DOI: 10.5281/zenodo.17404503
title: Data used in "Larval growth rate is not a major determinant of adult wing shape and eyespot size in the seasonally polyphenic butterfly Melanitis leda"

description: Each file has a sheet with 'readme' explaing the data in the columns.

Here are the key parts from the methods section of the published paper:

Overview

Three experiments were performed. The first experiment tested the effect of temperature on adult phenotype in a population from Ghana (Temperature Experiment). The second included effects of temperature and humidity using a population from South India (Temperature and Humidity Experiment). The third experiment tested for effect of host-plant species using the South-Indian population (Host Plant Experiment). While some methodological details differ, the data taken from these experiments are comparable.

In most cases, the parental butterflies were held in group cages so that eggs could not be attributed to particular mothers and were divided randomly over the treatments. When we did have eggs from specific mothers, we took special care to divide her offspring evenly over the treatments. In summary, caterpillars were reared on grasses growing in flower pots either from seeds or collected from the field (such as in Molleman, Halali & Kodandaramaiah, 2020b). The caterpillars were confined to the plants by placing the plants in fine-mesh sleeves. These sleeves were then kept in environmental test chambers that controlled temperature and humidity and had a 12/12 h light-dark schedule. Plants were watered every other day and were replaced once the majority of leaf material had been consumed or general plant quality had deteriorated. Once larvae reached the 5th instar, sleeves were inspected every 24 h for prepupae and pupae. Prepupae and pupae were kept individually in 100 mL cylindrical transparent containers until eclosion. Prepupae were left attached to the plant whenever possible: using adhesive tape, a piece of the plant where the prepupa was attached was fixed to the side or lid of the container, so that the prepupae could hang freely while pupating. The prepupal stage never exceeded one day, so pupation date of prepupa was the following day. Absorbent paper was placed at the bottom of the container. Hardened pupae were gently removed from the plant material to be weighed and sexed, and then placed back inside the container. Containers were kept upside down with the absorbent paper on the lid so that pupae and eclosing butterflies were easy to observe through the transparent bottoms. The sides of each of these containers were lined with a strip of paper on which the eclosing adult could climb to enable it to expand its wings. Containers were checked daily for adult eclosions. Butterflies were frozen one day after eclosion.

Data were collected on development time from egg hatching to pupation (larval development time), and from pupation to eclosion (pupal time). The wings of the enclosed butterflies were removed and photographed or scanned. For the Temperature Experiment, both left and right wings were photographed on the dorsal side, and for the other experiments, one wing in a pair was imaged on the dorsal and the other on the ventral side. Eyespots, forewing tip, and hindwing tails were measured using an ImageJ macro (Schindelin et al., 2015) and averaged between left and right wings when appropriate. For this study, we focused on the largest eyespot on the forewing to represent eyespot size and the forewing tip to represent wing shape. We used particular vein intersections as landmarks for our proxies for wing area and wingtip area 




Temperature experiment, Ghanaian population

We collected eggs from 14 females from a stock population established based on 80 females from Bobiri, Ghana (Cites collection and export permit 012068 issued by the Wildlife Division, Ghana), and used at most the third generation reared in the laboratory. Sleeves contained 5–30 larvae, with the average being ca. 20, which were fed ca. two-week-old corn seedlings. These sleeves were placed within environmental test chambers at 75% relative humidity. Larvae were reared at five temperatures: 19 °C, 22 °C, 25 °C, 28 °C and 31 °C. Because of unexpected results, a follow-up experiment was carried out at three temperatures: 22 °C, 25 °C, and 28 °C. Photographs were taken using a microscope imaging system (Leica DC200 digital camera with Leica MZ12 microscope).




Temperature and humidity experiment, Indian population

About fifty female M. leda butterflies were collected in Thiruvananthapuram, Kerala, South India, to provide eggs, and experiments were performed on three subsequent generations. Sleeves were set up with 15–17 caterpillars each, and were fed ca. two-week-old corn seedlings. Two environmental test chambers were used during subsequent experiments with combinations of four temperatures (19 °C, 21 °C, 27 °C, and 31 °C) and two levels of humidity (65% and 85% relative humidity). However, for technical reasons, a complete set of temperature and humidity combinations was not achieved so that the effect of humidity during rearing on adult phenotype could not be tested formally. The wings of the enclosed butterflies were scanned for phenotypic measurements (Konica Minolta, Bizhub 363, Osaka, Japan).




Host plant experiments, Indian population

The data presented here are based on the rearing of the fourth batch of larvae described in Molleman, Halali & Kodandaramaiah (2020b). In short, about fifty female M. leda butterflies were collected in Thiruvananthapuram to provide eggs. Larvae were then grown on potted plants belonging to 18 species of grass in an environmental test chamber with a constant temperature of 24 °C and a constant humidity of 69%. Photos of the wings of adults were taken with a Nikon D7000 camera under standard light inside a closed white styrofoam box with a shutter speed of 1/125 and an aperture of F14.

Measuring wing traits

Relative eyespot size was measured by dividing the area of the M3 eyespot by a proxy of total wing area. Wing shape was quantified as the area of the wing-tip triangle divided by the proxy for wing area Larval growth rate was calculated as (pupal mass)1/3/larval development time), following Tammaru & Esperk (2007). We first explored the distribution of growth rate, relative eyespot size, and wing shape for both sexes using density plots. While larval growth rate approached a normal distribution, relative eyespot size and wing shape were biased towards very small numbers and this could be corrected by using the square-root transformation.