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Change in significance of feeding during larval development in the yellow-spotted longicorn beetle, Psacotheahilaris
Journal of Insect Physiology 49 (2003) 975–981 www.elsevier.com/locate/jinsphys
Change in significance of feeding during larval development in the yellow-spotted longicorn beetle, Psacothea hilaris Yoshinori Shintani a,1, Florence N. Munyiri a, Yukio Ishikawa a,∗ a
Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Received 15 August 2002; received in revised form 7 July 2003; accepted 8 July 2003
Abstract Larvae of the west-Japan type yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae), show a long-day type photoperiodic response at 25 °C; under long-day conditions, larvae pupate after the fourth or fifth instar, while under shortday conditions, they undergo a few nonstationary supernumerary molts and eventually enter diapause. In the present study, the effect of food on the development and photoperiodic response of the larvae was examined with special reference to molting and pupation. Although the pupal body size was greatly affected by the food quality and the length of feeding, the critical day length for induction of metamorphosis at 25 °C was always between 13.5 and 14 h. Exposure to starvation of larvae reared on the standard diet revealed that the capability to pupate is acquired after a few days of feeding in the fourth instar. In the larvae that had acquired the capability to pupate, premature pupation was induced by exposure to starvation, indicating that feeding becomes dispensable long before it is normally terminated. 2003 Elsevier Ltd. All rights reserved. Keywords: Feeding; Starvation; Psacothea hilaris; Supernumerary molt; Metamorphosis; Premature pupation
1. Introduction The yellow-spotted longicorn beetle, Psacothea hilaris (Pascoe) (Coleoptera: Cerambycidae), is widely distributed in easternmost Asia, and infests Moraceae trees. This species is an important pest of sericulture in Japan because larvae bore tunnels in the trunks and adults feed on the leaves of the mulberry trees. Due to its large geographic variation in morphology, this insect is divided into many subspecies (Kusama and Takakuwa, 1984). One subspecies, P. hilaris hilaris, inhabits three of the four main islands of Japan, Honshu, Shikoku and Kyushu. Within this subspecies, two morphological types have been recognized, i.e. ‘east-Japan type’ and ‘westJapan type’. The two types are distinguished by the spot patterns on the pronotum of adults (Iba, 1980). The larval photoperiodic response of P. hilaris has Corresponding author. Tel.: +81-3-5841-5061; fax: +81-3-58415060. E-mail address: [email protected] (Y. Ishikawa). 1 Current address: Hokuriku Research Center, National Agricultural Research Center, Inada 1-2-1, Joetsu, Niigata 943-0193, Japan. ∗
0022-1910/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0022-1910(03)00167-7
been examined in detail in the west-Japan type population (Shintani et al., 1996a,b; Shintani and Ishikawa, 1997a,b, 1998). Larval development of this insect at 25 °C is under photoperiodic control with a critical day length between 13.5 and 14 h. Under long-day conditions, pupation occurs after the fourth or fifth instar, while under short-day conditions, the larvae undergo a few supernumerary molts and eventually enter diapause. Long-day strongly promotes metamorphosis; the larvae reared under short-day conditions until the fourth or later instars pupate in the current or next instar if the photoperiod is changed to long-day (Shintani and Ishikawa, 1998). As is the case with other cerambycid species, P. hilaris larvae do not move from one host plant to another; they have to complete their larval development in the trunk in which the eggs were deposited. Therefore, larval development largely depends on the nutritional value of the host tree, which varies from tree to tree. Furthermore, since all branches of mulberry trees are cut off in autumn, as is the practice in sericulture, the majority of larvae that are left in the cut off branches are confronted with deterioration of their food. However, it has been
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well documented that adults of P. hilaris emerge from the cut branches. P. hilaris larvae therefore must have mechanisms for adapting to the changes in food quality. In the present study, the effects of diet quality and starvation on the larval development were examined with special reference to the photoperiodic response. We also demonstrate that feeding becomes dispensable long before it is normally terminated.
2. Materials and methods 2.1. Insects Adults of P. hilaris were collected in the mulberry fields in Ino, Kochi Prefecture, Japan (33.5°N, 133.4°E) on 20 July, 1994. They were identified as west-Japan type by the yellowish severed spot pattern on the pronotum (Iba, 1980). The insects were reared in the laboratory following the method of Shintani et al. (1996a). The eggs deposited beneath the cortex of cut mulberry branches were collected and incubated on wet filter paper at 25 °C. The hatched larvae were individually placed in plastic Petri dishes (6 cm diameter) and supplied with artificial diet. The third, fourth and fifth laboratory generations were used for the experiments. 2.2. Preparation of artificial diets Artificial diets were prepared principally according to the method of Kawakami and Shimane (1985). To examine the effect of food quality on larval development, diets of different nutritional quality were made as follows. The two major ingredients, i.e. a commercial diet for the silkworm (dry type, Japan Chrollera Co., Tokyo) and the cellulose powder, were mixed in serially varied ratios: 50:50 (D50, standard diet), 25:75 (D25), 12.5:87.5 (D13) or 6.25:93.75 (D6). A sample of 200 g of such a mixture was thoroughly mixed with 10 g agar, 1 g sorbic acid, and 0.01 g chloramphenicol. The paste prepared by adding 600 ml of water was autoclaved at 121 °C for 10 min. 2.3. Effect of the quality of diet on the photoperiodic response and development of larvae The larvae were reared from hatching on one of the four artificial diets described above (D50, D25, D13, or D6) under one of the following photoperiods (light:darkness in h): 13L:11D, 13.5L:10.5D, 14L:10D, 14.5L:9.5D and 15L:9D. The diet was replaced with a fresh portion every four days. Larval development was monitored daily and the body weight was measured on the day of each ecdysis. When no larval molting or pupation was observed for 60 days in an instar, the lar-
vae were regarded as being in diapause, and their body weight was recorded. 2.4. Effect of starvation To examine the effect of starvation on larval development, the larvae that had been reared on D50 diet (D50larvae) were deprived of food at various ages under either 15L:9D (long-day) or diapause-inducing 12L:12D (short-day). In addition, to examine whether long-day cycles can override the destiny of diapause-conditioned larvae even under starved conditions, D50-larvae reared under 12L:12D were subjected to a photoperiodic change (15L:9D) concurrent with the start of starvation. All the experimental schemes in this section are summarized in Fig. 5 with the results. In the starvation experiments, the Petri dishes were changed from plastic to glass to prevent larvae from chewing the dishes. Body weight was measured at the start of starvation and when pupation occurred. 2.5. Responses of the fifth, sixth, and seventh instars to poor diet To examine if poor nutrition promotes metamorphosis, D50-larvae reared under 12L:12D until day 0 and day 8 of the fifth instar, day 0 and day 8 of the sixth instar, and day 0 of the seventh instar were subjected to three feeding regimes, i.e. normal feeding (D50 diet), semi-starvation (D6 diet) and complete starvation, and concurrently exposed to the 15L:9D photoperiod. 2.6. Photoperiodic response in starving larvae To test the effect of starvation on the photoperiodic response, the larvae reared on D50 diet under 12L:12D, 13L:11D, 13.5L:10.5D, 14L:10D and 15L:9D were deprived of food on day 4 of the fourth instar. 2.7. Analysis of data The data were analyzed by ANOVA unless otherwise stated, and the Tukey–Kramer multiple comparison test was applied if necessary. A nonparametric version of the ANOVA (the Kruskal–Wallis test) was applied to test the effect of food quality on the number of larval molts because normality of the data could not be assumed. Significance level was set at a = 0.05. 3. Results 3.1. Effect of food quality on mortality The larvae fed on nutritionally poor diets (D13 and D6) frequently perished in the early instars (first to third).
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When the data for various photoperiods were pooled, the mortality of the first to third instars in the D50, D25, D13 and D6 diet groups was 20%, 23%, 55% and 62%, respectively. Death in the first instar appeared to occur mostly at the beginning of feeding. After the third larval molting, death rarely occurred in any nutritional group; most of the larvae grew to the pupal stage or became diapause larvae. Data for the insects that died before becoming mature larvae were excluded from the analysis of photoperiodic response. 3.2. Effect of food quality on photoperiodic response of the larvae The nutritional quality of the diet in the range examined showed no effect on the photoperiodic responses of the P. hilaris larvae, the critical day length for induction of diapause being between 13.5 and 14 h (Fig. 1). Under longer day lengths (ⱖ14 h), pupation occurred in most individuals, whereas under shorter day lengths (ⱕ13.5 h), almost all the larvae remained as larvae. Under typical long-day conditions (15L:9D), most larvae pupated after the fourth or fifth instar, whereas under typical short-day conditions (13L:11D), the larvae entered diapause in the sixth to ninth instar (Fig. 2). Food quality showed no significant effect on the number of larval molts in either the case of pupation or diapause (the Kruskal–Wallis test, p ⬎ 0.05). Unlike the number of molts, the duration of the larval stage was largely prolonged with a decrease in food quality. Under 15L:9D, for example, the length of the larval stage increased significantly from 41.0 d in the D50 diet group to 57.4 d in the D6 diet group, when com-
Fig. 2. Number of larval instars in P. hilaris reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6) under a long-day (15L:9D) and under a short-day (13L:11D).
pared among individuals that pupated after the fourth instar (Fig. 3). 3.3. Effect of food quality on body size
Fig. 1. Photoperiodic responses of Psacothea hilaris larvae reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6). Responses of the larvae reared on the standard D50 diet until day 4 of the fourth instar and then completely starved are also shown (dotted line). Sample size was 17–29.
Food quality exerted a profound effect on the larval and pupal body size (Fig. 4). The body weight of the larvae after the third ecdysis was significantly different; the larvae reared on the D50 diet were more than twofold heavier than those reared on the D6 diet (Fig. 4a). The effect of food quality on body size was even more marked in pupae. The pupal body weight of individuals reared on the D6 diet was only about one-fourth the weight of those reared on the D50 diet, regardless of the number of larval instars the larvae underwent before pupation (Fig. 4b). This result suggests that the onset of metamorphosis is not triggered by a specific body size. As for the body size of the diapause larvae, the effect of food quality was relatively small; the body weight of the larvae reared on the D6 diet was reduced by about 50% in comparison with larvae on the D50 diet (Fig. 4c). This relatively large body size was attained not by the
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Fig. 3. Total larval period of P. hilaris reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6) under a long-day (15L:9D). Bars show the means and standard deviations. Bars with the same letter in the same last instar group do not differ significantly by the Tukey–Kramer multiple comparison test at the level of 5%. Sample sizes are shown inside the bars.
increase in the number of larval molts (Fig. 2), but by prolongation of each larval instar (data not shown). 3.4. The responses of larvae to starvation Since the onset of pupation is not triggered by a sizedependent mechanism, we sought possible developmental triggers. To this end, the larvae were deprived of food at various ages and their molting and pupation were investigated (Fig. 5). Starvation of the early instars (second or third) resulted in death within 15 days irrespective of the photoperiod. Interestingly, a high percentage of these early instar larvae accomplished one more larval molt before dying, when they were starved after a few days of feeding (L–L in Fig. 5a,b). The effect of starvation during the fourth or later instars depended on the photoperiod. Under 12L:12D, the fourth or later instar larvae tolerated starvation for long periods, but did not pupate except for one sixthinstar larva (Fig. 5a). In contrast, under 15L:9D (Fig. 5b), or when day lengths were switched from 12L:12D to 15L:9D on the day of food deprivation (Fig. 5c), the commitment to pupation occurred during the current or next instar (P and L–P in Fig. 5b,c). In both the photoperiodic regimes, no pupae were obtained among the larvae starved on the day of ecdysis to the fourth instar; 40–50% pupation resulted among larvae fed for 2 days; over 75% pupation resulted among larvae fed for 4 or more days. Thus, the critical stage for the destiny of larvae appeared to be around day 2 of the fourth instar. Both larval weight on the day of food deprivation and
Fig. 4. Body weight of P. hilaris reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6). (a) Body weight of the larvae at the third larval molting under a long-day (15L:9D). (b) Body weight of the pupae. Pupae were classified based on the larval instars they completed. (c) Body weight of larvae on day 60 of the diapausing instar under a short-day (13L:11D). Bars indicate standard deviations. Sample sizes are shown inside the bars.
the resulting pupal weight increased with the stage of food deprivation (Fig. 6). Within the fourth instar, the larval mass doubled from day 0 to day 2 and increased by another 20% during days 2–4 and 4–8 (Fig. 6), paralleling the increase in commitment to pupation (Fig. 5b,c). Hence, at least some feeding and growth during the fourth instar were necessary to commit larvae to pupation. The fifth-, sixth- or seventh-instar larvae were able to pupate without feeding, suggesting that feeding is not indispensable in the fifth and later instars (Fig. 5c). Regardless of the instar and photoperiod, a larval–larval molt never occurred when the larvae were exposed to starvation immediately after molting; some feeding was required for the next larval–larval molt to be induced.
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time required for induction of pupation. A poor diet, in addition to starvation, suppressed larval molting and promoted pupation (Table 1). This was clearly shown when the D50-larvae were subjected to the photoperiodic change on day 8 of the fifth instar; more than half of them underwent an additional molt and thereafter pupated, whereas when the food was also changed to the D6 diet or removed, the additional molt was significantly suppressed, in addition to shortening of the pre-pupation period in the individuals that pupated without an additional molt (Table 1). A similar phenomenon was observed in the cases of the sixth- and seventh-instar larvae (Table 1). 3.6. No change of photoperiodic response in starving larvae
Fig. 5. Responses of P. hilaris larvae to starvation at various ages. Larvae were reared on the standard D50 diet until the given age and thereafter the diet was removed. Larvae were kept continuously under (a) a short-day (12L:12D), (b) a long-day (15L:9D), or (c) the shortday was changed to long-day concurrently with food deprivation. Sample size was 20–31. P: pupated without larval molt. L-P: pupated after a larval molt. L-L: a larval molt occurred but did not pupate. L: no molt occurred.
After finding that starvation under the long-day condition promotes pupation, we examined whether this effect can override the photoperiodic control of diapause using the D50-larvae starved from day 4 of the fourth instar, i.e. soon after acquisition of the ability to pupate. We found the same critical day length (between 13.5 and 14 h) as in the normally fed or semi-starved larvae (Fig. 1, dotted line). Since the sensitivity to photoperiod is either absent or very low during the first to third instars (Shintani and Ishikawa, 1998), the response observed in this experiment can be regarded as that under starvation. Thus, it is concluded that the photoperiodic control of metamorphosis has precedence over the promotion of pupation by starvation.
4. Discussion
Fig. 6. The body weights of P. hilaris larvae at the time of food deprivation and of the resulting pupae. Larvae were reared on the standard D50 diet under 12L:12D until the given age and thereafter the food was removed concurrently with a photoperiodic change to 15L:9D. NP: no pupation occurred. Bars indicate standard deviations. Sample sizes are shown above the bars.
3.5. Poor diet promotes metamorphosis in the fifth and later instars As shown above, the fifth and later instars are able to pupate without any feeding. The response of such larvae to a sudden decrease in food quality or starvation was analyzed in terms of additional larval molting and the
One of the conspicuous characteristics of the cerambycid beetles is a large variation in adult body size. Field-collected P. hilaris adults have variable sizes even within a local population (more than 2.5-fold difference in the body length in extreme cases, unpublished data). The present study suggests that the variation in food quality is an important cause of this variation. The variation in body size is also caused by an intrinsic variation in the number of larval molts, which is largely affected by photoperiod (Shintani et al., 1996a,b) and temperature (Shintani and Ishikawa, 1997a; Watari et al., 2002). In particular, nonstationary supernumerary larval molts associated with diapause markedly contribute to the size variation (Shintani et al., 1996a). In several insects, food serves as a major diapauseregulating factor or a modifier of photoperiodic responses (Tauber et al., 1986). For example, the photoperiodic induction of diapause in the Colorado potato beetle, Leptinotarsa decemlineata, is enhanced when the insects feed on physiologically aged potato leaves (de Wilde et al., 1969; Hare, 1983). In P. hilaris, however,
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Table 1 Responses of fifth, sixth and seventh instar Psacothea hilaris larvae to poor diet and starvationa Diet/ageb
Pre-pupation period (days ± SD)
Additional larval molt induced (%)
Fifth, 0 Fifth, 8 Sixth, 0 Sixth, 8 Seventh, 0 Fifth, 0 D50 D6 Starvation
8 0 0
52 7 13
8 – 0
44 – 13
0 – 0
Fifth, 8
c
Sixth, 0
24.0 ± 2.8a 20.5 ± 3.1a 23.4 ± 2.4a 21.5 ± 3.2a 16.8 ± 3.9a – 16.3 ± 1.9b 15.4 ± 3.8b 17.0 ± 1.6b
Sixth, 8
Seventh, 0
24.0 ± 2.3a – 18.2 ± 1.5b
25.5 ± 2.6a – 18.5 ± 1.9b
– Not examined. Means with the same letter in the same column do not differ significantly by the t-test (sixth, 0; sixth, 8 and seventh, 0) or the Tukey-Kramer test for multiple comparisons (fifth, 0 and fifth, 8) at the level of 5%. Sample size was 21–31. a Larvae were reared on the D50 diet under 12L:12D until the given age and then transferred to 15L:9D and either fed the nutritionally poor D6 diet or starved. Controls were reared continuously on D50 diet with the photoperiodic change. b Instar, days in the instar. c The time from the start of experiment (photoperiodic change and/or starvation) to pupation. Data for individuals that pupated without an additional molt are presented.
the photoperiodic response curve and the number of larval molts were not affected by food quality in the range examined. Several insects exhibit the capability to undergo stationary or nonstationary supernumerary larval molts in response to extrinsic factors, such as starvation (e.g. Iwata and Nishimoto, 1985 for Lyctus brunneus) and crowded condition (e.g. Nakakita, 1982; Connat et al., 1991; Kotaki et al., 1993 for tenebrionids). Low humidity may be among the factors that enhance supernumerary molts in Sitophilus oryzae (e.g. Pittendrigh et al., 1997). However, photoperiod-induced nonstationary molts as observed in P. hilaris have been reported in few species (e.g. Gadenne et al., 1997 for Sesamia nonagrioides), and the biological significance of this phenomenon is not well understood. One possibility is that this is an adaptation for overwintering (Watari et al., 2002). The nonstationary supernumerary molting makes the larvae larger, and the larger larvae can be more adaptive for overwintering. In this regard, the presence of a threshold weight for successful overwintering needs to be investigated. Metamorphosis in many insects is triggered when a threshold size or weight is attained within a defined period during larval development (Sehnal, 1985 for review); at the beginning of the fifth instar in Manduca sexta (Nijhout, 1975, 1981) and Trichoplusia ni (Jones et al., 1981), and at the later part of the third instar in Blattella germanica (Tanaka, 1981). In these species, weight or size are useful predictors for the nature of the next molt (larval–larval or larval–pupal, nymphal–nymphal or nymphal–adult). A large developmental variability in P. hilaris made it difficult to analyze such size-related control of metamorphosis. However, it is now clear that the size is not a proximate cue for pupation in this species, although there is a threshold weight that the larvae must attain for a successful metamorphosis (see Fig. 6; Munyiri et al., 2003). Many insects have the ability to extend the last larval
instar in response to food deficiency, and wait for their critical size to be attained at a later time (e.g. Bradshaw and Johnson, 1995 for Wyeomyia smithii; Petersen et al., 2000 for M. sexta). In clear contrast to this phenomenon, some insects undergo premature pupation in response to food deprivation. Such a response was found in the wax moth (Sehnal, 1966) and was examined in detail in the dung beetle, Onthophagus taurus (Shafiei et al., 2001). Once the food resource (dung) of the beetle larvae is used up, locating additional resources is impossible. Those larvae that have reached a specific weight in the last (third) instar pupate and produce small adults. P. hilaris is the second beetle species in which the premature pupation in response to food shortage was clearly demonstrated. In the present study, however, the mechanism that controls the onset of metamorphosis could not be clarified, and remains to be studied. Acknowledgements We thank Mr. R. Ueda (Kochi Prefectural Sericultural Experiment Station) for collecting yellow-spotted longicorn beetles and two anonymous referees for many suggestions to improve the manuscript. We are grateful to Professor S. Tatsuki (Tokyo University) for encouragement throughout the course of this work. References Bradshaw, W.E., Johnson, K., 1995. Initiation of metamorphosis in the pitcher-plant mosquito: effects of larval growth history. Ecology 76, 2055–2065. Connat, J.L., Delbecque, J.P., Glitho, I., Delachambre, J., 1991. The onset of metamorphosis in Tenebrio molitor larvae (Insecta, Coleoptera) under grouped, isolated and starved conditions. Journal of Insect Physiology 37, 653–662. de Wilde, J., Bongers, W., Schooneveld, H., 1969. Effects of age on phytophagous insects. Entomologia Experimentalis et Applicata 12, 714–720.
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Change in significance of feeding during larval development in the yellow-spotted longicorn beetle, Psacothea hilaris Yoshinori Shintani a,1, Florence N. Munyiri a, Yukio Ishikawa a,∗ a
Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Received 15 August 2002; received in revised form 7 July 2003; accepted 8 July 2003
Abstract Larvae of the west-Japan type yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae), show a long-day type photoperiodic response at 25 °C; under long-day conditions, larvae pupate after the fourth or fifth instar, while under shortday conditions, they undergo a few nonstationary supernumerary molts and eventually enter diapause. In the present study, the effect of food on the development and photoperiodic response of the larvae was examined with special reference to molting and pupation. Although the pupal body size was greatly affected by the food quality and the length of feeding, the critical day length for induction of metamorphosis at 25 °C was always between 13.5 and 14 h. Exposure to starvation of larvae reared on the standard diet revealed that the capability to pupate is acquired after a few days of feeding in the fourth instar. In the larvae that had acquired the capability to pupate, premature pupation was induced by exposure to starvation, indicating that feeding becomes dispensable long before it is normally terminated. 2003 Elsevier Ltd. All rights reserved. Keywords: Feeding; Starvation; Psacothea hilaris; Supernumerary molt; Metamorphosis; Premature pupation
1. Introduction The yellow-spotted longicorn beetle, Psacothea hilaris (Pascoe) (Coleoptera: Cerambycidae), is widely distributed in easternmost Asia, and infests Moraceae trees. This species is an important pest of sericulture in Japan because larvae bore tunnels in the trunks and adults feed on the leaves of the mulberry trees. Due to its large geographic variation in morphology, this insect is divided into many subspecies (Kusama and Takakuwa, 1984). One subspecies, P. hilaris hilaris, inhabits three of the four main islands of Japan, Honshu, Shikoku and Kyushu. Within this subspecies, two morphological types have been recognized, i.e. ‘east-Japan type’ and ‘westJapan type’. The two types are distinguished by the spot patterns on the pronotum of adults (Iba, 1980). The larval photoperiodic response of P. hilaris has Corresponding author. Tel.: +81-3-5841-5061; fax: +81-3-58415060. E-mail address: [email protected] (Y. Ishikawa). 1 Current address: Hokuriku Research Center, National Agricultural Research Center, Inada 1-2-1, Joetsu, Niigata 943-0193, Japan. ∗
0022-1910/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0022-1910(03)00167-7
been examined in detail in the west-Japan type population (Shintani et al., 1996a,b; Shintani and Ishikawa, 1997a,b, 1998). Larval development of this insect at 25 °C is under photoperiodic control with a critical day length between 13.5 and 14 h. Under long-day conditions, pupation occurs after the fourth or fifth instar, while under short-day conditions, the larvae undergo a few supernumerary molts and eventually enter diapause. Long-day strongly promotes metamorphosis; the larvae reared under short-day conditions until the fourth or later instars pupate in the current or next instar if the photoperiod is changed to long-day (Shintani and Ishikawa, 1998). As is the case with other cerambycid species, P. hilaris larvae do not move from one host plant to another; they have to complete their larval development in the trunk in which the eggs were deposited. Therefore, larval development largely depends on the nutritional value of the host tree, which varies from tree to tree. Furthermore, since all branches of mulberry trees are cut off in autumn, as is the practice in sericulture, the majority of larvae that are left in the cut off branches are confronted with deterioration of their food. However, it has been
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well documented that adults of P. hilaris emerge from the cut branches. P. hilaris larvae therefore must have mechanisms for adapting to the changes in food quality. In the present study, the effects of diet quality and starvation on the larval development were examined with special reference to the photoperiodic response. We also demonstrate that feeding becomes dispensable long before it is normally terminated.
2. Materials and methods 2.1. Insects Adults of P. hilaris were collected in the mulberry fields in Ino, Kochi Prefecture, Japan (33.5°N, 133.4°E) on 20 July, 1994. They were identified as west-Japan type by the yellowish severed spot pattern on the pronotum (Iba, 1980). The insects were reared in the laboratory following the method of Shintani et al. (1996a). The eggs deposited beneath the cortex of cut mulberry branches were collected and incubated on wet filter paper at 25 °C. The hatched larvae were individually placed in plastic Petri dishes (6 cm diameter) and supplied with artificial diet. The third, fourth and fifth laboratory generations were used for the experiments. 2.2. Preparation of artificial diets Artificial diets were prepared principally according to the method of Kawakami and Shimane (1985). To examine the effect of food quality on larval development, diets of different nutritional quality were made as follows. The two major ingredients, i.e. a commercial diet for the silkworm (dry type, Japan Chrollera Co., Tokyo) and the cellulose powder, were mixed in serially varied ratios: 50:50 (D50, standard diet), 25:75 (D25), 12.5:87.5 (D13) or 6.25:93.75 (D6). A sample of 200 g of such a mixture was thoroughly mixed with 10 g agar, 1 g sorbic acid, and 0.01 g chloramphenicol. The paste prepared by adding 600 ml of water was autoclaved at 121 °C for 10 min. 2.3. Effect of the quality of diet on the photoperiodic response and development of larvae The larvae were reared from hatching on one of the four artificial diets described above (D50, D25, D13, or D6) under one of the following photoperiods (light:darkness in h): 13L:11D, 13.5L:10.5D, 14L:10D, 14.5L:9.5D and 15L:9D. The diet was replaced with a fresh portion every four days. Larval development was monitored daily and the body weight was measured on the day of each ecdysis. When no larval molting or pupation was observed for 60 days in an instar, the lar-
vae were regarded as being in diapause, and their body weight was recorded. 2.4. Effect of starvation To examine the effect of starvation on larval development, the larvae that had been reared on D50 diet (D50larvae) were deprived of food at various ages under either 15L:9D (long-day) or diapause-inducing 12L:12D (short-day). In addition, to examine whether long-day cycles can override the destiny of diapause-conditioned larvae even under starved conditions, D50-larvae reared under 12L:12D were subjected to a photoperiodic change (15L:9D) concurrent with the start of starvation. All the experimental schemes in this section are summarized in Fig. 5 with the results. In the starvation experiments, the Petri dishes were changed from plastic to glass to prevent larvae from chewing the dishes. Body weight was measured at the start of starvation and when pupation occurred. 2.5. Responses of the fifth, sixth, and seventh instars to poor diet To examine if poor nutrition promotes metamorphosis, D50-larvae reared under 12L:12D until day 0 and day 8 of the fifth instar, day 0 and day 8 of the sixth instar, and day 0 of the seventh instar were subjected to three feeding regimes, i.e. normal feeding (D50 diet), semi-starvation (D6 diet) and complete starvation, and concurrently exposed to the 15L:9D photoperiod. 2.6. Photoperiodic response in starving larvae To test the effect of starvation on the photoperiodic response, the larvae reared on D50 diet under 12L:12D, 13L:11D, 13.5L:10.5D, 14L:10D and 15L:9D were deprived of food on day 4 of the fourth instar. 2.7. Analysis of data The data were analyzed by ANOVA unless otherwise stated, and the Tukey–Kramer multiple comparison test was applied if necessary. A nonparametric version of the ANOVA (the Kruskal–Wallis test) was applied to test the effect of food quality on the number of larval molts because normality of the data could not be assumed. Significance level was set at a = 0.05. 3. Results 3.1. Effect of food quality on mortality The larvae fed on nutritionally poor diets (D13 and D6) frequently perished in the early instars (first to third).
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When the data for various photoperiods were pooled, the mortality of the first to third instars in the D50, D25, D13 and D6 diet groups was 20%, 23%, 55% and 62%, respectively. Death in the first instar appeared to occur mostly at the beginning of feeding. After the third larval molting, death rarely occurred in any nutritional group; most of the larvae grew to the pupal stage or became diapause larvae. Data for the insects that died before becoming mature larvae were excluded from the analysis of photoperiodic response. 3.2. Effect of food quality on photoperiodic response of the larvae The nutritional quality of the diet in the range examined showed no effect on the photoperiodic responses of the P. hilaris larvae, the critical day length for induction of diapause being between 13.5 and 14 h (Fig. 1). Under longer day lengths (ⱖ14 h), pupation occurred in most individuals, whereas under shorter day lengths (ⱕ13.5 h), almost all the larvae remained as larvae. Under typical long-day conditions (15L:9D), most larvae pupated after the fourth or fifth instar, whereas under typical short-day conditions (13L:11D), the larvae entered diapause in the sixth to ninth instar (Fig. 2). Food quality showed no significant effect on the number of larval molts in either the case of pupation or diapause (the Kruskal–Wallis test, p ⬎ 0.05). Unlike the number of molts, the duration of the larval stage was largely prolonged with a decrease in food quality. Under 15L:9D, for example, the length of the larval stage increased significantly from 41.0 d in the D50 diet group to 57.4 d in the D6 diet group, when com-
Fig. 2. Number of larval instars in P. hilaris reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6) under a long-day (15L:9D) and under a short-day (13L:11D).
pared among individuals that pupated after the fourth instar (Fig. 3). 3.3. Effect of food quality on body size
Fig. 1. Photoperiodic responses of Psacothea hilaris larvae reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6). Responses of the larvae reared on the standard D50 diet until day 4 of the fourth instar and then completely starved are also shown (dotted line). Sample size was 17–29.
Food quality exerted a profound effect on the larval and pupal body size (Fig. 4). The body weight of the larvae after the third ecdysis was significantly different; the larvae reared on the D50 diet were more than twofold heavier than those reared on the D6 diet (Fig. 4a). The effect of food quality on body size was even more marked in pupae. The pupal body weight of individuals reared on the D6 diet was only about one-fourth the weight of those reared on the D50 diet, regardless of the number of larval instars the larvae underwent before pupation (Fig. 4b). This result suggests that the onset of metamorphosis is not triggered by a specific body size. As for the body size of the diapause larvae, the effect of food quality was relatively small; the body weight of the larvae reared on the D6 diet was reduced by about 50% in comparison with larvae on the D50 diet (Fig. 4c). This relatively large body size was attained not by the
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Fig. 3. Total larval period of P. hilaris reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6) under a long-day (15L:9D). Bars show the means and standard deviations. Bars with the same letter in the same last instar group do not differ significantly by the Tukey–Kramer multiple comparison test at the level of 5%. Sample sizes are shown inside the bars.
increase in the number of larval molts (Fig. 2), but by prolongation of each larval instar (data not shown). 3.4. The responses of larvae to starvation Since the onset of pupation is not triggered by a sizedependent mechanism, we sought possible developmental triggers. To this end, the larvae were deprived of food at various ages and their molting and pupation were investigated (Fig. 5). Starvation of the early instars (second or third) resulted in death within 15 days irrespective of the photoperiod. Interestingly, a high percentage of these early instar larvae accomplished one more larval molt before dying, when they were starved after a few days of feeding (L–L in Fig. 5a,b). The effect of starvation during the fourth or later instars depended on the photoperiod. Under 12L:12D, the fourth or later instar larvae tolerated starvation for long periods, but did not pupate except for one sixthinstar larva (Fig. 5a). In contrast, under 15L:9D (Fig. 5b), or when day lengths were switched from 12L:12D to 15L:9D on the day of food deprivation (Fig. 5c), the commitment to pupation occurred during the current or next instar (P and L–P in Fig. 5b,c). In both the photoperiodic regimes, no pupae were obtained among the larvae starved on the day of ecdysis to the fourth instar; 40–50% pupation resulted among larvae fed for 2 days; over 75% pupation resulted among larvae fed for 4 or more days. Thus, the critical stage for the destiny of larvae appeared to be around day 2 of the fourth instar. Both larval weight on the day of food deprivation and
Fig. 4. Body weight of P. hilaris reared on diets of sequentially lowered nutritional quality (D50, D25, D13, D6). (a) Body weight of the larvae at the third larval molting under a long-day (15L:9D). (b) Body weight of the pupae. Pupae were classified based on the larval instars they completed. (c) Body weight of larvae on day 60 of the diapausing instar under a short-day (13L:11D). Bars indicate standard deviations. Sample sizes are shown inside the bars.
the resulting pupal weight increased with the stage of food deprivation (Fig. 6). Within the fourth instar, the larval mass doubled from day 0 to day 2 and increased by another 20% during days 2–4 and 4–8 (Fig. 6), paralleling the increase in commitment to pupation (Fig. 5b,c). Hence, at least some feeding and growth during the fourth instar were necessary to commit larvae to pupation. The fifth-, sixth- or seventh-instar larvae were able to pupate without feeding, suggesting that feeding is not indispensable in the fifth and later instars (Fig. 5c). Regardless of the instar and photoperiod, a larval–larval molt never occurred when the larvae were exposed to starvation immediately after molting; some feeding was required for the next larval–larval molt to be induced.
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time required for induction of pupation. A poor diet, in addition to starvation, suppressed larval molting and promoted pupation (Table 1). This was clearly shown when the D50-larvae were subjected to the photoperiodic change on day 8 of the fifth instar; more than half of them underwent an additional molt and thereafter pupated, whereas when the food was also changed to the D6 diet or removed, the additional molt was significantly suppressed, in addition to shortening of the pre-pupation period in the individuals that pupated without an additional molt (Table 1). A similar phenomenon was observed in the cases of the sixth- and seventh-instar larvae (Table 1). 3.6. No change of photoperiodic response in starving larvae
Fig. 5. Responses of P. hilaris larvae to starvation at various ages. Larvae were reared on the standard D50 diet until the given age and thereafter the diet was removed. Larvae were kept continuously under (a) a short-day (12L:12D), (b) a long-day (15L:9D), or (c) the shortday was changed to long-day concurrently with food deprivation. Sample size was 20–31. P: pupated without larval molt. L-P: pupated after a larval molt. L-L: a larval molt occurred but did not pupate. L: no molt occurred.
After finding that starvation under the long-day condition promotes pupation, we examined whether this effect can override the photoperiodic control of diapause using the D50-larvae starved from day 4 of the fourth instar, i.e. soon after acquisition of the ability to pupate. We found the same critical day length (between 13.5 and 14 h) as in the normally fed or semi-starved larvae (Fig. 1, dotted line). Since the sensitivity to photoperiod is either absent or very low during the first to third instars (Shintani and Ishikawa, 1998), the response observed in this experiment can be regarded as that under starvation. Thus, it is concluded that the photoperiodic control of metamorphosis has precedence over the promotion of pupation by starvation.
4. Discussion
Fig. 6. The body weights of P. hilaris larvae at the time of food deprivation and of the resulting pupae. Larvae were reared on the standard D50 diet under 12L:12D until the given age and thereafter the food was removed concurrently with a photoperiodic change to 15L:9D. NP: no pupation occurred. Bars indicate standard deviations. Sample sizes are shown above the bars.
3.5. Poor diet promotes metamorphosis in the fifth and later instars As shown above, the fifth and later instars are able to pupate without any feeding. The response of such larvae to a sudden decrease in food quality or starvation was analyzed in terms of additional larval molting and the
One of the conspicuous characteristics of the cerambycid beetles is a large variation in adult body size. Field-collected P. hilaris adults have variable sizes even within a local population (more than 2.5-fold difference in the body length in extreme cases, unpublished data). The present study suggests that the variation in food quality is an important cause of this variation. The variation in body size is also caused by an intrinsic variation in the number of larval molts, which is largely affected by photoperiod (Shintani et al., 1996a,b) and temperature (Shintani and Ishikawa, 1997a; Watari et al., 2002). In particular, nonstationary supernumerary larval molts associated with diapause markedly contribute to the size variation (Shintani et al., 1996a). In several insects, food serves as a major diapauseregulating factor or a modifier of photoperiodic responses (Tauber et al., 1986). For example, the photoperiodic induction of diapause in the Colorado potato beetle, Leptinotarsa decemlineata, is enhanced when the insects feed on physiologically aged potato leaves (de Wilde et al., 1969; Hare, 1983). In P. hilaris, however,
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Table 1 Responses of fifth, sixth and seventh instar Psacothea hilaris larvae to poor diet and starvationa Diet/ageb
Pre-pupation period (days ± SD)
Additional larval molt induced (%)
Fifth, 0 Fifth, 8 Sixth, 0 Sixth, 8 Seventh, 0 Fifth, 0 D50 D6 Starvation
8 0 0
52 7 13
8 – 0
44 – 13
0 – 0
Fifth, 8
c
Sixth, 0
24.0 ± 2.8a 20.5 ± 3.1a 23.4 ± 2.4a 21.5 ± 3.2a 16.8 ± 3.9a – 16.3 ± 1.9b 15.4 ± 3.8b 17.0 ± 1.6b
Sixth, 8
Seventh, 0
24.0 ± 2.3a – 18.2 ± 1.5b
25.5 ± 2.6a – 18.5 ± 1.9b
– Not examined. Means with the same letter in the same column do not differ significantly by the t-test (sixth, 0; sixth, 8 and seventh, 0) or the Tukey-Kramer test for multiple comparisons (fifth, 0 and fifth, 8) at the level of 5%. Sample size was 21–31. a Larvae were reared on the D50 diet under 12L:12D until the given age and then transferred to 15L:9D and either fed the nutritionally poor D6 diet or starved. Controls were reared continuously on D50 diet with the photoperiodic change. b Instar, days in the instar. c The time from the start of experiment (photoperiodic change and/or starvation) to pupation. Data for individuals that pupated without an additional molt are presented.
the photoperiodic response curve and the number of larval molts were not affected by food quality in the range examined. Several insects exhibit the capability to undergo stationary or nonstationary supernumerary larval molts in response to extrinsic factors, such as starvation (e.g. Iwata and Nishimoto, 1985 for Lyctus brunneus) and crowded condition (e.g. Nakakita, 1982; Connat et al., 1991; Kotaki et al., 1993 for tenebrionids). Low humidity may be among the factors that enhance supernumerary molts in Sitophilus oryzae (e.g. Pittendrigh et al., 1997). However, photoperiod-induced nonstationary molts as observed in P. hilaris have been reported in few species (e.g. Gadenne et al., 1997 for Sesamia nonagrioides), and the biological significance of this phenomenon is not well understood. One possibility is that this is an adaptation for overwintering (Watari et al., 2002). The nonstationary supernumerary molting makes the larvae larger, and the larger larvae can be more adaptive for overwintering. In this regard, the presence of a threshold weight for successful overwintering needs to be investigated. Metamorphosis in many insects is triggered when a threshold size or weight is attained within a defined period during larval development (Sehnal, 1985 for review); at the beginning of the fifth instar in Manduca sexta (Nijhout, 1975, 1981) and Trichoplusia ni (Jones et al., 1981), and at the later part of the third instar in Blattella germanica (Tanaka, 1981). In these species, weight or size are useful predictors for the nature of the next molt (larval–larval or larval–pupal, nymphal–nymphal or nymphal–adult). A large developmental variability in P. hilaris made it difficult to analyze such size-related control of metamorphosis. However, it is now clear that the size is not a proximate cue for pupation in this species, although there is a threshold weight that the larvae must attain for a successful metamorphosis (see Fig. 6; Munyiri et al., 2003). Many insects have the ability to extend the last larval
instar in response to food deficiency, and wait for their critical size to be attained at a later time (e.g. Bradshaw and Johnson, 1995 for Wyeomyia smithii; Petersen et al., 2000 for M. sexta). In clear contrast to this phenomenon, some insects undergo premature pupation in response to food deprivation. Such a response was found in the wax moth (Sehnal, 1966) and was examined in detail in the dung beetle, Onthophagus taurus (Shafiei et al., 2001). Once the food resource (dung) of the beetle larvae is used up, locating additional resources is impossible. Those larvae that have reached a specific weight in the last (third) instar pupate and produce small adults. P. hilaris is the second beetle species in which the premature pupation in response to food shortage was clearly demonstrated. In the present study, however, the mechanism that controls the onset of metamorphosis could not be clarified, and remains to be studied. Acknowledgements We thank Mr. R. Ueda (Kochi Prefectural Sericultural Experiment Station) for collecting yellow-spotted longicorn beetles and two anonymous referees for many suggestions to improve the manuscript. We are grateful to Professor S. Tatsuki (Tokyo University) for encouragement throughout the course of this work. References Bradshaw, W.E., Johnson, K., 1995. Initiation of metamorphosis in the pitcher-plant mosquito: effects of larval growth history. Ecology 76, 2055–2065. Connat, J.L., Delbecque, J.P., Glitho, I., Delachambre, J., 1991. The onset of metamorphosis in Tenebrio molitor larvae (Insecta, Coleoptera) under grouped, isolated and starved conditions. Journal of Insect Physiology 37, 653–662. de Wilde, J., Bongers, W., Schooneveld, H., 1969. Effects of age on phytophagous insects. Entomologia Experimentalis et Applicata 12, 714–720.
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