DOI: 10.5061/dryad.8sf7m0cwq
title: Epidermal club cell densities in fathead minnows: assessment of method and application to a case study

description: 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
description: 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 (<strong>Figure
 1</strong>). 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 (<strong>Figure
 1a</strong>). 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 (<strong>Figure 1b)</strong>. 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 R<sup>2</sup> 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 R<sup>2</sup> 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.
description: # 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