Microsporidian diversity is vast. There is a renewed drive to understand how microsporidian pathological, genomic, and ecological traits relate to their phylogeny. We comprehensively sample and phylogenetically analyse 125 microsporidian genera for which sequence data are available. Comparing these results with existing phylogenomic analyses, we suggest an updated taxonomic framework to replace the inconsistent clade numbering system, using informal taxonomic names: Glugeida (previously clades 5/3), Nosematida (4a), Enterocytozoonida (4b), Amblyosporida (3/5), Neopereziida (1), and Ovavesiculida (2). Cellular, parasitological, and ecological traits for 281 well-defined species are compared with identify clade-specific patterns across long-branch Microsporidia. We suggest that future taxonomic circumscriptions of Microsporidia should involve additional markers (SSU/ITS/LSU), and that a comprehensive suite of phenotypic and ecological traits help to predict broad microsporidian functional and lineage diversity.

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (10⁴ plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (10⁶ copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.

Microsporidia are intracellular parasites that infect a variety of animals, including humans. As highly specialized parasites, they are characterized by a number of unusual adaptations, many of which are manifested as extreme reduction at the molecular, biochemical, and cellular levels. One interesting aspect of reduction is the mitochondrion. Microsporidia were long considered to be amitochondriate, but recently a tiny mitochondrion-derived organelle called the mitosome was detected. The molecular function of this organelle remains poorly understood. The mitosome has no genome, so it must import all its proteins from the cytosol. In other fungi, the mitochondrial protein import machinery consists of a network series of heterooligomeric translocases and peptidases, but in microsporidia, only a few subunits of some of these complexes have been identified to date. Here, we look at targeting sequences of the microsporidian mitosomal import system and show that mitosomes do in some cases still use N-terminal and internal targeting sequences that are recognizable by import systems of mitochondria in yeast. Furthermore, we have examined the function of the inner membrane peptidase processing enzyme and demonstrate that mitosomal substrates of this enzyme are processed to mature proteins in one species with a simplified processing complex, Antonospora locustae . However, in Encephalitozoon cuniculi , the processing complex is lost altogether, and the preprotein substrate functions with the targeting leader still attached. This report provides direct evidence for presequencing processing in mitosomes and also shows how a complex molecular system has continued to degenerate throughout the evolution of microsporidia.

Summary: Glycolysis and oxidative phosphorylation are the fundamental pathways of ATP generation in eukaryotes. Yet in microsporidia, endoparasitic fungi living at the limits of cellular streamlining, oxidative phosphorylation has been lost: energy is obtained directly from the host or, during the dispersive spore stage, via glycolysis. It was therefore surprising when the first sequenced genome from the Enterocytozoonidae – a major family of human and animal‐infecting microsporidians – appeared to have lost genes for glycolysis. Here, we sequence and analyse genomes from additional members of this family, shedding new light on their unusual biology. Our survey includes the genome of Enterocytozoon hepatopenaei , a major aquacultural parasite currently causing substantial economic losses in shrimp farming, and Enterospora canceri , a pathogen that lives exclusively inside epithelial cell nuclei of its crab host. Our analysis of gene content across the clade suggests that Ent. canceri 's adaptation to intranuclear life is underpinned by the expansion of transporter families. We demonstrate that this entire lineage of pathogens has lost glycolysis and, uniquely amongst eukaryotes, lacks any obvious intrinsic means of generating energy. Our study provides an important resource for the investigation of host‐pathogen interactions and reductive evolution in one of the most medically and economically important microsporidian lineages.