Identification of semiochemicals attractive to Simulium vittatum (IS-7).

Many blackfly species (Diptera: Simuliidae) are economically important insect pests, both as nuisance biters and as vectors of pathogens of medical and veterinary relevance. Among the important blackfly pest species in North America is Simulium vittatum Zetterstedt sensu lato. The objective of this study was to identify compounds excreted by mammalian hosts that are attractive to host-seeking S. vittatum females. The attractiveness of putative compounds to colonized S. vittatum was tested through electrophysiological (electroantennography; n = 58 compounds) and behavioural (Y-tube assays; n = 7 compounds in three concentrations) bioassays. Five compounds were significantly attractive to host-seeking S. vittatum females: 1-octen-3-ol; 2-heptanone; acetophenone; 1-octanol, and naphthalene. These candidate compounds might be useful as attractants in traps that could be developed for use in alternative or complementary management tactics in programmes to suppress nuisance blackfly populations, or for the collection of samples in which to study the transmission ecology of pathogens transmitted by blackflies of the S. vittatum complex.

Elimination of human onchocerciasis: history of progress and current feasibility using ivermectin (Mectizan(®)) monotherapy.

We review and analyze approaches over a 65 year period that have proven successful for onchocerciasis control in several different epidemiological settings. These include vector control with the goal of transmission interruption versus the use of mass drug administration using ivermectin (Mectizan(®)) monotherapy. Ivermectin has proven exceedingly effective because it is highly efficacious against Onchocerca volvulus microfilariae, the etiological agent of onchocercal skin and ocular disease and the infective stage for the vector. For these reasons, the drug was donated by the Merck Company for regional control programs in Africa and the Americas. Recurrent treatment with ivermectin at semi-annual intervals also impacts adult worms and result in loss of fecundity and increased mortality. Using a strategy of 6-monthly treatments with high coverage rates, the Onchocerciasis Elimination Program for the Americas has interrupted transmission in seven of the thirteen foci in the Americas and is on track to eliminate onchocerciasis in the region by 2015. Treatments given annually or semi-annually for 15-17 years in three hyperendemic onchocerciasis foci in Mali and Senegal also have resulted in a few infections in the human population with transmission levels below thresholds postulated for elimination. Follow-up evaluations did not detect any recrudescence of infection or transmission, suggesting that onchocerciasis elimination could be feasible with Mectizan(®) treatment in some endemic foci in Africa.

Mosquito and arbovirus activity during 1997-2002 in a wetland in northeastern Mississippi.

The species composition and population dynamics of adult mosquitoes in a wetland near Iuka, MS, were analyzed over a 6-yr period (1997-2002) and reverse transcription-polymerase chain reaction (PCR) detection rates of arboviruses determined during five of those years. Blood meals of three likely vector species were identified using a PCR-based method that allows identification of the host to species. Culex erraticus (Dyar & Knab) composed 51.9% of the population during the 6-yr period with 295 females collected per trap night. Eastern equine encephalomyelitis (EEE) virus was detected in six genera of mosquitoes [Coquillettidia perturbans (Walker), Culex restuans Theobald, Culex salinarius Coquillett, Culex erraticus (Dyar & Knab), Anopheles crucians Wiedemann, Anopheles quadrimaculatus Say, Aedes vexans (Meigen), Ochlerotatus triseriatus Say, and Psorophora ferox Humboldt) with positive pools occurring in 1998, 1999, and 2002. Culiseta melanura Coquillett occurred at a low level (< 1%) and was not infected. Saint Louis encephalitis virus was detected once in a single pool of Cx. erraticus in 1998. Neither West Nile virus nor LaCrosse virus was found. Minimum infection rates per 1000 females tested of competent vectors of EEE virus were variable and ranged from 0.14 for Cx. erraticus to 40.0 for Oc. triseriatus. Thirty-nine species of birds were identified in the focus with blood-engorged mosquitoes found to contain meals (n = 29) from eight avian species. The majority of meals was from the great blue heron, Ardea herodias L. (n = 55%), but when bird abundance data were adjusted for avian mass, the brown-headed cowbird, Molothrus ater (Boddaert); blue jay, Cyanocitta cristata (L.); and northern mockingbird, Mimus polyglottos (L.), were overrepresented as hosts.

Evaluation of a recombinant salivary gland protein (thrombostasin) as a vaccine candidate to disrupt blood-feeding by horn flies.

The potential for controlling blood-feeding by the cattle pest, Haematobia irritans irritans (horn fly), was tested by vaccination against thrombostasin (TS), an inhibitor of mammalian thrombin that is released into skin during horn fly blood-feeding. The increase in blood meal size that occurred for flies feeding on sensitized non-vaccinated hosts was blocked and egg development in female flies was delayed when horn flies fed on rabbits and cattle immunized with recombinant TS. This demonstration of the impact of disrupting TS action by vaccination provides a novel approach toward control of this veterinary pest and offers a paradigm for limiting blood-feeding in other medically-important insect species.

Orientation of Onchocerca lienalis stiles (Filarioidea: Onchocercidae) microfilariae to black fly saliva.

Black flies (Simulium spp.) are intermediate hosts and vectors of parasitic nematodes belonging to the genus Onchocerca (Filarioidea Onchocercidae). Infection and subsequent transmission of infective third-stage larvae occur at the vertebrate host-skin interface. Experimental evidence presented here demonstrates that Onchocerca lienalis Stiles microfilariae orient to one or more components (microfilarial orientation factor [s]; MOF) in black fly saliva. MOFs may serve as a means for microfilariae to find and infect black flies during the act of blood-feeding. Directed movement through the host's skin to the bite site is necessary because Onchocerca spp. microfilariae do not circulate in the blood. The substance directing microfilarial orientation appears to be a salivary protein, but it is not the Simulium vittatum Zetterstedt erythema protein (SVEP) described from New World Simulium spp. These results support earlier field observations that associated increased numbers of cutaneous microfilariae with black fly feeding and indicate that a fundamental molecular mechanism linked to vector saliva may be key for the maintenance of the life cycle of Onchocerca spp. Salivary molecules that induce orientation of microfilariae to the bite site are potential targets for use in transmission-blocking vaccines to uncouple this primary vector infection step.

Thrombostasin: purification, molecular cloning and expression of a novel anti-thrombin protein from horn fly saliva.

Thrombostasin (TS), a novel protein found in the saliva of Haematobia irritans (horn fly), was purified by high-performance liquid chromatography from the saliva of field-collected insects. This protein, which inhibits thrombin, accounts for anti-clotting activity in horn fly saliva [J. Med. Entomol. 37 (2000) 416] and is the first purified anti-hemostatic factor described from the Stomoxyinae, a large group of blood-feeding insects that are major pests of livestock world-wide. The purified TS had an apparent molecular weight of 16.7 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed two isoelectric groups with isoelectric points (pIs) of approximately 4.6 and 4.8. Mass spectroscopy analysis, however, resulted in at least three major isoforms that range in mass from 9213 to 9274 Da. A 243-bp coding sequence was obtained from cDNA by using a degenerate primer deduced from the N-terminal sequence of the purified TS. The conceptual translation of the 243-bp sequence showed that the 81-amino-acid peptide, whose first 30 amino acids match those of the N-terminal sequence, had a predicted mass of 9213 Da with pI 4.14. A full-length TS cDNA was generated by rapid amplification of cDNA ends of the 5' and sequential polymerase chain reaction (PCR) amplification. It contained a 5'-end 12-bp segment preceding the putative ATG start codon, followed by a 54-bp sequence corresponding to a secretory signal and an additional 228-bp coding sequence preceding residues revealed by N-terminal sequencing of purified TS. The fidelity of the PCR-generated TS sequence was confirmed in genomic DNA and by biological activity of recombinant TS produced in a baculovirus expression system. Database comparisons revealed no homology between TS and other known molecules. Because of the paucity of other anti-hemostatic factors in horn fly saliva, TS may play a critical role in maintenance of the ectoparasitic lifestyle of horn flies.

Polymorphism of the thrombostasin gene in the horn fly (Haematobia irritans) revealed in a cDNA library and in genomic DNA.

Thrombostasin (TS) is a newly described thrombin-inhibiting protein isolated from the saliva of the horn fly (Haematobia irritans), a blood-sucking ectoparasite of cattle. This report provides a detailed characterization of the TS gene and the first analysis of the allelic complexity of a gene for an anti-hemostatic protein from a blood-feeding insect. Multiple point mutations at fixed positions in the TS gene were identified in a cDNA library prepared from mRNA isolated from horn fly salivary glands. When translated, the variant mRNAs would specify five biochemically active peptides that differ in molecular weight, isoelectric point and predicted secondary structure. Allelic variation with the same mutation pattern was revealed in the genomes of individual flies collected in the field and sampled from a long-standing laboratory colony. Approximately 60% of flies examined carried heterozygous alleles, including five additional alleles not found in the cDNA library. Comparative analysis of the allelic mutations and the predicted effects on secondary structures of the active proteins produced suggest that the TS gene may be undergoing evolutionary selection.

Simulium vittatum (Diptera: Simuliidae) and Lutzomyia longipalpis (Diptera: Psychodidae) salivary gland hyaluronidase activity.

Hyaluronidase activity in the salivary gland homogenates of Simulium vittatum (Zetterstedt) is described, and its optimal pH determined. Salivary activity was reduced significantly after a blood meal, indicating that it was secreted after blood feeding. Phlebotomus papatasi (Scopoli) also exhibited salivary hyaluronidase activity. These results indicate that hematophagous pool-feeding insects may secrete this enzyme to help the spread of salivary antihemostatic agents in the vicinity of the feeding lesion, and perhaps to increase the size of the feeding lesion itself. Additionally, this enzyme may affect local host immune reactions and promote arboviral transmission.

Horn fly (Diptera: Muscidae) saliva targets thrombin action in hemostasis.

The horn fly, Hematobia irritans (L.), is an important pest of livestock because the adult stage of both sexes are aggressive blood-feeders. Remarkably, even though horn fly adults feed recurrently on their hosts as ectoparasites, these flies lack the ADP-responsive antiplatelet aggregation and vasodilatory antihemostatic systems described for other blood-feeding Diptera. Horn fly salivary gland extracts do interfere with the normal coagulation process as demonstrated by the recalcification time assay. Using this as a baseline, the effects of saliva on recalcification time, activated partial thromboplastin time, prothrombin time, and thrombin time were measured to determine which arm(s) of the coagulation cascade might be impacted. Factor-deficient plasma assays also were used to measure possible perturbations in clotting. Gland-free saliva delayed the recalcification time as well as the activated partial thromboplastin time, prothrombin time, and thrombin time. Saliva also further delayed clotting times of plasmas deficient in factor V, factor VIII, and factor XIII, indicating that other factors in the coagulation cascade were inhibited. Although horn fly saliva did not alter the ability of deficient plasma reconstituted with factor X to clot, it did inhibit deficient plasma reconstituted with factor II (thrombin). Antithrombin activity in saliva was confirmed by its ability to interfere with thrombin hydrolysis of fibrinogen, its normal substrate, and by its inhibition of thrombin action on a chromagenic substrate that mimics the hydrolytic site of fibrinogen. Thus, horn fly saliva contains a factor that specifically targets thrombin, a key component in the coagulation cascade. While the biochemical mechanisms of inhibition may vary, this antihemostatic characteristic is shared with other zoophilic Diptera such as black flies, Simulium spp., and tsetse, Glossina morsitans morsitans Westwood, that feed on ungulates.

Blood-feeding strategy of Haematobia irritans (Diptera: Muscidae).

The economic impact on livestock production by Haematobia irritans (L.) is estimated to approach $1 billion per year in North America. However, there is little information regarding the blood-feeding strategy used by these insects. Information presented here shows that horn fly saliva interferes with the normal coagulation response as measured by the recalcification time assay. The relative anticoagulant activity on a per-gland basis was more than or equal to that reported for Simulium vittatum Zetterstedt, a common hematophagous black fly that also feeds on cattle. However, unlike S. vittatum, H. irritans salivary factors do not inhibit platelet aggregation using apyrase and have no detectable vasodilative activity. In this regard, the horn fly is strikingly different from blood-feeding species in the lower Diptera and shows a much more limited repertoire of antihemostatic factors.

Analyses of cDNA and recombinant protein for a potent vasoactive protein in saliva of a blood-feeding black fly, Simulium vittatum.

A cDNA was cloned from the salivary glands of a blood-feeding black fly Simulium vittatum. The encoded protein has been given the name Simulium vittatum erythema protein or SVEP, because of its ability to increase blood perfusion in skin capillaries, resulting in the well-characterized erythema of black fly bites. The full-length cDNA contains 548 base pairs which encode 152 amino acid residues of the nascent protein. Post-translational processing produces a mature, secreted protein of 133 residues with a molecular mass of 15.4 kDa. Recombinant SVEP (rSVEP) was produced in a baculovirus expression system and purified by a one-step reversed-phase HPLC procedure. Analyses of physical properties and biological potency demonstrated fidelity of rSVEP to the native protein. Recombinant SVEP relaxed rabbit aorta preparations when preconstricted with 2 micromol l-1 phenylephrine or 25 mmol l-1 K+ but not with 60 mmol l-1 K+. Further, the rSVEP-induced relaxation response of phenylephrine-constricted aorta was inhibited by glibenclamide (10 micromol l-1), suggesting that at least part of its action to relax smooth muscle may result from the opening of ATP-dependent K+ channels. SVEP is a novel salivary-gland-derived vasoactive protein that may be essential for blood feeding by black flies and could potentially enhance transmission of filarial parasites.

Comparison of black fly species (Diptera: Simuliidae) on an Amerindian reservation with a high prevalence of fogo selvagem to neighboring disease-free sites in the State of Mato Grosso do Sul, Brazil. The Cooperative Group on Fogo Selvagem Research.

Fogo selvagem is an autoimmune blistering skin disease that principally occurs among rural Brazilians living in geographically clumped disease foci. Exposure to hematophagous black flies possibly is related to the cause of the disease. We compared the occurrence, proportions, and richness of simuliid species immatures and the biting activity of adult females within a recently discovered, high prevalence focus of fogo selvagem, the Limão Verde Terena Reservation, to that of neighboring regions with no reported cases of fogo selvagem. Nine black fly species were collected from 12 stream sites during 5 trips to the fogo selvagem focus. The species showed longitudinal (upstream-downstream) trends in occurrence, proportions, and richness, and the abundance of simuliid immatures was greater at downstream sites. The most prevalent species at the focus, Simulium nigrimanum (Macquart), dominated the stream sites with highly abundant simuliid assemblages, and was the most common black fly in human bait collections. This species was absent or in very low numbers in neighboring valleys and villages that did not have cases of fogo selvagem.

Vector competence of select black fly species for vesicular stomatitis virus (New Jersey serotype).

Black flies collected from southern Arizona were evaluated for their vector competence to the Oaxaca and Camp Verde isolates of vesicular stomatitis virus (New Jersey serotype) (VSV-NJ). The Camp Verde isolate is the index isolate of the 1982-1983 VSV-NJ epizootic that infected humans and livestock in 14 western states. Previous experiments have shown that colonized Simulium vittatum females are competent laboratory vectors of both virus isolates. However, under controlled laboratory conditions, Simulium bivittatum and S. longithallum were found to be incompetent vectors of both virus isolates. After oral infections, the Oaxaca isolate replicated in 35% and 38% of S. bivittatum and S. longithallum, respectively, but did not disseminate to the salivary glands. Thus, virus was not detected in the saliva of either black fly species with either VSV-NJ isolate, indicating the presence of a midgut barrier. Simulium notatum was found to be a competent laboratory vector of both virus isolates. Infectious virions were detected in the saliva of 23% and 26% of S. notatum infected orally with the Oaxaca and Camp Verde VSV-NJ isolates, respectively. This study suggests that the black fly identified as S. bivittatum was probably not involved in virus dissemination during the 1982-1983 epizootic in the western United States. Because the geographic distribution of S. notatum is not known, its involvement in that epizootic remains obscure.

Black fly (Diptera:Simuliidae) salivary secretions: importance in vector competence and disease.

When blood-feeding, black flies introduce secretions into the feeding lesion that act in a coordinated manner on the 3 arms of the vertebrate hemostatic system (platelet aggregation, coagulation, and vasoconstriction). Apyrase activity inhibits platelet aggregation and is ubiquitous in the saliva of black flies, although activity per gland varies by species and has a positive association with anthropophagy. Anticoagulants target components in the final common pathway of the coagulation cascade, including factors V, Xa, and II (thrombin). The antithrombin salivary protein may exert a redundant effect by inhibiting the role of thrombin in platelet aggregation. Antithrombin presence and activity also varies among black fly species, and exhibits a positive correlation with zoophagy. Vasodilation of capillaries to increase blood supply to the feeding wound appears to be an important requirement for Simulium spp., because substantial erythema-inducing activity, has been demonstrated in salivary glands of all New World species examined. Salivary glands of Simulium ochraceum (Walker), a highly anthropophilic vector of Onchocerca volvulus (Leuckhart), contain greater vasodilator activity than several other species, including S. metallicum Bellardi, a secondary zoophagic vector of human onchocerciasis. Simulium vittatum Zetterstedt saliva affects immune cell responses and cytokine production. The ability of the saliva to modulate components of the host immune system provides an opportunity for enhancing transmission of pathogens during bloodfeeding. Thus, the likely possibility that effective pathogen transmission relies on vector saliva may complement present efforts aimed at target epitopes of O. volvulus or identify additional molecules to be investigated as part of a "river blindness" vaccine cocktail. Components in saliva also may enhance the transmission of other microbial agents either by a cofeeding process similar to that observed in ixodid ticks or through rupture of the labrum during escape of Onchocerca infective stage larvae. In a few instances, saliva of some Simulium spp. also has been associated with extensive tissue and organ pathology, including hemorrhagic shock and death. Pathologic signs associated with this syndrome indicate an enhanced antihemostatic activity in saliva.

Cellular hemolymph response of Simulium vittatum (Diptera:Simuliidae) to intrathoracic injection of Onchocerca lienalis (Filarioidea:Onchocercidae) microfilariae.

Hemolymph cellular changes in Simulium vittatum Zetterstedt in response to intrathoracic injection with Onchocerca lienalis Stiles were characterized by increased numbers of tissue fragments that contained cells embedded within a noncellular matrix. Cell numbers within the matrix fragments increased both for sham and microfilariae-injected files, indicating a blastogenic response to injection alone. Morphological characteristics of fixed, Giemsa-stained cells within these tissue fragments were most similar to those previously described for prohemocytes. The total population of freely circulating hemocytes collected 24 h after injection also responded to injection alone. Differential cell counts showed a complex pattern of changes that was influenced strongly by increased numbers of prohemocytes at 24 h in microfilariae-injected flies. Fat body fragments collected in hemocoel perfusates at 24 h were fewer when flies were maintained at 27 degrees C than at 21 degrees C. More fat body fragments were collected from microfilariae-injected flies than from control and sham-injected flies held at 27 degrees C. Injection of 1.25-5 micrograms of lipopolysaccharide per fly did not elicit similar hemolymph-associated matrix tissue and fat body changes, indicating that S. vittatum response to microfilaria infection and bacteria infection are likely to involve different mechanisms.

Molecular phylogeny and typing of blackflies (Diptera: Simuliidae) that serve as vectors of human or bovine onchocerciasis.

A subregion of the mitochondrial large subunit (16s) rRNA gene was amplified by polymerase chain reaction (PCR) from nine species of blackflies (Diptera: Simuliidae) which serve as natural or experimental vectors of human or bovine Onchocerca parasites. PCR products from each species of blackfly were tested by directed heteroduplex analysis (DHDA), and their genotypes established according to diagnostic banding patterns of the heteroduplex products. Three alleles of mitochondrial 16s rRNA were found to exist in members of the Simulium (Ewardsellum) damnosum sensu lato complex from West Africa, and two alleles were found in the Neotropical Simulium (Psilopelmia) ochraceum Walker complex and the Simulium (Simulium) metallicum Bellardi complex. Different single alleles were detected in Austrosimulium bancrofti, in English S.(S)noelleri and in two North American laboratory vectors: Simulium (Psilozia) vittatum Zetterstedt and S.(S.)decorum Walker. Phylogenetic analysis of 16s sequences indicated that blackflies from West Africa and the Americas formed distinct clades. Neotropical onchocerciasis vectors were found to be more closely related to Nearctic and Palaearctic non-vector Simulium species than to the African vectors of onchocerciasis.

Novel anticoagulant from salivary glands of Simulium vittatum (Diptera: Simuliidae) inhibits activity of coagulation factor V.

Anticoagulant activity was detected in fractions of a reversed phase-high-performance liquid chromatography (RP-HPLC) of salivary gland lysate from Simulium vittatum Zetterstedt. Using a plasma recalcification time assay, these fractions did not inhibit factor Xa or thrombin. HPLC-purified fractions showing the anticoagulant property were pooled and examined using the activated partial thromboplastin time test conducted on normal plasma and plasmas deficient in factors V, VIII, IX, XI, and XII. The anticoagulant prolonged the clotting time of all the plasmas, except plasma deficient in factor V. The detection of antifactor V activity, together with other anticoagulants reported from Simulium spp. indicates a feeding strategy that targets enzymes in the terminal portion of the coagulation cascade.

Occurrence of Lutzomyia anthophora (Diptera: Psychodidae) in Arizona.

Males and females of Lutzomyia anthophora Addis were collected by vacuum aspiration from woodrat, Neotoma albigula Hartley, and rock squirrel, Citellus variegatus Bailey, nests along Arivaca Creek in Pima County, Arizona. Additional flies were collected from the same location using CDC miniature light traps supplemented with CO2. These collections extend the recorded geographic distribution of this vector of Leishmania mexicana Biagi westward by approximately 724 km and place Lu. anthophora in a Sonoran desert habitat.

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status.

Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum, a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 +/- 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 +/- 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 +/- 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S. (Ps.) ochraceum (24.8 +/- 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum, a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S. (N.) exiguum (10.9 +/- 0.6 mU) than in Simulium (Notolepria) gonzalezi (5.9 +/- 1.9 mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O.volvulus.