Donald E. Champagne

Antihemostatic molecules from saliva of blood-feeding arthropods.

The ability to feed on vertebrate blood has evolved many times in various arthropod clades. Each time this trait evolves, novel solutions to the problem posed by vertebrate hemostasis are generated. Consequently, saliva of blood-feeding arthropods has proven to be a rich source of antihemostatic molecules. Vasodilators include nitrophorins (nitric oxide storage and transport heme proteins), a variety of peptides that mimic endogenous vasodilatory neuropeptides, and proteins that catabolize or sequester endogenous vasoconstrictors. A variety of platelet aggregation inhibitors antagonize platelet responses to wound-generated signals, including ADP, thrombin, and collagen. Anticoagulants disrupt elements of both the intrinsic and extrinsic pathways. Molecular approaches (termed ‘sialomics’) to characterize the full inventory of mRNAs transcribed in salivary glands have revealed a surprising level of complexity within a single species. Multiple salivary proteins may be directed against each component of hemostasis, resulting in both redundancy and in some cases cooperative interactions between antihemostatic proteins, as in the case of the Rhodnius prolixus apyrase (which hydrolyzes ADP) and Rhodnius platelet aggregation inhibitor 1 (which sequesters ADP). The complexity and redundancy of saliva ensures an efficient blood meal for the arthropod, but it also provides a diverse array of novel antihemostatic molecules for the pharmacologist.

The role of salivary vasodilators in bloodfeeding and parasite transmission

In this paper, Donald Champagne reviews the salivary vasodilators, points to effects of similar compounds that may be shared by the insect substances, and discusses the potential significance of these effects with regard to parasite transmission.

Saliva of the Yellow Fever mosquito, Aedes aegypti , modulates murine lymphocyte function

Saliva of many vector arthropods contains factors that inhibit haemostatic responses in their vertebrate hosts. Less is known about the effect of vector saliva on host immune responses. We investigated the effect of Aedes aegypti salivary gland extracts on antigen‐stimulated responses of transgenic OVA‐TCR DO11 mouse splenocytes in vitro. T‐cell proliferation was inhibited in a dose‐dependent manner, with greater than 50% inhibition at 0·3 salivary gland pair (SGP) equivalents/mL. LPS‐stimulated B‐cell proliferation was also inhibited. Secretion of the Th1 cytokines IL‐2 and IFN‐γ was reduced by 50% or more with 0·45–0·6 SGP/mL, as was secretion of the pro‐inflammatory cytokines GM‐CSF and TNF‐α, and the Th2 cytokine IL‐5. The Th2 cytokines IL‐4 and IL‐10 were similarly reduced with 0·6–2 SGP/mL. Inhibition of lymphocyte function involved modulation of viable T‐cells at low salivary gland extract (SGE) concentrations, and decreased viability at higher concentrations. Dendritic cells were not killed by salivary gland extracts at concentrations as high as 25 salivary gland pairs/mL, but secretion of IL‐12 was inhibited by 87% following exposure to 0·6 SGP/mL. Activity is present in saliva and extracts of female but not male salivary glands, and it is depleted from salivary glands of blood‐fed mosquitoes. The activity is denatured by boiling and by digestion with the protease papain, indicating a protein; gel filtration HPLC indicates a mass of about 387 kDa. These results suggest that A. aegypti saliva exerts a marked immunomodulatory influence on the environment at the bite site.

Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti

The gene encoding sialokinin I, the principal vasodilatory peptide of Aedes aegypti, has been isolated and characterized. Degenerate oligonucleotide primers based on peptide amino acid sequence were used to amplify a gene fragment from messenger RNA (mRNA) isolated from female salivary glands. The amplification product was used to probe a salivary gland complementary DNA (cDNA) library, and a number of corresponding cDNAs were isolated and their primary sequence determined. Analysis of the conceptual translation product of a 406‐bp cDNA indicates that sialokinin I is expressed as a preprosialokinin and is subsequently post‐translationally processed to the active peptide. Northern analysis revealed a 490‐bp transcription product expressed exclusively in female salivary glands, and hybridization in situ of probes to RNA in whole tissues localized gene expression to the medial lobe of female salivary glands. Screening of an Ae. aegypti genomic library with the cDNA resulted in the isolation of a clone containing the gene, designated Sialokinin I (Sia I). Comparison of the cDNA with the genomic clone reveals two introns of 62 bp and 833 bp. Primer extension analysis showed that several transcription initiation sites are present. Southern analysis of genomic DNA shows that Sia I is most probably a single‐copy gene. Similarities of the Sia I gene product with other genes are confined to the region encoding the active decapeptide.

Simukunin from the Salivary Glands of the Black Fly Simulium vittatum Inhibits Enzymes That Regulate Clotting and Inflammatory Responses

Background Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized. Methodology/Principal Findings Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or −170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (KD = 0.4 nM) and to the active site of FXa (KD = 3.07 nM). We propose the name “Simukunin” for this novel protein. Conclusions We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.

Simulidin: a black fly (Simulium vittatum) salivary gland protein with anti-thrombin activity

Abstract A protein purified from the salivary gland lysate of female Simulium vittatum was found to inhibit bovine α-thrombin. This protein is stable to heat, has a mass of 11,334 Da and is rich in threonine. Based on N-terminal sequencing for the first 35 amino acids, no significant sequence similarity with other proteins was detected, indicating that this salivary component may be unique in structure. Because of its source and its anti-hemostatic properties, this protein has been named simulidin.

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana

Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H(+)-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H(+)-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg(2+) and inhibited by NaF, as well as by IDP and Ca(2+). Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H(+)-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.

Experimental infection of two South American reservoirs with four distinct strains of Trypanosoma cruzi.

Trypanosoma cruzi (Tc), the causative agent of Chagas disease, is a diverse species with 2 primary genotypes, TcI and TcII, with TcII further subdivided into 5 subtypes (IIa-e). This study evaluated infection dynamics of 4 genetically and geographically diverse T. cruzi strains in 2 South American reservoirs, degus (Octodon degus) and grey short-tailed opossums (Monodelphis domestica). Based on prior suggestions of a genotype-host association, we hypothesized that degus (placental) would more readily become infected with TcII strains while short-tailed opossums (marsupial) would be a more competent reservoir for a TcI strain. Individuals (n=3) of each species were intraperitoneally inoculated with T. cruzi trypomastigotes of TcIIa [North America (NA)-raccoon (Procyon lotor) origin], TcI [NA-Virginia opossum (Didelphis virginiana)], TcIIb [South America (SA)-human], TcIIe (SA-Triatoma infestans), or both TcI and TcIIa. Parasitaemias in experimentally infected degus peaked earlier (7-14 days post-inoculation (p.i.)) compared with short-tailed opossums (21-84 days p.i.). Additionally, peak parasitaemias were higher in degus; however, the duration of detectable parasitaemias for all strains, except TcIIa, was greater in short-tailed opossums. Infections established in both host species with all genotypes, except for TcIIa, which did not establish a detectable infection in short-tailed opossums. These results indicate that both South American reservoirs support infections with these isolates from North and South America; however, infection dynamics differed with host and parasite strain.

Black fly salivary gland extract inhibits proliferation and induces apoptosis in murine splenocytes.

Black flies are known to be vectors of pathogens including Onchocerca volvulus, which causes human onchocerciasis, and Vesicular Stomatitis Virus. Their salivary secretion has been shown to contain a complex cocktail of anti‐haemostatic factors and immunomodulatory activities, which may contribute to efficient transmission of the pathogens. Black fly salivary gland extract (SGE) inhibits mitogen‐stimulated mouse splenocyte proliferation, including proliferation of both CD4+ and CD8+ T cells. The factor responsible for the inhibition was determined to be a protein (or protein complex) of a size larger than 50 kDa. Moreover, exposure to SGE results in activation of caspase 3 and characteristic morphological changes in CD4+ and CD8+ T cells, suggesting that induction of apoptosis could, at least in part, be responsible for this inhibition.

Effects of Plant Diet on Detoxification Enzyme Activities of Two Grasshoppers, Melanoplus differentialis and Taeniopoda eques

The polyphagous grasshoppers Melanoplus differentialis and Taeniopoda eques use different foraging patterns: over time M. differentialis tends to reduce the variety of host plants it feeds on and specialize on particular plants (diet components), whereas T. eques mixes host plants to achieve a very diverse diet. We tested the hypothesis that these differing behaviors are correlated with differing patterns of detoxification enzymes. The activities of midgut, fat body, and malpighian tubule detoxification enzymes were determined in last instars of the two grasshoppers, reared for five days on single-or mixed-plant diets. Significant differences in several cytochrome P450 activities and glutathione S-transferase were evident for nymphal grasshoppers feeding on different plant diets. However, the behavioral differences between the two species could not be explained by an underlying flexibility of detoxification response in M. differentialis, but lacking in T. eques. This is the first reported evidence that detoxification enzyme activities are affected by plant diet in polyphagous orthopterans.