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.

Functional characterization of the adipokinetic hormone in the pea aphid, Acyrthosiphon pisum.

Aphids are important plant phloem-sucking pests and detailed knowledge about the hormonal control of their metabolism can potentially contribute to the development of methods for their management. The insect metabolism is predominantly controlled by neuropeptides belonging to the adipokinetic hormone/red pigment-concentrating hormone family (AKH/RPCH). The main goal of this study was to obtain the sequence of AKH transcripts and analyze its expression in all polyphenic female forms of the pea aphid, Acyrthosiphon pisum. The neuropeptide is expressed in the brain of all female forms and in the ovaries of the both (wingless and winged) parthenogenetic forms. The form of active Acypi-AKH decapeptide was confirmed by the LC/MS and +ESI tandem mass spectrometry. The highest relative amount of Acypi-AKH was recorded in winged virginoparae. Furthermore, a potential role of this hormone when directly applied to the aphid was studied as well. Interestingly, no significant increase of trehalose in the wingless virginoparae after application of synthetic Acypi-AKH was detected. Yet this treatment did affect the level of protective polyol (mannitol) and furthermore led to increased activity of the detoxification enzyme glutathione S-transferase. The possible physiological function of AKH in A. pisum under the stress conditions is discussed.

Physiological and biochemical analysis of overwintering and cold tolerance in two Central European populations of the spruce bark beetle, Ips typographus.

Overwintering success is one of the key aspects affecting the development and outbreaks of the spruce bark beetle, Ips typographus (L.) populations. This paper brings detailed analysis of cold tolerance, and its influence on overwintering success, in two Central European populations of I. typographus during two cold seasons. Evidence for a supercooling strategy in overwintering adults is provided. The lower lethal temperature corresponds well to the supercooling point that ranges between -20 and -22°C during winter months. The supercooled state is stabilized by the absence of internal ice nucleators and by seasonal accumulation of a mixture of sugars and polyols up to the sum concentration of 900 mM. The cryoprotective function of accumulated metabolites is probably based on increasing the osmolality and viscosity of supercooled body fluids and decreasing the relative proportion of water molecules available for lethal formation of ice nuclei. No activity of thermal hysteresis factors (stabilizers of supercooled state) was detected in hemolymph. Lethal times for 50% mortality (Lts50) in the supercooled state at -5, -10 or -15°C are weeks (autumn, spring) or even months (winter), suggesting relatively little mortality caused by chill injury. Lts50 at -15°C are significantly shorter in moist (6.9 days) than in dry (>42 days) microenvironment because there is higher probability of external ice nucleation and occurrence of lethal freezing in the moist situation.

Gas chromatographic analysis of amino acid enantiomers in Carbetocin peptide hydrolysates after fast derivatization with pentafluoropropyl chloroformate.

A novel sample preparation protocol for gas chromatographic (GC) analysis of amino acid enantiomers in peptides was developed. It comprises traditional acid hydrolysis, a novel treatment of the analytes with a fluoroalkyl chloroformate and GC/FID separation of enantiomers on a chiral capillary column. The major improvements consist in that the derivatization step proceeds in organic-aqueous media within seconds and the amino acid derivatives are volatile enough to suit the temperature range of the chiral Chirasil-Val capillary column. The approach was found beneficial for chiral analysis of pharmaceutically important Carbetocin peptide.

Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus.

Overwintering adults of the spruce bark beetle, Ips typographus (L.) showed an unusually complex sugar/polyol cryoprotectant system. The major components of the multiple system were: glucose (177.6 mmolL(-1), March); trehalose (175.0 mmolL(-1), December); sorbitol (147.9 mmolL(-1), January); mannitol (81.2 mmolL(-1), March); and erythritol (40.7mmolL(-1), March) (in the parentheses, the maximum concentrations are shown and the month when they were reached). Other minor components were glycerol, fructose, threitol, myo-inositol, arabinitol and ribitol. Distinct seasonal patterns of accumulation/depletion in various components were found. Glycerol, trehalose and glucose started to accumulate first, during early autumn, when the air temperatures fluctuated between 20 and 0 degrees C, and diapause beetles continued in feeding. Glycerol was depleted, glucose remained stable and trehalose continued in accumulation during late autumn when the temperatures oscillated around 0 degrees C. During early winter severe frosts reaching -20 degrees C came, the beetles terminated their diapause and trehalose was partially depleted, while mannitol, sorbitol, fructose, threitol and erythritol started to accumulate. Cold weather continued also during late winter when the beetles remained quiescent. During this period, trehalose was re-accumulated, threitol and erythritol continued to increase, mannitol remained stable and sorbitol, fructose decreased. All cryoprotectans were finally cleared in the beetles which were spontaneously leaving bark during early spring. The seasonal maximum of total concentration of all cryoprotectants (578.2 mOsmol L(-1)) was reached in March. Such a concentration results in colligative depression of melting point of body fluids down by 1.08 degrees C only. It suggests that the potential cryoprotective effect of accumulated sugars and polyols was related rather to their non-colligative functions.

Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland, corpus allatum.

In the conventional view, the winter adaptation of membrane lipids is induced by temperature decrease. We propose that winter remodelling of membranes in Pyrrhocoris apterus is triggered by short-day photoperiod before the temperature decrease and changes caused by cold temperature represent the later phase of adaptation. The induction of diapause by short-day photoperiod results in an accumulation of phosphatidylethanolamine (PE) molecular species with C(16:0)/C(18:2) acyl chains esterified to sn-1/sn-2 positions of glycerol at the expense of C(18:0)/C(18:2). Proportions of C(16:0)/C(18:2)-PE are enhanced in short-day compared to long-day insects in both thoracic muscles (TM, 15.0 vs. 8.2%) and fat bodies (FB, 24.9 vs. 13.6 %). Proportions of C(16:0)/C(18:2)-PE are further enhanced during cold acclimation (to 26.5% in TM, 33.6 % in FB) at the expense of a more saturated species, C(18:0)/C(18:1)-PE. These changes are less prominent in phosphatidylcholines (PC). The effect of photoperiod seems to be mediated via the corpus allatum. Long-day non-diapause females deprived of their corpus allatum have the phospholipid molecular species profile similar to that found in short-day diapausing females. While the acyl chain remodelling is regulated by both photoperiod and temperature, the head group composition is regulated by temperature only. Similar to most other organisms, the level of PE is higher (50.3 vs. 43.5% in TM, 44.3 vs. 37.8% in FB) and that of PC is lower (35.9 vs. 40.2% in TM, 41.6 vs. 46.1 % in FB) at cold temperatures (/=16 degrees C). In contrast to a general rule, the PE is less unsaturated than PC. In both TM and FB, proportions of unsaturated/unsaturated molecular species are consistently high in PC (56.3-67.5% in TM, 59.2-66.6% in FB), while they are consistently low in PE (19.1-26.7% in TM, 12.1-15.1% in FB). An adaptive significance of changes in the phospholipid composition for the low temperature and/or dehydration stress is discussed in relation to known physical properties of phospholipids.

Entering diapause is a prerequisite for successful cold-acclimation in adult Graphosoma lineatum (Heteroptera: Pentatomidae).

In diapause adults of Graphosoma lineatum overwintering in a field-cage, high chill-tolerance (CT) developed gradually, within 5 months from August to December. In laboratory-acclimation experiments, the diapause state appeared to be an essential pre-condition for successful cold-acclimation and overwintering. First, diapause prevented elevation of the median supercooling point (SCP) by about 5.5 degrees C that accompanies the onset of reproductive activity in non-diapause specimens. Second, diapause allowed subsequent physiological changes resulting in cold-acclimation during a gradual (18-day) decrease of temperature from 25 to 0 degrees C. No, or very modest, cold-acclimation was observed in non-diapause specimens. Decrease of temperature led to a rapid loss of ca. 1/3 of the body water in both non-diapause and diapause specimens. Approximately 0.1 M of trehalose accumulated in tissues of diapause specimens only, and haemolymph osmolality rose from 347 mOsm (at 25 degrees C) to 444 mOsm after an 18-day cold-acclimation and to 764 mOsm during further storage at 0 degrees C for 100 days. Upon transfer of cold-acclimated diapause specimens back to 25 degrees C for one week (de-acclimation), the high CT was lost, the SCP elevated by about 2.5-3 degrees C, and the levels of trehalose, water content and haemolymph osmolality returned to pre-acclimation or non-diapause levels.

The agrobacterium tumefaciens C58-6b gene confers resistance to N(6)-benzyladenine without modifying cytokinin metabolism in tobacco seedlings

The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N(6)-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N(6)-benzyladenine-7-glucoside (80-92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

Beauverolides L and La from Beauveria tenella and Paecilomyces fumosoroseus.

New beauverolides L and La were isolated and identified from the entomopathogenic fungi, Beauveria tenella and Paecilomyces fumosoroseus. Their structures, cyclo-[3-hydroxy-4-methyldecanoyl-L-phenylalanyl-L-alanyl-D-leucyl ], and cyclo-[3-hydroxy-4-methyldecanoyl-L-phenylalanyl-L-alanyl-D-allo-i soleucyl] were deduced from HPLC and GC-mass spectrometric analyses of their hydrolysates and NMR and mass spectral data.