Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions1

In the last few years, genome‐based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.     

INTERACTIVE EFFECTS OF IRRADIANCE AND TEMPERATURE ON THE PHOTOSYNTHETIC PHYSIOLOGY OF THE PENNATE DIATOM PSEUDO‐NITZSCHIA GRANII (BACILLARIOPHYCEAE) FROM THE NORTHEAST SUBARCTIC PACIFIC1

This study examined how light and temperature interact to influence growth rates, chl a, and photosynthetic efficiency of the oceanic pennate diatom Pseudo‐nitzschia granii Hasle, isolated from the northeast subarctic Pacific. Growth rates were modulated by both light and temperature, although for each irradiance tested, the growth rate was always the greatest at ～14°C. Chl a per cell was affected primarily by temperature, except at the maximum chl a per cell (at 10°C) where the effects of light were noticeable. At both ends of the temperature gradient, cells displayed evidence of chlorosis even at low light intensities. Chl fluorescence data suggested that cells at 8°C were significantly more efficient in their photosynthetic processes than cells at 20°C, despite having comparable concentrations of chl. Cells at low temperature showed photosynthetic characteristics similar to high‐irradiance‐adapted cells. The decline of growth rates beyond the optimum growth temperature coincided with the cell's inability to accumulate chl in response to increasing temperature. The decline in photosynthetic ability at 20°C was likely due to a combination of high‐temperature stress on cellular membranes and a decline in chl. Our results highlight the important interactions between light and temperature and the need to incorporate these interactions into the development of phytoplankton models for the subarctic Pacific.     

RELATIONSHIPS BETWEEN GROWTH RATE,CELL SIZE,AND INDUCTION OF SPERMATOGENESIS IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYTA)1

Patterns of changes in cell size, growth rate, and the inducibility of spermatogenesis were followed in eight sub‐clones of two isolates of the centric diatom Thalassiosira weissflogii (Grunow) Fryxell & Hasle grown at saturating light. One isolate originated from Long Island Sound, New York, USA and the other originated from Jakarta Harbor, Indonesia. As expected from previous studies, oscillations between intervals of cell size reduction and cell size enlargement were observed for each sub‐clone. For both isolates, sperm were easily detected, but cells resembling eggs and auxospores were rarely observed and fertilization was not confirmed, suggesting that the observed cell size increases may have resulted from a combination of asexual cell enlargement and rare auxosporulation. The two isolates differed in their minimum and maximum sizes, and the threshold size for the induction of sperm formation. However, the two sets of isolated sub‐clones displayed comparable relationships between growth rate, sperm inducibility, and cell size relative to the minimum, maximum, and threshold sizes. Growth rate increased as cell size decreased during vegetative divisions until the threshold for sperm inducibility was crossed. Below the size threshold for sperm inducibility, growth rate declined as cell size continued to decrease. Smaller cells were susceptible to failure of normal cytokinesis and valve deposition, resulting in the formation of abnormally long and often multinucleate cells. Culture conditions may select against restoration of cell size via auxosporulation due to the relationship between growth rate and cell size.     

MOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Here, we describe the first system for genetic transformation of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, the only diatom for which a complete genome sequence is presently available. This method is based on microparticle bombardment followed by selection of transformants using the antibiotic nourseothricin. It exhibits the highest transformation efficiency compared with transformation systems for other diatom species. To achieve the high transformation efficiency, it is important to allow recovery of the bombarded T. pseudonana cells in non‐selective suspension culture before spreading on nourseothricin containing agar plates. It is demonstrated that T. pseudonana is readily susceptible to co‐transformation allowing for the simultaneous introduction of a non‐selective gene together with the selection marker gene. Both introduced genes are stably inherited even in the absence of the antibiotic selection pressure. We have developed two T. pseudonana‐specific expression vectors that can drive constitutive expression (vector pTpfcp) and inducible expression (vector pTpNR) of introduced genes. In combination with the available genome data the T. pseudonana transformation system is expected to provide a powerful tool for functional genomics in diatoms.     

A STRESS‐INDUCED PROTEIN ASSOCIATED WITH THE GIRDLE BAND REGION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYTA)1

We report the characterization of a cell‐surface protein isolated from the centric diatom Thalassiosira pseudonana Hasle and Heimdal. This protein has an apparent molecular weight of 150 kDa, is highly acidic, and is intimately associated with the cell wall. Although originally identified in cells experiencing copper toxicity, it is also induced by silicon and iron limitation but not by phosphate or nitrate limitation. Using immunofluorescence techniques, the 150‐kDa protein was localized to the girdle band region and covered the elongated girdle band region of morphologically aberrant cells suffering from copper toxicity. Although having biochemical similarities to girdle band associated proteins identified in pennate diatoms known as pleuralins, the 150‐kDa protein is not a sequence homolog and is predicted to have a number of unique features, such as chitin binding domains and a possible RGD cell attachment motif. Results presented here suggest that this protein is normally cell cycle regulated and may be involved in stabilizing cells during the division process.     

EFFECT OF SALINITY ON PSEUDO‐NITZSCHIA SPECIES (BACILLARIOPHYCEAE) GROWTH AND DISTRIBUTION1

Salinity varies widely in coastal areas that often have a high abundance of Pseudo‐nitzschia H. Peragallo. Pseudo‐nitzschia is abundant in Louisiana waters, and high cellular domoic acid has been observed in natural samples but no human illness has been reported. To assess the threat of amnesic shellfish poisoning (ASP), we examined the effect of salinity on Pseudo‐nitzschia occurrence in the field and growth in the laboratory with special emphasis on the salinity range where oysters are harvested (10–20 psu). In Louisiana coastal waters, Pseudo‐nitzschia spp. occurred over a salinity range of 1 to >35 psu, but they occurred more frequently at higher rather than lower salinities. Seven species were identified, including toxigenic species occurring at low salinities. In culture studies, seven clones of three species grew over a salinity range of 15 to 40 psu, some grew at salinities down to 6.25 psu, and most grew at salinities up to 45 psu. Tolerance of low salinities decreased from Pseudo‐nitzschia delicatissima (Cleve) Heiden to P. multiseries (Hasle) Hasle to P. pseudodelicatissima (Hasle) Hasle emend. Lundholm, Hasle et Moestrup. In conclusion, although Pseudo‐nitzschia was more prevalent in the field and grew better in the laboratory at higher salinities, it grew and has been observed at low salinities. Therefore, the probability of ASP from consumption of oysters harvested from the low salinity estuaries of the northern Gulf of Mexico is low but not zero; animal mortality events from toxin vectors other than oysters at higher salinity on the shelf are more likely.     

Occurrence of (24S)-24-methylcholesta-5, 22E-dien-3β-ol in the diatom Phaeodactylum tricornutum

The principal sterol of the marine diatom Phaedactylum tricornutum was identified as (24S)-24-methylcholesta-5,22E-dien-3β-ol. Two deuterium atoms were incorporated into this sterol when the diatom was cultured in the presence of [CD₃]methionine indicating a 24-methylene intermediate.     

Diatom Succession and Interaction in Littoral Periphyton and Plankton

Periphyton and plankton samples were collected at four littoral stations in a relatively shallow, eutrophic lake (Elk Lake, B.C., Canada) over a six month period from August 1967 to January 1968. The most abundant planktonic diatoms demonstrated a pattern of seasonal succession typical of temperate lakes, and all were present in the periphyton. This pattern was identical at all stations:Fragilaria crotonensis was dominant from August to October,Asterionella formosa in November and December, andMelosira italica (plusM. varians) in January.F. virescens, although never dominant, peaked in October. Periphyton communities were dominated byAchnanthes minutissima, Cocconeis placentula, F. crolonensis andF. virescens. Considerable station variation in successional patterns occurred over different exposure periods; station differences were least in those samples immersed for monthly intervals, and greatest in those immersed for a cumulative four-month period. Interaction between the phytoplankton and periphyton was illustrated by the occurrence of species common to both habitats. A decrease in cell numbers and percent abundance of these species in planktonic populations coincided with their increase in the periphyton, a relationship which appeared dependent on the breakdown of thermal stratification in November. For example, following turnover,F. crotonensis andA. formosa settled out of the plankton and correspondingly increased in percent abundance in the periphyton. This interdependence was less evident in the four-month samples, whereA. minutissima andC. placentula dominated throughout the entire period and appeared to out-compete the more typical planktonic components for diminishing substrate area. Species interaction or competition was accentuated as exposure duration and periphyton total cell standing crops increased and species diversity decreased, and appeared to account in part, for station differences in successional patterns.     

Valve morphogenesis in Diploneis smithii (Bacillariophyta)

Diploneis species have perhaps the most complex valve structure among pennate diatoms. The development of this structure was studied in Diploneis smithii and begins with the formation of a primary band, which then develops secondary arms at both poles and the center, as in the classic Chiappino–Volcani model of raphid diatom ontogeny. Spine‐like projections grow out from the primary band and secondary arms to establish the transapical ribs (virgae) of the mature valve and themselves develop spines, which are spaced first oppositely and then alternately and fuse with each other to delimit the stria pores. Subsequently, new pattern and structures develop both externally (formation of bifurcating projections that fuse to delimit the outer, sieve‐like layer of the valve) and internally (growth and fusion of flanges from the first‐formed ribs to create the longitudinal canals and deposition of a hymenate strip over the internal face of each stria). Comparisons are made with morphogenesis in other diatoms. Diploneis smithii ontogeny suggests how very slight developmental changes might have created the very variable external morphology of Diploneis species. It also indicates that the longitudinal canals of Diploneis and Fallacia have different origins, since the porous external wall is not formed as a unilaterally attached flap in Diploneis and the canal is internal to the first‐formed rib–stria system in Diploneis, but external to it in Fallacia.