The Wax Glands of the Cockroach (Blatta Germanica)

Resumido por el autor, E. H. Dusham.

Las glhdulas productoras de cera en la cucaracha (Blatta germanica). La hipodermis de 10s insectos ha sido descrita como formada por una sola capa de c6lulas. Una excepcidn se presenta en la pared dorsal del abdomen del macho de la cucaracha (Periplaneta orientalis), cuya hipodermis estd formada por dos o tres capas de c6lulas. El estudio de las paredes del abdomen ha demostrado que en esta especie dicha estructura existe, no solo en las paredes dorsal y ventral del macho, sino tambi6n en las de la hembra. El autor ha encontrado tambi6n una estructura semejante en Blatta germanica. La capa superior estA formada por cklulas hipod6rmicas semejantes a las que existen en otras regiones del cuerpo, en las que existen c6lulas hipodkrmicas tipicas. Las capas m&sprofundas estan formadas por c6lulas mucho mayores, con grandes nficleos vesiculares y citoplasma granuloso. Su aspecto irregular indica su naturaleza glandular, comprobada ademas por el hallazgo de canales pequefios que se extienden a trav6s de la cuticula que recubre a dichas glandulas, la cual presenta pequefios poros en vista superficial, y por medio de 10s cuales la secrecidn sale a la superficie. El analisis de dicha secrecidn, recogida en la cuticula, ha demostrado que esta formada de cera. En Blatta germanica estas glandulas se dis- tinguen por primera vez despues de la primera muda del tegu- mento, en las ninfas de dos dias, en las cuales algunas de las cblulas hipod6rmicas que ocupan ciertas Areas, aumentan de tamaiio y empujando a las c6lulas hipod6rmicas adyacentes dan a la hipodermis el aspecto de una capa poliestratificada. La hipodermis de la cucaracha, por esta causa, no difiere de la de 10s otros insectos.

Translation by Dr. Jose Nonidez, Columbia University. AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE RJBLIOQRAPHICSERVICE, NOYEMBER 18

THE WAX GLANDS OF THE COCKROACH (BLATTA GERMANICA)

E. H. DUSHAM Department of Entomology, Cornell University

ELEVEN FIGURES

INTRODUCTION The ability to secrete wax in one form or another is wide- spread among insects, being reported for the Libellulidae of the Odonata ; Notonectidae, Fulgoridae, Cercopidae, Psyllidae, Aphi- didae, Aleyrodidae, and Coccidae of the Hemiptera; Tortricidae and Papilionidae of the Lepidoptera ; Coccinellidae and Cur- culionidae of the Coleoptera; and the Tenthredinidae and Apidae of the Hjmenoptera. That this function should also be found in cockroaches is not surprising, for they are waxy in appearance, feel waxy when handled, and when thrown into water or other fluids float readily as if buoyed up by some oily covering, even after the air has been forced out of the tracheae by pressure: In fact, one of the difficulties encountered in fixing material is to submerge the specimens in fixing fluids, especially when these are used cold. On the other hand, no such difficulty is experienced when hot fluids are used, the hot liquid evidently melting the wax, thus allowing the insects to sink. Moreover, it is a well-known fact that the species in question is usually found in warm, damp places, kspecially in close prox- imity to sweating water pipes, its association with water pipes leading from the Croton Aqueduct in the vicinity of New York City earning for it the popular name of 'Croton bug.' Further- more, references to the literature of this species show that it has been carried for long distances in water mains without drown- 563 564 E. H. DUSHAM ing. That they have been able to live in moist places and also withstand submerging would lead one to suppose that their bodies were protected by some oily or waxy secretion.

HISTORICAL

The study of the wax glands of the cockroach is bound up in a controversy as to the number of layers in the hypodermis of these insects. In insects in general the body wall is relatively simple, consisting of an outer chitinous covering or cuticula, the hypodermis consisting of a single layer of cells, and the basement membrane, and as such, Miall and Denny described it in their work on the cockroach, in which they stated that the hypodermis consisted of a single layer of flattened cells, resting on a basement mebrane, each cell corresponding to a polygonal area of the overlying cuticula. In 1888, however, E. A. Minchin, while studying certain glands on the dorsal side of the cockroach, found that the hypodermis, at least on the dorsal side of the older stages of the male, consisted of two layers everywhere except in the intersegmental areas where only a single layer was present. In certain places he found that the cells of these layers had the appearance of giant cells with large nuclei, granular contents, and elongate processes. These he interpreted as ganglion cells. He found them scattered over each tergum, especially at the anterior portion of each tergite-that part which is overlapped by the preceding tergite-although they were also scattered throughout the region posterior to this part. Between these so-called ganglion cells he found the ordinary cells of the hypodermis. He therefore concluded that in the cockroach, at least, the hypodermis consisted of an upper layer of cells corresponding to the polygonal areas of the cuticula, and a lower very irregular layer, occasionally forming two layers, whose cells were modified to form nerve cells, and which were probably connected with setae where the terga were exposed. In 1889, Mingazzini made a detailed study of this apparently double-layered hypodermis in order to verify Minchin’s work, WAX GLANDS OF THE COCKROACH 565

examining the dorsal wall of the abdomen of males in different stages of development. He found that the hypodermis was not always made up of two layers of cells as Minchin described, but sometimes showed a single layer of cells, other times two or more layers. In the intersegmental areas, he found a single-layer of cells as Minchin described. Where the muscle bundles were inserted he also found a single layer of cells, modified in the usual way to form the characteristic muscle attachments. In other places the hypodermis consisted of a single layer of cells, usualiy of small size, or of two layers, the upper layer consisting of small cells, the lower one of much larger cells, or of several layers of cells arranged without order. Except for an occasional large cell at the surface among the smaller cells where a singlelayer of cells was present, the majority of the larger cells were below the smaller ones. He concluded that these cells were not to be considered as nerve cells, but as cells of an epithelial nature, derived from the upper layer of the hypodermis, but which had undergone en- largement and were no longer of use in the secretion of the cuticula. He was uncertain whether these large cells were hair- forming cells or gland cells, but because of the structure of the nucleus and protoplasm he favored the glandular view, the insects probably secreting an oily substance. He, therefore, concluded that the hypodermis of the cockroach did not differfrom that of other insects by the nature of the cells which composed it, but only because, as the insects increase in size, many hypodermal cells become specialized for a particular function, in- creased in size, took on a branching form, and were carried below the regular hypodermis, thus forming an apparently double- layered hypodermis. In 1909, Berlese, in commenting on this peculiar structure in the cockroach, stated that if the smaller cells were not merely infiltrated amoebocytes, they were to be regarded as a prolifera- tion of the hypodermis, and that possibly the larger cells had assumed another function. At least, he was sure that the condition was unique among insects and was still an open question. 566 E. H. DUSHAM

LOCATION AND STRUCTURE OF THE GLANDS

At the suggestion of Dr. W A. Riley, under whose direction this work was carried out, the writer undertook to settle this question. While the preceding investigations were made on Periplaneta orientalis, yet a study of Blatta germanica showed that similar conditions were present, and because of the abun- dance of the latter, this species was used almost entirely in the following work. In order to show the general distribution of the glands, the abdomen was cut from the anterior part of the body, and a slit through the chitin was made all around its margin, thus sepa- rating the dorsal from the ventral wall. From these respective parts the fat and muscle were removed as carefully as possible so as not to injure the delicate hypodermis. It was found more advisable to fix the entire abdomen before making the incision around the edge, because the greater rigidity after fixation facili- tated cutting, and prevented the parts from curling, as when fresh material was cut. Both dorsal and ventral abdominal walls were then stained with Grenacher’s borax carmine and Delafield’s haematoxylin, and mounted in balsam. Better results were obtained with the latter stain. The gland cells are so small that they can hardly be distinguished from the normal hypodermis except for the larger size of their nuclei, and these were rendered more conspicuous by using Delafield’s haematoxylin and destaining with acid alcohol until the nuclei stood out prominently, while the surrounding cytoplasm of the cells was but faintly tinged. The nuclei are even better accentuated by dipping the material in alkaline alcohol after destaining in acid alcohol, as they take on a deep blue coloration after such treatment. As the glands are most prominent at the anterior portion of each segment, and as this portion is covered by the posterior part of the preceding segment, some difficulty was encountered in making out their distribution. However, by injecting the abdomen with killing fluid by means of a hypodermic needle so that the intersegmental membranes were stretched taut on a plane with the rest of the tergite or sternite, all the glandular areas were WAX GLANDS OF THE COCKROACH 567 exposed. Bouin’s fluid proved the most practical for this pur- pose. When the parts were fully extended, they were plunged into hot killing fluid and the pressure maintained until the hot- killing fluid without and the killing fluid within had so fixed the material that the parts would remain extended when the pressure from the hypodermic needle was released. For histological study the material was fixed in Fleming’s (strong fluid), Gilson’s, Zenker’s, Bouin’s, and Dietrich’s fluids, Best cytological results were obtained with Fleming’s, although Dietrich’s and Bouin’s fluids gave almost as good results. With both of the latter‘more rapid penetration certainly was obtained. Gilson’s and Zenker’s fluids gave almost similar results. At first, material was imbedded entirely in paraffin, but when sections were cut, it was found that the chitin was very brittle and broke very readily, often catching on the edge of the knife and tearing gaps through the entire section. Because of this, sections were so mutilated that they were often useless, especially as the parts desired for study were immediately under the chitin. To obviate this difficulty several methods were tried either to soften the chitin or else to hold the parts in place so that they would maintain their normal position as nearly as possible. Metalinkoff’s method, by which the object was first h- bedded in paraffin in the usual way, and then all the paraffin scraped from the surface of the chitin, the object immersed in Eau de Javelle for twenty-four hours, and re-imbedded, did not give good results. But as this method was tried in only a few cases perhaps repeated trials would have proved more successful. Bedau’s ‘Seifenspiritus’ method gave no results, although fol- lowed as exactly as possible. The combined paraffin and celloidin method was also used. Three different modifications of this method were employed- Gilson’s rapid process, Apathy’s method, and Hoffman’s method. All three gave good results, and longitudinal sections through the entire abdomen, 5 micra thick, were easily obtained. Cross and longitudinal sections of the entire abdomen were cut from 4 to 10 micra in thickness. .These were fixed to slides by means of the Mayer albumen-and-water method. Instead, 568 E. H. DUSHAM

however, of first rubbing a drop of albumen on the slide and then adding water, three drops of the albumen were added to a watch- glass of distilled water, thoroughly mixed with it, and a layer of this was applied to the slide with a pipette. Sections were then placed on this and flattened by means of heat. By this method the least amount of albumen necessary remains on the slide, but always enough to hold the sections firmly in place. Sections were stained with Heidenhain’s iron haematoxylin and Delafield’s haematoxylin with eosin as a counterstain. Bet- ter cytological results were ,obtained with the former, especially after fixation with Fleming’s fluid, although good results were also obtained after fixation with Bouin’s fluid. However, for general histological work, Delafield’s haematoxylin and eosin gave very good preparations. Prepared mounts of the entire dorsal and ventral walls, sup- plemented by cross and longitudinal sections of the entire abdomen, showed that these modified hypodermal cells were present in each segment, not only on the dorsal side of the male, but on the ventral side also, and in the female as well as the male (figs. 1, 2, 3, and 4). Except in the intersegmental membranes, they are scattered over each tergite and sternite, being extremely abundant at the fore part of each segment laterad of the median line, especially that part of the segment which is overlapped by the preceding segment. Posterior to this region, i.e., where the tergite or sternite is not covered by the preceding segment, these modified cells are more scattered, occurring here and there among the normal hypodermal cells. Longitudinal sections show that in the intersegmental membranes there is but a single layer of unmodified hypodermal cells (fig. 8, i, and fig. 9, d). The nuclei of these cells are somewhat elongate and flattened, with their long axes parallel to the surface of the overlying cuticula; they are surrounded by but a small amount of cytoplasm, so slight in cases that it appears thread- like with the nuclei bulging out here and there. The nuclei themselves are very deeply stained and are fairly regularly arranged. WAX GLANDS OF THE COCKROACH 569

At the anterior end of each segment-that part which is overlapped by the preceding segment-the modified hypodermal cells are especially well developed, of ten producing the appearance of two or more layers of cells, no doubt due to the crowding of the individual cells, with the consequent displacement of their nuclei (figs. 8 and 9). This is best seen on the ventral side of the male where the cells are closely packed together so that there seems to be no order to their arrangement. In such places the cells also appear of different sizes, no doubt due to the fact that a single section did not pass through the middle of each cell. In other places they are but a single layer in thickness. These modified hypodermal cells possess large vesicular nuclei with relatively thick nuclear walls, and provided with a nucleolus and many deeply stained chromatin granules (fig. 10). Their cytoplasm is also coarsely granular in appearance, and contains several vacuoles. As a rule, they are cuboidal or columnar, except where crowding together his caused them to assume various forms. Scattered here and there between these cells and the overlying cuticula, or intercalated somewhat between them at their upper surface, are the non-glandular hypodermal cells with nuclei similar to those in the intersegmental areas. Posterior to that portion of the segment which is overlapped by the preceding segment, the normal hypodermal cells are in the majority, while here and there scattered among them are large modified cells similar to those occurring at the anterior end of the segment. Where such cells occur, the ordinary hypodermal cells are absent or else are somewhat crowded together. From the foregoing observations, therefore, it will be seen that the body wall of the cockroach does not differ materially from that of other insects, being composed of a cuticula, a hypodermis consisting of a single layer of cells, and a basement membrane. However, many of the hypodermal cells have become specialized, increasing in size, so that in places, particularly on the ventral side of the male, they present the appearance of two or more layers. The cytoplasm of these cells has become strongly granular while the nuclei have become large and vesicular. Their entire structure, therefore, indicates that they are secretory in

THE JOURNAL OF MORPHOLOOY, VOL. 31, NO. 3 570 E. H. DUSHAM nature, and are therefore gland cells. Their secretion passes to the exterior through very minute pores through the chitin (fig. 7). These are evident only in very thin sections, cut exactly perpendicular to the surface of the cuticula. Otherwise it is almost impossible to make them out. With favorable sections they show as a fine streaking of the cuticula’ (fig. 10, p). In portions of the cuticula stained black with osrnic acid the pores also show as minute white dots under an oil-immerson lens. The secretion passes through these pores to the exterior and without doubt spreads in a fine film over the outer surface of the cuticula. NATURE OF THE SECRETION

Mingazzini, in his work on Periplaneta orientalis, stated also that these modified cells were glandular in function and probably secreted an oily substance. 80 much did these cells resemble, both in appearance and position, those cells in Apis, Trigona, Bombus, Melepona, and Aphrophora, which were definitely known to secrete wax, that it occurred to the writer that possibly they might also perform this same function in the case of the cockroach. Tests were accordingly made to see if wax was present in these insects. A large number of the insects were cap- tured and thrown into a bottle of warmed chloroform. After remaining in this liquid for ten minutes, they were strained out before the chloroform could have had time to have penetrated into the interior of the insect and attacked the fat in the fat cells. The chloroform was then filtered and divided into two portions. From one of these portions the chloroform was entirely evaporated on a water-bath. When the chloroform hiid been entirely driven off, a brownish liquid was left which possessed the characteristic cockroach odor and which, on cooling, solidified into a greasy appearing substance. The next point was to find out whether this subsfance was grease or a wax. It is well-known that fatty oils, or fats treated with basic hydroxides, are decomposed into fatty acids and glycerol, this decomposition being hastened by using alcohol as a solvent for the alkali. Both the fatty acids and glycerol are WAX GLANDS OF THE COCKROACH 571 soluble in water, so that when water is added no precipitate would be formed. On the other hand, waxes, when similarly treated, yield smaller amounts of fatty acids and, instead of glycerol, give a large proportion of alcohol of the CnHanclseries, which is a solid body insoluble in water. Consequently, when water is added to these, a distinct precipitate is formed. The greasy appearing material obtained from the cockroaches was treated in this way. A small amount of pure beeswax was similarly treated as a check. In both cases, after the two sub- stances had been saponified and the liquid evaporated over st water-bath, a decided precipitate was obtained on the addition of water, more heavy in the case of beeswax, no doubt because more of the wax was present. It seemed, therefore, that the material secreted on the outside of the cockroach was wax. In order to substantiate the preceding conclusion, a series of solubility tests were made with the second portion of the chloroform in which the roacies had been dropped. These tests were similar to those carried out by Hollande ('14) in his research on the oenocytes of insects, when he demonstrated that the crystals found in these bodies were wax crystals. In order to obtain wax for these tests a few drops of the chloroform was placed on a glass slide and then subjected to a current of air. By this means the chloroform was quickly evaporated, leaving a small residue of wax, appearing as a whitish spot on the slide. Slides thus prepared were immersed in the various reagents for periods vary- ing from fifteen minutes to one-half hour. Observations were then made to see whether or not the material had been dissolved. Beeswax and wax from Pseudococcus citri dissolved in chloroform were used as checks. The results are as per table, page 572. The saponification and solubility tests therefore clearly indicate that wax is present on the body of the cockroach. There- fore, because wax is @resent on the bodies of these insects and glands are found in their abdomens resembling glands found in other insects which are known to secrete wax, it seems conclu- sive that these modified hypodermal cells are true wax glands. 572 E. H. DUSHAM

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COCKROACH PS E UDOCOCCUS REAGENT REEIWAX MATERIAL WAX - Insoluble Insoluble Insoluble Insoluble Insoluble Insoluble ...... Insoluble Insoluble Insoluble Alkaline soap. sol ...... Insoluble Insoluble Insoluble Citric acid...... Insoluble Insoluble Insoluble Tartaric acid...... Insoluble Insoluble Insoluble Oxalie acid ...... Insoluble Insoluble Insoluble Acetic acid...... Insoluble Insoluble Insoluble H2S04...... Insoluble Insoluble Insoluble HNO, ...... Insoluble Insoluble Insoluble HCL! ...... Insoluble Insoluble Insoluble Cold absol. alcohol...... Insoluble Insoluble Insqluble Acetone...... Insoluble Insoluble Insoluble Olive oil ...... Insoluble Insoluble Insoluble Cold alcoholic potash...... Insoluble rnsoluble Insoluble Ether ...... Soluble Soluble Soluble Chloroform...... Soluble Soluble Soluble Xylol ...... Soluble Soluble Soluble No coloration No coloration No coloration Sodium carbonate...... Insoluble Insoluble Insoluble Ammonium carbonate...... Insoluble Insoluble Insoluble

DEVELOPMENT OF THE GLANDS

Mingazzini, in his work on Periplaneta orientalis, found that these gland cells were less developed in the nymphs than in the adult. In such immature stages he found that sections showed only a single layer of cells, between which were observed fre- quently large modified cells. In Blatta germanica these gland cells were first distinguished during the first instar when the nymphs are two days old (fig. 11). At that time some of the normal hypodermal cells become modified. The nuclei which are deeply stained and densely granular, gradually increase in size still retaining their densely granular appearance. There\ is also an increase in the amount of cytoplasm around these nuclei, with the result that the adjacent normal hypodermal cells are crowded so that their regular arrangement is somewhat broken. The growth in size of the nucleus and the increase of cytoplasm also cause these cells to project below the level of the normal WAX GLANDS OF THE COCKROACH 573 hypodermis, so that they stand out rather conspicuously. Fi- nally, the cytoplasm becomes granular, the dense chromatin granules in the nucleus seem to assemble in large clumps, and the nucleolus, which up to this time could not be distinguished, becomes clearly visible. The transition from normal hypodermis to glandular cells can thus be readily observed in two-day-old nymphs. In the third instar the condition characteristic of the adult is attained, the cells being fully developed, and in some cases so closely packed $ogether as to simulate the appearance of two or more layers.

FUNCTION OF THE WAX The secretion of wax in insects serves various purposes-protection against enemies, cold, and moisture, a protection for eggs, an encasement for excreta, a lining for the larval burrow, etc. In the cockroach it probably serves as a protection, against moisture, for they live in protected places where other enemies are unable to attack them. On the other hand, their presence in warm, damp places intimately associated with water would seem to indicate that the wax was secreted to protect them from this. The fact that the wax possesses the characteristic ‘roachy’ odor suggested the idea that it might be a protection against enemies on account of its smell. However, an examination of beeswax and Pseudococcus wax showed that these also possessed characteristic odors. That of beeswax is well known. The odor from Pseudococcus was like mouldy wet leaves. That of the cockroach has already been described. From this it would seem that the wax secreted by each different family of insects had an odor ‘sui generis.’ The fact that the ‘roachy’ odor remains in roach-infested places for a considerable period of time after the roaches have been driven away or exterminated and that it is difficult to remove it from dishes and kitchen utensils may be accounted for by the fact that the wax remained adhering to these things over which they were accustomed to run. The writer wishes to acknowledge his sincere thanks.to Dr. W. A. Riley, whose advice and criticism have been invaluable and whose encouragement has made this work possible. 574 E. H. DUSHAM

BIBLIOGRAPHY

ALLEN 1910 Commercial organic analysis, vol. 2. BERLESE,A. 1909 Gli Insetti, pp. 470-471, 496-502. BUGNION,M. E. 1908 Glandes cirieres de Flata (Phromia) marginella (Ful-

goridae). Archiv des Sciences Physiques et Naturelles, T. 25. 9 CARLET,G. 1890 Sur les organes secreteurs et la secretion de la cire chez l’abeille. Comptes Rendus de 1’Academie des Sciences, Paris, T. 110. DEEGENER,P. 1912 Handbuch der Entomologie, S. 37-42. DREYLING,L. 1903 Vber die wachsbereitenden Organe der Honigbiene. Zool. Anz., Bd. 26. 1903 Weitere Mitteilungen uber die wachsbereitenden Organe der Honigbiene. Zool. Anz., Bd. 27. 1905 Beobachtungen uber die wachsbereitenden Organe bei den Hum- meln, nebst Bemerkungen uber die homologen Organe bei Trigonen. Zool. Anz., Bd. 29. 1905 Die wachsbereitenden Organe bei den geselliglebenden Bienen. Zool. Jahrb., Bd. 22. DUJARDIN,M. F. 1849 MBmoire sur 1’6tude microscopique de la cire. Annales des Sciences Naturelles, T. 12; 3d ser. GUILLBEAU,B. H. 1908 The origin of the froth in spittle insects. Amer. Nat., vol 42. HOLLANDE,A. 1914 Les cerodecytes ou oenocytes des insectes consideres ail point de vue biochimique. Archiv d’Anat. Micr. MAYER,P. 1891-93 Zur Kentniss von Coccus cacti. Mittheil. Zoolog. Station zu Neapel, Bd. 10. METALINKOFF,S. 1904 Sur un procede nouveau pour faire des coups micro- scopiques dans les animaux pourvu d’un tegument chitineux epais. Archiv Zool. Exp.; Notes et Revue, T. 4, ser. 2. MIALL, L. C., AND DENNY,A. 1886 The structure and life history of the cockroach. MINCHIN,E. A. 1888 Note on a new organ and on the structure of the hypodermis in Periplaneta orientalis. Jour. of Micr. Sci., N. S., vol. 29. MINGAZZINI,P. 1889 Recerche sulla struttura dell’ipodermide nella Peri- planeta orientalis. Atti della Reale Accademia dei Lincei, vol. 5, ser. 4. NUSSLIN,0. 1900 Zur Biologie der Schizoneuriden Gattung Mindarus Koch. Biol. Centralblatt, Bd. 20. OETTINGER,0. 1906 Uber die Drusentaschen am Abdomen von Periplaneta orientalis und Phyllodromia germanica. Zool. Anz., Bd. 30. PACKARD,A. S. 1898 Text-book of Entomology, pp. 361-366. SUNDWICK,E. E. 1907 uber das Wachs der Hummeln. Zeit. f. Physiol. Chemie, Bd. 53. VIALLANES,H. 1882 Histologie et developpement des Insectes. Annales des Sciences Naturelles, T. 14, ser. 6. WITLACZIL,E. 1885 Die Anatomie der Psylliden. Zeit. f. wiss. Zool., vol. 42. PLATES

575 PLATE I

EXPLANATION OF FIGURES 1 Dorsal abdominal surface of male, showing distribution of the wax glands. 2 Ventral abdominal surface of male, showing distribution of the wax glands.

576 WAX GLANDS OF THE COCKROACH PLATE 1 E. H. DUSHAM

577 PLATE 2

EXPLANATION OF FIGURES 3 Dorsal abdominal surface of female, showing distribution of the wax glands. 4 Ventral abdominal surface of female, showing distribution of the wax glands.

578 WAX GLANDS OF THE COCKROACH PLATE a E. H. DUBHAM

579 PLATE 3

EXPLANATION OF FIGURES 5 Surface view of the wax glands (focus below the non-glandular hypodermis). X 600. 6 Surface view of normal hypodermis of the intersegmental membrane. X WO. 7 Surface view of cuticula overlying the glandular areas showing pores. X 600. 8 Longitudinal section through a single intersegmental area on the dorsal surface of the female, lateral to the middle line, showing the appearance and location of the wax glands. i, intersegmental membrane; c, cuticula; h, hypodermis; g, wax glands; b, basement membrane. X 160. 9 Longitutinal section through a single intersegmental area on the ventral surface of the male, showing appearance and location of the wax glands. a, muscles; c, wax glands, b, basement membrane; d, intersegmental membrane; e, hypodermis; j, cuticula. X 160. 10 Portion of the body wall in the region of the wax glands, showing detailed structure. n, non-glandular hypodermis; p, pores; c, cuticula; v, vacuole; T, basement membrane. (No. 10 ocular, 1.8-mm. oil immersion.) 11 Longitudinal section through a single intersegmental area of a two-day- old nymph, showing the development of the gland. u, hypodermis; t, transform- ing hypodermal cell; s, fully-transformed cell. X 600.

580 WAX GLANDS OF THE COCKROACH PLATE 3 E. H. DUEHAM

6 7 5

10 11 581