As the saying goes, “When life gives you lemons, turn that tartness into little translucent balls in which to grow your young.” At least, that’s how the saying goes for a tiny insect called a cynipid wasp, whose larvae were recently discovered inducing plant growths called galls that contained acidity levels akin to lemons.

“This is exciting because it represents a novel defense system, one we haven’t seen before,” said Antoine Guiguet, an entomologist at Penn State and lead author on a paper about the discovery published today (March 1) in Biology Letters.

For decades, it has been known that most cynipid wasp species inject chemicals into leaves to induce oak trees to produce protective galls — or growths — around their larvae to ensure the safety of their developing offspring. The gall houses and feeds the insects during their larval development and serves a defensive function to ward off natural enemies. The galls eventually fall from the tree and the wasp larva eat their way out, leaving behind the little balls to decompose on the forest floor.

All this work takes chemistry and, until recently, the main defensive compounds identified in galls were tannins that accumulate on the gall’s surface, preventing damage by herbivores that might feed on the gall. In fact, the tannin levels are so high in oak galls that when crushed and soaked in water, they create a dark brown liquid that forms the base of a long-lasting ink — ink that was once used to write the U.S. Declaration of Independence, Constitution and Bill of Rights.

“It’s so fascinating because this is an animal using chemistry to force a plant to do its bidding,” said John Tooker, professor of entomology at Penn State and co-author on the study. “It's really a parasitic manipulation. The insect gets the plant to make the exact food it needs, which explains the nutritional hypothesis for why galls evolved, but undoubtedly, it has to be combined with a defense aspect, because if you have a good food source, other things are going to want to eat it.”

In their study, the researchers revealed a potentially novel manipulation of host-plant chemistry in the translucent oak gall, in which the wasp lowered the pH level of the interior of their developing gall to the acidity levels used by pitcher plants.

“We know that pH this low is rare in plants generally,” said Tooker. “And the pH that we measured was close to the acidic nature of what's inside a pitcher plant, which is about the same as a lemon. We are hypothesizing that the role of this is defense. Anything that wants to bore in there would be deterred by that acidic environment.”

The researchers examined the organic acid content of the translucent oak gall and compared it to fruits and other galls using mass spectrometry, an analytical technique used for the study of chemical substances. They found that malic acid, an acid with particularly high abundance in apples, represents 66% of the organic acid detected in the galls. The concentration of malic acid was two times higher than in other galls — and two times higher than apples. They also found that the pH of the gall was between 2 and 3, making it among the lowest pH levels found in plant tissues.

“Malic acid is a fundamental component of the metabolism of cells, so it’s there within the oak, within all plant and animal cells, just at a low concentration,” said Guiguet. “What’s amazing is this wasp is a capable of inducing its accumulation in the storage compartment in plant cells, called a vacuole.”

With a pH level below 3, the translucent oak gall is among the most acidic plant tissues measured to date. Until this discovery, only citrus fruit tissues were known to be capable of this extreme acidity, he explained

The researchers hypothesize the wasp could have developed acidic galls as an alternative strategy to the tannin accumulation observed in most other oak galls. Like tannins, low pH could decrease the efficiency of protein digestion in insects because caterpillar hindguts are highly alkaline, they said.

But unlike tannins, acidic environments could also prove efficient against parasitoid wasps, the main enemies of cynipid wasps, as it may degrade the tissue of the needle-like organ that species of female parasitoid wasps use to insert eggs into the gall.

“The molecular mechanism by which cynipid wasps induce galls remains a mystery,” said Guiguet. “Now we have added to this mystery by showing they have evolved with capability of altering pH.”

Other Penn State co-authors on the paper are Nathaniel McCartney, Andrew Deans, Jared Ali and Heather Hines. Kadeem J. Gilbert of Michigan State University is also a co-author on the study.

The work was supported in part by the National Science Foundation.