What is this virus that infects the phytoplankton?

A few years ago science writer Rose Eveleth joined us  on a research cruise in the North Atlantic. Rose answered readers questions about the interactions of E. hux and the virus that infects it – the organisms we work on at the mesocosm. Read her blog post about viruses that infect phytoplankton. 

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Viruses are the most abundant biological thing in the ocean, but we know very little about them. For a long time, people thought phytoplankton were very long-lived. They died when they were eaten, or floated down too deep, but otherwise scientists couldn’t figure out what would make the little plants die. Turns out viruses kill lots of phytoplankton.                       Read more

 

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What do oceanographers do? Sampling !

Sampling in the Whalen and Harvey Labs is in full swing. Our day begins at 0600 when members of both labs set out in a small boat to the mesocosm platform to collect the necessary water in acid-washed plastic carboys for all of our in-lab experiments. This happens in rain or shine, and we always hope for sunning warm weather, which is not always the case as in Norway, as residents can expect over 200 days of rain.

The Whalen Lab needs 60L of seawater from nutrient “replete” bags alone to prepare for the experiments ahead. We are interested in the molecule 2-heptyl-4-quinolone or HHQ for short. What is so special about HHQ….well, it is a known bacterial quorum sensing molecule – it helps bacteria talk with each other. Bacteria use special molecules to signal to other bacteria in the environment to coordinate behaviors – like turn on virulence genes, make antibiotics, or bioluminesce. Bacteria are always “listening” for these molecules with cell-surface receptors by monitoring their chemical environment in order to track changes in their numbers so they call collectively change their strategy at a moment’s notice.

At low densities of bacteria, quorum sensing molecules, like HHQ, diffuse away in the seawater, and therefore, are present at concentrations below the threshold required for detection. However, when bacteria are extremely abundant, the cumulative production of HHQ leads to locally high concentrations of the molecule in the microhabitat, thereby enabling detection by the bacterial community and inaction of a coordinated response.

 Up until now, HHQ was well known in the medical literature, having originally been discovered in the lungs of patients with severe bacterial infections. Here, HHQ can rapidly accumulate in the mucus surrounding bacterial cells and lead to the coordinated behavior including virulence factors necessary for a pathogenic lifestyle. Last year, we reported HHQ was present in cultures of marine bacteria, and were able to hunt down the genes responsible for this molecule’s production.

 We now know that HHQ does more than function simply as a communication molecule or “infochemical”. When we expose phytoplankton like Emiliania huxleyi, to low concentrations of HHQ, like 1 parts per billion, something interesting happens. This species of phytoplankton stops growing when exposed to low concentrations of HHQ and goes into what in laymen’s terms could be called stasis. The phytoplankton cell neither grows nor dies. This carries on for about three days, when finally death occurs.

 The Whalen and Harvey Labs are trying to understand how such a fundamental chemical used in bacterial communication might impact phytoplankton growth dynamics in the real world. That is where the Bergen Mesocosm’s come in. We have sufficient evidence for the effects of HHQ on individual species of phytoplankton, but we still don’t yet know how a community of phytoplankton and their bacterial buddies will respond in the presence of HHQ when we test this compound in the field. Our experiments for MesoHux2017 will address two general questions: (1) how does the bacterial community change during the course of E. huxleyi bloom in response to HHQ, and (2) how does phytoplankton physiology change at the molecular level in response to HHQ?

 To accomplish these aims, the Whalen lab has set out a very ambitious schedule of sample prep to obtain both DNA from bacterial communities to monitor how the “players” change in response to HHQ; and RNA which will show us what genes are being transcribed in response to HHQ exposure and how phytoplankton respond to bacterial chemical stressors. This requires us to filter lots of seawater from the mesocosm in a 10 deg C (50 deg F) on filters that can collect the material we want – 1 micron filter to retain phytoplankton and a 0.2 micron filter to get all the bacteria.

 Once we filter the water through special plastic filters, we pop these into special plastic tubes that can hold up to extreme cold conditions and drop them into our giant cryoshipper that can maintain a temperature of -120 deg Celsius for 50 days at a stretch. Back at Haverford College, both DNA and RNA will be isolated from filters and further processed to address the questions above.

Members of the Whalen/Harvey Labs have been working non-stop since their arrived, and everyone is pitching in to make the most of our limited time in the fjord. To keep spirits up, members take turns making dinner for the group of 8 scientists. We decided as a group to make a cake as a treat for celebrating our current sampling successes. Little did we know that the language barrier would be just an impediment to making a cake. For example. We thought we bought cake mix…turns out that was frosting. What we thought was frosting was actually custard mix. So we had to go back to the store to actually get cake mix. And the temperature the cake was baked at was for cupcakes and not a sheet pan. But in the end we figured it out and our “3 mistake cake” turned out awesome and the team was all smiles!

Signing off for now!

 Kristen Whalen

Mesocosms: The Middle World

Mesocosms- or a “medium world”- are at the heart of our fieldwork. Mesocosms are large enclosures that allow researchers to study the environment over a certain time period in a controlled way. The mesocosms we are working with are large bags, filled with natural seawater from the Norwegian fjord. The bags are closed off from the surrounding water and can be manipulated to test different hypotheses about how marine microbial communities function and respond to environmental factors. Mesocosm studies are useful tools to link patterns we see in laboratory cultures to the real world. Mesocosms truly represent a middle world for scientists!

One of our first tasks upon arriving to Norway was to setup and deploy the mesocosm bags. The bags are made of a thick, string reinforced plastic that are attached to floating frame and get filled from the bottom. The bags also have a detachable cone at the bottom 1 meter that will allow divers to collect sediments that have settled out (more on the importance of sediments later!). The cones were installed by professional commercial divers. The video below was captured by one of the divers during the time of deployment.

In Oceanography, you have to always expect the unexpected. During the mesocosm deployment, we had difficulty getting the bags, and the bottom settling cone, to sink. We had to come together to think of a quick solution to this problem. After trying several methods, taking multiple trips to the hardware store for weights,  and drilling several holes in the hoop frames, we were able to get the proper deployment!

Once the bags were deployed, we added nutrients (goodies for the phytoplankton!) to help give a jumpstart to a phytoplankton bloom. Three bags were excluded from nutrient additions (these are our control bags). Each bag was then equipped with bubblers to help mix the nutrient throughout the bag. Now we wait!

What do oceanographers do? They filter sea water

According to the National Oceanic and Atmospheric Administration (NOAA) , an oceanographer studies the ocean.  Some oceanographers study whales, sharks, fish and other large creatures, but we focus our research on microscopic life forms – phytoplankton and viruses. In order to collect enough of those tiny creatures, we need to concentrate large numbers of them in smaller volulme. We do this by filtering. We run a lot of sea water through filters that have very small pores and collect the tiny organisms that remain on the filters for analysis. When I say small pore size, I mean really small. We use a pore size of 1.2uM to collect phytoplankton cells. 1.2uM is 1.2/1,000,000 of a meter (or 0.00005 of an inch)* and 15 times smaller than the width of a thin human hair!  For other samples, such as viruses and non-living particles, we use an even smaller pore size – down to 0.02uM (those are the Anotop filters listed below). We will use thousands of filters to collect different samples while in Espegrend.

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Filters we shipped to the station for the project.

 

 

 

 

 

 

 

 

 

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Thanks to Sandra Lanman for her help with editing this post.

 

Espegrend – here we come!

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Espegrend, sometimes also called Espeland, is a Norwegian marine biological station. run by University of Bergen, Norway.  It is located about 20km south of Bergen.

Espeland has a number of specialized facilities and internationally well-known for is mesocosm facility.

From May 8 to June 2, 2017 a group of scientists (that’s us) will be at the station to study the interaction of the globally important phytoplankton Emiliania huxleyi (E. hux) with the viruses that infect and kill E. hux cells.  Read more about why and how we plan to to study this here.

We will post regular updates here  and we invite you to follow us on Facebook and Twitter

To see photos taken during a mesocosm experiment in 2008 in Espeland – click here.