The bacterial and fungal community involved in ambrosia beetle fungiculture remains poorly studied compared to the famous fungus-farming ants and termites. Here we studied microbial community dynamics of laboratory nests, adults, and brood during the life cycle of the sugarcane shot hole borer, Xyleborus affinis. We identified a total of 40 fungal and 428 bacterial operational taxonomic units (OTUs), from which only five fungi (a Raffaelea fungus and four ascomycete yeasts) and four bacterial genera (Stenotrophomonas, Enterobacter, Burkholderia, and Ochrobactrum) can be considered the core community playing the most relevant symbiotic role. Both the fungal and bacterial populations varied significantly during the beetle’s life cycle. While the ascomycete yeasts were the main colonizers of the gallery early on, the Raffaelea and other filamentous fungi appeared after day 10, at the time when larval hatching happened. Regarding bacteria, Stenotrophomonas and Enterobacter dominated overall but decreased in foundresses and brood with age. Finally, inferred analyses of the putative metabolic capabilities of the bacterial microbiome revealed that they are involved in (i) degradation of fungal and plant polymers, (ii) fixation of atmospheric nitrogen, and (iii) essential amino acid, cofactor, and vitamin provisioning. Overall, our results suggest that yeasts and bacteria are more strongly involved in supporting the beetle-fungus farming symbiosis than previously thought. IMPORTANCE Ambrosia beetles farm their own food fungi within tunnel systems in wood and are among the three insect lineages performing agriculture (the others are fungus-farming ants and termites). In ambrosia beetles, primary ambrosia fungus cultivars have been regarded essential, whereas other microbes have been more or less ignored. Our KEGG analyses suggest so far unknown roles of yeasts and bacterial symbionts, by preparing the tunnel walls for the primary ambrosia fungi. This preparation includes enzymatic degradation of wood, essential amino acid production, and nitrogen fixation. The latter is especially exciting because if it turns out to be present in vivo in ambrosia beetles, all farming animals (including humans) are dependent on atmospheric nitrogen fertilization of their crops. As previous internal transcribed spacer (ITS) metabarcoding approaches failed on covering the primary ambrosia fungi, our 18S metabarcoding approach can also serve as a template for future studies on the ambrosia beetle-fungus symbiosis.

A major protein component reactive with pooled human atopic sera was isolated from a lyophilized broth extract of Alternaria alternata 34-016. By successive chromatography on Whatman DE-52, Sephadex G-100 and Mono Q HR5/5, a low molecular weight antigen was obtained. Comparison with standard proteins on Sephadex G-100 indicated its molecular weight was 31 kD. Non-reduced samples run on SDS-PAGE showed a band at 29.2 kD which reacted strongly with human IgE. After reduction, it produced a doublet pattern on SDS-PAGE with MW 14.5 and 16.0 kD. The doublet pattern was confirmed by Western blotting with pooled human atopic sera. IEF of the protein showed a major component with a PI of 4.15 and two minor components at 4.25 and 4.40. Immunoblots of the IEF bands showed all three were reactive with human IgE. Ion exchange chromatography of the protein on Mono Q HR5/5 resulted in three resolved components, all of which are immunoreactive. Together with the IEF data, this suggests that there are several conformational or structural isoforms of this protein.

1989) Comparative studies of allergens from mycelia and culture media of four new strains of Alternaria tenuis , Grana, 28:1, 53-61,

Extracts of the mycelia were prepared and their biochemical and immunological properties were examined. Two isolates, IhlI 49632 and CBS 180.50 did not give a significant yield of mycelial mat, and the mycelial extract of ATCC 38810 was only weakly immunoreactive. In IgE immunoblots of sodium dodecylsulphate polyacrylamide electrophoresis gels, the remaining three extracts showed strongly reactive bands at 14, 17,25,35,41,47 and 97 kDa hlW and IhlI 96220 had two additional bands at 53 and 71 kDa. These extracts had 8 to 10 bands in isoelectric focusing with pl values between 3.75 to 5.2, the two predominant bands being at 4.5 and 5.2. In crossed -radioimmunoelectrophoresis using human atopic sera, IMI 96220showed one dominant and three minor allergens; IMI 49630 and CBS 121.47 had one dominant and one minor allergen. In direct radioallergosorbent tests with human atopie sera and passive cutaneous anaphylaxis tests with mouse IgE, Ih4I 49630 and 96220 were more potent than CBS 121.47. These results indicate that mycelial extracts of IMI 49630 and 96220 contain a potent but complex mixture of the major allergens of C. herbanmi and are preferred sources for a reference standard for this organism. Clmiosporiiirn is reported to be the most common airborne fungus in temperate zones (Solomon 1978) and in many countries the species C. herbarirni is a major fungal cause of inhalent allergy and allergic asthma in man (Nielsson & Aas 1976, Tarlo et al. 1988. Aukrust and Borch (1979) identified two major allergenic components of this organism by extensive CRIE studies, but significant biochemical and immunological differences have been reported among different strains (Sward-Nordmo et al. 1984). In the present investigation, six isolates of C. herboriitn were studied with the aim of identifying suitable isolate for a reference standard of this organism. The isolates, IMI 49630, IMI 49632, IMI 96220, CBS 121.47, CBS 180.50 and ATCC 38810 were examined for their antigenic and allergenic components by the techniques of crossed-radioimmunoelectrophoresis (CRIE) and SDS-PAGE immunoblotting. MATERIALS AND METHODS Preparation of mycelial extractsCultures of the isolates IhlI 49630. IMI 49632, IhlI 96220, CBS 121.47, CBS 180.50 and ATCC 38810 were grown on synthetic revised tobacco medium at 22°C for 4 weeks as reported earlier (Vijay et al. 1985). Dried mold pellicles that had been homogenized and defatted with acetone were extracted with 1:20 wlv Tris-buffer, pH 7.8, containing 0.15 M NaCl for 48h at 4°C. The suspension was centrifuged at 1000 g a t 4°C for 30 min, then at 30,000 g at 4°C for 20 min. The resulting supernatant was concentrated and diafiltered with water using an Amicon YM-5 membrane. The retentate was filtered through a 0.45 pm filter and lyophilized. A reference extract (RE) of C. herbanrrn was prepared from commercial mycelial material (Pharmacia, Uppsala, Sweden) in a similar way. Biochemical assaysProtein contents of the extracts were compared by a commercial dye-binding assay (Bio-Rad Laboratories), using bo...

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