INTRODUCTION

Damage in the sensory apparatus usually causes changes in animal behavior. In most cases, however, the distorted or inadequate behavior is corrected to some extent after a certain period. Such a “behavioral compensation” is essential for animals to survive in the natural environment. Behavioral plasticity is thought to arise from the plastic nature of the neural system. Therefore, investigation of the behavioral plasticity is one of the ways that could lead to better understanding of the basic mechanism of neural plasticity.

Crickets carry out an appropriate escape behavior in response to air disturbance (Bentley, 1975; Gras and Hörner, 1992; Kanou et al., 1999). This behavior is mediated by inputs from the mechanosensitive filiform hairs located on the surface of a pair of abdominal appendages called cerci (Bentley, 1975; Kanou et al., 1999). The information from cercal filiform hairs is integrated by some giant interneurons (GIs) in the central nervous system, which are supposed to be responsible for the elicitation of the escape behavior (Murphey et al., 1977; Kanou and Shimozawa, 1984; Jacobs et al., 1986). In adult crickets, ablation of one cercus results in changes in the response properties of GIs (Palka and Edwards, 1974; Tobias and Murphey, 1979; Levine and Murphey, 1980; Matsuura and Kanou, 1998a) and in the escape behavior (Kanou et al., 1999) in response to an air-motion stimulus.

Our previous studies revealed that neural and behavioral compensations in adult crickets occurred within 2 or 3 weeks after a unilateral cercal ablation (Matsuura and Kanou, 1998b; Kanou et al., 1999). In terms of behavioral compensation, however, the response rate and the escape direction showed different trends of recovery, i.e., the escape direction was almost perfectly compensated within 2 weeks after the ablation whereas the response rate measured even 3 weeks after the unilateral cercal ablation was less than half that of normal adults (approximately 20%; Kanou et al., 1999). Based on these results, we previously hypothesized that the information which regulates the response rate and the escape direction may be processed in different neural pathways (Kanou et al., 1999). If this were true, the response rate and the escape direction following a unilateral cercal ablation may show distinctive changes during postembryonic development. In order to explore this, we, in this study, first investigated the escape behavior in first-, third-, sixth- and last-instar nymphs. Next, we carried out a unilateral cercal ablation on the nymphs and investigated its effect on the air-puff-evoked escape behavior. Then, the ablated nymphs were reared until the final molt, and changes in the behavior was observed. Since a newly regenerated cercal bud was ablated at each molt, sensory inputs from a dissected cercus were totally absent during the development. The results indicate that an escape-eliciting potential of one cercus decreases, whereas the capacity for recovery of the response rate increases during postembryonic development.

MATERIALS AND METHODS

Analysis of the escape behavior

First, the response rate and the behavioral directionality of the first-, third-, sixth- and last-instar nymphs in response to the standard air puff were measured. Following the measurements, the left cercus of each insect was cut from the basal part. One day (24–36 hr) after the ablation, the second investigation of the air-puff-evoked escape behavior was carried out. Then, the nymphs were reared until the final ecdysis. During the postembryonic development, the new cercal bud that emerged at each molt was ablated within 24 hr postmolting. The wind-evoked escape behavior of the insects was investigated between 24 and 48 hr after the final molt.

The escape behavior elicited by an air puff was recorded on videotape, and later displayed on a CRT for analysis. During off-line analysis, the response rate and the direction of escape were measured. The response rate was calculated as the percentage of the number of the elicited escape responses relative to the number of trials. In this study, only a body motion accompanied by at least one leg step was counted as a “response”. The directionality of the escape was assessed from the relationship between the stimulus and response angles (Figs. 1A, B). The relationship between the angles was plotted on a graph, and a regression line and correlation coefficient were calculated for the parameters that assess the directionality of the escape (Fig. 1C). Five behavioral tests were conducted on each cricket, at 6-minute intervals. The off-line (videotape) analysis revealed that some insects had moved before administration of the air-puff stimulus. Such spontaneous locomotion was omitted from the data. Details of the behavioral analysis were the same as in the previous paper (Kanou et al., 1999).

Fig. 1

Methods for the investigation of escape directionality. Conventions in measuring stimulus (A) and response (B) angles. C: An example of the plot of escape directions for intact adult crickets. If crickets always escape 180° away from the incoming air puff, the regression line will show a 45° slope (y=x). In this illustrated case, the slope of regression line was 1.07 and the correlation coefficient was 0.96.

RESULTS

Escape behavior of intact nymphs

The escape behavior of intact cricket nymphs was first investigated using a standard air puff (3.0 m/sec). The response rates were 54.3, 58.5, 58.4 and 57.1% for the first-, third-, sixth- and last-instar nymphs, respectively (Table 1). There was no significant difference in the response rates of nymphs in different stages (P>0.05). Moreover, the response rate of nymphs at each developmental stage was not significantly different from that of intact adults (53.7±5.0%; mean ± SD: Kanou et al., 1999) (P>0.05).

Table 1

Summary of the wind-evoked escape behavior of intact nymphs

The slopes of regression lines were 0.73, 0.86, 0.89 and 0.88, and the correlation coefficients were 0.77, 0.89, 0.83 and 0.85 for the first-, third-, sixth- and last-instar nymphs, respectively (Table 1). In intact adults, the mean slope of the regression line and the correlation coefficient were 0.90 and 0.87, respectively (Kanou et al., 1999). Therefore, except for the first-instar nymphs, the parameters indicating the behavioral directionality of nymphs were almost identical with those of intact adults.

Effects of unilateral cercal ablation in nymphs

The response rates of the first-, third-, sixth- and last-instar nymphs measured one day after the left cercus ablation were 37.6, 19.1, 11.0 and 4.0%, respectively (Table 2). These response rates were significantly lower than those observed prior to the ablation (P<0.01) (Fig. 2). However, except for the last-instar nymphs, these response rates were significantly higher than those of adults (4.8–5.5 %; Kanou et al., 1999) measured one day after the unilateral cercal ablation (P<0.01). As the effect of unilateral cercal ablation on the response rate was less in younger insects, the efficacy of sensory inputs from one cercus for the elicitation of the escape behavior (escape-eliciting potential of one cercus) must decrease during postembryonic development. However, as mentioned above, the response rate of intact crickets is maintained almost 50–60 % throughout the developmental period (see discussion).

Table 2

Summary of the wind-evoked escape behavior of nymphal crickets one day after left cercus ablation

Fig. 2

Left ordinate and solid lines; response rates of nymphal crickets measured before and after a unilateral cercal ablation. Intact: response rates of intact nymphs measured at each developmental stage. 1-day: response rates of nymphs measured one day after the left cercus ablation. Final: response rates of the ablated insects measured after the final molt. Plotted from data shown in Tables 1, 2 and 3. Hatched area indicates the response rate of adult crickets measured one day after a unilateral cercal ablation (4.8–5.5 %; Kanou et. al., 1999). Right ordinate and dashed lines; the recovery ratios calculated by dividing each “Final” response rate by “1-day” response rate. The duration of each nymphal stage is 5–7 days.

The slopes of the regression lines measured one day after left cercus ablation were 0.55, 0.57, 0.72 and 0.20, and the correlation coefficients were 0.68, 0.74, 0.79 and 0.23 for the first-, third-, sixth- and last-instar nymphs, respectively (Table 2). Except for the sixth-instar nymphs, the slopes were significantly smaller than those measured at the same stage for intact nymphs (P<0.05). Following ablation, the angles of escape became smaller and showed large variations in most cases.

Behavioral compensation

The unilaterally cercal-ablated nymphs were reared to the adult stage and the response rate was measured after the final molt. During their developmental period, the cercal regenerates that emerged at each molt were removed. The response rates of crickets that underwent a left cercus ablation at the first-, third-, sixth- and last-instar nymphs were 41.3, 33.8, 28.7 and 12.2%, respectively (Table 3). The response rate of each group was higher than that measured one day after the ablation (statistically significant difference (P<0.01) was confirmed except for the insects reared from the first instar). Therefore, compensational recoveries in the response rate were confirmed in most cases. In order to assess the change of the capacity for recovery at each nymphal stage, the final response rate was divided by the response rate measured one day after the unilateral cercal ablation. The recovery ratios were calculated to be 1.09, 1.98, 2.61 and 3.05 for the first-, third-, sixth- and last-instar nymphs, respectively (Fig. 2). In spite of the short recovery time for the insects that underwent a unilateral cercal ablation in later stages, these insects showed a higher capacity for recovery (see discussion).

Table 3

Summary of the wind-evoked escape behavior after final ecdysis

The slopes of regression lines of the crickets whose left cercus was removed at the first-, third-, sixth- and last-instar nymphs were 0.85, 0.97, 0.61 and 0.31, respectively (Table 3). The corresponding correlation coefficients were 0.85, 0.90, 0.64 and 0.35 (Table 3). In the case of insects reared from the first and the third instar, the slopes of regression lines and correlation coefficients were both significantly larger than those measured one day after unilateral cercal ablation (P<0.05 or P<0.01; Table 3). Therefore, the directionality of escape demonstrated compensational changes in these insects. On the other hand, the insects that underwent unilateral cercal ablation from the sixth and the last instars did not show any compensational recovery in the escape direction. The time course of the recovery in the escape direction in nymphs may differ from that in adults (see discussion).

DISCUSSION

The response rates of intact nymphs were almost the same as those of intact adults (Table 1). However, the total number of cercal filiform hairs in crickets changes during postembryonic development, i.e., it increases at each molt (Palka and Edwards, 1974; Kanou et al., 1988). For example, an adult cricket is estimated to possess approximately 2000 filiform hairs (Chiba et al., 1992), whereas the number of hairs on a first-instar nymph is about 55 (Bentley, 1975; Knyazev and Popov, 1981). This suggests that each filiform sensory neuron of nymphs must drive postsynaptic neurons (e.g. GIs) more powerfully than in adults. In fact, despite the small number of cercal filiform hairs, some GIs in third-instar nymphs exhibited almost the same sensitivities as those of adults (Kanou et al., 1988). According to the increase in the number of filiform hairs during postembryonic development, the contribution of each filiform hair to the induction of the escape behavior must become smaller. In other words, the efficacy possessed by a small number of filiform sensory neurons in early stages of development may be distributed to a larger number of filiform sensory neurons that emerge in later developmental stages.

The effect of a unilateral cercal ablation on the response rate was lesser in nymphs than in adults, except for the last-instar nymphs. The younger the insects, the smaller the effect was (Table 2 and Fig. 2). For example, the response rate following ablation was 37.6 % for the first-instar nymphs (Table 2), whereas it was 4.8–5.5% for adults (Kanou et al., 1999; indicated by the hatched area in Fig. 2 in this paper). This appears somewhat inconsistent because the response rates before the unilateral cercal ablation were almost identical between adults and nymphs, i.e., 50–60 %. Based on the results derived from the behavioral experiments, we propose the following hypothesis to explain the functional changes in the escape-eliciting system during postembryonic development. In younger nymphs, the total sensory input from each cercus for the elicitation of an escape behavior (escape-eliciting potential of one cercus) is larger than in older nymphs and affects the neural circuit to induce the escape behavior almost independently. However, during postembryonic development, the escape-eliciting potential of each cercus gradually decreases; which is why the effect of a unilateral cercal ablation is larger in later-instar nymphs. On the other hand, the sensory inputs from each cercus must change to affect the escape-eliciting system not in an independent but in a facilitatory manner. As a result, a constant response rate is maintained throughout the postembryonic development (Fig. 3). Probably, the development of the facilitatory effect progresses in conjunction with the decrease in the escape-eliciting potential of each cercus during postembryonic development.

Fig. 3

Diagram showing hypothetical changes of the capacity for recovery of the response rate, escape-eliciting potential of one cercus and facilitation of inputs from both cercus. In intact crickets, escape-eliciting potential of one cercus gradually decreases during postembryonic development. Instead, facilitation of the inputs from each cercus becomes essential to determine the response rate. As a result, an identical response rate (white arrows) is maintained during the developmental period. When a unilateral cercal ablation is carried out (black arrow), no facilitation occurs hereafter. In such a situation, the response rate after the final molt is determined by the escape-eliciting potential of the remaining cercus (white circles) and the capacity for recovery of the response rate (black circles) at the time of ablation. The capacity for recovery of the response rate gradually increases during postembryonic development.

The response rates of unilaterally cercal-ablated crickets measured after the final molt were higher than those measured one day after the treatment (Fig. 2). Obviously, a compensational recovery occurred in the neural system to maintain the escape elicitation. The final response rate clearly depended on the period of cercal ablation, i.e., the longer the ablation period, the higher the final response rate (Table 3 and Fig. 2). However, it does not simply mean that a longer ablation period causes a higher degree of compensation because the response rate one day after the unilateral cercal ablation was higher in younger insects (Table 2 and Fig. 2). Furthermore, as the escape-eliciting potential of one cercus appears to gradually decrease during postembryonic development in normal crickets, the same process may occur in unilaterally cercal-ablated insects. Therefore, if the compensational recovery and the decrease occur simultaneously, we can hardly estimate the actual extent of the recovery of the response rate in the ablated insects. However, we assume that the instantaneous level of an escape-eliciting potential possessed by the remaining cercus at the time of unilateral cercal ablation is maintained until after the final molt because the sensory inputs from filiform hairs on the ablated cercus is totally absent thereafter. If this were true, we can roughly estimate the capacity for recovery of the response rate at each nymphal stage by dividing the final response rate by that at the time of unilateral cercal ablation. The estimated capacities for the recovery were 1.09, 1.98, 2.61 and 3.05 for the first-, third-, sixth- and last-instar nymphs, respectively (Fig. 2), and it was 3.8 in adults (18.2/4.8; Kanou et al., 1999). The capacity for recovery of the response rate gradually increases during postembryonic development. Therefore, the escape-eliciting potential of one cercus and the capacity for recovery of the response rate conversely change during postembryonic development (Fig. 3). The response rate after the final molt is likely to be determined by the escape-eliciting potential of the remaining cercus and the capacity for recovery of the response rate at the time of unilateral cercal ablation (Fig. 3).

In adults, the escape direction showed compensational recovery almost 2 weeks after a unilateral cercal ablation (Kanou et al., 1999). In those crickets that underwent unilateral cercal ablation from the first and the third instars, the escape direction was as accurate as that of normal crickets (present study). Since these crickets had more than 6 weeks before the final molt, the period must be sufficient for the compensational change in the escape direction. On the other hand, the compensational changes in insects that underwent ablation at the last nymphal stage were incomplete because these insects had only one week before the final molt. However, those that had a unilateral cercal ablation from the sixth instar exhibited no compensational change in escape direction despite the two-week period before the final molt. These results suggest that the compensational change in the escape direction progresses at different time courses for nymphs and adults.