422 PLASTOCAMY IN

8. Giese, A. C., Smith, S. G. & Berry-Dau, J. 1965. Effect of osmotic pressure, ionic imbalance and pH upon the regeneration rate of Blepharisma. Exp. Cell Res. 39, 145-60.

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273-6.

Plastogamy in Foraminif era: Glabratella ornatissima (Cushman) JERE H. LIPPS and MALCOLM G. ERSKIAN

Dept . of Geology and Institute of Ecology, Univ. of California, Davis 95616, and Bodega Marine Laboratory, Bodega Bay, Calif.

SYNOPSIS. The foraminiferan Glabratella ornatissima (Cush- man) undergoes plastogamy during reproduction. In this pro- cess, 2 (or rarely more) individuals join together by their apertural sides to mutually exchange gametes. The apertural sides and internal septa are dissolved, forming a single large brood chamber. After the zygotes grow to a 2- or 3-chambered stage, the young foraminifera are liberated from the enclosing parent tests.

Scanning electron micrographs and histochemically stained thin sections indicate that the reproductively mature individuals are firmly united by an organic membrane during plastogamy.

HE Foraminiferida and Gromida contain species that T undergo plastogamy at particular times during their life cycles (1, 6, 7, 8, 10, 14). The mechanism of this process has not been described completely for any species, altho the positioning of the individuals during it is docu- mented in detail (1, 11). The united tests were thought originally to result from asexual budding or division, and later, when the sexual process was understood, they were thought to be joined by “animal cement” and calcium carbonate (1 1) , or by an organic membrane (6, 8 ) . In this study, we have investigated the mechanism of plas- togamy in the foraminiferan Glubrutella ornatissima (Cush- man).

MATERIALS AND METHODS Several thousand living and dead specimens of Glabratella

ornatissima (Cushman) were collected from exposed, rocky-shore tidepools adjacent to the University of California’s Bodega Marine Laboratory, Bodega Bay, California. A quantity of algae and sediment was taken from the sides and bottoms of tidepools exposed at low tide, and washed in sea water over a coarse screen to separate the larger debris from the foraminifera. Living specimens were isolated from the washings by hand picking with a fine brush. These specimens were maintained in finger bowls thru which sea water from the Laboratory’s sea water system ran continually. The intake for the system is located near

This membrane is monolamellar, less than 1 p thick and has minute lumps on its surface. Biochemically it contains a non- sulfated acid mucopolysaccharide, which is unlike the mem- branes associated with CaC03 deposition lining the interior of foraminifera1 tests. The young foraminifera liberate themselves by secretion of an enzyme which degrades the membrane. The mechanism of plastogamy is interpreted to be an adaptation to life in turbulent waters that would decrease the chance of gamete union or zygote survival were the gametes released and fertilized freely.

the tidepools from which the specimens were collected. Observa- tions of the living specimens were continued for 2 months.

Other living specimens, freshly collected from the same tide- pools, were fixed in Bouin-Duboso (alcoholic Bouin’s) solution, dehydrated in serial transfers in ethanol, embedded in parafin, and finally sectioned at 7 p . Sections were stained with hematoxylin- eosin-Y, Alcian blue, periodic acid-Schiff (PAS), mercuric brom- phenol blue and azure A (15) . Others were embedded in wax directly. The sections were examined under a Zeiss Photo- microscope in transmitted and polarized light, and under phase contrast with magnifications to 1250X. Dead and air-dried specimens were also examined with a Stereoscan scanning electron microscope. The specimens were either coated with aluminum or left uncoated before examination.

RESULTS

Structure. Examination of living and dead populations revealed a dimorphism. One group consisted of larger, more conical and more thickly calcified individuals whereas another group consisted of smaller, lower individuals with flatter apertural sides. Members of this smaller group participated in plastogamy .

The apertural sides of the tests were sculptured with nodose ridges and intervening smooth grooves less than f / 4 the width of the ridges. These ridges and grooves

PLASTOGAMY IN FORAMINIFERA 423

Fig. 1. Plastogamy in Glabratella ornatissima (Cushrnan) from lD, E. Side view of 2 individuals ( t ) in plastogamy showing Bodega Bay, California. All illustrations are stereoscan scanning pseudopodial grooves ( p ) , ridges ( r ) and the uniting membrane electron micrographs. ( m ) . x 500.

1A. Apertural side of a reproductively mature gamont. X 100. 1F. Detail of uniting membrane ( m ) between tests ( t ) shown 1B. Plastogamous pair, attached by their apertural sides. x 100. in 1E. Note that it has cracked ( c ) on desiccation and that it 1C. Dissolved apertural side of gamont after completion of appears to have minute lumps ( 1 ) on its surface. x 2000.

plastogamy. x 100.

424 PLASTOGAMY IN FORAMINIFERA

radiated from the center of the side and terminated at the curved edges of the test (Fig. lA, B, C ) . The spiral sides were marked with pores in areas covering chambers but were nonperforate where internal septa joined the exterior wall.

Plastogamy. Our observations of the manoeuvers pre- ceding and during plastogamy confirmed Myers’ ( 1 1 ) previous ones on this and related species. Commonly 2 gamonts of unequal size participated in plastogamy, altho 3 or 4 united individuals have been observed in our material. The smaller of a pair was usually the more active, attempting to move under the test of the larger. When positioned with their flat apertural sides opposed, they rotated until they were closely appressed (Fig. 1B). The individuals were most commonly not centered directly on one another. They then secreted a membrane to bind themselves together.

The opposing apertural sides and internal chamber septa were dissolved prior to gametogenesis. Myers ( 11 ) reported that after the gametes are formed and exchanged, the zygotes grow to a 2- or 3-chambered stage. At this time the membrane binding the parent tests is dissolved and the young foraminifera are liberated.

Membrane. The membrane joining the plastogamous pairs was concave outward, lumpy in detail (Fig. lF) , monolamellar, and less than 1 p thick. Upon desiccation the membrane may crack (Fig. lE, F ) . Altho thin and fragile in appearance, the membrane binds the paired specimens so tightly that their forceful separation may break the calcite tests. In sodium hypochlorite the mem- brane was destroyed and the individuals fell apart freely. Examination in polarized light of untreated thin-sections revealed no calcium carbonate in the membrane itself, which was entirely organic.

Histochemical stains indicated that the membrane con- tained a nonsulfated acid mucopolysaccharide. Mercuric bromphenol blue gave a negative test for protein. Ehrlich’s hematoxylin-eosin-Y double stain was positive for eosin-Y but negative for hematoxylin, indicating an absence of chromatin. Azure A gave a positive test for non-specific acid substances but was negative for sulfated mucopoly- saccharides. PAS was negative, and Alcian blue was positive at pH 3, indicating a non-sulfonated acid muco- polysaccharide, probably of the uronic or sialir acid groups.

DISCUSSION The test dimorphism in Glabratella ornatissima is cor-

related with the life cycle, the agamonts being larger and more conical, and the gamonts being smaller and flatter. Other species that now can be assigned to Glabratella have similar dimorphism ( 11 ) . This variation in test structure has resulted in proliferation of names applied to this species and confusion in its generic assignment. Cushman (5) originally named the species and placed it in Discorbis on the basis of its gamonts. In the same paper he (5) named the agamont Pulvinulinella columbiensis. Bandy ( 2 ) examined Pleistocene material from Oregon and named the agamont Rotalia subcorpulenta and the gamont Dis- corbis columbiensis oregonensis. Various workers have as-

signed the species to any of several genera, including Dis- corbis, Eponides, Pulvinulinella, Rotalia or Trichohyalus, but never to Glabratella. The species is referred to Gla- bratella because of its general structure, the striated and rugose apertural sides, the lack of a clearly visible aperture, monolamellar septa and the radial wall structure in which the predominant direction of the optical C-axes of the calcite is perpendicular to the external test wall. These characters are recognized in recent taxonomic revisions as characteristic of Glabratella (9, 12).

The organic membrane is the only structure binding the tests together during plastogamy. The nodose ridges and smooth grooves have been thought to hold individuals together ( 12), but these structures cannot interlock. This observation indicates that striated apertural sides do not necessarily imply a plastogamous reproductive method as suggested for some fossils. In fact plastogamy occurs in some species which do not have ridges or grooves ( 1 ) . Plastogamy in the fossil record should be proven by paired tests, not by striated apertural sides. The grooves and ridges function as pseudopodial channels during vegeta- tive life when the organism is closely attached to a sub- stratum, and during the initial stages of plastogamy when the united individuals may move about. This function in foraminifera appears to be correlated with life in tur- bulent-water environments, such as reef flats or littoral areas.

The binding membrane, containing a nonsulfated acid mucopolysaccharide, differs from membranes previously reported lining the inside of chambers of other foramini- fera, which contain sulfated acid mucopolysaccharides. This could be expected as inner test linings are associated with the calcification process in which sulfated acid muco- polysaccharides participate (3) . Nonsulfated acid muco- polysaccharides are constituents of gel-like or semi-fluid intercellular connective tissue in many diverse groups of animals, and may be synthesized by bacteria as well (4, 1 3 ) . The presence of this substance in the membrane is con- sistent with its function during plastogamy.

A commonly identified nonsulfated acid mucopolysac- charide, hyaluronic acid, may be degraded by testicular and bacterial enzymes (endohexosaminidases) or by leech hyaluronidase (endoglucuronidase) . We assume that the young foraminifera are capable of synthesizing a similar enzyme during their escape from the parent tests. Myers ( 1 1 ) reported that the young foraminifera make their escape by contacting the membrane with their pseudopodia, which indicates that the enzyme is not liberated into the brood chamber, but is retained in vesicles in their cyto- plasm.

In summary, the following processes occur during plastog- amy.

1. Meeting and positioning of the foraminifera, apertural sides opposed.

2. Withdrawal of protoplasm into the tests and the syn- thesis of a nonsulfated acid mucopolysaccharide which binds the individuals together.

3. Dissolution of opposing apertural walls and the mutual exchange of gametes.

ACTION OF MYXIN ON Euglena 425

4.

5.

Formation of zygotes and their growth to a several- chambered stage. Synthesis of an enzyme by the young foraminifera which degrades the membrane permitting the release of the immature foraminifera.

Plastogamy appears to be an adaptation to life in turbulent waters that would decrease chances of gamete union or zygote survival were the gametes released and fertilized freely.

We thank N. D. Holland, Scripps Institution of Oceanography, for assistance with histochemical technics, and T. E. Everhart and M. Nemanic, University of California, Berkeley for scanning electron microscopic examination of our specimens. Supported by a grant from the Committee on Research, U. C. Davis.

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charides. Elsevier, kmsterdam.

5. Cushman, J. A. 1925. Recent foraminifera from British Columbia. Contr. Cushman Lab. Foram. Res. 1, 38-45. 6. Grell, K. G. 1958. Untersuchungen iiber die Fortpflanzung

und Sexualitat der Foraminiferen, 11. Rubratella intermedia. Arch. Protistenk. 102, 291-308. 7. - . 1958. Untersuchungen iiber die Fortpflanzung und

Sexualitat der Foraminiferen, 111. Glabratella sulcata. Arch. Protistenk. 102, 449-472. 8. - . 1967. Sexual reproduction in protozoa, in Chen,

T-T., Research in Protozoology, 2, 147-213, Pergamon Press, Oxford. 9. Loeblich, A. R., Jr. & Tappan, H. 1964. Sarcodina, chiefly

“thecamoebians” and Foraminiferida, in Moore, R. C., Treatise on Invertebrate Paleontology, Part C, Protista 2 . 10. Myers, E. H. 1935. The life history of Patellina corrugata

Williamson, a foraminifer. Calif . Univ. , Bull. Scripps Inst. Oceanogr.

11. - . 1940. Observations on the origin and fate of flagellated gametes in multiple tests of Discorbis (Foraminifera). J . Mar . Biol. Assoc. U .K . 24, 201-26.

12. Seiglie, G. A. & Bermudez, P. J. 1965. Monografia de la familia de foraminiferos Glabratellidae Geos 12, 15-65. 13. Stacey, M. & Barker, S. A. 1962. Carbohydrates o f living

tissues. Van Nostrand, London. 14. Weber, H. 1965. Uber die Paarung der Gamonten und

den Kerndualismus der Foraminifere Metarotaliella parva Grell. Arch. Protistenk. 108, 217-70. 15. Weesner, F. M. 1960. General zoological microtechnique.

Williams and Wilkins, Baltimore.

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J. PROTOZOOL. 16(3), 425-428 (1969).

Action of Myxin on the Chloroplast System of Euglena gracilis

D. R. McCALLA and WALDO BAERG

Dept . of Biochemistry, McMaster Univ., Hamilton, Ontario, Canada

SYNOPSIS. Myxin ( 1-hydroxy-6-methoxy-phenazine-5,lO-dioxide) , a wide spectrum antibiotic, inhibits chloroplast replication in Euglena gracilis strain Z at concentrations which have no effect upon growth or survival. Myxin also inhibits the synthesis of

ESULTS from several laboratories have shown that R the Euglena chloroplast system is extremely sensitive to a variety of antibiotics, mutagens and other agents. Some of these agents (e.g., UV light [lo] and nalidixic acid [8] are so highly selective that they cause complete and permanent loss of chloroplasts (“bleaching”) without affecting the growth of the organism. Similarly these agents do not inhibit the development of proplastids into chloroplasts when etiolated Euglena are illuminated. Other agents such as nitrosoguanidine ( 1 1 ) and derivatives of nitrofuran (12) are less selective. They bleach Euglena effectively but also inhibit growth and cell division tem- porarily and interfere with the differentiation of proplastids into chloroplasts.

We show here that myxin ( 1-hydroxy-6-methoxyphen- azine-5,lO-dioxide) , a wide spectrum antibiotic recently isolated from culture filtrates of a species of Sorangium (6, 13), bleaches Euglena at concentrations well below those required to affect growth. Myxin also inhibits the development of proplastids.

chlorophyll when etiolated Euglena are illuminated in resting medium. By analogy with its action on bacteria, it is suggested that myxin may cause selective inhibition of chloroplast nucleic acid synthesis.

MATERIALS AND METHODS Myxin was obtained thru the courtesy of Dr. R. Hochster,

Director, Cell Biology Institute, Canada Department of Agricul- ture, Ottawa. Euglena gracilis strain Z , originally obtained from Dr. S. H. Hutner, was maintained on tryptic soy agar slants. Cultures were grown for several cycles on pH 3.5 medium (2) or on Cramer-Myers medium (4) (pH 6.8) at 26 C. Illuminated cultures were exposed to light of 100-200 ft. candles from “cool white” fluorescent tubes. Details of the experimental procedures used have been published elsewhere (12). Except where other- wise stated, myxin was dissolved in medium, filtered thru type HA Millipore filters, and the solution diluted to the appropriate concentration with sterile medium. Media were inoculated im- mediately after addition of myxin. At appropriate intervals, samples were plated on tryptic soy agar and, after a week to 10 days incubation at about 26 C, scored for green and bleached colonies.

Cell mass was estimated by means of optical density measure- ments at 750 nm in a Bausch and Lomb “Spectronic 20” color- imeter.

The relative chlorophyll contents of the cells were determined by collecting cells from samples of the cultures and extracting each pellet with 4 ml of methanol at room temperature. After centrif- ugation of the mixture, the optical density of the clear super- natant was determined at 665 nm in the “Spectronic 20.”