The development of salivary tumours in Periplaneta americana (L.) as induced by duct ligation

J. Znsect Physiol., 1967, Vol. 13, pp.

137to 152. Pergamon

Press Ltd.

Printed in Great Britain

THE DEVELOPMENT OF SALIVARY TUMOURS PERIPLANETA AMERICANA (L.) AS INDUCED BY DUCT LIGATION*? DONALD Department

IN

J. SUTHERLAND

of Entomology and Economic Zoology, Rutgers University (Received

17 June

1966)

Abstract-The salivary glands of adult female cockroaches were subjected to duct ligation, unilateral duct ligation, or duct lesion and examined histologically at various times thereafter. Types of effects include (1) dissolution of acini, (2) acinar atrophy, (3) modified acinar atrophy with and without vesicle formation, and (4) haemocytic encapsulation and vesicle formation. Types 3 and 4 involve a proliferation of intercalated cells to form giant cells, which eventually yield the vesicular epithelium. Tumour incidence in acini is greatest after ligation; after duct lesion incidence is highest in the reservoirs. Experimental methods and tumours induced cause increased mortalities.

INTRODUCTION

THE subject of experimentally induced tumours in insects has been reviewed by SCHARRER and IACKHEAD (1950), HARKER(1963), and MATZ (1963). Experimental

methods include nerve severance (SCHARRER,1945; MATZ, 1961a, b; SUTHERLAND, of suboesophageal 1967a), duct ligation (SUTHERLAND,19631, transplantation ganglia (HARKER,1958), and chemical carcinogens (NOLANDand BAUMANN,1949 ; SCHLUMBERGER, 1952; SHORTINOet al., 1963 ; SUTHERLAND,1967b). The objective of this report is to describe the early and late developmental stages of tumours induced by salivary duct ligation, the possible causes of these tumours, and the r61e of haemocytes, which was not recognized earlier (SUTHERLAND,1963). The question has arisen as to whether the term ‘tumour’ should be applied to certain abnormal tissue growths in insects (CAMPBELL, 1959). However, as discussed by SCHARRER(1953), a utilization of the term does not imply that the growths are in every respect similar to neoplasms of vertebrates. The objective of a study of insect tumours is to elucidate the physiopathology of insects and the r81e of insects in comparative oncology. * This investigation was supported by Public Health Service Research Grant No. CA-07299 from the National Cancer Institute and also by an institutional grant from the American Cancer Society. f Paper of the Journal Series, New Jersey Agricultural Experiment Station, RutgersThe State University, Department of Entomology and Economic Zoology. 137

138

DONALD J. SUTHWLAND

METHODS Young adult female cockroaches, approximately 7 months after hatching, were generally used except where otherwise indicated. The method for ligation of the common salivary duct was the same as previously reported (SUTHERLAND,1963) with the exception that generally surgical thread (Code 110-S, J. A. DeKnatel and Son, Inc., N.Y.) was used. Ligation of one side of the gland or one reservoir was performed in a similar manner, posterior to the third cervical sclerite. Lesion of the duct was accomplished by slightly rupturing the integument and severing by means of micro-scissors. In those cockroaches in which only one side of the salivary system was operated on, examination was made on dissection to observe whether the opposite side was constricted by wound tissue. In experiments with implanted cellophane, small fragments of dialysis tubing were placed in the anterioventral portion of the thorax. Fragments were either sterilized in ethanol or autoclaved. Cockroaches were treated in groups of ten and maintained in plastic containers. Water was supplied in plastic, tight-fitting Petri dishes (Tri-State Plastics, Henderson, KY.), in the cover of which was drilled a hole to hold a short dental wick. Cockroaches were held in a 12 : 12 light : dark cycle and examined at specific intervals after operation or at death, Tissues were fixed in Carnoy’s, Bouin’s, or Helley’s fixative and stained with haematoxylin and eosin, Mallory’s triple stain, or Schiff’s reagent. RESULTS AND DISCUSSION Normal histology oj the salivary system

The normal histology of the salivary glands has previously been described (DAY, 1951; KESSEL and BEAMS,1963), but there are some further details which require mention here. Depending on the secretory activity, the nuclei of all cells in the gland system can vary in size so that often the nuclei of the parietal cells are larger than those of the zymogen cells. Carnoy’s fixative has been found to be best for nuclear detail, which generally is concealed, particularly by Mallory’s triple stain and to a lesser extent by haematoxylin. According to DAY (1951) the intracellular ductules and par&al cells always occur in pairs. KESSELand BEAMS(1963), however, report that usually each intracellular ductule opens within a single parietal cell, which generally possesses a single nucleus. In current studies the observations of Day generally are confirmed. Each branch of the intercalated ducts leads to an intracellular ductule, which divides and swells distally, each end within a single parietal cell (Fig. 1). The membrane separating the two parietal cells is frequently not visible and the ‘pairs’ of parietal cells may appear to be a single binucieate cell. Based on ultrastructure (KESSEL and BEAMS,1963), each distal swelling of an intracellular ductule within a parietal cell can be termed the microvillar bulb. The salivary reservoirs are always composed of two distinct layers of cells, which can be separated by placing ligated reservoirs, distended or contracted in a

DEVELOPMENT

OF SALXVARY

TUMOURS

IN

PERZPLANETA

AMh’RICANA

(L.)

139

hypotonic solution. The outer layer of cells appears to be connective tissue since it is continuous with that which covers the lobes of the glands. In these studies, there has been no evidence that cells from one layer of the reservoir wall migrate into the other layer (DAY, 1951).

Possibly the reservoirs are filled by back pressure of secretory products from other portions of the gland (DAY, 1951). In Peripluneta americana, however, the topography of the gland system and the large volume of a distended pair of reservoirs do not support this fully. Since both cellular layers stretch to the same degree in distension of the reservoir, it would seem that one layer of cells would be sufficient for this if the sole function of the reservoir were to receive and store salivary secretions. In addition, since at least the outer layer is rapidly permeable to water, possibly the reservoirs are at least partially filled by the osmotic pressure of concentrated salivary secretion entering them and, therefore, are an osmoregulator of the blood. Although DAY (1951) f ound no musculature associated with the ducts or reservoirs, a thin muscle, which arises on the thoracic dorsal area and inserts on the crop, also inserts several strands of muscle on the connective tissue layer at the junction of the reservoir and its duct. This muscle and its function will be described in a later report.

Acinar tumour development After ligation of the common salivary duct, most salivary systems apparently continue to function with the exception that the secretion cannot be discharged into the salivarium. Experiments, in which reservoirs have been filled as a result of cockroach starvation for 1 week prior to ligation with subsequent dissection after feeding, have shown the ligation method to be over 95 per cent effective in interrupting secretion flow. In the absence of ligation but under the same conditions of starvation and feeding the full reservoirs are quickly emptied. There are indications that there is a post-operative period during which some salivary systems are not fully functioning. In some groups dissected at various times after ligation, full reservoirs in 90 to 100 per cent of the cockroaches were observed only after 14 days. In some isolated cases, cockroaches have survived the operation, but the reservoirs have been compressed and emptied and the glands apparently normal even 2 months later. Undoubtedly the continued functioning of the system after ligation affects the type and speed of abnormal cellular events resulting. As early as 1 day after ligation, changes can be seen in the gross histology of the glands. The basophilic cytoplasm of the zymogen cells becomes broken into smaller patches and later becomes granular. The intercalated ducts close to the acini are distended, although the lumen is neither strongly basophilic nor acidophilic. The microvillar bulbs of the pa&al cells become swollen and more apparent (Fig. 2), and the cytoplasm around their basal portion is thicker and more evident. If pressure of contents is sufficiently great, the lobe of cytoplasm separating each pair of microvillar bulbs is flattened, and each pair of parietal cells may appear to be a binucleate cell with one large microvillar bulb. This is especially

140

DONALDJ. SUTHERLAND

true if cellular membranes are indistinct. The parietal nuclei generally increase in size, surpassing that of zymogen nuclei. The cytoplasm of the parietals, normally acidophilic, likewise increases in volume (Fig. 2), and in certain cases basophilic granules appear. If the pressure of contents within the intracellular ductules increases, the latter impart a vacuolated appearance to the acinus (Fig. 3). If the zymogens continue to secrete, large clear vacuoles appear (Fig. 4) which push aside the basophilic cytoplasm. Although normally the lumen contents of the intercalated ducts and reservoir are weakly acidophilic at the most, under conditions of ligation such contents subsequently become more acidophilic and sometimes contain large globules of strongly basophilic material. This possibly indicates that the salivary secretion originates as two distinct components which mix eventually. Subsequent to many of these events, the following types of abnormal tissue reactions result. 1. Dissolution of acini. In a small number of ligated glands, some acini undergo dissolution of zymogen and subsequently parietal cells, and a general release of cellular components into the haemolymph occurs (Figs. 5, 6). Cytoplasm of all cells remains acidophilic, and, since the zymogen cells are relatively inactive, the stimulus appears to be autolysis and not pressure of secretions. The nuclei of all cells atrophy and become pyknotic. No haemocytic encapsulation occurs. 2. Acinar atrophy. Atrophy is a common manifestation of senescence or disuse found in tissues of many organisms. It normally occurs at a low level in the salivary glands of cockroaches. However, its occurrence (Table 1) is much greater TABLE

I-PER

CENT

INCIDENCE

SUBJECTED

OF

TO LIGATION

TUMOURS OF THE

IN

THE

COMMON

SALIVARY SALIVARY

GLANDS

OF

COCKROACHES

DUCT

Months after ligation

Condition Tumorous Tumorous, with full reservoirs Normal, with full reservoirs Atrophied No. examined Of total tumorous, in

24 51 45 5

42 71 36 18

39 67 21 35

46 39 47 39

69 64 50 88

71 20 100 86

86 14 0 86

164

75

23

28

16

7

7

50 per cent glands only, 30 per cent reservoirs only, 20 per cent both.

ligated glands, and it may occur throughout the gland (Fig. 7 ; for normal gland see SUTHERLAND, 1963) or only in some lobes with and without other types of abnormal tissue reaction. Zymogen cells in an inactive state and with acidophilic cytoplasm decrease in size until they are completely lacking. The parietal cells including microvillar bulbs remain, and entire lobes of the gland can consist solely of parietal cells connected to intercalated ducts (Fig. 8). Parietal nuclei are large

DBVEL.OPMRNTOFEALSVARYTUMOURE IN PERIPLANETAAMERICANA

(L.)

141

and cytoplasm seems to increase, probably due to the atrophied gland being more compact. Although microvillae are present, it is not known if the parietal cells continue to function. Mitosis or haemocytic encapsulation does not occur. 3. Modifid a&tar atrophy with and without vesicleformation. This type is similar to the preceding type with the exception that, in addition to the loss of zymogen cells and maintenance of par&al cells, there occurs a proliferation (Fig. 9) of intercalated duct cells as in type 4. These new cells move toward the periphery of the acinus (Figs. 2, 10, 11). This occurs before complete disappearance of the zymogen cells, and the proliferating nuclei subsequently resemble those of the parietal cells. In contrast, however, the nuclear+ytoplasmic volume ratio is high. In advanced stages, with modification or loss of the parietal microvillar areas, it is difficult to distinguish cell types. Nuclei become more numerous and crowded, and cell boundaries often become indistinguishable. Frequently groups of two or more nuclei are so large and so close together (Figs. 2, 3, 11, 12) as to indicate multinucleate cells as a result of endomitosis. This is known to occur normally in certain glands of insects (GABE and ARTY, 1961). Haemocytes are infrequent, and haemocytic encapsulation does not occur. Although some acini remain in the above condition, others proceed toward vesicle formation (Fig. 13); both may exist in the same gland, but in the former there is no indication of development to the latter. Vesicle formation is characterized by autolysis of a few cells in the centre of the acinus, including some newly proliferated cells, and peripheral deployment of cells present to surround the area (Fig. 14). Mitosis rarely takes place at this time. Clumps of nuclei separate, apparently accompanied by cytoplasmic division, yielding mononucleate cells. Liquefaction occurs in the centre of the acini, and it is the pressure of this fluid together with that from other portions of the glands that pushes the new epithelium outward to form a rounded sphericle vesicle. Initially the contents are acidophilic, but this property is soon lost. The epithelium becomes stretched (Fig. 8), the nuclei elongate, but no cuticular lining is evident. Occasionally vesicles of adjacent acini, if they have a common intercalated duct, merge to form larger vesicles; in some cases a vesicle loses its connexion with the remaining acini and is held in place only by connective tissue. It is of interest that, throughout the development of type 3, apparently the connective tissue remains unchanged. 4. Haemocytic encupsulation and vesicle fortnation. This type of tissue reaction is the most common of all and is referred to in Table 1 as being tumorous. The tumours are readily recognized on gross examination; the acini are more compact, white, and contain deposits of brown inclusion cyst material (SUTHERLAND, 1963). In early stages the intercalated duct cells proliferate outwards toward the par&al cells as in type 3. This has also been seen in untreated glands but to a lesser degree. Normally the nuclear chromatin of the intercalated ducts is sparse compared to other nuclei in the gland. As a consequence, mitotic figures are observed only by close examination and do not equal the bold figures found in midgut nidi. Prophase and metaphase have been observed. The nuclei of the proliferating cells are more compact and elongate as they move outward along the

142

DONALD J.SUTHERLAND

intracellular ductule (Figs. 2, 10). Although they might easily be mistaken for haemocytes, which eventually appear, the proliferating cells often are paired, one on each side of the ductule as in type 3. In contrast to type 3, the zymogen cells remain, apparently secreting, and maintain turgor in the acinus. The new cells continue to move distally, their nuclear size increasing to that of the parietal nuclei, and rest finally adjacent to the parietals and around the periphery of the acinus. Often nuclei occur in groups of two or more (Fig. 11) and as many as fifteen, probably in multinucleate cells as a result of endomitosis. Haemocytic encapsulation can begin within 1 week after ligation and generally occurs after proliferation of intercalated duct cells is initiated. Normally, haemocytes can be found between the acini, resting on the acinar surface, or occurring in groups, al least in part due to conditions of dissection, its rapidity, and also to fixation (JONES, 1962). -Encapsulation of the acini begins almost exclusively by increased numbers of haemocytes appearing at the junction of the intercalated ducts and acini. A few haemocytes seem to penetrate the’acini intercellularly, but the majority remain outside adhering to the acinus and ducts. Additional haemocytes subsequently appear, adding to the process and eventually leading to final peripheral encapsulation of one or more acini. The immediate result of encapsulation is an apparent destruction of most cells of the acinus. This is particularly true of the zymogen cells. However, depending on the stage of type 4 at which encapsulation occurs, other cells (Fig. 15) remain, some of which are the product of intercalated duct cell proliferation. Some of these cells greatly increase in size (Fig. 16) and eventually undergo mitosis to yield a sphere of epithelium intermediate in the mass of encapsulating haemocytes (Figs. 17, 18). Central to this epithelium, the haemocytes and inclusion cyst material are broken down to form a clear liquid which becomes the contents of the sphere. Coincidentally, the haemocytes external to the epithelium apparently re-enter the haemolymph circulation, with the result that the vesicle formed is similar to that of type 3. Although the origins of the vesicular epithelium in types 3 and 4 probably are the same, the vesicles of type 4 differ from those of type 3 since in the former (1) a mesodermal sheath does not surround it and (2) the epithelium is not oriented by pressure of contents but by haemocytes. In the latter case the epithelium is not restricted to one or two acini but courses throughout the encapsulated mass to yield eventually one or more large vesicles. This can be so extensive as to replace more than one-half the original gland. As stated above, some doubt exists as to the exact origin of the new epithelium of the vesicles. Although it appears that multinucleate cells derived from the intercalated duct cells are its origin, some par&al cells, when lacking microvillar bulbs and definite cell boundaries, closely resemble them. Mitosis has been observed in pa&al cells, and, therefore, they cannot be dismissed as a possible source. The newly formed epithelium can be contiguous with the duct epithelium, but there is no evidence that the latter is the direct source. Also no evidence has been found that tracheal cells, haemocytes, or other cells from the haemolymph are the origin of the epithelium.

FIG. 1. Salivary gland 4 days after sham operation; P, parietal cell; Z, zymogen cell. F1c. 2. Salivary gland 2 days after ligation, with swollen parietal cells (SP) and nuclei from intercalated duct moving ( 7) toward periphery; Z, zymogen cell; D, intercalated duct; N, group of three non-parietal non-zymogenic nuclei. Fro. 3. Salivary gland 6 days after ligation, with vacuolated appearance due to swollen intracellular ductules (D); N, group of three nuclei. F1c. 4. Salivary gland 1 week after ligation, zymogen cells containing clear vacuoles (V).

FIG. 5. Salivary gland 1 month after ligation, with dissolution of acini and pyknosis of nuclei. FIG. 6. Salivary gland 2 weeks after lesion, with early dissolution of acini and appearance of round cells (C) of unknown origin, possibly from intercalated ducts. FIG. 7. Salivary gland 3"5 months after ligation, with advanced atrophy. FIG. 8. Salivary gland 3 months after ligation; zymogen cells are lacking, and acini are mainly composed of parietal cells (P) and intercalated duct cells. Adjacent lies a flattened vesicle, the lumen (L) of which is surrounded by new vesicular epithelium (E). FIG. 13. Salivary gland 6 months after ligation, with advanced atrophy and formation of vesicles (V).

O

I-

Fro. 9. Salivary gland 6 months after ligation, with mitosis of intercalated duct cells and movement ( >) toward the periphery of the acinus. Fxc. 10. Salivary gland 1 day after ligation, nuclei from intercalated duct cells moving ( - - ~ ) intercellularly toward periphery of acinus; P, parietal cells; Z, zymogen cells. FIG. 11. Salivary gland 1 month after duct lesion; group of 5 nuclei (N) moving ( - - ~ ) toward periphery of acinus; P, parietal cells. Fro. 12. Salivary gland, 2 days after ligation ; groups of two and three nuclei (N) are located at the periphery of the acinus.

! !~i

il!~

FIG. 14. Salivary gland 6 months after ligation with early formation of two vesicles; L, lumen of vesicles; N, giant nuclei at periphery; T, tri-lobed nucleus; M, mitosis. FIG. 15. Salivary gland 1 week after ligation, with encapsulation of acinus by haemocytes (H) and destruction of most cells in the acinus ; larger nuclei (N) within haemocyte mass are probably derived from intercalated ducts. FIG. 16. Salivary gland 3"5 months after ligation, with encapsulation of acinus by haemocytes (H) and appearance of giant cells. FIG. 17. Salivary gland 3"5 months after ligation, with encapsulation of acini by haemocytes (H) and the formation of vesicles with a new epithelium (E) from giant cells; D, inclusion cyst material and dead cells in the lumen of vesicles.

FIG. 18. Salivary gland 3-5 months after ligation, with encapsulation of acini by haemocytes (H) and formation of new epithelium (E) extending through several acini; D, inclusion cyst material and dead cells. FIG. 19. Salivary gland 2 weeks after duct lesion, indicating a normal duct (A) as compared to a duct with abnormal or mitotic nuclei (B). FI~. 20. Salivary gland 2 weeks after duct lesion; the epithelium (OE) of a large duct has been replaced by a new apparently identical epithelium (NE), H, haemocytes; L, lumen. Fie. 21. Salivary gland 1 day after ligation, granular inclusion cyst material (G) appearing in the cytoplasm of some cells.

DEVELOPMENT

OF SALIVARY TUMOURS

IN PERIPLANETA

AMERICANA

(L.)

143

Each vesicle seems to arise from a single rather than a multiple origin within an encapsulated acinus or group of acini, even when a giant vesicle is formed. The factors that govern this are not known, but it is apparent that the haemocytes act as a mould for the epithelium, providing a favourable gradient which governs the It would be of interest to know if such cells could direction of the proliferation. survive direct contact with the haemolymph and invade nearby organs. At present no information is available to indicate whether all tissues of type 4 eventually end in vesicle formation. The latter occurs 2 to 7 months after ligation. In ligation of the common salivary duct, vesicle incidence has been 14 per cent among those examined in that period. A higher incidence of 39 per cent has been observed for glands ligated on one side. Although incidence of the encapsulated stage of type 4 increases with time, vesicle formation does not. Possibly in some cases, encapsulation occurs too soon, and conditions are unfavourable for vesicle formation.

Duct and reservoir tumour development Salivary duct ligation also has an effect on other tissues in the salivary system. Studies on the ultrastructure of the secretory ducts by &3sEL and BEAMS (1963) have indicated that these ducts likewise contribute to the final secretory product of the gland. Under conditions of ligation large clear vacuoles sometimes appear basally to the infoldings of plasma membrane. The vacuoles increase in size and apparently coalesce to result in a separation of the basal and apical portions of the cells. A complete separation has not been observed, possibly indicating that some cells become inactive in the secretory process. In a few smaller ducts at a slight distance from their attachment to the acini, the nuclei stain densely and appear to be either abnormal or in the initial stages of mitosis (Fig. 19). In portions of the large ducts, nuclear hypertrophy occurs in advance of slight haemocyte involvement, but complete encapsulation and vesicle formation do not occur. In only one case has it been observed that the epithelium of the large ducts is destroyed and replaced (Fig. 20). Of all gland systems examined after ligation, reservoir tumours similar to type 4 acinar tumours have occurred in 50 per cent and are limited generally to distended reservoirs. Haemocytes gather, adhere to the reservoir surface, and subsequently encapsulate the reservoir. Prior to this there sometimes occurs a hypertrophy of nuclei in the outer mesodermal layer, but no mitosis has been observed in either of the two layers of cells. Haemocytes in large numbers can penetrate both cellular layers, and eventually large fragments of encapsulating haemocytes can be sloughed from the inner surface and be freely suspended in the reservoir liquid. Remarkably, darkening but no lysis occurs, and cellular detail of the fragments is maintained. Nuclear hypertrophy of the mesoderm in some cases continues and is sufficiently similar to be mistaken for newly developing epithelium. A definite new epithelium has been observed in a few cases; its exact source is not known.

144

DONALD J. SUTHERLAND

Inclusioncyst material

Brown material which appears in acinar tumours type 4 and often in reservoir tumours has been termed inclusion cyst material (SUTHERLAND,1963). Similar material has been observed in wound reactions in the mosquito (DAY and BENNETS, 1953) and the cockroach (DAY, 1952). In the latter, eschar appeared in the fore-, mid-, and hindgut, but a new non-cellular basophilic layer, continuous with the chitinous intima, was restricted to the fore- and hindgut. In salivary tumours, inclusion cyst material (ICM) is so compact that it is difficult to determine what parts of it are necrotic tissue (eschar) and possibly-new chitinous intima. Capabilities of producing the latter probably are possessed by all ectodermal tissues such as the salivary glands. ICM differs from wound eschar by being liquid during parts of its formation, while at other times it is solid and sections similarly to the integument. When large deposits of ICM are sectioned and examined for melanin (COWDRY,1948), only the core of the deposit is susceptible. The outer portions of such deposits are not susceptible to treatment with hot alcoholic KOH. Although haemocytes may contribute to melanization in many tumours (HARKJZR,1963), ICM in salivary glands arises as granules in the nuclei and cytoplasm prior to any apparent cellular destruction or haemocytic encapsulation (Fig. 21). Subsequently, haemocytes may contribute to the ICM deposits. Incidence of tumour development

The incidence of tumour development as induced by ligation of the common salivary duct is shown in Table 1. Cockroaches were either sacrificed or examined at death. The data are based on gross observations of tissues from 320 individuals at the time of dissection. Dead individuals whose tissues were too decayed for proper dissection are not included in the data. It is probable that tumour incidence would have been higher if such individuals had been examined and also if all salivary tissues had been examined histologically to determine cellular changes not grossly evident. The incidence of tumour development rises to approximately 70 per cent in 4 to 5 months, after which incidence continues to rise. Insufficient numbers of individuals have survived past 5 months to determine if incidence reaches 100 per cent. Previously (SUTHERLAND,1963) in similar experiments, tumour incidence in cockroaches treated at 9 months of age was 100 per cent after 1 month. Based on the hypothesis that the function of the reservoir is to store salivary secretion, stimulus for tumour development in ligated glands might possibly be hydrostatic pressure of the secretion. According to the data in Table 1, however, tumour development can proceed with or without a full reservoir, and a full reservoir after ligation does not ensure that salivary tumours develop. In addition, the percentages of full vs. empty reservoirs are similar to those found in untreated cockroaches at various times during a 24 hr cycle (unpublished experiments). Often in ligated glands the intercalated ducts and small secretory ducts are swollen due to secretion, while the reservoirs remain empty and compressed. It must be

DEVELOPMENT OF SALIVARY TUMOURS IN PERIPLANETA

AMERICANA

145

(L.)

concluded, therefore, that hydrostatic pressure, at least as is evidenced by a full and dilated reservoir, is not the direct stimulus leading to tumour development. Unilateral ligation of the salivary glands

The topography of the ducts of the salivary system (DAY, 1951) is such that it is possible with a single ligature to ligate the reservoir duct and the acinar salivary duct of one side of the salivary gland system. Since the ligature is placed posterior to the junction of the acinar duct and reservoir duct, the increase of fluids due to ligation and subsequent increase of hydrostatic pressure in one tissue is prevented from influencing the other tissue. The per cent tumour incidence of such ligation is presented in Table 2. The data are based on those individuals in which close examination revealed that the ducts on the untreated side of the gland were not closed by wound tissue from the nearby ligature. The abnormal cellular processes occurring after unilateral ligation are essentially the same as those described for common salivary duct ligation. However, tumour incidence (Table 2) in the ligated side or in both sides is generally lower. This TABLE 2--PER CENT TUMOURINCIDENCEIN SALIVARYGLANDSOF COCKROACHES SUBJECTED TO LIGATIONOF THE RIGHTSIDEOF THE GLAND Months after ligation Condition

Acini tumorous

Reservoir tumorous

Reservoir full

Acini atrophied

No. examined

Right only Left only Both sides Total Right only Left only Total Right only Left only Both sides Right only Left only Both sides

1

2

3

4-5

6-7

6 0 4 10 1 1 2 23 17 6 3 1 1

46 8 15 69 8 0 8 15 8 0 15 0 0

(%) 44 0 6 50 31 0 31 19 0 19 6 0 0

21 0 21 42 14 0 14 7 0 0 7 0 21

45 0 0 45 18 0 18 9 0 0 0 0 36

78

13

16

14

11

probably is due to the fact that the ligated reservoir cannot contribute to the tumour induction stimulus or that the ligated side does not fully function in the presence of fully functioning acini on the unligated side. Generally, tumours are limited to the ligated portion of the gland. However, 8 per cent of all examined showed progression of tumour development to involve both sides. Such low incidence indicates that either (1) haemocytic encapsulation

146

DONALDJ. SUTHERLAND

is a passive process in which the haemocyte receives the stimulus to encapsulate only upon coming in contact with the tissue to be encapsulated or (2) a specific chemical stimulus and gradient direct haemocytes to approach and encapsulate. The first of these possibilities would appear to be a very inefficient defence mechanism. The second is supported by observations of amoeboid movement of haemocytes in the cockroach, BZuberus (ARNOLD, 1959). In the 8 per cent in which both the ligated and non-ligated sides were tumorous, some vesicle formation, indicative of mitosis as in types 3 and 4 acinar tumours, occurred in the non-ligated portion. Since vesicle formation can be independent of haemocytic encapsulation, tumours in those portions are not the result of some error in stimulus for haemocytic encapsulation. Instead probably some non-cellular chemical stimulus caused this apparent metastasis. Lesion of the common salivary duct

To determine if salivary secretions escaping under hydrostatic pressure might be the stimulus for haemocytic encapsulation, two experimental methods were employed, lesion of the common salivary duct and injection of supernatants of salivary gland homogenates. When the common salivary duct is severed, the contents of the ducts and possibly the reservoir spill into the haemolymph. Within 24 hr haemocytes seal the severed end, with essentially the same effect as in duct ligation. The major difference is the absence of escaping secretion in the latter. The results of lesion (Table 3) d iff er f rom those of ligation, the incidence of tumorous acini being much TABLE Q-PER CENTINCIDENCE OF TUMOURS IN SALIVARY GLANDS

OF COCKROACHES SUBJECTED

TO LESION OF THE COMMON SALIVARY DUCT

Months after lesion Condition

4

1

2

3

4

Reservoirs tumorous Haemocytic fragments Reservoirs full Acini tumorous Acini atrophied

32 26 78 16 0

63 11 80 21 25

%’ 10 70 10 30

70 0 90

87 0 95

20

5

60

77

No. examined

50

47

10

10

22

lower. Probably during duct lesion nerves are severed which supply and stimulate acinar secretion, resulting in a reduction of both acinar activity and subsequently tumour development. Two nerves, laterally oriented to the common salivary duct, proceed posteriorly to the reservoirs and the acini. A detailed study of the nerves supplying the gland system is necessary to further clarify this aspect.

DIWELOPMENTOFSALIVARY

IN PERIPLANETA AMERICANA

TUMOURS

(L.)

147

In contrast to acinar tumours, lesion induces high tumour incidence in reservoirs. Among all those examined, incidence was 56 per cent in comparison to 18 per cent under conditions of ligation. Reservoir tumours induced by lesion in many cases invade tissue located near dilated reservoirs, such as the crop. Apparently when the duct is severed, the full reservoir empties, and its distended walls do not return to a compressed state. Instead the collapsed reservoir rests abnormally on other structures, and haemocytic encapsulation involves the reservoir and adjacent tissue. This effect of rapid emptying indicates that normally the reservoirs empty rather slowly and this is accompanied or controlled by the reservoirs returning to the compressed state. One effect often observed in reservoir tumours induced by lesion-is the appearance of haemocytic fragments, as previously described. The effect occurs less under duct ligation. In lesion experiments these fragments are found as soon as 2 months after operation. The haemocytes apparently penetrate both cellular layers of the reservoir, finally accumulating on the inner surface, from which the fragments are subsequently sloughed. Although some haemocytes may enter the reservoir by way of the severed duct prior to the latter’s encapsulation, there is no evidence that such haemocytes are a significant source of haemocytic fragments. Since acinar tumour incidence was not increased after duct lesion, apparently salivary secretion escaping into the haemolymph is not a stimulus for abnormal development. No abnormal tissue response was observed in insects injected with non-cellular gland homogenates, which supports this conclusion. Cockroach mortality

The per cent mortalities as caused by various experiments and subsequent abnormal tissue development are given in Table 4. Some bias has been introduced TABLET-MORTALITIES

OFC~CKROACHE~

SUB~XEDTO

VARIOUS TREATMENTS

AccumuIative y0 mortality/month Total Treatment

NO.

#

1

2

3

4

5

6

7

51

76

87

93

96

98

100

100

Duct ligation Duct ligation unilateral Duct lesion

210

31

117 78

15

30

52

69

81

90

38

61

78

90

94

100

Control

105

22

33

42

50

60

66

75

85

8

100

since the figures have been summarized from those individuals dying and do not include sacrificed individuals. Control groups in particular were sacrificed after 6 months, especially when treated groups serviced by a control group had completely succumbed. The data, therefore, are based on mortalities occurring during 8 months after treatment and solely indicate relative mortalities during that time.

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Even with a bias unfavourable to control groups, the latter survive longer than treated groups. Only minor differences exist in the mortality rates of groups subjected to lesion or ligation. The slightly greater rate for the former probably is due to the invasiveness of reservoir tumours. It is not known if the increased rates of mortalities due to lesion and ligation are caused by the tumorous tissues themselves or by their functional cessation. Possibly both factors are important, and operation effects may even extend to affecting the cockroach susceptibility to nematodes in the hindgut. The initial similarities between mortalities of control groups and those of unilateral ligation seem to indicate that functional cessation is an important cause of mortality. However, in the 2 to 8 months after operation, mortalities from unilateral ligation approach those of common duct ligation and lesion. If it is assumed that the sole function of the salivary gland system is to supply certain digestive enzymes, then one side of the gland could be expected to supply sufficient amounts. Therefore, increased mortalities at such times may be due to the presence of tumorous tissues and their effects on other tissues. If a more important function such as osmoregulation of the haemolymph can subsequently be shown for the reservoirs, functional cessation may prove to be a more important contributor to mortality. Implantation of cellophane

The reaction of insect tissues to wounding and the subsequent processes of repair have been studied in Aedes aegypti and Orosius argentatus (DAY and BENNET, 1953), in Periplaneta americana (ERILZIN, 1939; DAY, 1952), and in Rhodnius prolixus (WIGGLESWORTH,1937). Generally the wounded tissue, whether it is endodermal as in the midgut or ectodermal, regenerates and eventually completely reseals the wound. The haemocytes form an initial temporary seal at the wound site but apparently do not contribute to the regenerated tissue. Implantation of foreign objects into the haemolymph has been used by several authors to investigate the rdle of haemocytes. LAZARENKO(1925), using aseptic techniques, implanted sterile celloidin tubes into larvae of Orcytes nasicornis. After 94 days, an epithelium (‘des hypodermalen Epiteliums’) was formed around the tube, its source appearing to be either the epidermis of the integument or the tracheal epithelium. MATZ (1961a, b, 1963) has performed similar experiments with Locusta migratoria and Leucophaea maderae. However, an epithelium (pseudohypoderme; neo-hypoderme) formed only around cellophane which was not sterile. The source initially was attributed to oenocytoids and cells in the haemolymph, and subsequently to tracheal epithelium. Neither author described completely his sterile techniques nor used insects reared aseptically, and it is difficult to explain the conflicting results of Lazarenko and Matz except on the basis of species differences. Since a new epithelium is formed in certain salivary tumours after duct ligation, it was of interest to determine if a new epithelium formed around sterile and nonsterile cellophane implants in Periplaneta americana. Contra Matz, epithelium was formed around both non-sterile and sterile cellophane. In the latter, the

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cockroaches were not reared aseptically, but the integument around the implant area was washed in ethanol just prior to implantation. The only observed difference between the implants of sterile and non-sterile cellophane was the smaller number of haemocytes encapsulating the former. However, generally after 1 month, a new epithelial layer had been established. According to MATZ (1963), tracheal epithelium is the source of this layer in Leucophaea maderae. In Periplaneta growth of tracheae into the masses of haemocytes surrounding cellophane and tissue implants has been observed, supporting this view. In some sections the new epithelium appears to be continuous with that of an adjacent injured trachea. This is accompanied by the destruction of the side of the trachea closest to the cellophane implant. The new epithelium, however, lacks the taenidial lining of the trachea. In other cellophane implants bearing a new epithelium, no tracheae are observed and the epithelial origin is different. In such cases the epithelium arises from giant cells which develop in the area of large masses of basophilic substance. These generally occur at edges of the long axis of the cellophane where haemocytic encapsulation is greatest. The cells and their origin are deeply embedded in the encapsulating haemocytes, with no indication that they penetrate the tissue mass from some other source such as tracheae. The giant cells, probably containing polytene nuclei, bear some resemblance to those appearing in salivary acinar tumours. However, close examination reveals differences. The distinct layers in cellophane implants are (1) cellophane, (2) ‘chitinous’ (or eschar) layer originally cellular, (3) layer of cells with small nuclei, (4) dense basophilic layer, (5) epithelium, and (6) outer encapsulating haemocytes. Layers 3 and 4 are absent in salivary tumours but appear in foregut tumours induced by nerve lesion. Although tracheae may be the source of a new epithelium in some cellophane implants, other sources possibly exist. If the new epithelium is a true ectoderm, its source probably is not the mesodermal haemocytes. A more plausible source is ectodermal cells present in the haemolymph. Wound reaction vs. tumour development As stated previously, the reaction of insect tissues to mechanical wounding is characterized generally by a regeneration of the wounded tissue with haemocytes often functioning as a temporary seal of the wound area until regeneration is complete. The reaction to implanted materials is Iikewise a wound reaction if wounded tracheae are the source of new epithelium. If the source of the latter is certain cells in the haemolymph, the reaction is no longer due to wounding but instead to the presentation of a suitable solid substrate on which to base epithelium. In each, although the tissue is a response to a physical stimulus, undoubtedly biochemical factors are equally important as shown by WIGGLESWORTH (1937). In operations such as duct ligation, nerve lesion, and transplantation of tissue, the physical stimulus of operation is minor, and the ultimate tissue reactions are a response to a biochemical wound. In acinar tumours types 3 and 4 intercalated duct cells respond by proliferating, the hyperplasia being followed by the formation

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of giant probably multinucleate cells; subsequently an epithelium may be formed. This can, of course, be viewed as an attempt to repair the biochemical wound. However, in contrast to mechanical wounding, the repair of tissues is never fully attained. The zymogen and parietal cells are destroyed during tumour development and remaining cells are incapable of specialized development to replace them. More importantly, the new epithelium apparently is not identical with intercalated duct epithelium or ectoderm. As a result of mechanical and of biochemical wounding, haemocytic encapsulation of wounded tissues often takes place. Sometimes, as in acinar tumour type 3, haemocytic encapsulation does not occur. In Leucophaea, epithelial tumours are always invaded by haemocytes (histiocytes) according to MATZ(1961b). A major difference between encapsulation in physical wounding and in tumours due to biochemical wounding is that in the former the encapsulation is much more rapid, is in great part initially due to coagulation at the site of injury, and the haemocytes die or re-enter the haemolymph. In biochemical wounding, and also after injection of large particulate matter into the haemolymph, encapsulation is a slower process, the haemocytes adding gradually to the encapsulation, and orienting themselves around the area in a whirling pattern; eventually they die or re-enter circulation. Haemocytic encapsulation, therefore, remains as a defence mechanism of the insect, one which can act slowly or rapidly. The process by itself cannot be labelled as tumorous, nor can its presence or absence be a true indication of the presence or absence of tumorous tissues, at least in Periplaneta. This is,not only true of the salivary tumours described in this communication but also in rectal tumours induced by other means. Caution in the interpretation of haemocytic encapsulation should also extend to studies of metastasis of tumour transplants. This is particularly true of implantation into structures such as the salivary reservoir (MATZ, 1961a, Fig. 2), where apparent metastasis may be the result of operation injury or haemolymph clotting. Furthermore, in any classification of insect tumours, the term ‘haemocytic tumour’ should not be used to designate slow or rapid encapsulation; the term should be reserved for those cases where haemocytes actually have been shown to undergo tumorous change or where haemopoetic organs possibly are involved. At the current stage of research on insect tumours it is impossible to compare insect and vertebrate tumours. However, if a research stimulus is needed, one may superficially compare the abnormal growths of insect salivary glands with those of certain mammalian mammary glands as shown by DUNN (1959), and consider the occasional invasiveness of cockroach tumours (SCHARRERand LOCKHEAD,1950) and the recent indication that certain insect tumours are transmitted by nucleic acid extracts of tumours (MATZ et al., 1964). AckmwZedgmmts-The Miss

JEAN M.

TURENNE.

author

gratefully

acknowledges

the

valuable

assistance

of

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REFERENCES ARNOLDJ. W. (1959) Observations on living haemocytes in wing veins of the cockroach Blaberus &anteus (L.) (Orthoptera: Blattidae). Arm. ent. Sot. Am. 52, 229-236. CAMPBELLF. L., Ed. (1959) P~ytiZogy ofInsect DeveZopment. University of Chicago Press. COWDRYE. V. (1948) Laboratory Technique in Biology and Medic&, 2nd ed. Williams and Wilkins, Baltimore. DAY M. F. (1951) The mechanism of secretion by the salivary gland of the cockroach Periplaneta americana (L.). Aust.J. sci. Res. (B) 4, 136-143. DAY M. F. (1952) Wound healing in the gut of the cockroach Periplaneta. Aust. J. sci. Res. (B) 5, 282-289. DAY M. F. and BENNETSM. J. (1953) Healing of gut wounds in the mosquito Aedes aegypti (L.) and the leafhopper Orosius argentatus (Ev.). Au.st.J. biol. Sci. 6, 580-585. DUNN T. B. (1959) Morphology of mammary tumors in mice. In Physiopathology of Cancer 2nd ed. (Ed. by HOMBURGER F.), pp. 38-84. Hoeber, New York. ERMIN R. (1939) uber B au und Funktion der Lymphocyten bei Insekten (Perzplaneta americana L.). Z. Zellforsch.. 29, 613-669. GABE M. and ARVY L. (1961) In GlandCells (Ed. by BRACHETJ. and MIR~KY A. E.), Vol. 5, pp. l-88. Academic Press, New York. HARKERJ. E. (1958) Experimental production of midgut tumours in Per@aneta americana L. J. exp. Biol. 35, 251-259. HARKERJ. E. (1963) Tumours. In Insect Pathology (Ed. by STIUNHAUSE. A.), Vol. 1, pp. 191-213. Academic Press, New York. JONESJ. C. (1962) Current concepts concerning insect hemocytes. Am. Zoologist 2,209-246. K~~SEL R. G. and BEAMSH. W. (1963) Electron microscope observations on the salivary gland of the cockroach, Periplaneta amerixana. Zellforschung. 59, 857-877. L~NKO T. (1925) Beitrtige zur vergleichenden Histologie des Blutes und des Bindegewebes-II. Die morphologische Bedeutung der Blut- und Bindegewebeelement der Insekten. Z. mikrosk.-anat. Forsch. 3, 409499. MATZ G. (1961a) Tumeurs experimentales chez Leucophaea maderae F. et Locusta migratoria L. J. Insect Physiol. 6, 309-313. MATZ G. (1961b) Etude histologique des tumeurs experimentales chez Leucophaea maderae Fabr. et Locusta migratoria L. Bull. Sot. Zool. Fr. 86, 148-156. MATZ G. (1963) Reactions inflammatoires, cicatrisation et candrigenese chez les insectes. Bull. Sot. Zool. Fr. 88, 650-662. MATZ G., WEIL J. H., JOLY P., and EBEL J. P. (1961) Transmission de tumeurs chez les Insectes par un acide nucleique extrait des tumeurs. C. R. Acad. Sci., Paris 258, 4366-4368. NOLANDJ. L. and BAUMANNC. A. (1949) Effects of certain azo dyes upon the cockroach Bluttella germanica. Proc. Sot. exp. Biol. Med. 71, 365-368. SCHARRERB. (1945) Experimental tumors after nerve section in an insect. Proc. Sot. exp. Biol., N.Y. 60, 184-189. SCHARRERB. (1953) Insect tumors induced by nerve severance: incidence and mortality. Cancer Res. 13, 73-76. SCHARRERB. and LOCKHEADM. S. (1950) Tumors in the invertebrates: a review. Cancer Res. 10, 403-419. SCHLIJMBERGER H. G. (1952) A comparative study of the reaction to injury-I. The cellular response to methylcholanthrene and to talc in the body cavity of the cockroach (Periplaneta americana). Arch. Path. 54, 98-113. SCHORTINO T. J., C ANTWELLG. E., and ROBBINSW. E. (1963) Effect of certain carcinogenic 2-fluorenamine derivatives on larvae of the housefly, Musca domestica Linnaeus. g. Insect Path. 5, 489492.

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SUTHERLAND D. J. (1963) Experimentally induced tumours in Periplanetu arne~C~~na(L.). J. Insect Physiol. 9, 131-135. SUTHERLAND D. J. (1967a) Crop tumors induced by nerve lesion in Perifdaneta americana (L.). J. Inoert. Path. In press. SUTHERLAND D. J. (1967b) Effect of certain carcinogens on Peripluneta americana (L.). In preparation. WIGGLESWORTH V. B. (1937) Wound healing in an insect (Rhodnius prolixus Hemiptera). J. exp. Biol. 14, 364-381.