|Whiteman Laboratory at the University of Arizona||
After a month, our plants in the common garden, which have been pre-treated with the plant hormone SA, plant hormone JA, infected with one of three strains of Pseudmonas or a mock solution are ready for implantation with Scaptomyza larvae for our herbivory bioassay. The point of this experiment it to understand how prior infection with bacteria influences subsequent susceptibility to herbivory--much like prior infection in humans leads to secondary infections...This requires finding larvae from host plants that are then transplanted into experimental plants at Emerald Lake.
Above, just as in any garden, there is a lot of tedious tending that is required...here Nicolas Alexandre (University of Arizona undergraduate researcher) and Noah Whiteman (University of Arizona assistant professor) work to transplant a larva into a common garden plant.
This larva has just been placed on this leaf--hopefully it will burrow in within an hour and start feeding.
Noah prepares to pass Nic a larva for transplantation, but first, Nic wants to tell a joke.
Above, two days after we have transplanted the larva, it has fed on the leaf tissue and is ready to be removed from common garden (above).
Michelle Smith (above) was an extremely talented student from Florida State University who helped us process the hundreds of leaves and larvae...
Above, a meeting of the minds in the common garden (Nic, Noah and Cole). What this really shows is Noah, the most senior member of the team, needing to stand because his legs are falling asleep.
Above, Nic, Noah and Cole after a long, cold day at the common garden, prepare to head down valley to process leaves in the lab.
Above, Noah and Nic contemplate how to navigate along the very narrow road down from Schofield Pass to the Rocky Mountain Biological Laboratory (RMBL).
Above, back at RMBL in the Gothic Research Center, Nic places leaves with larvae on a sheet for processing. Each will be photographed and weighed.
It is very late in the season for finding Scaptomyza flies. So in hopes of catching a batch of late comers, we trekked to the highest point at North Pole Basin (~12,000 ft). The top most streams still had huge patches of Cardamine in bloom.
We used aspirators to catch the flies. The aspirators are constructed using two pieces of tubing attached to a glass bottle. When you breathe in through the tubing, a fly gets pulled in from the other side and is trapped in the container. In case you were wondering, there is a filter to prevent accidental inhalation. Unfortunately, after searching the Cardamine patches we didn't find many Scaptomyza.
It's a disappointing setback, but with any type of field work its always good to have a contingency plan. Nature will always do its own thing and won't necessarily coincide with the desired experimental conditions. Even if we can't get adult flies, we can still perform experiments with larvae. There are plenty of larvae to collect down by Emerald Lake.
Scaptomyza larvae normally feed inside Cardamine leaf tissue. However, they can crawl out and find new leaves to inhabit. It's possible that as the larvae feed, they may be tasting the isothiocyanates that form after hydrolysis of the glucosinolates. Since this is toxic to them, they may prefer to feed on Cardamine leaves with lower levels of toxins. We can present Scaptomyza larvae with Pseudomonas infected and uninfected leaves and see which ones they prefer.
We injected bacteria directly into the leaves of Cardamine. To do this we took advantage of natural structures in the plant leaves themselves. It is not obvious, but plants have microscopic openings on the underside of their leaves called stomata. The stomata are involved in gas exchange to intake carbon dioxide and expel oxygen. Fortunately for us, we can use these openings to force fluid into the leaf. Using a blunt syringe we can apply pressure to a volume of bacterial suspension and inject it straight into the leaves. The bacteria will pass through the stomata and profuse through the internal tissue. This method has the benefit of not damaging the outside of the leaves themselves. We will let the infections incubate for a few days to allow the plant to mount an anti-bacterial immune response. We will then observe how this affects herbivory by Scaptomyza.
To test the idea that bacterial infection might influence Scaptomyza larval growth in Cardamine, we will be infecting plants with different strains of bacteria. To maintain a more natural controlled environment for our experiment, we will be using strains of bacteria cultured from the same plants in the same area. These bacteria were isolated from inside the leaves of Cardamine from this region.
The bacteria were stored frozen until now. Living cells can be stored in the freezer for long periods of time; however, eventually they will lose viability as the cells continually die. Ice crystals forming inside the cell will rupture the cell membranes. Also, freezing ice can concentrate dissolved salts in parts of the cell and denature essential proteins. Nonetheless, slowing down chemical reactions in the cell will extend their lifespan. Keeping cells in the freezer is a delicate balance between preservation and minimizing damage.
Proper buffers added to the cells can help reduce the amount of cell death due to ice formation. Cryoprotectants, such as glycerol, can lower the freezing point so that ice crystals form less often. Crypresrevatives, such as polysaccharides or proteins, can form a protective layer around the cell membranes. These can extend the preservation time of frozen cells by upwards of 10 years.
When we need to use the bacteria, we can take a small swab from the frozen stock and add it to a warm nutrient broth. Any bacteria still living will quickly take advantage of nutritious media and start growing rapidly. Within a couple days we will have fresh healthy vials of the local bacteria to use for our experiments.
Our Cardamine for the Choice Experiment are growing well. We put them inside a butterfly tent to protect them from herbivores. Since Cardamine grows on the edges of streams and rivers, we placed our butterfly tent next to the Copper River. We also placed wet towels underneath the pots. This way there is plenty of moisture to keep our plants healthy. The plants are doing so well they have even started flowering. This is a good sign and we can go ahead with our study.
There is hardly a surface on the planet that hasn't been colonized by microorganisms, including inside other organisms. Just like we humans have resident microflora living inside our guts, plants are also home to innumerable bacteria living inside of them. The insides of large organisms house a rich resource of proteins, carbohydrates, and other nutrients.
It's not a stretch to think some bacteria might modify this internal environment to better suit their own needs. Could some bacteria influence plant metabolism to take care of their host, so the colonizers have a safe place to live? Alternatively, maybe some other bacteria weaken their host to make it easier to consume them? Even more fascinating is the possibility that some bacteria might attract herbivores to feed on their host and in the process help spread the infection to other plants? It's conceivable there are many types of interactions between the microscopic and macroscopic communities.
To understand these putative interactions, we first need to identify the types of bacteria that live inside of plants. To do this we collected leaves from a variety of plant species living in Gothic, CO. Since we are interested in the internally living bacteria, we first had to sterilize the external parts of the leaves. We washed each leaf in a series of water, ethanol, and bleach to clean off any bacteria living on the surface. We stored these leaves in the freezer at -80 degrees Celsius. This kills the cells but preserves their genetic material.
The genetic material in the leaves will be analyzed to identify the types of bacterial cells living inside. Genetic sequencing is useful because it samples all cells present in the tissue. Traditional methods of cataloging microbiome communities involved growing bacteria on agar plates. However, the limitation of this method is that only bacteria that are able to grow on agar will be identified. Many types of bacteria simply won't grow outside their normal host. By cataloging all the genetic material present in the tissue, all the bacteria can be accounted for whether or not it can be grown in the lab. The sequencing information can sort out plant cells from bacteria cells and assign an identification to all the bacterial species found.
In parallel with all our other experiments, we wanted to run another complimentary study. We wish to examine whether Scaptomyza flies will actually choose to lay their eggs on specific Cardamine plants. Do they have a preference? Are some plants more appealing than others?
One possibility is that different strains of bacteria living in leaves can exert an influence on insect herbivory. Bacterial infections can lower levels of jasmonic acid, a hormone that acts to increase defenses against insects. Specific bacterial strains may affect the regulation of jasmonic acid in various ways. Some may lower it a little. Others might lower it a lot. Others can even increase it by producing a phytotoxin that mimics jasmonic acid. It will be interesting to see is if Scaptomyza flies can notice these differences. It is possible the flies can taste the levels of isothiocyanates released and decide which plants to lay their eggs on. If a plant is producing less toxins, then the larvae can feed more freely on the plant tissue. Growth and survival of Scaptomyza larvae may depend on adult females making the right choice.
To run this experiment, we needed to get really young Cardamine with minimal fly damage. Since it is so late in the season, we had to hike to the very top of North Pole Basin to find plants that have just sprouted. The Cardamine we found are so young they haven't even flowered yet. We brought these plants back to the lab at RMBL. We will run a controlled experiment where we release Scaptomyza in a cage with the Cardamine and watch which ones get infested with the most larvae.
Infrared heating lamp
The great thing about working at the Rocky Mountain Biological Laboratory is that we can take advantage of some long term research installations. One of these is the Warming Meadow. The Warming Meadow was started in 1990 by John Harte to study the effects of climate change on alpine plant communities.
On one hillside outside of RMBL are set of sophisticated sensors and instruments. These sample the air (measuring moisture, temperature, etc.) and calculate continual changes in the atmosphere. A set of infrared lights then warm the plots to simulate the amount of heat that would be present if carbon dioxide levels were double what they currently are. This translates to a conservative estimate of 2 degrees Celsius increase.
The heaters have been continuously operating since the experiment started, and they keep the warming plots always 2 degrees Celsius higher than non-heated control plots. Predictably, snow melts sooner and the moisture content of the soil is lower in the warming plots.
What is amazing is that even with this seemingly small change in temperature, the plant communities in the Warming Meadow have been drastically altered. The alpine wildflowers that you would normally see are less abundant, and in their place you see a lot more sage brush and other dry-adapted vegetation. The difference is like having wet grassy meadows of the Colorado Rockies replaced with the dry chaparral of the California high desert. Since I am a native Californian, my allergies immediately peaked in response to the familiar plant antigens of my home state.
Unfortunately, what is happening in the Warming Meadows may soon be reflected in the rest of the Rocky Mountains. As global temperatures continue to rise, we may see a shift in local plant communities and a loss of the alpine wildflower meadows. What we are interested in is how this shift may also impact microbial communities.
Cardamine cordifolia (Bittercress) belongs to the order Brassicales, which includes mustard, horse radish, wasabi, brussels sprouts, capers, etc. The characteristic feature of this order is that they produce glucosinolate compounds. Toxins derived from these compounds cause the distinctive spicy or bitter flavors in these plants.
Glucosinolates are plant defense compounds that deter herbivores. The general structure of glucosinolates includes sulfur, nitrogen, and a glucose sugar. The difference in the R-side chain gives each mustard oil its own particular flavor and character. In the presence of the enzyme myrosinase, the glucose portion is hydrolyzed leaving a reactive isothiocyanate (mustard oil). Under normal conditions, glucosinolates and myrosinase are stored in different compartments. However, when plant tissue is damaged (as in the case of an insect feeding on the leaves), the enzyme is released and reacts with glucosinolates to release isothiocyanate mustard oils into the tissue.
The isothiocyanates are relatively toxic have been shown to be biocidal. Depending on the dose, everything from insects, worms, fungus, bacteria, to cancer cells can be killed by various mustard oils. Of course, in low doses we humans can find them delicious; but to something small like an insect, ingesting a mouthful of isothiocyanates is probably unpleasant. This deters some insects from feeding on Brassicales.
However, other insects have evolved ways of dealing with the toxic isothiocyanates. Some are generalist feeders and metabolize isothiocyanate the same way they deal with most other plant toxins. Nonetheless, this still benefits the plant because it takes valuable time and energy to break down plant toxins. This slows down insect feeding and growth rates. Other insects are specialist feeders and they can break down the mustard oils really quickly, or they can metabolize the glucosinolates even before they become toxic isothiocyanates. Scaptomyza is a specialist feeder on Cardamine.
To protect the nascent buds, we placed mesh tents over the young Cardamine to keep these insects out. Later on, we can control plant exposure to herbivores by selectively placing a set number of Scaptomyza larvae with the plants.
Antagonistic relationship between salicylic acid and jasmonic acid
We will be using two different plant hormones in our study, salicylic acid and jasmonic acid . These hormones are induced in response to biological stresses. Salicylic acid is up-regulated when bacteria infiltrate the plant tissue. This starts a cascade of events that produces proteins to attack and limit the growth of bacteria. On the other hand, jasmonic acid is released when herbivorous insects tear apart plant tissue. This starts a separate cascade that produces other proteins that discourage further feeding. Both arms of the plant immune response limit damage from pathogens and herbivores to keep the plant healthy.
Interestingly, these two immune pathways interact and antagonize one another. It's not completely clear if this is a form of adaptive regulation or an evolutionary constraint. However, when salicylic acid is induced, levels of jasmonic acid go down. Conversely, when jasmonic acid is induced, salicylic acid goes down. It may seem strange to have different immune response react adversely to each other, but in animals we see similar cross-talk happen in the immune system. Different arms of the mammalian immune system down-regulate each other as a means of regulation that keeps immune responses from spiraling out of control. A similar regulatory role between the different parts of plant immune responses may explain the the antagonism between Salicylic acid and Jasmonic acid.
This sets up a fascinating scenario for community interaction between insects that feed on plants and the bacterial communities that live in them. For instance, it's possible that Scaptomyza flies may prefer Cardamine plants that have been infected with bacteria to lay their eggs on. A plant that is infected with bacteria will have higher levels of salicylic acid and consequently lower levels of jasmonic acid. With lower levels of defense against herbivorous insects, the fly larvae will be free to dine on the plant tissue unimpeded. Whether or not Cardamine are attacked by Scaptomyza may depend on the level of hormones present in the plant.