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.

The effects of ivermectin on transmission of Onchocerca volvulus.

Ivermectin, given to onchocerciasis patients as a single oral dose of 200 micrograms per kilogram of body weight, substantially reduced the uptake of Onchocerca volvulus microfilariae by Simulium yahense, an efficient black fly vector of the parasite in the tropical rain forests of West Africa. Three months after treatment, patients given ivermectin infected flies at a significantly lower rate than those who had received diethylcarbamazine or placebo, thereby reducing the number of developing larvae in the vector population. This diminished rate of infectiousness was also evident 6 months after treatment. These results strongly suggest that ivermectin could be effective in interrupting transmission of Onchocerca volvulus for epidemiologically important periods of time.

Isolation and characterization of a coagulation factor Xa inhibitor from black fly salivary glands.

We have discovered and characterized a novel coagulation factor Xa inhibitor from the salivary gland of the black fly, Simulium vittatum. Salivary glands were surgically dissected from the flies and a crude salivary gland extract was tested for inhibition of a number of coagulation assays. The gland extract inhibited both thrombin and factor Xa. To purify further the factor Xa inhibitor, a factor Xa affinity column was utilized. Final purification of the black fly factor Xa inhibitor was achieved by reverse-phase C8 microbore high pressure liquid chromatography. Inhibition of factor Xa was nearly stoichiometric by the purified inhibitor with no inhibitor of thrombin detected. SDS-polyacrylamide gel electrophoresis indicated the inhibitor had a molecular weight of 18,000 and sequence analysis of the inhibitor revealed a blocked amino terminus. These data indicate that the blood-sucking black fly has evolved a highly potent inhibitor of mammalian coagulation factor Xa to disrupt its host normal hemostatic clotting mechanisms.

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.

Treatment of onchocerciasis. The ocular effects of ivermectin and diethylcarbamazine.

The ocular changes that occur with diethylcarbamazine treatment of onchocerciasis seriously restrict its usefulness. Ivermectin, a newly developed antifilarial drug, was compared with diethylcarbamazine for treatment of onchocerciasis in a double-masked, placebo-controlled trial. Thirty men with moderate to severe infection and ocular involvement were randomly assigned to receive ivermectin as a single oral dose (200 micrograms/kg), diethylcarbamazine (administered for eight days), or placebo. Detailed ocular examinations were performed serially over a 12-month period. Diethylcarbamazine treatment caused a marked increase in living and dead microfilariae in the cornea, punctate opacities, and limbitis during the first week of therapy. Ivermectin had no such effect. However, ivermectin therapy resulted in a long-term reduction in intraocular microfilariae comparable to that seen with diethylcarbamazine. Ivermectin appears to have few ocular complications and be a better-tolerated and more effective microfilaricidal agent than diethylcarbamazine for the treatment of onchocerciasis.

The gonotrophic cycle in Simulium ochraceum.

Oogenesis in Simulium ochraceum is characterized in terms of distinct stages of follicular development following emergence, sugar feeding, and completion of a blood meal. Changes in the ovary following the first oviposition are also described. This black fly species was found to be anautogenous and exhibited gonotrophic concordance. The formation of follicular dilatations after oviposition followed a distinct pattern with traces of cellular debris evident up to 144 hours. The presence of fructose, as detected by the cold Anthrone test, indicated that both parous and nulliparous flies routinely imbibe nectars as part of their foraging behavior. The availability and frequent use of nectar may contribute significantly to longevity of parous flies, hence increasing their vector potential.

Radiolabeling of Brugia malayi infective larvae in mosquitoes with 75Se-methionine and detection of these larvae in tissues of the Mongolian jird by autoradiography.

Through preliminary experiments, an effective method for radiolabeling Brugia malayi-infected mosquitoes in order to produce labeled infective Brugia larvae was developed. Starting on the 6th day after the infective blood meal, mosquitoes were fed a 7% sucrose solution containing 100 microCi/ml 75Se-L-methionine for 5 days. Infective larvae, retrieved 2 days after this labeling period, averaged 381 +/- 136 counts/min. Jirds were infected with these infective, labeled larvae either by allowing infected mosquitoes to feed on uninfected jirds for 30 min or by inoculating jirds subcutaneously in the groin with washed larvae recovered from mosquitoes. Jirds were killed at various times after infection and were sliced into approximately 0.5 mm thick sagittal sections, which were dried and placed on X-ray film. Autoradiograms were developed after 30-60 days at 5 degrees C. In a sample of 26 inoculated jirds, approximately 30% of the infecting larvae could subsequently be accounted for as Ag degrees foci on autoradiograms. The Ag degrees foci representing larvae were apparent up to 2.5 weeks after infection. In jirds infected by mosquito feeding, the Ag degrees associated with the feeding site persisted for more than 6 weeks after infection.

The effects of precocene II on reproduction and development of triatomine bugs (Reduviidae: Triatominae).

Precocene II is a botanically derived chemical that inhibits the production of juvenile hormone (JH) in insects. The effects of this anti-JH compound on molting and growth by Rhodnius prolixus and Triatoma dimidiata and reproduction in R. prolixus were tested and the efficacy of precocene II as a fumigant was assessed. Precocene II induced precocious metamorphosis in both species when applied by either contact exposure or fumigation, and this effect could be prevented by juvenile hormone replacement therapy. The dosage effective in inducing precocious metamorphosis in T. dimidiata was similar to the EC50 previously reported for R. prolixus. The morphology of precocious adultoid T. dimidiata of different instars was similar to Rhodnius adultoids of corresponding instars. However, T. dimidiata was more sensitive than Rhodnius to the molt-inhibiting effects of precocene II. Rhodnius nymphs were fully susceptible to the anti-JH action of precocene when exposed for 24 hours one week before feeding. Precocene II was highly toxic to adult female Rhodnius and treatment of newly emerged females prevented oogenesis.

Differential modulation of murine cellular immune responses by salivary gland extract of Aedes aegypti.

The ability of salivary gland extract (SGE) of Aedes aegypti to modulate cellular immune responses was investigated in a mouse model. Cytokine production was induced in naive and antigen-primed murine (BALB/c) spleen cells in vitro by stimulation with the T cell mitogen concanavalin A or the T cell-dependent antigen ovalbumin (OVA), respectively. Inclusion of Ae. aegypti SGE in in vitro culture with naive cells caused significant suppression of the cytokines interleukin-2 (IL-2) and interferon gamma in culture supernatants, while levels of other cytokines (IL-4 and IL-5) were unaffected by SGE. In contrast, SGE did not affect cytokine production by antigen-activated cells derived from OVA-primed mice. To determine whether SGE could inhibit the responsiveness of cells to exogenous cytokine stimuli, optimized quantities of lymphocyte growth factor cytokines IL-2 and IL-4 were added to SGE-treated spleen cells and the degree of cellular proliferation was determined. Cellular proliferation in response to IL-2 was markedly suppressed by prior exposure of cells to SGE, while the proliferative response to IL-4 was also affected by SGE but to a lesser extent. These results confirm that mosquito SGE can modulate host immune responses, and suggest that in Ae. aegypti modulation is directed primarily against cytokines associated with type 1 lymphocyte responses. The mode of immunomodulation and the possible relevance of these results to vector-borne disease research are discussed.

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.

Salivary apyrase in African and New World vectors of Plasmodium species and its relationship to malaria transmission.

The salivary gland activities of apyrase, an enzyme that prevents platelet aggregation by eliminating ADP, were compared among five members of the Anopheles gambiae species complex and An. albimanus. Within the An. gambiae group, An. quadriannulatus exhibited the lowest amount of enzyme activity at all pH levels measured. Apyrase activity could be separated into three groups at pH 7.5 and 8.0. The two most anthropophilic species (An. gambiae and An. arabiensis) exhibited higher activity at pH 9.0. Anopheles merus and An. melas, both saltwater taxa, and An. albimanus, a New World species, exhibited peak apyrase activity at pH 8.0. When the effects of divalent cations (Ca++, Mg++) on enzyme activity were compared at pH 8.5, apyrase activity in the presence of Mg++ could be separated into three levels. Anopheles gambiae and An. quadriannulatus exhibited reduced activity in the presence of Mg++. Anopheles arabiensis, An. merus, and An. melas displayed the highest relative levels of activity. Anopheles albimanus, with a Mg:Ca ratio of 0.80, was most similar to An. arabiensis. These biochemical differences suggest that different isoenzymes of apyrase have developed within the genus Anopheles.

Vasodilative activity in black fly salivary glands.

Salivary gland extracts of several Simulium spp. were shown to contain vasodilative activity as measured by the rapid and persistent induction of erythema in response to intradermal injection into rabbit skin. Total salivary gland activities were approximately equal for S. vittatum, S. metallicum, S. bivittatum, and S. argus (titers of 0.03-0.02 pairs of gland). Total gland activity in the highly anthropophilic species S. ochraceum, however, was an order of magnitude greater, with erythema produced by as little as 0.002 pairs of glands. Tests for physical stability of the activities from two species (S. vittatum and S. ochraceum) indicated that the vasodilators were proteinaceous and heat stable. A two-step, reversed-phase high-performance liquid chromatography (HPLC) procedure was developed that isolated both activities with similar elution patterns. Homogeneity of the purified protein from S. vittatum was confirmed by capillary gel electrophoresis. Electrospray ionization mass spectroscopy of the S. vittatum protein detected a mass of 15,351 daltons. Similarity in elution times of the proteins from a TSK HPLC column predict some structural similarities between the S. vittatum and S. ochraceum vasodilator proteins.

Seasonal trends and diurnal patterns of man-biting activity of four species of Guatemalan black flies (Simuliidae).

Collections and dissections of black flies from human subjects were carried out over a 13-month period at two onchocerciasis-endemic sites in Guatemala. Simulium ochraceum comprised 87% of all flies collected. Populations of this species peaked during the first part of the dry season in November, while populations of S. metallicum, S. callidum and S. downsi were highest during the rainy season from June-October. Diurnal patterns of host-seeking activity were most pronounced for S. ochraceum, with a sharp peak occurring between 0700 and 0900 hours each day. Parous ratio was lowest at this time (27%), and rose to a peak of 63% between 1200 and 1400 hours each day. Eighty-eight percent of all parous S. ochraceum collected during 1200-1400 hours had large dilatations in the tunica of the ovarioles resulting from recent oviposition, thus indicating that this species oviposits in the morning and immediately seeks the subsequent blood meal. Parous biting density (the product of the total number of flies biting at a given time and the corresponding parous ratio) showed two distinct diurnal peaks, one in the early morning characterized by low parous ratio and high total number of flies, and the other in the early afternoon characterized by high parous ratio and low total number of flies. Moreover, the diurnal pattern of biting density of filaria-infected S. ochraceum was similar to that of parous biting density.

Transmission of Venezuelan encephalitis virus by naturally infected Culex (Melanoconion) opisthopus.

During August 1977 two of 975 Culex (Melanoconion) opisthopus collected from an enzootic marsh habitat on the Pacific coast of Guatemala transmitted VE virus to hamsters. Eight VE strains were isolated from Cu. opisthopus. The minimal level of VE infection in this species during July-August 1977 at La Avellana, Guatemala was 1/128 (8/1,021), and the prevalence of Cu. (Mel.) opisthopus transmitting VE virus was 1/487 (2/975). This mosquito was the predominant species attacking humans at that time, suggesting that Cu. opisthopus is a vector of VE virus to man as well as a vector in enzootic cycles in Guatemala. These studies establish Cu. opisthopus as the third proven enzootic vector of VE virus.

Large scale production of the vertebrate infective stage (L3) of Onchocerca volvulus (Filarioidea: Onchocercidae).

Laboratory-derived Simulium yahense and S. sanctipauli females were used to produce large numbers of Onchocerca volvulus infective stage larvae (L3) for use in assessing the possible chemoprophylactic potential of ivermectin in the chimpanzee model. Engorgement rates and subsequent post-prandial survival were correlated with the time at which adult flies were offered a carbohydrate source following emergence and the age of flies that fed on microfiladermic volunteers.

Ecologic observations of Venezuelan encephalitis virus in vertebrates and isolations of Nepuyo and Patois viruses from sentinel hamsters at Pacific and Atlantic habitats in Guatemala, 1968-1980.

La Avellana and Puerto Barrios, two enzootic foci of Venezuelan encephalitis (VE) virus on the Pacific and Caribbean lowlands (respectively) of Guatemala have been studied over a 13-year period. Data from sentinel hamsters and guinea pigs and wild and domestic vertebrates are reported. VE virus strains were isolated from hamsters each period they were exposed during the rainy seasons 1968-1980 and at the end of the dry season 1974. Rates of isolation of VE virus ranged from 0.2%-5.7% hamster/days/exposure. All strains tested were free of epizootic virions. Although virus was isolated from sentinel guinea pigs, their deaths were not attributable to infection with VE virus. Antibody titers in 26 of 28 terrestrial mammals bled at La Avellana in 1971 were higher to enzootic than to epizootic VE strains. Thirty-seven percent of 109 residents of Puerto Barrios had antibody to VE virus. In 13 of 20 tested, antibodies were engendered by the enzootic strain. Nepuyo and Patois viruses were isolated from sentinel hamsters at both La Avellana and Puerto Barrios.

Effects of multiple monthly doses of ivermectin on adult Onchocerca volvulus.

This paper assesses the effects on adult Onchocerca volvulus of monthly doses of ivermectin (150 micrograms/kg) given over 4, 8, and 12 months to patients in Guatemala. Nodules were removed 4 months after the last dose; the adult O. volvulus were extracted by collagenase digestion, studied by histological techniques, and compared with worms from untreated patients. Twelve monthly doses killed a proportion of the adult worms (12% of males and 22% of females), leaving the remainder relatively unaffected and the females slowly resuming embryogenesis. After 8 and 12 doses, a number of female worms had resumed embryogenesis in 1 genital tract only, and in 1 female a total degeneration of 1 ovary was seen. Ivermectin also led to a marked drop in the number of male worms in nodules. No serious adverse reactions occurred and the treatment was well accepted.

The effect of multiple ivermectin treatments on infection of Simulium ochraceum with Onchocerca volvulus.

Ivermectin, a broad spectrum antiparasitic agent, interrupted the uptake of Onchocerca volvulus microfilariae by Simulium ochraceum from a group of human volunteers given multiple oral treatments of 200 micrograms/kg body weight. Two treatments, given at 7 month intervals, resulted in almost complete suppression of developing or infective larvae in the vector population for a 6 month period. The overall decline following 2 treatments was an order of magnitude lower than the pretreatment level. Ivermectin administration, in addition to the beneficial clinical effects, also could be useful for the control of human onchocerciasis as an independent measure or in conjunction with vector control.

Laboratory vector competence of Culex (Melanoconion) cedecei for sympatric and allopatric Venezuelan equine encephalomyelitis viruses.

Laboratory vector competence of Culex (Melanoconion) cedecei was examined for Venezuelan equine encephalomyelitis (VEE) viruses. Colonized adult female mosquitoes originating from a southern Florida population were given bloodmeals from viremic hamsters circulating various titers of 3 hemagglutination inhibition (HI) subtypes of VEE viruses. Following extrinsic incubation of about 3 weeks, mosquitoes were allowed to refeed on uninfected hamsters for transmission trials. Cx. cedecei was highly efficient in becoming infected with and transmitting its sympatric, HI subtype II "Everglades" virus. With bloodmeal titers of 10(0.9) chick embryo cell culture (CEC) plaque forming units (PFU), the infection rate was 9% and transmission occurred following extrinsic incubation. Infection rates were greater than or equal to 80% with oral doses of greater than or equal to 10(1.8), and all infected mosquitoes were capable of transmission following incubation. Cx. cedecei was also highly sensitive to infection with allopatric HI subtype IE Middle American VEE virus isolates. Infection rates were greater than or equal to 50% with bloodmeal titers undetectable by CEC assay. Rates were 100% with oral doses of greater than or equal to 10(0.8) CECPFU. Transmission rates were 100% in all experiments. Similar results were obtained with HI subtype IAB "epizootic" VEE virus isolates from the 1969 Middle American outbreak. Infection rates were 100% with oral doses of greater than or equal to 10(1.2), and transmission rates were 100% after extrinsic incubation. Comparisons with laboratory vector competence of the Middle American enzootic VEE virus vector, Culex (Melanoconion) taeniopus, are discussed.

Effects of three-month doses of ivermectin on adult Onchocerca volvulus.

Onchocerca volvulus worms in nodules from Guatemalan patients treated with four, eight, or 11 single doses of ivermectin (150 micrograms/kg of body weight) that were given once every three months were examined by routine histologic techniques and compared with worms in control nodules from untreated persons living in the same location over the same time periods. All treated nodules were removed four months after the last dose of ivermectin, i.e., 13, 25, or 34 months after the start of the trial. At the 25th and 34th months, i.e., after the eighth or eleventh doses of ivermectin, there were excess mortalities in female worms of 25.5% and 32.6%, respectively, over and above the levels in controls. Furthermore, the proportions of live females still producing scanty embryos up to the gastrula stage were only 7.7% and 18.2%, and no females were producing microfilariae. Ivermectin given at 3-month intervals also reduced significantly the mean numbers of live male worms in nodules, as well as the proportions of inseminated females. This regimen was effective in preventing embryogenesis to the microfilarial stage while, at the same time, it caused a slow but steady attrition of the adult worms.