Extracellular ice phase transitions in insects.

At temperatures below their temperature of crystallization (Tc), the extracellular body fluids of insects undergo a phase transition from liquid to solid. Insects that survive the transition to equilibrium (complete freezing of the body fluids) are designated as freeze tolerant. Although this phenomenon has been reported and described in many Insecta, current nomenclature and theory does not clearly delineate between the process of transition (freezing) and the final solid phase itself (the frozen state). Thus freeze tolerant insects are currently, by convention, described in terms of the temperature at which the crystallization of their body fluids is initiated, Tc. In fact, the correct descriptor for insects that tolerate freezing is the temperature of equilibrium freezing, Tef. The process of freezing is itself a separate physical event with unique physiological stresses that are associated with ice growth. Correspondingly there are a number of insects whose physiological cryo-limits are very specifically delineated by this transitional envelope. The distinction also has considerable significance for our understanding of insect cryobiology: firstly, because the ability to manage endogenous ice growth is a fundamental segregator of cryotype; and secondly, because our understanding of internal ice management is still largely nascent.

A 9 kDa antifreeze protein from the Antarctic springtail, Gomphiocephalus hodgsoni.

A 9 kDA antifreeze protein (AFP) was isolated and purified from the Antarctic springtail, Gomphiocephalus hodgsoni. By combining selective sampling procedures and a modified ice affinity purification protocol it was possible to directly isolate a single AFP protein without recourse to chromatographic separation techniques. Mass spectrometry identified a single 9 kDa component in the purified ice fraction. Intramolecular disulphide bonding was suggested by the presence of 12 cysteine residues. The specific amino acid composition is unique, particularly with regard to the presence of histidine (11.5%). But it also shows noticeable commonalities with insect AFPs in the abundance of cysteine (13.8%), while simultaneously hinting, through the presence of glycine (11.5%), that the metabolic building blocks of AFPs in Collembola may have a phylogenetically-determined component.

Freeze fitness in alpine Tiger moth caterpillars and their parasitoids.

The adaptive fitness of a freeze-tolerant insect may be mediated by both endogenous and exogenous interactions. The aim of the study presented here was to characterize the freeze tolerance of alpine Tiger moth caterpillars (Metacrias huttoni) and highlight two poorly explored indices of the potential attrition of fitness: (1) downstream development and reproduction; (2) parasitism. Caterpillars survived temperatures as low as -16°C and demonstrated >90% 72-h survival after exposures to -10°C. Two-week acclimations at 5, 10, and 20°C had no effect on body water content, haemolymph osmolality or survival of equilibrium freezing, but there was a significant elevation of the temperature of crystallization (T (c)) in those caterpillars acclimated to 5°C. Cell viability of fat body tissue was resilient to freezing (-10 to -16°C), but midgut and tracheal cells showed significant degradation. Pupation and eclosion were unaffected by freezing at -5 or -10°C. Likewise, there were no significant differences in egg production or the proportion of eggs that hatched between control and frozen insects. By contrast, the ability of tachinid larvae to survive freezing within their hosts means that parasitism plays an important role in regulating population size. Mean parasitism of caterpillars by tachinids was 33.3 ± 7.2%. Pupation and imago emergence of tachinids after host 'endo-nucleation' was >75%. Eclosed adult tachinids showed a non-significant increase in the incidence of wing abnormalities in relation to low temperature exposure.

Antifreeze proteins in the Antarctic springtail, Gressittacantha terranova.

Antarctic springtails are exemplars of extreme low temperature adaptation in terrestrial arthropods. This paper represents the first examination of such adaptation in the springtail, Gressittacantha terranova. Acclimatization state was measured in field-fresh samples over a 22-day period at the beginning of the austral summer. No evidence of temperature tracking was observed. Mean temperature of crystallization (T(c)) for all samples was -20.67 ± 0.32°C and the lowest T(c) recorded was -32.62°C. Ice affinity purification was used to collect antifreeze proteins (AFPs) from springtail homogenate. The purified ice fraction demonstrated both thermal hysteresis activity and recrystallisation inhibition. Growth-melt observations revealed that ice crystals grow normal to the c-axis (basal plane). Reverse-phased HPLC produce one clearly resolved peak (P1) and one compound peak (P2). Mass spectrometry identified the molecular mass of P1 as 8,599 Da. The P1 protein was also the most prominent in P2, although additional peptides of 6-7 KDa were also prominent. The main AFP of the Antarctic springtail, G. terranova has been isolated, although like other AFP-expressing arthropods, it shows evidence of expressing a family of AFPs.

Freezing in the Antarctic limpet, Nacella concinna.

The process of organismal freezing in the Antarctic limpet, Nacella concinna, is complicated by molluscan biology. Internal ice formation is, in particular, mediated by two factors: (a) the provision of an inoculative target for ice formation in the exposed mucus-secreting foot; and (b) osmoconformity to the marine environment. With regard to the first, direct observations of the independent freezing of pedal mucus support the hypothesis that internal ice formation is delayed by the mucal film. As to the second, ice nucleation parametrics of organismal tissue (head, midgut, gonad, foot) and mucus in both inter- and subtidal populations were characterized by high melting points (range=-4.61 to -6.29 degrees C), with only c.50% of a given sample osmotically active. At this stage it would be premature to ascribe a cryo-adaptive function to the mucus as the protective effects are more readily attributed to the physical properties of the secretion (i.e. viscosity) and their corresponding effects on the rate of heat transfer. As it is difficult to thermally distinguish between the freezing of mucus and the rest of the animal, the question as to whether it is tolerant of internal as well as external ice formation remains problematic, although it may be well suited to the osmotic stresses of organismal freezing.

Metabolomic fingerprint of cryo-stress in a freeze tolerant insect.

This study employed H-NMR spectroscopy to assay the metabolome of the high Arctic freeze-tolerant dipteran larvae, Heleomyza borealis, after recovery from exposure to a range of sub-zero temperature treatments. Our data demonstrate the resilience of freeze tolerance in individuals of this permanently freeze-tolerant species that were acclimated to summer temperatures (5 degree C): recovery of homeostasis after 48 h was not significantly disturbed by 2h exposures to -3, -12, or -20 degree C. Evidence of homeostatic perturbation to cryo-stress - both in terms of changes in specific metabolite concentrations as well as systemic changes in metabolism determined using multivariate pattern recognition techniques - was expressed almost entirely at a temperature coincident with the significant onset of mortality (-25 degree C) and considerably below the minimum winter temperatures of its over-wintering habitat (c.-12 degree C).

Plasticity in arthropod cryotypes.

Low-temperature acclimation and acclimatization produce phenotypic changes in arthropods at multiple levels of biological organization from the molecular to the behavioural. The role and function of plasticity - where a constitutive, reversible change occurs in the phenotype in response to low temperature - may be partitioned hierarchically at evolutionary scales according to cryoprotective strategy, at macrophysiological scales according to climatic variability, and at meso- and micro-scales according to ecological niche and exposure. In correspondence with these scales (which are interdependent rather than mutually exclusive), a hierarchical typology of interaction between thermal history and organism is proposed, descending, respectively, from what we define as 'cryotype' (class of cryoprotective strategy) to genotype and, ultimately, phenotype. Alternative (and sometimes complementary) strategies to plasticity include specialization, generalization, bet-hedging, cross-resistance and convergence. The transition of cryotypes from basal to derived states is a continuum of trait optimization, involving the fixation of plasticity and/or its alternatives.

Plasticity and superplasticity in the acclimation potential of the Antarctic mite Halozetes belgicae (Michael).

The plasticity of an organism's phenotype may vary spatially and temporally, and across levels of physiological organisation. Given the adaptive value of plasticity in heterogeneous environments, it might be expected that it will be expressed most in a phenotype's most significant adaptive suites; at high latitudes, one of these is low temperature adaptation. This study examines the phenotypic plasticity of cold acclimation in the Antarctic mite, Halozetes belgicae (Michael). Both plastic and 'superplastic' (extreme plasticity) acclimation responses were found. Plastic responses were evident in responses to laboratory acclimation and field acclimatisation. 'Superplasticity' was found in its ability to rapidly cold harden (RCH) at 0, -5 and -10 degrees C. For example, after just 2 h of acclimation at 0 degrees C, mites acclimated at 10 degrees C shifted their supercooling points (SCPs) by approx. 15 degrees C. In terms of the combined speed of induction and lowering of lethal temperature, this is the most potent RCH response yet reported for a terrestrial arthropod. RCH was also expressed in thermal activity thresholds. Mechanisms responsible for significant differences in recovery from chill torpor are unknown; however, analysis of gut nucleator abundance suggest that the dynamic management of supercooling potential is largely achieved behaviourally, via evacuation. Comparisons with the literature reveal that plasticity in this species varies latitudinally, as well as temporally. The high degree of plasticity identified here is coincident with H. belgicae's occupation of the most exposed spatial niche available to Antarctic terrestrial arthropods.

Recrystallisation temperature (Trc) in an Antarctic springtail.

Cryobiologists have traditionally assumed that the temperature of crystallisation (Tc) or supercooling point (SCP) of a chill-tolerant insect is not a stochastic event, i.e. that it is a biologically meaningful indicator of phenotypic characteristics, be they exogenous influences (e.g. acclimation/acclimatization) or endogenous factors (e.g. life history stage, moult state). Recent work by Wilson et al. (11) has suggested that SCPs--at least in non-biological samples--are more stochastic than previously thought. Here, this question is tested indirectly by the repetitive freezing of individuals of the Antarctic springtail, Cryptopygus antarcticus. The springtails were each supercooled ten times in succession to determine their re-crystallisation temperatures (Trc). SCPs were found to be deterministic i.e. related to their initial Tc. Despite the mortality of re-crystallised samples, 70 percent showed that less than 1 degree C difference between Tc and Trc1 and 95 percent showed less than 5 degree C difference. Tc and Trc1 were significantly correlated. Variability in re-crystallisation temperatures is hypothesised to be predominantly the result of differences in nucleator content and changes in body fluid osmolality during the experimental exposures. Factors affecting the relative variability of SCPs are discussed.