Effect of freeze temperature on ice formation and long-term survival of the woolly bear caterpillar (Pyrrharctia isabella)

Tissue ice content and post-freeze survival were documented for caterpillars of the arctiid moth Pyrrharctia isabella. Tissue ice content was inversely dependent on freeze temperature (−3 °C=24.4%, −6 °C=40.2%, −10 °C=48.7%) but values were substantially less than expected given hemolymph osmolality. Accumulation of glycerol (200-300 mM) in the hemolymph helped to colligatively reduce the amount of freezable water. Caterpillars engaged in locomotion within minutes after thawing but mortality occurred over the ensuing weeks, with the highest level (52.2%) occurring in the −10 °C fast thaw group. Pupation rates ranged between 45.7 to 52.4% of caterpillars in a test group. Adult emergence exceeded 60% of the pupae in the -3 and -6 °C test groups. Hence, P. isabella caterpillars survived ecologically relevant freezes and continued their life cycles to adulthood.     

Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs

Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about − 4 °C when cooled under dry conditions, but freezing of body fluids was readily induced at − 1 °C when in contact with external ice crystals. All three species survived freezing for 2 days at − 1 °C, and some A. rufus and A. lusitanicus also survived freezing at − 2 °C. ¹H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22 μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates.     

Temperature-dependent activity in early life stages of the stone crab Paralomis granulosa (Decapoda, Anomura, Lithodidae): A role for ionic and magnesium regulation?

Marine brachyuran and anomuran crustaceans are completely absent from the extremely cold (− 1.8 °C) Antarctic continental shelf, but caridean shrimps are abundant. This has at least partly been attributed to low capacities for magnesium excretion in brachyuran and anomuran lithodid crabs ([Mg²⁺]_HL = 20-50 mmol L^− 1) compared to caridean shrimp species ([Mg²⁺]_HL = 5-12 mmol L^− 1). Magnesium has an anaesthetizing effect and reduces cold tolerance and activity of adult brachyuran crabs. We investigated whether the capacity for magnesium regulation is a factor that influences temperature-dependent activity of early ontogenetic stages of the Sub-Antarctic lithodid crab Paralomis granulosa. Ion composition (Na⁺, Mg²⁺, Ca²⁺, Cl⁻, SO₄²⁻) was measured in haemolymph withdrawn from larval stages, the first and second juvenile instars (crabs I and II) and adult males and females. Magnesium excretion improved during ontogeny, but haemolymph sulphate concentration was lowest in the zoeal stages. Neither haemolymph magnesium concentrations nor Ca²⁺:Mg²⁺ ratios paralleled activity levels of the life stages. Long-term (3 week) cold exposure of crab I to 1 °C caused a significant rise of haemolymph sulphate concentration and a decrease in magnesium and calcium concentrations compared to control temperature (9 °C). Spontaneous swimming activity of the zoeal stages was determined at 1, 4 and 9 °C in natural sea water (NSW, [Mg²⁺] = 51 mmol L^− 1) and in sea water enriched with magnesium (NSW + Mg²⁺, [Mg²⁺] = 97 mmol L^− 1). It declined significantly with temperature but only insignificantly with increased magnesium concentration. Spontaneous velocities were low, reflecting the demersal life style of the zoeae. Heart rate, scaphognathite beat rate and forced swimming activity (maxilliped beat rate, zoea I) or antennule beat rate (crab I) were investigated in response to acute temperature change (9, 6, 3, 1, − 1 °C) in NSW or NSW + Mg²⁺. High magnesium concentration reduced heart rates in both stages. The temperature-frequency curve of the maxilliped beat (maximum: 9.6 beats s^− 1 at 6.6 °C in NSW) of zoea I was depressed and shifted towards warmer temperatures by 2 °C in NSW + Mg²⁺, but antennule beat rate of crab I was not affected. Magnesium may therefore influence cold tolerance of highly active larvae, but it remains questionable whether the slow-moving lithodid crabs with demersal larvae would benefit from an enhanced magnesium excretion in nature.     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

Feeding impairs chill coma recovery in the migratory locust (Locusta migratoria)

Low temperature causes loss of neuromuscular function in a wide range of insects, such that the animals enter a state known as chill coma. The ability to recover from chill coma (chill coma recovery time) is often a popular phenotype to characterise chill tolerance in insects. Chill coma in insects has been shown to be associated with a decrease in haemolymph volume and a marked increase in [K⁺], causing dissipation of K⁺ equilibrium potential and resting membrane potential. High potassium diet (wheat) has also previously been shown to increase haemolymph [K⁺] in Locusta migratoria leading to sluggish behaviour. The present study combined these two independent stressors of ion and water homeostasis, in order to investigate the role of K⁺- and water-balance during recovery from chill coma, in the chill sensitive insect L. migratoria. We confirmed that cold shock elicits a fast increase in haemolymph [K⁺] which is likely caused by a water shift from the haemolymph to the muscles and other tissues. Recovery of haemolymph [K⁺] is however not only reliant on recovery of haemolymph volume, as the recovery of water and K⁺ is decoupled. Chill coma recovery time, after 2 h at −4 °C, differed significantly between fasted animals and those fed on high K⁺ diet. This difference was not associated with an increased disturbance of haemolymph [K⁺] in the fed animals, instead it was associated with a slowed recovery of muscle [K⁺], muslce water, haemolymph [Na⁺] and K⁺equilibrium potential in the fed animals.     

Combined effects of temperature and cadmium exposure on haemocyte apoptosis and cadmium accumulation in the eastern oyster Crassostrea virginica (Gmelin)

Combined effects of acclimation temperature (12, 20 and 28 °C) and exposure to a toxic metal cadmium (Cd, 50 μg L⁻¹) on haemolymph parameters related to immune defense and metal transport were studied in a model marine bivalve, Crassostrea virginica. Acclimation to elevated temperatures resulted in higher plasma protein concentrations and increased Cd levels in oyster haemolymph plasma and haemocytes. Cd accumulation in haemocytes was linear over the 45 days of Cd exposure and accumulation rates were 0.10, 0.53 and 0.56 μg Cd g⁻¹ dry mass at 12, 20 and 28 °C, respectively. Percentage of blood Cd burden associated with haemocytes increased with increasing temperatures from 13–20% at 12 °C to 26–47% at 20 and 28 °C suggesting a higher role for cellular Cd transport at elevated temperatures. Cd levels in gills and hepatopancreas were positively correlated with Cd concentration in haemocytes, but accumulation rates were considerably faster, so that after 45 days of exposure Cd levels in gills and hepatopancreas were >10–20 times higher than in haemocytes. As a result of slow Cd accumulation possibly reflecting fast haemocyte turnover rates and/or exocytosis of Cd-containing granules, haemocytes in Cd-exposed oysters did not reach threshold Cd burdens required to trigger apoptosis. This suggests that haemocyte viability is not likely to contribute to immunosuppression in the environmentally relevant Cd range. In contrast, elevated temperature (28 °C) resulted in a significant increase in the percentage of apoptotic haemocytes compared to 12 or 20 °C supporting the notion that 28 °C is physiologically stressful for C. virginica. Overall, our study demonstrates strong effects of environmental temperature on haemocyte viability and other important blood parameters such as plasma protein content and metal transport capability which may mask potential Cd effects at environmentally relevant exposure levels.     

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.     

Partial biochemical characterization of α- and β-glucosidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae)

The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of α- and β-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of α- and β-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Activity of α- and β-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of α- and β-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Results showed that the optimal pH for α- and β-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for α- and β-glucosidase activity in the midgut was obtained as 45 °C. The addition of CaCl₂ (40 mM) decreased midgut β-glucosidase activity whereas α-glucosidase activity was significantly increased at this concentration. The α-glucosidase activity, in contrast to β-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive β-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.     

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves,straw sizes,and thawing rates

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (−10 °C/min) or a fast (−40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8 s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P < 0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P > 0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P < 0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P > 0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples thawed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P < 0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.     

Effect of varying salinity, temperature, ammonia and nitrous acid concentrations on nitrification of saline wastewater in fixed-bed reactors

Nitrification under changing salinities (0-9%), temperatures (6-50 °C), ammonia (0-5 g N L⁻¹) and nitrite concentrations (0-0.4 g N L⁻¹) was investigated in fixed-bed reactors. For all conditions ammonia oxidation rates (AOR) were lower than nitrite oxidation rates (NOR). AORs and NORs increased from 12.5 to 40 °C and were very low at 6 °C and almost zero at 50 °C. No recovery of nitrification was obtained after incubation at 50 °C, whereas nitrification was restorable after incubation at 6 °C. Ammonia concentrations of 5 g N L⁻¹ or nitrite concentrations up to 0.125 g N L⁻¹ decreased AOR to almost zero. AORs and NORs recovered if ammonia or nitrite was removed. At concentrations of 1 and 5 g N L⁻¹ ammonia AOR and NOR were inhibited by 50%, whereas 27 mg N/L nitrite inhibited AOR by 50%.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

Mononuclear and dinuclear mechanisms for catalysis of phosphodiester cleavage by alkaline earth metal ions in aqueous solution

In biological systems, enzymes often use metal ions, especially Mg²⁺, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg²⁺ and Ca²⁺ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (k_OH) of 4.1 × 10⁻⁴ M⁻¹s⁻¹ and 3.7 × 10⁻⁴ M⁻¹s⁻¹ in the presence of 0.30 M Mg²⁺ and Ca²⁺, respectively, compared to 8.3 × 10⁻⁷ M⁻¹s⁻¹ in the absence of divalent metal ion. Examining the dependence of k_OH on [M²⁺] at 50 °C indicates different kinetic mechanisms with Mg²⁺ utilizing a single ion mechanism and Ca²⁺ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k_OH values reveals a single Mg²⁺ catalyzes the reaction by 1800-fold whereas a single Ca²⁺ ion catalyzes the reaction by only 90-fold. The second Ca²⁺ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg²⁺ and Ca²⁺.     

Salinity induced changes in haemolymph osmolality and total metabolic rate of the mud crab Rhithropanopeus harrisii Gould, 1841 from Baltic coastal waters

The specific metabolic rate (SMR) and haemolymph osmolality (HO) of the mud crab Rhithropanopeus harrisii Gould, 1841 from Baltic brackish waters were measured at a habitat salinity of 7 psu (T = 15 °C, full air saturation) and after step-wise acclimation to a salinity range of 3-27 psu. Values of SMR at 7 psu varied between 0.40 and 3.89 J g^− 1 WW h^− 1 (n = 25, wet weight range 0.051-1.142 g) and were significantly (p < 0.05) related to the specimen's wet weight (WW) according to the power regression SMR = 0.94 WW^− 0.41 (R² = 0.68). The SMR of females did not differ significantly (p > 0.05) from those of males. When exposed to higher salinities, the SMR of R. harrisii decreased significantly (p < 0.05) and reached a minimum value at 23 psu (0.55 ± 0.05 J g^− 1 WW h^− 1, n = 6). Mean haemolymph osmolality at 7 psu amounted to 581 ± 26 mOsm kg^− 1 (n = 5) and was 2.9 times higher than that of the external medium. R. harrisii hyperosmoregulated its body fluids up to 24 psu (727 mOsm kg^− 1) at which salinity the isosmotic point was reached.     

Preservation at ultra-low temperature of in vitro cultured arbuscular mycorrhizal fungi via encapsulation–drying

At present, over 300 species of arbuscular mycorrhizal fungi (AMF) have been identified, most of which being stored in international collections. Their maintenance is mostly achieved in greenhouse via continuous culture on trap plants or in vitro in association with excised root organs. Both methods are work-intensive and for the former present the risk of unwanted contaminations. The in vitro root organ culture of AMF has become an alternative preventing contamination. Nevertheless, the risk for somaclonal variation during the sub-cultivation process cannot be excluded. A method for the long-term conservation that guarantees the stability of the biological material is thus highly demanded to preserve the microorganisms and their genetic stability. Here, 12 AMF isolates cultured in vitro in association with excised carrot roots were encapsulated in alginate beads and subsequently cryopreserved. Several protocols were tested taking into consideration culture age, alginate bead pre-drying, and rate of decrease in temperature. The viability of the AMF isolates was estimated by the percentage of potentially infective beads (%PIB) that measure the % of beads that contain at least one germinated propagule. Thermal behaviour of alginate beads was analysed by a differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. It was shown that the spore damage was directly related to ice formation during cryopreservation. The encapsulation and culture age were also determinant parameters for the successful cryopreservation. Irrespective of the AMF isolate, the optimal procedure for cryopreservation comprised five steps: (1) the encapsulation of propagules (i.e. spores and mycorrhizal root pieces) isolated from 5 m old cultures, (2) the incubation overnight in trehalose (0.5 M), (3) the drying during 48 h at 27 °C, (4) the cryopreservation in the freezer at −130 °C following a two-step decrease in temperature: a fast decrease (∼12 °C min⁻¹) from room temperature (+20 °C) to −110 °C followed by a slow decrease in temperature (∼1 °C min⁻¹) from −110 °C to −130 °C, and (5) the direct thawing in a water bath (+35 °C). The % PIB was above 70 % for all the isolates and even above 95 % for 11 out of the 12 isolates after several months of storage at ultra-low temperature. All the isolates kept their capacity to associate to an excised carrot root in vitro and to reproduce the fungal life cycle with the production of several hundreds to thousands of spores after 2 m. This method opens the door for the long-term maintenance at ultra-low temperature of AMF isolates within international repositories.     

Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics

Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately − 40 °C, with some individuals supercooling to as low as − 58 °C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to − 100 °C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of − 58 to − 70 °C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.     

Determination of ginsenoside Rg3 in human plasma and urine by high performance liquid chromatography–tandem mass spectrometry

Here we report a method capable of quantifying ginsenoside Rg3 in human plasma and urine. The method was validated over linear range of 2.5–1000.0 ng mL⁻¹ for plasma and 2.0–20.0 ng mL⁻¹ for urine using ginsenoside Rg1 as I.S. Compounds were extracted with ethyl acetate and analyzed by HPLC/MS/MS (API-4000 system equipped with ESI⁻ interface and a C₁₈ column). The inter- and intra-day precision and accuracy of QC samples were ≤8.5% relative error and were ≤14.4% relative standard deviation for plasma; were ≤5.6% and ≤13.3% for urine. The Rg3 was stable after 24 h at room temperature, 3 freeze/thaw cycles and 131 days at −30 °C. This method has been applied to pharmacokinetic study of ginsenoside Rg3 in human.     

Cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera

The cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera, was examined using measures of total body water content, supercooling point and mortality for a range of sub-zero temperature exposure regimes. The supercooling points for starved and fed nymphs were −13.1 ± 0.9 and −12.6 ± 1.6 °C, and freezing caused complete mortality. Above these temperatures, nymphs were cold tolerant to different degrees based on whether they were starved or given access to food and water for 24 h prior to exposure. The rate of cooling also had a significant effect on mortality. Very rapid cooling to −7 °C caused 84 and 87% mortality for starved and fed nymphs respectively, but this significantly decreased for starved nymphs if temperature declined by more ecologically realistic rates of 0.5 and 0.1 °C min⁻¹. These results are indicative of a rapid cold hardening response and are discussed in terms of the likely effects of cold nights and frost on first-instar nymphal survival in the field.     

Effect of freezing and dehydration on ion and cryoprotectant distribution and hemolymph volume in the goldenrod gall fly, Eurosta solidaginis

Extracellular freezing and dehydration concentrate hemolymph solutes, which can lead to cellular injury due to excessive water loss. Freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, may experience extreme cold and desiccation in winter. To determine whether larvae employ protective mechanisms against excessive cellular water loss we examined the effect of extracellular freezing and dehydration on hemolymph volume, and cryoprotectant and ion levels in the hemolymph. Dehydrated larvae or ones that had been frozen at −5 or −20 °C had a significantly smaller proportion of their body water as hemolymph (26.0-27.4%) compared to controls (30.5%). Even with this reduction in water content, hemolymph osmolality was similar or only slightly higher in frozen or dehydrated individuals than controls (908 mOsm kg⁻¹), indicating these stresses led to a reduction in hemolymph solutes. Hemolymph and intracellular content of ions remained largely unchanged between treatment groups; although levels of Mg⁺⁺ in the hemolymph were lower in larvae subjected to freezing (0.21 ± 0.01 μg mg⁻¹ dry mass) compared to controls (0.29 ± 0.01 μg mg⁻¹ dry mass), while intracellular levels of K⁺ were lower in groups exposed to low temperature (8.31 ± 0.21 μg mg⁻¹ dry mass). Whole body glycerol and sorbitol content was similar among all treatment groups, averaging 432 ± 25 mOsm kg⁻¹ and 549 ± 78 mOsm kg⁻¹ respectively. However, larvae subjected to dehydration and freezing at −20 °C had a much lower relative amount of cryoprotectants in their hemolymph (∼35%) compared to controls (54%) suggesting these solutes moved into intracellular compartments during these stresses. The correlation between reduced hemolymph volume (i.e. increased cellular water content) and intracellular movement of cryoprotectants may represent a link between tolerance of dehydration and cold in this species.     

Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus

The optimum conditions for the production of l-arabinose from debranched arabinan were determined to be pH 6.5, 75 °C, 20 g l⁻¹ debranched arabinan, 42 U ml⁻¹ endo-1,5-α-l-arabinanase, and 14 U ml⁻¹ α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75 °C, 20 g l⁻¹ sugar beet arabinan, 3 U ml⁻¹ endo-1,5-α-l-arabinanase, and 24 U ml⁻¹ α-l-arabinofuranosidase. Under the optimum conditions, 16 g l⁻¹l-arabinose was obtained from 20 g l⁻¹ debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l⁻¹ h⁻¹. This is the first reported trial for the production of l-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.     

Biochemical characterization of a 27 kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

Trichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The K_m and V_max values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323 mg ml⁻¹ and 0.315 U min⁻¹, respectively. The enzyme was strongly inhibited by Hg²⁺ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database.