The Impact of a Cypovirus on Parental and Filial Generations of Lymantria dispar L.

Recently, we found that the spongy moth Lymantria dispar L. is susceptible to infection by a Dendrolimus sibiricus cytoplasmic polyhedrosis virus (DsCPV-1). In the present study, we evaluated the pathogenicity of DsCPV-1 against L. dispar larvae and its impact on surviving insects after the infection. Offspring of virally challenged insects were tested for susceptibility to a stress factor (starvation). In addition, we used light microscopy and quantitative polymerase chain reaction (qPCR) to test the ability of DsCPV-1 to be transmitted vertically. We found insect mortality of the L. dispar parents following the infection was positively associated with DsCPV-1 dose. DsCPV-1 was lethal to second-instar L. dispar larvae with a 50% lethal dose (LD50) of 1687 occlusion bodies per larva. No vertical transmission of DsCPV-1 to offspring larvae was detected, while the majority of insect deaths among offspring larvae were caused by microsporidia (Vairimorpha lymantriae), which was harbored by the parents. The offspring of virally challenged parents exhibited a higher number of detected microsporidia compared to the control. Our findings suggest that the application of DsCPV-1 is effective in controlling pests in terms of transgenerational impact following virus exposure.

Effects of passages through an insect or a plant on virulence and physiological properties of the fungus Metarhizium robertsii.

Species of the genus Metarhizium are characterized by a multitrophic lifestyle of being arthropod parasites, rhizosphere colonizers, endophytes, and saprophytes. The process of adaptation to various organisms and substrates may lead to specific physiological alterations that can be elucidated by passaging through different hosts. Changes in virulence and cultivation properties of entomopathogenic fungi subcultured on different media or passaged through a live insect host are well known. Nevertheless, comparative in-depth physiological studies on fungi after passaging through insect or plant organisms are scarce. Here, virulence, plant colonization, hydrolytic enzymatic activities, toxin production, and antimicrobial action were compared between stable (nondegenerative) parent strain Metarhizium robertsii MB-1 and its reisolates obtained after eight passages through Galleria mellonella larvae or Solanum lycopersicum or after subculturing on the Sabouraud medium. The passaging through the insect caused similar physiological alterations relative to the plant-based passaging: elevation of destruxin A, B, and E production, a decrease in protease and lipase activities, and lowering of virulence toward G. mellonella and Leptinotarsa decemlineata as compared to the parent strain. The reisolates passaged through the insect or plant showed a slight trend toward increased tomato colonization and enhanced antagonistic action on tomato-associated bacterium Bacillus pumilus as compared to the parental strain. Meanwhile, the subculturing of MB-1 on the Sabouraud medium showed stability of the studied parameters, with minimal alterations relative to the parental strain. We propose that the fungal virulence factors are reprioritized during adaptation of M. robertsii to insects, plants, and media.

A New Cypovirus-1 Strain as a Promising Agent for Lepidopteran Pest Control.

Now more than ever researchers provide more and more evidence that it is necessary to develop an ecologically friendly approach to pest control. This is reflected in a sharp increase in the value of the biological insecticide market in recent decades. In our study, we found a virus strain belonging to the genus Cypovirus (Reoviridae); the strain was isolated from Dendrolimus sibiricus, possessing attractive features as a candidate for mass production of biological agents for lepidopteran-pest control. We describe the morphological, molecular, and ecological features of the new Cypovirus strain. This strain was found to be highly virulent to D. sibiricus (the half-lethal dose is 25 occlusion bodies per second-instar larva) and to have a relatively wide host range (infecting representatives of five families of Lepidoptera: Erebidae, Sphingidae, Pieridae, Noctuidae, and Lasiocampidae). The virus strain showed a strong interaction with a nontoxic adjuvant (optical brightener), which decreased the lethal dose for both main and alternative hosts, decreased lethal time, and may expand the host range. Moreover, we demonstrated that the insecticidal features were preserved after passaging through the most economically suitable host. By providing strong arguments for the possible use of this strain in pest control, we call on virologists, pest control specialists, and molecular biologists to give more attention to the Cypovirus genus, which may lead to new insights in the field of pest control research and may provide significant advantages to compare with baculoviruses and Bacillus thuringiensis products which are nowadays main source of bioinsecticides. IMPORTANCE In this article, we describe a newly discovered cypovirus strain that displays features ideally suited for the development of a modern biological insecticide: high potency, relatively broad host range, true regulating effect, flexible production (possibility to choose host species for production), interaction with enhancing adjuvants, and ecologically friendly. Based on an alignment of CPV genomes, we suggest that the enhanced host range of this new strain is the sequence of evolutionary events that occurred after coinfections involving different CPV species within the same host. These findings suggest that we need to positively reconsider CPVs as prospective agents as biocontrol products.

Laboratory Cultivation of Vairimorpha (Nosema) ceranae (Microsporidia: Nosematidae) in Artificially Infected Worker Bees.

Nosemosis type C is a dangerous and widespread disease of the adult European honey bee Apis mellifera and is caused by the spore-forming intracellular parasite Vairimorpha (Nosema) ceranae. The search for new ways of therapy for this disease is complicated due to the seasonal availability of V. ceranae-infected insects as well as the lack of a developed system for the pathogen's cultivation. By carrying out trials which used different infectious dosages of the parasite, spore storage protocols, host age, and incubation temperatures, we present a simple, safe, and efficient method of V. ceranae propagation in artificially infected worker bees in the laboratory. The method is based on feeding the groups of adult worker bees with microsporidian spores and insect maintenance in plastic bottles at 33 °C. The source of the spores originated from the cadavers of infected insects from the previous round of cultivation, in which the infective spores persist for up to six months. An analysis of five independent cultivation rounds involving more than 2500 bees showed that the proposed protocol exploiting the dosage of one million spores per bee yielded over 60 million V. ceranae spores per bee, and most of the spore samples can be isolated from living insects.

The microsporidium Nosema pyrausta as a potent microbial control agent of the beet webworm Loxostege sticticalis.

The microsporidium Nosema pyrausta is an important mortality factor of the European corn borer, Ostrinia nubilalis. The present study was aimed at N. pyrausta virulence testing to the beet webworm (BW), Loxostege sticticalis. This agricultural pest, L. sticticalis, was highly vulnerable to N. pyrausta. The parasite's spores were located in salivary glands, adipose tissue, and Malpighian tubules of the infected specimens. Infection was transmitted transovarially through at least 3 laboratory generations, in which BW fitness indices were lower than in the control, and moth emergence and fertility decreased prominently. Transovarial infection was most detrimental to female egg-laying ability, resulting in zero fertility in F3. When propagated in BW, the microsporidium tended to increase its virulence to L. sticticalis, as compared to the Ostrinia isolates. The parasite's ability to infect this host at low dosages and transmit vertically should guarantee its effective establishment and spread within BW populations. In conclusion, N. pyrausta is a promising agent against BW, which is a notorious polyphagous pest in Eurasia.

Susceptibility of the Gypsy Moth Lymantria dispar (Lepidoptera: Erebidae) to Nosema pyrausta (Microsporidia: Nosematidae).

The gypsy moth, Lymantria dispar, is a notorious forest defoliator, and various pathogens are known to act as natural regulators of its population density. As a widespread herbivore with a broad range of inhabited areas and host plants, it is potentially exposed to parasitic microorganisms from other insect hosts. In the present paper, we determined the susceptibility of gypsy moth larvae to the microsporidium Nosema pyrausta from the European corn borer, Ostrinia nubilalis. Gypsy moth samples from two localities of Western Siberia were used. N. pyrausta developed infections in the salivary gland and adipose tissue of gypsy moth prepupae and pupae, forming spore masses after 30 days of alimentary exposure to the second instar larvae. Among the experimental groups, the infection levels ranged from 0 to 9.5%. Effects of a covert baculovirus infection, phenylthiourea pretreatment and feeding insects on an artificial diet versus natural foliage were not significant in terms of microsporidia prevalence levels. Thus, L. dispar showed a low level of susceptibility to a non-specific microsporidium. It can be referred to as a resistant model host and not an appropriate substitute host for laboratory propagation of the microsporidium.

Genetic diversity of Loma acerinae (Microsporidia: Glugeida) from different fish hosts and localities - Short communication.

Loma acerinae is a xenoma-forming fish microsporidium described from common ruffe Gymnocephalus cernua (Perciformes: Percidae) and also found in Ponto-Caspian gobies (Gobiiformes: Gobiidae). This casts doubt on the strict host specificity of this parasite. The largest subunit RNA polymerase II (rpb1) was used as a genetic marker of the parasite isolated from six host species of Perciformes (G. cernua from the Baltic Sea), Atheriniformes (Atherina boyeri from the Azov Sea) and Gobiiformes (Neogobius spp. and Zosterisessor ophiocephalus from the Black Sea and Ponticola kessleri from the Caspian Sea basin). Two major rpb1 haplogroups were found with 98.5% identity between the groups. Notably, Haplogroup I was associated with Neogobius spp. samples (n = 6) only, whereas Haplogroup II included the samples from other host species (n = 7). These findings confirm the broad distribution and host range of L. acerinae, but also indicate that certain patterns of host-driven intraspecific polymorphism may exist. Furthermore, the study revealed low similarity between the ribosomal RNA gene sequences of L. acerinae and the type species, Loma morhua (as well as other species of the genus). This suggests loose genetic association within the genus, and may raise the need for the taxonomic revision of L. acerinae.

Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

Energy metabolism and its evolution in Microsporidia and allied taxa.

The reduction and specialization of the energy metabolism system is a common trait in the evolution of intracellular parasites. One group of fungi-related parasites, the Microsporidia, seems to have developed this trait far more than other eukaryotes. As an extreme adaptation for a parasitic lifestyle, some of them have completely lost the ability to synthesize ATP, relying heavily upon the metabolic processes of host cells to ensure their own development and reproduction. For a long time, only fragmentary data on the functioning and evolution of the energy metabolism system in microsporidia was available. However, the recent discovery of microsporidia-related microorganisms, the Cryptomycota and Aphelida, alongside with the genome sequencing and new data about basal groups in the Microsporidia has shed new light on this problem. Here, we review recent data about functioning of the energy metabolism system in microsporidia and closely related organisms, and discuss possible evolutionary pathways in the group.

Ultrastructure, molecular phylogeny, and prevalence rates of Alternosema bostrichidis gen. nov. sp. nov. (Microsporidia, Terresporidia), a parasite of Prostephanus truncatus and Dinoderus spp. (Coleoptera, Bostrichidae).

A new species and a new genus of a microsporidium Alternosema bostrichidis isolated from an adult Prostephanus truncatus in Mexico and from three species of the genus Dinoderus in Nigeria are described. The microsporidium is monomorphic, monoxenic, and develops in direct contact with host cell cytoplasm. The infection first appears with thoracic muscles, followed by a generalized invasion of the host. All developmental stages are diplokaryotic. Sporogony is disporoblastic. Mature spores are ovoid. Unfixed spores measure 3.7-4.2 × 2.0-2.6 µm, fixed and stained spores 3.5-5.0 × 2.4-2.8 µm. The polaroplast consists of dense lamellae and rare lamellae. The polar tube is slightly anisofilar, consisting of 11-17 coils, with 9-14 proximal (130 nm in diameter) and 2-3 distal coils (120 nm in diameter) arranged in one layer. Molecular phylogenetic analysis based upon a short portion of small-subunit ribosomal RNA gene (Genbank accession # KP455651) placed the new microsporidium within Liebermannia-Orthosomella lineage, which contains multiple undescribed parasites. In particular, A. bostrichidis showed maximal sequence similarity of 95% to Microsporidium sp. BBRE2 (# FJ755987) from Baikalian Diplacanthus brevispinus (Amphipoda: Acanthogammaridae) and Microsporidium sp. Comp CD Van 2 (# KC111784) from compost and soil in Canada. Frequent, devastating epizootics of laboratory cultures of A. bostrichidis support its potential as a biological control agent of grain borers.

A formal redefinition of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) and reassignment of species based on molecular phylogenetics.

The microsporidian genera Nosema and Vairimorpha comprise a clade described from insects. Currently the genus Nosema is defined as having a dimorphic life cycle characterized by diplokaryotic stages and diplosporoblastic sporogony with two functionally and morphologically distinct spore types ("early" or "primary" and "environmental"). The Vairimorpha life cycle, in addition to a Nosema-type diplokaryotic sporogony, includes an octosporoblastic sporogony producing eight uninucleate spores (octospores) within a sporophorous vesicle. Molecular phylogeny, however, has clearly demonstrated that the genera Nosema and Vairimorpha, characterized by the absence or presence of uninucleate octospores, respectively, represent two polyphyletic taxa, and that octosporogony is turned on and off frequently within taxa, depending on environmental factors such as host species and rearing temperature. In addition, recent studies have shown that both branches of the Vairimorpha-Nosema clade contain species that are uninucleate throughout their life cycle. The SSU rRNA gene sequence data reveal two distinct clades, those closely related to Vairimorpha necatrix, the type species for the genus Vairimorpha, and those closely related to Nosema bombycis, the type species for the genus Nosema. Here, we redefine the two genera, giving priority to molecular character states over those observed at the developmental, structural or ultrastructural levels and present a list of revised species designations. Using this approach, a series of species are renamed (combination novum) and members of two genera, Rugispora and Oligosporidium, are reassigned to Vairimorpha because of their phylogenetic position. Moreover, the family Nosematidae is redefined and includes the genera Nosema and Vairimorpha comprising a monophyletic lineage of Microsporidia.

Myxobolus pelecicola Voronin et Dudin 2015 is a junior synonym of Myxobolus ladogensis Rumyantsev et Schulman 1997 (Myxosporea: Myxobolidae) infecting the skeletal muscle of sichel Pelecus cultratus (Actinopterygii: Cyprinidae) in Russia.

Myxobolus pelecicola Voronin et Dudin, 2015 was recently described from the skeletal musculature of sichel Pelecus cultratus. However, another species, Myxobolus ladogensis Rumyantsev et Schulman, 1997, was described previously from the same host, displaying identical tissue localization and spore morphology as in M. pelecicola. Unfortunately, M. ladogensis was overlooked when M. pelecicola was described, resulting in the superfluous description of the latter species, which, according to the International Code of Zoological Nomenclature, is a junior synonym of M. ladogensis. The description of M. ladogensis is supplemented with SSU rDNA sequence analysis supporting the conspecificity with M. pelecicola. The closest relatives of Myxobolus ladogensis (syn. M. pelecicola) include several muscle-infecting Myxobolus spp. with sequence similarity below 97%.

Molecular Identification of a Densovirus in Healthy and Diseased Zophobas morio (Coleoptera, Tenebrionidae).

Zophobas morio is a tropical darkling beetle which is widely exploited for commercial large-scale insect growing. Outbreaks of a disease may occur causing total devastation of cultures. In the present paper, samples of diseased Z. morio were obtained and used for establishment of a laboratory model as they were found infective to the larvae of the same insect species from another source. It took about 1 month to develop symptoms of acute disease in mid-age larvae and about twice as much when younger larvae were used for infection. Affected larvae perished quickly, and within several days up to 90-100% of the colony could perish. Both in healthy and diseased larvae a virus was detected using PCR with degenerate primers specific for a gene coding for a non-structural protein (ORF3). The sequenced gene fragment (Genbank accession #MN732869) confirmed allocation of the virus to Densoviridae, with maximal similarity of 97.2% to Blatella germanica densovirus-like virus (#JQ320376) and 66.2% to B. germanica densovirus (#AY189948). Genomic DNA samples of Z. morio larvae from an independent colony devoid of symptoms of a disease were also positive for this virus with a slightly different (99.7% sequence similarity to the former sequence of the Z. morio densovirus) genotype (#MN732870).

Hexokinase as a versatile molecular genetic marker for Microsporidia.

Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3'-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

Infection of Chorthippus loratus (Orthoptera: Acrididae) with Liebermannia sp. (Microsporidia) in South-Western Russia.

Chorthippus loratus collected in Krasnodar Territory in 2017 was infected at 15% rate with a microsporidium possessing ovocylindrical binucleate spores, 2.6 × 1.2 µm in size. SSU RNA gene typing (Genbank accession # MH396491) showed its allocation to the genus Liebermannia. Degenerate primers based upon largest subunit RNA polymerase II (RPB1) sequences of closest relatives allowed amplifying the respective gene fragment of Liebermannia sp. (# MH396492). The present finding indicates worldwide distribution of the Liebermannia genus and parasitism in hosts with nonoverlapping geographic ranges (representing Neotropical versus Palearctic fauna), while previous observations were restricted to Acridoidea endemic for South America.

Detection of two Microsporidia pathogens of the European honey bee Apis Mellifera (Insecta: Apidae) in Western Siberia.

Two species of microsporidia, Nosema apis and Nosema ceranae, occur regularly and cause significant losses in apiculture throughout the world. N. ceranae is thought to be an emerging pathogen of the European honey bee which is replacing N. apis. Microscopic analysis of honey bees collected in Tyumen region, South-Western Siberia, suggested presence of two microsporidial pathogens slightly differing in spore size and shape. PCR detection using species-specific primer sets 312APIS and 218MITOC followed by PCR product sequencing confirmed the diagnosis of N. apis and N. ceranae, respectively. Microsporidia were present in private apiaries through 2008-2010, and among 21 colonies from 7 localities, two colonies were infected with both pathogens, while infections with N. apis only were detected in 8, and with N. ceranae only in 13 colonies. These data suggest that N. ceranae is widely spread in South-Western Siberia alongside with N. apis and is able to persist in the regions with average January temperatures below -18°C.

Vairimorpha ephestiae is a synonym of Vairimorpha necatrix (Opisthosporidia: Microsporidia) based on multilocus sequence analysis.

An isolate of the microsporidium Vairimorpha ephestiae (originally isolated from Ephestia kühniella) from collection of Prof. J. Weiser was propagated in a laboratory culture of Galleria mellonella. Only disporoblastic sporogony was observed and formation of octospores, characteristic of the genus Vairimorpha, never occurred. A partial nucleotide sequence of the small subunit rRNA gene (1247 bp) for this microsporidium showed 100% identity to the homologous sequences of Vairimorpha (Nosema) necatrix (Genbank accession # U11051 and # DQ996241), a microsporidium with a broad host range within the Lepidoptera. Sequence similarity of protein-coding genes (RPB1, HSP70 and actin) between V. ephestiae and V. necatrix was about 98-100%. The level of genetic polymorphism in the RPB1 locus between these two species was essentially the same as between isolates of V. necatrix. It is therefore concluded that V. ephestiae is in fact an isolate of V. necatrix and the former species should be synonymized with the latter. Though described later, V. necatrix has prevailing usage and its precedence over V. ephestiae is proposed to conserve stability and avoid confusion.

Greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) as a resistant model host for Nosema pyrausta (Microsporidia: Nosematidae).

Galleria mellonella fed 3 million Nosema pyrausta spores per larva showed 0 and 5% infestation rate at 30 °C and 24 °C, respectively. N. pyrausta virulence did not increase after passage through G. mellonella for three generations. When larvae were pretreated with phenylthiourea, Bacillus thuringiensis or combination of both, infection rates were 11%, 15% and 22%, respectively. Injection of untreated and potassium hydroxide-primed spores resulted in approximately 10% and 50% infection, respectively. G. mellonella is resistant to high dosages of N. pyrausta spores, serving as a prospective model of insect resistance to microsporidia, while host immunosuppression and/or spore activation increases success of the pathogen.

Natural infection of the beet webworm Loxostege sticticalis L. (Lepidoptera: Crambidae) with three Microsporidia and host switching in Nosema ceranae.

Three species of Microsporidia were identified from a population of the beet webworm Loxostege sticticalis at prevalence rates of 35, 4, and 3%. The most prevalent parasite (Tubulinosema sp.) was similar to Tubulinosema acridophagus (99.8% ssrDNA sequence similarity) and was also isolated from the parasitoid Lydella thompsoni (Diptera, Tachinidae) that emerged from the beet webworms. In laboratory assays, spores of this Tubulinosema sp. showed an infection rate of up to 80% for both L. sticticalis and Galleria mellonella larvae. The spores were viable after 12 months of storage in dried infected cadavers. The second most prevalent parasite was closely related to Nosema furnacalis and Nosema granulosis (98.7% similarity). Fresh spores showed a 50% infection rate under laboratory conditions. The third most abundant parasite was identified as the honeybee pathogen Nosema ceranae (100% ssrDNA and 95-100% IGS similarity). In the laboratory, fresh spores of N. ceranae isolated from beet webworm and honey bee were infective to L. sticticalis larvae at the rates of 5 and 2%, respectively.

Discovery of a novel microsporidium in laboratory colonies of Mediterranean cricket Gryllus bimaculatus (Orthoptera: Gryllidae): Microsporidium grylli sp. nov.

A microsporidium was found in a Mediterranean cricket Gryllus bimaculatus from a pet market in the UK and a lab stock at the Moscow Zoo (originating from London Zoo). The spores were ovoid, uninucleate, 6.3 × 3.7 µm in size (unfixed), in packets by of 8, 16, or 32. The spores were easily discharged upon dessication or slight mechanical pressure. The polar tube was isofilar, with 15-16 coils arranged in 1-2 rows. The polaroplast was composed of thin lamellae and occupied about one third of the spore volume. The endospore was 200 nm thick, thinning over the anchoring disc. The exospore was thin, uniform, and with no ornamentation. Phylogenetics based upon small subunit ribosomal RNA (Genbank accession # MG663123) and RNA polymerase II largest subunit (# MG664544) genes placed the parasite at the base of the Trachipleistophora/Vavraia lineage. The RPB1 locus was polymorphic but similar genetic structure and identical clones were found in both isolates, confirming their common geographic origin. Due to in insufficient ultrastructural data and prominent divergence from described species, the parasite is provisionally placed to the collective taxon Microsporidium.