Michael M. Martin

Tannin Assays in Ecological Studies: Lack of Correlation Between Phenolics, Proanthocyanidins and Protein-Precipitating Constituents in Mature Foliage of Six Oak Species

SummaryThere is no correlation between protein-precipitating capacity and either total phenolic or proanthocyanidin content of extracts of mature foliage from six species of oaks: Quercus alba (white oak), Q. bicolor (swamp white oak), Q. macrocarpa (bur oak), Q. palustris (pin oak), Q. rubra (red oak), and Q. velutina (black oak). It is argued that studies which probe the role of tannins in the selection and utilization of food by herbivores should include a protein-precipitation assay, since such an assay provides a measure of the property of tannins which is presumed to contribute to their utility as defensive compounds. A convenient modification of the bovine serum albumin (BSA) precipitation assay, which measures the amount of protein precipitated when a plant extract is added to a BSA solution, is described. Advantages of this procedure recommend its routine adoption in studies of the role of tannins in plant-herbivore interactions.

BIOCHEMICAL IMPLICATIONS OF INSECT MYCOPHAGY

CONTENTS I. Intmduction . . . . . . . . . 11. Arthropod-fungal associations . . . . . . . (I) Arthropods associated with sporophores . . . . (2) Arthropods in habitats which include fungal mycelium . (3) Insects involved symbiotically with fungi . . . . 111. Nutritive characteristics of fungal tissue . . . . . (I) Caloric value . . . . . . . . (2) Elemental composition . . . . . . . (3) The macronutrients of fungi . . . . . . (4) The micronutrients of fungi . . . . . . (5) summary. . . . . . . . . . IV. Digestive and metabolic requirements imposed on mycophagous species by the special characteristics of fungal tissue. . . . . . . . (I) Fungal polysaccharides . . . . . . . . . . (2) Fungal sterols . . . . . . . . . . . . (3) Urea and ammonia . . . . . . . . . . . (4) Secondary metabolites . . . . . . . . . . (5) Summary. . . . . . . . . . . . . V. Acquired digestive enzymes: a windfall of mycophagy . . . . . (I) Enzymes active against plant cell-wall polysaccharides . . . . (2) Enzymes active against fungal constituents. . . . . . . (3) Enzymes active against phenolic substances . . . . . . (4) Requirements imposed by the exploitation of acquired digestive enzymes . (5) Acquired digestive enzymes: a closing comment . . . . . VI. summary . . . . . . . . . . . . . VII. Acknowledgement . . . . . . . . . . . . VIII. References . . . . . . . . . . . . . i

Surfactants: their role in preventing the precipitation of proteins by tannins in insect guts

SummaryMuch more tannic acid or pin oak tannin is required to precipitate the abundant leaf protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), from Manduca sexta gut fluid adjusted to pH 6.5 than is required to precipitate this protein from an aqueous buffer at the same pH. This finding demonstrates that some characteristic of M. sexta gut fluid, in addition to its basicity, counteracts the potential of tannins to precipitate ingested proteins. Gut fluid of M. sexta has a surface tension of 36–39 dynes/cm, indicating the presence of surfactants. Lysolecithin and linoleoylglycine, surfactants known to be present in insect gut fluids, also interfere with the precipitation of RuBPC by tannins at pH 6.5. It is concluded that detergency is a widespread property of insect gut fluids that counteracts the potential of tannins to precipitate die ary proteins, and it is argued that there is no longer any justification for continuing to refer to tannins as digestibility-reducing-substances. Finding that there has been no formidable barrier to the evolution of mechanisms that counter a generalized antidigestive action by tannins is difficult to reconcile with the idea that reduced digestibility is an evolved anti-herbivore adaptation of apparent plants.

Cellulose Digestion in the Midgut of the Fungus-Growing Termite Macrotermes natalensis: The Role of Acquired Digestive Enzymes

The midguts of adult workers of the higher termite species Macrotermes natalensis contain the entire set of digestive enzymes required for the digestion of native cellulose. The Cx-cellulases and the β-glucosidases are produced, at least in part, by the termites own midgut epithelium and salivary glands. The C1-cellulases, on the other hand, are acquired by the termites when they feed on a fungus that grows in their nests. We propose that the involvement of acquired digestive enzymes could serve as the basis for a general strategy of resource utilization and further suggest that the acquisition of digestive enzymes may be a widespread phenomenon among mycophagous invertebrates.

Failure of tannic acid to inhibit digestion or reduce digestibility of plant protein in gut fluids of insect herbivores : Implications for theories of plant defense.

The rate of hydrolysis of the abundant foliar protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), in enzymatically active gut fluid ofManduca sexta larvae is very rapid and is unaffected by the presence of tannic acid, even when tannic acid is present in the incubation mixture in amounts in excess of the amount of RuBPC. When this protein is dissolved in the denatured gut fluids ofM. sexta larvae orSchistocerca gregaria nymphs, large amounts of tannic acid must be added to bring about the precipitation of significant quantities of protein. The ability of insect gut fluid to prevent the formation of insoluble tannin-protein complexes is due to the presence of surfactants. On the basis of our results and a review of the findings of other investigators, we argue that there is no evidence that tannins reduce the nutritional value of an insects food by inhibiting digestive enzymes or by reducing the digestibility of ingested proteins and, further, that the failure of tannins to interfere with digestion is readily explained on the basis of well-documented characteristics of the digestive systems of herbivorous insects. In challenging the currently popular notion that tannins are digestibility-reducing substances, we do not challenge the general utility of either the apparency theory or resource availability theory of plant defense. In debating the merits of these two analyses of plant-herbivore interactions, however, the demise of tannins as all-purpose, dose-dependent, digestibility-reducing defensive substances must be taken into account.

Cellulose digestion in insects

Abstract 1. 1. Cellulose digestion has been demonstrated in the Thysanura (Lepismatidae), Orthoptera (Cryptocercidae, Blattidae). Isoptera (Mastotermitidae. Kalotermitidae. Hodotermitidae, Rhinotermitdae, Termitidae). Coleoptera (Buprestidae. Anobiidae, Scarabaeidae, Cerambycidae), and Hymenoptera (Siricidae). 2. 2. In all but the scarab beetles, cellulose digestion is brought about by a complex of three types of enzymes (C1-cellulases, Cx-cellulases, and cellobiases), as in fungi. 3. 3. Many insects are able to synthesize their own Cx-cellulases and cellobiases, but few (if any) can synthesize C1-cellulases. 4. 4. Insects compensate for their inability to synthesize C1-cellulases by exploiting the cellulolytic potential of protozoa, bacteria, or fungi. 5. 5. The maintenance of permanent populations of hindgut protozoa, the maintenance of permanent populations of hindgut bacteria, and the ingestion of fungal cellulases are described as three distinct mechanisms by which insects have been shown to use the cellulolytic potential of microorganisms. 6. 6. A process in which ingested cellulolytic bacteria proliferate in one region of the gut at the expense of ingested cellulose, only to be digested and assimilated in a more posterior section, is a fourth possible mechanism by which insects might accomplish the digestion of cellulose with the help of microorganisms.

The effects of quantity and quality of diet nitrogen on the growth, efficiency of food utilization, nitrogen budget, and metabolic rate of fifth-instar Spodoptera eridania larvae (Lepidoptera: Noctuidae)

Relative growth rate and relative nitrogen accumulation rate for fifth-instar Spodoptera eridania larvae vary less than 20% on artificial diets in which protein content varies more than 250%, due to compensatory adjustments in consumption rate and changes in efficiencies of food and nitrogen utilization. The substitution of zein for two-thirds of the casein in a diet containing 26.0% protein results in a 25% decrease in both relative growth and nitrogen accumulation rates, due to reduced values of relative consumption rate, relative nitrogen consumption rate, approximate digestibility, approximate digestibility of nitrogen and efficiency of conversion of digested nitrogen. Although larval growth rate is relatively independent of diet nitrogen, larval composition is highly dependent upon both nitrogen quantity and quality. As diet nitrogen increases, larval nitrogen content increases and fat content decreases. Larvae on diets containing a mixture of casein and zein produce 3.53.9 times as much uric acid and respire at rates significantly higher than larvae on diets containing the same total amount of protein but lacking zein. Thus, there is a measurable metabolic cost associated with processing low quality protein. We conclude, however, that elevated metabolic rates of larvae on diets containing nutritionally unbalanced protein are not the cause of reduced growth rates. On these diets, growth is limited by an amino acid present in limiting quantities, and the elevated metabolic rate is due, at least in part, to increased synthesis of uric acid. Finally, we discuss the possibility that variation in amino acid profiles across host plant species might be a factor favouring specialization in insect herbivores. Key Word Index: Southern armyworm, Spodoptera eriaimia, growth, nutritional indices, food utilization, nitrogen budget, uric acid production, respiration rate, metabolic cost

Acquisition of Digestive Enzymes by Siricid Woodwasps from Their Fungal Symbiont

Larvae of the woodwasp, Sirex cyaneus, contain midgut digestive enzymes that enable them to utilize the major fungal and plant polysaccharides found in their food. At least two classes of enzymes, the Cχ-cellulases and the xylanases, are not produced by the larvae. Instead, larvae acquire these enzymes while ingesting tissue of Amylostereum chailletii, the fungal symbiont that occurs in the wood on which the larvae feed.

The digestion of protein and carbohydrate by the stream detritivore, Tipula abdominalis (Diptera, Tipulidae)

SummaryThe digestive system of larvae of Tipula abdominalis (Diptera, Tipulidae), a stream detritivore, is poorly adapted for the digestion of the major polysaccharides in its diet, but well adapted for the digestion of protein. These crane fly larvae are unable to digest the major cell wall polysaccharides of higher plants, i.e., cellulose, hemicellulose and pectin. The only polysaccharides toward which the midguts of T. abdominalis exhibited any activity were α-amylose and laminarin, indicating that polysaccharide digestion is restricted to α-1,4-and β-1,3-glucans. The most concentrated source of these two classes of carbohydrates in submerged leaf litter would be associated fungal tissue. The midgut of T. abdominalis is strongly alkaline throughout, with a maximum pH near 11.5 in a narrow zone near the midpoint. Proteolytic activity in the midgut is extraordinarily high, and the pH optimum for midgut proteolytic activity is above 11. We conclude that the high alkalinity and high proteolytic activity observed in T. abdominalis larvae are manifestations of a highly efficient protein-digesting system, a system of crucial importance to a nitrogen-limited organism which must derive its nitrogen from a resource in which much of the limited nitrogen present is in a “bound” form in complexes of proteins with lignins and polyphenols.

Antioxidant defenses in caterpillars: role of the ascorbate-recycling system in the midgut lumen.

This study demonstrates that an ascorbate-recycling system in the midgut lumen can act as an effective antioxidant defense in caterpillars that feed on prooxidant-rich foods. In tannin-sensitive larvae of the forest tent caterpillar, Malacosoma disstria (Lasiocampidae), ingested tannic acid is oxidized in the midgut lumen, generating significant quantities of peroxides, including hydrogen peroxide, which readily diffuses across cell membranes and is a powerful cytotoxin. By contrast, in the tannin-tolerant larvae of the white-marked tussock moth, Orgyia leucostigma (Lymantriidae), tannic acid oxidation and the generation of peroxides are suppressed. The superior defense of O. leucostigma against oxidative stress imposed by the oxidation of ingested polyphenols can be explained by the presence of higher concentrations of ascorbate and glutathione in the midgut lumen. In O. leucostigma at least 50% of the ingested ascorbate present in the anterior midgut is still present in the posterior midgut, whereas in M. disstria, only 10% of the ascorbate is present in the posterior half of the midgut. We propose that the maintenance of higher levels of ascorbate in the midgut lumen of O. leucostigma than in M. disstria is explained by the secretion of glutathione into the midgut lumen by O. leucostigma, thereby forming a complete ascorbate-recycling system. The concentration of glutathione in the midgut lumen of O. leucostigma is 3.5-fold higher than in M. disstria and more than double the concentration in the diet. Our results emphasize the importance of a defensive strategy in herbivorous insects based on the maintenance of conditions in the gut lumen that reduce or eliminate the potential prooxidant behavior of ingested phenols.