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The Architecture of the Accessory Reproductive Glands of the Male Desert Locust
TISSUE 8- CELL 1970 2 (2) 233-248 Pubi/shed by Oliver ,9 Boyd, Edinburgh, Printed in Great Britain
T H O M A S R. O D H I A M B O *
THE A R C H I T E C T U R E OF THE A C C E S S O R Y R E P R O D U C T I V E G L A N D S OF THE MALE DESERT LOCUST III. COMPONENTS OF THE MUSCULAR WALL ABSTRACT. An electron microscopic study of the sheath enclosing the accessory reproductive glands of the male desert locust has shown that it consists, for the most part, of a single myofibril, and that other tissues (nerve fibres, tracheal elements, and the fat body) are also associated with it to a greater or lesser extent. The myofibril has special features associated with the Z-bands, including the regular infolding and the attachment of the sarcolemma at the Z-bands, and the synapsing of nerve axons at these infoldings, which perhaps facilitate the rapid transmission of nerve impulses into the myofibril, The distribution of the T-systems and sarcoplasmic reticulum (SR) is described, and their relationship to the speed of action of the myofibril is discussed. The myofibril exhibits three distinct bands: the A-, I-, and Z-bands. In the A-band, each thick myofilament is surrounded by 10 to 12 thin filaments. This finding is related to similar findings in other arthropod visceral and slow-acting skeletal muscles. The basement membrane surrounding the glandular epithelium comprises t w o parts: the inner part, which is structureless and contains neutral mucopolysaccharide; and the outer part which contains numerous collagen-like fibrils and stains for acid mucopolysaccharide. This characteristic is considered in relation to the insertion and function of the myofibril.
Introduction
PREVIOUS investigations have given a conflicting picture of the histological composition of the outer sheath of the accessory reproductive glands (ARGs) and the anterior parts of the reproductive system of the male insect. Musgrave (1937) thought the contractile power of these parts resides in either the epithelial cells themselves or in their basement membrane; he apparently observed no musculature anterior to that found in the ductus ejaculatorius. Callahan & Cascio (1963) decided to call the seemingly non-striated sheath of the anterior parts of the male reproductive system 'smooth cir* Department of Entomology, UniversityCollege Nairobi, Kenya. Received 18 November1969,
cular muscle' simply because all these parts are highly contractile; they also argued that epithelial cells and the basement membrane are :not usually considered to possess contractile power. Other workers (e.g. Davey, 1958; Gregory, 1961, 1965; Cantacuz6ne, 1967; Odhiambo, 1969) have found a distinct, striated, muscular layer enveloping the ARGs and other anterior parts of the male reproductive system. These conflicting views serve to highlight the fact that little is known of the detailed structure of the visceral muscles in insects (Baccetti, 1963; Davey, 1964; Smith, Gupta & Smith, 1966; Anderson & Ellis, 1967). It is the purpose of the present paper to present some fine-structural studies on the rnusculature of the ARGs. Furthermore, observations on other components of the muscular sheath
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will also be presented, particularly as they may give indications on the probable control mechanisms for the peristaltic movements of these glands. Materials and Methods
Accessory reproductive glands of recently mature males of the desert locust, Schistocerca gregaria Forsk51, were used for these studies. The glands were prepared for electron microscopy as reported recently (Odhiambo, 1966). Briefly, the ARGs were fixed in 1% osmium tetroxide buffered with veronal acetate, embedded in Araldite, and thin sections double-stained in uranyl acetate and lead citrate. A Philips EM200 electron microscope operated at 60 kV was used to examine the material. Observations A number of tissues often or invariably envelop the glandular epithelium of the ARGs the basement membrane, connective tissue continuous with the basement membrane, striated muscle, fat body, nervous and tracheal elements. The ultrastructural interrelationships between these various tissues will be examined in the following paragraphs. BASEMENTMEMBRANE The basement membrane, which is about 0.5 l0t~ thick, immediately encloses the
glandular epithelium (Figs. 1, 4, 5). It consists essentially of two layers: an inner amorphous or structureless layer abutting onto the epithelium, and adjacent to this a more external layer containing numerous collagen-like fibrils (Figs. 4, 5). The 150 ~ thick, characteristically banded, collagen-like
fibrils exist as a meshwork of longitudinally, circumferentially, and obliquely oriented structures. A similar three-dimensional network of collagen-like fibrils has been noted in endocrine glands of cockroaches (Harper, Seifter & Scharrer, 1967). Smith & Treherne (1963) have reviewed the occurrence of collagen-like fibrils in insect neural lamella, and have found that the characteristic macroperiod banding of the fibrils depends on the species and the method of preparation adopted. The fibrils are embedded in a ground-substance of neutral mucopolysaccharide and they are arranged in a meshwork, thus making the neural lamella relatively inextensible. It is therefore of interest that in the locust ARGs the portion of the basement membrane immediately adjacent to the striated muscle has numerous collagen-like fibrils (Figs. 4, 5), perhaps imparting to this portion of the basement membrane an adequately rigid structure on which the muscle can act. Further information on the basement membrane of ARGs was revealed by histochemical techniques: (1) The basement membrane gives an intense PAS reaction, which is not altered by treatment with saliva. (2) The basement membrane also gives a positive test for both Millon's and Bonhag's (mercury-bromphenol) tests for protein. (3) Only the outer portion of the basement memb r a n e - t h e part that contains collagen-like fibrils--gives a positive reaction with the Alcian blue test for acid mucopolysaccharide. These tests indicate that the inner portion of the basement membrane probably consists of neutral mucopolysaccharide. Biochemical evidence suggests that such a structure would offer little resistance to the diffusion of various substances (including inulin), although
Fig, 1. Survey electron micrograph of part of an accessory gland (AG) and its muscular sheath. The sheath consists of a basement membrane (1) immediately surrounding the glandular epithelium, a muscle fibre which contains a single myofibril ( f ) , a n d a strand of fat body (FB) closely adherent to the latter. The myofibril has distinct, though irregular Z-bands (Z), to which are applied sarcolemmal infoldings (arrows). Note also the sarcoplasmic reticulum (sr), the connective tissue (*) in which the myofibril and fat body are embedded, and the thin basement membrane (2) surrounding the entire sheath. • 24,000. TISSUE 8: CELL 1970 2 (2)
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226 structure of the excretory system in adult N.
brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods
Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results
Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend
backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum
Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to
the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.
TISSUE 8- CELL 1970 2 (2)
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226 structure of the excretory system in adult N.
brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods
Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results
Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend
backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum
Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to
the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.
TISSUE 8- CELL 1970 2 (2)
ACCESSORY R E P R O D U C T I V E G L A N D S OF LOCUSTS there is a possibility that free anionic groups associated with the presence of collagen may lead to a difference in the ionic composition of the contents of the structure as compared to that of the haemolymph (Smith & Treberne, 1963). In regard to this latter complicating factor, Schubert (1964) states that protein/polysaccharide complexes in solution are highly diffuse and extend throughout the solution, with the result that such a 'domain' offers degrees of porosity to various molecules dependent rnore on their size and shape than on their ionic composition. If so, we may regard the basement membrane as a more or less porous structure. External to the basement membrane are extraglandular tissues imbedded in a 'connective tissue' continuous with the basement membrane (Fig. 8). This connective tissue typically contains collagen-like fibrils.
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Furthermore, it stains positively (with Alcian blue) for acid mucopolysaccharide. This latter finding is more in line with the histochemical characteristic of vertebrate connective tissue. Electron microscopic observations show collagen fibrils to be closely associated with the myofibrils. It is consequently of some interest that where the musculature surrounding an ARG is thick (Fig. 2), staining for acid mucopolysaccharide is particularly intense. Presumably, the relative diffusion-permeability of the basement membrane extends also throughout the so-called connective tissue of ARGs. If so, it is possible for fairly large molecules to pass from the haemolymph or the surrounding :fat body into the glandular epithelium by way of the intervening connective tissue and basement membrane.
Fig. 2. Survey electron micrograph of a thick muscular layer showing myofibrils disposed in various directions. Each myofibril is surrounded by a thin connective tissue layer (*). Note also the nucleus (n) of a myofibril and tracheoblasts (tb) distributed among the myofibrils. • 9,000, Fig, 3. Transverse section through the A-band of a myofibril, showing the configuration of myofilaments. A cisternal element of the sarcoplasmic reticulum (st) is located near the filaments. • 115,000.
Fig. 4. Longitudinal section of a myofibril to show the irregular arrangement of the Z-band (Z). The basement membrane ensheathing the muscle layer may contain collagen-like fibrils (arrows), especially when tracheoles (t) are attached to the muscle layer. Note also the sarcoplasm with its few organelles, including elongated mitochondria (m), and the sarcolemmal infoldings (cf. Fig. 1.). x 24,000. Fig. 5. Regionalization of the basement membrane of an accessory gland into an inner amorphous portion (x) adjoining the glandular epithelium, and an outer portion (y) containing numerous collagen-like fibrils (arrows) adjoining the muscle. • 24,000. Fig. 6. Myofibrils (f) adjoining a region of extensive sarcoplasm containing sparsely distributed elements of the reticulum (sr), mitochondria (m), and numerous ribosomes. The T-system (T) arises frequently as an invagination of the plasma membrane at the sarco]emmal infolding, The T-system and part of the sarcoplasmic reticulum are generally associated with the Z-bands (Z). Note the collagen-containing intercellular material (*) between adjacent muscle fibres. • 24,000, Fig. 7. Further illustrating the irregular configuration of the Z-band, consisting of a series of electron-dense zones (Z). Note the presence of dense lysosomelike bodies (*) in this region, x 24,000~ TISSUE 8 CELL 1970 2 (2)
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Fig, 8. A 'naked" axon (ax) in apparent synapse with a myofibril has synaptic vesicles concentrated (arrow) on the presynaptic membrane, Another myofibril, in transverse section, shows several invaginations of the plasma membrane to form the T-system (T). Note that the basement membrane (2) enveloping the whole muscular sheath has numerous collagen-like fibrils. The basement membrane of the gland is seen at 1. • 32,500.
A C C E S S O R Y R E P R O D U C T I V E G L A N D S OF LOCUSTS MUSCLE LAYER
A thin muscular layer forms the innermost extra-glandular element of each ARG. A characteristic feature of the muscle fibres comprising the muscular layer is that each consists of a single myofibril (Figs. 1, 2, 4, 9)~ This peculiarity in which the whole contractile portion of a muscle fibre is contained within a single myofibrfi has been described elsewhere only very recently, for instance around the intestine of larval Drosophila melanogaster (Sandborn et al., 1967) and around the seminal vesicle of the stick insect, Carausius morosus (Smith, Gupta & Smith, 1967). For most of the length of the ARGs the muscular layer is only one myofibril thick. But proximally, towards junction with the ejaculatory duct, the muscular layer becomes progressively thicker; near the junction it is obviously multi-fibrillar (Fig. 2). These myofibrils are usually arranged circumferentially around the ARGs, but in the thicker muscular regions there are also obliquely oriented fibrils (Fig. 2). Three bands are quite distinct in the ARG muscle: A-, I-, and Z-bands; other bands are not discerned. The myofilament configuration within the A-band is very different from that described in insect flight and locomotory muscles (see, for example, Smith, 1961; Ashhurst, 1967; Pringle, 1967; Reger & Cooper, 1967). The thick and thin filaments have a different relationship to each other in the A R G musculature: each thick filament is surrounded by 10 to 12 thin filaments. This feature has been noted in the few visceral muscles that have been adequately studied in other insects (Anderson & Ellis, 1967; Sandborn et al., 1967; Smith, Gupta & Smith, 1966); it has also been noted in the tibial extensor muscle of the lepidopteran, Achalarus lyciades (Reger & Cooper, 1967). It is possible that this feature is correlated with the comparatively slow-acting properties of these muscles. The Z-bands in the A R G muscle characteristically follow an irregular course, each band comprising a number of electron-dense TISSUE 8- CELL 1970 2 (2)
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zones (Figs. 1, 4, 6, 7). Since the myofibrils in a thickened muscular sheath are not all disposed in the same direction, the Z-bands in such multifibrillar regions are generally not in register. The sarcomere length is about 4.5 6-5t~, although in the functional seminal vesicle it may be as short as 3'5t~; the l-band is relatively narrow (about 0-5t, wide), and is not so obvious as in skeletal muscle. Although the sarcomere length is not as long as in some insect visceral muscles (Smith, Gupta & Smith, 1966), it is longer than it is in vertebrate skeletal muscle (H. E. Huxley, 1960). Each myofibril contains only one nucleus, located laterally in the muscle cell (Fig. 9). The sarcoplasm is extensive, and contains sparsely distributed reticular cisternae, occasional microtubules, numerous ribosomes, and is one of the two main regions (together with the [-band) that contain many mitochondria (Figs. 4, 6). The plasma membrane of the muscle cell is covered on the outside by a basement membrane that may occasionally have collagen-like fibrils, particularly where other extra-glandular tissues join it (Figs. 4, 13, 21). A striking feature of the ARG muscle is the regular invagination of the sarcolemma where the muscle sheath is thin (e.g. Fig. 4). Such infoldings are in register with the Zbands. There is a thickened, electron-dense specialization of the sarcolemma directly opposite each Z-band leaving a gap of less than 300 ~ between the two structures. This specialization may well be associated with the rapid transmission of nervous impulses to the myofibril, Probably associated with this function is the fairly extensive transverse tubular system (T-system). This arises as an invagination of the myofibrillar plasma membrane at faMy frequent intervals along the sarcomere, the tubular elements coursing in a transverse direction (Fig. 8) although there are also a few longitudinally oriented elements (Fig. 6). At the sarcolemmal infoldings electron-dense discs are present opposite the Z-band, and the T-system tubules generally originate here (Fig. 6). It is noteworthy that the other tubular system of
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the muscle cell, the longitudinally oriented sarcoplasmic reticulum, is very much reduced (Figs_ 3, 4, 6). Perhaps this gives some structural basis for the slower-acting response of this visceral muscle. NERVOUS SUPPLY Peristaltic waves of contraction of small amplitude, presumably the result of muscular activity, are observed in living ARGs, and they may be important in the regulation of the extrusion of secretory products. Probably participating in this regulation is the extensive ~letwork of nerve fibres found on the glands and originating from the last abdominal ganglion (Odhiambo, 1969). A nerve fibre, containing several axons ensheathed with glial cells, approaches a gland (Fig. 9), and then sends off several branches, thus forming a network over the gland. Individual axons eventually make synaptic contact with the fibres, one (Fig. 13) or more (Figs. 10, 15) axons synapsing with a single cell. An observation with probable significance for the transmission of nerve impulses to the myofibril is that the synapsing axons are usually located at the sarcolemmal infoldings (Figs. 10, 12, 15). This arrangement seems to add more weight to the suggestion that the sarcolemmal infolding is a specialization for the regulation of the activity of the A R G muscle. At any rate, the synapsing axons, which are 'naked' at their synapsing regions, are recognised by their possession of vesicles that on structural grounds have been identified here as 'synaptic vesicles' and also have many small mitochondria (Figs. 10, 11, 13-
15). They do not conspicuously contain any other organelles. In the greater part of the ARGs, the muscular layer comprises a single myofibril, disposed in a circular manner. Frequently, this layer is interrupted by gaps, and it has been observed that, in a few cases, axons are located in these gaps and appear to synapse directly with glandular epithelium (Fig. 14). It should be noted, however, that in these cases the synaptic vesicle-containing axons al-e separated from the epithelium by the thickness of the basement membrane material (Fig. 14); whereas in the usual case of a synapse between an axon and a muscle fibre the gap between the two plasma membranes is only about 150 ~ or less (Fig. 8). In regard to the first case, and according to Bullock & Horridge (1965), apposition of the axon with the glandular epithelium would not qualify for its being termed a synapse, since there is an intervening basement membrane. In a few synapses, it has been observed that synaptic vesicles are concentrated on the presynaptic membrane of the axon close to the synaptic cleft (Fig. 8). It has been suggested that such areas may represent the active points at which actual transmission of impulses is effected (De Robertis, 1964). OTaER TlSSUEELEMENTS The ARGs are well tracheated. Tracheoles are distributed superficially over the glands (e.g. in Fig. 4), but they may also be found between myofibrils where the muscle layer is multi-fibrillar (Figs. 2, 8). Occasionally, the tracheoles penetrate deep enough to be lodged within the basement membrane im-
Fig. 9. A nerve branch is shown here approaching gland 16 (the functional seminal vesicle), and sending several axons (ax), still enclosed in a glial sheath (arrows) towards the muscular wall (*) of the gland. • 11,000. Fig. 10. Nerve axons (ax) containing synaptic vesicles synapsing with a muscle fibre at a sarcolemmal infolding where the Z-band (Z) is also located, Note the accompanying tracheoblast (tb). • 22,500. Fig. 11. Axons (ax) and their accompanying gila] cells (g) ;junctional membranes of the latter are linked by septate desmosomes (arrows). Note the sparse synaptic vesicles (sv). x 44,000.
TISSUE ~ CELL 1970 2 (2)
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LEE
226 structure of the excretory system in adult N.
brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods
Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results
Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend
backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum
Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to
the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.
TISSUE 8- CELL 1970 2 (2)
ACCESSORY R E P R O D U C T I V E G L A N D S OF LOCUSTS mediately surrounding the glandular epithelium. Tracheoblasts contain numerous free ribosomes, some rnitochondria, welldefined Golgi apparatus, and some microtubular arrays; but elements of the endoplasmic reticulum are small and rare. The tracheoles themselves have characteristic taenidia, which have been described in detail elsewhere (Locke, 1964). Closely applied to the muscle layer surrounding the glandular epithelium is a portion of the fat body (Fig. 1), whose :fine structure has recently been described (Odhiambo, 1967). Only gland 16 (the functional seminal vesicle) of the locust A R G complex is regularly and completely surrounded by the fat body; the other glands are only partially enveloped. The close relation between the paragonadal fat body and the ARGs raises the question of whether or not the fat body elaborates precursors or prefabricated materials which are then passed onto the ARGs directly, a question which has been considered elsewhere (Odhiambo, 1969). Discussion
In the living state, locust ARGs exhibit peristaltic movements of very small amplitude which presumably participate in the regulation of the extrusion of secretory products and the mature sperm. From recent studies, it is clear that the various secretions which result in the formation of the spermatophore are poured into the ejaculatory duct in a very precise and regulated manner--both in terms of time and sequence (Gregory, 1961, 1965).
Such a mechanism presupposes the involvement of a neuromuscular apparatus. The observations reported here were aimed at adding to our knowledge of this apparatus as well as to an understanding of the association of other tissues with the glandular epithelium. The muscular and nervous systems associated with the locust ARGs show a number of striking features: (1) the entire contractile portion of the muscle fibre is contained within a single myofibril; (2) the sarcolemma is infolded at frequent and regular intervals along the myofibril, these infoldings generally occurring directly opposite and closely approximating the Zbands; at these contact areas, the sarcolemma is specialized into a thick, electrondense disc; (3) both the T-system and the sarcoplasmic reticulum (S.R.) are much reduced; (4) the T-system seems to be closely associated with the sarcolemmal infoldings, arising as fairly frequent invaginations of the myofibrillar plasma membrane; (5) each thick myofilament is surrounded by 10 to 12 thin filaments; (6) there are only three distinct bands in the myofibril (A-, i-, and Z-bands); and (7) axons containing synaptic vesicles usually synapse at the sarcolemmal infoldings. It seems, from some of the special features listed above (2, 4, 7), that the level of the Zband and its associated structures may welt be peculiarly adapted for the rapid dissemination of excitation to the rest of the cell. Like many other visceral muscles in insects, the locust A R G muscle is slow-acting
Fig, 12. A longitudinal section of a muscle fibre investing a homogeneous accessory gland, showing several sarcolemmal infoldings (arrows) and nerve elements located within the latter. Some of the axons (ax) contain synaptic vesicles, x 24,000. Fig. 13, An axon containing synaptic vesicles (sv) is broadly naked on the side adjoining the basement membrane (2) of the muscle. The vesicles are not concentrated on the presynaptic membrane. On either side of the axon lies a glial element (g), Note the outer basement membrane with distinct collagen-like fibrils (arrow). x 47,500. Fig. 14. T w o axons (ax) with numerous synaptic vesicles, adjoining the basement membrane (bm) of an accessory gland (AG). The glial sheath (g) is locally incomplete (arrow). x 37,500. TISSUE & CELL 1970 2 (2)
245
ODHIAMBO
246 (Davey, 1964). In those cases where the ultrastructure of the component myofibrils has been studied adequately, it has been observed that the T- and S,R. systems are much reduced compared to the situation in the wing muscles (Anderson & Ellis, 1967; von Hehn, 1967; Sandborn et al., 1967; Smith, Gupta & Smith, 1966). Since the same feature has been noted in other relatively slow-acting muscles (e.g. in tibial extensor muscle of a lepidopteran examined by Reger & Cooper, 1967), it seems likely that the reduction in these membrane systems is related to the speed of action of these muscles. Local activation experiments suggest that the Tsystem is responsible for triggering muscle contraction (A. F. Huxley, 1964; A. F. Huxley & Peachey, 1964). On the other hand, the S.R. is generally regarded as the site of Ca ~* binding and the source of the relaxing lector controlling ATPase activity during contraction (Hasselbach, 1964). Vertebrate smooth muscle has neither of the T- nor the S.R. systems, and this circumstance has been correlated with its slow-acting response (Peachey & Porter, 1959). It has therefore been proposed that the reduction of these membrane systems in insect visceral muscle may similarIy be related to their relatively slow-acting response (Smith, 1966; Anderson & Ellis, 1967). The increased ratio between the thick and thin myofilaments has recently been observed in other arthropod visceral muscles (Anderson & Ellis, 1967; Sandborn et al., 1967; Smith, Gupta & Smith, 1966), insect tibial extensor muscle (Reger & Cooper, 1967), and insect femoral muscle (Hagopian & Spiro, 1967). As yet there is no clear indication of the functional significance of this configuration, except perhaps to suggest that it probably results in more numerous cross-
linkages which may produce many more reactive sites between actin and myosin (Anderson & Ellis, 1967; Hagopian & Spiro, 1967) or add to the tensile strength of the myofibril (Anderson & Ellis, 1967). The observations reported here add a new dimension to the known structure of the basement membrane. It has been shown that the outer portion of the basement membrane has different histochemical and ultrastructural characteristics from that found in the inner half. In particular, the outer portion contains numerous collagen-like fibrils disposed in a three-dimensional manner. It is possible that this arrangement adds stability to the basement membrane against which the myofibril may act. Delphin (1963) has demonstrated the presence of neurosecretory granules in the ventral nerve cord of the desert locust. He suggested, from ligature experiments, that the granules are transported peripherally from the ganglia by way of the motor nerves. This suggestion seems to offer the possibility that neurosecretory hormones from the ventral nerve cord may take part in regulating the function or thc development of the ARGs. But, in spite of an extensive ultrastructural examination of nerve elements associated with the locust ARGs, I have failed to encounter any neurosecretory granules in any of the axons attached to the locust ARGs.
Acknowledgements I wish to thank the Uganda Government and the British Ministry of Overseas Development for research grants, Dr A. V. Grimstone of Cambridge University for electron microscope facilities, and Mr P. Lisamulla for technical assistance.
Fig. 1 5. At axe, an axon approaches the muscular sheath around the accessory gland, but is still ensheathed with a glial covering (g). The neighbouring axon (ax2) synapses with a muscle cell comprising a single fibril (f), across a narrow synaptic gap (arrow). Both axons contain synaptic vesicles (sv). Note the T-system invaginations (T) from the fiber plasma membranes. • 57,500.
TISSUE 8- CELL 1970 2 (2)
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References
ANDERSON, W. A. and ELLIS, R. A. 1967. A comparative electron microscope study of visceral muscle fibers in Cambarus, Drosophila and Lumbricus. Z. Zellforsch. mikrosk. Anat., 79, 581--591. ASnHURST, D. E. 1967. The fibrillar flight muscles of giant water-bugs: an electron-microscope study. J. Cell Sei., 2, 435444. BACCETrl, B. 1963. Sulla presenza di fibrille collagene in alcune membrane basali e nella tunica dei corpi grassi di un Ortottero. Monit. zool. ital., Firenze, 70-71, 361-367. BULLOCK, T. H. and IHORRIDGE,G. A. 1965. Structure and fimction in the nervous systems of invertebrates. London: W. H. Freeman & Co. CALLAHAN,P. S. and CASCIO, T. 1963. Histology of the reproductive tracts and transmission of sperm in the corn earworm, Heliothis zea. Ann. ent. Soc. Amer., 56, 535-556. CANTACUZENE,A.-M. 1967. Histologie des glandes annexes raffles de Schistocerea gregaria F. (Orthoptbres). Effet de l'allatectomie sur leur structure et leur activit6. C.R. Acad. Sci., Paris, 264, 93-96~ DArEr, K. G. 1958. The physiology o f reproduction in an insect, Rhodnius prolixus StdL Thesis, University of Cambridge. DArE'Z, K. G. 1964. The control of visceral muscles in insects. Adv. Insect Physiol., 2, 219-245. DEL~'HIN, F. 1963. Studies on neurosecretion in Schistocerca gregaria Forskdl (Orthoptera: Acridiidae). Thesis, University of London. D~ ROBERTIS, E. D. P. 1964. Histophysiology o f synapses and neurosecretion. London: Pergamon Press. GREGORY, G. E. 1961. The formation, function and fate o f the spermatophore in locusts, Thesis, University of Wales. GREGORY, G. E. I965. The formation and fate of the spermatophore in the African migratory locust, Locusta migratoria migratorioMes Reiche and Fairmaire. Trans. R. ent. Soe. Loud., 117, 33-66. ~JAGOPIAN,M. and SPIRO,D..1967. The sarcop)asmic reticulum and its association with the T system in an insect. J. Cell BioL, 32, 535 545. HARPER, E., S~[FTER, S. and SCHARRER, B. (1967). Electron microscopic and biochemical characterization of collagen in blattarian insects. J. Cell Sci., 33, 385-393. HASSELBACH, W. 1964. Relaxing factor and the relaxation of muscle. Progr. Biophys. molec. Biol., 14, 167-222. HUXLEY, A. F. 1964. The links between excitation and contraction. Proc. R. Soc. (B), 160, 486-488. HuxLEY, A. F. and PEACHEY, L. D. 1964. Local activation of crab muscle. J. Cell Biol., 23, 107A. HUXLEY, H. E. 1960. Muscle cells, in The Cell fed. J. Brachet and A. E. Mirsky), 4, 365481, London: Academic Press. LOCKE, M. 1964. The structure and formation of the integument in insects. In Physiology o f Insecta fed. M. Rockstein), 3, 379-470. New York: Academic Press. MUSGRAVE, A. J. 1937. The histology of the male and female reproductive organs of Ephestia kiihniella Zeller (Lepidoptera). I. The young imagines. Proc. zool. Soc. Loud., 107B, 337 364. ODI4TAMBO, T. R. 1966. The fine structure of the corpus allatum of the sexually mature male of the desert locust. J. Insect Physiol., 12, 819-828. ODHIAMBO, T. R. 1967. The fine structure and histochemistry of the fat body in the locust, Schistocerca gregaria, or. Cell Sci., 2, 235-242. ODH1AMBO, T. R. 1969. The architecture of the accessory reproductive glands of the male desert locust. 1. Types of glands and their secretions. Tissue & Cell 1, i55-182. PEACHE'[, L. D. and POra'CER, K. R. 1959. lntracellular impulse conduction in muscle cells. Science, N. Y., 129, 721-722. PmNGLE, J. W. S. 1967. The contractile mechanism of insect fibrillar muscle. Pro.er. Biophys. molec. Biol., 17, 1-60. REGER, J. F. and COOVER, D. P. 1967. A comparative study of the fine structure of the basalar muscle of the wing and the tibial extensor muscle of the leg of the lepidopteran Achalarus lyciades, J. Cell Sci., 33, 531-542. SANDBORN, E. B., DUCLOS, S., MESSIER, P.-E. and ROBERGE, J.-J. 1967. A typical intestinal striated muscle in Drosophila melanogaster. J. Ultrastruct. Res., 18, 695-702. SCHUBERT, M. 1964. Intercellular macromolecules containing polysaccharides. Biophy. J., 4, (Suppl.), 119-138. SM1TH, D. S. 1961. The structure of insect fibrillar muscle. J. Biophys. biochem. Cytol., 10, 123-158. SMtT~, D. S. 1966. The organization and function of the sarcoplasmic reticulum and T-system of muscle cells. Progr. Biophys. molec. BioL, 16, 107--142. SMrrH, D. S., GUPTA, B. L. and SM)TH, U. 1966. The organization and myofilament array of insect visceral muscle. J. Cell Sci., 1, 49-57. SMXZH, D. S. and TR~ERN~, J. E. 1963. Functional aspects of the organization of the insect nervous system. Adv. lnsect Physiol., 1,401-484. VON HE~N, G. 1967. Die Muskulatur des Eileiters yon Carausius morosus. I. Histologische Untersuchungen. Z. Zellforsch. mikrosk. Anot., 78, 51 i-545. TISSUE ~ CELL 1970 2 (.2)
T H O M A S R. O D H I A M B O *
THE A R C H I T E C T U R E OF THE A C C E S S O R Y R E P R O D U C T I V E G L A N D S OF THE MALE DESERT LOCUST III. COMPONENTS OF THE MUSCULAR WALL ABSTRACT. An electron microscopic study of the sheath enclosing the accessory reproductive glands of the male desert locust has shown that it consists, for the most part, of a single myofibril, and that other tissues (nerve fibres, tracheal elements, and the fat body) are also associated with it to a greater or lesser extent. The myofibril has special features associated with the Z-bands, including the regular infolding and the attachment of the sarcolemma at the Z-bands, and the synapsing of nerve axons at these infoldings, which perhaps facilitate the rapid transmission of nerve impulses into the myofibril, The distribution of the T-systems and sarcoplasmic reticulum (SR) is described, and their relationship to the speed of action of the myofibril is discussed. The myofibril exhibits three distinct bands: the A-, I-, and Z-bands. In the A-band, each thick myofilament is surrounded by 10 to 12 thin filaments. This finding is related to similar findings in other arthropod visceral and slow-acting skeletal muscles. The basement membrane surrounding the glandular epithelium comprises t w o parts: the inner part, which is structureless and contains neutral mucopolysaccharide; and the outer part which contains numerous collagen-like fibrils and stains for acid mucopolysaccharide. This characteristic is considered in relation to the insertion and function of the myofibril.
Introduction
PREVIOUS investigations have given a conflicting picture of the histological composition of the outer sheath of the accessory reproductive glands (ARGs) and the anterior parts of the reproductive system of the male insect. Musgrave (1937) thought the contractile power of these parts resides in either the epithelial cells themselves or in their basement membrane; he apparently observed no musculature anterior to that found in the ductus ejaculatorius. Callahan & Cascio (1963) decided to call the seemingly non-striated sheath of the anterior parts of the male reproductive system 'smooth cir* Department of Entomology, UniversityCollege Nairobi, Kenya. Received 18 November1969,
cular muscle' simply because all these parts are highly contractile; they also argued that epithelial cells and the basement membrane are :not usually considered to possess contractile power. Other workers (e.g. Davey, 1958; Gregory, 1961, 1965; Cantacuz6ne, 1967; Odhiambo, 1969) have found a distinct, striated, muscular layer enveloping the ARGs and other anterior parts of the male reproductive system. These conflicting views serve to highlight the fact that little is known of the detailed structure of the visceral muscles in insects (Baccetti, 1963; Davey, 1964; Smith, Gupta & Smith, 1966; Anderson & Ellis, 1967). It is the purpose of the present paper to present some fine-structural studies on the rnusculature of the ARGs. Furthermore, observations on other components of the muscular sheath
233
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ODHIAMBO
will also be presented, particularly as they may give indications on the probable control mechanisms for the peristaltic movements of these glands. Materials and Methods
Accessory reproductive glands of recently mature males of the desert locust, Schistocerca gregaria Forsk51, were used for these studies. The glands were prepared for electron microscopy as reported recently (Odhiambo, 1966). Briefly, the ARGs were fixed in 1% osmium tetroxide buffered with veronal acetate, embedded in Araldite, and thin sections double-stained in uranyl acetate and lead citrate. A Philips EM200 electron microscope operated at 60 kV was used to examine the material. Observations A number of tissues often or invariably envelop the glandular epithelium of the ARGs the basement membrane, connective tissue continuous with the basement membrane, striated muscle, fat body, nervous and tracheal elements. The ultrastructural interrelationships between these various tissues will be examined in the following paragraphs. BASEMENTMEMBRANE The basement membrane, which is about 0.5 l0t~ thick, immediately encloses the
glandular epithelium (Figs. 1, 4, 5). It consists essentially of two layers: an inner amorphous or structureless layer abutting onto the epithelium, and adjacent to this a more external layer containing numerous collagen-like fibrils (Figs. 4, 5). The 150 ~ thick, characteristically banded, collagen-like
fibrils exist as a meshwork of longitudinally, circumferentially, and obliquely oriented structures. A similar three-dimensional network of collagen-like fibrils has been noted in endocrine glands of cockroaches (Harper, Seifter & Scharrer, 1967). Smith & Treherne (1963) have reviewed the occurrence of collagen-like fibrils in insect neural lamella, and have found that the characteristic macroperiod banding of the fibrils depends on the species and the method of preparation adopted. The fibrils are embedded in a ground-substance of neutral mucopolysaccharide and they are arranged in a meshwork, thus making the neural lamella relatively inextensible. It is therefore of interest that in the locust ARGs the portion of the basement membrane immediately adjacent to the striated muscle has numerous collagen-like fibrils (Figs. 4, 5), perhaps imparting to this portion of the basement membrane an adequately rigid structure on which the muscle can act. Further information on the basement membrane of ARGs was revealed by histochemical techniques: (1) The basement membrane gives an intense PAS reaction, which is not altered by treatment with saliva. (2) The basement membrane also gives a positive test for both Millon's and Bonhag's (mercury-bromphenol) tests for protein. (3) Only the outer portion of the basement memb r a n e - t h e part that contains collagen-like fibrils--gives a positive reaction with the Alcian blue test for acid mucopolysaccharide. These tests indicate that the inner portion of the basement membrane probably consists of neutral mucopolysaccharide. Biochemical evidence suggests that such a structure would offer little resistance to the diffusion of various substances (including inulin), although
Fig, 1. Survey electron micrograph of part of an accessory gland (AG) and its muscular sheath. The sheath consists of a basement membrane (1) immediately surrounding the glandular epithelium, a muscle fibre which contains a single myofibril ( f ) , a n d a strand of fat body (FB) closely adherent to the latter. The myofibril has distinct, though irregular Z-bands (Z), to which are applied sarcolemmal infoldings (arrows). Note also the sarcoplasmic reticulum (sr), the connective tissue (*) in which the myofibril and fat body are embedded, and the thin basement membrane (2) surrounding the entire sheath. • 24,000. TISSUE 8: CELL 1970 2 (2)
LEE
226 structure of the excretory system in adult N.
brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods
Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results
Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend
backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum
Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to
the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.
TISSUE 8- CELL 1970 2 (2)
LEE
226 structure of the excretory system in adult N.
brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods
Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results
Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend
backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum
Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to
the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.
TISSUE 8- CELL 1970 2 (2)
ACCESSORY R E P R O D U C T I V E G L A N D S OF LOCUSTS there is a possibility that free anionic groups associated with the presence of collagen may lead to a difference in the ionic composition of the contents of the structure as compared to that of the haemolymph (Smith & Treberne, 1963). In regard to this latter complicating factor, Schubert (1964) states that protein/polysaccharide complexes in solution are highly diffuse and extend throughout the solution, with the result that such a 'domain' offers degrees of porosity to various molecules dependent rnore on their size and shape than on their ionic composition. If so, we may regard the basement membrane as a more or less porous structure. External to the basement membrane are extraglandular tissues imbedded in a 'connective tissue' continuous with the basement membrane (Fig. 8). This connective tissue typically contains collagen-like fibrils.
237
Furthermore, it stains positively (with Alcian blue) for acid mucopolysaccharide. This latter finding is more in line with the histochemical characteristic of vertebrate connective tissue. Electron microscopic observations show collagen fibrils to be closely associated with the myofibrils. It is consequently of some interest that where the musculature surrounding an ARG is thick (Fig. 2), staining for acid mucopolysaccharide is particularly intense. Presumably, the relative diffusion-permeability of the basement membrane extends also throughout the so-called connective tissue of ARGs. If so, it is possible for fairly large molecules to pass from the haemolymph or the surrounding :fat body into the glandular epithelium by way of the intervening connective tissue and basement membrane.
Fig. 2. Survey electron micrograph of a thick muscular layer showing myofibrils disposed in various directions. Each myofibril is surrounded by a thin connective tissue layer (*). Note also the nucleus (n) of a myofibril and tracheoblasts (tb) distributed among the myofibrils. • 9,000, Fig, 3. Transverse section through the A-band of a myofibril, showing the configuration of myofilaments. A cisternal element of the sarcoplasmic reticulum (st) is located near the filaments. • 115,000.
Fig. 4. Longitudinal section of a myofibril to show the irregular arrangement of the Z-band (Z). The basement membrane ensheathing the muscle layer may contain collagen-like fibrils (arrows), especially when tracheoles (t) are attached to the muscle layer. Note also the sarcoplasm with its few organelles, including elongated mitochondria (m), and the sarcolemmal infoldings (cf. Fig. 1.). x 24,000. Fig. 5. Regionalization of the basement membrane of an accessory gland into an inner amorphous portion (x) adjoining the glandular epithelium, and an outer portion (y) containing numerous collagen-like fibrils (arrows) adjoining the muscle. • 24,000. Fig. 6. Myofibrils (f) adjoining a region of extensive sarcoplasm containing sparsely distributed elements of the reticulum (sr), mitochondria (m), and numerous ribosomes. The T-system (T) arises frequently as an invagination of the plasma membrane at the sarco]emmal infolding, The T-system and part of the sarcoplasmic reticulum are generally associated with the Z-bands (Z). Note the collagen-containing intercellular material (*) between adjacent muscle fibres. • 24,000, Fig. 7. Further illustrating the irregular configuration of the Z-band, consisting of a series of electron-dense zones (Z). Note the presence of dense lysosomelike bodies (*) in this region, x 24,000~ TISSUE 8 CELL 1970 2 (2)
!! ....
i~il84~ i;ii!~i~i ....
Fig, 8. A 'naked" axon (ax) in apparent synapse with a myofibril has synaptic vesicles concentrated (arrow) on the presynaptic membrane, Another myofibril, in transverse section, shows several invaginations of the plasma membrane to form the T-system (T). Note that the basement membrane (2) enveloping the whole muscular sheath has numerous collagen-like fibrils. The basement membrane of the gland is seen at 1. • 32,500.
A C C E S S O R Y R E P R O D U C T I V E G L A N D S OF LOCUSTS MUSCLE LAYER
A thin muscular layer forms the innermost extra-glandular element of each ARG. A characteristic feature of the muscle fibres comprising the muscular layer is that each consists of a single myofibril (Figs. 1, 2, 4, 9)~ This peculiarity in which the whole contractile portion of a muscle fibre is contained within a single myofibrfi has been described elsewhere only very recently, for instance around the intestine of larval Drosophila melanogaster (Sandborn et al., 1967) and around the seminal vesicle of the stick insect, Carausius morosus (Smith, Gupta & Smith, 1967). For most of the length of the ARGs the muscular layer is only one myofibril thick. But proximally, towards junction with the ejaculatory duct, the muscular layer becomes progressively thicker; near the junction it is obviously multi-fibrillar (Fig. 2). These myofibrils are usually arranged circumferentially around the ARGs, but in the thicker muscular regions there are also obliquely oriented fibrils (Fig. 2). Three bands are quite distinct in the ARG muscle: A-, I-, and Z-bands; other bands are not discerned. The myofilament configuration within the A-band is very different from that described in insect flight and locomotory muscles (see, for example, Smith, 1961; Ashhurst, 1967; Pringle, 1967; Reger & Cooper, 1967). The thick and thin filaments have a different relationship to each other in the A R G musculature: each thick filament is surrounded by 10 to 12 thin filaments. This feature has been noted in the few visceral muscles that have been adequately studied in other insects (Anderson & Ellis, 1967; Sandborn et al., 1967; Smith, Gupta & Smith, 1966); it has also been noted in the tibial extensor muscle of the lepidopteran, Achalarus lyciades (Reger & Cooper, 1967). It is possible that this feature is correlated with the comparatively slow-acting properties of these muscles. The Z-bands in the A R G muscle characteristically follow an irregular course, each band comprising a number of electron-dense TISSUE 8- CELL 1970 2 (2)
241
zones (Figs. 1, 4, 6, 7). Since the myofibrils in a thickened muscular sheath are not all disposed in the same direction, the Z-bands in such multifibrillar regions are generally not in register. The sarcomere length is about 4.5 6-5t~, although in the functional seminal vesicle it may be as short as 3'5t~; the l-band is relatively narrow (about 0-5t, wide), and is not so obvious as in skeletal muscle. Although the sarcomere length is not as long as in some insect visceral muscles (Smith, Gupta & Smith, 1966), it is longer than it is in vertebrate skeletal muscle (H. E. Huxley, 1960). Each myofibril contains only one nucleus, located laterally in the muscle cell (Fig. 9). The sarcoplasm is extensive, and contains sparsely distributed reticular cisternae, occasional microtubules, numerous ribosomes, and is one of the two main regions (together with the [-band) that contain many mitochondria (Figs. 4, 6). The plasma membrane of the muscle cell is covered on the outside by a basement membrane that may occasionally have collagen-like fibrils, particularly where other extra-glandular tissues join it (Figs. 4, 13, 21). A striking feature of the ARG muscle is the regular invagination of the sarcolemma where the muscle sheath is thin (e.g. Fig. 4). Such infoldings are in register with the Zbands. There is a thickened, electron-dense specialization of the sarcolemma directly opposite each Z-band leaving a gap of less than 300 ~ between the two structures. This specialization may well be associated with the rapid transmission of nervous impulses to the myofibril, Probably associated with this function is the fairly extensive transverse tubular system (T-system). This arises as an invagination of the myofibrillar plasma membrane at faMy frequent intervals along the sarcomere, the tubular elements coursing in a transverse direction (Fig. 8) although there are also a few longitudinally oriented elements (Fig. 6). At the sarcolemmal infoldings electron-dense discs are present opposite the Z-band, and the T-system tubules generally originate here (Fig. 6). It is noteworthy that the other tubular system of
242
ODH1AMBO
the muscle cell, the longitudinally oriented sarcoplasmic reticulum, is very much reduced (Figs_ 3, 4, 6). Perhaps this gives some structural basis for the slower-acting response of this visceral muscle. NERVOUS SUPPLY Peristaltic waves of contraction of small amplitude, presumably the result of muscular activity, are observed in living ARGs, and they may be important in the regulation of the extrusion of secretory products. Probably participating in this regulation is the extensive ~letwork of nerve fibres found on the glands and originating from the last abdominal ganglion (Odhiambo, 1969). A nerve fibre, containing several axons ensheathed with glial cells, approaches a gland (Fig. 9), and then sends off several branches, thus forming a network over the gland. Individual axons eventually make synaptic contact with the fibres, one (Fig. 13) or more (Figs. 10, 15) axons synapsing with a single cell. An observation with probable significance for the transmission of nerve impulses to the myofibril is that the synapsing axons are usually located at the sarcolemmal infoldings (Figs. 10, 12, 15). This arrangement seems to add more weight to the suggestion that the sarcolemmal infolding is a specialization for the regulation of the activity of the A R G muscle. At any rate, the synapsing axons, which are 'naked' at their synapsing regions, are recognised by their possession of vesicles that on structural grounds have been identified here as 'synaptic vesicles' and also have many small mitochondria (Figs. 10, 11, 13-
15). They do not conspicuously contain any other organelles. In the greater part of the ARGs, the muscular layer comprises a single myofibril, disposed in a circular manner. Frequently, this layer is interrupted by gaps, and it has been observed that, in a few cases, axons are located in these gaps and appear to synapse directly with glandular epithelium (Fig. 14). It should be noted, however, that in these cases the synaptic vesicle-containing axons al-e separated from the epithelium by the thickness of the basement membrane material (Fig. 14); whereas in the usual case of a synapse between an axon and a muscle fibre the gap between the two plasma membranes is only about 150 ~ or less (Fig. 8). In regard to the first case, and according to Bullock & Horridge (1965), apposition of the axon with the glandular epithelium would not qualify for its being termed a synapse, since there is an intervening basement membrane. In a few synapses, it has been observed that synaptic vesicles are concentrated on the presynaptic membrane of the axon close to the synaptic cleft (Fig. 8). It has been suggested that such areas may represent the active points at which actual transmission of impulses is effected (De Robertis, 1964). OTaER TlSSUEELEMENTS The ARGs are well tracheated. Tracheoles are distributed superficially over the glands (e.g. in Fig. 4), but they may also be found between myofibrils where the muscle layer is multi-fibrillar (Figs. 2, 8). Occasionally, the tracheoles penetrate deep enough to be lodged within the basement membrane im-
Fig. 9. A nerve branch is shown here approaching gland 16 (the functional seminal vesicle), and sending several axons (ax), still enclosed in a glial sheath (arrows) towards the muscular wall (*) of the gland. • 11,000. Fig. 10. Nerve axons (ax) containing synaptic vesicles synapsing with a muscle fibre at a sarcolemmal infolding where the Z-band (Z) is also located, Note the accompanying tracheoblast (tb). • 22,500. Fig. 11. Axons (ax) and their accompanying gila] cells (g) ;junctional membranes of the latter are linked by septate desmosomes (arrows). Note the sparse synaptic vesicles (sv). x 44,000.
TISSUE ~ CELL 1970 2 (2)
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226 structure of the excretory system in adult N.
brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods
Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results
Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend
backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum
Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to
the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.
TISSUE 8- CELL 1970 2 (2)
ACCESSORY R E P R O D U C T I V E G L A N D S OF LOCUSTS mediately surrounding the glandular epithelium. Tracheoblasts contain numerous free ribosomes, some rnitochondria, welldefined Golgi apparatus, and some microtubular arrays; but elements of the endoplasmic reticulum are small and rare. The tracheoles themselves have characteristic taenidia, which have been described in detail elsewhere (Locke, 1964). Closely applied to the muscle layer surrounding the glandular epithelium is a portion of the fat body (Fig. 1), whose :fine structure has recently been described (Odhiambo, 1967). Only gland 16 (the functional seminal vesicle) of the locust A R G complex is regularly and completely surrounded by the fat body; the other glands are only partially enveloped. The close relation between the paragonadal fat body and the ARGs raises the question of whether or not the fat body elaborates precursors or prefabricated materials which are then passed onto the ARGs directly, a question which has been considered elsewhere (Odhiambo, 1969). Discussion
In the living state, locust ARGs exhibit peristaltic movements of very small amplitude which presumably participate in the regulation of the extrusion of secretory products and the mature sperm. From recent studies, it is clear that the various secretions which result in the formation of the spermatophore are poured into the ejaculatory duct in a very precise and regulated manner--both in terms of time and sequence (Gregory, 1961, 1965).
Such a mechanism presupposes the involvement of a neuromuscular apparatus. The observations reported here were aimed at adding to our knowledge of this apparatus as well as to an understanding of the association of other tissues with the glandular epithelium. The muscular and nervous systems associated with the locust ARGs show a number of striking features: (1) the entire contractile portion of the muscle fibre is contained within a single myofibril; (2) the sarcolemma is infolded at frequent and regular intervals along the myofibril, these infoldings generally occurring directly opposite and closely approximating the Zbands; at these contact areas, the sarcolemma is specialized into a thick, electrondense disc; (3) both the T-system and the sarcoplasmic reticulum (S.R.) are much reduced; (4) the T-system seems to be closely associated with the sarcolemmal infoldings, arising as fairly frequent invaginations of the myofibrillar plasma membrane; (5) each thick myofilament is surrounded by 10 to 12 thin filaments; (6) there are only three distinct bands in the myofibril (A-, i-, and Z-bands); and (7) axons containing synaptic vesicles usually synapse at the sarcolemmal infoldings. It seems, from some of the special features listed above (2, 4, 7), that the level of the Zband and its associated structures may welt be peculiarly adapted for the rapid dissemination of excitation to the rest of the cell. Like many other visceral muscles in insects, the locust A R G muscle is slow-acting
Fig, 12. A longitudinal section of a muscle fibre investing a homogeneous accessory gland, showing several sarcolemmal infoldings (arrows) and nerve elements located within the latter. Some of the axons (ax) contain synaptic vesicles, x 24,000. Fig. 13, An axon containing synaptic vesicles (sv) is broadly naked on the side adjoining the basement membrane (2) of the muscle. The vesicles are not concentrated on the presynaptic membrane. On either side of the axon lies a glial element (g), Note the outer basement membrane with distinct collagen-like fibrils (arrow). x 47,500. Fig. 14. T w o axons (ax) with numerous synaptic vesicles, adjoining the basement membrane (bm) of an accessory gland (AG). The glial sheath (g) is locally incomplete (arrow). x 37,500. TISSUE & CELL 1970 2 (2)
245
ODHIAMBO
246 (Davey, 1964). In those cases where the ultrastructure of the component myofibrils has been studied adequately, it has been observed that the T- and S,R. systems are much reduced compared to the situation in the wing muscles (Anderson & Ellis, 1967; von Hehn, 1967; Sandborn et al., 1967; Smith, Gupta & Smith, 1966). Since the same feature has been noted in other relatively slow-acting muscles (e.g. in tibial extensor muscle of a lepidopteran examined by Reger & Cooper, 1967), it seems likely that the reduction in these membrane systems is related to the speed of action of these muscles. Local activation experiments suggest that the Tsystem is responsible for triggering muscle contraction (A. F. Huxley, 1964; A. F. Huxley & Peachey, 1964). On the other hand, the S.R. is generally regarded as the site of Ca ~* binding and the source of the relaxing lector controlling ATPase activity during contraction (Hasselbach, 1964). Vertebrate smooth muscle has neither of the T- nor the S.R. systems, and this circumstance has been correlated with its slow-acting response (Peachey & Porter, 1959). It has therefore been proposed that the reduction of these membrane systems in insect visceral muscle may similarIy be related to their relatively slow-acting response (Smith, 1966; Anderson & Ellis, 1967). The increased ratio between the thick and thin myofilaments has recently been observed in other arthropod visceral muscles (Anderson & Ellis, 1967; Sandborn et al., 1967; Smith, Gupta & Smith, 1966), insect tibial extensor muscle (Reger & Cooper, 1967), and insect femoral muscle (Hagopian & Spiro, 1967). As yet there is no clear indication of the functional significance of this configuration, except perhaps to suggest that it probably results in more numerous cross-
linkages which may produce many more reactive sites between actin and myosin (Anderson & Ellis, 1967; Hagopian & Spiro, 1967) or add to the tensile strength of the myofibril (Anderson & Ellis, 1967). The observations reported here add a new dimension to the known structure of the basement membrane. It has been shown that the outer portion of the basement membrane has different histochemical and ultrastructural characteristics from that found in the inner half. In particular, the outer portion contains numerous collagen-like fibrils disposed in a three-dimensional manner. It is possible that this arrangement adds stability to the basement membrane against which the myofibril may act. Delphin (1963) has demonstrated the presence of neurosecretory granules in the ventral nerve cord of the desert locust. He suggested, from ligature experiments, that the granules are transported peripherally from the ganglia by way of the motor nerves. This suggestion seems to offer the possibility that neurosecretory hormones from the ventral nerve cord may take part in regulating the function or thc development of the ARGs. But, in spite of an extensive ultrastructural examination of nerve elements associated with the locust ARGs, I have failed to encounter any neurosecretory granules in any of the axons attached to the locust ARGs.
Acknowledgements I wish to thank the Uganda Government and the British Ministry of Overseas Development for research grants, Dr A. V. Grimstone of Cambridge University for electron microscope facilities, and Mr P. Lisamulla for technical assistance.
Fig. 1 5. At axe, an axon approaches the muscular sheath around the accessory gland, but is still ensheathed with a glial covering (g). The neighbouring axon (ax2) synapses with a muscle cell comprising a single fibril (f), across a narrow synaptic gap (arrow). Both axons contain synaptic vesicles (sv). Note the T-system invaginations (T) from the fiber plasma membranes. • 57,500.
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References
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