Do ciliates feel the stress?

To answer this and other questions about the response of protistan microzooplankton (ciliates) to “stressed” phytoplankton we used the Flow-Cam and an inverted microscope to enumerate and monitor live ciliates from all 12 mesocosms. We explored whether the microzooplankton assemblage changes with their prey, i.e. the “stressed” phytoplankton or whether the ciliates themselves are directly stressed by trace metal depletion or increased CO2.

 

Ciliates are very sensitive. They do not like to be pumped in water masses, squeezed or touched too much. Thus, we sampled mesocosms and fjord water in a very smooth way to keep the ciliates live until inspection with the Flow Cam and the microscope.

…Nancy and Jose on the way to the raft….

…Stella and Jose are carefully performing the sampling practice…

 

Back in the lab: The Flow-Cam is a very useful instrument to analyze live plankton, especially ciliates which are pretty sensitive to any fixative. In the Flow-Cam the live organisms are photographed and counted while flowing through a thin cuvette.

…when Stella was tired to feed the Flow Cam with new live samples Tanya took over…

 

At the beginning of the experiment we filled the mesocosms with fjord water, that was full of diatoms and we found some ciliates (orange frame)…day 1

 

In the middle of the experiment, all kinds of ciliates and phytoplankton developed, depending on different conditions in the mesocosms. Here you can see for example the control-mesocosm and the low iron-mesocosm on day 11. Ciliates are surrounded by a red frame.
…control mesocosm, day 11…

…low iron mesocosm, day 11…

 

The phytoplankton composition at the end of the experiment differed distinctly between the mesocosms. While the the control-mesocosm was full of small flagellates and large dinoflagellates (Ceratium longipes), the low-iron mesocosm was dominated by Emiliania huxleyi.
… control mesocosm, day 21…

…low iron mesocosm, day 21…

 

Most of the ciliates were gone at the end…were they stressed?

We will find it out when we analyse all the data…!

Cheers,

Stella & Tanya

Meso-DINNER


Farewell dinner all-together. We had a delicious dinner (as always). No cooking teams this time. All of us cooked a special dish and we had a great time, laughing, joking, and enjoying a lot!



Experiment Over

The experimental part is now over. Today has been the day of taking the bags out of the raft and clean them. Last samples have been taken and a whole load of analyses awaiting to be performed in our labs.

There were mainly three tasks today: 1) take the last total zooplankton from the bags, 2) take the bags out, 3) clean the bags. Although we could add another one: start packing the scientific material.

Part of the team already left. We wish all the best to Tanya, Nancy and Jose Antonio. Some others are leaving  tomorrow, and the rest, few days later, although a small number of us have still one month more to go.

When lids were taken out, it was quite obvious our success in producing differences in E. huxleyii bloom performance.

The “milky” look of some bags was due to a really high concentration of E. huxleyii cells (over 100,000 per mL).

Others were not able to bloom as much, as you can clearly observe in the following picture!

This was what we see outside, but now look  how the huge bags are deployed underwater.

 

For taking the zooplankton out of the bags, after manual resuspension, a net was immersed and pulled out. The water in the bags were released to the fiord, producing a semi natural bloom that was readily diluted.

The final feeling was satisfaction for carrying out a large experiment with nice preliminary and promising results. Not only science but people, weather, food and all help from the University of Bergen has been much appreciated and enjoyed.

A Sea of Tranquility

So far we are about to end the experiments and we are glad the weather has been really good overall. The time out of the lab we could enjoy beautiful landscapes, and eternal sunsets. This time of the year it is difficult to know by when the sunset is over and the sunrise has just started.

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Tiny Inhabitants of the Fjord

The water from the fjord contains a great diversity of  phytoplankton species. When the experiment started, there were mostly diatoms, but representatives of many other groups could be observed as well. Once nutrients were added to the mesocosms (nitrate and phosphate), Skeletonema costatum became the dominant species for a few days, but they started to disappear as soon as Emiliania huxleyii was growing exponentially reaching high numbers in some of the treatments but not in all of them. Certainly, E. huxleyii performes very different depending on CO2 conditions!

The large dinoflagellate Ceratium was also present in big numbers during a good part of the experiment

Pictures below were taken by Tanya Tsagaraki at the Electron Microscopy Lab of the University of Bergen (Norway)

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Grazers in a bag

We are also interested in  investigating possible differences in copepod feeding due to difference in prey physiology.  We want to test whether different pH and different iron availability will affect the specific feeding of the copepod Calanus finmarchicus on coccolithophores Emiliania huxleyi. We will perform gut content analysis on copepods incubated inside the mesocosms using E. huxleyi  specific primers and real time quantitative PCR (qPCR).

Copepods are collected in the fjord in front of the marine Station using a 333µm plankton net with non-filtering cod end. Mature females of Calanus finmarchicus are sorted and transferred inside plastic cages filled with filtered sea water . These plastic cages will be then incubated inside each mesocosm for 24 hours to allow copepods to feed. After this incubation period, each cage is  removed from the mesocosm,and  copepods are anesthetized for further processing.

We plan to perform a minimum of three experiments. One before the E. huxleyi bloom, one during the bloom and one in the post-bloom phase.

Taking daily water samples from the mesocosms

Every morning each bag is sampled by the sampling team. This team is changed every few days. Sampling requires highly clean procedures to avoid iron contamination of the “low iron” treatment. This includes a silicon tube from the bag to a carboy and vacuum membrane pumps at low pressure, so that organisms and gas content (CO2) are not affected. All the 16 25L carboys used and material is trace metal cleaned every day at night (rinsing with MQ-water, bath in HCL 10 % overnight and 4  rinses with MQ-water at about 6am next morning before we depart to the raft) , and careful manipulation includes gloves and plastic protection of all pieces.

Daily Rutine

These days are being long. The first team goes to the raft sampling at 7:00 and some do not stop working until 23:00 or even later. Every day, water samples are taken from the bags and kept in a temperature-controlled room  in the lab. Then, each subgroup starts its particular subsampling and processing, according to the plan. You can see it in the above tab “Planning“. The plan was fine tuned before the start of the experiments.

Meeting for fine tuning of the planning before experiments started.

Our colleagues form University of Bergen are taking nearly daily samples for cell counts by flow cytometry, and we have been able to observe the succession of  the different phytoplankton groups with time. The predominant species at the end, lucky enough, has been the so wished Emilinia huxleyii. The different treatments of CO2 and iron availability are starting to show clear differences.

Samples arriving from the raft.

Quick transport to the lab. All infrastructure is really close together making these routines really easy. See a panorama view of the station in an older post.

Samples stored in a cool room and immediately subsampled.

Some examples of the routines: Sample filtration for organic and inorganic carbon estimations.

Sample concentration for measuring carbon fixation by the C-14 method.

C-14 measurements in the isotope lab. A nice way to incubate samples at high irradiance without producing too much heat is to use LED lamps. They are giving a nice day-light spectrum, a good irradiance (even over 1000 micro moles m-2 s-1) and virtually no heat! An inexpensive and reliable light source!

Scintillation liquid being added to samples for radioactivity measurements of the photosynthetically-fixed C-14.

Sample processing for virus infecting E. huxleyii and metabolites.

Sample processing for daily chlorophyll fluorometric determinations.

Flow Cam direct observations of organisms living in the bags.

After dinner we always have a group meeting to comment on the day and revise and discuss key data obtained that very day. In the picture above , we just realised that Ehux was finally blooming. Satisfaction in our faces. One of the best moments we have had. Happy moments that sicence gives us!

Not everything is science

We also enjoy a lot having dinner together. We have organised cooking teams of three persons. Each team is in charge of making dinner for the rest. This way each one of us only have to cook every 3 days or so and we always eat well (too well…). We usually plan our menus for 4-5 days and whoever has a bit of time go shopping. The rest help dressing the table or cleaning.

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