Edward J. Buecher

Development and Maturation of Caenorhabditis Briggsae in Response To Growth Factor

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.

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

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.

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.

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

Techniques and media designed for nutritional studies of the free-living nematode Caenorhabditis briggsae were used in culture studies of Haemonchus contortus. A chemically defined medium supplemented with a proteinous growth factor was found to support partial development of Haemonchus contortus to the beginning of the fourth molt. Studies of stability of media at the higher incubation temperature required by the parasite are reported. Previous work has shown that the free-living nematode Caenorhabditis briggsae can be maintained in vitro in serial subculture when a proteinous growth factor in an activated form is added to a chemically defined basal medium. In the defined medium without this activated factor, C. briggsae in any of the four larval stages undergoes only one additional molt before development stops (Hansen, Buecher, and Yarwood, 1965). Activation of this factor was originally produced by freezing (Hansen, Sayre, and Yarwood, 1961) in the defined medium. Several additional and more useful methods have now been developed. Activation produced at the temperature of homeothermic animals is of particular interest (Hansen et al., 1964). One can now postulate that active growth factor is available to parasitic nematodes in vertebrate tissues and also that such an activated growth factor might be of use in cultivation of the parasitic stages in vitro. This paper describes activation and stabilization of growth factor by several different methods. C. briggsae was used as the test organism; the results were then applied to culture of Haemonchus contortus. We found that development of H. contortus into the fourth molt was supported by mixtures of defined medium and activated growth factor (Silverman, Hansen, and Buecher, 1965). MATERIALS AND METHODS

Sex Differentiation of Aphelenchus Avenae in Axenic 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.

Strongyloides fülleborni: environmental factors and free-living generations.

Abstract In fecal cultures of Strongyloides fulkborni inoculated with a known number of eggs, the proportion of filariform larvae to dioecious adults can be changed by changing the conditions of the cultures. Adults developed in thick feces. They also developed on thinly spread feces if carbon dioxide was increased and oxygen decreased. Successive generations of females occurred. These females reproduced only when males of the first generation were present. Females of the later generations differed slightly in morphology from those of the first generation.

Sulfhydryl Compounds Under Controlled Gas in Culture of Schistosoma mansoni Sporocysts

Summary Daughter sporocysts of S. mansoni cultured axenically in the presence of sulfhydryl compounds in a controlled gas environment underwent growth and development, in some cases culminating in the production of a new generation of daughter sporocysts. The effect occurred within a narrow range of concentrations of either dithiothreitol or a combination of cysteine and glutathione, together with lowering the oxygen level, but not when sulfhydryl or decreased oxygen were used alone. Sporocyst development was equivalent to that obtained monoxenically in association with cell cultures and greater than occurred in medium conditioned by cell cultures.

Alteration of Sex of Aphelenchus Avenae in Culture

In axenic cultures of a parthenogenetic strain of Aphelenchus avenae, the masculinizing effect of high temperature (30°) was prevented by mitomycin C. Puromycin reversibly inhibited maturation whereas hydroxyurea caused abnormal development in females. Males occurred at a lower temperature (28°) in axenic media containing ethanol and in monoxenic cultures with Rhizoctonia solani.

Axenic Culturing of Nematodes: an Easily Prepared Medium Containing Yeast Extract

An aqueous extract from ruptured bakers yeast added as a required supplement to a soy-peptone, Bacto-yeast solution constitutes an easily prepared, inexpensive and stable medium for the axenic culture of some nematodes. Cell walls and unbroken cells were removed by centrifugation and the supernatant was sterilized by filtration. Medium containing the supplement supported growth of three species of free-living nematodes and two species of Neoaplectana, a nematode parasite of insects.

Effects of mitomycin C on sex of Aphelenchus avenae (Nematoda) in axenic culture.

Summary Low levels of DNA inhibitor mitomycin C blocked a 30°C male induction system in the plant parasitic nematode Aphelenchus avenae. Instead, parthenogenetic females developed. These frequently lacked a fully formed vulva, but produced normal embryos. The stage at which this inhibitor was effective was the early second larval stage.