Many insects in temperate regions overwinter in diapause, during which they are cold hardy. In these insects, one of the metabolic adaptations to the unfavorable environmental conditions is the synthesis of cryoprotectants/anhydroprotectants. The aim of this study was to investigate the connection between the antioxidative system and synthesis of cryoprotectants (mainly glycerol) in diapausing larvae of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). At two periods during diapause (November and February), in three groups of insects (kept under field conditions; -12°C for two weeks; 8°C for two weeks), the activity of key enzymes of the antioxidative system and oxidative part of the hexose monophosphate shunt were measured: superoxide dismutase, catalase, non selenium glutathione peroxidase, glutathione S-transferase, glutathione reductase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, as well that of the antioxidative components: total glutathione and ascorbate, and dehydroascorbate reductase. There was a higher activity of antioxidative enzymes at the beginning of the diapause period (November) compared to late diapause (February), while glutathione and ascorbate were higher in February. Similarly, there was a lower activity of the hexose monophosphate shunt enzymes in February. Exposure of larvae to -12°C resulted in an elevation of hexose monophosphate shunt enzyme activity, especially in November. This was accompanied by a significant increase in glycerol content in February. Changes in ascorbate levels and dehydroascorbate reductase activity in both experimental groups (-12°C and 8°C) suggest a connection between the antioxidative system, metabolism during diapause and cold hardiness. Our results support the notion that antioxidative defense in larvae of Ostrinia nubilalis is closely connected with metabolic changes characteristic of diapause, mechanisms of cold hardiness involved in diapause and the maintenance of a stable redox state.
Conventional methods to preserve adult nematodes for taxonomic purposes involve the use of fixative or clearing solutions (alcohol, formaldehyde, AFA and lactophenol), which cause morphological alterations and are toxic. The aim of this study is to propose an alternative method based on glycerol-cryopreservation of nematodes for their subsequent identification. Adults of trichostrongylid nematodes from the abomasum of roe deer (Capreolus capreolus Linnaeus) were glycerol-cryopreserved and compared with those fixed in formaldehyde, fresh and frozen without cryoprotectans. Morphology, transparency and elasticity of the anterior and posterior portion of male nematodes were compared, especially the caudal cuticular bursa and genital accessories. The method presented is quick and easy to use, and the quality of nematode specimens is better than that of nematodes fixed by previously used fixatives. Moreover, glycerol cryopreserved nematodes can be stored for a long time at -20°C in perfect condition and they could be suitable for further analyses, such as histological or ultrastructural examinations.
Strong tolerance of freezing is an important strategy for insects living in extremely cold regions. They produce highly effective cryoprotectant systems consisting of ice-nucleating proteins and polyols, which enables tolerable freezing of the body fluid. Therefore, the measurement of the concentrations of polyols and the activity of ice nucleators in the haemolymph is an essential tool for describing tolerance to ice formation in insects occurring in particularly cold places. This study evaluates three parameters: insect body supercooling point (SCP), haemolymph glycerol content and the profile of haemolymph ice nucleating activity that characterize the strategies of cold adaptation and cold hardiness in two previously unstudied beetles, Chrysolina graminis graminis L. and Galerucella nymphaea L., inhabiting Yakutia (Russian Far East, latitude 62°N). The high SCP values, ice nucleating activity and survival of the chrysomelids after freezing indicate that both species are tolerant of freezing. According to the profiles of ice-nucleating activity, the haemolymph from C. graminis graminis is characterized by a higher nucleating potential than that from G. nymphaea. The glycerol level is also higher in C. graminis graminis. The results indicate that both species develop tolerance to low temperatures, but the cold hardiness potential of C. graminis graminis is greater than that of G. nymphaea. This was revealed by the survival test, in which beetles were frozen to a temperature of -22°C for 30 min; 86% of C. graminis graminis and 72% of G. nymphaea survived the test. Thus, the freeze-tolerance of these beetles seems to be based on the production of an integrated cryoprotectant system, the quality of which apparently influences the range of their cold resistance., Natalia G. Li., and Obsahuje bibliografii
Haemolymph levels of amino acids, sugars and glycerol were investigated in the tenebrionid Physadesmia globosa during dehydration and rehydration. The absolute amount of amino acid decreases during dehydration and increases during rehydration, indicating active regulation of this solute (the osmolal contribution of amino acids is large - approx. 25%). Changes in the amino acid content of the haemolymph during dehydration are not the result of interchange with soluble protein; the possibility exists during rehydration (between 1 h and 48 h). Trehalose and glucose are the only sugars found in appreciable quantity in the haemolymph of this species. Their osmolal contributions (total sugar: 2.6%), and contributions to osmoregulation, are not great. Glycerol is a minor osmolar effector in the haemolymph of Physadesmia, and changes in its levels do not contribute importantly to the regulation of haemolymph osmotic pressure.
A traditional method is reported for purification of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) from leaves of Amaranthus hypochondriacus L. with a high yield of 50 %, 135-fold purification, and specific activity of 900 mmol kg-1(protein) s-1. PEPC was purified from light-adapted leaves of A. hypochondriacus, involving 40-60 % ammonium sulphate fractionation, followed by chromatography on columns of DEAE-Sepharose, hydroxylapatite (HAP), and Seralose 6-B. The enzyme appeared as a single band on 10 % SDS-PAGE, with a molecular mass of about 100 kDa. Kinetic studies with purified enzyme confirmed the PEPC to be the light-form of the enzyme. Glycerol generally increased the stability of PEPC. The stability and storage of the purified enzyme was studied at temperatures of 4 °C, -20 °C, and liquid nitrogen. PEPC maintained its activity for up to 3 months upon storage with 50 % (v/v) glycerol in liquid nitrogen. and J. Gayathri, K. Parvathi, A. S. Raghavendra.
Dehydration of Onymacris unguicularis (Haag) for 10 days at 27°C resulted in a weight loss of 14.9%, and a 37% decrease in haemolymph volume. Although there was an overall decrease in the lipid content during this period, metabolic water production was insufficient to maintain total body water (TBW). Rehydration resulted in increases in body weight (6.2% of initial weight), TBW (to normality), and haemolymph volume (sub-normal at the end of rehydration). Despite an increase of 44.0 mg in the wet weight of O. unguicularis after drinking for 1h, there was little change in the water content at this time, although the total lipid content increased significantly. Increases in haemolymph osmolality, sodium, potassium, chloride, amino acid and total sugar concentrations during dehydration were subject to osmoregulatory control. No evidence of an active amino acid-soluble protein interchange was noted during dehydration or rehydration. Haemolymph trehalose levels were significantly increased at the end of rehydration (relative to immediate pre-rehydration values), indicating de novo sugar synthesis at this time. Osmotic and ionic regulation was evident during rehydration, but control of OP during haemolymph-dilution is poor and accomplished largely by the addition to the haemolymph of free amino acids and solute(s) not measured in this study. There was little mobilization of sodium and chloride ions from storage sites at this time. The lesser osmoregulatory ability of Onymacris unguicularis and perhaps earlier susceptibility to osmotic stress, a significantly high normal blood glycerol level (relative to other diurnal adesmiine tenebrionids), and a water storage mechanism associated with synthesis of fat, probably all contribute to the development of fog-basking behaviour in this species. Water gain in O. unguicularis during periods of relative drought is probably largely accomplished by a greater food consumption.