Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.
The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models.
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.
The intestinal microbiome is an essential component for maintenance of homeostasis, and involves both innate and adaptive immune responses1,2. The commensal microbiota community is characterized by different main commensal constituents: symbionts that confer beneficial effects by important immunomodulatory functions, and pathobionts that can have detrimental effects in genetically predisposed hosts and promote and trigger intestinal inflammation3,4. Many studies on symbionts and pathobionts and their influence on the host immune system have been published mainly studying adaptive immune responses.
Since these studies involve many animals for the investigations and the protection and replacement of animals used for experimentation is of increasing public interest, we seek to find a replacement model to allow for a screening of different bacterial immunogenic properties. Insects, especially Galleria mellonella, are a widely used replacement model in infection research. G. mellonella combines different advantages such as low costs and high throughput; it allows oral administration of bacteria, which is the natural exposure route, and it allows for systemic infection5,6. G. mellonella further enables incubation at 37 °C, which is the physiological body temperature of mammals and the optimum for bacterial virulence factor expression5. The main advantage of G. mellonella is the conserved innate immune system that enables the discrimination of self from non-self and encodes a variety of pattern recognition receptors like apolipophorin or the opsonin hemolin6,7. Upon microbe recognition, G. mellonella can trigger different downstream humoral immune responses. It can induce oxidative stress responses and secrete reactive oxygen species (ROS) which involves the activity of NOS (nitric oxidase synthase) and NOX (NADPH oxidase)6,8. In addition, G. mellonella activates a potent antimicrobial peptide (AMP) response, which results in the secretion of a mixture of different AMPs such as gloverin, moricin, cecropin or the defensin-like gallerimycin6,8,9,10. Generally, AMPs have quite broad host specificity against Gram-positive and Gram-negative bacteria and fungi and have to provide an potent response since insects are lacking any adaptive response10. Gloverin is an AMP active against bacteria and fungi and inhibits outer membrane formation6,11. Moricins exhibit their antimicrobial function against Gram-positive and Gram-negative bacteria by penetrating the membrane and forming a pore9,11. Cecropins provide activity against bacteria and fungi and permeabilize the membrane similarly like moricins9,10. Gallerimycin is a defensin-like peptide with anti-fungal properties9. Interestingly, it was found that the combination of cecropin and gallerimycin had a synergistic activity against E. coli10.
Due to their easy-to-use character G. mellonella larvae are an often used infection model to assess bacterial pathogenicity. In particular, studies in which data obtained from G. mellonella correlate with data obtained from mice support the strength of this alternative host model. It was found that the most pathogenic serotypes of Listeria monocytogenes in a mouse infection model lead also to higher mortality rates in G. mellonella after systemic infection. Further, less virulent serotypes turned out to be also less virulent in the G. mellonella model12. Similar observations have been made with the human pathogenic fungi Candida albicans. Virulence of different C. albicans strains has been assessed by systemic infection and subsequent monitoring of larval survival. Mouse avirulent strains were also avirulent or exhibited reduced virulence in G. mellonella, whereas the mouse virulent strains lead also to high larval mortality13. The G. mellonella model could further be used to identify type 3 secretion system pathogenicity factors of Pseudomonas aeruginosa14.
Since most investigations involving G. mellonella were focused on virulence factors using the systemic infection approach we were especially interested in providing a method suitable for the analysis of intestinal commensals in an oral force-feeding model in which we can apply a distinct dosage of bacteria per larvae and not only observe the larval mortality rate but analyze different hallmarks of innate immune responses to maintain intestinal homeostasis.
Our method helps to increase the use of G. mellonella as a replacement model since we combine the application of bacteria and the analysis of RNA expression. It is not only useful to strengthen the meaning of bacterial pathogenesis studies when including the analysis of immune responses after oral administration and not only the observation of mortality rates after systemic infection. Our methods allows for the analysis of immunogenic properties of bacterial non-pathogenic commensals since it is provides more complex conditions than cell culture by offering an intestinal barrier in a living organism.
1. G. mellonella rearing and preparation of the larvae for the experiments
NOTE: The cycle from egg to last instar larva takes approximately 5-6 weeks.
2. Cultivation and preparation of Bacteroides vulgatus and Escherichia coli for oral administration
3. Force-feeding of G. mellonella larvae with bacterial suspensions
4. Processing of orally administered larvae and RNA isolation
5. Quantification of the bacterial 16S copy numbers after force-feeding
NOTE: The copy numbers of the expressed bacterial 16S was determined using cDNA synthesized from the RNA extracted in section 4. Final quantification is calculated with the help of a standard curve of plasmid in which the 16S PCR fragment of either B. vulgatus or E. coli was cloned.
6. Determination of innate immune marker gene using quantitative RT-PCR
The G. mellonella hemolymph infection model in widely used to analyze the virulence factors of a huge variety of pathogens. Most measurements include the analysis of larvae mortality, which is a quite easy method. Nevertheless, this method does not allow conclusions about immune responses in general and link the results of G. mellonella immune responses with vertebrate immune mechanisms. The G. mellonella oral administration model on the other hand is only rarely used for oral infection or colonization of the larvae due to the difficulties to obtain exact infection dosage9. Further, only little is known about G. mellonella innate immune responses towards non-pathogenic bacteria especially mammalian intestinal commensals.
In contrast to pathogens, commensals challenge the host and trigger immune responses but the host immune system is able to maintain immune homeostasis. G. mellonella is able to clear the initial force-fed bacterial load until finally no bacteria were detectable anymore (Figure 2)8. The 16s gene copy numbers of both B. vulgatus and E. coli substantially decreased within 24 h.
We demonstrated that commensal-administered G. mellonella larvae induce RNA gene expression of different innate immunity marker genes: LPS-recognition molecules – apolipophorin (ApoIII) and hemolin (Figure 3A,B) were shown to be generally higher expressed in E. coli-administered larvae compared to B. vulgatus-administered larvae8. Further, marker gene expression of two kinds of antimicrobial molecules can be monitored. The production of reactive oxygen and nitrogen species (ROS/RNS) can be estimated by the measurement of Nos and Nox-4 gene expression which were demonstrated to be strongly upregulated upon E. coli force-feeding compared to B. vulgatus (Figure 4A,B)8. Furthermore, gene expression of antioxidative Gst could be observed (Figure 4C).8
In addition we showed that different antimicrobial peptide expression was induced stronger after E. coli administration than in response to B. vulgatus force-feeding. We observed upregulation of defensin-like gallerimycin peptide, LPS-interacting gloverin peptide, cecropin and moricin (Figure 5A,B,C,D)8.
Figure 1: Force-feeding setup using a microsyringe pump. A blunt-ended needle is adjusted into microsyringe pump which allows precise injection of bacteria. Please click here to view a larger version of this figure.
Figure 2: Persistence of bacterial load in Galleria mellonella larvae after force-feeding. Copy numbers of B. vulgatus– and E. coli-specific 16s rDNA genes were determined from 5 ng of cDNA at different time points using RT-PCR. Data points are shown with indication of the median. Modified from reference 8. Please click here to view a larger version of this figure.
Figure 3: Differential pattern recognition of bacteria by G. mellonella. The larvae were administrated with two different intestinal commensals, RNA was isolated after 1-6 h, and mRNA expression of LPS recognition molecule apolipophorin (ApoIII) (A) and hemolin (B) was determined. Data points represent geometric means with standard error of the mean (SEM)(p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).8 Please click here to view a larger version of this figure.
Figure 4: ROS marker gene expression after bacterial challenge. E. coli and B. vulgatus were force-fed and ROS defense marker gene expression was analyzed over time. Nos (A), Nox-4 (B) and Gst (C) mRNA expression was measured in isolated larval RNA. Data points represent geometric means with standard error of the mean (SEM)(p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Modified from reference 8. Please click here to view a larger version of this figure.
Figure 5: Commensal-induced defensin-like antimicrobial peptide expression in G. mellonella larvae and human epithelial cells. Larvae were orally administered with B. vulgatus or E. coli, immune responses were observed over time and RNA was isolated from larval individuals. gallerimycin (A), cecropin (B), gloverin (C), moricin (D) mRNA expression was determined. Data points represent geometric means with standard error of the mean (SEM)(p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Modified from reference 8. Please click here to view a larger version of this figure.
The G. mellonella model is a frequently used model to assess bacterial virulence factors in a systemic infection approach21. Since many pathogens and bacteria enter the host via the oral colonization or infection route, new insights need to be found to evaluate G. mellonella as a model for oral colonization and infection.
The possibility to rear G. mellonella between 15-37 °C is a great advantage since most mammalian models maintain body temperatures of 37 °C5. G. mellonella larvae can be purchased from different suppliers but the establishment of an own breeding population provides many advantages such as the absence of antibiotics that interfere with the assays, better estimation when to start experiments since the suppliers do not always provide larvae in a ready-to-use stage and stress responses are avoided due to transportation or temperature changes. Due to the temperature tolerance of G. mellonella the temperature range at which breeding can be performed is high. Higher temperatures lead to faster development of the larvae and according to the breeding temperature, we can estimate the lifecycle from egg to last instar larva. When larvae were selected for experiments, only pale and fast-moving individuals were chosen to avoid any stress and immune reactions to interfere with the experiments.
In order to establish the force-feeding model, it needs to be assured that the oral application was successful. Therefore, it was helpful to set up several trials for which a strong bromophenol dye was added to the solution intended for force-feeding. This helps to exclude any injured larvae and select for the larvae that have the blue dye only within their gut22.
Using this model, we found that G. mellonella larvae are useful to investigate innate immune response kinetics of certain marker genes. During the establishment of the oral administration model and the study of immune response kinetics we found gene expression to be not locally expressed in the midgut. First experimental trials to extract midgut RNA after oral administration of commensal bacteria did not provide conclusive results. Therefore, the immune responses were determined "globally" in whole individuals. These findings support the hypothesis of global recognition via intestinal receptors, transmission of the signal and triggering extraintestinal gene expression. Generally, G. mellonella is able to induce AMPs mainly in the fat body, but further in hemocytes and the intestinal system9. Since there is no precise information available about tissue-specific production of antimicrobial molecules in G. mellonella larvae after infection, the whole larval RNA was extracted from complete individuals and used for assaying RNA gene expression. A further advantage of whole larval RNA extraction is the complete containment of the living bacteria inside the gut and the possibility to quantify the bacterial load. The dissection of the gut could lead to the loss of bacteria due to preparation.
Since most G. mellonella research is performed on bacterial virulence traits we were especially interested if and how the larvae trigger immune responses towards non-pathogenic bacteria which are part of the mammalian microbiota. Recently, we showed that both G. mellonella and mammals share similar components of the innate immune response, which are homologous and evolutionary conserved. The nitric oxid synthase (Nos) and NADPH oxidase (Nox) genes share a high degree of similarity8. G. mellonella harbors further a defensin-like antimicrobial peptide gallerimycin which shares structural similarities with mammalian β-defensin 28.
Using the oral administration model it was possible to demonstrate differential bacterial recognition of either anti-inflammatory symbiotic B. vulgatus or pro-inflammatory pathobiotic E. coli. In addition downstream oxidative stress responses and antimicrobial peptide production were higher induced after E. coli administration compared to B. vulgatus administration8.
The authors have nothing to disclose.
This work was funded by the DFG (SPP1656), the DFG research training group 1708, the Bundesministerium für Bildung und Forschung (BMBF), and the German Center for Infection Research (DZIF).
1.5 mL tubes | Eppendorf | 0030120086 | |
100 bp DNA ladder | Thermo Fisher Scientific | 15628019 | |
1-Bromo-3-Chloropropane (BCP) | Sigma-Aldrich | B9673 | |
2 mL tubes | Eppendorf | 0030120094 | |
2x Mangomix | Bioline | BIO-25033 | Colony PCR |
50 mL tubes | Greiner Bio-One | 210 261 | |
Agarose | Biozym | 840004 | |
Beeswax | Mixed-Store.de | - | |
Brain heart infusion broth | Thermo Fisher Scientific | CM1135 | |
CloneJET PCR Cloning Kit | Thermo Fisher Scientific | K1232 | Cloning vector for 16S fragments |
Corn grits | Ostermühle Naturkost GmbH | 306 | Organic cultivation |
Difco LB Agar, Miller (Luria-Bertani) | Becton Dickinson | BD | |
Difoco LB Broth, Miller (Luria-Bertani) | Becton Dickinson | 244610 | |
DNA-free DNA Removal Kit | Thermo Fisher Scientific | 244510 | Dnase digestion |
Dried yeast | Rapunzel | - | Organic cultivation |
Dulbecco's Phosphate-Buffered Saline (DPBS) | Thermo Fisher Scientific | 14040 | |
Ethanol | VWR | 20821.330 | |
Glycerol | Sigma-Aldrich | W252506 | |
Honey | Ostermühle Naturkost GmbH | 487 | |
Isopropanol | VWR | 20842.330 | |
Lightcycler 480 Instrument II | Roche Molecular Systems | 5015278001 | |
LightCycler 480 Multiwell Plate 96, white | Roche Molecular Systems | 4729692001 | |
Manual Microsyringe Pump with Digital Display | World Precision Instruments | DMP | |
Micro-Fine+ U-100 insulin syringe 0.3 x 8 mm | Becton Dickinson | 324826 | Oral administration |
Mortar, unglazed | VWR | 410-9327 | |
Nanodrop | Thermo Fisher Scientific | 13-400-518 | |
Nuclease-free water | Thermo Fisher Scientific | 10977035 | |
Oxoid AnaeroGen sachets | Thermo Fisher Scientific | AN0025A | Quality and quantity of RNA |
PCR stripes | Biozym | 710970 | |
Pestle, unglazed grinding surface | VWR | 410-9324 | |
Phusion proof-reading enzyme | Thermo Fisher Scientific | F553S | |
Primers | Biomers | - | |
PureYield Plasmid Miniprep System | Promega | A1222 | |
QuantiFast SYBR Green PCR kit | Qiagen | 204056 | qPCR for bacterial copy number measurment |
QuantiFast SYBR Green RT-PCR Kit | Qiagen | 204156 | qRT-PCR for gene expression measurements |
QuantiTect Reverse Transcription Kit | Qiagen | 205311 | cDNA synthesis |
Qubit Assay Tubes | Thermo Fisher Scientific | Q32856 | |
Qubit dsHS DNA kit | Thermo Fisher Scientific | Q32851 | Quantification of plasmid and cDNA samples |
Qubit fluorometer | Thermo Fisher Scientific | Q33226 | Quantification of plasmid and cDNA samples |
RNase-ExitusPlus | AppliChem | A7153 | |
Rnasin Ribonuclease Inhibitor | Promega | N2511 | |
Skimmed milk powder | Sucofin | - | |
SYBR safe DNA Gel Stain | Thermo Fisher Scientific | S33102 | |
TRI reagent | Sigma-Aldrich | T9424 | |
Weighing boat | VWR | 10803-148 | |
Wheat meal | Ostermühle Naturkost GmbH | 6462 | Organic cultivation |