Pharyngeal pumping rate does not reflect lifespan extension in C. elegans transgenerational longevity mutants.

Epigenetic modifications must be reprogrammed with each new generation. In Caenorhabditis elegans , defects in histone methylation reprogramming allow for the transgenerational acquisition of longevity. For example, mutations in the putative H3K9 demethylase JHDM-1 extend lifespan after six to ten generations. We noticed that long-lived jhdm-1 mutants appear healthier than wild-type animals from the same generation. To quantify health, we compared the common metric of pharyngeal pumping rate at specific adult ages between early-gen populations with normal lifespans and late-gen populations with long lifespans. Longevity did not affect pumping rate, but long-lived mutants stop pumping at a younger age, suggesting a possible conservation of energy to extend lifespan.

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining.

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.