Isoenzyme variation in Aedes aegypti correlated with Dirofilaria immitis infectability.

From the Vero Beach strain of the mosquito Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae), substrains were selected for susceptibility (SS) and refractoriness (RR) to the dog heartworm Dirofilaria immitis (Leidy) (Filarioidea: Onchocercidae). These two lines and their reciprocal F1 hybrids were analysed for genetic variation at 14 enzyme loci, using polyacrylamide gel electrophoresis. Six of the enzyme loci showed variation (sample size 48 alleles/locus/line). Three of these were monomorphic in the refractory line but polymorphic in the susceptible, i.e. aconitase hydratase (Acoh), isocitrate dehydrogenase-1 (Idh-1) and phosphoglucomutase (Pgm). The other three loci, glucose-6-phosphate isomerase (Gpi), hexokinase-1 (Hk-1) and isocitrate dehydrogenase-2 (Idh-2), were polymorphic in both SS and RR lines and their hybrids. At two loci (Hk-1, Pgm) three alleles were detected, whereas the other polymorphic loci had only two alleles. For Hk-1, the most frequent allele was Hk-1(80) (0.563) in refractory and Hk-1(100) in the susceptible (0.521) and F1 hybrids. For Pgm the most frequent alleles were Pgm125 in the susceptible line (0.646) and Pgm100 in the F1 hybrids (0.563 and 0.604) and refractory line (1.000). The mean observed heterozygosity (Ho), the mean Hardy-Weinberg expected heterozygosity (He) and the mean number of alleles per locus in the refractory line were lower, but not significantly so, than in the susceptible line and their reciprocal F1 hybrids; the proportion of polymorphic loci was significantly lower in the refractory than in the susceptible line and their F1 hybrids. Within both lines all polymorphisms were in Hardy-Weinberg equilibrium, whereas significant departures from predicted frequencies were observed in SS x RR hybrids at four polymorphic loci (Acoh, Gpi, Hk-1, Pgm) and at three polymorphic loci (Acoh, Hk-1, Pgm) in RR x SS hybrids. The average Nei's and modified Rogers' genetic distances between the lines were 0.024 and 0.139, respectively. These electrophoretic data show that the refractory line (putatively lacking fi allele) can be distinguished from the susceptible line (fi/fi) and their hybrids (heterozygous fi) by isozyme marker frequencies, but it remains to be seen whether this difference is causal or chance linkage. In any case, this model system of Ae. aegypti/D. immitis provides opportunities to better understand and manipulate the molecular biology of filariasis transmission.

Aedes albopictus (Diptera: Culicidae): an experimental and natural host of Dirofilaria immitis (Filarioidea: Onchocercidae) in Florida, U.S.A.

Females of an Aedes albopictus (Skuse) colony from southeastern Florida, U.S.A., ingested low (22.9 +/- 3.2 mg/Female) and high (243.2 +/- 37.6 mf/Female) numbers of microfilariae from a dog infected with Dirofilaria immitis (Leidy). High mortality of females occurred during the first 4 d after infection regardless of the number of microfilariae ingested; daily mortality was almost negligible during 5-15 d after infection. Percentage of survival 15 d after infection was higher (63%) in females that ingested low numbers of microfilariae than those (15%) that ingested high numbers of microfilariae. The development of most of the D. immitis larvae was arrested in late L1 stage with some of the L1 stage larvae becoming melanized intracellularly in the Malpighian tubule cells of Ae. albopictus. Fifteen days after infection, development of D. immitis to the infective L3 stage occurred in only 10.9% of the surviving F1 and F2 Ae. albopictus that ingested low numbers of microfilariae, but in 94% of Anopheles quadrimaculatus Say ingesting similar numbers of microfilariae, as a control. Females of Ae. albopictus ingesting high numbers of microfilariae had more surviving females with L3 than those ingesting low numbers of microfilariae. The number of L3 larvae in the Malpighian tubules, hemocoel, head capsule and proboscis ranged from 1 to 37 per female, indicating the potential of Ae. albopictus to transmit D. immitis. Development of D. immitis larvae was not affected by co-infection with Ascogregarina taiwanensis (Lien & Levine), although both parasites infect the Malpighian tubules, the first intracellularly and the second extracellularly. After one generation of selection, a strain of Ae. albopictus susceptible to D. immitis developed 2.5 times more L3 than the parent strain. These results show that a small portion of the natural population of Ae. albopictus is susceptible to infection with D. immitis and that susceptibility may be increased rapidly by selection. The presence of developing Dirofilaria sp. larvae in the Malpighian tubules of field-caught females indicated that Ae. albopictus may be infected naturally with D. immitis in Florida.

Laboratory evaluation of biotic and abiotic factors that may influence larvicidal activity of Bacillus thuringiensis serovar. israelensis against two Florida mosquito species.

A technical powder of Bacillus thuringiensis serovar. israelensis (B.t.i.) (VectoBac TP, 5,000 international toxic units [ITU]/mg), an aqueous suspension (VectoBac 12AS, 1,200 ITU/mg), and a granular formulation (VectoBac CG, 200 ITU/mg) were tested in the laboratory under different biotic and abiotic, conditions for efficacy against larvae of saltwater (Aedes taeniorhynchus) and freshwater (Culex nigripalpus) mosquitoes. Second-, 3rd-, and 4th-instar larvae of Cx. nigripalpus were 1.3-3-fold more susceptible to both VectoBac TP and VectoBac 12AS than were the respective larval instars of Ae. taeniorhynchus. For each species, 2nd-instar larvae were several-fold more susceptible to these B.t.i. preparations than were the 4th instars. In test cups, larvae under lower densities exposed to B.t.i. concentrations sustained 5-9-fold higher mortalities than larvae under high-density conditions. VectoBac TP and VectoBac 12AS stayed in suspension for up to 24 h with similar larvicidal efficacy, which was greater at 32-35 degrees C than at 15-20 degrees C. At 60 degrees C maintained for 24 h, VectoBac 12AS was adversely affected 2-3-fold in terms of potency, but VectoBac TP was not affected. Significant loss of potency of both VectoBac 12AS and VectoBac TP occurred when exposed to 35-37 degrees C under high light intensity (140,000-170,000 lux) for 6 h. Increasing salinity levels from 0 (fresh water) to 50% sea water caused gradual efficacy declines of VectoBac 12AS and VectoBac TP against Ae. taeniorhynchus larvae. VectoBac CG caused insignificant initial and residual (up to 8 days) larval mortalities of both mosquito species. This formulation did not release the active ingredient of B.t.i. in any significant mosquito larvicidal concentration in surface layers of water, and the formulation was more effective in shallower water. Storage of all 3 formulations under constant laboratory and variable field conditions for up to 8 months did not produce detectable potency loss of these products.

Colonization of Culex nigripalpus theobald (Diptera: Culicidae) by stimulation of mating using males of other mosquito species.

Culex nigripalpus Theobald (Diptera: Culicidae) was recolonized successfully by cohabiting Aedes taeniorhynchus males or males of other mosquito species starting in a large outdoor cage under natural light-dark cycles and temperatures and ending in a 1-ft.3 cage under artificial light-dark cycles at 24 degrees C without added stimulation.

Toxicity of a phenyl pyrazole insecticide, fipronil, to mosquito and chironomid midge larvae in the laboratory.

Toxicity of a phenyl pyrazole insecticide, fipronil, to 4th-instar larvae of 6 species of colonized mosquitoes (Aedes aegypti, Ae. albopictus, Ae. taeniorhynchus, Anopheles quadrimaculatus, Culex nigripalpus, and Cx. quinquefasciatus) and 2 species of field-collected chironomid midges (Chironomus crassicaudatus and Glyptotendipes paripes) was evaluated in the laboratory. All mosquito species were highly susceptible with 48-h median lethal concentration (LC50) values ranging from 0.00043 ppm (Ae. taeniorhynchus and An. quadrimaculatus) to 0.023 ppm (Ae. albopictus). Chironomus crassicaudatus and G. paripes also were extremely susceptible (48-h LC50 of both species: 0.00042 ppm) to fipronil. Larval mortality checks of Ae. taeniorhynchus, Cx. nigripalpus, and G. paripes at 24 h and again at 48 h posttreatment revealed delayed activity of this compound against these species. First-instar larvae of Ae. albopictus and Cx. quinquefasciatus were significantly (P < 0.01) more susceptible to fipronil than the 4th-instar larvae of these mosquito species.

Vector ability of mosquitoes for isolates of Plasmodium elongatum from raptors in Florida.

Three isolates of Plasmodium elongatum were obtained from 3 species of raptors (red-tailed hawk [Buteo jamaicensis], bald eagle [Haliaeetus leucocephalus], and eastern screech owl [Otus asio]) from Florida using isodiagnostic techniques in Pekin ducks (Anas platyrhynchos). Six to 10 species of mosquitoes were tested for susceptibility to these 3 isolates. Complete development of the sporogonic cycle of the 3 isolates of P. elongatum occurred in 3 species of mosquitoes, Culex nigripalpus, Culex restuans, and Culex salinarius. The pattern of susceptibility was similar among the 3 isolates of P. elongatum in Cx. nigripalpus. Culex restuans and Cx. salinarius were significantly more susceptible than Cx. nigripalpus to the 3 isolates of P. elongatum tested. Culex nigripalpus transmitted all 3 isolates of P. elongatum from duck to duck both by bite and after intraperitoneal injection of sporozoites. Infections of the 2 isolates tested occurred in ducks after intraperitoneal injection of sporozoites from Cx. restuans and Cx. salinarius. The results suggest that these 3 Culex species are potential vectors of P. elongatum from raptors in Florida.

Plasmodium forresteri n. sp., from raptors in Florida and southern Georgia: its distinction from Plasmodium elongatum morphologically within and among host species and by vector susceptibility.

Plasmodium forresteri n. sp. naturally infects eastern screech-owls (Otus asio), great horned owls (Bubo virginianus), barred owls (Strix varia), bald eagles (Haliaeetus leucocephalus), red-shouldered hawks (Buteo lineatus), broad-winged hawks (Buteo platypterus), and red-tailed hawks (Buteo jamaicensis) in Florida and southern Georgia. Schizonts occur in mature or nearly mature erythrocytes, produce 2-6 merozoites arranged most commonly in fan or cruciform configuration, with mean dimensions among host species varying from 3.7 to 4.8 x 2.5 to 3.4 microns. Gametocytes are elongate, with mean dimensions among host species varying from 11.5 to 13.1 x 2.0 to 2.4 microns. One or both gametocyte margins are irregular and often crenulate. Gametocytes seldom fill the space between the erythrocyte nucleus and margin. Species characteristics were maintained in isodiagnostic Japanese quail (Coturnix japonica) and Pekin ducks (Anas platyrhynchos). In mosquito infection studies, only Culex restuans could support sporogony of P. forresteri, in contrast to Plasmodium elongatum of raptor origin that completed sporogony in both Cx. restuans and Culex nigripalpus.

Studies on a primaquine-tolerant strain of Plasmodium vivax from Brazil in Aotus and Saimiri monkeys.

A nonimmune American acquired an infection of Plasmodium vivax Type 1 malaria in Brazil in 1994. After returning to the U.S.A., he had a primary attack followed by 3 relapses. The primary attack and first 2 relapses were treated with a standard regimen of chloroquine, followed by 14 days of primaquine (15 mg/day). Following the third relapse, the primaquine treatment was extended to 28 days. No further relapses occurred. The lack of response to primaquine by this strain may recommend it as a suitable candidate for chemotherapeutic study if it can be adapted to an animal model. Anopheles quadrimaculatus mosquitoes infected by feeding on the patient during the first relapse were used to establish the strain in Aotus and Saimiri monkeys. Monkeys supported well the development of long-lasting parasitemia. Anopheles freeborni, Anopheles stephensi, and Anopheles gambiae mosquitoes were readily infected by feeding on the monkeys and by membrane feeding on diluted blood. Monkey-to-monkey transmission was obtained via the bites of infected mosquitoes and the intravenous injection of sporozoites dissected from salivary glands. This parasite is designated as the Brazil I/CDC strain of P. vivax.

Hemagglutinins in Anopheles quadrimaculatus, strains susceptible and refractory to Brugia malayi, and their role in the immune response to filarial parasites.

Hemagglutinins in the salivary gland extract and in the body fluid from strains of the mosquito, Anopheles quadrimaculatus, susceptible and refractory to the filarial parasite, Brugia malayi, had higher titers against Human A+, B- and O+, and sheep erythrocytes than against rabbit and jird erythrocytes. Hemagglutination activity in the body fluid was low in newly emerged females but increased and stabilized as they became older. Hemagglutination activity of the body fluid was not reduced by freezing at -20 degrees C, but it was destroyed following heating the body fluid to 60 degrees C and 100 degrees C for 45 min, indicating that the hemagglutinins are heat labile, and they are proteins or glycoproteins. Hemagglutinins in the salivary glands exhibited specificities for a broader range of carbohydrate moieties on the surface of Human A+ and sheep erythrocytes than those in the body fluid. Injections of specific carbohydrates in saline solution into B. malayi-infected females of the refractory strain of An. quadrimaculatus 24 hr after the infective blood meal showed that galactose, N-acetyl-D-galacto-samine, sorbose and mannose inhibited the increase in encapsulation (melanization) of L1 of B. malayi in the thoracic muscles of An. quadrimaculatus females when compared to those females injected with saline and other carbohydrates. The results suggest that hemagglutinins are present in the salivary gland extract and the body fluid of both strains of An. quadrimaculatus females and they may be involved in the immune response (encapsulation) to filarial parasites in An. quadrimaculatus.

Hemagglutinins in mosquitoes and their role in the immune response to Brugia malayi (Filarioidea:Nematoda) larvae.

Hemagglutinins were determined in six species of mosquitoes that are susceptible and refractory to Brugia malayi (Filarioidea: Nematoda). High titers of hemagglutinins were found in the salivary gland extract and in the body fluid of a completely refractory species, Aedes taeniorhynchus, and in partially refractory species, Anopheles quadrimculatus but low levels of hemagglutinins were also present in the body fluid of Aedes aegypti (Black-eye, Liverpool strain), a susceptible species. Hemagglutinating activity was not found in the other three completely refractory species of mosquitoes, Culex quinquefasciatus, Culex nigripalpus, and Aedes albopictus in which blood coagulated rapidly after ingestion. High titers of hemagglutinins in the salivary glands of Ae. taeniorhynchus and An. quadrimaculatus facilitated rapid movement of sheathed microfilariae from the midgut to the hemocoel. It is suggested that high titers of hemagglutinins present in the hemocoel bound to the glycoconjugates with exposed carbohydrate moieties present on the microfilarial sheaths and developing abnormal larvae (L1) in the thoracic muscle cells. These hemagglutinin-bound glycoconjugates formed capsules that subsequently stimulated the immune response and resulted in melanization of microfilarial sheaths and sheathed microfilariae in the hemocoel and intracellularly developing abnormal L1 in the thoracic muscles. Only minimal encapsulation and melanization of B. malayi microfilariae was observed in the hemocoel of the other four species of mosquitoes that lacked hemagglutinins in the salivary glands. The results suggest that tissue specific hemagglutinins are one of several factors of vector susceptibility/refractoriness through immune reactions (encapsulation, activation of prophenoloxidases).

Effect of Brugia malayi on the growth and proliferation of endothelial cells in vitro.

Athymic mice (C3H/HeN) parasitized by Brugia malayi develop massively dilated lymphatics. The lymphatic endothelial lining is perturbed, and numerous mononuclear and giant cells are closely apposed to the endothelium. The hyperplastic endothelial cells and low opening pressure of the lymphatics suggest abnormal multiplication of these cells may be important in the dilation. We studied the in vitro growth rate of human umbilical vein endothelial cells cultured with adult worms and microfilariae of B. malayi. The tetrazolium salt reduction assays were used to quantify possible direct mitogenic or inhibitory effects. The growth factor-induced proliferation of endothelial cells was significantly suppressed by 44-51% on day 1, 46-81% on day 3, and 45-79% on day 5 in cultures containing adult female worms, which had greater suppressor activity on endothelial cell proliferation than male worms, microfilariae, or soluble adult worm extract. Culture supernatant containing female worm excretory-secretory products significantly inhibited the growth and multiplication of cells, suggesting that adult female worms release antigens or proteins that have inhibitory activity on growth factors necessary for endothelial cell proliferation in vitro. Excess human recombinant epidermal growth factor and bovine brain extract partly reversed the inhibitory activity of worms in culture and restored the endothelial cell proliferation when incubated with worm culture supernatant. Indomethacin and BW 775Hcl failed to restore normal endothelial proliferation in the presence of female worms, suggesting that parasite-derived prostanoids and cyclooxygenase products did not cause the inhibition. Lymph from dilated lymphatics, but not serum from infected mice, increased the proliferation of cells in vitro. Together, these data demonstrate that excretory-secretory products of B. malayi parasites suppress vascular endothelial proliferation in vitro. Furthermore, increases in the number of these cells in vitro in the presence of lymph suggest that parasite-induced host factors may be important in modulating the degree of proliferation.

Regulatory cytokines in the lymphatic pathology of athymic mice infected with Brugia malayi.

To investigate whether Brugia malayi-induced lymphatic inflammation is due to production of pro-inflammatory cytokines, we determined the lymph and serum levels of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-alpha and granulocyte-macrophage colony stimulating factor (GM-CSF) using enzyme immunoassays. Serum from normal and infected mice did not show elevated cytokine concentrations. Samples of lymph from parasitized lymphatics had significantly increased levels of IL-1 (range = 6-1620 pg/ml), IL-6 (19-17,800 pg/ml), TNF-alpha (19-2000 pg/ml) and GM-CSF (4-275 pg/ml). The anti-inflammatory cytokine IL-4 (7-12 pg/ml) was in the normal range and no increase in interferon (INF)-gamma was detected in lymph samples. The data suggest that increased levels of mediators or cytokines localized in the lymphatics may be important contributors to massive lymphatic dilation and inflammation.

Lectin binding to extracellularly melanized microfilariae of Brugia malayi from the hemocoel of Anopheles quadrimaculatus.

Binding patterns of fluorescein isothiocyanate (FITC)- and gold-conjugated lectins to extracellularly melanized sheathed and exsheathed microfilariae of subperiodic Brugia malayi, isolated from and in situ in the abdominal hemocoel of Anopheles quadrimaculatus 72-hr postinfection, were examined. Five FITC-conjugated lectins [Helix pomatia agglutinin (HPA), Arachis hypogaea (peanut agglutinin-PNA), Triticum vulgaris (wheat germ agglutinin-WGA), Lens culinaris (lentil-LCH), and Concanavalin A (Con A)] with specificities for different carbohydrate moieties were tested for binding to isolated melanized microfilariae and observed with transmitted light and fluorescence microscopy. All five FITC-lectins bound strongly to the acellular material accompanying the melanin deposits on the surface of isolated melanized microfilariae. Significant inhibition of FITC-lectin binding occurred when lectins were preincubated with their complementary carbohydrates before testing. H. pomatia agglutinin binding was totally inhibited by N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. Other lectins were partially inhibited, such as PNA by galactose and lactose; WGA by N-acetylneuraminic acid; LCH by N-acetyl-D-glucosamine, mannose, glucose, and methyl alpha-D-mannopyranoside; and Con A by mannose and methyl alpha-D-mannopyranoside. Three gold-conjugated lectins (HPA, PNA, and Con A), examined by using transmission electron microscopy, bound to the outer surface of the acellular material associated with the melanin deposits on isolated melanized microfilarial sheaths and melanized microfilariae and to the remnants of lysed hemocytes found in the proximity of the melanized deposits. Con A in the presence of gold-labeled horseradish peroxidase, examined by using transmission electron microscopy, showed random binding within the melanized capsule formed around the microfilarial sheath in situ. These results indicate that the acellular material accompanying melanin deposits on melanized microfilarial sheaths and sheathed and exsheathed microfilariae contain several glycoconjugates with exposed carbohydrate moieties and are possibly glycoproteins. These glycoproteins could be the by-products of the activation of the prophenoloxidase by the microfilariae.

Comparison of migration and encapsulation of Brugia malayi microfilariae from the midgut to the hemocoel between Anopheles quadrimaculatus and Aedes aegypti.

Comparisons were made of migration and encapsulation of ingested sheathed microfilariae of Brugia malayi from the midgut into the hemocoel between Anopheles quadrimaculatus (refractory and susceptible strains to B. malayi) and Aedes aegypti (Black-eyed, Liverpool strain susceptible to B. malayi). Encapsulation and melanization of microfilarial sheaths and microfilariae occurred in both strains of An. quadrimaculatus and in Ae. aegypti. In both strains of An. quadrimaculatus, by 4 hr and by 24 hr after the ingestion of sheathed microfilariae of B. malayi in the infected bloodmeal, significantly more sheathed microfilariae penetrated the midgut and reached the hemocoel and thoracic muscles compared with those in Ae. aegypti. During the same time periods significantly more encapsulated and melanized microfilarial sheaths and a larger percentage of encapsulated and melanized microfilariae were observed in the hemocoel of both strains of An. quadrimaculatus than in Ae. aegypti. The results suggest that differences observed in the numbers of encapsulated and melanized microfilarial sheaths and percentages of melanized microfilariae between An. quadrimaculatus (both strains) and Ae. aegypti are due to different rates of penetration of the sheathed microfilariae from the midgut to the hemocoel.

Comparative toxicity of selected larvicides and insect growth regulators to a Florida laboratory population of Aedes albopictus.

Five organophosphates (OPs) (chlorpyrifos, chlorpyrifos methyl, fenthion, malathion, and temephos), 3 pyrethroids (bifenthrin, cypermethrin, and permethrin), and 2 microbial pesticides (Bacillus thuringiensis serovar.israelensis [B.t.i.] and Bacillus sphaericus) were tested as larvicides against a Florida Aedes albopictus population colonized in the laboratory. In addition, 3 insect growth regulators (IGRS) (diflubenzuron, methoprene, and pyriproxyfen) were evaluated. All OPs, except for malathion, were highly effective as indicated by low LC90s ranging from 0.0069 ppm (chlorpyrifos) to 0.026 ppm (fenthion); the larvae were considered tolerant to malathion (LC90 = 1.043 ppm). LC90 values of pyrethroids were: 0.0175 ppm (bifenthrin), 0.0079 ppm (cypermethrin), and 0.0031 ppm (permethrin). Commercial products of B.t.i., Vectobac and Bactimos were considered economically effective against Ae. albopictus larvae but products of B. sphaericus were ineffective (LC90s > 28 ppm). The IGRs showed exceptional activity. Pyriproxyfen (LC90 = 0.000376 ppm), was 2.23 and 21.5 times more toxic than diflubenzuron and methoprene, respectively. In general, toxicity ranking of chemicals and microbials tested was: IGRs > pyrethroids > OPs > microbials.

Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae.

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca2+. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p'-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, p-nitrophenyl p'-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediamine-tetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.

Evaluation of different medium supplements for in vitro cultivation of Brugia malayi third-stage larvae.

Growth and development of Brugia malayi (Nematoda: Filarioidea) third-stage larvae (L3) were compared in 5 medium supplements. The basic culture medium (NI) consisted of a 1:1 (v/v) mixture of NCTC-135 and Iscove's modified Dulbecco's medium, an antibiotic/antimycotic mixture, and 1 of the following 5 supplements: 25 mg/ml bovine albumin fraction-V (BAF), 10% fetal bovine serum (FBS), 10% commercially obtained human serum (CHS), 10-15% pooled human serum from hospital patients (PHS), and 10-15% human serum from a single individual (SHS). Cultures were maintained at 37 C in an atmosphere of 5% CO2 in air. NI-BAF and NI-CHS did not support molting of L3 to fourth-stage larvae (L4), whereas NI-FBS, NI-PHS, and NI-SHS did support molting of L3 to L4 but only the larvae in NI-SHS attempted the fourth molt. Growth and development of in vitro larvae in NI-PHS and NI-SHS were comparable to that observed in jirds for the first 28 days, after which the in vitro larvae lagged behind in vivo larvae. Optimal growth and development may be dependent on certain as yet unidentified components of specific human serum.

Characterization and localization of mosquito-gut receptors for trypsin modulating oostatic factor using a complementary peptide and immunocytochemistry.

The gut receptor of trypsin-modulating oostatic factor (TMOF), a decapeptide hormone that regulates trypsin biosynthesis in the mosquito gut, has been characterized. The binding of TMOF to mosquito gut membranes reached maximum at pH 7.4 and 24 degrees C. No binding was observed at pH 2.5 and the binding to the membranes declined rapidly at pH 8.0. At equilibrium, maximum binding to the receptor was observed at 60 min and 24 degrees C. A synthetic complementary decapeptide NH2-Ile-Leu-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-COOH (FOMT) for TMOF successfully competed with the gut receptor, and specifically bound TMOF (Kd = 4 microM and Kassoc = 2.5 x 10(5) M-1). TMOF binding to gut membranes was characterized with FOMT and a specific ELISA to the hormone at 24 and 72 h after blood feeding. Two classes of binding sites were found on the gut membrane; high affinity (Kd1 = 4.6 +/- 0.7 x 10(-7) M; Kassoc = 2.2 x 10(6) M-1 Bmax = 0.1 pmol/gut) and low affinity (Kd2 = 4.43 +/- 1 x 10(-6) M; Kassoc = 2.3 x 10(5) M-1; Bmax = 0.2 pmol/gut). The total binding sites for high and low affinity classes of TMOF per gut were estimated as 6.3 x 10(10) and 1.1 x 10(11) sites, respectively. Specific binding sites on the gut increased after the blood meal and were visualized by immunocytochemical staining. These results suggest that TMOF regulates trypsin biosynthesis by binding to specific receptor sites that are located on the mosquito gut, and that this receptor can be studied using a complementary peptide approach.

Ultrastructural comparison of extracellular and intracellular encapsulation of Brugia malayi in Anopheles quadrimaculatus.

Ultrastructural aspects of extracellular humoral encapsulation of microfilariae of Brugia malayi in the hemocoel of Anopheles quadrimaculatus were compared with those of intracellular encapsulation of first-stage larvae (L1) of the same parasite species, in the thoracic muscle cells of the same species of mosquito. The results showed that extracellular humoral encapsulation of microfilarial sheaths, and sheathed and exsheathed microfilariae, in the hemocoel of mosquitoes occurs around the parasite within the first 6 hr postingestion, apparently without initial participation of hemocytes. Hemocytes and their remnants were observed near the parasite during the first 6 hr postingestion. Within the next 24 hr, hemocytes attach to the initial humoral capsule. By contrast, intracellular encapsulation of L1S is initiated by the accumulation of a dense cytoplasmic layer derived from the infected thoracic muscle cell. Melanin deposits accumulate in this layer adjacent to the parasite cuticle, again without visible participation of hemocytes.

Intracellular development of subperiodic Brugia malayi influenced by mosquito thoracic muscle cells.

The in vitro development of 1-day-old intracellularly lodged larvae of Brugia malayi cultured in infected excised thoraces of selected susceptible and refractory strains of Aedes aegypti and Anopheles quadrimaculatus was compared with larvae reared in vivo. In susceptible mosquitoes, both in vitro and in vivo, larvae developed normally and abnormally. In refractory mosquitoes this pattern of both normal and abnormal development was also observed, except that comparatively fewer larvae developed to the infective third-stage larvae (L3) in vitro than in vivo and that more first-stage larvae (L1) were intracellularly melanized in vivo than in vitro. These studies indicate that factors in the thoracic muscle cells of the mosquito greatly affect the development of B. malayi microfilariae to L3. Intracellular melanization of L1 in An. quadrimaculatus, previously demonstrated in vivo, rarely occurred in vitro. These studies therefore suggest that refractoriness and melanization of B. malayi larvae in the thoraces of An. quadrimaculatus are controlled by two different and separate mechanisms.