The site of potato leafroll virus multiplication in its vector, Myzus persicae : an anatomical study

In search of the site of PLRV multiplication in its vector a detailed study was made of the anatomy of the aphid, <em>Myzus persicae</em> SULZ. The findings are summarized in the following lines:<em>Alimentary canal</em>The most anterior part of the alimentary canal is the food canal which is firmly interlocked by the maxillary stylets. From the stylets it passes into the pharyngeal duct which subsequently leads into the pharyngeal valve, pharyngeal pump, foregut, oesophageal valve, stomach, intestine, hindgut, and rectum to terminate into the anal opening (Figs. 3, 5, and 11).Each stylet originates from a retort-shaped organ (Fig. 4) and is attached by muscles to the tentorial bar, the maxillary sclerite (Fig. 31), and the pharynx floor (Fig. 8 no. 12-13). The further pathway of the stylets is described and figured (Figs. 7 no. 1, 2 and 3 no. 1-4). The stylet bundle shows in its way through the longitudinal labial groove a torsion of 180°.The pharyngeal duct is formed by the epipharynx and the hypopharynx lip (Fig. 7 no. 2-8). The epipharynx is marked by a thick sclerotized plate and reveals a median row of eight sensillum pores (Fig. 5). On each side of the epipharynx is an invagination (Fig. 7 no. 7-8), which runs as an arch from the third pore to the valve (Fig. 3). The cup-shaped part of the floor before the valve is provided with two sensillum pores (Figs. 5 and 8 no. 9-10).The pharyngeal duct is separated from the pharyngeal pump by a valve, of which both the dorsal wall and ventral wall are marked by two cuticular domeshaped prominences, the pharynx protuberances. The dorsal wall of the valve is controlled by two pairs of muscles, and each lateral side by only one muscle. In embryos the valve is open (Fig. 8 no. 10-12), but in larvae it is always found in a closed position (Fig. 8 no. 10'-11'). In the latter situation the opposite pharynx protuberances and the valve itself fit closely together. It is logical to suppose that the opened valve in the larvae has the same position as can be observed in embryos.The pharyngeal pump is controlled by 29 pairs of dorsal elevator muscles (Figs. 5, 6, and 8 no. 13-15). Close to the tentorial bar the floor of the pump is attached by muscles, one pair originating from the tentorium, and one pair from the piston of the salivary pump.The foregut consists of a single layer of squamous epithelia] cells which are lined with an intima forming a stellate narrow lumen (Fig. 32).The oesophageal valve marks the junction of the foregut and the stomach (Fig. 9). The inner layer is the continuation of the foregut into the stomach. The outer layer consists of cuboidal epithelial cells, The intima of the foregut continues in the region of the valve and terminates at the base of it.The anterior and posterior region of the stomach consists of cuboidal epithelial cells, while the middle region is occupied by tall, fingerlike columnar digestive cells (Figs. 9 and 10). The latter secrete material by constricting of apical cell parts. In some nuclei strongly basophilic clusters of chromatin material develop on the ninth day of the larva (Fig. 10 B'). The cells of the posterior region secrete material by forming of buds (Figs. 9 C and 10 C'). Both processes continue during larval life without any degeneration or multiplication of cells.The first part of the intestine is a narrow tube which passes into a broader one to terminate abruptly by constriction in the hindgut. In the first part the squamous cells form a stellate narrow lumen, while in the second division the strongly vacuolated cells are situated around a wide lumen (Fig. 11). It is suggested that the intestine is resorbtive in function in contrast to the stomach which is the secretory part of the midgut.The hindgut consists of long squamous epithelial cells containing many vesicles in which waxy droplets occur. These droplets, originating from degenerating fat cells, also occur in the lumen where they are released by minute projections situated at the apical surface of the cells (Fig. 26).On the ventral side the hindgut passes into the rectum whereas it continues dorsally a distance to terminate in a dorsal rectal sac (Fig. 12). During larval life the rectal sac gradually disappears to form with the posterior part of the rectum an almost straight duct. The rectum is built up of columnar cells having small vesicles liberating secretion into the lumen.During larval life the growth of the entire gut is due solely to enlargement of the epithelial cells. They retain their optical composition and persist unchanged into the adult stage. Cell divisions were never observed (Table 4).<em>Salivary gland</em>Each half of the system is composed of the principal and the accessory gland (Figs. 1 and 13). The common afferent duct forms a S-shaped flexure before entering the salivary pump (Figs. 5 and 19). At the place of entry the opening is controlled by two small muscles originating from the chitinous ridges. The exit opening from the pump chamber leads into the pumpstem which on its foot is provided with two sensillum pores. The pathway of the efferent salivary duct follows from Fig. 7 no. 5- 8. As well the cylinder as the piston are supplied with muscles.The accessory gland is composed of 3-4 cells of uniform size (Fig. 13 A). The basal part of these cells shows laminated structures and in the cytoplasm many branching canaliculi occur which cross the cuticular lining of the salivary duct (Fig. 14). It is suggested that the accessory glands excrete a watery suspension that during its discharge to the salivary pump is dehydrated by the duct cells to form the final viscous fluid. The viscous fluid, forming the salivary sheath, contains presumably waxy droplets. These droplets presumably render the stylet sheath its waterproofing.The principal gland is bilobed and each lobe contains six Deckzellen and fifteen Hauptzellen which are situated around the salivary duct. Considering the optical composition of the cytoplasm, the shape of the cell (Fig. 14), the size of the nucleus and nucleolus (Table 5), the six Deckzellen can be distinguished in two, and the fifteen Hauptzellen in six different types. During larval life the topographical position of all the gland celltypes is constant in each lobe (Table 6). Each cell has a canaliculum. which traverses the cuticular lining and the lumen of the salivary duct. The eight different celltypes (Fig. 13 A) contain a mass of secretory granules which are released by the cell into the canaliculi. It is supposed that the watery saliva originates from the principal gland.The distal part of the two lobes of each principal gland is connected by a myoepithelioid cell (Fig. 13 A and D). The bulk of the cytoplasm is occupied by myofibrils oriented in an interwoven pattern (Figs. 15, 16 A, and 17 A). At the periphery of this contractile mass some mitochondria and many vacuoles. occur. The surface of this cell, bounded by the four intercellular canaliculi of celltype 7 and the termination of both ducts, is strongly infolded. Injections with Evans blue via the cornicles in the larvae gave the evidence that haemolymph is pumped into the lumen of both ducts by pulsations of the myoepithelioid cell. Via the duct lumina it becomes transported through the salivary pump into the plant.In each principal gland the myoepithelioid cell is innervated by a paired nerve of the mediodorsal nervous system (Figs. 13 A, 17 B, and 18).The salivary duct is lined with endocuticle throughout its length. The apical surface of the duct epithelial cells bears regularly oriented microvilli (Fig. 16 B and C).During larval life cell divisions are never observed in the salivary gland system. However, the nuclei and their nucleoli increase in size (Table 5) as well as the cells (Fig. 14).<em>Mesodermal derivates</em>The mesodermal tissue is represented by a cell layer beneath the epidermis. It is composed of numerous fat cells between which basophilic mesodermal cells and connective tissue cells are evenly distributed (Figs. 20, 25, and 32 V).The first embryonic mesodermal cells are observed in the eldest embryo of a three days old larva in the area anterior of the future cornicles (Fig. 22, 1). During embryonic development the number of mesodermal cells continuously increases by cell divisions; the direction of cell divisions is from the site of origin both anteriad and posteriad (Fig. 22, 1-3). In the eldest embryo of a five days old larva the mesodermal cells lying in the area of origin start to develop into fat cells by the forming of lipid droplets in their cytoplasm (Fig. 22, 2). At the same time other embryonic mesodermal cells start to increase in size and become the final basophilic mesodermal cells (Fig. 22, bmc). During growth of the embryo all embryonic mesodermal cells develop into fat, basophilic mesodermal, and connective tissue cells. In the eldest embryo of a nine days old larva the mesodermal tissue has attained its final composition (Fig. 22, 4). After the embryonic mesodermal cells are differentiated into their derivates, no cell divisions take place anymore in these products (Fig. 22, 4-7).After transformation of the embryonic mesodermal cells into fat cells, they start to increase in size just like their nuclei and nucleoli. Lipid droplets increase both in number and in size (Fig. 22, 4 D). During coalescence of the lipid droplets (Fig. 22, 4 F) the first waxy droplets and rhomb-shaped crystals arise in the cytoplasm (Fig. 22, 4 G). The degeneration process of the fat cells is revealed by the appearance of vacuoles in the karyoplasm (Fig. 22, 4 H), after which the chromatin condenses and disintegrates into fragments (karyorhexis), granules (karyolysis), or becomes pyknotic. The nucleolus vacuolizes and dissolves peripherally and disappears completely. In the cytoplasm of the degenerating cells eosinophilic bodies arise. After breakup of the cell membrane the cell contents release into the haemolymph (Fig. 22, J-L). The degeneartion process described is already in full existence in the eldest embryo of a nine days old larva and proceeds during larval life.The enlargement of the basophilic mesodermal cells and their nuclei and nucleoli continues during larval life (Fig. 21). The cytoplasm becomes gradually acidophilic: and many minute vacuoles now arise. After the seventh day the chromatin starts to condense and the nuclear membrane becomes somewhat irregular to dissolve subsequently in the adult stage. The clotting chromatin is liberated into the vacuolized cytoplasm followed by a complete disintegration of the basophilic mesodermal cells.Some embryonic mesodermal cells change into the final connective tissue cells. They are much smaller than the other mesodermal derivates, and retain their original volume in the larval stage (Figs. 20 and 32 V). Their delicate cell extensions form a complicated network which support the mesodermal tissue. Moreover, these membranous filaments extend throughout the haemocoel and are connected with the various internal organs.The pericardial cells are derived from embryonic mesodermal cells. In the eldest embryo of a nine days old larva these cells have attained a structure and number, which persists during whole further life (Fig. 32). After larviposition these cells and their nuclei and nucleoli increase in size, but the total number of cells remains constant (Table 8). They are distributed along the entire dorsal vessel and connected with a branch of the medial dorsal nerve (Fig. 3). Furthermore there occur 2-3 pericardial cells on both lateral sides of each thoracic segment, and 3-4 lateral pericardial cells in the metathoracic segment (Figs. 20 and 24).The haemolymph is characterized by the absence of circulating haemocytes. On the other hand, the body cavity contains quite a number of waxy droplets during aphid's life. The droplets are released by the cell membrane of intact fat cells, or are liberated into the haemolymph after dissolving of the cell membrane. They are predominantly close to the visceral surface of the mesodermal tissue and especially in a high concentration around the hindgut and the accessory salivary glands. Many of them are distributed by the dorsal vessel. <em></em><em>Mycetome</em>The mycetome consists of two longitudinal bodies of syncytial tissues (Fig. 1) linked together dorsally of the hindgut (Fig. 26 P). The mycetocytes are completely filled with spherical microorganisms or symbionts which divide like bacteria. After the fifth day the larval mycetome starts to disintegrate in clusters of cells or in single mycetocytes. During larval life symbionts degenerate within the mycetocytes, and at the ninth day the cell membrane of some mycetocytes starts to dissolve after which intact and degenerating symbionts are liberated into the haemolymph (Fig. 26 P). They are distributed throughout the body cavity and occur in the mesodermal tissue among the degenerating fat cells as well as in the legs.<em>Oenocytes</em>The oenocytes are situated laterad at either side in the body cavity between the mesodermal tissue and the internal organs of the metathorax and the first five abdominal segments (Figs. 1, 25, and 27). They form a longitudinal row of 7-12 single cells and are anchored by membranes which originate from the connective tissue cells. They are recognizable because of their irregular or branched nuclei with granulated chromatin and a proportionally very big nucleolus. The cytoplasm is strongly vacuolated and many vacuoles contain granules (Fig. 21).<em>Circulatory system</em>The dorsal vessel lies just beneath the dorsal mesodermal tissue (Figs. 3, 25, and 27). The ventral wall of the funnel-shaped mouth is stretched transversally over the corpus allatum to which it is connected along the ventrolateral lines (Fig. 32). The dorsal wall of the mouth extends further anteriad and is attached to the corpora cardiaca (Fig. 28). Throughout its length the vessel pulsates vigorously and the anterior wider part is provided with three pairs of lateral ostia. In all coxae a tubular accessory pulsatile organ occurs.<em>Cornicles</em>The cornicles are in open connection with the body cavity and are filled with degenerating fat cells (Fig. 27). Each cornicle is closed at its top with a valvelike flap, which is controlled by the valve retractor muscle. Only in an erected position due to contraction of the elevator muscle, the cornicles can produce excretory material. This material is composed of degenerating fat cells, disintegrated nuclear material, waxy droplets, rhomb-shaped crystals, and incidently a basophilic mesodermal cell, while connective tissue cells have been never observed (Table 7). At the ninth day of the aphid's life the first intact and degenerating symbionts appear in the cornicle secretion. The fat cells of celltype J (Fig. 22), of which the cell membrane is still intact, are responsible for the hardening of the secretion after it has passed the cornicles. <em></em><em>Nervous system</em>The nervous system is described and figured (Figs. 3, 5, 28-31, and 32 P). The paired pharyngeal ganglions are situated ventrally of the tritocerebral lobes and frontolaterally of the pharyngeal pump on both sides of their retractor muscles (Figs. 3 and 30). Before the pharyngeal valve the two bilobed pharyngeal ganglions fuse together and this mass extends ventrally as the epipharyngeal and the hypopharyngeal gustatory organ. The central part of the epipharyngeal gustatory organ has on its periphery many neurons whose dendrites are connected with the pores of the eight sensilla in the pharyngeal roof (Fig. 5). Centrally each lateral lobe is occupied by a stellate invagination of the pharyngeal duct (Figs. 6 and 7 no. 8). From the wall of this invagination nerve fibres arise running to neurons situated mainly on the periphery of the lateral lobes. The hypopharyngeal gustatory organ lies beneath the pharyngeal valve. The neurons in this ganglion communicate with four sensillum pores, of which two of them are situated in the cup-shaped part of the floor of the pharyngeal duct (Fig. 8 no. 10 and 10'), while the other two are located on the foot of the salivary pumpstem (Fig. 7 no. 8 and Fig. 19).Endocrine glands. The paired corpora cardiaca are irregular- shaped bodies which are attached to the inner side of the dorsal aortic wall. Each gland is composed of 8-11 cells arranged around a neuropile mass from which nerves arise (Fig. 28).The corpus allatum is a single body containing 11-13 cells (Fig. 32). It is situated between the foregut and dorsal vessel behind the corpora cardiaca (Fig. 3).The paired thoracic glands consisting each of 8-11 cells, are situated in the vicinity of the tracheal trunks of the mesothoracic spiracle (Figs. 2 and 20). After the seventh day of larval life some cells start to degenerate, while on the ninth day all the cells are disintegrated into clusters of irregular spheres.The second part of the study deals with experimental and electron microscopic evidence on transport, multiplication, and release of PLRV.An attempt has been made to obtain information concerning the fate of the ingested virus in the aphid vector by recovering PLRV from dissected organs of viruliferous <em>M. persicae</em> larvae. The various organs were removed from the larvae and, after homogenization, tested for infectivity by injecting them into nonviruliferous aphids. As is shown in Table 11, the presence of PLRV could be demonstrated only in the contents of the alimentary canal, honeydew, haemolymph, fat cells presumably including basophilic mesodermal cells, and cornicle secretion. It may be concluded that a part of the ingested virus is transported from the gut's lumen into the haemolymph. After being circulated it multiplies in the fat cells. The remaining part of the ingested virus is egested with the honeydew. The negative results obtained with the salivary glands presented the evidence that the myoepithelioid cell, situated in the distal region of each principal salivary gland (Fig. 13 A), regulates the transport of PLRV from the haemolymph via the lumen of both internal salivary ducts into the sieve tubes.An electron microscopical investigation of the fat and basophilic mesodermal cells is given (Figs. 34-37). The cytoplasm of a fat cell is composed of cisternal rough-surfaced endoplasmic reticulum, ribosomes, mitochondria, and GOLGI bodies. During growth of the cell many lipid droplets are set off by the surrounding cytoplasm and become larger to coalesce subsequently into big ones. Simultaneously with the development and coalescence of the lipid droplets, glycogenlike particles, protein granules, multivesicular bodies, waxy droplets, and rhomb-shaped crystals arise in the cytoplasm. The degeneration process of the fat cells starts with the appearance of vacuoles in the karyoplasm after which the chromatin condenses and the nuclear envelope loses its doublemembrane structure. In a more advanced stage of degeneration the cytoplasmic material and the cell membrane disintegrates. In the disintegrating area of the cytoplasm arise cytolysomes which become larger by encapsulating mitochondria and other cytoplasmic organelles. After decomposition of the cell membrane the disintegrating cytoplasm is released into the haemolymph in which it dissolves completely.The ultrastructural organization of a basophilic mesodermal cell is quite similar to that of a young fat cell. They become larger and many membrane limited vacuoles are found in the cytoplasm. In the nucleus vacuolarlike structures develop, which is followed by condensation of the chromatin. During this process the nucleus becomes lobed with deep invaginations after which the nuclear envelope is dissolved leading to the release of the granulated chromatin material into the cytoplasm. The first degenerating basophilic mesodermal cells are observed only in a nine days old larva in the vicinity of the cornicles.The growth and degeneration of fat and basophilic mesodermal cells in viruliferous larvae show the same picture as that described for nonviruliferous ones (Figs. 21 and 22). In one instance a fat cell was found in a leafroll virus carrying larva containing an inclusion with dense particles measuring 23 nm in diameter with a hexagonal profile, and which presumably represent PLRV (Fig. 37 A).