Popul Ecol (2006) 48:159–166 DOI 10.1007/s10144-006-0253-4

ORIGINAL ARTICLE

Tomokazu Seko Æ Fusao Nakasuji Adaptive significance of egg size plasticity in response to temperature in the migrant skipper, Parnara guttata guttata (Lepidoptera: Hesperiidae)

Received: 29 July 2005 / Accepted: 16 December 2005 / Published online: 22 February 2006 The Society of Population Ecology and Springer-Verlag Tokyo 2006

Abstract Females of the migrant skipper, Parnara gut- Keywords Natural selection Æ Environmental cue Æ tata guttata, that are reared under lower temperatures Reproductive allocation Æ Leaf toughness Æ Fitness Æ lay smaller eggs. The adaptive significance of egg size Trade-off Æ Parnara guttata guttata plasticity in response to temperature is unknown in this species. We suggest, based on the following experimental results, that P. g. guttata uses temperature as an indirect cue to predict the host condition (leaf toughness) of the Introduction next generation. First, larvae were reared under the typical conditions of temperature and photoperiod Egg size is subject to selection because it has substantial experienced during the immature stages in the first, fitness effects on progeny. Larvae hatched from larger second, and overwintering (third) generations (LD 16:8 eggs have higher resistance to environmental stresses at 25C, LD 14:10 at 25C and LD 14:10 at 20C). Fe- such as larval competition, starvation, desiccation, and males reared under LD14:10 at 20C produced more, low temperature (Fox and Czesak 2000). However, there smaller eggs than those reared under LD14:10 and is often a trade-off between egg size and fecundity (Roff LD16:8 at 25C. Secondly, survival rates of first instar 1992; Cummins 1986; Braby 1994; Sinervo and Licht larvae derived from females reared under the three 1991; Heath et al. 1999). Life-history theory predicts photoperiod/temperature treatments were measured on that organisms inhabiting environments with relatively young soft rice leaves (‘‘soft’’), or tough, old rice leaves harsh conditions for the growth and survival of their (‘‘tough’’). Survival rates of hatchlings reared on soft offspring should produce fewer and larger offspring and tough leaves did not differ when females were reared (Sibly and Calow 1985). under LD16:8 and LD14:10 at 25C. However, hatch- Temperature is a particularly important and wide- ling survival was significantly higher on soft than on spread mediator of phenotypic variation in ectothermic tough leaves when females were reared under LD14:10 animals, resulting in predictable plastic changes in egg at 20C. Thirdly, we found that egg size plasticity in and body size (Fischer et al. 2003a). Egg size in ecto- response to temperature in P. g. guttata may be a therms commonly increases under colder conditions threshold response. Temperatures below 20C experi- (Ernsting and Isaaks 1997; Azevedo et al. 1996; enced during the immature stages may be effective for Yampolsky and Scheiner 1996; Blanckenhorn 2000; production of smaller and more eggs in the overwin- Fischer et al. 2003a). Much of this variation is probably tering generation of P. g. guttata. attributable to egg size plasticity. However, the adaptive significance of this response to temperature has not been investigated in most study systems (Azevedo et al. 1996; Blanckenhorn 2000; Fox and Czesak 2000). The migrant skipper, Parnara guttata guttata, typically has three generations per year in Japan and T. Seko (&) National Agricultural Research Center for Western Region, shows a large variation in egg size among generations 6-12-1 Nishifukatsu, Fukuyama 721-8514, Japan (Nakasuji and Kimura 1984). Nakasuji et al. (1986) and E-mail: sekot@affrc.go.jp Nakasuji and Ishii (1988) hypothesized that this varia- Tel.: +81-84-9234100 tion was the result of adaptation to the seasonal changes Fax: +81-84-9235219 in leaf toughness of host plants. Nakasuji and Kimura F. Nakasuji (1984) suggested that females use photoperiod as an Okayama University, Okayama, Japan indirect cue to determine the egg size of each generation 160 since this species lays smaller eggs under LD 16:8, but 1988; Ishii and Hidaka 1979). Overwintering individuals larger eggs under LD 14:10 at the same thermal condi- emerge from late May to early June, and are dark-col- tion. Nakasuji and Nakano (1990) compared the size of ored with a small body (overwintering and/or third eggs laid by females reared under LD 16:8, 14:10 and generation). Kidokoro (1992) suggested the possibility 12:12, corresponding to the photoperiod conditions of a return migration by adults of the overwintering during larval development of the three generations. generation from southern to northern Japan, but no However, the order of egg size among the three pho- clear evidence of spring migration has been found as yet. toperiods in the laboratory did not correspond with that P. g. guttata changes its habitat seasonally. The adults of of egg size in the field. On the other hand, the thermal the overwintering and first generations lay their eggs on environment experienced by larvae during development grasses in wet lowland areas, and the adults of the sec- has an effect on egg size in this species (Hareyama et al. ond generation lay their eggs in dry upland areas. The 1991). Females that developed under lower temperatures main host plant of the first two generations is the rice laid smaller eggs, in contrast to the common response to plant, Oryza sativa (Nakasuji 1982). The overwintering temperature in ectotherms. The adaptive significance of generation larvae feed primarily on Imperata cylindrical this response has remained unknown in P. g. guttata as (cogon grass), Miscanthus sinensis (eulalia), and Festuca well as in other species that show egg size plasticity in ovinna (sheeps fescue). Female adults of the overwin- response to temperature (but see Fischer et al. 2003b). tering generation and those of the first generation lay In the present study, three experiments were per- smaller eggs on grasses with soft leaves. These grasses formed to verify the possibility that the egg size plasticity grow in wet lowlands. On the other hand, females of the of P. g. guttata in response to temperature is related to a second generation lay larger eggs on grasses with tough seasonal adaptation, such as photoperiod. First, we leaves. Larvae hatched from these eggs have to feed on reared larvae under the combinations of temperature and tough leaves in dry uplands because grasses in wet photoperiod experienced by larvae in each of three gen- lowlands are unavailable in winter seasons (Nakasuji erations in the field. To elucidate the effect of tempera- 1982, 1987). Nakasuji and Kimura (1984) reported that ture on the reproductive allocation patterns, we larvae hatched from smaller eggs in the overwintering compared the size and number of eggs laid by females and first generations were not able to survive on tough among treatments (experiment 1). Predictions developed leaves in dry uplands. They also determined that egg size in previous work indicate that the reproduction of fewer is a function of the photoperiod under which the larvae but larger eggs is favored when host plant leaves are were raised, although as Roff (2002) suggests, the pho- tough. Therefore, survival rates of first instar larvae un- toperiod effect alone is not great enough to produce der each treatment were compared on both soft and naturally occurring eggs of such a size. tough rice leaves. A total fitness measure comprised of survival and fecundity was also compared among pho- toperiod/temperature treatments and among leaves of Study population and experimental design different degrees of toughness (experiment 2). Thirdly, to verify whether egg size plasticity in response to temper- Stock culture ature in P. g. guttata is a gradual or threshold response, the size of eggs laid by females was compared among the The stock culture of P. g. guttata used in this study different temperature treatments (experiment 3). originated from adults caught in Nara (western Japan; 3431¢N, 13544¢E) in July 2003. The stock culture was established using the method of Nakasuji and Honda Materials and methods (1979). Females were allowed to lay eggs in a plastic cage (25·35·35 cm3) with 1-mm mesh netting. The hatched Life history of P. g. guttata larvae were reared on 2-week-old rice seedlings that had been planted in plastic pots (10 cm in diameter and 8 cm Parnara guttata guttata inhabits the central and western in height) at 25C under a photoperiod of LD 16:8. The regions of Japan as the northern limit of its distribution. emerged adults were fed on a 10% honey solution, The adults in the central regions migrate in a south- which was supplied in artificial vinyl flowers (a plastic western direction during late August and September tube attached by a wire) (Nakasuji and Honda 1979), every year (Hiura 1973). P. g. guttata adults have mor- and they were allowed to mate and to lay eggs on rice phological traits that differ according to the season in seedlings. The stock culture of the fifth generation from which they emerge (Ishii and Hidaka 1979). Adults the field was used in this study. emerging in July under long day lengths are light-col- ored and have a small body (first generation). Adults Experiment 1: difference in the reproductive allocation emerging from late August to September under short patterns between size and number of eggs among three day lengths are dark-colored with a large body (second environmental conditions First instar larvae were generation). Day lengths shorter than 12.5 h induce randomly selected from the stock culture, and reared at diapause in larvae derived from female adults of the a density of 40 individuals per plastic container second generation in western Japan (Nakasuji and Ishii (25·17·8cm3) with rice seedlings under the following 161 three environmental conditions: 25C under LD 16:8, tip of soft or tough leaves. A piece of wet filter paper was 25C under LD 14:10, and 20C under LD 14:10 placed at the bottom of the cup. The number of second (hereafter, LD16:8Æ25C, LD14:10Æ25C, and instar larvae of each family was counted on soft and LD14:10Æ20C). These treatments correspond to the tough leaves when food was replenished at 2-day inter- natural field conditions during larval development of the vals. The survival rate of first instar larvae was calculated first generation, the second generation, and the third for soft and tough treatments for each family. The generation after overwintering, respectively. Another product of the number of eggs laid by each female and treatment (LD12:12Æ20C) that corresponds to the the survival rate of first instar larvae was defined as fit- environmental condition of the third generation before ness, and the fitness was compared among treatments overwintering was set up to investigate the effects of (LD16:8Æ25C, LD14:10Æ25C, and LD14:10Æ20C) on the diapause on egg size. At 18–24 h after pupation, pupae soft and tough plots. were weighed with a microbalance (4503MP6E, Sarto- rius, Tokyo). All pupae were placed at LD16:8Æ25C. Experiment 3: difference in egg size among several tem- After emerging, female adults were anesthetized at 4C perature regimes The size of eggs laid by females reared for 1 h and individually marked with a felt pen on their at 20, 22.5, 25, and 27C under LD 14:10 was compared. hind wing. Both male and female adults (20 individuals Egg size data at 20 and 25C are from experiment 1. For per sex) for each treatment were put together in the the conditions of 22.5 and 27C, larvae were reared by plastic cage with the stock culture and fed on 10% honey the same method described for experiment 1, and egg solution at LD16:8Æ25C. Each female that mated with a size data were obtained. male was immediately removed from the cage, and iso- lated in a cylindrical cage (8 cm in diameter, 18 cm in Analysis height) with honey solution and rice seedlings. The number of eggs laid on the rice plants was counted, and In experiments 1 and 3, ANOVA was performed to 30 eggs were randomly sampled from each female on the compare egg size among treatments, with egg size as the first or second day after mating. The diameter (d) and dependent variable, treatment as a fixed effect, and dam height (h) of these eggs were measured using a binocular as a random effect nested in treatment (SAS PROC microscope attached to the video-micrometer (VM-60, GLM, with RANDOM statement and TEST option). Olympus, Tokyo). The volume (V) of the egg was cal- The least square mean of egg size was also compared culated with the assumption that it was a half ellipsoid, among treatments by the two-tailed multiple t-test with using the formula: V=pd2h/6. Eggs laid by females Bonferroni correction following ANOVA. In experiment (families) derived from LD16:8Æ25C, LD14:10Æ25C, 2, the survival rate of first instar larvae was compared and LD14:10Æ20C were used in experiment 2 as follows. between soft and tough treatments using the Wilcoxon signed-ranks test. The Spearman rank correlation (r ) Experiment 2: difference in survival rate and fitness be- s was calculated to assess the relationship between egg size tween insects reared on the soft and tough rice leaves in and survival rate of first instar larvae on soft and tough each generation Seventy eggs laid by females derived leaves for each family within treatments. Two-way from each of the three treatments in experiment 1 were ANOVAs were performed to test the effects of leaf randomly selected and placed on a piece of wet filter toughness and whether there is an interaction in fitness paper in a petri dish (9 cm diameter, 2 cm height) until between treatment and leaf toughness. The effects of they hatched. The hatching rates of all treatments were mean pupal mass, fitness and number of eggs among more than 90%. Thirty hatchlings were reared on the soft treatments were determined by the two-tailed multiple or tough rice leaves (hereafter, soft and tough plots) t-test with Bonferroni correction following ANOVA in during the period of the first larval instar at experiments 1 and 2. The phenotypic correlation (r) LD16:8Æ25C. Soft leaves were obtained from 2-week-old between pupal mass of dam and egg size was calculated rice seedlings that had been planted in plastic pots in using Pearson’s product moment to assess the relation- July. Tough leaves were obtained from rice plants at the ship between female size and egg size for each treatment vegetative stage 6 weeks after transplanting in mid-June. in experiment 1. The significance was tested by trans- The toughness of soft and tough was quantified with a forming the correlation (r) to Fisher’s Z. digital force gauge (MODEL-1308, 9501B, 9502B, Aikoh Engineering, Tokyo). This was estimated as the force (in newtons) required for a 0.75-mm-diameter pin to pene- trate through the leaf. The tip of the pin was located on Results the leaf surface outside the vein. Two readings were taken from each of the 30 leaves sampled, for a total of 60 Reproductive allocation patterns between size observations in the categories of soft and tough. In the and number of eggs among three environmental field, the first instar larvae feed on soft rice leaves in the conditions (experiment 1) first generation, and tough ones in the second generation. Ten hatchlings were introduced into a transparent plastic The developmental period of larvae at LD12:12Æ20C cup (6 cm in diameter and 3 cm in height) and fed on the (male, 86.63±6.78 days; female, 86.39±6.80 days, 162

Table 1 ANOVA for the effects of treatment and dam within 0.065±0.033 and 0.166±0.093 N, respectively; ANO- treatment on egg size of P. g. guttata VA: F=63.34, P<0.0001). Survival rate was not dif- Source df MS FP ferent in hatchlings between the LD16:8Æ25C and LD14:10Æ25C treatments, which corresponded to the Treatment 3 0.14836 15.75 <0.0001 second and the overwintering generations in the field, Dam within treatment 61 0.00942 23.11 <0.0001 respectively (Table 3). However, hatchling survival on Error 1,885 0.00041 – – soft and tough leaves differed significantly in the Type III sums of squares were computed using the general linear LD14:10Æ20C treatment corresponding to the first model of SAS, PROC GLM, with RANDOM statement and TEST generation (Wilcoxon signed-ranks test, P<0.01). The option Spearman rank correlations (rs) between egg size and survival rate were positive on both tough and soft leaves mean ± SD) was much longer than that at in all treatments, but not significant (P>0.05). Fitness LD14:10Æ20C (male, 28.54±2.83 days; female, was compared among the LD16:8Æ25C, LD14:10Æ25C, 30.53±2.38 days). Larval diapause was therefore con- and LD14:10Æ20C treatments for soft and tough leaves firmed to occur at LD12:12Æ20C. ANOVA showed a (Fig. 1). Fitness was highest in the LD14:10Æ20C significant effect of treatment on egg size (Table 1, treatment, followed by LD16:8Æ25C and LD14:10Æ25C P<0.0001). Egg size was largest at LD14:10Æ25C, for both soft and tough, and the fitness under followed by LD16:8Æ25C, LD12:12Æ20C, and LD14:10Æ25C was significantly lower than that of the LD14:10Æ20C. Egg size was not significantly different other two treatments. There was no interaction between among the LD14:10Æ20C, LD12:12Æ20C, and treatment and leaf toughness (ANOVA: df=2, F=0.29, LD16:8Æ25C treatments (Table 2). Pupal mass did not P>0.05) in their effects on fitness. differ among LD 14:10Æ25C, LD 14:10Æ20C, and LD 12:12Æ20C for either sex. Pupae in these treatments were significantly larger than those at LD16:8Æ25C Egg size in several temperature regimes (experiment 3) (two-tailed multiple t-test with Bonferroni correction, P<0.0167). Fecundity was highest at LD12:12Æ20C, Egg size was significantly influenced by treatment followed by LD14:10Æ20C, LD16:8Æ25C, and (ANOVA: df=3, F=34.06, P<0.0001). Egg size did not LD14:10Æ25C (Table 2). The phenotypic correlation (r) differ between 20 and 22.5 C, or between 25 and 27 C between pupal mass of dam and egg size was signifi- (Fig. 2; two-tailed multiple t-test with Bonferroni cor- cantly positive for LD14:10Æ25C(r=0.82, P<0.01), rection, P>0.0167). However, eggs were significantly but was not significant in other treatments (P>0.05). larger at 25 and 27C than at 20 and 22.5C (Fig. 2).

Survival rate and fitness between the soft and tough rice-leaf treatments (experiment 2) Discussion

Leaf toughness was significantly different between soft We found that females of P. g. guttata reared at lower (n=30) and tough (n=30) leaves (mean±SD: temperatures during the immature stages produced

Table 2 Pupal mass, size, and number of eggs laid by females that were exposed to different temperature and photoperiod conditions during larval period

Treatments (corresponding Pupal mass (mg) (mean±SD) Eggs laid by female Egg size (mm3) generation in the field) (n) (mean±SD) Male Female Experimentala In the fieldb (least square (mean) mean±SE)

LD14:10Æ20C n=59 n=44 n=22 n=660 (Overwintering gen.)c 298.2±37.9a 330.0±42.8a 151.7±39.9a 0.183±0.0038a n=50 LD12:12Æ20C n=24 n=28 n=13 n=390 0.151 (Overwintering gen.)c 319.1±38.6a 333.1±32.3a 201.9±41.2b 0.186±0.0049a LD16:8Æ25C n=31 n=32 n=20 n=600 n=50 (1st gen.) 250.6±35.9b 298.5±31.9b 136.4±36.9ac 0.194±0.0040a 0.155 LD14:10Æ25C n=37 n=30 n=10 n=300 n=50 (2nd gen.) 296.8±38.9a 337.6±43.9a 104.1±22.7c 0.228±0.0056b 0.201

Means within a row followed by different letters are significantly different (P<0.0167, two-tailed multiple t-test with Bonferroni cor- rection) aStandard errors calculated using the Type III MS for dam within treatment as an error term (SAS, PROC GLM, with LSMEANS statement and STDERR option) bThese values were calculated from the data of Nakasuji and Kimura (1984) cLD14:10Æ20C and LD12:12Æ20C correspond to the natural field conditions during larval development of the overwintering generation after and before overwintering, respectively 163

Table 3 Comparison of survival rate of first instar larvae between tough and soft rice leaves

Treatment of the parenta LD14:10Æ20C LD16:8Æ25C LD14:10Æ25C

Number of familiesb n=18 n=19 n=10 Survival ratec [median (range)] Soft 0.97 (0.79–1.00) 0.97 (0.80–1.00) 0.95 (0.73–1.00) Tough 0.92 (0.63–1.00)** 0.97 (0.70–1.00)NS 0.96 (0.93–1.00)NS aHatchlings derived from each female in experiment 1 were used bSixty hatchlings per family in experiment 1 were divided into soft or tough cA statistical analysis of survival rate was conducted after arcsine transformation; **P<0.01, NS not significant

200 abab 200 aba

160 160

120 120 Fitness 80 Fitness 80

40 40

0 0 LD14:10.20 LD16:8.25 LD14:10.25 LD14:10.20 LD16:8.25 LD14:10.25 Treatment Treatment Fig. 1 Comparison of fitness among the treatments on tough and correction). Fitness is defined as the product of the number of eggs soft leaves. Means followed by different letters are significantly laid by each female and the survival rate of first instar larvae different (P<0.0167, two-tailed multiple t-test with Bonferroni

more and smaller eggs than those reared at higher large eggs on cogon grass. Thus, fitness of females temperatures (Table 2). Furthermore, survival rates of reared at LD14:10Æ25C may be highest among hatchlings derived from females reared at lower tem- treatments on cogon grass. peratures were significantly different on soft and tough Changes in host conditions may be important envi- leaves (Table 3). These data suggest that in addition to ronmental factors for the evolution of egg size plas- photoperiod, temperature is an important factor in ticity (e.g., Fox et al. 1999; Takakura 2004). Leaf influencing the optimal size and numbers of eggs in P. toughness of plants often increases in advanced growth g. guttata. Because first generation butterflies produce stages (Coley 1980). Leaves of rice and cogon grass on larger eggs, which should be more capable of devel- which P. g. guttata feeds are both softer in June than in oping on tough leaves as compared with overwintering October, but leaves of cogon grass are always tougher (third) generation ones, we predicted that the treat- than those of rice in the same season (Nakasuji 1987). ment corresponding to the first generation environ- Larvae in the first and second generation both feed on ment (LD 16:8Æ25C) would show the highest fitness rice, but rice leaves are softer in the first generation on tough leaves. Instead, we found that the fitness was than in the second. Therefore, hatchlings of P. g. gut- always highest in the LD14:10Æ20C treatment, tata in the first generation feed on the softest leaves of regardless of leaf toughness. However, Roff (2002) spring, while those in the overwintering generation feed compared fitness between development on rice and on on the tough leaves of autumn. In our study, the order cogon grass in P. g. guttata by using the data from of egg size among treatments tended to be consistent Nakasuji and Kimura (1984) and suggested that with that of egg size in the field (Table 2). These data selection should favor laying small eggs on rice but suggest that lower temperatures during the immature 164

aba b host quality was likely to have been higher in our study 0.28 than in the field. (22) (14) (10) (14) Two main types of plasticity have been recognized so far: graded responses expressed by allelic sensitivity and threshold responses expressed by gene regulation (Via 0.24 et al. 1995). Regulatory plasticity represents an active plastic response by the organism that is most likely ) 3 adaptive (Schlichting and Pigliucci 1998). Schlichting and Pigliucci (1995) suggested that this plasticity allows 0.20 anticipatory responses to environmental change. If temperature is an indirect cue used to predict the prog-

Egg size (mm eny’s habitat in each generation, egg size plasticity in response to temperature may be a threshold response in 0.16 P. g. guttata. In our study, egg size at 25 and 27C was considerably larger than at 20 and 22.5C (Fig. 2), but did not differ between 20 and 22.5C, or between 25 and 27C. This result suggests that plasticity is a threshold 0.12 20.0 22.5 25.0 27.0 response and a threshold is present between 22.5 and 25C. Temperature(°C) In the butterfly Bicyclus anynana, females lay larger Fig. 2 Comparison of egg size for larvae reared under LD14:10 at eggs at lower temperatures (Fischer et al. 2003a, 2004). 20, 22.5, 25, and 27C (least square mean±SE). Standard errors The response is adaptive because of the influence of calculated using the Type III MS for dam within treatment as an temperature on survival and development during error term (SAS, PROC GLM, with LSMEANS statement and immature stages (Fischer et al. 2003b). Selection favors STDERR option). Least square means within a row followed by the production of larger progeny at lower temperatures different letters are significantly different (two-tailed multiple t-test with Bonferroni correction, P<0.0167). Numbers in parentheses either to shorten development time, thereby reducing refer to the number of females for which egg size was measured at exposure to sources of mortality (Yampolsky and each temperature condition. The size of 30 eggs was measured per Scheiner 1996), or to decrease generation time (Perrin female 1988). However, in some species, it is unclear whether the responses of egg size to temperature are adaptations to temperature itself, non-adaptive physiological re- stages in the overwintering generation constitute an sponses to temperature, or whether temperature is used indirect cue for the production of smaller and more as a cue to predict some other environmental condition eggs in P. g. guttata. For many insects, timing of life- (Fox and Czesak 2000). The extent to which tempera- history events often changes in response to photoperiod ture-mediated natural selection vs. the environmental (e.g., Ishihara 2000; Zhou 2001; Tanaka et al. 1993). effect of temperature differentially affect traits at differ- However, the combination of photoperiod and another ent temperatures has generated debate between adaptive factor such as temperature is more reliable as an indi- (genetic) and developmental (nongenetic) hypotheses rect cue to predict various environmental conditions. (Stillwell and Fox 2005). Both photoperiod and temperature are used as cues for We believe, for several reasons, that egg size plasticity development and reproduction in some species (e.g., in response to temperature is unlikely to be a non- Chinnery and Williams 2003; Holmes et al. 1994; Kelly adaptive physiological response to temperature or an 2001). adaptation to temperature itself in P. g. guttata. First, In our study, females laid relatively larger eggs than some arthropods mature with a larger body size when those observed in the field (Table 2). Therefore, survival reared at lower temperatures (Moore and Folt 1993), rate or fitness of hatchlings on soft and tough leaves potentially resulting in an increase in egg size. However, might not perfectly correspond to the natural field sit- pupal mass did not differ between LD14:10Æ20C and uation. Two possibilities may explain the difference be- LD14:10Æ25C for either sex of P. g. guttata (Table 2). tween the findings in our study and the field. The first is Secondly, smaller eggs laid by females reared at lower that larvae in the overwintering generation encounter temperatures may result from the effect of lower tem- harsh temperatures in the winter season that were not perature during development, but fitness of females reproduced in our study. The second possibility relates reared at 20C was higher than that of females reared at to the difference in host plant quality. In our study, 25C on both soft and tough leaves. Total mass of eggs larvae were fed on young rice seedlings in all treatments was also significantly larger at LD16:8Æ20C than at of experiment 1. However, in the field, larvae of P. g. LD16:8Æ25CinP. g. guttata (T. Seko et al., unpub- guttata feed on old grasses of rice and cogon grass in the lished). Thirdly, larvae hatched from larger eggs in the second and overwintering generations. Generally, young overwintering generation may better withstand cold leaves are softer and have better nutritional value than stress in the winter season as compared with those in old leaves (Coley 1980; Scriber and Slanski 1981). Thus, other generations. However, larger eggs may have little 165 response to cold stress in P. g. guttata because the sur- Holmes JA, Beamish FWH, Seelye JG, Sower SA, Youson JH vival rate from hatching to emergence is high both at 20 (1994) Long-term influence of water temperature, photoperiod, and food-deprivation on metamorphosis of sea lamprey, Petr- and 25C under LD14:10 (T. Seko, F. Nakasuji, omyzon marinus. Can J Fish Aquat Sci 51:2045–2051 unpublished). Although the effects of lower tempera- Ishihara M (2000) Effect of variation in photoperiodic response tures in the winter season on survival and development on diapause induction and developmental time in the willow of eggs of different sizes is unknown, the host condition leaf beetle, Plagiodera versicolora. Entomol Exp Appl 96:27– 32 in the overwintering generation is more likely to be a Ishii M, Hidaka T (1979) Seasonal polymorphism of the adult rice- primary factor favoring larger eggs than temperature plant skipper, Parnara guttata guttata (Lepidoptera: Hesperii- itself. We cannot exclude nonadaptive physiological ef- dae) and its control. Appl Entomol Zool 14:173–184 fects of temperature in P. g. guttata because tempera- Kelly MS (2001) Environmental parameters controlling gameto- ture-mediated plasticity can be complex. However, our genesis in the echinoid Psammechinus miliaris. J Exp Mar Biol Ecol 266:67–80 findings suggest that the plasticity of egg size in response Kidokoro T (1992) Occurrence of rice-plant skipper, Parnara gut- to temperature is adaptive, and temperature combined tata guttata (Lepidoptera: Hesperiidae) in North Japan and with photoperiod provides predictable cues for the possibility of immigration from south Japan (in Japanese with environmental conditions that will be experienced by English summary). Jpn J Appl Entomol Zool 36:89–93 Moore M, Folt C (1993) Zooplankton body size and community offspring of the next generation. structure: effects of thermal and toxicant steress. TREE 8:178– 183 Acknowledgements We wish to thank Dr. M. Ishihara (Department Nakasuji F (1982) Seasonal changes in native host plants of a of Biological Science, University of Osaka Prefecture) for use of a migrant skipper, Parnara guttata Bremer Et Grey (Lepidoptera, digital force gauge. Two anonymous reviewers and an editor Hesperiidae). 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