Epidermal club cell densities in fathead minnows: assessment of method and application to a case study
Data files
Jan 16, 2024 version files 22.23 KB
-
data_for_dryad.xlsx
-
README.md
Abstract
Abstract
Many fishes possess epidermal club cells that are the presumptive source of chemical alarm cues and also play a role in innate immune defense. Club cell density has been estimated in some studies but a standardized method for quantifying these cells is lacking.
Here, we assess the repeatability of estimating club cell density in fathead minnows (Pimephales promelas). Thin-sectioned histological samples of fathead minnows were stained and mounted on slides and then digitally scanned for scoring. We estimated epidermal area using the segment tool in ImageJ to simulate the traditional method of scoring microscope slides using an ocular micrometer, where epidermal area was estimated by the lengths of straight-line segments of epidermal thickness and length of the tissue sample. The second approach measured epidermal area using the freehand tool in ImageJ.
The R2 value for repeated estimates of club cell density (club cells/mm2) ranged from 0.959 and 0.969, depending on the method used to estimate epidermal area. Measurement error in estimates of epidermal area was greater than measurement error in cell counts and the freehand tool was more repeatable than the segment tool as a method to measure epidermal area.
We applied these methods to test differences in club cell densities between two sources of fathead minnows; wild-caught fish versus lab-reared fish provided by the Environmental Protection Agency. Lab-reared fish had higher densities of club cells than wild-caught fish did, likely reflecting differences in body condition.epidermal club cells
README: Epidermal club cell densities in fathead minnows: assessment of method and application to a case study
Description of the Data and file structure
The excel file has two tabs. The first tab contains from the first counts, the second tab contains data from the second counts.
Explanation of column headings:
Slide = label on the slides we scored;
Wild1Lab2 = code for source population, 1 = wild-caught fathed minnows from a bait store, 2 = lab-reared fathead minnows from the EPA labs in Duluth, MN, USA;
pixels_100um_scale = using imageJ, the number of pixels in the image that spanned the scale marker for 100um;
pixels/mm = conversion factor for measurements in pixels to mm and mm2
CC = number of epidermal club cells
Area_by_FHTool_pixels = raw data in pixels for area of enclosed epidermal tissue using the freehand tool in imageJ;
AreaFHTmm = epidermal area based ondata from the freehand tool in mm2
Thickness = epidermal thickness in pixels
Thicknessmm = epidermal thickness in mm
Lengths_in_pixels_1st = length of first segment in pixels of the series of segments measuring the length of the histological seciton
2nd = length of the second segment inpixels
3rd = length of the third segment in pixels
4th = length of the fourth segment in pixels
5th = length of the fifth segment in pixels
6th = length of the sixth segment in pixels
7th = length of the sevenh segment in pixels
Total_Length_in_pixels = sum of segment lengths in pixels
Length_mm = total length of sample section converted to mm
AreaHxL = epidermal area in mm2 calculated as the product of epidermal height (thickness) by epidermal length
CCDensFHTool = club cell density per mm2 based on area calculated using the freehand tool
CCDensHxL = club cell density per mm2 based on area calculated by epidermal height x length
Methods
Sample preparation
Adult wild-caught fathead minnows collected from local prairie pothole lakes near Fargo, North Dakota, USA were obtained from a local bait dealer. Laboratory-reared fathead minnows were obtained from the U.S. Environmental Protection Agency (EPA) in Duluth, MN, USA. Fish were sacrificed using a lethal dosage of methane tricainesulfonate (~500 mg/L; IACUC protocol #A18054) and a 3-4 mm section of skin was excised from the nape from eight wild-caught minnows and 11 lab-reared minnows. Samples were fixed in 10% formalin solution for 24 h then set in parafilm for 8 h. Samples were stained with periodic acid Schiff reagent and counterstained with hematoxylin (PAS-H) and subsequently thin-sectioned and mounted on slides.
Digitization and estimation of club cell density
Slides were digitally scanned using a MoticEasyScan slide scanner using Plan Apochromatic objective (20X/0.75) with image detail equivalent to 40X lens. Club cells appear as large, unstained, circular cells within epithelial tissue (Figure 1). To assess the reliability and accuracy of estimates of club cell density, we scored the images twice using ImageJ software (Schneider et al. 2012). Images were enlarged to the optimal equilibrium between increasing visibility of structures without loss of resolution due to pixelation. We counted club cells visible along the entire length of epidermis visible in each image.
We used two methods to calculate epidermal area using ImageJ. The first method mimicked the method used in the pre-digital era (i.e., Wisenden & Smith 1997, 1998) when measurements were taken directly from a microscope slide using an ocular micrometer. We used the straight segment tool in ImageJ to measure the thickness of the epidermis in one location, typically near the center of the section, which seemed to represent typical thickness. Epidermal length was measured by summing several straight segments along the length of the sample, starting new segments as necessary to follow the curvature of the histological section (Figure 1a). The lengths of the segments were summed, then multiplied by epidermal thickness to estimate epidermal area. The second method was to use the freehand tool in ImageJ to trace along the outer edges of the epidermis and then used ImageJ to calculate the area of the enclosed area (Figure 1b). For both methods, each slide was scored twice, at separate sittings.
Data analysis
We assessed repeatability by regressing the estimates of club cell density from the second set of scores against the values obtained from the first set of scores. Perfect repeatable would show an R2 of 1. Because density is a ratio, error in estimates of club cell density could come from errors in counting the number of cells (numerator) or in estimating the area of epidermal tissue (denominator). Therefore, we calculated R2 values for second scores versus first scores for club cell counts and for estimates of epidermal area. We used Mann-Whitney U tests to compare club cell densities between wild-caught and lab-reared fathead minnows. The statistical package used was SPSS 28.0.