Functional characterization of the Frost gene in Drosophila melanogaster: importance for recovery from chill coma

Abstract

Background: Almost all animals, including insects, need to adapt to temperature fluctuations. The molecular basis of thermal adaptation is not well understood, although a number of candidate genes have been proposed. However, a functional link between candidate genes and thermal tolerance has rarely been established. The gene Frost (Fst) was first discovered when Drosophila flies were exposed to cold stress, but the biological function(s) of Fst has so far not been characterized. Because Fst is up-regulated after a cold stress, we tested whether it was essential for chill-coma recovery.

Methodology/principal findings: A marked increase in Fst expression was detected (by RT-PCR) during recovery from cold stress, peaking at 42-fold after 2 h. The GAL4/UAS system was used to knock down expression of Fst and recovery ability was assessed in transgenic adults following 12 h of chill coma at 0 degrees C. The ability to recover from cold stress (short-, medium- and long-term) was significantly altered in the transgenic adults that had Fst silenced. These findings show that Fst plays an essential role in the recovery from chill coma in both males and females.

Conclusions/significance: The Frost gene is essential for cold tolerance in Drosophila melanogaster and may play an important role in thermal adaptation.

Figure 1. Upregulation of Fst during cold stress and recovery.

White bars represent cold stressed treatment (S) at 0°C for 0.25 to 9 h and grey bars denote recovery (R) at 25°C for 0.5 to 8 h. Relative expressions are calculated using the 2^−ΔΔCt method. Expression levels of Fst are normalized against the housekeeping reference RpS20 and values are expressed as fold change relative to control (mean±SE; n = 4). The symbol (*) indicates when a value is significantly different from untreated controls (t-test).

Figure 2. Silencing the cold-inducible Fst expression impairs chill coma recovery in tub-GAL4-driven females.

(A) Expression of Fst mRNA in untreated (kept at 25°C) and recovering (2 h at 25°C after 12 h at 0°C) females. Expression levels of Fst are normalized against the housekeeping reference RpS20 and values are √1/x transformed (mean±CI; n = 3). The symbol (*) indicates when the level is significantly different in tub-GAL4/UAS-Fst versus tub-GAL4/+ females (t-test). (B) Comparison of temporal recovery curves in tub-GAL4/UAS-Fst (squares) versus tub-GAL4/+ (circles) females. Time to recover from chill coma was monitored in females recovering at 25°C after 12 h of cold stress at 0°C. Each dot represents the mean percentage (±SE); 45 females were tested per line. (C) Mortality rate in tub-GAL4/UAS-Fst versus tub-GAL4/+ females. Mortality was assessed in flies recovering for 24 h at 25°C after 12 h of cold stress at 0°C. Bars represents the percentage (±CI) derived from 150 females in each line. The symbol (*) indicates a significant difference between lines (Chi square test).

Figure 3. Silencing the cold-inducible Fst expression impairs chill coma recovery in tub-GAL4-driven males.

(A) Expression of Fst mRNA in untreated (kept at 25°C) and recovering (2 h at 25°C after 12 h at 0°C) males. Expression levels of Fst are normalized against the housekeeping reference RpS20 and values are √1/x transformed (mean±CI; n = 3). The symbol (*) indicates when the level is significantly different in tub-GAL4/UAS-Fst versus tub-GAL4/+ males (t-test). (B) Comparison of temporal recovery curves in tub-GAL4/UAS-Fst (squares) versus tub-GAL4/+ (circles) males. Time to recover from chill coma was monitored in males recovering at 25°C after 12 h of cold stress at 0°C. Each dot represents the mean percentage (±SE); 45 males were tested per line. (C) Mortality rate in tub-GAL4/UAS-Fst versus tub-GAL4/+ males. Mortality was assessed in flies recovering for 24 h at 25°C after 12 h of cold stress at 0°C. Bars represents the percentage (±CI) derived from 150 males in each line. The symbol (*) indicates a significant difference between lines (Chi square test).

Figure 4. Silencing the cold-inducible Fst expression impairs chill coma recovery in act-GAL4-driven females.

(A) Expression of Fst mRNA in untreated (kept at 25°C) and recovering (2 h at 25°C after 12 h at 0°C) females. Expression levels of Fst are normalized against the housekeeping reference RpS20 and values are √1/x transformed (mean±CI; n = 3). The symbol (*) indicates when the level is significantly different in act-GAL4/UAS-Fst versus act-GAL4/+ females (t-test). (B) Comparison of temporal recovery curves in act-GAL4/UAS-Fst (squares) versus act-GAL4/+ (circles) females. Time to recover from chill coma was monitored in females recovering at 25°C after 12 h of cold stress at 0°C. Each dot represents the mean percentage (±SE); 45 females were tested per line. (C) Mortality rate in act-GAL4/UAS-Fst versus act-GAL4/+ females. Mortality was assessed in flies recovering for 24 h at 25°C after 12 h of cold stress at 0°C. Bars represents the percentage (±CI) derived from 150 females in each line. The symbol (*) indicates a significant difference between lines (Chi square test). (D) Climbing activity monitored in act-GAL4/UAS-Fst versus act-GAL4/+ females. Measurements were taken in recovering females after 2, 4, 6 and 8 h at 25°C following 12 h at 0°C. Flies were categorized as fit (fast climbing) or recovering (slow climbing) or injured (no climbing). The symbol (*) indicate significant differences between lines (Chi square test, n = 70).

Figure 5. Silencing the cold-inducible Fst expression impairs chill coma recovery in act-GAL4-driven males.

(A) Expression of Fst mRNA in untreated (kept at 25°C) and recovering (2 h at 25°C after 12 h at 0°C) males. Expression levels of Fst are normalized against the housekeeping reference RpS20 and values are √1/x transformed (mean±CI; n = 3). The symbol (*) indicates when the level is significantly different in act-GAL4/UAS-Fst versus act-GAL4/+ males (t-test). (B) Comparison of temporal recovery curves in act-GAL4/UAS-Fst (squares) versus act-GAL4/+ (circles) males. Time to recover from chill coma was monitored in males recovering at 25°C after 12 h of cold stress at 0°C. Each dot represents the mean percentage (±SE); 45 males were tested per line. (C) Mortality rate in act-GAL4/UAS-Fst versus act-GAL4/+ males. Mortality was assessed in flies recovering for 24 h at 25°C after 12 h of cold stress at 0°C. Bars represents the percentage (±CI) derived from 150 males in each line. The symbol (*) indicates a significant difference between lines (Chi square test). (D) Climbing activity monitored in act-GAL4/UAS-Fst versus act-GAL4/+ males. Measurements were taken in recovering males after 2, 4, 6 and 8 h at 25°C following 12 h at 0°C. Flies were categorized as fit (fast climbing) or recovering (slow climbing) or injured (no climbing). The symbol (*) indicate significant differences between lines (Chi square test, n = 70).