CARBOHYDRATE MOVEMENT FROM AUTOTROPHS TO HETEROTROPHS IN PARASITIC and MUTUALISTIC SYMBIOSIS

Biol. Rev. (1969), 44, pp . 17-90

CARBOHYDRATE MOVEMENT FROM

MUTUALISTIC SYMBIOSIS AUTOTROPHS TO HETEROTROPHS IN PARASITIC AND

BY DAVID SMITH Department of Agriculture, University of Oxford

LEONARD MUSCATINE Department of Zoology, University of California, Los Angeles

AND DAVID LEWIS Department of Botany, University of Shefield

(Received I o May I 968)

I. Introduction . . . . . 11. Experimental study of carbohydrate

movement . . . . . ( I ) Demonstration of movement

to the heterotroph. . . (2) Methods of studying the char-

acteristics of carbohydrate movement . . . .

111. Algae and invertebrates . . . ( I ) Introduction. . . . (2) Demonstration of movement

to the heterotroph. . . (3) Characteristics of carbohy-

drate movement . . . (4) Fate of carbohydrate in the

heterotroph . . . . (5) Special aspects . . .

IV. Algae and fungi (lichens) . . ( I ) Introduction . . . (2) Demonstration of movement

to the heterotroph. . . (3) Characteristics of carbohy-

drate movement . . . (4) Fate of carbohydrate in the

heterotroph . . . . (5) Special aspects . . .

V. Autotrophic higher plants and fungi ( I ) Introduction. . . . (2) Demonstration of carbohy-

drate movement to the heterotroph . . . . .

(3) Characteristics of carbohydrate movement . . .

(4) The fate of carbohydrate in the heterotroph . . .

(5) Special aspects . . 2

CONTENTS 18 VI. Fungi and ‘saprophytic’ higher

plants . . . . . . 18 ( I ) Introduction. . . .

(2) Demonstration of carbohydrate movement to the hetcro- trophic higher plant . . 19

(3) T h e form in which carbohydrate moves and its fate in the heterotrophic higher plant . I9

20 (4) Special aspects . . . VII. Autotrophic higher plants and para-

sitic higher plants . . . . ( I ) Introduction. . . . 25

(2) Demonstration of carbohy- 26 drate movement . . .

(3) Characteristics of carbohydrate movement . . . 3 0

(4) Fate of carbohydrate in the 3’

33 heterotroph . . . . 33 (5) Special aspects . . . 34 VIII. Discussion . . . . .

( I ) Introduction. . . . (2) Development of surplus car-

bohydrate in the donor. . 35

(3) Mechanism of transfer froin donor to recipient. . . 40

(4) Preadaptations of donors and 40

44 recipients . . . . 44 (5) Efficiency of carbohydrate

transfer. . . . . (6) Factors other than carbohy-

45 drate movement . . . (7) General conclusions . .

47 IX. Summary . . . . . 49 X. Refcrences . . . . . 53 XI. Addendum . . . . .

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18 DAVID SMITH AND OTHERS

I. INTRODUCTION

When de Bary (1887) devised the term ‘symbiosis’ to describe certain examples of organisms living together, his concept clearly included both parasitic and mutualistic associations, and it is in this original sense that the term will be used here. Most books and review articles about mutualistic symbiosis have emphasized the descriptive rather than the experimental aspects, and have attempted little comparison between different types of associations (e.g. Schaede, 1962; Nutman & Mosse, 1963; Henry, 1966). For these and other reasons, symbiosis does not appear to be a particularly coherent subject with a nucleus of integrative themes.

Movement of substances between the component organisms has always been regarded as a primary feature of symbiosis. The purpose of this review is to consider the movement of a single class of compounds, carbohydrates produced in photosynthesis, from autotrophs to heterotrophs in mutualistic and parasitic symbiosis. The kinds of associations involved are those in which the heterotroph obtains carbohydrate from the living cells of the autotroph, and not from destruction of tissues and digestion of dead material, i.e. by biotrophic and not necrotrophic means. Only those associations for which adequate and relevant experimental studies exist will be discussed, and they are as follows: algae and invertebrates; algae and fungi (lichens); autotrophic higher plants and fungi; and autotrophic higher plants and parasitic higher plants. Also, symbioses between so-called saprophytic higher plants and fungi will be considered briefly, partly because they provide an example of carbohydrate flow in a reverse direction to that in associations of autotrophic higher plants with fungi, and partly because the ultimate source of carbohydrate may well be a living autotrophic higher plant. The term ‘carbohydrate’ will be used in the broad sense to include not only sugars, but also derivatives such as sugar alcohols including glycerol. Each association will be considered separately, and the common themes that emerge will be discussed in a concluding section.

Symbioses between autotrophs and heterotrophs not considered here include all those of green plants with bacteria and actinomycetes (root nodules, leaf nodules, etc.) and those of algae with vertebrates, higher plants and other algae. These are excluded because, with the exception of studies of leguminous plants infected by nodule bacteria, adequate experimental data are lacking, and not because of any assumption that there is no carbohydrate movement between the component organisms. In the case of legume nodules, there have been no studies of movement of photosynthate to the bacteria themselves (but see Bach, Magee & Burris, 1958; Pate, 1966).

11. EXPERIMENTAL STUDY OF CARBOHYDRATE MOVEMENT

The general methods for studying carbohydrate movement will be summarized in this section. Detailed results will be discussed in subsequent sections, as will special methods applicable only to particular associations together with any indirect evidence for carbohydrate movement.

Nearly all the direct experimental evidence for the movement of carbohydrates from autotrophs to heterotrophs in symbiotic systems has been derived from the use

Carbohydrate movement from autotrophs to heterotrophs ‘9 of 14C. This has usually been supplied to the photosynthetic partner as 14C0, in the light, but occasionally as sugars or related compounds.

( I ) Demonstration of movement to the heterotroph Two techniques, radioautography and dissection, have been widely applied to

demonstrate the movement of 14C-labelled compounds to the heterotroph. They differ essentially in that the former is, at best, only semi-quantitative, whereas the latter can be made an accurate qualitative and quantitative assay.

(a) Radioautography This technique has been applied to symbiosis of algae with invertebrates, and of

higher plants with both fungi and other higher plants. ‘ Macro-radioautography ’ may be distinguished from ‘micro-radioautography I. In the former, whole associations, such as leaves of higher plants with fungal associations, or stems with attached parasitic angiosperms, are exposed to film; in the latter, only sections of the associations are exposed, e.g. in studies of coelenterates with algae, and of higher plants with parasitic fungi.

The conventional process of preparing sections for ‘ micro-radioautography ’ re- moves substances soluble in the solvents used, and these may include not only inter- mediary metabolites and other substances of small molecular weight, but also some important storage compounds such as lipids. The extent of translocation may therefore be underestimated and misinterpreted if the evidence for it is based solely on ‘ micro- radioautography ’. Another disadvantage of this technique is that it often involves a long incubation of the symbiosis with lac. (b) Dissection

Dissection, which involves the separation of the heterotroph from the autotroph after exposure of the intact symbiosis to lac, has been employed in all associations under discussion. In this way, separate assays of the two partners may be made for total and specific radioactivity.

Dissection is used in a wide sense to include both partial and complete separation of alga-free epidermis from alga-laden gastrodermis of coelenterates ; alga-free medulla from alga-laden cortex of lichens; fungal sheath from core with Hartig Net of ectotrophic mycorrhizas, and superficial mycelium of powdery mildews from leaves with fungal haustoria.

Complete separations, in the sense that separate analyses of purely donor and purely recipient material are made, can be readily effected in associations of algae with some invertebrates, where the algae can be quantitatively recovered from homogenates of the associations (see z (b) below). Nearly complete separation can be achieved in associations of angiospermous parasites with stems or roots of their hosts, but the intimate haustorial region cannot be easily dissociated.

( 2 ) Methods of studying the characteristics of carbohydrate movement Investigations of the forms in which carbohydrate moves and of the quantitative

aspects of this movement are mainly restricted to two classes of symbiosis: ( a ) those 2-2


involving algae as donors of carbohydrate; and (b) those involving fungi as recipients. In the first group, much information has been derived from studies of algae directly isolated from the symbiotic system. In the second group, the technique that has been most exploited is the inhibition of uptake by the fungus of carbohydrate released by the autotrophic partner.

( a ) Direct isolation of algae from symbiotic associations Symbiotic algae may be obtained in quantity from both invertebrates and lichens

by differential centrifugation at low speeds of gently homogenized samples of the association. Detailed methods for invertebrates are described by Muscatine ( I 965, 1967) and for lichens by Drew & Smith (1967a), Richardson & Smith (1968b) and Richardson, Jackson Hill & Smith (1968) . Under appropriate conditions, such algae will fix 14C from NaH14C0, and release soluble organic photosynthate. The products in the medium may be isolated, identified and compared with the intracellular products to determine if release is the result of cell lysis or selective liberation. Quantitative recovery of all the algae is possible from associations with invertebrates such as Iiydra and Paramecium but, in the case of lichens, recovery may be as low as 5 yo.

(b ) Inhibition of uptake by fungus of carbohydrate released by the autotvoph This method was first developed for the study of lichen symbiosis by Drew & Smith

(1967b) and has become termed the ‘inhibition technique’. It involved incubating lichens on solutions of NaH1”CO, in the light, and including in the medium a high concentration (usually 1-2 %, W/V) of the non-radioactive form of the carbohydrate moving between the symbionts. The radioactive carbohydrate released from the alga is unable to compete for entry to the fungus with the much higher concentration of the non-radioactive form, and it then diffuses into the medium (Fig. I ) . The amount of 14C appearing in the medium is taken as a measure of the amount that would have moved between the symbionts. Correlated with the appearance of labelled carbohydrate in the medium is the inhibition of movement of 14C to entirely fungal regions of the thallus and the diminution of label appearing in specifically fungal metabolites. As well as the application of this technique to a wide range of lichens (Richardson & Smith, 1968a; Richardson, Smith & Lewis, 1967; Richardson et al. 1968), preliminary experiments show that it may also be successfully applied to fungal infections of higher plants (D.H. Lewis & C. Yuen, unpublished).

111. ALGAE AND INVERTEBRATES

( I ) Introduction (a) General

Approximately I 50 genera of invertebrates, representing eight phyla, possess autotrophic endosymbionts. A nearly complete list is given by Droop (1963) . Traditionally, the autotrophs have been grouped into three categories, without taxonomic significance. These are: ( a ) ‘zoochlorellae’, green symbionts inhabiting fresh water and a few marine invertebrates; ( b ) ‘zooxanthellae’, yellow-brown symbionts inhabiting marine

Carbohydrate movement from autotrophs to heterotrophs 21

invertebrates; and (c) ‘ cyanellae ’, blue-green symbionts in certain animals and plants. A more formal categorization is given in Table I.

(b) Structure and composition The following account is limited mainly to associations for which there are experi-

mental data on carbohydrate movement (and hence, associations of animals with ‘cyanellae’ will be excluded), and considers only those aspects which may be helpful in understanding the problem. A broader treatment, including the more historical and descriptive aspects, may be found in the reviews of Yonge (1957, 1964), Droop (1963), and McLaughlin and Zahl (1966).

AUTOTROPH SYMBIOTIC FUNGUS

( b )

Inhibited

‘*C-Carbohydrate ‘*’I

Fig. I . The inhibition, by exogenous supply of [12C]carbohydrate, of uptake by symbiotic fungi of [14C]carbohydrate derived from autotrophic hosts. In (a), no mobile [14C]carbohydrate accumulates in the medium, but much radioactivity reaches entirely fungal regions of the association so that specifically fungal metabolites become labelled. I n (b) much mobile [14C]carbohydrate accumulates in the medium, but little radioactivity reaches entirely fungal regions of the association, so that specifically fungal metabolites only become weakly labelled.

(i) Zoochlorellae: Chlorococcales. Coccoid ‘ Chlorella-like ’ algae occur as endosymbionts in many freshwater protozoans, sponges, hydras, and in some rhabdocoelous turbellarian flatworms. The majority are spherical or ellipsoidal with a cup-shaped chloroplast, pyrenoid, and a cell wall whose thickness varies in different associations (Oschman, 1967). The density of the flora can sometimes vary widely within an association. For example, Paramecium bursaria normally contains several hundred algae per cell, but mean values between I and over 1000 have been obtained in experiments, the size of the algal flora being controlled by interaction of symbiont and environmental factors (Siegel, 1960; Karakashian, 1963 ; Karakashian & Siegel, 1965 ; Karakashian & Karakashian, 1965).

The location of the alga within the host may vary in different associations. In fresh-


water sponges and sponge gemmules they occur primarily within the archcocytes (Brien, 1932; van Weel, 1949); 10-100 algae, each 2-3 ,u in diameter, may be found in these host cells (Lewin, 1966). In metazoan hosts the symbionts usually occur in specific cell types or body regions. In Chlorohydra viridissima (synonymous with Hydra viridis) algae are specific to the gastrodermal cells, any of which may contain I 5 to 25 symbionts, each 6-12 ,u in diameter. The degree of specificity of this location

Table I . Associations between algae and invertebrates Alga*

Class

I. Chlorophyceae

2. Prasinophyceae

Symbiont

Zoochlorellae

Order form Host animals Habitat References

Chlorococcales coccoid sponges, fresh Droop (1963) hydra, water protozoans, flatworms

turbellarian Oschmnn ( I 966) Pyramimonadales irregular acoelous marine Dorey ( I 965) ;

3. Chlorophyceae

I. Dinophyceae

2. Bacillariophyceae

I. Cyanophyceae

Siphonocladiales

Peridiniales

Bacillariales

Chroococcales

flatworms (Convoluta roscoffensis)

chloroplasts sacoglossan opisthobranch molluscs

Zooxanthellne coccoid sponges,

coelenterates molluscs

pennate, acoelous coccoid turbellarian

flatworms (Convoluta convolrrta)

Cyanellae variable protozoa

marine Kawaguti (1941); Kawaguti et al. (1965); Taylor, ( 1 968)

marine Droop (1963); McIaughlin & Zahl (1966)

(1965) marine Ax & Apelt

fresh Droop ( I 963) ; water Hall & Claus

(1963)

* As far as possible, the classification of algae adopted in this article is that of Christensen (1966).

has been demonstrated by Haynes & Burnett (1963), who noted that, when excised portions of gastrodermis regenerate into whole hydra, those cells which dedifferentiate and then become ectodermal cells invariably cast out their symbionts.

The algae reproduce within vacuoles of the host cell by formation of autospores. Daughter cells are segregated into separate vacuoles, while material believed to be the ruptured parent-cell wall persists for an indefinite period in a vacuole within the host. In some strains of green hydra the symbionts lack a pyrenoid (Oschman, 1967; Park, Greenblatt, Mattern & Merrill, 1967). Symbionts are inherited asexually in buds or sexually via the eggs (Brien & Reniers-Decoen, 1950).

Carbohydrate movement from autotrophs to hete-rotrophs 23 (ii) Zoochlorellae: Pyramimonadales. Keeble & Gamble (1907) recognized that the

green alga symbiotic with the marine acoelous turbellarian Convoluta roscoflensis differed from other zoochlorellae. They tentatively assigned it to the genus Carteria, noting that the motile free-living forms possessed four flagellae, a cell wall, a capsule, and an eyespot. However, it has recently been designated as Platymonas convolutae by Parke & Manton (1967). The extent to which this or related algae occur as symbionts of other invertebrates is not known.

Infection of C . roscoflensis takes place in each new generation by ingestion of free- living motile algae by newly hatched worms. The algae move from the area of ingestion to the peripheral regions of the host, where a small fraction appear in parenchymal digestive vacuoles, but the majority are intracellular in subepidermal cells. Examina- tion by electron microscopy (Dorey, 1965; Oschman, 1966; Sarfatti & Bedini, 1965; Oschman & Gray, 1965) show that the algae are bounded by their own thin plasma membrane, then by the animal cell vacuolar membrane, and then by the host cell membrane proper. The symbionts lack the cell wall, capsule, flagella, and eyespot. In the absence of a rigid cell wall, the symbionts assume an irregular shape, matching that of the confining space in the animal cells, and have fingerlike extensions, 1-2 p in diameter, which approach the ciliated outer layer of host epidermis. This feature increases the intimacy and area of contact with the host cytoplasm. Proximity to the highly active ciliated cells has given rise to the speculation that any carbohydrate from the algae would be available to cells with a presumably high energy requirement (Oschman, 1966).

(iii) Zoochlorellae: chloroplasts. A number of opisthobranch gastropods, particularly of the family Elysiidae (Order Sacoglossa), possess green symbionts (Buchner, 1965) which are smaller than the more widespread symbiotic Chlorococcales and are confined to cells which form the hepatic tubules. These tubules are numerous and permeate the folds of the respiratory chamber and parapodia. Electron-microscopic sections show that the symbionts in Elysia atroviridis (Kawaguti & Yamasu, 1965), E. viridis (Taylor, 1968) and those in Tridachia crispata and Placobranchus ianthobapsus ( R . K. Trench, unpublished; R. W. Greene, unpublished) possess internal stacks of lamellae resembling the thylakoids of chloroplasts. Kawaguti (1941) claims that when Placo- branchus ocellatus is illuminated, oxygen is produced in excess of that consumed. Further, the green bodies of P . ianthobapsus exhibit red fluorescence under ultraviolet light (Kawaguti, Yamamoto & Kamishima, 1965) and fix C140, in the light as do those of E. viridis (Taylor, 1968), T . crispata ( R . K . Trench, R. W. Greene & B. Bystrom, in preparation) and a species o f Tridachiella (L. Muscatine, unpublished).

The above evidence is consistent with the interpretation that the symbionts are functional chloroplasts. The gastropod hosts feed on siphonaceous algae such as Codium sp. (Kawaguti & Yamasu, 1965; Taylor, 1967) whose chloroplasts bear a striking resemblance to the symbionts, suggesting that they are acquired through the feeding process. The pigments of T . crispata and E . viridis have been compared with those of chloroplasts of a siphonaceous alga, and have been found to be identical (Trench et al. in preparation; Taylor, 1968).

(iv) Zooxanthellae. Since their initial description by Brandt ( I 88 I) , zooxanthellae


have run the gamut of classification and the majority have emerged unequivocally as members of the Dinophyceae (Peridiniales). The factors important in deciding their taxonomic position have been the successful culture of them, the observation in culture of biflagellated motile zoospores, and pigment analyses. For a complete review of the taxonomic history and present status, the papers of McLaughlin and Freudenthal should be consulted (see McLaughlin & Zahl, 1966). Zooxanthellae from the scyphozoan Cassiopeia sp. from the Caribbean have been designated as Symbiodinium microadriaticum (Freudenthal, 1962). Recently, Ax & Apelt (1965) demonstrated that the zooxanthellae in Convoluta convoluta (Abilg.) ( = C. paradoxa Oerst.) originate from infection by the free-living diatom Licmophora hyalina, a member of the Racillario- phyceae. Hence, zooxanthellae can no longer be regarded as belonging only to the Dinophyceae.

Zooxanthellae are abundant in a variety of marine invertebrates from temperate, tropical and polar waters, but are absent from freshwater invertebrates. They are of great ecological importance in tropical waters as the symbionts of reef-building corals. Droop (1963) lists most of the host genera which represent mainly Protozoa, Porifera (cf. also Sara, 1966), Coelenterata, Platyhelminthes, and Mollusca.

Light is undoubtedly a factor governing the vertical distribution of zooxanthellae in the ocean since the majority of the associations are encountered in the photosynthetic zone (upper 80 m.). However, anemones with zooxanthellae have been collected from 380 m. off Antarctica (Stephenson, 1910) and both anemones and corals with zooxanthellae have been dredged from a depth of 200 m. off Key Largo, Florida (McLaughlin & Zahl, 1959). The occurrence of zooxanthellae at such depths has led to the suggestion that they exist heterotrophically at the expense of the host. This should be investigated since zooxanthellae from the photosynthetic zone cannot survive darkness in situ for more than 50-75 days (Zahl & McLaughlin, 1959; Muscatine, 1961a), and attempts to grow them in darkness in vitro on organic carbon sources have been unsuccessful (McLaughlin & Zahl, 1959).

The symbionts are intracellular in the gastrodermal cells of most larval and adult coelenterates. Some exceptions to this include: those in the eggs of both the hydrozoan coral Millepora (Mangan, I 909) and the hydroid Myrionema amboinense (Fraser, 193 I ) ; those exclusively in the mesoglea of both Phymactis clematis, an intertidal anemone from Trinidad (Stephenson, 1910) and the scyphozoan Cassiopeia (Mayer," 1914; H. G. Smith, 1936; and those in the ectoderm of some coral planulae (Atoda, 1953). McLaughlin and Zahl reported the zooxanthellae of the anemone Condylactis as ' interstitial' in the gastrodermis. However, in the Pacific Coast anemone Anthopleura elegantissima they appear to be intracellular as judged from electron micrographs of gastrodermal cells and from phase-contrast microscopy of macerated gastrodermal tissues (Muscatine, 1961a, b) . The algae from the Jamaican reef corals are within special 'carrier cells' of the host gastrodermis (Goreau, 1961). Finally, Kawaguti (1964), on the basis of ultra-thin sections of the coral Oulastrea, interprets the location of the zooxanthellae as 'intercellular '.

* Actually Mayor, but misspelt in journal as Mayer.


Among the marine molluscs the best known association involving zooxanthellae is with bivalves of the family Tridacnidae (giant clams). The dorsal surface of the inner lobes of the mantle may appear chocolate-brown as a result of the presence of symbiotic zooxanthellae. These algae are about 7 ,IL in diameter and confined largely to the blood sinuses, where they are thought to be contained within amoeboid blood cells (Yowe, ‘936).

( c ) Supplementary techniques (i) Mass culture of various hosts. It is often difficult to obtain sufficient quantities of

symbiotic algae, especially those which have not yet been grown in culture. Large homogeneous populations of Paramecium bursaria (Karakashian, 1963), sponges (Rasmont, 1961 ; Miller, 1964) and C . viridissima (Loomis & Lenhoff, 1956; Muscatine & Lenhoff, 1965a) may be obtained using mass culture techniques. These provide very large numbers of host specimens of known nutritional, genetic, and developmental history under controlled laboratory conditions. These small hosts may be reproducibly sampled using conventional microbiological techniques and the algae (and host tissue) separated by homogenization and centrifugation.

(ii) Aposymbiotic hosts. Assessment of translocation in intact associations often requires the use of aposymbiotic controls (i.e. hosts of the same species without algae). Incubation in darkness is usually sufficient to rid hosts of their symbionts. Whitney (1907) used 0’5% glycerine to eliminate symbionts from green hydra. About 8 days are required for complete elimination. Shorter incubation times in glycerine yield ‘pale green’ hosts, the number of algae they contain being inversely proportional to the length of incubation. Pale green hydra have been used to assess the ‘critical mass’ of algae required to sustain growth and survival of the host (Muscatine & Lenhoff,

( 2 ) Demonstration of movement to the heterotroph ‘965b).

(u) Radioautography Radioautographic techniques have given evidence for movement of labelled carbon

from autotroph to heterotroph in the anemone Anthopleura elegantissima (Muscatine & Hand, 1958) in two reef-building corals from Jamaica (Goreau & Goreau, 1960), in the tridacnid bivalve Tridacna sp. (Goreau, Goreau & Yonge, 1965), and in the sacoglossan mollusc, Tridachia crispata (R. K. Trench, R. W. Greene & B. Bystrom, in preparation).

(b ) Dissection of the host and assay of algae-free tissues Quantitative separation of the alga-laden gastrodermis from alga-free epidermis

has been successfully carried out with the green hydra Chlorohydra viridissima (Muscatine & Lenhoff, 1963) and with excised tentacles of the sea anemone A . elegantissima (R. K. Trench, unpublished). Muscatine & Lenhoff (1963) allowed C. airi- dissima to incorporate 14C0, photosynthetically. At short intervals specimens were washed, decapitated, and treated with culture medium buffered at p H 2.5, which caused the mesoglea to dissociate and facilitated separation of tissue layers. These were


assayed separately for total and specific radioactivity to determine the extent of translocation to the epidermis. Similarly treated aposymbiotic C. viridissima served as controls for heterotrophic fixation of labelled carbon.

In the sacoglossan opisthobranch gastropod Placobranchus ianthohapsus the distribution of the tubules containing the symbiotic chloroplasts is so extensive, and the production of mucus by the animal so copious, that neither fresh dissection nor homogenization leads to clean separation of animal and plant constituents. K. W. Greene (unpublished) has studied translocation in P. ianthobapsus using disks of tissue taken from whole animals frozen on dry ice and then dissected in the frozen state.

( 3 ) Characteristics of carbohydrate movement

( a ) Form in which carbohydrate moves (i) Physiology of isolated algae. The form in which carbohydrate is translocated

cannot be observed directly in intact associations since the translocated material is altered as it is accumulated by the animal. However, inferences can be drawn from the nature of products liberated by isolated algae in vitro, and from analyses of translocated products in the animal tissues.

Table 2 . Form of carbohydrates released from algal symbionts to their hosts

Type of alga

Zoochlorellae

Zooxanthellae

Carbohydrates released to

Host animals host by alga

(a ) Protozoa, Paramecium bursaria maltose (b) Porifera, Spongilla lacustris glucose (c) Coelenterata

Chlorohydra viridissima (wild type) maltose C. viridissima (mutant) glucose

( d ) Platyhelminthes, unknown

( e ) Mollusca Convoluta roscoffensis

Placobranchus ianthobapsus unknown Tridacia crispata

(a) Coelenterata, Pocillopora damicornis, glycerol Anthopleura elegantissima, glycerol Zoanthus confertus, glycerol Fungia scutaria glycerol

(b) Mollusca, Tridacna crocea glycerol

References

Muscatine et a/ . (1967) Muscatine et al. (1967)

Muscatine (1965) Muscatine et al. (1967)

R. W. Greene (unpubl.) R. K. Trench (unpubl.) Muscatine ( I 967) Trench (unpubl.) Trench (unpubl.) Trench (unpubl.) Muscatine ( I 967)

The main products released during photosynthesis by symbiotic algae after isolation rom hosts are summarized in Table 2. In all cases, the principle extracellular product

is a soluble carbohydrate, and it is invariably different from the major intracellular rbohydrate. For example, zoochlorellae, regardless of host type, synthesize and retain

sucrose intracellularly, but excrete either maltose or glucose. Of the six strains of algae from Paramecium bursaria which have been studied, all liberate substantial extracellular maltose as do algae from wild-type Chlorohydra viridissima (Lenhoff strain C . 61). Some glucose is invariably detected in the medium surrounding algae

Carbohydrate movement from autotrophs to heterotrophs 27 from C. viridissima but this can be shown to be partly the result of maltase activity carried over from the host tissues during the isolation procedure; thus little glucose appears in the medium if the cells are thoroughly washed free of host tissue during isolation. On the other hand, the algae from the mutant C. viridissima do liberate glucose directly to the medium and in this respect resemble the algae from fresh water sponges. I t is uncertain if the algae in the mutant host are themselves wild type or mutant (Lenhoff, 1965). Since the intracellular photosynthetic products of the symbionts in witro are virtually identical with those of the algae in the intact association, it is concluded that the isolation procedure does not noticeably affect the nature of the products in short-term experiments. Alga-free tissues from wild type C. viridissima exhibit maltase activity and are rich in free glucose and glycogen. The inference is that maltose is translocated to the animal tissues and is immediately hydrolysed to glucose (Muscatine, 1965), or used in synthesis of glycogen and other substances.

Regardless of the host type, glycerol accounts for most of the carbon released by freshly isolated zooxanthellae in short-term experiments under appropriate conditions. Glucose is invariably excreted but only in minute quantities. I t is likely that glycerol is the form translocated in associations involving zooxanthellae, since the rate of liberation of glycerol in vitro is stimulated by the presence of some component of the host tissue (Muscatine, 1967), and in intact associations, such as corals, the animal tissues are rich in labelled glycerol derivatives, such as lipid, after the whole animal is incubated in the light with NaHC140, (L. Muscatine & E. Cernichiari, unpublished). The major intracellular soluble carbohydrate in zooxanthellae from Pocillopora and Tridacna is glucose, and only small quantities of glycerol are found-a further indication of the selective nature of glycerol release.

Von Holt and von Holt (1968b) state that a variety of radioactive products were released to the medium during photosynthesis by zooxanthellae isolated from Zoanthus spp. However, it is not clear that cell lysis did not occur in their experiments. Further, the experiments were carried out over a period of 3 hr.; R. K. Trench (unpublished) has shown in zooxanthellae isolated from Anthopleura elegantissima and Zoanthus sandwichensis that while most of the 14C released from algae immediately after isolation is in glycerol, the proportion of released 14C in glycerol progressively diminishes with time (similar phenomena also occur in isolated lichen algae, see Section 111).

(ii) Acquisition oj insoluble products by the host. I t has been suggested that insoluble carbohydrates may be acquired by the host as a result of: (a) degeneration and digestion of whole algal cells or their parts (Keeble & Gamble, 1907; Boschma, 1925; Gohar, 1940, 1948; Yonge, 1936; Oschman & Gray, 1964); or (b) sloughing off of unwanted material from healthy algae (Oschman, 1966; Kawaguti, 1965 ; McLaughlin, Zahl, Nowak & Marchisotto, 1963). For example, Keeble states that adult Convoluta do not feed and instead live off their algae. Similarly Gohar believes that soft corals of the family Xeniidae do not feed and instead derive all of their nutritional requirements from zooxanthellae. Yonge has concluded from histological observations that algae in Tridacna are digested. In all of these cases critical evidence is still lacking, and indeed it would be very difficult to obtain such evidence experimentally.

Oschman & Gray (1964, 1965) and Oschman (1966) observed in electron micro-


graphs of Convoluta what appeared to be particles of algae in vacuoles in the host epidermal cytoplasm. The algal nature of the particles was inferred from their resemblance to chloroplasts, having double membranes, matrix material, and starch grains. Since the algae have finger-like extensions reaching into the epidermis, trans- verse sections of these extensions could be interpreted as fragments of algae in vacuoles. Dorey (1965) concludes from a study of serial sections that the chloroplast is not fragmented but merely an extension of the parent algal body. Oschman and Gray further suggest that the tips of the algal extensions might be ‘pinched off into the epidermal cells in a phagocytic manner and digested intracellularly ’. Some algae appear in digestive vacuoles in the host parenchyma during the initial states of infection of the adult. In these, the algal cell wall, which is normally present in the free living forms, may be in various states of disorganization. Its ultimate disappearance from the symbionts has given rise to the speculation that it is assimilated by the host, perhaps assisted by enzymes secreted by the host (Oschman, 1966).

McLaughlin et al. (1963) reported that axenic cultures of zooxanthellae from Cassiopeia give rise to a mucoid substance insoluble in hot water and non-polar solvents which accumulates on and adheres to the surface of culture vessels. Its principal acid hydrolysis product was identified as glucose. Glucose was also obtained from hydrolysis of whole cells. The investigators speculate that this mucoid material may be a potential source of carbon for the host. However, it is not clear if this mucoid substance is produced by algae freshly isolated from the host, or whether it is a characteristic that develops in culture.

Kawaguti (1965) interprets electron micrographs of zooxanthellae from the coral Oulastrea as having a thin plasma membrane covered with an accumulation of double- membrane fragments presumed to be derived from the primary membrane. The fragments are thought by Kawaguti to be available for assimilation by the host.

I t may be concluded that there is no good evidence in the great majority of associations that digestion of algal cells or their insoluble products constitute a significant carbohydrate supply for the host.

(6 ) Quantitative aspects In Chlorohydra viridissima ca. 45-50y0 of the carbon fixed in photosynthesis moves

to the animal tissue (Eisenstadt, 1969); this was demonstrated using the technique of completely separating the algae from the animal tissue by centrifuging homogenates of the association. When just the ectodermal layer is removed by a dissection technique, Muscatine & Lenhoff (1963) showed that this tissue accumulated a fairly constant 10-12% of the 14C fixed during a 48 hr. period, so that the remainder of the 14C released by the algae was presumably retained in the gastrodermal cells associated with the algae. The epidermal tissues had a much higher specific activity than those of aposymbiotic controls (which fix carbon heterotrophically), confirming that a net translocation of photosynthate to the host had occurred.

Also using dissection techniques, R. K. Trench (unpublished) demonstrated translocation in excised tentacles of the anemone A. elegantissima. In a 10 hr. incubation the alga-free epidermis accumulated 29-33 yo of the total 14C fixed by the tentacles.

Carbohydrate movement from autotrophs to heterotrophs 29 Similarly, von Holt &von Holt (1968~) found that 24-40y0 of the total photosynthate in various coelenterates was in animal tissue after 3 hr. in the light. Chloroplast-free tissues of the opisthobranch Placobranchus ianthobapsus accumulate about 23-30 yo of the total activity in the animal after I hr. The specific activity of chloroplast-free tissues is several hundred-fold greater than in dark controls incubated for 12 hr. (R. W. Greene, unpublished). Little is known of how these rates vary with the nutritional state of the host or in response to other environmental stimuli. On the other hand, under virtually identical experimental conditions, different strains of algae, for example those from six strains of Paramecium bursaria, each exhibit their own intrinsic levels of excretion ranging from 5-85% of the total carbon assimilated in short experiments. Long-term quantitative kinetic studies of translocation in associations of algae with animals have not yet been reported.

( c ) Factors injuencing carbohydrate movement (i) p H . Excretion by isolated zoochlorellae is markedly influenced by the pH of the

medium (Muscatine, 1965). At pH 4-5 these algae release as much as 85y0 of their total fixed 14C, mainly as maltose, in 30 min. In more alkaline media, the amount of excretion declines to a minimum of 6-7y0, until at pH 7-5 and higher only small amounts of glycollic acid are produced and maltose can no longer be detected in the medium. This may reflect a pH optimum of a limiting enzymic step in the liberation of maltose. One might speculate that changes in intracellular p H might be mediated by the nutritional state of the host and might control the amount of material translocated from algae to host. By contrast, excretion of glucose by zoochlorellae from sponge is not pH-dependent (Muscatine, Karakashian & Karakashian, 1967).There have been no detailed studies of the effect of p H on excretion by zooxanthellae.

(ii) ‘Host’ factors. Although some glycerol is excreted by zooxanthellae isolated from their host and incubated in sea water, excretion is markedly increased when homogenized host tissue is added to the incubation mixture (Muscatine, 1967). In the case of zooxanthellae from Tridacna and the reef coral Pocillopora damicornis, a 16-fold increase in excretion was obtained in the presence of host homogenate. Excretion was proportional to the amount of homogenate added. Algae from a variety of other corals, alcyonaceans, zoanthids and anemones were also stimulated by homogenate from their own host, but to a lesser extent. Homogenized tissues from Tridacna had a stimulating effect on excretion by Pocillopora algae as did homogenized Pocillo- pora tissues on Tridacna algae. In both cases boiling the homogenate abolished its stimulatory property, as did frequent manipulation of the suspension or delaying its application. The primary effect of the homogenate is not yet known but pH, nutrient enrichment, and light-dark photoperiod were ruled out as possible environmental stimuli. R. Trench (unpublished) has demonstrated that host homogenate stimulates excretion by zooxanthellae from Anthopleura elegantissima. A homogenate of tissue from naturally occurring aposymbiotic anemones or from anemones whose algae were eliminated in darkness did not stimulate excretion. However, if an aposymbiotic anemone was reinfected with zooxanthellae, the host tissue then displayed stimulatory activity. The phenomenon of host stimulation of excretion may be unique


to zooxanthellae since zoochlorellae from hydra are unaffected by homogenates of their own host (Cernichiari, Muscatine & Smith, in press).

(iii) Starvation or reduced food intake by the host. There is some evidence that starvation or reduced intake of food may promote the translocation of carbohydrate to the host in the association of zoochlorellae with hydra. Stiven (1965) compared the growth efficiency (percentage of energy consumed as food that is converted to new protoplasm) of green and aposymbiotic Chlorohydra viridissima fed once per day and once per 2 days in light and dark. Analysis of variance of the data indicated that the green hydra have 42-60 yo higher efficiencies than aposymbiotic hydra when fed in the light. When food intake was reduced, the growth efficiency of green hydra increased. Assuming that the effect of the algae was through translocation of carbohydrate to the host, their contribution was judged to have doubled. Growth efficiency of aposymbiotic hydra was constant and independent of feeding rate and illumination. Similarly, growth of Paramecium bursaria is enhanced by light and by reducing the concentration of available bacterial food (Karakashian, I 963).

(4) Fate of carbohydrate in the heterotroph ( a ) Associations of zoochlorellae with coelenterates

Muscatine & Lenhoff ( I 963) investigated the fate of 14C excreted by zoochlorellae in green hydra by chemical fractionation of l*C-labelled alga-free epidermis. After 48 hr. of accumulation of 14C nearly half was alcohol-TCA insoluble, indicating that material was incorporated into proteins and high-molecular-weight carbohydrates. The remainder was largely in peptides and small molecules, and a fraction was recovered in the nucleic acids.

Chemical fractionation of the complete animal tissue after removal of algae from homogenates shows that the amount of labelled material in the hot TCA-soluble fraction (nucleic acids) is higher than that in the epidermal fraction alone. This may result from incorporation of labelled carbohydrate into the pentose moiety of the nucleic acids and may occur to a greater extent in the gastrodermis than in the epidermis (L. Muscatine, unpublished ; B. Roffman, personal communication). chroma- tography of an 80% ethanol extract of the animal tissues revealed appreciable amounts of free glucose. In view of the maltase activity in these tissues, this indicates that maltose from the algae is hydrolysed to glucose in the animal tissues. Glucose is then presumably metabolized to give a variety of compounds (Muscatine, 1965). Maltase activity has also been demonstrated in P . bursaria (Muscatine, Karakashian & Karakashian, 1967).

(b) Associations of zooxanthellae with coelenterates Utilization by the hosts of glycerol from zooxanthellae has not yet been thoroughly

investigated. Chromatograms of ethanol extracts of whole 14C-labelled corals and Tridacna show that a major portion of the labelled soluble material is lipid, suggesting that most of the glycerol is directed to lipid synthesis. After incubation of zooxanthellae with host homogenate, small amounts of labelled lipid appear in the medium in


addition to glycerol. This can be interpreted to mean that some glycerol is incorporated into lipid in vitro by the homogenate. Fractionation of alga-free coral tissue after incubation of whole corals in sea water containing NaH'*CO, for 24 hr. confirms that a substantial portion of the radioactivity is in the lipid fraction. However a significant amount is also recoverable from the protein fraction, suggesting that other pathways are available to translocated products (L. Muscatine & E. Cernichiari, unpublished).

Consequently, in both the zoochlorellae and zooxanthellae associations there is evidence that the translocated material is converted in the host to different substances. This may enhance the one-way flow of carbohydrate into the heterotroph.

( c ) Molluscs The fate of labelled carbon in the sacoglossan mollusc Tridachia crispata (in which

the symbionts are chloroplasts) has been followed by radioautography. The major site of incorporation appears to be the mucus-producing pedal gland (R. K. Trench, unpublished). This finding is similar to that of Goreau et al. (1965), who found that a substantial portion of the labelled carbon translocated from zooxanthellae to host tissue in Tridacna subsequently appeared in the crystalline style. The general interpretation is that, at least in molluscs, metabolic systems with a high turnover of carbohydrate, such as mucus glands, etc., draw most heavily on the translocated products of the symbionts.

( 5 ) Special aspects ( a ) Diferences between symbiotic and cultured algae

(i) Changes in pattern of carbohydrate release. It will be shown in the next section that some lichen algae exhibit a sharp decline in excretion after a short period in culture. No such decline is evident in zoochlorellae from Paramecium bursaria which have been in culture continuously for more than 3 years, and in one case (Loefer Strain) for 36 years. Zoochlorellae from Spongilla which have been in culture for 13 years exhibit very low levels of excretion, but whether or not this is the result of culture cannot be determined until freshly isolated cells are tested.

Zooxanthellae show marked changes in excretion after they are brought into pure culture. Whereas the symbiotic vegetative cells release glycerol, the cultured vegetative forms do not release soluble material but instead excrete insoluble mucoid substances as described above. Changes in pattern of excretion by symbiotic algae may begin within a short time of their isolation from their host (R. K. Trench, unpublished). The effect of host homogenate on cultured as distinct from freshly isolated algae has not yet been determined.

(ii) Changes in cell-wall characteristics. Symbiotic and cultured forms may also differ in the nature of their cell wall. Electron micrographs of Symbiodinium microadriaticum cultured from Cassiopeia show a complex cell membrane of unusual thickness and possessing intramembranal spaces; by contrast, sections of symbiotic cells do not show a stout wall (McLaughlin & Zahl, 1966). Platymonas possesses a cell wall in the free-living phase which is lost when the alga becomes symbiotic with Convoluta


(Oschman, 1966). Cell-wall ultrastructure of symbiotic and free-living forms of the chlorococcoid zoochlorellae do not exhibit any striking differences (Oschman, I 967).

(iii) Other dzflerences. Symbionts in culture may produce additional cell types. Zooxanthellae from Cassiopeia give rise to sixteen different morphological forms, including cysts, gametes, aplanospores, autospores, and motile gymnodinioid zoospores which eventually become sessile and cast off their flagella (Freudenthal, 1962). As described above, the Platymonas symbiont of Convoluta possesses flagella in culture which it loses in the symbiotic phase.

Zooxanthellae reproduce relatively slowly within the host by binary fission (Mc- Laughlin & Zahl, 1966), but in culture, isogametes may be produced (Freudenthal, I 962). In the host, S. microadriaticum chromosomes remain in continuous prophase but in culture they go into interphase (McLaughlin & Zahl, 1966).

(b) Importance of carbohydrate movement Although the extent to which invertebrate hosts can survive in nature in the alga-

free condition is not known, experimental studies in the laboratory indicate that the algae are of distinct survival value (Slobodkin, 1964; Stiven, 1965; Muscatine & Lenhoff, 1965 b). In quantitative experiments with aposymbiotic controls, Miller (1964) showed that starved sponge gemmules have lower rates of protein catabolism when algae were present, and Muscatine & Lenhoff (19656) obtained similar results with green hydra. The sea anemone Anthopleura elegantissima loses weight less rapidly during starvation if it contains algae (Muscatine, 1961a , b). In P. bursaria, the growth rate and population density increase when algae are present and, in the light, are enhanced when food is limited (Karakashian, 1963). All of these observations can be explained on the basis that carbohydrate from the autotroph can supplement the carbon requirements of the host during starvation. Alternative explanations are discussed in the next section.

Muscatine & Lenhoff (1965) demonstrated that with limited food or during starvation the growth rate and survival time of greenshydra always exceeded that of alga-free controls. Only when fed daily did green hydra and controls grow at nearly identical logarithmic growth rates. It was concluded that since food could serve in lieu of algae in promoting growth, the enhancing effect of algae was nutritional and not the result of waste removal or exchange of oxygen and carbon dioxide. This conclusion is further strengthened by observations that maltose released from the algae is accumulated and metabolized by the host tissues.

The degree to which the association between hydra and zoochlorellae is obligate can only be determined from growth requirements of aposymbiotic individuals. Even though alga-free hydra can be maintained in the laboratory, the association may still be obligate in nature where periods of starvation are frequently encountered (Welch & Loomis, 1924). This would explain why aposymbiotic adults have not yet been found in natural waters even though alga-free eggs are frequently produced by green hydra (Muscatine, unpublished). The general conclusion is that Carbohydrate movement is primarily nutritional in importance, and that algae are probably essential when food is limited or during starvation. Although it is reasonable to suppose that this

Carbohydrate movement from autotrophs to heterotrophs 33 conclusion may well apply to many of the other associations mentioned here, it must be remembered that critical quantitative experimental evidence is still lacking.

( c ) Other efJects of the ulgu on its host Geddes (1882) suggested that symbiotic algae could augment the well-being of their

animal hosts in several ways, including: ( I ) taking up carbon dioxide and producing oxygen during photosynthesis, thus facilitating host respiration and gas exchange ; and (2) taking up host excretory wastes such as ammonia, and phosphate (Yonge, 1964) thereby creating a less toxic micro-environment for the animal. These interactions undoubtedly take place to some extent in some associations but as yet there is little direct evidence that any of them are essential to the host. In fact, as pointed out earlier, they appear to be non-essential in the green hydra association. Further arguments are given by Droop (1963).

In some associations the autotrophs may subserve a role other than nutrition of the host. Goreau (1963) demonstrated that the presence of zooxanthellae in reef corals has a significant accelerating effect on the rate of calcification. Sugiura (1964) showed that the scyphomedusa Mustigius papua could not complete its life-cycle in the aposymbiotic condition. Finally, there is the possibility that the host may benefit from translocated substances other than carbohydrates. For example, Muscatine & Lenhoff (1965) showed by quantitative experiments that 20 yo of the algal flora in green hydra could favourably influence growth and survival to nearly the same extent as the total flora. It was suggested that co-factors translocated in trace amounts from relatively few algae might have the same effect as bulk translocation of carbohydrate. The possibility that algae supply growth factors to the host has also been suggested by Goreau & Goreau (1960). Consistent with this hypothesis is the observation that Coccomyxa sp., a green alga symbiotic with the lichen Peltigera aphthosa, releases biotin to a greater extent than free-living Chlorella pyrenoidosa (Bednar & Holm-Hansen, 1964). Other algae liberate nitrogenous compounds (cf. Muscatine, 1965) and the zooxanthellae from corals liberate traces of a ninhydrin-positive substance which may be a potential source of nitrogen for the host (Muscatine, 1967).

IV. ALGAE AND FUNGI (LICHENS)

( I ) Introduction (a ) General

There are approximately 17,000 species of lichens. The majority consist each of one particular alga in intimate association with one particular fungus. Only a few species contain more than one alga, and the rare instances where more than one fungus occurs are usually regarded as examples of specialized parasitism rather than multiple mutualistic symbiosis.

At least 26 genera of algae have been recorded (Ahmadjian, 1967b), all being either unicellular or simple filaments; according to Ahmadjian, the list of genera includes 8 of blue-green algae, 17 of green algae and I of yellow-green algae. The genera most frequently encountered are Trebouxia (unicellular, Chlorococcales), Trentepohlia

3 Biol. Rev. 44


(filamentous, Ulotrichales) and Nostoc (filamentous, Nostocales). In temperate regions, Trebouxia is by far the commonest, but the proportion of lichens with Trentepohlia increases in the tropics.

Almost all lichen fungi are ascomycetes, showing varying degrees of taxonomic relationship to a variety of free-living groups. There are also a very few basidiomycete lichen fungi. With very few possible exceptions, lichen fungi have not been found free-living in nature.

(b) Structure In the majority of lichens, the algae occur in a thin, well-defined layer just beneath

the thallus surface. Above the algal layer is usually a thin, tough cortex of fungal material, while beneath it is a thicker layer of more loosely arranged fungal hyphae, the medulla. Additional fungal tissues may often occur, such as a lower cortex, rhizinae, hypothallus, etc. In a small number of lichens the algae are not in an organ- ized layer but distributed throughout the thallus.

The proportion of the thallus occupied by algae is usually small. Bednar (1963) calculated from examination of thin sections of PeltiBera aphthosa that the alga (Coccomyxa) occupied about 5 % of the volume. Drew (1966) compared the chlorophyll contents of a known weight of thallus of Peltigera polydactyla with that of a known weight of algae (Nostoc) immediately after isolation from the lichen, and estimated that the alga comprised about 9% of the thallus dry weight.

The symbionts of lichens are in very close contact with each other, and in most species modified hyphae or hyphal branches are closely appressed to the algal cells. Penetration of some of the algal cells by fungal haustoria has been observed in many cases (cf. summaries in Ahmadjian, 1966, and Plessl, 1963), although it is often not clear from the published descriptions what proportion of the cells is affected. Dead algae are hardly ever observed in healthy thalli, so that it may be assumed even if penetration proves to be very widespread, it does not usually cause the death of the algal cells, and may well be simply a device which increases the surface area of intimate contact between the symbionts.

( c ) Special techniques Since lichens cannot yet be routinely synthesized from their two components, all

experiments on intact thalli must be made on material collected from the field. Apo- symbiotic algae and fungi may readily be cultured in the laboratory and a full account of techniques is given by Ahmadjian (1967~).

( 2 ) Demonstration of movement to the heterotroph Substantial movement of photosynthetically fixed carbon from the algal layer to

the fungal medulla has been demonstrated in several lichens in experiments involving dissection of thalli. In Peltigera polydactyla a technique has been developed in which an incision is made immediately beneath the algal layer and the thallus dissected into two separate tissue zones: the medulla, purely fungal in composition, and the ‘algal zone’, comprising the algal layer together with the upper cortex (Harley & Smith,

Carbohydrate movement from autotrophs to heterotrophs 35 1956; Smith, 1960; Smith & Drew, 1965). Using this technique, Smith and Drew showed that approximately 40% of all the 14C fixed in photosynthesis in the algal layer passed into the medulla during an experiment lasting 4 hr. Substantial amounts of fixed 14C must therefore have passed rapidly from alga to fungus within the algal layer.

Rapid movement of photosynthetically fixed 14C from algal layer to medulla was observed in two other lichens containing Nostoc, Peltigera horizontalis (Drew, 1966) and Lobaria scrobiculata (Richardson, Smith & Lewis, 1967) ; relatively fast movement was also observed in P. aphthosa, which contains Coccomyxa (Richardson, 1967). By contrast, in lichens with certain other algal genera, little movement of 14C could be detected, even over 24 hr. periods (Richardson et al. 1968, for Lobaria amplissima, which contains Myrmecia; Richardson, 1967, for Dermatocarpon, which contains Hyalococnrs, and for Roccella fuciformis, which contains Trentepohlia). The relatively slow movement in these species may be related to the fact that, as will be shown below, 14C movement between the symbionts is much slower than in lichens with Nostoc and Coccomyxa.

( 3 ) Characteristics of carbohydrate movement (a) Form in which carbohydrate moves

Table 3 lists those lichens in which the form of carbohydrate moving between the symbionts is known. In each case only one carbohydrate appears to move.

(i) Physiology of directly isolated algae. Immediately after isolation from lichen thalli, algae release appreciable amounts of carbohydrate from their cells. Drew & Smith (1967~) describe a method for the isolation of Nostoc from Peltigera polydactyla, Richardson & Smith (1968) for Trebouxia from Xanthoria aureola, and Richardson et al. (1968) for Coccomyxa from Peltkera aphthosa. T. G. A. Green (unpublished) has recently isolated Hyalococms from Dermatocarpon miniatum.

Virtually all the fixed I4C released from freshly isolated lichen algae is in the form of a single, simple carbohydrate ; in Nostoc this is glucose, in Trebouxia and Coccomyxa it is ribitol, and in Hyalococcus it is sorbitol. Evidence given below confirms that these are the principal forms in which fixed carbon moves between the symbionts in the thallus. It should be stressed that of the various compounds which accumulate 14C within the cells, only the carbohydrates mentioned above are released in quantity, indicating that release must involve some form of selective process. In Trebouxia, although some sucrose is formed within the cells, almost all is retained.

The rates of 14C release vary with different algae, being fastest in Nostoc, slowest in Trebouxia and intermediate in Coccomyxa. This is closely correlated with the different rates of 14C movement between symbionts found in lichens with these algae. (See below.)

I t is not known how the fungus induces the alga to release carbohydrates. A number of factors have been studied which can affect carbohydrate release in directly isolated algae; these may or may not be the same as those factors which are operative in the t hallus.

The pH of the medium has a marked effect on release of fixed 14C by Trebouxia 3 -2


cells directly isolated from thalli (Richardson, 1967; T. G. A. Green, unpublished). In buffered media at pH values below approximately 4.5, release of fixed 14C is stimulated, while above this level it decreases. When Trebouxia cells are incubated in un- buffered distilled water, a certain amount of release occurs, but it is accompanied by a sharp rise in pH which appears to cause a cessation of release. Green has shown

Table 3 . Carbohydrates moving from the alga to the fungus of lichens (after Richardson et al. 1968)

Algal symbiont

Class Genus

Chlorophyceae Trebouxia

Myrmecia

Coccomyxa

Trentepohlia

Hyalococcus

Cyanoph yceae Nostoc

Calothrix

Scy tonema

Lichen species

Lecanora conizaeoides Parmelia furfuracea P. saxatilis Umbilicaria pustulata Xanthoria aureola Dermatocarpon hepaticunr Lobaria amplissima L . laetevirens L. pulmonaria Peltigera aphthosa Solorina saccata Gyalecta cupularis Lecanactis stenhammarii Rocella fuciformis R . phycopsis Dermatocarpon fluviatile D. miniatum Collenia auriculatum Leptogium sp. Lobaria scrobicultata Peltigera canina P . horizontalis P. poiydactyla Sticta fiiliginosa Sticta sp. (Cyanicaudata group) Cephalodia of Lobaria amplissima Cephalodia of Peltigera aphthosa Cephalodia of Solorina saccata Lichina pygmaea

Coccocarpia sp.

Mobile carbohydrate

ribitol ribitol ribitol ribitol ribitol ribitol ribitol ribitol ribitol ribitol ribitol erythritol erythritol erythritol erythritol sorbitol sorbitol

glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose ?glucose/ ?glucosan glucose

that pretreatment at acid pH stimulates release from directly isolated Trebouxia. The effect of pH on other lichen algae remains to be studied, and even the situation in Trebouxia requires much further investigation. However, the balance of evidence at the moment would suggest that low pH is not the main factor causing release within the thallus. In order to induce the same level of release as occurs in the thallus, it is necessary to incubate directly isolated cells at about pH 4, but, when lichen thalli are incubated in weak buffer solutions, they tend to change the pH of the medium towards the region 5-6 and not towards more acid levels (Harley & Smith, 1956, for Peltigera

Carbohydrate movement from autotrophs to heterotrophs 37 pobdactyla; D. J. Hill (unpublished) for Xanthoria aureola). Furthermore, at the pH levels at which algal excretion is stimulated, fungal uptake of carbohydrates tends to be reduced. It is possible that ribitol excretion by Trebouxia may be dependent on a supply of hydrogen ions or reducing power at the cell surface, and this may explain some of the effects of pH on freshly isolated cells.

Follman (1960) demonstrated in plasmolysis experiments that the permeability to glucose of algae immediately after isolation from Cladonia furcata was greater than cells from a I-month-old culture. He suggested that ‘lichen substances’ such as usnic acid might increase the permeability of algal cells within a thallus, facilitating movement of substances to the fungus. Certainly, the function of most ‘lichen substances’- which are usually fungal products-is obscure. However, if any of them do stimulate carbohydrate excretion, evidence given previously shows that it would have to be achieved by altering selective rather than passive permeability. Richardson (1967) was not able to find any effect of parietin on excretion by Trebouxia, even though the parent thallus of Xanthoria aureola was rich in this substance. However, since parietin is a pigmented compound whose production is stimulated by light, it may well perform other functions.

(ii) Inhibition of uptake by fungus of carbohydrate released by alga. Some of the most useful methods of studying carbohydrate transfer between the symbionts of lichens are those based on the ‘inhibition technique’ first developed by Drew & Smith (1967b), and described above in Section II(2b). Drew and Smith give a detailed account of the use of this technique to prevent 14C-glucose released by the Nostoc in Peltigera polydactyla from entering the fungus. The concentration of 12C-glucose required in the medium to give a 50% inhibition of 1“-glucose movement into the fungus was approximately 0.004 M. Addition to the medium of 12C-hexoses other than glucose- even at concentrations as high as I yo (0.06 M)-failed to achieve any inhibition of 14C-glucose movement. The fungal uptake mechanism evidently had a very high specificity for glucose since uptake by the lichen of 14C-glucose from very low concentrations ( I O - ~ M) could not be prevented by much higher concentrations ( I O - ~ M)

of other hexoses. The only other compounds capable of preventing 14C-glucose movement into the fungus were the glucose derivatives 2-deoxyglucose and 3-methyl- glucose.

The ‘inhibition technique’ has been used to study twenty-seven lichen species (Richardson et al. 1968; Table 3). In lichens with green algae-where polyols are the principal form in which carbohydrate moves from alga to fungus-the specificity of the inhibition is not as great as that described above for glucose movement in Peltigera. Thus, in Xanthoria aureola, where the Trebouxia symbiont releases ribitol, externally supplied arabitol is as effective as ribitol in preventing entry of W-ribitol to the fungus. In Rocella, where erythritol is the mobile carbohydrate, ribose is almost as effective as erythritol. Presumably, the specificity of the inhibition closely reflects the specificity of the relevant carbohydrate uptake mechanism of the fungus.

One disadvantage of these techniques is that the excess of external carbohydrate inevitably distorts the normal pattern of metabolism in the lichen. In Peltigera polydactyla addition of I yo glucose to the medium results in a reduced net fixation of 14C,


together with an increased incorporation of 14C into the insoluble fraction. In the presence of 2-deoxy-glucose, there were also some marked differences in the pattern of 14C fixation.

Richardson et al. (1968) concluded from their survey of a range of lichens (summarized in Table 3) that the kind of carbohydrate moving between the symbionts depended on the kind of alga present. Representatives of eight different algal genera were involved-three Cyanophyceae and five Chlorophyceae. In the lichens with Cyanophyceae, glucose was the compound transferred, but it would be dangerous to draw general conclusions about symbiotic blue-green algae from this since all three genera involved (Nostoc, Calothrix and Scytonema) are in the same order (Oscilla- toriales), whereas representatives of other orders of the Cyanophyceae also occur in lichens. Further, there was a possibility that it was a glucosan, rather than glucose, which moves in the one Calothrix lichen investigated.

In each of the lichens with green algae, the mobile carbohydrate was always a polyol. The five genera involved were not all closely related: Trebouxia and Myrmecia (producing ribitol) belong to the Chlorococcales; Coccomyxa (ribitol), Hyalococcus (sorbitol) and Trentepohlia (erythritol) to the Ulotrichales. Polyols have not previously been found in any member of the Chlorophyceae other than those involved with lichens, although there has not yet been any systematic search for them. I t is therefore not clear what significance should be attached to the production of polyols by the lichen symbionts since it is not known if they are common products of terrestrial algae, from which lichen algae are presumably derived. However, it does seem as if polyol production is stimulated by some aspect of existence in the symbiotic state. As shown below, when Trebouxia is brought into pure culture, ribitol production is reduced, and in the case of Coccomyxa it disappears altogether. Possible reasons for this will be discussed later.

(iii) Short-term photosynthesis experiments. Studies of short-term photosynthesis in the thallus help to indicate which products are of algal rather than fungal origin. During photosynthesis of 14C0, by Peltigera polydactyla, the release of 14C-glucose by the Nostoc and its conversion to 14C-mannitol by the fungus is so rapid that it appears as if mannitol is the main product of photosynthesis. Only very small amounts of glucose can normally be found in the thallus. However, in very short term experiments it can be demonstrated that 14C-glucose is formed in the thallus within I min. of exposure to NaH14C0, but 14C-mannitol does not appear until after 2 min. (Drew & Smith, 1967b). In similar experiments with Xanthoria aureola, Bednar & Smith (1966) found that a pentitol, later identified as ribitol and shown to be an algal product by Richardson (1967), was the first carbohydrate to be formed in photosynthesis.

(b) Quantitative aspects Information about carbohydrate movement between the symbionts of lichens comes

mainly from experiments on the movement of 14C fixed in photosynthesis. Since the specific activity of the 14C-compounds was not known, there are no exact estimates of the amounts of carbohydrate moving. However, there is considerable indirect evidence that the movements may sometimes be substantial.

Carbohydrate movement from autotrophs to heterotrophs 39 In their survey of a range of lichens, Richardson et al. (1968) compared the rate at

which 14C moved between the symbionts under certain experimental conditions. They made an arbitrary grouping of lichens into four categories according to the time taken for fixed 14C to be released to the medium in the ‘inhibition’ experiments described in Section 3(a, ii) above. These categories were: ‘fast’ (2c-40O/~ of total fixed 14C in 3 hr.); ‘intermediate’ (10-20% in 3 hr.); ‘slow’ (2-4% in 3 hr., 20-45y0 in 24 hr.); and ‘very slow’ (1-2% in 3 hr., 5-1oy0 in 24 hr.). Although these groupings indicate the relative rates at which 14C moves between the symbionts, they do not necessarily reflect the real rate of carbohydrate movement. Thus, the same real rate of movement would appear ‘fast’ if the pool of mobile carbohydrate is very small and so becomes rapidly labelled with 14C, or ‘slow’ if the pool was large and took a long time to saturate with 14C. For example, under identical experimental conditions, Richardson & Smith (19686) showed that a pulse of 14C was transferred about 1 2 times more quickly in Peltigera polydactyla (a ‘fast’ lichen in which glucose is transferred) than in Xanthoria aureola (a ‘slow’ lichen in which ribitol is transferred). However, the glucose pool in P . polydactyla must be very small since the data of Smith (1963) indicates that the glucose content of thalli must be less than 0.05 yo dry weight, while the pool of mobile ribitol in X . aureola may be much larger because the total ribitol content may be up to about I yo dry weight (see Richardson & Smith, 1966). It is likewise significant that the concentration of 12C-glucose causing a 50% inhibition of 14C movement in P . polydactyla was 0.004 M (Drew & Smith, 1967b), while the concentration of ribitol which caused a similar degree of inhibition in X . aureola was higher, of the order of 0-02 M (D. J. Hill, unpublished).

The survey of Richardson et al. also showed that the rate of 14C movement depended on the kind of alga rather than the kind of carbohydrate. For example, while glucose was transferred in all lichens with Cyanophyceae, the movement of 14C was ‘slow’ in Calothrix lichens but ‘fast’ in the others. Similarly, in those species in which ribitol is transferred, movement in lichens with Coccomyxa was ‘intermediate’, but ‘slow’ in those with Trebouxiu and Myrmecia. The apparently strict relationship between the kind of alga and the rate of 14C movement was maintained even in those lichens where two different kinds of algae exist in the same thallus.

Dissection experiments described in Section ( 2 ) showed that the rate of 14C movement out of the algal layer into the medulla was correlated with measurements from ‘inhibition’ experiments of the rate of 14C movement between the symbionts. The rapid movement of 14C into the medulla of a ‘fast’ lichen such as P. polydactyla certainly implies that the supply of carbohydrate from the alga to the rest of the thallus is rapid. On the other hand, the very small amount of 14C translocation to the medulla in ‘slow’ lichens does not necessarily imply that the alga is a poor supplier of carbohydrate.

(c) Factors affecting carbohydrate movement (i) Light. Although a certain amount of carbohydrate movement can occur in the

dark (Smith, 1961; Smith & Drew, 1965) it is much more rapid in the light. D. J. Hill (unpublished) incubated material of X . aureola and P . polydactyla in the light in


14CO, for short periods, and then used the ‘inhibition technique’ to show that movement of the photosynthetically fixed 14C between the symbionts after transfer to darkness was approximately 10% of the value in the light.

(ii) p H . p H can influence movement both by affecting the rate of release of carbohydrate from algae and by affecting fungal uptake. For example, at low pH in X . aureola, algal release is stimulated but fungal uptake is partly inhibited so that some carbohydrate is released from the tissues to the medium (Richardson, 1967). A marked reduction in carbohydrate uptake at low p H by P. polydactyla had previously been observed by Harley & Smith (1956). At high pH, Richardson found that algal release was reduced in X . aureola, though fungal uptake seemed unaffected. Thus, movement appears to be reduced at the extremes of pH.

(4) Fate of carbohydrate in the heterotroph In all lichens so far investigated, carbohydrate from the alga accumulates in the

fungus initially as polyols. Mannitol is always present and in many cases is the main polyol formed. Arabitol is also frequently produced. In culture, the isolated fungus of X . aureola always contains mannitol, and arabitol only appears when grown on media containing ribitol. This suggests that arabitol is formed by the fungus of this lichen during the conversion of ribitol received from the alga to mannitol (Richardson & Smith, 1968b); the metabolic pathway suggested by Lewis & Smith (1967a) could be operative in this.

Nostoc isolated from P. polydactyla appears unable to metabolize mannitol, thus supporting the theory of Lewis & Harley ( 1 9 6 5 ~ ) that conversion of carbohydrate by the heterotroph to a form unavailable to the autotroph promotes the one-way flow of carbon in such symbiotic systems.

I t is not known if any of the green algae isolated from lichens can metabolize fungal polyols, apart from the observation of Quispel(1943) that growth in the light of some Trebouxia isolates was stimulated by mannitol. Although the green algae of lichens produce polyols themselves, they are different from those of the fungi, so it does not necessarily follow that they would always be able to utilize them.

( 5 ) Special aspects ( a ) Differences between symbiotic and cultured algae

(i) Changes in patterns of carbohydrate release. Immediately after isolation from the thallus, lichen algae can still release substantial amounts of photosynthetically fixed 14C, most of it as a single, simple carbohydrate (see Section IV (3a, i)). After growth in pure culture the proportion of fixed 14C that is released is very much smaller, and is no longer primarily in a single compound (Drew & Smith, 1967a, for Nostoc; Richardson & Smith, 1968b, Maruo, Hattori & Takahashi, 1965, and T. G. A. Green (unpublished) for Trebouxia; Richardson et al. 1968, for Coccomyxa). In the case of Nostoc and Coccomyxa the carbohydrates that are released in quantity by symbiotic cells (glucose and ribitol respectively) cannot be detected at all amongst the products released by cultured cells; in Trebouxia a small amount of ribitol can still be detected in the media of cultured cells.

Carbohydrate movement from autotrophs to heterotrophs 4' When directly isolated algae are suspended in distilled water the ability to release

fixed 14C declines rapidly and the identity of the substances released also changes quickly. Drew & Smith ( 1 9 6 7 ~ ) found that immediately after isolation Nostoc released 50% of the 14C fixed in 3 hr., virtually all of it as glucose. Two days later release had been reduced to 12% and the 14C released was now in compounds which remained on the base line of paper chromatograms, possibly polysaccharides. T. G. A. Green (unpublished) found a marked reduction in ribitol release from Trebouxia 12 hr. after isolation, while in Coccomyxa no release of ribitol could be detected after 3 days; in both cases, after ribitol release had declined, some 14C was then released in substances which similarly remained on the base line of paper chromatograms. It is important to stress that the algae showed no apparent decline in vigour during this period. Photosynthetic rates are not greatly changed, although the pattern of fixation alters (see below). Little or no detectable growth occurs during this period, so that the changes in carbohydrate release could not be explained in terms of the selection of one or two vigorously growing genotypes.

It was noted in Section IV ( 3 a , i) above that the decline in carbohydrate release by directly isolated Trebouxia suspended in distilled water could be associated with a rise in pH. However, pH is evidently not the sole factor governing release, since T. G. A. Green (unpublished) found that treatment of cultured Trebouxia at acid pH caused only a small rise in the proportion of fixed 14C released, while similar treatment of cultured Coccomyxa caused no increase at all. In neither case was more 14C released in ribitol.

(ii) Changes in the pattern of photosynthesis. The main product of photosynthesis of 14C0, in the symbiosis is often different from that in the isolated, cultured state. Drew & Smith ( 1 9 6 7 ~ ) showed that free glucose is the main product of photosynthesis of Nostoc in the thallus of Peltigera polydactyla, but in pure culture no free glucose is detectable. In Coccomyxa, free 14C-ribitol is the main product of photosynthesis in the thallus, but none is detectable in pure culture (Richardson, 1967; T. G. A. Green, unpublished). In Trebouxia, cells produce proportionately much more ribitol and much less sucrose than cultured forms. When directly isolated Trebouxia cease to release 14C-ribitol during suspension in distilled water, Green found no increase in 14C-ribitol within the cells, and none could be detected in the ethanol-insoluble material (an acid hydrolysate of this was found to be very largely composed of 14C-glucose).

A general feature shown by these algae (except Trebouxia) is that the proportion of carbon fixed into ethanol-insoluble material is less in the symbiotic than in the cultured state. The fixation of carbon mainly into soluble compounds by the symbiotic forms would have the obvious consequence of providing a reservoir of material potentially available to the fungus.

(iii) Changes in cell-wall characteristics. Lichen algae in symbiosis and in pure culture may differ in the nature of the cell wall. Drew & Smith (1967a) showed that Nostoc isolated from Peltkera polydactyla develops a mucilaginous sheath in culture which is either lacking or much reduced in the thallus. Ahmadjian (1959) found that cultured Trebouxia possessed a gelatinous sheath which was lacking in the symbiotic forms,


Some modifications to the cell wall may well be expected to result from the intimate contact invoIved in this kind of symbiosis. There is also a further significance in that a possible mechanism for carbohydrate excretion could arise by a modification of the mechanism for cell-wall synthesis in the alga. Thus, it could be envisaged that in a Peltigera thallus, Nostoc excretes glucose because of a modification in which hexose units normally destined to become part of the mucilaginous sheath are prevented from participating in polysaccharide synthesis and are released instead. However, it must be stressed that mucilage of blue-green algae yields a variety of carbohydrates on hydrolysis, while Nostoc in lichens releases only free glucose and no other carbohydrate.

The composition of the gelatinous sheath of Trebouxia is not known but, as mentioned above, Green could not detect ribitol in the ethanol-insoluble material of directly isolated Trebouxia which had ceased to release ribitol. Ribitol has not been found as a cell-wall component of any organism except bacteria, where it occurs as a component of teichoic acid. The role of modification of cell-wall metabolism in carbohydrate transfer between symbiotic organisms will be considered in more detail in the Discussion at the end of this article.

(iv) Other diflerences. Other differences exist between symbiotic and cultured forms of lichen algae. For example, Trebouxia forms motile zoospores freely in culture, but reproduces only by simple cell division in the thallus. Such differences stress that there are other aspects to the symbiosis besides carbohydrate transfer.

(b) Importance of carbohydrate movement between the symbionts of lichens The great majority of lichen fungi are not known to exist free-living in nature in

the absence of their algal symbionts, so that they appear to have an obligate dependence on them.

The fungus can probably absorb and utilize most of the dissolved organic nutrients which it encounters in its habitat. However, lichens usually live in conditions where nutrients are in extremely short supply, so that the fungus would have to derive most of its organic carbon requirements from the alga. Indeed, when lichens are in habitats where the nutrient supply becomes rich, they tend to disintegrate (Tobler, 1925). Similarly, one of the optimum conditions for the initiation of a lichen synthesis in culture is a very low level of nutrients (Ahmadjian, 1962). Thus, the absence of nutrients in the habitat may be a condition enforcing the obligatory nature of the lichen symbiosis.

The extent to which substances other than carbohydrates move from alga to fungus is not yet clear. Lichens containing blue-green algae can fix nitrogen (Scott, 1956), and it is reasonable to suppose that a movement of fixed nitrogen may occur from alga to fungus during this process, Bednar & Holm-Hansen (1964) showed that Coccomyxa isolated from Peltigera aphthosa excreted 17 times more biotin than a free-living alga, Chlorella pyrenoidosa. It is thus possible that the fungus may be dependent on the alga for a supply of certain growth factors, and this could also impose ‘obligateness’ on the association.


(c) Evolution of lichens Many algae release small amounts of substances from their cells as a normal aspect

of metabolism. I t is thus easy to visualize that certain kinds of fungi could develop loose ecological associations with terrestrial algae, existing saprophytically on their excretion products. Fungi can also excrete a variety of compounds, and if certain of them could stimulate carbohydrate excretion from the algae, then a higher degree of intimacy in the association might then presumably evolve. For example, many fungi can excrete organic acids, and it is known that acid pH can stimulate the excretion of organic matter from algae (e.g. Tolbert & Zill, 1956). Although it has been argued above that in the thallus low pH may not be the main factor inducing carbohydrate excretion, it might well have been important in the early evolution of lichen-like associations.

In considering how carbohydrate release from the lichen alga is induced, it is natural to think in terms of some ‘factor’ produced by the fungus. However, the role of the environment should not be excluded. For example, it may well be significant that in attempts to synthesize lichens artificially the rate of lichenization is increased if the cultures are subjected to a simple system of alternate wetting and drying (Ahmadjian, 1962); Quispel (1959) has also suggested that variations in humidity during synthesis might be an essential condition for its success. Since such variation in humidity occurs as a regular feature in the environment of almost all lichens, its effect on the physiology of lichen algae, and especially on carbohydrate release, is clearly worth investigation. Interaction between environment and fungus to produce particular conditions at the surface of the alga should also be considered.

It is remarkable that in the five genera of green algae from lichens so far examined, polyols are the main form in which carbohydrate moves from alga to fungus. This phenomenon may well hold a clue as to the origin of some groups of lichens. For example, Lewis & Smith (1967a) point out that there are several groups of free-living fungi which are probably ecologically dependent upon polyols, and notable amongst them are certain ‘sooty moulds’ which live on insect ‘honey-dews’. These excretions of scale insects and aphids are substrates which may be frequently rich in polyols (Fraser, 1937; Hackman & Trikojus, 1952; Lewis & Smith, 1967a). Many ‘sooty moulds’ (e.g. Capnodiaceae, Atichiaceae and Chaetothyriaceae) are classified as Locu- loascomycetes, the subclass to which lichen fungi with bitunicate asci are also referred (Richardson & Morgan- Jones, 1964). Furthermore, some species of ‘sooty moulds’, e.g. Chaetothyrium babingtonii, and other Loculoascomycetes, e.g. Cyrtidula ( = Der- matina) quercus and Leptorhaphis atomaria, have been recorded as being sometimes lichenized, i.e. associated with algae (Dennis, 1960).

These facts focus attention on ‘sooty moulds’ and their allies as a group of fungi that may have given rise to some of the lichens now classified with the Loculoasco- mycetes. Further, such fungi show a number of other physiological similarities to lichen fungi besides ecological dependence on polyols. Fraser (1937) summarizes some of the physiological properties of ‘sooty moulds’ as follows: great tolerance to high temperatures when dry, but a lesser resistance when moist; great powers of


resistance to desiccation; slow rates of growth; ability to grow in habitats which are only intermittently moist; and antibiotic activity such that, in some cases, several species may occur on the same leaf but the mycelia do not mix and the colonies remain distinct though in contact. All these attributes are also held by lichens (Smith, 1962). It is therefore not inconceivable that if selection favoured casual associations between algae potentially capable of excreting polyols and members of this group of fungi, permanent associations resembling lichens could be produced. ' Sooty moulds' merit closer physiological study, and it would be especially relevant to determine whether they stimulate the release of the photosynthetic products of free-living and cultured lichen algae.

Nevertheless, it must be remembered that since the algae and fungi of lichens are of diverse kinds, the development of this kind of symbiosis must have occurred on numerous distinct occasions during evolution. In so far as carbohydrate movement is concerned, it implies that if algal excretion is stimulated by a product of the fungus, then this feature must occur relatively commonly in a wide range of ascomycete fungi.

V. AUTOTROPHIC HIGHER PLANTS AND FUNGI

( I ) Introduction (a) General

The kinds of symbiosis to be considered are those of higher plants with fungi for which the association is ecologically obligate or nearly so. Only those for which sound experimental data exist will be discussed. These include infections of flowering plants by the parasitic rusts and smuts (Basidiomycetes-Uredinales and Ustilaginales), parasitic powdery mildews (Ascomycetes-Erysiphales) and mutualistic ectotrophic mycorrhizal fungi (mostly Basidiomycetes-Agaricales, Hymenogastrales and Sclero- dermatales). Although mycorrhizas have many features delimiting them from infections by these biotrophic parasites, both kinds of fungi derive carbohydrate from their hosts in an apparently similar manner, as will be shown below. The carbon nutrition of those fungi causing gross destruction of their hosts (i.e. necrotrophic parasites) will not be discussed. That of endotrophic mycorrhizal fungi associated with higher plants that are always autotrophic has yet to be investigated, e.g. those of the Ericaceae, and the vesicular-arbuscular endophytes of the Gramineae and other groups (see Harley, 1959).

( b ) Structure and composition Associations of higher plants with fungi differ from those of algae with the other

organisms discussed above in the relative proportion of autotroph to the heterotroph. In associations involving algae, the photosynthetic partner is less in both volume and dry weight than the fungus which frequently envelops it completely. By contrast, the photosynthetic partner of non-algal associations is by far the greater proportion of the whole. In the extreme, some biotrophic fungi simply form local lesions on the leaves of large trees.

Contacts between fungus and host may be either inter- or intracellular or both.

Carbohydrate movement from autotrophs to heterotrophs 45 In ectotrophic mycorrhizas, particularly characteristic of the young feeding roots of forest trees, a network of hyphae penetrates intercellularly into the cortex of the host root from the encircling sheath. There are also hyphae connecting the sheath with the surrounding soil. Intracellular haustorial penetration, common in the pathogenic infections, is rare or absent in mycorrhizas. In several pathogens, where intracellular penetration by haustoria does occur, electron-microscope examination of the region of contact shows a layer of material laid down by the host cytoplasm isolating the fungal hyphae from direct contact (e.g. Ehrlich & Ehrlich, 1963a, b; Peyton & Bowen, 1963; Berlin & Bowen, 1964). No such deposition occurs in mycorrhizal roots of Pinus radiata (Foster & Marks, 1966).

( 2 ) Demonstration of carbohydrate movement to the heterotroph ( a ) Indirect evidence and early 14C experiments

While fungi which are restricted to the aerial parts of their hosts must obviously obtain all their carbohydrates from the hosts, it is possible that mycorrhizal fungi, which retain hyphal connexion with the soil, might derive at least some of their carbohydrate saprophytically from the soil. Indirect evidence against this possibility was summarized by Harley (1959) and Lewis & Harley (1965 a). In culture, these fungi have a restricted power to break down the insoluble polymers usually present in soil and leaf litter, such as cellulose, pectin and lignin. Also, by comparison with free living soil fungi, they have a low competitive saprophytic ability. These two attributes, coupled with the frequent observation that degree of mycorrhizal infection is positively correlated with increasing light intensity and therefore photosynthetic ability, suggest that, like the aerial pathogens, ectotrophic mycorrhizal fungi derive their carbohydrate requirements from their hosts.

Direct evidence for such movements to mycorrhizal fungi was first presented by Melin & Nilsson (1957). They synthesized mycorrhizas with seedlings of Pinus sylvestris using the fungi Boletus variegatus and Rhixopogon roseolus, and then exposed them to 14C0, in the light. After a period to permit translocation, considerable amounts of radioactivity in excess of decapitated controls were found in the fungal sheaths. Later, Bjorkman (1960) demonstrated the movement of 14C, derived from glucose, from spruce (Picea excelsa) to its mycorrhizal fungus.

In earlier pioneering studies of carbon movement in pathogenic associations using gross counting as a measure of accumulation, Yarwood & Jacobsen (1955) clearly paved the way for more detailed investigations. They showed that more 14C accumulated in the infected than the uninfected sides of rusted bean leaves when 14C-sucrose was supplied to these leaves either directly by immersion or indirectly by translocation from other leaves.

( b ) Radioautography Table 4 summarizes radioautographic studies. Initially, there was controversy

whether accumulation was in fungal material or adjacent host cells. This has now been resolved and the macro-radioautographic experiments of Thrower (1965), who used


leaves of Trifolium subterraneum infected with Uromyces trifolii, illustrate a pattern which is probably common to many rusts. He first demonstrated that marked accumulation of carbon compounds supplied to the host occurred in the fungus 4 days after infection and before pustules could be seen. In a time-lapse study of movement of photosynthate from host to the fungus at the uredial stage, the following sequence could be discerned. Immediately after a 3 hr. period in 14C02 in the light there was high activity in the uredia, but this was attributed to dark fixation by fungal tissue. The uredia were surrounded by a zone of depressed activity. After a further period of 1 2 hr. a marked annulus of accumulation developed around the uredia in a region corresponding to that of the accumulation of starch which is characteristic of rust

Table 4. Radioautographic studies of movement of 14C from hosts to fungal pathogens

Forms in which C14 Pathogens Hosts supplied to host References

Erysiphe graminis Triticum compactum

Puccinia helianthi Helianthus annuus P. graminis

carbon dioxide; sugars; sugar lactones; sugar,

acids

Shaw et al. 1954; Shaw & Samborski, 1956;

~ ~ i ~ i ~ ~ ~ compactum aromatic and phenolic Shaw, 1961

Hordeum vulgare

T . dicoccum T . aestivum T . compactum T . compactum T . compactum T. dicoccum

carbon dioxide; glucose Wang, 1960 carbon dioxide

carbon dioxide von Sydow, 1966

von Sydow & Durbin, 1962

P. poarum Tussilago farfara carbon dioxide Lewis, Yuen & Breen (unpublished)

Uromyces trifolii Trifolium subterraneum carbon dioxide; glucose Thrower, I 965

I 1

infections. At this stage and up to 24 hr. the uredia lacked appreciable 14C. At 48, 72 and 96 hr. after removal from 14C02 a pronounced accumulation of radioactivity at the site of infection resulted in a much heavier labelling of the uredia than the leaflets of the host. Thrower also supplied [14C]glucose via the petiole to infected leaflets and obtained a similar accumulation within infected tissues, the major differences being the faster rate at which 14C was absorbed by the uredia and the less pronounced accumulation of radioactivity around the sites of infection. The results of Shaw & Samborski (1956), Shaw ( 1 9 6 1 ) and von Sydow ( 1 9 6 6 ~ ) using other fungi and hosts were essentiaIly similar. An interesting feature of the latter’s experiments was that a zone of enhanced activity around the rust pustules was present on the resistant ‘Khapli’ wheat, but absent on the susceptible variety ‘Little Club’. Other work (von Sydow & Durbin, 1962; von Sydow, 1966a) using micro-radioautographic techniques demonstrated that much accumulation of radioactivity occurred within fungal mycelium.

( c ) Dissection The movement of 14C supplied to host tissue into associated fungi has been studied

using dissection techniques in both mutualistic and parasitic symbiosis. In ectotrophic

Carbohydrate movement from autotrophs to heterotrophs 47 mycorrhizal roots of beech (Lewis, 1963; Lewis & Harley, 1 9 6 5 ~ ) and powdery mildew of barley (Edwards & Allen, 1966) the separation was into superficial fungus, and host with residual fungus (Hartig net and haustoria respectively). Both these investigations involved a study of the chemical fate of the 14C in the fungus and are discussed in more detail below (Section V(4)). In several other studies, particularly with rust infections, separation has been into healthy host and diseased host. This research which involved quantitative aspects of the carbohydrate movement is also discussed in the appropriate section below (Section V(3, b)).

(3) Characteristics of carbohydrate movement ( a ) Form in which carbohydrate moves

Unlike algae considered above, higher plants already possess a range of carbohydrates that are characteristically moved from photosynthetic to non-photosynthetic regions of the plant. A priori, there seems no reason why the contents of the normal translocation stream should not be available to symbiotic fungi, and evidence for this is presented in the next section. It therefore seems probably that sucrose, the most commonly translocated sugar, is the main carbohydrate source for symbiotic fungi. Direct evidence for this is sparse and, in view of the apparent induction by lichen fungi of ‘special’ carbohydrates which move from the alga, such alternatives must be enter- tained for infections of higher plants.

Lewis & Harley (1965 c) simulated translocation of 14C-sucrose through the host root to the fungus in excised beech mycorrhizas, and subsequently found low levels of r4C]sucrose in the fungal sheath, possibly suggesting this was the compound entering the fungus. However, contamination by some host tissue is possible in the dissection technique. A cleaner separation is possible in infections of barley by the powdery mildew, Erys+he graminis (Edwards & Allen, 1966). After short periods of exposure to 14C02 the fungal mycelium contained much [14C]sucrose, but the proportion of radioactivity in this compound declined with time, clearly indicating that sucrose was entering the fungus and was then being converted to fungal metabolites. A difficulty here is that sucrose is normally inverted by fungi before absorption and it is unlikely that resynthesis of sucrose from its constituent hexoses occurs in the fungus.

The ‘inhibition technique’, so valuable in studies with lichens, has, to date, been used only in one infection of higher plants (Lewis & Yuen, unpublished). In preliminary experiments they used disks cut from leaves of coltsfoot, Tussilago farfara, each infected by a single lesion of the aecial stage of the rust Pucciniapoarum. Samples of disks were exposed to 14C0, in the light for 3 hr. and then each disk was cut into half through the centre of the pustule. One set of halves was floated in the dark for 22 hr. on water and the other on a I yo [12C]sugar solution. On control water media only the fungal carbohydrates, mannitol and arabitol, appeared, most probably owing to contaminating spores. On [12C]sucrose, [14C]sucrose was most heavily labelled, indicating that this was probably the form of carbohydrate released from host cells. On media containing [12C]glucose or [12C]fructose, results were obtained suggesting that sucrose is inverted by the fungus before absorption.


Although all these data are consistent with sucrose being the mobile carbohydrate, much more detail is still required on the mechanism of transfer from host to fungus. Information is totally lacking in hosts where compounds other than sucrose are normally translocated.

(6 ) Quantitative aspects-the eflect of fungal infection on translocation within the host plant Although a fungal infection may be restricted to only a very small area of the host,

it may result in a substantial diversion of photosynthate from other regions to the site of infection. This completely disrupts the normal pattern of translocation of carbohydrates within the host plant.

Quantitative studies of this effect were first described by Shiroya et al. (1962) for mycorrhizal infections of pine and Zaki & Durbin (1962) for rust infections of bean. The results with mycorrhizas were later summarized by Nelson (1964). While the shoots of non-mycorrhizal plants exported only 5 yo of their photosynthate to the roots, those of mycorrhizal plants exported 54%. The root/shoot ratio was much higher in the mycorrhizal plants in this experiment, so that the increased movement of photosynthate to the roots might have been due to the greater root development rather than to mycorrhizal infection. However, further experiments under sterile conditions using plants of widely differing root/shoot ratios suggest that this is not so and that mycorrhizas do increase the ‘sink’ capacity of the root system.

The effects of rust infections on patterns of translocation in host plants have been described in detail by Doodson, Manners & Myers (1965) for Puccinia striiformis on wheat, by Livne & Daly (1966) and Pozsk & KirPly (1966) for Uromyces phaseoli on bean, and by Thrower & Thrower (1966) for Uromyces fabae on broad bean.

Doodson et al. used wheat plants in which the third leaf was infected with rust. For the first 80 days after infection, 14C0, techniques showed that healthy and diseased leaves exported similar percentages of photosynthate, but after 2 weeks diseased leaves only exported 0.4% during the 3 hr. experimental period compared with 21.o‘%, in controls. Similar effects were obtained when other leaves were infected and presented with 14C0,. The destination of what left the diseased third leaf was not affected either by the fungus or by infection in other leaves above or below it. However, within these other infected leaves, the principal destination of photosynthate arriving from the third leaf was around the pustules. When the time course of exit of photosynthate from healthy and diseased leaves was followed, healthy leaves exported 52*94/, in 4 hr. and 83.8% in 16 hr., whereas only 2.8% and 3‘4% respectively were translocated away from diseased third leaves, Doodson et al. conclude: ‘ In contrast to the marked retention of assimilate by an infected leaf, such a leaf was unable to distort the normal distribution by attracting assimilates from the other leaves.’

Similar experiments were conducted by Livne and Daly. They infected both of the lowest pair of bean leaves with Uromycesphaseoli and then presented one with 14C0,. Only 20/, of the fixed 14C left the leaf after 5$ hr., whereas an uninfected control exported over SO”/” during the same period; again a clear demonstration of the reten- tive nature conferred on the leaves by the fungus.

Carbohydrate movement from autotrophs to heterotrophs 49 In further experiments, Livne and Daly obtained somewhat different results from

Doodson et al. since they showed that infection of one leaf could influence translocation from another. They supplied I4CO1 to the next leaf above the lowest, and in uninfected plants over 50% of the fixed 14C was exported in j$ hr. The major destinations were: the youngest leaves ( I j%), stem (17'/,) and root (19%). Less than 1 % reached the lowest leaves. However, when the lowest leaves were infected, they received large quantities of photosynthate (32 %), principally at the expense of the youngest leaves, i.e. a marked reversal of the polarity of transport.

Investigations of U. fabae on broad bean by Thrower & Thrower (1966) confirmed these effects. They showed that the import of photosynthate from the third leaf was both prolonged and maintained at a higher level when this was diseased, with the result that import into the uninfected fifth leaf was decreased. When one of a pair of leaflets was infected, photosynthate moved into it from the opposite uninfected leaf- let-a situation not obtaining when both were uninfected. Thrower and Thrower suggested that a possible reason why infected bean leaves could attract assimilates from other leaves while infected wheat leaves could not (i.e. as in the experiments of Doodson et al.) was that there is a difference between monocotyledons and dicotyledons in possible routes of translocation.

It is interesting to note that infection of potato by the necrotrophic fungus Phyto- phthora infestans also caused an increased import to and decreased export from diseased leaves (Garraway & Pelletier, 1966). The experiments of Pozshr and Kirily are discussed below in the section concerned with the effects of plant hormones.

All the studies discussed here have been made with fungi producing localized infections. I t would be of interest to investigate patterns of translocation in systemically infected plants.

(4) The fate of carbohydrate in the heterotroph There is evidence from a variety of experiments that sugars arriving at sites of

infection from other parts of the host plant become rapidly converted to fungal carbohydrates. It is now generally accepted that the commonest and most abundant soluble carbohydrates in mycelia of most fungi (with the exception of phycomycetes) are trehalose and polyols, especially mannitol and arabitol. Where it is known that the host plant does not produce these carbohydrates, their formation at sites of infection and their incorporation of 14C from host sources are good indication of transfer of carbohydrate to the fungus.

Some of the earliest experiments which demonstrated this were carried out by Daly, Inman & Livne (1962). They fed [14C]glucose to healthy and diseased tissues (hypo- cotyls of safflower, Carthamus tinctorius, infected by Puccinia carthami; and leaves of bean, Phaseolus vulgaris, infected by Uromyces phaseoli). Sucrose, glucose and fructose were the only soluble carbohydrates to become labelled in healthy tissues, whereas in the diseased, three compounds, tentatively identified as trehalose, mannitol and arabitol, also became highly radioactive. At the flecking stages of the Uromyces infections the three fungal metabolites accounted for 44% of the total radioactivity and approximately 57 yo at the sporulation stage. Livne (1964) then investigated the

4 Biol. Rev. 44

5 0 DAVID SMITH AND OTHERS

[14CC]sugars formed during photosynthesis in 14C02 by healthy and rusted beans. When attached diseased leaves were fed WO, , fungal sugars (trehalose, mannitol and arabitol) accounted for 70.9 yo of the radioactivity in the soluble fraction, 28.7 ol0

being distributed between sucrose, glucose and fructose. In healthy leaves, these latter three sugars comprised 82.3% of the total.

Lewis (1963) and Lewis & Harley (196.5~-c) made similar studies on mycorrhizal roots of beech. They showed that mannitol and trehalose were present in mycorrhizal roots but absent in non-mycorrhizal. Mycorrhizas showed a marked synthesis of trehalose and mannitol from exogenous glucose, and mannitol from exogenous fructose. By contrast, non-mycorrhizal roots synthesized sucrose from both hexoses. In experiments where the translocation of [14C]sucrose to the host tissue of excised mycorrhizal roots was simulated, subsequent dissection of the fungal sheath from the host core tissue showed that the fungus could absorb sugar from the host, converting it mainly into trehalose and mannitol.

In experiments mentioned above, Edwards & Allen (1966) followed the movement of [I4C]photosynthate from barley leaves to the mycelium of the powdery mildew, Erysiphe graminis. Again, the fungus rapidly converted sucrose derived from the host to polyols and trehalose. Similarly, mannitol and, to a lesser extent, trehalose became labelled when the host-parasite complex of Puccinia graminis on wheat was exposed to [14C]sucrose (Lunderstadt, 1966), and mannitol when mycelium of flax rust, Melampsora h i , was exposed to [14C]glucose (Mitchell, 1964). D. H. Lewis, C. Yuen & S. J. Breen (unpublished) showed that when disks from leaves of coltsfoot, Tussilago farfara, infected by a single pustule of the spermagonial/aecial stage of Puccinia poarum, are exposed to 14C0, there is an initial synthesis of sucrose followed later by a conversion into mannitol and arabitol by the rust.

From their observations on beech mycorrhizas, Lewis (1963) and Lewis & Harley ( 1 9 6 5 ~ ) concluded that the rapid conversion of host sugars to fungal carbohydrates could serve to maintain a concentration gradient and so ensure a continued flow from the host to the fungus. This conclusion may also apply to many associations involving biotrophic parasites. For example, in addition to the cases described above, Table 5 lists a wide range of infected tissues which have been shown by chromatographic analysis to contain polyols characteristic of fungi; in the uninfected state, the host tissues do not contain the polyols mentioned.

In some of the earlier studies of fungal infections, no mention is made of the presence of polyols. However, it now appears likely that this may have been the result of misidentification of some carbohydrates, partly because of the lack of satisfactory analytical techniques. For instance, when Jain & Pelletier (1958) investigated the 14C sugars derived from 14C0, in healthy and diseased plants of wheat infected with the rust Puccinia graminis, the common fungal carbohydrates, trehalose, mannitol and arabitol, were not used as markers for their chromatograms. In the solvents used, trehalose would not have been readily distinguished from maltose, and could be their ‘unidentified a’. Mannitol would have run in the sedoheptulose region, and could have been their ‘unidentified c’. I t is significant in the light of the more recent work that both these chromatogram regions were more heavily labelled in diseased than in


Table 5 . The occurrence of acyclic polyhydric alcohols in tissues of higher plants infected by biotrophicparasites (unpublished data of D. H. Lewis, J . Webster and L. A. Thorpe)

(Nomenclature of plants after the folloNing authorities. Angiospermae: Dandy ( I 958) ; Uredinales: Wilson & Henderson (1966); Ustilaginales: Ainsworth & Sampson (1950); Erysiphales: Blumer (1967); other Ascomycetes: Dennis (1960).)

Class and Order

Basidiomycetes, Uredinales

Basidiomycetes, Ustilaginales

Ascomycetes, Clavicipitales

Ascomycetes, Taphrinales

Ascomycetes, Erysiphales

Basidiomycetes, Ustilaginales

Basidiomycetes, Agaricales

Species Fungal stage*

(a) Fungi with mannitol and arabitol Coleosporium tussilaginis Melampsorella symphyti M. caryophyllacearum Phragmidium bulbosum P. sanguisorbae Xenodocus carbonarius X . carbonarius Triphragmium filipendulae Puccinia violae P. malvacearum P. chaerophylli P. betonicae P. glomerata P. hieracii P. lapsanae P. punctiformis P . poarum P. caricina P . coronata P. recondita agropyrina P. r. holcina Tranzchelia anemones T. discolor Uromyces ficariae U. muscari Trachyspora intrusa Ustilago violacea U. violacea Sphacelotheca hydropiperis Epichloe typhina

Claviceps purpurea Taphrina deformans T. tosquinetii Sphaerotheca fuliginea Podosphaera clandestina P. clandestina P . lmcotricha Erysiphe graminis E. depressa

U. U. U. U./T. A. A.

Teliospores A. S./A. T. U./T. T. T. U./T. T. S./A. S./A. S./A. U. U. U. T. U./T. T. T. A. C. C. C.

conidial stroma sclerotia

asci present asci present

con i d i a 1 conidial conidial conidial conidial

perithecia present

(b) Fungi with mannitol and erythritol Ustilago longissima C. U. longissima C. U. hypodytes C. U. avenae C. U. nuda C. Urocystis anemones C. Ur. anemones C. Exobasidium vaccinii basidiospores

E. vaccinii basidiospores present

present

Host plant

Tussilago jarjara Synzphytum oficinale Cerastium tomentosum Rubus fruticosus Poterium sanguisorba Sanguisorba oficinalis S. oficinalis Filipendula vulgaris Viola sp. Malva sylvestris Myrrhis odorata Betonica oficinalis Senecio jacobaea Taraxacuni oficinale Lapsana coinmunis Cirsium arvense Tussilago farfara Urtica dioica Avena sativa Agropyron repens Holcus lanatus Anemone nemorosa Prunus domestica Ranunculus ficaria Endymion non-scriptus Alcheniilla xanthochlora Silene alba S. dioica Polygonum hydropiper Holcus mollis

Phalaris arundinacea Prunus persica Alnus glutinosa Taraxacum oficinale Filipendula ulmaria Crataegus monogyna Malus sylvestris Triticum sp. Arctium minus

Glyceria declinata G. maxima Elymus arenarius Arrhenatherum elatius Hordeum vulgare Anemone nemorosa Ranunculus repenr Vaccinium vitis-idaea

V. myrtillus

* S.= Spermagonial; A. = Aecial; U. = Uredial; T. = Telial; C. = Chlamydospores present.

4 -2

5 2 DAVID SMITH AND OTHERS

healthy tissue, whereas sucrose was less so. Some of the experiments of Wang (r960) also are open to similar criticism. In wheat infected with P. graminis and supplied with [14C]glucose, nearly half of the radioactivity was in a compound identified as mannose, but which was more probably mannitol. Increased levels of 14C over uninfected controls were found in compound MI, probably trehalose and not maltose, and in a compound of higher R, than fructose. No comment was made on this latter increase, which possibly represented accumulation of radioactivity in arabitol.

The fungi considered so far have all belonged to the Ascomycetes, Basidiomycetes or Fungi Imperfecti, in which polyols and trehalose are almost universally present (Lewis & Smith, 1 9 6 7 ~ ) . Since it is likely that most of these fungi can rapidly convert exogenously supplied sugars to their own carbohydrates, the conclusions of Lewis and Harley about the importance of this process in mycorrhizas can probably also be extended to biotrophic parasites.

In associations involving Phycomycetes, other mechanisms probably operate since no polyols have been found in those higher plant tissues infected with Phycomycetes that have been examined-four genera and nine species of Peronosporales, and three Synchytrium species of the Chytridiales (D. H. Lewis, J. Webster & L. A. Thorpe, unpublished). Metabolic conversions of carbohydrates have not been studied in these fungi.

The above discussion has dwelt entirely on soluble carbohydrates, but conversion of host sugars to insoluble compounds in the fungus must not be neglected. Thus, mycorrhizal roots show a rapid synthesis of r4C]glycogen from exogenous [14C]glucose while uninfected roots show a much lower level of incorporation of 14C into insoluble compounds (Lewis &Harley, 1965 b). Similar enhanced incorporation into the insoluble fraction has been noted for some tissues infected by certain rusts (Lunderstadt, 1966; von Sydow, 19663) though not for others (Livne, 1964). In their study of barley leaves infected with powdery mildew, Edwards & Allen (1966) also showed an increase in the labelling of the insoluble fraction of mycelia as compared with healthy host tissue. The importance of the synthesis of glycogen and other insoluble fungal storage products in promoting flow of carbohydrate from the host is in need of further investigation.

The biochemical mechanisms involved in the conversion of host sugars into both soluble and insoluble fungal carbohydrates also deserve further study. In general, when [14C]glucose is supplied to diseased tissue, various of the experiments described above show that trehalose becomes more heavily labelled than the polyols. The con- verse is true if [14C]sucrose is supplied-either directly, or indirectly via host photosynthesis of 14C0,. In beech mycorrhizas, exogenously supplied l4[C]fructose is converted to mannitol, and very little radioactivity appears in trehalose. The mechanism by which the fungi preferentially convert fructose moieties to mannitol and glucose moieties to trehalose is not known. The first suggestion of Lewis & Harley (1965b), that the fungus might have low phosphohexoisomerase activity, proved incorrect (Harley & Loughman, 1966). There may also be differences in fungal polysaccharide synthesis. For example, Lewis & Harley (1965a, b) demonstrated that glucose was a ready precursor of glycogen in mycorrhizal roots whereas fructose was not. In this

Carbohydrate movement from autotrophs to heterotrophs 53 context, it is of interest that fructose is frequently a much poorer carbon source than glucose for growth of mycorrhizal fungi in culture (see tables VIII and XI of Harley, I 959, for Tricholoma spp., Mycelium radicis atrovirens and Cenococcumgraniforme. The same holds for several Boletus spp. and Rhizopogon roseolus; B. W. Ferry, personal communication to Professor Harley).

( 5 ) Special aspects (a) General

The preceding sections show that photosynthate may be diverted from other regions of a host plant to the site of a fungal infection and there rapidly converted to fungal storage compounds. In order to present a comprehensive picture of the movement of carbohydrate from host to fungus, not only the mechanism of transfer to the fungus at the site of infection must be considered, but also how photosynthate becomes diverted to this site.

Livne & Daly (1966) point out that the accumulation of carbohydrate at sites of fungal infections and the attendant alterations in patterns of translocation within the host ‘appear to be in agreement with a classical concept of “source” and “sink” in determining movements of photosynthesized substances in higher plants ’. They are careful to add that use of the terms ‘source’ and ‘sink’ is only descriptive. Four lines of investigation throw some light on possible mechanisms by which the fungal region can act as a ‘sink’. Three are concerned essentially with local effects in the fungal region: lack of reciprocal flow of carbohydrate from heterotroph to autotroph; effects of the fungi on host cell walls; and permeability changes of host cells induced by the fungi. The fourth, changes in hormone balance, has been invoked in both accumulation of metabolites at infection sites and changes in patterns of translocation.

(b) Lack of reciprocal %ow of carbohydrate from heterotroph to autotroph If the conversion of host carbohydrates into trehalose, polyols and other specifically

fungal metabolites is an important aspect of the mechanism of transfer, the system would be even more efficient if these metabolites were not readily re-available to the donor. So far, there has only been one attempt to demonstrate this directly: Lewis & Harley ( 1 9 6 5 4 showed that mycorrhizal roots of beech could readily utilize exogenously supplied [14C]mannitol and [14C]trehalose, but the uninfected roots showed a very restricted ability to do so, especially of mannitol (14C0, production was the criterion of utilization in these experiments). However, there is important indirect evidence in that polyols are known to be only poorly metabolized in higher plants unless the plants normally contain them (Lewis & Smith, 1 9 6 7 ~ ) .

It therefore appears that the rapid conversion of host sugars to carbohydrates peculiar to the fungus not only serves to maintain a concentration gradient of host sugars into the mycelium, but also prevents reciprocal movement from fungus to host.

( c ) ESfects of fungi on host cell walls In the preceding section on lichens, the possibility was considered that the fungus

may derive carbohydrate by some interference with the synthesis of algal cell-wall


polysaccharides. A comparable possibility may be considered in ectotrophic mycorrhizas, as the following example shows.

Clowes (1954) suggested that the root-cap tissue in ectotrophic rnycorrhizas of beech is limited in extent, owing to decomposition of the outer cap cells within the fungal sheath. However, there is a general lack of cellulase or pectinase enzymes in mycorrhizal fungi, apart from a few notable exceptions such as Tricholoma jumosum and T . vaccinum, so that mycorrhizal roots may have limited root caps not because they are decomposed by the fungi, but because the fungi prevent the proper formation of cell walls in the daughter cells of the cap initials. Thus, if the fungi obtain carbohydrate, it would be through utilizing cell-wall precursors rather than by degrading fully formed complex polysaccharides. I t is especially relevant to this hypothesis that some fungal antibiotics, e.g. penicillin, exert their effect on bacteria by blocking some stage in the biosynthesis of cell-wall polysaccharides (see review by Reynolds, 1966). It remains to be seen whether other antibiotics are important in a similar role in the interactions between symbiotic fungi and their hosts.

( d ) Effects of fungi on the permeability of host cells Since the early studies of Thatcher and others, the idea that pathogenic fungi have

profound effects on the permeability of host cells has excited much interest (see reviews by Sempio, 1959, and Rubin & Artsikhovskaya, 1963). Moreover, some of the toxins produced by wilt-fungi cause selective increases in permeability, e.g. fusaric acid causes a greater leakage of monovalent cations than lycomarasmin, which itself has a greater effect on the leakage of some amino acids but not others (Linskens, 1955). The production of such compounds by symbiotic fungi and their effect on the selective permeability of host protoplasm to sugars are in need of fuller investigation.

The permeability changes induced in micro-organisms by antibiotics have been recently reviewed (Lampen, 1966; Brock, 1966). The effect of these compounds on higher plants is an aspect of host-parasite interaction which requires consideration, since streptomycin certainly has profound effects on the properties of membranes of higher plant cells, e.g. Saunders, Jenner & Blackman (1966), Venis & Blackman (1966).

( e ) Effects of fungi on the hormone balance of infected plants I t is beyond the scope of the present article to review the evidence that the trans-

location of metabolites in healthy higher plants is under hormone control (but see references in Pozsir & Kirily 1966, and Seth & Wareing, 1967). Experiments which rely on studying translocation patterns after local application of exogenous hormone suffer from the disadvantage that it is difficult to assess whether effects are due directly to the hormone per se, or indirectly through increased metabolic activity at the site of application. Nevertheless, there is an accumulation of data consistent with the notion of hormone-directed translocation, and that the alterations in patterns of translocation resulting from fungal infection are mediated by changes in the hormone balance of the healthy plant.

In the early studies of accumulation of radioactive metabolites at sites of fungal infection, Shaw & Samborski (1956) pointed out that their results resembled the

Carbohydrate movement from autotrophs to heterotrophs 55 process of accumulation at meristematic regions and postulated that diffusible substances might be responsible. Thrower (1965) has demonstrated that, following the application of kinetin to leaflets of Trifolium subterraneum, photosynthate accumulates in a manner comparable to infection by Uromyces trifolii. There is also considerable evidence for increased hormone content of infected tissues. Sequeira (1963) has reviewed the literature on this topic with regard to /3-indolyl-acetic acid (IAA) in diseased tissues, and the production of this auxin by mycorrhizal fungi was investigated by Ulrich (1960). Increased gibberellin levels have been recorded in rusted thistles by Bailiss & Wilson (1967), and increased cytokinin levels in rusted bean leaves by Kirily, El Hammady & Pozsir (1967). The production of zeatin, zeatin riboside and at least one other cytokinin by cultures of the mycorrhizal fungus, Rhizopogon roseolus, has recently been demonstrated by Miller (1967).

Pozsir & Kirhly (1966) have attempted to correlate patterns of phloem transport in healthy and rusted bean leaves with cytokinin levels. The movements of radioactive glucose, glycine, phosphate and sulphate were studied in healthy and diseased plants and the results were essentially the same as those of Livne & Daly (1966), in that translocation from uninfected to infected leaves was increased, but export from infected leaves almost abolished. A reversal in polarity of movement could also be achieved by removal of the terminal bud. Application of IAA to the cut apex had no effect on this reversal, but it could be prevented by application of kinetin or benzyl- adenine, which are synthetic cytokinins. Other effects of rusts similar to those produced by exogenous cytokinins, notably the maintenance of green islands on yel- lowing leaves and induction of senescence in other parts of the plant, certainly imply the involvement of such compounds in other aspects of the host-parasite complex as well as in control of patterns of translocation.

VI. FUNGI AND 'SAPROPHYTIC' HIGHER PLANTS

( I ) Introduction The so-called saprophytic higher plants include : liverworts such as Cryptothallus ;

pteridophyte prothalli such as Lycopodium, Psilotum and Ophioglossum; monocotyledons, e.g. members of the Burmanniaceae, Triuridaceae and such Orchidaceae as Neottia, Corallorhiza, Galeola and Epipogium; and dicotyledons such as Monotropa (Monotropaceae) and some members of the Gentianaceae and Pyrolaceae. These plants are all obligately mycotrophic, and are perhaps better regarded as being parasitic on their associated fungi rather than as saprophytes (cf. Terekhin, 1962). The structures of the associations have been reviewed by Harley (1959) and Meyer (1966).

Formerly, it was thought that these partnerships were simply of higher plant and fungus and it was generally accepted that the higher plant obtained its carbohydrate at the expense of its endophyte. Beginning with the observations of de Cordenoy, and Kusano (see Harley, 1959), considerable evidence has now accumulated that tri- partite arrangements, in which the fungus is associated either parasitically or mutual- istically with a second higher plant, are common. In this way, the so-called saprophytic complex of higher plant and endophyte is parasitic as far as carbohydrate is


concerned on another higher plant. This phenomenon was named epiplzytosis by Kuinen (1953), who clearly demonstrated that the endophyte of several epiphytic orchids was also parasitic on the plant to which the epiphytes were attached. In this way, it may well be the specificity of the common fungus which determines the specificity of many epiphytes for their supports. Bjorkman (1960) showed that the ect-endotrophic fungus of Monotropa hypopithys is simultaneously ectotrophic with associated conifers. He termed this situation epiparasitism. Campbell (1962, 1963, 1964) has described similar arrangements for three species of the non-green orchid genus Gastrodia. G. cunninghamii is epiparasitic on the roots of forest trees via Armillaria, G. minor on roots of Leptospermum scoparium and G. sesamoides on roots of Acacia melanoxylon, possibly via Fomes mastoporus.

( 2 ) Demonstration of carbohydrate movement to the heterotrophic higher plant ( a ) Associations of two organisms

Orchid seeds only germinate and develop if infected by mycorrhizal fungi or if artificially supplied with carbohydrate-clear indirect evidence that a function of the endophyte is to provide carbohydrate. The only experimental investigations of direct movement are those of S. E. Smith (1966, 1967) using orchid seedlings artificially infected with various isolates of Rhizoctonia spp. In associations between the fungus R . repens and seedlings of the orchid Dactylorchis purpurella, much greater growth of seedlings occurred when the fungus was growing on a cellulose medium than when no carbohydrate was supplied. When [14C]glucose was supplied to the fungal mycelium at a distance from the seedling, radioactivity could subsequently be detected in the seedling, showing that 14C compounds could be translocated along the hyphae into the seedling. Radiochemical analyses (Section VI (3) below) confirmed that movement was into tissues of the orchid and not just into fungal hyphae associated with it.

( b ) Associations of three organisms That non-green higher plants can obtain carbon compounds ultimately from sur-

rounding green plants has been demonstrated by Bjorkman (1960). He injected[l4C]- glucose into the trunks of spruce trees, Picea excelsa, and 4 days later radioactivity could be detected in adjacent plants of Monotropa. Since the two higher plants were not in direct contact, the 14C presumably moved along the fungal hyphae that linked them, for it did not move to other species of plant nearby.

(3) The form in which carbohydrate moves and its fate in the heterotrophic higher plant In the experiments of S. E. Smith (1967) mentioned above, the proportion of radio-

activity in individual sugars in the soluble fraction of infected seedlings (which were not green and photosynthetic) was determined at intervals up to 7 days after exposure to lac. The results were consistent with a translocation of trehalose along the fungal hyphae and its conversion into sucrose by the orchid tissue. This situation is the complete reverse of those described for beech mycorrhizas and for various pathogenic infections in Section V(3) and (4) above. Although mannitol was present in one strain

Carbohydrate movement from autotrophs to heterotrophs 57 of the fungus and incorporated 14C from the glucose, it did not appear to move into the seedling.

(4) Special aspects ( a ) The mechanism of carbohydrate transfer

An unresolved problem of these associations is the mechanism of carbohydrate transfer: does carbohydrate move in a biotrophic manner between living cells or does the higher plant only absorb the products of digested fungal hyphae in a necrotrophic manner? As Harley (1959) pointed out, ‘The digestive action of the host is, at least in part, an efficient defensive mechanism. It may also be nutritive.’ In either case, the orchid must be able to utilize the soluble sugars of the fungus. The ability of orchid seeds to germinate and grow on a variety of carbohydrates has recently been reviewed by Arditti (1967). Several species can grow satisfactorily on trehalose and this has also proved to be true for Dactylorchispurpurella (S. E. Smith, unpublished). Fewer species of those tested can utilize mannitol or other polyols. S. E. Smith suggests that the ‘sink’ capacity of the meristematic cells of the seedlings may be sufficient to promote flow of carbohydrate from the fungus, but that this may be accompanied by digestion.

( 6 ) Reciprocal movement of carbohydrate from ‘ saprophyte’ to fungus The ability of the fungus to grow from the orchid seedlings to a carbon-free medium

indicated that it may also be able to obtain carbohydrate from the orchid (S. E. Smith, I 967). Preliminary experiments with year-old, green, infected seedlings, however, showed no incorporation of 14C into fungal sugars following a 5 hr. period of photosynthesis in 14C0, and subsequent periods of 22 hr. in light or dark (S. E. Smith, J. L. Harley & D. H. Lewis, unpublished). These data suggest that reciprocal movement is, at the most, slow. The possibility and extent of two-way transfer in endotrophic mycorrhizas requires further investigation.

VII. AUTOTROPHIC HIGHER PLANTS AND PARASITIC HIGHER PLANTS

(I) Introduction Although many aspects of the biology of angiospermous parasites have been dis-

cussed recently (Hartel, 1959; Schmucker, 1959; Srinivasan & Subramanian, 1960; Ozenda, 1965 ; King, 1966), there has been no comprehensive review of the interchange of carbohydrates between hosts and these parasites. Ozenda lists fourteen families of flowering plants which contain nearly 150 genera and 2500 species of parasites. He omits the genus Krameria, a few species of which are also parasitic (Srinivasan & Subramanian, 1960). This large number of angiosperms contrasts with the single parasitic gymnosperm, Podocarpus ustus.

Some parasites completely lack chlorophyll, and so must derive all their carbohydrates from their hosts, although conclusive experimental proof is lacking in all cases. Other parasites do contain chlorophyll, and the question arises whether they are self-supporting with respect to carbohydrates, or even whether there is any export to the host. It is also possible that essential substances, not carbohydrates, may be required from a host by a green parasite.


Experimental studies of carbon movement or carbon metabolism are, at present, restricted to a few parasites of four families : Loranthaceae, Scrophulariaceae, Oro- banchaceae and Cuscutaceae. It is evident that different parasites depend on their hosts for carbohydrates to differing degrees, and it will be convenient to consider the taxonomic groups separately. Details of structure of the associations may be found in the reviews mentioned above.

The Loranthaceae possess about 1300 species, all parasites and all except three containing chlorophyll. They are usually attached to the aerial parts of the host by structures commonly regarded as modified adventitious roots which may often branch within the host.

The Scrophulariaceae is a large family of over 200 genera, most of which are completely autotrophic. Ozenda lists ten parasitic genera comprising some 500 species which contain chlorophyll, and a further six genera comprising 60 species which are probably entirely dependent on their hosts for carbohydrate. All are root parasites. A wide range of dependence on the host occurs, for some species can exist and flower away from a host whereas others require to be attached to reach maturity. The genus Striga is green and photosynthetic, but seed production can occur in S. asiatica in complete darkness when attached to a host plant (Rogers & Nelson, 1962).

The Orobanchaceae consists of 14 genera and 180 species, all non-green root parasites. This family is closely related to the Scrophulariaceae, and was formerly included in it. Indeed, from a physiological point of view, the Orobanchaceae re- presents the culmination of a series of increasing dependence on particular host plants, ranging from the unspecialized, facultative hemiparasitism of Odontites and continued through increasing specialization and obligate parasitism of other genera of the Scrophulariaceae such as Melampyrum and Tozzia.

The Cuscutaceae comprises about 170 species in the single genus Cuscuta. They mostly lack leaves and roots, and twine around the aerial part of their hosts. Although the Cuscutaceae were for a long time thought to be entirely heterotrophic, certain species have now been shown to be photosynthetic (Cuscuta gronowii, C. campestris and C . reflexa by MacLeod, 1961, 1962; C. indecora, C. campestris and C . approximata by Pattee, Allred & Wiebe, 1965; and C. australis, by Baccarini, 1966). Despite this ability and the fact that autotrophic development is possible in some species, e.g. C. pentagona (Zimmermann, 1962), many species undoubtedly derive much carbohydrate from their hosts.

Apart from the experimental studies of members of these four families, the carbohydrates of two further unrelated species have been analysed, Santalum album and Cassytha Jiliformis. The former, sandalwood, belongs to the Santalaceae, a family of some thirty genera and 400 species, all of which are probably root parasites; ten species of two genera are devoid of chlorophyll (Ozenda, 1965). Cassytha is the only parasitic genus of the otherwise autotrophic Lauraceae. I t comprises about twenty chlorophyllous species whose morphology closely resembles Cuscuta.


( 2 ) Demonstration of carbohydrate movement

( a ) Radioautography and dissection

(i) Loranthaceae. In the Loranthaceae, the mistletoe family, there appears to be little movement of carbohydrate between host and parasite in some associations, while in others it is considerable.

The possibility that species of Loranthus, Phoradendron and Viscum supply carbohydrates to their hosts has been much discussed (see Gill & Hawksworth, 1961). Skene (1934) quoted the experiments of Molisch and concluded that mistletoes could not export carbohydrate, whereas Kuijt (1964) has reviewed the evidence in favour of the opposite opinion. In particular, he concluded that where mistletoes live on defoliated hosts, translocation of photosynthates from parasite to the living tissues of the host must occur. However, such instances could have an alternative explanation. Defoliated trees, likely to contain abundant reserve carbohydrate in their stems, are less liable to die from lack of carbohydrate than from slow dehydration because a transpiration stream is absent. Parasitic mistletoes, by maintaining a transpiration pull through host tissues, may prevent such dehydration and so permit the leafless trunks to live off their reserves. Kuijt’s observation that no living branches extend above the mistletoe supports this explanation, since the transpiration stream would terminate at the parasite.

Experiments with 14C have recently clarified the problem. Working with Viscum album, a green parasite on Populus nigra, Seledzhanu and Galan-Fabian (1961) demonstrated that there was only very slight movement of 14C-photosynthate from host to parasite such that it could only be detected by prolonged radioautography of leaves of mistletoe. Similarly, reverse movement from parasite to host was minimal. These results have been clearly and dramatically confirmed for the related green mistletoes of the United States, Phoradendron spp. (Hull & Leonard, 19644 b; Leonard & Hull, 1965). Combinations of two leafy and one leafless species of Phoradendron with eight host plants (four angiosperm and four gymnosperm) were investigated by supplying W O , in the light to either host or mistletoes and analysing both tissues radioauto- graphically. In no case was any appreciable movement between host and parasite demonstrable even 2 weeks after exposure to 14C02. However, there was considerable translocation of photosynthate within each plant supplied with 14C02, the magnitude of this movement being dependent on season.

A completely contrasting picture emerged when Hull and Leonard made similar studies with two species of Arceuthobium, the dwarf mistletoes, infecting eight different conifers. Marked movement of r4C]photosynthate from host to parasite was demonstrated in all cases (Table 6), but no reciprocal movement from parasite to host was found, in contrast to the earlier reports of Rediske & Shea (1961). This discrepancy was attributed to a leakage of 14C0, in the experiments of Rediske and Shea which permitted its incorporation into assimilates by the host leaves. When such a possibility was eliminated, Hull and Leonard showed there was no movement to the host, and also none from the aerial parts of the parasite to its endophytic system.


Basipetal translocation in the parasite is therefore minimal and may be due to the lack of differentiated phloem tissue in the plant (Kuijt, 1955).

Measurements by Hull & Leonard (1964a) o f the radioactivity in host and parasite after exposure of the host to 14C02 confirmed their radioautographic studies. In associations involving Phoradendron, 2 weeks after exposure to 14C02, the foliage of

Table 6. Movement of I4C derived from I4CO2, [14C]urea or [14C]sugars from hosts to angiospermous parasites

Parasite Host Reference

Loranthaceae Arceuthobium americanum Pinus contorts*

P. murrayana*

Rediske & Shed, 1961 Hull & Leonard, 1964a Leonard & Hull, 1965

A. campylopodum Abies concolor A. magn@ca Pinus jeffreiy P. monophylla P. ponderosa P. sabiniana P. lambertiana

Hull & Leonard, 1964a Leonard & Hull, 1965

Scrophulariaceae Striga asiatica S . senegalensis Castilleja coccinea

Odontites verna

Cuscutaceae Cuscuta indecora C. campestris

Zea mays Sorghum vulgare Okonkwo, 1966a

Rogers & Nelson, 1959, 1962

Fragaria virginiana Antennaria neglecta Malcolm, 1966

Trifolium repens Hordeum vulgare Lolium perenne Ranunculus acris R. repens Lotus uliginoms Epilobium partiiflorum Govier, 1966 Chamaenerion angustifolium Scrophularia nodosa Ballota nigra Cirsium arvense Secale cereale Agrostis stolonifera

Govier & Harper, I 964, I 965

Govier et al. 1967

Medicago satiwa M. sativa

Littlefield et al. 1966

* Synonyms.

six host species had specific activities ranging from 91 to 575 c.p.m./mg. dry weight, while values for the shoots of their parasites ranged only from 0.2 to 1 .5 . In the extreme case the specific activity of leaves of %glans hindsii was nearly 3000-fold greater than that of infecting shoots of P.JEavescens var. macrophyllum, i.e. virtually no carbohydrate passed to these parasites. By contrast, in associations involving Arceuthobium, leaves of seven conifer hosts had specific activities o f 195 to 801 c.p.m./mg. dry weight, while shoots of the dwarf mistletoes reached 39 to 282. In infections of A. campylopodum

Carbohydrate movement from autotrophs to heterotrophs 61 on Pinus sabiniana the specific activities were almost equal (137 and 197 respectively), indicating rapid movement to the parasite.

Although these studies of Viscum, Phoradendron and Arceuthobium suggest differences along generic lines, it must be remembered that Viscum minimum lacks chlorophyll and, unlike V. album, must be totally dependent on its hosts.

(ii) Scrophulariaceae. The movement of 14C from host to parasite has been demonstrated by radioautography in all the species of Scrophulariaceae listed in Table 6 except Castilleja coccinea. Most workers studied only gross movement in whole plants, but Rogers & Nelson (1962) investigated movement from maize, Zea mays, to witch- weed, Striga asiatica, by micro-radioautography of sections of the haustorial regions; photosynthate in the phloem of the host was transferred to the parasite via the so- called ‘nucleus’ of the haustorium, a tissue lacking sieve tubes. In the case of C. coccinea, Malcolm (1966) continuously monitored the arrival of 14C in the parasite after r4C]fructose had been introduced into the host.

As in the Loranthaceae above, gross counting of separated parts of parasites and hosts has confirmed radioautographic evidence. Considerable movement of 14C to the parasite was shown after the host had been exposed to 14C02, r4C]urea or r4C]glucose in the following cases: Striga asiatica on Zea mays (Rogers & Nelson, 1962), Striga senegalensis on Sorghum vulgare (Okonkwo, 1966a) and Odontites verna on Trifolium repens or Festuca rubra (Govier & Harper, 1964; Govier, 1966). In both Strka species the specific activity (c.p.m./mg. dry wt.) of the parasite soon exceeded that of the roots of the host to which they were attached. Similar to the mistletoes described above, only very small quantities of radioactivity moved from parasites to hosts when 14C was fed to the parasites.

(iii) Cuscutaceae. Littlefield, Pattee & Allred (1966) showed by whole-plant radioautography that 14C moved from Medicago sativa (alfalfa) to two species of dodder, Cuscuta indecora and C. campestris (Table 6). Twenty-four hours after exposure of the host to 14C02, the specific activity of C. indecora reached a value higher than that of the treated alfalfa leaves. An earlier paper from this laboratory (Pattee et al. 1965) stated that, after attachment, the parasite and its host behaved as a single organism with free translocation between them. There is no evidence for this in their published radioautograph which shows no 14C in the host after 2 hr. fixation by the parasite and a further 48 hr. of translocation. A one-way transfer from host to parasite is also indicated by their further observation that 14C derived from one host plant could not pass to a second host to which the parasite was also attached (Littlefield et al. 1966).

( 3 ) Characteristics of carbohydrate movement (a ) Form in which carbohydrate moves

The nature of the carbohydrate transferred to angiospermous parasites from their hosts has not been examined by experiments comparable with those in the other symbiotic associations described above. Nevertheless, from evidence presented in Section (4) it seems most likely that those that do obtain carbohydrate from their hosts do so in the form of sucrose. Certainly, this was the most heavily labelled sugar in ethanolic extracts from tissues of hosts exposed to 14C02 (Hull & Leonard, 1964a;


Okonkwo, 1966a; Govier, Nelson & Pate, 1967) and was obtained in bleeding sap by Govier and co-workers from barley, one host of Odontites verna.

Okonkwo (1964, 19663, c ) concluded that the only compounds for which Striga senegalensis depended on its host were sugars, since seedlings could be successfully cultured on an inorganic medium supplemented only by sugars (glucose, fructose, sucrose, galactose, maltose, lactose, mannose, xylose and raffinose). Mannitol could not support growth but was not inhibitory as were ribose, sorbose and rhamnose. This failure of mannitol to support growth is discussed in Section ~ ( c ) .

(b) Quantitative aspects-the effect of infection on translocation within the host The derivation of photosynthate by angiospermous parasites from their hosts must

alter patterns of translocation within the hosts, but this has only been systematically investigated by Hull & Leonard ( 1 9 6 4 ~ ) and Leonard & Hull (1965) for mistletoes. Like symbiotic fungi, dwarf mistletoes (Arceuthobium) have profound effects on the pattern of translocation in their hosts. In studies of A. campylopodum on Abies concolor, particularly during the summer, the parasite was so powerful a ‘sink’ for host photosynthate that little was available for translocation beyond the infected areas. Less pronounced effects were found at other seasons when considerable movement beyond the site of infection occurred, but it was a general rule that infected branches of the host exported less than healthy ones. Although leaves of the host above the site of dwarf mistletoe infection were the main source of its nourishment, if these were accidentally or experimentally removed, a further influence of the parasite was to cause a flow of carbohydrate from the main stream into the lateral branch on which it was growing. This effect was particularly marked during the spring.

By contrast, the green mistletoes, Phoradendron, which do not obtain appreciable carbohydrate from their hosts, did not appear to affect the pattern of translocation within the hosts. In those cases where a limited amount of label did appear in green mistletoes, the possibility of passive movement from host xylem could not be eliminated.

No comparable studies have been made for species of Scrophulariaceae, but it is interesting that Odontites verna could not cause a diversion of photosynthate from flowering or fruiting shoots of a host (Govier, 1966; Govier et al. 1967). In such instances the natural ‘sink’ for photosynthate is presumably more powerful than the parasite.

( 4 ) Fate of carbohydrate in the heterotroph The immediate fate of labelled photosynthate derived from the host has only been

examined in Arceuthobium (Hull & Leonard, 1964a) and Odontites (Govier, 1966; Govier et al. 1967).

In the case of Arceuthobium campylopodum parasitic on Abies concolor, 20 days after exposure of the host to 14C02, most of the radioactivity in the endophytic system of the parasite was in glucose and fructose during summer and autumn. At other times of the year the pattern paralleled that in the stem of the host, with heaviest labelling in sucrose and oligosaccharides of the raffinose series. However, in the aerial shoots of the parasite, sucrose had the most radioactivity at all seasons, suggesting this was

Carbohydrate movement from autotrophs to heterotrophs 63 the compound translocated in the acropetal direction from the endophytic haustoria. Arceuthobium is therefore unlike symbiotic fungi in that host sugar is apparently not rapidly converted to other carbohydrates.

By contrast, in their studies of Odontites verna, Govier and co-workers concluded that although sucrose was the main compound translocated to the host root, it was not detectable in the sap or tissues of the hemiparasite. Instead, 14C derived from the host was distributed mainly into either amino acids or monosaccharides, depending both on the host and on the period of exposure to 14C0,. They considered that either sucrose did not move into the parasite, or it was rapidly converted to other compounds. Like them, we favour the latter hypothesis.

Govier et al. analysed the radioactivity in both the bleeding sap (which presumably reflects what is moving in the vascular system) and in ethanolic extracts (which reflects composition of all tissues) of host and parasite. A noteworthy feature of their experiments is that the ratio of 14C in fructose to glucose nearly always approximated to one, except in the sap of the parasite where there was always more 14C in fructose; the ratio was 10: I in associations with barley, and 1-5: I with clover. Although the authors do not comment on these differences, they may be significant. If the sugars in the bleeding sap are derived in part from the phloem, then fructose moieties may be preferentially translocated over glucose moieties. It is most unusual for free monosaccharides to move in the phloem, but their reduction products, sugar alcohols, frequently do (Lewis & Smith, 1967a). The possible importance of polyols in the metabolism of angiospermous parasites is discussed below. Most of the investigations of Govier et al. were primarily concerned not with carbohydrates but with the metabolism and movement of amino acids and amides in and between the hosts and Odontites. This most interesting and illuminating study is beyond the terms of reference of this review.

In symbiotic fungi, the importance of polyhydric alcohols as trapping agents for host photosynthate has already been emphasized (Sections IV(4) and V(4)). I t is therefore especially interesting that these compounds have been commonly identified in angiospermous parasites also, being especially abundant in the Scrophulariaceae (Table 7 ) . Polyols are also abundant in several species of the closely related Oroban- chaceae. Mannitol was not reported by Privat (1960) in his extensive analyses of Orobanche hederae, but the solvents and detection reagents used by him would not have revealed its presence.

The possible role of mannitol as a product into which there may be rapid conversion of sucrose from the host is indicated by Kiesel’s (1923) observation that Orobanche cumana contained 20 yo mannitol whereas its host, Helianthus annuus, contained none. Similarly, in a preliminary qualitative investigation, no polyols have been found in the hosts of Lathraea squamaria (Ulmusglabra) or L. clandestina (Corylus avellana and Populus sp.) (D. H. Lewis, unpublished), although the parasites are rich in them.

The carbohydrate composition of the parasite is presumably influenced to some extent by its host. In cases such as Arceuthobium, where no rapid conversion of host sugars to other carbohydrates occurs, the composition of the parasite is directly determined by the host. A similar situation may occur when compounds are transferred


via the xylem, a non-selective process. This may explain the observation of Plouvier (1953) that when mistletoe, Viscum album, grew on ash it contained mannitol, but not when it grew on other trees. Mannitol is not produced by V. album, but Trip, Nelson & Krotkov (1956) showed that it occurred in the xylem of American ash, Fraxinus americana. Even where it is likely that the parasite does convert host sugar to other compounds, the host may still determine the type of carbohydrate formed by the parasite. For example, Cuscuta reflexa contained dulcitol when it grew on species of Melia, Eugenia, Anacardium or Glycosmis (Subramanian & Nair, 1963, 1964b), but mannitol when the sandal, Santalum album, was the host (Subramanian & Nair, 1964a). The sandal is itself a parasite, but although its host was not named, Sreeni- visaya (1930) showed that sandal does not normally contain mannitol except when

Table 7 . The occurrence of sugar alcohols in angiospermous parasites Family Species Polyol References

Scrophulariaceae many, e.g. Rhinanthus mannitol Barker, 1955; Plouvier, 1963 several, e.g. Melampyrum dulcitol Barker, 1955 ; Plouvier, 1963

Orobanchdceae Orobanche cumana mannitol Kiesel, 1923 0. ramosa mannitol Haller, 1949 Lathraea squamaria mannitol Zellner, 191 3 L . clandestina mannitol D. H. Lewis (unpublished)

Santalaceae Santalum album mannitol Subramanian & Nair, 1964a Cuscutaceae Cuscuta rejlexa mannitol Subramanian & Nair, 1964a

Lauraceae Cassytha filiformis dulcitol Huzikawa et al. 1940 Loranthaceae Viscum album mannitol Plouvier, 1 9 5 3

C. reflexa dulcitol Subramanian & Nair, 1963, 19646

infected with the spike disease. As regards the other hosts of C . reflexa, Hackman & Trickojus (1952) have demonstrated that rjbitol occurs in species of Melia and Eugenia (as well as Maneifera, which is related taxonomically to Anarcardium and Citrus, which is related to Glycosmis).

Clearly, the metabolic importance of polyols in angiosperm parasites needs further study, especially in view of results, discussed below, which have shown that these compounds are major photosynthetic products in several parasitic members of the Scrophulariaceae.

( 5 ) Special aspects ( a ) Lack of reciprocal carbohydrate movement from parasites to their hosts

There is as yet no good evidence that photosynthetic parasites ever export carbohydrates to their hosts in any appreciable quantity, although it must be remembered that only a few of the several hundred green parasites have been investigated experimentally. Various mechanisms could be involved in preventing this reciprocal movement. Where phloem or comparable conducting tissue is continuous, the selective nature of phloem transport (Trip et al. 1965) may prevent movement of certain carbohydrates from host to parasite, and, more especially, from parasite to host. In addition to any anatomical barrier to reciprocal flow, the same biochemical barrier invoked

Carbohydrate movement from autotrophs to heterotrophs 65 for symbiotic fungi (Section V ( 5 b ) ) may apply. It is a remarkable coincidence that, on the one hand, such fungi utilize polyhydric alcohols as a trapping mechanism and, on the other, these compounds exist in quantity in many angiospermous parasites also.

The frequent occurrence of polyols in angiospermous parasites is not a feature distinguishing them from other angiosperms, for they also occur in many free-living families (Plouvier, 1963). However, the ability to metabolize polyols is perhaps restricted to those families which normally contain them (Lewis & Smith, 1967a) and this fact may have been important biochemically in the establishment of the parasitic habit. Free-living plants containing polyols would be ' pre-adapted' in the sense of being capable of converting host carbohydrates into compounds not readily re-utilized by the host (Lewis, 1963; Lewis & Harley, 1 9 6 5 ~ ) . In addition, polyols may be important in the non-metabolic role of maintaining a high osmotic pressure in the parasite, so facilitating transfer of water from the host plant (Lewis & Smith, 1 9 6 7 ~ ) . I t should, however, be stressed that certain angiospermous parasites, e.g. Arceutho- bium, which undoubtedly derive carbohydrate from their hosts do not contain polyols.

(b ) Photosynthesis and dark fixation in angiospermous parasites Angiospermous parasites which contain chlorophyll differ from all other carbon

recipients discussed in this article in that they themselves are photosynthetic and potentially capable of at least some autotrophic provision of their carbohydrate needs. The biochemical pattern of photosynthesis has only been examined in a few species but several interesting features have emerged. An especially important aspect is that, in several species, the destination of photosynthetically fixed carbon is different from the destination of carbon derived from the host. In the cases of Arceuthobium campylopodum, A. americanum and Cuscuta epithymum, this may have important implications in the wider context of general mechanisms of photo-assimilation of carbon dioxide.

Hull & Leonard (1964b) studied patterns of photosynthetic incorporation of 14C0, in Phoradendron and Arceuthobium, but only gave detailed data for the latter. Photo- synthesis in Phoradendron appeared to be of a normal pattern and 14C fixed from l4CO2 was mainly converted into an ethanol-insoluble form, 50% of which was starch. By contrast, only 10 yo of the 14C in the ethanol-insoluble fraction of Arceuthobium could be attributed to starch. In the ethanol-soluble material of both A . campylopodum and A. americanum a high proportion remained in the anionic fraction in contrast to the high level in sugars when 14C was derived from the host. Most of the l4C in the anionic fraction was in malic acid after 5 hr. photosynthesis, but became transformed with time to an unidentified phosphorylated reducing substance. In A. campylopodum a marked fixation into malic acid also took place in the dark and a similar transformation to the unidentified compound progressed during a subsequent 25 hr. period. It should be emphasized that the rate of photosynthesis by these dwarf mistletoes was low and never compensated for respiratory loss of carbon.

In Cuscuta the pattern of fixation varies with different species and with the age of tissue. The rate is low and values on a fresh weight basis for C. gronowii and C. campestris obtained by MacLeod ( 1 9 6 1 ) were only one-tenth of that observed in leaves of an autotrophic angiosperm, Pelargonium. The major soluble product in these

5 Biol. Rev. 44


species was sucrose, but no data were given concerning the proportions of radioactivity in various fractions. In C. australis, Baccarini (1966) found that 47% of the total 14C fixed in the light in I hr. by seedlings was in the neutral fraction of ethanol- soluble material. Sucrose was most heavily labelled, as in the species studied by MacLeod. However, a smaller proportion of radioactivity was found in the neutral fraction when the experimental material was stems or flowers of the parasite attached to a host (Medicago sativa) (27% and 36% respectively). In these cases the organic acid fraction contained most radioactivity. Detached stems gave very similar results to those from stems still attached to the host (Baccarini 1967). By contrast, after 24 hr. photosynthesis by C. epithymum, Ciferri & Poma (1963) found no radioactivity in phosphoglyceric acid or sugar phosphates, but in malate, citrate and acidic amino acids instead. Ribulose diphosphate carboxylase could not be detected and the light- stimulated fixation of carbon dioxide seemed to be mediated by either phosphopyruvate carboxylase or phosphopyruvate carboxy-transphosphorylase, two enzymes formerly more usually associated with non-photosynthetic dark fixation.

This major fixation into organic acids by species of Arceuthobium and Cuscuta is particularly interesting when viewed in the light of the new C-4 dicarboxylic acid pathway of photosynthesis formulated by Hatch & Slack (1966) for sugar cane; elaborated for several further monocotyledons by Hatch, Slack & Johnson (1967) and extended to a limited number of dicotyledons by Johnson & Hatch (1968). All species showing this pathway have low levels of ribulose diphosphate carboxylase and high levels of phosphopyruvate carboxylase (Slack & Hatch, 1967; Johnson & Hatch, 1968), features shared by C. epithymum.

The primary fixation into organic acids can only be demonstrated in the experiments of Hatch and co-workers by exposures of tissue to 14C0, for short periods of up to 4 sec. The light-stimulated fixation into organic acids by C. epithymum is apparently a continuous process, perhaps indicating that a consequence of parasitism has been the loss of the transcarboxylation enzyme linking the dicarboxylic acids via phosphoglyceric acid to sugar synthesis. Another essential enzyme of the Hatch and Slack pathway is phosphopyruvate synthetase (Hatch & Slack, 1967, 1968). A search for this enzyme in angiospermous parasites which appear to fix carbon dioxide primarily into organic acids may prove fruitful.

The destination of carbon fixed by their own photosynthesis in parasitic members of the Scrophulariaceae may also be different from that derived from their hosts. Okonkwo ( 1966a) stated that the principal photosynthetic product in Striga senegalensis was fructose. Although most radioactivity was undoubtedly in the fructose region of the chromatogram, the solvent used would not have separated fructose from hexi- tols, nor would the detection methods used for locating carbohydrates have reacted with them (Lewis & Smith, 1967b). The Scrophulariaceae, and particularly its parasitic members, are particularly rich in these compounds (see references in Barker, 1955, and Plouvier, 1963). It thus seems possible that the major photosynthetic compound was in fact a polyol. These compounds are known to be formed in photosynthesis in those plant tissues which contain them (Lewis & Smith, 1967a) and this has proved to be the case for the following species of Scrophulariaceae : Eziphrusiu offcin-

Carbohydrate movement from autotrophs to heterotrophs 67 a h , Odontites verna, Melampyrum pratense, Pedicularis sylvatica and Rhinanthus minor (D. H. Lewis, G. M. Mann & K. J. Mansfield, unpublished). Okonkwo did demonstrate that the fructose level in the aerial parasite was low, and this is further evidence that it was not the main product of photosynthesis. Nevertheless, as noted above, mannitol could not support the in vitro growth of Strka senegalensis. This may be due either to the fact that the endogenous polyol is dulcitol or that penetration of exogenous mannitol is difficult, a feature of the utilization of polyols discussed by Lewis & Smith ( 1 9 6 7 ~ ) .

The Orobanchaceae lacks chlorophyll, a trait recently confirmed for Orobanche hederae by Baccarini & Melandri (1967), who showed that the incorporation of I4CO, by this species followed the typical pattern of heterotrophic dark fixation, emphasizing the dependence of these parasites on their hosts for carbohydrates.

VIII. DISCUSSION

( I ) Introduction Evidence for appreciable carbohydrate movement has been found in all of the

associations described above, except those involving certain mistletoes. Since movement is always predominantly in one direction, the components of an association will, for convenience, be referred to as the donor (i.e, of carbohydrate) and the recipient.

The most valuable experimental techniques have been those employing 14C. They have shown that although the 14C becomes incorporated into a range of compounds in both donor and recipient, the bulk of the 14C that actually moves between the two organisms in most associations is in a single compound, a carbohydrate. This suggests that transference is mediated by a selective process that enables compounds essential to the donor to be retained within it. There are only a few associations in which transference occurs by a nonselective process, e.g. movement through xylem elements into certain angiosperm parasites such as Odontites.

The modifications required for the evolution of an efficient system of carbohydrate supply from donor to recipient are of two general kinds: (a ) a surplus of mobile carbohydrate must become available in the donor at its site of contact with the recipient- and this normally requires some modification of donor metabolism; and (b) some mechanism for the selective and efficient transfer of the carbohydrate to the recipient must be developed. These two kinds of modification will be discussed separately below.

( 2 ) Development of surplus carbohydrate in the donor A distinction must be made between donors which are algae and those which are

higher plants. Algae are nearly always completely encircled or enclosed by the recipient, whereas in higher plants only a small proportion of the surface is in contact. In the algae, the main requirement is therefore the generation of surplus mobile carbohydrate within the cells, while in higher plants a diversion of carbohydrate from other regions to the area of contact must be achieved.

5-2


( a ) Algal donors Some of the modifications resulting from existence in symbiosis become apparent

if the algal symbionts of invertebrates and lichens are compared with their free-living relatives. Especially in the case of lichens, the changes that occur when symbiotic algae are brought into isolated culture may yield further information. The main modifications resulting from existence in symbiosis appear to be as follows :

(i) Reduced growth rates. The growth rates of algae in symbiosis are often severely reduced, but there is no evidence of any marked reduction in rates of photosynthesis. Thus, if the recipient can reduce the growth rate of the algal donor without affecting its capacity for photosynthesis, conditions for the production of surplus carbohydrate by the donor would be created. In invertebrates, which reach a finite size, the algal population also ceases to increase. In lichens, although thalli are capable of infinite growth, the actual growth rate is extremely slow, measurable only in fractions of a millimetre per year in many crustaceous species. The limitation of the growth of algae in lichens is presumably due at least in part to a physical restriction by the encircling fungus. Such physical restrictions on the growth of algae in animals are not evident so other methods are presumably involved, such as possible limitations in the supply of some essential factor required by the algae. The possibility of production by the recipient of substances which inhibit algal cell-division cannot be ignored.

(ii) Diversion of fixed carbon from insoluble to soluble forms. In photosynthesis experiments, symbiotic algae typically fix most of the 14C into soluble compounds, while free-living algae fix mainly into insoluble products. The formation of excess soluble (and therefore potentially mobile) compounds has obvious advantages for efficient carbohydrate movement. It is not known how this modification is achieved. One possibility is that the reduction in growth rate noted above may result in restrictions in the amount of insoluble material (such as cell walls) that can be produced. However, alternative explanations must be sought for some algae. Thus, the Trebouxia symbiont of lichens still fixes similar amounts of 14C into soluble material whether it is in the thallus or in actively growing pure culture (Richardson & Smith, 1 9 6 8 ~ ) . By contrast, the Nostoc symbiont of Peltigera polydactyla fixes much more carbon into insoluble form after isolation (Drew & Smith, 1 9 6 7 ~ ) .

(iii) Fixation of more carbon into carbohydrates. As well as diverting more fixed carbon to soluble compounds, at least some symbiotic algae channel a greater proportion of total fixed carbon into carbohydrates. For example, although both cultured and symbiotic Trebouxia fix similar amounts of carbon into the soluble fraction, almost all of it accumulates in carbohydrates in symbiotic forms, but in the cultured forms appreciable amounts accumulate in amino and organic acids (Richardson & Smith, 1968h). Again, this modification could be a consequence of restriction in growth rate, since it is well known that in non-growing algae of a range of free-living species, the major assimilation product is a carbohydrate (Danforth, I 962).

(iv) Formation of ‘special’ carbohydrates. An unexpected feature in many symbiotic algae is that the form in which carbohydrate is transferred to the recipient is one

Carbohydrate movement from autotrophs to heterotrophs 69 which is absent or nearly absent in related free-living forms. In lichen algae and zooxanthellae, formation of the carbohydrate tends to cease if they are brought into isolated culture. Since it is believed that this phenomenon is relevant to the mechanism of transferring carbohydrate from donor to recipient, it will be discussed more fully below in Section (3) (b ) .

(b ) Higher plant donors In higher plants the products of photosynthesis are translocated from the leaves to

other organs in specialized vascular tissue, the phloem. There is now appreciable evidence that the pattern of translocation within the higher plant becomes modified as a result of fungal infection so that more carbohydrate is diverted to the site of infection. There is also some evidence that this occurs when the recipients are parasitic higher plants.

It is not known how the pattern of translocation within the higher plant becomes changed. Most theories of phloem transport hold that the predominant movement of carbohydrate is from ‘source’ (e.g. leaves) to ‘sink’ (e.g. regions of consumption or storage). The site of association with the recipient must therefore be able to act as a ‘sink’ powerful enough to compete with those in other regions of the host plant. A variety of mechanisms for achieving this was discussed in the section on association of higher plants with fungi, and they included modification of the hormone balance of the donor, and conversion of donor carbohydrate to other forms by the recipient so as to maintain a concentration gradient into the site of infection. The production of hormones by the recipient which could affect phloem transport in the donor is an obvious possibility that should be investigated in the case of parasitic higher plants. Further. it should be remembered that representatives of each of the major groups of plant hormones-auxins, cytokinins and gibberellins-have now been isolated from fungi.

Unlike algal donors, there is as yet no evidence of increased diversion of fixed carbon to soluble forms. The production of ‘special’ forms of carbohydrate for transfer has also not been found. The fact that vascular plants already possess characteristic translocatory carbohydrates may preclude the necessity for the induction of the ‘special’ carbohydrates that are formed by algal donors.

( 3 ) Mechanism of transfer from donor to recipient The mechanism of carbohydrate transfer from donor to recipient is not known for

any of the associations described above. It presumably involves two stages : movement out of the donor, and entry to the recipient. The extent to which these stages are directly linked is not known.

There could be two reasons, not mutually exclusive, why carbohydrate moves out of the donor: (a) changes in the permeability of the cell (evidence given previously suggests that it would have to involve ‘selective’ rather than ‘passive’ processes); and (b) modification of surface polysaccharide metabolism so that carbohydrate normally destined for cell wall synthesis is released instead. The relative importance of these alternatives is not known, and neither is the extent to which the recipient can directly


induce carbohydrate to move out of the donor. Evidence was given above that whereas some animal tissues contain ‘factors’ that cause their algal symbionts to excrete, this could not be demonstrated in others (Muscatine, unpublished); attempts to find such factors in lichens have also been unsuccessful so far (T. G. A. Green, unpublished). Certainly, the rapid uptake and utilization of carbohydrate by the recipient would set up concentration gradients promoting the flow of carbohydrate out of the donor.

A great deal of experimental evidence will be required for the various theories and possibilities to be evaluated. At present, it is only possible to consider a number of isolated topics for which there is some relevant data.

( a ) Carbohydrate excretion by symbiotic algae Most free-living algae can excrete soluble organic matter (e.g. see review of Fogg,

I 962). Carbohydrate excretion by symbiotic algae differs from this phenomenon in three main respects.

Firstly, symbiotic algae release a much greater proportion of fixed carbon during photosynthesis than other kinds of algae. For example, under identical experimental conditions, zoochlorellae directly isolated from Chlorohydra viridissima excreted up to 85 % of their fixed carbon, whereas Chlorella pyrenoidosa excreted only up to 3 yo (Muscatine, 1965). The lichen algae Trebouxia, Nostoc and Coccomyxa excrete ten to twenty times more fixed 14C immediately after isolation from lichen thalli than they do after a period in culture. In a survey of twenty-two species of free-living marine unicellular algae, Hellebust (1965) found that most species released 3-6% of photo- assimilated 14C; by contrast, Muscatine (1967) found that zooxanthellae isolated from corals and giant clams excreted 40%.

Secondly, much of the fixed carbon excreted by most symbiotic algae is in a single, simple carbohydrate and only a relatively small proportion is in other compounds. In free-living algae, a variety of compounds are usually prominent amongst the excretory products, and organic acids, polysaccharides and amino acids may often predominate. In his survey of excretion by marine algae, Hellebust found that in only a very few species was excretion limited almost exclusively to one compound.

Thirdly, as noted above, the carbohydrate that is excreted by symbionts is of a ‘special’ kind. In algal symbionts of invertebrates, it is invariably different from the major intracellular carbohydrate. Thus, zoochlorellae from Hydra produce sucrose intracellularly, but release maltose which only occurs in small amounts within the cells; zooxanthellae excrete glycerol, but glucose is the major intracellular product. In lichen symbionts the polyols produced by the green algae are either entirely absent in pure culture (as in Coccomyxa) or present in reduced amounts (as in Trebouxia); the blue-green algae excrete glucose, but this is not found free in either cultured or free-living forms.

The production of these ‘special ’ forms of carbohydrate undoubtedly holds some clue about the mechanism of transfer from donor to recipient. One or more of a variety of factors may be involved. In at least some algae, the form of carbohydrate excreted might be one which cannot easily be absorbed back into the cells ; thus, very few algae can utilize disaccharides such as maltose, so that its excretion by zoochlorellae

Carbohydrate movement from autotrophs to heterotrophs 7’ may promote the one-way transfer from donor to recipient. Unfortunately, there have been few investigations of the ability of symbiotic algae to utilize their excreted carbohydrates. Another possibility is that during periods of starvation, such as prolonged darkness, the transfer of carbohydrate to the recipient is restricted by stopping the pathway for producing ‘special ’ carbohydrates. This would represent a control mechanism which need not interfere with the ‘normal ’ intracellular carbohydrate metabolism, One might thus visualize that the ‘special’ carbohydrates are only produced when the supply of fixed carbon is in excess of the requirements of the alga. However, this hypothesis has yet to be verified experimentally.

It may at first seem surprising that although many symbiotic green algae produce sucrose intracellularly (e.g. zoochlorellae, Trebouxia) this is never the form in which carbohydrate is transferred to the recipient. Sucrose is one of the few mobile carbohydrates in higher plants, and may prove to be the commonest form to be transferred from higher plant donors to their recipients. Furthermore, free-living Chlorellu pyrenoidosa is able to excrete a certain amount of sucrose, especially under acid conditions (Tolbert & Zill, 1956). However, for reasons suggested above, if symbiotic algae possessed mechanisms for releasing sucrose in abundance to recipients, their intracellular soluble carbohydrate reserves might become vulnerable to serious depletion during periods of starvation.

Experimental investigations into factors affecting excretion by symbiotic algae have yielded a certain amount of information about the nature of the processes involved. The discovery of a ‘factor’ in tissues of corals and giant clams which stimulates glycerol excretion from their associated zooxanthellae is undoubtedly important. The failure to discover similar factors in other recipient tissues does not yet mean that they do not exist, since correct techniques may not have been used in the search for them. Excretion by some symbiotic green algae ( Trebouxia from lichens, zoochlorellae from Hydra) is very sensitive to pH and is strongly stimulated under acid conditions. In Hydra, the pH of the gastrodermal cells may well be a mechanism which controls carbohydrate release, but it is less certain that this is the operative mechanism in the lichen symbiosis. In some symbiotic green algae (e.g. zoochlorella from sponges and mutant Chlorohydra) excretion is not pH-dependent.

It is natural to suppose that the excretion mechanisms in different algae are not all the same, and require different stimuli to operate them. It is therefore remarkable that in those lichens with two different kinds of algae in the same thallus, the same fungus can induce glucose excretion from blue-green algae and ribitol excretion from green algae (Richardson et al. 1968).

(b) Cell-wall modi$cations It has been suggested above that a recipient might obtain carbohydrate from a donor

if the mechanism for cell-wall formation was modified so that carbohydrate otherwise destined for cell walls became available to the recipient.

Examination by various kinds of microscopy shows that the cell walls of donors in many kinds of associations have been structurally modified and reduced. For example, outer sheathing structures are often absent from the cell walls of lichen algae, while


endozoic algae may lack a cell wall altogether. The occurrence of such structural modifications does not of course prove that this is the process by which the recipient obtains carbohydrate, since removal or reduction of the cell wall may merely serve to bring the mechanisms of donor release and recipient uptake closer together.

It is now generally believed that material for plant cell walls is preformed internally, and then passed across the plasmalemma by a process of micropinocytosis (Pickett- Heaps & Northcote, 1966; Green & Jennings, 1967). Thus, if the recipient obtains carbohydrate from this extruded material it presumably has first to be broken down to its component sugars. Most plant cell walls yield a variety of carbohydrates on hydrolysis, yet donors typically release only one kind of carbohydrate. Further, in most associations, the kind of carbohydrate released is not known as a component of cell walls of the donor, i.e. glycerol released from zooxanthellae, maltose from zoochlorellae, and polyols from green algae of lichens. Indeed, in higher plants the restriction of the translocatory carbohydrates to sucrose, the raffinose series of oligosaccharides and polyols may be the result of selection of those carbohydrates not involved in polysaccharide synthesis (i.e. so that intercellular and intracellular movement is facilitated).

I t therefore seems unlikely that recipients obtain their major supply of carbohydrate from cell-wall material extruded on to the surface of donor cells. However, it is possible that modification of the processes of assembling cell-wall material within the donor might be the ultimate source of the excreted carbohydrate. Since the Golgi apparatus is believed to be intimately concerned with the formation of cell walls, a study of this and related organelles in donors may prove valuable.

It was noted earlier that some fungal antibiotics such as penicillin exert their effects on bacteria by blocking the biosynthesis of cell-wall polysaccharides. It remains to be seen whether other antibiotics are important in a similar role in the interactions between fungi and their higher plant or algal donors.

(c) The role of polyols Polyhydric alcohols (including glycerol) have featured prominently in the associa-

tions discussed here. It must of course be stressed that not all associations have involved polyols, and that many plants not involved in symbiotic or parasitic associations produce them. Nevertheless, it seems likely that some degree of correlation may exist; in angiosperms, polyols appear to be commoner in parasitic than in free-living species; the only terrestrial green algae known to be capable of polyol synthesis are those which are symbiotic with lichens; fungi, many of which are symbiotic or parasitic, have polyols as one of their predominant carbohydrates ; amongst the Dinophyceae, zooxanthellae appear to be the only members which synthesize glycerol in quantity. The existence of groups of plants rich in polyols which are not parasites or symbionts- such as the brown seaweeds-presumably reflects the fact that possession of polyols has a number of different advantages (Lewis & Smith, 1967a).

With regard to parasitism and symbiosis, two aspects of polyols will be considered. (i) Polyols as mobile carbohydrates. Tables 2 and 3 show that in many symbiotic

algae, carbohydrates are released to the recipient as polyols. It is not clear why polyols

Carbohydrate movement from autotrophs to heterotrophs 73 should feature so prominently. Possibly, since they cannot be formed into insoluble polymers, it may be easier for them to move out of cells without becoming involved in any of the mechanisms of polysaccharide synthesis. There are also other possibilities to be considered. Since polyols are neutral compounds, it may be easier for them to be transported in living systems; it is notable that in higher plants the only carbohydrates that can be transported in quantity in phloem except sugars of the sucrose-raffinose group are polyols. In the case of zooxanthellae, the host animals are rich in lipids. Since glycerol is an immediate precursor for lipid synthesis, the production of this rather than other carbohydrates by the zooxanthellae implies a degree of biochemical efficiency in the association.

(ii) Polyol formation by the recipient. Lewis & Harley ( 1 9 6 5 ~ ) suggested that rapid conversion of host sucrose to polyols and trehalose by the mycorrhizal fungus of beech promoted the flow of sugars from the host. Since the host roots could not utilize these fungal carbohydrates, their formation presumably ensured that the flow of carbohydrate would only be in one direction.

A similar system may operate in many other associations. Green plants which do not contain polyols are not usually able to metabolize them (Lewis & Smith, 1967a), while most fungal recipients and many angiosperm parasites contain abundant polyols. This system could be of particular importance in associations where the donor is a higher plant, and where there is a need to create a ‘sink’ at the site of attachment to the recipient.

The abundance of polyols in plants subject to some form of actual or potential water stress was noted by Lewis & Smith. The development of high polyol concentrations in the recipient may therefore be important in the non-metabolic role of maintaining a high osmotic pressure, so facilitating the transfer of water, and with it salts, from the host.

(4) Preadaptations of donors and recipients Symbiotic and parasitic organisms presumably possess characteristics which

originally predisposed them to becoming involved in the kind of association discussed here. For example, for reasons outlined above, Lewis and Harley suggested that the ability of fungi and some angiosperm parasites to synthesize polyols ‘ preadapted’ them to a symbiotic or parasitic existence. Such a concept could be extended to other examples where the recipient converts absorbed carbohydrate into a form unavailable to the donor, as with animals which convert glycerol from zooxanthellae into lipids. I t is of course not clear whether some characteristics develop before or after an association begins to evolve, and an example of this is the ability of ‘saprophytic’ orchids to utilize trehalose.

In the case of the very common lichen alga Trebouxia, Ahmadjian (1967b) has commented that its centrally placed, lobed chromatophore would have great selective value in the evolution of lichens compared to the parietal chromatophore of many other unicellular algae. He states : ‘ Haustoria of encroaching fungi. . .could then be embedded directly into, and be completely surrounded by, the assimilative and storage structure of the alga.’ However, Trebouxia is apparently not known in the free-living state, so that, as Ahmadjian himself implies, the position of the chromatophore is a


character which may have appeared during and not before the evolution of lichens. Similarly, isolated Trebouxia still fixes most of its carbon into soluble compounds, and still produces ribitol even after prolonged pure culture; these are also likely to be permanent modifications since other lichen algae lose such characteristics soon after isolation.

The ability of algae to excrete organic matter must have been an important factor in the development of symbiotic associations with both invertebrates and fungi. However, carbohydrate excretion by symbiotic algae differs in a number of respects from that known by free-living forms, and the ability to develop this type of excretion must have been a crucial factor in the selection of algal donors. It is of interest that members of the genus Chlorella can enter into symbiosis with a range of invertebrates but are only reported from three species of lichens (Ahmadjian, 19676). As regards the origin of lichen fungi, it was noted above (Section IV (5c) that ‘sooty moulds’ have several features that would preadapt them to becoming lichenized.

(5) Eficiency of carbohydrate transfer The mechanisms involved in the supply of carbohydrate from donor to recipient

appear to have developed a high level of efficiency in many of the associations described here. This is particularly exemplified by symbiotic algae, which can supply much if not all the carbohydrate requirements of their associations while only occupying a small volume of them. Present evidence suggests that at least a proportion of the productivity of the coral-reef ecosystem may be due ultimately to the zooxanthellae. It is significant that, although some animals can survive under artificial conditions without their algae, they are rarely found without them in nature.

It is to be expected that such symbiotic associations would evolve in a way which ensures optimum conditions for photosynthesis for their component algae, particularly with respect to such factors as CO, concentration, etc. However, an inevitable consequence for most symbiotic algae is that, being enclosed by the host, the amount of incident light they receive is reduced. It would not be surprising to find that symbiotic algae have relatively high rates of photosynthesis at low light intensities.

(6) Factors other than carbohydrate movement This review has dealt exclusively with carbohydrate movement. Obviously, in many

of the associations considered above, movement of other compounds also occurs, though relevant experimental evidence is scarce. It is important to remember that other mobile compounds could well have an effect on carbohydrate metabolism. For example, Muscatine & Lenhoff (1965 b) has suggested that certain experimental results on the size of the algal flora in Chlorohydra are explicable in terms of the movement of co-factors from donor to recipient which facilitated the carbohydrate metabolism of the latter. The pronounced excretion of biotin by the Coccomyxa symbiont of lichens (Bednar & Holm-Hansen, 1964) and the well-known biotin deficiency shown by lichen fungi in culture (Hale, 1961) might be another important indication of this possibility. Similarly, the requirements of mycorrhizal fungi in culture for vitamins, amino acids and other factors are naturally supplied by their hosts (Melin, 1963).

Carbohydrate movement from autotrophs to heterotrophs 75 Although the needs of the recipient for carbohydrate must have been a major factor

in the evolution of the kinds of association discussed here, other requirements of the organisms involved may have been important. For example, some parasitic higher plants obtain water and minerals as well as carbohydrate from their hosts. In symbioses involving blue-green algae, it is very likely that products of nitrogen fixation may also move to the recipient. Furthermore, the obligate dependence of some donor algae on their hosts-particularly as demonstrated by the difficulty of growing them in pure culture-presumably reflect requirements for some essential factor from the host.

The complex of physiological, morphological and behavioural modifications that occur when organisms enter into symbiosis may therefore have arisen in response to a variety of needs. Thus, it is important to remember changes in the carbohydrate metabolism of a symbiont or parasite might sometimes be connected with processes other than the development of a system of carbohydrate supply from donor to recipient.

(7) General conclusions In symbiotic and parasitic associations the component organisms interact with each

other, frequently so that each becomes modified in a variety of ways. A knowledge of how these interactions operate is fundamental to the understanding of mutualistic symbiosis and parasitism in general. This review has examined one aspect of these interactions which is not only one of the commonest, but also one of the simplest to study experimentally.

Table 8 summarizes the main features that have emerged from this comparative study of carbohydrate movement between living organisms in a variety of associations.

When algae enter into symbiosis, two of the most important modifications which occur are: ( a ) their metabolism changes so that surplus soluble carbohydrates are produced; and ( b ) a mechanism develops for the production and release in large quantities of a carbohydrate which is normally absent or nearly absent from non-symbiotic cells or related free-living algae. The generation of surplus carbohydrate may partly result from the growth rate being more restricted in symbiosis than the capacity for photosynthesis, but it is unlikely that this is the sole reason in at least some algae. The development of the mechanism for carbohydrate release is undoubtedly a basic and central feature in this kind of symbiosis, but it is not understood as yet; it differs in a number of respects from excretion by free-living algae. It is also not clear why the excreted carbohydrates are of a ‘special’ kind; it is conceivable that they are produced because the surface of algal cells may not have enzymes which could utilize them. Also, it is possible that since only ‘special’ carbohydrates can be released from cells, this may offer the alga a simple mechanism for controlling carbohydrate loss by controlling synthesis of the ‘special ’ carbohydrate.

In the case of higher plant donors, much experimental evidence is accumulating that association with both fungi and parasitic higher plants results in a partial diversion of the translocation stream towards the site of infection. It is not known how this occurs, and one of the difficulties is that the mechanism of translocation within healthy higher plants is itself not clearly understood; but there is some evidence that the hormone balance of the donor may become altered. Certainly, diversion of the


translocation stream could explain why localized pathogenic infections can have a generally debilitating effect on higher plants.

The role of the recipient in stimulating carbohydrate release from the donor is another vital feature in the understanding of symbiosis and parasitism. Factors exist in coral and giant clam tissue which stimulate the release of glycerol from their associated zooxanthellae; it will be of great interest to identify these factors, and to discover how far their production is characteristic of other kinds of recipients. The possibility

Table 8. Summary of carbohydrate movement from green plants to other organisms

DONOR RECIPIENT

ALGAE

Production of surplug carbohydrate

~

I. Growth rate restricted more than capacity for photosynthesis

2. Fixed carbon diverted from insoluble to soluble

3. ‘Special’ carbohydrates produced which are transferred to recipient

AUTOTROPHIC Translocation stream HIGHER becomes partly PLANTS diverted to site of

-I I Carbohydrate Immediate fate of

carbohydrate

glycogen, pentoses glucose

glycerol lipids, proteins Zoowaizthellae I polyols (erythritol, ribitol, sorbitol)

(? other translocated sugars)

Sucrose

Sucrose

+ polyols (arabitol, mannitol)

mannitol

c trehalose, glycogen, polyols (mannitol, arabitol, erythritd)

FUNGI

dulcitol) PLANTS

that hormones from fungi and parasitic higher plants can affect their autotrophic hosts is also worthy of investigation.

Table 8 illustrates that most recipients immediately convert carbohydrate into a form unavailable to the donor, and this may have undoubted value in promoting the one-way flow of carbohydrate out of the donor.

IX. SUMMARY

I . The bulk of the fixed carbon which moves from autotroph to heterotroph in most symbiotic associations is in a single compound, a carbohydrate. Techniques employing 14C have been most valuable for investigating this movement.

Carbohydrate movement from autotrophs to heterotrophs 77 2. Most ‘zoochlorellae ’ belong to the Chlorococcales, and they release carbohydrate

to the animal tissue as either glucose or maltose. In some molluscs, the ‘zoochlorellae’ are actually chloroplasts, possibly derived from siphonaceous algae. Although it is known that these chloroplasts supply photosynthetically fixed carbon to the animal tissue, the form of the carbon compounds which move is not known. In Convoluta roscoflensis the ‘ zoochlorellae ’ belong to the Pyramimonadales, but carbohydrate movement has not yet been directly studied in this association.

3. Most ‘zooxanthellae’ belong to the Dinophyceae. In associations involving coelenterates and molluscs, glycerol is the main carbohydrate moving to the animal. Homogenates of the host animal tissue stimulate excretion by isolated zooxanthellae. 4. In lichens, symbiotic blue-green algae release glucose to the fungus, but the

various genera of green algae that have been studied all release polyols (either erythritol, ribitol or sorbitol). Lichen fungi rapidly synthesize mannitol from all these compounds. When lichen algae are isolated into pure culture, they soon lose the ability to excrete carbohydrate, and intracellular production of the carbohydrate that is excreted either becomes much reduced, or ceases altogether.

5 . Mostly indirect evidence indicates that sucrose is the main carbohydrate moving from flowering plants to their associated symbiotic fungi. Diversion of the translocation stream towards the site of the association occurs. The fungi convert host sugars to their own carbohydrates, principally trehalose and polyols.

6. ‘ Saprophytic ’ higher plants are all obligately mycotrophic and receive carbohydrate from their associated fungi. In at least some associations, the fungus is simultaneously associated with an autotrophic higher plant, which is the ultimate source of carbohydrate for the association.

7. Some parasitic higher plants possess chlorophyll, but the extent to which they depend on their host for carbohydrate varies with different species. Green mistletoes evidently derive negligible carbon from their hosts, but other green parasites derive at least some. There is no evidence that any of the chlorophyll-containing parasites export carbohydrate back to their hosts. Parasitic higher plants which lack chlorophyll presumably derive all their carbohydrates from their hosts, but experimental investigations of this are scarce.

8. Comparison between different types of symbiotic association show that a number of common features emerge.

9. The algal symbionts of both invertebrates and lichens have, in comparison to free-living forms, reduced growth rates and greater incorporation of fixed carbon into soluble carbohydrates. They excrete a much greater proportion of their fixed carbon than free-living forms, and most of it is usually as a single carbohydrate. Particu- larly striking is the fact that the excreted carbohydrate is one which is either not the major intracellular carbohydrate, or one which ceases or nearly ceases to be produced in culture.

10. The translocation stream of autotrophic higher plants is diverted towards the site of association with either fungi or parasitic higher plants, but it is not known how this is achieved.

I I . In all associations, the cell walls of the autotroph become reduced or modified


at the site of contact with the heterotroph, but it seems likely that this is not directly connected with the mechanism of carbohydrate transfer between the symbionts.

12. In many associations, the heterotroph rapidly converts host sugars into other compounds (frequently into its own carbohydrates which are usually different from those of the host). This may serve to maintain a concentration gradient and so ensure a continued flow from the host.

13. Polyols feature prominently in symbiotic and parasitic associations, not only as the carbohydrates of many plant heterotrophs, but also as the form of carbohydrate released by both zooxanthellae and the green algae of lichens to their heterotrophic partners.

Our thanks are due to the many colleagues and research students who have allowed us to quote their unpublished results. We are especially grateful to Professor J. L. Harley, F.R.S., for his critical comments and advice on early drafts of this article.

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CARBOHYDRATE MOVEMENT FROM AUTOTROPHS TO HETEROTROPHS IN PARASITIC and MUTUALISTIC SYMBIOSIS

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