Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility

A protein spin label and lipid spin probes were used to study the temperature-dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20°C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12°C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14°C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.

     

Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods

The phase behavior of thylakoid polar lipids from plants sensitive to chilling injury was investigated by calorimetry, electron spin resonance spectroscopy of spin labels, and fluorescence intensity after labeling with trans-parinaric acid. The plants used were oleander (Nerium oleander), mung bean (Vigna radiata L. var Mungo), and tomato (Lycopersicon esculentum cv Grosse Lisse). For all plants the initiation temperature for the calorimetric exotherm was coincident (±1°C) with the transition determined by the increase in the temperature coefficient of spin label motion and fluorescence intensity of trans-parinaric acid. For oleander plants, grown at 45°C, the transition was at 7°C while for plants from the same clone, grown at 20°C, it was at −2°C. For mung bean and tomato the transition was between 9 and 12°C. The similarity in the transition detected by spin labeling and fluorescence intensity suggest that spin labels, like the fluorescent label trans-parinaric acid, preferentially partition into domains of ordered lipid. The coincidence of the temperature for initiation of the transition, determined by the three techniques, shows that each is a valid method of assessing a phase transition in membrane polar lipids.     

Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N–formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO^® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.     

Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer

Defferential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70). Hsc70 had endothermic trasitions with midpoints (Tm) at 59°C and 63°C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan. Combined with increased exposure at 60°C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70. An exothermic transition (Tm=66°C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes. An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c. A novel finding was an exothermic transition of Hsc70 begining at about 30°c (Tm=41°C). No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range. Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation. However, Hsc70 was protease-resistant at 20°C, sensitive at 40°C and resistant when returned to 20°C, indicating that this exotherm is associated with a reversible conformational change. As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK. For all of these substrates, the amount of complex formed increased with increasing termperature over the same range as the 41°C exotherm. It is proposed that a conformational change exposes polar and charged residues in Hsc70 Which subsequently become hydrated, resulting in an active chaperone. Hsc70 may be a thermal sensor that supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells. Thermal activation of Hsc70 may also have a role in acquired thermotolerance.     

Photoinduced Seed Germination of Oenothera biennis L: III. Analysis of the Postinduction Period by Means of Temperature

The postinduction period of Oenothera biennis L. seed germination was examined by temperature treatments. For all experiments, seeds received a standard 24 hour/24°C preinduction period and 12 hour/32°C photoinduction period. Germination is inhibited by postinduction temperatures above 32°C. When seeds are briefly incubated at 44°C and then transferred to 28°C, they germinate at a much lower percentage than 28°C controls. When thermally inhibited seeds are placed in the dark at 28°C for 20 hours, they can be promoted to germinate by a single pulse of red light. Seeds incubated at 12°C or below immediately after photoinduction enter a lag period in which they germinate slowly or not at all for a long time and then resume germination. The length of the lag period is exponentially related to the postinduction temperature. When seeds are incubated at a low temperature and then transferred to a warm temperature, they germinate much more rapidly than seeds not incubated at a low temperature. A model is proposed which is consistent with these and additional results. In the model, a germination promoter is irreversibly formed from a precursor and the synthesis of the precursor is favored at low temperatures and its degradation is favored at high temperatures.     

Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: III. Effect of Temperature and Cations on Protein Sulfhydryl Reactivity

Temperature and cations modified the reaction of barley (Hordeum vulgare L. cv Conguest) root plasma membrane protein sulfhydryl groups with N-4-(7-diethylamino-4-methylcoumarin-3-yl)-phenyl maleimide (CPM). The pseudo-first-order rate constants for the formation of fluorescent CPM-protein adducts increased as the temperature was raised above 30°C, suggesting changes in protein conformation. Monovalent [K(I), Na(I), L(I)] and certain divalent cations [Ba(II), Mg(II)] increased the reaction rates. Other divalent cations [Ca(II), Mn(II), Ni(II), Co(II), Sr(II), Cd(II), Hg(II)] decreased the rate of fluorescent adduct formation. Na(I) promoted and Ca(II) delayed the onset of the temperature-dependent increases in reaction rates. The results are discussed in terms of lipid-mediated, temperature-dependent changes in membrane protein conformation and ion-protein interactions.     

Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO₂ exchange or O₂ evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO₂ exchange and O₂ evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

Tolerance of photosynthesis to high temperature in desert plants

Winter- and summertime-active desert annual species were grown at different temperatures to assess their capacity for photosynthetic acclimation. Thermal stability of photosynthesis was determined from responses of chlorophyll fluorescence to increased temperature. Photosynthesis in winter ephemerals grown at 28°C/21°C became unstable close to 41°C in contrast to the summer annuals which were stable up to about 46°C. Growth at higher temperature (43°C/32°C) resulted in increases in thermal stability of 5 to 7°C for the winter annuals and 3 to 4°C for the summer annuals, showing that temperature can provide the primary stimulus for acclimation of the photosynthetic apparatus. The magnitude of these changes was very similar to the range of field values observed for the respective floras, indicating that the thermal acclimation response under field conditions was qualitatively similar to that occurring under controlled growth conditions. Perennial species, co-existing with these annuals in the desert, were on average more thermostable. The cacti were exceptionally heat stable, the threshold for fluorescence increase averaging 55°C.     

Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.     

Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth

Root temperature strongly affects shoot growth, possibly via “nonhydraulic messengers” from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25° and 35°C, respectively. Hydraulic conductance (L_p) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25°C, remaining high with further warming. In sorghum, the L_p of intact plants and of detached roots peaked at 35°C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L_p. Extreme temperatures greatly decreased ion flux, particularly K⁺ and NO₃⁻, to the xylem of detached roots of both species. Removing external K⁺ did not alter short-term K⁺ flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently “uncoupled” from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions.     

Site-specific fluorescence dynamics in an RNA ‘thermometer’ reveals the role of ribosome binding in its temperature-sensitive switch function

RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20–45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine–Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA.     

Chilling Sensitivity in Oryza sativa: The Role of Protein Phosphorylation in Protection against Photoinhibition

The effects of exposure to low temperature on photosynthesis and protein phosphorylation in chilling-sensitive and cold-tolerant plant species were compared. Chilling temperatures resulted in light-dependent loss of photosynthetic electron transport in chilling-sensitive rice (Oryza sativa L.) but not in cold-tolerant barley (Hordeum vulgare L.). Brief exposure to chilling temperatures (0-15°C, 10 min) did not cause a significant difference in photosynthetic O₂ evolution capacity in vivo between rice and barley. Analysis of in vivo chlorophyll fluorescence in chilling-sensitive rice suggests that low temperatures cause an increased reduction of the plastoquinone pool that could result in photoinhibitory damage to the photosystem II reaction centers. Analysis of ³²P incorporation into thylakoid proteins both in vivo and in vitro demonstrated that chilling temperature inhibited protein phosphorylation in rice, but not in barley. Low temperature (77 K) fluorescence analysis of isolated thylakoid membranes indicated that state I to state II transitions occurred in barley, but not in rice subjected to chilling temperatures. These observations suggest that protein phosphorylation may play an important role in protection against photoinhibition caused by exposure to chilling temperatures.     

Ubiquitin Pool Modulation and Protein Degradation in Wheat Roots during High Temperature Stress

Ubiquitin, a key component in an ATP-dependent proteolytic pathway, participates in the response of various eucaryotic organisms to high temperature stress. Our objective was to determine if ubiquitin serves a similar capacity for metabolizing altered proteins in higher plants during stress. Degradation of total proteins was measured, and ubiquitin pools (free versus conjugated) were extracted with an improved protocol from wheat (Triticum aestivum L. cv Len) roots treated at 22, 27, 32, 37, and 42°C for 1 hour and assayed by western blots and radioimmunoassays. Heat-shock protein synthesis was detected by in vivo labeling and autoradiography. Mean half-life of total root proteins decreased from 51 hours at 22°C to 23 hours at 40°C. Ubiquitin pools were extracted better and proteolysis was slowed more by the improved protocol than by a conventional procedure for plant proteins. Amounts of high molecular mass conjugates were elevated and levels of low molecular mass conjugates and free ubiquitin were depressed when roots were treated at 37 or 42°C than at lower temperatures; the same high temperatures also induced synthesis of heat-shock proteins. We concluded that high temperatures increase breakdown of root proteins, which are degraded via the ubiquitin proteolytic pathway. A conjugate with an apparent molecular mass of 23 kilodaltons was tentatively identified as an ubiquitinated histone.     

Aluminum and Temperature Alteration of Cell Membrane Permeability of Quercus rubra

This report extends research on Al-induced changes in membrane behavior of intact root cortex cells of Northern red oak (Quercus rubra). Membrane permeability was determined by the plasmometric method for individual intact cells at temperatures from 2 or 4 to 35°C. Al (0.37 millimolar) significantly increased membrane permeability to urea and monoethyl urea and decreased permeability to water. Al significantly altered the activation energy required to transport water (+32%), urea (+9%), and monoethyl urea (−7%) across cell membranes. Above 9°C, Al increased the lipid partiality of the cell membranes; below 7°C, Al decreased it. Al narrowed by 6°C the temperature range over which plasmolysis occurred without membrane damage. These changes in membrane behavior are explainable if Al reduces membrane lipid fluidity and kink frequency and increases packing density and the occurrence of straight lipid chains.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature

Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37°C and 33°C from 24 hours after transfection (including 19 hours recovery at 37°C in the normal growth medium), we found that a mild hypothermia (i.e., 33°C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33°C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method.     

TIME-TEMPERATURE RELATIONS IN THE INCUBATION OF THE WHITEFISH,COREGONUS CLUPEAFORMIS (MITCHILL)

1. Whitefish eggs incubated in aerated lake water at controlled tempera tures of 0°, 0.5°, 2°, 4°, 6°, 8°, 10°, and 12°C., failed to hatch at either 0° or 12°C. 0.6 per cent hatched alive at 10°C., 72.67 per cent hatched alive at 0.5°C., and an intermediate proportion hatched at intermediate temperatures. 2. The percentage of abnormal embryos which developed to the hatching stage varied directly with temperature between 4° and 12°, all embryos being abnormal at 12°C.; but none were abnormal at either 0.5°, or 2°C. Normal development predominated from 0.5 to 6°C. The highest proportion of embryos to hatch alive was 72.67 per cent at 0.5°C., which is, hence, the optimum temperature. 3. Total incubation time ranged from 29.6 days at 10°C. to 141 days at 0.5°C. 4. The time (T) required to attain any given stage of development is expressed in equations See PDF for Equation where temperature, t, is a negative exponent of the constant, A, whose value differs above or below 6°C., a critical temperature. Values of A above 6° fluctuate about 1.13; those of A below 6° fluctuate about 1.19 as a mean. 5. Applying Arrhenius'' equation µ values for the total incubation period are 27,500 below 6° and 27,100 above it. 6. The relative magnitude of A values of the exponential equation and µ values of Arrhenius'' equation show corresponding changes from one developmental period to another. 7. When plotted, thermal increments show cyclic variations, with maxima during periods of cleavage and of organogenesis. These may indicate the interaction of two separate sets of embryonic processes, which give a maximal response to temperature differences during these two separate periods. 8. Above 6°, µ values during the hatching process are distinct from those of developmental stages and are regarded as being due to the action of hatching enzymes.     

Diapause Induction and Termination in the Small Brown Planthopper,Laodelphax striatellus (Hemiptera: Delphacidae)

The small brown planthopper, Laodelphax striatellus (Fallén) enters the photoperiodic induction of diapause as 3rd or 4th instar nymphs. The photoperiodic response curves in this planthopper showed a typical long-day response type with a critical daylength of approximately 11 h at 25°C, 12 h at 22 and 20°C and 12.5 h at 18°C, and diapause induction was almost abrogated at 28°C. The third stage was the most sensitive stage to photoperiod. The photoperiodic response curve at 20°C showed a gradual decline in diapause incidence in ultra-long nights, and continuous darkness resulted in 100% development. The required number of days for a 50% response was distinctly different between the short- and long-night cycles, showing that the effect of one short night was equivalent to the effect of three long nights at 18°C. The rearing day length of 12 h evoked a weaker intensity of diapause than did 10 and 11 h. The duration of diapause was significantly longer under the short daylength of 11 h than it was under the long daylength of 15 h. The optimal temperature for diapause termination was 26 and 28°C. Chilling at 5°C for different times did not shorten the duration of diapause but significantly lengthened it when chilling period was included. In autumn, 50% of the nymphs that hatched from late September to mid-October entered diapause in response to temperatures below 20°C. The critical daylength in the field was between 12 h 10 min and 12 h 32 min (including twilight), which was nearly identical to the critical daylength of 12.5 h at 18°C. In spring, overwintering nymphs began to emerge in early March-late March when the mean daily temperature rose to 10°C or higher.