Involvement of bacteria in the development of fungal infections in the Colorado potato beetle.

Entomopathogenic fungi may interact with insects' symbiotic bacteria during infection. We hypothesized that topical infection with Beauveria bassiana may alter the microbiota of the Colorado potato beetle (CPB) and that these modifications may alter the course of mycoses. We used a model with two concentrations of conidia: (1) high concentration that causes rapid (acute) pathogenesis with fast mortality followed by bacterial decomposition of insects; (2) lower concentration that leads to prolonged pathogenesis ending in conidiation on cadavers. The fungal infections increased loads of enterobacteria and bacilli on the cuticle surface and in hemolymph and midgut, and the greatest increase was detected during the acute mycosis. By contrast, stronger activation of IMD and JAK-STAT signaling pathways in integuments and fat body was observed during the prolonged mycosis. Relatively stable (nonpathogenic) conditions remained in the midgut during both scenarios of mycosis with slight changes in bacterial communities, the absence of mesh and stat expression, a decrease in reactive oxygen species production, and slight induction of Toll and IMD pathways. Oral administration of antibiotic and predominant CPB bacteria (Enterobacteriaceae, Lactococcus, Pseudomonas) led to minor and mainly antagonistic effects in survival of larvae infected with B. bassiana. We believe that prolonged mycosis is necessary for successful development of the fungus because such pathogenesis allows the host to activate antibacterial reactions. Conversely, after infection with high concentrations of the fungus, the host's resources are insufficient to fully activate antibacterial defenses, and this situation makes successful development of the fungus impossible.

An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions.

One of the major pests of potato Solanum tuberosum L. in the temperate zone is the insect Colorado potato beetle (CPB). Most studies on the immunity and diseases of the CPB are conducted during active feeding stages. Nonetheless, there are fewer studies on resting stages, although these beetles spend most of their life cycle in a state of winter diapause (hibernation). In this work, a method for investigating CPB hibernation under natural conditions was developed and tested, offering an opportunity to collect a sufficient number of individuals in winter. In this article, CPB survival was assessed, and infectious agents at different stages of hibernation were identified. CPB mortality increased during the hibernation, reaching a maximum in April-May. Entomopathogenic fungi (Beauveria, Isaria, and Lecanicillium) and bacteria Bacillus, Sphingobacterium, Peribacillus, Pseudomonas, and Serratia were isolated from the dead insects. The survival rate of the beetles for the entire hibernation period was 61%. No frozen or desiccated beetles were found, indicating the success of the presented method.

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.

Tenuazonic acid alters immune and physiological reactions and susceptibility to pathogens in Galleria mellonella larvae.

Tenuazonic acid (TeA) is synthesized by phytopathogenic and opportunistic fungi and is detected in a broad range of foods. This natural compound is of interest in terms of toxicity to animals, but its mechanisms of action on insects are poorly understood. We administered TeA orally at different concentrations (0.2-5.0 mg/[gram of a growth medium]) to the model insect Galleria mellonella, with subsequent estimation of physiological, histological, and immunological parameters in different tissues (midgut, fat body, and hemolymph). Susceptibility of the TeA-treated larvae to pathogenic microorganisms Beauveria bassiana and Bacillus thuringiensis was also analyzed. The feeding of TeA to the larvae led to a substation delay of larval growth, apoptosis-like changes in midgut cells, and an increase in midgut bacterial load. A decrease in activities of detoxification enzymes and downregulation of genes Nox, lysozyme, and cecropin in the midgut and/or hemocoel tissues were detected. By contrast, genes gloverin, gallerimycin, and galiomycin and phenoloxidase activity proved to be upregulated in the studied tissues. Hemocyte density did not change under the influence of TeA. TeA administration increased susceptibility of the larvae to B. bassiana but diminished their susceptibility to B. thuringiensis. The results indicate that TeA disturbs wax moth gut physiology and immunity and also exerts a systemic action on this insect. Mechanisms underlying the observed changes in wax moth susceptibility to the pathogens are discussed.

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 ß-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.

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% (1.3-5×) 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 (2.8-3.5×) 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 (1.4×) of immune-competent plasmatocytes. Oral administration of symbiotic and entomopathogenic bacteria to the CPB larvae significantly decreased the THC (1.4×) after 24 h and increased (1.1-1.5×) 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. © 2022 Society of Chemical Industry.

Can Potato Plants Be Colonized with the Fungi Metarhizium and Beauveria under Their Natural Load in Agrosystems?

Beauveria and Metarhizium fungi are facultative plant endophytes that provide plant growth-stimulating, immunomodulatory, and other beneficial effects. However, little is known about the level of plant colonization by these fungi under natural conditions. We assessed the endophytic colonization of potatoes (Solanum tuberosum) with entomopathogenic fungi at their natural load in soils (102-104 colony-forming units per g). Microbiological analyses of soils and plant organs, as well as a metagenomic analysis of potato roots and leaves, were conducted in three locations in Western Siberia, consisting of conventional agrosystems and kitchen gardens. The fungi were isolated at a relatively high frequency from unsterilized roots (up to 53% of Metarhizium-positive plants). However, the fungi were sparsely isolated from the internal tissues of roots, stems, and leaves (3%). Among the genus Metarhizium, two species, M. robertsii and M. brunneum, were detected in plants as well as in soils, and the first species was predominant. A metagenomic analysis of internal potato tissues showed a low relative abundance of Beauveria and Metarhizium (<0.3%), and the communities were represented primarily by phytopathogens. We suggest that colonization of the internal tissues of potatoes occurs sporadically under a natural load of entomopathogenic fungi in soils. The lack of stable colonization of potato plants with Beauveria and Metarhizium may be due to competition with phytopathogens.

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.

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.

Interplay between Fungal Infection and Bacterial Associates in the Wax Moth Galleria mellonella under Different Temperature Conditions.

Various insect bacterial associates are involved in pathogeneses caused by entomopathogenic fungi. The outcome of infection (fungal growth or decomposition) may depend on environmental factors such as temperature. The aim of this study was to analyze the bacterial communities and immune response of Galleria mellonella larvae injected with Cordyceps militaris and incubated at 15 °C and 25 °C. We examined changes in the bacterial CFUs, bacterial communities (Illumina MiSeq 16S rRNA gene sequencing) and expression of immune, apoptosis, ROS and stress-related genes (qPCR) in larval tissues in response to fungal infection at the mentioned temperatures. Increased survival of larvae after C. militaris injection was observed at 25 °C, although more frequent episodes of spontaneous bacteriosis were observed at this temperature compared to 15 °C. We revealed an increase in the abundance of enterococci and enterobacteria in the midgut and hemolymph in response to infection at 25 °C, which was not observed at 15 °C. Antifungal peptide genes showed the highest expression at 25 °C, while antibacterial peptides and inhibitor of apoptosis genes were strongly expressed at 15 °C. Cultivable bacteria significantly suppressed the growth of C. militaris. We suggest that fungi such as C. militaris may need low temperatures to avoid competition with host bacterial associates.

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 fungal infection on insect physiology has mainly been studied for generalists (Metarhizium, Beauveria), but studies of specialized teleomorphic species, such as Cordyceps militaris, are rare. We conducted a comparative analysis of the immune reactions of the wax moth Galleria mellonella after injection with blastospores of C. militaris (Cm) and Metarhizium robertsii (Mr) in two doses (400 and 4000 per larva). Cm-injected insects died more slowly and were more predisposed to bacterial infections than Mr-injected insects. It was shown that Cm infection led to a predominance of necrotic death of hemocytes, whereas Mr infection led to apoptotic death of cells. Cm-infected insects produced more dopamine and reactive oxygen species compared to Mr-infected insects. Moreover, Cm injection led to weak inhibition of phenoloxidase activity and slight enhancement of detoxification enzymes compared to Mr-injected insects. Blastospores of Cm that were cultivated in artificial medium (in vitro) and proliferated in wax moth hemolymph (in vivo) were characterized by equal intensity of fluorescence after staining with Calcofluor White. In contrast, Mr blastospores that proliferated in the wax moth had decreased fluorescence intensity compared to Mr blastospores grown in medium. The results showed that insects combat Cm infection more actively than Mr infection. We suggest that Cm uses fewer universal tools of killing than Mr, and these tools are available because of specific interactions of Cm with hosts and adaptation to certain host developmental stages.

Bacterial decomposition of insects post-Metarhizium infection: Possible influence on plant growth.

Strains of entomopathogenic fungi may have substantial differences in their final stages of mycosis. Insect cadavers are usually overgrown with mycelium after colonization of the insect body, but in many cases, bacterial decomposition of the colonized hosts occurs. We used two Metarhizium robertsii strains in the work: Mak-1 (cadavers become overgrown with mycelium and conidia) and P-72 (cadavers decay after fungal colonization). We conducted a comparative analysis of gut and cadaver microbiota in Colorado potato beetle larvae using 16S rRNA gene sequencing after infection with these strains. In addition, we estimated the content of different forms of nitrogen in cadavers and the influence of cadavers on the growth of Solanum lycopersicum on sand substrates under laboratory conditions. It was shown that infections did not lead to a significant shift in the midgut bacterial communities of infected insects compared to those of untreated insects. Importantly, bacterial communities were similar in both types of cadaver, with predominantly enterobacteria. Decomposing cadavers (P-72) were characterized by increased nitrate and ammonium, and they had a stronger growth-promoting effect on plants compared to cadavers overgrown with mycelium and conidia (Mak-1). We also estimated the colonization and growth of plants after treatment with conidia of both strains cultivated on artificial medium. Both cultures successfully colonized plants, but strain P-72 showed stronger growth promotion than Mak-1. We propose that the use of deviant strains that are unable to sporulate on cadavers leads to a faster (though only passive) flow of nitrogen from killed insects to plants.

Combined effect of the entomopathogenic fungus Metarhizium robertsii and avermectins on the survival and immune response of Aedes aegypti larvae.

Combination of insect pathogenic fungi and microbial metabolites is a prospective method for mosquito control. The effect of the entomopathogenic fungus Metarhizium robertsii J.F. Bischoff, S.A. Rehner & Humber and avermectins on the survival and physiological parameters of Aedes aegypti (Linnaeus, 1762) larvae (dopamine concentration, glutathione S-transferase (GST), nonspecific esterases (EST), acid proteases, lysozyme-like, phenoloxidase (PO) activities) was studied. It is shown that the combination of these agents leads to a synergistic effect on mosquito mortality. Colonization of Ae. aegypti larvae by hyphal bodies following water inoculation with conidia is shown for the first time. The larvae affected by fungi are characterized by a decrease in PO and dopamine levels. In the initial stages of toxicosis and/or fungal infection (12 h posttreatment), increases in the activity of insect detoxifying enzymes (GST and EST) and acid proteases are observed after monotreatments, and these increases are suppressed after combined treatment with the fungus and avermectins. Lysozyme-like activity is also most strongly suppressed under combined treatment with the fungus and avermectins in the early stages posttreatment (12 h). Forty-eight hours posttreatment, we observe increases in GST, EST, acid proteases, and lysozyme-like activities under the influence of the fungus and/or avermectins. The larvae affected by avermectins accumulate lower levels of conidia than avermectin-free larvae. On the other hand, a burst of bacterial CFUs is observed under treatment with both the fungus and avermectins. We suggest that disturbance of the responses of the immune and detoxifying systems under the combined treatment and the development of opportunistic bacteria may be among the causes of the synergistic effect.

Changes in antifungal defence systems during the intermoult period in the Colorado potato beetle.

Susceptibility to the fungus Metarhizium robertsii and changes in host defences were evaluated in different stages of the intermoult period (4-6 h, 34-36 h and 84-86 h post moult in IV larval instars) of the Colorado potato beetle. A significant thickening of the cuticle during larval growth was accompanied by decreases in cuticle melanization, phenoloxidase activity and epicuticular hydrocarbon contents (C28-C32). At the same time, a decrease in the conidial adhesion rate and an increase in resistance to the fungus were observed. In addition, we recorded significant elevation of the encapsulation rate and total haemocyte counts in the haemolymph during the specified period. The activity of detoxification enzymes decreased in the haemolymph but increased in the fat body during larval growth. No significant differences in the fatty acid content in the epicuticle were observed. The role of developmental disorders in susceptibility to entomopathogenic fungi is also discussed.

Passive vectoring of entomopathogenic fungus Beauveria bassiana among the wax moth Galleria mellonella larvae by the ectoparasitoid Habrobracon hebetor females.

Females of the ectoparasitoid Habrobracon hebetor attack and envenomate numerous host individuals during oviposition. The vectoring of the entomopathogenic fungus Beauveria bassiana during the adhesion stage by ectoparasitoid females among the wax moth larvae Galleria mellonella was explored under laboratory conditions. Vectoring occurred both from infected parasitoids to wax moth larvae and from infected to healthy wax moth larvae by parasitoids. The efficacy of vectoring in both cases was dose dependent. Parasitoid females were unable to recognize infected larvae in a labyrinth test. In addition, the presence of H. hebetor females significantly (1.5-13 fold) increased the mycoses level in clusters of G. mellonella, with 40% of the larvae infected with fungal conidia. Envenomation by H. hebetor increased conidia germination on the cuticles of the wax moth larvae by 4.4 fold. An enhanced germination rate (2 fold) was registered in the n-hexane epicuticular extract of envenomated larvae compared to that of healthy larvae. Both envenomation and mycoses enhanced the phenoloxidase (PO) activity in the integument of G. mellonella and, in contrast, decreased the encapsulation rate in hemolymphs. We hypothesize that changes in the integument property and inhibition of cellular immunity provide the highest infection efficacy of entomopathogenic fungi with H. hebetor.

Effects of fluorine-containing usnic acid and fungus Beauveria bassiana on the survival and immune-physiological reactions of Colorado potato beetle larvae.

BACKGROUND: The search for compounds that interact synergistically with entomopathogenic fungi is aimed at enhancing the efficacy and stability of biological products against pest insects, for example, against the Colorado potato beetle (CPB). We hypothesized that fluorine-containing derivatives of usnic acid (FUA) might be candidates for the development of multicomponent bio-insecticides. The aim of this study was to analyze the co-influence of FUA and Beauveria bassiana on the survival and immune-physiological reactions of CPB larvae. RESULTS: Synergy between FUA and B. bassiana was observed after treatment of second, third and fourth larvae instars under laboratory conditions. Furthermore, synergy was observed in field trials in continental climate conditions in southeastern Kazakhstan. In a field experiment, the median lethal time was shortened three-fold, and cumulative mortality for 15 days increased by 36% in the combined treatment compared with a fungal infection alone. FUA treatment delayed larval development, decreased the total hemocyte count, and increased both the phenoloxidase activity in integuments and the detoxification enzyme rate in hemolymph. A combined treatment with fungus and FUA led to increases in the aforementioned changes. CONCLUSION: Toxicosis caused by FUA provides a stable synergistic effect between FUA and B. bassiana. The combination can be promising for the development of highly efficient products against CPB. © 2017 Society of Chemical Industry.

Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella.

This study examines how the dynamics of fungus-insect interactions can be modulated by temperature. The wax moth, Galleria mellonella, is a well-studied and important model insect whose larvae in the wild develop optimally at around 34 °C in beehives. However, surprisingly little research on wax moths has been conducted at relevant temperatures. In this study, the entomopathogenic fungus Metarhizium robertsii inflicted rapid and substantial mortality on wax moth larvae maintained at a constant temperature of 24 °C, but at 34 °C a 10 fold higher dose was required to achieve an equivalent mortality. The cooler temperature favored fungal pathogenicity, with condial adhesion to the cuticle, germination and hemocoel invasion all significantly enhanced at 24 °C, compared with 34 °C. The wax moth larvae immune responses altered with the temperature, and with the infective dose of the fungus. Enzyme-based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. A dramatic upregulation in the basal expression of galiomicin and gallerimycin was triggered by cooling, and this was augmented in the presence of the fungus. Profiling of the predominant insect epicuticular fatty acids revealed a 4-7 fold increase in palmetic, oleic and linoleic acids in larvae maintained at 24 °C compared with those at 34 °C, but these failed to exert fungistatic effects on topically applied fungus. This study demonstrates the importance of choosing environmental conditions relevant to the habitat of the insect host when determining the dynamics and outcome of insect/fungus interactions, and has particular significance for the application of entomopathogens as biocontrol agents.

Ecological preferences of Metarhizium spp. from Russia and neighboring territories and their activity against Colorado potato beetle larvae.

Thirty-four isolates of Metarhizium spp. from Russian collections were genotyped using 5' EF-1α gene sequence analysis. Four species were identified, of which M. robertsii and M. brunneum were the most frequent, whereas M. anisopliae and M. pemphigum were sporadic. Radial growth studies in the temperature range of 10-40°C revealed that growth at high temperatures (35-37.5°C) was inherent for M. robertsii isolates but not for M. brunneum isolates. In contrast, M. brunneum isolates were more active at cold temperatures (10°C) compared to M. robertsii. Virulence was evaluated against larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, under two regimes: humid (21°C, 80% relative humidity (RH)) and arid (31°C, 55% RH). M. brunneum isolates were less virulent compared to M.robertsii under both regimes. M. robertsii activity did not differ under the two regimes, but M. brunneum was less virulent under the arid regime compared to the humid one. A field experiment under natural conditions (steppe zone of Western Siberia) with daily ranges of 10-43°C and 13-98% RH showed that M. robertsii was significantly more active than M. brunneum against CPB larvae.

Immunological mechanisms of synergy between fungus Metarhizium robertsii and bacteria Bacillus thuringiensis ssp. morrisoni on Colorado potato beetle larvae.

The synergistic effect between the entomopathogenic fungus Metarhizium robertsii and a sublethal dose of the bacterium Bacillus thuringiensis ssp. morrisoni var. tenebrionis was studied in terms of immune defense reactions and detoxification system activity of the Colorado potato beetle, Leptinotarsa decemlineata, fourth instar larvae. Bacterial infection led to more rapid germination of fungal conidia on integuments. We found a significant decrease of cellular immunity parameters, including total hemocyte count and encapsulation response, under the influence of bacteria. Phenoloxidase activity in integuments was increased under bacteriosis, mycosis and combined infection compared to controls. However, phenoloxidase activity in the hemolymph was enhanced under bacteriosis alone, and it was decreased under combined infection. Activation of both nonspecific esterases and glutathione-S-transferases in the hemolymph was shown at the first day of mycosis and third day of bacteriosis. However, inhibition of detoxification enzymes was detected under combined infection. The suppression of cellular immunity and detoxification reactions in Colorado potato beetle larvae with a sublethal dose of bacteria is discussed as a reason for synergy between B. thuringiensis and M. robertsii.

Immune-physiological aspects of synergy between avermectins and the entomopathogenic fungus Metarhizium robertsii in Colorado potato beetle larvae.

The interaction between the entomopathogenic fungus Metarhizium robertsii and natural avermectin metabolites of the actinomycete Streptomyces avermitilis were investigated on Colorado potato beetle larvae. A synergy in the mortality of larvae was detected after simultaneous treatment with half-lethal doses of avermectins (commercial name actarophit) 0.005% and fungus (5×105conidia/ml). The treatment with avermectins led to rapid fungal colonization of the hemolymph. The defense strategies of insects infected by fungus and treated with avermectins and untreated insects were compared to investigate the mechanisms of this synergy. We have shown an increase in hemocytes, especially immunocompetent cells - plasmatocytes and granular cells in the initial stages of mycosis (third day post inoculation). In contrast, avermectins suppressed cellular immunity in hemolymph. Specifically, avermectins dramatically decreased the count of granular cells in larvae infected and uninfected with fungus. Apoptosis inducement and hemocyte necrosis under the influence of avermectins has been shown in vitro as one of the possible reasons for hemocyte mortality. In addition, avermectins enhanced the activity of phenoloxidases in integuments and hemolymph and increased the activity of glutathione-S-transferases activity in the fat body and hemolymph of infected and uninfected larvae, thereby intensifying the development of fungal infection by M. robertsii in Colorado potato beetle larvae. The combination of fungal infection and avermectins constitutes a new perspective for developing multicomponent bioinsecticides.