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. CuCl(2) and ZnCl(2) increase the activity of TLSOD2 and TLSOD3, while MnCl(2) 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.

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+.

First culture isolation of Borrelia lonestari, putative agent of southern tick-associated rash illness.

Southern tick-associated rash illness (STARI) is a Lyme disease-like infection described in patients in the southeastern and south-central United States, where classic Lyme disease is relatively rare. STARI develops following the bite of a lone star tick (Amblyomma americanum) and is thought to be caused by infection with an "uncultivable" spirochete tentatively named Borrelia lonestari. In this study, wild lone star ticks collected from an area where B. lonestari is endemic were cocultured in an established embryonic tick cell line (ISE6). The cultures were examined by dark-field microscopy for evidence of infection, and spirochete identity and morphology were evaluated by flagellin B and 16S rRNA gene sequence, by reaction to Borrelia-wide and B. burgdorferi-specific monoclonal antibodies, and by electron microscopy. Live spirochetes were first visualized in primary culture of A. americanum ticks by dark-field microscopy 14 days after the cell culture was inoculated. The sequences of the flagellin B and 16S rRNA genes of cultured spirochetes were consistent with previously reported sequences of B. lonestari. The cultured spirochetes reacted with a Borrelia-wide flagellin antibody, but did not react with an OspA antibody specific to B. burgdorferi, by indirect fluorescent antibody testing. Electron microscopy demonstrated organisms that were free and associated with ISE6 cells, with characteristic Borrelia sp. morphology. This study describes the first successful isolation of B. lonestari in culture, providing a much needed source of organisms for the development of diagnostic assays and forming a basis for future studies investigating the role of the organism as a human disease agent.

Serine proteinase inhibitors from eggs and larvae of tick Boophilus microplus: purification and biochemical characterization.

The present study describes the purification, characterization, and comparison of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs between the first day of hatching and the beginning of eclosion (defined as El, E2, and E3) and of larvae between the first day of eclosion and the infectant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCI buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from E1 to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant Ki determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and larvae (BmTls) were purified on trypsin-Sepharose column and analyzed by SDS-PAGE. The results showed a slight difference in protein pattern, with a protein band of 20 kDa in the E1 and E2 samples which did not appear in the other samples. The Ki for neutrophil elastase was 10-fold lower in L3 than E1. BmTI reverse-phase chromatography showed two and one major peaks in egg and larva samples, respectively. The N-terminal amino acid sequence of the L3 main peak from a C8 column showed a mix of BmTIs with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition activity suggest different roles for BmTIs during the development process.

Experimental teratogeny in the tick Hyalomma marginatum marginatum (Acari: Ixodida: Ixodidae): effect of high humidity on embryonic development.

The effect of 90% RH on the embryonic development of Hyalomma marginatum marginatum Koch was investigated at 25 degrees C. Under the influence of this factor, 2.1% dead eggs, 13.0% dead embryos, 6.9% abnormally hatched larvae, 0.2% larvae with malformations, and 77.8% normal larvae appeared. The embryos died during the cleavage of nuclei, the formation of the blastoderm, the formation of the germ band and its metamerization, and the differentiation of the leg anlagen. Egg hatch was also inhibited in various phases. Various kinds of anomalies were observed in larvae of Hyalomma m. marginatum. Most teratological changes (70.8%) occurred within the idiosoma. They were hetromorphose (32.6%), oligomely (15.4%), heterosymely (12.3%), symely (1.5%), atrophy (6.1%), and ectomely (3.1%). Anomalies within the gnathosoma occurred rarely (3.1%). As many as 26.2% larvae had composite anomalies (oligomely, heterosymely, atrophy) together. They contained various structures of the gnathosoma or idiosoma. These anomalies decreased the survival rate of the larvae. The investigations showed that during the formation of the blastoderm, the formation of the germ band and its metamerization the embryos have the largest susceptibility of being affected by high humidity. Some anomalies in specimens collected from nature may develop under influence of unfavorable humidity levels.

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.

[The impact of electromagnetic radiation at microwave frequency (9.8 HhZ)on the embryonic and postembryonic development of the tick Hyalomma asiaticum (Acarina, Ixodidae)]. / Vliianie élektromagnitnogo izlucheniia svch diapazona (9.8 ggts) na émbrional'noe i postémbrional'noe razvitie kleshcha Hyalomma asiaticum (Acarina, Ixodidae).

Low-power (20 microW/cm2) microwave-modulated radiation at a carrier frequency of 9.8 Hhz is shown to affect the course and specific features of ontogenesis of the ick H. asiaticum. The actin of microwave radiation on the development of H. asiaticum substantially depends on the frequency of microwave modulation of a signal and on the temperatures of an experiment. When the temperature is 22 degrees C, there is a significant suppression of development of fed larvae and nymphs after exposure to microwave radiation at modulated signal frequencies of 3 and 5 Hz/ The whole range of the tested modulation frequencies was 2 to 16 Hz. The hungry species of all developmental phases in H. asiaticum were virtually unresponsive to exposures. At 14 degrees C (a perithreshold temperature of H. asiaticum development), the action of microwave radiation changed from inhibitory to stimulating. At modulation frequencies of 3, 5 and 7 Hz, the proportion of hatching larvae was 42.5, 67.5 and 80.0%, respectively, and that of controls was 2.5%. Whether the size of a H.asiaticum population can be controlled by a radar that provokes the development of ticks before winter by its radiation is discussed.

Purification and characterization of beta-N-acetylhexosaminidase from bovine tick Boophilus microplus (Ixodide) larvae.

beta-N-Acetylhexosaminidase (HEX, E.C. 3.2.1.52) from larvae of the ixodid tick Boophilus microplus was purified to capillary zone electrophoresis homogeneity, and characterized. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-200, p-aminobenzyl-N-acetyl-beta-D-thioglucosamine affinity, and Mono-Q FPLC columns. Purification was about 1600-fold, with a yield of 10%, as determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme presented optimum pH 4.7, and optimum temperature 65 degrees C. The molecular weight of non-denatured enzyme was estimated as 127,000 by gel filtration chromatography, and 60,000 in SDS-PAGE. The tick hexosaminidase presented glycosyl residues, as evidenced by binding to Concanavalin-A. Among several p-nitrophenyl glycosides tested as substrate, HEX was active only on p-nitrophenyl-N-acetylglucosaminide and p-nitrophenyl-N-acetylgalactosaminide. The purified enzyme presented immunogenicity in rabbit, and the correspondent antibodies inhibited about 90% of its original, in vitro activity.

Immunization of rabbits against Hyalomma anatolicum anatolicum using homogenates from unfed immature ticks.

Antigens were prepared from unfed larvae and nymphs of H. a. anatolicum as homogenised antigens (HLAg and HNAg, respectively). Five rabbits each were inoculated, s.c. with 8.56 mg HLAg and 9.34 mg HNAg in 3 divided doses. Following immunisation rabbits developed significant level of protective immunity to infestation with adults of this species. Significant reduction in engorged percentage and weights of engorged females and egg masses were observed in females fed on immunized rabbits, compared to that of female ticks fed on control rabbits. The engorgement period was also increased significantly. However, conversion efficiency indices remained unaffected. Larval antigen immunized rabbits showed significant antibody level from 28-126 days while with HNAg elevated antibody levels were recorded up to 112 days. Further, the rabbits immunized with HLAg had elevated level of antibodies against HLAg, HNAg, and adult antigen in ELISA. But HNAg immunized rabbits had lower levels of antibodies against HLAg and HAAg as compared to values recorded against HNAg. Anti-HLAg and anti-HNAg sera recognised common antigenic bands of 97.4, 85, 66, 47.3, 42 and 31 kDa in homogenates of larvae, nymphs and adults.

Traffic of the tick embryo basic protein during embryogenesis 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 microM for leucine-p-nitroanilide. The involvement of the exopeptidase activity in the TEBP turnover after its translocation to the cytoplasm is discussed.

Rickettsia rickettsii growth and temperature-inducible protein expression in embryonic tick cell lines.

Rickettsia rickettsii has limited adverse effects on its arthropod vector, but causes severe disease in man. To model differences in host-parasite interaction, R. rickettsii growth and protein expression were examined at temperatures reflective of host environment in the tick cell lines DALBE3 and IDE2, the human endothelial cell line ECV304, and the African green monkey kidney cell line Vero76. At low multiplicities of infection, rickettsial titres increased 10(2)-10(3)-fold in all cell lines after incubation for 3 days at 34 degrees C. At higher multiplicities and with extended incubation, R. rickettsii showed enhanced survival in tick versus mammalian cells. No difference in rickettsial ultrastructure or protein profiles was detected between different host cell types. Rickettsial proteins of 42, 43, 48, 75 and 100 kDa are induced in tick cells shifted from 28 degrees to 34 degrees C, but not in cells maintained at 28 degrees C. This temperature response may be associated with expression of rickettsial determinants that are pathogenic to mammalian hosts.

Enzymes of delta 1-pyrroline-5-carboxylate metabolism in the camel tick Hyalomma dromedarii during embryogenesis. Purification and characterization of delta 1-pyrroline-5-carboxylate dehydrogenases.

The activity of P5C metabolizing enzymes: OAT, P5CR, PO, and P5CD, in the camel tick Hyalomma dromedarii has been followed throughout embryogenesis. The profiles of enzymatic activity showed clear differences in the four enzymes as the embryos grew older. During purification of P5CD to homogeneity the ion exchange chromatography steps lead to two separate forms (termed A and B) with different molecular weights (60,000-59,000 and 50,000-52,000 for the native and denatured enzymes, respectively), amino acid composition, Km for P5C and coenzymes, varying dehydrogenase activities with different substrate specificity when supplied with various aldehyde substrates. Both P5CD A and B exhibited sharp optima at pH 7.5. The effect of different divalent cations and competitive and noncompetitive inhibitors was examined. The changes in P5C metabolizing enzymes during embryogenesis suggest that H. dromedarii has the metabolic potential to convert ornithine into proline and glutamate.

Effects of ingested phytoecdysteroids in the female soft tick Ornithodoros moubata.

The effects of the ingestion of some phytoecdysteroids were studied in the soft tick Ornithodoros moubata. Supernumerary moulting and malformations of first leg pairs were obtained with 22-oxo-20-hydroxy-ecdysone, 20-hydroxyecdysone-22-acetate, and 20-hydroxyecdysone-22-benzoate. Egg-yield was reduced with 20-hydroxyecdysone-22-acetate and carthamosterone. Finally, drying-out of eggs was observed with carthamosterone and 22-deoxy-20,26-dihydroxyecdysone. In addition, we demonstrated that there is a correlation between the number of completed gonotrophic cycles and the impossibility of inducing supernumerary moulting.

Purification and characterization of a novel acid-soluble nuclear protein from developing embryos of the camel tick Hyalomma dromedarii (Acarina: Ixodidae).

A novel acid-soluble protein has been extracted from nuclei of developing embryos of H. dromedarii ticks and purified to homogeneity. This tick embryo basic protein (TEBP) was predominant during the cleavage stage of tick embryogenesis, whereas the complete set of histones was detectable at the late cleavage stage. The amount of TEBP reaches a maximum value at day 9 after oviposition. Thereafter, the original N-terminal dipeptide (leucine-serine) is eliminated. This coincides with the start of organogenesis. In spite of its low molecular mass, TEBP seems to be related to histone H1 in some properties such as solubility in perchloric acid and binding affinity to DNA. A task for the future will be to define the role of this protein as a counterpart of the histones for the genome organization during embryogenesis.

Pathways of regeneration of Haller's sensory organ during the life cycle of the tick Hyalomma asiaticum.

A study of regeneration in nymphs and adults of the Asiatic desert tick, Hyalomma asiaticum, from which forelegs had been amputated during the previous instar, revealed that regenerated Haller's sensory organs exhibited significant changes in structure. Adult regenerates possessed atavistic features in terms of the number and topography of different sensillar types. Regularity in these changes was similar to that demonstrated in regenerates of Ixodes ricinus (Leonovich and Belozerov [1992] Exp. Appl. Acarol., 15:59-79). Nymphal regenerates, by contrast, had larval features of the parent species. An hypothesis is presented about the ontogenesis of the complete, peripheral, polymodal sensory organ as a process in which the initial cell differentiation of cells begins at different starting points.

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.

[Teratologic changes complicating taxonomical studies of ticks (Acari: Ixodida)]. / Zmiany teratologiczne utrudniajace badania taksonomiczne kleszczy (Acari: Ixodida).

Various kinds of morphological anomalies, i.e. general (the changes in the shape and the asymmetry of body, the duplication of body, the nanism, the gigantism and the gynandromorphism), and local (oligomely, atrophy, polimely, heterosymely, symely, schistomely, ectomely, heteromorphose, disturbances in the structure of leg segments, cyclopy) occur in Ixodida. The anomalies within taxonomically important structures make the determination of tick species difficult or even impossible. Therefore, the anomalies deforming systematic features of different instars from Argasidae and Ixodidae families were first of all taken into account.

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.

Studies on the biology of Argas (A.) reflexus (Fabricius, 1794) (Acari: Ixodida: Argasidae). 2. Effect of alternating temperatures on embryonic development and egg hatch.

The paper presents the results of observations on the effect of temperature alterations between 9 degrees C and 30 degrees C every 6 and 12 hours, respectively, on the embryonic development and egg hatch of Argas (A.) reflexus. No effect of the frequency of temperature changes on the percentage of egg mortality, embryo mortality, abnormal egg hatching, or egg hatching into morphologically normal larvae was observed. The experiments showed that in changes temperature have a particularly detrimental effect on the eggs prior to blastulation.

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.