Supporting Online Material for

Transcription

1 Supporting Online Material for Evolution of a Polyphenism by Genetic Accommodation Yuichiro Suzuki* and H. Frederik Nijhout *To whom correspondence should be addressed. ys6@duke.edu Published February 6, Science, 65 (6) DOI:.6/science.8888 This PDF file includes: Materials and Methods Figs. S to S Tables S and S References

2 Supporting Online Materials Materials and methods Animals used The black mutant larvae arose spontaneously in a colony of wildtype M. sexta larvae in the early 97s. The black mutant larvae have been maintained in the lab in isolation from the wildtype larvae in parallel with the wildtype larvae. Selection and reaction norm measurements The timing of molts can be determined accurately by measuring the ratio of lengths of the fourth instar and fifth instar spiracles during apolysis from the fourth to fifth instar (). The response to heat-shock was greatest at -8 hrs before head capsule slippage, so selection on polyphenic and monophenic strains was conducted by rearing larvae at 5 C on a hr light (L):8hr dark (D) cycle, followed by heat-shock up to eight hours prior to head capsule slippage (HCS). Heat-shock was conducted in a lit incubator set at C for six hours. Larvae were fed on standard artificial diet. Reaction norms were determined by rearing the th generation organisms at various temperatures in temperature-controlled incubators. In order to avoid the confounding variable of photoperiodism, all measurements were conducted using hr L:8hr D cycle. The only exception was where we tested the effect of photoperiodism when we attempted to generate darker wildtype larvae at 5 C. In this case, a hr L:hr D cycle was used. Ligation experiments Ligations were performed by tying a piece of waxed dental floss around larvae anesthetized with carbon dioxide. For abdominal ligations, the ligation was performed on the th generation larvae, between the first and the second abdominal segments. For neck ligations, the ligation was performed on the th generation larvae, between the head capsule and the thorax. All ligations were performed three hours prior to HCS. For larvae undergoing heat-shock, they were heat-shocked immediately afterwards for six hours. To investigate the effects of methoprene application, different concentrations of methoprene were prepared in 95% ethanol and µl was applied to a spot on ligated th generation larvae on one side between the segments bearing the first and second prolegs. The ligations for these larvae were performed between the mesothorax and the metathorax six hours prior to HCS and the anterior portion was cut off to ensure the removal of hormone-secreting glands. We waited three hours to ensure that any endogenous JH was mostly cleared and at three hours prior to HCS, methoprene was applied. For those undergoing heat-shock, larvae were heat-shocked immediately for six hours. JH bioassay The level of JH was assayed using the black mutant JH bioassay (). JH was extracted using a modified version of JH extraction (). Hemolymph from generation larvae heat-shocked for three hours and those not heat-shocked were collected one hour after

3 HCS by cutting the tip of the first proleg. Hemolymph from three larvae were pooled and split into two tubes, each tube containing 75µl of hemolymph. To each tube, 87.5µl of methanol:water:8.n NH OH (concentration of :9:) solution was added and vortexed for five seconds. Upon addition of 97.5µl of isooctane to each tube, the tubes were vortexed for seconds and centrifuged for five minutes. The epiphase from the two tubes were collected and pooled into one single tube. Isooctane extraction was repeated and the epiphase was again added to the single tube. The isooctane was then evaporated under nitrogen gas and stored until use. For the bioassay, the extracted JH was redissolved in cyclohexane and applied to a spot on untreated black larvae, between the segments bearing the first and the second prolegs, at the time of HCS. Image of the spot was captured and the area of the color change was determined using Photoshop Elements (Adobe Systems Inc, San Jose, CA) and SigmaScan (Systat Software Inc, Richmond, CA). Five replicates were made for each of the two temperature treatments. All glassware used in this procedure was coated with Sigmacote (Sigma Cat# SL-, Sigma Chemical Co, St, Louis, MO).

4 Figure S. The heat-shock sensitive period for larval color change. Time is given as the number of hours prior to head capsule slippage (HCS) that the heat shock treatments began. All larvae were heat-shocked for six hours. (Error bars represent one standard error).

5 Figure S. Frequency distribution of the stock population (top; light blue) and postselection polyphenic (middle; green) and monophenic (bottom; black) strains reared at 8 C and heat-shocked at C. Stock population Generation polyphenic line Generation monophenic line

6 Figure S. Effect of heat-shock and abdominal ligation (blue bars = anterior; red bars = posterior). (* P<. compared to anterior monophenic coloration; # P<. compared to anterior coloration). Heat-shock * N=8 No heat-shock N=7 # N= N=7

7 Figure S. Effect of neck-ligation with heat-shock on polyphenic (green bars) and monophenic (black bars) larvae. Responses of unligated heat-shocked animals are provided for comparison. (* P<. compared to unligated polyphenic line). N=8 * N=9 N=8 N=5

8 Table S. The scoring system used to score methoprene-treated larvae (distinct from the scoring system used for other experiments; modified from ()). Score Description No color change. Very narrow spot at the segment boundary. Less than one-half segment wide. Green spot wider than one-half segment but localized to at most two segments Entire body completely green but dorsal-most surface has black dots between the white V-marks. Entire body completely green without the black dots on the dorsal-most surface.

9 Table S. The effect of methoprene treatment on thoracically-ligated larvae of the polyphenic and the monophenic lines with and without heat-shock. Monophenic (5 C) Methoprene concentration (log pg) Mean..9.. St. error..7.. Monophenic (Heat-shock) Mean St. error.... T-test Heat-shock v.s. 5 C Polyphenic (5 C) P=.5 P=.* N/S N/S.5 Mean...9. St. error.... Polyphenic (Heat-shock).5.5 Mean St. error..7.. T-test Heat-shock v.s. 5 C P=.58 P=. P=.5 P=.5

10 References. J. W. Truman, L. M. Riddiford, L. Safranek, J. Insect Physiol. 9, 95 (97).. R. E. Langelan, J. E. Fisher, K. Hiruma, S. R. Palli, L. M. Riddiford, Dev. Biol. 7, 8 ().. M. J. Fain, L. M. Riddiford, Biol. Bull. 9, 56 (975).. S. Niimi, S. Sakurai, J. Insect Physiol., 875 (997).