INSECTS

The seven-spotted ladybeetles, C. septempunctata, were taken from our laboratory colony in China Agricultural University, Beijing, P. R. China. Larvae were kept in plastic containers (7 cm in length × 4.5cm in width × 8 cm in height), reared with fresh bean aphids (Acyrthosiphon pisivorum) under a 16:8 h L:D photoperiod at 28–30 °C.

EFFECT OF ABLATION SITE, SIDE OF BODY, THORACIC LOCATION AND INSTAR ON REGENERATION FREQUENCY

To analyze the factors influencing the regeneration frequency and the length of the regenerated leg, the legs of lady beetle instars were ablated at the base of the tibia (half ablation) or at the base of the coxa (complete ablation) in 3 instars (2nd, 3rd and 4th), 2 body sides (left and right), and 3 thoracic locations (foreleg, middle leg, and hind leg) (Fig. 1). After anesthetization, the larvae were placed on double-sided tape, and the legs were ablated using a pair of micro-scissors. Then the ablated larvae were held and fed in the same conditions as before. The newly emerged adults were checked by microscope to determine whether and to what extent the ablated legs were regenerated. Each treatment was replicated 3 times, and each replication included 10 lady beetles.

EFFECT OF ABLATION SITE, THORACIC LOCATIONS AND INSTARS ON THE LENGTHS OF REGENERATED LEGS

The relative leg length (ratio of the length of the regenerated leg to that of the control) and total leg length (sum of the lengths of all segments) were calculated and analyzed based on measurements of the length of a line between the 2 endpoints of segments of adult leg. The legs of control groups were used as the base for analyzing the lengths of regenerated coxae, femurs, tibiae and tarsi of the treated adults.

The adults were stored in a solution of 70% ethanol and 15% glycerol to measure the lengths of regenerated legs by digital microscopy (VHX-1000, Keyence, Osaka, Japan). We scored regeneration as partial regeneration [regeneration into an incomplete leg with missing or fused segments (Fig. 2)], and as complete regeneration [regeneration into a complete leg with normal segments (Fig. 3)]. The relative leg lengths (the ratio of regenerated leg length to leg length of the normal contralateral leg) and total leg lengths (sum of the all segments lengths) were calculated and analyzed based on measurements of the length of a line between the 2 endpoints of the segments of each adult leg. The contralateral legs were used as the base of comparison (control) for analyzing the lengths of regenerated coxa, femur, tibia and tarsus as controls.

Fig. 1.

Complete ablation: larval leg of in C. septempunctata was ablated at the base of the coxa. Half ablation: larval leg was ablated at the base of the tibia. Scale bars equal 500 μm. This figure is shown in color in a supplementary document online as Suppl. Fig. 1 in Florida Entomologist 98(1) (March 2015) at  http://purl.fcla.edu/fcla/entomologist/browse.

Fig. 2.

The effects of half ablation on adult leg regeneration in C. septempunctata. (Left Foreleg): Phenotypes of the forelegs of adults derived from larvae whose left forelegs had been half ablated. No regeneration (NR) in the circle, partial regeneration (PR) and complete regeneration (CR) are shown. In the 2 columns on the right, PR' and CR' are color-level inversion images of the leg segments highlighted. Each scale bar equals 500 μm. This figure is shown in color in a supplementary document online as Suppl. Fig. 2 in Florida Entomologist 98(1) (March 2015) at  http://purl.fcla.edu/fcla/entomologist/browse.

MEASUREMENT OF END OF THE CRITICAL PERIOD FOR REGENERATION

In order to determine the time in stadium when the critical period for leg regeneration of C. septempunctata comes to an end, fourth instar larvae were chosen for the experiment because the second and third stadia were too short to precisely determine the end of the critical period for leg regeneration. We ablated the right midleg of a C. septempunctata 4th instar at base of the tibia every 12 h. The regeneration frequencies of tested insects ablated at different time points were recorded and compared. In all 9 treatments, each treatment was replicated 3 times and each replication contained 15 lady beetles.

Fig. 3.

The effects complete ablation on adult leg regeneration in C. septempunctata. (Left Foreleg): Phenotypes of the forelegs of adults derived from larvae whose left forelegs had been completely ablated. No regeneration (NR) in the circle, partial regeneration (PR) and complete regeneration (CR) are shown. In the 2 columns on the right, PR' and CR' are color-level inversion images of the leg segments highlighted. Each scale bar equals 500 μm. This figure is shown in color in a supplementary document online as Suppl. Fig. 3 in Florida Entomologist 98(1) (March 2015) at  http://purl.fcla.edu/fcla/entomologist/browse.

STATISTICAL ANALYSIS

The effects the ablated site, and the side of the body where the ablation occurred on the frequency of regeneration were analyzed by Student's t-test. We checked the data for normal distribution, subjected the data to one-way ANOVA, and separated the means by the post hoc Tukey's honest test of significance at the 5% level of statistical significance. All statistical analyses were performed using SPSS analysis software (20.0 for Windows; SPSS Inc., Chicago, USA).

MORPHOLOGY OF LEG REGENERATION

Adult legs were regenerated after larval legs had been ablated. Three phenotypes detected in both half ablation (Fig. 2) and complete ablation (Fig. 3) treatments were as follows: 1) no regeneration, i.e., no part of the leg was regenerated either during formation of adult or after adult emergence; 2) partial regeneration, i.e., at least one segment was regenerated, or some segments either were not regenerated, or they were regenerated but were fused together; 3) complete regeneration of legs with normal segments.

EFFECTS OF ABLATION SITE, LOCATION AND LARVAL INSTAR ON REGENERATION FREQUENCY

The regeneration frequency in half ablation (mean ± SE = 88.0 ± 2.0 %, n = 457) was significantly higher than that in complete ablation (66.0 ±3.1%, n = 424; t = 3.589, df = 28.334, P = 0.001; Fig. 4A). No significant difference occurred in ablation between both sides (half ablation, t = 0.036, df = 52, P = 0.971; complete ablation, t = 0.597, df = 52, P = 0.553; Fig. 4B) and among 3 locations (foreleg, midleg and hindleg; half ablation, F = 0.276, df = 2, 51, P = 0.760; complete ablation, F = 0.258, df = 2, 51, P = 0.774; Fig. 4C).

Fig. 4.

Regeneration frequencies (%) of the adult legs after either half ablation or complete ablation of larval legs (Means ± SE). A. Combined results; B. Results on either the left or the right side of the body. C. Results at various thoracic locations; and D. Results on the 2nd, 3rd, and 4th instar. Different letters indicate significant differences among the treatments (mean separation by Tukey's HSD, P < 0.05).

For half ablation, the regeneration frequencies were significantly different when the leg ablations were performed at the second, third or the fourth instars (F = 68.151, df = 2, 51, P < 0.001). For complete ablation, the tendency was similar to that in half ablation, and regeneration frequencies significantly decreased when legs of 4th instars were ablated (F = 92.461, df = 2, 51, P < 0.001; Fig. 4D).

LENGTH ANALYSIS OF REGENERATED LEGS

Lengths of Regenerated Legs in Half Ablation. The leg lengths of partial regeneration and complete regeneration were unaffected by the location of half ablation, i.e., the fore-, mid- or hind leg (partial regeneration, F = 0.636, df = 2, 24, P = 0.538; complete regeneration, F = 0.137, df = 2, 24, P = 0.872; Fig. 5A). Similarly, there were no significant differences among the 3 instars for both regeneration phenotypes (partial regeneration, F = 0.018, df = 2, 24, P = 0.982; complete regeneration, F = 0.027, df = 2, 24, P = 0.973; Fig. 5B).

Leg lengths differed significantly among partial regeneration, complete regeneration and the control (contralateral leg) phenotypes. However, leg lengths did not differ significantly between complete regeneration and the control phenotypes. Among the partial regeneration phenotypes, significantly shorter segments occurred mainly at the distal end of the tibia and the tarsus, which caused the whole leg to appear to be significantly shorter than in the control (F = 6.091, df = 2, 60, P = 0.004; Fig. 5C).

Lengtsh of Regenerated Legs in Complete Ablation. In the complete ablation treatments, the leg lengths of the partial and complete regeneration phenotypes were similar to those in the half ablation treatments. Ablation location (partial regeneration, F = 1.184, df = 2, 24, P = 0.323; complete regeneration, F = 0.013, df = 2, 24, P = 0.987; Fig. 5D) and larval instar (partial regeneration, F = 0.060, df = 2, 24, P = 0.942; complete regeneration, F = 0.004, df = 2, 24, P = 0.996; Fig. 5E) did not affect the lengths of regenerated legs in the complete ablation treatments. On the other hand, because of the occurrence of smaller or absent tibiae and fused tarsi in the partial regeneration phenotype, the regenerated legs were shorter than in the control and in the complete regeneration phenotype (F = 10.247, df = 2, 60, P < 0.001; Fig. 5F).

REGENERATION AND DEVELOPMENTAL COUPLING BETWEEN LARVAL AND ADULT STAGES.

In the partial regeneration phenotype, regenerated leg lengths in the complete ablation treatments were significantly shorter than in the half ablation treatments (F = 6.465, df = 2, 60, P = 0.003). In the complete regeneration phenotype, the lengths of regenerated leg segments both in the complete ablation treatments and the half ablation treatments were slightly shorter than the control (F = 0.071, df = 2, 60, P = 0.931).

Fig. 5.

Ratios (%) of lengths of regenerated legs to contralateral legs (means ± SE) after either partial or complete regeneration when oblation of larval legs was as follows: A. Half ablation at 3 thoracic locations (fore-, mid- and hind legs); and B. Half ablation in the 2nd, 3rd, and 4th instar. C. Half ablation showing lengths of regenerated coxa, femur, tibia, tarsus and entire leg. D. Complete ablation at 3 thoracic locations (fore-, mid- and hind leg); and E. Complete ablation in 3 instars (2nd, 3rd, 4th). F. Complete ablation showing length of regenerated coxa, femur, tibia, tarsus and entire leg. Different letters indicate significant differences (Tukey's HSD, P < 0.05) among the regenerated patternings within one segment or leg.

DETERMINATION OF THE END OF THE CRITICAL PERIOD FOR REGENERATION IN THE FOURTH INSTAR

The frequency of leg regeneration by some adults was small when the leg ablations were performed on fourth instars. Therefore, we further examined the critical period of ablation for leg regeneration in fourth instar C. septempunctata. The results showed that the nonregeneration began to be detected on the day 3.5 (92 ± 8.3%), and regeneration progressively declined at day 4 (50 ± 14%) and at day 4.5 (21 ± 11%) of the fourth instar's stadium, while all legs of larvae ablated before day 3.5 could regenerate in adult stage. So in the fourth instar, as the regeneration frequency on the 4th day became significantly low, the critical period for regeneration occurred on about day 3.5 (F = 13.574, df = 3, 8, P = 0.002; Fig. 6).

REGENERATION FREQUENCY AFFECTED BY THE DEGREE OF ABLATION AND INSTAR BUT NOT BY THORACIC LOCATION AND SIDE OF BODY

This is the first report of leg regeneration in C. septempunctata. Although some publications reported that larval legs of many species were able to regenerate after several molts (Bulliére & Bulliére 1985; Beauchemin et al. 1998; Tan et al. 2004), we found the initial appearance of the regenerated leg happened in prepupae and not in the larva. Our findings are inconsistent with previous studies that suggested that leg regeneration occurred just after the first molt after when larval leg had been ablated (Tan et al 2004; Bulliére 1985).

The leg phenotype suggested that the regeneration blastema possessed a memory of its position along the leg (Stocum 1984) that enabled it to retain the integrity of the leg, and that it guarantees that related functions of the mass of partially de-differentiated cells in the regeneration blastema result in the normal development of the regenerated leg (Tan et al. 2013). This is consistent with past studies of the ablation site in Eupolyphaga sinensis (Walker) (Blattaria: Polyphagidae) leg regeneration. Therefore we had surmised that in C. septempunctata leg regeneration could occur when leg ablations were made at different segments of the leg.

Fig. 6.

Determination of the end of the critical period for regeneration in the fourth instar. Adult leg regeneration frequencies (%) after the right midleg was ablated at various times in the 4th stadium.

Our study showed that the regeneration frequency of legs in half ablation was higher than in complete ablation (Fig. 4A), which suggested that leg regeneration was more difficult when greater amounts of the leg were missing. Our study also showed that there was a significantly lower regeneration frequency of the fourth instars compared to second and third instars (Fig. 4D). The result was consistent with previous studies that regenerative ability significantly decreased with increasing instar (Wang & Cai 2004; Angelini et al. 2009; Suzuki et al. 2009). These studies showed that, in each successive instar, the duration leading to non-regeneration between ablation and the end of the instar became progressively longer. However, ablation of legs at the different thoracic locations and body sides did not affect the regeneration frequency of the legs (Fig. 4B, 4C). These data indicated that thoracic location and side of the body could scarcely affect the generation rate of ablated legs.

LENGTHS OF REGENERATED LEGS WERE MEASURED TO PROVE DEVELOPMENTAL CONTINUITY BETWEEN LARVAL AND ADULT STAGES

We found that partially regenerated legs had shorter segments than completely regenerated legs in both ablation treatments. Especially shortened were the distal ends of the tibia and tarsus (Figs. 5C and 5F), which showed that leg regeneration proceeded along the proximo-distal axis. The lengths of partially regenerated legs in half ablation treatments were remarkably longer than in complete ablation treatments. This indicated that the de-differentiated regeneration blastema was not substantially involved in the patterning of the distal segments of the developing regenerated legs.

The regenerated segment at the point of ablation appeared to be stunted. Thus in the complete regeneration phenotype, leg lengths in both ablation treatments were slightly shorter than the control (contralateral legs) as was reported previously (Bohn 1970; Tan et al 2004). The data indicated that the alterations of larval leg morphology affected the morphology of the adult leg. There appeared to be a smooth transition between the larval appendages and the regenerated adult appendages. This implied that the larval leg patterning imposes a developmental constraint on the adult leg rather than allowing the adult leg to develop independently. The lengths of the regenerated legs were unaffected by the thoracic location of the ablated leg and side of body both in the half ablation and complete ablation treatments, which indicated that thoracic location and side of body could little affect the length of the regenerated leg.

DETERMINATION OF THE BOUNDARY OF THE CRITICAL PERIOD FOR REGENERATION

In our preliminary study, we found that there was a boundary between regeneration and non-regeneration in each instar. The terminal edge of the boundary was defined as the critical period for regeneration (Lüscher 1948). We found the critical period of fourth instar lady beetles was at day 3.5 in the fourth instar's stadium (Fig. 6). Some authors have suggested that there may be a correlation between the peak-hour of ecdysone and critical period as ecdysone had the function of regulating regeneration (Bulliére & Bulliére 1985; O'Farrell et al. 1956; Madhavan & Schneiderman 1969; Marks et al. 1970; Maleville & Reggi 1981), and this needs to be examined by additional research with C. septempunctata.

DEVELOPMENTAL CONTINUITY BETWEEN LARVAL AND ADULT LEG MORPHOLOGIES

Although the fundamental mechanism of developmental continuity between larval and adult legs in C. septempunctata is not clearly understood, superficially there appears to be a smooth transition between the larval and regenerated adult legs, and the latter developed with total dependence on the former. However, in lepidopterans, adult legs develop independently of larval legs and they actually arise from set-aside imaginal cells that reside in the larval legs and proliferate during the prepupal period rather than during the pupal period (Tanaka & Truman 2005). Thus developmental patterning of the leg in C. Septempunctata differs with that in many other arthropod species.

Our study showed that adult leg patterning of C. septempunctata was inherited from the larval leg, and that the patterning of the larval leg appears to have a developmental affect on the adult leg. For example, when a larval leg was ablated and the medial segments were removed, then this morphological alteration was also observed in the adult stage. Thus in the case of C. septempunctata 2 life-history stages (the larva and the adult) which must cope with somewhat different habitats may affect each other. The 2 distinct morphs may constrain each other not only in C. septempunctata but possibly also in most holometabolous insects.

Developmental features are major facilitators of evolution in that they allow the compartmentalization of the different stages of arthropods with complex life cycles (Moran 1994). Developmental continuity between larval and adult stages might be a constraint on evolutionary change. Many insect species have independently evolved imaginal discs including lepidopterans and fruit flies (Siegel & Fristrom 1978). This developmental feature has enabled the larval stage to be ecologically completely separated from the adult stage (Moran 1994; Truman 1999; Svacha 1992). However, through our anatomical experiments, we did not find imaginal discs in the larval legs of C. septempunctata, which cannot develop adult legs independently of the larval legs. Thus, in order to adapt rapid growth within the context of a short life cycle, the evolution of imaginal discs may play a critical role in complete dissociation of larval and adult structures in future evolutionary development.