Figure 3. Sulforaphane delays age-associated physiological decline. (A) L4 larvae were exposed to 100 μM sulforaphane (SF) or not (CO), and 20 worms per group were used for evaluation. Pharyngeal pumping was measured on days 6, 9 and 12 by the evaluation of the opening of the corpus (anterior pharynx, green arrow, image on the right) and the terminal bulb (red arrow), which contract and relax synchronously during pharyngeal pumping. The pump frequency per minute was calculated and is shown in the diagram on the left. (B) In L4 larvae treated as described above, the number of body bends (images on the right) per minute was evaluated (diagram on the left) to assess mobility. (C) At day 12 after sulforaphane treatment, blue autofluorescence, representing lipofuscin accumulation (images on the right), was detected by fluorescence microscopy. The blue autofluorescence in the figure indicates the accumulation of lipofuscin. The relative fluorescence intensity was evaluated by using ImageJ software, and the fluorescence of the untreated nematodes was set as 1. The scale bar indicates 0.1 mm. (D) L4 larvae were treated with sulforaphane (SF) or not (CO). Twenty-four hours later (Day 1), the number of eggs laid was counted under the microscope daily for 7 days. The number of eggs laid each day and the total number of eggs are shown in the diagram on the left. The green arrow in the diagram on the right indicates eggs in a worm, and the red arrow indicates eggs that are being laid by the worm. The data are shown as the mean ± SD as evaluated by Student's t test using Prism 6.0. *P< 0.05, **P < 0.01.