Hsueh, Yen-Ping et al. (2011) International Worm Meeting "Molecular mechanism governing interaction between nematodes and nematophagous fungi."

Schroeder, Frank, Schwarz, Erich, Sternberg, Paul, Hsueh, Yen-Ping, Forseth, Ry

[International Worm Meeting, 2011]

C. elegans shares the same ecological niche with their predators, nematophagous fungi, which are ubiquitous in the soil. The predator-prey relationship between nematodes and nematophagous fungi makes them an attractive model to study co-evolution. When exposed to one of the most common nematophagous fungi, Arthrobotrys oligospora, both C. elegans, and other nematode species were attracted to A. oligospora in chemotaxis assays. Furthermore, chemotaxis assays performed on partition plates demonstrated that volatile compounds produced by A. oligospora contribute to worm attraction. Genetic analysis and cell-specific laser ablation showed that the AWC neurons are required for chemotaxis toward A. oligospora in C. elegans. Using gas chromatography-mass spectrometry (GC-MS), we identified volatile organic compounds (VOCs) produced by A. oligospora that could potentially mediate worm-attraction. We will use single neuron transcriptome profiling and comparative genomics to identify potential G-protein coupled receptors (GPCRs) involved in the detection of A. oligospora. The candidate GPCRs will be expressed in heterologous systems to test for potential ligand-receptor specificity, providing insights into the function and evolution of GPCRs in nematodes.

Hsueh, Yen-Ping et al. (2013) International Worm Meeting "Nematophagous fungi eavesdrop on nematode pheromones, and lure their prey with attractive volatile organic compounds."

Schwarz, Erich, Xian, Zhaoying, Mahanti, Parag, Gronquist, Matthew, Zeng, Weihua, Sternberg, Paul, Mortazavi, Ali, Schroeder, Frank, Hsueh, Yen-Ping

[International Worm Meeting, 2013]

Nematophagous fungi are natural predators of soil-dwelling nematodes, and this predator-prey relationship makes them an attractive model to study co-evolution. How do microorganisms detect their metazoan prey, and how do prey respond to predators? We set out to investigate the cues that nematophagous fungi use to trigger morphogenesis of their nematode-trapping devices and how C. elegans responds to nematophagous fungi. We found that nematophagous fungi can detect and respond to ascarosides, small molecules produced by many nematodes that regulate nematode development and behavior. In response to ascarosides, Arthrobotrys oligospora and closely related nematophagous fungi induce morphogenesis of their nematode traps. Ascarosides thus represent a conserved molecular pattern used by nematophagous fungi to detect prey. Through RNA-seq analysis, we identified A. oligospora genes regulated by ascarosides and nematode exposure. On the other hand, C. elegans are attracted to A. oligospora. This attraction is, at least in part, mediated by volatile compounds. Gas chromatography and mass spectrometry revealed that volatile organic compounds produced by A. oligospora could attract nematodes. Genetic analysis and cell-specific laser ablation showed that AWC neurons are required for this behavior. To find genes involved in the AWC-mediated A. oligospora attraction, we performed single-cell RNA-seq of the AWCon neuron. We detected expression of 6,608 genes in AWC neurons, with 1,278 being AWC-enriched. Preliminary mutant screening revealed genes that have a function in AWC-mediated chemosensation.

Hsueh, Yen-Ping et al. (2015) International Worm Meeting "Fatal attraction: The olfactory neurons AWCs of Caenorhabditis elegans mediate attraction toward nematophagous fungus Arthrobotrys oligospora."

Schroeder, Frank C., Sternberg, Paul W., Schwarz, Erich M., Gronquist, Matthew R., Hsueh, Yen-Ping, Nath, Ravi D.

[International Worm Meeting, 2015]

We set out to investigate how C. elegans respond to their natural predator A. oligospora to gain insight into predator-prey coevolution. We found that C. elegans and several other nematode species are attracted to A. oligospora. This attraction is mediated by volatile compounds. Genetic analysis and cell-specific laser ablation showed that AWC neurons are required for this behavior. Gas chromatography-mass spectrometry identified several volatile organic compounds produced by A. oligospora. Some of these fungal odors were highly attractive to C. elegans when pure compounds were tested in chemotaxis assays. Furthermore, when C. elegans were adapted to A. oligospora culture, the attraction towards the fungal odors was significantly decreased, supporting that the compounds identified were genuine. Calcium-imaging showed that these compounds elicit a strong calcium response in the AWCon neuron in both wild-type and the unc-13 background, suggesting that the response observed is likely primary. To identify potential receptors for A. oligospora odors, we performed single-cell RNA-seq of the AWCon neuron. In total, 61GPCRs were detected, that are collectively likely to be less conserved and fast evolving among different Caenorhabditis species (Thomas and Robertson [2008], BMC Biol. 6, 42). Lastly, killing assays demonstrated that mutants defective in the AWC function have much higher survival rate compared to wild-type worms when encountered by A. oligospora.

Yang, Ching-Ting et al. (2017) International Worm Meeting "Surveying the species correlation between nematodes and nematode-trapping fungi in natural habitats."

Lin, Siou-Ying, Hsueh, Yen-Ping, Yang, Ching-Ting

[International Worm Meeting, 2017]

Nematodes and nematode-trapping fungi are both ubiquitously present in the environment. However, little is known about how these predatory fungi interact with their nematode-prey in nature. Our previous findings showed that the nematode-trapping fungi of the Arthrobotrys species eavesdrop on the nematode pheromone ascarosides as an indication of the presence of the nematode prey (Hsueh et al. 2013). We observed that different Arthrobotrys species exhibit different ascaroside-specificity and hypothesized that this might result from the co-evolution between the different species of nematodes and nematode-trapping fungi. To understand what species of nematodes and nematode-trapping fungi co-exist in the natural environment and to acquire material to test our hypothesis, we collected wild-isolates of nematodes and nematode-trapping fungi from different locations in Taiwan and determine the identity of the fungi and nematodes at the species or genus level based on their ITS or 18S rDNA sequences. In total, we successfully isolated both nematodes and nematode-trapping fungi from 13 of the 22 locations sampled. Seven species of Arthrobotrys have been identified to share the same niches with nematodes of the genera Oscheius, Cervidellus, Rhabditis, Caenorhabditis, Pelodera, Diploscapter, Meyerozyma, Acrobeloides, and Mesorhabditis, suggesting that Arthrobotrys species are likely to prey on a broad range of nematode species in nature. Hsueh YP, Mahanti P, Schroeder FC, Sternberg PW. 2013. Nematode-trapping fungi eavesdrop on nematode pheromones. Curr Biol 23: 83-86.

Lee, Ching-Han et al. (2021) International Worm Meeting "Organismal death triggered by oyster mushrooms via mitochondrial dysfunction"

Lee, Ching-Han, Hsueh, Yen-Ping

[International Worm Meeting, 2021]

The oyster mushroom Pleurotus ostreatus is a nematophagous basidiomycete that produces toxins to paralyze nematodes. We previously reported that P. ostreatus triggers a massive calcium influx and rapid cell necrosis in the neuromuscular system of C. elegans via its sensory cilia. However, how do the mushroom hyphae trigger rapid cell necrosis remains unclear. Here, we show that P. ostreatus induced calcium influx in the mitochondrial matrix, resulting in massive mitochondria enlargement within five minutes of hyphal contact. In addition, the ATP level dropped promptly in the pharyngeal muscle cells of the paralyzed nematodes. Moreover, we observed a calcium wave propagated across the mitochondria of hypodermis. Mutants exhibited muscle contraction defects restricted the calcium wave, suggesting that body wall muscle contraction contributed to the propagation of calcium wave in hypodermis. Furthermore, C. elegans mutants with disrupted ER-mitochondria contacts decreased the calcium influx in the mitochondria of pharyngeal muscle cells. Our findings illustrate that Pleurotus toxins trigger drastically ion imbalance and disrupt mitochondrial function, leading to energy failure and cell necrosis throughout the entire animal.

Chiang, Y. et al. (2017) International Worm Meeting "Wild-isolates of C. elegans exhibit variable attraction to the sex-pheromone mimicry compound produced by the nematode-trapping fungus A. oligospora."

Chiang, Y., Hsueh, Y.

[International Worm Meeting, 2017]

The nematode-trapping fungus A. oligospora produces odors that mimic sex and food cues to attract many nematode species (Hsueh et al. 2017). One of the compounds (MMB) that likely mimics male pheromone in Canerhbiditis nematodes is highly attractive to two lab strains of C. elegans (N2 and Hawaiian). To investigate whether this attraction is highly conserved among the wild-isolates, we analyzed the MMB chemotaxis behavior among the C. elegans wild isolates from the CeNDR collection. We found that MMB-attraction is highly polymorphic trait. Six out of forty wild-isolates tested exhibited weak attraction to MMB. Genetic analysis showed that this trait is controlled by QTL in some strains but a single locus in others. The high incidence of MMB-insensitive strains among the wild-isolates suggests that lost of MMB attraction might offer benefit to C. elegans in the natural environments. Hsueh YP, Gronquist MR, Schwarz EM, Nath RD, Lee CH, Gharib S, Schroeder FC, Sternberg PW. 2017. Nematophagous fungus Arthrobotrys oligospora mimics olfactory cues of sex and food to lure its nematode prey. eLife 6.

Sternberg, Paul W. et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "Evolution of nematode behavior"

Gronquist, Matthew R., Hsueh, Yen-Ping, Doma, Meenaskshi, Schroeder, Frank S., Nath, Ravi, Chow, Elly, Sternberg, Paul W., Leighton, Daniel

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

The molecular complexity of individual nematode neurons is likely evolved to overcome a severe constraint on neuronal cell number. This view is held up by transcriptional profiling of individual sensory and interneurons, and by the multiple roles many neurons play in behavior. To understand the evolution of nematode behavior, we need, in part, to understand the world they inhabit. Parasitic nematodes have evolved strikingly specialized host-location behaviors, such as the jumping of certain Steinernemas. On the other hand, free-living nematodes appear to inhabit a harsh world with rapidly changing environment and co-evolving predators. Predatory fungi such as Arthrobotrys oligospora attract nematodes and also sense their presence. We previously showed that Arthrobotrys can sense ascarosides, nematode secondary metabolites that serve as a variety of social cues. We are also studying how Arthrobotrys attracts C. elegans. Using GC-MS, we have identified odors that are naturally produced by this fungus, and found that they are attractive to C. elegans. At least some of these odors are sensed directly by the AWC sensory neuron based on calcium imaging studies. That C. elegans responds to chemicals and produce chemicals that lead to their death suggest the importance of these chemicals in their natural chemical ecology. The many odors that impact C. elegans olfactory neurons might help explain sensory neuron molecular biology, but interneurons seem to express as many genes. We have much to learn.

Yen, Kelvin et al. (2011) International Worm Meeting "Dissecting the interaction between insulin/IGF-1 and TOR signaling pathways."

Yen, Kelvin, Tissenbaum, Heidi

[International Worm Meeting, 2011]

Both the insulin/IGF-1 signaling and the TOR signaling pathways are known to regulate lifespan and feeding/fat in multiple species (1,2). Despite the fact that both pathways monitor energy levels and have been shown to have interconnected signaling cascades, lifespan analysis suggests that these pathways are independent of each other. We have analyzed single mutants and generated double mutant combinations of the insulin/IGF-1 pathway and the TOR pathway components. We have tested these strains for lifespan, lipid storage and protein phosphorylation status. We will present our data that suggest that there are multiple potential interaction points of the pathways. 1. Narasimhan, S., Yen, K., Tissenbaum, H. Converging Pathways in Lifespan Regulation. Current Biology 19, R657-R666 (2009). 2. Kapahi, P., et al. With TOR, Less Is More: A Key Role for the Conserved Nutrient-Sensing TOR Pathway in Aging. Cell Metabolism 11, 453-465 (2010).

Choi, Jae Im et al. (2017) International Worm Meeting "Identifying novel odor-receptor pairs in C.elegans."

Choi, Jae Im, Yoon, Kyoung-hye, Lee, Jin Il

[International Worm Meeting, 2017]

C. elegans is known to sense and respond to dozens of odors through G-protein coupled receptors expressed in three pairs of olfactory sensory neurons - AWA, AWB and AWC. In addition, worms display many olfactory behaviors including dose-dependent odortaxis, odor specificity, short and long-term odor memory, and odor-associative behaviors. One of these attractive odors, diacetyl, was shown to bind to its cognate receptor, ODR-10, by expressing the receptor in a heterologous cell line and demonstrating activity upon binding to diacetyl. However no other odor-receptor pairs have been definitively identified aside from ODR-10. In order to begin to identify new odor-GPCR matches in C. elegans, we conducted odortaxis assays to a set of 192 odors. We found that worms are highly attracted to 14% of these odors, mildly attracted to 10%, and show aversion to 7% of odors. We plan to identify which olfactory sensory neuron C. elegans uses to sense each of the odors using mutant analysis. To identify odor receptor pairs for AWC-sensed odors, we plan to express AWC-expressed ORs (Hsueh et al., 2016) in HEK293 mammalian cells, and determine their odor ligand using a cell-based reporter gene assay. We expect that these results will not only identify new odor-receptor pairs to study C. elegans olfactory behavior, but will contribute to our understanding of the genomic structure and evolution of olfactory receptors in the animal kingdom.

Leifer, Andrew et al. (2011) International Worm Meeting "Shining light on behavior: An optogenetic dissection of neural circuits in freely behaving C. elegans."

Samuel, Aravinthan, Leifer, Andrew, Clark, Christopher, Wen, Quan, Alkema, Mark, Fang-Yen, Christopher

[International Worm Meeting, 2011]

Optogenetics provides a promising new platform for conducting behavioral neuroscience investigations in the nematode Caenorhabditis elegans. The ability to precisely manipulate neural activity in individual neurons in a moving worm allows one to pinpoint the contribution of individual neurons to animal behavior. Previously we developed a high-resolution optogenetic illumination system capable of delivering arbitrary light patterns to targeted cells in a freely moving C. elegans (Leifer and Fang-Yen et al, Nature Methods, 2011). The system, called CoLBeRT, uses a high speed camera and custom computer vision software to monitor the motion of an unrestrained worm. As the worm swims or crawls, the system instructs a digital micromirror device to reflect patterns of blue or green laser light onto specific cellular targets expressing Channelrhdopsin-2 or Halorhodopsin. The system has sufficient accuracy and resolution to stimulate individual mechanosensory neurons while a worm swims. Here we have expanded the CoLBeRT system's capabilities to dissect the motor circuit, mechanosensory circuit and investigate the roles of command interneurons. The system is now able to generate illumination patterns with arbitrary amplitude waveforms and we have adapted the system to work with microfluidic devices. We have used the system to differentiate models of wave propagation in the motor circuit, to study habituation in the mechanosensory circuit and to begin an exploration of the command interneurons. Preliminary evidence suggests that stimulation of the command interneuron AVA modulates the worm's backward velocity.