DOI: 10.5061/dryad.mgqnk9963
title: Identifying the proximal cue(s) for pupal color variation in the bordered patch butterfly, Chlosyne lacinia (Geyer 1837; Lepidoptera: Nymphalidae)

description: Color is a multifaceted trait with many functions such as predator
 defense, thermoregulation, and immune response. We investigated pupal
 color variation in Chlosyne lacinia pupae, focusing on identifying the cue
 for increased melanization. Through laboratory experiments, we
 demonstrated pupae reared on black backgrounds exhibited significantly
 higher melanization compared to those on white backgrounds. Additionally,
 black pupae experienced longer developmental periods, suggesting a
 trade-off between defense and developmental time. Our findings support
 crypsis as a likely evolutionary driver for increased melanization in
 response to substrate color. We discuss potential implications for
 predator avoidance, immune response, and developmental costs associated
 with melanization. This study provides insights into the adaptive
 significance of pupal melanization in response to environmental cues,
 shedding light on the complex interplay between life history traits in
 butterflies.
description: <em>Identification of Black Pigment</em>
 Using the protocol for cuticular pigment extraction in Debecker
 et al. (2015), we used pupal exuviae from colony individuals to verify the
 black pigment as melanin. The pigment was extracted by adding 1 M NaOH/10%
 DMSO, of which the volume was adjusted to the mass of each exuvia
 (extraction volume in μL = 200 × exuvial mass in mg) after the exuviae had
 been homogenized twice for seven seconds. The samples were incubated at
 80°C for two hours in a water bath and then centrifuged at 12,000 g for 10
 minutes. The absorbance of the samples was read at 380 nm and compared to
 standard absorbance curves for melanin. <em>Cue
 Identification</em> Colony larvae of the
 third/fourth instar were selected for use in multiple experiments to
 identify the cue for pupal melanization. First, we conducted a shading
 experiment. We placed larvae in 32oz Styrofoam cups with white or black
 mesh over the opening held in place by the lid with a hole cut in the
 middle. A moist paper towel was placed at the bottom of each cup to
 maintain moisture levels and facilitate cleaning. Larvae were kept at room
 temperature (~23°C) under grow lights with an 18hrL:6hrD light cycle to
 mimic summer months. Larvae were fed <em>ad libitum</em> with
 <em>A. trifida</em> leaves with their cups checked and cleaned
 daily. There were 60 individuals with 30 in each treatment. Next, we
 tested the effects of larval density and temperature on pupal
 melanization. Using the same 32 oz Styrofoam cups, we tested 1 larva (low
 density), 3 larvae (medium density), and 6 larvae (high density) at each
 of two temperatures: 23°C and 30°C for a total of 60 individuals in this
 trial. 23°C were kept under grow lights and 30°C were housed in a Thermo
 Scientific P/N 3185051 Rev. 2 incubator at 30°C, ~30% humidity; both at a
 18hrL:6hrD light cycle. Common sunflowers were placed in each cup as food
 and were checked daily. We also tested the effects of wounding on pupal
 cuticular melanization. The same habitat conditions as the shading
 experiment were used, but for 20 larvae. We used a sterilized sewing
 needle to wound 10 of the 20 individuals and then documented the pupal
 coloration. All larvae from these trials (shading, temperature and
 density, and wounding) categorically resulted in 100% light-colored pupae,
 so we did not conduct any further experiments past the pilot trials
 (Supplemental Table 1). Additionally, we tested the
 effect of substrate color on pupal coloration. Twenty larvae were selected
 at random and placed in 32oz Styrofoam cups painted inside with either
 white or black non-toxic paint. A moist paper towel was placed in the
 bottom of each cup to keep the treatment environment moist and facilitate
 cleaning. A hole was cut into the plastic lid and mesh of the
 corresponding cup color was placed over the cup opening before the lid was
 placed on that cup covering almost the entire exposed surface area to the
 larva with the specified treatment color. The same methodology was used as
 the 30°C cups from the larval density and temperature experiment except
 larvae were fed with <em>A. trifida </em>leaves. Upon
 pupation, the date and color of the pupa were noted. On the second day of
 the pupal stage, a photograph was taken with an Olympus R-C-OLY-IM015
 digital camera using a standardized photography setup (Stevens et al.,
 2007). The day of eclosion was noted and each individual was sexed. The
 trial was repeated a second time to verify the results.
 The photographs were uploaded to ImageJ (ImageJ.JS v 0.4.0) and
 converted to a 2-D binary (black and white) image. Given the pupal
 coloration per individual is symmetrical, a lateral orientation of the
 left dorsal view of the pupa was used for the photographs (Fig. 1). Within
 the binary image, we obtained the information for the total number of
 pixels per pupa, the number of white pixels, and the number of black
 pixels. This information was used to create a percentage of melanization
 for each pupa. <em>Data Analysis
 </em> Using R (version 4.3.2) (R Core Team,
 2024), we conducted Generalized linear models (GLM) to test the effect of
 the substrate color (Treatment) and sex, and their interactions, on pupal
 development time, and percentage of melanization. Based on the output of
 these statistical tests, we conducted an AIC using the
 <em>AICcmodavg </em>package (Mazerolle, 2023) to select the
 model of best fit that explains the percent of melanization in pupae. We
 also conducted ANOVAs and GLMs to test if the percentage of melanization
 in pupae, sex, and larval color affected the pupal development
 time. Additionally, we wanted to test if there were
 effects of sex, percentage of melanization, and larval color on pupal
 developmental time. We used the<em> dplyr</em> package
 (Wickham et al., 2023) and <em>grep </em>function to subset
 the data for both white and black pupae. We then conducted GLMs to test
 these effects. Furthermore, we conducted a generalized linear regression
 analysis to test for the effect of pupal melanization on pupal
 developmental time.
description: # Identifying the proximal cue(s) for pupal color variation in the
 bordered patch butterfly, Chlosyne lacinia (Geyer 1837; Lepidoptera:
 Nymphalidae) ## Author Information A. Principal Investigator Contact
 Information Name: Jamie Phelps Institution: Louisiana State University
 Address: Baton Rouge, LA USA Email:
 [jphelp4@lsu.edu](mailto:jphelp4@lsu.edu) B. Associate or Co-investigator
 Contact Information Name: Luis Santiago-Rosario Institution: University of
 Minnesota Address: St. Paul, MN USA Email:
 [lsantiag@umn.edu](mailto:lsantiag@umn.edu) C. Associate or
 Co-investigator Contact Information Name: Morgan Kelly Institution:
 Louisiana State University Address: Baton Rouge, LA USA Email:
 [morgankelly@lsu.edu](mailto:morgankelly@lsu.edu) Date of data collection:
 2022-2023 Geographic location of data collection: South Texas, USA &
 Baton Rouge, LA USA ## SHARING/ACCESS INFORMATION Recommended citation for
 this dataset: Phelps, JM, Santiago-Rosario, LY, and Kelly MW. (2024).
 Identifying the proximal cue(s) for pupal color variation in the bordered
 patch butterfly, Chlosyne lacinia (Geyer 1837; Lepidoptera: Nymphalidae).
 Dryad Digital Repository
 [https://doi.org/10.5061/dryad.mgqnk9963](https://doi.org/10.5061/dryad.mgqnk9963) ## DATA & FILE OVERVIEW #### Pupal\_color\_master\_sheet\_w\_character\_vectors.csv Number of variables: 8 Number of cases/rows: 22 Variable List: * organism ID * treatment: reared in which substrate color * white pixels: # of white pixels in pupa image * black pixels: # of black pixels in pupa image * total # of pixels in pupa image * % black OR X..black: percent of black pixels * sex * date of pupation * date of eclosion * pupal duration: length (days) of pupal stage * larval color: what larval morph: rufa, nigra, or bicolor