Joan Stadler 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.

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.

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.

EFFECTS OF SURFACTANTS, pH, AND CERTAIN CATIONS ON PRECIPITATION OF PROTEINS BY TANNINS

Tannic acid and pin oak tannins precipitate large amounts of the abundant leaf protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), over a wide pH range (6.15–9.30) in the presence of sodium, potassium, magnesium, and calcium ions at concentrations comparable to those reported in the gut fluids of lepidopteran herbivores. The presence of lysolecithin, a surfactant known to be present in the gut fluids of some insects, significantly reduces the amount of RuBPC precipitated under these conditions. We conclude that high detergency is far more effective than high alkalinity in countering the potential protein-precipitating properties of tannins. We further conclude that tannins do not deserve the status they were once accorded as general, all-purpose, dose-dependent, antidigestive defensive chemicals. We also describe the application of the Schaffner-Weissman protein assay for studying the protein-precipitating capacity of plant extracts. This method is far superior to the one we have used in our earlier studies.

The Distribution and Origins of the Cellulolytic Enzymes of the Higher Termite, Macrotermes natalensis

All of the enzymes required for the digestion of native cellulose, as well as of xylan and pectin, are present in the gut fluids of adult workers of the fungus-growing termite, Macrotermes natalensis. Activity levels are highest in the midgut, suggesting that this portion of the gut, rather than the paunch, is the major site of polysaccharide digestion in this higher termite. The C1-enzymes, active against crystalline cellulose, are acquired by the termites when they feed upon the fungus nodules which grow on their fungus combs. The Cx-enzymes, active against noncrystalline cellulose and soluble derivatives and degradation products of cellulose, are derived in part from ingested fungal material, and in part they are produced internally by the termite, being secreted both by the midgut epithelium and the salivary glands. The nutritional dependence of the termites on their fungus gardens is explained in terms of their reliance upon the fungus nodules as a source of the critical C1-enzymes, which must be acquired before they are able to effect the digestion of cellulosic materials. It is proposed that a strategy of resource utilization based upon the acquisition of digestive enzymes which expand the range of natural substrates suitable for exploitation as nutrient sources may be a general one.

STUDIES ON ASSIMILATION, MOBILIZATION, AND TRANSPORT OF LIPIDS BY THE FAT BODY AND HAEMOLYMPH OF PYRRHOCORIS APTERUS

The incorporation and release of lipid in vitro by the fat body of adult female Pyrrhocoris apterus, and the lipid content and composition of the haemolymph were examined in relation to the cyclic growth and diminution of the fat body and the probable contribution of lipid from the fat body to the developing oocytes. The fat body released lipid at a constant rate during most of the reproductive cycle and in non-reproducing (diapausing or ovariectomized) females. On the other hand, the rate at which the fat body incorporated lipid in vitro was markedly reduced during the period of major oocyte growth (days 3-5 following the larval-adult ecdysis), while it remained high during the corres- ponding period after ovariectomy and was low in diapause. The change in rate of lipid incorporation appears to account for the change in the fat body from net accumulation of lipid (days l-3) to net loss of lipid (during rapid oiicyte growth on days 3-5). Several characteristics of lipid incorporation and mobilization in vitro by the Pyrrhocoris fat body are different from those of the few other insect species studied and from mammalian adipose tissues. In Pyrrhocoris, haemolymph or bovine serum albumin is required for the maximum rate of incorporation, which is ten times higher than that reported for other insects. The fat body of Pyrrhocoris is not dependent on an external source of glyceridide-glycerol for the esterification of fatty acids. Lipid is released at a much slower rate than found in other insect species. The primary class of lipid released is triglyceride, the form in which lipid is stored inthe fat body and which predominates among lipid classes in the haemolymph. The importance of the method used to label the fat body is discussed. The haemolymph of adult female Pyrrhocoris is normally filled with lipid droplets and lipid-filled cells which closely resemble fat body cells. It is suggested that the release of lipid-filled cells from the fat body constitutes one phase of the mobilization of fat body lipid.

The nutritional ecology of larvae of Alsophila pometaria and Anisota senatoria feeding on early- and late-season oak foliage

The larvae of Alsophila pometaria (Harr.), feeding on the young foliage of oak, has a higher relative growth rate (RGR) and relative nitrogen accumulation rate (RNAR) than the larvae of Anisota senatoria (J. E. Smith), feeding on the mature foliage of oak. Although the young oak foliage is more efficiently digested by A. pometaria (higher ADs), it is not more efficiently assimilated and used for growth (no difference in ECIs). Thus, the higher growth rate of A. pometaria is due entirely to a higher consumption rate (RCR and RNCR). Young foliage is significantly higher in nitrogen and water than mature foliage, but phenol and tannin levels are comparable in young and old foliage. A. pometaria consumes the foliage of different oak species at the same rate, independent of nitrogen content, while A. senatoria increases its consumption rate in response to decreased nitrogen levels. As a result, the growth rate of A. pometaria is directly related to leaf nitrogen content, while the growth rate of A. senatoria is independent of leaf nitrogen. The two species of insects have digestive systems that are very similar biochemically, and that are well‐designed for effective protein digestion. Tannins and phenols do not influence the nutritional indices of either species. We suggest that the major benefit of spring feeding is the availability of succulent, high‐nitrogen foliage, and not the avoidance of hightannin foliage. The spring feeder appears to have a feeding strategy that favors rapid growth at the expense of efficiency, while the late summer feeder has a strategy that favors efficiency over rate.

Lipid composition of fat body and its contribution to the maturing oöcytes in Pyrrhocoris apterus

Abstract The fat body of adult, female Pyrrhocoris apterus undergoes cyclic growth and diminution correlated with cycles of oocyte maturation. At all stages lipid accounts for 80 per cent of the dry weight of the fat body and the content of lipid, protein, and glycogen change in proportion to the changes in dry weight. The fatty acid composition of the lipid in fat body, ovaries, the whole insect, and the diet was examined by gas-liquid chromatography. Pyrrhocoris is similar to other insects in having triglyceride as the predominant class of lipid in the fat body but is distinctive in having an unusually high (more than 50 per cent) proportion of linoleate among its fatty acids, presumably owing to its diet. Reasons are presented for considering that little, if any, fatty acid is synthesized by the fat body and the ovaries during the reproductive cycle. During the first 5 days after the larval-adult ecdysis the Pyrrhocoris fat body first accumulates lipid and then releases it for utilization by the ovary. This temporary storage by the fat body compensates for the apparent inability of the insect to ingest a sufficient amount of lipid during vitellogenesis. The ovary normally utilizes dietary lipid as well during vitellogenesis; when the dietary supply was experimentally interrupted the amount of lipid released by the fat body increased. Storage of lipid in the fat body represents an important physiological adaptation since it frees the insect from dependence on a constant food supply during vitellogenesis.