Defined medium supporting development of cleansed Plasmodium berghei ookinetes in Anopheles stephensi.

Hamsters blood infected with Plasmodium berghei was cultured in vitro for the development of ookinetes. The ookinetes were separated from blood components, suspended in various defined media and fed to Anopheles stephensi through a membrane. The development of the oocysts and infective sporozoites was recorded. Mosquitoes infected with ookinetes suspended in L15 formulated into L15-B, L15-D (a medium specially modified for this purpose), IPL-41 or 199 media with no proteins added, developed at least as many oocysts as the control mosquitoes fed ookinetes suspended in blood. Ookinetes suspended in the L15-B medium yielded more oocysts than after feeding ookinetes suspended in L15-B with 5% casein. Sporozoites from mosquitoes maintained on blood, L15-B, L15-D, or L15-B with 5% casein were shown to be infective to hamsters. Mosquitoes fed ookinetes suspended in sucrose solutions showed very few oocysts, but the yield was increased when a blood meal was given 2-4 days after the infective meal. Some of the oocysts which had developed from the ookinetes suspended in artificial media were found to have degenerated. The described system could be potentially useful for a study of the interaction between the vector physiology and the parasite. The possible use of the system to learn which media should be developed in the future for in vitro cultivation of oocysts is discussed.

In vitro anti-mycoplasmal activity of amphotericin B methyl ester.

The polyene macrolide antibiotic amphotericin B (AB) and its chemically modified derivative amphotericin B methyl ester (AME) were tested for in vitro activity against Acholeplasma laidlawii, Spiroplasma citri and Mycoplasma gallisepticum. Both polyene macrolide preparations demonstrated anti-mycoplasmal activity. However, AME was mycoplasmacidal toward all three strains of mycoplasma at levels which previous studies have indicated would be permissible for most cell culture systems, whereas the levels of AB required for similar activity would be physiologically intolerable for tissue culture cells. In addition, AME was 100 fold more active than AB toward A. laidlawii, 10 fold more active than AB toward S. citri and demonstrated equivalent activity as AB toward M gallisepticum. The in vitro anti-mycoplasmal activity of AME and AB was directly correlated with polyene macrolide antibiotic levels and the number of treated mycoplasma.

Anopheles stephensi and Toxorhynchites amboinensis: aseptic rearing of mosquito larvae on cultured cells.

Aseptic larvae of Anopheles stephensi and Toxorhynchites amboinensis were reared on a continuous cell line (RU TAE 12 V) from the mosquito, T. amboinensis, that grew in suspension as multicellular vesicles. Surface-sterilized eggs were hatched in a 24-well plate containing 0.2 ml of Leibovitz's L-15 medium per well and incubated in a humidified atmosphere. Toxorhynchites amboinensis eggs of 36 hr or older were placed singly to assure hatching and avoid cannibalism. Hatching rates were over 80%. All larval instars were maintained in L-15 medium at 28 C with a 12-hr photoperiod. Anopheles stephensi larvae were reared in 25-cm2 tissue culture flasks containing 10 ml of L-15 medium with 30 to 50 first and second instar larvae or 10 third and fourth instar larvae per flask. Toxorhynchites amboinensis larvae remained in the 24-well plate in 1.5 ml of medium through the second instar; third instar larvae were kept in 12-well plates (3 ml of medium per well) and transferred to 25-cm2 flasks (10 ml per flask) when they reached the fourth instar. First and second instar A. stephensi larvae were fed cultured cells once, and third or fourth instar larvae twice a day. Toxorhynchites amboinensis larvae were fed vesicles once during the first 4 days after hatching, and every 1 or 2 days thereafter. Each A. stephensi larva consumed approximately 2 X 10(6) cells, and T. amboinensis larvae 10 times more cells before pupating. Anopheles stephensi pupated after 7 to 8 days and adults emerged during days 9 to 11. Pupation in T. amboinensis began on day 21 after hatching and adults emerged 5 days later. Cell lines isolated from A. stephensi larvae or embryos of the ticks Rhipicephalus sanguineus and Anocentor (Dermacentor) nitens supported only limited growth of A. stephensi larvae. Defibrinated hamster (Mesocricetus auratus) blood, though readily ingested, did not support the growth of A. stephensi whereas larvae reared on blood cells plus T. amboinensis cells showed limited growth.

The future of mosquito-borne diseases in the world.

The future of mosquito-borne diseases will depend on the improvements and implementation of chemotherapy and vaccination, as well as on biological and integrated control measures. Bacillus thuringiensis H-14, B. sphaericus, Lagenidium giganteum and other fungi are promising biological mosquito control agents. Other control measures include parasitoids, nematodes, larvivorous fish, Toxorhynchites mosquitoes, insect viruses, growth hormones, sex attractants, natural products, sanitation, and water management. Vector control should be combined with training of personnel and carried out on an international scale.

Factors affecting membrane feeding of Anopheles stephensi.

The impact of optimal temperature, osmotic pressure, and diet viscosity on the number of mosquitoes (Anopheles stephensi) feeding through a membrane, and on the size of the blood meal, was evaluated. An increase in diet viscosity decreased the number of imbibing mosquitoes, reduced the size of the ingested meal, and resulted in a slower rate of weight loss after feeding. The possible effect of these factors on the vectorial efficiency of mosquitoes is discussed.

Electron microscopy of the formation of wound tumor virus in abdominally inoculated insect vectors.

Fifth-instar nymphs of Agallia constricta leafhoppers were injected abdominally with extracts from root tumors confining wound tumor virus (WTV). The insects were sacrificed at predetermined intervals, their internal organs dissected, fixed, embedded, sectioned, stained, and examined in a Siemens Elmiskop I. Sequential stages in virus development were reconstructed from consecutive samples of fatbody tissues. Changes resulting from the infection were: (i) a viroplasm, i.e., an accumulation of electron-dense aggregates; (ii) the appearance at the periphery of the viroplasm of a few fully formed virus particles recognized as virions; (iii) the formation of increasing numbers of individual virions, not only at the periphery but also in the viroplasm; (iv) the engulfing of virions within multimembranous structures; and (v) the formation of virus microcrystals either at the sites of former viroplasms, or at some distance. These morphological findings indicate that, following abdominal inoculation of WTV, the plant-pathogenic virus develops within the cytoplasmic matrix proper of insect vector cells. In addition to the fatbody tissues, WTV was detected in the epidermis, muscles, and trachea of abdominally inoculated insects, demonstrating the systemic invasion of the mechanically infected arthropod host. No virus was found in the gut tissues.

Plaque assay of Heliothis zea baculovirus employing a mixed agarose overlay.

The nuclear polyhedrosis virus of Heliothis zea has been titrated in Heliothis zea cells by the plaque method, using 1 percent mixed agarose containing a mixture of Seakem and Ultra pure agarose. Visible plaques, formed 8 days postinfection, ranged in diameter from 0.5 to 2 mm. Dose-response experiments indicated that a single particle initiated the formation of a plaque. The titration of Heliothis zea baculovirus by the newly described plaque method provides an accurate technique for the determination of virus concentration.

Electron microscopy of a plant-pathogenic virus in the nervous system of its insect vector.

The central nervous system of wound tumor virus (WTV)-infected Agallia constricta was studied by electron microscopy to obtain information concerning the virus distribution in the nervous system. Wound tumor virions were mostly found in the cytoplasm of the ganglion cells and less frequently in the glial cells. WTV was occasionally observed in the perineurium cells, nerve axons, tracheoblasts, and lateral nerves. In the ganglion cells, virions appeared as individual isolated particles (V(1)), in tubular formation (V(2)), and occasionally in aggregates (V(3)). In the glial cells, the virions were mostly seen in the V(3) formation, and very seldom in the V(1) and V(2) formations. In the perineurium cells and tracheoblasts, only small V(3) formations were observed. The isolated virions were usually surrounded with polyribosomes, and often appeared around the foci of the viroplasm. Sometimes degenerating ganglion cells infected with the WTV were encountered. These damaged cells strongly indicated that WTV exerted a cytopathogenic effect on the nerve cells.

The effect of defined media, additive nutrients and metabolites on the development of the sporogony cycle of Plasmodium berghei in Anopheles stephensi.

Anopheles stephensi mosquitoes were infected with Plasmodium berghei by feeding on parasitaemic hamsters. After the infective blood meal they were separated into groups that were maintained on sugar solutions containing different additives. The numbers of oocysts developing in the various groups were then compared. When either casein, haemoglobin or foetal bovine serum was added to the sugar, the yield of oocysts was 1.6-2.1 times higher than that in controls fed only on sugar solutions. When either medium 199, Leibovitz's formulated L15 (L15-B) or 'B' compounds alone were added, the yield of oocysts was 2.3-2.9 times higher than that in the controls. The addition of p-aminobenzoic acid, hypoxanthine, methiolnine, or fractions of 'B' had no significant effect on the number of oocysts.

Electron microscopy of Friend murine leukemia virus in the mid-gut of experimentally infected mosquitoes.

After 30 and 78 hr, Friend murine leukemia virus (FLV) particles were detected by electron microscopy in the mid-gut lumen of the mosquitoes Aedes aegypti (Linnaeus) and Anopheles stephensi Liston which had fed on leukemia BALB/c mice infected with FLV. Various developmental stages of the virions were observed within and on the surface of ingested blood cells, particularly young erythroblasts, as well as free in the lumen after budding. These preliminary findings indicate that FLV continues to multiply in the mid-gut of these species for at least 3 days despite the action of digestive enzymes. Detailed studies are in progress to determine the fate of FLV in these mosquito species.