Arantes, Leticia et al. (2015) International Worm Meeting "Guarana (Paullinia cupana Mart) extends lifespan and modulates some gene expression levels in Caenorhabditis elegans."

Aschner, Michael, Machado, Marina, Soares, Felix, Zamberlan, Daniele, Arantes, Leticia

[International Worm Meeting, 2015]

Extensive research has focused on aging processes and compounds that can promote lifespan extension and delay age-associated diseases. Guarana (Paullinia cupana) is a Brazilian fruit traditionally consumed by Amazon indigenous communities and it is also an ingredient in many energy drinks. Although Guarana has some beneficial effects described, no studies could be identified on its aging-related effects. Thus, we designed a study using C. elegans to investigate the effects of Guarana in lifespan as well as some genetic mechanisms underlying its effects. Guarana ethanolic extract (GEE) powder was dissolved in water and spread on NGM plates with E. coli at 100, 500 and 1000 microg/ml final concentrations. Synchronized wild type L1 larvae were transferred to treatment plates at 20degC. Adults were transferred to fresh plates with GEE every other day and scored until death. Real-time qPCR was performed and pre-designed TaqMan primer sets were used to measure C. elegans sod-3, gst-4, gcs-1, daf-2, sir-2.1, hsf-1, hsp-16.2 and skn-1 expression in 1 and 10 days-old-adults. Median lifespan of treated worms was lengthened by 18% at 100 microg/ml and 36% at 500 and 1000 microg/ml. Maximal lifespan was increased by an average of 28% in the three concentrations of GEE. GEE significantly down regulated RNAm levels of daf-2, sir-2.1 and hsp-16.2 in 1 and 10-days-old-adults, gst-4 and hsf-1 only in 1 day-old-adults and skn-1 only in 10-days-old-adults. Expression of sod-3 and gcs-1 were not altered in both ages. Our study shows that GEE extends median and maximum lifespan of C. elegans and it is accompanied with a gene modulation related to antioxidant and aging pathways dependent on age. Despite additional and more systematic genetic analysis are necessary to establish the precise molecular pathways involved in these beneficial effects, our results contribute to the study of natural products' effects in vivo and also increases the knowledge of lifespan modulation process. .

Lehmen, Tassia Fontana et al. (2013) International Worm Meeting "Effect of chronic treatment with amphetamine in an experimental model using Caenorhabditis elegans."

Fachinetto, Roselei, Lehmen, Tassia Fontana, Soares, Felix A A, Puntel, Bruna, Gubert, Priscila

[International Worm Meeting, 2013]

Caenorhabditis elegans, a free-living soil nematode, has already been explored as a valuable bioindicator, since it is one of the best-characterized animal models at the genetic, physiological, molecular, and developmental levels. It has also been proved that C. elegans serves as an excellent candidate for studying the development and functions of nervous system and neurotoxicology. The worms have well characterized dopaminergic system, as well as eight neurons of this system located along the body. Amphetamine (AMPH) is a stimulant drug of the central nervous system that acts broadly on various behaviors. Whereas multiple lines of evidence implicate dopamine receptors in the mechanism of action of AMPH and other abused psychostimulants, the degree to which different receptors support AMPH-induced behaviors has not been completely elucidated. Because of this we decided to investigate the effects of amphetamine on the behavior of nematodes.L1 larva stage worms of wild type strain were treated with AMPH 1mM or water (control group) in agar plates until adulthood. We analyzed pharyngeal pumping rate, defecation cycle length, body bends and oviposition and egg production rates in adult worms using a microscope. Treatment with AMPH 1 mM promoted a reduction in the rates of pharyngeal pumping, oviposition and egg production. Our data demonstrates that the concentration of amphetamine tested can interfere the behaviors mentioned above without, however, changing the defecation cycle length and body bends, acting probably by different routes. However, more studies are needed to elucidate the different mechanisms of action that may be involved, using, for example, knockout strains to dopamine transporters and/or receptors.

Gubert, Priscila et al. (2013) International Worm Meeting "Metabolic effects of manganese in the nematode Caenorhabditis elegans.."

Bruna, Bruna, Aschner, Michael, Gubert, Priscila, Avila, Daiana, Soares, Felix, Lehmen, Tassia

[International Worm Meeting, 2013]

Manganese (Mn) is an essential metal since acts as a cofactor for many enzymes. However, exposure to high levels of this metal may lead to the development of psychomotor disorders , concomitant with dopaminergic (DAergic) neurodegeneration. Furthermore, Mn can interfere with lipid and carbohydrate metabolism. The sensitivity of the DAergic system to Mn is also evident in the nematode, Caenorhabditis elegans. Notably, the DAergic system is associated with the control of fat storage in the worm. Our aim was to evaluate whether Mn alters C. elegans fat storage and to identify putative pathways involved. Wild-type, dat-1::GFP and daf-16::GFP worms grown under normal conditions were exposed for 4h at larval stage L4 to NaCl or manganese chloride (MnCl2). Next, we determined worm behaviors as well as lipid storage (Nile Red vital labeling). Mn (45 (p<0.0001) and 60 mM (p<0.05)) caused an increase in Nile Red fluorescence absent DAergic neurodegeneration. Furthermore, Mn (45 and 60 mM) significantly reduced the worms' survival to 70% and 65%, respectively. The body bends and defecation cycles were not affected by Mn. Mn (30, 45 and 60 mM) also caused a significant decrease in pharyngeal pumping, egg-laying and egg-production behaviors. The Fork head transcription factor DAF-16 when activated can inhibit egg fertilization. Based on this observation we investigated DAF-16 localization in response to Mn. Mn led to nuclear translocation of DAF-16 immediately upon treatment, and at 24h post treatment DAF-16 was predominantly in the cytoplasm (inactivated) (p<0.05). Our results establish that Mn increase lipid storage in C. elegans and delays its reproduction. These effects may be associated with DAF-16 activation. Additional studies are necessary to confirm these hypotheses and verify changes in the lipid accumulation by alternative methods.

Belicard, Tony et al. (2011) International Worm Meeting "Evolution in viral sensitivity to the Orsay virus in natural C. elegans isolates."

Belicard, Tony, Felix, Marie-Anne

[International Worm Meeting, 2011]

We isolated the first natural viruses infecting Caenorhabditis nematodes: the Orsay virus in C. elegans isolate JU1580 and the Santeuil virus in C. briggsae JU1264 (Felix & al., 2011). We more recently found a third virus in C. briggsae JU1498 (Le Blanc virus). These viruses cause disorders in intestinal cells of their host and are horizontally transmitted. Their genomes are composed of two single-stranded positive RNA segments carrying 3 ORFs. One of them, the ORF d, has no homology with any known ORF (Felix & al., 2011). We aim to identify its role during infection. We thus cloned it and are currently expressing it in a JU1580 background in order to know whether it affects the anti-viral response of the worm. In order to evaluate natural variation in sensitivity to these viruses, we scored the susceptibility of natural isolates and standard laboratory strains of C. elegans and C. briggsae. The results reveal i) a species specificity of infection by each virus and ii) intraspecific variation in sensitivity within both species for their respective viruses. First, we found a species specificity of each virus for a specific Caenorhabditis host species. Indeed, the Santeuil and Le Blanc viruses do not infect JU1580, while the Orsay virus does not infect JU1264 and JU1498 Second, we evaluated the geographic and genetic distribution of Orsay virus susceptibility in a worldwide set of 25 C. elegans isolates representing wild genetic diversity. We measured the viral load by RT-qPCR. Preliminary results suggest that only a subset of isolates from the Old world are sensitive to the virus and none of the "New World". This diversity seems to be partially linked with their ability to perform a small RNA response that acts in anti-viral defense (Felix & al., 2011; poster by Nuez & Felix). We plan to determine the genetic architecture and identify the molecular basis for this intraspecific variation in Orsay virus susceptibility. One approach is to cross closely related sensitive and resistant strains to obtain Recombinant Inbred Lines. We will test the susceptibility to the virus in these lines in order to find loci involved in the last evolutionary event causing resistance/sensitivity to the virus. By identifying these loci, we will be able to describe the last step in the "arms race" between C. elegans and its natural virus.

Soares, Marcell et al. (2021) International Worm Meeting "toluene-induced bioenergetics changes generate early aging and decreased healthspan in caenorhabditis elegans"

Soares, Marcell, Baptista, Fabiane, Silveira, Tassia, Cordeiro, Larissa, Silva, Aline, Avila, Daiana, Soares, Felix

[International Worm Meeting, 2021]

aging is a normal and inevitable cellular physiological process, however, in current days, individual or environmental factors are contributors to early aging, thus generating an impaired healthspan. barker, in an ecologic psychology context mentioned that "the environment always exerts influences in the individual". if we adapt this to the toxicological sphere, daily we are exposed to many agents, whether intentional (drug's abuse) or in a non-intentional manner (environmental or occupational), that induce countless toxic effects to the organism (influence). toluene is a major solvent used in industry of paints, gasoline and adhesives, therefore, workers are the non-intentional target public. however, toluene is also a psychoactive substance and epidemiologic data have shown its abuse as the third mostly consumed in comparison to others drugs there are many studies reporting the short-term effects of toluene, nevertheless, long-term effects after end of exposure are scarce. based on that, the aim of our work was to investigate the long-term impact of airborne exposure to toluene using caenorhabditis elegans. we used two strains: n2 wild type and mutant pe255 feIs5 [sur-5p::luciferase::GFP + rol-6(su1006)] to measure atp levels. approximately 100 nematodes at l4 stage were exposed to toluene in a vapor chamber for 24h, mimetizing two scenarios of exposure: scenario 1 (mean conc. 792 ppm) and scenario 2 (mean conc. 1,094 ppm). the experiments were conducted 1, 48 and 96 hours after exposure. lipofuscin autofluorescence was measured as an aging bioindicator, neurobehaviors (head trashes, velocity and path length) to assess healthspan, and atp levels as mitochondrial function endpoint. we observed that worms exposed to both scenarios of exposure have shown increase of cellular lipofuscin accumulation, and that exposed worms demonstrated a progressive reduction of mobility in a significant manner. these findings corroborated with a decrease of mitochondrial functionality, demonstrated by atp levels reduction. here we demonstrate that toluene-induced early cellular aging can occur associate to accentuated motor loss that could be explained by a mitochondrial dysfunction, thus impacting on healthspan reduction. the literature supports this discovery, once long-term exposures either intentionally or occupationally are harmful to humans, however, here we observed that even long periods after withdrawal the toluene effects remain.

Arantes, Leticia P et al. (2013) International Worm Meeting "Protective effect of Paullinia cupana, the Guarana, on Caenorhabditis elegans under oxidative stress."

da Rocha, Joao, Machado, Marina, Arantes, Leticia P, Tassi, Cintia, Zamberlan, Daniele, Soares, Felix

[International Worm Meeting, 2013]

Paullinia cupana (Guarana) is a plant originally from Brazil with growing consumption in many countries. It has several reported functions, including antioxidant activity in vitro, and popularly has been linked to a delay in the aging process. However, few results from in vivo studies exist. Therefore, we used the well established model organism Caenorhabditis elegans to investigate the effects of chronic treatment with guarana fruit extract on oxidative stress in vivo. After synchronization, L1-larva stage wild type (WT) worms were transferred to agar plates seeded with E. coli OP50 and guarana extract in the concentrations of 100, 500 and 1000 mg/ml.To assessment of oxidative resistance, L4 WT treated worms were exposed to juglone 125 mM for 1 hour in M9 buffer and scored for viability in a microscope. In order to measure reactive species, L4 WT and TK22 (mev-1) worms treated with extract were exposed to CM-H2DCFDA 20 mM for 2 hours. Fluorescence was detected by a multiplate reader and images were taken in a fluorescent microscope. Expression of sod-3 was quantified after treatment through the fluorescence of L4 worms of CF1553 (sod-3::GFP) strain, under normal conditions and after exposure to juglone 50 mM, using a multiplate reader. Images were also taken. Our results showed that guarana extract protected worms against juglone-induced oxidative damage, increasing survival in 26% at 100 mg/ml and 60% at 500 mg/ml. The treatment with extract did not reduce the levels of reactive species under normal conditions but reduced the levels generated by TK22 strain, a respiratory chain defective mutant, with an overproduction of superoxide. The expression of sod-3 was not altered under normal conditions neither after juglone exposure. As aging and mortality are closely related to external stress, the effects of guarana extract demonstrated on C. elegans under oxidative stress are very important. More studies are needed to investigate the mechanisms of antioxidant protection and the effects on aging. Keywords: Antioxidants, Guarana, Oxidative stress, Paullinia cupana .

Machado, Marina et al. (2015) International Worm Meeting "Ilex paraguariensis reduced fat storage in Caenorhabditis elegans."

da Silva, Ingrid, da Silveira, Tassia, Machado, Marina, Zamberlan, Daniele, Arantes, Leticia, Soares', Felix

[International Worm Meeting, 2015]

Obesity is characterized by an excess of adipose tissue, and is associated with several disorders like health risks and increased mortality. Ilex paraguariensis (yerba mate) is a native tree species in South America, its aqueous extract is widely consumed every day by billions of people, and it is characterized for its high caffeine concentration, which is known for its thermogenic and stimulating proprieties in vitro. Thus, we decided to investigate the effect of chronic consumption of I. paraguariensis in vivo, using the nematode Caenorhabditis elegans (C. elegans) as a model. We treated L1 wild-type worms with 1 mg/mL of yerba mate until adulthood. First, we evaluated lipid accumulation using BODIPY labeling and behavioral changes. Ilex paraguariensis indeed decreased the fluorescence of BODIPY labeling in 63.36% compared to control. Subsequently, yerba mate did not change behaviors related to energetic balance, such as pharynx pumping, egg production, defecation cycles or movement. At least, we measure total body energy expenditure through oxygen consumption, and we found that yerba mate increases oxygen consumption in 70.16% compared to control. Our results suggest that I. paraguariensis induced fat store reduction in C. elegans. .

Byrnes, Catherine et al. (2021) International Worm Meeting "Nematode hunters: a citizen science approach to identifying new systems for the study of host-virus interactions"

Sowa, Jessica, Byrnes, Catherine

[International Worm Meeting, 2021]

C. elegans is a popular model organism that has proved very useful for studying the cell biology of intracellular infections. However, its use as a model for the study of host-virus interactions has been limited by the fact that only one natural viral pathogen of C. elegans has been identified to date (Felix and Wang, 2019; Franz et al., 2014). The goal of this project is to identify novel natural nematode viruses capable of infecting C. elegans by mobilizing ordinary citizens to collect wild nematodes. Studying the interactions of different types of viruses with their host's cells can provide new insights into cell biology and host-pathogen interactions. To date, only four viruses naturally infecting Caenorhabditis nematodes have been identified, and of those only one (Orsay virus) infects C. elegans (Felix et al., 2011; Frezal et al., 2019). In the past, identification of intracellular pathogens in wild-caught nematodes has relied on detection by microscopy of morphological changes caused by the infection (Felix et al., 2011; Troemel et al., 2008). This approach is relatively low throughput and requires an expert screener. Our approach instead uses a fluorescent reporter-based method, taking advantage of a set of genes which are expressed at low levels in basal conditions but highly upregulated during infection by intracellular pathogens (Bakowski et al., 2014; Reddy et al., 2017, 2019). Co-culturing infected nematodes together with C. elegans expressing these intracellular infection reporters produces fluorescence which is easily detected on a fluorescence dissecting microscope. By using this method on a large sampling of wild-caught nematodes, we hope to identify novel nematode viruses which can be transmitted to C. elegans. In the pilot phase of this project, we established protocols for wild nematode collection which require minimal supplies and can be performed at home by people with no particular science background after viewing a series of short training videos. We have successfully cultured wild nematodes from these samples in the lab, and have established systems for sample intake, expansion and frozen stocking of the strains, performing co-culture experiments, and sharing experimental results with the original collectors. In the fall of 2021, we hope to expand this project by partnering with educators at a variety of levels on a larger scale who would be interested in incorporating nematode hunting into their science curriculum.

Yin, Da et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "GENOMIC AND FUNCTIONAL CHARACTERIZATION OF GENOME SHRINKAGE IN CAENORHABDITIS"

Meyer, Barbara J., Haag, Eric S., Schartner, Caitlin M., Ralston, Edward J., Korf, Ian, Thomas, Cristel G., Yin, Da, Schwarz, Erich M.

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Sexual mode evolves rapidly in some eukaryotic lineages. This is expected to have pronounced consequences for population genetics, sexual differentiation and the nature and intensity of sexual selection, all of which may be reflected in the genome. C. elegans is a self-fertile species, derived recently from an obligately outcrossing male-female ancestor.This trait has evolved in at least two other species of the Elegans sub-genus, C. briggsae and C. sp. 11 (Kiontke et al. 2011). Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors (Thomas et al. 2012). This reproducibility may be stimulated by an interaction between partial selfing and segregation distortion affecting large indels in male meiosis (Wang et al. 2010). However, the size, location, and gene content of specific deletions remain unknown for any natural system. To characterize the process of genome shrinkage, we have produced a genome assembly from the closest known outcrossing relative of C. briggsae, C. nigoni (formerly C.sp. 9; Felix et al. 2014). The C. nigoni genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. nigoni contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. nigoni physical map. Genome-wide sequence alignment showed the majority of the size reduction is located on the two arms of the five autosomes. Using C. sp.5 as an outgroup, we are able to identify gene family reductions, as well as specific genes recently lost in the C. briggsae lineage. Finally, we present detailed characterization of a family of rapidly evolving proteins that were independently lost in C. elegans and C. briggsae, the MSS (male-specific secreted) family, We have characterized their temporal and spatial expression, and find they are likely to be transferred to the female reproductive tract. We are now developing assays to reveal potential physiological responses to MSS proteins in females, including using calcium imaging to localize putative responder cells in female reproductive tract. References Felix, M.-A., C. Braendle, et al. (2014). "A streamlined system for species diagnosis in Caenorhabditis (Nematoda: Rhabditidae) with name designations for 15 distinct biological species." PLoS One 9: e94723.Kiontke, K., M.-A. Felix, et al. (2011). "A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits." BMC Evol Biol 11: 339.Thomas, C. G., R. Li, et al. (2012). "Simplification and desexualization of gene expression in self-fertile nematodes." Curr Biol 22: 2167-2172.Wang, J., P. J. Chen, et al. (2010). "Chromosome size differences may affect meiosis and genome size." Science 329: 293.

Croydon-Sugarman, Melanie et al. (2009) International Worm Meeting "Geographic and genetic variation in fecundity under temperature stress in C. briggsae."

Prasad, Anisha, Croydon-Sugarman, Melanie, Cutter, Asher D.

[International Worm Meeting, 2009]

In C. briggsae, patterns of genetic diversity among strains from across the globe correlate perfectly with the geographic origin of the natural isolates, corresponding to clades of worms from temperate regions, the tropical circles of latitude, and near the equator (Cutter et al. 2006; Dolgin et al. 2007). Ecologically, these geographic regions differ dramatically in temperature regime, begging the question of whether heritable phenotypic differences might also conform to the geographic partitioning of variation in a potentially adaptive manner. An association between the temperature at which a particular isolate is optimally fecund and the temperature of the isolate''s clade of origin could indicate local adaptation and provide insight into C. briggsae ecology and evolution. To address this issue, we tested the thermal tolerance, as quantified by self-fecundity, of 10 wild-isolate strains originating from the three latitudinal regions when the strains were subjected to extreme high and low temperatures. Our results demonstrate a decline to zero progeny production at 32 deg C that was exhibited by worms from all three regions, indicating an upper fertile limit between 30 deg C and 32 deg C for C. briggsae as a species. However, at 30 deg C we observed a significant 4-fold difference in lifetime fecundity for strains from the Tropic circles of latitude clade compared to those of both the temperate and equatorial clades, suggesting a tolerance of the tropical isolates to higher temperatures. Ongoing work explores fecundity at low temperatures (12 deg C - 16 deg C) to test for heritable differences among strains at cooler temperatures. Cutter, A.D., M.A. Felix, A. Barriere & D. Charlesworth. 2006. Patterns of nucleotide polymorphism distinguish temperate and tropical wild isolates of Caenorhabditis briggsae. Genetics. 173: 2021-2031. Dolgin, E.S., M.A. Felix & A.D. Cutter. 2008. Hakuna nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C. briggsae. Heredity. 100: 304-315.