Hemolymph proteins in ticks

In comparison to insects and Crustacea, our knowledge of the predominant hemolymph proteins in ticks is minimal. The hemolymph protein most studied in ticks has been vitellogenin (Vg). Vg is synthesized by the tick fat body after female adults obtain a blood meal, is released into the hemolymph and is absorbed by developing oocytes as vitellin (Vn). Much of what we know about Vg is from studies of Vn. In general, the carbohydrate, lipid and amino acid composition is similar to insects except that in the tick, Vg contains heme, most likely from the digestion of host hemoglobin. In the American dog tick, Dermacentor variabilis, Vg is comprised of two native proteins and seven subunits on SDS-PAGE. Vg has been characterized in five tick species but the amino acid sequence is not yet available. Another predominant hemolymph protein, apparently a carrier protein (CP), has recently been studied in two tick species. This protein is found in the hemolymph of both male and females adults, in adult tissues outside of the hemolymph in some tick species, in coxal fluid of soft ticks and in whole body homogenates from eggs, larvae and nymphs. CP from the hard tick, D. variabilis, contains cholesterol, phospholipids, monoacylglycerides, triacylglycerides, free fatty acids, carbohydrate and heme. Under identical assay conditions, the analogous protein in the soft tick, Ornithodoros parkeri, did not contain heme. CP in the American dog tick consists of two subunits, one of which has 61% identity to the biliprotein, artemocyanin, from the fairy shrimp. CP is identical to a heme-lipoprotein (HeLp) from Boophilus microplus. The exact roles of CP and HeLp have not yet been fully determined, but they apparently are important in heme sequestration and as a storage depot for protein and lipid. Macroglobulin, lectin, antimicrobial, JH binding, JH esterase, and other tick hemolymph proteins are also discussed.     

Developmental profile, isolation, and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari: Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari: Argasidae)

A novel lipoglycoheme-carrier protein (CP) in the American dog tick, Dermacentor variabilis (Say) has been purified and characterized. CP was purified by native-PAGE from partially fed virgin females. CP has a density of 1.25 g/ml with a molecular weight of 200 K by native-PAGE and 340 K by gel filtration chromatography. CP is comprised of two majour subunits, 98 K and 92 K in molecular weight by SDS-PAGE. Separate amino acid composition of the two subunits indicated high contents of As(x), Gl(x) and leucine. However, the N-terminal amino acid sequence of the two subunits was only 13% identical. The lower molecular weight subunit showed 61% identity to artemocyanin (biliprotein) in fairy shrimps, 46% identity to minor vitellogenin in chickens and 13% identity to vitellin of the black-legged tick. No similarity match was found for the other subunit. CP is a lipoglycoheme-protein as indicated by selective staining of native-PAGE gel for lipids, carbohydrates and heme. Lipid analysis by thin layer chromatography revealed the presence of cholesterol, phospholipids, monoacylglycerides, triacylglycerides and free fatty acids. Heme associated with purified CP demonstrated a lambda(max) of 397.5 nm while the lambda(max) of crude hemolymph plasma was 402.5 nm. The presence of CP in whole body homogenates of eggs, unfed and fed larvae and fed nymphs as well as in the plasma of unfed and fed adults including vitellogenic females was demonstrated by native-PAGE. Although a protein of analogous size was not found in the soft tick, Ornithodoros parkeri Cooley, a high molecular weight protein (500 K) is the predominant plasma protein in both unfed and fed male and female adults of that species as determined by native-PAGE. Also, CP appears to function as a biliprotein which sequesters heme.     

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae)

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476 ± 14.5 kDa as compared to 590 ± 25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.     

Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 μM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1 mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 μM). The protein kinase A selective inhibitor Rp-cAMPS (50 μM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 μM dopamine increased cGMP levels two-fold in the presence of 1 mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 μM) impeded 10 mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca²⁺ channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca²⁺. Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca²⁺ influx.     

Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10

Bacillus licheniformis HS10 is a good biocontrol agent against Pseudoperonospora cubensis which caused cucumber downy disease. To identify and characterize the antifungal proteins produced by B.licheniformis HS10, the proteins from HS10 were isolated by using 30–60% ammonium sulfate precipitation, and purified with column chromatography on DEAE Sepharose Fast Flow, RESOURCE Q and Sephadex G-75. And the SDS–PAGE and MALDI-TOF/TOF-MS analysis results demonstrated that the antifungal protein was a monomer with molecular weight of about 55 kDa, identified as carboxypeptidase. Our experiments also showed that the antifungal protein from B. licheniformis HS10 had significantly inhibition on eight different kinds of plant pathogenic fungi, and it was stable with good biological activity at as high as 100 °C for 30 min and in pH value ranged from 6 to 10. The biological activity was negatively affected by protease K and 10 mM metal cations except Ca²⁺.     

Spatial and temporal factors affecting parasite genotypes encountered by hosts: Empirical data from American dog ticks (Dermacentor variabilis) parasitising raccoons (Procyon lotor)

The American dog tick (Dermacentor variabilis) is an important vector of numerous pathogens of humans and animals. In this study, we analysed population genetic patterns in D. variabilis at scales of the host individual (infrapopulation) and population (component population) to elucidate fine-scale spatial and temporal factors influencing transmission dynamics. We genotyped D. variabilis collected from raccoons (Procyon lotor) trapped in two habitat patches (located in Indiana, USA) which were spatially proximate (5.9 km) and limited in size (10.48 Ha and 25.47 Ha, respectively). Despite the fine spatial sampling scale, our analyses revealed significant genetic differentiation amongst component populations and infrapopulations (within each component population), indicating a non-random pattern of encountering tick genotypes by raccoons at both scales evaluated. We found evidence for male-biased dispersal in the ticks themselves (in one component population) and an age-bias in spatial scales at which raccoons encountered ticks in the environment. At the scale of the component population, our analyses revealed that raccoons encountered ticks from a limited number of D. variabilis family groups, likely due to high reproductive variance amongst individual ticks. Finally, we found evidence for a temporal effect with raccoons encountering ticks in the environment as “clumps” of related individuals. While the genetic structure of parasite populations are increasingly being investigated at small spatial scales (e.g. the infrapopulation), our data reveal that genetic structuring can originate at scales below that of the infrapopulation, due to the interaction between temporal and biological factors affecting the encounter of parasites by individual hosts. Ultimately, our data indicate that genetic structure in parasites must be viewed as a consequence of both spatial and temporal variance in host-parasite interactions, which in turn are driven by demographic factors related to both the host and parasite.     

Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps

Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from “fed diet” and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84 kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5′- and 3′-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061 bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca²⁺ -binding proteins. As predicted, NcSP84 exhibited Ca²⁺-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca²⁺ tended to decline discretely, depending on the concentration of CaCl₂ with which it was mixed for 1 h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca²⁺ ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.     

Kneallhazia carolinensae sp. nov., a microsporidian pathogen of the thief ant, Solenopsis carolinensis

A new species of microsporidia is described from adults of the thief ant, Solenopsis carolinensis, collected in Florida, USA. Morphological and genetic characterization of this new species showed that it is most closely related to the genus Kneallhazia and is therefore formally designated, Kneallhazia carolinensae sp. nov. Masses of ovoid, binucleate spores were localized to fat body of adult workers and measured 6.2 ± 0.1 × 3.1 ± 0.1 μm (fresh) and 6.0 ± 0.1 × 3.4 ± 0.1 μm (fixed). These spores were in direct contact with the cell cytoplasm and contained an isofilar polar filament with 12-15 coils. Blastn analysis revealed that the K. carolinensae 16S rDNA sequence exhibited 91% identity with the 16S rDNA gene of K. solenopsae. The morphological and sequence data support the conclusion that K. carolinensae is a novel microsporidian species distinct from K. solenopsae.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Purification and characterisation of a P-selectin-binding molecule from the salivary glands of Ornithodoros moubata that induces protective anti-tick immune responses in pigs

Ornithodoros moubata is an argasid tick that lives in Africa in wild and synanthropic habitats. It feeds on warthogs, domestic swine and humans, and is able to transmit severe diseases such as human relapsing fever and African swine fever. The elimination of O. moubata from the synanthropic surroundings should improve control of the diseases it transmits. Previous attempts to develop a vaccine against O. moubata showed that a salivary gland extract (SGE) induced a protective response that inhibited the feeding of the ticks by up to 60%. Our aim in the present work was to isolate and characterise the salivary antigens responsible for the protective immune response induced with the SGE. The work reported here describes the finding and partial characterisation of a tick salivary glycoprotein of 44 kDa (Om44) that binds host P-selectin, presumably preventing the adhesion of leucocytes and platelets to vessel walls, thus allowing the ticks to complete their feeding. Preliminary analysis indicated that Om44 is not a homologue of the mammalian PSGL-1s and lacks sialyl-Lewis^X, Lewis^X and Lewis^Y determinants but carries heparin, which is the P-selectin-binding motif. Om44 is not recognised by the pig immune system after natural contact with O. moubata, but it can be neutralised by specific vaccine-induced antibodies, resulting in feeding inhibitions of around 50% in adults and nymphs-4, 25% in nymphs-3, 5% in nymphs-2 and 0% in nymphs-1 when they are fed on Om44-vaccinated pigs. In addition, the fecundity of females was inhibited by up to 43.8%. Om44 provides new prospects for the development of new anti-tick vaccines.     

Functional properties of hemocyanin from Oncomelania hupensis, the intermediate host of Schistosoma japonicum

The gastropod mollusc, Oncomelania hupensis is a unique intermediate host for the human parasite Schistosoma japonicum. It is a primary factor for the epidemic of schistosomiasis and its distribution is consistent with the epidemic area of schistosomiasis. Here we report the functional properties of hemocyanin of O. hupensis (OhH), a copper-containing respiratory protein which was isolated from its hemolymph and purified by ammonium sulfate fractionation and ultracentrifugation. We identified the protein characters including UV absorption at 340 nm, copper content and quaternary structure. Furthermore, by induction of phenoloxidase and enzyme-linked immunosorbent assay we show that OhH exhibited o-diphenoloxidase activity after limited proteolysis, and shared carbohydrate epitopes with glycoconjugates of S. japonicum.     

Dermal gland secretion improves the heat tolerance of the brown dog tick, Rhipicephalus sanguineus, allowing for their prolonged exposure to host body temperature

Dermal glands (sensilla sagittiformia) secrete when brown dog ticks, Rhipicephalus sanguineus, are mechanically disturbed, presumably as a defensive mechanism. Recently, we observed that these glands secrete due to the pressure stimulation of engorgement. In this study, we examine how dermal gland secretion alters the physiology of R. sanguineus, particularly if this secretion is an important mechanism during blood feeding. The ability of ticks to retain water was not modified by dermal gland secretion, but heat tolerance was enhanced. Short-term heat shock was improved slightly (1 h at 50 °C to 1 h at 56 °C) and featured reduced injury responses and greater recovery after heat shock. When exposed to their host body temperature (37 °C) for prolonged periods, individuals that had secreted survived over 1 week longer than individuals that did not secrete. Dorsal application of squalene, the main component of dermal gland secretion, did not increase temperature tolerance, suggesting that the act of secreting rather than the physical properties of the secretion itself is responsible for the increase in heat tolerance. Based on our results, dermal gland secretion may be an essential mechanism in certain tick species (Amblyomma, Dermacentor, Hyalomma, Rhipicephalus, but not Ixodes) for tolerating body temperature and not succumbing to heat stress during the extended time periods of feeding on a mammalian host, serving as a mechanism to prevent heat damage from the host during feeding.     

Role of an aquaporin in the sheep tick Ixodes ricinus: Assessment as a potential control target

Ticks undergo tremendous osmoregulatory stress as they take on up to 100 times their body weight in blood, returning about 75% of the ingested water and ions via their saliva into the host. We postulated that water channels, or aquaporins, involved in this mass water transport might be good targets for acaricide development. An aquaporin (IrAQP1) identified in the sheep tick, Ixodes ricinus, was present only in tissues involved in mass water flux, namely the gut, rectal sac and especially abundant in the salivary glands. IrAQP1 was localised by in situ hybridisation in specific cell and acini types, possibly Type III acini, but absent from the type I acini that are responsible for rehydration of ticks in the non-feeding phase. Gene knockdown of IrAQP1 in isolated salivary glands completely inhibited dopamine-stimulated secretion. Further, IrAQP1 knockdown adult females had 50% reduced body weight gains over the first 5 days feeding on an artificial feeding apparatus and 21% at the point of engorgement on hosts. Haemolymph osmolarity was increased in the IrAQP1-knockdown ticks. Importantly, the blood volume ingested per body weight was reduced by 30%. Overall, it would appear that water passage from the gut to the saliva was disrupted and tick guts were simply too “full” to ingest more blood. However, double-stranded RNA interference of IrAQP1 did not affect mortality of the ticks which successfully fed to detachment at day 9. Overall, our data indicate that IrAQP1 plays a pivotal role in blood meal water handling through the gut and salivary gland, and although its disruption by double-stranded RNA interference dramatically affects feeding performance, ticks remained feeding on the host with subsequent potential pathogen transmission and, therefore, IrAQP1 is not a suitable candidate target for tick control.