Introduction to Pristionchus pacificus anatomy

Abstract Pristionchus pacificus has emerged as an important nematode species used to understand the evolution of development and behavior. While P. pacificus (Diplogasteridae) is only distantly related to Caenorhabditis elegans (Rhabditidae), both use an identical reproductive strategy, are easily reared on bacteria in Petri dishes and complete their life cycles within a few days. Over the past 25 years, several detailed light and electron microscopy studies have elucidated the anatomy of P. pacificus and have demonstrated clear homology to many cells in C. elegans. Despite this similarity, sufficient anatomical differences between C. elegans and P. pacificus have allowed the latter to be used in comparative evo-devo studies. For example, the stoma of P. pacificus contains a large dorsal tooth used during predation on other nematodes when supplementing its primarily bacterial diet. This review discusses the main anatomical features of P. pacificus with emphasis on comparison to C. elegans.

In 1988, the nematode Pristionchus pacificus was isolated from garden soil in Pasadena, CA . P. pacificus was originally studied as a comparative species to Caenorhabditis elegans ( Fig. 1). Both species are easily reared in the laboratory on bacterial lawns and have similar anatomies and life cycles . P. pacificus uses a facultative hermaphroditic reproductive strategy similar to C. elegans. Many of the laboratory tools developed for C. elegans research are readily transferable to P. pacificus (Pires da Silva, 2013). The amenability of P. pacificus to genetic manipulation and analysis has propelled this species forward as an important comparative model for understanding the molecular basis of evolution for diverse traits.
Hundreds of P. pacificus isolates and other related Pristionchus species from around the world have been isolated (McGaughran and Morgan, 2015). Due to its cosmopolitan distribution and the diversity of isolated populations, P. pacificus has emerged as a powerful model for studying evolutionary processes at the molecular level. Many P. pacificus isolates are found associated with beetles and data suggests a necromenic relationship wherein the nematode waits for the beetle to die and then feeds off of the rotting cadaver and associated microorganisms . However, others have proposed that the relationship between P. pacificus and beetles is phoretic -the nematodes use beetles as a means of transport to a new nutrient rich environment (Félix et al., 2018). P. pacificus is androdioecious, comprising both self-fertile hermaphrodites (XX) and occasional males (XO) that fertilize hermaphrodites. Males are usually less than 1% of the population; however, this fraction varies among P. pacificus isolates and environmental conditions (Morgan et al., 2017). The androdioecious system is valuable for genetic studies as selfing by hermaphrodites leads to genetically identical offspring, while the presence of males allows for crosses between distinct genotypes.

P. pacificus anatomy: Schroeder
Life cycle P. pacificus is easily maintained on bacterial cultures in the laboratory and frozen stocks can be kept for years (Pires da Silva, 2013). Development from fertilized egg to adult is completed in approximately four days which is only slightly longer than C. elegans ( Fig. 2)  . Following exposure to harsh environmental conditions, P. pacificus can form a stress-resistant dauer stage that can survive extended periods without feeding.
Unlike C. elegans, which hatches as a J1, P. pacificus does not emerge from its egg until J2 (Fig. 2). The presence of a distinct J1 stage in P. pacificus is marked by ecdysis (shedding of the cuticle) within the egg-shell (Fürst von Lieven, 2005) (Fig. 3). The shift in timing of P. pacificus hatch may allow for the development of moveable teeth within the stoma. A similar delay is seen in many plant-parasitic nematodes with moveable stylet mouthparts. Despite the delay in hatching, the generation time for P. pacificus is only slightly longer than C. elegans . Some events that occur in the post-hatch J1 of C. elegans occur prior to hatching in J1 P. pacificus. For example, migration of the Pn ectoblasts, which contribute to the post-embryonic development of the ventral nerve cord and vulva, occurs in the J1 of both species (Félix et al., 1999;Sulston and Horvitz, 1977). Unfortunately, the additional post-embryonic development and corresponding movement within the eggshell will make it challenging to describe the complete embryonic cell lineage of P. pacificus.

Cuticle
The cuticle of P. pacificus is a tough rigid extracellular matrix likely secreted by the underlying epithelial cells. Similar to C. elegans and other nematodes, collagen is a major biochemical component of the body wall (Kenning et al., 2004;Schlager et al., 2009). The cuticle of P. pacificus contains evenly spaced stippled longitudinal ridges along the length of the body (Fig. 4). Unlike the C. elegans alae, these longitudinal ridges are not restricted to the lateral field of the cuticle. Several tissues open to the outside through the cuticle including the stoma and anus. The excretory pore of P. pacificus lies on the ventral ridge near the terminal bulb of the pharynx. The lateral cuticle of P. pacificus hermaphrodites is also interrupted by a series of small pores that open to gland cells embedded within the lateral hypodermis  Sommer et al., 1996). These lateral gland cells are not present in C. elegans and their lineage in P. pacificus is unknown.
P. pacificus dauers tend to retain their L2 cuticle for extended periods of time. Retention of the L2 cuticle likely assists dauers in survival of adverse environmental conditions. P. pacificus dauers also secrete a waxy ester onto the cuticle surface that facilitates aggregation of dauers and the formation of 'dauer towers', which enhance the nematode's ability to attach to a beetle host (Penkov et al., 2014).

The epithelial system
The main epithelial system of P. pacificus consists of hypodermal syncytia. Similar to C. elegans, the hypodermis wraps around the body wall of the nematode alternating between thick regions with nuclei in the cords and a thin sheet-like region underlying the somatic muscles. The hypodermis is interrupted on the lateral ridges by a linear set of 15 epithelial 'seam' cells per side in the adult hermaphrodite, which is slightly less than the 16 homologous seam cells found in C. elegans (Cinkornpumin et al., 2014). Also, differing from C. elegans, the P. pacificus seam cells send processes from the apical membrane that extend away from the lateral midline.

The nervous system
As with C. elegans and other nematodes, the majority of P. pacificus neurons are located in the head and tail. The anterior nervous system of P. pacificus, including the pharynx and anterior amphid sensory neurons, have been reconstructed from serial-section electron microscopy (Bumbarger et al., 2013;Hong et al., 2019). The nervous system of P. pacificus is remarkably similar to that of C. elegans. Both the anterior amphid sensory sensilla and the pharyngeal nervous system have equivalent numbers of neurons in both species and many of these are obvious homologs based on their positions and structures (Bumbarger et al., 2013;Bumbarger and Riebesell, 2015;Hong et al., 2019). While both species have 12 pairs of amphid neurons, the sensory ending shape differs between some homologous neurons. For example, the P. pacificus amphids lack obvious winglike ciliated dendrites found in the AWA, AWB, and AWC neurons in C. elegans (Hong et al., 2019).
While the pharyngeal nervous system of both species comprises 20 neurons, the synaptic connectivity differs between these species (Bumbarger et al., 2013), although a more recent reanalysis of the C. elegans pharyngeal connectome suggests these differences may not be as great as originally reported (Cook et al., 2020). Wiring differences between the two species in the amphid and pharyngeal circuits may mediate behavioral differences (Bumbarger et al., 2013;Hong et al., 2019). Similarly, slight differences in neurotransmitter expression occur between P. pacificus and C. elegans neurons (Loer and Rivard, 2007). While there are differences reported in the number of neurons within the ventral nerve cord between C. elegans and P. pacificus, additional electron microscopy will be needed to determine the extent of neuronal differences between these species (Han et al., 2016).

The muscle system
The somatic body wall muscle of P. pacificus is platymyarian and similar in overall structure to C. elegans (Fig. 5). A basal lamina separates neurons and hypodermis from the body wall muscle. Typical to all nematodes, innervation of P. pacificus body wall muscles occurs through muscle arm processes extending to neurons (Bird and Bird, 1991). In addition to the body wall, non-striated muscles exist in the pharynx and surrounding the egg-laying apparatus and rectum.

The excretory system
The P. pacificus excretory system appears very similar to the C. elegans excretory system (Fig. 6). It consists of four cells-two excretory glands, a canal cell with bilaterally symmetrical longitudinal processes, and a duct cell. Similar to C. elegans, the P. pacificus CAN neuron travels alongside the canal cell processes (Carstensen et al., 2021). In P. pacificus, ablation of the CAN results in increased dauer formation, whereas in C. elegans ablation of CAN results in death (Mayer et al., 2015;White et al., 1986).

The coloemocytes
Both C. elegans and P. pacificus adult hermaphrodites contain six large scavenger cells called coelomocytes   that sit within the pseudocoelom body cavity (Jungblut and Sommer, 1998). For both species, two of the coelomocytes are generated post-embryonically (Photos et al., 2006).

The alimentary system
The stoma of P. pacificus comprises a cuticular lined cavity. Consistent with many nematodes in the Diplogastridae family, the P. pacificus stoma contains teeth used for predatory feeding of other nematodes (Fig. 7). The morphology of the stoma varies between two morphotypes called eurystomatous and stenostomatous that are specialized for predatory vs. microbial feeding, respectively (Serobyan et al., 2014). The eurystomatous (wide-mouthed) is characterized by a claw-like dorsal tooth and an opposing subventral tooth. The stenostomatous (narrow-mouthed) form has a less prominent triangle-shaped dorsal tooth and lacks the additional subventral tooth. The specific morphotype is determined through environmental conditions (Bento et al., 2010;Serobyan et al., 2013). P. pacificus dauers isolated from beetle hosts in the wild developed exclusively into eurystomatous adults (Renahan et al., 2021). The teeth of P. pacificus are attached to the anterior-most muscles of the pharynx, allowing for movement during feeding. Similar to C. elegans, the cuticle lined stoma is shed during each molt.
Posterior of the stoma, the P. pacificus pharynx is a muscular pump. While similar in gross morphology to the C. elegans pharynx, the P. pacificus pharynx uses a different pumping sequence to ingest food (Chiang et al., 2006). The P. pacificus pharynx lacks both the grinder and two gland cells found in the C. elegans terminal bulb; however, the three remaining gland cells of P. pacificus have evolved to occupy a larger fraction of the terminal bulb volume (Riebesell and Sommer, 2017).
The intestine connects to the posterior end of the pharynx. The lineage and a detailed description of the P. pacificus intestine are not yet available.

The reproductive system
The first genetic analyses of P. pacificus focused on the molecular genetics of vulva formation . Similar to C. elegans, the reproductive system of P. pacificus consists of a somatic gonad, the germ line, and the egg-laying apparatus; Figure 6: Transverse TEM micrograph through junction of excretory glands with canal cell and secretory vesicle being released in gland cell ampulla (arrowhead). Scale bars, 1 µm. Source: Ralf Sommer lab, Bumbarger13_2251. however, there is substantial divergence in the number of cells, overall shape, and developmental timing of the reproductive systems between C. elegans and P. pacificus (Kolotuev and Podbilewicz, 2004;Rudel et al., 2005).
Similar to C. elegans, the P. pacificus vulva is formed through a combination of cell division, migration and fusion from three ectoblastic vulval precursor P cells; however, the molecular signal controlling division differs between the species . Following induction, the P cells undergo divisions to form 20 vulval cells (two less than C. elegans) (Kolotuev and Podbilewicz, 2004). The vulval cells migrate to form a stack of rings at the location of the vulva and undergo cell-cell fusion to form stacked toroids. The timing, sequence and number of toroids differs between C. elegans and P. pacificus (Kolotuev and Podbilewicz, 2004).
The gonad of P. pacificus is didelphic comprising two arms converging on a vulva positioned near the center of the body. The somatic gonad consists of a uterus, spermatheca, sheath, and distal tip cells. The germline is a syncytium connected through a central rachis. Several features of the gonad in P. pacificus distinguish it from that in C. elegans (Rudel et al., 2005). Most obviously distinct in the P. pacificus gonad is the pretzel shape of the gonad arms, which extend from the ventral side to the dorsal side and back again (Fig. 8). The sheath cells wrap the proximal oocytes of both species but differ in number between species (4 pairs in P. pacificus, 5 in C. elegans). While both species have sperm storage organs called spermatheca, P. pacificus does not contain the connective uterine-spermatheca valve cells found in C. elegans and differs in number of cells (Rudel et al., 2005).