Introduction to Strongyloides stercoralis Anatomy

The S. stercoralis parasitic female resides in the host small intestine. An S. stercoralis parasitic female is long and slender, measuring approximately 2.4 mm in length and 37 µm in width (Schad, 1989; Lindo and Lee, 2001) (Figs. 2A, 3). S. stercoralis is sometimes called the threadworm because of its long, slender morphology (Lindo and Lee, 2001).

Figure 2:

Figure 3:

S. stercoralis free-living females are approximately 1.1 mm long and 62 µm wide, with a conical pointed tail (Schad, 1989) (Figs. 2B, 4A). For comparison, a C. elegans adult hermaphrodite is approximately 1 mm in length and 80 µm in width (Palikaras and Tavernarakis, 2013). S. stercoralis free-living adults were estimated to have approximately 740 somatic nuclei, excluding somatic nuclei in the gonad and intestine (Hammond and Robinson, 1994a); this is roughly similar to the 959 somatic nuclei, including somatic nuclei in the gonad and intestine, found in C. elegans adult hermaphrodites (Sulston and Horvitz, 1977; Wood, 1988). The S. stercoralis cuticle and body wall are transparent, allowing easy visualization of internal structures.

The S. stercoralis free-living adult male is smaller than the free-living adult female, measuring approximately 0.9 mm in length and 43 µm in width (Schad, 1989; Lindo and Lee, 2001) (Figs. 2C, 4B). Their broad tail is slightly curved and contains a pair of spicules used for insemination of the females (Fig. 4B).

Figure 4:

Rhabditiform larvae include all first-stage (L1) and second-stage (L2) larvae, as well as third-stage (L3) and fourth-stage (L4) larvae that are destined to become free-living adults (Figs. 5, 6). Rhabditiform larvae consume bacteria and are identified by the presence of a rhabditiform pharynx. A hatchling first-stage larva in the intestine is 180–240 µm long and 14–15 µm wide. By the time they exit the host in feces, the L1s are approximately 250 µm long and 17 µm wide (Schad, 1989). L1 larvae are rounded on the anterior end, and their tail is slender and pointed. L2 and L3 larvae are larger than L1 larvae, with similar internal structures. However, the gonad increases in cell number and definition through each larval stage (Schad, 1989; Speare, 1989) (Figs. 5, 6). The reproductive structures of the free-living male can be identified in early larval stages (Figs. 6A,B).

Figure 5:

Figure 6:

L2s that will develop into iL3s have two morphological characteristics that foretell their different developmental track (Schad, 1989). First, in preparation for the increase in cell number in the longer intestine of the parasitic female, L2s fated to develop into iL3s have 22 intestinal cells and 40 nuclei instead of the 22 nuclei seen in L2s fated to develop into free-living adults. Second, the rhabditiform pharynx is slightly extended, and the posterior portion is more glandular than the anterior portion, predicting the altered pharyngeal structure of the iL3 and parasitic female (Schad, 1989).

iL3s are also termed filariform larvae based on the structure of their pharynx. The morphology of the filariform larva is distinct from that of the other environmental stages, reflecting the anatomical changes required for host seeking and host invasion via skin penetration (Schad, 1989). iL3s are radially constricted, measuring approximately 560 µm in length but only approximately 16 µm in width. The filariform pharynx of the iL3s is approximately half of the body length, making this larva easy to identify under low magnification (Schad, 1989) (Fig. 7A). The tail of the S. stercoralis iL3 is tetrafurcated (Fig. 7B) (Nichols, 1956; Little, 1966a; Schad, 1989). The width of the iL3 increases substantially during the activation process as the pharynx expands and the larva resumes feeding (Fig. 7C).

Figure 7:

Third-stage autoinfective larvae (aL3s) are unique to S. stercoralis. These larvae develop within the same host as their parent parasitic female. They can be distinguished from iL3s based on their size (Fig. 8). The autoinfective larvae are shorter than filariform larvae, measuring approximately 270 µm in length and 11 µm in width (Humphreys and Hieger, 1979; Gocek et al., 1985; Kim et al., 2005). The aL3 pharynx is filariform and occupies approximately half of their length, like the pharynx of the iL3 (Kim et al., 2005). They also have the characteristic tetrafurcated tail of the filariform larvae (Schad et al., 1993; Kim et al., 2005).

Figure 8:

The body wall of nematodes comprises the cuticle, the outermost layer of extracellular matrix components; the hypodermis, or epithelial tissue, which secretes the cuticle components; and the muscular layer (Page and Johnstone, 2007). The cuticle is acellular and is composed of collagens, cuticulin, and assorted surface proteins (Politz and Philipp, 1992; Page et al., 2014). The S. stercoralis cuticle has not been studied in detail, so knowledge of the cuticle of Strongyloides species comes primarily from studies of the rat-parasitic species Strongyloides venezuelensis.

The cuticle of the S. venezuelensis parasitic female measures approximately 1 µm thick and consists of seven main layers: the outermost epicuticle, outer cortical, inner cortical, external medial, internal medial, fibrous, and basal layers (Martinez and de Souza, 1995) (Figs. 9A,B). The S. venezuelensis epicuticle is covered in a surface coat that interacts directly with host tissues (Martinez and de Souza, 1995). The surface coat of parasitic nematodes may contribute to their ability to evade the host immune system (Blaxter et al., 1992). The cortical, medial, and basal layers of the cuticle have networks of thick and thin fibers with globular structures in an irregular arrangement (Fig. 9B). The composition of the fibrous layer is similar, except that the fibers are found in a more parallel orientation (Fig. 9A). The spaces between the collagenous fibers of the cuticle are thought to be filled with fluid. This open mesh of fibers likely allows nutrients and secretory products to diffuse across the cuticle while still providing the nematode with structural support (Martinez and de Souza, 1995).

Figure 9:

The molecular composition of the iL3 cuticle differs from that of other life stages such that it is more resistant to environmental stress (Martinez and de Souza, 1997). The basic structure of the S. venezuelensis iL3 cuticle has five layers: the epicuticle, cortical, medial, fibrous, and basal layers. The width of the cuticle is approximately 300 nm (Martinez and de Souza, 1997). Like the parasitic females, the iL3s have a surface coat on the exterior of the cuticle that is approximately 12 nm deep (Martinez and de Souza, 1997). The iL3s of the rat parasite Strongyloides ratti also have a surface coat that disappears once the iL3s have penetrated host skin (Grove et al., 1984; Grove et al., 1987). The purpose of this coat and its molecular composition are unclear.

S. stercoralis iL3s have two pairs of lateral alae running the length of the worm and extending slightly past the tip of the tail (Figs. 10A–D). These alae give the iL3 a characteristic tetrafurcated tail, which can be used to differentiate S. stercoralis iL3s from hookworm iL3s (Nichols, 1956; Little, 1966a; Schad, 1989; Lindo and Lee, 2001; Riyong et al., 2020) (Fig. 10D). The alae likely function to increase the stability of the worm during rapid movement, consistent with the increased motility seen at this life stage (Schad, 1989; Lindo and Lee, 2001).

Figure 10:

The hypodermis of nematodes lies under the cuticle and surrounds the body. It has several functions, including establishment of the body plan, production of cuticle components, and storage of nutrients (Altun and Hall, 2009c; Wolkow et al., 2021). In recent literature, the hypodermis is sometimes called the epidermis because it derives from the ectoderm (Altun and Hall, 2009c). In C. elegans and the beetle-associated nematode Pristionchus pacificus, the fusion of specialized epithelial cells during development results in multinucleate syncytia that form the hypodermis (Altun and Hall, 2009c; Schroeder, 2021). Thicker parts of the hypodermis, sometimes called the hypodermal cords, contain the hypodermal nuclei. The dorsal and ventral hypodermal cords house the dorsal and ventral nerve cords, respectively, while the lateral hypodermal cords serve as conduits for the excretory canals (Chisholm and Xu, 2012; Basyoni and Rizk, 2016). Little is known about the specifics of the S. stercoralis hypodermis.

In general, the nervous system of S. stercoralis resembles that of C. elegans and other nematodes (Mendez et al., 2022). The structure of the anterior nervous system of S. stercoralis iL3s was determined by serial-section electron microscopy and found to resemble that of C. elegans, with several important differences (Ashton et al., 1995; Fine et al., 1997). Like C. elegans, S. stercoralis has paired amphid sensilla in the head that house many of the sensory neurons (Ashton et al., 1995) (Fig. 11). The amphid sensilla of the iL3s are open to the external environment, consistent with a role for sensory perception in host seeking and host invasion (Ashton et al., 1995). S. stercoralis has 13 pairs of amphid sensory neurons, in contrast to 12 pairs of amphid sensory neurons in C. elegans (Ashton et al., 1995). However, it remains unclear whether this is a true anatomical difference or a difference in nomenclature: the “extra” amphidial neuron in S. stercoralis has a short anterior process that terminates at the base of the amphidial channel and may correspond to the AUA neuron of C. elegans and P. pacificus, which is not considered a true amphidial neuron in these species (White et al., 1986; Ashton et al., 1995; Hong et al., 2019). The labial and cephalic sensilla of S. stercoralis, which are thought to be mechanosensory, are also morphologically similar to those of C. elegans, although there are some differences in the number of neurons per sensillum between the two species (Fine et al., 1997).

Figure 11:

Although the cell body positions of the anterior neurons are roughly conserved between C. elegans and S. stercoralis, dendritic morphology is less well conserved (Ashton et al., 1995; Fine et al., 1997). For example, the AFD thermosensory neurons of C. elegans have a distinctive finger-like dendritic structure that is not present in S. stercoralis (Ashton et al., 1995; Goodman and Sengupta, 2018). There is only one amphid neuron in S. stercoralis that has a complex dendritic structure, and this neuron was originally termed the ALD neuron due to its lamellar dendritic structure (Ashton et al., 1995) (Fig. 11). A more recent genetic and functional characterization of this neuron identified it as the homolog of C. elegans AFD and the primary thermosensory neuron of S. stercoralis; this neuron has now been renamed AFD for consistency with C. elegans nomenclature (Bryant et al., 2022).

S. stercoralis has a clearly visible nerve ring in the head, which comprises densely packed neuronal processes (Ashton et al., 1995; Fine et al., 1997). In C. elegans, the nerve ring contains processes from more than half of the neurons in the body and is the most synapse-rich region of the nervous system (Altun, 2017). The nerve ring of S. stercoralis has not been studied in detail.

In C. elegans, the pharyngeal nervous system controls the activity of the pharynx and is isolated from the somatic nervous system. The pharyngeal nervous system of S. stercoralis has not been studied.

The body wall muscle of nematodes is separated from the hypodermal layer by a basal lamina (Basyoni and Rizk, 2016). The S. stercoralis musculature is platymyarian (the muscle fibers are adjacent and perpendicular to the hypodermis) and meromyarian (comprised of eight longitudinal muscle cells) (Little, 1966a; Basyoni and Rizk, 2016). The pharyngeal musculature, as well as the muscle cells involved in reproduction, male spicule movement, egg-laying, and defecation, have not been studied in S. stercoralis.

The excretory system of nematodes has several functions, including maintenance of osmotic pressure, secretion of proteins into the environment, and elimination of waste (Sundaram and Buechner, 2016). The excretory system of S. stercoralis consists of two excretory canals running the length of the body along the lateral sides (Schad, 1989). A transverse duct connects the two canals to a single excretory cell, giving the S. stercoralis excretory system an H-shape like that of C. elegans (Schad, 1989,; Lindo and Lee, 2001; Sundaram and Buechner, 2016). The excretory cell is found posterior to the nerve ring, near the pharyngeal bulb (Nichols, 1956; Little, 1966a; Schad, 1989) (Fig. 12). A short canal ending in the excretory pore allows the contents collected by the excretory cell to be excreted into the environment (Lindo and Lee, 2001). The excretory pore opening is on the midventral line just posterior to the nerve ring, mid-pharynx (Schad, 1989; Lindo and Lee, 2001). The location of the excretory gland cells and neurons associated with the excretory system in S. stercoralis is unknown.

Figure 12:

Studies of the excretory/secretory (ES) proteins of S. stercoralis have identified a wide variety of proteins that could be important for manipulation of the host immune response (Marcilla et al., 2010; Varatharajalu et al., 2011; Marcilla et al., 2012; Cuesta-Astroz et al., 2017; Hunt et al., 2017; Culma, 2021). However, where ES proteins are produced, how they are released from the worm and how they function remains unclear. The role of the excretory system in ES release is an open question and could be instrumental in understanding S. stercoralis host-parasite interactions.

The coelomocyte system of S. stercoralis has not yet been characterized.

As in other nematodes, the stoma, or mouth opening, connects to the pharynx, which pumps food into the intestine. The stoma, pharynx, and intestine of S. stercoralis differ significantly in parasitic females, free-living larval and adult stages, and iL3s.

The stoma of the parasitic female can be used as an identifying feature for the different Strongyloides species (Little, 1966a, 1966b; Sato et al., 2008). In the case of S. stercoralis, the stoma is classified as angular and looks hexagonal when viewed en face (Little, 1966a). The stoma is surrounded by the circumoral elevation, a slightly elevated cuticle structure that consists of three paired lobes in S. stercoralis (Little, 1966a). Strongyloides procyonis, a parasite of raccoons, also has a hexagonal stoma shape (Little, 1966b; Sato et al., 2008). However, since S. procyonis does not infect humans, the hexagonal stoma can be used to differentiate S. stercoralis from the other human-infective species, Strongyloides fuelleborni, which has an X-shaped stoma (Little, 1966a).

The filariform (filament-shaped) pharynx of the S. stercoralis parasitic female extends up to one-third of the length of the body (Speare, 1989) (Fig. 3). The nerve ring encircles the pharynx, dividing it into an anterior one-fourth and posterior three-fourths (Schad, 1989). The expansion and contraction of the more muscular anterior portion of the pharynx together draws material into the stoma and the lumen of the pharynx (Lindo and Lee, 2001). The posterior region is glandular, with two subventral glands that deposit secretions into the pharyngeal lumen and one dorsal gland that deposits secretions near the mouth (Schad, 1989; Lindo and Lee, 2001). The exact functions and secretions of the pharyngeal gland cells are unclear, but it is possible these gland cells are a source of the ES products mentioned above. The pharynx connects to the intestine. The pharyngeal-intestinal junction is an easy-to-locate landmark a third of the way down the body (Fig. 3).

In nematodes, the intestine is responsible for the uptake of nutrients, disposal of waste products, and transport of signaling molecules (McGhee, 2007; Altun and Hall, 2009a). In general, the nematode intestine is composed of paired epithelial cells whose apical faces form the intestinal lumen and basal faces contact the body cavity (Altun and Hall, 2009a). Adherens junctions secure the epithelial cells together at the apical side (Altun and Hall, 2009a). The intestine is not anchored to the body wall; rather, it is anchored to the pharynx at the anterior end and the rectum at the posterior end (Altun and Hall, 2009a; Basyoni and Rizk, 2016). The intestine is lined with microvilli covered in a glycocalyx matrix (Altun and Hall, 2009a). The S. stercoralis parasitic female intestine is a thin tube with 40 uninucleate cells in two rows (Little, 1966a; Schad, 1989). It terminates near the tail in an anus that opens ventrally (Schad, 1989).

In free-living S. stercoralis females, the apical view of the stoma shows the oral opening to be dorsoventrally elongated, with two cephalic lobes extending above it (Little, 1966a). Each cephalic lobe has three papillae, small cuticle extensions that, based on knowledge of other nematodes and the lack of an opening to the external environment, are thought to be mechanosensory (Basyoni and Rizk, 2016). The paired amphid sensilla are located posterior to the papillae (Little, 1966a).

Free-living adults and other free-living life stages of S. stercoralis have a rhabditiform (rod-shaped) pharynx, in contrast to the filariform pharynx of the parasitic female and iL3. A rhabditiform pharynx is found in many bacterivorous nematodes, suggesting that S. stercoralis free-living adults feed on bacteria (Schad, 1989). In total, the rhabditiform pharynx of an S. stercoralis free-living adult is approximately 20% of the body length (Schad, 1989) (Fig. 4). The rhabditiform pharynx has three distinct regions: the procorpus, isthmus, and bulb (Schad, 1989). The anterior pharynx consists of the muscular procorpus, which attaches to the stoma. The isthmus connects the procorpus to the bulb containing the grinder. As in other nematodes, rhythmic contractions of the procorpus and the bulb draw bacteria into the pharyngeal lumen and pass it through the grinder into the intestine (Schad, 1989; Mango, 2007). Although the S. stercoralis grinder has not been studied in detail, the C. elegans grinder is composed of specialized cuticular structures that break up bacteria before it is released into the intestine (Altun and Hall, 2009b). The pharyngeal-intestinal valve is located approximately one-fifth of the way along the body in the adult females (Schad, 1989) (Fig. 4).

The intestine of the free-living life stages of S. stercoralis is composed of 22 uninucleate cells arranged in pairs, one dorsal and one ventral (Schad, 1989). It terminates near the tail, with the anal opening on the ventral midline (Schad, 1989). The intestinal cells are uninucleate but due to endoreplication–replication of the chromosomes without cell division–each nucleus may contain up to 16 times the haploid genomic material (16C) (Hammond and Robinson, 1994b). In contrast, the C. elegans intestine in adult hermaphrodites is composed of 20 cells with a total of 30–34 nuclei, with each nucleus containing up to 32C (McGhee, 2007; Altun and Hall, 2009a).

The stoma, rhabditiform pharynx, and intestine of the free-living male are as described for the free-living female (Schad, 1989).

The iL3s have a compressed mouth and pharynx, reflecting the fact that iL3s are nonfeeding, developmentally arrested third-stage larvae similar to dauer larvae of nonparasitic nematodes (Viney et al., 2005; Ashton et al., 2007; Stoltzfus et al., 2012; Crook, 2014). The intestine of the S. stercoralis iL3 differs morphologically from the free-living intestine. The morphological differences are evident starting with L2s that are destined to develop into iL3s. The L2 intestine is composed of 22 cells in the same paired arrangement as the free-living adult intestine (Little, 1966a; Schad, 1989). However, there are 40 nuclei present, since all pairs except the most anterior and posterior pairs undergo nuclear division (Little, 1966a; Schad, 1989). At the iL3 stage, the intestine still has 22 cells with a total of 40 nuclei, but the intestine constricts, the lumen closes, and the intestinal cells fill with lipid droplets (Little, 1966a; Schad, 1989). After an iL3 enters an appropriate host, it initiates activation, the process by which it exits developmental arrest and resumes growth and feeding (Mendez et al., 2022). As part of this process, the pharynx and intestine expand, and the pharynx begins to pump (Fig. 7C).

The L1 pharynx occupies the first third of the body, while the intestine occupies the remaining two-thirds. The L1 intestine has the same basic structure as that of free-living males and females (Schad, 1989).

S. stercoralis parasitic and free-living females are genetically identical; however, they are morphologically and functionally distinct life stages. Environmental, genetic, and host factors, including the immune status of the host, influence whether a female larva develops via the free-living or parasitic route (Viney et al., 2004; Viney and Lok, 2015). Parasitic females reproduce inside the host by parthenogenesis, generating a mix of male and female progeny (Hammond and Robinson, 1994a; Streit, 2008). Free-living females reproduce sexually, mating with free-living males to generate female progeny that will develop into iL3s (Yamada et al., 1991; Jaleta et al., 2017; Shao et al., 2017). Sex determination in S. ratti has been characterized as an XX/XO system with two pairs of autosomes and either a pair of X chromosomes in parasitic and free-living females or a single X chromosome in males (Harvey and Viney, 2001). S. stercoralis likely has the same XX/XO sex determination system, since it has the same number of chromosomes as S. ratti (Hammond and Robinson, 1994a).

The reproductive system of the S. stercoralis parasitic female is amphidelphic, comprising two ovaries with opposed arms (Schad, 1989) (Figs. 2–3). The vulva opens on the ventral side and is located approximately two-thirds down the length of the body (Fig. 3). The egg-containing uteri extend anterior and posterior from the vulva opening and lead into ovaries that reflex, with the distal ends of the arms found opposite the vulva (Fig. 3). Importantly, the arms of the uteri are straight and do not spiral around the intestine. This characteristic is diagnostic of S. stercoralis and S. ratti (Schad, 1989); other Strongyloides species, including S. fuelleborni, have spiral ovaries (Little, 1966a). The uteri of parasitic females contain eggs in a single row (Schad, 1989) (Figs. 2,3).

Like the S. stercoralis parasitic female, the S. stercoralis free-living female has an amphidelphic reproductive system consisting of opposed gonad arms that recurve around the intestine (Schad, 1989). The vulva opening is located at approximately the ventral midpoint of the body (Fig. 4A). The uteri extend from the vulva opening in opposite directions and then curve back toward the center of the worm to end on the dorsal side of the body directly opposite the vulva (Schad, 1989) (Fig. 4A). The distal arm of the gonad is largely composed of giant polyploid nondividing nuclei (Hammond and Robinson, 1994a). Proximal to the giant nuclei is a region with small, highly compact nuclei, followed by a region with nuclei undergoing meiosis. Even more proximally, differentiated oocytes are found (Hammond and Robinson, 1994a). The distal gonad is syncytial and consists of germ cell nuclei in common cytoplasm surrounding a central rachis (Fig. 13A). The syncytial gonad permits the introduction of exogenous DNA by microinjection to generate transgenics or knockouts (Castelletto et al., 2020).

Figure 13:

The S. stercoralis male reproductive system is morphologically simpler than that of the female–it consists of a single, straight tube that terminates at the cloaca (Schad, 1989) (Fig. 4B). The distal end of the testis is situated near the pharyngeal-intestinal junction and contains giant nuclei containing multiple copies of the genome (Hammond and Robinson, 1994a). The giant nuclei are followed by a region of small nuclei. The next visually distinct zone contains primary spermatocytes actively undergoing division to create spermatids, which will mature into sperm (Hammond and Robinson, 1994a). The copulatory organ of S. stercoralis, a pair of chitinous spicules, is found near the end of the tail and is extruded ventrally (Schad, 1989) (Fig. 4B). Situated in the cloaca, the spicules are connected to a second chitinous structure, the gubernaculum. Once a female has been encountered, the male coils its tail around the center of the female and uses the gubernaculum to both extend the spicules into the female vulva and retract them postcopulation (Schad, 1989). As in females, the syncytial nature of the distal gonad allows exogenous DNA to be introduced by microinjection (Shao et al., 2017) (Fig. 13B).

In the L1, the future reproductive system, or genital primordium, consists of about nine cells near the midbody. The number of cells in the gonad begins to expand around the time of the L2 molt. Thus, the cell count in the primordial gonad can be used to separate L1s from L2s (Lopez et al., 2000). A similar round of cell expansion occurs around the L3 molt (Lopez et al., 2000). Males and females are easily identifiable by the L3 stage based on the morphology of the tail and developing gonad structures (Figs. 6A–D). L3 and L4 females can be distinguished based on the development of the vulva. The L3 vulva comprises a group of cells in the mid-body on the ventral side, with the arms of the immature uteri extending posterior and anterior. The L4 vulva has a more distinct structure, including an invagination covered by the cuticle. The anterior and posterior uteri arms and ovaries are longer in the L4, and their structure is more defined (Figs. 6E–F).