Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado

Geographic distribution limits of ectothermal animals appear to be correlated with thermal tolerance thresholds previously identified from the onset of anaerobic metabolism. Transition to these critical temperatures was investigated in the spider crab (Maja squinado) with the goal of identifying the physiological processes limiting thermal tolerance. Heart and ventilation rates as well as PO(2) in the hemolymph were recorded on-line during progressive temperature change between 12 and 0 degrees C (1 degrees C/h) and between 12 and 40 degrees C (2 degrees C/h). Lactate and succinate were measured in tissues and hemolymph after intermediate or final temperatures were reached. High levels of hemolymph oxygenation suggest that an optimum range of aerobic performance exists between 8 and 17 degrees C. Thermal limitation may already set in at the transition from optimum to pejus (pejus = turning worse, progressively deleterious) range, characterized by the onset of a decrease in arterial PO(2) due to reduced ventilatory and cardiac performance. Hemolymph PO(2) values fell progressively toward both low and high temperature extremes until critical temperatures were reached at approximately 1 and 30 degrees C, as indicated by low PO(2) and the onset of anaerobic energy production by mitochondria. In conclusion, the limited capacity of ventilation and circulation at extreme temperatures causes insufficient O(2) supply, thereby limiting aerobic scope and, finally, thermal tolerance.     

Fetal and maternal body temperatures measured by radiotelemetry in near-term sheep during thermal stress.

Using implanted radiotelemeters, we have measured amniotic temperature and fetal lamb and pregnant ewe body temperatures continuously over the last 34 days of gestation and during conditions of thermal stress. Body temperature of the fetus was approximately 0.6 degrees C higher than that of the mother, and the fetomaternal temperature difference remained constant over the last 25 days of gestation, until the immediate prepartum period, when it rose. During exposure to mild heat stress (35 degrees C dry-bulb temperature, 24 degrees C wet-bulb temperature), ewe and fetal body temperatures rose, but fetal temperature rose at a slower rate. Thus the fetomaternal temperature gradient fell significantly in the initial exposure period. In an environment of 4 degrees C, body temperature of the pregnant ewes fell, but the fetomaternal gradient did not change significantly. During maternal fever, heat loss from the fetus was compromised; body temperature of the fetus rose more than that of the mother, and the fetomaternal temperature gradient rose significantly. We suggest that mild heat or cold exposure in pregnant animals constitutes little risk of fetal thermal stress. During maternal fever, however, the fetus may be at risk of thermal injury.     

The effects of hyperthermia and hyperthermia plus microwaves on rat brain energy metabolism

The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effect of temperature on adenosine triphosphate concentration [ATP] and creatine phosphate concentration [CP] was determined in the brains of rats that were maintained at 35.6, 37.0, 39.0, and 41.0 degrees C. At 37, 39, and 41 degrees C brain [ATP] and [CP] were down 6.0, 10.8, and 29.2%, and 19.6, 28.7, and 44%, respectively, from the 35.6 degrees C control concentrations. Exposure of the brain to 591-MHz radiation at 13.8 mW/cm2 for 0.5, 1.0, 3.0, and 5.0 min caused further decreases (below those observed for 30 degrees C hyperthermia only) of 16.0, 29.8, 22.5, and 12.3% in brain [ATP], and of 15.6, 25.1, 21.4, and 25.9% in brain [CP] after 0.5, 1.0, 3.0, and 5.0 min, respectively. Recording of brain reduced nicotinamide adenine dinucleotide (NADH) fluorescence before, during, and after microwave exposure showed an increase in NADH fluorescence during microwave exposure that returned to preexposure levels within 1 min postexposure. Continuous recording of brain temperatures during microwave exposures showed that brain temperature varied between -0.1 and +0.05 degrees C. Since the microwave exposures did not induce tissue hyperthermia, it is concluded that direct microwave interaction at the subcellular level is responsible for the observed decrease in [ATP] and [CP].     

Temperature-dependent development of cardiac activity in unrestrained larvae of the minnow Phoxinus phoxinus

The minnow (Phoxinus phoxinus) was raised up to the stage of swim bladder inflation at temperatures between 10 degrees C and 25 degrees C, and the time of development significantly decreased at higher temperatures. Accordingly, initiation of cardiac activity was observed at day 2 in 25 degrees C animals and at day 4 in 12.5 degrees C animals. Only a minor increase in body mass was observed during the incubation period, and, at the end of the incubation period, animals raised at 25 degrees C did not have a significantly lower body mass compared with animals raised at 15 degrees C. Metabolic activity, determined as the rate of oxygen consumption of a larva, increased from 3.3 to 19.5 nmol/h during development at 15 degrees C and from 5.6 to 47.6 nmol/h during development at 25 degrees C. Heart rate showed a clear correlation to developmental stage as well as to developmental temperature, but at the onset of cardiac activity, diastolic ventricular volume and also stroke volume were higher at the lower temperatures. Furthermore, stroke volume increased with development, except for the group incubated at 12.5 degrees C, in which stroke volume decreased with development. Initial cardiac output showed no correlation to incubation temperature. Although metabolic activity increased severalfold during development from egg to the stage of swim bladder inflation at 15 degrees C and at 25 degrees C, weight-specific cardiac output increased only by approximately 40% with proceeding development. At 12.5 degrees C, cardiac output remained almost constant until opening of the swim bladder. The data support the notion that oxygen transport is not the major function of the circulatory system at this stage of development. The changes in heart rate with temperature appear to be due to the intrinsic properties of the pacemaker; there was no indication for a regulated response.     

Role of core temperature as a stimulus for cold acclimation during repeated immersion in 20 degrees C water.

The relative importance of skin vs. core temperature for stimulating cold acclimation (CA) was examined by 5 wk of daily 1-h water immersions (20 degrees C) in resting (RG) and exercising (EG) subjects. Rectal temperature fell (0.8 degrees C; P < 0.05) during immersion only in RG. Skin temperature fell (P < 0.05) similarly in both groups. Physiological responses during cold-air exposure (90 min, 5 degrees C) were assessed before and after CA. Body temperatures and metabolic heat production were similar in both groups with no change due to CA. Cardiac output was lower (P < 0.05) in both groups post-CA (10.4 +/- 1.2 l/min) than pre-CA (12.2 +/- 1. 0 l/min), but mean arterial pressure was unchanged (pre-CA 107 +/- 2 mmHg, post-CA 101 +/- 2 mmHg). The increase in norepinephrine was greater (P < 0.05) post-CA (954 +/- 358 pg/ml) compared with pre-CA (1,577 +/- 716 pg/ml) for RG, but CA had no effect on the increase in norepinephrine for EG (pre-CA 1,288 +/- 438 pg/ml, post-CA 1,074 +/- 279 pg/ml). Skin temperature reduction alone may be a sufficient stimulus during CA for increased vasoconstrictor response, but core temperature reduction appears necessary to enhance sympathetic activation during cold exposure.     

Core threshold temperatures for sweating

To detect shifts in the threshold core temperature (Tc) for sweating caused by particular nonthermal stresses, it is necessary to stabilize or standardize all other environmental and physiological variables which cause such shifts. It is, however, difficult to cause progressive changes in Tc without also causing changes in skin temperature (Tsk). This study compares the technique of body warming by immersion in water at 40 degrees C, and subsequent body cooling in water at 28 degrees C, to determine the core threshold for sweating, with one by which Tc was raised by cycling exercise in air at 20 degrees C, and then lowered by immersion in water at 28 degrees C. The first of these procedures involved considerable shifts in Tsk upon immersion in water at 40 degrees C, and again upon transfer to water at 28 degrees C; the second procedure caused only small changes in Tsk. The onset of sweating at a lower esophageal temperature (Tes) during immersion in water at 40 degrees C (36.9 +/- 0.1 degrees C) than during exercise (37.4 +/- 0.3 degree C) is attributed to the high Tsk since Tes was then unchanged. Likewise, the rapid decline in the sweat rate during immersion at 28 degrees C had the same time course to extinction after the pretreatments. This related more to the Tsk, which was common, than to the levels or rates of change of Tes, which both differed between techniques. Tes fell most rapidly, and thus sweating was extinguished at a lower Tes, following 40 degrees C immersion than following exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

An analysis of the effect of changes in growth temperature on proteolysis in vivo in the psychrophilic bacterium Vibrio sp. strain ANT-300.

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the rate of protein degradation in vivo, measured at fixed temperatures, increased with elevation of the growth temperature. A shift in growth temperature induced a marked increase in this rate. Dialysed cell-free extracts hydrolysed exogenous insulin, globin and casein (in decreasing order of activity) but did not hydrolyse exogenous cytochrome c. Cells contained at least seven protease separated by DEAE-Sephacel chromatography, one of which was an ATP-dependent serine protease. The ATP-dependent proteolytic activity in extracts of cells incubated for 3 h at 16 degrees C after a shift-up from 0 degrees C increased to a level 36% and 17% higher than that of cells grown at 0 degrees C and 13 degrees C, respectively. A shift-down to 0 degrees C from 13 degrees C induced only a slight increase in the proteolytic activity. Extracts of all cells, whether exposed to temperature shifts or not, showed the same temperature dependence with respect to both ATP-dependent and ATP-independent protease activity. In all the extracts these proteases also exhibited the same heat lability. The ATP-dependent protease was inactivated by incubation at temperatures above 25 degrees C. There was an increase in ATP-independent protease activity during incubation at temperatures between 25 and 30 degrees C, but a decrease at 35 degrees C and higher. These results suggest that the marked increases in proteolysis in vivo, caused by a shift in temperature, may result not only from increases in levels of ATP-dependent serine protease(s) but also from increases in the susceptibility of proteins to degradation.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 degrees C/min while the ice nucleation temperature was varied between -3 and -10 degrees C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below -4 degrees C and cell survival exhibits an optimum at a nucleation temperature of -6 degrees C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at -3 degrees C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at -10 degrees C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in alpha-helical structures and a concomitant increase in beta-sheet structures starting at an onset temperature of approximately 48 degrees C.     

Survival of honey bee (Hymenoptera: Apidae) spermatozoa stored at above-freezing temperatures

The development of practical techniques for the storage of honey bee, Apis mellifera L., semen would significantly improve our ability to breed for desirable genotypes and maintain genetic diversity in populations. Artificial insemination of queens has been possible for some time, but the semen used is usually freshly collected, or held for < 1 wk at room temperature. I examined the limitations of spermatozoal survival at nonfrozen temperatures. Pooled, diluted semen was stored in sealed capillary tubes at room temperature (25 degrees C) or in a refrigerator set to 12 degrees C, for periods up to 1 yr. Survival of spermatozoa was assayed by a dual fluorescent staining technique using SYBR-14 and propidium iodide stains, which readily distinguishes live and dead cells. No significant loss of viable spermatozoa occurred within the first 6 wk. Between weeks 6 and 9, the percent live spermatozoa fell from 80 to 58%, and remained at that level until after 39 wk. By week 52, samples at room temperature, but not at 12 degrees C, fell to 18.9% live spermatozoa. Nonfrozen storage of honey bee semen has potential for short-term preservation of germplasm, however several factors need to be studied further to optimize survival rates.     

Dissolution of sucrose crystals in the anhydrous sorbitol melt

The dissolution of a sugar (sucrose as a model) with higher melting point was studied in a molten food polyol (sorbitol as a model) with lower melting point, both in anhydrous state. A DSC and optical examination revealed the dissolution of anhydrous sucrose crystals (mp 192 degrees C) in anhydrous sorbitol (mp 99 degrees C) liquid melt. The sucrose-sorbitol crystal mixtures at the proportions of 10, 30, 60, 100 and 150 g of sucrose per 100 g of sorbitol were heat scanned in a DSC to above melting endotherm of sorbitol but well below the onset temperature of melting of sucrose at three different temperatures 110, 130 and 150 degrees C. The heat scanning modes used were with or without isothermal holding. The dissolution of sucrose in the sorbitol liquid melt was manifested by an increase in the glass transition temperature of the melt and corresponding decrease in endothermic melting enthalpy of sucrose. At given experimental conditions, as high as 25 and 85% of sucrose dissolved in the sorbitol melt during 1 h of isothermal holding at 110 and 150 degrees C, respectively. Optical microscopic observation also clearly showed the reduction in the size of sucrose crystals in sorbitol melt during the isothermal holding at those temperatures.     

Temperature-dependence of rapid axonal transport in sympathetic nerves of the rabbit.

Stop-flow techniques were used to determine how temperature affected the axonal transport of dopamine-beta-hydroxylase (DBH) activity in rabbit sciatic nerves in vitro. These nerves were cooled locally to 2 degrees C for 1.5 hr, which caused a sharp peak of DBH activity to accumulate above the cooled region. Accumulated DBH was then allowed to resume migration at various temperatures. From direct measurements of the rate of migration, we found that the axonal transport velocity of DBH was a simple exponential function of temperature between 13 degrees C and 42 degrees C. Over this range of temperatures, the results were well described by the equation: V=0.546(1.09)T, where V is velocity in mm/hr, and T is temperature in degrees centigrade. The Q10 between 13 degrees and 42 degrees C was 2.33, and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 kcal. Transport virtually halted when temperature was raised to 47 degrees C, although only about half of the DBH activity disappeared during incubation at this temperature. Another transition occurred at 13 degrees C; below this temperature, velocity fell precipitously. This was not an artifact peculiar to the stop-flow system since the rate of accumulation of DBH activity proximal to a cold-block also decreased abruptly when the temperature above the block was reduced below 13 degrees C.     

Production of Spirulina biomass: Maintenance of monoalgal culture outdoors

The effects of sodium bicarbonate concentration, population density, and temperature on the maintenance of an outdoor monoculture of the cyanobacterium Spirulina platensis were studied. A clear response by Spirulina to the concentration of bicarbonate was evident, with 0.2M bicarbonate representing the lowest concentration in which a monoculture could be maintained. When the temperatures fell during the winter period to some 20-25 degrees C below the optimum for Spirulina, Chlorella sp. gradually increased and became the dominant species in the culture. Raising the temperature by covering the pond with transparent polyethylene resulted in a sharp decline in the population of Chlorella, and a gradual resumption of species dominance by Spirulina. In winter, there was an inverse relationship in the pond between the population density of Spirulina and the extent of contamination by Chlorella sp.; but no such effect was observed under field conditions at temperatures higher than 25 degrees C.     

Effect of thermoperiod on diapause intensity in Pyrrhocoris apterus (Heteroptera Pyrrhocoridae)

The intensity of adult diapause in Pyrrhocoris apterus was measured in two series of experiments as the duration of pre-oviposition period at a constant temperature of 25 degrees C after transfer from short (12L:12D) to long day conditions (18L:6D). Higher diapause intensity was induced with a thermoperiod than at constant temperatures. After the induction throughout larval instars 3-5 and during 4 weeks of adult life at short days and a thermoperiod of 25/15 degrees C the pre-oviposition period was 30+/-4 and 26+/-3 days. After induction at constant 25 degrees C the pre-oviposition period was 22+/-3 and 23+/-4 days, while after induction at constant 20 degrees C it was 17+/-4 and 19+/-4 days. Induction at a lower constant temperature of 20 degrees C was thus followed by a less intense diapause than the induction at a higher constant temperature of 25 degrees C. These counterintuitive results are discussed. The oxygen consumption rate measured at experimental temperatures prior to transfer from short to long days was higher at thermoperiodic conditions than at constant temperatures and it was similar at constant 20 and 25 degrees C. Thus, the oxygen consumption rate measured prior to the transfer was highest (indication of the least intense diapause) in the insects that showed later, after the transfer to long days, the longest pre-oviposition period (indication of the most intense diapause). Within the first two days after transfer to constant 25 degrees C, oxygen consumption rate measured at 25 degrees C decreased in the thermoperiodic insects, while it transiently increased in insects from constant 20 degrees C. Two days and later after the transfer, oxygen consumption rate was similar in all groups. Cold hardiness was not correlated with diapause intensity. The low lethal temperature in diapausing insects was correlated with the night temperature during diapause induction.     

Reef coral survival and mortality at low temperatures in the Arabian Gulf: new species-specific lower temperature limits

A series of cold fronts passing over the western Arabian Gulf from December 1988 to March 1989 produced the longest period of sustained low water temperatures ever recorded in a coral reef area. Sea water temperatures recorded on two reefs during this period provide new estimates of lower thermal limits for reef coral survival. Severe mortality of the corals Acropora pharaonis and Platygyra daedalea occurred at the northern site where minimum temperatures fell below 11.5°C on four consecutive days and mean daily temperatures were 13°C or less for more than 30 days. However, Porites compressa, the principal reef-former in this area, and various faviid corals initially showed only sub-lethal effects and appeared normal after six months. Corals were not damaged at the southern site, where minimum water temperature fell below 12.5°C for two consecutive days, but mean temperatures were 14°C or less for only 5 non-consecutive days.     

Effects on fetal and maternal body temperatures of exposure of pregnant ewes to heat, cold, and exercise.

We exposed Dorper-cross ewes at approximately 120-135 days of gestation to a hot (40 degrees C, 60% relative humidity) and a cold (4 degrees C, 90% relative humidity) environment and to treadmill exercise (2.1 km/h, 5 degrees gradient) and measured fetal lamb and ewe body temperatures using previously implanted abdominal radiotelemeters. When ewes were exposed to 2 h of heat or 30 min of exercise, body temperature rose less in the fetus than in the mother, such that the difference between fetal and maternal body temperature, on average 0.6 degrees C before the thermal stress, fell significantly by 0.54 +/- 0.06 degrees C (SE, n = 8) during heat exposure and by 0.21 +/- 0.08 degrees C (n = 7) during exercise. During 6 h of maternal exposure to cold, temperature fell significantly less in the fetus than in the ewe, and the difference between fetal and maternal body temperature rose to 1.16 +/- 0.26 degrees C (n = 9). Thermoregulatory strategies used by the pregnant ewe for thermoregulation during heat or cold exposure appear to protect the fetus from changes in its thermal environment.     

Effects of temperature gradients on in vitro maturation of bovine oocytes

The effect of temperature gradients on in vitro maturation of bovine oocytes was examined in this study. Six treatment groups were made by combining 3 different maturation periods (0 to 10 h, 10 to 18 h and 18 to 24 h) with 2 different culture temperatures (37.0 degrees C and 38.5 degrees C). The frequency of oocytes matured to the metaphase II stage was apparently gradually increased as the culture temperature was increased from 37.0 degrees C to 38.5 degrees C at 0, 10 and 18 h after the onset of culture (75.2 vs 80.5, 82.3 and 84.3%, respectively), but this difference was not significant. Neither was the minor decrease in the proportion of oocytes reaching metaphase II when the temperature was decreased from 38.5 degrees C to 37.0 degrees C at 10 and at 18 h after the onset of maturation (84.3 vs 82.4 and 78.0%, respectively). However, more oocytes cleaved (79.2%; P = 0.0653) and developed to morulae (43.6%; P = 0.0019) and blastocysts (27.4%; P = 0.1568) when they were in vitro matured at 38.5 degrees C between 0 and 10 h, and then at 37.0 degrees C from 10 to 24 h. Although only the morula group was statistically different, cleavage- (79.2 vs 69.8, 72.5, 74.2, 76.3, 74.3%, respectively) and blastocyst formation (27.4 vs 23.2, 24.6, 25.2, 19.6, 21.9%, respectively) from this group was the highest among the 6 treatments.     

Temperature effect on contractile activity of the Ambystoma dumerilii heart previously treated with isoproterenol

The spontaneous heart rate (HR) and ventricular (V) and atrium (A) tensions (T) were evaluated through isolated organ assays at different temperatures in hearts from Ambystoma dumerilii control and treated with isoproterenol (ISO) [(150 mg/kg i.p. each 24 h, for 3 days)] on days 1, 5, 30 and 90 after ISO. In control hearts, the HR increased and the T decreased when temperature was augmented. One day after ISO the HR (43-24%) and T (50-25%) decreased with respect to control, between 8 and 24 degrees C. Five, 30 and 90 days after ISO, HR showed a gradual recovery with similar effect when the temperature was changed; but the AT increased and VT decreased at temperatures between 8 and 12 degrees C and were only recovered at temperatures above 12 degrees C. Our results indicate that the HR recovers after ISO in A. dumerilii independently of temperature. The recovery of AT and VT is similar to HR at temperatures higher than 12 degrees C and the increases in VT could be compensating the decrease in VT caused by ISO, at temperatures lower than 12 degrees C. The changes in heart contractile activity of A. dumerilii after insult show the thermic plasticity that is observed in ectothermic vertebrates.     

Temperature dependent characteristics of intestinal glycyl-L-leucine dipeptidase in boreal zone fish

Three kinds of boreal zone fish were investigated for gastrointestinal glycyl-L-leucine (GL) dipeptide cleaving activity as a function of feeding stage and seasonal changes. The enzyme activity tested in the perch (Perca fluviatilis L.) intestine increased steadily during digestion and rapidly disappeared after completion. The temperature characteristics and the seasonal changes in dipeptide cleaving activity in pike perch (Stizostedion lucioperca L.) and bream (Abramis brama L.) were studied. In summer, the maximal activities in the pike perch and the bream were found at temperatures of 40 and 30 degrees C, respectively. In winter, the temperature of maximal activity in pike perch fell to only 30 degrees C, whereas no changes were observed in bream. The activation energies in bream and pike perch were several times lower in winter than in summer. Seasonal changes in the dipeptide cleaving activity at low temperature relative to that at the temperature of maximal activity were found. At high temperatures, the stability of the enzyme decreases in winter and increases in summer, but in the presence of a substrate the thermal stability of the enzyme increases both in winter and in summer. In our experiments, we found that in these fish, GL dipeptidase was unstable at 0 and -10 degrees C.     

The behaviour of log phase Escherichia coli at temperatures that fluctuate about the minimum for growth

AIMS: To investigate the behaviour of cold-adapted, log phase Escherichia coli exposed to temperatures that fluctuate below and above the minimum for growth. METHODS AND RESULTS: Log phase E. coli cultures were incubated at a constant temperature of 2, 4 or 6 degrees C or with temperatures allowed to increase from those temperatures for 35 min, to 10 degrees C, at 6-, 12- or 24-h intervals, as commonly occurs during retail display of chilled foods. At suitable intervals for each culture, the optical absorbance value was determined using a spectrophotometer, the forward angle light scatter was determined using a flow cytometer, and portions were spread on plate count agar for enumeration of colony forming units (CFU). Numbers of CFU decreased by 3 log units or increased by 1 log unit for cultures incubated at 6 degrees C for 17 days without or with temperatures fluctuations at < or =12-h intervals, respectively. Cells elongated when cultures were incubated at 4 or 2 degrees C with temperatures fluctuating at 6-h intervals, and at 6 degrees C at constant or fluctuating temperatures, but cells did not elongate in cultures incubated at a constant temperature of 2 or 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The minimum growth temperature of E. coli is assumed to be > or =7 degrees C. Elongated cells were able to divide when temperatures rose from 6 degrees C to above 7 degrees C for <45 min at < or =12-h intervals. Such temperature fluctuations may be experienced by chilled foods during defrosting cycles of retail display cases. The finding that cells behave differently under fluctuating than at constant temperatures may significantly affect understanding of appropriate temperatures for the safe storage of chilled foods and for predictive modelling of bacterial growth in such foods.     

Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar 'Deltapine 5415' bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30 degrees C/26 degrees C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22 degrees C/18 degrees C. However, endogenous gene silencing decreased at 30 degrees C/26 degrees C. There was an approximately 7 d delay in the onset of gene silencing at 22 degrees C/18 degrees C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.