We previously published methods to allow worm researchers to

(i) identify potentially useful Mos1 insertions from the NEMAGENETAG collection (MosLocator)

(ii) convert and/or update lists of genes (WormBase Converter; see Griffon and Ewbank, WBG 18 #4)

We’ve now added 2 more tools specifically designed for worm labs, with the aim of making life easier:

– ICeE, a worm-centered electronic notebook that facilitates storage of data and metadata. It comes pre-loaded with the CGC list of strains and the RNAi clones from the main collections.

– Clone Mapper, which does three things:
1. Map your clones: Automatically determines whether a clone taken from the Ahringer or Vidal RNAi library is what it is supposed to be, by mapping your clone sequences to an in-silico RNAi clone library.
2. Find Targets: Predicts gene targets for RNAi clones, on the basis of matching n-mer sequences, overcoming some of the limitations of current Wormbase predictions.
3. Retrieve Sequences: Allows single or multiple clone and transcript sequences to be retrieved.

Many people contributed to the elaboration of these tools, and we benefited from invaluable input from numerous members of the WormBase staff.

All these tools are freely available via our website. Feedback is welcome.

If you use any of them, please do cite the relevant publications:

MosLocator: Vallin et al., 2012 PMID: 22347378

Wormbase Converter: Engelmann et al.,  2011 PMID: 21602919

ICeE: Montanana et al., 2014 http://dx.doi.org/10.4161/worm.32160

Clone Mapper: Thakur et al., 2014 PMID: 25187039

Here, I present a simple script written in Perl for generating the necessary primers for use in GFP fusion experiments (Hobert, 2002; Reporter gene fusions) in C. elegans using multi sequence files. The program returns forward and reverse GFP fusion primers based on parameters determined by the user. These parameters include: length of search areas at the 5′ and 3′ ends of the input sequence; forward primer length, and reverse primer length (to be appended to the GFP specific oligo); and 3′ end GC clamp. The main feature of this script is that it works with files containing any number of FASTA formatted DNA sequences. The script uses the BioPerl SeqIO module (Stajich et al., 2002) to parse FASTA formatted sequences, and can easily be adapted to return primers from a large number of formats, including GenBank, EMBL, ABI, FASTQ, and KEGG (Stajich et al., 2002). The program returns primers with GC% between 40 and 60, and primer Tm values between 52°C and 68°C. The program selects against highly repetitive sequences composed of multiple tandem identical base pairs or multiple di-nucleotide repeats. The program also examines self-complementarity within each primer and enumerates a ‘selfie_score’ by generating reverse complement substrings of each primer and checking for pattern matches. Two ‘selfie_score’ values are returned for the fusion primer: the first one examines self-complementarity to the designed reverse primer only, and the second score checks complementarity between the designed portion and also the GFP specific portion.

The script is organized such that parameters for each primer (forward or reverse) can be easily modified (and improved) by the user independently, and contains comments describing each step. The program executes from the command line and the output provides tab separated features that prints to the screen and also writes to an output file: tab1: start position of primer; tab2: primer Tm; tab3: primer sequence; tab4: selfie_score {two scores for the fusion primer}; tab5: percent GC content.

This simple script might be of use to researchers working on multi-gene families, alternatively spliced transcripts, or really any type of large gene data set where GFP expression is desired. If only forward primers are desired the user can enter ‘zero’ for 3′ space to be sampled, and using such modifications to the code may provide further utility in traditional PCR experiments for large gene data sets. Hosted at SourceForge and freely available for download here – http://batchfusionprimerfetch.sourceforge.net/

Well, C. elegans is not really a meat eater, but if you read the recipe for Nematode Growth Medium (NGM) , you would be forgiven for thinking so. Bacto-peptone, contrary to what its name implies, is not a bacterial extract but is made from animal tissue. It is an enzymatic digest of beef and/or pork meat. Peptones were introduced into microbiology around 1880 by Naegeli and have become a standard ingredient for bacteriological culture media ever since.

It is not hard to imagine that bacto-peptone is not made out of prime steak or sirloin, but from meat that doesn’t sell. Because of the possible danger of bovine spongiform encephalopathy (BSE) coming from these tissues, BD Biosciences (a major bacto-peptone producer) began tracking the origin of their meat sources and requiring all bovine meat to come from BSE-free countries. Based on catalogue and lot numbers, a certificate of analysis can be requested that states the animal source and the different tissues that went into the production of your specific lot of bacto-peptone. One batch my lab used a couple of years ago had the lot-number 8052930 and was made out of bovine and porcine tissue from animals farmed in the US. It contained tissue categories I, II and IV which include: bone, bone marrow, mucosa, pancreas, brain, retina, pituitary gland, pineal gland, optic nerve, peripheral nerves, albumin, adipose tissue, cartilage, milk, prostate, ovary and plasma.

Incidentally, many of the tissues out of which this particular lot was made from, and probably most batches are made from, are tissues known to be involved in endocrine signaling. Not surprisingly, significant amounts of estrogen and estradiol have been found in peptone (Feldman and Krishnan, 1995) as well as at least 9 different bile acids (Kamekura et al., 1988), a class of acids to which dafachronic acids belongs (Motola et al., 2006).

How does this affect day-to-day C. elegans experiments? Using solvents in combination with NGM may have unintended consequences. The most widely used solvents, besides water, are ethanol and dimethyl sulfoxide (DMSO). DMSO is used in drug discovery and pharmacology because of its ability to pass through biological membranes and to carry other molecules with it. It is to be expected that DMSO will carry some of the constituents of bacto-peptone into C. elegans and potentially have some unintended consequences. If you use solvents like DMSO in your experiments in combination with NGM you should be at least aware of this possibility. Ideally, use a culture method that does not contain bacto-peptone. Otherwise, you may make your worms “eat some meat”, carnivorous indeed.

Talin localizes to cell adhesion sites, where it binds integrins, actin and vinculin (Petit and Thiery, 2000). In C. elegans, tln-1/talin is necessary for muscle cell attachment to the basal lamina via dense bodies (Lecroisey et al., 2007) and for gonad morphogenesis, which is driven by distal tip cell (DTC) migration (Wong and Schwarzbauer, 2012). In addition to the full-length transcript (tln-1a), two shorter splice isoforms (tln-1b, tln-1c) of the tln-1 transcript are predicted in C. elegans (Fig. 1A). Insights gained about specific tln-1 splice isoforms have implications for the functional profile of talin and roles of its various domains, which enhances our understanding of the mechanisms behind cell attachment and migration.

We confirmed tln-1b and tln-1c transcripts in N2 C. elegans cDNA through RT-PCR using primers targeted to their predicted unique exon sequences, indicating that they are transcribed (Fig. 1A). Based on conserved domain searches and sequence alignments, we identified domains encoded by the predicted amino acid sequences of tln-1b and tln-1c. TLN-1b contains the integrin-binding head domain and at least one vinculin binding site in the rod, while TLN-1c contains multiple actin and vinculin binding sites in the rod (Fig. 1B).

Knockdown of tln-1a using an RNAi construct that also targets tln-1c causes paralysis and DTC migration defects (Cram et al., 2003). To determine the individual roles of tln-1b and tln-1c, we performed RNAi feeding experiments using novel RNAi constructs that specifically targeted either tln-1b or tln-1c (Fig. 1A). tln-1b RNAi caused progressive total body paralysis in rrf-3(pk1426) nematodes: 97% of late-L4/adult animals were paralyzed by 55 hours of RNAi treatment (n=178). We next examined the localization of PAT-2/a-integrin using the JE2222 strain that carries the PAT-2::GFP transgene (Meighan and Schwarzbauer, 2007). Following tln-1b RNAi and paralysis, 66% of nematodes displayed aberrant muscle cell shape and PAT-2 localization (n=44), while 86% of nematodes showed disrupted actin filamentation (n=49). This suggests that tln-1b is important for muscle cell shape, PAT-2 localization, and cytoskeletal attachments. RNAi against tln-1c did not cause paralysis or muscle morphology defects.

Although loss of either tln-1b or tln-1c did not affect gonad morphology, double RNAi knockdown of tln-1b and ina-1/a-integrin produced a significantly greater proportion of defective gonad arms (p<0.05), including defects in migratory direction and early cessation, when compared to ina-1 reduction (Fig. 1C). The TLN-1b protein may interact with the INA-1 receptor during DTC migration. No synergistic effects were observed upon tln-1c + ina-1 double RNAi knockdown.

Our analyses indicate that tln-1b is important for muscle function and DTC migration, while specific functions of tln-1c remain to be elucidated. These findings suggest that multiple isoforms perform the biological roles of C. elegans talin and that the talin head domain is especially important for these processes. We propose that the structural simplicity of TLN-1b and its potential for strong integrin binding may be necessary for integrin clustering during adhesion formation, displacement of TLN-1a during adhesion disassembly, or integrin activation during dense body protein accretion, thus making it relevant for cell migration and muscle attachment.

The ability to sense and react to noxious high environmental temperature is critical for animals’ survival. C. elegans uses distinct escape and avoidance regimes to minimize exposure to noxious heat. It has been shown that C. elegans heat avoidance requires several polymodal nociceptors, such as the FLP neurons, ASH neurons and others, but many of the key molecules involved in C. elegans thermosensation must be identified (Liu et al., 2012). Moreover the neural circuit mechanisms by which thermosensory behavior is generated remain incompletely understood especially with respect to heat avoidance (Liu et al., 2012). Therefore we proposed that C. elegans behavior response to noxious heat includes adaptive changes in the activity of cholinergic synapses in neural circuit regulating worm’s movement (Jospin et al., 2009).

To test this hypothesis we used pharmacological analysis of noxious heat effect on steady state of cholinergic synapses of C. elegans in conditions of moderate rise in temperature tolerated by worms. Pharmacological analysis of steady state of C. elegans cholinergic synapses in vivo usually consists in measuring of locomotion sensitivity to ACh-esterase inhibitor aldicarb and nAChRs’ agonists levamisole and nicotine. In both cases enormous rise in ACh content caused by aldicarb or overactivation of nAChRs by their agonists induced locomotion disturbances. In numerous genetic investigations this analysis was used to identify molecular mechanisms of neuromuscular synapses’ function. In all these investigations aldicarb or levamisole sensitivity was measured by registration of worms’ full inability to move on agar plate after slight touch by needle (“paralysis”). In contrast, in our experiments performed in liquid medium (NG buffer) we registered swimming disturbances, such as failure of muscle contraction in undulatory pattern or inability to forward swimming during 10 seconds, induced by aldicarb, levamisole and nicotine in concentrations which are subthreshold for nematode paralysis. Aldicarb concentration effective to induce behavior disturbances in 50% of worms after 20-minutes exposure at 23°C was 32 µM, while such for 20-minutes exposure at 33°C was 15 µM.

C. elegans exposition to temperature 33°C didn’t cause disturbances in worms’ swimming induced by mechanical stimulus, but induced rapid (less than 30 minutes) rise in this behavior sensitivity to partial inhibition of ACh-esterase by aldicarb. These data indicate that noxious heat caused a rise of steady state cholinergic synaptic transmission in cholinergic synapses, involved in regulation of C. elegans swimming.

Temperature rise up to 33°C strongly elevated swimming sensitivity not only to aldicarb, but also to nAChRs’ agonists levamisole and nicotine. Concentrations of levamisole and nicotine effective to induce behavior disturbances in 50% of worms after 20-minutes exposure were respectively 62 µM and 710 µM at 23°C and respectively 15 µM and 320 µM at 33°C. Therefore it is evident that moderate heat stress activates steady state of synaptic transmission by sensitization of nAChRs, but not by stimulation of ACh release from neurons. Sensitization of nAChRs by noxious heat can be involved in rise of running away speed caused by heat stress.