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Interrelationships between yeasts and soil diplopods
Soil Bid. Biochem. Vol. 25, No. 8, pp. I1 1%I 126, 1993 Printed in Great Britain. All rights reserved
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INTERRELATIONSHIPS BETWEEN SOIL DIPLOPODS
0038-07 I7/93 $6.00 + 0.00 1993 Pergamon Press Ltd
YEASTS AND
B. A. BYZOV, Vu NGUYENTHANH and I. P. BABJEVA Department
of Soil Biology, Faculty of Soil Science, Lomonosov Moscow 119899, Russia
State University, Vorobjevy gory,
(Accepted 20 January 1993) Summary-Some functional interrelationships have been established between yeasts and the soil diplopods Pachyiu~uspaOipesC. L. Koch and GIomeris connexa C. L. Koch. Yeasts can serve as a food for diplopods when these animals are feeding on leaf litter. The digestion of yeast cells takes place mainly in the midgut as a result of enzymatic activity of the gut fluid. The midgut is a well-protected zone in which enzymatic activity prevents exogenic infection and thereby provides a steady-state environment for the yeast community. The hindgut is a region where specific yeasts can utilize readily-available carbon sources from leaf litter and hydrolyze uric acid-the end-product of nitrogen metabolism of the host animals. A model for yeast distribution in the gut of diplopods is described.
INTRODUCTION The list of microorganisms shown to be associated with soil invertebrates is still on the increase. Gram-
negative, Gram-positive bacteria and actinomycetes belonging to many genera have been isolated from the gut of insect larvae (Sxabb, 1974; Oertel and Schaller, 1984), woodlice (Griffiths and Wood, 1985; Ineson and Anderson, 1985; Ullrich et al., 1991), earthworms (Contreras, 1980; Ravasz et al., 1986; Shaw and Pawluk, 1986; Hossein et al., 1988) and diplopods (Jager et al., 1983; Marialigeti er al., 1985; Chu et al., 1987). Also the dominant microorganisms of the gut microbial community have been determined for many animals and often appear to be very specific and entirely different from that of the food and faeces of the animals. For example, it has been demonstrated that the gut of diplopods is inhabited mainly by facultative anaerobic Gram-negative bacteria of the families Enterobacteriaceae and Vibrionaceue (Byzov et al., 1988) as well as coryneform bacteria of the genus Promicromonospora (Chu et al., 1987; Byzov et al., 1989). The yeast community of the gut of soil invertebrates has been investigated by (Byzov er al., 1993) who demonstrated that there are specific ascomycete yeasts: Debaryomyces hansenii, Torulaspora delbrueckii and Zygowilliopsis californica (so called ‘DTZ’-group) in the gut of the diplopod Pachyiulus jlavipes; the composition and structure of the community was constant and remained unaltered under different feeding and rearing regimes. The actual functional role of a gut microbiota as well as types of zoomicrobial interactions are still poorly understood. Many of the difficulties in interpretation of results arise when only the dilution plate technique is used for population estimates.
We have tried to solve this problem by combining dilution plating with direct observation using light and scanning electron microscopes. In addition, we have modified the commonly-used scheme presuming short-term and static analysis of gut microbial community. Our approach was to change feeding regimes by amending food with pure cultures of yeasts and to estimate the changes in their populations as well as in the microbiota of the gut. MATERIALS
AND METHODS
Diplopodr and yeasts
Four species of soil diplopods were used: Pachyiulusflavipes C. L. Koch, were collected from leaf litter in fruit orchards, Crimea, Ukrainia; Glomeris connexa C. L. Koch, Leptoiulus polonicus Jawlowski and Megaphilium projectum kochi Verhoeff were collected from leaf litter of broadleaf forest, Mukachevo region, Ukrainia. The diplopods were reared in desiccators, in groups of about 50 animals, on fresh natural food substrates at 18-2O”C, before use in our experiments. The following yeast cultures were used: Debaryomyces hansenii, Torulaspora delbrueckii, Zygow caltfornica, illiopsis Pichia membranaefaciens, Trichosporon pullulans, Trichosporon cutaneum, Geothrichum candidum (isolated from the gut of the diplopod P.flavipes); Candida guilliermondii (isolated from the guts of the dead diplopod Glomeris connexa); Cryptococcus albidus and Candida scottii (isolated from leaf litter); Hyphopichia burtonii (isolated from cow manure); Succharomyces cerevisiae, Schizosaccharomyces starkeyi-hendricii, Rhodotorula rubru and Kloekera upis (from the yeast collection of
the Department University).
1119
of Soil Biology, Lomonosov
State
B. A. BYZOV et al.
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Feeding experiments
Five diplopods P. jlavipes were reared for 21 days on 1 g lots of sterile leaf litter or crystalline cellulose with natural moisture and amended separately with the yeasts D. hansenii, Candida guilliermondii or Candida scottii at initial concentrations of 1.8 x lo’, 7 x lo’, and 9.5 x lO’g-’ air-dry substratum, respectively, for 21 days. The initial inocula of yeasts in food estimated microscopically, was provided in a water suspension of cells serially washed three times. The substratum was renewed weekly to avoid contamination and then the number of yeasts was estimated in food, midgut, hindgut and freshly collected faecal pellets by the dilution plate method as described by Byzov et al. (1993) on 7, 14 and 21 days after the start of feeding. Three replicates of each diet were used. To elucidate the participation of the yeast Candida guilliermondii in any pathogenic processes the following experiment was carried out. Samples of five diplopods (G. connexa) were fed sterile leaf litter enriched by yeasts in increasing amounts either 104 5 x 106, 10’ or lo8 cells g-’ After 7 days feeding, the gut yeasts were estimated using dilution plating and scanning electron microscopy. Sterile leaf litter was used as a control. Three replicates of each yeast concentration were used. Estimation of yeast-lysing bacteria
To estimate the composition and number of yeastlysing microorganisms in the gut and faecal pellets of diplopods, a medium with living yeast cells was used (30 g I-’ (NH4)rS04, 1 g l-’ KH2P04, 1 g 1-l MgSO,, Difco agar). After autoclaving, the medium was cooled to 4045°C and enriched with 5 x lo6 cells ml-’ of D. hansenii, which had been washed three times in distilled water. The yeast cells were the only carbon source in the medium. Estimation of yeast-lysing activity of midgut fluid
To quantify the fate of yeast, cells passing through the gut of diplopods, the midguts of ten animals were carefully removed aseptically to retain the digestive fluid inside the gut tube. The digestive fluid was collected and divided into lOOm1 portions. Pure cultures of D. hansenii, Torulaspora delbrueckii, Trichosporon pullulans, Candida scottii, Geothrichum candidum and Cryptococcus albidus were added to the
digestive fluid at a concentration
of 10’ cells ml-’ The
fluid inoculated by yeasts was then incubated at 37°C. Cell morphology was examined by light microscopy every 2 h for 10 h. Yeasts suspended in distilled water were used as a control and the experiment was replicated twice. The ability of yeasts to grow on uric acid
To estimate the ability of all the yeast strains isolated from leaf litter, guts of diplopods and their faecal pellets to grow on uric acid, the following media were used: (1) log I-’ uric acid (the only source of carbon), 6.7 gl-’ Yeast Nitrogen Base Difco, 20 g I-’ Difco agar; (2) 1 g I-’ uric acid (only source of nitrogen), 11.3 g 1-l Yeast Carbon Base Difco, 2Ogl-’ Difco agar; (3) IOgl-’ uric acid (source of both carbon and nitrogen), 20 g I-’ Difco agar. The following nutrients were added to the all media: 0.2 g 1-l K,HPG,, 0.1 g I-’ KH2PG,, 0.2 g I-’ MgS0,7H,O, 0.2gl-* NaCl, 0.1 gl-’ K,SO,, 50ml of yeast water as a source of vitamins. Uric acid was autoclaved separately. Glucose and (NH&SO, were used as controls in first and second experiments respectively. The ability of yeasts to utilize uric acid was estimated by counting numbers of typical colonies and clearing zones around inocula. Scanning electron microscopy
The yeast community in the gut of diplopods was studied using scanning electron microscopy JSM-2 (‘Jeol’). The inner surfaces of midgut epithelium and hindgut cuticle were examined. The samples for electron microscopy were prepared as described by Guzev et al. (1986). RESULTS
Yeasts as a food for diplopods
Diplopods fed on sterile cellulose amended with the yeasts D. hansenii, Candida scottii and Candida guilliermondii (Table 1). Over 21 days feeding, there was a considerable decrease in the number of yeasts in faecal pellets. However, Candida scottii was eliminated to a greater extent (about >99%) than D. hansenii and Candida guilliermondii (about 90 and 92%, respectively). After 21 days of continuous feeding, yeast cells provided with cellulose were found in the midgut (17 x lo4 cfu g-l) as well as in the hindgut (43 x lo4 cfug-‘) and faecal pellets (160 x 104 cfug-‘)
Table 1. Number of colony forming units of yeasts in the food and faecal pellets of diplopods. Pachiulus &oipes, after 7, 14 and 21 days continuous feeding on sterile crystalline cellulose amended with Candida guilliermondii, Candida scorlii or Debwyomyces hansenii. Standard error,
Yeast species on crystalline cellulose
Faecal pellets Food
Candrda
guilliermondii
82k
10
Candida
scotfii
93k
16
Debarvomvces
hansenii
15+4
7 days
14 days
21 days
42 f
45+
6.6 f
29
II
0.5 *0.1
3.4 f 0.5
0.2 + 0.1
9.0 f 2.3
1.3
0.1 1.6kO.l
0.5 f
Interrelationships
between yeasts and soil diplopods
Fig. 1. Scanning electron micrographs of destroyed asci with one ascospore inside (arrows) of yeasts, supposedly belonging to Debaryomyces genus in the midgut (A, B); yeast cells colonizing the gut wall surface in the midgut (C), and the cuticula in the hindgut (D) of diplopods, Pachyiulus Jauipes, feeding on natural leaf litter. Bars indicate for (A) 6pm; (B) 2nm ; (C) and (D) 20pm.
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3. A. BYZOVet al.
Fig. 2. Scanning electron micrographs of native vegetative yeast cells (A) and yeast cells at different stages of the digestion of cell walls (arrows) (B)-(D) in the midgut of diplopods, Pachyiulusflavips, feeding on natural leaf litter. Bars indicate 6 pm.
Interrelationships between yeasts and soil diplopods
1123
Table 2. Number of colony forming units of yeasts in the midgut, hindgut and faccal pellets of diplopods, Pachyiulw@zt@es, after 21 days continuous feeding on sterile crystalline ceIlulose amended with Candid0 guilliermondii, Candida scottii or Deharyomyces hattsenii. Standard error, a=3 Number of yeasts, cfu x IO g-’ dry wt Yeast species on crystalline allulosc
Food
Midgut
Candid0 guilliermondii(a)
8200 + IOOOa 3.4 f IDTZ*
Candida scottii(b) Debarvomvces hansenii(cl
9600~16OOb
0
150000t400c
17 + ISDTZ
Hindgut
Faecal pellets
9.1 + 0.6DTZ 4.8 f I .8a 5.4 + 1.8DTZ 43 + IODTZ
660 f 13oa 50 f IOb 16Of10c
lDTZ-for explanation see text. tThe same letters indicate the same yeast cultures.
only when diplopods had been fed on D. hansenii. Candida gzdlliermondii could be recognized only in the hindgut and Candida scottii only in the excrements of diplopods. On the other hand yeasts of the DTZ-group (0. hansenii, Torulaspora delbrueckii, 2. californica) constantly occurred at high densities in the hindgut (1-3 x lo4 cfu per gut, i.e. 2@-50 times higher than the normal level), but fewer than in excrements (Table 2). To elucidate the digestion mechanisms, yeastlysing bacteria were estimated in the gut and excrements of diplopods in the same experiment. Living yeast cells were added to the medium to select these gut bacteria. Yeast-lysing microorganisms (mainly actinomycetes) were isolated only from the hindgut and faecal pellets (Table 3). However, the number of actinomycetes was even less when diplopods fed on cellulose amended with yeasts compared with yeast-free food and did not exceed lo3 cfu per gut. With light microscopy it was shown that after 10 h incubation in gut fluid, about 60% of asci of D. hansenii and Torulaspora delbrueckii were destroyed and ascospores were released. This compares with only 2% in the control. The results obtained by the use of scanning electron microscopy corresponded well with results from light microscopy. The destroyed asci, supposedly belonging to Debaryomyces sp., were found in the midgut of diplopods [Fig. 1 (A), (B)]. In Fig. 2 native vegetative yeast cells [Fig. 2 (A)] and different digestion stages of these cells [Fig. 2 (B)-(D)] that occurred in the midgut of diplopods are shown. In about 70% of Candida scottii cells, the internal contents were destroyed with no vacuoles or lipid granules visible. Also, no daughter bud cells were
observed. There was practically no change observed in the control (incubation in water). However, the gut fluid did not affect the morphology of Cryptococc~ albidus and Trichosporon pulhdans. Only in the case of Geothrichum candidzun was growth stimulation of cells noted. Mutualistic
interaction between yeasts and diplopodr
Practically all of the gut isolates utilized uric acid as their only source of nitrogen as well as many litter inhabitants; only two yeast isolates could utilize uric acid as a source of nitrogen as well as a carbon (Table 4). Some yeast strains produced lytic zones whereas other did not. Yeasts as pathogenic agents
Considerable increases in yeast density in the guts of the diplopods Glomeris connexa, Leptoizdur polonicus and Megaphilium projectzun were found when the animals had been fed on natural leaf litter that had not been renewed for 3 months. It has been demonstrated with scanning electron microscopy that yeasts mainly colonize the hindgut of freshly-collected diplopods Glomeris connexa [Fig. 1 (D)], their number being about lo3 Cells mm-’ (IO’ Cells per gut). Only a few cells were found in the midgut [Fig. 1 (C)l. Three months later the number of yeasts in the gut appeared to be ca 100 times higher than at the start of the experiments. Yeasts colonized both the midgut and hindgut of diplopods. At this time the majority of animals were dead. Candida guilliermondii dominated in the yeast community of the gut. It is possible that Candida guilliermondii might have acted as a pathogen. When diplopods originally fed on sterile crystalline cellulose enriched by Candida guilliermondii were
Table 3. Number of colony forming units of yeast-lysing bacteria in the food, midgut, hindgut and faecal pellets of diplopods, Pachyitdw&@es, after 21 days continuous feeding on sterile crystalline ccllulosc, sterile leaf litter or sterile crystalline cellulose amended with yeasts, Candida gtdlliermondii, Candido scottii and Deharyomyces hattsenii. Standard error, n = 3 Number of yeast-lysing bacteria, cfu x Io-‘g-’ dry wt Food
Midgut
Sterile leaf litter
0
0
54’*8
Crystalline cellulose (CC) Car&& guilIiermondii on CC Candiak scottii on CC Deharyomyces hansenii on CC
0 0 0 0
0 0 0 0
41 f 29 f 49 f 45*
Type of food substrates
ND = not determined.
Hindgut 4 3 26 10
Faecal pellets 320 k 20 96+ I5 II+11
ND ND
B. A. BYZOV et al.
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Table 4. The ability of yea& strains to grow on media with uric acid as the SOIFC or N source
c
source
Spwies, strains
1
2
I
2
I
2
Debaryomyces hansenii G-43, G-45, G-46, G-47, G-48, G-56 Z. cali/ornica G-68, G-83, G-87(2) Z. californica G-126 Z. calfirnica G-130 Torulaspora delbrueckii G- I30 Torulaspora delbrueckii G-55 Torulaspora delbrueckii G-72, G-80 Pichia membranaefacietw G-74, G-125, G-131, G-104 Candida guilliermondii G-24, G-14 Kloekera apis G-106 K. apis G-137 K. apis G-61 Trichosporon cutaneurn G-l 13 Trichosporon cutaneum G-5 Trichosporon cu~aneum G-79 Trichosporon pdlulans G-90, G-l 15 Trichosporon pullulan~ G- 118 Cryptococcus albidus G-71, G-117, G-116 Rhodotorula rubra G-3, G-34 Rh. r&a G-111 Candida scottii G-50 Saccharomyces cerevisiae G-7, G-2 Schizosaccharomyces starkeyi-henrichii G-95(2) Hyphopichia burtonii G-97 Geothrichum candidurn G-IO Geothrichwn sp. G-102
d
-
+ +
-
-
d
-
-
-
-
+
_
-
-
d -
-
+ + +
-
-
_ -
+
-
d
-
+
-
d
I
d
-
-
d
-
-
-
N source
+
C, N source
-
7
+ +
-
-
+
-
d
-
+ +
+ -
-
-
-
+
f
+
+
+
+
-
-
+
+
-
-
+
+
-
-
+ +
+ -
-
-
+ -
+ -
-
-
+ + + -
+ + -
-
+ -
-
-
-
-
-
-
-
+ -
+
-
-
-
1= growth:+ = active growth; d = delayed growth; - = absence of growth. 2 - lytic zones: + = good visible; - = absence of uric acid lysis.
given a second dose, Candida guilliermondii numbers increased considerably on the inner surface of gut tissue in all the parts of the digestive tract-up to 5 x lo5 Cells g-l (or 5 x Iti per gut) according to our plate counts (Fig. 3). The midguts of the diplopods were colonized to a lesser extent than the foreguts and hindguts, up to the fourth dose. In the control, only the foreguts of the animals were colonized by Candida guilliermondii and their number did not exceed 102 cells g-1. Similar results were obtained with direct microscopic observations. However, the numbers of yeasts counted were ca 100 times higher than with plate
counting. With the highest infecting dose of Candida guilliermondii, 75% of the experimental animals died 7 days after feeding ceased. DISCUSSION
The results indicate that there are at least three types of interactions between yeasts and soil diplopods. This is a trophic interaction, in which yeasts can serve as a food for diplopods when they are supplied with leaf litter. Actually, there were only very few living yeast cells in the midgut in contrast to large
lindgut
Fig. 3. Numberof colony formingunits of yeast in the foregut,midgutand hindgutof diplopods,Glomeris connexa, feedingon sterilecrystallinecellulose inoculated with different amounts of Candtduguilliermondii.
1125
Interrelationships between yeasts and soil diplopods
and uric acid that are transported into the hindgut space (Bignell, 1984). Carbohydrates are also transported there as part of the food. Thus, there are all the essential nutrients in the hindgut to promote yeast growth. In their turn, yeasts reutilize uric acid which is toxic for animals thereby providing gut bacteria with nitrogen-containing compounds. The hypothesis concerning the ability of gut microbial symbionts in some terrestrial insects to utilize uric acid, the main end-product of nitrogen metabolism, has been stated by Koch (1967) and Baker et al. (1970). However, it had not been confirmed experimentally. A model of yeast distribution and activity in the gut of diplopods is presented in Fig. 4. According to this model the midgut is the site where lysis and digestion of ingested yeasts take place. Here also nutrients from lysed microbial cells are absorbed as well as labile organic compounds in leaf litter. The animal’s own enzymes (or microbial autolytic enzymes possibly controlled by a phenomenon of
numbers of yeasts in the hindgut. However, if the hindgut yeasts (Debaryomyces hansenii, Candida guilliermondii) pass through the midgut as exogenic substratum, they largely disappear. The fate of true litter inhabitants (Candida scottii) passing through the gut is the same. No increase of yeast-lysing actinomycetes was found in the gut of diplopods when animals were fed on yeast-amended substratum. So, microorganisms cannot be considered as a main factor in the digestion of ingested yeasts. On the other hand, gut fluid from the midgut appeared to be a very active medium in which yeasts cell walls can be destroyed. Thus, the digestion of yeasts cells takes place mainly in the middle part of the gut system apparently as a result of enzymatic activity of the gut fluid. There are also mutualistic interactions when the host organism (diplopods) provides yeasts with a favourable place of residence, namely the hindgut. The Malpighian tubules excrete mineral compounds ---------_-
_-_-_-_-_
Foregut
7
t-------
Midgut
Digestive
enzymes
Nutrients absorbtion
+
C
Peritrophic membrane
//
-Malpighian
tubules:
uric acid, PO:; so:; K+, Na+
Cl,
.--_
Exogenic yeasts Hindgut
Intestinal yeasts Lysed yeasts
Uric acid #l~I~__Peristaltic
Faecal pellets
movement
0@?
Fig. 4. The model of yeast distribution in the gut of diplopods, Pachyiulus@@es.
B. A. BYZOVet al.
1126
induced autolysis) are responsible for yeast destruction. The midgut is also a specific buffer zone in which enzymatic activity can protect the residential gut yeast community against external infection. The hindgut is a natural fermenter. It is inhabited by mixed cultures of resident yeasts-the DTZgroup, which can be considered as typical gut microbiota (Byzov et al., 1993). These bacteria are absent from the food of diplopods, but occur in the midgut in fewer numbers. It means that a portion of the hindgut yeast biomass probably moves into the midgut due to antiperistaltic movement (endogenic feeding). It was found that all of the gut yeasts could utilize uric acid as the sole source of nitrogen, thus, uric acid is supposedly one of the source of nitrogen supporting yeast growth in the hindgut of diplopods. Uric acid is hydrolysed extracellularly by uricase and decomposition products are consumed by both yeasts and bacteria. There is still no evidence that other hindgut microorganisms can hydrolyse uric acid, although their great diversity has been demonstrated in the hindgut of diplopods by Byzov et al. (1988, 1993). Thus, there are two possible functions for yeasts in the guts of diplopods: reutilization of uric acid, the toxic end-product of nitrogen metabolism; and endogenic feeding of diplopods. This mutualistic interaction can change into a parasitic state if the natural protective ability of the midgut is disturbed. It can happen if unnaturally large amounts of gut yeasts are introduced, following removal of natural microbiota. The yeasts, Candidu guilliermondii, which normally inhabit the hindgut of the diplopod Glomeris connexa, can cause the disease when their natural numbers are increased lOO-fold. To be killed by yeasts, a total colonization of the midgut epithelium is necessary. This can happen naturally during the long-term rearing of diplopods under laboratory conditions. Finally, we would like to stress that this work should be considered as a first step in the understanding of mechanisms and types of yeast-invertebrate interactions. Acknowledgements-We gratefully thank Professor D. G. Zvyagintsev, head of the Department of Soil Biology, Lomonosov State University, Moscow, for many useful comments. We would like to thank Dr V. S. Guzev, head of the Electron Microscopy laboratory, for his consultations in scanning microscopy. We also wish to thank Professor J. M. Anderson from the University of Exeter and Dr Lynne Boddy from the University of Wales, College of Cardiff, Wales, who corrected the English text. We are grateful to Dr Ju. B. Byzova from the Institute of Evolutionary Morphology and Ecology of Animals RAN, Moscow, for her help in sampling and identification of animals. REFERENCES
Baker J. M., Laidlaw R. A. and Smith G. A. (1970) Wood breakdown and nitrogen utilization by Anobiumpuctatum Deg. feeding on Scats pine sapwood. Holzforschung 24, 45-53.
Bignell D. E. (1984) The arthropod gut as an environment for microorganisms. In Invertebrate-Microbial Inter actions (J. M. Anderson, A. D. M. Rayner and D. W. H. Walton, Eds), pp. 205-228. Cambridge University Press, Cambridge. Byzov B. A., Dobrovolskaya T. G. and Chernjakovskaya T. F. (1989) Dynamics of microbial communities of the saprotroph zoomicrobial complex. Abstract 10th International Symposium on Soil Biology, Keszthely, Hungary. Byzov B. A., Dobrovolskaya T. G. and Zvyagintsev D. G. (1988) Microorganisms in the gut and faeces of Glomeris connexa C. L. Koch and Leptoiulus polonicus Jawlowski (Diplopoda) Abstract 10th International colloq. Soil Zoology, 7-13 August 1988, Bangalore, India. Byzov B. A., Vu Nguyen Thanh and Bab’eva I. P. (1993) Yeasts associated with soil invertebrates. Biology & Fertility of Soils. In press. Byzov B. A., Dobrovolskaja T. G., Chemjakovskaja T. F. and Zenova G. M. (1993) Bacterial communities associated with soil diplopods. Pedobiologia. In press. Chu T. L., Szabo I. M. and Szabo I (1987) Nocardioform gut actinomycctes of Glomeris hexasticha Brandt. (Diplopoda). Biology & Fertility of Soils 3, 113-116. Contreras E. (1980) Studies on the intestinal actinomycete flora of Eisenia lucens (Annehda : Oligochaeta). Pedobiologia 20, 411416.
Griffiths B. S. and Wood S. (1985) Microorganisms associated with the hindgut of Oniscus asellw (Crustacea : Isopoda). Pedobiologia 28, 377-38 1. Guzev V. S., Byzov B. A., Guzeva L. H. and Zvyagintsev D. G. (1986) Scaniruyushaja electronnaja mikroskopija pri izuchenii vzaimodeystvija mikroorganismov s bespozvonochnymi. In Ekologicheskaja rol mikrobnych metabolitov, pp: 212-231. M, izd-vo Mask., un-ta. Hossein E. A.. Ziczi A.. Contreras E. and Szab6 I. M. (1988) Occurrence and roie of nocardioform actinomycetes in the gut of earthworms. Abstract 10th International colloq. Soil Zoology, 7-13 August 1988, Bangalore, India. Ineson P. and Anderson J. M. (1985) Aerobically isolated bacteria associated with the gut and faeces of the litter feeding macroarthropods Oniscus aselluF and Glomeris marginata. 843-849.
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Jager K., Marialigeti K., Hauck M. and Barabas G. (1983) Promicromonospora enterophila sp. nov., a new species of monospore actinomycetes. International Journal of Systematic Bacteriology 33, 525-531.
Koch A. (1967) Ins&s and their endosymbionts. In Symbiosis (S. M. Henrv. Ed.) Vol. 3. Academic Press. London. Marialigeti K., Con&ms’E., Barabas G., Heydrich M. and Szabo I. M. (1985) True intestinal actinomycetes of millipedes (Diplopoda). Journal of Inoertebrate Pathology 45, 120-121.
Oertel B. and Schaller G. (1984) Symbiose von Insekten mit Mikroorganismen. Ein Uberblick. Biol. Rdsch 22, 303-329.
Ravasz K., Ziczi A., Contreras E., Szell V. and Szabo I. M. (1986) Uber die Darmaktinomyceten-Gemeinschaften einiger Regenwurm-Arten. Opusc ula Zoologie 22, 85-102.
Shaw C. and Pawluk S. (1986) Faecal microbiology of Octalasium tyrtaeum, Aporrectodea turgida and Lumbricus terrestris and its relation to the carbon budgets of three artificial soils. Pedobiologia 29, 377-389. Szabo I. M. (1974) Microbial Communities in a ForestRendsina Ecosystem. Akademiai Kiado, Budapest. Ullrich B., Starch V. and Schaiker H. (1991) Bacteria on the food, in the intestine and on the faeces of the woodlouse Oniscus asellus (Crustacea : Isopoda). Species composition and nutritive value. Pedobiologia 35, 41-51.
Copyright 0
INTERRELATIONSHIPS BETWEEN SOIL DIPLOPODS
0038-07 I7/93 $6.00 + 0.00 1993 Pergamon Press Ltd
YEASTS AND
B. A. BYZOV, Vu NGUYENTHANH and I. P. BABJEVA Department
of Soil Biology, Faculty of Soil Science, Lomonosov Moscow 119899, Russia
State University, Vorobjevy gory,
(Accepted 20 January 1993) Summary-Some functional interrelationships have been established between yeasts and the soil diplopods Pachyiu~uspaOipesC. L. Koch and GIomeris connexa C. L. Koch. Yeasts can serve as a food for diplopods when these animals are feeding on leaf litter. The digestion of yeast cells takes place mainly in the midgut as a result of enzymatic activity of the gut fluid. The midgut is a well-protected zone in which enzymatic activity prevents exogenic infection and thereby provides a steady-state environment for the yeast community. The hindgut is a region where specific yeasts can utilize readily-available carbon sources from leaf litter and hydrolyze uric acid-the end-product of nitrogen metabolism of the host animals. A model for yeast distribution in the gut of diplopods is described.
INTRODUCTION The list of microorganisms shown to be associated with soil invertebrates is still on the increase. Gram-
negative, Gram-positive bacteria and actinomycetes belonging to many genera have been isolated from the gut of insect larvae (Sxabb, 1974; Oertel and Schaller, 1984), woodlice (Griffiths and Wood, 1985; Ineson and Anderson, 1985; Ullrich et al., 1991), earthworms (Contreras, 1980; Ravasz et al., 1986; Shaw and Pawluk, 1986; Hossein et al., 1988) and diplopods (Jager et al., 1983; Marialigeti er al., 1985; Chu et al., 1987). Also the dominant microorganisms of the gut microbial community have been determined for many animals and often appear to be very specific and entirely different from that of the food and faeces of the animals. For example, it has been demonstrated that the gut of diplopods is inhabited mainly by facultative anaerobic Gram-negative bacteria of the families Enterobacteriaceae and Vibrionaceue (Byzov et al., 1988) as well as coryneform bacteria of the genus Promicromonospora (Chu et al., 1987; Byzov et al., 1989). The yeast community of the gut of soil invertebrates has been investigated by (Byzov er al., 1993) who demonstrated that there are specific ascomycete yeasts: Debaryomyces hansenii, Torulaspora delbrueckii and Zygowilliopsis californica (so called ‘DTZ’-group) in the gut of the diplopod Pachyiulus jlavipes; the composition and structure of the community was constant and remained unaltered under different feeding and rearing regimes. The actual functional role of a gut microbiota as well as types of zoomicrobial interactions are still poorly understood. Many of the difficulties in interpretation of results arise when only the dilution plate technique is used for population estimates.
We have tried to solve this problem by combining dilution plating with direct observation using light and scanning electron microscopes. In addition, we have modified the commonly-used scheme presuming short-term and static analysis of gut microbial community. Our approach was to change feeding regimes by amending food with pure cultures of yeasts and to estimate the changes in their populations as well as in the microbiota of the gut. MATERIALS
AND METHODS
Diplopodr and yeasts
Four species of soil diplopods were used: Pachyiulusflavipes C. L. Koch, were collected from leaf litter in fruit orchards, Crimea, Ukrainia; Glomeris connexa C. L. Koch, Leptoiulus polonicus Jawlowski and Megaphilium projectum kochi Verhoeff were collected from leaf litter of broadleaf forest, Mukachevo region, Ukrainia. The diplopods were reared in desiccators, in groups of about 50 animals, on fresh natural food substrates at 18-2O”C, before use in our experiments. The following yeast cultures were used: Debaryomyces hansenii, Torulaspora delbrueckii, Zygow caltfornica, illiopsis Pichia membranaefaciens, Trichosporon pullulans, Trichosporon cutaneum, Geothrichum candidum (isolated from the gut of the diplopod P.flavipes); Candida guilliermondii (isolated from the guts of the dead diplopod Glomeris connexa); Cryptococcus albidus and Candida scottii (isolated from leaf litter); Hyphopichia burtonii (isolated from cow manure); Succharomyces cerevisiae, Schizosaccharomyces starkeyi-hendricii, Rhodotorula rubru and Kloekera upis (from the yeast collection of
the Department University).
1119
of Soil Biology, Lomonosov
State
B. A. BYZOV et al.
1120
Feeding experiments
Five diplopods P. jlavipes were reared for 21 days on 1 g lots of sterile leaf litter or crystalline cellulose with natural moisture and amended separately with the yeasts D. hansenii, Candida guilliermondii or Candida scottii at initial concentrations of 1.8 x lo’, 7 x lo’, and 9.5 x lO’g-’ air-dry substratum, respectively, for 21 days. The initial inocula of yeasts in food estimated microscopically, was provided in a water suspension of cells serially washed three times. The substratum was renewed weekly to avoid contamination and then the number of yeasts was estimated in food, midgut, hindgut and freshly collected faecal pellets by the dilution plate method as described by Byzov et al. (1993) on 7, 14 and 21 days after the start of feeding. Three replicates of each diet were used. To elucidate the participation of the yeast Candida guilliermondii in any pathogenic processes the following experiment was carried out. Samples of five diplopods (G. connexa) were fed sterile leaf litter enriched by yeasts in increasing amounts either 104 5 x 106, 10’ or lo8 cells g-’ After 7 days feeding, the gut yeasts were estimated using dilution plating and scanning electron microscopy. Sterile leaf litter was used as a control. Three replicates of each yeast concentration were used. Estimation of yeast-lysing bacteria
To estimate the composition and number of yeastlysing microorganisms in the gut and faecal pellets of diplopods, a medium with living yeast cells was used (30 g I-’ (NH4)rS04, 1 g l-’ KH2P04, 1 g 1-l MgSO,, Difco agar). After autoclaving, the medium was cooled to 4045°C and enriched with 5 x lo6 cells ml-’ of D. hansenii, which had been washed three times in distilled water. The yeast cells were the only carbon source in the medium. Estimation of yeast-lysing activity of midgut fluid
To quantify the fate of yeast, cells passing through the gut of diplopods, the midguts of ten animals were carefully removed aseptically to retain the digestive fluid inside the gut tube. The digestive fluid was collected and divided into lOOm1 portions. Pure cultures of D. hansenii, Torulaspora delbrueckii, Trichosporon pullulans, Candida scottii, Geothrichum candidum and Cryptococcus albidus were added to the
digestive fluid at a concentration
of 10’ cells ml-’ The
fluid inoculated by yeasts was then incubated at 37°C. Cell morphology was examined by light microscopy every 2 h for 10 h. Yeasts suspended in distilled water were used as a control and the experiment was replicated twice. The ability of yeasts to grow on uric acid
To estimate the ability of all the yeast strains isolated from leaf litter, guts of diplopods and their faecal pellets to grow on uric acid, the following media were used: (1) log I-’ uric acid (the only source of carbon), 6.7 gl-’ Yeast Nitrogen Base Difco, 20 g I-’ Difco agar; (2) 1 g I-’ uric acid (only source of nitrogen), 11.3 g 1-l Yeast Carbon Base Difco, 2Ogl-’ Difco agar; (3) IOgl-’ uric acid (source of both carbon and nitrogen), 20 g I-’ Difco agar. The following nutrients were added to the all media: 0.2 g 1-l K,HPG,, 0.1 g I-’ KH2PG,, 0.2 g I-’ MgS0,7H,O, 0.2gl-* NaCl, 0.1 gl-’ K,SO,, 50ml of yeast water as a source of vitamins. Uric acid was autoclaved separately. Glucose and (NH&SO, were used as controls in first and second experiments respectively. The ability of yeasts to utilize uric acid was estimated by counting numbers of typical colonies and clearing zones around inocula. Scanning electron microscopy
The yeast community in the gut of diplopods was studied using scanning electron microscopy JSM-2 (‘Jeol’). The inner surfaces of midgut epithelium and hindgut cuticle were examined. The samples for electron microscopy were prepared as described by Guzev et al. (1986). RESULTS
Yeasts as a food for diplopods
Diplopods fed on sterile cellulose amended with the yeasts D. hansenii, Candida scottii and Candida guilliermondii (Table 1). Over 21 days feeding, there was a considerable decrease in the number of yeasts in faecal pellets. However, Candida scottii was eliminated to a greater extent (about >99%) than D. hansenii and Candida guilliermondii (about 90 and 92%, respectively). After 21 days of continuous feeding, yeast cells provided with cellulose were found in the midgut (17 x lo4 cfu g-l) as well as in the hindgut (43 x lo4 cfug-‘) and faecal pellets (160 x 104 cfug-‘)
Table 1. Number of colony forming units of yeasts in the food and faecal pellets of diplopods. Pachiulus &oipes, after 7, 14 and 21 days continuous feeding on sterile crystalline cellulose amended with Candida guilliermondii, Candida scorlii or Debwyomyces hansenii. Standard error,
Yeast species on crystalline cellulose
Faecal pellets Food
Candrda
guilliermondii
82k
10
Candida
scotfii
93k
16
Debarvomvces
hansenii
15+4
7 days
14 days
21 days
42 f
45+
6.6 f
29
II
0.5 *0.1
3.4 f 0.5
0.2 + 0.1
9.0 f 2.3
1.3
0.1 1.6kO.l
0.5 f
Interrelationships
between yeasts and soil diplopods
Fig. 1. Scanning electron micrographs of destroyed asci with one ascospore inside (arrows) of yeasts, supposedly belonging to Debaryomyces genus in the midgut (A, B); yeast cells colonizing the gut wall surface in the midgut (C), and the cuticula in the hindgut (D) of diplopods, Pachyiulus Jauipes, feeding on natural leaf litter. Bars indicate for (A) 6pm; (B) 2nm ; (C) and (D) 20pm.
1121
1122
3. A. BYZOVet al.
Fig. 2. Scanning electron micrographs of native vegetative yeast cells (A) and yeast cells at different stages of the digestion of cell walls (arrows) (B)-(D) in the midgut of diplopods, Pachyiulusflavips, feeding on natural leaf litter. Bars indicate 6 pm.
Interrelationships between yeasts and soil diplopods
1123
Table 2. Number of colony forming units of yeasts in the midgut, hindgut and faccal pellets of diplopods, Pachyiulw@zt@es, after 21 days continuous feeding on sterile crystalline ceIlulose amended with Candid0 guilliermondii, Candida scottii or Deharyomyces hattsenii. Standard error, a=3 Number of yeasts, cfu x IO g-’ dry wt Yeast species on crystalline allulosc
Food
Midgut
Candid0 guilliermondii(a)
8200 + IOOOa 3.4 f IDTZ*
Candida scottii(b) Debarvomvces hansenii(cl
9600~16OOb
0
150000t400c
17 + ISDTZ
Hindgut
Faecal pellets
9.1 + 0.6DTZ 4.8 f I .8a 5.4 + 1.8DTZ 43 + IODTZ
660 f 13oa 50 f IOb 16Of10c
lDTZ-for explanation see text. tThe same letters indicate the same yeast cultures.
only when diplopods had been fed on D. hansenii. Candida gzdlliermondii could be recognized only in the hindgut and Candida scottii only in the excrements of diplopods. On the other hand yeasts of the DTZ-group (0. hansenii, Torulaspora delbrueckii, 2. californica) constantly occurred at high densities in the hindgut (1-3 x lo4 cfu per gut, i.e. 2@-50 times higher than the normal level), but fewer than in excrements (Table 2). To elucidate the digestion mechanisms, yeastlysing bacteria were estimated in the gut and excrements of diplopods in the same experiment. Living yeast cells were added to the medium to select these gut bacteria. Yeast-lysing microorganisms (mainly actinomycetes) were isolated only from the hindgut and faecal pellets (Table 3). However, the number of actinomycetes was even less when diplopods fed on cellulose amended with yeasts compared with yeast-free food and did not exceed lo3 cfu per gut. With light microscopy it was shown that after 10 h incubation in gut fluid, about 60% of asci of D. hansenii and Torulaspora delbrueckii were destroyed and ascospores were released. This compares with only 2% in the control. The results obtained by the use of scanning electron microscopy corresponded well with results from light microscopy. The destroyed asci, supposedly belonging to Debaryomyces sp., were found in the midgut of diplopods [Fig. 1 (A), (B)]. In Fig. 2 native vegetative yeast cells [Fig. 2 (A)] and different digestion stages of these cells [Fig. 2 (B)-(D)] that occurred in the midgut of diplopods are shown. In about 70% of Candida scottii cells, the internal contents were destroyed with no vacuoles or lipid granules visible. Also, no daughter bud cells were
observed. There was practically no change observed in the control (incubation in water). However, the gut fluid did not affect the morphology of Cryptococc~ albidus and Trichosporon pulhdans. Only in the case of Geothrichum candidzun was growth stimulation of cells noted. Mutualistic
interaction between yeasts and diplopodr
Practically all of the gut isolates utilized uric acid as their only source of nitrogen as well as many litter inhabitants; only two yeast isolates could utilize uric acid as a source of nitrogen as well as a carbon (Table 4). Some yeast strains produced lytic zones whereas other did not. Yeasts as pathogenic agents
Considerable increases in yeast density in the guts of the diplopods Glomeris connexa, Leptoizdur polonicus and Megaphilium projectzun were found when the animals had been fed on natural leaf litter that had not been renewed for 3 months. It has been demonstrated with scanning electron microscopy that yeasts mainly colonize the hindgut of freshly-collected diplopods Glomeris connexa [Fig. 1 (D)], their number being about lo3 Cells mm-’ (IO’ Cells per gut). Only a few cells were found in the midgut [Fig. 1 (C)l. Three months later the number of yeasts in the gut appeared to be ca 100 times higher than at the start of the experiments. Yeasts colonized both the midgut and hindgut of diplopods. At this time the majority of animals were dead. Candida guilliermondii dominated in the yeast community of the gut. It is possible that Candida guilliermondii might have acted as a pathogen. When diplopods originally fed on sterile crystalline cellulose enriched by Candida guilliermondii were
Table 3. Number of colony forming units of yeast-lysing bacteria in the food, midgut, hindgut and faecal pellets of diplopods, Pachyitdw&@es, after 21 days continuous feeding on sterile crystalline ccllulosc, sterile leaf litter or sterile crystalline cellulose amended with yeasts, Candida gtdlliermondii, Candido scottii and Deharyomyces hattsenii. Standard error, n = 3 Number of yeast-lysing bacteria, cfu x Io-‘g-’ dry wt Food
Midgut
Sterile leaf litter
0
0
54’*8
Crystalline cellulose (CC) Car&& guilIiermondii on CC Candiak scottii on CC Deharyomyces hansenii on CC
0 0 0 0
0 0 0 0
41 f 29 f 49 f 45*
Type of food substrates
ND = not determined.
Hindgut 4 3 26 10
Faecal pellets 320 k 20 96+ I5 II+11
ND ND
B. A. BYZOV et al.
1124
Table 4. The ability of yea& strains to grow on media with uric acid as the SOIFC or N source
c
source
Spwies, strains
1
2
I
2
I
2
Debaryomyces hansenii G-43, G-45, G-46, G-47, G-48, G-56 Z. cali/ornica G-68, G-83, G-87(2) Z. californica G-126 Z. calfirnica G-130 Torulaspora delbrueckii G- I30 Torulaspora delbrueckii G-55 Torulaspora delbrueckii G-72, G-80 Pichia membranaefacietw G-74, G-125, G-131, G-104 Candida guilliermondii G-24, G-14 Kloekera apis G-106 K. apis G-137 K. apis G-61 Trichosporon cutaneurn G-l 13 Trichosporon cutaneum G-5 Trichosporon cu~aneum G-79 Trichosporon pdlulans G-90, G-l 15 Trichosporon pullulan~ G- 118 Cryptococcus albidus G-71, G-117, G-116 Rhodotorula rubra G-3, G-34 Rh. r&a G-111 Candida scottii G-50 Saccharomyces cerevisiae G-7, G-2 Schizosaccharomyces starkeyi-henrichii G-95(2) Hyphopichia burtonii G-97 Geothrichum candidurn G-IO Geothrichwn sp. G-102
d
-
+ +
-
-
d
-
-
-
-
+
_
-
-
d -
-
+ + +
-
-
_ -
+
-
d
-
+
-
d
I
d
-
-
d
-
-
-
N source
+
C, N source
-
7
+ +
-
-
+
-
d
-
+ +
+ -
-
-
-
+
f
+
+
+
+
-
-
+
+
-
-
+
+
-
-
+ +
+ -
-
-
+ -
+ -
-
-
+ + + -
+ + -
-
+ -
-
-
-
-
-
-
-
+ -
+
-
-
-
1= growth:+ = active growth; d = delayed growth; - = absence of growth. 2 - lytic zones: + = good visible; - = absence of uric acid lysis.
given a second dose, Candida guilliermondii numbers increased considerably on the inner surface of gut tissue in all the parts of the digestive tract-up to 5 x lo5 Cells g-l (or 5 x Iti per gut) according to our plate counts (Fig. 3). The midguts of the diplopods were colonized to a lesser extent than the foreguts and hindguts, up to the fourth dose. In the control, only the foreguts of the animals were colonized by Candida guilliermondii and their number did not exceed 102 cells g-1. Similar results were obtained with direct microscopic observations. However, the numbers of yeasts counted were ca 100 times higher than with plate
counting. With the highest infecting dose of Candida guilliermondii, 75% of the experimental animals died 7 days after feeding ceased. DISCUSSION
The results indicate that there are at least three types of interactions between yeasts and soil diplopods. This is a trophic interaction, in which yeasts can serve as a food for diplopods when they are supplied with leaf litter. Actually, there were only very few living yeast cells in the midgut in contrast to large
lindgut
Fig. 3. Numberof colony formingunits of yeast in the foregut,midgutand hindgutof diplopods,Glomeris connexa, feedingon sterilecrystallinecellulose inoculated with different amounts of Candtduguilliermondii.
1125
Interrelationships between yeasts and soil diplopods
and uric acid that are transported into the hindgut space (Bignell, 1984). Carbohydrates are also transported there as part of the food. Thus, there are all the essential nutrients in the hindgut to promote yeast growth. In their turn, yeasts reutilize uric acid which is toxic for animals thereby providing gut bacteria with nitrogen-containing compounds. The hypothesis concerning the ability of gut microbial symbionts in some terrestrial insects to utilize uric acid, the main end-product of nitrogen metabolism, has been stated by Koch (1967) and Baker et al. (1970). However, it had not been confirmed experimentally. A model of yeast distribution and activity in the gut of diplopods is presented in Fig. 4. According to this model the midgut is the site where lysis and digestion of ingested yeasts take place. Here also nutrients from lysed microbial cells are absorbed as well as labile organic compounds in leaf litter. The animal’s own enzymes (or microbial autolytic enzymes possibly controlled by a phenomenon of
numbers of yeasts in the hindgut. However, if the hindgut yeasts (Debaryomyces hansenii, Candida guilliermondii) pass through the midgut as exogenic substratum, they largely disappear. The fate of true litter inhabitants (Candida scottii) passing through the gut is the same. No increase of yeast-lysing actinomycetes was found in the gut of diplopods when animals were fed on yeast-amended substratum. So, microorganisms cannot be considered as a main factor in the digestion of ingested yeasts. On the other hand, gut fluid from the midgut appeared to be a very active medium in which yeasts cell walls can be destroyed. Thus, the digestion of yeasts cells takes place mainly in the middle part of the gut system apparently as a result of enzymatic activity of the gut fluid. There are also mutualistic interactions when the host organism (diplopods) provides yeasts with a favourable place of residence, namely the hindgut. The Malpighian tubules excrete mineral compounds ---------_-
_-_-_-_-_
Foregut
7
t-------
Midgut
Digestive
enzymes
Nutrients absorbtion
+
C
Peritrophic membrane
//
-Malpighian
tubules:
uric acid, PO:; so:; K+, Na+
Cl,
.--_
Exogenic yeasts Hindgut
Intestinal yeasts Lysed yeasts
Uric acid #l~I~__Peristaltic
Faecal pellets
movement
0@?
Fig. 4. The model of yeast distribution in the gut of diplopods, Pachyiulus@@es.
B. A. BYZOVet al.
1126
induced autolysis) are responsible for yeast destruction. The midgut is also a specific buffer zone in which enzymatic activity can protect the residential gut yeast community against external infection. The hindgut is a natural fermenter. It is inhabited by mixed cultures of resident yeasts-the DTZgroup, which can be considered as typical gut microbiota (Byzov et al., 1993). These bacteria are absent from the food of diplopods, but occur in the midgut in fewer numbers. It means that a portion of the hindgut yeast biomass probably moves into the midgut due to antiperistaltic movement (endogenic feeding). It was found that all of the gut yeasts could utilize uric acid as the sole source of nitrogen, thus, uric acid is supposedly one of the source of nitrogen supporting yeast growth in the hindgut of diplopods. Uric acid is hydrolysed extracellularly by uricase and decomposition products are consumed by both yeasts and bacteria. There is still no evidence that other hindgut microorganisms can hydrolyse uric acid, although their great diversity has been demonstrated in the hindgut of diplopods by Byzov et al. (1988, 1993). Thus, there are two possible functions for yeasts in the guts of diplopods: reutilization of uric acid, the toxic end-product of nitrogen metabolism; and endogenic feeding of diplopods. This mutualistic interaction can change into a parasitic state if the natural protective ability of the midgut is disturbed. It can happen if unnaturally large amounts of gut yeasts are introduced, following removal of natural microbiota. The yeasts, Candidu guilliermondii, which normally inhabit the hindgut of the diplopod Glomeris connexa, can cause the disease when their natural numbers are increased lOO-fold. To be killed by yeasts, a total colonization of the midgut epithelium is necessary. This can happen naturally during the long-term rearing of diplopods under laboratory conditions. Finally, we would like to stress that this work should be considered as a first step in the understanding of mechanisms and types of yeast-invertebrate interactions. Acknowledgements-We gratefully thank Professor D. G. Zvyagintsev, head of the Department of Soil Biology, Lomonosov State University, Moscow, for many useful comments. We would like to thank Dr V. S. Guzev, head of the Electron Microscopy laboratory, for his consultations in scanning microscopy. We also wish to thank Professor J. M. Anderson from the University of Exeter and Dr Lynne Boddy from the University of Wales, College of Cardiff, Wales, who corrected the English text. We are grateful to Dr Ju. B. Byzova from the Institute of Evolutionary Morphology and Ecology of Animals RAN, Moscow, for her help in sampling and identification of animals. REFERENCES
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