- Reptile freeze tolerance: Metabolism and gene expression


Table 1. Assessment of reptile freezing survival

Common name Freezing tolerance Best measured freezing survival References
Turtle hatchlings
Chrysemys picta marginata Midland painted turtle Good 3–11 days at −2 to −2.5 °C [12] and [84]
1 day at −4 °C
Chrysemys picta bellii Western painted turtle Good 3–7 days at −2.5 °C [12], [30] and [64]
3–4 days at −2 °C [64] and [65]
Emydoidea blandingii Blanding’s turtle Good 3 days at −3.5 °C [29] and [30]
7 days at −2.5 °C
Malaclemys terrapin Diamondback terrapin Good 7 days at −2.5 °C [22] and [30]
Terrapene ornata Ornate box turtle Good 3–7 days at −2.5 °C [22] and [30]
Graptemys geographica Map turtle Poor 50% survival 3 days at −2.5 °C [2], [22] and [30]
Chelydra serpentine Snapping turtle Poor 60% survival 3 days at −2.5 °C [22] and [30]
Trachemys scripta elegans Red-eared slider Poor 4 h at −4 °C [13] and [30]
60% survival 3 days at −2.5 °C

Turtle adults
Terrapene carolina Box turtle Good 2–3 days at Tb as low as −3.6 °C [15], [19] and [82]
2 days at −2 °C

Lacerta vivipara European common lizard Good 1–3 days at −3 °C [17], [87] and [88]
−4 °C for 2 h
Podarcis muralis Wall lizard Poor Up to 2 h at Tb −0.6 to −1.0 [14]
Podarcis sicula Italian wall lizard Poor 40 min at −3 °C, 60 min at −2.2 °C [5]

Thamnophis sirtalis Garter snake Poor 3–5 h at −2.5 °C, 6 h at −3.3 °C [11] and [16]
48 h at Tb −0.8 to −1.2
Vipera berus Boreal adder Poor 2–3 h at −3.1 °C [1]

Note. Temperatures are ambient in most cases or body temperature (Tb) where indicated.


Table 2. Genes identified by DNA array screening as freeze and anoxia up-regulated in liver and heart of hatchling painted turtles, C. p. marginata

Expression ratio, stress:control (fold up-regulation)
Liver Heart
Iron binding proteins
Ferritin heavy chain 1.6–2.6 1.9–2.3
Ferritin light chain 3.5 2.0–2.8
Transferrin receptor 2 1.8–2.1 1.6–2.0
Hemoglobin α 1.7 1.8
Hemoglobin β 1.7–1.9 1.6–1.9

Antioxidant proteins
Glutathione peroxidase 1 1.8–2.4 2.2–2.5
Glutathione S-transferase M5 2.7 1.7–1.9
Glutathione S-transferase A2 2.2 1.8
Peroxiredoxin 1 1.7–2.1 1.6

Serpin C1 1.6–3.6
Serpin D1 2.3
Serpin G1 1.6

Eggs collected in Algonquin Park, Ontario in June were laboratory-incubated until hatching in September. Hatchlings were transferred to plastic boxes containing damp sphagnum moss and temperature was lowered in stages to 5–7 °C where turtles were maintained for 4 months (mean animal mass 4.34 g ± 0.44 SD, n = 43). Experiments done in winter compared three groups. Control animals were sampled directly from the 5–7 °C incubator. For freezing, turtles were held at −2.5 °C in trays lined with damp paper toweling and were sampled 5 h post-nucleation (based on a mean chilling time before nucleation of 30 min as determined from thermistors attached to the plastrons of some individuals). Mean body temperature was −1.2 °C at sampling and ice was evident in the extremities and brain cavity. For anoxia, plastic jars with a layer of damp paper towel on the bottom were set in crushed ice and then flushed with nitrogen gas (introduced via syringe port) for 2  min. Turtles (four per jar) were added and N2 flushing was continued for 20 min followed by sealing the jars and returning to the 5–7 °C incubator for 4 h. Nitrogen flushing was reconnected while individuals were removed for dissection. Excised tissues were frozen in liquid nitrogen and transferred to −80 °C for storage. RNA extraction and quality assessment were as described previously [32] and then samples were sent to the Microarray Center of the University Health Network (UHN; Toronto, ON) for screening. RNA samples were labeled by the UHN indirect (amino-allyl) labeling protocol (10 μg total RNA per sample) and hybridized to UHN Hum19k6ss arrays. Images were acquired using the ScanArray4000 (Perkin Elmer) scanner and quantified using ArrayVision (v8.0, Imaging Research). The data were imported into GeneTraffic (Iobion Informatics) for normalization (LOWESS, sub-grid), filtering and visualization. The expression ratio, stress:control, indicates the fold up-regulation indicated by the screening. Hemoglobin α was also identified as freeze responsive in C. p. marginata by cDNA library screening.


Source: Cryobiology Volume 52, Issue 1 , February 2006, Pages 1-16


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