Μm-filtered Seawater Research Articles

Till Death Do Us Part: Stable Sponge-Bacteria Associations under Thermal and Food Shortage Stresses

Sporadic mass mortality events of Mediterranean sponges following periods of anomalously high temperatures or longer than usual stratification of the seawater column (i.e. low food availability) suggest that these animals are sensitive to environmental stresses. The Mediterranean sponges Ircinia fasciculata and I. oros harbor distinct, species-specific bacterial communities that are highly stable over time and space but little is known about how anomalous environmental conditions affect the structure of the resident bacterial communities. Here, we monitored the bacterial communities in I. fasciculata (largely affected by mass mortalities) and I. oros (overall unaffected) maintained in aquaria during 3 weeks under 4 treatments that mimicked realistic stress pressures: control conditions (13°C, unfiltered seawater), low food availability (13°C, 0.1 µm-filtered seawater), elevated temperatures (25°C, unfiltered seawater), and a combination of the 2 stressors (25°C, 0.1 µm-filtered seawater). Bacterial community structure was assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene sequences and transmission electron microscopy (TEM). As I. fasciculata harbors cyanobacteria, we also measured chlorophyll a (chl a) levels in this species. Multivariate analysis revealed no significant differences in bacterial T-RFLP profiles among treatments for either host sponge species, indicating no effect of high temperatures and food shortage on symbiont community structure. In I. fasciculata, chl a content did not significantly differ among treatments although TEM micrographs revealed some cyanobacteria cells undergoing degradation when exposed to both elevated temperature and food shortage conditions. Arguably, longer-term treatments (months) could have eventually affected bacterial community structure. However, we evidenced no appreciable decay of the symbiotic community in response to medium-term (3 weeks) environmental anomalies purported to cause the recurrent sponge mortality episodes. Thus, changes in symbiont structure are not likely the proximate cause for these reported mortality events.

Simultaneous estimation of viral lysis and protozoan grazing on bacterial mortality using a modified virus-dilution method

A modified dilution method designed to simultaneously estimate bacterial mortality due to viral infection and protozoan grazing was developed and compared with the standard dilution protocol. Various fractions of original seawater (non-filtered) and 1.0 μm-filtered seawater (grazer-free) collected from coastal waters in Hokkaido, Japan, were diluted with 10 kDa filtered seawater (virusfree) to set up 4 gradients of predator–prey interaction, and monitored every 12 h for bacterial abundance during a 48 h incubation. In more diluted fractions, bacterial abundance increased rapidly, and a good linear fit with a negative slope (as mortality) was found between the apparent growth rate and fractions of original water. The resulting slopes of the regression in samples prepared from the original seawater were significantly higher than grazer-free samples, which denotes both viral lysis and protozoan grazing in the former, and only viral lysis in the latter. Ranges of specific lytic and grazing rate were 0.53 to 0.98 and 0.05 to 0.13 d–1, respectively, and lytic pressure accounted for 87 to 91% of the total mortality. Comparison of our method (using virus-free diluent) with the standard dilution protocol (using 0.2 μm diluent) showed significant differences in slope and y-intercept (potential growth rate without mortality) found at 0.26 d–1 and 0.88 d–1, respectively. The above results suggest that using the standard dilution protocol might underestimate instantaneous growth rate, particularly in environments where lytic pressure is relatively high.

The role of particles on the biogeochemical cycling of domoic acid and its isomers in natural water matrices

The adsorption of dissolved domoic acid (DA) and its geometrical isomers was assessed in aqueous solutions containing various types of particles. In one series of experiments carried out in coastal seawater, detectable net adsorption of 100 nM DA occurred only onto natural seawater particles (unfiltered seawater) and 0.5 g L −1 chromatographic silica (18%) in 0.2 μm-filtered seawater. Some net adsorption (<5%) also occurred in the 0.5 g L −1 suspension of estuarine sediment and 0.5 g L −1 solution of humic acid in filtered seawater. No losses were seen in 0.5 g L −1 suspensions of illite, kaolinite, montmorillonite, and silica sand. Biological degradation accounted for small losses (8–10%) in filtered seawater without particles. A second series of experiments using organic-free, <5 μm fractions of kaolinite and montmorillonite in deionized water (DIW) demonstrated that ∼70% of DA adsorbed onto kaolinite, but only 5% onto montmorillonite. Geometrical isomers of DA ( iso-DA D, E, and F) showed negligible adsorption (0–8%) onto a variety of particles in filtered seawater, suggesting that major ions in seawater neutralize electrostatic attractions between particles and DA isomers. These results suggest that DA and its isomers are relatively hydrophilic and not particle reactive. Our data suggest that photochemical and biological degradation of dissolved DA and its isomers appears to occur in bulk surface seawater and its transport to bottom sediments must be mainly biologically driven.

Evidence of Trichodesmium viral lysis and potential significance for biogeochemical cycling in the oligotrophic ocean

The planktonic cyanobacterium Trichodesmium spp. is a globally important diazotroph, fixing at least 80 Tg N yr -1 in tropical waters. Despite its biogeochemical importance, the mechanisms of Trichodesmium mortality, and the means by which its fixed N enters upper levels of the food web, are poorly understood. Potential virus-like particle (VLP) production by Trichodesmium spp. was observed in both culture (IMS101) and field samples from the tropical North Pacific Ocean, in oceanic waters around the Hawaiian Islands. VLP observed by TEM in IMS101 lysate were approximately 56 nm wide and untailed, and VLP with similar morphology were observed in tissue treated with mit- omycin C. A most-probable number cultivation technique (utilizing bacterized cultures of Tricho- desmium) detected moderate abundances of Trichodesmium-infecting cyanophage (605 to 9750 ml -1 infecting only 1 cultured strain) in 0.2 µm-filtered seawater samples from surface, subsurface and deep chlorophyll maximum samples. Estimation of mortality from virus production was not possible due to rapid VLP release in the first 6 h of dilution incubations. Rather, an indirect approach using burst size (determined from mitomycin C treatment of washed trichomes resuspended in virus-free seawater), decay rate of VLP (from latter part of virus production incubations) and average cyanophage titer was used to estimate mortality. These conservative calculations suggested that 0.3 to 6.5% d -1 (mean = 1.65 ± 0.99% d -1 ) of trichomes could potentially be lysed by viruses, represent- ing the release of approximately 3 to 64% fixed N d -1 . These estimates are based upon a steady-state maintenance of observed VLP abundance, which in nature could be from lysogeny, pseudolysogeny, carrier state, or successive lytic infection. Viral lysis therefore may represent a significant novel mechanism of N release from Trichodesmium spp., even in non-bloom conditions.

Dissolved organic carbon contamination induced by filters and storage bottles

To optimize experimental protocol for marine dissolved organic carbon (DOC) studies in laboratory conditions, we examined the potential DOC releases from filters of different qualities as well as those from different types of bottles during 10 day-storage of Milli-Q water and 0.2 μm-filtered seawater. The results showed that utilization of 47-mm circle filters including polycarbonate (0.2 and 1 μm-pore size), polytetrafluoroethylene (PTFE: 0.5 μm-pore size) and aluminum oxide (0.2 and 0.02 μm-pore size) may induce significant DOC contamination ranging from 12 to 42 μM C for the first hundred milliliters of the filtrated solutions. However, filtrations of 2 to 4 l of Milli-Q water decreased the DOC blank of the filters to 2–5 μM C. As expected, precombusted (450°C, 6 h) glass fiber filters rinsed with 0.5–3 l of Milli-Q water produced very low blanks (1–4 μM C). We found that 10 day-storage (15°C, in the dark) of Milli-Q water and 0.2 μm-filtered seawater (250 ml) into 1 l-polyethylene, polycarbonate and perfluoalkoxy (PFA)-Teflon bottles, initially cleaned with HCl 1% and abondantly rinsed with copious amounts of Milli-Q water as well as in precombusted glass bottles (450°C, 6 h) induced very low DOC contamination of the solutions (0–5 μM C). Although complete DOC-free contamination experiment is difficult to reach particularly in shipboard conditions, these results strongly suggest that appropriate cleaning procedure of the material is essential to reach blank level compatible with DOC studies in marine environments.

A combined microcosm and mesocosm approach to examine factors affecting survival and mortality of Pseudomonas fluorescens Ag1 in seawater

The survival of Pseudomonas fluorescens Ag1 in seawater of Roskilde Fjord (Denmark) was evaluated by a series of laboratory microcosm and field-based mesocosm experiments. In sterile seawater microcosms, culturability of Ag1 was negatively influenced by high salinity (34 versus 8.5‰). In microcosms with 0.2 μm-filtered seawater, addition of a carbon + nitrogen + phosphorus nutrient mixture was needed to induce proliferation of Ag1. In nutrient-amended microcosms the Ag1 population maintained viability, as determined by the direct viable counts method, at a level close to 100%. In natural water microcosms, Ag1 decreased by two to three orders of magnitude in three days. Field experiments in 5300-litre seawater enclosures demonstrated a less pronounced decline. The observed average decline rate agreed well with the calculated average predation potential of heterotrophic nanoflagellates using fluorescence-labelled Ag1 as prey. During the experiment, mesocosms were amended with nutrients as in microcosms to attempt induction of Ag1 cell proliferation in situ, but the decrease rate of Ag1 remained unchanged. Viability remained above 25% throughout the experiment, emphasizing that the decline of Ag1 did not result from extensive cell death. The combination of micro and mesocosms proved useful as a test scenario for fate studies of microorganisms introduced into an aquatic environment.

A combined microcosm and mesocosm approach to examine factors affecting survival and mortality of Pseudomonas fluorescens Ag1 in seawater

The survival of Pseudomonas fluorescens Ag1 in seawater of Roskilde Fjord (Denmark) was evaluated by a series of laboratory microcosm and field-based mesocosm experiments. In sterile seawater microcosms, culturability of Ag1 was negatively influenced by high salinity (34 versus 8.5‰). In microcosms with 0.2 μm-filtered seawater, addition of a carbon + nitrogen + phosphorus nutrient mixture was needed to induce proliferation of Ag1. In nutrient-amended microcosms the Ag1 population maintained viability, as determined by the direct viable counts method, at a level close to 100%. In natural water microcosms, Ag1 decreased by two to three orders of magnitude in three days. Field experiments in 5300-litre seawater enclosures demonstrated a less pronounced decline. The observed average decline rate agreed well with the calculated average predation potential of heterotrophic nanoflagellates using fluorescence-labelled Ag1 as prey. During the experiment, mesocosms were amended with nutrients as in microcosms to attempt induction of Ag1 cell proliferation in situ, but the decrease rate of Ag1 remained unchanged. Viability remained above 25% throughout the experiment, emphasizing that the decline of Ag1 did not result from extensive cell death. The combination of micro and mesocosms proved useful as a test scenario for fate studies of microorganisms introduced into an aquatic environment.

Chitinase activity in the crystalline style of the american oyster Crassostrea virginica

1. 1. Presence of chitinase in Crassostrea virginica crystalline styles was established. 2. 2. Specific activity of chitinase tended to increase towards the anterior portion of the style. 3. 3. Chitinase activity was not stimulated by chitin additions to 1 μm filtered sea water. 4. 4. Kaolin additions to the diet resulted in doubling chitinase specific activity.