Computational protocol: Concerted evolution of body mass and cell size: similar patterns among species of birds (Galliformes) and mammals (Rodentia)

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Protocol publication

[…] Prior to dissection, animals were deprived of food for at least 12 h and then weighed to the nearest 0.01 g (small rodents), 0.1 g (birds), or 1 g (rats and coypu). We took blood samples from each animal with heparinized glass capillary tubes (Medlab, Raszyn, Poland) to prepare blood smears. For Galliformes, blood was taken from the brachial vein, and in rodents, from the caudal vein or jugular vein (only coypus). Blood smears were dried and fixed with methanol (Avantor Performance Materials Poland S. A, Gliwice, Poland) and then stained with Gill's III Hematoxylin (Merck, Darmstadt, Germany) and a 1% ethanol solution of Eosin Y (hereafter, 1% Eosin Y; Analab, Warszawa, Poland) for birds or with 1% Eosin Y for mammals.After removing feathers or shaving the coat, we collected a skin sample from between the scapulae along the dorsum. We dissected out the middle part of the trachea, the central part of the right lobe of the liver, the descending part of the duodenum and the whole right kidney. The tissue samples were fixed in a 10% buffered solution of formaldehyde (Bio Optica, Milano, Italy). Then, they were dehydrated in ethanol (Linegal Chemicals, Warszawa, Poland), cleared in ST Ultra (Leica, Wetzlar, Germany) and embedded in Paraplast Plus (Leica). Serial sections (4 µm thick) were cut with a motorized rotary microtome (Hyrax M55; Zeiss, Oberkochen, Germany). Slides were stained with Gill's III Hematoxylin and 1% Eosin Y and mounted with CV Ultra (Leica).Erythrocytes in blood smears, tracheal chondrocytes, hepatocytes and duodenal enterocytes were photographed at a resolution of 0.033 µm per pixel under a light microscope (Eclipse 80i; Nikon, Tokyo, Japan) equipped with a camera (Digital Sight, Nikon) and Lucia Measurement image acquisition software (Lim Laboratory Imaging, Praha, Czech Republic) using a 100×-magnification oil immersion objective. Cells from kidney proximal tubules and epithelial skin cells were photographed at a resolution of 1 µm per pixel using a 40×-magnification objective on an automatized light microscope (BX 51 VS; Olympus, Tokyo, Japan) equipped with a digital camera (XC10, Olympus) and dotSlide (Olympus) image acquisition software. The use of two microscopic systems, including one that was automated, helped to expedite the digitization of microscopic slides and did not bias our results because we consistently used the same system to analyze a given tissue type in all animals.We used image analysis software to measure cells: ImageJ from the National Institutes of Health (USA) for JPEG images from the Nikon camera and cellSens from Olympus for a specialized image format obtained from the Olympus camera. We outlined 60 randomly chosen erythrocytes per animal and calculated their areas (µm2), which was our measure of erythrocyte size. For birds, we used the same method to measure the areas of erythrocyte nuclei. We outlined randomly chosen lacunae in chondrocytes and calculated their areas (µm2), which was used as a measure of chondrocyte size. If chondrocytes occurred in isogenic groups, we measured one chondrocyte per group. Cell borders in the remaining tissues were often not clearly visible. Following the methods developed by and , ), we measured the areas of cell groups in tissue samples from the liver, duodenum and kidney (µm2) and the lengths of longitudinal transects of cell groups in skin (µm) samples. After counting the nuclei within the measured areas or along each transect, we calculated the average cell size by dividing the area or transect length by the number of nuclei. To outline areas for measurement, demarcation lines between hepatocytes in liver samples were drawn equidistance between neighboring nuclei. In duodenum samples, we considered only enterocytes in the epithelial mucous membrane in the middle part of villi. We used the basement membrane and the apical surfaces of cells as the lower and upper borders of layers, respectively, and two cell nuclei at two ends of the layer as the beginning and the end of the layer. In kidney samples, we outlined the cross-sectional areas of proximal tubules (without the lumen). In skin samples, we considered epithelial cells of the basal layer, which formed longitudinal transects. The ends of a transect were defined by two nuclei, one at each end of the linear transect of nuclei. We measured the following number of cells per individual ‒ birds: 52-71 in trachea, 88-274 in livers, 46-257 in duodenums, 332-521 in kidneys and 2-102 in skin; mammals: 37-89 in trachea, 70-238 in livers, 37-295 in duodenums, 9-600 in kidneys, 13-166 in skin. Finally, we calculated the average size of each cell type (and the mean size of nuclei in bird erythrocytes) for each animal. Additionally, we calculated mean karyoplasmic ratios for erythrocytes in each bird.Information about C-values, where C-value is the amount of DNA in a haploid cell (pg), in the studied species was obtained from an online database (). If more than one estimate of the C-value was available for a species, we calculated the mean C-value. We found data on C-value in two species of birds and four species of mammals. Therefore, the data for birds were used only for descriptive purposes, whereas the data for mammals were analyzed statistically. […]

Pipeline specifications

Software tools ImageJ, cellSens
Application Microscopic phenotype analysis
Chemicals Oxygen