Co-option of an Astacin Metalloprotease Is Associated with an Evolutionarily Novel Feeding Morphology in a Predatory Nematode.

Animals consume a wide variety of food sources to adapt to different environments. However, the genetic mechanisms underlying the acquisition of evolutionarily novel feeding morphology remain largely unknown. While the nematode Caenorhabditis elegans feeds on bacteria, the satellite species Pristionchus pacificus exhibits predatory feeding behavior toward other nematodes, which is an evolutionarily novel feeding habit. Here, we found that the astacin metalloprotease Ppa-NAS-6 is required for the predatory killing by P. pacificus. Ppa-nas-6 mutants were defective in predation-associated characteristics, specifically the tooth morphogenesis and tooth movement during predation. Comparison of expression patterns and rescue experiments of nas-6 in P. pacificus and C. elegans suggested that alteration of the spatial expression patterns of NAS-6 may be vital for acquiring predation-related traits. Reporter analysis of the Ppa-nas-6 promoter in C. elegans revealed that the alteration in expression patterns was caused by evolutionary changes in cis- and trans-regulatory elements. This study suggests that the co-option of a metalloprotease is involved in an evolutionarily novel feeding morphology.

Efficient visual screening of CRISPR/Cas9 genome editing in the nematode Pristionchus pacificus.

CRISPR/Cas9 genome editing has been applied to a wide variety of organisms, including nematodes such as Caenorhabditis elegans and Pristionchus pacificus. In these nematodes, genome editing is achieved by microinjection of Cas9 protein and guide RNA into the hermaphrodite gonads. However, P. pacificus is less efficient in CRISPR/Cas9 genome editing and exogenous gene expression. Therefore, it takes considerable time and effort to screen for target mutants if there are no visual markers that indicate successful injection. To overcome this problem, co-injection markers (gRNA for Ppa-prl-1, which induces the roller phenotype, and Ppa-egl-20p::turboRFP, a plasmid expressing a fluorescent protein) have been developed in P. pacificus. By selecting worms with the roller phenotype or turboRFP expression, screening efficiency is substantially increased to obtain worms with desired mutations. Here, we describe a step-by-step protocol for the visual screening system for CRISPR/Cas9 genome editing in P. pacificus. We also describe technical tips for microinjection, which is difficult for beginners. This protocol will facilitate genome editing in P. pacificus and may be applied to other nematode species.

Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus.

CRISPR/Cas9 genome-editing methods are used to reveal functions of genes and molecular mechanisms underlying biological processes in many species, including nematodes. In evolutionary biology, the nematode Pristionchus pacificus is a satellite model and has been used to understand interesting phenomena such as phenotypic plasticity and self-recognition. In P. pacificus, CRISPR/Cas9-mediated mutations are induced by microinjecting a guide RNA (gRNA) and Cas9 protein into the gonads. However, mutant screening is laborious and time-consuming due to the absence of visual markers. In this study, we established a Co-CRISPR strategy by using a dominant roller marker in P. pacificus. We found that heterozygous mutations in Ppa-prl-1 induced the roller phenotype, which can be used as an injection marker. After the co-injection of Ppa-prl-1 gRNA, target gRNA, and the Cas9 protein, roller progeny and their siblings were examined using the heteroduplex mobility assay and DNA sequencing. We found that some of the roller and non-roller siblings had mutations at the target site. We used varying Cas9 concentrations and found that a higher concentration of Cas9 did not increase genome-editing events. The Co-CRISPR strategy promotes the screening for genome-editing events and will facilitate the development of new genome-editing methods in P. pacificus.

Different combinations of serotonin receptors regulate predatory and bacterial feeding behaviors in the nematode Pristionchus pacificus.

Feeding behavior is one of the most fundamental behaviors in animals, and regulation of this behavior is critical for proper food intake. The nematode Pristionchus pacificus exhibits dimorphism in feeding behavior, bacterial feeding and predatory feeding on other nematodes, and the latter behavior is assumed to be an evolutionarily novel behavior. Both types of feeding behavior are modulated by serotonin; however, the downstream mechanism that modulates these behaviors is still to be clarified. Here, we focused on serotonin receptors and examined their expression patterns in P. pacificus. We also generated knockout mutants of the serotonin receptors using the CRISPR/Cas9 system and examined feeding behaviors. We found that Ppa-ser-5 mutants and the Ppa-ser-1; Ppa-ser-7 double mutant decreased predation. Detailed observation of the pharyngeal movement revealed that the Ppa-ser-1; Ppa-ser-7 double mutant reduces tooth movement, which is required for efficient predatory feeding. Conversely, Ppa-ser-7 and Ppa-mod-1 mutants decreased bacterial feeding. This study revealed that specific combinations of serotonin receptors are essential for the modulation of these distinct feeding behaviors, providing insight into the evolution of neural pathways to regulate novel feeding behavior.

Serotonergic modulation of feeding behavior in Caenorhabditis elegans and other related nematodes.

Serotonin is a conserved neuromodulator that controls feeding behavior in response to environmental inputs in a wide range of species, including the nematode, Caenorhabditis elegans. To understand the detailed mechanism and evolution of serotonergic neuromodulation, the feeding behaviors of C. elegans and related species have been studied intensively because of their simple neural anatomy and genetic manipulability. C. elegans shows patterned movements of a feeding structure called the pharynx, and serotonin modulates feeding rhythms via several serotonin receptors expressed in pharyngeal motor neurons and muscles. Environmental inputs and physiological states like food signals, starvation, and heat affect the activity of serotonergic neurons and downstream neural pathways. We focus on serotonergic neural pathways in the feeding behavior of C. elegans and other nematodes, neuromodulation between environmental inputs and behavioral outputs, and their evolutionary path.