Comparative virulence of Nosema plodiae and Nosema heterosporum in the Indian meal moth, Plodia interpunctella

When larvae of the Indian meal moth, Plodia interpunctella, were fed diets containing spores of Nosema plodiae, the number that survived to the adult stage decreased and the rate of adult emergence was retarded as the concentration of spores was increased; all surviving adults were infected. Also, when larvae were reared on diets containing spores of Nosema heterosporum, the number that survived to the adult stage decreased as the concentration of spores was increased; however, no relationship was apparent between concentration of spores and the rate of adult emergence. The LC₅₀'s of N. plodiae and N. heterosporum were 8.09 × 10⁶ and 4.52 × 10³ spores/g diet, respectively, which confirmed preliminary observations regarding the relative virulence of the two species of Nosema to Indian meal moth larvae.     

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA)

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689 bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.     

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.     

Transovarian transmission of Nosema plodiae in the Indian-meal moth, Plodia interpunctella

Twenty-five adult female Plodia interpunctella infected with Nosema plodiae laid 856 eggs in laboratory tests; 12.5% of the eggs were infected transovarially. The highest level of transmission by an individual female was 14 infected eggs of 27 laid (51.8%); the lowest level of transmission observed was 1 of 43 eggs (2.3%). All stages of N. plodiae were not transmitted with equal frequency; moreover, most eggs harbored predominently only the trophozoite stage of the pathogen. Approximately 80% of the infected eggs contained trophozoites almost exclusively; about 13.1% contained about an equal number of spores and trophozoites, and about 6.5% contained mainly spores. Histological observations indicated that infections may be initiated in nurse cells and subsequently transferred to associated oocytes.     

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.     

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.     

Inactivation and mechanisms of chlorine dioxide on Nosema bombycis

Biological tests demonstrated that the inactivation of Nosema bombycis (N. bombycis) spores by chlorine dioxide (ClO₂) occurs very fast and is highly sensitive. The lowest effective inactivation dosage and time was 15 mg/mL for 30 min. The inactivation of spores was additionally verified by using double color fluorescence stain and spore germination testing. A series of biological changes, including a large number of substrates that were leaked out from the spores included proteins, DNA, polysaccharide, K⁺, and Ca²⁺, occurred a short time after N. bombycis spores were treated with ClO₂. In addition, the lipid of spores was disrupted and ATPase activity was inhibited, which resulted in the destruction of the inner structure of the spores.     

Spore Dimorphism in a Microsporidan Isolate*

A microsporidan isolate currently considered to represent a mixed infection of Nosema necatrix Kramer, 1965 and Thelohania diazoma Kramer, 1965 was subjected to cultivation in hosts held at various temperatures. The ratio of the Nosema (monospore) to the Thelohania (octospore) forms at these temperatures was found to vary from 1:1 at 16 C to 1:0 at 32 C. Isolation technics using mechanical, temperature and temporal methods separated monosporous from octosporous forms for inoculation purposes. However, microscopic examination of hosts receiving these inocula revealed the presence of both monospores and octospores. Electrophoretic analysis of monospores and monospore-octospore mixtures indicated equivalent hydrophobic protein spectra. These observations suggest that this isolate has the ability to produce either single spores or spores in groups of eight. This microsporidan was not considered a member of the genus Stempellia since spores in groups of 2 or 4 were not observed. Retention of the name Nosema necatrix Kramer is suggested.     

Impact of Vairimorpha necatrix and Vairimorpha sp. (Microspora: Microsporida) on Bonnetia comta (Diptera: Tachinidae) within Agrotis ipsilon (Lepidoptera: Noctuidae) hosts

The effect of Vairimorpha necatrix and Vairimorpha sp. on Bonnetia comta developing within pathogen-treated black cutworm, Agrotis ipsilon, hosts was studied. Parasitism by B. comta did not interfere with the near 100% mortality of A. ipsilon caused by either Vairimorpha species. Both microsporidia decreased the number of B. comta able to pupate from a. ipsilon hosts, the days required for adult parasitoid eclosion, and the weights of the puparia. Only in the V. necatrix treatments, however, were these effects significant (P < 0.05) as determined by Duncan's (1955) multiple range test. The impact of each microsporidium on the parasitoid increased with an increase in the concentration of spores to which A. ipsilon was exposed. Both Vairimorpha species had a more detrimental effect on female B. comta than they did on male B. comta. Histological examination of B. comta maggots within V. necatrix- and Vairimorpha sp.-infected A. ipsilon showed the spores primarily restricted to the gut lumen. In older B. comta, dissected from dead Vairimorpha-infected hosts, the guts containing spores were much distended, some to the point that most other parasitoid tissues were absent. This suggests that the detrimental impact the host microsporidian infections had on the parasitoid were related to a nutritional deficiency caused by the accumulation of nondigestable spores in the parasitoid's gut lumen.     

A moderate level of hypovirulence conferred by a hypovirus in the avocado white root rot fungus,Rosellinia necatrix

Two isolates of Rosellinia necatrix (Rn118-8 and Rn480) have previously obtained from diseased avocado trees in commercial orchards of the coastal area in southern Spain. Rn118-8 and Rn480 have weak virulence on avocado plants, and are infected by R. necatrix hypovirus 2 (RnHV2). In this work, the possible biological effects of the hypovirus on R. necatrix were tested. First, RnHV2 was transmitted from each of Rn118-8 and Rn480 to a highly virulent, RnHV2-free isolate of R. necatrix (Rn400) through hyphal anastomosis, using zinc compounds which attenuate the mycelial incompatibility reactions and allow for horizontal virus transfer between vegetatively incompatible fungal strains. Next, we carried out an analysis of growth rate in vitro and a virulence test of these newly infected strains in avocado plants. We obtained five strains of Rn400 infected by RnHV2 after horizontal transmission, and showed some of them to have lower colony growth in vitro and lower virulence on avocado plants compared with virus-free Rn400. These results suggest that R. necatrix isolates infected by RnHV2 could be used as novel virocontrol agents to combat avocado white root rot.     

Nosema ceranae, a new parasite in Thai honeybees

Adult workers of Apis cerana, Apis florea and Apis mellifera from colonies heavily infected with Nosema ceranae were selected for molecular analyses of the parasite. PCR-specific 16S rRNA primers were designed, cloned, sequenced and compared to GenBank entries. The sequenced products corresponded to N. ceranae. We then infected A. cerana with N. ceranae spores isolated from A. florea workers. Newly emerged bees from healthy colonies were fed 10,000, 20,000 and 40,000 spores/bee. There were significant dosage dependent differences in bee infection and survival rates. The ratio of infected cells to non-infected cells increased at 6, 10 and 14 d post infection. In addition, hypopharyngeal glands of bees from the control group had significantly higher protein concentrations than infected groups. Bees infected with 40,000 spores/bee had the lowest protein concentrations. Thus, N. ceranae isolated from A. florea is capable of infecting another bee species, impairing hypopharyngeal gland protein production and reducing bee survival in A. cerana.     

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.     

Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee

The incidence of nosemosis has increased in recent years due to an emerging infestation of Nosema ceranae in managed honey bee populations in much of the world. A real-time PCR assay was developed to facilitate detection and quantification of both Nosema apis and N. ceranae in both single bee and pooled samples. The assay is a multiplexed reaction in which both species are detected and quantified in a single reaction. The assay is highly sensitive and can detect single copies of the target sequence. Real-time PCR results were calibrated to spore counts generated by standard microscopy procedures. The assay was used to assess bees from commercial apiaries sampled in November 2008 and March 2009. Bees from each colony were pooled. A large amount of variation among colonies was evident, signifying the need to examine large numbers of colonies. Due to sampling constraints, a subset of colonies (from five apiaries) was sampled in both seasons. In November, N. apis levels were 1212 ± 148 spores/bee and N. ceranae levels were 51,073 ± 31,155 spores/bee. In March, no N. apis was detected, N. ceranae levels were 11,824 ± 6304 spores/bee. Changes in N. ceranae levels were evident among apiaries, some increasing and other decreasing. This demonstrates the need for thorough sampling of apiaries and the need for a rapid test for both detection and quantification of both Nosema spp. This assay provides the opportunity for detailed study of disease resistance, infection kinetics, and improvement of disease management practices for honey bees.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

Sclerotization as a long-term preservation method for Rosellinia necatrix strains

This work describes a simple protocol for longterm preservation of strains of Rosellinia necatrix based on sclerotia production combined with storage at 4°C in liquid substrate, without affecting the growth and pathogenic characteristics of the fungal isolates recovered. The sclerotization process was set up in both liquid and solid media, and the sclerotia-like structures (pseudosclerotia) obtained were preserved in liquid media or water at 4°C. R. necatrix pseudosclerotia viability after 6 years of preservation at 4°C was confirmed by growth and microscopic characteristics, with no differences when compared with the fungal strains routinely preserved by periodic transfers. Additionally, pathogenicity on avocado plants by the preserved R. necatrix strains showed no difference from those preserved by periodic transfers. The albino strain used in this study should continue to be preserved by periodic subculturing.     

Estimation of the influence of selected products on co-infection with N. apis/N. ceranae in Apis mellifera using real-time PCR

To protect the world’s honey bee population many scientific centres are searching for products and methods that control nosemosis. Real-time PCR was used to assess infection level in worker bees infected with Nosema spp. in bee colonies co-infected with Nosema apis and Nosema ceranae after the administration of three products (Nozevit, ApiHerb and ApiX) and sugar syrup. The study was conducted in the field condition therefore there was no possibility to affect the number of spores in the selected material. The study demonstrated considerable differences in the number of spores of individual Nosema spp. in the analysed samples of bees. HSD Tukey’s test showed that the statistically significant effect on limiting the N. apis invasion had ApiX (p – 0.049). Nozevit, Apiherb and syrup showed no statistically significant effect on reducing the amount of N. apis spores. The same test showed that the statistically significant effect on limiting the N. ceranae invasion had: Nozevit (p – 0.014), Apiherb (p – 0.032), ApiX (p – 0.034) and syrup (p – 0.033). There was no statistically significant decrease in the N. ceranae spores in the control group.     

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.     

Factors controlling in vitro hatching of Vairimorpha plodiae (Microspora) spores and their infectivity to Plodia interpunctella,Heliothis virescens, and Pieris brassicae

Factors which influence the hatching of spores and proliferation of stages of the microsporidium Vairimorpha plodiae in two susceptible insects, Plodia interpunctella and Heliothis virescens, and one nonsusceptible insect, Pieris brassicae, were investigated. Spores hatched in 0.1 and 1 m KCl solutions when subjected to a change in pH, from pH 11 to pH 8. K⁺ was essential for hatching; NaCl solutions were not effective. Ca²⁺ and Mg²⁺ inhibited hatching, and calcium and magnesium chelating agents enhanced it. All three insect species had alkaline midgut contents and smooth, fragile peritrophic membranes. Spores hatched inside the midguts of all three insect species (P. interpunctella: maximum rate, 92.5%; H. virescens, 91.5%; P. brassicae, 82%). Sporoplasms were observed in the midgut epithelial and associated tracheole cells of P. brassicae. Both H. virescens and P. brassicae became infected when injected intrahemocoelically with spores.     

Identification and Isolation of Two Ascomycete Fungi from Spores of the Arbuscular Mycorrhizal Fungus Scutellospora castanea

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.     

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.