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The ecdysone receptor(1), which is a heterodimer of EcR and the retinoic acid receptor (RXR) homolog, Ultraspiracle (USP), has been well studied in the evolutionarily advanced and derived insects, the flies and moths. It is less well characterized in more primitive insect orders such as the Orthoptera, which include the grasshoppers and locusts. Following our previous isolation from Locusta migratoria (Lm) of a shorter RXR isoform (now called LmRXR-S), the isolation of a second, longer isoform (LmRXR-L) that appears to have characteristics of a ligand-modulated nuclear receptor is reported here. Transcripts for both isoforms, as well as LmEcR, were detected in embryos and in females during oocyte maturation. After expression in E. coli, both LmRXR-S and LmRXR-L form heterodimers with recombinant LmEcR in vitro which bind the active ecdysteroid, ponasterone A. Binding was only weakly competed for by ecdysone agonists that are known to be toxic to more advanced insects, suggesting functionally significant divergence in EcR ligand binding domains. In contrast, the DNA binding domain of LmEcR is less divergent and a protein complex, presumably LmEcR/LmRXR, that bound the ecdysone response element, IR-1, was detected in locust nuclear extracts. Because of reports of juvenile hormone (JH III) binding to Drosophila USP and the observed in silico RXR-like ligand-binding site in LmRXR-L, the recombinant proteins were also tested for binding to JH III. Neither LmRXR isoform, alone or in combination with LmEcR, bound JH III at nanomolar concentrations.

Adult flies of Drosophila melanogaster were examined for their tolerance to direct (cold-shock) and indirect chilling (chilling) injury. An exponential increase in mortality with low temperatures occurs. Chilling tolerance (T ch ) at 4 or 0°C was 6.3 days or 37.91 h for 50% survival (LT 50 ), respectively, while the cold-shock tolerance (LT 50 of T cs ) at −7°C was only 1.5 h. The increase in cold-shock tolerance (T cs ) at −7°C by acclimation at 4, or 0°C was rapid: the LT 50 increased about 50% with only 2 h of treatment in comparison with the highest LT 50 obtained by acclimation. An equilibrium point (T eq ), the optimal acclimation time for maximal survival of chilling and/or cold-shock, was calculated from the survival data of experiments designed to assess T ch and T cs . The T eq was found to be the half time of the acclimation period which resulted in 90% mortality at the chilling temperature (LT 90 of T ch ). Selection for tolerance to cold-shock or chilling over several generations resulted in a significant increase in T cs or T ch , respectively. Higher contents of glycogen and total proteins were observed in the cold selected lines than in the control line. Exposure to chilling temperatures eventually depleted the energy reserves, but the highest concentrations of triacylglycerols and proteins were observed at the acclimation time with the highest T cs . This suggests that systemic regulation in these energetic reserves may be an important part of the cold-hardening mechanisms to cope with the fluctuations of low temperatures causing cold-shock or chilling injury.

Treatment of Drosophila melanogaster adults with an inhibitor of protein synthesis led to a decrease in intrinsic cold-shock tolerance, but no difference in the rapid cold hardening response, which is apparent only if a period at 4 degrees C precedes the cold stress. Increases in energy reserves, including proline, were found in lines of flies selected for resistance to chilling injury. Since an increase in proline levels has been associated with overwintering in insects, and for salt and cold tolerance in plants, an RNase protection assay was developed to assess changes in transcript abundance for two genes encoding enzymes important for proline metabolism, pyrroline 5-carboxylate reductase and proline oxidase. The mRNA levels did not change in response to low temperature, but the high level of pyrroline 5-carboxylate reductase transcript is consistent with the interpretation that a large proline pool is important for Drosophila metabolism and survival during cold stress.

After 5 days at the restrictive temperature (29.5°C) adult Drosophila females heterozygous for the dominant temperature-sensitive mutation, L(3)3DTS, have an ecdysteroid level of about half that in mutant females at 22°C and subsequently become completely sterile due to the inviability of progeny embryos. The lethal phase of progeny from mutant females varies depending upon the length of time DTS-3 females are kept at a sublethal temperature of 27°C. Thus, the DTS-3 mutation shows a maternal effect, and a deficiency of ecdysteroids or ecdysteroid-induced gene products may be responsible for progeny lethality. This lethality cannot be attributed to a deficit in the products of the hormonally-regulated yolk polypeptide genes however, since yolk polypeptide mRNA and protein levels are not reduced in DTS-3 females at the restrictive temperature.