Electron-Microscopic Studies on Theileria ovis Rodhain, 1916: Development of Kinetes in the Gut of the Vector Tick, Rhipicephalus evertsi evertsi Neumann, 1897, and Their Transformation Within Cells of the Salivary Glands

SYNOPSIS. Gamogony of Theileria ovis Rodhain occurs within the gut of nymphs of the tick Rhipicephalus evertsi evertsi Neumann. After molting, spherical and ovoid parasites are found within the intestinal cells of the ticks. These stages are thought to be zygotes, because they undergo a transformation process leading ultimately (in 3 days) to the formation of a motile stage, the kinete , the fine structure of which is very similar to that of the ookinetes of the hemosporidia. The kinete leaves the gut cells of the tick and penetrates the salivary gland cells where it produces infective stages (the sporozoites ). These stages may be transmitted to sheep during the next blood meal of the tick. The developmental processes of T. ovis are compared to those of Hemosporina.     

Theileria parva: variation in the infection rate of the vector tick, Rhipicephalus appendiculatus

The variation in Theileria parva infection rates of experimental batches of adult Rhipicephalus appendiculatus ticks, used during the course of several years, was examined. It was found that considerable variation occurred, but that this could not always be correlated with the piroplasm parasitaemia in the cattle on which the ticks engorged as nymphs. Statistical analysis showed that the infection rate of ticks fed on cattle with a parasitaemia of 41–50 per cent was significantly higher than that of ticks fed on cattle with lower parasitaemias. A number of experiments were then carried out in which one or several factors of this aspect of the host-parasite relationship remained constant whilst others were altered. None of these factors was seen to play a major part in the variation. Finally, randomly selected groups of 10 ticks which had dropped engorged as nymphs from the same animal on the same day were examined. The variation observed even in these groups was so great that it was concluded that the infection rate could depend on a factor such as the juxtaposition of possibly-infected gut epithelial cells and developing salivary glands during the nymphal moult.     

Ultrastructural demonstration of succinic dehydrogenase and cytochrome oxidase activity in sporozoites of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in salivary glands of tick vectors (Rhipicephalus bursa, Hyalomma anatolicum excavatum)

Salivary gland stages ("sporozoites") of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in female ixodid ticks were studied for ultracytochemical activity of the respiratory enzymes, succinic dehydrogenase (SDH), and cytochrome oxidase. Both SDH and cytochrome oxidase were demonstrated in the sporozoites and the mitochondria in these stages. Identified in this way the final reaction product of SDH was located mainly at the inner side of the mitochondrial boundary, though it was also visible in the internal space of the organelle. Cytochrome oxidase activity always was confined to the wall of mitochondria. This enzyme was demonstrated also in the erythrocyte stage of B. ovis. The cytochemical results indicate respiratory potential of the piroplasmean stages studied. Cristate or typical protozoan mitochondria have not been observed in sporozoites of Babesia or Theileria. This report is the first demonstration of mitochondria, or mitochondrialike activity in Babesia.     

Markers of cell death in salivary glands of males of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae)

The salivary glands of Rhipicephalus sanguineus males at stages: unfed (control), at day seven post-attachment, and at days three and seven post-detachment from the host were examined using methods of enzymatic analysis and cell viability. At these stages of feeding, different staining patterns were observed in the cells of type IV, III, II and I acini, which were affected by degeneration in this sequence. Acid phosphatase reaction was inversely proportional to that of ATPase, while ATPase reaction was proportional to membrane integrity. Salivary gland cells of unfed males exhibited intact nucleus and plasma membrane, suggesting that the acid phosphatase detected may participate in the normal physiology of acini. In males at day seven post-attachment, intact membranes were observed in almost all types of acini, as well as stronger reaction for acid phosphatase, nuclear changes, and decrease in ATPase reaction, changes associated with the degenerative process. At days three and seven post-detachment degeneration progress, being observed loss of membrane integrity, nuclear changes, prominent decrease in ATPase reaction, and an increase in acid phosphatase reaction in the first case and a decreased of it at day seven post-detachment from the host. During cell death, alterations occurred in the following sequence: a) nuclear changes, b) loss of ATPase reaction, c) loss of integrity of the plasma membrane, and d) increase of acid phosphatase. The latter might be associated with the late degradation of cytoplasmic remnants, characterizing the process of cell death in glands of R. sanguineus males as atypical or non-classic apoptosis.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Endocannabinoids and related fatty acid amides,and their regulation,in the salivary glands of the lone star tick

The salivary glands and saliva from the lone star tick Amblyomma americanum (L.) were analyzed for the presence of the two endogenous agonists of cannabinoid receptors, N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as of the anandamide congener, N-palmitoylethanolamine (PEA), an anti-inflammatory and analgesic mediator that is inactive at cannabinoid receptors. Two very sensitive mass-spectrometric techniques were used for this purpose. Both 2-AG and PEA, as well as other N-acylethanolamines (NAEs), were identified in salivary glands, but anandamide was below detection. The levels of 2-AG were considerably higher in the salivary glands of partially fed than replete females. Ex vivo gland stimulation with arachidonic acid increased the levels of 2-AG, but not of PEA or other NAEs, and caused the formation of anandamide and of the potent analgesic compound N-arachidonoylglycine. Instead, the amounts of anandamide, 2-AG and PEA were not influenced by treatment of salivary glands with dopamine, which stimulates saliva secretion. The possible biosynthetic precursors of anandamide, PEA and other NAEs were also detected in salivary glands, whereas only PEA was detected in tick saliva. These data demonstrate for the first time that the salivary glands of an obligate ectoparasite species can make endocannabinoids and/or related congeners with analgesic and anti-inflammatory activity, which possibly participate in the inhibition of the host defense reactions.     

Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi.

Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.     

Cytochemical studies on the localization of 5'-nucleotidase in the acinar cells of the rat salivary glands.

The cytochemical localization of 5'-nucleotidase (5'-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5'-AMPase and non-specific alkaline phosphatase (beta-GPase) possessed different pH optima. The cytochemical distribution of the reaction products from the 5'-AMPase activity was distinct from those of beta-GPase. 5'-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. beta-GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5'-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5'-AMPase and beta-GPase. The results indicate that 5'-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Light microscopic studies on the development of Theileria annulata (Dschunkowsky and Luhs, 1904) in Hyalomma anatolicum excavatum (Koch, 1844). II. The development in haemolymph and salivary glands (author's transl)]

Fully differentiated kinetes, average length 17.6 micrometer, appeared in the haemolymph of engorged nymphs usually 17 to 20 days after repletion. Kinetes were observed at first in the salivary glands on day 18 after repletion. The kinetes then transformed into fission bodies of about 10 micrometer in diameter, mainly in type III alveoli and less frequently in type II alveoli. The fission bodies grew up to a size of about 20 micrometer after several divisions of their nucleus. At this time the ticks moulted and no further development occurred until activation. Shortly before infestation the salivary glands began to proliferate, and rapid growth of the fission bodies was observed, especially in young ticks where development of 'infective particles' ('sporozoites') was concluded within two days. Development in feeding adult ticks apparently occurred in four major steps: (1) Division of primary fission bodies (sporonts) into numerous secondary fission bodies ('primary sporoblasts'), (2) division of secondary fission bodies into tertiary fission bodies ('secondary sporoblasts'), (3) production of particles ('sporozoites') by tertiary fission bodies and release of particles into the saliva, and (4) degeneration of fission bodies and their host cell but further release of particles. The host cell was stimulated to giant growth, thus its diameter increased, on average, from 15 to 110 micrometer. Heavy infections resulting from parasitaemias of greater than 40% caused disease and mortality in the tick population. Development was much retarded by aging. In ticks starved for six months 'sporozoites' did not develop before day five to seven of infestation. 'Sporozoites' did not develop before day five to seven of infestation. 'Sporozoites' may not develop at all in six to nine month old female ticks during the infestation period. The significance of the described developmental stages of T. annulata was discussed and a sexual generation postualted. The hypothetic development of T. annulata in its tick vector was illustrated.     

Ultrastructural characteristics of the salivary glands of the taiga tick, Ixodes persulcatus (Ixodidae). I. The granulosecreting alveoles of the fasting female

Two types of granulosecreting alveoles were found in salivary glands of hungry females by means of electron microscopy of ultrafine sections. Alveoles of the IInd type occur in the anterior helf of the gland. They are not numerous and consist of three types of secretory cells (A, B, C) surrounding the inneralveolar cavity. The secretory cells are separated from each other and from the basal membrane by the strands of the epithelial cells P. Three types of spherical inclusions were found in the secretory cells. They differ in size, electron density and intensity of staining of half-fine sections with toluidin blue. The apical cytoplasmatic membrane of secretory cells bears numerous microvilli. Alveoles of the IIIrd type, which constitute the main mass of the gland tissue, have a narrow slit-like inneralveolar cavity. The basal part of the alveole is formed by 3--4 large cells filled with large spherical electron-transparent vacuoles of the secretion. The apical part of the alveole is occupied by 9 to 11 cells E, whose cytoplasm is filled with numerous flat cisternae of granular endoplasmatic reticulum and small and medium secretory vacuoles of different electron density. Alveoles of the IInd and IIIrd type of I. persulcatus are not identical with those of Hyalomma asiaticum, Boophilus microplus and other members of the subfamily Amblyomminae.