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Function of the salivary glands of the cockroach, Nauphoeta cinerea
J. Insect Physiol., 1971, Vol. 17, pp. 2069 to 2084. Pergamon Press. Printed in Great Britain
FUNCTION OF THE SALIVARY GLANDS OF THE COCKROACH, NAUPHOETA CINEREA K. P. BLAND Department
and C. R. HOUSE
of Veterinary Physiology, Royal (Dick) School of Veterinary University of Edinburgh, Edinburgh EH9 1QH (Received
3 June
Studies,
1971)
Abstract-The salivary glands of Nauphoeta cinerea consist of four cell types: peripheral cells, central cells, and secretory and non-secretory duct cells. The structure and histochemistry of these cells are described. The peripheral cells are probably responsible for the primary transport of water and ions into the gland whereas the non-secretory duct cells may alter the ionic concentrations in the saliva as it passes down the ducts. The central cells secrete amylase and probably also the other enzymes (invertase, maltase, and protease) found in the gland. Lactase and lipase were not detected in the gland although the latter enzyme was present in the gut. A mucous component containing sialoglycans is produced by the secretory duct cells; these cells also contain a large concentration of tryptophan although certain other amino acids (tyrosine, arginine, and cystine) are absent. INTRODUCTION WHILE investigating
the electrical properties of the cells of the cockroach salivary gland, it became necessary to know the function of the individual cell types. A review of the literature revealed confusion both in the terminology and in the functions of the different cells (see Table 1). This investigation is an attempt to clarify the situation in the cockroach, Nauphoeta cinerea (Oliv.). MATERIALS
AND
METHODS
Light microscopy Fresh salivary glands from Nuuphoeta cinerea were removed by dissection and fixed in various fixatives (10% formalin, Bouin’s fluid, Carnoy’s fluid, 2.5% glutaraldehyde in sucrose and phosphate buffer). After wax embedding, 5 or 10 TV wax sections were prepared, mounted, and stained with either Erhlich’s Haematoxylin and Eosin, Mallory’s Triple Stain, or Bismark Brown. Only fixation in buffered 2.5% glutaraldehyde retained stainable secretory material in the central cells. Electron microscopy Fresh phosphate
salivary glands were fixed for 1 hr in 2.5% glutaraldehyde in 0.05 M buffer with S.5Oh sucrose added, followed by 30 min in 1% osmium 2069
2070
K. P. BLAND AND C. R. HOUSE
FUNCTION
OF COCKROACH SALIVARY GLAND
2071
tetroxide. Following dehydration and clearing in propylene oxide, the tissue was embedded in Araldite. Thin sections were stained with uranyl acetate. Enzyme
characterisation
Identification of the enzymes present in the salivary glands was performed using methods based on those of BALDWINand BELL (1955). The salivary glands and reservoirs were removed from four cockroaches, rinsed in cockroach Ringer solution, * and ground up with a little acid-washed silver sand in 0.5 ml distilled water. The filtered extract was then tested for enzyme activity; each test was performed at least twice. Amylase. The extract was incubated with 2% starch solution at 40°C for 5 min. Any degradation of the starch solution was monitored by testing samples of it at intervals with acidified iodine solution. Change of the normal blue colour of the starch-iodine mixture to claret indicates its breakdown to dextrins and maltose due to the action of amylase. Maltuse. Further incubation as above, with total loss of colour reaction to iodine would indicate further degradation to glucose due to the action of maltase. The generation of glucose was tested by the preparation of the osazone of the product of prolonged incubation (and those of glucose and maltose as controls). Maltosazone is very soluble in hot water but glucosazone is practically insoluble. Invertase or sucrase. The extract was incubated with 1% sucrose solution at 56°C for 5 min. The production of the reducing sugars, glucose, and fructose, was indicated by Fehling’s test for reducing sugars. Lactase. The extract was incubated with 1% lactose solution at 56°C for 30 min. Glucose production was assessed by the preparation of the osazone. Lactosazone is very soluble in hot water but glucosazone is not. Lipase. Incubation of the extract for 30 min at 40°C with olive oil dissolved in ethanol and made just alkaline with 2% sodium carbonate will yield acidic products (i.e. fatty acids) if lipase is present. Extracts of the guts of four cockroaches were prepared by a similar method and tested as controls. Protease. Extracts of both salivary glands and gut were incubated for several hours at 40°C with calcified milk made just acid with O-01 N Hydrochloric acid. Precipitation indicated the presence of protease. Histochemistry
Unless otherwise stated all histochemical tests were from PEARSE(1968) and 5 TVwax-embedded sections used following fixation in 2.5% glutaraldehyde in phosphate and sucrose buffer. This fixative was chosen because it retained the secretion in the central cells. The size and nature of the gland prevented the use of frozen sections. The histochemical tests used were: Molybdatc-benzidine method for phosphate (GLICK, 1949); Bracco and Curti’s benzidine reaction for sulphate; saturated aqueous thionine for metachromasia; *One 1. contains: 160 mM NaCl, 10 mM KCl, 5 mM CaCl,, 2 mM NaHCO,, and
0.1 mM NaH,PO,.
2072
K. P.
BLAND AND
C. R. HOUSE
1.5% aqueous Toluidine Blue for metachromasia; 1.5% aqueous Toluidine Blue after mild methylation in O-1 N HCl in methanol at 37°C for 2 hr; Periodic acidSchiff’s reagent method for 1 : 2 glycol groups; 1% Alcian Blue in 3% acetic acid (pH 2.5); 1% Al cian Blue in 0.1 N WC1 (pH 1-O); Ravetto’s Bial reaction for sialic acid ; Sudan Black and Sudan IV Red for lipids (air-dried whole mounts only) ; Bensley and Gersh’s Millon reaction for tyrosine; Baker’s modified Sakaguchi reaction for arginine; Adams’ dimethylaminobenzaldehyde (DMAB)-nitrite method for tryptophan; Adams’ DMAB-nitrite after performic acid oxidation; Hammett and Chapman’s nitroprusside reaction for sulphydryl groups (fresh whole glands) ; Adams and Sloper’s performic acid-Alcian Blue method for cystine ; modified Tremblay’s method for amylase. Both the original method of TREMBLAY (1963) for amylase and the modified method of TREMBLAYand CHARE~T(1968) were tried on fresh frozen sections, frozen sections of glands embedded in gelatin, and fresh whole glands. These methods confirmed the presence of amylase but the labile nature of the central cell secretion made these methods unsatisfactory for localization of the enzyme. Thus, starch films were prepared according to TREMBLAY (1963) and using an operating microscope with substage lighting, fresh glands were dissected in cockroach Ringer to obtain pieces of either acinus or duct. These pieces were rinsed briefly in cockroach Ringer solution and placed at carefully marked loci on the starch film. After 10 min incubation at room temperature the tissue was rinsed off the slide with distilled water and the starch film made blue with dilute iodine solution. Areas of starch film underlying sites containing amylase/maltase activity were indicated by an absence of blue colour. RESULTS General anatomy and terminology The salivary glands of N. cinerea are a branched duct system; the smallest ducts terminating in spherical glandular acini and the large main ducts joining separately the main duct of the reservoirs. The anatomy and function of the reservoirs has been described by SUTHERLAND and CHILLSEYZN(1968). The glands basically consist of four types of cells (Fig. 1). The ducts are composed of either non-secretory or secretory duct cells. The latter only occur in close proximity to the acinus and are clearly identified by the presence of large masses of acidophilic secretion within them. The remaining two types occur in the acini. The bulk of each acinus is occupied by large central cells predominantly filled with basophilic secretion whereas the major part of the periphery is occupied by numerous small peripheral cells containing ductules. Of these four cell types only the central cells and the secretory duct cells appear to elaborate secretion. The secretory behaviour of these two cell types differs considerably. The secretory duct cells elaborate a dense secretion which can be synthesized, stored, and released. On the other hand, the central cells undergo cyclical synthesis and release. Initially the central cells elaborate a densely staining secretion which then appears to undergo progressive dilution. The diluted
FUNCTION
OF COCKROACH
SALIVARY GLAND
2073
secretion is then released from the cell. Throughout these events the cytoplasm is acidophilic. The cytoplasm then becomes basophilic and enters a regenerative stage prior to again producing secretion. Throughout the bulk of the regenerative stage the vacuoles left by the released secretion are apparent. Within an acinus the cells are at differing stages of production, secretion, and regeneration.
\
Alcells/
Cent
FIG. 1. Schematic diagram, based on electronmicrographs, of an acinus from the salivary gland of the cockroach, IV. cinereu. The four cell types are shown-only the central cells and the secretory duct cells elaborate a secretion. The sequence A to F shows central cells in different stages of secretory activity: A, a completely depleted cell, with regeneration of the endoplasmic reticulum just beginning; B, regeneration of the endoplasmic reticulum complete; C, production of droplets of secretion underway; D and E, cells full with secretion; F, following release of the secretion the residual empty structures disappear to give rise to a cell like that shown in A.
Electron microscopical anatomy Peripheral cell. Each cell contains an intracellular ductule which is lined with numerous microvilli (Fig. 2A). The shape of these cells is pyramidal and they occur frequently in pairs. Underneath the basement membrane the plasma membrane undergoes considerable infolding. There are many mitochondria within the cytoplasm and numerous dense glycogen granules are also present. The endoplasmic reticulum does not constitute a prominent component of the cytoplasm, and Golgi bodies are observed only occasionally. KIESEL and BEAMS (1963) reported that they found a ‘few large, dense structures . . ., composed, in part, of concentric membranous lamellae' ; however, these bodies are also evident not only in the peripheral cells (Fig. 2B) but also in all other cell types in our sections.
2074
K. P.
BLAND AND
C. R. HOUSE
Central cell. In contrast to the peripheral cell, the basal surface of this cell occupies a relatively minor fraction of the acinar surface, and, moreover, it is not extensively infolded. However, the apical plasma membrane has numerous microvilli on its invaginated surface (Fig. 2C). The endoplasmic reticulum is prominent in both a rough and vacuolar form and the cell also contains Golgi bodies. There are few mitochondria except in regions adjacent to the lumen of the duct. The large secretory droplets contain granular material which is not dense ; neither is the density of the droplets uniform. These droplets often possess a limiting membrane and since they have been observed in the duct lumen in the proximity of the central cells, their release must occur by exocytosis (Fig. 2D). It is consistent with both the electron microscopical observations and the histological findings that the synthesis of the secretory material in the population of central cells is asynchronous (Fig. 3A). Secretory duct cell. The basal plasma membrane shows no pronounced infolding although the apical membrane does invaginate (Fig. 3B). The lumen of the duct at this level is lined with a layer of chitin which is electron opaque. The chitin lining continues into the acinar lumen until it reaches the ductules of the peripheral cells and the apical surface of the central cells where it ends. This lining does not make intimate contact with the plasma membrane of these cells. There are numerous mitochondria in the secretory duct cells and these tend to be associated with the apical rather than the basal surfaces. Although the endoplasmic reticulum is not well developed, these cells contain large droplets of dense granular material. Unlike the secretion from the central cells, the dense material from the secretory duct cells becomes more granular and appears to dissolve immediately on its release. Non-secretory duct cell. It is in the neighbourhood of the acinus only that the duct cells perform a secretory function and the non-secretory .duct cells occupy the major portion of the length of the ducts. The apical plasma membrane of the nonsecretory duct cells is highly invaginated in its position underneath the chitin lining (Fig. 3D). In agreement with KESSEL and BEAMS(1963) we have found that
FIG. 2A. Portion of a typical peripheral cell showing the numerous microvilli (MV) surrounding the lumen of the ductule (D). Note the deep and complicated The cell infoldings of the surface adjacent to the basement membrane. contains numerous mitochondria (MC) and glycogen granules (G). FIG. 2B. Portion of another peripheral cell with the same features as in Fig. 2A. In addition a few fragments of endoplasmic reticulum (ER) and some of the lamellated structures (K), noted by KESSEL and BEAMS(1963), are present. FIG. 2C. The apical portions of three central cells forming a duct. Note the numerous microvilli (MV) on the apical surface of the central cells and the chitin lining (C) within the duct. Several large droplets of secretion (Sl) are apparent along with numerous mitochondria (MC) associated with the apical portion of the central cells. FIG. 2D. A longitudinal section of a duct similar to that in Fig. 2C. Large droplets of secretion (Sl) are present in the duct lumen and prominent septate desmosomes (SD) are present joining adjacent central cells.
2075
2076
FIG. 3.
FUNCTION OFCOCKROACH SALIVARY GLAND
2077
the infoldings of this membrane form a web-like structure of vacuolar spaces (Fig. 3D). There is also a great deal of infolding at the basal surface. Numerous mitochondria are found between the infoldings of the basal plasma membrane (Fig. 3C). The endoplasmic reticulum is not prominent but the cytoplasm contains numerous very small dense granules, possibly ribosomes. Connexions between cells. Septate desmosomes, similar to those described by KE~SEL and BEAMS(1963), h ave been observed between central cells and their neighbours, namely peripheral cells, secretory duct cells and other central cells. Such connexions have been observed also between some peripheral cells and secretory duct cells. All these connexions occur in the vicinity of the lumen of the gland. The secretory and non-secretory duct cells are also joined to each other by septate desmosomes. Usually there is considerable coiling of such connexions and this is particularly prominent at the junction between the non-secretory duct cells. However, the septate desmosomes between central cells are sometimes short and uncoiled. Trucheoles and innervation. Both intracellular and extracellular tracheoles have been observed in association with all cell types. The gland is richly innervated and the exact form and function of this innervation is the subject of a current study. Enzyme production
In extracts of salivary glands and reservoirs, amylase, maltase, and invertase were readily demonstrable although lactase appeared to be absent. Lipase could not be detected in the salivary glands although it was present in the gut. On the other hand, protease was present in both the glands and the gut, although the amount in the former is very small since only mild precipitation occurred and that not until some 14 hr after precipitation with gut extracts. Histochemical characterixation of secretions
The histochemical
findings are summarized
in Table 2.
FIG. 3A. Portions of three central cells in different stages of secretory activity. Cell 1 is in early stages of the formation of the secretory product and shows the regeneration of abundant endoplasmic reticulum (ER): Cell 2 contains large droplets of secretion (Sl). After the release of the secretion, the endoplasmic reticulum (ER) starts to reform as in Cell 3. FIG. 3B. Transverse section of part of a duct showing secretory duct cells. Note the large dense droplets of secretion (S2) and the infoldings of the apical plasma membrane lying beneath the chitin lining (C) of the duct.
FIG. 3C. The basal region of a non-secretory duct cell. Numerous elongated mitochondria (MC) lie within the folds of the basal plasma membrane underneath the basement membrane (BM). FIG. 3D. The apical region of a non-secretory duct cell. Adjacent to the chitin lining (C) of the duct, the apical plasma membrane is contorted into numerous small vacuole-like infoldings (V). The mitochondria (MC) in this part of the cell are not elongated neither are they closely associated with the apical infoldings. Septate desmosomes (SD) are apparent joining the cells.
Test
2---HISTOCHEMICAL
REACTIONS
Sudan methods
Modified Tremblay
(amylase) (+)
++++ (pinkish)
-
Hammett and Chapman’s nitroprusside Performic acid-Alcian Blue (-SS) Adams’ DMAB-nitrite (tryptophan) DMAB-nitrite (after oxidation)
(-3-I)
-
Bensley and Gersh’s Millon (tyrosine) Modified Sakaguchi (arginine)
++++ -t
-
(+)
-
Secretion
GLANDS
OF THE CENTRAL
Secretory duct cells
OF THE SECRETION
y Metachromasia (alcohol-stable) /3Metachromasia (alcohol-labile) p Metachromasia (after methylation) Alcian Blue at pH 1.0 (- SO, mucosubstances) Alcian Blue at pH 2.5 (acid mucopolysaccharide) Periodic acid-Schiff’s reagent (1 : 2 glycol) Ravetto’s bial (sialic acid)
Bracco and Curti’s benzidine (- SOa) Molybdate-benzidine ( - PO3
TABLE
SECRETORY
+ (T) -
++ ++ ++ -
(+)
++ + -
-
+:
(1) -
(+) 1+) I +
-
-
+ + I
-
Secretion
at max.
SALIVARY
-
-
-
J
Secretion voided
CELLS OF THE COCKROACH
Central cells
DUCT
-
little and dense
AND
sd EC 8 Ki
c,
5
g
F
F .‘d
FUNCTION
OF COCKROACH
SALIVARY
GLAND
2079
Secretion of secretory duct cells Irrespective of the fixative employed the secretory products of the duct cells showed a strong positive reaction with the periodic acid-Schiff’s technique, indicative of the presence of 1 : 2 glycosidic linkages. The secretion also gave a distinct colouration with Ravetto’s bial method for identifying high concentrations of sialic acid. This secretion, therefore, probably consists in part of sialoglycans (glucosaminoglycans). The neutral nature of sialoglycans would explain their failure to react with Alcian Blue (pH 1.0 or 2.5) or to give metachromasia with Toluidine Blue or thionine. There was another component to the duct cell secretion which was more labile and was lost by fixation in unbuffered fixatives. This component showed a strong reaction with the DMAB-nitrite method for tryptophan. Absence of the reaction after performic acid oxidation confirmed that tryptophan was responsible since such oxidation is thought to be specific for the indole ring (HUMASON, 1967). However, after oxidation the deep blue colour was replaced by a pale pink. This may indicate a positive Morgan-Elson reaction for N-acetylated hexosamines (MORGAN and ELSON, 1934). The high tryptophan content is unusual and appeared to occur in the absence of stainable amounts of the amino acids; tyrosine (Millon’s reaction), arginine (Sakaguchi method), and cystine (Adams and Sloper’s performic acid-Alcian Blue method). Secretion of central cells The secretion of the central cells also gave a positive reaction with periodic acid-Schiff’s method indicating the presence of 1 : 2 glycosidic linkages. There was, however, in addition a distinct but weak staining with Alcian Blue at pH 2.5 which indicates the presence of weakly acidic mucopolysaccharide (glycosaminoglucuronoglycan). Since no staining took place with Alcian Blue at pH 1-O it is improbable that strongly acidic sulphated mucosubstances are present. This conclusion was supported by the absence of a response to Bracco and Curti’s benzidine method for sulphate. Furthermore, the secretion showed alcohollabile p metachromasia with 1*5”/0 aqueous Toluidine Blue and saturated aqueous thionine. Such metachromasia usually indicates the presence of a high concentration of weakly acidic carboxyl (or possibly phosphoryl) groupings. Mild methylation (37°C for 2 hr) of the tissue with 0.1 N HCl in methanol destroyed this metachromasia. This process was once thought to block only the carboxyl groups but is now known to be less specific (PEARSE, 1968). Thus the central cell secretion appears to be composed in part of acidic carboxyl-containing carbohydrate. The modified Tremblay’s method indicated that the amylase activity of the salivary glands resided in the cells of the acinus as opposed to the secretory or non-secretory duct cells. As the central cells are the only acinar cells of a secretory nature, they are probably responsible for the production of this enzyme (and possibly the other enzymes produced by the gland). This central cell production of amylase was supported by the fact that during the acidophilic stage of their
2080
K. P.
BLAND AND
C. R.
HOUSE
secretory cycle, the secretion stained positively for tyrosine (Millon’s reaction), arginine (Sakaguchi method), cystine (Adams and Sloper’s performic acid-Alcian Blue method), cysteine (Hammett and Chapman’s nitroprusside method), and tryptophan (DMAB-nitrite method). Performic acid oxidation which is specific for the indole ring, destroyed the reaction of the central cells for tryptophan. Each of these amino acids amounts to more than 4 per cent (by weight) of human salivary amylase (DIXON and WEBB, 1958).
DISCUSSION Detection of amylase, maltase, invertase, and protease in the salivary glands of N. cinerea conforms with the findings in other species (see Table 3). The failure to detect lactase and lipase in this species using methods similar to those employed by RAYCHAUDHURI and GHOSH (1964) to demonstrate these enzymes in Periplaneta awicana may indicate a species difference. The predominance of amylase in the saliva of cockroaches shows an interesting parallel to the situation in mammals. However, unlike the latter, lysozyme is unlikely to be produced by the cockroach salivary gland in view of this enzyme’s ability to slowly hydrolyse chitin (BERGERand WEISER, 1957). The central cells appeared to be the source of the amylase and possibly the other enzymes produced by the salivary glands because: (a) The central cell secretion had an electron microscopical structure suggesting protein. (b) The central cells contained abundant rough endoplasmic reticulum which is usually associated with active protein synthesis. (c) The secretion has a protein nature as it stained positive for the amino-acids; tyrosine, arginine, cystine, cysteine, and tryptophan. Cockroach amylase was shown by WIGGLESWORTH(1927) to be activated by chloride ions in a similar manner to human salivary amylase (DIXON and WEBB, 1958). This common characteristic may indicate a structural similarity between the two enzymes, at least in the active regions. The above amino acids each constitute more than 4 per cent by weight of human salivary amylase (DIXON and WEBB, 1958). (d) Th e modification of Tremblay’s method for amylase indicated the presence of amylase in the central cells. Weakly acidic carbohydrate material also occurs in the secretion of the central cells and it is this that has probably caused the confusion about the role of these cells (see Table 1). The similar labile nature of both the carbohydrate and the protein fractions of the secretion suggests that some binding exists between these substances. The staining properties of the central cell secretion indicate the presence of mucoprotein, a finding which would support the suggestion that these cells produce mucin. However, the protein part of the secretion is the enzyme amylase, thus cockroach salivary amylase may be similar to the fl-amylase of wheat, which contains a polysaccharide component essential for its activity (MCILROY, 1948). Since this form of amylase is not usually considered to be present in animals and furthermore is not activated by chloride ions (LONG, 1961), the carbohydrate is probably present solely to stabilize the enzyme.
FUNCTION
OF COCKROACH
SALIVARY
GLAND
2081
2082
K. P. BLAND AND C. R. HOUSE
The structure of the peripheral cells is similar to that of the cells in the distal portion of the salivary glands of CuZZiphora erythrocephulu, Meig. (OSCHMANand BERRIDGE, 1970). Each secretory cell in the distal region of that gland possesses a branching system of canaliculi opening into the lumen and Oschman and Berridge found that these cells elaborated a fluid which contained approximately 140 mM KC1 and 10 mM NaCl/l. In Calliphora, these cells are also responsible for the secretion of amylase since they contain a relatively prominent rough endoplasmic reticulum, free ribosomes, Golgi complexes, and secretory droplets. From the structural similarity between the cockroach peripheral cells and CuZZiphora secretory cells the former are probably concerned with the transport of ions and water from the haemolymph into the lumen of the gland. Moreover, the movement of ions and water is apparently the sole function of the peripheral cells since, unlike Caltiphoru secretory cells, they do not contain either abundant endoplasmic reticulum or secretory droplets. In N. cinereu the mucous component of the saliva appears to be primarily produced by the secretory duct cells. The secretion of these cells contains sialoglycans but little or no stainable protein. The only previous mention of this type of secretory cell was made by RAYCHAUDHURIand GHOSH (1964). These workers noted that in P. americana the columnar epithelia lining of the ‘ductules’ contained granules suggesting ‘glandular activity’ but did not comment further about them. The nature of the tryptophan-rich and more labile component of the duct cell secretion is unresolved. This high level of reactivity to the DMAB-nitrite method in the apparent absence of other amino acids could be ascribed to 5-hydroxytryptamine (S-HT) which also reacts in this system (PEARSE, 1968). However, a histochemical fluorescence method for 5-HT based on the ninhydrin method of JEPSON and STEVENS (1953) did not reveal a high concentration of 5-HT in the secretory duct cells. No fluorescence is given by tryptophan with this method. Following performic acid oxidation the DMAB-nitrite method replaced the intense blue reaction for tryptophan with a faint but distinct reddish-purple colour. This colour was probably due to acetylhexosamines in the sialoglycans reacting with DMAB in a manner similar to that described by MORGANand ELSON (1934) for the The positive tryptophan reaction per se was not due to assay of acetylglucosamine. hexosamines because: (a) Chitin (composed almost entirely of acetylglucosamine) did not stain, However, PEARSE (1968) ob served that in the histochemical use of DMAB for amino sugars no reaction is obtained with insect chitin. (b) In vitro assessment of the specificity of the DMAB-nitrite method with tryptophan and N-acetylglucosamine confirmed that only tryptophan reacted. However, both tryptophan and N-acetylglucosamine (pretreated by boiling in 1 N KOH) gave identically coloured products with 5% DMAB in concentrated HCl-the initial product was bright purple which rapidly faded to dull purple-red. Nitrite oxidation gave the characteristic dark blue of carboline blue with tryptophan but produced only a yellowish colour with N-acetylglucosamine. The non-secretory duct cells may be concerned with the re-absorption of certain ions from the saliva or with the secretion of other ions into the saliva. They
FUNCTIONOF COCKROACH SALIVARYGLAND
2083
have a similar structure to that of the re-absorptive cells in the proximal part of Calliphora salivary gland (OSCHMANand BERRIDGE, 1970) where potassium ions are re-absorbed and sodium ions are secreted into the lumen. The particular features, which are common to both of these types of cell, are the pronounced infoldings of the apical and basal plasma membranes in association with numerous mitochondria and the absence of secretory droplets. According to Oschman and Berridge the proximal region of Calliphora salivary gland reduces the osmolarity of Preliminary analyses of the the saliva to a value which is hypotonic to haemolymph. saliva in the reservoir of cockroach gland indicates that the concentrations of potassium and sodium ions are approximately identical (20 mM/l) and somewhat similar to those in the saliva of Calliphora when the proximal re-absorption of potassium is at a maximum. Acknowledgements-We wish to thank Miss J. M. MACKNESSfor the preparation of the electron microscopical sections and Dr. A. W. G. MANNINGof the Zoology Department for providing specimens of N. cinerea. One of us (C. R. H.) IS indebted to the Science Research Council for financial support. REFERENCES BALDWIN E. and BELL D. J. (19.55) Cole’s Practical Physiological Chemistry, 10th ed. Heffer, Cambridge. BAXH S. (1858) Untersuchungen iiber das chylopoetische und uropoetische System der Blatta orientalis. Sitzungsberichte der Kaiserlichen Akademie der Wissenschaften (Muth.Nut. Classe). Wien 33, 234-260. BERGER L. R. and WEISER R. S. (1957) The ,&gl ucosaminidase activity of egg-white lysozyme. Biochim. biophys. Acta 26, 517421. DAY M. F. (1949a) The distribution of alkaline phosphatase in insects. Aust.J. sci. Res. (B) 2, 31-41. DAY M. F. (1949b) The occurrence of mucoid substances in insects. Aust. J. sci. Res. (B)
2, 421427. DAY M. F. (1951) The mechanism of secretion by the salivary gland of the cockroach Periplaneta americana (L). Aust.J. sci. Res. (B) 4, 136-143. DAY M. F. and PO~NING R. F. (1949) A study of the process of digestion in certain insects. Aust. J. sci. Res. (B). 2, 175-215. DIXON M. and WEBB E. C. (1958) Enzymes. Longmans, Green, London. GLICK D. (1949) Techniques of Histo- and Cytochemistry. Interscience, New York. HUMASONG. L. (1967) Animal Tissue Techniques, 2nd ed. Freeman, San Francisco. JEPSON J. B. and STEVENSB. J. (1953) A Auorescent test for seratonin and other tryptamines. Nature, Lond. 172, 772. KESSEL R. G. and BEAMS H. W. (1963) Electron microscope observations on the salivary gland of the cockroach, Periplaneta americana. 2. Zellforschung. 59, 857-877. LEBEDEFF A. (1899) cited by DAY (1951). LONG C. (ed.) (1961) Biochemist’s Handbook. Spon, London. MCILROY R. J. (1948) The Chemistry of the Polysaccharides. Arnold, London. MORGANW. T. J. and ELSON L. A. (1934) A calorimetric method for the determination of N-acetylglucosamine and N-acetylchrondrosamine. Biochem. J. 28, 988-995. OSCHMANJ. L. and BERRIDGE M. J. (1970) Structural and functional aspects of salivary fluid secretion in Calliphora. Tissue and Cell 2, 281-310. PEARSEA. G. E. (1968) Histochemistry, Theoretical and Applied, 1,3rded. Churchill, London.
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PLATEAU F. (1876) Note sur les phCnom&nes de la digestion chez la Blatte americaine (Periplaneta americana L.) Bull. Acad. roy. Belg. (2), 41, 1206-1233. RAYCHAUDHURI D. N. and GHOSHS. K. (1964) Study on the histophysiology of the salivary apparatus of Periplaneta americana Linn. Zool. Anz. 173, 227-237. SUTHERLAND D. J. and CHILLSEYZNJ. M. (1968) Function and operation of the cockroach salivary reservoir. J. Insect Physiol. 14, 21-31. SWINCLEH. S. (1925) Digestive enzymes in an insect. OhioJ. Sci. 25, 209-218. TREMBLAYG. (1963) The localization of amylase activity in tissue sections by a starch film method. J. Histochem. Cytochem. 11, 202-206. TREMBLAY G. and C&REST J. (1968) Modified starch film method for the histochemical localization of amylase activity. J. Histochem. Cytochem. 16, 147-148. WIGGLESWORTHV. B. (1927) Digestion in the cockroach-II. The digestion of carbohydrates. Biochem. J. 21, 797-811.
FUNCTION OF THE SALIVARY GLANDS OF THE COCKROACH, NAUPHOETA CINEREA K. P. BLAND Department
and C. R. HOUSE
of Veterinary Physiology, Royal (Dick) School of Veterinary University of Edinburgh, Edinburgh EH9 1QH (Received
3 June
Studies,
1971)
Abstract-The salivary glands of Nauphoeta cinerea consist of four cell types: peripheral cells, central cells, and secretory and non-secretory duct cells. The structure and histochemistry of these cells are described. The peripheral cells are probably responsible for the primary transport of water and ions into the gland whereas the non-secretory duct cells may alter the ionic concentrations in the saliva as it passes down the ducts. The central cells secrete amylase and probably also the other enzymes (invertase, maltase, and protease) found in the gland. Lactase and lipase were not detected in the gland although the latter enzyme was present in the gut. A mucous component containing sialoglycans is produced by the secretory duct cells; these cells also contain a large concentration of tryptophan although certain other amino acids (tyrosine, arginine, and cystine) are absent. INTRODUCTION WHILE investigating
the electrical properties of the cells of the cockroach salivary gland, it became necessary to know the function of the individual cell types. A review of the literature revealed confusion both in the terminology and in the functions of the different cells (see Table 1). This investigation is an attempt to clarify the situation in the cockroach, Nauphoeta cinerea (Oliv.). MATERIALS
AND
METHODS
Light microscopy Fresh salivary glands from Nuuphoeta cinerea were removed by dissection and fixed in various fixatives (10% formalin, Bouin’s fluid, Carnoy’s fluid, 2.5% glutaraldehyde in sucrose and phosphate buffer). After wax embedding, 5 or 10 TV wax sections were prepared, mounted, and stained with either Erhlich’s Haematoxylin and Eosin, Mallory’s Triple Stain, or Bismark Brown. Only fixation in buffered 2.5% glutaraldehyde retained stainable secretory material in the central cells. Electron microscopy Fresh phosphate
salivary glands were fixed for 1 hr in 2.5% glutaraldehyde in 0.05 M buffer with S.5Oh sucrose added, followed by 30 min in 1% osmium 2069
2070
K. P. BLAND AND C. R. HOUSE
FUNCTION
OF COCKROACH SALIVARY GLAND
2071
tetroxide. Following dehydration and clearing in propylene oxide, the tissue was embedded in Araldite. Thin sections were stained with uranyl acetate. Enzyme
characterisation
Identification of the enzymes present in the salivary glands was performed using methods based on those of BALDWINand BELL (1955). The salivary glands and reservoirs were removed from four cockroaches, rinsed in cockroach Ringer solution, * and ground up with a little acid-washed silver sand in 0.5 ml distilled water. The filtered extract was then tested for enzyme activity; each test was performed at least twice. Amylase. The extract was incubated with 2% starch solution at 40°C for 5 min. Any degradation of the starch solution was monitored by testing samples of it at intervals with acidified iodine solution. Change of the normal blue colour of the starch-iodine mixture to claret indicates its breakdown to dextrins and maltose due to the action of amylase. Maltuse. Further incubation as above, with total loss of colour reaction to iodine would indicate further degradation to glucose due to the action of maltase. The generation of glucose was tested by the preparation of the osazone of the product of prolonged incubation (and those of glucose and maltose as controls). Maltosazone is very soluble in hot water but glucosazone is practically insoluble. Invertase or sucrase. The extract was incubated with 1% sucrose solution at 56°C for 5 min. The production of the reducing sugars, glucose, and fructose, was indicated by Fehling’s test for reducing sugars. Lactase. The extract was incubated with 1% lactose solution at 56°C for 30 min. Glucose production was assessed by the preparation of the osazone. Lactosazone is very soluble in hot water but glucosazone is not. Lipase. Incubation of the extract for 30 min at 40°C with olive oil dissolved in ethanol and made just alkaline with 2% sodium carbonate will yield acidic products (i.e. fatty acids) if lipase is present. Extracts of the guts of four cockroaches were prepared by a similar method and tested as controls. Protease. Extracts of both salivary glands and gut were incubated for several hours at 40°C with calcified milk made just acid with O-01 N Hydrochloric acid. Precipitation indicated the presence of protease. Histochemistry
Unless otherwise stated all histochemical tests were from PEARSE(1968) and 5 TVwax-embedded sections used following fixation in 2.5% glutaraldehyde in phosphate and sucrose buffer. This fixative was chosen because it retained the secretion in the central cells. The size and nature of the gland prevented the use of frozen sections. The histochemical tests used were: Molybdatc-benzidine method for phosphate (GLICK, 1949); Bracco and Curti’s benzidine reaction for sulphate; saturated aqueous thionine for metachromasia; *One 1. contains: 160 mM NaCl, 10 mM KCl, 5 mM CaCl,, 2 mM NaHCO,, and
0.1 mM NaH,PO,.
2072
K. P.
BLAND AND
C. R. HOUSE
1.5% aqueous Toluidine Blue for metachromasia; 1.5% aqueous Toluidine Blue after mild methylation in O-1 N HCl in methanol at 37°C for 2 hr; Periodic acidSchiff’s reagent method for 1 : 2 glycol groups; 1% Alcian Blue in 3% acetic acid (pH 2.5); 1% Al cian Blue in 0.1 N WC1 (pH 1-O); Ravetto’s Bial reaction for sialic acid ; Sudan Black and Sudan IV Red for lipids (air-dried whole mounts only) ; Bensley and Gersh’s Millon reaction for tyrosine; Baker’s modified Sakaguchi reaction for arginine; Adams’ dimethylaminobenzaldehyde (DMAB)-nitrite method for tryptophan; Adams’ DMAB-nitrite after performic acid oxidation; Hammett and Chapman’s nitroprusside reaction for sulphydryl groups (fresh whole glands) ; Adams and Sloper’s performic acid-Alcian Blue method for cystine ; modified Tremblay’s method for amylase. Both the original method of TREMBLAY (1963) for amylase and the modified method of TREMBLAYand CHARE~T(1968) were tried on fresh frozen sections, frozen sections of glands embedded in gelatin, and fresh whole glands. These methods confirmed the presence of amylase but the labile nature of the central cell secretion made these methods unsatisfactory for localization of the enzyme. Thus, starch films were prepared according to TREMBLAY (1963) and using an operating microscope with substage lighting, fresh glands were dissected in cockroach Ringer to obtain pieces of either acinus or duct. These pieces were rinsed briefly in cockroach Ringer solution and placed at carefully marked loci on the starch film. After 10 min incubation at room temperature the tissue was rinsed off the slide with distilled water and the starch film made blue with dilute iodine solution. Areas of starch film underlying sites containing amylase/maltase activity were indicated by an absence of blue colour. RESULTS General anatomy and terminology The salivary glands of N. cinerea are a branched duct system; the smallest ducts terminating in spherical glandular acini and the large main ducts joining separately the main duct of the reservoirs. The anatomy and function of the reservoirs has been described by SUTHERLAND and CHILLSEYZN(1968). The glands basically consist of four types of cells (Fig. 1). The ducts are composed of either non-secretory or secretory duct cells. The latter only occur in close proximity to the acinus and are clearly identified by the presence of large masses of acidophilic secretion within them. The remaining two types occur in the acini. The bulk of each acinus is occupied by large central cells predominantly filled with basophilic secretion whereas the major part of the periphery is occupied by numerous small peripheral cells containing ductules. Of these four cell types only the central cells and the secretory duct cells appear to elaborate secretion. The secretory behaviour of these two cell types differs considerably. The secretory duct cells elaborate a dense secretion which can be synthesized, stored, and released. On the other hand, the central cells undergo cyclical synthesis and release. Initially the central cells elaborate a densely staining secretion which then appears to undergo progressive dilution. The diluted
FUNCTION
OF COCKROACH
SALIVARY GLAND
2073
secretion is then released from the cell. Throughout these events the cytoplasm is acidophilic. The cytoplasm then becomes basophilic and enters a regenerative stage prior to again producing secretion. Throughout the bulk of the regenerative stage the vacuoles left by the released secretion are apparent. Within an acinus the cells are at differing stages of production, secretion, and regeneration.
\
Alcells/
Cent
FIG. 1. Schematic diagram, based on electronmicrographs, of an acinus from the salivary gland of the cockroach, IV. cinereu. The four cell types are shown-only the central cells and the secretory duct cells elaborate a secretion. The sequence A to F shows central cells in different stages of secretory activity: A, a completely depleted cell, with regeneration of the endoplasmic reticulum just beginning; B, regeneration of the endoplasmic reticulum complete; C, production of droplets of secretion underway; D and E, cells full with secretion; F, following release of the secretion the residual empty structures disappear to give rise to a cell like that shown in A.
Electron microscopical anatomy Peripheral cell. Each cell contains an intracellular ductule which is lined with numerous microvilli (Fig. 2A). The shape of these cells is pyramidal and they occur frequently in pairs. Underneath the basement membrane the plasma membrane undergoes considerable infolding. There are many mitochondria within the cytoplasm and numerous dense glycogen granules are also present. The endoplasmic reticulum does not constitute a prominent component of the cytoplasm, and Golgi bodies are observed only occasionally. KIESEL and BEAMS (1963) reported that they found a ‘few large, dense structures . . ., composed, in part, of concentric membranous lamellae' ; however, these bodies are also evident not only in the peripheral cells (Fig. 2B) but also in all other cell types in our sections.
2074
K. P.
BLAND AND
C. R. HOUSE
Central cell. In contrast to the peripheral cell, the basal surface of this cell occupies a relatively minor fraction of the acinar surface, and, moreover, it is not extensively infolded. However, the apical plasma membrane has numerous microvilli on its invaginated surface (Fig. 2C). The endoplasmic reticulum is prominent in both a rough and vacuolar form and the cell also contains Golgi bodies. There are few mitochondria except in regions adjacent to the lumen of the duct. The large secretory droplets contain granular material which is not dense ; neither is the density of the droplets uniform. These droplets often possess a limiting membrane and since they have been observed in the duct lumen in the proximity of the central cells, their release must occur by exocytosis (Fig. 2D). It is consistent with both the electron microscopical observations and the histological findings that the synthesis of the secretory material in the population of central cells is asynchronous (Fig. 3A). Secretory duct cell. The basal plasma membrane shows no pronounced infolding although the apical membrane does invaginate (Fig. 3B). The lumen of the duct at this level is lined with a layer of chitin which is electron opaque. The chitin lining continues into the acinar lumen until it reaches the ductules of the peripheral cells and the apical surface of the central cells where it ends. This lining does not make intimate contact with the plasma membrane of these cells. There are numerous mitochondria in the secretory duct cells and these tend to be associated with the apical rather than the basal surfaces. Although the endoplasmic reticulum is not well developed, these cells contain large droplets of dense granular material. Unlike the secretion from the central cells, the dense material from the secretory duct cells becomes more granular and appears to dissolve immediately on its release. Non-secretory duct cell. It is in the neighbourhood of the acinus only that the duct cells perform a secretory function and the non-secretory .duct cells occupy the major portion of the length of the ducts. The apical plasma membrane of the nonsecretory duct cells is highly invaginated in its position underneath the chitin lining (Fig. 3D). In agreement with KESSEL and BEAMS(1963) we have found that
FIG. 2A. Portion of a typical peripheral cell showing the numerous microvilli (MV) surrounding the lumen of the ductule (D). Note the deep and complicated The cell infoldings of the surface adjacent to the basement membrane. contains numerous mitochondria (MC) and glycogen granules (G). FIG. 2B. Portion of another peripheral cell with the same features as in Fig. 2A. In addition a few fragments of endoplasmic reticulum (ER) and some of the lamellated structures (K), noted by KESSEL and BEAMS(1963), are present. FIG. 2C. The apical portions of three central cells forming a duct. Note the numerous microvilli (MV) on the apical surface of the central cells and the chitin lining (C) within the duct. Several large droplets of secretion (Sl) are apparent along with numerous mitochondria (MC) associated with the apical portion of the central cells. FIG. 2D. A longitudinal section of a duct similar to that in Fig. 2C. Large droplets of secretion (Sl) are present in the duct lumen and prominent septate desmosomes (SD) are present joining adjacent central cells.
2075
2076
FIG. 3.
FUNCTION OFCOCKROACH SALIVARY GLAND
2077
the infoldings of this membrane form a web-like structure of vacuolar spaces (Fig. 3D). There is also a great deal of infolding at the basal surface. Numerous mitochondria are found between the infoldings of the basal plasma membrane (Fig. 3C). The endoplasmic reticulum is not prominent but the cytoplasm contains numerous very small dense granules, possibly ribosomes. Connexions between cells. Septate desmosomes, similar to those described by KE~SEL and BEAMS(1963), h ave been observed between central cells and their neighbours, namely peripheral cells, secretory duct cells and other central cells. Such connexions have been observed also between some peripheral cells and secretory duct cells. All these connexions occur in the vicinity of the lumen of the gland. The secretory and non-secretory duct cells are also joined to each other by septate desmosomes. Usually there is considerable coiling of such connexions and this is particularly prominent at the junction between the non-secretory duct cells. However, the septate desmosomes between central cells are sometimes short and uncoiled. Trucheoles and innervation. Both intracellular and extracellular tracheoles have been observed in association with all cell types. The gland is richly innervated and the exact form and function of this innervation is the subject of a current study. Enzyme production
In extracts of salivary glands and reservoirs, amylase, maltase, and invertase were readily demonstrable although lactase appeared to be absent. Lipase could not be detected in the salivary glands although it was present in the gut. On the other hand, protease was present in both the glands and the gut, although the amount in the former is very small since only mild precipitation occurred and that not until some 14 hr after precipitation with gut extracts. Histochemical characterixation of secretions
The histochemical
findings are summarized
in Table 2.
FIG. 3A. Portions of three central cells in different stages of secretory activity. Cell 1 is in early stages of the formation of the secretory product and shows the regeneration of abundant endoplasmic reticulum (ER): Cell 2 contains large droplets of secretion (Sl). After the release of the secretion, the endoplasmic reticulum (ER) starts to reform as in Cell 3. FIG. 3B. Transverse section of part of a duct showing secretory duct cells. Note the large dense droplets of secretion (S2) and the infoldings of the apical plasma membrane lying beneath the chitin lining (C) of the duct.
FIG. 3C. The basal region of a non-secretory duct cell. Numerous elongated mitochondria (MC) lie within the folds of the basal plasma membrane underneath the basement membrane (BM). FIG. 3D. The apical region of a non-secretory duct cell. Adjacent to the chitin lining (C) of the duct, the apical plasma membrane is contorted into numerous small vacuole-like infoldings (V). The mitochondria (MC) in this part of the cell are not elongated neither are they closely associated with the apical infoldings. Septate desmosomes (SD) are apparent joining the cells.
Test
2---HISTOCHEMICAL
REACTIONS
Sudan methods
Modified Tremblay
(amylase) (+)
++++ (pinkish)
-
Hammett and Chapman’s nitroprusside Performic acid-Alcian Blue (-SS) Adams’ DMAB-nitrite (tryptophan) DMAB-nitrite (after oxidation)
(-3-I)
-
Bensley and Gersh’s Millon (tyrosine) Modified Sakaguchi (arginine)
++++ -t
-
(+)
-
Secretion
GLANDS
OF THE CENTRAL
Secretory duct cells
OF THE SECRETION
y Metachromasia (alcohol-stable) /3Metachromasia (alcohol-labile) p Metachromasia (after methylation) Alcian Blue at pH 1.0 (- SO, mucosubstances) Alcian Blue at pH 2.5 (acid mucopolysaccharide) Periodic acid-Schiff’s reagent (1 : 2 glycol) Ravetto’s bial (sialic acid)
Bracco and Curti’s benzidine (- SOa) Molybdate-benzidine ( - PO3
TABLE
SECRETORY
+ (T) -
++ ++ ++ -
(+)
++ + -
-
+:
(1) -
(+) 1+) I +
-
-
+ + I
-
Secretion
at max.
SALIVARY
-
-
-
J
Secretion voided
CELLS OF THE COCKROACH
Central cells
DUCT
-
little and dense
AND
sd EC 8 Ki
c,
5
g
F
F .‘d
FUNCTION
OF COCKROACH
SALIVARY
GLAND
2079
Secretion of secretory duct cells Irrespective of the fixative employed the secretory products of the duct cells showed a strong positive reaction with the periodic acid-Schiff’s technique, indicative of the presence of 1 : 2 glycosidic linkages. The secretion also gave a distinct colouration with Ravetto’s bial method for identifying high concentrations of sialic acid. This secretion, therefore, probably consists in part of sialoglycans (glucosaminoglycans). The neutral nature of sialoglycans would explain their failure to react with Alcian Blue (pH 1.0 or 2.5) or to give metachromasia with Toluidine Blue or thionine. There was another component to the duct cell secretion which was more labile and was lost by fixation in unbuffered fixatives. This component showed a strong reaction with the DMAB-nitrite method for tryptophan. Absence of the reaction after performic acid oxidation confirmed that tryptophan was responsible since such oxidation is thought to be specific for the indole ring (HUMASON, 1967). However, after oxidation the deep blue colour was replaced by a pale pink. This may indicate a positive Morgan-Elson reaction for N-acetylated hexosamines (MORGAN and ELSON, 1934). The high tryptophan content is unusual and appeared to occur in the absence of stainable amounts of the amino acids; tyrosine (Millon’s reaction), arginine (Sakaguchi method), and cystine (Adams and Sloper’s performic acid-Alcian Blue method). Secretion of central cells The secretion of the central cells also gave a positive reaction with periodic acid-Schiff’s method indicating the presence of 1 : 2 glycosidic linkages. There was, however, in addition a distinct but weak staining with Alcian Blue at pH 2.5 which indicates the presence of weakly acidic mucopolysaccharide (glycosaminoglucuronoglycan). Since no staining took place with Alcian Blue at pH 1-O it is improbable that strongly acidic sulphated mucosubstances are present. This conclusion was supported by the absence of a response to Bracco and Curti’s benzidine method for sulphate. Furthermore, the secretion showed alcohollabile p metachromasia with 1*5”/0 aqueous Toluidine Blue and saturated aqueous thionine. Such metachromasia usually indicates the presence of a high concentration of weakly acidic carboxyl (or possibly phosphoryl) groupings. Mild methylation (37°C for 2 hr) of the tissue with 0.1 N HCl in methanol destroyed this metachromasia. This process was once thought to block only the carboxyl groups but is now known to be less specific (PEARSE, 1968). Thus the central cell secretion appears to be composed in part of acidic carboxyl-containing carbohydrate. The modified Tremblay’s method indicated that the amylase activity of the salivary glands resided in the cells of the acinus as opposed to the secretory or non-secretory duct cells. As the central cells are the only acinar cells of a secretory nature, they are probably responsible for the production of this enzyme (and possibly the other enzymes produced by the gland). This central cell production of amylase was supported by the fact that during the acidophilic stage of their
2080
K. P.
BLAND AND
C. R.
HOUSE
secretory cycle, the secretion stained positively for tyrosine (Millon’s reaction), arginine (Sakaguchi method), cystine (Adams and Sloper’s performic acid-Alcian Blue method), cysteine (Hammett and Chapman’s nitroprusside method), and tryptophan (DMAB-nitrite method). Performic acid oxidation which is specific for the indole ring, destroyed the reaction of the central cells for tryptophan. Each of these amino acids amounts to more than 4 per cent (by weight) of human salivary amylase (DIXON and WEBB, 1958).
DISCUSSION Detection of amylase, maltase, invertase, and protease in the salivary glands of N. cinerea conforms with the findings in other species (see Table 3). The failure to detect lactase and lipase in this species using methods similar to those employed by RAYCHAUDHURI and GHOSH (1964) to demonstrate these enzymes in Periplaneta awicana may indicate a species difference. The predominance of amylase in the saliva of cockroaches shows an interesting parallel to the situation in mammals. However, unlike the latter, lysozyme is unlikely to be produced by the cockroach salivary gland in view of this enzyme’s ability to slowly hydrolyse chitin (BERGERand WEISER, 1957). The central cells appeared to be the source of the amylase and possibly the other enzymes produced by the salivary glands because: (a) The central cell secretion had an electron microscopical structure suggesting protein. (b) The central cells contained abundant rough endoplasmic reticulum which is usually associated with active protein synthesis. (c) The secretion has a protein nature as it stained positive for the amino-acids; tyrosine, arginine, cystine, cysteine, and tryptophan. Cockroach amylase was shown by WIGGLESWORTH(1927) to be activated by chloride ions in a similar manner to human salivary amylase (DIXON and WEBB, 1958). This common characteristic may indicate a structural similarity between the two enzymes, at least in the active regions. The above amino acids each constitute more than 4 per cent by weight of human salivary amylase (DIXON and WEBB, 1958). (d) Th e modification of Tremblay’s method for amylase indicated the presence of amylase in the central cells. Weakly acidic carbohydrate material also occurs in the secretion of the central cells and it is this that has probably caused the confusion about the role of these cells (see Table 1). The similar labile nature of both the carbohydrate and the protein fractions of the secretion suggests that some binding exists between these substances. The staining properties of the central cell secretion indicate the presence of mucoprotein, a finding which would support the suggestion that these cells produce mucin. However, the protein part of the secretion is the enzyme amylase, thus cockroach salivary amylase may be similar to the fl-amylase of wheat, which contains a polysaccharide component essential for its activity (MCILROY, 1948). Since this form of amylase is not usually considered to be present in animals and furthermore is not activated by chloride ions (LONG, 1961), the carbohydrate is probably present solely to stabilize the enzyme.
FUNCTION
OF COCKROACH
SALIVARY
GLAND
2081
2082
K. P. BLAND AND C. R. HOUSE
The structure of the peripheral cells is similar to that of the cells in the distal portion of the salivary glands of CuZZiphora erythrocephulu, Meig. (OSCHMANand BERRIDGE, 1970). Each secretory cell in the distal region of that gland possesses a branching system of canaliculi opening into the lumen and Oschman and Berridge found that these cells elaborated a fluid which contained approximately 140 mM KC1 and 10 mM NaCl/l. In Calliphora, these cells are also responsible for the secretion of amylase since they contain a relatively prominent rough endoplasmic reticulum, free ribosomes, Golgi complexes, and secretory droplets. From the structural similarity between the cockroach peripheral cells and CuZZiphora secretory cells the former are probably concerned with the transport of ions and water from the haemolymph into the lumen of the gland. Moreover, the movement of ions and water is apparently the sole function of the peripheral cells since, unlike Caltiphoru secretory cells, they do not contain either abundant endoplasmic reticulum or secretory droplets. In N. cinereu the mucous component of the saliva appears to be primarily produced by the secretory duct cells. The secretion of these cells contains sialoglycans but little or no stainable protein. The only previous mention of this type of secretory cell was made by RAYCHAUDHURIand GHOSH (1964). These workers noted that in P. americana the columnar epithelia lining of the ‘ductules’ contained granules suggesting ‘glandular activity’ but did not comment further about them. The nature of the tryptophan-rich and more labile component of the duct cell secretion is unresolved. This high level of reactivity to the DMAB-nitrite method in the apparent absence of other amino acids could be ascribed to 5-hydroxytryptamine (S-HT) which also reacts in this system (PEARSE, 1968). However, a histochemical fluorescence method for 5-HT based on the ninhydrin method of JEPSON and STEVENS (1953) did not reveal a high concentration of 5-HT in the secretory duct cells. No fluorescence is given by tryptophan with this method. Following performic acid oxidation the DMAB-nitrite method replaced the intense blue reaction for tryptophan with a faint but distinct reddish-purple colour. This colour was probably due to acetylhexosamines in the sialoglycans reacting with DMAB in a manner similar to that described by MORGANand ELSON (1934) for the The positive tryptophan reaction per se was not due to assay of acetylglucosamine. hexosamines because: (a) Chitin (composed almost entirely of acetylglucosamine) did not stain, However, PEARSE (1968) ob served that in the histochemical use of DMAB for amino sugars no reaction is obtained with insect chitin. (b) In vitro assessment of the specificity of the DMAB-nitrite method with tryptophan and N-acetylglucosamine confirmed that only tryptophan reacted. However, both tryptophan and N-acetylglucosamine (pretreated by boiling in 1 N KOH) gave identically coloured products with 5% DMAB in concentrated HCl-the initial product was bright purple which rapidly faded to dull purple-red. Nitrite oxidation gave the characteristic dark blue of carboline blue with tryptophan but produced only a yellowish colour with N-acetylglucosamine. The non-secretory duct cells may be concerned with the re-absorption of certain ions from the saliva or with the secretion of other ions into the saliva. They
FUNCTIONOF COCKROACH SALIVARYGLAND
2083
have a similar structure to that of the re-absorptive cells in the proximal part of Calliphora salivary gland (OSCHMANand BERRIDGE, 1970) where potassium ions are re-absorbed and sodium ions are secreted into the lumen. The particular features, which are common to both of these types of cell, are the pronounced infoldings of the apical and basal plasma membranes in association with numerous mitochondria and the absence of secretory droplets. According to Oschman and Berridge the proximal region of Calliphora salivary gland reduces the osmolarity of Preliminary analyses of the the saliva to a value which is hypotonic to haemolymph. saliva in the reservoir of cockroach gland indicates that the concentrations of potassium and sodium ions are approximately identical (20 mM/l) and somewhat similar to those in the saliva of Calliphora when the proximal re-absorption of potassium is at a maximum. Acknowledgements-We wish to thank Miss J. M. MACKNESSfor the preparation of the electron microscopical sections and Dr. A. W. G. MANNINGof the Zoology Department for providing specimens of N. cinerea. One of us (C. R. H.) IS indebted to the Science Research Council for financial support. REFERENCES BALDWIN E. and BELL D. J. (19.55) Cole’s Practical Physiological Chemistry, 10th ed. Heffer, Cambridge. BAXH S. (1858) Untersuchungen iiber das chylopoetische und uropoetische System der Blatta orientalis. Sitzungsberichte der Kaiserlichen Akademie der Wissenschaften (Muth.Nut. Classe). Wien 33, 234-260. BERGER L. R. and WEISER R. S. (1957) The ,&gl ucosaminidase activity of egg-white lysozyme. Biochim. biophys. Acta 26, 517421. DAY M. F. (1949a) The distribution of alkaline phosphatase in insects. Aust.J. sci. Res. (B) 2, 31-41. DAY M. F. (1949b) The occurrence of mucoid substances in insects. Aust. J. sci. Res. (B)
2, 421427. DAY M. F. (1951) The mechanism of secretion by the salivary gland of the cockroach Periplaneta americana (L). Aust.J. sci. Res. (B) 4, 136-143. DAY M. F. and PO~NING R. F. (1949) A study of the process of digestion in certain insects. Aust. J. sci. Res. (B). 2, 175-215. DIXON M. and WEBB E. C. (1958) Enzymes. Longmans, Green, London. GLICK D. (1949) Techniques of Histo- and Cytochemistry. Interscience, New York. HUMASONG. L. (1967) Animal Tissue Techniques, 2nd ed. Freeman, San Francisco. JEPSON J. B. and STEVENSB. J. (1953) A Auorescent test for seratonin and other tryptamines. Nature, Lond. 172, 772. KESSEL R. G. and BEAMS H. W. (1963) Electron microscope observations on the salivary gland of the cockroach, Periplaneta americana. 2. Zellforschung. 59, 857-877. LEBEDEFF A. (1899) cited by DAY (1951). LONG C. (ed.) (1961) Biochemist’s Handbook. Spon, London. MCILROY R. J. (1948) The Chemistry of the Polysaccharides. Arnold, London. MORGANW. T. J. and ELSON L. A. (1934) A calorimetric method for the determination of N-acetylglucosamine and N-acetylchrondrosamine. Biochem. J. 28, 988-995. OSCHMANJ. L. and BERRIDGE M. J. (1970) Structural and functional aspects of salivary fluid secretion in Calliphora. Tissue and Cell 2, 281-310. PEARSEA. G. E. (1968) Histochemistry, Theoretical and Applied, 1,3rded. Churchill, London.
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PLATEAU F. (1876) Note sur les phCnom&nes de la digestion chez la Blatte americaine (Periplaneta americana L.) Bull. Acad. roy. Belg. (2), 41, 1206-1233. RAYCHAUDHURI D. N. and GHOSHS. K. (1964) Study on the histophysiology of the salivary apparatus of Periplaneta americana Linn. Zool. Anz. 173, 227-237. SUTHERLAND D. J. and CHILLSEYZNJ. M. (1968) Function and operation of the cockroach salivary reservoir. J. Insect Physiol. 14, 21-31. SWINCLEH. S. (1925) Digestive enzymes in an insect. OhioJ. Sci. 25, 209-218. TREMBLAYG. (1963) The localization of amylase activity in tissue sections by a starch film method. J. Histochem. Cytochem. 11, 202-206. TREMBLAY G. and C&REST J. (1968) Modified starch film method for the histochemical localization of amylase activity. J. Histochem. Cytochem. 16, 147-148. WIGGLESWORTHV. B. (1927) Digestion in the cockroach-II. The digestion of carbohydrates. Biochem. J. 21, 797-811.