DOI: 10.5061/dryad.dv41ns1xz
title: Melanic pigmentation and light preference within and between two Drosophila species

description: Environmental adaptation and species divergence often involve suites of
 co-evolving traits. Pigmentation in insects presents a variable, adaptive,
 and well-characterized class of phenotypes for which correlations with
 multiple other traits have been demonstrated. In Drosophila, the
 pigmentation genes ebony and tan have pleiotropic effects on flies’
 response to light, creating the potential for correlated evolution of
 pigmentation and vision. Here we investigate differences in light
 preference within and between two sister species, Drosophila americana and
 D. novamexicana, which differ in pigmentation in part because of evolution
 at ebony and tan, and occupy environments that differ in many variables
 including solar radiation. We hypothesized that lighter pigmentation would
 be correlated with a greater preference for environmental light, and
 tested this hypothesis using a habitat choice experiment. In a first set
 of experiments, using males of D. novamexicana line N14 and D. americana
 line A00, the light-bodied D. novamexicana was found slightly but
 significantly more often than D. americana in the light habitat. A second
 experiment, which included additional lines and females as well as males,
 failed to find any significant difference between D. novamexicana-N14 and
 D. americana-A00. Additionally, the other dark line of D. americana (A04)
 was found in the light habitat more often than the light-bodied D.
 novamexicana-N14, in contrast to our predictions. However, the lightest
 line of D. americana, A01, was found substantially and significantly more
 often in the light habitat than the two darker lines of D. americana, thus
 providing partial support for our hypothesis. Finally, across all four
 lines, females were found more often in the light habitat than their more
 darkly-pigmented male counterparts. Additional replication is needed to
 corroborate these findings and evaluate conflicting results, with the
 consistent effect of sex within and between species providing an
 especially intriguing avenue for further research.
description: <i>Fly lines</i>
 <i>Drosophila americana </i>lines A04, A00, and A01,
 and <i>Drosophila novamexicana</i> line N14 were ordered from
 the Cornell University Drosophila Stock Center (Table 1), and maintained
 at Whitman College on Nutri-Fly Instant fly food (Genesee Scientific, San
 Diego, CA, U.S.A.). Flies were maintained at ambient light, on benches
 adjacent to windows. Within <i>D.
 americana</i>, A01 is the lightest line that has been documented to
 date, and it contains a <i>novamexicana</i>-like (functionally
 “light”) allele linked to the <i>tan</i> gene, while the dark
 A00 line contains functionally “dark” alleles at both
 <i>ebony</i> and <i>tan</i>  (Wittkopp et al.,
 2009). The dark A04 line is functionally uncharacterized, although it is
 phenotypically very similar to line A00 (Table 1). <i>Drosophila
 novamexicana</i>-N14 is the best characterized line of its species
 (Wittkopp et al., 2009; Cooley et al., 2012), but is actually somewhat
 dark relative to the range of variation within <i>D.
 novamexicana</i> (see Davis and Moyle, 2019 for images of lighter
 lines). <i>Experimental
 overview</i> Mixed-species trials were performed
 in fall 2017, summer 2018, and spring 2019. For each trial, twenty male
 flies were placed in each cage: ten on each side, with five <i>D.
 americana</i>-A00 and five <i>D. novamexicana</i>-N14 on
 each side (Fig. 2A). This number was selected as being easily countable by
 eye. The number of flies in the “light” habitat was counted at 12 pm
 daily, for six days per trial. In 2019, an additional 4 pm data collection
 time was added to assess the effect of time of day on fly
 behavior. Single-taxon trials were performed in the
 spring, summer, and fall of 2020, across five separate rounds of data
 collection. For each trial, ten flies were placed in each cage: five on
 each side, with each cage containing flies from a single line (Fig. 2A).
 The number of flies in the “light” habitat was counted at 12 pm daily, for
 six days per trial. Both males and females were tested in the 2020
 experiments, but each cage contained only a single sex. Due to the
 COVID-19 pandemic, data collection by two of the experimenters was split
 between work done at Whitman College and work done at the students’ homes.
 In each case, the data were coded as two separate experiments based on
 their locations. <i>Cage
 construction</i> To provide alternate light
 environments for the behavioral choice experiments, cages were constructed
 using small, transparent betta fish tanks with a dark plastic divider
 (Fig. 2B). All outer sides of half of each cage were covered in two layers
 of duct tape to create a dark environment. Uniform holes ¼” in diameter
 were drilled into the dividers, allowing flies to pass between the light
 and dark sides of the cages. The dividers were locked in place by hot
 glue, sealing them to the insides of the cages. Clear tape was used on the
 inside of the lids to prevent flies from escaping through air-holes. Each
 side of the container had identical plastic caps filled with synthetic fly
 food to sustain the flies throughout the trial period. Only enough water
 was added to the synthetic fly food to slightly saturate it, to prevent
 the buildup of excess condensation in the cages.
 <i>Selection of flies for behavioral
 trials</i> To ensure that flies used in the
 behavioral trials were no more than one week old, all adult flies were
 transferred out of the collecting vials one week prior to each trial. On
 the day of the trial, the collecting vials – containing flies which had
 eclosed within the past week – were chilled at 4°C to immobilize the
 flies. Genital morphology was used to sex the flies, since these species
 lack both sex combs and sex-specific pigmentation patterns. Flies of a
 single sex and taxon were sorted in sets of five into empty test tubes.
 The vials were kept off ice so liveliness could be evaluated once they
 warmed up. This was to ensure they had not been damaged and could fly and
 move normally. Flies that appeared old, deformed, or injured were also
 returned to the main population. Once collected and checked for
 liveliness, flies were re-immobilized by chilling on ice to facilitate
 transfer and were then poured into each side of the cage. The lids were
 secured with clear tape. <i>Data collection in
 the behavioral trials</i> In 2017, fly cages were
 placed in a darkened room under a greenhouse grow light set on a 12-hour
 timer. Due to concerns that the artificial light was creating warm
 temperatures, in all subsequent experiments, fly cages were instead placed
 on a table about a meter away from a large window, exposing them to
 natural sunlight. Each trial was run for six
 consecutive days. At 12 pm every day, the number and species of flies in
 the light side of each cage were recorded. In the mixed-species
 experiments, this was done by looking for the number of dark-bodied flies
 (<i>D. americana</i>-A00) and light-bodied flies (<i>D.
 novamexicana</i>-N14) present in the light side of the cage. In
 2019, a second observation period at 4 pm was added. At
 the end of each trial, cages were placed in a freezer at -20 °C for one
 hour to immobilize the flies. This allowed us to remove the lid and more
 thoroughly look for missing or dead flies. The data from cages with dead
 or missing flies were excluded from analysis. We disposed of the flies and
 cleaned the cages with ethanol. <i>Temperature
 evaluation</i> In the 2019 experiment, a
 temperature control study was set up to test for a temperature difference
 between the light and dark sides of the cages. The wire probes of Fluke 52
 II dual input digital thermometers (Everett, WA) were placed in both the
 light and dark sides of two empty cages. We recorded the temperature
 reading of each side of each cage, at noon and 4 pm daily for six
 days. <i>Statistical analyses</i>
 To test for differences in fly light preference, a generalized
 linear model was fitted using the glm() command in RStudio 1.3.1093,
 “Apricot Nasturtium,” within the lme4 package. We assumed a Poisson
 distribution for the dependent variable, which was the number of flies on
 the light side of the cage. Independent variables included a fixed effect
 of taxon; a fixed effect of sex in experiments that included both male and
 female flies; a fixed effect of time of day for comparisons between 12 pm
 and 4 pm; a random effect of cage, to account for the repeated
 measurements made on each cage; and a random effect of experiment to
 account for the fact that multiple rounds of data collection were
 performed, at different times and by different groups of
 experimenters. A paired t-test in R was used to
 determine whether there was a significant temperature difference between
 the light and dark sides of the cages.
 <i>Genotyping</i> At the end of
 the 2020 experiments, the flies were visually inspected to verify
 homogeneity of pigmentation within each line. To further confirm that the
 lines had not interbred over the course of the experiments, one female fly
 of each line was sequenced at both the <i>tan</i> and
 <i>ebony</i> genes. DNA was extracted using the Omega E.Z.N.A.
 Tissue DNA Kit (Norcross, GA, U.S.A.) and eluted in 50 uL of water.
 Partial sequence was amplified from the <i>tan</i> gene using
 primers 5’- CCGATGCCTGTTCCATTAAC-3’ and 5’- GGCGGCTTGTATTTACCAAA-3’, and
 from the <i>ebony</i> gene using primers
 5’-AGCCCGAGGTGGACATCA-3’ and 5’GTATGGGTCCCTCGCAGAA-3’, with G-Biosciences
 Taq DNA Polymerase (St. Louis, MO, U.S.A.). Thirty cycles of PCR were
 performed with a 54°C annealing temperature and a 30-second extension
 time. PCR product purity and concentration were estimated from a 1%
 agarose gel. Samples were sequenced using both forward and reverse primers
 by Eton Biosciences (San Diego, CA, U.S.A.). Sequences were compared to
 sequences of <i>D. americana</i> and <i>D.
 novamexicana</i> obtained from GenBank and from Cooley et al.
 (2012). Alignments were created in Geneious R9 (https://www.geneious.com).
description: “Experiment” – character variable – unique name given to each
 round of experiments. Differences across experiments include year, season,
 identity of the researcher, whether the data were collected at a student’s
 home (during the Sars-CoV-2 pandemic – marked “home”) - and which lines
 and sexes were included. “Taxon” – character
 variable. N14 = <i>Drosophila
 novamexicana</i> (light pigment) A01 =
 <i>Drosophila americana </i>(moderate pigment)
 A00 = <i>Drosophila americana</i> (dark
 pigment) A04 = <i>Drosophila americana
 </i>(dark pigment) “Unique CageID” –
 numeric variable. Flies were counted once per day, for six days per cage.
 Each cage is assigned a unique ID number. “Day” – count
 variable. Records observation day, with Day 1 being ~24 hours after flies
 were placed in the cage. “FliesInLight” – count
 variable. Number of flies observed on the light side of the cage. (Number
 on the dark side could not be observed directly.)
 “Imputed?” – a comments column, marking two experimental days in
 which observations were not obtained for a set of cages. The data were
 instead imputed, by fitting a line to (Flies in Light) as a function of
 (Day) for the collection of cages with missing data.