Role of Aerobic Microbial Populations in Cellulose Digestion by Desert Millipedes
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1982, p. 000-000 Vol. 44, No. 2
0099-2240/82/080001-00$02.00/0
Role of Aerobic Microbial Populations in Cellulose Digestion
by Desert Millipedes
ELSA C. TAYLOR
Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131
Received 15 January 1982/Accepted 22 April 1982
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I examined the role of aerobic microbial populations in cellulose digestion by
two sympatric species of desert millipedes, Orthoporus ornatus and Comanchelus
sp. High numbers of bacteria able to grow on media containing cellulose,
carboxymethyl cellulose, or cellobiose as the substrate were found in the
alimentary tracts of the millipedes. Enzyme assays indicated that most cellulose
and hemicellulose degradation occurred in the midgut, whereas the hindgut was
an important site for pectin degradation. Hemicellulase and 0-glucosidase in both
species and possibly Cx-cellulase and pectinase in 0. ornatus were of possible
microbial origin. Degradation of [14C]cellulose by millipedes whose gut floras
were reduced by antibiotic treatment and starvation demonstrated a reduction in
14Co2 release and 14C assimilation and an increase in 14C excretion over values
for controls. It appears that the millipede-bacterium association is mutualistic and
makes available to millipedes an otherwise mostly unutilizable substrate. Such an
association may be an important pathway for decomposition in desert ecosys-
tems.
In desert ecosystems, rates of decomposition Cellulose decomposition is effected by three
are limited by available water, nitrogen, and classes of enzymes: C1-enzymes (active upon
carbon (see literature cited in reference 20). crystalline cellulose), Cx-erizymes (active upon
Decomposition and nutrient cycling are there- noncrystalline cellulose and soluble derivatives
fore key processes affecting primary production or degradation products of cellulose), and ,B-
in these arid regions. Cellulose decomposition, a glucosidases or cellobiases (active upon cellu-
complex process mediated by a series of en- biose) (28). Because any of these enzymes may
zymes, is carried out by a wide variety of be produced by microflora or invertebrates, it is
organisms. In soils, decomposition can be ac- important in quantifying cellulolytic activity to
complished directly through the activities of trace the origin of these enzymes.
fungi (21) or aerobic and anaerobic bacteria The present study compares the levels of
capable of degrading cellulose to glucose and a activity, origin, and ultimate function of cellulo-
mixture of acids (25). Indirect degradation is lytic enzymes found in two sympatric species of
believed to be effected by the production of desert millipedes. Orthoporus ornatus (Spiro-
enzymes by microorganisms in invertebrate ani- streptidae) is a large, desiccation-resistant milli-
mal alimentary tracts. Evidence of this is often pede (13) that forages over a broad area. Coman-
conflicting and inconclusive, owing to difficul- chelus sp. (Atopetholidae) is smaller, less
ties in culturing bacteria and distinguishing en- desiccation-resistant, and more restricted in for-
zymes of microbial origin from those of inverte- aging area and habitat. The digestive tract of 0.
brate origin. Nevertheless, some cellulases have ornatus is composed of a small foregut (FG),
been fairly conclusively shown to originate in larger midgut (MG), and very large, soil-filled
invertebrate animals (26; see references 23, 24, hindgut (HG) which usually averages a little
and 38 for more conclusive evidence) and bacte- over half the length of the animal (Fig. 1). In
ria (11, 30; see references 14, 16, 17, 36, 37, and Comanchelus sp., the MG is large and soil filled,
41 for definite evidence). Studies of some milli- whereas the HG is much smaller, although it is
pede species have indicated that cellulose is also usually packed with soil.
digested during passage through the intestinal The following questions are posed in this
tract (7, 34); however, the origin of cellulolytic paper. (i) Are bacteria in the guts of either or
enzymes is unknown. In another millipede spe- both species capable of utilizing cellulose? (ii) If
cies, ingestion of a cellulose diet has been shown cellulolytic enzymes are present in detectable
to result in midgut bacterial population develop- amounts in the guts of these millipedes, in what
ment (2). part of the gut is such activity found? (iii) Is the
281282 TAYLOR APPL. ENVIRON. MICROBIOL.
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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 it lose plates with 1% hexadecyltrimethyl ammonium
associated with intestinal tissue, indicating that bromide (22). Zones of clearing around or under
these enzymes have a millipede origin? (iv) Do colonies (which were scraped from the plates before
bacteria, through cellulose degradation, make flooding) constituted a positive result, and those mor-
available to millipedes otherwise unutilizable photypes which showed clearing were further charac-
terized morphologically (5). (Results are based upon
substrates, or are bacteria and millipedes com- growth on CM-cellulose or cellulose medium, since
peting for the same food sources? some cultures would not grow on nutrient agar.)
The midsegment width (diameter of the middle of
MATERIALS AND METHODS the millipede as measured with calipers) of each milli-
Study site. Millipedes were collected from a site at pede was measured, and then the FG, MG, and HG
the base of the volcanic escarpment immediately were extracted under sterile conditions and triturated
northwest of Albuquerque, N.M. This Chihuahuan separately in crucibles with 10 ml of sterile distilled
desert site is covered with large, dark, basalt boulders water. Initial dilutions were shaken for 40 min on a
and a mixture of shrubs, mainly Gutierrezia sarothrae, wrist action shaker (time predetermined from the
Atriplex canescens, and Rhus trilobata (see reference standard curve of number of colonies versus time
35 for a more complete description of habitat). Before shaken). Serial dilutions of each initial dilution were
each experiment, both species of millipedes were spread on three replicate plates, and 1-ml aliquots
collected from the same habitat. Each species was were added to five tubes for estimates of most proba-
then maintained separately in the laboratory on habitat ble numbers. All inoculated media were incubated
soil and detritus food until used. aerobically at 30°C (mean daily field temperature dur-
Bacterial isolation. Selective media were used to ing feeding season) and 60% relative humidity for 14
isolate bacteria capable of producing any of the three days (cellulose), 4 days (CM-cellulose), or 3 days
cellulases. Agar plates contained Skinner cellulose (cellobiose). To facilitate counting, I air dried cellulose
medium B (33) with Hoagland trace element solution plates for 2 h and then flooded them with dilute (1:10
and either 1% Whatman powdered cellulose with 1.5% [vol/vol]) Safranin for 5 min, which yielded dark-
agar and cycloheximide (0.1 mg ml-') to inhibit fungi orange colonies on a pale-orange background. Direct
(12) or 0.5% carboxymethyl cellulose (CM-cellulose) microscopic counts were made in a Petroff-Hausser
with 1% agar (22). Whatman cellulose was purified by counting chamber.
method of Leedle and Hespell (27). Because cellulose To standardize data for millipedes of different sizes,
settled during solidification in the petri dishes, the I measured the midsegment width of five individuals of
hardened agar disks were inverted before inoculation, each species. The FG, MG, and HG were extracted
which gave bacteria a higher concentration of cellu- and then dried at 60°C under vacuum for 48 h. A linear
lose. Medium for estimates of most probable numbers regression of the midsegment width with the weight of
contained Skinner cellulose medium B, Hoagland dried gut tissue plus contents gave standard curves
trace element solution, 1% cellobiose, and phenyl red with high correlations.
indicator. Enzyme assays. To assess the presence in millipedes
Medium containing Skinner cellulose medium B, of cellulolytic enzymes and other enzymes capable of
Hoagland trace element solution, and 1.5% agar but no degrading plant polymers, I maintained both species
cellulose, CM-cellulose, or cellobiose served as a separately in the laboratory for 2 weeks before assay
control. In addition, I isolated numerically dominant and gave them normal field detritus. Four 0. ornatus
morphotypes from cellulose and CM-cellulose plates, and five Comanchelus sp. were pooled to make each
purified them by three subculturings, and then tested sample. FGs of millipedes in each sample were re-
them for the presumptive ability to breakdown CM- moved and triturated in a crucible with 0.001 M
cellulose by flooding 9-day old cultures on CM-cellu- phosphate buffer, pH 7.0. For MG and HG samples,VOL. 44, 1982 CELLULOSE DIGESTION BY DESERT MILLIPEDES 283
gut parts were slit open, and the contents were flushed To determine the probable level of decrease in flora,
out with buffer and then triturated for assay of en- I dissected the guts of four additional millipedes of
zymes. (Hereafter, MC will refer to MG contents and each species that had received the starvation-antibiot-
HC to HG contents.) Tissue was flushed with two ic treatment and determined bacterial levels by the
additional 10-ml aliquots of buffer (subsequently dis- techniques described above.
carded) and then triturated in a 10-ml aliquot of fresh Uniformly labeled, powdered ['4C]cellulose (specif-
buffer. (Hereafter, MT will refer to MG tissue and HT ic activity, 7.7 ,uCi mg-'; dose, 1.4 mg per six milli-
to HG tissue.) All homogenates were centrifuged pedes) was triturated with a glass rod in a small (75 by
(10,000 x g; 4°C; 20 min), and the supernatant fluid 12 mm) test tube; 0.15 ml of molten sterile agar was
was then passed through columns of PD-10 Sephadex then added (potato dextrose for 0. ornatus and treha-
(5-cm bed height) with 0.001 M phosphate as the lose-fructose for Comanchelus sp. [final concentra-
elution buffer. The protein-containing eluate fractions tions: sugars, 0.5% each; agar, 1.5%]). Preliminary
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were then pooled for use. preference tests with various prepared media and
Substrates were suspended in 0.1 M buffers that mixtures of sugar and agar in distilled water indicated
reflected the mean pH of the gut part being assayed: that 0. ornatus readily ingested potato dextrose agar,
for FG, phosphate buffer (pH 6.0); for MT and MC, whereas Comanchelus sp. preferred a combination of
sodium acetate buffer (pH 5.5); and for HT and HC, trehalose and fructose (unpublished data). Cellulose
Tris buffer (pH 8.5) (31). Assay procedures were those was dispersed into the agar by mixing on a Vortex
of Martin and Martin (29). To determine the presence mixer; drops were then quickly placed on tin foil with
of Cx-cellulase, hemicellulase, and pectinase, I made a pipette. Each millipede (individuals were in separate
1% suspensions of CM-cellulose, locust bean gum, Tupperware containers) was then offered a drop to eat.
and citrus pectin in appropriate buffers. A 0.3-ml The cellulose could not be powdered finely enough to
portion of substrate was incubated with 0.3 ml of be evenly dispersed throughout the agar, so the drops
enzyme for S to 60 min at 35°C. I terminated incuba- (which also differed in size) did not contain a uniform
tion by adding 0.6 ml of 3,5-dinitrosalicylic acid rea- quantity of cellulose. In addition, individual millipedes
gent (Bernfield reagent; 6) and heating the mixture in a did not always ingest all of the agar drop offered. As a
boiling water bath for 5 min. A 0.9-ml portion of water result of these problems, each millipede ingested a
was then added, and the optical density at 540 nm of different and undetermined quantity of cellulose.
Bernfield reagent-reducing sugar complex was deter- As soon as a millipede had eaten, it was placed in an
mined. Controls were run with enzyme denatured by experimental flask in a train in which CO2 could be
heating in a boiling water bath for 15 min. trapped. The flask in which a millipede with reduced
I determined the presence of Cl-cellulase by incu- flora was placed contained a moist soil-detritus layer
bating the enzyme with microcrystalline cellulose in which had been autoclaved (20 lb/in2, 220°C, 1 h) and
buffer (50 mg ml-'). A drop of toluene, to inhibit cooled before the millipede (surface sterilized by being
bacterial growth, was added before incubation for 24 dipped in 2% Lysol for 20 s) was added. This flask was
to 27 h with shaking at 35°C. Incubation was terminat- preceded in the train by an air filter (15) to prevent
ed by rapid filtration through Celite; the assay condi- bacteria from entering as the air was bubbled through
tions were those described above. the train and into the cocktail at the approximate rate
A 3.32 mM solution of p-nitrophenyl-3-D-glucoside of 30 ml s-1. "CO2 was trapped for 6-h periods in a
was used to determine the presence of aryl-f-glucosi- cocktail containing 55% toluene, 39o ethylene glycol
dase. A 0.5-ml portion of the enzyme was incubated monomethyl ether, 5.5% ethanolamine, and 0.5% PPO
with 0.5 ml of substrate for 5 to 60 min at 35°C. (2,5-diphenyloxazole). Every 12 h, all fecal pellets and
Incubation was terminated by the addition of 1 ml of 1 5 ,ul of hemolymph were taken. Hemolymph was
M NH40H-NH4Cl buffer (pH 9.8), and the optical mixed in a 99.5% toluene-0.5% PPO cocktail. Fecal
density at 420 nm of liberated nitrophenol was deter- pellets were air dried, triturated, and suspended in a
mined. Controls were run with enzyme denatured as Cab-o-sil cocktail (99.5% toluene, 0.5% PPO, 4% cab-
described above. o-sil). The experiment was terminated after 54 h, when
For the determination of the amount of protein animals were dissected and two portions of fat body
present in each sample, the Bradford protein assay per millipede were removed, weighed, solubilized in
(10) was used to run enzyme extracts and protein NCS solubilizer, and mixed with a 99% toluene-0.5%
standards containing bovine albumin. PPO cocktail. Radioactivity was determined with a
Radioisotope assay. Each species of millipede was model LS230 liquid scintillation counter (Beckman
divided into two groups: insects with reduced flora and Instruments, Inc.). Corrections were made for back-
control insects. Gut floras were reduced by a combina- ground radiation levels and for counting efficiency and
tion of starvation and antibiotic treatment. Individuals quenching among cocktails and among vials of each
that were newly emerged from dormancy in the soil cocktail type (40).
and that had just started to eat were collected and Control animals were treated in a similar manner
starved in the laboratory for 7 days (0. ornatus) or 5 except that starvation of these millipedes, which had
days (Comanchelus sp.). Preliminary tests indicated been eating in the field for 2 weeks before collection,
that these lengths of time caused a gut flora reduction was for 36 h. (Preliminary tests indicated that this
in each species without imposing undue physiological length of time rendered millipedes hungry enough to
stress. During the last 36 h, individuals consumed eat readily without affecting population levels of gut
drops of 10%o dextrose-water (0. ornatus) or 10% flora.) Antibiotics were omitted from the sugar-water
fructose-water (Comanchelus sp.) (sugars were cho- drops, millipedes were dipped in distilled water, and
sen on the basis of species preference) with 0.044 mg the experiment was run under unsterile conditions.
each of tetracycline and chlortetracycline per g of Statistics. Bacterial and enzyme data for differences
millipede weight. between both species and within each species were284 TAYLOR APPL. ENVIRON. MICROBIOL.
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A B A B A B
FIG. 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 of nies on plates with cellulose sources, indicating
variance. Where differences were found, the Newman- that most of the colonies on plates with cellulose
Kuels multiple range test was employed. Radioisotope or CM-cellulose were probably using these car-
data and degree of flora reduction effected by antibiot- bon sources. There was no significant difference
ic-starvation treatment were analyzed by the Wilcox- between millipede species in overall numbers of
on rank sums test (46).
Chemicals. All chemicals were purchased from Sig- bacteria counted by direct microscopic counts
ma Chemical Co., with the following exceptions: the or cultivated (Fig. 3A).
agar was from BBL Microbiology Systems; the potato In pooling results for 0. ornatus organisms
dextrose agar was from Difco Laboratories, PD-10 isolated from all media, I found that the HG had
Sephadex columns were from Pharmacia Fine Chemi- the highest number of bacteria per milligram,
cals, Inc., microcrystalline cellulose was from Poly- followed by the MG and then the FG (all signifi-
science, [14C]cellulose and NCS solubilizer were from cantly different at P < 0.001; Fig. 2). In pooling
Amersham-Searle, Cab-o-sil and ethanolamine were data for gut parts and testing for density differ-
from Eastman Kodak Co., and toluene was from
Fisher Scientific Co. ences among media, I found that cellobiose
supported larger numbers of bacteria (P < 0.05)
than did cellulose or CM-cellulose, which
RESULTS ranked equally. Each substrate supported the
Bacteria. All data were standardized as de- growth of the same number of FG bacteria
scribed above so that results could be expressed (Table 1). Cellobiose supported significantly
as the number of bacteria per milligram of gut higher numbers of MG and HG bacteria than did
tissue plus contents. Both millipede species con- cellulose or CM-cellulose (probability:VOL. 44, 1982
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CELLULOSE DIGESTION BY DESERT MILLIPEDES
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8
285
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FIG. 3. Direct microscopic counts of total numbers of bacteria from millipede guts. Values are for each
milligram of gut part tissue plus contents. A, Gounts of normal flora. Results for 10 of each millipede species are
shown. B, Gounts after gut flora reduction by starvation and antibiotics. Results for four of each millipede
species are shown. Each vertical bar represents one standard error.
highest numbers of bacteria in Comanchelus sp. could be expected given its more restricted
grew in cellobiose media, followed by cellulose foraging area, Comanchelus sp. had a far smaller
and then CM-cellulose media (all significant at P diversity of bacteria than did 0. ornatus.
< 0.05). Equal numbers of FG bacteria grew in Enzymes. Results of total enzyme activity
all three substrates (Table 1). The number of MG assays indicated that both millipede species had
bacteria growing in cellobiose media was greater the capacity to degrade cellulose and other plant
than the numbers growing in cellulose and CM- polymers and that the MG was the main site for
cellulose media (P < 0.025; P < 0.001, respec- cellulose and hemicellulose (but not necessarily
tively), whereas HG bacteria that grew in cello- pectin) degradation in both millipede species
biose outnumbered those that grew in CM- (Table 3). The amount of pectin degradation in
cellulose only (P < 0.005). the 0. ornatus HG was surprisingly high. CG-
Direct microscopic counts showed that for cellulase activity was higher in 0. ornatus than
each millipede species (Fig. 3A), there was less in Comanchelus (P < 0.0001); otherwise, total
bacteria in the FG than in the MG and HG, enzyme activity levels for the entire alimentary
which had equal numbers (P < 0.05). tract were the same for both species. Assay
Many types of bacteria were able to establish results for separated gut tissue and contents are
zones of clearing on CM-cellulose (Table 2). expressed as micromoles of equivalent reducing
Many of these were, in addition, able to grow on sugar liberated per microgram of protein in
media with cellulose, although no attempt was enzyme homogenate per unit of time so that the
made to determine whether cellulose was uti- results could be standardized with the least
lized, owing to difficulties in seeing clearing on amount of bias: the millipedes used were of
such thick plates and to the drying of the plates different sizes, and expressing results on a per-
during the long incubation period required. As milligram-of-gut-weight basis (versus per micro-
gram of protein) would have strongly biased
TABLE 1. Differences among bacteria from each results toward the FG, MT, and HT, owing to
gut part in ability to grow on three types of mediaa their low weight, compared with the high weight
of soil present in the MC and HC (Table 4).
Gut p Growth of
0. ornatus on: Growth of
Comanchelus sp. on:
C1-cellulase in 0. ornatus was significantly
higher in MC than in HT (P < 0.001), FG (P <
FG CB, CEL, CMC CEL, CMC, CB 0.05), or MT and HC (P < 0.05) (Table 5). (For
MG CB,CELCMC CB, CEL, CMC clarity of discussion gut contents will be consid-
HG CB, CEL, CMC CB, CEL, CMC ered as a separate gut part.) Activity of Cx-
a Underlined variables are not significantly different cellulase in MC was higher than those in HT,
at P < 0.05. Relative values are given in the text. CB, MT, FG, and HC (probability:286 TAYLOR APPL. ENVIRON. MICROBIOL.
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 Motility
tion' ri tion' stain
3d White 1 1 3 - Streptococci in sheaths
4d 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 - +
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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 than dase and hemicellulase were present in extreme-
HT (probability:VOL . 44, 1982 CELLULOSE DIGESTION BY DESERT MILLIPEDES 287
TABLE 3. Total enzyme activity of each gut part TABLE 4. Enzyme levels found in millipede gut
tissue and contents
Enzyme and gut Total enzyme activity (mean ± SE)'
part 0. ornatus Comanchelus sp. Enzyme and gut Enzyme activity (mean + SE)'
part 0. ornatus Comanchelus, sp.
Cl-cellulase
FG 1.18 ± 0.12 0.61 ± 0.09 C1-cellulase
MG 7.33 ± 0.57 2.87 ± 0.76 FG 24.90 ± 8.87 142.99 + 112.96
HG 1.68 ± 0.26 0.76 ± 0.10 MT 30.49 ± 2.49 27.25 + 5.78
MC 94.75 ± 11.56 78.26 + 28.32
Cx-ceHulase HT 9.73 ± 2.74 61.59 ± 23.74
FG 24.72 ± 3.16 3.98 ± 2.48 HC 34.57 ± 5.65 14.18 ± 8.72
64.97 ± 10.12
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MG 275.01 ± 18.16
HG 38.85 ± 3.84 3.44 ± 2.11 Cx-cellulase
FG 13.54 ± 3.92 8.23 t 8.23
1-Glucosidase MT 10.37 ± 0.67 7.56 t 1.97
FG 16.11 ± 1.81 4.69 ± 0.17 MC 69.14 ± 3.71 16.83 t 1.99
MG 234.22 ± 14.72 271.39 ± 41.85 HT 1.64 ± 1.01 0.00 ± 0.00
HG 28.87 ± 2.26 6.92 ± 1.33 HC 15.84 ± 2.88 0.00 ± 0.00
Hemicellulase 1-Glucosidase
FG 47.89 ± 8.61 16.06 ± 1.87 FG 8.64 ± 1.41 10.73 t 1.63
MG 317.18 ± 81.43 666.60 ± 47.70 MT 2.38 ± 0.29 3.85 t 0.54
HG 35.19 ± 2.72 9.67 ± 0.52 MC 55.23 ± 8.03 88.84 ± 2.29
HT 2.17 ± 0.14 1.79 ± 1.11
Pectinase HC 10.92 ± 0.68 8.36 ± 0.99
FG 31.54 ± 9.59 18.82 ± 8.03
MG 196.22 ± 27.75 66.88 ± 7.25 Hemicellulase
HG 223.48 ± 52.01 42.75 ± 8.41 FG 28.32 ± 5.54 38.60 ± 9.56
MT 6.36 ± 0.61 9.59 ± 0.88
a Values for C1-Cx-cellulases, hemicellulase, and MC 78.56 ± 4.63 225.05 t 10.51
pectinase are expressed as micromoles of reducing HT 3.19 ± 0.84 2.13 ± 2.13
sugar (x103) liberated per animal per minute. Values HC 13.03 ± 2.58 6.64 ± 4.46
for [-glucosidase are expressed as micromoles of
nitrophenol (x103) liberated per animal per minute. Pectinase
FG 17.53 ± 4.48 47.48 ± 20.11
MT 9.37 ± 1.39 9.36 ± 2.54
ments could be made [Table 7].) Impaired MC 46.62 ± 6.34 16.46 ± 1.20
cellulose degradation was shown by a decrease HT 12.42 ± 3.93 12.57 + 3.70
in assimilation of label into hemolymph, a de- HC 85.24 ± 44.34 54.03 ± 16.60
crease in production of 14Co2, and an increase in a Values are expressed as follows: for Cl-cellulase,
the amount of label excreted. Levels of 4CO2 micromoles of maltose (x105) liberated per microgram
decreased over time, indicating that most of the of protein per hour; for Cx-cellulase, hemicellulase,
label was either degraded or passed through the and pectinase, micromoles of maltose (x105) liberated
gut during the course of the experiment. The per microgram of protein per minute; for 13-glucosi-
percentage of total label assimilation in control dase, micromoles of nitrophenol liberated per micro-
gram of protein per minute.
animals ranged from one-fifth to one-third, the
range of assimilation efficiency found by Woo-
ten and Crawford (44) for 0. ornatus (i.e., 22.8 Increase in bacterial growth on media with
± 2.65% for 0. ornatus and 26.58 + 3.33% for cellulose, CM-cellulose, or cellobiose as the
Comanchelus sp.). Values for animals with re- source of carbon and energy over growth on
duced flora were significantly (P < 0.05) lower: control media indicates that the millipedes con-
16.25 ± 2.37% for 0. ornatus and 17.48 ± 3.24% tained bacteria which could produce the Cl- and
for Comanchelus sp. Cx-cellulases and P-glucosidase necessary to
degrade cellulose. The bacteria in the FG were
DISCUSSION probably present as a result of ingestion and are
Both millipede species are detritivores: they therefore not part of the resident flora. During
feed on soil, plant litter, and other items found winter, the number of FG bacteria varies greatly
on the soil surface. In addition, 0. ornatus from individual to individual, whereas the MG
grazes on the bark of bushes in its habitat (45). and HG of all individuals retain large popula-
These feeding patterns enable both species to tions (unpublished data).
acquire varied microbial floras (fungal flora of Numerical differences between bacteria cul-
0. ornatus are discussed in another article tured and bacteria counted by direct microscop-
[35a]). ic counts indicate that many bacteria were not288 TAYLOR APPL. ENVIRON. MICROBIOL.
TABLE 5. Differences in enzyme production in each gut parta
Gut part producing enzyme
Enzyme
0. ornatus Comanchelus sp.
C1-cellulase MC HC MT FG HT MC HT FG MT HC
CG-cellulase MC HC FG MT HT MC MT FG HT HC
P-Glucosidase MC HC FG MT HT MC FG HC MT HT
Pectinase HC MC FG HT MT HC FG MC HT MT
Hemicellulase MC FG HG MT HT MC FG MT HC HT
a
Underlined variables are not significantly different at P < 0.05. Relative values are given in the text.
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cultured by the methods employed. A large ornatus. However, although large numbers of
number of these may be strict anaerobes, which bacteria were found in the Comanchelus HG,
have been found in the digestive tracts of other the low levels of enzymatic activity, particularly
invertebrates (8, 39). In particular, the large, CG-cellulase activity, indicate that this is not an
moist, soil-packed HG of 0. ornatus and the MG important site of cellulose degradation. The abil-
and HG of Comanchelus sp. could quite possi- ity to degrade locust bean gum, which repre-
bly contain numerous anaerobic sites. Strict sents one category of hemicellulose (galacto-
anaerobes were not screened for in the present mannan), demonstrates the capacity to degrade
study. some but not all categories of hemicellulose
Both bacterial and enzyme data indicate that which may be present in millipede food.
cellulose degradation occurred in the HG of 0. It is possible that hemicellulase and P-glucosi-
dase are of microbial origin in both millipede
species; CG-cellulase in the MG of 0. ornatus
TABLE 6. Reduction in gut flora per milligram of may also be of microbial origin. Both species
gut tissue plus contents of antibiotic-treated versus appear to be able to produce some pectinase
control (36-h starvation) millipedes in various which may, in addition, be produced by micro-
ecological groups organisms. Most cellulose and hemicellulose di-
Ecological group and Gut % Reduction Significance gestion occurs in the MG; pectin degradation
millipede species part (P value)' appears to occur primarily in the HG, which is
Cellulose degraders generally considered (at least in the case of
0. ornatus FG 66.51 NS insects) to function only in water and ion absorp-
MG 99.57 0.009 tion and to play no direct part in digestion (43).
HG 99.28 0.006 Recent work, however, indicates that bacterial
fermentation products produced in the HG of
Comanchelus sp. FG 97.18 0.02 cockroaches and the proctodeal dilation in scar-
MG 99.36 0.009 abid larvae can be absorbed from these sites into
HG 89.73 NS the hemolymph (4, 9).
Microorganisms present during incubation of
CM-cellulose degraders the enzyme assay mixture may have contributed
0. ornatus FG 81.45 NS some variance. This was particularly true for C1-
MG 99.86 0.006 cellulase because of the 24- to 27-h incubation.
HG 99.28 0.006 Microscopic examination of the assay mixture
revealed that although toluene inhibited microbi-
Comanchelus sp. FG 99.75 0.006 al growth, there were still large numbers of
MG 97.83 0.02 bacteria present. This may account for the ex-
HG 80.86 0.03 tremely high C1-cellulase activity in the Coman-
chelus FG.
Cellobiose degraders It is difficult to establish beyond doubt wheth-
0. ornatus FG 97.77 NS er these enzymes were of millipede or microbial
MG 99.92 0.006 origin. The fact that the activity in the gut
HG 99.34 0.006 contents was far higher than that in tissue may
point to a microbial origin. However, one cannot
Comanchelus sp. FG 99.64 NS determine whether the low activity found in the
MG 99.94 0.006 tissue is due to millipede production or the
HG 99.08 0.06 incomplete flushing of gut contents from the
aProbability based on Z statistic for Wilcoxon rank tissue. In addition, since a secretory cell usually
sums test. NS, Not significant. produces and secretes its product constantly,VOL. 44, 1982 CELLULOSE DIGESTION BY DESERT MILLIPEDES 289
TABLE 7. Distribution of "4C in C02, hemolymph, fat body, and fecal pellet sampled
14C 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 0.01
Hemolymphb 1.09 ± 0.27 0.51 ± 0.07 0.01 3.51 ± 0.70 1.75 ± 0.19 0.006
Fat bodyc 0.10 ± 0.02 0.09 ± 0.01 NSd 0.52 ± 0.19 0.26 ± 0.04 NS
Fecal pellete 27.36 ± 5.73 52.34 ± 0.75 0.01 12.51 ± 1.76 39.09 ± 4.16 0.006
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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 be The fact that dependence on the Cl- and C,,-
lower in tissue than in contents. How great a cellulases is greater than dependence on ,-
differential would indicate a microbial rather glucosidase may be due to the nature of the food
than a millipede origin of enzymes is not known. ingested. The degree to which detritus is degrad-
For more conclusive determination of the enzy- ed by free-living microorganisms, particularly
matic origins, characteristics of enzymes from fungi, often determines the palatability of the
millipedes and cultured bacteria or levels of food to millipedes (19, 32). It may be, therefore,
activity in gut contents before and after treat- that much of the food that these millipedes
ment with antibiotics could be compared. ingest has already been somewhat degraded by
The importance of the microbial gut flora to free-living flora possessing the necessary Cl-
cellulose degradation is clearly suggested by the and C,-cellulases. This would also be a form of
radioisotope results. Increase in production of millipede dependence on microbially produced
"CO2 could be due to increased respiration by enzymes.
the millipedes or the bacteria. The increase in The association between each species of milli-
label in the hemolymphs of control animals, pedes and its gut flora may be mutualistic.
however, does indicate that the products of Bacteria, through the production of cellulolytic
microbial degradation are assimilated by the enzymes, make available to millipedes other-
millipedes as well as by bacteria. Production of wise unutilizable substrates, which could be of
any of the three cellulases by bacteria could be crucial importance to millipede survival in des-
the important factor in allowing millipedes to erts where production of detritus is low (42). The
assimilate cellulose. The variability in body fat millipedes, in turn, provide for bacteria an envi-
results could have been due to the short duration ronment with regulated moisture, temperature,
of the experiment or to the fact that small, and pH and supply the bacteria with a constant
randomly selected portions were assayed, rather flow of substrates to degrade. This observation
than the total fat body. Comparison of the is supported by the fact that the gut contains far
percentage of assimilated label excreted by con- higher numbers of bacteria per gram than does
trol animals with the percentage excreted by the surrounding soil (unpublished data), indicat-
animals with reduced flora indicates that the ing that the gut provides a habitat in which
already low assimilation efficiency (although it is bacteria can grow and multiply. Further evi-
relatively high for a detritivore [44]) is further dence is provided by the finding that soil and
decreased by a reduction in gut flora. litter bacteria increase in number during passage
Comanchelus sp. and 0. ornatus are distantly through the digestive tract of the millipede spe-
related (different orders), yet both have evolved cies Glomeris marginata (1).
a strong dependence upon microorganisms for In summary, the results indicate that both
digestive enzymes. This may be a universal millipede species contained bacteria capable of
phenomenon among millipedes, as other species utilizing cellulose. Enzymes necessary to de-
are also believed to rely upon microbial enzymes grade cellulose and other plant polymers were
(3, 34). Alternatively, this may be a function of present and may, in some instances, have been
living in a desert ecosystem where nutrients and of microbial origin. Reduction in gut flora result-
moisture are at low levels (18) and energetically ed in decreased assimilation of cellulose by
costly to obtain. Use of acquired enzymes would millipedes, indicating that products of microbial
decrease some of the expense of feeding for the cellulose degradation were utilized by milli-
millipede. pedes.290 TAYLOR APPL. ENVIRON. MICROBIOL.
This type of association has important impli- vol. 1, p. 291-292. Blackwell Scientific Publishers, Lon-
cations for nutrient cycling in deserts. Rates of don.
13. Crawford, C. S. 1972. Water relations in a desert milli-
decomposition are controlled by temperature, pede (Orthoporus ornatus, Girard) (Spirostreptidae).
moisture, and proximity of nutrients necessary Comp. Biochem. Physiol. 42A:521-535.
to sustain decomposer organisms (25). Condi- 14. Cruden, D. L., and A. J. Markovetz. 1979. Carboxy-
tions favorable to the activities of free-living soil methyl cellulose decomposition by intestinal bacteria of
cockroaches. App. Environ. Microbiol. 38:369-372.
microorganisms may be sporadic and of short 15. Elsworth, R. 1969. Treatment of process air for deep
duration. Any association which would enhance culture. Methods Microbiol. 1:123-136.
the creation of a favorable decomposer situation 16. Evans, W. A. L., and E. G. Jones. 1962. Carbohydrases in
could greatly increase the rate of mineralization the alimentary tract of the slug Arion ater L. Comp.
Biochem. Physiol. 5:149-160.
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and the production of humic compounds. Thus, 17. French, J. R. J. 1975. The role of termite hindgut bacteria
the presence of invertebrate-microbe associa- in wood decomposition. Mater. Org. 10:1-13.
tions could be of particular value during seasons 18. Friedman, E. I., and M. Galun. 1974. Desert algae, li-
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be of greater importance in more extreme des- the humicative function of Diplopoda and Isopoda. Acta
erts than in moderate deserts (35b). This funda- Biol. 6:257-271.
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ACKNOWLEDGMENTS gland.
21. Gray, T. R. G., and S. T. Williams. 1971. Soil Micro-
I thank E. Arguello and J. Washburn for technical assist- organisms. Oliver and Boyd, Edinburgh.
ance. C. S. Crawford, M. M. Martin, D. E. Caldwell, J. 22. Hankin, L., and S. L. Anagnostakis. 1977. Solid media
Trujillo, F. W. Taylor, W. W. Whitford, J. A. Wiens, and containing carboxymethyl cellulose to detect Cx-cellulase
G. V. Johnson are gratefully acknowledged for review of the activity of microorganisms. J. Gen. Microbiol. 98:109-
manuscript. 115.
This research was supported by grants from the Graduate 23. Hartenstein, R. R. 1964. Feeding, digestion, glycogen and
Research Allocations Committee and the Graduate School at the environmental conditions of the digestive system of
the University of New Mexico. Oniscus ascellus. J. Insect Physiol. 10:611-621.
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