nematode Panagrellus redivivus were incubated in. Ringer's solution and tap water, ammonium was the major end product of N metabolism (Wright 1975). In.
OIKOS 40: 75-80. Copenhagen 1983
Organic and inorganic nitrogenous losses by microbivorous nematodes in soil R. V. Anderson, W. D. Gould, L. £. Woods, C. Cambardella, R. £. Ingham and D. C. Coleman
Anderson, R. V,, Gould, W, D,, Woods, L, E,, Cambardella, C , Ingham, R, E, and Coleman, D, C, 1983, Organic and inorganic nitrogenous losses by microbivorous nematodes in soil, - Oikos 40: 75-80, To examine the relationship between primary and secondary decomposers and N mineralization in soil, amino acid-N, NH4-N, and population dynamics were measured in a soil microcosm system employing: (a) bacteria, and (b) bacteria + microbivorous nematodes. Appreciable amino acid-N (19 ng amino acid-N/g soil) was detected in the presence of nematodes at day 5 of the experiment. However, after 35 d the nitrogenous waste products of the nematodes shifted to primarily NH4-N because of food stress and a shift in the nematode population structure. More N was mineralized in the system containing nematodes and bacteria than in the system containing bacteria alone. R. V. Anderson, Dept of Biological Sciences, Western Illinois Univ., Macomb, IL 61455, USA. W. D. Gould, Agriculture Canada, Research Station, Lethbridge, Alberta T1J4B1, Canada. L. E. Woods, High Plains Grassland Research Station, USDA/ARS, 8408 Hildreth Road, Cheyenne, WY 82001, USA. C. Cambardella, R. E. Ingham and D. C. Coleman (correspondence). Natural Resource Ecology Lab. and Dept of Zoology/Entomology, Colorado State Univ., Fort Collins, CO 80523, USA. flnn npoBepKH cooTHoueHHH Mewgjy aKTHBHocTBm nepBH^HtJx H BToppwHtJx pa3pyii]nTenea H MHHepanHsaujiefi aaoTa B no'CBe HccneuoBajm nHHaMHKy aMHHOKHCnoTHoro a3OTa H HHHaMHKy nonynHLutfi B ncMBeHHUx MHKpoKCoex, BKJicHaKiiiHX: a / 6aKTePHH, H 6 / 6aKTepnH + HeMaTCinfcJ-MHKpo6cKl>arH. 3aMeTHoe KarB«ecTBO aMHHOKHCjiOTHoro a3OTa 6hino KOHcraTHpOBaHO B npHcyTCTBHH HeMaTon Ha rurrbifi ceHb ontJT a . OHHaKO, Mepe3 35 uneti B npoayKTax 5enKOBoro o6MeHa HeMaToa HaMernncH rrmvrr B CTopoHy a^/^1OHHflHO^o a3OTa B pe3yjibTaTe rnnueBoro crrpecca H CUBHT B CTpyKType norrynHUHfii HSMaTon. B CHcrreMe, ccmep)Kam6fi H»«TOfl, H 6aKTepHH MH6Qnbiue a3OTa, ^esA B CHCTeMe, conepwameH TonbKo
Accepted 3 March 1982 © OIKOS 0030-1299/83/010075-06 $02-50/0
75 OIKOS 40:1 (1983)
1. Introduction
Soil fauna grazing on microbial populations influence nutrient dynamics and ecosystem activity (Anderson et al. 1981a). Although affected by a number of soil characteristics, i.e., soil texture (Elliott et ai. 1980), most soil fauna organisms mineralize nutrients (Anderson et al. 1981b) and some have been shown to directly influence nutrient levels in plants (Elliott et al. 1979). Thus an understanding of nutrient release by soil fauna in relation to soil fertility status (BS§th et al. 1978) is important to the development of the association between plants and the soil community. Nematodes, as one of the dominant soil organisms, may account for a large turnover of N (Nielsen 1949, Twinn 1974). Unfortunately, the quantities of labile nitrogen in these studies were not well known. In a soil microcosm experiment reported by Coleman et al. (1977), significantly more N was mineralized as ammonium in the presence of bacterial-feeding nematodes than with bacteria alone. However, in a similar experiment (Woods et al. 1982) no N was mineralized with bacteria or bacteria and microbivorous nematodes. This inconsistency was attributed to differences in the population development of the nematodes in the two experiments. Ammonium levels increased only when nematode populations declined and became dominated by "dauer" juveniles or intrauterine juvenile production, indicating food stress. Nematodes have a high C:N ratio [8:1 to 12:1 (Myers and Krusberg 1965)] relative to their food source [3:1 to 4:1 (Hunt et al. 1977)] and thus must always release N as a waste product. Although nematodes are considered ammonetelic, Rogers (1969) reported that amino acids and peptides accounted for as much as 49% of the total N excreted by free-living and parasitic nematodes. Many studies of nematode excretion must be viewed with discretion, since the nematodes are often placed in stressful environments during monitoring and losses of some excreted compounds may be experimentally induced (Wright and Newall 1976). Addressing this problem, Rothstein (1963) cultured the bacterial-feeding nematode Caenorhabditis briggsae in an axenic medium in which the nematodes grew and reproduced. Amino acids comprised a considerable portion of the excreted N. Large-scale excretion of amino acids seemed to be a common trait among nematodes, but it was not apparent why free-living nematodes, such as C. briggsae, should lose potentially useful metabolites. When healthy individuals of the bacterial-feeding nematode Panagrellus redivivus were incubated in Ringer's solution and tap water, ammonium was the major end product of N metabolism (Wright 1975). In addition, the ratio of ammonium to amino acids in the solution increased with time of starvation. This suggests that apparent excretion of amino acids represents the release of nonassimilated material due to incomplete
76
utilization of ingested material (Rothstein 1963, Lee and Atkinson 1977). When bacterial food supplies are plentiful in the soil, nematodes may be expected to discharge amino acids in substantial amounts. As bacterial populations decrease because of nematode feeding or environmental factors, the nematodes begin to starve and protein eatabolism for maintenance energy requirements leads to increased ammonium excretion by the nematodes. Bacteria requiring amino acids are preferentially stimulated in the rhizosphere (Lochhead and Thexton 1947). Amino acids have been detected in plant root exudates (Rovira and McDougall 1967), but the role of nematodes in amino acid production in the rhizosphere has not been examined. The objective of this experiment was to determine whether mierobivorous nematodes cultured in soil release significant amounts of amino acids, particularly when food is plentiful, as may be the case in the rhizosphere. 2. Materials and methods 2.1. Soil microcosms Gnotobiotic soil microcosms consisting of 20 g soil in 50-ml Erienmeyer flasks were used. The soil was an Olney sandy loam (Aridic Argiustoll) with nutrient levels of 11.3 |.ig g~' soil bicarbonate-extractable P and 20 ^g g-' soil N H : - N , 0.06% N, 1.2% organic matter, and pH 6.4. The soil was brought to field capacity (15% w/v), incubated for 24 h, and then autoclaved for 1 h. The incubation/autoclaving procedure was repeated three times and the soil was then air dried. Initial and long-term sterility checks on nutrient agar (Difco) showed that viable organisms were not present. Soil from each microcosm was also cheeked on nutrient agar for contamination at the time of sampling. Two of the 96 microcosms were found to be contaminated and were excluded from data analysis. 2.2. Amendments The microcosms were amended with 500 pig glucose-C g-' soil and 60 \ig NH^-N [as (NH4).SO4] g-\ Two biological treatments were used: bacteria alone with an initial inoculum of 10'' bacteria {Pseudomonas paucimobilis) per g soil and bacteria plus gnotobiotically-cultured nematodes {Mesodiplogaster Iheritieri). The same bacterial level was used and 10 nematodes per g soil were added as a mixed culture of adults and juveniles. Three microcosms of each biological treatment and uninoculated controls were sampled at twoday intervals. The first sampling date was one day after nematode inoculation. Nematodes were added one day after bacterial inoculation, which allowed bacterial populations to reach sufficient levels to support nematode population growth. On each sampling date
OIKOS 40:1 (19«3)
bacterial numbers, nematode numbers as adults and juveniles, NH4-N, and total amino acids were determined. Nitrate was not monitored, as P. paucimobilis is not a nitrifier. Nematode and bacterial numbers were determined by Baermann extraction and plate counts, respectively (Anderson et al. 1978). Soil NH4-N was extracted with a 10:1 (v:w) ratio of 2.0 M KCl to soil. After being shaken for 30 min, the extracts were filtered and analyzed for NH4-N in Conway microdiffusion dishes (Stanford et al. 1973). 2.3. Amino nitrogen Amino acid-N analysis used a modification of Sanger's (1945) technique. Four g of soil were extracted with 40 ml of 2 M KCl. After shaking for 30 min, 2 ml of sample were treated with 8 ml of 1% NaBO. and 0.8 ml FDNB reagent (0.13 ml l-fluoro-2-4-dinitrobenzene dissolved in 10 ml absolute ethanol) and incubated at 60°C for 30 min, then quickly cooled. The optical density of the solution was determined at 380 nm and the amino acid content determined from a standard curve prepared with mixed amino acids. Per cent recovery was 12% (range 7-17.7%), as determined from recovery of known additions of amino acids added to the soil prior to extraction. Values were corrected based on this factor. Low recovery is possibly a result of amino acids complexing with free radicals released during autoclaving the soil and is not unusual. 2.4. Data analysis Data analysis included ANOVA and Tukey's honest significant difference (HSD) mean separation test (Kirk 1968) for the first 9 sample dates of bacterial numbers and the first 4 sample dates of amino acid-N. The Q values representing significant differences between means are included in Figs 1 and 3. The subscript beside each Q value indicates the number of means that can be compared simultaneously with that O value. For example, in Fig. 1 the O2 is used to compare any two means on any one day, while the Ois value can be used to compare any of the 18 means for the first 9 days with any of the others, regardless of day or treatment. All significant differences are presented as P < 0.05.
10
20
30
TIME(Doys)
Fig. I. Bacterial numbers in soil microcosms \\ith bacteria alone or bacteria plus a bacterial-feeding nematode. Q Q Bacteria treatment. • • Nematode and bacteria treatment.
populations had also declined. Nematode populations remained high, even after reduction of bacterial numbers, but were dominated by "dauer"" juveniles or females with intrauterine juvenile development (Fig. 2). Population fluctuations were marked by peaks produced when internally produced juveniles began breaking free of the female cuticle (day 11). This population response to food stress has been observed in agar cultures (Anderson and Coleman 1981). Theoretically, nematode grazing pressure can account not only for the reduction in bacterial numbers but also N release. Based on determinations of nematode
1000-1
3. Results and discussion 3.1. Population responses Bacterial populations in the treatment grazed by nematodes peaked earlier (on day 5) than those in the ungrazed treatment (Fig. 1). By day 7 however, nematodes had significantly reduced the number of bacteria in the grazed treatment. Bacteria in the grazed treatment remained significantly less than in the ungrazed treatment until day 21, when the ungrazed OIKOS 40:1 (1983)
TIME (Days)
Fig. 2. Adult and juvenile nematode numbers in soil microcosms with bacteria and bacterial-feeding nematodes. o O adult. • • juvenile. 77
Tab. 1. Biomass (ng C
g • soil ') and nitrogen (^g N
g • soil"') content of nematodes and bacteria in soil incubations.
Organism Nematodc-C
1.3 Bactcrial-C (grazed) . . . . 2 8 6 Bactcrial-C (ungrazed) .. 134 Nematodc-N 0.1 Bacterial N (grazed) . . . . 57.2 Bacterial N (ungrazed) .. 26.8
Day 17.8 75.2 363 88 213 286 1.8 7.5 72.6 17.6 42.6 57.2
62.5 79 290 6.3 15.8 58
II
13
15
17
19
46.1 88 484 4.6 17.6 96.8
91.0 125 198 9.1 25 39.6
70.7 125 231 7.1 25 46.2
57.4 110 229 5.7 22 45.8
29.5 106 154 2.9 21.2 30.8
biomass carbon (Coleman et al. 1978) and bacterial biomass carbon [2.2 x 10"^ \ig C/bacterium (Bryant et al. ]982)] and the assumptions of C:N ratios of 10 for nematodes and 5 for bacteria (Woods et al. 1982), changes in biomass and nitrogen between sampling dates (Tab. I) can be examined. Between days 5 and 7, when maximum nitrogen release began in the grazed system, bacterial biomass decreased by 275 [ig C and nematode biomass increased by 57.4 ^g C. Using an estimated production efficiency for this nematode (feeding on bacteria) of 0.04 (Coleman et al. 1978), the nematodes would have consumed 1445 \.ig bacterial C, or about 5 times as many bacteria as reflected by the bacterial population decline. It is likely that many of the consumed bacteria were not digested. Bacteria, after a transit of bacteriophagic nematodes' digestive tracts, may have a survival rate as high as 93% (Smerda et al. 1971). Increased bacterial productivity as a result of greater nutrient availability due to grazing is another factor contributing to the difference between calculated consumption and actual bacterial population declines. Reduction of bacterial biomass is well within the range of that expected from bacterial-feeding nematode assimilation efficiencies (approximately 20%) in the literature (Marchant and Nicholas 1974, Nicholas and Viswanathan 1975). In the ungrazed system, bacterial biomass did not decline until day 13, after the biomass had reached 484 [.ig on day 11 (Tab. 1). Since only 500 [ig glucose-C had been added to the microcosms, even with utilization of some native soil-C, the system had probably become C limited. This occurred earlier in the grazed system, where nematode-mediated nutrient availability increased bacterial turnover. It should be noted that both grazed and ungrazed systems eventually had similar biologically bound C and N levels (Tab. 1).
31.7 128 132 3.2 25.6 26.4
23
25
27
29
32
35
22.9 88 95 2.3 17.6 19
12.6 92 88 1.3 18.4 17.6
23.4 114 90 2.3 22.8 18
18.8 92 95 1.9 18.4 19
38.6 59 88 3.9 11.8 17.6
23.6 92 57 2.4 18.4 11.4
|j.g between days 5 and 7. Therefore, some N must also have been released in uric acid, proteins (Rogers 1969), or dead nematodes and shed cuticles (Woods et al. 1982). These organic compounds would eventually be broken down as the system became C limited and additional N would be mineralized as ammonium. Thus, systems with bacteriophagic nematodes not only should have a high C utilization (a result of nematode-bacterial interactions) but also should show a more rapid and significantly higher mineralization of N as ammonium than in identical systems without these grazers. The difference in NH4-N levels between the grazed and ungrazed treatments would continue until metabolic C limitations resulted in increased mineralization of NH4-N by the bacteria in the ungrazed treatment. The amino acid concentration in the microcosms was dependent on the population development of both the nematodes and bacteria. Amino acids are not likely to occur as a nematode waste product when the nematode population is starving. In contrast, under conditions of "luxury consumption", some elimination of amino acids from incompletely digested bacterial cells is possible.
3.2. Nitrogen dynamics
In the grazed system, where biomass decreased, N mineralization should have increased proportionately, since less N was biologically immobilized. Between days 5 and 7 total biomass (nematodes and bacteria) had decreased by 217.6 |ig C. Assuming the C:N ratios previously given, organism-bound nitrogen would have decreased by 49.3 pig N (Tab. 1). However, N measured in amino acids or NH4 increased by only approximately 15 78
Fig. 3. Amino acid-N and NH4-N in soil microcosms with bacteria or bacteria plus a bacterial-feeding nematode. • - - • NH4-N Bacteria alone. D- -D NH4-N Nematodes and bacteria, o O AA-N Bacteria alone. • • AA-N Nematodes and bacteria. OIKOS 40:1 (1983)
Amino acid-N in the grazed treatment on day 5 of the experiment was significantly higher than in the ungrazed treatment (Fig. 3). As the nematodes began to starve (after day 7), as evidenced by a population dominated by "dauer" juveniles and low bacterial numbers, an increasing amount of the returned N was NH4-N (Fig. 2). On the fifth day after inoculation, when bacterial numbers began to be affected by nematode grazing, yet nematode populations were still expanding, NH4-N and amino acid-N retum were nearly equal (Fig. 3). Free amino acid concentrations in soil are generally 2-4 |xg N/g soil, but addition of glucose and potassium nitrate can increase amino acid concentration to 100 \ig N/g soil (Paul and Schmidt 1961). Very httle amino acid-N was released by the bacteria alone in our experiment, the highest concentration (— 2 [ig N/g soil) occurring on day 5 (Fig. 3). On later days, however, almost all of the N retumed was NH4-N. Thus, the large amount of amino acid N observed in amended soils by Paul and Schmidt (1961) may have originated from nematodes or other grazers, rather than from bacteria. The source of the amino acids may be ruptured bacterial cells defecated from nematodes feeding under luxury consumption conditions. The system with nematodes also released significantly more NH4-N from day 9 onward and at a higher rate [note difference in slopes (Fig. 3)] than the ungrazed system. In the grazed system, N levels reached a plateau and approached the initial concentrations added to the microcosms. In the ungrazed system, bacteria gradually returned the N, probably as a result of increasing carbon limitation (Woods et al. 1982), since all the glucose is generally utilized in the first 2-4 days (Anderson et al. 1981b). 3.3. Implications for nntrient cycling This shift in composition of nitrogenous waste has two important implications for nutrient cycling in soils. First, the amino acids provide both an N and C source for decomposers. This may stimulate decomposition of other substrates. When environmental conditions such as temperature or moisture become limiting or the C substrate is in short supply, decomposer populations begin to decline. Nematode waste products then shift to an NHVN-rich material, which can be taken up by plants or decomposers. The nematodes potentially maintain a nutrient flux under varied environmental conditions affecting both primary decomposers and producers.
Acknowledgements - We thank Ms C. Ramsey for technical assistance. This work was supported by NSF grant numbers DEB 78-11201 and 79-19683, to Colorado State University.
OIKOS40:1 (1983)
References Anderson, R. V. and Coleman, D.C. 1981. Population development and interactions between two species of bacteriophagic nematodes. - Nematologica 27: 6-19. - , Elliott, E. T., McClellan, J. F., Coleman, D. C , Cole, C. V. and Hunt, H. W. 1978. Trophic interactions in soils as they affect energy and nutrient dynamics. III. Biotic interactions of bacteria, amoebae, and nematodes. - Microb. Ecol. 4: 361-371. - , Coleman, D. C. and Cole, C. V. 1981a. Effects of saprotrophic grazing on net mineralization. - In: Clark, F. E. and Rosswall, W. (eds). Terrestrial nitrogen cycles. Ecol. Bull. (Stockholm) No. 33, pp. 201-216. - , Coleman, D . C , Cole, C. V. and Elliott, E. T. 1981b. Effect of the nematodes Acrobeloides sp. and Mesodiplogaster Iheritieri on substrate utilization and nitrogen and phosphorus mineralization in soil. - Ecology 62: 549—555. Biith, E., Lohm, U., Lundgren, B., Rosswall, T., Soderstrom, B., Sohlenius, B. and Wiren, A. 1978. The effect of nitrogen and carbon supply on the development of soil organism populations and pine seedlings: a microcosm experiment. - Oikos 31: 153-163. Bryant, R. J., Woods, L. E., Coleman, D. C , Fairbanks, B. C , McClellan, J. F. and Cole, C. V. 1982. Interactions of bacterial and amoebal populations in soil microcosms with fluctuating moisture content. - Appl. Environ. Microbiol. 43: 747-752. Coleman, D. C , Cole, C. V.. Anderson, R. V.. Blaha. M., Campion, M. K., Clarholm, M.. Elliott, E. T.. Hunt, H. W., Schaefer, B. and Sinclair. J. 1977. Analysis of rhizosphere-saprohage interactions in terrestrial ecosystems. — In: Lohm, U. and Persson, T. (eds). Soil organisms as components of ecosystems. Ecol. Bull. (Stockholm) No. 25. pp. 299-309. - . Anderson. R. V., Cole, C. V., Elliott, E. T., Woods, L. and Campion, M. K. 1978. Trophic interactions is soils as they affect energy and nutrient dynamics. IV. Flows of metabolic and biomass carbon. - Microb. Ecol. 4: 373-380. Elliott, E. T., Coleman, D. C. and Cole, C. V. 1979. The influence of amoebae on the uptake of nitrogen by plants in gnobiotic soil. — In: Harley, J. L. and Russell, R. Scott (eds). The soil-root interface. Academic Press, London, pp. 221-229. - , Anderson, R. V.. Coleman, D. C. and Cole. C. V. 1980. Habitable pore space and microbial trophic interactions. — Oikos 35: 327-335. Hunt, H. W., Cole, C. V., Klein, D. A. and Coleman, D. C. 1977. A simulation model for the effect of predation on bacteria in continuous culture. — Microb. Ecol. 3: 259-278. Kirk, R. E. 1968. Experimental design: Procedures for the behavioral sciences. - Brooks/Cole, Belmont. California. Lee, D. L. and Atkinson, H. J. 1977. Physiology of nematodes, 2nd ed. — Columbia Univ. Press, New York. Ltichhead, A. G. and The.xton, R. H. 1947. Qualitative studies of soil micro-ornanisms. VII. The ""rhizosphere effect" in relation to the amino acid nutrition of bacteria. — Can. J. Res. 25: 20-26. Marchant, R. and Nicholas. W. L. 1974. An energy budget for the free-living nematode Petodera (Rhabditidae). — Oecologia (Berl.) 16: 237-252. Myers, R. F. and Krusberg, L. R. 1965. Organic substances discharged by plant parasitic nematodes. — Phytopathol. 55: 429—437. Nicholas, W. L. and Viswanathan, S. 1975. A study of the nutrition of Caenorhabditis briggsae (Rhabditidae) fed on ^''C and ^^P-labelled bacteria. - Nematologica 21: 385-400. Nielsen, C. O. 1949. Studies of the soil microfauna. II. Soil inhabiting nematodes. - Nat. Jutlandica 2: 1-131.
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Paul, E. A. and Schmidt, E. L. 1961. Formation of free amino acids in rhizosphere and nonrhizosphere soil. - Soil Sei. Soc. Am. Proc. 25: 359-362. Rogers, W. P. 1969. Nitrogenous components and their metabolism: Acanthocephala and Nematoda. - In: Florkin, M. and Scheer, B. T. (eds). Chemical zoology, vol. III. Academic Press, New York, pp. 3 7 9 ^ 2 8 . Rothstein, M. 1963. Nematode biochemistry. III. Excretion products. - Cump. Biochem. Physiol. 9: 51—59. Rovira, A. D. and McDougall, B. M. 1967. Microbiological and biochemical aspects of the rhizosphere. — In: McLaren, A. D. and Peterson, G. H. (eds). Soil biochemistry, vol. 1. Marcel Dckkcr, New York, pp. 4 1 7 ^ 6 3 . Sanger, F. 1945. The free amino groups of insulin. — Biochem. 39: 507-515. Smerda, S. M., Jensen, H. J. and Anderson, A. W. 1971. Escape of Salmonellae from chlorination during ingestion of Pristionchus Iheritieri (Nematoda: Diplogasterinae). - J. Nematol. 3: 201-204.
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Stanford, G., Carter, J. N., Simpson, L. M. and Schwaninger, D. E. 1973. Nitrate determination by a modified Conway microdiffusion method. - J. Assoc.Off. Anal. Chem. 56: 1365-1368. Twinn, D.C. 1974. Nematodes. - In: Dickinson, C. H. and Pugh, G. J. F. (eds). The biology of plant litter decomposition. Academic Press, New York, pp. 421-465. Woods, L. E., Cole, C. V., Elliott, E. T., Anderson, R. V. and Coleman, D. C. 1982. Nitrogen transformations in soil as affected by bacterial-microfaunal interactions. - Soil Biol. Biochem. 14: 93-98. Wright, D. J. 1975. Elimination of nitrogenous compounds by Panagrellus redivivus, Goodey 1945 (Nematoda:Cephalobidae). - Comp. Biochem. Physiol. 52B: 247-254. - and Newall, D. R. 1976. Nitrogen excretion, osmotic and ionic regulation in nematodes. - In: Croll, N. A. (ed.). The organization of nematodes. Academic Press, New York, pp. 163-210.
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