The genus Strongyloides contains around fifty species of parasitic nematodes that infect hosts ranging from reptiles to humans, with most species having a narrow host range consisting of one or a few host species (Speare, 1989; Viney and Lok, 2015). For example, the threadworm Strongyloides stercoralis infects humans, non-human primates, dogs, and cats, while the closely related species Strongyloides ratti infects rats (Viney, 2006; Viney and Kikuchi, 2017; Wulcan et al., 2019). The first description of S. stercoralis infections in humans was in 1876, when soldiers returning from the region that is now Vietnam began suffering from severe diarrhea (Bavay, 1877). S. stercoralis infections in humans cause strongyloidiasis, a disease that has been termed a “disease of disadvantage” due to its prevalence in low-resource settings with poor sanitation infrastructure (Beknazarova et al., 2016). Current estimates place S. stercoralis infections at over 600 million people worldwide, primarily in tropical and subtropical regions (Buonfrate et al., 2020). However, due to diagnostic challenges such as low larval count in stool specimens and low parasite burden (Ericsson et al., 2001; Greaves et al., 2013; Czeresnia and Weiss, 2022), the true prevalence of S. stercoralis is likely underreported.

S. stercoralis infects hosts as developmentally arrested, all-female, infective third-stage larvae (iL3s). The iL3s invade hosts primarily through skin penetration but also orally through passive ingestion (Czeresnia and Weiss, 2022). Within the host, the iL3s develop into parasitic adults and ultimately establish an infection in the host small intestine. Most of the progeny of the parasitic females exit the host in feces, develop into iL3s in the environment, and subsequently infect a new host. However, some of the female progeny complete their life cycle within the same host, a phenomenon that is termed autoinfection.

The severity of S. stercoralis infections among humans depends in part upon the health of the infected individual. S. stercoralis infections can be asymptomatic in healthy individuals, or they can cause gastrointestinal and pulmonary distress as well as eosinophilia (Czeresnia and Weiss, 2022). Cycles of autoinfection in healthy patients can result in chronic strongyloidiasis, which can persist undetected for decades. In contrast, infections are often fatal in immunosuppressed individuals or individuals infected with certain viruses such as human T-lymphotropic virus 1 (HTLV-1) (Czeresnia and Weiss, 2022). In these cases, the population of parasites engaging in the autoinfective cycle can increase rapidly, leading to hyperinfection and disseminated strongyloidiasis (Czeresnia and Weiss, 2022; Herbert et al., 2022).

The development of Strongyloides species into genetically tractable model organisms has been bolstered by several aspects of their life cycle and morphology. Both S. stercoralis and S. ratti can be cultured in the lab by passaging them through mammalian hosts. S. stercoralis can be passaged through dogs as a natural host or gerbils as a laboratory host (Lok, 2007), and S. ratti can be passaged through its natural host, the rat (Viney and Kikuchi, 2017). In addition, Strongyloides species and the closely related Parastrongyloides species are unique among parasitic nematodes in that they are capable of cycling through one or a limited number of free-living generations (Schad, 1989). In the case of S. stercoralis and S. ratti, they can cycle through multiple consecutive parasitic generations but only a single free-living generation (Mendez et al., 2022) (SIntroFIG 1). Free-living Strongyloides adults are similar in size and morphology to Caenorhabditis elegans adults and can be cultured on the standard media that is used to culture C. elegans, making the free-living stages easily accessible for experimentation. Moreover, like C. elegans adults, Strongyloides free-living adults have a syncytial gonad. Thus, exogenous DNA can be introduced into Strongyloides free-living adults by intragonadal microinjection to generate transgenics and knockouts in their progeny using techniques adapted from C. elegans (Lok, 2007; Castelletto et al., 2020; Castelletto and Hallem, 2021; Mendez et al., 2022). Except for the Strongyloides and Parastrongyloides species, other mammalian-parasitic nematodes lack a free-living generation and as a result are less easily amenable to genetic manipulation. Thus, both S. stercoralis and S. ratti have become genetic model systems for the study of parasitic nematode biology.

2 S. stercoralis Life Cycle

S. stercoralis parasitic adult females colonize the mucosal layer of the host small intestine. They produce eggs via mitotic parthenogenesis and deposit them in the mucosal epithelium, usually in the crypts of Leiberkühn (Dionisio et al., 2000). The eggs hatch into male and female post-parasitic first-stage larvae, which then make their way into the host intestinal lumen (Little, 1966). All male (XO) larvae pass into the environment with the feces and develop into free-living adults. The female (XX) post-parasitic larvae have three potential developmental routes: 1) They can pass into the environment with the feces and execute four molting cycles to become free-living adults. The free-living male and female adults reproduce sexually in the environment to produce all-female larvae that are restricted to becoming iL3s. 2) They can pass into the environment and develop through two larval stages to become iL3s. 3) They can develop through two larval stages within the host intestine into autoinfective third-stage larvae (aL3), which subsequently develop to adulthood inside the same host (SIntroFIG 1). The ability to alternate between homogonic (direct to parasitism) and heterogonic (indirect to parasitism) life cycles is unique to nematodes in the genus Strongyloides and Parastrongyloides, and the ability to complete the life cycle within the same host through the generation of autoinfective larvae is thought to be unique to S. stercoralis (Grant et al., 2006; Nutman, 2017; Czeresnia and Weiss, 2022).

S. stercoralis iL3s are developmentally arrested third-stage larvae that resemble C. elegans dauer larvae (Viney et al., 2005; Ashton et al., 2007; Stoltzfus et al., 2012; Crook, 2014). The iL3s are soil-dwelling, and must navigate their environment to find and invade a host (Mendez et al., 2022). Once inside the host, the iL3s exit developmental arrest in a process called activation and migrate through the body to the lungs (Genta and Gomes, 1989; Czeresnia and Weiss, 2022). During this migration, the worms are thought to molt into fourth-stage larvae (Schad, 1989). They pass through the capillaries into the alveoli, causing tissue damage. Eventually, the larvae are coughed up and swallowed into the digestive system. Once the larvae have passed through the stomach and into the proximal small intestine, the duodenum, the larvae molt into reproductive parasitic adult females. Larvae undergoing autoinfective development penetrate through the host intestinal wall or perianal skin, and then follow the same migratory route as iL3s, ending up as parasitic adults in the small intestine (Czeresnia and Weiss, 2022). In some cases, migrating larvae may bypass the lungs and arrive in the intestine via other routes (Schad et al., 1989; Mansfield et al., 1995).

3 Anatomy

3.1 Body Shape

Parasitic female
The Strongyloides 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) (SIntroFIG 2A & SIntroFIG 3A). S. stercoralis is commonly called the threadworm because of its long, slender morphology (Lindo and Lee, 2001). The morphology of the S. ratti parasitic female resembles that of the S. stercoralis parasitic female (SIntroFIG 3B).

Free-living female
Strongyloides free-living females resemble C. elegans hermaphrodites in size and morphology. S. stercoralis free-living females are approximately 1.1 mm long and 62 µm wide, with a conical pointed tail (Schad, 1989) (SIntroFIG 2B & SIntroFIG 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 840 somatic nuclei (Hammond and Robinson, 1994a), which is slightly less than the 959 somatic nuclei found in C. elegans adult hermaphrodites (Wood, 1988). As is the case for C. elegans, the Strongyloides cuticle and body wall are transparent, allowing easy visualization of internal structures.

Free-living male
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 (SIntroFIG 2C & SIntroFIG 4B). Their broad tail is slightly curved and contains a pair of spicules used for insemination of the females (SIntroFIG 4B). The reproductive structures of the free-living male can be identified in early larval stages (SIntroFIG 5A-B).

Rhabditiform larva
Rhabditiform larvae include first-stage (L1) and second-stage (L2) larvae that are destined to become either free-living or parasitic adults, as well as third-stage (L3) and fourth-stage (L4) larvae that are destined to become free-living adults (SIntroFIG 5). The 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 that are destined to become free-living adults are essentially larger versions of the L1s (Schad, 1989). In contrast, 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).

Filariform larva
iL3s are also termed filariform larvae based on the structure of their pharynx. The morphology of the filariform larva is quite 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 40% of the body length, making this larva easy to identify under low magnification (Schad, 1989) (SIntroFIG 6A). The tail of the S. stercoralis iL3 is notched (SIntroFIG 6B). The width of the iL3 increases substantially during the activation process as the pharynx expands and the larva resumes feeding (SIntroFIG 6C).

3.2 Organs and Tissues

3.2.1 Cuticle

The body wall of nematodes is comprised of 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). Studies of the Strongyloides cuticle have focused on parasitic females and iL3s, since these two stages interact with the host. Similar in structure to the cuticle of other nematodes, the Strongyloides cuticle is acellular and is composed of collagens, cuticulin, and assorted surface proteins (Politz and Philipp, 1992; Martinez and de Souza, 1995; Martinez and de Souza, 1997; Page and Johnstone, 2007). In the iL3, paired lateral alae run the length of the worm and create the notched tail (Nichols, 1956; Schad, 1989).

Parasitic female
Our knowledge of the Strongyloides parasitic female cuticle 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) (SIntroFIG 7A-B). The epicuticle is covered in a surface coat that interacts directly with host tissues (Martinez and de Souza, 1995). The surface coat of parasitic nematodes is thought to be critical for the ability of parasitic nematodes to evade the host immune system (Blaxter et al., 1992). Furthermore, in some species, the nematode appears to shed surface coat proteins once inside the host, resulting in a dynamic coat composition that escapes host antibodies (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 (SIntroFIG 7B). The composition of the fibrous layer is similar, except that the fibers are found in a more parallel orientation (SIntroFIG 7A). 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).

Filariform larva
The molecular composition of nematode cuticles varies across life stages (see C. elegans Hermaphrodite Cuticle and Dauer Cuticle). Like the cuticle of C. elegans dauer larvae, the cuticle of the iL3 is more resistant to the environment than that of other life stages (Martinez and de Souza, 1997). The iL3s have a distinctive cuticle with two pairs of lateral alae running the length of the worm and extending slightly past the tip of the tail (SIntroFIG 8A-D). These alae give the iL3 a characteristic notched tail, which can be used to differentiate S. stercoralis iL3s from hookworm iL3s (Nichols, 1956; Schad, 1989; Lindo and Lee, 2001; Riyong et al., 2020) (SIntroFIG 8D). 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). The basic structure of the cuticle of a Strongyloides iL3 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 wide (Martinez and de Souza, 1997). Once the iL3s have penetrated through host skin, the surface coat disappears (Grove et al., 1984; Grove et al., 1987). The purpose of this coat and its molecular composition are unclear.

3.2.2 Epithelial System

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 (see C. elegans Hermaphrodite Hypodermis and Aging Hypodermis). In recent literature, the hypodermis is sometimes called the epidermis because it derives from the ectoderm (Hermaphrodite Hypodermis). 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 (Hermaphrodite Hypodermis; Introduction to P. pacificus). 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 Strongyloides hypodermis, except that the dorsal and ventral cords are reduced in size compared to C. elegans and P. pacificus, and the lateral cords are larger than the dorsal-ventral cords (Little, 1966).

3.2.3 Nervous System
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 a pair of amphid sensilla in the head that house many of the sensory neurons (SIntroFIG 9) (Ashton et al., 1995). 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).

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 (SIntroFIG 9) (Ashton et al., 1995). 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).

The pharyngeal nervous system of S. stercoralis has not yet been studied.

3.2.4 Muscle System

As in other nematodes, the body wall muscle of Strongyloides is separated from the hypodermal layer by a basal lamina (Basyoni and Rizk, 2016). Strongyloides musculature is platymyarian (the muscle fibers are adjacent and perpendicular to the hypodermis) and meromyarian (comprised of eight longitudinal muscle cells) (Little, 1966; 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 Strongyloides.

3.2.5 Excretory System

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 (SIntroFIG 10) (Nichols, 1956; Little, 1966; Schad, 1989) . 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.

Studies of the excretory/secretory (ES) proteins of Strongyloides have identified a wide variety of proteins that could be important for manipulation of the host immune response (Marcilla et al., 2010; Soblik et al., 2011; 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 and how they are released from the worm is unclear. The role of the excretory system in ES release is an open question and could be instrumental in understanding Strongyloides host-parasite interactions.

3.2.6 Coelomocyte System

The coelomocyte system of Strongyloides has not yet been characterized.

3.2.7 Alimentary System

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 Strongyloides differ significantly in parasitic females, free-living females, and iL3s.

Parasitic female
The stoma of the parasitic female can be used as an identifying feature for the different Strongyloides species (Little, 1966; Sato et al., 2008). In the case of S. stercoralis, the stoma is classified as angular and looks hexagonal when viewed en face. The stoma is surrounded by the circumoral elevation, a slightly elevated cuticle structure that consists of three paired lobes in S. stercoralis. Strongyloides procyonis, a parasite of raccoons, also has a hexagonal stoma shape (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 füelleborni.

The filariform (filament-shaped) pharynx of the parasitic female extends up to one-third of the length of the body (Speare, 1989). 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 in Strongyloides are unclear, but it is possible that these secretions play a role in host invasion or immune modulation. The pharynx connects to the intestine. In parasitic females, the intestine is a thin tube comprised of forty cells in two rows (Schad, 1989). It terminates near the tail in an anus that opens ventrally (Schad, 1989). The pharyngeal-intestinal junction is an easy-to-locate landmark a third of the way down the body.

As in other nematodes, the intestine is responsible for the uptake of nutrients, disposal of waste products, and transport of signaling molecules (McGhee, 2007; see also C. elegans Hermaphrodite Intestine). 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 (IntFIG 3). Adherens junctions secure the epithelial cells together at the apical side (IntFIG 5C-F). 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 (IntFIG 1; Basyoni and Rizk, 2016). The intestine is lined with microvilli covered in a glycocalyx matrix (IntFIG 5C-F). The Strongyloides parasitic female intestine has 40 uninucleate cells with unspecified genomic content (Little, 1966; Schad, 1989). In at least some Strongyloides species, the central region of the intestine may be a syncytium (Colley, 1970).

Free-living female
In free-living females, the apical view of the stoma shows two cephalic lobes extending above the mouth. Each cephalic lobe has three papillae, small cuticle extensions that are thought to have mechanosensory function. The paired amphid sensilla are located posterior to the papillae (Ashton et al., 1995; Fine et al., 1997; Ashton et al., 1999).

Free-living adults and other free-living life stages of Strongyloides 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 Strongyloides 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). 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. 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 Strongyloides 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 (see C. elegans Hermaphrodite Pharynx; PhaFIG 2). The pharyngeal-intestinal valve is located approximately one-fifth of the way along the body in the adult females (Schad, 1989).

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; C. elegans Hermaphrodite Intestine).

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

Filariform larva
The iL3s have a compressed mouth and pharynx, reflecting the fact that iL3s are non-feeding, developmentally arrested third-stage larvae similar to free-living dauer larvae (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 intestine (Little, 1966; Schad, 1989). However, there are 40 nuclei present since all pairs except the most anterior and posterior pairs undergo nuclear division (Little, 1966; 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, 1966; Barrett, 1968; 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 (Mimori et al., 1982) (SIntroFIG 6C).

Rhabditiform larva
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).

3.2.8 Reproductive System

Sex determination in S. ratti has been well 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). Parasitic and free-living females are genetically identical; however, they are morphologically and functionally distinct life stages. Environmental, genetic, and host factors influence whether a female worm develops via the free-living or parasitic route (Viney and Lok, 2015). Similarly, the proportion of free-living males in the population is environmentally controlled – while males are determined by their XO genotype, X chromosome elimination depends on environmental signals sensed by the parasitic female (Streit, 2008; Streit, 2014). The percentage of males in two-day-old fecal cultures isolated from chronically infected humans was reported to range from 4% to 45% (Hammond and Robinson, 1994a). Free-living males and females reproduce sexually to generate female progeny that will develop into iL3s. This all-female generation arises because free-living males produce mature sperm that are solely X-containing; nullo-X sperm are eliminated post-meiotically (Dulovic et al., 2022). In contrast, parasitic females reproduce inside the host by parthenogenesis (Streit, 2008).

Parasitic female
The reproductive system of the parasitic female is amphidelphic, comprised of two ovaries with opposed arms (Schad, 1989) (SIntroFIG 2 & SIntroFIG 3). The vulva opens on the ventral side and is located approximately two-thirds down the length of the body. The egg-containing uteri extend anterior and posterior from the vulva opening and lead into ovaries that reflex, with the ends of the arms found opposite the vulva. 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. füelleborni, have spiral ovaries (Little, 1966). The uteri of parasitic females contain relatively few eggs in a single row (Schad, 1989) (SIntroFIG 2 & SIntroFIG 3).

Free-living female
Like the parasitic female, the 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. 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. In contrast to the uteri of parasitic females, the uteri of free-living females are densely packed with eggs (Schad, 1989) (SIntroFIG 4A). The germline of Strongyloides free-living females has been studied primarily in S. ratti and the ruminant parasite Strongyloides papillosus. Morphologically, the Strongyloides germline resembles that of C. elegans. However, many differences between the two species exist on a cellular level. In C. elegans, the distal tip of each gonad arm contains a distal tip cell (DTC), and Notch signaling from the distal tip cell causes the distal germ cells to undergo mitosis. As the germ cells move away from the DTC, they switch from mitosis to meiosis and begin to differentiate into gametes (C. elegans Hermaphrodite Germline; GermFIG 3A-D). In Strongyloides, although there is a DTC-like cell, the distal arm of the gonad is largely composed of giant polyploid nondividing nuclei (Kulkarni et al., 2016). Germ cell mitosis occurs only in the young larval germline, not the L4 or adult germline. 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 (Kulkarni et al., 2016). As in C. elegans, the distal germline is syncytial and consists of germ cell nuclei in common cytoplasm surrounding a central rachis (SIntroFIG 11A). However, in contrast to the C. elegans rachi, the Strongyloides rachi do not extend the entire length of the distal gonad (Kulkarni et al., 2016). The syncytial gonad permits the introduction of exogenous DNA by microinjection to generate transgenics or knockouts (Castelletto et al., 2020).

Free-living male
The Strongyloides male reproductive system is morphologically simpler than that of the female – it consists of only a single, straight tube that terminates at the cloaca (Schad, 1989) (SIntroFIG 4B). The distal end of the testis is situated near the pharyngeal-intestinal junction and contains giant nuclei containing multiple copies of the genome. The giant nuclei are followed by a region of small nuclei (Dulovic et al., 2022). The next visually distinct zone contains primary spermatocytes actively undergoing division to create spermatids, which will mature into sperm (Dulovic et al., 2022). The copulatory organ, a pair of chitinous spicules, is found near the end of the tail and is extruded ventrally. 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 post-copulation (Schad, 1989). The cellular organization of the Strongyloides male gonad resembles that of the female gonad (Kulkarni et al., 2016), and as in females, the syncytial nature of the distal germline allows exogenous DNA to be introduced by microinjection (SIntroFIG 11B) (Shao et al., 2017).

Rhabditiform larva
In the L1, the reproductive system consists of only a few cells near the midbody. The gonads begin to develop in the L2s, and their cell count can be used to separate L2s from L3s (Lopez et al., 2000). Males and females are easily identifiable by the L3 stage based on the morphology of the tail and developing gonad (SIntroFIG 5A-D). L3 and L4 females can be separated based on their developing vulvas. The L3 vulva comprises a group of cells in the mid-body on the ventral side, with the arms of the developing uteri extending posterior and anterior. The L4 vulva has a more defined 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 (SIntroFIG 5E-F).

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