In vivo propagation of a microsporidan pathogenic to insects

Equivalent numbers of spores were produced when the microsporidan Nosema necatrix was propagated in either Trichoplusia ni or Heliothis zea. Maximum spore production was obtained at an inoculum level of 1 × 10⁵ spores/ml. Larvae inoculated 5 days post-hatching contained 1.6 × 10⁹ spores/gram larva after an incubation period of 21 days. Temperature optima for the parasite are 21–26°C in both hosts.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.     

Effect of prolonged storage of spores on field applications of Nosema locustae (Microsporida: Nosematidae) against grasshoppers

Spores of Nosema locustae that were freshly prepared, stored in water at ?10°C for 8 months to 3 years, or stored in cadavers for 1 year at ?10°C were applied on bran at the rate of 10⁹ spores/1.5 lb bran per acre. Applications of fresh spores resulted in higher density reductions and higher incidence of infection among survivors than applications of spores stored in water or cadavers in both a complex of grasshoppers predominated by a single species and in a mixed species complex. Density reductions due to treatment with fresh spores were similar in the two populations, but the mixed species complex had a lower incidence of infection than the complex in which one species predominated. Applications of fresh spores reduced grasshopper densities in both complexes to levels below the economic thresholds.     

Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line

This paper describes the replication and serial passage of Heliothis zea nuclear polyhedrosis virus (NPV) in a H. zea cell line. It was demonstrated that long-term serial passages of the H. zea NPV in homologous host cell culture decreased both the total number of polyhedral inclusion bodies (PIBs) produced and the infectivity of the supernatant as measured by TCID₅₀. The growth curve indicated that infectious material was released from cells 24 hr postinfection (p.i.) and approached a maximal titer 3 days p.i. The kinetics of H. zea NPV decay at 4°, 27°, and 37°C were determined. Infectivity was not detected after 3 weeks at 37°C, but approximately 10^3.5 TCID₅₀/ml activity was still present after 3 and 8 weeks storage at 27° and 4°C, respectively. Electron microscopy confirmed the presence of single embedded virions in the inoculated cells.     

The influence of storage temperature on spore viability of Mattesia trogodermae (Protozoa: Neogregarinida)

The viability of Mattesia trogodermae spores stored at different temperatures was assessed by the percentage infection induced in 30-day-old Trogoderma glabrum larvae. Exposure to 73°C and higher temperatures for 30 min was lethal to the spores. Spores stored at ?19°C survived better than those stored at 26.7°, 3.5°, or ?30°C.     

The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.     

A novel milky disease organism from Australian scarabaeids: Field occurrence,isolation, and infectivity

A novel milky disease organism has been found causing disease in Aphodius tasmaniae and other scarabaeid larvae in the field in Australia. The sporangium is exceptionally long, measuring 10.5 × 1.5 μm, with a small central spore, measuring 1.0 × 0.6 μm. The vegetative cell is about half the size of the sporangium. The disease was easily transmitted by injection of spores into the hemocoel, with typically milky symptoms developing in 2–4 weeks. Spores will form in vivo at temperatures down to 12°C. For A. tasmaniae third-instar larvae, the ID₅₀ by injection was 3 × 10² spores/larva, yet no infection resulted when larvae were reared in peat containing up to 10⁸ spores/g, i.e., the disease was not successfully transmitted per os. All 10 species of scarabaeids tested were susceptible to the disease when spores were injected; however, all attempts to infect larvae per os were unsuccessful. In vitro culture was also unsuccessful.     

The use of a microinjection procedure for large-scale production of the microsporidian Nosema eurytremae in Pieris brassicae

Nosema eurytremae, a microsporidian parasite of Malaysian trematodes, was injected at the rate of 1 × 10⁴ spores/larva into Pieris brassicae. The larvae, which subsequently pupated, were incubated at 25 to 26°C and on harvesting 19 days later yielded an average of 6 × 10⁸ spores/pupa. This was equivalent to 60,000 times the initial dose. Purity of filtered, washed spore suspensions ranged from 80 to 99% with up to 20% host debris.     

Interactions of the microsporidium Vairimorpha necatrix with a bacterium,virus, and fungus in Heliothis zea

Interactions between Vairimorpha necatrix and three other pathogens of Heliothis zea were evaluated with dose-mortality studies in order to find a synergistic combination which could be tested for field control of H. zea. The effect of the microsporidium combined with the bacterium Bacillus thuringiensis was at least additive, with indications of synergism. The interaction between V. necatrix and Heliothis nuclear-polyhedrosis virus was antagonistic except that the highest microsporidian dose overcame the antagonism with a resultant independent action. The interaction between V. necatrix and the fungus Nomuraea rileyi was additive, though response varied some-what with different proportions of the two pathogens. Even though none of these interactions is likely to be valuable in microbial control, V. necatrix has the potential to synergize or antagonize any biological or chemical agent that acts on the midgut epithelium of host insects.     

Proceedings: Preservation of rust fungi in liquid nitrogen

Spores of rust fungi can be expected to retain viability without loss of infectivity for at least several years when stored in liquid nitrogen (?196 °C). Addition of liquid suspending media is harmful and not necessary. Some rust fungi experience cold-induced dormancy when exposed to less than 0 °C for a minute or longer but germinability is dependably restored on applying a heat shock by heating the spores to 40 °C for at least 15 sec during or after thawing. Most rust fungi are not sensitive to moisture content at the time of freezing but Puccinia striiformis must be vacuum dried before freezimg. The need for heat shock may not show up until several days after thawing. All of the rust strains tested to date have retained their properties to the extent tested and for the duration of storage. Data are available for up to 11 years. Preliminary experiments to preserve saprophytic mycelial cultures of P. graminis have so far failed, with and without use of 10% glycerol and 5% DMSO. The successful preservation of rust spores has made feasible the development of a collection of living rust fungi at ATCC beginning in 1965 and which now has over 80 strains in 20 species in 7 genera.     

Lyophilization, vacuum drying, and subsequent storage of Nosema pyrausta spores

Methods of preserving Nosema pyrausta spores were developed by subjecting laboratoryreared, nondiapausing larvae of the European corn borer, Ostrinia nubilalis, infected with N. pyrausta to different techniques of lyophilization and vacuum drying. Such material was still viable when it was stored in an airtight container and held at ?12°C for 12 months, or when it was stored in a vacuum desiceator and held at 22–24°C for 6 months.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

Host age and pathogen dosage impact cyst morphogenesis in the invertebrate pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae)

Helicosporidium sp. is a pathogenic alga that replicates in the hemolymph of various invertebrate hosts. Morphogenesis of the infectious life stage, the cyst, occurs in the infected host, but to date cannot be induced in vitro. Using larvae of the heterologous host Helicoverpa zea, we examined potential factors influencing pathogenicity and in vivo cyst production of the alga and the impact of infection on host survival. Factors tested were cyst dosage administered per os (ranging from 10² to 10⁵ cysts per larva) and host age at exposure (third, fourth, and fifth larval instar). Cyst production occurred between 7 and 13 days after treatment, regardless of host age at treatment. Increasing dosage increased both percent infection and mortality, but cyst production did not track the total infection response. Increasing host age at exposure mitigated dosage effects on infection and mortality and also elevated cyst production in later-treated larvae. Only the highest dosage produced a significant decrease in the overall time to death. Moderate cyst dosages and later host ages were most effective at regenerating Helicosporidium cysts.     

Persistence of spores of Pleistophora schubergi (Cnidospora: Microsporida) in the field,and their application in microbial control

Spores of Pleistophora schubergi, when applied to oak trees in the field at 2 × 10⁸ spores/ml with a uv protectant, “Shade,” infected 88% of Anisota senatoria larvae at 4 days after spray application. Spores without the uv protectant infected only 10% of the larvae at 4 days after application. When the spores were applied at the rate of 2 × 10⁸ and 2 × 10⁷ spores/ml in the field, 96 and 72% of the A. senatoria larvae and 100 and 100% of the Symmerista canicosta larvae were infected 14 days after spray application.     

Pathologie d’une microsporidiose de l’arpenteuse du chou,Trichoplusia ni [Lep.: Noctuidae], parVairimorpha necatrix

Résumé Les larves deTrichoplusia ni (Hübner) infectées parVairimorpha (=Nosema) necatrix (Kramer) présentent des sympt?mes chroniques, semi-chroniques et aigus selon les doses de spores deV. necatrix (5 à 500, 5.10³ à 5.10⁵ et 5.10⁶ par larve, respectivement) ingérées par larve.V. necatrix parasite principalement le corps adipeux et les tissus des muscles quand les larves ont des sympt?mes chroniques, tandis que les larves manifestent les sympt?mes aigus lorsque les microsporidies attaquent principalement l’intestin moyen. L’histopathologie de la maladie est étudiée.

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Summary Infection of larvae ofTrichoplusia ni (Hübner) byVairimorpha, (=Nosema) necatrix was classified as chronic, semi-chronic and acute symptoms depending on the quantity of spores (5 to 500, 5×10³ to 5×10⁵, and 5×10⁶ per larva, respectively) ingested per larva.V. necatrix infects mainly the fat body and some muscle tissue of larvae with chronic symptoms and mainly midgut tissue of larvae with acute symptoms.V. necatrix caused death in 3 to 4.5 days when a larva ingested 5×10⁶ spores. The histopathology of the disease was studied.

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Ce travail a fait l’objet d’une thèse de 3^e cycle, présentée à l’Université de Paris VI en mars 1977, parWei Hsuang Chu.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

The significance of temperature during sporulation on the biology of pycnidiospores of Mycosphaerella ligiilicola

The germination, infectivity and survival of pycnidiospores obtained from cultures of Mycosphaerella ligulicola grown at 15 and 26 °C were compared. Spores formed at 26° (‘26° spores’) were less able to germinate at low relative humidities and showed a narrower temperature range for maximum germination after 6 h. At high spore densities 26° spores showed self-inhibition of germination and, over a range of lower densities, growth of their germ tubes was checked, which resulted in lower infection of leaf discs compared with 15° spores in which this phenomenon did not occur. The fungus could be recovered from un-sterile compost over a longer period after inoculation with 15° spores. Only after storage at a temperature well below zero was there a difference in viability between 15° and 26° spores. It is thought that the potential advantage of producing larger numbers of spores at 26° would be realized only under optimum conditions for dispersal and infection. The smaller number of spores produced at 15° are likely to be successful under natural conditions.     

A new mycosis of larval lobster (Homarus americanus).

Spodoptera exempta larvae were reared on semisynthetic maize diet. Pathogenicity studies were undertaken on first- to fifth-instar larvae fed a high dosage of Nosema necatrix spores. Larvae from the earlier instars were most susceptible to the microsporidan and also developed bacteriosis. A cytoplasmic polyhedrosis virus (CPV) was evident in some infected larvae but not in controls. The development of N. necatrix is redescribed using the light microscope. A disporoblastic life cycle was evident at 25°C and both a disporoblastic and an octosporoblastic life cycle at 20°C. The implications of the occurrence of bacteriosis and CPV and the possible biological significance of the two sporogonic sequences are discussed. The taxonomic position of N.necatrix is reviewed and, after comparison with existing species of the genera Nosema and Parathelohania, it is placed in the new genus Vairimorpha. The implications of polymorphism are discussed in relation to the classification of the Microsporida.     

Infection of the corn earworm, Heliothis zea, with Nosema acridophagus and Nosema cuneatum from grasshoppers: Relative virulence and production of spores

Per os inoculations of 4- to 6-day-old larvae of the corn earworm, Heliothis zea, with suspensions containing 10⁶ spores of Nosema acridophagus or 10⁴, 10⁵, and 10⁶ spores of Nosema cuneatum retarded the growth and development of the larvae. Migratory grasshoppers, Melanoplus sanguinipes, inoculated with N. acridophagus produced fewer spores than similarly inoculated corn earworms, but spore production was similar in these insects when they were inoculated with N. cuneatum. Standard bioassay procedures showed that spores of both microsporidians were some-what more virulent when they were produced in corn earworms than when they were produced in grasshoppers. Spores of these microsporidians might be produced more efficiently in corn earworm larvae than in grasshoppers.     

Effect of desiccation level and storage temperature on green spore viability of Osmunda japonica

In order to effectively preserve green spores, which have relatively higher water content and lose viability more quickly than non-green spores, we studied the effect of desiccation level and storage temperature on Osmunda japonica spores. The water content of fresh spores was 11.20%. After 12 h desiccation by silica gel, the water content decreased to 6% but spore viability did not change significantly. As the desiccation continued, the decrease in water content slowed, but spore viability dropped. For almost all storage periods, the effects of storage temperature, desiccation level, and temperature × desiccation level were significantly different. After seven days of storage, spores at any desiccation level stored at 4 °C obtained high germination rates. After more than seven days storage, liquid nitrogen (LN) storage obtained the best results. Storage at −18 °C led to the lowest germination rates. Spores stored at room temperature and −18 °C all died within three months. For storage at 4 °C and in LN, spores desiccated 12 and 36 h obtained better results. Spores without desiccation had the highest germination rates after being stored at room temperature, but suffered the greatest loss after storage at −18 °C. These results suggest that LN storage is the best method of long-term storage of O. japonica spores. The critical water content of O. japonica spores is about 6% and reduction of the water content to this level improves outcome after LN storage greatly. The reason for various responses of O. japonica spores to desiccation and storage temperatures are discussed.