Traffic of the tick embryo basic protein during embryongenesis of the camel tick Hyalomma dromedarii (Acari: Ixodidae)

The tick embryo basic protein (TEBP) is present in the nucleus as a counterpart of histones at early embryonic stages of the tick Hyalomma dromedarii. The sharp drop in the TEBP nuclear level and elimination of the N-terminal dipeptide (leucine–serine) between days 12 and 15 after oviposition suggested the transport of TEBP to the cytoplasm for protein turnover. The traffic of TEBP during tick embryogenesis was examined. The level of TEBP was detected in the cytoplasm from the different embryonic stages by the established enzyme-linked immunosorbent assay (ELISA) and confirmed by immunoblotting. At day 12, a 2-fold increase in the cytoplasmic TEBP level coincided with its decrease in the nucleus. This result indicates that TEBP starts to leave the nucleus for the cytoplasm at day 12. The changes in the cytoplasmic leucine aminopeptidase (LAP)-specific activity were followed during tick embryogenesis. The LAP activity started to increase at day 12 and reached its maximum level at day 21. The enzyme displayed an optimum pH between 7.5 and 8.8 and a K_m value of 0.5 M for leucine-p-nitroanilide. The involvement of the exopeptidase activity in the TEBP turnover after its translocation to the cytoplasm is discussed.     

Ultrastructure of the nymphal integument in the camel tick Hyalomma (Hyalomma) dromedarii (Ixodoidea: Ixodidae)

The ultrastructure of the nymphal integument in the ixodid tick Hyalomma (Hyalomma) dromedarii is compared for stages of development during and after feeding, and up to the first step of molting, apolysis. The integument comprises a cuticular layer and underlying epidermal cells. The body cuticle, which consists of both sclerotized and non-sclerotized parts, is divided into an outer, thin epicuticle, and an inner, thick, fibrillar procuticle. Pore canals in the procuticle are continuous with wax canals which traverse the epicuticle. As feeding progresses, the parallel, extensible epicuticular folds disappear due to the gut filling with ingested blood. The procuticular zone, however, becomes subdivided into an exocuticle, similar to the previously seen procuticle, and a lamellate endocuticle. Pore canals lose their parallel pattern and appear to have become deformed by stretching of the cuticle. The flat epidermal cells grow asynchronously during feeding; their cytoplasm becomes packed with well-developed rough endoplasmic reticulum (RER), while the cell apices project long microvilli extending deep into the procuticle. The RER undergoes ultrastructural changes indicating synthetic activity. Dense material released through the microvilli may serve to lyse the endocuticle and thus cause separation of the cuticle from the epidermis during apolysis. The lysed area, the exuvial cavity, is filled with lysed components which are probably withdrawn by endocytosis into the apical coated vesicles which appear in epidermal cells. Two types of integumental glands, which may participate in wax production, are observed in this study. The ultrastructure of their previously undescribed cuticular ducts is described, in addition to other hypodermal structures including epidermis-muscle attachments and sensory receptors.     

Fine structure of the Gene's organ in the camel tick Hyalomma (Hyalomma) dromedarii (Ixodoidea: Ixodidae)

Gene's organ of the camel tick Hyalomma (Hyalomma) dromedarii is located in the anterodorsal region of the body cavity ventrad to the scutum. It consists of a short stalk, dividing posteriorly into 2 pairs of horns and then into tubular glands. In unfed ticks, the epithelial layer of both the stalk and horns is lined internally by 2 cuticular layers; an inner, thin, greatly folded, dense layer surrounds the organ main lumen, and an outer, thick, slightly folded, less dense layer abuts the cell apices. Only the inner cuticular layer extends into the horn posterior region and appears perforated with numerous pore canals and covered with fine, cuticular projections. The horn and tubular glands epithelium is structurally consistent with a secretory function that apparently increases as feeding progresses. During oviposition, the inner cuticular layer unfolds and inflates into a pair of balloonlike structures that evert through the organ external aperture to receive and manipulate each egg as it is laid, coating it with a waxy layer that prevents desiccation. The fine cuticular projections may have a function in gripping the eggs as they leave the vagina. This organ appears to be everted by hydrostatic pressure from the hemolymph and is retracted by muscles.     

Isolation and properties of two forms of thrombin inhibitor from the nymphs of the camel tick Hyalomma dromedarii (Acari: Ixodidae)

Two forms of the nymphal thrombin inhibitors (NTI) 3.2 kDaand 14.9 kDa were purified by chromatography on CM-cellulose,Sephacryl S-300 and Sephadex G-50 columns and designated NTI-1 and NTI-2respectively. The NTI-2 turned out to be homogenous monomeric protein in bothnative-PAGE and denatured SDS-PAGE with M(r) value of 14.9 kDaapproximately and its pI value ranged from 7.2 to 7.5. The NTI-1 and NTI-2displayed anticoagulant activity since they prolonged both the activatedpartialthromboplastin time (APTT) and the prothrombin time (PT) of the camel plasma ina concentration-dependent manner. The potency of NTI-1 toward thrombin was5-fold higher than that toward FXa, while NTI-2 was 3-fold active toward FXathan thrombin. However, both of them did not inhibit any of the other examinedproteases. The types of inhibition of thrombin by NTI-1 and NTI-2 were non-competitive and competitive with inhibition constants (Ki) values of 11.7M and 211 nM respectively. One binding site wasdeduced on thrombin for each inhibitor.     

Adenosine deaminase from camel tick Hyalomma dromedarii: purification and characterization

Adenosine deaminase is involved in purine metabolism and is a key enzyme for the control of the cellular levels of adenosine. Adenosine deaminase activity showed significant changes during embryogenesis of the camel tick Hyalomma dromedarii. From the elution profile of chromatography on DEAE-sepharose, three forms of enzyme (ADAI, ADAII and ADAIII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass of adenosine deaminase ADAII was 42 kDa for the native enzyme and represented a monomer of 42 kDa by SDS-PAGE. The enzyme had a pH optimum at 7.5 and temperature optimum at 40°C with heat stability up to 40°C. ADAII had a K _m of 0.5 mM adenosine with higher affinity toward deoxyadenosine and adenosine than other purines. Ni²⁺, Ba²⁺, Zn²⁺, Li²⁺, Hg²⁺ and Mg²⁺ partially inhibited the ADAII. Mg²⁺ was the strongest inhibitor by 91% of the enzyme's activity.     

Catalase from larvae of the camel tick Hyalomma dromedarii

Catalase plays a major role in protecting cells against toxic reactive oxygen species. Here, Catalase was purified from larvae of the camel tick Hyalomma dromedarii and designated TLCAT. It was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Sephacryl S-300 and CM-cellulose columns. Gel filtration and SDS-PAGE of the purified TLCAT indicated that the protein has a native molecular weight of 120 kDa and is most likely a homodimer with a subunit of approximately 60 kDa. The Km value of TLCAT is 12 mM H₂O₂ and displayed its optimum activity at pH 7.2. CaCl₂, MgCl₂, MnCl₂ and NiCl₂ increased the activity of TLCAT, while FeCl_2, CoCl₂, CuCl₂ and ZnCl₂ inhibited the activity of TLCAT. Sodium azide inhibited TLCAT competitively with a Ki value of 0.28 mM. The presence of TLCAT in cells may play a role in protecting H. dromedarii ticks against oxidative damage. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of untraditional methods to control them.     

Hyalomma dromedarii (Acari: Ixodoidea: Ixodidae): Central and peripheral nervous system anatomy

TheHyalommadromedarii central nervous system, the synganglion, is an integrated nerve mass concentrated around the esophagus and formed by fusion of a small anterodorsal supraesophageal part an a large posteroventral subesophageal part. The supraesophageal part consists of the protocerebrum including a pair of optic ganglia, a pair of cheliceral ganglia, a pair of pedipalpal ganglia, and the stomodeal pons. The subesophageal part includes four paired pedal ganglia and the complex opisthosomatic ganglion. The peripheral nervous system includes the following pairs of nerves: optic, cheliceral, pedipalpal, primary and accessory (histologically traced); also unpaired pharyngeal and recurrent nerves, four pairs of pedal nerve trunks, each with a hemal branch, and two pairs of opisthosomatic nerves. Each peripheral nerve is traced distally to the innervation site. The salivary glands are innervated anteriorly by branches of the pedipalpal nerve and medially by branches of the hemal nerves associated with the third pedal nerves.Reprint request should be sent to: Medical Zoology Department, NAMRU-3, Fleet Post Office, New York 09527, U.S.A.     

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40 ± 2 kDa, 67 ± 1.5 kDa and 45 ± 2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl₂ and ZnCl₂ increase the activity of TLSOD2 and TLSOD3, while MnCl₂ increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.     

alpha-Amylase from developing embryos of the camel tick Hyalomma dromedarii

alpha-Amylase activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of alpha-amylase III to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). alpha-Amylase III with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of alpha-amylase III was 106 kDa for the native enzyme, composed of two subunits of 43 and 66 kDa, respectively. alpha-Amylase had a value of 10 mg starch/ml. Varying alpha-amylase activity was detected when supplied with various substrates. alpha-Amylase III had a temperature optimum at 40 degrees C with heat stability up to 50 degrees C, and a pH optimum of 7.0. The enzyme activity was activated by CaCl2, MgCl2 and NaNO3, but not activated by NaCl, p-CMB, N-ethylmaleimide and iodoacetamide. EDTA and beta-mercaptoethanol strongly inhibited activity.     

Hyaluronidase isoforms from developing embryos of the camel tick Hyalomma dromedarii

Changes in hyaluronidase activity in the camel tick Hyalomma dromedarii were followed throughout embryogenesis. Peak activity of the enzyme on days 21 and 24 during development was accompanied with a complete organization of larvae before hatching on day 27. During purification of hyaluronidase to homogeneity, ion exchange chromatography lead to four forms (HAase1, 2, 3 and 4). HAase2 and HAase4 with highest purity and specific activities after chromatography on Sephacryl S-200. The apparent molecular masses of HAase2 and HAase4 were 25 and 40 kDa, respectively. HAase2 and HAase4 had the same pH optimum of 3.6 and Km values of 0.3 and 0.34 mg/mL hyaluronic acid, respectively. Cleaving activities of HAase2 and HAase4 were demonstrated in the order: hyaluronic acid>chondroitin sulphate A>chondroitin sulphate C>chondroitin sulphate mixed>chondroitin sulphate B>heparin, low M.Wt>heparin. HAase2 and HAase4 had the same temperature optimum (40 degrees C) with heat stability up to 40 degrees C. H. dromedarii HAase2 and HAase4 had broad plateau of NaCl requirement with optimum activity recorded at 0.15 and 0.3 M NaCl, respectively. HAase2 and HAase4 were inhibited by Ca2+, Fe3+, Co2+ and Hg2+ and enhanced by Mg2+ and Mn2+.     

Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii

Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl₂, and inhibited by CaCl₂, MgCl₂, NiCl₂, and ZnCl₂.     

Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae)

Owing to the need to combat the spread of chemical acaricide resistance in ticks, we evaluated the efficacy of a mixture of the entomopathogenic fungus Beauveria bassiana AT17 and acaricides for the control of Hyalomma anatolicum anatolicum in China. A mixture of B. bassiana AT17 at the concentration of 10⁸ conidia/mL and the synthetic pyrethroid deltamethrin at concentrations of 2500, 250, 25, 5, 2.5, 0.5 and 0.25 ppm was tested in vitro. The germination capability, vegetative growth, conidia production, and viability of B. bassiana AT17 were assessed and the efficacy of the mixture in killing engorged H. anatolicum anatolicum females was measured. High mortality rates were achieved when the entomopathogen was combined with different concentrations of deltamethrin. Neither B. bassiana AT17 nor deltamethrin alone at the same concentrations could cause the higher mortality rates seen with the combination. In addition the combination killed the ticks more rapidly than did either agent alone (3–5 days more rapidly). Our results indicate that the mixture of B. bassiana AT17 and deltamethrin has potential as a new type of reagent for integrated control of H. anatolicum anatolicum.     

Fine structure of muscles in the tickHyalomma (Hyalomma) dromedarii (Ixodoidea: Ixodidae)

Skeletal and visceral muscles are distinguished in the unfed nymphHyalomma (Hyalomma) dromedarii according to position, structure and function. The skeletal muscles include the capitulum, dorsoventral and leg oblique muscles. Their muscle fibres have the striated pattern of successive sarcomeres whose thick myosin filaments are surrounded by orbitals of up to 12 thin actin filaments. The cell membrane invaginates into tubular system (T) extending deeply into the sarcoplasm and closely associated to cisternae of sarcoplasmic reticulum (SR). The T and SR forming two-membered dyads are considered to be the main route of calcium ions whose movements are synchronized with the motor impulse to control contraction and relaxation in most muscles. Two types of skeletal muscle fibres are recognized, and are suggested as representing different physiological phases.In the visceral-muscle fibres investing tick internal organs, the actin and myosin filaments are slightly interrupted, and the T and SR are well demonstrated. Both skeletal and visceral muscles are invaginated by tracheoles and innervated by nerve-axons containing synaptic vesicles.     

Effect of blood meal and mating on the genital tract ultrastructure in the female camel tickHyalomma (Hyalomma) dromedarii (Ixodoidea: Ixodidae)

The genital tract ultrastructure in the femaleHyalomma (Hyalomma) dromedarii is described during feeding and mating and up to oviposition. The vagina, consisting of vestibular (VV) and cervical (CV) regions, is formed of an epithelium lined internally with a folded cuticular layer and surrounded externally by muscle layers. These facilitate the passage of endospermatophores containing sperm into the receptaculum seminis (RS), and ova to the exterior. A pair of tubular accessory glands (AG) opening at the junction of VV and CV consist of an epithelial layer of undifferentiated cells. As feeding progresses, these cells synthesise their granular secretion which lubricates the egg surface during its passage through VV. The RS, opening anteriorly into the CV, consists of cuboidal cells lined with a thin cuticular layer. These cells become rich in glycogen and lipid vacuoles, possibly acting as a source of energy for various cell activities including granule synthesis and exocytosis. The granules discharge their contents into intercellular spaces distributed throughout the RS wall and communicate with the main lumen via narrow channels. The cell secretion may dissolve the endospermatophore wall to release sperm, while their lysosome-like structures may function in the breakdown of endospermatophoric material taken up by pinocytosis. The connecting tube (CT) opens into the CV anterodorsally and leads into the common oviduct (COV) posteriorly. It consists of an epithelium lined by a closely-adhering cuticular layer, giving the tube lumen the appearance of an undulate labyrinth with a complicated configuration. No secretory activity in the CT has been observed before, during, or after feeding. The paired, non-cuticular oviducts, extending from the ovary and fusing anteriorly to form COV, consist of an epithelium poor in cell organelles. At the final stages of feeding the cell cytoplasm contains large, phagosomal vacuoles penetrated by sperms, in addition to micropinocytotic vesicles which serve to break down the seminal fluid and other materials. The basal membrane is infolded giving characteristic features, which increase dramatically during oviposition, of epithelia involved in ion and water transport. The oviducal secretion may function as a tanning agent to harden the egg-shell and may also act as a lubricant for egg passage.     

Towards a diagnostic view of Hyalomma (Hyalomma) aegyptium (Acari,Ixodidae)

The diagnostic characters of larval, nymphal and adult Hyalomma aegyptium (L., 1758) based on specimens from the territory of most part of the area are given. In the diagnoses of immature stages, was used the characters, which were formerly tested by the author for diagnostics of other Hyalomma species occurring in the former USSR. Commonly used characters and those which were revealed by the author as useful for the majority of Euhyalomma Filippova, 1984 and Hyalommina Schulze, 1919 species, have been used in the diagnoses of male and female. Differential diagnosis of Hyalomma aegyptium. Female: genital orifice as wide arch with straight posterior margin (fig. 2, 5); vestibular part of vagina funnel-like, greatly swollen (fig. 2, 5); setae of alloscutum stick-like, tapering in apical one (figs 2, 3, 4); second segment of palps with proximal narrowing (figs 3, 1, 2); spurs of coxae I widely separated, triangular, wide, subequal in size (fig. 3, 5). Male: any grooves of conscutum absent, except short and pit-like cervical ones (fig. 4); punctation sparce and impressive (fig. 4); adenal shields short and wide, without inner branch, posterior part widened, anteromedian margin straight (fig. 5, 4); spurs of coxae I widely separated, triangular, wide, subequal in size (fig. 6, 5). Nymph: posteromedian setae of alloscutum stick-like and, as a rule, with indented apices (fig. 7, 2); spurs of coxae I large, median spur as equilateral triangle and shorter than lateral one (fig. 7, 8); spurs of coxae II-IV well developed, with acute apices (fig. 7, 8). Larva: posterior part of scutum (behind the eyes) heavy elongated, its apex straight, postero-lateral incisions weakly developed (fig. 8, 1); spurs of coxae I as equilateral triangle in shape and with rounded apices, spurs of coxae II-III very large (fig. 8, 5).     

Fine structure of the sight organs in the tickHyalomma (Hyalomma) dromedarii (ixodoidea: Ixodidae)

The paired eyes in adultsHyalomma (Hyalomma) dromedarii consists of a cuticular, transparent, convex lens extending downward as an inner, hemispherical, semitransparent projection, and underlying group of photoreceptor cells. The lateral cell cytoplasm consists mainly of interrupted cisternae of rough and smooth endoplasmic reticulum, while the cell middle side is packed with rhabdomeric microvilli. The cell basal regions become function axons forming the optic nerve. These cells are adapted to distinguish between light and darkness.     

Detection of new Francisella-like tick endosymbionts in Hyalomma spp. and Rhipicephalus spp. (Acari: Ixodidae) from Bulgaria

We report on the identification of two new Francisella-like endosymbionts (FLEs) found in three different tick species from Bulgaria. The FLEs were characterized by 16S rRNA and tul4 gene sequencing and seem to lack the molecular marker RD1. These two new taxa seem to be facultative secondary endosymbionts of ticks.     

Visual potentialities of the tick Hyalomma asiaticum asiaticum (Ixodidae)

Ethological experiments with the desert tick Hyalomma asiaticum asiaticum are conducted. It is shown that tick inhabiting open landscapes discovers his host using his eyes. Since the eyes of this species have a poor resolving possibility, black objects with white strips drawn on it are less attractive for the tick. When the tick scans an area looking for host, the principal optical axis of his eyes is directed not higher than 20 degrees. In allows the tick to discover objects having angular sizes of 5 degrees and greater.     

Morphology of the Eyes in adult Hyalomma Truncatum Ticks (Acari: Ixodidae)

The eyes of Hyalomma truncatum ticks are morphologically similar in both sexes. They appear aspaired hemispherical structures situated dorsally between the first and second pair of legs on thelateral scutal margin. Each eye consists of a lens and photoreceptor cells, which are separated by afine fibrillar layer and the hypodermis. The lens contains numerous channels which open beneaththe epicuticle, converge uninterruptedly to the interior of the lens and end below the fine fibrillarlayer. The inner closure of the lens is formed as an oval plateau with a cone-like projectionsituated caudolaterally and eccentrically to the longitudinal axis of the lens. The hypodermis is asingle layer of cells, situated immediately below the fine fibrillar layer. Beneath the hypodermis,directly below the cone-like projection of the inner lens are the photoreceptor cells localized in arosette-like arrangement. Facing the hypodermis, each photoreceptor cell is provided withnumerous microvilli. The microvilli consistently border the microvilli regions of other cells atdifferent angles but are always oriented at a right angle to the lenticular channels. Thephotoreceptor cells are unipolar neurons, whose axons arise from the basal portion of the cell andjoin to nerve fibre bundles forming the optic nerve. It is concluded that the eyes in adult H.truncatum ticks possess all the structures necessary to perceive and conduct light stimuli andvisual signals.