Comparative Genomics of Strictly Vertically Transmitted, Feminizing Microsporidia Endosymbionts of Amphipod Crustaceans

Abstract

Microsporidia are obligate intracellular eukaryotic parasites of vertebrates and invertebrates. Microsporidia are usually pathogenic and undergo horizontal transmission or a mix of horizontal and vertical transmission. However, cases of nonpathogenic microsporidia, strictly vertically transmitted from mother to offspring, have been reported in amphipod crustaceans. Some of them further evolved the ability to feminize their nontransmitting male hosts into transmitting females. However, our understanding of the evolution of feminization in microsporidia is hindered by a lack of genomic resources. We report the sequencing and analysis of three strictly vertically transmitted microsporidia species for which feminization induction has been demonstrated (Nosema granulosis) or is strongly suspected (Dictyocoela muelleri and Dictyocoela roeselum), along with a draft genome assembly of their host Gammarus roeselii. Contrary to horizontally transmitted microsporidia that form environmental spores that can be purified, feminizing microsporidia cannot be easily isolated from their host cells. Therefore, we cosequenced symbiont and host genomic DNA and devised a computational strategy to obtain genome assemblies for the different partners. Genomic comparison with feminizing Wolbachia bacterial endosymbionts of isopod crustaceans indicated independent evolution of feminization in microsporidia and Wolbachia at the molecular genetic level. Feminization thus represents a remarkable evolutionary convergence of eukaryotic and prokaryotic microorganisms. Furthermore, a comparative genomics analysis of microsporidia allowed us to identify several candidate genes for feminization, involving functions such as DNA binding and membrane fusion. The genomic resources we generated contribute to establish Gammarus roeselii and its microsporidia symbionts as a new model to study the evolution of symbiont-mediated feminization.

Keywords: Wolbachia; endosymbiont; feminization; microsporidia; strict vertical transmission.

Fig. 1

Phylogenetic tree of microsporidia (left) along with genome assembly completeness (BUSCO, middle) and features (right). The three strictly vertically transmitted and feminizing species sequenced in this study (Nosema granulosis, Dictyocoela muelleri, and Dictyocoela roeselum) are bolded. The strictly vertically transmitted albeit nonfeminizing species (Hamiltosporidium magnivora) is underlined. All other species undergo horizontal transmission to varying degrees. Bootstrap and SH-aLRT values (%) are indicated at each node of the tree. Scale bar indicates changes per site. *Species used to create the microsporidia BUSCO reference set. ^pPartially assembled genome.

Fig. 2

Repeat content of the Nosema granulosis (A) and Dictyocoela muelleri (B) genome assemblies. Long interspersed nuclear elements (LINE), long terminal repeat retrotransposons (LTR), DNA transposons (DNA), and other repeats are shown. Unmasked regions correspond to nonrepeated regions of the genome.

Fig. 3

Prevalence of orthogroups shared between Nosema granulosis and other microsporidia, but that are not present in any other Nosema genome. Strictly vertically transmitted and feminizing species are shown in blue. Strictly vertically transmitted albeit nonfeminizing genomes are shown in green. Other species are shown in gray. The x axis indicates the percentage of orthogroups shared with N. granulosis out of all orthogroups identified in each species.

Fig. 4

Maximum-likelihood phylogenetic tree of malacostraca based on 12 single-copy nuclear genes extracted from nine sequenced genomes available in NCBI and Gammarus roeselii (this study). Pink, species reported to be infected by feminizing endosymbionts (microsporidia in G. roeselii and Wolbachia in the two isopods). Bootstrap values (%) are indicated at each node. Scale bar indicates changes per site.