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Insects
Cold hardening has also been observed in insects such as the fruit fly and diamondback moth. The insects use rapid cold hardening to protect against cold shock during overwintering periods. Overwintering insects stay awake and active through the winter while non-overwintering insects migrate or die. Rapid cold hardening can be experienced during short periods of undesirable temperatures, such as cold shock in environment temperature, as well as the common cold months. The build up of cryoprotective compounds is the reason that insects can experience cold hardening. Glycerol is a cryoprotective substance found within these insects capable of overwintering. Through testing, glycerol requires interactions with other cell components within the insect in order to decrease the body's permeability to the cold. When an insect is exposed to these cold temperatures, glycerol rapidly accumulates. Glycerol is known as a non-ionic kosmotrope forming powerful hydrogen bonds with water molecules. The hydrogen bonds in the glycerol compound compete with the weaker bonds between the water molecules causing an interruption in the makeup of ice formation. This chemistry found within the glycerol compound and reaction between water has been used as an antifreeze in the past, and can be seen here when concerning cold hardening. Proteins also play a large role in the cryoprotective compounds that increase ability to survive the cold hardening process and environmental change. Glycogen phosphorylase(GlyP) has been a key protein found during testing to increase in comparison to a controlled group not experiencing the cold hardening. Once warmer temperatures are observed the process of acclimation begins, and the increased glycerol along with other cryoprotective compounds and proteins are also reversed. There is a rapid cold hardening capacity found within certain insects that suggests not all insects can survive a long period of overwintering. Non-diapausing insects can sustain brief temperature shocks but often have a limit to which they can handle before the body can no longer produce enough cryoprotective components. Inclusive to the cold hardening process being beneficial for insects survival during cold temperatures, it also helps improve the organism s performance. Rapid cold hardening (RCH) is one of the fastest cold temperature responses recorded. This process allows an insect to instantly adapt to the severe weather change without compromising function. The Drosophila melanogaster (common fruit fly) is a frequently experimented insect involving cold hardening. A proven example of RCH enhancing organisms performance comes from courting and mating within the fruit fly. It has been tested that the fruit fly mated more frequently once RCH has commenced in relation to a controlled insect group not experiencing RCH. Most insects experiencing extended cold periods are observed to modify the membrane lipids within the body. Desaturation of fatty acids are the most commonly seen modification to the membrane. When the fruit fly was observed under the stressful climate the survival rate increased in comparison to the fly prior to cold hardening. In addition to testing on the common fruit fly, Plutella xylostella (diamondback moth) also has been widely studied for its significance in cold hardening. While this insect also shows an increase in glycerol and similar cryoprotective compounds, it also shows an increase in polyols. These compounds are specifically linked to cryoprotective compounds designed to withstand cold hardening. The polyol compound is freeze-susceptible and freeze tolerant. Polyols simply act as a barrier within the insect body by preventing intracellular freezing by restricting the extracellular freezing likely to happen in overwintering periods. During the larvae stage of the diamondback moth, the significance of glycerol was tested again for validity. The lab injected the larvae with added glycerol and in turn proved that glycerol is a major factor in survival rate when cold hardening. The cold tolerance is directly proportional to the build up of glycerol during cold hardening.

Cold hardening of insects improves the survival rate of the species and improves function. Once environmental temperature begins to warm up above freezing, the cold hardening process is reversed and the glycerol and cryprotective compounds decrease within the body. This also reverts the function of the insect to pre-cold hardening activity.