Survival of Chlorophyceae Ingested by Saprozoic Nematodes I PATRICIA A. LEAKE" AND HAROLD J. JENSEN Abstract: The saprozoic nematode, Pristionchus lheritieri ingested cells of four species of unicellular Chlorophyceae (grass-green algae) including Chlamydomonas reinhardi and unidentified species of A n- kistrodesmus, Chlamydornonas and Scenedesmus. Additional tests with Ankistrodesmus sp. and Chlamy- domonas sp., indicated cells of Ankistrodesmus survived passage through the alimentary canal and were subsequently cultured, while viable cells of Chlarnydomonas were only occasionally recovered. Key Words: Pristionchus lheritieri, Ingestion, Survival, Chlorophyceae, Algae, Chlamydomonas reinhardi, Chlamydomonas sp., Ankistrodesmus sp., Scenedesmus sp. In 1960, Chang, Berg and Clarke (1) Algae used in this study were Chlamydom- investigated the role of pathogenic micro- onas reinhardi, Dangeard, unidentified spe- organisms in municipal water supplies and cies of Ankistrodesmus, Chlamydomonas, demonstrated that two species of saprozoic and Scenedesrnus. The algae were cultured nematodes (Cheilobus quadrilabiatus and in glass jars which contained 100 or 150 ml Diplogaster nudicapitatus) ingested a number of mineral culture media. Two such media of human pathogenic microorganisms which were used: H. C. Bold's Culture Solution #8 thus survived excessive chlorination treat- (5) and Bristol's Solution as modified by ments. Recently, studies by Jensen (3) H. C. Bold (6). Cultures were aerated by a showed that saprozoic nematodes (principally small air pump which also reduced sedimen- P. lheritieri) ingested various plant patho- tation and kept the nutrients mixed. Clear genic bacteria and fungal spores which sur- plastic covers on the jars permitted penetra- vived passage through the alimentary canal. tion of 225 ft-c surface intensity from fluores- Apparently, there has been little or no inves- cent tubes suspended above the cultures. tigation of possible interrelationships of sap- Photoperiods were 8 a.m. to 5 p.m. and rozoic nematodes and algae. 11:30 p.m. to 1:30 a.m. daily. New cultures were started every three weeks by aseptic MATERIALS AND METHODS transfer of a bit of inoculum to fresh media. A saprozoic nematode, Pristionchus lheri- A semi-solid mineral medium was pre- tieri, (Maupas) Paramonov, was cultured in pared by the addition of agar to Bold's Cul- Petri dishes on nutrient agar medium (3 g ture Solution. Petri dishes of this agar were beef extract, 5 g peptone, 15 g agar in one inoculated with several drops of algal culture liter distilled water). An unidentified Pseu- suspension. Then 50 to 60 nematodes were domonas sp. bacterial contaminant present added and permitted to feed 4 to 5 days. on the cuticle or in the intestine provided a The same photoperiods were used for these food source. These cultures were easily main- plates as were used for cultures of algae in tained by transferring small plugs of agar liquid media. Whole, water-mounted nema- containing nematodes to new plates every 6 todes from these plates were examined micro- or 7 days. scopically for ingested cells. Received for publication 4 May 1970. To determine viability of ingested algal 1 This study was supported by National Science Foundatioo. cells, the nematodes were first surface-steri- Undergraduate Research Participation Grant GY-6141. Technical paper No. 2886, Oregon Agricultural Experi- lized either by thorough washing in distilled ment Station, Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331. water or by rinsing 20 min in 20 ppm (resid- e Present address of senior author: 805 Allen Avenue, Ash- tabula, Ohio 44004. ual) chlorine solution and then examined 351 352 Journal of Nematology, Vol. 2, No. 4, October 1970 for algae adhering to the cuticle. Surface- covered from both the intestinal contents sterilized nematodes were crushed with a dis- and feces of P. lheritieri, but viable cells of secting needle and the intestinal contents Chlamydomonas sp. rarely were recovered. were transferred to sterile agar and observed At least 30 min exposure to 40 ppm chlo- for growth of algae. Other surface-sterilized rine was necessary to completely kill cells of nematodes were placed on agar to test for Chlamydomonas sp. which had clumped to- viable algae voided in the feces. gether in a palmelloid cyst. If aeration was Several experiments were conducted to adequate in the liquid algal culture, however, determine the optimum time and concentra- the cells remained single and were effectively tion of chlorine lethal to species of Ankistro- killed by 20 ppm chlorine for 20 rain. Pre- desmus and Chlamydomonas. Similar data liminary work with Ankistrodesmus sp. indi- available for P. lheritieri (2) indicate excep- cated that exposure to 20 ppm for 15 rain tional tolerance to this biocide. A thick algal was lethal. suspension was mixed with chlorine solutions to give the desired concentrations of residual DISCUSSION chlorine ranging from 0.60 ppm to 100 ppm. P. lheritieri can ingest cells of four uni- Samples of these mixtures were taken at cellular Chlorophyceae: C. reinhardi, and timed intervals (ranging from 2 to 60 min unidentified species of Ankistrodesmus, after adding the algae) and added to a sodium Chlamydomonas, and Scenedesmus. Further- thiosulfate solution (final concentration = more, it seems quite possible that the diges- 1%) to neutralize the chlorine. A 2 ml sam- tive enzymes or physical-chemical factors ple of each treatment was plated on Bold's in the alimentary canal of P. lheritieri are agar and incubated under the same conditions selectively detrimental to algae. Our studies for the liquid algal cultures. indicated Ankistrodesmus survived passage through the alimentary canal while Chlamy- RESULTS domonas rarely survived. Since there is no Microscopic examination of nematodes detailed information on digestive enzymes after 4 to 5 days feeding on algal cultures or other physical-chemical features in the revealed that all four algae were ingested. alimentary canal of this microbivorous nem- Numbers of cells ingested varied from a few atode (4), further investigation is clearly scattered throughout the intestine lumen to indicated. Another comparative study of tol- great concentrations packed in the anterior eration to these various factors by Ankistro- intestine near the esophagus or in the pos- desmus and Chlamydomonas might well be terior portion near the cloaca or rectum. enlarged to include C. reinhardi, Scenedes- Photomicrographs were obtained of each of mus, other unicellular Chlorophyceae and the four algae in nematode intestine (Fig. 1, possibly some of the blue-green algae. Our A-D). results suggest that P. lheritieri is a selective Two techniques used to check viability agent in the distribution of unicellular algae. of ingested algal cells gave parallel results. Investigations involving the chlorination Viable cells of .4nkistrodesmus sp. were re- of algae are only preliminary and thus no FIG. 1. Photomicrographs of Pristionchus Iheritieri showing a section of the body with the intesti- nal tract containing various ingested algae (Note arrows). A. Chlarnydomonas sp.; B. Ankistrodesmus sp.; C. Chlamydomonas reinhardi; D. Scenedesmus sp. ALGAE IN NEMATODES • Leake, Jensen 353 354 Journal of Nematology, Vol. 2, No. 4, October 1970 general conclusion can be reached. Previous free-living nematodes isolated from water work by Jensen (3) and results presented supplies. Amer. J. Med. and Hyg. 9:136. 2. CrtA~aNao, A. 1968. Transmission of plant above indicate, however, that algal cells in pathogenic bacteria and a bacteriophage of the intestine of saprozoic nematodes would Agrobacterium tumefaciens (Smith and be unharmed by concentrations of chlorine Townsend) Conn. by a saprozoic nematode, Diplogaster lheritieri Maupas, 1919. Ph.D. which would be lethal to exposed cells. Thesis, Oregon State University, Corvallis, Hence, an alga highly tolerant of the diges- Oregon. 74 pp. tive enzymes of P. lheritieri could survive 3. JENSEN, H. J. 1967. Do saprozoic nema- todes have a significant role in epidemiology during ingestion in an otherwise lethal en- of plant diseases? Plant Dis. Rep. 51:98- vironment, while an alga with low tolerance 102. would not survive. Again, the need of further 4. LEE, D.L. 1965. The Physiology of Nema- todes. University Reviews in Biology. W.H. investigation in this direction is indicated. Freeman, San Francisco. 154 pp. 5. RotmE, W. 1948. Environmental Require- LITERATURE CITED ments of Fresh-Water Plankton Algae. Symb. Bot. Upsal. 10:1. 1. CHANG, S. L., G. BERG, AND N. A. CLARKE. 6. S'rARR, R.C. 1964. The Culture Collection 1960. Survival and protection against of Algae at Indiana University. Amer. J. chlorination of human enteric pathogens in Bot. 51:1013-1044. .