Survival of the first stage larva of the metastrongyloid nematode Elaphostrongylus rangiferi under various conditions of temperature and humidity

First-stage larvae of E. rangiferi kept in water at 50°C died within 80 minutes, while at 6° the last larvae died between day 180 and 210. The time it took to reach 1_x= 0.5 (half of the larvae dead) at various temperatures between 6° and 50° was well described by the exponential function y = 614.6e^−0.15x, giving a value of 615 days to reach 1_x= 0.5 at 0°C. There was no clear decrease in the survival of larvae frozen at −20° in faeces and in water, and at −80° in faeces after 360 days. When subjected to repealed freezing and thawing, all larvae died within 77 days. When kept in air at RH = 20% and 22°C, all larvae died within 11 days, while when frozen (−20°C) in air at RH approx. 0%, 1_x stayed at approx. 0.5 from day 5 to day 16.     

Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae,Eurosta solidaginis (Diptera,Tephritidae)

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall (Solidago sp.). In late September, when the water content of the galls was high (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at -4 and -6 degrees C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures > or = -6 degrees C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as -4 degrees C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization (T(c)) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.     

Low temperature storage of larvae and synchronization of adult emergence in the predatory midge Aphidoletes aphidimyza.

Diapause larvae of Aphidoletes aphidimyza were stored at a temperature of 3 degrees C under continuous darkness for up to 7 months with survival rates above 50%; after storage for 1 year the survival rate dropped to 12%. Diapause was terminated in the majority of individuals within 120 days of chilling under storage conditions. Brief exposure (10-60 s) to the vapor of n-hexane appeared to be a useful alternative to chilling for the termination of diapause. The larvae with terminated diapause required, on average, an additional 31 days at 22 degrees C and long-day conditions in order to reach the adult stage. The 10-90% adult emergence spanned a period of 21.1 days. When the larvae with terminated diapause were exposed to 30 degrees C for 1 week after the end of low temperature storage, the survival rate was not affected, the average "time-to-adult" shortened moderately to 28 days, and the synchrony of adult emergence improved considerably to 10 days. Low temperature storage of nondiapause larvae resulted in a decrease in survival from 98 to 31% during the first 60 days of storage. Nondiapause larvae did not enter diapause during low temperature storage and, as a consequence, the adults emerged relatively rapidly (after 14-15 days) and synchronously (within 2-3 days) after the end of storage. Directions for future research, which might bring further improvement in low temperature storability and synchrony of adult emergence in A. aphidimyza, are proposed.     

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at −80°C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60–70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.     

Effects of oxygen deficiency on survival and glycogen content of Chironomus anthracinus (Diptera,Chironomidae) under laboratory and field conditions

Growth and glycogen content of Chironomus anthracinus in Lake Esrom, Denmark was examined during summer stratification in 1992 and 1993. Simultaneously, effects of oxygen deficiency on glycogen utilization and survival were experimentally studied. The population consisted of almost fullgrown 4th instar larvae in 1992 and 2nd and 3rd instar larvae in 1993. Growth rate and glycogen content changed as hypolimnetic oxygen deficiency increased. During a 1st phase of stratification dry weight and glycogen content increased (2nd and 3rd instars) or was almost constant (4th instar) but decreased significantly during the following 2nd phase. This change from growth to degrowth and utilization of endogenous glycogen reserves correlated with a change in the thickness of the microxic layer (<0.2 mg O₂ 1^–1) above the sediment surface. The layer increased from 2–3 m in phase 1 to 4–5 m in phase 2, and we suggest that this deteriorated the oxygen conditions and resulted in a change in larval energy metabolism from fully aerobic during the 1st phase to partly anaerobic in the 2nd phase. During the 2nd phase larval metabolism was estimated at less than 20% of normoxic rate. Experimental exposure of the larvae to anoxia indicated highly different survival of young larvae (2nd and 3rd instars) and older larvae (large 4th instars). The morality of young larvae was 50% after three days in anoxia at 10 °C, whereas only 25% of the older larvae had died after 3–4 weeks under similar conditions. Extending the treatment, however, resulted in increased death rate of the 4th instar larvae with only 10% surviving after seven weeks. The anaerobic metabolism of 4th instar larvae as estimated from glycogen degradation at 10 °C was 5% of normoxia in the interval from 0–5 days but 1.5% in the interval from 20–25 days. It is concluded that survival of C. anthracinus in anoxia is very limited, but traces of oxygen in the environment allowing for faint aerobic metabolism prolong the survival time of the larvae from a few days (2nd and 3rd instars) or a few weeks (4th instar) to probably 3–4 months.     

Water balance in the sugarbeet root maggot Tetanops myopaeformis,during long‐term low‐temperature storage and after freezing

The sugarbeet root maggot Tetanops myopaeformis Röder (Diptera: Ulidiidae) can be stored in moist sand at 4–6 °C for up to 5 years and is freeze‐tolerant. The majority of stored larvae survive in a state of post‐diapause quiescence and the remainder are in a multi‐year diapause. The present study aims to determine larval water content and water loss rates in diapausing and low‐temperature stored larvae. Body water content ranges from 57% to 70.1%. Two distinct groupings of larvae are revealed based on dry weights. The first group consists of the diapausing larvae and larvae stored for 1 year. This group has significantly higher dry weights than the second grouping, which consists of the larvae stored for 2 and 3 years. There are no significant differences within each group. Larval water losses follow a first‐order kinetic relationship with time. Larvae stored for 2 years lose water at a significantly higher rate than diapausing larvae. Larvae exhibit no active water uptake at storage temperatures. A freezing event does not induce a significant decrease in wet weights, nor does it increase larval water loss rates. These results indicate that metabolic water and the microclimate during storage are key factors enabling the long‐term survival of T. myopaeformis larvae during low‐temperature storage, and may provide insights for maintaining other insect species under similar conditions.     

Effects of summer frost exposures on the cold tolerance strategy of a sub-Antarctic beetle

The sub-Antarctic beetle Hydromedion sparsutum (Coleoptera, Perimylopidae) is common locally on the island of South Georgia where sub-zero temperatures can be experienced in any month of the year. Larvae were known to be weakly freeze tolerant in summer with a mean supercooling point (SCP) around -4 degrees C and a lower lethal temperature of -10 degrees C (15min exposure). This study investigated the effects of successive freezing exposures on the SCP and subsequent survival of summer acclimatised larvae. The mean SCP of field fresh larvae was -4.2+/-0.2 degrees C with a range from -1.0 to -6.1 degrees C. When larvae were cooled to -6.5 degrees C on 10 occasions at intervals of 30min and one and four days, survival was 44, 70 and 68%, respectively. The 'end of experiment' SCP of larvae surviving 10 exposures at -6.5 degrees C showed distinct changes and patterns from the original field population depending on the interval between exposure. In the 30min interval group, most larvae froze between -6 and -8 degrees C, a depression of up to 6 degrees C from the original sample; all larvae were dead when cooling was continued below the SCP to -12 degrees C. In the one and four day interval groups, most larvae froze above -6 degrees C, showing no change as a result of the 10 exposures at -6.5 degrees C. As with the 30min interval group, some larvae froze below -6 degrees C, but with a wider range, and again, all were dead when cooled to -12 degrees C. However, in the one and four day interval groups, some larvae remained unfrozen when cooled to -12 degrees C, a depression of their individual SCP of at least 6 degrees C, and were alive 24h after cooling. In a further experiment, larvae were cooled to their individual SCP temperature at daily intervals on 10 occasions to ensure that every larva froze every day. Most larvae which showed a depression of their SCP of 2-4 degrees C from their day one value became moribund or died after six or seven freezing events. Survival was highest in larvae with SCPs of -2 to -3 degrees C on day one and which froze at this level on all 10 occasions. The results indicate that in larvae in which the SCP is lowered following sub-zero exposure, the depression of the SCP is greatest in individuals that do not actually freeze. Further, the data suggest that after successive frost exposures in early winter the larval population may become segregated into two sub-populations with different overwintering strategies. One group consists of larvae that freeze consistently in the temperature range from -1 to -3 degrees C and can survive multiple freeze-thaw cycles. A second group with lower initial SCPs (around -6 degrees C), or which fall to this level or lower (down to -12 degrees C) after freezing on one or more occasions, are less likely to freeze through extended supercooling, but more likely to die if freezing occurs.     

The development and overwintering survival of free-living larvae of Haemonchus contortus in Sweden

Five complimentary studies were undertaken with the overall aim to examine the ability of free-living stages of Haemonchus contortus to over-winter and tolerate cold stress. Two studies deal with the development and long-term survival of eggs and infective larvae of two geographically different isolates (Kenya and Sweden). Eggs and larvae were monitored in climatic chambers at temperatures that fluctuated daily between -1 degrees C and 15 degrees C, or at constant temperatures of 5 degrees C and 15 degrees C. The development from egg to larvae was dependent on temperatures over 5 degrees C. The long time survival was favoured at lower temperatures. Furthermore, the overwintering capacity of the free-living stages of these isolates was estimated under Swedish field conditions. Two groups of lambs were experimentally infected with different isolates, and kept separated on previously ungrazed plots. In early May the following year, two parasite-naive tracer lambs were turned out on each of the plots to estimate the pick up of overwintered larvae. This experiment was replicated in central and southern Sweden. In addition, two experiments were performed in 2003 on pasture previously grazed by naturally infected sheep. One trial was on a pasture in southern Sweden grazed by a commercial flock, where extreme numbers of H. contortus were found towards the end of the grazing season 2002. The other study was on a pasture plot in central Sweden grazed by a hobby flock in 2002, where three of six lambs died due to haemonchiasis. Overwintered H. contortus was recorded on three of four experimental sites. Worm burdens were in all instances extremely low. No differences in development and survival were found between the isolates. Consequently, overwintering on pasture is of no practical significance in the transmission of H. contortus between grazing-seasons in Sweden.     

Frozen Storage of Tritrichomonas foetus for 5.6 Years*

SYNOPSIS. Tritrichomonas foetus was cultivated in Diamond's medium without phosphates or agar; 10% glycerol was added to the medium and the protozoa were frozen slowly and stored at ?28 or ?95 C. About half of the trichomonads died within the first day at either temperature. Thereafter, they continued to die off slowly during storage at ?28, and none remained alive at or after 1024 days. New cultures could not be initiated with them after 64 days. At ?95, almost as many trichomonads were alive after 256 days as after 1 day. Thereafter, however, they decreased in numbers, but 11% were still alive after 2048 days (5.6 years). The survivors appeared in good condition, and new cultures were readily initiated with them.     

Superparasitism and host discrimination byEphedrus cerasicola [Hym.: Aphidiidae], an aphidiid parasitoid ofMyzus persicae [Hom.: Aphididae]

Myzus persicae (Sulzer) at different densities (1 to 120 aphids) on a paprika plant in a 1.8 dm³ cage were exposed at 21°C to single females of the parasitoidEphedrus cerasicola Stary. Three different exposure periods were used: 1 hr, 6 hrs, 24 hrs. The aphids were dissected 6 days after parasitization. Superparasitism was measured as number of hosts superparasitized and number of parasitoid larvae per aphid. It attained a maximum around host density=5 and decreased with increasing host density and decreasing exposure time. Almost no superparasitism occurred during the 1st hour. A non-random larval distribution indicated thatE. cerasicola discriminates between unparasitized and parasitized aphids, but probably not between aphids with different numbers of parasitoid eggs. Larvae in superparasitized aphids developed slower than single larvae. The supernumerary larvae died during the 1st instar, probably killed by chemical means since no physical attacks between larvae have been observed.     

Resistance to ivermectin by Haemonchus contortus in goats and calves.

Efficacy of ivermectin on susceptible or resistant populations of the parasitic nematode Haemonchus contortus was determined in cattle and goats held in a barn. Goats were each infected with 3000 infective, ivermectin-susceptible or -resistant H. contortus larvae on day 0 and reinfected with 2000 infective larvae on day 24. Goats were treated orally with 600 micrograms kg-1 ivermectin on day 31. No significant differences were detected in blood packed cell volume (PCV) or total protein (TP), prepatent period, or epg among the four groups of goats that were each infected with one of four parasite strains (one susceptible, three resistant). There were no differences among the four parasite strains in the numbers of infective larvae that developed to the third larval stage from fecal cultures or in the viability of cultured infective larvae when held in the laboratory at 27 +/- 1 degrees C for 14 weeks. After treatment with ivermectin, there were significant differences among the parasite strains in PCV, TP, and epg. Total worm counts were reduced by 94 to 97% with three times the recommended dose. Immature and adult Skrjabinema ovis were also present in two treated goats. In a second test, one goat infected once with 10,000 infective larvae of a resistant strain of H. contortus and then treated with nine doses of ivermectin, increasing from 500 to 2000 micrograms kg-1 over a period of 133 days, had 35 adult worms at necropsy. In a third test, three calves were readily infected with an ivermectin-resistant strain of H. contortus from goats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation of infective larvae of Dipetalonema viteae.

infective larvae of Dipetalonema viteae produced infections in Mongolian jirds (Meriones unguiculatus) after storage of infected ticks (Ornithodoros tartakovskyi) in the presence of dimethyl sulfoxide (DMSO, 5%) for 7 or 595 days in liquid nitrogen (-196 C). Infectivity of these larvae was only partially impaired. Microfilaremias of test jirds were generally lower than those of control jirds given nonfrozen larvae; however, the majority of test jirds developed microfilarial counts suitable for use in infecting ticks. In contradistinction, larvae frozen free of the tick failed to retain infectivity. Apparently the tick, in conjunction with DMSO, protects the larvae during freezing and thawing.     

The effect of temperature on energy distribution during the last-larval stadium of the female house cricket, Acheta domesticus

The distribution of energy during the last stadium of the house cricket at two temperatures was the main theme of this study. Food consumption, growth, and oxygen consumption were greater in the first half of the stadium at both 25 and 35°C. An RQ > 1 indicated the conversion of carbohydrates to lipids during the first half of the instar at both temperatures. The duration of the stadium increased from 6 days at 35°C to 14 days at 25°C. The same maximal weight, protein content and lipid content were attained at both 25 and 35°C. A weight loss (mostly in stored lipids) after the midstadium peak weight was greater at the lower temperature. The absorption efficiency and the production of metabolic wastes were not affected by temperature, but the metabolic efficiency was much higher at 35 than at 25°C during the first half as well as the latter half of the stadium. Although during the first half of the stadium more energy was ingested, absorbed, and made available for growth at 25 than at 35°C, only slightly more growth occurred at 25°C. During the last half of the stadium less energy was ingested at 25 than at 35°C, and much more growth occurred at 35°C because of the even greater heat loss at 25 than at 35°C. Therefore at a lower temperature cricket larvae eat slightly more and reach the same maximal weight as at a higher temperature, but they end up smaller because they waste more energy during the extended duration of the stadium at the lower temperature.     

Long-term storage of blood samples at freezing temperatures in the presence of a cryoprotectant for hemiglobin assay

Changes in Hi levels in experimentally prepared blood samples during storage at various temperatures were studied. When whole blood in which Hi levels were elevated by sodium nitrite was stored unfrozen, rapid reduction of Hi was observed within 24 hr even at 0 degrees C. When whole blood or a diluted hemolysate was stored frozen for a week or longer, considerable formation of Hi by autoxidation was observed, the formation at -20 degrees C being much more significant than that at -30 degrees C. On the other hand, addition of an equal volume of the cryoprotectant solution of Rowe et al. to blood almost completely inhibited this Hi formation during freezing storage until at least 30 days. Thus, a new method for long-term storage of blood samples for Hi assay was devised.     

Denaturation of Soybean Protein by Freezing

When a solution of soybean acid-precipitated or 11S protein was frozen and stored at ?1 to ?5°C, the protein became partially insoluble after thawing. Ultracentrifugation and disc-electrophoresis of freeze-stored 11S protein solution after removing insoluble components revealed that new components which may be aggregates or associates of the 11S component were formed. When concentrated and stored at 5°C, disc-electrophoresis of 11S component showed that associates were formed. Mercaptoethanol could dissolve the insoluble protein and also convert the associates to the original 11S component. NEM–11S was not insolubilized by frozen storage at ?5°C or storage at 5°C after being concentrated. From these facts it can be concluded that denaturation of soybean protein by freezing may be caused by intermolecular reactions through S-S bonds as a result of concentration by freezing. This may suggest a mechanism of the formation of sponge-like texture in kori-tofu which is made by frozen storage of soybean curd for 15 to 20 days at ?1 to ?3°C.     

Anhydrobiosis increases survival of trichostrongyle nematodes

This study demonstrates that infective-stage larvae of 2 trichostrongyle ruminant gastrointestinal nematodes, Haemonchus contortus and Trichostrongylus colubriformis, can enter into anhydrobiotic states when completely desiccated. Larvae of control trichostrongyle species, Heligmosomoides polygyrus and Nippostrongylus brasiliensis, that infect mice were unable to survive desiccation or to enter into anhydrobiosis. Ruminant larvae were able to survive up to 7 desiccation/rehydration cycles, and, during anhydrobiosis, metabolic activity was decreased and survival of the larvae was prolonged both in the laboratory and in the field. Relative humidity had no effect on ruminant larval survival after anhydrobiosis compared with controls. Temperature had a significant effect, 85.8 +/- 2.3% of larvae in anhydrobiosis could survive low temperatures (0 C) that killed all control larvae. Metabolic activity, measured by changes in lipid content and CO2 respiration, was significantly lower in larvae that entered anhydrobiosis compared with controls (P < 0.05). In field experiments using open-meshed chambers under ambient environmental conditions, larvae in anhydrobiosis had significantly higher survival rates in the field compared with controls (P < 0.05) during summer and winter trials. These data suggest that anhydrobiosis in ruminant larvae promotes survival at freezing temperatures, decreases metabolic activity, and prolongs survival under natural field conditions.     

A preliminary study of the effect of microclimate on third-stage larvae of Haemonchus contortus and Haemonchus placei on irrigated pasture.

Assessments were made on the influence of several microclimatic variables on the availability of third-stage larvae of Haemonchus contortus and Haemonchus placei on four strata of irrigated Kikuyu pasture. Three replicates of these pasture samples were collected on 18 sample days over 12 months and the log10 mean counts of the larvae recovered were analysed by a step-wise regression model. Predictors for the log counts of the four strata for the two nematode species included relative humidity, illumination, air temperature and windspeed. The effect of air temperature on larvae of both Haemonchus species was similar; as air temperature increased, the number of larvae on pasture increased. The inverse was true for windspeed; as windspeed increased larval counts decreased. For H. contortus, relative humidity increased as the number of larvae increased on all strata except upper herbage. The R2 values ranged from 0.11 to 0.21 for H. contortus and from 0.04 to 0.12 for H. placei. Under the conditions of this study, only 21% of the effect on H. contortus and 12% on H. placei third-stage larvae on pasture can be explained by microclimatic conditions.     

Cryopreservation of semen in the dog: use of ultra-freezers of -152 degrees C as a viable alternative to liquid nitrogen

This experimental work was carried out to validate the use of a -152 degrees C ultra-low temperature freezer to freeze and store canine semen. The semen of three dogs was pooled and processed to obtain a final dilution with a concentration of 100 x 10(6) spermatozoa/mL, glycerol at 5% and Equex at 0.5%. Then, four freezing protocols were tested to evaluate the cryosurvival of sperm at 1, 7, 30, 60 and 120 days after freezing: (I) semen was frozen and stored in liquid nitrogen; (II) semen was frozen in liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (III) semen was frozen in the vapour of liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (IV) semen was frozen and stored in the ultra-low freezer at -152 degrees C. Data were statistically analyzed by repeated measures analysis of variance to determine the effect of the freezing protocol and time on the sperm characteristics assessed. The percentages of sperm motility and of dead/live spermatozoa were similar throughout the experimental period, with no significant differences (P < 0.05) to be observed between four different freezing techniques tested. At 120 days after freezing, the percentage of abnormal cells and the percentage of sperm cells with abnormal acrosome were not significantly different between the freezing techniques. Although the number of dogs used was slightly low, in vitro results of this preliminary study showed that the use of ultra-freezers at -152 degrees C to freeze and store canine semen could be a viable alternative to liquid nitrogen.     

A novel anthelmintic model utilizing jirds, Meriones unguiculatus, infected with Haemonchus contortus

Currently, no in vivo laboratory model is available for evaluating anthelmintics against the important ruminant helminth Haemonchus contortus. This report outlines a novel anthelmintic assay utilizing immunosuppressed (0.02% hydrocortisone in feed) jirds, Meriones unguiculatus, infected with H. contortus. Immunosuppressed jirds were inoculated with approximately 1,000 exsheathed infective larvae of H. contortus, treated per os on day 10 postinoculation (PI), and necropsied on day 13 PI. Each stomach was removed, opened longitudinally, incubated in distilled water at 37 C for 5 hr, fixed in formaldehyde solution, and stored for subsequent examination. Stomach contents were examined using a stereomicroscope (15-45x). A variety of standard anthelmintics has been evaluated in the model; modern broad-spectrum ruminant anthelmintics (benzimidazoles, febantel, ivermectin, levamisole hydrochloride, and milbemycin D) are active uniformly and in most cases at doses (mg/kg) comparable to those required for efficacy against H. contortus in ruminants. This model provides an important new tool to assess preliminarily the activity of experimental drugs against H. contortus in vivo prior to studies in ruminants and also may provide a useful tool for studying host-parasite interactions for H. contortus.     

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.