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The cuticle as a template for growth in Rhodnius prolixus
J. Insect Physic& 1971, Vol. 17, $9. 2421 to 2434. Pergamon Press. Printed in Great Britain
THE CUTICLE AS A TEMPLATE FOR GROWTH IN RHODNIUS PROLIXUS H. C. BENNET-CLARK Department of Zoology, The University, Edinburgh EH9, 3 JT (Received 16 March 1971; revised 13 May 1971) Abstract-The variation in size that occurs in Rhodnius has been examined and it is found that while the size of the head in any instar is almost constant, the dimensions of the abdomen vary as Vweight. The meal that is taken is 5.7 times the unfed weight, suggesting that the abdomen stretches by a standard ratio regardless of size. In the adult the size of the abdomen is neither constant nor varies as $/weight and the size is difficult to predict from the size of the fifth instar larva. Experiments with dilute meals and constraints on the size of the cuticle showed that the size of the larval abdomen is affected by the size of the cuticle in the previous instar; the effect of nutrition on the size of the head, however, is far less great. Experiments with fifth instar bugs showed that the size of the abdomen of the adult was directly related to the size of the larval abdomen at about the time of the deposition of the epicuticle. A model has been suggested to describe the growth of Rhodnius which prop poses that the size in the next instar is the product of the existing size and a local growth factor. The local growth factor may change at metamorphosis and is probably expressed at the cellular level in terms of cell density and the area of epicuticle deposited per cell. INTRODUCTION
BEFORE the ecdysis of an insect, an initial phase of mitotic activity of the epidermis is followed by the deposition of the epicuticle. Shortly after its deposition, the epicuticle expands and becomes folded (WIGGLESWORTH, 1933). Before ecdysis, a considerable thickness of presumptive exocuticle is laid down and, after the expansion that accompanies ecdysis, this may be tanned and so stabilizes the size of the newly ecdysed insect at the size to which it has expanded during ecdysis. At ecdysis the exocuticle becomes briefly and reversibly plastic (BENNETCLARK, 1961; COTTRELL, 1962) and so offers little limitation to the expansion that occurs. Instead, it seems likely that the limit to expansion is reached when the folds in the epicuticle have been pulled out (WIGGLESWORTH, 1933; BENNETCLARK, 1963). The size that is attained after ecdysis is thus determined by the degree to which the epicuticle is expanded after its deposition but before ecdysis. Studies of the surface sculpturing of the epicuticle of insects (HINTON, 1970) support the view that the gross folding of the epicuticle is pulled out flat at the ecdysial expansion; minor surface irregularities remain and, with pre-ecdysial thickening or tanning, or merely from the elasticity of the epicuticle, form the fine surface sculpturing that may occur. 2421
2422
H. C. BENNET-CLARK
The mechanical events that occur at ecdysis merely manifest a previously determined pattern of epicuticular folding. Where patterning of insects has been studied, it has been shown that there are general and local factors (WIGGLESWORTH, 1940a) and that there are directional gradients in various parts of the body (LOCKE, 1959, 1966) which imply that there is an inherent control of pattern in Rhodnius that is largely independent of such factors as the rate of development or the nutrition of the insect. This however is not the case in Diptera (ALPATOV, 1930). Where the effect of nutrition has been examined, the relation between the initial and final size of growing tracheae has been shown to be complex (LOCKE, 1958), and it was shown that the control of the increase of diameter was different from the control of the increase of length. It is easy to envisage a growth mechanism that follows a standard programme of epicuticular expansion in the pharate phase of each instar and, indeed, this is the simplest explanation for the logarithmic growth that occurs in many insects (c$ WIGGLESWORTH,1965). In the larvae of many insects such as most Endopterygota and Hemiptera, the abdominal cuticle is extensible and development of the next instar may occur after an abdominal extension which must merely exceed a certain threshold (WIGGLESWORTH,1933). Such a cuticle clearly presents a variable surface on which the epidermal cells divide and form the new cuticle. This paper examines the idea that the old cuticle might act as a template for the formation of the new cuticle. In this context I am following the Shorter Oxford Dictimary definition that a template is ‘used as a gauge or guide in bringing any piece of work to the desired shape’.
MATERIALS
AND METHODS
The bug Rhodnius prolixus has been used for the experimental parts of this work. The culture has been maintained for many years at Edinburgh but probably originated from the London School of Hygiene and Tropical Medicine. As such, the culture has probably been maintained for 40 years, or about 100 generations, and is inbred and homogeneous. Bugs are routinely fed on the shaved belly of a rabbit. As it is difficult to feed more than about 80 fourth instar or 40 fifth instar larvae at one time, experiments have been designed about batches of bugs of these numbers. Within any batch there is some variation in the weight, the meal size, and the time taken to feed; the normal bug feeds till it is replete at which stage the abdominal cuticle is shiny and apparently all epicuticular folds are flattened. Following the meal, considerable diuresis occurs and upwards of 50 per cent of the weight of the meal is excreted within 24 hr (WIGGLESWORTH, 1931). If the meal is larger than a critical size, the bug ecdyses to the next instar, from the fourth instar in 13 to 15 days and from the fifth instar to the adult in 22 to 25 days at 25°C the temperature at which I have kept my animals. Bugs may alternately be fed on small quantities of heparinized blood using an artificial feeder described earlier (BENNET-CLARK, 1962).
THE CUTICLE AS A TEMPLATE
FOR GROWTH
IN RHODNIUS
PROLIXUS
2423
At the start of an experiment, bugs are taken 14 days after ecdysis and are weighed, marked on the prothorax with identifying spots of paint (using a variation of the international resistor colour code), and are then measured using a calibrated microscope with a measuring eyepiece. For the measurement, the. bugs were held on a sticky slide made by winding Scotch tape sticky-side-out around a glass slide, one surface of which was attached to another slide. Bugs held thus did not appear to be damaged in any way and were unable to move during the measurement. Bugs were confined in a tared container and weighed on an Oertling single pan balance. Linear measurements under 5 mm were recorded to the nearest 0.05 mm and larger measurements to + 0.05 mm. Weights were rounded to the nearest milligram but, for experiments, bugs were batched into groups at 1 mg intervals with third instar, 2 mg intervals with fourth instar, and 3 mg intervals with the fifth instar unfed larvae. After feeding the bugs were reweighed and replaced in numbered bottles. Where appropriate, newly fed bugs were also measured using the. microscope. Subsequent measurements were made where appropriate to the experiment. Observations have also been made with the cockroach Periplaneta americana. This insect has a far smoother cuticle than Rhodnius and so it is easy to see the hexagonal patches of epicuticle laid down by the individual epidermal cells. Measurements of cell density have been made using a microscope fitted with either 8 or 4 mm objectives and counts have been made with the use either of a haemocytometer slide or of a calibrated eyepiece graticule. RESULTS
Size of normal Rhodnius The length of the head, the length of the abdomen, and the width of the fourth abdominal tergite of Rhodnius were measured using batches of unfed bugs of the same age. The head was chosen as an easily measurable part of the body that does not change in size during the course of an instar and the abdomen because of the twofold linear stretch that accompanies feeding (BENNET-CLARK, 1963). The results are shown in Fig. 1 where the width or length of the organ is plotted against the unfed weight, 2 weeks after ecdysis. Various points emerge; the head of a heavy bug is somewhat longer than that of a light one but significantly less than would be expected if length cc +/weight. The length and width of the larval abdomen are both nearly proportional to +/weight which in itself is suggestive since the abdomen accommodated the meal. After metamorphosis, the abdominal cuticle becomes sclerotized and inextensible. The length and width of the abdomen of the adult no longer show the same dependence on the weight of the bug and there is sexual dimorphism in the length of the abdomen. It appears that the hard parts of the larval stages obey Dyar’s law in that there is a standard size for the instar and a standard factor for its increase at each ecdysis. The size of the abdomen varies continuously as the q/weight in the larva but changes abruptly at metamorphosis when it is neither dependent on a power of the weight nor is constant.
2424
I-1. C.
BENNET-CLARK
a-
1
0
20
Weight
2
I
1
40
GO
weeks
after
1
I
80 ecdysis,
100
/
120
mg
FIG. 1. Graph showing the relationship
between the weight and the linear dimensions of various organs for third, fourth, and fifth instar larval and adult Rhodnius measured 14 days after ecdysis. The symbols indicate the mean value and the vertical line the 95 per cent confidence limits where these exceed the size of the symbol. (0 refers to fifth instar bugs resulting from feeding fourth instar bugs with 25 per cent blood described in The effect of large dilute blood meals.
When larvae of known size are fed to repletion, the weight when fed directly is proportional to the unfed weight (Fig. 2). For bugs fed 14 days after the preceding ecdysis, the normal full meal is 5.7 times the unfed weight or the fed weight is 6.7 times the unfed weight. This appears to hold for the second to fifth instar larvae; the first instar larva takes a larger meal of up to eleven times the unfed weight (BUXTON, 1930). Since fed weight is proportional to unfed weight and since abdomen width is proportional to $‘unfed weight it follows that the stretched size of the abdomen after feeding is proportional to the unstretched size of the abdomen or that abdominal distension is dependent on the unfed size of the abdomen. The effect of large dilute blood meals A batch of 20 19 mg fourth instar Rhodnius was taken. Half were fed on whole heparinized ox blood and the other half were fed 25 per cent heparinized ox blood
THE CUTICLE AS A TEMPLATE
FOR GROWTH
IN RHODNIUS
PROLIXUS
2425
in Ringer-Locke. In both batches, the weight of the meal was similar but the bugs fed 25 per cent blood excreted about 75 per cent of the weight of the meal in the
Unfed
weight
of larva,
mg
FIG. 2. Graph showing the relationship between the fully fed weight and the unfed weight of second, third, fourth, and fifth instar larval Rho&&s weighed and fed 14 days after ecdysis. The graph also shows the bugs examined in The efJect of large dilute blood meals. The vertical lines show 95 per cent confidence limits of the means.
first day compared with 45 per cent in the controls. After ecdysis, the bugs were weighed and measured; the results are summarized in Table 1. The length of the head is slightly but significantly smaller in the experimental group and the dimensions of the abdomen are closer to those of the normal fourth instar bugs than to fifth instar bugs (the dimensions are plotted on Fig. 1) and show the same dependence on the unfed weight of the insect. This suggests that the initial distension is not important in determining the size of the head or the abdomen in the next instar. Roth groups of bugs were fed in the fifth instar. The experimental group fed to repletion but took a significantly smaller meal than the controls (Table 1) which was appropriate to bugs of their weight (Fig. 2). A second experiment using 23 mg fourth instar bugs gave similar results. There was no significant difference between the experimental and the control group in the time between the meal and ecdysis but the length of the head was significantly smaller in the experimental group. This suggests that the effect of the content of the meal is nutritional rather than the result of a change in the time course of moulting. The e#ect
of abdomen
size during
the mouking
cycle
When the normal fifth instar Rhodnius metamorphoses to the adult, the abdominal cuticle changes in character. The abdomen of the adult is relatively
H. C. BENNET-CLARK
2426
longer than that of the larva and the dimensions are neither proportional to q/weight nor are they constant (Fig. 1). This suggests that the relation between the dimensions of the adult and of the preceding larva is complex. TABLE ~-COMPARISON OF THE DIMENSIONS OF NORMALFIFTH INSTARLARVALRhodnius WITH THOSERESULTING FROMFEEDING25 PER CENTBLOODIN THE FOURTHINSTAR*
Head length (mm) Abdomen width (mm) segment 4 Abdomen length (mm) Unfed weight as fifth instar (mg) Replete weight in fifth instar (mg)
Fed 25% blood
Control, whole blood
2.96 + 0.075, n = 9 4.38 + 0.17, n = 9
3.13 kO.06, n = 10 4.92 z! 0.20, n = 10
6.37 + 0.19, n = 9 25.2 + 2.2, n = 9
6+34+0*15, it = 10 40.9 + 3.9, n = 10
167f44,
n= 8
278 + 61,1z = 9
* The size of the experimental groups and standard deviations of the means are quoted. In all cases the probability of similarity between experimental and control groups is less than 0.001.
A batch of fifth instar bugs of different weights was fed to repletion, after measurement of the various dimensions. After ecdysis, the adults were measured. The relation between the width of the larval abdomen and that of the adult is plotted in Fig. 3. The relation can be empirically described by the formula, width in adult = 0.9 x width in larva+2
mm.
The fact that the relation is indirect suggests that the unfed dimensions of the larva may not be important in determining the size of the adult. A homogeneous batch of 30 21 mg fourth instar bugs was taken. One side of the abdominal tergites was painted using Belco cellulose paint in one group of 10 bugs before feeding so that that region could not distend normally. Another group of 10 bugs was fed to repletion and one side of the abdominal tergites was painted immediately, before the cuticle had started to contract; this maintained the stretch. The third group formed a normal control. After ecdysis, the experimental fifth instar bugs were asymmetrical. Those painted before the meal were narrower and shorter than the controls and those painted while distended were longer and wider than the controls. All bugs were marked and fed at 28 days. After the adult ecdysis, they were measured again. The width of the fourth abdominal tergite, measured from the lateral margin to the centre line in the fifth instar is plotted against the width of the same tergite after metamorphosis in Fig. 4. It will be seen that again the relation between the larval and the adult dimension is not direct, and that the relation between the two sides of the same bug closely follows the previously found relationship here
THE
CUTICLE
AS A TEMPLATE
Abdomen
FOR
width,
GROWTH
unfed
IN
5th
RtT0IlNIU.S
instar,
PROLIXUS
2427
mm
FIG. 3. Graph showing the relationship between the width of the fourth abdominal tergite in the fifth instar larva and the adult of Rhodnius. The relationship is empirically described by the straight line y = 0.9 x + 2 mm.
-\ ,1/ y=O.9x+I
2
5 72 3
0
I
I I
I
I 2
[7
Control Painted unfed one
side, 05 4th Untreated side of same bug + Painted replete one wde. (IS 4th b Untreated side of same bug
I
Width of abdomen, edge to center, unfed 5th
I 3 instar,
I 4
mm
4. Graph showing the relationship between the width from edge to centre line of the fourth abdominal tergites of fifth instar and adult RRhodnius.The points show the treated or control sides of bugs that have been made asymmetrical in the fourth instar. The relationship is empirically described by the straight line y = 0.9 x+1 nun. FIG.
II. C. BENNET-CLARK
2428
modified to semiwidth in adult = O-9 x semiwidth in larva+ 1 mm. The effect of this relationship is that the adults are far less, asymmetrical than the larvae from which they developed. Another experiment was performed using a heterogeneous group of fifth instar Rhodnius weighing between 35 and 55 mg. One group of 10 bugs was painted over one side of the abdominal tergites before feeding, another group was painted over one side when replete, a third group was painted along one side before feeding, and the anus was blocked so that the stretch was maintained until ecdysis. A fourth group acted as control. The results are shown in Fig. 5 where the width from the margin to the centre line of the abdomen of the adult is plotted against this width in the fifth instar.
Side polnted
unfed
Untreafed~side p Side
Width
of abdomen,
edge to centre,
painted
i
Untreated
?
nnus
A
Side
unfed
rsple:
side
blocked pmnied
5th
unfe
instar,
mm
FIG. 5. Graph showing the relationship between the width from the edge to the centre line of the fourth abdominal tergite of fifth instar and adult Rhodnius. The points show the treated or control sides of bugs that have been made asymmetrical by treatment in the fifth instar. The treated sides approach the direct relationships y = x or y = 2x shown by straight lines. For clarity the relationship for the control is shown by dashes.
For clarity, the width in the control group has not been plotted but the relationship was the same as in Fig. 3. The untreated sides of all the experimental animals that
THECUTICLEASA TEMPLATE FORGROWTH IN RHODNIUS
2429
PROLIXUS
survived are similar to those of the controls but where the larval cuticle has been prevented from stretching, the width of the adult abdomen is nearly the same as that in the preceding instar. Where the stretch has been maintained, the width of the adult cuticle becomes nearly twice that of the larva; during the stretch that accompanies feeding, there is a twofold increase in the width of the cuticle (BENNETCLARK, 1962). It is clear that the effect of experimental control of the width of the abdomen in the fifth instar is more direct than that of similar control in the fourth instar, and further reinforces the view that the initial size of the cuticle is not important but that the experience during the moulting cycle may be significant. To examine this, a homogeneous group of 40 to 43 mg fifth instar bugs was fed. Some were removed before feeding had been completed but others were allowed to feed fully. The length and width of the abdomen was measured immediately, at 6 days and at 18 days after the meal. The newly fed abdomen is circular in section, by 6 days is oval, and by 18 days the tergites are almost flat, so a correction factor must be applied in each to calculate the circumference from the measured diameter. By drawing optical sections of the cross-section of the abdomen, and measuring the diameter and circumference of these drawings, empirical factors have been found. For newly fed bugs, the diameter must be multiplied by 1.45 to give the circumference of the tergites, at 6 days the factor is 1.25, and at 18 days the factor is 1.05. Results are plotted on Fig. 6, which shows the circumference of the
-8
-
c
6 .%
I--
: .: a, n
Unfed
5th
instar
:: 0 -4
Mitosis
%
-1mogo
2
Epicutick 0
I
4I
I
/ 8 Days
deposition ! 1 I2
I
after
I 16
1
1 20
1
E P 3
24O
feeding
FIG. 6. Graph showing the width of the stretched cuticle for fifth instar larval Rho&&s immediately after feeding and at 6 and 18 days. The bugs were of the same initial size but were allowed to feed to different extents. The time at which events preceding ecdysis occur is derived from WIGGLE~WORTH (1933).
abdominal fourth tergite for 5 bugs fed different sized meals. Although the initial width after feeding varies from 1.6 to 2.3 times the unfed width, the width after 18 days shows far less variation and the width after ecdysis is closely similar to that at 18 days after feeding. This bears out the contention in The effect of large dilute blood meals that the initial distension is not important in determining the subsequent size after ecdysis.
H. C. BENNET-CLARK
2430
In another experiment the width of the fourth abdominal tergite and the overall length of the abdomen were measured in a batch of 10 fifth instar bugs. The bugs were fed with different sized meals and the width and length of the abdomen were measured 18 days after the meal and after ecdysis to the adult. The ratio between the dimension in the adult and that in the unfed larva or in the larva 18 days after feeding has been calculated. The results are shown in Table 2. TABLE 2-RATIOS BETWEENTHE DIMENSIONS OF UNFEDFIFTH INSTARLARVAEOR OF LARVAE18 DAYSAFTERTHE MEALANDTHE DIMENSIONS OF THE ADULT*
Unfed larva : unfed adult Width of fourth abdominal tergite (mm) Length of male abdomens (mm) Length of female abdomens (mm) Probability, males : females
1 : I.30
kO.10, n = IO
Fed larva at 18 days : unfed adult 1 : 0.997 f 0.027, n = 10
1 : I.542 f 0.056, n = 5 1 : I.608 + 0.088, n = 5
1 : I.125 kO.026, n = 5 1 : I.190 kO.028, n = 5
P = 0.2
P = 0.01
* The standard deviation and size of the sample are shown.
If the ratio between the unfed larval and adult dimensions is compared with the ratio between the fed larval and adult dimensions, it is found that the prediction of the width of the abdomen is significantly worse from measurement of the unfed larva than that from measurement made 18 days after feeding. Similarly, the difference between the length of the abdomen in male and female which appears at metamorphosis can be predicted if the sex of the fifth instar larva and the length of the abdomen is known 18 days after feeding but not from length of the abdomen of the unfed larva. Epidermal cell area in Rhodnius and Periplaneta It is possible to see polygonal patches on the forewings and abdominal tergites of Rhodnius but these are not clearly enough defined to be easily counted. A tentative measure of the average area of each patch is 200 pm2 on the forewing and 70 to 100 pm2 on the fourth abdominal tergite. This figure agrees adequately with an estimate of 80 pm2 derived for the area of each epidermal cell from other sources (WIGGLESWORTH, 1963). The wing bud of the fifth instar larva is about 3 mm long and the forewing of the adult is about 12 mm long; the wing increases in surface area about sixteenfold while that of the abdomen increases about l*Zfold at metamorphosis. It is clear that the major part of this difference is the result of a greater increase in cell number in the wing bud than the abdomen. It is easy to see a polygonal pattern on the surface of Periplaneta americana and to verify, by staining, that there is a close correspondence between the number of epidermal nuclei and the number of polygons at the epicuticular surface. Counts
THE CUTICLE AS A TEMPLATE
FOR GROWTH
IN RHODNIUS
PROLIXUS
2431
made on the last instar larva gave an average area of 155 pm2 for cells of the mesothoracic wing bud and 180 pm2 for cells of the third abdominal tergite. In the adult, the area on the mesothoracic wing was 235 pm2, on the third abdominal tergite 155 pm2, and on the fore femur 210 pm 2. There was a significant difference between the average area on the wing and on the abdomen. As the wing expands alt least twentyfold in area at the last ecdysis, it is clear that the major part of this expansion is due to increase in epidermal cell number. DISCUSSION
It has been shown that the size of Rhodnius can be changed in a subsequent instar by the experience in the preceding instar. Of the effects that have been noted, the effect of nutrition seems to be quite small and only alters the subsequent size by 6 per cent. Far larger effects can be produced by altering the size of the existing cuticle around the time of cuticular deposition. At metamorphosis in Rhodkus, epidermal mitoses occur from the fifth to fifteenth days and the epicuticle appears at ‘about the 16th day from feeding. It becomes slightly folded during the 17th and 18th days and then the chitinous endocuticle is formed’ (WIGGLESWORTH, 1933). It is around this time that the relation between the size of the larval and the expanded adult cuticle becomes direct regardless of the size of the meal and this bears out the earlier contention that the shape and size of an insect is defined by the degree to which the epicuticle can be unfolded during the ecdysial expansion (BENNET-CLARK, 1963). The present work bears out a suggestion that is derived from WIGGLESWORTH’S o’bservations (1933, 1963) that the epidermal cells divide to a constant density per unit area and that this epidermis then deposits an epicuticle that is folded in a standard manner. The epidermis thus uses the existing cuticle as a template to restrict mitosis so the post-ecdysial size is defined by some factor of the preelcdysial size, the expansion factor of the epicuticle. This expansion factor varies from organ to organ, being about 1.1 for the length of the head in the last moult but 1.0 for the width of the abdomen, and is different along different axes, being 1-O across the abdomen and 1.125 or l-190 along the length of the abdomen. The directionality of the expansion factor is expected in view of the longitudinal pattern gradients that occur in Rhodnius cuticle (LOCKE, 1959,1966). It is not known what causes mitosis to stop, merely that it ceases after a certain density has been reached. It is not known whether cells in the epidermis of Rhodnius divide along preferred axes, merely that the density reaches a constant value per unit area; Bouligand has pointed out to me that the mechanism that I have proposed here will only be controlled in the presence of differential growth if the axes of mitosis are defined as well as the cell density. The present work suggests a model to describe the increase in size that occurs at an ecdysis (Fig. 7a). This model may be expressed in two ways: Length after ecdysis = length at the time of epicuticle formation x local growth factor
(1)
2432
H. C. BENNET-CLARK
or Length after ecdysis = epidermal cell number after mitosis x length of epicuticle deposited per epidermal cell.
(2)
This model may be modified by a nutritional factor but this appears to be far less important than the size of the cuticular template which either provides the reference length or the limit to cell number. The shape and area of epicuticle deposited by each cell is fairly constant on regions of the body such as abdominal tergites and, though the fundamental shape is a hexagon, this may be distorted (HINTON, 1970). This observation and the measurements of cell area made here support the assumptions made above that there is a local growth factor or a specific area of epicuticle per epidermal cell. An interesting situation arises in the regeneration of legs in Rhodnius (L~~SCHER, 1948). The tibia will regenerate a new tarsus at its apex; when this tarsus is complete, it is smaller than normal but its size increases at each instar by the same proportion as the normal tarsus. Meanwhile, the tibia from which the tarsal epidermis has differentiated grows in a complex manner and it appears that the redifferentiation of a length of the tibia causes a lasting increase in the growth rate of the remainder. This is probably a special case but the growth of the regenerated tarsus is readily explained on the template model proposed here. The model described above does not explain allometric growth unless it can be shown either that the area of epicuticle deposited varies from organ to organ or that the mitotic activity of cells varies from point to point during development. The latter seems to be a major factor in view of the observation that cell lines are preserved as clusters in the development of mesothoracic imaginal discs in Drosophila (MURPHY and TOKUNAGA,1970). These clusters may have different mitotic rates and specific directions of growth and it is significant that the mitotic rate is highest in the part of the mesonotal disc, that gives rise to the largest part of the mesonotum of the imago and smallest in the region bordering the wing insertion which is a small area in the imago. Similarly, where extra growth occurs as in the lateral pleat or in the wings of the imaginal Rhodnius there is excessive cell division (WIGGLESWORTH,1963). A further problem arises with the growth of the wings of Drosophila in different conditions (ALPATOV,1930). If the larvae are underfed, the wings are smaller but the cells are also smaller than normal. If the flies are reared at lS”C, the wings
are larger and the cells larger. There is, however, a direct linear relation between cell area and wing length, not, as might be expected between cell area and wing area. This suggests that the area of epicuticle deposited by each cell may depend on the extent of mitosis of the clone in its specific direction and further suggests that the degree of differentiation of a clone of cells may increase as the cell number increases; an analogous situation to this is found in many exopterygote insects in the differentiation of wing buds and has been shown also in the morphological changes that accompany abnormal metamorphosis in Rhodnius (WIGGLESWORTH, 1940a, b, 1963). While such a mechanism would also be consistent with one that
TNE CUTICLE
AS A TEMPLATEFORGROWTHIN
RHODNIUS PROLIXUS
2433
maintained se~ental gradients, it is inconsistent with the simple template model (Fig. 7a) proposed here and requires a second-order term for its description (Fig. 7b).
Mitosis to standard density
(a)
Before
Ecdysis and expansion
Epicuticol deposition
moult
ib)
Area of epicuticte’ per cell is constant
Mitosis standard
to density
Area of epicuticie per cell depends
On Cell “0.
New size and old size X cell no. increase
FIG. 7. Diagrams of the way in which growth may be determined in insects. (a) All epidermal cells deposit a similar folded area of epicuticle regardless of previous experience. This describes the condition in the abdomen of Rhodnius. (b) Epidermal cells differentiate, depending on previous mitotic experience, and so, where the number increases by a larger proportion, a larger area of epicuticle is deposited by each cell. This is similar to the condition in the wings of Dyo~o~h~~a and possibly in the regenerating legs of Rhodnius.
The growth of Rhodnius can be described accurately using the cuticle of one instar as a template for the formation of a folded cuticle of the next instar. This mechanism is of an attractive simplicity but restricts the range of morphological changes that can occur. Where a pupal stage is found between the larval and the adult stages, greater changes can occur at metamorphosis but the control of size and form becomes more difficult and the mechanism by which the control is effected becomes more complex in the absence of a continuous mechanical template. ~c~~o~~ed~e~e~~s-Dr* Y. ROULIGANDdrew my attention to the importance of the axis of cell division in this problem and by so doing greatly broadened my interest and approach to the discussion. REFERENCES ALPATOV W. Vv. (1930) Phenotypical variation in body and cell size of Drosophila melanogas&r. Biof. Br.&., Woods Hob 58, 85-103. EEN~-CLA~ R. C. (1961) The mechanics of feeding in the bug ~od~~~ prolixzls St&h Ph.D. Thesis, University of Cambridge. BENNET-CLARK H. C. (1962) Active control of the mechanical properties of insect cuticle. J. Insect Physiol. 8, 627-633.
2434
H. C. BENNET-CLARK
BENNET-CLARKH. C. (1963) The relation between epicuticular folding and the subsequent size of an insect. J. Insect Physiol. 9, 43-46. BUXTONP. A. (1930) The biology of a blood-sucking bug, Rhodnius prolixus. Trans. R. ent. Sot. Land. 78, 227-234. COTTRELL C. B. (1962) The imaginal ecdysis of blowflies. Evidence for a change in the mechanical properties of the cuticle at expansion. J. exp. Biol. 39, 449-458. HINTON H. E. (1970) Some little known surface structures. Symp. R. ent. Sot. Lond. 5, 41-58. LOCKE M. (1958) The co-ordination of growth in the tracheal system of insects. Q. JZ microsc. Sci. 99, 373-391. LOCKEM. (1959) The cuticular pattern in an insect, Rhodnius prolixus Stil. J. exp. Biol. 36, 459-477. LOCKE M. (1966) The cuticular pattern in an insect. The behaviour of grafts in segmented appendages. J. Insect. Physiol. 12,397-402. L~~SCHER M. (1948) The regeneration of legs in Rhodniusprolixus (Hemiptera). J. exp. Biol. 25, 334-343. MURPHY C. and TOKUNAGAC. (1970) Cell lineage and the dorsal mesothoracic disc of Drosophila. J. exp. 2001. 175, 197-220. WIGGLESWORTH V. B. (1931) Physiology of excretion in a blood-sucking insect Rhodnius pvolixus (Hemiptera, Reduviidae)-I. Composition of the urine. J. exp. Biol. 8, 411427. WIGGLESWORTHV. B. (1933) The physiology of the cuticle and of ecdysis in Rhodnius proZixus (Triatomidae, Hemiptera); with special reference to the function of the oenocytes and of the dermal glands. Q. Jl microsc. Sci. 76, 269-318. WIGGLESWORTH V. B. (1940a) Local and general factors in the development of pattern in Rhodnius prolixus (Hemiptera). J. exp. Biol. 17, 180-200. WIGGLE~WORTHV. B. (1940b) The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J. exp. Biol. 17, 201-222. WIGGLESWORTH V. B. (1963) The action of moulting hormone and juvenile hormone at the cellular level in Rhodnius prolixus. J. exp. Biol. 40, 231-245. WIGGLESWORTHV. B. (1965) The Principles of Insect Physiology, chapter 3. Methuen, London.
THE CUTICLE AS A TEMPLATE FOR GROWTH IN RHODNIUS PROLIXUS H. C. BENNET-CLARK Department of Zoology, The University, Edinburgh EH9, 3 JT (Received 16 March 1971; revised 13 May 1971) Abstract-The variation in size that occurs in Rhodnius has been examined and it is found that while the size of the head in any instar is almost constant, the dimensions of the abdomen vary as Vweight. The meal that is taken is 5.7 times the unfed weight, suggesting that the abdomen stretches by a standard ratio regardless of size. In the adult the size of the abdomen is neither constant nor varies as $/weight and the size is difficult to predict from the size of the fifth instar larva. Experiments with dilute meals and constraints on the size of the cuticle showed that the size of the larval abdomen is affected by the size of the cuticle in the previous instar; the effect of nutrition on the size of the head, however, is far less great. Experiments with fifth instar bugs showed that the size of the abdomen of the adult was directly related to the size of the larval abdomen at about the time of the deposition of the epicuticle. A model has been suggested to describe the growth of Rhodnius which prop poses that the size in the next instar is the product of the existing size and a local growth factor. The local growth factor may change at metamorphosis and is probably expressed at the cellular level in terms of cell density and the area of epicuticle deposited per cell. INTRODUCTION
BEFORE the ecdysis of an insect, an initial phase of mitotic activity of the epidermis is followed by the deposition of the epicuticle. Shortly after its deposition, the epicuticle expands and becomes folded (WIGGLESWORTH, 1933). Before ecdysis, a considerable thickness of presumptive exocuticle is laid down and, after the expansion that accompanies ecdysis, this may be tanned and so stabilizes the size of the newly ecdysed insect at the size to which it has expanded during ecdysis. At ecdysis the exocuticle becomes briefly and reversibly plastic (BENNETCLARK, 1961; COTTRELL, 1962) and so offers little limitation to the expansion that occurs. Instead, it seems likely that the limit to expansion is reached when the folds in the epicuticle have been pulled out (WIGGLESWORTH, 1933; BENNETCLARK, 1963). The size that is attained after ecdysis is thus determined by the degree to which the epicuticle is expanded after its deposition but before ecdysis. Studies of the surface sculpturing of the epicuticle of insects (HINTON, 1970) support the view that the gross folding of the epicuticle is pulled out flat at the ecdysial expansion; minor surface irregularities remain and, with pre-ecdysial thickening or tanning, or merely from the elasticity of the epicuticle, form the fine surface sculpturing that may occur. 2421
2422
H. C. BENNET-CLARK
The mechanical events that occur at ecdysis merely manifest a previously determined pattern of epicuticular folding. Where patterning of insects has been studied, it has been shown that there are general and local factors (WIGGLESWORTH, 1940a) and that there are directional gradients in various parts of the body (LOCKE, 1959, 1966) which imply that there is an inherent control of pattern in Rhodnius that is largely independent of such factors as the rate of development or the nutrition of the insect. This however is not the case in Diptera (ALPATOV, 1930). Where the effect of nutrition has been examined, the relation between the initial and final size of growing tracheae has been shown to be complex (LOCKE, 1958), and it was shown that the control of the increase of diameter was different from the control of the increase of length. It is easy to envisage a growth mechanism that follows a standard programme of epicuticular expansion in the pharate phase of each instar and, indeed, this is the simplest explanation for the logarithmic growth that occurs in many insects (c$ WIGGLESWORTH,1965). In the larvae of many insects such as most Endopterygota and Hemiptera, the abdominal cuticle is extensible and development of the next instar may occur after an abdominal extension which must merely exceed a certain threshold (WIGGLESWORTH,1933). Such a cuticle clearly presents a variable surface on which the epidermal cells divide and form the new cuticle. This paper examines the idea that the old cuticle might act as a template for the formation of the new cuticle. In this context I am following the Shorter Oxford Dictimary definition that a template is ‘used as a gauge or guide in bringing any piece of work to the desired shape’.
MATERIALS
AND METHODS
The bug Rhodnius prolixus has been used for the experimental parts of this work. The culture has been maintained for many years at Edinburgh but probably originated from the London School of Hygiene and Tropical Medicine. As such, the culture has probably been maintained for 40 years, or about 100 generations, and is inbred and homogeneous. Bugs are routinely fed on the shaved belly of a rabbit. As it is difficult to feed more than about 80 fourth instar or 40 fifth instar larvae at one time, experiments have been designed about batches of bugs of these numbers. Within any batch there is some variation in the weight, the meal size, and the time taken to feed; the normal bug feeds till it is replete at which stage the abdominal cuticle is shiny and apparently all epicuticular folds are flattened. Following the meal, considerable diuresis occurs and upwards of 50 per cent of the weight of the meal is excreted within 24 hr (WIGGLESWORTH, 1931). If the meal is larger than a critical size, the bug ecdyses to the next instar, from the fourth instar in 13 to 15 days and from the fifth instar to the adult in 22 to 25 days at 25°C the temperature at which I have kept my animals. Bugs may alternately be fed on small quantities of heparinized blood using an artificial feeder described earlier (BENNET-CLARK, 1962).
THE CUTICLE AS A TEMPLATE
FOR GROWTH
IN RHODNIUS
PROLIXUS
2423
At the start of an experiment, bugs are taken 14 days after ecdysis and are weighed, marked on the prothorax with identifying spots of paint (using a variation of the international resistor colour code), and are then measured using a calibrated microscope with a measuring eyepiece. For the measurement, the. bugs were held on a sticky slide made by winding Scotch tape sticky-side-out around a glass slide, one surface of which was attached to another slide. Bugs held thus did not appear to be damaged in any way and were unable to move during the measurement. Bugs were confined in a tared container and weighed on an Oertling single pan balance. Linear measurements under 5 mm were recorded to the nearest 0.05 mm and larger measurements to + 0.05 mm. Weights were rounded to the nearest milligram but, for experiments, bugs were batched into groups at 1 mg intervals with third instar, 2 mg intervals with fourth instar, and 3 mg intervals with the fifth instar unfed larvae. After feeding the bugs were reweighed and replaced in numbered bottles. Where appropriate, newly fed bugs were also measured using the. microscope. Subsequent measurements were made where appropriate to the experiment. Observations have also been made with the cockroach Periplaneta americana. This insect has a far smoother cuticle than Rhodnius and so it is easy to see the hexagonal patches of epicuticle laid down by the individual epidermal cells. Measurements of cell density have been made using a microscope fitted with either 8 or 4 mm objectives and counts have been made with the use either of a haemocytometer slide or of a calibrated eyepiece graticule. RESULTS
Size of normal Rhodnius The length of the head, the length of the abdomen, and the width of the fourth abdominal tergite of Rhodnius were measured using batches of unfed bugs of the same age. The head was chosen as an easily measurable part of the body that does not change in size during the course of an instar and the abdomen because of the twofold linear stretch that accompanies feeding (BENNET-CLARK, 1963). The results are shown in Fig. 1 where the width or length of the organ is plotted against the unfed weight, 2 weeks after ecdysis. Various points emerge; the head of a heavy bug is somewhat longer than that of a light one but significantly less than would be expected if length cc +/weight. The length and width of the larval abdomen are both nearly proportional to +/weight which in itself is suggestive since the abdomen accommodated the meal. After metamorphosis, the abdominal cuticle becomes sclerotized and inextensible. The length and width of the abdomen of the adult no longer show the same dependence on the weight of the bug and there is sexual dimorphism in the length of the abdomen. It appears that the hard parts of the larval stages obey Dyar’s law in that there is a standard size for the instar and a standard factor for its increase at each ecdysis. The size of the abdomen varies continuously as the q/weight in the larva but changes abruptly at metamorphosis when it is neither dependent on a power of the weight nor is constant.
2424
I-1. C.
BENNET-CLARK
a-
1
0
20
Weight
2
I
1
40
GO
weeks
after
1
I
80 ecdysis,
100
/
120
mg
FIG. 1. Graph showing the relationship
between the weight and the linear dimensions of various organs for third, fourth, and fifth instar larval and adult Rhodnius measured 14 days after ecdysis. The symbols indicate the mean value and the vertical line the 95 per cent confidence limits where these exceed the size of the symbol. (0 refers to fifth instar bugs resulting from feeding fourth instar bugs with 25 per cent blood described in The effect of large dilute blood meals.
When larvae of known size are fed to repletion, the weight when fed directly is proportional to the unfed weight (Fig. 2). For bugs fed 14 days after the preceding ecdysis, the normal full meal is 5.7 times the unfed weight or the fed weight is 6.7 times the unfed weight. This appears to hold for the second to fifth instar larvae; the first instar larva takes a larger meal of up to eleven times the unfed weight (BUXTON, 1930). Since fed weight is proportional to unfed weight and since abdomen width is proportional to $‘unfed weight it follows that the stretched size of the abdomen after feeding is proportional to the unstretched size of the abdomen or that abdominal distension is dependent on the unfed size of the abdomen. The effect of large dilute blood meals A batch of 20 19 mg fourth instar Rhodnius was taken. Half were fed on whole heparinized ox blood and the other half were fed 25 per cent heparinized ox blood
THE CUTICLE AS A TEMPLATE
FOR GROWTH
IN RHODNIUS
PROLIXUS
2425
in Ringer-Locke. In both batches, the weight of the meal was similar but the bugs fed 25 per cent blood excreted about 75 per cent of the weight of the meal in the
Unfed
weight
of larva,
mg
FIG. 2. Graph showing the relationship between the fully fed weight and the unfed weight of second, third, fourth, and fifth instar larval Rho&&s weighed and fed 14 days after ecdysis. The graph also shows the bugs examined in The efJect of large dilute blood meals. The vertical lines show 95 per cent confidence limits of the means.
first day compared with 45 per cent in the controls. After ecdysis, the bugs were weighed and measured; the results are summarized in Table 1. The length of the head is slightly but significantly smaller in the experimental group and the dimensions of the abdomen are closer to those of the normal fourth instar bugs than to fifth instar bugs (the dimensions are plotted on Fig. 1) and show the same dependence on the unfed weight of the insect. This suggests that the initial distension is not important in determining the size of the head or the abdomen in the next instar. Roth groups of bugs were fed in the fifth instar. The experimental group fed to repletion but took a significantly smaller meal than the controls (Table 1) which was appropriate to bugs of their weight (Fig. 2). A second experiment using 23 mg fourth instar bugs gave similar results. There was no significant difference between the experimental and the control group in the time between the meal and ecdysis but the length of the head was significantly smaller in the experimental group. This suggests that the effect of the content of the meal is nutritional rather than the result of a change in the time course of moulting. The e#ect
of abdomen
size during
the mouking
cycle
When the normal fifth instar Rhodnius metamorphoses to the adult, the abdominal cuticle changes in character. The abdomen of the adult is relatively
H. C. BENNET-CLARK
2426
longer than that of the larva and the dimensions are neither proportional to q/weight nor are they constant (Fig. 1). This suggests that the relation between the dimensions of the adult and of the preceding larva is complex. TABLE ~-COMPARISON OF THE DIMENSIONS OF NORMALFIFTH INSTARLARVALRhodnius WITH THOSERESULTING FROMFEEDING25 PER CENTBLOODIN THE FOURTHINSTAR*
Head length (mm) Abdomen width (mm) segment 4 Abdomen length (mm) Unfed weight as fifth instar (mg) Replete weight in fifth instar (mg)
Fed 25% blood
Control, whole blood
2.96 + 0.075, n = 9 4.38 + 0.17, n = 9
3.13 kO.06, n = 10 4.92 z! 0.20, n = 10
6.37 + 0.19, n = 9 25.2 + 2.2, n = 9
6+34+0*15, it = 10 40.9 + 3.9, n = 10
167f44,
n= 8
278 + 61,1z = 9
* The size of the experimental groups and standard deviations of the means are quoted. In all cases the probability of similarity between experimental and control groups is less than 0.001.
A batch of fifth instar bugs of different weights was fed to repletion, after measurement of the various dimensions. After ecdysis, the adults were measured. The relation between the width of the larval abdomen and that of the adult is plotted in Fig. 3. The relation can be empirically described by the formula, width in adult = 0.9 x width in larva+2
mm.
The fact that the relation is indirect suggests that the unfed dimensions of the larva may not be important in determining the size of the adult. A homogeneous batch of 30 21 mg fourth instar bugs was taken. One side of the abdominal tergites was painted using Belco cellulose paint in one group of 10 bugs before feeding so that that region could not distend normally. Another group of 10 bugs was fed to repletion and one side of the abdominal tergites was painted immediately, before the cuticle had started to contract; this maintained the stretch. The third group formed a normal control. After ecdysis, the experimental fifth instar bugs were asymmetrical. Those painted before the meal were narrower and shorter than the controls and those painted while distended were longer and wider than the controls. All bugs were marked and fed at 28 days. After the adult ecdysis, they were measured again. The width of the fourth abdominal tergite, measured from the lateral margin to the centre line in the fifth instar is plotted against the width of the same tergite after metamorphosis in Fig. 4. It will be seen that again the relation between the larval and the adult dimension is not direct, and that the relation between the two sides of the same bug closely follows the previously found relationship here
THE
CUTICLE
AS A TEMPLATE
Abdomen
FOR
width,
GROWTH
unfed
IN
5th
RtT0IlNIU.S
instar,
PROLIXUS
2427
mm
FIG. 3. Graph showing the relationship between the width of the fourth abdominal tergite in the fifth instar larva and the adult of Rhodnius. The relationship is empirically described by the straight line y = 0.9 x + 2 mm.
-\ ,1/ y=O.9x+I
2
5 72 3
0
I
I I
I
I 2
[7
Control Painted unfed one
side, 05 4th Untreated side of same bug + Painted replete one wde. (IS 4th b Untreated side of same bug
I
Width of abdomen, edge to center, unfed 5th
I 3 instar,
I 4
mm
4. Graph showing the relationship between the width from edge to centre line of the fourth abdominal tergites of fifth instar and adult RRhodnius.The points show the treated or control sides of bugs that have been made asymmetrical in the fourth instar. The relationship is empirically described by the straight line y = 0.9 x+1 nun. FIG.
II. C. BENNET-CLARK
2428
modified to semiwidth in adult = O-9 x semiwidth in larva+ 1 mm. The effect of this relationship is that the adults are far less, asymmetrical than the larvae from which they developed. Another experiment was performed using a heterogeneous group of fifth instar Rhodnius weighing between 35 and 55 mg. One group of 10 bugs was painted over one side of the abdominal tergites before feeding, another group was painted over one side when replete, a third group was painted along one side before feeding, and the anus was blocked so that the stretch was maintained until ecdysis. A fourth group acted as control. The results are shown in Fig. 5 where the width from the margin to the centre line of the abdomen of the adult is plotted against this width in the fifth instar.
Side polnted
unfed
Untreafed~side p Side
Width
of abdomen,
edge to centre,
painted
i
Untreated
?
nnus
A
Side
unfed
rsple:
side
blocked pmnied
5th
unfe
instar,
mm
FIG. 5. Graph showing the relationship between the width from the edge to the centre line of the fourth abdominal tergite of fifth instar and adult Rhodnius. The points show the treated or control sides of bugs that have been made asymmetrical by treatment in the fifth instar. The treated sides approach the direct relationships y = x or y = 2x shown by straight lines. For clarity the relationship for the control is shown by dashes.
For clarity, the width in the control group has not been plotted but the relationship was the same as in Fig. 3. The untreated sides of all the experimental animals that
THECUTICLEASA TEMPLATE FORGROWTH IN RHODNIUS
2429
PROLIXUS
survived are similar to those of the controls but where the larval cuticle has been prevented from stretching, the width of the adult abdomen is nearly the same as that in the preceding instar. Where the stretch has been maintained, the width of the adult cuticle becomes nearly twice that of the larva; during the stretch that accompanies feeding, there is a twofold increase in the width of the cuticle (BENNETCLARK, 1962). It is clear that the effect of experimental control of the width of the abdomen in the fifth instar is more direct than that of similar control in the fourth instar, and further reinforces the view that the initial size of the cuticle is not important but that the experience during the moulting cycle may be significant. To examine this, a homogeneous group of 40 to 43 mg fifth instar bugs was fed. Some were removed before feeding had been completed but others were allowed to feed fully. The length and width of the abdomen was measured immediately, at 6 days and at 18 days after the meal. The newly fed abdomen is circular in section, by 6 days is oval, and by 18 days the tergites are almost flat, so a correction factor must be applied in each to calculate the circumference from the measured diameter. By drawing optical sections of the cross-section of the abdomen, and measuring the diameter and circumference of these drawings, empirical factors have been found. For newly fed bugs, the diameter must be multiplied by 1.45 to give the circumference of the tergites, at 6 days the factor is 1.25, and at 18 days the factor is 1.05. Results are plotted on Fig. 6, which shows the circumference of the
-8
-
c
6 .%
I--
: .: a, n
Unfed
5th
instar
:: 0 -4
Mitosis
%
-1mogo
2
Epicutick 0
I
4I
I
/ 8 Days
deposition ! 1 I2
I
after
I 16
1
1 20
1
E P 3
24O
feeding
FIG. 6. Graph showing the width of the stretched cuticle for fifth instar larval Rho&&s immediately after feeding and at 6 and 18 days. The bugs were of the same initial size but were allowed to feed to different extents. The time at which events preceding ecdysis occur is derived from WIGGLE~WORTH (1933).
abdominal fourth tergite for 5 bugs fed different sized meals. Although the initial width after feeding varies from 1.6 to 2.3 times the unfed width, the width after 18 days shows far less variation and the width after ecdysis is closely similar to that at 18 days after feeding. This bears out the contention in The effect of large dilute blood meals that the initial distension is not important in determining the subsequent size after ecdysis.
H. C. BENNET-CLARK
2430
In another experiment the width of the fourth abdominal tergite and the overall length of the abdomen were measured in a batch of 10 fifth instar bugs. The bugs were fed with different sized meals and the width and length of the abdomen were measured 18 days after the meal and after ecdysis to the adult. The ratio between the dimension in the adult and that in the unfed larva or in the larva 18 days after feeding has been calculated. The results are shown in Table 2. TABLE 2-RATIOS BETWEENTHE DIMENSIONS OF UNFEDFIFTH INSTARLARVAEOR OF LARVAE18 DAYSAFTERTHE MEALANDTHE DIMENSIONS OF THE ADULT*
Unfed larva : unfed adult Width of fourth abdominal tergite (mm) Length of male abdomens (mm) Length of female abdomens (mm) Probability, males : females
1 : I.30
kO.10, n = IO
Fed larva at 18 days : unfed adult 1 : 0.997 f 0.027, n = 10
1 : I.542 f 0.056, n = 5 1 : I.608 + 0.088, n = 5
1 : I.125 kO.026, n = 5 1 : I.190 kO.028, n = 5
P = 0.2
P = 0.01
* The standard deviation and size of the sample are shown.
If the ratio between the unfed larval and adult dimensions is compared with the ratio between the fed larval and adult dimensions, it is found that the prediction of the width of the abdomen is significantly worse from measurement of the unfed larva than that from measurement made 18 days after feeding. Similarly, the difference between the length of the abdomen in male and female which appears at metamorphosis can be predicted if the sex of the fifth instar larva and the length of the abdomen is known 18 days after feeding but not from length of the abdomen of the unfed larva. Epidermal cell area in Rhodnius and Periplaneta It is possible to see polygonal patches on the forewings and abdominal tergites of Rhodnius but these are not clearly enough defined to be easily counted. A tentative measure of the average area of each patch is 200 pm2 on the forewing and 70 to 100 pm2 on the fourth abdominal tergite. This figure agrees adequately with an estimate of 80 pm2 derived for the area of each epidermal cell from other sources (WIGGLESWORTH, 1963). The wing bud of the fifth instar larva is about 3 mm long and the forewing of the adult is about 12 mm long; the wing increases in surface area about sixteenfold while that of the abdomen increases about l*Zfold at metamorphosis. It is clear that the major part of this difference is the result of a greater increase in cell number in the wing bud than the abdomen. It is easy to see a polygonal pattern on the surface of Periplaneta americana and to verify, by staining, that there is a close correspondence between the number of epidermal nuclei and the number of polygons at the epicuticular surface. Counts
THE CUTICLE AS A TEMPLATE
FOR GROWTH
IN RHODNIUS
PROLIXUS
2431
made on the last instar larva gave an average area of 155 pm2 for cells of the mesothoracic wing bud and 180 pm2 for cells of the third abdominal tergite. In the adult, the area on the mesothoracic wing was 235 pm2, on the third abdominal tergite 155 pm2, and on the fore femur 210 pm 2. There was a significant difference between the average area on the wing and on the abdomen. As the wing expands alt least twentyfold in area at the last ecdysis, it is clear that the major part of this expansion is due to increase in epidermal cell number. DISCUSSION
It has been shown that the size of Rhodnius can be changed in a subsequent instar by the experience in the preceding instar. Of the effects that have been noted, the effect of nutrition seems to be quite small and only alters the subsequent size by 6 per cent. Far larger effects can be produced by altering the size of the existing cuticle around the time of cuticular deposition. At metamorphosis in Rhodkus, epidermal mitoses occur from the fifth to fifteenth days and the epicuticle appears at ‘about the 16th day from feeding. It becomes slightly folded during the 17th and 18th days and then the chitinous endocuticle is formed’ (WIGGLESWORTH, 1933). It is around this time that the relation between the size of the larval and the expanded adult cuticle becomes direct regardless of the size of the meal and this bears out the earlier contention that the shape and size of an insect is defined by the degree to which the epicuticle can be unfolded during the ecdysial expansion (BENNET-CLARK, 1963). The present work bears out a suggestion that is derived from WIGGLESWORTH’S o’bservations (1933, 1963) that the epidermal cells divide to a constant density per unit area and that this epidermis then deposits an epicuticle that is folded in a standard manner. The epidermis thus uses the existing cuticle as a template to restrict mitosis so the post-ecdysial size is defined by some factor of the preelcdysial size, the expansion factor of the epicuticle. This expansion factor varies from organ to organ, being about 1.1 for the length of the head in the last moult but 1.0 for the width of the abdomen, and is different along different axes, being 1-O across the abdomen and 1.125 or l-190 along the length of the abdomen. The directionality of the expansion factor is expected in view of the longitudinal pattern gradients that occur in Rhodnius cuticle (LOCKE, 1959,1966). It is not known what causes mitosis to stop, merely that it ceases after a certain density has been reached. It is not known whether cells in the epidermis of Rhodnius divide along preferred axes, merely that the density reaches a constant value per unit area; Bouligand has pointed out to me that the mechanism that I have proposed here will only be controlled in the presence of differential growth if the axes of mitosis are defined as well as the cell density. The present work suggests a model to describe the increase in size that occurs at an ecdysis (Fig. 7a). This model may be expressed in two ways: Length after ecdysis = length at the time of epicuticle formation x local growth factor
(1)
2432
H. C. BENNET-CLARK
or Length after ecdysis = epidermal cell number after mitosis x length of epicuticle deposited per epidermal cell.
(2)
This model may be modified by a nutritional factor but this appears to be far less important than the size of the cuticular template which either provides the reference length or the limit to cell number. The shape and area of epicuticle deposited by each cell is fairly constant on regions of the body such as abdominal tergites and, though the fundamental shape is a hexagon, this may be distorted (HINTON, 1970). This observation and the measurements of cell area made here support the assumptions made above that there is a local growth factor or a specific area of epicuticle per epidermal cell. An interesting situation arises in the regeneration of legs in Rhodnius (L~~SCHER, 1948). The tibia will regenerate a new tarsus at its apex; when this tarsus is complete, it is smaller than normal but its size increases at each instar by the same proportion as the normal tarsus. Meanwhile, the tibia from which the tarsal epidermis has differentiated grows in a complex manner and it appears that the redifferentiation of a length of the tibia causes a lasting increase in the growth rate of the remainder. This is probably a special case but the growth of the regenerated tarsus is readily explained on the template model proposed here. The model described above does not explain allometric growth unless it can be shown either that the area of epicuticle deposited varies from organ to organ or that the mitotic activity of cells varies from point to point during development. The latter seems to be a major factor in view of the observation that cell lines are preserved as clusters in the development of mesothoracic imaginal discs in Drosophila (MURPHY and TOKUNAGA,1970). These clusters may have different mitotic rates and specific directions of growth and it is significant that the mitotic rate is highest in the part of the mesonotal disc, that gives rise to the largest part of the mesonotum of the imago and smallest in the region bordering the wing insertion which is a small area in the imago. Similarly, where extra growth occurs as in the lateral pleat or in the wings of the imaginal Rhodnius there is excessive cell division (WIGGLESWORTH,1963). A further problem arises with the growth of the wings of Drosophila in different conditions (ALPATOV,1930). If the larvae are underfed, the wings are smaller but the cells are also smaller than normal. If the flies are reared at lS”C, the wings
are larger and the cells larger. There is, however, a direct linear relation between cell area and wing length, not, as might be expected between cell area and wing area. This suggests that the area of epicuticle deposited by each cell may depend on the extent of mitosis of the clone in its specific direction and further suggests that the degree of differentiation of a clone of cells may increase as the cell number increases; an analogous situation to this is found in many exopterygote insects in the differentiation of wing buds and has been shown also in the morphological changes that accompany abnormal metamorphosis in Rhodnius (WIGGLESWORTH, 1940a, b, 1963). While such a mechanism would also be consistent with one that
TNE CUTICLE
AS A TEMPLATEFORGROWTHIN
RHODNIUS PROLIXUS
2433
maintained se~ental gradients, it is inconsistent with the simple template model (Fig. 7a) proposed here and requires a second-order term for its description (Fig. 7b).
Mitosis to standard density
(a)
Before
Ecdysis and expansion
Epicuticol deposition
moult
ib)
Area of epicuticte’ per cell is constant
Mitosis standard
to density
Area of epicuticie per cell depends
On Cell “0.
New size and old size X cell no. increase
FIG. 7. Diagrams of the way in which growth may be determined in insects. (a) All epidermal cells deposit a similar folded area of epicuticle regardless of previous experience. This describes the condition in the abdomen of Rhodnius. (b) Epidermal cells differentiate, depending on previous mitotic experience, and so, where the number increases by a larger proportion, a larger area of epicuticle is deposited by each cell. This is similar to the condition in the wings of Dyo~o~h~~a and possibly in the regenerating legs of Rhodnius.
The growth of Rhodnius can be described accurately using the cuticle of one instar as a template for the formation of a folded cuticle of the next instar. This mechanism is of an attractive simplicity but restricts the range of morphological changes that can occur. Where a pupal stage is found between the larval and the adult stages, greater changes can occur at metamorphosis but the control of size and form becomes more difficult and the mechanism by which the control is effected becomes more complex in the absence of a continuous mechanical template. ~c~~o~~ed~e~e~~s-Dr* Y. ROULIGANDdrew my attention to the importance of the axis of cell division in this problem and by so doing greatly broadened my interest and approach to the discussion. REFERENCES ALPATOV W. Vv. (1930) Phenotypical variation in body and cell size of Drosophila melanogas&r. Biof. Br.&., Woods Hob 58, 85-103. EEN~-CLA~ R. C. (1961) The mechanics of feeding in the bug ~od~~~ prolixzls St&h Ph.D. Thesis, University of Cambridge. BENNET-CLARK H. C. (1962) Active control of the mechanical properties of insect cuticle. J. Insect Physiol. 8, 627-633.
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BENNET-CLARKH. C. (1963) The relation between epicuticular folding and the subsequent size of an insect. J. Insect Physiol. 9, 43-46. BUXTONP. A. (1930) The biology of a blood-sucking bug, Rhodnius prolixus. Trans. R. ent. Sot. Land. 78, 227-234. COTTRELL C. B. (1962) The imaginal ecdysis of blowflies. Evidence for a change in the mechanical properties of the cuticle at expansion. J. exp. Biol. 39, 449-458. HINTON H. E. (1970) Some little known surface structures. Symp. R. ent. Sot. Lond. 5, 41-58. LOCKE M. (1958) The co-ordination of growth in the tracheal system of insects. Q. JZ microsc. Sci. 99, 373-391. LOCKEM. (1959) The cuticular pattern in an insect, Rhodnius prolixus Stil. J. exp. Biol. 36, 459-477. LOCKE M. (1966) The cuticular pattern in an insect. The behaviour of grafts in segmented appendages. J. Insect. Physiol. 12,397-402. L~~SCHER M. (1948) The regeneration of legs in Rhodniusprolixus (Hemiptera). J. exp. Biol. 25, 334-343. MURPHY C. and TOKUNAGAC. (1970) Cell lineage and the dorsal mesothoracic disc of Drosophila. J. exp. 2001. 175, 197-220. WIGGLESWORTH V. B. (1931) Physiology of excretion in a blood-sucking insect Rhodnius pvolixus (Hemiptera, Reduviidae)-I. Composition of the urine. J. exp. Biol. 8, 411427. WIGGLESWORTHV. B. (1933) The physiology of the cuticle and of ecdysis in Rhodnius proZixus (Triatomidae, Hemiptera); with special reference to the function of the oenocytes and of the dermal glands. Q. Jl microsc. Sci. 76, 269-318. WIGGLESWORTH V. B. (1940a) Local and general factors in the development of pattern in Rhodnius prolixus (Hemiptera). J. exp. Biol. 17, 180-200. WIGGLE~WORTHV. B. (1940b) The determination of characters at metamorphosis in Rhodnius prolixus (Hemiptera). J. exp. Biol. 17, 201-222. WIGGLESWORTH V. B. (1963) The action of moulting hormone and juvenile hormone at the cellular level in Rhodnius prolixus. J. exp. Biol. 40, 231-245. WIGGLESWORTHV. B. (1965) The Principles of Insect Physiology, chapter 3. Methuen, London.