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Anatomy, histology, and secretions of the salivary glands of Sphaerodema rusticum (F.) (Hemiptera: Belostomatidae)
Int. J. Insect Morphol. & Embryol. 4 (1): 35-47. 1975. Pergamon Press. Printed in Great Britain.
ANATOMY, HISTOLOGY, AND SECRETIONS OF THE SALIVARY GLANDS OF SPHAERODEMA R U S T I C U M (F.) (HEMIPTERA" BELOSTOMATIDAE) MAHMUD-UL AME~N and NINA ABEt)IN Department of Zoology, University of Dacca, Dacca 2, Bangladesh
(Accepted 23 July 1974) Abstract--The salivary glands of Sphaerodema rusticum consist of paired principal
and accessory glands. Each principal gland is made up of a superior and an inferior lobe; the latter is differentiated into 3 regions. The accessory glands are vesicular anteriorly and tubular posteriorly. The salivary ducts originate from the hilus of the principal glands; the accessory gland ducts also open into the hilus. The salivary ducts of 2 sides meet within the salivarium to form a very short common duct. Each lobule of the principal glands consists of a syncytial epithelium, enclosing a large lumen. The lobules open either into the central longitudinal canal or into another lobule. The inferior lobe homogenate contained protease and amylase, and that of the superior lobe only amylase. The accessory glands showed weak amylase activity; they also contained a watery fluid in the vesicular part. Index descriptors (in addition to those in the title): Salivary enzymes. INTRODUCTION THE SHAPE and size of the salivary glands are highly variable in different insects and in some, notably the Hemiptera, they are made up of several lobes with different structure and function. In most insects the glands are acinous, but in some they are tubular. Baptist (1941) listed the more important works on hemipteran salivary glands up to 1939, and recorded the anatomy, histology, and enzyme contents of the salivary glands of 20 species of Hemiptera. Nuorteva (1954, cited in Bronskill et al., 1958) made a similar study on 8 species of wheat-injuring Hemiptera, Southwood (1955, cited in Bronskill et al., 1958) on terrestrial Heteroptera, and Bronskill et aL (1958) on Oncopeltusfasciatus. Some of the other works on the hemipteran salivary glands are those of Negi (1934) on Laccifer lacca, Quayum (1959) on Ranatra, Rastogi (1961) on Sphaerodema rusticum, Khanna (1964) on Dysdercus koenigi, Quayum (1966) on D. cingulatus, Bose and Sen 0969) on Tessarotoma javanica, and Saxena and Chada (1971) on Schizaphis graminum. The giant water bug, S. rusticum is a common aquatic insect in Bangladesh. It is found among weeds in shallow water zone of ponds, lakes, canals, and other bodies of standing water. The bugs pass their entire life cycle in water. They are gregarious in habit, clinging to partially or totally submerged aquatic weeds like Hydrilla. They are carnivorous, feeding on small fish and other aquatic animals. They have large salivary glands. In this paper the anatomy and histology of the salivary glands of S. rusticum, and the nature and localization of certain digestive enzymes in various regions of the glands have been reported. Our observations differ on certain points from those of Rastogi (1961). 35
36
MAHMUD-UL AMEEN a n d NINA ABEDIN
MATERIALS
AND
METHODS
Only adults of S. rusticum were studied. The bugs were collected from various ponds and artificial lakes in Dacca city. They were kept in small aquaria with tap water and aquatic weeds, and were fed on small fish collected from the drains. For anatomical observations, the insects were dissected either in saline, or in distilled water under a stereoscopic binocular microscope. To facilitate observations the dissected glands were stained in methylene blue, borax carmine, or cotton blue (aniline blue) in lactophenol and then studied under a compound microscope. For histological work the glands were fixed either in Carnoy's, or Bouin's fluid; the former gave better results. The tissues were dehydrated in grades of ethyl alcohol, and embedded in paraffin wax through benzene, sectioned at 6-7/z, and stained in Delafield's haematoxylin and eosin or in Bremer's haematoxylin without counterstaining. The sections were mounted in canada balsam. Tissues that were not immediately embedded were stored in 1% Parloidin (a substitute for Celloidin) in methyl benzoate; when necessary these tissues were embedded in paraffin wax through benzene. Dissections for histological work were made directly in the fixative as the dissections took a long time due to the very hard integument, especially in the thoracic region. The drawings were made with the help of a camera lucida.
Detection of the digestive enzymes Protease. The presence ofprotease was determined by the gelatin slide method of Pickford and Dorris (1934). Different lobes of the salivary glands from 4 insects were isolated in normal saline and kept in watch glasses cooled by keeping them on ice. To this a drop of phosphate buffer solution (pH 7-5) and a drop of toluene were added. Homogenates of the different lobes were prepared with 0.5 ml of phosphate buffer in a glass homogeniser. Three separate drops of the homogenate were placed on each gelatin slide; another gelatin slide with 3 separate drops of buffer solution only was used as the control slide. The slides were then incubated for 2 hr at 37°C. Amylase and disaccharidases. The technique of Swingle (1928, in Strivastava, 1957) was followed for the detection of amylase and disaccharidases. Three percent, I'I/o, and 0.5"/,, solutions of starch, and 1o//o and 0.5% solutions of sucrose, maltose, lactose, and glucose were prepared with distilled water in conical flasks. Eight drops of toluene were added to each flask to prevent bacterial fermentation. Different lobes of the salivary glands of the 5 insects were separated and placed on cooled watch glasses with a drop of phosphate buffer (pH 6.5) and toluene. Homogenates of the different lobes were prepared separately with 0-5 or 1 ml of the buffer solution and placed in test tubes. One ml of the starch or disaccharide substrate was added to each test tube containing the homogenates. Control tubes contained only the starch or disaccharide substrate and 0.5 or I ml of the buffer. The tubes were then incubated for l hr at 37°C in a water bath. After incubating, 1-2 drops of Lugol's iodine solution was added to the test tubes containing starch substrate and the homogenate; colour change in the tubes indicated the presence or absence of amylase activity. Osazone test was carried out to determine the presence of disaccharides. Formation of glucose osazone crystals in the incubated homogenates would indicate disaccharidase activity. Lipase. For the detection of lipase the method given by Baldwin and Bell (1955) was
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F).
37
followed. However, Bromophenol Blue was used as the indicator instead of Phenol Red. Phosphate buffer ofpH 7.5 was used. Experiments were repeated with 10, 5, and 2 drops of olive oil into 4, 2, and 1 ml of ethyl alcohol respectively. One and 0.5 ml of the homogenate of each lobe was used for these tests. The incubation period was 10-20 min and 1 hr.
OBSERVATIONS
Gross anatomy
The salivary glands of S. rusticum consist of a pair of principal and accessory glands (Fig. l), lying in the thoracic cavity on either side of the alimentary canal. Both the principal and accessory glands receive rich tracheal supply. The glands lie more or less free in the body cavity except for the tracheal attachments.
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Acc D
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t
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i
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-ACe GI Inf L - -
1
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FIGS. 1-2. Salivary glands of S. rusticum. I. Principal and accesssory glands with their ducts (of one side only). 2. Accessory gland (magnified).
38
MAHMUD-UL AMEEN and NINA ABEDIN
Acc.D. Acc. Gl. Ant. Lb. C.D. Cent. L.C. Hi. Inf. L. Lb. Mid. Lb. N Neck Post. Lb. Sal. D. Sal. Sy. Secon.
-= = = ---= -----
Secr. Sup. L. Tr. Tub. Pt. Ves. Pt.
----
Abbreviations used in figures Accessory gland duct. Accessory gland. Anteriormost lobule of inferior lobe. Common salivary duct within salivary syringe, Central longitudinal canal. Hilus. Inferior lobe of principal salivary gland. Lobule of the inferior lobe. Lobules of middle region of inferior lobe. Nucleus. Elongated neck of a lobule opening directly into central longitudinal canal. Lobules of posterior clumped region of inferior lobe. Salivary duct. Salivary syringe. A lobule opening into a second Iobule instead of opening into central longitudinal canal. Secretory material in lumen of lobule. Superior lobe of principal salivary gland. Trachea. Posterior tubular part of accessory gland. Anterior vesicular part of accessory gland.
Principal gland and its ducts The principal gland is a bilobed structure (Figs. 1, 3). Unlike in other Heteroptera (Baptist, 1941; Bronskill et al., 1958) the smaller lobe remains above the larger lobe in S. rusticum. Therefore, the former is designated as the superior and the latter as inferior lobe in this report instead of the usual anterior and posterior lobe designations. The superior lobe is about 4 m m long. The inferior lobe is about 5.5 m m long and extends backwards up to the anterior end o f the midgut. Each superior lobe is connected to the inferior lobe by a very short duct. This duct emerges transversely from the anterior half o f the superior lobe and meets the main salivary duct as it emerges from the inferior lobe, their meeting point forming a hilus (Fig. 4). The main salivary duct extends from the hilus forward and cephalad. The duct is about 5.2 m m long. The 2 main salivary ducts from the 2 principal glands meet in the head region to form a very short c o m m o n duct within the salivarium (Fig. 5). Another duct proceeds cephalad from the hilus, and makes a sharp kink near the cephalothoracicjunction and then proceeds backwards to open into the accessory gland (Fig. 1). The total length o f this accessory gland is about 5.5 mm. Both the superior and inferior lobes are made up of many globular acini or lobules, which open into a central longitudinal canal. This canal in turn opens into the hilus. Owing to this arrangement the principal gland appears like a bunch of grapes. The lobules are more or less uniform in shape and size in the superior lobe. In the inferior lobe (Figs. 1, 3), on the other hand, the lobules have become differentiated into 3 types - - t h e 2 anteriormost lobules of the inferior lobe are larger than the others, usually bluish in colour, and about 0.6 m m in diameter. The 2 large lobules open directly into the hilus. The middle region of the inferior lobe consists of lobules having a diameter of about 0 . 3 ~ 0 " 5 mm. The posterior region is made up o f small and compactly arranged lobules. Accessorv glands The accessory gland is about 8'4 mm long and differentiated into 2 parts (Figs. 1, 2). The anterior part is vesicular, spindle-shaped, transparent, and about 2 m m long. Posteriorly,
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F.)
39
this part tapers into a tubular structure, somewhat opaque and cream-coloured, which ends blindly. The tubular part is about 6.4 mm long, and is arranged in a zig-zag manner attached to the gut wall by tracheal branches. /_ ~J
Said / . AccD
Sup.L Ant Lb
MidLb
0.[
Post.Lb.
FIG. 3. S. rusticum. Principal salivary gland. Salivary syringe The salivary syringe is a conical, transparent, and cuticularized structure, lying below the hypopharynx. The 2 salivary ducts end inside this syringe (Fig. 6) and the syringe in turn opens into the salivary canal inside the rostrum. Histology The acini or lobules consist of a thin outer membrane surrounding the salivary epithelium, enclosing a large lumen. The diameter of the lumen varies from about 600/z in the 2 anteriormost lobules of the inferior lobe to about 110/z in the posterior lobules. However, the usual diameter of most of the lobules is about 200-250/~. The epithelium is one layered and
40
MAHMUD-UL AMEEN and NINA ABEDIN
¢
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I
q
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J S
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Lb.
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/ ~i,', ' L
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....
- Lb.
6 4
Flu. 4. S. rusticum. Part of L.S. of principal salivary gland showing hilus in inferior lobe
and the ducts.
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaeroderna Rusticum (F.)
41
syncytial, containing several nuclei with nucleoli (Figs. 7, 8). It is not uniform in thickness throughout the lobules. The height of the epithelium varies from 12 to 73/z. The sections show that the lobules open either into the central longitudinal canal through a narrow neck or they may open into another lobule (Fig. 9) when a large number of lobules are closely aggregated together.
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\,
FIGS. 5-6. S. rusticum. 5. Formation of short common salivary duct within salivary syringe. 6. Salivary syringe. Sections of the central longitudinal canal show that it consists of a thin syncytial epithelium. It stains lighter than the epithelium of the iobules. The diameter of the canal is about 42 /z. Sectioning of the salivary and accessory ducts, and the accessory gland is rather difficult, and good preparations were not available for study. The secretions stain differently and vary in nature in different regions of the inferior lobe; the secretion in the posterior region is highly basophilic, and appears as a homogenous mass under lower magnification, whereas in other regions the secretion is eosinophilic and granular. In the superior lobe the secretion is grandular and eosinophilic throughout the entire lobe. A comparison of the sections of salivary glands from starved and fed insects show definite secretory changes in the fed insects. The nucleoplasm appears like a network and the nuclear membrane disappears. The cytoplasm appears to be in a state of reorganization around the nucleus, and the epithelium becomes thicker than usual, as high as 73/z compared to 12-30/z in starved insects. Secretion droplets, surrounded by the plasmalemma of the cells, were given out from the apical border of the cells (Fig. 10). In the classical sense the secretion has to be classified as of 'merocrine' type; but following Shubnikova's (1967) classification, the secretion seems to be of 'lemmocrine' type. The freshly secreted material appears different in form and colour from that which has been secreted sometime earlier. Insects starved for a long time show degeneration of glandular epithelium of the lobules, the cytoplasm appears to have moved towards the lumen, separating from the surrounding membrane. The same situation was observed repeatedly in all sections indicating that it was not due to bad preparation.
42
MAHMUD-UL AMEEN and NINA ABEDIN
• "'.. ::': :-;.i"i';.i." ".3" ..
. .,.:...~
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FiG. 7. S. rusticum. Part of L.S. of superior lobe of salivary gland.
Digestive enzymes in the salivary glands Protease. The inferior lobe of the principal gland showed strong protease activity. Clear circular rings were formed around all the 3 drops of homogenate on the gelatin slide. No activity was indicated on either of the slides containing homogenate from the superior lobes of the principal glands or the accessory glands, Amylase• Tests with 3%, starch solution gave no indication of amylase activity. However, when tested with 1% starch solution as substrate, the inferior lobe homogenate indicated strong and the superior lobe homogenate showed weak amylase activity. Further tests with 0.5°/,, starch solution showed strong amylase activity in both the inferior and superior lobes of the principal gland; and the accessory gland homogenate indicated weak amlyase activity. Tests with 0.5% starch solution and homogenates from the 3 different regions of the inferior lobe gave interesting results. On addition of Lugol's Iodine solution, the contents of each of the test tubes containing homogenates of the 2 anterior most lobules and the posterior clumped region turned mauve, whereas the contents of the tube containing the homogenate of the middle region became colourless.
Anatomy, Histology, and Secretions of the Salivary Glands of S p h a e r o d e m a R u s t i c u m (F.)
~.~~ "
"'
"
.,~,,:,,
'
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43
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FIG. 8. S. r u s t i c u m . Part of L.S. of inferior lobe of salivary gland.
Disaccharidases and lipase. Tests with 1% and 0.5% solutions of sucrose, maltose, and lactose as substrate at pH 6.5 gave negative results. Similarly tests with 10, 5, and 2 drops o f olive oil a t p H 7.5 g a v e n e g a t i v e r e s u l t s f o r l i p a s e a c t i v i t y in t h e v a r i o u s p a r t s o f t h e salivary glands. T h e d a t a o n t h e o c c u r r e n c e o f d i g e s t i v e e n z y m e s in t h e v a r i o u s l o b e s o f t h e s a l i v a r y g l a n d s o f S . r u s t i c u m h a v e b e e n s u m m a r i z e d in T a b l e 1. TABLE | .
O C C U R R E N C E OF DIGESTIVE ENZYMES IN VARIOUS LOBES OF SALIVARY G L A N D S OF S .
r u s t i c u m (F)
Principal gland Enzyme Protease Amylase Maltase Sucrase Lactase Lipase
Accessory gland Superior lobe
Inferior lobe
-÷ ÷ . ----
~+ t-- ÷ .
.
-t .
--
-- no activity, + weak activity, - - + strong activity.
---
44
M A H M U D - U L AMEEN a n d N I N A ABEDIN
:L 0 0 C~l
.C.
i ~i~,~
~:, ~i'
OZk !i!~ i¸¸i ):: ~~)ii:¸::~ 'i~ iii!,~i::~i¸~~:i~i!i~ili¸ii~i!~! ~: IO
FiGs. 9-10. S. rusticum. 9. T.S. of inferior lobe of salivary gland. 10. A salivary gland cell showing secretion.
DISCUSSION
Bugnion and Popoff (1910, cited in Baptist, 1941) and Dokroscky (1931, cited in Bronskill et al., 1958) considered a bilobed principal gland with a unilobular accessory gland as the primitive condition in Heteroptera. The situation in S. rusticum also shows the same condition.
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F.)
45
From a comparison of the present work with that described by Baptist (1941) it was found that in terms of arrangement and appearance, the salivary glands of S. rusticum closely resemble the salivary glands of Nepa cinerea. However, in S. rusticum the duct connecting the superior and inferior lobes emerges from an anteromedial position, instead of arising anteriorly from the inferior lobe as in Nepa (Baptist, 1941). That the 2 anteriormost lobules of the posterior lobes of Nepa were somewhat larger than the other lobules (Baptist, 1941) agrees with the condition in S. rusticum; but Baptist (1941) did not mention any further differentiation in the rest of the lobules of the posterior lobe of salivary gland in Nepa. Our observations on S. rusticum differ considerably from those described by Rastogi (1961). His (1961, Fig. 3) description and diagram of the salivary glands do not show clearly the differentiation among the lobules of the inferior lobe as shown in this paper. According to him the principal glands are aciniform and the acini decrease in size and number posteriorly. He also noted that the principal ducts of the salivary glands open in the salivary syringe and that the 2 ducts from the 2 sides do not join in the head. However, we found that they do meet in the head region forming a very short common salivary duct within the salivarium (Fig. 5). Rastogi's (1961, Fig. 3) diagram shows that 2 independent ducts issue from the 2 lobes of the principal gland which unite and continue as the main salivary duct; the accessory duct branches out from the main duct. This is quite different from our observations noted earlier and illustrated in Figs. 1, 3, and 4. Rastogi (1961) observed that the acini of the principal salivary glands were pyriform pockets, which opened into the central canal of the gland. In the present investigation it was observed that although most of the lobules opened into the central canal, nevertheless some of the peripheral lobules open into a second lobule instead of the central canal (Fig. 9). This is probably necessary for greater production and storage of the enzymes necessiated by the rapacious nature of the insects. Quick killing of the prey and extra intestinal digestion would need a large amount of enzyme secretion. Although not determined experimentally, observation on the feeding of S. rusticum indicate the presence of extra-intestinal digestion. After catching the fish, provided for feeding, with their forelegs the bugs push their stylets into the fish body; after feeding is completed parts of the prey (fish) become transparent, indicating that the tissue of that region has been dissolved during feeding by the bug. The anatomically differentiated regions of the inferior lobe also show differentiation in their staining and nature of the secretory products. These differences may be due to functional differences as was indicated by enzyme analysis. Tests showed that amylase was present in all the anterior, middle, and posterior lobules of the inferior lobe. The breakdown of starch to monosaccharides was indicated only in the middle lobules by the colourless end product in Lugol's iodine test. Whereas in the anterior and posterior lobules the mauve colour of the Lugol's iodine test indicated that the original starch substrate has been broken down to oligosaccharide stage only. This would indicate presence of oligosaccharidase in the middle lobules and its absence in the anteriormost and posterior lobules. But the tests for disaccharidases indicated absence of maltase, sucrase, and lactase. This might be explained either by assuming that the oligosaccharidase present was other than those for which tests were performed. Alternatively, the methods employed for the detection of oligosaccharidases were not fine enough to detect the small quantities of the oligosaccharidases present. As in Nepa (Baptist, 1941) and Ranatra (Quayum, 1959) the accessory glands consist of a vesicular and tubular part. However, in S. rusticum the tubular region is comparatively longer than that of Nepa or Ranatra. The transparency of the vesicular part shows that it
46
MAHMUD-UL AMEEN and NINA ABEDIN
acts only as a reservoir, and in freshly dissected insects it is usually distended due to the presence of a watery fluid. Goodchild (1966), in reviewing the evolutoin of the alimentary canal in the Hemiptera, noted that the salivary glands of the Heteroptera consist of a pair of basically two-lobed main glands and a pair of accessory glands terminating in thin walled vesicles. He thought that the function of such accessory glands in the zoophagous families may be that of recirculating water from the gut to ensure copious flow of saliva to wash out the dissolved internal organs of the prey. Baptist (1941) observed that the accessory glands of Heteroptera produced only a watery secretion and did not find any enzyme in it. Miles and Helliwell (1961) showed that an oxidase other than cytochrome oxidase was present in the accessory glands and salivary ducts of Aphanus sordidus. Bronskill et al. (1958) found slight enzyme (protease) activity in the accessory glands of Oncopeltusfasciatus. The present work also showed slight enzyme (amylase) activity in the accessory glands of S. rusticum.
Baptist (1941) noted that only 2 types of enzymes are found in any particular gland; the enzymes were always related to the type of food consumed, and were those concerned with the digestion of that particular component of the food, which was present in the greatest proportion. Bronskill et al. (1958) opposed the view by showing that 4 enzymes, viz., amylase, protease, invertase, and lipase were present in the salivary glands of O. fasciatus. During the present work only 2 enzymes, viz., protease and amylase were found in S. rusticum; but the enzyme analysis was performed at a p H of 7.5 only for protease and lipase, and at a pH of 6-5 only for amylase and disaccharidases. Therefore, the possibility cannot be ruled out that other enzyme also may be present. Ford (1962) reported the presence of lipase, protease, a - - and fl-- glucosidases, and a weak amylase in Dysdercus fasciatus. The findings of Bronskill et al. (1958), Ford (1962), and the presence of amylase in the basically carnivorous S. rusticum contradict the generalization made by Baptist (1941) that the enzymes or complement of enzymes present in the salivary glands are directly related to diet. Baptist (1941) and Day and Waterhouse (1953) concluded that there was no evidence that different materials were secreted by the various lobes of hemipterous salivary glands. This was contradicted by Bronskill et al. (1958) and the present findings also showed that the same lobe of the salivary glands may produce widely different secretions. Thus in S. rusticum the inferior lobe of the principal gland showed both protease and amylase activity. Acknowledgements--We are thankful to Dr. (Mrs.) Anwara Begum of this Department for criticism of the manuscript and to Dr. S. H. Chowdhury, Department of Zoology, University of Chittagong for valuable suggestions, critical reading of the manuscJipt, and translation of the Russian text. The observations contained in this paper formed a part of an M.Sc. Thesis submitted by the second author to the University of Dacca.
REFERENCES BALDWIN, E. and D. J. BELL1955. Cole's Practical Physiological Chemistry, 10th Ed., Heifer, Cambridge. BAPTIST,B. A. 1941. The morphology and physiology of the salivary glands of Hemiptera: Heteroptera. Quart. J. Microsc. Sci. 83: 91-139. BosE, K. C. and A. K. SEN. 1969. The morphology of the alimentary canal and salivary apparatus of Tessarotoma javanica Thunb. (Hemiptera: Pentatomidae). J. ZooL Soc. lndia 18: 94-103. (Not seen in original) BRONSKILL,J. F., E. H. SALKELD,and W. G. FRIEND. 1958. Anatomy, histology, and secretions of salivary glands of the large milkweed bug, Oneopeltusfaseiatus (Dallas) (Hemiptera : Lygaeidae). Can. J. Zool. 36: 961-68.
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F.)
47
DAY, M. F. and D. F. WATERHOUSE. 1953. Functions of the alimentary system, pp. 299-310. In K. D. ROEDER (Ed) Insect Physiology, Wiley, New York. FORD, J. B. 1962. Studies on the digestive processes of Dysdercus fasciatus Sign. Ann. AppL Biol. 50: 355. GOODCHILD, A. J. P. 1966. Evolution of the alimentary canal in the Hemiptera. Biol. Rev. (Cambridge) 41 : 97-140. ](HANNA, S. 1964. The digestive system of Dysdercus koenigi (Fabr.) (Hemiptera): (Pyrrhocoridae). Indian J. Entomol. 26: 46-61. MILES, P. W. and A. HELLIWELL. 1961. Oxidase activity in the saliva of a plant-bug. Nature (Lond.) 192: 374-75. NEGI, P. S. 1934. The alimentary canal, its appendages, salivary glands and the nervous system of the adult female lac Laccifer lacca, Kerr (Coccidae). Bull. Entomol. Res. 25: 541-46. PICKFORD, G. E. and F. DORRIS. 1934. Micro-methods for the detection of proteases and amylases. Science (Wash., D.C.) 80: 317-19. QUAYUM, M. A. 1959. Some studies of the salivary glands in Hemiptera (Heteroptera). M.Sc. Thesis,
Univ. Dacca, Bangladesh. QUAYUM, M. A. 1966. Studies on the salivary glands of Dysdercus cingulatus--Fabr. (Hemiptera). Pakistan J. Sci. Res. 18(2): 111-17. RASTOGI, S. 1961. The anatomy of the digestive organs of Sphaerodema rusticum Fabr. (Heteroptera: Belostomatidae). Proc. Rajasthan Acad. Sci. 8: 60-78. SAXENA, P. N. and H. L. CHADA. 1971. The green bug, Schizaphis graminum. 2. The salivary gland complex. Ann. Entomol. Soc. Amer. 64(4): 904-12. SHUBNIKOVA,E. A. 1967. Cytology and Cytophysiology of Secretory Process (in Russian). Moscow State Univ. Publications. SRIVASTAVA,P. D. 1957. Studies on the choice of food-plant and certain aspects of the digestive physiology of the larvae and adults of Athalia lugens proxima (Klug) and Epilachna vigintioctopunctata (F.). Bull. Entomol. Res. 48: 289-97.
ANATOMY, HISTOLOGY, AND SECRETIONS OF THE SALIVARY GLANDS OF SPHAERODEMA R U S T I C U M (F.) (HEMIPTERA" BELOSTOMATIDAE) MAHMUD-UL AME~N and NINA ABEt)IN Department of Zoology, University of Dacca, Dacca 2, Bangladesh
(Accepted 23 July 1974) Abstract--The salivary glands of Sphaerodema rusticum consist of paired principal
and accessory glands. Each principal gland is made up of a superior and an inferior lobe; the latter is differentiated into 3 regions. The accessory glands are vesicular anteriorly and tubular posteriorly. The salivary ducts originate from the hilus of the principal glands; the accessory gland ducts also open into the hilus. The salivary ducts of 2 sides meet within the salivarium to form a very short common duct. Each lobule of the principal glands consists of a syncytial epithelium, enclosing a large lumen. The lobules open either into the central longitudinal canal or into another lobule. The inferior lobe homogenate contained protease and amylase, and that of the superior lobe only amylase. The accessory glands showed weak amylase activity; they also contained a watery fluid in the vesicular part. Index descriptors (in addition to those in the title): Salivary enzymes. INTRODUCTION THE SHAPE and size of the salivary glands are highly variable in different insects and in some, notably the Hemiptera, they are made up of several lobes with different structure and function. In most insects the glands are acinous, but in some they are tubular. Baptist (1941) listed the more important works on hemipteran salivary glands up to 1939, and recorded the anatomy, histology, and enzyme contents of the salivary glands of 20 species of Hemiptera. Nuorteva (1954, cited in Bronskill et al., 1958) made a similar study on 8 species of wheat-injuring Hemiptera, Southwood (1955, cited in Bronskill et al., 1958) on terrestrial Heteroptera, and Bronskill et aL (1958) on Oncopeltusfasciatus. Some of the other works on the hemipteran salivary glands are those of Negi (1934) on Laccifer lacca, Quayum (1959) on Ranatra, Rastogi (1961) on Sphaerodema rusticum, Khanna (1964) on Dysdercus koenigi, Quayum (1966) on D. cingulatus, Bose and Sen 0969) on Tessarotoma javanica, and Saxena and Chada (1971) on Schizaphis graminum. The giant water bug, S. rusticum is a common aquatic insect in Bangladesh. It is found among weeds in shallow water zone of ponds, lakes, canals, and other bodies of standing water. The bugs pass their entire life cycle in water. They are gregarious in habit, clinging to partially or totally submerged aquatic weeds like Hydrilla. They are carnivorous, feeding on small fish and other aquatic animals. They have large salivary glands. In this paper the anatomy and histology of the salivary glands of S. rusticum, and the nature and localization of certain digestive enzymes in various regions of the glands have been reported. Our observations differ on certain points from those of Rastogi (1961). 35
36
MAHMUD-UL AMEEN a n d NINA ABEDIN
MATERIALS
AND
METHODS
Only adults of S. rusticum were studied. The bugs were collected from various ponds and artificial lakes in Dacca city. They were kept in small aquaria with tap water and aquatic weeds, and were fed on small fish collected from the drains. For anatomical observations, the insects were dissected either in saline, or in distilled water under a stereoscopic binocular microscope. To facilitate observations the dissected glands were stained in methylene blue, borax carmine, or cotton blue (aniline blue) in lactophenol and then studied under a compound microscope. For histological work the glands were fixed either in Carnoy's, or Bouin's fluid; the former gave better results. The tissues were dehydrated in grades of ethyl alcohol, and embedded in paraffin wax through benzene, sectioned at 6-7/z, and stained in Delafield's haematoxylin and eosin or in Bremer's haematoxylin without counterstaining. The sections were mounted in canada balsam. Tissues that were not immediately embedded were stored in 1% Parloidin (a substitute for Celloidin) in methyl benzoate; when necessary these tissues were embedded in paraffin wax through benzene. Dissections for histological work were made directly in the fixative as the dissections took a long time due to the very hard integument, especially in the thoracic region. The drawings were made with the help of a camera lucida.
Detection of the digestive enzymes Protease. The presence ofprotease was determined by the gelatin slide method of Pickford and Dorris (1934). Different lobes of the salivary glands from 4 insects were isolated in normal saline and kept in watch glasses cooled by keeping them on ice. To this a drop of phosphate buffer solution (pH 7-5) and a drop of toluene were added. Homogenates of the different lobes were prepared with 0.5 ml of phosphate buffer in a glass homogeniser. Three separate drops of the homogenate were placed on each gelatin slide; another gelatin slide with 3 separate drops of buffer solution only was used as the control slide. The slides were then incubated for 2 hr at 37°C. Amylase and disaccharidases. The technique of Swingle (1928, in Strivastava, 1957) was followed for the detection of amylase and disaccharidases. Three percent, I'I/o, and 0.5"/,, solutions of starch, and 1o//o and 0.5% solutions of sucrose, maltose, lactose, and glucose were prepared with distilled water in conical flasks. Eight drops of toluene were added to each flask to prevent bacterial fermentation. Different lobes of the salivary glands of the 5 insects were separated and placed on cooled watch glasses with a drop of phosphate buffer (pH 6.5) and toluene. Homogenates of the different lobes were prepared separately with 0-5 or 1 ml of the buffer solution and placed in test tubes. One ml of the starch or disaccharide substrate was added to each test tube containing the homogenates. Control tubes contained only the starch or disaccharide substrate and 0.5 or I ml of the buffer. The tubes were then incubated for l hr at 37°C in a water bath. After incubating, 1-2 drops of Lugol's iodine solution was added to the test tubes containing starch substrate and the homogenate; colour change in the tubes indicated the presence or absence of amylase activity. Osazone test was carried out to determine the presence of disaccharides. Formation of glucose osazone crystals in the incubated homogenates would indicate disaccharidase activity. Lipase. For the detection of lipase the method given by Baldwin and Bell (1955) was
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F).
37
followed. However, Bromophenol Blue was used as the indicator instead of Phenol Red. Phosphate buffer ofpH 7.5 was used. Experiments were repeated with 10, 5, and 2 drops of olive oil into 4, 2, and 1 ml of ethyl alcohol respectively. One and 0.5 ml of the homogenate of each lobe was used for these tests. The incubation period was 10-20 min and 1 hr.
OBSERVATIONS
Gross anatomy
The salivary glands of S. rusticum consist of a pair of principal and accessory glands (Fig. l), lying in the thoracic cavity on either side of the alimentary canal. Both the principal and accessory glands receive rich tracheal supply. The glands lie more or less free in the body cavity except for the tracheal attachments.
t !l
Acc D
!
t
~
: :~--
So~L
i
Ves
-ACe GI Inf L - -
1
TubPt
]r.
FIGS. 1-2. Salivary glands of S. rusticum. I. Principal and accesssory glands with their ducts (of one side only). 2. Accessory gland (magnified).
38
MAHMUD-UL AMEEN and NINA ABEDIN
Acc.D. Acc. Gl. Ant. Lb. C.D. Cent. L.C. Hi. Inf. L. Lb. Mid. Lb. N Neck Post. Lb. Sal. D. Sal. Sy. Secon.
-= = = ---= -----
Secr. Sup. L. Tr. Tub. Pt. Ves. Pt.
----
Abbreviations used in figures Accessory gland duct. Accessory gland. Anteriormost lobule of inferior lobe. Common salivary duct within salivary syringe, Central longitudinal canal. Hilus. Inferior lobe of principal salivary gland. Lobule of the inferior lobe. Lobules of middle region of inferior lobe. Nucleus. Elongated neck of a lobule opening directly into central longitudinal canal. Lobules of posterior clumped region of inferior lobe. Salivary duct. Salivary syringe. A lobule opening into a second Iobule instead of opening into central longitudinal canal. Secretory material in lumen of lobule. Superior lobe of principal salivary gland. Trachea. Posterior tubular part of accessory gland. Anterior vesicular part of accessory gland.
Principal gland and its ducts The principal gland is a bilobed structure (Figs. 1, 3). Unlike in other Heteroptera (Baptist, 1941; Bronskill et al., 1958) the smaller lobe remains above the larger lobe in S. rusticum. Therefore, the former is designated as the superior and the latter as inferior lobe in this report instead of the usual anterior and posterior lobe designations. The superior lobe is about 4 m m long. The inferior lobe is about 5.5 m m long and extends backwards up to the anterior end o f the midgut. Each superior lobe is connected to the inferior lobe by a very short duct. This duct emerges transversely from the anterior half o f the superior lobe and meets the main salivary duct as it emerges from the inferior lobe, their meeting point forming a hilus (Fig. 4). The main salivary duct extends from the hilus forward and cephalad. The duct is about 5.2 m m long. The 2 main salivary ducts from the 2 principal glands meet in the head region to form a very short c o m m o n duct within the salivarium (Fig. 5). Another duct proceeds cephalad from the hilus, and makes a sharp kink near the cephalothoracicjunction and then proceeds backwards to open into the accessory gland (Fig. 1). The total length o f this accessory gland is about 5.5 mm. Both the superior and inferior lobes are made up of many globular acini or lobules, which open into a central longitudinal canal. This canal in turn opens into the hilus. Owing to this arrangement the principal gland appears like a bunch of grapes. The lobules are more or less uniform in shape and size in the superior lobe. In the inferior lobe (Figs. 1, 3), on the other hand, the lobules have become differentiated into 3 types - - t h e 2 anteriormost lobules of the inferior lobe are larger than the others, usually bluish in colour, and about 0.6 m m in diameter. The 2 large lobules open directly into the hilus. The middle region of the inferior lobe consists of lobules having a diameter of about 0 . 3 ~ 0 " 5 mm. The posterior region is made up o f small and compactly arranged lobules. Accessorv glands The accessory gland is about 8'4 mm long and differentiated into 2 parts (Figs. 1, 2). The anterior part is vesicular, spindle-shaped, transparent, and about 2 m m long. Posteriorly,
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F.)
39
this part tapers into a tubular structure, somewhat opaque and cream-coloured, which ends blindly. The tubular part is about 6.4 mm long, and is arranged in a zig-zag manner attached to the gut wall by tracheal branches. /_ ~J
Said / . AccD
Sup.L Ant Lb
MidLb
0.[
Post.Lb.
FIG. 3. S. rusticum. Principal salivary gland. Salivary syringe The salivary syringe is a conical, transparent, and cuticularized structure, lying below the hypopharynx. The 2 salivary ducts end inside this syringe (Fig. 6) and the syringe in turn opens into the salivary canal inside the rostrum. Histology The acini or lobules consist of a thin outer membrane surrounding the salivary epithelium, enclosing a large lumen. The diameter of the lumen varies from about 600/z in the 2 anteriormost lobules of the inferior lobe to about 110/z in the posterior lobules. However, the usual diameter of most of the lobules is about 200-250/~. The epithelium is one layered and
40
MAHMUD-UL AMEEN and NINA ABEDIN
¢
Ant. Lb.
I
q
\,
" ...;~:,
J S
Acc. D Sal. D
Lb.
E E
',,
/ ~i,', ' L
,,..
.....~ ~--.-. -
....
- Lb.
6 4
Flu. 4. S. rusticum. Part of L.S. of principal salivary gland showing hilus in inferior lobe
and the ducts.
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaeroderna Rusticum (F.)
41
syncytial, containing several nuclei with nucleoli (Figs. 7, 8). It is not uniform in thickness throughout the lobules. The height of the epithelium varies from 12 to 73/z. The sections show that the lobules open either into the central longitudinal canal through a narrow neck or they may open into another lobule (Fig. 9) when a large number of lobules are closely aggregated together.
E E e,l 0
\,
FIGS. 5-6. S. rusticum. 5. Formation of short common salivary duct within salivary syringe. 6. Salivary syringe. Sections of the central longitudinal canal show that it consists of a thin syncytial epithelium. It stains lighter than the epithelium of the iobules. The diameter of the canal is about 42 /z. Sectioning of the salivary and accessory ducts, and the accessory gland is rather difficult, and good preparations were not available for study. The secretions stain differently and vary in nature in different regions of the inferior lobe; the secretion in the posterior region is highly basophilic, and appears as a homogenous mass under lower magnification, whereas in other regions the secretion is eosinophilic and granular. In the superior lobe the secretion is grandular and eosinophilic throughout the entire lobe. A comparison of the sections of salivary glands from starved and fed insects show definite secretory changes in the fed insects. The nucleoplasm appears like a network and the nuclear membrane disappears. The cytoplasm appears to be in a state of reorganization around the nucleus, and the epithelium becomes thicker than usual, as high as 73/z compared to 12-30/z in starved insects. Secretion droplets, surrounded by the plasmalemma of the cells, were given out from the apical border of the cells (Fig. 10). In the classical sense the secretion has to be classified as of 'merocrine' type; but following Shubnikova's (1967) classification, the secretion seems to be of 'lemmocrine' type. The freshly secreted material appears different in form and colour from that which has been secreted sometime earlier. Insects starved for a long time show degeneration of glandular epithelium of the lobules, the cytoplasm appears to have moved towards the lumen, separating from the surrounding membrane. The same situation was observed repeatedly in all sections indicating that it was not due to bad preparation.
42
MAHMUD-UL AMEEN and NINA ABEDIN
• "'.. ::': :-;.i"i';.i." ".3" ..
. .,.:...~
.:
.. , . . . . . . .
.,.
:if,~ ~. ; •: •
,..,
.' ' . "
' ,',F"'"
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.:
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...
~:::.:
: ',
~,'
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. . . . ...
..-.-'.i.:".•"..'~ .."
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7
FiG. 7. S. rusticum. Part of L.S. of superior lobe of salivary gland.
Digestive enzymes in the salivary glands Protease. The inferior lobe of the principal gland showed strong protease activity. Clear circular rings were formed around all the 3 drops of homogenate on the gelatin slide. No activity was indicated on either of the slides containing homogenate from the superior lobes of the principal glands or the accessory glands, Amylase• Tests with 3%, starch solution gave no indication of amylase activity. However, when tested with 1% starch solution as substrate, the inferior lobe homogenate indicated strong and the superior lobe homogenate showed weak amylase activity. Further tests with 0.5°/,, starch solution showed strong amylase activity in both the inferior and superior lobes of the principal gland; and the accessory gland homogenate indicated weak amlyase activity. Tests with 0.5% starch solution and homogenates from the 3 different regions of the inferior lobe gave interesting results. On addition of Lugol's Iodine solution, the contents of each of the test tubes containing homogenates of the 2 anterior most lobules and the posterior clumped region turned mauve, whereas the contents of the tube containing the homogenate of the middle region became colourless.
Anatomy, Histology, and Secretions of the Salivary Glands of S p h a e r o d e m a R u s t i c u m (F.)
~.~~ "
"'
"
.,~,,:,,
'
~ .~
.,~
~R~..-.._,.~?,'~
',~
Secr.
..'.
i¢
43
:.
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'~1!
FIG. 8. S. r u s t i c u m . Part of L.S. of inferior lobe of salivary gland.
Disaccharidases and lipase. Tests with 1% and 0.5% solutions of sucrose, maltose, and lactose as substrate at pH 6.5 gave negative results. Similarly tests with 10, 5, and 2 drops o f olive oil a t p H 7.5 g a v e n e g a t i v e r e s u l t s f o r l i p a s e a c t i v i t y in t h e v a r i o u s p a r t s o f t h e salivary glands. T h e d a t a o n t h e o c c u r r e n c e o f d i g e s t i v e e n z y m e s in t h e v a r i o u s l o b e s o f t h e s a l i v a r y g l a n d s o f S . r u s t i c u m h a v e b e e n s u m m a r i z e d in T a b l e 1. TABLE | .
O C C U R R E N C E OF DIGESTIVE ENZYMES IN VARIOUS LOBES OF SALIVARY G L A N D S OF S .
r u s t i c u m (F)
Principal gland Enzyme Protease Amylase Maltase Sucrase Lactase Lipase
Accessory gland Superior lobe
Inferior lobe
-÷ ÷ . ----
~+ t-- ÷ .
.
-t .
--
-- no activity, + weak activity, - - + strong activity.
---
44
M A H M U D - U L AMEEN a n d N I N A ABEDIN
:L 0 0 C~l
.C.
i ~i~,~
~:, ~i'
OZk !i!~ i¸¸i ):: ~~)ii:¸::~ 'i~ iii!,~i::~i¸~~:i~i!i~ili¸ii~i!~! ~: IO
FiGs. 9-10. S. rusticum. 9. T.S. of inferior lobe of salivary gland. 10. A salivary gland cell showing secretion.
DISCUSSION
Bugnion and Popoff (1910, cited in Baptist, 1941) and Dokroscky (1931, cited in Bronskill et al., 1958) considered a bilobed principal gland with a unilobular accessory gland as the primitive condition in Heteroptera. The situation in S. rusticum also shows the same condition.
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F.)
45
From a comparison of the present work with that described by Baptist (1941) it was found that in terms of arrangement and appearance, the salivary glands of S. rusticum closely resemble the salivary glands of Nepa cinerea. However, in S. rusticum the duct connecting the superior and inferior lobes emerges from an anteromedial position, instead of arising anteriorly from the inferior lobe as in Nepa (Baptist, 1941). That the 2 anteriormost lobules of the posterior lobes of Nepa were somewhat larger than the other lobules (Baptist, 1941) agrees with the condition in S. rusticum; but Baptist (1941) did not mention any further differentiation in the rest of the lobules of the posterior lobe of salivary gland in Nepa. Our observations on S. rusticum differ considerably from those described by Rastogi (1961). His (1961, Fig. 3) description and diagram of the salivary glands do not show clearly the differentiation among the lobules of the inferior lobe as shown in this paper. According to him the principal glands are aciniform and the acini decrease in size and number posteriorly. He also noted that the principal ducts of the salivary glands open in the salivary syringe and that the 2 ducts from the 2 sides do not join in the head. However, we found that they do meet in the head region forming a very short common salivary duct within the salivarium (Fig. 5). Rastogi's (1961, Fig. 3) diagram shows that 2 independent ducts issue from the 2 lobes of the principal gland which unite and continue as the main salivary duct; the accessory duct branches out from the main duct. This is quite different from our observations noted earlier and illustrated in Figs. 1, 3, and 4. Rastogi (1961) observed that the acini of the principal salivary glands were pyriform pockets, which opened into the central canal of the gland. In the present investigation it was observed that although most of the lobules opened into the central canal, nevertheless some of the peripheral lobules open into a second lobule instead of the central canal (Fig. 9). This is probably necessary for greater production and storage of the enzymes necessiated by the rapacious nature of the insects. Quick killing of the prey and extra intestinal digestion would need a large amount of enzyme secretion. Although not determined experimentally, observation on the feeding of S. rusticum indicate the presence of extra-intestinal digestion. After catching the fish, provided for feeding, with their forelegs the bugs push their stylets into the fish body; after feeding is completed parts of the prey (fish) become transparent, indicating that the tissue of that region has been dissolved during feeding by the bug. The anatomically differentiated regions of the inferior lobe also show differentiation in their staining and nature of the secretory products. These differences may be due to functional differences as was indicated by enzyme analysis. Tests showed that amylase was present in all the anterior, middle, and posterior lobules of the inferior lobe. The breakdown of starch to monosaccharides was indicated only in the middle lobules by the colourless end product in Lugol's iodine test. Whereas in the anterior and posterior lobules the mauve colour of the Lugol's iodine test indicated that the original starch substrate has been broken down to oligosaccharide stage only. This would indicate presence of oligosaccharidase in the middle lobules and its absence in the anteriormost and posterior lobules. But the tests for disaccharidases indicated absence of maltase, sucrase, and lactase. This might be explained either by assuming that the oligosaccharidase present was other than those for which tests were performed. Alternatively, the methods employed for the detection of oligosaccharidases were not fine enough to detect the small quantities of the oligosaccharidases present. As in Nepa (Baptist, 1941) and Ranatra (Quayum, 1959) the accessory glands consist of a vesicular and tubular part. However, in S. rusticum the tubular region is comparatively longer than that of Nepa or Ranatra. The transparency of the vesicular part shows that it
46
MAHMUD-UL AMEEN and NINA ABEDIN
acts only as a reservoir, and in freshly dissected insects it is usually distended due to the presence of a watery fluid. Goodchild (1966), in reviewing the evolutoin of the alimentary canal in the Hemiptera, noted that the salivary glands of the Heteroptera consist of a pair of basically two-lobed main glands and a pair of accessory glands terminating in thin walled vesicles. He thought that the function of such accessory glands in the zoophagous families may be that of recirculating water from the gut to ensure copious flow of saliva to wash out the dissolved internal organs of the prey. Baptist (1941) observed that the accessory glands of Heteroptera produced only a watery secretion and did not find any enzyme in it. Miles and Helliwell (1961) showed that an oxidase other than cytochrome oxidase was present in the accessory glands and salivary ducts of Aphanus sordidus. Bronskill et al. (1958) found slight enzyme (protease) activity in the accessory glands of Oncopeltusfasciatus. The present work also showed slight enzyme (amylase) activity in the accessory glands of S. rusticum.
Baptist (1941) noted that only 2 types of enzymes are found in any particular gland; the enzymes were always related to the type of food consumed, and were those concerned with the digestion of that particular component of the food, which was present in the greatest proportion. Bronskill et al. (1958) opposed the view by showing that 4 enzymes, viz., amylase, protease, invertase, and lipase were present in the salivary glands of O. fasciatus. During the present work only 2 enzymes, viz., protease and amylase were found in S. rusticum; but the enzyme analysis was performed at a p H of 7.5 only for protease and lipase, and at a pH of 6-5 only for amylase and disaccharidases. Therefore, the possibility cannot be ruled out that other enzyme also may be present. Ford (1962) reported the presence of lipase, protease, a - - and fl-- glucosidases, and a weak amylase in Dysdercus fasciatus. The findings of Bronskill et al. (1958), Ford (1962), and the presence of amylase in the basically carnivorous S. rusticum contradict the generalization made by Baptist (1941) that the enzymes or complement of enzymes present in the salivary glands are directly related to diet. Baptist (1941) and Day and Waterhouse (1953) concluded that there was no evidence that different materials were secreted by the various lobes of hemipterous salivary glands. This was contradicted by Bronskill et al. (1958) and the present findings also showed that the same lobe of the salivary glands may produce widely different secretions. Thus in S. rusticum the inferior lobe of the principal gland showed both protease and amylase activity. Acknowledgements--We are thankful to Dr. (Mrs.) Anwara Begum of this Department for criticism of the manuscript and to Dr. S. H. Chowdhury, Department of Zoology, University of Chittagong for valuable suggestions, critical reading of the manuscJipt, and translation of the Russian text. The observations contained in this paper formed a part of an M.Sc. Thesis submitted by the second author to the University of Dacca.
REFERENCES BALDWIN, E. and D. J. BELL1955. Cole's Practical Physiological Chemistry, 10th Ed., Heifer, Cambridge. BAPTIST,B. A. 1941. The morphology and physiology of the salivary glands of Hemiptera: Heteroptera. Quart. J. Microsc. Sci. 83: 91-139. BosE, K. C. and A. K. SEN. 1969. The morphology of the alimentary canal and salivary apparatus of Tessarotoma javanica Thunb. (Hemiptera: Pentatomidae). J. ZooL Soc. lndia 18: 94-103. (Not seen in original) BRONSKILL,J. F., E. H. SALKELD,and W. G. FRIEND. 1958. Anatomy, histology, and secretions of salivary glands of the large milkweed bug, Oneopeltusfaseiatus (Dallas) (Hemiptera : Lygaeidae). Can. J. Zool. 36: 961-68.
Anatomy, Histology, and Secretions of the Salivary Glands of Sphaerodema Rusticum (F.)
47
DAY, M. F. and D. F. WATERHOUSE. 1953. Functions of the alimentary system, pp. 299-310. In K. D. ROEDER (Ed) Insect Physiology, Wiley, New York. FORD, J. B. 1962. Studies on the digestive processes of Dysdercus fasciatus Sign. Ann. AppL Biol. 50: 355. GOODCHILD, A. J. P. 1966. Evolution of the alimentary canal in the Hemiptera. Biol. Rev. (Cambridge) 41 : 97-140. ](HANNA, S. 1964. The digestive system of Dysdercus koenigi (Fabr.) (Hemiptera): (Pyrrhocoridae). Indian J. Entomol. 26: 46-61. MILES, P. W. and A. HELLIWELL. 1961. Oxidase activity in the saliva of a plant-bug. Nature (Lond.) 192: 374-75. NEGI, P. S. 1934. The alimentary canal, its appendages, salivary glands and the nervous system of the adult female lac Laccifer lacca, Kerr (Coccidae). Bull. Entomol. Res. 25: 541-46. PICKFORD, G. E. and F. DORRIS. 1934. Micro-methods for the detection of proteases and amylases. Science (Wash., D.C.) 80: 317-19. QUAYUM, M. A. 1959. Some studies of the salivary glands in Hemiptera (Heteroptera). M.Sc. Thesis,
Univ. Dacca, Bangladesh. QUAYUM, M. A. 1966. Studies on the salivary glands of Dysdercus cingulatus--Fabr. (Hemiptera). Pakistan J. Sci. Res. 18(2): 111-17. RASTOGI, S. 1961. The anatomy of the digestive organs of Sphaerodema rusticum Fabr. (Heteroptera: Belostomatidae). Proc. Rajasthan Acad. Sci. 8: 60-78. SAXENA, P. N. and H. L. CHADA. 1971. The green bug, Schizaphis graminum. 2. The salivary gland complex. Ann. Entomol. Soc. Amer. 64(4): 904-12. SHUBNIKOVA,E. A. 1967. Cytology and Cytophysiology of Secretory Process (in Russian). Moscow State Univ. Publications. SRIVASTAVA,P. D. 1957. Studies on the choice of food-plant and certain aspects of the digestive physiology of the larvae and adults of Athalia lugens proxima (Klug) and Epilachna vigintioctopunctata (F.). Bull. Entomol. Res. 48: 289-97.