Wild Animal Migration As a Potential Threat of Introduction of New Viruses into Russia.

The SARS-CoV-2 pandemic has shown how serious the problem of re-emerging zoonotic infections is for our existence. Migrations of animals, which are natural reservoirs of a particular virus, play a colossal role in the spread of pathogens to new territories. Examples are the migrations of both land animals (carnivores, rodents, and ungulates) and many marine mammals (pinnipeds and cetaceans). Yet the most interesting from the point of view of the speed and range of the spread of viral infections are migrations associated with flights. In nature, these can be migrations of insects, bats, and, of course, birds. Unfortunately, there are very few studies on the migration of these animals in Russia. Considering the problems related to climate change and other environmental factors, it is important to obtain up-to-date data on the changing animal migration routes and, as a consequence, to develop domestic equipment, particularly transmitters, to fix them.

Effect of the Entomopathogenic Fungus Beauveria bassiana on the Development of Faba Bean (Vicia faba) Diseases in the Field Conditions.

Several ascomycetous entomopathogenic fungi, including species in the genera Beauveria, are plant symbionts/endophytes and are termed as endophytic insect-pathogenic fungi. It was shown that the fungus Beauveria bassiana (BBK-1 strain) successfully colonized Vicia faba bean plants in laboratory and field conditions of Western Siberia. The B. bassiana reisolate passed through the plants had significantly higher antagonistic activity against phytopathogens in comparison with the primary stem of entomopahogenic fungi. Pre-sowing faba bean seeds treatment reduced the level of infection of the seed material with phytopathogens, significantly decrease the development and prevalence of root rot disease. A decrease in the disease development index (chocolate spot, powdery mildew, fusariosis and other spots diseases) was found as a result of the use of B. bassiana. The effectiveness and prolonged action of B. bassiana on plants opens up new opportunities both in the creation of biological products and in molecular-genetic research and selection of certain pairs of plants and fungi based on the principle of the greatest synergy.

A Neurotoxic Insecticide Promotes Fungal Infection in Aedes aegypti Larvae by Altering the Bacterial Community.

Symbiotic bacteria have a significant impact on the formation of defensive mechanisms against fungal pathogens and insecticides. The microbiome of the mosquito Aedes aegypti has been well studied; however, there are no data on the influence of insecticides and pathogenic fungi on its structure. The fungus Metarhizium robertsii and a neurotoxic insecticide (avermectin complex) interact synergistically, and the colonization of larvae with hyphal bodies is observed after fungal and combined (conidia + avermectins) treatments. The changes in the bacterial communities (16S rRNA) of Ae. aegypti larvae under the influence of fungal infection, avermectin toxicosis, and their combination were studied. In addition, we studied the interactions between the fungus and the predominant cultivable bacteria in vitro and in vivo after the coinfection of the larvae. Avermectins increased the total bacterial load and diversity. The fungus decreased the diversity and insignificantly increased the bacterial load. Importantly, avermectins reduced the relative abundance of Microbacterium (Actinobacteria), which exhibited a strong antagonistic effect towards the fungus in in vitro and in vivo assays. The avermectin treatment led to an increased abundance of Chryseobacterium (Flavobacteria), which exerted a neutral effect on mycosis development. In addition, avermectin treatment led to an elevation of some subdominant bacteria (Pseudomonas) that interacted synergistically with the fungus. We suggest that avermectins change the bacterial community to favor the development of fungal infection.

Genetic diversity and geographical distribution of the Siberian subtype of the tick-borne encephalitis virus.

The tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is currently subdivided into three main subtypes-the European (TBEV-Eu), the Far-Eastern (TBEV-FE), and the Siberian (TBEV-Sib). The TBEV-Sib is the most common subtype and found in all regions where TBEV was detected, except for Central and Western Europe. Currently, four genetic lineages have been described within TBEV-Sib. In this study, detailed analysis of TBEV-Sib genetic diversity, geographic distribution, phylogeography and divergence time of different TBEV-Sib genetic lineages based on E gene fragments, complete genome sequences, and all currently available data in the GenBank database was performed. As a result, a novel Bosnia lineage within the TBEV-Sib was identified. It was demonstrated that the Zausaev lineage is the most widely distributed among the TBEV-Sib lineages, and was detected in all studied regions except the Far East. The Vasilchenko lineage was found from Western Siberia to the Far East. The Baltic lineage is presented from Europe to Western Siberia. The Obskaya lineage was found only in Western Siberia. TBEV strains from a newly described Bosnia lineage were detected in Bosnia, the Crimean peninsula, Kyrgyzstan and Kazakhstan. The greatest divergence of the TBEV-Sib genetic variants was observed in Western Siberia. Within the TBEV-Sib, the Obskaya lineage diverged from the common ancestor the earliest, after that the Bosnia lineage was separated, then the Baltic lineage, and the Zausaev and Vasilchenko lineages diverged most recently.

The Change in the Entomopathogenic Properties in Streptomycin Resistant Bacillus thuringiensis.

Streptomycin-resistant strains (StrR) of the entomopathogenic bacteria Bacillus thuringiensis ssp. galleriae (Btg) have been obtained. Assessment of growth rate of Btg 69-6 colonies revealed significant difference between the initial strain StrS sensitive to antibiotics and StrR. Decrease in susceptibility of instar IV larvae of Galleria mellonella to Btg 69-6 StrR by a factor of eight compared to Btg 69-6 StrS has also been recorded. In Btg 190 StrR, the insecticidal activity decreased by a factor of five. In StrR, the biochemical properties changed after acquisition of resistance compared to the initial strain.

[Phenotypic and genetic changes of entomopathogenic ascomycete Beauveria Bassiana under passaging through various hosts].

Phenotypic and genetic estimations of entomopathogenic ascomycete B.bassiana (strain Sar-31) after 6-passaging through four hosts were shown. Increasing of virulence, changes in morpho-cultural characteristics and variations in Inter Simple Sequence Repeats (ISSR) assay between initial and reisolated cultures were registered. Six passages of entomopathogenic ascomycete Beauveria bassiana (strain Sar-31) through four hosts (Galleria mellonella, Tenebrio molitor, Leptinotarsa decemlineata, Locusta migratoria) and following estimation of phenotypic and genetic differences of the initial strain and reisolated cultures were conducted. The passaging of strain through certain host led to increasing of virulence for both this host and other test-insects. Unidirectional changes of morpho-cultural characteristics: colonies pigmentation and relief strengthening, increasing of conidia production and lipolytic activity were registered in all passaged cultures. Genetic analysis with 6 ISSR markers revealed variations between initial and reisolated cultures in 3 markers. Taken together, the results of this study help us understand potential ways of fungi strains changes during epizootic process and possibilities of ISSR assay applying for investigation of pathogen transmission.

[COMPARATIVE ANALYSIS OF THE IMMUNE REACTIONS IN COLORADO POTATO BEETLE LARVAE UNDER DEVELOPMENT OF MYCOSES CAUSED BY METARHIZIUM ROBERTSII, M. BRUNNEUM AND M. PEMPHIGI].

A comparative investigation of humoral and cellular immune response in larvae of Colorado potato beetle Leptinotarsa decemlineata was conducted under development of mycoses caused by entomopatho- genic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found highly suscep- tible to M. robertsii, M. brunneum and less susceptible to M. pemphigi. The susceptibility to the fungi was not correlated with the rate of conidia germination in epicuticular extracts of larvae. A non-specific for Colorado beetle pathogen M. pemphigi did not cause significant changes in the immune response and did not result in colonization of larvae hemocoel. Infection with M. robertsi and M. brunneum led to an increase in total hemocyte count at the initial stages of mycoses (day 2) followed by a sharp decrease on day 3. The strongest decrease was observed for the immunocompetent cells - plasmatocytes and granu- locytes. Enhanced phenoloxidase activity in hemolymph and cuticle was found on days 2 and 3 after in- fection. These changes in immune reactions correlated with the level of virulence of the strains. Thus, the immune response in Colorado potato beetle larvae is an important factor determining differences in the development of mycoses caused by different Metarhizium species.

A comparative study on microbiota from the intestine of Prussian carp (Carassius gibelio) and their aquatic environmental compartments, using different molecular methods.

AIMS: The aim of this study was to evaluate, via various molecular methods, the possible correlations between microbial community structure of Prussian carp and the environmental compartments of their habitat. METHODS AND RESULTS: Microbial communities in the intestine and environmental compartments were studied using PCR-screening, cloning and next-generation high-throughput sequencing of the 16S ribosomal RNA genes. The 16S rDNA metagenomic sequencing showed higher bacterial diversity in comparison with clone libraries, while group-specific PCR showed positive detection of nine bacteria phyla. Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria and Actinobacteria were most abundant both in the intestine and habitat environments. The comparative analyses reveal that the bacterial community in the Prussian carp intestine is most similar to that identified from the chironomid. CONCLUSIONS: This study demonstrated some differences between molecular methods and showed advantages and limitations associated with them. These differences have the potential to reduce bias in results obtained from analysis of the community structure. The advantages of each molecular technique can be used for a better understanding of microbial diversity. The microbiota of Prussian carp intestine is most similar to those from the chironomids. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated the diversity of the intestinal microbiota in an economically important aquaculture species, the Prussian carp (Carassius gibelio). The results provide significant information to discuss possible functions of these bacteria for further understanding of Prussian carp health.

Feeding habits and ontogenic changes in digestive enzyme patterns in five freshwater teleosts.

Feeding habits and the activity of digestive enzymes (total alkaline proteases, α-amylase and lipase) from dace Leuciscus leuciscus, roach Rutilus rutilus, Prussian carp Carassius auratus gibelio, perch Perca fluviatilis and pikeperch Sander lucioperca fry were studied in the Malye Chany Lake-Kargat Estuary (western Siberia, Russia). The diet of fry from all studied species was mainly composed of chironomid larvae and zooplanktonic organisms (i.e. cladocera and copepoda), whereas carnivorous species such as P. fluviatilis and S. lucioperca also preyed on fry from other fishes while detritus and microalgae were also important in the diet of ommivorous species. When comparing diet similarity (Sørensen-Dice index, Q(S)) among fry at different stages of development, both omnivorous and carnivorous species showed a high level of similarity (0.67 < Q(S) < 0.89 and 0.73 < Q(S)< 0.89, respectively). Diet similarity values were in agreement with the overall digestive activity profile analysed by cluster analysis. Diet similarity suggested potential trophic competition when zooplanktonic and benthic prey began to decline towards autumn. The analysis of pancreatic digestive enzymes revealed a correlation among their activities and fry feeding habits with α-amylase:total proteases (A:P) values higher than 1 in omnivorous species and lower (A:P ≤ 1) in carnivorous species.

Contributions of cellular and humoral immunity of Galleria mellonella larvae in defence against oral infection by Bacillus thuringiensis.

In this study the cellular and humoral immune reactions of the Greater wax moth Galleria mellonella have been investigated during bacterial infection caused by oral administration of Bacillus thuringiensis. Two different dose strengths were investigated to assess the contribution of immune parameters to induced Bt resistance. Low-dose (sublethal LC15) infection resulted in significantly elevated haemolymph phenoloxidase and lysozyme-like activity, enhanced phagocytic activity of haemocytes, and increased encapsulation responses in infected larvae at 48 and 72 h post infection. Higher doses of Bt (half-lethal LC50) also triggered significantly elevated haemolymph phenoloxidase and lysozyme-like activity, but decreased the coagulation index and activity of phenoloxidase in haemocytes of infected larvae. In both types of infection, the pool of circulating haemocytes became depleted. The importance of cellular and humoral immune reactions in induced insect resistance to intestinal bacterial infection Bt is herein discussed.

[Insecticidal and immunosuppressive effect of ascomycete Cordyceps militaris on the larvae of the Colorado potato beetle Leptinotarsa decemlineata].

The immunosuppressive and insecticidal activity of cultures of the entomopathogenic fungus Cordyceps militaris on the larvae of the Colorado potato beetle Leptinotarsa decemlineata has been established for the first time. It was found that the peroral effect of the fungal culture resulted in dose-dependent decrease in survival, delayed in development time and molting, decreases in the total hemocyt counts, increased activity of phenoloxidases in the hemolymph, and reduced activity of the enzyme in the cuticle, as well as increased sensitivity of larvae to the fungus Beauveria bassiana at the level of the synergistic effect.

More than a colour change: insect melanism, disease resistance and fecundity.

A 'dark morph' melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.

[An increase in the immune system activity of the wax moth Galleria mellonella and of the Colorado potato beetle Leptinotarsa decemlineata under effect of organophosphorus insecticide].

There has been performed evaluation of the effect of the organophosphorus insecticide (pirimifos-methyl) on some components of the insect immune response. The cellular (a change of the number of hemocytes and of intensity of incapsulation) and the humoral (a change of phenoloxidase activity) components of the immune response were studied in larvae of representatives of two orders--the Colorado potato beetle (Leptinotarsa decemlineata, Chrysomelidae, Coleoptera) and the wax moth (Galeriia mellonella, Pyralidae, Lepidoptera). The action of the insecticide has been found to lead to stimulation of immune reactions (an increase of phenoloxidase activities and of intensity of incapsulation, a rise of the number of hemocytes) at the contact treatment of both sublethal and the half-lethal doses of pirimifos-methyl.

The effects of dietary nickel on the detoxification enzymes, innate immunity and resistance to the fungus Beauveria bassiana in the larvae of the greater wax moth Galleria mellonella.

In this study, we tested the effects of dietary nickel on the activity of glutathione S-transferase (GST), esterases, phenoloxidase, and encapsulation in the haemolymph of larvae of the greater wax moth Galleria mellonella. We also explored the effects of dietary nickel on larval resistance to infection by the fungus Beauveria bassiana. Larvae fed a low dose of nickel (10 µg g(-1)) had significantly higher GST, phenoloxidase activity and encapsulation responses than controls fed on a nickel-free diet. We also found that larvae fed a sublethal dose of nickel (50 µg g(-1)) had increased GST, esterase activity and encapsulation rates but decreased phenoloxidase activity. Although, a sublethal dose of dietary nickel enhanced innate immunity, we found that this reduced resistance against the real pathogen. Our results suggest that enhanced immunity and detoxification enzyme activity of insects may not be beneficial to resistance to fungal infection. It appears that there is a trade off between different resistance mechanisms in insects under different metal treatments.

The effect of Habrobracon hebetor venom on the activity of the prophenoloxidase system, the generation of reactive oxygen species and encapsulation in the haemolymph of Galleria mellonella larvae.

The cellular and humoral immune reactions in haemolymph of the wax moth Galleria mellonella larvae naturally injected by venom of ectoparasitic wasp Habrobracon hebetor were analyzed. A strong decline of phenoloxidase (PO) activity in the haemolymph and the number of haemocytes with PO activity of envenomated wax moth was observed. In addition, it has been shown that the rate of capsule melanization in the envenomated larvae was half that of the control. Also production of reactive oxygen species (ROS) in the haemolymph of envenomated larvae decreased. The obtained data casts light on the suppression of the main immune reactions in G. mellonella larvae during natural envenomation by H. hebetor.

[Activity of detoxifying and antioxidant enzymes in the pond snail Lymnaea stagnalis (gastropoda: pulmonata) during invasion by trematode cercariae].

A comparative analysis of changes of activities of detoxifying and antioxidant enzymes was carried out in the body homogenate of the pond snail Lymnaea stagnalis. Juvenile snails with the shell size of 4-6 mm were infested with cercariae of one (Echinoparyphium aconiatum or E. recurvatum or Moliniella anceps) or two (E. aconiatum +M. anceps or E. aconiatum + E. recurvatum or E. recurvatum + M. anceps) trematode species. It has been revealed that activities of detoxifying and antioxidant enzymes in the body of the snail L. stagnalis change at invasion with trematodes and that the character of the changes depends on the stage of development of the trematodes in the host body, the variant of infestation (monoinvasion or mixed infestation), and the species of the parasite. The first 2 h after penetration of the trematode cercariae into the molluse tissues is accompanied by an increase of activities of the detoxifying and antioxidant enzymes. A long coexistence of metacercariae in the host body (no less than for 13 days) is accompanied by a decrease of activities of non-specific esterases and glutathione-S-transferase and by an increase of superoxide dismutase.

[Generation of reactive oxygen species and activity of antioxidants in larva hemolymph of galleria mellonella (L.) (lepidoptera: piralidae) at development of process of encapsulation].

Activities of enzymatic antioxidants (superoxide dismutase, glutathione-S-transferase, and catalase) have been determined in hemocytes and generation of reactive oxygen species (ROS) has been studied in lymph of larvae of the wax moth Galleria mellonella at development of the process of encapsulation of nylon implants. It has been established that as soon as 15 min after piercing of cuticle with implant the capsule is formed on its surface. The active melanization of the capsule has been shown to last for 4 h. There have been shown a statistically significant increase of the ROS generation in lymph and a decrease of activities of enzymatic antioxidants in hemocytes of the insects after the implant incorporation. The authors suggest that the key role in maintenance of the oxidation-reduction balance in hemolymph at development of the incapsulation process is played by the lymph non-enzymatic antioxidants.