Eder L. Hansen

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.

In vitro cultivation procedures for parasitic helminths: recent advances.

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.

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

Publisher Summary This chapter describes the in vitro cultivation of various stages of parasitic worms. It emphasizes the application of this technique for elucidation of problems concerning parasite physiology and development. The chapter considers axenic culture of free-living and insect or plant parasitic nematodes. It also reviews the effect of variety of environmental conditions that must be satisfied before successful adaptation of a parasitic helminth to an in vitro culture can be achieved. Environmental conditions considered include (1) host stimuli and trigger mechanisms, (2) precise conditions required during successive stages of development, (3) factors influencing development and organogenesis as distinct from maintenance or survival, (4) immunological inhibition, (5) the toxicity and elimination of metabolic waste products, and (6) pretreatment factors acting on the parasite before cultivation. Several techniques along with trigger mechanisms of various kinds and media and various conditions are presented. The chapter concludes with description of recent studies concerning trematodes, cestodes, nematodes, and applications of metazoan in vitro cultivation procedures.

AXENIC HELMINTH CULTURES AND THEIR USE FOR THE PRODUCTION OF ANTIPARASITIC VACCINES

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.

Continuous axenic culture of free-living nematodes.

Research on immunologic resistance of vertebrate animals to metazoan parasites has encompassed a wide variety of infections including cestode, trematode, and nematode parasites. In this presentation, special emphasis is placed on nematode parasites of the families Metastrongylidae and Trichostrongylidae. These helminths parasitize domestic animals and in some cases, man. In general, the parasitic nematodes go through a life cycle involving a free-living stage that takes place in the host’s fecal excrement. Helminth eggs or embryos are passed out in the feces, where they develop and grow, molting twice, into ensheathed third-stage larvae. If the infective third-stage larva is ingested by a suitable host, the larva exsheaths, casting off the extra cuticle retained from the second free-living ecdysis. Inside the host, the larva enters the parastic or histotropic stage and undergoes a further two molts in the host’s tissues or in intimate contact with the intestinal mucosa. After the fourth and final molt, the worm matures, copulates, and eggs are produced and excreted into the host’s intestinal lumen to passed out in the feces. Work of the past thirty years on the mechanism of immunity to infection with parasitic helminths indicates that the sources of antigen and the site of antibody inhibition occur during growth and development of the histotropic stages. More specifically, it has been shown that inhibition of parasite development in resistant hosts is evident at the first, and to a lesser extent, during the second parasitic ecdysis. The functional antigens, which have been incriminated as important stimulants of the host immune response, are apparently elaborated during certain transitory stages (Silverman & Patterson, 1960). The stages which so far have been identified as particularly involved in the immune stimulating process are the molting third to fourth stage and the molting fourth and early fifth (or young adult) stage (Silverman, 1965a). Antigens prepared from these stages obtained by in vivo (Silverman & Patterson, 1960) and in vitro (Silverman, Poynter & Podger, 1962; Silverman, 1965a) means have proved to be effective in inducing resistance to a number of different parasites in animals. Work is presently under way in several laboratories to translate these promising results into practical vaccination procedures in veterinary and human medicine. This paper proposes to deal with some of the problems involved in the large-scale axenic culture of the histotropic stage of helminths for the production of antigen to be used in antiparasitic vaccines.

Development of Haemonchus contortus in media designed for studies on Caenorhabditis briggsae.

A new method for continuous thin film axenic culture of nematodes involving a minimum of handling permits attainment of larger populations and higher proportions of adults than is attained in test tube cultures with identical media. The increase in population may be attributable to improved physiological conditions which enhance gas exchange. These observations suggest that results obtained in test tube cultures have led to an underestimation of the nutritional adequacy of the medium.