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Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi
Tissue & Cell, 1999 31 (3) 264–273 © 1999 Harcourt Publishers Ltd Article no. tice.1999.0057
Tissue&Cell
Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi C. K. Moreira-Ferro, O. Marinotti, A. T. Bijovsky
Abstract. Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.
Keywords: salivary glands, mosquito, ultrastructure, apyrase, alpha-glucosidase, lysozyme
Introduction Mosquitoes’ salivary glands are being investigated because of their important role in the transmission of pathogenic organisms to man and domestic animals. The adult female salivary glands are the final destination of several parasites before they are transmitted to the vertebrate hosts through the saliva (James & Rossignol, 1991; James, 1994). In addition, several biological activities related to hematophagy are found in the mosquitoes’ saliva. The salivary glands produce and secrete several molecules with anti-hemostatic and anti-inflammatory activities (Law et al., 1992). Among
Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil
Received 18 February 1999 Accepted 7 June 1999 Correspondence to: Cristina K. Moreira-Ferro, Departamento de Parasitologia, ICB, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374. CEP 05508–900. São Paulo, SP, Brazil. Tel.: 55 11 8187272; Fax: 55 11 8187417; E-mail: [email protected]
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these molecules, apyrase was identified as the enzyme responsible for the inhibition of platelet aggregation (Ribeiro et al., 1984a, 1984b, 1985; Cupp et al., 1994; Marinotti et al., 1996). Although the main focus of analysis of mosquitoes’ salivary glands results from their role in blood feeding and the transmission of pathogenic agents, these organs are also involved with sugar feeding. Mosquito saliva helps in the solubilization, ingestion and digestion of oligosaccharides found mostly in the nectar of flowers, plant exudates and fruit juices (Yuval, 1992). A salivary alpha-glucosidase is probably responsible for the oligosaccharide digestion in the mosquito crop (Marinotti & James, 1990; Marinotti et al., 1996). Another compound also found in mosquito saliva is a lysozyme that provides protection against bacterial invasion of the mouthparts and crop (Rossignol & Lueders, 1986; Moreira-Ferro et al., 1998). Morphological aspects of mosquitoes’ salivary glands were described for Aedes aegypti, Anopheles stephensi and Culex pipiens (Orr et al., 1961; Wright, 1969; Janzen & Wright, 1971; Barrow et al., 1975). The glands from these
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species are similar, consisting of three lobes connected to a main salivary duct. The salivary glands of males and females are morphologically distinct, with the male glands being smaller than those in the female. The lateral lobes of female glands have at least two distinct secretory regions, the proximal and distal portions, separated by a non-glandular region often referred to as the intermediate portion (James, 1994). The medial lobe appears to have only the intermediate and distal portions. Anopheles darlingi is an important malaria vector in Brazil and other Latin America countries. This mosquito species is responsible for the transmission of more than 90% of the malaria in Brazil, affecting approximately 500 000 people a year (PAHO, 1998). However, few studies have been conducted with this species mostly due to the absence of mosquito colonies adapted to the captivity. In this paper we describe the morphology of An. darlingi salivary glands and report the differential distribution of salivary components within the glands of female mosquitoes.
Materials and methods Insects Anopheles darlingi adult mosquitoes were captured in Rondonia State, Brazil. The salivary glands were dissected and immediately processed as described below. Nomarski microscopy The salivary glands were dissected under stereoscopic microscope in 0.15 M NaCl and fixed for 1 h in 0.1 M sodium phosphate buffer, pH 7.2, containing 4% (w/v) paraformaldehyde at room temperature. The glands were settled onto slides and studied with an Axiophot Zeiss microscope. Electron microscopy Salivary glands dissected as described above were fixed overnight at 4°C in 0.1 M cacodylate buffer, pH 7.2, containing 1 mM CaCl2, 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde. Following post-fixation with 1% (w/v) OsO4, samples were dehydrated in graded ethanol, transferred to propylene oxide, and embedded in Spurr resin. Thick sections (200–300 nm) were stained with toluidine blue for light microscope studies. Thin sections (60–70 nm), stained with uranyl acetate and lead citrate, were examined in a JEOL 100 CX electron microscope. Lysozyme enzymatic activity assay Lysozyme activity assays were conducted on Petri dishes with a 1% agarose gel containing 1 mg/ml of Micrococcus lisodeiktius (Sigma) in 0.067 M sodium phosphate buffer, pH 7.0, 0.05 M sodium chloride (ionic strength=0.125), as described by Moreira-Ferro et al. (1998). Samples of salivary glands homogenate were applied to pre-formed 2 mm diameter wells and the dishes were incubated for 16 h at 37°C. The lysozyme activity creates a clear zone in the bacteria layer.
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Fig. 1 Adult An. darlingi salivary glands. (a) Paired female salivary glands, each with one medial lobe (ML) and two lateral lobes (LL). Extraacinar ducts emerge from the anterior part of each gland and joint to form the common duct (CD) that ends anteriorly through the proboscis. (b) Paired male salivary glands, each with a single lobe (L). CD: common duct. Differential interference contrast. a: X 40; b: X 70.
Apyrase enzymatic activity assay Apyrase activity was determined as previously described by Marinotti et al. (1996). 1 µl of salivary glands homogenate was incubated at 37°C with 99 µl of 50 mM Tris-HCl buffer, pH 9.0, containing 100 mM NaCl, 5 mM CaCl2, 2 mM Adenosine S’-Diphosphate (ADP) and 20 mM β-mercaptoethanol in a flat-bottom microtiter plate. After 15 min, the reaction was interrupted by addition of 2 µl of reducing reagent (0.02% 1-amino-2-naphtol-4-sulfonic acid, 0.12% sodium bisulfite, 0.12% sodium sulfite) and 25 µl of 1.25 M H2SO4 containing 1.25% ammonium molybdate. The activity was determined colorimetrically in an ELISA reader at 630 nm. The standard curve for inorganic phosphorous produced was determined according to Fiske & Subarrow (1925). Enzymatic assay for alpha-glucosidase activity detection Alpha-glucosidase activity was determined by the glucose oxidase-peroxidase method (Dahlqvist, 1968). The reactions were carried out in flat-bottom microtiter plates. 10 µl of salivary glands homogenate were incubated for 30 min at 37°C with 10 µl of 100 mM phosphate buffer, pH 7.0, containing 0.1 M sucrose. After this, 150 µl of 0.5 M TrisHCl, pH 7.0, containing 25 U glucose oxidase, 10 mg Odianisidine, 0.0125% Triton X-100 dissolved in 95% ethanol and 2.5 U peroxidase were added and the reaction was incubated at 37°C for 1 h. The activity was determined colorimetrically in an ELISA reader at 405 nm. A glucose concentration standard curve was used to determine the amount of glucose produced in the reaction.
Results The salivary glands of adult females are paired organs lying in the thorax on either side of the esophagus. They have a single medial and two lateral lobes (Fig. 1a). Atypical salivary glands with more than three lobes were occasionally seen. The salivary glands of males consist of a single small
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Fig. 2 Lateral and medium lobes of the salivary glands of adult female An. darlingi. (a) Image taken with differential interference contrast. The most proximal region displays a filigree aspect (arrow). (PL): proximal portion of lateral lobe with the cuticular salivary canal (CC). DL: distal portion of lateral lobe with the salivary canal (C), M: medial lobe X 250. (b) Semithin section of the proximal portion of lateral lobe. In this region, a single layer of epithelial cells surrounds the salivary canal (arrowhead), lined by a cuticle. The cells are occupied mostly by large secretory cisternae filled with a finely flocculent product (S). It is possible to recognize a cisternae with a mottled pattern of secretion (*), illustrating the absence of transition zone between the two glandular regions. X 490. (c) Semithin section of the distal portion of lateral lobe. A single layer of epithelial cells surrounds the larger salivary canal (arrowhead). Observe the mottled pattern of the secretory product (S). X 490. (d) Semithin section of medium lobe. Epithelial cells contain homogeneous secretory masses (S). No salivary canal can be detected. X 490.
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Fig. 3 Electron micrograph of a proximal lobe of an adult female gland. The epithelial cells contain a well-developed and reticulated rough endoplasmic reticulum (R) and a nucleus (N) with large masses of condensed chromatin. Short microvilli (arrow) protrude into the secretory cavities. A thin basal membrane (arrowhead) encompasses the cell periphery. X 10 400. Fig. 4 Detail of a cell from the proximal-lateral lobe showing various mitochondria (M) located among the vesiculated cisternae of rough endoplasmic reticulum (R). The secretory cavity, filled with finely granular secretion, shows a few transverse sections of microvilli (arrow). Basal membrane surrounds the outside of the gland (arrowhead). X 32 000. Fig. 5 Detail of a cell from the proximal-lateral lobes showing the salivary canal (C) surrounded by at least four epithelial cells joined by long gap junctions (arrowheads). The cells in this section vary in their structure: cell A reveals a cisterneiform endoplasmic reticulum; cell B shows a darker cytoplasm also with a prominent reticulum; cells C and D have an endoplasmic reticulum with a reticular pattern and numerous free ribosomes. The salivary canal is surrounded by a thick cuticle perforated by numerous and irregular canals (arrows). A filamentous meshwork and granular material similar to the secretion product fills the periductal space (*). X 21 300. Fig. 6
High magnification of Figure 5 showing the coarse granularity of the canal cuticle. X 41 000.
lobe (Fig. 1b). The lobes are acinar structures organized as a simple epithelium surrounded by a thin basal membrane. There are no other external structures of the organ, and they are immersed in situ in the hemolymph. Fat body tissue is frequently adhered to the gland (Figs 1a & 2a). The lateral lobe of each female salivary gland is composed of two secretory regions, proximal and distal, while the medial lobe has only one region (Figs 1a & 2). A region without acini is seen at the most proximal portion of the salivary glands (Fig. 2a). The unicellular epithelium surrounds a central salivary canal. In the proximal portions of the lateral lobes, the canal is surrounded by a cuticle (Fig. 2b). In the distal portion of the lateral lobes and in the medium lobe, the canal does not have the cuticular wall. Extra-acinar ducts emerge from the anterior part of each gland and join to form the common salivary duct (Fig. 1a) that extends to the exterior through the proboscis of the mosquitoes. Light microscopy of transverse sections of the proximal-lateral lobes reveals eight to ten cells surrounding the canal (Fig. 2b). Each cell has a glass form, with most of the cytoplasm and the nucleus localized to the basal end, and a finely granulated secretory material occupying the interior of the glass. Electron microscopy reveals that the secretory masses have a finely filamentous aspect and seem to push the nucleus and the cytoplasmic structures to the periphery of the gland. The entire gland is surrounded by a thin basal membrane (Figs 3 & 4). The cell cytoplasm contains few mitochondria with prominent cristae dispersed between a rough endoplasmic reticulum with very irregular and enlarged cisternae (Figs 3 & 4). The structure of the endoplasmic reticulum varies among the cells of the proximal region, probably due to asynchronous secretory activity (Fig. 5). Nuclei are irregular in form and have conspicuous nucleoli and large masses of condensed chromatin (Fig. 3). Secretory material is surrounded by delicate septa formed by two adjacent cells, frequently joined by long gap junctions (Fig. 5). Short microvilli project into the secretory masses (Figs 3, 4 & 5). In the proximal region, the salivary canal contains a coarsely-granulated cuticular wall that is perforated by irregular canals (Figs 5 & 6). A filamentous meshwork surrounds the canal and an irregular periductal space separates the cells from the canal. Occasionally, the apical tip of the epithelial cells is limited by the filamentous meshwork
although an enlarged periductal space filled with flocculent material, identical and continuous with the secretory product, can also be observed (Fig. 5). There is no transition zone between the proximal and distal regions of the lateral lobe, and cells characteristic of the proximal region are in intimate contact with cells of the distal region (Fig. 2b). The distal region of the lateral lobes consists of a glandular epithelium surrounding an enlarged salivary canal, which does not have a cuticular wall. The cellular architecture is similar to that of the proximal region, however, the secretory material is denser, and has a mottled appearance (Fig. 2c). Electron microscopy of the cells of the distal-lateral lobe reveals a dense cytoplasm with small and abundant mitochondria among a reticulate, rough endoplasmic reticulum. The endoplasmic reticulum occupies most of the basal cell cytoplasm and the lateral extensions between the secretory material cisternae (Figs 7 & 8). The secretory material is highly condensed, and has a mottled pattern. There is a uniformly electrondense ring at the periphery, which contains small cytoplasmic extensions and vesicles (Figs 7 & 8). In some samples, intracytoplasmic masses of dense secretory material not limited by membranes were seen (Fig. 8). Nuclei show condensed chromatin masses and prominent nucleoli similar to those of the proximal lobe (Fig. 7). The medial lobe shows the same general epithelial architecture as the lateral lobes, but the cells accumulate a uniform and highly electrondense secretory material (Figs 2d, 9 & 10). The cytoplasm of the medial lobe shows abundant cisternae of rough endoplasmic reticulum and numerous plate-like mitochondria (Fig. 10). Nuclei are irregular with large nucleoli (Fig. 9). The most proximal portion of the glands shows a filigree aspect in light microscopy (Fig. 2a). Electron microscopy reveals two well-defined cellular types in this region. One type consists of cells with numerous and deep membrane infoldings that extend from the basal region to the periductal space (Fig. 11). The infoldings contain a high number of mitochondria and almost no cytoplasm (Fig. 12). Nuclei with small nucleoli are confined to the peripheral region. A highly dense and ruffled cuticular wall limits the salivary canal, and its lumen contains a finely filamentous material (Fig. 11). The other cell type in the most proximal region of the salivary
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Fig. 7 Electron micrograph of reticulated cells from the distal-lateral lobe. Epithelial cells contain a reticulated rough endoplasmic reticulum (R), and large nuclei with prominent nucleoli (Nu). Secretory cavities (S) contain coarsely granulated material. X 9200. Fig. 8 High magnification of cells in the distal-lateral lobe. The secretory mass shows a coarse granulated aspect with a peripheral condensed ring pierced with vesicles (arrows). Numerous vesicles also perforate an intracytoplasmic patch of condensed secretory material (*) that is encircled by numerous mitochondria (M). X 11 200. Fig. 9 Electron micrograph of cells from the medial lobe. Nuclei (N) have conspicuous nucleoli. Secretory cavities (S) contain dark homogeneous material with irregular borders. X 9300. Fig. 10 High magnification of a portion of the medial lobe. Dense cytoplasm is encompassed by a well-developed endoplasmic reticulum with a reticulated pattern (R). X 31 500.
glands shows numerous mitochondria, some of them with mixed cristae and tubular invaginations, and abundant cisternae of smooth endoplasmic reticulum (Fig. 13).
The biochemical analyses of the salivary glands of female An. darlingi revealed the presence of apyrase, alphaglucosidase and lysozyme activities (Table 1). Apyrase
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Fig. 11 The most proximal region of the glands has cells with a filigree aspect surrounding the salivary canal with a ruffled cuticular wall (C). Cells display numerous and deep infoldings of membrane extended from the basal region to the periductal space. The infoldings contain a high number of mitochondria and almost no cytoplasm. Small nuclei (N) are marginal. The arrowhead points to the basal membrane. Two cells of the proximal portion (P) are included in this section. X 4350. Fig. 12 High magnification of Figure 11 showing the limit between the secretory cells (P) and the most proximal cells (*). Observe the abundant mitochondria (arrowheads) with prominent cristae occupying the membrane infoldings in the proximal cells. X 12 100. Fig. 13
Section through the most proximal region of lobes showing a cell with numerous tubular-mitochondria (arrowheads). X 7800.
activity was detected by the production of inorganic phosphorous following ADP hydrolysis. The activity determined at pH 9.0 (optimum pH), was 6.4±1.4 nmol of inorganic
phosphorous/ minute/ salivary glands pair (X±standard deviation, n=8 samples, each sample containing 3 salivary glands pairs). The analysis of the distribution of this enzyme
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Table 1 Distribution of apyrase, α-glucosidase and lysozyme within Anopheles darlingi salivary glands.
Apyrasea α-glucosidaseb Lysozymec
Lateral lobe distal portion
Lateral lobe proximal portion
Medium lobe
66% 16% +
12% 76% +++
22% 8% ++
aApyrase activity was determined by inorganic phosphate production from ADP hydrolysis as described by Marinotti et al. (1996) (n=24 females); bα-glucosidase activity was determined by the release of glucose from sucrose by the glucose oxidase-peroxidase method (Dahlqvist, 1968) (n=5 females); cLysozyme activity was detected as described by Moreira-Ferro et al. (1998). Plates containing 1% agarose gel in phosphate buffer 67 mM, pH 6.2/ NaCl 0.05 M/ 4 mg/ml Micrococus lisodeiktius were incubated with salivary gland extracts at 37°C (n=4). The total activity in the glands was set as 100%
showed that 66% of the apyrase is accumulated in the distal portion of the lateral lobes (Table 1). The activity of alpha-glucosidase detected by the production of glucose from the sucrose hydrolysis at pH 7.0 (optimum pH), was 0.07±0.02 µg of glucose/ minute/ salivary glands pair (X±standard deviation, n=17 mosquitoes). Most of this enzyme, 76%, is accumulated in the proximal portion of the lateral lobes (Table 1). Lysozyme activity was detected in salivary glands homogenates by a qualitative assay, the lysis of M. lisodeiktius, in which the enzyme creates clear zones in the bacteria layer. The highest activity of the An. darlingi salivary lysozyme was found at pH 6.2. Analysis of the distribution showed that this enzyme is mainly accumulated in the proximal portion of the lateral lobes, with some activity in the medium lobe (Table 1). The salivary glands of adult males are tubular paired organs located in the same position of the female glands. Each gland has only one small lobe (Fig. 1b). The general array of the cells is similar to that described for the female gland.
Discussion The morphological studies of female An. darlingi salivary glands showed that they are similar to the salivary glands of Aedes, Culex and Anopheles species (Orr et al., 1961; Wright, 1969; Janzen & Wright, 1971; Barrow et al., 1975). However, some differences were observed. In the salivary glands of female Ae. aegypti, Culex quinquefasciatus and Culex tritaeniorhynchus, the cuticular canal extends throughout the full length of all the three lobes (Clements, 1992). In An. darlingi, as described for An. stephensi (Wright, 1969), only the proximal portion of the lateral lobes has a cuticular canal. In addition, we did not find an intermediate region at the boundary of the proximal and distal portions of the lateral lobes, as was described for other mosquito species (Wright, 1969; Janzen & Wright, 1971). In An. darlingi, both secretory regions are continuous and both cellular types are adjoining (see Fig. 2a). In addition, a
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region without acini was observed at the most proximal regions of the gland, close to the point where the duct emerges from the acini. Our ultrastructural analysis of the lateral lobes showed that the cellular architecture is similar in both proximal and distal portions, although specific morphological differences were seen. The proximal portion has a cisterneiform rough endoplasmic reticulum while the distal portion presents a reticulated one. We observed different densities of rough endoplasmic reticulum among cells of the proximal portion of the lateral lobes suggesting that they have asynchronous secretory activity, as described by Wright (1969). On the other hand, cells of the distal portion, as well as that of the medium lobe, have a uniform pattern of the endoplasmic reticulum. The different secretory pattern is possibly correlated with their participation in sugar and blood feeding. The proximal portion of the lateral lobes has a secretion product with a high hydration pattern and a finely filamentous texture, possibly due to coagulation of dispersed proteins and carbohydrates complexes. The secretory product from the distal portion has a mottled pattern that suggests a coarse coagulation of a moderately hydrated secretory material. The high electron density when stained with uranyl/lead, is probably due to its high protein content. The secretory material of the medial lobe is uniformly dense suggesting a high hydration state and a significant content of proteins. Wright (1969) observed a similar pattern of secretory materials in the salivary glands of An. stephensi. In agreement with our findings, the observations of Orr et al. (1961) showed that in female Ae. aegypti, the three secretory regions of the salivary glands produce different materials, each one displaying variable affinity for histochemical dyes. Products from the lateral lobes are mainly carbohydrate and protein complexes, while those produced by the medial lobe are mainly mucopolysaccharides. In addition to these morphological variances, differences in the carbohydrate composition of the basal lamina from the different regions of female mosquito salivary glands were described (Perrone et al., 1986; Molyneux et al., 1990). Since the basal lamina is produced by the adjacent cell, these differences may reflect the functional and biochemical properties of the morphologically-distinct portions of the salivary glands. Cytoplasmic projections into the secretory masses, called microvilli by Wright (1969), are more abundant in the proximal portion. The same was described in An. stephensi and Ae. aegypti salivary glands (Wright, 1969; Janzen & Wright, 1971). We observed intracytoplasmic masses of dense secretory material that are not bound by membrane only in the cells of the distal portion of the lateral lobes of An. darlingi salivary glands. Wright (1969) described such masses in the salivary glands of An. stephensi, suggesting that they could originate from Golgi-independent secretion. An. darlingi possess a non-glandular region in the most proximal portion of the gland, close to the point where the duct emerges from the acini. Two main cellular types are apparent in this region. One type has the typical morphology
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of solute-linked water transporter cells with numerous mitochondria in extensive cell membrane infoldings that may play a role in water and ion transportation, as proposed for the intermediate portions of An. stephensi and Ae. aegypti salivary glands (Wright, 1969; Janzen & Wright, 1971). The other cellular type exhibits agranular endoplasmic reticulum and mitochondria with mixed tubular and plate-like cristae, resembling cells involved in lipid synthesis. Synthesis of prostaglandins and other eicosanoids was described for ticks and is related to their blood-feeding ability as well as their competence as vectors of some parasites (reviewed by Bowman et al., 1996). A recent work described the presence of different prostaglandin molecules in the saliva of Amblyomma americanum (Pedibhotla et al., 1997). Cells in the apical region of female An. darlingi salivary glands could be involved in the production of prostaglandin-like molecules. However, there are no reports yet of the presence of prostaglandins or other eicosanoids in mosquitoes salivary glands or saliva. Previous work with Ae. aegypti and Aedes albopictus indicates that the proximal portions of the lateral lobes of mosquitoes’ salivary glands synthesize and accumulate molecules that help in sugar solubilization, ingestion and digestion (Clements, 1992; Marinotti et al., 1996). The MalI gene expressed exclusively in the cells of the proximal portion of the Ae. aegypti salivary glands encodes an alphaglucosidase that is secreted during mosquito sugar feeding. This enzyme hydrolyzes preferentially sucrose, the major oligosaccharide found in the nectar of flowers and other natural sugar sources (James et al., 1989; Marinotti & James, 1990). The distal portion of the lateral lobes is involved with the synthesis and accumulation of molecules necessary for mosquito blood feeding. An apyrase, for example, is synthesized and accumulated in the distal portion of Ae. aegypti and Ae. albopictus salivary glands (Rossignol et al., 1984; Marinotti et al., 1996). This enzyme is secreted by the salivary glands only during blood-feeding. The salivary glands of Ae. aegypti have a bacteriolytic activity (Rossignol & Lueders, 1986; Pimentel & Rossignol, 1990), that shows the same distribution as the alphaglucosidase, being accumulated preferentially in the proximal lateral lobes. More recently, the bacteriolytic activity found in the salivary glands of An. darlingi was identified as a lysozyme, and it has been suggested that this molecule could help the mosquitoes to prevent infections of the mouthparts (Moreira-Ferro et al., 1998). Using these three enzymatic activities (alpha-glucosidase, apyrase and lysozyme) as markers of the salivary glands functions (sugar feeding, blood feeding and prevention of mouthparts infection, respectively), we analyzed the salivary glands of the malaria vector An. darlingi. Our results show that both morphological and biochemical aspects of the An. darlingi salivary glands follow the same pattern as that described for the other studied mosquitoes. Marinotti et al., (1990) showed in Ae. aegypti that there is a selective decrease in the activity of the alpha-glucosidase, but not in the apyrase, following a sugar meal. Following a
blood meal, both alpha-glucosidase and apyrase showed a reduction on their activities. These observations are in agreement with our morphological data about asynchronous secretory activity in the cells of the proximal region, related to sugar feeding and other activities, while the cells of the distal portion, that have their secretion regulated by blood meal, display synchronous activity. Our data reinforce the idea that the general (morphological and functional) architecture of female mosquito salivary glands is conserved among the mosquito subfamilies. Ae. aegypti and Aedes triseriatus secrete tachykinin-like vasodilators (Champagne & Ribeiro, 1994), while An. albimanus mosquitoes secrete catechol-oxidase molecules that have the same function (Ribeiro & Valenzuela, 1999). The same is true for anticoagulant activities, Ae. aegypti secretes an anti-factor Xa, while Anopheles gambiae mosquitoes contain an anti-thrombin in the saliva (Stark & James, 1996). Despite the fact that different molecules perform the same salivary functions in mosquitoes’ subfamilies, in all of them the proximal portion is involved with sugar feeding while the distal portion is implicated with blood feeding. The salivary glands of male and female An. darlingi mosquitoes are morphologically different. While three distinct lobes comprise the female glands, the male glands have a single small lobe. It was described that the salivary glands, of male Aedes and Culex mosquitoes are composed of three identical lobes, all of them formed by cells morphologically similar to that of the female proximal portion of the lateral lobes (Stark & James, 1996). Ultrastructural studies of the male glands are currently in progress in our laboratory. ACKNOWLEDGEMENTS We thank the following: Dr Anthony A. James for critical evaluation of this manuscript; Susana Pessoa de Lima, Cassiano Pereira Nunes and Kendi Okuda for technical assistance; Luiz Marcelo Aranha Camargo, Luis Herman Soares Gil and Mauro Toledo Marrelli for supplying mosquitoes; and Lynn Olson for helping in the retyping. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and UNDP/ World Bank/WHO special programme for research and training in tropical diseases to O. Marinotti. C.K. MoreiraFerro is recipient of a FAPESP fellowship. A.T. Bijovsky and O. Marinotti are staff members of the Departamento de Parasitologia, ICB, USP and O. Marinotti is a CNPq fellow. REFERENCES Barrow, P.M., McIver, S.B. and Wright, K.A. 1975. Salivary glands of female Culex pipiens: morphological changes associated with maturation and blood-feeding Can. Entomol. 107, 1153–1159. Bowman, A.S., Dillwith, J.W. and Sauer, J.R. 1996. Tick salivary prostaglandins: presence, origin and significance. Parasitol. Today 12, 388–396. Champagne, D. and Ribeiro, J.M.C. 1994. Sialokinins I and II: Two salivary tachykinins from the Yellow Fever mosquito, Aedes aegypti. Proc. Natl. Acad. Sci. USA 91, 138–142. Clements, A.N. 1992. The biology of mosquitoes. Development, nutrition and reproduction. Chapman & Hall, London, Vol. 1, 251–262.
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Cupp, E.W., Cupp, M.S. and Ramberg, F.B. 1994. Salivary apyrase in African and New World vectors of Plasmodium species and its relationships to malaria transmission. Am. J. Trop. Med Hyg. 50, 235–240. Dahlqvist, A. 1968. Assay of intestinal dissacharidases. Analyt. Biochem. 22: 99. Fiske, C.H. and Subarrow, Y. 1925. The colorimetric determination of phosphorus J. Biol. Chem. 6, 375–497. James, A.A. 1994. Salivary glands of vector mosquitoes. Bull. Inst. Pasteur 92, 133–150. James, A.A., Blackmer, K. and Racioppi, J.V. 1989. A salivary glandspecific maltase-like gene of the vector mosquito Aedes aegypti. Gene 75, 73–83. James, A.A. and Rossignol, P.A. 1991. Mosquito salivary glands: parasitological and molecular aspects. Parasitol. Today 7, 267–271. Janzen, H.G. and Wright, K.A. 1971. The salivary glands of Aedes aegypti (L): an electron microscope study. Can. J. Zool. 49, 1343–1345. Law, J.H., Ribeiro, J.M. and Wells, M.A. 1992. Biochemical insights derived from insect diversity. Ann. Rev. Biochem. 61, 87–111. Marinotti, O. and James, A.A. 1990. An alpha-glucosidase in the salivary glands of the vector mosquito Aedes aegypti. Insect Biochem. 6, 619–623. Marinotti, O., James, A.A. and Ribeiro, J.M.C. 1990. Diet and salivation in female Aedes aegypti mosquitoes. J. Insect Physiol. 36, 545–548. Marinotti, O., Brito, M. and Moreira, C.K. 1996. Apyrase and alphaglucosidase in the salivary glands of Aedes albopictus. Comp. Biochem. Physiol. 113B, 675–679. Molyneux, D.H., Okolo, C.J. and Lines, J.D. 1990. Variation in fluorescein-labelled lectin staining of salivary glands in the Anopheles gambiae complex. Med. Vet. Entomol. 4, 459–462. Moreira-Ferro, C.K., Daffre, S., James, A.A. and Marinotti, O. 1998. A lysozyme in the salivary glands of the malaria vector Anopheles darlingi. Insect Mol. Biol. 7, 257–264. Orr, C.W., Hudson, A. and West, A.S. 1961. The salivary glands of Aedes aegypti. Can. J. Zool. 39, 265–272. PAHO. 1998. In: Health in the Americas, scientific publication. PanAmerican Health Organization (PAHO), Brazil 123–145.
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Pedibhotla, V.K., Sauer, J.R. and Stanley-Samuelson, D.W. 1997. Prostaglandin biosynthesis by salivary glands isolated from the lone star tick, Amblyomma americanum. Insect Biochem. Mol. Biol. 27(3), 255–261. Perrone, J.B., Demaio, J. and Spielman, A. 1986. Regions of mosquito salivary glands distinguished by surface lectin-binding characteristics. Insect Biochem. 16, 313–318. Pimentel, G.E. and Rossignol, P.A. 1990. Age dependence of salivary bacteriolytic activity in adult mosquitoes. Comp. Biochem. Physiol. 96B, 549–551. Ribeiro, J.M.C., Rossignol, P.A. and Spielman, A. 1984a. Role of mosquito saliva in blood vessel location. J. Exp. Biol. 108, 1–7. Ribeiro, J.M.C., Sarkis, J.F., Rossignol, P.A. and Spielman, A. 1984b. Salivary apyrase of Aedes aegypti: characterization and secretory fate. Comp. Biochem. Physiol. 79B, 81–86. Ribeiro, J.M.C., Rossignol, P.A. and Spielman, A. 1985. Salivary gland apyrase determines probing time in anopheline mosquitoes. J. Insect. Physiol. 31, 689–692. Ribeiro, J.M.C. and Valenzuela, J.G. 1999. Purification and cloning of the salivary peroxidase/catechol oxidase of the mosquito Anopheles albimanus. J. Exp. Biol. 202, 809–816. Rossignol, P.A. and Lueders, A.M. 1986. Bacteriolytic factor in the salivary glands of Aedes aegypti. Comp. Biochem. Physiol. 83B, 819–822. Rossignol, P.A., Ribeiro, J.M. and Spielman, A. 1984. Increased intradermal probing time in sporozoite-infected mosquitoes. Am. J. Trop. Med. Hyg. 33, 17–20. Stark, K.R. and James, A.A. 1996. The salivary glands of disease vectors. In: Beaty, B.J. and Marquardt, W.C. (eds) The Biology of disease vectors. University Press of Colorado, Colorado, 333–348. Wright, K.A. 1969. The anatomy of salivary glands of Anopheles stephensi Liston. Can. J. Zool. 47, 579–587. Yuval, B. 1992 The other habit-sugar feeding by mosquitoes. Bull. Soc. For Vector Ecology 17, 150–156.
Tissue&Cell
Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi C. K. Moreira-Ferro, O. Marinotti, A. T. Bijovsky
Abstract. Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.
Keywords: salivary glands, mosquito, ultrastructure, apyrase, alpha-glucosidase, lysozyme
Introduction Mosquitoes’ salivary glands are being investigated because of their important role in the transmission of pathogenic organisms to man and domestic animals. The adult female salivary glands are the final destination of several parasites before they are transmitted to the vertebrate hosts through the saliva (James & Rossignol, 1991; James, 1994). In addition, several biological activities related to hematophagy are found in the mosquitoes’ saliva. The salivary glands produce and secrete several molecules with anti-hemostatic and anti-inflammatory activities (Law et al., 1992). Among
Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil
Received 18 February 1999 Accepted 7 June 1999 Correspondence to: Cristina K. Moreira-Ferro, Departamento de Parasitologia, ICB, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374. CEP 05508–900. São Paulo, SP, Brazil. Tel.: 55 11 8187272; Fax: 55 11 8187417; E-mail: [email protected]
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these molecules, apyrase was identified as the enzyme responsible for the inhibition of platelet aggregation (Ribeiro et al., 1984a, 1984b, 1985; Cupp et al., 1994; Marinotti et al., 1996). Although the main focus of analysis of mosquitoes’ salivary glands results from their role in blood feeding and the transmission of pathogenic agents, these organs are also involved with sugar feeding. Mosquito saliva helps in the solubilization, ingestion and digestion of oligosaccharides found mostly in the nectar of flowers, plant exudates and fruit juices (Yuval, 1992). A salivary alpha-glucosidase is probably responsible for the oligosaccharide digestion in the mosquito crop (Marinotti & James, 1990; Marinotti et al., 1996). Another compound also found in mosquito saliva is a lysozyme that provides protection against bacterial invasion of the mouthparts and crop (Rossignol & Lueders, 1986; Moreira-Ferro et al., 1998). Morphological aspects of mosquitoes’ salivary glands were described for Aedes aegypti, Anopheles stephensi and Culex pipiens (Orr et al., 1961; Wright, 1969; Janzen & Wright, 1971; Barrow et al., 1975). The glands from these
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species are similar, consisting of three lobes connected to a main salivary duct. The salivary glands of males and females are morphologically distinct, with the male glands being smaller than those in the female. The lateral lobes of female glands have at least two distinct secretory regions, the proximal and distal portions, separated by a non-glandular region often referred to as the intermediate portion (James, 1994). The medial lobe appears to have only the intermediate and distal portions. Anopheles darlingi is an important malaria vector in Brazil and other Latin America countries. This mosquito species is responsible for the transmission of more than 90% of the malaria in Brazil, affecting approximately 500 000 people a year (PAHO, 1998). However, few studies have been conducted with this species mostly due to the absence of mosquito colonies adapted to the captivity. In this paper we describe the morphology of An. darlingi salivary glands and report the differential distribution of salivary components within the glands of female mosquitoes.
Materials and methods Insects Anopheles darlingi adult mosquitoes were captured in Rondonia State, Brazil. The salivary glands were dissected and immediately processed as described below. Nomarski microscopy The salivary glands were dissected under stereoscopic microscope in 0.15 M NaCl and fixed for 1 h in 0.1 M sodium phosphate buffer, pH 7.2, containing 4% (w/v) paraformaldehyde at room temperature. The glands were settled onto slides and studied with an Axiophot Zeiss microscope. Electron microscopy Salivary glands dissected as described above were fixed overnight at 4°C in 0.1 M cacodylate buffer, pH 7.2, containing 1 mM CaCl2, 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde. Following post-fixation with 1% (w/v) OsO4, samples were dehydrated in graded ethanol, transferred to propylene oxide, and embedded in Spurr resin. Thick sections (200–300 nm) were stained with toluidine blue for light microscope studies. Thin sections (60–70 nm), stained with uranyl acetate and lead citrate, were examined in a JEOL 100 CX electron microscope. Lysozyme enzymatic activity assay Lysozyme activity assays were conducted on Petri dishes with a 1% agarose gel containing 1 mg/ml of Micrococcus lisodeiktius (Sigma) in 0.067 M sodium phosphate buffer, pH 7.0, 0.05 M sodium chloride (ionic strength=0.125), as described by Moreira-Ferro et al. (1998). Samples of salivary glands homogenate were applied to pre-formed 2 mm diameter wells and the dishes were incubated for 16 h at 37°C. The lysozyme activity creates a clear zone in the bacteria layer.
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Fig. 1 Adult An. darlingi salivary glands. (a) Paired female salivary glands, each with one medial lobe (ML) and two lateral lobes (LL). Extraacinar ducts emerge from the anterior part of each gland and joint to form the common duct (CD) that ends anteriorly through the proboscis. (b) Paired male salivary glands, each with a single lobe (L). CD: common duct. Differential interference contrast. a: X 40; b: X 70.
Apyrase enzymatic activity assay Apyrase activity was determined as previously described by Marinotti et al. (1996). 1 µl of salivary glands homogenate was incubated at 37°C with 99 µl of 50 mM Tris-HCl buffer, pH 9.0, containing 100 mM NaCl, 5 mM CaCl2, 2 mM Adenosine S’-Diphosphate (ADP) and 20 mM β-mercaptoethanol in a flat-bottom microtiter plate. After 15 min, the reaction was interrupted by addition of 2 µl of reducing reagent (0.02% 1-amino-2-naphtol-4-sulfonic acid, 0.12% sodium bisulfite, 0.12% sodium sulfite) and 25 µl of 1.25 M H2SO4 containing 1.25% ammonium molybdate. The activity was determined colorimetrically in an ELISA reader at 630 nm. The standard curve for inorganic phosphorous produced was determined according to Fiske & Subarrow (1925). Enzymatic assay for alpha-glucosidase activity detection Alpha-glucosidase activity was determined by the glucose oxidase-peroxidase method (Dahlqvist, 1968). The reactions were carried out in flat-bottom microtiter plates. 10 µl of salivary glands homogenate were incubated for 30 min at 37°C with 10 µl of 100 mM phosphate buffer, pH 7.0, containing 0.1 M sucrose. After this, 150 µl of 0.5 M TrisHCl, pH 7.0, containing 25 U glucose oxidase, 10 mg Odianisidine, 0.0125% Triton X-100 dissolved in 95% ethanol and 2.5 U peroxidase were added and the reaction was incubated at 37°C for 1 h. The activity was determined colorimetrically in an ELISA reader at 405 nm. A glucose concentration standard curve was used to determine the amount of glucose produced in the reaction.
Results The salivary glands of adult females are paired organs lying in the thorax on either side of the esophagus. They have a single medial and two lateral lobes (Fig. 1a). Atypical salivary glands with more than three lobes were occasionally seen. The salivary glands of males consist of a single small
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Fig. 2 Lateral and medium lobes of the salivary glands of adult female An. darlingi. (a) Image taken with differential interference contrast. The most proximal region displays a filigree aspect (arrow). (PL): proximal portion of lateral lobe with the cuticular salivary canal (CC). DL: distal portion of lateral lobe with the salivary canal (C), M: medial lobe X 250. (b) Semithin section of the proximal portion of lateral lobe. In this region, a single layer of epithelial cells surrounds the salivary canal (arrowhead), lined by a cuticle. The cells are occupied mostly by large secretory cisternae filled with a finely flocculent product (S). It is possible to recognize a cisternae with a mottled pattern of secretion (*), illustrating the absence of transition zone between the two glandular regions. X 490. (c) Semithin section of the distal portion of lateral lobe. A single layer of epithelial cells surrounds the larger salivary canal (arrowhead). Observe the mottled pattern of the secretory product (S). X 490. (d) Semithin section of medium lobe. Epithelial cells contain homogeneous secretory masses (S). No salivary canal can be detected. X 490.
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Fig. 3 Electron micrograph of a proximal lobe of an adult female gland. The epithelial cells contain a well-developed and reticulated rough endoplasmic reticulum (R) and a nucleus (N) with large masses of condensed chromatin. Short microvilli (arrow) protrude into the secretory cavities. A thin basal membrane (arrowhead) encompasses the cell periphery. X 10 400. Fig. 4 Detail of a cell from the proximal-lateral lobe showing various mitochondria (M) located among the vesiculated cisternae of rough endoplasmic reticulum (R). The secretory cavity, filled with finely granular secretion, shows a few transverse sections of microvilli (arrow). Basal membrane surrounds the outside of the gland (arrowhead). X 32 000. Fig. 5 Detail of a cell from the proximal-lateral lobes showing the salivary canal (C) surrounded by at least four epithelial cells joined by long gap junctions (arrowheads). The cells in this section vary in their structure: cell A reveals a cisterneiform endoplasmic reticulum; cell B shows a darker cytoplasm also with a prominent reticulum; cells C and D have an endoplasmic reticulum with a reticular pattern and numerous free ribosomes. The salivary canal is surrounded by a thick cuticle perforated by numerous and irregular canals (arrows). A filamentous meshwork and granular material similar to the secretion product fills the periductal space (*). X 21 300. Fig. 6
High magnification of Figure 5 showing the coarse granularity of the canal cuticle. X 41 000.
lobe (Fig. 1b). The lobes are acinar structures organized as a simple epithelium surrounded by a thin basal membrane. There are no other external structures of the organ, and they are immersed in situ in the hemolymph. Fat body tissue is frequently adhered to the gland (Figs 1a & 2a). The lateral lobe of each female salivary gland is composed of two secretory regions, proximal and distal, while the medial lobe has only one region (Figs 1a & 2). A region without acini is seen at the most proximal portion of the salivary glands (Fig. 2a). The unicellular epithelium surrounds a central salivary canal. In the proximal portions of the lateral lobes, the canal is surrounded by a cuticle (Fig. 2b). In the distal portion of the lateral lobes and in the medium lobe, the canal does not have the cuticular wall. Extra-acinar ducts emerge from the anterior part of each gland and join to form the common salivary duct (Fig. 1a) that extends to the exterior through the proboscis of the mosquitoes. Light microscopy of transverse sections of the proximal-lateral lobes reveals eight to ten cells surrounding the canal (Fig. 2b). Each cell has a glass form, with most of the cytoplasm and the nucleus localized to the basal end, and a finely granulated secretory material occupying the interior of the glass. Electron microscopy reveals that the secretory masses have a finely filamentous aspect and seem to push the nucleus and the cytoplasmic structures to the periphery of the gland. The entire gland is surrounded by a thin basal membrane (Figs 3 & 4). The cell cytoplasm contains few mitochondria with prominent cristae dispersed between a rough endoplasmic reticulum with very irregular and enlarged cisternae (Figs 3 & 4). The structure of the endoplasmic reticulum varies among the cells of the proximal region, probably due to asynchronous secretory activity (Fig. 5). Nuclei are irregular in form and have conspicuous nucleoli and large masses of condensed chromatin (Fig. 3). Secretory material is surrounded by delicate septa formed by two adjacent cells, frequently joined by long gap junctions (Fig. 5). Short microvilli project into the secretory masses (Figs 3, 4 & 5). In the proximal region, the salivary canal contains a coarsely-granulated cuticular wall that is perforated by irregular canals (Figs 5 & 6). A filamentous meshwork surrounds the canal and an irregular periductal space separates the cells from the canal. Occasionally, the apical tip of the epithelial cells is limited by the filamentous meshwork
although an enlarged periductal space filled with flocculent material, identical and continuous with the secretory product, can also be observed (Fig. 5). There is no transition zone between the proximal and distal regions of the lateral lobe, and cells characteristic of the proximal region are in intimate contact with cells of the distal region (Fig. 2b). The distal region of the lateral lobes consists of a glandular epithelium surrounding an enlarged salivary canal, which does not have a cuticular wall. The cellular architecture is similar to that of the proximal region, however, the secretory material is denser, and has a mottled appearance (Fig. 2c). Electron microscopy of the cells of the distal-lateral lobe reveals a dense cytoplasm with small and abundant mitochondria among a reticulate, rough endoplasmic reticulum. The endoplasmic reticulum occupies most of the basal cell cytoplasm and the lateral extensions between the secretory material cisternae (Figs 7 & 8). The secretory material is highly condensed, and has a mottled pattern. There is a uniformly electrondense ring at the periphery, which contains small cytoplasmic extensions and vesicles (Figs 7 & 8). In some samples, intracytoplasmic masses of dense secretory material not limited by membranes were seen (Fig. 8). Nuclei show condensed chromatin masses and prominent nucleoli similar to those of the proximal lobe (Fig. 7). The medial lobe shows the same general epithelial architecture as the lateral lobes, but the cells accumulate a uniform and highly electrondense secretory material (Figs 2d, 9 & 10). The cytoplasm of the medial lobe shows abundant cisternae of rough endoplasmic reticulum and numerous plate-like mitochondria (Fig. 10). Nuclei are irregular with large nucleoli (Fig. 9). The most proximal portion of the glands shows a filigree aspect in light microscopy (Fig. 2a). Electron microscopy reveals two well-defined cellular types in this region. One type consists of cells with numerous and deep membrane infoldings that extend from the basal region to the periductal space (Fig. 11). The infoldings contain a high number of mitochondria and almost no cytoplasm (Fig. 12). Nuclei with small nucleoli are confined to the peripheral region. A highly dense and ruffled cuticular wall limits the salivary canal, and its lumen contains a finely filamentous material (Fig. 11). The other cell type in the most proximal region of the salivary
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Fig. 7 Electron micrograph of reticulated cells from the distal-lateral lobe. Epithelial cells contain a reticulated rough endoplasmic reticulum (R), and large nuclei with prominent nucleoli (Nu). Secretory cavities (S) contain coarsely granulated material. X 9200. Fig. 8 High magnification of cells in the distal-lateral lobe. The secretory mass shows a coarse granulated aspect with a peripheral condensed ring pierced with vesicles (arrows). Numerous vesicles also perforate an intracytoplasmic patch of condensed secretory material (*) that is encircled by numerous mitochondria (M). X 11 200. Fig. 9 Electron micrograph of cells from the medial lobe. Nuclei (N) have conspicuous nucleoli. Secretory cavities (S) contain dark homogeneous material with irregular borders. X 9300. Fig. 10 High magnification of a portion of the medial lobe. Dense cytoplasm is encompassed by a well-developed endoplasmic reticulum with a reticulated pattern (R). X 31 500.
glands shows numerous mitochondria, some of them with mixed cristae and tubular invaginations, and abundant cisternae of smooth endoplasmic reticulum (Fig. 13).
The biochemical analyses of the salivary glands of female An. darlingi revealed the presence of apyrase, alphaglucosidase and lysozyme activities (Table 1). Apyrase
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Fig. 11 The most proximal region of the glands has cells with a filigree aspect surrounding the salivary canal with a ruffled cuticular wall (C). Cells display numerous and deep infoldings of membrane extended from the basal region to the periductal space. The infoldings contain a high number of mitochondria and almost no cytoplasm. Small nuclei (N) are marginal. The arrowhead points to the basal membrane. Two cells of the proximal portion (P) are included in this section. X 4350. Fig. 12 High magnification of Figure 11 showing the limit between the secretory cells (P) and the most proximal cells (*). Observe the abundant mitochondria (arrowheads) with prominent cristae occupying the membrane infoldings in the proximal cells. X 12 100. Fig. 13
Section through the most proximal region of lobes showing a cell with numerous tubular-mitochondria (arrowheads). X 7800.
activity was detected by the production of inorganic phosphorous following ADP hydrolysis. The activity determined at pH 9.0 (optimum pH), was 6.4±1.4 nmol of inorganic
phosphorous/ minute/ salivary glands pair (X±standard deviation, n=8 samples, each sample containing 3 salivary glands pairs). The analysis of the distribution of this enzyme
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Table 1 Distribution of apyrase, α-glucosidase and lysozyme within Anopheles darlingi salivary glands.
Apyrasea α-glucosidaseb Lysozymec
Lateral lobe distal portion
Lateral lobe proximal portion
Medium lobe
66% 16% +
12% 76% +++
22% 8% ++
aApyrase activity was determined by inorganic phosphate production from ADP hydrolysis as described by Marinotti et al. (1996) (n=24 females); bα-glucosidase activity was determined by the release of glucose from sucrose by the glucose oxidase-peroxidase method (Dahlqvist, 1968) (n=5 females); cLysozyme activity was detected as described by Moreira-Ferro et al. (1998). Plates containing 1% agarose gel in phosphate buffer 67 mM, pH 6.2/ NaCl 0.05 M/ 4 mg/ml Micrococus lisodeiktius were incubated with salivary gland extracts at 37°C (n=4). The total activity in the glands was set as 100%
showed that 66% of the apyrase is accumulated in the distal portion of the lateral lobes (Table 1). The activity of alpha-glucosidase detected by the production of glucose from the sucrose hydrolysis at pH 7.0 (optimum pH), was 0.07±0.02 µg of glucose/ minute/ salivary glands pair (X±standard deviation, n=17 mosquitoes). Most of this enzyme, 76%, is accumulated in the proximal portion of the lateral lobes (Table 1). Lysozyme activity was detected in salivary glands homogenates by a qualitative assay, the lysis of M. lisodeiktius, in which the enzyme creates clear zones in the bacteria layer. The highest activity of the An. darlingi salivary lysozyme was found at pH 6.2. Analysis of the distribution showed that this enzyme is mainly accumulated in the proximal portion of the lateral lobes, with some activity in the medium lobe (Table 1). The salivary glands of adult males are tubular paired organs located in the same position of the female glands. Each gland has only one small lobe (Fig. 1b). The general array of the cells is similar to that described for the female gland.
Discussion The morphological studies of female An. darlingi salivary glands showed that they are similar to the salivary glands of Aedes, Culex and Anopheles species (Orr et al., 1961; Wright, 1969; Janzen & Wright, 1971; Barrow et al., 1975). However, some differences were observed. In the salivary glands of female Ae. aegypti, Culex quinquefasciatus and Culex tritaeniorhynchus, the cuticular canal extends throughout the full length of all the three lobes (Clements, 1992). In An. darlingi, as described for An. stephensi (Wright, 1969), only the proximal portion of the lateral lobes has a cuticular canal. In addition, we did not find an intermediate region at the boundary of the proximal and distal portions of the lateral lobes, as was described for other mosquito species (Wright, 1969; Janzen & Wright, 1971). In An. darlingi, both secretory regions are continuous and both cellular types are adjoining (see Fig. 2a). In addition, a
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region without acini was observed at the most proximal regions of the gland, close to the point where the duct emerges from the acini. Our ultrastructural analysis of the lateral lobes showed that the cellular architecture is similar in both proximal and distal portions, although specific morphological differences were seen. The proximal portion has a cisterneiform rough endoplasmic reticulum while the distal portion presents a reticulated one. We observed different densities of rough endoplasmic reticulum among cells of the proximal portion of the lateral lobes suggesting that they have asynchronous secretory activity, as described by Wright (1969). On the other hand, cells of the distal portion, as well as that of the medium lobe, have a uniform pattern of the endoplasmic reticulum. The different secretory pattern is possibly correlated with their participation in sugar and blood feeding. The proximal portion of the lateral lobes has a secretion product with a high hydration pattern and a finely filamentous texture, possibly due to coagulation of dispersed proteins and carbohydrates complexes. The secretory product from the distal portion has a mottled pattern that suggests a coarse coagulation of a moderately hydrated secretory material. The high electron density when stained with uranyl/lead, is probably due to its high protein content. The secretory material of the medial lobe is uniformly dense suggesting a high hydration state and a significant content of proteins. Wright (1969) observed a similar pattern of secretory materials in the salivary glands of An. stephensi. In agreement with our findings, the observations of Orr et al. (1961) showed that in female Ae. aegypti, the three secretory regions of the salivary glands produce different materials, each one displaying variable affinity for histochemical dyes. Products from the lateral lobes are mainly carbohydrate and protein complexes, while those produced by the medial lobe are mainly mucopolysaccharides. In addition to these morphological variances, differences in the carbohydrate composition of the basal lamina from the different regions of female mosquito salivary glands were described (Perrone et al., 1986; Molyneux et al., 1990). Since the basal lamina is produced by the adjacent cell, these differences may reflect the functional and biochemical properties of the morphologically-distinct portions of the salivary glands. Cytoplasmic projections into the secretory masses, called microvilli by Wright (1969), are more abundant in the proximal portion. The same was described in An. stephensi and Ae. aegypti salivary glands (Wright, 1969; Janzen & Wright, 1971). We observed intracytoplasmic masses of dense secretory material that are not bound by membrane only in the cells of the distal portion of the lateral lobes of An. darlingi salivary glands. Wright (1969) described such masses in the salivary glands of An. stephensi, suggesting that they could originate from Golgi-independent secretion. An. darlingi possess a non-glandular region in the most proximal portion of the gland, close to the point where the duct emerges from the acini. Two main cellular types are apparent in this region. One type has the typical morphology
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of solute-linked water transporter cells with numerous mitochondria in extensive cell membrane infoldings that may play a role in water and ion transportation, as proposed for the intermediate portions of An. stephensi and Ae. aegypti salivary glands (Wright, 1969; Janzen & Wright, 1971). The other cellular type exhibits agranular endoplasmic reticulum and mitochondria with mixed tubular and plate-like cristae, resembling cells involved in lipid synthesis. Synthesis of prostaglandins and other eicosanoids was described for ticks and is related to their blood-feeding ability as well as their competence as vectors of some parasites (reviewed by Bowman et al., 1996). A recent work described the presence of different prostaglandin molecules in the saliva of Amblyomma americanum (Pedibhotla et al., 1997). Cells in the apical region of female An. darlingi salivary glands could be involved in the production of prostaglandin-like molecules. However, there are no reports yet of the presence of prostaglandins or other eicosanoids in mosquitoes salivary glands or saliva. Previous work with Ae. aegypti and Aedes albopictus indicates that the proximal portions of the lateral lobes of mosquitoes’ salivary glands synthesize and accumulate molecules that help in sugar solubilization, ingestion and digestion (Clements, 1992; Marinotti et al., 1996). The MalI gene expressed exclusively in the cells of the proximal portion of the Ae. aegypti salivary glands encodes an alphaglucosidase that is secreted during mosquito sugar feeding. This enzyme hydrolyzes preferentially sucrose, the major oligosaccharide found in the nectar of flowers and other natural sugar sources (James et al., 1989; Marinotti & James, 1990). The distal portion of the lateral lobes is involved with the synthesis and accumulation of molecules necessary for mosquito blood feeding. An apyrase, for example, is synthesized and accumulated in the distal portion of Ae. aegypti and Ae. albopictus salivary glands (Rossignol et al., 1984; Marinotti et al., 1996). This enzyme is secreted by the salivary glands only during blood-feeding. The salivary glands of Ae. aegypti have a bacteriolytic activity (Rossignol & Lueders, 1986; Pimentel & Rossignol, 1990), that shows the same distribution as the alphaglucosidase, being accumulated preferentially in the proximal lateral lobes. More recently, the bacteriolytic activity found in the salivary glands of An. darlingi was identified as a lysozyme, and it has been suggested that this molecule could help the mosquitoes to prevent infections of the mouthparts (Moreira-Ferro et al., 1998). Using these three enzymatic activities (alpha-glucosidase, apyrase and lysozyme) as markers of the salivary glands functions (sugar feeding, blood feeding and prevention of mouthparts infection, respectively), we analyzed the salivary glands of the malaria vector An. darlingi. Our results show that both morphological and biochemical aspects of the An. darlingi salivary glands follow the same pattern as that described for the other studied mosquitoes. Marinotti et al., (1990) showed in Ae. aegypti that there is a selective decrease in the activity of the alpha-glucosidase, but not in the apyrase, following a sugar meal. Following a
blood meal, both alpha-glucosidase and apyrase showed a reduction on their activities. These observations are in agreement with our morphological data about asynchronous secretory activity in the cells of the proximal region, related to sugar feeding and other activities, while the cells of the distal portion, that have their secretion regulated by blood meal, display synchronous activity. Our data reinforce the idea that the general (morphological and functional) architecture of female mosquito salivary glands is conserved among the mosquito subfamilies. Ae. aegypti and Aedes triseriatus secrete tachykinin-like vasodilators (Champagne & Ribeiro, 1994), while An. albimanus mosquitoes secrete catechol-oxidase molecules that have the same function (Ribeiro & Valenzuela, 1999). The same is true for anticoagulant activities, Ae. aegypti secretes an anti-factor Xa, while Anopheles gambiae mosquitoes contain an anti-thrombin in the saliva (Stark & James, 1996). Despite the fact that different molecules perform the same salivary functions in mosquitoes’ subfamilies, in all of them the proximal portion is involved with sugar feeding while the distal portion is implicated with blood feeding. The salivary glands of male and female An. darlingi mosquitoes are morphologically different. While three distinct lobes comprise the female glands, the male glands have a single small lobe. It was described that the salivary glands, of male Aedes and Culex mosquitoes are composed of three identical lobes, all of them formed by cells morphologically similar to that of the female proximal portion of the lateral lobes (Stark & James, 1996). Ultrastructural studies of the male glands are currently in progress in our laboratory. ACKNOWLEDGEMENTS We thank the following: Dr Anthony A. James for critical evaluation of this manuscript; Susana Pessoa de Lima, Cassiano Pereira Nunes and Kendi Okuda for technical assistance; Luiz Marcelo Aranha Camargo, Luis Herman Soares Gil and Mauro Toledo Marrelli for supplying mosquitoes; and Lynn Olson for helping in the retyping. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and UNDP/ World Bank/WHO special programme for research and training in tropical diseases to O. Marinotti. C.K. MoreiraFerro is recipient of a FAPESP fellowship. A.T. Bijovsky and O. Marinotti are staff members of the Departamento de Parasitologia, ICB, USP and O. Marinotti is a CNPq fellow. REFERENCES Barrow, P.M., McIver, S.B. and Wright, K.A. 1975. Salivary glands of female Culex pipiens: morphological changes associated with maturation and blood-feeding Can. Entomol. 107, 1153–1159. Bowman, A.S., Dillwith, J.W. and Sauer, J.R. 1996. Tick salivary prostaglandins: presence, origin and significance. Parasitol. Today 12, 388–396. Champagne, D. and Ribeiro, J.M.C. 1994. Sialokinins I and II: Two salivary tachykinins from the Yellow Fever mosquito, Aedes aegypti. Proc. Natl. Acad. Sci. USA 91, 138–142. Clements, A.N. 1992. The biology of mosquitoes. Development, nutrition and reproduction. Chapman & Hall, London, Vol. 1, 251–262.
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