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Scanning electron microscopy of the Rhodnius neglectus (Hemiptera) labial salivary glands after starvation
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Scanning electron microscopy of the Rhodnius neglectus (Hemiptera) labial salivary glands after starvation Bruno Konig Junior*, Telma Sumie Masuko and Bertha Rosenberg** * Department of Anatomy, Institute of Biomedial Sciences of the Sao Paulo University. P.O. Box 66208, BR-05389-970 Sao Paulo - SP, Brasil, ** Department of Morphology, Faculty of Dentistry State University of Sao Paulo in Sao Jose dos Campos, Brasil
Summary. Labial salivary glands are found in the majority of insects. They are relatively large, extend back into the thorax, and in Rhodnius, they are cherry red in color due to a pigment derived from traces of hemoglobin absorbed form the gut. In most insects they are acinous shaped, with long excretion channels that present differentiated regions which from salivary reservoirs. The glands may be relatively simple or complexly branched and convoluted. In Rhodnius they are described as being unilobed with no traces of division. The main duct leaves the gland at its anterior extremity. The acini have different kinds of cells but all of them are seen as sources of secretion. Our material has a different shape due to the fact that the animals spent 20 days under starvation conditions. New data are also obtained through treatment with collagenase and HCl. The importance of the study of these glands lies in the fact that it will further understanding of the transmission of Chagas' disease.
Key words: Salivary glands - Rhodnius - Collagenase SEM
Introduction The type of secretion produced by the salivary glands of insects is quite variable and so are the size and shape of the structures. Amylase and invertase are the enzymes most often encountered in insect salivary glands. In many blood sucking insects the saliva possesses poisonous or irritant substances *Visiting Professor at the Department of Anatomy, Medical University of LUbeck, Germany, granted by the Alexander von HumboldtStiftung Correspondence to: B. Konig Junior
~~I S~MPER ~
Ann Anat (1993) 175: 411-416 Gustav Fischer Verlag lena
and, in some ofthem, Cornwall and Patton (1914) have detected a powerful anticoagulin. In most insects the labial glands are the functional salivary glands. The saliva serves to lubricate the mouth and production is increased if the food is dry (Wigglesworth 1927). The variable form in different incects was already detected by Bugnion and Popoff (1908) and Faure (1910), and in some insects like Hemiptera the glands are made up of several lobes with quite different structures and functions. This is also confirmed by Baptist (1941). In 1972 Wigglesworth confirmed previous observations (Kni.ippeI1881), and in describing Rhodnius, he observed that the glands were cherry red in color due to the presence of 'haemalbumin' , a pigment derived from traces of haemoglobin absorbed from the gut. Generally, the glands which are most highly developed in insects have an outlet tube that ends in a pocket of the ventral wall of the head between the base of the free part of the labium and the base of the hypopharynx (Snodgrass 1935). They are usually situated in the thorax, but may lie in the head or extend to the abdomen. A reservoir is frequently developed from the conducting part of the system. Mellanby (1936) gives a description of the development of the gland in Rhodnius, but no accessory gland is mentioned.
Material and methods Six salivary glands from four mosquitoes were dissected after 20 days of starvation and fixed in a 2 % paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (PH 7.4) solution at a low temperature for 3 days. Digestion was performed by using HCI 8 N for 40 minutes in a water bath, with temperatures ranging from 45 DC to 60 DC, and then in a solution of collagenase type II (10 mglIO ml)
Fig. I . The bilobular appearance is evident. The opened acini show an empty inner space. Some vessels and a few nerves can be distinguished. x 225. Fig. 2. With higher magnification, acini, vessels, nerves, excretion channels (some of them convoluted (arrow» and some collagenous tissue that was not digested can be seen. X 540. Fig. 3. At a still higher magnification, some channels are seen to be very large, and long in this panoramic view, when compared to the size of the acini (arrow). X 675.
412
for 3 to 5 hours at a temperature of 37 °e. After rinsing in distilled water, dehydration through a graded series of ethanol and transfer to isoamyl acetate followed. After critical point dehydration the samples were mounted on aluminium stubs and sputtered with gold palladium. Scanning electron microscopy and photography were performed at the University of Sao Paulo, Brazil, Faculty of Medicine, Pathologic Anatomy, and at the Institute of Anatomy of the Medical University of Liibeck, Germany.
Results Probably due to the starvation, the labial salivary glands of Rhodnius were paler than usual. The characteristic red color could not be seen. However, the glands remained large in size, extending to the thorax. They looked unilobed during dissection, and the main duct left the gland at its anterior extremity. The shape of the structure was acinar with long excretion channels, and as the connective tissue was removed a division of the gland in two lobes could be seen (Fig. I). After removal of the connective tissue, the acini were revealed to be separated from each other and covered by a basement membrane. A great number of vessels and a few nerves could be distinguished. The excretion channels might show a convoluted aspect (Figs. 2 and 3), and in some regions they seemed very large compared to the size of the acini. Each acinus had one excretion duct that joined neighboring ones to constitute a wider one (Fig. 4). At higher resolutions (Fig. 5) some little granules could be seen at random in the interacinar and excretion duct spaces, and some structures of a different kind seemed to unite one acinus to a neighboring one (Fig. 6). When observed in profile, some acini followed by their ducts had a conical shape. The basement membrane of acini and tubuli was exposed, and the filamentous layer became evident with a velcro-like rough structure (Fig. 7). Intercellular junctions and myoepithelial cells, if present, could not be seen with the method employed. In some areas there were some expansions that could be interpreted as, apparently at least, communications among acini and excretory ducts (Fig. 8). The acini that were open showed divisions in the interior corresponding to some kind of fenestrated membranes. No secretion granula were seen. The glandular epithelium was not evident as a whole.
Discussion In many insects, the labial salivary glands have other functions than the production of salvia. In some they produce silk. We know through research performed by Cornwall and Patton (1914) that in Rhodnius the saliva contains amylase and invertase, and a powerful anticoagulin as in other blood-sucking insects in order to make their work easier. Haridass and Avanthakrishnan (1981) describe hemophagus triatomidae and state that the unilobed gland produces only anticoagulants that ensure an uninterruped flow of blood from the vertebrates hosts.
The labial glands of Rhodnius are described as cherry red in color when observed macroscopically (Wigglesworth 1972). We suppose that it is due to the 20 day starvation period that our insects have a paler gland. Apparently the size of the glands remained unchanged, since they reached the thorax as normally described. The main duct was in the anterior extremity of the gland, as mentioned by the authors specified in the bibliography. In spite of the fact that the gland has been described as being unilobed, our material showed that this appearance is only due to the collagenous capsule that surrounds the gland. Once this is removed, it can be seen that the gland has two lobes, an anterior and a posterior. This fact was already detected by Baptist (1941), who deduced that the gland in Rhodnius must have two lobes, an anterior and a posterior, due to the position of the issuing principal duct. The minute vesicle and secondary duct described as an accessory gland was not seen in our material. Our observations are the same as those made by MeJlanby (1936), who studied the development of the gland but does not mention an accessory one. However, it must be kept in mind that our material was taken from mosquitoes that had been starved for 20 days. It is classical knowledge that the glandular epithelium always consists of a single layer of glandular cells around a central lumen. The cytoplasm has secretion granules and collecting vacuoles. In insects no doubt these collecting vacuoles are well developed as an adaptation to the exceptionally large size of the cell and store up quite a quantity of secretion. It could be that the opened acini are empty in our material due to the fact that normally secreted granules are built up in the cytoplasm to the zymogen-granule stage, transformed into secretion and transferred to the collecting vacuoles. When the collecting vacuoles are filled, the glandular synthesis proceeds as far as the zymogen-granule stage and is stored up as such in the cytoplasm (Baptist 1941). Due to a lack of food, our animals did not reach this stage of salivary production. Describing the salivary gland of the desert locust, Kendall (1969) mentions the presence of different cells. Linking this to our observations, it is interesting to notice the presence of pigment cells, that are pinkish red, lie free in the hemocoel and are attached to each acinus by connective tissue. In general, the interacinar spaces of our material look somewhat wider than normal. The excretion ducts are very large when compared to the acini in some regions, and salivary reservoirs were not encountered,. These differences are the result of the physiologic stage of the gland; the acini are empty but the excretion channels still contain the product. As mentioned above, the macroscopic size of the gland appears normal. The little granules that, in higher resolutions, can be found at random in the interacinar and excretion duct spaces, may correspond to the small dense masses, distinct from secretion or zymogen granules, which were also described by Baptist (1941). They may represent some kind of reserve material, as well as other structures that are found running from one acinus to the other.
413
Fig. 4. Some acini (A) with corresponding excretion channels (C) that come together to form a wider one (*). x 2.610. Inset x 3.780. Fig. 5. Little granules found at random (arrow), the basement membrane of an acinus (A), excretion channels (C), one of which is convoluted (CC) and some kind of mucous collections (MC) can be seen. x 3.150. Inset x 3.780. Fig. 6. A kind of folded linkage between glandular structures: Acini (A) and excretion channel (C). Little granules, as in Figure 5, can be observed. x4.050.
414
Fig. 7. The basement membrane is fibrous or granular in different areas of the same acinus. The fibrous part looks like a collagen meshwork (F); the granular part like a kind of velvet (G). (A) acinus; (C) excretion channel. X 4.860 (7 A). X 7.650 (7B). Fig. 8. Expansions link acini to acini and to excretion channels. As in Figure 5, little granula are interspersed in the interacinar and excretion channel spaces. X 8.200.
415
Using our technique, after digestion the basement membrane is exposed in its filamentous layer that displays a velcro-like rough structure. The appearance of the basement membrane was described in accordance with the paper published by Watanabe, Koriyarna and Yamada (1992) in High Resolution Scanning Microscopy. They describe the membrane as consisting of a meshwork structure made of a fine net of collagen fibrils.
References Baptist BA (1941) The morphology and physiology of the salivary glands of Hemiptera heteroptera. Quart J Micr Sci 83: 91-139 Bugnion E, Popoff N (1910) Appareil salivaire des Hemipteres. Arch Anat Microsc 11: 435-456 Cornwall JW, Patton WS (1914) Salivary secretion of commoner blood sucking insects and ticks. Ind J Med Res 2: 569-596 FaunS-Fremiet E (1910) Glandes labiaies des Hydrocorises. Ann Sci Nat Zool (ser. 9) 12: 217-240
Kendall MD (1969) The fine structure of the salivary glands of the Desert Locust, Schistocerca gregaria Forskal. Z Zellforsch 98: 399-420 Harridass ET, Avanthakrishnan TN (1981) Functional morphology of the salivary system in some Reduviidae (Insecta heteroptera). Proc Indian Acad Sci Sect B 90: 145-160 Kniippel A (1886) Salivary glands of insects. Arch Naturgesch 52: 271-303 Mellanby H (1936) Embryology of Rhodnius prolixus. Quart J Micr 79: 140-149 Snodgrass RE (1935) Pricipies of insect morphology. McGraw Hill, New York Watanabe I, Koriyama Y, Yamada E (1992) High resolution scanning electron microscopic study of the mouse submandibular salivary gland. Acta Anat 143: 59-66 Wigglesworth VB (1972) Determination of pH, by colorimetric method, of insect saliva. Biochem J 21: 791-811 Wigglesworth VB (1972) The principles of Insect Physiology. 7th edn. Chapman and Hall, London Accepted January 14, 1993
416
Scanning electron microscopy of the Rhodnius neglectus (Hemiptera) labial salivary glands after starvation Bruno Konig Junior*, Telma Sumie Masuko and Bertha Rosenberg** * Department of Anatomy, Institute of Biomedial Sciences of the Sao Paulo University. P.O. Box 66208, BR-05389-970 Sao Paulo - SP, Brasil, ** Department of Morphology, Faculty of Dentistry State University of Sao Paulo in Sao Jose dos Campos, Brasil
Summary. Labial salivary glands are found in the majority of insects. They are relatively large, extend back into the thorax, and in Rhodnius, they are cherry red in color due to a pigment derived from traces of hemoglobin absorbed form the gut. In most insects they are acinous shaped, with long excretion channels that present differentiated regions which from salivary reservoirs. The glands may be relatively simple or complexly branched and convoluted. In Rhodnius they are described as being unilobed with no traces of division. The main duct leaves the gland at its anterior extremity. The acini have different kinds of cells but all of them are seen as sources of secretion. Our material has a different shape due to the fact that the animals spent 20 days under starvation conditions. New data are also obtained through treatment with collagenase and HCl. The importance of the study of these glands lies in the fact that it will further understanding of the transmission of Chagas' disease.
Key words: Salivary glands - Rhodnius - Collagenase SEM
Introduction The type of secretion produced by the salivary glands of insects is quite variable and so are the size and shape of the structures. Amylase and invertase are the enzymes most often encountered in insect salivary glands. In many blood sucking insects the saliva possesses poisonous or irritant substances *Visiting Professor at the Department of Anatomy, Medical University of LUbeck, Germany, granted by the Alexander von HumboldtStiftung Correspondence to: B. Konig Junior
~~I S~MPER ~
Ann Anat (1993) 175: 411-416 Gustav Fischer Verlag lena
and, in some ofthem, Cornwall and Patton (1914) have detected a powerful anticoagulin. In most insects the labial glands are the functional salivary glands. The saliva serves to lubricate the mouth and production is increased if the food is dry (Wigglesworth 1927). The variable form in different incects was already detected by Bugnion and Popoff (1908) and Faure (1910), and in some insects like Hemiptera the glands are made up of several lobes with quite different structures and functions. This is also confirmed by Baptist (1941). In 1972 Wigglesworth confirmed previous observations (Kni.ippeI1881), and in describing Rhodnius, he observed that the glands were cherry red in color due to the presence of 'haemalbumin' , a pigment derived from traces of haemoglobin absorbed from the gut. Generally, the glands which are most highly developed in insects have an outlet tube that ends in a pocket of the ventral wall of the head between the base of the free part of the labium and the base of the hypopharynx (Snodgrass 1935). They are usually situated in the thorax, but may lie in the head or extend to the abdomen. A reservoir is frequently developed from the conducting part of the system. Mellanby (1936) gives a description of the development of the gland in Rhodnius, but no accessory gland is mentioned.
Material and methods Six salivary glands from four mosquitoes were dissected after 20 days of starvation and fixed in a 2 % paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (PH 7.4) solution at a low temperature for 3 days. Digestion was performed by using HCI 8 N for 40 minutes in a water bath, with temperatures ranging from 45 DC to 60 DC, and then in a solution of collagenase type II (10 mglIO ml)
Fig. I . The bilobular appearance is evident. The opened acini show an empty inner space. Some vessels and a few nerves can be distinguished. x 225. Fig. 2. With higher magnification, acini, vessels, nerves, excretion channels (some of them convoluted (arrow» and some collagenous tissue that was not digested can be seen. X 540. Fig. 3. At a still higher magnification, some channels are seen to be very large, and long in this panoramic view, when compared to the size of the acini (arrow). X 675.
412
for 3 to 5 hours at a temperature of 37 °e. After rinsing in distilled water, dehydration through a graded series of ethanol and transfer to isoamyl acetate followed. After critical point dehydration the samples were mounted on aluminium stubs and sputtered with gold palladium. Scanning electron microscopy and photography were performed at the University of Sao Paulo, Brazil, Faculty of Medicine, Pathologic Anatomy, and at the Institute of Anatomy of the Medical University of Liibeck, Germany.
Results Probably due to the starvation, the labial salivary glands of Rhodnius were paler than usual. The characteristic red color could not be seen. However, the glands remained large in size, extending to the thorax. They looked unilobed during dissection, and the main duct left the gland at its anterior extremity. The shape of the structure was acinar with long excretion channels, and as the connective tissue was removed a division of the gland in two lobes could be seen (Fig. I). After removal of the connective tissue, the acini were revealed to be separated from each other and covered by a basement membrane. A great number of vessels and a few nerves could be distinguished. The excretion channels might show a convoluted aspect (Figs. 2 and 3), and in some regions they seemed very large compared to the size of the acini. Each acinus had one excretion duct that joined neighboring ones to constitute a wider one (Fig. 4). At higher resolutions (Fig. 5) some little granules could be seen at random in the interacinar and excretion duct spaces, and some structures of a different kind seemed to unite one acinus to a neighboring one (Fig. 6). When observed in profile, some acini followed by their ducts had a conical shape. The basement membrane of acini and tubuli was exposed, and the filamentous layer became evident with a velcro-like rough structure (Fig. 7). Intercellular junctions and myoepithelial cells, if present, could not be seen with the method employed. In some areas there were some expansions that could be interpreted as, apparently at least, communications among acini and excretory ducts (Fig. 8). The acini that were open showed divisions in the interior corresponding to some kind of fenestrated membranes. No secretion granula were seen. The glandular epithelium was not evident as a whole.
Discussion In many insects, the labial salivary glands have other functions than the production of salvia. In some they produce silk. We know through research performed by Cornwall and Patton (1914) that in Rhodnius the saliva contains amylase and invertase, and a powerful anticoagulin as in other blood-sucking insects in order to make their work easier. Haridass and Avanthakrishnan (1981) describe hemophagus triatomidae and state that the unilobed gland produces only anticoagulants that ensure an uninterruped flow of blood from the vertebrates hosts.
The labial glands of Rhodnius are described as cherry red in color when observed macroscopically (Wigglesworth 1972). We suppose that it is due to the 20 day starvation period that our insects have a paler gland. Apparently the size of the glands remained unchanged, since they reached the thorax as normally described. The main duct was in the anterior extremity of the gland, as mentioned by the authors specified in the bibliography. In spite of the fact that the gland has been described as being unilobed, our material showed that this appearance is only due to the collagenous capsule that surrounds the gland. Once this is removed, it can be seen that the gland has two lobes, an anterior and a posterior. This fact was already detected by Baptist (1941), who deduced that the gland in Rhodnius must have two lobes, an anterior and a posterior, due to the position of the issuing principal duct. The minute vesicle and secondary duct described as an accessory gland was not seen in our material. Our observations are the same as those made by MeJlanby (1936), who studied the development of the gland but does not mention an accessory one. However, it must be kept in mind that our material was taken from mosquitoes that had been starved for 20 days. It is classical knowledge that the glandular epithelium always consists of a single layer of glandular cells around a central lumen. The cytoplasm has secretion granules and collecting vacuoles. In insects no doubt these collecting vacuoles are well developed as an adaptation to the exceptionally large size of the cell and store up quite a quantity of secretion. It could be that the opened acini are empty in our material due to the fact that normally secreted granules are built up in the cytoplasm to the zymogen-granule stage, transformed into secretion and transferred to the collecting vacuoles. When the collecting vacuoles are filled, the glandular synthesis proceeds as far as the zymogen-granule stage and is stored up as such in the cytoplasm (Baptist 1941). Due to a lack of food, our animals did not reach this stage of salivary production. Describing the salivary gland of the desert locust, Kendall (1969) mentions the presence of different cells. Linking this to our observations, it is interesting to notice the presence of pigment cells, that are pinkish red, lie free in the hemocoel and are attached to each acinus by connective tissue. In general, the interacinar spaces of our material look somewhat wider than normal. The excretion ducts are very large when compared to the acini in some regions, and salivary reservoirs were not encountered,. These differences are the result of the physiologic stage of the gland; the acini are empty but the excretion channels still contain the product. As mentioned above, the macroscopic size of the gland appears normal. The little granules that, in higher resolutions, can be found at random in the interacinar and excretion duct spaces, may correspond to the small dense masses, distinct from secretion or zymogen granules, which were also described by Baptist (1941). They may represent some kind of reserve material, as well as other structures that are found running from one acinus to the other.
413
Fig. 4. Some acini (A) with corresponding excretion channels (C) that come together to form a wider one (*). x 2.610. Inset x 3.780. Fig. 5. Little granules found at random (arrow), the basement membrane of an acinus (A), excretion channels (C), one of which is convoluted (CC) and some kind of mucous collections (MC) can be seen. x 3.150. Inset x 3.780. Fig. 6. A kind of folded linkage between glandular structures: Acini (A) and excretion channel (C). Little granules, as in Figure 5, can be observed. x4.050.
414
Fig. 7. The basement membrane is fibrous or granular in different areas of the same acinus. The fibrous part looks like a collagen meshwork (F); the granular part like a kind of velvet (G). (A) acinus; (C) excretion channel. X 4.860 (7 A). X 7.650 (7B). Fig. 8. Expansions link acini to acini and to excretion channels. As in Figure 5, little granula are interspersed in the interacinar and excretion channel spaces. X 8.200.
415
Using our technique, after digestion the basement membrane is exposed in its filamentous layer that displays a velcro-like rough structure. The appearance of the basement membrane was described in accordance with the paper published by Watanabe, Koriyarna and Yamada (1992) in High Resolution Scanning Microscopy. They describe the membrane as consisting of a meshwork structure made of a fine net of collagen fibrils.
References Baptist BA (1941) The morphology and physiology of the salivary glands of Hemiptera heteroptera. Quart J Micr Sci 83: 91-139 Bugnion E, Popoff N (1910) Appareil salivaire des Hemipteres. Arch Anat Microsc 11: 435-456 Cornwall JW, Patton WS (1914) Salivary secretion of commoner blood sucking insects and ticks. Ind J Med Res 2: 569-596 FaunS-Fremiet E (1910) Glandes labiaies des Hydrocorises. Ann Sci Nat Zool (ser. 9) 12: 217-240
Kendall MD (1969) The fine structure of the salivary glands of the Desert Locust, Schistocerca gregaria Forskal. Z Zellforsch 98: 399-420 Harridass ET, Avanthakrishnan TN (1981) Functional morphology of the salivary system in some Reduviidae (Insecta heteroptera). Proc Indian Acad Sci Sect B 90: 145-160 Kniippel A (1886) Salivary glands of insects. Arch Naturgesch 52: 271-303 Mellanby H (1936) Embryology of Rhodnius prolixus. Quart J Micr 79: 140-149 Snodgrass RE (1935) Pricipies of insect morphology. McGraw Hill, New York Watanabe I, Koriyama Y, Yamada E (1992) High resolution scanning electron microscopic study of the mouse submandibular salivary gland. Acta Anat 143: 59-66 Wigglesworth VB (1972) Determination of pH, by colorimetric method, of insect saliva. Biochem J 21: 791-811 Wigglesworth VB (1972) The principles of Insect Physiology. 7th edn. Chapman and Hall, London Accepted January 14, 1993
416