Two new species of Bacillidium (Microsporidia) from coelomocytes of Branchiura sowerbyi (Oligochaeta: Naididae) in China

Bacillidium spp. exclusively infect oligochaetes and these microsporidian pathogens are typically characterized by their rod-shaped spores. Seven Bacillidium spp. are presently reported from different organs of oligochaetes. Here, we describe two new Bacillidium species, Bacillidium sinensis n. sp. and Bacillidium branchilis n. sp., from coelomocytes of Branchiura sowerbyi. This is the first report of Bacillidium spp. in oligochaetes from China. Both species of Bacillidium elicit the formations of opaque xenoma-like lesions in coelomocytes of the host. A diplokaryotic nucleus occurs in all life stages of these two new Bacillidium species. Mature spores of B. sinensis are 15.9 +/- 0.6 (14.7-17.1) mu m long (average +/- standard error, range, n = 50) and 2.5 +/- 0.1 (2.3-2.7) mu m wide in fresh preparations. A new type of exospore (sixteen-layered exospore) is discovered from B. sinensis n. sp. which is distinctly different from B. branchilis n. sp., and other Bacillidium spp. (double-layered exospore) reported previously. These two Bacillidium species are morphologically distinguished from each other and all Bacillidium spp. described previously in terms of hosts, infection sites, spore size, spore wall or polar filament thickness. BLASTn searches indicated that these two new microsporidian parasites are surprisingly most similar to Janacekia tainanus (94.76% for B. sinensis and 90. 2% for B. branchilis) isolated from the fat body of midge larva (Kiefferulus tainanus). Phylogenetic analysis demonstrates that the two novel taxons cluster with J. debaisieuxi, J. tainanus, and Bacillidium sp. within the Jirovecia-Bacillidium-Janacekia clade. Other available 18S rRNA gene sequences for microsporidia that infect oligochaetes include J. sinensis, B. vesiculoformis, Neoflabelliforma aurantiae, and Bacillidium sp., but these do not form a single cluster with B. sinensis and B. branchilis, but are instead dispersed through the clade. Based on the ultrastructural features and molecular characteristics, two new species within the genus Bacillidium, B. sinensis n. sp. and B. branchilis n. sp., are designated.