The development of a successful in vitro rearing system for mermithids has been slow because of a lack of readily available material. Consequently, most basic in vitro research has been done with R. culicivorax. Another major problem involves the nature of food ingestion in mermithids. Unlike most nematodes, mermithids lack a functional gut and must rely on transcuticular uptake of nutrients. Since these nematodes feed only during their parasitic phase while in the hemocoel of the host, nutritional products must be in a form that can be readily absorbed through a selective and delicate cuticle (Poinar and Hess, 1977; Rutherford and Webster, 1974). Thus, they are very sensitive to mechanical damage and to hypo- and hypertonic changes.
In initial studies with R. culicivorax, Sanders et al. (1973) reported growth of 1-4 mm in length after 15 days and 5-7 mm after 25 days and some stichosome and trophosome development when preparasites were placed in Schneider's Drosophila medium with 10% fetal calf serum at pH of 6.5-7.0 and 25'C. Growth rates were about one-third that of nematodes grown in vivo.
More recently, Castillo et al. (1982) tested more than 50 combinations of vertebrate and invertebrate tissue culture media and microbiological media. Slow growth and limited development of internal structures were obtained with various supplemented Grace's tissue culture media and Schneider's Drosophila media. Nematodes attained a stage of development after 3-4 weeks comparable to that attained in the host after 4-5 days.
The most successful attempts to rear R. culicivorax in vitro were reported by Finney (1981). She reported that the best results were obtained using Grace's medium containing 10% fetal calf serum at 26'C with osmotic pressure adjusted to 240 mosm and pH 6.4-6.5. Slow growth of the nematodes occurred over a 6-week period during which only juvenile females developed, but storage material in the trophosome was lacking. Finney has since reduced the time to reach parasitic maturity to 3 weeks with the trophosome filled with material. However, she was still unable to produce male nematodes (Chapman and Finney, 1982).
In an important breakthrough, Fassuliotis and Creighton (1982) were successful in rearing F. leipsandra, a parasite of agricultural pests, in vitro. After unsuccessful attempts with several media, the researchers found that juveniles of F. leipsandra would develop to the postparasitic stage on Schneider's Drosophila medium containing fetal bovine serum. To complete the life cycle the nematodes had to be transferred to a solid substrate which preconditioned them and triggered the mechanism necessary for the final molt to the adult stage. In vitro development took about I week longer than the time it takes F. leipsandra to be reared in vivo. This accomplishment may aid greatly in determining if economical in vitro rearing methods for other mermithids are practical.
