399 Introduction More than 15·years ago, we reported a rapid cold-hardening (RCH) response that protects various species of insects against cold shock (non-freezing) injury (Chen et al., 1987; Lee et al., 1987). The RCH response is manifested not only in measured survival but also in enhanced success in courtship, mating and fecundity (Rinehart et al., 2000; Shreve et al., 2000). Recently, we demonstrated that RCH also allows an organism’s overall cold tolerance to track changes in environmental temperature, as would occur in natural diurnal thermal cycles (Kelty and Lee, 1999, 2001). The distribution of the terrestrial chironomid Belgica antarctica extends further south than any other free-living holometabolous insect and, since Antarctic vertebrates are essentially marine, it is the largest terrestrial animal in Antarctica (Usher and Edwards, 1984; Sugg et al., 1983). This endemic species is sporadically dispersed, though locally abundant, on the west coast of the Antarctic Peninsula and its islands. Detailed accounts of the life history and ecology of B. antarctica are provided by Convey and Block (1996), Sugg et al. (1983), Usher and Edwards (1984) and references cited therein. Briefly, its two-year life cycle includes four larval stages, and overwintering may occur in any instar. Larvae feed on moss, terrestrial algae, particularly Prasiola crispa, plant and animal detritus and microorganisms. Pupation and adult emergence occur in spring and summer. Like many insects living in wind-swept alpine and oceanic habitats, the adults are wingless. Adults live for fewer than 10·days. Like the male swarms of winged midges in temperate and arctic regions, mating occurs in aggregations of flightless males. Females mate within one day of eclosion and lay several clusters of eggs within 1–2·days. Although ambient air temperatures may reach winter lows of –40°C on Anvers Island, B. antarctica survives freezing to only –15°C (Baust and Lee, 1981), a relatively modest level of cold tolerance compared with many alpine and polar insects. Thermal buffering within its microhabitat explains this apparent anomaly; at 1·cm depth, substrate temperatures Rapid cold-hardening (RCH) is well known to increase the tolerance of chilling or cold shock in a diverse array of invertebrate systems at both organismal and cellular levels. Here, we report a novel role for RCH by showing that RCH also increases freezing tolerance in an Antarctic midge, Belgica antarctica (Diptera, Chironomidae). The RCH response of B. antarctica was investigated under two distinct physiological states: summer acclimatized and cold acclimated. Summer-acclimatized larvae were less cold tolerant, as indicated by low survival following exposure to –10°C for 24·h; by contrast, nearly all cold- acclimated larvae survived –10°C, and a significant number could survive –15°C. Cold-acclimated larvae had higher supercooling points than summer larvae. To evaluate the RCH response in summer-acclimatized midges, larvae and adults, maintained at 4°C, were transferred to –5°C for 1·h prior to exposures to –10, –15 or –20°C. RCH significantly increased survival of summer-acclimatized larvae frozen at –10°C for 1·h compared with larvae receiving no cold-hardening treatment, but adults, which live for only a week or so in the austral summer, lacked the capacity for RCH. In cold- acclimated larvae, RCH significantly increased freeze tolerance to both –15 and –20°C. Similarly, RCH significantly increased cellular survival of fat body, Malpighian tubules and gut tissue from cold-acclimated larvae frozen at –20°C for 24·h. These results indicate that RCH not only protects against non-freezing injury but also increases freeze tolerance. Key words: rapid cold-hardening, freezing tolerance, Chironomidae, Belgica antarctica. Summary The Journal of Experimental Biology 209, 399-406 Published by The Company of Biologists 2006 doi:10.1242/jeb.02001 Rapid cold-hardening increases the freezing tolerance of the Antarctic midge Belgica antarctica Richard E. Lee, Jr 1, *, Michael A. Elnitsky 1 , Joseph P. Rinehart 2 , Scott A. L. Hayward 2,3 , Luke H. Sandro 1 and David L. Denlinger 2 1 Department of Zoology, Miami University, Oxford, OH 45056, USA, 2 Department of Entomology, Ohio State University, 318 W. 12th Avenue, Columbus, OH 43210, USA and 3 School of Biological Sciences, Liverpool University, Crown Street, Liverpool, L69 7ZB, UK *Author for correspondence (e-mail: leere@muohio.edu) Accepted 16 November 2005 THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY