Robert R. Rojas

Comparative separation of low-molecular-weight carbohydrates and polyols by high-performance liquid chromatography: radially compressed amine modified silica versus ion exchange

Abstract A radially compressed silica column (modified with tetraethylenepentamine) was compared to an ion-exchange high-performance liquid chromatographic column for the separation of carbohydrates and polyols. Both columns afford an approximate molecular weight elution sequence but in opposite order. The silica system is characterized by very long life, low cost, high resolution and high linear sample capacity. The ion-exchange column gave greater sensitivity and resolved ethanol from carbohydrates and polyols, but was relatively short-lived, more expensive, had to be run at elevated temperatures for best results, is only partially compatible with automated chromatographic systems and has a somewhat smaller useful sample range than the modified silica system.

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Abstract Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of 14 CO 2 evolution were determined after injection of [ 14 C]1-glucose, [ 14 C]6-glucose, and [ 14 C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [ 14 C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [ 14 C]glucose than did early autumn larvae.

An Evaluation of Eluent Recycling and Column Life for HPLC Analysis of Carbohydrates

Abstract The length of service of amine modified silica columns for the automated analysis of carbohydrates in food and beverages typically exceeds 500 sample injections. Excessive accumulation of carbohydrate in the recirculated eluent results in a gradual decrease in peak height. Detector response is independent of injection volume over the range of 1–50 μl. These results suggest critical parameters for use in automated carbohydrate analysis systems.

Juvenile hormone:modulation of cryoprotectant synthesis in Eurosta solidaginis by a component of the endocrine system

Abstract The role of juvenile hormone as a possible modulator of insect cold-hardiness has been investigated in the gall fly larvae of Eurosta solidaginis. The 3rd-instar larva is freezing tolerant and survives the rigours of winter by producing carbohydrate cryoprotective agents (i.e. glycerol and sorbitol). Since cold-hardening frequently occurs in conjuntion with developmental processes under endocrine control, a study of these possible interrelationships was made. Third-instar larvae collected during autumn were allatectomized by ligation of the head. Experimental larvae received topical applications of synthetic juvenile hormone, methoprene, a potent juvenile hormone analogue, or precocene II, an anti-juvenoid. Following treatment, all larvae were then subjected to one of two temperature acclimation protocols at 5 or 15°C for up to 5 days. Ligation resulted in a 20% decrease in glycerol and up to a 40% increase in sorbitol levels vs controls following 5°C exposure. At 15°C, deprivation yielded a 13% decrease in glycerol and up to a 47% increase in sorbitol. Ligation resulted in decreased glycerol levels independent of temperature especially in early autumn. Cryoprotectant synthesis/accumulation was responsive to juvenile hormone replacement with peak sensitivity observed in October. Ligated larvae were generally insensitive to juvenile hormone replacement in September and December. These responses were temperature dependent; polyol accumulation was reduced by 50% at +5°C and enhanced by 22% at +15°C (2 and 8 μg glycerol/mg larvae, respectively). This study provides the first evidence suggesting an association between juvenile hormone and regulation of cryoprotectant accumulation in insects.

Juvenile hormone modulation of insect cold hardening: Ice-nucleating activity

Abstract Data are presented offering the first evidence for probable endocrine involvement in the control of cold hardening in Eurosta solidaginis . Juvenile hormone (JH) deprivation experiments in which the corpora allata were removed by head ligation resulted in a loss of supercooling (SC) capacity in larvae collected over 2 years. This loss of supercooling capacity is indicative of synthesis of organismal pools of ice-nucleating agents (INA). Larval sensitivity to JH removal (ligation) on SC is seasonally dependent. For example, in 1983, larvae were most sensitive in October, secondarily so in September, and relatively insensitive in December regardless of acclimation temperature. While in 1984, larvae acclimated to +5 °C were most sensitive in November, secondarily so in October, and relatively insensitive in December, and larvae acclimated to +15 °C were most sensitive in October and December and least so in November. Supercooling point elevations as great as 7 °C over controls were observed with maximal responses occurring within 1 day following ligation. In 1983, juvenile hormone replacement following ligation generally resulted in an expansion of supercooling capacity when compared to controls. As with ligation, the sensitivity to JH replacement on SC was seasonally dependent: September and October larvae being the most sensitive with December larvae being insensitive. Larvae collected in 1984 were given a greater dose of JH than those in the previous year and showed no significant change in SC over ligated-acetone controls. Hormone analog potentiation experiments in which unligated larvae collected in 1983 and acclimated to +5 °C were given methoprene resulted in depression of supercooling points for September and October larvae. JH titres appear to play an important role in the regulation of SC capacity in E. solidaginis larvae.

Invertebrate germplasm cryopreservation: potential, problems, and prospects

Although the demand for long-term storage of invertebrate germplasm has never been more evident than it is now, specific knowledge of such storage is sparse. To date, germplasm has been stored mainly by those concerned with plant and domestic animal breeding or with type culture collections for protozoans. In the research laboratory, the continuous rearing of numerous strains of insects and other invertebrates is costly and labor intensive. Catastrophic loss and genetic drift are also important considerations when an organism such as an insect is to be used as a biological control agent and is reared under factorylike conditions. Cryopreservation, or cold storage, provides a feasible alternative to continuous rearing while circumventing the aforementioned difficulties. However, the unique reproductive and environmental adaptations characteristic of invertebrates often preclude the use of much of the conventional cryobiological methodology. This report focuses on research needs for invertebrate cryopreservation and suggests some possible routes for gaining success in the long-term storage of invertebrate germplasm.