Morphological, ultrastructural, and molecular identification of a new microsporidian pathogen isolated from Crepidodera aurata (Coleoptera, Chrysomelidae)

A new microsporidian pathogen isolated from Crepidodera aurata was identified based on morphological and ultrastructural characteristics, coupled with a molecular phylogenetic analysis. The spores of the microsporidian pathogen were slightly curved in shape, and measured 2.44–3.55 μm in length and 1.25–1.55 μm in width (n = 50). Its ultrastructure is characteristic of monokaryotic groups. All lifecycle stages of the pathogen, including meronts, sporonts, sporoblasts, and mature spores, are monokaryotic. The spore has 6–8 windings of the polar filament. Morphological and ultrastructural characteristics of the lifecycle stages place it within the family Unikaryonidae. However, the phylogenetic tree constructed on the 16S rRNA gene sequence analysis indicates that the pathogen is closely related to the Nosema/Vairimorpha clade of microsporidia. Therefore, we have classified the microsporidian of C. aurata in the tentative group Microsporidium in order to avoid creating an unnecessary or incorrect new genus/species

New nephridiophagid genera (Fungi, Chytridiomycota) in a mallow beetle and an earwig

Nephridiophagids are unicellular fungi (Chytridiomycota) that infect the Malpighian tubules of insects. Most species have been found in cockroach hosts and belong to the genus Nephridiophaga. Three additional genera have been described from beetles and an earwig. Here, we characterise morphologically and molecular phylogenetically the nephridiophagids of the European earwig Forficula auricularia and the mallow beetle Podagrica malvae. Their morphology and life cycle stages resemble those of other nephridiophagids, but their rRNA gene sequences support the existence of two additional genera. Whereas the earwig nephridiophagid (Nephridiochytrium forficulae gen. nov. et sp. nov.) forms a sister lineage of the Nephridiophaga cluster, the mallow beetle nephridiophagid (Malpighivinco podagricae gen. nov. et sp. nov.) represents the earliest divergent lineage within the nephridiophagids, being sister to all other species. Our results corroborate the hypothesis that different insect groups harbour distinct nephridiophagid lineages

Identifizierung der endo- und ectosymbiontischen Bakterien und cellulolytischen Aktivitäten der symbiontischen Flagellaten der australischen Termite Mastotermes darwiniensis

The lower wood-feeding Australian termite Mastotermes darwiniensis Froggatt (Fig. 1) is the only living member of the family Mastotermitidae. The complex symbiotic hindgut flora consists of protozoa (formerly named Archaezoa; Cleveland & Grimstone 1964; Brugerolle & al. 1994; Berchtold & König 1995; Fröhlich & König 1999a, b), bacteria (Berchtold & König 1996; Berchtold & al. 1999), archaea (Fröhlich & König 1999a, b) and yeasts (Prillinger & al. 1996; Schäfer & al. 1996). The digestive system of Mastotermes darwiniensis consists of the foregut with the crop and the gizzard, the midgut, and the hindgut (Noirot & Noirot-Timothée 1969; 1995). The hindgut consists of five segments (P1 – P5): the proctodeal segment, the enteric valve, the paunch, the colon and the rectum. The paunch is the main microbial fermentation chamber, but the colon also contains microorganisms. The paunch is subdivided into a dilated thin-walled region (P3a) and a thick walled more tubular region (P3b) (Fig. 1c). In the case of Mastotermes darwiniensis oxygen diffusion gradients could be detected up to 100 μm below the epithelium (Berchtold & al., 1999).Termiten sind mit die wichtigsten holzabbauenden Insekten. Die Darmmikrobiota spielt eine unverzichtbare Rolle im Abbau der Nahrung. Sie besteht aus Bakterien, Archaebakterien (Archaea), Flagellaten und Hefen. Die einzigartigen Flagellaten der Termiten sind sehr früh in der Evolution der Eukaryoten abgezweigte Einzeller, die zu den Preaxostyla (Oxymonadida) und Parabasalia (Cristamonadida, Spirotrichonymphida, Trichomonadida, Trichonymphida) gehören. Die australische Termite Mastotermes darwiniensis ist die einzige heute noch lebende Art der primitiven Termitenfamilie Mastotermitidae. In der Gärkammer im Hinterdarm leben die vier größeren Flagellaten Koruga bonita, Deltotrichonympha nana, Deltotrichonympha operculata und Mixotricha paradoxa. Weiterhin kommen die zwei kleineren Flagellaten Metadevescovina extranea (Cristamonaden) und Pentatrichomonoides scroa (Trichomonaden) vor. Die Flagellaten selbst sind Wirte von ecto- und endosymbiontischen Prokaryoten. Von Zellextrakten der nichtkultivierbaren größeren Flagellaten wurden zwei Endoglucanasen mit einer ähnlichen apparenten Masse von 36 kD isoliert. Sie zeigten signifikante Homologie zu termiteneigenen Cellulasen. Die entsprechenden Gene wurden nicht im mRNA-Pool der Flagellaten gefunden, sondern in den Speicheldrüsen von Mastotermes darwiniensis. Das deutet darauf hin, dass die intestinalen Flagellaten auch die Wirtsenzyme für die Cellulosehydrolyse benutzten. Andererseits besitzen mindestens Koruga- und Deltotrichonympha-Species auch eigene Cellulasegene. Im Darminhalt der Termiten wurden allerdings auch drei Cellulasen nachgewiesen, die von den Flagellaten stammen sollten

Long rDNA amplicon sequencing of insect-infecting nephridiophagids reveals their affiliation to the Chytridiomycota and a potential to switch between hosts

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota—a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed

On the Enigmatic Hook of the Metaradiophryids (Alveolata, Ciliophora)

The astomatous metaradiophryids are ciliates which live endosymbiotically in earthworms (Annelida, Lumbricidae). Their prominent hook apparatus is demonstrated in detail in light micrographs of living organisms as well as in scanning electron micrographs of Parducz-fixed cells. Since it was first observed, this structure has been interpreted as a ‘holdfast’ organelle preventing ciliates from being expelled prematurely from the intestine of the worm along with its excrements. No active movement of the hook has been reported in earlier papers or in our recent studies. Nevertheless, a detailed description of different parts of the hook apparatus exists in older literature, including a hypothesis on how these elements interact with each other to function as a holdfast device – without any experimental evidence. The suspected mode of function of this structure is questioned and critically discussed

New insights into the coevolutionary history of termites and their gut flagellates: Description of Retractinympha glossotermitis gen. nov. sp. nov. (Retractinymphidae fam. nov.)

Lower termites harbor diverse consortia of symbiotic gut flagellates. Despite numerous evidence for co-cladogenesis, the evolutionary history of these associations remains unclear. Here, we present Retractinymphidae fam. nov., a monogeneric lineage of Trichonymphida from Serritermitidae. Although Retractinympha glossotermitis gen. nov. sp. nov. morphologically resembles members of the genus Pseudotrichonympha, phylogenetic analysis identified it as sister group of the Teranymphidae. We compared morphology and ultrastructure of R. glossotermitis to that of Pseudotrichonympha and other Teranymphidae, including the so-far undescribed Pseudotrichonympha solitaria sp. nov. from Termitogeton planus (Rhinotermitidae). Like all Teranymphidae, R. glossotermitis is a large, elongated flagellate with a bilaterally symmetric rostrum, an anterior, flagella-free operculum, and an internal rostral tube. However, it is readily distinguished by the length of its rostral flagella, which never exceeds that of the postrostral flagella, and its retractable anterior end. Inclusion of the hitherto unstudied Stylotermes halumicus (Stylotermitidae) in our survey of trichonymphid flagellates in Neoisoptera confirmed that the combined presence of Heliconympha and Retractinympha and absence of Pseudotrichonympha is unique to Serritermitidae. The close phylogenetic relatedness of Heliconympha in Serritermitidae to the spirotrichosomid flagellates in Stolotermitidae provides strong support for their acquisition by horizontal transmission

Nosema pieriae sp. n. (Microsporida, Nosematidae): A New Microsporidian Pathogen of the Cabbage Butterfly Pieris brassicae L. (Lepidoptera: Pieridae)

A new microsporidian pathogen of the cabbage butterfly, Pieris brassicae is described based on light microscopy, ultrastructural characteristics and comparative small subunit rDNA analysis. The pathogen infects the gut of P. brassicae. All development stages are in direct contact with the host cell cytoplasm. Meronts are spherical or ovoid. Spherical meronts measure 3.68 ± 0.73 × 3.32 ± 1.09 µm and ovoid meronts 4.04 ± 0.74 × 2.63 ± 0.49 µm. Sporonts are spherical to elongate (4.52 ± 0.48 × 2.16 ± 0.27 µm). Sporoblasts are elongated and measure 4.67 ± 0.60 × 2.30 ± 0.30 µm in length. Fresh spores with nuclei arranged in a diplokaryon are oval and measure 5.29 ± 0.55 µm in length and 2.31 ± 0.29 µm in width. Spores stained with Giemsa’s stain measure 4.21 ± 0.50 µm in length and 1.91 ± 0.24 µm in width. Spores have an isofilar polar filament with six coils. All morphological, ultrastructural and molecular features indicate that the described microsporidium belongs to the genus Nosema and confirm that it has different taxonomic characters than other microsporidia infecting Pieris spp

New insights into the coevolutionary history of termites and their gut flagellates: Description of Retractinympha glossotermitis gen. nov. sp. nov. (Retractinymphidae fam. nov.)

Lower termites harbor diverse consortia of symbiotic gut flagellates. Despite numerous evidence for co-cladogenesis, the evolutionary history of these associations remains unclear. Here, we present Retractinymphidae fam. nov., a monogeneric lineage of Trichonymphida from Serritermitidae. Although Retractinympha glossotermitis gen. nov. sp. nov. morphologically resembles members of the genus Pseudotrichonympha, phylogenetic analysis identified it as sister group of the Teranymphidae. We compared morphology and ultrastructure of R. glossotermitis to that of Pseudotrichonympha and other Teranymphidae, including the so-far undescribed Pseudotrichonympha solitaria sp. nov. from Termitogeton planus (Rhinotermitidae). Like all Teranymphidae, R. glossotermitis is a large, elongated flagellate with a bilaterally symmetric rostrum, an anterior, flagella-free operculum, and an internal rostral tube. However, it is readily distinguished by the length of its rostral flagella, which never exceeds that of the postrostral flagella, and its retractable anterior end. Inclusion of the hitherto unstudied Stylotermes halumicus (Stylotermitidae) in our survey of trichonymphid flagellates in Neoisoptera confirmed that the combined presence of Heliconympha and Retractinympha and absence of Pseudotrichonympha is unique to Serritermitidae. The close phylogenetic relatedness of Heliconympha in Serritermitidae to the spirotrichosomid flagellates in Stolotermitidae provides strong support for their acquisition by horizontal transmission

Defensive behavior is linked to altered surface chemistry following infection in a termite society

The care-kill response determines whether a sick individual will be treated or eliminated from an insect society, but little is known about the physiological underpinnings of this process. We exploited the stepwise infection dynamics of an entomopathogenic fungus in a termite to explore how care-kill transitions occur, and identify the chemical cues behind these shifts. We found collective responses towards pathogen-injected individuals to vary according to severity and timing of pathogen challenge, with elimination, via cannibalism, occurring sooner in response to a severe active infection. However, injection with inactivated fungal blastospores also resulted in increased albeit delayed cannibalism, even though it did not universally cause host death. This indicates that the decision to eliminate an individual is triggered before pathogen viability or terminal disease status has been established. We then compared the surface chemistry of differently challenged individuals, finding increased amounts of long-chained methyl-branched alkanes with similar branching patterns in individuals injected with both dead and viable fungal blastospores, with the latter showing the largest increase. This coincided with the highest amounts of observed cannibalism as well as signs of severe moribundity. Our study provides new mechanistic insight into the emergent collective behaviors involved in the disease defense of a termite society

Stress corrosion crack initiation testing with tapered specimens in high-temperature water – results of a collaborative research project

The applicability of an accelerated test technique using tapered tensile specimens for investigating thestress corrosion crack (SCC) initiation behaviour of structural materials in high-temperature water wasassessed in the framework of a European collaborative research project (MICRIN – MItigation of CRackINitiation). The main advantage of using a tapered geometry is, that in a single test a stress gradient isobtained through the gauge length, and therefore a stress threshold for SCC initiation can bedetermined in a reasonable timeframe. This method was used to investigate two different materialsthat were known to be susceptible to SCC in light water reactor environment: a high-Si stainlesssteel and a Ni-base weld metal (Alloy 182). The results of the international test programmeconfirmed that the tapered specimen test methodology could be used to identify a SCC initiation stress threshold, albeit that significant scatter was present in the data