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Cellobiase activity in three species of Australian termites
Insect Biochem., Vol. 10, pp. 563 to 567. © Pergamon Press Ltd. 1980. Printed in Great Britain.
0020-1700/80/1001--0563 $02.00/0
CELLOBIASE ACTIVITY IN THREE SPECIES OF AUSTRALIAN TERMITES SANDRA E. McEWEN, M. SLAYTOR and R. W. O'BRIEN Department of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia (Received 13 December 1979)
A~traet--The cellobiase of the higher termite Nasutitermes exitiosus was located in the foregut (8%) and the midgut (92%). In N. walkeri 97% of the activity was in the midgut, 2% in the foregttt and 1% in the mixed segment. Most of the cellobiase activity in the lower termite Coptotermes lacteus was in the hindgut (78%) and the midgut (21%) with traces (1%) in the foregut. Removal of the protozoa and spirochaetes by oxygen treatment resulted in loss of most of the cellulase and cellobiase from the hindgnt within 28 hr of treatment; the midgut enzymes were unaffected. Isolated protozoa had cellulase and cellobiase of high specific activity and accounted for most of the total ceUulolytic activity in the hindgut. These results indicate that the enzymes in the midgut are secreted by all three termites, whereas the enzymes in the hindgut (C. lacteus only) are of protozoal origin. The optimum pH for cellobiase from N. exitiosus (foregut and midgut).ranged from 4.5 to 7.0; the C. lacteas enzymes had an optimum activity at pH 5.0-6.0. The pH ofN. exitiosas gut ranged from 2.0 to 2.8 (foregut), 2.8 to 3.8 (rectum), and from 6.8 to 7.5 (midgut and mixed segment). The pH of the midgut and hindgut of C. lacteus was 6.8--7.4 and foregut was 3.8--4.4. The significance of the gut pH on cellobiase activity is discussed. Crude preparations of C. lacteus cellobiase (both midgut and hindgut) were inactivated at 62°C and were competitively inhibited by 1,5-gluconolactone. Key Word Index: Termites, cellobiase, cellulase, protozoa, Nasutitermes exitiosus, Nasutitermes walkeri, Coptotermes lacteas
INTRODUCTION THE DIGESTION of cellulose in termites has been the subject of many investigations mainly to determine the role of the gut micro-organisms in the initial hydrolysis of cellulose. A major product from the action of cellulase on cellulose is the disaccharide cellobiose, which can be hydrolysed to glucose by cellobiase or non-specific//-glucosidases. Cellobiase activity has been found in the guts of worker termites of Trinervitermes trinervoides (PoTTS and HEWITT, 1973) and in the guts of workers, larvae and soldiers of Hodotermes mossambicus (RETIEFF and HEWITT, 1973). In H. mossambicus the greatest concentration of the enzyme activity was found in the hindgut of the larvae and in the midgut of the soldiers. Bacteria, capable of utilising cellobiose as a carbon source, have been isolated from various termites (EuTICK et al., 1978a; THAYER, 1976), but their role in cellobiose metabolism is not known. In this paper we describe the occurrence and distribution of cellobiase activity in the gut of the higher termites, Nasutitermes exitiosus and N. walkeri, and of cellulase and cellobiase activity in the lower termite Coptotermes lacteus. MATERIALS AND METHODS
collected at Greenwich, N.S.W. The termites were maintained in the laboratory as described by EUTICKet al. (1976). Worker caste termites were used in all experiments. Cellobiase assay The guts of twenty-five termites were dissected into foregut, midgut, mixed segment (posterior midgut + anterior hindgut, found only in the higher termites, N. exitiosus and N. walkerO, and hindgut. The segments were homogenized separately in 2 ml 0.1 M acetate buffer, pH 5.0, in a Ten Broeck homogenizer and diluted to a final volume of 2.5 ml. The homogenates were centrifuged at 20,000 g for 10 min and the supernatants were assayed for cellobiase activity. All operations were carried out at 0--4°C. The supernatants (0.5 ml) were incubated with 1.0 ml 1% (w/v) cellobiose in 0.1 M acetate buffer, pH 5.0, for 1 hr at 37°C. The enzyme activity was linear over this period. The amount of glucose released was measured by the glucose oxidase method (Sigma Tech. Bulletin No. 510) with the enzymes dissolved in 0.5 M Tris buffer, pH 7.0, to inhibit disaccharidases in the glucose---oxidase preparation (DAmOXrtST, 1968). The cellobiase activity was corrected by subtracting the residual activity present in the extract after it had been heated for 10 rain at 100°C. The protein content was determined by the method of LOWRYet al. (1951 ) using bovine gamma-globulin (Sigma) as a standard. One unit of cellobiase activity is defined as the amount of enzyme which produced 1 mg of glucose per hr per ml of extract. Specific activity is defined as mg glucose produced per hr per mg protein. The optimum pH of the cellobiase of the various gut sections was determined by assaying the enzyme, as described above, using Mcllvaine citrate-phosphate buffers.
Termites Ground nests of the lower termite C. lacteus Froggatt were Cellulase assay collected at Bilpin, N.S.W., and the Ourimbah State Forest, Gut segments were homogenized with 2.5 ml of near Wyong, N.S.W. Ground nests of the higher termite N. exitiosus Hill were collected from the Ku-ring-gai National 0.1 M acetate buffer, pH 5.5, and centrifuged at Park, near Sydney and arboreal nests of N. walkeri Hill were 20,000 g for 10 rain. The supernatants (0.5 ml) were incubated 563
564
SANDRAE. McEWEN, M. SLAYTORAND R. W. O'BRIEN
with 1.0 ml of 1% (w/v) sodium carboxymethylcellulose for 30 min at 37°C. The incubation mixture was assayed for reducing sugar according to the method of SOMOGW(1952) using glucose as a standard. One unit of cellulase activity is defined as the amount of enzyme which produced 1 mg of reducing sugar (expressed as glucose) per hr per ml of extract. Specific activity is defined as mg of glucose produced per hr per mg of protein. Total nitrogen determination Ten termites were digested according to the Kjeldahl method using potassium sulphate and mercuric oxide (JACoBS, 1965) and the ammonia produced was assayed by the phenol/hypochlorite method (JAcoBs, 1965). pH of the termite gut Termites were fed wood impregnated with pH indicators as described by EUTICKet al. (1976). After 24 hr, the guts were dissected and examined for location and colour of the dyes. The guts were then treated with acid or alkali to ensure that the indicators had retained their acid-base colour change. The pH indicators used are listed in Table 1. Although no attempt was made to determine whether the indicators were toxic to the termite symbionts, guts of treated termites were similar to those of untreated termites and did not show any signs of shrinkage, which occurs if gut protozoa are killed by starvation or oxygen treatment. Removal of protozoa Protozoa and spirochaetes were killed by subjecting termites to oxygen in a pressure vessel at 2.53 × 102 kPa for 2 hr. The survival of protozoa and spirochaetes was determined by dissecting out the gut of several termites into distilled water and examining the suspension under phasecontrast microscopy. Bacteria were isolated and counted as described by EUT]CK et al. (1978b). Plates were incubated under anaerobic conditions for 48 hr at 30°C using a BBL Gas-Pak system (Becton, Dickinson and Co., Cockeysville, Maryland, U.S.A.) in an anaerobe jar. Isolation of protozoa The paunch section of the hindgut was ruptured and the protozoa allowed to diffuse into 0.05 ml of the medium of TRAGER (1934). The paunch was then washed with an additional 0.05 ml of the same medium and added to the original suspension. The protozoa from twenty termites were separated from the other gut contents by allowing them to settle and decanting the medium. They were washed twice by resuspension in fresh medium. The extract and the washes were examined separately under phase-contrast microscopy for the presence of bacteria, spirochaetes, protozoa and gut food particles. The protozoa were then homogenized in 0.1 M acetate buffer, pH 5.0, in a Ten Broeck homogenizer and diluted to a final volume of 4 ml with the same buffer. The homogenate was centrifuged at 20,000 g for 10 min at 5°C and the superantant was used for enzyme assays.
RESULTS Following our success in measuring the distribution of cellulase activity in the different sections of the guts o f N . exitiosus and C. lacteus (O'BRtEN et al., 1979), it became of interest to study the distribution of cellobiase in the guts of these termites to obtain a better overall understanding of cellulose digestion. Initially the effect of p H on cellobiase activity of extracts of N. exitiosus and C. lacteus was determined (Fig. 1). tn extracts o f the foregut and midgut of N. exitiosus, maximal activity was observed over the p H range of 4.5-7.0. In extracts of C. lacteus, maximal
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Fig. 1. Effect ofpH on cellobiase activity in the gut sections of (a) N. exitiosus and (b) C. lacteus, using Mcllvaine citrate-phosphate buffers. O, Foregut; I-q, Midgut; &, Hindgut. Points on the curve represent the average of two determinations, both giving the same pH optima. activity occurred over a narrower pH range, from about 5.0 to 6.0. A p H optimum of 5.0--5.5 seemed low when compared with the p H of the gut which is around neutrality (EUTICK et al., 1976). For this reason, the p H of the gut of N. exitiosus and C. lacteus was measured. The results (Table 1) show that the foregut and rectum of both species were very acid, whereas the pH of other sections of the gut was indeed near neutrality. The distribution of cellobiase (measured at pH 5.0) throughout the gut of N. exitiosus, N. walkeri and C. lacteus is shown in Table 2. Most of the ceUobiase activity (92%) of N. exitiosus was located in the midgut, with no activity in the mixed segment or hindgut sections. A similar distribution of cellobiase activity was observed in N. walkeri, with 97% of the total activity in the midgut. In C. lacteus, only 1% of the total activity was found in the foregut, whereas 21% was found in the midgut, and 78% in the hindgut where the protozoa and the bacteria are located. To determine the origin of the cellobiase, the protozoa (and the spirochaetes) were killed by treating the termites with oxygen. The termites were then assayed for cellulase and cellobiase activity. The conditions required to kill the protozoa and spirochaetes, but not the termites, were determined experimentally since BREADY and FRIEDMAN (1963) reported that oxygen above a partial pressure of 50% in carbon dioxide was poisonous, not only to gut protozoa, but to termites as well. Two hours was the minimum time required to kill all the protozoa and spriochaetes using 100% oxygen at 2.53 x 10 z kPa pressure. Preliminary experiments showed that 80-85% of C. lacteus were alive three days after this
1.08 _+ 0.52 2.37 +_ 0.74 --
Foregut Midgut M i x e d segment Hindgut
6.8-7.5
7.0-7.5
> 4.4 > 5.4 > 6.8 ~ 7.0 7.0-8.0
< 8.0
Midgut
7.0-7.5
7.0-7.5
_>4.4 a 6.5-6.8 ~ 7.0 7.0-8.0
< 8.0
~6.8
7.0-7.5
>_4.4 >_ 5.4 < 6.8 ~ 7.0 < 7.0
< 8.0
Paunch colon
2.8-3.8
< 6.6
_> 3.2 < 3.8 < 5.2 < 6.0 > 2.0
< 2.8
Rectum
4.4 3.8 5.2 6.0 2.0
3.8-4.4
< 6.6
> < < < >
> 2.8
Foregut
0.01 _+ 0.006 0.11 _+ 0.01 ---
N. exitiosus* Units§ 8 _+ 5 92 + 6 --
~o" 1.14 + 0.68 7.55 -+ 1.38 0.14 _+ 0.06
Sp. act.
--
0.02 _+ 0.01 1.11 + 0.35 0.01 _+ 0.005
C e l l o b i a s e activity N. walkeri't Units
Sp. act. 3.67 _____0.64 25.86 + 4.57 -25.86 _+ 3.55
2 + 1.8 97 + 2 1__+0.3 --
6.8-7.4
a
> 4.4 > 5.4 > 6.8 ~ 7.0 7.0-7.4
< 8.0
0.05 + 0.02 1.17 + 0.18 -4.10 _+ 0.30
C. lacteus* Units
6.8-7.4
~ 7.0
> 4.4 > 5.4 > 6.8 ~ 7.0 7.0-7.4
< 8.0
C. lacteus Paunch Midgut colon
~o
T a b l e 2. D i s t r i b u t i o n o f cellobiase activity in the gut o f N. exitiosus, N. walkeri a n d C. lacteus
2.0-2.8
< 6.6
< 3.2 < 3.8 < 5.2 < 6.0 _>2.0
< 2.8
Foregut
*Activities were d e t e r m i n e d on four different g r o u p s o f termites (_+ S.E.M.). t Activities were m e a n values o b t a i n e d f r o m t w o e x p e r i m e n t s (_+ range). :~Activities were d e t e r m i n e d o n seven different g o u p s o f termites (_+ S.E.M.). § Sp. act = Specific activity (defined in the text), U n i t s also defined in text. "Values are percentage of the total activity e x p r e s s e d as units.
Sp. act.§
1.2-2.8 8.0-9.6 3.2-4.4 3.8-5.4 5.2-6.8 6.0-7.6 1.0-2.0 7.0-8.6 6.6-8.0
pH Range
G u t Section
a I n d e t e r m i n a t e result.
Estimated pH
P h e n o l red
Methyl orange B r o m o c r e s o l green Bromocresol purple B r o m o t h y m o l blue Cresol red
T h y m o l blue
Indicator
N. exitiosus Mixed segment
T a b l e 1. p H o f the gut o f N. exitiosus a n d C. lacteus
1 +__ 0.3 21 + 3 -78 +__ 3
Vo
2.8-3.2
< 6.6
< 3.2 < 3.8 < 5.2 < 6.0 >_ 2.0
> 2.8
Rectum
566
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SANDRAE. MCEwEN, M. SLAYTORAND R. W. O'BRIEN
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Table 3. Cellulase and cellobiase activity in extracts of gut protozoa
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13.5 3.3 4.4
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were 2.82 mM and 200/~mol glucose per hr per mg protein, respectively. The competitive inhibitor, 1,5gluconolactone, increased the K,, of both enzymes fourfold and had no effect on the V~x o f the midgut enzyme, but decreased that of the hindgut enzyme to 143 #mol glucose per hr per mg protein.
48
hr
Fig. 2. Effect of oxygen treatment of cellulase (0) and cellobiase (O) activity in (a) the midgut and (b) the hindgut of C. lacteus. Gut extracts were assayed before oxygen treatment (0 hr), and at various times after 2 hr oxygen treatment. The experiment was carried out twice giving essentially the same result. The results in this figure are from one of these experiments.
treatment. Plating of the gut contents of oxygentreated termites as described in Materials and Methods showed that the bacterial numbers (except spirochaetes) were unaffected by the oxygen treatment. The effect of oxygen treatment on cellulase and cellobiase activity in the hindgut of C. lacteus is shown in Fig. 2. The decline in cellobiase activity was apparent 3 hr after oxygen treatment and continued to decline, until at 28 hr only 15% of the original activity remained. Cellulase activity underwent a similar decrease and was completely lost by 48 hr. When these data were plotted as specific activity the trend was the same (data not shown). Termites lost only 0.03% of their total nitrogen showing that the decline in enzyme activity was not due to excretion of gut contents, and hence protozoa, as a consequence of the oxygen treatment. Midgut enzyme activities over 48 hr were not significantly affected by the treatment (Fig. 2). The protozoa were isolated intact and essentially free of other microorganisms and termite gut contents. Washing caused some lysis but substantial cellulase and cellobiase activity was, nevertheless, detected in protozoal extracts (Table 3). The data shown in Table 3 and the results of oxygen treatment indicate that there are two cellobiases, one of protozoal and one of termite origin. As judged by experiments using crude extracts of the midgut and hindgut, the two enzymes from C. lacteus exhibit very similar properties. Thus they have similar pH profiles (Fig. 1) and are both inactivated after heating for 10 rain at 62°C. Kinetic studies showed some differences in the enzymes. The midgut cellobiase had a K m of 4.17 mM and a Vmax of 143/~mol glucose per hr per mg protein, whereas these values for the hindgut enzyme
DISCUSSION The results obtained with N. exitiosus and N. suggest that, in these higher termites, cellobiase activity is not due to the gut bacteria. Cellobiase activity in both termites is confined mainly to the midgut, with small amounts (1-3%) in the foregut. As the foregut and midgut are devoid of bacteria, it is probable that the enzyme in the midgut is secreted by the epithelium. Secretion from salivary glands may account for the small amounts of cellobiase in the foregut; while traces of cellobiase activity found in the mixed segment of N. walkeri were probably due to passage of the enzyme from the midgut. It is concluded that cellobiase in both N. exitiosus and N. walkeri is of termite origin. MARTIN and MARTIN (1978) also observed in the higher termite, Macrotermes natalensis, that fl-glucosidase activity was mainly in the midgut, with only traces of activity in the paunch. On the other hand, POTTS and HEWITT (1973) found in Trinervitermes trinervoides, another higher termite, that cellobiase activity was evenly distributed between the m!dgut and the paunch of the hindgut; the latter activity was attributed to hindgut bacteria. KOVOOR (1969) found considerable variation in cellobiase activity in Microcerotermes edentatus with workers from one nest showing activity only in the midgut, whereas those from another nest showed activity in midgut and hindgut. Of the total cellobiase in C. lacteus, 21% is found in the midgut. This enzyme must be of termite origin since the midgut does not contain any microbiota. Further, this enzyme cannot come from the hindgut as the proctodeal valve prevents reflux of hindgut contents to the midgut (NoIROT and NOIROTTIMOTHI~E,1969). In the hindgut the protozoa and/or spirochaetes produced all of the cellulase and 92% of the cellobiase, since after the protozoa and spirochaetes had been killed, these enzyme activities dropped almost to zero. Since the specific activities of cellulase and cellobiase in protozoal extracts were similar to those of whole hindgut extracts, it is suggested that both enzymes in the hindgut are produced only by the protozoa. walkeri
Cellobiase of termites
567
DAHLQVISTA. (1968) Assay of intestinal dissacharidases. Analyt. Biochem. 22, 99-107. EUTICK M. L., O'BRIEN R. W. and SLAYTORM. (1976) Aerobic state of gut of Nasutitermes exitiosus and Coptotermes lacteus, high and low caste termites. J. Insect Physiol. 22, 1377-1380. EUTICR M. L., O'BRIEN R. W. and SLAYTORM. (1978a) Bacteria from the gut of Australian termites. Appl. environ. Microbiol. 35, 823-828. Euaacg M. L., VEIVERSP., O'BRIENR. W. and SLAVXORM. (1978b) Dependence of the higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lacteus on their gut flora. J. Insect Physiol. 24, 363-368. JAcoas S. (1965) The determination of nitrogen in biological materials. In Methods of Biochemical Analysis (Ed. by GLICKD.), Vol. 13, pp. 241-263. Interscience, New York. KOOVORJ. (1970) Presence d'enzymes cellulolytiques dans l'intestin d'un termite suprrieur Microcerotermes edentatus (Was.). Ann. Sci. natn. ZooL 12, 65-71. LOWRYO. H., ROSEBROUGHN. J., FARRA. U and RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MARTIN M. S. and MARTIN J. S. (1978) Cellulose digestion in the midgut of the fungus-growing termite Macrotermes natalensis: The role of acquired digestive enzymes. Science, Wash. 199, 1453-1455. MOORE B. P. (1969) Biochemical studies in termites. In Biology of Termites (Ed. by KPdSHNAK. and WEESr~ER F. M.), Vol. 1, pp. 407-432. Academic Press, New York. NOIROT C. and NOIROT--TIMOTr~EC. (1969) The digestive system. In Biology of Termites (Ed. by KRISnNAK. and WEESNERF. J.), Vol. 1, pp. 49-88, Academic Press, New York. O'BRIENG. W., VEIVERSP. C., MCEWENS. E., SLAVTORM. and O'BRIENR. W. (1979) The origin and distribution of cellulase in the termites, Nasutitermes exitiosus and Coptotermes lacteus, lnsect Biochem. 9, 619-625. POTrS R. C. and HEWITTP. H. (1973) The distribution of intestinal bacteria and cellulase activity in the harvester termite, Trinervitermes trinervoides (Nasutitermitinae). lnsecte Soc. 20, 215-220. RETIEFFL. W. and HEWITTP. H. (1973) Digestive fl-glycosidases of the harvester termite, Hodotermes mossambicus: Properties and distribution. J. Insect Physiol. 19, 1837-1847. SONOGY1M. (1952) Notes on sugar determination. J. biol. Chem. 195, 19-22. THAYERD. W. (1976) Facultative wood-digesting bacteria from the hindgut of the termite Reticulitermes hespersus. J. gen. Microbiol. 95, 287-296. TRACEYM. V. and YOUATTG. (1958) Cellulase and chitinase in two species of Australian termites. Enzymologia 19, 70-72. Acknowledgements--We thank Dr. J. A. L. WATSON, CSIRO Division of Entomology, Canberra for confirming TRAGERW. (1932) A cellulase from the symbiotic intestinal flagellates of termites and of the roach Cryptocercus our termite identification and Miss P. C. VEIVERSfor nitrogen punctulatus. Biochem. J. 26, 1762-1771. estimations. This research was supported by a grant from the TRAGERW. (1934) The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other Australian Research Grants Committee. termite protozoa. Biol. Bull. mar. biol. Lab., Woods Hole 66, 182-190. YAMXNM. A. (1978) Axenic cultivation of the cellulolytic REFERENCES flagellate Trichomitopsis termopsidis (Cleveland) from the termite Zootermopsis. J. Protozool. 25, 535-538. BREADYJ. K. and FRIEDMANS. (1963) Oxygen poisoning of YAMINM. A. and TRAGERW. (1979) Cellulolytic activity of an axenically-cultivated termite flagellate, Trichomitopsis the termite, Reticulitermesflavipes (Kollar), and protection termopsidis. J. gen. Microbiol. 113, 417--420. by carbon dioxide. J. Insect Physiol. 9, 337-347.
The possibility of proctodeal feeding can also be discounted as the specific activity of cellobiase in the foregut should be the same as that of hindgut and midgut, i.e. seven times greater than the observed value and the percentage of the total activity in the foregut would be greater than the observed value of 1%. The results obtained with C. lacteus are consistent with those of O'BRIEN et al. (1979). These authors concluded that protozoa were responsible for most of the cellulase in the hindgut of C. lacteus, and that the termite also secreted a cellulase of its own. Cellulase activity has been detected in cultured protozoa (TRAGER, 1932; YAMIN and TRAGER, 1979). Because the gut bacteria were not killed by oxygen treatment, which caused such a dramatic decrease in cellulase and cellobiase activity in the hindgut of C. lacteus, it appears that bacteria do not participate in the degradation of cellulose in this termite, which is consistent with the failure of EUTICK et al. (1978a) to isolate cellulose-degrading bacteria from C. lacteus. While TRACEYand YOUATT(1958) and O'BRIEN et al. (1979) have noted that C. lacteus had a higher cellulase activity than N. exitiosus, and the present study has shown the same for cellobiase in the two species, cellulose utilization in N. exitiosus is more efficient than in C. lacteus (MOORE, 1969). Inner mound matrix material from nests of N. exitiosus contains about 6% of residual cellulose, whereas C. lacteus nests retain at least 18%. The differences in efficiency may lie in the pH optima of the cellobiase in the gut of workers of both species. The broad pH optimum of cellobiase in iV. exitiosus would allow the enzyme to work at near optimal activity in all sections of the gut. In C. lacteus the foregut and midgut enzymes have an optimum pH for activity well removed from the pH of the gut which would result in the enzymes working at about 40% of their maximum rate. This apparent lack of efficiency of the termite enzymes would ensure the passage of cellulose to the hindgut to provide the carbon source for the protozoa. Some termite protozoa appear to have a specific requirement for cellulose (TRAGER, 1934; YAMIN, 1978). It is not possible to determine whether the hindgut enzyme is maximally active since the pH of the interior of the protozoa is unknown.
18 10:5
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0020-1700/80/1001--0563 $02.00/0
CELLOBIASE ACTIVITY IN THREE SPECIES OF AUSTRALIAN TERMITES SANDRA E. McEWEN, M. SLAYTOR and R. W. O'BRIEN Department of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia (Received 13 December 1979)
A~traet--The cellobiase of the higher termite Nasutitermes exitiosus was located in the foregut (8%) and the midgut (92%). In N. walkeri 97% of the activity was in the midgut, 2% in the foregttt and 1% in the mixed segment. Most of the cellobiase activity in the lower termite Coptotermes lacteus was in the hindgut (78%) and the midgut (21%) with traces (1%) in the foregut. Removal of the protozoa and spirochaetes by oxygen treatment resulted in loss of most of the cellulase and cellobiase from the hindgnt within 28 hr of treatment; the midgut enzymes were unaffected. Isolated protozoa had cellulase and cellobiase of high specific activity and accounted for most of the total ceUulolytic activity in the hindgut. These results indicate that the enzymes in the midgut are secreted by all three termites, whereas the enzymes in the hindgut (C. lacteus only) are of protozoal origin. The optimum pH for cellobiase from N. exitiosus (foregut and midgut).ranged from 4.5 to 7.0; the C. lacteas enzymes had an optimum activity at pH 5.0-6.0. The pH ofN. exitiosas gut ranged from 2.0 to 2.8 (foregut), 2.8 to 3.8 (rectum), and from 6.8 to 7.5 (midgut and mixed segment). The pH of the midgut and hindgut of C. lacteus was 6.8--7.4 and foregut was 3.8--4.4. The significance of the gut pH on cellobiase activity is discussed. Crude preparations of C. lacteus cellobiase (both midgut and hindgut) were inactivated at 62°C and were competitively inhibited by 1,5-gluconolactone. Key Word Index: Termites, cellobiase, cellulase, protozoa, Nasutitermes exitiosus, Nasutitermes walkeri, Coptotermes lacteas
INTRODUCTION THE DIGESTION of cellulose in termites has been the subject of many investigations mainly to determine the role of the gut micro-organisms in the initial hydrolysis of cellulose. A major product from the action of cellulase on cellulose is the disaccharide cellobiose, which can be hydrolysed to glucose by cellobiase or non-specific//-glucosidases. Cellobiase activity has been found in the guts of worker termites of Trinervitermes trinervoides (PoTTS and HEWITT, 1973) and in the guts of workers, larvae and soldiers of Hodotermes mossambicus (RETIEFF and HEWITT, 1973). In H. mossambicus the greatest concentration of the enzyme activity was found in the hindgut of the larvae and in the midgut of the soldiers. Bacteria, capable of utilising cellobiose as a carbon source, have been isolated from various termites (EuTICK et al., 1978a; THAYER, 1976), but their role in cellobiose metabolism is not known. In this paper we describe the occurrence and distribution of cellobiase activity in the gut of the higher termites, Nasutitermes exitiosus and N. walkeri, and of cellulase and cellobiase activity in the lower termite Coptotermes lacteus. MATERIALS AND METHODS
collected at Greenwich, N.S.W. The termites were maintained in the laboratory as described by EUTICKet al. (1976). Worker caste termites were used in all experiments. Cellobiase assay The guts of twenty-five termites were dissected into foregut, midgut, mixed segment (posterior midgut + anterior hindgut, found only in the higher termites, N. exitiosus and N. walkerO, and hindgut. The segments were homogenized separately in 2 ml 0.1 M acetate buffer, pH 5.0, in a Ten Broeck homogenizer and diluted to a final volume of 2.5 ml. The homogenates were centrifuged at 20,000 g for 10 min and the supernatants were assayed for cellobiase activity. All operations were carried out at 0--4°C. The supernatants (0.5 ml) were incubated with 1.0 ml 1% (w/v) cellobiose in 0.1 M acetate buffer, pH 5.0, for 1 hr at 37°C. The enzyme activity was linear over this period. The amount of glucose released was measured by the glucose oxidase method (Sigma Tech. Bulletin No. 510) with the enzymes dissolved in 0.5 M Tris buffer, pH 7.0, to inhibit disaccharidases in the glucose---oxidase preparation (DAmOXrtST, 1968). The cellobiase activity was corrected by subtracting the residual activity present in the extract after it had been heated for 10 rain at 100°C. The protein content was determined by the method of LOWRYet al. (1951 ) using bovine gamma-globulin (Sigma) as a standard. One unit of cellobiase activity is defined as the amount of enzyme which produced 1 mg of glucose per hr per ml of extract. Specific activity is defined as mg glucose produced per hr per mg protein. The optimum pH of the cellobiase of the various gut sections was determined by assaying the enzyme, as described above, using Mcllvaine citrate-phosphate buffers.
Termites Ground nests of the lower termite C. lacteus Froggatt were Cellulase assay collected at Bilpin, N.S.W., and the Ourimbah State Forest, Gut segments were homogenized with 2.5 ml of near Wyong, N.S.W. Ground nests of the higher termite N. exitiosus Hill were collected from the Ku-ring-gai National 0.1 M acetate buffer, pH 5.5, and centrifuged at Park, near Sydney and arboreal nests of N. walkeri Hill were 20,000 g for 10 rain. The supernatants (0.5 ml) were incubated 563
564
SANDRAE. McEWEN, M. SLAYTORAND R. W. O'BRIEN
with 1.0 ml of 1% (w/v) sodium carboxymethylcellulose for 30 min at 37°C. The incubation mixture was assayed for reducing sugar according to the method of SOMOGW(1952) using glucose as a standard. One unit of cellulase activity is defined as the amount of enzyme which produced 1 mg of reducing sugar (expressed as glucose) per hr per ml of extract. Specific activity is defined as mg of glucose produced per hr per mg of protein. Total nitrogen determination Ten termites were digested according to the Kjeldahl method using potassium sulphate and mercuric oxide (JACoBS, 1965) and the ammonia produced was assayed by the phenol/hypochlorite method (JAcoBs, 1965). pH of the termite gut Termites were fed wood impregnated with pH indicators as described by EUTICKet al. (1976). After 24 hr, the guts were dissected and examined for location and colour of the dyes. The guts were then treated with acid or alkali to ensure that the indicators had retained their acid-base colour change. The pH indicators used are listed in Table 1. Although no attempt was made to determine whether the indicators were toxic to the termite symbionts, guts of treated termites were similar to those of untreated termites and did not show any signs of shrinkage, which occurs if gut protozoa are killed by starvation or oxygen treatment. Removal of protozoa Protozoa and spirochaetes were killed by subjecting termites to oxygen in a pressure vessel at 2.53 × 102 kPa for 2 hr. The survival of protozoa and spirochaetes was determined by dissecting out the gut of several termites into distilled water and examining the suspension under phasecontrast microscopy. Bacteria were isolated and counted as described by EUT]CK et al. (1978b). Plates were incubated under anaerobic conditions for 48 hr at 30°C using a BBL Gas-Pak system (Becton, Dickinson and Co., Cockeysville, Maryland, U.S.A.) in an anaerobe jar. Isolation of protozoa The paunch section of the hindgut was ruptured and the protozoa allowed to diffuse into 0.05 ml of the medium of TRAGER (1934). The paunch was then washed with an additional 0.05 ml of the same medium and added to the original suspension. The protozoa from twenty termites were separated from the other gut contents by allowing them to settle and decanting the medium. They were washed twice by resuspension in fresh medium. The extract and the washes were examined separately under phase-contrast microscopy for the presence of bacteria, spirochaetes, protozoa and gut food particles. The protozoa were then homogenized in 0.1 M acetate buffer, pH 5.0, in a Ten Broeck homogenizer and diluted to a final volume of 4 ml with the same buffer. The homogenate was centrifuged at 20,000 g for 10 min at 5°C and the superantant was used for enzyme assays.
RESULTS Following our success in measuring the distribution of cellulase activity in the different sections of the guts o f N . exitiosus and C. lacteus (O'BRtEN et al., 1979), it became of interest to study the distribution of cellobiase in the guts of these termites to obtain a better overall understanding of cellulose digestion. Initially the effect of p H on cellobiase activity of extracts of N. exitiosus and C. lacteus was determined (Fig. 1). tn extracts o f the foregut and midgut of N. exitiosus, maximal activity was observed over the p H range of 4.5-7.0. In extracts of C. lacteus, maximal
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.-.:_:\:
o I 5
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I 5
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I 7
I 8
pH
Fig. 1. Effect ofpH on cellobiase activity in the gut sections of (a) N. exitiosus and (b) C. lacteus, using Mcllvaine citrate-phosphate buffers. O, Foregut; I-q, Midgut; &, Hindgut. Points on the curve represent the average of two determinations, both giving the same pH optima. activity occurred over a narrower pH range, from about 5.0 to 6.0. A p H optimum of 5.0--5.5 seemed low when compared with the p H of the gut which is around neutrality (EUTICK et al., 1976). For this reason, the p H of the gut of N. exitiosus and C. lacteus was measured. The results (Table 1) show that the foregut and rectum of both species were very acid, whereas the pH of other sections of the gut was indeed near neutrality. The distribution of cellobiase (measured at pH 5.0) throughout the gut of N. exitiosus, N. walkeri and C. lacteus is shown in Table 2. Most of the ceUobiase activity (92%) of N. exitiosus was located in the midgut, with no activity in the mixed segment or hindgut sections. A similar distribution of cellobiase activity was observed in N. walkeri, with 97% of the total activity in the midgut. In C. lacteus, only 1% of the total activity was found in the foregut, whereas 21% was found in the midgut, and 78% in the hindgut where the protozoa and the bacteria are located. To determine the origin of the cellobiase, the protozoa (and the spirochaetes) were killed by treating the termites with oxygen. The termites were then assayed for cellulase and cellobiase activity. The conditions required to kill the protozoa and spirochaetes, but not the termites, were determined experimentally since BREADY and FRIEDMAN (1963) reported that oxygen above a partial pressure of 50% in carbon dioxide was poisonous, not only to gut protozoa, but to termites as well. Two hours was the minimum time required to kill all the protozoa and spriochaetes using 100% oxygen at 2.53 x 10 z kPa pressure. Preliminary experiments showed that 80-85% of C. lacteus were alive three days after this
1.08 _+ 0.52 2.37 +_ 0.74 --
Foregut Midgut M i x e d segment Hindgut
6.8-7.5
7.0-7.5
> 4.4 > 5.4 > 6.8 ~ 7.0 7.0-8.0
< 8.0
Midgut
7.0-7.5
7.0-7.5
_>4.4 a 6.5-6.8 ~ 7.0 7.0-8.0
< 8.0
~6.8
7.0-7.5
>_4.4 >_ 5.4 < 6.8 ~ 7.0 < 7.0
< 8.0
Paunch colon
2.8-3.8
< 6.6
_> 3.2 < 3.8 < 5.2 < 6.0 > 2.0
< 2.8
Rectum
4.4 3.8 5.2 6.0 2.0
3.8-4.4
< 6.6
> < < < >
> 2.8
Foregut
0.01 _+ 0.006 0.11 _+ 0.01 ---
N. exitiosus* Units§ 8 _+ 5 92 + 6 --
~o" 1.14 + 0.68 7.55 -+ 1.38 0.14 _+ 0.06
Sp. act.
--
0.02 _+ 0.01 1.11 + 0.35 0.01 _+ 0.005
C e l l o b i a s e activity N. walkeri't Units
Sp. act. 3.67 _____0.64 25.86 + 4.57 -25.86 _+ 3.55
2 + 1.8 97 + 2 1__+0.3 --
6.8-7.4
a
> 4.4 > 5.4 > 6.8 ~ 7.0 7.0-7.4
< 8.0
0.05 + 0.02 1.17 + 0.18 -4.10 _+ 0.30
C. lacteus* Units
6.8-7.4
~ 7.0
> 4.4 > 5.4 > 6.8 ~ 7.0 7.0-7.4
< 8.0
C. lacteus Paunch Midgut colon
~o
T a b l e 2. D i s t r i b u t i o n o f cellobiase activity in the gut o f N. exitiosus, N. walkeri a n d C. lacteus
2.0-2.8
< 6.6
< 3.2 < 3.8 < 5.2 < 6.0 _>2.0
< 2.8
Foregut
*Activities were d e t e r m i n e d on four different g r o u p s o f termites (_+ S.E.M.). t Activities were m e a n values o b t a i n e d f r o m t w o e x p e r i m e n t s (_+ range). :~Activities were d e t e r m i n e d o n seven different g o u p s o f termites (_+ S.E.M.). § Sp. act = Specific activity (defined in the text), U n i t s also defined in text. "Values are percentage of the total activity e x p r e s s e d as units.
Sp. act.§
1.2-2.8 8.0-9.6 3.2-4.4 3.8-5.4 5.2-6.8 6.0-7.6 1.0-2.0 7.0-8.6 6.6-8.0
pH Range
G u t Section
a I n d e t e r m i n a t e result.
Estimated pH
P h e n o l red
Methyl orange B r o m o c r e s o l green Bromocresol purple B r o m o t h y m o l blue Cresol red
T h y m o l blue
Indicator
N. exitiosus Mixed segment
T a b l e 1. p H o f the gut o f N. exitiosus a n d C. lacteus
1 +__ 0.3 21 + 3 -78 +__ 3
Vo
2.8-3.2
< 6.6
< 3.2 < 3.8 < 5.2 < 6.0 >_ 2.0
> 2.8
Rectum
566
3
>
SANDRAE. MCEwEN, M. SLAYTORAND R. W. O'BRIEN
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1.0
°
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Protozoa Empty hindgut Hindgut contents minus protozoa
/\/
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Table 3. Cellulase and cellobiase activity in extracts of gut protozoa
I
.dr
I
dr
0
I
I 5
I 8
I
I 28
13.5 3.3 4.4
21.6 _+ 3.1 3.5 _+ 1.1 4.7 + 0.9
* Data from one experiment. t Data from two experiments (___ range) ++Sp. act. = Specific activity (defined in text).
(b)
°
2
Cellobiaset Sp. act.:~
I
1.0
O
Cellulase* Sp. act.++
/-
"
were 2.82 mM and 200/~mol glucose per hr per mg protein, respectively. The competitive inhibitor, 1,5gluconolactone, increased the K,, of both enzymes fourfold and had no effect on the V~x o f the midgut enzyme, but decreased that of the hindgut enzyme to 143 #mol glucose per hr per mg protein.
48
hr
Fig. 2. Effect of oxygen treatment of cellulase (0) and cellobiase (O) activity in (a) the midgut and (b) the hindgut of C. lacteus. Gut extracts were assayed before oxygen treatment (0 hr), and at various times after 2 hr oxygen treatment. The experiment was carried out twice giving essentially the same result. The results in this figure are from one of these experiments.
treatment. Plating of the gut contents of oxygentreated termites as described in Materials and Methods showed that the bacterial numbers (except spirochaetes) were unaffected by the oxygen treatment. The effect of oxygen treatment on cellulase and cellobiase activity in the hindgut of C. lacteus is shown in Fig. 2. The decline in cellobiase activity was apparent 3 hr after oxygen treatment and continued to decline, until at 28 hr only 15% of the original activity remained. Cellulase activity underwent a similar decrease and was completely lost by 48 hr. When these data were plotted as specific activity the trend was the same (data not shown). Termites lost only 0.03% of their total nitrogen showing that the decline in enzyme activity was not due to excretion of gut contents, and hence protozoa, as a consequence of the oxygen treatment. Midgut enzyme activities over 48 hr were not significantly affected by the treatment (Fig. 2). The protozoa were isolated intact and essentially free of other microorganisms and termite gut contents. Washing caused some lysis but substantial cellulase and cellobiase activity was, nevertheless, detected in protozoal extracts (Table 3). The data shown in Table 3 and the results of oxygen treatment indicate that there are two cellobiases, one of protozoal and one of termite origin. As judged by experiments using crude extracts of the midgut and hindgut, the two enzymes from C. lacteus exhibit very similar properties. Thus they have similar pH profiles (Fig. 1) and are both inactivated after heating for 10 rain at 62°C. Kinetic studies showed some differences in the enzymes. The midgut cellobiase had a K m of 4.17 mM and a Vmax of 143/~mol glucose per hr per mg protein, whereas these values for the hindgut enzyme
DISCUSSION The results obtained with N. exitiosus and N. suggest that, in these higher termites, cellobiase activity is not due to the gut bacteria. Cellobiase activity in both termites is confined mainly to the midgut, with small amounts (1-3%) in the foregut. As the foregut and midgut are devoid of bacteria, it is probable that the enzyme in the midgut is secreted by the epithelium. Secretion from salivary glands may account for the small amounts of cellobiase in the foregut; while traces of cellobiase activity found in the mixed segment of N. walkeri were probably due to passage of the enzyme from the midgut. It is concluded that cellobiase in both N. exitiosus and N. walkeri is of termite origin. MARTIN and MARTIN (1978) also observed in the higher termite, Macrotermes natalensis, that fl-glucosidase activity was mainly in the midgut, with only traces of activity in the paunch. On the other hand, POTTS and HEWITT (1973) found in Trinervitermes trinervoides, another higher termite, that cellobiase activity was evenly distributed between the m!dgut and the paunch of the hindgut; the latter activity was attributed to hindgut bacteria. KOVOOR (1969) found considerable variation in cellobiase activity in Microcerotermes edentatus with workers from one nest showing activity only in the midgut, whereas those from another nest showed activity in midgut and hindgut. Of the total cellobiase in C. lacteus, 21% is found in the midgut. This enzyme must be of termite origin since the midgut does not contain any microbiota. Further, this enzyme cannot come from the hindgut as the proctodeal valve prevents reflux of hindgut contents to the midgut (NoIROT and NOIROTTIMOTHI~E,1969). In the hindgut the protozoa and/or spirochaetes produced all of the cellulase and 92% of the cellobiase, since after the protozoa and spirochaetes had been killed, these enzyme activities dropped almost to zero. Since the specific activities of cellulase and cellobiase in protozoal extracts were similar to those of whole hindgut extracts, it is suggested that both enzymes in the hindgut are produced only by the protozoa. walkeri
Cellobiase of termites
567
DAHLQVISTA. (1968) Assay of intestinal dissacharidases. Analyt. Biochem. 22, 99-107. EUTICK M. L., O'BRIEN R. W. and SLAYTORM. (1976) Aerobic state of gut of Nasutitermes exitiosus and Coptotermes lacteus, high and low caste termites. J. Insect Physiol. 22, 1377-1380. EUTICR M. L., O'BRIEN R. W. and SLAYTORM. (1978a) Bacteria from the gut of Australian termites. Appl. environ. Microbiol. 35, 823-828. Euaacg M. L., VEIVERSP., O'BRIENR. W. and SLAVXORM. (1978b) Dependence of the higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lacteus on their gut flora. J. Insect Physiol. 24, 363-368. JAcoas S. (1965) The determination of nitrogen in biological materials. In Methods of Biochemical Analysis (Ed. by GLICKD.), Vol. 13, pp. 241-263. Interscience, New York. KOOVORJ. (1970) Presence d'enzymes cellulolytiques dans l'intestin d'un termite suprrieur Microcerotermes edentatus (Was.). Ann. Sci. natn. ZooL 12, 65-71. LOWRYO. H., ROSEBROUGHN. J., FARRA. U and RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MARTIN M. S. and MARTIN J. S. (1978) Cellulose digestion in the midgut of the fungus-growing termite Macrotermes natalensis: The role of acquired digestive enzymes. Science, Wash. 199, 1453-1455. MOORE B. P. (1969) Biochemical studies in termites. In Biology of Termites (Ed. by KPdSHNAK. and WEESr~ER F. M.), Vol. 1, pp. 407-432. Academic Press, New York. NOIROT C. and NOIROT--TIMOTr~EC. (1969) The digestive system. In Biology of Termites (Ed. by KRISnNAK. and WEESNERF. J.), Vol. 1, pp. 49-88, Academic Press, New York. O'BRIENG. W., VEIVERSP. C., MCEWENS. E., SLAVTORM. and O'BRIENR. W. (1979) The origin and distribution of cellulase in the termites, Nasutitermes exitiosus and Coptotermes lacteus, lnsect Biochem. 9, 619-625. POTrS R. C. and HEWITTP. H. (1973) The distribution of intestinal bacteria and cellulase activity in the harvester termite, Trinervitermes trinervoides (Nasutitermitinae). lnsecte Soc. 20, 215-220. RETIEFFL. W. and HEWITTP. H. (1973) Digestive fl-glycosidases of the harvester termite, Hodotermes mossambicus: Properties and distribution. J. Insect Physiol. 19, 1837-1847. SONOGY1M. (1952) Notes on sugar determination. J. biol. Chem. 195, 19-22. THAYERD. W. (1976) Facultative wood-digesting bacteria from the hindgut of the termite Reticulitermes hespersus. J. gen. Microbiol. 95, 287-296. TRACEYM. V. and YOUATTG. (1958) Cellulase and chitinase in two species of Australian termites. Enzymologia 19, 70-72. Acknowledgements--We thank Dr. J. A. L. WATSON, CSIRO Division of Entomology, Canberra for confirming TRAGERW. (1932) A cellulase from the symbiotic intestinal flagellates of termites and of the roach Cryptocercus our termite identification and Miss P. C. VEIVERSfor nitrogen punctulatus. Biochem. J. 26, 1762-1771. estimations. This research was supported by a grant from the TRAGERW. (1934) The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other Australian Research Grants Committee. termite protozoa. Biol. Bull. mar. biol. Lab., Woods Hole 66, 182-190. YAMXNM. A. (1978) Axenic cultivation of the cellulolytic REFERENCES flagellate Trichomitopsis termopsidis (Cleveland) from the termite Zootermopsis. J. Protozool. 25, 535-538. BREADYJ. K. and FRIEDMANS. (1963) Oxygen poisoning of YAMINM. A. and TRAGERW. (1979) Cellulolytic activity of an axenically-cultivated termite flagellate, Trichomitopsis the termite, Reticulitermesflavipes (Kollar), and protection termopsidis. J. gen. Microbiol. 113, 417--420. by carbon dioxide. J. Insect Physiol. 9, 337-347.
The possibility of proctodeal feeding can also be discounted as the specific activity of cellobiase in the foregut should be the same as that of hindgut and midgut, i.e. seven times greater than the observed value and the percentage of the total activity in the foregut would be greater than the observed value of 1%. The results obtained with C. lacteus are consistent with those of O'BRIEN et al. (1979). These authors concluded that protozoa were responsible for most of the cellulase in the hindgut of C. lacteus, and that the termite also secreted a cellulase of its own. Cellulase activity has been detected in cultured protozoa (TRAGER, 1932; YAMIN and TRAGER, 1979). Because the gut bacteria were not killed by oxygen treatment, which caused such a dramatic decrease in cellulase and cellobiase activity in the hindgut of C. lacteus, it appears that bacteria do not participate in the degradation of cellulose in this termite, which is consistent with the failure of EUTICK et al. (1978a) to isolate cellulose-degrading bacteria from C. lacteus. While TRACEYand YOUATT(1958) and O'BRIEN et al. (1979) have noted that C. lacteus had a higher cellulase activity than N. exitiosus, and the present study has shown the same for cellobiase in the two species, cellulose utilization in N. exitiosus is more efficient than in C. lacteus (MOORE, 1969). Inner mound matrix material from nests of N. exitiosus contains about 6% of residual cellulose, whereas C. lacteus nests retain at least 18%. The differences in efficiency may lie in the pH optima of the cellobiase in the gut of workers of both species. The broad pH optimum of cellobiase in iV. exitiosus would allow the enzyme to work at near optimal activity in all sections of the gut. In C. lacteus the foregut and midgut enzymes have an optimum pH for activity well removed from the pH of the gut which would result in the enzymes working at about 40% of their maximum rate. This apparent lack of efficiency of the termite enzymes would ensure the passage of cellulose to the hindgut to provide the carbon source for the protozoa. Some termite protozoa appear to have a specific requirement for cellulose (TRAGER, 1934; YAMIN, 1978). It is not possible to determine whether the hindgut enzyme is maximally active since the pH of the interior of the protozoa is unknown.
18 10:5
- G