Seasonal patterns of the thermal response in relation to sugar and polyol accumulation in overwintering adults of elm leaf beetle, Xanthogaleruca luteola (Coleoptera: Chrysomelidae)

Elm leaf beetle, Xanthogaleruca luteola (Muller), is one of the key pests of elm trees that survive winter in reproductive diapause in sheltered locations. Overwintering adults of the elm leaf beetle showed a complex sugar/polyol cryoprotectant system. The major components of the multiple systems were glucose, myo-inositol and trehalose. In this study, we investigated the seasonal profile of low molecular weight compounds and glycogen in natural population and also in response to thermal constant regimes (5 and 15 °C). Among these components, a remarkable seasonal pattern of accumulation/depletion was observed in myo-inositol over the course of hibernation with the development of diapause progress. Incubating at 5 °C only elicited a strong response in myo-inositol synthesis during diapause. It suggests that the elm leaf beetle accumulates myo-inositol not only in relation to entering diapause but also in response to low temperatures and their interactions. The laboratory acclimation experiments showed that adults exposed to 15 °C had no chance for accumulation of low molecular weight carbohydrate even during diapause. The results of this study illustrated that overwintering adults of elm leaf beetle produce myo-inositol as the primary substance which plays a specific role in some biochemical adjustments in overwintering adults of X. luteola.     

轮纹异痂蝗卵的过冷却能力与其体内水分和生化物质含量的关系

【目的】低温是影响昆虫生长发育和存活的关键因子之一。为明确轮纹异痂蝗Bryodemella tuberculatum dilutum卵的抗寒性及其机理, 研究了其过冷却能力与其体内水分及其他抗寒性相关生化物质含量的关系。【方法】采用热电偶法测定了轮纹异痂蝗卵的过冷却点,同时采用烘干法、残余法、氨基酸自动分析仪和高效液相色谱法分别测定了其卵的含水量、脂肪、氨基酸及小分子糖醇的含量。【结果】土壤含水量对轮纹异痂蝗滞育前卵的含水量、脂肪含量（鲜重）及过冷却点有极显著的影响(P<0.01)。随着土壤含水量从4%增加到13%,卵含水量从45.12%上升到55.25%,卵脂肪含量（鲜重）从10.39%下降到9.39%,而过冷却点从-30.11℃上升到-25.69℃。不同发育阶段卵的过冷却点、含水量、脂肪、氨基酸及小分子糖醇含量存在极显著差异(P<0.01)。从卵产下当天至120 d,卵过冷却点从-26.78℃下降至-30.18℃,含水量从51.93%下降至46.69%,脂肪含量分别从9.99%（鲜重）和17.37%（干重）上升至13.92%(鲜重)和25.29%（干重）。滞育卵的过冷却点显著低于滞育前卵的过冷却点。从卵中共检测到17种氨基酸,其中5种与过冷却点存在显著的相关关系(P<0.05)。卵过冷却点随着甘氨酸和脯氨酸含量的升高而降低,而随着胱氨酸、亮氨酸及天冬氨酸含量的增加而升高。随着卵的发育,海藻糖、甘油、肌醇和山梨醇含量逐渐上升,而葡萄糖和果糖含量逐渐下降。在卵发育过程中,海藻糖和甘油的含量显著高于其他4种物质的含量。卵过冷却点与上述6种小分子糖醇均存在显著的相关关系,其中与海藻糖和葡萄糖的相关性最强。过冷却点随海藻糖、甘油、肌醇和山梨醇含量的升高而降低,而随葡萄糖和果糖含量的升高而上升。【结论】轮纹异痂蝗卵在发育过程中,通过降低含水量,积累脂肪、脯氨酸、甘氨酸及海藻糖、甘油、肌醇和山梨醇等抗寒物质,从而提高其过冷却能力。     

Differences in egg thermotolerance between tropical and temperate populations of the migratory locust Locusta migratoria (Orthoptera: Acridiidae)

The migratory locust Locusta migratoria L., which is widely distributed throughout the world, exhibits within- and between-population variation in cold tolerance. To understand physiological adaptation in populations, we studied the genetic basis of thermotolerance in Hainan (tropical) and Liaoning (temperate) populations and measured expression of Hsp70 and Hsp90 mRNA in both populations at low (0 degrees C) and high temperatures (40 degrees C). Phenotypic variation of thermotolerance is heritable. Heritable characteristics differed among different stages of locust egg development, as well as among different measures of thermotolerance. Nuclear genetic factors, rather than cytoplasmic factors, contribute to differences in cold tolerance between the tropical and temperate populations of the migratory locust; for heat tolerance, maternal effects were involved in three stages of egg development. Expression of Hsp90 mRNA was induced in temperate population after heat shock (40 degrees C x 12h), whereas expression of Hsp70 and 90 was induced in tropical population after cold shock (0 degrees C x 12h). We suggest that thermotolerance of locust eggs has a complex genetic basis and heat shock proteins may be involved in differences of thermotolerance between locust populations.     

Thermoperiodic acclimations enhance cold hardiness of the eggs of the migratory locust

Treatment of thermoperiods that simulate the patterns of natural occurrence is most efficient in enhancing cold hardiness. To examine the effects of different thermoperiods on cold hardiness of eggs in the migratory locust, Locusta migratoria (L.), the survival rates, cryoprotectant levels and three hsps expressions in mid-stage eggs (7-day-old) were measured after the eggs were subjected to three different thermoperiod regimes, : short (2 day), long (10 day), and nature-mimicking thermoperiodic acclimation. The thermoperiodic acclimations resulted in the highest egg survival rates in both the short and the long period acclimation groups in comparison with the groups treated with constant temperatures. The egg survival of nature-mimicking thermoperiod groups was significantly higher than those of constant temperature groups for the same acclimation duration. The survival rate of eggs under single daily thermoperiod was higher than that of multiple daily thermoperiods. The concentration of cryoprotectants (myo-inositol, trehalose, mannitol and sorbitol) and the expression levels of hsp20.5, hsp70, and hsp90 all increased in thermoperiodic acclimation eggs.     

Changes in Sugar Content during Cold Acclimation and Deacclimation of Cabbage Seedlings

The relationship between freezing tolerance and sugar contentin cabbage seedlings was investigated. Seedlings exposed tonon-freezing low temperature (5 °C) acquired freezing tolerancedown to -6 °C. The degree of freezing tolerance increasedwith duration of exposure to low temperature (up to 10 d). Sucrose,glucose, fructose and myo -inositol were detected as solublesugars in cabbage leaves, and all soluble sugars, except formyo -inositol, and starch increased gradually during cold acclimationsuch that their levels were positively correlated with the degreeof freezing tolerance. The induced freezing tolerance was attributednot to ontogenetic changes but to cold acclimation. However,the induced freezing tolerance was lost after only 1 d of deacclimationat control temperatures, and this change was associated witha large reduction in sugar content. These results reveal that the sugar content of cabbage leavesis positively correlated with freezing tolerance. Brassica oleracea L.; cabbage; cold acclimation; deacclimation; freezing tolerance; sugars     

POLYOL ACCUMULATION IN IN VITRO SYSTEMS OF BOMBYX EGGS

The mechanism of polyol accumulation in diapausing Bombyx eggs, conversion of [6-¹⁴C] glucose-6-phosphate into polyols and other neutral sugars was investigated in in vitro reaction systems. When a crude homogenate or a press juice of the eggs was incubated with [6-¹⁴C]glucose-6-P, the labelled trehalose, sorbitol and glycerol accumulated in the reaction mixture. In the press juice incubation system of developing eggs at day 1, ¹⁴C-sorbitol was detected in appreciable amounts, but it decreased rapidly with the development of the embryos. When the press juice was prepared from eggs in diapause, the formation of ¹⁴C-sorbitol was 3–5 times greater in eggs at early stages (day 2 to day 4) than in developing eggs.     

Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs

To examine the relationship between cooling rate and cold hardiness in eggs of the migratory locust, Locusta migratoria, the survival rates and cryoprotectant levels of three embryonic developmental stages were measured at different cooling rates (from 0.05 to 0.8 degrees C min(-1)) in acclimated and non-acclimated eggs. Egg survival rate increased with decreasing cooling rate. The concentration of cryoprotectants (myo-inositol, trehalose, mannitol, glycerol, and sorbitol) increased in non-acclimated eggs, but varied significantly in response to different cooling rates in acclimated eggs. The acclimation process (5 degrees C for 3 days) did not increase eggs resistance to quick cooling ("plunge" cooling and 0.8 degrees C min(-1)). Earlier stage embryos were much more sensitive than later stage embryos to the same cooling rates. Time spent at subzero temperatures also had a strong influence on egg survival.     

Metabolism of sugars to polyols in the boll weevil

When fed to starved adults of Anthonomus grandis, several pentoses and hexoses were metabolized to the corresponding polyols (sugar alcohols). Xylitol, galactitol, arabitol, ribitol, rhamnitol, mannitol, and sorbitol were metabolites of d-xylose, d-galactose and lactose, d-arabinose, d-ribose, l-rhamnose, d-mannose, and d-glucose and d-fructose, respectively. l-Sorbose was not metabolized to a polyol. Large quantities of xylitol and galactitol and intermediate amounts of arabitol, ribitol, and rhamnitol accumulated while only small amounts or traces of mannitol and sorbitol were detected. The limited accumulation of sorbitol in the glucose- and fructose-fed weevils probably was caused by the rapid metabolism of sorbitol to glucose, fructose, trehalose, and glycogen. Each of the ingested sugars, the corresponding polyols, and trehalose were present in the weevil haemolymph. Most of the polyols had never before been detected as metabolites in an insect.     

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5 M DMSO, 1 M sucrose, 0.5 M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H₂O₂ was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.     

Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco : High Amounts of Sorbitol Lead to Necrotic Lesions

We analyzed transgenic tobacco (Nicotiana tabacum L.) expressing Stpd1, a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple, under the control of a cauliflower mosaic virus 35S promoter. In 125 independent transformants variable amounts of sorbitol ranging from 0.2 to 130 μmol g⁻¹ fresh weight were found. Plants that accumulated up to 2 to 3 μmol g⁻¹ fresh weight sorbitol were phenotypically normal, with successively slower growth as sorbitol amounts increased. Plants accumulating sorbitol at 3 to 5 μmol g⁻¹ fresh weight occasionally showed regions in which chlorophyll was partially lost, but at higher sorbitol amounts young leaves of all plants lost chlorophyll in irregular spots that developed into necrotic lesions. When sorbitol exceeded 15 to 20 μmol g⁻¹ fresh weight, plants were infertile, and at even higher sorbitol concentrations the primary regenerants were incapable of forming roots in culture or soil. In mature plants sorbitol amounts varied with age, leaf position, and growth conditions. The appearance of lesions was correlated with high sorbitol, glucose, fructose, and starch, and low myo-inositol. Supplementing myo-inositol in seedlings and young plants prevented lesion formation. Hyperaccumulation of sorbitol, which interferes with inositol biosynthesis, seems to lead to osmotic imbalance, possibly acting as a signal affecting carbohydrate allocation and transport.     

Thermostability Engineering of Fungal Phytases Using Low-Mr Additives and Chemical Crosslinking

With the ultimate goal to develop preparations of phytase (myo-inositol hexakisphosphate phosphohydrolase) with improved thermal resistance for inclusion in animal feed, several thermostabilization approaches were investigated with a set of fungal (Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, and Aspergillus niger phytase) and consensus phytases. Screening of different low-M_r additives revealed that polyethylene glycols increase the thermostability of all phytases in a molecular weight-dependent fashion. The polyols ribitol, xylitol (C₅ sugars) and sorbitol (C₆ sugar) also improved their thermostability, whereas polyols containing more or less carbon atoms, such as glycerol, erythritol and mannoheptulose, showed only minor effects. The stabilizing effects of PEGs and polyols were concentration dependent. In a second series of experiments, crosslinking of the carbohydrate chains of A. fumigatus and consensus phytase using sodium periodate and adipic acid dihydrazide resulted in the formation of oligomeric forms, which may explain the observed thermostability enhancement of 10–15d`C.     

Cold-induced vasodilation and vasoconstriction in the finger of tropical and temperate indigenes

While heat acclimatization reflects the development of heat tolerance, it may weaken an ability to tolerate cold. The purpose of this study was to explore cold-induced vasodilation (CIVD) responses in the finger of tropical indigenes during finger cold immersion, along with temperate indigenes. Thirteen tropical male indigenes (subjects born and raised in the tropics) and 11 temperate male indigenes (subjects born and raised in Japan and China) participated. Subjects immersed their middle finger at 4.3±0.8 °C water for 30 min. Rectal temperature, skin temperatures, finger skin blood flow, blood pressure and subjective sensations were recorded during the test. The results showed that: (1) the tropical group demonstrated a lower minimum (T_min), maximum (T_max) and mean finger temperature (T_mean) compared to those of the temperate group (P<0.05); (2) seven tropical indigenes demonstrated a late-plateau type of CIVD pattern, which is characterized by a pronounced 1st vasoconstriction and a single CIVD with a faint and weak 2nd vasoconstriction, whereas no temperate indigene demonstrated the late-plateau type; and (3) the hand temperature at the end of finger immersion was 3 °C lower in the tropical than the temperate group (P<0.05). These results indicate that tropical indigenes have less active responses of arterio-venous anastomoses in the finger and weaker vasoconstrictions after the first CIVD response during finger cold immersion, which can be considered as being more vulnerable to cold injury of the periphery in severe cold.     

Carbohydrate Metabolism in Drought-Stressed Leaves of Pigeonpea (Cajanus cajan)

Pigeonpea is a tropical grain-legume, which is highly dehydrationtolerant. The effect of drought stress on the carbohydrate metabolismin mature pigeonpea leaves was investigated by withholding waterfrom plants grown in very large pots (50 kg of soil). The moststriking feature of drought-stressed plants was the pronouncedaccumulation of D-pinitol (1D-3-methyl-chiro-inositol), whichincreased from 14 to 85 mg g^–1 dry weight during a 27d stress period. Concomitantly, the levels of starch, sucroseand the pinitol precursors myo-inositol and ononitol all decreasedrapidly to zero or near-zero in response to drought. The levelsof glucose and fructose increased moderately. Drought stressinduced a pronounced increase of the activities of enzymes hydrolysingsoluble starch (amylases) and sucrose (invertase and sucrosesynthase). The two anabolic enzymes sucrose phosphate synthase(sucrose synthetic pathway) and myo-inositol methyl transferase(pinitol synthetic pathway) also showed an increase of activityduring stress. These results indicate that pinitol accumulatedin pigeonpea leaves, because the carbon flux was diverted fromstarch and sucrose into polyols. Key words: Drought, polyols, pinitol, sucrose, starch, pigeonpea     

Enhanced Trehalose Accumulation and Desiccation Survival of Entomopathogenic Nematodes Through Cold Preacclimation

Limited storage stability is a major obstacle to further expansion of the use of entomopathogenic nematodes for pest control. Progress has been made that Steinernema carpocapsae can now be stored under partial anhydrobiosis for up to 6 months at 25°C and 10 months at 5°C in a water-dispersible granular (WG) formulation. However, other species have been more difficult to store in the WG formulation due to migration of nematodes out of the granules and sensitivity of some species to desiccation directly at cold temperatures. As acclimation to cold induces trehalose accumulation (a major cryo- and desiccation protectant) in many invertebrates, it was hypothesized that cold preacclimation of entomopathogenic nematodes will enhance their survival in the WG formulation at cold temperatures. This hypothesis was tested using a temperate species Steinernema feltiae , a subtropical species S. carpocapsae , and a tropical species Steinernema riobrave possessing different thermal niche breadths and reproduction temperature optima. Cold acclimation of infective juveniles increased trehalose accumulation in all three species and the amount of trehalose accumulated was both temperature and species dependent. Trehalose content reached at its peak after 6 days at 5°C in S. feltiae (82.28 μg/mg dry weight), after 10 days at 10°C in S. carpocapsae (94.16 μg/mg dry weight) and after 6 days at 15°C in S. riobrave (47.58 μg/mg dry weight). Cold preacclimation at 5°C for 2 days enhanced desiccation survival of S. feltiae in 25% glycerol (osmotic desiccation) at both 5 and 25° and of S. carpocapsae and S. riobrave only at 5°C. Non-cold acclimated S. carpocapsae and S. riobrave were extremely sensitive to desiccation directly at 5°C in 25% glycerol, resulting in over 98% mortality within 6 days, but S. feltiae was more sensitive to desiccation at 25°C than at 5°C. Cold preacclimation increased survival of all the three species in the WG formulation at both 5 and 25°C. The survival of S. riobrave at 5°C in the WG formulation was positively correlated with the length of preacclimation period at 5°C (R 2 = 0.99) and with the amount of trehalose accumulated during cold preacclimation (R 2 = 0.81). These results support the hypothesis that cold preacclimation enhances desiccation survival of entomopathogenic nematodes at cold temperatures and the increased survival correlates well with the increased trehalose accumulation. Results also demonstrate that cold preacclimation can be used as a tool to enhance survival of nematodes in the formulations with reduced water activity.     

Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-Symport of numerous substrates, including myo-inositol, glycerol, and ribose

Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5–green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H⁺-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.