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Gut biota profiles in Bt infected and uninfected R and S line larvae. Profile of the bacterial community in midguts from fourth instar larvae from both resistant and susceptible lines on the second day post Bt infection. Values are averaged across 4 independent control (uninfected) and 4 infected samples of each line. (A) Bacteria classified by phylum and (B) Comparison of community, classified by class, from infected and uninfected R and S line larvae (p < 0.01, p < 0.001 compared with infected insects from the corresponding line).
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Microevolutionary mechanisms of resistance to a bacterial pathogen were explored in a population of the Greater wax moth, Galleria mellonella, selected for an 8.8-fold increased resistance against the entomopathogenic bacterium Bacillus thuringiensis (Bt) compared with a non-selected (suspectible) line. Defense strategies of the resistant and susce...
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Context 1
... expression of AMPs, IMPI, stress and inflammation management genes compared with the S larvae (3-6-fold change) (Fig. 1B, Table S1). Furthermore, in comparing the midgut with the fat body, the R line expression of growth factors was signifi- cantly higher (p < 0.01) but AMPs / immunity and stress management significantly lower (p < 0.05) (SI Fig. ...
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... S and R line larvae revealed that bacterial communities were domi- nated by only a few phyla, with over 99.5% of the community being represented by 4 phyla (average rel- ative abundance values averaged across all uninfected larvae): Firmicutes (80.7 § 6.3%), Proteobacteria (11.8 § 4.5%), Actinobacteria (3.9 § 1.6) and Bacter- oidetes (3.1 § 1.1) (Fig. ...
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... of both lines with Bt led to a shift in domi- nance from the Firmicutes (80.7 § 6.3%) to the Proteo- bacteria (86.3 § 2.6%) (p < 0.001) (Fig. 4A, 4B). Uninfected R line had significantly more Enterobacter than the S larvae, however, upon infection with Bt the levels were both much elevated but to the same degree (SI, Fig. 6). Pseudomonas was present at similar but low levels in uninfected R and S larvae, but Bt infection resulted in opposite effects on the 2 lines. In the case of ...
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... was detected, while there was an increase in the S line relative to the uninfected insects (p < 0.05, Fig. 6). Phenomena common to both lines were the disappearance of several genera (e.g. Micrococcineae) post-infection and a huge shift in dominance from Enterococcus (Gram Cve) in unin- fected to Enterobacter (Gram ¡ve) in infected insects ( Fig. 4; Fig. 6). No Bacillaceae were detected in unin- fected R and S lines but small amounts (2-3%) were detected post-infection (Fig. 6). Most striking was the significant reduction in richness and diversity of bacte- rial communities in the midgut of the infected R line, because such changes were not observed in the S line (Fig. 5). In the ...
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The human pathogenic bacteria Bacillus cereus , Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germinatio...
Citations
... There have been limited published studies on how Bt toxins or Bt impact the microbiota in insect guts [36]. In Galleria mellonella and P. xylostella, both Cry toxins and Bt infection can dramatically decrease the variety and titer of gut microbes [37,38]. On the other hand, a research project carried out by Jiang et al. [39] involving honeybees demonstrated that the presence of genetically modified maize pollen expressing Cry Bt did not have a significant effect on the diversity of symbiotic bacteria in their gut. ...
... This leads to the activation of antimicrobial peptides, melanization, and stem cell growth as the host tries to combat the harm caused by Bt infection [40,41]. Exposure to harmful pathogens like Bt toxins leads to dysbacteriosis, which triggers the activation of antimicrobial peptide genes and oxidative stress [20,37,42]. Maintaining a balance in the gut microbiome is crucial, and these factors play a key role in achieving this [43]. ...
... The diversity analysis revealed a reduction in the diversity and richness of intestinal bacteria in the Bt-tolerant larvae compared with the susceptible strain. Similarly, Dubovskiy et al. [37] revealed a decrease in both the variety and quantity of microorganisms in the intestines of a Bt-resistant strain of the Greater wax moth, G. mellonella, which are vast and plentiful. Exposure to Cry1Ab/2Ab toxins resulted in a significant alteration in the makeup of the intestinal bacteria with a decrease in the overall load of symbiotic bacteria in Locusta migratoria [87]. ...
Bacillus thuringiensis (Bt) represents one of the most economical biopesticides to date. It produces toxins with insecticidal activity against many agricultural pests, including members of the genus Spodoptera. However, Bt tolerance leads to inefficiency in biological control. To overcome this problem, discovering the hidden cause(s) for the evolution of insect tolerance against Bt is of great importance. We hypothesized that changes in the gut microbiota due to the frequent application of Bt is one of those hidden causes. To investigate this hypothesis, we studied the effect of Bt Cry1c application on the Spodoptera littoralis larval gut microbiota in both Bt-susceptible and Bt-tolerant populations. The results revealed changes in the diversity and abundance of gut bacterial composition between the susceptible and tolerant populations. A high abundance of Enterococcaceae was detected in the tolerant population. Interestingly, Cry1c tolerance eliminates the bacterial genera Klebsiella and Serratia from the larval midgut. These changes may confirm the mechanism developed by Spodoptera larvae to counteract Bt Cry1c toxicity. Understanding the B. thuringiensis–gut microbiota interaction may help in improving biocontrol strategies against agricultural pests to overcome the evolution of tolerance.
... However, with the large-scale cultivation of genetically modified crops, the frequency of resistance in cotton bollworm has significantly increased worldwide (Shelton et al., 2002;Jin et al., 2015;Ahmad et al., 2019). Research indicates that the development of cotton bollworm resistance is largely attributed to internal microorganisms and indigenous gut bacteria increasing the tolerance of larvae to B. thuringiensis toxin (Takatsuka and Kunimi, 2000;Dubovskiy et al., 2016). In addition, the resistance of larvae to the entomopathogenic bacterium B. thuringiensis is affected by the maturity of the host plants. ...
Yeast is one of the important symbiotic flora in the insect gut. However, little is known about the gut yeast in Helicoverpa armigera (Lepidoptera: Noctuidae) under various dietary conditions. The composition and function of the intestinal yeast community also remain unclear. In this research, we explored the composition of yeast microorganisms in H. armigera larvae under different feeding environments, including apple, pear, tomato, artificial diet (laboratory feeding), Urtica fissa, Helianthus annuus, and Zinnia elegans (wild environment) using high-throughput sequencing. Results showed that a total of 43 yeast OTU readings were obtained, comprising 33 yeast genera and 42 yeast species. The yeast genera with a total content of more than 5% were Hanseniaspora (36.27%), Moesziomyces (21.47%), Trichosporon (16.20%), Wickerhamomyces (12.96%) and Pichia (6.38%). Hanseniaspora was predominant when fed indoors with fruits, whereas Moesziomyces was only detected in the wild group (Urtica fissa, Helianthus annuus, Zinnia elegans) and the artificial diet group. After transferring the larvae from artificial diet to apple, pear and tomato, the composition of intestinal yeast community changed, mainly reflected in the increased relative abundance of Hanseniaspora and the decreased abundance of Trichosporon. Simultaneously, the results of α diversity index indicated that the intestinal yeast microbial diversity of H. armigera fed on wild plants was higher than that of indoor artificial feeding. PCoA and PERMANOVA analysis concluded that there were significant differences in the gut yeast composition of H. armigera larvae on different diets. Our results confirmed that gut yeast communities of H. armigera can be influenced by host diets and may play an important role in host adaptation.
... Finally, an imbalance in the gut microbiota may alter the activity of digestive enzymes and food digestibility, as well as reduce host viability and breeding [8][9][10]. Toxicosis progression is often accompanied by an increased count of bacteria belonging to certain groups and an imbalance in the microbiota [11][12][13][14]. The presence of certain microbiota in the gut definitely facilitates the host's adaptation to different diets and metabolites [15][16][17] and plays a significant role in toxin degradation [18][19][20]. ...
Simple Summary
The insect gut microbiota plays a crucial role in the host’s resistance to pathogenic microorganisms and toxins. Resident microorganisms may persist for a long time and maintain a certain location due to the synthesis of antimicrobial agents (including antibiotics). We consider it one of the adaptation mechanisms of microorganisms in various communities. Not only do the uncontrolled use of antibiotics and changes in environmental conditions drastically alter the microbiota structure and give rise to resistant microorganisms, but they also cause a number of alterations in the host’s physiology and its sensitivity to pathogens. This study opens up new prospects for further research into antibiotic-resistant symbiotic microorganisms, their benefits for persistence in the dynamic environment of the insect’s gut, and the conditions necessary for changing their strategy and manifestation of virulent properties.
Abstract
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.
... Any observed effects on the immune response were as a result of the presence of this genus of bacteria. A previous study also found only Enterococcus species in the G. mellonella gut (52); however, more recently published studies have also identified, along with Enterococcus but in lower abundances, Enterobacter, Bacillus, Halomonas, Shewanella, Pseudomonas, Variovorax, Staphylococcus, Serratia, and Escherichia-Shigella present in the late-larval instar of G. mellonella (28,41,(53)(54)(55)(56). With a more diverse microbiota, shifts in the dominance between bacterial groups post-pathogen infection have been observed. ...
... With a more diverse microbiota, shifts in the dominance between bacterial groups post-pathogen infection have been observed. In particular, studies led by Dubovskiy et al. (41) and Grizanova (2022) found that Enterobacter (a genus of Gramnegative bacteria) maintained a minor presence in the gut community of G. mellonella at basal conditions, but proliferated during infection by Btg to overwhelm and dominate the community. Similar shifts have been observed post-fungal infection with Beauveria bassiana (54). ...
... In addition, we recognize that the immune response is modulated by additional genes than those tested in this study, as well as molecules not traditionally considered "immune genes" but which are also important for the immune response (41,73). Therefore, a wider view using a larger transcriptomic approach may provide a more comprehensive understanding of the immune system stimulation by the gut microbiota both before and during infections. ...
Understanding the intricate interplay between the gut microbiota and the immune response in insects is crucial, given its diverse impact on the pathogenesis of various microbial species. The microbiota’s modulation of the host immune system is one such mechanism, although its complete impact on immune responses remains elusive. This study investigated the tripartite interaction between the gut microbiota, pathogens, and the host’s response in Galleria mellonella larvae reared under axenic (sterile) and conventional (non-sterile) conditions. The influence of the microbiota on host fitness during infections was evaluated via two different routes: oral infection induced by Bacillus thuringiensis subsp. galleriae ( Btg ), and topical infection induced by Metarhizium robertsii ( Mr ). We observed that larvae without a microbiota can successfully fulfill their life cycle, albeit with more variation in their developmental time. We subsequently performed survival assays on final-instar larvae, using the median lethal dose (LD 50 ) of Btg and Mr . Our findings indicated that axenic larvae were more vulnerable to an oral infection of Btg ; specifically, a dose that was calculated to be half-lethal for the conventional group resulted in a 90%–100% mortality rate in the axenic group. Through a dual-analysis experimental design, we could identify the status of the gut microbiota using 16S rRNA sequencing and assess the level of immune-related gene expression in the same group of larvae at basal conditions and during infection. This analysis revealed that the microbiota of our conventionally reared population was dominated entirely by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two antimicrobial peptides (AMPs)—gallerimycin and gloverin—in the conventional larvae compared with the axenic larvae. Furthermore, Enterococcus mundtii , isolated from the gut of conventional larvae, showed inhibition activity against Btg in vitro . Lastly, other immune effectors, namely, phenoloxidase activity in the hemolymph and total reactive oxygen/nitrogen species (ROS/RNS) in the gut, were tested to further investigate the extent of the stimulation of the microbiota on the immune response. These findings highlight the immune-modulatory role of the Enterococcus- dominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host’s response to oral and topical infections.
... However, the microbiota diversity was significantly lower in ACB-AbR in the present study and D. virgifera Bt-resistant strains [16], but was higher in C. suppressalis Cry1Ca resistant strains [21]. In addition, Bt strain infection significantly reduced the richness and diversity of the gut microbiota in the Galleria mellonella-resistant strains [47]. The Cry1Ac treatment significantly reduced the bacterial diversity and changed the composition of the microbiota in P. xylostella [19]. ...
Bacillus thuringiensis is an effective entomopathogen, and its crystal toxin expressed in transgenic crops has been widely used for pest control. However, insect resistance risk is the main threat to the continued successful utility of Bt crops. Several studies reported the role of midgut microbiota in Bt resistance, but the mechanism remains controversial. In the present study, using high-throughput sequencing of the bacterial 16S ribosomal RNA gene, we surveyed the midgut bacterial flora of Ostrinia furnacalis from one Bt-susceptible (ACB-BtS) and two Bt-resistant (ACB-AbR and ACB-FR) strains and explored the mortality of O. furnacalis after eliminating the gut bacteria. Gut bacterial diversity in Bt-resistant strains was significantly lower in Bt-resistant than in Bt-susceptible strains. Ordination analyses and statistical tests showed that the bacterial community of ACB-AbR was distinguishable from ACB-BtS. The genus Halomonas was dominated in ACB-BtS, but the unclassified_Enterobacteriaceae was the most enriched genus in ACB-AbR and ACB-FR. Furthermore, interactions of the bacterial community are more complex in Bt-resistant strains than in Bt-susceptible strains. Moreover, the mortalities of ACB-AbR and ACB-BtS strains treated by the Cry1Ab toxin were significantly reduced after eliminating the gut bacteria. Our findings suggest that Bt stressors structured in the midgut bacterial community and the microbiota have the potential to regulate the Bt-induced killing mechanism.
... Our data showed that the diversity of microbial communities was significantly reduced in M. separata feeding on insect-resistant transgenic maize, but the relative abundance of E. mundtii and E. cloacae was significantly increased. The large-scale variations in bacterial communities were consistent with previous reports (Dubovskiy et al. 2016;Paddock et al. 2021). The changes in the of microbial community caused by Bt crop treatment were detrimental to insect growth and development, which might be an important insecticidal mechanism of Bt toxins acting on target insects. ...
Although the gut microbiota has been well reported to contribute to the pest insecticidal activity of Bacillus thuringiensis (Bt) toxin, the underlying mechanism remains largely unknown. Here, bioassay, RNA-seq, and 16S rRNA sequencing were employed to explore the potential mechanism by which gut microbiota enhance the virulence of Bt maize to Mythimna separata. Two groups of neonate larvae were fed with the leaves of conventional maize (CK group) and insect-resistant transgenic maize (Bt group), respectively. The results indicated that body weight, body length, and survival rate of M. separata in the Bt group were significantly reduced. Additionally, the induced growth retardation further led to normal mating failure and fecundity deprivation of M. separata in Bt group. The 16S rRNA sequencing results indicated that exposure to insect-resistant transgenic maize significantly decreased the diversity of the bacterial community and increased the relative abundance of Enterobacter cloacae and Enterococcus mundtii by 153.02-folds and 2.08-folds, respectively. Reintroduction of these two bacteria significantly increased the sensitivity of M. separata to Bt toxin. Transcriptome results revealed that numerous genes related to Notch, Hippo, Wnt, amino acid synthesis, and fatty acid metabolism pathways were significantly down-regulated in the Bt group, whereas almost all Bt toxin receptor aminopeptidase N genes were up-regulated. This study revealed that exposure to insect-resistant transgenic maize reduced gut bacterial diversity and altered the gene expression of M. separata. Furthermore, E. cloacae and E. mundtii enhanced the susceptibility of M. separata to Bt toxin. Our findings on the interaction between Bt toxins, gut microbiota, and the host insect will contribute to the development of pest management strategies and the promotion of transgenic crops in China.
Graphical abstract
... An investigation revealed that the diversity of bacterial microbiota in the tolerant Aedes aegypti larvae is reduced after Bt infection (Tetreau et al., 2018). In the Bt-resistant line of Galleria mellonella, Bt infection significantly reduced the diversity and abundance of the gut microbiota, possibly due to secreted antimicrobial factors in the resistant insects (Dubovskiy et al., 2016). The gut bacteria also affect Bt HD-1 insecticidal activity against Plodia interpunctella (Hübner) (Orozco-Flores et al., 2017). ...
The gut microbiota is essential for the growth and development of insects, and the intestinal immune system plays a critical role in regulating the homeostasis of intestinal microorganisms and their interactions with pathogenic bacteria. Infection with Bacillus thuringiensis (Bt) can disrupt the gut microbiota of insects, but the regulatory factors governing the interaction between Bt and gut bacteria are not well understood. Uracil secreted by exogenous pathogenic bacteria can activate DUOX-mediated reactive oxygen species (ROS) production, which helps maintain intestinal microbial homeostasis and immune balance. To elucidate the regulatory genes involved in the interaction between Bt and gut microbiota, we investigate the effects of uracil derived from Bt on gut microbiota, and host immunity using a uracil deficient Bt strain (Bt GS57△ pyrE ) obtained by homologous recombination. We analyze the biological characteristics of the uracil deficient strain and found that the deletion of uracil in Bt GS57 strain changed the diversity of gut bacteria in Spodoptera exigua , as investigated using Illumina HiSeq sequencing. Furthermore, qRT-PCR analysis showed that compared with Bt GS57 (control), the expression of the SeDuox gene and the level of ROS were significantly decreased after feeding with Bt GS57△ pyrE . Adding uracil to Bt GS57△ pyrE restored the expression level of DUOX and ROS to a higher level. Additionally, we observed that PGRP-SA , attacin, defensin and ceropin genes were significant different in the midgut of S. exigua infected by Bt GS57 and Bt GS57△ pyrE , with a trend of increasing first and then decreasing. These results suggest that uracil regulates and activates the DUOX-ROS system, affects the expression of antimicrobial peptide genes, and disturb intestinal microbial homeostasis. We preliminarily speculate that uracil is a key factor in the interaction between Bt and gut microbiota, and these findings provide a theoretical basis for clarifying the interaction between Bt, host, and intestinal microorganisms, as well as for gaining new insights into the insecticidal mechanism of B. thuringiensis in insects.
... Quantitative reverse transcription-PCR (qRT-PCR) was performed with the SYBR green method in a reaction mixture volume of 15 mL, containing 0.5 mL of cDNA sample, 10 mM forward and reverse primers, and 1Â GoTaq qPCR master mix (Promega). The primers and conditions for denaturation, annealing, and extension for each pair of primers are listed in Table S1 (53,65,87,88). ...
For many years, Streptococcus anginosus has been considered a commensal colonizing the oral cavity, as well as the gastrointestinal and genitourinary tracts. However, recent epidemiological and clinical data designate this bacterium as an emerging opportunistic pathogen. Despite the reported pathogenicity of S. anginosus, the molecular mechanism underpinning its virulence is poorly described. Therefore, our goal was to develop and optimize efficient and simple infection models that can be applied to examine the virulence of S. anginosus and to study host-pathogen interactions. Using 23 S. anginosus isolates collected from different infections, including severe and superficial infections, as well as an attenuated strain devoid of CppA, we demonstrate for the first time that Dictyostelium discoideum is a suitable model for initial, fast, and large-scale screening of virulence. Furthermore, we found that another nonvertebrate animal model, Galleria mellonella, can be used to study the pathogenesis of S. anginosus infection, with an emphasis on the interactions between the pathogen and host innate immunity. Examining the profile of immune defense genes, including antimicrobial peptides, opsonins, regulators of nodulation, and inhibitors of proteases, by quantitative PCR (qPCR) we identified different immune response profiles depending on the S. anginosus strain. Using these models, we show that S. anginosus is resistant to the bactericidal activity of phagocytes, a phenomenon confirmed using human neutrophils. Notably, since we found that the data from these models corresponded to the clinical severity of infection, we propose their further application to studies of the virulence of S. anginosus.
... This suggests an initial level of environmental stress placed on the larvae, which over time, the insect acclimates to. The higher levels of viable bacteria in the faeces may be due to impaired antimicrobial peptides (AMP) synthesis upon ionising radiation treatment (Dubovskiy et al., 2016). This effect of environmental stress leading to Fig. 7. Ionising radiation affected encapsulation in Galleria mellonella. ...
Larvae of the greater wax moth Galleria mellonella are common pests of beehives and commercial apiaries, and in more applied settings, these insects act as alternative in vivo bioassays to rodents for studying microbial virulence, antibiotic development, and toxicology. In the current study, our aim was to assess the putative adverse effects of background gamma radiation levels on G. mellonella. To achieve this, we exposed larvae to low (0.014 mGy/h), medium (0.056 mGy/h), and high (1.33 mGy/h) doses of caesium-137 and measured larval pupation events, weight, faecal discharge, susceptibility to bacterial and fungal challenges, immune cell counts, activity, and viability (i.e., haemocyte encapsulation) and melanisation levels. The effects of low and medium levels of radiation were distinguishable from the highest dose rates used – the latter insects weighed the least and pupated earlier. In general, radiation exposure modulated cellular and humoral immunity over time, with larvae showing heightened encapsulation/melanisation levels at the higher dose rates but were more susceptible to bacterial (Photorhabdus luminescens) infection. There were few signs of radiation impacts after 7 days exposure, whereas marked changes were recorded between 14 and 28 days. Our data suggest that G. mellonella demonstrates plasticity at the whole organism and cellular levels when irradiated and offers insight into how such animals may cope in radiologically contaminated environments.
... A series of signalling cascades occurs between insect and fungal molecules produced in battle during the binding and invading of entomopathogenic fungal conidia to an insect integument (Mukherjee and Vilcinskas, 2018). Moreover, G. mellonella produces antimicrobial peptides (AMP) that regulates gut microbiota and the higher levels of viable bacteria in the faeces may be due to impaired AMP synthesis upon UV-treatment (Dubovskiy et al., 2016). Therefore, our study, while using an insect model that lacks an adaptive immune system, complements those conclusions from mammalian and other insect studies, suggesting the existence of a novel integument-gut axis in insects. ...
For humans, acute and chronic overexposure to ultraviolet (UV) radiation can cause tissue damage in the form of sunburn and promote cancer(s). The immune-modulating properties of UV radiation and health-related consequences are not well known. Herein, we used the larvae of the wax moth (Galleria mellonella) to determine UV-driven changes in cellular components of innate immunity. From immune cell (haemocyte) reactivity and the production of antimicrobial factors, these insects share many functional similarities with mammalian cellular innate immunity. After exposing insects to UVA or UVB for up to two hours, we monitored larval viability, susceptibility to infection, haemolymph (blood) physiology and faecal discharge. Prolonged exposure of larvae to UVB coincided with decreased survival, enhanced susceptibility to bacterial challenge, melanin synthesis in the haemolymph, compromised haemocyte functionality and changes in faecal (bacterial) content. We contend G. mellonella is a reliable in vivo model for assessing the impact of UV exposure at the whole organism and cellular levels.
