Fig 1.
The life cycle of H. glycines.
H. glycines hatch as mobile second-stage juveniles (J2s), migrate to the host roots, infect and initiate feeding. Following the initiation of feeding, the body diameter increases and the nematodes become sedentary. J3s remain sedentary and continue feeding. During late J3, we are able to distinguish sexes. Following the molt to J4, males begin remodeling into a vermiform shape while females continue to feed and grow in width. Following the final molt, adult males are fully mobile and seek out females to inseminate. Adult females remain immobile and continue to feed. Each life stage is not represented to scale.
Fig 2.
Body wall muscles atrophy during sedentary stages.
Fluorescent micrographs of phalloidin-stained H. glycines. The J2 body wall muscles (image from head region) contain actin-enriched striated filaments obliquely oriented to the longitudinal axis. In J3, the diameter of muscle filaments is smaller than in J2 and lacks a well-defined organization. The phalloidin-stained body wall muscle filaments are thicker in J4 males compared with J3. In the adult male, the body wall muscles contain additional filaments, but are otherwise similar to J2 body wall muscles. In J4 females, the size of the actin-enriched muscle filaments is further reduced compared to J3s. Also see S1 Fig. Scale bars, 5 μm.
Fig 3.
H. glycines body wall muscle undergoes ultrastructural changes during development.
Transverse TEM sections of body wall muscle in the midbody of H. glycines (left) and with pseudocolor overlay (right). (A) Mobile J2 H. glycines have intact muscle separated from the cuticle by a thin layer of epidermis. (B) Sedentary J3 muscle shows signs of atrophy including fewer thick and thin filaments and a thickening of epidermis. (C) J4 male muscle comprise distinct sarcomeres, but with reduced numbers of thick filaments and an epidermal thickness similar to J3s. (D) J4 females undergo further atrophy of sarcomeres and increased thickening of the epidermis beyond that seen in J3s. (E) The muscles of adult males are similar in orientation and size to J2 muscle. Scale bar, 1 μm.
Table 1.
Ultrastructural measurements of H. glycines.
Fig 4.
Motor neurons degenerate in the ventral nerve cord during H. glycines development.
Lateral and sub-ventral micrographs of DAPI-stained mobile J2 (top) and sedentary J4 female (bottom), respectively. VNC neuronal nuclei (arrowheads) are highly condensed fluorescent puncta. VNC nuclei in immobile J4 females deviate from the linear pattern seen in mobile J2s.
Fig 5.
J4 male H. glycines undergo extensive remodeling.
(A) DIC micrograph of J4 male remodeling from sausage-shaped J3 to vermiform adult. At this stage the male is wrapped within the J3 cuticle. (B) Transverse TEM micrograph of J4 male at approximate location of box in (A). Note that the TEM micrographs are not of the same animal as in (A). TEM demonstrate the various parts of the male during J4 are wrapped within a thick extracellular matrix. (C) High magnification of boxed region in (B) showing the VNC (purple) with synaptic vesicles and adjacent body wall muscle (green). The epidermis (tan) at this time point is much thicker than during the mobile J2 or the adult male stage (see Table 1). Scale bar, 1 μm.
Table 2.
Developmental changes to the ventral nerve cord in sedentary and mobile nematodes.
Fig 6.
hg-UNC-25 synthesizes GABA in H. glycines.
The hg-unc-25 cDNA from H. glycines partially rescues the C. elegans unc-25(e156) mutant. In wild-type C. elegans, GABAergic neurons are detected in the head and ventral nerve cord (arrowheads) using anti-GABA staining. In the C. elegans unc-25(e156) mutant, GABA is not produced and GABAergic neurons cannot be detected using anti-GABA staining. A plasmid construct containing hg-unc-25 cDNA driven by the C. elegans unc-25 promotor partially rescues the C. elegans unc-25(e156) mutant as seen by GABA-immunoreactivity (arrowheads). Arrows indicate the expression of coel::RPF used as a co-injection marker. Scale bars, 10 μm.
Fig 7.
hg-unc-25 is down regulated and fewer GABA-immunoreactive neurons are detected at the sedentary stages.
(A) Relative mean ± standard error expression of hg-unc-25, based on RT-qPCR. Data were normalized to J2 with hg-far-1 used as an internal control. Treatments with different letters are statistically different at α = 0.05 as determined by ANOVA. (B) Lateral left view cartoon (top) and representative fluorescent micrographs of GABA-immunostained J2 H. glycines sections (bottom). GABA immunostaining was conducted on bisected animals to facilitate penetration. The location of individual neurons was determined by their position relative to landmarks such as the esophagus (purple) and the primordial gonad (blue). GABA-immunoreactive neurons (green) are located surrounding the nerve ring, between the metacorpus and the esophageal glands, and along the VNC. (C) Ventral view fluorescent micrograph of anti-GABA stained H. glycines J4 female. Scale bars, 10 μm.
Fig 8.
The independently evolved sedentary nematode M. incognita undergoes muscle atrophy during development.
Fluorescent micrographs of phalloidin-stained Meloidogyne incognita mobile J2s (A) and sedentary post-infection stage (B). Similar to H. glycines, M. incognita undergoes cell-specific muscle atrophy with only remnant body wall muscle stripes (arrowheads) apparent in sedentary stages. Scale bar, 10 μm.