JOURNAL OF BACTERIOLOGY, June 1972, p. 1163-1170Copyright O 1972 American Society for Microbiology

Vol. 110, No. 3Printed in U.S.A.

Ultrastructure of Nematode-Trapping Fungi'C. E. HEINTZ AND DAVID PRAMER

Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey 08903

Received for publication 3 March 1972

Capture cells differ ultrastructurally from vegetative cells in the nematode-trapping fungi, Dactylella drechslerii, Monacrosporium rutgeriensis and Ar-throbotrys dactyloides, which capture prey by means of adhesive knobs, adhe-sive hyphal networks, and constricting rings, respectively. Adhesive knobs andadhesive networks contain dense inclusions not found in hyphal tips or subap-ical regions of the vegetative hyphae, and rough- and smooth-surfaced mem-branes are abundant in these trap cells. The fine structure of constricting ringsdiffers from that of adhesive traps, and it is altered by closure. In the open con-figuration, there are membrane-bound inclusions, labyrinthine networks, andelectron-lucent regions between the protoplasts and cell wall, all localized onthe luminal side of the ring cells. After closure, these features no longer areevident and the cytoplasm of trap cells stains less densely.

There are almost 100 species of fungi thattrap and kill nematodes. Some capture prey onseemingly undifferentiated hyphae that aremucilaginous, many produce networks of adhe-sive hyphal branches or stalked adhesiveknobs, and others form constricting rings thattrap nematodes by occlusion. Adhesive trapsresult from the differentiation of branchedhyphae into structures that secrete a stickycoat. Constricting rings are fashioned fromthree cells at the end of a short stalk. When anematode enters the trap lumen, the ring cellsinstantly swell to three times their normalvolume, constricting the prey so that it cannotescape (2, 6, 10). Interest in nematode-trap-ping fungi intensifies as the search for alterna-tives to chemical pesticides continues, and wehave used electron microscopy to provide astructural basis for understanding the mecha-nism by which fungal predators are able tocapture and consume their prey.

MATERIALS AND METHODSCultures and growth conditions. The fungi

examined were Dactylella dreschslerii, Monacro-sporium rutgeriensis, and Arthrobotrys dactyloides,which capture nematodes by means of adhesiveknobs, adhesive hyphal networks, and constrictingrings, respectively (Fig. 1). They were grown at 28 Cfor 5 to 6 days on cornmeal extract agar at pH 6 andwere then supplied with the nematode Panagrellusredivivus (15) as prey. Incubation was continued,

'Paper of the Journal Series, New Jersey AgricultureExperiment Station.

and the fungal cultures were examined periodicallyfor traps and trapped nematodes. Precacious activitywas observed in 6 to 24 hr and was usually concen-trated in a band 5 to 10 mm from the periphery ofthe fungal colonies.

Electron microscopy. Traps and trapped nema-todes were prepared for electron microscopy byflooding petri dish cultures with an iced solution ofeither 5% glutaraldehyde and 4% formaldehyde in0.1 M cacodylate buffer, pH 7.1, prepared by themethod of Karnovsky (7), or 3% glutaraldehyde and0.5% OSO4 in 0.1 M cacodylate buffer, pH 7.0, pre-pared by the method of Franke et al. (3). After briefexposure to the fixative, small pieces of mycelium-bearing agar from areas where traps were most abun-dant were excised from the plates and transferred tovials containing the same iced fixative. Fixation wasfor 1 hr on ice when the glutaraldehyde-formalde-hyde solution was used and for 2 hr on ice in thedark when the glutaraldehyde-OsO4 solution wasused. In either case, the cells then were washed 10 to12 times in iced 0.1 M cacodylate buffer and post-fixed for 2 hr on ice in 1% OSO4 in 0.1 M cacodylatebuffer. During a second series of buffer rinses whichfollowed the OsO4 postfixation step, the materialgradually was warmed to room temperature, brieflyrinsed in distilled water, and then soaked in 0.5%aqueous uranyl acetate for 3 hr. After a brief rinse indistilled water, the material was dehydrated thorugha graded series of alcohols followed by absolute ace-tone and infiltrated in an ascending series of ace-tone-resin mixtures. The resin used for infiltrationand embedding the cells was the Araldite 6005 mix-ture described by Richardson et al. (12). After po-lymerization of the blocks at 60 C for 48 hr, selectedtraps and trapped nematodes were sectioned,stained with lead citrate (11), and examined at 80 kvin a JEM 7A/120 electron microscope.

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FIG. 1. Diagrammatic representation of the ad-hesive and mechanical traps shown in the electronmicrographs. The dotted lines indicate the planes inwhich the specimens were sectioned. A, Adhesiveknobs of Dactylella drechslerii. B, Adhesive hyphalnetwork of Monacrosporium rutgeriensis. C-E, Con-stricting rings of Arthrobotrys dactyloides. C, (a)Side view of an unconstricted (open) ring. 7he dottedline indicates the plane of sectioning of the speci-mens shown in Fig. 4; (b) front view of an open ring.R = ring cell, St = stem cell, L = trap lumen. D,Side (a) and front (b) views of a constricted (closed)ring. E, Side view of a nematode (Ne) in a constrictedring. 7he dotted line indicates the plane of section-ing of the specimens shown in Fig. -5.

RESULTSThe ultrastructure of hyphal tips of nema-

tode-trapping fungi (Fig. 2A) does not differsignificantly from that described (4, 5) forother fungi, and the septa (Fig. 2B) they formare of the ascomycetous type (1).The cells that comprise the traps differ from

vegetative cells, however, and they also differfrom one another depending on whether theycapture prey by adhesion or occlusion. Forexample, adhesive knobs of D. drechslerii aremultinucleate and contain characteristic inclu-sions of moderate electron density (Fig. 3A, B,D). The inclusions range in size from 0.1 to 0.4,um, and the number found in sections of dif-ferent knobs varies.The ultrastructural organization of the adhe-

sive hyphal network of M. rutgeriensis is sim-ilar to that of D. drechslerii in that cells com-

prising the traps are multinucleate and con-tain electron-dense inclusions 0.2 to 0.3 um indiameter (Fig. 3C, E). The adhesive cells pro-duced by both D. drechslerii and M. rutger-iensis contain numerous profiles of rough- andsmooth-surfaced membranes (Fig. 3A and E,respectively).The protoplasmic organization of con-

stricting rings is quite different from thatfound in adhesive traps. Moreover, the ultra-structure of the constricting ring differs in theopen and closed configurations. Three featurescharacterize the ring cells of A. dactyloideswhen the trap is open or unconstricted (Fig.4A). The cytoplasm stains more intensely thanthat of the stem or other vegetative cells, andmembrane-bound electron-dense inclusions 0.1to 0.2 ,um in diameter are localized beneaththe plasma membrane on the inner (luminal)side of the ring only. The membranes sur-rounding the inclusions are similar structurallyto the plasma membrane and capable of fusingwith it (Fig. 4B). As a result of this process,the contents of the inclusions are expelledfrom the cytoplasm, and the surface area of theplasma membrane is increased. The inclusionsalso appear able to fuse with each other toform a labyrinthine matrix enclosed by involu-tions of the expanded and invaginated plasmamembrane (Fig. 4C). The third feature thatcharacterizes open ring cells is an electron-lu-cent region between the plasma membraneand cell wall on the luminal side of the ring(Fig. 4A, D) which at times contains a floccu-lent material of unknown composition (Fig.4D).A number of ultrastructural changes are evi-

dent after trap closure: the cytoplasm of thering cells stains less densely, and the mem-brane-bound inclusions, labyrinthine networks,and electron-lucent regions which characterizethe open ring cells no longer are evident (Fig.5A, B). The outer fibrillar portion of the ringcell wall is ruptured, but the expanded proto-plast remains enclosed by the plasma mem-brane and inner wall material (Fig. 5B). Thecells that comprise the constricted ring pressagainst the cuticle of a trapped nematode, andthe fungus wall conforms to the contours of theanimal surface (Fig. 5B). One or more of thecells in contact with captured prey form pene-tration hyphae (Fig. 5C) which enter the nema-tode, ramify throughout the carcass (Fig. 5A,C), and utilize its contents.

DISCUSSIONTrap cells of predacious fungi are vegetative

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FIG. 2. Electron micrographs of vegetative hyphae of Arthrobotrys dactyloides. The markers represent 1rim. A, Hyphal tip showing the characteristic cluster of apical vesicles (A V). B, Simple septa with associatedWoronin bodies (W) are found in the subapical regions of the hyphae.

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FIG. 3. See page 1169 for legend.

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ULTRASTRUCTURE OF NEMATODE-TRAPPING FUNGI

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ULTRASTRUCTURE OF NEMATODE-TRAPPING FUNGI

hyphae which are specialized for snaring prey.The ultrastructural features which make thevarious types of trap cells distinct from vege-tative cells and from each other were deter-mined. Moreover, by comparing the fine struc-ture of cells that comprise constricting rings inthe open and closed configurations, insight wasgained into how these cells are able to undergolarge, rapid, and irreversible increases involume and remain viable.

Cells that comprise adhesive traps of D.dreschlerii and M. rutgeriensis have certaindistinct features in common. They are multi-nucleate and contain dense inclusions andaccumulations of membranous profiles. Be-cause membranes play an important role insecretory processes (8), their presence in abun-dance here suggests a function in the forma-tion or transport of the adhesive that is se-creted from the traps. Since the dense inclu-sions are found only in cells that compriseadhesive traps, they may be sites where theadhesive is localized prior to secretion.The constricting ring of nematode-trapping

fungi provides a system for studying ultra-structural changes associated with large andrapid increases in cell surface and volume.Since there is a threefold increase in cell size

during the 0.1 second required for trap closure(9), there is need for additional wall and mem-brane to accommodate the enlarged cell sur-face. This may be met either by stretching ofexisting structures or by the incorporation ofpreformed wall and membrane materials. Thebrief time required for trap closure makes denovo synthesis an unlikely explanation. Thefibrillar outer portion of the cell wall ruptures,but the remaining inner portion appears tostretch. However, the ultrastructural polariza-tion of cells that comprise open rings suggestsan additional mechanism for providing theincreased surface required to retain the ex-panded protoplast. The labyrinthine structureproduced by fusion of the membrane-boundinclusions with each other and with the plasmamembrane may represent reserve surface thatis preformed and able on demand to accommo-date an expanding protoplast. Others (5, 14)have shown that surface increases are medi-ated by fusion of membrane-bound inclusionswith the plasma membrane. Since the outerdiameter of individual traps measured prior toand after constriction is unchanged (6, 13; J.Balan, personal communication, 1971), it ap-pears that cells expand into the ring openingonly. The convoluted network of electron-

FIG. 3. Electron micrographs showing the intracellular organization of adhesive traps. The markers repre-sent 1 gm unless otherwise specified. A, Club-shaped adhesive knobs of Dactylella drechslerii are multinu-cleate (N) and contain moderately dense inclusions (1) and numerous profiles of rough endoplasmic retic-ulum (ER). B, Another knob of D. drechslerii has many inclusions (1). The number of inclusions in an adhe-sive knob may be a reflection of the relative maturity of the cell. C, The multinucleate (N) adhesive hyphalnetwork of Monacrosporium rutgeriensis contains inclusions (I) similar to those found in knobs of D. drechs-lerii. D, A portion of the cell shown in Fig. 3A at higher magnification to show differences in size of the inclu-sions. The marker represents 5 gm. E, A portion of Fig. 3C at higher magnification to show the numeroussmooth-surfaced cisternal membranes (C) which are found in these cells.

FIG. 4. Electron micrographs of unconstricted (open) traps of Arthrobotrys dactyloides. A, This portion ofan open trap includes parts of two ring cells (R) and the stem cell (St) by which the ring is attached to themycelium. The cytoplasm of ring cells typically stains more densely than that of the stem cell. On the lu-minal side of the cells that comprise the ring, membrane-bound dense inclusions (I) are concentrated imme-diately beneath the plasma membrane and there is an electron-lucent region (ELR) that separates theplasma membrane and the fibrillar cell wall. The marker represents 1 Am. B, At higher magnification, theasymmetrically staining plasma membrane (PM) and the membranes enclosing the inclusions appear similarand are capable of fusion (arrows). The marker represents 10 Aim. C, An accumulation of the inclusion con-tents beneath the invaginated plasma membrane (PM) results in the formation of a moderately dense laby-rinthine matrix (LM) outside the cytoplasm. The membrane-bound inclusions apparently also are capable offusing with each other (arrows). The marker represents 5 um. D, The electron-lucent region (ELR) is mostobvious in cross sections of unconstricted traps. The marker represents 1 Mm.

FIG. 5. Electron micrographs of constricted (closed) traps of Arthrobotrys dactyloides. The markers repre-sent 1 Mm. A, This longitudinal section through part of a trapped nematode (Ne) shows portions of two con-stricted ring cells (R) in cross section. Fungal hyphae (H) are present in the nematode, the contents of whichhave undergone partial dissolution. B, At higher magnification, it may be seen that the expanded trap cellsare tightly pressed against the cuticle (Cu) of the nematode. Following constriction, the fibrillar cell wall ofthe rings is ruptured (arrows) and a less electron-dense cell wall (CW) encloses the inflated protoplast. Theelectron-lucent region between the protoplast and cell wall and the labyrinthine matrix are not observed insections of constricted rings. Vacuoles of varying sizes occur both in the open (see Fig. 4A) and constrictedrings of A. dactyloides. C, Penetration hypha (PH) formed from a constricted ring cell (R) has entered thenematode (Ne) after rupturing the cuticle.

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dense material and plasma membrane local-ized at the site of change on the inner surfaceof the ring cells is, therefore, in the proper po-sition to accommodate any need of the ex-panding cells for additional surface.

In the constricting ring, the electron-lucentregion is a suitable site for membrane fusionand development of the labyrinthine structure.Additionally, it may contain materials whichcontribute to the wall when the ring expands.It may serve also as a reservoir of fluid im-bibed by the protoplast as it expands (9). Cellsof traps in the closed configuration stain lessdensely than in the open configuration, pos-sibly because condensed cytoplasm is dilutedby water uptake into the expanding proto-plast.The suggested mechanism of ring constric-

tion is consistent with the observations made:it predicts that the membrane-bound inclu-sions and labyrinthine networks will be con-sumed and the electron-lucent region will dis-appear on closure. Moreover, it is essentiallyirreversible and anticipates the reported (9)inability of inflated ring cells to recover andregain their original dimensions. It does notreduce viability or prevent further growth andpenetration of prey by cells that comprise theconstricting ring.

ACKNOWLEDGMENTSWe thank D. James Morre and Charles E. Bracker for

their suggestions and critical evaluation of this manuscript.This investigation was supported by Public Health

Service grant RR-7058.

LITERATURE CITED1. Bracker, C. E. 1967. Ultrastructure of fungi. Annu. Rev.

Phytopathol. 5:343-374.2. Duddington, C. L. 1957. The friendly fungi. Faber and

Faber, London.3. Franke, W. W., S. Krien, and R. M. Brown. 1969. Si-

multaneous glutaraldehyde-osmium tetroxide fixationwith postosmication. Histochemie 19:162-164.

4. Grove, S. N., and C. E. Bracker. 1970. Protoplasmicorganization of hyphal tips among fungi: vesicles andspitzenk6rper. J. Bacteriol. 104:989-1009.

5. Grove, S. N., C. E. Bracker, and D. J. Morre. 1970. Anultrastructural basis for hyphal tip growth in Pythiumultimum. Amer. J. Bot. 57:245-266.

6. Higgins, M. L., and D. Pramer. 1967. Fungal morpho-genesis: ring formation and closure by Arthrobotrysdactyloides. Science 155:345-346.

7. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehydefixative of high osmolality for use in electron micros-copy. J. Cell Biol. 27:137A-138A.

8. Mollenhauer, H. H., and D. J. Morre. 1966. Golgi appa-ratus and plant secretion. Annu. Rev. Plant Physiol.17:27-46.

9. Muller, H. G. 1958. The constricting ring mechanism oftwo predacious hyphomycetes. Trans. Brit. Mycol.Soc. 41:341-364.

10. Pramer, D. 1964. Nematode-trapping fungi. Science 144:382-388.

11. Reynolds, E. S. 1963. The use of lead citrate at high pHas an electron-opaque stain in electron microscopy. J.Cell Biol. 17:208-212.

12. Richardson, K. C., L. Jarett, and E. H. Finke. 1960.Embedding in epoxy resins for ultrathin sectioning inelectron microscopy. Stain Technol. 35:313-323.

13. Schmidt-Hoensdorf, F., and E. Ehrentreich. 1957. Un-tersuchungen uber die Vernichtung von Strongyli-denlarven durch "Raubpilze" der gattung, Arthrobo-trys. Zentrallbl. Veterinarmed. 4:389-402.

14. Van Der Woude, W. J., D. J. Morre, and C. E. Bracker.1971. Isolation and characterization of secretory vesi-cles in germinated pollen of Lilium longiflorum. J.Cell Sci. 8:331-351.

15. Winkler, E. J., and D. Pramer. 1961. A chamber for cul-turing and collecting the nematode Panagrellus redi-vivus. Nature (London) 192:472-473.

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