17-year cicadas, 2004 |
Every 17 years around Washington DC, millions of 1-2 inch
long insects crawl out of small holes in the ground that they’ve dug, metamorphose into giant cicadas then fly
into windows, smash onto car windshields, carpet sidewalks, and generally
make a sensational showing. These are the 17-year cicadas, species Magicicada [some refer to these as locusts, but they're not], one of the most understudied
insects because it can only really be investigated every 17 years. 2004 was the
last eruption of this brood of insects in the east. They’ll be back out next
year, 2021!
Brown branch tips chewed by cicadas |
Geophysical Lab summer intern Hilary Christensen was a young
biology major from Carleton College and fit the bill for someone to take the
lead on using stable isotopes for studying the diet and nutrition of these
small beasts. Our study plan was to collect specimens in two locations—the
grounds of the Lab and Hilary’s hometown in Pennsylvania. She sampled seven
specimens in each spot, as well as leaves from nearby trees, that most likely
hosted the locusts underground for the past 17 years. She also collected 50
wing samples in each location from newly expired cicadas that died after a very
brief mating period.
Magicicada cicadas
survive on xylem fluids—the fluid found in a plant’s vascular system that
brings water from the roots up to the branches and leaves. They do this while
surviving deep underground, associated with just one tree, for 17 years! But
they have a major nutritional problem. Xylem fluids, which we measured, don’t
have a full complement of the amino acids needed to build the cicada’s body. In
particular, the xylem fluid we measured lacked several essential amino acids,
those that animals can’t make themselves and must get from their food. How were
they surviving on this minimal diet for 17 years?
Many insects have a similar problem because they eat leaves
with low protein amounts or like the cicada have inadequate food sources. They
get by, and do so with great vigor, by hosting
microbes in their guts that convert xylem fluids or leaf material into
amino acids that are then used by the organism to build structural tissues.
When Hilary and I analyzed the amino acid isotope patterns in the cicadas, they
did not match with amino acids from leaves taken from area trees, and several
weren’t present at all in the xylem fluid.
We concluded then that gut microbes supplied all of the cicadas’
essential amino acids--valine, isoleucine, and lysine--as well as significant
portions of the other amino acids. It took us 7 years to publish the work. By
that time, I’d thought further about this with postdoc Seth Newsome. We were
developing a collaboration in 2008 with Carlos Martinez del Rio, an ecologist
from the University of Wyoming with a specialty in combining ecology with
physiology.
Carlos had a student who had grown tilapia on controlled
diets with varying amounts of protein. The idea was that with lower amounts of
protein in their diet, the fish would need to synthesize all of their nonessential
amino acids rather than obtain them directly from their food. With increasing
amounts of protein, the hypothesis was that amino acids would be routed
directly from the protein in their diet into fish tissues. It was assumed that
the essential amino acids would show little change and would have the same
isotope pattern as their diet.
Carlos Martinez del Rio and Seth, Geophysical Lab 2008 |
When the data was collected and the results inspected, we
were surprised to see that in no case did the essential amino acids have the
same isotope patterns as their diet. At low concentrations of dietary protein,
50-70% originated from another source—the
microbes in the tilapia’s guts. With very high protein diets, even then bacteria
were producing 10-20% of the essential amino acids. We wrote the paper, as we
all do, with the slant that we had figured this out before we made the
measurements. That might sound cocky to the non-scientist, but if you write a
paper describing your discovery “path”, reviewers always seem to ask you to
revise it. This work was also published in 2011.
Seth moved on from the Geophysical Lab to the University of
Wyoming to continue working with Carlos. While there, he also branched out and
began independent work with grad student Nathan Wolf. In the first of many
experiments with laboratory mice, Seth and Nathan grew mice on a diet with
varying amounts of fat (i.e. lipids). The protein the mice were fed originated
from C3 plants, while the fats were corn oil, corn being a C4
plant. (See former blog posts on what these mean). Because the stable isotope
signatures of compounds from these plants are very different, we could tell if
the mice were using protein or fat to build muscle tissue.
This experiment was designed to answer an argument in the
isotope community about the role of lipids in analyzing and interpreting the
isotope patterns in whole tissues. Half of the stable isotope ecology community
extracted and discarded the lipids in animal and diet tissues, finding them an
unnecessary problem in interpreting the isotope data. Others, particularly
people who studied animals that had fat heavy diets, knew that fats were
important and needed to be part of the analyses. Our experiments confirmed that
even in mice, the carbon atoms in dietary fat were re-used to produce mouse
muscle.
Not only did the mouse use fats to make the nonessential
amino acids in their tissues, but we also found strong evidence that some of
that carbon from fat ended up in the essential amino acids as well. We
concluded in this work that gut microbes produced valine and isoleucine, two of
the amino acids made by microbes in the tilapia experiment. A second experiment
with tilapia was conducted in 2012 to investigate hydrogen isotope patterns.
When the results were completed, it was—once again—clear that essential amino
acids coming from the rumps of microbes, more often than not, were
supplementing animal diet.
Now at the University of New Mexico, Seth, colleague Tina
Vesbach, and I submitted an NSF pre-proposal to continue the work. It was
rejected as not making the mark of a detailed enough project. We completed
another study with mice, and used that data for a second submission to NSF. The
study was finally funded and is currently underway!
Our model of how microbe amino acids get into tissues |
My fourth paper on this subject with those
results that convinced NSF to fund our work was recently published in the Proceedings of the Royal Society B, a
prestigious journal with a wide audience. The manuscript had been rejected,
however, from PNAS, Science Advances,
Functional Ecology, and ISME
before we re-submitted it in December 2019 to the Royal Society journal. After
three full rounds of review, it’s now out.
People are sometimes consumed with new diet crazes, like the
current keto-diet fad. The keto-diet requires you to give up all carbs—no
potatoes, no bread, and even no fruit! Certainly, no sugar—no cookies, soda or
ice cream. Drastic changes like this to human diet must come with changes in the
types of bacteria living in a person’s gut. Our isotope work is now linked to
changes in the gut microbial community with changes in diet. In our work with
mice, we found—once again—that valine and isoleucine were made by microbes
30-60% of the time. We also documented a shift in the type of microbe from
Bacteroidetes to Firmicutes as dietary sugar increased. We don’t know yet
exactly how this seemingly simple process happens or what those particular
microbes are actually doing.
Proc. Royal Society B: microbes contribute a lot |
The bottom line is one that I believe will be increasingly
important in using amino acid isotope patterns for tracing animal diets. I
suspect that in the coming year more researchers will have a closer look at
their data and realize that those microscopic
beasts in animal guts are key to more than has been generally recognized.
Stay tuned for our new results using position-specific
isotope labels, micro-capturing of individual species, and more realistic
diets.