Evangeline A. Yarwood

Biochemical aspects of the nutrition of Caenorhabditis briggsae.

Abstract In an attempt to establish a biochemical and biological tool of multiple potential, we have sought to define the absolute and limited nutritional requirements of Caenorhabditis briggsae. The following has thus far been done: (1) a chemically defined basal medium has been obtained, which sustains life and partial growth but not reproduction, (2) certain biological supplements, which enable the organism to reproduce, have been comparatively evaluated, (3) unexplained freeze-activation of one such supplement has been observed, and (4) through the use of analogues, preliminary suggestions have been derived regarding the action of certain metabolites.

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

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

Development and Maturation of Caenorhabditis Briggsae in Response To Growth Factor

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).

Ficoll activation of a protein essential for maturation of the free-living nematode Caenorhabditis briggsae.

Cultural characteristics of Caenorhabditis briggsae were examined in a medium composed of a chemically defined basal medium and a supplement consisting of a proteinous growth factor. In each of the separate components used as a medium, larvae developed through only one molt. Development was resumed when the medium was restored to completeness. In the complete medium maturation was slower and reproduction somewhat reduced compared with that in monoxenic cultures with E. coli.

Axenic cultivation of the dioecious nematode Panagrellus redivivus.

Summary The sucrose polymer Ficoll at the proper concentration activates the proteinaceous growth factor necessary for continuous cultivation of the free-living nematode, C. briggsae, in a chemically denned medium. It increases the activity that is produced by other activating procedures. It increases uniformity of maturation, allows greater stability at 37°C, and produces a mixture with growth factor that can be readily lyophilized. Its use thus greatly extends the usefulness of the growth factor.

Growth of nematodes in defined medium containing hemin and supplemented with commercially available proteins.

The free living nematode Panagrellus redivivus has been maintained in serial axenic culture for 6 months. P. redivivus was freed from bacteria in a semi-automatic device using antimicrobial agents. Cultures have been established and maintained monoxenically on nutrient agar slants with Escherichia coli, and axenically in a liquid medium containing soya peptone, yeast extract and heated liver extract. Cultures of P. redivivus have been maintained by serial subcultures in a chemically defined medium supplemented with purified protein growth factor (40 μg/ml) under the defined conditions designed for nutritional studies with the protandrous hermaphrodite, Caenorhabditis briggsae (Rhabditidae) . P. redivivus appears to be obligately dioecious. Reproduction has not been impaired by cultivation in liquid media. In marked contrast to C. briggsae, P. redivivus is able to survive 4° C for 3 months.

The life cycle of Adelina cryptocerci sp.nov., a coccidian parasite of the roach Cryptocercus punctulatus

It has been shown that several free-living nematodes, the insect parasite Neoaplectana carpocapsae, and the plant parasite Aphelenchus avenae, will grow and reproduce in axenic culture in defined medium containing hemin and supplemented with γ-globulin. In addition, other commercially available proteins were tested with Caenorhabditis briggsae and A. avenae. Activity was less than that of liver growth factor. Proper precipitation of the protein was important in determining the effectiveness of the supplements for C. briggsae. Hemin added to growth factor had no stimulatory effect. These results extend the range of proteins suitable as supplements for growing nematodes.

Differential nutritional requirements for reproduction of two strains of Caenorhabditis elegans in axenic culture.

1. The occurrence of Adelina cryptocerci in the host, Cryptocercus punctulatus , and its probable means of natural transmission are discussed. 2. Artificial infection experiments were carried out and furnished material for determination of succession of stages in the life cycle of the organism. The cycle is briefly as follows: ( a ) Mature cysts are ingested by the hosts with tissues of infected roaches. Sporozoites enter the hosts tissues by passing through the midgut wall. ( b ) The first schizogony, in which merozoites are produced, ocurs in the fat-bodies between the sixteenth and twenty-fifth days after ingestion of cysts. ( c ) In the second schizogonial generation two types of schizogony produce merozoites and gametoblasts, respectively, between the twenty-fifth and twenty-ninth days following infection. There is evidence that both male and female gametoblasts are formed from a single schizont. ( d ) Male and female gametoblasts become associated during the growth period of the latter. When fully grown, they are surrounded by a membrane, the gametocyst. The male gametoblast undergoes 2 divisions, forming 4 microgametes, of which 1 fertilizes the macrogamete and the other 3 lie unused between the gametocyst and the first oocyst, which is formed shortly after fertilization. A second oocyst is formed before the first division of the synkaryon. ( e ) By repeated divisions of the synkaryon a multinucleate sporont is formed. This divides into uninucleate sporoblasts. Each sporoblast divides once, forming a sporocyst containing 2 sporozoites. Mature cysts occurred in experimentally infected roaches from 40 days onwards. 3. The stages in the life cycle are described in detail. 4. Evidence for zygotic meiosis in Adelina cryptocerci is presented. 5. The effect of the parasite on the host is discussed. This investigation has been made at the suggestion of Dr Harold Kirby, Jr., and under his direction and that of Dr C. A. Kofoid. I wish to express here my grateful appreciation to both for their interest and many valuable suggestions and criticisms.

Sex Differentiation of Aphelenchus Avenae in Axenic Culture

Caenorhabditis elegans (Maupas) ist eine selbstbefruchtende protandrische zweigeschlechtliche Art. Zwei Varietaten von verschiedenen geographischen Gebieten, die als Bergerac und Bristol nach ihren Herkunftsgebieten benannt werden, wurden isoliert. Es wurden Unterschiede in ihren Nahrungsanspruchen gefunden, wie durch den Entwicklungsumfang ihrer Kulturen in verschiedenen Nahrboden bewiesen werden konnte.

Schistosoma mansoni: emergence of progeny-daughter sporocysts in monoxenic culture.

In axenic cultures of a parthenogenetic strain of Aphelenchus avenae males developed at 30° C and at 28° under 5-20% CO2. The sex conversion was studied in individuals in hanging drops and test tube cultures, with environmental conditions changed during development of the egg or early hatched larvae. Maleness was not associated with retardation of rate of development. No intersexes were seen.