Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata , survive cryopreservation in liquid nitrogen

Abstract

The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (−196°C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.insect freeze tolerance | cold hardiness | glass transition | metabolomics T emperate and polar insects overwinter at subzero body temperatures (1-3), which makes them suitable models for studying the principles of survival in animal cells, tissues, and complex organisms at cryothermic conditions. The drosophilid fly, Chymomyza costata (Zetterstedt) (Diptera: Drosophilidae), appears to be a highly promising model. It is distributed over a cool-temperate, Holarctic area (4) and fully grown third instar larvae enter facultative winter diapause in response to long night length and/or low temperature (5). Diapause larvae supercool to between −15°C and −25°C (6) but can survive freezing to −100°C provided they undergo cold acclimation (1 mo at 4°C), are inoculated by external ice at −2°C, and are cooled at a slow rate of 0.1°C∕ min (7,8). Diapause, cold-acclimated larvae of C. costata were successfully cryopreserved at the temperature of liquid nitrogen (N 2 ) (−196°C) for 1 h (9). To our knowledge, larva of C. costata represents the most complex metazoan organism that has reportedly survived after submergence in liquid N 2 in a fully hydrated state. Physiological mechanisms underlying this unique capacity remain poorly understood. Inoculative freezing and a slow cooling rate allow time for osmoti...

Conversion of the chill susceptible fruit fly larva ( Drosophila melanogaster ) to a freeze tolerant organism

“…After mating, survivors produced viable offspring. This result closely resembles the situation in C. costata larvae, where combination of diapause and elevated concentrations of proline support the highest survival to the adult stage after freezing (20). Feeding the trehalose-or glycerol-augmented diets had no significant effect on larval freeze 2.…”

“…2) at the cellular level (23.0% survival), but the survivors had little ability to continue developing; i.e., to pupariate (3.2%) and to metamorphose into adults (2.3%, or 8 of 343). This observation corresponds well to the situation in nondiapausing larvae of C. costata, where artificially increased levels of proline promote the occurrence of freeze tolerance but at the cellular and tissue levels only (20). A strong synergism between FTR-quiescence and cryopreservation by proline was observed in treatment v. These larvae showed high survival at the cellular level (61.0%), 14.1% of them pupariated, and 9.1% (42 of 462) metamorphosed into adults.…”

“…Proline fulfils the criteria for suitable exogenous cryoprotective compound, at least in drosophilid larvae (20). First, proline can penetrate into tissues when added to the diet (20, Dataset S3).…”

“…We have previously shown that high freeze tolerance in these larvae is based on two principal prerequisites. First, larvae enter into a state of diapause and second, during the cold acclimation process, they accumulate high levels of the free amino acid L-proline-an innate cryoprotectant (proline in further text) (20).In this study, we investigated whether the principles underlying high freeze tolerance in C. costata could apply to chill-susceptible D. melanogaster. We acclimated the fruit fly larvae at low temperatures including the fluctuating thermal regime (FTR) inducing dormancy-a low temperature quiescence state.…”

“…We have previously shown that high freeze tolerance in these larvae is based on two principal prerequisites. First, larvae enter into a state of diapause and second, during the cold acclimation process, they accumulate high levels of the free amino acid L-proline-an innate cryoprotectant (proline in further text) (20).…”