Life history and physiology of the red alga, Gelidium coulteri, in unialgal culture

Aquaculture, 61 (1987) 281-293 Elsevier Science Publishers B.V., Amsterdam

281 -

Printed

in The Netherlands

Life History and Physiology of the Red Alga, Gelidium coulteri, in Unialgal Culture BRUCE A. MACLER’

and JOHN A. WEST

Department of Botany, University of California, Berkeley, CA 94720 (U.S.A.) (Accepted

20 October 1986)

ABSTRACT Macler, B.A. and West, J.A., 1987. Life history and physiology in unialgal culture. Aquaculture, 61: 281-293.

of the red alga, Gelidium coulteri,

Gelidium coulteri Harv., an agarophyte of commercial importance, was grown in unialgal culture in natural seawater under various environmental conditions. Growth rate, reproductive status, morphology and agar production for whole plants under each condition were determined over 2month periods. Reproduction could be induced in these plants by (1) limiting them for nutrients, then relieving the limitation with addition of Provasoli’s enrichment, or (2) increasing photon flux densities (PFD ) from < 50 ,uE m-* s-l to 150 PE m-’ s-l. The sexual life history was completed in 2 months. Red light inhibited tetraspore germination vs. white light, but blue and green light did not. Vegetative tetrasporophytes and gametophytes showed no differences in their growth responses to environmental changes. Growth rates increased with increasingPFD and were optimal at 150-250 PE m-2 SK’. Growth inhibition and bleaching were seen at 400 PE m-’ s-‘. Agar yield on a dry weight basis increased with increasing PFD to 32-35% at 150 PE me2 s-’ and remained constant at higher PFDs. Plants grown at PFD -=z50 PE me2 s-i were linear with little branching. Increasing PFD to 150 PE mm2 s-’ stimulated branching within 1 week. Growth rate increased with increasing temperature to a maximum at 20-27°C. Temperatures above 31 “C were lethal. Agar yield was constant at 32-35% dry weight with respect to temperature to 31 ‘C, at which agar yields increased to 40%. Growth was optimal at pH 7.5-8.5. Tissue necrosis and cell death occurred at pH below 7.0. Plants formed superficial carbonate deposits at pH above 9.0. Agar yields were constant from pH 6.0 to 9.0. Plants grown in unfortified seawater showed declines in chlorophyll content and growth rate to nearly zero after 3 weeks. Phycobiliprotein levels decreased to zero after 12 days. Agar yields of these plants were 40-60% higher than in plants grown with nutrient supplementation. Subsequent 0.36 mM nitrate addition led to a recovery of chlorophyll and phycobiliprotein in 2-3 days.

INTRODUCTION

Members of the genus Gel&urn are well known for the quality of their phycocolloid, agar. For this product, natural populations of these plants have been ‘To whom reprint requests should be addressed. 0 1987 Elsevier

Science Publishers

B.V.

282

harvested for many years. As is typical of field-collected material, the quality and quantity of Gelidium has varied markedly between locations and over time, causing commercial problems of inconsistent agar quality and availability. In addition, the increasing demand for agar has led to an over-exploitation of this genus. In response, efforts are being made to use Gelidium in intensive mariculture systems (Hansen, 1980). Based on these results and the results from other mariculture systems (reviewed by Mathieson, 1980), it is clear that a better understanding of the life history and physiological requirements of Gelidium is necessary to proceed further with its domestication. While extensive work on the physiological requirements of macroalgae in culture has been reported for several red algae, including the carrageenophyte, Chondru.s crispus Stack. (Neish et al., 1977; Simpson et al., 1978; Simpson and Shacklock, 1979)) and the agarophyte genus, Grucilaria (Edelstein, 1977; Lapointe and Ryther, 1979; Bird et al., 1981)) the physiological requirements of Gelidium in culture have been little studied. Light, temperature and water movement were examined in G. filicinum, G. lingulatum and G. spinulosum (Santelices et al., 1981) and the related species, Pterockzdiu caerulescens, P. cupillucea and Gelidiella ucerosa (Santelices, 1978). Mairh and Rao (1978) examined daylength and media effects on growth rate in Gelidium pusillum. Bird (1976) examined nitrogen uptake rates in G. nudifrons. Hansen (1980) examined photosynthetic 0, evolution as a function of temperature and light. Environmental effects on sporeling development were reported by Correa et al. (1985). It is important to note that plants for the above experiments were from field collections of diverse individuals. Variation in age, environmental exposure, nutrient status, physical location and season can yield large differences in physiological responses. Santelices and Stewart (1985) provide an extensive account of the taxonomit status of Gelidium species and include a discussion of the important vegetative and reproductive features. It does not appear, however, that any Gelidium species have been cultured through their complete life history. In order to expand the work on Gelidium and to consider means to maximize growth and agar production in culture, we initiated studies on the life history, growth rate, agar yield and reproduction as affected by various environmental and nutritional conditions of unialgal culture. To eliminate plant-to-plant variation, clones of a single individual were used. In this paper, we report the basic life history and physiological requirements of Gelidium coulteri in culture. MATERIALS AND METHODS

Gelidium coulteri (2604) isolated into unialgal culture was from a collection by J.A. West at Campo Costa Rica in northern Baja California on 12 October 1981. It had been in culture for approximately 8 months prior to the initiation of these experiments. Whole, vegetative plants 2-4 cm long and less than 3

283

months old were used for all experiments. Five pm filtered, steam-sterilized natural seawater adjusted to 30%0 salinity with glass-distilled water or reagentgrade NaCl was used. For general culturing, half-strength (20 ml/l) Provasoli’s enrichment (PES) was added (McLachlan, 1973). Cultures were maintained in 15-1 aerated carboys at densities of 0.3-1.5 g fresh weight/l. Media were changed weekly. Light at various photon flux densities (PFD ) was provided by “cool-white” fluorescent bulbs and plastic window screen. Prior to experiment, plants were maintained at 150pE m-’ s-l and 16:8 L:D daylength. PFD was measured with a Li-Cor LI 185 photometer. Temperature-controlled incubators were used for all temperature-related experiments, otherwise cultures were at 25-26’ C. Growth was measured by changes in fresh weight. Plants were blotted dry and weighed immediately. Measurements were made every 4-7 days. Growth rate was calculated and reported as the exponential growth rate, ,B= In (final weight/initial weight ) /days. For experiments varying PFD, temperature or pH, G. coulteri was cultured in 4-l half-strength PES in flasks bubbled with water-filtered air; 50-75 whole plants with a combined initial wet weight of about 500 mg were grown per flask. For experiments examining spore germination and thallus morphology, plants were cultured in 300-ml deep culture dishes (Pyrex No. 3250) without aeration; lo-15 plants with a combined initial wet weight of about 100 mg were grown in each dish. Culture pH was adjusted daily. Standard conditions were 150 PE me2 s-’ PFD, 25°C pH 8.1 and 30%0 salinity. Four cultures were grown for each experimental condition. Cultures were maintained for 2 months or until plants died. The method of agar extraction was modified from Craigie and Leigh (1978). Plant tissue was dried at 60’ C overnight and ground in dry ice with a mortar and pestle. Tissue was rehydrated in 0.02% sodium acetate in water for 30-60 min, then autoclaved for 60 min. The hot solution was filtered through Whatman GF/C paper under N, gas. The residue was re-extracted as above and the filtrates combined. These were reduced in volume by gentle heating, then mixed with 3 volumes of 100% ethanol to precipitate the agar. The agar was collected by centrifugation, washed in 80% ethanol, 95% ethanol, dried overnight at 60 ’ C and weighed immediately. The method to extract the red algal phycobiliprotein was modified from Glazer et al. (1982). Fresh material was blotted, weighed, frozen and ground with dry ice and fired quartz sand with a mortar and pestle. The ground material was extracted three times in 0.05 M phosphate buffer, pH 7.0, containing 1 mM sodium azide and 1.2% (v:v) Triton X-100 detergent. The combined extracts were centrifuged at 8000 g for 10 min. The supernatant fraction was assayed spectroscopically from 350 to 600 nm. Quantitation was determined from relative peak heights of R-phycoerythrin at 568,544 and 499 nm.

284 TABLE 1 Spore germination as a function of light quality. PFD was 25 ,!AEm-2 s-’ for all conditions. Values are average percentages f SE of three cultures of 50 spores each Light

Germination

Red (600-750 nm) Blue (435-490 nm) Green (480-560 nm) White

17f12 72+16 68 It 8 74+ 14

( %)

Chlorophylls were quantified from DMSO/methanol rial by the method of Duncan and Harrison (1982).

extracts of fresh mate-

RESULTS

Life,history

Carpospores and tetraspores were isolated and grown separately. Successful germination occurred at PFD < 100 ,uE rnp2 s-l. Above 100 PE mm2 s-l PFD, the spores bleached and died within 1 week. In aerated culture when spores were kept in suspension (unattached) they also failed to germinate. Red light (600-750 nm) at 25 ,uE mm2 s-’ PFD inhibited spore germination and spore development some 75% relative to white light; blue (435-490 nm) and green (480-560 nm) light at 25 ,BE mV2 s-l PFD did not (Table 1, Fig. la). Early sporeling development of G. coulteri was similar to that described and illustrated in the classic work of Inoh (1947) on spore germination in red algae. After release and settling, the spore developed a wall and the protoplast formed a new cell (germ tube), thus leaving an empty spore wall (Fig. lb). The new cell then underwent a bipolar division to form an elongate, non-pigmented, rhizoidal cell and a larger, pigmented cell that established the erect main axis (Fig. lc) . After this cell divided into a series of smaller cells and cell enlargement was initiated, an elongate blade with a uniaxial apical meristem was established and secondary rhizoids developed (Fig. Id). If undisturbed, the resulting sporelings attached themselves to culture dishes with rhizoids. If loosened from the culture dish, sporelings did not reattach. Unattached, free-floating plants grew radially as highly branched spheres (Fig. le) , while attached plants developed as tufts consisting of numerous upright blades growing from horizontal stolons (Fig. If). Unattached sporelings grew faster and reproduced more rapidly than did attached plants. Maximum exponential growth rate for unattached plants was 15; maximum rate for attached plants was 2.5. All plants remained vegetative at PFD< 100 ,BE me2 s-l or at tempera-

b

-

e

Fig. 1. Development of Gelidium coulteri. (a) Sporeling development under red and blue light. PFD was 25 fi m-’ s-l. Left: red (660-750 nm) light. Right: blue (480-500 nm) light. Bar equals 1 cm. (b) Early germination showing an empty spore and its derivative cell. Bar equals 25 pm. (c) Sporeling with developing blade and rhizoid. Bar equals 25 pm. (d) Early development of flattened unbranched blade with a uniazial apical meristem and several rhizoids. Bar equals 50 pm. (e) Morphology of free-floating plant in laboratory culture. Bar equals 1 cm. (f) Morphology of attached, field-collected plant. Bar equals 1 cm. (g) Thallus morphology under low (50 fi me2 s-l) PFD. Bar equals 1 cm. (h) Change in morphology after 1 week at 150 $3 m-’ s-l PFD. Bar equals 1 cm.

tures < 20’ C. Unattached gametophytes and tetrasporophytes cultured at 150 PE m-’ s-’ PFD, 26’ C, and 16:8 L:D reached reproductive maturity as a single flattened blade when 1-2 mm in length (Fig. 2a, b, c) and within 2 weeks after

Fig. 2. Reproductive development of Gelidium coulteri. (a) Laboratory-cultured reproductive tetrasporophyte, longitudinal section. Bar equals 25 pm. (b) Reproductive male sporeling, surrounded by discharged spermatia in a viscous matrix. Bar equals 100 pm. (c ) Reproductive female sporeling. Note trichogynes in insert. Bar equals 100 pm. (d) Mature carposporophyte on female with persistent trichogynes. Bar equals 100 pm.

287

spore germination. Mature (3-5 cm) vegetative plants could be induced to become reproductive under two conditions: (1) when initially grown at PFD < 5OpE me2 s-l, 25”C, 16:8 L:D and in half-strength PES, then grown at 150 ,uE me2 s-’ PFD, 212 4% became reproductive within 4 weeks; (2 ) when plants were starved for nitrogen 2-3 weeks in unenriched seawater at 150 PE rnp2 s-l 25’ C and 16:8, then fortified with PES, 27 2 5% became reproductive within weeks. Vegetative plants stressed by temperatures above 30°C salinities < 20%0, or pH < 6.0 or > 9.0, did not form reproductive structures either under these conditions or when these stresses were removed by return to standard conditions after 1-2 weeks. Reproduction did not occur in plants cultured in 12:12 L:D or shorter daylengths, or at daylengths of 20:4 and greater. Reproduction was not observed in plants cultured in red, blue or green light over a 3-month period at 16:8 L:D. Mature plants continued vegetative growth when sexual. Reproductive sporelings (l-5 mm long), however, grew very little vegetatively, but continued to produce additional reproductive structures for at least 2-4 months. Reproduction occurred only when flattened bladelets developed on upright axes arising from the decumbent cylindrical stolons, or in very young sporelings, when the single main axis became flattened by bilateral growth. Reproductively mature bladelets often produced one or more lateral branches that, in turn, became flattened and then formed new reproductive structures. Anatomical preparations showed that tetrasporangial and spermatangial formations were basically as described by Fan (1961) . Reproductive structures were borne in the uppermost portion of the flattened blades. In the male gametophyte a spermatangial sorus was formed on both sides of the bladelet and released spermatia continuously in a viscous matrix (Fig. 2b). In female gametophytes the structure of the unfertilized carpogonial branch, supporting cell and nutritive filaments, was as described by Fan (1961) . We made no anatomical observations on postfertilization development. The upper 0.2 mm of any female bladelet usually bore 10 or more functional trichogynes simultaneously on both sides (Fig. 2c, d) , but only a single cytocarp was observed. The unfertilized trichogynes persisted for at least 2 weeks during carposporophyte development. Both carpospores and tetraspores were released in clusters within mucilaginous matrices adhering to the parent tissue and often germinated in situ. A sexual life history was completed in 2 months. Both vegetative and reproductive branches formed numerous unicellular hairs on the cortex of the actively growing region near the apex when transferred to fresh medium and higher PFD ( > 50 PE me2 s-’ ) . Hair formation in Gelidium is discussed briefly by Dromgoole and Booth (1985).

4’

Photon flux, temperature and pH

Experiments to assess the effects of varying PFDs on growth rate and agar yield examined PFD from 10 to 600 ,uE rnp2 s-l with other conditions stand-

Photon

flux density

(p Ernw2 s- ’ )

Fig. 3. Photon flux density vs. growth rate and percent agar yield. Closed circles, exponential growth rate, p; open circles, agar yield.

ardized as described in “Methods”. Growth rates were maximal over the range 150-250 fi m-’ s-l (Fig. 3). Decreased growth rate, loss of chlorophyll and phycobiliproteins, and eventual tissue necrosis were observed at fluxes above 3OOpE mm2 s-‘. Growth rates were inhibited at fluxes below 100 @ m-’ s-l, although plants appeared otherwise healthy. Percent agar increased with increasing PFD to 32% at 150 PE m-’ s-l, then remained constant at higher fluxes. Plants grown at PFD > 50 PE m-’ s-l were highly branched (Fig. le) . Morphology of plants grown at PFD < 50 ,uE me2 s-’ was stoloniferous with little branching (Fig. lg) . When such plants were then placed at 150 ,uE me2 s-l, branching was observed within 1 week (Fig. lh) . Plants were also grown at a series of temperatures from 5-35 ‘C. Increasing temperature led to higher growth rates up to a maximum in the range 20-27 ’ C (Fig. 4). Inhibition leading to plant death was seen at temperatures above 30°C. At these temperatures, tissues bleached completely in 3-4 days and growth rates became negative. Agar yields were nearly constant at 32-35% over the viable temperature range. When plants were cultured at lethal temperatures, percent agar increased to a maximum of about 40% dry weight, presumably from loss of other cellular materials. The effects of pH were assessed by growing plants at pH of 5.0-10.0. Increasing growth rates were seen from pH 6.0 to 7.5 (Fig. 5)) with maximum growth at pH 8.0, the ambient pH of natural seawater. Plants grown at pH 5.0 became bleached and necrotic within 4-5 days. Plants grown at pH 9.0 or above became covered with a thin layer of CaCO, granules. Little variation in agar yields was seen from pH 6.0 to 9.0. Agar yields were 50% and 65% lower at pH 5.0 and 10.0, respectively.

289

-7

r

7; 70 + F

6.0

-

fs

5.0

-

g

4.0

-

.-Fi

3.0

-

2 Q)

2.0-

iz

0‘ 0

,

,

,

,

5

IO

15

20

Temperature

25

30

(“(2)

Fig. 4. Temperature vs. growth rate and percent agar yield. Closed circles, exponential ,u; open circles, agar yield.

growth rate,

Nitrogen studies

Plants cultured in seawater without PES enrichment eventually bleached from red to green-yellow unless the seawater was replaced daily. This bleaching corresponded with decreases in chlorophylls and phycobiliproteins. Recovery of chlorophylls and phycobiliproteins in bleached plants was observed to occur

,

,

,

,

0

PH Fig. 5. pH vs. growth rate and percent agar yield. Closed circles, exponential circles, agar yield.

growth rate, P; open

290

TABLE 2 Growth rates and percent agar yields from G. coulteri grown in PES deficient in nitrogdn, phosphorous and/or vitamins. Values are averages f SE of four cultures of lo- 15 plants each Nitrate

Phosphate

Vitamins

Growth rate’ (PI

Agar yield (%drY wt.1

+ + + +

+ + + + -

+ + + + -

9.9k0.6 9.6 + 0.7 llf1.6 1Ozkl.O 6.4 + 0.8 6.3-e0.6 5.8f0.6 6.0 k 0.4

32+3 28+3 26zt3 27+2 42+5 36_+4 40+4 4243

-

‘p = Exponential growth rate based on wet weight (see ‘Materials and Methods’).

within 3 days after adding half-strength PES (336 ,BMtotal N) to the media. When plants were cultured in PES lacking N (as nitrate and ammonium), phosphorus (as phosphate) or the required vitamins biotin, thiamine and cyanocobalamin, growth rates were less relative to controls for all cultures lacking N, but remained nearly the same in cultures lacking phosphorus or O--O

growth

rote

o-o

chlorophyll

X--X

phycobilins

.+ .c .0 + c Q,

0 L

40-

20 -

0

2

4

6

Starvation

8

IO

x-x-x-x12 14

(days)

Fig. 6. Effects of nitrogen starvation. Plants were previously cultured in half-strength PES, then placed in unenriched seawater.

291

vitamins (Table 2). Agar yield increased 40-60% in plants starved for nitrogen, but was not affected by phosphorus or vitamin starvation. To assess this further, an experiment was undertaken to starve G. coulteri for N and follow changes in growth rate, chlorophyll and phy~obiliprotein levels. Such starved plants showed a 50% decrease in phycobiliproteins after 4 days in unenriched seawater and no detectable phycobiliproteins after 11 days (Fig. 6). A 50% decrease in chlorophylls was seen after 7 days. Growth rate was reduced 50% after 8 days and decreased to nearly zero after 3 weeks. Agar yield increased from 26% at the beginning of starvation to a maximum of 42% after 3 weeks. DISCUSSION

Among the many considerations for the successful mariculture of seaweeds, the provision of seedstock and the de~nition of culture conditions stand as most important. The research reported here was designed to determine the life history and characterize the physiological requirements of G. coulteri when grown in unialgal culture. The findings here are generally consistent with those reported for other Gelidium species (Bird, 1976; Cooper and Johnstone, 1944; Correa et al., 1985; Hansen, 1980; Mairh and Rao, 1978; Santelices, 1978; Santelices et al., 1981) and those observed in this laboratory for G. fZoridu~u~ Taylor and PterocZudiutenuis Okamura (B.A. Macler, unpublished data, 1983 1. By culturing in the optimal range for all environmental conditions, G. coulteri was maintained for more than a year at exponential growth rates > 10 for vegetative material. These values are four-fold higher than those reported by Hansen (1980) for tank-cultured G. coulteri and perhaps 40-fold that seen for plants in the field. G. coulteri has an isomorphic life history that can be completed in laboratory culture in 2 months. The induction regimes for sexual reproduction, involving release of plants from nutrient or light limitation, suggest that G. coulteri is a plant that reproduces under enhanced growth conditions, rather than under limiting conditions. This would be consistent with spring reproduction in the field, as would the requirement for daylengths greater than 12:12L:D. The low ( < 100 ,uE mW2s-l) light requirements for spore germination and sporeling development must be considered for long-term culture of reproductive plants. Reproduction in GeZidiu~ had not previously been shown to be daylength or light quality dependent and further work will be required to define the photoperiod and phytochrome system. Plants may become reproductive in the tetrasporophyte or gametophyte phase as l-2 mm sporelings. This may have implications in terms of fieldcollected material, where the gametoph~ic forms are unknown (T. DeCew, personal communication, 1986) and such small-sized plants are seldom col-

lected. In this light it should be noted that these small forms could be maintained reproductively in culture at 1-5 mm lengths for months at a time. These reproductively adult plants did not grow to the typical “adult” size, but continued to form additional reproductive structures. In the field, such plants might be rapidly overgrown or obscured by vegetative plants. The relative ease with which G. coulteri could be induced to form spores would be a benefit in producing “seedstock” for mariculture. We have chemically mutated both carpospores and tetraspores from Gelidium species and isolated a variety of morphological and biochemical mutant strains (B.A. Macler, unpublished data, 1983,1984). This may allow the development of seedstock enhanced for properties such as growth rate, pigment biosynthesis, agar quality and quantity, and pathogen resistance. The use of cloned material was an advantage in that replication from experiment to experiment was highly consistent. Clones enhanced for growth rate were stable for at least 3 years. G. coulteri occurs naturally in the field at water temperatures of 8-17” C. In laboratory culture, this species grew optimally from 20-27’ C with growth rates about three-fold those observed for typical field temperatures, suggesting that this environmental factor would be of major importance in mariculture schemes. Optimal growth rate was observed at pH 7.5-8.5. This was maintained by aeration to provide CO, and TRIS buffer, so that carbon limitation was not observed. At higher and lower pH, growth was inhibited, but plants remained viable from pH 6.0-10.0. This may be from carbon limitation (Raven, 1970; Lucas and Berry, 1985) via shifts in the CO,: HCO,- : CO,“- ratio. In unaerated and unbuffered culture systems, pH decreased over time (B.A. Macler, unpublished data, 1984)) indicating depletion of carbon. For other nutrients, N was most obviously limiting over days to weeks in culture. P and vitamins did not appear to limit growth over 2 months. From a mariculture perspective, elimination of vitamins from nutrient supplements may significantly reduce bacterial contamination. ACKNOWLEDGEMENTS

This work was supported by Ocean Genetics, Inc. and the National Science Foundation (Grant # CE-8360570)) for which the authors express their gratitude. We also wish to thank George I. Matsumoto and Andrew M. Dienstfrey for their capable assistance.

REFERENCES Bird, K.T., 1976. Simultaneous assimilation of ammonium and nitrate by Gelidium nudifrons (Gelidiales: Rhodophyta) . J. Phycol., 12: 238-241. Bird, K.T., Hanisak, M.D. and Ryther, J., 1981. Chemical quality and production of agars extracted from Gracikvia tikuahiae grown in different nitrogen enrichment conditions. Bot. Mar., 24: 441-444.

293 Correa, J., Avila, M. and Santelices, B., 1985. Effects of some environmental factors on growth of sporelings in two species of Gelidium (Rhodophyta) . Aquaculture, 44: 221-227. Cooper, N.C. and Johnstone, G.R., 1944. The seasonal production of agar in Gelidium cartilagineum, a perennial red alga. Am. J. Bot., 31: 638-640. Craigie, J.S. and Leigh, C., 1978. Carrageenans and agars. In: J.A. Hellebust and J.S. Craigie (Editors), Handbook of Phycological Methods. Physiological and Biochemical Methods. Cambridge University Press, Cambridge, pp. 109-132. Dromgoole, F. and Booth, W., 1985. The structure and development of hairs on the thallus of Gelidium caulacantheum J. Ag. N. 2. J. Mar. Freshwater Res., 19: 43-48. Duncan, M.J. and Harrison, P.J., 1982. Comparisons of solvents for extracting chlorophylls from marine macrophytes. Bot. Mar., 25: 445-447. Edelstein, T., 1977. Studies on Gracilaria sp: experiments on inocula incubated under greenhouse conditions. J. Exp. Mar. Biol. Ecol., 30: 249-259. Fan, K.-C., 1961. Morphological studies of the Gelidiales. Univ. Calif. Publ. Bot., 32: 315-368. Glazer, A.N., West, J.A. and Chan, C., 1982. Phycoerythrins as chemotaxonomic markers in red algae: a survey. Biochem. Syst. Ecol., 10: 203-215. Hansen, J.E., 1980. Physiological considerations in the mariculture of red algae. In: I.A. Abbott, M.S. Foster and L.F. Eklund (Editors), Pacific Seaweed Aquaculture. California Sea Grant College Program, pp. 80-91. Inoh, S., 1947. Kaiso no hassei (Development of Marine Algal Spores). Published by author, Tokyo, 255 pp. Lapointe, B.E. and Ryther, J.H., 1979. The effects of nitrogen and seawater flow rate on the growth and biochemical composition of Gracilaria foliifera var. angustissima in mass outdoor cultures. Bot. Mar., 22: 529-537. Lucas, W.J. and Berry, J.A. (Editors), 1985. Inorganic Carbon Uptake by Aquatic Photosynthetic Organisms. Am. Sot. Plant Physiol., Rockville, MD, 494 pp. Mairh, O.P. and Rao, P.S., 1978. Culture studies on Gelidium pusillum (Stackh.) LeJolis. Bot. Mar., 21: 169-174. Mathieson, A.C., 1980. Seaweed cultivation: a review. In: C.J. Sindermann (Editor), Proc. Sixth U.S.-Japan Meeting on Aquaculture, Santa Barbara, California. U.S. Dept. Commerce Rep. NMFS Circ. 442, pp. 25-66. McLachlan, J., 1973. Growth mediamarine. In: J.R. Stein (Editor), Handbook of Phycological Methods. Culture Methods and Growth Measurements. Cambridge University Press, Cambridge, pp. 25-51. Neish, A.C., Shacklock, P.F., Fox, C.H. and Simpson, F.J., 1977. The cultivation of Chondrus crispus. Factors affecting growth under greenhouse conditions. Can. J. Bot., 55: 2263-2271. Raven, J.A., 1970. Exogenous inorganic carbon sources in plant photosynthesis. Bot. Rev., 45: 167-221. Santelices, B., 1978. Multiple interaction of factors in the distribution of some Hawaiian Gelidiales (Rhodophyta). Pac. Sci., 32: 119-147. Santelices, B. and Stewart, J., 1985. Pacific species of Gelidium Lamouroux and other Gelidiales (Rhodophyta) , with keys and descriptions to the common or economically important species. In: I. Abbott and J. Norris (Editors), Taxonomy of Economic Seaweeds. California Sea Grant College Program. Report No. T-CSGCP-011, pp. 17-27. Santelices, B., Oliger, P. and Montalve, S., 1981. Production ecology of Chilean Gelidiales. Proc. Int. Seaweed Symp., 10: 351-356. Simpson, F.J., Neish, A.C., Shacklock, P.F. and Robson, D.R., 1978. The cultivation of Chondrus crispus. Effect of pH on growth and production of carrageenan. Bot. Mar., 21: 229-235. Simpson, F.J. and Shacklock, P.F., 1979. The cultivation of Chondrw crispus. Effect of temperature on growth and carrageenan production. Bot. Mar., 22: 295-298.