Developmental profiles of ecdysteroids, ecdysteroid receptor mRNAs and DNA binding properties of ecdysteroid receptors in the Ixodid tick Amblyomma americanum (L.)

Total body ecdysteroid titers were determined at specific stages during the larval and nymphal life of Amblyomma americanum (L.). One ecdysteroid peak was observed following the completion of larval apolysis. However, two distinct ecdysteroid peaks occurred at a comparable stage in the nymphal molting cycle. The first occurred following apolysis and the second peak occurred at about the time of ecdysis. When whole body profiles of EcR and RXR mRNAs were examined during the molting cycle using RT-PCR, the expression of both AamEcR and AamRXR mRNAs was shown to be correlated with the ecdysteroid titer. Using an electrophoretic gel mobility shift assay, it was demonstrated that AamEcR*AamRXR1, but not AamEcR*AamRXR2, exhibits broad DNA binding specificity, forming complexes with a variety of synthetic direct repeat and palindromic nuclear response elements with the half-site consensus AGGTCA. These data suggest that functional differences may exist between the AamRXR1 and AamRXR2 proteins.     

Ultrastructure of Haller's organ in the tick Amblyomma americanum (L.)

Summary Haller's organ on the tarsus of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) was studied by scanning and transmission electron microscopy. It consists of a distal bristle group, (the anterior pit), and a proximal capsule which encloses several sensilla. The seven sensilla of the anterior pit (A1–A7) are all thick-walled and multi-innervated (2–9 neurons), but at least three different types can be differentiated. Sensilla A1 and A2 possess large, plugged pores (>1000 Å) and are the only sensilla with branching dendrites. A3 and A5 are characterized by a spoke-wheel arrangement of the cuticle wall and very fine pores (100–200 Å) penetrating the spokes centrally; A4, A6, and A7 do not exhibit any pore system but a single opening at the bristle tip is assumed.The capsule contains seven thin-walled, blunt-tipped sensilla, and several non-sensory cuticular projections (pleomorphs). All of these sensilla possess large plugged pores in the cuticle wall and numerous dendritic branches of several neurons (3–5) in the lumen. Glandular openings were found inside the capsule; their significance is discussed.The fine structure of Haller's organ supports the functions postulated by Lees (1948), namely olfaction for the capsule and humidity reception (among others) for the anterior pit.This research was supported in part by the Office of Naval Research, and by NIH Training grant ES 00069. Paper no. 3459 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

Fine structural analysis of palpal receptors in the tick Amblyomma americanum (L.)

Summary Palps of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) were studied by scanning and transmission electron microscopy. The terminal palp segment (IV) bears the so-called palpal organ, a cluster of 10 short, blunt-tipped sensilla. All sensilla (except for the center sensillum) receive a dual innervation: 2 mechanoreceptive dendrites which terminate in the socket membrane plus several chemoreceptive dendrites (4–12) which enter the lumen. The thick-walled cuticular shaft possesses 2–3 small pore openings (100 Å) below the tip, thus establishing communication between dendrites and environment. Two structurally different types of palpal sensilla exist: The A-type has a characteristic doublelumen and always contains 4 dendrites, the B-type features a single lumen and a specially layered cuticular shaft with 6–12 dendrites. The fine structure of the tick palpal receptors corresponds closely to that of known contact chemoreceptors in insects.This research was supported in part by a contract with the Office of Naval Research (R. C. Axtell, principal investigator), and by NIH Training grant ES 00069. Paper no. 3700 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

Diversity of Rickettsiales in the Microbiome of the Lone Star Tick,Amblyomma americanum

Ticks are important vectors for many emerging pathogens. However, they are also infected with many symbionts and commensals, often competing for the same niches. In this paper, we characterize the microbiome of Amblyomma americanum (Acari: Ixodidae), the lone star tick, in order to better understand the evolutionary relationships between pathogens and nonpathogens. Multitag pyrosequencing of prokaryotic 16S rRNA genes (16S rRNA) was performed on 20 lone star ticks (including males, females, and nymphs). Pyrosequencing of the rickettsial sca0 gene (also known as ompA or rompA) was performed on six ticks. Female ticks had less diverse microbiomes than males and nymphs, with greater population densities of Rickettsiales. The most common members of Rickettsiales were “Candidatus Rickettsia amblyommii” and “Candidatus Midichloria mitochondrii.” “Ca. Rickettsia amblyommii” was 2.6-fold more common in females than males, and there was no sequence diversity in the sca0 gene. These results are consistent with a predominantly vertical transmission pattern for “Ca. Rickettsia amblyommii.”     

A dopamine sensitive adenylate cyclase in the salivary glands of Amblyomma americanum (L.)

1. Enzyme activity, basal or dopamine-stimulated (10 microM), was linear with time to 25 min and with protein concentration to 0.8 mg protein/ml of final assay volume. Activity was maximal between pH 7.0 and 7.5. 2. Mg2+ maximally stimulated basal or dopamine-sensitive adenylate cyclase activity at about 4 mM. 3. Adenylate cyclase had a Km of 0.042 mM for ATP and maximum velocities for basal and dopamine-stimulated activity of 107 and 179 pmol cyclic AMP formed/mg protein per min, respectively. 4. Half-maximal stimulation of the enzyme occurred at about 4.2 x 10(-7) M dopamine with the threshold being less than 10(-9) M. Dopamine increased the Vmax but had no effect on the Km of ATP. 5. Eighty-five to 90% of the adenylate cyclase activity was found in the particulate fraction. 6. Calcium ion produced a marked inhibition of adenylate cyclase activity above 0.04 mM and half-maximal inhibition occurred near 0.1-0.2 mM.     

Highly Prevalent Coxiella sp. Bacterium in the Tick Vector Amblyomma americanum

Laboratory-reared and field-collected Amblyomma americanum ticks were hosts of a Coxiella sp. and a Rickettsia sp. While the Coxiella sp. was detected in 50 of 50 field-collected ticks, the Rickettsia sp. was absent from 32% of ticks. The Coxiella sp. showed evidence of a reduced genome and may be an obligate endosymbiont.     

Localization and Visualization of a Coxiella-Type Symbiont within the Lone Star Tick, Amblyomma americanum

A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.     

Amblyomma americanum (L.): protein kinase C-independent fluid secretion by isolated salivary glands.

Protein kinase C activity was partially purified from tick salivary glands by fast protein liquid chromatography anion-exchange chromatography. Enzyme activity was stimulated by Ca2+, phosphatidylserine, and diacylglycerol with the highest activity observed in the presence of all three modulators. Enzyme activity was inhibited by a synthetic pseudosubstrate peptide with an amino acid sequence resembling the protein kinase C substrate phosphorylation site. The protein kinase C activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), when added to whole in vitro salivary glands previously prelabeled with 32P, stimulated the phosphorylation of salivary gland proteins. Activators of protein kinase C (phorbol ester or OAG) did not stimulate fluid secretion by isolated tick salivary glands. OAG and phorbol ester had only minimal affects on the ability of dopamine to stimulate secretion by isolated salivary glands and dopamine's ability to increase salivary gland cyclic AMP.     

Molecular cloning of cAMP-dependent protein kinase catalytic subunit isoforms from the lone star tick, Amblyomma americanum (L.)

The salivary glands of ixodid ticks are central to tick feeding and to survival during off-host periods. They produce and secrete a number of molecules critical to maintaining the complex host-vector interface and to maintaining osmotic balance. We have previously shown that a cyclic AMP-dependent protein kinase (cAPK) is involved in the mechanism of salivary gland secretion. We have now cloned cDNAs encoding three isoforms of the catalytic subunit (cAPK-C) of the cAPK from Amblyomma americanum, which are probably produced from alternative RNA processing of a single cAPK-C gene. The cDNAs contain unique N-termini of variable lengths that are linked to a common region containing the alpha A helix, catalytic core, and a C-terminal tail. The common region is highly similar to both insect and vertebrate cAPK-Cs. We have examined mRNA profiles in whole ticks and in isolated salivary glands throughout feeding and find that a single cAPK-C isoform is expressed in the salivary glands of both unfed and feeding females.     

The Genome Sequence of Lone Star Virus,a Highly Divergent Bunyavirus Found in the Amblyomma americanum Tick

Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing <61% overall amino acid identity with any other bunyavirus. Despite this sequence diversity, LSV was found by phylogenetic analysis to be part of a well-supported clade that includes members of the Bhanja group viruses, which are most closely related to SFSTV/HRTV. The genome sequencing of LSV is a critical first step in developing diagnostic tools to determine the risk of arbovirus transmission by A. americanum, a tick of growing importance given its expanding geographic range and competence as a disease vector. This study also underscores the power of deep sequencing analysis in rapidly identifying and sequencing the genomes of viruses of potential clinical and public health significance.     

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick,Amblyomma americanum (L.)

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.     

Permeability of tarsal sensilla in the tick Amblyomma americanum L. (Acarina, Ixodidae)

Ticks were submerged in silver-protein solution, prior to fixation for electron microscopy, in order to trace the pathway of molecules in supposed tarsal chemoreceptors. Sensilla with radially arranged cuticular canals (100-200 A in diameter) leading to the centrally located dendrites show silver granules inside the canals and in the central lumen, thus directly making contact with the dendrites. Sensilla with large, plugged pores (1200 A) exhibit an accumulation of silver granules in the pore openings but no granules (about 50 A in diameter) were observed penetrating into the lumen. Apparently silver granules could diffuse in, but not through the material which suspends the pore plugs. It is suggested that this material corresponds to the 'pore tubules' in insect olfactory sensilla and that it may play an essential role in transmitting a chemical stimulus from the environment to the dendrites.     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.     

Adenosine-3',5'-monophosphate in salivary glands of unfed and feeding female lone star ticks,Amblyomma americanum (L.)

1. Basal levels of cAMP in salivary glands of female lone star ticks were found to be about 5 pmoles/mg protein during all stages of feeding.2. Glands stimulated with 10⁻⁵M dopamine and 10⁻⁵M dopamine plus theophylline exhibited significant increases in cAMP/mg protein.3. After stimulation by 10⁻⁵ M dopamine was removed, cAMP decreased faster in glands from slowly feeding ticks (< 200 mg) than in glands of rapidly feeding ticks ( > 200 mg).     

Analysis of lipids in the salivary glands of Amblyomma americanum (L.): Detection of a high level of arachidonic acid

Analysis of lipids in salivary glands of the lone star tick, Amblyomma americanum, demonstrated that arachidonic acid (20:4, n-6) comprises 8% of all fatty acids identified by gas chromatography. The occurrence of arachidonic acid and other C₂₀ polyunsaturated fatty acids in tick salivary glands was confirmed by gas chromatography-mass spectrometry. Arachidonate is located entirely in the phospholipid fraction and is associated exclusively with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Salivary glands stored and frozen for several months had a similar lipid composition as freshly dissected salivary glands, with the exception of a small amount of free arachidonic acid and an increase in lysophosphatidylcholine. Incubation of salivary gland homogenates with snake venom phospholipase A₂ showed that most saturated fatty acids are esterified in the sn-1 position of PC and PE, with the unsaturated fatty acids in the sn-2 position. Approximately 75% of arachidonic acid is in the sn-2 position of PC and PE, adding support to the hypothesis that arachidonic acid is released into the cytoplasm after activation of a phospholipase A₂ for subsequent metabolism to prostaglandins and/or other eicosanoids. © 1993 Wiley-Liss, Inc.     

The role of inositol 1,4,5-trisphosphate in mobilizing calcium from intracellular stores in the salivary glands of Amblyomma americanum (L.)

Isolated tick salivary glands, permeabilized with digitonin in the presence of the Ca²⁺ uptake inhibitors, sodium azide and vanadate, released Ca²⁺ in response to 20 μM inositol-1,4,5-trisphosphate (IP₃). Inositol-1-phosphate (IP₁) and inositol-1,4-bisphosphate (IP₂) appeared to stimulate an uptake of Ca²⁺ into whole glands. Inositol-1,4,5-trisphosphate caused release of Ca²⁺ from a 100,000 g microsome enriched pellet; however, IP₁ and IP₂ were ineffective in stimulating an uptake or efflux of Ca²⁺. The combined 900 and 11,500 g pellets showed no significant release of Ca²⁺ in response to addition of IP₃. Inositol-1,4,5-trisphosphate concentrations as low as 1 μM are capable of stimulating a significant release of Ca²⁺ from microsomes. Results suggest that intracellular Ca²⁺ is mobilized from microsomal intracellular stores in response to agonists which increase cytosolic IP₃ in tick salivary glands. Results also suggest a possible role for IP₁ and IP₂ or both in stimulating an uptake of Ca²⁺ into vanadate and azide-insensitive intracellular pools.