The WBG

An online publication service of WormBook

Dorsal Dauers and Directionally Dysfunctional dex-1 Dumpies

Caroline D. Beshers1,2, Kristen M. Flatt1,3 and Nathan E. Schroeder1,3
1 Crop Sciences Department, University of Illinois Urbana-Champaign
2 Neuroscience Department, Oberlin College, Ohio
3 Neuroscience Program, University of Illinois Urbana-Champaign
Correspondence to: Caroline Beshers ([email protected])
The dauer life stage in wild-type C. elegans exhibits morphological and behavioral characteristics that differentiate it from non-dauers (Hu, 2007). In addition to the commonly observed radial shrinkage, quiescence and lack of pharyngeal pumping, we noted a tendency of wild-type dauers to lie dorsoventrally when mounted on agarose slides, as opposed to laterally like adults. To quantify this observation, we mounted wild-type dauers and L3s on increasing concentrations of agarose with 0.1 M levamisole. We found that 75-90% of wild-type dauers mounted with levamisole lie dorsoventrally regardless of agarose concentration (Fig. 1). Very few (2.5-5%) non-dauer animals were positioned dorsoventrally. There was no significant difference in positioning between dauers that still retained their L2d cuticle and those that were unsheathed.  We then utilized a non-anesthetic method of immobilization by mounting dauers on 10% agarose with 10 micron polystyrene microbeads (Kim et al., 2013). Interestingly, only 15% of wild type dauers lie dorsoventrally when mounted using the microbeads. We hypothesized that the radial shrinkage and the presence of lateral alae in wild-type dauer cuticles may cause anesthetized dauers to roll to a dorsoventral position, but that “conscious” dauers immobilized with microbeads would remain lateral, despite the presence of alae. To test this, we examined dex-1 mutant dauers. We recently found that dex-1(ns42) dauers are defective for dauer alae formation and radial constriction leading to a dumpy dauer phenotype. We found that dex-1 dauers are significantly more likely to lay laterally than wild-type dauers (p=0.0013) (Fig. 1) suggesting that dauer alae or overall body dimensions regulate the anesthetized “sleeping” position.

Figures
Figure 1: This chart illustrates the difference in percentage of N2 dauers, N2 L3s and dex-1 dauers lying dorsoventrally across slide methods with increasing concentrations of agarose using levamisole (lev) and 10% agarose using polystyrene microbeads (beads). The N2 dauers consistently (75-90%) lay dorsoventrally, while 2-5% of non dauer animals lay dorsoventrally. N2 dauers lay dorsoventrally significantly more than dex-1 dauers (p=.0013).


Figure 2:

References

Hu, P.J. Dauer (August 08, 2007), WormBook, ed. The C. elegans Research Community, WormBook  PubMed

Kim E, Sun L, Gabel CV, Fang-Yen C (2013) Long-Term Imaging of Caenorhabditis elegans Using Nanoparticle-Mediated Immobilization. PLoS ONE 8(1): e53419.   PubMed

CelegansChrome: a Google Chrome Browser Extension for C. elegans Research

Damien M. O’Halloran1,2
1Department of Biological Sciences, George Washington University
2Institute for Neuroscience, George Washington University
Correspondence to: Damien M. O’Halloran ([email protected])

Browser extensions are small software programs that enhance the user’s online experience by creating greater browser functionality. Here, I present a Caenorhabditis elegans specific extension for Google Chrome browsers. The software is built from four files: 1) an HTML file called celegansChrome.html that generates the user interface; 2) a JavaScript file called celegansChrome.js that contains objects of C. elegans gene data; 3) a JSON file that contains a manifest of the program contents called manifest.json; and 4) a small image file of a C. elegans hermaphrodite called icon.png that gets loaded into the browser toolbar. The software is freely available at the Google Chrome Web Store by simply clicking the add to Chrome link to load the extension into the browser:

chrome.google.com/webstore/detail/celeganschrome/ipnlfcanpnhkhljmminidjndijjmheib

The user can enter a gene common name (e.g., egl-4) and the Chrome extension can provide the user with C. elegans gene data from any website or even while off-line. Often, it’s convenient to retrieve linkage data or interactome data for a specific C. elegans gene while browsing different webpages or reading journal articles online – this extension provides a frictionless interface for the user to retrieve these data without having to open a new tab and search another website or paper for these data. Currently, the extension provides the user with linkage data for C. elegans genes, gene interaction data, human othology data to a C. elegans gene, and also a gene overview using the WormBase (Harris et al., 2014) RESTful API (only this feature requires internet connectivity). The linkage data loaded into the C. elegans Chrome extension was mined from WormMart WS220 (www.wormbase.org/), while the human ortholog data is from the InParanoid program database (Sonnhammer and Ostlund. 2015). The interaction data were downloaded from the WormBase version WS237 ftp site, and the overview of gene data relies on the WormBase RESTful API. The WormBase RESTful API provides access to gene data by creating a URI using standard HTTP requests that is unique to each gene – the unique identifier being a WBID. The C. elegans Chrome extension takes as input the C. elegans gene common name and maps it to the corresponding WBID to generate the unique URI.

This simple browser extension operates as a client-side program (with the exception of the overview option), and thus provides very fast and seamless data retrieval for C. elegans gene data while browsing any site online.

References

Harris TW, Baran J, Bieri T, Cabunoc A, Chan J, et al., (2014). WormBase 2014: New views of curated biology. Nucleic Acids Res. 42: D789-793.  PubMed

Sonnhammer EL, and Ostlund G, (2015). InParanoid 8: Orthology analysis between 273 proteomes, mostly eukaryotic. Nucleic Acids Res. 43: D234-239.  PubMed

New lab announcement: Kevin Collins

Kevin Collins
1Department of Biology University of Miami, Coral Gables FL
Correspondence to: Kevin Collins ([email protected])

I started my independent research group from August 2014 in the Department of Biology at the University of Miami in Coral Gables, Florida. Our fundamental goal is to understand how neurons communicate in circuits to establish an appropriate level of activity that produces a robust, stable behavior. Our approach is to analyze in detail a model neural circuit that controls egg-laying behavior in C. elegans. We are taking advantage of the optical clarity and powerful genetics in this experimental system to literally watch the activity of every cell in the circuit in behaving animals using fluorescent Ca2+ reporters, and also to manipulate their activity using optogenetic tools. Using mutations and transgenes to discover and alter molecular signaling events between cells, we are determining how the complex pattern of activity in a circuit creates a coherent, regulated behavior. We expect these studies will reveal general principles of neurotransmitter signaling and neural circuit function with applications to understanding the human nervous system and its dysfunction in disease.

Website:  http://www.as.miami.edu/biology/people/faculty/kevin-collins/

Ph.D. student and Postdoc positions are currently available.  To apply, please send a CV and description of research interests to [email protected]

Nematicidal activity of Fungal sp. US14 and Aspergillus terreus extracts on C. elegans

Piyush Prati1, Nidhi Rastogi1, Supra Sharma1, Jitendra Kumar2 and Brenda Prasad1
1Microbial & Molecular Genetics Lab., Department of Botany/Biotechnology, Patna University, Patna-800 005, India, 2Buck Institute for Research on Aging, Novato CA

Correspondence to: Birendra Prasad ([email protected])

Many species of nematodes are known that are pathogenic as well as toxigenic for plants and many of them live as a parasite on the host plant causing severe crop damage and economic loss (Burglin et al., 1998). Although chemicals are available to control the harmful nematodes, they have their own set of deleterious effects such as decrease in soil fertility, impact on non target beneficial fauna, decrease in crop yield, and toxic effects on farmers. As fungi are known to have nematocidal activity (Khan et al., 2003), they may be used as a potential biological control agent.

In order to examine the effect of extra-cellular fungal extract as a nematicidal agent, C. elegans was used as a model system. In this study, we choose two Indian fungal isolates: (1) Aspergillus terreus, and (2) Fungal sp. US14, for its nematicidal potential on C. elegans. Fungal sp. US14 showed only 96% sequence similarity with Purpureocillium lilacinum with regards to its internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS2) sequences. So, it might be a new species and was deposited at NCBI in the name of Fungal sp. US14 (NCBI accession number-JN802258.1).

We carried out mortality assays on C. elegans using seven-day old grown-culture supernatant (extra-cellular culture filtrate) of both fungi. Synchronised L1 larvae of wild-type C. elegans were transferred with and without fungal supernatants,  followed by incubation at 15oC. Mortality % of larvae was determined up to 48 hours by counting the dead larvae in the presence of fungal supernatant as compared to control animals which were grown in M9 buffer. We counted the number of dead larvae at 24 hours and 48 hours. The fungal supernatant of US14 killed 29±10.5% larvae at 24 hours and 93±1.9% at 48 hours (p<0.0001) compared to control larvae. The fungal supernatant of Aspergillus terreus did not kill larvae (Figure 1), and was almost similar to the control assay, suggesting its nonpathogenecity towards C. elegans.

Some of the study shows that the nematicidal activity of the fungus may be attributed to the presence of chitinase, protease, and lipases in the culture supernatant. The chitinase activity destroys the cuticle of nematodes and causes mortality (Miller and Sands, 1977). Furthermore, chitinase activity of nematophagous fungi has been previously correlated with their pathogenicity to some plant-parasitic nematodes (Stirling and Mankau, 1979). All together, the Fungal sp. US14 appeared as a promising candidate for biological control of nematodes.

Figures
Figure 1: Effect of culture supernatant of Fungal sp. US14 and Aspergillus terreus on C. elegans. L1 larvae were incubated with both fungal supernatants and M9 buffer separately. For motility assay, dead larvae were counted at an interval of 24 hours and 48 hours. At 48 hours, the supernatant of fungal sp. US14 showed 93±1.9% mortality in comparison to control and fungal supernatant of Aspergillus terreus (p<0.0001).

References

Bürglin TR, Lobos E, and Blaxter ML. (1998). Caenorhabditis elegans as a model for parasitic nematodes. Int. J. Parasitol. 28, 395-411. PubMed

Khan A, Williams K, and Nevalainen H. (2003). Testing the nematophagous biological control strain Paecilomyces lilacinus 251 for paecilotoxin production. FEMS Microbiol. Lett. 227, 107-111. PubMed

Miller, PM and Sands, DC. (1977). Effects of hydrolytic enzymes on plant-parasitic nematodes. J. Nematol. 9, 192-197. PubMed

Stirling, GR and Mankau, R. (1979). Mode of parasitism of Meloidogyne and other nematode eggs by Dactylella oviparasitica. J. Nematol. 11, 282-288. PubMed

Live worm respiration in 24-well format

Beverley M. Dancy1, Ernst-Bernhard Kayser1, Margaret M. Sedensky1 and Philip G. Morgan1
1Center for Developmental Therapeutics, Seattle Children’s Research Institute, Seattle WA

Correspondence to: Beverley M. Dancy ([email protected])

This is a protocol for the determination of oxygen consumption rate (OCR) in living C. elegans using the Seahorse apparatus with 24-well plates. OCR provides a measurement of respiration, which can be altered by genetic or pharmacologic manipulation of the mitochondrial respiratory chain. Although the Seahorse was originally designed for use with ex vivo cells, it can readily be used in vivo with living C. elegans by following our simple procedure,2 described here for the analysis of four samples side-by-side.

Synchronize each of the four samples of worms. We allow 200 adults to lay eggs for 4 h on a 10 cm plate by picking 200 adults to a 10 cm plate, kill the adults, then study the F1s as day 1 adults. Program the Seahorse Bioscience XF24 Extracellular Flux Analyzer with the following protocol: 1- calibrate probes; 2- loop 10 times; 3- mix 2 min; 4- time delay 2 min; 5- measure 2 min; 6- loop end. Furthermore, the internal heater should be switched off to allow the instrument to be close to room temperature. Hydrate the probes in the extracellular flux assay kit by adding 1 ml of Seahorse calibrant solution to each well and incubating overnight at 37 oC . Load this into the Seahorse on the day of the experiment for calibration.

Rinse the worms off the plate and wash three times using gravity separation in M9 and resuspend each washed sample in 2 ml. Get a rough estimate of the concentration of worms by pipetting 5 drops of 20 µl each per sample onto an empty dish and counting under the dissecting scope. There should be about 2 worms per drop. In the Seahorse assay plate, place 500 µl M9 into four blank wells. At least 30 min after the worms were first rinsed, pipet the four worm samples into a total of 500 ul M9 in each of the other wells with between 25 and 150 worms per well and five replicates of each sample. We use a low retention tip cut to create a wider opening, and found that picking worms into the wells with a platinum wire leads to unstable results.

When prompted by the instrument, replace the calibration plate with the worm plate. At the end of the run, count the actual number of worms per well; for this purpose, we capture images on a camera-fitted dissecting scope. Average the background-corrected OCR (pMol O2/min) across the 10 time-points for each well, then normalize the OCR to the actual number of worms in each well. Calculate the average and standard deviation of OCR per worm across the five replicates for each sample. A moderate amount of variation is expected between time-points, wells, plates, and days: we therefore use a minimum of 10 time points of 5 wells, and on at least 2 plates and/or days, to get a meaningful result.

1 For a more detailed protocol with additional tips, please contact Beverley at [email protected].

2 We acknowledge other labs using Seahorse for C. elegans who provided some tips when we began this work, including Laurent Mouchiroud in Johan Auwerx’s lab at Ecole Polytechnique Fédérale de Lausanne, David Raizen at University of Pennsylvania, and Paul Brookes at University of Rochester Medical Center.

3 We thank José Antonio Mari ([email protected]) for putting together the graphics in Figure 1.

Figures
Figure 1: Overview of the Seahorse workflow. The four worm samples (e.g., different mutants, drug treatments, food sources) are represented by navy, yellow, green, and pink colors. The blank (no worm) wells are clear.3
Figure 2: OCR is linearly related to the number of worms per well. In this experiment N2 were synchronized by picking the parents, grown at 20 oC on NGM+OP50, assayed at day 1 of adulthood, and measured in 16 wells of one Seahorse plate in M9 at 22 oC. The average oxygen consumption rate was 13.8 ± 2.5 picomoles per minute per worm. The average across 57 wells (on 6 Seahorse plates, on 5 days) was 10.2 ± 4.6.