José M. C. Ribeiro

Role of saliva in tick/host interactions

Although several hosts mount efficient anti-tick immunity, natural tick/host associations are characterized by inefficient or non-existent anti-tick immunity. The absence of efficient anti-tick immunity in natural hosts could result from either host immune incompetence or the ectoparasites successful evasion of the hosts immune response. In this review I discuss data supporting the immune-evasion hypothesis and discuss its consequences to tick/host interactions.

Function and Evolution of a Mosquito Salivary Protein Family

Saliva of blood-sucking arthropods contains a complex and diverse mixture of antihemostatic, antiinflammatory, and immunomodulatory compounds. The D7 salivary family of proteins is abundantly expressed in blood-feeding Diptera and is distantly related to the odorant-binding protein superfamily. In mosquitoes, two subfamilies exist, the long and short D7 proteins. Ticks and kissing bugs evolved salivary lipocalins that act as efficient scavengers of biogenic amines, and a similar function was postulated for the D7 proteins. Accordingly, we expressed the five members of the small D7 family of the African malaria vector Anopheles gambiae and a D7 long form from Aedes aegypti and showed by isothermal microcalorimetry, a modified and very sensitive non-equilibrium chromatography/spectrum distortion method, and by smooth muscle bioassay that four of these five short D7 proteins and the D7 long form bind serotonin with high affinity, as well as histamine and norepinephrine. The nonbinding D7 protein is poorly expressed in the salivary glands and appears to be on the path to becoming a pseudogene. Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties. It appears that counteracting biogenic amines is of strong adaptive value in the convergent evolution of arthropods to hematophagy. This adaptation has been solved independently in ticks, bugs, and mosquitoes by co-option of either member of the lipocalin or, as shown here, by the odorant-binding protein families.

Saliva of the tick Ixodes dammini inhibits neutrophil function

Pilocarpine-induced saliva of adult female Ixodes dammini ticks inhibits the function of peritoneal-derived rat neutrophils, as measured by anaphylatoxin-induced aggregation, FMLP-induced granule enzyme secretion, zymosan-induced superoxide secretion, and phagocytosis of Borrelia burgdorferi spirochetes. Inhibition ranged from 40 to 80% when saliva was diluted 20 times into the assay medium. This neutrophil-inhibiting activity of I. dammini saliva may aid in tick feeding and facilitate pathogen transmission.

Ixodes dammini: salivary anti-complement activity

Saliva of the tick Ixodes dammini prevents hemolysis of rabbit erythrocytes by the human alternative pathway of complement. Deposition of C3b to activating surfaces and concomitant C3a release are inhibited. C3b deposition to activating surfaces is inhibited regardless the origin (humans, rat, mouse, guinea pig, and hamster) of the serum. The inhibitor elutes as a single peak upon gel filtration, with an apparent molecular weight of 49,000. Salivary anti-complement may contribute to successful feeding of I. dammini in their natural hosts.

The role of vector saliva in transmission of arthropod-borne disease.

Blood-sucking arthropod disease vectors all share one important feature: while probing for blood in the vertebrate hosts skin they salivate into the wound they create. Recent studies on the pharmacological properties of vector saliva have revealed an array of activities that are potentially beneficial to both the vector and to the pathogen. These observations may help explain why certain vectors and pathogens have co-evolved. In this article, Richard Titus and Jose Ribeiro discuss the role vector saliva may play in disease transmission, and the prospects for its use in the control of arthropod-borne pathogens.

Purification, Cloning, and Expression of an Apyrase from the Bed Bug Cimex lectularius A NEW TYPE OF NUCLEOTIDE-BINDING ENZYME

An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purifiedC. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of ∼40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectulariuscDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with nativeC. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized bothC. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum.C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the genome of the nematode C. elegansand in mouse and human expressed sequence tags from fetal and tumor EST libraries.

The D7 family of salivary proteins in blood sucking diptera

The D7 subfamily of salivary proteins is widespread in blood sucking Diptera and belongs to the superfamily of pheromone/odourant binding proteins. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. In the present work we report the sequence of seventeen novel D7 proteins, and propose an evolutionary scenario for the appearance of the several forms of this protein, based on a total of twenty‐one sequences from Culex quinquefasciatus, Aedes aegypti, Anopheles gambiae, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.

Ixodes dammini: Salivary anaphylatoxin inactivating activity

Saliva of the tick, Ixodes dammini, antagonizes anaphylatoxin, abolishing both the effects of anaphylatoxin on guinea pig ileum preparations regularly stimulated with histamine and the local edema caused by intradermal injection of anaphylatoxin into guinea pigs. Saliva of these ticks, however, did not modify polymorphonuclear leukocyte aggregation induced by anaphylatoxin. Bradykinin and lysil-bradykinin were inactivated, but angiotensin I, angiotensin II, and substance P were not affected. Amino acids were released rapidly following incubation of saliva with bradykinin, but slowly from des-arg-9-bradykinin. These results suggest the presence of a salivary carboxypeptidase with specificity for terminal basic amino acids. Such activity may inactivate anaphylatoxin and bradykinin at the site of tick attachment.

Amblyomma americanum: Characterization of salivary prostaglandins E2 and F2α by RP-HPLC/bioassay and gas chromatography-mass spectrometry

Secretagogue-induced saliva of the tick, Amblyomma americanum (L.) was fractionated by reversed-phase-high-performance liquid chromatography (RP-HPLC) and bioassayed in smooth muscle preparations. Material with retention times of authentic prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) were found to cause contraction of preparations of rat colon and rat stomach strips. Gas chromatography-mass spectra of selected ions of both HPLC-purified fractions confirmed the existence of PGE2 and PGF2 alpha. Bioassay of individual samples obtained from ticks stimulated to salivate with pilocarpine, dopamine + theophiline, or dopamine + theophiline + GABA indicated that all these secretagogues induced similar amounts of prostaglandin secretion, averaging 469 ng PGE2/ml. These pharmacological doses of prostaglandin are hypothesized to assist in tick feeding by inducing vasodilation and/or other pharmacological events in their hosts.

Diet and salivation in female Aedes aegypti mosquitoes

Salivary glands of the female mosquito, Aedes aegypti, contain an α-glucosidase in their proximal lobes and an apyrase in the distal lobes. Following a liquid sugar meal, there is a significant reduction in the activity of the salivary glucosidase, but no significant reduction in the activity of the apyrase. Following a blood meal, both enzyme activities are significantly reduced. The rate of accumulation of both enzymes after a blood meal differs. The glucosidase activity returns to nonfed control values within 1 h of the blood meal and increases to 3 times the control values after 2 days. Salivary apyrase activity remains significantly lower than control values for up 3 h after the blood meal, reaches control values by 24 h and becomes significantly (30%) higher than control values 2 days after the blood meal. Total salivary gland protein levels remain significantly lower than control values for up to 3 h after a blood meal and return to control values at 24 h. A neuro-humoral model is postulated to explain the results.