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Cotton bollworm Helicoverpa armigera is one of the most polyphagous and cosmopolitan pests. Intracellular endosymbionts are widespread in Lepidoptera, often playing an important role in their dynamics. The prevalence of endosymbionts of cotton bollworm in Russia was not investigated. Cotton bollworm larvae and adults were collected in 2018–2020 in Krasnodar Area, and in Voronezh and Saratov Regions (from 131 to 170 insects) and analyzed by PCR using sets of group-specific primers for baculoviruses (locus lef8), bacteria of the genus of Wolbachia (locus wsp), and microsporidia (locus SSU rRNA). Level of infection with baculoviruses was 16 % for the sample of 32 individuals collected in Temryuk District of Krasnodar Area in 2018. The infection rate of the entire sample of 170 individuals was 2.9 %. The lef8 locus demonstrated 98.7–99.6 % of sequence similarity to the nuclear polyhedrosis virus isolates from the cotton bollworm and American bollworm. Among the tested 131 insects, bacteria of the genus of Wolbachia were not detected. PCR screening for microsporidia revealed one positive larvae among 19 insects collected in Krasnoarmeysk District of Krasnodar Area in 2019, which corresponded to the prevalence of 5 %. Partial sequencing of the genes coding for SSU rRNA and largest subunit RNA polymerase II made it possible to identify the new isolate as N. bombycis.

Beet webworm Loxostege sticticalis is a notorious pest widely distributed on the territory of Eurasia. Its outbreaks cause severe damage to crops in Russia and China. Here Wolbachia infection is reported for the first time in L. sticticalis. Larvae were sampled in Rostov, Saratov, Irkutsk Regions and Republic of Buryatia in 2005–2013. Primers targeting the wsp gene were used for the PCR screening of Wolbachia. Among 148 larvae, 35 were Wolbachia positive. Wolbachia prevalence rate ranged from 21 to 40 % in the Asian and from 0 to 47 % in the European part of Russia. The combined sample subsets were compared for European versus Asian part of Russia and 2005–2009 versus 2010–2013 timeframes. The prevalence rates of Wolbachia were not significantly different between two parts of Russia, but the endosymbiont presence (estimated for the total dataset) increased with time within the observation period.

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).

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.

Analysis of the prevalence rates of the endosymbiotic bacterium of the Wolbachia genus in the populations of corn borers of the Ostrinia genus was carried out. Samples of genomic DNA were extracted from field-collected larvae. The PCR analysis for the presence of Wolbachia infection was performed independently using three diagnostic loci: 16SrRNA, gatB, and fbpA. A positive signal for Wolbachia was obtained for 13.5% of the Ostrinia nubilalis larvae (N = 141) and 31.9% of the Ostrinia scapulalis larvae (N = 138). The infection rate ranged from 2.9% (N = 34) to 65.8% (N = 38) in different geographical populations. At the same time, a significantly higher infection rate of O. scapulalis than O. nubilalis was observed in three out of four habitats. It was also found that the Wolbachia infection was more common for O. scapulalis individuals feeding on hemp than for those collected from mugwort.

A comparison of the small subunit rRNA gene sequences of the microsporidian isolates from corn borers of the Ostrinia genus from Russia and Belarus indicates that they belong to the Nosema pyrausta species. In order to analyze genetic polymorphism, the nucleotide sequences of the molecular clones of the rRNA intergenic spacer (IGS) of the N. pyrausta isolates from Ostrinia nubilalis and Ostrinia scapulalis (Krasnodar territory), as well as from O. nubilalis (Gomel region, Belarus), are determined. Based on the information obtained and available in Genbank, a high level of divergence of the IGS locus between the isolates of different species and populations of microsporidia, as well as between the molecular clones within individual isolates of parasites, is shown.

Background . Endosymbiotic bacteria of the genus Wolbachia are widespread in arthropods and often cause reproductive abnormalities in lepidopteran insects, including corn borers of the genus Ostrinia . Wolbachia - Ostrinia is a promising model for studies of parasite-host interactions yet parasite prevalence in natural insect host populations remains unknown. Materials and Methods . Molecular genetic screening and statistical analysis is applied to evaluate prevalence rates of Wolbachia in sympatric populations of two corn borer species. Individual genomic DNA samples were extracted from last instar larvae collected in nature from different forage plants. For each sample of DNA showing positive signal with insect-specific primers the detection is performed using three diagnostic loci of Wolbachia : 16SrRNA, gatB and fbpA. Results . Wolbachia -positive signal is obtained for 13.5% larvae of Ostrinia nubilalis (N = 141) and 31.9% larvae of Ostrinia scapulalis (N = 138). In different localities the Wolbachia prevalence ranged from 2.9% (N = 34) to 65.8% (N = 38). Significantly higher rates of Wolbachia prevalence in insects from mugwort and hemp ( O. scapulalis ) as compared to those from corn ( O. nubilalis ) are revealed in three out of four localities. Conclusions . Endosymbiotic bacteria of the genus Wolbachia are revealed in natural populations of corn borers for the first time for Eastern Europe. The prevalence rates can be high and this should be taken into consideration when reproductive isolation is examined in population of these hosts as well as establishment of laboratory cultures is performed.

Background. Microsporidia are ubiquitous parasites of animals, most abundant in arthropods and fishes. Many species of these parasites are important from standpoints of medicine, veterinary and agriculture. Microsporidium Nosema pyrausta is an important disease agent in corn borer populations causing adverse effect on host fitness. Genotyping of this parasite is necessary for proper species identification and intraspecific polymorphysm studies. Materials and Methods. Microsporidia-infected larvae of corn borers of the genus Ostrinia were recovered from Krasnodar Territory in Russia and Gomel Region in Belarus. Small subunit ribosomal RNA (SSU rRNA) was amplified and sequenced directly, while intergenic spacer (IGS) was amplified, cloned and sequenced (1-5 clones per sample) for four isolates of microsporidia. Sequences were aligned and compared using standard bioinformatics tools (Clustal W and BLAST). Results. SSU rRNA genotyping showed allocation of all four isolates to N. pyrausta with 100% identity to each other and 99.7% similarity to Nosema bombycis, the type species of the genus Nosema. High levels of IGS sequence variation (61-74%) is observed both between isolates of different species and populations of microsporidia as well as between molecular clones within parasite isolates from individual hosts. Conclusion. N. pyrausta is widespread in corn borer populations and its genetic structure is complicated, as in other species of these parasites. Further studies of molecular markers are needed for genetic differentiation of geographic isolates of N. pyrausta.

The experiments were aimed at evaluation of the effect of feeding with wheat colonized by the fungus Beauveria bassiana on the development of locusts. The seeds were treated with fungal conidia and 90 days after sowing, the plant parts were fed to II instar nymphs of laboratory colonies of migratory and desert locusts within the period of 18 days. The mortality levels between experimental and control (fed with untreated wheat) groups were not significantly different though a tendency of an increase in variants with contaminated fodder was observed. Two weeks after the beginning of the bioassay, insect mortality was 20-30% and 15% in control. As many as 50% of migratory locust and 40% of desert locust cadavers were covered by a fungal mycelium. Sequencing of two diagnostic DNA loci has confirmed attribution of the reisolate to the genotype of the initial Beauveria bassiana strain BBK-1. It can be therefore concluded that the endophyte fungus is capable of causing classical pathogenesis of mycosis in locust nymphs. Moreover, the nymphal development was retarded by feeding with the fungus-colonized plants.

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.

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.

Genomic DNA samples from larvae of the beet webworm Loxostege sticticalis collected in the south-western Russia were used to amplify mitochondrial cytochrome oxidase unit I (COI) gene. In a small proportion of samples, the sequenced product showed considerable heterogeneity due to admixture of a minor sequence. A preliminary BLAST analysis of a 100-bp-long fragment of this minor sequence showed its maximal similarity to the COI gene region of Cotesia, a genus of braconid larval endoparasitoids of Lepidoptera. An additional primer was designed to specifically amplify ca 300 bp of the COI gene region from Braconidae. As many as seven of 25 samples were positive by PCR. Sequencing of the amplified products in all these samples showed nucleotide sequence identity to the COI region of Cotesia vestalis (Cotesia plutellae) and the presence of two molecular haplotypes among individual parasitoid samples.

Population dynamics of the beet webworm Pyrausta sticticalis L. was studied in 2003–2005 in Krasnodar Territory (Slavyansk-on-Kuban District) during the period of low population density of the pest. In the period examined, the adult death rate was high, fecundity was low; the low rate of hatchability and high rate of mortality was typical of larvae reared in the laboratory. Analysis of life tables has demonstrated that the observed population density of adults could be explained only by a constant flow of migrants. Low viability of insects from the local population could be explained by the infection with pathogenic microorganisms, including viruses of the Polyhedrosis and Granulosi groups of Baculovirus, and microsporidia, including Nosema sticticalis and Microsporidium sp. Reproduction of the pest can also be influenced by unfavorable weather conditions reducing population density in the subsequent generation. It is demonstrated that the model of the beet webworm population density dynamics based on indices of female fertility, changes in the offspring viability, and the rate of infection with microsporidia is more reliable than models based only on meteorological factors.

Populations of European corn borer (Ostrinia nubilalis Hubner) from Krasnodar Territory (Southwestern Russia) become regularly infected with Nosema-like microsporidia. To identify the parasite, it was subjected to electron microscopy and small subunit ribosomal RNA (SSU rRNA) gene sequencing. The spore ultrastructure of the parasite was highly similar to Nosema bombycis from China and Nosema pyrausta from the USA. The nucleotide sequence of SSU rRNA gene was identical to a microsporidium isolated from O. nubilalis in southern France (GenBank accession no. HM566196) and closely related to Nosema bombycis (no. AY209011, 99.7 % sequence similarity) from Bombyx mori of Chinese origin and N. pyrausta (no. AY958071) from O. nubilalis of North American origin. As the molecular haplotype of SSU rRNA is fixed for the parasite infecting O. nubilalis across Europe and N. pyrausta was initially described in France as Perezia pyraustae (Paillot CR Acad Sci Paris 185: 673–675, 1927), we conclude that the parasite examined under the present study correspond to the type isolate of N. pyrausta. The microsporidium from O. nubilalis in North America (no. AY958071) corresponds therefore to a closely related, yet distinct haplotype.