Pathogens of the oak processionary moth Thaumetopoea processionea: Developing a user-friendly bioassay system and metagenome analyses for microorganisms.

The oak processionary moth (OPM) Thaumetopoea processionea is a pest of oak trees and poses health risks to humans due to the urticating setae of later instar larvae. For this reason, it is difficult to rear OPM under laboratory conditions, carry out bioassays or examine larvae for pathogens. Biological control targets the early larval instars and is based primarily on commercial preparations of Bacillus thuringiensis ssp. kurstaki (Btk). To test the entomopathogenic potential of other spore-forming bacteria, a user-friendly bioassay system was developed that (i) applies bacterial spore suspensions by oak bud dipping, (ii) targets first instar larvae through feeding exposure and (iii) takes into account their group-feeding behavior. A negligible mortality in the untreated control proved the functionality of the newly established bioassay system. Whereas the commercial Btk HD-1 strain was used as a bioassay standard and confirmed as being highly efficient, a Bacillus wiedmannii strain was ineffective in killing OPM larvae. Larvae, which died during the infection experiment, were further subjected to Nanopore sequencing for a metagenomic approach for entomopathogen detection. It further corroborated that B.wiedmannii was not able to infect and establish in OPM, but identified potential insect pathogenic species from the genera Serratia and Pseudomonas.

Advancing pathogen surveillance by nanopore sequencing and genotype characterization of Acheta domesticus densovirus in mass-reared house crickets.

Rapid and reliable detection of pathogens is crucial to complement the growing industry of mass-reared insects, in order to safeguard the insect colonies from outbreak of diseases, which may cause significant economic loss. Current diagnostic methods are mainly based on conventional PCR and microscopic examination, requiring prior knowledge of disease symptoms and are limited to identifying known pathogens. Here, we present a rapid nanopore-based metagenomics approach for detecting entomopathogens from the European house cricket (Acheta domesticus). In this study, the Acheta domesticus densovirus (AdDV) was detected from diseased individuals using solely Nanopore sequencing. Virus reads and genome assemblies were obtained within twenty-four hours after sequencing. Subsequently, due to the length of the Nanopore reads, it was possible to reconstruct significantly large parts or even the entire AdDV genome to conduct studies for genotype identification. Variant analysis indicated the presence of three AdDV genotypes within the same house cricket population, with association to the vital status of the diseased crickets. This contrast provided compelling evidence for the existence of non-lethal AdDV genotypes. These findings demonstrated nanopore-based metagenomics sequencing as a powerful addition to the diagnostic tool kit for routine pathogen surveillance and diagnosis in the insect rearing industry.

A practical guide and Galaxy workflow to avoid inter-plasmidic repeat collapse and false gene loss in Unicycler's hybrid assemblies.

Generating complete, high-quality genome assemblies is key for any downstream analysis, such as comparative genomics. For bacterial genome assembly, various algorithms and fully automated pipelines exist, which are free-of-charge and easily accessible. However, these assembly tools often cannot unambiguously resolve a bacterial genome, for example due to the presence of sequence repeat structures on the chromosome or on plasmids. Then, a more sophisticated approach and/or manual curation is needed. Such modifications can be challenging, especially for non-bioinformaticians, because they are generally not considered as a straightforward process. In this study, we propose a standardized approach for manual genome completion focusing on the popular hybrid assembly pipeline Unicycler. The provided Galaxy workflow addresses two weaknesses in Unicycler's hybrid assemblies: (i) collapse of inter-plasmidic repeats and (ii) false loss of single-copy sequences. To demonstrate and validate how to detect and resolve these assembly errors, we use two genomes from the Bacillus cereus group. By applying the proposed pipeline following an automated assembly, the genome sequence quality can be significantly improved.

Elucidating the genomic history of commercially used Bacillus thuringiensis subsp. tenebrionis strain NB176.

Bacillus thuringiensis subsp. tenebrionis (Btt) produces a coleopteran-specific crystal protoxin protein (Cry3Aa δ-endotoxin). After its discovery in 1982, the strain NB125 (DSM 5526) was eventually registered in 1990 to control the Colorado potato beetle (Leptinotarsa decemlineata). Gamma-irradiation of NB125 resulted in strain NB176-1 (DSM 5480) that exhibited higher cry3Aa production and became the active ingredient of the plant protection product Novodor® FC. Here, we report a comparative genome analysis of the parental strain NB125, its derivative NB176-1 and the current commercial production strain NB176. The entire genome sequences of the parental and derivative strains were deciphered by a hybrid de novo approach using short (Illumina) and long (Nanopore) read sequencing techniques. Genome assembly revealed a chromosome of 5.4 to 5.6 Mbp and six plasmids with a size range from 14.9 to 250.5 kbp for each strain. The major differences among the original NB125 and the derivative strains NB176-1 and NB176 were an additional copy of the cry3Aa gene, which translocated to another plasmid as well as a chromosomal deletion (~ 178 kbp) in NB176. The assembled genome sequences were further analyzed in silico for the presence of virulence and antimicrobial resistance (AMR) genes.

Identification of a new nucleopolyhedrovirus isolated from the olive leaf moth, Palpita vitrealis, from two locations in Egypt.

The olive leaf moth (jasmine moth), Palpita vitrealis (Lepidoptera: Crambidae), is an important insect pest of olives in several Mediterranean countries. A new alphabaculovirus was isolated from diseased larvae of P. vitrealis in Egypt, first in Giza in spring 2005 and again in Marsa Matrouh in 2019.The larvae exhibited typical symptoms of a baculovirus infection. Light and scanning electron microscopy studies revealed polyhedral occlusion bodies. Transmission electron microscopy of ultrathin sections of purified OBs revealed virions with multiple embedded nucleocapsids. The identity of the two virus isolates was confirmed by sequencing the partial polyhedrin and lef-8 genes, and sequence comparison suggested a relationship to group I alphabaculoviruses. Therefore, this virus was termed Palpita vitrealis nucleopolyhedrovirus (PaviNPV). Whole genome sequencing of the PaviNPV isolate from Giza (Gz05) revealed a genome of 117,533 bp, 131 open reading frames (ORFs) and four homologous repeat (hr) regions. Phylogenetic reconstruction and genetic distance analyses using 38 core genes indicated that PaviNPV should be considered to belong to a novel species within the genus Alphabaculovirus. In bioassays, PaviNPV was highly virulent against second-instar larvae of P. vitrealis. The study reports a novel baculovirus that might have potential as a biological control agent of the olive leaf moth.

Cross-Resistance of the Codling Moth against Different Isolates of Cydia pomonella Granulovirus Is Caused by Two Different but Genetically Linked Resistance Mechanisms.

Cydia pomonella granulovirus (CpGV) is a widely used biological control agent of the codling moth. Recently, however, the codling moth has developed different types of field resistance against CpGV isolates. Whereas type I resistance is Z chromosomal inherited and targeted at the viral gene pe38 of isolate CpGV-M, type II resistance is autosomal inherited and targeted against isolates CpGV-M and CpGV-S. Here, we report that mixtures of CpGV-M and CpGV-S fail to break type II resistance and is expressed at all larval stages. Budded virus (BV) injection experiments circumventing initial midgut infection provided evidence that resistance against CpGV-S is midgut-related, though fluorescence dequenching assay using rhodamine-18 labeled occlusion derived viruses (ODV) could not fully elucidate whether the receptor binding or an intracellular midgut factor is involved. From our peroral and intra-hemocoel infection experiments, we conclude that two different (but genetically linked) resistance mechanisms are responsible for type II resistance in the codling moth: resistance against CpGV-M is systemic whereas a second and/or additional resistance mechanism against CpGV-S is located in the midgut of CpR5M larvae.

Infection effects of the new microsporidian species Tubulinosema suzukii on its host Drosophila suzukii.

Microsporidian infections of insects are important natural constraints of population growth, often reducing lifespan, fecundity and fertility of the infected host. The recently discovered Tubulinosema suzukii infects Drosophila suzukii (spotted wing drosophila, SWD), an invasive pest of many fruit crops in North America and Europe. In laboratory tests, fitness effects on larval and adult stages were explored. High level infection after larval treatment caused up to 70% pupal mortality, a decreased lifespan and a 70% reduced oviposition of emerging adults in biparental infection clusters. A shift to higher proportion of female offspring compared to controls suggested a potential parthenogenetic effect after microsporidian infection. A clear sex-linkage of effects was noted; females were specifically impaired, as concluded from fecundity tests with only infected female parents. Additive effects were noted when both parental sexes were infected, whereas least effects were found with only infected male parents, though survival of males was most negatively affected if they were fed with T. suzukii spores in the adult stage. Although most negative effects on fitness parameters were revealed after larval treatment, infection of offspring was never higher than 4%, suggesting limited vertical transmission. For that reason, a self-reliant spread in natural SWD populations would probably only occur by spore release from cadavers or frass.

Molecular and morphological characterisation of a novel microsporidian species, Tubulinosema suzukii, infecting Drosophila suzukii (Diptera: Drosophilidae).

A microsporidium showing morphological characteristics typical of a Tubulinosema species was discovered in Drosophila suzukii. All developmental stages were diplokaryotic and grew in direct contact with the host cell cytoplasm. Spores from fresh preparations were ovoid to slightly pyriform and measured 4.29 × 2.47 µm in wet mount preparations. The spore wall consisted of a 125 nm thick endospore covered by a double layered exospore of 39 nm and 18 nm. The polar filament measured 67 µm in length, was slightly anisofilar and was arranged in ten coils in one or rarely two rows. The two posterior coils were 95 nm in diameter while the anterior coils were 115 nm in diameter. Early developmental stages were surrounded by electron-dense, 35.3 nm diameter, surface ornaments scattered over the membrane. Tubular elements with diameters of approximately 75 nm were seen attaching to the periphery of meronts and sporonts. Tissues infected included fat body, midgut and muscle. A 1915 bp rDNA fragment, covering the small subunit (SSU), the internal transcribed spacer (ITS) and the 5' end of the large subunit ribosomal DNA, was amplified by PCR and sequenced. Phylogenetic analyses of the SSU rDNA fragment revealed closest relationship to Tubulinosema pampeana (Host: Bombus atratus, South America) and Tubulinosema loxostegi (Host: Loxostege sticticalis, ubiquitous), but using the complete dataset of SSU-ITS-LSU rDNA genes revealed T. hippodamiae (Host: Hippodamiae convergens) as the most closely related species. Based on the morphological and genetic features a new species, Tubulinosema suzukii sp. nov., is proposed for this microsporidium isolated from D. suzukii.

Interaction of Phthorimaea operculella granulovirus with a Nosema sp. microsporidium in larvae of Phthorimaea operculella.

An antagonistic effect of a microsporidium (Nosema sp.) infection on the virulence of Phthorimaea operculella granulovirus (PhopGV) was recorded in potato tuber moth (Phthorimaea operculella) larvae with mixed infections. When the P. operculella colony was infected at a high rate (42.8-100%) with the microsporidium, it was less susceptible to the isolate PhopGV-GR1.1. A virus concentration 1.89 × 105 higher was necessary to cause the same level of mortality produced in the P. operculella colony when it was uninfected or had a low level of infection with the microsporidium (0-30%). This antagonistic effect was driven by a Nosema isolate (termed Nosema sp. Phop) that was purified from microsporidian-infected P. operculella individuals. The purified microsporidium was characterised by morphological features, including size, filament coils and different developmental stages using transmission electron microscopy (TEM). On the molecular level, the partial cistron rDNA information of the small ribosomal subunit (SSU), internal transcribed spacer (ITS), and the large ribosomal subunit (LSU) were identified. Phylogenetic analyses revealed that the newly described microsporidium belongs to the "true Nosema" clade. Partial sequence information of the RNA polymerase II largest subunit (RPB1) suggested that Nosema bombycis is the closest relative (98% identity). The morphological and phylogenetic characteristics suggest that it is an isolate of N. bombycis. Interactions of microsporidia and betabaculoviruses are rarely described in the literature, although mixed infections of different pathogens seem to be rather common events, ranging from antagonistic to mutualistic interactions. The observed antagonistic relationship between the Nosema sp. and PhopGV-GR1.1 showed that pathogen interactions need to be considered when single pathogens are applied to insect populations in the context of biological control of insect pests.

Persistence of root-colonizing Pseudomonas protegens in herbivorous insects throughout different developmental stages and dispersal to new host plants.

The discovery of insecticidal activity in root-colonizing pseudomonads, best-known for their plant-beneficial effects, raised fundamental questions about the ecological relevance of insects as alternative hosts for these bacteria. Since soil bacteria are limited in their inherent abilities of dispersal, insects as vectors might be welcome vehicles to overcome large distances. Here, we report on the transmission of the root-colonizing, plant-beneficial and insecticidal bacterium Pseudomonas protegens CHA0 from root to root by the cabbage root fly, Delia radicum. Following ingestion by root-feeding D. radicum larvae, CHA0 persisted inside the insect until the pupal and adult stages. The emerging flies were then able to transmit CHA0 to a new plant host initiating bacterial colonization of the roots. CHA0 did not reduce root damages caused by D. radicum and had only small effects on Delia development suggesting a rather commensal than pathogenic relationship. Interestingly, when the bacterium was fed to two highly susceptible lepidopteran species, most of the insects died, but CHA0 could persist throughout different life stages in surviving individuals. In summary, this study investigated for the first time the interaction of P. protegens CHA0 and related strains with an insect present in their rhizosphere habitat. Our results suggest that plant-colonizing pseudomonads have different strategies for interaction with insects. They either cause lethal infections and use insects as food source or they live inside insect hosts without causing obvious damages and might use insects as vectors for dispersal, which implies a greater ecological versatility of these bacteria than previously thought.

Electron Microscopy Methods for Virus Diagnosis and High Resolution Analysis of Viruses.

The term "virosphere" describes both the space where viruses are found and the space they influence, and can extend to their impact on the environment, highlighting the complexity of the interactions involved. Studying the biology of viruses and the etiology of virus disease is crucial to the prevention of viral disease, efficient and reliable virus diagnosis, and virus control. Electron microscopy (EM) is an essential tool in the detection and analysis of virus replication. New EM methods and ongoing technical improvements offer a broad spectrum of applications, allowing in-depth investigation of viral impact on not only the host but also the environment. Indeed, using the most up-to-date electron cryomicroscopy methods, such investigations are now close to atomic resolution. In combination with bioinformatics, the transition from 2D imaging to 3D remodeling allows structural and functional analyses that extend and augment our knowledge of the astonishing diversity in virus structure and lifestyle. In combination with confocal laser scanning microscopy, EM enables live imaging of cells and tissues with high-resolution analysis. Here, we describe the pivotal role played by EM in the study of viruses, from structural analysis to the biological relevance of the viral metagenome (virome).

The genome sequence of Agrotis segetum granulovirus, isolate AgseGV-DA, reveals a new Betabaculovirus species of a slow killing granulovirus.

The European isolate Agrotis segetum granulovirus DA (AgseGV-DA) is a slow killing, type I granulovirus due to low dose-mortality responses within seven days post infection and a tissue tropism of infection restricted solely to the fat body of infected Agrotis segetum host larvae. The genome of AgseGV-DA was completely sequenced and compared to the whole genome sequences of the Chinese isolates AgseGV-XJ and AgseGV-L1. All three isolates share highly conserved genomes. The AgseGV-DA genome is 131,557bp in length and encodes for 149 putative open reading frames, including 37 baculovirus core genes and the per os infectivity factor ac110. Comprehensive investigations of repeat regions identified one putative non-hr like origin of replication in AgseGV-DA. Phylogenetic analysis based on concatenated amino acid alignments of 37 baculovirus core genes as well as pairwise distances based on the nucleotide alignments of partial granulin, lef-8 and lef-9 sequences with deposited betabaculoviruses confirmed AgseGV-DA, AgseGV-XJ and AgseGV-L1 as representative isolates of the same Betabaculovirus species. AgseGV encodes for a distinct putative enhancin, distantly related to enhancins from other granuloviruses.

Metschnikowia cf. typographi and other pathogens from the bark beetle Ips sexdentatus - Prevalence, histological and ultrastructural evidence, and molecular characterization.

Ips sexdentatus (six-spined engraver beetle) from Austria and Poland were dissected and examined for the presence of pathogens. Specimens collected in Austria were found to contain the ascomycetous fungus Metschnikowia cf. typographi. Infection rates ranged from 3.6% to 26.8% at different collection sites. M. cf. typographi infected midguts were investigated by histological, ultrastructural and molecular techniques. Extraordinary ultrastructural details are shown, such as ascospores with bilateral flattened flanks resembling alar rims at both sides of their attenuating tube-like ends. These have not yet been described in other yeast species. Molecular investigations showed a close phylogenetic relationship to the fungi Metschnikowia agaves and Candida wancherniae. Presence of the entomopathogenic fungus Beauveria bassiana found in Austria was confirmed both morphologically and molecularly. The eugregarine Gregarina typographi was diagnosed most frequently. Infection rates of all I. sexdentatus specimens ranged from 21.4% to 71.9% in Austria and 54.1% to 68.8% in Poland. Other entomopathogenic protists, bacteria, or viruses were not detected.

Ultrastructural characterization and multilocus sequence analysis (MLSA) of 'Candidatus Rickettsiella isopodorum', a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda).

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation 'Candidatus Rickettsiella isopodorum' is introduced for this new lineage of isopod-associated Rickettsiella bacteria.

On the susceptibility of the box tree moth Cydalima perspectalis to Anagrapha falcifera nucleopolyhedrovirus (AnfaNPV).

The box tree moth Cydalima perspectalis is an invasive insect pest in many European countries. Caterpillars of this species cause widespread damage on box tree plants. In this study, a new opportunity to control this pest with the baculovirus Anagrapha falcifera nucleopolyhedrovirus (AnfaNPV) was investigated. Initially, AnfaNPV was identified to infect larvae of Diaphania nitidalis by determining the partial nucleotide sequence of the three highly conserved genes polh, lef-8 and lef-9 of the infection causing agent. Two AnfaNPV isolates, termed Dn10 and BI-235, were then propagated in larvae of C. perspectalis and purified by sucrose density centrifugation. A bioassay using leaf disks of box tree was established to evaluate the virulence of both AnfaNPV isolates to neonate C. perspectalis larvae. After 7days, the mortality of larvae was scored and median lethal concentrations (LC50) were determined to 7.8×10(5)OBs/ml for isolate BI-235 and 2.3×10(6)OBs/ml for isolate Dn10 by using probit analysis. Thus, AnfaNPV BI-235 was significantly more virulent to neonate C. perspectalis larvae than Dn10 based on a three times higher LC50 value. Additionally, light and transmission electron microscopic investigations verified high rates of infection in fat body, epidermis and tracheal matrix of C. perspectalis by both AnfaNPV isolates BI-235 and Dn10. In conclusion, the performed laboratory experiments indicate the susceptibility of C. perspectalis to AnfaNPV.

Multilocus sequence analysis (MLSA) of 'Rickettsiella agriotidis', an intracellular bacterial pathogen of Agriotes wireworms.

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests that affect numerous crops globally. A new bacterial specimen identified in diseased wireworms had previously been shown by microscopy and 16S ribosomal RNA (rRNA) gene-based phylogenetic reconstruction to belong to the taxonomic genus Rickettsiella (Gammaproteobacteria) that comprises intracellular bacteria associated with and typically pathogenic for a wide range of arthropods. Going beyond these earlier results obtained from rRNA phylogenies, multilocus sequence analysis (MLSA) using a four marker scheme has been employed in the molecular taxonomic characterization of the new Rickettsiella pathotype, referred to as 'Rickettsiella agriotidis'. In combination with likelihood-based significance testing, the MLSA approach demonstrated the close phylogenetic relationship of 'R. agriotidis' to the pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae', i.e., subjective synonyms of the nomenclatural type species, Rickettsiella popilliae. 'R. agriotidis' forms, therefore, part of a Rickettsiella pathotype complex that most likely represents the species R. popilliae. As there are currently no genetic data available from the R. popilliae type strain, the respective assignment cannot be corroborated directly. However, an alternative taxonomic assignment to the species Rickettsiella grylli has been positively ruled out by significance testing. MLSA has been shown to provide a more powerful tool for taxonomic delineation within the genus Rickettsiella as compared to 16S rRNA phylogenetics. However, the limitations of the present MLSA scheme for the sub-species level classification of 'R. agriotidis' and further R. popilliae synonyms has been critically evaluated.

Occurrence and Prevalence of Insect Pathogens in Populations of the Codling Moth, Cydia pomonella L.: A Long-Term Diagnostic Survey.

About 20,550 larvae, pupae and adults of the codling moth, Cydia pomonella L., were diagnosed for pathogens during long-term investigations (1955-2012) at the Institute for Biological Control in Darmstadt, Germany. The prevailing entomopathogens diagnosed in these studies were insect pathogenic fungi, especially Beauveria bassiana and Isaria farinosa, the microsporidium, Nosema carpocapsae, the Cydia pomonella granulovirus (CpGV), as well as mostly undetermined bacteria. While the CpGV was observed exclusively in larvae and pupae from laboratory colonies or from field experiments with this virus, entomopathogenic fungi were most frequently diagnosed in last instars in autumn and in diapausing larvae and pupae in spring. B. bassiana was identified as the major fungal pathogen, causing larval prevalences of 0.9% to 100% (mean, about 32%). During prognostic long-term studies in larvae and adults of C. pomonella, N. carpocapsae was diagnosed in codling moth populations from various locations in Germany. The mean prevalence generally ranged between 20% and 50%. Experiments revealed that the fecundity and fertility of microsporidia-infected female adults were significantly reduced compared to healthy ones. The results underpin the importance of naturally occurring microbial antagonists and represent a base for further ecological studies on developing new or additional biological and integrated control strategies.

A Rickettsiella bacterium from the hard tick, Ixodes woodi: molecular taxonomy combining multilocus sequence typing (MLST) with significance testing.

Hard ticks (Acari: Ixodidae) are known to harbour intracellular bacteria from several phylogenetic groups that can develop both mutualistic and pathogenic relationships to the host. This is of particular importance for public health as tick derived bacteria can potentially be transmitted to mammals, including humans, where e.g. Rickettsia or Coxiella act as severe pathogens. Exact molecular taxonomic identification of tick associated prokaryotes is a necessary prerequisite of the investigation of their relationship to both the tick and possible vertebrate hosts. Previously, an intracellular bacterium had been isolated from a monosexual, parthenogenetically reproducing laboratory colony of females of the hard tick, Ixodes woodi Bishopp, and had preliminarily been characterized as a "Rickettsiella-related bacterium". In the present molecular taxonomic study that is based on phylogenetic reconstruction from both 16 S ribosomal RNA and protein-encoding marker sequences complemented with likelihood-based significance testing, the bacterium from I. woodi has been identified as a strain of the taxonomic species Rickettsiella grylli. It is the first time that a multilocus sequence typing (MLST) approach based on a four genes comprising MLST scheme has been implemented in order to classify a Rickettsiella-like bacterium to this species. The study demonstrated that MLST holds potential for a better resolution of phylogenetic relationships within the genus Rickettsiella, but requires sequence determination from further Rickettsiella-like bacteria in order to complete the current still fragmentary picture of Rickettsiella systematics.

Multilocus sequence typing (MLST) for the infra-generic taxonomic classification of entomopathogenic Rickettsiella bacteria.

The genus Rickettsiella comprises intracellular bacterial pathogens of a wide range of arthropods that are currently classified in four recognized species and numerous further pathotypes. However, both the delineation of and the synonymization of pathotypes with species are highly problematic. In the sequel of a previous phylogenomic study at the supra-generic level, nine selected genes - the 16S and 23S rRNA genes and the protein-encoding genes dnaG, ftsY, gidA, ksgA, rpoB, rpsA, and sucB - were evaluated for their potential as markers for the generic and infra-generic taxonomic classification of Rickettsiella-like bacteria. A methodological approach combining phylogenetic reconstruction with likelihood-based significance testing was employed on the basis of sequence data from the species Rickettsiella grylli and Rickettsiella popilliae, pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae'. This study provides the first multilocus sequence typing (MLST) data for the genus Rickettsiella and identifies two new genetic markers, gidA and sucB, for the infra-generic classification within this taxon. In particular, aforesaid genes were found more reliable and informative markers than the corresponding 16S rRNA-encoding sequences that failed to produce strictly significant infra-generic taxonomic assignments. However, gidA- and sucB-based phylogenies were consistent with the currently accepted view of species delineation and species-pathotype synonymization within the genus Rickettsiella.

Genetic and electron-microscopic characterization of Rickettsiella bacteria from the manuka beetle, Pyronota setosa (Coleoptera: Scarabaeidae).

Larvae of manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae) cause pasture damage in New Zealand by feeding on the roots of grasses. Surveys for potential biocontrol agents revealed a putative disease, expressed as whitened larvae of one of the outbreak species, Pyronota setosa. Microbial diagnosis indicated an intracoelomic, intracellular infection, and intracellular bacteria have been identified with subcellular structures characteristic of infection by Rickettsiella-like microorganisms. These bacteria were rod-shaped, often slightly bent with a mean of 628 nm in length and 220 nm in width. Numerous associated protein crystals of variable size and shape occurred within round to oval shaped "giant bodies" either singly or as clusters of smaller crystals. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the manuka beetle pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the pathotype designation 'Rickettsiella pyronotae' is proposed to refer to this organism. Moreover, genetic analysis makes it likely that--on the basis of the currently accepted organization of the genus Rickettsiella--this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.