The role of the symbiotic fungus in the digestive metabolism of two species of fungus-growing ants.

Leaf-cutting ants live in an obligatory symbiosis with a fungus which they grow on fresh leaves harvested by workers. This study attempts to clarify the respective role of ants and fungus in the degradation of plant material, in order to highlight the evolutionary basis of this mutualistic association. The symbiotic system of two ant species, Acromyrmex subterraneus subterraneus and Acromyrmex crassispinus, was investigated. To identify the digestive carbohydrases, a total of 19 specific and synthetic plant material substrates were tested on workers from different castes (major and minor), larvae and fungus. Extracts of A. subterraneus and A. crassispinus workers showed high enzymatic activity particularly on starch, maltose, sucrose and alpha-1,4 glucoside. Larvae degraded starch, sucrose, maltose but also laminarin, and all the detected activities were higher than those found for workers. The symbiotic fungus of A. subterraneus was mostly active on laminarin, xylan and cellulose, while the symbiotic fungus of A. crassispinus was mostly active on laminarin, starch, maltose and sucrose. The enzymatic activities of ants and fungus belonging to the same symbiotic system tended not to overlap, suggesting that the association is highly evolved and of an ancient origin.

Mannan-degrading enzymes purified from the crop of the brown garden snail Helix aspersa Müller (Gastropoda Pulmonata).

Two mannan-degrading enzymes were purified from the crop of the terrestrial snail Helix aspersa Müller. The crude extracts were taken from dormant (for 4 months) snails. The enzymes were a betaD-mannanase of 37.4 +/- 0.3 kDa (EC 3.2.1.78) and a betaD-mannosidase of 77.8 +/- 1.9 kDa (EC 3.2.1.25). Both enzymes degraded insoluble mannan, releasing either mannose only (beta-mannosidase) or oligosaccharides, possibly mannotriose and mannopentaose (beta-mannanase). The beta-mannanase had a typical endo-activity pattern, while the beta-mannosidase was an exoenzyme. The incubation of both enzymes with mannan increased the catalysis by 83%. The best synergy was found with 75% mannosidase combined with 25% mannanase. The beta-mannanase also hydrolysed beta-linked heteromannans and its affinity for different galactomannans was studied. The Km values, varying from 2.89 +/- 0.47 mg x ml(-1) to 0.26 +/- 0.01 mg x ml(-1), revealed the inhibitory effect of the alphaD-galactosyl residues released. The beta-mannosidase was acidic (optimum pH = 3.3) and heat-sensitive (50% residual activity at 42 degrees C after 5 min of pre-incubation), while the beta-mannanase remained stable until 48.5 degrees C (50% residual activity) and over a pH range of 4.3-7.5. The properties of these mannanolytic enzymes are discussed in this paper compared with those purified in other gastropods and in a bacterium, Enterococcus casseliflavus, a quite similar strain previously isolated from this snail intestine. The occurrence of thermostable enzymes in H. aspersa digestive tract could be a zootechnic parameter of great importance for snail farming. J. Exp. Zool. 290:125-135, 2001.

Metabolic evolution in alpha-amylases from Drosophila virilis and D. repleta, two species with different ecological niches.

alpha-Amylases from Drosophila virilis and D. repleta were partially purified by ion exchange chromatography. The two amylases share common characteristics for pH and cations effects, although with slight differences. D. virilis has optimal activity at pH 6.6 and D. repleta at pH 7.2. Calcium, sodium, and potassium cations activate amylolytic activity in both species but Ba2+ has an activation effect in D. repleta only. In contrast, there are major differences in thermal offbility and kinetics among amylases of the two species. D. virilis amylase is much more stable at high temperature and the optimal temperatures are very different between the two species, respectively, 45 degrees C and 30 degrees C for D. virilis and D. repleta. alpha-Amylase activity using different substrates is greater on starch than on glycogen in both species and still higher on amylose for D. virilis, the nonfungus feeder species. alpha-Amylase of D. repleta, the mycophagous species, has a better affinity to amylopectin and glycogen. Such differences in substrate specificity suggest adaptation to different resources in these species living in different habitats. Metabolic evolution seems to have occurred through a "tradeoff" between kinetic effectiveness and the nature of substrate, with a higher Vmax on amylose for D. virilis and a lower K(m) on glycogen for D. repleta.

Purification and properties of the xylanases from the termite Macrotermes bellicosus and its symbiotic fungus Termitomyces sp.

Four xylanases were purified, two from the termite Macrotermes bellicosus workers (XIT and X2T) and two from its symbiotic fungus Termitomyces sp. (X1Mc and X2Mc). The analysis of the step required for the purification of X1T and X1Mc and the comparison of their different properties suggested that xylanases X1T and X1Mc were the same enzyme, X1. The determination of the reducing sugars by TLC revealed that X1 was an endoxylanase (EC 3.2.1.8) and X2T and X2Mc were endoxylanases (EC 3.2.1.37). The apparent molecular weights of the three xylanases, determined by SDS-polyacrylamide gel electrophoresis, were 36 kDa for X1, 56 kDa for X2T and 22.5 kDa for X2Mc. The optimal pH of the three xylanases was approximately 5.5, and Km values determined with birchwood xylan as substrate were 0.2% for X1, 0.1% for X2T and 0.3% for X2Mc, showing a high affinity for this substrate. The three enzymes differed also by their thermal stability.