Eukaryotic microbes, principally fungi and labyrinthulomycetes, dominate biomass on bathypelagic marine snow.

In the bathypelagic realm of the ocean, the role of marine snow as a carbon and energy source for the deep-sea biota and as a potential hotspot of microbial diversity and activity has not received adequate attention. Here, we collected bathypelagic marine snow by gentle gravity filtration of sea water onto 30 µm filters from ~1000 to 3900 m to investigate the relative distribution of eukaryotic microbes. Compared with sediment traps that select for fast-sinking particles, this method collects particles unbiased by settling velocity. While prokaryotes numerically exceeded eukaryotes on marine snow, eukaryotic microbes belonging to two very distant branches of the eukaryote tree, the fungi and the labyrinthulomycetes, dominated overall biomass. Being tolerant to cold temperature and high hydrostatic pressure, these saprotrophic organisms have the potential to significantly contribute to the degradation of organic matter in the deep sea. Our results demonstrate that the community composition on bathypelagic marine snow differs greatly from that in the ambient water leading to wide ecological niche separation between the two environments.

Dragon kings of the deep sea: marine particles deviate markedly from the common number-size spectrum.

Particles are the major vector for the transfer of carbon from the upper ocean to the deep sea. However, little is known about their abundance, composition and role at depths greater than 2000 m. We present the first number-size spectrum of bathy- and abyssopelagic particles to a depth of 5500 m based on surveys performed with a custom-made holographic microscope. The particle spectrum was unusual in that particles of several millimetres in length were almost 100 times more abundant than expected from the number spectrum of smaller particles, thereby meeting the definition of "dragon kings." Marine snow particles overwhelmingly contributed to the total particle volume (95-98%). Approximately 1/3 of the particles in the dragon-king size domain contained large amounts of transparent exopolymers with little ballast, which likely either make them neutrally buoyant or cause them to sink slowly. Dragon-king particles thus provide large volumes of unique microenvironments that may help to explain discrepancies in deep-sea biogeochemical budgets.

A simple separation method for downstream biochemical analysis of aquatic microbes.

In order to study the chemical composition of aquatic microbes it is necessary to obtain completely separated fractions of subpopulations. Size separation by filtration is usually unsuccessful because the smaller group of organisms contaminates the larger fractions due to being trapped on filter surfaces of nominally much larger pore sizes. Here we demonstrate that a simple sucrose density separation method allowed us to separate microorganisms of even subtle size differences and to determine their bulk biochemical composition (proteins, polysaccharides+nucleic acids, and lipids). Both autotrophs and heterotrophs (through anaplerotic pathways) were labeled with (14)C-bicarbonate for biochemical fractionation. We provided proof of concept that eukaryotic microbes could be cleanly separated from prokaryotes in cultures and in field samples, enabling detection of differences in their biochemical makeup. We explored methodological issues regarding separation mechanisms, fixation, and pre-concentration via tangential flow filtration of oligotrophic marine waters where abundances of microorganisms are comparably low. By selecting an appropriate centrifugal force, two processes (i.e., isopycnal and rate-zonal separation) can be exploited simultaneously resulting in finely-separated density fractions, which also resulted in size separation. Future applications of this method include exploration of the stoichiometric, biochemical and genetic differences among subpopulations of microbes in a wide variety of aquatic environments.

Role of macroscopic particles in deep-sea oxygen consumption.

Macroscopic particles (>500 mum), including marine snow, large migrating zooplankton, and their fast-sinking fecal pellets, represent primary vehicles of organic carbon flux from the surface to the deep sea. In contrast, freely suspended microscopic particles such as bacteria and protists do not sink, and they contribute the largest portion of metabolism in the upper ocean. In bathy- and abyssopelagic layers of the ocean (2,000-6,000 m), however, microscopic particles may not dominate oxygen consumption. In a section across the tropical Atlantic, we show that macroscopic particle peaks occurred frequently in the deep sea, whereas microscopic particles were barely detectable. In 10 of 17 deep-sea profiles (>2,000 m depth), macroscopic particle abundances were more strongly cross-correlated with oxygen deficits than microscopic particles, suggesting that biomass bound to large particles dominates overall deep-sea metabolism.

Re-evaluation of the EUK516 probe for the domain eukarya results in a suitable probe for the detection of kinetoplastids, an important group of parasitic and free-living flagellates.

Two frequently used universal eukaryote probes, EUK1209 and EUK516, are not consistent with one branch of the eukaryotic phylogenetic tree, the Kinetoplastida, which has undergone rapid evolution of their small subunit rRNA gene. Kinetoplastids include medically important parasitic organisms (e.g. Trypanosoma, Leishmania) and free-living flagellates that occur in all aquatic environments and in soils (e.g. Bodo, Neobodo, Rhynchomonas). A modified probe presented here as KIN516, now based on the kinetoplastid sequence, provides a strong signal with Neobodo designis, Leishmania donovani, and Trypanosoma cruzi using the catalyzed reporter deposition protocol. EUK516 and KIN516 function as competitor probes, thereby greatly increasing discriminatory power when used in combination. The probe pair was tested in field samples collected in a freshwater pond in Norfolk, the mesohaline Elizabeth River, Norfolk, Virginia, and a tropical lagoon in Belize. The combined probes bound to 58-84% of organisms identified as eukaryotic based on having large DAPI-stained nuclei. The contribution of kinetoplastids to total eukaryotes (positive signal of EUK516+KIN516) was much higher in marine samples (ca. 17%) than in either the freshwater or brackish water sites (<0.2%).