The Role of the Haemocytes in the Growth and Moulting of An Insect, Rhodnius Prolixus (Hemiptera)

The chief organs in the abdomen in Rhodnius are the gut and Malpighian tubes, the heart and pericardial cells, the gonads, muscles, nerves and sense organs, the fat body, dermal glands, oenocytes and the haemocytes of various kinds. The gonads are not necessary for growth; the oenocytes and the dermal glands reach the height of their secretory activity long after growth in the epidermis has begun (Wigglesworth, 1933, 1948) and may therefore probably be excluded. In the present work attention has been concentrated on the pericardial cells and haemocytes. These tissues cannot readily be removed by surgical means. The experiments have consisted in observing the effects of injected substances which are specifically taken up by the cells in question—on the assumption that cells laden with such substances may thereby be ‘blocked’ and precluded from performing their other functions.

Most of the experiments have been done on the 4th-stage larva of Rhodnius. Injection is effected by holding the insect down with plasticene, cutting through the tibia of one of the hindlegs, inserting the abruptly tapered end of a graduated capillary tube, and forcing in by compressed air a measured volume of fluid. The capillary pipette is withdrawn while the tibia is compressed with forceps and the cut end sealed with paraffin wax. In this way known volumes can be injected without loss and with no mortality.

Fourth-stage larvae at 1 day after feeding were injected with 5 mm.³ of 0·02% trypan blue in Ringer’s solution. Some were fixed with Camoy’s fluid 6 days later : the pericardial cells were filled with masses of the dye but there was none in the haemocytes. Mitosis was actively in progress in the epidermis. Those not dissected moulted at the usual time, 13-14 days after feeding. This small amount (1 μg.) of trypan blue is wholly taken up by the pericardial cells and causes no delay in moulting.

When 4th-stage larvae at 1 day after feeding were injected with 5 mm.³ of 0·2% trypan blue (10μg.) and dissected 7 days later, not only were the pericardial cells filled with the dye, but the haemocytes were massed together in great clumps and within these cells the dye was segregated into vacuoles. There was no sign of mitosis in the epidermis, where it normally begins 4 days after feeding. At 15 days there was still no mitosis in the epidermis ; the haemocytes had increased enormously in number. At 21 days mitosis had begun and moulting was in progress; it would probably have been completed about a week later. There was therefore a delay of about 2 weeks in the onset of moulting.

The results with trypan blue suggest that blockage of the pericardial cells alone does not affect moulting, but blockage of the haemocytes causes delay or temporary inhibition. This has been confirmed by the injection of indian ink, which is taken up only by the haemocytes. Stick ink was made up in boiled Ringer’s solution to give a suspension of a density suitable for pen drawing, and 5 mm.³ were injected into 4th-stage larvae 1 day or 2 days after feeding. Among twelve insects the time required for moulting ranged from 21 to 50 days.

A third substance injected was iron saccharate, a 2% solution freshly prepared in Ringer’s solution being used. This is taken up by the pericardial cells and by the haemocytes. 5 mm.³ injected at 1 day after feeding will delay moulting for 1-3 weeks beyond the normal 14 days.

It is clear from the foregoing results that blockage of the haemocytes with trypan blue, indian ink, or iron saccharate will delay moulting. Is this effect due to interference with the general processes of intermediary metabolism, or to some specific action upon the initiation of mitosis and moulting in the epidermis?

This has been tested by injecting the substances at different times after feeding. Fig. 1 shows the effect of blockage of the haemocytes with iron saccharate at each day after feeding, from the first to the eighth. The average time required for moulting (at 25° C.) is 14 days. When injections were made on the first, second or third days after feeding, there was a delay ranging from 1 to 3 weeks. But injection on the fourth day and later caused no delay whatever. (There may be a slight delay of 24 hr. or so after the seventh or eighth day, due perhaps to the operation.) Injection of an equivalent amount of Ringer’s solution at 1 day after feeding caused no delay in moulting.

Injection of full-strength indian ink on successive days gave similar results. Batches of six larvae were injected as follows:

At 1 day after feeding: moulted at 20, 24, 30, 32, 37, 66 days

At 2 days after feeding: moulted at 19, 19, 21, 25, 35, 54 days

At 3 days after feeding: moulted at 29, 33, 39, 41, 78 (1 never moulted)

At 4 days after feeding: moulted at 15, 15, 15, 15, 17, 17 days

At 5 days after feeding: moulted at 15, 15, 15, 15, 15, 17 days

Fig. 2 shows an experiment in which 5 mm.⁸ of a weak cloudy suspension of indian ink was injected: delay in moulting was very slight, but once more it was sharply limited to the first 3 days after feeding.

Clearly there is an abrupt change of some kind between the third and fourth days. The fourth day is the day on which mitosis in the epidermis usually begins and the visible process of moulting can be said to have started. It would appear that blockage of the haemocytes acts specifically upon the initiation of growth.

Moulting in Rhodnius can be prevented or delayed by a number of different procedures: (i) Removal of the neurosecretory cells of the brain by decapitation (Wigglesworth, 1934, 1940). (ii) Exclusion of the thoracic gland by isolation of the sabdomen (Wigglesworth, 1952a). (iii) Exposure to temperatures above 35° C. (Wigglesworth, 19520). (iv) Blockage of the haemocytes. Each of these procedures inhibits (or delays) moulting only when carried out before a certain ‘critical period ‘. In the hope of throwing further light on these processes the critical period in each type of experiment was compared, using batches of twelve insects from the same culture, kept in the same incubator at 25° C. The results are shown in Fig. 3 and may be summarized as follows.

Decapitation (Fig. 3 A). Of those decapitated 1 and 2 days after feeding, none moult. By 3 days after feeding, 80% moult; that is, most of the insects have already secreted sufficient of the brain factor to activate the thoracic glands. By 4 days, 100 % moult.

Isolated abdomen (Fig. 3B). By 3 days, although 80% have liberated sufficient activating factor from the brain (see Fig. 3 A), only 20% have secreted enough thoracic gland hormone for the isolated abdomen to moult. By 4 days, moulting of the isolated abdomen is still limited to 40%. By 5 days, 100% moult.

Thus nearly 2 days are required between the liberation of an adequate quantity of the activating secretion from the brain and the establishment of independence in the abdomen: by the end of 3 days the brain can be dispensed with in 80%; but not until the end of 5 days can the thorax be dispensed with in 100%.

Blockage of the haemocytes (Fig. 3 C). In the blockage of the haemocytes there is a very sharp ‘critical period’ between the end of the third and the end of the fourth days. At the end of 3 days 100% show delayed moulting: in none have the haemocytes accomplished their task. At this time 80% can moult without the brain (Fig. 3 A). The haemocytes cannot therefore be necessary for the secretion of the brain factor.

At this same time the isolated abdomen can moult in 20% (Fig. 3B). In a preliminary note (Wigglesworth, 1955a) this was taken as evidence that activation of the thoracic gland is independent of the haemocytes and that these must therefore intervene after the thoracic gland hormone has been liberated. Further experiments (see below) have shown that this conclusion was not justified.

Transfer to 36° C. (Fig. 3 D). The ‘critical period’ for transfer from 25 to 36° C. does not agree exactly with any of the other three, but it comes closest to the critical period for moulting in the isolated abdomen. That would suggest that although the high temperature may well have an adverse effect on the secretion of the brain factor (for example, the thoracic glands show no signs of secretory activity in larvae kept at 36° C. from the time of feeding), it can also arrest moulting by interfering with the secretion of the thoracic gland hormone or with its action upon the epidermis.

This conclusion is supported by the observation that the 4th-stage larva, decapitated at 6 days after feeding, will induce moulting in another 4th-stage larva, decapitated at 1 day after feeding, to which it is joined in parabiosis at 25° C.—but if the insects after joining are placed at 36° C., although the larva decapitated at 6 days will moult, it will not induce moulting in its partner.

In general, these comparisons of the timing of the ‘critical periods’ as determined by different operative procedures support the conclusion that the haemocytes are concerned in one of the later steps in the hormonal initiation of moulting; but it is not possible from them alone to decide whether the haemocytes are involved in the production of the thoracic gland hormone or in the action of this upon the epidermis.

The cells of the thoracic gland show a cycle of secretory activity during moulting (Wigglesworth, 1952a). In the 4th-stage larva at 1 day after feeding the nuclei are relatively small and the cytoplasm shrunken and inconspicuous (Fig. 4A). By 4 days after feeding the nuclei have become enlarged and stain more deeply and they are surrounded by more or less dense cytoplasm which is vacuolated at the periphery. Large numbers of haemocytes are closely associated with the thoracic gland cells (Fig. 4C).

In 4th-stage larvae injected with iron saccharate (5 mm.³ of 2% solution at 1 day after feeding) the nuclei have become similarly enlarged and lobulated by 4 days after feeding. There has also been some increase in the vacuolated cytoplasm around them; but both nuclei and cytoplasm stain far less deeply than in the normal insect (Fig. 4B). Haemocytes are almost absent (see p. 658).

These observations suggest that although there is some activation of the thoracic gland cells even when the haemocytes are blocked, the haemocytes are necessary if the cells are to develop their full secretory activity.

(i)Active thoracic glands were removed from 5th-stage larvae at 10 days after feeding. One gland from each pair was implanted into a decapitated 4th-stage larva (1 day after feeding) which had just been injected with 5 mm.³ of 2% iron saccharate; the other gland of the pair was implanted into a similar 4th-stage larva which had received no iron saccharate.

A single gland proved to be rather too small a dose, and only about 30 % of the insects moulted. But there was no difference between the two groups: four out of twelve survivors in the control group moulted, and three out of ten survivors in the injected group; and moulting began at about the same time in all of them. It would appear that moulting induced by the implantation of a fully active thoracic gland is not delayed by blockage of the haemocytes.

(ii) The converse experiment consisted in implanting into the decapitated 4th-stage larva a pair of thoracic glands removed from a 5th-stage larva, 10 days after feeding, into which 10 mm.³ of 2% iron saccharate had been injected at 1 day after feeding. As a control, similar 4th-stage larvae received a pair of thoracic glands taken from normal jth-stage larvae 10 days after feeding.

In the control series there were twelve survivors of which nine were induced to moult. Among those receiving thoracic glands from the larvae with blocked haemocytes, there were ten survivors of which only one moulted. Blockage of the haemocytes with iron saccharate has prevented the development of full secretory activity in the thoracic glands.

A brief account of the haemocytes in Rhodnius has already been published (Wiggles-worth, 1933), and a more detailed study will be published elsewhere; but a short description is necessary here. For the most part the cells are not floating freely in the circulating haemolymph, but are loosely adherent to the basement membranes. The following four types have been recognized.

• (i) Proleucocytes. Small rounded forms with only a thin layer of darkly staining cytoplasm around the nucleus; non-phagocytic; often dividing; almost certainly the precursors of the amoebocytes and perhaps other types.

• (ii) Amoebocytes. Highly pleomorphic; rounded, spindle-shaped, irregular or spread out in an attenuated form so as to merge imperceptibly into the basement membrane ; usually furnished with numerous filamentous pseudopodia. These cells contain rounded, oval or rod-like deposits of mucopolysaccharide; they provide the substance of the basement membranes and connective tissue sheaths which have the same staining reactions and histochemical properties (Wigglesworth, 19556). The amoebocytes are actively phagocytic.

• (iii) Oenocytoids. Rounded or oval disk-shaped cells with small flattened nuclei. The cytoplasm is clear and homogenous or contains very minute vacuoles ; the cell membrane sharply defined and refractile with no pseudopodia. The oenocytoids are non-phagocytic.

• (iv) Lipocytes. Cells resembling small fat-body cells lying free in the body cavity. They are non-phagocytic and contain deposits of fat, glycogen and protein like the main fat-body cells.

  In addition to these, two further types can be distinguished.

• (v) Large granular cells. May be rounded or oval, but more often spindle-shaped. Nucleus large and vesicular with a very large round nucleolus; the cytoplasm filled with granules or small vacuoles.

• (vi) Large non-granular cells. Usually spindle-shaped and often drawn out into filaments at either pole ; resemble the large granular cells but the cytoplasm more or less hyaline.

Types (v) and (vi) are relatively very few in number; they are connected to the amoebocytes (ii) by intermediate types. But they lack the mucopolysaccharide deposits characteristic of the amoebocytes and they are either non-phagocytic, or very feebly phagocytic, for particles of indian ink or iron saccharate.

Since moulting is delayed by the injection of materials that are taken up by the phagocytic cells we are interested here chiefly in the amoebocytes. They are best studied by cutting along the margins of the abdomen and removing the tergites and sternites with the underlying heart, muscles, fat body and tracheal system in situ. These parts may be mounted fresh in Ringer’s solution containing suitable vital dyes, or fixed with Camoy’s or Bouin’s fixatives and mounted whole after staining for 3 min. with Prenant’s ferric trioxyhaematein ; or they may be cut, preferably in tangential sections, and stained in various ways. During the days which follow feeding, mitotic figures are plentiful among the proleucocytes, and the haemocytes of all types increase enormously in number.

Fig. 5 A shows typical amoebocytes at 3 days after feeding when stained supra-vitally with gentian violet in Ringer’s solution and mounted fresh. The cytoplasm shows a faint diffuse pinkish blue staining; it contains rounded or rod-shaped inclusions staining a slate-blue colour; there may be occasional clear vacuoles.

Fig. 5 B shows amoebocytes similarly treated at 4 days after feeding. The average size of the cells is increased. In very many of them the cytoplasm is now almost completely filled with clear non-staining vacuoles, the blue-staining deposits being compressed between them. There are still some amoebocytes which are not vacuolated, but the difference between the majority of amoebocytes at 3 and 4 days after feeding is very striking. Similar vacuolated cells can be seen as late as 7 days after feeding at least.

Fig. 6 A and B show typical amoebocytes on the third and fourth days after feeding, as seen in sections fixed with Bouin’s fixative and stained with Masson’s trichrome stain. In the fixed preparations the cells are smaller and the filamentous pseudopodia are not well preserved. At 3 days the nuclei stain red, the cytoplasm has a finely granular mauve colouring and contains rounded or rod-shaped deposits staining bright green. A few of the amoebocytes contain a single small vacuole. At 4 days the cytoplasm stains quite a deep purple but is often vacuolated and many cells are completely filled with vacuoles with the green-staining deposits appearing flattened between them.

Fresh preparations treated with other vital dyes and whole mounts and sections variously stained all give the same result : there is a striking change in the amoebocytes between 3 days (when injection of indian ink etc. delays moulting) and 4 days (when moulting is no longer delayed by injections).

Fourth-stage larvae were injected with the standard quantity of 5 mm.³ of 2% iron saccharate at different times after feeding. Then 1 day later the tergites and stemites were dissected off, fixed in Bouin’s fixative and stained for iron by immersion in ammonium sulphide and subsequent treatment with 2-4-dinitrosoresorcinol (Humphrey, 1935), and counterstained with Prenant’s ferric trioxyhaematein.

After injection at 1 day after feeding the amoebocytes are no longer dispersed over the surface of the various organs but are massed in clumps containing 100-500 or more contiguous cells. These cells are filled with iron deposits, mostly in the form of rounded droplets (Fig. 7A). A few of the cells are breaking down with the liberation of iron-containing droplets and little spheres of cytoplasm. The proleucocytes and oenocytoids contain no iron deposits.

After injection at 3 days the amoebocytes are massed as before and are filled with similar deposits of iron (Fig. 7B). Many more of these cells are now disintegrating and setting free cytoplasmic droplets. The figure shows an oenocytoid (now enlarging somewhat), a proleucocyte and a non-phagocytic giant haemocyte.

After injection at 4 days the amoebocytes are again mostly aggregated, though a larger proportion remains scattered. They are larger than at 3 days and appear to contain less iron. This is deposited in crescentic form over the surface of the clear vacuoles which more or less fill the cells (Fig. 7C). Occasional cells can be seen to be breaking down—but far fewer than after injection at 3 days. The oenocytoids are perhaps becoming still further enlarged. The figure also shows a large spindle-shaped haemocyte with a small deposit of iron.

After injection at 5 and 6 days the haemocytes are more scattered and show less tendency to form clumps. Vacuolated cells are present but are not so evident as at 4 days. Few of the cells are breaking down.

Similar results are obtained after the injection of indian ink. When injected at 2 days after feeding, and fixed 1 day later the amoebocytes are so densely packed with spherical droplets filled with the ink that even the nuclei are barely visible (Fig. 8 A). Aggregation into clumps occurs, but is not so evident as after the injection of iron saccharate.

When injected at 3 days, many of the cells are filled with the ink as at 2 days; but there are more cells which are not quite so densely packed with the ink droplets (Fig. 8B). There is very little break-down among the cells such as occurs after the injection of iron.

After injection at 4 days, the haemocytes are more scattered; they are slightly larger than at 3 days and contain much less indian ink. This is no longer densely packed but is diffuse and often separated by the vacuoles which fill the cells (Fig. 8C).

There are occasional vacuolated cells with relatively little ink after injection at 3 days, and occasional cells with dense deposits of ink at 4 days ; but the figures and descriptions indicate the state of the majority of the cells present on these days. The results confirm the observations on the histology of the normal haemocytes in showing a striking change in the secretory activity of the amoebocytes between the third and fourth days after feeding.

Butenandt & Karlson (1954) have recently succeeded in isolating in crystalline form from pupae of the silkworm, an active principle which will induce puparium formation in Calliphora and which, as proved by Williams (Butenandt & Karlson, 1954) will bring about renewed development in the diapausing pupa of Platysamia. Through the kindness of the German authors 1 have been able to test the activity of their hormone on Rhodnius.

The hormone was made up in saline at a concentration of 50 p.g. or 100 p.g. per ml. This was injected, by the method outlined above, into 4th-stage larvae at 1 day after feeding. The larvae were then decapitated. The 4th-stage larva at this time weighs about 80 mg., most of this weight being due to the blood meal in the stomach. Doses ranging from 0·025 to 0·25 μg. were given to twelve insects, but all the results were negative.

A dose of 0·25 μg. was then given at 1 day after feeding and repeated at daily intervals for 3 days, making a total of 0·75 μg. The two larvae so treated were dissected at 5 days after feeding; mitosis was actively in progress in the epidermis.

This experiment was repeated on two further larvae, one of which received a total of 0·75 μg. of hormone, the other 0·5 fig. By 25 days both had completed the formation of the new cuticle and the digestion of the old. The new cuticle (as was to be expected in the absence of the corpus allatum) was of the adult type.

The active principle from the silkworm pupa will thus induce moulting in the decapitated Rhodnius. In his tests in Platysamia Williams found that diapause could be terminated and development induced by injection of the hormone into the isolated abdomen. This experiment also has been carried out on Rhodnius. Four 4th-stage larvae at 1 day after feeding were injected with a single dose of 1·0μ.g. of the hormone and the abdomen immediately isolated by a ligature through the metathorax as previously described (Wigglesworth 1952a). All four were induced to start moulting, although they died or were dissected and fixed before the process was complete. Six larvae were given 0·75 fig. in a single dose and treated in the same way : one died, moulting was induced in all the remaining five.

Since their hormone was effective in the isolated abdomen Butenandt & Karlson (1954) suggested that it is probably the active principle of the thoracic gland. We have seen that moulting induced by implantation of the fully active thoracic gland is not delayed or inhibited by blockage of the haemocytes. It was therefore of interest to test the crystalline hormone in the same way.

With this object four 4th-stage larvae were decapitated at 1 day after feeding. Two of them were given a single dose of 1 μg. oíthe hormone and two were given the same dose of hormone plus 5 mm.³ of 2% iron saccharate. All started to moult at about the same time: the characteristic changes which become visible in the integument of the normal insect at 7 days after feeding had become apparent by 5 days, that is, by 4 days after the injection. Moulting was complete in 17-20 days.

This experiment was repeated with indian ink. Four 4th-stage larvae decapitated at 1 day after feeding were given 1 μg. of the hormone, and two of them received in addition 5 mm.³ of a strong suspension of indian ink. Moulting had clearly begun in all four insects by 6 days after feeding and went forward at approximately the same rate in all of them.

Thus, blockage of the haemocytes causes no delay in the onset of moulting induced by the crystalline hormone of Butenandt & Karlson. This agrees with the suggestion that this hormone is the factor normally produced by the thoracic gland.

The study of the ‘critical period’ for transfer from 25 to 36° C. in the 4th-stage larva (p. 653) suggests that the high temperature may not only arrest the secretion of the thoracic gland hormone but may interfere with the subsequent action of this hormone in the abdomen.

Four 4th-stage larvae were decapitated at 1 day after feeding and each was given a single injection of 1 μ tg. of hormone. Two were placed at 25° C. and two at 36° C. At 12 days after feeding moulting was well advanced in those at 25° C., and by 20 days both had moulted to the adult type. Those placed at 36° C. showed no sign of moulting at 12 days; at 20 days both had died but in neither had moulting commenced. This result confirms the earlier conclusion that high temperature prevents the initiation of mitosis in the epidermis, even when the necessary hormones are already present.

Throughout the study of growth hormones in Rhodnius it has always been maintained that the ‘moulting hormone’ might prove to be a complex of successive secretions. It would now appear that the neurosecretory cells in the brain, the amoebocytes, and the thoracic glands constitute a secretory chain necessary for the initiation of growth. But there is no reason to suppose that the story is yet complete. The oenocytoids and other non-phagocytic haemocytes are undergoing cycles of activity; the part they are playing in the intermediary metabolism of growth remains to be proved. The role of the pericardial cells is very incompletely understood. And it is possible that there may be some division of labour among the epidermal cells themselves.

How the amoebocytes intervene in the moulting process is not known. They might be concerned in transporting the hormone from the neurosecretory cells to the thoracic gland. But some degree of activation of the thoracic gland occurs even when the amoebocytes are blocked. It is perhaps more probable that they are secreting some raw material that is necessary for the full activity of the thoracic glands. Alternatively, it is possible that the brain factor is removed from the blood by the phagocytic activity of the amoebocytes; or, as Prof. C. M. Williams has suggested to me, it is conceivable that during their disturbed metabolism the amoebocytes might inactivate the brain factor in some other way.

The demonstration of the part played by the amoebocytes in moulting raises the question of their possible role in wound healing. The histological changes in wound healing in Rhodnius are the same as those in normal moulting (Wigglesworth, 1937). These changes can go forward in the decapitated insect. It was therefore suggested (Wigglesworth, 1954) that the epidermis might be responsible for the localized production of factors comparable with those produced centrally in the normal process of moulting. Now it is characteristic of wound healing that there is a large aggregation of haemocytes around the site of injury. It may be that they are concerned in this local process of hormone production. (It may also be that the large local increase in haemocytes after wounding may be concerned in the persistent rise in the respiration rate which Schneiderman & Williams (1953) have observed to follow such injuries.)

One further point calls for comment. Discussions on the function of the haemocytes have generally been based on the assumption that a given type of blood cell has a single function. It is quite clear that the amoebocytes in the blood of Rhodnius have several functions : they are active phagocytes ; they produce the mucopolysaccharides which go to form the basement membranes and connective tissues (Wigglesworth, 1955 b); and they produce the droplets of secretion which appear to be essential for the full activity of the thoracic gland. This multiplicity of function in a single cell is equally evident in the fat body cells and in the epidermal cells of insects.

I am indebted to Prof. A Butenandt and Dozent Dr P. Karlson of the University of Tübingen for a supply of their crystalline hormone.