A method of visualizing and assessing deposits of aerially sprayed insect microbes

A technique is described for visualizing and assessing deposits of various types of insect microbes on membrane filter surface. It is proposed as an accurate method of determining coverage of entomopathogenic microorganisms sprayed in a tower or from an aircraft in microbial control operations. The actual numbers of spores and/or crystals and number of International Units deposited per unit area, spore: crystal ratio, microbial impact droplet size, and spore viability of Bacillus thuringiensis have been determined with simulated aerial applications. The morphological integrity of different types of insect pathogens is easily distinguishable with the technique.Field trials with nuclear polyhedrosis virus of the spruce budworm, Choristoneura fumiferana, demonstrate the utility of the method in insect pest control by aerially applied microbial agents.     

Bacillus thuringiensis HD-73 Spores Have Surface-Localized Cry1Ac Toxin: Physiological and Pathogenic Consequences

Spores from Cry(sup+) strains of Bacillus thuringiensis bound fluorescein isothiocyanate-labeled antibodies specific for the 65-kDa activated Cry 1Ac toxin, whereas spores from Bacillus cereus and Cry(sup-) strains of B. thuringiensis did not. The Cry(sup+) spores could be activated for germination by alkaline conditions (pH 10.3), whereas Cry(sup-) spores could not. Once the surrounding exosporia had been removed or permeabilized, Cry(sup+) spores were able to bind the toxin receptor(s) from insect gut brush border membrane vesicle preparations, and their germination rates were increased ca. threefold in the presence of brush border membrane vesicles. A model is presented whereby in the soil the Cry toxins on the spore surface are protected by the exosporium while in the gut they are exposed and available for binding to the insect receptors. This model explains why the disulfide-rich C terminus of the cry genes is so highly conserved even though it is removed during the processing of the protoxin to the activated toxin. It also highlights the trade-off resulting from having Cry toxins located on the spore surface, i.e., decreased spore resistance versus enhanced insect pathogenesis.     

Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season

Two media were developed which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form. Using these media B. thuringiensis was shown conclusively for the first time to exist in an active form on the phylloplane. The profile of its appearance in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Three simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers a considerable diversity of cry toxin gene types was found in isolates that had been recovered in vegetative form ('vegetative isolates') with the majority possessing multiple delta-endotoxin genes while some had only one of those tested. Bioassays against a lepidopteran insect of purified delta-endotoxins showed that they were no more potent than those from a laboratory-adapted strain. PCR primers for an internal region of the vip3A gene produced amplification in 70% of the vegetative isolates compared to 25% of the laboratory-adapted strains tested.     

Some Effects of Diflubenzuron on Growth and Sporogenesis in Streptomyces spp

Diflubenzuron, an insect growth regulator that blocks chitin deposition in insect cuticles, was tested for its effects on morphogenesis of Streptomyces spp. Use of diflubenzuron resulted in reduced dominance of spore hairs, reduced the width of the outer wall, and prevented formation of the inner spore wall in S. bambergiensis. In S. coelicolor, diflubenzuron altered the structure of the fibrillar pattern of spore envelopes. Exposure to diflubenzuron resulted in small increases in exported protein and in a ca. 20% increase in chitinase in both Streptomyces spp.     

Infection of Tribolium castaneum with Bacillus thuringiensis: Quantification of Bacterial Replication within Cadavers,Transmission via Cannibalism,and Inhibition of Spore Germination

Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.     

Observations on the method of transmission of internal boll disease of cotton by the cotton stainer-bug

Results are presented to show that all the rules of proof for insect transmission of Nematospora by Dysdercus spp. have now been satisfied: viz. a close association of the insect with diseased plants in nature; visitation of healthy plants by the insect under conditions suitable for transmission of the disease; the presence of the pathogen in or on the insect in nature or following visitation to a diseased plant; and experimental production of the disease by insect visitation under controlled conditions. Hence it is claimed that Dysdercus spp. are proved vectors of internal boll disease of cotton.
Fungal material as spores or mycelium is carried as an external contaminant on the mouth-parts, although it is located in the deep stylet pouches, where protection is afforded and spore germination is possible.
The fungus is cast off with the exuviae during moulting, but recontamination from exuviae or other sources in the environment occurs.
The transmission is hence purely mechanical with the insect obligatory only in its function as a syringe to inject the fungus which is otherwise unable to reach its substrate. Insect and fungus can develop independently and the insect is apparently unharmed by the presence of the fungus.
Nematospora spores also reach the intestine but do not appear to remain viable there and do not germinate.
The fungus is believed to enter and leave the insect by the same route. The spores are well adapted in shape to reach the stylet pouches of the insect.
No alternative morphological form of Nematospora gossypii within the insect has been found.
The results do not lead to methods of control of the disease beyond those in practice-trapping of insects, destruction of alternative host plants, and the alteration of time of planting to avoid insect attack.     

Entomophthora species with E. muscae-like primary spores on two new insect orders, Coleoptera and Hymenoptera

Adults of Cantharis livlda (Coleoptera: Cantharidae) and of Torymus druparum (Hymenoptera: Torymidae) were found naturally infected by fungi from the Entomophthorales in Denmark. The morphology of the primary spores of the two fungi clearly showed that they belong to the genus Entomophthora s.str. No species from this genus has been reported so far from these insect orders. With respect to spore size and number of nuclei per spore, the fungi fall within the range of species from the E. muscae complex, known only from Diptera. A transfer of the fungus from T. druparum to Psila rosae (Diptera: Psilidae) was however possible. The findings thus confirm a significant widening of the host range of species within the E. muscae complex.     

Some Effects of Diflubenzuron on Growth and Sporogenesis in Streptomyces spp.

Diflubenzuron, an insect growth regulator that blocks chitin deposition in insect cuticles, was tested for its effects on morphogenesis of Streptomyces spp. Use of diflubenzuron resulted in reduced dominance of spore hairs, reduced the width of the outer wall, and prevented formation of the inner spore wall in S. bambergiensis. In S. coelicolor, diflubenzuron altered the structure of the fibrillar pattern of spore envelopes. Exposure to diflubenzuron resulted in small increases in exported protein and in a ca. 20% increase in chitinase in both Streptomyces spp.     

The yeast spore wall enables spores to survive passage through the digestive tract of Drosophila

In nature, yeasts are subject to predation by flies of the genus Drosophila. In response to nutritional starvation Saccharomyces cerevisiae differentiates into a dormant cell type, termed a spore, which is resistant to many types of environmental stress. The stress resistance of the spore is due primarily to a spore wall that is more elaborate than the vegetative cell wall. We report here that S. cerevisiae spores survive passage through the gut of Drosophila melanogaster. Constituents of the spore wall that distinguish it from the vegetative cell wall are necessary for this resistance. Ascospores of the distantly related yeast Schizosaccharomyces pombe also display resistance to digestion by D. melanogaster. These results suggest that the primary function of the yeast ascospore is as a cell type specialized for dispersion by insect vectors.     

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.     

Ultrastructure and chemical composition of the ballistospore wall ofConidiobolus obscurus

The ballistospores of the entomopathogenConidiobolus obscurus are spheroidal cells with a papilla corresponding to the zone of attachment on the sporophore. They are covered by a mucus responsible for the attachment of the spore to the insect. Chemical and cytochemical investigations of the nature of the wall components have been undertaken to better understand fungus-insect interactions in entomopathology. β(1→3)-Glucans and chitin together represented the main components of the wall which did not contain chitosan and uronic acids. Transmission electron microscopy revealed that the spore wall was composed of a thick electron-lucent inner layer and a thin outer electron-dense layer, the latter being absent at the papilla region. The spore is covered by a mucilaginous layer that upon spore impact on a substratum, is dispersed and forms a halo around the spore. Shadow replicas of the discharged spores showed that they are covered by rodlets except on the papilla which displayed a chitinous, microfibrillar structure. The ontogeny of the rodlets deposited on the surface of young spores was characterized by a progressive organization of separate rodlets and then a clustering of the rodlets in fascicles. Shadow replicas and chemical and enzymatic investigations of the halo surrounding discharged spores showed that the mucus was composed of long β(1→3)-glucan microfibrils embedded in amorphous proteins partly covered by rodlets discharged from the spore surface.     

Identification of a Nosema bombycis (Microsporidia) spore wall protein corresponding to spore phagocytosis

Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.     

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 cm(2):ml versus 4.2 cm(2):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 x 10(4) resting spores/ml.     

Morphogenetic pathway of spore wall assembly in Saccharomyces cerevisiae

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.     

两种昆虫激素对鹿儿岛被毛孢表观形态的影响

昆虫激素不仅能调控昆虫的生长发育,而且也是昆虫免疫系统的重要调节因子,因此当虫生真菌入侵昆虫寄主体腔并定殖时可能会受其影响。但定殖过程中虫生真菌与昆虫激素之间的直接作用关系尚不清楚。本研究利用不同浓度的蜕皮激素(20E)和保幼激素(JH)模拟鳞翅目昆虫家蚕体内的主要激素水平,对虫生真菌鹿儿岛被毛孢Hirsutella satumaensis进行培养,观察其表观形态特征的变化。结果表明,两种昆虫激素对鹿儿岛被毛孢的表观形态均有不同程度的影响。添加20E和JH培养后,鹿儿岛被毛孢菌落直径和菌丝生物量的变化差异不大;孢子的萌发率稍有下降;而菌落产生的色素和产孢量与对照组相比则呈现显著性变化。菌落色素圈和分生孢子的粘液厚度会随两种激素浓度的增加而增大,而产孢量随激素浓度增加呈先增加后降低的趋势,这说明鹿儿岛被毛孢定殖昆虫血体腔的过程中两种昆虫激素会抑制真菌的生长,而虫生真菌会适应性地做出应答反应,这对于进一步理解虫生真菌在昆虫血体腔中的定殖机理有重要意义。     

Viable spore count as an index of effective dose of Bacillus thuringiensis

Leaf samples from Cercis occidentalis trees, collected 5–75 hr after application of varying concentrations of a Bacillus thuringiensis formulation, Dipel, were used as dose sources for third instar Schizura concinna larvae. Dose, defined as logarithmically transformed, estimated viable spore counts, and duration of time the formulation was on the leaf prior to sampling were considered in combination as predictors of insect mortality. By use of the multiple logistic model both factors were found to be significant predictors of insect mortality (P < 0.0001). Available methods for assessing goodness of fit indicate that the multiple logistic model adequately fits these data.     

Bacterial endospores and their significance in stress resistance

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface

The exosporium is the outermost layer of the Bacillus anthracis spore. The predominant protein on the exosporium surface is BclA, a collagen-like glycoprotein. BclA is incorporated on the spore surface late in the B. anthracis sporulation pathway. A second collagen-like protein, BclB, has been shown to be surface-exposed on B. anthracis spores. We have identified sequences near the N-terminus of the BclA and BclB glycoproteins responsible for the incorporation of these proteins into the exosporium layer of the spore and used these targeting domains to incorporate reporter fluorescent proteins onto the spore surface. The BclA and BclB proteins are expressed in the mother cell cytoplasm and become spore-associated in a two-step process involving first association of the protein with the spore surface followed by attachment of the protein in a process that involves a proteolytic cleavage event. Protein domains associated with each of these events have been identified. This novel targeting system can be exploited to incorporate foreign proteins into the exosporium of inactivated, spores resulting in the surface display of recombinant immunogens for use as a potential vaccine delivery system.     

Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro

Entomophthora thripidum is an obligate biotrophic insect pathogenic fungus that grows as protoplasts within the hemocoel of thrips. Prior to penetration through the insect cuticle and spore formation at the insect surface the protoplasts switch to hyphal growth. In vitro, the differentiation to hyphal growth was a prerequisite for the subsequent formation of infectious spores and was detected 10-20 days after inoculation. E. thripidum secreted a factor that autoinduced the differentiation to hyphal growth. The discovery of this activity inducing hyphal growth made possible the reliable production of spores, the infection of host insects and the consecutive re-isolation of the fungus from the infected insects.     

Production and efficiency of protozoa

Despite the chronic and debilitative nature of the infection they cause, several species of microsporidia and neogregarines offer a good potential as microbial control agents, particularly against insect pests of high economic thresholds. Techniques for mass production of protozoa have usually involved per os, inoculation or injection of the protozoa into their usual or alternate hosts. The spores are harvested subsequently from heavily infected host tissues by grinding, filtration, and differential centrifugation. Although fresh spores are used in most field tests, the spores of many species can be stored with high survival either frozen or in water at low temperatures (0–4°C) for up to several months. Sunlight or ultraviolet (UV) radiation is a serious factor limiting spore persistence. However, the protozoa do not appear to be significantly limiting spore persistence. However, the protozoa do not appears to be significantly more susceptible to UV radiation than other insect pathogens and persistence can be prolonged with UV protectants. Most field tests with protozoa have involved the application of spores in sprays and have usually resulted in a high degree of infection in the target host species. The potential for control of few species has been improved by formulation of spores in to baits, and the potential of other species will likely increase if suitable bait formulation can be devised in the future. One species, Nosema locustae, formulated as a bait, has been successfully used to control grasshoppers on rangelands. Limited laboratory and field studies have also suggested that increased short-term control might be obtained if candidate protozoan species can be combined with certain insecticides. While recent and increased efforts have been devoted to assess the potential of protozoa as microbial control agents, potential hazards to nontarget organism have been investigated for only three species. Their close relation taxonomically to protozoa pathogenic for mammals will necessitate careful evaluation of the safety of candidate control species for nontarget organisms.