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C. elegansis a free-living nematode that must adapt to a wide range of environments including both aerobic and anaerobic conditions. To survive in low oxygen,C. eleganscan use an unusual form of anaerobic respiration that relies on rhodoquinone (RQ) as an alternative electron carrier. Parasitic nematodes like hookworm and whipworm also require rhodoquinone-dependent metabolism (RQDM) to survive in the highly anaerobic conditions in the human gut. Understanding how RQDM is regulated inC. elegansmay thus identify new ways to combat these closely-related major human pathogens. We previously established a simple movement-based assay for RQDM inC. elegans. In this study, we tested a panel of wild-type isolates ofC. elegansin our RQDM assay and find substantial variation in their ability to use RQDM. We carried out a genome-wide association study (GWAS) to identify loci that affect RQDM — this identified a single major QTL on the right arm of Chromosome III. We used RNAi to test almost all genes within the QTL region for involvement in RQDM and found one gene,wah-1, that strongly modulates RQDM-dependent recovery inC. elegans. WAH-1 is a mitochondrial flavoprotein that affects the electron transport chain, consistent with a role in RQDM. We show thatwah-1expression varies between isolates due to major changes inwah-1transcript structures and this correlates tightly with variation in RQDM. Finally, we show that there is similar complexity towah-1transcription in parasitic nematodes and thatwah-1transcript structures change as parasites shift from aerobic to anaerobic, RQ- requiring metabolism. We thus conclude that reducedwah-1expression correlates with increased ability to survive in conditions where RQDM is essential.

Soil transmitted helminths (STHs) are major human pathogens that infect over a billion people. Resistance to current anthelmintics is rising and new drugs are needed. Here we combine multiple approaches to find druggable targets that are essential for RQ-dependent metabolism, an unusual form of anaerobic metabolism which STHs need to survive in their host. We identified 25 genes predicted to act in RQ-dependent metabolism from sensing hypoxia to RQ synthesis — this includes components of the kynurenine pathway we previously showed to be essential for RQ synthesis (Del Borrello et al., 2019). We found 9 genes to be required — since all have host orthologues, we used comparative genomics and structural modeling to identify those with helminth-specific active sites and found 4 such targets. These 4 high confidence targets open up the possibility of in silico screens to identify STH-specific inhibitors of these enzymes as new anthelmintics.

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In aerobic environments they use ubiquinone (UQ) but in anaerobic environments inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. The switch to RQ synthesis is driven by substrate selection by the polyprenyltransferase COQ-2 but the mechanisms underlying this substrate choice are unknown. We found that helminths make twocoq-2isoforms,coq-2aandcoq-2e, by alternative splicing. COQ-2a is homologous to COQ2 from other eukaryotes but the COQ-2e-specific exon is only found in species that make RQ and its inclusion changes the enzyme core. We show COQ-2e is required for RQ synthesis and for survival in cyanide inC. elegans. Crucially, we see a switch from COQ-2a to COQ-2e as parasites transition into anaerobic environments. We conclude that under anaerobic conditions helminths switch from UQ to RQ synthesis via alternative splicing ofcoq-2.

Parasitic helminths infect over a billion humans. To survive in the low oxygen environment of their hosts, these parasites use unusual anaerobic metabolism. This requires Rhodoquinone (RQ), an electron carrier that is made by very few animal species — crucially it is not present in any parasitic hosts. RQ synthesis is thus an ideal target for anthelmintics but little is known about how RQ is made and no drugs are known to block RQ synthesis.C.elegansmakes RQ and can use RQ-dependent metabolic pathways — here, we useC.elegansgenetics to identify the pathway for RQ synthesis and show thatC.elegansrequires RQ for survival in hypoxic conditions. Finally, we establish a robust assay for drugs that block RQ-dependent metabolism. This study identifies for the first time how RQ is made in any animal and establishes a novel assay that can drive the development of a new class of anthelmintic drugs.