DAF-16 and Δ9 desaturase genes promote cold tolerance in long-lived Caenorhabditis elegans age-1 mutants

Abstract

In Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS) pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ(9) desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ(9) desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546) mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546) mutants is predominantly due to the Δ(9) desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress.

Figure 1. Survival curves following direct transfer from 20°C to 4°C.

Survival curves at 4°C for A) wild type (N2), age-1(hx546) mutants, daf-16(mu86) mutants and age-1(hx546); daf-16(mu86) double mutants, and B) wild type (N2) and age-1(hx546) mutant adults and wild type (N2) dauers (n = 90–100 per genotype and stage).

Figure 2. Contribution of Δ9 desaturase genes to survival following direct transfer from 20°C to 4°C.

Survival curves at 4°C for A) wild type (N2), age-1(hx546) mutants, fat-6(tm331); fat-7(wa36) double mutants and age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants (n = 90–100 per genotype), B) wild type (N2) and age-1(hx546) mutant controls on empty vector HT115 bacteria (N2(e) and age-1(e)), and fat-6(tm331) mutants, fat-7(wa36) mutants, age-1(hx546); fat-6(tm331) double mutants and age-1(hx546); fat-7(wa36) double mutants on fat-5 RNAi bacteria (n = 50–60 per genotype on empty vector HT115 bacteria, n = 90–100 per genotype on fat-5 RNAi bacteria), C) wild type (N2) and age-1(hx546) mutant controls on empty vector HT115 bacteria, fat-6(tm331); fat-7(wa36) double mutants and age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants on fat-5 RNAi bacteria, and fat-5(tm420) mutants and age-1(hx546); fat-5(tm420) double mutants on fat-6/fat-7 RNAi bacteria (n = 50–60 individuals per genotype/treatment).

Figure 3. Cold coma recovery as a measure of cold tolerance.

Variation in recovery times among wild type (N2), age-1(hx546) mutants, fat-6(tm331); fat-7(wa36) double mutants and age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants following 6 hours cold shock at 4°C (n = 95–100 individuals per genotype, observed over 2 separate blocks).

Figure 4. Subcellular localization of DAF-16::GFP in response to cold stress.

A) Subcellular distributions of DAF-16::GFP were categorised with a score from 1 (left) with no nuclear localization to 4 (right) with complete nuclear localization. B) The mean proportion of age-1(+) adults and age-1(hx546) mutant adults which displayed categories 1–4 of DAF-16::GFP localisation at 20°C (left) and 4°C (right). Error bars represent standard errors of the means (n = 100–120 per genotype and treatment observed over 3 separate blocks).