Relative assimilations of 14C-labelled microbial tissues and 14C-plant fibre ingested with leaf litter by the millipede Glomeris marginata under experimental conditions

Saif &al. Rio&em. Vol. 21. NO. 6, pp. 819-827, 1989 Printed in Great Britain.All rightsreserved

Copyrightc

0038-0717,89 S3.00+ 0.00 1989PergamonPressptc

RELATIVE ASSIMILATIONS OF “C-LABELLED MICROBIAL TISSUES AND “C-PLANT FIBRE INGESTED WITH LEAF LITTER BY THE MILLIPEDE GLOMERIS MARGINA TA UNDER EXPERIMENTAL CONDITIONS D. E. BIGNELL* Department of Zoology, Westfield College, University of London, Hampstead. London NW3 7ST. England (Accepfed 12 lcmuary 1989) Summary-Reference strains of four bacteria (Esc!zerichiuco/i, Erwinia herbicola. Pseudomonas syrhgae and Bacillus subfilir) and the fungus hfucor hiem& were grown in pure liquid cultures containing “C-thymidine, “C-glucose or a “C-amino acid mixture and harvested for feeding to the miiiipedc Glomeris marginam after topical application to oak or beech leaf litter discs. After 24 h the assimilation of ingested microbial tissue by the animals was determined from the partitioning of radiolabel between carcass, haemoiymph, evolved CO,, faecal pellets and the residual gut contents. The ‘*C-amino acid mixture was found to give the greatest absolute level of labelling of microbial cells and assimilation efficiencies with this source of radiolabel were: &&erichia coli, .72.2 f 0.9%; Erwinia herbicolu, 95.9 + 2.3%; Pseudomwtas syingae. 93.2 f 2.2%; Bacillus subtilis, 82.6 f 5.0%; and Mucor hiemalis, 73.5 _+1.5%. The assimilation of t4C-leaf fibres (lab&led from ‘*CO& determined by a similar method, was 18.8 f 3.3% on beech discs and 29.6 * 4.8% on oak. Previous incubation of “C-leaf fibre with various bacteria, including the ceiluia~-~reting C~fu~baga bufchi~o~~, failed to change the calculated ~imiiation by millipedes signifi~ntiy. The results support a proposition that microorganisms colonizing leaf litter are a valuable nut~tional resource to detritivorous animals such as millipedes and are assimilated with high efficiency. Scanning electron microscopy of the food, gut contents and faeces of GIomerb marginala confirmed that colonizers were stripped from leaf litter on ingestion, but also indicated that recolonization of faecal pellets by bacteria occurred during or immediately after transit of the hindgut.

INTRODUCl’lON It has become well established that microbial conditioning of deciduous tree litter is an important factor governing its palatability to macroarthro~d detritivores (e.g. Dunger, 1958; Healey and Swift, 1971; Edwards, 1974; Swift, Heal and Anderson, 1979; Hassall and Rushton, 1984). On the assumption that freshly fatlen litter retains the principal antiherbivore defences of the parent plant, it has been argued that colonization in the initial phase of decomposition physically weakens the leaves by softening (Hassall and Rushton, 1984), accelerates the degradation and loss of polyphenolic feeding inhibitors (Satchel1 and Lowe, 1967; Edwards, 1974) or improves its nutrient status for potential consumers by the accumulation of biovolume and immobili~tion of high concentrations of phosphorus (Anderson, 1973; Neuhauser and Hartenstein, 1978; Swift ef al., 1979; Rushton and Hassall, 1983). Evidence that litter colonizers may also produce exo-enzymes which assist the digestion of detritus by animals is available from the work of Hassall and Jennings (1976) and from general considerations of myco-

‘Present address: School of Biological Sciences, Queen Mary College, University of London, Mile End, London Et 4NS. England.

phagy (Martin, 1979,1984). The importance of determining the factors which are responsible for the selective feeding behaviours of macrofauna is underlined by the demonstration that their presence in soil broadens microbial diversity and indirectly stimulates the mineralization of N and major cations (Anderson and Ineson, 1984, Anderson et al., 1983, 1985; Visser, 1985). The biochemical, biological and structural heterogeneity of litter is such that crude chemical and gravimetric assessments of assimilation, although quite frequently reported, fail to identify precisely the fractions of the resource which are utilized by the macroarthropod (Anderson and Bignell, 1982). It is, however, speculated that microbial tissue is preferentially digested and absorbed, while the lignocellulose components of plant detritus pass through the alimentary canaf relatively unchanged (e.g. Reyes and Tiedje, 1976; Hassall and Rushton, 1984, Ineson and Anderson, 1985). In terrestrial macrodecomposers the evidence to support such ‘a theory of microbial stripping is no more than circumstantial, but the selective utilization of colonizing microorganisms is well ‘documented for many equivalent invertebrates in aquatic systems (e.g. Baker and Bradnam, 1976; Berrie, 1976; Barlocher and Kendrick, 1981; Findlay, Meyer and Smith, 1984.1986). This advance has been obtained by the use of 14C- or ‘H-precursors to mark ingested microbial tissue, or else from the biovolume 819

820

D. E. Btcaizrr

differences following the direct examination of food and faecal materials (Baker and Bradnam, i976; Taylor and Sullivan, 1984). In terrestrial macroarthropod detritivores, passage of leaf litter through the gut is accompanied by an increase in bacterial biomass and a (temporary) loss of fungi (Anderson and Bigneil, 1980; Ineson and r\nderson, 1985; Visser, 1985). Clearly, this could merely represent a conversion of fungal carbon into bacterial tissue without a net benefit to the animal host, particularly since the hindgut and fecal bacteria are different from those characteristic of uningested leaf litter; further resolution is therefore dependent on finding a technique that will indentify any component of microbial ceils entering the midgut which is subsequently incorporated into host tissue following digestion. Labeiling of bacteria and fungi with radioisotopes would appear to provide by far the greatest sensitivity and also, depending on the chemical identity of the initial tracer, the best opportunity to achieve specificity; but owing to the prolonged nature of the microbial conditioning process it is difficult to prepare an experimental meal in which only the microorganisms are labelled, but where their distribution and abundance in the plant detritus corresponds to that which is likely to occur under natural conditions. Further problems arise from the heavy quenching of low-energy p-emitters in liquid scintillation systems by pigments, poiyphenoii~ and other constituents of leaf litter, and from self-absorption by insoluble lignoceilulose. Hence a high specific activity is generally required for labelied microbial tissues to generate assimilation data of acceptable accuracy. Anderson and Bignell (1982) determined the shortterm uptake of non-labile, non-living residues of plant materials in Glomeris marginuta by applying ‘JC-labelicd fibre topically to unlabelled standard discs of well-rotted oak litter and determining the partitioning of label between carcass, haemoiymph, evolved CO?, faeces and residual gut contents after 24 h. Assimilation efficiency (AE) was calculated as the sum of counts in body tissues and CO,, divided by the total of all counts recovered (the latter assumed to represent the radioactivity ingested). As this technique most probably fails to allow the radioisotope to reach its equilibrium body burdens (Southwood, 1978), the conception of AE it embodies is somewhat simplistic but the method does offer the advantage of confining the label to a single component of the diet before ingestion and of accommodating the selective consumption of non-vascular portions of the leaf lamina (at the expense of veins), which is characteristic of millipede feeding. Topical application of specific labeiied materials to leaf litter discs therefore permits an assessment of their assimilation, at least on a comparative basis. I report the USCof the technique to determine the assimilation of ‘-‘C-iabclied bacteria, “C-fungus and ‘dC-piant fibre by G. marginuta under experimental laboratory conditions. Uptake of microbial tissue into the animal after feeding was found to be more than three times that of plant fibre. Prior incubation of ldC-fibre with bacteria, including a ceilulolytic species, did not increase its assimilation significantly on subsequent ingestion by the millipedes.

MATERIALS AND METHODS

Animals G. marginuta (Dipiopoda : Glomeridae) were coiiected from chalk downiand and maintained in iaboratory stock cultures at 1S’C with a mixture of well rotted, moist oak and beech litters. Experimental animals (wherever possible individuals weighing more than 20 mg) were separated from stocks 24 h before use and kept without leaf litter in small containers. Defaecation during this period was found to empty the gut of most existing food material. Determinations of palatability and assimilation were carried out in circular glass pots, 8 cm dia and 5 cm deep, containing about I cm of moistened Plaster of Paris substratum and sealed with an airtight plastic lid. A small uncapped glass vial was pressed into the substratum during preparation, to stand upright in the centre of the chamber after setting. Where appropriate this contained 0.3 ml of I M NaOH as CO, absorbant, inaccessible to the millipedes. Five repiicate pots were used for each determination, each containing five animals and five prepared leaf litter discs. Preliminary experiments showed that a I: I ratio of animals to discs ensured that the amount of unconsumed food after a 24 h exposure was a minimum and that most ingested material completed a transit of the gut and was voided as faecal pellets within this time. Refeetion of faeces was not observed in G. marginata. Labelling of bacteria, fungi and leaf jbre

Static liquid cultures of the bacteria Esherichia coli (NCIB 8113). Errvinia herbicola (East Maliing DC 93), Pseudomonas syringae (East Mailing P 53) and Bucillus subtilis (NCIB 3610) were initiated in loosely capped Sterlin specimen tubes containing 20ml of phosphate-buffered salt solution (KzHPOd; KH,PO, 3 g I-‘; MgSO,+7H,O, 0.1 g I-‘; pH,],SO,, I g I-‘), 0.3 ml of the concentrated, acidified trace mineral solution of Powell and Errington (1963) and a variable quantity, between 0.1 and 0.8 ml, of a concentrated ‘dC-iabelied organic growth substrate. The substrates employed were 20% “C-(U)-glucose, 0.5$ ml-‘; 10% “C-(U)-amino acid mixture (source algal ceil hydroiysates), 0.5 PC ml-‘; IO% “C-(U)-thymidine, 1.0 PC ml-‘. Inoculation was achieved by suspending bacterial cells from a nutrient agar slope in a small quantity of 0.1% peptone water and adding this to the incubation mixture. Incubation temperature was 25°C. Experiments with uniabeiled cultures had established that population growth was exponential between 6 and 22 h after inoculation for E. coli, Er. herbicof and P. syringue and between 20 and 32 h for 3. subtilis. To determine the relative inco~orations of each label and the effects on this of the volume of concentrated organic growth substrate added to the culture medium, 22 h populations of E. colt’ were harvested by centrifugation, washed with several changes of 0.1% peptone water and finally washed in distilled water until no radioactivity could be detected in the supernatant. The final precipitate was suspended in 2.3 ml of distilled water and divided into two aiiquots. 0.3 ml were dissolved in about I.5 ml of Protosol tissue solubilizer and counted by liquid

Assimilation of microbes by millipedes scintillation spectroscopy in a toluene-based cocktail. The remaining 2.0mi were diluted with an equal volume of biuret reagent and protein determined by optical density at 54Onm (Racusen and Johnson, 1961). Casein protein was used as standard. The fungus Mucor hiemalis was cultured in 9cm crystallizing dishes containing 200mi of a static liquid medium based on the formulation of Levi and Cowling (1969). but modified by the addition of 5 ml of a 10% solution of “C-(U)-glucose or “C-(U)amino acid mixture (specific activities 0.5 PC ml-‘) in place of uniabelied glucose and asparagine. The medium was inoculated with one or two pieces of myceiium from a malt extract agar slope and held at 25°C for several days until a hyphai mat had formed across the entire surface of the liquid. 14C-leaf Iibre was prepared from Canna &r&co at Amersham International, as described by Anderson and Bigneli (1982). The crude fibres were added to excess of a 50:50 (w/w) mixture of uniabelled, baiimilled ashiess filter paper and uniabeiied hemiceiiulose (Bigneil, i977), pulverized in a Wig-i-Bug dental amalgamator and after thorough turning in an etectric rolling mill were extracted exhaustively with hot 80% ethanol to remove unincorporated radioactivity. For application to leaf litter discs, about 100 mg of the extracted residue was suspended in 2 ml of cold distilled water. Pre~~ratjon and use of leaf litter discs Weil aged beech and oak titters were collected al Whiteleaf Cross, Princes Risborough, Bucks and Hembury Woods, Buckfastleigh, Devon and stored in large plastic bags at 7°C. Leaves were selected subjectively as most likely to be palatable to G. marginata (lighter colour, greater thickness and relative dampness were the principal criteria) and lightly brushed to remove superficial detritus. Then i cm discs were cut from the lamina, stored temporarily over a substratum of moist plaster of Paris and transferred individually to I cm wells cut in slabs of 2% tap water agar. Bacterial ceils. iabetled and harvested as described above, were suspended in i ml of distilled water. A single drop (about 40~1) of this suspension was placed on the surface of each leaf disc by means of a vertically held Pasteur pipette and spread evenly over the disc surface with a sterile Pt wire loop. Excess suspension drained away rapidly into the surrounding agar, leaving a monolayer coating of bacterial cells [Fig. 2(c)]. Labelled fungal mycelium was harvested by decanting the contents of the crystallizing dish into a 9 cm dia Buchner funnel containing a No. I grade litter paper. Excess 0.1% peptone water was passed through the funnel and the fungal mat carefufly unfolded with a needle to lie evenly over the surface of the filter paper. Additional washing was carried out with dir&ted water until the filtrate was free of detectable radioactivity and the fitter paper, with adherent hyphal mat, was lifted out with forceps and drained on absorbant tissue paper. The I cm discs were cut from the hyphal mat by gentle pressure with a cork borer, taking care that the underlying filter paper was not penetrated. each manoeuvered flat onto a broad spatula with a needle and finally slid from the spatula onto the surface of

82t

a leaf disc placed in an agar well. After the absorption of excess fluid by the agar the hyphal disc and leaf iamina adhered tightly together and were consumed jointly by foraging millipedes without discrimination. Additional discs were coated topically with “C-leaf fibre (Anderson and Bigneii, 1982). The suspensions of labelled bacterial cells and labelled leaf fibre were such that approx. 2mg of solid material were deposited on each disc. While the specific activity of the microbial tissue varied with species and with the iabeiling technique, that of the leaf fibre was constant at approx. 4.5 PC g-‘, some four times greater than the highest specific activity achieved in bacterial cells and twice the highest level obtained in the hyphai mat of M. hiemu~js. Immediately before the introduction of coated leaf discs the millipedes were removed momentarily from their sealed glass pots and 5 discs placed evenly around the circumference of the container on the Plaster of Paris substratum, with the coating uppermost. The animals were placed one by one between the discs, 0.3 ml of 1 M NaOH and a sliver of filter paper as wick were placed in the central upright vial and the resealed pots held in the dark for 24 h at 15°C. In most cases about 90% of the disc material would be consumed during this period and a substantiai proportion voided as faecat pellets. Additional incubation did not lead to the consumption of the remaining leaf litter tissues. Ex~~rnents with coated, uniabeifed leaf litter discs had shown that the addition of a coating of microbial tissue or leaf fibre did not effect the palatability of discs. Thus the dry weights of faeces voided and discs uneaten were not significantly different for any microbial or leaf-fibre coating in five replicates (faeces: FS,Z4= 2.37, P > 0.1; uneaten leaf tissue, F,,, = 1.89. P > 0.1). Prior incubation of “C-teaf j&e In additional experiments oak leaf litter discs received successive topical applications of “C-teaf fibre and an untabeiied suspension of bacterial ceils prepared from static liquid cultures. These were E. coli, Er. herbicolu and P. syringae, grown as described above, and a fourth species, Cytophaga hutchinsonii (NCIB 9469) which was cultured in a liquid cellulose medium based on the formulation of Eggins and Pugh (1962). The double coated discs were maintained in agar wells for a further 24 h of incubation, as described above. Counting of radioactivity in fueces, animal tissues and gut contents Following incubation of the animals with labelled teaf litter, faeces were collected from the culture pots with fine forceps, added to 2 ml of distilled water in a McCartney bottle containing a plastic bead of approx. 5 mm dia. and homogenized for 5 min by vigorous mechanical shaking. 0.1 ml of the resulting suspension was added to 1Oml of a dioxan-based scintillation cocktail and this mixture gelled by the addition of excess Cabosii gelatinizing powder, accompanied by vigorous shaking. To collect haemolymph (and minimize the loss of this fluid during subsequent dissection) each animal was punctured through the ventrolateral cuticle and emerging fluid drawn off in a capillary pipette. Samples of

822

D. E. BIGNELL

haemol~ph obtained in this manner were pooled for each replication and added to 0.5 ml Protosol in a scintillation vial. When the haemolymph was completely dissolved, 10 ml of toluene-based scintillation fluid were added. The entire alimentary canal was then removed from each animal by ventral dissection, added to a pool in 2 ml of distilled water, homogenized and prepared for liquid scintillation counting as described for faeces above. The carcasses remaining were also pooled and extracted for 24 h in 2ml Protosol at room temperature. 0.1 ml aliquots of this solution were added to IO ml of toluene-based scintillation fluid. 0.1 ml of the COz absorbant was counted in IS ml of TppE (toluene and ethanolbased) ~intillation cocktail of Fox fl976). Quench corrections and empirical determination counting eficiency

of

Counting efficiencies were determined by the external standard ratio method, following the construction of quench correction curves for dioxan-based scintillation cocktails (using the synthetic faecal ash of Fox, 1976) and toluene-based cocktails (using chloroform), respectively. Internal standards of “C-glucose added to unlabelled carcass extracts and aqueous suspensions of faeces and gut contents before the scintillant gave the following empirical counting efficiencies (6 replicates): carcass extract, 68.2 + 10.2%; faeces, 49.3 t 6.0%; alimentary canal, 57.3 f 5.2%. In a further experiment E. coli cells were labelled with “C-(U)-glucose, harvested and suspended in distilled water as described above. 0.1 ml aliquots of the suspension were dissolved in Protosol and counted in toluene-based cocktail while additional aliquots were added to 2 ml of an aqueous faecal suspension and counted, after gelling, in the dioxan-based scintillation mixture. For bacterial suspensions of equal specific activity, the mean counts obtained in the presence of homogenized faeces were 78% of those given by cells dissolved in Protosol (3 replications), with counting efficiencies by reference to the quench correction curves of 58.7 and 58.3% respectively. The deficit of 22% of the counts in the dioxan system was attributed to self-absorption by suspended faecal materials. The use of tissue solubilizer and faeces together, in any combination with either scintillation cocktail, gave rise to heavy colour quenching and in consequence an unacceptably low counting efficiency. Calculation of assimilation eficiency

The formula of Anderson and Bignell (1982) was used, giving assimilation efficiency (AE) as dis min-’ in haemolymph + dis min-’ in carcass f dis min-’ as CO, total dis mine* recovered This assumes that the total radioactivity recovered from the animals, their faeces and CO* is equal to the total radioactivity ingested, and also that the calculated ratio is independent of the absolute quantity of radioactivity in the food materials. Scanning electron microscopy

Fragments of beech litter, cut discs, samples of the gut contents and faecal pellets were pre-fixed by

exposure to OsO, vapour in a closed Petri-dish for l-2 h and then transferred to a cold 2.5% solution of glutaraldehyde in 0.1 Mcacodylate buffer for a further 24 h. Following dehydration in acetone and critical point drying, specimens were coated with gold and examined in a Cambridge Model 600 scanning electron microscope. RESULTS Choices of radiolabel andgrowth conditions for experimental microorganisms

In view of the importance of achieving a high level of labelhng in ingested microbial cells to offset inaccuracies introdu~d by quenching, experiments were conducted with E. coli to compare the relative effectiveness of 14C-amino acids and “C-thymidine as precursors. On the assumption that assimilation by the millipedes would be independent of the quantity of microbial tissue ingested under the conditions of the experiment, a satisfactory label was judged to be one which gave the greatest absolute incorporation rather than just the highest specific activity. Figure l(a) shows that when labelled glucose, amino acid mixture and thymidine were added singly to culture solutions as the sole organic substrate, this criterion was met by the amino acid mixture which increased the absolute radioactivity in harvested cells in proportion to its ~on~ntration in the solution. Thymidine incorporation was poor, both in absolute terms and as a specific activity. Figure l(b) shows that incorporation of “C-amino acid mixture was enhanced when 0.5% of unlabelled glucose was also present in the culture medium. In the reverse combination the incorporation of “C-glucose was reduced in the presence of unlabelled amino acid mixture and specific activity was also less than that achieved when labelled glucose alone was employed. For these reasons the “C-amino acid mixture was chosen as the most satisfactory precursor for the routine labelling of microbial tissues, but used in the presence of unla~lled glucose. To examine the possibility that each labelled precursor would be incorporated differentially into fractions of the microbial tissue and might therefore produce a different value for assimilation by the millipede, cultures of E. coli were labelled separately with each of the “C-substrates available and harvested for feeding to millipedes with leaf-letter discs. To compensate for the relatively poor incorporation of ‘JC-thymidine, this precursor was supplied to the growing cells at a specific activity of 4fiC ml-‘. Assimilation tissues

oj

‘%-ieaf

fibre

and

“CC-microbiai

Table I summa~zes the partitioning of radioactivity in millipedes and the calculated assimilation efficiencies, following the consumption of beech leaflitter discs coated with “C-leaf fibre and various ‘*C-labeiled microbial tissues. The main feature of the data is the wide disparity between the assimilation of the leaf libre and that of any microbial tissues, however labelled. In no case is the calculated assimilation efficiency of a microbial tissue less than three times that of the leaf-fibre, with most comparisons involving bacteria giving a ratio of four times or

Assimilation

ol microbes

by millipedes

VoWme of labellad growth substrate added I ml 1 Fig. I. Labelling characteristicsof E. c~li in a static liquid culture at 27°C. The total radioisotope incorporated (continuous lines) and spcific activities (broken lines) achieved after 24 h incubation are shown for three organic carbon substrates (‘4C-glucosc. +; “C-amino acid mixture. A; “C-thymidine. 1) and five concentrations (as volume concentrated growth substrate added to 20.3 ml of buffered salt solution with trace clmcnts), In (a) each growth substrate is ad&d singly to separate cultures of the bacterium. Irr (b) the indOrp(ation of “C-glucoseand “C-zttiirtO arid mixtrrre is shown in the pteance of 0.1 ml 10% casein hydrolysate solution and 0.1 ml 20% unlabelled glucose solution. respectively. Labelled growth substrates were: 20% glucose (“C-glucose. O.SpC ml-‘); 10% casein hydrolysate

(“C-amino

acid mixture, 0.5 pC ml-‘):

IO% thymidine (“C-thymidine

more. Alrhough some caution is necessary in inttrpre-

tation. owing to the absence of information about the diskibution of radiolabels within each material. the data appear to establish a primefacie case for the preferential digestion of microbial tissue entering the alimentary canal in comparison with olher components of the diet. Without an assessment of the total C requirements of the animal it is not possible to say whether microbial tissue represents the primary nutritional resource available to a macroarthropod detritivore, but a facility to utilize the material efficiently, when present in the food, is clearly established. Further scrutiny of the results shows that the variance of dis min- ’ by the replicate groups of millipedes was generally much greater than that of the final calculated assimilation efficiencies, even

Table

I. Distribution

Dir mine’ in

Led fibrc Erc~rickia rdi E cd &. coii B. herbide P. 1,.GigIN 8. x&M&T M. Irlem&

M. liiemiliir

where the same organism (E. COU) is labelltd with different substrates or where the same “C-precursor is employed for different organisms. Thus while the total dis min-’ consumed differ, presumably a reflection of variations in microbial growth between cultures and in the palatability of leaf-litter discs, the proportion of label incorporated into animal tissues is more constant. This supports the proposition that assimilation is independent of microbial biovolume within broad limits. The proportion of ingested label appearing in respired CQ was considerably higher when “C-fungal tissue was consumed, although the overall assimilation efficiency of the fungal tissue was lower than that for bacteria, excepting E. cd labelled with ‘dC-glucose and “C-amino acids. Since the biovolume of the mycelial mat applied topically to the leaf-litter discs would exceed that of the equivalent

or radioactivity fallowing ingestion of bcceh leaf liuer discs singly coated with “C-leaf fibre or “C-labelled microbial tiss~. Analysts took tdaa 24 h after Dresentalion of the discs to lhc mum&.

“C-substrate Locationof label

I pC ml-‘).

for labcUing CD, Glucose Amino acid mixture Tbylnidjnc Amino acid mixture Amino xid mixture Amino a&l mixlurc Glluosc Amino acid mixture

hacmolymph and tissues

IX369f

4956 6554 + 1473 3001 + 477 7458 f 3187 27.004 f 15.832 IS.645 l 4337

I i .2f 2f 2505 11,454* 9% 22.354 f 4810

Dir min-’

in gut

To1al

Dis min -’

and fact-%

as co,

7W45 * 24.875 23 54 k 249 1221 * I98 1084 f 542 902 5 141 1113*373 2389 2 913

361 + 214 683 f 240 155*95 to4*9s 325 ? 270 264*64 131 f I33

7849f 1438

5928 f 1438

12.206 2 2673

1I .245 f

2533

dir mix’ inncsicd 96,674 k 9594 f 4377 f 864s f 28.23 I + t 7.022 * 13,732 f

27.640 Id72 583 3144 15.525 4139

2901 25.24Qk 2433

46.005 f 97 I I

Assimilation Significance cflicicncy group f < 0.06 (%) 34.9 2

18.3 2 3.3 4.7 72.2 * 0,9 86.2 * 9.0 95.9 5 2.3 93.2 * 2.2 E2.6 2 5.0

a, b a, b a. b. c. d c c. e d

69.2 i 7.1

b

73.5 i 1.5

b

Mean + I SD of the radiotracer is sbom. correcti for quenching. self-absorption and background (dis min-‘t. of = 5 (dc&rminarionsL 5 animals/determination. The Real column shows a non-panmtiric compar&n of the mc;rnr af asrimihcion efficiency by STP [Sokal and Rohlr. 1969). MCW’S with the IBIIK Icttcr arc not significantly diffcrcnt (f’ c 0.051.

824

D. E. B~GNELL Table 2. Uptake of radioactivity from ingested “C-leaf ftbre following 24 h incubation with bacterial suspensions. Analyses took t&cc 24 h after urescntation of oak kaf-litter discs to the animals. Total dis mitt-’ ingested

Material and treatment

“C-leaf tibre only “C-leaf “C-leaf “C-leaf “C-leaf “C-leaf

fibre fibrc ftbre ftbre tibre

incubated incubated incubated incubated incubated

with with with with with

bacteriological Ringers’ CyroplMsa hurchiamii &. coli &r. herbicola P. winaae

101,826 ?I 26.473 89.374 i: 4989 124.388 t: 19,904 88,468 t 9S88 70.309 + 17,962 73.486 rt 13.964

Dis mitt-’ in CO?, hacmolymph and tissues 29,370 24,922 36,664 24.408 24,236 23.254

Assimiktion efticiency (%I

+- 5426 k 3300 k 16.488 f: 7641 + 13. I69 f 7%5

Mean + I SD of radiotracer corrected for quenching, self-absorption and background (dis mitt-‘). (determinations). 5 animals/determination.

monolayer of bacterial ceils (contending to the dominance of fungi in nature), more nutrient of fungai origin may be available to the metabolic pools of the millipede, proportionately, than substances released by the lysis of bacterial cells. Thus the conditions of the experiment may favour the use of more fungal material for respiration than bacterial derivatives, but these unequal contributions would also be expected in nature since fungi are the more abundant early colonizers of leaf litter (Swift er al., 1979). The proportion of total CO* generated by the permanent hindgut flora of the millipede is unknown. Some signifi~nt differences were observed between the respective assimilation efficiencies of the four species of bacteria examined. Most notable were the very high efficiencies achieved for Er. herbicola and P. syringae, two species which would be expected to colonize natural leaf litters, exceeding the assimilation of any other labelied material excepting the cells of E. coli prepared with ‘“C-thymidine (where the variance was rather large). No significant differences in assimilation efficiency could be demonstrated for E. coli labelied with “C-glucose, “Camino acids or “C-thymidine, or for M. hiemufis iabelled with “C-glucose or 14C-amino acids. This suggests that extensive lysis of both bacterial and fungal tissues occurs after ingestion by the millipede. Prior incubution bacteria

of

‘%-leaf

jibre

with

unlubelled

To examine the possibility that extracellular enzymes secreted by microbial colonizers of leaf litter enhanced the digestibility of the structural polysaccharides in the millipede gut, labeiled leaf-fibre was applied to oak litter discs and incubated with cultures of Cytophuga sp., E co/i, Er. herbicola and P. syringae for 24 h before presentation to the animals. No significant differences in assimilation of the fibre were observed after these treatments (F,, = 0.269;

29.6 k 28.6 F 28.2 + 27.1 f 32.2 + 30.8 +

4.8 2.7 8.1 5.6 11.1 5.7

n= 5

P > 0.05). but the uptake of radiolabel into animal tissue, with or without added bacteria, was greater when the “C-fibre was presented on oak discs than beech (Table 2). The present figure for the assimilation efficiency of labelled leaf fibre presented on oak was 29.6 rf: 4.8% (without added bacteria, but with an incubation of 24 h imposed before consumption by the animals) and is comparable with the value of 35.8 rt 3.4% given by Anderson and Bignell (1982) for leaf fibre on oak discs. Scanning eiectron microscopy

A brief scanning survey of uningested food materials, gut contents and faecal pellets was carried out to examine the nature and dist~bution of microbes on the surfaces of natural and topically labelled leaf litters and to verify the inference of the assimilation data that extensive iysis of fungal and bacterial tissue occurs in the mesenteron [Fig. 2, (a-f)]. The survey confirmed that fungal mycelium is dominant in naturally conditioned beech litter, although the hyphae are largely confined to the leaf surface and do not seem to penetrate into the internal tissues [Fig. 2, (a-c)]. Short rods or coccoid forms of bacteria may be found singly or in small groups both on the leaf surface and as internal colonizers of the palisade tissues [Fig. 2 (b, d)]. Topical application of a bacterial suspension was generally effective in producing a monolayer of labelled cells on the leaf surface, although in greater density than the natural bacterial flora [Fig. 2(c)]. Examination of the gut contents after passage through the mesenteron (i.e. in the extreme anterior hindgut) showed that microbial cells were largely absent from the fragments of plant tissue, suggesting that they had been digested or physically removed from the leaf surfaces and internal spaces [Fig. 2(e)]. However, during transit of the remaining portion of the hindgut there is an extensive recolonization by rods which achieve high density in the faecal pellets [Fig. Z(f)].

(Fig. 2 opposite) Fig. 2. Scanning electron microscope survey of the food, gut contents and faecal pellets of G. marginafa. (a) Low power view of the surface of a well-rotted beech leaf showing a well-established superficial fungal mycelium x 115. (b) At a higher magnification there is a sprinkling of bacterial alls in small groups.

Arrow shows a portion of fungal hypha collapsed by the preparative technique x 2060. (c) A monolayer of E. coli cells on a cut leaf-litter disc, prepared by topical application of a drop of cell suspension x 187.5. (d) The colonization of palisade cells by coccoid bacteria in an intact beech litter leaf mechanically fractured after critical-point drying x 2250. (e) Fragments of ingested beech leaf litter from the anterior hindgut. showing the absence of adherent organisms x 2060. (f) Similar fragments from a faecal pellet showing an extensive proliferation of rod form bacteria x 1950.

Assimilation of microbes by millipedes

825

826

D. E. BIGXLL DISCUSSION

The

results are consistent with the view that microbial colonizers of decomposing leaf litter are digested in the millipede gut and make a contribution to the animal’s nutrition, but it remains unclear whether this contribution is restricted to the release and uptake of immobilized nutrients (chiefly N and P) or provides, in addition, a signi~cant source of labile organic C. Although the calculated assimilation efficiency of YXeaf fibre (in the range l&32%) is low, the abundance of this and similar materials in conditioned litter may be sufficient for the animal to extract an adequate amount of carbohydrate by weak digestive processing, possibly confined to the degradation of hemicellulosic fractions (Reyes and Tiedje, 1976; Anderson and Bignell, 1982). Estimates of microbial biovolume (based on direct observation) in decomposing leaf litter equivalent to that used in the present work are available from the work of R. 0. Okelo (1987, unpublished observations). Biovolumes in well-rotted (8-month-old) oak litter ranged to IS-2.0mm’ g-’ dry litter for bacteria, ~Ornrn~ g-i for fungal hyphae and spores (combined) and SO-1000 mm3 g-i for protozoa and nematodes (also combined). The last estimates, although very variable, are surprisingly high and may indicate that the eukaryotic microfauna colonizing leaf litter under some circumstances represents a potential nutritional resource for macroarthropods greater than that of the bacteria and fungi combined. The evidence of Anderson and Ineson (1983), that the flux of organic N into ledchates of litter that accompanies macroarthropod feeding consists only in small part of nitrogenous material derived from animal excretion, suggests that microbe-stripping in the millipede gut does not result in even the temporary sequestration of the bulk of the immobilized N pool. There appears, therefore, to be a distinction between the fates of microbial C and N once conditioned litter is ingested. This makes it unlikely that intestinal microorganisms compete directly with the host for the more readily digestible substrates (Reyes and Tiedjc, 1976), but would support the view that the proliferation of indigenous organisms in the hindgut and faeces is made possible in part by the release of nutrients in the mesenteron. The very high assimilation efficiencies for ingested microbial tissues obtained in this study and the microscopical demonstration that leaf litter fragments leaving the mesenteron are apparently devoid of adherent organisms is thus consistent with the observation of Ineson and Anderson (1985) that the hindgut and faecal flora of G, rrrnrginarn is made up from bacteria which do not occur in uningested litter. While a complete transit of the gut causes an overall increase in the numbers of bacteria associated with leaf litter (Anderson and Bignell, 1980). there is little difference in gross chemical composition between food and faeces (Van der Drift, 1950; Gere, 1956; Dunger, 1958; Edwards. 1974). The use of over-simplified techniques of biolabelling to determine nutrient fluxes in decomposcr chains has been widely criticized (Sorokin, 1968; Conover and Francis, 1973; Hollisbaugh et al., 1980; Taylor and Sullivan. 1984). In particular. there is concern that readily metabolized “C-substrates (such

as “C-glucose or ‘$C-amino acids) distribute the label too widely in biochemical pathways, with subsequent losses or direct transfer to other members of the microbial community in the course of normal exudation processes. Recycling of the label was unlikely to be of significance in my experiments where a single cultured ~croorganism was applied topically to the leaf Iitter disc and in excess of natural ~pulation density, but losses by exudation would call into question the extent to which the calculated assimilation efficiency represented a proportionate lysis of ingested cells. However, confirmation that significant lysis of microbial cells does indeed occur in the mesenteron is provided by the electron microscopy and by the observation that when E. coli was labelled with iJC-thymidine, the calculated assimilation efficiency was not significantly different from that achieved when “‘C-glucose or “C-amino acids were employed as substrate. Thus it was not considered necessary to use a hot pulse-cold chase technique of microbial labelling, such as r~ommended by Taylor and Sullivan (1984), to enhance the retention of the “C-marker. Some advantages of the topical application of harvested, labelled cells to leaf-litter discs are that existing ‘*C-exudates are removed by washing and subsequent draining of the discs in agar wells and that the microbial tissues enter the animal’s gut with the same physical orientation as labelled plant fibres and are thus comparably accessible to digestive enzymes. While assimilation may be overestimated in comparison with completely natural food material, the relative differences in uptake between the bacteriat and fungal species and between all the microorganisms and the plant fibre retain some validity. Since fungal biovolume generally exceeds that of bacteria in conditioned leaf litter (Swift rt nl., 1979; R. 0. Okelo, unpublished observations), it is unfortunate that assimilation data are available for just a single species, M. hietnalis. Other species of fungi were examined as candidates for biolabelling (Coriolus uersicolor, Trichodermu riride and Pythium rammaniumcm) but were rejected either because of pellicle formation in culture or because the harvested mycelium was sufficiently thick to enable the animals to graze it from the surface of the leaf litter disc. leaving the latter uningested, However, the demonstration that a number of bacteria are assimilated is useful in emphasizing the potential importance of coprophagy, a habit that is known to occur in many millipedes and woodlice, although it was not observed in G. murginuta under the conditions of these experiments. thank Drs J. M. Anderson and P. Ineson for helpful discussions. Isolates of litter fungi were kindly furnished by Dr M. J. Swift. Acknowk~~~~menrs-I

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