The presence of a mycangium in European Sinodendron cylindricum (Coleoptera: Lucanidae) and the associated yeast symbionts

Abstract

Part of the exoskeleton of some wood-inhabiting insects is modified to form a mycangium, which is a specialized organ used to convey fungal spores or yeasts to their offspring. Although most stag beetles (Coleoptera: Lucanidae) are known to have female-specific mycangia and associated yeast symbionts, the evolutionary origin of the mycangium in this group remains unresolved. Here, we report the presence of a mycangium and associated yeast symbionts in the European horned stag beetle Sinodendron cylindricum (L.), which belongs to an ancestral clade of the Lucanidae. The mycangium of S. cylindricum is shown to be female-specific and have the same developmental origin as that of other stag beetles. A total of five yeast strains were isolated from adult mycangia and larval gut of S. cylindricum Of these, we suggest that SICYAM1 is an undescribed yeast with taxonomic novelty, and have identified SICYLG3 as the xylose-fermenting yeast Scheffersomyces insectosa using nuclear ribosomal RNA and ITS sequences. The remaining three yeast strains, SICYAM2, SICYLG1, and SICYLG2, were assigned to the genus Sugiyamaella Yeast density in the adult mycangium was lower than that of the more evolutionarily advanced stag beetles, the European Lucanus cervus (L.) and Dorcus parallelipipedus (L.), which were also examined in this study. No living yeasts were isolated from the adult guts. However, a third instar larva of S. cylindricum harbored 10(4)-10(6) living yeasts in each gut region, which suggests that gut yeasts play an important role in these wood-feeding larvae.

Keywords: evolution; gut flora; insect–fungal associations; saproxylic insects; vertical transmission.

Fig. 1.

Adults and a larva of S. cylindricum. (a) Male, (b) female, (c) third instar larva, (d) sampling locations in France and Switzerland. (e, f) Male reproductive organs and the absence of mycangium. Arrow indicates the location of a mycangium in a female. (g, h) Female reproductive organs and the presence of mycangium (arrows). Removed hindgut is indicated by dotted lines. (I, j) Larval gut and cecum-like sacs. ag, accessory gland; cs, cecum-like sac; fg, foregut; hg, hindgut; mcg, mycangium; mg, midgut; mpt, Malpighian tubule, ov, ovary; ovd, (main) oviduct; sp, spermatheca; spg, spermathecal gland; ts, testis. Scale bars: (c, i) 5 mm; (d) 100 km; (e–h, j) 1 mm.

Fig. 2.

Morphological traits of the yeasts isolated from S. cylindricum. (a, b) Colonies on a PDA plate medium where the homogenate of female mycangium was spread. Two types of colonies are found on the plate. (c) SICYAM1 (unclassified yeast from adult mycangium), (d) SICYAM2 (Sugiyamaella sp. from adult mycangium), (e) SICYLG3 (Scheffersomyces sp. from larval gut). Scale bars: (a) 10 mm; (b) 5 mm; (c–e) 50 μm.

Fig. 3.

Phylogenetic placement of the yeasts isolated from S. cylindricum and other lucanid species. The yeast strains isolated in this study are shown in bold. Asterisks after the accession numbers indicate the DNA sequences determined in this study. The two numbers on each branch represent bootstrap values (1,000 replications) by using neighbor-joining (NJ) and ML methods. See also the footnotes in Table 2 for the definition of strain names of yeast symbionts.