Lethargus-a sleep-like stage occurring during C. elegans larval development-is regulated by some of the same mechanisms that regulate sleep in mammals and fruit flies, suggesting that lethargus can be used to study sleep. To monitor the behavioral quiescence of 24 nematodes simultaneously, we have developed the Caenorhabditis-in-Drop (CiD) method, which entails automated videography of worms housed individually in 125 nL aqueous droplets on a polydimethylsiloxane (PDMS) surface and covered with mineral oil. We used the CiD method to demonstrate reduced quiescence in
kin-2 mutants, and to reproduce previous observations of increased quiescence in animals over-expressing LIN-3C (Van Buskirk and Sternberg 07). We found that
egl-4 mutants, which have reduced quiescence when housed on an agar surface (Raizen et al, 08), had no change in lethargus quiescence in CiD. This indicates that the chamber used to house the animal can affect behavior and raises a note of caution in the interpretation of both positive and negative results. In comparison to behavior on an agar surface, animals in CiD were more active and showed reduced survival, with most animals dead after one week. Housing the worms in a 10-fold higher volume (1250 nL) of aqueous solution on a PDMS surface increased survival in comparison to survival in 125-nL droplets, indicating that reduced longevity is not explained by the liquid habitat, by exposure to mineral oil, or by exposure to the PDMS surface. Surprisingly, animals survived longer in 125-nL droplets that previously housed a worm for a week.