Non-Mendelian inheritance of mosquito susceptibility to infection with Brugia malayi and Brugia pahangi.

The mode of inheritance of susceptibility or refractoriness of insect vectors to medically important pathogens such as those causing malaria or filariasis is usually believed to follow normal Mendelian laws and to involve a single pair of alleles. In this report, experiments are described that demonstrate another mode of inheritance of mosquito susceptibility to filarial parasites. Crosses were made between susceptibile and refractory species of the Aedes Scutellaris complex, and the hybrid and backcross progeny were tested for susceptibility to infection by Brugia malayi and Brugia pahangi. The data indicate that inheritance follows a non-Mendelian pattern indicative of extrachromosomal factors inherited through the maternal parent.

A mallophaga, Trinoton anserinum, as a cyclodevelopmental vector for a heartworm parasite of waterfowl.

The biting louse Trinoton anserinum serves as the intermediate host in the life cycle of the filarial heartworm, Sarconema eurycerca. Microfilariae, second-, and third-stage larvae were dissected from 39 of 89 lice infesting whistling swans, Cygnus columbianus columbianus, in North America and mute swans, Cygnus olor, in the Black Sea, U.S.S.R. Infective third-stage larvae obtained from lice collected from heartworm-parasitized whistling swans were injected subcutaneously into each of two hand-reared, nonparasitized mute swan cygnets. Both of these birds developed heartworm infections, one becoming microfilaremic at 14 weeks. The results of this study provide conclusive evidence that a mallophagan serves as a natural cyclodevelopmental vector of a filarial parasite.

Dispersal and other population parameters of Aedes aegypti in an African village and their possible significance in epidemiology of vector-borne diseases.

Dispersal of Aedes aegypti aegypti adults within Shauri Moyo, an African village in the Rabai area north of Mombasa, Kenya, was studied using the mark-release-recapture method. A total of 920 mosquitoes were captured and uniquely marked, of which 828 (90%) were released and 332 (40%) recaptured. A great majority of mosquitoes were recaptured once, but some individuals were recaptured up to 10 times. Most females visited 1 or 2 houses (40.8%, 44.9%, respectively), but there were females that visited as many as 5 houses. Mosquito movement among houses of the village is summarized in tables. Dispersal ability of Aedes aegypti females was d = 0.592 and males d = 0.433. The maximum distance females and males moved per 24 hr was 154 m and 113 m, respectively. The mean distance rate was 57.0 m/day for females and 44.2 m/day for males. The size of the mosquito population at Shauri Moyo village was estimated to be 331.1 +/- 146.5 using the Lincoln index method, 533.3 by the Fisher and Ford method, 270.3 +/- 179.5 by Bailey's triple catch method and 380.1 +/- 92.9 by the Jolly-Seber method. The mean female and male survival was 10.7 and 5.8 days, respectively. The maximum survival of females was 42 days and males 18 days. The daily survival of females and males estimated by the Fisher and Ford method was 0.825 and 0.693, respectively. The daily survival estimated using the Jolly-Seber method was 0.852 for females and 0.532 for males. Longevity is discussed in relation to extrinsic incubation periods of yellow fever and dengue viruses. The recapture rates, survivorship, and size of the village population indicate that the Ae. aegypti population in Shauri Moyo village is fairly stable. Presence or absence of blood in the stomach, and developmental stages of the ovaries were measured, showing that the majority of females fed twice and some fed as many as 3 times during one gonotrophic cycle.

The degree of susceptibility and levels of infection in ten different strains of Aedes polynesiensis Marks infected with subperiodic Brugia malayi and Brugia pahangi.

Ten strains of Aedes polynesiensis were infected with subperiodic Brugia malayi and Brugia pahangi. Susceptibility to B. malayi ranged from 92.1--100%, and susceptibility to B. pahangi from 97.5--100%. Further analysis showed significant differences in the numbers of third-stage larvae both between parasites and between strains within parasites. Because of the high levels of susceptibility, it appears that Ae. polynesiensis provides an even better laboratory model for vector-parasite studies than the strains of Ae. aegypti currently used.

Transmission of onchocerciasis by local black flies on the Firestone Rubber Plantation, Harbel, Liberia.

A quantitative description of the transmission dynamics of onchocerciasis on the Firestone Rubber Plantation, Harbel, Liberia is presented. The putative vector, Simulium yahense, comprised 98.5% of the flies examined. Diurnal vector-host contact was unimodal, expressing a distinct peak between 0800 and 1100 hours, particularly during the wet season. Perennial breeding occurs in the Firestone biotope, and is manifested by a mean annual daily landing rate of 75 flies per person per day. S. yahense is considered an efficient vector by nature of its anthropophily, moderate infective worm burden (4.3 infective larvae per infective fly) and its spatial and temporal ubiquitousness. Onchocerciasis transmission peaked during the dry season, when 73.5% of the annual transmission occurred. The mean annual transmission potential for the study area was estimated to be 1,425 infective larvae per person. The significance of onchocerciasis as a public health problem on the Plantation is discussed, and the potential for limiting transmission is considered.

A field method for the procurement of infective larvae of Onchocerca volvulus.

Natural vectors of onchocerciasis (S. yahense) were collected. Ninety-eight were intrathoracically inoculated with 30 freshly obtained skin-dwelling microfilariae. Of the 82 flies dissected, 56 were infected and 28 harbored infective larvae. A total of 193 infective O. volvulus larvae were obtained. The mean infective worm burden was 6.9.

The prevalence of onchocerciasis on selected divisions of the Firestone Rubber Plantation, Harbel, Liberia.

A survey for the prevalence of onchocerciasis was conducted on selected divisions of the Firestone Rubber Plantation, Harbel , Liberia, in November 1980. Divisions 19, 22, 23, and 36 are located in the north-central portion of the plantation, and were selected to complement transmission studies which were performed there in 1979 and 1980. Biopsy of the calf, iliac crest, and shoulder was done on a total of 583 individuals. No lateral difference in microfilarial density was observed. A prevalence of 80.8% was found. Prevalence appeared to be equal between the sexes, and to plateau above 35 years of age. Of those presenting positive skin biopsies, 20.2% had palpable onchocercomata . The geometric mean wet weight microfilarial density/mg of skin for the population sampled was 19. Onchocerciasis is considered hyperendemic in the Harbel area. Further investigations are warranted to define its dimensions in relation to clinical sequelae on the plantation.

Identification of larval and adult Simulium yahense and Simulium sanctipauli based on species-specific enzyme markers and their distribution at different breeding habitats in Central Liberia.

Human-biting adults and late instar larvae of the Simulium damnosum complex from four ecologically different simuliid breeding habitats in the Firestone Rubber Plantation at Harbel, Liberia, were identified morphologically and the monthly species composition of each site was recorded. Samples of the predominant species found at each site were assayed electrophoretically for species-specific variants of phosphoglucomutase (PGM) and trehalase (TRE). Enzyme identifications of flies and larvae were compared with morphological identifications to determine the accuracy of field identifications relying upon morphological characters. Enzyme identifications confirmed the accuracy of over 98% of the adult female identifications. S. yahense was found to be the predominant human-biting species at each site over the 10 months of sampling, with S. sanctipauli comprising a small percentage of the biting fly population. Species-specific larval enzymes confirmed the accuracy of more than 96% of the larval identifications. S. yahense was the predominant larval species found in smaller, more shaded, cooler breeding waters, while S. sanctipauli predominated in the single large watercourse that was sampled. Normally allopatric, mixed populations of these two larval species were found to exist at all sites, but sympatry occurred primarily during the wet season months of May-October. Biting activity of S. sanctipauli was found to be greatest during wet season months, and generally reflected the increase of S. sanctipauli in the larval populations of habitats dominated by S. yahense. The low human-biting activity of S. sanctipauli at all sites and during times which fostered large populations of S. sanctipauli larvae may be an indication of this specie's zoophilic tendency. Circumstantial evidence of hybridization, the expression of PGM and TRE species-specific variants for both species, was found in adults and larvae morphologically identified as S. yahense. The frequency of this "hybrid" condition, based upon PGM and TRE, was calculated to be comparable to the frequency of hybridization as determined by larval chromosome inversions.

Quantitative studies of onchocerciasis transmission by Simulium yahense and Simulium sanctipauli in the Firestone Rubber Plantation at Harbel, Liberia.

Black fly vectors of onchocerciasis from three ecologically different Simuliid breeding habitats in the Firestone Rubber Plantation at Harbel, Liberia, were surveyed by human-biting collections conducted at weekly intervals over a 13-month period. Black flies were identified morphologically and the monthly and seasonal contribution of different vector species to the transmission of Onchocerca volvulus at each site was determined. Simulium yahense was identified as the predominant vector species at each site. Greatest populations of this species occurred during wet season (May-Oct), but its impact on transmission of onchocerciasis was most profound during dry season (Nov-Apr) when parity, infection, and infectivity were high. S. sanctipauli was the only other vector species captured, but biting populations of this species were small, and during wet season confined primarily to the vicinity of its breeding site in the Farmington River. Dry season populations of S. sanctipauli were also characterized by lower human-biting rates, and by higher rates of parity, infection, and infectivity. Monthly transmission potentials at each site were attributed primarily to S. yahense, with peak monthly transmission occurring during the dry season months of January-April. Against the WHO standard of 100 as a "tolerable" annual level of onchocerciasis transmission, annual transmission potentials for the three sampling sites were 94, 1,877, and 4,900, with highest values being calculated for S. yahense breeding sites.

Induction of protective immunity against larval Onchocerca volvulus in a mouse model.

BALB/cBYJ mice were immunized against larval Onchocerca volvulus by subcutaneous injection of normal, irradiated, or freeze-thaw-killed Onchocerca sp. larvae. The mice received challenge infections of O. volvulus third-stage larva (L3) contained in diffusion chambers implanted subcutaneously. At two-weeks postinfection, the diffusion chambers were removed and larval survival was assessed. When mice were immunized a single time with 35-krad-irradiated or normal O. volvulus L3, there was a significant reduction in the survival of challenge parasites. However, there was little or no reduction in challenge worm survival when mice were immunized a single time with freeze-thaw-killed O. volvulus L3 or fourth-stage larva (L4), or irradiated O. lienalis L3. When a second dose of freeze-thaw killed O. volvulus L3 or irradiated O. lienalis L3 was administered, there was a significant reduction in parasite survival in immunized mice. Immunization with O. volvulus L4 or a combination of L3 and L4 failed to confer protection. These results demonstrate that mice can be immunized against larval O. volvulus and that diffusion chambers are an efficient method for studying protective immunity to this parasite in a mouse model.

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.

Ivermectin prophylaxis against experimental Onchocerca volvulus infection in chimpanzees.

Ivermectin was tested for possible prophylactic action against the third and fourth larval stages (L3 and L4) of Onchocerca volvulus inoculated into chimpanzees. The infective larvae (L3) were obtained from laboratory-raised black flies. Eighteen chimpanzees were inoculated, each with approximately 250 L3. Six were treated with ivermectin (200 micrograms/kg) on the day of inoculation, 6 were treated with ivermectin on day 28, and 6 were not treated. Monthly skin snips were taken for the next 30 months to detect patent infection. One of the chimpanzees treated with ivermectin on the day of infection developed a patent infection as did 4 of the 6 treated at day 28 and 4 of the 6 control animals. The results suggest that ivermectin may have a partial in vivo effect against the L3 of O. volvulus but has no effect against later larval stages of the parasite.

Effect of mass treatment of a human population with ivermectin on transmission of Onchocerca volvulus by Simulium yahense in Liberia, West Africa.

The impact of mass treatment with ivermectin on the intensity of Onchocerca volvulus transmission by the black fly (Simulium yahense) was evaluated on the Liberian Agricultural Company rubber plantation in Liberia, West Africa. The adult pre-treatment prevalence of onchocerciasis was greater than 80%, and the average intensity of infection was 5.35 mf/mg of skin. The drug was administered at 2 annual intervals, reaching 58-60% of the approximately 14,000 people living in 73 camps. Landing/biting catches of black flies made in central and peripheral plantation zones indicated similar fly activity before and after ivermectin treatment (man hr index of 2.1 and 2.4 within the plantation, and 10 and 10.9 outside the plantation, respectively). The number of infected flies with developing larvae (L1, L2, L3 stages) of O. volvulus in treated areas was reduced by 93.4-95%; the number of infective flies with L3 larvae was reduced by 81.7-89.3%. Parasite loads of infected (L1, L2) and infective flies (L3 stages only) outside the plantation also decreased by 86.8% and 80%, respectively. Monthly transmission potential (MTP) showed a similar decrease: from 22.9 to 5.8 (74.6% reduction) in the treated area, and from 210 to 158.8 (24.4% reduction) in untreated areas. Mass treatment with ivermectin efficiently controlled, and at least temporarily interrupted, transmission of Onchocerca volvulus by black fly vectors.

Aedes (Gymnometopa) mediovittatus (Diptera: Culicidae) as an experimental vector of Brugia pahangi and B. malayi (Spirurida: Filariidae).

To test the susceptibility of Aedes (Gymnometopa) mediovittatus to infection with Brugia pahangi and Brugia malayi, females originating from the suburbs of San Juan, Puerto Rico, were fed on infected gerbils (Meriones unguiculatus). On average, 39.2% of the Ae. mediovittatus females became infected with L3 larvae of B. pahangi and 47.4% with B. malayi. The average number of infective L3 larvae of B. pahangi and B. malayi dissected from mosquitoes was 2.6 +/- 1.2 and 2.9 +/- 1.0, respectively. The largest number of L3 in a single mosquito was 16. After 10 d of development in the mosquitoes, L3 larvae of both Brugian species were found in greatest number in the thorax, in lesser number in the head/proboscis, and in least number in the abdomen. Ae. mediovittatus may serve as a useful laboratory model for the study of genetic susceptibility and refractoriness of mosquito vectors to filarial parasites.

Duration of larval development of Simulium yahense (Diptera: Simuliidae) under natural conditions.

The duration of Simulium yahense Vajime & Dunbar larval development on a dam spillway in Harbel, Liberia, was observed to make accurate decisions regarding the frequency of larvicide treatments against this onchocerciasis vector. Larval development required a minimum of 10-12 d from eclosion to first pupation. Initial larvicidal treatment for S. yahense control would require a treatment cycle of 7 d. Once suppression of adult and larval populations is achieved, a 9-12-9-12 day treatment cycle could be adopted.

Brugia malayi microfilariae (Nematoda: Filaridae) enhance the infectivity of Venezuelan equine encephalitis virus to Aedes mosquitoes (Diptera: Culicidae).

We examined the potentially conflicting effects that microfilarial (MF) enhancement of viral infectivity and MF-induced mortality in mosquitoes have on the vectorial capacity of Aedes aegypti (L.), Aedes triseriatus (Say), and Aedes taeniorhynchus (Wiedemann) for Venezuelan equine encephalitis virus (VEE) when mosquitoes feed on gerbils co-infected with Brugia malayi (Buckley). Groups of mosquitoes were fed on gerbils that were either dually infected (VEE plus B. malayi MF) or singly infected (VEE only). Mosquito mortality was recorded daily, and 5-8 d later, surviving mosquitoes were assayed for disseminated viral infection. The contrasting effects of MF enhancement and MF-induced mortality differed among mosquito species and were determined by the nature and consequences of MF penetration through the mosquito midgut, but not to differences in mosquito susceptibilities to parenterally introduced virus. In Ae. aegypti, MF-induced mortality was high and tended to eliminate any significant effect of MF enhancement. In Ae. triseriatus, MF-induced mortality was low, and feeding on dually infected hosts resulted in 9 times as many mosquitoes with disseminated viral infections as did feeding on singly-infected hosts. In Ae. taeniorhynchus, MF-induced mortality was extremely high, yet under our experimental conditions, feeding on a dually infected hosts resulted in nearly 30 times as many disseminated infections as did feeding on singly infected hosts. The final outcome on vectorial capacity depended on the specific combination of MF, virus, and mosquito species involved. Therefore, future efforts toward understanding MF enhancement should be directed toward mosquito-virus-parasite species combinations that occur together in nature.

Diurnal biting activity and transmission of Onchocerca volvulus (Filariata: Onchocercidae) by Simulium yahense (Diptera: Simuliidae) in Liberia.

To determine the influence of meterological factors on the diurnal biting cycle of Simulium yahense Vajime & Dunbar (a member of the Simulium damnosum Theoblad complex), we captured host-seeking females as they landed on the exposed lower legs of humans in the Harbel area of Liberia. Biting activity was greatest during the morning hours and was characterized by a unimodal harmonic curve. Although meteorological conditions had no decisive influence on the unimodal pattern of diurnal biting activity, hourly variation in the number of S. yahense captured at human bait was affected by meterological factors (i.e., the diurnal pattern of S. yahense biting activity is regulated by an internal clock, but the number of bits during any given hour is in response to meterological conditions). The transmission of Onchocerca volvulus (Leuckart) was highest during the morning hours when high transmission potentials were in phase with peak human activity and served to maintain the hyperendemicity of onchocerciasis in the Harbel area.

Estimates of population size, dispersal, and longevity of domestic Aedes aegypti aegypti (Diptera: Culicidae) by mark-release-recapture in the village of Shauri Moyo in eastern Kenya.

Estimates of the absolute size of a domestic population of Aedes aegypti aegypti (L.) were made in the Rabai area of Kenya, based on a single release, followed by either single or repeated recaptures. From single recapture within 24 h of release, the size of the female Ae. a. aegypti population in the Shauri Moyo village was estimated by the Lincoln index to be 365. Using a single release and repeated recaptures, population size was estimated by Jackson's positive method to be 337. Depletion of the unmarked females by daily removal sampling provided us with two additional, direct estimates of the size of the village population (451 by Kano's method and [464 +/- 18.9 SEM] by the Moran-Zippin method). The longevity of marked females was at least 9 d. The day-to-day movement of marked mosquitoes revealed the dispersal pattern of Ae. a. aegypti among houses clustered within different distance zones. Marked mosquitoes reached all houses of the village within 24 h of release. After 5 d, the dispersion of both marked and unmarked mosquitoes among houses was similar.

Ultrastructure of infective larvae (L3) of Onchocerca volvulus (Nematoda: Filarioidea) developed in Simulium yahense in Liberia.

Third-stage infective larvae of Onchocerca volvulus were examined to elucidate the ultrastructure and the interrelations of the stoma, esophagus, intestine, and nervous system. The alimentary canal involves a cuticularized stoma with a triradiate lumen that is continuous with a similar triradiate lumen in the muscular region of the esophagus. The lumen wall may be laterally appressed or opened into a stellate form in the glandular region. Posteriad from the esophagointestinal valve, the cylindroid lumen becomes partially occluded with microvilli formed by the evaginations of the apical membranes of the intestinal epithelium. Cross sections, through this region reveal that groups of 5 radiating epithelial cells are joined near the lumen surface by junctional complexes. The alimentary canal terminates via a rectal valve and channel supported by somatic and neural cells. The central nervous system consists of a nerve ring that surrounds the muscular region of the esophagus. Related neurons support chemoreceptors and tactoreceptors of sensilla and the extensive coelomyarian and meromyarian somatic muscles. Extensive accumulations of glycogen rosettes are present in many of the muscle and hypodermal cells.

Development of Onchocerca volvulus (Filarioidea: Onchocercidae) in the West African black fly Simulium yahense (Diptera: Simuliidae) in Liberia.

Simulium yahense black flies infected with microfilaria of Onchocerca volvulus were kept in a defined insectary environment in Liberia, West Africa. A daily sample of infected flies was dissected for larvae developing in the thoracic muscles and examined for growth in stadial development. Microfilariae ingested by black flies transformed to the L1 larval stage without molting. Successive larval development included molting to the L2 stage and, finally, to the L3 stage, which was infective in humans. The cephalic cap, consisting of a laterally located hook and central stoma, occurs in the first larval stage. The caudal appendix and the laterally located anal opening are apparent in the L1 larva. In the L2 stage, the cephalic cap is lost and the large circular stoma becomes surrounded with elevated flaps. The caudal appendix was lost after larvae molted to the L3 stage, and in its place, 3 terminal papillae developed. Sense organs, such as 2 opposing phasmids and 8 papillae that were arranged into 2 circles, developed in the cephalic region of the L3 larva. The evidence of pathological consequences due to the presence of the L3 larva in the fly host are illustrated and discussed.