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Histochemical staining of the complex carbohydrates of the midgut of the mosquito, Culex tarsalis coquillett
Insect Biochem. Vol. 16, No. 4, pp. 667-675, 1986
0020-1790/86 $3.00+0.00 Pergamon Journals Ltd
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HISTOCHEMICAL STAINING OF THE COMPLEX CARBOHYDRATES OF THE MIDGUT OF THE MOSQUITO, C U L E X T A R S A L I S COQUILLETT EDWARD J. HOUK,* JAMES L. HARDY and ROBERT E. CHILES Naval Biosciences Laboratory, School of Public Health, University of California, Berkeley, CA 94720, U.S.A. (Received 5 July 1985; revised and accepted 17 September 1985)
Abstraet--Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic-glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic-glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic-glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed. Key Word Index: Mosquito, Culex tarsalis, midgut histochemistry, complex carbohydrates
INTRODUCTION The mosquito midgut represents the principal site of nutrient absorption and is an integral component within an osmo-regulatory system in conjunction with the Malpighian tubules and the hindgut (Berridge and Oschman, 1972). The luminal and abluminal surfaces of the midgut are involved in the above tasks respectively. Actually, the insect midgut epithelial cell has three distinct functional surfaces: microvilli, apical intercellular junctional complex and basolateral plasma membrane (PM) with its associated basal lamina (BL). Electron microscopic cytochemical analysis of glycoproteins and glycosaminoglycans (GAGs) have concentrated on these surfaces and their proximal extracellular spaces (Dailai, 1970; Andries, 1972; Cheung and Marshall, 1973; Reinhardt and Hecker, 1973; Kitajima, 1975; Mello and Viana, 1977; Francois, 1978; Gutierrez and Burgos, 1978; Humbert, 1979). The luminal (microvillar) surface of the insect midgut has not been thoroughly studied. Primary emphasis has been the detection of vic-glycol saccharides along this surface using conventional staining methods [e.g. Thiery's silver protein (AgP), phosphotungstic acid (PTA) and silver methenamine (AgM); Thiery, 1967; Dallai, 1970; Andries, 1972; Reinhardt and Hocker, 1973; Mello and Viana, 1977; Gutierrez and Burgos, 1978; Humbert, 1979]. In addition, a complex staining system of concana*Address correspondence to: Dr E. J. Houk, Naval Biosciences Laboratory, Naval Supply Center, Oakland, CA 94625, U.S.A.
valin A-horseradish peroxidase-diaminobenzidineosmium tetroxide, specific for D-mannosides, stained the microvilli of Tomocerus minor (Humbert, 1979). The apical midgut epithelial intercellular junctional complex has as its primary component a junction unique to the invertebrates, the smooth septate junction (zonula continua; Noirot and Noirot-Timothee, 1967). Since the insect midgut is devoid of a so-called terminal web, the smooth septate junction apparently provides both cell to cell adhesion and occlusive properties to the apical intercellular space. Histochemical studies of this junction indicate positive staining by AgP (Dallai, 1970; Reinhardt and Hecker, 1973; Humbert, 1979) and also PTA, both acidic (Dallai, 1970; Humbert, 1979) and ethanolic (Reinhardt and Hocker, 1973). The basolateral surface, including the BL, has not been effectively studied. In fact, most reports ignore the PM and concentrate on the BL. PTA has been the primary study tool for staining the BL with conflicting results, from minimal but positive (Dallai, 1970) through completely negative staining (Reinhardt and Hocker, 1973; Humbert, 1979). The importance of cell surface glycoproteins and glycolipids in the selective uptake of many biologically active substances, such as hormones, toxins and infectious agents, has been pointed out (Quinton and Philpott, 1973; Grinnell et al., 1975; Hughes, 1975; Quaglia et al., 1976). The infection of mosquitoes by parasites, especially viruses, is mediated by selective adsorption within the midgut epithelium (Chamberlain and Sudia, 1961; Hardy et al., 1983). Recent studies of the mosquito, Culex tarsalis, and western equine encephalomyelitis virus have indicated that
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Table I. Histochemical staining of Culex tarsalis midgut epithelial cell surface complex carbohydrates Basolateral Intercellular plasma Basal Staining Staining method Microvilli junction membrane lamina specificity Ruthenium red Polyanionic En blot' + * + glycosaminoglycans Post stain + + (GAGs) Tannic acid + + Polyanionic GAGs Alcian blue En bloc + + Polyanionic GAGs Post stain +/+ + Phosphotungstic acid Controversial En bloc + + + (a) Positive charges Post stain-chromic acid (b) Protonated Post stain-periodic acid + + + hydroxyl groups Periodic acid-thiosemicarbazide silver protein + + Vie-glycols Silver methenamine + +/V/c-glycols Lanthanum + +/Polyanionic GAGsphosphate affinity Diaminobenzidine-osmium . . . . . Polyanionic GAGssulfate specific High iron diamine Polyanionic GAGssulfate specific *Electron opaque deposit observed.
infection of the m o s q u i t o has a genetic basis. Resistance to viral infection a p p e a r s to reside with a midgut associated a d s o r p t i o n barrier ( H a r d y e t a l . , 1983). This study characterizes the histochemistry of the complex glycoconjugates o f m o s q u i t o m i d g u t epithelial surfaces t h r o u g h electron microscopic examination. In the future, we hope to determine if a relationship exists between the complex glycoconjugates of the m o s q u i t o midgut a n d susceptibility a n d / o r refractoriness o f the m o s q u i t o to arboviruses.
Lanthanum hydroxide (100 mg) was dissolved in a minimum volume of concentrated HC1 and then brought to approximate neutrality (i.e. slight precipitation with NaOH). This solution was then mixed with 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) to a final concentration of 0.5% lanthanum hydroxide (w/v). Mosquito midguts were dissected directly into this solution and fixed for 2 hr in the same. The midguts were rinsed twice (15 min) in cacodylate buffer followed by a 15 min rinse in 0.2 M phosphate buffer (Houk, 1977). Subsequent steps of osmication, dehydration and embedding (Houk, 1977) were in lanthanum-free solutions. Two methods specific for sulfated GAGs were applied to the mosquito midgut: HID (Spicer et al., 1978) and diaminobenzidine-osmium tetroxide (Monga et al., 1972).
MATERIALS AND M E T H O D S
Maintenance of mosquitoes, tissue processing and electron microscopic examination procedures have been described (Houk, 1977). All of the staining methods used for complex carbohydrates, except for tannic acid (TA) and high iron diamine (HID), have been reviewed by Hayat (1975; Table 1). Ruthenium red (RR) staining was either by the en bloc method of Luft (1964) and as a post stain of 1% RR (w/v) in 0.1 M NHaOH (Kobayashi and Asboe-Hansen, 1971). TA (2% w/v) staining was in either the 2% glutaraldehyde primary fixation or in the 1% osmium tetroxide post fixative (Singley and Solursh, 1980). Alcian blue (AB) staining was en bloc (Behnke and Zelander, 1970) and as a post stain of 0.1% AB (w/v) in 3% acetic acid (Tadano and Yamada, 1978). This sections post stained with AB were also post stained with lead citrate (Reynolds, 1963). PTA was also utilized either en b l o c or as a post stain. The en bloc staining was 1% PTA (w/v) in 70% ethanol overnight (Bloom and Aghajanian, 1968). The post staining method was 1% (w/v) PTA in 10% chromic acid (ChO; w/v) with (Tsuchiya and Ogawa, 1973) or without periodic acid (PA) oxidation (Roland et al., 1972). Two post staining methods specific for v/c-glycols were also examined (Rambourg and Leblond, 1967; Thiery, 1967). The silver methenamine (AgM) method (Rambourg and Leblond, 1967) was examined both with and without PA oxidation. AgP staining with PA oxidation and thiosemicarbazide (TSC) as the bridging agent examined using all appropriate controls (Thiery, 1967).
RESULTS Ruthenium
red (RR)
R R b o t h e n b l o c (Fig. I) a n d as a post stain (Fig. 2) stained the BL a n d the P M intensely (Table 1). T h e staining reaction was confined to the outer m e m b r a n e leaflet (Fig. 2). N o staining was a p p a r e n t in the intercellular j u n c t i o n s or along the microvillar m e m b r a n e (Fig. 3). Tannic
acid (TA )
M i d g u t extracellular surface staining with T A was a n exact duplication o f R R staining (Table 1). Intense staining was observed within the BL a n d along the P M , while intercellular j u n c t i o n s a n d microvilli were devoid o f stain. Alcian
blue (AB)
A B staining of P M (Fig. 4) a n d microvilli (Fig. 5) was d u b i o u s at best. However, the BL was distinctly stained, with small particulate deposits including adjacent muscle b a s e m e n t m e m b r a n e (Fig. 4). Post staining with A B revealed distinct particulate deposits within the BL a n d along the P M (Fig. 6). Particulate deposits were also a p p a r e n t along the surface of microvilli but n o t within intercellular junctions (Fig. 7). En bloc
Plate 1. Fig. 1. En bloc RR stains the basal lamina (BL) and the basolateral plasma membrane (PM). x 33,500. Fig. 2. RR stains material associated only with the outer membrane leaflet (OML) of the basolateral plasma membrane; inner membrane leaflet (IML) is unstained; basal lamina (BL). x 95,000. Fig. 3. RR does not stain microvilli (M) nor intercellular junctions (IJ). x 37,500. Fig. 4. Particulate staining within the midgut basal lamina (BL) is faint and unclear; no evidence of basolateral plasma membrane (PM) staining with en bloc AB. x 44,500. 669
Plate 2. Fig. 5. Microvilli (M) are devoid of particulate reaction product after en bloc staining with AB; intercellular junction (I J). x 27,500. Fig. 6. Midguts post stained with AB and lead citrate reveal enhanced particulate staining (arrowheads) along the basolateral plasma membranes (PM) and within the basal lamina (BL). x 69,500. Fig. 7. Microvilli (M) and intercellular junctions of AB and lead citrate post stained midguts reveal no evidence of staining, x 51,000. Fig. 8. En bloc ethanolic PTA stains the intercellular junction (I J), but not the microvilli (M). x 65,500. 670
Plate 3. Fig. 9. Intense staining of the basal lamina (BL) and finely localized staining of the basolateral plasma membrane (PM) after en bloc ethanolic PTA. x 56,500. Fig. 10. Microvilli (M) staining intensely with PA-ChO-PTA, negligible staining of all but the immediately luminal subjacent areas (arrowheads) of the intercellular junction (IJ). x 52,000. Fig. 11. PA-ChO-PTA intensely stains the basal lamina (BL) and the basolateral plasma membrane (PM). × 34,000. Fig. 12. Finely particulate silver deposits within the intercellular junction (IJ) after PA-TSC-AgP staining, x 41,500. 671
Plate 4. Fig. 13. Elimination of PA oxidation from the AgP method of Thiery (1967) results in a distinct distribution of reaction product within the plasma membrane of the intercellular junction (I J) space. x 64,000. Fig. 14. AgM staining of microvillar membranes (M) is sparse but specific. × 62,500. Fig. 15. Lanthanum specifically adsorbs to the microvillar surface (M) and apical regions of the intercellular junction (IJ). × 36,500.
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Midgut histochemistry We attempted RR post staining of en bloc AB stained midguts. The intense staining characteristic of RR (Figs 1 and 2) was not observed within the BL nor the PM. This would indicate that AB was indeed bound to the RR staining sites within these structures even though electron microscopic detection of AB staining was dubious within the PM (Fig. 6). Phosphotungstic acid (PTA ) En bloc PTA (e-PTA) staining was most evident in the intercellular junction (Fig. 8) and the BL (Fig. 9). Limited staining along the PM was observed (Fig. 9). ChO-PTA did not reveal additional staining sites nor enhance staining of the midgut extracellular surfaces (Table 1). Treatment of midgut tissue with 1% PA prior to ChO-PTA completely altered the staining pattern. Intense staining was apparent along the microvillar surface (Fig. 10), PM and BL (Fig. 11). Periodic acid-thiosemicarbazide-silver protein ( P A TSC-AgP) Deposition of extremely fine silver particles w a s apparent within the intercellular junction, along with enhanced electron opacity of microvillar membranes (Fig. 12). No staining was apparent within the BL; rather indistinct staining was observed along the PM (Table 1). As reported by Dallai (1970), substantial staining of the intercellular junction and microvillar membranes was apparent without PA oxidation (Fig. 13). However, the staining was distinctly different; TA-TSC-AgP allowed delineation of interseptal spaces within the continuous junction (Fig. 12) while TSC-AgP did not (Fig. 13). Silver methenamine ( AgM) As contrasted with the PA-TSC-AgP staining of microvilli (Fig. 12), AgM staining yielded large silver particles with a somewhat sparse distribution (Fig. 14). Specific staining of the BL, PM and intercellular junction was not observed (Table 1). Lanthanum Lanthanum stained microvilli (Fig. 15) and those immediately subjacent areas of the intercellular junctions, while staining of the more basal areas of the intercellular junctional complex, BL and PM was sporadic and appeared not to be specific (Table 1). Diaminobenzidine-osmium tetroxide (DAB-Os) Enhanced electron opacity was apparent only within the intercellular junction and this was considered to be nonspecific (Table 1). High iron diamine (HID) The HID method appeared as nonspecific granular deposits on all membrane systems (Table 1). DISCUSSION
The cellular surfaces of the mosquito midgut epithelium possess a unique histochemistry, when compared to vertebrate gastrointestinal absorptive cells a tissue with analogous function (Jersild and Crawford, 1978; Spicer et al., 1981; Yamamoto, 1982). The apical surface was stained sparingly by all polyanionic specific methods tested (Table 1); the only I.B. 16/4-,--E
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electronegative character is attributed to phosphoglycoconjugates as revealed through lanthanum and rather sparse AB staining. Neutral v/c-glycols were the predominant polysaccharide component of the apical surface; positive staining by PAChO-PTA, AgM and PA-TSC-AgP. The intercellular junctional spaces contained fixed positive charges as revealed by en bloc PTA staining and, in addition, some neutral vie-glycols (i.e. positive to PA-TSC-AgP). The PM system was anionic, as was the BL; positive staining by RR, TA and AB. Two vertebrate epithelial cells parrot the staining pattern observed for mosquito midgut: dark cells of the renal collecting tubules of guinea pigs and mitochondriarich cells of the toad urinary bladder (Spicer et al., 1981). The mosquito midgut is involved in nutrient absorption and osmoregulation (Berridge and Oschman, 1972). The luminal and abluminal surfaces of the mosquito midgut are involved in the above tasks respectively. Thus, one should suspect the histochemical staining reactions of these two surfaces would be different. However, it is surprising that the histochemistry of the mosquito midgut is different from the functionally analogous vertebrate gastrointestinal tract (Cook and Stoddart, 1973; Jersiid and Crawford, 1978; Spicer et al., 1982; Yamamoto, 1982); the microvillar surface being very basophilic in the vertebrate. Two possible explanations for the neutral staining characteristics of the mosquito midgut microvillar surface might be suggested: (1) the need to sequester proteins from vertebrate blood may require a neutral surface and/or (2) the neutral polysaecharide surface coat may be insensitive to proteases secreted into the lumen of the midgut by the insect during digestion of the bloodmeal. Pipa and Cook (1958) indicated that the microvillar surface of the midgut epithelium of bloodfeeding, sucking lice is predominantly neutral polysaccharide. Gutierrez and Burgos 0978) report that the microvillar surface of Triatoma infestans, another bloodfeeding insect, has a very weak electronegative character associated with phosphoglycolipids. Again, the predominant polysaccharide component of the intestinal microvillar surface in T. infestans was neutral. It appears plausible that physiological function, rather than coincidence, determines the similar staining characteristics with regard to the lack of a definitive anionic component associated with the microvillar surface in these three bloodfeeding insects. A minor anionic component was detected along the mosquito microvillar surface, as revealed by lanthanum staining (Fig. 15; Table i). Several investigators have observed staining of cellular surfaces by lanthanum (Doggenweiler and Frenk, 1965; Lesseps, 1967; Revel and Karnovsky, 1967; Overton, 1968; Shea, 1971). The specific nature of the lanthanum reaction has not been determined. However, Lesseps (1967), Overton (1968) and Shea (1971) were able to lessen lanthanum staining by pretreatment of their tissue preparations with phospholipase C, thus implicating phospholipds as the site of lanthanum binding. We plan to investigate the effects of phospholipase C treatment on lanthanum staining of the mosquito midgut microvilli.
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The intercellular junctional complex of mosquito midgut epithelia is a smooth septate junction (Reinhardt and Hecker, 1973; Houk, 1977); a junction unique to invertebrates. It apparently serves two purposes: (1) cell to cell cohesion and (2) an occlusion barrier to paracellular diffusion. Most authors agree on the former function but the latter has been the subject of scrutiny (Satir and Gilula, 1973; NoirotTimothee and Noirot, 1980; Lane and Skaer, 1980). The differential staining of extrajunctional apical and basolateral plasma membrane regions we observed for the mosquito midgut epithelium would substantiate cell to cell cohesion within the junction and point to restricted intramembrane diffusion between these two regions. As a barrier to paracellular diffusion, the smooth septate junction remains anomalous. Reinhardt and Hecker (1973) and the e-PTA data presented herein (Fig. 8) would indicate the presence of fixed positive charges, presumably alkaline proteins and/or glycoproteins, within this junction. These positive charges could provide an electrostatic barrier to the paracellular diffusion of cationic materials (e.g. Na ÷ and K +) through the junctional complex. However, a number of investigators have been able to demonstrate penetration into septate junctions of both smooth and pleated sheet morphology by exogenous tracers (see Lane and Skaer, 1980; Noirot-Timothee and Noirot, 1980). One criticism of these studies is that the observed penetration of the exogenous tracer could be an artifact of fixation and incubation conditions. Hematophagous insects present an interesting model for the investigation of the paracellular diffusion barrier function for smooth septate junctions. As an example, the female mosquito periodically ingests large quantities of blood and as such probably allows for the testing of smooth septate junctions as barriers under conditions of extreme physical stress. Houk and Hardy (1981, 1982) reported the diffusion of ingested material (i.e. bloodmeal components) into apical intrajunctional spaces of the mosquito midgut smooth septate junction; an in vivo event. Manipulation of the composition of the bloodmeal (i.e. varying ratios of rabbit serum and defibrinated rabbit blood) led to the conclusion that hemoglobin or another component of whole blood was responsible for the observed staining within the smooth septate junction. The mosquito, Cx. tarsalis, apparently represents an extreme example since other mosquitoes examined (i.e. Culex pipiens and Aedes dorsalis) do not appear to allow bloodmeal materials to diffuse into intrajunctional spaces (Houk, unpublished observations). Intrajunctional diffusion would appear to be dependent upon the hydrostatic pressure exerted by the ingested bloodmeal in producing a separation of adjacent cells; perhaps dependent upon the ability of individual mosquitoes to control the amount of material ingested. As such the basis for the barrier function of smooth septate junctions is most likely the greatly extended diffusion pathway dependent upon the number and geometric arrangement of intrajunctional septa (Filshie and Flower, 1977). In summary, the luminal surface of the mosquito midgut epithelium is primarily composed of neutral
polysaccharides with the only anionic staining characteristic tentativity attributed to phosphoglycoproteins and/or phosphoglycolipids. The smooth septate junction contains fixed positive charges in addition to some vic-glycols. The basolateral membranes and the basal lamina, based on their affinity for RR and TA, contain an abundance of negative charges, almost exclusively carboxyl groups of G A G s and glycoproteins. The effects of chemical and biochemical modification of the midgut epithelial surfaces are currently being investigated. In addition, we are pursuing lectin binding studies and specific probes for charge distribution. We hope that these studies will allow some insight into the mechanism(s) of mosquito midgut-arbovirus interaction. Acknowledgements--This research was supported by a U.S. Army Contract/Grant, DAMD 17-77-C-7018, U.S. Army Medical Research and Development Command, Washington, D.C. and by the Office of Naval Research. REFERENCES
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Pipa R. L. and Cook E. F. (1958) The structure and histochemistry of the connective tissue of the sucking lice. J. Morph. 103, 353-386. Quaglia A., Sabelli B. and Villani L. (1976) Studies on the intestine of Daphnidae (Crustacea, Cladocera) ultrastructure of the midgut of Daphnia magna and Daphnia obtusa. J. Morph. 150, 711-725. Quinton P. M. and Philpott C. W. (1973) A role for anionic sites in epithelial architecture. J. Cell Biol. 56, 787-796. Rambourg A. and Leblond C. P. (1967) Electron microscope observations on the carbohydrate-rich cell coat present at the surface of cells in the rat. J. Cell Biol. 32, 27-53. Reinhardt C. and Hecker H. (1973) Structure and function of the basal lamina and of the cell junctions in the midgut epithelium (stomach) of female Aedes aegypti L. (Insecta, Diptera). Acta trop. 30, 213-236. Reynolds E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. Roland J. C., Lembi C. A. and Morre D. J. (1972) Phosphotungstic acid-chromic acid as a selective electrondense stain for plasma membrane of plant cells. Stain Technol. 47, 195-200. Satir P. and Gilula N. B. (1973) The fine structure of membranes and intercellular communication in insects. A. Rev. Ent. 18, 143-166. Shea S. M. (1971) Lanthanum staining of the surface coat of ceils. J. Cell Biol. 51, 611~20. Singley C. T. and Solursh M. (1980) The use of tannic acid for the ultrastructural visualization of hyaluronic acid. Histochemistry 65, 93-102. Spicer S. S., Hardin J. H. and Setser M. E. (1978) Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedures. Histochem. J. 10, 435-452. Spicer S. S., Baron D. A., Sato A. and Schulte B. A. (1981) Variability of cell surface glycoconjugates--relation to differences in cell function. J. Histochem. Cytochem. 29, 994-1002. Tadano Y. and Yamada K. (1978) The histochemistry of complex carbohydrates in the ovarian follicles of adult mice. Histochemistry 57, 203-215. Thiery J. P. (1967) Mise en evidence des polysaccharides sur soupes fines en microscopie electronique. J. Microsc. 6, 987-1018. Tsuchiya A. and Ogawa K. (1973) Ultracytochemistry of the periodic acid (PA)--phosphotungstic acid (PTA) reaction. J. Electron Microsc. 22, 290. Yamamoto T. (1982) Ultrastructural basis of intestinal absorption. Arch. hist. jap. 45, 1-22.
0020-1790/86 $3.00+0.00 Pergamon Journals Ltd
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HISTOCHEMICAL STAINING OF THE COMPLEX CARBOHYDRATES OF THE MIDGUT OF THE MOSQUITO, C U L E X T A R S A L I S COQUILLETT EDWARD J. HOUK,* JAMES L. HARDY and ROBERT E. CHILES Naval Biosciences Laboratory, School of Public Health, University of California, Berkeley, CA 94720, U.S.A. (Received 5 July 1985; revised and accepted 17 September 1985)
Abstraet--Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic-glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic-glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic-glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed. Key Word Index: Mosquito, Culex tarsalis, midgut histochemistry, complex carbohydrates
INTRODUCTION The mosquito midgut represents the principal site of nutrient absorption and is an integral component within an osmo-regulatory system in conjunction with the Malpighian tubules and the hindgut (Berridge and Oschman, 1972). The luminal and abluminal surfaces of the midgut are involved in the above tasks respectively. Actually, the insect midgut epithelial cell has three distinct functional surfaces: microvilli, apical intercellular junctional complex and basolateral plasma membrane (PM) with its associated basal lamina (BL). Electron microscopic cytochemical analysis of glycoproteins and glycosaminoglycans (GAGs) have concentrated on these surfaces and their proximal extracellular spaces (Dailai, 1970; Andries, 1972; Cheung and Marshall, 1973; Reinhardt and Hecker, 1973; Kitajima, 1975; Mello and Viana, 1977; Francois, 1978; Gutierrez and Burgos, 1978; Humbert, 1979). The luminal (microvillar) surface of the insect midgut has not been thoroughly studied. Primary emphasis has been the detection of vic-glycol saccharides along this surface using conventional staining methods [e.g. Thiery's silver protein (AgP), phosphotungstic acid (PTA) and silver methenamine (AgM); Thiery, 1967; Dallai, 1970; Andries, 1972; Reinhardt and Hocker, 1973; Mello and Viana, 1977; Gutierrez and Burgos, 1978; Humbert, 1979]. In addition, a complex staining system of concana*Address correspondence to: Dr E. J. Houk, Naval Biosciences Laboratory, Naval Supply Center, Oakland, CA 94625, U.S.A.
valin A-horseradish peroxidase-diaminobenzidineosmium tetroxide, specific for D-mannosides, stained the microvilli of Tomocerus minor (Humbert, 1979). The apical midgut epithelial intercellular junctional complex has as its primary component a junction unique to the invertebrates, the smooth septate junction (zonula continua; Noirot and Noirot-Timothee, 1967). Since the insect midgut is devoid of a so-called terminal web, the smooth septate junction apparently provides both cell to cell adhesion and occlusive properties to the apical intercellular space. Histochemical studies of this junction indicate positive staining by AgP (Dallai, 1970; Reinhardt and Hecker, 1973; Humbert, 1979) and also PTA, both acidic (Dallai, 1970; Humbert, 1979) and ethanolic (Reinhardt and Hocker, 1973). The basolateral surface, including the BL, has not been effectively studied. In fact, most reports ignore the PM and concentrate on the BL. PTA has been the primary study tool for staining the BL with conflicting results, from minimal but positive (Dallai, 1970) through completely negative staining (Reinhardt and Hocker, 1973; Humbert, 1979). The importance of cell surface glycoproteins and glycolipids in the selective uptake of many biologically active substances, such as hormones, toxins and infectious agents, has been pointed out (Quinton and Philpott, 1973; Grinnell et al., 1975; Hughes, 1975; Quaglia et al., 1976). The infection of mosquitoes by parasites, especially viruses, is mediated by selective adsorption within the midgut epithelium (Chamberlain and Sudia, 1961; Hardy et al., 1983). Recent studies of the mosquito, Culex tarsalis, and western equine encephalomyelitis virus have indicated that
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Table I. Histochemical staining of Culex tarsalis midgut epithelial cell surface complex carbohydrates Basolateral Intercellular plasma Basal Staining Staining method Microvilli junction membrane lamina specificity Ruthenium red Polyanionic En blot' + * + glycosaminoglycans Post stain + + (GAGs) Tannic acid + + Polyanionic GAGs Alcian blue En bloc + + Polyanionic GAGs Post stain +/+ + Phosphotungstic acid Controversial En bloc + + + (a) Positive charges Post stain-chromic acid (b) Protonated Post stain-periodic acid + + + hydroxyl groups Periodic acid-thiosemicarbazide silver protein + + Vie-glycols Silver methenamine + +/V/c-glycols Lanthanum + +/Polyanionic GAGsphosphate affinity Diaminobenzidine-osmium . . . . . Polyanionic GAGssulfate specific High iron diamine Polyanionic GAGssulfate specific *Electron opaque deposit observed.
infection of the m o s q u i t o has a genetic basis. Resistance to viral infection a p p e a r s to reside with a midgut associated a d s o r p t i o n barrier ( H a r d y e t a l . , 1983). This study characterizes the histochemistry of the complex glycoconjugates o f m o s q u i t o m i d g u t epithelial surfaces t h r o u g h electron microscopic examination. In the future, we hope to determine if a relationship exists between the complex glycoconjugates of the m o s q u i t o midgut a n d susceptibility a n d / o r refractoriness o f the m o s q u i t o to arboviruses.
Lanthanum hydroxide (100 mg) was dissolved in a minimum volume of concentrated HC1 and then brought to approximate neutrality (i.e. slight precipitation with NaOH). This solution was then mixed with 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) to a final concentration of 0.5% lanthanum hydroxide (w/v). Mosquito midguts were dissected directly into this solution and fixed for 2 hr in the same. The midguts were rinsed twice (15 min) in cacodylate buffer followed by a 15 min rinse in 0.2 M phosphate buffer (Houk, 1977). Subsequent steps of osmication, dehydration and embedding (Houk, 1977) were in lanthanum-free solutions. Two methods specific for sulfated GAGs were applied to the mosquito midgut: HID (Spicer et al., 1978) and diaminobenzidine-osmium tetroxide (Monga et al., 1972).
MATERIALS AND M E T H O D S
Maintenance of mosquitoes, tissue processing and electron microscopic examination procedures have been described (Houk, 1977). All of the staining methods used for complex carbohydrates, except for tannic acid (TA) and high iron diamine (HID), have been reviewed by Hayat (1975; Table 1). Ruthenium red (RR) staining was either by the en bloc method of Luft (1964) and as a post stain of 1% RR (w/v) in 0.1 M NHaOH (Kobayashi and Asboe-Hansen, 1971). TA (2% w/v) staining was in either the 2% glutaraldehyde primary fixation or in the 1% osmium tetroxide post fixative (Singley and Solursh, 1980). Alcian blue (AB) staining was en bloc (Behnke and Zelander, 1970) and as a post stain of 0.1% AB (w/v) in 3% acetic acid (Tadano and Yamada, 1978). This sections post stained with AB were also post stained with lead citrate (Reynolds, 1963). PTA was also utilized either en b l o c or as a post stain. The en bloc staining was 1% PTA (w/v) in 70% ethanol overnight (Bloom and Aghajanian, 1968). The post staining method was 1% (w/v) PTA in 10% chromic acid (ChO; w/v) with (Tsuchiya and Ogawa, 1973) or without periodic acid (PA) oxidation (Roland et al., 1972). Two post staining methods specific for v/c-glycols were also examined (Rambourg and Leblond, 1967; Thiery, 1967). The silver methenamine (AgM) method (Rambourg and Leblond, 1967) was examined both with and without PA oxidation. AgP staining with PA oxidation and thiosemicarbazide (TSC) as the bridging agent examined using all appropriate controls (Thiery, 1967).
RESULTS Ruthenium
red (RR)
R R b o t h e n b l o c (Fig. I) a n d as a post stain (Fig. 2) stained the BL a n d the P M intensely (Table 1). T h e staining reaction was confined to the outer m e m b r a n e leaflet (Fig. 2). N o staining was a p p a r e n t in the intercellular j u n c t i o n s or along the microvillar m e m b r a n e (Fig. 3). Tannic
acid (TA )
M i d g u t extracellular surface staining with T A was a n exact duplication o f R R staining (Table 1). Intense staining was observed within the BL a n d along the P M , while intercellular j u n c t i o n s a n d microvilli were devoid o f stain. Alcian
blue (AB)
A B staining of P M (Fig. 4) a n d microvilli (Fig. 5) was d u b i o u s at best. However, the BL was distinctly stained, with small particulate deposits including adjacent muscle b a s e m e n t m e m b r a n e (Fig. 4). Post staining with A B revealed distinct particulate deposits within the BL a n d along the P M (Fig. 6). Particulate deposits were also a p p a r e n t along the surface of microvilli but n o t within intercellular junctions (Fig. 7). En bloc
Plate 1. Fig. 1. En bloc RR stains the basal lamina (BL) and the basolateral plasma membrane (PM). x 33,500. Fig. 2. RR stains material associated only with the outer membrane leaflet (OML) of the basolateral plasma membrane; inner membrane leaflet (IML) is unstained; basal lamina (BL). x 95,000. Fig. 3. RR does not stain microvilli (M) nor intercellular junctions (IJ). x 37,500. Fig. 4. Particulate staining within the midgut basal lamina (BL) is faint and unclear; no evidence of basolateral plasma membrane (PM) staining with en bloc AB. x 44,500. 669
Plate 2. Fig. 5. Microvilli (M) are devoid of particulate reaction product after en bloc staining with AB; intercellular junction (I J). x 27,500. Fig. 6. Midguts post stained with AB and lead citrate reveal enhanced particulate staining (arrowheads) along the basolateral plasma membranes (PM) and within the basal lamina (BL). x 69,500. Fig. 7. Microvilli (M) and intercellular junctions of AB and lead citrate post stained midguts reveal no evidence of staining, x 51,000. Fig. 8. En bloc ethanolic PTA stains the intercellular junction (I J), but not the microvilli (M). x 65,500. 670
Plate 3. Fig. 9. Intense staining of the basal lamina (BL) and finely localized staining of the basolateral plasma membrane (PM) after en bloc ethanolic PTA. x 56,500. Fig. 10. Microvilli (M) staining intensely with PA-ChO-PTA, negligible staining of all but the immediately luminal subjacent areas (arrowheads) of the intercellular junction (IJ). x 52,000. Fig. 11. PA-ChO-PTA intensely stains the basal lamina (BL) and the basolateral plasma membrane (PM). × 34,000. Fig. 12. Finely particulate silver deposits within the intercellular junction (IJ) after PA-TSC-AgP staining, x 41,500. 671
Plate 4. Fig. 13. Elimination of PA oxidation from the AgP method of Thiery (1967) results in a distinct distribution of reaction product within the plasma membrane of the intercellular junction (I J) space. x 64,000. Fig. 14. AgM staining of microvillar membranes (M) is sparse but specific. × 62,500. Fig. 15. Lanthanum specifically adsorbs to the microvillar surface (M) and apical regions of the intercellular junction (IJ). × 36,500.
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Midgut histochemistry We attempted RR post staining of en bloc AB stained midguts. The intense staining characteristic of RR (Figs 1 and 2) was not observed within the BL nor the PM. This would indicate that AB was indeed bound to the RR staining sites within these structures even though electron microscopic detection of AB staining was dubious within the PM (Fig. 6). Phosphotungstic acid (PTA ) En bloc PTA (e-PTA) staining was most evident in the intercellular junction (Fig. 8) and the BL (Fig. 9). Limited staining along the PM was observed (Fig. 9). ChO-PTA did not reveal additional staining sites nor enhance staining of the midgut extracellular surfaces (Table 1). Treatment of midgut tissue with 1% PA prior to ChO-PTA completely altered the staining pattern. Intense staining was apparent along the microvillar surface (Fig. 10), PM and BL (Fig. 11). Periodic acid-thiosemicarbazide-silver protein ( P A TSC-AgP) Deposition of extremely fine silver particles w a s apparent within the intercellular junction, along with enhanced electron opacity of microvillar membranes (Fig. 12). No staining was apparent within the BL; rather indistinct staining was observed along the PM (Table 1). As reported by Dallai (1970), substantial staining of the intercellular junction and microvillar membranes was apparent without PA oxidation (Fig. 13). However, the staining was distinctly different; TA-TSC-AgP allowed delineation of interseptal spaces within the continuous junction (Fig. 12) while TSC-AgP did not (Fig. 13). Silver methenamine ( AgM) As contrasted with the PA-TSC-AgP staining of microvilli (Fig. 12), AgM staining yielded large silver particles with a somewhat sparse distribution (Fig. 14). Specific staining of the BL, PM and intercellular junction was not observed (Table 1). Lanthanum Lanthanum stained microvilli (Fig. 15) and those immediately subjacent areas of the intercellular junctions, while staining of the more basal areas of the intercellular junctional complex, BL and PM was sporadic and appeared not to be specific (Table 1). Diaminobenzidine-osmium tetroxide (DAB-Os) Enhanced electron opacity was apparent only within the intercellular junction and this was considered to be nonspecific (Table 1). High iron diamine (HID) The HID method appeared as nonspecific granular deposits on all membrane systems (Table 1). DISCUSSION
The cellular surfaces of the mosquito midgut epithelium possess a unique histochemistry, when compared to vertebrate gastrointestinal absorptive cells a tissue with analogous function (Jersild and Crawford, 1978; Spicer et al., 1981; Yamamoto, 1982). The apical surface was stained sparingly by all polyanionic specific methods tested (Table 1); the only I.B. 16/4-,--E
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electronegative character is attributed to phosphoglycoconjugates as revealed through lanthanum and rather sparse AB staining. Neutral v/c-glycols were the predominant polysaccharide component of the apical surface; positive staining by PAChO-PTA, AgM and PA-TSC-AgP. The intercellular junctional spaces contained fixed positive charges as revealed by en bloc PTA staining and, in addition, some neutral vie-glycols (i.e. positive to PA-TSC-AgP). The PM system was anionic, as was the BL; positive staining by RR, TA and AB. Two vertebrate epithelial cells parrot the staining pattern observed for mosquito midgut: dark cells of the renal collecting tubules of guinea pigs and mitochondriarich cells of the toad urinary bladder (Spicer et al., 1981). The mosquito midgut is involved in nutrient absorption and osmoregulation (Berridge and Oschman, 1972). The luminal and abluminal surfaces of the mosquito midgut are involved in the above tasks respectively. Thus, one should suspect the histochemical staining reactions of these two surfaces would be different. However, it is surprising that the histochemistry of the mosquito midgut is different from the functionally analogous vertebrate gastrointestinal tract (Cook and Stoddart, 1973; Jersiid and Crawford, 1978; Spicer et al., 1982; Yamamoto, 1982); the microvillar surface being very basophilic in the vertebrate. Two possible explanations for the neutral staining characteristics of the mosquito midgut microvillar surface might be suggested: (1) the need to sequester proteins from vertebrate blood may require a neutral surface and/or (2) the neutral polysaecharide surface coat may be insensitive to proteases secreted into the lumen of the midgut by the insect during digestion of the bloodmeal. Pipa and Cook (1958) indicated that the microvillar surface of the midgut epithelium of bloodfeeding, sucking lice is predominantly neutral polysaccharide. Gutierrez and Burgos 0978) report that the microvillar surface of Triatoma infestans, another bloodfeeding insect, has a very weak electronegative character associated with phosphoglycolipids. Again, the predominant polysaccharide component of the intestinal microvillar surface in T. infestans was neutral. It appears plausible that physiological function, rather than coincidence, determines the similar staining characteristics with regard to the lack of a definitive anionic component associated with the microvillar surface in these three bloodfeeding insects. A minor anionic component was detected along the mosquito microvillar surface, as revealed by lanthanum staining (Fig. 15; Table i). Several investigators have observed staining of cellular surfaces by lanthanum (Doggenweiler and Frenk, 1965; Lesseps, 1967; Revel and Karnovsky, 1967; Overton, 1968; Shea, 1971). The specific nature of the lanthanum reaction has not been determined. However, Lesseps (1967), Overton (1968) and Shea (1971) were able to lessen lanthanum staining by pretreatment of their tissue preparations with phospholipase C, thus implicating phospholipds as the site of lanthanum binding. We plan to investigate the effects of phospholipase C treatment on lanthanum staining of the mosquito midgut microvilli.
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The intercellular junctional complex of mosquito midgut epithelia is a smooth septate junction (Reinhardt and Hecker, 1973; Houk, 1977); a junction unique to invertebrates. It apparently serves two purposes: (1) cell to cell cohesion and (2) an occlusion barrier to paracellular diffusion. Most authors agree on the former function but the latter has been the subject of scrutiny (Satir and Gilula, 1973; NoirotTimothee and Noirot, 1980; Lane and Skaer, 1980). The differential staining of extrajunctional apical and basolateral plasma membrane regions we observed for the mosquito midgut epithelium would substantiate cell to cell cohesion within the junction and point to restricted intramembrane diffusion between these two regions. As a barrier to paracellular diffusion, the smooth septate junction remains anomalous. Reinhardt and Hecker (1973) and the e-PTA data presented herein (Fig. 8) would indicate the presence of fixed positive charges, presumably alkaline proteins and/or glycoproteins, within this junction. These positive charges could provide an electrostatic barrier to the paracellular diffusion of cationic materials (e.g. Na ÷ and K +) through the junctional complex. However, a number of investigators have been able to demonstrate penetration into septate junctions of both smooth and pleated sheet morphology by exogenous tracers (see Lane and Skaer, 1980; Noirot-Timothee and Noirot, 1980). One criticism of these studies is that the observed penetration of the exogenous tracer could be an artifact of fixation and incubation conditions. Hematophagous insects present an interesting model for the investigation of the paracellular diffusion barrier function for smooth septate junctions. As an example, the female mosquito periodically ingests large quantities of blood and as such probably allows for the testing of smooth septate junctions as barriers under conditions of extreme physical stress. Houk and Hardy (1981, 1982) reported the diffusion of ingested material (i.e. bloodmeal components) into apical intrajunctional spaces of the mosquito midgut smooth septate junction; an in vivo event. Manipulation of the composition of the bloodmeal (i.e. varying ratios of rabbit serum and defibrinated rabbit blood) led to the conclusion that hemoglobin or another component of whole blood was responsible for the observed staining within the smooth septate junction. The mosquito, Cx. tarsalis, apparently represents an extreme example since other mosquitoes examined (i.e. Culex pipiens and Aedes dorsalis) do not appear to allow bloodmeal materials to diffuse into intrajunctional spaces (Houk, unpublished observations). Intrajunctional diffusion would appear to be dependent upon the hydrostatic pressure exerted by the ingested bloodmeal in producing a separation of adjacent cells; perhaps dependent upon the ability of individual mosquitoes to control the amount of material ingested. As such the basis for the barrier function of smooth septate junctions is most likely the greatly extended diffusion pathway dependent upon the number and geometric arrangement of intrajunctional septa (Filshie and Flower, 1977). In summary, the luminal surface of the mosquito midgut epithelium is primarily composed of neutral
polysaccharides with the only anionic staining characteristic tentativity attributed to phosphoglycoproteins and/or phosphoglycolipids. The smooth septate junction contains fixed positive charges in addition to some vic-glycols. The basolateral membranes and the basal lamina, based on their affinity for RR and TA, contain an abundance of negative charges, almost exclusively carboxyl groups of G A G s and glycoproteins. The effects of chemical and biochemical modification of the midgut epithelial surfaces are currently being investigated. In addition, we are pursuing lectin binding studies and specific probes for charge distribution. We hope that these studies will allow some insight into the mechanism(s) of mosquito midgut-arbovirus interaction. Acknowledgements--This research was supported by a U.S. Army Contract/Grant, DAMD 17-77-C-7018, U.S. Army Medical Research and Development Command, Washington, D.C. and by the Office of Naval Research. REFERENCES
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