Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kvir20 Virulence ISSN: 2150-5594 (Print) 2150-5608 (Online) Journal homepage: https://www.tandfonline.com/loi/kvir20 From moths to caterpillars: Ideal conditions for Galleria mellonella rearing for in vivo microbiological studies Adeline L. Jorjão, Luciane D. Oliveira, Liliana Scorzoni, Lívia Mara A. Figueiredo-Godoi, Marcia Cristina A. Prata, Antonio Olavo C. Jorge & Juliana C. Junqueira To cite this article: Adeline L. Jorjão, Luciane D. Oliveira, Liliana Scorzoni, Lívia Mara A. Figueiredo-Godoi, Marcia Cristina A. Prata, Antonio Olavo C. Jorge & Juliana C. Junqueira (2018) From moths to caterpillars: Ideal conditions for Galleria�mellonella rearing for in�vivo microbiological studies, Virulence, 9:1, 383-389, DOI: 10.1080/21505594.2017.1397871 To link to this article: https://doi.org/10.1080/21505594.2017.1397871 © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Accepted author version posted online: 13 Nov 2017. Published online: 01 Mar 2018. Submit your article to this journal Article views: 1181 View Crossmark data Citing articles: 7 View citing articles https://www.tandfonline.com/action/journalInformation?journalCode=kvir20 https://www.tandfonline.com/loi/kvir20 https://www.tandfonline.com/action/showCitFormats?doi=10.1080/21505594.2017.1397871 https://doi.org/10.1080/21505594.2017.1397871 https://www.tandfonline.com/action/authorSubmission?journalCode=kvir20&show=instructions https://www.tandfonline.com/action/authorSubmission?journalCode=kvir20&show=instructions http://crossmark.crossref.org/dialog/?doi=10.1080/21505594.2017.1397871&domain=pdf&date_stamp=2017-11-13 http://crossmark.crossref.org/dialog/?doi=10.1080/21505594.2017.1397871&domain=pdf&date_stamp=2017-11-13 https://www.tandfonline.com/doi/citedby/10.1080/21505594.2017.1397871#tabModule https://www.tandfonline.com/doi/citedby/10.1080/21505594.2017.1397871#tabModule PROTOCOL From moths to caterpillars: Ideal conditions for Galleria mellonella rearing for in vivomicrobiological studies Adeline L. Jorj~aoa, Luciane D. Oliveiraa, Liliana Scorzonia, L�ıvia Mara A. Figueiredo-Godoia, Marcia Cristina A. Pratab, Antonio Olavo C. Jorgea, and Juliana C. Junqueiraa aDepartment of Biosciences and Oral Diagnosis, Institute of Science and Technology, S~ao Paulo State University (UNESP), S~ao Jos�e dos Campos, S~ao Paulo, Brazil; bEmpresa Brasileira de Agropecu�aria (Embrapa Gado de Leite), Juiz de Fora, Minas Gerais, Brazil ARTICLE HISTORY Received 10 May 2017 Revised 23 October 2017 Accepted 24 October 2017 ABSTRACT Galleria mellonella is a well-accepted insect model for the study of pathogen-host interactions and antimicrobial compounds. The main advantages of this model include the low cost of maintenance, the fast life cycle, the possibility of using a large number of caterpillars and the innate immune system, which is evolutionarily conserved relative to mammals. Because of these advantages, different research groups have been working to implement the rearing of G. mellonella in laboratory conditions. This protocol describes our experience in the rearing of G. mellonella caterpillars for experimental infection models and the influence of different artificial diets on developmental and physiological parameters. Here, we suggest a diet composition that benefits the life cycle of G. mellonella by accelerating the larval phase length and increasing the caterpillar weight. This diet also stimulated the immune system of G. mellonella by increasing the hemolymph volume and hemocyte concentration. In addition, our rearing protocol generated caterpillars that are more resistant to infection by Staphylococcus aureus, Escherichia coli and Candida albicans. A standard G. mellonella rearing protocol is fundamental to minimize external influences on the results, and this simple and easy protocol can support researchers starting to rear G. mellonella. KEYWORDS experimental model; Galleria mellonella; in vivo study; rearing Introduction Mammalian models are the gold standard for in vivo assays; however, ethical problems, high cost and spe- cialized training requirements have led scientists to develop alternatives for in vivo experimentation. The greater wax moth, Galleria mellonella, has been well- accepted by the scientific community as an experimen- tal model for infection with different bacterial and fun- gal species.1–8 This model provides useful information on virulence mechanisms, pathogenesis, and antimi- crobial efficacy.4,9–11 In the last few decades, studies have shown a positive correlation between results obtained in G. mellonella and mammalian models.12–15 Furthermore, this insect model has numerous advan- tages over other in vivo models, such as low cost, the possibility of large-scale breeding, the speed and ease of test execution and interpretation, the ability to incu- bate caterpillars at temperatures between 25�C and 37�C and the evolutionary conservation relative to mammals.16,17 Another advantage of the G. mellonella model is the multiple options available for the facile inoculation of the pathogen.18,19 Most studies introduce the infection by injecting a standardized microbial inoculum;20 however, the infection can also be performed via ingestion or oral delivery.21 Galleria mellonella has a wide geographic distribu- tion and lives naturally in beehives, causing serious problems for the apicultural industry.22 This insect has four developmental stages: egg, caterpillar, pre-pupae/ pupa and adult insect (Fig. 1). The eggs are white to light pink in color and take between 5 and 8 days (24- 27�C) to develop and became caterpillars. The caterpil- lars are creamy-white and measure 1–23 mm.22,23 This stage takes 6–7 weeks at 28–32�C. During this time, caterpillars undergo 8–10 molting stages and spin silk threads across all stages, but only the last instar spins a cocoon.22,23 When the caterpillar ceases feeding and still presents some motility, during the formation of the coccon, characterizes the intermediate stage of the pre-pupae.24 Next, the caterpillars became pupae, which are dark reddish brown and immobilized in cocoons. From pupa to moth takes 1 to 8 weeks, and the insect does not eat throughout this time. Moths are a reddish brown and pale cream color, are active CONTACT Liliana Scorzoni liliscorzoni@yahoo.com.br Universidade Estadual Paulista, J�ulio de Mesquita Filho- Avenida Francisco Jos�e Longo, 777 S~ao Jos�e dos Campos/S~ao Paulo 12245-000, Brazil. © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/ 4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. VIRULENCE 2018, VOL. 9, NO. 1, 383–389 https://doi.org/10.1080/21505594.2017.1397871 http://crossmarksupport.crossref.org/?doi=10.1080/21505594.2017.1397871&domain=pdf mailto:liliscorzoni@yahoo.com.br http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ https://doi.org/10.1080/21505594.2017.1397871 http://www.tandfonline.com during the night and are able to lay 50–150 eggs.22,23,25,26 Male moths are slightly smaller and ligh- ter in color than females. Additionally, female moths present a bifurcated proboscis and a labial palps pro- jecting forward (beak-like appearance).26 Therefore, the labial palps are peaked in females and rounded in males (Fig. 2). The entire life cycle takes approximately 40 days depending on environmental conditions like temperature and food supply.22,23,25,26 The caterpillars of this insect obtain nutrients natu- rally from honey, pupal skins, pollen and beeswax.27 However, the use of a poor artificial diet can cause developmental problems and sometimes death. Previous studies compared natural diets and artificial diets from different sources for G. mellonella rearing and evaluated biological parameters like larval weight, larval phase duration, pupal weight and duration, lon- gevity, egg productivity, immunity and others.22,27–29 Here, we suggest a diet that provides G. mellonella a short life cycle, an increase in hemolymph volume and hemocyte concentration, and a higher caterpillar sur- vival when compared to other diets. Since G. mello- nella is a well-accepted infection model, there is increasing motivation for the use of this alternative animal; however, depending on the country, the avail- ability of these caterpillars for purchase for experimen- tation is limited. Based on this, research centers have started rearing G. mellonella. This study aims to develop a protocol for G. mellonella rearing and to establish a diet that will provide better development of caterpillars in order to obtain a standardized model for future microbiological research. Materials Reagents � Brain-heart Infusion Broth (BHI, Difco) � Sabouraud Dextrose Broth (SDB, Difco) � Phosphate-Buffered Saline (PBS) � Glass or plastic containers with holes in the cover (Moths cage) � Plastic or metal containers with holes in the cover (Caterpillar’s cage) � Cloth-type voile � Cardboard � Filter paper � Spatula � Diet components described in Table 1 � Wax blocks Equipament � Incubator (BOD, Eletrolab) � Hamilton syringes (10 mL capacity, Reno) � Scale (Sartorius) � Centrifuge (MPW-350) Figure 1. Different developmental stages of Galleria mellonella. Eggs (1), approximately 10-day-old caterpillar (2), approximately 20-day- old caterpillar (3), 25-35-day-old caterpillar (4 and 5), approximately 40-day-old caterpillar (last larval stage) (6), pre-pupae and pupae (7 and 8), adult moths (9). Figure 2. Galleria mellonella male (A) and female (B). 384 A. L. JORJeAO ET AL. � Automatic Cell Counter (Countess® Automated Cell Counter-Invitrogen) � Spectrophotometer (B582, Micronal) Methods To initiate G. mellonella rearing in the laboratory of Micro- biology and Immunology at the Institute of Science and Technology (ICT/Unesp/Brazil), we acquired some cater- pillars from Empresa Brasileira de Pesquisa Agropecu�aria (Embrapa, Juiz de Fora, Brazil) that were kindly provided by Dr. Marcia Prata. The caterpillars were fed with 3 differ- ent artificial diets of high nutritional quality as described in Table 1. To prepare diets 1, 2 and 3, all components were weighed and mixed well using a spatula. Pieces of wax blocks were added to supplement the diet. The composi- tions of diets 2 and 3 were based on the protocol described by Mead et al.30 and Brighenti et al.,31 respectively. Procedure of Galleria mellonella rearing For G. mellonella rearing, we established a detailed pro- tocol (Fig. 3) that covers all life stages of this insect model as follows: (1) A total of 10 to 30 caterpillars in the last larval stage (and/or already pupae) were placed in plas- tic/glass containers (+/¡ 30 cm high) with holes in the covers. CAUTION: Use dark containers or protect the containers from light. There is no need to add an exact number of male or female moths. Male moths can fertilize multiple females; moreover, the number of eggs that females lay is high. (2) To support egg-laying, a layer of filter paper was placed in the top of the container followed by a piece of cloth-type voile. The containers were incu- bated at room temperature for 1–3 weeks until most of the moths became old or died. CAUTION: Although pupae and moths do not need to be fed, a little food should be added inside the container to avoid the starvation of very small caterpillars that could appear before egg removal. (3) Twice a week, the filter paper with the eggs was replaced with a new one. The removed filter paper was transferred to a plastic container with a cover with several holes. A paper towel was placed between the cover and the container to prevent small caterpillars from getting out, and wax and Figure 3. Process for Galleria mellonella rearing. Table 1. Dietary components evaluated for Galleria mellonella rearing. Diet 1 Diet 2 Diet 3 20 g of brown sugar 300 g oat flakes 250 g of corn meal 80 g of glycerol 300 g of whole wheat flour 150 g of yeast extract 400 g of powder milk 60 g dried yeast 100 g of soy flour 120 g yeast extract 120 mL of glycerol 100 g of powder milk 200 g of whole wheat flour 120 ml of honey 200 g of honey 200 g of wheat bran beeswax blocks 200 g of glycerol 200 g of wheat germ beeswax blocks beeswax blocks VIRULENCE 385 food were provided. The egg containers were incu- bated at 28�C for approximately 20 days and checked once a week to monitor the caterpillar growth. CAUTION: Eggs are very sensitive and may burst if pressed; extreme care is needed when transferring the eggs. (4) After 20 days, when the caterpillars have reached a sufficient size to be handled (approximately 1 cm), the container was cleaned to remove the webs and cocoons. Then, the caterpillars were separated according to size (small, medium and large, respec- tively, with 1.0, 1.5 and 2.0 cm) and transferred to containers with holes in the covers and food. The caterpillars were maintained at 28�C. Experimental design Evaluation of the effects of nutritious diets on the development of G. mellonella caterpillars To evaluate the effects of diets on the life cycle of G. mel- lonella, 250 eggs were monitored daily to determine the larval phase duration (period from egg to the last larval stage) and the percentage of pupae. Moreover, to study the effects of diet on weight, 30 caterpillars fed different diets were monitored daily. Evaluation of nutritious diets on the physiology of G. mellonella caterpillars For this analysis, we determined the hemolymph volume and hemocytic density of each G. mellonella caterpillar. The hemolymph was collected as described by Harding et al.32 using a pre-chilled Eppendorf tube to avoid the clotting process. The volume of hemolymph collected was measured with a micropipette. To measure hemo- cytic density, the number of cells in the hemolymph was estimated by counting viable cells using Trypan blue in an Automatic Cell Counter. Evaluation of the effects of nutritious diets on the response of G. mellonella to infections with S. aureus, E. coli and C. albicans The susceptibility of G. mellonella caterpillars to micro- bial infection was determined with survival curve assays. Initially, a standardized suspension containing 108 cells/mL was prepared for each strain of microor- ganism: S. aureus ATCC 6538, E. coli ATCC 25922 and C. albicans ATCC 18804. The caterpillars were infected with each microorganism and incubated at 37�C. The survival rate was monitored 18 h post-infection and subsequently every 24 h for the next 7 days. The caterpillars were considered dead when there was no movement after being touched with forceps. Statistical analysis The results were analyzed using GraphPad Prism 6.0 (La Jolla CA, USA), and statistics were generated with ANOVA and Tukey’s post-test. The survival curves were analyzed by the Log-rank test (Mantel-Cox). The signifi- cance level adopted in all analyzes was P < 0.05. Antecipated results Initially, the parameters of the life cycle of caterpillars fed with three different diets were evaluated (Table 2). Diets 1 and 2 increased Galleria mellonella larval stage approxi- mately 10 days when compared to the groups fed diet 3. The caterpillar weights in the three different diet groups were compared as well and there were no significant dif- ferences between the weights of the caterpillars fed with diet 1 or diet 2. However, the weights of the caterpillars fed with diet 3 were higher than the weights of either other group. The analysis of the influence of diet on pupae formation demonstrated that caterpillars fed with diet 1 had a significantly higher percent pupae formation than the other two groups. The lowest percentage of pupae formation was observed in the group fed diet 2. The physiological parameters of G. mellonella fed differ- ent diets were also evaluated. The hemolymph volume and hemocyte density of caterpillars fed diet 3 showed higher values compared to the groups fed diets 1 and 2 (Table 3). The survival rates of caterpillars infected with S. aureus, E. coli and C. albicans are also shown in Table 3. Although no significant difference among the groups for any of the microorganisms studied was observed, the greatest survival rates were found in the groups fed diet 3 (Fig. 4). Discussion and problem handling This study aimed to establish a protocol for G. mellonella rearing that could generate caterpillars to be used as model hosts for experimental infections. It is known that some protocol characteristics are crucial to obtain a culture of G. mellonella with good developmental cycles and the ability Table 2. Larval life cycle parameters of Galleria mellonella groups fed different diets. Diet Larval phase (days) Weight (g) Pulpal (%) Diet 1 42 § 1.53a 0.277 § 0.031a 15.2 § 1.70a Diet 2 49 § 2.52b 0.278 § 0.019a 4 § 1.29b Diet 3 35 § 2.00c 0.365 § 0.033b 8.4 § 0.84c Mean values and standard deviations (§SD) of three independent experi- ments are represented. Different lowercase letters represent significant differences among the groups, according to Tukey’s test (P < 0.05). 386 A. L. JORJeAO ET AL. to generate a large number of healthy and active caterpil- lars. Therefore, we tested three different diets for feeding G. mellonella caterpillars, evaluated their development, physio- logical characteristics and response to infections. To evaluate the development of G. mellonella, we mea- sured the duration of the larval phase and the weight of caterpillars at the last instar. We noticed that caterpillars fed with diet 3 exhibited a shorter larval phase and a higher weight compared to those fed diets 1 and 2. Diet quality has been demonstrated to affect the development of caterpillars, and suitable diets require different nutrients, such as carbohydrates, protein and lipids.26,29 A weak diet results in a long life cycle with a short ovipo- sition period and a lengthy egg incubation period.27 In our study, the composition of diet 3 was the most appro- priate to obtain a short larval phase and, consequently, to generate healthier caterpillars. A fast life cycle has been correlated with the ability to grow and reproduce.33 In the analysis of the development of G. mellonella, we also quantified the percentage of pupae formed. Since we observed a shorter larval phase on diet 3, we expected to find a greater percentage of pupae with this diet. How- ever, diet 3 resulted in 8.4% of pupa being formed, which was between the groups fed diet 1 (15.2%) and diet 2 (4%). Intraspecific factors, such as competition for food and cannibalism (of vulnerable early instars and late instars), may have affected the survival of caterpillars.26 In addition to the diet, temperature and relative humidity are also crucial to the entire life cycle of G. mel- lonella. According to different authors, a temperature range of 25–33�C, as used in our study, ensures an ade- quate length of the G. mellonella life cycle.22,23,25,34 How- ever, we did not monitor humidity during our experiments. Kwada et al.26 reported that a humidity average of 29–33% appears appropriate for the develop- ment of the G. mellonella life cycle. In this study, we also considered an adequate diet one that resulted in caterpillars with suitable amounts of hemolymph and hemocyte concentrations for in vivo experimentation. Among the different diets tested, indi- viduals in the diet 3 group had the highest hemolymph volume and concentration of hemocytes. The hemo- lymph of G. mellonella is responsible for substance and nutrient transport and is composed of six types of hemo- cytes (prohemocytes, plasmatocytes, granulocytes, coa- gulocytes, spherulocytes and oenocytoids) with different functions, including phagocytosis.35 These hemocytes are responsible for cellular immune responses in G. mel- lonella, such as nodule formation and encapsulation.35 Therefore, our results proved that the composition of diet has a significant effect on the G. mellonella immune system. The influence of diet on the insect immune sys- tem was also reported by Siva-Jothy e Thompson36 who found that food deprivation can decrease the immune response activity. In another study, Krams et al.28 dem- onstrated that the composition of the diet is related to immune system activity. A very high-energy diet can Figure 4. Survival curves of Galleria mellonella fed different diets and infected with Staphylococcus aureus (A), Escherichia coli (B) and Candida albicans (C). No significant difference was observed among the groups fed diets 1, 2 or 3 for each microorganism: S. aureus (P = 0.7367), E. coli (P = 0.4010), and C. albicans (P = 0.5027). Table 3. Immune system parameters of Galleria mellonella groups fed different diets. Survival (%) Diet Hemolymph volume (mL) Hemocyte concentrations (x106/mL) S. aureus E. coli C. albicans Diet 1 20 § 4.57a 2.4 § 0.29a 41.67 33.33 33.33 Diet 2 23 § 5.29a 2.8 § 0.17a 41.67 41.67 25.00 Diet 3 48 § 4.20b 5.7 § 0.43b 50.00 58.33 41.67 Mean values and standard deviations (§SD) of three independent experiments are represented. Different lowercase letters represent significant differences among the groups, according to Tukey’s test (P < 0.05). VIRULENCE 387 promote the fast development of body weight, but the immune system can have several deficiencies. Matura- tion is necessary for the immune system to increase the metabolism of caterpillars.28 This fact emphasizes the importance of standardizing the diets during the devel- opment of G. mellonella caterpillars to promote inter- laboratory comparisons. In addition, we evaluated the susceptibility of caterpil- lars to infection with different microorganisms, since this assay gives information important for microbiolog- ical studies. For all the diets tested in our study, it was possible to monitor the infection process up to 7 days, including the infections induced by gram-positive and gram-negative bacteria, as well as a fungal pathogen. According to Champion et al.,37 in the G. mellonella infection model, the survival rate needs to be monitored for up to 5 days post-infection to allow for the calcula- tion of a maximum half lethal dose. In this study, we ver- ified that the survival of caterpillars infected with bacteria and yeast increased 8–25% when they were fed with diet 3 relative to diets 1 and 2. It is known that nutrition is an important parameter to be considered for rearing G. mellonella for microbiological studies.38 Ban- ville et al.38 demonstrated that starvation before experi- mentation could decrease microorganism susceptibility because it causes immune response depletion. Therefore, our results suggested that diet 3 activates the immune response of G. mellonella and makes this insect more resistant to infection by bacteria and yeast. In summary, the protocol described here is simple, easy and can support the rearing of G. mellonella. Addi- tional questions and tips can be found in the Table 4. According to our results, diet 3 showed the best condi- tion for rearing G. mellonella for microbiological studies because this diet provided a short life cycle, increased caterpillar weight and immune system activation. In addition to the physiological benefits, the ingredients for this diet are easy to purchase, and the diet is inexpensive and easy to prepare. Based on this, the standardization of parameters related to rearing as well as the protocol for in vivo experimentation can improve inter-laboratory comparisons and provide reliable results for experiments using G. mellonella. Acknowledgments This work was supported by the following Brazilian organiza- tions: The Fundaç~ao de Apoio �a Pesquisa do Estado de S~ao Paulo (FAPESP) 2015/09770-9 and the Coordenaç~ao de Aper- feiçoamento de Pessoal de N�ıvel Superior (CAPES). Funding S~ao Paulo Research Foundation (FAPESP) ID: 2015/09770-9 References 1. Scorzoni L, de Lucas MP, Mesa-Arango AC, Fusco-Almeida AM, Lozano E, Cuenca-Estrella M, Mendes-Giannini MJ, Zaragoza O. Antifungal efficacy during Candida krusei infection in non-conventional models correlates with the yeast in vitro susceptibility profile. PLoS One. 2013;8: e60047. doi:10.1371/journal.pone.0060047. 2. Achterman RR, Smith AR, Oliver BG, White TC. Sequenced dermatophyte strains: growth rate, conidiation, drug susceptibilities, and virulence in an invertebrate model. Fungal Genet Biol. 2011;48:335–41. doi:10.1016/j. fgb.2010.11.010. 3. Junqueira JC. Galleria mellonella as a model host for human pathogens: recent studies and new perspectives. Virulence. 2012;3:474–6. doi:10.4161/viru.22493. 4. Garc�ıa-Rodas R, Casadevall A, Rodr�ıguez-Tudela JL, Cuenca-Estrella M, Zaragoza O. Cryptococcus neoformans capsular enlargement and cellular gigantism during Galle- ria mellonella infection. PLoS One. 2011;6:e24485. doi:10.1371/journal.pone.0024485. 5. Diago-Navarro E, Chen L, Passet V, Burack S, Ulacia-Her- nando A, Kodiyanplakkal RP, Levi MH, Brisse S, Kreis- wirth BN, Fries BC. Carbapenem-resistant Klebsiella pneumoniae exhibit variability in capsular polysaccharide and capsule associated virulence traits. J Infect Dis. 2014;210:803–13. doi:10.1093/infdis/jiu157. 6. Alghoribi MF, Gibreel TM, Dodgson AR, Beatson SA, Upton M. 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Change the filter paper with the eggs more often. How many male and female moths are necessary for egg position? There is no need to add an exact number of male or female moths. Male moths can fertilize multiple females; moreover, the number of eggs that females lay is high. How can the eggs be removed from the filter paper? It is not necessary to remove the eggs from the filter paper because they are very sensitive. Transfer the paper with the eggs as described. 388 A. L. JORJeAO ET AL. https://doi.org/10.1371/journal.pone.0060047 https://doi.org/10.1016/j.fgb.2010.11.010 https://doi.org/10.1016/j.fgb.2010.11.010 https://doi.org/10.4161/viru.22493 https://doi.org/10.1371/journal.pone.0024485 https://doi.org/10.1093/infdis/jiu157 https://doi.org/10.1371/journal.pone.0101547 https://doi.org/10.1128/IAI.73.7.3842-3850.2005 Galleria mellonella as a heterologous host. Fungal Biol. 2011;115:1279–89. doi:10.1016/j.funbio.2011.09.005. 9. 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VIRULENCE 389 https://doi.org/10.1016/j.funbio.2011.09.005 https://doi.org/10.3109/13693786.2012.737031 https://doi.org/10.1016/j.ijantimicag.2016.05.012 https://doi.org/10.3389/fmicb.2016.00290 https://doi.org/10.1016/j.ijantimicag.2016.07.025 https://doi.org/10.1242/dmm.019901 https://doi.org/10.1093/infdis/jiu441 https://doi.org/10.1111/j.1574-695X.2002.tb00617.x https://doi.org/10.1111/j.1574-695X.2002.tb00617.x https://doi.org/10.1007/s11046-007-9082-z https://doi.org/10.1016/j.bbadis.2013.03.008 https://doi.org/10.4161/viru.1.6.12985 https://doi.org/10.4161/viru.1.6.12985 https://doi.org/10.1007/978-1-4419-5638-5_10 https://doi.org/10.1007/978-1-4419-5638-5_10 https://doi.org/10.1016/j.jip.2009.09.005 https://doi.org/10.3896/IBRA.1.52.1.10 https://doi.org/10.1016/0305-0491(73)90205-8 https://doi.org/10.3390/insects8020061 https://doi.org/10.3390/insects8020061 https://doi.org/10.1111/1744-7917.12132 https://doi.org/10.1016/0022-1910(86)90137-X https://doi.org/10.1016/S0531-5565(03)00159-1 https://doi.org/10.1016/j.femsre.2003.09.002 https://doi.org/10.1016/j.femsre.2003.09.002 https://doi.org/10.1080/21505594.2016.1203486 https://doi.org/10.4161/viru.21972 Abstract Introduction Materials Reagents Equipament Methods Procedure of Galleria mellonella rearing Experimental design Evaluation of the effects of nutritious diets on the development of G. mellonella caterpillars Evaluation of nutritious diets on the physiology of G. mellonella caterpillars Evaluation of the effects of nutritious diets on the response of G. mellonella to infections with S. aureus, E. coli and C. albicans Statistical analysis Antecipated results Discussion and problem handling Acknowledgments Funding References