Confirmation and identification of parasitic stages of obligate endobionts (Harpellales) in blackflies (Simuliidae) by means of rRNA sequence data

Over the last 35 y, the life cycle of endosymbiotic gut fungi (Harpellales) has been expanded to include cyst stages associated with the developing ovaries. Ovarian cysts (chlamydospore stages) have been identified after germination and production of asexual trichospores in vitro, but germination is not always successful, and spores exhibit morphological variation. Sequence data (for partial 18S and 28S rRNA genes) were generated for these putative stages of Harpellales using ungerminated cysts from adult blackflies and germinated cysts associated with field-collected blackfly egg masses. Cladistic analyses of the 18S and 28S rRNA sequences confirmed that ovarian cysts are stages in the life cycle of Harpellales. Ungerminated cysts, from a blackfly collected from New York state were identified as Pennella simulii and two samples from Newfoundland Prosimulium mixtum adults were identified as Harpella melusinae. Cysts with bipolar germ tubes, associated with field-collected Simulium egg masses from Newfoundland, were also identified as H. melusinae. Two other samples of cysts could not be matched with available sequences of gut fungi from larval hosts. The potential use of this approach to identify pathogenic stages associated with adult ovaries or field-collected egg masses among other host groups is highlighted and promoted as a tool to test the hypothesis that ovarian cysts are a dispersal stage common to all genera of Harpellales.