The effect of feeding status on sexual attractiveness of Ixodes ricinus (Acari: Ixodidae) females.

Sexual attractiveness of unengorged, semiengorged and fully engorged females of both field and laboratory populations of Ixodes ricinus was studied under laboratory conditions by means of a computerised video tracking system. A male and a female were allowed to walk freely in a glass arena during 1 h and their behavioural interactions were observed. Obtained results revealed that the feeding status of L. ricinus females affects their sexual attractiveness. The highest attractiveness was observed in engorged females, the lowest in unengorged females of the field population. Copulation occurred in all experimental groups with the highest frequency observed in the unengorged virgin laboratory females. Despite engorged females being highly attractive for males on distance, the frequency of copulation was low. Differences between field and laboratory populations were found in both behavioural parameters and frequency of copulation.

Nymphal sexual dimorphism in the sheep tick Ixodes ricinus (Acari: Ixodidae).

Unfed nymphs of Ixodes ricinus (L.) can be divided into two morphological groups according to the length of idiosoma, scutum, hypostome and palpal segment III, and the number of dorsal alloscutal setae. Specimens of greater body dimensions and more numerous dorsal alloscutal setae moulted predominantly into females. The frequency of different nymphal length categories in field-collected ticks followed a normal distribution. The length of unfed nymphs correlates well with the length (r = 0.7248 +/- 0.0711, P < 0.001) and weight (r = 0.6519 +/- 0.0782, P < 0.001) of engorged nymphs, however, it varies in ticks of different origin. In field-collected ticks, freshly engorged female nymphs were 2.30-2.94 mm long, male nymphs 2.14-2.46 mm long. Feeding period (P < 0.05) and premoulting period (P < 0.001) were significantly longer in female nymphs both in field-collected and laboratory-derived I. ricinus. The engorgement weight was found to be the best criterion for differentiation of male and female nymphs of ixodid ticks. In field-collected nymphs engorged on BALB/c mice, 98.6% of females moulted from nymphs weighting more than 3.60 mg, while in laboratory-derived ticks, 98.4% of females emerged from nymphs of 3.42 mg body mass or more.

Cuticular hydrocarbon variation and progeny phenotypic similarity between laboratory breeds of allopatric populations of Argas (persicargas) persicus (oken) (acari: argasidae).

Cuticular hydrocarbons of laboratory breeds of four Argas (Persicargas) persicus (Oken) population samples were studied by gas chromatography. Cuticular hydrocarbon patterns were used to determine Nei's genetic distances among populations, and their average heterozygosity. Sixteen n-alkanes, 37 monomethylalkanes and 26 dimethylalkanes were identified. Mostly quantitative differences were recorded between populations, rarely among males and females of the same population. A low genetic distance (0.0278-0.0781) together with a prominent degree of average heterozygosity (60.37-66.98%) were recorded in the population samples studied. When crossbreeds with adult specimens from the same or different geographical origins are performed, all larval progenies closely correlated within themselves and with their parents in hydrocarbon pattern. A slight matrocliny occurred in larvae coming from crosses of the same geographical source. From cross data it is postulated that hydrocarbons in A. persicus are inherited under a two dominant alleles hypothesis.

Immunosuppression and feeding success of Ixodes ricinus nymphs on BALB/c mice.

The effect of repeated infestations of Ixodes ricinus (L.) nymphs on BALB/c mice was studied. Four successive infectations resulted in an increase of tick feeding success. Tick yield and mean engorged weight increased and the length of the feeding period was reduced significantly (P < 0.05-0.01). The increase of specific anti-tick antibodies was not significant (P > 0.05). The blastogenic response of spleen lymphocytes to T-cell mitogens (Con A and PHA-P) was unimpaired or slightly enhanced, whereas the response to B-cell activators (LPS and PWM) was suppressed, as was the total antibody generation in vitro. The numbers of mast cells in murine skin at the tick attachment sites slightly decreased during the third infestation. The suppression of B-cell competence and of antibody generation, together with decrease of skin mast cell numbers in tick attachment sits, are considered to be responsible for enhancement of tick feeding success.

Present state of research on ticks (Ixodoidea) in the Czech Republic.

Tick research in the Czech Republic started developing rapidly after World War II and was directed to the faunistics and taxonomy, biology, ecology and behaviour, physiology and genetics, disease transmission, host-parasite relationships and control. Altogether 15 tick species were reported from the territory of the Czech Republic. Most studies in biology, ecology and virus transmission were dedicated to Ixodes ricinus, but biology and ecology of Dermacentor reticulatus, I. laguri and I. hexagonus were also studied. Many studies were done on argasid ticks, mostly on pheromonal communication of Argas persicus and immunology of feeding of A. polonicus. Population variability of both these species was also studied. The present research on ticks is mostly directed to study of interaction among hosts, ticks and pathogens on the humoral, cellular and molecular level.

Present state of research on house dust mites (Pyroglyphidae) in the Czech Republic.

Systematic research on house dust mites in the Czech Republic started in the early seventies, when mite fauna of human dwellings and distribution of pyroglyphid mites were studied. Special attention was paid to old-age-pensioners' homes and children's sanatoria. Pyroglyphidae were found also in the city air and in the dust of city pavements, especially in localities with high occurrence of domestic pigeons. While D. pteronyssinus and D. farinae mostly occurred in permanently inhabited flats, Euroglyphus maynei prevailed in recreation houses and some hospitals. Autumn increase of population density occurred in both, D. pteronyssinus and D. farinae populations, however, the number of protonymphs was remarkable higher than that of other developmental stage in D. farinae population. Special rearing technique was developed for the mass-culture of mites and a Czechoslovak patent was assigned to the technology of allergen preparation for the diagnostics and the desensibilization of patients with house-dust allergies.

Determination of purine bases and nucleosides by conventional and, microbore high-performance liquid chromatography and gas chromatography with an ion-trap detector.

A reversed-phase high-performance liquid chromatographic method has been developed for the analysis of purine and pyrimidine bases, uric acid and nucleosides largely relating to the purine synthetic and degradation metabolic pathways, with particular attention to the separation of hypoxanthine, xanthine and guanine. Complete separation and quantitation of the purines has been accomplished in the nanogram-microgram scale on conventional 4.6 mm I.D. columns with a standard gradient HPLC instrumentation as well as on 1 mm I.D. microbore columns with a dedicated isocratic micro-HPLC system using a dioxane-sodium acetate buffer. For the definite identification of components in excreta of ticks a GC-MS method has been described involving formation and GC of the trimethysilyl derivatives on a 25-m DB-5 column directly coupled with an ion trap detector. The methods are demonstrated on the analysis of the purine metabolites having an assembly pheromone effect on argasid ticks.

Antigens and glycoproteins of larvae, nymphs and adults of the tick Ixodes ricinus.

Protein components of homogenates of unfed larvae and nymphs of Ixodes ricinus (L.), and of ovary, haemolymph, Malpighian tubules, rectal ampulla, fat body, integument, salivary glands and midgut of partially fed adult females were studied for their antigenicity and carbohydrate moieties using immunoblotting and lectin affinity blotting (LAB) techniques. Comparing the individual anti-larval, anti-nymphal and anti-adult immune sera for their capacity to recognize the specific and trans-stadially cross-reactive antigenic proteins, larval feeding induced the most effective humoral response. The majority of immunogens recognized by rabbit anti-tick immune sera are glycoproteins. Most of the glycosylated antigens were modified with N-type glycans; however, O-type glycans were also demonstrated in some antigens. The correlation of the type of glycosylation with antigenicity, and the sharing of common antigenic epitopes by various tissues, are discussed.

Humoral and cellular immune response of BALB/c mice to repeated infestations with Ixodes ricinus nymphs.

The capacity of female BALB/c mice to mount an immune response and effective resistance to repeated infestations with I. ricinus nymphs was studied. An anamnestic antibody response and transient in vitro responsiveness of spleen lymphocytes to tick antigens were demonstrated in repeatedly infested mice. On the other hand, the response to concanavalin A--a T-cell mitogen, was suppressed at the same time. In the presented experiment, BALB/c mice did not manifest tick resistance after three successive infestations (with a reinfestation period of 2 weeks). The possibility of an infestation-dependent modulation of immune response in BALB/c mice is discussed.

Age-dependent immune response of chickens to feeding by Argas persicus larvae.

Chickens of the dwarf Velsum breed acquired immunity to Argas persicus larvae during repeated infestations with 100 larval ticks in 2-week intervals, when primarily infested at the age of 10 weeks. This immunity was manifested by 74.6% and 75.9% rejection of larvae during secondary and tertiary infestations (P < 0.01), respectively, in relation to controls. The mean weight reductions observed were 16.5% and 12.2% in secondary and tertiary infestations, respectively (P < 0.05). Weak immunity also occurred after secondary and tertiary infestations in chickens infested first at the age of 6 and 8 weeks, but not in chickens aged 2 or 4 weeks at first infestation. No changes were observed in mean feeding periods of larvae engorged on chickens of all age groups (P > 0.05). A significant increase in the leucocyte counts in peripheral blood (P < 0.01 or P < 0.05), mostly of lymphocytes and heterophil granulocytes, followed each infestation in chickens of all age groups. Specific anti-tick circulating antibodies were detected by ELISA in the sera of 10-week-old chickens after primary infestations, in the 4-8-week chicken groups following secondary infestations when aged 6-10 weeks, and in the 2-week chicken group only after tertiary infestations, when the chickens reached the age of 6 weeks. A 53 kDa protein was recognized in the whole-body extract of larval ticks by immune serum during immunoblotting. This was probably the antigenic protein responsible for eliciting the circulating antibodies. The acquired immunity to ticks seen in chickens aged 6 weeks or more may be connected with maturation of the bursa of Fabricius and the development of complete immunocompetence, which is believed to occur in chickens at the age of 4-5 weeks.

Effect of repeated infestations of BALB/c mice with Ixodes ricinus nymphs on tick-borne encephalitis virus infection.

The effect of repeated infestations of BALB/c mice with Ixodes ricinus (L.) nymphs on tick borne encephalitis (TBE) virus infection was studied. Enhancement of nymphal feeding, occurring in noninfected mice during the quaternary infestations, was less apparent or absent in female nymphs engorged on TBE virus infected mice. The mice infected with TBE virus during quaternary tick infestation survived significantly longer (P < 0.01) than mice infected with TBE virus during the primary tick infestation. The mean titre of virus in murine blood (determined by plaque assay) was significantly lower (P < 0.01) and the number of nymphs acquiring virus was reduced (P < 0.05) when feeding on hosts infected during the quaternary infestation. The results indicate that repeated infestations of I. ricinus nymphs on BALB/c mice, although enhancing tick feeding, reduced infection with TBE virus when inoculated intraperitoneally.

Identification of xanthine and hypoxanthine as components of assembly pheromone in excreta of argasid ticks.

In addition to guanine, xanthine and hypoxanthine were identified in white spherules in excreta of five species of Argas and Ornithodoros ticks by a reverse-phase high-performance liquid chromatography (HPLC) and a gas chromatographic method with mass spectrometric detection (GC/MS). The mutual relationships of these purines in excreta of Argas (Persicargas) persicus were found to be less than 1.5% for hypoxanthine, less than 9.0% for xanthine and 89.8-98.6% for guanine. In excreta of other species, the relationships of purines were similar, with the exception of Argas (A.) reflexus and A. (A.) polonicus, where the amount of hypoxanthine was rather elevated. Uric acid was also identified in some cases. The assembly efficacy of xanthine and hypoxanthine is similar to that of guanine, but xanthine significantly enhances the assembly efficacy of commercial guanine when mixed in ratio of about 1:25. Thus, xanthine seems to be the second important component of assembly pheromone of argasid ticks.

Lectin binding analysis of Argas polonicus tissue glycoproteins.

Proteins of the Malpighian tubules (MT), midgut tissue (MG), salivary glands (SG), internal reproductive organs (RO), epidermis (EP), cerebral ganglion (CG), rectal ampulla (RA) and larval homogenate (LA) of Argas (Argas) polonicus were studied for their antigenicity and lecin affinity using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, lectin affinoblotting and enzyme-linked lectin sorbentassay (ELLSA) techniques. A glycoprotein of 305 kDA was found in all tissues studied. All low molecular weight antigenic proteins recognized by anti-larval immune pigeon serum, except for one of 35 kDA, i.e. the 19-, 21-, 23-, 27-, 34-, and 46- kDa proteins, were shown to be glycoproteins. The glycosylation was shown to be N-linked in all of these antigens, but O-type glycosylation was also demonstrated in the 34-kDa glycoprotein. The correlation between the glycosylation and antigenicity of these proteins is also discussed.

Artificial immunization of pigeons against Argas polonicus (Ixodoidea, Argasidae).

Two subcutaneous injections of salivary gland antigen (SGA) or larval homogenate (LH) at 2-week intervals induced a resistance in pigeons to Argas (Argas) polonicus Siuda, Hoogstraal, Clifford and Wassef larvae and induced anti-tick antibodies. The number of larvae rejected after LH immunization was significantly higher compared to SGA immunization but lower than the number of larvae rejected after two natural infestations at 2-week intervals. The antibody titre reached a peak on day 6 following the first inoculation of LH, and 11-13 days after SGA inoculation. The maximum antibody titre was recorded 6 days after a second challenge for both antigens. The highest antibody titre was reached after the first inoculation with LH but only after the second inoculation with SGA. The sera of pigeons immunized either with SGA or LH cross-reacted with the other antigen as demonstrated by ELISA. SDS-PAGE and immunoblot studies demonstrated several differences in the protein profiles of these antigens, the presence of 34 and 35 kdal proteins in SGA and their absence in LH.

Population variability of some quantitative characters in Argas polonicus larvae (Acarina: Argasidae).

The nature of variability of quantitative morphometrical characters was studied in larvae of two local populations of Argas (Argas) polonicus Siuda, Hoogstraal, Clifford et Wassef, 1979 collected in Czechoslovakia and Poland. Statistically significant differences in five quantitative characters studied, in which the larvae of both wild populations differed from one another, disappeared during three generations of laboratory rearing. The variability of these characters was lower in laboratory populations than in field collected ticks. The results of hybridization experiments and analysis of variability of larvae of individual populations and parental pairs suggest that rather adaptive than genetic variation is involved. The genetic component of the variation is inferior and is expressed probably by dominant relations between alleles of the same locus, or by different types of non-allelic interactions.

Antibody-mediated response of pigeons to Argas polonicus larval feeding and characterization of larval antigen.

Circulating antibodies to larval Argas polonicus antigen detected in the blood of pigeons by means of ELISA reach their highest level 3-6 days post-tick attachment. During 6-8 days post infestation when most larvae detach from their host, there is an abrupt drop of the antibody level in blood followed by second peak at day 10-15. During the secondary and subsequent infestations the dynamics of the antibody production is analogous, but the maximum absorbance values found are higher with each following infestation. This is in direct correlation with the growth of immune resistance of hosts. The transfer of immunoglobulins of resistant pigeons produces in naive hosts a partial resistance in a statistically significant (P less than 0.01) reduction of the number of engorged larvae, in the shortening of larval feeding period and in the decrease of their mean weight after feeding. However this resistance was significantly (P less than 0.01) less expressed than in naturally resistant pigeons during secondary infestation. The protracted effect on the duration of premoulting period and the percentage of moulted larvae manifested in larvae after secondary infestation was not apparent in molecular weight of approximately 19, 21, 23, 27, 45 and 165 kilodaltons, were recognized by serum of resistant pigeons.

Cutaneous and blood leucocyte response of pigeons to larval Argas polonicus feeding.

Dynamics of granular leucocyte response in peripheral blood of naive and sensitized pigeons infested with Argas polonicus larvae was only slightly dissimilar. In both cases a marked increase of heterophil and eosinophil counts and only a mild increase of basophil count were recorded at 6 hours post-infestation. In naive hosts the basophil count increased again at 96 hours post-infestation, but in sensitized hosts it did so as early as 48 hours post-tick attachment and was also accompanied by an increase of eosinophil levels 72 and 96 hours post-infestation. Cutaneous response of sensitized hosts at the tick feeding site was characterized by a large heterophil accumulation at 24 hours post-infestation, by an increased eosinophil count at 48 hours and basophil counts at 72 and 96 hours post-infestation. In primary hosts lesions were characterized by a mild increase of heterophil count later than 48 hours post-infestation and by slight eosinophil accumulations at 24 and 48 hours post-tick attachment, as well as basophil accumulation as late as 96 hours post-infestation. Cutaneous lesions of sensitized hosts were accompanied by apparent inflammatory changes which were mild and sometimes missing in primary hosts. Cytotoxic and degenerative alterations of basophils and other granulocytes were observed as well as vesiculation of secretory granules of basophils. Cutaneous response of sensitized hosts to larval A. polonicus feeding can be characterized as a cutaneous basophil hypersensitivity.

Acquired resistance of pigeons to Argas polonicus larvae.

The acquired resistance of pigeons to Argas polonicus manifests itself in an adverse effect on the feeding and further development of larvae. After five subsequent infestations, the rejection of larvae reached 90-95% and the mean weight of fed larvae dropped down from 2.18 mg to 1.72 mg in the Polish tick population. There appeared also a tendency to more rapid feeding in larvae from later infestations. Also the percentage of moulted larvae decreased, probably as a result of insufficient blood meal taken by larvae on repeatedly challenged hosts. The hosts sensitized by larvae of Polish tick population manifested a higher tick resistance than those sensitized by larvae of Czechoslovak population. No adverse effect has been observed in males and females after repeated infestation of sensitized pigeons. Anti-tick antibodies were detected in the serum of pigeons sensitized by larvae with the aid of counter current immunoelectrophoresis using extracts from whole larvae and from salivary glands of adults as antigen. No antibodies were detected in the serum of pigeons sensitized by adult ticks.

Identity of Argas (Argas) polonicus populations in Czechoslovakia and Poland.

The author made a morphological and biological comparison between the population of Argas (A.) polonicus Siuda, Hoogstraal, Clifford et Wassef, 1979 from Poland and the population of that species from Czechoslovakia, denoted in previous author's papers as Argas (A.) vulgaris Filippova, 1961. The comparison of morphometric features as well as the results obtained in the studies of those argasids in SEM have demonstrated some statistically significant differences which the author evaluates as intraspecific adaptive variability and does not attach any taxonomic importance to them. Experimental hybridization of P1 and F1 generations in both populations has not suggested any genetic incompatibility. A few differences in the developmental cycle of homogamic crosses in the Polish tick population is likely to be the consequence of the adaptation of this population to different living conditions. Due to the lack of taxonomically valuable differences the author includes the members of the Czechoslovak population of these argasids in the species A. polonicus.