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Formation of tumorlike lesions in the cockroach Leucophaea maderae after nerve severance
JOURNAL
OF
INVERTEBRATE
Formation
PATHOLOGY
of Tumorlike
13,
167-187
Lesions
(1969)
in the Cockroach
after Nerve
Leucophaecs
mderae
Severance1
RONALD L. TAYLOR Center
for Pathobiology,
University Received
of CalifornZa, August
19,
Irvine,
California
92664
1968
Severance of the recurrent nerve in the Madeira cockroach, Leucophaea maderae, induced the formation of lesions in the alimentary canal-lesions which were similar to those obtained by earlier investigators using the same technique on the same animal. Whereas these have been generally considered to be neoplasms, they are interpreted here to be inflammatory lesions. Recurrent nerve severance prevents passage of food from the crop to the midgut and, since feeding apparently is not inhibited, results in an abnormally engorged foregut. The hypothesis is made that the resultant stagnation throughout the alimentary canal provided ideal circumstances for the proliferation of certain microorganisms-microorganisms which infected the gut epithelium, which was stressed and possibly injured by the overdistension and/or accumulated secretions. Regardless of the involvement of microorganisms (which were involved in many but apparently not all lesions), blood cells infiltrated the injured areas and established inflammatory foci. The presence of blood cells in the lesions was established both by light and electron microscopy. Additional observations are made on the histology and fine structure of the lesions and on other possible mechanisms by which denervation may induce tumorlike lesions.
INTRODUCTION
tary canal (esophagus, crop, gastric ceca, anterior midgut) and the salivary complex ( salivary glands and salivary reservoirs). Following the same procedure, Matz (1963b) induced similar lesions in the grasshoppers Locusta migratoria and Schistocerca gregaria, the dragonfly Aeschna stick Dixippus c yanea, and the walking morosus. Subsequently, Hema (1966) and Sutherland (personal communication to Matz, 1966) induced “tumors” in the cockroach Peripluneta americana although earlier Sutherland ( 1963) and Scharrer (personal communication to Sutherland, 1963) were unable to do so. Hema (1966) also induced “tumors” in the cockroach Stylopyga sp. Not all workers have been as successful at inducing lesions by nerve severance as have those authors cited above. Johansson and Schreiner (1966) found cryptogenic
In the Madeira cockroach, Leucophaea maderae, the recurrent nerve, which belongs to the autonomic nervous system, originates in the frontal ganglion, passes between the brain and pharynx, is intimately associated with the corpora cardiaca, gives off branches to the salivary glands and salivary reservoir, divides into two main branches in the thoracic region, and finally ramifies into thin branches which appear to terminate in the region of the gastric ceca (Scharrer, 1945). By severance of this nerve in L. maderae, Scharrer ( 1945, 1949, 1953a,b) and Matz (1961) induced lesions which they interpreted to be neoplasms in the anterior portion of the alimen1 This investigation was supported by Public Health Service Research Grant No. 5358 (to E. A. Steinhaus) from National Institute of Allergy and Infectious Diseases.
“tumors” 167
in
the
alimentary
canal
of
the
168
TAYLOR
German cockroach, Blatella germanica, and reported that recurrent nerve severance had no effect on the frequency of their occurrence. Edwards et al. (1967) induced lesions in the salivary complex by ligation of the salivary ducts of the cockroaches P. americana, Blabera gigantea, and Gramphodorina portentosa, and noted that severance of the recurrent nerve had no influence on the frequency of their formation. Other workers have severed recurrent nerves in non-tumor-related research. For example, Day (1951) and Mills (1967) severed the recurrent nerve in P. americana; Engelmann (1966) severed the nerve in L. maderae; Fraenkel and Hsiao (1962) severed it in the blowfly Calliphora, and Dethier and Gelperin (1967) in the black blowfly, Phormia regina. As noted above, however, these experiments were not in connection with “tumor” induction, and no reference was made to lesions resulting from the nerve intervention. The only other nerves severed in “tumor” induction studies were the efferent splanchogenital nerves of the last ganglion in the ventral nerve cord of L. maderae and L. migratoria (Matz, 1963a,b). In these cases, lesions developed in the posterior part of the midgut, the hindgut, and the genital ducts. Although the lesions obtained by nerve severance are commonly called “tumors,” as if they bore a strict homology to vertebrate neoplasms, their precise nature is far from determined. It was in an attempt to clarify their nature that the work reported here was undertaken. MATERIALS
AND METHODS
For these experiments Madeira cockroaches (Leucophaea maderae) that had been adults for 4 to 82 days were first immobilized by hypothermia. Using aseptic techniques the anterior portion of the crop was exposed, and one branch of the re-
current nerve was severed in 31 cockroaches (18 females and 13 males) and both branches in 14 cockroaches (8 females and 6 males). All of the animals were maintained at 2627°C and 60-75% relative humidity. Roaches were dissected and observed for pathological changes at specified intervals or while moribund (or dead if necrosis hadn’t progressed too far). In order to determine the incidence of naturally occurring lesions, 25 male and 25 female cockroaches were examined at an adult age of 2 weeks to 9 months. Tissues for light-microscopic examination were fixed in Zenker’s fluid, dehydrated via ethanol, embedded in Paraplast, sectioned at 610 u, and stained with hematoxylin and eosin. For electron-microscopic examination tissues were fixed for 1.5 hr in cold Dalton’s chromeosmium tetroxide at pH 7.2. Embedding was done in Epon after dehydration in ethanol. Sections were cut with a Sorvall MT-2 Porter-Blum ultramicrotome equipped with glass knives and were then treated with uranyl acetate and lead citrate to enhance contrast. An RCA EMU-3G electron microscope was used for examining the sections. RESULTS
Naturally Of
Occurring Lesions
the control (unoperated) animals (10 males and 12 females) had no lesions of any kind. Some of the animals which did have lesions commonly had more than one type. Twenty-six roaches (52% ) had pigmented lesions of the Malpighian tubules of the type described below under “Malpighian Tubule Lesions.” In any one roach pigmentation generally occurred in only a few tubules and then only in a portion of the affected tubules; pigmentation varied from a light brown to black. Small pigmented masseswere seen in the fat body of 14% of the roaches (3 males and 4 females). Tumefactive ( = tumorlike) lesions
447,
LESIONS
LN COCKROACH
were observed in 8% of the roaches. One male was found with six pigmented, tumorlike lesions in the wall of the hindgut and a pigmented lesion in the wall of a salivary reservoir. The latter lesion was surrounded by a whitish, opaque mass which histological sections of similar lesions have revealed to be infiltrating hemocytes in the process of encapsulating necrotic material. Two females had pigmented tumefactive lesions in the wall of the esophagus, one with two such lesions and the other with six. A final animal was observed to have pathological ovaries. Experimentally
Induced
Lesions
Mortality resulting from the surgical procedures generally occurred within the first 5 days after nerve severance. Such roaches died of a septicemia caused by Serratia marcescens, a bacterium which is a normal inhabitant in the alimentary canal of the cockroaches in our colony of L. maderae. It is harmless until it gains access to the hemocoel and then it multiplies rapidly, resulting in the rapid death of the host. No lesions could be found in the roaches dying within this s-day period. Mortality during this period was clearly higher among the males; whereas only 19% of the females died within the first 5 days post-nerve severance, 84~~ of the males died within this period. My experience has shown that female L. maderae in general, regardless of the treatment, are hardier than males. Mortality was also higher among those cockroaches with both branches of the recurrent nerve severed than among those with only one branch severed. It is possible though that this result may be due to the additional trauma induced by the search for and severance of the second branch, rather than to a direct result of the nerve interruption. Of the initial 45 cockroaches with severed nerves, 24 lived beyond 5 days and, as two
AFTER
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SEVERANCE
of these were too necrotic for dissection, only 22 roaches were considered in this study. Nineteen of these roaches were female (14 had one branch of the recurrent nerve severed and 5 had both branches severed) and 3 were males (all with one branch severed). Eight of these roaches died at periods from 6 to 195 days postnerve severance for various reasons (e.g., cannabilism and septicemia) and were examined as soon after death as possible. The other 14 were sacrificed and examined at intervals from 30 to 195 days post-nerve severance. Lesions resembling (even remotely) tumorlike masses were found in nine roaches, with some roaches having more than one lesion. Five roaches were found with lesions of the salivary reservoir, five with lesions of the crop, two with lesions of the hindgut, and two with lesions of the tracheae. Also, many roaches were found with darkened (brown to black) Malpighian tubules and/or with numerous small brown to black spots evenly spaced throughout the fat body. These Malpighian tubule and fatbody lesions have all the characteristics of simpIe encapsulation reactions and are not considered tumorlike. Due to the small number of animals involved in the experiments, no significant relationship can be established between the incidence of lesions, the number of branches of the recurrent nerve severed, and the sex of the animals. It should be noted that only 417, of the roaches surviving beyond 5 days developed tumorlike lesions, which is little more than half the incidence obtained by Scharrer (1945) and Matz (1961). Their data, however, are based on hundreds of nerve severances and therefore should be statistically more valid. Malpighian
Tubule
Lesions
The Malpighian tubules in operated animals are frequently enlarged, darkened (brown to black), and brittle. The number
TAYLOR
KEY
FOH
CR, blood chondrion;
ALL
FIGUHES:
cell granule; NC, nucleolus;
BB, HC, NU,
fibrous banded body; CU, cuticle; hemocoel: HE, hemocyte( s); IM, nucleus; TM, tunica muscularis.
EP, epithelimn; CL, intercellular material;
gut lumen; MI, mito-
LESIONS
IN
COCKROACH
of tubules affected in any one animal varies considerably. In some animals only a few (or portions of a few) are affected whereas in others the majority are affected; in the latter instance the animals’ normal excretory processes would undoubtedly be severely impeded. Microscopic examination shows that the normal cellular architecture is completely destroyed (Figs. 1, 2). In cross section the pathological tubules are seen to consist of a sometimes granular, sometimes hyaline central region which is either eosinophilic or amber-colored (yellow to brown) or a mottled mixture of both. This central region is often surrounded by a thick, well-delimited, dark amber-colored “ring” containing nuclei in various stages of pyknosis, karyorrhexis, and karyolysis. These central necrotic regions are encapsulated by two or more layers of spindleshaped hemocytes with elongated nuclei. Two or more Malpighian tubules are often loosely linked by blood cells among a basophilic fibrous network (Figs. 1,2). The blood cells of the network, which are apparently responsible for the deposition of the fibers, are not all alive; many are in various stages of necrosis. As they die, as do most animal cells, they become increasingly eosinophilic. Their nuclei undergo pyknosis, and finally karyolysis often karyorrhexis, until they completely disappear. The dead blood cell is an eosinophilic mass of coagulated protein, which in some lesions may lose its eosinophilia, become refractory to
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stains, and finally become a hardened, amber-colored mass. Although Malpighian-tubule lesions occur in unoperated animals, the incidence of the lesions is higher and more tubules are affected in experimental animals. An increased incidence of Malpighian-tubule lesions also results from treatments other than nerve severance (Taylor, 1969), and therefore in the present case is not believed to be a direct result of the nerve severance. The Malpighian tubules are apparently sensitive indicators of disturbances in the animal’s homeostasis. Exactly why they should necrotize is not known. Fat-Body
Lesions
The fat body in experimental animals is frequently speckled with minute brownblack spots. In histological section, these spots are seen to consist of a central, necrotic, amber-colored hyaline core encapsulated by several layers of spindle-shaped blood cells with elongated nuclei and dense eosinophilic cytoplasm (Fig. 3). Some pyknotic and karyolytic nuclei can be seen within the central core. At the periphery of the capsule the blood cells become more rounded and merge with the surrounding fat body. Similar lesions, although in lower incidence, were found in unoperated animals. They have also been found in animals receiving injections of various materials, the incidence somehow varying with the nature and quantity of injected material (unpub-
1, 2. Cross sections of Malpighian tubule lesions. Note central regions of necrosis encapsuFIGS. 1-S. lated by hemocytes. Also note hemocytes linking neighboring, necrotic tubules. 120 x and 290 X, respectively. 3. Section through a typical fat-body lesion consisting of a central necrotic core encapsu4. Section through a tracheal lesion. Fragmented tracheal intima (arrows) lated by hemocytes. 290 X. is surrounded by unidentified cells, which are in turn encapsulated by a thin layer of hemocytes. 150 X. 5, 6. Section through an apparently old lesion (delimited by arrows) in the wall of the hindgut. See text 7. Section through a crop lesion showing the for description of lesion. 40 x and 470 X, respectively. hypertrophied epithelium, the thickened cuticle, and the cuticlelike material-apparently secreted by the 290 X. 8. Gross appearance of cockroach epidermis-which is enclosing microorganisms (arrow). with dorsal body wall removed exposing greatly distended crop and two tumorlike lesions (arrows). Figures 11 through 21 were prepared from the lower lesion shown here. Approximately actual size; scale is in centimeters.
I72
TAYLOR
lished observations). It is my belief that small foreign particles, microorganisms, or #dead cells become encapsulated by blood cells which subsequently become affixed to the fat body. Once pigmented and hardened the encapsulated material is effectively isolated. Encapsulated
Trachea
Tracheae were frequently severed during the nerve severance procedures and the cut ends were subsequently encapsulated. In histological section the tracheae appear fragmented ( Fig. 4). Their cuticular intima is eosinophilic except near the lumen where it is amber-colored. The normal tracheal intima is not so darkened. The tracheae are surrounded by rounded and highly vacuolated cells with irregularly shaped nuclei and distinct cell walls. The origin and nature of these cells was not determined although it is suggested that they may be hyperplastic tracheablasts. These latter cells are completely encapsulated by spindleshaped hemocytes with elongate nuclei and dense eosinophilic cytoplasm. Numerous fibers are present within the capsule. Hindgut
Lesions
Hindgut lesions were observed in two animals. One lesion consisted of typical amber-colored material and blood cells in various stages of necrosis. The entire mass was adhering to the muscularis and did not seem to involve the gut epithelium. Two essentially identical lesions were found in the hindgut of a second animal. Juxtaposed to a relatively normal appearing cuticle and its underlying epithelium is a scablike mass of completely necrotic tissue which is separated from the gut lumen by an amber-colored second cuticle (Figs. 5, 6). The portion of the scablike layer nearest the integument is eosinophilic, whereas that in contact with the second cuticle is ambercolored and is refractory to stains. The gut wall is folded in all areas except that of the
lesion, where such folding is apparently prevented by the amber-colored portions of the lesion which are hardened. Such hardening possibly results from a process similar to sclerotization in the normal cuticle. This lesion can be interpreted as the healed remains of a lesion that may have existed at one time in the integument. Through normal wound-healing processes ( Lazarenko, 1924; Wigglesworth, 1937; Day, 1952) the epithelial cells peripheral to the lesion migrated beneath the dead and dying cells until a continuous sheet of epithelial cells had formed. The new epithehum then secreted a normal cuticle, thereby isolating the necrotic material between the new and the old cuticles. I have observed that hardening and darkening commonly, though not always, occurs in necrotic tissues. The darkening of the old cuticle is possibly due to the diffusion from dying blood cells of substances responsible for hardening and darkening. In a hindgut lesion induced by an oxyuroid nematode (Taylor, 1968), I noted darkening of the cuticle over the lesion, and where folds in the gut epithelium brought the cuticle of normal epithelium in contact with (or in close proximity to) the abnormally darkened cuticle over the lesion, the outer portion of the normal cuticle darkened also. Foregut
Lesions
No lesions were found in the esophagus of experimental roaches although crop lesions were found in 5 animals. Inexplicably, nearly the opposite situation occurred in unoperated animals, viz., no crop lesions and two esophageal lesions were observed. The first of two crop lesions to be described consists in part of hypertrophied epithelial cells with enlarged nuclei (Fig. 7). The cuticle over these cells is thicker and more basophilic than normal. Adhering firmly to the cuticle in the gut lumen is an amber-colored, hardened, hyaline material which appears to be cuticular in nature.
LESIONS
IN
COCKROACH
Unidentified microorganisms are enclosed in pockets in this material. Perhaps for some unknown reason the cells became hyperactive (thus their hypertrophied appearance) and secreted excess cuticle (the amber-colored material) which inadvertently incorporated the microorganisms. Alternatively, perhaps the cells became hyperactive in response to the microorganisms and secreted the abnormal cuticle as a defense mechanism. Although the size of a cockroach’s crop varies with the quantity of food it contains, it is often abnormally enlarged after recurrent nerve severance. The crop of one roach approximately 4 months post-nerve severance was greatly distended with food, appearing approximately three times larger than normal (Fig. 8). Several tumescent, whitish lesions could be clearIy seen on the surface of the crop. The two largest ones measured 2 and 3.5 mm in diameter. In contrast with the norma situation the cuticle involved in the lesions was hard, brown-black, and shiny. One-half of the second largest lesion was removed and processed for electron microscopy, and the other half, including the remainder of the crop, was processed for light microscopy. For purposes of comparison the histological appearance of normal crop is shown in Figs. 9 and 10. The cuticle in the lesion in contrast to the normal situation is ambercolored (Figs. 11-13). Next to the cuticle is an alveolar or spongy appearing layer which extends across most of the lesion and which is hard and pigmented similarly to the cuticle (Fig. 13). Adjacent to the cuticle are pockets of at least two different types of microorganisms (Fig. 12), one of which is clearly a bacterium. Enclosing these pockets is a region of dying blood cells with pyknotic and karyolytic nuclei. This region is eosinophilic in areas most distant from the microorganisms, and, progressing towards the pockets of microorganisms, it first becomes refractory to staining
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and then amber-colored. These changes are believed to represent progressive stages in the death of the blood cells. This region is sharply delimited from surrounding tissues by a dark amber-colored layer (Fig. 11) . The remainder of the lesion consists of hyperplastic epithelial cells (or epithelioid infiltrating spindle-shaped blood cells), blood cells, and widely separated and broken up muscles of the tunica muscularis. Only one mitotic figure was seen among the hyperplastic epithelial cells ( Fig. 14). The ultrastructure of the tunica muscularis of this lesion has been described and compared with normal crop muscle (Taylor, 1967). To summarize briefly, the Z lines appeared fragmented and irregular in width and the myofilaments in the region of the I bands appeared disoriented (Fig. 15); there was no well-developed sarcoplasmic reticulum and no distinct mitochondria although there were numerous oval-shaped vacuoles containing sparse flocculent material which may represent disintegrating mitochondria; most significant was the finding of fibrous banded structures between groups of abnormal myofibrils. These fusiform-shaped structures were not present in normal crop muscle and were of uncertain significance. For a more complete description of this pathology and a discussion of the possible collagenous nature of the fibrous banded bodies, see Taylor ( 1967). The normal epithelial cells are rich in glycogen, endoplasmic reticulum, and Golgi zones-characteristics of cells of high metabolic activity. The normal ultrastructure is similar to that described for the gut of other insects (Beams and Anderson, 1957; NoirotTimothke and Noirot, 1965; Staubli et al., 1966). Considerable necrosis was evident in the pathological “epithelium.” The chromatin material of many cells was for the most part clumped along the nuclear membrane (Figs. 16, 17). Interchromatinic granules were aggregated along the interface between the chromatin and the inter-
174
TAYLOR
I?IGS. 9-14. 9, 10. Cross section through normal micrographs of lower crop lesion shown in Fig. 8. tion through lesion at different level from Fig. rounded by dying and dead hemocytes. 1’20 X. layer next to cuticle. 290 X. 14. Section through
crop. 40 x and 290 X, respectively. 11-14. Light 11. See text for description of lesion. 40 X. 12. Sec11. Note pockets of microorganisms (arrows) sur13. Section through crop lesion showing “alveolar” crop lesion showing mitotic figure. 1050 X.
LESIONS
IN
COCKROACH
Figures 15 to 21 are electron FIGS. 15, 16. structure of tunica muscularis showing fibrous rows ) 23,900 X. 16. Most necrotic portion swollen and barely recognizable mitochondria,
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micrographs of lower lesion shown in Fig. 8. 15. Ultrabanded body. Note also disoriented myofilaments (arof lesion. Note clumping of chromatin in nuclei, and numerous vacuoles and vesicles. 7150 X.
176 chromatic substance. The extremely prominent in cells and contained a fine rial. Numerous vacuoles
TAYLOR
latter material was the most necrotic filamentous mateand vesicles were
present in the necrotic cells (Figs. 16, 18), some of which contained sparse filamentous material of unknown composition. The origin of the vacuoles and vesicles is not clear
FIGS. 17, 18. 17. Note clumping of chromatin in nucleus. Also with distinct granules. 8850 X. 18. Note vacuoles, vesicules, abnormal latter is recognized by its distinct cytoplasmic granules. 5450 X.
note “round” mitochondria,
blood cell packed and hemocyte. The
LESIONS
IN
COCKROACH
although some probably resulted from pathological changes in the endoplasmic reticulum. The mitochrondria are barely recognizable in most cells, appearing similar to the mitochrondria of the tunica muscularis, i.e., they are swollen, their cristae are disrupted, and they contain a sparse flocculent material (Figs. 16, 18). In some of the less necrotic cells, viz., those cells nearest the muscularis, small accumulations of glycogen granules are present (Fig. 19). Some, though not many, myelin figures are present in the abnormal tissue (Fig. 20) ; their presence is indicative of membrane degeneration. A constant feature of the lesion at all levels of the epithelium is the presence of hemocytes, which are identified by their distinct cytoplasmic organelles (Figs. 17-19, 21). (A more detailed description of these blood cell organelles and their possible function will form the subject of a subsequent publication. ) Often these are the only cells retaining normal architecture within an otherwise degenerating area. The organelles are often seen loose within the degenerating area but demonstrate no signs of degeneration themselves. Salivary
Reservoir Lesions
Being diverticulae of the foregut, the salivary reservoirs are of ectodermal origin and constructed similarly to the foregut. The tunica muscularis, however, consists of only scattered muscle fibers, and the cuticle is loosely lamellated, apparently providing for slippage as the reservoir enlarges and shrinks. The reservoir is completely transparent when filled with saliva. Lesions of the salivary reservoir were found in five roaches, an incidence considerably higher than in unoperated animals. Inexplicably, all lesions occurred in the right reservoir only. Two of these lesions are described below. One cockroach was found to have a pigmented (brown-black) spot, 0.5-l mm diameter, in the wall of the right salivary
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reservoir. A small (about 1 mm long), whitish, lobular mass was suspended from the pigmented spot into the saliva. Several smaller whitish lesions (some with brownblack pigment spots) were also present on the same reservoir, one of which was processed for electron microscopy. In histological section, the larger lesion is seen to be quite complex (Fig. 22). A thin layer of blood cells is adhering firmly to the epithelium ( Figs. 22 , 23 ) . Some of the blood cells are in small whorls around amber-colored cores of hyaline material. Immediately adjacent to the cuticular intima of the reservoir is a layer of loosely adhering cells with large nuclei; these cells, the nature of which was not determined, are situated in a fibrous network. Adjacent to this region is a scablike layer consisting of completely necrotic, hardened and darkened (amber-colored) blood cells which merges gradually with a region of eosinophilic blood cells containing pyknotic and karyolytic nuclei. Some of the cells contain small granules of amber-colored material. At the periphery of the lesion, the cuticle appears to separate-one portion continuing over the epithelial cells beneath the lesion in a relatively normal manner and the other portion passing over the lesion. The lobular mass projecting into the saliva consists of large numbers of an unidentified microorganism trapped between widely separated lamellae of the cuticle (Figs. 23,24). The microorganisms were spherical, ca. 1 p in diameter, and transparent with asymmetrically thickened walls. The means by which the lesion came to assume the above form is not known although one may speculate that it developed in a manner similar to the following: The microorganisms somehow invaded the reservoir wall and established an inflammatory focus disrupting the normal continuity of the epithelium. The cuticle, being flexible and nonliving, was damaged only slightly if at all. Normal wound repair processes
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TAYLOR
FIGS. 19-21. 19. Region of lesion bordering the muscularis. Hernocytes, glycogen (arrow), and numerous mitochondria can be seen. 5450 X. 20. Myelin figure in necrotic tissue. 81,900 X. 21. Blood cell granules. The presence of these organelles in tissue indicates the presence of hemocytes. 14,700 X,
FIGS. 22-25. Figures 22-24 are light micrographs of salivary reservoir lesion. 22. Section through center of lesion. Normal salivary wall appears in lower left portion of photograph. Detail of lesion is better shown in .Fig. 23. 120 X. 23. Section through entire lesion showing part of “sack” of microorganisms (arrow) projecting into reservoir lumen. See text for description of lesion. 290 X. 24. Unidentified microorganisms in “sack” shown in Fig. 23. 1170 x . Figures 25-30 are electron micrographs of salivary reservoir lesion immediately adjacent to lesion shown in Figs. 22-24. 25. Note irregularly shaped nuclei, large dense chromatin aggregates, blood cell granules, irregularly shaped mitochondria, and apparent contraction of nuclear contents away from nuclear membrane. Also note the amorphous, finely granular and fibrillar intercellular material. 6100 x .
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TAYLOR
began whereby the epithelial cells multiplied around the lesion and migrated beneath it, eventually reestablishing continuity. When the new cuticle was secreted, the wound healing process was essentially complete with the necrotic tissue effectively cast off although still contained between the old and new cuticles. This situation would give the appearance at the edges of the lesion of the cuticle having “separated.” The microorganisms, apparently not contained, and feeding in the saliva, continued to multiply between the lamellae of the old cuticle, forming the lobular mass described earlier. This interpetation is purely speculative although it fits the observations and the phenomena known to occur in wound healing ( Wigglesworth, 1937). The ultrastructure of the neighboring salivary-reservoir lesion differs considerably from that of the crop lesion already described. The only similarity is the involvement of blood cells in both lesions. Two cell types are present in the salivary-reservoir lesion and their plasma membranes are fragmented and highly irregular. One cell type has a high nuclear-to-cytoplasmic ratio (Figs. 25, 26). Its nucleus is irregularly shaped and consists almost entirely of large dense chromatin aggregates between which is a less dense interchromatic substance containing randomly distributed interchromatinic granules. The nuclear contents appear to have condensed or contracted away from the nuclear membrane on one or more sides, forming large electron-lucid vacuoles. (At lower magnifications these nuclei would probably appear pyknotic. ) The cytoplasm of these cells contains irregularly shaped mitochrondria, free ribosomes, and numerous vesicles. The other cell type contains a nucleus with a highly irregular and indistinct nuclear membrane and with chromatin evenly distributed throughout the nucleoplasm, i.e., there are no variations in density of the nuclear sap (Figs. 27, 28 ) . Nucleolar material can be seen in the
nucleus in some sections. The prominent features of the cytoplasm are numerous vesicles, mitochondria, and blood cell granules. (These are the same granules that are seen within the blood cells of the crop lesion. ) Also present within the cell are various-sized deposits of a membranebounded material that is relatively dense and finely granular and fibrillar. This material is also present as large interconnecting deposits between the cells of the lesion (Figs. 25-27). The blood cell granules appear to play a role in the formation of this material as they can be seen in what appear to be transitional stages. They appear to lose their internal structure and to coalesce with other similar granules, forming membrane-bounded deposits within the cell (Figs. 29, 30) ; these then fuse with the plasma membrane and extrude their contents. It should be noted that the mitochondria of these cells are not swollen or with indistinct cristae as in the crop lesion. The blood cell granules are morphologically very similar to granules in the hemocytes of Limulus which are involved in the clotting process (Dumont et al., 1966). Perhaps a process akin to clotting has taken place within this lesion. It is interesting in this connection that when Day (1952) injected an anticoagulant (0.01 M ascorbic acid) into P. anrericana, the number of hemocytes infiltrating and becoming involved in a wound was reduced. The suggestion here is that clotting or a related phenomenon occurs normally at some stage in the wound repair process. This phenomenon may explain the adhesion of hemocytes at an injury site. The electron-microscopic observations on the salivary reservoir lesion cannot be reliably compared with the light-microscopic observations on the neighboring lesion. The two lesions appeared different grossly, the Epon-embedded lesion lacking the pigmentation as well as the lobular mass which
LESIONS
FIGS. 2628. Note shaped mitochondria, membranes. Compare
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apparent contraction of nuclear contents away from nuclear membrane, irregularly and intercellular material. 9300 X . 27, 28. Note nuclei with indistinct nuclear with nuclei in Figs. 25 and 26. Also note intercellular material. 9300 X.
182
FIGS. 29, 30. Suggested stages granular and fibrillar intercellular 16,100 x.
TAYLOR
in transition of the material: I (earliest
blood stage)
cell granules through IV
into (fully
the amorphous, finely transformed granule).
LESIONS
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COCKROACH
hung into the salivary reservoir of the Paraplast-embedded specimen. The largest salivary reservoir lesion found measured 1.5-2 mm in diameter and approximately 0.5 mm in depth. Grossly, it appeared black and tracheated. In cross section, this lesion is seen to consist of several lavers or regions which merge more or less gradually into each other ( Figs, 31-34). The portion of the lesion nearest the cavity of the reservoir is completely necrotic, hardened, and darkened (amber colored), and includes the disrupted lamellae of the cuticle (layer A, Fig. 32). This layer merges with a strongly eosinophilic layer of dying blood cells containing pyknotic and karyolytic nuclei (layer B, Figs. 32-34). Next is a single layer of large weakly staining cells with large nuclei, followed by a layer of loosely adhering spindle-shaped cells (layers C and D, respectively, Figs. 32-34). The latter layer separates those parts of the lesion described above from the main mass of the lesion, which appears to consist entirely of blood cells (layer E, Figs. 32-34). Many of these cells are in “whorls,” some of which have encapsulated necrotic cells and amber-colored deposits. Tracheoles are present within this living mass of blood cells. Those cells nearest the hemocoel contain eosinophilic granules of diverse sizes. The incomplete single layer of large cells between the eosinophilic mass of dying blood cells and the loosely adhering spindleshaped cells are possibly epithelial cells. After injury the epithelial cells around a lesion will migrate through the inflammatory mass until epithelial continuity is restored (Wigglesworth, 1937). Cuticle is then secreted, resulting in the isolation of the damaged and necrotic tissues from the hemocoel. In the case of the lesion here described, epithelial continuity has not been completelv established, and new cuticle has not been deposited. The spindle-shaped cells immediately adjacent to this layer may be forming a new basement membrane.
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183
DISCUSSION
Vertebrates and invertebrates alike undergo localized tissue reactions to injury or injurious agents, and with this as our definition of inflammation (Steinhaus, 1949) we are able to apply the term to all animals. Admittedly, invertebrate inflammatory lesions are not inflamed in the vertebrate sense of the word (i.e., red or hot) although I know of no study to see if there is an elevation of temperature in an invertebrate lesion. I will use the term inflammation whenever referring to localized tissue reactions to injury or injurious agents (including reactions to microorganisms, parasites, foreign bodies, etc.). The difficulties attendant to the differentiation between neoplasia, inflammation, and wound repair in invertebrates are commonly voiced. One of these difficulties has to do with the fact that lesions of diverse etiology frequently consist, at least in part, of cells which differ significantly in general morphology and cytology from any of the animal’s “normal” cells. These “abnormal’ cells, it is therefore commonly believed, must be neoplastic. Hemocytes, however, infiltrate various inflammatory and wound repair processes, and in so doing alter their form. Day ( 1952), for example, studied wound healing in the gut of the cockroach, P. americana, and noted that the hemocytes “ . . . undergo striking changes in shape when they become incorporated into the wound tissue. _ . . The hemocytes may become flattened, elongate, or spheroidal, depending upon their position.” Wigglesworth ( 1937) studied wound healing in the body wall of Rhoclnius ~rrolixus and noted that in the later stages of the wound healing process it was impossible to distinguish the infiltrating hemocytes from the hyperplastic epidermal cells. Schlumberger ( 1952) noted the epithelioid character of hemocytes in a reaction to injury in the cockroach, P. americana, and Dawe et al. (1967) made a
184
TAYLOR
similar observation in the cockroach, L. maderae. Therefore the presence of cells in a lesion which differ significantly in morphology from the normal hemocytes and from
normal cells of the affected tissue is not sufficient evidence for neoplasia. Also, one must be careful in the interpretation of certain cytological character-
FIGS. 31-34. Large salivary reservoir lesion at increasing magnification. 40 X, 150 X, 290 X, 470 X, respectively. 31. Section through entire lesion, 32-34. A, Completely necrotic, hardened, and amber-colored portion of lesion; B, strongly eosinophilic layer of dying blood cells; C, single layer of large, weakly staining cells believed to be regenerated epidermal cells; D, layer of spindle-shaped cells believed to be forming a new basement membrane; E, healthy aggregated hemocytes.
LESIONS
IN
COCKROACH
istics commonly associated with neoplastic cells. Wigglesworth ( 1937), for example, observed the following conditions in wound tissue in R. prolixus: numerous mitoses, polyploidy, irregular mitoses, greatly enlarged nuclei, multipolar mitotic figures, pyknotic nuclei, etc. Wigglesworth and Schlumberger occasionally observed hyperplastic accumulation of epithelial cells at certain stages of the wound healing process. In P. americana. injected with talc or methylcholanthrene, Schlumberger observed (although rarely) ova whose follicle cells had “. . . enlarged to form giant cells with bizarre, irregular pyknotic nuclei. . , .” Although the lesions studied by the abovecited authors had many characteristics suggestive of neoplasms, they clearly were not. Jones ( 1969) noted the involvement of hemocytes in a variety of anomalous lesions reported originally to be tumors. The point here is to emphasize the difficulty of differentiating between neoplastic and inflammatory lesions and to suggest the possibility that many authors understandably may have described lesions as neoplastic which were in fact inflammatory. When the difficulty exists, the simplest possible interpretation of the lesions which is consistent with the data should be sought. The simplest interpretation of the lesions described in this report is that they represent processes of inflammation and repair. The evidence does not demand, or even strongly suggest, that the lesions be interpreted as having resulted from a neoplastic transformation. Assuming that they are inflammatory foci, the question is immediately raised, “What is the possible connection between severance of the recurrent nerve and the development of inflammatory lesions, especially at locations distant from the point of surgical intervention?” The answer to this question, I think, is not too difficult. Dethier and Gelperin ( 1967) have recently shown that the black blowfly, Phormia Fegina, overeats when the recurrent nerve is
AFTER
NERVE
SEVERANCE
185
severed. Their work indicates that severance of the recurrent nerve may prevent the brain from receiving impulses from stretch receptors in the gut, impulses which are necessary to inhibit feeding behavior after the animal has sufficiently fed. Davey and Treherne (1963) and Englemann (1966) have shown that severance of the recurrent nerves in P. americana and L. maderae prevents passage of food from the crop into the midgut. If after recurrent nerve severance the cockroach both overeats and fails to pass the food on to the midgut, the result would be an abnormally engorged foregut. Scharrer (1945) herself noted that the anterior portion of the alimentary canal in nervesevered cockroaches was frequently filled with an abnormally large quantity of food. As noted earlier in this paper, I have made the same observation. One can hypothesize that the resultant stagnation throughout the alimentary canal would provide ideal circumstances for proliferation of certain microorganisms. These microorganisms might then infect the gut epithelium, particularly when it is stressed and possibly injured by overdistension and/or accumulated secretions. I noted microorganisms associated with many of the lesions. However, regardless of the involvement of microorganisms, blood cells would be expected to infiltrate such injured areas and establish inflammatory foci. An interesting test of the above hypothesis would be to sever the recurrent nerve of a group of roaches and then starve them. (I have determined that starved roaches will live up to 86 days.) In this way engorgement of the crop with the resultant stress and stagnation could not occur. Predictably, the incidence of lesions in these animals should be less than in recurrent nerve-severed animals that are optimally fed. The electron-microscopic observations reported in this paper confirm the existence of blood cells within the lesions. (The ob-
I86
TAYLOR
servations were too limited to base additional conclusions on them. Further study will be necessary to determine the significance of the differences in fine structure between the crop and salivary reservoir lesions.) Schlumberger questioned the neoplastic nature of the lesions described by Scharrer and noted that in several of the tumors “. . , hemocytes appear to figure prominently, suggesting that the epithelial proliferation is hyperplasia accompanying a reaction to injury.” From Scharrer’s later publications concerning L. maderae tumors, it appears that she was less certain than earlier of the neoplastic nature of the lesions induced by nerve severance. For example, “The nature of these abnormal growths is largely obscure,” (Scharrer, 1953a), and “The term ‘tumor’ as used here does not imply that the growths under investigation are in every respect of the same nature as neoplasms in vertebrates” (Scharrer, 195313). Hema ( 1966)) working with the American cockroach, P. americana, noted that regeneration of the recurrent nerve set in soon after nerve severance, and if restoration was accomplished, the tumorous mass regressed somewhat. Possibly regeneration restores normal function to the recurrent nerve and thus to the gut. With a return to normal conditions the lesions could then heal, resulting in the observed “regression” of the lesion. I do not put this hypothesis forward as the only interpretation of the nature of the lesions obtained by nerve severance but simply as a plausible alternative to the tumor hypothesis. I have to admit that although none of the lesions described in this paper convincingly demonstrate the characteristics which in toto constitute true tumors, and though I do not believe that any of them are true tumors, I cannot categorically state that none of them is. Other hypothesized mechanisms by which denervation may induce tumorlike
lesions are activation of occult viruses (Scharrer, 1959) and interruption of flow of some humoral factor to tissues supplied by the nerve (Scharrer, personal communication to Sutherland, 1963). Also, one cannot discount the direct effects of operation injury on the induction of tumorlike lesions. Depending on the level at which the nerve is cut, one can easily sever the dorsal vessel (“heart”) or injure the foregut, salivary complex, or the tracheae. With regard to the latter, Schlumberger (1952) noted that when large tracheae are damaged during surgery their lumina may become occluded and such blockage may interfere with oxygen exchange in the tissues and lead to “infarction.” According to Schlumberger the gut epithelium is particularly susceptible to this form of injury. As noted earlier, I observed severed and occluded tracheae in several animals. Whether or not this type of injury could be involved in the development of the lesions awaits determination. ACKYXOWLEDGMENTS I would like to express my sincere appreciation to Miss Paula Butler, Miss Regina Zeikus, and Mr. William Freckleton for their expert technical assistance. REFERENCES BEAMS, H. W., AND ANDERSON, E. 1957. Light and electron microscope studies on the striated border of the intestinal epithelial cells of insects. J. MorphoZ., 100, 601-619. DAVEY, K. G., AND TREHERNE, J. E. 1963. Studies on crop function in the cockroach (Peripheta americana L. ). II. The nervous control of crop-emptying. .I. Exptl. Biol., 40, 775-780. DAWE, C. J., MORGAN, W. D., AND SLATICK, bl. S. 1967. Cellular response of a cockroach Leucophaea maderae to transplants of cell culture lines of vertebrates. Federation Proc., 26,1698-1706. DAY, M. F. 1951. The mechanism of secretion by the salivary gland of the cockroach Periplaneta americana (L. ). Australian J. Sci., Res. (B), 4, 136-143.
LESIONS
DAY,
IN
COCKROACH
M. F. 1952. Wound healing in the gut of the cockroach Periplaneta. Australian J. Sci., Res. ( B ), 5, 282-289. DETHIER, V. G., AND GELPERIN, A. 1967. Hyperphagia in the blowfly. J. Exptl. Biol., 47, 191-200. DUMONT, J. N., ANDERSON, E., AND WINNER, G. 1966. Some cytologic characteristics of the hemocytes of Limulus during clotting. J. Morph& 119, 181-208. EDWARDS, J. S., KORAL, J., AND RUDY, G. 1967. Pathological changes following salivary duct ligature in cockroaches. J. Invertebrate Pathol., 9, 160-163. ENGELMANN, F. 1966. Control of intestinal proteolytic enzymes in a cockroach. Naturwissenschaften, 4, 113-114. FFIAENKEL, G., AND HSIAO, C. 1962. Hormonal and nervous control of tanning in the fly. Science, 138, 27-29. HEMA, P. 1966. Induced gut tumours in cockroaches. Curr. Sci. (India), 35, 624-626. JOHANSSON, A. S., AND SCHREINER, B. 1966. Intestinal tumors in the German cockroach Blattella germanica L. Nature, 212, 845. JONES, J. C. 1969. Hemocytes and the problem of tumors in insects. Natl. Cancer Inst. Monogr. 30 ( to be published). LAZARENKO, F. M. 1924. Histological observations on the healing of integument wounds in insects. Izvestiya of the Biological Scientijic Research Institute and Biological Station. Perm State University. 2, 389-398. (In RusSian). MATZ, G. 1961. Tumeurs experimentales chez Leucophaea maderae F. et Locusta migratoria L. J. insect Physiol., 6, 309-313. MATZ, G. 1963a. Le cancer chez les insectes. Bull. Assoc. Philomath. Ahace Lorraine, 11, 281-284. MATZ, G. 1963b. R&actions inflammatoires, cicatrisation et canc&igen&se chez les insectes. Bull. Sot. Zool. France, 88, 660-662. MATZ, G., WEIL, J.-H., JOLY, P., AND EBEL, J.-P. 1966. Transmission of tumors in Locusta migratoria Linnaeus by nucleic acid extracted from the tumors. J. Incertebrate Pathol., 8, 8-13. MULLS, R. R. 1967. Control of cuticular tanning in the cockroach: bursicon release by nervous stimulation. J. Insect Physiol., 13, 815-820.
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NOIROT-TIMOTHI~E, C., AND NOIROT, CH. 1965. L’intestin moyen chez la reine des termites supCrieurs &de au microscope klectronique. Ann. Sci. Nat. Zool. Biol. Animals, 7, 185-208. SCHARRER, B. 1945. Experimental tumors after nerve section in an insect. Proc. Sot. Exptl. Bid. Med., 60, 184-189. SCHARRER, B. 1949. Tumor mortality and sex in Leucophaea maderae. Anat. Record, 105, 624-625. SCHARRER, B. 1953a. Metabolism and mortality in insects with gastrointestinal tumors induced by nerve severance. J. Natl. Cancer Inst., 13, 951-954. SCHARRER, B. 195313. Insect tumors induced by nerve severance: incidence and mortality. Cancer Res., 13, 73-76. SCHARRER B In “Physiology of Insect , . 1959. Development” (F. L. Campbell, ed.). p. 129. Univ. Chicago Press, Chicago, Ill. SCHARRER, B., AND LOCHHEAD, M. S. 1950. Tumors in the invertebrates: a review. Cnncer Res., IO, 403-419. SCHLUMBERGER, H. G. 1952. A comparative study of the reaction to injury. A.M.A. Arch. Pathol., 54, 98-113. ST~JBLI, W., FREYVOGEL, T. A., AND SUTER, J. 1966. Structural modification of the endoplasmic reticulum of midgut epithelial cells of mosquitoes in relation to blood intake. J. Microscopic, 5, 189-204. STEINHAUS, E. A. 1949. “Principles of Insect Pathology,” 757 pp. McGraw-Hill, New York. SUTHERLAND, D. J. 1963. Experimentally induced tumors in Periplaneta americana L. 1. Insect Physiol., 9, 131-135. TAYLOR, R. L. 1967. A fibrous banded structure in a crop lesion of the cockroach, Leucophaea maderae. f. Ultrastruct. Res., 19, 130-141. TAYLOR, R. L. 1968. Tissue damage induced by an oxyuroid nematode, Leidynema sp., in the hindgut of the Madeira cockroach, Leucophaea maderae. J. Invertebrate Pathol., 11, 214-218. 1969. Formation of tumorlike TAYLOR, R. L. lesions in the cockroach Leucophaea maderae after decapitation. NatZ. Cancer Inst. Monogr. 30, (to be published). 1937. Wound healing in WIGGLESWORTH, V. B. an insect (Rhodnius ~TOUXUS Hemiptera). J. Exptl. Biol., 14, 364-381.
OF
INVERTEBRATE
Formation
PATHOLOGY
of Tumorlike
13,
167-187
Lesions
(1969)
in the Cockroach
after Nerve
Leucophaecs
mderae
Severance1
RONALD L. TAYLOR Center
for Pathobiology,
University Received
of CalifornZa, August
19,
Irvine,
California
92664
1968
Severance of the recurrent nerve in the Madeira cockroach, Leucophaea maderae, induced the formation of lesions in the alimentary canal-lesions which were similar to those obtained by earlier investigators using the same technique on the same animal. Whereas these have been generally considered to be neoplasms, they are interpreted here to be inflammatory lesions. Recurrent nerve severance prevents passage of food from the crop to the midgut and, since feeding apparently is not inhibited, results in an abnormally engorged foregut. The hypothesis is made that the resultant stagnation throughout the alimentary canal provided ideal circumstances for the proliferation of certain microorganisms-microorganisms which infected the gut epithelium, which was stressed and possibly injured by the overdistension and/or accumulated secretions. Regardless of the involvement of microorganisms (which were involved in many but apparently not all lesions), blood cells infiltrated the injured areas and established inflammatory foci. The presence of blood cells in the lesions was established both by light and electron microscopy. Additional observations are made on the histology and fine structure of the lesions and on other possible mechanisms by which denervation may induce tumorlike lesions.
INTRODUCTION
tary canal (esophagus, crop, gastric ceca, anterior midgut) and the salivary complex ( salivary glands and salivary reservoirs). Following the same procedure, Matz (1963b) induced similar lesions in the grasshoppers Locusta migratoria and Schistocerca gregaria, the dragonfly Aeschna stick Dixippus c yanea, and the walking morosus. Subsequently, Hema (1966) and Sutherland (personal communication to Matz, 1966) induced “tumors” in the cockroach Peripluneta americana although earlier Sutherland ( 1963) and Scharrer (personal communication to Sutherland, 1963) were unable to do so. Hema (1966) also induced “tumors” in the cockroach Stylopyga sp. Not all workers have been as successful at inducing lesions by nerve severance as have those authors cited above. Johansson and Schreiner (1966) found cryptogenic
In the Madeira cockroach, Leucophaea maderae, the recurrent nerve, which belongs to the autonomic nervous system, originates in the frontal ganglion, passes between the brain and pharynx, is intimately associated with the corpora cardiaca, gives off branches to the salivary glands and salivary reservoir, divides into two main branches in the thoracic region, and finally ramifies into thin branches which appear to terminate in the region of the gastric ceca (Scharrer, 1945). By severance of this nerve in L. maderae, Scharrer ( 1945, 1949, 1953a,b) and Matz (1961) induced lesions which they interpreted to be neoplasms in the anterior portion of the alimen1 This investigation was supported by Public Health Service Research Grant No. 5358 (to E. A. Steinhaus) from National Institute of Allergy and Infectious Diseases.
“tumors” 167
in
the
alimentary
canal
of
the
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TAYLOR
German cockroach, Blatella germanica, and reported that recurrent nerve severance had no effect on the frequency of their occurrence. Edwards et al. (1967) induced lesions in the salivary complex by ligation of the salivary ducts of the cockroaches P. americana, Blabera gigantea, and Gramphodorina portentosa, and noted that severance of the recurrent nerve had no influence on the frequency of their formation. Other workers have severed recurrent nerves in non-tumor-related research. For example, Day (1951) and Mills (1967) severed the recurrent nerve in P. americana; Engelmann (1966) severed the nerve in L. maderae; Fraenkel and Hsiao (1962) severed it in the blowfly Calliphora, and Dethier and Gelperin (1967) in the black blowfly, Phormia regina. As noted above, however, these experiments were not in connection with “tumor” induction, and no reference was made to lesions resulting from the nerve intervention. The only other nerves severed in “tumor” induction studies were the efferent splanchogenital nerves of the last ganglion in the ventral nerve cord of L. maderae and L. migratoria (Matz, 1963a,b). In these cases, lesions developed in the posterior part of the midgut, the hindgut, and the genital ducts. Although the lesions obtained by nerve severance are commonly called “tumors,” as if they bore a strict homology to vertebrate neoplasms, their precise nature is far from determined. It was in an attempt to clarify their nature that the work reported here was undertaken. MATERIALS
AND METHODS
For these experiments Madeira cockroaches (Leucophaea maderae) that had been adults for 4 to 82 days were first immobilized by hypothermia. Using aseptic techniques the anterior portion of the crop was exposed, and one branch of the re-
current nerve was severed in 31 cockroaches (18 females and 13 males) and both branches in 14 cockroaches (8 females and 6 males). All of the animals were maintained at 2627°C and 60-75% relative humidity. Roaches were dissected and observed for pathological changes at specified intervals or while moribund (or dead if necrosis hadn’t progressed too far). In order to determine the incidence of naturally occurring lesions, 25 male and 25 female cockroaches were examined at an adult age of 2 weeks to 9 months. Tissues for light-microscopic examination were fixed in Zenker’s fluid, dehydrated via ethanol, embedded in Paraplast, sectioned at 610 u, and stained with hematoxylin and eosin. For electron-microscopic examination tissues were fixed for 1.5 hr in cold Dalton’s chromeosmium tetroxide at pH 7.2. Embedding was done in Epon after dehydration in ethanol. Sections were cut with a Sorvall MT-2 Porter-Blum ultramicrotome equipped with glass knives and were then treated with uranyl acetate and lead citrate to enhance contrast. An RCA EMU-3G electron microscope was used for examining the sections. RESULTS
Naturally Of
Occurring Lesions
the control (unoperated) animals (10 males and 12 females) had no lesions of any kind. Some of the animals which did have lesions commonly had more than one type. Twenty-six roaches (52% ) had pigmented lesions of the Malpighian tubules of the type described below under “Malpighian Tubule Lesions.” In any one roach pigmentation generally occurred in only a few tubules and then only in a portion of the affected tubules; pigmentation varied from a light brown to black. Small pigmented masseswere seen in the fat body of 14% of the roaches (3 males and 4 females). Tumefactive ( = tumorlike) lesions
447,
LESIONS
LN COCKROACH
were observed in 8% of the roaches. One male was found with six pigmented, tumorlike lesions in the wall of the hindgut and a pigmented lesion in the wall of a salivary reservoir. The latter lesion was surrounded by a whitish, opaque mass which histological sections of similar lesions have revealed to be infiltrating hemocytes in the process of encapsulating necrotic material. Two females had pigmented tumefactive lesions in the wall of the esophagus, one with two such lesions and the other with six. A final animal was observed to have pathological ovaries. Experimentally
Induced
Lesions
Mortality resulting from the surgical procedures generally occurred within the first 5 days after nerve severance. Such roaches died of a septicemia caused by Serratia marcescens, a bacterium which is a normal inhabitant in the alimentary canal of the cockroaches in our colony of L. maderae. It is harmless until it gains access to the hemocoel and then it multiplies rapidly, resulting in the rapid death of the host. No lesions could be found in the roaches dying within this s-day period. Mortality during this period was clearly higher among the males; whereas only 19% of the females died within the first 5 days post-nerve severance, 84~~ of the males died within this period. My experience has shown that female L. maderae in general, regardless of the treatment, are hardier than males. Mortality was also higher among those cockroaches with both branches of the recurrent nerve severed than among those with only one branch severed. It is possible though that this result may be due to the additional trauma induced by the search for and severance of the second branch, rather than to a direct result of the nerve interruption. Of the initial 45 cockroaches with severed nerves, 24 lived beyond 5 days and, as two
AFTER
NERVE
169
SEVERANCE
of these were too necrotic for dissection, only 22 roaches were considered in this study. Nineteen of these roaches were female (14 had one branch of the recurrent nerve severed and 5 had both branches severed) and 3 were males (all with one branch severed). Eight of these roaches died at periods from 6 to 195 days postnerve severance for various reasons (e.g., cannabilism and septicemia) and were examined as soon after death as possible. The other 14 were sacrificed and examined at intervals from 30 to 195 days post-nerve severance. Lesions resembling (even remotely) tumorlike masses were found in nine roaches, with some roaches having more than one lesion. Five roaches were found with lesions of the salivary reservoir, five with lesions of the crop, two with lesions of the hindgut, and two with lesions of the tracheae. Also, many roaches were found with darkened (brown to black) Malpighian tubules and/or with numerous small brown to black spots evenly spaced throughout the fat body. These Malpighian tubule and fatbody lesions have all the characteristics of simpIe encapsulation reactions and are not considered tumorlike. Due to the small number of animals involved in the experiments, no significant relationship can be established between the incidence of lesions, the number of branches of the recurrent nerve severed, and the sex of the animals. It should be noted that only 417, of the roaches surviving beyond 5 days developed tumorlike lesions, which is little more than half the incidence obtained by Scharrer (1945) and Matz (1961). Their data, however, are based on hundreds of nerve severances and therefore should be statistically more valid. Malpighian
Tubule
Lesions
The Malpighian tubules in operated animals are frequently enlarged, darkened (brown to black), and brittle. The number
TAYLOR
KEY
FOH
CR, blood chondrion;
ALL
FIGUHES:
cell granule; NC, nucleolus;
BB, HC, NU,
fibrous banded body; CU, cuticle; hemocoel: HE, hemocyte( s); IM, nucleus; TM, tunica muscularis.
EP, epithelimn; CL, intercellular material;
gut lumen; MI, mito-
LESIONS
IN
COCKROACH
of tubules affected in any one animal varies considerably. In some animals only a few (or portions of a few) are affected whereas in others the majority are affected; in the latter instance the animals’ normal excretory processes would undoubtedly be severely impeded. Microscopic examination shows that the normal cellular architecture is completely destroyed (Figs. 1, 2). In cross section the pathological tubules are seen to consist of a sometimes granular, sometimes hyaline central region which is either eosinophilic or amber-colored (yellow to brown) or a mottled mixture of both. This central region is often surrounded by a thick, well-delimited, dark amber-colored “ring” containing nuclei in various stages of pyknosis, karyorrhexis, and karyolysis. These central necrotic regions are encapsulated by two or more layers of spindleshaped hemocytes with elongated nuclei. Two or more Malpighian tubules are often loosely linked by blood cells among a basophilic fibrous network (Figs. 1,2). The blood cells of the network, which are apparently responsible for the deposition of the fibers, are not all alive; many are in various stages of necrosis. As they die, as do most animal cells, they become increasingly eosinophilic. Their nuclei undergo pyknosis, and finally karyolysis often karyorrhexis, until they completely disappear. The dead blood cell is an eosinophilic mass of coagulated protein, which in some lesions may lose its eosinophilia, become refractory to
AFTER
NERVE
SEVERANCE
171
stains, and finally become a hardened, amber-colored mass. Although Malpighian-tubule lesions occur in unoperated animals, the incidence of the lesions is higher and more tubules are affected in experimental animals. An increased incidence of Malpighian-tubule lesions also results from treatments other than nerve severance (Taylor, 1969), and therefore in the present case is not believed to be a direct result of the nerve severance. The Malpighian tubules are apparently sensitive indicators of disturbances in the animal’s homeostasis. Exactly why they should necrotize is not known. Fat-Body
Lesions
The fat body in experimental animals is frequently speckled with minute brownblack spots. In histological section, these spots are seen to consist of a central, necrotic, amber-colored hyaline core encapsulated by several layers of spindle-shaped blood cells with elongated nuclei and dense eosinophilic cytoplasm (Fig. 3). Some pyknotic and karyolytic nuclei can be seen within the central core. At the periphery of the capsule the blood cells become more rounded and merge with the surrounding fat body. Similar lesions, although in lower incidence, were found in unoperated animals. They have also been found in animals receiving injections of various materials, the incidence somehow varying with the nature and quantity of injected material (unpub-
1, 2. Cross sections of Malpighian tubule lesions. Note central regions of necrosis encapsuFIGS. 1-S. lated by hemocytes. Also note hemocytes linking neighboring, necrotic tubules. 120 x and 290 X, respectively. 3. Section through a typical fat-body lesion consisting of a central necrotic core encapsu4. Section through a tracheal lesion. Fragmented tracheal intima (arrows) lated by hemocytes. 290 X. is surrounded by unidentified cells, which are in turn encapsulated by a thin layer of hemocytes. 150 X. 5, 6. Section through an apparently old lesion (delimited by arrows) in the wall of the hindgut. See text 7. Section through a crop lesion showing the for description of lesion. 40 x and 470 X, respectively. hypertrophied epithelium, the thickened cuticle, and the cuticlelike material-apparently secreted by the 290 X. 8. Gross appearance of cockroach epidermis-which is enclosing microorganisms (arrow). with dorsal body wall removed exposing greatly distended crop and two tumorlike lesions (arrows). Figures 11 through 21 were prepared from the lower lesion shown here. Approximately actual size; scale is in centimeters.
I72
TAYLOR
lished observations). It is my belief that small foreign particles, microorganisms, or #dead cells become encapsulated by blood cells which subsequently become affixed to the fat body. Once pigmented and hardened the encapsulated material is effectively isolated. Encapsulated
Trachea
Tracheae were frequently severed during the nerve severance procedures and the cut ends were subsequently encapsulated. In histological section the tracheae appear fragmented ( Fig. 4). Their cuticular intima is eosinophilic except near the lumen where it is amber-colored. The normal tracheal intima is not so darkened. The tracheae are surrounded by rounded and highly vacuolated cells with irregularly shaped nuclei and distinct cell walls. The origin and nature of these cells was not determined although it is suggested that they may be hyperplastic tracheablasts. These latter cells are completely encapsulated by spindleshaped hemocytes with elongate nuclei and dense eosinophilic cytoplasm. Numerous fibers are present within the capsule. Hindgut
Lesions
Hindgut lesions were observed in two animals. One lesion consisted of typical amber-colored material and blood cells in various stages of necrosis. The entire mass was adhering to the muscularis and did not seem to involve the gut epithelium. Two essentially identical lesions were found in the hindgut of a second animal. Juxtaposed to a relatively normal appearing cuticle and its underlying epithelium is a scablike mass of completely necrotic tissue which is separated from the gut lumen by an amber-colored second cuticle (Figs. 5, 6). The portion of the scablike layer nearest the integument is eosinophilic, whereas that in contact with the second cuticle is ambercolored and is refractory to stains. The gut wall is folded in all areas except that of the
lesion, where such folding is apparently prevented by the amber-colored portions of the lesion which are hardened. Such hardening possibly results from a process similar to sclerotization in the normal cuticle. This lesion can be interpreted as the healed remains of a lesion that may have existed at one time in the integument. Through normal wound-healing processes ( Lazarenko, 1924; Wigglesworth, 1937; Day, 1952) the epithelial cells peripheral to the lesion migrated beneath the dead and dying cells until a continuous sheet of epithelial cells had formed. The new epithehum then secreted a normal cuticle, thereby isolating the necrotic material between the new and the old cuticles. I have observed that hardening and darkening commonly, though not always, occurs in necrotic tissues. The darkening of the old cuticle is possibly due to the diffusion from dying blood cells of substances responsible for hardening and darkening. In a hindgut lesion induced by an oxyuroid nematode (Taylor, 1968), I noted darkening of the cuticle over the lesion, and where folds in the gut epithelium brought the cuticle of normal epithelium in contact with (or in close proximity to) the abnormally darkened cuticle over the lesion, the outer portion of the normal cuticle darkened also. Foregut
Lesions
No lesions were found in the esophagus of experimental roaches although crop lesions were found in 5 animals. Inexplicably, nearly the opposite situation occurred in unoperated animals, viz., no crop lesions and two esophageal lesions were observed. The first of two crop lesions to be described consists in part of hypertrophied epithelial cells with enlarged nuclei (Fig. 7). The cuticle over these cells is thicker and more basophilic than normal. Adhering firmly to the cuticle in the gut lumen is an amber-colored, hardened, hyaline material which appears to be cuticular in nature.
LESIONS
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Unidentified microorganisms are enclosed in pockets in this material. Perhaps for some unknown reason the cells became hyperactive (thus their hypertrophied appearance) and secreted excess cuticle (the amber-colored material) which inadvertently incorporated the microorganisms. Alternatively, perhaps the cells became hyperactive in response to the microorganisms and secreted the abnormal cuticle as a defense mechanism. Although the size of a cockroach’s crop varies with the quantity of food it contains, it is often abnormally enlarged after recurrent nerve severance. The crop of one roach approximately 4 months post-nerve severance was greatly distended with food, appearing approximately three times larger than normal (Fig. 8). Several tumescent, whitish lesions could be clearIy seen on the surface of the crop. The two largest ones measured 2 and 3.5 mm in diameter. In contrast with the norma situation the cuticle involved in the lesions was hard, brown-black, and shiny. One-half of the second largest lesion was removed and processed for electron microscopy, and the other half, including the remainder of the crop, was processed for light microscopy. For purposes of comparison the histological appearance of normal crop is shown in Figs. 9 and 10. The cuticle in the lesion in contrast to the normal situation is ambercolored (Figs. 11-13). Next to the cuticle is an alveolar or spongy appearing layer which extends across most of the lesion and which is hard and pigmented similarly to the cuticle (Fig. 13). Adjacent to the cuticle are pockets of at least two different types of microorganisms (Fig. 12), one of which is clearly a bacterium. Enclosing these pockets is a region of dying blood cells with pyknotic and karyolytic nuclei. This region is eosinophilic in areas most distant from the microorganisms, and, progressing towards the pockets of microorganisms, it first becomes refractory to staining
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and then amber-colored. These changes are believed to represent progressive stages in the death of the blood cells. This region is sharply delimited from surrounding tissues by a dark amber-colored layer (Fig. 11) . The remainder of the lesion consists of hyperplastic epithelial cells (or epithelioid infiltrating spindle-shaped blood cells), blood cells, and widely separated and broken up muscles of the tunica muscularis. Only one mitotic figure was seen among the hyperplastic epithelial cells ( Fig. 14). The ultrastructure of the tunica muscularis of this lesion has been described and compared with normal crop muscle (Taylor, 1967). To summarize briefly, the Z lines appeared fragmented and irregular in width and the myofilaments in the region of the I bands appeared disoriented (Fig. 15); there was no well-developed sarcoplasmic reticulum and no distinct mitochondria although there were numerous oval-shaped vacuoles containing sparse flocculent material which may represent disintegrating mitochondria; most significant was the finding of fibrous banded structures between groups of abnormal myofibrils. These fusiform-shaped structures were not present in normal crop muscle and were of uncertain significance. For a more complete description of this pathology and a discussion of the possible collagenous nature of the fibrous banded bodies, see Taylor ( 1967). The normal epithelial cells are rich in glycogen, endoplasmic reticulum, and Golgi zones-characteristics of cells of high metabolic activity. The normal ultrastructure is similar to that described for the gut of other insects (Beams and Anderson, 1957; NoirotTimothke and Noirot, 1965; Staubli et al., 1966). Considerable necrosis was evident in the pathological “epithelium.” The chromatin material of many cells was for the most part clumped along the nuclear membrane (Figs. 16, 17). Interchromatinic granules were aggregated along the interface between the chromatin and the inter-
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I?IGS. 9-14. 9, 10. Cross section through normal micrographs of lower crop lesion shown in Fig. 8. tion through lesion at different level from Fig. rounded by dying and dead hemocytes. 1’20 X. layer next to cuticle. 290 X. 14. Section through
crop. 40 x and 290 X, respectively. 11-14. Light 11. See text for description of lesion. 40 X. 12. Sec11. Note pockets of microorganisms (arrows) sur13. Section through crop lesion showing “alveolar” crop lesion showing mitotic figure. 1050 X.
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Figures 15 to 21 are electron FIGS. 15, 16. structure of tunica muscularis showing fibrous rows ) 23,900 X. 16. Most necrotic portion swollen and barely recognizable mitochondria,
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micrographs of lower lesion shown in Fig. 8. 15. Ultrabanded body. Note also disoriented myofilaments (arof lesion. Note clumping of chromatin in nuclei, and numerous vacuoles and vesicles. 7150 X.
176 chromatic substance. The extremely prominent in cells and contained a fine rial. Numerous vacuoles
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latter material was the most necrotic filamentous mateand vesicles were
present in the necrotic cells (Figs. 16, 18), some of which contained sparse filamentous material of unknown composition. The origin of the vacuoles and vesicles is not clear
FIGS. 17, 18. 17. Note clumping of chromatin in nucleus. Also with distinct granules. 8850 X. 18. Note vacuoles, vesicules, abnormal latter is recognized by its distinct cytoplasmic granules. 5450 X.
note “round” mitochondria,
blood cell packed and hemocyte. The
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although some probably resulted from pathological changes in the endoplasmic reticulum. The mitochrondria are barely recognizable in most cells, appearing similar to the mitochrondria of the tunica muscularis, i.e., they are swollen, their cristae are disrupted, and they contain a sparse flocculent material (Figs. 16, 18). In some of the less necrotic cells, viz., those cells nearest the muscularis, small accumulations of glycogen granules are present (Fig. 19). Some, though not many, myelin figures are present in the abnormal tissue (Fig. 20) ; their presence is indicative of membrane degeneration. A constant feature of the lesion at all levels of the epithelium is the presence of hemocytes, which are identified by their distinct cytoplasmic organelles (Figs. 17-19, 21). (A more detailed description of these blood cell organelles and their possible function will form the subject of a subsequent publication. ) Often these are the only cells retaining normal architecture within an otherwise degenerating area. The organelles are often seen loose within the degenerating area but demonstrate no signs of degeneration themselves. Salivary
Reservoir Lesions
Being diverticulae of the foregut, the salivary reservoirs are of ectodermal origin and constructed similarly to the foregut. The tunica muscularis, however, consists of only scattered muscle fibers, and the cuticle is loosely lamellated, apparently providing for slippage as the reservoir enlarges and shrinks. The reservoir is completely transparent when filled with saliva. Lesions of the salivary reservoir were found in five roaches, an incidence considerably higher than in unoperated animals. Inexplicably, all lesions occurred in the right reservoir only. Two of these lesions are described below. One cockroach was found to have a pigmented (brown-black) spot, 0.5-l mm diameter, in the wall of the right salivary
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reservoir. A small (about 1 mm long), whitish, lobular mass was suspended from the pigmented spot into the saliva. Several smaller whitish lesions (some with brownblack pigment spots) were also present on the same reservoir, one of which was processed for electron microscopy. In histological section, the larger lesion is seen to be quite complex (Fig. 22). A thin layer of blood cells is adhering firmly to the epithelium ( Figs. 22 , 23 ) . Some of the blood cells are in small whorls around amber-colored cores of hyaline material. Immediately adjacent to the cuticular intima of the reservoir is a layer of loosely adhering cells with large nuclei; these cells, the nature of which was not determined, are situated in a fibrous network. Adjacent to this region is a scablike layer consisting of completely necrotic, hardened and darkened (amber-colored) blood cells which merges gradually with a region of eosinophilic blood cells containing pyknotic and karyolytic nuclei. Some of the cells contain small granules of amber-colored material. At the periphery of the lesion, the cuticle appears to separate-one portion continuing over the epithelial cells beneath the lesion in a relatively normal manner and the other portion passing over the lesion. The lobular mass projecting into the saliva consists of large numbers of an unidentified microorganism trapped between widely separated lamellae of the cuticle (Figs. 23,24). The microorganisms were spherical, ca. 1 p in diameter, and transparent with asymmetrically thickened walls. The means by which the lesion came to assume the above form is not known although one may speculate that it developed in a manner similar to the following: The microorganisms somehow invaded the reservoir wall and established an inflammatory focus disrupting the normal continuity of the epithelium. The cuticle, being flexible and nonliving, was damaged only slightly if at all. Normal wound repair processes
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FIGS. 19-21. 19. Region of lesion bordering the muscularis. Hernocytes, glycogen (arrow), and numerous mitochondria can be seen. 5450 X. 20. Myelin figure in necrotic tissue. 81,900 X. 21. Blood cell granules. The presence of these organelles in tissue indicates the presence of hemocytes. 14,700 X,
FIGS. 22-25. Figures 22-24 are light micrographs of salivary reservoir lesion. 22. Section through center of lesion. Normal salivary wall appears in lower left portion of photograph. Detail of lesion is better shown in .Fig. 23. 120 X. 23. Section through entire lesion showing part of “sack” of microorganisms (arrow) projecting into reservoir lumen. See text for description of lesion. 290 X. 24. Unidentified microorganisms in “sack” shown in Fig. 23. 1170 x . Figures 25-30 are electron micrographs of salivary reservoir lesion immediately adjacent to lesion shown in Figs. 22-24. 25. Note irregularly shaped nuclei, large dense chromatin aggregates, blood cell granules, irregularly shaped mitochondria, and apparent contraction of nuclear contents away from nuclear membrane. Also note the amorphous, finely granular and fibrillar intercellular material. 6100 x .
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began whereby the epithelial cells multiplied around the lesion and migrated beneath it, eventually reestablishing continuity. When the new cuticle was secreted, the wound healing process was essentially complete with the necrotic tissue effectively cast off although still contained between the old and new cuticles. This situation would give the appearance at the edges of the lesion of the cuticle having “separated.” The microorganisms, apparently not contained, and feeding in the saliva, continued to multiply between the lamellae of the old cuticle, forming the lobular mass described earlier. This interpetation is purely speculative although it fits the observations and the phenomena known to occur in wound healing ( Wigglesworth, 1937). The ultrastructure of the neighboring salivary-reservoir lesion differs considerably from that of the crop lesion already described. The only similarity is the involvement of blood cells in both lesions. Two cell types are present in the salivary-reservoir lesion and their plasma membranes are fragmented and highly irregular. One cell type has a high nuclear-to-cytoplasmic ratio (Figs. 25, 26). Its nucleus is irregularly shaped and consists almost entirely of large dense chromatin aggregates between which is a less dense interchromatic substance containing randomly distributed interchromatinic granules. The nuclear contents appear to have condensed or contracted away from the nuclear membrane on one or more sides, forming large electron-lucid vacuoles. (At lower magnifications these nuclei would probably appear pyknotic. ) The cytoplasm of these cells contains irregularly shaped mitochrondria, free ribosomes, and numerous vesicles. The other cell type contains a nucleus with a highly irregular and indistinct nuclear membrane and with chromatin evenly distributed throughout the nucleoplasm, i.e., there are no variations in density of the nuclear sap (Figs. 27, 28 ) . Nucleolar material can be seen in the
nucleus in some sections. The prominent features of the cytoplasm are numerous vesicles, mitochondria, and blood cell granules. (These are the same granules that are seen within the blood cells of the crop lesion. ) Also present within the cell are various-sized deposits of a membranebounded material that is relatively dense and finely granular and fibrillar. This material is also present as large interconnecting deposits between the cells of the lesion (Figs. 25-27). The blood cell granules appear to play a role in the formation of this material as they can be seen in what appear to be transitional stages. They appear to lose their internal structure and to coalesce with other similar granules, forming membrane-bounded deposits within the cell (Figs. 29, 30) ; these then fuse with the plasma membrane and extrude their contents. It should be noted that the mitochondria of these cells are not swollen or with indistinct cristae as in the crop lesion. The blood cell granules are morphologically very similar to granules in the hemocytes of Limulus which are involved in the clotting process (Dumont et al., 1966). Perhaps a process akin to clotting has taken place within this lesion. It is interesting in this connection that when Day (1952) injected an anticoagulant (0.01 M ascorbic acid) into P. anrericana, the number of hemocytes infiltrating and becoming involved in a wound was reduced. The suggestion here is that clotting or a related phenomenon occurs normally at some stage in the wound repair process. This phenomenon may explain the adhesion of hemocytes at an injury site. The electron-microscopic observations on the salivary reservoir lesion cannot be reliably compared with the light-microscopic observations on the neighboring lesion. The two lesions appeared different grossly, the Epon-embedded lesion lacking the pigmentation as well as the lobular mass which
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FIGS. 2628. Note shaped mitochondria, membranes. Compare
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apparent contraction of nuclear contents away from nuclear membrane, irregularly and intercellular material. 9300 X . 27, 28. Note nuclei with indistinct nuclear with nuclei in Figs. 25 and 26. Also note intercellular material. 9300 X.
182
FIGS. 29, 30. Suggested stages granular and fibrillar intercellular 16,100 x.
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in transition of the material: I (earliest
blood stage)
cell granules through IV
into (fully
the amorphous, finely transformed granule).
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hung into the salivary reservoir of the Paraplast-embedded specimen. The largest salivary reservoir lesion found measured 1.5-2 mm in diameter and approximately 0.5 mm in depth. Grossly, it appeared black and tracheated. In cross section, this lesion is seen to consist of several lavers or regions which merge more or less gradually into each other ( Figs, 31-34). The portion of the lesion nearest the cavity of the reservoir is completely necrotic, hardened, and darkened (amber colored), and includes the disrupted lamellae of the cuticle (layer A, Fig. 32). This layer merges with a strongly eosinophilic layer of dying blood cells containing pyknotic and karyolytic nuclei (layer B, Figs. 32-34). Next is a single layer of large weakly staining cells with large nuclei, followed by a layer of loosely adhering spindle-shaped cells (layers C and D, respectively, Figs. 32-34). The latter layer separates those parts of the lesion described above from the main mass of the lesion, which appears to consist entirely of blood cells (layer E, Figs. 32-34). Many of these cells are in “whorls,” some of which have encapsulated necrotic cells and amber-colored deposits. Tracheoles are present within this living mass of blood cells. Those cells nearest the hemocoel contain eosinophilic granules of diverse sizes. The incomplete single layer of large cells between the eosinophilic mass of dying blood cells and the loosely adhering spindleshaped cells are possibly epithelial cells. After injury the epithelial cells around a lesion will migrate through the inflammatory mass until epithelial continuity is restored (Wigglesworth, 1937). Cuticle is then secreted, resulting in the isolation of the damaged and necrotic tissues from the hemocoel. In the case of the lesion here described, epithelial continuity has not been completelv established, and new cuticle has not been deposited. The spindle-shaped cells immediately adjacent to this layer may be forming a new basement membrane.
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DISCUSSION
Vertebrates and invertebrates alike undergo localized tissue reactions to injury or injurious agents, and with this as our definition of inflammation (Steinhaus, 1949) we are able to apply the term to all animals. Admittedly, invertebrate inflammatory lesions are not inflamed in the vertebrate sense of the word (i.e., red or hot) although I know of no study to see if there is an elevation of temperature in an invertebrate lesion. I will use the term inflammation whenever referring to localized tissue reactions to injury or injurious agents (including reactions to microorganisms, parasites, foreign bodies, etc.). The difficulties attendant to the differentiation between neoplasia, inflammation, and wound repair in invertebrates are commonly voiced. One of these difficulties has to do with the fact that lesions of diverse etiology frequently consist, at least in part, of cells which differ significantly in general morphology and cytology from any of the animal’s “normal” cells. These “abnormal’ cells, it is therefore commonly believed, must be neoplastic. Hemocytes, however, infiltrate various inflammatory and wound repair processes, and in so doing alter their form. Day ( 1952), for example, studied wound healing in the gut of the cockroach, P. americana, and noted that the hemocytes “ . . . undergo striking changes in shape when they become incorporated into the wound tissue. _ . . The hemocytes may become flattened, elongate, or spheroidal, depending upon their position.” Wigglesworth ( 1937) studied wound healing in the body wall of Rhoclnius ~rrolixus and noted that in the later stages of the wound healing process it was impossible to distinguish the infiltrating hemocytes from the hyperplastic epidermal cells. Schlumberger ( 1952) noted the epithelioid character of hemocytes in a reaction to injury in the cockroach, P. americana, and Dawe et al. (1967) made a
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similar observation in the cockroach, L. maderae. Therefore the presence of cells in a lesion which differ significantly in morphology from the normal hemocytes and from
normal cells of the affected tissue is not sufficient evidence for neoplasia. Also, one must be careful in the interpretation of certain cytological character-
FIGS. 31-34. Large salivary reservoir lesion at increasing magnification. 40 X, 150 X, 290 X, 470 X, respectively. 31. Section through entire lesion, 32-34. A, Completely necrotic, hardened, and amber-colored portion of lesion; B, strongly eosinophilic layer of dying blood cells; C, single layer of large, weakly staining cells believed to be regenerated epidermal cells; D, layer of spindle-shaped cells believed to be forming a new basement membrane; E, healthy aggregated hemocytes.
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istics commonly associated with neoplastic cells. Wigglesworth ( 1937), for example, observed the following conditions in wound tissue in R. prolixus: numerous mitoses, polyploidy, irregular mitoses, greatly enlarged nuclei, multipolar mitotic figures, pyknotic nuclei, etc. Wigglesworth and Schlumberger occasionally observed hyperplastic accumulation of epithelial cells at certain stages of the wound healing process. In P. americana. injected with talc or methylcholanthrene, Schlumberger observed (although rarely) ova whose follicle cells had “. . . enlarged to form giant cells with bizarre, irregular pyknotic nuclei. . , .” Although the lesions studied by the abovecited authors had many characteristics suggestive of neoplasms, they clearly were not. Jones ( 1969) noted the involvement of hemocytes in a variety of anomalous lesions reported originally to be tumors. The point here is to emphasize the difficulty of differentiating between neoplastic and inflammatory lesions and to suggest the possibility that many authors understandably may have described lesions as neoplastic which were in fact inflammatory. When the difficulty exists, the simplest possible interpretation of the lesions which is consistent with the data should be sought. The simplest interpretation of the lesions described in this report is that they represent processes of inflammation and repair. The evidence does not demand, or even strongly suggest, that the lesions be interpreted as having resulted from a neoplastic transformation. Assuming that they are inflammatory foci, the question is immediately raised, “What is the possible connection between severance of the recurrent nerve and the development of inflammatory lesions, especially at locations distant from the point of surgical intervention?” The answer to this question, I think, is not too difficult. Dethier and Gelperin ( 1967) have recently shown that the black blowfly, Phormia Fegina, overeats when the recurrent nerve is
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severed. Their work indicates that severance of the recurrent nerve may prevent the brain from receiving impulses from stretch receptors in the gut, impulses which are necessary to inhibit feeding behavior after the animal has sufficiently fed. Davey and Treherne (1963) and Englemann (1966) have shown that severance of the recurrent nerves in P. americana and L. maderae prevents passage of food from the crop into the midgut. If after recurrent nerve severance the cockroach both overeats and fails to pass the food on to the midgut, the result would be an abnormally engorged foregut. Scharrer (1945) herself noted that the anterior portion of the alimentary canal in nervesevered cockroaches was frequently filled with an abnormally large quantity of food. As noted earlier in this paper, I have made the same observation. One can hypothesize that the resultant stagnation throughout the alimentary canal would provide ideal circumstances for proliferation of certain microorganisms. These microorganisms might then infect the gut epithelium, particularly when it is stressed and possibly injured by overdistension and/or accumulated secretions. I noted microorganisms associated with many of the lesions. However, regardless of the involvement of microorganisms, blood cells would be expected to infiltrate such injured areas and establish inflammatory foci. An interesting test of the above hypothesis would be to sever the recurrent nerve of a group of roaches and then starve them. (I have determined that starved roaches will live up to 86 days.) In this way engorgement of the crop with the resultant stress and stagnation could not occur. Predictably, the incidence of lesions in these animals should be less than in recurrent nerve-severed animals that are optimally fed. The electron-microscopic observations reported in this paper confirm the existence of blood cells within the lesions. (The ob-
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servations were too limited to base additional conclusions on them. Further study will be necessary to determine the significance of the differences in fine structure between the crop and salivary reservoir lesions.) Schlumberger questioned the neoplastic nature of the lesions described by Scharrer and noted that in several of the tumors “. . , hemocytes appear to figure prominently, suggesting that the epithelial proliferation is hyperplasia accompanying a reaction to injury.” From Scharrer’s later publications concerning L. maderae tumors, it appears that she was less certain than earlier of the neoplastic nature of the lesions induced by nerve severance. For example, “The nature of these abnormal growths is largely obscure,” (Scharrer, 1953a), and “The term ‘tumor’ as used here does not imply that the growths under investigation are in every respect of the same nature as neoplasms in vertebrates” (Scharrer, 195313). Hema ( 1966)) working with the American cockroach, P. americana, noted that regeneration of the recurrent nerve set in soon after nerve severance, and if restoration was accomplished, the tumorous mass regressed somewhat. Possibly regeneration restores normal function to the recurrent nerve and thus to the gut. With a return to normal conditions the lesions could then heal, resulting in the observed “regression” of the lesion. I do not put this hypothesis forward as the only interpretation of the nature of the lesions obtained by nerve severance but simply as a plausible alternative to the tumor hypothesis. I have to admit that although none of the lesions described in this paper convincingly demonstrate the characteristics which in toto constitute true tumors, and though I do not believe that any of them are true tumors, I cannot categorically state that none of them is. Other hypothesized mechanisms by which denervation may induce tumorlike
lesions are activation of occult viruses (Scharrer, 1959) and interruption of flow of some humoral factor to tissues supplied by the nerve (Scharrer, personal communication to Sutherland, 1963). Also, one cannot discount the direct effects of operation injury on the induction of tumorlike lesions. Depending on the level at which the nerve is cut, one can easily sever the dorsal vessel (“heart”) or injure the foregut, salivary complex, or the tracheae. With regard to the latter, Schlumberger (1952) noted that when large tracheae are damaged during surgery their lumina may become occluded and such blockage may interfere with oxygen exchange in the tissues and lead to “infarction.” According to Schlumberger the gut epithelium is particularly susceptible to this form of injury. As noted earlier, I observed severed and occluded tracheae in several animals. Whether or not this type of injury could be involved in the development of the lesions awaits determination. ACKYXOWLEDGMENTS I would like to express my sincere appreciation to Miss Paula Butler, Miss Regina Zeikus, and Mr. William Freckleton for their expert technical assistance. REFERENCES BEAMS, H. W., AND ANDERSON, E. 1957. Light and electron microscope studies on the striated border of the intestinal epithelial cells of insects. J. MorphoZ., 100, 601-619. DAVEY, K. G., AND TREHERNE, J. E. 1963. Studies on crop function in the cockroach (Peripheta americana L. ). II. The nervous control of crop-emptying. .I. Exptl. Biol., 40, 775-780. DAWE, C. J., MORGAN, W. D., AND SLATICK, bl. S. 1967. Cellular response of a cockroach Leucophaea maderae to transplants of cell culture lines of vertebrates. Federation Proc., 26,1698-1706. DAY, M. F. 1951. The mechanism of secretion by the salivary gland of the cockroach Periplaneta americana (L. ). Australian J. Sci., Res. (B), 4, 136-143.
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M. F. 1952. Wound healing in the gut of the cockroach Periplaneta. Australian J. Sci., Res. ( B ), 5, 282-289. DETHIER, V. G., AND GELPERIN, A. 1967. Hyperphagia in the blowfly. J. Exptl. Biol., 47, 191-200. DUMONT, J. N., ANDERSON, E., AND WINNER, G. 1966. Some cytologic characteristics of the hemocytes of Limulus during clotting. J. Morph& 119, 181-208. EDWARDS, J. S., KORAL, J., AND RUDY, G. 1967. Pathological changes following salivary duct ligature in cockroaches. J. Invertebrate Pathol., 9, 160-163. ENGELMANN, F. 1966. Control of intestinal proteolytic enzymes in a cockroach. Naturwissenschaften, 4, 113-114. FFIAENKEL, G., AND HSIAO, C. 1962. Hormonal and nervous control of tanning in the fly. Science, 138, 27-29. HEMA, P. 1966. Induced gut tumours in cockroaches. Curr. Sci. (India), 35, 624-626. JOHANSSON, A. S., AND SCHREINER, B. 1966. Intestinal tumors in the German cockroach Blattella germanica L. Nature, 212, 845. JONES, J. C. 1969. Hemocytes and the problem of tumors in insects. Natl. Cancer Inst. Monogr. 30 ( to be published). LAZARENKO, F. M. 1924. Histological observations on the healing of integument wounds in insects. Izvestiya of the Biological Scientijic Research Institute and Biological Station. Perm State University. 2, 389-398. (In RusSian). MATZ, G. 1961. Tumeurs experimentales chez Leucophaea maderae F. et Locusta migratoria L. J. insect Physiol., 6, 309-313. MATZ, G. 1963a. Le cancer chez les insectes. Bull. Assoc. Philomath. Ahace Lorraine, 11, 281-284. MATZ, G. 1963b. R&actions inflammatoires, cicatrisation et canc&igen&se chez les insectes. Bull. Sot. Zool. France, 88, 660-662. MATZ, G., WEIL, J.-H., JOLY, P., AND EBEL, J.-P. 1966. Transmission of tumors in Locusta migratoria Linnaeus by nucleic acid extracted from the tumors. J. Incertebrate Pathol., 8, 8-13. MULLS, R. R. 1967. Control of cuticular tanning in the cockroach: bursicon release by nervous stimulation. J. Insect Physiol., 13, 815-820.
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NOIROT-TIMOTHI~E, C., AND NOIROT, CH. 1965. L’intestin moyen chez la reine des termites supCrieurs &de au microscope klectronique. Ann. Sci. Nat. Zool. Biol. Animals, 7, 185-208. SCHARRER, B. 1945. Experimental tumors after nerve section in an insect. Proc. Sot. Exptl. Bid. Med., 60, 184-189. SCHARRER, B. 1949. Tumor mortality and sex in Leucophaea maderae. Anat. Record, 105, 624-625. SCHARRER, B. 1953a. Metabolism and mortality in insects with gastrointestinal tumors induced by nerve severance. J. Natl. Cancer Inst., 13, 951-954. SCHARRER, B. 195313. Insect tumors induced by nerve severance: incidence and mortality. Cancer Res., 13, 73-76. SCHARRER B In “Physiology of Insect , . 1959. Development” (F. L. Campbell, ed.). p. 129. Univ. Chicago Press, Chicago, Ill. SCHARRER, B., AND LOCHHEAD, M. S. 1950. Tumors in the invertebrates: a review. Cnncer Res., IO, 403-419. SCHLUMBERGER, H. G. 1952. A comparative study of the reaction to injury. A.M.A. Arch. Pathol., 54, 98-113. ST~JBLI, W., FREYVOGEL, T. A., AND SUTER, J. 1966. Structural modification of the endoplasmic reticulum of midgut epithelial cells of mosquitoes in relation to blood intake. J. Microscopic, 5, 189-204. STEINHAUS, E. A. 1949. “Principles of Insect Pathology,” 757 pp. McGraw-Hill, New York. SUTHERLAND, D. J. 1963. Experimentally induced tumors in Periplaneta americana L. 1. Insect Physiol., 9, 131-135. TAYLOR, R. L. 1967. A fibrous banded structure in a crop lesion of the cockroach, Leucophaea maderae. f. Ultrastruct. Res., 19, 130-141. TAYLOR, R. L. 1968. Tissue damage induced by an oxyuroid nematode, Leidynema sp., in the hindgut of the Madeira cockroach, Leucophaea maderae. J. Invertebrate Pathol., 11, 214-218. 1969. Formation of tumorlike TAYLOR, R. L. lesions in the cockroach Leucophaea maderae after decapitation. NatZ. Cancer Inst. Monogr. 30, (to be published). 1937. Wound healing in WIGGLESWORTH, V. B. an insect (Rhodnius ~TOUXUS Hemiptera). J. Exptl. Biol., 14, 364-381.