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

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

Can Insects Develop Resistance to Insect Pathogenic Fungi?

This paper presents new, important information on the microevolution of insect resistance to the insect pathogenic fungus Beauveria bassiana which will have far-reaching implications for the development of insect pathogenic fungi as biological control agents. We placed successive generations of a melanic population of the Greater wax moth, Galleria mellonella, under constant selective pressure from the insect pathogenic fungus, Beauveria bassiana. Enhanced fungal resistance was observed and larvae from the 25th generation were studied in detail to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. There are 3 novel, core findings from the study:1.Antifungal resistance in these insects is pathogen species-specific, and probably arises through trans-generational immune priming. The resistance was less obvious in earlier generations, suggesting subtle cumulative changes that are only fully apparent in the 25th generation. 2.The insect’s fecundity is already pushed close to minimum by its melanic phenotype. Therefore, the additional drain on resources required to boost antifungal defence still more, comes not from further compromising life history traits but via a re-allocation of the insect’s immune defences. Specifically during B. bassiana infection, systemic (fat body and hemocoel) responses, particularly the expression of antimicrobial peptides, are damped down in favour of a tailored repertoire of enhanced responses in the integument (cuticle and epidermis) – the foremost and most important barrier to natural fungal infection. 3.A previously-overlooked range of putative stress-management factors are activated during the specific response of selected insects to B. bassiana. This too occurs primarily in the integument, and contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses during the battle between host and pathogen.No other study to date has examined so many genes in this context. Indeed, we show that the epidermis has a great capacity to express defense and stress-management genes as well as the fat body (which is the main tissue producing antimicrobial peptides and has been the traditional focus of attention). We therefore propose a “be specific / fight locally / de-stress” model to explain resource allocation and defence priorities for insects selected for superior resistance to insect-pathogenic fungi. However, we also show that these insects are less fecund and probably at no evolutionary advantage in the wild, implying that the risk is small of biological control agents failing in the field

Expression of Immunity- and Stress-Related Genes during an Intermolt Period in the Colorado Potato Beetle

Different developmental stages of insects may be dissimilar in immunity functioning. Additionally, the stages often inhabit diverse environments with specific microbial communities. In the Colorado potato beetle, a strong increase in resistance to entomopathogenic fungi is observed during the intermolt period of last-instar larvae, but mechanisms of this change are insufficiently understood. We studied changes in the expression of immunity- and stress-related genes in the fat body and integument during this intermolt period by quantitative PCR. By the end of the instar, there was upregulation of transcription factors of Toll, IMD, and Jak–Stat pathways as well as genes encoding metalloprotease inhibitors, odorant-binding proteins, and heat shock proteins. Nonetheless, the expression of gene LdRBLk encoding β-lectin did not change during this period. Most of the aforementioned genes were upregulated in response to Metarhizium robertsii topical infection. The expression alterations were more pronounced in recently molted larvae than in finishing feeding larvae and in the integument compared to the fat body. We believe that upregulation of immune-system- and stress-related genes at the end of the intermolt period is an adaptation caused by migration of larvae into soil, where the probability of encountering entomopathogenic fungi is high

Comparative analysis of the immune response of the wax moth Galleria mellonella after infection with the fungi Cordyceps militaris and Metarhizium robertsii

Entomopathogenic fungi form different strategies of interaction with their insect hosts. The influence of funga

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

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

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