The enigmatic protist Nephromyces

487 BioSysterns, 14 (1981) 487--490 Elsevier/North-Holland Scientific Publishers Ltd. THE ENIGMATIC PROTIST NEPHROMYCES* MARY BETH SAFFO Department of Biology, Swarthmore College, Swarthmore, PA 19081, U.S.A. (Received June 3rd, 1981) This paper reviews current knowledge concerning the identity of Nephromycez, a collection o f fungus-like cells found in the renal sac of all molgulid tunicates thus far examined. The following has been demonstrated: (1) The cells called Nephromyces do exist, and are n o t produced by their tunicate hosts, but are something foreign to these animals. (2) Many forms of "Nephromyces" cells are present simultaneously in the renal sac of adults of each molguUd species. Despite their extraordinarily eclectic appearance, all these cells are part of the same life cycle. (3) Initial ultrastructural data indicate that Nephromyces is a eukaryote. They also suggest that, despite the speculations of early authors, Nephromyces may not be a Chytridiomycete. "Nephromyces Giard must be regarded as extremely doubtful urdem new evidence becomes available." D.J. Alderman (1976) Nephromyces is the name given by Giard (1888) to a collection of largely colorless cells found in the ductless "renal sac" of molgalid tunicates, a cosmopolitan family of marine chordate animals. As their genus appellation suggests, these cells show a broad resemblance to fungi in their lack of conspicuous pigmentation, and in the filamentous forms which comprise a major part of the array of Nephromyces cells. Whether these cells have any real relation to known fungal groups, however, is not at all clear. A century after its first descriptions (De Lacaze-Duthiers, 1874; Giard, 1888), the identity and phylogenetic relationships of Nephromyces remain thoroughly in doubt. Each of the earlier students of Nephromyces have addressed the question of the identity of Nephromyces, with a variety of conclusions. De Lacaze-Duthiers (1874) described a diverse *This paper is based on an invited presentation made at the Conference o n Cellular Evolution (Fourth International Meeting of the Society for Evolutionary Protistology) held 31 May--3 June 1981 at the University of Maryland's Donaldson Brown Conference Center, Port Deposit, Maryland, USA. array of cells from the renal sac lumen of Molgula occulta. He considered that some of these cells ("confervoid filaments") resembled gregarines. Describing (without drawings!) cells from the renal sac lumen of M. occulta and M. complanata, Giard (1888) considered that these "Nephromyces" were chytrids, as did Harant (1930). Buchner (1930, 1965) also found Nephromyces cells in the renal sac of various molgulids; with Claussen he concluded that "Nephromyces is a lower fungus, which has no relatives, not even distant ones, among [other groups of lower fungi] ". Not surprisingly, these scanty, inconsistent descriptions of peculiar cells in a bizarre habitat (the renal sac: Saffo, 1976, 1978a; Saffo and Lowenstam, 1978) have met with skepticism among mycologists. Johnson and Sparrow (1961) concluded: "In view of the conflicting concepts of species, almost complete lack of uniformity in what little illustrative matter has been published, and failure of the author of the genus to describe adequately its members, the taxa must remain extremely doubtful". We at least know now that Nephromyces 0303-2647/81/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd. 488 If Nephromyces cells are universally and exclusively present in molgulids, isNephrornyces perhaps not a microorganism at all, but merely a collection of cells produced by the tunicate host? Since Molgula manhattensis can bc raised (for a time) without Nephromyces, and experimentally inoculated with Nephromyces (Saffo and Davis, 1982), it is now clear that Nephromyces are not tunicate cells, but something foreign to their host. One of the most puzzling features of Nephromyces is the diversity in appearance cells exist. Certainly, there are cells in the renal sac lumen of molgulids. In all adults of all molgulid species thus far examined (six species of two genera: Saffo, 1978b, 1980, 1981a) from the Atlantic, Pacific and Gulf Coasts, the renal sac invariably contains cells with a broad resemblance to the "Nephromyces" of earlier authors. Thus far, no adult molgulid has been found which does not possess Nephromyces, and Nephromyces cells have never been recognized anywhere other than in molgulids. ~ REFRACTILE I ~ ~ Z (OOSPORES) / / ~ f CENTR~VACUOLE /~ ~ ~,. ,~ / • i ~5~Jm Fig. 1. Putative life cycle of Nephrornyces from Molgula manhattensis. 489 of its cells. In adult molgulids, all of the following cell types (Fig. 1) are generally present simultaneously (Saffo, 1978b, 1982): (1) filaments with large central vacuoles, up to 125 um in length; (2) filaments without vacuoles, 15--500 ~m in length; occasionally, some of these are spindleshaped; (3) filaments with irregularly-shaped, "amoeboid-like" boundaries, around 50 ~m in length; (4) rosette~shaped sporangia, about 20 ~m in diameter; (5) posteriorly biflagellate zoospores (cells 3--4 ~m in length), which are discharged from the sporangia; (6) thickwalled "doughnuts", reminiscent of mammalian red blood cells in shape, but closer in size (3 u m frontal diameter) to prokaryotes. Although not evident in adult hosts, the earliest cell evident in the renal sac lumen of Nephromyces-inoculated M. manhattensis is an amoeboid cell about 5 ~m in diameter (Saffo, 1978b, 1980, unpublished). Each of these cell types is reminiscent of different microorganisms (Saffo, 1982): most of the filaments (with and without vacuoles) generally resemble phycomycetous fungi, while the spindle-shaped filaments and irregularly-bounded filaments are suggestive of mycetozoans; the amoeboid cell is suggestive of protozoans or mycetozoans; the "doughnuts" resemble bacteria (especially Flectobacillus: Sieburth, 1979) more than they do most protists; the sporangia resemble those of the mycetozoan thraustochytrids in light microscope morphology and behaviour (Goldstein, 1963), although the posteriorly biflagellate zoospores which emerge from them differ strikingly from those of thraustochytrids. Most paradoxically, the zoospore -traditionally an important taxonomic character in flagellate groups -- in Nephrornyces is the most obviously peculiar structure of this organism. The diminutive size and central refractile globule characteristic of Nephromyces zoospores are reminiscent of chytrid zoospores. Other features (an apical beak broadly reminiscent, at the light microscope level, of a haptonema: Saffo, 1982) suggest a resemblance to some groups of algae. However, the Nephromyces zoospore has two posteriorly directed, apparently whiplash flagella of equal length (Saffo, 1980) -- an arrangement unlike any comparable cells among algae, protozoa, fungi or mycetozoans. Remarkably, all evidence thus far supports the view that, in each host species, all these diverse cell types belong to a single life cycle (Saffo, 1982, unpublished), and that Nephro. myces from different molgulid hosts are rather clearly related to each other. All molgulid species possess a similar array of Nephromyces forms, with only minor variations from species to species (Saffo, 1982). After inoculation of the renal sac of an uninfectedM, manhattensis, Nephromyces cells appear not simultaneously, but in a predictable sequence (Saffo, 1981b). Inoculation with a single type (the "doughnuts") leads to infection of the renal sac with all the other Nephromyces forms (Saffo, unpublished). A ultrastmctural study of Nephromyces, recently begun, is obviously crucial to illumination of the systematics of Nephromyces. Thus far, ultrastructural data have confirmed that Nephromyces is (despite the problematic "doughnuts") a eukaryote: filaments possess mitochondria, endoplasmic reticulum, and membrane-bounded nuclei: zoospores possess membrane-bounded nuclei and typically eukaryotic 9+2 flagella. If mitochondria are the descendants of endosymbionts, the taxonomic clues suggested by variations in their ultrastructure must be interpreted with caution. Nevertheless, mitochondrial structure has some promise as a phylogenetic marker (Taylor, 1978). Nephro. myces filaments have tubular mitochondrial cristae (Saffo, unpublished). Tubular cristae are present in cells of Oomycetes, Hyphochytriomycetes and Plasmodiophoromycetes among the flagellated fungi, but significantly, not in the Chytridiomycetes which have flattened mitochondrial cristae. Persistence of tubular mitochondrial cristae in Nephrornyces cells, regardless of environment or developmental stage (Vickerman, 1974), would make it difficult to support the hypothesis that Nephromyces is a chytrid, regardless of the suggestions of earlier authors. 490 Is Nephromyces, then, a new major taxon with no close relations to any known group of protists? Might its phylogeneticaUy eclectic appearance suggest relationships among the array of protists to which its various life history stages show hazy resemblance? Or, might Nephromyces in fact have close affinities with a known protistan group, despite its perplexing morphology? Whatever the answer, Nephromyces is bound to enlarge our understanding of protist evolution -- by adding another peculiar taxon to the motley kingdom Protista, by suggesting new relationships among known protists, or by leading to a modification of the diagnostic characters presently used in our attempts to interpret protist phylogeny. If foreign to molgulids, Nephromyces cells do seem -- from their possible ubiquitous distribution among molgulids -- to have been associated with molgulids for a very long time. Although not necessarily mutualistic, the Nephromyces.molgulid association is presumably, at least, an intimately coevolved one. Nephromyces is bound also, then, to provide clues to the biological role of their eccentric habitat -- the renal sac -- and increase our understanding of the biology of their abundant, but little-known hosts. Acknowledgements T h e research s u m m a r i z e d h e r e has been s u p p o r t e d b y a Miller Research Fellowship (University o f California, B e r k e l e y ) , a Steps toward I n d e p e n d e n c e fellowship (Marine Biological L a b o r a t o r y ) , a Cottrell College Science G r a n t (Research C o r p o r a t i o n ) and an A m e r i c a n Philosophical S o c i e t y grant. I t h a n k L. S t a t h o p l o s and W. Davis f o r technical assistance. C o n t r i b u t i o n No. 167 o f t h e Tallahassee, S o p c h o p p y and G u l f Coast Marine Biological Association. References Alderman, D.J., 1976, Fungal diseases of marine animals, in: Recent advances in aquatic mycology, E.B. Gareth-Jones (ed.) (John Wiley, New York) pp. 223--260. Buchner, P., 1930, Tier und Pflanze in symbiose (second edition) (Borntraeger, Berlin) pp. 749-753. Buchner, P., 1965, Endosymbiosis of animals with plant micro-organisms (trans. B. Mueller and F.H. Foeckler) (Wiley.lnteracience, New York) pp. 614--616. De Lacaze-Duthiers, H., 1874, Histoire des Ascides simples des CStes de France. I. Arch. Zool. Exp. Gen., 3,309--311. Giard, A., 1888, 8ur les Nephromyces, genre nouveau de champignons parasites du rein des Molgulid~es. C.R. Acad. Sci. Paris, 106, 1180--1182. Goldstein, S., 1963, Development and nutrition of new species of Thraustochytrium. Am. J. Bot., 50,271--279. Harant, H., 1931, Contribution i l'histoire naturelle des ucides et de leura parasites. 2. Chytridin~es. Ann. Inst. Oc~mnogr. Monaco, 8,349--352. Johnson, T.W., Jr. and F.K. Sparrow, Jr., 1961, Fungi in oceans and estuaries (Cramer, Weinheim) pp. 355--356. Saffo, M.B., 1976, Studies on the renal sac of Molgula manhattends DeKay (Ascidiacea, Tunicata, phylum Chordata). Ph.D. Thesis, Stanford University, 128 pp. Saffo, M.B., 1978a, Studies on the renal sac of the escidian Molgula manhattensis. J. Morphol., 155, 287--310. Saffo, M.B., 1978b, Symbionts in the renal sac of the escidian Molgula. Am. Zool., 18, 665. Saffo, M.B., 1980, Nephromyces, renal sac endosymbiont of molgulid tunicates. Am. Zool. 20,640. Saffo, M.B., 1982, Nephromyees, endosymbiont of molgulid tunicates. I. Distribution within the Molgulidae. Bio!. Bull. in press. Saffo, M.B. and W.L. Davis, 1982, Nephromyces Giard, endo6ymbiont of molgulid ascidian tunicates. II. Modes of infection of Molgula manhattensis by Nephromyces. Biol. Bull. in press. Saffo, M.B. and H.A. Lowenstam, 1978, Calcareous deposits in the renal sac of a molgulid tunicate. Science 200, 1166--1168. Sieburth, J.McN., 1979, Sea microbes (Oxford, New York) pp. 287--290. Taylor, F.J.R., 1978, Problems in the development of an explicit phylogeny of the lower eukaryotes. BioSystems 10, 67--89. Vickerman, K., 1974, The ultrestructure of pathogenic flagellates, in: Trypanosomiesis and Leishmaniesis with special reference to Chages' disease (Associated Scientific Publishers, New York) pp. 171--198.