Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10³, 10⁵, 10⁷, and 10⁹ spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10⁹ spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10⁶ spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.     

Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simuliidae)

The pathogenesis and midgut histopathology which resulted when larvae of the blackfly, Simulium vittatum, were exposed to Bacillus thuringiensis at various temperatures and periods of exposure were investigated. The onset of mortality was studied at 10°, 15°, 19°, and 24°C. For each 4–5°C increase in temperature above 15°C, the onset of mortality was shortened by 24 hr. Exposures as brief as 15 min to 10 ppm of a whole spore preparation resulted in an average mortality of 29% in late-instar larvae. Mortality increased sharply for exposures up to 3 hr, approaching a maximum of 80%.The gross signs of disease included cessation of feeding and tetany with brachytosis. The tissue most affected was the midgut epithelium in the regions of the gastric caeca and posterior stomach. The formation of cytoplasmic vacuoles followed by cell lysis and/or sloughing were very apparent in moribund larvae. Death resulted without bacteremia.     

Importance of spores,crystals, and δ-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae

Physical methods were used to produce spores containing impurities of 0.02–0.05% crystals and crystals containing impurities of 0.001–0.01% spores from cultures of Bacillus thuringiensis. In Galleria mellonella larvae, these preparations from varieties galleriae, aizawai, and wuhanensis were only moderately active compared to 1:1 mixtures of spores and crystals. Spores of an acrystalliferous aizawai mutant were inactive and did not contain a polypeptide of the same size as the potent M_r 138000 δ-endotoxin present in spores and crystals of all three wild-type strains. Thus, this polypeptide probably contributed to the moderate activity of wild-type spores. Spore impurities in the crystal preparation were killed by γ irradiation without harming the crystals. The crystals without live spores were virtually inactive (LC₅₀s, ca. 10¹⁰ crystals/g insect food). Addition of 10³ spores to 10⁸ crystals/g food (0.001% spores) increased the mortality of larvae from 0 to 36%, and addition of 10⁴ spores (0.01% spores) killed 64% larvae. Thus, the addition of low levels of spores increased the potency of crystals in G. mellonella from virtually zero to moderate levels, suggesting that the live spore impurities in the crystal preparations were responsible for the observed moderate potency of crystals before γ irradiation, a view supported by a reduction of potency of crystal preparations following admixture of streptomycin to the insect food. In contrast to the results with G. mellonella, crystals were ca. 30 times as active as spores in Pieris brassicae larvae. Many authors have found crystals purified by physical methods to be highly active in a range of lepidopterous hosts. The present work indicates that the role of the spore impurities in these species may need further investigation. Absence of live spores of B. thuringiensis may impair the control of some insect species feeding on spore-free products and on microorganisms or plants into which endotoxins have been introduced by genetic manipulation.     

Modeling horizontal transmission of microsporidia infecting gypsy moth,Lymantria dispar (L.), larvae

We developed a simulation model that describes the horizontal transmission of three different microsporidia, Endoreticulatus schubergi, Nosema lymantriae and Vairimorpha disparis and their insect host, the gypsy moth, Lymantria dispar. The model describes the stage specific development and mortality of uninfected, latently infected or infectious hosts, the food consumption, the infection by spore-laden feces of E. schubergi and N. lymantriae and by spore-laden cadaver of N. lymantriae and V. disparis. Model results were compared to percent infection of L. dispar test larvae published in earlier studies using caged oak trees and potted oak-plants. When feces were selected as the source of spores for transmission of E. schubergi or N. lymantriae, the model estimated a percent infection in susceptible larvae that was in the range of the experimental studies. When spore-laden cadavers were the source of spores of N. lymantriae or V. disparis, the model did not correctly predict the experimentally measured percent infection in susceptible larvae. The most critical points of the simulation model are exact calculation of spore release, mortality and exact determination of the transmission coefficients when cadavers were included as a source for microsporidian infection.     

Nosema ceranae Can Infect Honey Bee Larvae and Reduces Subsequent Adult Longevity

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.     

Effect of gut bacterial isolates from Apis mellifera jemenitica on Paenibacillus larvae infected bee larvae

The probiotic effects of seven newly isolated gut bacteria, from the indegenous honey bees of Saudi Arabia were investigated. In vivo bioassays were used to investigate the effects of each gut bacterium namely, Fructobacillus fructosus (T1), Proteus mirabilis (T2), Bacillus licheniformis (T3), Lactobacillus kunkeei (T4), Bacillus subtilis (T5), Enterobacter kobei (T6), and Morganella morganii (T7) on mortality percentage of honey bee larvae infected with P. larvae spores along with negative control (normal diet) and positive control (normal diet spiked with P. larvae spores). Addition of gut bacteria to the normal diet significantly reduced the mortality percentage of the treated groups. Mortality percentage in all treated groups ranged from 56.67% up to 86.67%. T6 treated group exhibited the highest mortality (86.67%), whereas T4 group showed the lowest mortality (56.67%). Among the seven gut bacterial treatments, T4 and T3 decreased the mortality 56.67% and 66.67%, respectively, whereas, for T2, T6, and T7 the mortality percentage was equal to that of the positive control (86.67%). Mortality percentages in infected larval groups treated with T1, and T5 were 78.33% and 73.33% respectively. Most of the mortality occurred in the treated larvae during days 2 and 3. Treatments T3 and T4 treatments showed positive effects and reduced mortality.     

Soil burrowing Muscina angustifrons (Diptera: Muscidae) larvae excrete spores capable of forming mycorrhizae underground

Muscina angustifrons (Diptera: Muscidae) is a mycophagous species that exploits a variety of fungi, including ectomycorrhizal fungi. Larvae of this species have been shown to feed on sporocarps (including spores), and full-grown larvae leave sporocarps and pupate 0–6?cm below the soil surface. In this study, we examined whether M. angustifrons larvae are capable of transporting ectomycorrhizal fungal spores and enhancing ectomycorrhiza growth on host-plant roots. Full-grown larvae were found to move horizontally 10–20?cm from their feeding sites and burrow underground. These wandering larvae retained ectomycorrhizal fungal spores in their intestines, which were excreted following relocation to underground pupation sites. Excreted spores retained germination and infection capacities to form ectomycorrhiza on host-plant roots. In the infection experiments, ectomycorrhizal fungal spores applied in the vicinity of underground host-plant roots were more effective in forming ectomycorrhiza than those applied to the ground surface, suggesting that belowground transportation of spores by M. angustifrons larvae could enhance ectomycorrhizal formation. These results suggested that M. angustifrons larvae act as a short-distance spore transporter of ectomycorrhizal fungi.     

Factors influencing the pathogenicity and development of Mattesia trogodermae infecting Trogoderma glabrum larvae

Factors that regulate development of Mattesia trogodermae in Trogoderma glabrum were defined, and their quantitative effects were determined. The rate of and the extent to which spore formation proceeds is strictly governed by temperature. More spores are produced at 30° than at 25°C and very low numbers of spores are formed when the incubation temperature is 35°C. When insects are incubated at 35°C for 1–10 days and transferred to 30°C for the remainder of the 30-day experiment, spore production capacity gradually declines with increasing time at 35°C. Two hypotheses are proposed for this phenomenon. Larval size also regulates the extent of spore production, larger larvae having greater potential for spore development. This is not influenced by dosage. Spore production in pupae and adults was always retarded.Dosage and environmental conditions which influence the virulence of M. trogodermae were investigated. These studies show that rates of mortality are higher at higher temperatures. Low doses of spores result in longer LT50's than do high doses at 25° and 30°C. No differences in rates of mortality were found between different doses at 35°C.     

Effects of a novel microsporidium on the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae)

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (<3.0%). Laboratory studies were conducted by orally exposing both larvae and adults of O. sulcatus to varying concentrations of Canningia sp. spores. Larval bioassays at a variety of dosages (0, 10, etc.) were performed to evaluate pathogen infectivity, larval survival and growth. Adult bioassays (dosages: 0, 10, etc.) were performed to evaluate longevity, fecundity and mechanisms of vertical pathogen transmission. Larvae and adults were infected in all spore treatments. Larval growth was significantly reduced at dosages above 10 spores/larva. Adults infected at all dosages experienced high levels of mortality and fecundity was reduced to zero. Greenhouse trials were performed to determine if larvae feeding in soil acquired infections when spores were topically applied as a drench application (0, 10⁵, 10⁶, 10⁷ spores/pot). Established larvae feeding on plant roots in pots developed infections when exposed to drench treatments of 10⁶ and 10⁷ spores/pot after 14-21 days. Canningia sp. is an acute pathogen of O. sulcatus infective to both larvae and adults. Topically applied spores also infected larvae feeding on roots in soilless potting media, suggesting the possibility of using this pathogen in a microbial control program.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

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.     

Nonoccluded virus-like particles in the mollusc Agriolimax reticulatus (Stylommatophora: Limacinae)

A novel slide-culture technique was used to study germination and outgrowth of Bacillus popilliae spores without disturbing the microenvironment. Infective spores formed in larvae required 24 hr to begin outgrowth, whereas noninfective spores from colonies initiated outgrowth in 12 hr. Dissolution of the paraspore coincided with outgrowth and not with germination of the attached spore. Germination and outgrowth were asynchronous events and required several days in all cell populations, except for free spores.     

Germination of Bacillus thuringiensis var. israelensis spores in the gut of Aedes larvae (Diptera: Culicidae)

In laboratory experiments, germination and growth of Bacillus thuringiensis israelensis in the gut of Aedes aegypti and A. vexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of living larvae and in cadavers was estimated by plaing homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40–140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells, was found in cadavers. In A. aegypti larvae, germination and growth of B. thuringiensis israelensis in the larval gut was photographically documented.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Entomopathogenic fungi disturbed the larval growth and feeding performance of Ocinara varians (Lepidoptera: Bombycidae) larvae

Abstract Feeding experiments using three strains of entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea were conducted with newly moulted 3rd–5th instar Ocinara varians Walker larvae in the laboratory. The mortality of larvae immersed individually in spore suspension (1 × 10⁷ spores/mL) of all the strains was ≥ 80% except 5th instar larvae treated with M. anisopliae which transformed into pupae, but did not result in adult emergence. The growth (total body mass), consumption, relative consumption rate and relative growth rate, were reduced at all three larval stages, while developmental time was extended in infected larvae with concurrent significant increase in approximate digestibility in infected larvae. Conversion of digested food (ECD) and ingested food (ECI) values declined in infected larvae as compared to the healthy larvae (control). The 5th instar larvae treated with M. anisopliae showed higher ECD and ECI values than control. Based on mortality and growth inhibition it can be suggested that all the studied fungal strains have a high potential for biocontrol and could be developed into biocontrol agents against O. varians.     

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.     

Growth, survival, and starvation resistance of Colorado squawfish larvae

Synopsis Growth and survival of Colorado squawfish, Ptychocheilus lucius, larvae under fluctuating 18, 22, and 26° C (5° C diel fluctuations) and constant 18, 22, 26° C, and 30° C temperature conditions and ration size corresponding to 12.5, 28,64,142, 320 brine shrimp nauplii fish^–1 day^–1 determined from laboratory experiments. Growth was optimal at 31° C and high at temperatures of 26° C to 30° C, at the highest food abundance. Lowest growth was under lowest food rations and highest temperatures. Growth of Colorado squawfish larvae declined substantially at temperatures < 22° C. Neither growth nor survival was significantly different between fluctuating or constant regimes. Survival of Colorado squawfish larvae was highest (95%) at 26.2° C and 235 nauplii fish^–1 day^–1 and high at temperatures of 20 to 30° C with food abundance > 180 nauplii fish^–1 day^–1. Survival was lowest when food abundance was low and temperature was high. Highest mortality occurred more than 20 days after experiments began and mortalities occurred sooner in higher than lower temperatures. Colorado squawfish larvae denied food for 5, 10, or 15 d after first feeding could have begun (6 d), had survival greater than 87 % which was equivalent to continuously fed controls. Survival of fish denied food for 17.5 d after feeding could have begun declined from 84% before feeding to 57% after feeding. Point of no return was estimated between 17.5 and 20 d. Colorado squawfish have relatively high starvation resistance. Low, stable flows that simulate natural hydrographs may enhance growth, survival, and recruitment of early life stages of Colorado squawfish by increasing water temperature and food abundance in regulated rivers of the Colorado River basin.