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Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori
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Journal of Insect Physiology 52 (2006) 807–815 www.elsevier.com/locate/jinsphys
Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori Shinji Nagata, Hiromichi Nagasawa Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 26 January 2006; received in revised form 26 April 2006; accepted 27 April 2006
Abstract Continuous observations of larvae of the silkworm, Bombyx mori, revealed that feeding occurred at regular intervals throughout larval development. To investigate possible factors influencing meal-timing, the behaviours of diet-deprived Bombyx larvae were also analysed. Diet-deprivation resulted in longer durations of the first meals after diet replacement, but did not affect feeding patterns. Furthermore, long-term diet-deprivation promoted wandering behaviour and a consequent delay in feeding after diet replacement. Under dietdeprivation conditions, meal-starts appeared to be inducible by defecation and physical stimulation. However, stimulation-induced mealstarts were dependent on the time elapsed since the larvae’s previous meals. Provided that more than 1 h had elapsed since their previous meals, larvae could be induced to feed by defecation and tapping. At less than 1 h post-meal, larvae were less likely to begin feeding after defecation or physical stimulation. Activated locomotions such as wandering and feeding were observed in the long-term diet-deprived larvae only after diet blocks were replaced, while long-term diet-deprived larvae did not show activated locomotion during the absence of diet blocks. Collectively, these data suggest that a combination of elevated locomotion activity and the presence of diet may be necessary for the initiation of feeding in diet-deprived larvae. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bombyx mori; Diet-deprivation; Feeding behaviour; Lepidoptera; Silkworm
1. Introduction Patterns of insect feeding have been extensively documented to characterize factors controlling the timing of feeding initiation (meal-start) and termination. Continuous observations of various insects indicate that many insect species have patterned feeding cycles. In the locust, Locusta migratoria, meals occur at regular intervals (Blaney et al., 1973; Simpson, 1982), and are controlled by many factors (Simpson, 1981) including volumetric regulation of the hindgut (Simpson, 1983), nutrient control (Simpson and Abisgold, 1985; Simpson and Raubenheimer, 1993), previous meal size (Simpson and Ludlow, 1986), and food supply (Raubenheimer and Simpson, 1990). Feeding behaviour in L. migratoria is also influenced by the
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E-mail address: [email protected] (S. Nagata). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.04.008
defecation cycle (Simpson, 1995), which likely contributes to hindgut volumetric control of feeding behaviour. These results suggest that patterns of feeding behaviour are structured by a combination of endogenous and exogenous oscillations involving a number of factors such as nutritional imbalance and somatic volumetric constraints. Feeding patterns have also been observed in other lepidopteran insects such as the tobacco hornworm, Manduca sexta (Reynolds et al., 1986; Bowdan, 1988a, b; Timmins et al., 1988; Bernays and Woods, 2000), the woolly bear caterpillar, Grammia geneura (Bernays and Singer, 1998) and the corn earworm, Helicoverpa armigera (Barton Browne and Raubenheimer, 2003). Studies on feeding behaviour in M. sexta demonstrated that meal onset is strongly related to the non-feeding ‘‘intermeal’’ periods directly preceding and following meals (Bernays and Woods, 2000). In addition, feeding patterns of Manduca larvae raised on artificial diet were found to be different from those of caterpillars fed plant leaves
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(Reynolds et al., 1986; Bernays and Woods, 2000). Generally, in the lepidopteran species investigated to date, the timing of meal-starts are influenced by the duration of the previous meal and the time spent in the previous intermeal period. The silkworm, Bombyx mori, is a monophagous lepidopteran insect which has been domesticated for more than seven thousand years. The physiology of this species has been studied extensively due to the economic importance of silk production over the centuries. Structural identification of dietary components essential for feeding in Bombyx larvae (Hamamura, 1959; Hamamura et al., 1962) was followed years later by detailed investigations on the relationships between dietary components and growth (Ito, 1980). Further studies on food preferences and optimal nutrient levels for maximum larval growth and silk production eventually led to the development of an artificial diet. Today, B. mori can be raised entirely on artificial diet from the first to the last larval instars. Despite the abundance of nutritional and behavioural studies on B. mori, few continuous observation studies have been conducted on this species’ feeding patterns. The present study was designed to generate data on Bombyx larval feeding behaviours through continuous observations of larvae throughout larval development. In addition to this analysis, the behaviours of starved larvae were also observed to determine how diet-deprivation affects feeding behaviour. Finally, the study examined defecation and physical stimulation in Bombyx larvae as possible triggers for feeding (Simpson, 1995).
survivor plots were analysed from the behavioural data, and feeding bout criteria were determined from the inflection points in the plotted curves. In this paper, we used ‘‘wandering’’ as a term of behaviour that larvae move around or toward artificial diet blocks, which is different from general usage of wandering observed in caterpillars just prior to pupal ecdysis. 2.3. Diet-deprivation of larvae Larvae were starved by diet-deprivation. To avoid any possible influences of faecal pellet odours on feeding behaviour, faeces were frequently removed from the containers throughout the study. Diet-deprivation was initiated in synchronously growing fifth-instar, day 2 larvae (2.770.3 g), following the end of a regular feeding period. As a control group, larvae of similar weight (2.770.3 g) fed ad libitum were also observed. In the present study, we defined ‘‘short-term diet-deprivation’’ as diet-deprivation of less than 24 h, and ‘‘long-term diet-deprivation’’ as dietdeprivation of 4 days. 2.4. Tapping experiment (physical stimulation experiment) Synchronously developing fifth-instar, day 2 larvae were lightly tapped above the rectum near the cercus using the head of a pen. Behaviours of the larvae were observed for 30 min after tapping. 2.5. Statistical analysis
2. Materials and methods 2.1. Animals Eggs of the silkworm, B. mori (hybrid strain, Kinshu Showa), were purchased from a silkworm eggproducing company, UEDA SANSHU Ltd. (Ueda, Japan). Larvae were reared in plastic containers at 2572 1C with 7075% relative humidity, and long-day lighting conditions (16L:8D). Larvae were reared on SILKMATE 2S artificial diet purchased from NIPPON NOSAN Co. Ltd. (Yokohama, Japan). Larvae were provided with fresh diet on a daily basis. All larvae were staged to synchronize their growth. 2.2. Observation of feeding behaviour Only populations of larvae in synchronous growth were observed. During observations, each larva was placed in a plastic container facing a 3 cm3 block of artificial diet. Larvae were spaced within the container so as not to disrupt the feeding behaviour of other animals. Larvae were observed for 6 h daily throughout all five larval stages. A 5 magnifying glass was utilized for easier viewing of mouthpart movements of first- to third-instar larvae. Log-
Data in Fig. 2 were analysed to goodness of fit of the negative binomial distribution as described by Reynolds et al. (1986). In Fig. 2A, data longer than bout criterion were analysed by the goodness of fit of the negative binomial distribution. The goodness of fit to the negative binomial distributions in individual data was confirmed by w2 test as shown in Fig. 2A. Data of Fig. 2B were also analysed by the goodness of fit of the negative binomial distributions. The individual data fitted to the negative binomial distribution were confirmed by w2 test as shown in Fig. 2B. In Fig. 3, the arcsine-transformed values of percentile data were analysed by one-way analysis of variance (ANOVA). A P-value of less than 0.05 was considered to be statistically significant. In analysis of Fig. 3, we did not compare the values of the larvae at first day (day 0) and last day of each instar, since those values could exclude from the parametric test as the non-normal distributions in those values ðPo0:05Þ. Data in Fig. 4 were statistically analysed by one-way ANOVA with a P-value of less than 0.05 as to be statistically significant. In Figs. 5 and 6, non-parametric tests were utilized to compare pairs of observed parameters. In Tables 1 and 2, the comparison of each category was performed by w2 test.
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3. Results 3.1. General feeding behaviour in Bombyx larvae We continuously observed 10 larvae fed ad libitum throughout the larval life cycle from hatching to pupation. The observed larvae showed very similar behavioural patterns in both feeding ratios and feeding durations. The observed animals fed at regular intervals except during the quiescent period immediately preceding ecdysis (Fig. 1).
1st instar
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During non-quiescent states, larvae exhibited regular, repetitive feeding patterns. Log-survivor plot analyses confirmed that feeding occurred in regular, repetitive sequences. In fifth-instar, day 2 larvae, log-survivor plots of the gaps between feedings clearly showed two distinct populations of nonfeeding bouts: a long, quiescent intermeal period and a short resting period within a meal (Fig. 2A). ‘‘Bout criterion,’’ the longest pause in feeding that would still be considered part of a meal was inspected from the
Day1 Day2 Day3 Day4
2nd instar Day0 Day1 Day2 Day3 3rd instar
Day0 Day1 Day2 Day3 Day4
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Day0 Day1 Day2 Day3 Day4 Day5 Day6
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Day0 Day1 Day2 Day3 Day4 Day5 Day6 Day7
0
60 120 Arbitrary observation time (minutes)
180
Fig. 1. Arbitrary alignment of feeding behaviour patterns of Bombyx mori larvae throughout larval development. Data were obtained from multiple 6-h observation periods. Filled boxes represent the time spent in feeding, open boxes represent the time spent in wandering, and blank areas indicate periods of no locomotion activity (quiescent states). Representative data for each developmental day from the observed synchronously developing larvae are shown. Since the alignments were arranged to show the similar pattern throughout the larval development, the time is arbitrary and only the time scale is significant.
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810
insect #1
100
Number of gaps between feeding behaviours
50
insect #3
insect #2
χ2 (df6)=4.90
χ2 (df5)=7.635
P > 0.05
P > 0.05
insect #4
insect #5
χ2 (df6)=4.93
χ2 (df8)=9.75
P > 0.05
χ2 (df8)=8.37
P > 0.05
P > 0.05
10 5
1 0
20
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80 100 0
insect #6 100
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insect #7
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20
insect #8
χ2 (df5)=11.0
50
20
χ2 (df7)=7.96
P > 0.05
60
80 100
0
insect #9 χ2 (df9)=7.98
P > 0.05
40
20
60
80
insect #10 χ2 (df6)=7.58
χ2 (df6)=6.86
P > 0.05
40
P > 0.05
P > 0.05
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1 0
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40
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80 100 0
20
40
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80
0
20
(A)
100
insect #1
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insect #2 χ2
40 60 80 100 120 Gap duration (minutes)
0
insect #3
(df7)=10.0
χ2
P > 0.05
P > 0.05
40
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80
0
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insect #4 χ2
(df14)=12.5
20
40
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80 100
insect #5 χ2
(df5)=8.04
P > 0.05
χ2 (df8)=7.41
(df11)=8.596
P > 0.05
P > 0.05
Number of feeding behaviours
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1 0 100
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0
insect #6
50
10
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insect #7
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10
insect #8
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insect #9
χ2 (df4)=3.03
χ2 (df8)=12.2
χ2 (df9)=4.30
χ2 (df10)=2.47
P > 0.05
P > 0.05
P > 0.05
P > 0.05
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20
insect #10 χ2 (df11)=11.1 P > 0.05
10 5
1 0
(B)
10
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0
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10 20 Feeding duration (minutes)
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20
Fig. 2. (A) Log-survivor plots fitted to the intermeal duration times of 10 fifth-instar, day 2 larvae. The arrow represents the bout criterions. Data longer than bout criterion were used to analyse the goodness of fit of the negative binomial distribution. Each individual calculated w2 value by w2 test was described in each plot. (B) Log-survivor plots fitted to the time spent in feeding behaviour of 10 fifth-instar, day 2 larvae. Each individual calculated w2 value by w2 test was described in each plot, confirming the goodness of fit of the negative binomial distribution of data.
log-survivor plots to be 92.578.5 s. This implies that during a given meal larvae would pause, then resume feeding within approximately 90 s. The goodness of fit of the data longer than bout criterion in each plot showed a fitted negative binomial distribution ðw2 40:05Þ, indicating that the meals would start at the regular intervals.
We also analysed the durations of meals using logsurvivor plots. All 10 observed animals exhibited similar log-survivor plots with convex curves and no discontinuous points (no bout criterion) (Fig. 2B). The goodness of fit of data in each plot of feeding period showed a fitted negative binomial distribution ðw2 40:05Þ, also indicating that
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811
25
Feeding ratio (%)
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a a
a a
a
a,b
b a,b
a,b a,b
a,b
a,b
b
a,b a,b
a
15 0 1 2 3 0 1 2 3 0 1 2 3 4 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 1st instar 2nd instar 3rd instar 4th instar 5th (last) instar Larval development (days)
Bombyx larvae show similar meal duration. There were few differences in log-survivor plots obtained from Bombyx larvae at any stage of growth, indicating that Bombyx larvae feed at regular intervals throughout larval development. Next, to investigate changes in meal duration during larval development, the proportion of time spent in meals was calculated (Fig. 3). Although the data showed no significant differences within each larval stage (ANOVA, P40:1, n ¼ 10), differences of proportion of time spent in meals were observed on day 4 and 5 of fourth-instar larvae and day 3 and 4 of fifth-instar larvae compared with larvae at early developmental stages (first- and second-instar larvae and day 1 of third-instar larvae). In general, the proportion of time spent in meals accrued gradually with larval growth during each instar, except for the last (fifth) instar. During the fifth-instar, the proportion of time spent feeding reached the maximum on day 4, then gradually declined until they entered into quiescent states in preparation for pupal ecdysis. Cyclic patterned feeding behaviours were observed in all developmental stages. 3.2. The effect of short-term diet-deprivation on the feeding cycle To examine the effects of diet-deprivation on the structure of feeding patterns and the probability of mealstarts, feeding behaviours of diet-deprived larvae were observed. Artificial diet blocks were removed from containers of fifth-instar, day 2 larvae for periods ranging from 0.5 to 24 h. Diet-deprivation significantly lengthened the time spent in the first meal after diet replacement, particularly in the larvae that experienced diet-deprivation for more than 6 h (ANOVA, Po0:05) (Fig. 4). The maximum increase in first meal duration was observed in larvae deprived of diet for 12 h, spending an average of 45.477.4 min in the first
Duration of first meal after diet-replacement (minutes)
Fig. 3. The proportion of time spent in feeding behaviour during observations of larvae fed ad libitum throughout larval development. On the first and last days of each instar, the ratio of feeding behaviours to non-feeding behaviours was drastically reduced as many of the larvae were in quiescent states. Each point was plotted using data from 10 larvae (mean7SEM). Means with different letters denote significantly different one-way ANOVA, Po0:05. In comparison, the data of the first and last day of each instar were not used for statistical analysis because of their non-normal distributions.
60 b
b
50 b 40 a,b
30 a 20 10 0
4
8 12 16 20 Diet-deprivation period (hours)
24
Fig. 4. First meal duration in the diet-deprived larvae after diet replacement. Each plot (mean7SEM) is derived from observations of 20 diet-deprived animals. The diet-deprivation periods ranged from 30 min to 24 h. Means with different letters denote significantly different one-way ANOVA, Po0:05.
meal compared with the usual 17.972.9 min spent in meals in larvae fed ad libitum. Despite the significant increase in meal duration in the first meal after diet-replacement, dietdeprived larvae resumed normal feeding patterns in subsequent meals (data not shown). 3.3. The effect of long-term diet-deprivation on feeding behaviour As with short-term diet-deprived larvae (6–24 ), longterm diet-deprivation (4 days) resulted in prolonged first meals following diet replacement. Interestingly, most of the larvae that experienced long-term diet-deprivation did not start feeding immediately after diet replacement, but
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initially wandered around the artificial diet blocks delaying feeding up to 3 min after diet replacement (Fig. 5). In most of these larvae, the delay before the first bite ranged from 0 to 3 min, whereas the first bite generally occurred within 1 min in short-term (12 h) diet-deprived larvae (Mann– Whitney test, Po0:05). A few long-term diet-deprived larvae wandered around the reintroduced diet blocks for more than 10 min before feeding. In addition, larvae that experienced long-term diet-deprivation spent more time wandering during the intermeal period following the first meal after diet replacement than short-term diet-deprived larvae (Mann–Whitney test, Po0:001) (Fig. 6). 3.4. The effect of physical stimulation on feeding behaviours During the continuous observations, larvae were occasionally observed to begin wandering or feeding following defecation during intermeal periods. Defecation appeared to trigger wandering or feeding in larvae immediately prior to meals, but defecation was not associated with wandering or feeding if it occurred immediately after a meal. To investigate the link between feeding behaviour and defecation, larvae were closely observed during the intermeal periods. The mean intermeal durations of approximately 90 min were divided into three segments: the ‘‘post-
(%) 15 Proportion of time spent in wandering behaviour
812
10
5
24 hours
4 days
Diet-deprivation period Fig. 6. The proportion of time spent in wandering behaviour during the intermeal period between the first and second meals after diet replacement in 24-h and 4-day diet-deprived larvae. Bars represent means7SEM.
Table 1 Effect of defecation on feeding behaviour
Feeding larvae Observed larvae
Post-feeding
Inter-feeding
Pre-feeding
2 30
5 26
19 23
Time until first bite after diet-replacement (seconds)
90
60
30
0 24 hours 4 days Diet-deprivation period Fig. 5. Time spent in wandering behaviour until the start of the first meal after diet replacement in 24-h and 4-day diet-deprived larvae. A total of 15 and 20 diet-deprived larvae were observed for 24 h and 4 days, respectively. Each dot represents the time interval before a larva initiated feeding.
feeding’’ segment (0–30 min after a meal), the ‘‘interfeeding’’ segment (30–60 min after a meal) and the ‘‘prefeeding’’ segment (more than 60 min after a meal). Most larvae that defecated during the pre-feeding segment began to feed immediately, whereas larvae that defecated during the post-feeding and inter-feeding segments were much less likely to start feeding (w2(d.f.2) ¼ 40.2, Po0:00001) (Table 1). This result indicated that defecation immediately prior to a regular feeding period caused a physiological change in the larvae that may direct to the meal. To examine whether general physical stimulation would also induce feeding behaviour, larvae fed ad libitum were lightly tapped along the cercus above the rectum during an intermeal period. Immediately following the tapping, all larvae initiated wandering behaviour. Within 3 min, some larvae resumed their original quiescent states, while others started to feed. Larvae were also tapped at 30, 60 and 90 min following a meal (corresponding to the post-, inter-, and prefeeding segments as defined above). More than 80% of the larvae tapped during the pre-feeding segment and a small proportion that were tapped during the inter-feeding segment started wandering or feeding. The larvae tapped during the post-feeding segment did not initiate feeding behaviours (w2(d.f.2) ¼ 33.1, Po0:00001) (Table 2).
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Feeding larvae Observed larvae
Post-feeding
Inter-feeding
Pre-feeding
0 20
8 20
18 20
4. Discussion In the present study, we observed that feeding occurred at regular intervals in Bombyx larvae throughout larval development. Previously, Hirao and Yamaoka (1981) recorded the feeding of Bombyx larvae using a short circuit actograph that detected the current between the larval body and an artificial diet block or mulberry leaf. While the short circuit actograph monitored the cyclic feeding patterns of fifth-instar, day 4 larvae, feeding behaviours of larvae at other developmental stages have not been studied or statistically analysed. The current investigation demonstrated that such feeding behaviour patterns are maintained throughout larval development. Furthermore, through continuous observation of the animals, additional behaviours were documented which would be difficult to detect by a short circuit actograph. Behavioural rhythms have also been observed in larvae of other lepidopteran species: M. sexta (Reynolds et al., 1986; Bowdan, 1988a, b; Timmins et al., 1988; and Bernays and Woods, 2000), G. geneura, (Bernays and Singer, 1998) and H. armigera (Barton Browne and Raubenheimer, 2003). Log-survivor plot analyses revealed that feeding behaviour patterns of these insects are structured by regularly occurring meals. Also, log-survivor plots of the gaps between feeding behaviours included a bout criterion ranging from 1.5 to 3.5 min in those insects. The results of this study generated a feeding bout criterion value of 92.578.5 s for Bombyx larvae indicating that Bombyx feeding patterns are similar to other lepidopteran larvae (Fig. 1). Interestingly, the feeding patterns of Manduca larvae on plant leaves differ from those of Manduca larvae raised on artificial diet (Reynolds et al., 1986; Bowdan, 1988a, b; Timmins et al., 1988; Bernays and Woods, 2000). First, the amount of diet intake by larvae fed plant leaves is significantly less than the intake of artificial diet by domesticated larvae. Second, the time spent in feeding behaviour in Manduca larvae fed plant leaves is significantly longer than that of larvae fed artificial diet. Cyclic patterns of feeding behaviour were also observed in Manduca larvae fed artificial diet, but not in larvae feeding on plant leaves. Such differences in behavioural patterns have also been recorded for Bombyx larvae (Hirao and Yamaoka, 1981), although statistical analyses have not been performed. The results of this study revealed that patterns of feeding behaviour in Bombyx larvae that were raised on artificial diet resembled those of domesticated Manduca larvae that were fed artificial diet but not to
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Manduca larvae that were fed plant leaves. Since the feeding behavioural differences were observed even in Bombyx and Manduca larvae fed plant leaves in the laboratory observation (Hirao and Yamaoka, 1981; Reynolds et al., 1986), the differences in feeding patterns between larvae fed artificial diet and larvae fed plant leaves may be due to differences in diet contents. The feeding pattern of Manduca larvae in the field might be more complicated due to the additional influences such as environmental stress or predation (Bernays and Woods, 2000). Short-term diet-deprivation increased the duration of the first meal after diet replacement and increased the time spent wandering during the subsequent intermeal period. Long-term diet-deprivation also resulted in enhanced locomotion activity. This suggests that diet-deprivation stimulates the locomotion systems of Bombyx larvae. Prolonged feeding and activated locomotion have also been associated with starvation in the larvae of the gypsy moth, Porthetria dispar. P. dispar exhibits silking behaviour which helps it to secure itself to food substrates. Dietdeprivation in P. dispar larvae triggered silking behaviour (Leonard, 1967). Although Bombyx larvae also exhibit silking behaviour while on a food source, increase of silking behaviour was not observed in diet-deprived larvae. Long-term, diet-deprived Bombyx larvae, however, showed increased wandering behaviour after diet-replacement, indicating that reduced gut volume might be associated with increased locomotion activity. Elevated locomotion activity in starved larvae in the presence of diet may also result in increased foraging behaviour, probably due to the stimulation of chemical attractants from diet. In Heliothis zea, it has been proposed that nutritional imbalance results in altered levels of neurotransmitters in the central nervous system (Cohen et al., 1988), implying that diet-deprivation may change the level of neurotransmitters in Bombyx larvae, elevating the locomotion activity for foraging. The fact that long-term diet-deprived Bombyx larvae did not begin wandering or foraging until food was replaced indicates that starvation-associated locomotion activity might be controlled by chemical attractants in food. Their quiescence in the absence of food also suggests that longterm diet-deprivation might induce a reduced metabolic state in the larva that would minimize energy loss or nutrient depletion. It is unclear, however, whether locomotion activity and the initiation of feeding behaviour are controlled by the same factors. During observations of Bombyx larvae after meals, we noticed that in some larvae, defecation might be involved in the initiation of feeding behaviour during the pre-feeding segment. In the locust, feeding behaviour is related to defecation behaviour (Simpson and Ludlow, 1986). It is reasonable that defecation triggers feeding behaviour through hindgut volumetric control, since the intake of diet or the entry of diet into the gut would increase gut volume. The complemented volume of the gut by feeding would, ultimately, supply the lost nutrients in the body. In
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the present study, Bombyx larvae did not start feeding behaviours after defecation when defecation occurred immediately following a meal, suggesting that the larvae just after feeding were different from the larvae just prior to regular meals. Therefore, in both locust and Bombyx larvae, the rejection or acceptance of food appears to be strongly related to the time elapsed since the previous meal. Thus, the data indicate that meal-start process may be required for the physical stimulation such as defecation. Also, the fact that the starved larvae did not show the locomotion activity in the absence of diet block suggested chemical attractants of diet influence synergistically on the initiation of feeding behaviour in Bombyx larvae. Accurate elucidation of the factors influencing mealstarts in insects is a complicated issue. Intrinsic modulators and exogenous factors may collaterally affect feeding behaviour. Further investigations into the patterns of behaviour related to nutrient control and ingest ratio (Ueda and Suzuki, 1967; Horie et al., 1976, 1978) will aid in broadening our understanding of feeding behaviour in domesticated Bombyx larvae. With the genetic and genomic information of this species now available (Mita et al., 2003, 2004; Xia et al., 2004) detailed molecular studies utilizing this data will gradually clarify the complex mechanisms underlying the feeding behaviours of the silkworm. Acknowledgements This work was partly supported by the Graduate School of Agricultural and Life Sciences, the University of Tokyo. The authors wish to thank Ms. Jennifer S.I. for critical reading and checking English description. We also thank the members of our laboratory for generous supports and helpful discussions and continuous encouragements. References Barton Browne, L., Raubenheimer, D., 2003. Ontogenetic changes in the rate of ingestion and estimates of food consumption in fourth and fifth instar Helicoverpa armigera caterpillars. Journal of Insect Physiology 49 (1), 63–71. Bernays, E.A., Singer, M.S., 1998. A rhythm underlying feeding behaviour in a highly polyphagous caterpillar. Physiological Entomology 23, 295–302. Bernays, E.A., Woods, H.A., 2000. Foraging in nature by larvae of Manduca sexta influenced by an endogenous oscillation. Journal of Insect Physiology 46, 825–836. Blaney, W.M., Chapman, R.F., Wilson, A., 1973. The pattern of feeding of Locusta migratoria (L.) (Orthoptera, Acrididae). Acrida 2, 119–137. Bowdan, E., 1988a. The effect of deprivation on the microstructure of feeding by the tobacco hornworm caterpillar. Journal of Insect Behavior 1, 31–50. Bowdan, E., 1988b. Microstructure of feeding by tobacco hornworm caterpillar, Manduca sexta. Entomologia Experimentalis et Applicata 47, 127–136. Cohen, R.W., Friedman, S., Waldbauer, G.P., 1988. Physiological control of nutrient self-selection in Heliothis zea larvae: the role of serotonin. Journal of Insect Physiology 34, 935–940.
Hamamura, Y., 1959. Food selection by silkworm larvae. Nature 183, 1746–1747. Hamamura, Y., Hayashiya, K., Naito, K., Matsuura, K., Nishida, J., 1962. Food selection by silkworm larvae. Nature 194, 754–755. Hirao, T., Yamaoka, K., 1981. Analysis of feeding behaviour in the silkworm, Bombyx mori-short circuit type actograph. Journal of Sericultural Science of Japan 50, 335–342. Horie, Y., Inokuchi, T., Watanabe, K., Nakasone, S., Yanagawa, H., 1976. Quantitative study on food utilization by the silkworm, Bombyx mori, through its life cycle I. economy of dry matter, energy, and carbon. Bulletin of the Sericultural Experiment Station 26, 411–442. Horie, Y., Inokuchi, T., Watanabe, K., 1978. Quantitative study of food utilization by the silkworm, Bombyx mori, through its life cycle II. economy of nitrogen and amino acids. Bulletin of the Sericultural Experiment Station 27, 531–578. Ito, T., 1980. Application of artificial diets in sericulture. Japan Agricultural Research Quarterly 14, 163–168. Leonard, E.D., 1967. Silking behavior of the gypsy moth, Porthetria dispar. Canadian Entomology 99, 1145–1149. Mita, K., Morimyo, M., Okano, K., Koike, Y., Nohata, J., Kawasaki, H., Kadono-Okuda, K., Yamamoto, K., Suzuki, M.G., Shimada, T., Goldsmith, M.R., Maeda, S., 2003. The construction of an EST database for Bombyx mori and its application. Proceedings of the National Academy of Sciences, USA 100 (24), 14121–14126. Mita, K., Kasahara, M., Sasaki, S., Nagayasu, Y., Yamada, T., Kanamori, H., Namiki, N., Kitagawa, M., Yamashita, H., Yasukochi, Y., Kadono-Okuda, K., Yamamoto, K., Ajimura, M., Ravikumar, G., Shimomura, M., Nagamura, Y., Shin-I, T., Abe, H., Shimada, T., Morishita, S., Sasaki, T., 2004. The genome sequence of silkworm, Bombyx mori. DNA Research 11 (1), 27–35. Raubenheimer, D., Simpson, S.J., 1990. The effect of simultaneous variation in protein digestible carbohydrate and tannic acid on the feeding behavior of larval Locusta migratoria (L.) and Schistocerca gregaria (forskal). I. Short-term studies. Physiological Entomology 15, 219–233. Reynolds, S.E., Yeomans, M.R., Timmins, W.A., 1986. The feeding behaviour of caterpillars (Manduca sexta) on tobacco and on artificial diet. Physiological Entomology 11, 39–51. Simpson, S.J., 1981. An oscillation underlying feeding and a number of other behaviours in fifth-instar Locusta migratoria nymphs. Physiological Entomology 6, 315–324. Simpson, S.J., 1982. Patterns in feeding: a behavioural analysis using Locusta migratoria nymphs. Physiological Entomology 7, 325–336. Simpson, S.J., 1983. The role of volumetric feedback from the hindgut in the regulation of meal size in fifth-instar Locusta migratoria nymphs. Physiological Entomology 8, 451–467. Simpson, S.J., 1995. The control of meals in chewing insects. In: Chapman, R.F., de Boer, J. (Eds.), Regulatory Mechanisms of Insect Feeding. Chapman & Hall, New York, pp. 137–156. Simpson, S.J., Abisgold, J.D., 1985. Compensation by locusts for changes in dietary nutrients: behavioral mechanisms. Physiological Entomology 10, 443–452. Simpson, S.J., Ludlow, A.R., 1986. Why locusts start to feed?: a comparison of causal factors. Animal Behavior 34, 480–496. Simpson, S.J., Raubenheimer, D., 1993. The central role of the haemolymph in the regulation of nutrient intake in insects. Physiological Entomology 18, 395–403. Timmins, W.A., Bellward, K., Stamp, A.J., Reynolds, S.E., 1988. Food intake, conversion efficiency and feeding behaviour of tobacco hornworm caterpillar given artificial diet of varying nutrient and water content. Physiological Entomology 13, 303–314. Ueda, S., Suzuki, K., 1967. Studies on the growth of the silkworm, Bombyx mori, L. I. Chronological changes of the amount of food ingested and digested, bodyweight and water content of body, and their mutual relationships. Bulletin of the Sericultural Experiment Station 22, 33–74. Xia, Q., Zhou, Z., Lu, C., Cheng, D., Dai, F., Li, B., Zhao, P., Zha, X., Cheng, T., Chai, C., Pan, G., Xu, J., Liu, C., Lin, Y., Qian, J., Hou,
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Journal of Insect Physiology 52 (2006) 807–815 www.elsevier.com/locate/jinsphys
Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori Shinji Nagata, Hiromichi Nagasawa Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 26 January 2006; received in revised form 26 April 2006; accepted 27 April 2006
Abstract Continuous observations of larvae of the silkworm, Bombyx mori, revealed that feeding occurred at regular intervals throughout larval development. To investigate possible factors influencing meal-timing, the behaviours of diet-deprived Bombyx larvae were also analysed. Diet-deprivation resulted in longer durations of the first meals after diet replacement, but did not affect feeding patterns. Furthermore, long-term diet-deprivation promoted wandering behaviour and a consequent delay in feeding after diet replacement. Under dietdeprivation conditions, meal-starts appeared to be inducible by defecation and physical stimulation. However, stimulation-induced mealstarts were dependent on the time elapsed since the larvae’s previous meals. Provided that more than 1 h had elapsed since their previous meals, larvae could be induced to feed by defecation and tapping. At less than 1 h post-meal, larvae were less likely to begin feeding after defecation or physical stimulation. Activated locomotions such as wandering and feeding were observed in the long-term diet-deprived larvae only after diet blocks were replaced, while long-term diet-deprived larvae did not show activated locomotion during the absence of diet blocks. Collectively, these data suggest that a combination of elevated locomotion activity and the presence of diet may be necessary for the initiation of feeding in diet-deprived larvae. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bombyx mori; Diet-deprivation; Feeding behaviour; Lepidoptera; Silkworm
1. Introduction Patterns of insect feeding have been extensively documented to characterize factors controlling the timing of feeding initiation (meal-start) and termination. Continuous observations of various insects indicate that many insect species have patterned feeding cycles. In the locust, Locusta migratoria, meals occur at regular intervals (Blaney et al., 1973; Simpson, 1982), and are controlled by many factors (Simpson, 1981) including volumetric regulation of the hindgut (Simpson, 1983), nutrient control (Simpson and Abisgold, 1985; Simpson and Raubenheimer, 1993), previous meal size (Simpson and Ludlow, 1986), and food supply (Raubenheimer and Simpson, 1990). Feeding behaviour in L. migratoria is also influenced by the
Corresponding author. Tel.: +81 3 5841 5135; fax: +81 3 58418022.
E-mail address: [email protected] (S. Nagata). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.04.008
defecation cycle (Simpson, 1995), which likely contributes to hindgut volumetric control of feeding behaviour. These results suggest that patterns of feeding behaviour are structured by a combination of endogenous and exogenous oscillations involving a number of factors such as nutritional imbalance and somatic volumetric constraints. Feeding patterns have also been observed in other lepidopteran insects such as the tobacco hornworm, Manduca sexta (Reynolds et al., 1986; Bowdan, 1988a, b; Timmins et al., 1988; Bernays and Woods, 2000), the woolly bear caterpillar, Grammia geneura (Bernays and Singer, 1998) and the corn earworm, Helicoverpa armigera (Barton Browne and Raubenheimer, 2003). Studies on feeding behaviour in M. sexta demonstrated that meal onset is strongly related to the non-feeding ‘‘intermeal’’ periods directly preceding and following meals (Bernays and Woods, 2000). In addition, feeding patterns of Manduca larvae raised on artificial diet were found to be different from those of caterpillars fed plant leaves
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(Reynolds et al., 1986; Bernays and Woods, 2000). Generally, in the lepidopteran species investigated to date, the timing of meal-starts are influenced by the duration of the previous meal and the time spent in the previous intermeal period. The silkworm, Bombyx mori, is a monophagous lepidopteran insect which has been domesticated for more than seven thousand years. The physiology of this species has been studied extensively due to the economic importance of silk production over the centuries. Structural identification of dietary components essential for feeding in Bombyx larvae (Hamamura, 1959; Hamamura et al., 1962) was followed years later by detailed investigations on the relationships between dietary components and growth (Ito, 1980). Further studies on food preferences and optimal nutrient levels for maximum larval growth and silk production eventually led to the development of an artificial diet. Today, B. mori can be raised entirely on artificial diet from the first to the last larval instars. Despite the abundance of nutritional and behavioural studies on B. mori, few continuous observation studies have been conducted on this species’ feeding patterns. The present study was designed to generate data on Bombyx larval feeding behaviours through continuous observations of larvae throughout larval development. In addition to this analysis, the behaviours of starved larvae were also observed to determine how diet-deprivation affects feeding behaviour. Finally, the study examined defecation and physical stimulation in Bombyx larvae as possible triggers for feeding (Simpson, 1995).
survivor plots were analysed from the behavioural data, and feeding bout criteria were determined from the inflection points in the plotted curves. In this paper, we used ‘‘wandering’’ as a term of behaviour that larvae move around or toward artificial diet blocks, which is different from general usage of wandering observed in caterpillars just prior to pupal ecdysis. 2.3. Diet-deprivation of larvae Larvae were starved by diet-deprivation. To avoid any possible influences of faecal pellet odours on feeding behaviour, faeces were frequently removed from the containers throughout the study. Diet-deprivation was initiated in synchronously growing fifth-instar, day 2 larvae (2.770.3 g), following the end of a regular feeding period. As a control group, larvae of similar weight (2.770.3 g) fed ad libitum were also observed. In the present study, we defined ‘‘short-term diet-deprivation’’ as diet-deprivation of less than 24 h, and ‘‘long-term diet-deprivation’’ as dietdeprivation of 4 days. 2.4. Tapping experiment (physical stimulation experiment) Synchronously developing fifth-instar, day 2 larvae were lightly tapped above the rectum near the cercus using the head of a pen. Behaviours of the larvae were observed for 30 min after tapping. 2.5. Statistical analysis
2. Materials and methods 2.1. Animals Eggs of the silkworm, B. mori (hybrid strain, Kinshu Showa), were purchased from a silkworm eggproducing company, UEDA SANSHU Ltd. (Ueda, Japan). Larvae were reared in plastic containers at 2572 1C with 7075% relative humidity, and long-day lighting conditions (16L:8D). Larvae were reared on SILKMATE 2S artificial diet purchased from NIPPON NOSAN Co. Ltd. (Yokohama, Japan). Larvae were provided with fresh diet on a daily basis. All larvae were staged to synchronize their growth. 2.2. Observation of feeding behaviour Only populations of larvae in synchronous growth were observed. During observations, each larva was placed in a plastic container facing a 3 cm3 block of artificial diet. Larvae were spaced within the container so as not to disrupt the feeding behaviour of other animals. Larvae were observed for 6 h daily throughout all five larval stages. A 5 magnifying glass was utilized for easier viewing of mouthpart movements of first- to third-instar larvae. Log-
Data in Fig. 2 were analysed to goodness of fit of the negative binomial distribution as described by Reynolds et al. (1986). In Fig. 2A, data longer than bout criterion were analysed by the goodness of fit of the negative binomial distribution. The goodness of fit to the negative binomial distributions in individual data was confirmed by w2 test as shown in Fig. 2A. Data of Fig. 2B were also analysed by the goodness of fit of the negative binomial distributions. The individual data fitted to the negative binomial distribution were confirmed by w2 test as shown in Fig. 2B. In Fig. 3, the arcsine-transformed values of percentile data were analysed by one-way analysis of variance (ANOVA). A P-value of less than 0.05 was considered to be statistically significant. In analysis of Fig. 3, we did not compare the values of the larvae at first day (day 0) and last day of each instar, since those values could exclude from the parametric test as the non-normal distributions in those values ðPo0:05Þ. Data in Fig. 4 were statistically analysed by one-way ANOVA with a P-value of less than 0.05 as to be statistically significant. In Figs. 5 and 6, non-parametric tests were utilized to compare pairs of observed parameters. In Tables 1 and 2, the comparison of each category was performed by w2 test.
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3. Results 3.1. General feeding behaviour in Bombyx larvae We continuously observed 10 larvae fed ad libitum throughout the larval life cycle from hatching to pupation. The observed larvae showed very similar behavioural patterns in both feeding ratios and feeding durations. The observed animals fed at regular intervals except during the quiescent period immediately preceding ecdysis (Fig. 1).
1st instar
809
During non-quiescent states, larvae exhibited regular, repetitive feeding patterns. Log-survivor plot analyses confirmed that feeding occurred in regular, repetitive sequences. In fifth-instar, day 2 larvae, log-survivor plots of the gaps between feedings clearly showed two distinct populations of nonfeeding bouts: a long, quiescent intermeal period and a short resting period within a meal (Fig. 2A). ‘‘Bout criterion,’’ the longest pause in feeding that would still be considered part of a meal was inspected from the
Day1 Day2 Day3 Day4
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Day0 Day1 Day2 Day3 Day4 Day5 Day6
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Day0 Day1 Day2 Day3 Day4 Day5 Day6 Day7
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60 120 Arbitrary observation time (minutes)
180
Fig. 1. Arbitrary alignment of feeding behaviour patterns of Bombyx mori larvae throughout larval development. Data were obtained from multiple 6-h observation periods. Filled boxes represent the time spent in feeding, open boxes represent the time spent in wandering, and blank areas indicate periods of no locomotion activity (quiescent states). Representative data for each developmental day from the observed synchronously developing larvae are shown. Since the alignments were arranged to show the similar pattern throughout the larval development, the time is arbitrary and only the time scale is significant.
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insect #1
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insect #3
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χ2 (df6)=4.90
χ2 (df5)=7.635
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χ2 (df6)=4.93
χ2 (df8)=9.75
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insect #9 χ2 (df9)=7.98
P > 0.05
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insect #10 χ2 (df6)=7.58
χ2 (df6)=6.86
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insect #4 χ2
(df14)=12.5
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insect #5 χ2
(df5)=8.04
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(df11)=8.596
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Number of feeding behaviours
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Fig. 2. (A) Log-survivor plots fitted to the intermeal duration times of 10 fifth-instar, day 2 larvae. The arrow represents the bout criterions. Data longer than bout criterion were used to analyse the goodness of fit of the negative binomial distribution. Each individual calculated w2 value by w2 test was described in each plot. (B) Log-survivor plots fitted to the time spent in feeding behaviour of 10 fifth-instar, day 2 larvae. Each individual calculated w2 value by w2 test was described in each plot, confirming the goodness of fit of the negative binomial distribution of data.
log-survivor plots to be 92.578.5 s. This implies that during a given meal larvae would pause, then resume feeding within approximately 90 s. The goodness of fit of the data longer than bout criterion in each plot showed a fitted negative binomial distribution ðw2 40:05Þ, indicating that the meals would start at the regular intervals.
We also analysed the durations of meals using logsurvivor plots. All 10 observed animals exhibited similar log-survivor plots with convex curves and no discontinuous points (no bout criterion) (Fig. 2B). The goodness of fit of data in each plot of feeding period showed a fitted negative binomial distribution ðw2 40:05Þ, also indicating that
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811
25
Feeding ratio (%)
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a a
a a
a
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b
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a
15 0 1 2 3 0 1 2 3 0 1 2 3 4 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 1st instar 2nd instar 3rd instar 4th instar 5th (last) instar Larval development (days)
Bombyx larvae show similar meal duration. There were few differences in log-survivor plots obtained from Bombyx larvae at any stage of growth, indicating that Bombyx larvae feed at regular intervals throughout larval development. Next, to investigate changes in meal duration during larval development, the proportion of time spent in meals was calculated (Fig. 3). Although the data showed no significant differences within each larval stage (ANOVA, P40:1, n ¼ 10), differences of proportion of time spent in meals were observed on day 4 and 5 of fourth-instar larvae and day 3 and 4 of fifth-instar larvae compared with larvae at early developmental stages (first- and second-instar larvae and day 1 of third-instar larvae). In general, the proportion of time spent in meals accrued gradually with larval growth during each instar, except for the last (fifth) instar. During the fifth-instar, the proportion of time spent feeding reached the maximum on day 4, then gradually declined until they entered into quiescent states in preparation for pupal ecdysis. Cyclic patterned feeding behaviours were observed in all developmental stages. 3.2. The effect of short-term diet-deprivation on the feeding cycle To examine the effects of diet-deprivation on the structure of feeding patterns and the probability of mealstarts, feeding behaviours of diet-deprived larvae were observed. Artificial diet blocks were removed from containers of fifth-instar, day 2 larvae for periods ranging from 0.5 to 24 h. Diet-deprivation significantly lengthened the time spent in the first meal after diet replacement, particularly in the larvae that experienced diet-deprivation for more than 6 h (ANOVA, Po0:05) (Fig. 4). The maximum increase in first meal duration was observed in larvae deprived of diet for 12 h, spending an average of 45.477.4 min in the first
Duration of first meal after diet-replacement (minutes)
Fig. 3. The proportion of time spent in feeding behaviour during observations of larvae fed ad libitum throughout larval development. On the first and last days of each instar, the ratio of feeding behaviours to non-feeding behaviours was drastically reduced as many of the larvae were in quiescent states. Each point was plotted using data from 10 larvae (mean7SEM). Means with different letters denote significantly different one-way ANOVA, Po0:05. In comparison, the data of the first and last day of each instar were not used for statistical analysis because of their non-normal distributions.
60 b
b
50 b 40 a,b
30 a 20 10 0
4
8 12 16 20 Diet-deprivation period (hours)
24
Fig. 4. First meal duration in the diet-deprived larvae after diet replacement. Each plot (mean7SEM) is derived from observations of 20 diet-deprived animals. The diet-deprivation periods ranged from 30 min to 24 h. Means with different letters denote significantly different one-way ANOVA, Po0:05.
meal compared with the usual 17.972.9 min spent in meals in larvae fed ad libitum. Despite the significant increase in meal duration in the first meal after diet-replacement, dietdeprived larvae resumed normal feeding patterns in subsequent meals (data not shown). 3.3. The effect of long-term diet-deprivation on feeding behaviour As with short-term diet-deprived larvae (6–24 ), longterm diet-deprivation (4 days) resulted in prolonged first meals following diet replacement. Interestingly, most of the larvae that experienced long-term diet-deprivation did not start feeding immediately after diet replacement, but
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initially wandered around the artificial diet blocks delaying feeding up to 3 min after diet replacement (Fig. 5). In most of these larvae, the delay before the first bite ranged from 0 to 3 min, whereas the first bite generally occurred within 1 min in short-term (12 h) diet-deprived larvae (Mann– Whitney test, Po0:05). A few long-term diet-deprived larvae wandered around the reintroduced diet blocks for more than 10 min before feeding. In addition, larvae that experienced long-term diet-deprivation spent more time wandering during the intermeal period following the first meal after diet replacement than short-term diet-deprived larvae (Mann–Whitney test, Po0:001) (Fig. 6). 3.4. The effect of physical stimulation on feeding behaviours During the continuous observations, larvae were occasionally observed to begin wandering or feeding following defecation during intermeal periods. Defecation appeared to trigger wandering or feeding in larvae immediately prior to meals, but defecation was not associated with wandering or feeding if it occurred immediately after a meal. To investigate the link between feeding behaviour and defecation, larvae were closely observed during the intermeal periods. The mean intermeal durations of approximately 90 min were divided into three segments: the ‘‘post-
(%) 15 Proportion of time spent in wandering behaviour
812
10
5
24 hours
4 days
Diet-deprivation period Fig. 6. The proportion of time spent in wandering behaviour during the intermeal period between the first and second meals after diet replacement in 24-h and 4-day diet-deprived larvae. Bars represent means7SEM.
Table 1 Effect of defecation on feeding behaviour
Feeding larvae Observed larvae
Post-feeding
Inter-feeding
Pre-feeding
2 30
5 26
19 23
Time until first bite after diet-replacement (seconds)
90
60
30
0 24 hours 4 days Diet-deprivation period Fig. 5. Time spent in wandering behaviour until the start of the first meal after diet replacement in 24-h and 4-day diet-deprived larvae. A total of 15 and 20 diet-deprived larvae were observed for 24 h and 4 days, respectively. Each dot represents the time interval before a larva initiated feeding.
feeding’’ segment (0–30 min after a meal), the ‘‘interfeeding’’ segment (30–60 min after a meal) and the ‘‘prefeeding’’ segment (more than 60 min after a meal). Most larvae that defecated during the pre-feeding segment began to feed immediately, whereas larvae that defecated during the post-feeding and inter-feeding segments were much less likely to start feeding (w2(d.f.2) ¼ 40.2, Po0:00001) (Table 1). This result indicated that defecation immediately prior to a regular feeding period caused a physiological change in the larvae that may direct to the meal. To examine whether general physical stimulation would also induce feeding behaviour, larvae fed ad libitum were lightly tapped along the cercus above the rectum during an intermeal period. Immediately following the tapping, all larvae initiated wandering behaviour. Within 3 min, some larvae resumed their original quiescent states, while others started to feed. Larvae were also tapped at 30, 60 and 90 min following a meal (corresponding to the post-, inter-, and prefeeding segments as defined above). More than 80% of the larvae tapped during the pre-feeding segment and a small proportion that were tapped during the inter-feeding segment started wandering or feeding. The larvae tapped during the post-feeding segment did not initiate feeding behaviours (w2(d.f.2) ¼ 33.1, Po0:00001) (Table 2).
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Feeding larvae Observed larvae
Post-feeding
Inter-feeding
Pre-feeding
0 20
8 20
18 20
4. Discussion In the present study, we observed that feeding occurred at regular intervals in Bombyx larvae throughout larval development. Previously, Hirao and Yamaoka (1981) recorded the feeding of Bombyx larvae using a short circuit actograph that detected the current between the larval body and an artificial diet block or mulberry leaf. While the short circuit actograph monitored the cyclic feeding patterns of fifth-instar, day 4 larvae, feeding behaviours of larvae at other developmental stages have not been studied or statistically analysed. The current investigation demonstrated that such feeding behaviour patterns are maintained throughout larval development. Furthermore, through continuous observation of the animals, additional behaviours were documented which would be difficult to detect by a short circuit actograph. Behavioural rhythms have also been observed in larvae of other lepidopteran species: M. sexta (Reynolds et al., 1986; Bowdan, 1988a, b; Timmins et al., 1988; and Bernays and Woods, 2000), G. geneura, (Bernays and Singer, 1998) and H. armigera (Barton Browne and Raubenheimer, 2003). Log-survivor plot analyses revealed that feeding behaviour patterns of these insects are structured by regularly occurring meals. Also, log-survivor plots of the gaps between feeding behaviours included a bout criterion ranging from 1.5 to 3.5 min in those insects. The results of this study generated a feeding bout criterion value of 92.578.5 s for Bombyx larvae indicating that Bombyx feeding patterns are similar to other lepidopteran larvae (Fig. 1). Interestingly, the feeding patterns of Manduca larvae on plant leaves differ from those of Manduca larvae raised on artificial diet (Reynolds et al., 1986; Bowdan, 1988a, b; Timmins et al., 1988; Bernays and Woods, 2000). First, the amount of diet intake by larvae fed plant leaves is significantly less than the intake of artificial diet by domesticated larvae. Second, the time spent in feeding behaviour in Manduca larvae fed plant leaves is significantly longer than that of larvae fed artificial diet. Cyclic patterns of feeding behaviour were also observed in Manduca larvae fed artificial diet, but not in larvae feeding on plant leaves. Such differences in behavioural patterns have also been recorded for Bombyx larvae (Hirao and Yamaoka, 1981), although statistical analyses have not been performed. The results of this study revealed that patterns of feeding behaviour in Bombyx larvae that were raised on artificial diet resembled those of domesticated Manduca larvae that were fed artificial diet but not to
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Manduca larvae that were fed plant leaves. Since the feeding behavioural differences were observed even in Bombyx and Manduca larvae fed plant leaves in the laboratory observation (Hirao and Yamaoka, 1981; Reynolds et al., 1986), the differences in feeding patterns between larvae fed artificial diet and larvae fed plant leaves may be due to differences in diet contents. The feeding pattern of Manduca larvae in the field might be more complicated due to the additional influences such as environmental stress or predation (Bernays and Woods, 2000). Short-term diet-deprivation increased the duration of the first meal after diet replacement and increased the time spent wandering during the subsequent intermeal period. Long-term diet-deprivation also resulted in enhanced locomotion activity. This suggests that diet-deprivation stimulates the locomotion systems of Bombyx larvae. Prolonged feeding and activated locomotion have also been associated with starvation in the larvae of the gypsy moth, Porthetria dispar. P. dispar exhibits silking behaviour which helps it to secure itself to food substrates. Dietdeprivation in P. dispar larvae triggered silking behaviour (Leonard, 1967). Although Bombyx larvae also exhibit silking behaviour while on a food source, increase of silking behaviour was not observed in diet-deprived larvae. Long-term, diet-deprived Bombyx larvae, however, showed increased wandering behaviour after diet-replacement, indicating that reduced gut volume might be associated with increased locomotion activity. Elevated locomotion activity in starved larvae in the presence of diet may also result in increased foraging behaviour, probably due to the stimulation of chemical attractants from diet. In Heliothis zea, it has been proposed that nutritional imbalance results in altered levels of neurotransmitters in the central nervous system (Cohen et al., 1988), implying that diet-deprivation may change the level of neurotransmitters in Bombyx larvae, elevating the locomotion activity for foraging. The fact that long-term diet-deprived Bombyx larvae did not begin wandering or foraging until food was replaced indicates that starvation-associated locomotion activity might be controlled by chemical attractants in food. Their quiescence in the absence of food also suggests that longterm diet-deprivation might induce a reduced metabolic state in the larva that would minimize energy loss or nutrient depletion. It is unclear, however, whether locomotion activity and the initiation of feeding behaviour are controlled by the same factors. During observations of Bombyx larvae after meals, we noticed that in some larvae, defecation might be involved in the initiation of feeding behaviour during the pre-feeding segment. In the locust, feeding behaviour is related to defecation behaviour (Simpson and Ludlow, 1986). It is reasonable that defecation triggers feeding behaviour through hindgut volumetric control, since the intake of diet or the entry of diet into the gut would increase gut volume. The complemented volume of the gut by feeding would, ultimately, supply the lost nutrients in the body. In
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the present study, Bombyx larvae did not start feeding behaviours after defecation when defecation occurred immediately following a meal, suggesting that the larvae just after feeding were different from the larvae just prior to regular meals. Therefore, in both locust and Bombyx larvae, the rejection or acceptance of food appears to be strongly related to the time elapsed since the previous meal. Thus, the data indicate that meal-start process may be required for the physical stimulation such as defecation. Also, the fact that the starved larvae did not show the locomotion activity in the absence of diet block suggested chemical attractants of diet influence synergistically on the initiation of feeding behaviour in Bombyx larvae. Accurate elucidation of the factors influencing mealstarts in insects is a complicated issue. Intrinsic modulators and exogenous factors may collaterally affect feeding behaviour. Further investigations into the patterns of behaviour related to nutrient control and ingest ratio (Ueda and Suzuki, 1967; Horie et al., 1976, 1978) will aid in broadening our understanding of feeding behaviour in domesticated Bombyx larvae. With the genetic and genomic information of this species now available (Mita et al., 2003, 2004; Xia et al., 2004) detailed molecular studies utilizing this data will gradually clarify the complex mechanisms underlying the feeding behaviours of the silkworm. Acknowledgements This work was partly supported by the Graduate School of Agricultural and Life Sciences, the University of Tokyo. The authors wish to thank Ms. Jennifer S.I. for critical reading and checking English description. We also thank the members of our laboratory for generous supports and helpful discussions and continuous encouragements. References Barton Browne, L., Raubenheimer, D., 2003. Ontogenetic changes in the rate of ingestion and estimates of food consumption in fourth and fifth instar Helicoverpa armigera caterpillars. Journal of Insect Physiology 49 (1), 63–71. Bernays, E.A., Singer, M.S., 1998. A rhythm underlying feeding behaviour in a highly polyphagous caterpillar. Physiological Entomology 23, 295–302. Bernays, E.A., Woods, H.A., 2000. Foraging in nature by larvae of Manduca sexta influenced by an endogenous oscillation. Journal of Insect Physiology 46, 825–836. Blaney, W.M., Chapman, R.F., Wilson, A., 1973. The pattern of feeding of Locusta migratoria (L.) (Orthoptera, Acrididae). Acrida 2, 119–137. Bowdan, E., 1988a. The effect of deprivation on the microstructure of feeding by the tobacco hornworm caterpillar. Journal of Insect Behavior 1, 31–50. Bowdan, E., 1988b. Microstructure of feeding by tobacco hornworm caterpillar, Manduca sexta. Entomologia Experimentalis et Applicata 47, 127–136. Cohen, R.W., Friedman, S., Waldbauer, G.P., 1988. Physiological control of nutrient self-selection in Heliothis zea larvae: the role of serotonin. Journal of Insect Physiology 34, 935–940.
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