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.

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+.

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.

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.

Modeling embryo development and emergence of Boophilus annulatus (Acari: Ixodidae).

Guanine accumulation in Boophilus annulatus (Say) at 15 temperatures (3-42 degrees C) showed embryonic development from 9 to 42 degrees C. Guanine concentrations steadily increased over the period of development. Eggs at 17-36 degrees C achieved hatch (requiring 54-12 d, respectively), and eclosion dates were estimated for larvae developing at 12 degrees C (day 172) and 14 degrees C (day 154) using linear regression. Development rates from 12 to 36 degrees C are described by a six-parameter biophysical model for poikilothermic organisms which defines three temperature development phases characterized by low-temperature (TL, 284.7 degrees K or 11.7 degrees C) and high-temperature (TH, 307.7 degrees K or 34.7 degrees C) enzyme inactivation and a linear region (RHO25, 0.049 day-1) of no temperature inhibition. A model of emergence distribution was derived by fitting a Weibull function to a single distribution representative of the normalized emergence distribution at each temperature.

Biochemical effects of juvenile hormone III on the tick, Argas (Persicargas) arboreus (Acari: Argasidae), during embryogenesis.

The biochemical effects of juvenile hormone III (JH III) on developing embryos from treated female Argas (Persicargas) arboreus Kaiser, Hoogstraal and Kohls were examined. Exogenous JH III resulted in a decrease in total proteins (P less than 0.001) only during the first 2 d of embryogenesis. There was no significant difference (P greater than 0.05) between RNA and DNA content in eggs from control and JH III-treated females. No significant difference (P greater than 0.05) was observed between control and JH III eggs in their lipid or phospholipid contents throughout embryogenesis. A total of 14-17 protein bands and 6-8 glycoprotein bands were separated by electrophoresis during embryogenesis of A. arboreus with some differences in mobility ratio between bands from control and JH III eggs. Differences in activity and isozyme patterns of malic acid, lactic acid, glucose 6-phosphate dehydrogenase, acid phosphatase, and alkaline phosphatase were not observed during embryogenesis of control and JH III-treated A. arboreus. Differences were observed in esterase activity.

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.

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.

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.

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.

Influence of microclimate on the life cycle of the common tick Ixodes ricinus (L.) in an open area in comparison with forest habitats.

Under conditions of the South-Moravian region of Pannonian climate (Valtice near Breclav), the life cycle of the common tick Ixodes ricinus (L.) was studied and a continous recording of main elements of microclimate (temperature and humidity) was carried out in an open grassy area. Simultaneously the process of hibernation was studied in four soil layers (surface, depths of 10, 20 and 30 cm). Observations were assessed by mathematiccal-statistical tests and compared with the results obtained by the same methods in the forest biotope (tipe of thermophilic oak forest)and in the ecotone of forest margin (Daniel et al. 1976). The ticks are able to complete the developmental cycle also in the open grassy areas, where during the vegetation period the development proceeds more quickly than in the forest but with considerably higher losses. In the discussion the conclusions are compared with literary data from other parts of Czechoslovakia.

A rapid spectrophotometric method for the monitoring of embryonic development in ticks (Acarina: Ixodoidea).

A rapid spectrophotometric method for the monitoring of embryonic development in Boophilus decoloratus (Koch, 1844) is described. The method is based on a quantitative assessment of guanine, the prinicipal end-product of nitrogenous metabolism in ticks, which is gradually built up and stored in the rectal sac during embryonic development of the larvae. A study of the growth of tick embryos under constant temperature conditions and 6 different humidity conditions demonstrated that embryonic development was dependent on the water content of the eggs at the time of oviposition. When eggs lost more than 35% of their initial mass through evaporation, nitrogenous metabolism (as indicated by guanine production) was seriously affected and embryos died.

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.

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.

[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.

[Disturbances in the process of hatching of larvae Argas (A) reflexus (Fabr.) (Acari: Ixodida: Argasidae) exposed to various thermal and humidity conditions]. / Zaklócenia procesu wylegu larw Argas (A.) reflexus (Fabr.) (Acari: Ixodida: Argasidae) pod wplywem róznych warunków termicznych I wilgotnosciowych.

The most cases of abnormal hatch larvae (40.2%) of A. reflexus were in relative humidity 90% and temperature 25 degrees C; the least (0-2.8%)--at the low relative humidity levels (10 and 30%) in all examined temperatures of experiments. The rise of relative humidity caused the greater number of the disturbances of hatch larvae. The most often +non-separation of integument eggs from body of larvae was observed, what made impossible normal formation of legs and made difficult locomotive faculty.

[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.

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.