Comparative aspects of plant cell wall digestion in insects

.4nimal Feed Science and Technology, 32 ( 1991 ) 101-118

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Elsevier Science Publishers B.V., Amsterdam

Comparative aspects of plant cell wall digestion in insects R.A. P r i n s I a n d D . A . K r e u l e n 2 ~Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren (The Netherlands) 2P.O. Box 128, 9750 AC Haren (The Netherlands)

ABSTRACT Prins, R.A. and Kreulen, D.A., 1991. Comparative aspects of plant cell wall digestion in insects. Anim. Feed Sci. Technol.. 32:101-118. Although many phytophagous and wood-eating invertebrates form their own cellulases, there is an overwhelmingvariety of symbioses between plant- and wood-utilisinginsects and microorganisms. In one type of symbiosis (endosymbiosis), insects (rhinoceros beetle, cockroach, lower termites) host cellulolytic protozoa and/or bacteria in the hindgut. In a number of insect taxa (higher termites, cerambycid beetles) cellulose digestion is aided by the ingestion of fungal tissues for the delivery of certain components of the cellulase complex needed to complement the host enzymes for action on crystalline forms of cellulose. While the bulk of the plant lignin is not significantlydegraded under the conditions characteristic of gut contents of herbivores, some groups of insects are thought to be able to digest a part of this noncarbohydrate polymer.

INTRODUCTION

The role played by the gut flora in the digestion of plant food polysaccharides in most groups of invertebrates other than the groups of insects discussed here, remains unknown or vague at best. Many fresh water or marine invertebrates which use lignocelluloses from wood or other plant parts are free of gut microbes and the presence of endogenously produced host cellulases has been postulated in a number of cases. Much information has been accumulated recently on the origin of the enzymes involved in plant cell wall hydrolysis in the insect gut. Large differences between groups of insects have become apparent: some insects use intestinal microorganisms for this purpose, others acquire microbial enzymes by ingesting fungi, and other groups form at least part of the cellulase complex by themselves. However, not all phytophagous insects utilise the plant cell wall. 0377-8401/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.

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PHYTOPHAGOUS INSECTS WHICH DO NOT UTILISE THE PLANT CELL WALL

There are several families of beetles which eat bark, phloem tissue and wood, but are unable to digest the cellulose they consume. Also, no evidence for a significant digestion of cellulose has been observed in foliage-feeding insects (adults or larvae of Orthoptera, Coleoptera or Lepidoptera) or in detritusfeeding insects (Plecoptera, Coleoptera, Diptera, Trichoptera). In the migratory grasshopper (Melanoplus sanguinipes; Orthoptera, Acrididae) raised on barley shoots and bran, extensive indigenous populations of luminal and epimural bacteria were found in the gut which seemed to be predominantly facultative anaerobes. No significant populations of obligately anaerobic bacteria were found. This is consistent with the rapid rate of passage of digesta: egestion times of solid digesta ranged from 3.0 to 5.7 h. Acetic acid was the only acid found in all sections of the gut and in the haemolymph (Mead et al., 1988 ). The grasshopper probably does not digest cellulose. No epimural bacteria were found in the midgut but this has also been found for the cockroach (Bracke et al., 1979). The reason is the continuous secretion of the chitinous peritrophic membrane from the epithelium which surrounds the digesta during movement through the gut. Attachment of bacteria to the luminal but not the epithelial side of the peritrophic membrane was found in the common house cricket (Acheta domestica) by Ulrich et al. ( 1981 ). Payne and Davidson (1974) failed to detect the presence of anaerobic bacteria in the gut of the locust, Schistocerca gregaria. INSECTS CAPABLE OF DIGESTING CELLULOSE

Cellulose digestion has been demonstrated in representatives of widely different taxonomic groups: silverfish, cockroaches and various wood-eating insects such as woodroaches (Orthoptera), lower and higher termites (Isoptera), various beetles (Coleoptera) and wood wasps (Hymenoptera). The values found for the proportion of cellulose digested in these groups of insects are sometimes extremely high, especially for silverfish, roaches and termites (see Table 1, modified from Martin, 1983 ). Several groups of insects are able to synthesize endo-glucanases (Cx-cellulase; E.C. 3.2.1.4) and cellobiases (E.C. 3.2.1.21 ), as these enzymes are present in extracts of their salivary glands and/or midgut tissues. However, with the possible exception of the silverfish (Lasker and Giese, 1956 ), insects never appear to be capable of forming cellobiohydrolases (Ct-cellulase; E.C. 3.2.1.91 ). This problem has been solved with an overwhelming variety of symbioses between phytophagous insects, notably wood-inhabiting insects and microorganisms. One major type of symbiosis is an endosymbiosis, an association in which the microorganisms live either intraceUularly or extracellularly in their host

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TABLE 1 Cellulose digestion in some insects Species

Cellulose digestibility (%)

Silverfish Lower termites Higher termites Beetles (Anobiidae) (Scarabaeidae) (Cerambycidae) Wood wasps

72-87 74-99 91-97 31-49 68 12-57 22-31

~Approximate cellulose digestibility determined by comparing the cellulose contents of food and faeces. From Martin ( ! 983).

(Koch, 1967 ). This concerns the omnivorous and/or phytophagous insects which have an extracellular location of their symbionts in fermentation chambers in the gut. In these associations, the insects most often harbour the microorganisms in their hindgut, which forms an enlarged compartment for the fermentation of cellulose and other plant polysaccharides. In contrast to the situation in various groups of vertebrate herbivores, foregut symbioses in insects are very uncommon. The absorption of unfermented nutrients at a high rate is probably required to meet the energy and nutrient requirements of the insect host. Microbial colonisation of the midgut is less common, not only because this is the main site for action of the enzymes of the host, but also because in many insects a peritrophic membrane is formed. However, in some insects with a peritrophic membrane, ectopefitrophic colonisation of the midgut occurs, e.g. by actinomycctes in the soil-feedingtermite Procubitermes aburiensis (Bignell et al., 1980a,b) or by an endospore-forming rod in larvae of the midge Xylotopus par (Kaufman et al., 1986 ). Two forms of endosymbiosis have been rccognised: (i) by exploitation of cellulolytic hindgut flagellate protozoa: this strategy is used by woodroaches and lower termites (see Section Insects with gut-flagellates as cellulolytic symbionts); (ii) cooperation with cellulolytic hindgut bacteria: this strategy is used by the cockroach, by scarab beetles and, to an unknown extent, by higher termites (see Section Insects with gut bacteria as their cellulolytic endosymbionts). A third possible mechanism is mentioned by Martin (1983) who refers to the older idea that endosymbiotic microorganisms carried in specialized cells or organs by many insects would be involved in the digestion of cellulose, but no convincing evidence for this idea has ever been produced. The other major type of symbiosis (ectosymbiosis) occurs when the microorganisms live outside the body of their host, although they may be stored temporarily in special organs of ectodermal origin for the purpose of dissem-

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ination (Francke-Grossman, 1967). The microbes associated with these mostly wood-boring and/or bark-feeding insects are yeasts and filamentous fungi. The insects may cultivate the fungi outside their body in herbaceous or woody material as substrate or they may exploit independentlyoccurring fungi. The fungal tissue is ingested for its compensatory C ~-activity and this strategy is called digestion with "acquired fungal enzymes" (Martin, 1987). In the most advanced cases the fungus is used as the sole food, in other cases the fungus is eaten together with wood particles. Confirmed ectosymbiotic associations are found among various groups of beetles, wasps and termites (see Section Ectosymbiosis: acquired fungal enzymes). Ingestion of (hemi) ceUulolytic bacteria which would then proliferate in one region of the gut at the expense of ingested (hemi)cellulose has been observed in various insects, but the significance of this solution to the cell wall problem is not known and will not be discussed here. General information on the microbiology and biochemistry of the interactions between wood-eating and other phytophagous insects, their gut endosymbionts and cellulose-degrading fungi is given by Martin (1983), Anderson et al. (1984), Breznak (1984), Jones (1984), Smith and Douglas (1987) and Orpin and Anderson (1988). In most of the studies the evidence for cellulose digestion comes from the demonstration of an enzymatic capacity to degrade various cellulosic substrates, but in other cases the evidence was derived from the observation that animals survived on a diet of pure cellulose. In still other cases, the evidence was based on the evolution of 14CO2 and incorporation of labelled carbon into insect tissues following the ingestion of U-14C-cellulose, or cellulose digestion was calculated from a comparison of cellulose contents of food and faeces. In the enzyme tests, activity towards microcrystalline cellulose is usually explained as an indication that the entire cellulase complex required to degrade native cellulose is present, while activity toward carboxymethylceUulose (CMC) indicates the presence of endoglucanases (Cx-cellulases), which attack soluble degradation products of cellulose or the amorphous regions in cellulose preparations. Activity towards larchwood xylan indicates the presence of enzymes required to degrade arabino-4-O-methylglucuronoxylans,a major class of hemicelluloses present in both hardwoods and softwoods. On the other hand, the demonstration of these enzymatic activities in gut contents of insects does in itself not necessarily prove that these enzymes really act on the food in the animal. For this, correlation between enzyme levels and cellulose breakdown still has to be demonstrated (see the case for the woodlouse, Kukor and Martin, 1986b). In addition to the problem of tapping the energy in the cell wall polysaccharides, there is the problem of the extremely low nitrogen contents in wood. It has become clear that endosymbiosis with microorganisms in the hindgut is instrumental in helping to overcome this problem by a variety of solutions ( 1 ) Fixation of nitrogen by gut microbes.

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(2) Recycling of uric acid and other N-products of insect metabolism. (3) Absorption of microbial N by the host. (4) N from microbes is taken up with food colonised by microorganisms. ENDOSYMBIOSIS WITH PROTOZOA AND BACTERIA

Insects with gut flagellates as cellulolytic symbionts: woodroaches and lower termites Woodroaches Woodroaches (Cryptocercus sp. ), in contrast to ordinary cockroaches, have hypermastigote and oxyrnonad (but not trichomonad) flagellate endosymbionts for the utilisation of plant cell walls (Cleveland et al., 1934; Yamin, 1979 ). In the woodroach there is a remarkable synchrony in the sexual cycle of the protozoa and the synchronous moulting of the host (Cleveland, 1960 ). Lower termites Termites (order Isoptera) are near relatives of the cockroaches and live in colonies in a highly developed social organisation based on castes. The 2000 species of termites, of which most live in the tropics, can be divided into seven families, six of which comprise the "lower" termites, while the seventh family is that of the Termitidae or "higher" termites. While many termites thrive on sound wood, other species prefer leaves, roots, grasses, dung of herbivores, humus, fungi-containing soil, or partially decayed wood. The many groups of termites have in common a diet that is rich in lignocelluloses and poor in nitrogen. Termites are thought of as "oligonitrotrophic saprovores" (Breznak, 1984), which can dominate the processes of decomposition and nutrient cycling in the tropics where their biomass density sometimes can be very high ( 10-20 g m-2). Excellent reviews on the fascinating biochemical aspects of symbiosis between termites and their intestinal microflora have been published by O'Brien and Slaytor (1982) and Breznak ( 1982, 1984). Food enters the foregut (stomodeum), which is composed of the crop and the muscular gizzard. It then enters the midgut (mesenteron) and finally the hindgut (proctodeum). The latter can be divided into paunch, colon and rectum and it is the paunch region which is of most importance for the fermentation of plant food components. The absorption of nutrients liberated by the enzymes of the animal takes place in the mesenteron, the main site for nutrient absorption in most insects. An enteric valve prevents refluxing of hindgut contents to the midgut. The digesta are finally submitted to microbial attack in the hindgut, from where the fermentation products (mainly acetate ) are absorbed. The mean retention time of digesta in the hindgut is estimated to be around 2 4 - 2 6 h. Urine enters from the Malpighian tubules at the precise junction of midgut

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and hindgut. The N-containing detoxification products in the urine of the insect therefore enter the hindgut, arc subjected to microbial attack and may serve as the insect's source of N. Those termites that practice proctodcal trophallaxis probably absorb from their midgut amino acids and other nutrients from the solubilized hindgut microbes. In higher termites there may be a so-called "mixed segment" formed by an elongation of the midgut, such that the lumen of the digestive tube is bounded on one side by the mesenteron and on the other side by the proctodcum. The chemical environment in the gut is roughly similar in lower and higher termites, although exceptions have been found among soil-feedinghigher termites where extreme high pH values have been found in midgut and hindgut (see Breznak, 1984). In general, a near neutral pH (6-7.5) is found in the regions where microbes are found (hindgut and midgut), aerobic conditions in the midgut (especially when not an important site for microbes) and anaerobic conditions with low redox potentials ( - 2 3 0 to - 2 7 0 mY) in the heavily colonised hindgut, where strict anaerobes abound. The facultative and strict anaerobic bacteria consume oxygen and thus maintain anaerobiosis in the hindgut (Veivers et al., 1982). Feeding antibiotics causes destruction of the hindgut spirochaetes and protozoa, and results in an aerobic hindgut. Exposure to hyperbaric oxygen atmosphere also kills the anaerobic symbionts. Killing the hindgut microflora results in a collapse of the colonisation resistance offered by the anaerobic population. In soil-feedingtermites the reducing conditions are milder, and microaerobic to aerobic conditions prevail, suggesting that chemical solubilisation, hydrolysis of lignocellulosic complexes and breakdown by (micro)aerobic microorganisms may be of nutritional importance (Bignell et al., 1979). Very high digestion coefficients (up to 99%) for cellulose and hemicelluloses have been measured for wood-feedingtermites together with very high assimilation efficiency: 54-93%. Some digestion and/or solubilisation (up to about 10%) of wood lignin occurs in the higher termite Nasutitermes exitiosus (Cookson, 1988), while little or no lignin degradation has been found with the lower termites Mastotermes darwiniensis and Coptotermes acinaciformis (Cookson, 1987b ). Enzymatic destruction of some of the lignin in the termite gut would be one of the explanations for the high cellulose digestibility. Both lower and higher termites form their own cellulases, but lower termites in addition make use of their symbiotic gut flagellates for the digestion of lignocellulosic food.

Cell wall digestion by lower termites. It has long been known - since the experiments of Cleveland in the 1920s - that lower termites will not survive on wood after removal of their hindgut protozoa. Mauldin ¢t al. (1981 ) and Carter and Mauldin (1981) showed that the wood of various species of American trees when offered in no-choice situations was toxic to termite in-

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testinal protozoa and/or to the host. Wood from Magnolia grandiflora, llex opaca and Morus alba eliminated the flagellates from the termite Reticultermes flavipes without killing the host. Defaunated lower termites will not survive for long on standard lignoceUulose diets, while reinfection with the flagellates prolongs or restores survival. This is in contrast to the situation in ruminants which do not need their rumen protozoa for the utilisation of plant material as food (Demeyer, 1981 ). Although chiefly depending on flagellates for the fermentation of plant cell walls, both bacteria and protozoa are found in the hindgut of lower termites. The ratio of protozoa to bacteria is much higher in the termite hindgut than in the rumen, e.g. in the termite Reticulotermesflavipes (4 X 104 protozoa and 3 × 106 bacteria per termite) it is 1000 times higher than in the rumen. Of the 521 known species of lower termites, 205 have been studied in relation to their gut flagellates, together with two of the three species of the related "wood'-roach Cryptocercus. A total of 434 species and subspecies of trichomonad (175 species), oxymonad (68 spp.) and hypermastigote (191 spp.) of flagellates (Protozoa) have been reported from the intestines of the six families of lower termites (Yamin, 1979). The family Kalotermitidae is the only one hosting flagellates of the order Oxymonadida. Yamin (1978) was able to obtain axenic cultures of the cellulolytic flagellate Trichomitopsis termopsidis from the termite Zootermopsis by using autoclaved rumen fluid as a supplement in media containing antibiotics, cellulose and serum. The flagellates did not grow with clarified rumen fluid or with heat-killed cells of several known bacterial species as a supplement. When cellulose was replaced by other carbohydrates no growth occurred either. CeUulolytic activity of the flagellate was demonstrated in vivo and in vitro (Yamin and Trager, 1979). Crude extracts of the flagellate possessed endo-fl1,4-glucanase and ceUobiase activity and activity against crystalline cellulose. These results were confirmed and extended by Odelson and Breznak (1985b). Thus, the proof that hindgut flagellates are not dependent on their endosymbiontic prokaryotes for cellulose digestion was finally delivered, settling a debate of > 40 years since the pioneering work by authors such as L.R. Cleveland, W. Trager and R.E. Hungate (for references see Breznak, 1984). The cellulase complex produced by the lower termite Coptotermes lacteus itself (Hogan et al., 1988a) also consists of an endo-fl-l,4-glucanase (E.C. 3.2.1.4) and a true cellobiase (E.C. 3.2.1.21 ). The cellulases present in the hindgut of Coptotermes lacteus proved to be much more complex, probably because the cellulase activity is from more than one species of flagellate. Very little of the termite's own cellulase enters the hindgut and it was concluded that the enzymes of the host are perhaps endocytosed from the gut. The cellulases in the hindgut therefore are from gut bacteria and/or protozoa. In lower termites acetic acid is the chief fermentation product found in the hindgut (94-99% of the volatile fatty acids (VFA) concentrations ranging

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between 50 and 80 mM). Acetate, hydrogen and carbon dioxide are the chief fermentation products of cellulose metabolism in Trichomitopsis termopsidis and the acetic acid is used by the termite as its chief energy source: 77-100% of the energy requirement of the termites could be met by oxidation of acetate from the hindgut (Yamin, 1980; Odelson and Brcznak, 1985a). Of the cellulose carbon fermented by the hindgut flagellates (nonaxenic) 10-30% could not be recovered as fermentation products. This fermentation pattern is different from the rumen protozoa which under non-methanogenic conditions form at least lactate and butyratc in addition to acetate, hydrogen and carbon dioxide (Hungatc, 1966; Pfins and van Hoven, 1977; van Hoven and Prins, 1977). A later study (Odelson and Breznak, 1985a) showed that the putatively axenic strain of Trichomitopsis termopsidis 6057 of Yamin still contained methanogenic bacteria. When this strain of Trichomitopsis termopsidis subsequently was "cured" of methanogenic activity with bromoethanesulphonate, the nutritional and growth characteristics of the cured derivative (strain 6057C) remained the same as that of the parent strain: both required foetal bovine serum and bicarbonate for good growth and particulate forms of cellulose; the growth of both strains was markedly improved by cells of a certain strain ofBacteroides sp. The nature and the precise role of the nutrients used from the bacteria are still not known. In the same study, Trichomitopsis termopsidis appeared to grow faster and to higher cell densities when co-cultured with the H2-using Methanospirillum hungatii. It would seem that in vivo methanogenic bacteria probably arc narrowly associated with the flagellates, keeping the partial pressure of hydrogen low and allowing a fermentation pattern in which acetate dominates. However, only low concentrations of methanogens are present in the termite hindgut Zootermopsis angusticollis. The methanogens arc mainly associated as epibionts or cndosymbionts with the small cellulolytic flagellate Trichomitopsis termopsidis (discussed above ) and the non-cellulolytic flagellates Tricercomitus termopsidis and Hexamastix termopsidis, but remarkably enough not with the cellulolytic hydrogen-producing flagellate Trichonympha, the organism responsible for most of the cellulolysis in this termite (Lee et al., 1987). Also, a smaller proportion of the cellulose carbon is vented as methane from the gut than from the rumen of the termite, probably as a result of the presence of acetogenic bacteria (Breznak and Switzer, 1986). Conclusive evidence for acctogenesis was given by Breznak and Switzer (1986) but the process was already inferred from the stoichiometry of fermentation products and the hydrogen and methane emission rates of Reticulotermesflavipes (Odelson and Breznak, 1983 ). Bacterial reduction of carbon dioxide to acetate rather than to methane is thus the main terminal electron sink reaction providing enough acetate to support one-third of the entire respiratory requirement (Breznak and Switzer, 1986). At least one of the organisms responsible is the newly discovered species Sporome-

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dusa termitida, which not only uses carbon dioxide but also other substrates (lactate, methanol, methoxylated aromatics) for the production of acetate. Not the affinity for hydrogen, but mixotrophic growth (simultaneous use of hydrogen and organic substrates) was thought to be the explanation for the ability of these acetogens to outcompete the methanogens (Breznak et al., 1988). Thus, despite the importance of the flagellates in plant cell wall breakdown, hindgut bacteria (Bacteroides sp., methanogens, acetogens, spirochaetes, Bacillus, Streptococcus, Staphylococcus sp., etc. ) also play important roles in the fermentation (Breznak, 1984). ( 1 ) Some are responsible for the fixation of nitrogen, the transformations of various N-compounds from the insect's urine to ammonia which serves as a nitrogen source for the hindgut microflora. (2) The bacteria are essential to the nutrition of the flagellates (Yamin, 1981; Odelson and Breznak, 1985a). (3) Bacteria help to create the environmental conditions for flagellate growth: oxygen removal by microaerobic, facultative or even by strict anaerobic bacteria; the facilitation of cellulose hydrolysis by fermentation of the liberated sugars by several fermentative types, in turn aided by hydrogen removal as a result of the activities of methanogens and acetogens. (4) Bacteria could be involved in lignin breakdown (of some significance in higher termites).

Insects with gut bacteria as their cellulolytic endosymbionts: cockroaches, beetles and higher termites Cockroaches Cockroaches are ancient and generally omnivorous insects with a hindgut microbial population as complex as that of the termite hindgut or the rumen. Cockroaches such as the omnivorous and opportunistically feeding American cockroach Periplaneta americana primarily have bacteria as hindgut symbionts. Owing to the presence of chitinous spines in the cockroach (and termite) hindgut there is a very large area for attachment of bacteria and other microorganisms. The wall and spines are covered with a thick layer of microbes, many of which have not been observed elsewhere (Bracke et al., 1979; Cruden et al., 1979; Cruden and Markovetz, 1980, 1981 ). Hindgut contents of the cockroach Eublaberus posterius contain higher concentrations of polyphosphate and poly-fl-hydroxybutyrate than rumen contents; both compounds are energy storage materials of the gut symbionts (Cruden et al., 1983; Cruden and Markovetz, 1984). Upon culture, facultative anaerobes such as Klebsiella, Citrobacter and Enterobacter sp. are found, but the anaerobic count is 100 times higher (Cruden and Markovetz, 1984). While metronidazole killed the hydrogen-producing anaerobic bacteria in

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Periplaneta americana, the health of the mature insect was not affected, but stunting occurred in subaduR animals maintained on the antibiotic from hatching (Bracke et al., 1978 ). Cruden and Markovetz (1979) found that intestinal bacteria from two cockroach species on a laboratory rodent diet had carboxymethyl cellulase (CMC-ase) activity. Counts on CMC-ase were < 1% of the total count on a non-selective medium, while counts from the hindgut contents were much higher than counts from midgut material. Counts on plates incubated anaerobically were 10-fold higher than on plates incubated aerobically. The significance of the fermentation of cellulose and hemicellulose in the alimentary canal of the American cockroach was demonstrated in an experimental study. From artificial diets containing '4C-cellulose and '4C-hemicellulose, ~4C-CO2was produced during the first 300 h after ingestion with evolution starting at 20-40 h after feeding (Bignell, 1977). The labelled CO2 generated over the 300 h represented 73% of the cellulose and 48% of the hemicellulose ingested. The degradation of ~4C-celluloseand ~4C-hemicellulose is sensitive to antibiotics (Bignell, 1977).

Beetles Larvae of Oryctes nasicornis (Coleoptera, Scarabaeidae) ingest food that has already been degraded partially by soil fungi (Bayon, 1981a) and digest two-thirds of the cellulose remaining in the rotted woodpulp with the help of their gut bacteria (Roessler, 1961; Bayon, 198 la). The breakdown of plant cell wall carbohydrates by bacteria starts in the mesenteron, but is of more importance in the proctodeal dilatation by bacteria (see fig. 1 in Bayon, 198 l b). The process can be seen by following the ultrastructural modifications in the primary and secondary plant cell wall as seen with the electron microscope (Bayon, 1981a ). Incubation of gut segments after injection with ~4C-cellulosedemonstrates that both the mesenteron and the proctodeal dilatation are sites of cellulolysis, but the latter site is twice as active in larvae fed with a fermenting mixture of wood shavings and cattle dung (dung-wood) (Bayon and Mathelin, 1980). While the food mass stays for about 8.5 h in the alkaline mesenteron (oH 9-11, Bayon, 1980a,b ), rates of uptake of VFA (chiefly acetic acid) from this organ are low (approx. 2.4 gmol VFA h -~ per animal), while VFA accumulate to concentrations (25 mM) that are believed (Bayon, 1981a) to inhibit the bacterial fermentation of cell walls. In the more anaerobic proctodeum (pH 7.2-8.4) food is retained for about 6 h, but concentrations of bacteria are 100 times higher than in the woodpulp and 50 times higher than in the mesenteron. Rates of uptake of VFA are higher here ( ~ 5 #tool VFA h - ~per animal), while VFA accumulates to a lesser extent (7.2 raM). The proctodeum possesses various forms of spines and protrusions which are believed

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TABLE 2 Proportions of VFA (%) in gut contents of Oryctes nasicornis larvae Larvae fed with Dung-wood

Acetic Propionic Iso-butyric Butyric Iso-valeric

a-cellulose

Midgut

Proctodeal dilatation

Midgut

Proctodeal dilatation

66 32 1 0.5 0.5

81 17 1 0.5 0.5

63 33 1 0.5 0.5

88 10.5 0.5 0.5 0.5

From Bayon and Mathelin (1980).

to aid in the retention of bacteria as well as in the regulation of ingesta flow (Bayon, 1971, 1981b). The micro-environmental conditions in the lumen of midgut and proctodeal dilatation are different. The redox potential in the midgut varies from + 50 to - l0 mV, but the proctodeum contents are characterised by a potential between - 4 0 and - 100 mV (Bayon and Etievant, 1980). In one larva fed on dung-wood, the midgut produced 2.4X 10 -6 mol VFA h - i upon incubation, whereas the proctodeal dilatation produced 5 X 10 - 6 tool h - i (Bayon and Mathelin, 1980). Table 2 shows the percentage distribution of the VFA in gut contents of the Oryctes nasicornis larvae. The VFA pattern in the haemolymph strongly resembled that of the intestinal contents (Bayon, 1980a), but the concentrations were much lower. The chief fermentation product acetic acid is used immediately by the animal (Bayon and Mathelin, 1980). While the VFA are absorbed by the gut epithelium (Bayon, 1980b), methane is vented as a waste product. No methane can be detected in the mesenteron, but active methanogenesis takes place in the proctodeum (Bayon and Etievant, 1980). Animals fed on sawdust produced between 308 and 372 nmol methane h - i, corresponding to a rate of 34-41 nmol h - t g- i liveweight. Animals fed on pure a-cellulose released between 343 and 380 nmol methane h - 1 corresponding to a rate of 38-48 nmol methane h - i g- i liveweight. These rates are in the same range as those observed for the xylophagous cockroach Cryptocercus and for the lower termite Reticulitermes (Breznak, 1975 ).

Higher termites: probably not dependent on symbionts for cellulose digestion Because of the popularity of the symbiosis between (lower) termites and flagellate protozoa, it is not widely appreciated that in the hindgut of the higher

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termites (family Termitidae), which constitute roughly 75% of all termite species, it is mainly bacteria that are present, while protozoa - when they occur - are seen in low density only and are not ceUulolytic (Hogan et al., 1988a,b). As active cellulolytic bacteria were never encountered in higher termites in sufficient numbers to be of importance and because cellulases which act on crystalline cellulose are produced by the insects themselves, it is now thought that higher termites are not dependent on symbionts for the utilisation of cellulose and lignocellulosic feeds (Hogan et al., 1988a,b). In fact, it is well established (O'Bden and Slaytor, 1982 ) that both higher and lower termites make their own cellulases. These enzymes are restricted to the salivary glands, foregut and midgut in the lower termite Coptotermes lacteus and the higher termite Nasutitermes exitiosus (O'Brien et al., 1979; McEwen et al., 1980). Glucose is produced for absorption in the midgut. The cellulase complex produced by the higher termite Nasutitermes walkeri is secreted predominantly in the anterior region of the midgut (Hogan et al., 1988b), where 90% of the components of cellulase activity: exo-fl- 1,4-glucosidase, l/-1,4-glucosidase and endo-fl-l,4-glucanase (but mainly the latter two, see Schulz et al., 1986) are found. Some cellulase activity is secreted by the salivary glands. Traces of the insect's cellulases that are not resorbed are found in the paunch. No bacterial cellulases were found in the paunch of either Nasutitermes walkeri or Nasutitermes exitiosus. Cellulose digestion is therefore accomplished solely by the termite's own cellulases. Among possible functions of the hindgut bacteria there is a possibility that acetate formed in the paunch fermentation from food oligosaccharides or sugars liberated in the midgut act as an energy source for the absorption of minerals and amino acids from the hindgut. Another possible function of the microflora could be the degradation of low-molecular-weightlignin components liberated in the gizzard by fine mechanical action, as is seen, for example, in the higher termite Nasutitermes exitiosus (Cookson, 1988 ). Studies on the digestion of lignin in the past were usually based on the Klason lignin method and the results may have been overestimates as the increase in acid-solubility of the lignin was measured rather than actual lignin degradation (Cookson, 1987a). A better test is the ability to degrade (14C)lignins from wood labelled with (14C)-precursors either in the side chain or in the aromatic ring (Butler and Buckerfield, 1979 ). Up to about 10% of the (14C)-lignin from Acer rubrum wood was recovered as CO2 after giving this wood to Nasutitermes exitiosus and the lignin breakdown was inhibited severely by several antibiotics (especially by metronidazole) or by exposing the insect to an atmosphere of 100% 02 (Cookson, 1987a,b; 1988). Lignin degradation was only marginally higher in starved termites, probably because lignin became progressively more resistant to degradation after passage through the termite's gut. The ability of Nasutitermes exitiosus to degrade lignin diminished when the animals were maintained under standard laboratory conditions. Under such conditions compositional changes in the gut mi-

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croflora are observed, while methane production and N-fixation are lowered. Survival and lignin degradation were optimal at relative humidities of 9096%. ECTOSYMBIOSIS: A C Q U I R E D F U N G A L E N Z Y M E S

Fungus-growing termites Among the higher termites there is one subfamily (Macrotermitiae) whose members cultivate wood-degradingfungi as a food source. These termites primarily eat the fungus combs which are enclosed in the chambers of the termite nest. The species Macrotermes natalensis acquires Cl-cellulase from the nodules of its associated fungus Termitomyces (Martin and Martin, 1978, 1979). Likewise, for Macrotermes muelleri and its symbiotic fungus Termitomyces sp., their carbohydrate hydrolysing enzymes, the purification of the cellulases from fungus and termite as well as the synergism observed between these cellulases on crystalline cellulose are described by Rouland et al. (1988a-e).

Woodwasps (Siricidae and Xiphydriidae) Adult female woodwasps (Sirex cyaneus) lay their eggs in dying and dead standing trees, while they simultaneously inoculate the wood with a mass of fungal oidia which are maintained in special pouches within the egg-laying apparatus. The fungus permeates the surrounding wood and the larvae ingest the softened wood and the fungal hyphae. The larvae derive Cx-cellulase and xylanase from the fungus Amylostereum chailletii, the fungal symbiont that occurs in the wood on which the larvae feed (Kukor and Martin, 1983 ).

Beetles of the families Cerambycidae, Anobiidae, Buprestidae, Lymexylonidae and Scolytidae Wood-boring larvae of the ccrambycid beetles assimilate 12-57% of the ingested cellulose (Martin, 1983 ). At one time it was believed that cndosymbiont yeasts were responsible for the digestion of wood polysaccharides (Buchner, 1928 ), but this hypothesis was convincingly ruled out in the 1930s, while no evidence was ever presented to show the involvement of cellulolytic gut bacteria. However, larvae of the (cerambycid) balsam fir sawyer, Monochamus marmorator, assimilated over 50% of the dry matter and 25% of the cellulose of fungus-infected balsam fir (Abies balsamea). These insects were shown to acquire the capacity to digest cellulose by actively ingesting fungal cellulases, while eating wood infected with Trichoderma harzianum (Kukor and Martin, 1986a). The larvae by themselves do not secrete the full comple-

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ment of cellulases, but probably only Cx-cellulase and xylanase. A more or less similar situation is seen in the aspen borer Saperda calcarata. These larvae, which do not normally include fungal tissue or fungal enzymes in their diet and are not normally able to digest cellulose, can be transformed into cellulose-digesters by adding the fungal enzymes to their food (Kukor and Martin, 1986c). After finding that ingested fungal enzymes were also responsible for the digestion of cellulose in xylophagous larvae of four other species of cerambycid beetles (Bellamira scalaris (Lepturinae), Graphisurus fasciatus (Lamiinae), Orthosoma brunneum ( Prioninae ) and Perandra brunnea ( Parandrinae) ), Kukor et al. ( 1988 ) argued that ingested fungal enzymes are probably responsible for cellulose digestion in many, perhaps even all, xylophagous larvae from the three coleopteran families Anobiidae, Buprestidae and Cerambycidae. Unlike the fungus-growing termites and siricid woodwasps (Martin and Martin, 1978, 1979; Kukor and Martin, 1983), these beetles are not known to maintain specific associations with particular strains of species of fungi. However, in the ambrosia beetles, constituting more than 1500 species ofxylomycetophagous and phloecophagous (bark-feeding) beetles of the family Scolytidae, with main genera Xyleborus and Xylosandrus, Fusarium solani seems to be the dominant filamentous fungal symbiote (Norris, 1979; Beaver, 1989). Aromia moschata, a cerambycid beetle, the larvae of which feed on Safix sp. wood was found to contain anaerobic facultative cellulolytic bacteria, but the meaning of this finding is not clear as the possible role of fungal cellulases was not determined (Andreoni et al., 1987). This short review of the literature on plant cell wall digestion in insects shows that the array of solutions found for the "problem" of cellulose digestion in these invertebrates is fascinating. It should be realised that the physiological grouping of insects according to their symbiotic relationships with various groups of microorganisms and/or their enzymes could be too simplistic. It may very well be that further studies will disclose that even more solutions are possible and indeed present in nature. Examination of the experimental approaches and techniques used by scientists in the study of plant cell wall digestion in insects, suggests that there is little exchange between these workers and those who study rumen function. The high extent of cellulose digestion in some groups of insects (termites) and the possible relationship with a (limited) oxidation of lignin in these animals deserve the attention of researchers trying to improve the utilisation of lignocellulosic feeds in animal husbandry. REFERENCES Anderson, J.M., Rayner, A.D.M. and Walton D.W.H. (Editors), 1984. Invertebrate-microbial interactions. Cambridge University Press, Cambridge, 349 pp.

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Andreoni, V., Baggi, G., Campana, M. and Suss, L., 1987. Gut microbiota ofwood-catingAromia moschata. Ann. Microbiol., 37:81-90. Bayon, C., 1971. La cuticule proctodeale de la larve d'Oryctes nasicornis L. (Coleopteres Scarabeides). Etude au microscope 61ectronique/l balayage. J. Microsc., 11: 353-370. Bayon, C., 1980a. Volatile fatty acids and methane production in relation to anaerobic carbohydrate fermentation in Oryctes nasicornis larvae (Coleoptera, Scarabaeidae). J. Insect. Physiol., 26: 819-828. Bayon, C., 1980b. Transit des aliments et fermentations continues dans le tube d'une larve xyiophage d'Insecte: Oryctes nasicornis (Coleoptera, Scarabeidae). C.R. Acad. Sci. Ser. D, 290:1145-1147. Bayon, C., 1981a. Modifications ultrastructurales des parois v6g6tales dans le tube digestif d'une larve xylophage Oryctes nasicornis (Coleoptera, Scarabaeidae): r61e des bact6ries. Can. J. Zool., 59: 2020-2029. Bayon, C., 1981b. Ultrastructure de l'6pithelium intestinal et flore pari6tale chez la iarve xylophage d'Or.vctes nasicornis L. (Coleoptera: Scarabaeidae). Int. J. Insect. Morphol. Embryol.. 10: 359-371. Bayon, C. and Etievant, P., 1980. Methanic fermentation in the digestive tract ofa xylophagous insect: Oryctes nasicornis L. larva (Coleoptera: Scarabaeidae). Experientia, 36:154-155. Bayon, C. and Mathelin, J., 1980. Carbohydrate fermentation and by-product absorption studied with labelled cellulose in Oryctes nasicornis larvae (Coleoptera: Scarabaeidae). J. Insect. Physiol., 26: 833-840. Beaver, R.A., 1989. Insect-fungus relationships in the bark and ambrosia beetles. In: N. Wilding, N.M. Collins, P.M. Hammond and J.F. Webber (Editors), Insect-Fungus Interactions. Academic Press, London, pp. 121-143. Bigneli, D.E., 1977. An experimental study of cellulose and hemiceilulose degradation in the alimentary canal of the American cockroach. Can. J. Zool., 55: 579-589. Bignell, D.E., Oskarsson, H. and Anderson, J.M., 1979. Association of actinomycete-like bacteria with soil-feeding termites (Termitidae, Termitinae). Appl. Environ. Microbioi., 37: 339-342. Bignell, D.E., Oskarsson, H. and Anderson, J.M., 1980a. Distribution and abundance of bacteria in the gut of a soil-feeding termite Procubitermes aburiensis (Termitidae, Termitinae ). J. Gen. Microbiol., 117" 393-403. Bignell, D.E., Oskarsson, H. and Anderson, J.M., 1980b. Colonisation of the epithelial face of the peritrophic membrane and the ectoperitrophic space by actinomyctes in a soil-feeding termite. J. Invert. Pathol., 36: 426-428. Bracke, J.W., Cruden, D.L. and Markovetz, A.J., 1978. Effect ofmetronidazole on the intestinal microflora of the American cockroach, Periplaneta americana L. Antimicrob. Agents Chemother., 13:115-120. Bracke, J.W., Cruden, D.L. and Markovetz, A.J., 1979. Intestinal microbial flora of the American cockroach, Periplaneta americana L. Appl. Environ. Microbiol., 38: 945-955. Breznak, J.A., 1975. Symbiotic relationships between termites and their intestinal microbiota. In: D.H. Jennings and D.L. Lee (Editors), Symbiosis. Cambridge University Press, Cambridge, pp. 559-580. Breznak, J.A., 1982. Intestinal microbiota of termites and other xylophagous insects. Ann. Rev. Microbiol., 36: 323-343. Breznak, J.A., 1984. Biochemical aspects of symbiosis between termites and their intestinal microbiota, In: J.M. Anderson, A.D.M. Rayner and D.W.H. Walton (Editors), Invertebrate-Microbial Interactions. Cambridge University Press, Cambridge, pp. 174-203. Breznak, J.A. and Switzer, J.M., 1986. Acetate synthesis from H2 plus CO2 by termite gut microbes. Appl. Environ. Microbiol., 52: 623-630.

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Breznak, J.A., Switzer, J.M. and Seitz, H.-J., 1988. Sporomedusa termitida sp. nov., and H2/ CO2-utilizing acetogen isolated from termites. Arch. Microbiol., 150: 282-288. Buchner, P., 1928. Holzern[ihrung und Symbiose. Springer, Berlin, 64 pp. Butler, J.H.A. and Buckerfield, J.C., 1979. Digestion oflignin by termites. Soil Biol. Biochem., 1l: 507-51 I. Carter, F.L. and Mauldin, J.K., 1981. Responses of Reticulitermesflavipes to extracts from antitermitic hardwoods. Mater. Organism., 16:175-188. Cleveland, L.R., 1960. The centrioles of Trichonympha from termites and their functions in reproduction. J. Protozool., 7: 326-341. Cleveland, L.R., Hall, S.R., Sanders, E.P. and Collier, J., 1934. The wood-feeding roach Cryptocercus, its protozoa, and the symbiosis between protozoa and roach. Mere. Am. Acad. Arts Sci., 17:185-342. Cookson, L.J., 1987a. Influence of laboratory maintenance, relative humidity and coprophagy on ( 14C)lignin degradation by Nasutitermes exitiosus. J. Insect Physiol., 33: 683-687. Cookson, L.J., 1987b. ~4C-lignin degradation by three Australian termite species. Isoptera: Mastotermitidae, Rhinotermitidae, Termitidae. Wood Sci. Technol., 21:11-25. Cookson, L.J., 1988. The site and mechanism of ~4C-lignin degradation by Nasutitermes exitiosus. J. Insect Physiol., 34: 409-414. Cruden, D.L. and Markovetz, A.J., 1979. Carboxymethyl cellulose decomposition by intestinal bacteria of cockroaches. Appl. Environ. Microbiol., 38: 369-372. Cruden, D.L. and Markovetz, A.J., 1980. A thick-walled organism isolated from the cockroach gut by using a spent medium technique. Appl. Environ. Microbiol., 39: 261-264. Cruden, D.L. and Markovetz, A.J., 198 I. Relative numbers of selected bacterial forms in different regions of the cockroach hindgut. Arch. Mikrobiol., 129:129-134. Cruden, D.L. and Markovetz, A.J., 1984. Microbial aspects of the cockroach hindgut. Arch. Mikrobiol., 138: 131-139. Cruden, D.L., Gorrell, T.E. and Markovetz, A.J., 1979. Novel microbial and chemical components of a specific black band region in the cockroach hindgut. J. Bacteriol., 140: 687-698. Cruden, D.L., Durbin, W.E. and Markovetz, A.J., 1983. Utilization of PPi as an energy source by a Clostridium sp. Appl. Environ. Microbiol., 46:1403-1408. Demeyer, D.I., 198 I. Rumen microbes and digestion of plant cell walls. Agric. Environ., 6: 295337. Francke-Grossman, H., 1967. Ectosymbiosis in wood-inhabiting insects. In: S.M. Henry (Editor), Symbiosis, Vol. II. Associations of Invertebrates, Birds, Ruminants, and Other Biota. Academic Press, New York/London, pp. 141-205. Hogan, M.0 Schulz, M.W., Slaytor, M., Czolij, R.T. and O'Brien, R.W., 1988a. Components of termite and protozoal cellulases from the lower termite, Coptotermes lacteus Frogatt. Insect. Biochem., 18:45-51. Hogan, M., Veivers, P.C., Slaytor, M. and Czolij, R.T., 1988b. The site of cellulose breakdown in higher termites (Nasutitermes walkeri and Nasutitermes exitiosus). J. Insect. Physiol., 34: 891-899. Hungate, R.E., 1966. The Rumen and its Microbes. Academic Press, New York/London, 533 PP. Jones, C.G., 1984. Microorganisms as mediators of plant resource exploitation by insect herbivores. In: P.W. Price, C.N. Slobodchikoffand W.S. Gaud (Editors), A New Ecology. Wiley, New York, pp. 53-59. Kaufman, M.G., Pankratz, H.S. and Klug, M.J., 1986. Bacteria associated with the ectoperitrophic space in the midgut of the larva of the midge Xylotopus par (Diptera: Chironomidae). Appl. Environ. Microbiol., 51: 657-660. Koch, A., 1967. Insects and their endosymbionts. In: S.M. Henry (Editor), Symbiosis, Vol. IL

PLANT CELL WALL DIGESTION IN INSECTS

1 17

Associations of Invertebrates, Birds, Ruminants, and Other Biota. Academic Press, New York/London, pp. 1-106. Kukor, J.J. and Martin, M.M., 1983. Acquisition of digestive enzymes by siricid woodwasps. Science, 220: 1161-1163. Kukor, J.J. and Martin, M.M., 1986a. Cellulose digestion in Momochamus marmorator Kby. (Coleoptera: Cerambycidae): role of acquired fungal enzymes. J. Chem. Ecol., 12: 10571070. Kukor, J.J. and Martin, M.M., 1986h. The effect of acquired microbial enzymes on assimilation efficiency in the common woodlouse, Tracheoniscus rathkei. Oecologia, 69: 360-366. Kukor, J.J. and Martin, M.M., 1986c. The transformation of Saperda calcarata (Coleoptera: Cerambycidae) into a cellulose digester through the inclusion of fungal enzymes in its diet. Oecologia, 71: 138-141. Kukor, J.J., Cowan, D.P. and Martin, M.M., 1988. The role of ingested fungal enzymes in cellulose digestion in the larvae ofcerambycid beetles. Physiol. Zool., 61: 364-371. Lasker, R. and Giese, A.C., 1956. Cellulose digestion in the silverfish Ctenolepisma lineata. J. Exp. Biol., 33: 542-553. Lee, M.J., Schreurs, P.J., Messer, A.C. and Zinder, S.H., 1987. Association of methanogenic bacteria with flagellated protozoa from a termite hindgut. Curr. Microbiol., 15: 337-341. Martin, M.M., 1983. Cellulose digestion in insects. Comp. Biochem. Physiol., 75A: 313-324. Martin, M.M., 1987. Invertebrate-Microbial Interactions: Ingested Fungal Enzymes in Arthropod Biology. Cornell University Press, Ithaca, New York, 148 pp. Martin, M.M. and Martin, J.S., 1978. Cellulose digestion in the midgut of the fungus-growing termite Macrotermes natalensis: the role of acquired digestive enzymes. Science, 199: ! 4531455. Martin, M.M. and Martin, J.S., 1979. The distribution and origins of the cellulolytic enzymes of the higher termite, Macrotermes natalensis. Physiol. Zool., 52: I 1-2 I. Mauldin, J.K., Carter, F.L. and Rich, N.M., 1981. Protozoan populations of Reticulitermes flavipes (Kollar) exposed to heartwood blocks of 21 American tree species. Mater. Organism., 16: 15-28. McEwen, S.E., Slaytor, M. and O'Brien, R.W., 1980. Cellobiase activity in three species of Australian termite. Insect Biochem., 10: 563-567. Mead, L.J., Khatchatourians, G.G. and Jones, G.A., 1988. Microbial ecology of the gut in laboratory stocks of the migratory grasshopper, Melanoplus sanguinipes (Fab.) (Onhoptera: Acrididae). Appl. Environ. Microbiol., 54:1174-1181. Norris, D.M., 1979. The mutualistic fungi of xyleborini beetles. In: L. Batra (Editor), InsectFungus Symbiosis, Nutrition, Mutualism, and Commensalism. Wiley, New York, pp. 5363. O'Brien, G.W., Veivers, P.C., McEwen, S.E., Slaytor, M. and O'Brien, R.W., 1979. The origin and distribution of cellulase in the termites, Nasutitermes exitiosus and Coptoterme.~ lacteus. Insect. Biochem., 9: 619-625. O'Brien, R.W. and Slaytor, M., 1982. Role of microorganisms in the metabolism of termites. Aust. J. Biol. Sci., 35: 239-262. Odelson, D.A. and Breznak, J.A., 1983. Volatile fatty acid production by the hindgut microbiota of xylophagous termites. Appl. Environ. Microbiol., 45:1602-1613. Odelson, D.A. and Breznak, J.A., 1985a. Nutrition and growth characteristics of Trichomitopsis termopsidis, a cellulolytic protozoan from termites. Appl. Environ. Microbiol., 49:614-62 I. Odelson, D.A. and Breznak, J.A., 1985b. Cellulase and other polymer-hydrolyzing activities of Trichomitopsis termopsidis, a symbiotic protozoan from termites. Appl. Environ. Microbiol., 49: 622-626. Orpin, C.G. and Anderson, J.M., 1988. The animal environment. In: J.M. Lynch and J.E. Hobbie

l 18

R.A.PRINSANDD.A.KREULEN

(Editors), Micro-organisms in Action: Concepts and Applications in Microbial Ecology. Blackwell, Oxford, pp. 163-192. Payne, D.W. and Davidson, L.M., 1974. Cellulose digestion in the locust, Schistocerca gregaria. J. Entomol. Ser. A, 48: 213-215. Prins, R.A. and van Hoven, W., 1977. Carbohydrate fermentation by the rumen ciliate lsotricha prostoma. Protistologica, 13: 549-556. Roessler, M.E., 1961. Ernaehrungsphysiologische Untersuchungen an Scarabaeidenlarven (Oryctes nasicornis L., Melolontha melolontha L. ). J. Insect. Physiol., 6: 62-80. Rouland, C., Renoux, J. and Petek, F., 1988a. Purification and properties of two xylanases from Macrotermes muelleri (Termitidae, Macrotermitinae) and its symbiotic fungus Termitomyces sp. Insect Biochem., 18:709-715. Rouland, C., Civas, A., Renoux, J. and Petek, F., 1988b. Purification and properties of cellulases from the termite Macrotermes Muelleri (Termitidae, Macrotermitinae) and its symbiotic fungus Termitomyces sp. Comp. Biochem. Physiol., 91 B: 449-458. Rouland, C., Civas, A., Renoux, J. and Petek, F., 1988c. Synergistic activity of the enzymes involved in cellulose degradation, purified from Macrotermes muelleri and from its symbiotic fungus Termitomyces sp. Comp. Biochem. Physiol., 91B: 459-465. Rouland, C., Mora, Ph. and Renoux, J., 1988d. Essai d'interpr6tation de la symbiose digestive chez Macrotermes muelleri (Termitidae, Macrotermitinae). Actes Colloq. Insect. Soc., 4: lll-ll8. Rouland, C., Cararas, C., Mora, Ph. and Renoux, J., 1988e. Comparaison entre les osidases du termite Macrotermes muelleri et celles de son champignon symbiotique Termitomyces sp. C.R. Acad. Sci. Paris, 306(III): 115-120. Schulz, M.W., Slaytor, M., Hogan, M. and O'Brien, R.W., 1986. Components of ceilulase from the higher termite, Nasutitermes walkeri. Insect. Biochem., 16: 929-932. Smith, D.C. and Douglas, A.E., 1987. The Biology of Symbiosis. Edward Arnold, London, 302 pp. Ulrich, R.G., Buthala, D.A. and Klug, M.J., 198 I. Microbiota associated with the gastrointestinal tract of the common house cricket Acheta domestica. Appl. Environ. Microbiol., 4 l: 246-254. Van Hoven, W. and Prins, R.A., 1977. Carbohydrate fermentation by the rumen ciliate Dasytricha ruminantium. Protistolngica, 13: 599-606. Veivers, P.C., O'Brien, R.W. and Slaytor, M., 1982. Role of bacteria in maintaining the redox potential in the hindgnt of termites and preventing entry of foreign bacteria. J. Insect Physiol., 28:947-95 I. Yamin, M.A., 1978. Axenic cultivation of the cellulolytic flagellate Trichomitopsis termopsidis (Cleveland) from the termite Zootermopsis. J. Protozool., 25: 535-538. Yamin, M.A., 1979. Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grasse and Hypermastigida Grassi & Foa reported from lower termites (Isoptera families Mastotermitidae, Kalotermitidae, Hodotermitidae, Termopsidae, Rhinotermitidae, and Serritermitidae) and from the wood-feeding roach Cryptocercus (Dictyoptera: Cryptocercidae). Sociobiology, 4: 3-119. Yamin, M.A., 1980. Cellulose metabolism by the termite flagellate Trichomitopsis termopsidis. Appl. Environ. Microbiol., 39: 859-863. Yamin, M.A. and Trager, W., 1979. Cellulolytic activity of an axenically-cultivated termite flagellate, Trichomitopsis termopsidis. J. Gen. Microbiol., l 13:417-420.