Go to: Photo Gallery mainpage | Storey homepage | Carleton University homepage
INVERTEBRATE
COLD HARDINESS
In the Storey lab we study
the mechanisms of cold hardiness used by invertebrate animals including
terrestrial insects and mollusks that live in the marine intertidal zone.
Freeze tolerant marine mollusks can also live without oxygen for long times so
for information on both freezing and anoxia tolerance of these animals, go to
our Anoxia webpage.
|
Eurosta
solidaginis |
Epiblema scudderiana |
Littorina littorea |
Geukensia demissus |
COLD HARDY INSECTS
In the Storey lab we work
on two species of cold hardy insects that live
in stem galls on goldenrod.
Both spend the winter as mature
larvae within galls but they have opposite strategies for surviving the winter.
Larvae of Eurosta
solidaginis, the gall fly, are freeze
tolerant. They freeze solid when outdoor temperatures fall below about -8°C but
happily survive with as much as 65% of their body water turned to ice. Larvae
of Epiblema
scudderiana, the gall moth, use the
freeze avoidance strategy, packing their bodies with antifreezes so that they
can stay liquid down to nearly -40°C.
Below are photos of both
species and their galls. This is followed by information on the Life
Histories of the
two species and an explanation of the different insect options for Surviving
the Winter.
Visit NatureNorthZine for classroom experiments that you can do with freeze
tolerant insects.
Visit the Solidago gall homepage at
|
Picture 1. Examples of ball galls containing Eurosta solidaginis larvae and
elliptical galls containing Epiblema
scudderiana larvae. Galls are found on the stems of goldenrod.
|
Picture 2. Top: Eurosta solidaginis, the goldenrod gall fly,
overwinters as a third instar larva in the central cavity of ball galls. Bottom: Epiblema scudderiana, the goldenrod
gall moth, overwinters as a final instar caterpillar in elliptical galls.
|
Picture 3. Closer look at a Eurosta
larva in its gall. The larva eats and grows all summer in the central cavity.
Just before settling down for the winter, the larva eats a tunnel to the
surface of the gall, leaving only the “skin” of the gall in place. Then it
returns to the central cavity and enters diapause (a form of dormancy) for
the winter.
|
|
Picture 4. Closer look at Epiblema
in its gall. The larva eats and
grows in the central cavity all summer but retreats downward into the
hollowed out stem for the winter where it also settles into diapause.
|
Picture 4. Eurosta solidaginis. The larva winters in the central cavity but in this
photo is partly in the tunnel leading to the gall surface. Black mouth parts
are seen on the left.
|
Picture 6. Epiblema scudderiana in its winter position tucked into the hollow stem
below the main gall cavity.
|
|
Picture 7. There is only 1 Eurosta larva in each gall
but double galls on one stem occur quite often -- the most we have ever found
on one stem is 4!!
|
Picture 8. Double Eurosta ball galls in late October
after frost has killed the plant.
|
|
|
Picture 9. Double Epiblema
galls are less common. Here the goldenrod plant is still green in
mid-September.
|
Picture 10. Galls are well camouflaged on the dying plants in
late October.
|
|
|
Picture 11. The normal exit hole (left) made by the fly escaping
in the spring is very neat but chickadees tear open the gall (right) to eat
the high calorie larvae, a favorite winter food.
|
Picture 12. Downy woodpeckers start to attack the galls in
late October as soon as the larvae have finished their tunnels. They leave
much neater holes than the chickadees. |
Picture 13. In late April, the Eurosta larvae pupate. Epiblema
pupate a little later. |
|
Picture 14. Adult fly Eurosta solidaginis
|
Picture 15. Adult fly
|
Picture 16. Adult fly on JM Storey |
|
Picture 17. The adult Epiblema moth is about 1 cm long. |
Picture 18. Adult Epiblema
moth on a gall showing the exit hole.
a |
Picture 19. Exit holes at the top of elliptical galls l |
|
Picture 20. |
August field showing full size green galls
on blooming goldenrod, Solidago sp. In the background, brown galls on dead stems are the remains of last year's crop. |
|
Picture 21.
|
August field showing two of the main
wildflowers blooming in late summer - goldenrod and purple asters. The ball galls shine in the sunlight and are easy to spot. |
|
Picture 22. Mature larvae of the hermit flower beetle, Osmoderma
eremicola (Knoch)(Coleoptera, Scarabaeidae).
|
These giant larvae (4-5 cm) live in the
heartwood of decaying deciduous trees in eastern |
LIFE HISTORIES of GOLDENROD GALL
INSECTS
Eurosta solidaginis (Fitch) (Diptera, Tephritidae) is the larva
of the goldenrod gall fly. Eggs are
laid in the growing tips of goldenrod plants in the spring and when they hatch
the larvae bore into the center of the stem and start to eat. Secretions of the
larvae mimic plant hormones and cause the plant to form a ball gall around them
and to stock the inside of the gall with cells that are high in nutrients which
the larvae eat. Third instar larvae reach maximum size by early autumn. They
bore a tunnel out to near the surface of the gall (leaving just the epithelium
layer) and then settle back into the center of the gall to spend the winter.
Downy woodpeckers and chickadees will tap on galls until they find this tunnel
and then dive in the get the juicy larva which is a high fat winter treat for
them. Once settled in the center of the gall, the larvae respond to autumn cues
(shorter days, cooler temperatures, senescence of the plant) by preparing for
winter. They accumulate 2 cryoprotectants, glycerol and sorbitol, and increase
the supercooling point of their body fluids by adding ice nucleators that
stimulate freezing of the larvae whenever temperature drops below about -8 to
-10°C. The larvae survive freezing and can endure the conversion of up to about
65 % of their total body water in the extracellular ice. They endure multiple
freeze/thaw cycles over the winter and can survive to at least -30°C in
southern
Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae) is
the caterpillar of the goldenrod gall moth. Its life cycle is much the same as that of Eurosta. It lives
inside an elliptical gall on the stem of goldenrod. The gall is much more
camouflaged than that of Eurosta with wood-grain like scars on the
outside but frequently the presence of the gall makes the plant above it split
out into more bushy appearance so you can often find the galls by looking at
the appearance of the plant. The final instar larva overwinters. It moves out
of the main gall cavity and down into the hollowed out stem below where it fits
snugly and vertically in a head up position. The caterpillar lines the interior
of the stem and gall with silk which probably helps to act as a barrier to
water or ice penetration. Epiblema's strategy for cold hardiness is
freeze avoidance and by mid-winter the larvae in the
Eurosta solidaginis can be attacked by parasites and predators in their
galls. Two species of wasps lay their eggs in the galls and the wasp larvae eat
the gall fly larvae. One species (Eurytoma obtusiventris) lays its egg
right inside the gall fly larva and eats it from the inside; it causes the gall
fly larva to form a pupa in the autumn (instead of the spring) and then winters
inside the pupal case. The other wasp larva (Eurytoma gigantea) eats up
the whole gall fly and takes its place in the gall cavity. This wasp larva is
also cream-colored, like the gall fly larva, but is a bit smaller and has
distinctly pointed ends. Typically, too, once the gall fly larva is dead, the
interior cavity of the gall degenerates and goes brown and partly hollow
compared with the white and solid interior (except for the central cavity that
just fits the larva) if the gall fly larva is alive. The third is a beetle
larva (Mordellestina unicolor) which is described as an accidental
predator but again eats the gall fly larva from the outside. The beetle larva
looks like a small caterpillar with distinct legs.
Epiblema scudderiana also have parasites but we know less about these. However,
when we took larvae out of galls in the winter and left them at room
temperature in hopes that they would pupate, they often did not pupate.
Instead, several small worm-like larvae would come out of each Epiblema.
|
Picture 14. For more info on "Parasites and predators" see the Solidago gall homepage |
Eurosta solidaginis and its parasites Top: Eurosta - 2nd and 3rd instars Left: Eurytoma gigantea, wasp larva which eats Eurosta Right: Eurytoma obtusiventris, wasp larva which
makes Eurosta pupate prematurely Bottom: Mordellistena convicta, beetle larva that
sometimes eats Eurosta Photo: John Baust |
|
|
From
left to right: (a) Eurosta solidaginis 3rd instar larva, (b) Eurosta
are triggered to pupate in the
autumn when parasitized by Eurytoma obtusiventris, (c) Eurytoma
gigantea, and (d) Mordellistena convicta. Photo: Jan Storey |
TEMPERATURES
To start with, lets define temperatures
because scientists always use the Celcius scale. On the Celsius scale, 0°C is
the freezing point of water and 100°C is the boiling point of water. This
compares with the Fahrenheit scale where 32°C is the freezing point of water
and 212°C is the boiling point.
A home refrigerator is generally at 4-5°C and
your home freezer is usually between -15 to -20°C.
STRATEGIES OF
INSECT COLD HARDINESS
For insects there are 5 main ways of dealing
with winter temperatures that fall below the freezing point of water (below
0°C).
1. "heat up the house"
This in only done by
honeybees. Bees generate heat by shivering and with thousands of bees in a colony
and a huge supply of stored food (honey) they can afford to huddle and shiver
all winter and keep the colony at a comfortably warm temperature that is a lot
higher than the outdoor air temperature.
2. "get out of town"
Some insects migrate
to get away from cold temperatures; for example, Monarch butterflies fly to
Other insects find
places to hide for the winter where they will never experience subzero (below
0°C) temperatures. Some go underground far enough so that they are below the
frostline and some spend the winter in ponds and streams in an aquatic life
stage. For example, dragonflies spend the winter as aquatic larvae and then
transform into the adult flying form in the spring/summer.
3. Spend the winter as a simple life stage --
usually an egg.
Eggs are easier to
protect than is a multicellular larva or adult insect and many insects are
present only as the egg stage during the winter. Some insect eggs can survive
extensive dehydration and spend the winter in a very dry form. If you have little
or no free water in your system, ice crystals can't form and you can't be
harmed by freezing. Other insects pack their eggs with cryoprotectants such as
glycerol or sorbitol. This creates such a syrupy solution that water in the egg
can't freeze. Eggs can also reduce their metabolism to near zero and many can
be in such deep dormancy that they can survive not only over a whole winter,
but sometimes for many years.
4. Freeze avoidance
Many insects
happily remain liquid even at temperatures that are well below 0°C. We call
this strategy "freeze avoidance". We always think that water freezes
at 0°C but in reality, both plain water itself and the water in an animal's
body can often be cooled to much lower temperatures before ice forms.
Microscopic droplets of very pure water can be cooled to -40°C before they
freeze. Water "normally" freezes at 0°C because the growth of ice
crystals is "seeded" by the presence of some particle or surface that
helps to line up the water molecules into the crystal shape. Once an initial
microscopic nucleus of ice is formed, more and more water molecules quickly
join the crystal and high speed crystal growth is triggered. The trick,
therefore, is to keep your body fluids from coming into contact with ice
crystals themselves or with molecules or particles that can act as nucleators
to seed crystal growth.
Insects in general
are good at this trick of "supercooling"
- staying liquid when their body temperature is below zero. This is because of
the small body size of most insects (the smaller the better for supercooling)
and the strong, waxy cuticle that coats the insect's body which provide
excellent waterproofing and also prevents external ice from coming into contact
with body fluids. Even in summer, most insect won't freeze until at least -5°C
or even lower. In the winter, many insects activate adaptations that push
supercooling even farther. Insects that spend the winter near the surface of
the ground (e.g. on the forest floor or in the soil under your lawn) where they
will be covered by a blanket of snow, can generally supercool to -15 or -20°C
without freezing. Insects that spend the winter high up in trees or in other
exposed sites can often supercool to -40°C.
The main
adaptations that increase an insect's ability to supercool and avoid freezing
are: (a) make antifreeze proteins, and (b) make cryoprotectants. Antifreeze proteins function by
binding to microscopic ice crystals as they start to form in the insect's body
fluids. Binding prevents the crystal from growing any larger. Most insects that
spend the winter in insulated places such as under the snow, only have to add
antifreeze proteins to their body fluids to get all the protection that they
need. This is because its almost always a lot warmer under the snow than above
it. Lots of insects and other animals can stay active under the snow at
temperatures that rarely fall below -5 to -8°C even though air temperatures
above the snow may be -20 or -30°C.
Insects that live
in very exposed sites also produce cryoprotectants. In insects the most common
cryoprotectant is glycerol which is a 3-carbon
polyhydric alcohol. Glycerol is a longer version of ethylene glycol, the
2-carbon polyhydric alcohol that we use as antifreeze in the radiator and
windshield washer fluid of cars. Several other types of polyols as well as some
kinds of sugars are also used as cryoprotectants by some species. The freezing
point of water decreases in proportion to the concentration of dissolved
substances in it so the more cryoprotectant that you can pack into your body
fluids, the lower the temperature at which you will freeze. Pack in enough and
the insect won't freeze at the normal winter temperatures that it encounters.
The key advantages of glycerol as a cryoprotectant are that it is highly
soluble in water, is not toxic to metabolism, and can be easily synthesized by
the insect. The concentrations of glycerol can be so high in some insect
species in winter that as much as 20-25% of the insect's total body mass is
glycerol! Glycerol also has another action and that is that it is very good at
binding to water and, for insects that live in exposed sites such as in trees,
this helps the animals to avoid death due to desiccation over the long winter
months when they are exposed to dry, cold air.
5. Freeze tolerance
Two of the
disadvantages of the freeze avoidance strategy that we talked about above are
(1) if your antifreeze is only good to -20°C and temperature drops to -25°C,
you freeze and die, and (2) you can only stay supercooled if you can avoid
contact with ice crystals -- even if your antifreeze is good to -20°C, if you
touch ice crystals your body fluids will be seeded and you will freeze and die.
So, the freeze avoidance strategy is a gamble -- insects gamble that
temperatures won't fall too low and that they are sufficiently protected so
that they won't come in contact with external ice. For example, many insect
pick dry spots to hibernate in such as under the bark of trees and many spin a
waterproof cocoon around themselves or have a very thick wax cuticle on their
body to block contact with ice.
Other types of
insects have "given up" on the idea of avoiding freezing and figured
out instead how to accept freezing yet not die. It's a much more difficult
strategy to use because freezing requires many complex adaptations. You might
be interested to know, however, that the methods used by insects to protect
their tissues during freezing are the same ones that are used in medicine to
freeze and preserve many types of cells and tissues to save them for transplant.
Things like blood, sperm, embryos, skin, heart valves and corneas can be
frozen, stored for many years, and then thawed and transplanted.
So,
to survive freezing, here's what an insect has to do.
1. Ice is allowed to grow only outside of cells, never
inside the cells. So ice will form within the insect's body, surrounding all
the internal tissues, and ice will grow through all the blood vessels of the
insect. So, the animal is never completely frozen because the insides of its
cells are always liquid. Only about 65% of the insect's total body water
freezes -- the rest stays liquid inside cells. If ice grows inside of cells it
kills them because the crystals cause way too much damage to the inside
structure of the cells.
2. Most insects are naturally good at supercooling but if
an animal begins to freeze after it has supercooled to a low temperatures (say
-10°C or lower), the speed of ice formation is so high that the insect may be
killed or severely injured. So, freeze tolerant insects use nucleators to help them "seed" their body
fluids so that they start to freeze somewhere between 0 and -10°C. Sometimes
they do this by letting their bodies be seeded through contact with external
ice in their environment and sometimes they produce special proteins (called ice nucleating proteins) that trigger ice formation.
You might be interested to know that the ice nucleating proteins that are
produced by some kinds of bacteria are used by ski resorts to "seed"
freezing when making artificial snow.
3. Freeze tolerant insects also have antifreeze proteins. Isn't that odd??? Why would they
need a protein that stops ice crystals from growing? The reason is that small
ice crystals can be harmless but big ones can do some serious damage. When
something is frozen for a long time, the ice undergoes a strange process called
recrystallization. Small ice crystals reform
themselves into larger and larger crystals and eventually the crystals could
get so big that they harm the delicate tissues of the frozen animal. So
antifreeze proteins are used to keep small crystals small. The best example of
recrystallization that you can see is in the ice cream in your home freezer. It
is all smooth and creamy when you first open it but if you leave it in the
freezer for a long time, you will find that big feathery ice crystals have
separated from the ice cream.
4. To keep the inside of cells liquid, freeze tolerant
insects pack them up with glycerol or other cryoprotectants.
So, instead of preventing your entire body from freezing, like freeze avoiding
insects do, the insects that survive freezing only focus on protecting the
insides of their cells with cryoprotectants.
5. When ice crystals grow outside of cells (in what is
called extracellular spaces), they set up a stress on cells that sucks water out
of the cells so that the amount of water inside cells decreases and the cells
shrink in size. The high cryoprotectants inside the cells help to hold onto the
intracellular water and keep the cells from shrinking too much. Other
protective molecules are also made strengthen the membranes
that surround the cells so that the membranes do not break either when the
cells shrink during freezing or when they swell again during thawing.
6. When the extracellular water of an animal freezes,
this includes its blood. Frozen blood cannot deliver nutrients to cells so all
of the tissues of the animal have to stay alive during the freeze by relying
only on the fuels that are inside each cell and without the oxygen that is normally delivered from the outside.
Oxygen is delivered by the blood in mammals (like us) but in insects oxygen is
delivered to each tissue by tiny tubes called trachea that lead inwards from
small holes (called spiracles) that are all over the insect's body. When an
insect freezes, oxygen delivery stops for 2 reasons: (a) the muscles that help
to keep air flow moving through the trachea freeze, and (b) the extracellular
fluid at the end of the trachea freezes. So a frozen animal has to live without
oxygen for the whole time that it is frozen and this can be a challenge.
Animals that can survive freezing have developed adaptations that help them
live longer without oxygen.
Go to: Photo Gallery mainpage | Storey homepage | Carleton University homepage