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Cellulose digestion in termites and cockroaches: What role do symbionts play?
Comp. Blochem.Physiol.Vol. 103B,No. 4, pp. 775--784,1992 Printed in Great Britain
0305-0491192 $5.00+ 0.00 © 1992PergamonPress Ltd
MINI REVIEW CELLULOSE DIGESTION IN TERMITES AND COCKROACHES: WHAT ROLE DO SYMBIONTS PLAY? MICHAEL SLAYTOR Department of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia (Tel. 02 692-2597; Fax 02 692-4571) (Received 7 May 1992; accepted 26 June 1992) Abstraet--l. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts. 2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of micrc~ organisms, on a diet of crystalline cellulose. Under these conditions the RQ is I, indicating that the cockroach is surviving on glucose produced by endogenous cellulase. 3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO2 in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is suflident for their needs. 4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers. 5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-fl-l,4-glueanase (EC 3.2.1.4) and fl-l,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-fl-l,4glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-fl-l,4-glucanasecomponents are active against both crystalline cellulose and carboxymethylcellulose. 6. There is no evidence that hacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.
INTRODUCTION The study of cellulose digestion in termites and cockroaches has been plagued by misconceptions of the role of symbionts in the process. In spite of the demonstration in insects, and many other invertebrates which live on cellulose, that the major components of cellulase activity are present, which is normally sufficient proof of their endogenous nature, there has been little recognition that cellulose digestion is endogenous and does not have to involve symbionts. The demonstration in 1924 that the termite Zootermopsis sp. has cellulolytic Protozoa in its hindgut and that these flagellates are essential for the termite's survival (Cleveland, 1924) is the cornerstone of the thesis that insects do not secrete cellulase and are dependent on symbionts for cellulose digestion. This thesis, coupled with the general belief that cellulase activity is restricted to fungi, bacteria and Protozoa, has led research workers on fruitless searches for bacterial cellulases and to propose the biochemically unprecedented concept of acquired enzymes. One of the major obstacles to accepting that insects are not dependent on symbionts for cellulose digestion is the almost universal presence of microorganisms in the digestive tract. A bacteria-free gut as in the case of the marine isopod Limnoria lignorum is
a rare exception (Boyle and Mitchell, 1978). Entomologists would do well to recall the reluctance with which cellulase activity in L. lignorum has been accepted as being endogenous. Early reports, contemporary with Cleveland's work on termites, failed to find cellulase activity (using a four-week incubation at 32°C with sawdust) or any evidence of Protozoa in the gut in this obvious wood-eating organism; the speculation was that bacteria were involved in digestion (Yonge, 1927). A re-examination of the problem 25 years later showed that cellulase activity was present in crude extracts of the midgut caeca and that no symbiotic bacteria were present in the gut (Ray and Julian, 1952). This was not accepted as conclusive because, in 1957, it was proposed that marine fungi must be responsible (Meyers and Reynolds, 1957) but this was eliminated two years later (Ray and Stuntz, 1959). Finally, in 1978, 51 years after Yonge's initial observations, scanning electron microscopy confirmed that the gut was completely free of microorganisms and thus the cellulase must be endogenous (Boyle and Mitchell, 1978). That this is still not universally accepted can be deduced from the statement that L. lignorum "acquires useful digestive enzymes from its food" (Martin, 1987). It is still widely believed that insects feeding on cellulose are dependent on symbionts for cellulose
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MICHAEL SLAYTOR
digestion although support for this belief has grown more tentative since the topic was last reviewed by Martin in this journal (1983) and more recently elsewhere (Martin, 1991). The loss of confidence has been caused by the reluctant acceptance that endogenous endo-fl-l,4-glucanase (EC 3.2.1.4) and fl-l,4-glucosidase (EC 3.2.1.21) activities are produced in insects (Martin, 1991). The centre of the argument that insects are dependent on symbionts for cellulose digestion is that an exo-fl-l,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) component is essential for the hydrolysis of crystalline cellulose. As there has been no demonstration of this component in an insect, it is argued that it must be supplied by a symbiotic Protozoa, bacteria or fungi. But is this component essential for a cellulase to be capable of producing glucose from crystalline cellulose? The infrequency with which it has been found in other systems, e.g. bacterial cellulases (Rapp and Beermann, 1991), suggests that insects also may not need it. We believe that a critical evaluation of the available data leads to the conclusion that termites and cockroaches which feed on cellulose produce cellulase in the salivary glands and midgut to hydrolyse it. In other words, cellulase is an endogenously produced digestive enzyme secreted primarily in the midgut like the enzymes needed for starch or sucrose hydrolysis. This review is primarily concerned with our work over the last 15 years but also with a critical evaluation of other published data on cellulose digestion in termites and cockroaches. The definition of cellulase, which will be used in this review, is one which will proudce glucose from crystalline cellulose. It should be noted that a ceUulase thus defined will include a t - 1,4-glucosidase component. CHOICE OF TERMITES AND COCKROACHES
Primarily our choice of termites and cockroaches has been determined by local availability but together they cover the major issues in insect cellulose digestion. Thus, we have worked on Mastotermes darwiniensis, the most primitive termite and the one most closely allied to the cockroaches (McKittrick, 1965), and Coptotermes lacteus as representatives of the lower termites with cellulolytic Protozoa. M. darwiniensis is a destructive and opportunistic feeder; it will attack live and dead wood as well as sugar cane and flour (Hill, 1942). C. lacteus, an abundant mound-builder in the cooler regions around Sydney, is xylophagous. In Sydney, the commonest higher termites are the arboreal Nasutitermes walkeri and the mound-building N. exitiosus. Both of these species contain only bacterial symbionts and feed primarily on sound dead wood though colonies of N. walkeri are frequently found in living trees. Large colonies of N. walkeri can be used for multiple sampling without apparent disruption of the colony. The procedure is akin to rubber tapping and ensures a ready supply of freshly collected termites. The only exotic species we have studied are the African higher termites belonging to the Macrotermitinae, Macrotermes subhyalinus and M. michaelsent. Their fascination is the symbiotic role of the fungus garden they cultivate and its function in digestion of the grass the termites harvest.
Additionally, unlike the majority of termites, the midgut of M. subhyalinus is the largest (65% by volume) section of the gut (Miihlemann et al., 1992). The close phylogenetic relationship between termites and cockroaches, coupled with the larger size of cockroaches, invites termitologists to work on cockroaches as well. Thus Cleveland extended his work on lower termites to the wood-eating cockroach Cryptocercus punctulatus (Cryptocercidae) which also contains cellulolytic Protozoa (Cleveland et al., 1934). There are no Australian representatives of the Cryptocercidae family. Instead, we have chosen three cockroaches belonging to the Blaberidae family, concentrating on Panestheia cribrata which feeds on rotting wood. Unlike the termite gut, its gut is essentially a straight tube containing nothing comparable to the paunch and does not need symbionts to enable it to survive on crystalline cellulose and, like the lower termite M. darwiniensis, can survive indefinitely on starch (Scrivener et al., 1989). The cockroaches, Geoscapheus dilatatus and Calolampra elegans, also Blaberidae, are morphologically similar to P. cribrata but have different feeding habits: G. dilatatus is a detritovore living in burrows and feeding on dead Eucalyptus spp. leaves and C. elegans is an opportunistic feeder which can attack seedlings of commercial crops. ROLE OF BACTERIA AND PROTOZOA IN CELLULOSE DIGESTION
All the cockroaches and termites we have studied have microorganisms in their gut. However, the cockroaches basically have a tube-like gut with a concentration of microbiota a 1000-fold lower than that found in termites and there is no indication of a fermentation chamber comparable to the termite paunch. More importantly, the microbiota in the gut of P. cribrata can be removed by antibiotic treatment without affecting mortality or the ability to live on crystalline cellulose (Scrivener et al., 1989). With one exception, G. dilatatus, the insects we have studied can be maintained on crystalline cellulose. G. dilatatus is a fastidious feeder and will only thrive on its normal diet of dried eucalyptus leaves. While this indicates that the insect (and/or its symbionts) contains cellulase activity, it does not prove that the insect cellulase is endogenous unless the activity is unaffected by the removal of all microorganisms. While this can be done with P. cribrata, it is not possible with termites. There is no disputing that termites have symbionts in their hindgut, but there is no evidence that the few bacteria found in other parts of the gut have any symbiotic role. There is no simple way to define any role for these bacteria as their selective removal is not possible without affecting those in the hindgut, and there have been no reports of bacterial isolates from regions of the gut other than the hindgut. All termites contain bacteria and the majority (75%) of termites are higher termites which contain only bacteria in their hindgut. Studies on the higher termite N. exitiosus (Czolij et al., 1985) and the lower termite Reticulitermes flavipes (Breznak and Pankratz, 1977) give some idea of the variety of bacterial morphotypes which are found in the paunch. Additionally, R. flavipes, as a lower termite,
Cellulose digestion in termites and cockroaches has an impressive collection of Protozoa (Breznak and Pankratz, 1977), but it is wise to remember, when considering the origin of cellulase activity in the gut, that the microbiota are largely restricted to the hindgut. For example, in N. walkeri the number of bacteria in the foregut and midgut represent 0.02*/0 of the total (Schulz et al., 1986) and are absent in these regions in C. lacteus (Hogan et al., 1988b). That the bacteria are essential for survival is not in dispute, e.g. antibiotic defaunation of N. exitiosus reduces life expectancy to about 13 days, comparable to that caused by starvation (Eutick et al., 1978). Some roles for the bacteria in termites which have been identified are: maintaining the redox of the gut and protecting the gut from invasion by foreign bacteria (Veivers et al., 1982b), acetogenesis (Breznak et al., 1988), nitrogen fixation (Lovelock et al., 1985) and methanogenesis (Odelson and Breznak, 1985). It has not proved possible to assign them a role in cellulose degradation. The lack of reports on bacterial cellulase activity in termites, coupled with the ease with which protozoan cellulase activity has been demonstrated in the lower termites, has a clear message: bacteria are not directly involved in cellulose degradation in termites. The significance of reports such as that of the isolation of the cellulolytic bacterium Micrococcus roseus from Odontotermes obesus (Sarkar and Upadhaya, 1990) is difficult to assess as no attempt has been made to correlate this activity with that found in the termite. No details are given of the bacterial numbers within the termite, of their location or of the amount of cellulase activity present in that location. When considering the role of Protozoa and bacteria in cellulose digestion in termites it is instructive to make a biochemical comparison of the role of symbionts between ruminants and termites. The structure of the ruminant gut is such that any food entering the rumen is immediately available to the symbionts. This means that end-products of metabolism must include gluconeogenic substrates for the ruminant. Bacterial products, such as lactate and suceinate, clearly fulfil this function (Dehority, 1991). In contrast, in the insect gut, endogenous cellulases and absorption are found predominantly in the midgut, a region largely devoid of symbionts. Thus, any glucose in the diet, or produced by endogenous cellulases or other polysaccharidases, will be absorbed in the midgut. Presumably the rate of glucose uptake in the midgut is dependent on the needs of the termite and surplus glucose could be used in the hindgut. The glucose present in the hindgut of Macrotermes michaelseni (Veivers et al., 1991) could have originated in this way. Other termites we have studied, Nasutitermes sp. and Coptotermes sp., have undetectable amounts of glucose in the hindgut. If glucose is produced and absorbed as required in the midgut, there is no need for the end-products of symbiont metabolism in the paunch to contain gluconeogenic substrates and this indeed is the case. There are no reports of the presence of gluconeogenic substrates in the paunch being produced by symbionts. This is indirect evidence for their existence in other parts of the gut. Acetate is the major endproduct in the termites we have studied but formate has been found in others (Brauman et al., 1990).
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Acetate in the hindguts of M. darwiniensis and P. cribrata can be transported across the gut wall (Hogan et al., 1985), converted into acetyt-CoA in the tissues by acetyl-CoA synthetase (O'Brien and Breznak, 1984) and thence used for energy purposes or lipid synthesis. We consider that the symbiotic role, if any, of the Protozoa is currently unknown. Removal of the Protozoa results in the death of the termite (Eutick et al., 1978) though it should be noted that methods which remove Protozoa, such as 02 or metranidazole treatment, also remove spirochaetes. It is possible that it is the spirochaetes and not the Protozoa which are essential for lower termite survival, as has been established for the survival of the higher termite N. exitiosus (Eutick et al., 1978). But why do the termites die if they have enough endogenous cellulase activity in the midgut to produce enough glucose in vitro from crystalline cellulose to account for the in vivo rate of CO2 production? An apparent exception to this paradox is M. darwiniensis where the four large species of Protozoa can be removed by placing the termites on a starch diet (Veivers et al., 1983). These Protozoa are strictly cellulolytic; their removal results in the loss of the cellulase activity in the paunch. These partially defaunated termites can survive indefinitely on starch but die if transferred to a cellulose diet (Veivers et al., 1983). The role of the Protozoa in the symbiosis is never discussed in detail. Familiar statements in textbooks such as the digestion of wood by termites depends on the protozoa in their guts, in a mutually beneficial association (Stryer, 1988) are accepted uncritically and summarize entrenched attitudes. Such statements have two separate messages: firstly, that termites have no endogenous cellulase and, secondly, that they need Protozoa in a symbiotic relationship to digest cellulose. We believe that the first message is wrong but the second is more difficult to assess. Three cellulolytic flagellates have been isolated from termites, Trichomitopsis termopsidis from R. flavipes (Trager, 1932) and Zootermopsis sp. (Yamin, 1978) and Trichonympha sphaerica from Zootermopsis sp. (Yamin, 1981). Crude extracts of T. termopsidis hydrolysed crystalline cellulose to glucose and possessed both endo-fl-1,4-glucanase and t-1,4-glucosidase activities but it was not possible to say whether an exo-fl-l,4glucanase component was present (Odelson and Breznak, 1985). T. termopsidis (Yamin and Trager, 1979) and T. sphaerica (Yamin, 1981) convert cellulose to acetate, CO2 and H 2. Presumably/n vivo the last two products are bacterially converted to acetate. At present, all that can be said is that acetate produced by protozoan fermentation is available for energy purposes or for lipid synthesis. ROLE OF FUNGI IN CELLULOSE DIGESTION
Fungi are found in the gut content of termites but do not appear to be involved in cellulose digestion as their removal by fungicide treatment, e.g. as in N. exitiosus, affects neither the life span nor the cellulose activity (Eutick et aL, 1978). Alone of the xylophagous insects we have studied, P. cribrata lives in and feeds on rotting timber, so it was necessary to provide answers to the following two questions: (i)
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MICHAELSLAYTOR
3
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2
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1
O
50
75
IO 0
Elution volume (rot)
Fig. 1. Elution profiles of endo-fl-l,4-glucanase activities from fungal nodules and from young and old major workers of M. michaelseni on Bio-Gel® P-60. For the sake of clarity, zero value activities are indicated at the leading and trailing edge of each peak. The shaded portion indicates the overlap of fungal endo-fl-1,4-glucanase activity with that of the termite. A unit of endo-fl-1,4-glucanase activity is defined as mg reducing sugar (expressed as glucose equivalents) per termite or per mg fungal material (fresh wt) per hr. I , Fungal nodules; O, young major workers; ©, old major workers (Veivers et al., 1991).
does the cockroach produce a cellulase which can hydrolyse crystalline cellulose? and (ii) does it ingest fungal enzymes to assist in cellulose digestion as the Macrotermitinae are alleged to do? The removal of microbiota without affecting the ability to live on crystalline cellulose answers the first question (Scrivener et al., 1989) and the failure to find any fungal enzymes in the gut of P. cribrata answers the second question (Scrivener and Slaytor, 1992a). We believe that damp rotting wood is a more congenial environment for the cockroach and is easier to chew. The family Macrotermitinae are higher termites which maintain a complex social order based on caste, age and feeding habits and which cultivate a symbiotic fungus garden which is essential for the termite's survival. The nature of the symbiosis has not been established but pertinent to this review is the suggestion that it provides a "missing" component, i.e. a cellobiohydrolase, in the termite's cellulase (Martin, 1987). The addition of this component "completes" the cellulase in the gut with the result that the termite can then utilize cellulose. Given the mechanisms which animals use to destroy foreign proteins, it has always seemed surprising how readily the idea of acquired enzymes has been accepted. The idea is based on a comparative study of the cellulases found in the midgut of Macrotermes natalensis and the fungal nodules from the fungus garden. The evidence is slender. On the basis of both feeding experiments, which made no attempt to recreate the complexity of the nest or the highly specialized feeding habits of the worker castes (Martin and Martin, 1978), and experiments in which extracts were electrofocussed on polyacrylamide gels, it was claimed that fungal cellobiohydrolase activity was present in the midgut. The products from crystalline cellulose were not identified beyond reducing sugars (Martin and Martin, 1979). Electrofocussing did not separate cellobiohydrolase and endo-fl- 1,4-glucanase activities and it could be argued equally that there is
an endo-fl-l,4-glucanase present in the midgut with activity against both carboxymethylcellulose (CMC) and crystalline cellulose, as has been found with purified proteins in M. miilleri (Rouland et al., 1988). A difficult result to explain was the absence of fungal /3-1,4-glucosidase activity in the midgut. The proposal that the fungal enzymes were modified by limited proteolysis has never been substantiated (Martin and Martin, 1979). Our work on fungal and termite cellulases associated with Macrotermes subhyalinus and M. michaelseni is incomplete (Veivers et al., 1991) but considerably more advanced than that on which the original hypothesis was advanced (Martin and Martin, 1978). We can supply no evidence to support the hypothesis of acquired fungal enzymes in these termites. Our main approaches have been to correlate the cellulase activity in the fungus with the eating habits of the termites and to resolve the components of the fungal and termite cellulases so that the fungal components are detectable in gut extracts (Veivers et al., 1991). Most (99%) of the fungal activity is associated with the fungal nodules and these are only eaten by the young major and young minor workers (Badertscher et al., 1983; Darlington, 1986). There is no indication of cellobiohydrolase activity but there are at least four endo-fl-l,4-glucanase and two fl-l,4glucosidase activities in the fungal nodules associated with M. michaelseni. Based on the partial resolution of the endo-/~-l,4-glucanase activities of the fungal nodule and the termite, our results show that it is possible that 9% of the endo-fl-1,4-glucanase activity in young major workers in M. michaelseni could be supplied by the fungus, but we regard this as unlikely as the major endo-fl-l,4-glucanase present in the fungal nodules cannot be detected in the termite gut (Veivers et al., 1991). This important result is clearly seen in Fig. 1. Equally damaging to the hypothesis of acquired enzymes is the fact that the rate of eating, i.e. the acquisition of fungal enzymes, could only
Cellulose digestion in termites and cockroaches
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Fig. 2. Distribution of endo-fl-1,4-glucanase and ~t-amylaseactivities in the anterior midgut of N. walkeri. Endo-/~-l,4-glucanase activity (solid bars) estimated as reducing sugars produced from CMC; or-amylase activity (shaded bars) estimated as reducing sugars produced from amylose (Hogan et al., 1988a). account for 0.03% of the total cellulase activity of the young major workers of M. michaelseni. It is unlikely that ingested fungal enzymes would be cumulative and stable. This is an important consideration when evaluating the claim that the fungal endo-/L1,4glucanase constitutes 60% of the endo-~-l,4glueanase activity in the gut of M. miilleri (Rouland et al., 1988). No correlation was made in this work between the enzyme activity in the termite and that in the fungal material the termites ate. There were problems in explaining the absence of fungal /~glucosidase in the gut extracts as Martin and Martin (1979) had found earlier with M. natalensis. No fungal ~-l,4-glucosidase activity could be detected in /~-l,4-glucosidases from M. michaelseni. This is not surprising as there is an extremely small amount of fl-l,4-glucosidase activity in the fungus. There is no need to invoke lack of secretion of the fungal enzyme as has been done with M. mfilleri (Rouland et al., 1988).
LOCATION
AND
FATE
OF
HYDROLYTIC
ENZYMES
In all termites and cockroaches we have studied, cellulase activity has been found to be largely restricted to the foregut and midgut. Exceptions are the small and variable amounts of cellulase activity in the salivary glands and substantial amounts of protozoan cellulase in the hindgut of the lower termites M. darwiniensis and C. lacteus. In studying the location and fate of hydrolytic enzymes in the gut we have used the technique of serial sectioning of the gut, which has enabled us to demonstrate that in the higher termite N. walkeri cellulase and a-amylase activities are largely confined to the anterior midgut and are dearly of termite origin as this region is largely devoid of bacteria (Hogan et aL, 1988a): the distribution of endo-/~-l,4-glucanase and ~t-amylase activities in the anterior midgut is shown in Fig. 2. In the closely related N. exitiosus,/~-l,4-glucosidase was distributed between the foregut (8%) and the midgut (92%) (McEwen et al., 1980). The distribution of ¢ndo-/~-l,4-glucanase (and fl-l,4-glucosidase) in the
midgut of P. cibrata is similar (Fig. 3) to that in N. walkeri but the crop contains at least 50% of the enzyme activities (Scrivener et al., 1989). The distribution of cellulase in G. dilatatus and C. elegans is similar to that in P. cibrata (Zhang et al., 1992). The restriction of the cellulase components in the midgut to the anterior region is intriguing and obviously part of the mechanism which ensures conservation of the endogenous enzymes, as these are absent from the hindgut. As it is found in both termites and cockroaches, it would appear to be a general mechanism as ct-amylase activity is similarly restricted in N. exitiosus (Hogan et al., 1988a) and P. cribrata (Scrivener et al., 1989). At present, we do not know how it is achieved. Mechanisms we have considered and rejected on the basis of experimental evidence are resorption, inhibition or dilution (Hogan et al., 1988a). The endogenous origin of the cellulase has been confirmed using enzyme localization studies with -1,4-glucosidase in N. walkeri (Hogan et al., 1988a). The epithelial cells contain about 16% of the midgut activity. Within these cells, the activity is restricted to the anterior ventriculus and associated mainly with the plasmalemma. This is comparable to M. natalensis where 17% of the midgut endo-fl-l,4-glucanase
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L e n g t h (mm)
Fig. 3. Distribution of ~-l,4-glucosidase activity along the posterior ventriculus and the hindgut of P. cribrata. Measurements are from the foregut-midgut and midgut-hindgut junctions (Scrivener et aL, 1989).
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MICHAEL SLAYTOR Table 1. Carbohydrase activities in the salivary glands of termites and cockroaches
Termites Mastotermes darwiniensis Macrotermes michaelseni Old major workers Young major workers Cockroaches Panesthia cribrata Geoscapheus dilatatus Calolampra elegans
Cellulase
Endo-fl-l,4glucanase
66
168
fl-l,4Glucosidase 87
1.2 0.1
8.0 10.4
0.4 0.5
6.3 0.7 1.0
3.5 3.1 2.9
0.05 O. 1 0.7
~t-Amylase 537
Maltase
Invertase
5
40
Reference Veivers et al., 1982 Vieivers et al., 1991
Zhang et al., 1991 Zhang et al., 1992 Zhang et al., 1992
Activities are expressed as percentages relative to activity in the foregut and midgut.
activity is found in the tissues (Martin and Martin, 1979) but lower than T. trinervoides where the relative amount is 40% (Potts and Hewitt, 1973). In P. cribrata about 6% of the activities of the midgut cellulase components are in the epithelium (Scrivener et al., 1989). HYDROLYTIC ENZYMES OTHER THAN CELLULASES
Termites and cockroaches have a bewildering variety of enzymes, other than cellulase, which are involved in the hydrolysis of polysaccharides and disaccharides. There is rarely a report on the absence of one of these hydrolytic enzymes as the following studies of Chararas and co-workers in termites show. Thus, in the lower termite Calotermes flavicollis, a range of hydrolytic activities is found in the salivary glands, the midgut and in the flagellates in the hindgut (Chararas et al., 1984; Chararas et al., 1985). Similar results have been reported in the midgut and hindgut of the higher termite Nasutitermes lujae with the majority of the activities being in the midgut (Chararas and Noirot, 1988). Attempted correlations with diet are more difficult as comparative studies of termites show (Rouland et al., 1986a). Similar studies on the cockroaches, P. cribrata, G. dilatatus and C. elegans show that all enzymes necessary to hydrolyse the carbohydrate components of their diets are secreted in the midgut, although the relative amounts of the enzyme activities vary between species (Zhang et al., 1992). What is most interesting as far as this review is concerned is that these activities are always secreted with endo-fl-1,4-glucanase and fl-1,4glucosidase activities, strengthening the argument that cellulase is endogenous. The conclusion can also be drawn that all of these enzyme activities are secreted regardless of diet. Why, for instance, does P. cribrata produce sufficient ~t-amylase and maltase to survive on starch (Scrivener et al., 1989), a minor component of its normal food (Zhang et al., 1992)? An even clearer example is invertase in P. cribrata: there is no sucrose in its diet (Zhang et al., 1992). CELLULASES IN TERMITES AND COCKROACHES
Digestion in insects begins with the mixing of food and saliva so it is not surprising that cellulase is found, together with other carbohydrates, in the salivary glands. Importantly for this review, the salivary glands of termites are free of microorganisms (Czolij and Slaytor, 1988) and enzymes found there
must be endogenous. The criticism that there is the possibility of contamination with gut enzymes during dissection (Martin, 1983) is not acceptable with larger insects such as M. darwiniensis and cockroaches. With one exception, M. darwiniensis (Veivers et al., 1982a), the amount of activity in the salivary glands relative to that in the foregut and midgut is small (Table 1) though as the rate of secretion of the saliva is not known, it is not possible to assess the quantitative significance of the salivary enzymes. As expected, the major activities are generally those associated with initial breakdown such as endo-fl-1,4glucanase rather than those producing glucose from initial breakdown products like cellodextrins. The midgut, the site of glucose absorption, tends to be the major site of secretion of fl-l,4-glucosidase. There is no detailed description of a salivary gland cellulase presumably because of the low amounts of activity. Most reports on cellulase activity in the salivary glands are preliminary and it is usually not possible to relate the activity to that in the midgut. One exception is the work of Martin and Martin who have shown by electrofocussing that the endo-fl-1,4glucanase from the salivary glands of M. natalensis is multicomponent and at least three of the four activities are distinct from those secreted in the midgut tissues (Martin and Martin, 1979). All the endo-fl1,4-glucanase activities present in the midgut of P. cribrata are present in the salivary glands while only small amounts of fl-l,4-glucosidase activity can be detected (Scrivener and Slaytor, 1992b). A summary of our work on the distribution of cellulase, and its endo-fl-l,4-glucanase and fl-l,4glucosidase components in crude extracts of the guts of termites and cockroaches is shown in Table 2. In all species examined we have found cellulase activity, i.e. activity capable of hydrolysing crystalline cellulose, in the gut. There is a remarkable consistency in the results: the predominant site of cellulase activity in higher termites and cockroaches is the midgut. In those species with a large crop, there is a significant amount of activity in the foregut. These results are in complete agreement with studies on other termites. In Trinervitermes trinewoides, an African member of the family Nasutiterminae, 90% of the total endo-fl-l,4glucanase activity is found in the foregut, midgut and mixed segment, all regions which are virtually free of bacteria (Potts and Hewitt, 1973); the midgut has approximately 70 % of the total endo-fl-1,4-glucanase activity (Potts and Hewitt, 1973). The midgut is also the region of highest activity in two other members of the Macrotermitinae, Microcerotermes edentatus
781
Cellulose digestion in termites and cockroaches Table 2. Distribution of c¢llulase and its components in termites and cockroaches Cellulase Foregut + midgut Hindgut Termites Mastotermes darwiniensis Coptotermes lacteus Nasutitermes exitiosus Nasutitermes walkeri Macrotermes michaelseni Old major workers Young major workers Cockroaches Panesthia cribrata Geoscapheus dilatatus Calolampra elegans
17 17
83 83
ne 99
Endo-fl - 1,4-glucanase Foregut + midgut Hindgut
Reference
36
64
67
33
ne I
51 92 99
49 8 1
22 100 98
78 -2
> 99 99.7
< 1 <0.1
97 95
3 5
99 95
1 5
Veivers et al., 1991 Veivers et al., 1991
100 98
0 2
99 99.3 99.5
1 0.7 0.5
98 99.4 99
2 0.6 I
Scrivener et al., 1989 Zhang et al., 1992 Zhang et al., 1992
(Kovoor, 1970) and Macrotermes natalensis (Martin and Martin, 1979). Preliminary results on the demonstration of cellulase activity have been extended by the partial resolution of the midgut and hindgut cellulases from C. lacteus (Hogan et al., 1988b), the cellulases from the higher termites N. walkeri (Schulz et al., 1986) and M. michaelseni (Veivers et al., 1991) and the complete resolution of the major endo-//-1,4-glucanase components from P. cribrata (Scrivener and Slaytor, 1992b). The cellulases from all these insects were similar, consisting of a number of endo-fl-l,4glucanase and fl-l,4-glucosidase activities. For example, the cellulase from N. walkeri consists of at least five components: two major and one minor endo-//-1,4-glucanase and one major and one minor//-1,4-glucosidase. The major endo-/~-l,4glucanase peaks of activity were not resolved from one another or from the minor peak of fl-l,4glucosidase activity. Finally, the minor endo-fl-l,4glucanase activity did not separate from the major fl-l,4-glucosidase activity (Schulz et al., 1986). The cellulases from the salivary glands, foregut and midgut of C. lacteus were similar, though distinct from the cellulase of N. walkeri in consisting of endo-fl-l,4-glucanase and fl-l,4-glucosidase components (Hogan et al., 1988b). The fl-l,4-glucosidase component has some activity against crystalline cellulose; it has an apparent minimum Mr of 106 and can form oligomers. The presence of cellulolytic Protozoa in the paunch of the lower termite, M. darwiniensis and C. lacteus, accounted for the relatively smaller percentage of the total activity in the midgut of these species. The protozoan cellulase(s) from the hindgut are discussed later but it is important to note here that they are chromatographically distinct from the midgut cellulase. Three groups, including our own, have attempted to resolve Macrotermes cellulases and those from their associated fungus gardens. The fungal cellulases have already been discussed. The cellulases from M. michaelseni have been partially resolved by gel chromatography (Veivers et al., 1991) and those from M. natalensis by electrofocussing (Martin and Martin, 1979). The midgut enzyme from both termites consists of a number of endo-fl-l,4giucanase and fl-l,4-glucosidase activities. It is difficult to make a direct comparison with the celluCBPB 103/4.--C
fl - 1,4-Glucosidas¢ Foregut + midgut Hindgut
Veivers et al., 1983 Hogan et al., 1988 McEwen et al., 1980 McEwen et al., 1980 Hogan et al., 1988
lase from M. miilleri as freeze-dried material was used (Rouland et al., 1988) and no reports on the stability of the celhilase under freeze-drying have been published. Crude extracts of young major and young minor workers of both M. subhyalinus and M. michaelseni lose 80% of endo-fl-l,4-glucanase activity on freeze-drying though the /]-l,4ghicosidase activities are not affected (Veivers, unpublished results). Additionally, Rouland and coworkers purified the main components rather than analyse what was present, as had been attempted with the endo-fl-l,4-glucanases from M. natalensis (Martin and Martin, 1979) and M. michaelseni (Veivers et al., 1991). Two endo-fl-l,4-glucanase activities present in a ratio of 3:2 were purified (Rouland et al., 1988). Both activities were active predominantly against CMC and, to a minor extent, against crystalline cellulose, and hence are endo-fl1,4-glucanase activities; the major activity is considered to be of fungal origin (Rouland et al., 1988). There are two fl-l,4-glucosidase activities but no true cellobiohydrolases as claimed (Rouland et al., 1986b). The most detailed study which has been made of an insect cellulase is that from P. cribrata (Scrivener and Slaytor, 1992b). Unlike Limnoria species, where the gut is completely devoid of microorganisms, they are found throughout the gut of P. cribrata; most (74%), including all the Protozoa which occur in low numbers (,,, 500 per cockroach), are in the midgut (Scrivener et al., 1989). The concentration of microbiota is 1000-fold lower than in termites and there is no suggestion of a fermentation chamber in the hindgut. Our preliminary results showed that defaunated cockroaches can survive for at least 12 weeks (the length of the study) on crystalline cellulose with no decrease in cellulase activity or change in the respiratory quotient (Scrivener et al., 1989). The cellulase is a multi-component system, comprising at least six endo-fl-l,4-glucanases (two major, low molecular weight proteins and at least four higher molecular weight components present in low quantities), one major (90%) and one minor (10%) fl-l,4-glucosidase. The two major endo-fl-l,4-giucanase activities, which have been purified to homogeneity, are the dominant proteins in the foregut and midgut, together comprising more than 40% of the protein of the gut contents (Scrivener and Slaytor, 1992b).
782
MICHAELSLAYTOR
MECHANISM OF CELLULOSEDIGESTIONIN TERMITES AND COCKROACHES
Can the cellulases produce sufficient glucose for the insect's needs, and how is this achieved if there is no cellobiohydrolase component? We have demonstrated that, with P. cribrata maintained on crystalline cellulose, the in vitro rate of glucose production by crude gut extracts is of the same order as the in vivo rate of CO2 production (Scrivener et al., 1989). A similar result is found in N. walkeri when the rate of CO2 production in freshly collected termites is compared to the rate of glucose production from crude gut extracts (Veivers and Slaytor, unpublished results). If there is no exo-fl-l,4-glucanase component, then the endo-fl-l,4-glucanase components must be active against crystalline cellulose. The purified endo-fl-l,4-glucanase activities from M. mfilleri are active against crystalline cellulose although, as expected, the activity is low compared with that against CMC (Rouland et al., 1988). The products are cellotriose and cellobiose which are substrates for fl-l,4-glucosidase. Similarly, the two major endo-fl-l,4-glucanase activities from P. cribrata are active against crystalline cellulose (Scrivener and Slaytor, 1992b). The low activity (1/3000 of that against CMC), although clearly sufficient for the insect's needs, may be the reason for the large amount of endo-fl-l,4-glucanase activity in the gut. All the endo-p-l,4-glucanase activities in N. walkeri are active against crystalline cellulose (Veivers and Slaytor, unpublished results). One of the minor endo-fl-1,4-glucanase activities in the midgut of C. lacteus produces glucose from crystalline cellulose (Hogan et al., 1988b). This activity is free from any fl-l,4-glucosidase activity. There is no indication that the protozoan cellulase(s) from C. lacteus contain a cellobiohydrolase or that one is needed. Two of the endo-fl-l,4-glucanase activities produce glucose in significant amounts from CMC, while another produces glucose from crystalline cellulose (Hogan et al., 1988b) indicating that it is an exo-fl-l,4-glucosidase (EC 3.2.1.74).
they consist of multiple endo-fl-l,4-glucanase and fl-l,4-glucosidase components. There is no evidence that an exo-fl-l,4-glucanase is present or is necessary as all the cellulases produce sufficient glucose from crystalline cellulose. The predominant sites where cellulase activity is found in termites and cockroaches (the salivary glands, the foregut and the midgut) are also the sites for the secretion of other hydrolytic enzymes generally considered endogenous such as amylase, maltase, invertase and hemicellulase. Carbohydrase activities are largely absent from the hindgut, the region of the gut associated with the highest number of microorganisms. The only exceptions occur in the lower termites which, additionally, have cellulase activity in the hindgut associated with cellulolytic Protozoa. There is no evidence for the presence of bacterial cellulases in the higher termites or the cockroaches. In the Macrotermitinae, the symbiotic role of the fungus garden must be regarded as unknown in Macrotermes subhyalinus and M. michaelseni. Its contribution to the termite cellulase, if any, is minimal as the termite secretes its own cellulase in the midgut, Are the termites and wood-eating cockroaches special cases? We believe not and consider that cellulose-digesting invertebrates all use essentially the same mechanism of producing their own cellulase independent of any symbionts. Endogenous cellulase in Limnoria has already been discussed; another example is the lepidopteran Philosamia ricini where the gut microbiota can be removed without affecting the cellulase activity (Pant and Ramana, 1989). Our current work is directed to purifying a termite ceilulase, sequencing the major endo-fl-1,4-glucanase activity from P. cribrata and refining our preliminary work on enzyme localization in the midguts of both termites and cockroaches. REFERENCES
CONCLUSIONS
Badertscher S., Gerber C. and Leuthold R. H. (1983) Polyethism in food supply and processing in termite colonies of Macrotermes subhyalinus (Isoptera). Behav. Ecol. Sociobiol. 12, 115-I 19. Boyle P. J. and Mitchell R. (1978) Absence of microorganisms in crustacean digestive tracts. Science 21111,
Cellulase, as defined in this review, is an enzyme complex capable of producing glucose from crystalline cellulose. Complementary to this definition is the proposition that if an insect can live on crystalline cellulose then it must have the required cellulase. We have demonstrated this with all termites we have studied, and the cockroach, P. cribrata. The only exception is the fastidious feeder G. dilatatus. Further, if an insect can live on crystalline cellulose in the absence of symbionts then the cellulase must be endogenous. This has been demonstrated with P. cribrata. The conclusion is that P. cribrata secretes an endogenous cellulase and is not dependent on symbionts for cellulose digestion. Termites can live on crystalline cellulose and secrete cellulase in regions which are either devoid of microorganisms or contain very small numbers of microorganisms. On this evidence it is concluded that cellulase secreted in the salivary glands, the foregut and midgut of all termites is endogenous. The cellulases found are all similar:
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Mfihlemann R., Bignell D. E., Leuthold R. H., Slaytor M. and Veivers P. C. (1992) Morphological, physiological and biochemical indicators of the role of gut flora in four worker castes of the fungus-growing termite Macrotermes subhyalinus. J. Insect Physiol. (in preparation). O'Brien R. W. and Breznak J. A. (1984) Enzymes of acetate and glucose metabolism in termites. Insect Biochem. 14, 639-643. Odelson D. A. and Breznak J. A. (1985) Nutrition and growth characteristics of Trichomitopsis termopsidis, a cellulolytic protozoan from termites. Appl. environ. Microbiol. 49, 614-621. Pant R. and Ramana D. (1989) Cellulolytic activity in a phytophagous lepidopteran insect Philosamia ricini: the origin of the enzymes. Insect Biochem. 19, 269-276. Potts R. C. and Hewitt P. H. (1973) The partial purification and some of the properties of the cellulase from the termite Trinervitermes trinervoides (Nasutermitinae). Comp. Biochem. Physiol. 47, 317-326. Rapp P. and Beermann A. (1991) Bacterial cellulases. In Biosynthesis and Biodegradation of Cellulose (Edited by Halgler C. H. and Weimer P. J.), pp. 535-597. Marcel Dekker, New York. Ray D. L. and Julian J. R. (1952) Occurrence of cellulase in Limnoria. Nature 169, 32-33. Ray D. L. and Stuntz D. E. (1959) Possible relation between marine fungi and Limnoria attack on submerged wood. Science 129, 93-94. Rouland C., Chararas C. and Renoux J. (1986a) ]~tude comparte des osidases de trois esptces de termites africains ~i rtgime alimentaire diff6rent. C. r. Acad. Sci. Paris 302, 341-345. Rouland C., Civas A., Renoux J. and Petek F. (1988) Purification and properties of cellulases from the termite Macrotermes miilleri (Termitidae, Macrotermitinae) and its symbiotic fungus Termitomyces sp. Comp. Biochem. Physiol. 91B, 449~,58. Rouland C., Mora P., Matoub M., Renoux J. and Petek F. (1986b) Etude comparative entre la fl-glucosidase prtsente dans le tube digestif du termite Macrotermes miilleri et la fl-glucosidase du champiguon symbiotique Termitomyces sp. Acres. Coll. Insectes Soc. 3, 109-118. Sarkar A. and Upadhaya S. N. (1990) Cellulases of Micrococcus roseus from Odontotermes obesus gut. In Microbiology in Poecilotherms (Edited by Ltsel R.), pp. 83-86. Elsevier, Amsterdam. Schulz M. W., Slaytor M., Hogan M. E. and O'Brien R. W. (1986) Components of cellulase from the higher termite, Nasutitermes walkeri. Insect Biochem. 16, 929-932. Scrivener A. M. and Slaytor M. (1992a) Cellulose digestion in Panesthia cribrata Sanssure. Does fungal cellulase play a role? Comp. Biochem. Physiol. (in preparation). Scrivener A. M. and Slaytor M. (1992b) Purification and properties of major components of endogenous celhilase from Panesthia cribrata Saussure. Insect Biochem. On preparation). Scrivener A. M., Slaytor M. and Rose H. A. (1989) Symbiont-independent digestion of cellulose and starch in Panesthia cribrata Saussure, an Australian wood-eating cockroach. J. Insect Physiol. 35, 935-941. Stryer L. (1988) Biochemistry, p. 343. W. H. Freeman, New York. Trager W. (1932) A cellulase from the symbiotic intestinal flagellates of termites and of the roach, Cryptocercus punctulatus. Biochem. J. 26, 1762-1771. Veivers P. C., Miihiemann R., Slaytor M., Leuthold R. H. and Biguell D. E. (1991) Digestion, diet and polytheism in two fungus-growing termites: Macrotermes subhyalinus Rambur and M. michaelseni Sjestedt. J. Insect Physiol. 37, 675--682. Veivers P. C., Musca A. M., O'Brien R. W. and Slaytor M. (1982a) Digestive enzymes of the salivary glands and
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gut of Mastotermes darwiniensis. Insect Biochem. 12, 35-40. Veivers P. C., O'Brien R. W. and Slaytor M. (1982b) Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria. J. Insect Physiol. 28, 947-951. Veivers P. C., O'Brien R. W. and Slaytor M. (1983) Selective defaunation of Mastotermes darwiniensis and its effects on cellulose and starch metabolism. Insect Biochem. 13, 95-101. Yamin M. A. (1978) Axenic cultivation of the ceilulolytic flagellate Trichomitopsis termopsidis (Cleveland) from the termite Zootermopsis. J. Protozool. 25, 535-538.
Yamin M. A. (1981) Cellulose metabolism by the flagellate Trichonympha from a termite is independent of endosymbiotic bacteria. Science 211, 58-59. Yamin M. A. and Trager W. (1979) Cellulolytic activity of an axenically-cultivated termite flagellate, Trichomitopsis termopsidis. J. gen. Microbiol. 13, 417-420. Yonge C. M. (1927) The absence of a cellulase in Limnoria. Nature 119, 855. Zhang J., Scrivener A. M., Slaytor M. and Rose H. A. (1992) Diet and carbohydrase activities in three cockroaches, Geoscapheus dilatatus Saussure, Calolampra elegans Roth and Panesthia cribrota Saussure. Comp. Biochem. Physiol. (submitted).
0305-0491192 $5.00+ 0.00 © 1992PergamonPress Ltd
MINI REVIEW CELLULOSE DIGESTION IN TERMITES AND COCKROACHES: WHAT ROLE DO SYMBIONTS PLAY? MICHAEL SLAYTOR Department of Biochemistry, The University of Sydney, Sydney, N.S.W. 2006, Australia (Tel. 02 692-2597; Fax 02 692-4571) (Received 7 May 1992; accepted 26 June 1992) Abstraet--l. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts. 2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of micrc~ organisms, on a diet of crystalline cellulose. Under these conditions the RQ is I, indicating that the cockroach is surviving on glucose produced by endogenous cellulase. 3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO2 in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is suflident for their needs. 4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers. 5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-fl-l,4-glueanase (EC 3.2.1.4) and fl-l,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-fl-l,4glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-fl-l,4-glucanasecomponents are active against both crystalline cellulose and carboxymethylcellulose. 6. There is no evidence that hacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.
INTRODUCTION The study of cellulose digestion in termites and cockroaches has been plagued by misconceptions of the role of symbionts in the process. In spite of the demonstration in insects, and many other invertebrates which live on cellulose, that the major components of cellulase activity are present, which is normally sufficient proof of their endogenous nature, there has been little recognition that cellulose digestion is endogenous and does not have to involve symbionts. The demonstration in 1924 that the termite Zootermopsis sp. has cellulolytic Protozoa in its hindgut and that these flagellates are essential for the termite's survival (Cleveland, 1924) is the cornerstone of the thesis that insects do not secrete cellulase and are dependent on symbionts for cellulose digestion. This thesis, coupled with the general belief that cellulase activity is restricted to fungi, bacteria and Protozoa, has led research workers on fruitless searches for bacterial cellulases and to propose the biochemically unprecedented concept of acquired enzymes. One of the major obstacles to accepting that insects are not dependent on symbionts for cellulose digestion is the almost universal presence of microorganisms in the digestive tract. A bacteria-free gut as in the case of the marine isopod Limnoria lignorum is
a rare exception (Boyle and Mitchell, 1978). Entomologists would do well to recall the reluctance with which cellulase activity in L. lignorum has been accepted as being endogenous. Early reports, contemporary with Cleveland's work on termites, failed to find cellulase activity (using a four-week incubation at 32°C with sawdust) or any evidence of Protozoa in the gut in this obvious wood-eating organism; the speculation was that bacteria were involved in digestion (Yonge, 1927). A re-examination of the problem 25 years later showed that cellulase activity was present in crude extracts of the midgut caeca and that no symbiotic bacteria were present in the gut (Ray and Julian, 1952). This was not accepted as conclusive because, in 1957, it was proposed that marine fungi must be responsible (Meyers and Reynolds, 1957) but this was eliminated two years later (Ray and Stuntz, 1959). Finally, in 1978, 51 years after Yonge's initial observations, scanning electron microscopy confirmed that the gut was completely free of microorganisms and thus the cellulase must be endogenous (Boyle and Mitchell, 1978). That this is still not universally accepted can be deduced from the statement that L. lignorum "acquires useful digestive enzymes from its food" (Martin, 1987). It is still widely believed that insects feeding on cellulose are dependent on symbionts for cellulose
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MICHAEL SLAYTOR
digestion although support for this belief has grown more tentative since the topic was last reviewed by Martin in this journal (1983) and more recently elsewhere (Martin, 1991). The loss of confidence has been caused by the reluctant acceptance that endogenous endo-fl-l,4-glucanase (EC 3.2.1.4) and fl-l,4-glucosidase (EC 3.2.1.21) activities are produced in insects (Martin, 1991). The centre of the argument that insects are dependent on symbionts for cellulose digestion is that an exo-fl-l,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) component is essential for the hydrolysis of crystalline cellulose. As there has been no demonstration of this component in an insect, it is argued that it must be supplied by a symbiotic Protozoa, bacteria or fungi. But is this component essential for a cellulase to be capable of producing glucose from crystalline cellulose? The infrequency with which it has been found in other systems, e.g. bacterial cellulases (Rapp and Beermann, 1991), suggests that insects also may not need it. We believe that a critical evaluation of the available data leads to the conclusion that termites and cockroaches which feed on cellulose produce cellulase in the salivary glands and midgut to hydrolyse it. In other words, cellulase is an endogenously produced digestive enzyme secreted primarily in the midgut like the enzymes needed for starch or sucrose hydrolysis. This review is primarily concerned with our work over the last 15 years but also with a critical evaluation of other published data on cellulose digestion in termites and cockroaches. The definition of cellulase, which will be used in this review, is one which will proudce glucose from crystalline cellulose. It should be noted that a ceUulase thus defined will include a t - 1,4-glucosidase component. CHOICE OF TERMITES AND COCKROACHES
Primarily our choice of termites and cockroaches has been determined by local availability but together they cover the major issues in insect cellulose digestion. Thus, we have worked on Mastotermes darwiniensis, the most primitive termite and the one most closely allied to the cockroaches (McKittrick, 1965), and Coptotermes lacteus as representatives of the lower termites with cellulolytic Protozoa. M. darwiniensis is a destructive and opportunistic feeder; it will attack live and dead wood as well as sugar cane and flour (Hill, 1942). C. lacteus, an abundant mound-builder in the cooler regions around Sydney, is xylophagous. In Sydney, the commonest higher termites are the arboreal Nasutitermes walkeri and the mound-building N. exitiosus. Both of these species contain only bacterial symbionts and feed primarily on sound dead wood though colonies of N. walkeri are frequently found in living trees. Large colonies of N. walkeri can be used for multiple sampling without apparent disruption of the colony. The procedure is akin to rubber tapping and ensures a ready supply of freshly collected termites. The only exotic species we have studied are the African higher termites belonging to the Macrotermitinae, Macrotermes subhyalinus and M. michaelsent. Their fascination is the symbiotic role of the fungus garden they cultivate and its function in digestion of the grass the termites harvest.
Additionally, unlike the majority of termites, the midgut of M. subhyalinus is the largest (65% by volume) section of the gut (Miihlemann et al., 1992). The close phylogenetic relationship between termites and cockroaches, coupled with the larger size of cockroaches, invites termitologists to work on cockroaches as well. Thus Cleveland extended his work on lower termites to the wood-eating cockroach Cryptocercus punctulatus (Cryptocercidae) which also contains cellulolytic Protozoa (Cleveland et al., 1934). There are no Australian representatives of the Cryptocercidae family. Instead, we have chosen three cockroaches belonging to the Blaberidae family, concentrating on Panestheia cribrata which feeds on rotting wood. Unlike the termite gut, its gut is essentially a straight tube containing nothing comparable to the paunch and does not need symbionts to enable it to survive on crystalline cellulose and, like the lower termite M. darwiniensis, can survive indefinitely on starch (Scrivener et al., 1989). The cockroaches, Geoscapheus dilatatus and Calolampra elegans, also Blaberidae, are morphologically similar to P. cribrata but have different feeding habits: G. dilatatus is a detritovore living in burrows and feeding on dead Eucalyptus spp. leaves and C. elegans is an opportunistic feeder which can attack seedlings of commercial crops. ROLE OF BACTERIA AND PROTOZOA IN CELLULOSE DIGESTION
All the cockroaches and termites we have studied have microorganisms in their gut. However, the cockroaches basically have a tube-like gut with a concentration of microbiota a 1000-fold lower than that found in termites and there is no indication of a fermentation chamber comparable to the termite paunch. More importantly, the microbiota in the gut of P. cribrata can be removed by antibiotic treatment without affecting mortality or the ability to live on crystalline cellulose (Scrivener et al., 1989). With one exception, G. dilatatus, the insects we have studied can be maintained on crystalline cellulose. G. dilatatus is a fastidious feeder and will only thrive on its normal diet of dried eucalyptus leaves. While this indicates that the insect (and/or its symbionts) contains cellulase activity, it does not prove that the insect cellulase is endogenous unless the activity is unaffected by the removal of all microorganisms. While this can be done with P. cribrata, it is not possible with termites. There is no disputing that termites have symbionts in their hindgut, but there is no evidence that the few bacteria found in other parts of the gut have any symbiotic role. There is no simple way to define any role for these bacteria as their selective removal is not possible without affecting those in the hindgut, and there have been no reports of bacterial isolates from regions of the gut other than the hindgut. All termites contain bacteria and the majority (75%) of termites are higher termites which contain only bacteria in their hindgut. Studies on the higher termite N. exitiosus (Czolij et al., 1985) and the lower termite Reticulitermes flavipes (Breznak and Pankratz, 1977) give some idea of the variety of bacterial morphotypes which are found in the paunch. Additionally, R. flavipes, as a lower termite,
Cellulose digestion in termites and cockroaches has an impressive collection of Protozoa (Breznak and Pankratz, 1977), but it is wise to remember, when considering the origin of cellulase activity in the gut, that the microbiota are largely restricted to the hindgut. For example, in N. walkeri the number of bacteria in the foregut and midgut represent 0.02*/0 of the total (Schulz et al., 1986) and are absent in these regions in C. lacteus (Hogan et al., 1988b). That the bacteria are essential for survival is not in dispute, e.g. antibiotic defaunation of N. exitiosus reduces life expectancy to about 13 days, comparable to that caused by starvation (Eutick et al., 1978). Some roles for the bacteria in termites which have been identified are: maintaining the redox of the gut and protecting the gut from invasion by foreign bacteria (Veivers et al., 1982b), acetogenesis (Breznak et al., 1988), nitrogen fixation (Lovelock et al., 1985) and methanogenesis (Odelson and Breznak, 1985). It has not proved possible to assign them a role in cellulose degradation. The lack of reports on bacterial cellulase activity in termites, coupled with the ease with which protozoan cellulase activity has been demonstrated in the lower termites, has a clear message: bacteria are not directly involved in cellulose degradation in termites. The significance of reports such as that of the isolation of the cellulolytic bacterium Micrococcus roseus from Odontotermes obesus (Sarkar and Upadhaya, 1990) is difficult to assess as no attempt has been made to correlate this activity with that found in the termite. No details are given of the bacterial numbers within the termite, of their location or of the amount of cellulase activity present in that location. When considering the role of Protozoa and bacteria in cellulose digestion in termites it is instructive to make a biochemical comparison of the role of symbionts between ruminants and termites. The structure of the ruminant gut is such that any food entering the rumen is immediately available to the symbionts. This means that end-products of metabolism must include gluconeogenic substrates for the ruminant. Bacterial products, such as lactate and suceinate, clearly fulfil this function (Dehority, 1991). In contrast, in the insect gut, endogenous cellulases and absorption are found predominantly in the midgut, a region largely devoid of symbionts. Thus, any glucose in the diet, or produced by endogenous cellulases or other polysaccharidases, will be absorbed in the midgut. Presumably the rate of glucose uptake in the midgut is dependent on the needs of the termite and surplus glucose could be used in the hindgut. The glucose present in the hindgut of Macrotermes michaelseni (Veivers et al., 1991) could have originated in this way. Other termites we have studied, Nasutitermes sp. and Coptotermes sp., have undetectable amounts of glucose in the hindgut. If glucose is produced and absorbed as required in the midgut, there is no need for the end-products of symbiont metabolism in the paunch to contain gluconeogenic substrates and this indeed is the case. There are no reports of the presence of gluconeogenic substrates in the paunch being produced by symbionts. This is indirect evidence for their existence in other parts of the gut. Acetate is the major endproduct in the termites we have studied but formate has been found in others (Brauman et al., 1990).
777
Acetate in the hindguts of M. darwiniensis and P. cribrata can be transported across the gut wall (Hogan et al., 1985), converted into acetyt-CoA in the tissues by acetyl-CoA synthetase (O'Brien and Breznak, 1984) and thence used for energy purposes or lipid synthesis. We consider that the symbiotic role, if any, of the Protozoa is currently unknown. Removal of the Protozoa results in the death of the termite (Eutick et al., 1978) though it should be noted that methods which remove Protozoa, such as 02 or metranidazole treatment, also remove spirochaetes. It is possible that it is the spirochaetes and not the Protozoa which are essential for lower termite survival, as has been established for the survival of the higher termite N. exitiosus (Eutick et al., 1978). But why do the termites die if they have enough endogenous cellulase activity in the midgut to produce enough glucose in vitro from crystalline cellulose to account for the in vivo rate of CO2 production? An apparent exception to this paradox is M. darwiniensis where the four large species of Protozoa can be removed by placing the termites on a starch diet (Veivers et al., 1983). These Protozoa are strictly cellulolytic; their removal results in the loss of the cellulase activity in the paunch. These partially defaunated termites can survive indefinitely on starch but die if transferred to a cellulose diet (Veivers et al., 1983). The role of the Protozoa in the symbiosis is never discussed in detail. Familiar statements in textbooks such as the digestion of wood by termites depends on the protozoa in their guts, in a mutually beneficial association (Stryer, 1988) are accepted uncritically and summarize entrenched attitudes. Such statements have two separate messages: firstly, that termites have no endogenous cellulase and, secondly, that they need Protozoa in a symbiotic relationship to digest cellulose. We believe that the first message is wrong but the second is more difficult to assess. Three cellulolytic flagellates have been isolated from termites, Trichomitopsis termopsidis from R. flavipes (Trager, 1932) and Zootermopsis sp. (Yamin, 1978) and Trichonympha sphaerica from Zootermopsis sp. (Yamin, 1981). Crude extracts of T. termopsidis hydrolysed crystalline cellulose to glucose and possessed both endo-fl-1,4-glucanase and t-1,4-glucosidase activities but it was not possible to say whether an exo-fl-l,4glucanase component was present (Odelson and Breznak, 1985). T. termopsidis (Yamin and Trager, 1979) and T. sphaerica (Yamin, 1981) convert cellulose to acetate, CO2 and H 2. Presumably/n vivo the last two products are bacterially converted to acetate. At present, all that can be said is that acetate produced by protozoan fermentation is available for energy purposes or for lipid synthesis. ROLE OF FUNGI IN CELLULOSE DIGESTION
Fungi are found in the gut content of termites but do not appear to be involved in cellulose digestion as their removal by fungicide treatment, e.g. as in N. exitiosus, affects neither the life span nor the cellulose activity (Eutick et aL, 1978). Alone of the xylophagous insects we have studied, P. cribrata lives in and feeds on rotting timber, so it was necessary to provide answers to the following two questions: (i)
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MICHAELSLAYTOR
3
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75
IO 0
Elution volume (rot)
Fig. 1. Elution profiles of endo-fl-l,4-glucanase activities from fungal nodules and from young and old major workers of M. michaelseni on Bio-Gel® P-60. For the sake of clarity, zero value activities are indicated at the leading and trailing edge of each peak. The shaded portion indicates the overlap of fungal endo-fl-1,4-glucanase activity with that of the termite. A unit of endo-fl-1,4-glucanase activity is defined as mg reducing sugar (expressed as glucose equivalents) per termite or per mg fungal material (fresh wt) per hr. I , Fungal nodules; O, young major workers; ©, old major workers (Veivers et al., 1991).
does the cockroach produce a cellulase which can hydrolyse crystalline cellulose? and (ii) does it ingest fungal enzymes to assist in cellulose digestion as the Macrotermitinae are alleged to do? The removal of microbiota without affecting the ability to live on crystalline cellulose answers the first question (Scrivener et al., 1989) and the failure to find any fungal enzymes in the gut of P. cribrata answers the second question (Scrivener and Slaytor, 1992a). We believe that damp rotting wood is a more congenial environment for the cockroach and is easier to chew. The family Macrotermitinae are higher termites which maintain a complex social order based on caste, age and feeding habits and which cultivate a symbiotic fungus garden which is essential for the termite's survival. The nature of the symbiosis has not been established but pertinent to this review is the suggestion that it provides a "missing" component, i.e. a cellobiohydrolase, in the termite's cellulase (Martin, 1987). The addition of this component "completes" the cellulase in the gut with the result that the termite can then utilize cellulose. Given the mechanisms which animals use to destroy foreign proteins, it has always seemed surprising how readily the idea of acquired enzymes has been accepted. The idea is based on a comparative study of the cellulases found in the midgut of Macrotermes natalensis and the fungal nodules from the fungus garden. The evidence is slender. On the basis of both feeding experiments, which made no attempt to recreate the complexity of the nest or the highly specialized feeding habits of the worker castes (Martin and Martin, 1978), and experiments in which extracts were electrofocussed on polyacrylamide gels, it was claimed that fungal cellobiohydrolase activity was present in the midgut. The products from crystalline cellulose were not identified beyond reducing sugars (Martin and Martin, 1979). Electrofocussing did not separate cellobiohydrolase and endo-fl- 1,4-glucanase activities and it could be argued equally that there is
an endo-fl-l,4-glucanase present in the midgut with activity against both carboxymethylcellulose (CMC) and crystalline cellulose, as has been found with purified proteins in M. miilleri (Rouland et al., 1988). A difficult result to explain was the absence of fungal /3-1,4-glucosidase activity in the midgut. The proposal that the fungal enzymes were modified by limited proteolysis has never been substantiated (Martin and Martin, 1979). Our work on fungal and termite cellulases associated with Macrotermes subhyalinus and M. michaelseni is incomplete (Veivers et al., 1991) but considerably more advanced than that on which the original hypothesis was advanced (Martin and Martin, 1978). We can supply no evidence to support the hypothesis of acquired fungal enzymes in these termites. Our main approaches have been to correlate the cellulase activity in the fungus with the eating habits of the termites and to resolve the components of the fungal and termite cellulases so that the fungal components are detectable in gut extracts (Veivers et al., 1991). Most (99%) of the fungal activity is associated with the fungal nodules and these are only eaten by the young major and young minor workers (Badertscher et al., 1983; Darlington, 1986). There is no indication of cellobiohydrolase activity but there are at least four endo-fl-l,4-glucanase and two fl-l,4glucosidase activities in the fungal nodules associated with M. michaelseni. Based on the partial resolution of the endo-/~-l,4-glucanase activities of the fungal nodule and the termite, our results show that it is possible that 9% of the endo-fl-1,4-glucanase activity in young major workers in M. michaelseni could be supplied by the fungus, but we regard this as unlikely as the major endo-fl-l,4-glucanase present in the fungal nodules cannot be detected in the termite gut (Veivers et al., 1991). This important result is clearly seen in Fig. 1. Equally damaging to the hypothesis of acquired enzymes is the fact that the rate of eating, i.e. the acquisition of fungal enzymes, could only
Cellulose digestion in termites and cockroaches
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Fig. 2. Distribution of endo-fl-1,4-glucanase and ~t-amylaseactivities in the anterior midgut of N. walkeri. Endo-/~-l,4-glucanase activity (solid bars) estimated as reducing sugars produced from CMC; or-amylase activity (shaded bars) estimated as reducing sugars produced from amylose (Hogan et al., 1988a). account for 0.03% of the total cellulase activity of the young major workers of M. michaelseni. It is unlikely that ingested fungal enzymes would be cumulative and stable. This is an important consideration when evaluating the claim that the fungal endo-/L1,4glucanase constitutes 60% of the endo-~-l,4glueanase activity in the gut of M. miilleri (Rouland et al., 1988). No correlation was made in this work between the enzyme activity in the termite and that in the fungal material the termites ate. There were problems in explaining the absence of fungal /~glucosidase in the gut extracts as Martin and Martin (1979) had found earlier with M. natalensis. No fungal ~-l,4-glucosidase activity could be detected in /~-l,4-glucosidases from M. michaelseni. This is not surprising as there is an extremely small amount of fl-l,4-glucosidase activity in the fungus. There is no need to invoke lack of secretion of the fungal enzyme as has been done with M. mfilleri (Rouland et al., 1988).
LOCATION
AND
FATE
OF
HYDROLYTIC
ENZYMES
In all termites and cockroaches we have studied, cellulase activity has been found to be largely restricted to the foregut and midgut. Exceptions are the small and variable amounts of cellulase activity in the salivary glands and substantial amounts of protozoan cellulase in the hindgut of the lower termites M. darwiniensis and C. lacteus. In studying the location and fate of hydrolytic enzymes in the gut we have used the technique of serial sectioning of the gut, which has enabled us to demonstrate that in the higher termite N. walkeri cellulase and a-amylase activities are largely confined to the anterior midgut and are dearly of termite origin as this region is largely devoid of bacteria (Hogan et aL, 1988a): the distribution of endo-/~-l,4-glucanase and ~t-amylase activities in the anterior midgut is shown in Fig. 2. In the closely related N. exitiosus,/~-l,4-glucosidase was distributed between the foregut (8%) and the midgut (92%) (McEwen et al., 1980). The distribution of ¢ndo-/~-l,4-glucanase (and fl-l,4-glucosidase) in the
midgut of P. cibrata is similar (Fig. 3) to that in N. walkeri but the crop contains at least 50% of the enzyme activities (Scrivener et al., 1989). The distribution of cellulase in G. dilatatus and C. elegans is similar to that in P. cibrata (Zhang et al., 1992). The restriction of the cellulase components in the midgut to the anterior region is intriguing and obviously part of the mechanism which ensures conservation of the endogenous enzymes, as these are absent from the hindgut. As it is found in both termites and cockroaches, it would appear to be a general mechanism as ct-amylase activity is similarly restricted in N. exitiosus (Hogan et al., 1988a) and P. cribrata (Scrivener et al., 1989). At present, we do not know how it is achieved. Mechanisms we have considered and rejected on the basis of experimental evidence are resorption, inhibition or dilution (Hogan et al., 1988a). The endogenous origin of the cellulase has been confirmed using enzyme localization studies with -1,4-glucosidase in N. walkeri (Hogan et al., 1988a). The epithelial cells contain about 16% of the midgut activity. Within these cells, the activity is restricted to the anterior ventriculus and associated mainly with the plasmalemma. This is comparable to M. natalensis where 17% of the midgut endo-fl-l,4-glucanase
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L e n g t h (mm)
Fig. 3. Distribution of ~-l,4-glucosidase activity along the posterior ventriculus and the hindgut of P. cribrata. Measurements are from the foregut-midgut and midgut-hindgut junctions (Scrivener et aL, 1989).
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MICHAEL SLAYTOR Table 1. Carbohydrase activities in the salivary glands of termites and cockroaches
Termites Mastotermes darwiniensis Macrotermes michaelseni Old major workers Young major workers Cockroaches Panesthia cribrata Geoscapheus dilatatus Calolampra elegans
Cellulase
Endo-fl-l,4glucanase
66
168
fl-l,4Glucosidase 87
1.2 0.1
8.0 10.4
0.4 0.5
6.3 0.7 1.0
3.5 3.1 2.9
0.05 O. 1 0.7
~t-Amylase 537
Maltase
Invertase
5
40
Reference Veivers et al., 1982 Vieivers et al., 1991
Zhang et al., 1991 Zhang et al., 1992 Zhang et al., 1992
Activities are expressed as percentages relative to activity in the foregut and midgut.
activity is found in the tissues (Martin and Martin, 1979) but lower than T. trinervoides where the relative amount is 40% (Potts and Hewitt, 1973). In P. cribrata about 6% of the activities of the midgut cellulase components are in the epithelium (Scrivener et al., 1989). HYDROLYTIC ENZYMES OTHER THAN CELLULASES
Termites and cockroaches have a bewildering variety of enzymes, other than cellulase, which are involved in the hydrolysis of polysaccharides and disaccharides. There is rarely a report on the absence of one of these hydrolytic enzymes as the following studies of Chararas and co-workers in termites show. Thus, in the lower termite Calotermes flavicollis, a range of hydrolytic activities is found in the salivary glands, the midgut and in the flagellates in the hindgut (Chararas et al., 1984; Chararas et al., 1985). Similar results have been reported in the midgut and hindgut of the higher termite Nasutitermes lujae with the majority of the activities being in the midgut (Chararas and Noirot, 1988). Attempted correlations with diet are more difficult as comparative studies of termites show (Rouland et al., 1986a). Similar studies on the cockroaches, P. cribrata, G. dilatatus and C. elegans show that all enzymes necessary to hydrolyse the carbohydrate components of their diets are secreted in the midgut, although the relative amounts of the enzyme activities vary between species (Zhang et al., 1992). What is most interesting as far as this review is concerned is that these activities are always secreted with endo-fl-1,4-glucanase and fl-1,4glucosidase activities, strengthening the argument that cellulase is endogenous. The conclusion can also be drawn that all of these enzyme activities are secreted regardless of diet. Why, for instance, does P. cribrata produce sufficient ~t-amylase and maltase to survive on starch (Scrivener et al., 1989), a minor component of its normal food (Zhang et al., 1992)? An even clearer example is invertase in P. cribrata: there is no sucrose in its diet (Zhang et al., 1992). CELLULASES IN TERMITES AND COCKROACHES
Digestion in insects begins with the mixing of food and saliva so it is not surprising that cellulase is found, together with other carbohydrates, in the salivary glands. Importantly for this review, the salivary glands of termites are free of microorganisms (Czolij and Slaytor, 1988) and enzymes found there
must be endogenous. The criticism that there is the possibility of contamination with gut enzymes during dissection (Martin, 1983) is not acceptable with larger insects such as M. darwiniensis and cockroaches. With one exception, M. darwiniensis (Veivers et al., 1982a), the amount of activity in the salivary glands relative to that in the foregut and midgut is small (Table 1) though as the rate of secretion of the saliva is not known, it is not possible to assess the quantitative significance of the salivary enzymes. As expected, the major activities are generally those associated with initial breakdown such as endo-fl-1,4glucanase rather than those producing glucose from initial breakdown products like cellodextrins. The midgut, the site of glucose absorption, tends to be the major site of secretion of fl-l,4-glucosidase. There is no detailed description of a salivary gland cellulase presumably because of the low amounts of activity. Most reports on cellulase activity in the salivary glands are preliminary and it is usually not possible to relate the activity to that in the midgut. One exception is the work of Martin and Martin who have shown by electrofocussing that the endo-fl-1,4glucanase from the salivary glands of M. natalensis is multicomponent and at least three of the four activities are distinct from those secreted in the midgut tissues (Martin and Martin, 1979). All the endo-fl1,4-glucanase activities present in the midgut of P. cribrata are present in the salivary glands while only small amounts of fl-l,4-glucosidase activity can be detected (Scrivener and Slaytor, 1992b). A summary of our work on the distribution of cellulase, and its endo-fl-l,4-glucanase and fl-l,4glucosidase components in crude extracts of the guts of termites and cockroaches is shown in Table 2. In all species examined we have found cellulase activity, i.e. activity capable of hydrolysing crystalline cellulose, in the gut. There is a remarkable consistency in the results: the predominant site of cellulase activity in higher termites and cockroaches is the midgut. In those species with a large crop, there is a significant amount of activity in the foregut. These results are in complete agreement with studies on other termites. In Trinervitermes trinewoides, an African member of the family Nasutiterminae, 90% of the total endo-fl-l,4glucanase activity is found in the foregut, midgut and mixed segment, all regions which are virtually free of bacteria (Potts and Hewitt, 1973); the midgut has approximately 70 % of the total endo-fl-1,4-glucanase activity (Potts and Hewitt, 1973). The midgut is also the region of highest activity in two other members of the Macrotermitinae, Microcerotermes edentatus
781
Cellulose digestion in termites and cockroaches Table 2. Distribution of c¢llulase and its components in termites and cockroaches Cellulase Foregut + midgut Hindgut Termites Mastotermes darwiniensis Coptotermes lacteus Nasutitermes exitiosus Nasutitermes walkeri Macrotermes michaelseni Old major workers Young major workers Cockroaches Panesthia cribrata Geoscapheus dilatatus Calolampra elegans
17 17
83 83
ne 99
Endo-fl - 1,4-glucanase Foregut + midgut Hindgut
Reference
36
64
67
33
ne I
51 92 99
49 8 1
22 100 98
78 -2
> 99 99.7
< 1 <0.1
97 95
3 5
99 95
1 5
Veivers et al., 1991 Veivers et al., 1991
100 98
0 2
99 99.3 99.5
1 0.7 0.5
98 99.4 99
2 0.6 I
Scrivener et al., 1989 Zhang et al., 1992 Zhang et al., 1992
(Kovoor, 1970) and Macrotermes natalensis (Martin and Martin, 1979). Preliminary results on the demonstration of cellulase activity have been extended by the partial resolution of the midgut and hindgut cellulases from C. lacteus (Hogan et al., 1988b), the cellulases from the higher termites N. walkeri (Schulz et al., 1986) and M. michaelseni (Veivers et al., 1991) and the complete resolution of the major endo-//-1,4-glucanase components from P. cribrata (Scrivener and Slaytor, 1992b). The cellulases from all these insects were similar, consisting of a number of endo-fl-l,4glucanase and fl-l,4-glucosidase activities. For example, the cellulase from N. walkeri consists of at least five components: two major and one minor endo-//-1,4-glucanase and one major and one minor//-1,4-glucosidase. The major endo-/~-l,4glucanase peaks of activity were not resolved from one another or from the minor peak of fl-l,4glucosidase activity. Finally, the minor endo-fl-l,4glucanase activity did not separate from the major fl-l,4-glucosidase activity (Schulz et al., 1986). The cellulases from the salivary glands, foregut and midgut of C. lacteus were similar, though distinct from the cellulase of N. walkeri in consisting of endo-fl-l,4-glucanase and fl-l,4-glucosidase components (Hogan et al., 1988b). The fl-l,4-glucosidase component has some activity against crystalline cellulose; it has an apparent minimum Mr of 106 and can form oligomers. The presence of cellulolytic Protozoa in the paunch of the lower termite, M. darwiniensis and C. lacteus, accounted for the relatively smaller percentage of the total activity in the midgut of these species. The protozoan cellulase(s) from the hindgut are discussed later but it is important to note here that they are chromatographically distinct from the midgut cellulase. Three groups, including our own, have attempted to resolve Macrotermes cellulases and those from their associated fungus gardens. The fungal cellulases have already been discussed. The cellulases from M. michaelseni have been partially resolved by gel chromatography (Veivers et al., 1991) and those from M. natalensis by electrofocussing (Martin and Martin, 1979). The midgut enzyme from both termites consists of a number of endo-fl-l,4giucanase and fl-l,4-glucosidase activities. It is difficult to make a direct comparison with the celluCBPB 103/4.--C
fl - 1,4-Glucosidas¢ Foregut + midgut Hindgut
Veivers et al., 1983 Hogan et al., 1988 McEwen et al., 1980 McEwen et al., 1980 Hogan et al., 1988
lase from M. miilleri as freeze-dried material was used (Rouland et al., 1988) and no reports on the stability of the celhilase under freeze-drying have been published. Crude extracts of young major and young minor workers of both M. subhyalinus and M. michaelseni lose 80% of endo-fl-l,4-glucanase activity on freeze-drying though the /]-l,4ghicosidase activities are not affected (Veivers, unpublished results). Additionally, Rouland and coworkers purified the main components rather than analyse what was present, as had been attempted with the endo-fl-l,4-glucanases from M. natalensis (Martin and Martin, 1979) and M. michaelseni (Veivers et al., 1991). Two endo-fl-l,4-glucanase activities present in a ratio of 3:2 were purified (Rouland et al., 1988). Both activities were active predominantly against CMC and, to a minor extent, against crystalline cellulose, and hence are endo-fl1,4-glucanase activities; the major activity is considered to be of fungal origin (Rouland et al., 1988). There are two fl-l,4-glucosidase activities but no true cellobiohydrolases as claimed (Rouland et al., 1986b). The most detailed study which has been made of an insect cellulase is that from P. cribrata (Scrivener and Slaytor, 1992b). Unlike Limnoria species, where the gut is completely devoid of microorganisms, they are found throughout the gut of P. cribrata; most (74%), including all the Protozoa which occur in low numbers (,,, 500 per cockroach), are in the midgut (Scrivener et al., 1989). The concentration of microbiota is 1000-fold lower than in termites and there is no suggestion of a fermentation chamber in the hindgut. Our preliminary results showed that defaunated cockroaches can survive for at least 12 weeks (the length of the study) on crystalline cellulose with no decrease in cellulase activity or change in the respiratory quotient (Scrivener et al., 1989). The cellulase is a multi-component system, comprising at least six endo-fl-l,4-glucanases (two major, low molecular weight proteins and at least four higher molecular weight components present in low quantities), one major (90%) and one minor (10%) fl-l,4-glucosidase. The two major endo-fl-l,4-giucanase activities, which have been purified to homogeneity, are the dominant proteins in the foregut and midgut, together comprising more than 40% of the protein of the gut contents (Scrivener and Slaytor, 1992b).
782
MICHAELSLAYTOR
MECHANISM OF CELLULOSEDIGESTIONIN TERMITES AND COCKROACHES
Can the cellulases produce sufficient glucose for the insect's needs, and how is this achieved if there is no cellobiohydrolase component? We have demonstrated that, with P. cribrata maintained on crystalline cellulose, the in vitro rate of glucose production by crude gut extracts is of the same order as the in vivo rate of CO2 production (Scrivener et al., 1989). A similar result is found in N. walkeri when the rate of CO2 production in freshly collected termites is compared to the rate of glucose production from crude gut extracts (Veivers and Slaytor, unpublished results). If there is no exo-fl-l,4-glucanase component, then the endo-fl-l,4-glucanase components must be active against crystalline cellulose. The purified endo-fl-l,4-glucanase activities from M. mfilleri are active against crystalline cellulose although, as expected, the activity is low compared with that against CMC (Rouland et al., 1988). The products are cellotriose and cellobiose which are substrates for fl-l,4-glucosidase. Similarly, the two major endo-fl-l,4-glucanase activities from P. cribrata are active against crystalline cellulose (Scrivener and Slaytor, 1992b). The low activity (1/3000 of that against CMC), although clearly sufficient for the insect's needs, may be the reason for the large amount of endo-fl-l,4-glucanase activity in the gut. All the endo-p-l,4-glucanase activities in N. walkeri are active against crystalline cellulose (Veivers and Slaytor, unpublished results). One of the minor endo-fl-1,4-glucanase activities in the midgut of C. lacteus produces glucose from crystalline cellulose (Hogan et al., 1988b). This activity is free from any fl-l,4-glucosidase activity. There is no indication that the protozoan cellulase(s) from C. lacteus contain a cellobiohydrolase or that one is needed. Two of the endo-fl-l,4-glucanase activities produce glucose in significant amounts from CMC, while another produces glucose from crystalline cellulose (Hogan et al., 1988b) indicating that it is an exo-fl-l,4-glucosidase (EC 3.2.1.74).
they consist of multiple endo-fl-l,4-glucanase and fl-l,4-glucosidase components. There is no evidence that an exo-fl-l,4-glucanase is present or is necessary as all the cellulases produce sufficient glucose from crystalline cellulose. The predominant sites where cellulase activity is found in termites and cockroaches (the salivary glands, the foregut and the midgut) are also the sites for the secretion of other hydrolytic enzymes generally considered endogenous such as amylase, maltase, invertase and hemicellulase. Carbohydrase activities are largely absent from the hindgut, the region of the gut associated with the highest number of microorganisms. The only exceptions occur in the lower termites which, additionally, have cellulase activity in the hindgut associated with cellulolytic Protozoa. There is no evidence for the presence of bacterial cellulases in the higher termites or the cockroaches. In the Macrotermitinae, the symbiotic role of the fungus garden must be regarded as unknown in Macrotermes subhyalinus and M. michaelseni. Its contribution to the termite cellulase, if any, is minimal as the termite secretes its own cellulase in the midgut, Are the termites and wood-eating cockroaches special cases? We believe not and consider that cellulose-digesting invertebrates all use essentially the same mechanism of producing their own cellulase independent of any symbionts. Endogenous cellulase in Limnoria has already been discussed; another example is the lepidopteran Philosamia ricini where the gut microbiota can be removed without affecting the cellulase activity (Pant and Ramana, 1989). Our current work is directed to purifying a termite ceilulase, sequencing the major endo-fl-1,4-glucanase activity from P. cribrata and refining our preliminary work on enzyme localization in the midguts of both termites and cockroaches. REFERENCES
CONCLUSIONS
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Cellulase, as defined in this review, is an enzyme complex capable of producing glucose from crystalline cellulose. Complementary to this definition is the proposition that if an insect can live on crystalline cellulose then it must have the required cellulase. We have demonstrated this with all termites we have studied, and the cockroach, P. cribrata. The only exception is the fastidious feeder G. dilatatus. Further, if an insect can live on crystalline cellulose in the absence of symbionts then the cellulase must be endogenous. This has been demonstrated with P. cribrata. The conclusion is that P. cribrata secretes an endogenous cellulase and is not dependent on symbionts for cellulose digestion. Termites can live on crystalline cellulose and secrete cellulase in regions which are either devoid of microorganisms or contain very small numbers of microorganisms. On this evidence it is concluded that cellulase secreted in the salivary glands, the foregut and midgut of all termites is endogenous. The cellulases found are all similar:
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Yamin M. A. (1981) Cellulose metabolism by the flagellate Trichonympha from a termite is independent of endosymbiotic bacteria. Science 211, 58-59. Yamin M. A. and Trager W. (1979) Cellulolytic activity of an axenically-cultivated termite flagellate, Trichomitopsis termopsidis. J. gen. Microbiol. 13, 417-420. Yonge C. M. (1927) The absence of a cellulase in Limnoria. Nature 119, 855. Zhang J., Scrivener A. M., Slaytor M. and Rose H. A. (1992) Diet and carbohydrase activities in three cockroaches, Geoscapheus dilatatus Saussure, Calolampra elegans Roth and Panesthia cribrota Saussure. Comp. Biochem. Physiol. (submitted).