Robert R. L. Guillard

Culture of Phytoplankton for Feeding Marine Invertebrates

These pages describe relatively simple and reliable methods for the culture of marine phytoplankton species useful for feeding marine invertebrates. The methods suffice for the most fastidious algae now routinely cultivable, and simplifications indicated for less demanding species are easily made; for example, omission of silicate for plants other than diatoms. Certain modifications of techniques, ancillary methods, and precautions will be treated briefly because questions often arise concerning them, but documentation will be minimal and hopefully restricted to publications readily available.

YELLOW-GREEN ALGAE WITH CHLOROPHYLLIDE C1,2

Chlorophyllide c (chlorophyll c) wax found in axenic or unialgal cultures of 5 members of the class Xanthophyceae and in 2 members of the class Raphidophyceae (Chloromonadophyceae).

MEDIA FOR THE CULTURE OF OCEANIC ULTRAPHYTOPLANKTON

A medium (K) developed for culturing fastidious oceanic phytoplankton has been tested using recently isolated ultraphytoplankton clones representing at least seven different algal classes. The medium was designed to satisfy as completely as possible the nutritional requirements of this diverse group of phytoplankters. Important aspects are the addition of selenium, the inclusion of both nitrate and ammonium, an increased level of chelation and a moderate level of pH buffering. The seawater‐based version of this medium has been tested on 200 clones of which 186 grew reliably. A synthetic counterpart (AK) was tested on 40 of the more difficult clones and 27 grew well; 13 grew not all. While neither medium meets the exacting nutritional needs of all the ultraphytoplankton forms tested, they are excellent for most oceanic clones and are very successful for the isolation and establishment in culture of new oceanic phytoplankton clones.

Hydrocarbons of marine phytoplankton

The hydrocarbon contents of 23 species of algae (22 marine planktonic), belonging to 9 algal classes, were analyzed. The highly unsaturated 3,6,9,12,15,18-heneicosalhexaene predominates in the Bacillariophyceae, Dinophyceae, Cryptophyceae, Haptophyceae and Euglenophyceae. Rhizosolenia setigera contains n-heneicosane, presumably derived from the hexaolefin by hydrogenation. Two isomeric heptadecenes have been isolated: the double bond is located in 5-position in the bluegreen alga Synechococcus bacillaris and in 7-position in 2 green algae. Our complete analyses are discussed in the context of earlier data; some generalizations appear no longer valid. Hydrocarbon analysis of marine algae should provide a tool for the investigation of the dynamics of the marine food chain. Knowledge now available provides the background needed for distinguishing between hydrocarbons of recent biogenic origin and hydrocarbon pollutants from fossil fuels.

AQUIL: A CHEMICALLY DEFINED PHYTOPLANKTON CULTURE MEDIUM FOR TRACE METAL STUDIES12

The medium Aquil and its variations have been successfully used for trace metal studies of marine phytoplankton (diatoms and dinoflagellates) over the past three years. Here, the recipes, the methods of preparation and the chemical composition of Aquil are presented in detail. To permit complete definition of chemical speciation of the various components as calculated from thermodynamic equilibria, trace element contamination is controlled and the formation of precipitates and adsorbates is avoided. It is established that Aquil is suitable for physiological experiments with a variety of marine phytoplankters representing all major phyla. Modifications of the basic recipe and design of chemically defined media in general are discussed.

Saturated and unsaturated hydrocarbons in marine benthic algae

Saturated and olefinic hydrocarbons were determined in 24 species of green, brown and red benthic marine algae from the Cape Cod area (Massachusetts, USA). Among the saturated hydrocarbons, n-pentadecane predominates in the brown and n-heptadecane in the red algae. A C17 alkyleyclopropane has been identified tentatively in Ulvalactuca and Enteromorpha compressa, two species of green algae. Mono-and diolefinic C15 and C17 hydrocarbons are common. The structures of several new C17, C19 and C21 mono-to hexaolefins have been elucidated by gas chromatography, mass spectrometry and ozonolysis. In fruiting Ascophyllum nodosum, the polyunsaturated hydrocarbons carbons occur exclusively in the reproductive structures. The rest of the plant contains n-alkanes from C15 to C21. A link between the reproductive chemistry of benthic and planktonic algae and their olefin content is suggested. An intriguing speculation is based on Paffenhöfers (1970) observation that the sex ratio of laboratory reared Calanus helgolandicus depends upon the species of algae fed to the nauplii. The percentage of males produced correlates with our analyses of heneicosahexaene in the algal food. Our analyses of the hydrocarbons in benthic marine algae from coastal environments should aid studies of the coastal food web and should enable us to distinguish between hydrocarbon pollutants and the natural hydrocarbon background in inshore waters.

SELECTIVE PREDATION BY FAVELLA EHRENBERGII (TINTINNIA) ON AND AMONG DINOFLAGELLATES

In culture, the tintinnid Favella ehrenbergii requires dinoflagellates as food. Of the dinoflagellates tested, strain Gymno, Gonyaulax tamarensis, G. polyedra, and Heterocapsa sp. are good foods and Prorocentrum mariaelebouriae is a poor food. Amphidinium carterae, which produces choline-like substances, is not eaten.Favella recognizes dinoflagellates with very different sizes and morphologies as prey. In mixtures of dinoflagellates and non-dinoflagellates, Favella selectively preys on dinoflagellates. Cryptophytes, haptophytes, chrysophytes, diatoms, prasinophytes, and chlorophytes of suitable size are consumed in small amounts, if at all.

A polyunsaturated hydrocarbon (3, 6, 9, 12, 15, 18-heneicosahexaene) in the marine food web

An olefinic hydrocarbon (all-cis-3,6,9,12,15,18-heneicosahexaene, “HEH”)_was islated from marine planktonic plants and animals. Its structure was established by ultraviolet, infrared and mass spectrometry in combination with chemical techniques. The olefin occurs in many species of marine planktonic algae and is probably derived from the corresponding docosahexaenoic acid. Rhincalanus nasutus accumulates HEH nonselectively from its algal food together with the triglyceride lipids. Other, related copepods contain little or no HEH even when grown in cultures of algae that provide R. nasutus with that olefin. The presence of HEH in marine vertebrates suggests that, within the animal lipids, hydrocarbons are remarkably stable. This work has practical implications for studies of the marine food web and of marine pollution with persistent chemicals.

GROWTH AND THE PRODUCTION OF EXTRACELLULAR SUBSTANCES BY TWO STRAINS OF PHAEOCYSTIS POUCHETI1,2

Clones of Phaeocystis were isolated from winter surface waters off Woods Hole, Massachusetts, and from the tropical Atlantic near Surinam, South America. Northern strains survived only up to 14 C, while the tropical strain survived only as low as 17 C. Colony shapes of the northern and tropical clones differed somewhat, but the motile and non‐motile single cells of both strains seemed identical in the light microscope. By current taxonomic criteria both strains belong to the species P. poucheti (Hariot) Lagerheim.

Thoracosphaera heimii (Lohmann) Kamptner Is a Dinophyte: Observations on Its Morphology and Life Cycle

Abstract Thirty clones of Thoracosphaera heimii (Lohmann) Kamptner have been isolated and cultured from oceanic waters of the western North Atlantic and Gulf of Mexico. Observations on the life cycle and morphology of one clone isolated from the Sargasso Sea are reported herein. The calcareous cell wall is present in the coccoid, vegetative life phase. Reproduction is accomplished by the formation of transitory aplanospore or planospore stages or occasionally by binary fission of weakly calcified cells. The planospore is non-thecate and Gymnodinium-like with an undulating transverse flagellum and a whip-like longitudinal flagellum. All life stages possess chloroplasts and a nucleus with continually condensed chromosomes. The planospore morphology and the dinocaryotic nucleus demonstrate that T. heimii is a dinophyte and not a coccolithophorid. The taxonomic affinity and classification of T. heimii within the Dinophyceae is discussed and a new order Thoracosphaerales Tangen, ord. nov. is proposed for primarily coccoid marine dinoflagellates that possess a calcified cell wall in the vegetative life phase.