Morphogenesis and the role of microtubules in synchronous populations of Saprolegnia zoospores

~EXPEI~IiV[ENTALMYCOLOGYi, 9-29 (1977)

Morphogenesis and the Role of Microtubules in Synchronous Populations of Saprolegnia Zoospores SHIRLEY

A. HOLLOWAY

AND I. ]~RENT HEATH

Department of Biology, Yor]~ University, ~700 Keele Street, Downsview, Toronto, Ontario, Canada M3J 1P3 Received June 9, 1976

]-~OLLOWAY,S. A., AND HEATH, I. B., 1977. Morphogenesis and the role of microtubules in synchronous populations of Saprolegnia zoospores. Experime, tal Mycology 1, 9-29. Synchronous populations of secondary zoospores of an isolate of the Oomycete Saprolegnia (probably S.ferax) were obtained with a temperature-shock treatment. Light microscopy and serial section ultrastructural analysis of these, and the primary zoospores, provided a detailed description of their microtubular systems and the changes in these systems during the morphogenic processes which accompanied secondary zoospore exeystment and encystment. In both zoospore types there was a correlation between the arrangement of the cytoplasmic microtubule systems and zoospore morphology, but the asymmetrical arrangement of microtubules in the symmetrical secondary zoospores showed that morphogenesis is complex. Because there was a close temporal correlation between the presence of maturity in both cytoplasmic microtubule arrangement and secondary zoospore shape, the microtubules are probably the major morphogenie agents. There were two locationally and morphologically distinct sets of microtubules in the primary zoospores and seven sets in the secondary zoospores. Because the sets were synthesized and broken down at different times the spores must possess a complex system for control of microtubule polymerization, a simple monomei~polymer-common pool of subunits system is inadequate. Flagella were synthesized at rates up to 2 gm/min. Their breakdown in primary zoospores and cysts involved loss of functional interdoublet and central pair links followed by fragT~entation prior to ultimate disappearance. INDEX DESCRIPTORS : microtubules; morphogenesis; zoospores; flagella; oomycetes; cell synchronization ; Saprolegnia. I n numerous cell t y p e s various developmental processes and shape changes are correlated with c o n c o m i t a n t changes in arrays of microtubules. While a considerable a m o u n t of information has been acc u m u l a t e d on the control of microtubule polymerization in vitro, v e r y little is k n o w n a b o u t the control processes inside a cell (see reviews of Tilney, 1971; Olmsted a n d Borisy, 1973; Hepler and Palevitz, 1974; and Snyder and 3~[cIntosh, 1976). During its asexual life cycle the aquatic Oomycete, Saprolegnia, p r e d i c t a b l y goes t h r o u g h a sequence of cell shape changes during which p y r i f o r m and reniform zoospores are gen-

Copyright ~ 1977 by Academic Press, Inc. All rights of reproduction in a n y form reserved.

erated, and flagella are developed and asynchronously r e t r a c t e d ( G a y and Greenwood, 1966; C r u m p and Branton, 1966; H e a t h and Greenwood, 1971 ; Holloway a n d H e a t h , 1974). Analogy with other 0 o m y c e t e zoospores Suggests t h a t the different zoospore shapes m a y be correlated with different arrays of cytoplasmic microtubules ( H e a t h and Greenwood, 1971; Reichle, 1969a; H o c h and Mitchell, 1972; H e a t h , 1976) and Held (1972) has suggested t h a t loss of zoospore shape is correlated with depolymerization of microtubules. T h u s this predictable, n a t u r a l l y controlled, syst e m could be a good model in which to

10

HOLLOWAY AND HEATH

study the control mechanisms of in vivo microtubule assembly. However, dynamic processes cannot be adequately investigated unless large synchronous populations of cells can be obtained. Furthermore, before the mechanisms of control can be worked out, it is essential to have an accurate and detailed description of the correlated changes in morphology and microtubule arrays. The objective of the present paper is to report the results of our attempt to devise an adequate synchronization procedure, then to describe in detail the cytoplasmic mierotubule system of the various spore types, and then correlate development and loss of this system with the various spore morphologies. MATERIALS AND METHODS

Organism. The species of Saprolegnia used in these experiments was isolated by the first author and was found to differ from all species of Saprolegnia described in Seymour (1970). A brief description of its taxonomic characteristics is given in Holloway and Heath (1977), where it is coneluded to be S. ferax as defined by Seymour (1970). Synchronization procedure. Vegetative colonies were induced to produce primary zoospores by transfer from a nutrient medium to a dilute salts solution as described previously (I-Iolloway and Heath, 1974). Primary zoospores and primary cysts were collected at about 4 h after the first sporangiM discharge. The myeelium was removed by filtering the cultures through cheesecloth and collecting the spores on a 5-~m Millipore filter (all primary zoospores encysted during this procedure). The cysts were then transferred to a small volume of dilute salts solution and kept at 3°C for 8 h. Genesis of secondary zoospores from the primary cysts was initiated by transfer to 24°C. In order to measure the degree of synchronization of

the developing secondary zoospore population, samples were taken from the gently swirled suspension every 15 rain and the number of cysts, motile zoospores, encysting zoospores, and germinating secondary cysts were counted. Not less than 80 cells were counted in each sample. Micrographs of selected stages were made using Nomarski interference contrast optics and an electronic flash unit. Electron microscopy. Cells in the desired stage of development were fixed in situ for 10 rain by addition to the medium of an equal volume of isothermal 3% glutaraldehyde in M/15 phosphate buffer (pH 7.0). Primary zoospores and cysts were filtered through cheesecloth, collected on a 5-~m Millipore filter, mixed with a drop of 2% agar solution on a warm slide, and allowed to set as a thin film containing dispersed cells. The secondary zoospores and cysts were collected by centrifugation and processed as a pellet without addition of agar. All samples were post fixed in 1%, buffered, osmium tetroxide for 1 h, dehydrated in ethanol, and embedded in Epon resin. For sectioning of the primary zoospores and cysts, single cells showing the desired morphology were selected from the polymerized resin wafers and serially sectioned, whereas the secondary cells were examined from serial sections of the pelleted material. All sections were stained with uranyl acetate and lead citrate. RESULTS

Synchronization The composition of the zoospore-cyst suspension during the observation period following cold shock is shown in Fig. 1. Motile secondary zoospores were observed within 15 rain of initiation (i.e., return to room temperature, 24°C) and 98% of the population was swimming after 1.75 h. The secondary zoospores swam for approximately 1 h before encysting. After a quiescent period of as little as 15 rain the

100t<

MORPHOGENESIS IN Saprolegnia ZOOSPORES secondary cysts germinated into vegetative hyphae. On the basis of these results it was possible to predict the composition of cell suspensions at any given time. Therefore, in the following work, samples for observation of primary zoospores and cysts were taken 8 h after transfer of colonies to dilute salts solution (prior to the cold shock), exeysting secondary spores 15 min after initiation, motile secondary zoospores after 1.75 h, encysting secondary zoospores after 2.5 h, and germinating secondary cysts after 4 h.

90

8O

70

60

1

2

5 Hours

Light Microscopy

11

if)

FIG. 1. Composition of spore population after Primary zoospore morphology and en- induction of secondary zoospore excystment by cystment have been described for this iso- transfer of cells from 3 to 24°C (time 0). Open late previously (Holloway & Heath, 1974) circles, cysts; X, motile secondaryzoospores; closed and thus will not be described here except circles, germinatingcysts. to note that the rounding off of the spore at encystment takes approximately 1 s. A traeted with an 8-s cycle as soon as the typical secondary zoospore excystment se- spore emerged from the cyst. Initially after exeystment the zoopores quence is shown in Figs. 2a-i. A localized had no flagella and were approximately bulge appeared on the primary cyst wall oblate with the nucleus and wev towards (Fig. 2a). This bulge extended to form a one end. Flagella developed rapidly and short tube (the exit tube) about 3 ~m in beat with an ineffectual but regular sine diameter (Fig. 2b). The tip of the tube ruptured to allow emergence of the cyto- wave during elongation (Figs. 2g-h). As plasm (Fig. 2b). Emergence of the eyto- the flagella developed, the cell typically plasm was slow until the nuclear region became reniform and developed a longipassed the exit tube (up to 5 min) (Fig. 2c). tudinal groove (Figs. 2h-i). The flagella The bulk of the cytoplasm then left the were inserted in the groove just behind the cyst in one, two, or three pulses, each wev. Occasionally, flagellar elongation was lasting from 2 to 10 s. (Figs. 2d-f). The completed before any change in spore shape rate of movement during this phase varied occurred (Figs. 2j-m) and sometimes the from approximately 0.2 to 2.0 ~m/s. Mea- spore became reinform before any flagella surements from three exeysting sequences appeared. Usually, however, the two procshowed no change in cyst diameter from esses were coincidental, with the 20-~m-long pre-bulge to completion of excystment, the flagella being synthesized in approximately figures being 34, 35, and 36 mm for the 10 min at a rate of 2 pm/min. After elongathree spores as measured on the prints tion was complete, the flagella twitched (print magnification, X3500). Typically, violently for up to 4 min before coordinated one water expulsion vacuole (wev) I per beating commenced and the zoospore swam spore was located near the nuclear region away. After swimming for 45 to 60 min, swimwhere it rhythmically expanded and conming became erratic and the flagella de1Abbreviationused : wev,water expulsionvacuole. veloped beads. These were the first two

12

HOLLOWAY AND HEATH

MORPHOGENESIS IN Saprolegnia ZOOSPORES outward events in the encystment process illustrated in Fig. 3. The zoospore then rotated about its long axis (Fig. 3a), occasionally becoming motionless for a few seconds while the flagella were held rigid (Fig. 3b). Finally, rotation stopped completely and, as the longitudinal groove disappeared, the straightened flagella assmned an apical position (Figs. 3c-d) before the spore rounded off completely (Fig. 3e) and shed its flagella (Figs. 3f-g). The process from the end of the rotary movement to completion of rounding off and loss of flagella was extremely rapid; typically, it took less than 2 s. While the flagella were usually shed, occasionally one would wrap around the spore (Fig. 3h) and gradually disappear from view, presumably by resorption. The wev ceased to function at the time of flagellar loss and rapidly disappeared.

Electron Microscopy Primary zoospores. The formation and arrangement of microtubules and the interkinetosomal fibers during differentiation of the primary zoospores of another isolate of S. ferax have been described previously (Heath and Greenwood, 1971) and have not been repeated for this isolate since observations of sporangia and zoospores revealed no differences between the two isolates. In the mature zoospore, two sets of cytoplasmic microtubules were present; the perinuclear cone and the outer cone. Both sets had one end of each microtubule inserted in osmiophilic material which surrounded the bases of the kinetosomes (Fig. 4). The component microtubules of both cones occurred only as singlets. The perinuclear cone was formed during zoosporegenesis (Fig. 5; Heath and Greenwood, 1971) and occurred as an evenly spaced

13

array diverging from the kinetosomes and running adjacent to the envelope of the nuclear beak (i.e., the apex of the pyriform nucleus) (Figs. 4 and 5). The outer cone was more divergent and ran through the cytoplasm closer to the plasmalemma (Fig. 4) for much of the length of the spore (up to approximately 8 ~m). The two fagella (one whiplash and one Flimmer bearing) developed from the kinetosomes and were borne a~ the apex (anterior) of the pyriform J zoospor V Rounding off of the zoospore at encystment w£s associated with loss of the outer cone of; microtubules because these were absent i'h very young cysts in which flagellar deplolymerization was not complete. However, the perinuclear cone mierotubules persisted until the nucleus rounded up, which occurred after the axonemes had disappeared and a mature cyst cell wall had been deposited (not illustrated). The rounded nucleus was associated with very few microtubules which were much shorter (less than 1 ~m) than those found in the perinuclear cone of the mature zoospore (up to approximately 5 ~m). Flagellar axoneme degeneration involved a number of stages. At encystment, one or both flagella developed "beads' which were formed by coiling of the axoneme beneath the flagellar membrane (Fig. 6). Displacement of the central pair of microtubules sometimes occurred in these coiled axonemes (Fig. 6). Once inside the cyst, the axonemal doublet microtubules separated from one another (Fig. 10) but did not break into shorter fragments until sometime after they were shortened to 8 ~m. Further displacement and fragmentation then occurred (Fig. 7), and when only 1.7-pro-long doublet fragments could be found, they had a wavy appearance (Fig. 8). At this stage the transition region above the terminal plate was still intact (Fig. 9), but it disappeared

FIG. 2. E×cystmentand shape generation of secondaryzoospores. (a-i) A typical excystmentsequence as described in the text. The arrow indicates the nucleus. X]900. (jm) An excysted zoospo~:ein which the flagellahave developed prior to reniform shape generation. X2500.

14

HOLLOWAY AND HEATH

FIG. 3. (a-g) A typical encystment sequence for a secondary zoospore as described in fhe text. X 1427. (h) An encysting secodary zoospore in which one of the flagella wrapped around the spore (arrow) and was absorbed into the spore. X1321. after t h e nucleus r o u n d e d off a n d did r e a p p e a r until s h o r t l y before flagellar v e l o p m e n t in t h e s e c o n d a r y zoospore. s h o w n in Fig. 9, t h e k i n e t o s o m e s of

not deAs the

n e w l y e n c y s t e d p r i m a r y zoospores often had a bent appearance. Secondary zoospores. I n order to first introduce the terminology, the development

MORPHOGENESIS IN Saprolegnia ZOOSPORES of the microtubule root system, and i t s associated structures, will be described after an account of the morphology and arrangement of these components in the ~ . u r e secondary zoospore. ']?he kinetosomes were located in the protuberance approximately one-third of the way back from the anterior end of the groove which ran along the side of the zoospore. From the vicinity of these kinetosomes, radiating from structures to be described later, there were seven sets of cyto: plasmic microtubular roots (Fig. 11) the lengths of which are given in Table 1. The octet was typically composed of a ribbon of eight (occasionally six, seven, or nine) microtubules ~'hich lacked any associated osmiophilic material along most of their length (Figs. 12-16) and which failed to exhibit any intertubule cross-bridges after extensive careful observation of transverse sections. The octet was located along one side of the groove (Fig. 14), posterior to the kinetosomes. Along the other side of the groove there was a pair of somewhat widely spaced microtubules, the posterior doublet (Figs. 11, 14, and 32). The anterior portion of the groove was lined on one side by another pair of microtubules, the anterior doublet (Fig. 11), and on the other side by the triplet (Figs. 11, 24-28, 30, 31, and 33-36). The triplet was invariably composed of three adjoining microtubules which were backed by a band of osmiophilie material to which they were cross]inked at 27-nm intervals by osmiophilic cross-bridges (Figs. 25, 28, 30, and 31; unpublished micrographs). The angle between the octet and triplet roots was approximately 170 ° with this obtuse angle facing away from the nucleus. Abutting onto one side of the microtubules of the triplet root were a number of single microtubules termed the ribs (Figs. 12, 30, and 31). These ribs were evenly spaced at 30-nm (center-to-center) intervals and extend across the cytoplasm only on the triplet side of the groove (Figs. 11, 12, 30, and 31). Arising between the kinetosomes

15

\

\

:FIG. 4. Diagram showing the disposition of the nucleus (N), kinetosomes (k), interkinctosomal osmiophilic material (solid black), and microtubules (straight lines) traced from 25 sections in a series of 50 through the appropriate region of part of a motile primary zoospore. The microtubules between the large arrowheads are those of the outer cone, while those indicated by small arrowheads are part of the perinuclear cone population. X29,700. was the intermediate bundle, which was a group of single microtubules diverging from one another as they crossed the protuberance and ran through the cytoplasm, over the wev, toward the plasmalemma on the appropriate side of the zoospore (Figs. 11 and 32). As in the primary zoospore, the secondary zoospore also contained a group of microtubules, the perinuclear cone, which diverged from the bases of the kinetosomes over the surface of the nuclear beak (Figs. 11, 25, 26, 28, 38, and 39). All of the above microtubular roots, with the exception of the ribs, were connected to one another, and to the kinetosomes, by a complex array of osmiophilic structures, shown diagrammatically in Fig. 37. The kinetosomes were interconnected by two striate fibers, a short one at their bases and a larger one higher up (Figs. 24-27, 33, and 37). The octet was attached to a multilayered fibrous structure, the octet buttress (Figs. 12, 13, 15, 19, and 37). This buttress lay adjacent to the basal third of the whiplash kinetosome, to which it was attached

16

I-IOLLOWAY A N D H E A T H

MORPHOGENESIS IN Saprolegnia ZOOSPORES

/

17

\

® FIG. 11. Diagrammatic representation of the microtubular root system of the mature, reniform, secondary zoospore. Arrow indicates direction of swimming; the Flimmer bearing flagellum is carried anteriorly. by narrow connections at both the base a n d t h e m o r e d i s t a l r e g i o n (Figs. 13, 15, 33, a n d 37). T h e s h o r t b a s a l s t r i a t e fiber interconnecting the kinetosomes merged w i t h t h e m o s t b a s a l o c t e t c o n n e c t i o n (Figs. 33 a n d 37). A n e x t e n s i o n of t h e o c t e t b u t t r e s s w a s c o n n e c t e d to t h e d i s t a l r e g i o n of t h e F l i m m e r k i n e t o s o m e (Fig. 19). T h e b a s e of t h e w h i p l a s h k i n e t o s o m e w a s also c o n n e c t e d to t h e n u c l e a r b e a k b y a striated

strap w h i c h w a s a p p r e s s e d to t h e n u c l e a r e n v e l o p e (Figs. 22, 29, a n d 37) a n d w h i c h j o i n e d t o o s m i o p h i l i c m a t e r i a l on t h e o c t e t (Fig. 13) a n d a r o u n d t h e b a s e of t h e k i n e t o s o m e (Fig. 29). T h e t r i p l e t w a s conn e c t e d t o t h e b a s e of t h e F l i m m e r k i n e t o s o m e b y t h e f u s i o n of i t s o s m i o p h i l i c b a c k i n g m a t e r i a l w i t h a r i n g of m a t e r i a l s u r rounding the cartwheel-containing portion of t h e k i n e t o s o m e (Figs. 24-27, 33, 34, a n d

FIG. 5. Portion of a developing sporangium showing the arrangement of the perinuclear cone mierotubules running over the apex of the pyriform nucleus (N) from the base of the kinetosome (k). The kinetosomes did not develop further beyond the telTninal plate than the length arrowed here until flagellum development, after cytoplasmic cleavage. X48,800. Fie. 6. Portion of a flagellar bead on an encysting primary zoospore. Note the localized displacement of the central pair of microtubules (arrow) in only one part of the axoneme which was coiled, and thus, sectioned twice in this section. X72,300. FIG. 7. Part of a recently encysted primary spore showing sections of dispersed doublet microtubules (large arrows) and a central pair (small arrow). The central pair extended through 12 sections and was thus about 0.8 t~m long. X45,700. FIG. 8. Section of a recently encysted primary spore at a late stage in axoneme degeneration. Note the wavy appearance of the doublet microtubules, the maximum extent of which are shown in this section. X38,900. FIG. 9. Detail of part of a prima~:y cyst in which axoneme degeneration had progressed further than shown in Fig. 8. Note the intact transition zone structures (arrowed region) and the characteristically wavy kinetosome (k) adjacent to the nucleus (N). X57,300. FIG. 10° An~early stage in axoneme degradation in a primary cyst. Note the doublet micmmbuies (arrow) substantially displaced from the rest of the axoneme. X35,900.

18

HOLLOWAY AND HEATH

TABLE 1 Changes in the Microtubule Root Systems during the Life Cycle of Saprolegnia Secondary Zoospores~ Developmental stage

Triplet (t~m)

Ribs Octet (~m) (complete) (t~m)

Posterior doublet (urn)

Anterior doublet (t~m)

Intermediate bundle (um)

I. Excysting zoospores

a. before nucleus passes exist pore (of. Fig. 40A) b. after nucleus passes exit pore 2. Premature zoospores a. oblate, no groove, kinetosomes apical (cf. Fig. 40B) b. reniform, groove, ldnetosomes lateral 3. Mature zoospores (cf. Fig. 40C) 4. Encysting zoospores a. axoneme broken but flagellum still attached no groove (cf. Fig. 40D) b. axoneme shed or retracted no cell wall, cell spherical

2.53 4- 0.202 (3)

0.82 4- 0.381 (3)

0.06 (1)

--

0.14 (1)

2.54 (1)

0.60 5.10"*

?

--

?

3.35 (1)

3.93 (1)

0.26 (1)

--

?

5.18 (1)

6.12 (1)

?

?

1.22 4- 0.032 (4)

2.51 4- 0.075 (4)

3.40 (1) 3.76 4- 0.035 (13)

-{-

q- 5.19 4- 0.303 4.95 4- 0.124 (lO) (7)

3.68 (1) 3.21 4- 0.112 (5)

--

1.06

0.36

(i)

(1)

0.91 4- 0.512 0.71 4- C.082 (5) (5)

--

--

?

0.60 (1)

a 4-, standard error of the mean; ( ) , number of observations; **, extension of two tubules only; ?, a quantity which was not measured; q-, feature present; - , feature absent. Average lengths (t~m) of the various mierotubular structures are given.

37). W e h a v e t e r m e d this ring t h e triplet buttress. T h e m i e r o t u b u l e s of t h e interm e d i a t e b u n d l e a n d a n t e r i o r a n d posterior d o u b l e t s diverged f r o m a r a t h e r a m o r p h o u s region of osmiophilie m a t e r i a l w h i c h ext e n d e d f r o m t h e basal region of t h e w h i p lash k i n e t o s o m e (Figs. 33, 34, a n d 37). T h e perinuelear cone of m i e r o t u b u l e s inserted into m o r e a m o r p h o u s m a t e r i a l a r o u n d t h e basal regions of b o t h k i n e t o s o m e s (Figs. 24-28). I n a d d i t i o n to t h e a b o v e - m e n t i o n e d i n t e r c o n n e c t i o n s , t h e kinetosomes, in their m o r e distal regions, were s u r r o u n d e d b y conspicuous clear zones (Figs. 15 a n d 16) w h i e h were t r a v e r s e d b y p o o r l y defined osmiophilic c o n n e c t i o n s to t h e p l a s m a l e m m a (Fig. 1 6 ) .

T h e changes in t h e a b o v e - d e s c r i b e d m i c r o t u b u l a r r o o t s y s t e m during t h e exc y s t m e n t a n d e n c y s t m e n t of t h e zoospore are illustrated d i a g r a m m a t i e a ] l y in Fig. 40, described q u a n t i t a t i v e l y in T a b l e 1, a n d explained in m o r e detail as follows. Exeystment. P r i o r to e x e y s t m e n t , d u r i n g t h e life of t h e p r i m a r y cyst, a n u m b e r of changes o c c u r r e d in t h e osmiophilie s t r u c tures which intereonneeted the kinetosomes a n d r o o t systems. T h e striate fibers r e m a i n e d u n c h a n g e d b u t t h e osmiophilie m a t e r i a l s u r r o u n d i n g t h e base of t h e F l i m m e r k i n e t o s o m e in t h e p r i m a r y zoospore b e c a m e m o r e osmiophilie a n d clearly defined ( c o m p a r e Figs. 5 a n d 25). T h e

MORPttOGENESIS IN S~prolegn~aZOOSPORES osmiophilic material surrounding the base of the intermediate bundle became apparent when the base of the bundle itself appeared, but its generation could be a minor repositioning of some of the general osmiophilic material around the bases of the kinetosomes of the primary zoospore kinetosomes. However, two totally new structures with discrete morphology were assembled in the primary cyst prior to excystment; these were the octet buttress (Fig. 38) and the striate strap (Fig. 22). Once formed, all the above structures underwent no detected change throughout the excystment process to zoospore maturity. In contrast to the above situation, the various root systems underwent numerous changes during excystment and shape development. The perinuclear cone microtubules elongated again (Figs. 38 and 22) (after their shortening when the nucleus of the primary zoospore rounded up after encystment as described earlier) and the nucleus became pyriform (Fig. 22) prior to exeystment. Apart from a narrowing and elongation of the nuclear beak at maturity (not measured in detail) these roots did not change further. The triplet developed to approximately 70% of its mature length prior to excystment (Fig. 38, Table 1) and thus only elongated a little more as the zoospore excysted and developed its mature shape (Table 1). The octet, posterior doublet, and intermediate bundle were all detectable, but as only fractions of their mature length in the pre-excystment stage (Figs. 38 and 40, Table I). They continued to elongate in the oblate, excysted spore (Fig. 32, Table I), but the attainment of their full length, plus the formation of the anterior doublet and ribs, were all coincident with the development of the mature shape of the excysted zoospore (Fig. 40, Table 1). In addition to the above changes in the root systems during excystment there were two changes in arrangement which were unexpected. Before excystment and in the mature zoospore, the kinetosomes

19

enclosed an angle of about 130 °, but during excystment this angle was temporarily reduced as the cell squeezed through the exit pore (data deduced from serial sections of the cells shown in Figs. 39, 28, and 34). Similarly, the angle between the octet and triplet roots prior to excystment and at maturity was approximately 170 °, as noted earlier, but this also appeared to change during excystment, because in the recently emerged spore shown in Fig. 37 it was found to be about 140 ° . Ultrastructural observations showed that flagellar axonemes did not extend more than approximately 0.1 ~m beyond the terminal plate of the kinetosomes in all celis examined before these plates had become attached to the plasmalemma (e.g., Figs. 22 and 23). These observations also confirmed the light microscope observations showing that flagellar elongation could occur either before, or after, attainment of the reinform spore morphology and the concomitant maturation of the flagellar root systems (e.g., cells shown in part in Figs. 28 and 29). Encystment. The first sign of impending encystment in the secondary zoospore was beading of the flagella. After the zoospore rounded up, the flagellar axonemes broke just above the terminal plates (Fig. 17) and the flagella were typically shed. Occasionally, one flagellum wrapped around the zoospore. Subsequent merging of flagellar and cell membranes (Figs. 18-20) brought the axoneme into the cytoplasm and the flagellar membrane became part of the plasmalemma (Figs. 19 and 20). Evidence for membrane fusion was provided by the apparently granular material, which lines part of the flagellar membrane (Fig. 17), coming to lie under the spore plasmalemma (Fig. 20). There was no evidence for significant axonemal depolymeri, ization prior to shedding or resorption of flagella. Loss of the mature zoospore shape was coincident with substantial changes in the

20

ItOLLOWAY AND:ttEATH

lvIORPHOGENESIS IN mierotubular root systems. In cells which had become rounded, as opposed to reni' form, b u t which had still not lost their flagella, the ribs and anterior doublet were absent, the intermediate bundle was reduced to approximately 0.2 ~m of microtubules which were associated with osmiophilie material, and the octet and posterior doublet were reduced to less t h a n 20% of their m a t u r e length (Fig. 40 and Table 1). These changes must have t a k e n place in less t h a n 1 min because t h a t is the maxim u m length of time observed to have elapsed between rounding off and loss of flagella (Fig. 3 and earlier sections). During the shortening of the octet in these cells there was a preferential clustering of ribosomes along the octet (Fig. 21). After loss of the flagella, the triplet, remaining posterior doublet, octet, and later, the nuclear cone roots, all slowly shortened and disappeared during and after the depo-

Saprolegnia-ZOOSPORES

21

sition of the cyst wall. The osmiophilic fibers, buttresses, etc. around the kinetosomes also vanished before germination of the secondary cyst. DISCUSSION

Synchronization Procedure Because the s y n e h r o n y of zoospore release from the sporangia is poor and the motile period of the p r i m a r y zeospores is short, the procedure employed in this work is of little value for the s t u d y of large populations of p r i m a r y zoospores. However, it does permit the production of substantial quantities of secondary zoospores at predictable stages of development and is thus useful for the s t u d y of these spores. The system is not as good as t h a t reported b y Olson and Fuller (1971) for Allomyces b u t their system has not yet been worked out for biflagellate species. Thus, the pres-

]PIG. 12. Longitudinal (parallel with the groove) section through the kinetosome region of a mature secondary zoospore. Note arrangement of octet (o), octet buttress (ob), Flimmer flagellum kinetosome (k), triplet (t), and ribs (r). X61,400. Fro. 13. Transverse section of the proximal portion of the octet (only five microtubules present in this plane of section) showing details of the octet buttress (ob) and its connections to the striated strap (the strap itself is out of the plane of section) (large arrow) and the whiplash flagellum kinetosome (k) (small arrow). Nucleus is at left (N). X68,250. FIG. 14. Transverse section of the posterior part of the groove of a mature secondary zoospore showing the whiplash flagellum, the octet (o), and the posterior doublet (small arrows). X78,300. Fins. 15 and 16. Two of a series of sections through a whiplash flagellum kinetosome (k) of a secondary zoospore showing the octet, octet buttress (ob) and its connection to the kinetosome (large arrow), and the conspicuous clear zone around the kinetosome (brackets). Note the illdefined osmiophilic structures radiating across the clear zone (small arrows). X78,300. FIG. 17. Portion of a secondary zoospore which had rounded up and was presumably about to shed its flagella at the time of encystment. Note the granular material underlying the flagellar membrane (small arrow) and the break in the axoneme below the transition zone (large arrow). )432,300. FIG. 18. Section of a secondary zoospore which was retracting its flagellum at the time of fixation. Note the coiled axoneme in the bead and the internalized axoneme (arrow). X29,400. FIG. 19. Part of a secondary zoospore which was encysting at the time of fixation. Only one axoneme, that of the Flimmer flagellum Ewitnessed by the attached Flimmer hairs (small arrow)J was withdrawn and it had broken between the transition zone and terminal plate of the kinetosome (medium arrow). Note octet (o), triplet (t), and the connection between the octet buttress and the kinetosome (large arrow). X27,600. FIG. 20. Section sei~ial to that shown in Fig. 19, showing internalized axoneme with granular material similar to that shown in Fig. 17 underlying the plasmalemma (arrow), thus indicating fusion between flagellar membrane and plasmalemma. X34,600. FIG. 21. Recently, encysted secondary spore showing many ribosomes (arrow) clustered along the degenerating octet. X72,300.

22

HOLLOWAY AND HEATH

NIORPHOGENESiS IN Saprolegnia ZOOSPORES ent procedure is a significant contribution to the s t u d y of Oomycetes, especially the diplanetie genera such as Saprolegnia.

Excystment Mechanism The precise mechanism of excystment remains unclear. We could detect no evidence for the involvement of small vacuoles as suggested b y C r u m p and B r a n t o n (1966), nor does their suggestion t h a t the violent p0stemergence twitching plays a major role in zoospore release seem likely since it occurred in zoospores which had already separated from the cyst. Clearly, it has another cause or role. I t is unlikely t h a t expansion of the wev generates a n y propulsive force because it is located in the anterior part of the cell. The " b u b b l e " mechanism proposed b y Webster and Dennis (1967) to explain the analogous sporangium discharge system of Pythium also seems improbable because t h e y propose t h a t (a) initial discharge is caused b y sporangium (or in this case, c y s t ) c o n t r a c tion which did not occur in these c y s t s and (b) final discharge is driven b y surface tension when the extruded cytoplasm has

23

a larger volume t h a n t h a t remaining in the sporangium (cyst). Point b is not consistent with the present observations because the rapid emergence phase begins when considerably less t h a n half the total cytoplasm has emerged. We suggest t h a t amoeboid movement, using the exit tube as a substrate against which force can be generated, is a more probable mechanism to

explain exeystment. At least the rate of exeystment (up to 2 ~m/s) is comparable to t h a t observed in Amoeba proteus (up to 3 pro/s; Mast and Stahler, 1937) b u t this does not, of course, prove homology of mechanism.

Morphology-Microtubule Correlations The close correlation between nuclear beak shape and the arrangement of the perniculear cone mierotubules, together with the close spacing between the nuclear envelope and these microtubules suggest t h a t the microtubules act as an exoskeleton for the apex of the nucleus in b o t h p r i m a r y and secondary zoospores. I n addition, their apparent a t t a c h m e n t to the kinetosomes suggests t h a t they anchor these to the

Flo. 22. A primary cyst at an early stage of secondary zoospore excystment. The nucleus (N) is pyriform. The striated strap (large arrow) and perinuclear cone microtubules (small arrow) are evident. Note the kinetosome (k) and the developing exit tube (lower right). The kinetosomes were no longer than shown in this section. X15,900. Fro. 23. An excysting secondary spore at a later stage than that shown in Fig. 22. The nucleus had assumed an apical position in the oblate cell and the kinetosomes (k) had come to lie with their terminal plates (tp) close to the cell membrane. Note numerous perinuclear cone microtubules close to the nucleus (N). The kinetosome was no longer than shown here. X74,600. Fins. 24-27. Serial sections through the bases of the kinetosomes (k) of an excysting secondary zoospore. Note the numerous, single, perinucIear cone microtubules connected to osmiophilic material around the bases of the kinetosomes. The osmiophilic backing material of the triplet (t) is evident in Figs: 24-26 and its connection to the triplet buttress (tb) is shown in Fig. 25. The striate fiber connecting the bases of the kinetosomes is arrowed in Fig. 26, while a portion of the osmiophilic material associated with the base of the intermediate bundle is labeled (i) in Fig. 27. The tip of the nuclear beak is to the left (N). X67,800. FIG. 28. The kinetosome region of a recently excysted secondary zoospore which had developed flagella but was still oblate in shape (cf. Fig. 2j). The triplet (t), octet (o), and perinuclear cone microtubtfles are all present but ribs were absent. Note part of the triplet buttress (arrow) around the kinetosome. X41,900. FIG. 29. Portion of a newly excysted secondary zoospore which had attained the reniform shape but whose flagella had not developed beyond the stage shown on the left of this picture. Note the striated strap (s) adjacent to the nuclear beak (N) and connected to the whiplash kinetosome (k), the osmiophilio material at the base of the intermediate bundle (i), and the ribs (r). X46,000.

24

HOLLOWAY'AND HEATH m

MORPHOGENEStS IN nucleus (cf. H e a t h , 1975). T h e shortening a n d loss of these microtubules after encystment, when the nucleus rounds off, supports their hypothesized exoskeletal role. T h e outer cone of microtubules in the p r i m a r y zoospores also a p p e a r to be cytoskeletal because their formation, and demise, is closely correlated with the development, and loss of, the p y r i f o r m shape of the spore. Their a r r a n g e m e n t also correlates with the p y r i f o r m shape of the spore. I n addition, t h e y too p r o b a b l y h a v e a role in flagellar anchorage. T h e m o r p h o l o g y - m i c r o t u b u l e correlations in the secondary zoospores are more complex t h a n those of the p r i m a r y zoospores. T h e general a r r a n g e m e n t of the microtubules in the m a t u r e zoospore correlates reasonably well with zoospore m o r phology with one m a j o r exception. W h e n viewed along the long axis of the spore (i.e., along the groove), the spore is app a r e n t l y s y m m e t r i c a l yet the ribs are only located on one side of the anterior p a r t of the spore and the i n t e r m e d i a t e bundle is likewise a s y m m e t r i c a l l y located near the middle of the other side of the spore. T h u s the shape assumed b y the spore is generated in a more complex m a n n e r t h a n simply b y draping c y t o p l a s m and m e m b r a n e s over the m i c r o t u b u l a r root system. Nevertheless, the close correlation between f o r m a t i o n and subsequent less of the m a t u r e shape a n d the m a t u r e c o m p l e m e n t of cytoplasmic microtubules is too striking to be reason-

Saprolegnla ZOOSPORES

25

® Fin. 37. Diagrammatic reconstruction of the interconnections between the various microtubule root systems, the kinetosomes, and their associated fibers and osmiophilic connections. The diagram was prepared from a model viewed in the direction of the arrow, as shown on the inset (i.e., from above and to one side of the groove). For clarity, the per;nuclear cone microtubules are omitted, and the osmiophilic material into which the intermediate bundle, etc., insert has been displaced to the right. It should connect at its base with the base of the whiplash kinetosome as shown in Fig. 34. N, nucleus; S, striated strap; sf, striate fibers; O, octet; T, triplet; Bo, octet buttress; Bt, triplet buttress ; Kw and Kf, whiplash and Flimmer kinetosomes, respectively; I, intermediate bundle; A, anterior doublet; P, posterior doublet. ably explained b y other t h a n a cause a n d effect relationship. However, the a s y m m e t r y of the microtubules along either portion of the s y m m e t r i c a l groove (i.e., octet vs triplet and ribs) clearly suggests t h a t the details of the m i c r o t u b u l a r root s y s t e m h a v e other significance t h a n a cytoskeletal role. T h e m o s t obvious explanation is the

FIGs. 30 and 31. Transverse sections from a series through the anterior part of the groove of a mature secondary zoospore. The triplet (t), with cross bridges to its backing osmiophilic material (arrows), clearly only has ribs (r) connected to one side of its microtubules. Note details of the transition zone of the Flimmer bearing flagellum (cf. Heath and Greenwood, 1971). Fig. 30, X58,400; Fig. 31, X46,400. Fro. 32. A mature secondary zoospore viewed from above, down into the groove. The arrangement of the kinetosomes, octet (o), triplet (t), intermediate bundle (i), and posterior doublet (p) can all be seen. Note the various interconnections between the kinetosomes and buttresses. X35,520. FIGS. 33-36. Serial sections of the region shown in Fig. 32 showing the interrelationships between the whiplash (kw) and Flimmer (kf) kinetosomes, octet buttress (ob), triplet buttress (tb), striate fibers (arrows, Fig. 33), and osmiophilic material into which the intermediate bundle and posterior and anterior doublets insert (i). Figs. 24, 25, 27, X61,400; Fig. 26, X46,100.

26

HOLLOWAY AND HEATK

%1 J

® FIG. 38. Superimposed tracings of 15 sections in a series of 22 through the kinetosomes (k) and mierotubular ~oQts of an excysting, immature secondary zoospore. The octet (seen in side view, thus appearing as only one mierotubule), triplet, posterior doublet (arrow), and intermediate bundle (brackets) were sectioned in their entirety; thus, the represented lengths are the maximum lengths present in the spore. The tip of the nuclear beak (N) is indicated by the stippling. The unmarked microtubules (lines) are primarily those of the perinuclear cone. The osmiophilie connections between the kinetosome, triplet, and octet are shown as solid black areas. X30,200. need for differentially strong systems to anchor the flagella and absorb and dissipate the reaction to their beating. Given the a s y m m e t r y of the flagella themselves ( w h i p lash versus Flimmer) some a s y m m e t r y in the microtubular root system might be anticipated, b u t the observed complexity cannot be adequately comprehended until a m u c h b e t t e r understanding of flagella and motile cell mechanics is available. Two aspects of the development of the cytoplasmic microtubule system deserve more attention. T h e changes in b o t h the interkinetosomal and the octet-triplet angles during excystment and m a t u r e shape generation are evidence t h a t the osmiophilic structures interconnecting kinetos0mes and microtubules are flexible despite their

intuitively ".obvious" rigidity. The consequence of this apparent change in flexibility ~ i t h 0 u t concomitant morphological changes (of the osmiophilic structures) is t h a t if the osmiophilie structures are responsible for maintaining the arrangement of the microtubular root systems, as seems probable, there must be some system for modulating their flexibility properties. Conversely, the observations could be explained if the osmiophflic structures had only a directing influence during microtubule formation and some other undetected system acting on the roots and kinetosomes themselves was responsible for the arrangement of the roots. At present we cannot resolve this problem. T h e second m a j o r point is t h a t because the different microtubular roots and the

MORPHOGENESIS IN Saprolegnia ZOOSPORES

27

microtubules of the flagellar axonemes are polymerized and depolymerized at different times within a single cell there is clearly a finely t u n e d system of effecting individual microtubule polymerization control. Obviously a crude m o n o m e r - p o l y m e r c o m m o n pool of subunits is not a n adequate control system. There are a n u m b e r of mechanisms which m a y be invoked to explain this, b u t since the present work does little to differentiate between possible models t h e y will not be described here. However, it is of interest to note that, a m o n g the cytoplasmic microtubules, the first to form and last C

~® Fro. 40. Diagrammatic reconstruction of the correlated changes in shape and microtubular root system in an excysting (A and B), mature (C), encysting (D), and encysted (E) secondary zoospore. Details are given in the text and in Table 1. i~ intermediate bundle; o, octet; t, triplet; p, posterior doublet; a, anterior doublet; r, ribs; F, Flimmer bearing flagellum, W, whiplash flagellum; N, nucleus.

,.

n

/ f

®

FIG. 39. Reconstruction of the major microtubular root systems of a recently excysted secondary zoospore which had begun to develop flagella (F)but which still had an oblate shape and an immature root system. The illustrated data encompassed the entire length of the octet and triplet and was derived from 45 sections in a series of 90 through the spore~ Not every section in the series of 45 was photographed, h~nce the gaps in"the octet. The figures on the cell (solid and dotted lines), nuclear (N), and wev outlines and :other structures denote the section numbers in which :that outline or structure occurred. They are included to indicate the third dimension 'of the diagram. X 15,250.

to break down are those which are close to, and p r o b a b l y crosslinked to, other structures (i.e., the osmiophilic backing material of the triplet and the most proximal regions of the posterior doublet and intermediate bundle and the nuclear envelope for the perinuclear cone microtubu]es). Such associations m a y require more time to synthesize and m a y also confer resistance to depolymerizing influences.

Comparisons with Other Oomycete Microtubular Roots The cytoplasmic microtubule system described here for the secondary zoospore

28

HOLLOWAY AND HEATH

differs from that described in similarly shaped zoospores of other oomycetes such as Phytophthora (Reichle, 1969a), Aphanomyces (Hoch and Mitchell, 1972), and Lagenidium (Bland and Amerson, 1973). While some of the reported differences are undoubtedly real [-e.g., the number of microtubules in the octet equivalent of Aphanomyces is 12 (Hoch and Mitchell, 1972)J, the major differences, such as those between the asymmetry of the octet, triplet, and ribs shown here and the symmetrical array diagrammed in Phytophthora by Reichle (1969a) may be less real because in fact none of the figures in either Reichle (1969a), Hoch and Mitchell (1972), or Bland and Amerson (1973) demonstrate a root system which differs in symmetry from that reported here. Serial section analysis is essential to describe the threedimensional arrangement of the microtubule systems of these spores adequately. If such an analysis does prove differences, then of course such differences could well have phylogenetic and taxonomic significance, but at present the lack of complete detailed studies on other oomycete zoospores renders comparisons rather futile.

Flagella Formation and Loss The formation of flagella in Saprolegnia does not differ substantially from the process found in other organisms with the exception that it is significantly faster at 2.0 urn/rain compared with the 0.5 um/min for Naegleria (Kowit and Fulton, 1974), 0.4 #m/rain for Chlamydomonas (Rosenbaum et al., 1969), 0.64 ~m/min for Peranema (Tamm, 1967), and 1.3 #m/rain for in vitro microtubule polymerization (Binder et al., 1975). During the processes of fiagellar loss, the coiling of the axoneme in the beads is comparable with the coiling reported for trypsinized axonemes (Summers and Gibbons, 1971). This, together with the observation of central pair displacement in the beads and doublet separation

after axoneme withdrawal, strongly suggests that the various intermicrotubule linkages are the first things to be disrupted in the flagellum degeneration process. As previously suggested (Holloway and Heath, 1974), axoneme depolymerization prior to retraction into the cytoplasm probably occurs because no more than 8 #m of axoneme was ever found in recently encysted zoospores. However, subsequently fragmentation of doublets occurred prior to final depolymerization of the microtubules. It is tempting to conclude that such fragmentation is enzymatically controlled since prorein synthesis is needed for axonemal disintegration in Blastocladiella (Soll and Sonneborn, 1971). Occasional resorption of flagella in the secondary zoospores, as shown here, has also been reported for Phytophthora (Reichle, 1969b). We regard this as evidence for changes in membranes permitting membrane fusions. The cause of such changes is probably adverse environmental factors of unknown nature. CONCLUSIONS The demonstrated correlations between zoospore shape and microtubule arrangement and the observations of temporally asynchronous formation of various different microtubules together with the achievable degree of synchrony and high concentration of microtubules in the secondary zoospores make them excellent specimens for further analysis of the control mechanisms of microtubule polymerization. Such investigations are currently under way. ACKNOWLEDGMENTS We are pleased to acknowledge grants from the National Research Council of Canada to I. B. H., a National Research Council postgraduate scholarship to S. A. It., and the excellent secretarial assistance of Dorothy Gunning. This work was submitted by S. A. H. in partial fulfillment of the Ph.D. requirements of York University.

M O R P H O G E N E S I S I N Saprolegnia ZOOSPORES REFERENCES

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29

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