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\begin{document}

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



\author[1]{Artur Trzebny}%
\author[2]{Anna Slodkowicz-Kowalska}%
\author[3]{Becnel James}%
\author[3]{Sanscrainte Neil}%
\author[1]{Miroslawa Dabert}%
\affil[1]{Adam Mickiewicz University}%
\affil[2]{Poznan University of Medical Sciences Faculty of Medicine I}%
\affil[3]{USDA-ARS Center for Medical Agricultural and Veterinary Entomology}%


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  \date{March 15, 2025}


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\begin{abstract}
Microsporidia are obligate intracellular eukaryotic parasites that
infect nearly all animal groups, including humans. The most common
molecular methods for Microsporidia detection rely on species-targeting
qPCR or end-point PCR using group-specific primers. However, these
methods could be not specific enough or fail in case of mixed
infections. We developed a method for parallel detection of both
microsporidian infection and the host species. We designed new primer
sets: one specific for the classical Microsporidia (targeting
hypervariable V5 region of ssu rDNA), and a second one targeting a
shortened fragment of the COI gene (standard metazoan DNA-barcode); both
markers are well suited for a NGS approach. The analysis of ssu rDNA
dataset representing 607 microsporidian species (120 genera) indicated
that the V5 region enables identification of \textgreater{}98\% species
in the dataset (596/607). To test the method, we used microsporidians
that infect mosquitoes in natural populations. Using mini-COI data, all
field-collected mosquitoes were unambiguously assigned to seven species;
among them almost 60\% of specimens (127/212) were positive for at least
11 different microsporidian species, including a new microsporidian ssu
rDNA sequence (Microsporidium sp. PL01). Phylogenetic analysis of
Microsporidium sp. PL01 ssu rDNA showed that this species belongs to one
of the two main clades in the Terresporidia. In addition, the level of
microsporidian mixed infections was relatively high (9.4\%). The numbers
of sequence reads for the OTUs suggest that the occurrence of
\emph{Nosema} spp. in co-infections could benefit them; however, this
observation should be re-tested using more intensive host sampling. The
proposed method for detection of Microsporidia can be applied to all
types of DNA extracts, including medical and environmental samples.%
\end{abstract}%



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