A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae)

Authors: TRZEBNY, ARTUR - Adam Mickiewicz University; SLODKOWICZ-KOWALSKA, ANNA - University Of Warsaw; BECNEL, JAMES - Retired ARS Employee; Sanscrainte, Neil; DABERT, MIROSLAWA - Adam Mickiewicz University

Submitted to: Molecular Ecology Resources
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2020
Publication Date: 6/9/2020
Citation: Trzebny, A., Slodkowicz-Kowalska, A., Becnel, J., Sanscrainte, N.D., Dabert, M. 2020. A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae). Molecular Ecology Resources. 00:1-19. https://doi.org/10.1111/1755-0998.13205.
DOI: https://doi.org/10.1111/1755-0998.13205

Interpretive Summary: Microsporidia are parasites that infect the cells of many different hosts, from insects to fish to mammals. Scientists at the Center for Medical, Agricultural and Veterinary Entomology have worked with university collaborators in Poland to develop new molecular biology methods that can detect microsporidia as well as identify the mosquito host by examining short DNA sequences. This method was tested using several known mosquito species and microsporidia that infect mosquitoes. Field collected mosquitoes were also screened and microsporidia were found in almost 60% of the samples. These methods can be used to provide enhanced surveillance for mosquitoes that transmit pathogens to man and animals.

Technical Abstract: Microsporidia are a large group of obligate intracellular parasites that infect a wide spectrum of hosts. Since microsporidia are studied mostly as zoonotic and/or waterborne agents of human disease or veterinary parasites, the real species diversity in this group probably is largely unknown. Here we present a new method for parallel detection of microsporidia and their hosts based on a metabarcoding approach. We developed a microsporidia-specific primer set which amplifies the V5 region of the ssu rRNA gene. For host species identification, we proposed a short fragment of the standard COI barcode; both markers are well suited for a next generation sequencing approach. To test this method, we used as a model several microsporidia infectious to mosquitoes. The sensitivity of our metabarcoding method is generally 100 spores/mL, and as low as 10 spores/mL for Encephalitozoon hellem. We found that 10,000 reads per sample are required to identify all microsporidian diversity in the tested host. The non-target sequences never exceeded 12% of quality-filtered sequence data obtained in our sequencing experiments. Using COI sequence data, all mosquitoes were unambiguously assigned to the proper species. Among them, almost 60% of individuals were positive for microsporidian DNA. Overall, in field collected mosquitoes we found 11 microsporidian species belonging to five genera. In addition, our metabarcoding approach allowed us to detect a relatively high level (9.81%) of co-occurrence of DNAs representing different microsporidian species in the same host individual. Moreover, we noticed that some Nosema spp. were found only in co-infections and a correlation was observed in increasing numbers of sequence reads representing co-infecting species.