A medium for the maintenance of Chironomus tentans salivary glands in vitro

Abstract A synthetic medium based upon the chemical composition of fourth instar Chironomus haemolymph was formulated for the in vitro culture of Chironomus tentans salivary glands. Salivary glands maintained in the medium for up to 4 days appeared morphologically normal. Secretion-free glands, obtained from pilocarpine-treated larvae, accumulated proteinaceous material in the gland lumen and exhibited a 46% increase in total gland protein after 24 hr in the medium. Cycloheximide almost totally inhibited the accumulation of secretion material and the increase in total gland protein by cultured glands. Glands cultured for up to 4 days continued to incorporate 14 C-leucine into acid-insoluble total protein and 3 H-uridine into total RNA, but at reduced levels. The incorporation of both isotopes was almost completely inhibited by cycloheximide. Autoradiographic squash preparations of glands pulse-labelled with 3 H-thymidine after 3 days in culture revealed a normal pattern of asynchronous chromosomal DNA replication. Glands cultured for up to 4 days exhibited 3 H-uridine incorporation into nucleoli and into distinct chromosomal regions which corresponded with sites of cytochemically demonstrable acidic protein. The chromosomes of cultured glands appeared morphologically and cytochemically normal, except for some regression of the Balbiani rings. Addition of ecdysterone to media containing glands previously cultured for 3 days resulted in puff induction at the IV-2-B chromosomal locus.