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

A Diet with Amikacin Changes the Bacteriobiome and the Physiological State of <i>Galleria mellonella</i> and Causes Its Resistance to <i>Bacillus thuringiensis</i>

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

The effect of mixtures of Bacillus thuringiensis-based insecticide and multiple nucleopolyhedrovirus of Lymantria dispar L. in combination with an optical brightener on L. dispar larvae

This study evaluated the efficacy of the commercially available insecticide Lepidocide based on Bacillus thuringiensis var. kurstaki and Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) and their combination with an optical brightener to control L. dispar L. Efficacy against both second and fourth instar L. dispar larvae was evaluated, and the type of interaction between the tested components was determined using second instar L. dispar larvae. Most combinations of Lepidocide and LdMNPV containing a 5 mg ml(-1) optical brightener had synergistic effects, and their mixtures were most effective in reducing the number of second instar larvae. In contrast, mixtures containing Lepidocide and LdMNPV with an optical brightener caused significantly lower mortality of fourth instar L. dispar larvae than mixtures without Lepidocide. This result suggests that an increased concentration of Lepidocide in a mixture containing LdMNPV and an optical brightener leads to an antagonistic effect on insect mortality. The possible reasons for the differences in the observed effects of the components on the second and fourth instar L. dispar larvae may be associated with the resistance of fourth-instar larvae to the antifeedant effect of B. thuringiensis

Citrobacter freundii, a natural associate of the Colorado potato beetle, increases larval susceptibility to Bacillus thuringiensis

BACKGROUND We assume that certain representatives of gut microflora mediate immune changes during dysbiosis, accelerating septicemia caused by Bacillus thuringiensis. RESULTS Co-introduction of Citrobacter freundii with Bacillus thuringiensis var. tenebrionis (morrisoni) (Bt) led to an increase in Colorado potato beetle (CPB) larval mortality to 69.0% (1.3-5x) and a synergistic effect was observed from day 1 to day 6. Ultrathin sections of the CPB midgut showed autophagosome formation and partial destruction of gut microvilli under the influence of Bt, which was accompanied by pronounced hypersecretion of the endoplasmic reticulum with apocrine vesicle formation and oncotic changes in cells under the action of C. freundii. The destruction of gut tissues was accompanied by suppression of detoxification processes under the action of the bacteria and a decrease (2.8-3.5x) in the concentration of lipid oxidation products during Bt infection. In the first hours post combined treatment, we registered a slight increase in the total hemocyte count (THC) especially a predomination (1.4x) of immune-competent plasmatocytes. Oral administration of symbiotic and entomopathogenic bacteria to the CPB larvae significantly decreased the THC (1.4x) after 24 h and increased (1.1-1.5x) the detoxifying enzymes level in the lymph. These changes are likely to be associated with the destruction of hemocytes and the need to remove the toxic products of reactive oxygen species. CONCLUSION The obtained results indicate that feeding of C. freundii and B. thuringiensis to the CPB larvae is accompanied by tissue changes that significantly affect the cellular and humoral immunity of the insect, increasing its susceptibility to Bt

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

Microbial community structure in a host–parasite system: the case of Prussian carp and its parasitic crustaceans

Aims: The aim of the study was to investigate the skin microbiota of Prussian carp infested by ectoparasites from the genera Argulus and Lernaea. Methods and Results: Associated microbiota of skin of Prussian carp and ectoparasites were investigated by sequencing of the V3, V4 hypervariable regions of 16S rRNA using Illumina MiSeq sequencing platform. Conclusions: According to the Spearman rank correlation test, the increasing load of ulcerations of the skin of Prussian carp was weakly negatively correlated with reduction in the abundance of the following taxa: Acrobacter, bacteria C39 (Rhodocyclaceae), Rheinheimera, Comamonadaceae, Helicobacteraceae and Vogesella. In this study, the microbiota of ectoparasites from the genera Lernaea and Argulus were characterized for the first time. The microbiota associated with L. cyprinacea was significantly different from microbial communities of intact skin mucosa of both infested and uninfested fish and skin ulcers (ADONIS, P ≤ 005). The microbiota associated with parasitic crustaceans L. cyprinacea were dominated by unclassified bacteria from Comamonadaceae, Aeromonadaceae families and Vogesella. The dominant microbiota of A. foliaceus were represented by Flavobacterium, Corynebacterium and unclassified Comamonadaceae. Significance and Impact of the Study: Results from these studies indicate that ectoparasites have the potential to alter skin microbiota, which can play a possible role in the transmission of secondary bacterial infections in fish, caused by pathogenic bacteria

Influence of Bacillus thuringiensis and avermectins on gut physiology and microbiota in Colorado potato beetle: Impact of enterobacteria on susceptibility to insecticides.

Gut physiology and the bacterial community play crucial roles in insect susceptibility to infections and insecticides. Interactions among Colorado potato beetle Leptinotarsa decemlineata (Say), its bacterial associates, pathogens and xenobiotics have been insufficiently studied. In this paper, we present our study of the survival, midgut histopathology, activity of digestive enzymes and bacterial communities of L. decemlineata larvae under the influence of Bacillus thuringiensis var. tenebrionis (morrissoni) (Bt), a natural complex of avermectins and a combination of both agents. Moreover, we estimated the impact of culturable enterobacteria on the susceptibility of the larvae to Bt and avermectins. An additive effect between Bt and avermectins was established regarding the mortality of the larvae. Both agents led to the destruction of midgut tissues, a decrease in the activity of alpha-amylases and alkaline proteinases, a decrease in the Spiroplasma leptinotarsae relative abundance and a strong elevation of Enterobacteriaceae abundance in the midgut. Moreover, an elevation of the enterobacterial CFU count was observed under the influence of Bt and avermectins, and the greatest enhancement was observed after combined treatment. Insects pretreated with antibiotics were less susceptible to Bt and avermectins, but reintroduction of the predominant enterobacteria Enterobacter ludwigii, Citrobacter freundii and Serratia marcescens increased susceptibility to both agents. We suggest that enterobacteria play an important role in the acceleration of Bt infection and avermectin toxicoses in L. decemlineata and that the additive effect between Bt and avermectin may be mediated by alterations in the bacterial community