Enhancement of Hounsfield unit distribution in cone-beam CT images for adaptive radiation therapy: Evaluation of a hybrid correction approach

PurposeThis study aims to evaluate the accuracy of a hybrid approach combining the histogram matching (HM) and the multilevel threshold (MLT) to correct the Hounsfield Unit (HU) distribution in cone-beam CT (CBCT) images.Methods and MaterialsCBCT images acquired for ten prostate cancer patients were processed by matching their histograms to those of deformed planning CT (pCT) images obtained after applying a deformable registration (DR) process. Then, HU values corresponding to five tissue types in the pCT were assigned to the obtained CBCT images (CBCT_HM-MLT). Finally, the CBCT_HM-MLT images were compared to the deformed pCT visually and using different statistical metrics.ResultsThe visual assessment and the profiles comparison showed that the high discrepancies in the CBCT images were significantly reduced when using the proposed approach. Furthermore, the correlation values indicated that the CBCT_HM-MLT were in good agreement with the deformed pCT with correlation values ranging from 0.9893 to 0.9962. In addition, the root mean squared error (RMSE) over the entire volume was reduced from 64.15 ± 9.50 to 51.20 ± 6.76 HU. Similarly, the mean absolute error in specific tissue classes was significantly reduced especially in the soft tissue-air interfaces. These results confirmed that applying MLT after HM worked better than using only HM for which the correlation values were ranging from 0.9878 to 0.9955 and the RMSE was 55.95 ± 10.43 HU.ConclusionEvaluation of the proposed approach showed that the HM + MLT correction can improve the HU distribution in the CBCT images and generate corrected images in good agreement with the pCT.     

Accuracy of target delineation by positron emission tomography-based auto-segmentation methods after deformable image registration: A phantom study

PurposeDiagnostic positron emission tomography and computed tomography (PET/CT) images can be fused to the planning CT images by a deformable image registration (DIR). The aim of this study was to evaluate the standardized uptake value (SUV) and target delineation on deformed PET images.MethodsWe used a cylindrical phantom and removable inserts of four spheres (16–38 mm in diameter) and three ellipsoids with a volume equal to the 38-mm-diameter sphere (S38) in each. S38 was filled with 18F-fluorodeoxyglucose activity, and then PET/CT images were acquired. The contours of S38 were generated using original PET images by PET auto-segmentation (PET-AS) methods of (1) SUV2.5, (2) 40% of maximum SUV (SUV40%max), and (3) gradient-based (GB), and were deformed to the other inserts by DIR. We compared the volumes and the SUV_max with the generated contours using the deformed PET images.ResultsThe SUV_max was slightly decreased by DIR; the mean absolute difference was −0.10 ± 0.04. For SUV2.5 and SUV40%max, the differences in S38 volumes between the original and deformed PET images were less than 5%, regardless of deformation type. For the GB, the contoured volumes obtained from deformed PET images were larger than those of the original PET images for the deformation type of ellipsoids. When the S38 was deformed to the 16-mm-diameter sphere, the maximum volume difference was −22.8%.ConclusionsAlthough SUV fluctuations by DIR were negligible, the target delineation on deformed PET images by the GB should be carefully considered owing to the distortion of intensity profiles.     

A novel tool for assessing the correlation of internal/external markers during SGRT guided stereotactic ablative radiotherapy treatments

IntroductionAn in-house developed tool was implemented and validated to investigate the skin surface, hepatic dome, and target displacement for stereotactic ablative radiotherapy (SABR) of thoracic/abdominal lesions using a Surface Guided Radiation Therapy (SGRT) system combined with 4D- images.Materials and methodsFourteen consecutive patients with tumors near the hepatic dome undergoing SABR treatments were analyzed. For each patient, a planning 4D-CT and five 4D-CBCT images were acquired. The C-RAD technology was also used to register/monitor the position of the skin reference point (SRP) as an external marker representative of patient breathing. The 4D images were imported in the developed tool, and the absolute maximum height (P_max,dome) of the hepatic dome on the ten respiratory phases was semi-automatically detected. Similarly, the contour of the skin surface was extracted in correspondence with the SRP position. The tool has been validated using an ad hoc modified moving phantom with pre-selected amplitudes and numbers of cycles. The Pearson correlation coefficients and Bland-Altman plots were calculated.ResultsThere was a strong correlation between the skin motion amplitude based on 4D-CBCT and the C-RAD in all the patients (0.90 ± 0.08). Similarly, the mean ± SD of Pearson correlation coefficients of skin and P_max,dome movements registered by 4D-CT and 4D-CBCT were 0.90 ± 0.05 and 0.94 ± 0.05, respectively. The mean ± SD of Pearson correlation coefficients comparing the skin and P_max,dome displacements within each imaging modality were 0.88 ± 0.05 and 0.90 ± 0.05 for 4D-CT and 4D-CBCT, respectively. The SRP displacement during the set-up imaging and the treatment delivery were similar in all the investigated patients. Similar results were obtained for the ad hoc modified phantom in the preliminary validation phase.ConclusionThe strong correlation between the tumor/ hepatic dome and skin displacements confirms that the SGRT approach can be considered appropriate for intra- and inter-fraction motion management in SABR therapy.     

A quantitative evaluation of deformable image registration based on MV cone beam CT images: Impact of deformation magnitudes and image modalities

Background and purposeTo evaluate the impact of deformation magnitude and image modality on deformable-image-registration (DIR) accuracy using Halcyon megavoltage cone beam CT images (MVCBCT).Materials and methodsPlanning CT images of an anthropomorphic Head phantom were aligned rigidly with MVCBCT and re-sampled to achieve the same resolution, denoted as pCT. MVCBCT was warped with twenty simulated pre-known virtual deformation fields (T_i, i = 1–20) with increasing deformation magnitudes, yielding warped CBCT (wCBCT). The pCT and MVCBCT were registered to wCBCT respectively (Multi-modality and Uni-modality DIR), generating deformation vector fields V_i and V_i′ (i = 1–20). V_i and V_i′ were compared with T_i respectively to assess the DIR accuracy geometrically. In addition, V_i, T_i, and V_i′ were applied to pCT, generating deformed CT (dCT_i), ground-truth CT (G_i) and deformed CT′ (dCT_i′) respectively. The Hounsfield Unit (HU) on these virtual CT images were also compared.ResultsThe mean errors of vector displacement increased with the deformation magnitude. For deformation magnitudes between 2.82 mm and 7.71 mm, the errors of uni-modality DIR were 1.16 mm ~ 1.73 mm smaller than that of multi-modality (p = 0.0001, Wilcoxon signed rank test). DIR could reduce the maximum signed and absolute HU deviations from 70.8 HU to 11.4 HU and 208 HU to 46.2 HU respectively.ConclusionsAs deformation magnitude increases, DIR accuracy continues to deteriorate and uni-modality DIR consistently outperformed multi-modality DIR. DIR-based adaptive radiotherapy utilizing the noisy MVCBCT images is only conditionally applicable with caution.     

Feasibility of image quality improvement for high-speed CBCT imaging using deep convolutional neural network for image-guided radiotherapy in prostate cancer

PurposeHigh-speed cone-beam computed tomography (CBCT) scan for image-guided radiotherapy (IGRT) can reduce both the scan time and the exposure dose. However, it causes noise and artifacts in the reconstructed images due to the lower number of acquired projection data. The purpose of this study is to improve the image quality of high-speed CBCT using a deep convolutional neural network (DCNN).MethodsCBCT images of 36 prostate cancer patients were selected. The CBCT images acquired at normal scan speed were defined as CBCT_100%. Simulated high-speed CBCT images acquired at twofold and fourfold scan speed were created, which were defined as CBCT_50% and CBCT_25%, respectively. The image quality of the CBCT_50% was treated as the requirement for IGRT in this study because previous studies reported that its image is sufficient with respect to IGRT. The DCNN model was trained to learn direct mapping from CBCT_25% to the corresponding CBCT_100%. The performance of the DCNN model was evaluated using the sixfold cross-validation method. CBCT images generated by DCNN (CBCT_25%+DCNN) were evaluated for voxel value accuracy and image quality.ResultsThe DCNN model can process CBCT_25% of a new patient within 0.06 s/slice. The CBCT_25%+DCNN was comparable to the CBCT_50% in terms of both voxel value accuracy and image quality.ConclusionsWe developed a DCNN model to remove noise and artifacts from high-speed CBCT. We emphasize that it is possible to reduce exposure to one quarter and to increase the CBCT scan speed by a factor of four.     

Evaluation of target volume and dose accuracy in intrafractional cases of lung cancer based on 4D-CT and 4D-CBCT images using an in-house dynamic thorax phantom

BackgroundThis study aimed to evaluate the target volume and dose accuracy in intrafraction cases using 4-dimensional imaging modalities and an in-house dynamic thorax phantom. Intrafraction motion can create errors in the definition of target volumes, which can significantly affect the accuracy of radiation delivery. Motion management using 4-dimensional modalities is required to reduce the risk.Materials and methodsTwo variations in both breathing amplitude and target size were applied in this study. From these variations, internal target volume (ITVs) contoured in 10 phases of 4D-CT (ITV₁₀), average intensity projection (AIP), and mid-ventilation (Mid-V) images were reconstructed from all 4D-CT datasets as reference images. Free-breathing (FB), augmentation free-breathing (Aug-FB), and static images were also acquired using the 3D-CT protocol for comparisons. In dose evaluations, the 4D-CBCT modality was applied before irradiation to obtain position correction. Then, the dose was evaluated with Gafchromic film EBT3.ResultsThe ITV₁₀, AIP, and Mid-V provide GTVs that match the static GTV. The AIP and Mid-V reference images allowed reductions in ITVs and PTVs without reducing the range of target movement areas compared to FB and Aug-FB images with varying percentages in the range of 29.17% to 48.70%. In the dose evaluation, the largest discrepancies between the measured and planned doses were 10.39% for the FB images and 9.21% for the Aug-FB images.ConclusionThe 4D-CT modality can enable accurate definition of the target volume and reduce the PTV. Furthermore, 4D-CBCT provides localization images during registration to facilitate position correction and accurate dose delivery.     

A new strategy for craniospinal axis localization and adaptive dosimetric evaluation using cone beam CT

Background and AimComputational complexities encountered in craniospinal irradiation (CSI) have been widely investigated with different planning strategies. However, localization of the entire craniospinal axis (CSA) and evaluation of adaptive treatment plans have traditionally been ignored in CSI treatment. In this study, a new strategy for CSI with comprehensive CSA localization and adaptive plan evaluation has been demonstrated using cone beam CT with extended longitudinal field-of-view (CBCT_eLFOV).Materials and MethodsMulti-scan CBCT images were acquired with fixed longitudinal table translations (with 1 cm cone-beam overlap) and then fused into a single DICOM-set using the custom software coded in MatLab™. A novel approach for validation of CBCT_eLFOV was demonstrated by combined geometry of Catphan-504 and Catphan-604 phantoms. To simulate actual treatment scenarios, at first, the end-to-end workflow of CSI with VMAT was investigated using an anthropomorphic phantom and then applied for two patients (based on random selection).ResultsThe fused CBCT_eLFOV images were in excellent agreement with planning CT (pCT). The custom developed software effectively manages spatial misalignments arising out of the uncertainties in treatment/setup geometry. Although the structures mapped from pCT to CBCT_eLFOV showed minimal variations, a maximum spatial displacement of up to 1.2 cm (and the mean of 0.8 ± 0.3 cm) was recorded in phantom study. Adaptive plan evaluation of patient paradigms showed the likelihood of under-dosing the craniospinal target.ConclusionOur protocol serves as a guide for precise localization of entire CSA and to ensure adequate dose to the large and complex targets. It can also be adapted for other complex treatment techniques such as total-marrow-irradiation and total-lymphoid-irradiation.     

Dose warping performance in deformable image registration in lung

PurposeIt is unclear that spatial accuracy can reflect the impact of deformed dose distribution. In this study, we used dosimetric parameters to compare an in-house deformable image registration (DIR) system using NiftyReg, with two commercially available systems, MIM Maestro (MIM) and Velocity AI (Velocity).MethodsFor 19 non-small-cell lung cancer patients, the peak inspiration (0%)-4DCT images were deformed to the peak expiration (50%)-4DCT images using each of the three DIR systems, which included computation of the deformation vector fields (DVF). The 0%-gross tumor volume (GTV) and the 0%-dose distribution were also then deformed using the DVFs. The agreement in the dose distributions for the GTVs was evaluated using generalized equivalent uniform dose (gEUD), mean dose (D_mean), and three-dimensional (3D) gamma index (criteria: 3 mm/3%). Additionally, a Dice similarity coefficient (DSC) was used to measure the similarity of the GTV volumes.ResultsD_mean and gEUD demonstrated good agreement between the original and deformed dose distributions (differences were generally less than 3%) in 17 of the patients. In two other patients, the Velocity system resulted in differences in gEUD of 50.1% and 29.7% and in D_mean of 11.8% and 4.78%. The gamma index comparison showed statistically significant differences for the in-house DIR vs. MIM, and MIM vs. Velocity.ConclusionsThe finely tuned in-house DIR system could achieve similar spatial and dose accuracy to the commercial systems. Care must be taken, as we found errors of more than 5% for D_mean and 30% for gEUD, even with a commercially available DIR tool.     

Evaluation of the Block Matching deformable registration algorithm in the field of head-and-neck adaptive radiotherapy

Background and purposeTo compare the accuracy of the Block Matching deformable registration (DIR) against rigid image registration (RIR) for head-and-neck multi-modal images CT to cone-beam CT (CBCT) registration.Material and methodsPlanning-CT and weekly CBCT of 10 patients were used for this study. Several volumes, including medullary canal (MC), thyroid cartilage (TC), hyoid bone (HB) and submandibular gland (SMG) were transposed from CT to CBCT images using either DIR or RIR. Transposed volumes were compared with the manual delineation of these volumes on every CBCT. The parameters of similarity used for analysis were: Dice Similarity Index (DSI), 95%-Hausdorff Distance (95%-HD) and difference of volumes (cc).ResultsWith DIR, the major mean difference of volumes was −1.4 cc for MC, revealing limited under-segmentation. DIR limited variability of DSI and 95%-HD. It significantly improved DSI for TC and HB and 95%-HD for all structures but SMG. With DIR, mean 95%-HD (mm) was 3.01 ± 0.80, 5.33 ± 2.51, 4.99 ± 1.69, 3.07 ± 1.31 for MC, TC, HB and SMG, respectively. With RIR, it was 3.92 ± 1.86, 6.94 ± 3.98, 6.44 ± 3.37 and 3.41 ± 2.25, respectively.ConclusionBlock Matching is a valid algorithm for deformable multi-modal CT to CBCT registration. Values of 95%-HD are useful for ongoing development of its application to the cumulative dose calculation.     

Development of a dynamic deformable thorax phantom for the quality management of deformable image registration

The purpose of this study was to develop a novel dynamic deformable thorax phantom for deformable image registration (DIR) quality assurance (QA) and to verify as a tool for commissioning and DIR QA.The phantom consists of a base phantom, an inner phantom, and a motor-derived piston. The base phantom is an acrylic cylinder phantom with a diameter of 180 mm. The inner phantom consists of deformable, 20 mm thick disk-shaped sponges. To evaluate the physical characteristics of the phantom, we evaluated its image quality and deformation. DIR accuracies were evaluated using the three types of commercially DIR software (MIM, RayStation, and Velocity AI) to test the feasibility of this phantom. We used different DIR parameters to test the impact of parameters on DIR accuracy in various phantom settings. To evaluate DIR accuracy, a target registration error (TRE) was calculated using the anatomical landmark points.The three locations (i.e., distal, middle, and proximal positions) had different displacement amounts. This result indicated that the inner phantom was not moved but deformed. In cases with different phantom settings and marker settings, the ranges of the average TRE were 0.63–15.60 mm (MIM). In cases with different DIR parameters settings, the ranges of the average TRE were as follows: 0.73–7.10 mm (MIM), 8.25–8.66 mm (RayStation), and 8.26–8.43 mm (Velocity). These results suggest that our phantom could evaluate the detailed DIR behaviors with TRE. Therefore, this is indicative of the potential usefulness of our phantom in DIR commissioning and QA.     

Divergence of thermal physiological traits in terrestrial breeding frogs along a tropical elevational gradient

Critical thermal limits are thought to be correlated with the elevational distribution of species living in tropical montane regions, but with upper limits being relatively invariant compared to lower limits. To test this hypothesis, we examined the variation of thermal physiological traits in a group of terrestrial breeding frogs (Craugastoridae) distributed along a tropical elevational gradient. We measured the critical thermal maximum (CT_max; n = 22 species) and critical thermal minimum (CT_min; n = 14 species) of frogs captured between the Amazon floodplain (250 m asl) and the high Andes (3,800 m asl). After inferring a multilocus species tree, we conducted a phylogenetically informed test of whether body size, body mass, and elevation contributed to the observed variation in CT_max and CT_min along the gradient. We also tested whether CT_max and CT_min exhibit different rates of change given that critical thermal limits (and their plasticity) may have evolved differently in response to different temperature constraints along the gradient. Variation of critical thermal traits was significantly correlated with species’ elevational midpoint, their maximum and minimum elevations, as well as the maximum air temperature and the maximum operative temperature as measured across this gradient. Both thermal limits showed substantial variation, but CT_min exhibited relatively faster rates of change than CT_max, as observed in other taxa. Nonetheless, our findings call for caution in assuming inflexibility of upper thermal limits and underscore the value of collecting additional empirical data on species’ thermal physiology across elevational gradients.     

Commissioning and validation of commercial deformable image registration software for adaptive contouring

PurposeTo report the commissioning and validation of deformable image registration(DIR) software for adaptive contouring.MethodsDIR (SmartAdapt®v13.6) was validated using two methods namely contour propagation accuracy and landmark tracking, using physical phantoms and clinical images of various disease sites. Five in-house made phantoms with various known deformations and a set of 10 virtual phantoms were used. Displacement in lateral, anterio-posterior (AP) and superior-inferior (SI) direction were evaluated for various organs and compared with the ground truth. Four clinical sites namely, brain (n = 5), HN (n = 9), cervix (n = 18) and prostate (n = 23) were used. Organs were manually delineated by a radiation oncologist, compared with the deformable image registration (DIR) generated contours. 3D slicer v4.5.0.1 was used to analyze Dice Similarity Co-efficient (DSC), shift in centre of mass (COM) and Hausdorff distances Hf_95%/avg.ResultsMean (SD) DSC, Hf_95% (mm), Hf_avg (mm) and COM of all the phantoms 1–5 were 0.84 (0.2) mm, 5.1 (7.4) mm, 1.6 (2.2) mm, and 1.6 (0.2) mm respectively. Phantom-5 had the largest deformation as compared to phantoms 1–4, and hence had suboptimal indices. The virtual phantom resulted in consistent results for all the ROIs investigated. Contours propagated for brain patients were better with a high DSC score (0.91 (0.04)) as compared to other sites (HN: 0.84, prostate: 0.81 and cervix 0.77). A similar trend was seen in other indices too. The accuracy of propagated contours is limited for complex deformations that include large volume and shape change of bladder and rectum respectively. Visual validation of the propagated contours is recommended for clinical implementation.ConclusionThe DIR algorithm was commissioned and validated for adaptive contouring.     

Critical thermal maxima of juvenile alligator gar (Atractosteus spatula,Lacépède, 1803) from three Mississippi‐drainage populations acclimated to three temperatures

Local adaptation may cause thermal tolerance to vary between nearby but distinct populations of a species. During the summer of 2013, alligator gar Atractosteus spatula spawned from broodstock collected from three populations within the Mississippi River drainage separated by a 5° latitudinal gradient were acclimated to three temperatures (25, 30, and 35°C). Ten fish from each population were acclimated at each temperature. CT_Max was determined at each temperature for each population, using five fish for each population‐acclimation temperature pairing. CT_Max for each population‐acclimation temperature pairing was compared using two‐factor anova . CT_Max increased significantly with acclimation temperature (F_2,40 = 600.5, P < 0.001) but population had no significant effect (F_2,40 = 1.882, P = 0.166). Temperature tolerance appears to be consistent across populations of alligator gar, with no evidence of local adaptation.     

Effects of autonomic blockade on acute thermal tolerance and cardioventilatory performance in rainbow trout,Oncorhynchus mykiss

Predicted future increases in global temperature may impose challenges for ectothermic animals like fish, but the physiological mechanisms determining the critical thermal maximum (CT_max) are not well understood. One hypothesis suggests that impaired cardiac performance, limited by oxygen supply, is an important underlying mechanism. Since vagal bradycardia is suggested to improve cardiac oxygenation and adrenergic stimulation may improve cardiac contractility and protect cardiac function at high temperatures, we predicted that pharmacological blockade of cardiac autonomic control would lower CT_max. Rainbow trout was instrumented with a flow probe and a ventilation catheter for cardioventilatory recordings and exposed to an acute thermal challenge until CT_max following selective pharmacological blockade of muscarinic or β-adrenergic receptors.Contrary to our prediction, CT_max (~26 °C) was unchanged between treatments. While β-adrenergic blockade reduced heart rate it did not impair cardiac stroke volume across temperatures suggesting that compensatory increases in cardiac filling pressure may serve to maintain cardiac output. While warming resulted in significant tachycardia and increased cardiac output, a high cholinergic tone on the heart was observed at temperatures approaching CT_max. This may represent a mechanism to maintain scope for heart rate and possibly to improve myocardial contractility and oxygen supply at high temperatures. This is the first study evaluating the importance of autonomic cardiac control on thermal tolerance in fish. While no effects on CT_max were observed, this study raises important questions about the underlying mechanisms determining thermal tolerance limits in ectothermic animals.     

Free-to-use DIR solutions in radiotherapy: Benchmark against commercial platforms through a contour-propagation study

PurposeA contour propagation study has been conducted to benchmark three algorithms for Deformable Image Registration (DIR) freely available online against well-established commercial solutions.MethodsElastiX, BRAINS and Plastimach, available as modules in the open source platform 3DSlicer, were tested as the recent AAPM Task group 132 guidelines proposes. The overlap of the DIR-mapped ROIs in four computational anthropomorphic phantoms was measured. To avoid bias every algorithm was left to run without any human interaction nor particular registration strategy. The accuracy of the algorithms was measured using the Dice Similarity Coefficient (DSC) and Mean Distance to Conformity (MDC) metrics. The registration quality was compared to the recommended geometrical accuracy suggested by AAPM TG132 and to the results of a large population-based study performed with commercial DIR solutions.ResultsThe considered free-to-use DIR solutions generally meet acceptable accuracy and good overlap (DSC > 0.85). Mild failures (DSC < 0.75) were detected only for the smallest structures. In case of extremely severe deformations acceptable accuracy was not met (MDC > 3 mm). The morphing capability of the tested algorithms equals those of commercial systems when the user interaction is avoided. Underperformances were detected only in cases where a specific registration strategy is mandatory to obtain a satisfying match.ConclusionsAll of the considered algorithms show performances not inferior to previously published data and have the potential to be good candidates for use in the clinical routine. The results and conclusions only apply to the considered phantoms and should not be considered to be generally applicable and extendable to patient cases.     

Fluctuating environments impact thermal tolerance in an invasive insect species Bactrocera dorsalis (Diptera: Tephritidae)

The incidence and severity of environmental stressors associated with global climate change are increasing and insects frequently face variability in temperature and moisture regimes at variable spatio-temporal scales. Coincidental with this, is increased thermal and hydric stress on insects as warming increases vapour pressure deficit (VPD), the drying power of the air. While the effects of mean temperatures on fitness are widely documented, fluctuations in both temperature and relative humidity (RH) are largely unexplored. Here, we investigated the effects of dynamic temperature and RH fluctuations (around the mean [28°C; 65% RH]) on low and high thermal tolerance of laboratory-reared adult invasive Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), measured as critical thermal minima (CT_min), critical thermal maxima (CT_max), chill coma recovery time (CCRT) and heat knockdown time (HKDT). Our results show that increased environmental amplitude significantly influenced low and high temperature responses and varied across traits tested. The highest amplitude (δ12°C; 28% RH) compromised CT_min, CCRT and HKDT traits while enhancing CT_max. Similarly, acclimation to δ3°C; 7% RH compromised both low (CT_min and CCRT) and high (CT_max and HKDT) fitness traits. Variations in fitness reported here indicate significant roles of combined thermal and moisture fluctuations on B. dorsalis fitness suggesting caveats that are worthy considering when predicting species responses to climate change. These results are significant for B. dorsalis population phenology, management, quantifying vulnerability to climate variability and may help modelling future biogeographical patterns.     

Effect of dietary supplementation of l-tryptophan on thermal tolerance and oxygen consumption rate in Cirrhinus mrigala fingerlings under varied stocking density

A 60 day feeding trial was conducted to study the effect of dietary l-tryptophan on thermal tolerance and oxygen consumption rate of freshwater fish, mrigala, Cirrhinus mrigala reared under ambient temperature at low and high stocking density. Four hundred eighty fingerlings were distributed into eight experimental groups. Four groups each of low density group (10 fishes/75 L water) and higher density group (30 fishes/75 L water) were fed a diet containing 0, 0.68, 1.36 or 2.72% l-tryptophan in the diet, thus forming eight experimental groups namely, Low density control (LC) (basal feed +0% l-tryptophan); LT₁ (basal feed+0.68% l-tryptophan); LT₂ (basal feed+1.36% l-tryptophan); LT₃ (basal feed+2.72% l-tryptophan); high density control (HC) (basal feed+0% l-tryptophan); HT₁ (basal feed+0.68% l-tryptophan); HT₂ (basal feed+1.36% l-tryptophan); and HT₃ (basal feed+2.72% l-tryptophan) were fed at 3% of the body weight. The test diets having crude protein 34.33±0.23 to 35.81±0.18% and lipid 423.49±1.76 to 425.85±0.31 K Cal/100 g were prepared using purified ingredients. The possible role of dietary l-tryptophan on thermal tolerance and oxygen consumption rate was assessed in terms of critical thermal maxima (CT_Max), critical thermal minima (CT_Min), lethal thermal maxima (LT_Max) and lethal thermal minima (LT_Min). The CT_Max, CT_Min, LT_Max and LT_Min values were found to be significantly higher (p<0.05) in the treatment groups with CT_Max 42.94±0.037 (LT₂); LT _Max 43.18±0.070 (LT₂); CT_Min 10.47±0.088 (LT₂) and LT_Min 9.42±0.062 (LT₃), whereas the control group showed a lower tolerance level. The same trend was observed in the high density group (CT_Max 42.09±0.066 (LT₃); LT_Max 4₃ 23±0.067 (HT₃); CT_Min 10.98±0.040 (HT₃) and LT_Min 9.74±0.037 (HT₃). However, gradual supplementation of dietary l-tryptophan in the diet significantly reduced the oxygen consumption rate in both the low density group (Y=−26.74x+222.4, r²=0.915) and the high density group (Y=−32.96x+296.5, r²=0.8923). Dietary supplementation of l-tryptophan at a level of 1.36% improved the thermal tolerance level and reduced the oxygen consumption rate in C. mrigala fingerlings.     

The cost of ventilation in birds measured via unidirectional artificial ventilation

The highly derived mechanism birds use to ventilate their lungs relies on dorsoventral excursions of their heavily muscled sternum and abdominal viscera. Our expectation of the level of mechanical work involved in this mechanism led us to hypothesize that the metabolic cost of breathing is higher in birds than in other tetrapods. To test this theory, we used unidirectional artificial ventilation (UDV) to stop normal ventilatory movements in guinea fowl (Numida meleagris L.) at rest and during treadmill locomotion at three speeds. Oxygen consumption was measured during normal breathing and UDV, and the difference was used to approximate the cost of ventilation. Contrary to our prediction, metabolism increased when ventilatory movements ceased during UDV at rest. Although we do not understand why this occurred we suspect that UDV induced a homeostatic mechanism to counteract the loss of carbon dioxide. Nevertheless, across all running speeds, metabolism decreased significantly with UDV, indicating a minimum cost of ventilation during running of 1.43 ± 0.62% of total running metabolism or 0.48 ± 0.21 mL O₂ (L ventilated)^? 1. These results suggest that the metabolic cost of ventilation is low in birds and that it is within the range of costs reported previously for other amniotes.     

Thermal adaptation generates a diversity of thermal limits in a rainforest ant community

The Thermal Adaptation Hypothesis posits that the warmer, aseasonal tropics generates populations with higher and narrower thermal limits. It has largely been tested among populations across latitudes. However, considerable thermal heterogeneity exists within ecosystems: across 31 trees in a Panama rainforest, surfaces exposed to sun were 8 °C warmer and varied more in temperature than surfaces in the litter below. Tiny ectotherms are confined to surfaces and are variously submerged in these superheated boundary layer environments. We quantified the surface CT_min and CT_maxs (surface temperatures at which individuals grew torpid and lost motor control, respectively) of 88 ant species from this forest; they ranged in average mass from 0.01 to 57 mg. Larger ants had broader thermal tolerances. Then, for 26 of these species we again tested body CT_maxs using a thermal dry bath to eliminate boundary layer effects: body size correlations observed previously disappeared. In both experiments, consistent with Thermal Adaptation, CT_maxs of canopy ants averaged 3.5–5 °C higher than populations that nested in the shade of the understory. We impaled thermocouples in taxidermy mounts to further quantify the factors shaping operative temperatures for four ant species representing the top third (1–30 mg) of the size distribution. Extrapolations suggest the smallest 2/3rds of species reach thermal equilibrium in <10s. Moreover, the large ants that walk above the convective superheated surface air also showed more net heating by solar radiation, with operative temperatures up to 4 °C higher than surrounding air. The thermal environments of this Panama rainforest generate a range of CT_max subsuming 74% of those previously recorded for ant populations worldwide. The Thermal Adaptation Hypothesis can be a powerful tool in predicting diversity of thermal limits within communities. Boundary layer temperatures are likely key to predicting the future of Earth's tiny terrestrial ectotherm populations.