Effects of a high cholesterol diet on chill tolerance are highly context-dependent in Drosophila.

Chill susceptible insects are thought to be injured through different mechanisms depending on the duration and severity of chilling. While chronic chilling causes "indirect" injury through disruption of metabolic and ion homeostasis, acute chilling is suspected to cause "direct" injury, in part through phase transitions of cell membrane lipids. Dietary supplementation of cholesterol can reduce acute chilling injury in Drosophila melanogaster (Shreve et al., 2007), but the generality of this effect and the mechanisms underlying it remain unclear. To better understand how and why cholesterol has this effect, we assessed how a high cholesterol diet and thermal acclimation independently and interactively impact several measures of chill tolerance. Cholesterol supplementation positively affected tolerance to acute chilling in warm-acclimated flies (as reported previously). Conversely, feeding on the high-cholesterol diet negatively affected tolerance to chronic chilling in both cold and warm acclimated flies, as well as tolerance to acute chilling in cold acclimated flies. Cholesterol had no effect on the ability of flies to remain active in the cold or recover movement after a cold stress. Our findings support the idea that dietary cholesterol reduces mechanical injury to membranes caused by direct chilling injury, and that acute and chronic chilling are associated with distinct mechanisms of injury. Feeding on a high-cholesterol diet may interfere with mechanisms involved in cold acclimation, leaving cholesterol augmented flies more susceptible to chilling injury under some conditions.

Locust gut epithelia do not become more permeable to fluorescent dextran and bacteria in the cold.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown whether the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

Cold-induced immune activation in chill-susceptible insects.

Chilling injuries in chill-susceptible insects, such as the model dipteran Drosophila melanogaster, have been well-documented as a consequence of stressful low-temperature exposures. Cold stress also causes upregulation of genes in the insect immune pathways, some of which are also upregulated following other forms of sterile stress. The adaptive significance and underlying mechanisms surrounding cold-induced immune activation, however, are still unclear. Here, we review recent work on the roles of reactive oxygen species, damage-associated molecular patterns, and antimicrobial peptides in insect immune signaling or function. Using this emerging knowledge, we propose a conceptual model linking biochemical and molecular causes of immune activation to its consequences during and following cold stress.

Warm periods in repeated cold stresses protect Drosophila against ionoregulatory collapse, chilling injury, and reproductive deficits.

In some insects, repeated cold stresses, characterized by warm periods that interrupt a sustained cold period, have been found to yield survival benefits over continuous cold stresses, but at the cost of reproduction. During a cold stress, chill susceptible insects like Drosophila melanogaster suffer from a loss of ion and water balance, and the current model of recovery from chilling posits that re-establishment of ion homeostasis begins upon return to a warm environment, but that it takes minutes to hours for an insect to fully restore homeostasis. Following this ionoregulatory model of chill coma recovery, we predicted that the longer the duration of the warm periods between cold stresses, the better a fly will endure a subsequent chill coma event and the more likely they will be to survive. We also predicted, however, that this recovery may lead to reduced fecundity, possibly due to allocation of energy reserves away from reproduction. Here, female D.melanogaster were treated to a long continuous cold stress (25 h at 0 °C), or experienced the same total time in the cold with repeated short (15 min), or long (120 min) breaks at 22 °C. We found that warm periods in general improved survival outcomes, and individuals that recovered for more time in between cold periods had significantly lower rates of injury, faster recovery from chill coma, and produced greater, rather than fewer, offspring. These improvements in chill tolerance were associated with mitigation of ionoregulatory collapse, as flies that experienced either short or long warm periods better maintained low hemolymph [K+]. Thus, warm periods that interrupt cold periods improve cold tolerance and fertility in D. melanogaster females relative to a single sustained cold stress, potentially because this time allows for recovery of ion and water homeostasis.