The Regeneration of Legs in Rhodnius Prolixus (Hemiptera)

Each insect was kept separately in a glass tube and fed separately, so that the complete individual history was known. Apart from the dates of moulting and feeding, the records included the measurements of all parts of the amputated and the control leg in each instar. The exuviae, on which all external details of the regenerating leg can be seen, were prepared together with the regenerated leg of the adult so that every stage in regeneration could be seen in a single preparation (Pl. 9). All experiments were carried out on the second pair of legs. No significant differences could be detected between the regeneration potencies of the different legs. As a rule the insects were kept at a temperature of 25° C. and at a relative humidity of 50–60%.

The possibilities of regeneration in a leg of Rhodnius are summarized in Text-fig. 1. The operation, which is carried out at the time of feeding, is shown in the first column. The other three columns show the regeneration resulting after the next three moults. The figure is valid for any nymphal stage, no striking differences in the regeneration potencies of the different nymphal stages being detectable.

If a leg is amputated through the coxa (a), the remaining part of the coxa is eliminated and no regeneration occurs. After amputation between coxa and trochanter (b), the coxa remains intact but no regeneration occurs. If the leg is cut off through the trochanter (c), a part of the latter persists. Amputation through the femur (d) causes the elimination of the remaining part of the femur and of a part of the trochanter; as a rule no regeneration takes place after this, but occasionally a small rudiment with end-claws appears at a late stage. If the leg is cut off between femur and tibia (e and f), there are two possibilities : either the whole femur and a part of the trochanter are eliminated (e), or the femur remains intact after the next moult (f). In the latter case regeneration begins to be visible after the second moult when a small rudimentary tibia is formed. A more complete leg is regenerated after the third moult. After an amputation through the tibia (g, h, i) regeneration is more rapid and begins after the next moult. The figure shows that the capacity for regeneration increases gradually the more distally the amputation level lies. If the proximal part (g) of the tibia is amputated no tarsus is formed after the next moult, while a tarsus of one segment is formed after amputation in the middle of the tibia (h) and a tarsus with two segments after amputation in the distal part of the tibia.

We may conclude from this account that Rhodnius, unlike Phasmids and Mantids, is not capable of complete regeneration but only of partial regeneration of its legs. The regeneration potencies appear only at the level of the femur-tibia joint and thence increase gradually in a distal direction. This does not mean that if the tip of the leg is cut off there will be a quantitative maximum of regeneration, but differentiation of the regenerated partw ill be more advanced the more distal the operation.

In order to see how regeneration proceeds after it has been initiated by amputation we may study two typical cases.

In Pl. 9 A the regeneration of case 59r is shown from the first instar to the adult. This is a photograph of the exuviae of each nymphal instar and of the leg of the adult. The leg was amputated between femur and tibia at the time of feeding. After the first moult nothing has happened except that the wound is completely closed and the cuticle of the whole femur is much thinner, softer and less darkly pigmented. A short tibia has been formed after the second moult, and after the third moult the regenerated part consists of a tibia and a tarsus of one segment with claws. It is only after the fourth moult that the regenerated leg is complete with a tarsus of two segments. But in the following adult stage the tarsus of the regenerated leg still consists of two segments, while that of the control leg has acquired its third segment. Regeneration is therefore incomplete; the regenerated leg has not reached the length of the control leg and one tarsal segment is missing. It is also noteworthy that after five moults the cuticle of the whole leg, i.e. even that of the femur which has not been regenerated, is much softer and clearer than that of the control leg.

The measurements of the regenerating leg and of the control leg in the same case (59r) are shown in Text-fig. 2A. The times of moulting and feeding are also indicated in this graph, in which the absolute lengths of the different parts of the leg are given in micrometer units. Regeneration and normal growth are combined in this figure, and I have therefore drawn in Text-fig. 2 B the length of the regenerating leg as a ratio of the length of the control leg. The changes due to normal growth have therefore been eliminated in this figure. The figure shows that after the first moult not only is there no regeneration but the femur decreases relatively in length, and only after the second moult is the excessive growth of regeneration evident. This continues until the fifth nymphal instar is reached, when this hypertrophy comes to an end, although the regenerated leg is still far shorter than the control. It seems to be a general rule that regeneration ceases after the fourth moult if the greater part of the tibia is amputated. If only a short part is amputated, regeneration is terminated earlier, but the regenerated leg will never be quite as long as the control.

A further case (58g) in which regeneration was much more rapid is shown in Pl. 9B and in Text-fig. 2C, D. In the first nymphal instar the tibia was amputated in the middle at the time of feeding. After the next moult a tarsus with claws was already present. After the second moult the tarsus consisted of two segments, and in the adult the tarsus of the regenerated leg, as in the control leg, consisted of three segments. Here, therefore, we have a case of complete regeneration although the normal length is not reached (Text-fig. 2D). In this case regeneration also comes to an end after the fourth moult, but the excessive growth begins at once while in the first case it appears only at the second moult.

Text-fig. 3 shows a comparison of some similar cases in which the leg has been amputated at different levels of the tibia in the first nymphal instar. The figure shows that the maximum rate of regeneration occurs after the first or the second moult when the tibia has reached a length of 10 –15 % of the whole length of the leg. An increase of 20% and more is then possible between successive moults.

It has been pointed out already that there are no striking differences in the regeneration potencies of the various nymphal stages. This might be illustrated by some experiments carried out on first, third and fifth nymphal instars. The tibia was amputated near the middle at the time of feeding. The figures in Table 1 give the relative increase in length of the regenerating legs after the first post-amputation moult. There is great variability between the individuals at the same nymphal instar. The average relative increase is greater in the late than in the early instars, but since in the former moulting occurs only after a longer period, we find that the relative increase per day is actually less, although of course more tissue is regenerated during the whole period between successive moults. The difference in regenerative capacity is significant, but there is no significant difference in the degree of differentiation of the regenerated leg after the moult which follows the amputation.

As was first pointed out by Schaxel & Adensamer (1923), the differentiation of the regenerated leg is dependent on the time which elapses between the amputation and the next moult. In spite of its obvious significance, however, the relationship has never been investigated experimentally. For this purpose Rhodnius is an ideal insect, since the time of moulting is dependent on the time of feeding and can therefore be controlled (Buxton, 1930).

The legs of a number of third instar nymphs were amputated through the middle of the tibia at different times before and after feeding. All stages of regeneration from an unhealed wound to a well-differentiated tarsus of two segments resulted. This is shown in Text-fig. 4. If the moult occurs 2 or 3 days after the amputation, the wound does not heal properly, though the edges of the new cuticle are gradually drawn together. Some haemolymph will flow from the wound and close it by coagulation. After a moult occurring 4 days after the amputation, the wound is perfectly healed and a continuous cuticle is formed. With an interval of 6 days very small claws begin to appear at the end of the tibia, while a slight separation between tibia and tarsus can be detected with an interval of 9 days. With 11 days a diminutive tarsus is clearly separated from the tibia. With 15 days the tarsus is much longer but still consists of one segment only. A tarsus of two segments is formed with an interval of 18 days.

The increase in length of the regenerated leg after different time intervals is shown graphically in Text-fig. 5. The maximum rate of regeneration is reached when moulting occurs 20 days after the operation, i.e. if the operation has been carried out about 5 days before feeding. If the amputation is performed earlier, regeneration is less efficient. It is therefore very probable that 5 days after the amputation the activation by the initiating factor is in full operation, so that a new activation by feeding will fall at just the right time to produce excessive growth. The height of cell activity during wound healing in Rhodnius also occurs 5 days after the injury (Wigglesworth, 1937). The same phenomenon has also been demonstrated in tadpoles of the anuran Xenopus laevis which have been kept without food for a certain time. Five days after the tails of these tadpoles had been amputated, mitotic activity among the cells of the stump reach a peak (Lüscher, 1946).

Since the time of moulting is dependent upon the temperature, it is obvious that this factor also influences regeneration. It was expected that regeneration would be accelerated by high temperatures to the same extent as the moulting processes. But experiment has shown that the degree of acceleration is much more rapid in the case of regenerative growth. Three lots of third instar nymphs were fed and their legs amputated through the middle of the tibia. They were then exposed to temperatures of 17, 25 and 30° C. The results are shown in Table 2. The figures show clearly that a high temperature is very favourable for regeneration while a low temperature of 17° C. is unfavourable, even though the time which is available for regeneration is relatively long. The regeneration processes are therefore more dependent on temperature than the moulting processes.

We have already mentioned the fact that the regeneration potencies increase progressively as the amputation level passes towards the tip of the leg. This obviously can only be true for the differentiation of the regenerated part and not for the quantitative regenerative growth. In order to investigate the relation of the amputation level to the regenerative growth, the legs of a number of fourth instar nymphs were amputated at different levels of the tibia at the time of feeding. Text-fig. 6 shows the results of this experiment. It gives the total increase in length of the tibia and the tarsus, i.e. the regenerating part which is formed in the remaining portion of the tibia, and represents the sum of regenerative and normal growth. A maximum is reached.when the stump of the tibia has a length of 40 micrometer units. From this point the curve continues horizontally. From this we may conclude that the whole growth, normal and regenerative, does not require more than a part of the tibia which is 40 units long, and that only the distal part (40 units long) of the tibial stump takes part in regeneration.

If we deduct from this growth increment the values for normal growth (which fall on a straight line), we obtain a curve with a definite maximum of regenerative growth when the length of the remaining part of the tibia is about 35 micrometer units (Text-fig. 7). We may suppose that there are two factors limiting regenerative growth: (i) the volume of the tibial stump and (ii) the length of the missing part, i.e. only a certain proportion of the growth required for replacement of the missing part will be accomplished. Obviously there must be a zone where both limiting factors (they may be thought of as gradients) are equal, and in this zone there will be a maximum of regenerative growth. The limiting factors may be drawn as straight lines as has been done in Text-fig. 7. According to this figure the first limiting factor, the space factor, would be about 200%, i.e. a regenerative increase of not more than 200% is possible in the remaining part of the tibia. The second limiting factor would be about 76%, i.e. not more than 76% of the required regenerative growth for the re-establishment of the normal length can be accomplished in the time available.

This conception of two limiting factors is not in accordance with our first conclusion, namely, that only the distal part of the remaining portion of the tibia takes part in growth and regeneration. At the present moment it is not possible to decide which conception is correct. Nevertheless, as there is no evidence for normal growth taking place only in a part of the tibia, the conception of the two limiting factors may prove a useful working hypothesis.

As Rhodnius has been shown to be capable of a partial regeneration of its legs, this function has now been demonstrated in an hemipteran. Very probably partial regeneration is also possible in many other insects. It also occurs, for example, in the milkweed bug Oncopeltus fasciatus, though the extent of regeneration is relatively slight (unpublished).

Partial regeneration becomes more complete the more distally the amputation level lies. The leg of Rhodnius may be regarded as possessing a gradient of regenerative potencies which is highest in the end of the tarsus. In this insect, therefore, the mechanism seems to be quite different from that in Phasmids where, according to Friedrich (1930), regeneration is much quicker if the leg is autotomized in the trochanter than if it is amputated at a more distal level. In Phasmids, however, regeneration and the correlated mechanism of autotomy are highly specialized acquisitions. In Rhodnius we may be dealing with a very primitive form of regeneration.

Regeneration in Rhodnius is dependent on the time interval between the amputation and the next moult, and almost any state of differentiation of the regenerated part can be produced by varying the time relations. A maximum of regeneration has been found when the insects were fed 5 days after the operation, probably when the initiating factor has reached its highest activity. Regeneration is also dependent on temperature. It can be almost entirely suppressed by low temperature while it is considerable at high temperatures.

The differentiation of the regenerated leg is also dependent on the amputation level. After amputation of part of the tibia the regenerative growth seems to depend on the volume of the tibia that remains and on the length of the missing part, only a certain proportion of which can be regenerated.

For future work on the factors governing regeneration we may conclude that it will be very important to keep the insects at a constant temperature, and to amputate the leg always at the same level and at the same time in relation to the time of feeding. But even so there may be considerable individual differences, so that it will be necessary to perform an extensive series of experiments and to analyse the results statistically.

1. Partial regeneration of the legs is possible in Rhodnius prolixus.

2. The regeneration potencies appear at the level of the femur-tibia joint and increase gradually in a distal direction.

3. Regeneration usually ceases after the fourth post-amputation moult.

4. No striking differences in regenerative capacity can be detected either in the different legs on one nymphal instar or in the legs of different nymphal instars. There is, however, considerable individual variation.

5. Regeneration is dependent on the time which elapses between the amputation and the next moult.

6. The regeneration processes have a higher temperature coefficient than the moulting processes.

7. A conception of two limiting factors is briefly discussed.