Specialized proteins in gall fly larvae create channels that push freezing water out of their cells and let natural anti‑freeze flow into them.

Introduction

In fields and gardens around the world, the tips of goldenrods sparkle with tiny pops of yellow along many spindly branches. Over 100 species of these flowers exist, and most grow natively in North America. They’re late bloomers, ideal for sustaining late-season pollinators.

But they’re not just good for bees and butterflies. Throughout much of North America and especially in central regions, Goldenrod gall flies depend on some of these plants for their winter homes. In the spring, adult female gall flies lay eggs on newly sprouted stems. When an egg hatches, the larva chews its way into the stem. As a result, the stem develops a fat knot called a gall that becomes the larva’s shelter. It’s believed (though not known for sure), that the gall is a result of hormone-driven growth, stimulated by the larva’s saliva. The goldenrod, otherwise indifferent to its guest, continues to grow upward, eventually blooming and dying off in the fall. The larva stays enclosed within the gall throughout winter until warm temperatures stimulate it to transition to a pupa and then adult.

It might seem like wintering inside of a walnut-shaped abode would insulate the larva from the cold, but it doesn’t. Goldenrod gall fly larvae are “freeze-tolerant,” meaning they can freeze down to temperatures of -112°F (-80°C) during winter then thaw in the spring completely uninjured.

Goldenrod blooms
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Goldenrod typically blooms in late summer and flowers can last until October. Female gall flies lay eggs in their stems when they emerge in spring. 

Goldenrod gall harboring a gall fly
Image: Anita Gould / CC BY NC - Creative Commons Attribution + Noncommercial

When the egg hatches, the larva burrows into the stem. Although not completely understood how, it is believed that the saliva from the larva’s chewing stimulates hormonal growth to create the fat walnut‑shaped gall.

Goldenrod gall on the plant (left), gall cut open showing the larval chamber inside (right)
Image: Kent McFarland / CC BY NC - Creative Commons Attribution + Noncommercial

The gall remains on the stem throughout fall and winter (left), and the larva will live in the center chamber (right). One of the last things the gall fly larva does before winter is to burrow a tunnel close to the edge to make it easy to emerge as an adult in the spring (this larva’s tunnel isn’t seen here, but the cross section of the gall reveals the thickness through which it much make its way).

Adult Goldenrod Gall Fly
Image: Scott King / CC BY - Creative Commons Attribution alone

Warmer spring temperatures stimulate the larva to transition to pupa and then to a winged‑adult, when it will emerge and abandon its home. As an adult it lives a mere two weeks, breeding and beginning the cycle anew.

The Strategy

If you’ve ever left a glass bottle full of water in the freezer, you learned the hard way that water expands when it freezes. Living cells are essentially membranes filled with water, and most would rupture (and die) if frozen. So how do gall flies survive?

The answer depends on how water moves in and out of cells. Water can diffuse across cellular membranes, driven to find an equilibrium between what’s inside the cell and what’s outside. Or water can pass through a class of s called aquaporins that work like doorways. These long snake-like proteins—with five loops connecting six that cross cell membranes—open pores, through which water can flow.

The Structure of Aquaporins
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All aquaporins have similar structures where 6 helices bridge the cell membranes connected by 5 loops (A through E).

All living organisms, including humans, have aquaporin proteins, but they only impart freeze-tolerance to certain species of insects, fish, reptiles, and amphibians. Scientists at Miami University proved that these proteins are critical to freeze protection; when they inhibited the proteins’ function, the gall fly tissue could no longer survive extreme cold. As a result, the researchers concluded these aquaporin channels push water from cells before it can freeze, expand, and rupture cell membranes, which would cause the cells to burst and die.

In a later study, the same researchers verified this pushing effect, demonstrating that the aquaporin channels in gall flies allow water to flow across the membrane nine times faster than it would by diffusion. Furthermore, the number of these specialized channels increases in larval cells as the temperature cools. When the scientists compared protein abundance in October to that in December, they found the colder weather added almost 40% more aquaporins throughout the insect’s body. Perhaps most importantly, they measured the highest quantity in the brain—the organ that needs the most freeze-protection to ensure survival.

To augment the insect’s freeze protection, a related group of proteins called aquaglyceroporins open channels for glycerol to enter cells. Glycerol is similar to glycol—the main component in car engine antifreeze—and both are alcohols. Unlike a bottle of water, a bottle of vodka in the freezer is fine because alcohols tend to have very low freezing points. So while the aquaporin channels pump out water to protect membranes from rupturing, aquaglyceroporin channels pump in more “anti-freeze” to slow the rate and reduce the extent of freezing.

The Potential

Much of science fiction assumes that humans will one day be frozen in pods to endure eons spent in intergalactic travel without aging. While this may or may not be possible in the distant future, there are ways to improve how we freeze human biological material that could actually save many lives right here on Earth.

With the exception of plasma, most of the components in donated blood cannot be frozen. Although red blood cells can be frozen for emergency use, thawing them is expensive and requires trained staff and specialized equipment.

 

Unfortunately, human organs cannot be frozen without destroying their cells. Consequently, a kidney can be stored for only 30 hours before it becomes unviable.

And freezing organs for transplanting isn’t an option. Unfortunately, human organs cannot be frozen without destroying their cells. Consequently, a can be stored for only 30 hours before it becomes unviable. A or lasts just 12 hours, while a heart or lung must be transplanted within only six hours.

The inability to store organs long-term keeps the supply of them low, and makes it difficult to get them to patients in remote, sparsely populated areas. The World Health Organization estimates that only 10% of patients across the world who need organ transplants actually receive them. Each year in the U.S., approximately 50,000 people are added to organ transplant waiting lists while over 700,000 deaths occur among those waiting.

If we can learn from the gall fly and other freeze-tolerant animals how they survive, it could help humans protect each other—and other living things—from many more dangers than just those of a long winter.

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Last Updated February 16, 2021