora decreased the ability of both millipedespecies to degrade and assimilate cellulose.(Since each millipede ingested different and un-known amounts of the powdered cellulose-agarmixture, results are expressed as percentage oftotal label trapped by each method. Percentresults do not represent whole-organism per-centages but were used as a means of standard-izing counts so that comparisons between treat-

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TABLE 3. Total enzyme activity of each gut part

Enzyme and gut Total enzyme activity (mean ± SE)'part 0. ornatus Comanchelus sp.

Cl-cellulaseFG 1.18 ± 0.12 0.61 ± 0.09MG 7.33 ± 0.57 2.87 ± 0.76HG 1.68 ± 0.26 0.76 ± 0.10

Cx-ceHulaseFG 24.72 ± 3.16 3.98 ± 2.48MG 275.01 ± 18.16 64.97 ± 10.12HG 38.85 ± 3.84 3.44 ± 2.11

1-GlucosidaseFG 16.11 ± 1.81 4.69 ± 0.17MG 234.22 ± 14.72 271.39 ± 41.85HG 28.87 ± 2.26 6.92 ± 1.33

HemicellulaseFG 47.89 ± 8.61 16.06 ± 1.87MG 317.18 ± 81.43 666.60 ± 47.70HG 35.19 ± 2.72 9.67 ± 0.52

PectinaseFG 31.54 ± 9.59 18.82 ± 8.03MG 196.22 ± 27.75 66.88 ± 7.25HG 223.48 ± 52.01 42.75 ± 8.41

a Values for C1-Cx-cellulases, hemicellulase, andpectinase are expressed as micromoles of reducingsugar (x103) liberated per animal per minute. Valuesfor [-glucosidase are expressed as micromoles ofnitrophenol (x103) liberated per animal per minute.

ments could be made [Table 7].) Impairedcellulose degradation was shown by a decreasein assimilation of label into hemolymph, a de-crease in production of 14Co2, and an increase inthe amount of label excreted. Levels of 4CO2decreased over time, indicating that most of thelabel was either degraded or passed through thegut during the course of the experiment. Thepercentage of total label assimilation in controlanimals ranged from one-fifth to one-third, therange of assimilation efficiency found by Woo-ten and Crawford (44) for 0. ornatus (i.e., 22.8± 2.65% for 0. ornatus and 26.58 + 3.33% forComanchelus sp.). Values for animals with re-duced flora were significantly (P < 0.05) lower:16.25 ± 2.37% for 0. ornatus and 17.48 ± 3.24%for Comanchelus sp.

DISCUSSIONBoth millipede species are detritivores: they

feed on soil, plant litter, and other items foundon the soil surface. In addition, 0. ornatusgrazes on the bark of bushes in its habitat (45).These feeding patterns enable both species toacquire varied microbial floras (fungal flora of0. ornatus are discussed in another article[35a]).

TABLE 4. Enzyme levels found in millipede guttissue and contents

Enzyme and gut Enzyme activity (mean + SE)'part 0. ornatus Comanchelus, sp.

C1-cellulaseFGMTMCHTHC

Cx-cellulaseFGMTMCHTHC

1-GlucosidaseFGMTMCHTHC

HemicellulaseFGMTMCHTHC

PectinaseFGMTMCHTHC

24.90 ± 8.8730.49 ± 2.4994.75 ± 11.569.73 ± 2.74

34.57 ± 5.65

13.54 ±10.37 ±69.14 ±1.64 ±

15.84 ±

8.64 ±2.38 ±

55.23 ±2.17 ±10.92 ±

28.32 ±6.36 ±

78.56 ±3.19 ±13.03 ±

3.920.673.711.012.88

1.410.298.030.140.68

5.540.614.630.842.58

17.53 ± 4.489.37 ± 1.39

46.62 ± 6.3412.42 ± 3.9385.24 ± 44.34

142.99 + 112.9627.25 + 5.7878.26 + 28.3261.59 ± 23.7414.18 ± 8.72

8.23 t7.56 t16.83 t0.00 ±0.00 ±

10.73 t3.85 t

88.84 ±1.79 ±8.36 ±

38.60 ±9.59 ±

225.05 t2.13 ±6.64 ±

47.48 ±9.36 ±16.46 ±12.57 +

8.231.971.990.000.00

1.630.542.291.110.99

9.560.88

10.512.134.46

20.112.541.203.70

54.03 ± 16.60

a Values are expressed as follows: for Cl-cellulase,micromoles of maltose (x105) liberated per microgramof protein per hour; for Cx-cellulase, hemicellulase,and pectinase, micromoles of maltose (x105) liberatedper microgram of protein per minute; for 13-glucosi-dase, micromoles of nitrophenol liberated per micro-gram of protein per minute.

Increase in bacterial growth on media withcellulose, CM-cellulose, or cellobiose as thesource of carbon and energy over growth oncontrol media indicates that the millipedes con-tained bacteria which could produce the Cl- andCx-cellulases and P-glucosidase necessary todegrade cellulose. The bacteria in the FG wereprobably present as a result of ingestion and aretherefore not part of the resident flora. Duringwinter, the number of FG bacteria varies greatlyfrom individual to individual, whereas the MGand HG of all individuals retain large popula-tions (unpublished data).Numerical differences between bacteria cul-

tured and bacteria counted by direct microscop-ic counts indicate that many bacteria were not

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TABLE 5. Differences in enzyme production in each gut partaGut part producing enzyme

Enzyme0. ornatus Comanchelus sp.

C1-cellulase MC HC MT FG HT MC HT FG MT HCCG-cellulase MC HC FG MT HT MC MT FG HT HC

P-Glucosidase MC HC FG MT HT MC FG HC MT HTPectinase HC MC FG HT MT HC FG MC HT MT

Hemicellulase MC FG HG MT HT MC FG MT HC HTa Underlined variables are not significantly different at P < 0.05. Relative values are given in the text.

cultured by the methods employed. A largenumber of these may be strict anaerobes, whichhave been found in the digestive tracts of otherinvertebrates (8, 39). In particular, the large,moist, soil-packed HG of 0. ornatus and the MGand HG of Comanchelus sp. could quite possi-bly contain numerous anaerobic sites. Strictanaerobes were not screened for in the presentstudy.Both bacterial and enzyme data indicate that

cellulose degradation occurred in the HG of 0.

TABLE 6. Reduction in gut flora per milligram ofgut tissue plus contents of antibiotic-treated versus

control (36-h starvation) millipedes in variousecological groups

Ecological group and Gut % Reduction Significancemillipede species part (P value)'

Cellulose degraders0. ornatus FG 66.51 NS

MG 99.57 0.009HG 99.28 0.006

Comanchelus sp. FG 97.18 0.02MG 99.36 0.009HG 89.73 NS

CM-cellulose degraders0. ornatus FG 81.45 NS

MG 99.86 0.006HG 99.28 0.006

Comanchelus sp. FG 99.75 0.006MG 97.83 0.02HG 80.86 0.03

Cellobiose degraders0. ornatus FG 97.77 NS

MG 99.92 0.006HG 99.34 0.006

Comanchelus sp. FG 99.64 NSMG 99.94 0.006HG 99.08 0.06

aProbability based on Z statistic for Wilcoxon ranksums test. NS, Not significant.

ornatus. However, although large numbers ofbacteria were found in the Comanchelus HG,the low levels of enzymatic activity, particularlyCG-cellulase activity, indicate that this is not animportant site of cellulose degradation. The abil-ity to degrade locust bean gum, which repre-sents one category of hemicellulose (galacto-mannan), demonstrates the capacity to degradesome but not all categories of hemicellulosewhich may be present in millipede food.

It is possible that hemicellulase and P-glucosi-dase are of microbial origin in both millipedespecies; CG-cellulase in the MG of 0. ornatusmay also be of microbial origin. Both speciesappear to be able to produce some pectinasewhich may, in addition, be produced by micro-organisms. Most cellulose and hemicellulose di-gestion occurs in the MG; pectin degradationappears to occur primarily in the HG, which isgenerally considered (at least in the case ofinsects) to function only in water and ion absorp-tion and to play no direct part in digestion (43).Recent work, however, indicates that bacterialfermentation products produced in the HG ofcockroaches and the proctodeal dilation in scar-abid larvae can be absorbed from these sites intothe hemolymph (4, 9).Microorganisms present during incubation of

the enzyme assay mixture may have contributedsome variance. This was particularly true for C1-cellulase because of the 24- to 27-h incubation.Microscopic examination of the assay mixturerevealed that although toluene inhibited microbi-al growth, there were still large numbers ofbacteria present. This may account for the ex-tremely high C1-cellulase activity in the Coman-chelus FG.

It is difficult to establish beyond doubt wheth-er these enzymes were of millipede or microbialorigin. The fact that the activity in the gutcontents was far higher than that in tissue maypoint to a microbial origin. However, one cannotdetermine whether the low activity found in thetissue is due to millipede production or theincomplete flushing of gut contents from thetissue. In addition, since a secretory cell usuallyproduces and secretes its product constantly,

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CELLULOSE DIGESTION BY DESERT MILLIPEDES 289

TABLE 7. Distribution of "4C in C02, hemolymph, fat body, and fecal pellet sampled14C distribution (mean % ± SE) in:

Source 0. ornatus Comanchelus sp.Control Reduced flora P value Control Reduced flora P value(n 6) (n 5)' vle (n =6) (n =5)0 au

CO2 70.64 ± 5.89 47.06 ± 0.77 0.01 82.62 ± 2.77 58.89 ± 3.10 0.01Hemolymphb 1.09 ± 0.27 0.51 ± 0.07 0.01 3.51 ± 0.70 1.75 ± 0.19 0.006Fat bodyc 0.10 ± 0.02 0.09 ± 0.01 NSd 0.52 ± 0.19 0.26 ± 0.04 NSFecal pellete 27.36 ± 5.73 52.34 ± 0.75 0.01 12.51 ± 1.76 39.09 ± 4.16 0.006

0 The gut flora of one individual had clearly not been reduced so it was excluded from calculations.b Results are for a total of 20 ,u of hemolymph.c Results are means per milligram of fat body assayed.d NS, Not significant.I Means for five fecal pellets produced. Mean number of fecal pellets produced: 0. ornatus, 41.82;

Comanchelus sp., 80.75.

one would expect the level of activity to belower in tissue than in contents. How great adifferential would indicate a microbial ratherthan a millipede origin of enzymes is not known.For more conclusive determination of the enzy-matic origins, characteristics of enzymes frommillipedes and cultured bacteria or levels ofactivity in gut contents before and after treat-ment with antibiotics could be compared.The importance of the microbial gut flora to

cellulose degradation is clearly suggested by theradioisotope results. Increase in production of"CO2 could be due to increased respiration bythe millipedes or the bacteria. The increase inlabel in the hemolymphs of control animals,however, does indicate that the products ofmicrobial degradation are assimilated by themillipedes as well as by bacteria. Production ofany of the three cellulases by bacteria could bethe important factor in allowing millipedes toassimilate cellulose. The variability in body fatresults could have been due to the short durationof the experiment or to the fact that small,randomly selected portions were assayed, ratherthan the total fat body. Comparison of thepercentage of assimilated label excreted by con-trol animals with the percentage excreted byanimals with reduced flora indicates that thealready low assimilation efficiency (although it isrelatively high for a detritivore [44]) is furtherdecreased by a reduction in gut flora.Comanchelus sp. and 0. ornatus are distantly

related (different orders), yet both have evolveda strong dependence upon microorganisms fordigestive enzymes. This may be a universalphenomenon among millipedes, as other speciesare also believed to rely upon microbial enzymes(3, 34). Alternatively, this may be a function ofliving in a desert ecosystem where nutrients andmoisture are at low levels (18) and energeticallycostly to obtain. Use of acquired enzymes woulddecrease some of the expense of feeding for themillipede.

The fact that dependence on the Cl- and C,,-cellulases is greater than dependence on ,-glucosidase may be due to the nature of the foodingested. The degree to which detritus is degrad-ed by free-living microorganisms, particularlyfungi, often determines the palatability of thefood to millipedes (19, 32). It may be, therefore,that much of the food that these millipedesingest has already been somewhat degraded byfree-living flora possessing the necessary Cl-and C,-cellulases. This would also be a form ofmillipede dependence on microbially producedenzymes.The association between each species of milli-

pedes and its gut flora may be mutualistic.Bacteria, through the production of cellulolyticenzymes, make available to millipedes other-wise unutilizable substrates, which could be ofcrucial importance to millipede survival in des-erts where production of detritus is low (42). Themillipedes, in turn, provide for bacteria an envi-ronment with regulated moisture, temperature,and pH and supply the bacteria with a constantflow of substrates to degrade. This observationis supported by the fact that the gut contains farhigher numbers of bacteria per gram than doesthe surrounding soil (unpublished data), indicat-ing that the gut provides a habitat in whichbacteria can grow and multiply. Further evi-dence is provided by the finding that soil andlitter bacteria increase in number during passagethrough the digestive tract of the millipede spe-cies Glomeris marginata (1).

In summary, the results indicate that bothmillipede species contained bacteria capable ofutilizing cellulose. Enzymes necessary to de-grade cellulose and other plant polymers werepresent and may, in some instances, have beenof microbial origin. Reduction in gut flora result-ed in decreased assimilation of cellulose bymillipedes, indicating that products of microbialcellulose degradation were utilized by milli-pedes.

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APPL. ENVIRON. MICROBIOL.

This type of association has important impli-cations for nutrient cycling in deserts. Rates ofdecomposition are controlled by temperature,moisture, and proximity of nutrients necessaryto sustain decomposer organisms (25). Condi-tions favorable to the activities of free-living soilmicroorganisms may be sporadic and of shortduration. Any association which would enhancethe creation of a favorable decomposer situationcould greatly increase the rate of mineralizationand the production of humic compounds. Thus,the presence of invertebrate-microbe associa-tions could be of particular value during seasonswhen environmental conditions are inimical tothe activities of free-living microbes and couldbe of greater importance in more extreme des-erts than in moderate deserts (35b). This funda-mental process would play a key role in makingdeserts habitable for higher trophic levels.

ACKNOWLEDGMENTSI thank E. Arguello and J. Washburn for technical assist-

ance. C. S. Crawford, M. M. Martin, D. E. Caldwell, J.Trujillo, F. W. Taylor, W. W. Whitford, J. A. Wiens, andG. V. Johnson are gratefully acknowledged for review of themanuscript.This research was supported by grants from the Graduate

Research Allocations Committee and the Graduate School atthe University of New Mexico.

LITERATURE CITED

1. Anderson, J. M., and D. E. Bignell. 1980. Bacteria in thefood, gut and faeces of the pill millipede, Glomeris mar-ginata. Soil Biol. Biochem. 12:251-254.

2. Baleux, B., and C. P. Vivarls. 1975. Etude preliminaire dela flora bacterienne intestinale de Schizophyllum sabulo-sum var rubriper lat (Myriapoda, Diplopoda). Soc. Zool.France 99:771-779.

3. Bano, K., D. J. Bagyaraj, and R. V. Krishnamoorthy.1976. Feeding activity of millipede, Jonespeltis splendidusVerhoeff and soil humification. Proc. Indian Acad. Sci.133:1-11.

4. Baylor, C., and J. Mathelln. 1980. Carbohydrate fermenta-tion and by-product absorption studied with labelled cellu-lose in Orycetes nasicornis larvae (Coleoptera: Scarabaei-dae). J. Insect Physiol. 26:833-840.

5. Benson, H. J. 1973. Microbiological applications, 2nd ed.,p. 145. William C. Brown Co. Publisher, Dubuque, Iowa.

6. Bernfield, D. 1955. Amylases a and P. Methods Enzymol.1:149-150.

7. Bocock, K. L. 1963. Digestion and assimilation offood byGlomeris, p. 85-91. In J. Doeksen and J. Van der Drift(ed.), Soil organisms. North-Holland Publishing Co., Am-sterdam.

8. Bracke, J. W., D. L. Cruden, and A. J. Markovetz. 1978.Effect of metronidazole on the intestinal microflora of theAmerican cockroach, Periplaneta americana. Antimi-crob. Agents Chemother. 13:115-120.

9. Bracke, J. W., and A. J. Markovetz. 1980. Transport ofbacterial end products from the colon of Periplanetaamericana. J. Insect Physiol. 26:85-89.

10. Bradford, M. M. 1976. A rapid and sensitive method forquantitation of microgram quantities of protein utilizingthe principle of protein-dye binding. Anal. Biochem.72:248-254.

11. Breznak, J. A. 1975. Symbiotic relationships betweentermites and their intestinal microbiota, p. 559-580. InD. H. Jennings and D. L. Lee, Sympiosis XXIX. Cam-bridge University Press, Cambridge, England.

12. Buxton, A., and G. Fraser. 1977. Animal microbiology,

vol. 1, p. 291-292. Blackwell Scientific Publishers, Lon-don.

13. Crawford, C. S. 1972. Water relations in a desert milli-pede (Orthoporus ornatus, Girard) (Spirostreptidae).Comp. Biochem. Physiol. 42A:521-535.

14. Cruden, D. L., and A. J. Markovetz. 1979. Carboxy-methyl cellulose decomposition by intestinal bacteria ofcockroaches. App. Environ. Microbiol. 38:369-372.

15. Elsworth, R. 1969. Treatment of process air for deepculture. Methods Microbiol. 1:123-136.

16. Evans, W. A. L., and E. G. Jones. 1962. Carbohydrases inthe alimentary tract of the slug Arion ater L. Comp.Biochem. Physiol. 5:149-160.

17. French, J. R. J. 1975. The role of termite hindgut bacteriain wood decomposition. Mater. Org. 10:1-13.

18. Friedman, E. I., and M. Galun. 1974. Desert algae, li-chens and fungi, p. 166-212. In G. W. Brown, Jr. (ed.),Desert biology, vol. 2. Academic Press, Inc., New York.

19. Gere, G. 1956. The examination of the feeding biology andthe humicative function of Diplopoda and Isopoda. ActaBiol. 6:257-271.

20. Godall, D. W., and R. A. Perry (ed.). 1979. Arid-landecosystems: structure, functioning and management, vol.1. IBP 16. Cambridge University Press, Cambridge, En-gland.

21. Gray, T. R. G., and S. T. Williams. 1971. Soil Micro-organisms. Oliver and Boyd, Edinburgh.

22. Hankin, L., and S. L. Anagnostakis. 1977. Solid mediacontaining carboxymethyl cellulose to detect Cx-cellulaseactivity of microorganisms. J. Gen. Microbiol. 98:109-115.

23. Hartenstein, R. R. 1964. Feeding, digestion, glycogen andthe environmental conditions of the digestive system ofOniscus ascellus. J. Insect Physiol. 10:611-621.

24. Holden, M., and M. V. Tracey. 1950. A study of enzymesthat can break down tobacco-leaf components. 2. Diges-tive juice of Helix on defined substrates. Biochem. J.47:407-414.

25. Imshenetsky, A. A. 1968. Decomposition of cellulose inthe soil, p. 234-255. In T. R. G. Gray and D. Parkinson(ed.), The Ecology of Soil Bacteria. University of TorontoPress, Toronto.

26. Lasker, R., and A. C. Giese. 1956. Cellulose digestion bythe silverfish Ctenolepisma lineata. J. Environ. Biol.33:542-553.

27. Leedle, J. A., and R. B. Hespell. 1980. Differential carbo-hydrate media and anaerobic replica plating techniques indelineating carbohydrate-utilizing subgroups in rumenbacterial populations. Appl. Environ. Microbiol. 39:709-719.

28. Martin, M. M., and J. S. Martin. 1978. Cellulose diges-tion in the midgut of the fungus-growing termite Macro-termes natalensis: the role of acquired digestive enzymes.Science 199:1453-1455.

29. Martin, M. M., and J. S. Martin. 1978. The distributionand origins of the cellulolytic enzymes of the highertermite, Macrotermes natalensis. Physiol. Zool. 52:11-21.

30. Nielsen, C. 0. 1962. Carbohydrases in soil and litterinvertebrates. Oikos 13:200-215.

31. Nunez, F. S., and C. S. Crawford. 1976. Digestive en-zymes of the desert millipede Orthoporus ornatus (Girard)(Diplopoda: Spirostreptidae). Comp. Biochem. Physiol.55A:141-145.

32. Sakwa, W. N. 1972. A consideration of the chemical basisfor food preference in millipedes, p. 329-346. In G. N.Blower (ed.), Third International Congress of Myriapo-dology, Manchester. Academic Press, Inc., London.

33. Skinner, F. A. 1971. The isolation of soil Clostridia, p. 57-80. In D. A. Shapton and R. G. Board (ed.), Isolation ofanaerobes. Society of Applied Bacteriology technical se-ries 5. Academic Press, Inc., New York.

34. Striganova, B. R. 1969. Cellulose decomposition in theintestine of the millipede Pachyiulusfoetidissimus (Mur.)(Juolidae, Diplopoda). Doklady Akad. Nauk SSSR190:703-705. (In Russian.)

290 TAYLOR

on July 3, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

CELLULOSE DIGESTION BY DESERT MILLIPEDES 291

35. Taylor, E. C. 1979. Seasonal distribution and abundanceof fungi in two desert grassland communities. J. AridEnviron. 2:295-312.

35a.Taylor, E. C. 1982. Fungal preference by a desert milli-pede Orthorporus ornatus (Spirostreptidae). Pedobiologia23:329-334.

35b.Taylor, E. C., and C. S. Crawford. 1982. Microbial gutsymbionts and desert detritivores. Sci. Rev. Arid ZoneRes. 1:37-52.

36. Tetrault, P. A., and W. L. Weis. 1937. Cellulose decom-position by a bacterial culture from the intestinal tract oftermites. J. Bacteriol. 33:95.

37. Thayer, D. W. 1978. Carboxymethyl cellulase producedby facultative bacteria from the hind-gut of the termiteReticulitermes hesperus. J. Gen. Microbiol. 106:13-18.

38. Tracey, M. V. 1951. Cellulase and chitinase of earth-worms. Nature (London) 167:776-777.

39. Ulrich, R. G., D. A. Buthala, and M. J. Klug. 1981. Mi-crobiota associated with the gastrointestinal tract of thecommon house cricket, Acheta domestica. Appl. Envi-ron. Microbiol. 41:246-254.

40. Wang, C. H., D. L. Willis, and W. D. Loveland. 1975.

Radiotracer methodology in the biological, environmentaland physical sciences, p. 274. Prentice-Hall, Inc., Engle-wood Cliffs., N.J.

41. Wharton, D. R. A., M. L. Wharton, and J. E. Lola. 1965.Cellulase in the cockroach, with special reference toPeriplaneta american (L.). J. Insect Physiol. 11:947-959.

42. Whitford, W. G., M. Bryant, G. Ettershank, J. Etter-shank, and P. F. Santos. 1980. Surface litter breakdown ina Chihuahuan desert ecosystem. Pedobiologia 4:243-245.

43. Wigglesworth, V. B. 1974. The principles of insect physi-ology, 7th ed, p. 476-552. Halstead Press, New York.

44. Wooten, R. C., Jr., and C. S. Crawford. 1975. Food,ingestion rates and assimilation in the desert millipedeOrthoporus ornatus (Girard) (Diplopoda). Oecologia20:231-236.

45. Wooten, R. C., Jr., C. S. Crawford, and W. A. Riddle.1975. Behavioral thermoregulation of Orthoporus ornatus(Diplopoda: Spirostreptidae) in three desert habitats.Zool. J. Linn. Soc. 57:59-74.

46. Zar, J. H. 1974. Biostatistical analysis. Prentice-Hall,Inc., Englewood Cliffs, N.J. 620 pp.

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