Beets, Isabel, Dalzell, Johnathan, Lee, Junho, Yang, Heeseung, Van Bael, Sven, Vandewyer, Elke, Kieswetter, Amanda, Boelen, Rose, Cockx, Bram, Temmerman, Liesbet
[
International Worm Meeting,
2021]
Emerging evidence suggests that behavioral changes associated with host finding of pathogenic nematodes may be regulated by neuropeptides. Hence, we started from peptidomic discovery in such a species, Steinernema carpocapsae, to direct functional discovery. We observe numerous similarities between the well-studied C. elegans peptidome and the S. carpocapsae peptidome, which we further exploit to understand neuropeptidergic contributions to regulating nictation, an evolutionary conserved behavior for foraging in (these) nematodes. An in-house method based on acidified methanol was used to extract endogenous neuropeptides of Steinernema carpocapsae infective juveniles, a life stage similar to the C. elegans dauer stage. Neuropeptide identification was done by state-of-the-art UHPLC-MS/MS. We detected 30% (139) of the predicted peptidome in these infective juveniles, which provides a resource for comparison with the C. elegans dauer peptidome (in house). We hypothesized that nictation-relevant peptides will be abundant in infective juveniles and/or dauers, and prioritized these for functional assays. Out of several tested target genes, we found at least one neuropeptidergic signaling system that is involved in modulation of nictation behavior, which we assayed using microdirt arenas. In addition, using phylogenetic analyses, we aim to get a better global understanding of conserved peptidergic signaling systems in parasitic and free living nematodes. Steinernema spp. are used as eco-friendly alternative for chemicals to combat pest insects. Knowledge on neuropeptidergic regulation of host-finding strategies will help understand how entomopathogenic nematodes regulate their behavior. This should contribute to improving their applicability and host specificity in the field.
[
International C. elegans Meeting,
1999]
We would like to reconstruct the history of the C. elegans species at the genome level, therefore we sampled the genomes of four natural isolates (strain CB4857 isolated in Claremont, California, RC301 from Freiburg, Germany, TR403 from Madison, Wisconsin and AB1 from Adelaide, Australia) for single nucleotide polymorphisms (SNPs). Random genomic DNA fragments from the 4 strains were shotgun cloned and sequenced. There was no selection for transcribed or non-transcribed regions of the genome. In total we sequenced 1572 clones resulting in over 1 Mb of sequence information. The sequences are compared to the canonical Bristol N2 sequence to ask the question whether the clone maps to a unique sequence, and -if so- whether it contains polymorphisms. Once a SNP is identified we check other strains for the presence of the same polymorphism by PCR amplification and sequence analysis. In an initial experiment we found approximately one SNP per 3000 bp sequenced. The SNPs are randomly spread over the genome. Based on these observations we expect to find approximately 500 SNPs, one in every 200 kb. In the initial experiment we found, as expected, that several SNPs initially detected in one strain were also present in some but not all other strains. For example: a T in the Australian AB1, is a G at the same position in Bristol N2 in cosmid K10D2 at position 27946, and we found it to be like AB1 in the Californian CB4857 strain and the German RC301 strain, while the TR403 strain from Wisconsin resembles the Bristol N2 strain. Thus different patches of the genome have different ancestors. With our high density SNP map we will generate a genome map for each isolate which will show how each genome is patched together from a limited set of parental strains. The SNP's will be added to ACeDB, and can also be used as markers on the genetic map. They can be recognised by PCR followed by sequencing, but we also found that the SNPs we looked at could be visualised by SSCP analysis. We thank Jane Rogers and Amanda McMurray for their assistance in sequencing the clones.