Use of electrolyte leakage to assess floral damage after freezing.

Premise: With growing interest in the impact of false springs on plant reproduction, there is the need to develop reliable, high-throughput methods for assessing floral freezing damage. Here we present a method for use with floral tissue that will facilitate more comparative work on floral freezing tolerance in the future. Methods and Results: We examined the effectiveness of a modified electrolyte leakage protocol to assess floral freezing damage. By comparing data from temperature response curves to an estimate of visual tissue damage, we optimized the protocol for different floral types and improved the signal-to-noise ratio for floral data. Conclusions: Our modified protocol provides a quick and straightforward method for quantifying floral freezing damage that can be standardized across floral types. This method allows for cross-species comparisons and can be a powerful tool for studying broad patterns in floral freezing tolerance.

Formulation and Validation of an Extended Sigmoid Emax Model in Pharmacodynamics.

PURPOSE OR OBJECTIVE: Drug concentration-response curves (DRCs) are crucial in pharmacology for assessing the drug effects on biological systems. The widely used sigmoid Emax model, which accounts for response saturation, relies heavily on the effective drug concentration ( E D 50 ). This reliance can lead to validation errors and inaccuracies in model fitting. The Emax model cannot generate multiple DRCs, raising concerns about whether the dataset is fully utilized. METHODS: This study formulates an extended Emax (eEmax) model designed to overcome these limitations. The eEmax model generates multiple DRCs from a single dataset by using various estimated α ' s ∈ 0,100 , while keeping E D α fixed, rather than estimating an E D 50 value as in the Emax model. RESULTS: This model effectively captures a broader range of concentration-response behavior, including non-sigmoidal patterns, thus providing greater flexibility and accuracy compared to the Emax model. Validation using various drug-response data and PKPD frameworks demonstrates the eEmax model's improved accuracy and versatility in handling concentration-response data. CONCLUSIONS: The eEmax model provides a robust and flexible method for drug concentration-response analysis, facilitating the generation of multiple DRCs from a single dataset and reducing the possibility of validation errors. This model is particularly valuable for its ease of use and its capability to fully utilize datasets, providing its potential in PKPD modeling and drug discovery.

A Skin Testing Strategy for Non-IgE-Mediated Reactions Associated With Vancomycin.

BACKGROUND: Vancomycin infusion reaction (VIR), reportedly mediated through Mas-Related G Protein-Coupled Receptor-X2, is the primary vancomycin-induced immediate drug reaction. Clinically, distinguishing the underlying drug-induced immediate drug reaction mechanisms is crucial for future treatment strategies, including drug restriction, re-administration, and pretreatment considerations. However, the lack of validated diagnostic tests makes this challenging, often leading to unnecessary drug restriction. OBJECTIVE: To determine whether intradermal tests (IDTs) and, separately, the basophil activation test (BAT) differentiate VIR from vancomycin-tolerant subjects. METHODS: This was a cross-sectional study of vancomycin-exposed adults with and without a history of VIR. Data on demographics, allergy-related comorbidities, history of vancomycin exposures, and VIR characteristics were collected. IDT with vancomycin was performed. IDT dose-response EC50, IDT-related local symptoms, and BAT results were compared between groups. RESULTS: A total of 11 VIR and 10 vancomycin-tolerant subjects were enrolled. The most reported VIR symptoms were pruritus (82%), flushing (82%), hives (46%), angioedema (27%), and dyspnea (19%). The IDT dose-response mean EC50 was 328 µg/mL (95% CI, 296-367) in the VIR versus 1166 µg/mL (95% CI, 1029-1379) in the tolerant group (P < .0001). All VIR subjects reported IDT-related local pruritus compared with 60% of tolerant subjects (P = .0185). The %CD63+ basophils were consistently less than 2%, without significant differences between groups (P < .54). CONCLUSIONS: Variations in skin test methodologies could help identify other immediate drug reaction mechanisms beyond IgE. This skin test protocol holds the potential for identifying VIR, particularly in cases where patients have received multiple drugs while BAT is insufficient. Future studies will validate and delineate its predictive value, assessing the risk of VIR.

Analytical solutions for cavity contraction in strain-softening materials with linear or exponential strength decay.

This paper presents an analytical investigation into the contraction of spherical and cylindrical cavities excavated in strain-softening rock masses obeying the Mohr-Coulomb or Tresca yield criterion, with linear or exponential uniaxial compressive strength decay. The derivation of the ground response curves is based on the simplifying assumption that the strains inside the plastic zone are completely plastic. This significantly simplifies the mathematical formulation, enabling the derivation of closed-form solutions. An alternative simplifying approach which partially neglects the elastic strains inside the plastic zone and which is commonly adopted in the literature, is also examined. The accuracy of the simplified solutions is evaluated by comparing their predictions with rigorous solutions obtained by numerical finite-difference analyses. The investigation demonstrates that the proposed closed-form solutions represent a significant improvement on those based on the commonly-made simplifying assumption involving partial neglect of elastic strains.

Hyperbolic discounting underpins response curves of mammalian avoidance behaviour.

As humans clear natural habitat, they are brought into increased conflict with wild animals. Some conflict is direct (e.g. elevated exposure of people to predators), some indirect (e.g. abandoning suitable habitat because of human activity). The magnitude of avoidance is expected to track frequency of human activity, but the type of response is an open question. We postulated that animals do not respond passively to increased disturbance nor does response follow a power law; instead, their ability to estimate magnitude leads to 'discounting' behaviour, as in classic time-to-reward economic models in which individuals discount larger value (or risk) in more distant time. We used a 10-year camera dataset from southern California to characterize response curves of seven mammal species. Bayesian regressions of two non-discounting models (exponential and inverse polynomial) and two discounting models (hyperbolic and harmonic) revealed that the latter better fit response curves. The Arps equation, from petroleum extraction modelling, was used to estimate a discount exponent, a taxon-specific 'sensitivity' to humans, yielding a general model across species. Although discounting can mean mammal activity recovers rapidly after disturbance, increased recreational pressure on reserves limits recovery potential, highlighting a need to strike a balance between animal conservation and human use.

Stimulus-Response Plots as a Novel Bowel-Sound-Based Method for Evaluating Motor Response to Drinking in Healthy People.

Constipation is a common gastrointestinal disorder that impairs quality of life. Evaluating bowel motility via traditional methods, such as MRI and radiography, is expensive and inconvenient. Bowel sound (BS) analysis has been proposed as an alternative, with BS-time-domain acoustic features (BSTDAFs) being effective for evaluating bowel motility via several food and drink consumption tests. However, the effect of BSTDAFs before drink consumption on those after drink consumption is yet to be investigated. This study used BS-based stimulus-response plots (BSSRPs) to investigate this effect on 20 participants who underwent drinking tests. A strong negative correlation was observed between the number of BSs per minute before carbonated water consumption and the ratio of that before and after carbonated water consumption. However, a similar trend was not observed when the participants drank cold water. These findings suggest that when carbonated water is drunk, bowel motility before ingestion affects motor response to ingestion. This study provides a non-invasive BS-based approach for evaluating motor response to food and drink, offering a new research window for investigators in this field.

A novel approach to assess the health risk of aryl hydrocarbon receptor-bound contaminants via inhalation exposure using CYP1A1 expression as a biomarker.

Polycyclic aromatic hydrocarbons (PAHs) and dioxins are potential causes of multiple diseases by activating the aryl hydrocarbon receptor (AhR) pathway. Health risk assessment of chemicals primarily relies on the relative potency factor (RPF), although its accuracy may be limited when solely using EC50 values. The induction of cytochrome P4501A1 (CYP1A1) serves as a biomarker for AhR activation and is an integrator of dioxin-like toxicity. Here, we present a method for evaluating the risks associated with AhR activation using mathematical models of dose-CYP1A1 induction. The dose-effect curves for certain PAHs and dioxins, including Ant, BghiP, 1,2,3,4,7,8-HxCDD, and others, exhibited a non-classical S-shaped form. The toxic equivalent factor (TEF) profiles revealed a broad range of toxic equivalent factor values. The TEFs for PAHs ranged from approximately 0.01 to 6, with higher values being observed when the concentration was less than 10-10 M, with the exceptions of Ace, Phe, and BghiP. Most congeners of dioxins got the lowest TEF value at around 10-10 M, ranging from 0.04 to 1.00. The binding affinity of AhR to ligands did not display a strong correlation with the EC50 of CYP1A1 expression, suggesting that the AhR-mediated effects of PAHs and dioxins are not fixed but instead fluctuate with the dose. Air samples acquired from a parking area were used to compare the proficiency of RPF and our current approach. In the current method, naphthalene and chrysene were the primary contributors of PAHs to AhR-mediated risks in parking lots air samples, respectively. However, the contributions of naphthalene and chrysene could be disregarded in the RPF approach.

Phase Delays between Mouse Globus Pallidus Neurons Entrained by Common Oscillatory Drive Arise from Their Intrinsic Properties, Not Their Coupling.

A hallmark of Parkinson's disease is the appearance of correlated oscillatory discharge throughout the cortico-basal ganglia (BG) circuits. In the primate globus pallidus (GP), where the discharge of GP neurons is normally uncorrelated, pairs of GP neurons exhibit oscillatory spike correlations with a broad distribution of pairwise phase delays in experimental parkinsonism. The transition to oscillatory correlations is thought to indicate the collapse of the normally segregated information channels traversing the BG. The large phase delays are thought to reflect pathological changes in synaptic connectivity in the BG. Here we study the structure and phase delays of spike correlations measured from neurons in the mouse external GP (GPe) subjected to identical 1-100 Hz sinusoidal drive but recorded in separate experiments. First, we found that spectral modes of a GPe neuron's empirical instantaneous phase response curve (iPRC) elucidate at what phases of the oscillatory drive the GPe neuron locks when it is entrained and the distribution of phases at which it spikes when it is not. Then, we show that in this case the pairwise spike cross-correlation equals the cross-correlation function of these spike phase distributions. Finally, we show that the distribution of GPe phase delays arises from the diversity of iPRCs and is broadened when the neurons become entrained. Modeling GPe networks with realistic intranuclear connectivity demonstrates that the connectivity decorrelates GPe neurons without affecting phase delays. Thus, common oscillatory input gives rise to GPe correlations whose structure and pairwise phase delays reflect their intrinsic properties captured by their iPRCs.

Dose‒Response Effects of Patient Engagement on Anxiety and Depression in a Cognitive-Behavioral Intervention: Secondary Analysis of a Pilot Randomized Controlled Trial and a Clinical Controlled Trial.

Understanding the dose‒response relationship between patient engagement in cognitive behavioral therapy (CBT) and health outcomes is critical for developing and implementing effective CBT programs. In studies of CBT interventions, patient engagement is measured only at a single time point, and outcomes are typically assessed before and after the intervention. Examination of the dose‒response relationship between patient engagement in CBT and outcomes is limited. It is unclear whether a dose‒response relationship exists between patient engagement in on-site CBT intervention and anxiety and depression in people living with HIV (PLWH). If present, does this dose‒response relationship occur early or later in the intervention? This study aimed to address this gap by examining the dose‒response relationships between patient engagement and anxiety and depression in CBT interventions among PLWH. Utilizing data from a pilot randomized trial (10 participants) and a clinical controlled trial (70 participants), our secondary analysis spans baseline, 3-month, and 6-month assessments. Both trials implemented the nurse-led CBT intervention. Cluster analysis identified two groups based on on-site attendance and WeChat activity. Patients with good adherence (6-10 times) of on-site attendance exhibited significantly lower anxiety and depression scores at 3 months (ß = 1.220, P = 0.047; ß = 1.270, P = 0.019), with no significant differences observed at 6 months. WeChat activity did not significantly influence anxiety or depression scores. The findings highlight a significant short-term dose‒response relationship, endorsing nurse-led CBT interventions for mental health in PLWH. Organizational strategies should focus on incentivizing and facilitating patient engagement, particularly through enhancing WeChat features.

A novel method to estimate the response of habitat types to nitrogen deposition.

Increasing nitrogen depositions adversely affect European landscapes, including habitats within the Natura2000 network. Critical loads for nitrogen deposition have been established to quantify the loss of habitat quality. When the nitrogen deposition rises above a habitat-specific critical load, the quality of the focal habitat is expected to be negatively influenced. Here, we investigate how the quality of habitat types is affected beyond the critical load. We calculated response curves for 60 terrestrial habitat types in the Netherlands to the estimated nitrogen deposition (EMEP-data). The curves for habitat types are based on the occurrence of their characteristic plant species in North-Western Europe (plot data from the European Vegetation Archive). The estimated response curves were corrected for soil type, mean annual temperature and annual precipitation. Evaluation was carried out by expert judgement, and by comparison with gradient deposition field studies. For 39 habitats the response to nitrogen deposition was judged to be reliable by five experts, while out of the 41 habitat types for which field studies were available, 25 showed a good agreement. Some of the curves showed a steep decline in quality and some a more gradual decline with increasing nitrogen deposition. We compared the response curves with both the empirical and modelled critical loads. For 41 curves, we found a decline already starting below the critical load.

Topography-mediated light environment regulates intra-specific seasonal and diurnal patterns of photosynthetic plasticity and plant ecophysiological adaptation strategies.

Due to substantial topographic variations in the Himalaya, incident solar radiation in the forest canopy is highly unequal. This results in significant environmental differences at finer scales and may lead to considerable differences in photosynthetic productivity in montane forests. Therefore, local-scale ecophysiological investigations, may be more effective and instructive than landscape-level inventories and models. We investigated leaf ecophysiological differences and related adaptations between two Quercus semecarpifolia forests in aspect-mediated, significantly varying light regimes in the same mountain catchment. Seasonal and diurnal rates of photosynthesis (A) were significantly higher in south aspect (S) than the north (N). Although temperature was a key contributor to seasonal fluctuations in photosynthetic physiology, photoperiod significantly determined intraspecific variations in seasonal and diurnal plasticity of leaf ecophysiological traits between the two topography-mediated light environments. The regression model for A as a function of stomatal conductivity (gsw) explained the critical role of gsw in triggering photosynthetic plasticity as an adaptive function against varying environmental stresses due to seasonal solar differences. We also examined, modifications in chlorophyll content between the two light regimes across seasons to determine the chlorophyll adaptation strategy. The N aspect had higher leaf chl a, b, and chl a + b and a lower chl-allocation ratio (a/b) than S, which helped to optimize the required light reception in the photoreaction centers for improved photosynthetic performance. The leaf light response curves for A and gsw were observed against varying incident photosynthetic photon flux densities (0-2000 mol.m2 s-1 PPFD) for both aspects. We found that the same species developed significantly distinct light response strategies and photosynthetic capacities in S than in N for the given magnitudes of PPFD. Such acquired ecophysiological adaptations owing to varying light environments may provide significant clues for understanding the impact of future climate change on Himalayan tree species.

Encoding Genetic Circuits with DNA Barcodes Paves the Way for High-Throughput Profiling of Dose-Response Curves of Metabolite Biosensors.

Metabolite biosensors, through which the intracellular metabolite concentrations could be converted to changes in gene expression, are widely used in a variety of applications according to the different output signals. However, it remains challenging to fine-tune the dose-response relationships of biosensors to meet the needs of various scenarios. On the other hand, the short read length of next-generation sequencing (NGS) has greatly limited the design capability of sequence libraries. To address these issues, we describe a DNA trackable assembly method, coupled with fluorescence-activated cell sorting and NGS (Sort-Seq), to achieve the characterization of dose-response curves in a massively parallel manner. As a proof of the concept, we constructed a malonyl-CoA biosensor library containing 5184 combinations with six levels of transcription factor dosage, four different operator positions, and 216 possible upstream enhancer sequence (UAS) designs in Saccharomyces cerevisiae BY4700. By using Sort-Seq and machine learning approach, we obtained comprehensive dose-response relationships of the combinatorial sequence space. Therefore, our pipeline provides a platform for the design, tuning, and profiling of biosensor response curves and shows great potential to facilitate the rational design of genetic circuits.

Radiprodil, a selective GluN2B negative allosteric modulator, rescues audiogenic seizures in mice carrying the GluN2A(N615S) mutation.

BACKGROUND AND PURPOSE: GRIN-related disorders are neurodevelopmental disorders caused by mutations in N-methyl-D-aspartate receptor (NMDAR) subunit genes. A large fraction of these mutations lead to a 'gain of function' (GoF) of the NMDAR. Patients present with a range of symptoms including epilepsy, intellectual disability, behavioural and motor. Controlling seizures is a significant unmet medical need in most patients with GRIN-related disorders. Although several hundred GRIN mutations have been identified in humans, until recently none of the mouse models carrying Grin mutations/deletions showed an epileptic phenotype. The two recent exceptions both carry mutations of GluN2A. The aim of this study was to assess the efficacy of radiprodil, a selective negative allosteric modulator of GluN2B-containing NMDARs, in counteracting audiogenic seizures (AGS) in a murine model carrying the GluN2A(N615S) homozygous mutation (Grin2aS/S mice). EXPERIMENTAL APPROACH: Grin2aS/S mice were acutely treated with radiprodil at different doses before the presentation of a high-frequency acoustic stimulus commonly used for AGS induction. KEY RESULTS: Radiprodil significantly and dose-dependently reduced the onset and severity of AGS in Grin2aS/S mice. Surprisingly, the results revealed a sex-dependent difference in AGS susceptibility and in the dose-dependent protection of radiprodil in the two genders. Specifically, radiprodil was more effective in female versus male mice. CONCLUSION AND IMPLICATIONS: Overall, our data clearly show that radiprodil, a GluN2B selective negative allosteric modulator, may have the potential to control seizures in patients with GRIN2A GoF mutations. Further studies are warranted to better understand the sex-dependent effects observed in this study.

A Longitudinal Dose-Response Curve Between Leisure-Time Physical Activity and the Prevalence of Diabetes Based on the Different Levels of Cognitive Function Among Older Adults.

This study investigated a dose-response relationship between Leisure-Time Physical Activity participation (LTPA) and the risk of diabetes and a comparison of the risk across different cognitive function groups among older adults. The Health and Retirement Study data were used from 2012 to 2020 (n = 18 746). This study conducted a Cox Proportional Hazard Regression to investigate the Dose-Response Curve between the prevalence of diabetes and the covariates following a level of LTPA participation. The result presented that the Odds Ratio continuously decreased as the level of LTPA participation increased. Among the three cognitive function groups, the high group (OR = .43, P < .05) and the mid group (OR = .71, P < .05) had a larger negative slope coefficient than the low group. This study found that LTPA participation reduces the risk of diabetes and gives evidence for the importance of cognitive function in reducing the prevalence of diabetes.

Tolerance to mild shading levels in cattail as related to increased photosynthesis and changes in its leaf area and anatomy.

Shading is an environmental factor that has been little investigated regarding its effects on emergent aquatic plants. Typha domingensis Pers. is an emergent macrophyte that demonstrates some plasticity for self-shading, and as it can shade other species in the same area, the effect of shading on its traits deserves further investigation. The objective of the present study was to evaluate the gas exchange, leaf anatomy, and growth of T. domingensis cultivated under increasing shading intensities. The plants were collected and propagated in a greenhouse, and the clones were subjected to four shading intensities: 0% (unshaded), 35%, 73%, and 83% shading created by black nets. Growth traits, clonal production, photosynthesis, transpiration, and leaf anatomy were evaluated. The 73% and 83% shading promoted the death of all plants, but all plants survived in the 35% and unshaded treatments. Compared with the unshaded treatment, the 35% shading treatment promoted a higher photosynthetic rate and greater transpiration, supporting increased growth and production of clones. The increase in the photosynthetic rate in the 35% shading was related to the increase in leaf area which increased the photosynthesis of the whole plant. The 73% and 83% treatments inhibited the development of photosynthetic parenchyma and stomata in T. domingensis, leading to a drastic reduction in photosynthesis and energy depletion. Therefore, T. domingensis does not tolerate intense shading, but its photosynthetic characteristics and growth are favored by mild shading, a factor that may be of great importance for its competitiveness and invasive behavior.

Effects of Rainfall Exclusion Treatment on Photosynthetic Characteristics of Black Locust in the Sub-Humid Region of the Loess Plateau, China.

The mesic-origin species Robinia pseudoacacia L. (black locust) is widely planted in the semiarid and sub-humid areas of the Loess Plateau for the reforestation of vegetation-degraded land. Under the scenario of changing precipitation patterns, exploring the response of photosynthesis to drought allows us to assess the risk to sustainable development of these plantations. In this study, paired plots were established including the control and a treatment of 30% exclusion of throughfall (since 2018). The photosynthetic characteristics were investigated using a portable photosynthesis system for four periods in the full-leaf growing season of 2021-2022, the fourth and fifth years, on both treated and controlled sampling trees. Leaf gas exchange parameters derived from diurnal changing patterns, light response curves, and CO2 response curves showed significant differences except for period II (9-11 September 2021) between the two plots. The photosynthetic midday depression was observed in 2022 in the treated plot. Meanwhile, the decline of net photosynthetic rate in the treated plot was converted from stomatal limitation to non-stomatal limitation. Furthermore, we observed that black locust adapted to long-term water deficiency by reducing stomatal conductance, increasing water use efficiency and intrinsic water use efficiency. The results demonstrate that reduction in precipitation would cause photosynthesis decrease, weaken the response sensitivity to light and CO2, and potentially impair photosynthetic resilience of the plantations. They also provide insights into the changes in photosynthetic functions under global climate change and a reference for management of plantations.

More N fertilizer, more maize, and less alfalfa: maize benefits from its higher N uptake per unit root length.

Root plasticity is fundamental to soil nutrient acquisition and maximizing production. Different soil nitrogen (N) levels affect root development, aboveground dry matter accumulation, and N uptake. This phenotypic plasticity is well documented for single plants and specific monocultures but is much less understood in intercrops in which species compete for the available nutrients. Consequently, the study tested whether the plasticity of plant roots, biomass and N accumulation under different N levels in maize/alfalfa intercropping systems differs quantitatively. Maize and alfalfa were intercropped for two consecutive years in large soil-filled rhizoboxes and fertilized with 6 different levels of N fertilizer (0, 75, 150, 225, 270, and 300 kg ha-1). Root length, root surface area, specific root length, N uptake and yield were all increased in maize with increasing fertilizer level, whereas higher N rates were supraoptimal. Alfalfa had an optimal N rate of 75-150 kg ha-1, likely because the competition from maize became more severe at higher rates. Maize responded more strongly to the fertilizer treatment in the second year when the alfalfa biomass was much larger. N fertilization contributes more to maize than alfalfa growth via root plasticity responses. Our results suggest that farmers can maximize intercropping yield and economic return by optimizing N fertilizer management.

Changes in Photosynthetic Characteristics between Green-Leaf Poplar Linn. "2025" and Its Bud-Sporting Colored-Leaf Cultivars.

Colored-leaf poplar is increasingly popular due to its great ornamental values and application prospects. However, the photosynthetic characteristics of these colored-leaf cultivars have not been well understood. In this study, the photosynthetic differences between green-leaf poplar Populus deltoids Linn. "2025" (L2025) and colored-leaf cultivars 'Zhonghong poplar' (ZHP), 'Quanhong poplar' (QHP), and 'Caihong poplar' (CHP) were investigated on several levels, including chloroplast ultrastructure observation, photosynthetic physiological characteristics, and expression analysis of key genes. The results showed that the photosynthetic performance of ZHP was basically consistent with that of L2025, while the ranges of light energy absorption and efficiency of light energy utilization decreased to different degrees in CHP and QHP. A relatively low water use efficiency and high dark respiration rate were observed in QHP, suggesting a relatively weak environmental adaptability. The differences in chloroplast structure in different colored-leaf poplars were further observed by transmission electron microscopy. The disorganization of thylakoid in CHP was considered an important reason, resulting in a significant decrease in chlorophyll content compared with other poplar cultivars. Interestingly, CHP exhibited extremely high photosynthetic electron transport activity and photochemical efficiency, which were conductive to maintaining its relatively high photosynthetic performance. The actual quantum yield of PSII photochemistry of ZHP was basically the same as that of QHP, while the relatively high photosynthetic performance indexes in ZHP suggested a more optimized photosynthetic apparatus, which was crucial for the improvement of photosynthetic efficiency. The differential expressions of a series of key genes in different colored-leaf poplars provided a reasonable explanation for anthocyanin accumulation and specific photosynthetic processes.

Weak and strong phase response curves of the onion fly circadian clock at temperature changes of 1 °C and 4 °C.

With increasing soil depth, the amplitude and phase of the daily temperature cycle decreases and is delayed, respectively. The onion fly, Delia antiqua, which pupates at a soil depth of 2-20 cm, advances the eclosion phase of its circadian clock as the temperature amplitude decreases. This "temperature-amplitude response" compensates for the depth-dependent phase delay of the temperature change and ensures eclosion in the early morning. To clarify the physiological mechanisms that induce a temperature-amplitude response, we performed phase-resetting experiments using a 12-h high- or low-temperature pulse with an amplitude of 1 °C or 4 °C. Based on the results obtained, four phase transition curves and four phase response curves were constructed. These curves show that the phase of the eclosion clock shifted more as the magnitude of the temperature change increased. The 24-h temperature cycle delayed, rather than advanced, the phase of the D. antiqua circadian eclosion rhythm. Therefore, we propose that a small phase delay is caused by a small temperature amplitude at a deep site in the soil and a large phase delay is caused by a large temperature amplitude at a shallow site, leading to the temperature-amplitude response exhibited by D. antiqua.