The WBG

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M9 and nlp-29: standard buffer can induce antimicrobial peptide gene expression

Nathalie Pujol1 and Jonathan Embank1
1Centre d’Immunologie de Marseille-Luminy, INSERM U1104, Marseille, France

Correspondence to: Jonathan Ewbank (ewbank@ciml.univ-mrs.fr)

The production of antimicrobial peptides (AMPs) is arguably the most conserved aspect of innate immunity in all organisms. For the last few years, much of our research has been focused on understanding the regulation of the related nlp and cnc AMP gene families in C. elegans. This has turned out to be surprisingly complex. They generally have very dynamic developmental expression profiles. This may be one reason why AMP genes frequently appear in lists of differentially regulated genes when two strains of worms are compared: even minor difference in the speed of development are sufficient to result in marked differences in AMP gene expression when samples are harvested at the same time rather than at a precise developmental stage.

Various stimuli affect the expression of different AMP genes. We’ve worked most with nlp-29, often relying on a standard reporter transgene, with GFP under the control of the nlp-29 promoter. Like the other members of the family, nlp-29 is expressed in hyp7. In young adults, nlp-29 expression is induced by infection with the fungus Drechmeria coniospora and by physical injury (Pujol et al., 2008). It is also induced by osmotic shock (Pujol et al., 2008). On the other hand, many stimuli that trigger the expression of stress-response genes, including starvation, heat-shock, heavy metals, or drugs like tunicamycin that induce an unfolded protein response do not up-regulate nlp-29 expression in young adults (Couillault et al., 2012). It is also, and this is a point we have tried to rectify in the literature (Wong et al., 2007), not induced by intestinal bacterial pathogens such as Serratia marcescens.

We have found that incubation of worms in M9 can also induce the expression of the nlp-29 reporter gene. After as little as 4 hours, GFP levels are increased by 2-fold, and leaving worms in M9 (with OP50) for 24 hours can provoke an increase of up to 20-fold (Figure 1). We carried out tests to see whether this increase was attributable to a single component of M9. Preliminary assays suggest that this isn’t the case (Figure 2), but we haven’t pursued our investigation further. Nor have we measured the expression level of other AMPs, but note that several of the genes in the nlp cluster appear to be controlled in the same way as nlp-29 (Pujol et al., 2008).

Our results do, however, suggest that as far as AMP gene expression is concerned, caution is needed when interpreting the results of any studies that involve incubation of worms in M9 for prolonged periods. We’ve reviewed our previously published results and do not believe it has been a problem. Nevertheless, we now routinely replace M9 with 50 mM NaCl.

Figures



Figure 1: Red and green fluorescence visualized simultaneously in transgenic worms (wt;frIs7) carrying pnlp-29::GFP and pcol-12::dsRed reporter constructs after incubation in water (left) and M9 (right) for 24 hr.


Figure 2: Ratio of green to red fluorescence of wt;frIs7 worms incubated in water, 50 mM NaCl or solutions containing the different components of M9 buffer.

A new Daf (DAuer Flatulence) phenotype?

Nathan E. Schroeder1 and Maureen M. Barr1
1Department of Genetics, Rutgers, The State University of New Jersey, Piscataway NJ

Correspondence to: Maureen M. Barr (barr@biology.rutgers.edu)

While the anatomy of the adult hermaphrodite has been carefully dissected, there are numerous gaps in our knowledge of dauer anatomy. Dauers are known to be relatively resistant to environmental insults compared with non-dauer stages. This is attributed to both changes in cuticle structure and occlusion of the stoma during dauer (Cassada and Russell, 1975). The analogous infective stages of certain parasitic nematode species form an anal plug, potentially sealing the alimentary canal at the posterior end (Vincent et al., 1979). It has been suggested that C. elegans dauers may produce a similar structure (Wolkow and Hall, 2011).

Here we present evidence demonstrating that that the anus is not occluded during the dauer stage of C. elegans. During the course of microscopic examination with DIC Nomarski optics, we regularly observe the production of bubbles emanating from the anuses of wild-type C. elegans dauers (Movie 1). Bubbles are frequently seen outside the anus immediately following examination of a newly mounted dauer. We typically mount dauers on 2-5% agarose in M9 buffer amended with 0.1 M levamisole. To test whether expulsion was a result of levamisole exposure, we also mounted dauers on 10% agarose with microsphere beads with no anesthetic (See Fang-Yen et al., WBG 18:1) and found that 25/39 dauers produced bubbles.

This finding poses several questions:

1) Does this action serve a homeostatic function similar to that seen with the pumping of the excretory pore during dauer (Nelson and Riddle, 1984)? Our preliminary data suggests no difference in expulsion following exposure to a series of M9 dilutions.

2) What is the chemical nature of the bubbles? Our tongue-in-cheek title suggests a gaseous phase. Indeed, the bubbles are less dense than the surrounding media. Dauers mounted ventral-side down will produce bubbles that rise to the dorsal surface. However, this difference in density and the relative stability of the bubbles could also suggest a lipid based excretion.

3) Are there neuronal changes in dauers which control this expulsion? While changes to the anterior nervous system are known to occur in dauer (Albert and Riddle, 1983), there are likely additional changes which mediate other dauer-specific processes.

Figures


Figure 1: Ventral view of wild-type dauer repeatedly expelling bubbles from its anus. Numerous bubbles have already accumulated around the posterior end of the animal.
Click thumbnail video to play movie.

References

Albert PS, and Riddle DL. (1983). Developmental alterations in sensory neuroanatomy of the Caenorhabditis elegans dauer larva. J. Comp. Neurol. 219, 461-481. PubMed

Cassada RC, and Russell RL. (1975). The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev. Biol. 46, 326-342. PubMed

Nelson FK, and Riddle DL. (1984). Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery. J. Exp. Zool. 231, 45-56. PubMed

Vincent AL, Frommes SP, and Ash LR. (1979). Ultrastructure of the rectum of infective-stage Wuchereria bancrofti (Nematoda: Filarioidea). J. Parasitol. 65, 246-252. PubMed

Wolkow C, and Hall DH. (2011). The Dauer Cuticle. In WormAtlas. PubMed

Cloning of che-6 and che-7

Heidi K. Smith1 and Oliver Hobert1
1Department of Biochemistry & Molecular Biophysics, Columbia University Medical Center, HHMI, New York NY

Correspondence to: Oliver Hobert (or38@columbia.edu)

Our lab is interested in elucidating signal transduction mechanisms in the ASE salt receptor neurons (Ortiz et al., 2009). 35 years ago, Lewis and Hodgkin described single alleles of two chemosensory mutants, che-6 and che-7 (Lewis and Hodgkin, 1977). We analyzed these mutants using current standard chemotaxis protocols and found that both mutants have a similar, though not identical, spectrum of chemotaxis defects.

che-6(e1126) animals have intermediate defects in chemotaxis to NaCl, which is sensed by both ASEL and ASER, but are strongly defective in chemotaxis to individual ions that are sensed by either ASEL or ASER, i.e. sodium (ASEL) and chloride (ASER). che-7(e1128) animals are also defective in odortaxis to diacetyl but not benzaldehyd.

Similarly, che-7(e1128) animals have intermediate defects in NaCl chemotaxis, but are strongly defective in chemotaxis to sodium, chloride and lithium. che-7(e1128) animals are defective in odortaxis to both benzaldehyd and diacetyl.

che-6 had previously been linked to LGIV and che-7 to LGVII. We did whole genome sequencing of the che-6 and che-7 mutant strains. We found a missense mutation in the cyclic nucleotide-gated ion channel cng-4/C23H5.7 in che-6(e1126) mutant animals and a missense mutation in the innexin gene inx-4 in che-7(e1128) mutant animals. In both cases, the mutant phenotype can be rescued with genomic DNA pieces that encompass the respective genomic loci. Moreover, independently isolated knockout alleles (ok2373 for inx-4/che-7 and tm5036 for cng-4/che-6) showed a similar spectrum of chemotactic defects.

While we currently have no specific hypothesis in regard to che-7 function, we speculate that che-6 forms a heteromeric CNG channel complex together with tax-2 and tax-4 in the ASE neurons. This is based on the notion that CNG channels are generally thought to be tetramers composed of three different subunits, two beta subunits and two different alpha subunits (Zheng and Zagotta, 2004). In the case of the ASE neurons, we speculate the CNG complex to contain two TAX-2 beta subunits and the alpha subunits TAX-4 and CHE-6.

References

Ortiz CO, Faumont S, Takayama J, Ahmed HK, Goldsmith AD, Pocock R, McCormick KE, Kunimoto, H, Iino Y, Lockery S and Hobert, O. (2009). Lateralized gustatory behavior of C.elegans is controlled by specific receptor-type guanylyl cyclases. Curr. Biol. 19, 996-1004.  PubMed

Lewis JA, and Hodgkin JA. (1977). Specific neuroanatomical changes in chemosensory mutants of the nematode Caenorhabditis elegans. J. Comp. Neurol. 172, 489-510.  PubMed

Zheng J, and Zagotta WN. (2004). Stoichiometry and assembly of olfactory cyclic nucleotide-gated channels. Neuron 42, 411-21.  PubMed

Gnotobiological analysis of entomopathogenic nematode/bacterium symbiotic associations on ENGM media leading to some co-evolutionary conclusions

András Fodor1, Byron Adams2, Erzsébet Böszörményi3, Richard Ffrench-Constant4, Éva Lehoczky1, Judit Marokházi5 and István Venekei5
1Institute of Plant Protection, Georgikon Faculty, University of Pannonia, Keszthely, Hungary, 2Department of Biology, Brigham Young University, Provo UT, 3National Public Health and Medical Officer Service of Hungary, Ráckeve, Hungary, 4Biosciences, University of Exeter, Cornwall Campus, Penryn UK, 5Department of Biochemistry, Faculty of Natural Sciences, Eötvös Loránd University, Budapest, Hungary

Correspondence to: András Fodor (fodorandras@yahoo.com)

We have recently established an agar based media (ENGM) amenable to use in genetic studies of entomopathogenic nematodes (Fodor et al., WBG 18:2). Herein we present some results confirming the practical usefulness of this media. Three recognized Photorhabdus species (P. asymbiotica, P. temperata and P. luminescens) include subspecies, which, apart from P. asymbiotica ssp. asymbiotica are obligate endosymbionts of different heterorhabditid nematodes, (Peat et al., 2010). We used six different enzymes (AluI, TaqI, MseI, HinfI, RsaI, and DdeI) to establish the PhastSystem PAGE RFLP patterns (as phenotypes) of the intergenic spacer sequences (IGS) within the 16S – 23S rRNA operon of the natural EPB symbionts (Pamjav et al., 1999). Gnotobiological analysis (culturing nematodes on each others’ symbionts and evaluating the results) were carried out on ENGM to delineate partner specificities and host ranges. The study covers majority but not all of the known respective symbiotic complexes. We described Symbiotic Association (SA) Groups within which the symbiotic partners could not (NESA-G) or could (RESA-G1 & 2) reciprocally be exchanged. In one SA group (RESA-G3) the option for symbiotic partner exchange is occasional. Both partners of NESA-G1 are monophyletic (H. indica and P. luminescens ssp. akhurstii strains). All EPN partners in RESA-G1 (H. bacteriophora) and in RESA-G2 (H. megidis North Western European strains) are monophyletic, but the EPB partners are polyphyletic. In RESA-G3 both the EPN (H. megidis, H. marelatus some H. bacteriophora strains) and the EPB partners are polyphyletic. The data of exhibiting gnotobiological promiscuity related to exceptional symbiont exchanges between H. marelata, H. megidis, H. bacteriophora and H. downesii strains suggest that alternative co-evolutionary pathways might be initiated by fixation of a novel natural Heterorhabditis/Photorhabdus symbiotic association. Once an EPN/EPB SA established the evolutionary fate of the bacterial, but the nematode partner became unambiguously determined and canalized. We concluded that the Heterorhabditis/Photorhabdus co-evolution is a “one-way street”.

Figures



Figure 1: Phylogenetic relations between Photorhabdus strains that could reciprocally be exchanged between a given group of Heterorhabditis nematodes forming non-overlapping symbiotic units (Symbions).

References

Pamjav H, Triga D, Buzás Z, Vellai T, Lucskai A, Adams B, Reid AP, Burnell A, Griffin C, Glazer I, Klein MG, and Fodor A. (1999). Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism of internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes. Electrophoresis 20, 1266-1273. PubMed

Peat SM, Ffrench-Constant RH, Waterfield NR, Marokházi J, Fodor A, and Adams, BJ. (2010). A robust phylogenetic framework for the bacterial genus Photorhabdus and its use in studying the evolution and maintenance of bioluminescence: a case for 16S, gyrB, and glnA. Mol. Phylogenet. Evol. 57, 728–740. PubMed

Entomopathogenic nematodes: from sequence to biology is a one-way street?

András Fodor
1Department of Animal Sciences, Georgikon Faculty, University of Pannonia, Keszthely, Hungary

Correspondence to: András Fodor (fodorandras@yahoo.com)

The establishing of the complete genome sequence of the first animal, C. elegans had been preceded by a two-decade long thoughtful phenotypic and genetic analysis of the organism. As a consequence, from the sequence information we could go back to the biology, and vice versa. We believed that the genomic information from the model organism C. elegans would ease to explore the biology of other nematodes, like the entomopathogenic Heterorhabditis and Steinernema species. It was hoped that if we determine the genomic sequences of these nematodes and compare them scientifically with that of C. elegans it will help to explore the differences and similarities at the level of the organisms. Although the genome of H. bacteriophora has been sequenced by a large consortium and was compared professionally to that of C. elegans, it may not replace the thoughtful genetic analysis of that species. There are some conclusions that could not be drawn simply from the sequence information without a functional aspect. Let me provide two examples of unsolved problems which might not been over-bridged by the comparative sequence analysis.

  1. Thank for the thoughtful genetic analysis of the Riddle laboratory, the genetic pathway of dauer-larva formation and recovery in C. elegans is well known. The dauer stage is of a vital importance for entomopathogenic nematodes. Surprisingly enough, there are hardly any genes related to C. elegans daf (C) and daf (def) genes were found in H. bacteriophora. Despite the sequence information, the genetic analysis of the dauerlarva formation and recovery in H. bacteriophora cannot be avoided.
  1. Thank for the pioneer work followed by a thoughtful genetic analysis at the highest intellectual level of the forth member of the three-member Nobel- laureate team, Jonathan Hodgkin, the sex determination and dosage compensation in C. elegans is completely known. Disappointingly, hardly any of the genes of playing key role in these processes could be found in the H. bacteriophora genome.

Both examples indicate that similar biological problems could be solved by different genes in closely related species. The classical genetic analysis of biological functions cannot exclusively be replaced by the sequence analysis. In the absence of the proper classical genetic information the road from sequence to biology is only a one-way street with a dead end.

References

Adhikari BN, Lin CY, Bai X, Ciche TA, Grewal PS, Dillman AR, Chaston JM, Shapiro-Ilan DI, Bilgrami AL, Gaugler R, Sternberg PW, and Adams BJ. (2009). Transcriptional profiling of trait deterioration in the insect pathogenic nematode Heterorhabditis bacteriophora. BMC Genomics. 10:609.   PubMed

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