Regular monitoring of estuarine and coastal areas at fixed points was carried out to investigate changes in biomass and species composition of thraustochytrids, which are colorless heterotrophs in the class Labyrinthulomycetes, Stramenopiles. Cell number counts using the most probable number (MPN) method with pine pollen baiting showed that 1 or 2 conspicuous peaks in abundance (termed 'thraustochytrid spikes') occurred between spring and late summer in most sampling years. Changes in thraustochytrid biomass had no correlation with phytoplankton abundance, although spikes seemed to occur after reductions in salinity and associated rapid rises of water temperature. The average cell density at the estuary site, excluding values during spike periods, was 4670 cells l −1 . Although thraustochytrid biomass was only 1.59% that of bacterial biomass, the fixed energy (as biomass) transferred directly from thraustochytrids to zooplankton was estimated to be 15.9% of that transferred from bacterioplankton via phagotrophic protists. This is because, per the theory of energy efficiency, energy transfer between trophic levels only creates 10% of the net production in the next trophic level relative to the first. The phylogenetic identification of established strains revealed an unexpectedly high diversity of thraustochytrids, including 10 unidentified lineages. A similar seasonal succession of phylogenetic groups was observed in each year of sampling. The differences in thraustochytrids isolated at each monitoring site and date suggest that habitat segregation may occur as a result of differences in environmental factors such as water temperature, salinity, and nutrient sources.

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo r ), driven with an ubiquitin or an EF-1␣ promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo r marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo r mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single-or double-crossover homologous recombination. Finally, the fatty acid ⌬5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-␥-linolenic acid (C 20:3n-6 ) and eicosatetraenoic acid (C 20:4n-3 ), substrates for ⌬5 desaturase, and a decrease of arachidonic acid (C 20:4n-6 ) and eicosapentaenoic acid (C 20:5n-3 ), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.

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