DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

PHYSIOLOGY OF SEA ICE DIATOMS. I. RESPONSE OF THREE POLAR DIATOMS TO A SIMULATED SUMMER-WINTER TRANSITION1

Three axenic polar sea ice diatom cultures were subjected to a 30 day simulated summer-winter transition in which light and temperature were decreased and salinity was increased to mimic seasonal changes previously reported for ice-covered polar seas. The diatoms responded to these changes by a reduction in cellular metabolism as indicated by: 1) A decline in growth rate and photosynthetic rate; 2) a decrease in cellular ATP; and 3) the storage and subsequent utilization of endogenous carbon reserves. In addition, heterotrophic potential of the three clones increased by as much as 60-fold. In some cases, the decrease in light intensity characteristic of the onset of polar winter was alone sufficient to trigger these physiological changes.     

Culturing Selenastrum capricornutum (Chlorophyta) in a synthetic algal nutrient medium with defined mineral particulates

Algal nutrient studies in chemically-defined media typically employ a synthetic chelator to prevent iron hydroxide precipitation. Micronutrient-particulate interactions may, however, significantly affect chemical speciation and hence biovailability of these nutrients in natural waters. A technique is described by which Selenastrum capricornutum Printz (Chlorophyta) may be cultured in a medium where trace metal speciation (except iron) is controlled, not by organic chelation, but by sorption onto titanium dioxide. Application of this culturing protocol in conjunction with results from sorption studies of nutrient ions on mineral particles provides a means of studying biological impacts of sorptive processes in aquatic environments.     

Environmental conditions for Diaphorina citri Kuwayama (Hemiptera: Liviidae) take‐off

Environmental factors that influence flight activity of Diaphorina citri Kuwayama (Hemiptera: Liviidae) may have implications for Huanglongbing spread and management. In this work, four studies were conducted to evaluate the effect of environmental conditions on D. citri take‐off. In the first, insects were transferred to sweet orange seedlings and confined inside an acrylic cage to verify the take‐off periodicity and the effect of environmental factors on this process. In the second, take‐off temperature threshold was estimated by recording the number of insects that initiated flight from a platform when subjected to gradual temperature increases from 15 to 39°C. In the other studies, we evaluated the effect of different photoperiods and temperature regimes (third study) and of constant temperatures (fourth study) on the propensity for D. citri flight. Insects were confined in clear plastic bottle cages with tubes of 50 ml placed on the cab, to collect emerged adults that initiated flights. Results showed that a small portion of the tested population (maximum 10%) tends to take off from plants and this behaviour is more prevalent in the afternoon (14:00–16:00 h), coinciding with daytimes of lower humidity and higher thermal amplitude. Adults that were submitted to lower temperatures (18°C) and short light periods (10 h) showed less propensity to flight. In contrast, at constant 27°C, the insects were more prone to flight, and this result was confirmed when individuals were submitted to increases in temperature, indicating that 27.14°C is the take‐off temperature threshold of D. citri. Results show that temperature plays an important role in the flight activity of D. citri and suggest that control measures of the insect may be more effective in the morning and in temperatures below 27°C, when the probability to take off from a host is lower.     

The cryptochrome family of photoreceptors

CRY1, the primary photoreceptor responsible for blue light-mediated inhibition of hypocotyl elongation in Arabidopsis thaliana, has been characterized. The properties of CRY1, and those of the related protein CRY2, and their relationship to the photolyase family of flavoproteins are discussed.     

A proteolytic enzyme, trypsin, for immunofluorescence observation of microtubules in algal cells

The usefulness of a proteolytic enzyme, trypsin, in immunofluorescence microscopy was confirmed in algal ceils. Flagella of Umspora penicilliformis zoo-spores were visible using an anti-β tubulin antibody after trypsin treatment, and the cortical microtubules of vegetative cells could also be clearly detected. Interestingly, centrioles that were not detected in the control observation appeared in gametophyte cells of Acrosiphonia duriuscula and Monostroma angicava using the trypsin treatment.     

MALE GAMETES OF ATRACTOMORPHA ECHINATA HOFFMAN (CHLOROPHYCEAE)1

Many naked gametes are produced in each fusiform, male gametangium of Atractomorpha echinata Hoffman and are liberated through irregularly shaped pores in the gametangial wall. They are typically biflagellate, pyriform or fusiform in shape, 6-11 μm long, and only a few micrometers wide. A mature male gamete is characterized by: (i) a nucleus with condensed chromatin and no nucleoli, (ii) a reduced, starch filled chloroplast occupying a posterior position, and (iii) a cup shaped eyespot consisting of a single layer of plastoglobuli. The flagellar apparatus includes two types of flagellar roots alternating in a cruciate pattern. One type consists of two microtubules, while the other consists of microtubules of varying number, usually eight or nine, but rarely as many as eleven. The paired basal bodies are connected anteriorly by a broad, striated distal fiber; there is no dense apical cap as reported in Sphaeroplea sperm. A unique structure, consisting of three layers of small subunits (6–8 nm diameter) arranged in a paracrystalline array, is positioned beneath each basal body. Based on the structure of its male gametes, Atractomorpha clearly demonstrates affinity with the chlorophycean rather than the ulvaphycean line of evolution. Moreover, if phylogenetic affinities for the Sphaeropleaceae are to be sought among other groups of green algae, the Chlorococcales appears the most promising candidate.     

MARINE CRYPTOMONAD STARCH FROM AUTOLYSIS OF GLYCEROL-GROWN CHROOMONAS SALINA1

The extracellular starch released by autolysis of glycerol-grown Chroomonas salina (Wislouch) Butcher was purified and chemically characterized. It contained 99%D-glucose, formed a blue complex (λmax at 605 nm) with iodine, and showed an optical rotation value more positive than that of potato starch. Methylation analysis established the presence of 1,4- and 1,4,6-glucoside linkages in the proportion 15:1. These properties indicate the cryptomonad starch to be an iodophilic α-(1,4)-glucan, composed of ca. 30% amylose with amylopectin, and broadly resembling potato, Chilomonas and other unicellular algal starches.     

Oxytetracycline age validation of an adult shortfin mako shark Isurus oxyrinchus after 6 years at liberty

This study presents findings on an oxytetracycline injected adult male shortfin mako Isurus oxyrinchus recaptured in waters off of southern California after 6 years at liberty. During the period at liberty, the vertebral band‐pair deposition rate was validated at one per year. This result indicates that from a time at or near sexual maturity, male I. oxyrinchus in the north‐east Pacific Ocean exhibit a band‐pair deposition rate of one band pair per year, while deposition rates for juveniles in the area have been validated at two band pairs per year.