| Summary: | The ice fly Diamesa steinboecki Goetghebuer, 1933 (Diptera: Chironomidae: Diamesinae) is exclusive to glacier-fed streams in the East Palaearctic region and is threatened by extinction due to global warming and glacier retreat. To date, no data are available on its thermal tolerance or ability to develop a heat shock response (HSR) or involve other biomarkers when exposed to higher-than-natural temperatures (i.e., >4–6 °C). Our study aimed to investigate the warmth resistance of IV-instar larvae of D. steinboecki in terms of (1) ability to survive heat shock and (2) gene expression of four genes known to be involved in the detoxification/stress response (cytochrome p450 (Cyp450), heat shock protein 70 (hsp70), hsp70 with intron and heat shock protein cognate 70 (hsc70)). Larvae were exposed to short-term shocks for 1 h at increasing temperatures (26, 28, 30, 32, 34, 36, 38, and 40 °C) to estimate the lethal temperature, obtaining high values (LT10 = 38.1 °C, LT50 = 39.2 °C, LT99 = 40.3 °C), suggesting a strong heat resistance up to 38 °C and a very rapid decline in survival thereafter. Moreover, gene expression analysis by real-time PCR was performed on larvae from the control (at 2 °C) and larvae found alive after the previous treatment at 26, 28, 30, 32, 34, 36, and 38 °C. Modulation of the expression was observed only for hsc70 and hsp70 genes. Specifically, hsc70 resulted in constitutive overexpression, even at 26 °C when all larvae were found alive without evidence of suffering. By contrast, hsp70 showed up and downregulation according to the specific temperature, suggesting the activation of an HSR at 28 °C, when some larvae were found alive but suffering (almost paralyzed). The results suggest that, based on LTs, D. steinboecki is more thermally tolerant than other Diamesa species (e.g., D. tonsa) from cold freshwaters, but, as in these, hsp70 and hsc70 are involved in surviving short-term heat shock. This makes the ice fly from the Alps different from Belgica antarctica and other cold-adapted ...
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