- Email: [email protected]
Soil macroinvertebrates, aldehyde oxidase, catalase, cellulase and peroxidase
0038-07 17/82/040387-05SO3.00/0 Copyright 8 1982 Pergamon Press Ltd
Soil Biol. Biochem. Vol. 14, pp. 387 to 391, 1982 Printed in Great Britain. All rights reserved
SOIL MACROINVERTEBRATES, ALDEHYDE OXIDASE, CATALASE, CELLULASE AND PEROXIDASE ROY HARTENSTEIN School of Biology, Chemistry and Ecology, State University College of Environmental Science and Forestry, Syracuse, NY 13210, U.S.A. (Accepted
20 September 1981)
Summary-Catalase and cellulase activities were found in each of 13 macroinvertebrates (soil isopods, diplopods, molluscs and oligochaetes) studied, peroxidase in all but one, aldehyde oxidase only in isopods and molluscs, and polyphenoloxidase in none. Optimum pH was obtained for each enzyme present in each species; specific activity at optimum pH was determined monthly from June 1976 to October 1977 except during cold months when the invertebrates had moved into subterranean sites. Specific activity of cellulase was significantly highest in molluscs, followed by oligochaetes, isopods and diplopods, the latter two having equal levels. This reflects a corresponding hierarchy in cellulose degradation per unit weight of invertebrate, and suggests that, within a given period, the taxon with the highest product of biomass per unit soil area or volume times specific activity may play a dominant role, amongst the four invertebrate taxa, in cellulolysis. Specific activity of peroxidase was significantly greater in earthworms than in any of the other three groups, which exhibited similar levels. This suggests a dominant role for earthworms in the condensation stage of the humification process. Specific activity of catalase was also highest in the oligochaetes.
INTRODUCTION
Little information is available on the specific contributions of soil isopods, diplopods, molluscs and oligochaetes to the heterotrophic decomposition process. An indisputable function is comminution of organic debris; the resulting increase in surface area facilitates, at least transiently, degradative activities of microbes. An example of this is the increase in rate of oxygen uptake and nucleic acid concentration in wormcasts versus sludge from which the castings were derived (Hartenstein and Hartenstein, 1981). In addition to comminution, the foregoing soil invertebrates are capable of demethylating and decomposing simple aromatic molecules, such as vanillin, but not complex molecules, such as synthetic lignins known as dehydrogenated polymerizates (Neuhauser et al., 1974, 1978; Neuhauser and Hartenstein, 1976a, b). My objective was to obtain quantitative data relating to certain enzymological capabilities of soil isopods, diplopods, molluscs and oligochaetes with respect to degradation or turnover of organic matter in general. Cellulase (EC 3.2.1.4) was studied because large amounts of cellulose are ingested by each of these taxa. Peroxidase (EC 1.11.7) was examined because of its possible role in detoxication of xenobiotics (Bartha et al., 1968), its function in protection of host plants against pathogens (Kosuge, 1969), its catalytic role in humification (Schnitzer, 1976), and its widespread presence in soil invertebrates (Wurzinger and Hartenstein, 1974). Aldehyde oxidase (EC 1.2.3.1) was considered because it is present in some soils (Libbert and Paetow, 1962) and may be implicated in oxidation of aldehydes, which are known to accumulate during heterotrophic decomposition, as reviewed
by Patrick (1971). The activity of catalase (EC 1.11.1.6) was measured because of its possible involvement in destroying H202 which might be present in some ingesta of the invertebrates, since HzOl is secreted by some fungi into decaying wood (Koenigs, 1974); and because higher amounts of organic matter in aerated soils are generally correlated with higher concentrations of catalase (Kiss et al., 1975). Tyrosinase or polyphenoloxidase (EC 1.10.3.1) was tested since it is generally implicated, like peroxidase, in the humification process (Mathur and Schnitzer, 1978).
387
METHODS AND MATERIALS
Invertebrates (Table l), collected from local woodlots, were used within a day. At least five animals of each species were pooled, homogenized in 50m~ potassium phosphate, pH 7.0, in a chilled mortar with a pestle, and centrifuged 10 min at 10,000 g. Optimum pH’s of the enzymes in the supernatant from initial samples of invertebrates were determined in buffers adjusted to 0.1 ionic strength with KCl. Enzyme activities of monthly samples (June-October, 1976 and April-October 1976; a total of 12 sampling dates) were tested at optimum pH (Table 2) at 25°C. Aldehyde oxidase, catalase and peroxidase activities are given in international units mg- ’ protein, while cellulase is expressed as (100) (set 20 min- 1 reaction mg- ’ protein), where set refers to time for control methylcellulose minus time required for enzyme-treated methylcellulose to pass the lower reference point on the viscometer. Catalase activity was tested with 27m~ Hz02 (Maehly and Chance, 1954), aldehyde oxidase with 0.27 mM vanillin (Hartenstein, 1973a), and peroxidase with 10 mM methyl hydrogen peroxide
388
ROY HARTENSTEIN Table 1. Enzymes present in invertebrates and pH of maximum rate or optimum range Enzymes Invertebrates
Aldehyde oxidase
Apheloria coriacea (Koch) Narceus americanus (Beauvios) Pseudopolydesmus serratus (Say)
ND’ ND ND 7.8
Catalase
Cellulase
Peroxidase
5.8-7.2(7.0)’ 6.48.4(7.0) 5.2-8.4(7.0)
4.8-6.4(4.6) 4.6 4.6
5.2-9.0(5.2) 4.68.4(5.8) 4.8-9.0(7.2)
7.8
5.8&7.q6.4) 7&9.0(7.2) 5.8%8.4(6.4)
4.&6.4(4.6) 4X&5.8(4.6) 4&5.8(4.6)
6/l-8.6(7.8) 4.6-7.9(7.8) 5.8%8.6(7.8)
7.8 7.8 7.8 ND
5.69.8(7.0) 5.69.4(7.0) 5.8-9.8(7.0) 5.8%10.4(7.0)
4.&5.8(4.6) 4-65.2(4.6) 4.&5.2(4.6) 4.65.2(4.6)
4.2-7.0(5.2) 4&7.0(5.2) l.23 ND
ND ND
5.8%10.4(7.8) 5.69.6(7.8)
[email protected](4.6) 4&5.8(4.6)
5.8%7.8(5.8) 4.67.8(5.8)
Diplopoda
Isopoda Armadillidium wlgare (Zatreille) Oniscus asellus L. Tracheoniscus rathkei (Brant)
7.8
Mollusca Arion hortensis Ferusac Deroceras reticulatum (Muller) Osychilus draparnaldi (Beck) Philomycus carolinianus (Bose)
Oligochaeta Eisenia,fbetida (Savigny) Lumhricus terrestris L.
’ Not detectable. ’ Values in parenthesis denote pH in graphic plot at which reaction rate appeared to be maximum. The two values which specify the range are points at which the reaction was 50% of the maximum rate. 3 Weak and sometimes questionable activity was found.
and 2rn~ guaiacol (Hartenstein, 1973b). The foregoing activities were measured kinetically in a Gilford Model 2@OO spectrophotometer on a 25.4cm chart. Cellulase activity was measured viscometrically with 2.5% 400 poise methylcellulose (Fisher Chem. Co.) in Ostwald viscometers. All measurements were duplicated; and additionally if the difference between the two values was not within about 5% of the mean. No inhibitor of potential microbial growth was used in the assays in view of the high relative centrifugal force
used to prepare the enzyme extracts, the short duration of assay, and retention of extracts at 5°C when not in cuvettes or viscometers. RESULTS Presence of enzyme activity and optimum pH range
Tyrosinase or polyphenoloxidase (based on reaction of extracts with L-dihydroxyphenylalanine at pH 7.0 and capability of measuring changes in optical
Table 2. Enzyme activities for individual species June-October, 1976 and April-October, on 12 data points, one for each month of assay
Invertebrates
1977. Values are % k SE, based
Aldehyde oxidase x1.U. ’ x 10,000 f SE
Enzymes’ Catalase x1.U. & SE
Cellulase XUnit x 100 f SE
ND3 ND ND
16 k4 16 If: 3 19 * 3
28 + 7t 12 + 2tt 28 * 5t
27 + 6 27 + 9 46 + 13
39 f 7 37 & 6 31 * 5
39 + 8 31 & 10 41 *9
75 * 13t 50 + 811 38 k 7tt
41 + 5 37 f 6 51 _+7
Peroxidase x1.U. x 10,000 f SE
Diplopoda A. coriacea N. americanus P. serratus
Isopoda A. uulgare 0. asellus T rathkei
Mollusca A. D. 0. P.
hortensis reticulatium draparnaldi carolinianus
22 f 2 34 f 6 26 + 3 ND
256 k 140 f 184 + 321 +
45t 19tt 21tt 56t
206 k 39tt 269 f 48tt 136 _+ lot 108 + 26t
28 _+4t 23 + 4t IO f. 2tt ND
Oligochaeta E. foetida L. terrestris 0. tyrtaeum
ND ND ND
238 + 39 314 + 58 432 f 80
153 f 21t 161 + 26t 93 * 14tt
1 Significant differences at 0.05 level denoted within a taxonomic group by constrasting between groups see text and Table 3. ’ International units, specific activity. 3 Not detectable.
494 * 94 378 + 45 551 + 78 superscripts;
for differences
389
Enzymes in soil invertebrates Phyletic diflerences
PH Fig. 1. Specific activity of aldehyde oxidase from 0. asellus in relation to pH. Acetate (0) and potassium phosphate (0).
in any of the invertebrates over 1 h in reaction vessels with up to 1 mg protein ml- ‘. Aldehyde oxidase was present only in each of the isopods and in three of the four molluscs (Table 1); maximum reactivity was seen in all cases at pH 7.8 (Fig. 1). Cellulase was active within a relatively-narrow acid range and its activity was greatest at pH 4.6 for most of the invertebrates (Table 1). Peroxidase and catalase reacted maximally over a relatively-wide range of pH, precluding the possibility in some cases of determining the most favorable value (Fig. 2). Peroxidase activity occurred maximally at an acid pH in the earthworms, molluscs and two of the three millipedes, and at an alkaline pH in the third millipede and the three isopods. Catalase activity was fastest at neutral pH in extracts from diplopods and molluscs, near-neutral with respect to isopods, and at an alkaline pH for earthworms. density of 10d3 units) was not detectable
B
Fig. 2. Specific activity of peroxidase from T rathkei (A) and catalase from E. foetida (B) in relation to pH. Acetate (0), potassium phosphate (0). Tris (hydroxymethyl) aminomethane (A), and glycine (M).
No significant difference was found for specific activity of aldehyde oxidase between species of isopods or between species of molluscs (Table 2). Nor was a significant difference obtained in comparing the three species of isopods as a single group against the four species of molluscs as a group (Table 3). Significant differences were found for specific activity of catalase between species of molluscs, but not between species of diplopods, isopods or earthworms (Table 2). Among the molluscs, A. hortensis and P. carolinianus exhibited higher values than D. reticulaturn and 0. draparnaldi. Earthworms collectively had a significantly higher specific activity than molluscs, which in turn displayed higher values then the diplopods and isopods, the latter two groups having statistically similar levels (Table 3). Significant interspecific differences were found for cellulase within each of the four major groups of invertebrates (Table 2). For each group as a whole, the specific activity of cellulase was greatest in mollusts, followed by earthworms, and significantly lower but statistically equal in isopods and diplopods (Table 3). Significant interspecific differences were found for peroxidase among the molluscs, but not within any of the other three invertebrate groups (Table 2). The specific activity of this enzyme was significantly greater in the earthworms than in the other three groups, which had statistically similar levels (Table 3).
DISCUSSION Specific activity rather than total number of enzymes units was used to evaluate the potential enzymological capabilities of the invertebrates; this
choice was forced into the study on technical grounds. The conclusions drawn, however, are valid, since the enzymes studied are soluble, as opposed to particulate. Activity levels can thus be compared directly from one invertebrate to another. Several comments are in order with respect to the results. First, the specific activity of catalase was very low in the diplopods and isopods, and very high in the molluscs and oligochaetes. Whether any biochemical or pedobiological correlation can be associated with this observation remains to be determined. Second, the specific activity of cellulase was significantly higher in the molluscs and oligochaetes than in isopods and diplopods. This suggests that the former two groups, especially if predominant in biomass, may be more effective than the latter two in degrading cellulosic components which fall to the ground. Isopods consume significantly larger quantities of decaying leaves per unit body weight than do diplopods, though possibly lesser quantities of wood (Neuhauser and Hartenstein, 1978). Possibly these organisms play a predominant role in trituration relative to oligochaetes and molluscs. Third, whatever the functions of the invertebrate peroxidases may be, oligochaetes displayed a higher specific activity than any of the other three invertebrate groups. It is not known whether the earthworm peroxidase is a single enzyme or a family of enzymes, some of which, like myeloperoxidase and salivary per-
ROY HARTFNSTEIN
390
Table 3. Enzyme activities for major groups of macroinvertebrates. same dimensions as in Table 2
Values have the
Enzymes’ Invertebrates
N2
Diplopoda Isopoda Mollusca Oligochaeta
36 36 48 36
Aldehyde oxidase
Catalase
Cellulase
Peroxidase
ND3 35 + 3 29 k 3
17 * 2t 37 * 5t 219 k 30tt
24 + 3t 54 k 6t 203 _+22tt
34 + 6t 43 * 3t 21 * 2t
ND
328 k 37ttt
138 k 12ttt
474 + 44tt
1 Significance at 0.05 level denoted by contrasting superscripts. * Values of X + SE are based on 12 data points, one for each month of assay, for
three species of diplopods collectively, three isopods, four molluscs and three oligochaetes. 3 Not detectable. oxidase of vertebrates, may catalyze microbicidal deaminase activities (Klebanoff, 1975), while others, such as vertebrate hepatic peroxidase of horseradish peroxidase, catalyze relatively non-specific (Saunders et al., 1964) reactions in which two electrons are removed from one or a pair of molecules in a two step sequence (Hartenstein, 1973b). Possibly both types of actions are effected, though neither crayfish peroxidase nor peroxidases from other invertebrates including Dermestes spp and isopods (unpublished data) have been shown to be capable of deaminating amino acids or proteins. If the function of the earthworm peroxidases is to catalyze non-specific substrate oxidations, it remains to be determined whether the peroxidases are effective solely within the animal’s body or within the castings as well. Kozlov (1965) reported peroxidase in earthworm castings but no attempt was made to determine whether the source was earthworm or microbial tissue. Nonetheless, since earthworms turn over more soil than any of the other groups studied here, their higher level of specific activity of peroxidase and high level of cellulase suggest a predominant role in the heterotrophic decomposition process and humification. The very low peroxidase activity in diplopods may be due to the presence of cyanide in their extracts (Wurzinger and Hartenstein, 1974), or perhaps it is simply present in small amounts, as in isopods and molluscs (Table 2). Fourth, the presence of aldehyde oxidase in isopods and certain molluscs raises the question of whether these organisms perform certain functions in the heterotrophic decomposition process which earthworms and diplopods are incapable of. Aldehyde oxidase reacts relatively nonspecifically with numerous aldehydes and purines, as does xanthine oxidase (Krenitsky et al., 1972); the latter is either not present or below the limit of detection in the four invertebrate groups studied here (Wurzinger and Hartenstein, 1974). The function of aldehyde oxidase in isopods and molluscs remains to be determined. Finally, in a study attempting to assess contributions of soil animals to soil enzymology, Kozlov (1965) reported (1) the presence of catalase and polyphenoloxidase, but not peroxidase, in caterpillar (Dendrolimus sibiricus) excrement, (2) more catalase, polyphenoloxidase and peroxidase activity in soil from an ant (Formicafusca) nest than from nearby soil, and (3) as much polyphenoloxidase, peroxidase and catalase
in extracts from soil inhabited by Eisenia nordenskioldi as in extracts from this earthworm (Kozlov’s comparisons were based on weight of material tested and the polyphenoloxidase activity was based on oxidation of reduced ascorbic acid). My findings and those of Kozlov, suggest that in addition to trituration, soil invertebrates contribute somewhat to enzy mological reactions in soil. Acknowledgements-Research was supported by the National Science Foundation. Technical help was provided by Dr E. F. Neuhauser, Mr F. Flack, and Mr D. Craig.
REFERENCES BARTHAR., LINKEH. A. B. and PRAMERD. (1968) Pesticide transformations: Production of chloroazobenzenes from chloroanilines. Science 161, 582-283. HARTENSTEIN R. (1973a) Characteristics of a uartiallv Durified aldehyde bxidask from the crayfish dambat&-bartoni. Comparative 87-93.
Biochemistry
and Physiology
45B,
HARTENSTEIN R. (1973b) Characteristics of a peroxidase from the fresh-water crayfish Cambarus bartoni. Comparative Biochemistry and Physiology 45B, 749-762.
HARTENSTEIN R. and HARTENSTEIN F. (1981) Physicochemical changes effected in activated sludge by the earthworm Eisenia foetida. Journal of Environmental Quality 10, 377-382. KISS S., DRXGAN-BULARDA M. and RADULESCUD. (1975) Enzymes accumulated in soil. Advances in Agronomy 27, 25-87.
KLEBANOFF S. J. (1975) Antimicrobial systems of the polymorphonuclear leucocyte. In The Phagocytic Cell in Host Resistance (J. A. Bellanti and D. H. Dayton, Eds), pp. 45-59. Raven Press, New York. KOENIGSJ. W. (1974) Production of hvdroaen oeroxide bv wood-rotting‘ fungi in wood and -its &riation with weight loss, depolymerization and pH changes. Archives of Microbiology 99, 129-145.
KOSUGET. (1969) The role of phenolics in host response to infection. Annual Review ofPhytopathology 7, 145-222. KOZLOVK. (1965) ijber die Rolle der Bodenfauna bei der Anreichung vob Fermenten im Boden. Pedobiologia 5, 14G145.
KRENITSKY T. A., NEIL S. M., ELION G. B. and HITCHINGS G. H. (1972) A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Archives of Biochemistry and Biophysics 150, 585-599. LIEBERTE. and PAETOWW. (1962) Untersuchungen iiber die enzymatische Hydrolyse von Indol-3-acetonitril und
Enzymes in soil invertebrates Oxydation von Indol-3-aldehyd in Erdboden und in rijher Milch. Flora, Jena 152,540-544. MAEHLYA. C. and CHANCEB. (1954) The assay of catalases and peroxidases. Methods of B~ochemicu~ Analysis 1, 357-424. MATHURS. P. and SCHNITZERM. (1978) A chemical and spectroscopic characterization of some synthetic analogues of humic acids. Journal. Soil Science Society of America 42, 591-596.
NEUHAUSER E. F. and HARTENSTEIN R. (1976a) On the presence of 0-demethylase activity invertebrates. Comparative B~ochemisfry and Physiology 536, 37-39. NEUHAUSER E. F. and HARTENSTEIN R. (1976b) Degradation of phenol, cinnamic and quinic acid in the terrestrial crustacean, Oniscus asellus. Soil Biology & Biochemistry 8, 95-98. NEUHALJSER E. F. and HARTENSTEIN R. (1978) Phenolic content and palatability of leaves and wood to soil isopods and diplopods. Pedob~o~og~uIS, 99-109. NEUHAUSER E. F., YOUMELLC. and HARTENSTEIN R. (1974)
391
Degradation of benzoic acid in the terrestrial crustacean, Oniscus asellus. Soil Biology & Biochemistry 6, 101-107. NEUHAUSER E. F., HARTENSTE~N R. and CONNORSJ. (1978) The role of soil macroinvertebrates in the degradation of vanillin, cinnamic acid, and lignins. Soil Biology & Biochemistry 10, 431-435.
PATRICK Z. A. (1971) Phytotoxic substances associated with the decomposition in soil of plant residues. Proceedings. Soil Science Society of America 111, 13-18. SAUNDERSB. C., HOLMES-SIEDLE A. G. and STARK B. P. (1964) Peroxidase. ~utterworths, Washington. SCHNITZERM. (1976) The chemistry of humic substances. In Environmental Biogeochemistry, Vol. 1, (J. 0. Nriagu, Ed.), pp. 89-107. Ann Arbor Publishers, Ann Arbor. WURZINGERK. H. and HARTENSTEIN R. (1974) Phylogeny and correlations of aldehyde oxidase, xanthine oxidase, xanthine dehydrogenase and peroxidase in animal tissues. Comparative Biocbem~stry and Phys~o~og~l488, 171-185.
Soil Biol. Biochem. Vol. 14, pp. 387 to 391, 1982 Printed in Great Britain. All rights reserved
SOIL MACROINVERTEBRATES, ALDEHYDE OXIDASE, CATALASE, CELLULASE AND PEROXIDASE ROY HARTENSTEIN School of Biology, Chemistry and Ecology, State University College of Environmental Science and Forestry, Syracuse, NY 13210, U.S.A. (Accepted
20 September 1981)
Summary-Catalase and cellulase activities were found in each of 13 macroinvertebrates (soil isopods, diplopods, molluscs and oligochaetes) studied, peroxidase in all but one, aldehyde oxidase only in isopods and molluscs, and polyphenoloxidase in none. Optimum pH was obtained for each enzyme present in each species; specific activity at optimum pH was determined monthly from June 1976 to October 1977 except during cold months when the invertebrates had moved into subterranean sites. Specific activity of cellulase was significantly highest in molluscs, followed by oligochaetes, isopods and diplopods, the latter two having equal levels. This reflects a corresponding hierarchy in cellulose degradation per unit weight of invertebrate, and suggests that, within a given period, the taxon with the highest product of biomass per unit soil area or volume times specific activity may play a dominant role, amongst the four invertebrate taxa, in cellulolysis. Specific activity of peroxidase was significantly greater in earthworms than in any of the other three groups, which exhibited similar levels. This suggests a dominant role for earthworms in the condensation stage of the humification process. Specific activity of catalase was also highest in the oligochaetes.
INTRODUCTION
Little information is available on the specific contributions of soil isopods, diplopods, molluscs and oligochaetes to the heterotrophic decomposition process. An indisputable function is comminution of organic debris; the resulting increase in surface area facilitates, at least transiently, degradative activities of microbes. An example of this is the increase in rate of oxygen uptake and nucleic acid concentration in wormcasts versus sludge from which the castings were derived (Hartenstein and Hartenstein, 1981). In addition to comminution, the foregoing soil invertebrates are capable of demethylating and decomposing simple aromatic molecules, such as vanillin, but not complex molecules, such as synthetic lignins known as dehydrogenated polymerizates (Neuhauser et al., 1974, 1978; Neuhauser and Hartenstein, 1976a, b). My objective was to obtain quantitative data relating to certain enzymological capabilities of soil isopods, diplopods, molluscs and oligochaetes with respect to degradation or turnover of organic matter in general. Cellulase (EC 3.2.1.4) was studied because large amounts of cellulose are ingested by each of these taxa. Peroxidase (EC 1.11.7) was examined because of its possible role in detoxication of xenobiotics (Bartha et al., 1968), its function in protection of host plants against pathogens (Kosuge, 1969), its catalytic role in humification (Schnitzer, 1976), and its widespread presence in soil invertebrates (Wurzinger and Hartenstein, 1974). Aldehyde oxidase (EC 1.2.3.1) was considered because it is present in some soils (Libbert and Paetow, 1962) and may be implicated in oxidation of aldehydes, which are known to accumulate during heterotrophic decomposition, as reviewed
by Patrick (1971). The activity of catalase (EC 1.11.1.6) was measured because of its possible involvement in destroying H202 which might be present in some ingesta of the invertebrates, since HzOl is secreted by some fungi into decaying wood (Koenigs, 1974); and because higher amounts of organic matter in aerated soils are generally correlated with higher concentrations of catalase (Kiss et al., 1975). Tyrosinase or polyphenoloxidase (EC 1.10.3.1) was tested since it is generally implicated, like peroxidase, in the humification process (Mathur and Schnitzer, 1978).
387
METHODS AND MATERIALS
Invertebrates (Table l), collected from local woodlots, were used within a day. At least five animals of each species were pooled, homogenized in 50m~ potassium phosphate, pH 7.0, in a chilled mortar with a pestle, and centrifuged 10 min at 10,000 g. Optimum pH’s of the enzymes in the supernatant from initial samples of invertebrates were determined in buffers adjusted to 0.1 ionic strength with KCl. Enzyme activities of monthly samples (June-October, 1976 and April-October 1976; a total of 12 sampling dates) were tested at optimum pH (Table 2) at 25°C. Aldehyde oxidase, catalase and peroxidase activities are given in international units mg- ’ protein, while cellulase is expressed as (100) (set 20 min- 1 reaction mg- ’ protein), where set refers to time for control methylcellulose minus time required for enzyme-treated methylcellulose to pass the lower reference point on the viscometer. Catalase activity was tested with 27m~ Hz02 (Maehly and Chance, 1954), aldehyde oxidase with 0.27 mM vanillin (Hartenstein, 1973a), and peroxidase with 10 mM methyl hydrogen peroxide
388
ROY HARTENSTEIN Table 1. Enzymes present in invertebrates and pH of maximum rate or optimum range Enzymes Invertebrates
Aldehyde oxidase
Apheloria coriacea (Koch) Narceus americanus (Beauvios) Pseudopolydesmus serratus (Say)
ND’ ND ND 7.8
Catalase
Cellulase
Peroxidase
5.8-7.2(7.0)’ 6.48.4(7.0) 5.2-8.4(7.0)
4.8-6.4(4.6) 4.6 4.6
5.2-9.0(5.2) 4.68.4(5.8) 4.8-9.0(7.2)
7.8
5.8&7.q6.4) 7&9.0(7.2) 5.8%8.4(6.4)
4.&6.4(4.6) 4X&5.8(4.6) 4&5.8(4.6)
6/l-8.6(7.8) 4.6-7.9(7.8) 5.8%8.6(7.8)
7.8 7.8 7.8 ND
5.69.8(7.0) 5.69.4(7.0) 5.8-9.8(7.0) 5.8%10.4(7.0)
4.&5.8(4.6) 4-65.2(4.6) 4.&5.2(4.6) 4.65.2(4.6)
4.2-7.0(5.2) 4&7.0(5.2) l.23 ND
ND ND
5.8%10.4(7.8) 5.69.6(7.8)
[email protected](4.6) 4&5.8(4.6)
5.8%7.8(5.8) 4.67.8(5.8)
Diplopoda
Isopoda Armadillidium wlgare (Zatreille) Oniscus asellus L. Tracheoniscus rathkei (Brant)
7.8
Mollusca Arion hortensis Ferusac Deroceras reticulatum (Muller) Osychilus draparnaldi (Beck) Philomycus carolinianus (Bose)
Oligochaeta Eisenia,fbetida (Savigny) Lumhricus terrestris L.
’ Not detectable. ’ Values in parenthesis denote pH in graphic plot at which reaction rate appeared to be maximum. The two values which specify the range are points at which the reaction was 50% of the maximum rate. 3 Weak and sometimes questionable activity was found.
and 2rn~ guaiacol (Hartenstein, 1973b). The foregoing activities were measured kinetically in a Gilford Model 2@OO spectrophotometer on a 25.4cm chart. Cellulase activity was measured viscometrically with 2.5% 400 poise methylcellulose (Fisher Chem. Co.) in Ostwald viscometers. All measurements were duplicated; and additionally if the difference between the two values was not within about 5% of the mean. No inhibitor of potential microbial growth was used in the assays in view of the high relative centrifugal force
used to prepare the enzyme extracts, the short duration of assay, and retention of extracts at 5°C when not in cuvettes or viscometers. RESULTS Presence of enzyme activity and optimum pH range
Tyrosinase or polyphenoloxidase (based on reaction of extracts with L-dihydroxyphenylalanine at pH 7.0 and capability of measuring changes in optical
Table 2. Enzyme activities for individual species June-October, 1976 and April-October, on 12 data points, one for each month of assay
Invertebrates
1977. Values are % k SE, based
Aldehyde oxidase x1.U. ’ x 10,000 f SE
Enzymes’ Catalase x1.U. & SE
Cellulase XUnit x 100 f SE
ND3 ND ND
16 k4 16 If: 3 19 * 3
28 + 7t 12 + 2tt 28 * 5t
27 + 6 27 + 9 46 + 13
39 f 7 37 & 6 31 * 5
39 + 8 31 & 10 41 *9
75 * 13t 50 + 811 38 k 7tt
41 + 5 37 f 6 51 _+7
Peroxidase x1.U. x 10,000 f SE
Diplopoda A. coriacea N. americanus P. serratus
Isopoda A. uulgare 0. asellus T rathkei
Mollusca A. D. 0. P.
hortensis reticulatium draparnaldi carolinianus
22 f 2 34 f 6 26 + 3 ND
256 k 140 f 184 + 321 +
45t 19tt 21tt 56t
206 k 39tt 269 f 48tt 136 _+ lot 108 + 26t
28 _+4t 23 + 4t IO f. 2tt ND
Oligochaeta E. foetida L. terrestris 0. tyrtaeum
ND ND ND
238 + 39 314 + 58 432 f 80
153 f 21t 161 + 26t 93 * 14tt
1 Significant differences at 0.05 level denoted within a taxonomic group by constrasting between groups see text and Table 3. ’ International units, specific activity. 3 Not detectable.
494 * 94 378 + 45 551 + 78 superscripts;
for differences
389
Enzymes in soil invertebrates Phyletic diflerences
PH Fig. 1. Specific activity of aldehyde oxidase from 0. asellus in relation to pH. Acetate (0) and potassium phosphate (0).
in any of the invertebrates over 1 h in reaction vessels with up to 1 mg protein ml- ‘. Aldehyde oxidase was present only in each of the isopods and in three of the four molluscs (Table 1); maximum reactivity was seen in all cases at pH 7.8 (Fig. 1). Cellulase was active within a relatively-narrow acid range and its activity was greatest at pH 4.6 for most of the invertebrates (Table 1). Peroxidase and catalase reacted maximally over a relatively-wide range of pH, precluding the possibility in some cases of determining the most favorable value (Fig. 2). Peroxidase activity occurred maximally at an acid pH in the earthworms, molluscs and two of the three millipedes, and at an alkaline pH in the third millipede and the three isopods. Catalase activity was fastest at neutral pH in extracts from diplopods and molluscs, near-neutral with respect to isopods, and at an alkaline pH for earthworms. density of 10d3 units) was not detectable
B
Fig. 2. Specific activity of peroxidase from T rathkei (A) and catalase from E. foetida (B) in relation to pH. Acetate (0), potassium phosphate (0). Tris (hydroxymethyl) aminomethane (A), and glycine (M).
No significant difference was found for specific activity of aldehyde oxidase between species of isopods or between species of molluscs (Table 2). Nor was a significant difference obtained in comparing the three species of isopods as a single group against the four species of molluscs as a group (Table 3). Significant differences were found for specific activity of catalase between species of molluscs, but not between species of diplopods, isopods or earthworms (Table 2). Among the molluscs, A. hortensis and P. carolinianus exhibited higher values than D. reticulaturn and 0. draparnaldi. Earthworms collectively had a significantly higher specific activity than molluscs, which in turn displayed higher values then the diplopods and isopods, the latter two groups having statistically similar levels (Table 3). Significant interspecific differences were found for cellulase within each of the four major groups of invertebrates (Table 2). For each group as a whole, the specific activity of cellulase was greatest in mollusts, followed by earthworms, and significantly lower but statistically equal in isopods and diplopods (Table 3). Significant interspecific differences were found for peroxidase among the molluscs, but not within any of the other three invertebrate groups (Table 2). The specific activity of this enzyme was significantly greater in the earthworms than in the other three groups, which had statistically similar levels (Table 3).
DISCUSSION Specific activity rather than total number of enzymes units was used to evaluate the potential enzymological capabilities of the invertebrates; this
choice was forced into the study on technical grounds. The conclusions drawn, however, are valid, since the enzymes studied are soluble, as opposed to particulate. Activity levels can thus be compared directly from one invertebrate to another. Several comments are in order with respect to the results. First, the specific activity of catalase was very low in the diplopods and isopods, and very high in the molluscs and oligochaetes. Whether any biochemical or pedobiological correlation can be associated with this observation remains to be determined. Second, the specific activity of cellulase was significantly higher in the molluscs and oligochaetes than in isopods and diplopods. This suggests that the former two groups, especially if predominant in biomass, may be more effective than the latter two in degrading cellulosic components which fall to the ground. Isopods consume significantly larger quantities of decaying leaves per unit body weight than do diplopods, though possibly lesser quantities of wood (Neuhauser and Hartenstein, 1978). Possibly these organisms play a predominant role in trituration relative to oligochaetes and molluscs. Third, whatever the functions of the invertebrate peroxidases may be, oligochaetes displayed a higher specific activity than any of the other three invertebrate groups. It is not known whether the earthworm peroxidase is a single enzyme or a family of enzymes, some of which, like myeloperoxidase and salivary per-
ROY HARTFNSTEIN
390
Table 3. Enzyme activities for major groups of macroinvertebrates. same dimensions as in Table 2
Values have the
Enzymes’ Invertebrates
N2
Diplopoda Isopoda Mollusca Oligochaeta
36 36 48 36
Aldehyde oxidase
Catalase
Cellulase
Peroxidase
ND3 35 + 3 29 k 3
17 * 2t 37 * 5t 219 k 30tt
24 + 3t 54 k 6t 203 _+22tt
34 + 6t 43 * 3t 21 * 2t
ND
328 k 37ttt
138 k 12ttt
474 + 44tt
1 Significance at 0.05 level denoted by contrasting superscripts. * Values of X + SE are based on 12 data points, one for each month of assay, for
three species of diplopods collectively, three isopods, four molluscs and three oligochaetes. 3 Not detectable. oxidase of vertebrates, may catalyze microbicidal deaminase activities (Klebanoff, 1975), while others, such as vertebrate hepatic peroxidase of horseradish peroxidase, catalyze relatively non-specific (Saunders et al., 1964) reactions in which two electrons are removed from one or a pair of molecules in a two step sequence (Hartenstein, 1973b). Possibly both types of actions are effected, though neither crayfish peroxidase nor peroxidases from other invertebrates including Dermestes spp and isopods (unpublished data) have been shown to be capable of deaminating amino acids or proteins. If the function of the earthworm peroxidases is to catalyze non-specific substrate oxidations, it remains to be determined whether the peroxidases are effective solely within the animal’s body or within the castings as well. Kozlov (1965) reported peroxidase in earthworm castings but no attempt was made to determine whether the source was earthworm or microbial tissue. Nonetheless, since earthworms turn over more soil than any of the other groups studied here, their higher level of specific activity of peroxidase and high level of cellulase suggest a predominant role in the heterotrophic decomposition process and humification. The very low peroxidase activity in diplopods may be due to the presence of cyanide in their extracts (Wurzinger and Hartenstein, 1974), or perhaps it is simply present in small amounts, as in isopods and molluscs (Table 2). Fourth, the presence of aldehyde oxidase in isopods and certain molluscs raises the question of whether these organisms perform certain functions in the heterotrophic decomposition process which earthworms and diplopods are incapable of. Aldehyde oxidase reacts relatively nonspecifically with numerous aldehydes and purines, as does xanthine oxidase (Krenitsky et al., 1972); the latter is either not present or below the limit of detection in the four invertebrate groups studied here (Wurzinger and Hartenstein, 1974). The function of aldehyde oxidase in isopods and molluscs remains to be determined. Finally, in a study attempting to assess contributions of soil animals to soil enzymology, Kozlov (1965) reported (1) the presence of catalase and polyphenoloxidase, but not peroxidase, in caterpillar (Dendrolimus sibiricus) excrement, (2) more catalase, polyphenoloxidase and peroxidase activity in soil from an ant (Formicafusca) nest than from nearby soil, and (3) as much polyphenoloxidase, peroxidase and catalase
in extracts from soil inhabited by Eisenia nordenskioldi as in extracts from this earthworm (Kozlov’s comparisons were based on weight of material tested and the polyphenoloxidase activity was based on oxidation of reduced ascorbic acid). My findings and those of Kozlov, suggest that in addition to trituration, soil invertebrates contribute somewhat to enzy mological reactions in soil. Acknowledgements-Research was supported by the National Science Foundation. Technical help was provided by Dr E. F. Neuhauser, Mr F. Flack, and Mr D. Craig.
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