Predacious Activity of the Nematode-Destroying Fungus, Arthrobotrys Oligospora, on Preparasitic Larvae of Cooperia Oncophora and on Soil Nematodes

Proc. Helminthol. Soc. Wash. 53(2), 1986, pp. 237-243

Predacious Activity of the Nematode-destroying Fungus, Arthrobotrys oligospora, on Preparasitic Larvae of Cooperia oncophora and on Soil Nematodes

P. NANSEN,1 J. GR0NVOLD,1 Sv. AA. HENRIKSEN,2 AND J. WOLSTRUP3 1 Royal Veterinary and Agricultural University, Institute of Hygiene and Microbiology, 13 Biilowsvej, DK-1870 Frederiksberg C, Denmark 2 State Veterinary Serum Laboratory, 27 Biilowsvej, DK-1870 Frederiksberg C, Denmark and 3 Royal Veterinary and Agricultural University, Dept. of Microbiology and Microbial Ecology, 21 Rolighedsvej, DK-1958 Copenhagen V, Denmark

ABSTRACT: Comparisons were made among larval stages of Cooperia oncophora and the soil nematodes Rhab- ditis wohlgemuthi and Panagrellus redivivus in their abilities to induce the nematode-destroying fungus Arthro- botrys oligospora Fres. to form traps in vitro. No difference in the potential of the various nematodes to induce fungal traps were found. Regardless of the nematode species or stage, the fungus trapped with same efficiency. In all tests the soil nematodes and first- and second-stage C. oncophora larvae were immobilized and killed soon after entrapments. However, the third-stage C. oncophora larvae continued to struggle vigorously in the traps for more than 20 hours. This is explained possibly by the inability of the fungus to penetrate the nematode's outer cuticle from the previous molt.

The nematode-destroying fungus, Arthrobo- etc. Factors effecting trap formation and predac- trys oligospora Fres, is possibly the most com- ity in this particular nematode-destroying fungus monly encountered of the predacious fungi in have been intensively studied by Nordbring- Danish agricultural soils (Shepherd, 1961). To Hertz and her group (cf. Nordbring-Hertz, 1977; ensnare nematodes the predacious fungi must Nordbring-Hertz and Jansson, 1984). develop the characteristic organs of capture, The ability of animal-parasitic nematodes to which in the case of A. oligospora are three-di- induce the capturing devices in the fungi has not mensional networks of sticky hyphal anasto- been studied in detail. In these experiments the mosing loops. morphogenic potentials of the 3 external larval Undoubtedly, the capturing devices of the pre- stages of Cooperia oncophora, a trichostrongylid dacious fungi have primarily evolved to trap soil nematode parasite of cattle, will be compared nematodes, but it has been demonstrated that with that of nonparasitic, free-living nematodes, some species and stages of animal-parasitic i.e., Rhabditis wohlgemuthi and Panagrellus re- nematodes may be effectively trapped as well vividus. Also, the experiments allowed for a di- (Descazeaux, 1939; Roubaud and Deschiens, rect comparison of the efficiency of the fungus 1941; Soprunov, 1958; Pandey, 1973; Granvold to capture animal-parasitic nematodes as op- et al., 1985; Gruner et al., 1985). This is perhaps posed to the free-living nematodes. We included not surprising in the light of prevailing views on all 3 developmental stages of C. oncophora: The the evolution of parasitism, which suggest that first 2 stages (Lj and L2) are bacteria feeders with most zooparasitic nematodes have derived from a rhabditoid esophagus, and in many respects free-living ancestors belonging to the soil-dwell- they are comparable with their free-living ances- ing rhabditids (Anderson, 1984). Furthermore, tors. However, third and infective larval stage as already pointed out by Drechsler (1941), cap- (L3) is encased in the cast cuticle of the second turing organs of many fungi are such that they molt and cannot feed. The filariform esophagus allow little discrimination in their choice of prey. and the ensheathing cuticle may to some extent In pure culture, and probably also in a nema- protect the stage from the surrounding environ- tode-free natural environment, many predacious ment. fungi do not form traps and they behave as sap- rophytes. In A. oligospora the transition from a Materials and Methods saprophytic to a predacious phase of growth may Arthrobotrys oligospora be induced by the presence of live nematodes, The strain of A. oligospora Fres. (ATCC 24927) was nematode metabolites, peptides and amino acids, grown and kept on corn meal agar (CMA) adjusted to

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Copyright © 2011, The Helminthological Society of Washington 238 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY pH 7 as described by Lysek and Nordbring-Hertz (1981). At each time interval nematodes were also counted This medium allowed good mycelial growth and conid- (x 20). Only the normal, freely moving individuals were ial formation but not trap development. Test petri enumerated. In order to account for "natural" deaths dishes, 3.2 cm in diameter, were each filled with ap- among the nematodes the average numbers of such proximately 4 ml of CM A and inoculated with 3-5- free individuals recorded in the test dishes were ex- week-old fungal cultures. Using a metal cork borer (5 pressed as a percentage of those in fungus-free control mm in diameter), circular agar plugs were cut from the petri dishes. In addition, on several occasions the con- fungal lawn and placed with mycelium down in the tact between individual worms and traps was studied center of the CMA petri dish. in closer detail with higher magnification (100 x). Both stock and test cultures were maintained at 100% Two series of experiments were conducted: Series A humidity at room temperature (20-2 3°C) in daylight. included R. wohlgemuthi and the 3 developmental Under these conditions the mycelium usually reached stages of C. oncophora. Series B differed in that P. the periphery of the agar in 4-5 days. Grid lines on the redivivus replaced R. wohlgemuthi. In addition, at the bottom of each petri dish facilitated the counting of end of series B, the L3 of C. oncophora larvae were nematodes and measuring trap development. added to the dishes where traps were already present and induced by the other nematodes. Free-living nematodes Panagrellus redivivus was cultured in flasks contain- Results ing a soy peptone-liver extract medium (Nordbring- Hertz, 1972). Rhabditis wohlgemuthi was cultivated on Figure 1 (Series A) and Figure 2 (Series B) show serum agar plates (Monrad, pers. comm.). These nema- that the rates of trap development were virtually todes were harvested from approximately 1 -week-old independent of the type of nematode added. Af- cultures, using the Baermann funnel technique, and ter 3-6 hours traps were formed in some nema- washed several times by alternate centrifugations and resuspensions in sterile water. The resulting suspen- tode treatments; by 6 hours traps were formed sions contained both adult and juvenile nematodes. in all nematode-fungus combinations. Over subsequent hours all treatments exhibited an almost Parasitic nematodes parallel increase in number of traps. The treat- Eggs ofCooperia oncophora were harvested from the feces of a calf carrying an experimental monospecific ment receiving nematode-free supernatants had infection of the nematode. Larvae were allowed to de- no traps. velop in the feces by a cultivation procedure of Hen- Also, Figures 1 and 2 show that the decline in riksen and Korsholm (1983) and were isolated by a the numbers of the free migratory nematodes modified Baermann technique. By starting cultures at started 3-6 hours after they were added to the different intervals it was possible to have batches of dishes. The observation coincided with the ini- Ll5 L2, and L3 larvae simultaneously available. The state of development was checked in each case by mi- tiation of trap formation. After 9 hours the ma- croscopical examination. Prior to use in the experi- jority of the nematodes were trapped and at 15 ments the larvae were washed by serial centrifugations hours there was an almost complete absence of and resuspensions in sterile water. freely migrating individuals. Checks using higher Experimental procedure magnification (100x) revealed that the majority To compare the ability of the nematodes to induce of immobile nematodes were trapped. However, capture organs (hyphal loops) and to become trapped R. wohlgemuthi (Series A) seemed to present an in such organs, their suspensions were added to 4-day- exception in that a few migratory juveniles were old cultures of A. oligospora. A drop of each nematode suspension, which had been adjusted to 100-150 per observed at the end of the experiment. drop, was added to each of 3 test and 3 control dishes. During the experiments, the L, and L2 stages Counts of free-living nematodes included both adults of C. oncophora on the fungus-free control dishes and juveniles. To check whether any nematode-free decreased 10-25% in numbers. Both species of substance of the inoculum would induce traps, fungus CMA dishes were exposed to 1 drop of the supernatant the free-living nematodes increased slightly in of each of the final nematode suspensions. numbers towards the end of the experiment. Starting with 3-hour intervals and ending with 6-hour On close examination all treatments showed intervals, for a 27-hour period, test dishes were ex- nematodes migrating over the entire agar sur- amined at lOOx using a binocular microscope. Trap faces of the dishes. On the fungus dishes they formation began as a stout branch erecting from a vege- tation hypha. Subsequently it grew and curled back so migrated in close physical contact with the hy- its tip anastomosed with the parent hypha or with an phal networks and occasionally caused slight adjacent trap already formed. Only traps that formed movements of the hyphal system. After the traps completely closed loops, either in isolated position or had developed it was noticed that casual nema- more commonly as part of complex, three-dimensional networks, were counted. Five randomly selected fields tode contacts did not necessarily result in im- were counted in each dish for a total of 15 fields. The mediate capture. Sometimes nematodes were seen average number of traps per mm2 was calculated. to move into the loops and then escape by sud-

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Figure 1. Series A: Loops per mm2 and trapping efficiency of A. oligospora exposed to first-, second- and third-stage C. oncophora larvae and to R. wohlgemuthi (juveniles and adults). Average number of free individuals on fungus dishes expressed as a percentage of that on fungus-free control dishes.

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Figure 2. Series B: Loops per mm2 and trapping efficiency of A. oligospora exposed to first-, second- and third-stage C. oncophora larvae and to P. redivivus (juveniles and adults). Average number of free individuals on fungus dishes expressed as a percentage of that on fungus-free control dishes. Notice that figures on the scale for P. ra/jviviw-induced loops are higher than those of the other scales.

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 53, NUMBER 2, JULY 1986 241 denly retracting, curling up, and circumventing 100 the trap. All nematodes exhibited this interesting 80 behavior but inevitably most were trapped. However, once the prey were ensnared their 60 EXPOSED TO subsequent fate was sealed. Free-living nema- C. ONCOPHORA !_., 40 todes and LI and L2 stages of C. oncophora wrig- gled up to a few hours, after which they became 20 paralyzed, and fungal hyphae could be seen inside their bodies. The L3 stage of C, oncophora, 0 on the other hand, struggled vigorously for a much 20 40 60 80 100 120 MINUTES longer period of time and some few of them even 100 succeeded in breaking the hyphal nets. Single 80 individuals were seen freely moving on the sub- EXPOSED TO strate with fungal loops around their bodies with 60 attached hyphal branches. Before long these C.ONCOPHORA L2 nematodes were recaptured in other traps. The 40 L3 of C. oncophora continued to wriggle in their 20 traps, some for more than 20 hours after their capture. 0 In Series B when the original nematodes were 20 40 60 80 100 120 MINUTES all caught, a suspension of L3 C. oncophora was 100 added to 1 of each type of test dish and to 1 80 control dish. Figure 3 shows that all test dishes EXPOSED TO possessed a rapid and high trapping efficiency in 60 that all nematodes were caught within approxi- C.ONCOPHORA L3 mately 1 hour. In the previously P. redivivus- 40 exposed dish, traps were particularly numerous 20 and the capture of L3 was instantaneous. On closer inspection it was noticed again that larvae 0 struggled violently, but after 24 hours roughly only 15% remained active. 100 Discussion In these experiments the capability of C. oncophora larvae to induce traps in the predacious hyphomycete,A oligospora, was comparable with that of 2 free-living soil nematodes, R. wohlgemuthi and P. redivivus. The loops developed only in the presence of living nematodes, but our observations do not allow us to make conclusions 20 40 60 80 100 120 as to the nature of the morphogenic stimulus. MINUTES Nevertheless, others have suggested that the di- Figure 3. Trapping of third-stage C. oncophora lar- rect physical contact of nematodes with the fun- vae on A. oligospora dishes previously exposed to first-, second- and third-stage C. oncophora larvae, or to P. gal hyphae, or influence from metabolic products redivivus (juveniles and adults). The figure presents excreted by the living nematodes, or both, are number of free individuals on fungus dishes expressed responsible (cf. Nordbring-Hertz, 1977; Nord- as a percentage of that on fungus-free control dishes. bring-Hertz and Jansson, 1984). Among the developmental stages of C. oncophora the morphogenic capability of the ensheathed L3 was nematode was the direct intimate contact with comparable with that of the preceding and met- the hyphae. In fact, this was our observation un- abolically more active stages (L[ and L2). This is der the microscope. noteworthy and may perhaps suggest that the Drechsler (1941) studied the specificity of a predominant stimulus of this animal parasitic particular fungus to a particular nematode and

Copyright © 2011, The Helminthological Society of Washington 242 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY stated that as a rule a predacious fungus traps ceeded. In line with this adhesion to third-stage and digests nematodes to a large extent indepen- larvae of 4 species of ruminant, trichostrongylids dently of their species or genus position. This has did not in any case result in infection. later been confirmed by others (Soprunov, 1958; The present and previous results suggest that Barren, 1977). Our finding that various stages of under natural conditions in organic matters A. two soil and one animal parasitic nematode are oligospora and related fungi may be capable of trapped with comparable efficiency is, therefore, trapping free-living stages of animal parasitic not surprising. In A. oligospora and related species nematodes such as trichostrongylids. This may the firmness of attachment of soil nematodes to take place in the soil surrounding cow pats where the trap is believed to be highly dependent on parasitic larvae may be abundant and persist over an adhesive produced by the fungus, and exper- longer periods of time (Al Saqur et al., 1982; imentally a lectin has been shown to mediate the Grenvold, 1984), or it may take place even in binding of nematodes (Nordbring-Hertz and the dung itself since A. oligospora has also been Mattiasson, 1979). In our study A. oligospora isolated from such material (Shepherd, 1956; So- was just as effective in trapping C. oncophora as prunov, 1958; Duddington, 1962). Studies should soil nematodes; thus, we might suspect that an be made to elucidate possible, already existing adhesive was also important for capture of the relationships between nematode-destroying fun- parasite. gi and animal-parasitic nematode larvae in the A. oligospora invades and digests free-living natural feces/soil ecosystems, although we are nematodes within a few hours after capture. Ini- aware that such studies involve great technical tially after dissolving the cuticle a mycelial branch and interpretative difficulties. penetrates into the body of the nematode and In a recent paper we discussed the potential forms a so-called infection bulb from which hy- use of the fungus in controlling nematodes of phae grow and fill the entire length of the body. ruminants (Grenvold et al., 1985). Death of the nematode often seems faster than can be explained by the hyphal growth alone and Literature Cited consequently it has been suggested that a toxin Al Saqur, L, K. Bairden, J. Armour, and G. Gettinby. secreted by the fungus paralyzes the prey (Shep- 1982. Population study of bovine Ostertagia spp. infective larvae on herbage and in soil. Research herd, 1955; Olthof and Estey, 1963). In the pres- in Veterinary Science 32:332-337. ent study hyphal branches were observed both Anderson, R. C. 1984. The origins of zooparasitic in free-living nematodes and in L! and L2 C. nematodes. Canadian Journal of Zoology 62:317- oncophora, and presumably the fungus handled 328. Barren, G. L. 1977. The nematode-destroying fungi. these nematodes in the same way. Canadian Biological Publications. Pages 1-140. L3 larvae of C. oncophora, on the other hand, Descazeaux, J. 1939. Action des champignons Hy- continue to wriggle in their traps over an ex- phomycetes predateur sur les larves de certain tended period of time and at least within our nematodes parasites des ruminants. Bulletin de la observation period hyphal growth was not seen Societe de Pathologic Exotique 32:457-459. Drechsler, C. 1941. Predacious fungi. Biological Re- inside them. The cast cuticle from the previous views of the Cambridge Philosophical Society 16: molt may protect the larvae. Because neither my- 265-290. celia nor paralyzing toxins can enter the worm, Duddington, C. L. 1962. Predacious fungi and the it continues to struggle. This hypothesis finds control of eelworms. In Duddington, C. L. and J. D. Carthy, eds. Viewpoints in Biology. Vol. 1. support in the observations by Nordbring-Hertz Butterworths, London. and Stalhammar-Carlemalm (1978) that A. oli- Gnmvold, J. 1984. The influence of earthworms on gospora does not penetrate the cuticle of dead P. the transmission of infective larvae of bovine tri- redivivus. An interesting parallel may also be chostrongyles. Ph.D. Thesis. Royal Veterinary and drawn to a recent study by Jansson et al. (1985) Agricultural University, Copenhagen. , H. Korsholm, J. Wolstrup, P. Nansen, and S. on the endoparasitic fungus Meria coniospora. A. Henriksen. 1985. Laboratory experiments to They found that adhesion of conidia to the first- evaluate the ability of Arthrobotrys oligospora to stage larvae of newly hatched eggs of the rodent destroy infective larvae of Cooperia species, and trichostrongylid Nematospiroides dubius result- to investigate the effect of physical factors on the growth of the fungus. Journal of Helminthology ed in infection whereas adhesion to third-stage 59:119-125. larvae did not. However, if the ensheathing cu- Gruner, L., M. Peloille, C. Sauve, and J. Cortet. 1985. ticle of the latter was removed, infection suc- Survie et conservation de Factivite predatrice

vis-a-vis de nematodes trichostrongylides apres tode-trapping fungus shows lectin-mediated host- ingestion par des Ovins de trois hyphomycetes microorganism interaction. Nature 281:477-479. predateurs. Comptes Rendus de 1'Academie des -, and M. Stalhammar-Carlemalm. 1978. Cap- Sciences, Paris 300(III):525-528. ture of nematodes by Arthrobotrys oligospora, an Henriksen, S. A., and H. Korsholm. 1983. A method electron microscope study. Canadian Journal of for culture and recovery of gastrointestinal stron- Botany 56:1297-1307. gyle larvae. Nordisk Veterinaermedicin 35:429- Olthof, T. H. A., and R. H. Estey. 1963. A nema- 430. toxin produced by the nematophagous fungus Ar- Jansson, H.-B., A. Jeyaprakash, and B. M. Zucker- throbotrys oligospora Fres. Nature 197:514-515. inann. 1985. Differential adhesion and infection Pandey, V. S. 1973. Predatory activity of nematode of nematodes by the endoparasitic fungus Meria trapping fungi against the larvae of Tricho- coniospora (Deuteromycetes). Applied and Envi- strongylus axei and Ostertagia ostertagi: a possible ronmental Microbiology 49:552-555. method of biological control. Journal of Helmin- Lysek, G., and B. Nordbring-Hertz. 1981. An en- thology 47:35-48. dogenous rhythm of trap formation in the nemato- Roubaud, E., and R. Deschiens. 1941. Essais relatifs phagous fungus Arthrobotrys oligospora. Planta a la prophylaxie de 1'anguillulose du mouton par 152:50-53. 1'usage des hyphomycetes predateur de sol. Nordbring-Hertz, B. 1972. Scanning electron mi- Comptes Rendus des Seances de la Societe de Bio- croscopy of the nematode-trapping organs in Ar- logie, Paris 135:687-690. throbotrys oligospora. Physiologia Plantarum 26: Shepherd, A. M. 1955. Formation of the infection 279-284. bulb in Arthrobotrys oligospora Fres. Nature 175: . 1977. Nematode-induced morphogenesis in 475. the predacious fungus Arthrobotrys oligospora. . 1961. Nematode-trapping fungi in Danish Nematologica 23:443-451. agricultural soils. Horticultura 15:94-96. , and H.-B. Jansson. 1984. Fungal develop- Soprunov, F. F. 1958. Predacious hyphomycetes and ment, predacity, and recognition of prey in nema- their application in the control of pathogenic tode-destroying fungi. Pages 327-333 in M. J. Klug nematodes. Academy of Sciences of the Turkmen and C. A. Reddy, eds. Current Perspectives in Mi- SSR, Ashkhabad. Pages 1-292. (English transla- crobial Ecology. American Society for Microbi- tion 1966 by Israel Program for Scientific Trans- ology, Washington, DC. lations, Jerusalem.) , and B. Mattiasson. 1979. Action of a nema-