Pharyngeal Pumping Assay v1

The pharynx is a is a neuromuscular pump found at the anterior end of the alimentary tract, consisting of 20 muscles and 20 neurons. A proper feeding rate in worms is coordinated by the precise timing of pharyngeal movements, with one complete cycle of synchronous contraction and relaxation of the corpus and terminal bulb termed a “pump”. A simple way to measure C. elegans feeding is to count how many times worms pump in a minute (pumps per minute). Movement of the grinder (in the terminal bulb) can easily be observed using a stereomicroscope, and because cycles of contraction/relaxation are synchronised along the pharynx, pumps per minute can be measured simply by counting grinder movements.

Worms find PEZO-1’s function easy to swallow

JGP study finds that the C. elegans orthologue of the PIEZO family is a mechanosensitive ion channel that regulates pharyngeal pumping and food sensation.

Neuropeptide signalling shapes feeding and reproductive behaviours in male C. elegans

Sexual dimorphism occurs where different sexes of the same species display differences in characteristics not limited to reproduction. For the nematode Caenorhabditis elegans, in which the complete neuroanatomy has been solved for both hermaphrodites and males, sexually dimorphic features have been observed both in terms of the number of neurons and in synaptic connectivity. In addition, male behaviours, such as food-leaving to prioritise searching for mates, have been attributed to neuropeptides released from sex-shared or sex-specific neurons. In this study, we show that the lury-1 neuropeptide gene shows a sexually dimorphic expression pattern; being expressed in pharyngeal neurons in both sexes but displaying additional expression in tail neurons only in the male. We also show that lury-1 mutant animals show sex differences in feeding behaviours, with pharyngeal pumping elevated in hermaphrodites but reduced in males. LURY-1 also modulates male mating efficiency, influencing motor events during contact with a hermaphrodite. Our findings indicate sex-specific roles of this peptide in feeding and reproduction in C. elegans, providing further insight into neuromodulatory control of sexually dimorphic behaviours.

Pharyngeal timing and particle transport defects in Caenorhabditis elegans feeding mutants

The nematode C. elegans uses rhythmic muscle contractions and relaxations called pumps to filter, transport, and crush food particles. A number of feeding mutants have been identified, including those with slow pharyngeal pumping rate, weak muscle contraction, defective muscle relaxation, and defective grinding of bacteria. Many aspects of these pharyngeal behavioral defects and how they affect pharyngeal function are not well understood. For example, the behavioral deficits underlying inefficient particle transport in ‘slippery’ mutants have been unclear. Here we use high speed video microscopy to describe pharyngeal pumping behaviors and particle transport in wild-type animals and in feeding mutants. Different ‘slippery’ mutants exhibit distinct defects including weak isthmus contraction, failure to trap particles in the anterior isthmus, and abnormal timing of contraction and relaxation in pharyngeal compartments. Our results show that multiple deficits in pharyngeal timing or contraction can cause defects in particle transport.

miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging

Muscle function relies on the precise architecture of dynamic contractile elements, which must be fine-tuned to maintain motility throughout life. Muscle is also plastic, and remodeled in response to stress, growth, neural and metabolic inputs. The conserved muscle-enriched microRNA, miR-1, regulates distinct aspects of muscle development, but whether it plays a role during aging is unknown. Here we investigated Caenorhabditis elegans miR-1 in muscle function in response to proteostatic stress. mir-1 deletion improved mid-life muscle motility, pharyngeal pumping, and organismal longevity upon polyQ35 proteotoxic challenge. We identified multiple vacuolar ATPase subunits as subject to miR-1 control, and the regulatory subunit vha-13/ATP6V1A as a direct target downregulated via its 3′UTR to mediate miR-1 physiology. miR-1 further regulates nuclear localization of lysosomal biogenesis factor HLH-30/TFEB and lysosomal acidification. Our studies reveal that miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact muscle function and health during aging.

C. elegans PEZO-1 is a Mechanosensitive Channel Involved in Food Sensation

PIEZO channels are force sensors essential for physiological processes including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, expressed in several tissues including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive channel and contributes to pharyngeal function remains unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recordings to establish that pezo-1 is expressed in the pharynx, including a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food quality. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the pezo-1 isoform G (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.

Cholinergic signalling at the body wall neuromuscular junction couples to distal inhibition of feeding in C. elegans

Complex biological functions within organisms are frequently orchestrated by systemic communication between tissues. In the model organism C. elegans, the pharyngeal and body wall neuromuscular junctions are two discrete structures that control feeding and locomotion, respectively. These distinct tissues are controlled by separate, well-defined neural circuits. Nonetheless, the emergent behaviours, feeding and locomotion, are coordinated to guarantee the efficiency of food intake. We show that pharmacological hyperactivation of cholinergic transmission at the body wall muscle reduces the rate of pumping behaviour. This was evidenced by a systematic screening of the cholinesterase inhibitor aldicarb’s effect on the rate of pharyngeal pumping on food in mutant worms. The screening revealed that the key determinant of the inhibitory effect of aldicarb on pharyngeal pumping is the L-type nicotinic acetylcholine receptor expressed in body wall muscle. This idea was reinforced by the observation that selective hyperstimulation of the body wall muscle L-type receptor by the agonist levamisole inhibited pumping. Overall, our results reveal that body wall cholinergic transmission controls locomotion and simultaneously couples a distal inhibition of feeding.

Pharyngeal Pumping and Tissue-Specific Transgenic P-Glycoprotein Expression Influence Macrocyclic Lactone Susceptibility in Caenorhabditis elegans

Macrocyclic lactones (MLs) are widely used drugs to treat and prevent parasitic nematode infections. In many nematode species including a major pathogen of foals, Parascaris univalens, resistance against MLs is widespread, but the underlying resistance mechanisms and ML penetration routes into nematodes remain unknown. Here, we examined how the P-glycoprotein efflux pumps, candidate genes for ML resistance, can modulate drug susceptibility and investigated the role of active drug ingestion for ML susceptibility in the model nematode Caenorhabditis elegans. Wildtype or transgenic worms, modified to overexpress P. univalens PGP-9 (Pun-PGP-9) at the intestine or epidermis, were incubated with ivermectin or moxidectin in the presence (bacteria or serotonin) or absence (no specific stimulus) of pharyngeal pumping (PP). Active drug ingestion by PP was identified as an important factor for ivermectin susceptibility, while moxidectin susceptibility was only moderately affected. Intestinal Pun-PGP-9 expression elicited a protective effect against ivermectin and moxidectin only in the presence of PP stimulation. Conversely, epidermal Pun-PGP-9 expression protected against moxidectin regardless of PP and against ivermectin only in the absence of active drug ingestion. Our results demonstrate the role of active drug ingestion by nematodes for susceptibility and provide functional evidence for the contribution of P-glycoproteins to ML resistance in a tissue-specific manner.