A Diet with Amikacin Changes the Bacteriobiome and the Physiological State of Galleria mellonella and Causes Its Resistance to Bacillus thuringiensis.

Environmental pollution with antibiotics can cause antibiotic resistance in microorganisms, including the intestinal microbiota of various insects. The effects of low-dose aminoglycoside antibiotic (amikacin) on the resident gut microbiota of Galleria mellonella, its digestion, its physiological parameters, and the resistance of this species to bacteria Bacillus thuringiensis were investigated. Here, 16S rDNA analysis revealed that the number of non-dominant Enterococcus mundtii bacteria in the eighteenth generation of the wax moth treated with amikacin was increased 73 fold compared to E. faecalis, the dominant bacteria in the native line of the wax moth. These changes were accompanied by increased activity of acidic protease and glutathione-S-transferase in the midgut tissues of larvae. Ultra-thin section electron microscopy detected no changes in the structure of the midgut tissues. In addition, reduced pupa weight and resistance of larvae to B. thuringiensis were observed in the eighteenth generation of the wax moth reared on a diet with amikacin. We suggest that long-term cultivation of wax moth larvae on an artificial diet with an antibiotic leads to its adaptation due to changes in both the gut microbiota community and the physiological state of the insect organism.

Links between Soil Bacteriobiomes and Fungistasis toward Fungi Infecting the Colorado Potato Beetle.

Entomopathogenic fungi can be inhibited by different soil microorganisms, but the effect of a soil microbiota on fungal growth, survival, and infectivity toward insects is insufficiently understood. We investigated the level of fungistasis toward Metarhizium robertsii and Beauveria bassiana in soils of conventional potato fields and kitchen potato gardens. Agar diffusion methods, 16S rDNA metabarcoding, bacterial DNA quantification, and assays of Leptinotarsa decemlineata survival in soils inoculated with fungal conidia were used. Soils of kitchen gardens showed stronger fungistasis toward M. robertsii and B. bassiana and at the same time the highest density of the fungi compared to soils of conventional fields. The fungistasis level depended on the quantity of bacterial DNA and relative abundance of Bacillus, Streptomyces, and some Proteobacteria, whose abundance levels were the highest in kitchen garden soils. Cultivable isolates of bacilli exhibited antagonism to both fungi in vitro. Assays involving inoculation of nonsterile soils with B. bassiana conidia showed trends toward elevated mortality of L. decemlineata in highly fungistatic soils compared to low-fungistasis ones. Introduction of antagonistic bacilli into sterile soil did not significantly change infectivity of B. bassiana toward the insect. The results support the idea that entomopathogenic fungi can infect insects within a hypogean habitat despite high abundance and diversity of soil antagonistic bacteria.

Academ Virus, a Novel Hantavirus in the Siberian Mole (Talpa altaica) from Russia.

To date, six hantavirus species have been detected in moles (family Talpidae). In this report, we describe Academ virus (ACDV), a novel hantavirus harbored by the Siberian mole (Talpa altaica) in Western Siberia. Genetic analysis of the complete S-, M-, and partial L-genomic segments showed that ACDV shared a common evolutionary origin with Bruges virus, previously identified in the European mole (Talpa europaea), and is distantly related to other mole-borne hantaviruses. Co-evolution and local adaptation of genetic variants of hantaviruses and their hosts, with possible reassortment events, might have shaped the evolutionary history of ACDV.

Geographic Distribution and Phylogeny of Soricine Shrew-Borne Seewis Virus and Altai Virus in Russia.

The discovery of genetically distinct hantaviruses (family Hantaviridae) in multiple species of shrews, moles and bats has revealed a complex evolutionary history involving cross-species transmission. Seewis virus (SWSV) is widely distributed throughout the geographic ranges of its soricid hosts, including the Eurasian common shrew (Sorex araneus), tundra shrew (Sorex tundrensis) and Siberian large-toothed shrew (Sorex daphaenodon), suggesting host sharing. In addition, genetic variants of SWSV, previously named Artybash virus (ARTV) and Amga virus, have been detected in the Laxmann's shrew (Sorex caecutiens). Here, we describe the geographic distribution and phylogeny of SWSV and Altai virus (ALTV) in Asian Russia. The complete genomic sequence analysis showed that ALTV, also harbored by the Eurasian common shrew, is a new hantavirus species, distantly related to SWSV. Moreover, Lena River virus (LENV) appears to be a distinct hantavirus species, harbored by Laxmann's shrews and flat-skulled shrews (Sorex roboratus) in Eastern Siberia and far-eastern Russia. Another ALTV-related virus, which is more closely related to Camp Ripley virus from the United States, has been identified in the Eurasian least shrew (Sorex minutissimus) from far-eastern Russia. Two highly divergent viruses, ALTV and SWSV co-circulate among common shrews in Western Siberia, while LENV and the ARTV variant of SWSV co-circulate among Laxmann's shrews in Eastern Siberia and far-eastern Russia. ALTV and ALTV-related viruses appear to belong to the Mobatvirus genus, while SWSV is a member of the Orthohantavirus genus. These findings suggest that ALTV and ALTV-related hantaviruses might have emerged from ancient cross-species transmission with subsequent diversification within Sorex shrews in Eurasia.

Fungus Metarhizium robertsii and neurotoxic insecticide affect gut immunity and microbiota in Colorado potato beetles.

Fungal infections and toxicoses caused by insecticides may alter microbial communities and immune responses in the insect gut. We investigated the effects of Metarhizium robertsii fungus and avermectins on the midgut physiology of Colorado potato beetle larvae. We analyzed changes in the bacterial community, immunity- and stress-related gene expression, reactive oxygen species (ROS) production, and detoxification enzyme activity in response to topical infection with the M. robertsii fungus, oral administration of avermectins, and a combination of the two treatments. Avermectin treatment led to a reduction in microbiota diversity and an enhancement in the abundance of enterobacteria, and these changes were followed by the downregulation of Stat and Hsp90, upregulation of transcription factors for the Toll and IMD pathways and activation of detoxification enzymes. Fungal infection also led to a decrease in microbiota diversity, although the changes in community structure were not significant, except for the enhancement of Serratia. Fungal infection decreased the production of ROS but did not affect the gene expression of the immune pathways. In the combined treatment, fungal infection inhibited the activation of detoxification enzymes and prevented the downregulation of the JAK-STAT pathway caused by avermectins. The results of this study suggest that fungal infection modulates physiological responses to avermectins and that fungal infection may increase avermectin toxicosis by blocking detoxification enzymes in the gut.

Geographical distribution and hosts of the cestode Paranoplocephala omphalodes (Hermann, 1783) Lühe, 1910 in Russia and adjacent territories.

Paranoplocephala omphalodes is a widespread parasite of voles. Low morphological variability within the genus Paranoplocephala has led to erroneous identification of P. omphalodes a wide range of definitive hosts. The use of molecular methods in the earlier investigations has confirmed that P. omphalodes parasitizes four vole species in Europe. We studied the distribution of P. omphalodes in Russia and Kazakhstan using molecular tools. The study of 3248 individuals of 20 arvicoline species confirmed a wide distribution of P. omphalodes. Cestodes of this species were found in Microtus arvalis, M. levis, M. agrestis, Arvicola amphibius, and also in Chionomys gud. Analysis of the mitochondrial gene cox1 variability revealed a low haplotype diversity in P. omphalodes in Eurasia.

Parasitoid envenomation alters the Galleria mellonella midgut microbiota and immunity, thereby promoting fungal infection.

Gut bacteria influence the development of different pathologies caused by bacteria, fungi and parasitoids in insects. Wax moth larvae became more susceptible to fungal infections after envenomation by the ectoparasitoid Habrobracon hebetor. In addition, spontaneous bacterioses occurred more often in envenomated larvae. We analyzed alterations in the midgut microbiota and immunity of the wax moth in response to H. hebetor envenomation and topical fungal infection (Beauveria bassiana) alone or in combination using 16S rRNA sequencing, an analysis of cultivable bacteria and a qPCR analysis of immunity- and stress-related genes. Envenomation led to a predominance shift from enterococci to enterobacteria, an increase in CFUs and the upregulation of AMPs in wax moth midguts. Furthermore, mycosis nonsignificantly increased the abundance of enterobacteria and the expression of AMPs in the midgut. Combined treatment led to a significant increase in the abundance of Serratia and a greater upregulation of gloverin. The oral administration of predominant bacteria (Enterococcus faecalis, Enterobacter sp. and Serratia marcescens) to wax moth larvae synergistically increased fungal susceptibility. Thus, the activation of midgut immunity might prevent the bacterial decomposition of envenomated larvae, thus permitting the development of fungal infections. Moreover, changes in the midgut bacterial community may promote fungal killing.

Variability of whipworm infection and humoral immune response in a wild population of mole voles (Ellobius talpinus Pall.).

Restricted mobility and spatial isolation of social units in gregarious subterranean mammals ensure good defence mechanisms against parasites, which in turn allows for a reduction of immunity components. In contrast, a parasite invasion may cause an increased adaptive immune response. Therefore, it can be expected that spatial and temporal distribution of parasites within a population will correlate with the local variability in the host's immunocompetence. To test this hypothesis, the intra-population variability of a whipworm infestation and the humoral immune response to non-replicated antigens in mole voles (Ellobius talpinus Pall.), social subterranean rodents, was estimated. Whipworm prevalence in mole voles increased from spring to autumn, and this tendency was more pronounced in settlements living in natural meadows compared to settlements in man-made meadows. However, humoral immune response was lowest in animals from natural meadows trapped in autumn. Since whipworm infestation does not directly affect the immunity of mole voles, the reciprocal tendencies in seasonal dynamics and spatial distribution of whipworm abundance and host immunocompetence may be explained by local deterioration of habitat conditions, which increases the probability of an infestation.

Reappraisal of Hydatigera taeniaeformis (Batsch, 1786) (Cestoda: Taeniidae) sensu lato with description of Hydatigera kamiyai n. sp.

The common cat tapeworm Hydatigera taeniaeformis is a complex of three morphologically cryptic entities, which can be differentiated genetically. To clarify the biogeography and the host spectrum of the cryptic lineages, 150 specimens of H. taeniaeformis in various definitive and intermediate hosts from Eurasia, Africa and Australia were identified with DNA barcoding using partial mitochondrial cytochrome c oxidase subunit 1 gene sequences and compared with previously published data. Additional phylogenetic analyses of selected isolates were performed using nuclear DNA and mitochondrial genome sequences. Based on molecular data and morphological analysis, Hydatigera kamiyai n. sp. Iwaki is proposed for a cryptic lineage, which is predominantly northern Eurasian and uses mainly arvicoline rodents (voles) and mice of the genus Apodemus as intermediate hosts. Hydatigera taeniaeformis sensu stricto (s.s.) is restricted to murine rodents (rats and mice) as intermediate hosts. It probably originates from Asia but has spread worldwide. Despite remarkable genetic divergence between H. taeniaeformis s.s. and H. kamiyai, interspecific morphological differences are evident only in dimensions of rostellar hooks. The third cryptic lineage is closely related to H. kamiyai, but its taxonomic status remains unresolved due to limited morphological, molecular, biogeographical and ecological data. This Hydatigera sp. is confined to the Mediterranean and its intermediate hosts are unknown. Further studies are needed to classify Hydatigera sp. either as a distinct species or a variant of H. kamiyai. According to previously published limited data, all three entities occur in the Americas, probably due to human-mediated introductions.

Effects of parasites and antigenic challenge on metabolic rates and thermoregulation in northern red-backed voles (Myodes rutilus).

Perturbations in host energetics are considered to be an essential pathway for parasite impact on host fitness. However, direct estimations of parasite-induced variations in basal metabolic rates of vertebrate hosts have so far provided contradictory results. The energy requirements of immunity and other vital functions may be compromised in energy-demanding conditions in comparison to comfortable conditions; therefore, in our study performed on the wild red-backed vole, Myodes rutilus, we compared the values of indices that reflect metabolic and thermoregulatory responses to acute cooling in individuals that had been naturally infected by gut helminths or Ixodes persulcatus taiga ticks to individuals with no signs of infestation. To consider the possible effects of an acquired immune response on host energetics, we also injected some of the tested individuals with sheep red blood cells (SRBC). Red-backed voles infected by the nematode Heligmosomum mixtum injected with SRBC showed significantly lower cold-induced maximum oxygen consumption than the saline control. Additionally, individuals infected with H. mixtum showed significantly lower oxygen consumption during the final minute of the 15-min acute cooling period and a significantly greater decline in body temperature than individuals free from helminths. In individuals concurrently infected by H. mixtum and the cestodes Arostrilepis horrida, these indices did not differ from helminth-free individuals. The number of ticks simultaneously parasitizing the voles at the moment of capture correlated positively with their SMR. Our results suggest that even natural parasites produce deleterious effects on host aerobic capacity and thermoregulatory abilities, although the effects of different parasites might not be additive.

Genetic diversity of Echinococcus spp. in Russia.

In Russia, both alveolar and cystic echinococcoses are endemic. This study aimed to identify the aetiological agents of the diseases and to investigate the distribution of each Echinococcus species in Russia. A total of 75 Echinococcus specimens were collected from 14 host species from 2010 to 2012. Based on the mitochondrial DNA sequences, they were identified as Echinococcus granulosus sensu stricto (s.s.), E. canadensis and E. multilocularis. E. granulosus s.s. was confirmed in the European Russia and the Altai region. Three genotypes, G6, G8 and G10 of E. canadensis were detected in Yakutia. G6 was also found in the Altai region. Four genotypes of E. multilocularis were confirmed; the Asian genotype in the western Siberia and the European Russia, the Mongolian genotype in an island of Baikal Lake and the Altai Republic, the European genotype from a captive monkey in Moscow Zoo and the North American genotype in Yakutia. The present distributional record will become a basis of public health to control echinococcoses in Russia. The rich genetic diversity demonstrates the importance of Russia in investigating the evolutionary history of the genus Echinococcus.

Seewis virus: phylogeography of a shrew-borne hantavirus in Siberia, Russia.

BACKGROUND: Hantaviral antigens were originally reported more than 20 years ago in tissues of the Eurasian common shrew (Sorex araneus), captured in European and Siberian Russia. The recent discovery of Seewis virus (SWSV) in this soricid species in Switzerland provided an opportunity to investigate its genetic diversity and geographic distribution in Russia. METHODS: Lung tissues from 45 Eurasian common shrews, 4 Laxmann's shrews (Sorex caecutiens), 3 Siberian large-toothed shrews (Sorex daphaenodon), 9 pygmy shrews (Sorex minutus), 28 tundra shrews (Sorex tundrensis), and 6 Siberian shrews (Crocidura sibirica), captured in 11 localities in Western and Eastern Siberia during June 2007 to September 2008, were analyzed for hantavirus RNA by reverse transcription-polymerase chain reaction. RESULTS: Hantavirus L and S segment sequences, detected in 11 S. araneus, 2 S. tundrensis, and 2 S. daphaenodon, were closely related to SWSV, differing from the prototype mp70 strain by 16.3-20.2% at the nucleotide level and 1.4-1.7% at the amino acid level. Alignment and comparison of nucleotide and amino acid sequences showed an intrastrain difference of 0-11.0% and 0% for the L segment and 0.2-8.5% and 0% for the S segment, respectively. Phylogenetic analysis, using neighbor-joining, maximum-likelihood, and Bayesian methods, showed geographic-specific clustering of SWSV strains in Western and Eastern Siberia. CONCLUSIONS: This is the first definitive report of shrew-borne hantaviruses in Siberia, and demonstrates the impressive distribution of SWSV among phylogenetically related Sorex species. Coevolution and local adaptation of SWSV genetic variants in specific chromosomal races of S. araneus may account for their geographic distribution.