Light and electron microscopy studies of the midgut and salivary glands of second and third instars of the horse stomach bot, Gasterophilus intestinalis.

A morphological study of the midgut and salivary glands of second and third instars of Gasterophilus intestinalis (De Geer) (Diptera: Oestridae) was conducted by light, scanning and transmission electron microscopy. The midgut is anteriorly delimited by a proventriculus, without caeca, and is composed of posterior foregut and anterior midgut tissue from which a double-layered peritrophic matrix is produced. The midgut can be divided into anterior, median and posterior regions on the basis of the structural and physiological variations of the columnar cells which occur along its length. Two other types of cell were identified: regenerative cells scattered throughout the columnar cells, and, more rarely, endocrine cells of two structural types (closed and open). Different secretion mechanisms (merocrine, apocrine and microapocrine) occur along the midgut epithelium. Abundant microorganisms are observed in the endoperitrophic space of the anterior midgut. The origin and nature of these microorganisms remain unknown. No structural differences are observed between the second and third instar midguts. The salivary glands of G. intestinalis second and third instars consist of a pair of elongated tubular structures connected to efferent ducts which unite to form a single deferent duct linked dorsally to the pharynx. Several intermediate cells, without cuticle, make the junction with the salivary gland epithelium layer. Cytological characteristics of the gland epithelial cells demonstrate high cellular activity and some structural variations are noticed between the two larval stages.

The life-cycle of the bont tick Amblyomma hebraeum in vitro.

The life-cycle of the hard tick Amblyomma hebraeum was completed in vitro by feeding all life-stages of the tick through silicone membranes on bovine blood from an abattoir. Ticks were placed in a simple feeder membranes on bovine blood from an abattoir. Ticks were placed in a simple feeder consisting of a honey jar containing the blood with a glass tube insert (o.d. 42 mm) across the end of which the membrane was stretched. This feeding unit was held in a water bath (38 degrees C). Larvae and nymphs fed on a membrane (< 90 microns thick) made of silicone reinforced with Kodak lens cleaning paper, and adults on a silicone membrane (0.5 mm thick) reinforced with Terylene netting. To control microbial growth, gentamicin (5 micrograms/ml) and nystatin (100 i.u./ml) were added to the weekly open-collected blood, which was manually defibrinated. The blood was changed twice daily for nymphs and three times for adults and larvae. Attachment of ticks was induced with combinations of host hair, tick faeces, a bovine pelage extract and a synthetic aggregation-attachment pheromone mixture. The in vitro life-cycle started with unengorged "natural" adults, which had moulted from nymphs fed on steer. The life-cycle closed with unengorged, first in vitro generation adults which had moulted from nymphs fed in vitro. Although the feeding and development of larvae and nymphs were similar to in vivo controls, females fed and developed poorly in vitro. The toxicity of the systemic acaricide Ivermectin for nymphs of A. hebraeum was confirmed using the in vitro feeding method.

Ecdysteroid titre and metabolism and cuticle deposition during embryogenesis of the ixodid tick Amblyomma hebraeum (Koch).

Three embryonic cuticles are formed before larval cuticle deposition during embryonic development of Amblyomma hebraeum. The quantity of radioimmunoassay-positive material varied between 50 and 200 pg ecdysone equivalents per mg, but no significant peaks were detected. Maternally incorporated [3H]-20-hydroxyecdysone and [3H]-ecdysone contained in freshly laid eggs appear to be conjugated to C-22 fatty acid esters and 3 alpha epimers of those esters, and, thus, appear doubly inactivated. In addition, ecdysone is converted to an unknown product called 2'. The role of these maternally derived ecdysteroids is unknown.

Cuticle alkanes of honeybee larvae mediate arrestment of bee parasiteVarroa jacobsoni.

The ectoparasitic miteVarroa jacobsoni invades worker brood cells of the honeybeeApis mellifera during the last 20 hr before the cells are sealed with a wax cap. Cuticle extracts of 8-day-old worker honeybee larvae occupying such brood cells have an arrestment effect on the mite. The mites run for prolonged periods on the extract, systematically returning onto the stimulus after touching the borders of the treated area. Mites increase walking speed and path straightness in response to increasing doses of a nonpolar fraction of the cuticle extract. Saturated straight-chain odd-numbered C19-C29 hydrocarbons were identified by thin-layer argentation chromatography and gas chromatography-mass spectrometry as the most active constituents, with branched alkanes also contributing to the arrestment effect of this active fraction. Analysis of the behavior responses to syntheticn-alkanes indicate that the response is probably based on a synergism between the different alkane components of the fraction rather than to an individual compound.

Contrasting effects of sera from rabbits and cattle infested with ticks on the in vitro feeding performance of the tick Rhipicephalus appendiculatus.

The in vitro feeding activity of partially engorged Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae) females fed on sera from uninfested hosts was compared to that of ticks fed serum of hosts which had previously been infested with ticks. Although ticks fed best on sera from bovid hosts which had no prior exposure to this ectoparasite, two infestations of a bovid had no significant effect on the acceptability of its serum. In contrast, ticks fed sera from rabbits which had twice been infested with ticks gained significantly less weight than those fed serum obtained from the same animals before the infestations. Clearly there is a difference between natural host resistance and that of laboratory animals.

Metabolism of [3H]-ecdysone in embryos and larvae of the tick Ornithodoros moubata.

The fate of [3H]-ecdysone ([3H]-E) was investigated in hanging drop cultures of embryos and larvae of the tick Ornithodoros moubata using HPLC. The hormone was metabolized more slowly during described periods of increasing endogenous ecdysteroid (ES) titers than during periods of low titers except for young embryos. Three different classes of metabolites were produced: 1) apolar products (AP) corresponding to C-22 fatty acid ester conjugates of E and, in some cases, of 20-hydroxyecdysone (20E), 2) unidentified polar products (PP) more polar than E, one peak of which had the same retention times as 20,26-dihydroxyecdysone, and finally, 3) 20E verified by comigration of cold standards on RP-18 and silica columns. Hydroxylation of E to 20E first became evident in cultures of 2 day old embryos and was present in all cultures of older animals. Highest production of free 20E occurred during increasing endogenous ES titers in embryos and during the ES peak in larvae. Conjugation of E to AP occurred in all stages investigated, but was more pronounced during periods of low endogenous ES titers, and may correspond to a detoxification mechanism. In contrast, PP were produced during high 20E production in embryos and during periods of high and decreasing endogenous titers in larvae.

Cuticle deposition and ecdysteroid titers during embryonic and larval development of the argasid tick Ornithodoros moubata (Murray, 1877, sensu Walton, 1962) (Ixodoidea:Argasidae).

Timing of embryonic and larval molts at the ultrastructural level and presence of ecdysteroids (ES) during embryonic and larval development of the argasid tick Ornithodoros moubata were studied. Embryonic "cuticles" A, B, and C were deposited 24-30 hr, 48-56 hr, and 6 days after oviposition, respectively. Deposition of the larval cuticulin layer started on Day 8 of embryonic development and procuticle deposition continued after hatching until apolysis of the larval cuticle 40 hr posthatch. Plaques of cuticulin formed on tips of microvilli 48-56 hr after hatching and procuticle was deposited until after ecdysis. Radioimmunoassay (RIA) was used to determine the ES titer in methanolic extracts of various ages of embryos and larvae. No peaks of RIA-positive material were detected during deposition of envelopes A, B, and C. However two peaks of ES were observed during embryonic development, one which coincided with the shortening of the germ band and a second which coincided with the deposition of the larval epicuticle on Day 8. During larval development, a peak of ES was observed on Day 3 (48-56 hr posthatch) and was correlated with nymphal epicuticle deposition. HPLC-RIA revealed that these last two peaks consisted mainly of 20-hydroxyecdysone together with a small quantity of ecdysone. Conjugated RIA positive material was present in freshly laid eggs and an augmentation of this esterase hydrolysable material was noted at the appearance of each ES peak. Thus the embryos did not appear to be hydrolyzing the maternal apolar conjugates to release ES during embryonic development; on the contrary, they appeared to be conjugating the newly synthesized hormones.

Biosynthesis, production site, and emission rates of aggregation-attachment pheromone in males of twoAmblyomma ticks.

The aggregation-attachment pheromone componentso-nitrophenol (ONP) and methyl salicylate (MS) in maleAmblyomma variegatum ticks appeared after three days of feeding on the host and reached high values after about six days. Variable quantities of 1.3-7.3 µg ONP and about 0.6 µg MS were present within ticks. ONP and MS were released at the high rates of 300-1800 ng/hr and 20-600 ng/hr per male tick, respectively. After a temporary decrease, males continued to emit at high rates after nearby attachment of females. InA. hebraeum, ONP showed a similar pattern, but with a delay of about a day. A male, which had fed during 14 days, contained about 2 µg and released 225-280 ng/hr. Emission in forcibly detached males of both species dropped rapidly to low levels of less than 10 ng/hr per tick. Host skin and tick feces in the vicinity of feeding males were pheromoneimpregnated. The very high emission rates are consistent with the observations that the pheromone is an important component of the host-location mechanism of conspecifics. ONP and MS are produced in the dermal glands type 2 associated with the ventrolateral cuticle.

Metabolism of ecdysteroids in the female tick Amblyomma hebraeum (Ixodoidea, Ixodidae): accumulation of free ecdysone and 20-hydroxyecdysone in the eggs.

[3H]-20-hydroxyecdysone ([3H]-20E) injected into Amblyomma hebraeum females 7 days before the beginning of oviposition, viz. at the beginning of vitellogenesis, was converted to 3 polar peaks of unknown nature called 1, 2 and 3, and to apolar conjugates AP1, AP2 and AP3. AP2 have the same retention times as the esters of 20E with long chain fatty acids described in Ornithodoros moubata (Diehl et al. 1985). However, principally unmetabolized 20E was incorporated into the ovaries, and 16% of the injected labelling was recovered in the eggs, 3/4 being free 20E. When 20E was injected during oviposition, it was not converted to the polar products but only to the apolar products. At this time, 76% of the total radiolabel injected accumulated in the egg-batch, principally in the form of free unmetabolized 20E. After injection of [3H]-ecdysone ([3H]-E), the three polar metabolites 1', 2' and 3', probably 20-deoxy homologues of 1, 2 and 3 described above were always produced irrespective of the time of injection. In addition, E was metabolized to 20E and to the apolar conjugates AP1, AP2, and AP3. E, 20E and peak 2' were incorporated into the ovary within the first day after injection. These 3 compounds were found in freshly laid eggs in variable proportions, the quantity of E decreasing with time while 20E and peak 2' increased. At the end of oviposition, ca. 60% of the injected radiolabel had been incorporated into the eggs. Apolar products and polar metabolites accumulating in the body were apparently not used as a source of free hormone for the eggs. Our results with tritiated ecdysteroids confirm our data concerning endogenous ecdysteroids of the eggs of A. hebraeum (Connat et al. 1985). This species, in contrast to 2 other female ticks, Ornithodoros moubata and Boophilus microplus, incorporates free E and 20E instead of ecdysteroid conjugates into its eggs. The role of these free ecdysteroids remains to be elucidated.

Metabolism of [3H]ecdysone by isolated tissues of the female ixodid tick Amblyomma hebraeum (Ixodoidea; Ixodidae).

Malpighian tubules, gut, ovaries and carcasses of the adult female tick Amblyomma hebraeum were incubated in vitro in the presence of 2 microM [3H]ecdysone. Organs and media were separately extracted after 6, 24 and 48 h incubations and the patterns of ecdysone metabolites were analyzed by HPLC. Esterase-susceptible apolar metabolites similar to the AP2 already described in the soft tick Ornithodoros moubata and thus presumably corresponding to the same conjugates (C-22 esters with fatty acids) were rapidly produced in all tissues investigated. They were mainly found within the organs but they were also released into the medium to some extent. By contrast, less apolar metabolites corresponding to the AP1 esters were mainly found in the media. Malpighian tubules and gut were the most active organs regarding the conversion of ecdysone into 20-hydroxyecdysone (20E). However, only low quantities of 20E were formed, reaching respectively 12.5% and 11.6% of the total metabolites after 48 h incubations. In the carcass and in the ovary the formation of 20E was only a minor pathway (1.7% and 3.1% of the total metabolites after 48 h). In ovaries we observed a massive conversion of ecdysone into 3-epiecdysone, which (as in insects) presumably proceeded through the intermediate formation of 3-dehydroecdysone. These two compounds were identified among the metabolites by CI/D mass spectrometry. The 3-epimer was released into the media, in contrast with the AP2 which were essentially stored within ovaries. Epimerization was also realized to some extent by carcasses, and again the epimer was released into the culture media. The different pathways are compared with those found in other tick species and in insects, and the significance of the various metabolites is discussed.

Ornithodoros moubata: spermateleosis and secretory activity of the sperm.

Cytological aspects of spermateleosis in the tick Ornithodoros moubata were studied by electron microscopy. During spermateleosis, detachment of the operculum from the outer sheath of the prospermium results from the fusion of the plasma and the cisternal membranes. The fusion occurs between the shoulder of the acrosomal vesicle and the electron-dense layer of the operculum. A factor inducing vitellogenesis and egg-laying is secreted by the sperm cell after spermateleosis, and begins after the cell is almost completely devaginated. In vitro, fully devaginated spermiophores secrete most of this factor during the first 12 hr of incubation. The vitellogenesis-inducing activity of the secretion is sensitive to proteinase K (EC 3.4.21.14) digestion and correlates with the presence of two high-molecular-weight proteins in the sperm cell incubation medium.

Metabolism of ecdysteroids during the vitellogenesis of the tick Ornithodoros moubata (Ixodoidea, Argasidae): accumulation of apolar metabolites in the eggs.

The fate of injected [3H]ecdysone [( 3H]E) and 20-hydroxy-[3H]ecdysone [( 3H]20E) has been investigated in the female tick Ornithodoros moubata (Murray, 1877; sensu Walton, 1962). When injected into fed mated vitellogenic females, [3H]E is converted into [3H]20E and two apolar classes of metabolites, AP1 and AP2. Injected [3H]20E is directly converted into AP1 and AP2. AP2 is incorporated into the ovaries in a high proportion and at the end of the vitellogenic cycle represents about 25% of the total label recovered from the animal. The fate of labeled hormones injected into virgin females which perform an abortive vitellogenic cycle is quite similar. However, the ovaries incorporated less of the AP2 products. Ovaries of mated females cultured in vitro in the presence of [3H]E are able to produce [3H]20E and AP2. AP2 is incorporated, while [3H]20E is mainly found in the medium. Ovaries of virgin females presented a slower rate of transformation and of incorporation of the label. Labeled AP2 is recovered in freshly laid eggs and AP1 in the females after oviposition. AP1 and AP2 can produce [3H]20E, [3H]E, and other minor polar peaks when submitted to hydrolysis by esterase. It is concluded that the female O. moubata possesses a special enzymatic mechanism for transformation of ecdysteroids into apolar products and for selective incorporation of AP2 into the ovaries. These products are present in the freshly laid eggs and could play a role during embryogenesis.

The ultrastructure of Rickettsia slovaca in naturally infected females of the tick Dermacentor marginatus.

The ultrastructure of Rickettsia slovaca in naturally infected females of the tick Dermacentor marginatus is similar to that of other members of the spotted fever group rickettsiae. R. slovaca was found to multiply in all organs, tissues and cells examined. It is primarily found free in the cytoplasm; occasionally it may also invade the cell nucleus. Special attention was paid to the rickettsiae in the salivary gland. They multiply in all cells of the three acini types I-III and also in the duct cells. The amount of rickettsiae eliminated via the saliva reached values of about 10(4)EID50/ml.

Regulation of body volume by salivation in a tick challenged with fluid loads.

Injection into the hemolymph of 1.2% NaCl, 11.2% sucrose, 2.3% urea (all approximately isosmotic to hemolymph), or distilled water induced salivary fluid secretion in the ixodid tick Amblyomma hebraeum Koch. Saline gave the largest response at high doses. Injection of hyperosmotic NaCL into the hemolymph did not induce salivation but led to the drinking of distilled water in amounts sufficient to dilute the salt load to isosmolarity. Atropine only partially inhibited salivation induced by NaCl, sucrose, and distilled water. Reserpine markedly inhibited salivation induced by NaCl. We propose that at least two sensory pathways (one cholinergic, one not) converge on the secretory nerve. The physiological significance of the cholinergic pathway is not known. The other pathway probably mediates the regulation of hemolymph volume, possibly via stretch receptors, but its transmitter is not known.

Na, K-ATPase in the salivary gland of the ixodid tick Amblyomma hebraeum (Koch) and its relation to the process of fluid secretion.

Total and ouabain-sensitive ATPase activities were determined in the salivary glands of ticks throughout the feeding cycle. Activities were very low in unfed specimens. In the glands of feeding females, the activities rose until a maximum was reached for both ATPase components at approximately 200 mg. The activities remain low in males throughout the feeding period. These findings are discussed in relation to the fluid secretory process of the salivary glands.