Conserved behavioral and genetic mechanisms in the pre-hatching molt of the nematode Pristionchus pacificus

During development, juvenile nematodes undergo four molts. Although the number of molts appears to be constant within the Nematoda, the timing of the first molt can occur either before or after hatching. A previous study indicates that, as in some parasitic nematode lineages, a pre-hatching juvenile stage also exists in Diplogastrid nematodes. A detailed description of these sequence of events has yet to be shown for any single species.

To delineate the timing of the pre-hatching molt in the beetle-associated Pristionchus pacificus, we tracked individual mid-J1 stage worms inside the eggshell through the J1-J2 transition and hatching. We found that active movement ended 21 hours after egg-laying, followed by lethargus and hatching. We inferred that lethargus behavior represents the onset of the first molt, which precedes each post-hatching molt in C. elegans and P. pacificus. The onset of the J1-J2 molt was also marked by the upregulation of the P. pacificus molting marker Ppa-pnhr-1. We further corroborated the pre-hatching molt with the isolation of two genetic mutants that exhibited aberrant molting both inside the egg and after hatching, as characterized by protracted and often-aborted shedding of the old cuticle.

Our results describe in detail the pre-hatching juvenile molt in P. pacificus, provide strong visual evidence of a pre-hatching molt, and show support for common genetic mechanisms regulating molting in the pre-hatching and post-hatching developmental stages. Our findings support the hypothesis that the evolution of pre-hatching development in Diplogastrid nematodes is likely due to a heterochronic shift between the timing of the first molt and hatching.