The Impact of Long-Term Hypoxia on the Antioxidant Defense System in the Siberian Frog Rana amurensis.

The Siberian frog Rana amurensis has a uniquely high tolerance to hypoxia among amphibians, as it is able to withstand several months underwater with almost no oxygen (0.2 mg/liter) vs. several days for other studied species. Since it was hypothesized that hypoxia actives the antioxidant defense system in hypoxia-tolerant animals, one would expect similar response in R. amurensis. Here, we studied the effect of hypoxia in the Siberian frog based on the transcriptomic data, activities of antioxidant enzyme, and content of low-molecular-weight antioxidants. Exposure to hypoxia upregulated expression of three relevant transcripts (catalase in the brain and two aldo-keto reductases in the liver). The activities of peroxidase in the blood and catalase in the liver were significantly increased, while the activity of glutathione S-transferase in the liver was reduced. The content of low-molecular-weight antioxidants (thiols and ascorbate) in the heart and liver was unaffected. In general, only a few components of the antioxidant defense system were affected by hypoxia, while most remained unchanged. Comparison to other hypoxia-tolerant species suggests species-specific adaptations to hypoxia-related ROS stress.

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.

The endosymbiotic bacterium Wolbachia (Rickettsiales) alters larval metabolism of the parasitoid Habrobracon hebetor (Hymenoptera: Braconidae).

Infection of intestinal tissues with Wolbachia has been found in Habrobracon hebetor. There are not many studies on the relationship between Habrobracon and Wolbachia, and they focus predominantly on the sex index of an infected parasitoid, its fertility, and behavior. The actual role of Wolbachia in the biology of Habrobracon is not yet clear. The method of complete eradication of Wolbachia in the parasitoid was developed here, and effects of the endosymbiont on the host's digestive metabolism were compared between two lines of the parasitoid (Wolbachia-positive and Wolbachia-negative). In the gut of Wolbachia+ larvae, lipases' activity was higher almost twofold, and activities of acid proteases, esterases, and trehalase were 1.5-fold greater than those in the Wolbachia- line. Analyses of larval homogenates revealed that Wolbachia+ larvae accumulate significantly more lipids and have a lower amount of pyruvate as compared to Wolbachia- larvae. The presented results indicate significant effects of the intracellular symbiotic bacterium Wolbachia on the metabolism of H. hebetor larvae and on the activity of its digestive enzymes.

Effects of passages through an insect or a plant on virulence and physiological properties of the fungus Metarhizium robertsii.

Species of the genus Metarhizium are characterized by a multitrophic lifestyle of being arthropod parasites, rhizosphere colonizers, endophytes, and saprophytes. The process of adaptation to various organisms and substrates may lead to specific physiological alterations that can be elucidated by passaging through different hosts. Changes in virulence and cultivation properties of entomopathogenic fungi subcultured on different media or passaged through a live insect host are well known. Nevertheless, comparative in-depth physiological studies on fungi after passaging through insect or plant organisms are scarce. Here, virulence, plant colonization, hydrolytic enzymatic activities, toxin production, and antimicrobial action were compared between stable (nondegenerative) parent strain Metarhizium robertsii MB-1 and its reisolates obtained after eight passages through Galleria mellonella larvae or Solanum lycopersicum or after subculturing on the Sabouraud medium. The passaging through the insect caused similar physiological alterations relative to the plant-based passaging: elevation of destruxin A, B, and E production, a decrease in protease and lipase activities, and lowering of virulence toward G. mellonella and Leptinotarsa decemlineata as compared to the parent strain. The reisolates passaged through the insect or plant showed a slight trend toward increased tomato colonization and enhanced antagonistic action on tomato-associated bacterium Bacillus pumilus as compared to the parental strain. Meanwhile, the subculturing of MB-1 on the Sabouraud medium showed stability of the studied parameters, with minimal alterations relative to the parental strain. We propose that the fungal virulence factors are reprioritized during adaptation of M. robertsii to insects, plants, and media.

Special Issue on "Entomopathogenic Fungi: Ecology, Evolution, Adaptation": An Editorial.

Entomopathogenic endophytic ascomycetes are the most widespread and commercially promising fungi and are used to solve many problems in basic and applied research in ecology, evolution, and agricultural sciences [...].

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.

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.

Identification of the Ricin-B-Lectin LdRBLk in the Colorado Potato Beetle and an Analysis of Its Expression in Response to Fungal Infections.

Ricin-B-lectins (RBLs) have been identified in many groups of organisms, including coleopterans insects, particularly the Colorado potato beetle Leptinotarsa decemlineata (LdRBLs). We hypothesized that one of these LdRBLs (LdRBLk) may be involved in the immune response to fungal infections. We performed a theoretical analysis of the structure of this protein. Additionally, the expression levels of the LdRBlk gene were measured in L. decemlineata in response to infections with the fungi Metarhizium robertsii and Beauveria bassiana. The expression levels of LdRBlk in the L. decemlineata cuticle and fat body were increased in response to both infections. The induction of LdRBlk expression was dependent on the susceptibility of larvae to the fungi. Upregulation of the LdRBlk gene was also observed in response to other stresses, particularly thermal burns. Elevation of LdRBlk expression was frequently observed to be correlated with the expression of the antimicrobial peptide attacin but was not correlated with hsp90 regulation. Commercially available ß-lectin of ricin from Ricinuscommunis was observed to inhibit the germination of conidia of the fungi. We suggest that LdRBLk is involved in antifungal immune responses in the Colorado potato beetle, either exerting fungicidal properties directly or acting as a modulator of the immune response.

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.

Galleria mellonella larvae fat body disruption (Lepidoptera: Pyralidae) caused by the venom of Habrobracon brevicornis (Hymenoptera: Braconidae).

The ability of Habrobracon brevicornis venom to elevate the nutritional suitability of a host by affecting the host larvae fat body condition was studied. To understand whether H. brevicornis crude venom impacts the host biochemical profile, the concentrations of total lipids and main sugars in the host larvae lymph were analyzed. All measurements were carried out during the first 3 days after envenomation. A significant increase in the lipid level was fixed only on the second day after envenomation. A significant increase in the total trehalose count was detected during all 3 days, while a significant increase in glucose concentration was noted only on the first day. Well-observed disruptions were fixed in thin and semithin sections of the G. mellonella larval fat body starting from the second day after envenomation. Significant increases in both phospholipase A2 and C enzyme activity as well as acid proteases were detected in the wax moth fat body after envenomation during all experimental times. At the same time, imbalances in the antioxidant system, including changes in the activities of superoxide dismutase, peroxidases, catalase, and glutathione-S-transferase, were detected. The reliable increase in the expression of the gene encoding Hsp70 was fixed both for 24 and 48 h after envenomation, while a reliable increase in the expression of the gene encoding inhibitor of apoptosis protein was detected only 24 h after wax moth larvae envenomation. Considering the absence of DNA fragmentation, the imbalance in the "ROS/antioxidants" system, and the increased activity of phospholipases and acid proteases in the fat body cells from envenomated wax moth larvae, we can hypothesize that the fat body disruption occurs in a necrotic manner. The results of the work expand the knowledge about the biochemical aspects of interaction between ectoparasitoids and their hosts.

Analysis of Anopheles messeae s.l. intron gene polymorphism associated with imidacloprid resistance.

Due to their high solubility and stability, neonicotinoid insecticides are able to accumulate in water bodies, affecting aquatic organisms. The aims of this study were to evaluate resistance (LC50 ) of Anopheles messeae s.l. (Anopheles messeae and An. daciae) to the neonicotinoid imidacloprid and to search for genetic markers associated with insecticide resistance. The LC50 values of these species in the collections during 2017 and 2018 were indistinguishable and were in the range of 0.027-0.051 mg/l. In general, the LC50 values of the mosquitoes were comparable with values of other mosquito species of the Anopheles and Culex genera. Gene polymorphisms of the variations in intron lengths and the presence of restriction sites in introns that were potentially associated with the metabolism of insecticides were studied. Polymorphisms of the studied genes in the pair of closely related species considered overlapped, but allele frequencies were different. Part of the genetic variants arose due to insertions of repetitive elements of the genome. Two variants of the cytochrome P450 gene Cyp6AG1 in An. daciae were associated with increased resistance to imidacloprid. Possible side effects of selection on insecticide resistance in blood-sucking mosquitoes are discussed.

Entomopathogenic fungi decrease Rhizoctonia disease in potato in field conditions.

Rhizoctonia potato disease is widespread in the world and causes substantial yield and quality losses in potato. This study aimed to evaluate the efficacy of entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana in the inhibition of potato Rhizoctonia complex disease. The efficacy of the entomopathogenic fungi M. robertsii and B. bassiana in the defense of potato against Rhizoctonia disease (stem cancer, black scrulf and other forms of manifestation on tubers) was estimated under field conditions in Western Siberia. Preplanting treatment of the tubers with B. bassiana decreased Rhizoctonia disease in the stems and stolons. At the same time, treatment with M. robertsii did not cause a decrease in Rhizoctonia disease in these organs. However, both fungi decreased the sclerotium index on the tubers of new crops. We demonstrated two mechanisms of inhibition of Rhizoctonia solani by M. robertsii and B. bassiana, including (1) direct effect, expressed as inhibition of R. solani sclerotium formation in cocultivation assays, and (2) indirect effect, which is associated with increased peroxidase activity in potato roots under the influence of colonization by entomopathogenic fungi. We suggest that the treatment of seed tubers with B. basiana can effectively manage Rhizoctonia disease during the plant vegetative season and that both fungi significantly improve the quality of the new tuber crop.

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.

The effect of trematode infection on the markers of oxidative stress in the offspring of the freshwater snail Lymnaea stagnalis.

Most invertebrate species exhibit immunological responses that can inactivate and eliminate penetrating parasites. Such immune responses in particular involve the formation of potentially toxic reactive oxygen species (ROS). We explored the immune capabilities of the first-generation (F1) offspring of naturally infected freshwater snails, Lymnaea stagnalis, in response to infection by trematode cercariae under laboratory conditions. The rates of ROS formation and peroxidase activity in the hemolymph of the F1 offspring of L. stagnalis parents infected by an asexual stage of trematodes were significantly higher than in F1 offspring of uninfected parents. Compared to offspring from uninfected parents, the growth rate of F1 snails from infected parents was higher, but survival was lower. After infection of F1 snails by trematode cercariae of Echinoparyphium aconiatum under laboratory conditions, the rate of ROS formation and peroxidase activity in the hemolymph of F1 offspring of uninfected parents increased compared to control snails. This pattern persisted throughout the entire 3-week observation period. In contrast, the rate of ROS formation in the hemolymph of F1 snails from infected parents after experimental infection by E. aconiatum cercariae did not differ from controls, and peroxidase activity even decreased. Thus, trematode parthenitae infection of parents could alter the immune response of their offspring.

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.