Isolation and Cultivation of Auanema Nematodes

Auanema rhodensis n. sp. (strain SB347) was originally isolated from blood-engorged deer ticks (Ixodes scapularis) that were used as bait for nematodes. The ticks were placed in the upper layer of the soil in Kingston (University of Rhode Island), R.I., United States, in September 2001 by E. Zhioua (W. Sudhaus, pers. comm.). Subsequently, a laboratory culture of A. rhodensis SB347 was established by W. Sudhaus^{14 (link)}. A. freiburgensis n. sp. (strain SB372) was isolated from a dung pile in Freiburg, Germany, in August 2003 by W. Sudhaus. Both strains have been kept in the laboratory on NGM plates seeded with Escherichia coli OP50, as is standard for C. elegans²⁶ and preserved cryogenically (e.g. at the NYU Rhabditid Collection).
Both species produce males and females, and hermaphrodites after passage through the dauer stage^{19 (link)}. The genders were collected separately as follows. In A. rhodensis, most female embryos are produced by their mother within the first 15 hours of adulthood^{19 (link), 22 (link)}. To obtain females, dauer juveniles were placed individually on a small agar plate seeded with OP50 and cultured at 20 °C until adulthood. After these hermaphrodites oviposited 25 eggs or fewer, they were removed. The F1 generation developed into adult females. To obtain hermaphrodites, dauer juveniles were transferred from old cultures onto seeded NGM plates and collected with the Baermann funnel technique after they reached adulthood. Males were hand-picked from 3- to 7-day-old cultures. For A. freiburgensis, females were obtained by letting hermaphrodites self-fertilize on individual plates. Most self-progeny under these conditions are either female or male. Hermaphrodites were obtained by isolating dauer juveniles from crowded plates and letting them develop into adults.