Ellsworth C. Dougherty

Introduction to axenic culture of invertebrate Metazoa: a goal.

As consulting editor, I saw and now take the opportunity to amalgamate concepts scattered in the papers that follow. My hope also is to bring a certain unity to the heterogeneous studies contributed by the imaginative and resourceful workers who created the program of the conference on which this monograph is based. Axenict cultivation, the rearing of one or more individuals of a single species on a nonliving medium (Baker and Ferguson, 1942; Dougherty, 1953a), dominates microbiology. Axenic cultures, usually referred to as “pure cultures,” are fundamental to identification of many protists (bacteria, algae, fungi, and protozoa) and to their exploitation in research. By contrast, cultivation of the Metazoa brings to my mind the Mesozoic, teeming with spectacular, untamed beasts-a latter-day Mesozoic, of course, for man is loose in its jungles, which he has thinly peopled for many years. ‘‘ Germ-free ” rearing of metazoa (namely, vertebrates) was attempted in the late Nineteenth Century (Baker and Ferguson, 1942; Reyniers and Trexler, 1943), but only in recent years have systematic efforts a t rearing a variety of species been rewarding. The importance of such studies is well expressed by Kidder (1953) in his review of invertebrate nutrition. Still, i t would be misleading to ignore our debt to pioneer efforts of European workers, beginning with a Russian and especially including the French. Thus Bogdanov (1908), in partly equivocal experiments, was apparently first to rear an insect (bluebottle flies of the genus Calliphora) from egg to adulthood axenically. His studies were followed by definitive work, largely with CaZZiphora, of Eugitne Wollman (1911) of Paris, France, who died a t the hands of the Nazis during World War 11. About ten years after Wollman’s report, a Swiss student, Zimmermann (1921), working in Geneva, Switzerland, under &mile Guyknot (the latter French by origin), recorded the first culture of an invertebrate metazoon through successive generations. The organism was a nematode,

AXENIC CULTIVATION OF CAENORHABDITIS BRIGGSAE (NEMATODA: RHABDITIDAE) WITH UNSUPPLEMENTED AND SUPPLEMENTED CHEMICALLY DEFINED MEDIA*

known in English as the vinegar eel, for which the scientific name is Turbatrix aceti. Recent workers (Ells and Read, 1952; Nicholas, 1956) have not realized the same results, perhaps because of failing

Genetic control of differential heat tolerance in two strains of the nematode Caenorhabditís elegans.

In 1944 Margaret Briggs Gochnauer (Gochnauer and McCoy, 1954) isolated the free-living, self-fertilizing, hermaphroditic soil nematode Caenorhabditis briggsae.1 Since that time it has been maintained and intensively studied in the laboratory, first by Gochnauer (Briggs, 1946) and then by Dougherty and his co-workers (for example, Dougherty and Hansen, 1956~) in the United States, by Nigon and Dougherty (1949; 1950) in France, and by Nicholas (in press) in England. A major objective of this work has been, and continues to be, development of a chemically defined medium8 capable of supporting the indefinite axenic culture of C. briggsae and related rhabditid nematodes. Such a medium would open unexplored vistas on the physiology-especially the biochemistry of nutrition and metabolism-of nematodes. From the studies that would then become possible, new generalizations on metazoan physiology could be confidently anticipated. Despite much work on this problem, a defined medium capable of supporting the indefinite axenic culture of C. briggsae has yet to be devised. The best result realized has been the slow rearing of isolated larvae to reproductive adults in certain media, with slight development of the F1 progeny in one of them (Dougherty and Hansen, 1956~). Yet one has only to add relatively small amounts of suitable tissue extracts to such media to obtain an excellent response: rapid growth and repeated subculture, with no diminution of vigor, insofar a

Synxenic and Attempted Axenic Cultivation of Rotifers

tested. Four successive transfers, with at least eight generations, were made on a defined medium (GS-25)1/ plus a low level (1 per cent) of a liver preparation, Liver Protein Fraction C (LPF-C).

Axenic Cultivation of Caenorhabditis briggsae (Nematoda: Rhabditidae). V. Maturation on Synthetic Media.

Two strains (Bergerac and Bristol) of the nematode Caenorhabditis elegans, with different temperature tolerances, were reared axenically at 23� to 25�C. The Bergerac strain is heat sensitive (that is, it is sterile at maturity), whereas the Bristol strain is heat resistant (that is, it matures and reproduces normally). Hybrid hermaphrodites (F1), produced by crossing Bristol males and Bergerac hermaphrodites, are heat tolerant. Heat sensitivity segregates as a simple Mendelian recessive in the F2 and F3 generations.

The B-vitamins required by Caenorhabditis briggsae (Rhabditidae).

Three species of rotifers have now been grown synxenically and, to a limited extent, axenically. Brachionus variabilis thrives in suitable media containing Chlorella pyrenoidosa and a bacterial species. Lecane inermis and Philodina acuticornis var. odiosa are bacteriophagous, the former doing best with two bacterial species (dixenically), the latter doing well with Escherichia coli alone (monoxenically).

A list of the trichostrongylid lungworms (Phylum Nematoda) and a key to the six genera.

Summary 1. Newly hatched larvae of Caenorhabditis briggsae can grow to adulthood and F1 larvae can be produced on a completely defined synthetic medium (GM-8) consisting of 18 L-amino acids, D-glucose, 4 ribonucleotides plus thymine, choline, i-inositol, ascorbic acid, 17 “trace” vitamins and growth factors, and a mixture of salts. 2. Media (GM-9 and −11) like GM-8 except for certain omissions (and minor additions to restore acid-base balance and salt levels) also support maturation and reproduction. 3. In the case of GM-11, omitted substances include the 8 “non-essential” amino acids. 4. The presence of a trace amount of the standard liver medium used for the indefinite axenic cultivation of C. briggsae is markedly stimulatory to the synthetic media, and some F1 larvae mature and produce F2 larvae when liver medium is present at only 0.032% of its standard strength. 5. For sustained growth, however, C. briggsae requires the presence of much higher levels of liver medium—between 10 and 50% of the standard level used for stock cultures. 6. Evidence is here presented that the liver medium contains 2 requirements, or sets of requirements, not present in the synthetic media, at least in adequate amounts or proportions.

AXENIC CULTIVATION OF CAENORHARDITIS BRIGGSAE (NEMATODA: RHABDITIDAE) WITH CHEMICALLY UNDEFINED SUPPLEMENTS; COMPARATIVE STUDIES WITH RELATED NEMATODES*

School of General Studies, Australian National University, Canberra, A.C.T., Australia; Laboratory of Comparative Biology, Kaiser Foundation Research Institute, Richmond, California, U.S.A. ; Department of Nutrition, College of Agriculture, University of California, Berkeley, California, U.S.A., respectively. To find which of the B vitamins are required by Caenorhabditis briggsae Dougherty & Nigon, it was cultured axenically in a known medium together with chick embryo extract. The concentration of each vitamin added to the medium was varied, and each was omitted in turn. Omitting thiamine, ribloflavin, folic acid, calcium pantothenate, niacinamide and pyridoxine was deleterious.

The genera and species of the subfamily Rhabditinae Micoletzky, 1922 (Nematoda): a nomenclatorial analysis-including an addendum on the composition of the family Rhabditidae Örley, 1880.

In recent papers I have given, in relatively compact form, lists of the genera and species of metastrongylid lungworms; these publications are in turn summarized in a paper which follows (Dougherty, 1948). However, the trichostrongylid lungworms, constituting the subfamily Skrjabingylinae Skriabin, 1933, have not been completely treated, although in a recent work (Dougherty, 1945), in which certain lungworms were formally transferred from the Metastrongylidae to the Trichostrongylidae, the Skrjabingylin genera were listed without critical appraisal, and in the same paper the genus Crenosoma Molin, 1861, was reviewed in detail; subsequently the genus Dictyocaulus Railliet & Henry, 1907, has also been reviewed (by Dougherty, 1946).