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Ultrastructural changes in the abdominal midgut of the mosquito, Culiseta melanura, during the gonotrophic cycle
TISSUE AND CELL, 1990 22 (6) 895-909 © 1990 Longman Group UK Ltd.
0040-8166/90/0022-0895/$10.00
S. C. WEAVER*l" and T. W. SCOTT*
ULTRASTRUCTURAL CHANGES IN THE A B D O M I N A L M I D G U T OF THE MOSQUITO, CULISETA MELANURA, DURING THE GONOTROPHIC CYCLE Keywords: Mosquito, Insecta, midgut, ultrastructure, epithelium, Culiseta melanura ABSTRACT. Abdominal midguts of the mosquito, Culiseta melanura, were examined by light and electron microscopy 1 hr-14 days days after blood feeding. Epithelial cells were drastically altered from columnar to squamous in form after engorgement, and returned to columnar by day 4 after feeding. Accumulation of mitochondria along brush borders of digestive cells, followed by the appearance of large secondary lysosomes, accompanied blood digestion. Evidence was obtained that myelin-like material in the lysosomes, probably the result of mitochondrial autolysis, is extruded into the lumen. Digestive cells resumed their pre-blood meal appearance by 10-14 days post-engorgement. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. Other midgut cells containing large, microvilli-lined apical cavities were identified in most specimens. No evidence of division or differentiation was obtained for any cell types.
and are termed digestive-absorptive cells (Snodgrass, 1935), while minority popuThe midgut of m o s q u i t o e s is the initial barrier lations of endocrine and regenerative cells to infection e n c o u n t e r e d by many vectorcan be distinguished based on ultrastructure borne pathogens. F o r arthropod-borne (Brown et al., 1985). H o w e v e r , specific funcviruses, the abdominal or posterior midgut tions of each cell type remain undefined. epithelium is b e l i e v e d to be the site of initial Culiseta melanura is the enzootic mosquito replication during m o s q u i t o infection (Hardy vector of the togavirus, eastern equine et al., 1983). K n o w l e d g e of the ultrastructure encephalomyelitis ( E E E ) virus. Mosquito of this organ is t h e r e f o r e of special siginfection is believed to occur following nificance for interpreting early events in the engorgement on a viremic host, when virus mosquito-virus interaction. infects epithelial cells in the abdominal Previous studies of mosquito midgut ultramidgut. E E E virus disseminates from the structure (Bertram and Bird, 1961; H e c k e r midgut within 1-2 days and infects salivary et al., 1971, 1974; H o u k , 1977; Reinhardt glands via h e m o l y m p h , resulting in transand Hecker, 1973; Staubli et al., 1966) mission when an infected mosquito refeeds described a single layer of columnar epion a susceptible v e r t e b r a t e (Scott et al., 1984; thelial cells with an apical brush border and Scott and Weaver, 1989; W e a v e r et al., 1990). a basal lamina. T h e majority of epithelial W e recently d e m o n s t r a t e d that infection cells were morphologically indistinguishable with E E E virus causes cytopathic changes in the midgut of Cs. m e l a n u r a ( W e a v e r et al., * Department of Entomology, The Universityof Mary- 1988). This raised questions regarding norland, College Park, Maryland 20742, U.S.A. "t Department of Microbiology and Immunology, The mal midgut processes which accompany the Medical College of Pennysylvania, Philadelphia, Penn- gonotrophic cycle. The present study was sylvania 19129, U.S.A. implemented to describe morphological information on these processes and to proReceived 19 July 1990. 895 Introduction
WEAVER AND SCOTT
896 vide the first ultrastructural information of the midgut of a mosquito in the Culiseta genus.
Materials and Methods Mosquitoes Cs. melanura mosquitoes were obtained from a colony established in 1967 from adult females collected in Farmington, Connecticut, U . S . A . (Wallis & W h i t m a n , 1969) or were collected as 4th instar larvae in the P o c o m o k e swamp, Maryland, U . S . A . during 1987. All mosquitoes were r e a r e d as previously described (Scott et al., 1984) at 2 4 26°C. A d u l t females were maintained on 5 10% aqueous sucrose for 7-14 days poste m e r g e n c e and offered 1-day-old chicks for blood feeding; chicks were restrained and exposed to groups of 25-75 mosquitoes for one h o u r in 3.8 liter plastic cages at 24-26°C and 80% relative humidity. Following chick exposure, mosquitoes were sedated by chilling and sorted according to e n g o r g e m e n t status. Fully e n g o r g e d individuals were retained in 3.8 liter cages at 2 4 26°C, 80% relative humidity, and provided 5% aqueous sucrose and ovipositional sites. Microscopy Two field collected and two colony mosquitoes were aspirated from cages, chilled, and prepared for microscopic examination at selected intervals following blood feeding: 12 hr, 24 hr, 48 hr, 3 days, 4 days, 5 days, 7 days, 10 days, and 14 days. Mosquitoes were fixed by intrathoracic inoculation of ca. 0.5/A of fixative (glutaraldehyde, 0 . 8 % ; paraformaldehyde, 4%; sodium cacodylate, 0.1 M,
p H 7 . 3 ) at 5°C. Following inoculation, the a b d o m e n was cut from the thorax with a razor blade, the last abdominal segment severed, and the a b d o m e n immersed in fixative at 5°C for 12hr or more. Prior to further processing, a b d o m e n s were cut in half transversely. Following post-fixation in 1% osmium tetroxide for 36 hr at 5°C, a b d o m e n s were stained en bloc in 0.5% uranyl acetate (methanolic 30%) for 4 hr and dehydrated in a graded series of acetone. Spurr's resin was used for embedding, and thick sections of 0.5-1.0 pm were cut using glass knives. Thick sections were stained with toluidine blue and examined by bright field, phase contrast, or Nomarski optics. Thin sections of 60-90 nm were cut with a diamond knife, stained with lead citrate, and photographed on a Zeiss EM10 or Phillips EM300 transmission electron microscope at 60 or 8 0 K V electron acceleration. A t least four planes of transverse section were examined for each incubation interval.
Histochemistry Lysosomes were identified histochemically by cerium-based localization of acid phosphatase. M o s q u i t o midguts were dissected into 2% glutaraldehyde, 0.1 M sodium cacodylate, p H 7.3 at 5°C, and fixed for 1 hr. Following three washes in 0.1 M sodium cacodylate, midguts were incubated in a cerium-based medium for acid phosphatase (Robinson and Karnovsky, 1983). Following substrate reaction, midguts were washed three times in cacodylate buffer, post-fixed, dehydrated and e m b e d d e d as described above.
Abbreviations BL - basal lamina, BR - basal labyrinth, EC - endocrine cell, FB - fat body, G - Golgi complex, L - lipid droplet, LB - lamellar body, M - mitochondria, MU - muscle, MV - microvilli, N - nucleus, PM - peritr0phic membrane, RC - regenerative cell, Z - Zonula continua. Fig. 1. Midgut epithelium of unengorged, wild-caught Cs. melanura7 days after emergence. Note regenerative cell, highlyconvoluted basal labyrinth, basal lamina, and longitudinal muscle cell. x7200. Fig. 2. Wild caught mosquito, unengorged. Note characteristic whorls of rough endoplasmic reticulum (unlabeled arrows), numerous Golgi complexcs and a lamellar body or myelin-like figure, x 19,000. Fig. 3. Intercellular junctions between epithelial cells of an unengorged colony mosquito. Note the zonula continua and a tight junction (unlabelled arrow). ×50,000. Fig. 4. Zonula continua (arrows), colony mosquito, 3 days after engorgement. ×76,000.
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Results
intercellular spaces were less apparent and were located only in the most basal portion of the epithelium. The large volume of blood in the midgut lumen compressed the epithelium against the abdominal fat body (Fig. 6). Chick blood cells in the midgut lumen were separated from the epithelium by a band of amorphous, acellular material (Fig. 6). Cells at the anterior and posterior ends of the abdominal midgut appeared more cuboidal following engorgement. Basal and apical margins of these cells remained similar in appearance to those of unengorged mosquitoes.
Unfed mosquitoes Most epithelial cells in the abdominal midgut of unengorged mosquitoes were cuboidal to columnar in shape, with an apical brush border of microvilli and a basal lamina (Fig. 1). These cells were consistent in appearance with digestive-absorptive cells (Snodgrass, 1935). Cytoplasm of these cells included numerous Golgi complexes, mitchondria, ribosomes, and rough endoplasmic reticulum (RER), sometimes in concentric whorls (Fig. 2). Some lamellar bodies or myelin-like figures were present in the apical cytoplasm (Fig. 2), while lipid droplets were primarily found in the basal region. Nuclei contained large amounts of heterochromatin and a prominent, centrally located nucleolus (Fig. 1). Intercellular spaces of ca. 0.1~).5#m were present in basal regions of the epithelium, along with a highly convoluted basolateral plasma membrane, or basal labyrinth. Apical intercellular junctions appeared identical to the zonula continua or continuous junction described in other insects (Noirot and Noirot-Timothee, 1967) (Figs 3, 4). These junctions rarely appeared septate, and desmosomes and intermediate junctions were never observed. Tight junctions were present in some sections (Fig. 3).
24 hr post-engorgement Following 24 hr of blood digestion and an apparent decrease in midgut luminal volume, central regions of the abdominal midgut epithelium thickened, and cells assumed a cuboidal form (Fig. 7). Mitochondria were noticeably concentrated in a band adjacent to the brush border. A peritrophic membrane measuring 320-1020nm in thickness was present on the luminal side of the epithelium, and luminal blood contents were amorphous in the periphery, suggesting digestion of blood components. Concentric whorls of R E R remained in the apical cytoplasm (Fig. 7).
1-2 hr post-engorgement Midgut cell morphology was drastically altered after blood feeding. Cells in central portions of the posterior midgut assumed a squamous appearance, and basal margins became more regular in appearance (Figs 5, 6). Microvilli were compressed and irregular in form, due to tissue stretching, while the basal lamina was straightened. Intercellular junctions appeared unchanged, although
2 days post-engorgement Epithelial cells remained cuboidal in shape between days i and 2 post-engorgement (Fig. 8). Apical mitochondria were even more densely packed adjacent to the microvilli, and the cytoplasm was densely packed with RER. The PM measured 570-1210nm in thickness and was sometimes separated slightly from the epithelium. Peritrophic membrane formation in Cs. rnelanura has
Fig. 5. Colony mosquito, 1-2 hr after blood feeding. Note straightened basal lamina, basal lipid droplets and irregular microvilli below, x21,000. Fig. 6. Colony mosquito, 1-2 hr after engorgement. Note squamous appearance and closely opposed fat body trophocytes above. Amorphous, acellular matcrial (unlabeled arrows) separate epithelium from chick blood cells, x4200. Fig. 7. Wild-caught mosquito, 24 hr after engorgement. Note the euboidal appearance of epithelial cell, presence of the peritrophic mcmbrame, and row of mitochondria lining the brush border (unlabelled arrows), x8500.
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Fig. 8. Wild-caught mosquito, 2 days after engorgement. Note concentration of apical mitochondria adjacent to microvilli (arrows), and basal endocrine cell. x8500.
Fig. 9. Wild-caught mosquito, 4 days after engorgement. Note basal nuclei and large secondary lysosomes (unlabelled arrows) in apical regions, x3200. Fig. 10. Secondary lysosomes, colony mosquito, 4 days after engorgement. Note the abundance of lamellar bodies and lipid droplets. ×13,000. Fig. 11. Apical cytoplasm, wild-caught mosquito, 5 days after engorgement. Note large lipid droplets and numerous secondary lysosomes (unlabelled arrows), x2900.
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been described in greater detail elsewhere (Weaver and Scott, 1990). Chick blood remained in the lumen of all mosquitoes examined, with intact cells discernable only in the center of the blood bolus.
3-5 days post-engorgment Luminal volume of the posterior midgut decreased greatly due to excretion of the blood bolus between days 3 and 4. Thus, epithelial cells returned to a columar form by day 4 (Fig. 9). Nuclei were basally situated, and the basal lamina resumed a highly convoluted form. Basal intercellular spaces extended more than half the distance from the basal lamina to the brush border. Large inclusions containing an abundance of lamellar bodies or myelin-like figures, as well as lipid droplets and other unidentified debris, appeared in the apical cytoplasm (Figs 9, 10). Many cells also contained free lipid droplets occupying large volumes (Fig. 11). Golgi complexes and rough endoplasmic reticulum were reduced in abundance relative to numbers observed in cells 2-3 days after blood feeding. Numerous mitochondria, varying greatly in appearance, were dispersed throughout the cytoplasm of absorptive cells on days 4-5 (Figs 12, 13). In some cells, apical mitochondria were vesicular in appearance; the most vesicular of these were often similar in appearance to adjacent lamellar bodies showing myelin-like structure (Figs 12, 13). Lamellar bodies were also observed in the midgut lumen on days 4-5; they extended from the apical plasma mem-
brane through the microvilli and into the lumen (Figs 14-15). Membranes of these luminal lamellar bodies sometimes extended into the apical cytoplasm, but were not continuous with microvillar membranes (Fig. 16). Lamellar bodies, similar in appearance to those observed in cytoplasm, were present in the abdominal midgut lumen on days 514 (Fig. 17).
Day 7-14 post-engorgement Ultrastructural features of absorptive cells fixed 7 days after engorgement were similar to those of days 4-5. Luminal lamellar bodies were still observed in the midgut on day 7. By days 10-14, the epithelium appeared nearly indistinguishable from that of unfed mosquitoes (Fig. 18). Lamellar inclusions in apical cytoplasm were relatively small and mitochondria appeared normal and distributed throughout the cytoplasm.
Other cell types At least three cell types could be clearly distinguished from digestive-absorptive cells in the posterior midgut epithelium. All were present throughout the gonotrophic cycle. Small, basally situated regenerative cells were seen throughout the posterior midgut (Fig. 1). Regenerative cells often appeared crescent shaped, and were usually lower in cytoplasmic density than digestive cells. Mitotic figures were never seen in regenerative cells or in any other cell types in the midgut epithelium. Endocrine cells were also identified
Fig. 12. Colony mosquito, 5 days after engorgement. Note apical lamellar bodies and mitochondria. :<4600. Fig. 13. Apical cytoplasm, colony mosquito, 4 days after engorgement. Note vesicularappearing mitochondria with cristae in periphery only. x8600. Fig. 14. Wild-caught mosquito, 5 days after engorgement. Lamellar bodies or myelin-like figures (arrows) in midgut lumen within microvilli of epithelium (E). x21,000. Fig. 15. Wild-caught mosquito, 5 days after engorgement. Lamellar body (arrows) cxtcnding from epithelial cell (E) into midgut lumen, x31,000. Fig. 16. Wild-caught mosquito, 5 days after engorgement. Junction of luminal membranous material with epithelium. Note membrane extending into the apical cytoplasm (arrows). x 100,000. Fig. 17. Wild-caught mosquito, 7 days after engorgement. Lamellar bodies within the abdominal midgut lumen. Note membranous material extending between microvilli (arrow). x 26,000.
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Fig. 18. Colony mosquito, 14 days after engorgement. Note extensive basal labyrynth extending into the epithelium, convoluted basal lamina, and longitudinal muscles. ×9200.
throughout the epithelium (Fig. 8). These cells were characterized by the presence of secretory granules and were usually low in cytoplasmic density relative to digestiveabsorptive cells. Endocrine cells extended to the apical side of the epithelium in some sections, while serial sections revealed that some did not reach the brush border. These
usually contained an open, microvilli-lined space at their apical end. Unidentified cells containing large microvilli-lined cavities or bodies of up to 25/~m in diameter were observed in various regions of the midgut (Figs 19-20). These cells occurred in the epithelium of all mosquitoes examined. The cavities contained amorphous
Fig. 19. Microvilli-lined cavity, colony mosquito, 3 days after engorgement. Note fibrous material (F) in center, and lack of microvilli on apical plasma membrane adjacent to cavity (unlabeled arrow), x5800. Fig. 20. Microvilli-lined cavity, colony mosquito, 7 days after engorgement. Notc dense, amorphous matter in cavity, with small droplets dispersed among the microvilfi. × 18,000.
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and fibrous material of varying density and u n k n o w n identity. Dense, amorphous material was observed between microvilli inside cavities, but this material was n o t identified in any form in the cytoplasm (Fig. 20). These cavities were always m e m b r a n e limited and surrounded by cytoplasm on all sides; despite intensive serial sectioning, the m e m b r a n e surrounding these cavities was observed to be continuous with the apical cell mem-
brane. The placement of these cavities varied from apical to central. Nuclei of these cells were basally situated, and secretory granules or other distinguishing organelles were not identified in the cytoplasm. Large amounts of R E R were present in the cytoplasm, as well as n u m e r o u s mitochondria. Some cells containing cavities did not have apical microvilli typical of the adjacent cells (Fig. 19).
Fig. 21. Apical cytoplasm, colony mosquito, day 3, acid-phosphatasehistochemistry.Note primary lysosomesstained with cerium (arrows) along with large numbers of mitochondria. x 26,000. Fig. 22. Colony mosquito, day 6, acid phosphatase histochemistry.Note cerium deposition within secondary lysosomes (arrows) containing membranous material and other debris. x23,000.
M I D G U T D U R I N G G O N O T R O P H I C CYCLE
Acid phosphatase localization
Acid phosphatase was detected in lysosomes of midgut epithelial cells during all stages of the gonotrophic cycle. In unengorged mosquitoes, primary lysosomes were detected infrequently within the apical cytoplasm. During blood digestion, primary lysosomes appeared to increase in number and were abundant in apical cytoplasm of digestive cells by day 3 (Fig. 21). These lysosomes were interspersed with numerous mitochondria. By 4-5 days after engorgement, acid phosphatase was detected within secondary lysosomes containing myelin-like material, lipid droplets and unidentified debris (Fig. 22). These secondary lysosomes were less frequently observed on days 10 and 14. Acid phosphatase was not identified in cells containing microvilli-lined cavities.
Discussion
Examination of midguts from unengorged mosquitoes revealed ultrastructural features generally consistent with those previously reported for other mosquito species (Bertram and Bird, 1961; Houk et al., 1979; Hecker et al., 1971; Staubli et al., 1966). Minor differences were observed between Cs. melanura ultrastructure and that of other mosquito species. For example, in Cs. melanura, RER whorls were present both before and after blood feeding, in contrast to those in Ae. aegypti, which unfold following blood feeding (Bertram and Bird, 1961), and those in Cx. tarsalis, which appear only after blood feeding (Houk, 1977). Another difference from previous studies was the absence of desmosomes in Cs. melanura, which have been reported in midguts of Aedes (Bertram and Bird, 1961; Reinhardt and Hecker, 1974) and Culex (Houk, 1977) mosquitoes. Hecker (1977) reported a lack of desmosomes and intermediate junctions in 2 species of Anopheles and concluded that these intercellular junctions are not essential for tissue integrity during the severe stretching which accompanies blood feeding by mosquitoes. Our results are consistent with that view. The accumulation of mitochondria along the brush border of absorptive cells 1-3 days after blood feeding (Figs 7, 8) agrees with previous studies (Houk, 1977). This dis-
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tribution suggests that mitochondria may concentrate in the apical cytoplasm in order to supply oxidative energy required for blood digestion. The appearance, 4-7 days after blood feeding, of swollen mitochondria and what appear to be transitional forms with lamellar bodies, suggests an autolytic process. The appearance of large numbers of apical, acid-phosphatase-containing primary lysosomes in Cs. melanura midgut cells and later formation of secondary lysosomes containing myelin-like material, supports this view. We hypothesize that the large numbers of mitochondria appearing adjacent to the brush border, and presumably recruited or generated after blood feeding, may no longer be needed following cessation of blood digestion 3-4 days after engorgement. Thus, elimination of un-needed mitochondria in this manner may be advantageous for reasons of organelle parsimony or metabolic efficiency. Autolytic breakdown of mitochondria has been described for a variety of cell types and usually involves lysosomes (Luzikov, 1985). An alternative explanation for these abnormal mitochondrial forms is that artifactual alterations appeared due to oxygen deprivation (Munn, 1974). However, many mitochondria within the same or nearby cells showed normal morphology (see Fig. 12), suggesting that specimen preparation was not responsible for these changes. The large acid phosphatase-containing secondary lysosomes appearing in apical cytoplasm of digestive cells (Figs 9-11, 23) have been described in other mosquito species (Houk, 1977; Hecker et al., 1971), as well as other hematophagous diptera (de Priester, 1971) and insects in general (Shodgrass, 1935). Our results suggest that the lamellar bodies which comprise some of the lysosome contents may be derived from autolytic mitochondrial degeneration and possibly other membrane material associated with endocytosis and blood digestion. The finding of lamellar bodies extending from the apical cytoplasm into the lumen on days 57 (Figs 14-17), combined with the drastic reduction in secondary lysosome size and numbers by 11-14 days after blood feeding (Fig. 18), suggests that contents of autolysosomes may be eliminated into the midgut lumen. This form of organelle turnover was proposed by de Priester (1971) for the midgut
WEAVER AND SCOTT
908 of the fly Calliphora erythrocephala, and exocytosis of secondary lysosomes through a brush border has been described for rat proximal tubule kidney cells (Maunsbach, 1966). Snodgras (1935) described three forms of midgut digestive cell disintegration: 1) rupture of apical plasma membranes and release of 'granular material' into the lumen, 2) 'budding' of apical cytoplasmic droplets into the lumen and 3) extrusion nucleated cells into the lumen. The second form has been described for Cs. melanura, although its occurrence is predominant in the anterior midgut (Weaver and Scott, 1990), while the third form was described for mosquitoes undergoing cytopathologic changes following infection with E E E virus, but is rare in uninfected mosquitoes (Weaver et al., 1988, Weaver and Scott, 1990). To our knowledge, the first form has not been described for Cs. melanura or other mosquitoes. Our findings support Snodgrass's (1935) view that cytoplasmic 'budding' is the predominant form of midgut digestive cell disintegration in adult insects. The extrusion of lamellar bodies into the midgut lumen, which we report herein, was probably not detectible prior to widespread use of the electron microscope, and therefore overlooked by Snodgrass (1935). In the present study, regenerative cells observed in the midgut epithelium never showed transitional forms or mitotic figures, suggesting that epithelial cell turnover is low in adult female Cs. melanura during the gonotrophic cycle. Previous studies examining E E E virus-infected midguts undergoing cytopathic loss of many epithelial cells also revealed no signs of cell regeneration (Weaver et al., 1988). This brings into question whether regenerative cells have any epithelial cell replacement function in midguts of mature adult mosquitoes. Our results are consistent with Hecker's (1977) report that midgut cell replacement is rare in mosquitoes. The abdominal midgut cells containing
large cavities lined with microvilli (Figs 1920) are unlike any described in the literature for mosquitoes. They bear superficial resemblance to 'closed' mosquito endocrine cells which have on their apical tip a clear, vesiculated area lined by microvilli (Brown et al., 1985; Hecker et al., 1971). However, the cells we describe contain cavities many times larger than those described for endocrine cells during the present or past studies. They also contain no secretory granules. These Cs. melanura midgut cells bear some resemblance to goblet cells in the embryonic midgut of the tobacco hornworm, Manduca sexta (Hakim et al., 1988), which form closed cavities not yet open to the midgut lumen. The cells we describe also are closed to the lumen, although they lack the apical 'valve' structure seen in Manduca. They also lack elongated mitochondria, which line the cavity of many insect goblet cells (Smith, 1968). The presence of relatively large amounts of R E R in Cs. melanura midgut cells containing these cavities is suggestive of secretory role. However, further work is required to determine the cavity contents of these cells and to evaluate their function. In summary, abdominal midgut epithelial cells underwent alterations in morphology and organelle content associated with blood digestion. By day 14 post-engorgement, the epithelium resumed its pre-fed appearance. Mitochondria accumulated along the apical brush border 1-2 days after blood feeding and underwent autolytic degradation, involving lysosomes, after blood digestion. Unidentified cells containing large, microvilli-lined cavities, were seen throughout the midgut. Acknowledgements
We thank Les Lorenz for excellent technical assistance, and Mark Brown for critical review of the manuscript. This work received financial support from NIH grants AI26787 and AI22119, and the Maryland Agricultural Experiment Station.
References
Bertram, D. S. and Bird, R. G. 1961. Studieson mosquito-bornevirusesin their vector. I. The normal fine structure of the midgut epithelium of adult female Aedes aegypti L. and the functional significanceof its modifications followinga bloodmeal. Trans. R. Soc. trop. Med. Hyg., 55, 404-423. Brown, M. R., Raikhel, A. S. and Lea, A. O. 1985. Ultrastructureof midgut endocrinecellsin the adult mosquito, Aedes aegypti. Tissue & Cell 17, 709-721.
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Hardy, J. L., Houk, E. J., Kramer L. D. and Reeves, W. C. 1983. Intrinsic factors affecting vector competence of mosquitoes for arboviruses. Ann. Rev. Entomol. 28, 22%262. Hecker, H., Freyvogel, T. A., Briegel, H. and Steiger, R. 1971. Ultrastructural differentiation of the midgut epithelium in female Aedes aegypti L. (Insecta, Diptera). Acta Trop., 28, 80-105. Hecker, H., Brun, R., Reinhardt, C. and Burri, P. H. 1974. Morphometric analysis of the midgut of female Aedes aegypti L. (Insecta, Diptera) under various physiological conditions. Cell Tiss. Res., 152, 31-49. Hecker, H. 1977. Structure and function of midgut epithelial cells in Culicidae mosquitoes (Insecta, Diptera). Cell Tiss. Res., 184, 321-341. Houk, E. J. 1977. Midgut ultrastructure of Culex tarsalis (Diptera: Culicidae) before and after a bloodmeal. Tissue & Cell, 9, 103-118. Luzikov, V. N. 1985. Mitochondrial biogenesis and breakdown. 362 pp. Consultants Bureau (Plenum), New York. Maunsbach, A. B. 1966. Observations on the ultrastructure and acid phosphatase activity of the cytoplasmic bodies in rat kidney proximal tubules. J. Ultrastr. Res., 16, 197-238. Munn, E. A. 1974. The structure of mitochondria. 463 pp. Academic Press, New York. Noirot, CH. and Noirot-Timothee, C. 1967. Un nouveau type de jonction intercellulaire (zonula continua) dans l'intestin moyen des Insectes. C. r. hebd. Seanc. Acad. Sei. Paris,264, (D), 2796-2798. 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. Robinson, J. M. and Karnovsky, M. J. 1983. Ultrastructural localization of several phosphatases with cerium. J. Histochem. Cytochem., 31, 1197-1208. Scott, T. W., Hildreth, S. W. and Beaty, B. J. 1984. The distribution and development of eastern equine encephalitis virus in its enzootic mosquito vector, Culiseta melanura. Am. J. Trop. Med. Hyg., 33, 300-310. Scott, T. W. and Weaver, S. C. 1989. Eastern equine encephalomyelitisvirus: epidemiology and evolution of mosquito transmission. Ado. Virus Res., 37, 277-328. Snodgrass, R. E. 1935. Principles of Insect Morphology. 667 pp. McGraw-Hill, New York. Staubli, W., Freyvogel, T. A. and Sutter, J. 1966. Structural modification of the endoplasmic reticulum of the midgut epithelial cells of mosquitoes in relation to blood intake. J. Microscopic, 5, 18%204. Wallis, R. C. and Whitman, L. 1969. Colonization of Culiseta melanura (Coquillett) in the laboratory. Mosquito News 29, 255-258. Weaver, S. C., Scott, T. W., Lorenz, L. H., Lerdthusnee, K. and Romoser, W. S. 1988. Togavirus-associated pathologic changes in a natural mosquito vector. J. Virology 62, 2083-2090. Weaver, S. C. and Scott, T. W. 1990. Peritrophic membrane formation and cellular turnover in the midgut of Culiseta melanura (Diptera:Culicidae) during the genotrophic cycle. J. Med. Entomol. 27, 86%873. Weaver, S. C. and Scott, T. W. and Lorenz, L. H. 1990. Patterns of eastern equine encephalomyelitisvirus infection in Culiseta melanura (Diptera:Culicidae), J. Med. Entomol. 27, 878-891.
0040-8166/90/0022-0895/$10.00
S. C. WEAVER*l" and T. W. SCOTT*
ULTRASTRUCTURAL CHANGES IN THE A B D O M I N A L M I D G U T OF THE MOSQUITO, CULISETA MELANURA, DURING THE GONOTROPHIC CYCLE Keywords: Mosquito, Insecta, midgut, ultrastructure, epithelium, Culiseta melanura ABSTRACT. Abdominal midguts of the mosquito, Culiseta melanura, were examined by light and electron microscopy 1 hr-14 days days after blood feeding. Epithelial cells were drastically altered from columnar to squamous in form after engorgement, and returned to columnar by day 4 after feeding. Accumulation of mitochondria along brush borders of digestive cells, followed by the appearance of large secondary lysosomes, accompanied blood digestion. Evidence was obtained that myelin-like material in the lysosomes, probably the result of mitochondrial autolysis, is extruded into the lumen. Digestive cells resumed their pre-blood meal appearance by 10-14 days post-engorgement. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. Other midgut cells containing large, microvilli-lined apical cavities were identified in most specimens. No evidence of division or differentiation was obtained for any cell types.
and are termed digestive-absorptive cells (Snodgrass, 1935), while minority popuThe midgut of m o s q u i t o e s is the initial barrier lations of endocrine and regenerative cells to infection e n c o u n t e r e d by many vectorcan be distinguished based on ultrastructure borne pathogens. F o r arthropod-borne (Brown et al., 1985). H o w e v e r , specific funcviruses, the abdominal or posterior midgut tions of each cell type remain undefined. epithelium is b e l i e v e d to be the site of initial Culiseta melanura is the enzootic mosquito replication during m o s q u i t o infection (Hardy vector of the togavirus, eastern equine et al., 1983). K n o w l e d g e of the ultrastructure encephalomyelitis ( E E E ) virus. Mosquito of this organ is t h e r e f o r e of special siginfection is believed to occur following nificance for interpreting early events in the engorgement on a viremic host, when virus mosquito-virus interaction. infects epithelial cells in the abdominal Previous studies of mosquito midgut ultramidgut. E E E virus disseminates from the structure (Bertram and Bird, 1961; H e c k e r midgut within 1-2 days and infects salivary et al., 1971, 1974; H o u k , 1977; Reinhardt glands via h e m o l y m p h , resulting in transand Hecker, 1973; Staubli et al., 1966) mission when an infected mosquito refeeds described a single layer of columnar epion a susceptible v e r t e b r a t e (Scott et al., 1984; thelial cells with an apical brush border and Scott and Weaver, 1989; W e a v e r et al., 1990). a basal lamina. T h e majority of epithelial W e recently d e m o n s t r a t e d that infection cells were morphologically indistinguishable with E E E virus causes cytopathic changes in the midgut of Cs. m e l a n u r a ( W e a v e r et al., * Department of Entomology, The Universityof Mary- 1988). This raised questions regarding norland, College Park, Maryland 20742, U.S.A. "t Department of Microbiology and Immunology, The mal midgut processes which accompany the Medical College of Pennysylvania, Philadelphia, Penn- gonotrophic cycle. The present study was sylvania 19129, U.S.A. implemented to describe morphological information on these processes and to proReceived 19 July 1990. 895 Introduction
WEAVER AND SCOTT
896 vide the first ultrastructural information of the midgut of a mosquito in the Culiseta genus.
Materials and Methods Mosquitoes Cs. melanura mosquitoes were obtained from a colony established in 1967 from adult females collected in Farmington, Connecticut, U . S . A . (Wallis & W h i t m a n , 1969) or were collected as 4th instar larvae in the P o c o m o k e swamp, Maryland, U . S . A . during 1987. All mosquitoes were r e a r e d as previously described (Scott et al., 1984) at 2 4 26°C. A d u l t females were maintained on 5 10% aqueous sucrose for 7-14 days poste m e r g e n c e and offered 1-day-old chicks for blood feeding; chicks were restrained and exposed to groups of 25-75 mosquitoes for one h o u r in 3.8 liter plastic cages at 24-26°C and 80% relative humidity. Following chick exposure, mosquitoes were sedated by chilling and sorted according to e n g o r g e m e n t status. Fully e n g o r g e d individuals were retained in 3.8 liter cages at 2 4 26°C, 80% relative humidity, and provided 5% aqueous sucrose and ovipositional sites. Microscopy Two field collected and two colony mosquitoes were aspirated from cages, chilled, and prepared for microscopic examination at selected intervals following blood feeding: 12 hr, 24 hr, 48 hr, 3 days, 4 days, 5 days, 7 days, 10 days, and 14 days. Mosquitoes were fixed by intrathoracic inoculation of ca. 0.5/A of fixative (glutaraldehyde, 0 . 8 % ; paraformaldehyde, 4%; sodium cacodylate, 0.1 M,
p H 7 . 3 ) at 5°C. Following inoculation, the a b d o m e n was cut from the thorax with a razor blade, the last abdominal segment severed, and the a b d o m e n immersed in fixative at 5°C for 12hr or more. Prior to further processing, a b d o m e n s were cut in half transversely. Following post-fixation in 1% osmium tetroxide for 36 hr at 5°C, a b d o m e n s were stained en bloc in 0.5% uranyl acetate (methanolic 30%) for 4 hr and dehydrated in a graded series of acetone. Spurr's resin was used for embedding, and thick sections of 0.5-1.0 pm were cut using glass knives. Thick sections were stained with toluidine blue and examined by bright field, phase contrast, or Nomarski optics. Thin sections of 60-90 nm were cut with a diamond knife, stained with lead citrate, and photographed on a Zeiss EM10 or Phillips EM300 transmission electron microscope at 60 or 8 0 K V electron acceleration. A t least four planes of transverse section were examined for each incubation interval.
Histochemistry Lysosomes were identified histochemically by cerium-based localization of acid phosphatase. M o s q u i t o midguts were dissected into 2% glutaraldehyde, 0.1 M sodium cacodylate, p H 7.3 at 5°C, and fixed for 1 hr. Following three washes in 0.1 M sodium cacodylate, midguts were incubated in a cerium-based medium for acid phosphatase (Robinson and Karnovsky, 1983). Following substrate reaction, midguts were washed three times in cacodylate buffer, post-fixed, dehydrated and e m b e d d e d as described above.
Abbreviations BL - basal lamina, BR - basal labyrinth, EC - endocrine cell, FB - fat body, G - Golgi complex, L - lipid droplet, LB - lamellar body, M - mitochondria, MU - muscle, MV - microvilli, N - nucleus, PM - peritr0phic membrane, RC - regenerative cell, Z - Zonula continua. Fig. 1. Midgut epithelium of unengorged, wild-caught Cs. melanura7 days after emergence. Note regenerative cell, highlyconvoluted basal labyrinth, basal lamina, and longitudinal muscle cell. x7200. Fig. 2. Wild caught mosquito, unengorged. Note characteristic whorls of rough endoplasmic reticulum (unlabeled arrows), numerous Golgi complexcs and a lamellar body or myelin-like figure, x 19,000. Fig. 3. Intercellular junctions between epithelial cells of an unengorged colony mosquito. Note the zonula continua and a tight junction (unlabelled arrow). ×50,000. Fig. 4. Zonula continua (arrows), colony mosquito, 3 days after engorgement. ×76,000.
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Results
intercellular spaces were less apparent and were located only in the most basal portion of the epithelium. The large volume of blood in the midgut lumen compressed the epithelium against the abdominal fat body (Fig. 6). Chick blood cells in the midgut lumen were separated from the epithelium by a band of amorphous, acellular material (Fig. 6). Cells at the anterior and posterior ends of the abdominal midgut appeared more cuboidal following engorgement. Basal and apical margins of these cells remained similar in appearance to those of unengorged mosquitoes.
Unfed mosquitoes Most epithelial cells in the abdominal midgut of unengorged mosquitoes were cuboidal to columnar in shape, with an apical brush border of microvilli and a basal lamina (Fig. 1). These cells were consistent in appearance with digestive-absorptive cells (Snodgrass, 1935). Cytoplasm of these cells included numerous Golgi complexes, mitchondria, ribosomes, and rough endoplasmic reticulum (RER), sometimes in concentric whorls (Fig. 2). Some lamellar bodies or myelin-like figures were present in the apical cytoplasm (Fig. 2), while lipid droplets were primarily found in the basal region. Nuclei contained large amounts of heterochromatin and a prominent, centrally located nucleolus (Fig. 1). Intercellular spaces of ca. 0.1~).5#m were present in basal regions of the epithelium, along with a highly convoluted basolateral plasma membrane, or basal labyrinth. Apical intercellular junctions appeared identical to the zonula continua or continuous junction described in other insects (Noirot and Noirot-Timothee, 1967) (Figs 3, 4). These junctions rarely appeared septate, and desmosomes and intermediate junctions were never observed. Tight junctions were present in some sections (Fig. 3).
24 hr post-engorgement Following 24 hr of blood digestion and an apparent decrease in midgut luminal volume, central regions of the abdominal midgut epithelium thickened, and cells assumed a cuboidal form (Fig. 7). Mitochondria were noticeably concentrated in a band adjacent to the brush border. A peritrophic membrane measuring 320-1020nm in thickness was present on the luminal side of the epithelium, and luminal blood contents were amorphous in the periphery, suggesting digestion of blood components. Concentric whorls of R E R remained in the apical cytoplasm (Fig. 7).
1-2 hr post-engorgement Midgut cell morphology was drastically altered after blood feeding. Cells in central portions of the posterior midgut assumed a squamous appearance, and basal margins became more regular in appearance (Figs 5, 6). Microvilli were compressed and irregular in form, due to tissue stretching, while the basal lamina was straightened. Intercellular junctions appeared unchanged, although
2 days post-engorgement Epithelial cells remained cuboidal in shape between days i and 2 post-engorgement (Fig. 8). Apical mitochondria were even more densely packed adjacent to the microvilli, and the cytoplasm was densely packed with RER. The PM measured 570-1210nm in thickness and was sometimes separated slightly from the epithelium. Peritrophic membrane formation in Cs. rnelanura has
Fig. 5. Colony mosquito, 1-2 hr after blood feeding. Note straightened basal lamina, basal lipid droplets and irregular microvilli below, x21,000. Fig. 6. Colony mosquito, 1-2 hr after engorgement. Note squamous appearance and closely opposed fat body trophocytes above. Amorphous, acellular matcrial (unlabeled arrows) separate epithelium from chick blood cells, x4200. Fig. 7. Wild-caught mosquito, 24 hr after engorgement. Note the euboidal appearance of epithelial cell, presence of the peritrophic mcmbrame, and row of mitochondria lining the brush border (unlabelled arrows), x8500.
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Fig. 8. Wild-caught mosquito, 2 days after engorgement. Note concentration of apical mitochondria adjacent to microvilli (arrows), and basal endocrine cell. x8500.
Fig. 9. Wild-caught mosquito, 4 days after engorgement. Note basal nuclei and large secondary lysosomes (unlabelled arrows) in apical regions, x3200. Fig. 10. Secondary lysosomes, colony mosquito, 4 days after engorgement. Note the abundance of lamellar bodies and lipid droplets. ×13,000. Fig. 11. Apical cytoplasm, wild-caught mosquito, 5 days after engorgement. Note large lipid droplets and numerous secondary lysosomes (unlabelled arrows), x2900.
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been described in greater detail elsewhere (Weaver and Scott, 1990). Chick blood remained in the lumen of all mosquitoes examined, with intact cells discernable only in the center of the blood bolus.
3-5 days post-engorgment Luminal volume of the posterior midgut decreased greatly due to excretion of the blood bolus between days 3 and 4. Thus, epithelial cells returned to a columar form by day 4 (Fig. 9). Nuclei were basally situated, and the basal lamina resumed a highly convoluted form. Basal intercellular spaces extended more than half the distance from the basal lamina to the brush border. Large inclusions containing an abundance of lamellar bodies or myelin-like figures, as well as lipid droplets and other unidentified debris, appeared in the apical cytoplasm (Figs 9, 10). Many cells also contained free lipid droplets occupying large volumes (Fig. 11). Golgi complexes and rough endoplasmic reticulum were reduced in abundance relative to numbers observed in cells 2-3 days after blood feeding. Numerous mitochondria, varying greatly in appearance, were dispersed throughout the cytoplasm of absorptive cells on days 4-5 (Figs 12, 13). In some cells, apical mitochondria were vesicular in appearance; the most vesicular of these were often similar in appearance to adjacent lamellar bodies showing myelin-like structure (Figs 12, 13). Lamellar bodies were also observed in the midgut lumen on days 4-5; they extended from the apical plasma mem-
brane through the microvilli and into the lumen (Figs 14-15). Membranes of these luminal lamellar bodies sometimes extended into the apical cytoplasm, but were not continuous with microvillar membranes (Fig. 16). Lamellar bodies, similar in appearance to those observed in cytoplasm, were present in the abdominal midgut lumen on days 514 (Fig. 17).
Day 7-14 post-engorgement Ultrastructural features of absorptive cells fixed 7 days after engorgement were similar to those of days 4-5. Luminal lamellar bodies were still observed in the midgut on day 7. By days 10-14, the epithelium appeared nearly indistinguishable from that of unfed mosquitoes (Fig. 18). Lamellar inclusions in apical cytoplasm were relatively small and mitochondria appeared normal and distributed throughout the cytoplasm.
Other cell types At least three cell types could be clearly distinguished from digestive-absorptive cells in the posterior midgut epithelium. All were present throughout the gonotrophic cycle. Small, basally situated regenerative cells were seen throughout the posterior midgut (Fig. 1). Regenerative cells often appeared crescent shaped, and were usually lower in cytoplasmic density than digestive cells. Mitotic figures were never seen in regenerative cells or in any other cell types in the midgut epithelium. Endocrine cells were also identified
Fig. 12. Colony mosquito, 5 days after engorgement. Note apical lamellar bodies and mitochondria. :<4600. Fig. 13. Apical cytoplasm, colony mosquito, 4 days after engorgement. Note vesicularappearing mitochondria with cristae in periphery only. x8600. Fig. 14. Wild-caught mosquito, 5 days after engorgement. Lamellar bodies or myelin-like figures (arrows) in midgut lumen within microvilli of epithelium (E). x21,000. Fig. 15. Wild-caught mosquito, 5 days after engorgement. Lamellar body (arrows) cxtcnding from epithelial cell (E) into midgut lumen, x31,000. Fig. 16. Wild-caught mosquito, 5 days after engorgement. Junction of luminal membranous material with epithelium. Note membrane extending into the apical cytoplasm (arrows). x 100,000. Fig. 17. Wild-caught mosquito, 7 days after engorgement. Lamellar bodies within the abdominal midgut lumen. Note membranous material extending between microvilli (arrow). x 26,000.
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Fig. 18. Colony mosquito, 14 days after engorgement. Note extensive basal labyrynth extending into the epithelium, convoluted basal lamina, and longitudinal muscles. ×9200.
throughout the epithelium (Fig. 8). These cells were characterized by the presence of secretory granules and were usually low in cytoplasmic density relative to digestiveabsorptive cells. Endocrine cells extended to the apical side of the epithelium in some sections, while serial sections revealed that some did not reach the brush border. These
usually contained an open, microvilli-lined space at their apical end. Unidentified cells containing large microvilli-lined cavities or bodies of up to 25/~m in diameter were observed in various regions of the midgut (Figs 19-20). These cells occurred in the epithelium of all mosquitoes examined. The cavities contained amorphous
Fig. 19. Microvilli-lined cavity, colony mosquito, 3 days after engorgement. Note fibrous material (F) in center, and lack of microvilli on apical plasma membrane adjacent to cavity (unlabeled arrow), x5800. Fig. 20. Microvilli-lined cavity, colony mosquito, 7 days after engorgement. Notc dense, amorphous matter in cavity, with small droplets dispersed among the microvilfi. × 18,000.
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and fibrous material of varying density and u n k n o w n identity. Dense, amorphous material was observed between microvilli inside cavities, but this material was n o t identified in any form in the cytoplasm (Fig. 20). These cavities were always m e m b r a n e limited and surrounded by cytoplasm on all sides; despite intensive serial sectioning, the m e m b r a n e surrounding these cavities was observed to be continuous with the apical cell mem-
brane. The placement of these cavities varied from apical to central. Nuclei of these cells were basally situated, and secretory granules or other distinguishing organelles were not identified in the cytoplasm. Large amounts of R E R were present in the cytoplasm, as well as n u m e r o u s mitochondria. Some cells containing cavities did not have apical microvilli typical of the adjacent cells (Fig. 19).
Fig. 21. Apical cytoplasm, colony mosquito, day 3, acid-phosphatasehistochemistry.Note primary lysosomesstained with cerium (arrows) along with large numbers of mitochondria. x 26,000. Fig. 22. Colony mosquito, day 6, acid phosphatase histochemistry.Note cerium deposition within secondary lysosomes (arrows) containing membranous material and other debris. x23,000.
M I D G U T D U R I N G G O N O T R O P H I C CYCLE
Acid phosphatase localization
Acid phosphatase was detected in lysosomes of midgut epithelial cells during all stages of the gonotrophic cycle. In unengorged mosquitoes, primary lysosomes were detected infrequently within the apical cytoplasm. During blood digestion, primary lysosomes appeared to increase in number and were abundant in apical cytoplasm of digestive cells by day 3 (Fig. 21). These lysosomes were interspersed with numerous mitochondria. By 4-5 days after engorgement, acid phosphatase was detected within secondary lysosomes containing myelin-like material, lipid droplets and unidentified debris (Fig. 22). These secondary lysosomes were less frequently observed on days 10 and 14. Acid phosphatase was not identified in cells containing microvilli-lined cavities.
Discussion
Examination of midguts from unengorged mosquitoes revealed ultrastructural features generally consistent with those previously reported for other mosquito species (Bertram and Bird, 1961; Houk et al., 1979; Hecker et al., 1971; Staubli et al., 1966). Minor differences were observed between Cs. melanura ultrastructure and that of other mosquito species. For example, in Cs. melanura, RER whorls were present both before and after blood feeding, in contrast to those in Ae. aegypti, which unfold following blood feeding (Bertram and Bird, 1961), and those in Cx. tarsalis, which appear only after blood feeding (Houk, 1977). Another difference from previous studies was the absence of desmosomes in Cs. melanura, which have been reported in midguts of Aedes (Bertram and Bird, 1961; Reinhardt and Hecker, 1974) and Culex (Houk, 1977) mosquitoes. Hecker (1977) reported a lack of desmosomes and intermediate junctions in 2 species of Anopheles and concluded that these intercellular junctions are not essential for tissue integrity during the severe stretching which accompanies blood feeding by mosquitoes. Our results are consistent with that view. The accumulation of mitochondria along the brush border of absorptive cells 1-3 days after blood feeding (Figs 7, 8) agrees with previous studies (Houk, 1977). This dis-
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tribution suggests that mitochondria may concentrate in the apical cytoplasm in order to supply oxidative energy required for blood digestion. The appearance, 4-7 days after blood feeding, of swollen mitochondria and what appear to be transitional forms with lamellar bodies, suggests an autolytic process. The appearance of large numbers of apical, acid-phosphatase-containing primary lysosomes in Cs. melanura midgut cells and later formation of secondary lysosomes containing myelin-like material, supports this view. We hypothesize that the large numbers of mitochondria appearing adjacent to the brush border, and presumably recruited or generated after blood feeding, may no longer be needed following cessation of blood digestion 3-4 days after engorgement. Thus, elimination of un-needed mitochondria in this manner may be advantageous for reasons of organelle parsimony or metabolic efficiency. Autolytic breakdown of mitochondria has been described for a variety of cell types and usually involves lysosomes (Luzikov, 1985). An alternative explanation for these abnormal mitochondrial forms is that artifactual alterations appeared due to oxygen deprivation (Munn, 1974). However, many mitochondria within the same or nearby cells showed normal morphology (see Fig. 12), suggesting that specimen preparation was not responsible for these changes. The large acid phosphatase-containing secondary lysosomes appearing in apical cytoplasm of digestive cells (Figs 9-11, 23) have been described in other mosquito species (Houk, 1977; Hecker et al., 1971), as well as other hematophagous diptera (de Priester, 1971) and insects in general (Shodgrass, 1935). Our results suggest that the lamellar bodies which comprise some of the lysosome contents may be derived from autolytic mitochondrial degeneration and possibly other membrane material associated with endocytosis and blood digestion. The finding of lamellar bodies extending from the apical cytoplasm into the lumen on days 57 (Figs 14-17), combined with the drastic reduction in secondary lysosome size and numbers by 11-14 days after blood feeding (Fig. 18), suggests that contents of autolysosomes may be eliminated into the midgut lumen. This form of organelle turnover was proposed by de Priester (1971) for the midgut
WEAVER AND SCOTT
908 of the fly Calliphora erythrocephala, and exocytosis of secondary lysosomes through a brush border has been described for rat proximal tubule kidney cells (Maunsbach, 1966). Snodgras (1935) described three forms of midgut digestive cell disintegration: 1) rupture of apical plasma membranes and release of 'granular material' into the lumen, 2) 'budding' of apical cytoplasmic droplets into the lumen and 3) extrusion nucleated cells into the lumen. The second form has been described for Cs. melanura, although its occurrence is predominant in the anterior midgut (Weaver and Scott, 1990), while the third form was described for mosquitoes undergoing cytopathologic changes following infection with E E E virus, but is rare in uninfected mosquitoes (Weaver et al., 1988, Weaver and Scott, 1990). To our knowledge, the first form has not been described for Cs. melanura or other mosquitoes. Our findings support Snodgrass's (1935) view that cytoplasmic 'budding' is the predominant form of midgut digestive cell disintegration in adult insects. The extrusion of lamellar bodies into the midgut lumen, which we report herein, was probably not detectible prior to widespread use of the electron microscope, and therefore overlooked by Snodgrass (1935). In the present study, regenerative cells observed in the midgut epithelium never showed transitional forms or mitotic figures, suggesting that epithelial cell turnover is low in adult female Cs. melanura during the gonotrophic cycle. Previous studies examining E E E virus-infected midguts undergoing cytopathic loss of many epithelial cells also revealed no signs of cell regeneration (Weaver et al., 1988). This brings into question whether regenerative cells have any epithelial cell replacement function in midguts of mature adult mosquitoes. Our results are consistent with Hecker's (1977) report that midgut cell replacement is rare in mosquitoes. The abdominal midgut cells containing
large cavities lined with microvilli (Figs 1920) are unlike any described in the literature for mosquitoes. They bear superficial resemblance to 'closed' mosquito endocrine cells which have on their apical tip a clear, vesiculated area lined by microvilli (Brown et al., 1985; Hecker et al., 1971). However, the cells we describe contain cavities many times larger than those described for endocrine cells during the present or past studies. They also contain no secretory granules. These Cs. melanura midgut cells bear some resemblance to goblet cells in the embryonic midgut of the tobacco hornworm, Manduca sexta (Hakim et al., 1988), which form closed cavities not yet open to the midgut lumen. The cells we describe also are closed to the lumen, although they lack the apical 'valve' structure seen in Manduca. They also lack elongated mitochondria, which line the cavity of many insect goblet cells (Smith, 1968). The presence of relatively large amounts of R E R in Cs. melanura midgut cells containing these cavities is suggestive of secretory role. However, further work is required to determine the cavity contents of these cells and to evaluate their function. In summary, abdominal midgut epithelial cells underwent alterations in morphology and organelle content associated with blood digestion. By day 14 post-engorgement, the epithelium resumed its pre-fed appearance. Mitochondria accumulated along the apical brush border 1-2 days after blood feeding and underwent autolytic degradation, involving lysosomes, after blood digestion. Unidentified cells containing large, microvilli-lined cavities, were seen throughout the midgut. Acknowledgements
We thank Les Lorenz for excellent technical assistance, and Mark Brown for critical review of the manuscript. This work received financial support from NIH grants AI26787 and AI22119, and the Maryland Agricultural Experiment Station.
References
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Hardy, J. L., Houk, E. J., Kramer L. D. and Reeves, W. C. 1983. Intrinsic factors affecting vector competence of mosquitoes for arboviruses. Ann. Rev. Entomol. 28, 22%262. Hecker, H., Freyvogel, T. A., Briegel, H. and Steiger, R. 1971. Ultrastructural differentiation of the midgut epithelium in female Aedes aegypti L. (Insecta, Diptera). Acta Trop., 28, 80-105. Hecker, H., Brun, R., Reinhardt, C. and Burri, P. H. 1974. Morphometric analysis of the midgut of female Aedes aegypti L. (Insecta, Diptera) under various physiological conditions. Cell Tiss. Res., 152, 31-49. Hecker, H. 1977. Structure and function of midgut epithelial cells in Culicidae mosquitoes (Insecta, Diptera). Cell Tiss. Res., 184, 321-341. Houk, E. J. 1977. Midgut ultrastructure of Culex tarsalis (Diptera: Culicidae) before and after a bloodmeal. Tissue & Cell, 9, 103-118. Luzikov, V. N. 1985. Mitochondrial biogenesis and breakdown. 362 pp. Consultants Bureau (Plenum), New York. Maunsbach, A. B. 1966. Observations on the ultrastructure and acid phosphatase activity of the cytoplasmic bodies in rat kidney proximal tubules. J. Ultrastr. Res., 16, 197-238. Munn, E. A. 1974. The structure of mitochondria. 463 pp. Academic Press, New York. Noirot, CH. and Noirot-Timothee, C. 1967. Un nouveau type de jonction intercellulaire (zonula continua) dans l'intestin moyen des Insectes. C. r. hebd. Seanc. Acad. Sei. Paris,264, (D), 2796-2798. 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. Robinson, J. M. and Karnovsky, M. J. 1983. Ultrastructural localization of several phosphatases with cerium. J. Histochem. Cytochem., 31, 1197-1208. Scott, T. W., Hildreth, S. W. and Beaty, B. J. 1984. The distribution and development of eastern equine encephalitis virus in its enzootic mosquito vector, Culiseta melanura. Am. J. Trop. Med. Hyg., 33, 300-310. Scott, T. W. and Weaver, S. C. 1989. Eastern equine encephalomyelitisvirus: epidemiology and evolution of mosquito transmission. Ado. Virus Res., 37, 277-328. Snodgrass, R. E. 1935. Principles of Insect Morphology. 667 pp. McGraw-Hill, New York. Staubli, W., Freyvogel, T. A. and Sutter, J. 1966. Structural modification of the endoplasmic reticulum of the midgut epithelial cells of mosquitoes in relation to blood intake. J. Microscopic, 5, 18%204. Wallis, R. C. and Whitman, L. 1969. Colonization of Culiseta melanura (Coquillett) in the laboratory. Mosquito News 29, 255-258. Weaver, S. C., Scott, T. W., Lorenz, L. H., Lerdthusnee, K. and Romoser, W. S. 1988. Togavirus-associated pathologic changes in a natural mosquito vector. J. Virology 62, 2083-2090. Weaver, S. C. and Scott, T. W. 1990. Peritrophic membrane formation and cellular turnover in the midgut of Culiseta melanura (Diptera:Culicidae) during the genotrophic cycle. J. Med. Entomol. 27, 86%873. Weaver, S. C. and Scott, T. W. and Lorenz, L. H. 1990. Patterns of eastern equine encephalomyelitisvirus infection in Culiseta melanura (Diptera:Culicidae), J. Med. Entomol. 27, 878-891.