Common use of Injections Clause in Contracts

Injections. The numbers of pupae used for the hormone injection experiment are given in table 3.4. Pupal survival was significantly lower for 20-hydroxyecdysone (ecdysone from here onwards) injected animals compared to untreated pupae at both extreme temperatures, whilst at 22.5ºC, no difference in pupal survival was observed (table 3.4). Probability of survival increased with increasing pupal weight, which was a highly significant covariate (p < 0.001) in the logistic regression at all temperatures. With an increase in pupal weight of 10 mg, survival probability increased by 1.13 at 27ºC, 1.20 at 22.5ºC and 1.26 at 18ºC. There were no interactions between pupal weight and treatment. Pupal survival probability did not differ between the sexes, but the significance levels of the covariate pupal weight were reduced to p < 0.05 (females are larger than males). The effects of treatment remained unchanged. Because we did not know the sex of all unhatched pupa, incorporating this factor decreased the power of the test. Survival of untreated control pupae differs across temperatures (χ2 = 15.3, df = 2, p < 0.001), and is lowest at 18ºC (table 3.4). The other two temperatures do not differ in survival (χ2 = 0.4, df = 1, p = 0.53). We could not incorporate pupal weight, because it differed between temperatures (F2,2540 = 478.1, p < 0.0001; pattern: 22.5ºC > 18ºC > 27ºC). temp. injection N emerged N dead % emerged odds ratio [95% C.I.] 18ºC ecdysone 10 36 22 *** 0.03 [0.01-0.10] injected controls 6 5 55 ns 1.55 [0.27-9.52] untreated controls 599 91 87 22.5ºC ecdysone 172 16 91 ns 0.54 [0.14-1.63] injected controls 62 2 97 ns 3.15 [0.63-36.1] untreated controls 1041 88 92 27ºC ecdysone 125 55 69 *** 0.13 [0.06-0.27] injected controls 50 5 91 ns 2.41 [0.77-10.1] untreated controls 901 87 91 ecdysone injection saline injection ecdysone effect 18ºC 22.5ºC 27ºC eyespot size relative to not injected 0.02 -0.02 -0.04 ecdysone injection saline injection ecdysone effect pupal time relative to not injected (days) 0.8 -1.6 Figure 3.4 Effects of injection of physiological salt or ecdysone on pupal time and eyespot size at different temperatures. Effects of ecdysone are derived by subtracting effects of physiological salt injection from ecdysone injection. At 27ºC, selection lines reacted differently to injection per se, hence the two bars (lighter bar for SLOW, see text for more information). At all temperatures, pupae injected with physiological salt had significantly smaller eyespots than the untreated animals. Pupal time also tended (significant at 22.5ºC only) to be longer for saline injected butterflies (figure 3.4). The effect of injection per se might, therefore, counteract potential effects of the hormone implying that ecdysone injection has a twofold effect: one due to the injection treatment per se, and one resulting from the hormone. We can disentangle these two effects by subtracting the injection effects, as measured by the effects of physiological salt injection. At 27ºC, selection lines differed in their change in pupal time as a reaction to saline injection (significant line × injection interaction): lines with a SLOW component of selection showed a significant increase in pupal time after physiological salt injection, whilst partly FAST selected and unselected lines did not (figure 3.4). Because genetic background influenced the effect of injection per se, we corrected pupal time at 27ºC using separate correction factors for the SLOW lines, and for the FAST and unselected lines, and used those data for all further analyses. Pupal time significantly decreased for the ecdysone-treated animals at all temperatures (figure 3.4). Although pupal time increased with pupal weight, the mass of a pupa did not affect the effects of ecdysone and/or injection (no significant interaction terms, p > 0.10). Only at 22.5ºC was there an interaction between selection line and hormone treatment (F1,1188 = 2.46, p = 0.044; other factors in the model were: line, sex, line × sex, pupal weight and replicate[line]). The decline in pupal time was significantly less for SLOW selected groups than for partly FAST selected lines (contrast, t = 2.36, p = 0.018); this is clearest for males in figure 3.5. However, the sex by treatment interaction was not significant (F1,1188 = 1.52, p = 0.22). Eyespot size increased under the influence of ecdysone at 18ºC (trend, F1,287 = 2.89, p = 0.09) and at 22.5ºC (F1,547 = 8.91, p = 0.003), but hormone treatment did not alter eyespot size at 27ºC (F1,454 = 0.08, p = 0.77), see figure 3.4. There were no interactions between eyespot size and hormone treatment. males females 10 pupal time (days) E N E N Figure 3.5 Effects of ecdysone, corrected for effect of injection (denoted as E) on pupal time (± standard error) versus not treated animals (N), for males and females at 22.5ºC. Selection lines are: FAST DRY (○), FAST WET (●), SLOW DRY (□), SLOW WET (■), and UNSELECTED (×).

Injections. The numbers of pupae used for the hormone injection experiment are given in table 3.4. Pupal survival was significantly lower for 20-hydroxyecdysone (ecdysone from here onwards) injected animals compared to untreated pupae at both extreme temperatures, whilst at 22.5ºC, no difference in pupal survival was observed (table 3.4). Probability of survival increased with increasing pupal weight, which was a highly significant covariate (p < 0.001) in the logistic regression at all temperatures. With an increase in pupal weight of 10 mg, survival probability increased by 1.13 at 27ºC, 1.20 at 22.5ºC and 1.26 at 18ºC. There were no interactions between pupal weight and treatment. Pupal survival probability did not differ between the sexes, but the significance levels of the covariate pupal weight were reduced to p < 0.05 (females are larger than males). The effects of treatment remained unchanged. Because we did not know the sex of all unhatched pupa, incorporating this factor decreased the power of the test. Survival of untreated control pupae differs across temperatures (χ2 = 15.3, df = 2, p < 0.001), and is lowest at 18ºC (table 3.4). The other two temperatures do not differ in survival (χ2 = 0.4, df = 1, p = 0.53). We could not incorporate pupal weight, because it differed between temperatures (F2,2540 = 478.1, p < 0.0001; pattern: 22.5ºC > 18ºC > 27ºC). temp. injection N emerged N dead % emerged odds ratio [95% C.I.] 18ºC ecdysone 10 36 22 *** 0.03 [0.01-0.10] injected controls 6 5 55 ns 1.55 [0.27-9.52] untreated controls 599 91 87 22.5ºC ecdysone 172 16 91 ns 0.54 [0.14-1.63] injected controls 62 2 97 ns 3.15 [0.63-36.1] untreated controls 1041 88 92 27ºC ecdysone 125 55 69 *** 0.13 [0.06-0.27] injected controls 50 5 91 ns 2.41 [0.77-10.1] untreated controls 901 87 91 ecdysone injection saline injection ecdysone effect A 18ºC 22.5ºC 27ºC eyespot size relative to not injected 0.02 -0.02 -0.04 ecdysone injection saline injection ecdysone effect B pupal time relative to not injected (days) 0.8 -1.6 Figure 3.4 Effects of injection of physiological salt or ecdysone on pupal time and eyespot size at different temperatures. Effects of ecdysone are derived by subtracting effects of physiological salt injection from ecdysone injection. At 27ºC, selection lines reacted differently to injection per se, hence the two bars (lighter bar for SLOW, see text for more information). At all temperatures, pupae injected with physiological salt had significantly smaller eyespots than the untreated animals. Pupal time also tended (significant at 22.5ºC only) to be longer for saline injected butterflies (figure 3.4). The effect of injection per se might, therefore, counteract potential effects of the hormone implying that ecdysone injection has a twofold effect: one due to the injection treatment per se, and one resulting from the hormone. We can disentangle these two effects by subtracting the injection effects, as measured by the effects of physiological salt injection. At 27ºC, selection lines differed in their change in pupal time as a reaction to saline injection (significant line × injection interaction): lines with a SLOW component of selection showed a significant increase in pupal time after physiological salt injection, whilst partly FAST selected and unselected lines did not (figure 3.4). Because genetic background influenced the effect of injection per se, we corrected pupal time at 27ºC using separate correction factors for the SLOW lines, and for the FAST and unselected lines, and used those data for all further analyses. Pupal time significantly decreased for the ecdysone-treated animals at all temperatures (figure 3.4). Although pupal time increased with pupal weight, the mass of a pupa did not affect the effects of ecdysone and/or injection (no significant interaction terms, p > 0.10). Only at 22.5ºC was there an interaction between selection line and hormone treatment (F1,1188 = 2.46, p = 0.044; other factors in the model were: line, sex, line × sex, pupal weight and replicate[line]). The decline in pupal time was significantly less for SLOW selected groups than for partly FAST selected lines (contrast, t = 2.36, p = 0.018); this is clearest for males in figure 3.5. However, the sex by treatment interaction was not significant (F1,1188 = 1.52, p = 0.22). Eyespot size increased under the influence of ecdysone at 18ºC (trend, F1,287 = 2.89, p = 0.09) and at 22.5ºC (F1,547 = 8.91, p = 0.003), but hormone treatment did not alter eyespot size at 27ºC (F1,454 = 0.08, p = 0.77), see figure 3.4. There were no interactions between eyespot size and hormone treatment. males females 10 pupal time (days) E N E N Figure 3.5 Effects of ecdysone, corrected for effect of injection (denoted as E) on pupal time (± standard error) versus not treated animals (N), for males and females at 22.5ºC. Selection lines are: FAST DRY (○), FAST WET (●), SLOW DRY (□), SLOW WET (■), and UNSELECTED (×).