High Nutritional Conditions Influence Feeding Plasticity in Pristionchus pacificus and Render Worms Non-Predatory

Abstract

Developmental plasticity, the ability of a genotype to produce different phenotypes in response to environmental conditions, has been subject to intense studies in the last four decades. The self-fertilising nematode Pristionchus pacificus has been developed as a genetic model system for studying developmental plasticity due to its mouth-form polyphenism that results in alternative feeding strategies with a facultative predatory and non-predatory mouth form. Many studies linked molecular aspects of the regulation of mouth-form polyphenism with investigations of its evolutionary and ecological significance. Also, several environmental factors influencing P. pacificus feeding structure expression were identified including temperature, culture condition and population density. However, the nutritional plasticity of the mouth form has never been properly investigated although polyphenisms are known to be influenced by changes in nutritional conditions. For instance, studies in eusocial insects and scarab beetles have provided significant mechanistic insights into the nutritional regulation of polyphenisms but also other forms of plasticity. Here, we study the influence of nutrition on mouth-form polyphenism in P. pacificus through experiments with monosaccharide and fatty acid supplementation. We show that in particular glucose supplementation renders worms non-predatory. Subsequent transcriptomic and mutant analyses indicate that de novo fatty acid synthesis and peroxisomal beta-oxidation pathways play an important role in the mediation of this plastic response. Finally, the analysis of fitness consequences through fecundity counts suggests that non-predatory animals have an advantage over predatory animals grown in the glucose-supplemented condition.

Keywords: Pristionchus pacificus; developmental plasticity; fatty acids; glucose; nutrition; polyphenism.

Figure 1

Mouth‐form polyphenism of P. pacificus, and the experimental design for supplementation studies. (a) Differential interference contrast (DIC) images of the eurystomatous (Eu) and the stenostomatous (St) mouth morphs. The Eu morph has a wide buccal cavity, a hook‐like dorsal tooth (on the left, outlined), and a sub‐ventral tooth. The narrow‐mouthed stenostomatous (St) morph has a flint‐shaped dorsal tooth (on the left, outlined). Scale bar is 5 μm. (b) Illustration of the general experimental design using supplementation with monosaccharides and fatty acids.

Figure 2

Effect of oleic acid and glucose supplementations on mouth‐form plasticity and lipid storage in P. pacificus. (a) Concentration‐dependent effect of oleic acid and glucose on mouth‐form plasticity. N ≥ 3 biological replicates per condition for each concentration. Each faint data point represents a replicate (plate), with 30 animals per replicate being scored for mouth‐form percentage (Eu%). Error bars represent SEM. (b) Oil Red O absorbance (RawIntDen/body area) obtained from worms grown in oleic acid, glucose, and respective control conditions. N = 15 per condition. Each faint data point represents an individual worm. P values are obtained from Welch two sample t‐test and two‐sample t‐test for oleic acid and glucose comparisons, respectively. Bars represent the mean values of all samples in each condition. Error bars represent s.d. (c) Representative images of ORO‐quantified worms, indicating lipid storage profile. Images are acquired from the blue channel in grayscale. Lipid droplets appear dark. Scale bar is 50 μm.

Figure 3

Transcriptome analysis. (a) An illustration of RNA extraction. (b) Overrepresented KEGG pathways of differentially expressed genes for oleic acid and glucose conditions. Statistical analysis by Fisher's exact test with multiple testing corrections (Bonferroni corrected p < 0.05).

Figure 4

Significance of delta‐9 desaturase activity in glucose supplementation‐induced mouth‐form plasticity. (a) Schematic representation of the essential activity of delta‐9 desaturases, facilitating fat storage by converting saturated fatty acids into monounsaturated fatty acids. Sterol regulatory element binding protein 1 (SBP‐1) activates delta‐9 desaturases. (b) A simplified phylogenetic tree of Pristionchus delta‐9 desaturase‐like genes (Ppa‐pddl‐1, Ppa‐pddl‐2, Ppa‐pddl‐3, Ppa‐pddl‐4), and C. elegans delta‐9 desaturases (fat‐5, fat‐6 and fat‐7). Triangle denotes P. pacificus genes which are constitutively expressed during development (Baskaran et al. 2015). The phylogenetic tree is drawn based on the tree in Figure S3. (c) Eu percentages of CRISPR mutants of Ppa‐pddl‐1, Ppa‐pddl‐3, Ppa‐pddl‐4 and wild type animals in control and glucose conditions. N ≥ 2 biological replicates per strain for each condition. Bars represent mean values of all replicates. Error bars represent s.d. (d) Eu percentages of wild type, tu2028(Ppa‐pddl‐1), and CRISPR repaired‐tu2028 strains in control and glucose conditions. N ≥ 3 biological replicates per strain for each condition. (c,d) Each faint data point represents a replicate (plate), with 30 animals per plate being scored for mouth‐form percentage (Eu%). (e) ORO absorbance (RawIntDen/body area) obtained from wild type, tu2028(Ppa‐pddl‐1), and CRISPR repaired‐tu2028 strains grown on control and glucose conditions. N = 15 per strain for each condition. Each faint data point represents an individual worm. (d,e) Error bars represent s.e.m. (f) Representative images of ORO‐quantified strains from control (‐glucose) and glucose ( + glucose) conditions. Images are acquired from the blue channel in grayscale. Lipid droplets appear dark. Scale bar is 100 μm.

Figure 5

Response of peroxisomal beta‐oxidation mutants to glucose‐supplemented diet. (a) Schematic of peroxisomal beta‐oxidation pathway, indicating duplicate copies in P. pacificus for dhs‐28 and daf‐22 genes relative to C. elegans. (b) Eu percentages of peroxisomal beta‐oxidation mutants and wild type strains in control and glucose conditions. N ≥ 2 biological replicate per strain for each condition. Each faint data point represents a replicate (plate), with 30 animals per plate being scored for mouth‐form percentage (Eu%). (c) ORO absorbance (RawIntDen/body area) obtained from peroxisomal beta‐oxidation mutants and wild type strains grown on control and glucose conditions. N = 15 per strain for each condition. Each faint data point represents an individual worm. (b,c) Error bars represent s.e.m. (d) Representative images of ORO‐quantified strains from control (‐glucose) and glucose ( + glucose) conditions. Images are acquired from the blue channel in grayscale. Lipid droplets appear dark. Scale bar is 100 μm.

Figure 6

Role of plasticity switch genes in glucose supplementation‐induced mouth‐form plasticity. (a) Schematic of the mouth‐form gene regulatory network, indicating different modules with genetic and epigenetic components. (b) Eu percentages of plasticity switch mutants and wild type strains in glucose and control conditions. N = 3 biological replicates per strain for each condition. Each faint data point represents a replicate (plate), with 30 animals per plate being scored for mouth‐form percentage (Eu%). (c) ORO absorbance (RawIntDen/body area) measured in plasticity switch mutants and wild type strains grown on control and glucose conditions. N = 15 per strain for each condition. Each faint data point represents a worm. (b,c) Error bars represent s.e.m.

Figure 7

Fecundity of different morphs after glucose supplementation. (a) Illustration for the experimental design to study fecundity of Eu and St animals obtained from the glucose‐supplemented condition. (b) Daily fecundity of Eu and St animals. Error bars represent s.e.m. (c) Overall fecundity of Eu and St animals. Bars represent mean values of all individuals for each morph. Error bars represent s.d. P value is obtained from Wilcoxon rank sum test. (b,c) N = 34 per morph. Each faint data point represents a worm.