DOI: 10.5061/dryad.wdbrv15tm
title: Resilin distribution and abundance in Apis mellifera across biological age classes and castes

description: The presence of resilin, an elastomeric protein, in insect vein joints
 provides the flexible, passive deformations that are crucial to flapping
 flight. This study investigated the resilin gene expression and
 autofluorescence dynamics among Apis mellifera (honey bee) worker age
 classes and drone honey bees. Resilin gene expression was determined via
 ddPCR on whole honey bees and resilin autofluorescence was measured in the
 1m-cu, 2m-cu, Cu-V, and Cu2-V joints on the forewing and the Cu-V joint of
 the hindwing. Resilin gene expression varied significantly with age, with
 resilin activity being highest in the pupae. Autofluorescence of the 1m-cu
 and the Cu-V joints on the ventral forewing and the Cu-V joint on the
 ventral hindwing varied significantly between age classes on the left and
 right sides of the wing, with the newly emerged honey bees having the
 highest level of resilin autofluorescence compared to all other groups.
 The results of this study suggest that resilin gene expression and
 deposition on the wing is age-dependent and may inform us more about the
 physiology of aging in honey bees.  
description: Specimen Collection
 Honey bees were collected from
 Bountiful Blossoms Bee Company, LLC in Glenwood, IA, USA from May–June,
 2019. Biological age class was estimated on-site using class-specific
 characteristics and behavioral norms. Specifically, purple-eyed pupae were
 collected for the pupae class, representing a distinct age class of
 approximately 7–10 days post-oviposition, workers actively emerging out of
 cells as the newly emerged class, workers actively tending to the nest as
 nest honey bees, and workers returning to the hive with pollen on their
 legs as foragers. Drones at the entrance of the hive were collected for
 drone specimens. Honey bees were collected from 5 different hives for each
 class. For gene expression analysis, honey bees were immediately placed in
 RNAlater and then stored at −80°C until further use. For autofluorescent
 analysis of resilin on wings, honey bees were collected in wooden cages
 and then frozen at −20 °C until further use. Resilin
 Autofluorescence Dissection and Slide
 Preparation For each
 honey bee, the wings were dissected along the axillary sclerites. Each
 wing was first swabbed with 70% ethanol and then secondly with a solution
 of 50% Triton X and 50% distilled water to clean off pollen and other
 particles on the wing. The fluorescence of resilin is sensitive to pH: in
 neutral and alkaline media, the excitation/excitation maxima are 320/420
 nm, while in an acidic media, they are 285/420 nm. The wash protocol of
 ethanol, Triton X-100, and distilled water resulted in wings being exposed
 to a primarily neutral solution (pH 7.2–7.3), which was taken into account
 when selecting the best filter for autofluorescence detection. After being
 swabbed, the wings were covered and dried. The microscope slides were
 cleaned using 70% ethanol and wiped to prevent dust contamination. The
 wings were arranged, polyurethane adhesive was swabbed along the edges of
 the microscope slide, and another microscope slide was placed on
 top.  Microscopy Wings were observed using fluorescence
 microscopy on an Olympus BX61 microscope (Olympus Scientific Solutions
 Americas Corp., Waltham, MA, USA) equipped with an aniline blue filter
 (excitation maxima: 377 nm; emission maxima: 409 nm). Images were taken
 with an Olympus SC100 Color Camera that was connected to the microscope.
 Initial images of resilin were taken with samples and underwent tests to
 determine the exposure that allowed for the greatest clarity of resilin
 across multiple joints and focal planes without saturation. All images
 were taken at 20× magnification and 450 ms exposure. The dorsal, ventral,
 left, and right sides of all wings were imaged at the first medio-cubital
 (1m-cu), second medio-cubital (2m-cu), cubito-vannal (Cu-V), and second
 cubito-vannal (Cu2-V) joints on the forewing and the cubito-vannal (Cu-V)
 joint on the hindwing. Image Analysis All images were blinded by a third party
 and uploaded to Image J version 15.2 (U.S. National Institutes of Health,
 Bethesda, Maryland, USA). The scale was set and the following measurements
 were selected: area, minimum and maximum gray value, mean gray value, and
 integrated density. The picture identification code, honey bee and hive
 identification code, and orientation were recorded, and scale bars were
 burned into each image. The autofluorescent resilin was manually
 traced three times and the average for each measurement was found. Then
 three selections of the background were taken and the average for each
 measurement was found. The corrected total cell fluorescence (CTCF)
 equation, a quantitative fluorescence imaging analysis, was used to
 calculate the final measurement: CTCF = Mean Integrated Density − (Mean
 area of the cell x Mean Gray Value of the background).
 CTCF can be used for any fluorescent material within a restricted area.
 CTCF calculates the fluorescence through the integrated density
 measurement, which finds the summation of gray pixel values within a
 selection and multiplies it by the area of the selection. Average CTCF was
 calculated because it incorporates the area and intensity of resilin
 fluorescence into one standard number while simultaneously subtracting out
 background fluorescence. Average CTCF was measured in relative
 fluorescence units (RFU). RFU is a standard measure of fluorescence
 intensity. ddPCR Droplet digital PCR was completed
 according to the manufacturer’s protocol using the Bio-Rad QX200 ddPCR
 System. Pro-resilin gene expression analysis was used to make inferences
 about resilin deposition timing as it related to worker age classes.
 Pro-resilin is the precursor protein that gets transported to the
 subcuticular space before crosslinking to form a functional resilin
 protein network. The concentration of cDNA was normalized to a
 housekeeping gene, eukaryotic translation initiation factor 3 subunit C
 (eIF3-S8). Primers and
 probes were utilized at concentrations of 300 nM and 150 nM, respectively.
 A PCR was performed using the C1000 Touch thermocycler (Bio-Rad
 Laboratories). The thermocycler was programmed with the following
 settings: (1) 10 min at 95 °C, (2) 30 s at 94 °C, (3) 1 min at 57.5 °C,
 (4) repeat steps 2 and 3 for a total of 39 cycles, (5) 10 min at 98°C, and
 (6) 20 min at 4 °C. Additionally, a ramp rate of 2.5 °C was applied to
 Steps 2–4. Samples were then transferred to the Bio-Rad QX200 Droplet
 Reader. The output from the droplet reader was analyzed using
 Quantasoft
 software version 1.7 (Bio-Rad
 Laboratories, Hercules, CA, USA)to determine the copy number of target
 sequences.
description: # Resilin distribution and abundance in *Apis mellifera* across biological
 age classes and castes
 [https://doi.org/10.5061/dryad.wdbrv15tm](https://doi.org/10.5061/dryad.wdbrv15tm) This dataset contains wing joint autofluorescence data and resilin gene expression data from honey bees. Autofluorescence and resilin gene expression were determined in multiple classes of workers and drone honey bees in this experiment. Significant differences in gene expression and autofluorescence were discovered among different groups examined, with higher rates of resilin expression and autofluorescence generally detected in the youngest age class examined. ## Description of the data and file structure There are two tabs in this dataset. Tab 1 includes the autofluorescence data and Tab 2 includes the gene expression data. In both tabs, the data are sorted by sample ID, bee age class/caste and hive ID. In the autofluorescence tab, the wing joint ID is given, along with the CTCF value. In the gene expression tab, the relative gene expression level is given. ## Sharing/Access information N/A ## Code/Software R code performed during statistical analyses of these data can be found here: https://github.com/andrewckraemer/ResilinAnalysis\_2023Apr18\_ACK