Larval feeding performance of two Neotropical Ornithodoros ticks (Acari: Argasidae) on reptiles

This paper determines the feeding performance of the larvae of two Neotropical soft tick species namely Ornithodoros rostratus Aragão, 1911 and O. puertoricensis Fox, 1947 on reptiles (Gekkonidae) using rabbits, mice and guinea pigs to provide comparisons with feeding features on mammals. O. puertoricensis produced a larval feeding rate of 63% on reptiles, while that of O. rostratus was only 20%. But the final success (attaching + feeding) was similar, 12.4% for O. puertoricensis and 10.4% for O. rostratus. The feeding time was also very different for both species. In O. puertoricensis, detachment begins at 16th day and lasts until day 27. In O. rostratus detachment begins at 1.5 h and lasts until day 10. These values of feeding on reptiles are different from those obtained on mammals (average 5.6 days for O. puertoricensis and 2.9 for O. rostratus).     

Isolation and characterization of an anticoagulant from the salivary glands of the tick, Ornithodoros savignyi (Acari: Argasidae)

An inhibitor of activated coagulation factor X (fXa) was isolated from salivary gland extracts prepared from Ornithodoros savignyi using a two-step procedure, involving reversed-phase high-performance liquid chromatography (RP-HPLC) and diethylaminoethyl (DEAE) ion-exchange chromatography. From its behaviour during DEAE chromatography it could be deduced that it possesses an acidic pI (4.6). Capillary zone electrophoresis (CZE) of the purified inhibitor showed it to be homogeneous. The molecular mass was determined as 12 kDa using capillary gel electrophoresis (CGE) and as 7183.4 using laser desorption mass spectrometry (LDMS). The N-terminal amino acid sequence (residues 1–12) was determined and found to share a 66% identity with tick anticoagulant peptide (TAP). The O. savignyi peptide is a slow, tight-binding inhibitor of fXa (K _i=0.83±0.10 nM). The interaction of the fXa-inhibitor was found to be competitive and dependent on ionic strength. Preliminary investigations show that the inhibitor may be specific for fXa.Deceased.     

Cypermethrin induction of vitellogenesis and ovarian development in unfed adult female Ornithodoros moubata (Acari: Argasidae)

Summary

Vitellogenesis in ticks is known to be induced by engorgement and mating. In this paper, the synthetic pyrethroid cypermethrin (CyM) is shown to induce production of yolk protein precursor, vitellogenin (Vg), and ovarian development in unengorged mated adult female Ornithodoros moubata. The levels of Vg found in the hemolymph and ovarian development induced by CyM were dose-dependent. i.e., CyM doses of more than 0.2 and 1.0 μg/tick were needed for significant increase of Vg titer in the hemolymph and yolk deposition in oocytes, respectively. Immunological and electrophoretical analyses of Vg and Vitellin (Vn) induced by CyM were identical with those induced by engorgement. Vg titer induced by CyM in unengorged females followed approximately the same time course as that in the normal engorged females. However, Vg titer induced by CyM continued to increase after day 8 and reached a maximum (95 μg/μ1) on day 10 after treatment, while Vg titer induced by engorgement decreased again after reaching a maximum (60 μg/μ1) on day 6, correlated with yolk Vn deposition in oocytes. Ovarian development induced by even high doses (10 or 20 μg/tick) of CyM was slow compared to normal development stimulated by engorgement. Oviposition was not observed in females treated with CyM.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata

Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti-rabbit IgG (anti-human IgG) but not with anti-human IgG (anti-rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post-engorgement, and remained high for over 4 months during and after oviposition. 125I-labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).     

Respiratory metabolism of the soft tick,Ornithodoros turicata (Dugès)

The rate of oxygen consumption was investigated in fed larval, nymphal and adult Ornithodoros turicata ticks and in starved nymphal and adult ticks. Oxygen consumption rate of fed adult ticks increased with increasing temperature. The metabolic rate of adult ticks was affected by starvation whereby starved adult ticks showed a significantly lower oxygen consumption than their fed counterparts. The oxygen consumption rate of fed female ticks was significantly higher than that of fed males but, there was no significant difference between the oxygen consumption rates of starved female versus starved male ticks. Oxygen consumption of fed larvae was significantly greater than those of fed first through third instar nymphs. Fed and starved nymphal ticks as well as fed adult ticks ventilated continuously. In contrast, starved adults ventilated discontinuously. The ability to reduce metabolic rate, plus the capability to ventilate discontinuously allow O. turicata adults to cope with prolonged starvation.     

Ornithodoros sawaii (Ixodida: Argasidae) Larvae Collected from Hydrobates monorhis on Sogugul and Gaerin Islands,Jeollanam-do (Province), Republic of Korea

The 65th Medical Brigade and Public Health Command District-Korea, in collaboration with the Migratory Bird Research Center, National Park Research Institute, conducted migratory bird tick surveillance at Sogugul and Gaerin Islands (small rocky bird nesting sites), Jeollanam-do (Province), Republic of Korea (ROK), on 30 July and 1 August 2009. Breeding seabirds captured by hands in their nesting burrows were banded, identified to species, and carefully examined for ticks during the nesting season. A total of 9 Ornithodoros sawaii larvae were removed from 4 adult Hydrobates monorhis (Swinhoe’s storm petrel). The identification of the larvae of O. sawaii collected from migratory seabirds were molecularly confirmed using mitochondrial 16S rDNA primer sets.     

Identification of anticoagulant activities in the salivary glands of the soft tick,Ornithodoros savignyi

Salivary gland extracts of the sand tampan, Ornithodoros savignyi, prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) significantly in a concentration-dependent manner. There was also a pronounced inhibition of human activated factor Xa (fXa) by salivary gland extracts. The salivary gland extracts inhibited chromogenic assays specific for both fXa and thrombin. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the salivary gland proteins followed by elution of specific areas or bands from a polyvinylidene difluoride (PVDF)-membrane, showed that various anticoagulant factors are present when screened by means of the APTT assay. The most active component was associated with a band of M _r of 14 kDa. Partial purification of this component was achieved using isoelectric focusing (IEF) and size-exclusion highperformance liquid chromatography (HPLC).     

The Mechanism of Oviposition in Argas (Persicargas) Walkerae (Acari: Argasidae)

The process of oviposition in A. walkerae was observed and found to be a sequence of exactly coordinated, interlocking events independent of the age of ticks. Egg-laying always commenced with numerous dorso-ventral movements of the capitulum. The pedipalps were spread during the last dorso-ventral movement and lowered to the ventral body wall embracing the genital aperture on both sides. Simultaneously, the cuticular sac of Gene's organ emerged and immediately everted maximally. Immediately afterwards the vestibulum vaginae prolapsed from the genital aperture touching the cuticular sac. The vestibulum vaginae handed over an egg to the cuticular sac after a brief contact. Then the vestibulum vaginae invaginated, the pedipalps closed and the cuticular sac was retracted. Finally, the capitulum very frequently moved up and down and the free egg was pushed in front of the ticks. The total time of laying an egg averaged 27min in both 4-week-old and 12-month-old ticks. In 4-week and 12-month-old ticks, egg-laying was preceded by a mean preoviposition period of 6.1 days and 7.5 days, respectively, while the mean oviposition was completed in 14 and 10 days and the average egg totals were 119 and 103.     

Identification and expression analysis of an actin gene from the soft tick, Ornithodoros moubata (Acari: Argasidae)

Actin genes are found in all living organisms and highly conserved in various animals as shown by numerous studies on actin gene expression and function. Because of this ubiquitous nature of actin, it is often used as an internal control in gene expression studies. To clarify the suitability of actin gene as an internal control in soft ticks, isolation and expression analyses of an actin gene from Ornithodoros moubata was performed. An actin gene of Ornithodoros moubata (OmAct2, GenBank accession no. AB208021) with 1,131 bp and 376 amino acid residues was identified. The homology of OmAct2 with other arthropod actin genes was greater than 80% in nucleotides and 99% in amino acids. OmAct2 gene was classified as a cytoskeletal actin type by absence of muscle-specific amino acids commonly found in insects and ubiquitous expression in all stages and both sexes. Southern blot revealed that O. moubata has four to seven actin genes. In addition, actin expression analyzed by real-time PCR before and after blood feeding was not significantly different indicating OmAct2 is an appropriate internal control for the analysis of gene expression in these ticks.     

Juvenile hormone-like substances can induce vitellogenesis in the tick Ornithodoros moubata (Acarina: Argasidae)

Topical application of different juvenile hormone analogs (JHA) or of a mixture of stereoisomers of insect juvenile hormone (JH) 1 and 3 to fed virgin female Ornithodoros moubata immediately after feeding induced vitellogenesis and egg-laying in up to 70% of treated females. In controls only 13.7% oviposited. The eggs were sterile, with abnormal shape, but their number versus the weight of engorged females was normal or sometimes greater than in mated females. However, preoviposition period was longer than in mated females.

It was more difficult to induce egg-laying by similar topical applications 100 days after feeding of virgin females. A maximum of 58% of ovipositing females was obtained with a very high dosage of JH mixture (500 fig). Injection of this mixture into the females was more potent; 15 to 50 fig induced oviposition in about 60% of the females. The preoviposition period was also longer than in control females.

Our results suggest the presence of a JH-like substance which is involved in the hormonal control of vitellogenesis. However, since natural isomers of JH were much less efficient than isomeric mixtures or JHA, we suppose that the natural tick hormone does not correspond to JH, but rather to a JH-like substance.     

Immunocytochemical mapping of FMRFamide-like peptides in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis

FMRFamide-like immunoreactivity was studied in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis using immunocytochemistry based on the peroxidase-antigeroxidase method. FMRFamide-like immunoreactive cells are widely distributed in various regions of the tick synganglion including protocerebral, cheliceral, stomodeal, palpal, pedal I–IV, and opisthosomal regions in both species. However, there is one layer of immunoreactive cells located on the dorsal surface of the postoesophageal part of the synganglion that is found only in D. variabilis. Besides the immunoreactivity within the cell body and its axons, the neuropile and the neural lamella (the extracellular sheath of the synganglion) are rich in immunoreactive materials. Some coxal muscles are innervated by the FMRFamide-like immunoreactive processes of the nerve from the pedal ganglion.     

Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae)

We previously purified and determined the partial amino acid sequence of a 4 kDa peptide having high homology with scorpion defensin from the hemolymph of adult fed female soft ticks, Ornithodoros moubata. In this study, the full length sequences of two defensin isoforms were obtained. Deduced amino acid sequences reveal a precursor protein of 73 amino acid residues with a mature portion consisting of 37 amino acid residues. This mature peptide contains six cysteine residues conserved in the same location as other invertebrate defensins. Phylogenetic analysis reveals that Ornithodoros defensin is most closely related to scorpion defensin and other more ancient arthropods. Ornithodoros defensin mRNA is constitutively expressed and up-regulated by blood-feeding and bacterial injection. Ornithodoros defensin gene expression occurs mainly in the midgut. This is the first report of the cloning and gene expression of an antibacterial peptide from the Acari.     

Vitellogenin synthesis,processing and hormonal regulation in the tick,Ornithodoros parkeri (Acari:Argasidae)

This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri. Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [³⁵S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.     

Ticks (Acari: Ixodoidea: Argasidae, Ixodidae) of Chile

The tick species recorded from Chile can be listed under the following headings: (1) endemic or established: Argas keiransi Estrada-Peña, Venzal and Gonzalez-Acuña, A. neghmei Kohls and Hoogstraal; Ornithodoros amblus Chamberlin; Otobius megnini (Dugès); Amblyomma parvitarsum Neumann; A. tigrinum Koch; Ixodes auritulus Neumann; I. chilensis Kohls; I. cornuae Arthur, I. sigelos Keirans, Clifford and Corwin; I. stilesi Neumann; I. uriae White; Rhipicephalus sanguineus Koch. (2) Probably established or endemic: Argas miniatus Koch; Ornithodoros spheniscus Hoogstraal, Wassef, Hays and Keirans; Ixodes abrocomae Lahille; I. neuquenensis Ringuelet; I. pararicinus Keirans and Clifford. (3) Doubtfully established: Argas reflexus Fabricius; Ornithodoros talaje (Guérin-Méneville). (4) Exotic: Amblyomma argentinae Neumann; A. latum Koch, Rhipicephalus (=Boophilus) microplus (Canestrini). (5) Erroneously identified as present in Chile: Amblyomma americanum (Linnaeus); A. maculatum Koch; A. varium Koch; Ixodes conepati Cooley and Kohls; I. frontalis (Panzer); I. ricinus (Linnaeus); Margaropus winthemi Karsch. (6) Nomina nuda: Argas reticulatus Gervais; Amblyomma inflatum Neumann; Ixodes lagotis Gervais. Hosts and localities (including new records) are presented. Argas neghmei, O. amblus, O. megnini, I. uriae and R. sanguineus may cause severe injury to their hosts, including humans. The Chilean Ixodes fauna is unique to the Neotropical Zoogeographic Region, and additional research is needed in order to understand the biological importance of these species.This revised version was published online in May 2005 with a corrected cover date.     

Induction of egg maturation and oviposition in the tick Ornithodoros parkeri (Acari: Argasidae)

Hemocoelic injection and vaginal insertion of selected male reproductive and non-reproductive tissue homogenates into fed-virgin female Ornithodoros parkeri stimulated varying degrees of ovum maturation and/or oviposition during 14 and 30 day observation periods, respectively. Mean times for oviposition and mean numbers of eggs laid per ovipositing female receiving hemocoelic injections of male reproductive tissue homogenates did not differ significantly from fed-mated controls. In addition, hemocoelic injection of male salivary glandular homogenate induced oviposition, yet synganglial homogenate did not. Although vaginal insertion induced both ovum maturation and oviposition, the effect was not as pronounced as when similar doses were administered by hemocoelic injection. These results indicate that a complex interrelated series of precopulatory and copulatory stimuli are necessary for oviposition to occur in fed O. parkeri.     

Effects of precocenes on vitellogenesis in the adult female tick,Ornithodoros moubata (Acari: Argasidae)

Vitellogenin (Vg) concentrations in the hemolymph and ovarian development were studied inOrnithodoros moubata after treatment with precocenes 1 (P1) and 2 (P2). Precocene was dissolved in acetone or DMSO and topically applied to the dorsal surface of ticks: (1) at adult ecdysis; (2) 24 h before engorgement; (3) immediately after engorgement; and (4) 24 h after engorgement. Subsequently, P1 and P2 were dissolved in olive oil and injected through the gonopore into the body cavity 24 h after engorgement. Vitellogenin concentration was measured on days 5 and 10 after engorgement and ovarian development was scored on day 10, 20 or 30. Oviposition was also recorded and the average weight of eggs laid by females was determined. No differences in concentration of Vg in the hemolymph occurred between the control ticks and ticks treated topically or by injection with P1 and P2. Precocene did not suppress the synthesis of Vg inO. moubata. However, oviposition was reduced in ticks that survived repeated treatment with high doses of P2 dissolved in acetone.     

A comparison of Chryseobacterium indologenes pathogenicity to the soft tick Ornithodoros moubata and hard tick Ixodes ricinus

A yellow-pigmented Gram-negative bacterium, Chryseobacterium indologenes, was found in the gut contents of about 65% of soft ticks Ornithodoros moubata from a perishing laboratory colony. The isolated putative pathogen, C. indologenes, was susceptible to cotrimoxazol and addition of this antibiotic (Biseptol 480) to the blood meal significantly decreased the tick mortality rate. The artificial infection of healthy O. moubata by membrane feeding on blood contaminated with C. indologenes was lethal to all ticks at concentrations 10(6) bacteria/ml. On the contrary, a similar infection dose applied to the hard tick Ixodes ricinus by capillary feeding did not cause significant mortality. Examination of guts dissected from infected O. moubata and I. ricinus revealed that C. indologenes was exponentially multiplied in the soft tick but were completely cleared from the gut of the hard ticks within 1 day. In both tick species, C. indologenes were found to penetrate from the gut into the hemocoel. The phagocytic activity of hemocytes from both tick species was tested by intrahaemocoelic microinjection of C. indologenes and evaluated by indirect fluorescent microscopy using antibodies raised against whole bacteria. Hemocytes from both tick species displayed significant phagocytic activity against C. indologenes. All O. moubata injected with C. indologenes died within 3 days, whereas the increase of the mortality rate of I. ricinus was insignificant. Our results indicate that hard ticks possess much more efficient defense system against infection with C. indologenes than the soft ticks. Thus, C. indologenes infection has the potential to be a relevant comparative model for the study of tick immune reactions to transmitted pathogens.     

Identification of extrinsic blood coagulation pathway inhibitors from the tick Ornithodoros savignyi (Acari: Argasidae)

The salt BaSO(4) selectively adsorbs two proteins from crude Ornithodoros savignyi salivary gland extract. They co-purify during reversed-phase HPLC, but can be separated by hydrophobic-interaction chromatography. Their molecular masses are 9333 and 9173Da. The 9.3kDa protein was designated BSAP1 and the 9.1kDa protein BSAP2. Their amino acid compositions show significant differences, in particular the presence of seven and eight cysteine residues in BSAP1 and BSAP2, respectively. The proteins do not contain gamma-carboxyglutamic acid, hydroxyproline, or hydroxylysine. The proteins do not inhibit the intrinsic coagulation cascade, but inhibit the extrinsic pathway. The observed inhibition is not due to inhibition of factor VII. Both proteins bind to membranes. BSAP1 binds neutral and negatively charged membranes more strongly than BSAP2. Its affinity for negative membranes is, however, much lower than for neutral membranes. In contrast, BSAP2 binds both membranes equally strongly. The binding of the proteins to the membranes was significantly lowered upon pre-incubation with Ca(2+).     

An experimental assessment of the tampan tick Ornithodoros moubata as vector of hepatitis B virus

Wild-caught and colonized tampan ticks, Ornithodoros moubata (Murray), were fed on hepatitis B virus (HBV)-positive blood-means in a series of four experiments. Hepatitis B surface antigen (HBsAg) persisted in nymphal and adult ticks for up to 779 days, while the epsmark antigen (HBeAg) persisted in mature nymphs up to 13 days, in adult males up to 11 days and in adult females up to 16 days. HBsAg was transmitted trans-stadially through two moults during the life cycle but transovarial transmission did not occur. The surface antigen was transmitted by two out of fifteen single ticks into 0.4 ml aliquots of HBV-negative blood, although six groups of ticks failed to transmit into 5.5 ml aliquots of blood: this antigen was not transmitted to hamsters. HBsAg was detected in samples of the ticks' coxal and rectal fluid secretions always at the infecting feed and usually at the second feed. HBeAg was only detected in one of two samples of coxal fluid collected at the infecting feed. The results as a whole indicate that no biological multiplication of virus occurs in O.moubata but that mechanical transmission from ticks to man could occur by: (i) contamination of a person when crushing infected ticks; (ii) infection by bite; (iii) contamination with coxal fluid, especially by scratching bites. This is thought to take place among the Kavango tribe in their village huts in north-eastern Namibia where infestations of infected O.moubata occur.