A molecular tool for detection and tracking of a potential indigenous Beauveria bassiana strain for managing emerald ash borer populations in Canada.

Emerald ash borer is an invasive species from Asia. Beauveria bassiana strain L49-1AA is being tested for the control of emerald ash borer in Canada, using an autocontamination trapping system. We have developed a simplified allele discrimination polymerase chain reaction (PCR) assay to screen B. bassiana strain, L49-1AA from other Beauveria species by targeting the inter-strain genetic differences in 5' end of EF1-α gene of the genus Beauveria. A single nucleotide polymorphism (SNP) site, T→C was identified only in L49-1AA and was used to develop a simplified allele discrimination polymerase chain reaction (PCR) assay based on a modified allelic inhibition of displacement activity (AIDA) approach for distinguishing B. bassiana L49-1AA from all background Beauveria isolates. The SNP site was employed to design inner primers but with a deliberate mismatch introduced at the 3' antepenultimate from the mutation site in order to maximize specificity and detection efficiency. Amplification was specific to L49-1AA without cross-reaction with DNA from other Beauveria strains. In addition, the designed primers were also tested against environmental samples in L49-1AA released plots and observed to be highly efficient in detecting and discriminating the target strain, L49-1AA from both pure and crude DNA samples. This new method can potentially allow for more discriminatory tracking and monitoring of released L49-1AA in our autocontamination and dissemination projects for managing EAB populations. Additionally, the modified-AIDA format has potential as a tool for simultaneously identifying and differentiating closely related Beauveria species, strains/isolates as well as general classification of other pathogens or organisms.

Comment on "Invasive harlequin ladybird carries biological weapons against native competitors".

Conclusions about the nontarget effects of putatively invasive pathogens should be based on biologically relevant data. We disagree that the research experiments on a microsporidium isolated from Harmonia axyridis conducted by Vilcinskas et al. (Reports, 17 May 2013, p. 862) can explain the decline of native coccinellid species in the absence of such data.

Characterization and virulence of Beauveria spp. recovered from emerald ash borer in southwestern Ontario, Canada.

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is an invasive wood boring beetle that is decimating North America's ash trees (Fraxinus spp.). To find effective and safe indigenous biocontrol agents to manage EAB, we conducted a survey in 2008-2009 of entomopathogenic fungi (EPF) infecting EAB in five outbreak sites in southwestern Ontario, Canada. A total of 78 Beauveria spp. isolates were retrieved from dead and mycosed EAB cadavers residing in the phloem tissues of dead ash barks, larval frass extracted from feeding galleries under the bark of dead trees. Molecular characterization using sequences of the ITS, 5' end of EF1-α and intergenic Bloc region fragments revealed that Beauveria bassiana and Beauveria pseudobassiana were commonly associated with EAB in the sampled sites. Based on phylogenetic analysis inferred from ITS sequences, 17 of these isolates clustered with B. bassiana, which further grouped into three different sub-clades. However, the combined EF1-α and Bloc sequences detected five genotypes among the three sub-clades. The remaining 61 isolates clustered with B. pseudobassiana, which had identical ITS sequences but were further subdivided into two genotypes by variation in the EF1-α and Bloc regions. Initial virulence screening against EAB adults of 23 isolates representing the different clades yielded 8 that produced more than 90% mortality in a single concentration assay. These isolates differed in virulence based on LC(50) values estimated from multiple concentration bioassay and based on mean survival times at a conidia concentration of 2×10(6) conidia/ml. B. bassiana isolate L49-1AA was significantly more virulent and produced more conidia on EAB cadavers compared to the other indigenous isolates and the commercial strain B. bassiana GHA suggesting that L49-1AA may have potential as a microbiological control agent against EAB.

Towards the development of an autocontamination trap system to manage populations of emerald ash borer (Coleoptera: Buprestidae) with the native entomopathogenic fungus, Beauveria bassiana.

Emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) is an invasive species from Asia that was discovered in North America Canada, in 2002. Herein, we describe studies to develop an autocontamination trapping system to disseminate Beauveria bassiana to control beetle populations. The standard trap for emerald ash borer in Canada is a light green prism trap covered in an insect adhesive and baited with (Z)-3-hexenol. We compared of green multifunnel traps, green intercept panel traps (both with and without fluon coating) and green prism traps for capturing emerald ash borer in a green ash plantation. The coated green multifunnel traps captured significantly more males and more females than any other trap design. We examined the efficacy of two native B. bassiana isolates, INRS-CFL and L49-1AA. In a field experiment the INRS-CFL isolate attached to multifunnel traps in autocontamination chambers retained its pathogenicity to emerald ash borer adults for up to 43 d of outdoor exposure. Conidia germination of the INRS-CFL isolate was >69% after outdoor exposure in the traps for up to 57 d. The L49-1AA isolate was not pathogenic in simulated trap exposures and the germination rate was extremely low (<5.3%). Mean (+/- SEM) conidia loads on ash borer adults after being autocontaminated in the laboratory using pouches that had been exposed in traps out of doors for 29 d were 579,200 (+/- 86,181) and 2,400 (+/- 681) for the INRS-CFL and the LA9-1AA isolates, respectively. We also examined the fungal dissemination process under field conditions using the L49-1AA isolate in a green ash plantation. Beetles were lured to baited green multifunnel traps with attached autocontamination chambers. Beetles acquired fungal conidia from cultures growing on pouches in the chambers and were recaptured on Pestick-coated traps. In total, 2,532 beetles were captured of which 165 (6.5%) had fungal growth that resembled B. bassiana. Of these 25 beetles were positive for the L49-1AA isolate.

A new Encephalitozoon species (Microsporidia) isolated from the lubber grasshopper, Romalea microptera (Beauvois) (Orthoptera: Romaleidae).

We describe a new microsporidian species, Encephalitozoon romaleae n. sp., isolated from an invertebrate host, the grasshopper Romalea microptera, collected near Weeks Island, Louisiana, and Jacksonville, Florida. This microsporidian is characterized by specificity to the gastric caecae and midgut tissues of the host and a life cycle that is nearly identical to that of Encephalitozoon hellem and Encephalitozoon cuniculi. Mature spores are larger (3.97 x 1.95 microm) than those of other Encephalitozoon species. Polar filament coils number 7 to 8 in a single row. Analysis of the small subunit (SSU) rDNA shows that E. romaleae fits well into the Encephalitozoon group and is a sister taxon to E. hellem. This is the first Encephalitozoon species that has been shown to complete its life cycle in an invertebrate host.

Phylogenetic characterization of Encephalitozoon romaleae (Microsporidia) from a grasshopper host: relationship to Encephalitozoon spp. infecting humans.

Encephalitozoon species are the most common microsporidian pathogens of humans and domesticated animals. We recently discovered a new microsporidium, Encephalitozoon romaleae, infecting the eastern lubber grasshopper Romalea microptera. To understand its evolutionary relationships, we compared partial gene sequences of alpha- and beta-tubulin and methionine aminopeptidase 2 enzyme from this and related species. We also analyzed the rRNA internal transcribed spacer. Based on tubulin and MetAP-2 gene phylogenetic analysis, E. romaleae clustered with the Encephalitzoon group with strong bootstrap support (>99%). Within the Encephalitozoon clade, E. romaleae clustered with Encephalitozoon hellem for both the beta-tubulin and MetAP-2 phylogenies based on ML tree. The alpha-tubulin based ML tree, however, placed the new microsporidium closer to Encephalitozoon cuniculi. The rRNA internal transcribed spacer region of E. romaleae has 91% homology with E. hellem.

Testing intra-hemocelic injection of antimicrobials against Encephalitozoon sp. (Microsporidia) in an insect host.

Encephalitozoon spp. are the primary microsporidial pathogens of humans and domesticated animals. In this experiment, we test the efficacy of four commercial antimicrobials against an Encephalitozoon sp. in an insect host by intra-hemocelic injection. All four antimicrobials, viz., thiabendazole, quinine, albendazole, and fumagillin, significantly reduced but did not eliminate microsporidia spore counts in the grasshopper host. Among these four drugs, thiabendazole was most effective in reducing the microsporidia spore level up to 90%, followed by quinine (70%), albendazole (62%), and fumagillin (59%). No control or quinine-treated animals died, whereas 45% of albendazole animals died. Despite the high mortality induced by albendazole, this drug significantly reduced spore counts, a result not seen in previous per os trials. Among the treatment groups, grasshoppers injected with thiabendazole lost a significant mass. Our study suggests that quinine and related alkaloids should be further examined for antimicrosporidial activity.

Effect of four antimicrobials against an Encephalitozoon sp. (Microsporidia) in a grasshopper host.

Encephalitozoon spp. are the primary microsporidial pathogens of humans and domesticated animals. In this experiment, we test the efficacy of 4 commercial antimicrobials against an Encephalitozoon sp. infecting a grasshopper (Romalea microptera) host. Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts (93% and 88% respectively), whereas spore counts of grasshoppers fed quinine produced a non-significant 53% reduction in spores, and those fed streptomycin a non-significant 29% increase in spores, compared to the control. We observed a moderate dose-response effect for thiabendazole, whereby spore count decreased as drug consumption increased. No thiabendazole-treated animals died, whereas 27% of streptomycin-treated animals died, suggesting that thiabendazole was not toxic at the doses administered. The deaths among streptomycin-treated animals may have been caused by drug toxicity, parasite burden, or both. Although fumagillin and thiabendazole significantly reduced spore counts, in no individual was the pathogen totally eliminated. Our data confirm that microsporidia are difficult to control and that fumagillin and thiabendazole are partially effective antimicrobials against this group. Our study suggests that quinine and related alkaloids should be further examined for antimicrosporidial activity, and streptomycin should be examined as a possible enhancer of microsporidiosis.

Efficacy of eleven antimicrobials against a gregarine parasite (Apicomplexa: Protozoa).

BACKGROUND: The Apicomplexa are a diverse group of obligate protozoan parasites infesting a wide range of invertebrate and vertebrate hosts including humans. These parasites are notoriously difficult to control and many species continue to evolve resistance to commercial antibiotics. In this study, we sought to find an effective chemotherapeutic treatment against arthropod gregarines (Apicomplexa), and to identify candidate compounds for testing against other groups of protozoan parasites. METHODS: We tested eleven commercial antibiotics against a gregarine parasite of Romalea microptera grasshoppers. Infected insects were fed daily, lettuce containing known amounts of specific antibiotics. On Days 15 or 20, we measured the number of gregarines remaining in the digestive tract of each grasshopper. RESULTS: Treatment with metronidazole and griseofulvin in host insects significantly reduced gregarine counts, whereas, gregarine counts of insects fed, albendazole, ampicillin, chloramphenicol, fumagillin, quinine, streptomycin, sulfadimethoxine, thiabendazole or tetracycline, were not significantly different from the controls. However, albendazole produced a strong, but non-significant reduction in gregarine count, and streptomycin exhibited a non-significant antagonistic trend. CONCLUSION: Our results confirm that gregarine infections are difficult to control and suggest the possibility that streptomycin might aggravate gregarine infection. In addition, the insect system described here, provides a simple, inexpensive, and effective method for screening antibiotics.

New insect system for testing antibiotics.

New and efficient methods to screen antibiotics are needed to counter increased antibiotic resistance in pathogens and the emergence of new diseases. Here we report a new insect model for screening antibiotics in vivo using the grasshopper Romalea microptera. The system is inexpensive, efficient, and flexible, avoids animal-welfare problems, and can be used to test against most major pathogenic groups. We employed this system to test 11 commercial antibiotics against a pathogenic Encephalitozoon species (Microsporidia). Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts, whereas spore counts of grasshoppers fed with albendazole, ampicillin, chloramphenicol, griseofulvin, metronidazole, sulfadimethoxine, or tetracycline were not significantly different from the infected controls. Quinine produced a distinct, but nonsignificant, reduction in spores, and streptomycin a nonsignificant increase in spores. Although 2 antibiotics significantly reduced spore counts, in no case was the pathogen totally eliminated. This study demonstrates the validity of this system as a method to screen antibiotics. It also corroborates the difficulty researchers and physicians have had in treating microsporidia infections, and suggests that quinine and related alkaloid compounds should be further examined as possible therapeutic agents against this group of ubiquitous pathogens. In addition, streptomycin and related compounds should be tested to determine if this widely used antibiotic enhances microsporidiosis.