Yu, Y. et al. (2019) International Worm Meeting "A rapid untargeted technique for identifying activated compounds in living systems."

Yu, Y., Le, H.

[International Worm Meeting, 2019]

In metabolism, most compounds that undergo chemical transformations get activated as thiol esters with Coenzyme A, which then undergo diverse biosynthetic transformations. Such compounds include intermediates of fatty acid metabolism1 and ascarosides2 in C. elegans, and it is likely that the vast majority of activated metabolites have remained unknown. Using conventional metabolomics protocols, such thiol esters will either undergo hydrolysis or react with another molecules in the worm lysate, e.g. proteins, and become invisible in LCMS analysis. We developed a protocol to capture CoA-derivatives and other activated intermediates using a simple trapping reagent, hydroxylamine (NH2OH), Treatment with hydroxylamine during preparation of worms for metabolomic analysis converts thiol esters into the corresponding hydroxamic acids, which can be easily detected and identified using high-resolution LC-MS. Using a new software platform we have developed for comparative metabolomics in worms and other model systems, Metaboseek (https://metaboseek.com/ ), changes in the production of several 1,000 activated intermediates and other metabolites in different mutants or in response to different environmental conditions can be assessed. I will present results using these approaches for the comparison of the activated metabolome of several fatty acid metabolism mutants. 1. Stryer, L. (1995). "Fatty acid metabolism." In: Biochemistry (Fourth Ed.). New York: W.H. Freeman and Company. pp. 603-628. ISBN 0 7167 2009 4. 2. Von Reuss, S. "Comparative Metabolomics Reveals Biogenesis of Ascarosides, a Modular Library of Small-Molecule Signals in C. elegans." J. Am. Chem. Soc. 2012, 134, 1817-1824

Frezal, Lise et al. (2015) International Worm Meeting "Caenorhabditis briggsae and its two natural viruses, coevolution in a 'menage a trois'."

BRESARD, Gautier, Felix, Marie-Anne, Frezal, Lise

[International Worm Meeting, 2015]

We study the natural coevolution between Caenorhabditis briggsae and its two recently described RNA viruses called Santeuil and Le Blanc (1, 2). The main advantage of this system is to combine the access to wild host and virus populations with powerful molecular tools and experimental evolution designs. We characterized the incidence of the two C. briggsae viruses in France and found that they are found in sympatry. By monitoring the viral RNAs in wild-caught C. briggsae isolates using Fluorescent In Situ Hybridization, we demonstrated that the Le Blanc and Santeuil viruses could coexist in one host population, one animal and one intestinal cell. Molecular variation of the wild-caught viruses was assessed by sequencing their two RNA molecules. While both viruses' diversities are geographically structured, we detected balancing selection on the RNA-dependent RNA polymerase (RdRp) locus in one local Santeuil population. Despite the frequent incidence of coinfection in the wild, we found no evidence for genetic exchange (recombination or RNA reassortment) between the Santeuil and Le Blanc viruses. However, we found clear evidence for RNA reassortment between different Santeuil virus variants. Finally, we investigated natural variation in C. briggsae resistance to each virus. We tested a set of wild isolates -representative of C. briggsae worldwide diversity- for their sensitivity to the Santeuil and Le Blanc viruses. While temperate C. briggsae genotypes are generally susceptible to both viruses, the tested tropical C. briggsae genotypes are resistant to both viruses. Most interestingly, two Japanese C. briggsae genotypes show specific resistance to the Le Blanc virus. To understand the genetic basis of the general and virus-specific resistances of C. briggsae, we carried out a QTL-mapping approach using recombinant inbred lines between AF16 and HK104 (3) and identified a main QTL region on chromosome IV responsible for the variation in resistance to Santeuil virus infection.(1) Felix, Ashe, Piffaretti et al. 2011 PloS Biology. (2) Franz et al. 2012 Journal of Virology. (3) Ross et al. 2011 PLoS Genetics..

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Alkan , Cigdem et al. (2017) International Worm Meeting "Interactions between Caenorhabditis nematodes and viruses infecting them naturally."

Ruaud, Albane, Alkan , Cigdem, BRESARD, Gautier, Felix, Marie-Anne

[International Worm Meeting, 2017]

The discovery of RNA viruses that naturally infect C. elegans and C. briggsae serves as an ideal model system to study antiviral immunity and host-pathogen co-evolution. The Orsay virus only infects C. elegans whereas Santeuil and Le Blanc viruses only infect C. briggsae. Intraspecifically, within both species we found a wide variation in viral sensitivity, as well as a positive correlation among wild isolates in sensitivity to both viruses in C. briggsae. An exception to this correlation is the C. briggsae strain HK104, which is specifically resistant to Le Blanc virus but sensitive to Santeuil virus. Taking advantage of this natural variation in the host, we use a genetic approach from the host side and use Recombinant Inbred Lines (RILs) to first map the recombinant genomic regions participating to the resistance/sensitivity in a general and/or specific manner. The RILs were phenotyped for the sensitivity to the relevant viruses using Fluorescent In Situ Hybridization (FISH). The genotype (SNP markers from pool sequencing) and phenotype (resistance/sensitivity from FISH) data were used to perform QTL analysis. Several Near Isogenic Lines (NILs) were created by introgressing the candidate regions. C. briggsae AF16 is resistant to both Santeuil and Le Blanc viruses while C. briggsae HK104 is specifically sensitive to the Santeuil virus. Using AF16xHK104 Advanced Intercrossed RILs (AIRILs) (Ross et al. 2011), two QTLs were detected on chromosomes III and IV for Santeuil virus sensitivity. The NILs in the AF16 background confirm both candidate regions. C. briggsae JU1498 is sensitive to both Santeuil and Le Blanc viruses. Using JU1498xHK104 RILs, a QTL on chromosome II was detected and is being introgressed. Once candidate polymorphisms associated with the virus sensitivity/resistance are identified, we will test them by RNAi knockdown, transformation rescue and/or CRISPR-mediated gene replacement.

BRESARD, Gautier et al. (2013) International Worm Meeting "Specificity of interaction between Caenorhabditis and their natural viruses."

Felix, Marie-Anne, BRESARD, Gautier

[International Worm Meeting, 2013]

Species involved in host-pathogen relationships exert selective pressures on each other. This co-evolution situation results in an arms race between host and pathogen, which may lead to specialisation of their interactions. We recently found three related horizontally-transmitted RNA viruses that naturally infect C. elegans or C. briggsae, called Orsay, Santeuil and Le Blanc viruses (Felix et al. 2011, Franz et al. 2012). Here we study their specificity for C. elegans vs. C. briggsae, and at the intraspecific level in C. briggsae. We first used viral filtrates to infect a set of C. elegans and C. briggsae isolates, and measured by RT-PCR the virus ability to replicate. We find that the Orsay virus can infect C. elegans but not C. briggsae, whereas Santeuil and Le Blanc viruses infect C. briggsae, but not C. elegans. Thus, each virus shows specificity toward one of these two Caenorhabditis species. Given that C. briggsae can be infected by two viruses, we then measured viral replication after infection of C. briggsae isolates by either Santeuil or Le Blanc viruses, using RT-qPCR. We observed 1) wide variation among C. briggsae isolates; 2) correlation between the sensitivities to each virus; 3) an exception to the correlation. Schematically, C. briggsae isolates can be separated into two groups: sensitive isolates, in which the viruses replicate efficiently; and resistant ones, in which the viruses either disappear or are barely maintained. Strikingly, all sensitive strains belong to the temperate C. briggsae clade, raising the possibility that sensitivity is derived within this clade. The exception to the correlation in sensitivity is HK104, a temperate-clade isolate from Japan. HK104 is sensitive to the Santeuil virus, but resistant to Le Blanc. This result opens the possibility to study specificity of host-pathogen interactions through genetic analysis.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Frezal, Lise et al. (2013) International Worm Meeting "Microevolution and coexistence of Santeuil and Le Blanc viruses in Caenorhabditis briggsae. ."

Frezal, Lise, Felix, Marie-Anne

[International Worm Meeting, 2013]

We recently found three viruses, Orsay, Santeuil and Le Blanc, which naturally infect Caenorhabditis nematodes (1,2). These ss(+)RNA viruses cause intestinal cell symptoms and are horizontally transmitted. Whereas C. elegans can so far only be infected by the Orsay virus, European C. briggsae genotypes are susceptible to both Santeuil and Le Blanc viruses, and both viruses have been found in the same locations. This vulnerability of C. briggsae to two viruses enables studies of in vivo viral competition and of the mechanisms driving their short-term evolution, as well as the impact of their competition on worm fitness. RNA viruses may evolve rapidly through both high mutation rates and recombination events. The impact of recombination widely varies from one viral species to another but in all cases, for recombination to occur, different virus types have to infect the same host cell. The first step is thus to assess whether different virus species can co-infect the same worm population, the same animal and the same cell. By using quantitative RT-PCR, we demonstrate that the Le Blanc and Santeuil viruses can coexist in a worm population, even when originally introduced at widely different concentrations. The two viruses are jointly maintained over 10 worm generations. We presently investigate the co-infection at the whole organism and single cell levels by tracking the viral RNAs in co-infected worms using Fluorescent In Situ Hybridization. 1- Felix, Ashe, Piffaretti et al. 2011 PloS biology. 2- Franz et al. 2012 Journal of virology.

Rasika Kalamegham et al. (2004) East Coast Worm Meeting "EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a mammalian acyl transferase"

Wendy Hanna-Rose, Rasika Kalamegham

[East Coast Worm Meeting, 2004]

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no putative transmembrane domains, it is localized at the apical membrane of cells where it is expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression as opposed to normal apical membrane localization. The corresponding Serine residue is conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by restoring membrane localization to EGL-26n481 via addition of alternative membrane localization signals.

Schwartz, Hillel et al. (2013) International Worm Meeting "Developing Heterorhabditis nematodes as an experimental system for the study of mutualistic symbiosis."

Schwartz, Hillel, Sternberg, Paul

[International Worm Meeting, 2013]

Entomopathogenic nematodes of the genus Heterorhabditis are insect killers that live in mutually beneficial symbiosis with pathogenic Photorhabdus bacteria. Photorhabdus is rapidly lethal to insects and to other nematodes, including C. elegans, but is required for Heterorhabditis growth in culture and for the insect-killing that defines the entomopathogenic lifestyle. The symbiosis between Heterorhabditis and Photorhabdus offers the potential to study the molecular genetic basis of their cooperative relationship. We developing tools to make such studies more feasible: we have been studying multiple nematodes of the genus Heterorhabditis and developing tools for the molecular genetic analysis of Heterorhabditis bacteriophora. Many species of Heterorhabditis and variants of Photorhabdus have been isolated; some pairings show specificity in their ability to establish a symbiotic relationship. To better understand these interactions and other variations in the lifestyles of Heterorhabditis, we have sequenced H. indica, H. megidis, H. sonorensis, and H. zealandica; a H. bacteriophora genome sequence is available. A comparison of these closely related species may help us to identify mechanisms that regulate the response to bacterial interactions and to find variations that correlate with differences in lifestyle or bacterial compatibility. In addition to genomics, we are developing H. bacteriophora as a laboratory organism. H. bacteriophora grows well on plates, has been reported to be susceptible to RNAi and transgenesis, and can develop as a selfing hermaphrodite, and so should be a powerful system for the molecular genetic study of the aspects of biology to which it is uniquely well suited, most prominently symbiosis. This potential is severely diminished by inconvenient sex determination: the self-progeny of hermaphrodites are mostly females with some males; at low density, their progeny are almost exclusively females. We have screened for and isolated a constitutively hermaphroditic mutant for use in molecular genetic studies of symbiosis. This mutant also offers the opportunity to explore the basis of hermaphrodite sex determination in H. bacteriophora.

Schwartz H et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Towards a SNP map for Heterorhabditis bacteriophora"

Sternberg P, Schwartz H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Heterorhabditis bacteriophora is a species of insect-parasitic nematode that lives in mutually beneficial symbiosis with pathogenic Photorhabdus luminescens bacteria. P. luminescens bacteria are lethal to insects and to other nematodes, including the soil nematode Caenorhabditis elegans, but are required for H. bacteriophora growth. The symbiosis between H. bacteriophora and P. luminescens therefore offers the potential to study the molecular genetic basis of their cooperative relationship. We are interested in developing tools to make such studies more feasible; in particular, we propose to generate tools for genetic mapping in H. bacteriophora. We will test independent isolates of H. bacteriophora to ensure that the isolates are cross-fertile. We will then examine the abilities of these isolates to grow on and to become infected with different wild-type and mutant Photorhabdus bacteria. From these tests we will select an isolate on which we will use next-generation high-throughput sequencing technology for the purpose of refining the existing draft genome sequence and for the creation of a SNP map. We anticipate that this SNP map will enable us and the wider insect-parasitic nematode community to identify induced mutations and natural variations affecting the interactions between H. bacteriophora nematodes and pathogenic Photorhabdus bacteria.