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.     

Detection of relationships among microsporidan isolates by electrophoretic analysis: hydrophilic extracts

Hydrophilic spore proteins were extracted from Nosema sp. and Nosema trichoplusiae. These proteins were subjected to electrophoretic analysis. The resulting electrophoretic spectra were found to be unstable when (1) two genera of hosts were used for spore propagation, (2) hosts were reared at a variety of temperatures, (3) protein was extracted from spores stored for different periods of time, or (4) spore incubation period was varied. Comparison of the major bands obtained from spore protein of the isolates indicated no overlap in relative migration values. Although variation in spectra was observed, the use of major band patterns indicate electrophoretic analysis of hydrophilic spore protein can provide characters useful in the separation and identification of microsporidan isolates. Nosema sp. and Nosema trichoplusiae are not considered to be closely related phylogenetically.     

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.     

Detection of relationships among microsporidan isolates by electrophoretic analysis: hydrophobic extracts

Hydrophobic spore proteins were extracted from 11 microsporidan isolates obtained from 9 species of insects for which these microorganisms are pathogenic. Hydrophobic protein spectra were found to be stable when (1) two different genera of hosts were used for spore propagation, (2) hosts were reared at a variety of temperatures, or (3) protein was extracted from spores harvested at different stages of sporogenesis. Five consistent and distinct electrophoretic spectra were observed. Spectrum I was represented by 6 isolates including Nosema necatrix, Thelohania diazoma, Nosema plodiae, and Nosema sphingidis; spectrum II by Pleistophora sp; Spectrum III by Nosema whitei; spectrum IV by Thelohania legeri; and spectrum V by Nosema trichoplusia. The highly reproducible nature of these analyses indicated polyacrylamide gel disc electrophoresis of hydrophobic extracts can be used for the identification of Microsporida. Moreover, these analyses do not support the present classification, based mainly on the number of spores in a pansporoblast, inasfar as (1) some species of Nosema have the same pattern (I) as a species of Thelohania and (2) two species of Nosema have different patterns (III and V) in contrast to the Nosema species showing pattern I.     

Nosema takapauensis n.sp., a microsporidan parasite of larvae of Costelytra zealandica (Coleoptera: Scarabaeidae) in New Zealand

Morphological and biological features of a microsporidan protozoan parasite of larvae of Costelytra zealandica collected at Takapau, southern Hawkes Bay, are described and evaluated taxonomically. The parasite multiplies in the fat body of all larval instars, causing massive tissue disintegration in advanced infection resulting in the retardation of development and ultimately death before pupation. The microsporidan forms one spore per sporont, and therefore belongs to genus Nosema’, it is considered to be specifically distinct from its nearest congener, N. melolonthae.     

Temperature-Dependent Spore Dimorphism in Burenella dimorpha (Microspora: Microsporida)

Burenella dimorpha, a microsporidian parasite of the tropical fire ant, Solenopsis geminata, produces two morphologically distinct types of spores. The binucleate free spores (spores not bound by a pansporoblast membrane) develop normally at temperatures at least as low as 20°C and as high as 32°C. The uninucleate octospores (spores bound in octets by a pansporoblast membrane), however, develop in a restricted range of temperature. Octospores constituted 35.9%± 2.6 of the spores in 25 pupae held at 28°C. Raising the temperature to 30°C reduced octospores to < 1% of the total spore population. Lowering the temperature to 25° or 22°C reduced the octospore population to 8.5%± 6.5 or 0.4 ± 0.5, respectively. Inhibition of octospore development was complete at 20°C. In contrast, the octospores of Vairimorpha necatrix and Vairimorpha plodiae are reported to be abundant at 16°C and 21°C, respectively. The critical event blocked in octospore development may be meiosis, as evidenced by an abundance of binucleate sporonts in the octospore sequence of development, and absence of more advanced sporogonic stages in hosts held at inhibitory temperatures. Free spore size is not affected by temperature although yield may be slightly reduced at elevated temperature.     

Growth of Nosema algerae in Pig Kidney Cell Cultures*

SYNOPSIS. Nosema algerae, a microsporidan parasite of mosquitoes, can infect pig kidney cell cultures. Spores germinated in the culture medium, infected the cells within 30 min of germination, multiplied, and produced spores. The early developmental stages in the N. algerae life cycle are described.     

Dissemination of the biocontrol agent Vairimorpha necatrix by the spined soldier bug, Podisus maculiventris

The ability of the spined soldier bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae), to disseminate infective forms of two lepidopteran pathogens, Vairimorpha necatrix (Kramer) (Microspora: Microsporidia) and Lacanobia oleracea granulovirus (LoGV) was investigated. Individual female P. maculiventris that had fed on Lacanobia oleracea L. (Lepidoptera: Noctuidae) larvae, infected with V. necatrix, excreted approximately 6 × 10⁸V. necatrix spores during the subsequent 7 days. Excreted spores were fed to L. oleracea larvae, causing 100% mortality, indicating that the spores remained viable after passing through the gut of the predator. Podisus maculiventris that had fed on V. necatrix or LoGV‐infected larvae were allowed to defecate on the foliage of tomato plants, prior to the infestation of the plants with L. oleracea or Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) larvae. This proved to be an effective way of infecting the pest larvae with the pathogens, particularly when five predatory bugs were used per plant. After 20 days, the number of S. littoralis and L. oleracea surviving on the plants was reduced by 75% and 61%, respectively. Female P. maculiventris maintained on V. necatrix‐infected prey showed reduced egg production and longevity, whilst those fed on LoGV‐infected prey showed only reduced egg production. The potential for P. maculiventris to disseminate insect pathogens is discussed in the context of improved biological control of lepidopteran pests.     

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.     

A serological comparison of some species of microsporida.

Double immunodiffusion techniques were used to investigate the taxonomic relationships between six different microsporidian isolates. Microsporidia used included Nosema bombycis, N. algerae, N. plodiae, and three organisms morphologically similar to N. necatrix. Antigens were extracted from spores after disruption in an MSK Braun cell homogenizer. Cross-reactions were seen between N. plodiae and two of the N. necatrix isolates, while the third N. necatrix, N. bombycis, and N. algerae were antigenically unrelated. One of the N. necatrix isolates revealed temperature-related antigenic differences, but no antigenic differences resulted from aging spores for 5 months before disruption.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

Nosema algerae: Infection of the white mouse by a mosquito parasite

Spores of Nosema algerae, a microsporidan parasite of mosquitoes, were subcutaneously injected into the ears, tail, and feet of white mice. Infections were transient and localized at the injection sites. Spore germination tests in blood plasma indicated that it is unlikely that spores injected by an infected mosquito bite would result in an infection.     

THE LIFE CYCLE OF BANGIA FUSCOPURPUREA IN CULTURE. I. EFFECTS OF TEMPERATURE AND PHOTOPERIOD ON THE MORPHOLOGY AND REPRODUCTION OF THE BANGIA PHASE12

The Bangia phase of Bangia fuscopurpurea was grown in laboratory culture in a variety of photoperiod and temperature regimes. Plants of the Bangia phase grown from 2 types of asexual spores, monospores and conchospores, exhibited growth differences under similar growing conditions. Plants derived from monospores grew more rapidly and matured earlier than those derived from carpospores. Day length and temperature were found to significantly influence growth rule, maturation, and plant size. Long day lengths resulted in more rapid growth in filament length and diameter and earlier spore formation and spore release. Maximum filament length was observed in a 12/12 hr light-dark cycle at 15 C. Spore formation and release were delayed by decreasing day length or temperature. Temperature and photoperiod were also found to influence the type of spores produced by the Bangia phase. When grown at 22 C, the Bangia phase produced only monospores, which reproduced the Bangia phase. At 9 C, with photoperiods of 11 hr or more of light, the Bangia phase produced carpospores which gave rise to the alternating Conchocelis phase. The conditions under which sporogenesis occurred determined the spore type differentiated.     

Stemphylium botryosum f. lactucae- its developmentjn vifro and its pathogenicity to lettuce leaves

Stemphylium botryosum f. lactucae, incitant of a leaf-spot disease of stored lettuce, was found to be relatively restricted in its host range. Cross-inoculations with spore suspension of this fungus failed to induce symptoms in any of the host plants tested, except carrot. Among isolates of S. botryosum from various hosts, only the isolate from carrot induced slight symptoms on lettuce. While mycelial growth of the lettuce isolate was confined to the range 13–37 ^oC spores germinated at more extreme temperatures. The optimum temperature for germination and for radial growth on PDA was found to be between 25 and 30 ^oC. Wet spores were quickly inactivated at 50 ^oC, whereas more than 40 % of dry spores withstood a 24 h exposure to that temperature. Only the outer leaves of lettuce responded readily to inoculation with a spore suspension, the required incubation period being 3 days at 25 ^oC. Symptoms developed less readily on bruised leaves. Relative humidity approaching saturation was necessary for prompt and typical infection, notably during the 24 h following inoculation. Short dry periods (60 % r.h.) interposed at a later stage, while somewhat inhibitory, did not prevent infection.     

Fixation of microsporidian spores for electron microscopy

Fresh and frozen spores of the microsporidia Nosema apis and Nosema bombi were fixed using various fixatives at different times and temperatures. Paraformaldehyde and technical formaldehyde gave results comparable to or better than glutaraldehyde. Increased fixation temperature improved the fixation of spores from terrestrial hosts. Freezing did not destroy the cytology of the spore.     

Porphyra monospore system (Bangiales, Rhodophyta): A model for the developmental biology of marine plants

We have developed an experimental system of cohort monospores from clonal culture of leafy gametophytes in Porphyra yezoensis Ueda (strain TU-1). This system is quite different from traditional systems for algal protoplast experimentation, which require expensive enzymatic treatment and utilize an ineffective method of preservation. Cohort monospores were obtained by utilizing a mode of asexual reproduction in the culture strain (monospores) and artificial regulation (thallus length, temperature, light, etc.) of monospore release. When the leafy gametophytes that formed monospores were frozen at - 20°C in a cryoprotective solution composed of 5% DMSO and 5% dextran in 100% seawater, about 98% survived for 3 months. When stored at 5°C without cryoprotectants, these leafy gametophytes could be kept without monospore release for 1 week. Maximum monospore yield was about 3000 spores per 100 gametophytes, and germination rate was about 70%, This system will accelerate developmental biology studies in Porphyra.     

Internal ultrastructure of spores of microsporidans isolated from the Silkworm,Bombyx mori

Nosema bombycis, two Nosema spp., and a Pleistophora sp. were propagated in the silkworm and the fine structures of their spores were studied. The morphology of the polaroplast, the appearance of the nucleus, and the number of coils in the polar filament differed among the spores of the four species. The spores of the three Nosema species, however, had several identical components; e.g., the polaroplast was made up of two parts, they had two nuclei, and the ribosome arrangement was similar. On the other hand, the spore of Pleistophora sp. had a polaroplast composed of three parts, a single nucleus, and ribosomes arranged around the polar filament. Thus the fine structures of the spore differentiate microsporidan species.     

Interactions of the hyperparasitoidsCatolaccus aeneoviridis [Hym.: Pteromalidae] andSpilochalcis side [Hym.: Chalcididae] with the microsporidansNosema heliothidis andN. Campoletidis

The interactions of the hyperparasitoidsCatolaccus aeneoviridis (Girault) andSpilochalcis side (Walker) with the microsporidansNosema heliothidis Lutz & Splendor andN. campoletidis Brooks & Cranford are described. Neither hyperparasitoid species was infected upon parasitization of pupae of the primary parasitoidCampoletis sonorensis infected withN. heliothidis. However, upon development inCampoletis pupae infected withN. campoletidis, adults ofC. aeneoviridis were infected systemically; trassovarian transmission of the microsporidan to F₃ individuals was also shown.S. side was also infected byN. campoletidis but in this hyperparasitoid, microsporidan development was arrested in the sporoblast stage. Mature spores were not observed in infectedS. side adults and the microsporidan was not transmitted transovarially. The interactions of the hyperparasitoids with the two microsporidans were not demonstrably detrimental as data on the developmental period and adult longevity of infected or exposed hyperparasitoids were similar to those of control individuals.

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Résumé Les auteurs décrivent les interactions entre deux hyperparasitoides,Catolaccus aeneoviridis etSpilochalcis side, et les microsporidies,Nosema heliothidis etN. campoletidis. Aucune des espèces hyperparasitoides ne fut infectée quand elles ont parasité des nymphes du parasitoide primaire,Campoletis sonorensis infecté parN. hehothidis. Cependant, les adultes deC. aeneoviridis furent infectés systématiquement lors qu'ils se développèrent dans des nymphes deCampoletis infectées parN. campoletidis; la transmission transovarienne de la microsporidie jusqu'aux individus de la F3 a été également démontrée.S. side a été aussi infecté parN. campoletidis mais chez cet hyperparasitoide le développement de la microsporidie a été arrêté au stade de sporoblaste. Les spores mures n'ont pas été observées dans les adultes deS. side et la microsporidie n'est pas transmise par la voie transovarienne. Les interactions entre les hyperparasitoides et les deux microsporidies n'ont pas d'effet nuisible démontrable puisque la durée de développement et la longévité des adultes chez les hyperparasitoides contaminés ou exposés sont comparables à celles observées chez les témoins.

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Paper No. 4123 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina.     

Observations on the Spores of Pleistophora gigantea (Thélohan, 1895) Swellengrebel, 1911, a Microsporidan Parasite of the Fish Crenilabrus melops*

SYNOPSIS. After 1914 protozoologists have generally agreed that Pleistophora gigantea (Thélohan, 1895) Swellengrebel, 1911, Ichthyosporidium giganteum (Thélohan, 1895) Swarczewsky, 1914, and I. phymogenes Caullery and Mesnil, 1905, are identical. Because no polar filament was found in the spores, however, some authors have followed Swarczewsky in considering this species to be a haplosporidan, while others have persisted in thinking it a microsporidan. Using preserved material that Swellengrebel saved from a tumor on which he based his studies, we have found a polar filament in the spores both with the PAS reaction and with the electron microscpe. This new information removes the only basis for the doubt which some authors have entertained, that Thélohan and Sweliengrebel correctly considered the parasite to belong to the Microsporida. Since Pleistophora gigantea is believed to be identical with I. phymogenes, recently selected by Sprague as type species of genus Ichthyosporidium Caullery and Mesnil, 1905, then Ichthyosporidium, originally assigned to the Haplosporida, must be regarded as a microsporidan genus. Whether it is distinct from all other microsporidan genera is a matter needing further consideration.