Dissecting the teratogenic effects of an insect host pheromone on Pristionchus pacificus embryos

Developmental toxicity can be caused by biotic and abiotic factors known as teratogens. Given the success of nematodes in inhabiting most of the Earth's known ecological niches, we know very little about the possible environmental teratogens that can adversely affect early nematode development inside their tough eggshells. Plant parasitic nematodes can suffer delayed hatching and irreversible arrested development when bathed in solutions of volatile fatty acids released during plant decomposition. Human-made environmental toxins, notably plasticizers in PVC, are known to cause embryonic and other developmental defects. Other elements, including barium and mercury, are also known to cause a reduced body size and abnormal vulvas in exposed C. elegans embryos. Quite unexpectedly, the volatile host pheromone of the nematode Pristionchus pacificus can irreversibly arrest early stage embryos but it is unclear how and what developmental stage embryos succumb to permanent arrest. To characterize the threshold of sensitivity to the oriental beetle pheromone ZTDO, we determined the minimum exposure duration and developmental window that would result in arrested embryos. While most unexposed embryos hatch as J2 larvae after 24 hours, we found that exposing embryos to ZTDO for as short as 3 hours during gastrulation can arrest embryonic development permanently. We referred to these active embryos that develop abnormally and do not hatch as "zombryos." To further determine whether the beetle pheromone arrests embryonic development indirectly by inhibiting gas exchange, or directly by entering the eggs' shells and disrupting normal developmental processes, we utilized the membrane-specific lipophilic dye FM®4-64 known to stain the membrane layers of pre-anaphase oocytes in C. elegans. We found that FM®4-64 stained the plasma membranes of embryonic tissues, suggesting that the ZTDO pheromone likely penetrates the eggshell and its permeability barrier. By contrast, in unexposed embryos only the outermost vitelline layer of the eggshell is stained by FM®4-64. As further support that ZTDO acts on embryonic development directly, we show that non-exposed P. pacificus embryos raised in an anaerobic chamber result in delayed hatching but otherwise normal phenotype, and thus limiting gas-exchange in the embryo does not phenocopy the effects of ZTDO exposure. These results support the hypothesis that ZTDO may disrupt the permeability barrier and interfere with cellular functions, rather than preventing gas exchange. To determine the genetic factors involved in ZTDO-induced arrest, we conducted an EMS mutagenesis for resistance to ZTDO. The strongest ZTDO-arrest resistant allele, zar-5, shows a delayed in hatching when exposed to ZTDO but has a superficially normal phenotype. The zar-5 eggshell also appears be more resistant to ZTDO since FM®4-64 stains only the eggshell of zar-5 embryos. This study challenges our stereotype about the impermeable nematode egg and shows that the nematode embryo is sensitive to a host pheromone that acts as a developmental teratogen.