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An ultrastructural study of the anal papillae of aedes campestris larvae and of the hind gut of Aedes… Meredith, Joan 1971

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AN UXTRASTRUCTURAL STUDY OF THE 'ANAL PAPILLAE OF AEDES CAMPESTRIS LARVAE AND OF THE HIND GUT OF AEDES CAMPESTRIS AND AEDES AEGYPTI LARVAE by JOAN MEREDITH B. Sc. U n i v e r s i t y of B r i t i s h Columbia, 1°66 A t h e s i s submitted i n p a r t i a l f u l f i l l m e n t of the requirements f o r the degree of MASTER OF SCIENCE . i n the Department of Zoology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1971. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of Brit ish Columbia Vancouver 8, Canada ABSTRACT i The basic morphology and ultrastructure of the anal papillae of a saline water mosquito larva (Aedes campestris) have been investigated under two physiological conditions: (a) normal hyperosmotic external medium and (b) dilute hyposmotic medium. The ultrastructure of these organs suggests that they are not rudimentary, but rather are made up of cells that are morphologically specialized for transport. The anal papillae appear active in both the normal and dilute media and possible functions under the two physiological conditions are discussed. No major qualitative or quantitative differences were observed with the large change in external medium. This suggests that physiologically-demonstrated adaptive changes may require only minor structural alterations. The morphology and ultrastructure of the hind gut of A. campestris larvae were compared under two physiological conditions; normal (requiring hyporegulation) and dilute (requiring hyperegulation) external medium. No differences which might be associated with hyporegulation were observed. The rectum of A. campestri s was compared to that of Aedes aegypti. The rectum in the former is composed of two regions, an anterior and posterior rectum, while in Aedes aegypti, an exclusively freshwater mosquito larva, the rectum has only a single region. The rectal epithelia in both insects studied have morphological specializations for water and ion transport, and a consideration of quantative differences suggests that the posterior rectum is unique to Aedes campestris and hence could be responsible for the ability of this species to produce hyperosmotic urine. When the posterior rectal epithelium is compared to similar transport-ing epithelia in previously-studied terrestrial insects, several ultra-i i structural differences were noted. It is suggested that the mechanism of hyperosmotic urine production in saline water insects may be different from that in terrestrial insects. Several possible mechanisms of hyperosmotic urine production are discussed. TABLE OF CONTENTS Page GENERAL INTRODUCTION x PART I: THE ULTRASTRUCTURE OF THE ANAL PAPILLAE OF A 3 BRACKISHWATER MOSQUITO LARVA, AEDES CAMPESTRIS INTRODUCTION k MATERIALS AND METHODS T Light Microscopy 7 Electron Microscopy 7 RESULTS 10 Light Microscopy and General Morphology 10 (a) Animals Acclimated to Normal Hyperosmotic XQ Media (b) Animals Acclimated to Dilute Hyposmotic i u Media Electron Microscopy lU (a) Animals Acclimated to Normal Hyperosmotic i i * Media (b) Animals Acclimated to Hyposmotic Media 26 DISCUSSION 30 Fixation 30 Basic Ultrastructure 31 Comparison of Anal Papillae taken from Salt- 31+ and Freshwater Mosquito Larvae Ultrastructural Changes in Relation to 36 Physiological State PART I I : T H E MORPHOLOGY AND ULTRASTRUCTURE OF T H E HIND GUT OF A BRACKISHWATER (AEDES CAMPESTRIS) AND A FRESH-WATER (AEDES A E G Y P T I T M O S Q U I T O LARVA INTRODUCTION MATERIALS AND METHODS RESULTS Light Microscopy and General Morphology (a) Larvae of Aedes campestris acclimated to normal hyperosmotic media. (b) Larvae of Aedes campestris acclimated to dilute hyposmotic media. (c) Larvae of Aedes aegypti reared in fresh water. Electron Microscopy (a) Larvae of Aedes campestris acclimated to normal hyperosmotic media. (b) Larvae of Aedes campestris acclimated to dilute hyposmotic media. (c) Larvae of Aedes aegypti reared in fresh water. DISCUSSION (a) The morphology of the ileum and anal portion of the hind gut and associated tissues in Aedes campestris larvae. (b) The morphology and ultrastructure of the rectum of salt- and freshwater mosquito larvae. (c) The mechanism of hyperosmotic urine production SUMMARY REFERENCES Page Ul 42 43 44 44 44 52 54 54 54 76 78 81 81 82 89 95 97 LIST OF TABLES Chemical composition of GR 2 lake water Comparison of Anal Papillae from Fourth-stage Larvae of Aedes campestris Adapted to Hyperosmotic and Hyposmotic Media Effect of Different Fixatives on Anal Papillae of Aedes campestris Larvae A Comparison of Anal Papillae from Salt-(Aedes campestris) and Freshwater Species of Mosquito Larvae Dimensions of the Hind gut in Aedes campestris Fourth-stage Larvae in mm. Width of the Epithelium and Striated Border in the Anterior and Posterior Recta of Aedes  campestris Larvae Maintained in Normal and Hyposmotic External Media The Ultrastructure of Some Transporting Epithelia Involved in Regulation as Related to the Habitat and Regulatory Ability of Various Insects VI LIST OF PLATES Plate Page I Diagrams of the morphology and ultrastructure of 12 anal papilla and associated cell types in Aedes campestris larvae II Light micrographs of the anal papilla and associated 13 cell types in Aedes campestris larvae III Electron micrographs of the general integument on the 16 ventral surface of the anal segment IV Electron micrographs of the anal papilla cell attached 19 to a collar cell V Electron micrograph of the anal papilla cell layer of 21 a larva adapted to normal medium VI Electron micrographs of the apical region of the anal 22 papilla cell VII Electron micrographs of the basal region of the anal 23 papilla cell VIII Electron micrograph of the anal papilla cell layer of 25 a larva adapted to dilute medium IX A diagram to illustrate the organization of the hind h5 gut in Aedes campestris larvae and the morphology of the different cell types X Diagram of the morphology and ultrastructure of the k6 anterior rectum of A. campestris larva XI Diagram of the morphology and ultrastructure of the kl posterior rectum in A. campestris larva XII Diagram of the morphology and ultrastructure of the 48 rectum of Aedes aegypti larva XIII Light micrographs of the regions of the hind gut ^9 in Aedes campestris and Aedes aegypti larvae XIV Diagram summarizing the basic ultrastructural features 50 of the rectal pad (papilla) in some adult dipteran and orthopteran species XV Electron micrographs of the ileal epithelium 55 V l l Plate Page XVI Electron micrographs taken at higher magnification 57 of the ileal epithelium XVII A low-power electron micrograph of the ileum and 58 micrographs showing details of the muscle coat in the ileal region and the ileal epithelium XVIII Electron micrographs of nerves and muscles seen 60 in association with the hind gut of Aedes campestris XIX A low-power electron micrograph of the anterior 62 rectal epithelium of A. campestris larva XX Electron micrographs of the apical and basal 63 borders of the anterior rectum XXI Details of the apical lamellae in the posterior and 6b anterior rectum of A. campestris larva XXII Electron micrographs of the hind gut of A. campestris 65 larva in the region where the anterior rectum joins the junctional tissue XXIII Electron micrographs of the junctional tissue 66 XXIV Electron micrographs of the posterior rectum in 69 A. campestris larva XXV Electron micrographs of the apical and basal borders TO of the posterior rectal epithelium XXVI Electron micrographs of the apical and basal borders T l of the posterior rectal epithelium XXVII Electron micrographs of closely-apposed basal 73 borders of adjacent posterior rectal cells XXVIII Electron micrographs of assorted tracheae and a 75 tracheole XXIX Electron micrographs of the anal portion of the hind 77 gut in A. campestris larva XXX Electron micrographs of the rectum in Aedes aegypti larva 79 v i i i ACKNOWLEDGEMENTS I would l i k e t o thank Dr. J.E. P h i l l i p s f o r h i s s u p e r v i s i o n of t h i s r e s e a r c h and a s s i s t a n c e i n the p r e p a r a t i o n of t h i s t h e s i s . I would l i k e t o thank Dr. A.B. Acton f o r h i s v a l u a b l e ($20 +) and p a t i e n t c r i t i c i s m of micrographs and Mr. L a s l o Veto f o r h i s a s s i s t a n c e i n photography. I acknowledge w i t h a p p r e c i a t i o n the he l p o f Dr. G.G.E. Scudder i n c r i t i c i s m o f the manuscript and the help of Mr. David M a r t i n , Miss P a t r i c i a C o l l e n and Mrs. B r i d i e Byrne i n i t s p r e p a r a t i o n . GENERAL INTRODUCTION Apart from mammals and birds, insects are the only group of animals capable of producing hyperosmotic urine. Since they do not possess any arrangement analagous to the counter-current system of the vertebrate kidney, hyperosmotic urine must be produced in a different manner amongst insects. This mechanism of hyperosmotic urine production in both terrestrial and saltwater insects is still largely speculative. The approach used in this study was to compare the morphology and ultrastructure of the excretory systems of the larvae of two closely related mosquito species. The animals studied were Aedes campestris, a brackishwater mosquito that is able to produce hyperosmotic urine and Aedes aegypti, a freshwater species that is unable to tolerate high salinities and can produce only hyposmotic urine. In addition, A. campestris when in a dilute medium, can also produce hyposmotic urine and hence this animal may be compared under two different physiological states. It was hoped that such a comparison might indicate the particular modifications of cell structure that are required for production of hyperosmotic urine and thereby provide information on which physiological models might be proposed. More specifically, the excretory system responsible for maintain-ing a constant internal environment in mosquito larvae consists of two parts; five blind-ended Malpighian tubules lying free in the body cavity and opening into the gut at the posterior end of the midgut, and the hindgut. The Malpighian tubules produce a primary excretory fluid that is isosmotic to the blood but which has an entirely different composition. Hence the effect of tubule secretion is to alter rather than maintain the 2 internal environment (Shaw and Stobbart, 1963). The primary excretory fluid secreted by the Malpighian tubules, plus the digested material, passes through the hindgut to the rectum where selective reabsorption takes place to produce the final excreta (Stobbart and Shaw, 1964). While osmotic pressure changes do occur in the Malpighian tubule fluid in response to changes in the osmotic pressure of the surrounding fluid, the most important organ in maintaining constant hemolymph osmotic pressures and ion concentrations is the rectum (Ramsay, 1950). Part II of this study is concerned with a comparison between the recta of Aedes campestris larvae in concentrated saline and dilute media and Aedes aegypti in fresh water. In addition to the excretory system, an external modification of the integument, the anal papillae, constitute an extra-renal mechanism of ionic regulation. Since these organs had not been studied ultra-structural ly and were considered to be rudimentary in saltwater forms (Shaw and Stobbart, 1963) the opportunity to study these organs was taken. The role of the anal papillae of Aedes campestris larvae in normal and dilute media is considered in Part I. PART I THE ULTRASTRUCTURE OF THE ANAL PAPILLAE OF A BRACKISHWATER MOSQUITO LARVA, AEDES CAMPESTRIS It INTRODUCTION Both physiological and morphological studies have been carried out on the anal papillae of freshwater mosquito larvae. As early as 1933, Wigglesworth suggested that "anal gills" function in maintaining water balance rather than in respiration (Wigglesworth, 1933a, 1933b). Chloride uptake from dilute solutions via the anal papillae (Koch, 1938; Stobbart, 1967) as well as sodium, potassium (Ramsay, 1953; Treherne, 1954; Stobbart, 1960, 1965, 1967), and phosphate (Hasset and Jenkins, 1951) has been demonstrated. Active transport must be postulated for sodium (Stobbart, 1960), chloride (Stobbart, 1965) and by analogy probably potassium (Shaw and Stobbart, 1963). Morphological studies on the anal papillae of freshwater mosquito larvae have been carried out by Copeland (1964) and Sohal and Copeland (1966). These anal papillae are characterized by extensive infoldings of the apical (facing the external media) and basal (facing the blood space) plasma membranes. Numerous mitochondria are seen in close association with the apical infoldings. These specialized features are characteristic of other tissues involved in transport; e.g. the hind-gut and rectal epithelium of various insects (Berridge and Gupta, 1967; Oschman and Wall, 1969; Noirot and Noirot-Timothee, 1966a, b) and the vertebrate kidney, intestine and gall bladder, (Fawcett, 1966; Diamond and Tormey, 1966). Thus morphological as well as physiological evidence indicates that the anal papillae are actively engaged in transport. Until recently, anal papillae of saltwater mosquito larvae were thought to be rudimentary (Beadle, 1939; Ramsay, 1950; Shaw and Stobbart, 5 1963). The only physiological studies were on Aedes detritus by Beadle (1939) and Ramsay (1950). A. detritus can survive a large range of external salinities from distilled water to 10% sodium chloride (Beadle, 1939) and can produce urine hyperosmotic to the blood in sea water or hyposmotic to the blood in fresh water (Ramsay, 1950). Regulation at the environmental extremes was assumed to be solely due to the ability of the rectum to produce hypo- or hyperosmotic urine as required (Ramsay, 1950). In general then early studies provide no evidence for extrarenal regulation in any of the saltwater insects (reviewed by Stobbart and Shaw, 1964). Recent work, however, (Phillips & Meredith, 1969) has shown that anal papillae in Aedes campestris larvae function in ionic regulation. Thus this work is the first demonstration of extrarenal regulation in saltwater insects. Initial experiments established that the tolerance limits and pattern of ionic and osmotic regulation for Aedes campestris were similar to those for A. detritus. After a suitable period of adaptation, net uptake of sodium and chloride via the anal papillae of A. campestris was demonstrated from dilute external solutions (0.5 and 5 mM/l NaCl but not 0.05 mM/l NaCl). Measurements of the electropotential difference across the papilla epithelium indicated that the uptake of both sodium and chloride was by active transport. These experiments suggested that active uptake of Na+ and Cl" was turned on or induced following transfer of larvae from normal hyperosmotic medium to fresh water. Moreover, preliminary observations suggest that the direction of transport across the anal papillae may be reversed in hyperosmotic media (i.e. ions are secreted into the external medium) in a manner reminiscent of euryhaline fish $iz.e gills (Philpott and Copeland, 1963). Changes in V (Wigglesworth 1933a, 1938) and ultrastructure (Sohal and Copeland, 1966) of anal papillae in freshwater mosquito larvae have been correlated to changes in the salinity of the external medium. The present study was designed to provide independent ultrastructural evidence of the transport capacity of anal papillae in A. campestris and to determine if any ultrastructura changes could be observed in association with the physiologically demonstrated changes in transport activity in different media. 7 MATERIALS AND METHODS Larvae of Aedes campestris were collected from a small alkaline lake (GR2) near Clinton, British Columbia, during the months of May and June. They were maintained at 4°C in normal lake water which has a composition as shown in Table I. Fourth-instar larvae were acclimated for at least one week at approximately 10°C in either (a) normal lake water (0.6 osM/1) which is hyperosmotic to the insect hemolymph (0.32 osM/1), or (b) lake water diluted 100-fold with distilled water (ca. 0.01 osM/1), which is hyposmotic to the hemolymph. Light Microscopy A. campestris larvae were taken and the posterior four abdominal segments removed, fixed in Bouin's fluid or formol-sucrose (Culling, 1963), dehydrated and embedded in paraplast. In some cases the hind gut including the anal papillae was dissected out and fixed. Transverse and longitudinal sections were cut (5-7 u thick) and stained with Ehrlich's hematoxylin and eosin (Culling, 1963), chrome hematoxylin (Gomori, 1941), or Mailory's triple stain (Pantin, 1949). In addition, thick sections (0.5 - 1 p thick) of Epon-embedded material were stained with alkaline toluidine blue (Pea/se, 1964) or observed unstained using phase contrast microscopy. Electron Microscopy Initially the hind gut with anal papillae attached was dissected out, or the anal segment with the papillae was removed, and the tissues were fixed. These methods often resulted in imperfect penetration of the fixative. Better results were obtained when the papillae were fixed separately allowing fixative to penetrate both sides of the tissue. TABLE.I CHEMICAL COMPOSITION OF GR 2 LAKE WATER I 0 n Concentration mM/l Na+ 478 K+ 12 Ca + + 0.02 M g++ 0.5 CT 81 S04= 11 HCO3-, C 0 3 = 380 The pH of the lake water is 10.2 9 When anal papillae were prepared alone, the initial stages of fixation were carried out in funnels lined with filter paper. This facilitates handling of small pieces of tissue. Seven fixatives of varying osmolarity were tried. Basically, tissues were fixed 0.25 - 2 hours at room temperature in a 6% solution of glutaraldehyde (Sabatini ejt al_., 1963) buffered at pH 7.4 with Sorensen's phosphate buffer (Culling, 1963). They were then washed 15 minutes with buffer and post fixed in a 1% phosphate-buffered solution of osmium tetroxide for 15 minutes. The osmolarity of the glutaraldehyde fixative was varied by adding sucrose, making final concentrations of 1%, 5%, 10%, 14% and 15% or by adding glucose bringing the final osmolarity of the fixative to 0.35 osM (similar to the insect hemolymph). In the case of Karlsson and Schultz solution (1965), sodium chloride was added to bring the osmolarity of the fixative vehicle to 0,44 osM. The osmolarity of the osmium tetroxide fixative was not adjusted. Some tissues were fixed in a 40% solution of osmium tetroxide in carbon tetrachloride, and washed in many changes of carbon tetrachloride (Afzelius, 1959). Fixation was followed by rapid dehydration in a graded series of ethanols and finally propylene oxide. Infiltration of the embedding medium, Epon 812, was carried out over a period of 8 hours with tissues alternately being gently agitated and placed under vacuum to remove air bubbles. Final embedding was done in flat aluminum dishes. © Thin sections (800A)were cut on a LKB 'Ultrotome', picked up on uncoated copper grids, and stained with uranyl acetate (a saturated solution in 70% ethanol) for 15 minutes and lead citrate (Reynolds, 1963) for 30 minutes. Observations were made using a Hitachi HU-11A electron microscope. RESULTS Light Microscopy and General Morphology (a) Animals Acclimated to Normal Hyperosmotic Media. In the fourth-stage larvae of Aedes campestris the four anal papillae are external conical structures which surround the anus. These anal papillae are about 130 ju in length and 90 p at their widest diameter (Plate 1-1,4; II-A,B). They are formed by a single layer of cuboidal cells, 20 p in depth, enclosing the hemocoel. In the light microscope, a densely-staining, homogeneous cuticle layer can be seen on the external surface of the cells. Mitochondria appear as granules throughout the cell, especially near the basal (hemocoel) side where the intensity of staining is less. Very faint striations can sometimes be seen below the apical (cuticular) surface. Oval-shaped nuclei have a granulated appearance, and are about 13ju in diameter and 20u in length. The minimum distance between the centres of adjacent nuclei seen in cross-section is 32u and in longitudinal-section 37u. These measurements make it possible to roughly estimate the number of nuclei per anal papilla. Considering the anal papilla as a cylinder with a height of 128u and a diameter of 90u, the number of nuclei present is not more than 30. Nucleoli and lateral cell borders between nuclei were not visible. The blood space is often filled with a lightly-staining, granular substance and occasionally blood cells are present. Tracheolar cells can be seen within the hemocoel usually attached to the basal surface of the papilla cells. Cells of the anal papillae are directly attached to a short collar (22.5u in length) of slightly thinner cells (14u in thickness) that form a constriction at the base of the papilla (Plate I-l,3;II-Bl These cells have a less clearly defined Cell Types ABBREVIATIONS USED IN PLATES Ultrastructural features (Cont A axon . 9 Golgi apparatus AC anal papilla cell h hemocoel AP anal portion i infolding AR anterior rectum in inclusion BC blood cell is intercellular space CC collar cell 1 lamellae, apical G glial cell lb lateral border Gl general integument lu lumen J junctional cell m mitochondria I ileum mf area of membrane fusion M muscle cell mt microtubules M' developing muscle cell N nucleus PR posterior rectum ng neurosecretory granules TC Tracheal cell 0 osmophilic granules P particulate coat Ultrastructural features Pi protein-like inclusion pt peri trophic membrane am apical plasma membrane r endoplasmic reticulum b basement membrane sb sperical body bi basal infolds sc sub-cuticular layer bm basal plasma membrane sd septate desmosome c cuticle st striated border ch chromatin t trachea chb chromatin bands u unit membrane cm circular muscle V vacuole e external medium vs vesicle system * cytoplasmic space 12 Plate 1-1 Plate 1-2 Plate 1-3 Plate I-k Diagram to i l l u s t r a t e the relative anatomical position, extent, and dimensions of the anal papilla and associated c e l l types in longitud-ina l section (X800). Diagram of the ultrastructure of the general integument on the ventral surface of the anal segment (X5.000). Diagram of the ultrastructure of a collar c e l l (X2,l+00). Diagram of the ultrastructure of an anal papilla c e l l (X2,H00). 13 Plate II-A A light micrograph showing a longitudinal section through an anal papilla. Note the s t r i -ated border on the apical surface of the c e l l layer ( X T 5 0 ) . Plate II-B Same as Plate II-A. Note the collar cells at the base of the papilla joined to the general integument (X1,000). 14 cuticular layer and a more homogeneous cytoplasm. The collar cells are in turn attached to the general integument (1.8u in thickness) covering the ventral surface of the anal segment (Plate 1-2, II-B). Cells making up this sheet are overlaid by a cuticle of varying thickness. (b) Animals Acclimated to Dilute Hyposmotic Media. It has been reported that the anal papillae of Aedes aegypti exhibit hypertrophy or atrophy in response to osmotic pressure changes in the external environment (Wigglesworth, 1933a, 1938) and to feeding (Stobbart, 1959, 1960). As a crude indication of functional differences under two environmental conditions the length and widest diameter of anal papillae from hyposmotic media were compared to anal papillae from hyperosmotic media (Table II). The width (0.11 +0.02 mm) and length (0.20 +0.02 mm) of anal papillae from dilute (hyposmotic) media were larger than the width (0.10 + 0.01 mm.)and length (0.17 +_ 0.02 mm) of anal papillae from normal (hyperosmotic) media. These differences were not statistically significant. Total cell thick-ness and % depth of apical infolds were not compared statistically since variances in the measurements of anal papillae from the two conditions were inhomogeneous. Measurement of a larger sample size might serve to make the variances homogeneous as well as show significant differences. Electron Microscopy (a) Animal Acclimated to Normal Hyperosmotic Media. Cells of the general integument on the ventral surface of the anal segment are flattened and covered on the external surface with a cuticle layer about 0.5u thick (Plate 1-2; Plate III-A.B). On the basal side a closely applied basement membrane, 40mu thick, underlies the tissue. The TABLE II Comparison of Anal Papillae from Fourth-Stage Larvae of Aedes campestris Adapted to Hyperosmotic and Hyposmotic Media Measurement Media Mean Range Standard Deviation Standard Error No. of Animals No. of Observations Maximum width (mm.) Total length (mm} Cell thickness Depth of apical infolds as a % of total cell thickness Hyperosmotic 0.10 Hyposmotic 0.11 Hyperosmotic 0.14 Hyposmotic 0.20 Hyperosmotic 19 Hyposmotic 27 Hyperosmotic 15 Hyposmotic 32 0.07 -0.10 0.09 -0.22 0.12 -0.16 0.16 -0.22 14 - 24 18 - 36 0.01 0.02 0.02 0.02 2 8 13 - 19 2.1 22 - 47 11.0 .0 05 .0 05 ,0 06 .0 06 0.89 4.0 0.87 4.5 4 4 4 4 5 5 1 4 7 12 7 12 5 5 6 6 16 Plate III-A Transverse section through the general integument on the ventral surface of the anal segment. An electron micrograph showing smooth apical and basal membranes, and small mitochondria which occur relatively infrequently (X20,000). Plate III-B Same as Plate III-A (X17,000). cuticle varies in thickness and in favourable sections the following layers can be differentiated (terminology after Locke, 1961, 1964). On the outer surface there is a well defined membrane-like cuticulin layer (20 mu) under-lying which is a homogeneous dense layer, 40 mu, together making up the epicuticle. The discontinuous, finely-granular precipitate sometimes seen on the outside of the cuticulin layer is thought to be a contaminant rather than cement or wax layers. The bulk of the cuticle is made up of endo-cuticle (0.3ju thick) in which there are sometimes seen a variable number of oriented lamellae or fibre layers (Plate III-A). Below this and adjacent to the apical plasma membrane is a granular subcuticular layer (Schmidt's layer; O.lu thick; Plate III-A) now thought to be similar to the endocuticle but with unoriented fibres (Locke, 1961). The apical plasma membrane (14mu thick) is smooth or somewhat microvillate, but otherwise unmodified as is also the basal plasma membrane. Lateral cell borders are long and tortuous, sometimes with an intercellular space basally (Plate III-A). Towards the apical surface, lateral plasma membranes of adjacent cells are closely apposed and joined by at least one septate desmosome. The degree of dilation of the intercellular space may be dependent on the osmolarity of the fixative used. When such spaces were large the basement membrane could be seen to partially extend into them (Plate III-A). Mitochondria are small (0.2ju in diameter) and not numerous, their profiles making up about 1.5% of the total cell area seen in micrographs. Endoplasmic reticulum is found in this tissue as well as occasional randomly-oriented microtubules. Occasionally a system of small vesicles is seen (Plate III-B). The cytoplasmic ground substance is relatively dense and nuclei, being very large (4JJ in diameter^ cause an increase in cell thickness where present. The cells of the collar region, attaching the anal papilla to the general integument, differ from cells of the latter region in that they are 2-3 times thicker and have numerous oriented microtubules (Plate 1-3; Plate IV-A.B) as well as a cuticle of very irregular thickness (Plate IV-A). A cuticulin layer can sometimes be distinguished over a rather thick endo-cuticle. Embedded in this layer and often concentrated below the cuticulin layer are osmophilic granules, 25-40mu in diameter, (Plate IV-A). No lamellae are seen in the endocuticle nor can a subcuticular layer be distinguished. The apical and basal plasma membranes are relatively straight but the lateral cell borders are long and winding. All inter-cellular attachments between two collar cells, or between collar cells and the general integument or anal papillae cells, are by means of a series of septate desmosomes. Septate desmosomes consist of areas in which lateral plasma membranes run strictly parallel and are separated by a space 10 mu in diameter. Septa (11 mu thick) traverse this space every 14 m/i. Mitochondria are only moderately numerous, their profiles comprising about 9% of the cell areas in micrographs, and are subject to osmotic shock when dilute fixatives are used. Golgi bodies consisting of 3-4 stacks of parallel cisternae 120 A0 with vesicles 270 A0 in diameter at either end, are frequently found (Plate IV-B). Microtubules occur in large numbers throughout the cell and may be grouped into bundles of increased electron density. These bundles tend to be oriented perpendicular to the apical surface of the cell. Nuclei are large and finely granular with condensed chromatin throughout. Endoplasmic reticulum is abundant (Plate IV-B). Anal papillae beyond the collar region consist of one cell type An electron micrograph of a transverse section through the base of an anal papilla c e l l attached to a collar c e l l . The collar c e l l contains large numbers of microtubules and smooth apical and "basal membranes (X25,000). Same as Plate IV-A. Note the presence of Golgi bodies (X17,200). A portion of the l a t e r a l border between the collar and anal papilla c e l l seen in Plate IV-A at high-er magnification showing a septate desmosome (X125,000). with certain pronounced morphological specializations (Plate IV-A; V-A; VI-A,B.C). The cuticle is similar to that seen in the general integument. The apical membrane, however, is greatly infolded and forms a series of long parallel sheets or lamellae (Plate VI-A,B; 1-4). Each lamella (400 A0) encloses an extracellular space 70 A0 wide. Each parallel membrane has the usual unit-membrane structure of two electron-dense leaflets (30 A0 thick) separated by a space 20 A0 wide. On the cytoplasmic surface of the apical membrane there is a particulate coat about 80 A0 thick. Particles making up the coat are about 90 A0 in diameter separated by a 30-40 A0 space (Plate VI-B). Lamellae when closely packed are separated by a cytoplasmic space about 100 A0 wide. In cross-section, lamellae have predominantly sheet-like rather than cylindrical profiles (Plate VI-C). The basal membrane is also folded, the deep infoldings extending as an interconnected canalicular system into the cell to the level of the apical infoldings (Plate V-A). These basal infoldings are generally dilated and only occasionally appear to be filled with a fine precipitate (Plate VII-A,B). A basement membrane lines the hemocoel side of the anal papillae without entering the basal infoldings. No lateral cell borders are seen. Tracheal end cells about 0.6u in diameter containing tracheoles 0.5u in diameter occur frequently in anal papilla cells, reaching the base of the apical infoldings (Plate V-A). They are invested with their own basement membrane and contain many microtubules and a few small mitochondria. The anal papilla cells are rich in mitochondria, profiles occupying about 30% of the total cell area seen in micrographs. These mitochondria are dense structures about 300mu in diameter with regular parallel 21 Plate V-A An electron micrograph of a transverse section through the anal papilla c e l l layer of a larva adapted to normal, hyperosmotic medium. Note the elaboration of the apical and basal membranes, the large numbers of mitochondria, and ri c h supply of tracheoles (X13,300). 22 P l a t e VI-A The a p i c a l border of the ana l p a p i l l a c e l l seen i n P l a t e V-A at increased m a g n i f i c a t i o n showing the e l a b o r a t i o n of the a p i c a l membrane and i t s c l o s e a s s o c i a t i o n w i t h mitochondria (X21,H00). P l a t e VI-B D e t a i l of the a p i c a l l a m e l l a e showing the par-a l l e l arrangement of i n f o l d i n g s w i t h a p a r t i c u l a t e coat on the cytoplasmic surface of the membrane ( X L 7 5 . 0 0 0 ) . P l a t e VI-C An e l e c t r o n micrograph of a t a n g e n t i a l s e c t i o n through the a p i c a l r e g i o n o f an a n a l p a p i l l a c e l l . Note t h a t the l a m e l l a e have s h e e t - l i k e r a t h e r than c y l i n d r i c a l p r o f i l e s . Note a l s o the swollen m i t o c h o n d r i a as evidence of poor f i x a t i o n (X21,U00). 23 Plate VII-A An electron micrograph of a transverse section through the "basal side of the anal papilla c e l l layer. Note two effects of bad fixation; swollen, empty mitochondria and the apparent retraction spaces caused by shrinking of cytoplasm to areas immediately surrounding c e l l organelles • (X21,V00). Plate VII-B An electron micrograph of a transverse section through the basal side of an anal papilla c e l l including part of an unidentified c e l l containing large vesicles and inclusions. There i s a large spherical body in the hemocoel (X21',U00). cristae (40mu wide) separated by a space 20mu. The cristae are usually oriented perpendicular to the long axis of the mitochondria and extend across the entire width of the organelle (Plate V-A; VI-A). The papilla cell cytoplasm is relatively dense with a few profiles of endoplasmic reticulum, some microtubules, and numerous ribosomes. As in the general integument, small vesicle systems occur in anal papilla cells. Nuclei are finely granular structures with irregularly-condensed clumps of chromatin. Small vacuoles (440mu in diameter) filled with electron-opaque material, possibly lipid, occur occasionally in this tissue (Plate VIII-A). The blood space of the anal papillae may be filled with a finely granular precipitate and sometimes epithelial cells enclosing one or more tracheae occur in the hemocoel near the basal border of the papilla cells. These cells in addition to being immediately surrounded by their own basement membrane are loosely connected to the wall of the papilla by an extension of the basement membrane underlying the papilla cells. In one case the space between the wall of the anal papilla and the tracheoles was filled with a less dense, coarser precipitate than was the main hemocoel space. In the case of another papilla the hemocoel contained a large, spherical, membrane-bound body, 9.9u in diameter, filled with a dense granular material (Plate VII-B). The wall of the anal papilla was displaced and near the body was a cell containing large irregularly-shaped inclusions having a density resembling protein. In addition there were electron-opaque vacuoles, 3u in diameter, in this cell. No muscle or connective tissue cell layers were seen in association with the anal papill Anal papillae proved to be difficult tissues to fix for electron 25 Plate VTII-A An electron micrograph of a transverse section through the anal papilla c e l l layer of a larva adapted to dilute, hyposmotic medium. Note the same general organization as found in anal pap-i l l a e from larvae maintained in normal, hyper-osmotic medium (X13,300). microscopy. The foregoing description is based on results obtained from tissues fixed in a number of different fixatives. No one method gave entirely satisfactory or consistent results. Table III is a compilation of the effects of various fixatives used. One of the most common criteria for good fixation is the appearance of mitochondria. In anal papillae fixed in Karlsson and Schultz solution or in glutaraldehyde solutions containing 5% or less sucrose, artifacts often appeared ranging from swollen, empty-looking mitochondria to ruptured organelles (Plate VI-C; VII-A). Another criterion of good fixation is the appearance of cytoplasmic ground substance. It should be finely and evenly precipitated. In only one case (fixation with glutaraldehyde plus 1% sucrose) did this appear to be coarsely precipitated hence not well fixed (Plate V-A). The most variable feature is the appearance of basal infoldings and the distension of related channels. No consistent trends are discernable throughout the series of fixatives used for tissues acclimated to either hyposmotic or normal media. These features are variable among individual animals and within one anal papilla. Using fixatives of extreme osmolarity (1% or 15% sucrose added) anal papillae from larvae in hyperosmotic media exhibit distension of the basal infoldings throughout the tissue and the cytoplasm is retracted to areas immediately surrounding the cell organelles (Plate VII-A). With fixatives of intermediate osmolarity basal infoldings have various degrees of dilation in animals reared in the same medium. (b) Animals Acclimated to Hyposmotic Media. Generally anal papillae cells from larvae acclimated to hyposmotic media (Plate VIII-A) appear similar to those from hyperosmotic media (Plate V-A). No major differences were seen in the apical infoldings. The frequency, appearance and distribution of both basal infoldings and TABLE III Effect of Different Fixatives on Anal Papillae of Aedes campestris Larvae Fixative and Animals Concerned Mitochondria Cytoplasmic Ground Substance Basal Infolds Apical Infolds 0-1.2% glucose (0.35 osM.) Animals acclimated to Normal and Hyposmotic media Karlsson and Schultz Normal media only swollen, empty, often burst as above moderately dense, finely precipitated as above extensive uniform Normal infolds form spaces which are wide and empty, cytoplasm appears contracted Hyposmotic spaces not widely open extensive uniform Normal spaces not widely open 1% sucrose Normal and Hyposmotic media 5% sucrose Normal and Hyposmotic media 14-15% sucrose Normal and Hyposmotic media swollen, dense, may be burst Normal irregularly shaped, not swollen Hyposmotic swollen, dense not burst dense, not burst moderately dense coarsely precipitated moderately dense finely precipitated as above extensive uniform Normal infolds form a series of interconnected channels dilated at intervals Hyposmotic cytoplasm appears contracted leaving wide spaces filled with a fine precipitate extensive uniform Normal and Hyposmotic media infolds form a series of interconnected channels extensive Normal cytoplasm appears contracted, infolds form spaces which are wide and empty Hyposmotic infolds form a series of interconnected channels uniform /over. TABLE III (Cont'd.) * 10% sucrose - fixation very poor with little or no preservation of cell detail ** Afzelius fixative - fixation and penetration poor. Excessive hardening with the result tissues impossible to section. ro CD mitochondria were similar in larvae acclimated to both external media. DISCUSSION Fixation One of the most noticeable effects of some fixation procedures in this study was the swelling and rupturing of mitochondria. This phenomenon has been reported in most types of cell injury and is one of the first signs of necrosis (Rouiller, 1960; Trump, Goldblatt and Stowell, 1965). An attempt to achieve more rapid penetration of fixative by using a 40% solution of osmium tetroxide in carbon tetrachloride (Afzelius, 1959) failed owing to excessive hardening of the tissues and poor infiltration of the embedding media. There is evidence that swollen mitochondria are indicative of osmotic disorder within the cell (Opie, 1948; Rouiller, 1960). Sjostrand (1953, 1956) stressed the importance of osmolarity of fixatives. Maunsbach (1966) suggested that a fixative solution that is isotonic or slightly hypertonic to the tissue, is preferable to one that is hypotonic. When the osmolarity of the fixative solution was increased to 0.35 osM/1, a value slightly above the osmotic pressure of the hemolymph (0.32 osM/1) and below normal external media (0.6 osM/1) swelling artifacts were still observed. Similarly, when sodium chloride was used to increase the osmolarity of the fixative vehicle to 0.44 osM/1 (Karlsson and Schultz, 1965), mitochondria stil l ruptured. A problem arises in measuring the intra-cellular osmotic pressure (Robinson, 1961) which in the case of anal papillae of A. campestris may be different from both the internal environ-ment (hemolymph) and the external media. Hence, the adjustment of the fixative in this way does not necessarily produce a fixative isosmotic to the tissue. Since then the choice of isosmocity is somewhat arbitrary, if a series of fixatives of varying osmolarities is used and the effects of extreme hypo- and hyperosmocity are determined, then criteria of good fixation can be established and the best fixative of intermediate osmolarity employed (Caulfield, 1957). Since glutaraldehyde is believed to produce cross-links in protein, thus stabilizing cell structure (Trump and Ericsson, 1965), the osmolarity of the post-fixative solution is not considered critical, and in this study no sucrose was added to the osmium tetroxide fixative. When sucrose was added to the glutaraldehyde solutions in the following concentrations: 1%, 5%, 10% and 14% and 15%, fixation was improved using intermediate fixative osmolarities, although no one fixative gave entirely satisfactory results for all areas of the anal papillae cells under all experimental conditions (Table III). The following criteria were used as evidence of good fixation. Ideally fixed cells of anal papillae should have dense, unswollen mitochondria with cristae extending throughout the organelle, complete plasma membranes, and a uniform endoplasmic reticulum. All infolded membranes should be well preserved. The cytoplasm should be finely and evenly precipitated and, in areas near the basal infoldings, should not give the impression of being retracted to areas immediately surrounding cell organelles. Basic Ultrastructure The ultrastructure of anal papilla cells from fourth-instar larvae of Aedes campestris suggests that these cells are engaged in ion transport. The large increase in surface area produced by both apical and basal infoldings as well as the system of interconnecting canaliculi extending throughout the cell are morphological features characteristic of trans-porting cells. The large number of mitochondria and extensive tracheation support this conclusion. This same general organization is found in the rectal epithelium of A. campestris and A. aegypti larvae (Phillips and Meredith, in preparation), in the anal papillae of freshwater mosquitoes (Copeland, 1964; Sohal and Copeland, 1966), in the ileac pad cells of Cenocorixa bifida (Jarial and Scudder, 1970), and in the paunch cells of Cephalotermes rectangularis (Noirot and Noirot-Timothee, 1967). Apical membrane infoldings, usually in addition to lateral membrane infoldings, are found in the recta of several insects; Calliphora (Gupta and Berridge, 1966a,b), Cephalotermes (Noirot and Noirot-Timothee, 1966b), Periplaneta (Oschman and Wall, 1969), Schistocerca (Jarial, Irvine and Phillips, un-published observation). Apical infoldings are believed to be physiologically analogous to microvilli (Waterhouse and Wright, 1960) which are found in a wide variety of tissues: in insects, in the mid-gut, Malpighian tubules, vas deferens, oocytes, and spermatheca (Smith, 1968), and in vertebrates in such tissues as the intestine, choroid plexus, epidydimis (Fawcett, 1966), kidney (Fawcett, 1958; Schmidt-Nielsen and Davis, 1968), gall bladder Diamond and Tormey, 1966) and stomach (Ito, 1961). Basal infoldings occur in a variety of insect tissues, in the hind gut (Grimstone e_t al_., 1968; Wessing, 1967), midgut and Malpighian tubules (Smith, 1968). They also occur in the salt gland of marine birds (Doyle, 1960) and in the chloride cell of teleosts (Philpott and Copeland, 1963; Philpott, 1965). Such vertebrate epithelia as kidney tubules, submaxillary gland, choroid plexus, the ciliary body of the eye (Pease, 1956) and stomach (Ito, 1961) also possess an elaborated basal plasma membrane. All these tissues are specialized for transepithelial transport. The particulate coat seen on the outer surface of the apical plasma membrane may also play a role in transport. It is very similar to the coat found in the rectal papillae of Calliphora (Gupta and Berridge, 1966b), and it has been suggested that membrane paritcles may be concerned with supplying energy or enzymes necessary for transport. Similar coats have been found on the apical infoldings in the rectum of A. campestris and A. aegypti larvae (Meredith and Phillips, in preparation), in the hind-gut of Cephalotermes (Noirot and Noirot-Timothee, 1966b) and Periplaneta (Oschman and Wall, 1969), and Cenocorixa (Jarial and Scudder, 1970). Cytoplasmic coats occur on the lateral membranes in the rectal papillae of Aedes aegypti adults (Hopkins, 1967) and on the basal membranes in the mixed segment of the mid-gut of Cephalotermes (Noirot, Noirot-Timothee, and Kovoor, 1967a). The other cell types observed, the collar cell and epithelial cell of the general integument, are unlikely to function in epithelial transport. The apical and basal plasma membranes are unmodified. The lateral cell borders are long and tortuous with intercellular connections in the form of septate desmosomes similar to those found in Hydra (Wood, 1959; Overton, 1963). Septate desmosomes may function to give mechanical strength, provide intercellular communication, or block passive leakage between cells (Lowenstein and Kanno, 1964; Lowenstein e_t al_., 1965). There is no physiological evidence of transport across the general integument (Phillips and Meredith, 1969). The presence of bundles of microtubules in the collar cells suggests that these cells function to maintain structural rigidity and provide strong attachment for anal papillae to the general body surface. The dense osmophilic granules that are deposited in the cuticle of the collar cells could be pigment granules, possibly similar to those seen in Galena cuticle (Locke, 1961). The presence within the blood space of an anal papilla of a large spherical body and a cell containing electron-dense inclusions is unexplained. The vacuoles and dense inclusions, in addition to the smooth basal plasma membrane of the cell, suggest that it is not part of the wall of the anal papilla, but rather is a separate cell, possibly a hemocyte. There is a basal or external lamina adjacent to the basal surface of the cell that would seem to indicate that the cell is more or less stationary however. It should be noted that this basal lamina is not as thick as the basement membrane underlying the anal papilla cell (terminology after Fawcett, 1966) and could represent a mucopolysaccharide layer. Hemocytes have been known to produce such layers (Locke, 1964). Basal laminae have been found associated with sessile cells of mesenchymal origin (Fawcett, 1966). Hence if the hemocyte was non-motile such a lamina could be laid down. Crystals have been found in insect hemolymph (Jones, 1964), and in this case the spherical body may represent uric acid. Uric acid, a common component of hemolymph, which is stored as a potassium salt, precipitates on acidification (possibly by the fixative) (Stobbart and Shaw, 1964). Comparison of Anal Papillae taken from Salt- and Freshwater Mosquito Larvae Anal papillae from saline water mosquito larvae appear very similar to those from freshwater larvae. Ultrastructural details are compared in Table IV. The features of the freshwater forms are taken from the work of Wigglesworth (1933a, b, c), Copeland (1964) and Sohal and Copeland (1966). Anal papillae from A. campestris larvae are shorter, narrower and made up of thicker cells than those from freshwater mosquito species. In all these species the epithelial cells are characterized by systems of parallel lamella-like apical infoldings perpendicular to the cuticular surface, and basal infoldings which may dilate to form a series of inter-35 TABLE IV A Comparison of Anal Papillae from Salt-(Aedes campestris) and Freshwater Species of Mosquito Larvae Measurement Total length Maximum width Cell thickness Apical infoldings Length as a % of total cell thickness Thickness of one lamella Particulate coat on cytoplasmic surface of lamellae Distance between lamellae Mitochondria area of profiles as a % of total cell area seen in micrographs Average diameter of profiles Width of.cristae Distance between cristae Arrangement and extent of cristae Basal infolds distribution Aedes campestris 140 M (hyperosmotic media) 200 (hyposmotic media) 100 JJ (hyperosmotic media) 110 .M (hyposmotic media) 19 ju (hyperosmotic media) 15% (hyperosmotic media) 32% (hyposmotic media) 40 nju present 7 mu degree of dilation 300 mjj 40 mju 18 mjj parallel and perpendicular to the long axis, extending through-out the organelle throughout the cell to the level of the apical lamellae variable Freshwater Species 1 500 w 1 160 ju 8 j J 2 a > b 1 9 / i 2 a 53 nyj 2 a' b not reported 3 mu 2 10% 2 a > b 500 mu 2 a,b 30 rmj 2 a' b 23 mu 2 a , b irregular, with 2 a' b fingerprint whorls, not extending throughout the organelle throughout the cell to the level of the apical lamellae variable 2 a,b 2a,b Wigglesworth (1933a) 2 Measurements made from micrographs of aCopeland (1964) bSohal and Copeland (1966) connected channels throughout the cell. It appears that in papilla cells of saltwater larvae there are more mitochondria and that mitochondrial cristae are more densely and regularly packed than in papilla cells of freshwater larvae. Very close associations of membrane stacks and mitochon-dria as described by Copeland (1964) were not conspicuous in A. campestris. Lateral borders were not reported in freshwater species (Wigglesworth, 1933a, b, c; Copeland, 1964; Sohal and Copeland, 1966), nor were they observed in this study. Beadle (1939) noted that anal papillae from saltwater larvae had a thicker cuticle and more vacuolated appearance than did those from fresh water. These differences were not confirmed in this electon microscope examination. Ultrastructural Changes in Relation to Physiological State Anal papillae of A. campestris maintained in hyperosmotic (normal) and hyposmotic (diluted) external media were compared and no major differences found. Owing to the variability observed within one animal, among individuals, and with different fixatives, caution must be used in relating slight morphological differences to differences in physiological state as a result of acclimation to different media. Generally cells of larvae from both external media have the same organization and both appear active. Anal papillae from larvae in hyposmotic media are slightly larger than those from larvae in hyperosmotic media. Tentatively it is suggested that the cell thickness as well as the apical membrane development is greater in anal papillae from larvae in hyposmotic media than hyperosmotic media. On the basis of the number of animals measured however, these quantitative differences were not statistically significant. In a previous study, Sohal and Copeland (1966) studied ultra-structural changes in anal papillae of _A. aegypti reared under external salinities of 0.039, 0.67, and 1.07% sodium chloride. These media are hyposmotic isomotic, and hyperosmotic respectively to the hemolymph. Since A. aegypti is unable to produce a urine that is hyperosmotic to the blood (Ramsay, 1950), the hyperosmotic media exceeds the upper limit of tolerance (Wigglesowrth, 1933c). Accordingly, such dystrophic changes as reduced numbers of mitochondria and endoplasmic reticulum profiles, separation of cuticle and plasma membrane, and the presence of cytolysome-like bodies and vacuoles, were noted in these anal papillae. When anal papillae from A. aegypti reared in isosmotic and hyposmotic media were compared, two major differences were reported by Sohal and Copeland (1966). Under isosmotic conditions, apical infoldings extended less deeply into the cell and were widely separated. In addition, the numbers of mitochondria and endoplasmic reticulum profiles were reduced. These differences were not quantified nor was statistical information given. Ultrastructural changes have been related to physiologically demonstrated transport activity in other epithelia. In response to osmotic stress (Ernest and Ellis, 1969), cells of the secretory epithelium of avian salt glands increase both their surface area, by lateral and basal membrane infoldings, and the number of mitochondria. The rectal epithelial cells of Calliphora are characterized by infoldings of the lateral plasma membranes. The intercellular spaces formed by these infoldings are normally highly dilated but under conditions of minimal fluid transport eg. fasting and starved flies, they are completely collapsed. Under conditions of maximal fluid transport (flies injected with hypotonic xylose solution) the spaces become grossly distended (Berridge and Gupta, 1967). Similarly in proximal and distal tubules of various reptilian kidneys, lateral intercellular spaces are closed when no fluid transport occurs, and open when fluid transport is occurring (Schmidt-Nielsen and Davis, 1968). After a blood meal, when maximal fluid transport is occurring, cells of the rectal papillae in A. aegypti undergo the following morphological changes; intercellular spaces expand, membranes become more closely associated with mitochondria, glycogen content of the cytoplasm increases, and the apical membrane withdraws from the cuticle (Hopkins, 1967). In the teleost Fundulus heteroclitus it has been suggested that the chloride cell found in the g i l l , oral and opercular epithelia (Burns and Copeland, 1950), might have an osmoregulatory role, the excretion of salts in saltwater environments and absorption of salts from freshwater environments (Philpott and Copeland, 1963). The apical membrane of this cell develops into an apical cavity filled with an amorphous material in response to salt-water adaptation. In addition, the tubular system of agranular endoplasmic reticulum which extends throughout the cell becomes slightly distended apically and filled with amorphous material. In freshwater-adapted animals, the apical plasma membrane of the chloride cells does not develop ~ cavities but rather has coated microprojections exposed to the external media. In none of these studies are the ultrastructural changes measured quantitatively. Tormey and Diamond (1967) found that when transport was experimentally inhibited in rabbit gall bladder, lateral cell spaces collapsed. This change was measured, found to be statistically significant and the lateral cell spaces considered to be a result of fluid transport. More recently it has been found that in toad urinary bladder however, that lateral spaces will enlarge under conditions of no net transfer of water when vasopressin is added (DiBona and Civan, 1969). This suggests a greater degree of complexity than previously supposed may exist and should be considered in interpreting ultrastructural changes. Ito (1961) in studying the gastric mucosa from various vertebrates found a system of plasma membrane infoldings forming an intracellular canalicular system. So much variation was observed in the patency of this system that no correlation could be made with either the physiological state (hibernation, starvation, etc.), or the secretory cycle of HCl. Oschman and Wall (1969) could find no correlation between distension of lateral intercellular spaces (or other ultrastructural changes) and physiological state of activity in the rectum of the cockroach. In the present study, large quantitative changes (100-fold) in the concentration of the external media, were not accompanied by major changes in the ultrastructure of the anal papillae. This may indicate that the physiologically-demonstrated adaptive changes (Phillips and Meredith, 1969) require minor structural alterations such as permeability changes or activation of enzyme systems already present in the membranes. In hyposmotic media then, both ultrastructural and physiological evidence suggests that the anal papillae function to take up salts from the dilute external media. In hyperosmotic media, it is doubtful whether anal papillae are functioning as in hyposmotic media. Yet their ultrastructure suggests that the tissue is stil l very active and still involved in ion transport. It is possible that the anal papillae in hyperosmotic media are functioning to remove excess salt from the hemolymph. Such a situation would be similar to that suggested for teleost fish gills (Copeland, 1950; Philpott and Copeland, 1963). In A. campestris, one physiological observation supports this concept of transport reversal (Phillips and Meredith, 1969). If animals with and without anal papillae are reared in isosmotic Ringer's solution, then placed in hyperosmotic sodium chloride solutions (430 mM/l NaCl) and after 42 hours blood chloride levels measured, these levels are found to be significantly higher in those animals without anal papillae. If anal papillae are active in both extremes of external environ-ment then the least physiological activity could be expected when animals are maintained in isotonic Ringers. It would be interesting to look at the ultrastructure of anal papillae under this condition to determine whether there is less morphological specialization for transport. PART II THE MORPHOLOGY AND ULTRASTRUCTURE WATER (AEDES CAMPESTRIS) AND A MOSQUITO OF THE HIND GUT OF A BRACKISH-FRESHWATER (AEDES AEGYPTI) LARVA INTRODUCTION Aedes campestris larvae can tolerate a large range of external salinities from distilled water to the equivalent of 70% sea water (Phillips, unpublished results). In dilute media, water tends to enter the body primarily by drinking and secondarily by passive flux through the body wall. The animals must then eliminate water and maintain ions. Under conditions of dilute external media, A. campestris larvae produce a urine that is hyposmotic to the blood. In concentrated media the problems are reversed. The drinking rate increases and ions tend to enter the body through the gut and body wall. As in terrestrial insects the animal is now faced with eliminating excess salt while retaining water by producing a urine hyperosmotic to the blood. Larvae of Aedes aegypti, a closely related species, cannot tolerate high salinities. This is due to their inability to produce a hyperosmotic urine. In all media then, they produce hyposmotic urine (Shaw and Stobbart, 1963). In this study, the morphology and ultrastructure of the recta of A. campestris and A. aegypti were compared to determine whether the ability to produce a hyperosmotic urine could be related to any distinctive ultra-structural features present in the saltwater form but absent from the fresh-water form. In addition, the morphology of the rectum of A. campestris was compared under conditions of normal (hyperosmotic) and diluted (hyposmotic) external media to determine if any changes occurred that might be related to hyperosmotic and hyposmotic urine production. MATERIALS AND METHODS The animals and techniques used for the study of the morphology and ultrastructure of the hind-gut of A. campestris larvae were the same as those used in the study of the anal papillae, Part I. A. aegypti larvae were taken from a laboratory culture maintained in fresh water. For electron microscopy the hind-gut was dissected out and fixed as before. RESULTS Light Microscopy and General Morphology (a) Larvae of Aedes campestris acclimated to normal hyperosmotic media. The hind gut of A. campestris larvae consists of four major regions: the ileum, anterior rectum, posterior rectum, and the anal portion (Plate IX). Table V lists measurements made from live dissections of overall lengths and widths of the hind gut regions. The ileum is a long (about 1.6 mm.), narrow (0.15 mm.) region, made up of a single layer of epithelial cells 2-4 u in width with irregularly-shaped nuclei about 2 JJ by 8 u (Plate XIII-A). The luminal or apical surface of this cell layer is covered with a cuticle. Completely surrounding the epithelial cell layer is a coat of circular muscle of varying thickness 6-24 p. This coat appears to be made up of two layers; a loose lightly-staining layer 3 /J in diameter, and a compact layer 3-21 u thick. There are a few longitudinal fibres on the outside of the circular muscle. In the posterior region, the ileum joins the anterior rectum directly. At this point the hind gut widens to 0.52 mm. in overall diameter and the muscle coat becomes reduced to small circular bands 5 u in diameter (Plate XIII-B,C). These circular muscle bands are surrounded by a basement membrane which extends as a sheet between the bands over the basal surface of the cells giving the rectum a shiny appearance in live dissections. The anterior rectum extends for about 0.68 mm. and is made up of a single layer of about 100 epithelial cells about 35 JLI in width, with nuclei 14 u in diameter centrally placed in the cell (Plate X-A). The lumen is lined with a cuticle underneath which is a striated border 5-7 u in depth. Joining the anterior and posterior rectum is a short collar, 0.04 mm. long, of narrow cells, 3.6 JJ in depth (Plate A diagram to i l l u s t r a t e the organization of the hind gut in Aedes campestris larvae and the morphology of the different c e l l types as i t appears in the light microscope in longitudinal section (X50; XI,000). A diagram of a -transverse section of the anterior rectum of A_^  campestris larva as i t appears in the light microscope (XUOO). A diagram of the ultrastructure of cells making up the anterior rectum in A_^  campestris larvae (XI,000). A diagram of a transverse section of the posterior rectum of A^ campestris larva as i t appears in the light microscope (x"+00). A diagram of the ultrastructure of ce l l s making up the posterior rectum in X campestris larvae (XI,000). 1*8 P l a t e XII-A A diagram of a t r a n s v e r s e s e c t i o n of the rectum of Aedes aegy p t i l a r v a as i t appears i n the l i g h t microscope (X1,000). P l a t e XII-B A diagram of the u l t r a s t r u c t u r e of c e l l s making up the rectum of A^ aeg y p t i l a r v a e (XI.300). 1*9 Plate XIII-(A-E) Light micrographs of transverse sections of the hind gut i n Aedes campestris l a r v a ; A-ileum (X1,000), B-anterior rectum (X875), C-junctional region (X500), D-anal portion (X1,000), and E-posterior rectum (X200); and i n Aedes aegypti l a r v a , F-rectum (X700). 50 P l a t e XIV A diagram summarizing the b a s i c u l t r a s t r u c t u r a l f e a t u r e s of the r e c t a l pad ( p a p i l l a ) i n some a d u l t d i p t e r a n and orthopteran species (taken from P h i l l i p s , 1 9 7 0 ) . TABLE V Dimensions of the Hind gut in Aedes campestris Fourth-Stage Larvae in mm. Region Total Length Total Width Ileum 1.6 0-15 Anterior rectum 0.68 0.52 Posterior rectum 0.76 0.48 Anal portion 1.05 0.10 XIII-C). The nuclei of these cells are very large, oval-shaped structures 20 p by 6 p with a nucleolus and a few clumps of chromatin. There is a very narrow cuticle lining the lumen, and the cytoplasm of these cells is homogeneous. The third major region of the hind gut, the posterior rectum, is slightly thinner (0.48 mm.) and longer (0.76 mm.) than the anterior rectum. As in the anterior rectum, there are small bands of circular muscle (Plate XIII-E). The single epithelial layer making up the posterior rectum is about 62 u in diameter, and is usually thrown into a number of ridges (Plate XI-A). Nuclei, 20 p in diameter, usually lie at the base of these ridges, in the basal region of the cell. A striated border, about 36 p in depth, beneath a cuticle characterizes the luminal side. There are about 108 epithelial cells in the posterior rectum. The posterior rectum joins the anal portion of the hind gut, a narrow tube, 0.10 mm. in width, that extends to the anal opening. It is about 1.1 mm. long and is made up of a very thin, highly convoluted layer of epithelial cells, 2 p in diameter, which is surrounded by a thick coat of circular muscle (12 p thick) (Plate XIII-D). As in the ileum a few longitudinal fibers may be seen on the outside of the circular muscle coat. The lumen is lined with cuticle and nuclei of the epithelial cells and are relatively large, 8 p in diameter, and i rregularly-shaped. (b) Larvae of Aedes campestris acclimated to dilute hyposmotic media. When A. campestris larvae, acclimated to hyposmotic media are compared to those acclimated to hyperosmotic media, no qualitative differences are found in the morphology of the recta. Table VI is a comparison of measurements made on these recta. A change in the external media does not appear to greatly affect the total cell, width or the width of the striated border in either the anterior or posterior rectum TABLE VI Width of the Epithelium and Striated Border in the Anterior and Posterior Recta of Aedes campestris Larvae Maintained in Normal and Hyposmotic External Media Media Normal Region Anterior rectum Cell Width M 32.2 + 8.5 (SD) # obser. 36 Striated Border Width JU 5.5 + 1.8 (SD) # obser. 36 |" Border] Width x 100% Lcell J Hyposmotic Anterior rectum 38.9 + 7.1 (SB") # obser. 24 6.1 + 2.4 (SF) # obser. 24 17% Normal Posterior rectum 61.5 + 8.0 (SD) # obser. 31 36.8 + 7.0 (SDJ # obser. 31 60% Hyposmotic Posterior rectum 63.1 + 9.7 (SD) # obser. 13 33.8 + 8.5 (SDT # obser. 13 63% # obser. = number of observations SD = standard deviation of A. campestris larvae. (c) Larvae of Aedes aegypti reared in fresh water. The hind gut of A. aegypti consists of only three parts; an ileum, rectum, and anal portion. The first and last regions are similar to those of A. campestris. The rectum however is smaller, 0.2 mm. in diameter and 0.5 mm. long, and is not divided into anterior and posterior regions. It is made up of a single layer of epithelial cells which varies greatly in thickness (5-15 u) and is often thrown into irregular folds (Plate XIII-F). Nuclei are large, up to 14 u across, and are irregularly-shaped (Plate XII-A). As in A. campestris, the muscle coat is reduced to small circular bands. A thin cuticle covers the luminal surface and in some regions a striated border can be distinguished, extending into the cell for a depth of about 5 yj. Lateral cell borders are sometimes very apparent in the rectum, due to abrupt changes in the density of the cytoplasm of adjacent cells. There are about 320 epithelial cells in the rectum. Electron Microscopy (a) Larvae of Aedes campestris acclimated to normal hyperosmotic media. The epithelial cells of the ileum are flattened forming a narrow sheet of cells that is thrown into complex folds by a muscular coat (Plate XV-A,B). The cuticle, which covers the apical surface, has a relatively constant thickness, 0.2 ju, and consists of two layers: the epicuticle (20 mjj thick) made up of a membrane-like cuticulin layer on the surface and a homogeneous dense layer immediately underneath, and the endocuticle (180 mjj thick) in which 2 or 3 lamellae can occasionally be distinguished (Plate XV-B). A sub-cuticular layer is not observed. The apical plasma A transverse section of the i l e a l epithelium and part of the muscle coat. An electron micrograph showing overall organization of the c e l l layers especially the extensive late r a l borders in the epithelium (X7 , 8 0 0 ) . As Plate XV-A. Note the peritrophic membrane and folded nucleus (X9 . 000 ) . membrane is without specialization, as is also the basal plasma membrane (Plate XVI-A,B). The basal surface of the epithelial cells is always covered by a basement membrane, 20-30 mu in thickness, even when the cell layer is highly infolded. Lateral cell membranes are long and winding. Near the apical surface, the lateral membranes have several septate desmosomes (Plate XVI-B). Basally, the lateral plasma membranes are strictly parallel without modifications for attachment. The basement membrane does not enter the intercellular space. The nuclei of the ileal cells are large, extending almost the entire cell width, and hence are often infolded just as is the cell layer (Plate XV-B). The chromatin within the nuclei is finely and evenly precipitated. The cytoplasm of these cells shows no unusual features (Plates XV-A,B; XVI-A,B; XVII-C). There are profiles of endoplasmic reticulum, free ribosomes, and a few randomly-oriented microtubules. The mitochondria are small and dense, about 90 mu by 440 mu, with a few irregular cristae, 23 mu in width. They are not numerous, their profiles occupying only about 3% of the total cell areas seen in micrographs. Surrounding the ileum is a relatively thick coat of circular muscle (Plate XVII-B ). Each muscle bundle is surrounded by a basement membrane 30 mju thick and within the same muscle bundle myofilaments can often be seen in tangential and cross-section. A banding pattern is present in tangential sections and within the dense regions of this pattern, smooth-membraned vesicles that are probably part of the sarcoplasmic reticulum, occur. At regular intervals, the sarcolemma is deeply indented on either side of the muscle fibre to the level of the myofilaments. These indentations are lined with basement membrane and do not appear to be in register with any muscle bands. In addition 57 Plate XVI-A An electron micrograph taken at higher magnif-ication of the i l e a l epithelium showing a r e l -atively large nucleus and centrally placed nuc-leolus (X15,000). Plate XVI-B As Plate XVI-A. Note the unspecialized apical and basal plasma membranes as well as the septate desmosomes in the l a t e r a l plasma membranes (Xl8,000). 58 Plate XVII-A Plate XVII-B Plate XVII-C A detail of the muscle coat in the i l e a l region showing t i g h t l y - and loosely-packed layers of muscle fibres in A. campestris larva maintained in hyposmotic medium (X9,400). A low-power electron micrograph of a transverse section of the ileum. Note the regular indenta-tions of the sarcolema in the thick muscular coat (X4J00). A detail of the i l e a l epithelium. Note the Golgi body (X12,000). to indentations there are infoldings of the sarcolemma approximately every 1 u along its length. These infoldings are formed by a narrow pocketing of the plasma membrane. Each infolding, about 170 A0 in diameter, is rarely expanded, and is of variable length, 0.3 - 1.5 p long. The cytoplasm of the muscle bundles is sparse and organelles are confined to the outside of the muscle fibre. There are numerous small mitochondria, prominent Golgi bodies, and a few free ribosomes (Plate XV-A). No polysomes or profiles of rough endoplasmic reticulum are observed. Sometimes empty spaces occur within the cytoplasm, giving the impression that some material, possibly glycogen, has been leached out during fixation. There are both thick and thin filaments that appear in cross section to have a variable arrangement, each myosin filament being surrounded by from 6 to 8 actin filaments. In some areas there appears to be another muscle layer adjacent to the epithelium, containing only a few myofibrils in the periphery of the cell (Plate XVII-A). Small bundles of thick and thin filaments occur without transverse bands. Free ribosomes occur within the cytoplasm and sometimes within the myofilament bundles. The cytoplasm is generally sparse, containing only a few small mitochondria and some irregularly-shaped membrane fragments. No polysomes are seen. Bundles of axons are often seen near the muscle bundles or adjacent to the basal surface of the gut (Plate XVIII-A,B). The axons shown here have sparse cytoplasm filled with microtubules or neurotubules (200 A0 in diameter) and in one case a few large electron-lucid vesicles 800A° across (Plate XVIII-A). No synaptic vesicles (250-450A0, according to Smith, 1968) were observed. Glial cells surround some of the axons shown and there is also a glial cell nucleus present with coarsely but 60 Plate XVIII-A An electron micrograph showing nerves and muscles seen in association with the hind gut of A. campestris larva maintained in normal medium. Note the G l i a l cells surrounding the axon (X9,000). Plate XVIII-B As Plate XVIII-A. Neurosecretory granules and microtubules are present in two of the axon profiles (X23;000). evenly precipitated chromatin. The glial cell cytoplasm (Plate XVIII) filled with granules, possibly glycogen, and contains some dense mito-chondria. Axon profiles sometimes contain mitochondria and occasionally neurosecretory granules (73-120 mu in diameter; Plate XVIII-B). Tracheal end cells are not often seen in this region of the hind-gut and are never seen to penetrate the ileal cells. In the lumen of the ileum, the peri trophic membrane appears as a highly convoluted, dense band of granular precipitate, 60 mu thick (Plate XV-B). A less dense band, 185 mu across, lies outside the thinner band, largely filling the area between the convolutions. Finally, encircling both inner layers without infolding is a third poorly-defined band about 250 mu across having a density similar to that of the middle band In all layers there are microfibres. The next region of the hind-gut, the anterior rectum, is made up of a single epithelial layer that directly attaches to the ileum. In contrast to the ileal cells, epithelial cells of the anterior rectum are thicker (8X) and have several specialized features. The cuticle varies in thickness and ranges from 0.22-0.70 JJ, average 0.42 u (Plate XIX-A; XX-A; XXII-B; XXIII-A). A cuticulin layer (14 rmj thick) together with a homogeneous dense layer (50-120 mu thick) make up the epicuticle. Embedded in this layer are a few osmophilic granules (Plate XXII-B). The endocuticle lying below the epicuticle is fairly regular in thickness (280 mu) and has 3 or 4 lamellae. Perhaps the most distinctive feature of the anterior rectal cuticle is the presence of a homogeneous, dense, subcuticular layer beneath the endocuticle (Plate XIX-A, XX-A, XXIII-A). It has the most variable thickness of all the cuticle layers and is always present in this region. The apical membrane is modified into a 6a Plate XIX-A A low-power electron micrograph of a transverse section of the anterior rectal epithelium of A. campestris larva maintained in normal medium. Note the apical and basal infolds as well as a relatively straight, unspecialized l a t e r a l bor-der (X12 ,900) . An electron micrograph taken at higher magnification of the apical border of the anter-ior rectal epithelium. Note the subcuticular layer and condensed chromatin with a banding pattern (X9,500). An electron micrograph of the basal border of the anterior rectum. Note the basal infolding (X12.900). Plate XXI-A A detail of the apical lamellae in the posterior rectum of A. campestris larva main-tained in normal medium showing the particulate cytoplasmic coat CX150,000). Plate XXI-B A detail of the apical lamellae in the anterior rectum of Aj^ campestris larva main-tained in dilute medium. Note the particulate coat (X75.000). An e l e c t r o n micrograph of a t r a n s v e r s e s e c t i o n of the h i n d gut of ah A. Campestris l a r v a maintained i n normal medium i n the r e g i o n where the a n t e r i o r rectum j o i n s the j u n c t i o n a l t i s s u e . The e p i t h e l i a l c e l l l a y e r i s h i g h l y f o l d e d at t h i s p o i n t and the j u n c t i o n a l t i s s u e appears t o surround a p o r t i o n of the a n t e r i o r r e c t a l e p i t h e l i u m (X12 , 9 0 0 ) . As P l a t e XXII-A. Note the winding l a t e r a l bor-ders j o i n i n g the a n t e r i o r r e c t a l and j u n c t i o n a l e p i t h e l i a (X12 . 9 0 0 ) . Plate XXIII~A An electron micrograph of the hind <rat showing junctional tissue and anterior rectum. Note the differences in cuticle thickness, apical membrane elaboration and cytoplasmic density (X12,900). Plate XXIII-B An electron micrograph of a transverse section of junctional tissue. In this micrograph the junc-tional tissue appears to surround a basal portion of the posterior rectal epithelium CX12,900) 67 series of lamellae (Plate XX-A; XXIII-A) similar to those seen in the anal papillae (Part I). These lamellae occupy 17-19% of the total cell depth. Each lamella (Plate XXI-B) has the same basic structure as described for the lamellae in anal papillae. In the anterior rectal cells, the extra-cellular space enclosed in each infolding is slightly less than in the anal papillae (40 A° as compared to 70 A°) and the particulate coat is somewhat thicker (100 A* as compared to 80 A*). Lamellae are therefore 400 A* in thickness and are separated by a cytoplasmic space at least 70 A0 wide. The apical infoldings form the striated border seen in the light microscope. Although mitochondria occasionally occur between the infoldings, associations between mitochondria and lamellae are not conspicuous. The basal plasma membrane is very highly infolded to form a complex interconnecting canalicular system (Plate XIX-A, XX-B, XXII-A) extending throughout the cell to the level of the apical lamellae just as in the anal papillae(Part I). The degree of dilation of this system is variable. A basement membrane, 40 )i covers the basal surface of the epithelial cells and may extend into the basal infoldings (Plate XIX-A). Lateral plasma membranes are relatively unmodified with septate desmosomes in the apical region. In the basal region the lateral membranes have a few dilations. Mitochondria are dense oval structures often oriented with their long axes perpendicular to the apical cell surface. They are about 600 mu by 100 my in cross section with regular parallel cristae (19 mu thick separated by a space 3 mu in width) extending throughout the organelle generally at right angles to the long axis. Mitochondria in the anterior rectum comprise about 22% of the total cell area seen in micrographs and show no preferential distribution in the cytoplasm. They are sensitive to osmotic shock and may appear swollen and empty (Plate XX-A). Nuclei have chromatin precipitated in dense clumps with some evidence of a banding pattern in the larger clumps (Plate XX-A). The cytoplasm exhibits no unusual features. There are a few profiles of endoplasmic reticulum and some randomly-oriented microtubules. A diagram of the main ultra-structural features of the anterior rectum is shown in Plate X-B. Cells of the anterior rectum occur very close to the cells of the posterior rectum. However, at no time are these cells directly attached. Rather both the anterior and posterior rectum are attached to a collar made up of 2 or 3 relatively unspecialized cells (Plate XXII-A; XXIII-A,B). The attachments are by long winding lateral cell borders having a series of septate desmosomes (Plate XXII-B). The cells of the junctional tissue have a cuticle on the luminal side that is considerably thinner (0.17 u) than that found in the anterior rectum. There is no subcuticular layer. The apical plasma membrane is straight and the basal membrane only slightly infolded. The cytoplasm is very dense with microtubules and much endoplasmic reticulum. Nuclei are large with finely precipitated chromatin. There are only a few small mitochondria. Sometimes in the lumen of the gut in this region the remnants of the peritrophic membrane can be seen (Plate XXIII-B). The posterior rectum is similar to the anterior rectum in general organization (Plate XXIV-C; XXV-A.B). The cuticle covering the apical surface is usually slightly thinner than in the anterior rectum and is less variable in thickness (0.36 ju thick). A distinct subcuticular layer is not always apparent, nor are lamellae within the endocuticle always discernable. The apical plasma membrane is again elaborated into lamellae that are much more extensive than those occurring in the anterior rectum or anal papillae (Plate XXIV-B.C; XXV-A, XXVI-A). 69 Plate XXIV-A An electron micrograph showing the basal border of the posterior rectum and the pos-i t i o n of the nucleus in A. campestris larva maintained in normal medium. A muscle, bundle with a large nucleus l i e s close to the basal "border (X5',000). Plate XXIV-B An electron micrograph showing a narrow region of the posterior rectal epithelium in which the apical lamellae occupy the entire c e l l depth (X3.300). Plate XXIV-C A survey electron micrograph of the posterior rectal epithelium. Note the very extensive elab-oration of the apical plasma membrane into lamellae, the relatively straight l a t e r a l plasma membrane, and the preferential distribution of mitochondria (X2,400) . Plate XXV-A An electron micrograph of the apical border of the posterior rectal epithelium. Note the very extensive elaboration of the apical plasma membrane into lamellae and the relatively straight l a t e r a l border (X10 ,200) . Plate XXV-B An electron micrograph of the basal border of the posterior rectum. Note the basal infolds and the associated nerve axon ( X l l , 2 0 0 ) . Plate XXVI-A As Plate XXV-A (X5,000). Plate XXVI-B As Plate XXV-B. Note the bundles of tig h t l y -packed microtubules Cx6,ooo). Plate XXVI-C A portion of a microtubule-bundle in Plate XXVI-B seen at higher magnification (X2U,000). The lamellae of the posterior rectum are slightly thicker than other apical lamellae (450 A°) (Plate XXI-A) and are often more tightly packed, the least cytoplasmic space between lamellae being about 40 A°. The O particulate coat is slightly thicker (130 A ) but particles making up the coat are much the same size and occur at the same spacing as previously e described). Lamellae enclose an extracellular space about 25 A wide. In some areas these lamellae extend throughout the entire cell to the basal border (Plate XXIV-B). The basal plasma membrance is infolded as in the anterior rectum, but the canalicular system thus formed is less extensive in the posterior rectum (Plate XXV-B). A basement membrane, 30 mu thick, covers the basal surface and may enter the basal infolds for a short distance. Lateral cell borders are as previously described and are relatively straight (Plate XXIV-C, XXV-A, XXVI-A). The posterior rectal epithelium is infolded into a variable number of ridges usually about 5. The effect of these infolds is to bring part of the basal borders of adjacent cells into close apposition (Plate XXVT-B, XXVII-A,B). Thus there is a narrow channel formed that is lined with basement membrane. Basal infolds open into this channel along its length and towards the luminal end, give the appearance of radiating outwards from the closed end of the channel. Nuclei are irregular in shape and usually lie at the base of the indentations (Plate XXIV-A,C). Chromatin is coarsely precipitated without showing a banding pattern as in the anterior rectum. All areas of the cytoplasm contain numerous dense mitochondria but these are pre-dominantly concentrated in the regions of apical infoldings (Plate XXIV-C, XXVI-A). They occupy about 24% of the total cytoplasmic area seen in micrographs. Their average diameter is about 320 mu with a range from 200-450 nju. In the apical region of the cell mitochondria are elongated Plate XXVTI^A An electron micrograph showing two closely-apposed "basal borders of adjacent posterior rectal c e l l s . Note the retention of the basement membrane on the surface of both basal borders (30.1,000). Plate XXVII-B As Plate XXVII-A. Note the penetration of tracheae between the cells and the radiation basal infolds from the apex of the fold in the c e l l layer (XT,000). structures up to 2.9 u in length, lying between, and in close association with, the apical lamellae. Cristae about 15 mu thick, separated by about 5 mu, extend throughout the mitochondria, not always regularly oriented, so that within the same organelle, longitudinal, tangential and cross-sections of cristae can often be seen (Plate XXV-B). In cross-section cristae are sometimes circular, indicating that they are finger-like projections of the inner mitochondrial membrane. The cytoplasm of posterior rectal cells is dense, containing relatively large amounts of endoplasmic reticulum and free ribosomes, as well as some randomly-oriented micro-tubules. In the basal region of the cell there are areas of closely packed parallel microtubules. These microtubule bundles usually lie oriented in a basal-apical direction (Plate XXVI-B.C). Plate XI-B is a diagram of the main ultrastructural features of the posterior rectum. Tracheae and tracheoles are commonly found in association with the anterior and posterior rectum (Plate XXVIII-A-D). Tracheae are large (greater than lu in diameter), cuticle-lined tubes surrounded by epithelial cells (Plate XXVIII-A). The cuticular lining is thrown into helical folds or taenidia (70 mju in diameter) within which runs a taenidial thread. The cuticular layer is made up mainly of a cuticulin layer. Tracheae are found close to the basal border of the rectal cells, between muscle bands and rectal cells (Plate XXVUI-D), or penetrating between rectal cells (Plate XXVIII-C). The tracheal cells contain large numbers of microtubules running parallel to the tracheae, a few small mitochondria, and profiles of smooth endoplasmic reticulum. The plasma membrane is smooth and surrounded by a basement membrane 40-50 mu thick. The plasma membrane of the rectal cell surrounding the tracheolar cell may be infolded. Tracheoles (about 0.3u in diameter) occur within the 75 Plate XXVTII-(A-D) Electron micrographs of assorted tracheae; A-(X10 ,800) , C~(X2,li00), and D-(X3 , 700 ) , and tracheole; B-(X25,200). rectal tissue (Plate XXVIII-B) either between the epithelial cells or as ihpushings into the cells. They are always surrounded by tracheal end cells which are in turn invested with a basement membrane hence are essentially extracellular. Tracheoles may have fine taenidia 60 mjj in depth similar in structure to those of the tracheae, or they may have no annulations, in which case the cuticular lining of the tube consists only of a cuticulin layer 30 rmj thick. Tracheal end cells are similar to cells surrounding tracheae in that they have oriented microtubules and a few small mitochondria. The posterior rectum joins the anal portion of the hind gut directly (Plate XXIX-A). In this region at the apical side of the tissue there are many finger-like projections of cuticle usually fil l e d with narrow projections of cytoplasm. Together with major infolding of the entire cell layer these projections may form a sphincter. Cells of the anal portion have relatively dense cytoplasm and contain much endoplasmic reticulum, Golgi bodies and commonly myelin figures (Plate XXIX-B). With the exception of these features they are identical to cells of the ileum. Mitochondria comprise about 6% of the total cell area seen in micrographs. No tracheae or tracheoles were seen in association with this region. Like the ileum, the anal portion is surrounded by a thick circular muscle coat. (b) Larvae of Aedes campestris acclimated to dilute hyposmotic media. No qualitative differences were found between larvae acclimated to normal and dilute media. A survey of a limited number of sections did not suggest large quantitative differences worth statistical comparison. TT Plate XXIX-A An electron micrograph of a transverse section of the hind-gut of A. campestris larva maintained in normal medium. Section cut at the level of the join between the posterior rectum and anal portion. The epithelial layer is high-l y folded at this point forming projections that almost occlude the lumen (XT,500). Plate XXIX-B An electron micrograph of a transverse section of the anal portion of the hind-gut of A. campes- t r i s larva maintained in normal medium. Note the unspecialized apical and basal plasma mem-branes (XT,500). (c) Larvae of Aedes aegypti reared in fresh water. The ultrastructural organization of cells in the rectum of Aedes  aegypti is similar to that in the anterior rectum of A. campestris larvae (Plate XXX-A,B). The cuticle covering the apical side is made up of the usual layers, epicuticle (90 nyj thick) and endocuticle (75-220 mjj thick). A subcuticular layer is sometimes present. The apical membrane is elaborated as before, but lamellae are considerably farther apart and e not as regular. Even when tightly packed, lamellae are 190 A apart and may be as much as 230 mu apart. They enclose an extracellular space about 35 A thick. A particulate coat 70 A thick covers the cytoplasmic surface 0 of the apical membrane. Thus each lamella is about 335 A thick. The basal plasma membrane is infolded to about 30% of the total cell depth. Both the extent and the degree of dilation of the basal infolds are variable however. The infolded basal plasma membrane is covered on the external surface with a basement membrane 30 mp thick. Lateral membranes are relatively straight with septate desmosomes apically. There are a few free ribosomes, profiles of endoplasmic reticulum and a few unoriented microtubules. Tracheoles 0.25u in diameter are seen closely apposed to the basal surface of the rectum but have not been seen within the epithelial tissue. Mitochondria comprise about 13% of the cell area seen in micrographs and are dense oval structures about 200 mp across, up to 970 mu long, with parallel cristae 19 mu thick separated by a space of 10 mjj. Cristae have no constant orientation among organelles. The lumen of the rectum is filled with poorly preserved oval bodies 200 mjj by 700 mjj in size. The epithelium of A. aegypti is thrown into folds by circular muscle bands and, as in the posterior rectum of A. campestris larvae, the basal borders of adjacent cells are closely apposed forming what appears to be a central canal. Plate XII-B is a diagram of the main ultra-An electron micrograph of a transverse section of the rectum of Aedes aegypti larva reared in freshwater. Note the apical and basal infoldings (X13,300), As Plate XXX-B. Note the relatively straight l a t e r a l membrane as well as the closely-apposed basal borders of two adjacent c e l l s (X13,300). structural features of the rectum of Aedes aegypti. DISCUSSION (a) The morphology of the ileum and anal portion of the hind gut and associated tissues in Aedes campestris larvae. Both the ileum and anal portion of the hind gut consist of relatively unspecialized epithelium. Apical and basal membranes are straight, there are few mitochondria, and the tracheation is not extensive, thus suggesting l i t t l e or no reabsorptive function. There is however in both regions a thick muscular coat surrounding the gut, suggesting that the principal function of these regions is the movement of excreted material. At the anterior end of the anal portion, the epithelium is modified into a valve-like structure which probably serves to open and close the rectum, thus controlling the time during which reabsorption can occur. The peritrophic membrane in the lumen of the ileum is a complex structure made up of three separate layers. Initial observations on the peritrophic membrane showed that i t is usually made up of a few fibri l s more or less regularly arranged, while chemically i t resembles inner cuticle layers and is made up of protein and chitin (Mercer and Day, 1952). Richards and Richards (1969) found that in the larvae of A. aegypti the fully differentiated peritrophic membrane consists of three or four fibrous layers separated by granular material. In Calliphora the peritrophic membrane is a similarly complex structure with three very distinct layers (Smith, 1968). It is most probable that the junctional tissue merely provides a strong but flexible attachment between the anterior and the posterior rectum. Although the cell layer is thin i t is joined to the rectal epithelium by septate desmosomes for a considerable distance along the lateral membrane. The circular and longitudinal muscles observed in this study present no unusual features. As in all insect muscle they are striated and probably in vivo contain glycogen. In some cases i t appears that glycogen has been removed during fixation. Each myosin filament is surrounded by 6 - 8 actin filaments. The increase of actin to myosin filament ratio as well as a somewhat irregular array is reported to be associated with slower muscle activity (Smith, 1968). The sparse tracheation supports this conclusion. The muscle fibre shown in Plate VI is probably not fully developed. Spiro and Hagopian (1967) report that in early stages of muscle development there is an aggregation of thick and thin myofilaments into small nonstriated bundles. In addition, free ribosomes and polysomes are present throughout the cytoplasm. Although no polysomes are seen in this muscle fibre, small filament bundles as well as free ribosomes are present. Nerve bundles seen in association with the hind gut appear to be similar to other insect nerves (Smith and Treherne, 1963). Most axons seen are probably motor neurons inervating the nearby muscle bundles. Occasionally electron-dense granules 730-1200 A in diameter are seen in axons. They are similar to neurosecretory granules (800 -1100 A) in Periplaneta (Smith and Treherne, 1963). No axon terminals were seen in the rectal epithelium of A. campestris and there is nothing known about hormonal control of excretion in this insect. The involvement in such control of neurosecretory granules seen adjacent to the rectal epithelium of other insects had been previously suggested (Maddrell, 1962; Delphin, 1963; Wall and Ralph, 1962). (b) The morphology and ultrastructure of the rectum of salt- and freshwater mosquito larvae. The ultrastructure of the rectum in A. campestris and A. aegypti suggests that this organ functions in ion and water transport, supporting the physiological evidence. The general organization of the rectal epithelium is similar to that of the anal papillae (Part I). The extensive infolding of the apical and basal membranes, the straight lateral membranes joined by septate desmosomes on the apical side, the particulate coat on the apical membrane, the relatively large numbers of mitochondria and extensive tracheation are all features associated with transporting tissues (see Part I for references). Table VII is a comparison of some of these features in various insect tissues. An unusual feature of the posterior rectal cells is the presence of bundles of microtubules in the basal area. Such bundles are not found in the anterior rectum. Since cells making up the posterior rectum are relatively large and subject to peristaltic contractions i t may be that the microtubules serve to maintain structural rigidity in the epithelial cells. Bundles of microtubules have been described in the rectal epithelium of Tenebrio (Grimstone et al_., 1968). Bundles occur near the lateral cell border and are oriented parallel to i t . Microtubules are not tightly packed in these bundles however, and do not appear similar to those in A. campestris. Microtubule bundles are also characteristic of rectal cells in the wood louse Oniscus ascellus (Witkus, Grillo and Smith, 1969). In this case the microtubules are tightly packed and run parallel to the long axis of the cell. It is suggested that they function to maintain structural support in an unusually large cell and in the alignment of cell organelles. Large numbers of microtubules are observed in other rectal cells but they are not arranged in bundles. In this study, the rectum of A. campestris is found to have two regions. Information obtained at an ultrastructural level, indicates that the general TABLE VII The Ultrastructure of Some Transporting Epithelia Involved in Regulation as as Related to the Habitat and Regulatory Ability of Various Insects Reference Insect Habitat Regulatory Transporting Apical Basal Lateral ability tissue membranes membranes membranes Copeland (1964) This study Culex quinque fasciatus larvae Aedes campestris larvae fresh hyporegulation anal water papillae fresh hyper- or anal and hyporegulation papillae brackish water infolded extensively into infolded lamellae as above* as above Not reported Not seen This study Aedes campestris larvae as above as above anterior rectal epithelium as above* as above septate desmosomes apically, slight dilations basally, not infolded, no conspicuous associations with mitochondria This study This study Aedes campestris larvae Aedes aegypti larvae as above as above fresh water hyposmotic urine only posterior rectal epithel ium rectal epithelium very infolded, extensively borders of infolded into adjacent cells as above lamellae * infolded into lamellae * may become closely apposed by folding of the cell layer as above as above Jarial & Scudder (1970) Cenocorixa fresh hyposmotic bifida adult water urine only ileac pad cel Is as above * infolded present 00 -fr-/over TABLE VII (Cont'd.) Reference Insect Habitat Regulatory ability Transporting tissue Apical membranes Basal membranes Lateral membranes Oschman and Wall (1969) Hopkins (1967) Gupta and Berridge (1966a,b) Jarial, Irvine and Phillips (in prep-aration) Periplaneta amencana adult terrest-rial hyperosmoti c uri ne rectal pads Aedes aegvjDti adult Cal1i phora  erythrocephala adult as above as above as above as above rectal papillae as above Schistocerca as above as above gregaria adult rectal pads infolded into lamellae* straight septate desmosomes apically and basally, extensively infolded (close associations with mitochondria) intercellular spaces thus formed communicate with spaces surrounding tracheae which in turn open into the hemocoel at restricted points irregularly infolded into microvilli infolded into lamellae* relatively straight straight infolded into lamellae as above except inter-cellular spaces open into a central canal at a restricted point near papilla tip-* as above except inter-cellular spaces enter an infundibular space (between cortical and medullary cells) which in turn communicates with the hemocoel via a one-way valve straight as above except inter-cellular spaces open into a subepithelial space (between primary and secondary cells) which in turn communicate with the hemocoel where tracheae penetrate the,^ secondary cell layer ^ TABLE VII (Cont 'd.) Reference Insect Habitat Regulatory Transport- Apical Basal Lateral a b i l i t y ing t issue membranes membranes membranes Noirot and Cephalotermes ter res t -Noirot- rectangularis r i a l Timothee adult (1966b) hyperosmotic urine rectal papi l lae i n f o l d e d i n t o lamellae not not' reported reported Bacetti Aiolopus as as above (1962) strepans above Latr\ adult Wessing Drosophila as as above (1967) melanogaster above adult as above as above win folded into lamellae i r r e gu l a r l y infolded into m i c r o v i l l i s t ra ight extensively infolded forming i n t e r c e l l u l a r channels associated with mitochondria extensively as above in fo lded, c lo se l y associated with mitochondria * Indicates the presence of a cytoplasmic coat CO organization of the two regions is similar and that they differ mainly in the overall cell thickness and in the extent of apical and, to a lesser degree, basal infoldings (Table VIII). Lateral cell borders are present between the cells but are obscured at a light microscope level by membrane infoldings. Only one type of rectal epithelium is found in A. aegypti, the general organization of which is again similar to that of A. campestris. Using measurements of the depth of apical infoldings relative to total cell depth to compare anterior and posterior rectal tissues from A. campestris larvae with rectal tissue from A. aegypti (Table VIII), i t is concluded that the rectal epithelium of the freshwater mosquito larva is similar to the anterior rectal epithelium of the saltwater mosquito. This suggests therefore that the posterior rectum is unique to A. campestris larvae and hence possibly associated with the ability to produce hyperosmotic urine. Ramsay (1950) was able to distinguish by light microscope two regions in the rectum of Aedes detritus larvae, another saltwater mosquito species. The anterior region was found to be made up of a thin layer of cells bearing striations in the basal region. Nuclei were pushed to the extreme basal edge of the cells. Cells of the posterior rectum were thicker and had a distinct striated border on the apical side. Nuclei were centrally placed. No lateral cell borders were seen. The rectum of A. campestris appears to closely resemble the rectum of A. detritus and the relatively greater extent of basal infoldings seen in the anterior rectum of A. campestris at an ultrastructural level may account for Ramsay's observation of a striated border in this region in A. detritus. In the same study, Ramsay found that the rectum of A. aegypti consisted of only one region having a striated border on the luminal side. On the basis of TABLE VIII A Comparison of Rectal Epithelia from Aedes campestris and Aedes aegypti Animal and Tissue Cell depth M Apical Lamel1ae depth ju % Lamellae depth % Mitochondria ** A. campestris anterior rectum (66 observations) 35 + 9 SD 6 + 2 SD 18 + 7 SD 22 A. campestris posterior rectum (59 observations) 62+8 SD 36 + 8 SD 61 + 13 SD 24 A. aegypti rectum 18 15 - 30 13 Percentages independently determined (i.e. not from values given for cell and lamellae depth) ** The area occupied by profiles of mitochondria expressed as a percentage of total cytoplasmic areas seen in electron micrographs SD = standard deviation his light microscope observations, Ramsay concluded that this rectal epithelium most closely resembled the posterior rectum of A. detritus. (c) The mechanism of hyperosmotic urine production. Ramsay (1950) showed that in the larvae of A. detritus in sea water, osmotic work resulting in the formation of hyperosmotic urine occurs in the rectum. Thus the hemolymph is maintained hyposmotic to the surrounding medium. In dilute media, salts are reabsorbed from the rectum against large concentration gradients and excess water is eliminated to maintain the hemolymph hyperosmotic to the medium. It has been established that the general pattern of osmotic and ionic regulation in A. campestris is very similar to that in A. detritus (Phillips and Meredith, 1969). The mechanism of hyperosmotic urine production in saltwater insects is unknown. The process has been studied in detail in some terrestrial insects such as Schistocerca, Calliphora and Periplaneta. In Calliphora (Gupta and Berridge, 1966a, b; Berridge and Gupta, 1968) the rectal pouch is a complex structure consisting of four rectal papillae that project into the lumen. They are joined by junctional cells to a thin layer of relatively unspecialized rectal epithelial cells. Rectal papillae are made up of a single layer of cortical cells covering a medulla. The cortical cells are larger with infolded lateral plasma membranes that form a complex series of intercellular channels often closely associated with mitochondria. At the apical and basal surfaces of the cortical cells the lateral plasma membranes are tightly joined by septate desmosomes. The intercellular channels are therefore largely shut off from the lumen and the hemocoel. They communicate with the space between the cortex and medulla only at the tip of the papillae. This space in turn opens into the hemocoel only at the base of the papilla via a one-way valve (Gupta and Berridge, 1966a). Rectal pads of the desert locust Schistocerca exhibit a comparable structure. Two types of cells are present; primary cells which make up the rectal pad and secondary cells which form a cell layer lying on the hemocoel side of the pad. Lateral membranes of the primary cells are joined apically and basally by septate desmosomes. Within the pad cells, they form interdigitations each containing a mitochondrion and are in some regions dilated to form intercellular channels. These channels communicate with larger spaces containing tracheoles surrounding basement membranes and thus eventually with the space between primary and secondary cells. This space in turn communicates through a limited number of channels with the hemocoel. The secondary cells of the locust, unlike the medulla cells of the blowfly, have a specialized system of apical infolds often associated with mitochondria (Jarial, Irvine and Phillips, in preparation). The rectum of the cockroach (Oschman and Wall, 1969) exhibits an identical arrangement to the locust rectum, with the exception that there are no secondary cells present. The organization of epithelial cells in the recta of typical terrestrial, freshwater and saltwater insects can be seen by comparing Plates XIV, XII-B, XI-B (Plate XIV taken from Phillips, 1970). No information concerning which tissues in Aedes campestris larvae might be functioning to produce hyperosmotic urine in normal media (but non-functional in dilute media) can be gained from a comparison of ultra-structure in different physiological states since no major ultrastructural changes can be observed. A comparison of the recta of salt-and freshwater forms of Aedes however, suggest that the posterior rectal tissue is unique to A. campestris and might therefore be associated with the ability to produce hyperosmotic urine. A. aegypti larvae possess only one type of rectal tissue (similar to the anterior rectal tissue of A. campestris) and do not have the ability to produce hyperosmotic urine. The ultra-structure of the posterior rectum of A. campestris differs in several aspects from that of the recta of terrestrial insects previously studied: (a) There is a much greater development of the apical membrane into lamellae. The apical lamellae represent the major membrane infoldings. (b) The lateral membranes are relatively straight and closed by septate desmosomes at the apical end only. In terrestrial forms the lateral membranes form the major component of membrane infoldings. (c) Most of the mitochondria are associated with the apical rather than the lateral membranes, (d) Extensive infolding of the basal membrane (forming an intracellular canalicular system) seen in A. campestris is not generally observed in terrestrial forms except Drosophila (Wessing, 1967). (e) As in the cockroach there is only a single layer of epithelium, but in A. campestris this is only of one cell type in the posterior region in contrast to two-four types seen in terrestrial insect recta. Two models of fluid transport, the double membrane model (Curran, 1960; Patalk et aj_., 1963), and the local osmosis hypothesis (Diamond, 1965, 1968) may be applicable to terrestrial insect recta i f the models are modified to allow for solute recycling (Berridge and Gupta, 1966a; Oschman and Wall, 1969; Phillips, 1969, 1970). The double membrane model supposes that the cell has two membranes of different reflection coefficients or permeabilities. One membrane is selective and highly permeable to water, but not to solutes, while the other is nonselective and highly permeable to water and solute. If then there is a solute pump maintaining the interior of the cell at a high osmotic pressure, water will diffuse into the cell causing a hydrostatic pressure which in turn causes water and solute to flow out across the nonselective membrane. The solute is then reabsorbed by a solute pump. The local osmosis model (Diamond, 1965, 1968) requires ion transport into long narrow restricted channels. Water that follows by osmosis then creates a hydrostatic pressure and causes fluid to flow down the channels. If the solute is then reabsorbed across a second water-impermeable membrane, water could be moved across the epithelial layer as a whole against a concentration gradient, without proportional solute movement. The double membrane model could be applied to A. campestris. The apical membrane could be selective, and i f a solute pump were located on the nonselective basal or lateral membranes, then the osmotic pressure within the cell could be maintained at a higher level than in the lumen. Water would be drawn in from the lumen causing a high hydrostatic pressure within the cell. Laminar flow of fluid would result through the non-selective membrane in the basal infolds through the basal canalicular system and into the hemocoel. Solutes could be recycled by the solute pump. However, the disposition of a mitochondria does not favour this model. Alternatively, a solute pump could be located on a nonselective apical membrane to maintain a high osmotic pressure in the subintimal space. The concentration of mitochondria at the apical border suggests this is the site of osmotic work. Water would then be drawn in from the lumen across a selectively permeable intima or cuticle and forced by hydrostatic pressure across the apical membrane. Diamond's model (as modified by Phillips, 1969; Osman and Wall, 1969) requires long narrow restricted channels which in terrestrial insects 'could be the spaces between the lateral membranes that communicate with fi r s t the spaces around the tracheae, then with the space between the primary and secondary (or medullary cells) and finally with the hemocoel where tracheae enter the rectal pads. Lateral membrane elaboration in close association with mitochondria appears to be characteristic of terrestrial insects (Table VII). In A. campestris such a complex system of lateral channels does not exist. The space formed between closely apposed basal membranes as the posterior rectal epithelium is folded into ridges, may constitute a long channel. It is not known whether these ridges and channels are constant features of the posterior rectum. They could however function by secreting solute in at the luminal side of the channel. Water would then follow by osmosis, fluid would flow towards the hemocoel and i f solutes were reabsorbed at the basal end of the channels, hyperosmotic urine would result. Only about 30% of the total basal border forms long channels and only about 40% of the basal infolds open into these channels, while the remainder open directly into the hemocoel. These channels are not unique to A. campestris but have also been seen in A. aegypti. Thus they are equally well developed in a species which cannot produce hyperosmotic urine. The cytoplasmic spaces between apical lamellae might represent comparable channels. If solutes were pumped into the apical end of the spaces causing water to follow, and then secreted through a water-impermeable membrane at the basal end, hyperosmotic urine could result. It has always been assumed that saltwater insects produce hyperosmotic urine in the same way as do terrestrial insects (Stobbart and Shaw, 1964) although this has never been experimentally verified. On the basis of the present ultrastructural observation there is reason to believe that the process of hyperosmotic urine formation might be basically different in saltwater insects. Unlike terrestrial forms, these insects have no need to conserve water but only to eliminate salts. Therefore water could be obtained with salts by drinking and the posterior rectum might simply secrete salts into the lumen through a water-impermeable cell layer. In support of this suggestion, i t might be noted that the organization of the rectal epithelium of adult mosquitoes is different from that of the aquatic larval form and corresponds to the organization found in other terrestrial insects. The mechanism of hyperosmotic urine production cannot be determined from the result of this study. The posterior rectal epithelium with very extensive apical infoldings is present in the brackish water species but absent in the freshwater species of mosquito larvae and may be responsible for hyperosmotic urine production. This urine production does not require gross structural changes in the rectum since no ultrastructural differences were found between the recta of A. campestris adapted to fresh (producing hyposmotic urine) and brackish water (producing hyperosmotic urine). The absence of infolded lateral membranes associated with mitochondria and forming long intercellular channels with restricted exits (a feature characteristic of the terrestrial insect recta that have been studied) suggests that the mechanism of hyperosmotic urine production may be basically different in terrestrial and brackish water insects. SUMMARY 1. The basic morphology and ultrastructure of anal papillae of Aedes  campestris larvae adapted to normal and dilute media have been described. 2. Anal papillae of A. campestris larvae appear to be active and are characterized by several features: (a) extensive infolding of the apical membrane into lamellae, (b) a particulate coat on the cytoplasmic surface of the apical lamellae, (c) extensive infolding of the basal membrane into a canalicular system throughout the ce l l , and (d) relatively large numbers of mitochondria and tracheoles. 3. The ultrastructure of anal papillae suggests that these organs are involved in transport activity and appear to be functioning in both normal and dilute media. 4. No qualitative or statistically-significant quantitative changes were observed in anal papillae with changes in the external media and i t is suggested that physiologically-demonstrated adaptive changes may require minor structural alterations. 5. The morphology and ultrastructure of the hind gut of A. campestris larvae adapted to normal and dilute media is described. The hind gut consists of four major regions, the ileum, anterior rectum, posterior rectum, and the anal portion. 6. Cells making up the anterior and posterior rectum have the same specialized features found in anal papillae (in addition to straight lateral membranes joined apically by septate desmosomes) which again suggests these tissues are engaged in water and ion transport activity. 7. 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