MICHEGAN STATE UNIVERSITY Evelyn Anne Horenstein 1965 THESIS LIBRARY Michigan Statﬂ University

PHOTOMORPHGGENI’ESIS EN A NEW AQUATEC E’UNGUS BLASTQCLQMELLA BRHANNECA

That. {01’ Hm Degree a? DB. D. MICHEGAN STATE UNIVERSITY Evelyn Anne Horenstein 1965 THESIS LIBRARY Michigan Statﬂ University

This is to certify that the

' thesis entitled

Photomorphogenesis in a new aquatic fungus, Blastocladiella britannica

presented by

Evelyn Anne Horenstein

has been accepted towards fulfillment of the requirements for

Eh . D. degree in _an:an.y_

WOW Major professor

Date May 6, 1955

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ABSTRACT

PHOTOMORPHOGENESIS IN A NEW AQUATIC FUNGUS BLASTOCLADIELLA BRITANNICA

by Evelyn Anne Horenstein

The developmental history in pure culture of a single—

spore isolate of a Blastocladiella was investigated in some

detail. No other species in the Blastocladiaceae which has been subjected to rigorous examination displays such a high degree of morphological variability--a variability which is probably phenotypically controlled. This characteristic led to the isolation of several distinct substrains, one of which was incapable of producing resistant sporangia and

another (strain B 101) produced them in abundance. As a consequence of these and other observations, this single- celled fungus was designated a new species, B. britannica°

In strain B 101, formation of resistant Sporangia was

affected by several environmental factors, but most strikingly by white light. When grown on agar media, it produces:

(a) in the dark, about 90-100% brown, thick-walled resistant

sporangia (RS) with a generation time of about 65 hr.; (b) in white light, nearly colorless, thin-walled (TW) Sporangia with a generation time of about 30 hr. However, neither the

absence nor the presence of light is required continuously t r «v- 6:- . . v..-

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(ll Evelyn Anne Horenstein

throughout the entire growth period for the genesis of one or the other of these morphological forms° The organism's early stages of development are quite plastic; cells which start their growth in the light, and which are, therefore, on the TW pathway, can be induced to revert to RS types by eliminating the light. Conversely, cells which start growth in the dark (i.e. along the RS pathway) can be transformed into TW types by exposure to light. In both instances, however, a stage in development is reached beyond which addition or withdrawal of illumination can no longer effect morphogenetic reversal. At this point of no return, the cells have become committed to one pattern or the other.

To study photomorphogenesis at a chemical level, it was necessary to grow the organism in large quantities under conditions which induced reproducible morphological uniformity and which could be controlled as precisely as possible.

Consequently, a method was devised for growing synchronized, single generations of B. britannica (a million or more cells at a time) uniformly suspended in agitated media, wherein the effects of light and dark on morphogenesis were demonstrable as well as the reversal of morphogenesis by alteration of the light and dark regime before a point of no return.

Up to a stage just preceding the end of the generation time of a TW sporangium, no morphological differences are discernible under the light microscope between it and a

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dark-grown developing RS at a corresponding chronological age. Yet, within the next 1-2 hr., the entire protoplast of a TW is cleaved into hundreds of uninucleate, uniflagellate motile spores, and this new generation of cells is then discharged. On the other hand, the thalli growing in the dark have reached only the half-way point in their ontogeny; they continue to enlarge for several hours thereafter and then gradually differentiate into mature RS.

In synchronous cultures, dry weight/cell increases exponentially at the same rate in light and dark. On the other hand, the capacity for uptake of glucose by cells of various ages grown in the dark exceeds that of light-grown cells. Furthermore, just as the course of development can be reversed by excluding or supplying light before their respective points of no return, so, too, their capacities for glucose uptake can be similarly reversed. However, the point of no return for glucose uptake precedes the point of no return for morphogenesis by several hours. The light-

sensitive glucose uptake by B. britannica may be a factor in the determination of the ultimate morphology of this organism. PHOTOMORPHOGENESIS IN A NEW AQUATIC FUNGUS

BLASTOCLADIELLA BRITANNICA

Evelyn Anne Horenstein

A THESIS

Submitted to Michigan State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1965 ACKNOWLEDGMENTS

I am very grateful to Dr. L. G. Willoughby who graciously sent us the culture of Blastocladiella britannica, thus affording me the opportunity to study this organism.

Thanks are due to Professor William M. Seaman, who kindly provided the Latin translation for the diagnosis of Blastocladiella britannica.

For his capable guidance, his never-failing en- couragement and expressions of confidence, and his enthusiastic support, I should like to express my very deepest gratitude to Professor Edward C. Cantino who, more than anyone, is responsible for the successful completion of this thesis.

***************

ii To

Mother, Dad, and Don

iii TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

LITERATURE REVIEW...... 4

Description of the Blastocladiaceae with Emphasis on Unique Characteristics of the Family. . . . 4 The motile spore ...... 6 The resistant sporangium ...... 10 Life Cycles in the Blastocladiales ...... 12 Brachyallomyces type ...... 12 Cystogenes type...... 12 Euallomyces type ...... 15 Differentiation of the Resistant Sporangium in Blastocladiella emersonii...... 15 Induction of resistant sporangium formation. . 15 Formulation of a hypothesis...... 15 Biochemistry of morphogenesis...... 19 Period of exponential growth ...... 22 Period of differentiation...... 27

MATERIALS AND METHODS...... 32

Culture Procedures ...... 52 Analytical Procedures...... 37

OBSERVATIONS AND EXPERIMENTAL...... 41

Conditions for Optimal Growth...... 41 Morphological Variability...... 42 Pure Strains of TW Colonies...... 47 The RS Colonial Strain ...... 50 Life history ...... 50 Effect of temperature on RS formation. . . . . 51 Effect of bicarbonate on RS formation. . . . . 51 Effect of glucose on RS formation...... 52 Effect of light on RS formation. . . . . 54 Isolation of RS strains yielding 100% individual RS plants ...... 54 Development of Synchronous Cultures of Resistant Sporangia...... 60 Growth and Morphological Characteristics of Syn- chronized Single Generation Cultures . . . . . 68

iv TABLE OF CONTENTS - Continued

Page

Growth pattern in the dark 70 Growth pattern in the light. 71 Point of no return in development. 76 Glucose Uptake Capacity. . . . 79 Reversal of GUC...... 81 The point of no return for GUC 82

DISCUSSION . . o ......

The Taxonomic Position of Blastocladiella britan-

nica O O O O O O I O O O O 85 The Aquatic Phycomycetes and Biological Research Morphogenesis in Blastocladiella britannica. 95 Morphological variability. 95 Photomorphogenesis . . . . 97

SUMMARY...... 101

LIST OF REFERENCES ...... 102

APPENDICES ...... 108 LIST OF TABLES

TABLE Page

Lactic acid production by B. britannica. . . . 41

II Morphological types produced by B. britannica. 46

III Relationships among volume per cell, weight per cell, and weight per unit volume of TW cells at maturity...... 76

IV GUC and intracellular pools...... 117

Effect of phosphate on GUC ...... 119

Effect on GUC of different sugars during growth ...... 120

VII Effect on GUC of the presence of other sugars. 122

vi LIST OF FIGURES

FIGURE Page

1. Life cycles in the Blastocladiales ...... 14

2. Enzyme reversal in B. emersonii...... 24

5. Comparative activities of isocitritase in syn— chronously developing OC and RS plants of B. emersonii during ontogeny ...... 26

Transformations in total RNA during morphogenesis of RS...... 28

Transformations in composition of RNA during . sol morphogenes1s of RS...... 28

Changes in protein nitrogen, plant volume, and dry wt. during RS morphogenesis in B. emersonii. 50

Increase in chitin and melanin content during RS differentiation in B. emersonii ...... 50

Changes in lactate pool, glucose-6-ph05phate dehydrogenase, glucose~consumption, and poly— saccharide pool during RS differentiation in B. emersonii ...... 50

Developmental potentialities in B. britannica. . 45

10. Size of spores from the TW and RS strains. . . . 48

11. Effect of different concentrations of PYG (2% agar) on the composition of populations derived from spores of TW colonies ......

12. Effect of different concentrations of PYG (2% agar) on the composition of populations derived from spores of RS colonies ...... 55

15. Effect of different concentrations of glucose (in PYG) on RS formation ...... 55

14. Effects of semi-anaerobic conditions on the composition of populations derived from spores of RS colonies ...... 55

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[14

LIST OF FIGURES - Continued

FIGURE Page

15. Effect of light on RS plant development. . . . . 55

16. The relation between duration of light and RS formation on medium PYG...... 56

17. Results of attempts to select populations of vigorous RS producers...... 59

18. Growth curves of individual RS plants...... 59

19. Incidence of RS after transfer from liquid to solid media...... 64

20. Growth of RS cells in the dark ...... 72

21. Effect of pH upon the growth rate of TW cells in the light...... 72

22. Effect of population density upon the growth of TW cells ...... 74

25. Effect of population density upon the volume of TW cells at generation time...... 74

24. Effect of population density upon the generation time of TW cells ...... 75

25. Relationship between the volume of the TW cell at generation time and its dry wt. per unit volume at this time...... 75

26° Points of no return for RS and TW cells. . . . . 78

27. Cell types resulting from exposure to different light-dark regimes ...... 78

28. Increase in dry wt. of synchronous cultures. . . 80

29. The glucose uptake capacity of light-grown and dark-grown cells at different stages in ontogeny at 24 ...... 80

50. The reversal of glucose uptake capacity. . . 84

51. The point of no return for glucose uptake capacity ...... 84

viii LIST OF PLATES

PLATE Page

Blastocladiella britannica ...... 45

II B. britannica grown in light and dark...... 61

III Selected stages in development of TW and RS cells...... 69

ix LIST OF APPENDICES

APPENDIX Page

I List of Abbreviations...... 107

II Attempts at RS Germination ...... 110

III Ballons d'essai...... 116 INTRODUCTION

As a result of an ecological investigation of the lower

saprophytic fungi from Esthwaite Water in the Lake District

of England, Willoughby (1959) isolated the first species of

Blastocladiella from Great Britain or the continent of

Europe; all previously reported species were obtained in the

Western Hemisphere. He suggested that we might be interested

in this fungus and sent us pure cultures from one of his

single spore isolates.

Because I had been associated with him for a number of years in his research on Blastocladiella emersonii, Dr.

Cantino thought I might enjoy and profit from the experience of working with a different organism. The idea was that I would have a look at this new water mold; that is, grow it, observe its characteristics, and determine if, indeed, it was a new Blastocladiella. At the outset, I expected that

this new creature would be thoroughly domesticated and all pertinent observations would be completed within a few months, after which time I would resume my work with B. emersonii.

My naiveté has long since vanished; there were very few observations that could be made without qualifications.

As a result, I have been 'looking at' this organism ever since. Along with these qualifications came questions, arising at an ever accelerating rate. I was very soon forced into making a choice concerning what aspects of these riddles

I was going to pursue. Being especially intrigued by the

degree of variability in developmental potential displayed

by this microfungus--the frustrations far exceeding the

intrigue at times--I chose to exploit this particular attribute

for morphogenetic studies. Past experience with B. emersonii was also a significant influencing factor in that I had

acquired an appreciation for these tiny plants as valuable

tools in such investigations.

Notwithstanding the fact that the Blastocladiellas, for

example, are one- and two-celled plants and have been rele-

gated to a poSition on one of the lower branches of the phylo-

genetic tree, they exhibit what appears to be a highly

developed system of regulatory mechanisms enabling them to

alter their morphogenetic pattern in response to various

environmental conditions. Massive cultures of synchronously

growing plants can be obtained with ease and almost any phase

of their development can be followed throughout a single

generation. Transitions from one moment to the next, includ-

ing many of the dynamic aspects attached thereto, can be

analyzed in a complete organism--not merely an amputated organ or tissue. Those of us who have worked with the water molds

feel that they have much to offer to studies of growth and differentiation.

This thesis represents essentially a progress report on what has been accomplished to date on Blastocladiella britannica,

including its establishment as a new species. Only a beginning has been made, for after a number of years an understanding of its nature and behavior has but advanced to the point where it now provides a more enlightened notion of what questions to ask of it. LI TERATURE REVI EW

Description of the Blastocladiaceae with Emphasis on Unique Characteristics of the Family

The group commonly referred to as the aquatic Phycomy-

cetes comprises the most primitive of the true fungi. In

spite of the fact that they are such an ubiquitous and myriad lot (see Sparrow, 1960), relatively little, aside from

taxonomic descriptions, is known of most of them. There are

only a few genera, primarily in the family Blastocladiaceae,

about which an abundance of information has become available.

The genus Blastocladia was first described by Reinsch

(1878) with the discovery of B. pringsheimii. In the original diagnosis, he placed this organism in the Saprolegniales prob—

ably because of the presence of what he mistakenly thought were oogonia, the female reproductive organs which are regu-

larly formed in this group. In 1909 the order Blastocladiales was proposed by Petersen (1909) to incorporate this single genus. This decision was based on two characteristics-~the

lack of cellulose in the walls of Blastocladia and the absence of sexual reproduction, both well established characters of the Saprolegniales. Since that time, three additional genera have been added to the family Blastocladiaceae which typifies the order. They are Allomyces (Butler, 1911), Blastocladiella

(Matthews, 1957), and Blastocladigpsis (Sparrow, 1950).

These are all saprophytes and can be found in damp soils and slow moving bodies of fresh water, growing on organic debris of a diverse variety (Sparrow, 1960). It is not unusual for them to be associated with great numbers of microflora and microfauna, sometimes forming dense pustules surrounded by a slimy layer of microorganisms.

The family is characterized by the following general features of morphology and growth habit. The very distinctive motile, uninucleate spore (swarmer), after a period of swimming, settles down and germinates. A delicate germ tube emerges and proliferates into a branched system of rhizoids which seem to serve, in the main, to anchor the growing plant to its substratum.* Shortly after the initiation of the rhizoidal system, the chitinous-walled thallus begins to develop. The extent of this development is highly variable and, like the final architecture, is dependent upon the genus-

For the simplest type of morphology we look to ELEEEQ‘ cladiella, which displays a determinate system of growth.

At the end of its growing period (i.e. when there is no fur— ther increase in size), the greater part of the plant body becomes delimited from a basal portion by a septum and is converted into a single reproductive structure: either a

*- It has often been stated that the rhizoids also serve as nutrient gathering devices although no evidence has ever been offered to substantiate this assertion. colorless, thin-walled eporangium or a brown, thick-walled resistant sporangium.

Blastocladia, which is also characterized by a determin—

ate growth pattern, does however, develop into a more complex

structure which may be branched or lobed and on which are usually formed several thin-walled sporangia and/or resistant

sporangia.

Blastocladiopsis does not differ radically from Blasto-

cladia and is differentiated from it by certain features of its resistant sporangium.

The genus Allomyces, on the other hand, manifests an

indeterminate system of growth characterized by extensive dichotomously branched hyphae, on the ends of which develop the reproductive organs.

None of these fungi is septate (although Allomyces dis-

plays 'pseudosepta'), except when the reproductive structures become partitioned from the rest of the plant; thus, they are coenocytic organisms, with their many nuclei dispersed throughout the cytoplasm of the thallus. At maturity, the entire protoplasmic content of the multinucleate sporangium under— goes progressive cleavage into uninucleate cells which are then liberated through one or more papillae or discharge tubes as motile spores, ready to begin the cycle once again.

The motile spore

The spore is a motile cell measuring about 5 to 12 u in length and slightly less in width, the exact dimensions varying with the species. It possesses a single, posterior, whiplash flagellum (Sparrow, 1960), the length of which is several times that of the spore body. Numerous observations made with the light microscope resulted in the following composite picture of the Blastocladiaceous spore.

1) Near the posterior end of the spore is found the single prominent nucleus with its nucleolus.

2) Surrounding the apical portion of the nucleus and extending much beyond, making it the most conspicuous inclu— sion in the spore, is the nuclear cap. This organelle, which is typical of the Blastocladiales and some Chytridiales, has long been referred to as a 'foodbody' (another example of mycological guesswork) although its function was not under- stood (Barrett, 1912; Emerson, 1941; Sparrow, 1960).

5) In some Species of Blastocladiella, a body along one

side of the nuclear apparatus is consistently observed.

Harder and Sargel (1958) first referred to it as the

'seitenkorper' in their description of B. variabilis.

4) There are also many smaller organelles, including refractile lipid granules and vacuoles (and in Blastocladiella

emersonii, some very small 'gamma' particles which stain with

* the Nadi reagent ; Cantino and Horenstein, 1956a).

More recent studies employing the electron microscope, cytochemical techniques, and chemical analyses have elucidated

*- This color reaction results from the oxidation of di- methyl-p-phenylenediamine in the presence of ornaphthol and is catalyzed by oxidative enzymes. the situation enormously and provided us with a more sophisti- cated picture of the spore. The results of these investigations are summarized below.

1) Electron photomicrographs of gametes of Allomyces macrogynus and spores of Blastocladiella emersonii have shown

the flagellum of both species to be structurally similar to that of many other motile cells (Hoffman-Berling, 1959).

There are two central fibrils surrounded by nine outer ones, all enclosed within the flagellar sheath (Blondel and Turian,

1960; Cantino et al., 1965). Fourteen years ago, Cantino

(1951) observed the retraction of the flagellum into the body of the spore upon germination of B. emersonii. More recently,

electron photomicrographs verified the presence of the flagellum within the cell after retraction, where it was extended along almost the entire inner periphery of the spore wall (Lovett, unpublished).

2) Results obtained with various cytochemical techniques led Turian (1955; 1958) to conclude that the nuclear caps of Allomyces gametes represent a localized accumulation

of RNA. Additional evidence was offered by Blondel and

Turian (1960), who reported that RNA-containing particles

(presumably ribosomes) concentrated in the areas surrounding the nuclei in the mature gametangia of A. macrogynus during the genesis of the caps, just as gametes were being cleaved.

Electron photomicrographs suggested that the nuclear caps of

Allomyces gametes and Blastocladiella spores are similar.

Virtually all of the-electron dense particles in the swarmers

of B. emersonii are contained within its nuclear cap (Cantino

et al., 1965; Lovett, 1965). Chemical analyses of 'clean'

nuclear caps isolated from the spores of B. emersonii cor-

roborated what was implied by the electron photomicrographs

(Lovett, 1965). The caps represented 69% of the total RNA

of the cell and they, themselves, were composed of 57% protein

and 65% RNA. These data, in addition to other evidence,

support the concept that this organelle is a unique package

of ribosomes. However, relative to ontogeny, this aggregation

of ribosomes is a temporary phenomenon; at the onset of germination of both the spore of Blastocladiella and the

zygote of Allomyces (in which the nuclear caps from the two participating gametes have fused), the membrane bounding the nuclear cap disintegrates and the ribosomes are dispersed throughout the cytoplasm (Turian, 1958; Blondel and Turian,

1960; Lovett, 1965; Cantino and Lovett, 1964).

5) Electron microscopy has revealed another structural

similarity between Allomyces gametes and Blastocladiella

spores. The double membrane which separates the nuclear cap

from the nucleus is perforated by pores, thus possibly perm mitting exchange of materials between these two structures

(Turian and Kellenberger, 1956; Cantino et al., 1965).

These seem to be the first documented instances of such intimate contact between the bulk of the DNA and RNA of a cell

(Cantino and Lovett, 1964). 1O

4) These same studies (Cantino et al., 1965) provided evidence that the everpresent 'sidebody' in B. emersonii is a giant mitochondrion, the gnly_mitochondrion in the spore:

This somewhat cup-shaped body extends upward from the vicinity of the flagellum at the base of the cell to about

2/5 of the distance on one side of the swarmer. The mitochondrion is penetrated by the flagellum which appears to be attached to it yia_one or more rootlet-like appendages. In view of the fact that the flagellum propels the spore while it is actively swimming, one would expect that a great deal of energy is being expended during this process. As a matter of fact, the endogenous 002 of the spores is close to 100 at this time (McCurdy and Cantino, 1960). This, then, would be a most convenient location for the energy source needed for motility. The revelation of a solitary mitochondrion in the spore of B. emersonii is a deviation from the situation in the gamete of Allomyces macrogynus where several mito-

chondria, of smaller dimensions, have been detected (Turian and Kellenberger, 1956; Blondel and Turian, 1960).

The resistant sporangium

It is not exceptional that aquatic microorganisms have evolved an expedient, usually in the form of a specialized resistant structure, to 'guarantee' their survival under otherwise intolerable environmental situations. The Blasto- cladiales have evolved the resistant Sporangium. Instead of 11 developing a thin—walled sporangium, the fungus produces one possessing a thick chitinous wall (Lovett and Cantino, 1960a) which becomes impregnated with melanin (Emerson and Fox,

1940; Cantino and Horenstein, 1955). After its fabrication, the resistant sporangium remains in a state of dormancy until conditions become favorable for the release of spores.

Resistant sporangia of Allomyces have retained their viability

after twenty years of desiccation (Emerson, 1954), and

B. emersonii for almost as long.

It had been assumed by some for many years, but never shown experimentally, that meiosis occurred in the zygote of the aquatic fungi. In 1949 Emerson and Wilson (1949; Wilson.

1952) made the first detailed study of meiosis in a water mold and offered conclusive evidence that in Euallomyces, meiosis occurs in the resistant sporangium. This demonstration of the site of meiosis placed Allomyces in a unique category

of filamentous fungi, in which vegetative hyphae are truly diploid. To distinguish between the two types of swarmers liberated from the sporophytic plant, Emerson (1950) proposed the term 'mitospores' for diploid spores resulting from mitotic divisions and liberated from thin-walled sporangia, and 'meiospores' for haploid spores resulting from meiotic divisions and liberated from resistant sporangia. The site of meiosis, if it occurs at all, has not been established for either Blastocladia or Blastocladiella.

Life Cycles in the Blastocladiales

Emerson (1941) assigned subgeneric names to Allomyces

on the basis of three different types of life cycles which it displayed. Inasmuch as these three patterns circumscribe all the known life cycles of the other genera as well,

Sparrow (1960) adopted Emerson's nomenclature to include the entire order Blastocladiales. This precedent will be con— tinued in the following description.

Brachyallomyces type

The simplest of the life histories is the Brachyallomyces

type (Fig. 1). This comprises an asexual (or sporophytic) generation only, and is exhibited by such Species as Blasto- cladia pringsheimii, Blastocladiella emersonii, and Allomyces

anomolous. Spores released from both thin-walled sporangia and resistant sporangia develop directly once again into a new generation of sporophytes.

Cystogenes type

The organisms displaying the Cystogenes life cycle under~

go both sexual and asexual reproduction (Fig. 1). The asexual is much the dominant phase and is similar to the Brachy- allomyces type as far as the Spores released from the thin-

walled sporangium are concerned. But the spores from the re~ sistant sporangium encyst shortly after discharge; the cyst constitutes the gametophyte. Isogametes emerge from the cyst, and the zygote resulting from sexual fusions develops once 15 again into a sporophyte. Organisms of this sort have not been studied extensively; therefore, more detailed information is lacking. Allomyces cystogenus and Blastocladiella gystogena

are two species displaying such behavior.

Euallomyces type

This third type is one in which there is an alteration of equivalent Sporophytic and gametophytic generations (Fig. 1).

The sporophyte is morphologically the same as in the two previous types. The basic growth pattern of the gametophyte is similar to the sporophyte except when the reproductive cells are differentiated. In Allomyces, gametangia are formed in

pairs on the same thallus. One member of the pair develops into a female gametangium, much like the colorless thin-walled sporangium in appearance. The other member, the male, in addition to being different in size than the female (smaller or larger, depending on the species), reveals, at maturity, orange protoplasm resulting mainly from the presence of gamma carotene in lipoidal bodies (Emerson and Fox, 1940). The arrangement of these paired gametangia is species specific.

In A. macrogynus, the orange male gametangium is terminal

(epigynous) and in A. arbuscula, the male is subterminal

(hypogynous). The motile female gametes liberated from colorless gametangia resemble mito— or meiospores in morphology, while male gametes are orange in color, somewhat smaller in size, and more rapid in motion. Male and female gametes

(often from the same plant) fuse to form a biflagellate zygote. _ 3.. 3. . VI 3 1| ‘ IA H. Aﬁ" an- v. Z a. “a r" Cu SﬁAv

‘, .l..4t li.~v:- iilu‘l ¢. nil: 14

L » Spores i Thin-walled sporangium Sporophyte~<:::::::: Brachyallomyces t Resistant sporangium 4, Spores

- Spores

l, Thin-walled sporangium Sporophyte <::::::; T Resistant sporangium

Zygote Spores Cystogenes

1 Gametes Gametophytic cyst

MitOSpores

Thin-walled sporangium Sporophyte <:::::::; Resistant Sporangium (K i, Meiospores Euallomyces

Gametophyte

3' Gametangium 3 Gametangium

51 Gametes 9 Gametes

Zygote

Fig. 1. Life cycles in the Blastocladiales

Following a brief swimming period, the zygote develops into a new sporophytic generation. Copulation between two aniso- planogametes was described by Kniep (1929) in the first accounts of sexuality in Allomyces. This is a phenomenon that hitherto had not been known to occur among the fungi; up to the present, such fusions are, to my knowledge, still un- known outside of the Blastocladiales. The description of

Blastocladiella variabilis (Harder and Sérgel, 1958) renders

the life cycle of this organism similar to Euallomyces; however, the male and female gametangia are on individual thalli by virtue of its monocentric nature.

This type of life cycle, then, is representative of_a group of Blastocladiales which is distinctive among the aquatic fungi: (a) the sporophyte is an independent diploid generation; (b) the resting stage is an asexually formed resistant sporangium; (c) the zygote germinates immediately after its formation.

Differentiation of the Resistant Sporangium in Blastocladiella emersonii

Induction of resistant sporangium formation

Emerson (1954) has said of resistant Sporangia (RS),

"Observing their highly modified structure, noting their special function, and recalling that they are the locus of the critical reduction divisions in Allomyces, one can immediately

recognize their intrinsic interest." But until relatively 16 recent times, not much attention was focused on this alternate developmental structure. Perhaps one of the reasons is that when some of these fungi were brought into the laboratory and divested of all other creatures commonly sharing their habitat, RS were rarely seen. This is what happened when

Emerson and Cantino isolated Blastocladia for the first time,

preparative to carrying out studies on nutrition and physiology

(Emerson and Cantino, 1948). When grown in pure culture under aerobic conditions, RS of B.Apringsheimii were unobtainable.

It wasn't until pure tank C02 was bubbled through the medium, in which the pH was maintained between 5.5-5.8, that these structures were produced.

A few years later a new species of Blastocladiella,

B. emersonii, was isolated by Cantino (1951). With this organism too, difficulty was met in attempts to obtain RS.

When swarmers were inoculated onto agar media, the first generation population consisted solely of thin-walled, ordinary colorless (OC)* plants. When each of these OC plants matured and discharged swarmers, hundreds of new plants grew, clustered around the now evacuated parent Sporangium. Among each of the second generation clones, varying numbers of RS appeared. It turned out that C02, as bicarbonate, played a fundamental role in RS induction in B. emersonii, just as

* 0C sporangia are equivalent to thin-walled sporangia, but since ’OC' is entrenched in the literature on B. emersonii, its use will be continued in the discussion of this species.

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with Blastocladia pringsheimii, although its effective concen-

tration was of an entirely different magnitude and the opti-

mum pH range was not so narrow. Blastocladiella did not

tolerate C02 concentrations above 5%; however, by incorporat— ing 10'2 M bicarbonate into the nutrient agar, the resultant

first generation populations consisted of 95-100% RS (Cantino,

1952). When the bicarbonate was omitted, the population

invariably consisted, instead, of 100% OC plants. Here, then,

was an organism whose genotype endowed it with the capacity to

develop into one of two distinct morphological types--either

a smooth, thin-walled OC sporangium or a pitted, thick-walled,

brown RS (with a longer generation time)--depending on the

absence or presence of bicarbonate, respectively. Bicarbonate

acted in some way to effect a profound modification in a

morphogenetic pattern which, in nature, could literally Spell

the difference between perpetuation or extinction of the

Species.

Formulation of a hypothesis

In the earliest studies, it was found that under certain nutritional conditions where bicarbonate alone was insufficient to induce RS in B. emersonii, the addition of obketoglutarate

in conjunction with bicarbonate did induce their formation.

Subsequent experiments, centered around the possible involvement of orketoglutarate, produced the following results

(Cantino, 1951): (a) biotin, which has been implicated in l

a...

18 oxidative decarboxylation reactions (Lichstein, 1960), prevented RS formation in the presence of aﬁketoglutarate;

(b) arsenite (an inhibitor of oxidative decarboxylations) and semicarbazide-(a keto reagent) augmented the effect of orketoglutarate in RS induction. These same metabolic inhibitors prevented the germination of mature RS at the identical concentrations that facilitated their formation; (c) when

OC plants were bathed in bicarbonate solution, their internal pool of diketoglutarate increased.

These observations led to the notion that bicarbonate somehow interfered with the normal operation of the Krebs cycle by preventing the oxidative decarboxylation of orketoglutarate and thereby caused it to accumulate within the plant (Cantino,

1955).

When OC plants are grown in the presence of glucose, under certain conditions, fermentation to lactic acid is the predominant dissimilatory mechanism (Cantino, 1951). Although homogenates of these same cells reveal the presence of all the enzymes associated with the Krebs cycle, it seemed unlikely that it functioned as the major energy source for the plants' requirements under these conditions. Cantino suggested that in B. emersonii, the role of this cycle lay elsewhere, such

as the focal point for shunt mechanisms emenating from the accumulated oPketoglutarate and ultimately leading to the genesis of an RS. This notion was strengthened by the examination of a mutant strain of B. emersonii which was incapable

19 of forming RS, even in the presence of bicarbonate. This

strain was devoid of aconitase and orketoglutarate dehydrogen-

ase, thus the two successive decarboxylations in the Krebs cycle could not take place (Cantino and Hyatt, 1955a).

As a consequence of the preceding discoveries, the

following hypothesis was formulated:

1) Increased concentrations of bicarbonate interfere with the normal operation of the Krebs cycle.

2) The effect of this obstruction is to set in motion

shunt mechanisms leading to the genesis of RS.

5) The primary locus for the triggering of these mechanisms, at the biochemical level, is at the orketoglutarate area of metabolism.

Biochemistry of morphogenesis

Intensive investigation was now directed toward the elucidation of the "biological Significance of the 'trigger mechanism' in terms of causal biochemical reactions involved”

(Cantino, 1955). Comparative studies revealed the following distinguishing features associated with mature RS plants, as compared to mature OC plants: (a) de novo synthesis of y-carotene, melanin, and at least one new soluble protein fraction (Cantino, 1961a); (b) deposition of increased amounts of chitin and fat (Lovett and Cantino, 1960b): (c) disappearance of cytochrome oxidase and two soluble protein fractions

(Cantino, 1961a); (d) drastic reduction, if not a complete loss, of enzymatic activity associated with the Krebs cycle 20 except for a TPN—specific isocitric dehydrogenase which, in the presence of bicarbonate, mediates the reductive carboxylation of orketoglutarate (Cantino and Horenstein, 1955):

(e) decrease in the free amino acid pool, particularly a sharp drop in tyrosine (Lovett and Cantino, 1960b).

To test the hypothesis that new pathways, leading to the genesis of RS, derived from orketoglutarate, the melanin synthesizing system was selected for examination. The ob- jective was-to look for the presence of a polyphenol oxidase as well as a possible coupling to orketoglutarate. Such an enzyme was found in homogenates of RS plants; cell-free preparations oxidized tyrosine, catechol, and dihydroxyphenyl- alanine (Cantino and Horenstein, 1955). These activities were not present in preparations made from CC plants. More- over, this polyphenol oxidase, which seemed to function as a terminal oxidase in lieu of the cytochrome system which. dis- appeared in RS, could be coupled to either oxygen or TPN (but not DPN). The addition of drketoglutarate to the reaction mixture accelerated the enzymatic oxidation of catechol and tyrosine. It was postulated that the TPN, which served as a hydrogen acceptor in the tyrosinase reaction, was generated during the carboxylation of onetoglutarate to isocitrate.

amino acidypool \ isocitrate tyrosine fumarate TPN

succinate «ATPNH orketoglutarate melanin block C02 21

From the inception of this Work, it had been known that in order for B. emersonii to develop along the RS pathway, bicarbonate need not be present throughout the entire growth period. There is a point of no return in ontogeny--during the exponential growth period--before which the dual potentiality of the fungus is maintained. This means that plants which have been growing in the presence of bicarbonate, and therefore developing along the RS pathway, can be reversed to the OC pathway by removal of the bicarbonate. Beyond this point, the plants' fate has been sealed. This point of no return, therefore, signifies that the changeover to the metabolic machinery responsible for the fabrication of RS is complete and that differentiation is irrevocable.

During the first 40% of their generation time, thalli developing along the RS pathway appear to be morphologically identical to OC thalli during a comparable period in their ontogeny. However, it seemed plausible to assume that certain mechanisms are put into play ty exogenous bicarbonate long before the morphological changes become discernible. In order to determine exactly what changes were occurring, methods were developed for obtaining synchronously developing mass cultures of both OC plants (McCurdy and Cantino, 1960; Goldstein and Cantino, 1962) and RS plants (Lovett and Cantino, 1960b).

This means of growing populations of B. emersonii, in which

108 to 1010 plants are all precisely the same age and at the same developmental stage, made it possible to follow the 22 dynamics-of-various systems throughout a single generation.

Investigations-of the relation between early biochemical events and later morphogenetic events were undertaken. In such RS cultures,-the~point of no return occurs at 45% of its

84 hour generation time (56 hr. at 240). This time also de- notes the termination of exponential growth (increase in Size) and the concurrent formation of a crosswall which delimits the sporangium from the basal portion of the thallus. The final

57% of the generation time is the period of RS differentiation.

Period of exponential growth. Compared to an 0C plant, an RS plant exhibits a 46% decrease in its exponential growth rate (based on dry wt./cell) and an abrupt drop in Qoe—-the latter, by the end of exponential growth, amounts to a 90% reduction. These changes are, in themselves, not the cause of RS morphogenesis since decreases in both growth rate and respiration can be brought about by other means without concomitant RS production. Nevertheless, they are clearly effects induced by bicarbonate. One would expect that oxygen consumption would drop, for during this period the Krebs cycle comes to a virtual standstill (judging from changes in enzyme activity), no glucose is being consumed, and a reserve pool of polysaccharide is being accumulated.

It was assumed a priori that any metabolic systems inti~ mately involved in RS morphogenesis and turned on by bicarbonate would be operative before the expression of the morphogenetic events they regulated, and that these systems would be 25 reversible before the point of no return but not afterwards.

Cultures of RS were harvested at various ages, and two relevant enzyme systems were examined during the exponential growth period. There was a 6500-fold increase in isocitric dehydrogenase but-only a 650-fold increase in orketoglutarate dehydrogenase-activity; that is, the activity/cell of the latter enzyme rose to only 10% of that of isocitric dehydro— genase. This, considered together with the 90% reduction in oxygen consumption, is evidence for the presumed mode of operation of the bicarbonate trigger mechanism. Moreover, when bicarbonate was removed before 45% of the generation time had elapsed, orketoglutarate dehydrogenase increased the isocitric dehydrogenase activity decreased, both of them approach— ing the level found in OC plants (Fig. 2). After the point of no return, the activities of these two enzymes remained un— affected by the removal of bicarbonate.

McCurdy and Cantino (1960) demonstrated the operation of a previously postulated isocitritase system in B. emersonii, as well as a glycine-alanine transaminase. These two systems apparently operate in sequence to remove isocitrate as it is formed and thus prevent the carboxylation of asketoglutarate from bogging down.

TPNH +-orketoglutarate + C02 -—ﬁ>—isocitrate + TPN

isocitritase glycine g1 oxylatSZ/+ succinate >' tr ansaminase ‘

qr- POINT OF NO RETURN

\. 6000

o I o F' \ z ISOCITRATE \ jg \

ACTIVITY a.’ \ \ a: I au: it 3000 0

RELATIVE

KETOGLUTARATE

48 AGE (HRS)

Fig.2 Reversal of enzyme activity during morphogenetic reversal in By_emersonii.

The activity of isocitric dehydrogenase and aC-ketoglutaric dehydrogenase per RS plant is shown for different stages of development. The total activities per plant at the Spore stage were assigned values of 1.0, and all other total activities at different stages of deve10pment were related to the Spore level. The dotted lines represent the changes in activity induced by the removal of bicarbonate (from Lovett and Cantino, 1961). 25

The activity of these enzymes was followed in both OC and RS.

plants. The specific activity of isocitritase in OC plants

decreases during the first half of the generation time and

rises again during the second half, reaching the initial level

at the time the protoplast is being cleaved into spores once

again (Fig. 5-left). On the other hand, isocitritase activity

in RS plants rises sharply at about 40% of the generation time-~

the region of the point of no return--and then drops. Trans-

aminase activity follows approximately the same pattern.

When these data are expressed on a per plant basis rather than

on a dry weight or soluble protein basis, a strikingly dif-

ferent picture obtains (Fig. 5-right)f For the first half of

the generation time of an OC plant, there is essentially no

synthesis of isocitritase; that which was present in the Spore

is diluted as the plant increases in volume many-fold. But

under the influence of bicarbonate, synthesis of isocitritase

is induced immediately in RS plants. The metabolic scheme

which appears on page 25 illustrates the importance of this

enzyme system for the RS story.

The protein pool has also been shown to undergo consider-

able reorganization during exponential growth. There are quantitative differences in the patterns between young OC

plants and young RS plants. When bicarbonate is withdrawn before the point of no return, the protein composition of the

*These figures are reproduced to illustrate the importance of using a 'per cell' approach, possible only with synchronized cultures. Conclusions as drawn above could not have been made otherwise.

re \ \ 1 \ ( 3°”) J s NIEIOHd . lNV'Id/Slan CU \“ 1 ,

0c 26 'SW/Slan

1 I l

20 60 I00 20 60 IOO % GENERATION TIME

Fig.3. Comparative activities of isocitritase in synchronously develOping DC and RS plants of B. emersonii during ontogeny. left. units/mg. protein right. units/plant (redrawn from Cantino. 1961a) 27

RS plant partially reverts to approximate that of the OC

plant (Cantino and Goldstein, 1962); these protein changes

could conceivably correspond to enzyme changes (vide supra).

Finally, there are certain conversions of RNA that deserve mention. In his analyses, Cantino (1961b) separated

this nucleic acid into two fractions--a NaCl soluble RNA

(RNASOl) and a NaCl insoluble RNA (RNAinSOl). Up to 29 hours

(55% of generation time), the RNA complement of an RS plant

is composed solely of the RNAsol variety. At 29 hours, the

Synthesis of RNA. begins and continues throughout most of insol the remaining generation time, maintaining a constant base

composition-~all four nucleotides occur in almost equimolar

amounts. Net accumulation of RNAsol' which is continuous

from shortly after Spore germination (the exact time has not

been established), begins to decrease at the point of no re-

turn. Hence, a qualitative as well as a quantitative change

in RNA is initiated just before the critical point (Fig. 4).

The above observations, as well as others not mentioned, Show

that there are metabolic differences between young RS and OC

plants. It is also obvious that none of the changes induced by bicarbonate prior to the point of no return prevents an RS

plant from reverting to an 0C plant upon removal of the in-

ducer.

Period of differentiation. After 56 hours of growth,

the developing RS is obliged to differentiate all the way

into the thick~walled dormant structure. During this time, '00 <~ PNR

RNA

RNA/PLANT TOTAL X 1 I n

36 60 83 I00 Fig.4 1 GENERATION TIME

Percent generation time

moles/loom 2‘

719,-? - ‘00. I"

Fig.4. Transformations in total RNA during morphogenesis of R3. (PNR 3 point of no return)

Fig.5. Transformations in molar composition of RNA during morphogenesis ‘ sol of R5. (data from Cantino, 1961b) 29 however, there is no further increase in the volume of the sporangium. In fact, the only visible change is a gradual darkening and thickening of the wall, for only 20% and 47% of the final complement of melanin and chitin, respectively, has thus far been laid down. But the degree of internal activity belies this apparent quiescent situation. Enzyme systems are increasing, leveling off, or decreasing in activity. To illustrate, representative data have been selected from the literature and replotted as percent of maximum activity or content, to make them comparable (Figs. 6. 7, 8).

Although alterations in the RNA composition of the RS plant begin to show up at the end of exponential growth, the magnitude of these changes can best be appreciated by examining them in the differentiating sporangium. Immediately following the point of no return, the RNA which begins to 501' decrease, undergoes a qualitative change; by the end of the differentiation process, the RNAsol which remains in the mature RS, is different in composition from the original

RNA'SOl of the young plant. It now, like the RNAi is com- nsol’ posed of equimolar amounts of nucleotides (Fig. 5).

At this point, the ultimate function of the RS should be considered. Although very little is known about the physiology of the RS plant during its interim state of

'dormancy', this sporangium converts its entire protoplasmic contents into a new generation of Spores when conditions again K30 A 1; V r L—x l .3 WOWIXVW

5C) 50 :I O %

L Fig.8

Ioo " I00 X GENERATION TIME Fig.6. Changes in protein nitrogen(a), plant volume(b), and dry wt.(c)/plant during RS morphogenesis in B. emersonii. (For this and subsequent figures, maximum level for each item was set at 100, and all other data related thereto.)

Fig.7. Increase in chitin(a) and melanin(b) content/plant during RS differentiation in B.~emersonii.

Fig.8. Changes in lactate pool(a), glucose-B-phOSphate dehydrogenase(b), glucose consumption(c), and polysaccharide pool(d)/plant during RS differentiation in B. emersonii.

(Above data were recalculated from Cantino and Lovett, 1960; Lovett and Cantino, 1960b; Cantino and Goldstein, 1961.)

becom- This RV‘ use H— I): (I.

'(J O (U {I} g ’4. ’1 , 'i .

51 become amenable. Let us recall the structure of the nuclear cap in the motile spore. It consists of a package of ribosomes and represents 69% of the total RNA of the spore.

This bit of data, in conjunction with the fact that the newly formed RNA. accounts for 65% (Fig. 4) of the total 1nsol RNA in a mature RS plant, led Cantino (1961b) to suggest that

RNA is that which is incorporated into nuclear caps insol during spore formation.

This section represents a much-condensed summation of what is known relative to the biochemical basis of RS morpho— genesis in B. emersonii. It is hoped that it will serve a useful purpose as a point of reference, as well as a point of departure, for the work that has begun on photomorphogenesis in B. britannica and which will be delineated in the sections to follow. MATERIALS AND METHODS

Culture Procedures

Stock cultures

All pure cultures of Blastocladiella britannica were

derived from the single Spore isolate sent to us by

Willoughby. Stock cultures were maintained on Petri dishes

of Difco PYG agar (1.25 gm. peptone,1.25 gm. yeast extract,

5 gm. glucose, and 20 gm. agar/liter H20; pH 6.8). Plates were routinely inoculated every 10 to 15 days as follows: a thin layer of surface agar (ca. 5 to 5 mm2) bearing a few

dozen mature plants was placed in 8 ml. sterile H20 at 220

C. A suspension of motile spores was obtained in 4 to 8 hr.

and a few drops of this suspension were distributed uniformly

over the agar surface of plates, prepared at least one day

in advance. These cultures were incubated at 220. As a rule, for security purposes, two sets of plates were main~

tained; the duplicate was stored at 50 C after initiation of growth at 22°.

Morphological observations

Early observations on the morphology of B. britannica were made on plants cultured on PYG agar, unless otherwise

specified. The populations ranged from ca. 500 to 500

52 55 individuals. This small number of plants, well dispersed, reduced the effects of interaction (pH changes, metabolic products,-etc.) to a presumably insignificant level and also facilitated the analysis of entire populations.

Illuminated cultures

In all experiments-involving illumination of cultures growing on agar media, the incident level of light (fluorescent;

Sylvania, Standard, Cool White) at the surface of the population was 180 f.c. at 220. Dark-grown control plates were covered with aluminum foil.

Static liquid cultures

Early studies, during which optimum conditions for growth were established, involved the use of static, multiple generation, liquid cultures grown in Difco PYG broth, pH 6.8

(same as above, minus the agar). For all experiments, a few drops of .04% bromcresol purple were incorporated into the medium and a constant pH was held by intermittent neutralization with NaOH. Cultures were harvested by vacuum filtration onto filter paper through a Bﬁchner funnel and washed with H2O, the total wash amounting to twice the volume of spent medium.

During harvesting and washing, the plant material was kept in suspension in a small volume of liquid to insure rapid filtration, efficient washing, and uniform density of cells.

After the final wash, the plant material was sucked 'dry'. 54

The portion of the mat not removed for dry weight determinations was immediately stored at -200 for subsequent

analyses.

Dry weight determinations

The plant material, which was easily peeled from the

filter paper, was weighed at once (for a wet weight value).

Either the entire mat or a measured portion of it was then dried to a constant weight at 900 in a vacuum oven using tared papers or weighing bottles.

Synchronized, single generation, liquid cultures

Preparation of inocula. The preparation of a large inoculum of swimming spores, with which synchronized cultures were begun, is described below. The method has been used many times and, if followed carefully, can be relied upon to yield reproducible results.

From a stock plate of RS strain #101B (isolation of this strain is described in a later section), maintained at 50

(usable for a few weeks after preparation if sealed with rubber rings to prevent dehydration), a block of agar bearing

15 to 20 plants was cut out and transferred to 5 ml. of H20 at 220. Several thousand swimming spores were liberated within

12 to 24 hr. The time required varied with the age of the stock culture.

A sample of the suspension (ca. 100 swimming spores) was transferred to a standard (9 cm. diam.) Petri dish of 55

PYG/5 (Difco PYG broth, one-third the recommended strength, supplemented with 1.5% Difco agar), distributed over the entire surface, and incubated in the dark at 220. Approximately

100 mature plants of uniform Size were formed 48 hr. later.

The plates were flooded with 5 ml. of H20 at 220.

Under these conditions, the cells liberated their complement of swarmers (average: 400 swarmers/mature plant) in 5 to 4 hr. Thus, the available spore population became ca. 4 x 104.

The spores above were distributed among 5 or 4 new plates of PYG/5 and the process was repeated. In this way, suspensions containing 5 x 105 or more Spores/ml. were easily obtained.

From these, roughly 5 x 105 Spores were transferred to each of 10 large (14 cm. diam.) Petri dishes of PYG/5 agar and incubated at 220 for 56 hr. With such high population densities, the average generation time, from Spore stage to mature cell bearing discharge tubes (Plate I,c), was 56 rather than the 48 hr. described above for sparse pOpulations of cells.

Finally, each plate was flooded with 25 ml. of H20 and placed at 220. After 5 to 4 hr., there were dense populations of actively swimming swarmers. Following a careful microscopic examination for possible contaminants, the spore suspensions were pipetted from the plates and filtered through sterile semi-crimped rapid filter paper. Any mature plants which might have been dislodged from the agar, as well as

56 young germlings, were trapped behind on the paper while swarmers went through unhindered. The heavy swarmer sus~ pension (5 x 105 to 10s cells/ml.) thus obtained served as the inoculum with which synchronized liquid cultures were begun. A rough estimate of the population density was obtained turbidimetrically with a Klett-Summerson colorimeter and 420 mu filter (1 Klett unit = about 105 spores/ml.; this value was corrected for background absorption by a low- speed centrifugation of the swarmers)- An accurate determination of the viable cell count was always made by transr ferring samples, after serial dilutions of the spore sus— pension, to plates of PYG and counting the mature cells produced.*

Growth of cultures. The standard medium finally developed consisted of Difco PYG broth (5.5 gm./liter) contain~ ing citric acid, 5.2 x 10'3 M and NagHPO4, 7.2 x 10‘3 M.

(Allowance was always made for the anticipated volume of spore inoculum, with the actual volume of H20 used reduced accordingly). The pH after autoclaving was 5.6. Media

(600 ml. in a 1 liter flask or 1200 ml. in a 2 liter flask) were inoculated so as to yield 5 x 104 to 7 x 104 Spores/ml. and incubated in a H20 bath at 24 a; 0.050. Cultures were

*- Comparisons made between plate counts and those done with a Coulter Counter (acquired too recently to be used during the course of the work described in this thesis) indicated a very high correspondence. 57

aerated vigorously with HZO—saturated air through flowmeters

(2000 ml./min. for 2 liter flasks and 1500 ml./min. for 1

liter flasks), and either illuminated from below with white

light (KEN-RAD, fluorescent, cool-white; 500 f.c. at the bottom surface of the cultures) or kept dark with aluminum

foil.

Cultures were sampled at intervals for size measure~ ments (50 cells per sample); plants in the population were uniform. For example, size measurements of a typical sample yielded the following: mean diam., 48.5 p; standard deviation,

5.21 p; distribution curve symmetrical about the mean with 95.4% within mean :.I-_ 2 standard deviations; 99.54% within mean.i 5 standard deviations. The organisms were harvested with suction on filter paper and washed (as described earlier

for static cultures). Dry weights were determined at 900 in vacuo. The weight/cell was calculated from the dry weights

and number of cells per culture vessel. Throughout ontogeny, the plant was almost a perfect Sphere (Plate I); thus, the weight of the cell per unit volume was directly calculable (i.e., the rhizoids presumably introduced a negligible error into the calculation).

Analytical Procedures

Lactic acid analysis

Spent culture media were acidified and extracted with

ether; the extracts were analyzed according to Ryan's (1958) microdiffusion method. 58

Carotenoid analysis

Mature plants-were homogenized in acetone, the homo- genate extracted with n-hexane, and the spectrum of the latter taken with a Beckman model DU spectrophotometer.

Chitin analysis

Cells were-digested with NaOH and HCl as was done with

B. emersonii (Lovett and Cantino, 1960b). Hydrolysates were

freed of HCl and analyzed for hexosamines with the indole-HCl reaction of Dische and Borenfreund (1950), before and after deamination with HNOQ. They were also examined chromato- graphically using Whatman #1 paper, pyridine-ethyl acetate- water (11:40:6) in the tank (Fischer and Nebel, 1955), and an111ne diphenylamine and nlnhydrin sprays (Rglucose for glucosamine, 0.66; for galactosamine, 0.57).

Melanin analysis

Dried, powdered plants were digested in 0.5 N KOH

(15 mgn/ml.; 2 hr. at 1000). The absorption spectrum of the extract was measured in the Beckman spectrophotometer.

Soluble polysaccharide analysis

This was determined according to the procedure used by

Cantino and Goldstein (1961) for B. emersonii. In brief, cell homogenates were treated with trichloroacetic acid and, after centrifugation, soluble polysaccharide was precipitated by addition of ethanol. The alcohol-insoluble fraction was 59

analyzed for glucose following HCl hydrolysis. Glucose was determined with glucose oxidase (Glucostat Reagent from

Worthington Biochemical Corp., Freehold, New Jersey) accord-

ing to Washko and Rice (1961).

Determination ofpglucose uptake capacity

For these particular determinations, cells were har— vested somewhat differently than described previously. The entire population from a synchronously growing culture was

filtered through an ordinary glass funnel and washed with citric acid-phosphate buffer (same concentration as used in medium). In the process, the fungus was concentrated into the bottom of the cone of the filter paper. The funnel was then placed over a calibrated cylinder, a small hole was poked through the bottom of the paper, and the cells--easily dislodged--were washed quantitatively into the cylinder with buffer and made up to a desired volume. The density of the final suspension ranged from 0.5 x 106 to 1.4 x 106 cells/ml.

Throughout the procedure, cells were readily maintained in suspension and unclumped. Replicate samples were removed for dry weight determinations.

Ten and 15 ml. samples of the above cell suSpension were placed in test tubes at 240 and the volume was adjusted to

19 ml. with citrate-phOSphate buffer. At zero time, one ml. of buffer containing 50 uM glucose was added to each tube

(this concentration was selected because higher levels yielded no increase in glucose consumption while lower levels tended 40 to depress its uptake). A fine stream of air served to keep the contents agitated and the cells suspended throughout a

45 min. incubation. For this time period, glucose uptake by

such preformed plants was not affected by the presence or

absence of light; therefore, these incubations were carried out under ordinary light conditions of the laboratory. At the end of the incubation, cells were filtered off and washed with H20. The washings and filtrate were combined and frozen as were the cells. Glucose was determined with glucose oxidase (see section on polysaccharide analysis). Glucose uptake capacity was calculated from glucose concentrations in the filtrate before and after incubation. OBSERVATIONS AND EXPERIMENTAL

Conditions for Optimal Growth

Early studies, using static liquid cultures of multiple generations, revealed that growth was prolific over a broad pH plateau (6-8); therefore, subsequent studies were carried out at pH 6.8. The optimum temperature for growth extended along a plateau between 16—250 with a sharp drop-off below

140 and above 280. Like so many of the water molds (Cantino,

1955; Cantino and Turian, 1959), Blastocladiella britannica

can be a vigorous lactic acid producer. Cultures in static

liquid media must be neutralized periodically to insure a constant pH. Furthermore, the yield of lactic acid/unit weight of organism is the same whether or not glucose is present in the medium (Table I). This suggests a strong tendency toward a fermentative mode of metabolism, irrespective of the nature of the food supply (nitrogenous or carbohydrate).

TABLE I. Lactic acid production by B. britannica

Medium PYG PYG minus glucose

Wet weight 19.1 gm. 4.6 gm.

Lactic acid re- 177.5 mg./1. 45 mg./l. covered from or or spent medium 9.5 mg./gm.wet wt.9.8 mg./gm. wet wt-

41 42

Morphological Variability

Concurrently with the foregoing studies, the lineage of many isolates from subcultures of the original single spore isolate was followed on agar media through as many as fifteen generations, with environmental conditions maintained as constant as possible. As a result, it became clear that Blasto- cladiella britannica displayed tremendous morphological variability, both quantitative and qualitative, whether populations were derived from a single plant or from a number of plants of the same morphological variety. To illustrate, let us look at a typical plate--one that had been inoculated about seven days earlier with hundreds of Spores liberated from a single sporangium (Fig. 9). Such a plate usually consisted of the following:

1) individual cells with a resistant sporangium (RS) having the brown, pitted, thick wall typical of this genus.

2) individual cells with a thin-walled (TW) sporangium of a barely perceptible light yellow color, most easily seen when they are crowded together, such as in colonies (see below).

5) RS colonies composed of both RS and TW sporangia.

4) TW colonies composed exclusively of TW sporangia.

However, when this same plate was examined five days earlier, at the time the culture was but two days old, every plant had the appearance of a TW individual. These young plants, 45

TW PLANT l . ”'7' < V

PLACED IN H10

[POPULATION 0F SPORESJ (IS/6’ rv

STREAKED ON PYG AGAR

RS PLANT rTW PLANT INCAPABLE OF INCAPABABLE OF GERMINATION GERMINATION IN SITU 1

TV! PLANT GERMINATING IN SITU

% ‘E‘t‘i (-) K RS COLONYj— j—TK ﬂ TW COLONY

SELECTION

7. a M dg’é} TT‘EI

d! PURE STRAIN OF TW COL ONIES ~

Fig.9 DeveIOpmental potentialities in 8 britannica. 44 therefore, had matured into either individual TW plants, individual RS, or into TW plants which discharged Spores in Situ. It was the third alternative which gave rise to the

RS and TW colonies, both comprised of second generation plants.

Those TW sporangia which retained their individuality after seven days, were capable of liberating motile spores only if they were removed from the agar and submerged in water; it was just such a plant which gave rise to the progeny described above. In contrast, RS did not discharge Spores, either in situ or when placed in water: (More will be said regarding

RS in a later section.) Finally, when plates were inoculated with spores derived from the cells of a TW or RS colony (rather than from an individual TW Sporangium), the same kind of variability in the resultant population was obtained (Fig. 9).

It should be mentioned that the picture just presented, although a frequent one, was not a consistent one. That is, only a proportion of the hundreds of plates examined revealed the presence of all four types of progeny (Table II). Often, only three of the four types appeared, sometimes only two, but never a population consisting of a single morphological variety.

Even within these four major groups, morphological variants appeared; a few are cited at this time though detailed studies remain to be done. For example, thalli of TW plants generally bore an extensive rhizoidal system causing

such plants to have a very shaggy appearance ('shaggy' plants,

Plate I,g); however, strains of TW plants were selected which 45

a-a--—————~“~'

Plate I Blastocladiella britannica

a. Plants of B. emersonii grown in liquid media; note typical, elongated basal stalks. x ca. 55. b. Plants of B. britannica grown in liquid media; note lack of stalks. x ca. 55. c. Mature plant of B. britannica with developing dis— charge tubes. x ca. 115. d. Resistant sporangium of B. britannica; ruptured to illustrate pitted surface. x ca. 560. e. Portion of D, enlarged. x ca. 1290. f. 'Smooth' TW plant of B. britannica x ca. 115. 'Shaggy' TW plant of B. britannica x ca. 115. 46

* TABLE II. Morphological types produced by B. britannica

% of trials in which population exhibited

Progeny from RS plants RS colonies TW plants TW colonies

TW plants 40 50 95 40

RS colonies 65 45 75 55

TW colonies 15 5 100 85

*These data were derived from plates which were examined when they were at least 7 days old; therefore, the populations consisted of first generation TW plants )which did not discharge in situ), RS plants, and second generation TW and RS colonies which resulted from first generation TW plants which did discharge in situ. Several plates were inoculated with spores discharged from plants of every age category (2—6, 7-11, 12-16, 17-21, 22-26, 27-51 days), using cultures of different histories selected at random over a 6 month period.

bore a sparse rhizoidal system, giving them a relatively smooth appearance ('smooth' plants, Plate I,f). It should be emphasized that such strains do not Simply represent the extremes of a morphological distribution curve, for intermediates were seldom seen. At the same time, it was also possible to select out two different RS-producing strains, one with RS averaging 98 x 101 u, and the other 122 x 124 u; again, few intermediates were ever seen.

Because of such clear-cut, inherent heterogeneity in

B. britannica, certain strains were repeatedly and selectively 47

subcultured until they ‘bred true'; these were used in the

following studies.

Pure Strains of TW Colonies

The data in Table II had revealed an obvious tendency

for TW colonies, in contrast to TW individuals and RS colon-

ies, to yield TW clones a very high percentage of the time.

After successive subculture, swarmers from these clones gave

rise to low percentages of RS plants and RS colonies. By

repeated selection from TW clones, strains were obtained which produced only TW colonies which bred true: When solid media were inoculated with swarmers from such TW colonies, the

first generation plants discharged spores to produce new

clones which were identical in composition to the parental

TW clones, i.e., RS plants were never formed. While their

temperature range for growth was normal, their generation

time was ca. 120-140 hr. as compared with ca. 40 hr. for ordinary TW plants which gave rise to RS colonies. Perhaps most surprising was the fact that the Size of the spores produced by this pure TW colonial strain was ca. 4/5 the Size of those liberated by the parental RS colonial strains (Fig. 10).

TW clones gradually developed a yellow pigmentation which reached maximum intensity at 220 when they were ca. 10 days old. The solubility and absorption spectrum of extracts

(peaks at ca. 456, 465, and 492 mu in hexane) left little doubt that the material contained one or more carotenoids,

OOI I NOIIV'IndOd TW STRAIN AV.- 6.9)1 DIAM. TW COLONIES -- ‘ Q ‘ > O O I \ \ s

‘\ 48

d0 ‘x ‘0 \ " \ I— ‘\ ‘5 / ‘ \TVI PLANTS

09 . \ I— \ \ \ \ O \O \

A l l l ‘ 5.6 o I I t 11111 3-3 7.0 7.7 0.4 OJ 9.. l0.5 'IINoILUTEo v2 AV.INAN. InI I14 V0 INC 0 STRENGTH or Pve

Fig.10. Size of Spores from the TN and RS Fig.11. Effect of different concentrations of strains. (Fixed with 1% osmic acid and PYG (2% agar) on the composition of popu- stained with gentian violet.) Each division lations derived from Spores of TN colonies. on the vertical axis represents 5 Spores. 49 a fact quite consistent with the known distribution of such pigments in the Blastocladiaceae (Emerson and Fox, 1940;

Cantino and Hyatt, 1955b; Cantino and Horenstein, 1956b;

Turian and Cantino, 1959).

Inasmuch as first generation plants of Blastocladiella emersonii could be induced to form RS by the addition of bicarbonate to PYG agar (cf. LITERATURE REVIEW), attempts were made to induce B. britannica to do the same.— In spite of

repeated trials with a variety of bicarbonate concentrations, the results were uniformly negative.

Then, because the concentration of PYG had affected the incidence of resistant sporangia in the RS strain (yiQ§_ infra), the pure TW colonial strain was grown on serial dilutions of the basal PYG medium. Although the number of TW clones increased as the concentration of PYG decreased

(Fig. 11), neither RS plants nor RS clones were ever produced.

Similarly, modifications of the glucose concentration in PYG

(from 0.5% to zero) did not induce RS formation.

Finally, because of the profound effects of illumination upon the RS strain (vide infra), cultures of the TW colonial

strain were incubated on PYG in light and dark. The composition of populations grown in the light was 40% TW colonies, 17% TW plants, and 45% non-viable, as compared with 10%, 90%, and nil, respectively, for those grown in the dark. Once again, no RS plants were produced. Thus, it was concluded that this pure TW colonial strain, incapable of producing RS 50 plants even though it was derived from a parental culture which had this-ability, had suffered a permanent metabolic lesion of some sort in its cytoplasmic and/or chromosomal machinery. For this reason, it should serve as a valuable tool in studies on morphogenesis in B. britannica.

The RS Colonial Strain

Life histopy

As was Shown in Table II, an assortment of progeny can be derived from an RS colony which is composed of varying proportions of RS plants and TW plants (Fig. 9). It should be emphasized at the start that there has been a total lack of success in inducing even one RS plant to discharge spores.

Innumerable attempts have been made, using sporangia of many ages (5 days to 14 months) grown under a wide variety of conditions and maintained on wet agar media, soil, desiccated

filter paper strips, and liquid cultures (nutrient media, soil media, hemp seeds in water, etc.). Furthermore, RS were subjected to all of the usual treatments (heat shocks, cold treatments, wetting agents, fat solvents, nutrients, etc.) plus many unusual treatments, but with a singular lack of success (see APPENDIX II). Thus, in the sections which follow, any reference to spore discharge from an RS colony will always mean that it is only the TW plants in such a colony which

liberate spores. 51

Effect of temperature on RS formation

The earliest Observations with the original culture and its progeny suggested that temperature had a differential effect on RS formation. Subsequent experiments revealed that when spores derived from RS clones were incubated on

PYG agar at 12, 16, 19, 22, and 250 until all first generation plants had reached maturity, individual RS plants were seldom produced at 12, 16, or 190. However, at 22 and 250, up to half of the total population consisted of RS plants: Clearly,

RS formation was favored by elevated temperatures.

Effect of bicarbonate on RS formation

Reasoning once again from the studies of the bicarbonate trigger mechanism in Blastocladiella emersonii, B. britannica was grown on various concentrations of bicarbonate, along with B. emersonii for comparison, and incubated for 10 days at

220. The results revealed two important facts. First, the tolerance of B. britannica for bicarbonate was quite different

from that of B. emersonii; between 6 x 10‘3 M and 2 x 10‘2 M,

the range within which B. emersonii grows well and is induced

to produce RS, growth of B. britannica was inhibited com—

pletely. Second, at 4 x 10'3 M bicarbonate and below,

B. britannica did grow well, but RS formation was greatly de—

layed rather than promoted in comparison with the time . required for RS development in the absence of added bicarbonate. Finally, even total lack of atmospheric C02, which does affect the development of B. emersonii (Cantino and

Horenstein, 1959), had no detectable effect on populations of B. britannica.

Just as with the TW colonial strain, progressive dilu- tion of the PYG medium caused a Shift in the composition of populations derived from the RS colonial strain of B. britan- gig§_(Fig. 12). But once again, when bicarbonate was incorporated into 1:10 PYG, the higher concentrations of bicarbonate as used above inhibited growth, while lower concentrations reduced RS formation. Thus, in its response to bicarbonate,EL britannica differs strikingly from B. emersonii.

Effect of_glucose on RS formation

Alteration of the concentration of glucose in PYG had a remarkable effect upon RS formation. The number of individual RS plants rose from 26% at double-strength glucose to ca. 90% when glucose was entirely eliminated; conversely, individual TW plants dropped from 56% to ca. 10%. There was little change in the usual small number of colonies (Fig. 15).

On the other hand, alteration of the concentration of peptone and/or yeast in PYG had no pronounced effect on the incidence of RS plants.

Because of the effect of glucose, cultures were grown under reduced oxygen tensions. For example, under a flowing stream of nitrogen (part. pres. of 02, ca. 0.1 mm. Hg)

Bppbritannica exhibited no detectable growth. Under somewhat higher partial pressures of 02 (e.g. under mineral oil),

OOI OOI I I RS COLONIES RS PLANTS NOILV'IﬂdOd NOIlV'IﬂdOd 08 OS .. T I... W PLANTS I L -; a o 09 09 :10 :IO 55 x I I I I Of I I 3 RS PLANTS I 09 I t I I ‘5 I I I I I I I I I I I 0 \ I T W PLANTS

OZ \ I OZ I I- O I \\ ”s O \ ~“

*-_.

o ‘ ...... O , LL I I .‘u ...... Q I

r 1RR/ woo ss 33 o UNOILUTED I12 I14

STRENGTH OF PYG GLUCOSE CONC, G OF NORMAL)

Fl .12. Effect of different concentrations of PYG Fig.13. Effect of different concentrations of (2% agar) on the composition of pOpulations glucose (in PYG) an R3 formation. The low in- derived from Spores of RS colonies. cidence of colonies, which was not plotted, remained approximately constant in all dilutions. (Normal glucose concn. in PYG = 3 gm./l.) 54

growth did occur, but relative to aerobic controls individual

RS plants decreased while non-viable plants and RS colonies

increased (Fig. 14).

Effect of light on RS formation

Finally, because-visible light accelerated RS formation

in B. emersonii (Cantino, 1957) when bicarbonate was present,

B. britannica was also grown in the presence and absence of

light. Fig. 15 illustrates the dramatic effect obtained.

B. britannica, grown in the absence of light, responded morpho-

genetically as B. emersonii does in the presence of bicarbOnate;

i.e., by differentiating into resistant sporangia.

Consequently, the question immediately arose: throughout what

period in ontogeny was light needed for formation of TW plants

(or, conversely, inhibition of RS plants)? The results (Fig.

16) of an experiment designed to test this revealed that plants

could be exposed to light for about 28 hrs. and still retain

their capacity for developing into RS when placed in the dark.

Beyond this point of no return, a change in the light regime

had no effect.

_;§olation of RS strains yielding 100% gguiividual RS plants

As was mentioned earlier, attempts to effect germination

0f mature RS plants have been totally frustrated. On the

other hand, it ya§_possible to induce Spore discharge in

'YOung', potential, RS plants; the methods evolved to accom-

Plish this were essential to subsequent experimental studies 55

Fig.14 SHADED - CONTROL

CO '- SOLID - UNDER OIL

POPULATION

OF N

3:3 I-=' 2!a a:3: J j 2 )4 a o. 0J a g‘ v3 3 O 03 c I- 0 t.-

Fig.15

g- :I §

3 .: SHADED-DARK " 0_ some -LIoNT

_ I \7 \ ‘\ 2- _ a\ as \ J \ .\

Fig.14. Effects of semi-anaerobic conditions on the composition of populations derived from Spores of RS colonies. 24 hr. following inoculation, plates were covered with mineral oil 5 mm. deep and incubated for 5 days.

Fig.15. Effect of light on RS plant develOpment. Inoculated plates of PYG were incubated in the light (or dark) for 3 days. Each bar represents the percentage of RS plants in a population derived from Spores of a single RS colony. 56

H30

POPULATION

PLANTS

4!)

RS N 20 I I l I 0 IO 20 30 4O 50

EXPOSURE TO LIGHT (HRS)

Fig.16. The relation between duration of light and RS formation on medium PYG. Control plates were incubated in the dark for 50 hr.; all others were eXposed to 180 f.c. of white light for different times and then covered with aluminum foil for the remainder of the 50 hr. growth period. Each point is an average value for duplicate cultures. 57

of B. britannica in particular, and they may be applicable

to other members of the Blastocladiaceae in general. It has

already been stressed that the usual population of mature

plants on PYG agar is a heterogeneous and variable one.

However, when this population is still young, i.e., 50 to 40 hr. old at 22°, all plants have essentially the same appear-

ance; it is possible to induce spore cleavage and discharge

in many of them if they are simply removed from the agar

and placed in water. Thus, after isolating individual thalli

in this age group, inducing spore discharge, and inoculating

these spores onto PYG agar plates, the population which re-

sulted was, when mature, usually as variable as the parent population. During the course of many such experiments, however, populations derived from spores of single plants occasionally consisted of a very high percentage of RS plants.

This suggested that the young isolated plants from which these

spores had come may well have been potential RS plants, and that they would have developed thus had they been permitted to reach maturity. Having assumed that it might be possible to obtain a pure RS strain (i.e., populations of 100% individual RS plants) if the 'right' plants were selected, this approach was continued on an expanded scale.

Many 55 hr. Old plants were isolated at random, placed individually into water in depression slides, and the discharged swarmers inoculated onto PYG agar. Then, when these first generation populations were 55 hr. old, 5 to 10 58 individual thalli were again isolated from each plate, and the process was repeated. And, indeed, populations were finally obtained which, when grown in the dark to maturity, consisted of 100% RS individuals! Tabulation of the progeny derived from a random sample of such individual plants

(Fig. 17) revealed that selection from one end of the distribu— tion curve was being achieved. Typical growth curves at 16 and 220 for such RS strains which produced only individual

RS plants are shown in Fig. 18. The maintenance of these strains presented a serious problem because populations had to be subcultured every 55 hr., i.e., before they developed into mature RS which, as has been pointed out, do not germinate. However, the difficulty was partially circumvented by maintaining populations of these potential RS plants in an immature state for several weeks through the simple expedient of incubating them at 50. Thus, plants were isolated from the 50 population whenever they were needed for initiation of subcultures of the pure strain of RS plants.

At this time it is not known if the swarmers derived from young, potential RS plants are the same, genotypically or phenotypically, as those which should emerge from the same plant in its 'fully mature' state, that is, a resistant

Sporangium. Until the means for germinating mature RS plants is at hand, this question will remain unresolved. 59

I00

the

MATLRE

PLANTS

inward

plants. 90

(22°),

(“—1 diameter

I SO

shrink

hr. I (HRS)

average

I 70

ca.

individual PLANTS

the

begins

OF PLANTS/

line) AGE

curves

NATURE 50

represents Beginning

(solid

o line). 40

Growth

point

wall

plants. 22’ I

(dotted

Each OZI oo 09 09 09 OZ 20

Id I'NVKI'AV Fig.18.

epor-

eporangia

populations

single

population

select

from

number to

POPULATION

Each

shown.

progeny

refers

attempts

PLANTS

producers.

populations

the

frequency

consisted

Results vigorous

counted

producing angium;

AON3003Ud Fig.17. 60

Development of Synchronous Cultures of Resistant Sporangia

To recapitulate, a strain of Blastocladiella britannica

was selected from the various morphological types which, when grown on PYG agar medium, culminates in the formation of either a hyaline thin-walled cell or a thick—walled, brown, pitted resistant sporangial cell. Unlike its near relative,

B. emersonii, these alternate morphogenetic pathways in

B. britannica are not controlled by the presence or absence of exogenous bicarbonate. However, an environmental factor which does have a profound effect on differentiation on solid media is white light; TW cells are formed in its presence and RS cells are formed in its absence (Plate II). In order to study this phenomenon at a biochemical level, it was essential to learn to grow the organism in large quantities under conditions which permitted reproducible morphological uniformity, and which could be precisely controlled. The following section is devoted to the elaboration of methods for growing synchronized, single generation, mass cultures of B. britannica, uniformly suspended in agitated media wherein the effects of light and dark on morphogenesis were demonstrable.

It soon became apparent that to procure mass populations of RS cells in liquid culture, vastly more was entailed than simply growing plants in PYG broth in flasks instead of on

PYG agar in Petri dishes; Special methods had to be devised. 61

Plate II B. britannica grown in light and dark.

A plate of PYG agar was inoculated with spores, covered with black paper from which the letters "BB” had been cut out, and exposed to 500 f.c. of white light. The white "dots" represent TW cells and some small, second generation colonies derived therefrom. The dark RS cells occur in great abundance in the darker areas in the picture but are not visible because of the black background. Stray light diffusing beyond the confines of the letter cutouts, was responsible for the occurrence of some scattered TW cells among the RS cells. 62

It was not until 22 months and 72 experiments later (involv-

ing some 500 cultures) that this goal was achieved to any degree Of satisfaction.

When liquid PYG was used, light- and dark-grown cultures behaved in identical fashion; i.e., they discharged

swarmers. The first goal, then, was to find a medium in which at least §gme_plants would develop into RS. This, it was reasoned, would provide a clue and a starting point.

It was known from work already described that aeration of the cultures was desirable for two reasons: first, growth was much more prolific and second, plants were maintained in

suspension, resulting in more uniformity. Over a two to three day incubation period, there was considerable evaporation of the medium; this led to adoption of a system whereby incoming air was first passed through water, thus saturating it and reducing evaporation to a minimum. B. britannica grew well enough under these conditions; but, no RS1

It was recalled from work with static cultures that RS occasionally were seen in flasks which had NaOH in the side arm (used for neutralization). Perhaps alkali absorbed metabolic C02 which might have been inhibitory to RS morphogenesis. Experiments in which NaOH was used as a C02 trap indicated that this was not so.

Then, in attempts to extrapolate from observations made from plate cultures, two flasks of FY broth (PYG minus glucose) were inoculated with spores and incubated, one in the 65

light and one in the dark. At the end of the growth period,

there was no evidence of RS in either one of these flasks.

From these same cultures however, small samples had been removed at various times during growth and transferred onto

PYG agar plates which, in turn, were incubated in the dark.

This was done to ascertain if B. britannica, by growing under

these conditions, had actually lost the potential to develop

along the RS pathway. The plates made from illuminated liquid

cultures produced the expected results. The proportion of RS

in the mature populations on these plates was reciprocally related to the duration of previous exposure to light; the

longer the exposure, the fewer the RS. Unfortunately, the plants transferred from dark—grown cultures to plates responded in the same manner (Fig. 19): Cells removed from liquid cultures (both light and dark) during the first 15 hr. of growth produced high percentages of RS plants on agar media. But beyond 15 hr. the number of RS on plates rapidly decreased.

These results seemed to indicate complete independence of any

influence of light.

The same kind of experiment was carried out using PYG broth + bicarbonate (5 x 10"3 M). In this instance the re-

sults were quite different (Fig. 19). The dark-grown plants retained the ability to develop into RS when transferred to

agar plates, while light-grown plants gradually lost this capacity. And yet, those cells which were left to mature in unilluminated liquid cultures did not form RS; instead, they 64

g a x x o 5 "' r ‘------.-‘\O \ I DM:*’¢”‘ I - dz/I”””””T”——--. ° ‘. Pro-Incas I

'I (I) O " I LT >DK _ I’Y IAL ‘5 IDK > DUI T7, I \

" LT + on . POPULATION

IN 40

RS 20 9:, l L l lIn_____d. O 5 IO I5 20 25 AGE OF PLANTS AT TRANSFER

Fig.19. Incidence of RS after transfer from liquid to solid media. Small samples of cells from light- and dark-grown broth cultures (DY and PYG + HCD ') of various ages were inoculated on PYG plates which were then incubated in the dark. when mature, pOpulations were scored for R8. 65 behaved like the light-grown cells by developing into TW plants and discharging Spores at about 50 hr. of age.

To eliminate the contingency that metabolic products, inhibitory to RS morphogenesis, accumulated in liquid cultures, the following test was made. Spent media, in which

B. britannica had produced only mature TW sporangia in the dark, were filtered and autoclaved. Agar plates prepared therefrom were inoculated with Spores and incubated in the dark. The resulting populations consisted of 95% RS plants; therefore, inhibition by non-volatile, heat-stable metabolic products was at least ruled out.

Aerating cultures with various concentrations of gases and mixtures of gases with air, including N2 and C02, was tried; none of these induced RS formation.

The possibility that the agar component in PYG plates was exerting some physical effect which might be responsible for the photomorphogenetic response of B. britannica was considered next. Small quantities of agar (.01 - .1% y§_2% on plates) were incorporated into liquid media. Only TW plants developed (in light and dark), but at the higher concentrations, agar tended to extend the generation time of dark- grown TW cells. However, a 58 hr. old culture containing

.05% agar did contain some RS. With reference to this particular experiment, it must be added that no attempt had been made to maintain neutral conditions, and by the end Of the growth period, the pH in all the flasks had dropped from 6.8 66 to ca. 4.85. Although only a few RS were observed, this was

a straw to grasp at--low pH with agar added to the medium.

But very soon thereafter, it was concluded from further experiments that agar was not a physical factor in photomorphogenesis, and its use was discontinued. On the other hand, reduced pH was still a condition to be reckoned with.

Incorporation of a citric acid-phosphate buffer into the medium to lower the pH proved to be extremely satis-

factory. At the concentration most favorable for growth, its buffering capacity was quite adequate for maintaining a constant pH (i.0.2 unit) during the entire incubation period, thus eliminating the necessity for adjusting it periodically. An unexpected but important fringe benefit

accrued when the citrate-phosphate buffer was used. Plants remained nicely suspended as individual cells rather than in

small clumps which often was the case in all of the unbuffered media used theretofore.

Notwithstanding the lower pH values, the appearance of

RS was still an inconsistent if not infrequent event. All

during this exploratory period, the RS strain continued to produce ca. 100% RS when grown in the dark on solid media.

Once again, the possible involvement of agar came to mind, but this time in terms of chemical effects. Plates made with purified agar (Difco) failed to alter the composition of mature populations. This indicated the improbability that

impurities in agar were responsible for RS induction; 67 however, the chemical constitution of agar itself was questioned. Because it is composed of a sulfuric acid ester of a linear galactan, the unlikely possibility was considered that B. britannica might utilize the galactose moiety

of this polymer in preference to the glucose in the medium.

This idea was contemplated because in some cases more RS had been produced on plates in the complete absence of glucose than in its presence (Fig. 15). A new series of experiments was launched, and for the first time reproducible results were obtained. Liquid cultures in which 0.5% galactose was added to PYG broth, as well as those in which 0.5% galactose was added in place of the glucose, gave 50-70% RS. These media were buffered with citrate-phosphate to different levels over a pH range of 4.9-5.5. After some modifications in buffer concentration and determination of the most suitable inoculum size (number of swarmers), a system was at hand wherewith Single generations of synchronously developing thalli of B. britannica could be mass produced. By exposing

cultures to light, populations consisting of 100% TW Sporangia resulted; elimination of light resulted in 90-100% RS.

At this point, galactose was replaced by glucose and the same satisfactory results were obtained. Although it served in working out the details of the conditions necessary for RS cultures, galactose was not a requisite for their production.

Instead, it was a critical pH range that was required in liquid culture: The procedure ultimately adopted for 68 cultivating large numbers (107 — 109) of B. britannica is described in MATERIALS AND METHODS. Unless otherwise indicated, RS substrain B101wwas used in all subsequent work.

Growth and Morphological Characteristics of Synchronized Single Generation Cultures

The growth and morphology of Blastocladiella britannica in liquid culture did not differ perceptibly from that on

agar media. The uninucleate spore (ca. 5 x 7 u), following

a short Swimming period, rounded up and began to germinate

(PLATE III). A slender germ tube emerged which branched in root-like fashion culminating in an extensive system of rhizoids. Concomitantly, the thallus enlarged symmetrically into a single-celled spherical plant.* Until shortly before the end of the generation time of a light-grown TW sporangium, no morphological differences were discernible microscopically between it and a dark-grown potential RS at a corresponding chronological age. Yet, within the next 1-2 hr., the entire protoplast of a TW plant cleaved into hundreds of uninucleate, uniflagellate motile spores and a new generation of cells was then discharged through newly formed discharge tubes. On the other hand, the thalli grown in the dark had reached only the half—way point in their ontogeny; they continued to enlarge

* In these respects the morphology of B. britannica differs from that of B. emersonii. The latter is characterized by a somewhat cylindrical form as well as septation of the basal portion as the plant approaches maturity (PLATE I), thus making B. emersonii a two- celled organism. 69

.. it? an? 'r’ 4.x!”

Plate III Selected stages in development of TW and RS cells.

Up to 28 hr., cells grown in light or dark were identical in appearance. After 28 hr., morphological differences began to appear. Light-grown thalli developed into typical TW cells with discharge tubes (51 hr.), and dark-grown thalli developed into thick-walled, brown RS (55, 50, and 65 hr.). Note that in these synchronized cultures the rhizoidal system arose from one locus; this contrasted Sharply with results obtained when B. britannica was grown on solid media, where rhizoids arose at several places on the surface of the cell. 70 for several hours thereafter and then gradually differentiated into mature RS.

For TW sporangia in liquid culture, generation time is defined as the time at which 20% of the population of such cells produce discharge tubes. Essentially all of the cells in a population produce such tubes within one hour. This re- flects the high degree of synchrony obtainable in a Single generation culture. The generation time of RS grown in liquid culture is defined as 65 hr. Since RS cells do not discharge spores, this time was selected because it represents the point at which no further change in morphology and degree of pigmentation is microscopically evident.

Growth pattern in the dark

In the absence of illumination, a population of Spores gave rise to 90-100% RS cells. The volume/cell increased exponentially (Fig. 20) up to about 50% of the generation time of the cell. After this, the chitinous wall quickly thickened and, simultaneously, the cell began to decrease slightly in diameter (compare photographs of 65 hr. cell and

50 hr. cell in PLATE III). Glucosamine was the only hexos- amine found in chromatograms of cell wall hydrolysates. This was true for plants grown in media containing glucose as well as those grown with galactose as the sole carbohydrate source; acid hydrolysates of the latter contained no detectable galactosamine. For a mature TW cell which weighed 1.68 x 10‘2 ug, the chitin content was 9.28 x 10‘4 ug or approximately 5% 71 of its dry weight. RS cells contained about 10% more chitin/ mg. dry wt. than TW cells. Concomitantly with increased chitin synthesis, a melanin-like pigment was deposited in the wall; its absorption spectrum was linear between 400 and

600 mu with a slope of 0.00298. This compared well with the

slope of 0.00285 for B. emersonii (Cantino and Horenstein,

awn-«Iii? . 1955) and 0.0027 for Neurospora crassa (Schaeffer, 1955).

At maturity, the cell was a typical blastocladiaceous, brown, pitted, resistant sporangium. The 2 hr. delay before an increase in volume began (Fig. 20) is a real lag; it involves, among other things, a brief swimming stage, followed by loss of the Spores' single flagellum. The growth pattern shown in Fig. 20 was obtained with population densities of 2 x 104 to 9 x 104 cells/ml.; outside of this range, complications may arise (vide infra).

Growthppattern in the light

In the presence of continuous illumination, only TW cells were produced. Within the population density range mentioned above for RS cells, and at a suitable pH (e.g., pH

5.6 along plateau in Fig. 21), the volume/cell appeared to increase exponentially (Fig. 22) after a lag of 2 to 5 hours.

It is worth noting that this growth rate was 40-fold greater than that of similar cells grown in the same medium, but without aeration and agitation. Although the growth rate was not affected by variations in population density (within the range shown in Fig. 22), the final volume of the mature TW cell was 72

00 45

o A t) / b 0 .7

IpsI/CELL 09’

U U

o/ :AW

VOLUME 0‘ O

L06

25 Fig.20

3:) :5 howls

PLATEAU;

b /"0 ‘—TTpI4 ss.—5I9 1

RATE

;

GROWTH

RELAnVE

Fig.21

5.5

Fig.20. Growth of RS cells in the dark. Each point is an average for 50 measurements derived from 5 experiments.

Fig.21. Effect of pH upon the growth rate of TN cells in the light. Relative rates were derived from the linear slepe of plots of log volume per cell vs. time; that obtained at pH 4.85 was set at 1.0, and other rates were related to it. 75

affected; note the arrows in Fig. 22, which indicate cell

generation time.

This phenomenon is seen more clearly in Fig. 25, where the relationship between population size and the volume of

a mature TW cell is delineated (the greater the population

size, the smaller the volume of the cell at maturity). This

appeared to be due to the fact that the generation time of the cell (but not the rate of volume change during exponential growth as in Fig. 22) was a function of population Size (Fig. 24); in general, the smaller the population size, the greater was the generation time.

This being the case, one might expect to find that the dry weight per unit volume would be constant, irrespective of the final Size of the mature TW cell. This however, was not so (Fig. 25); the greater the volume of a mature TW cell, by virtue of an extended generation time, the smaller was its dry weight per unit volume. In fact, the total dry weight of a mature TW cell tended to be constant, regardless of its final volume (TABLE III).

Thus, within the range of the population densities studied, increasing the number of plants per unit volume of medium did not affect the exponential rate at which such cells increased in volume, nor did it greatly affect the final weight of the mature cell. However, it did decrease the duration of the final enlargement stage of the cell and, therefore, the Size of the cell at maturity. 74

55 - Fig.22 o s— I as x Io4 CELLS/ML 0/ so - go ‘_ 3.5 x Io‘ CELLS/ML

4— 519on4 CELLS/ML 45 I’ 4 +— 9.9 XIO CELLS/ML o . A .1 O In 40' / 0 .~ \ 7‘ 3 0 LI.)1 / 3 35 " °

°> / o (D O. 3,, , / o/°

2.5 I-

20 L a A J A

0 IO 20 so so so

”Ob-s

Fig.23 O 0

U 2 '7 5 o b 2 III 0 0 .— ‘ o .J .1 0 \ 3 o ”i .. 45-

“I g 0 o.I > g o .I

b ‘0 l l I n . L .

o s x Io‘ I05 Is no" ZXIO‘ POPULATION DENSITY (CELLS/ML)

Fig.22. Effect of pOpulation density upon the growth of TN cells. Within the range shown, increases in population density extended the generation time (see arrows) of the cell but not the rate at which it increased in volume during exponential growth.

Fig.23. Effect of pOpulation density upon the volume of TN cells at generation time. voL./cELL AT oEN. TIME ()1 3 x Io3I - GENERATION TIME (HRS) 35 30. 25l- I00- so- 0

0 D

D I D b I I D . D Fig.25. Fig.24.

o Fig.24 Fig.25 l o o weight generation Effect Relationship TN a 5x I Io‘ A POPULATION cell 20 any l per of A L IIIIT/n3 at 75 A pOpulation DENSIYV time unit generation a I05 AT O\ between L I GEN. 0 0 ICCLLS/ILI of volume 25 A SINGLE Av. mac SHOWING l TN A or the density A (”4): 2 time cells. EXPTS. at Ismos on A A volume LIMITS > I08) this a and EXPTS. 50 A upon A I time. its of .- 2XI05 the the dry o

TABLE III. Relationships among volume per cell, weight per cell, and weight per unit volume of TW cells at maturity

rr I :_=

Volume per cell Weight per cell Weight per unit ( x 108 ) volume ( x 1013)

I43 am . 9m o/Ma

42,800 1.07 2.5 47,100 1.10 2.5 50,800 1.10 2.2 61,500 1.15 1.9 65,000 1.14 1.8 76,000 1.12 1.5

*Grown at 240 on the standard medium with population densities varied over the range of 2 x 104 to 9 x 104 cells/ml. to induce the differences in the final volumes per cell.

Point of no return in development

In synchronized single generation cultures of B. britan- pigg, containing between 5 x 104 and 5 x 104 cells/ml., the generation time of light-grown TW sporangia occurred at ca.

50 to 52 hr. at 240 (Fig. 24). The generation time for dark- grown RS has been defined as 65 hr. However, neither the presence nor the absence of light was required continuously throughout the entire growth period for genesis of one or the other of these morphological forms. The organism was quite plastic during the early stages of development. When a synchronized population of illuminated cells growing in the

standard medium was transferred to complete darkness before a 77 critical period in development, all cells on the photomorphogenetic pathway leading to TW cells up to that time developed into brown, pitted RS cells. If the culture was transferred to the dark environment after this period, inevitably only TW cells formed. This point of no return was rather sharp and occurred at ca. 65% of the normal generation time of the

TW plant (Fig. 26). Similarly, synchronized populations of cells growing in the dark (and thus on the way to RS plants) could be induced to develop into TW cells only if they were subjected to illumination before a point of no return (45-50% of the normal generation time of an RS cell). However, if illumination was delayed until the last possible moment—- i.e., until this point of no return was reached--the generation time of the induced TW plants was usually prolonged by a few hours; if it was delayed beyond this point, TW plants were no longer formed while RS appeared instead. Thus, on the basis of the combinations tried, there were three different regimes of light and dark which yielded only TW Sporangia and three which yielded only RS (Fig. 27). In effect, there appeared to be two non-overlapping periods (18—20 hr. and 28-50 hr.) in the life Span of B. britannica during which light exerted its sharp effect on morphogenesis.‘ Light was obligatory for TW Sporangia formation only during one or the other of these periods--or alternatively, RS formation was incumbent on the absence of light during either one of these two periods. 78

LATION

Q 0

IN 0) O

CELLS

RS. 4O

20 Fig.26

1 k

20 40 60 80 I00 _ lJGHT GELLs TRANSP To om-o- x GE" .TNI ' E AT wNIN. C DARK CELLS TRANSF.T0 LT-o-

- L . . 1"

40 50 60 AGE (HRS) Fig.2?

Fig.26. Points of no return for R8 and TN cells. Beyond these points, transfer of cells to an illuminated environment or a dark environment, reSpectively, can no longer cause the cells to revert to the alternate morphogenetic pathway.

Fig.27. Coll types resulting from exposure to different light-dark regimes. The generation times of the TwS (thin-walled Sporangia) are represented by the heavy vertical bars. (See text for details.) 79

Although the first visible signs of photomorphogenesis were not detectable microscopically until just before the generation time of a TW cell was reached, it was clear from the above reversal studies that light modified the cells of

B. britannica in some manner during an earlier stage in their ontogeny. Since a parameter associated with growth rates might have reflected these changes, a detailed search was made for possible differences in cell weights. However, in both light- and dark-grown cultures, the dry weight/cell increased exponentially at an identical rate (following the

2-5 hr. lag after spore inoculation) up to 50 hr.; i.e., the pattern was not affected by illumination (Fig. 28). These data complemented the earlier experiments (Fig. 20, 22) which indicated that the rate of exponential increase in the volume of a growing plant was also independent of light and darkness.

Glucose Uptake Capacipy

Because the aforementioned growth indices were not photo- responsive, a physiological criterion was selected for examini- nation. X-ray diffraction studies, electron microscopy, and chemical analyses (Nabel, 1959; Frey, 1950; Aronson and

Preston, 1960; Fuller, 1960) have established that the cell walls of the Blastocladiales consist predominantly of chitin.

Thus, since the glucose chain is a building block in the chitin backbone, glucose or glucose derivatives must play a role in the metabolism of Blastocladiella britannica. This reasoning

L00 some Fig.29 "30 (,ONWI C! S? 'I'IBO/lM . ’- 0 H30 I DARK 80 .n—O A80 (.Ol x or!) L0

'OTE) "j LIGHT

02 1 l I I J 1 lo 1 I

O 5 l0 IS 20 25 30 IO 20 3O 4O AGE OF CELLS (HRS) AGE OF CELLS (HRS)

Fig.28. Increase in dry wt. of synchronous cultures. The dry wt./cell increases exponentially in the light and dark at 24°. Each point is an average for at least 3 different cultures, 29 of them light-grown and 42 dark-grown. Population densities were within the ranges 3-15 X 10 cells/ml. medium.

Fig.29. The glucose uptake capacity of light-grown and dark-grown cells at different stages in ontogeny at 24°. 81 led to glucose uptake studies to determine whether or not light exerted an effect on it. Freshly harvested cells of various ages were incubated in glucose and their glucose uptake capacity (GUC) was measured. GUC is defined as the amount of glucose consumed per cell in 45 min. under the conditions Specified in the MATERIALS AND METHODS section. In striking contrast to the immunity of the cell's dry weight and volume to illumination, its GUC gig_respond. The previous history of the cell--i.e., whether it was grown in light or darkness--had a definite effect on its capacity to consume glucose under-non-growing conditions (Fig. 29). The rate of increase of GUC was slightly greater for dark-grown than light-grown cells; in both cases, it was exponentiaF up to 26 hr. (i.e., 81% of the generation time for TW plantd and 40% of the generation time for RS). At 26 hr., the GUC of a light-grown cell leveled off and then dropped precipi- tously just before its generation time. A dark-grown cell also exhibited a change in its GUC pattern, albeit a different one;

GUC continued to rise to ca. 50 hr., after which it rose at a much lower rate up to ca. 65% of the RS generation time.

Then, it too dropped sharply (Fig. 29).

Reversal of GUC

Experiments were done to ascertain if the GUC of a dark- grown cell could be reversed (i.e., reduced) to the GUC of a light-grown cell, just as morphogenesis itself could be reversed. First, cultures were grown in the dark for 18 hr. 82

(this time was selected because it corresponded to one of the photosensitive periods). These same cultures were then illuminated, and after various additional periods of growth in the light (up to 50 hr.), the cells were harvested and their GUC determined. The thesis was this: if light-depressed

GUC is a causal factor for the light-induced differentiation of a TW plant, then the effect of light on GUC Should occur before the photomorphogenetic response. The results of these experiments on GUC reversal are delineated in Fig. 50, where they are compared with the GUC values for control cultures grown continuously in either light or dark. It is apparent that with relatively short exposures to light before the point of no return for morphogenesis, values for GUC were practically the same as those of cells grown continuously in the dark for the same total period of time; but, with increasing exposures to light, GUC levels were progressively depressed and closely approached those of light-grown cells (Fig. 50). It is important to note, however (as shall be seen in retrospect), that complete reversal of GUC to the level associated with a

TW plant should not have been expected, because it turned out that 18 hr. was just a little beyond the point of no return for GUC .

The point of no return for GUC

The morphological point of no return for light-grown

TW cells and dark-grown RS has been established. The following 85 experiments-were—designed to define-the point of no return for the accumulation in the cell of GUC itself. Plants were grown in the dark for varying lengths of time and then transferred to light; after 50 hr. of growth, all of them were harvested and their GUC was determined. The results (Fig. 51) show that when cells were grown from the outset in the dark but then illuminated before 12 hr. had elapsed (40% of the generation time of a TW plant), they retained the same low

GUC characteristic of cells grown continuously in the light for 50 hr. But, once beyond 40% of the cell's generation time, increased exposures to darkness before providing the dosage of light resulted in increased levels of GUC; these values gradually approached the typically high GUC of a cell grown continuously in the dark.

While these data do not provide conclusive evidence, they are strongly suggestive that TW yg RS morphogenesis is a

*- function of GUC.

* During the course of the foregoing investigations and further testing of the notion proposed above, some provocative leads, which should be pursued, were brought to light. They seem important enough to deserve mention and are briefly re— corded in APPENDIX III. 84

Fig.30

IZOP S

a) O

I0“)

(no O) O our. .5 9

20 l 22 2‘4 ' is 28‘30 AGE or «us II-RSI AT Guc OETm

I20 " Fig.31 °

IOO

IO‘)

O) O

(no

5 O

GUC. O N

0 6 I2 I8 24 SO HRS IN DARK BEFORE TRANSFER TO LIGHT 0 20 4'0 60 so loo 1 OF T.W. GENERATION TIME

Fig.30. The reversal of glucose uptake capacity. Following growth in the dark for 18 hr., plants were illuminated and after various additional periods of growth in the light, their CUC was determined. The results obtained are compared to controls which were grown exclusively in the 1ight(L) or in the dark(D).

Fig.31. The point of no return for glucose uptake capacity. Cells were grown in the dark for different periods of time and then transferred to light; after a total growth period of 30 hr., their GUC was determined.

DISCUSSION

The Taxonomic Position of Blastocladiella britannica

Willoughby (1959) suspected that his isolate of Blasto-

cladiella, on general morphological grounds, was more closely

related to B,.Simplex Matthews (1957) and B, laevisperma

Couch and Whiffen (1942) than to other species of Blasto- cladiella (cf. Sparrow, 1960) but that, on the basis of spore

size, it more nearly resembled B. emersonii (Cantino and Hyatt,

1955c). With Willoughby's observations and my own experiments in mind, most other described species of Blastocladiella

do seem to be sufficiently different as not to merit further

attention for taxonomic purposes; but B. stuebenii Couch and Whiffen (Stﬁben, 1959; Couch and Whiffen, 1942), whose super-

ficial morphology is similar to that of B. britannica, does

deserve consideration.

Inasmuch as I have assumed the responsibility for pro-

claiming B. britannica a new species, I feel compelled to

testify in favor of its well-defined character on the basis

of the additional parameters that have resulted from my work; thus, Willoughby's (1959) conclusions will be used as a convenient point of departure.

First of all, additional conclusive evidence that an

unmistakable distinction exists between B. emersonii and

85 86

B. simplex, on the one hand, and B. britannica on the other,

can now be provided.

1) When grown submerged in liquid culture or beneath the surface of solid media (PYG, 220) both B. emersonii and

B. simplex always produce long, cylindrical basal cells bearing a terminal rhizoidal system (PLATE I,a). Furthermore, even on the surface of PYG agar, shorter but distinct stalks

are also always produced. Under identical conditions

B. britannica never produces a detectable stalk or basal cell;

instead, in the fully grown plant, the rhizoids are attached

at numerous points on the spherical thallus (PLATE I,b) .

These comparative observations alone, made under controlled

and reproducible conditions, provide sufficient morphological evidence that B. britannica is not identical with B. emersonii

or B. simplex.

2) In addition, under these same laboratory conditions, the resistant sporangia of B. emersonii and B. simplex

germinate with ease; those of B. britannica do not.

5) Finally, and again under controlled conditions where populations have ample 'lebensraum', both E. emersonii and

B. simplex always liberate spores via pores, each resulting

from deliquescence of a papilla, while B. britannica always does so through obvious exit tubes of different lengths

(Plate I,c).

Having eliminated B. emersonii and B. Simplex from

consideration, there remain only B. laevisperma and

B. stuebenii. Here, it was necessary to resort to a comparison of personal observations of B. britannica with the data available in the literature about B. laevisperma and

B. stuebenii, as follows:

1) The living Spores of B. britannica average 4.5-6 x

7.5-11 u (Willoughby, 1959); fixed preparations are not

Significantly different (av. 7.5 x 9.4 u). Even the spores of the substrain of TW colonies, which are distinctly smaller than those of the parental 'wild type', measure 6 x 7.7 u.

On the other hand, the spores of B. laevisperma (Couch and

Whiffen, 1942) are 5.8-4.6 x 6-6.5 u, and those of B. stuebenii

(Stﬁben, 1959) average 5.5 x 4.8 u.

2) The resistent sporangia of B. laevisperma (Couch and

Whiffen, 1942) and B. stuebenii (Stﬁben, 1959) have no pits;

those of B. britannica always bear very small pits (ca. 1 u

diam.; of PLATE I,d,e).

5) The resistant sporangia of B. laevigperma are reported to germinate after a short 'rest period' (Couch and

Whiffen, 1942). The resistant sporangia of B. stuebenii, when grown on dilute peptone media (Stﬁben, 1959), whereon the thickness of the wall was minimized, also germinated with regularity. 'The resistant sporangia of B. britannica are not produced on such dilute peptone media. But, under the various conditions under which resistant Sporangia have been induced to form, germination was never obtained. 88

Thus, B. laevigperma must be eliminated from consider-

ation.

However, in its spherical nature, its profuse rhizoidal system, and its development from an enlarged reproductive rudiment derived from the swollen body of a spore, B. steubenii does display some characteristics in common with B. britannica.

A few more distinguishing criteria of a general physiological nature, therefore, will help to rule it out of the picture.

All references to B. stuebenii are taken from Stﬁben (1959).

1) The optimum temperature for growth of B. stuebenii on peptone (and other) media is 540, whereas that for

B. britannica is 16-260 on peptone media; indeed, growth does

not occur above 520 under any condition.

2) Under crowded conditions on agar media, B. stuebenii produces colonies in which plants tend to become distinctly elongated; furthermore, in such crowded conditions, resistant sporangia are produced in the centers of the densely populated colonies while thin—walled plants are produced on the periphery; B. britannica, under these sorts of conditions,

behaves in diametrically opposite fashion; the thalli never elongate, resistant sporangia are produced round the edges of thickly populated colonies, and thin-walled plants are produced in the center.

5) Finally, colonies of B. britannica on peptone media

gradually accumulate an unmistakable yellow color, due (at least in part) to synthesis of carotene; Stﬁben never reported 89

the appearance of such a pigment in colonies of B. stuebenii.

It seems justifiable, therefore, to designate this

fungus a new Species. The diagnosis follows.

Blastocladiella britannica sp. nov.

Thallus sporangium parietibus tenuibus colore subflavo (carotene), aut parietibus crassis brunneum puteis praeditum perdurans. In culture liquida vel solida et nutriente sporangium semper sphaericum, sine stipite et cum patente compositione rhizoidali in locis multis thalli surgente. In officina in cultura pura thalli sporangiales perdurantes 98-140 u diametro, pariete 4.2 u. Genesis sporangiorum perdurantium par NaHCOa non creantur- Germinatio sporangiorum perdurantium et sexus non cognoscitur. Thalli parietibus tenuibus 80- 180 u diametro sporas posterius uniflagellatas, typice blastocladeaceas, 7.5 x 9.4 u per paucos tubos emittunt. Flagellum 20 u longitudine. In basi solide progenies ex sporis maxime varia potestate crescendi, alios thallos parietibus tenuibus qui sporas in situ generent, alios thallos parietibus tenuibus qui sporas solum in aqua generant, alios thallos sporangiales perdurantes ferens. Evolutio praeter morem inclinata ad effectum lucis manifestae. Luce absente omnes fere sporae thallos sporangiales perdurantes generant, negue thallos parietibus tenuibus. Collata et discreta per L. G. Willoughby ex Aqua Esthwaitensi (Regio Lacuum), Angliae, 1958; typus est Herb I. M. I. No. 84599.

Thallus a thin-walled sporangium with light yellow pigmentation (carotene) or a thick-walled, brown, minutely pitted, resistant sporangium. In liquid or solid nutrient media, always spherical, without stalk, and with extensive rhizoidal system arising at many points on thallus. Under controlled conditions in pure culture, resistant sporangial plants 98—140 u diameter, with wall 4.2 u thick. Genesis of resistant Sporangia not induced by bicarbonate.

Germination of resistant sporangia and sexuality not known. 90

Thin-walled plants 80-180 u diameter, liberating posteriorly uniflagellate, typically blastocladiaceous spores, 7.5 x

9.4 u, through several tubes. Flagellum 20 u long. On solid substrata, progeny from spores extremely variable in developmental potential, yielding thin-walled plants liberating spores in situ, thin-walled plants liberating spores only when placed in water, and resistant sporangial plants.

Development extraordinarily susceptible to influence of visible light. In absence of light, almost all Spores produce resistant sporangial plants instead of thin-walled plants.

Type. Collected and isolated by L. G. Willoughby from

Esthwaite Water (Lake District), Great Britain, 1958.

Herb. I. M. I. No. 84599 (slide and preserved material).

The Aquatic Phycomycetes and Biological Research

Foster (1949), in the introduction to his book, pointed out that up until thirty or forty years ago, the study of fungi had largely been relegated to the plant pathologist who was interested in these organisms solely from a practical point of view. Since that time, the higher fungi have found a secure place in the laboratory where they are playing a number of productive roles. Because of their many and diverse metabolic patterns, they have served the biochemist admirably in his efforts to extend and complete some of the dead end roads on metabolic maps as well as to provide new and alternate routes. 91

There were those who recognized that certain fungi, because of their particular methods of reproduction and the ease with which they could be handled, had much to contribute to a better understanding of genetic mechanisms.

From among the fruits of their efforts came the inception of a new approach to the study of genetics, the biochemical approach. The classic series of experiments by Beadle and

Tatum with Neurospora provided impetus for the current wide-

spread interest in microbial genetics; this, in turn, has led to much insight into the primary action of genetic material.

On the other hand, the ubiquitous aquatic Phycomycetes are still, for the most part, an undomesticated group of organisms whose special attributes have yet to be exploited.

Their diversity as well as their many perversities are among the reasons they have not yet found themselves a solid branch on the phylogenetic tree. Some of them resemble certain algal forms while others are much like some protozoan flagellates. Considered from almost any point of view, they compose a heterogeneous assemblage.

Morphologically, the thallus may be a very small structure (in some, less than 10 u in diameter) whose entire mass is converted into a reproductive body. At the opposite morphological extreme are organisms whose vegetative form is characterized by an indeterminate system of growth, pre— sumably limited only by the availability of nutrients. 92

Probably in no other group of organisms does one find such an array of sexual reproductive methods. Although this means of propogation is not universal, among those species in which it does take place it may involve the fusion of two hyphal tips, fusion of motile gametes (either isogametes or anisogametes), or the penetration of a non-motile gamete by a motile gamete, not to mention variations on these basic patterns.

From a nutritional point of View, these fungi also display great differences in synthetic capacities. For example, only some of the Chytridiales are able to utilize nitrate as the sole nitrogen source while all of them are able to use ammonia. Among the Blastocladiales, the capacity to use nitrate has been lost completely; whereas most of them can use ammonia, others must be supplied organic forms.

These are just a few of the areas in which aquatic fungi harbor countless opportunities for biologists who have questions to ask. Their suitability as experimental tools cannot be overemphasized. To use a specific example,

I would like to extol the beauty of Blastocladiella britannica as a tool for developmental studies, disregarding for the moment the particular nature of its morphogenetic behavior.

It is, at one and the same time, a single cell as well as a complete organism; in it, differentiation occurs in the absence of cell division. 95

Techniques were worked out whereby mass quantities of synchronously developing, single generation plants are easily cultured under controlled environmental conditions and without the imposition of a synchronizing agent. Such cultures permit a breakdown of the growth cycle into numerous subdivisions. All of the plants harvested at any one time during the course of the life cycle are the same size and at the same morphological stage of development; these characteristics undoubtedly reflect a high degree of synchrony of their physiological and biochemical machinery.

This contrasts to most other microbial systems in which analyses have been limited to a cross-section of variously- aged cell types; under these circumstances, any data collected represent the physiological stage of the average cell in a mixed population, not an individual cell.

Scherbaum (1960) suggests that this is the primary reason why so little is known of the biochemistry of microbial life cycles. The techniques developed for B. britannica permit the study of the differentiation process as it is taking place. It becomes possible to follow the course of the appearance and disappearance of an enzyme, turnover of various intracellular components and pools, changes in metabolic pathways and finally, a correlation of these events with morphological changes--hopefully in terms of cause and effect. Because the number of plants included in any one analysis is always known and because the cells are all 94 identical, data can be related to a single plant. This obviates the necessity to base results on dry weight or soluble protein content--characteristics which, in themselves, are constantly changing at different rates. Many of the data for B. britannica (and B. emersonii), and their

significance, could not have been obtained by any other means. In addition, because B. britannica is practically a

perfect sphere during all of its life cycle (following the swarmer stage), even more precise measurements can be made, such as those based on a single unit of volume or surface area. Such measurements could have heuristic value in assigning a locale to certain activities of the organism.

In Spite of these virtues, to date only B. emersonii

(McCurdy and Cantino, 1960; Lovett and Cantino, 1960b;

Goldstein and Cantino, 1962), Rhizophlyctis rosea (Smith

and Lovett, unpublished), and B. britannica (present work)

of the ubiquitous, non-filamentous water molds have been grown synchronously in sufficient quantity to permit develop— mental analysis in chemical terms. This is regrettable, for any information derived from organisms at this organi- zational level could contribute to a better understanding of the more intricate morphogenetic mechanisms at a higher level of complexity. To quote Edds (1958):

. . . if we have no adequate chemical or physical explanations for the 'Simpler' unitary events of morphogenesis at the cellular level . . . then it is for the moment unreasonable to hope, and scien— tifically unprofitable to seek, for such explanations 95

of morphogenesis at the larger and more complex levels of tissues and organs. Previous experience forces the recognition that the most rapid progress will be made in any physico-chemical analysis if the simplest possible morphogenetic systems are selected and then subjected to intensive exploration.

Morphogenesis in Blastocladiella britannica

Morphological variability

For experimental studies of morphogenesis, we could not have been provided with a more handsome little organism than Blastocladiella britannica. No other species in the

Blastocladiales which has been subjected to rigorous examination displays such a high degree of morphological variability-~a variability apparently under phenotypic control (sensu Emerson, 1950). Three principal observations

lead to this presumption: first, true gametic copulation has never been detected, thus supposedly precluding segre- gation of causative factors via meiosis; second, and perhaps more convincing, a population of uninucleate Spores, each originating from a single plant (which, in turn, is derived

from a single uninucleate spore), can exhibit a wide gamut of diversity*; and third, this developmental potential is strikingly modified by environmental conditions such as nutrients, temperature, and in particular, light.

*This observation echoes the dilemna confronting those who are attempting to explain morphogenesis at more complex levels of organization; i.e., how can cells with presumably identical nuclear inheritance be so different in structure and function? 96

Disregarding polyploidy as a cause, it is possible,

therefore, that either organized control centers or other

less Specialized devices may be responsible for this morpho-

logical variation, and that they are seated in the cyto-

plasm of the cell; furthermore, it seems a good guess that,

following the final cleavage of the protoplast into hundreds

of spores, this machinery is distributed in random fashion resulting in a swarmer population which varies over a wide range in its content of these hypothetical cytoplasmic

factors. The data in Fig. 17 lend credence to this hypothe-

sis, for they clearly illustrate that these plants were

selected from that portion of the population in which these

cytoplasmic control devices, whatever their exact nature, were present in very high or very low concentration;

selection of the correct alternative would be contingent on whether the potentiality for formation of RS is associated with a relative abundance or relative lack of these hypo~ thetical factors.

Finally, if this concept is tentatively accepted, then, it would follow that the activity and/or reproductive rate of these components could be influenced by external

conditions, and the most impressive effect so far demon-

strable would be that exerted by light (as depicted in

Fig. 15); in this instance, the population possessed a high

capacity for formation of RS plants in the dark, and this

capacity was almost completely nullified by exposure to

light. 97

The concept of cytoplasmic control however, is not unique to this particular fungus, nor is it by any means a new concept. Over a period of many years, the role of the cytoplasm in the determination of the ultimate fate of many organisms has been repeatedly demonstrated. According to Markert (1964), in a discussion of the role of chromosomes in development, only one of the various explanations currently being propounded for the diversification and specialization of cells during development seems to be the likely one, and that is that the cytoplasm which is allocated to daughter cells during division is qualitatively different.

At the moment, this notion regarding B. britannica is founded on tenuous correlations and indirect evidence, but ample precedence has accumulated for assigning such vital regulatory powers to the cytoplasmic components of the cells in the Blastocladiaceae. In B. emersonii, Allomyces arbuscula, and A. macrogynus (see Cantino and Turian, 1959

for discussion), the distribution of cytoplasmic particles, whose concentration in the cell is subject to environmental modification, has been correlated with biosynthesis of y-carotene and, concomitantly, the capacity for morphological and sexual differentiation.

Photomorphogenesis

Mohr (1962) has defined photomorphogenesis as "the control which can be exerted by visible light over growth, 98

development, and differentiation of a plant, independent of photosynthesis.“ I chose to investigate the nature of photomorphogenesis in the RS strain of B. britannica in physiological terms, thus hoping to provide a basis for the morphological events which occur under alternate conditions

of illumination.

In RS cultures of the close relative B. emersonii,

there are two immediate consequences related to the presence of the RS inducer, bicarbonate: a rapid decline in oxygen consumption and a sharp reduction in the growth rate (as measured by both dry weight and volume increases). The lowered growth rate persists throughout the entire exponential growth period, although the mature RS is considerably larger than the mature OC plant. In contrast, darkness (the RS inducer) does not at all influence the rate of growth nor the final size attained by RS of B. britannica (PLATE III, also, compare Fig. 20 with Fig. 22)l In fact, there is no mopphological parameter that can be used to distinguish between a developing RSlplant and a developing TW plant until the TW generation time has been reached. It appears, then, that the two different inducers responsible for RS formation do not trigger the same mechanisms, at least in terms of early observable events. It therefore becomes necessary to look elsewhere for the primary events incited by light.

9(- 0xygen consumption data are not available for B. britannica.

Comparative descriptions of RS and TW cells have disclosed that, in addition to structural differences, RS

cells also have an extended generation time (65 hr., _§

50 hr. for the TW cell). This means that formation and discharge of spores from RS plants must be delayed. In

fact, this is a sine qua non for RS morphogenesis; RS dif-

ferentiation, at the morphological level, is not initiated until afpgp 50 hr. Therefore, sporogenesis by TW cells

and morphogenesis of RS are not concomitant events.

However, the metabolic systems which actuate these processes undoubtedly operate long before their morphological mani~

festations and may very well be associated with one another.

Relative to this, the results obtained by exposure of

B. britannica to various light-dark regimes provided grist

for conjecture (Fig. 27). By eliminating light prior to

18 hr., TW plants are induced to differentiate into RS cells; however, TW plants subjected to light for more than 20 hr. can no longer be so reversed morphologically. Under these particular light-dark regimes, the mechanisms responsible

for light-induced sporogenesis become irreversible between

18~20 hr. When the opposite light regime is imposed-~i.e., a period of darkness preceding light--the point of no return

for RS differentiation occurs between 28-50 hr. Plants growing in the dark up to 28 hr. can be made to follow the

TW pathway by transfer to the light; however, after 50 hr.

in the dark they are committed to the RS pathway. 100

Since photo-induced sporogenesis and dark-induced RS

morphogenesis are allied, the relationship should be

traceable to a-common metabolic system. Moreover, some

portion of this system Should display photosensitivity;

the glucose uptake capacity of the cell did sol The de-

pressing effect of light on GUC was evident before the photomorphogenetic response. This would be expected if sporo-

genesis was dependent on light-suppressed GUC. This pre-

sumption was reinforced by the results of attempts to reverse GUC. Not only was it possible to reverse the GUC

of dark-grown plants so that it very closely approached the

GUC level associated with plants grown in the presence of

illumination, but the point at which this reversal could

no longer be completely effected preceded the morphological point of no return.

The data provided by these experiments do not, of

course, furnish proof that a cause-and-effect relationship

exists between the magnitude of GUC and the selection of

one of the alternate morphogenetic pathways. However, the data are consistent with the present hypothesis: namely,

(a), that although there is considerable latitude in the

GUC level which permits a cell to develop into a TW

sporangium, a significantly higher minimum level of GUC must be attained by a cell before it can develop into an

RS; and (b), that visible light, in some fashion, controls this level of GUC. SUMMARY

1. The developmental history of a single spore isolate

of a Blastocladiella was investigated in pure culture.

Its wide range of morphological variability led to the

isolation of several distinct substrains, one of which

produced resistant sporangia in abundance. In the

latter, formation of resistant sporangia was completely

inhibited by white light. The fungus was designated a

new species, B. britannica.

A method was evolved for growing synchronous, single

generations of B. britannica. In such cultures, the

all-or-none effect of light and dark upon differentiation

of thin-walled cells and resistant sporangial cells,

respectively, was demonstrated, and the point of no

return for both morphological pathways defined.

The glucose uptake capacity of dark-grown cells was

Shown to exceed that of light-grown cells. Photo-

sensitive glucose uptake may be a factor in the deter-

mination of the ultimate morphology of B. britannica.

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Markert, C. L. (1964). The role of chromosomes in development. 25rd Symp. Soc. Study Dev. Growth. pp. 1-9. Academic Press,-New York. Matthews, V. D. (1957). A new genus of the Blastocladiaceae. J. of the Elisha Mitchell~sci. Soc. 55: 191-195.

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108 APPENDIX I

List of Abbreviations ribonucleic acid deoxyribonucleic acid triphosphopyridine nucleotide (NADP) diphosphopyridine nucleotide (NAD) resistant sporangium ordinary colorless (refers to the thin- walled sporangium of Blastocladiella emersonii)

thin-walled (equivalent to OC of

B. emersonii)

glucose uptake capacity

109 110

incubated 220 22

220

Continued at at sets

at 220 and 16

at incubated incubated

incubated Conditions replicate

_ .7

ml. 52 TW

10 H20 and incubated

with which into GERMINATION 11, in discharged RS RS sets 2, H20 H20 H20 AT

plates had for then

APPENDIX into into into 1

flooded at days, H20 plants RS

RS Treatment RS replicate ATTEMPTS

—————————————————————————————————————————————————————————— ______

______

and old

days

28,

old and

old

hr.

151,

26,

89 days

and

plates

days old

128, plates old

plates plates plates

124,

and

6: 57 PYG

11, History

PYG

and

124, days days

102,

and

from from

5, 75,

5, 5

81, 55,

old

179

154 5:

HRS 111

at at

agar

22° 220

hr.

160 160 in

sets:

220 20 H20 of Washed

added

sets;

then then

for at of replicate incubated

plants Continued

block

set set dish. wash,

vol.

min., min.,

and

replicate

20 50 10—20 small

22°. duplicate ‘m- -... final Petri intervals H20 Incubated .‘h large

50°- 50°—

500+

50°—

22° 16° hr. -L—n-.rn~__a.~

fresh 6)

small 5)

set

Set

4) 2)

Placed

5) Incubated Incubated

16°.

Conditions Incubated 1 1) bearing After

with p--—-—-—————----—-—-

for

H20

into

old old

H20

incubated

days

into

days

then

Plates

Treatment RS

when

90.

months

age

11 15

12 15

14 treated

(continued)

82,

old

plates plates

72,

old old

days

PYG

PYG History

58,

when

days

from

from 25 27

age 59 42

and

57 desiccated 52,

APPENDIX

112

—1

Petri

min.

and

bearing

After

small

H20

agar

220

220 220

22°.

220. into Continued

added

at at

block

Washed

plants

wash,

final

incubated

intervals ————————————————————————————

Placed

Incubated Incubated Incubated

dish. Conditions 12-20 )—

for buffer

6.5

10‘4, solution

10"2 10-3,

H20 pH 6.6 7.7 phosphate

at 10'7

into 10's,

old old

(molarity)

10-2,

HCl bicarbonate M715

incubated

5 10‘s,

days

into into

into 1orl,

days

48 80 8.0 8.5

then (molarity)

Plates 10‘s,

1, 10‘s, RS

Treatment RS RS ———————————————————————————————

-1

ﬂ—-—————-—-—-————————————————-—dr——-—-——-———————-———-—----—4

for

old;

old

temp. days

(continued)

day

25 month

old old

room

filter

8 II plates plates

plates

= old

when

and

and PYG PYG

PYG

days

days days days

History

5 4

days

days 57 18

———————————————————————— from

from 64

from stored

—————————————————————————

41 1,5,

APPENDIX - Dried

[paper

115

4 16°.

at: add-

H20;

with at 22°. at

H20.

sets set

with

plate

min.,

washed, Continued

with

22°

Washed

stand

22° incubated

duplicate duplicate

surface surface

let

and and

off. treatment,

16 H20

flooded

Conditions ether; ed

Incubated

Incubated Incubated

Covered After Incubated

poured ‘

+-—-————————————————-—-———--———

solu- 5, ether

ether 2,

solution

.0001% .0001%

solution 1,

extract

20 £3

0.001%

0.0001% 0.00001% 0.000001%

.001,

.001, for

yeast

Tween

peptone petroleum

petroleum min.

.01,

.01, 10

into into into

into into

.1,

0.1% 0.01%

tion

1%. and Treatment

RS 10%

RS treated

RS RS

RS L_____---_---__-___-______n__-___-_-_-__-__---_-____----_-

old

days

then days

stored

days 45

paper

old then

old

for =

incubated

old;

days, days

days (continued)

incubated incubated

32 old

days

old old

days

filter II days

days,

plates plates

plates

days temp.

old,

for

old, old,

for

on 52 58

PYG PYG

History PYG

days days days

220

22°

1 days

10 1

room

69 75

days RS days

from

from from

5 at at

at at

——————————————————————————————————————————————————————— ______at

Dried 57 5 APPENDIX

When

114

and

22°;

H20

H201 22°.

5xé

wash—

at 20,

hr., at

added added

incubated

hr.,

10,

Continued

22°. 22°;”

chitinase, 1

Washed

placed

and

for

placed 220

hr.

washed, washed,

added

incubated

Tween

600

set:

at H20,

1 then

hr.

hr. at

5x;

5 5

H20;

added

incubated

22°.

min.,

after

and

and 50

added

ed,

duplicate

Conditions

Incubated incubated

Heated After

Treated

washed

and fungal solutions

solution solution

(Streptomyces)

NaOCl NaOCl

Tween

into into

into eatment

different

.6% .06% fungal 2

chitinase

chitinase chitinase Tr

10% RS RS RS ______

88,

old

days

when

78,

old

temp. days

age

old;

treafed

152 114

152 116

(continued) 64,

plants

days

old

room

plates plates

58, 98

and

days

PYG

days History PYG

and 55,

55,

days

when

stored

from

age

96,

Desiccated

desiccated

APPENDIX 41,

41, when

)

115

hr.

for

at 22 at

1 1

40,

set set

5x,

H20. ether

20,

H20.

After

temp.

washed, with

for

incubated

22°. pet.

washed

room

added 5x

Incubated

60°

and

duplicate

removed, with

min., chitinase.

H20.

H20 washed

washed,

min.; added

and added

Incubated

22°.

hr.

16°.

Treated

Incubated

Incubated Heated Incubated

16°.

Conditions then Added

NaOCl

solution

ether

NaOCl

.6%

0.6%

.06%

into

and

chitinase

petroleum

Treatment RS RS

______

45 45

old

days when

for for days

old old

85 85 86

O age

1 1 100

treated 100

(continued)

96 plants

at at days

days

planSs plates

and 80

48 PYG

= =

days History

55, when

day-old 4 62 25 27 59

day-old

from

age 41, incubated 57

days incubated

days desiccated

APPENDIX

APPENDIX III

Ballons d'essai

GUC and its relation to certain cellular components

A few years ago, there was found in Blastocladiella

emersonii a considerable quantity of an ethanol-insoluble, glycogen-like polysaccharide which yielded only glucose upon acid hydrolysis (Cantino and Goldstein, 1961).

B. britannica possesses a similar polysaccharide. For this reason, in one of the GUC experiments plants were analyzed for intracellular polysaccharide as well as free glucose, both before and after incubating in glucose. As part of the same experiment, the spent media in which the cells were grown were analyzed for glucose disappearance during growth. The results are tabulated in Table IV. The data are suggestive but until the results of later experiments with C14-labeled glucose have been scrutinized, no un- equivocal conclusions can be drawn. However, some of the suggestions calling for further consideration are as follows:

1) B. britannica does not appear to accumulate a

large free glucose pool, either during growth with glucose as the carbohydrate source or during incubation of pre- grown cells in a glucose solution. The increase in free glucose following GUC experiments accounted for only 0.5-

5% of the glucose consumed.

116 117

hr.

.0441

.0552 6.67

7.06 5.50

-5.5% 55% 50

118.9

where

plants

hr.

.0515

.0158 5.0 5.54

5.17 57.7%

except

47.9 127%

104,

Dark—grown

hr.

.0595

1.17 1.85 46.4%

22 51.2

406%

hr. .171

.0517

5.85 5.52 9.6%

5.96

75.0 50 values

459% ants

all hr.

.0567 .0568

2.58

2.79 5.08

54.8%

50.5 26 10.4%

basis; pools

nght—growngpl

hr. .969 .0247

per-cell 9.2%

1.75

a 22

45.5% 55.6

expressed.

from intracellular

are is

growth

and

pool

free

poly-

pool

glucose

GUC consumed

values

during

plants

glucose

incubation

glucose percentage

of IV. glucose

glucose All

saccharide

Polysaccharide

Increase Free Beforegglucose

Increase Free

GUC

After Age Glucose

medium TABLE

118

2) The polysaccharide content of B. britannica

accounts for a substantial portion of the dry weight of the

fungus. This level does not remain constant throughout ontogeny; it might be related to GUC and, by extension, to photomorphogenesis.

Effect of phosphate on GUC

Another aspect that deserves in-depth investigation

involves the function of inorganic phosphorous in glucose uptake. Certain experiments intimated that it might play

a role of primary importance in the mechanism of glucose consumption by B. britannica. In these studies, some cells

were grown in the usual manner in PYG broth made up in citrate-phosphate buffer, pH 5.6; other cells were grown in PYG broth minus the phosphate, where the pH was adjusted with NaOH. Growth was equivalent in both media. With such cells GUC studies were done in the citrate-phOSphate buffer

and in citrate minus the phosphate. The results of two of these experiments, in which 29 hr. old dark-grown cells were used, are shown in Table V. The presence of inorganic phosphate during growth enhanced GUC when these cells were placed in a non-growing situation. Inclusion of phosphate during GUC incubations augmented this effect. In what capacity this influence was exerted on GUC remains to be

fathomed. 119

TABLE V. Effect of phosphate on GUC

GUC (ug x 105) Grown in PYG + Incubated in glucose + Exp.#1 Exp.#2

citrate citrate 47 56.5

citrate citrate-phosphate 59 79

citrate-phosphate citrate 69 54

citrate-phosphate citrate-phosphate 88 105

Effect of different sugars on GUC

Effect during_growth. It has already been seen that

certain conditions during growth affected the GUC of

B. britannica. One of these conditions was the presence or absence of illumination; another was the presence or

absence of inorganic phosphorous. What effect, if any, would qualitative and/or quantitative variations in the

carbohydrate source during growth have on the fungus'

capacity to consume glucose under non-growing conditions?

A first attempt to answer this question indicated that the

effects were considerable (TABLE VI). In addition, galactose and glucosamine markedly reduced the size of the

cells (TABLE VI). Although the plants grown in the presence of galactose had a normal appearance, those cells grown in glucosamine appeared very abnormal. This latter TABLE VI. Effect on GUC of different sugars during growth

Carbohydrate supplied Surface area GUC/u2 Polys./cell Polys./u3 during growth of plant (u (ug x 105) (ug x 10°) (ug x 105) (ug x 108)

0.5% glucose (control) 4640 25.9 5.2 57.1

0.05% glucose 5711 18.9 5.5 40.8 1.0

0.5% galactose 2415 5.76 1.6 9.2 0.85 120

0.55% glucosamine 1760 0.86 0.5

28 hr. old dark-grown plants were used in above experiment. 121 observation could well account for the very low GUC displayed by these cells. Plants grown in the presence of

1/10 the standard glucose concentration were slightly larger than the control cells but otherwise had a normal appearance. Because of the difference in size of the plants grown with different sugars, GUC was also calculated on a surface area basis (TABLE VI). This indicated that the cells which grew with galactose as the sugar source did not have their GUC impaired as much as it seemed at first glance.

The values obtained from polysaccharide analyses of these cells following incubation in glucose are also listed in TABLE VI.

Effect during GUC incubations. When an equimolar

amount of galactose, glucosamine, of onmethyl-D-glucoside was added simultaneously with glucose to a suspension of

28 hr. old dark-grown plants, there was no inhibition of

GUC during the course of a one hr. incubation period

(TABLE VII). In fact, there was a ca. 20% stimulation of GUC by oﬁmethyl-D-glucoside. This observation is not without precedent; Rogers and Yu (1962) reported that certain non- inhibitors of galactose uptake in Escherichia coli strain

A increased its uptake by as much as 15%.

However, when B. britannica was preincubated in the

presence of glucosamine, there was complete inhibition of

GUC. Smith (1960), in describing hexokinase inhibition in 122

105) in

x have

0 (ug 20.4

19.4 14.6 18.1

15.6 11.4

15.6

tubes

of time; GUC

seems

hr.

set

one

refrigerator

last

ca. the

glucose

sugars

run

for the

ml.

incubations

hr.

were

other time

glucose

1.0

stored

GUC

stored of

-)(- the

ml. for

added

were

by been

1.0

reactions

before

dark

had presence

min. that used.

orCHg-D—glucoside at

added

50 the

Only the

glucosamine* galactose

cells

suspensions

of means ml. were ml.

ml.

min.

after

cells GUC.

50 GUC 1.0

mixture

cell

grown these 1.0

1.0 This

sugar on

+ +

the their

after been

each pre-grown

reaction Effect

of had

glucose glucose

glucose glucose glucose glucose H20, glucosamine, incubated,

in meantime. EM ml. ml. ml.

ml. ml. ml.

ml. ml.

Storing

the

therefore, diminished

VII.

Cells was

* *- ' * Sugars 1.0 1.0

1.0 1.0 1.0 1.0

1.0

1.0 TABLE

125

Spirochaeta recurrentis, noted that glucosamine inhibited

enzyme activity and furthermore, for maximum inhibition,

it had to be added before the glucose.

It would be out of place to draw any conclusions on

the basis of these few experiments, but this does seem to be an area which might prove fruitful upon intensive in-

vestigation.