Eimeria tenella is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the E. tenella sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study E. tenella mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that E. tenella sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from E. tenella has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.

A substance that sexualizes planarians, an ancestral group of parasitic flatworms, is widely present in planarians and parasitic flatworms. Here, we present a protocol for extracting and purifying the active fraction with sex-inducing activity. We describe steps for homogenization of flatworms, sample concentration, open column chromatography, and reverse-phase high-performance liquid chromatography. We then detail a feeding bioassay to confirm sex-inducing activity. The obtained active fraction may positively affect parasitic flatworm sexual maturation and can be tested by adding it into the culture media. For complete details on the use and execution of this protocol, please refer to Sekii et al. (2023).1

Ubiquinone (UQ) is a lipophilic electron carrier that functions in the respiratory and photosynthetic electron transfer chains of proteobacteria and eukaryotes. Bacterial UQ biosynthesis is well studied in the gammaproteobacterium Escherichia coli, in which most bacterial UQ‐biosynthetic enzymes have been identified. However, these enzymes are not always conserved among UQ‐containing bacteria. In particular, the alphaproteobacterial UQ biosynthesis pathways contain many uncharacterized steps with unknown features. In this work, we identified in the alphaproteobacterium Rhodobacter capsulatus a new decarboxylative hydroxylase and named it UbiN. Remarkably, the UbiN sequence is more similar to a salicylate hydroxylase than the conventional flavin‐containing UQ‐biosynthetic monooxygenases. Under aerobic conditions, R. capsulatus ΔubiN mutant cells accumulate 3‐decaprenylphenol, which is a UQ‐biosynthetic intermediate. In addition, 3‐decaprenyl‐4‐hydroxybenzoic acid, which is the substrate of UQ‐biosynthetic decarboxylase UbiD, also accumulates in ΔubiN cells under aerobic conditions. Considering that the R. capsulatus ΔubiD‐X double mutant strain (UbiX produces a prenylated FMN required for UbiD) grows as a wild‐type strain under aerobic conditions, these results indicate that UbiN catalyzes the aerobic decarboxylative hydroxylation of 3‐decaprenyl‐4‐hydroxybenzoic acid. This is the first example of the involvement of decarboxylative hydroxylation in ubiquinone biosynthesis. This finding suggests that the C1 hydroxylation reaction is, at least in R. capsulatus, the first step among the three hydroxylation steps involved in UQ biosynthesis. Although the C5 hydroxylation reaction is often considered to be the first hydroxylation step in bacterial UQ biosynthesis, it appears that the R. capsulatus pathway is more similar to that found in mammalians.