Insect Tissue Culture Medium Research Articles

Complete development of mosquito phases of the malaria parasite in vitro.

Methods for reproducible in vitro development of the mosquito stages of malaria parasites to produce infective sporozoites have been elusive for over 40 years. We have cultured gametocytes of Plasmodium berghei through to infectious sporozoites with efficiencies similar to those recorded in vivo and without the need for salivary gland invasion. Oocysts developed extracellularly in a system whose essential elements include co-cultured Drosophila S2 cells, basement membrane matrix, and insect tissue culture medium. Sporozoite production required the presence of para-aminobenzoic acid. The entire life cycle of P. berghei, a useful model malaria parasite, can now be achieved in vitro.

In Vitro Formation of Resting Spores by the Insect Pathogenic Fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace’s insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm2:ml versus 4.2 cm2:ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 × 104 resting spores/ml.

In Vitro Production of Hyphal Bodies of the Mite Pathogenic Fungus Neozygites floridana

The fungus Neozygites floridana is a path- ogen commonly associated with the cassava green mite (CGM), Mononychellus tanajoa, and the two- spotted spider mite (TSM), Tetranychus urticae, in South America. Difficulty in culturing N. floridana in vitro has hampered its development as a bioacaricide. A Colombian isolate from TSM was multiplied in TSM and hyphal bodies from infected specimens were inoculated into 18 mycological and tissue cul- ture media. Grace's insect tissue culture medium plus 0.33% lactalbumin hydrolysate and 0.33% yeastolate (GS) afforded vigorous multiplication of hyphal bod- ies (over 106 /mL). The number of hyphal bodies increased with aeration. Final concentration of hy- phal bodies was not affected by fetal bovine serum (FBS), but growth was somewhat enhanced. Hyphal bodies produced germ tubes when transferred from GS medium to Grace's medium plus 10% FBS and shaken at 200 rpm for 24 h. The germinated hyphal bodies, when placed on water agar, produced pri- mary conidia from which capilliconidia were pro- duced. These capilliconidia were infective to both TSM and CGM.

In vitro production of hyphal bodies of the mite pathogenic fungus Neozygites floridana

The fungus Neozygites floridana is a pathogen commonly associated with the cassava green mite (CGM), Mononychellus tanajoa, and the two-spotted spider mite (TSM), Tetranychus urticae, in South America. Difficulty in culturing N. floridana in vitro has hampered its development as a bioacaricide. A Colombian isolate from TSM was multiplied in TSM and hyphal bodies from infected specimens were inoculated into 18 mycological and tissue culture media. Grace's insect tissue culture medium plus 0.33% lactalbumin hydrolysate and 0.33% yeastolate (GS) afforded vigorous multiplication of hyphal bodies (over 106 /mL). The number of hyphal bodies increased with aeration. Final concentration of hyphal bodies was not affected by fetal bovine serum (FBS), but growth was somewhat enhanced. Hyphal bodies produced germ tubes when transferred from GS medium to Grace's medium plus 10% FBS and shaken at 200 rpm for 24 h. The germinated hyphal bodies, when placed on water agar, produced primary conidia from which capilliconidia were produced. These capilliconidia were infective to both TSM and CGM.

In vitro cultivation of the entomopathogenic fungus Entomophthora thripidum: isolation, growth requirements, and sporulation

Entomophthora thripidum Samson, Ramakers & Oswald (Zygomycota, Entomophthorales) was isolated from Thrips tabaci Lindeman (Thysanoptera, Thripidae; onion or potato thrips). Vegetative fungal cells were released into culture medium by opening surface sterilized insects and multiplied in Grace's insect tissue culture medium supplemented with yeastolate, lactalbumin hydrolysate and 10% fetal bovine serum (FBS). These cells grew as protoplasts (no staining for cell wall) and aggregated to form pellets that were only observed in vitro. After exhaustion of the medium, walled mycelium formed which produced forcibly ejected spores when transferred onto water agar.

In vitro Cultivation and Sporulation of Neozygites parvispora (Zygomycetes: Entomophthorales)

Summary Neozygites parvispora was isolated from Tbrips tabaci infesting leek plants. In vitro cultivation of two isolates was achieved using supplemented Grace's insect tissue culture medium complemented with fetal bovine serum (FBS) and pre-treated lepidopteran hemolymph. This medium composition enabled exponential cell growth as rod-shaped hyphal bodies with a length over diameter ratio of 4.5-5.0. Both FBS and hemolymph were mandatory for sustained growth. The growth requirements indicate the presence of one or several growth factors in both the hemolymph and FBS. Absence of FBS resulted in growth arrest with subsequent cell lysis. Omission of hemolymph led to rounding and size decrease of the hyphal bodies, some of which formed septated germ tubes in a later stage. Eventually, cell death occurred under such conditions. Hyphal bodies from in vitro culture were capable of capilliconidia formation after entrapment in alginate pellets. This suggests the requirement of a liquid/air interface, which triggers primary sporulation and subsequent capilliconidium formation.

Myrmecomyces annellisae gen. nov., sp. nov. (Deuteromycotina: Hyphomycetes), an endoparasitic fungus of fire ants, Solenopsis spp. (Hymenoptera: Formicidae)

Two strains of a parasitic fungus, differing only in assimilation of cellobiose and colonial morphology on some media, occur in the haemolymph of fire ants in the United States and Argentina. In the host, obclavate yeast cells bud from a narrow base at the larger pole. A true, septate mycelium without clamp connections readily develops in vitro , and has been observed on rare occasions in the host. The hyphal septum has a simple, central pore. Morphology in insect tissue culture medium closely resembles that in the host; however, in simpler media the cells and hyphae are pleomorphic and often only mycelial. Chlamydospores are produced; otherwise, sporulation has not been observed. The morphological, cultural, and physiological characteristics are described, and the fungus is named as a new species, Myrmecomyces annellisae , representing a new genus. Gross pathology, histopathology, or changes in host behaviour have not been observed; however, parasitized hosts appear to succumb more readily to stress.

Effects of Medium Alterations on In vitro Development of Brugia malayi Larvae in Cultured Mosquito Thoraces

A recent study showed that 1-day-old, intracellularly lodged larvae of Brugia species develop in vitro to the infective third-stage larvae (L3) in excised thoraces of susceptible mosquitoes in the diphasic insect tissue culture medium containing a nutrient agar base overlaid with a 1:1 mixture of Schneider's Drosophila medium and Grace's insect cell culture medium supplemented with 20% fetal bovine serum (FBS) and antimicrobial agents. In the present investigation, the diphasic culture medium was used to evaluate the effects of medium alterations on the development of 1-day-old, intracellularly lodged larvae of subperiodic Brugia malayi in excised thoraces of Aedes aegypti to the L3. One-day-old larvae developed to the L3 in medium without nutrient agar base, at pH 7.0 and pH 7.5, in Hanks' balanced salt solution (HBSS) and in HBSS supplemented with bovine albumin fraction-V (BAF-V). These larvae also developed in the absence of FBS in the overlay medium, in overlay medium containing 5-20% FBS, in medium components obtained from different sources, in serum free Sf-900 (GIBCO) medium, and when FBS is replaced by BAF-V in the overlay medium. The percentage of L3 was not increased substantially in infected excised thoraces of mosquitoes when nutrient supplements, such as folic acid, p-aminobenzoic acid, glucose, lipid concentrate, hemin, or reduced glutathione, were added to the overlay medium containing BAF-V. These results suggested that 1-day-old, intracellularly lodged larvae developed to the L3 in infected excised thoraces of mosquitoes at almost the same rate as in intact mosquito, when excised thoraces were maintained alive under optimal conditions in a culture medium.

In vitro development of Brugia pahangi and Brugia malayi in cultured mosquito thoraces

In vitro cultivation of Brugia pahangi and subperiodic Brugia malayi one-day old larvae to infective stage larvae (L 3) within thoraces excised from Aedes aegypti (Black Eye, Liverpool) and Anopheles quadrimaculatus was attempted. The mosquito thoraces were excised under aseptic conditions, 24 h after a blood meal on either B. pahangi- or B. malayi-infected jirds. The excised thoraces were washed aseptically and inoculated into a diphasic media. A nutrient agar base was overlaid with either Grace's insect cell culture medium or Schneider's Drosophila medium or a mixture (1:1) of these two media. Each overlay medium contained a 1 × concentration of antibiotic/antimycotic mixture plus 20% fetal bovine serum. The excised thoraces provided the intracellular milieu for development of Brugia larvae. In Grace's or Schneider's insect tissue culture medium alone, the filaria larvae of both species developed only to the second larval stage after 12 days; whereas, in a mixture (1:1) of Grace's and Schneider's media, some one-day old larvae of both Brugia species developed to the infective larval (L 3) stage after 12 days. However, large numbers of both species of larvae developed to the infective larval stage when, prior to providing an infective blood meal, the mosquitoes of both species were fed 1 × concentration of antibiotic/antimycotic mixture in a 10% sucrose solution containing 0.1% p-aminobenzoic acid for 6 days. These results showed for the first time that if one-day old Brugia larvae are confined intracellularly in excised thoraces, they can then develop in insect tissue culture media without adding a feeder layer of mosquito cells or conditioning the media with mosquito cell lines.

Survey of Entomopathogenic Fungi Naturally Infecting Cereal Aphids (Homoptera: Aphididae) of Irrigated Grain Crops in Southwestern Idaho

A four-year field survey of wheat, barley, and corn generated data on 10 species of fungal pathogens, including eight Entomophthorales and two Hyphomycetes recovered from 2,930 cadavers of aphids infesting grains grown under irrigation in southwestern Idaho. Seven species of cereal aphids, including the Russian wheat aphid, Diuraphis noxia (Mordvilko); the potato aphid, Macrosiphum euphorbiae (Thomas); Metopolophium dirhodum (Walker); the corn leaf aphid, Rhopalosiphum maidis (Fitch); the bird-cherry oat aphid, R. padi (L.); the greenbug, Schizaphis graminum (Rondani); and the English grain aphid, Sitobion avenae (F.), were recorded as hostsof one or more species of entomopathogenic fungi. The fungi were identified as Pandora neoaphidis (Remaudiere & Hennebert) Humber, Conidiobolus obscurus (Hall & Dunn) Remaudiere & Keller, C. coronatus (Costantin), C. thromboides Drechsler, Entomophthora chromaphidis Burger & Swain, Neozygites fresenii (Nowakowski) Remaudiere & Keller, Zoophthora radicans (Brefeld) Batko, Z. occidentalis (Thaxter) Batko, Beauveria bassiana (Balsamo) Vuillemin, and Verticillium lecanii (Zimmermann) Viegas. P. neoaphidis was the most prevalent species annually in populations of M. dirhodum and D. noxia and also frequently infected other hosts. Conidiobolus spp. were secondary to P. neoaphidis in occurrence, commonly infecting several aphid species. E. chromaphidis, N. fresenii, Z. radicans , and Z. occidentalis were recovered only occasionally and on fewer aphid species. Two Hyphomycetes, B. bassiana and V. lecanii , infected aphids in the field at very low levels compared with the Entomophthorales. All species, except N. fresenii and Z. occidentalis , were isolated from their aphid hosts with agar media (SDAY or SEMA, or both) or liquid insect tissue culture medium.

Production, stabilization and regeneration of protoplasts of Zoophthora radicans using a commercial enzyme

Wall-less protoplasts have been prepared from both mycelia and conidia of Zoophthora radicans , using the commercial lytic enzyme, Novozym 234. Optimal conditions for protoplast release were 2 h incubation (23–25°C) of mycelia in a solution containing 1–2 mg enzyme per ml of 0·2 m phosphate buffer (pH 6·0), with 0·6 m -KCl to control the osmotic potential at approximately −30 bar. Greatest productivity of protoplasts (5000/mg wet weight of mycelium) was obtained from 1-d-old mycelia grown in either glucose-tryptone-yeast extract, Gibco 199 (with Hank's salts), or Grace's insect tissue culture medium. After release of protoplasts in enzyme solution, their subsequent stability was greatest (60% survival after 24 h) when protoplasts had been produced in a lytic mixture containing KCl as an osmotic stabilizer and subsequently resuspended in a solution buffered at pH 6·0 with 0·6 m mannitol as the osmotic stabilizer. When protoplasts were returned to growth media, cell walls were quickly regenerated and the fungus commenced to grow. Some protoplasts produced a chain of yeast-like cells, but the majority developed hyphae. Using these techniques, protoplasts were also released from hyphae of Entomophaga aulicae and Erynia neoaphidis .

Production, stabilization and regeneration of protoplasts of Zoophthora radicans using a commercial enzyme

Wall-less protoplasts have been prepared from both mycelia and conidia of Zoophthora radicans, using the commercial lytic enzyme, Novozym 234. Optimal conditions for protoplast release were 2 h incubation (23–25°C) of mycelia in a solution containing 1–2 mg enzyme per ml of 0·2 m phosphate buffer (pH 6·0), with 0·6 m-KCl to control the osmotic potential at approximately −30 bar. Greatest productivity of protoplasts (5000/mg wet weight of mycelium) was obtained from 1-d-old mycelia grown in either glucose-tryptone-yeast extract, Gibco 199 (with Hank's salts), or Grace's insect tissue culture medium. After release of protoplasts in enzyme solution, their subsequent stability was greatest (60% survival after 24 h) when protoplasts had been produced in a lytic mixture containing KCl as an osmotic stabilizer and subsequently resuspended in a solution buffered at pH 6·0 with 0·6 m mannitol as the osmotic stabilizer. When protoplasts were returned to growth media, cell walls were quickly regenerated and the fungus commenced to grow. Some protoplasts produced a chain of yeast-like cells, but the majority developed hyphae. Using these techniques, protoplasts were also released from hyphae of Entomophaga aulicae and Erynia neoaphidis.

Methods for the development of cell lines from insects

This paper describes techniques for the development of continuous cell lines from insect embryos and other tissues. Methods include how to obtain aseptic primary tissues, how to dissociate the tissue, and then how to maintain the primary culture until cells grow consistently. The relative advantages of different tissues (embryos, ovaries, etc.) in establishing insect cell lines are discussed and several insect tissue culture media are described.

In vitro cultivation of Herpetosoma trypanosomes in insect cell tissue culture media.

The cultivation of Herpetosoma trypanosomes in insect tissue culture media supplemented with foetal calf serum is described. Trypanosoma lewisi and T. musculi, which can be grown in blood agar media, were compared with four other species of Herpetosoma trypanosomes, T. microti, T. evotomys, T. grosi and T. nabiasi, in their growth in Schneider's Drosophila medium, Grace's, Mitsuhashi-Maramorosch, RPMI 1640, TCM 199 and nutrient blood agar media. Schneider's Drosophila and Grace's media supplemented with 20% foetal calf serum proved the most suitable media for growth of all parasites except T. nabiasi from rabbits which was not successfully established. Primary cultures were passaged after approximately 3 weeks and were maintained to continuously produce metacyclic trypomastigotes which produced less virulent infections although they maintained their infectivity to their respective hosts. The growth patterns in culture and morphology of the parasites are described.

Effect of mutagen on cultured Schistosoma mansoni.

Although lightly homogenized three-week-old Schistosoma mansoni incubated in Mitsuhashi and Maramorosch insect tissue culture medium or the medium of Weller and Wheeldon produced adherent cell layers, continued growth of these cells did not occur. Non-adherent cells obtained by trypsinization also failed to produce long term cell cultures even after the addition of a range of growth factors. The possibility of producing tumour-like schistosome cells by the use of the mutagen ethyl methane sulphonate was therefore examined. Four-hour exposure of three-week-old schistosomes caused in some worms (a) large fluid filled 'ballooning', which also occurred in adult males, (b) enlargement of the gut, (c) increase in numbers of large round cells within the worms and (d) tissue outgrowths. It is suggested that these effects of mutagen offer new approaches to obtaining permanent schistosome cell cultures.

Bat trypanosome models for Trypanosoma cruzi

Amongst the Trypanosomatidae, the sub- genus Schlzotrypanum is relatively homogeneous. It comprises about 6 species of Trypanosoma, which are stenoxenous (or monoxenous) parasites of bats (Chiroptera), with only Trypanosoma cruzi having escaped this host restriction to become euryxenous, able to infect a wide range of mammals in- cluding man. Comparative biochemical and genetic studies on T. cruzi and its stenoxenous relatives could help to ex- plain the important and widespread phenomenon of host specificity, and re- cent work supports the earlier suggestion that bat trypanosomes such as T. d~on~sfi could serve as useful laboratory models for the more dangerous T. cruzi i . Two members of the subgenus Schizo- trypanum, T. (S.) dionl~;ii and T. (S.) vesper- tihonls 2 parasitize bats (Chiroptera) in Eng- land (Fig. I). The prevalence of infection is probably over 50%, and three bat species have been found infected: pipistrelles (Piplstrellus plpistrellus) (with both trypanosome species), noctules (Nyctalus noctula) and Leisler's bat (N. letslen) (both with T. vespertihonis only). The trypano- somes are transmitted in the faeces of the bat bedbug Cimex pipistrelli 3, but there is no evidence that the trypanosomes harm their host. T. dionisii will also develop and multiply in Rhodnius proh~<us, although it fails to establish permanent infections4; this could be useful as a simple teaching model for T. cruzi transmission. The trypanosomes grow readily Jh vitro (Fig. I) in a variety of simple culture media such as Grace's insect tissue culture medium supplemented with 30% (v/v)in- activated foetal bovine serum s . Both species also infect several lines of tissue cul- ture cells in vitro. Susceptible cells include mouse L cells 6, HeLa cells 6, unstimulated mouse peritoneal macrophages61 and 'buf- falo' (= bison)lung (BL) cells 7 (Fig. 2). Both species of trypanosome have been biochemically characterized by DNA bouyant density 8, isoenzyme elec- trophoresis 8, and polypeptide profiles 9. T. dionisii, at least, seems to be strictly re- stricted to chiropteran hosts ~h vivo m, and there is no evidence that either species can infect man, although this has not, of course, been investigated experimentally. It there- fore seems a reasonable assumption that T. dion~siiis a safe organism to handle in the laboratory without the need to take special precautions I L ELISA investigation has revealed considerable cross-reactivity between T. dionis#, T. vespe~lionls and T. cruzi 12. T. dionisii and 7-. vespertilionis them- selves share at least two ¢omrnon anti-

Isolation and Culture of Entomophthora gammae, a Fungal Parasite of Noctuid Larvae

Entomophthora gammae (Weiser) was isolated by allowing conidia from infected soybean looper, Pseudoplusia includens (Walker), larvae to shower onto egg yolk agar. Fungal and bacterial contaminants were always present, but at 20 @*C, E. gammae grew fast enough to allow the subculture of hyphal bodies to clean medium. Once isolated, the fungus was cultured on Czapek solution agar, potato dextrose agar, Sabouraud dextrose agar, and Sabouraud maltose agar, all containing 0.2% yeast extract, and on Grace's insect tissue culture medium. It did not grow on Wolf's medium, potato agar or potato dextrose agar without yeast extract. The fungus grew most rapidly in culture when maintained at 25 @*C and 100% RH. Conidia on glass slides held at 25 @*C germinated only at 100% RH. A gradual increase in conidium size occurred over a 10-month period of continuous culture. Attempts to infect larvae with the cultured fungus were unsuccessful.

Tetrahymena dimorpha sp.nov. (Hymenostomatida: Tetrahymenidae), a new ciliate parasite of Simuliidae (Diptera) with potential as a model for the study of ciliate morphogenesis

A new species of hymenostome ciliate, Tetrahymena dimorpha sp.nov., is described. This ciliate occurs as a parasite in the haemocoel of larval, pupal and adult Simuliidae (Diptera). In larval hosts the total number of parasites never exceeds about 240 and the infection is benign. Within larval hosts the ciliates are large and broadly oval and possess an unusually wide range of somatic kineties, from 30 to 66; moreover a variable proportion of these kineties are characteristically disorganized, being incomplete, meandering or branched. Metamorphosis of the host to the adult fly is accompanied by a dramatic increase in the number of ciliates, which reach pathogenic intensity. Adult hosts may contain up to 19000 ciliates and the flies soon die from this heavy burden. Associated with ciliate population growth during host metamorphosis is a startling morphological transformation of the ciliates themselves. In adult hosts the ciliates are smaller and pyriform in shape and the cortex is greatly modified; the total number of somatic kineties is considerably reduced and has a limited range of 19—22. Most significantly, the kineties are ordered with typical tetrahymenine precision. By application of appropriate culture conditions to ciliates isolated from any host stage, either of the distinctive morphological forms of T. dimorpha may be selectively induced in vitro . In bacterized infusions, ciliates are produced that have the general form and cortical characteristics of those found naturally in adult hosts. Sterile culture in serum-supplemented Mitsuhashi and Maramorosch insect tissue culture medium produces a population showing features characteristic of ciliates from larval hosts. Sterile culture in proteose-peptone-yeast-extract medium results in populations exhibiting concurrent dimorphism, even after cloning. The extreme nature and multiple facets of the dimorphism together with the ease of its manipulation in vitro afford opportunities for the experimental investigation of many problems, particularly those related to cell surface patterning in ciliates, and these possibilities are discussed in relation to current concepts of ciliate morphogenesis.

Metacyclogenesis of Trypanosoma cruzi in vitro: a simplified procedure

A simplified procedure for obtaining high levels of infective, metacyclic trypomastigotes of a Brazilian strain of Trypanosoma cruzi in vitro is described. Epimastigotes cultured in Yaeger's Liver Infusion Tryptose Medium, when transferred to Grace's Insect Tissue Culture Medium (GMA) supplemented with 10% foetal bovine serum, yielded over 90% metacyclic forms. Decreasing or eliminating the serum from GMA adversely affected growth but did not decrease levels of transformation, thus providing a chemically defined culture system for analysing factors influencing metacyclogenesis of T. cruzi.

In vitro studies of Coelomomyces punctatus from Anopheles quadrimaculatus and Cyclops vernalis

Attempts to grow mycelium of Coelomomyces punctatus from Anopheles quadrimaculatus larvae were made using more than 50 combinations of known vertebrate and invertebrate tissue culture media and microbiological media. Growth and/or differentiation of mycelium into sporangia were observed in several media. Significant growth of hyphal fragments and differentiation into young resting sporangia occurred in conditioned Mitsuhashi-Maramorosch insect tissue culture medium. This medium was conditioned by growth for 3 weeks in it of Varma's Anopheles stephensi tissue culture cells and was supplemented with 20% heat-inactivated fetal bovine serum and a synthetic tripeptide, glycyl-histidyl-lysine. Limited growth and elongation of lateral hyphal branches and subsequent development into resting sporangia with typical outer wall markings and pigmentation of mature forms were observed in a modified brain-heart infusion medium. Some media stimulated hyphae to develop into smooth-walled, spherical bodies of size and appearance typical of young sporangium initials but with no further maturity. In most media, no growth or development of mycelium occurred, but the fungus remained alive for 2–4 weeks. Mycelium of C. punctatus dissected from Cyclops vernalis did not grow and develop in any of the media utilized. However, in one case the mycelium differentiated into gametes shortly after dissection into modified brain-heart infusion medium.