Search

A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.

Transgenes are widely used throughout molecular biology for numerous applications. In

Horizontal gene transfer (HGT) enables the acquisition of novel traits via non-Mendelian inheritance of genetic material. HGT plays a prominent role in the evolution of prokaryotes, whereas in animals, HGT is rare and its functional significance is often uncertain. Here, we investigate horizontally acquired cellulase genes in the free-living nematode model organism Pristionchus pacificus. We show that these cellulase genes 1) are likely of eukaryotic origin, 2) are expressed, 3) have protein products that are secreted and functional, and 4) result in endo-cellulase activity. Using CRISPR/Cas9, we generated an octuple cellulase mutant, which lacks all eight cellulase genes and cellulase activity altogether. Nonetheless, this cellulase-null mutant is viable and therefore allows a detailed analysis of a gene family that was horizontally acquired. We show that the octuple cellulase mutant has associated fitness costs with reduced fecundity and slower developmental speed. Furthermore, by using various Escherichia coli K-12 strains as a model for cellulosic biofilms, we demonstrate that cellulases facilitate the procurement of nutrients from bacterial biofilms. Together, our analysis of cellulases in Pristionchus provides comprehensive evidence from biochemistry, genetics, and phylogeny, which supports the integration of horizontally acquired genes into the complex life history strategy of this soil nematode.

A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.

Animals are associated with a diverse bacterial community that impacts host physiology. It is well known that nutrients and enzymes synthesized by bacteria largely expand host metabolic capacity. Bacteria also impact a wide range of animal physiology that solely depends on host genetics through direct interaction. However, studying the synergistic effects of the bacterial community remains challenging due to its complexity. The omnivorous nematode Pristionchus pacificus has limited digestive efficiency on bacteria. Therefore, we established a bacterial collection that represents the natural gut microbiota that are resistant to digestion. Using this collection, we show that the bacterium Lysinibacillus xylanilyticus by itself provides limited nutritional value, but in combination with Escherichia coli, it significantly promotes life-history traits of P. pacificus by regulating the neuroendocrine peptide in sensory neurons. This gut-to-brain communication depends on undigested L. xylanilyticus providing Pristionchus nematodes a specific fitness advantage to compete with nematodes that rupture bacteria efficiently. Using RNA-seq and CRISPR-induced mutants, we show that 1-h exposure to L. xylanilyticus is sufficient to stimulate the expression of daf-7-type TGF-β signaling ligands, which induce a global transcriptome change. In addition, several effects of L. xylanilyticus depend on TGF-β signaling, including olfaction, body size regulation, and a switch of energy allocation from lipid storage to reproduction. Our results reveal the beneficial effects of a gut bacterium to modify life-history traits and maximize nematode survival in natural habitats.

Nematodes such as Caenorhabditis elegans are powerful systems to study basically all aspects of biology. Their species richness together with tremendous genetic knowledge from C. elegans facilitate the evolutionary study of biological functions using reverse genetics. However, the ability to identify orthologs of candidate genes in other species can be hampered by erroneous gene annotations. To improve gene annotation in the nematode model organism Pristionchus pacificus, we performed a genome-wide screen for C. elegans genes with potentially incorrectly annotated P. pacificus orthologs. We initiated a community-based project to manually inspect more than two thousand candidate loci and to propose new gene models based on recently generated Iso-seq and RNA-seq data. In most cases, misannotation of C. elegans orthologs was due to artificially fused gene predictions and completely missing gene models. The community-based curation raised the gene count from 25,517 to 28,036 and increased the single copy ortholog completeness level from 86% to 97%. This pilot study demonstrates how even small-scale crowdsourcing can drastically improve gene annotations. In future, similar approaches can be used for other species, gene sets, and even larger communities thus making manual annotation of large parts of the genome feasible.

The plant-parasitic nematode, Pratylenchus penetrans, is a major constraint to red raspberry (Rubus idaeus) production. To determine the impact of P. penetrans on the establishment and productivity of eight raspberry cultivars, R. niveus, and R. leucodermis, plants were grown in fumigated and nonfumigated soil. Then, soil and root populations of P. penetrans and plant productivity (vigor, cane height, biomass, and yield) were monitored over 2 years. In a separate experiment, the role that soil type plays in mediating P. penetrans populations and raspberry establishment was investigated. At 6 and 12 months after planting, population densities of P. penetrans were lower in fumigated than in nonfumigated soil; this trend continued 18 months after planting. All cultivars and R. leucodermis were found to be extremely susceptible to damage caused by P. penetrans. By the end of the experiment, yield was the most sensitive indicator of plant productivity among cultivars, with losses in nonfumigated soil ranging from 63 to 100% of those observed in fumigated soil. However, there was no difference in the productivity of R. niveus plants grown in nonfumigated versus fumigated soils. Results also indicated that the damage potential of P. penetrans was greater on sandy loam versus silt loam soils. The total plant biomass of ‘Meeker’ raspberry was 46% lower in a sandy loam soil containing P. penetrans compared with soil without P. penetrans; this trend was not observed consistently in a silt loam soil. Despite the fact that soil fumigation is increasingly more limited by regulations, this study shows the utility of fumigation in reducing P. penetrans populations for a sufficient period of time to ensure that newly planted raspberry seedlings can become successfully established.

The success of all plant-parasitic nematodes is dependent on the completion of several complex behaviors. The lesion nematode Pratylenchus penetrans is an economically important parasite of a diverse range of plant hosts. Unlike the cyst and root-knot nematodes, P. penetrans moves both within and outside of the host roots and can feed from both locations. Adult females of P. penetrans require insemination by actively moving males for reproduction and can lay eggs both within and outside of the host roots. We do not have a complete understanding of the molecular basis for these behaviors. One candidate modulator of these behaviors is the neurotransmitter serotonin. Previous research demonstrated an effect of exogenously applied serotonin on the feeding and male mating behaviors of cyst and root-knot nematodes. However, there are no data on the role of exogenous serotonin on lesion nematodes. Similarly, there are no data on the presence and function of endogenous serotonin in any plant-parasitic nematode. Here, we establish that exogenous serotonin applied to P. penetrans regulates both feeding and sex-specific behaviors. Furthermore, using immunohistochemistry and pharmacological assays, our data suggest that P. penetrans utilizes endogenous serotonin to regulate both feeding and sex-specific behaviors.