Temporal plasticity in cold hardiness and cryoprotectant contents in northern versus temperate Colias butterflies (Lepidoptera: Pieridae).

　　BACKGROUND: Butterflies Colias hyale and C. palaeno differ in distribution, habitat, voltinism and cold hardiness. OBJECTIVE: To compare changes in supercooling point (SCP) and cryoprotectant concentration(CPAc) of outdoor-reared caterpillars. MATERIALS AND METHODS: SCP was measured with a thermocouple and CPAc by gas chromatography monthly during winter. RESULTS: Seasonal changes in SCP and CPAc in overwintering larvae followed the pattern of change in ambient temperature. Only in warm November, SCP was low and CPAc high in boreal univoltine C. palaeno, whereas the opposite appeared in temperate multivoltine C. hyale. In mild December, SCP increased and CPAc decreased in C. palaeno, and acclimation reappeared in cold January. Both species differed in monthly cryoprotectant profiles, regarding both constitutive and inducible compounds. CONCLUSION: Seasonal pattern of SCP/CPAc enables C. palaenoto survive early frosts, but the costs of repeated acclimation during mild winters may set southern or low altitude limits of its distribution.

Overwintering of the boreal butterfly Colias palaeno in central Europe.

BACKGROUND: Colias palaeno (Linnaeus, 1761) (Lepidoptera: Pieridae) is a butterfly with boreal distribution with declining populations in peat bogs and subalpine habitats in Central Europe. OBJECTIVE: We investigated the cold tolerance of overwintering caterpillars from one mountain population from Czech Republic (960m a.s.l.) and one alpine population from Italy (2000m a.s.l.). METHODS: We measured supercooling point (SCP), lower lethal temperature (LLT) and content of cryoprotectants. RESULTS: The caterpillars were freeze-avoiding, with lower LLT close to their very low SCP (-25 to -27 degree C). The mountain population accumulated high concentrations of glycerol (5% fresh mass) and sugars (trehalose 0.8%, glucose 0.2%), while the Italian alpine population only moderate amounts of glycerol (0.3%) and sugars (trehalose 0.5%, glucose 0.3%) without effect on their cold hardiness. Larvae that overwintered at +5 degree C had a lower body mass than those overwintering in natural conditions, indicating a metabolic weight loss, but both groups survived equally well. CONCLUSION: We hypothesize that the high concentration of glycerol contributes to the high desiccation tolerance.

Effect of thermoperiod on diapause intensity in Pyrrhocoris apterus (Heteroptera Pyrrhocoridae).

The intensity of adult diapause in Pyrrhocoris apterus was measured in two series of experiments as the duration of pre-oviposition period at a constant temperature of 25 degrees C after transfer from short (12L:12D) to long day conditions (18L:6D). Higher diapause intensity was induced with a thermoperiod than at constant temperatures. After the induction throughout larval instars 3-5 and during 4 weeks of adult life at short days and a thermoperiod of 25/15 degrees C the pre-oviposition period was 30+/-4 and 26+/-3 days. After induction at constant 25 degrees C the pre-oviposition period was 22+/-3 and 23+/-4 days, while after induction at constant 20 degrees C it was 17+/-4 and 19+/-4 days. Induction at a lower constant temperature of 20 degrees C was thus followed by a less intense diapause than the induction at a higher constant temperature of 25 degrees C. These counterintuitive results are discussed. The oxygen consumption rate measured at experimental temperatures prior to transfer from short to long days was higher at thermoperiodic conditions than at constant temperatures and it was similar at constant 20 and 25 degrees C. Thus, the oxygen consumption rate measured prior to the transfer was highest (indication of the least intense diapause) in the insects that showed later, after the transfer to long days, the longest pre-oviposition period (indication of the most intense diapause). Within the first two days after transfer to constant 25 degrees C, oxygen consumption rate measured at 25 degrees C decreased in the thermoperiodic insects, while it transiently increased in insects from constant 20 degrees C. Two days and later after the transfer, oxygen consumption rate was similar in all groups. Cold hardiness was not correlated with diapause intensity. The low lethal temperature in diapausing insects was correlated with the night temperature during diapause induction.

Snow white and the seven dwarfs: a multivariate approach to classification of cold tolerance.

Two main cold hardiness strategies of insects - freeze tolerance in some species, and overwintering in a supercooled state without tolerance of freezing in many others - were recently reclassified. However, I present several problems with the current systems. My suggested classification is based on clearer definitions of the causes of cold injury. I recognize three main mortality factors: freezing of body liquids, cold shock, and cumulative chill injury. Presence or absence of each of these factors produce eight combinations. I have named the eight classes after Snow White and the Seven Dwarfs to avoid nomenclatural confusion. Some of these classes are probably not used as tactics against cold injury by any insect species. Other classes contain so many species that they might be reclassified in more detail, using values of supercooling point and other quantitative parameters. However, widely comparable parameters, like the upper limit of cold injury zone and the sum of injurious temperatures are still rarely published, thus we still lack comprehensive data for multivariate analyses. Every cold hardiness strategy should be characterized by a meaningful class or subclass together with the physiological, biochemical, and behavioural mechanisms employed by the insects. I also point out the existence of strategies that combine two tactics - either a switching strategy (during preparation for winter, population "chooses" which tactic will be used), or a dual strategy (individuals are ready to use one of the tactics depending on the prevailing environmental conditions).

Chill tolerance in the tropical beetle Stenotarsus rotundus.

The tropical beetle Stenotarsus rotundus (Endomychidae) survived chilling at mildly low temperatures (above +5 degree C). With upper limit of cold injury zone (ULCIZ, the highest temperature that causes cold injury) well above freezing point, the supercooling ability (mean supercooling point - SCP; -11 degree C) has no cryoprotective importance. Mortality increases rapidly between -9 and +5 degree C, dependent on accumulated dose of chilling (sum of injurious temperatures - SIT; 2 degree-days below ULCIZ). The cold hardiness traits found in this species are by-products of deep diapause, and may serve as pre-adaptation for expansion into cooler regions.

Temperature Regulation of Supercooling and Gut Nucleation in Relation to Diapause of Pyrrhocoris apterus (L.) (Heteroptera)

The heteropteran Pyrrhocoris apterus (L.) does not survive freezing of its body fluids; there is a good correlation between values of survival at subzero temperatures and the supercooling point (SCP), i.e., the temperature at which body fluids start to freeze. The decrease of the SCP and thus the increase in cold hardiness is regulated by photoperiod and temperature. The relative importance of these factors depends on the physiological state of the insect. The SCP is about -7°C at the onset of prediapause and a decrease of about 4-5°C is associated with the development of the diapause syndrome in adults; these processes both are induced by a short-day photoperiod with temperature playing a secondary role. The induction of the diapause syndrome is a prerequisite for the subsequent decrease of the SCP by about 5-6°C during cold acclimation. An intermediate temperature of 15°C, or fluctuating outdoor temperatures and short-day photoperiods, are more suitable for the decrease of SCP than 5°C in continuous darkness. The sensitivity to photoperiod gradually disappears during the development of diapause; after the termination of diapause around the winter solstice the SCP irreversibly increases at a high temperature of 26°C even if exposed to a short-day photoperiod. The SCPs of hemolymph, gut, fat body, and gonads were compared to whole-body SCP. The gut was identified as the primary site of ice nucleation because its SCP value was very similar to the value for the whole body in both short-day and long-day insects. The SCPs of other organs, including the hemolymph, were always lower than the whole body SCP. Food was not a source of ice nucleating agents because the SCP of freshly ecdysed adults remained high after 2 weeks of starvation. In contrast, feeding was a prerequisite for the decrease of the SCP during prediapause. In postdiapause insects, the SCP increased at high temperatures in spite of the absence of food.

Effects of starvation on body composition and cold tolerance in the collembolan Orchesella cincta and the isopod Porcellio scaber.

The effects of long-term starvation on the body composition of the isopod Porcellio scaber (Latreille) and the collembolan Orchesella cincta (L.) were studied, by determining the body composition in starved and fed animals. A period under summer conditions (19 degrees C, 75% RH and L/D 16/8 photoperiod), was followed by a period under winter conditions (5 degrees C, 75% RH and LD 6/18 photoperiod). O. cincta was held under summer conditions for 3weeks, during which its protein and lipid content decreased, while its water content increased. In P. scaber, the same occurred during the 6weeks they were kept under summer conditions. During subsequent weeks under winter conditions, changes in cold tolerance of the animals were investigated. Cold tolerance and haemolymph osmolality were measured once a week. Starved animals had lower cold tolerance than fed ones. For P. scaber a decreased haemolymph osmolality was found in starved animals compared to fed ones. This is assumed to be caused by a combination of the consumption of carbohydrates out of the haemolymph and of protein reserves and the accumulation of body water. O. cincta appeared to be capable of osmoregulation, as haemolymph osmolality did not differ between starved and fed animals, despite differences in body water content. Decreased cold tolerance in starved animals of both species may be caused by increased water content or, more probably, by the decrease in reserves needed to produce cryoprotective substances.