TYPE Review PUBLISHED 20 October 2022 DOI 10.3389/fmicb.2022.991678 Drosophila melanogaster as a OPEN ACCESS model to study innate immune EDITED BY Axel Cloeckaert, Institut National de Recherche pour memory l’Agriculture, l’Alimentation et l’Environnement (INRAE), France Marta Arch1,2† , Maria Vidal1,2,3,4† , Romina Koiffman1,5† , REVIEWED BY Sofia J. Araújo, Solomon Tibebu Melkie1,5 and Pere-Joan Cardona1,2,3,4,6* University of Barcelona, Spain 1 Tuberculosis Research Unit, Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain, Tiina Susanna Salminen, 2 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Spain, Tampere University, Finland 3 Comparative Medicine and Bioimage Centre of Catalonia (CMCiB), Germans Trias I Pujol Research Bruno Lemaitre, Institute (IGTP), Badalona, Spain, 4 Microbiology Department, Laboratori Clínic Metropolitana Nord, Swiss Federal Institute of Technology Germans Trias i Pujol University Hospital, Badalona, Spain, 5 UCBL, UnivLyon, Université Claude Lausanne, Switzerland Bernard Lyon 1 (UCBL1), Villeurbanne, France, 6 Centro de Investigación Biomédica en Red en *CORRESPONDENCE Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Madrid, Spain Pere-Joan Cardona pj.cardona@gmail.com † These authors have contributed equally to this work Over the last decades, research regarding innate immune responses has SPECIALTY SECTION This article was submitted to gained increasing importance. A growing body of evidence supports the Infectious Agents and Disease, notion that the innate arm of the immune system could show memory a section of the journal traits. Such traits are thought to be conserved throughout evolution and Frontiers in Microbiology provide a survival advantage. Several models are available to study these RECEIVED 11July 2022 ACCEPTED 03 October 2022 mechanisms. Among them, we find the fruit fly, Drosophila melanogaster. This PUBLISHED 20 October 2022 non-mammalian model has been widely used for innate immune research CITATION since it naturally lacks an adaptive response. Here, we aim to review the latest Arch M, Vidal M, Koiffman R, Melkie ST and Cardona P-J (2022) Drosophila advances in the study of the memory mechanisms of the innate immune melanogaster as a model to study response using this animal model. innate immune memory. Front. Microbiol. 13:991678. doi: 10.3389/fmicb.2022.991678 KEYWORDS COPYRIGHT Drosophila melanogaster, innate immune memory, trained immunity, tolerance, © 2022 Arch, Vidal, Koiffman, Melkie resistance, infection and Cardona. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original Introduction publication in this journal is cited, in accordance with accepted academic The immune system can be generally divided into an innate and an adaptive arm practice. No use, distribution or reproduction is permitted which does (Medzhitov and Janeway, 2000). For long, the dogma in immunology has been that the not comply with these terms. innate immune response is rapid and non-specific, while the adaptive immune response is slower, but antigen-specific and led to long-term immunological memory. However, it is unlikely for a crucial trait like immune memory to be restricted to the adaptive arm of the immune response when more than 95% of species do not rely on this immune system and many studies have provided pieces of evidence that some vaccines and infections protect against secondary exposure in a specific or unspecific way (Hirano et al., 2011; Conrath et al., 2015; Reimer-Michalski and Conrath, 2016). Up to date, several models have been proposed for studying innate immune memory, such as plants, vertebrates, and invertebrates (Netea et al., 2011, 2020). Frontiers in Microbiology 01 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 The fruit fly Drosophila melanogaster has been recognized as as a result of deepening their study (also listed in Box 1). an outstanding model to study host-pathogen interactions and The concept of “trained immunity” was first proposed by immunity (Lemaitre and Hoffmann, 2007; Buchon et al., 2014; Netea et al. (2011), being referred as a long-lasting functional Galenza and Foley, 2019; Aromolaran et al., 2021). Over the reprogramming of innate immune cells after exposure to a years, several infection models have been evaluated in the fruit microorganism or an inflammatory stimulus that lasts over fly, so a deep understanding of the molecular mechanisms taking time and leads to an altered response toward a second place in the host after infection has been gained [Extensively challenge. Nevertheless, this reprogramming has also been seen reviewed lately in Younes et al. (2020) and Michael Harnish et al. in epithelial stem cells, showing faster mobilization and higher (2021)]. induction of interferon-stimulated genes during secondary In this review, we have first compiled the latest insights challenges (Naik et al., 2017, 2018). The first exposure of the regarding the host’s immune response to consecutive challenges host to the challenge is defined by the authors in the field and the subsequent acquisition of innate immune memory. as priming (Cooper and Eleftherianos, 2017; Sheehan et al., Secondly, we outlined the relevant characteristics that make 2020). As perfectly detailed in the review by Bindu et al. (2022), of D. melanogaster a good model for understanding innate the innate immune reprogramming happening after priming immune memory and the methods available to assess it both is given via epigenetic and metabolic modifications of trained orally and systematically. cells which can lead either to an increase or a decrease in immunity (Boraschi and Italiani, 2018). The mechanisms of this reprogramming are not completely understood yet, though Innate immune memory some evidence supports the existence of multiple regulators (Ghisletti et al., 2010; Smale et al., 2014; Fanucchi et al., 2019; Definition and mechanisms of innate Natoli and Ostuni, 2019). In addition, new studies have also immune memory suggested changes in DNA methylation patterns and changes in cellular metabolism as a mediator of trained immunity An increasing number of evidences that infections or (Donohoe and Bultman, 2012; Cheng et al., 2014; Arts et al., exposure to microbial-derived compounds can induce not 2016; Verma et al., 2017; Das et al., 2019; Penkov et al., 2019). only specific protection against reinfection but also non- RNA interference (RNAi) was recently put in light as specific protection against a subsequent challenge with another an important immune priming mechanism which confers pathogen have been described in mice models (Di Luzio and an adaptive antiviral response (Tassetto et al., 2017). This Williams, 1978; Krahenbuhl et al., 1981; Muñoz et al., 2010; mechanism is based on a post-transcriptional gene regulation Marakalala et al., 2013; Ribes et al., 2014). It has also been mechanism by which small interfering RNAs (siRNAS) induce described that vaccination with bacille Calmette-Guerin (BCG) the sequence-specific degradation of homologous messenger conferred protection against a range of infectious diseases in RNA (mRNA) sustaining the antiviral effect along time after both mice (Van’t Wout et al., 1992; Kaufmann et al., 2018; first exposure (Meng and Lu, 2017). In addition to the Covián et al., 2019; Witschkowski et al., 2020) and humans cell-autonomous immunity conferred to the infected cell, (Garly et al., 2003; Aaby et al., 2011; Biering-Sørensen et al., this antiviral RNAi signals have shown its ability to spread 2017; Rieckmann et al., 2017; Arts et al., 2018; Walk et al., 2019) systematically conferring innate immune memory (Tassetto as well as induced antitumoral effects through the activation et al., 2017). This antiviral mechanism and its priming capacity of innate immune cells like monocytes and macrophages have been reported in vertebrates, invertebrates and plants, (Villumsen et al., 2009; Stewart and Levine, 2011; Buffen although the spreading mechanisms differ between taxa (Meng et al., 2014; Redelman-Sidi et al., 2014). The non-specificity of and Lu, 2017; Tassetto et al., 2017; Cooper and Van Raamsdonk, this protection, the shortened and reversible protective effect 2018; Gourbal et al., 2018; Javdat and Tamara, 2020). (Nankabirwa et al., 2015; Netea et al., 2016; Dominguez-Andres Despite this, the term trained immunity is also used and Netea, 2019) and the evidence that plants and invertebrates, by various authors to refer to the immune phenotype the which only rely on innate immunity, also showed greater cells obtain after a primary challenge which allows them to protection against reinfections (Dimopoulos et al., 2002; Moret react quicker and stronger to secondary one, thus producing and Siva-Jothy, 2003; Rodrigues et al., 2010; Norouzitallab an increase in the immune response to a stimulus (Netea et al., 2016; Reimer-Michalski and Conrath, 2016; Melillo et al., et al., 2020); instead, other authors suggest more accurate 2018; Lanz-Mendoza and Contreras-Garduño, 2022) revealed terms which describe this phenomenon such as “non-specific that immunological adaptation may also occur on the innate acquired resistance,” “potentiation,” or “trained potentiation” immunity. (Boraschi and Italiani, 2018). On the other hand, although The nomenclature regarding the innate immune memory this reprogramming of innate immunity to an inflammatory acquisition process is still controversial. We describe below the profile provides a great advantage in host defense, it may also basic concepts used in this field and how they have evolved be detrimental in the context of chronic inflammatory diseases. Frontiers in Microbiology 02 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 BOX 1 Innate immune memory related terms. Trained innate immunity/Innate immune memory Long-lasting functional reprogramming of innate immune cells after exposure to a microorganism or an inflammatory stimulus that leads to an altered response toward subsequent challenges (Netea et al., 2011, 2016). Priming Phenomenon of memory induction which consists of the exposure of dead microbes or sublethal doses of live pathogens to the host in order to activate the innate immune response (Cooper and Eleftherianos, 2017; Sheehan et al., 2020). Innate immune reprogramming Transcriptional and epigenetic modifications, accompanied and regulated by induced metabolic changes, happening in innate immune cells when exposed to stimulus (Boraschi and Italiani, 2018; Bindu et al., 2022). Non-specific acquired resistance/Trained potentiation Priming-induced enhancement of the innate immune response to subsequent challenges (Boraschi and Italiani, 2018). Trained tolerance Priming-induced downregulation of the innate immune response to subsequent challenges (Boraschi and Italiani, 2018). As compensatory mechanism it has also been shown the Resistance and tolerance as opposite reaction, being the priming-induced downregulation, consequences of innate immune which results in a reduced response after a secondary stimulus memory (Cooper and Eleftherianos, 2017; Boraschi and Italiani, 2018; Uribe-Querol and Rosales, 2020). Authors will be referring as Upon infection, there are two major strategies to fight “tolerance” or “trained tolerance” to this also considered innate disease: resistance and tolerance. Relating these two types of memory phenomenon (Boraschi and Italiani, 2018). Tolerance host’s responses to the adaptive programs exposed by Divangahi is also induced and maintained by epigenetic changes. Analysis et al. (2020) trained immunity and priming would relate to an of this mechanism revealed that lipopolysaccharides (LPS) increased host resistance to reinfection, and tolerance would administration could induce alterations in the chromatin that refer to a tolerogenic host response to reinfection. In this silenced pro-inflammatory genes but not antimicrobial effector regard, resistance strategies will aim at killing the pathogen or genes (Seeley and Ghosh, 2017; Hajishengallis et al., 2019; Lajqi inhibit its proliferation (Boraschi and Italiani, 2018). However, et al., 2021; Figure 1). mounting an immune response is energetically costly, leading Whether adaptation of innate immune cells will enhance to a reduction in nutrient storage, growth, and reproduction in or reduce the immune response after a second challenge will the organism. This suggests a strong link between immunity depend on the dose and the duration of the first stimulus as and host fitness (Stearns, 1989; Schwenke et al., 2016). The well as on the kind of stimuli which not necessarily has to be health status of a host does not always depend on the number the same source in both challenges (Divangahi et al., 2020). of pathogens it can bear. Instead, it depends on the ability Based on the functional status of these cells before the second to reduce the effects of the damage and stress (Chow and challenge, Divangahi et al. (2020) published a review with an Kagan, 2018). Tolerance strategies will reduce the negative effect on fitness caused by the infection, but will not have an updated definition of the different adaptive programs a cell can impact on pathogen fitness (Howick and Lazzaro, 2017). Since incorporate. In addition to trained immunity and tolerance, the mechanisms of disease tolerance do not reduce pathogen in which the immune genes expression return to basal levels load, they should promote the transmission of the infectious after the first stimulus is removed and the response after the agent in the population. Some pathogens like Salmonella second challenge is increased or decreased, respectively, these enterica typhimurium induce tolerance to increase their spread. authors also define differentiation and priming as two adaptive However, throughout evolution, reducing infections, tolerance programs affecting a cell innate immune response. The change and resistance mechanisms became highly interconnected of an immature cell into its mature counterpart is defined as (Howick and Lazzaro, 2017). For example, by increasing host cell differentiation and often comes together with morphological fitness, tolerance responses will aim at reducing tissue damage changes; although differentiation can occur during homeostatic and thus, allowing the resistance mechanisms to work more conditions, differentiated cells can be trained as well after potently (Martins et al., 2019). Besides, by increasing host infection or vaccination (Lavin et al., 2014). On the other hand, fitness, tolerance mechanisms give time to induce resistance when the first stimulus changes the state of the innate immune strategies to eliminate the pathogen (Chow and Kagan, 2018; cells and the active gene expression does not return to basal Sheehan et al., 2020). Disease tolerance can also be important levels before the second challenge it is named “priming,” and in cases where the immune response is the one generating the often the response to the second stimulus is synergistic with the damage, for example, in the case of sepsis (Chow and Kagan, first one (Divangahi et al., 2020; Figure 1). 2018). In some situations, the activation of one pathway can Frontiers in Microbiology 03 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 FIGURE 1 Changes after the induction of innate immune memory. Priming is characterized by an activation of gene expression in innate immune cells that is sustained over time and does not return to basal levels before the second challenge. Often the response to the second stimulus is synergistic with the first one. In trained immunity the gene expressions levels return to basal levels when the first stimulus is removed, but epigenetic changes persist favoring a faster and higher immune response in subsequent infections. Finally, tolerance is the opposite to trained immunity, where after activation by a first stimulus and returning to basal levels the immune response is reduced in subsequent challenges. The flat dotted line represents the basal activation levels of the innate immune response in absence of infection. Adapted from Divangahi et al. (2020) and Netea et al. (2020). contribute to tolerance and resistance mechanisms. Autophagy, features with vertebrates including immune cascades, signal for example, can reduce pathogen burden, thus contributing to transduction pathways, and transcriptional regulators (Younes disease resistance. However, in other contexts, it can contribute et al., 2020), as well as a significant amount of well-conserved to disease tolerance by reducing endothelial barrier destruction homologs of disease-causing genes in humans (Bier, 2005). (Chow and Kagan, 2018). Evidence suggests that different These facts make the fruit fly a good model for studying innate organs have different tolerance capacities since they can be more immune responses and gene functionality in basic research. susceptible to damage or have a higher tissue replication rate, As in vertebrates, D. melanogaster immune system is also allowing a higher reparation rate. Moreover, the consequences divided into humoral and cellular responses. The main mode of tissue damage can vary according to the tissue. For instance, of action of the humoral response in D. melanogaster is the damages in the endothelium can compromise vascular integrity production of antimicrobial peptides (AMPs). After a systemic and lead to ischemia and tissue necrosis, whereas skin damage infection, AMPs are released into the hemolymph where they may not be life-threatening (Medzhitov et al., 2012). persist for several days and can protect the flies against a second exposure to the pathogen (Boman et al., 1972). AMPs can act synergically or be highly specific depending on the Drosophila melanogaster as a pathogen (Imler and Bulet, 2005; Hanson et al., 2019, 2022; Hanson and Lemaitre, 2020). This response is mediated by model for studying innate immune the fat body, which is the equivalent of the liver in mammals memory and represents the main immune-responsive organ in the fly (Lemaitre and Hoffmann, 2007). Barrier epithelial cells are also Drosophila melanogaster innate able to secrete AMPs and reactive oxygen species (ROS) in immune response response to a localized infection (Tzou et al., 2000). Besides this, lower levels of AMPs are also expressed from the hemocytes, The use of D. melanogaster as a model has provided a muscles, Malpighian tubules, and neuronal tissues (Charroux huge insight into the mechanisms of action of the innate and Royet, 2009; Badinloo et al., 2018). Three main signaling immunity, as insects rely solely on this type of response thus pathways have been described to play a role in the regulation avoiding the variability that adaptive mechanisms imply. In of immune genes induced after infection. The Toll and the addition, D. melanogaster presents a high degree of conserved immune deficiency (Imd) pathways regulate the majority of Frontiers in Microbiology 04 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 immune genes, including the production of AMPs. The Toll of vertebrates’ macrophages and neutrophils (Stramer et al., pathway is activated in response to the detection of lysine 2005). Melanization is another predominant immune response (Lys)-type peptidoglycan of Gram-positive bacteria, and β- in insects based on the production and release of melanin glucans of yeasts and fungi, as well as through the sensing around intruding microorganisms (Christensen et al., 2005). of danger signals, like microbial proteases, or abnormal cell This response is involved in wound healing and hemolymph death. On the other hand, the Imd pathway is activated coagulation, and is mediated by the crystal cells (Ashida and through the detection of diaminopimelic acid (DAP)-type Brey, 1995; Söderhäll and Cerenius, 1998; Tang, 2009; Leitão peptidoglycan from Gram-negative and certain Gram-positive et al., 2020; Younes et al., 2020). Lastly, lamellocytes are bacteria (Buchon et al., 2014; Figure 2). The study done by differentiated from plasmatocytes when the cell-mediated innate Kutzer and Armitage (2016) revealed that the Imd and Toll immune response is induced upon infection with parasitoid pathways have different kinetics since the AMPs triggered by wasp, wounding or artificially by genetic induction, and are those pathways were observed at different times after infection. in charge of the encapsulation of foreign bodies that are too An infection of flies with a gram-negative bacterium such as large to be phagocytosed, as well as melanization (Kounatidis E. coli that triggered the Imd pathway showed the highest AMP and Ligoxygakis, 2012; Honti et al., 2014; Dudzic et al., 2015; concentration after 6 h. In contrast, when challenged with a Vlisidou and Wood, 2015; Leitão et al., 2020). gram-positive bacterium like Lactococcus lactis, thus triggering In Drosophila, the mechanisms by which a septic infection the Toll pathway, the peak of AMPs was observed 24 h post- causes a systemic response controlled by the fat body have been infection. These differences in immune pathways contribute well-characterized, even though these types of infections are to the differences in the clearance of different pathogens rare. However, although the so-called natural infections take (Kutzer and Armitage, 2016). In addition, a different pattern of place constantly, the epithelial immune response remains less activation is also observed between males and females (Duneau characterized. It is known that this local response is mainly D. F. et al., 2017; Belmonte et al., 2020). Finally, Drosophila also regulated by the Imd-pathway (Onfelt Tingvall et al., 2001) has a complete yet more simple JAK/STAT signaling pathway, and is shaped against different microbes via the JAK/STAT and which is involved in diverse biological processes, including early the JNK signaling pathways (Wagner et al., 2009). In 2013, a and late development, innate immunity, germ-cell adhesion, microarray analysis performed by Gendrin et al. (2013) showed and inhibition of apoptosis (De Gregorio et al., 2001; Boutros that the induction of Imd-dependant genes varies substantially et al., 2002). This pathway also contributes to the immune among tissues with only very few “universal genes” being response by inducing the transcription of thioester-containing expressed in the fat body, the gut, and the trachea (Gendrin et al., protein genes and Turandot stress genes, which, both play a 2013). This set of genes includes mainly AMPs and pathway role in Drosophila defense against pathogens (Lagueux et al., components. 2000; Agaisse et al., 2003; Dostálová et al., 2017). In addition, Specifically in the gut, local immunity includes physical and a communication axis between the humoral pathway Imd and chemical barriers as well as a cellular response (Buchon et al., JAK/STAT which controls fly antiviral immune response has 2013). ROS are induced by two enzymes: dual oxidase (Duox) is been recently described (Shen et al., 2022). stimulated through pathogen-derived uracil and peptidoglycan Cell-mediated innate immunity of Drosophila comprises of (Lee et al., 2013) and NADPH oxidase (Nox) is induced by immune blood cells, which can be found freely circulating in microbiota-derivate lactate (Iatsenko et al., 2018). In the gut, the hemolymph or associated with diverse tissues. These cells, Duox-derived ROS are mainly involved in immune response which are collectively called hemocytes, can be differentiated and repair tissue damage, while Nox-derived ROS regulate according to their morphology and immunological functions epithelial renewal (Iatsenko et al., 2018). However, excessive in plasmatocytes, lamellocytes and crystal cells (Lanot et al., production of ROS is ultimately toxic to the host and induces 2001; Hartenstein, 2006; Hultmark and Andó, 2022). Recently, epithelial cell death and early ageing, thus Duox expression a new class of cells have been identified in Drosophila, the is tightly regulated through the p38 mitogen-activated protein primocytes, whose functions are not fully elucidated, but they kinase–activating transcriptional factor 2 (p38 MAPK–Atf2) are thought to regulate the larval hematopoietic organ which, in pathway (Ha et al., 2009b). The uracil secreted by some turn, controls the hematopoiesis of other hemocytes (Hultmark pathogenic bacteria activates the phospholipase Cβ (PLCβ), and Andó, 2022). In addition, the presence and proportion of which in turn activates the p38MAPK-Atf2 pathway. The these cells may vary depending on the developmental stage PLCβ pathway also promotes the release of Ca2+ from the the animal (Honti et al., 2014). Phagocytosis represents a endoplasmic reticulum to bind and activate Duox (Ha et al., fundamental process of the innate immune response and in 2009a). In response to commensals, the levels of both uracil and the maintenance of tissue homeostasis. In D. melanogaster peptidoglycan are lower and the concentration of cytosolic Ca2+ this process is mainly performed by plasmatocytes, which is reduced, thus Duox activity is kept minimal (Ha et al., 2009b). represent up to 90% of the total circulating hemocytes Several studies have proved that an interorgan (Wang et al., 2013) and appear to perform the functions communication occurs in D. melanogaster by proving that Frontiers in Microbiology 05 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 FIGURE 2 Immune recognition of microorganisms in Drosophila. The two major pathways that sense bacteria and fungi in fruit flies are the Toll pathway (left) and the immune deficiency (Imd) pathway (right). Both pathways function in the fat body for the production of antimicrobial peptides (AMP) by activating the expression of NF-κB-like factors, which are highly conserved among species. In addition, the Imd pathway also functions in the epithelial surfaces. The Imd pathway is activated when DAP-type peptidoglycan from gram-negative bacteria, and some gram-positive, binds to Peptidoglycan Recognition Proteins (PGRPs), and this activation leads to the generation of AMP and synthesis of Duox enzyme for the production of reactive oxygen species (ROS). Gram-positive bacteria contain lys-type peptidoglycan, which is recognized by PGRP-SA and Gram Negative Bacteria Protein (GNBP) 1, and GNBP3 binds to β-glucans of yeasts and fungi, leading to the activation of the Toll pathway. This pathway can also be triggered by danger signals like proteases or abnormal cell death that activates the protease Persephone (Psh). In all cases, the activation of the Toll pathway triggers a proteolytic cascade that activates the protease Spätzle- processing enzyme (SPE). This protein will cleave Spätzle (Spz). As a result of the activation of the Toll pathway, the transcription factors Dorsal-related immunity factor (Dif) or Dorsal (Dl) will translocate to the nucleus, thus inducing the expression of AMP genes like drosomycin. Similarly, the activation of the Imd pathway induces the nuclear translocation of the transcription factor Relish (Rel) and induction of the expression of AMP genes, such as diptericin. The generation of ROS is induced by the activation of Duox in the presence of uracil. This is caused by the activation of a G protein-coupled receptor (GPCR), which promotes the release of calcium from the endoplasmic reticulum. This signaling pathway, together with the activation of the Imd pathway, contributes to the expression of the Duox enzyme during infection. Atf2, activating transcription factor 2; Dredd, death-related ced-3/Nedd2-like caspase; Fadd, FAS-associated death domain ortholog; Gαq, G protein αq-subunit; IKK, inhibitor of NF-κB kinase; MKK3, MAPK kinase 3; modSP, modular serine protease; Tak1, TGFβ-activated kinase 1. Adapted from Buchon et al. (2014) and Younes et al. (2020). Frontiers in Microbiology 06 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 local intestinal infections are able to induce a systemic response is suggested to use the microinjection method. Here, a needle in the fat body (Basset et al., 2000; Foley and O’Farrell, 2003; is charged with a specific volume of pathogen solution, and Wu et al., 2012). The simplest explanation would be that when using a pumped injector, the solution is introduced in the an ingested pathogen is able to break the intestinal epithelial abdomen or thorax, entering directly into the haemolymph, thus barrier and enter into the hemolymph, circulating immune cells generating a systemic infection. Although more reproducible, phagocyte them and activate a systemic immune response in this method is slower and requires specific equipment. Assessing the fat body, as observed for the bacteria-derived peptidoglycan the circumstances, an oral infection could be desirable. Unlike (PGN) (Charroux et al., 2018). However, some studies revealed the injecting methods, feeding results in local infection primarily Ecc15 is not detectable in the hemolymph of larvae after in the gut epithelium (Lee et al., 2018). However, it should ingestion, thus proving that it is unlikely that the bacteria be noted that in oral infections the infective dose is also a directly interact with the hemocytes in the hemolymph (Basset limitation, as it is variable and difficult to monitor (Troha and et al., 2000; Foley and O’Farrell, 2003). Although the specific Buchon, 2019). mechanisms involved have not been extensively explored yet, In order to assess innate immune memory acquisition, flies it has been shown that gut-derived ROS might interact with have to be primed by exposing them to either a sublethal dose circulating hemocytes and regulate diptericin production in of a pathogen, a heat-killed pathogen or a pathogen-derived the fat body cells (Amcheslavsky and Ip, 2012). Other studies material previously to the second challenge (lethal infection) also showed a correlation between gut-expressed PGRP-LE (Sheehan et al., 2020). Priming can also be performed both with the levels of AMPs expression in the fat body (Paredes orally and systematically, making sure in the last one that both et al., 2011; Bosco-Drayon et al., 2012). Finally, a cross-talk challenges are done one on each side of the fly body (Kutzer between muscles and immunity via JAK/STAT activation has et al., 2019). The protective effect is then assessed by comparing also been described (Yang and Hultmark, 2016). This interorgan survival and pathogen loads between primed and non-primed communication mechanisms have been recently extensively flies in response to the second challenge. reviewed (Liu and Jin, 2017; Figure 3). Other methods to assess the immunological mechanisms behind the innate immune memory acquisition in D. melanogaster, include analysis of gene expression of AMPs Methods to assess innate immune by RT-qPCR, RNAi, or RNA-seq; protein levels quantitation, memory in Drosophila melanogaster phagocytic or melanization activities and release of ROS upon infection (reviewed in Troha and Buchon, 2019). Measuring Several methods have been developed that aided in the expression levels of AMPs together with survival could bring understanding of the processes related to innate immune surprising results, as it has been observed by several authors memory in the D. melanogaster model. According to the that on certain occasions, there were no changes in survival research question and the experimental design, different kinds after oral administration of non-pathogenic bacteria, yet levels of infection models could be used. For inducing a systemic of AMPs were increased (Patrnogic et al., 2018). In addition, infection there are several methods available; the most used ones on a genetic level, the process of innate immune memory are needle pricking and injection pumping or microinjection is thought to be associated with epigenetic changes in the (Troha and Buchon, 2019). Needle pricking involves dipping chromatin. To evaluate such changes, methyl marks in histone a needle into a solution containing the pathogen, which is H3 can be measured, as well as the concentration of enzymes later used to prick anesthetized flies. Inoculation can be done associated with chromatin remodeling, like histone lysine in the abdomen or thorax, yet minimal damage should be methyltransferases (Bonnet et al., 2019). generated. Since a wound is produced, infection is first local and Last but not least, several studies have taken advantage of later disseminates systemically, allowing to assess both types of the multiple genetic-editing tools available in the Drosophila infections. This type of inoculation is quick; hence this method model to assess the mechanisms of innate immune acquisition can be used for the infection of large amounts of flies. However, (Prakash and Khan, 2022). Most studies have used Toll- and a limitation of this method is that the infection dose can be Imd-impaired mutants to validate the role of these key pathways variable. in the defense against subsequent infections (Apidianakis et al., In addition, to be able to observe dissemination and systemic 2005; Pham et al., 2007; Aymeric et al., 2010; Christofi and infection, very small number of bacteria should be inoculated Apidianakis, 2013; Wen et al., 2019; Cabrera et al., 2022). by needle pricking to minimize the effect of the local infection Other studies have used fly lines unable to mount a proper (Apidianakis and Rahme, 2009). Therefore, some authors agree cellular immune response (Christofi and Apidianakis, 2013; that needle pricking would be most appropriate injection Chakrabarti and Visweswariah, 2020), as well as ROS-deficient method for highly virulent bacteria such as Pseudomonas individuals (Chakrabarti and Visweswariah, 2020). In addition, aeruginosa and Staphylococcus aureus (Apidianakis and Rahme, this model also offers the possibility to study wild populations to 2009; Lee et al., 2018). In case a more precise dose is needed, it assess whether host environment accounted for the difference Frontiers in Microbiology 07 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 FIGURE 3 Schematic representation of the interorgan communication during infection in Drosophila melanogaster. In response to ingested pathogens, these microorganisms might either break the gut epithelium themselves or release peptidoglycan (PGN) from their cell walls into the hemolymph. The enterocytes (ECs) are also able to release reactive oxygen species (ROS) in response to tissue damage. These circulating signals are sensed by the hemocytes located close to the gut epithelium. Hemocyte-expressed Decapentaplegic (Dpp) and Unpaireds (Upds) during early stages of the infection activate the intestinal stem cells (ISCs) proliferation to cope with tissue damage. Hemocytes-derived Upds also induce accumulation of the transcription factor dFOXO in the fat body via JAK/STAT pathway. Activation of dFOXO induces expression of AMPs and a systemic metabolic dysregulation that mobilizes energy resourced toward phagocytic cells. Finally, infection-induced Upd3 in enterocytes activate the JAK/STAT signaling pathway in visceral muscles (VM), which in turns promote ISCs proliferation, as well as activation of the close-located hemocytes. in resistance patterns observed after infection with the same to subsequent infections neither with the same pathogen nor pathogen (Corby-Harris and Promislow, 2008). with S. pneumoniae (Pham et al., 2007). Similar studies were performed with P. aeruginosa. Priming flies with the avirulent Systemic induction of innate immune memory P. aeruginosa strain, CF5, revealed that both Toll and Imd in Drosophila melanogaster pathways and phagocytosis were necessary for the induction of Few studies have been performed in D. melanogaster protection against subsequent infections with the more virulent addressing this topic (Table 1). Pham et al. (2007) found strain PA14 and that heat-killed CF5 did confer protection that the first exposure to a non-lethal dose of Streptococcus although this was shorter (Apidianakis et al., 2005; Christofi and pneumoniae or to the heat-killed bacteria conferred protection Apidianakis, 2013). against a second exposure to lethal doses of the same pathogen. Some authors have evaluated the protection conferred This response was found to last the rest of the fly’s life, by exposure of D. melanogaster to an avirulent Escherichia to be specific and to be mediated by the Toll pathway coli against subsequent infection in a short period of time; together with phagocytes, but not the Imd pathway or AMPs interestingly, protection on both occasions is driven by the (Pham et al., 2007). They also showed that a wide range presence of AMPs from the Imd signaling pathway in fly of heat-killed bacteria, including S. typhimurium, Listeria hemolymph prior to challenge (Apidianakis et al., 2005; monocytogenes, and Mycobacterium marinum, which are known Aymeric et al., 2010). More recent studies have gone deeper to be potent immune activators, did not confer protection into the mechanisms of these innate immune adaptations. Frontiers in Microbiology 08 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 Chakrabarti and Visweswariah defined the ROS production In D. melanogaster oral induction of immune memory and accumulation in hemocytes after an injury as the key has also been assessed (Table 1). Contrary to what happens regulators for the induction of the Toll pathway, which in turn systematically, a transstadial assay in which larvae were exposed confers protection to subsequent infections with Enterococcus to DCV showed an increase in the tolerance to infections faecalis (Chakrabarti and Visweswariah, 2020). Nevertheless, with the same virus in adulthood. This protection was shown this protection conceived by the wound lasted a maximum of to be species-specific as well as RNAi pathway dependent 5 days. On the other hand, when Drosophila was primed with (Mondotte et al., 2018). Specificity of protection was also tested a low dose of E. faecalis, the protection against the second by Wen et al. (2019) by oral priming of D. melanogaster with infection with the same pathogen was observed to las for at heat-killed bacteria. This study evidenced an heterogeneous least 7 days (Cabrera et al., 2022). More insights on the innate protection gram-positive/gram-negative-specific (Wen et al., immune memory acquisition were described by Tassetto et al. 2019). In addition, this study also found differences among the (2017) when D. melanogaster was systemically infected with levels of AMPs expression between males and females. Females Sindbis virus (SINV). They found that the hemocytes acquired showed higher levels of AMPs after short-term oral priming with immunological memory in the form of stable virus-derived killed bacteria, which translated to higher protection against complementary DNA and that they were able to systemically subsequent infection. On the other hand, further research has disseminate it through RNAi-containing exosomes. proven that a previous encounter with non-pathogenic bacteria Lack of protection after priming has also been documented. on the diet failed to protect fruit flies from an infection For example, systemic injection of killed Lactococcus lactis, with entomopathogenic bacteria (Patrnogic et al., 2018). To Pseudomonas entomophila, or Providencia burhodogranariea delve into the mechanisms of fly immunity, priming of flies did not confer the expected protection against subsequent through diet has also been carried out by administering either homologous infections (Kutzer et al., 2019; Acuña Hidalgo and suppressors or inducers of the immune response, Juvenile Armitage, 2022); just as systemic infections with a low dose of Hormone (JH) or cyclic dinucleotide (CDN), respectively Drosophila C Virus did not protect against subsequent challenge (Schwenke and Lazzaro, 2017; Segrist et al., 2021). Results of with a lethal one (Longdon et al., 2013). Considering all of these studies showed the impact of the hormonal pathway in the above, protection in D. melanogaster is achieved differently D. melanogaster in the regulation of the immune response depending on the priming agent. This protection seems to (Schwenke and Lazzaro, 2017), as well as the possibility of be correlated with the immunity pathways activated by this bacterial-derived CDNs to induce immunity in microbiota- priming species that allows protection, specific or not, against deficient flies (Segrist et al., 2021). the challenge (Pham et al., 2007; Aymeric et al., 2010; Christofi As another memory mechanism, Madhwal et al. (2020) and Apidianakis, 2013). showed that the systemic elevation of GABA levels neuronally upon olfactory stimulation of D. melanogaster (via oral), Oral induction of innate immune memory in specifically promoted pre-differentiation of lamellocytes and Drosophila melanogaster thus, a more efficient cellular response when challenged Drosophila melanogaster and mammalian intestines are with Leptopilina boulardi parasitoid wasp. Moreover, the similar both in structure and function, but D. melanogaster protection of D. melanogaster by oral priming has not only has a more simple microbiota composed of only 2–30 bacterial been proven against subsequent infections. Jacqueline et al. species (Wong et al., 2011). However, as seen in mammals, the (2020) showed that previous exposure to Pectobacterium microbiota can also influence the immune response and shape carotovorum protected flies with a cancer-inducing genotype; intestinal function and development in insects (Ryu et al., 2008; results evidenced the importance of both Diptericin and Liu et al., 2017; Capo et al., 2019). Drosomycin in tumor cell death, and thus, in tumor regression. Previous research done in which non-pathogenic or heat- All these findings suggest that protection provided by an oral killed bacteria were administrated by food to different insects exposure to a particular microorganism or antigenic agent showed, in certain cases, an increased level of AMPs or increased is possible in D. melanogaster. However, further studies on survival. For example, pre-exposure with non-pathogenic E. coli the mechanisms of immunity are needed in search of more can protect larvae of tobacco hornworm Manduca sexta from a consistent evidence of immunological memory. Photorhabdus luminescens infection (Eleftherianos et al., 2006). Moreover, feeding the larvae of cabbage looper Trichoplusia ni Transgenerational induction of innate immune with non-pathogenic E. coli and Micrococcus luteus has shown memory to increase the antibacterial activity (Freitak et al., 2007). These It has also been described in insects that innate immune studies serve as a background to establish that microorganisms memory can pass down from primed parent individuals to the ingested with the diet, even if non-pathogenic, can trigger an offspring (Dhinaut et al., 2018; Vilcinskas, 2021). This kind immune response that could reduce the risk of severe disease of memory is currently named as Transgenerational Immune from other infections. Priming (TgIP) and has been studied in a wide range of insects, Frontiers in Microbiology 09 frontiersin.org Frontiers in Microbiology Arch et al. TABLE 1 Studies assessing innate immune memory in Drosophila melanogaster. References Priming species Challenge Time in between Cell wall component Measures Evidence of Main results species (priming/2nd challenge) protection Systemic priming Apidianakis P. aeruginosa CF5 P. aeruginosa PA14 3, 6, 12, 24 h DAP/DAP Survival and Yes - Best protection 6 h after priming and lasts for >24 h. et al., 2005 (avirulent) gene expression - Toll and Imd pathways act synergistically to trigger Avirulent E. coli P. aeruginosa PA14 6h DAP/DAP Survival Yes protection. Avirulent S. aureus P. aeruginosa PA14 6h LYS/DAP Survival No Avirulent P. aeruginosa PA14 6h β-glucan/DAP Survival No Cryptococcus neoformans Pham et al., 2007 S. pneumoniae (low S. pneumoniae 1–14 days LYS/LYS Survival Yes - Priming with S. pneumoniae favors survival reducing dose) bacterial load. Protection is long lasting and species-specific. hk S. pneumoniae S. pneumoniae 1–14 days LYS/LYS Survival and Yes - Phagocytes specifically drives protection to CFUs subsequent infection. - Toll pathway is required for the primed response but not Imd. hk S. typhimurium S. typhimurium 7 days DAP/DAP Survival No 10 hk L. monocytogenes L. monocytogenes 7 days LYS/LYS Survival No hk M. marinum M. marinum 7 days Mycolic acid Survival No Beauveria bassiana B. bassiana 7 days β -glucan/β -glucan Survival Yes (low dose) S. pneumoniae (low All the pathogens 7 days LYS/DAP and LYS Survival No dose) Mixture (hk E. coli, S. pneumoniae 7 days DAP and LYS and β -glucan/LYS Survival No M. luteus, B. bassiana) Mixture + hk S. pneumoniae 7 days DAP and LYS/LYS Survival Yes S. pneumoniae Aymeric et al., Avirulent E. coli Xenorhabdus 1 day DAP/DAP Survival and Yes - Increased survival of primed flies 1 day before 2010 nematophila or gene expression challenge, but not when coinfected. 10.3389/fmicb.2022.991678 P. luminescens P. luminescens phoP P. luminescens 1 day DAP/DAP Survival and Yes - Protection mediated by the Imd-related AMPs in the mutant gene expression haemolymph previously to challenge (Continued) frontiersin.org Frontiers in Microbiology Arch et al. TABLE 1 (Continued) References Priming species Challenge Time in between Cell wall component Measures Evidence of Main results species (priming/2nd challenge) protection Systemic priming Christofi and P. aeruginosa CF5 P. aeruginosa PA14 11 days DAP/DAP Survival Yes - Short-lasting protective effect Apidianakis, (avirulent) - Relish and to a less extend Dif are important for 2013 protection. - Immune priming 2 days pre-infection do not depend on phagocytosis. hk P. aeruginosa CF5 P. aeruginosa PA14 2, 5 or 7 days DAP/DAP Survival Yes - Immune priming 5 days pre-infection depends (avirulent) heavily on phagocytosis. Longdon et al., Drosophila C Virus DCV (lethal dose) 3 days – Survival and No - Priming had no effect on flies’ survival. 2013 (DCV) (low dose) viral load - Mortality correlated with viral presence in the fly. Kutzer et al., hk L. lactis L. lactis 7 days LYS/LYS Survival, CFUs No - P. entomophila priming increased bacterial load at 2019 and gene 1 day post-challenge and survival at 28 days expression post-challenge although non-significantly. - Generally, authors found no effect of the priming on survival, resistance to infection or fecundity - In surviving flies, L. lactis becomes a persistent 11 hk P. entomophila P. entomophila 7 days DAP/DAP Survival, CFUs No and gene infection but P. entomophila is cleared expression Acuña Hidalgo hk or CH2O-treated L. lactis 7 days LYS/LYS Survival and No - Priming did not confer any significant advantages to and Armitage, L. lactis CFUs infected flies. 2022 - Bacterial load 1 day post-challenge showed bimodal hk or CH2O-treated P. burhodogranariea 7 days DAP/DAP Survival and No distribution, while 7 days post-challenge was P. burhodogranariea CFUs unimodally distributed. Cabrera et al., E. faecalis (low dose) E. faecalis 7 days LYS7LYS Survival, CFUs Yes - Priming with a low dose of E. faecalis favors survival 2022 and gene at least 7 days later although this increase in survival is expression not linked with a clearance of the bacteria. - Phagocytosis is needed to mount a primed response to subsequent infection. - Both Imd and Toll pathway are dispensable for the primed response. Oral priming 10.3389/fmicb.2022.991678 Mondotte et al., DCV (larval stage) DCV (adult flies) 7–8 days (Transstadial – Survival and Yes - Priming larvae with DCV showed increased tolerance 2018 assay) viral load when challenged in adulthood. - Transstadial immune priming is RNAi-dependent, virus and sequence-specific. frontiersin.org (Continued) Frontiers in Microbiology Arch et al. TABLE 1 (Continued) References Priming species Challenge Time in between Cell wall component Measures Evidence of Main results species (priming/2nd challenge) protection DCV (larval stage) Cricket Paralysis 7–8 days (Transstadial – Survival and Yes Virus (CrPV) and assay) viral load Flock House Virus (FHV) (adult flies) Patrnogic et al., Mixture (E. coli and P. luminescens or 1 day DAP and LYS/DAP Survival and No - Imd pathway was upregulated after challenge in flies 2018 M. luteus) Photorhabdus gene expression previously primed with both mixtures. asymbiotica Mixture (hk E. coli P. luminescens or 1 day DAP and LYS/DAP Survival and No - Toll pathway was upregulated after challenge in flies and hk M. luteus) P. asymbiotica gene expression previously primed with live mixture, but not the hk. - Oral priming did not favor survival after challenge. Wen et al., 2019 hk E. coli E. faecalis or 1 day DAP/LYS and DAP Survival and Yes - Gram-positive/Gram-negative-specific protection. P. aeruginosa gene expression hk S. aureus E. faecalis or 1 day LYS/LYS and DAP Survival and Yes - Priming with hk S. aureus showed sexual dimorphism P. aeruginosa gene expression in protection against infection. - Toll and Imd pathways act synergistically to trigger protection. Transgenerational priming 12 Bozler et al., L. heterotoma L. heterotoma or Transgenerational assay – Survival, blood Yes - Longer survival in the offspring of primed parental 2020 parasitoid wasps L. victoriae cells counts and flies is characterized by enhanced cellular response (parental flies) parasitoid wasps gene expression mainly mediated by a rapid lamellocytes production. (offspring) - The cellular phenotype in the offspring corresponds with a downregulation of PGRP-LB gene in maternal flies. - There were educed infection rates in the offspring of exposed flies. Leitão et al. Leptopilina boulardi L. boulardi Transgenerational assay – Estimation of Yes - The parasitism increased resistance due to a cellular (2020) parasitoid wasps parasitoid wasps encapsulation immune system activation which leads to (parental flies) (offspring) ratio and gene encapsulation over generations. expression Mondotte et al., SINV SINV Transgenerational assay – Survival, Yes - D. melanogaster transmits antiviral immunological 2020 luciferase memory to their progeny. activity and viral -Virus and sequence-specific protection, but RNAi load independent. 10.3389/fmicb.2022.991678 Others Schwenke and Methoprene Hk Providencia – -/DAP Survival, CFUs No - Priming suppressed the induction of AMPs. Lazzaro, 2017 (synthetic JH analog) rettgeri (oral) and gene - JH and Sex Peptide (SP) favor reproduction over (oral) expression protection, suppressing resistance to infection. frontiersin.org (Continued) Frontiers in Microbiology Arch et al. TABLE 1 (Continued) References Priming species Challenge Time in between Cell wall component Measures Evidence of Main results species (priming/2nd challenge) protection Chakrabarti and Wound E. faecalis (systemic) 2, 5, or 7 days -/LYS Survival and Yes - ROS production in hemocytes is key to activate Visweswariah, ROS production JAK/STAT and Toll which in turn conferred protection 2020 against subsequent infections. - Wound confers short-lasting protection against subsequent infections. Jacqueline et al., P. carotovorum (oral Cancer induction – DAP/- Tumor size and Yes - Infection could have a protective role through the 2020 and cutaneous) gene expression production of Diptericin and Drosomycin that increase tumor cell death in flies primed with P. carotovorum, but no with B. bassiana. B. bassiana (oral and Cancer induction – β-glucan/- Tumor size and No - Toll and Imd pathways act synergistically to trigger 13 cutaneous) gene expression protection. Madhwal et al. Wasp-odor food L. boulardi – – Cellular immune Yes - Those flies pre-conditioned with WOF before (2020) (WOF) parasitoid wasp response infection showed primed their immune response when challenged by elevating their systemic GABA levels which, in turn, promote the pre-differentiation of lamellocytes. Segrist et al., Cyclic dinucleotide DCV or SINV (oral) 0 days – Survival and Yes - Oral, but not systemic ingestion of CDNs protect flies 2021 (CDN) (oral) gene expression against systemic virus infection and induce gene expression in the gut of antibiotic-treated flies. - Protection is dSTING- and dTBK1- dependent. - Toll and Imd pathways act synergistically to trigger protection. HK, heat-killed, CFUs, colony forming units. CH2O-treated (treated with formaldehyde). 10.3389/fmicb.2022.991678 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 although only a few used the Drosophila model. However, in tolerance among hosts types (Råberg et al., 2008; Ayres and the exact mechanism is not yet fully understood, as well as Schneider, 2012). how many generations it lasts (Sheehan et al., 2020). One of In ecology and evolutionary biology, this description of the possible mechanisms is the transgenerational transfer of how specific individuals respond to different environmental microbial elicitors, in which bacteria or bacterial fragments conditions is known as the “reaction norm” and considers the ingested by female individuals are translocated to their eggs different pathogen burdens that can be applied to infections following the same route as bacterial symbionts, thus providing (Råberg et al., 2007). Depending on the animal model, host the offspring with the capacity to mount a more specific innate fitness can be measured differently. In mammals, the most often immune response against these bacteria (Herren et al., 2013; measured effects of infection on host health are anemia and Freitak et al., 2014; Knorr et al., 2015). However, some studies weight loss and they are plotted against the peak pathogen also provide evidences of paternal TgIP in insects which are density (Råberg et al., 2008). In insects, counting the Bacillary thought to be induced by epigenetic modifications transferred Load Upon Death (BLUD, in case of bacterial infections) is to the germ cells and transmitted to the offspring (Soubry et al., interesting as dead caused by disease is correlated with the 2014; Zenk et al., 2017). ability of the pathogen to proliferate and the ability of the Using the D. melanogaster model, Bozler et al. (2020) host to react against that infection. Therefore, as explained, the showed that increased maternal Peptidoglycan Recognition maximal bacterial load that flies can cope with before death Protein LB (PGRP-LB) expression levels after exposure to represents a measure of host disease tolerance (Duneau D. et al., parasitic wasp correlated with a more successful immune 2017). Moreover, the Set Point Bacterial Load (SPBL), meaning response to the parasite in the offspring mainly mediated by measuring the colony forming units (CFUs) at different time a rapid cellular activation. Same implication of the cellular points, is also a useful observation to dissect the mechanism immune response in TgIP was observed in flies primed with taking place throughout the course of infection. Studies done with D. melanogaster aim to measure bacillary load against another parasitic wasp species (Leitão et al., 2020). Mondotte inoculation dose as a measure of resistance (Corby-Harris et al., et al. (2020) also described an antiviral transgenerational 2007; Kutzer et al., 2019; Wen et al., 2019), and both host’s health immune priming in both D. melanogaster and the mosquito or fecundity as the host-fitness parameter against inoculation Aedes aegypti after parental priming with different single dose to measure tolerance (Kutzer et al., 2019). stranded RNA viruses with specificity in the protection of the When using this linear model, four different patterns progeny for several generations. On the contrary, it has also been could be observed. A low survival with low pathogen load seen how the offspring inherited indirect costs associated with could explain the metabolic costs associated with mounting an the immune response to the infection of the progenitors, having immune response. When a high mortality is associated with a shorter lifespans (Linder and Promislow, 2009). high bacterial load, it might be due to the high costs of fighting a virulent microorganism. High survival rates associated with Measuring immune memory readouts: A low pathogen loads suggest that a robust immune response is Drosophila melanogaster approach present, and the host is resistant to the infection. In contrast, Once flies are primed, measurement of tolerance and a high survival with high bacterial loads suggests a tolerant resistance to the subsequent infection is needed to assess phenotype in which the host can survive longer since it does not the acquisition of innate immune memory. In this regard, need to use sources to clear the infection (Corby-Harris et al., different theoretical models have been used along time (Råberg 2007; Kutzer and Armitage, 2016; Duneau D. et al., 2017; Box 2). et al., 2008; Lefèvre et al., 2011; Gupta and Vale, 2017). The In contrast to the linear models, Gupta and Vale proposed a application of these models is not absolute and aspects such 4-parameter non-linear model in D. melanogaster to estimate as the experimental model used, its lifespan, or the possibility disease tolerance with more detail (Gupta and Vale, 2017). While of carrying out a certain test along the course of infection, linear approaches bring light to the rate at which hosts lose will determine which model is the most appropriate at each health, non-linear approaches are useful to clarify the dose that moment. In the literature, resistance is defined as the inverse causes this lost in health or the severity of the infection (Gupta of the pathogen concentration (number of parasites per host or and Vale, 2017). per unit tissue); when all the other variables are equal, a lower Y-intercept means the host is more resistant. Tolerance, on the contrary, is usually defined as the slope of a linear regression Discussion model when plotting host fitness against infection intensity in 2- dimensional health-by-microbe space; the flatter the slope, Over the years, scientists have used the D. melanogaster the higher the tolerance (Råberg et al., 2008). If the slope varies model to study innate immunity. However, the mechanisms among groups, such that the fitness of some hosts declines faster behind the induction of innate immune memory in this with increasing inoculation doses, this means there is variation host model are still not fully elucidated. Previous research Frontiers in Microbiology 14 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 BOX 2 Patterns associated with an infection according to linear models. Metabolic cost of immunity Low survival and low pathogen load. Insufficient immune response against a virulent microorganism Low survival and high pathogen load. Resistant phenotype High survival and low pathogen load. Tolerant phenotype High survival and high pathogen load. has shown that a former encounter of D. melanogaster with plantaginis received a diet containing a low dose of Serratia heat-killed, not pathogenic bacteria or low dose of pathogens marcescens or non-pathogenic E. coli. Larvae primed with could confer protection from future infections with pathogenic S. marcescens were later protected from an otherwise lethal microorganisms (Pham et al., 2007; Cooper and Eleftherianos, injection of the same pathogen. This, however, was not the 2017; Patrnogic et al., 2018). case of larvae primed with E. coli. This study suggests that Interestingly, this mechanism cannot be generalized, so pathogen recognition at the midgut level can be important certain parameters have to be standardized first to have a for conferring systemic immunity (Mikonranta et al., 2014). better comparison of the results. Susceptibility to infection Moreover, other examples include the oral administration of in flies can vary according to age, sex, genetics, feeding and peptidoglycans from the cell wall of P. aeruginosa, or heat-killed physiological and environmental factors, such as temperature, resources, humidity, time of the day or light; or even according P. aeruginosa conferred protection against a following infection to the presence of other infections or single-nucleotide with P. aeruginosa in silkworms. However, silkworms receiving polymorphisms (SNPs) that could confer greater vulnerability heat-killed S. aureus did not show an increased survival after to certain pathogens (Merkling and Van Rij, 2015; Duneau D. infection with P. aeruginosa (Miyashita et al., 2015). This review F. et al., 2017; Kutzer et al., 2019; Troha and Buchon, 2019). underlines the lack of standardized method for assessing the However, the advantage of this model is that experiments can type of protection provided by priming the host with different be repeated easily and in an affordable manner. Such a quantity stimuli, as each author focused on different parameters, such of factors regulating fly immune response explains why not as survival, pathogen load or gene expression. However, the every challenge with a non-lethal dose of a pathogen induces assessment of all three parameters would be of interest in order protection from further encounters, and why the duration of this to define the mechanism behind innate immune memory. protection is variable (Hillyer, 2016; Hajishengallis et al., 2019), Immune priming in D. melanogaster has been studied although it was suggested that it might pass to the progeny on several occasions. And, although the results verify the (Mondotte et al., 2020). The differences on the kinetics of the great variability that the fly presents in terms of protection, different immune pathways that are activated upon infection, they allow us to obtain an increasingly precise overall view as well as the sexual dimorphism that exists when mounting an immune response, also influence the acquisition of innate of the mechanisms behind trained immunity. Most of the immune memory. In addition, a different pattern of activation studies conclude that a certain specificity between the priming is observed between sexes (Duneau D. F. et al., 2017; Belmonte pathogen, the innate immune response triggered by the host and et al., 2020), which translate with different protection levels the protection that this provides against subsequent infections. against a second exposure to the pathogen (Wen et al., 2019). This specificity has been observed at species level (Pham As tolerance and resistance confer protection to a host at et al., 2007) and sequence-specific in viruses (Mondotte et al., different levels when infected, several models to measure their 2020). Fewer studies have also shown less specific protection acquisition have been described (Råberg et al., 2008; Lefèvre e.g., between Gram-negative/Gram-positive species (Wen et al., et al., 2011; Gupta and Vale, 2017). Nevertheless, the choice 2019). Nevertheless, in those studies evidencing protection of the model will depend such as the nature of the host against subsequent infections is not easy to discriminate if this or the answers we want to obtain. Recent research on this protection is due to innate immune memory acquisition or topic pointed out that tolerance and resistance mechanisms due to the fact that the immunity is still stimulated in the can have a differential importance throughout the course of flies at the time of the second challenge (Chambers et al., the infection. For example, during the early phases of Listeria 2019). Further studies which test maximum times in between monocytogenes infection in mice, resistance mechanisms were predominant, whereas tolerance was more important during the priming and challenge, as well as the maximum duration of last stages. This also points out the fact that both mechanisms protection would help to elucidate this. In addition, it would be are interconnected (Kutzer et al., 2019). Examples of immune interesting to perform more priming tests in this animal model priming that could protect insects from future infections to further elucidate the specificity of protection and the ability to were seen when the larvae of wood tiger moth Parasemia transmit this protection to offspring. In addition, oral priming Frontiers in Microbiology 15 frontiersin.org Arch et al. 10.3389/fmicb.2022.991678 prior to the second challenge has been poorly studied in this Spanish Government-FEDER Funds through PI17/01511 experimental model. A more exhaustive study of the subject grant, and the “CIBER Enfermedades Respiratorias” would be interesting in order to screen protective components Network (CIBERES). RK, registered in the EMJMD against infection with the possibility of further translation to LIVE (Erasmus + Mundus Joint Master Degree Leading other animal models. The use of Drosophila to assess oral International Vaccinology Education), co-funded by the priming might be very useful considering the possibility to EACEA (Education, Audiovisual and Culture Executive Agency, study local adaptation of wild-caught strains, which are in award 2018-1484) of the European commission, receives a constant contact with different pathogen-rich environments scholarship from the EACEA. (Corby-Harris and Promislow, 2008). Bearing all of the above in mind, it is still a challenge to decipher innate immune memory mechanisms induced after infection with a pathogen and further research is needed. As Conflict of interest selecting the ideal model is crucial for future developments, non-mammalian models, especially D. melanogaster, are a good The authors declare that the research was conducted in the alternative to deep into its study. absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it Publisher’s note for publication. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated Funding organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or This research was funded by “La Caixa” Foundation claim that may be made by its manufacturer, is not guaranteed (ID 100010434), under agreement LCF/PR/GN16/10290002, or endorsed by the publisher. References Aaby, P., Roth, A., Ravn, H., Napirna, B. M., Rodrigues, A., Lisse, I. M., Humans through the Induction of Cytokines Associated with Trained Immunity. et al. (2011). Randomized trial of BCG vaccination at birth to low-birth-weight Cell Host Microbe 23, 89–100.e5. children: Beneficial nonspecific effects in the neonatal period?. J. Infect. 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