Preservation of aerial conidia and biomasses from entomopathogenic fungi Beauveria brongniartii and Metarhizium anisopliae during lyophilization

In this study, we assessed the stability provided by different formulations to aerial conidia or biomasses (conidia, blastospores, and mycelia) of Beauveria brongniartii and Metarhizium anisopliae subjected to lyophilization. First, the impact of the freezing and drying processes on spore survival was evaluated. Whereas unprotected B. brongniartii spores showed high cryosensitivity, those of M. anisopliae were markedly harmed by the drying process. Then, the protective efficiency of 14 excipients was systematically evaluated and optimized regarding required concentrations. Fructose, glucose, and saccharose significantly enhanced viabilities for B. brongniartii and M. anisopliae spores following lyophilization, especially as a result of their cryoprotective effects. In addition, the effect of various bulking agents on spore survival was studied and dextran 4 was selected to enhance the physical properties of the lyophilized products. The combination of fructose and dextran 4 was further applied to prepare lyophilized biomasses of both fungi. In comparison to freshly harvested biomasses, the lyophilized products showed similar growth rates and a comparable production of virulent secondary metabolites such as destruxin A, destruxin B, or oosporein, suggesting their applicability as biological control agents.     

A redescription of Nosema herpobdellae (Microspora: Nosematidae), a parasite of the leech Erpobdella octoculata (Hirudinea: Erpobdellidae)

Nosema herpobdellae was recorded in populations of the leech Erpobdella octoculata from lakes in northwest England and North Wales and is redescribed using light and transmission electron microscopy. It differes from N. glossiphoniae in the nature of the infection and tissues parasitized and from N. tractabile in its larger spore size, longer polar filament, in the angle of the anterior coils of the polar filament to the spore long axis, and apparently in its developmental cycle. The infection was found in a massive xenoma, in the connective tissue surrounding the gut, which was presumed to be formed from a single hypertrophied cell. Its developmental cycle included merogony and sporogony.     

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.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Persistence of Bacillus thuringiensis parasporal crystal insecticidal activity in soil

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai (H-serotype 7), strain HD137, streptomycin-resistant mutant were added to acidic (pH 5.0) natural and autoclaved soil and incubated at ?0.10 MPa, 25°C. Populations of B. thuringiensis in both soil treatments showed exponential rates of mortality which were represented by linear regression, the loss of viability being greater in natural than autoclaved soil. In natural soil, parasporal crystal insecticidal activity was lost at a complex, nonexponential rate. The initial, rapid decrease of activity gradually slowed, and the level of activity stabilized at 10% of the original inoculum level after 250 days incubation, until the cessation of sampling at >2 years. In autoclaved soil no significant (P > 0.2) loss of parasporal crystal insecticidal activity was detected over the same period, which suggested that soil microorganisms were responsible for the loss of crystal insecticidal activity in the natural, nonsterilized soil. The rate of loss of crystal activity in natural soil correlated well with assay data reported in the literature using Galleria mellonella, which measures the combined activity of spore and crystal. In autoclaved soil correlation was poor, probably due to variability in the bioassay data.     

Zschokkella (Protozoa: Myxosporida) in macrourid fishes

Six species of Zschokkella, three new, were recovered from eleven genera and forty-three species of macrourid fishes found in both the Atlantic and Pacific Oceans and many adjacent seas. The constancy in spore size and shape, over great distances and among many hosts, was remarkable.     

Davallia (Polypodiales: Davalliaceae) macrofossils from Early Miocene Otago (New Zealand) with in situ spores

Fossil fern fronds referable to the extant fern genus Davallia (Polypodiales: Davalliaceae) bearing sporangia with in situ spores are described from the Early Miocene Foulden Maar diatomite deposit, Otago, New Zealand. The fronds are the first published Southern Hemisphere macrofossil record for the family and provide valuable palaeoclimate data supporting warm conditions in Early Miocene New Zealand. The matching of Davallia fronds to the form spore taxon Polypodiisporites radiatus shows that the genus has had a long, apparently continuous history throughout late Cenozoic New Zealand.     

Virulence of entomopathogenic Fungi (Fungi imperfecti) for the adults of Acanthoscelides obtectus (Coleoptera: Bruchidae)

Tests with the bean weevil, Acanthoscelides obtectus, in which the hosts were exposed indirectly to various dilutions of conidia of four entomopathogenic fungi showed that mortality was a function of the concentration of the inoculum. In these tests a given spore suspension was sprayed on the internal surfaces of a Petri dish. Adult weevils of a known age were placed in the dish, held there for 24 hr, then removed and kept at 20°C. After 20 days, the host mortality was determined. From the data obtained, it was possible to trace a probit regression line of the mortality in relation to the increasing spore concentration. Infection was observed in hosts exposed to a concentration of approximately 5 × 10⁶ spores/ml up to a maximum of about 1 × 10⁹ spores/ml. The A. obtectus was susceptible to infection by spores of Beauveria bassiana, B. tenella, Metarrhizium anisopliae, and Paecilomyces fumoso-roseus.     

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.     

Transmission and infectivity of spores of Burenella dimorpha (Microsporida: Burenellidae)

Burenella dimorpha infects the tropical fire ant, Solenopsis geminata, producing two morphologically distinct types of spores. A binucleate, nonpansporoblast membrane-bounded (NPMB) spore develops in and destroys the hypodermis, rupturing the cuticle in the pupal stage. A uninucleate, pansporoblast membrane-bounded (PMB) spore develops in the fat body. Adult ants cannibalize ruptured pupae but do not ingest spores. Instead, the spores and particulate foods are diverted to the infrabuccal cavity, formed into an infrabuccal pellet, and fed to fourth-instar larvae only. This larval instar is the only stage in the life cycle of S. geminata that is vulnerable to infection. NPMB spores are infective, but PMB spores do not extrude their polar filaments in the larval gut and are expelled in the meconium upon pupation.     

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.     

A microsporidian pathogen of the poroporo stem borer,Sceliodes cordalis (Dbld) (Lepidoptera: Pyralidae): Effects on adult reproductive success

Larvae of Sceliodes cordalis were infected over a range of ages and doses with Nosema sp. (NSC). The resulting spore loads in adults ranged from 9.3 × 10⁵ to 8.9 × 10⁷ spores and varied with the age of the larva at infection and with the dose ingested. Infection caused small reductions in female weight, mating success, potential fecundity, longevity, and egg viability. The magnitude of spore loads in female moths was not found to influence any of these factors apart from potential fecundity in very low weight moths. The cumulative effects of infection reduced natality in the laboratory colony from 11,400 to 6000 hatching eggs per 100 females. Laboratory reared, infected females were smaller, had a lower longevity, a reduced spore load, and were more fecund (eggs per milligram adult weight) than infected females from a field population. Manipulation of NSC in a biological control program is not considered to be of any value.     

双色真藓的组织培养和发育研究

对双色真藓(Bryum dichotomum Hedw.)的孢子发育过程及愈伤组织的诱导和培养进行了研究。结果表明,双色真藓孢子萌发和原丝体发育属于典型的真藓型。将双色真藓原丝体接种在含有2.0 mg L-1的硅酸钠和3.0 mg L-1 6-BA的MS固体培养基上,可诱导双色真藓原丝体分化为愈伤组织。愈伤组织在含有2.0 mg L-1的硅酸钠、1.0 mg L-12,4-D和1.0 mg L-1 6-BA的MS固体培养基上可以长期继代培养。而愈伤组织在含有2.0 mg L-1的硅酸钠、1.0 mg L-1 2,4-D和1.0 mg L-1 6-BA的MS液体培养基中可以悬浮培养,且生长迅速,培养28 d达到接种鲜重的9.25倍。     

Relative stability of biological properties in encapsulated strains of Staphylococcus aureus after freezing and freeze-drying

The relative stability of the biological properties of three encapsulated strains of Staphylococcus aureus was compared after preservation for 1 year in two different vehicles, 10% glycerol and 15% honey and at two different temperatures, ?30 and ?80 °C. A third method of preservation was by lyophilization in 10% skim-milk plus 0.1% glutamic acid and 2% honey. Comparison with control stock cultures maintained by bimonthly subcultivation on brain heart infusion (BHI) agar slants indicated that viability of the organisms was best preserved in 15% honey. When freezing and freeze-drying were compared, superiority was achieved by the latter. Quantitative activities of acid phosphatase, DNase, and coagulase remained constant in all subcultures. Also, while no loss of virulence for mice was observed with these methods, some did occur with the stock subcultures on BHI agar slants. Concerning relative salt tolerance of the strains in these preparations, the lyophilized organisms surpassed the frozen ones. However, when lyophilizing time was prolonged, yellow pigmentation corresponding to β-carotene decreased. Finally, both frozen and lyophilized organisms maintained stable characteristics of growth type in serum-soft agar.     

Novel Myxobolus and Ellipsomyxa species (Cnidaria: Myxozoa) parasiting Brachyplatystoma rousseauxii (Siluriformes: Pimelodidae) in the Amazon basin,Brazil

We describe two novel myxosporean parasites from Brachyplatystoma rousseauxii, an economically important freshwater catfish from the Amazon basin, Brazil. Myxobolus tapajosi n. sp., was found in the gill filaments of 23.5% of 17 fish, with myxospores round to oval in frontal view and biconvex in lateral view: length 15 (13.5–17) μm and width 10.7 (9.6–11.4) μm; polar capsules equal, length 5.8 (4.6–7.1) μm and width 3 (2.3–3.8) μm containing polar tubules with 6–7 turns. Ellipsomyxa amazonensis n. sp. myxospores were found floating freely or inside plasmodia in the gall bladder of 23.5% of fish. The myxospores were ellipsoidal with rounded extremities: length 12.8 (12.3–13.6) μm and width 7.6 (6.7–8.7) μm; with two equal, slightly pyriform polar capsules, length 3.8 (3.8–4.0) μm and width 3.1 (2.5–3.4) μm, containing polar tubules with 2–3 turns. We combined spore morphometry, small-subunit ribosomal DNA data, specific host, and phylogenetic analyses, to identify both of these parasites as new myxozoan species. Maximum likelihood and Bayesian analyses showed that Myxobolus tapajosi n. sp. clustered in a basal branch in a subclade of parasites from exclusively South American pimelodid fishes. Ellipsomyxa amazonensis n. sp. clustered within the marine Ellipsomyxa lineage, but we suspect that although the parasite was collected in freshwater, its hosts perform a large migration throughout the Amazon basin and may have become infected from a brackish/marine polychaete host during the estuary phase of its life.     

Biological evaluation of the systematic validity of the African Argas (Persicargas) Arboreus and the Asian-Australian A. (P.) Robertsi (Ixodoidea: Argasidae)

Khalil G. M., Hoogstraal H. and Oliver J. H. Jr. 1980. Biological evaluation of the systematic validity of the African Argas (Persicargas) arboreus and the Asian-Australian A.(P.)robertsi (Ixodoidea: Argasidae). International journal for Parasitology10: 253–259. The closely related A. (P.)arboreus (Ethiopian Faunal Region) and A. (P.) robertsi (Oriental and Australian Faunal Regions) interbreed readily and produce partially fertile progeny. Intrahybrid crosses are much less productive than P₁ homogamic crosses. Results of backcrosses indicate that the progeny of the robertsi ♂ × arboreus ♀ cross are less fertile than the progeny of the reciprocal cross. The fertility of F₁ hybrid progeny is lower than that of the P₁ pure species as expressed in lower female fecundity, egg hatch, and/or viability of immature stages. These 2 species probably originated from a common ancestor and geographic isolation caused genetic incompatibility.     

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.     

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log₁₀ reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.