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.

Cloning and sequence of a gene for the homologue of the stearoyl CoA desaturase from salivary glands of the tick Amblyomma americanum.

A 1488 base pair cDNA clone has been isolated from a cDNA library made from salivary glands from 3-day feeding adult female ticks. The sequence of this cDNA suggests it is the gene for the tick homologue of the stearoyl CoA desaturase. This gene is expressed in eggs and all feeding stages of the adult examined, but appears to be transcribed to an 8 kb mRNA as well as a 1.5 kb mRNA. Because ticks have the ability to synthesize monounsaturated fatty acids and demonstrate a large increase in salivary monounsaturated fatty acids during tick feeding, we hypothesize that stearoyl CoA desaturase may be a key enzyme in the morphogenesis of tick salivary glands during feeding.

Cloning and sequence of a gene for a homologue of the C subunit of the V-ATPase from the salivary gland of the tick Amblyomma americanum (L).

A 1084 base pair partial cDNA showing similarity to the C subunit of the vacuolar ATPase (V-ATPase) was isolated on a clone from a cDNA library made from salivary glands from 3-day-old feeding adult Amblyomma americanum (L.) female ticks. The 5' end was completed using primer extension and the two pieces joined to form a complete cDNA of 1373 bp. This mRNA is expressed in embryos and the salivary glands of unfed adults and adult females at all stages of feeding. Specific inhibitors of the V-ATPase decrease the rate of dopamine-stimulated secretion of isolated salivary glands, but not as much as ouabain, an inhibitor of the Na+, K+ ATPase, indicating that a V-ATPase may participate in the mechanism of salivary fluid secretion in A. americanum, but the volume of saliva secreted is more dependent on an active Na+, K+ ATPase.

Tick salivary gland physiology.

The multifunctional, morphologically complex salivary glands are essential to the biological success of ticks and are intricately involved in the transmission of pathogens. They are innervated, and there is convincing evidence that dopamine is a neurotransmitter at the neuroeffector junction controlling fluid secretion. As feeding progresses, the rate of salivary fluid secretion increases greatly, enabling the ixodid tick to concentrate the bloodmeal by returning excess water and ions to the host. Saliva in feeding ticks is rich in bioactive components and exhibits a range of pharmacological properties. Factors identified in saliva or salivary glands include cement to help anchor the mouthparts to the host, various enzymes and inhibitors, histamine agonists and antagonists, prostaglandins, antihemostatic factors, and immuno-modulating factors. A secretion from the salivary glands allows ticks to absorb water from the air during the lengthy periods off their hosts. The physiology of this remarkable organ provides a striking example of strategies that have evolved to meet the challenge of a unique parasitic life style.

Cell membrane receptors and regulation of cell function in ticks and blood-sucking insects.

Immunoglobulins cross the midgut epithelium and enter the haemolymph of many blood-feeding arthropods without losing their immunological properties. Antigens essential to the survival of the blood-sucking arthropods which may be affected by the small amounts of specific antibody that cross the gut epithelium include membrane receptors or other factors which regulate cell function. Membrane receptors implicated in transmembrane signalling in response to specific neural and endocrine factors fall into three major classes: (1) gated ion channels, (2) agonist-stimulated tyrosine kinases and (3) receptors that interact with GTP-binding (G) proteins. Examples of all three types have been found in insects and ticks. A dopamine receptor interacts with a G-protein essential for controlling fluid secretion by the salivary glands of ixodid ticks. Another receptor in the ixodid tick salivary gland binds a neuropeptide from the tick synganglion and stimulates turnover of plasma membrane phosphoinositides, but its mechanisms of transmembrane signalling and function remain elusive. Another large class of membrane receptors are those concerned with endocytosis. Examples of receptor-mediated endocytosis include incorporation of vitellogenin by developing oocytes in mosquitoes and ticks and uptake of lysed blood-meal components by digest cells of the tick gut. Many cell membrane receptors and possibly hormones could serve as targets for vaccines in blood-feeding insects and ticks. The major challenge is to identify and characterize essential internal receptors and cellular components that are accessible to and affected by specific antibodies that are introduced into the body of blood-feeding arthropods.

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.

Oral secretion elicited by effectors of signal transduction pathways in the salivary glands of Amblyomma americanum (Acari: Ixodidae).

The abilities of various effectors of signal pathways in tick salivary glands to stimulate oral secretions in partially fed Amblyomma americanum (L.) female ticks were compared. Pilocarpine stimulated secretion the most rapidly following tick stimulation, but its effectiveness declined with time and subsequent injections. Secretion rates induced by dopamine and theophylline increased with time of collection and additional injections and were as effective as pilocarpine after 60 min. Activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA), and 1-oleoyl-2-acetyl-sn-glycerol inhibited dopamine, and theophylline stimulated secretion. gamma-amino-butyric acid either by itself or in combination with other drugs had no effect on volume of oral secretions. Peptides were visible in the oral secretion stimulated by the various drugs after separation and staining in SDS-polyacrylamide gels. More peptides were present in the oral secretion obtained from the smallest partially fed (slow feeding) ticks. In some ticks, proteins were observed in oral secretions obtained from the same tick at one collection time but not another in response to the same pharmacological agent.

Putative new expression of genes in ixodid tick salivary gland development during feeding.

Poly(A+) mRNA-enriched fractions from salivary glands of partially fed Amblyomma americanum female ticks were translated in vitro with a rabbit reticulocyte translation system. Translated proteins were labeled with [35S]methionine, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and identified by autoradiography. Thirty major identifiable polypeptides with molecular weights ranging from 14 to 136 kDa were synthesized from mRNA isolated from salivary glands of ticks weighing less than 100 mg. Polypeptides that comigrated at the same molecular weight were translated by mRNA from ticks at a more advanced stage of feeding (more than 300 mg) as were 8 others with molecular weights of 31, 71, 91, 106, 113, 118, and 128 kDa. Results demonstrated that differential gene expression may be stimulated in the developing salivary glands as the tick feeds.

Protein phosphorylation and control of tick salivary gland function.

Tick salivary glands are controlled by nerves, dopamine being a neurotransmitter at the neuroeffector junction. Dopamine and cyclic AMP (cAMP) stimulate fluid secretion by isolated salivary glands. Dopamine activates an adenylate cyclase to increase intracellular cAMP within the female salivary glands. Phosphoproteins whose levels of phosphate are affected by cAMP-dependent protein kinase have been identified in subcellular fractions. Protein(s) phosphorylated by cAMP appears to activate protein phosphatase in the salivary glands. Another phosphorylation pathway appears to act through protein kinase C because of an ability of phorbol esters (known activators of protein kinase C) to stimulate the phosphorylation of proteins, and an ability of a peptide factor in tick brain to metabolize salivary-gland phosphoinositides, an event that often precedes activation of protein kinase C. Because cAMP modulates brain-factor-stimulated formation of inositol phosphates (products of phosphoinositide breakdown) an interrelationship between the two pathways seems likely. Evidence of regulatory processes, including protein phosphorylation/dephosphorylation reactions, will provide a basis for helping assess the physiological significance of secretory products and the role of the salivary glands in disease transmission.