Immunological studies have generally tended to favour the use of murine models, such as those based on rats. Nevertheless, due to the costly and labor-intensive nature of using such animals, and the necessity of maintaining large populations to acquire statistically significant data, there is an increasing demand for alternative models. One such possibility involves the use of those based on invertebrates (1). Comparative genome analyses in insects and other invertebrates have revealed a multitude of homologous genes to those found in humans, which encode proteins responsible for pathogen recognition or signal transduction. Hence, models based on insects such as Drosophila melanogaster, Blattella germanica, Galleria mellonella, Culex quinquefasciatus and Bombyx mori are becoming increasingly popular in studies on the virulence of microorganisms and host immunity (2).

Currently, the G. mellonella model (also called the wax worm or moth) is increasing in popularity in biological research. Several attributes of the larvae confer advantages as models: they are cost-effective to rear in abundance, straightforward to utilize, and require no specialized laboratory equipment for maintenance (3). The considerable size of the final instar larvae (12-20 mm) simplifies manipulation and enhances the ease of collecting tissue/hemolymph samples for analysis. Furthermore, administering test substances to the larvae is straightforward via food, topical application, or injection. The short life cycle of G. mellonella, approximately seven to eight weeks, makes them ideal for large-scale studies, especially since female wax moths can deposit around 1500 eggs in a single reproductive cycle. The temperature range at which these insects can be cultivated is crucial; unlike many other alternative invertebrate models, they thrive in a broad temperature range (18 - 37°C), with the length of the cycle influenced by temperature fluctuations (4). This characteristic is particularly valuable in immunological research as it enables mapping of temperature variations within mammalian bodies.

G. mellonella serves as a model host for human pathogens such as Bacillus cereus (5), Candida albicans (6, 7), Listeria monocytogenes (8), and Staphylococcus aureus (9). Additionally, this insect is a popular model in research on entomopathogenic fungi activity (10) like Conidiobolus coronatus (Entomophthorales), a soil fungus pathogenic to insects and occasionally humans. In immunocompromised patients, particularly in tropical climates, C. coronatus can cause chronic infections, known as rhinofacial mycosis, characterized by invasion of adjacent skin and subcutaneous tissue in the face and nose, leading to deformity (11). C. coronatus as an entomopathogen selectively attacks various species of insects (12). Previous research on four medically significant fly species (Calliphora vicina, Calliphora vomitoria, Lucilia sericata (all Diptera: Calliphoridae), and Musca domestica (Diptera: Muscidae)) has revealed that while pupae exhibit resistance to C. coronatus infection, the adult flies are susceptible (13). The larvae of C. vicina have a thick cuticle that serves as a highly effective barrier against C. coronatus and exhibits poor degradation by proteases in vitro. The protective function of the cuticle is reinforced by the hemolymph, which has demonstrated effective antiproteolytic capabilities. However, the immune response of C. vicina is generally weak, marked by hemocytes with low phagocytic and encapsulating activity, an inefficient polyphenol oxidase (PO) system, and hemolymph with low lysozyme activity (14). On the contrary, despite possessing both humoral and cellular components in their immune systems, G. mellonella larvae are susceptible to C. coronatus infection due to their comparatively thin and readily degradable cuticle (15). Understanding the immune system’s response during infection is crucial for preventing and managing its effects.

The immunological system of G. mellonella larvae exhibits significant structural and functional resemblance to the innate immune response of mammals. Just as mammalian skin serves as a barrier to pathogens, the insect cuticle plays a similar role. Furthermore, the hemolymph of insects shares some similarities with mammalian blood, as both contain immunocompetent cells (16). While literature data did not confirm the presence of acquired immunity seen in mammals, which is characterized by the production of specific antibodies, they possess the capability to synthesize and secrete a range of antimicrobial peptides (AMPs) into the hemolymph (17). The humoral immune response can manifest through processes such as melanization, clotting, as well as production of reactive oxygen species (ROS) (18).

In G. mellonella, the cellular immune system rely on phagocytosis, nodulation, and encapsulation reactions and based on the presence of five distinct types of hemocytes, each contributing uniquely to the immune response. Plasmatocytes and granulocytes, the predominant cells, are known for their active phagocytic role. Conversely, oenocytoids, spherulocytes, and prohemocytes, though less explored, seem to have minor involvement in immune responses (19, 20).

While the factors governing immune responses in mammals are relatively well studied, there is little literature data describing these processes in insect. Nonetheless, it is widely described that their humoral immune responses mostly rely on antimicrobial peptides (AMPs) releasing from the fat body, which is mediated through pathways such as Toll, IMD (immune deficiency) and JAK-STAT (Janus kinase-signal transducer and activator of transcription). Toll signaling pathway is induced by Gram-positive bacteria and fungi, while Gram-negative bacteria mostly activate the IMD pathway (21).

The first stage of defence mechanisms in mammals are identification of molecular patterns detected in pathogens; in this phase the most important role plays the toll-like receptors (TLRs) present in dendritic cells, macrophages, and granulocytes, produced in hematopoietic stem cells.

Upon binding to a pathogen ligand, TLRs trigger a cascade of signaling pathways which are conserved from insects to plants and humans. They ultimately activate NF-κB (Nuclear Factor kappa B) which, in mammalian cells, leads to the induction of cytokine genes and the establishment of innate immunity (22). Within dendritic cells, TLR signals trigger the production of type I interferons (IFN α/β), which subsequently establish an antiviral state within host cells. Moreover, TLR signals elicit the release of both pro-inflammatory cytokines like IFNs, IL-1, TNF-α, and IL-12, and anti-inflammatory cytokines such as IL-10 and IL-6. Among these, IL-12 and IL-10 serve as connectors bridging early innate responses to specific immune responses (23).

The IMD pathway is very similar to the mammalian TNF-α pathway, a key regulator of vertebrate immunity and metabolism. Two well-described/examinated cellular reactions to TNF-α production include the initiation of apoptosis and the stimulation of transcription processes promoting cell survival. TNF-α-induced apoptosis is characterized by the activation of caspase cascades, leading to cell death through the cleavage of specific cellular substrates. Furthermore, TNF-α triggers the activation of two transcription factors: NF-κB and activating protein 1 (AP-1). Among these, NF-κB plays a crucial role in preventing apoptosis and ensuring cell survival by stimulating the expression of genes involved in inflammation, cell growth, and signal regulation (24).

The JAK-STAT pathway is one of the best understood signal transduction cascades. It is known to be universal and essential to cytokine receptor signaling. Almost 50 cytokine receptors realize their signals through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Activation of this pathway induces cell proliferation, differentiation, migration, and apoptosis (25, 26).

The three main pathways for regulating cytokine activity in the insect immune system (Toll, IMD, JAK-STAT) have analogous equivalents in mammals. Therefore, we hypothesize that cytokine-like proteins may also be present in insects. The main goal of this study is to confirm the presence of eighteen cytokine-like molecules in G. mellonella hemocytes following C. coronatus infection.

The effects of C. coronatus infection on the amounts of eighteen cytokine-like proteins (IL-1α, IL-1β, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-β, GM-CSF, M-CSF, G-CSF) in hemocytes were examined in three groups of G. mellonella larvae. In the experimental phase of the research, fungus-treated insects were employed. The larvae were divided into two groups, both of which underwent a 24-hour incubation period with the fungus. First one, named F24, was larvae immediately taken do the experiments after exposition to fungus, the second one (F48) the fungus-infected larvae were incubated additionally 24h on sterile petri dish and then used in experiments. The control insects were incubated with sterile Sabouraud agar medium. Following collection, all samples were subjected to examination by fluorescence microscopy, flow cytometry and ELISA.

In vertebrates, the responses to exogenous and endogenous insults, the tissue repair mechanisms and the restoration of tissue homeostasis are controlled by cytokines; as such they are regarded as the major regulators of immune processes (29, 30). Primarily these compounds are secreted by immunocytes, their function is believed to be mediated through interactions with particular cytokine receptors. Research on the functional similarities between the innate (non-adaptive) host defenses of vertebrates and invertebrates suggests that invertebrates possess soluble cytokine-like mediators that regulate inflammatory responses to infection or injury (31).

Although the topic of cytokines has been widely discussed in vertebrates, especially mammals (32), there is little information about cytokine-like proteins in invertebrates, and even less in insects (33). The present study examined the presence of 18 cytokine-like proteins (IL-1α, IL-1β, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-β, GM-CSF, M-CSF, G-CSF) in the hemolymph of G. mellonella after infection with the entomopathogenic fungus C. coronatus. The research was carried out using three methods: fluorescence microscopy, flow cytometry and ELISA tests.

Thirteen of the tested proteins (G-CSF, GM-CSF, M-CSF, TNF-β, IFN-gamma, TNF-α, IL-1 β, IL-3, IL-6, IL-7, IL-15, IL-17, IL-19) were found in the hemolymph and hemocytes of G. mellonella; however, the results regarding the influence of infection differed according to the detection method. Fluorescence microscopy identified higher levels of G-CSF, GM-CSF, M-CSF, IL-3, IL-15, IL-1 β, IL-6, IL-19 in both F24 and F48 compared to controls. Flow cytometry analysis showed an increase in M-CSF, GM-CSF, IL-1 β, IL-8 and IL-19 for both post-infection samples compared to controls; in addition, ELISA assays indicated higher levels of M-CSF, GM-CSF and IL-1 α in the infected samples. These differences may result from the sample preparation method. All subpopulations of hemocytes can be detected by flow cytometry. However, fluorescence microscopy requires several washes during sample preparation, which may remove everything other than adherent subpopulations of hemocytes, i.e. plasmatocytes and granulocytes. In addition, ELISA tests are based on whole hemolymph, i.e. homogenized cells and plasma; however, this hemolymph was collected directly from the larvae and not subjected to in vitro cell culture.

Initial proteomic analysis conducted with the BLASTP tool revealed potential homology between three human cytokines (IL-1α, IL-17 and M-CSF) and uncharacterized proteins from G. mellonella, whose sequences were deposited in databases. For a more comprehensive assessment of the homology between human cytokines and insect cytokine-like proteins, a complete proteomic analysis is required. Our previous studies, in which analyzes were performed using 2D electrophoresis, Western Blot and LC-MS-MS/MS protein identification, confirmed 33% protein sequence coverage of G. mellonella IFN-γ- like protein to IFN-γ sequence from Homo sapiens (28). We are currently undertaking a proteomic analysis of larval hemolymph, utilizing FAIM (high-field asymmetric waveform ion mobility spectrometry) measurements and assessments through the Swissprot Eukaryota databases. We have obtained initial results into the identification of IL-17 and IL-19, which are undergoing further refinement for subsequent publication.

Previous immunocytochemical studies have confirmed the presence of cytokine-like proteins in various invertebrate species belonging to the Mollusca (34), Nematoda (35), Annelida (36), Tunicata (37) and Insecta (38). Of these, IL-1-, IL-6- and TNF-like molecules have been found to predominate in inter alia annelids, molluscs, echinoderms and protochordates (31, 39).

The IL-1-like cytokine was among the earliest-discover cytokine-like molecules in invertebrates, isolated from the coelomic fluid and coelomocytes of the starfish Asterias forbesi (40). In addition, molecules cross-reacting with antibodies against the chemokines IL-8, IL-1, IL-6 and IL-2, and against transforming growth factor (TGF)-β1, have been identified in molluscs (34, 41). Moreover, IL-2-like activity was observed in protochordates and echinoderms, specifically in deuterostome invertebrates possessing a hematopoietic organ and T-like cells (42). Finally, genes encoding TGF-β have been detected in the genome of cnidarians, such as the sea anemone Aiptasia pallida, and the cytokine was found to depress certain immune reactions, including nitric oxide production (43, 44).

Comparative studies indicated the presence of cytokine-like molecules in invertebrate and also similarities on genome level in both invertebrates and mammals, for example the research indicated the homology of mammalian messenger RNA (mRNA) to IL-1b mRNA isolated from cerebral ganglion of the protochordate Stylea plicata (45). In addition, PCR studies on Manduca sexta using primers based on shared vertebrate IL-1 protein sequences identified a product demonstrating 35% homology with sheep, rat, rabbit, cow, mouse and human IL-1α, IL-1β or IL-1-receptor antagonist (IL-1-ra) (46).

Few studies have reported the occurrence of these proteins in insects. However, a sequence homologous to an interferon consensus response element has been reported in the diptericin promoter of Drosophila (47), and a cytotoxic molecule (Gallysin 2) that may be an analogue of TNF has been isolated in the lepidopteran G. mellonella (48). Non-activated granular cells from G. mellonella or hemocytes from Estigmene acraea larvae have demonstrated strong positive reactions to anti-IL-1α and TNF-α polyclonal antibodies (pAb), while a less positive reaction was noted for G. mellonella plasmatocytes (49). In addition, TNF-like molecules have been observed in the plasmatocytes and granular cells of Calliphora vomitoria hemocytes (50). The presence of cytokine-like proteins in insects is also confirmed by our present findings; however, our study is the first to examine such a wide range of cytokines from different groups.

Similarly, very little is known of the effect of cytokine-like molecules on insects. Earthworm coelomocytes exhibited higher phagocytosis in response to recombinant human IL-12 and IFN-γ, whereas blue mussel hemocytes displayed increased stress and reduced phagocytosis when stimulated with TNF-α. Similarly, insect (D. melanogaster) cells demonstrated stimulation upon exposure to recombinant human IL-8, resulting in an increase in phagocytic cells (39). In addition, hemocytes of the fly Calliphora vomitoria demonstrated TNF-like molecule expression following encapsulation;the authors propose that his may serve as a chemoattractant (50). Finally, a defence complex consisting of IL-1-like molecule and phenol oxidase, the enzyme responsible for melanization, has been reported in the hemolymph of the insect Manduca sexta (51).

Infection by C. coronatus and the administration of its metabolites (harman and norharman) has been found to affect the insect immune system. Harman and norharman increase the phagocytic activity of hemocytes and the level of serotonin (5-HT) in G. mellonella hemolymph (15, 52). Our previous data identified the presence of certain heat shock proteins (HSPs), viz. HSP90, HSP70, HSP60 and HSP27, in G. mellonella hemocytes: HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 present in trace amounts, while HSP60 and HSP27 were elevated in the F24 and F48 groups, and HSP90 in F48 alone; the fungal infection had no effect on HSP70 levels (53). Both HSPs and serotonin are involved in regulating the functioning of the immune system. Heat shock proteins are activators of the innate immune system, and are capable of inducing pro-inflammatory cytokine production by mammalian macrophages (54). In contrast, 5-HT reduced IFN-γ and IL-17, and increased IL-10 production by T lymphocytes (55).

Insects mostly rely on their cuticular, humoral, and cellular defenses to combat fungal pathogens (56).

It is possible that the cytokine-like molecules present in insect hemolymph also inhibit fungal infection. In mammals, the role of cytokines in fungal infection is widely described: the cells of the host innate immune system recognize fungal organisms by their cell wall components, which act as pathogen‐associated molecular patterns (PAMPs). The PAMPs bind to, and are recognized by, pattern‐recognition receptors (PRRs), including Toll‐like receptors (TLRs), on the surface of host cells (57, 58).

The phagocytic activity directed against fungi takes place through oxidative and non‐oxidative mechanisms, and can be increased by opsonins and T‐cell‐derived cytokines (59). The production and activation of mature phagocytic cells from hematopoietic progenitor cells is stimulated by a group of glycoproteins known as hematopoietic growth factors (HGFs). Of the HGFs, the most relevant to antifungal host defenses are granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and macrophage colony‐stimulating factor (M‐CSF) (60). Our present findings indicate that these cytokines are elevated in G. mellonella hemocytes after fungal infection, while our previous studies confirm that C. coronatus metabolites (harman and norharman) stimulate phagocytosis in these cells (52).

HGFs have been proven to provide defense against fatal fungal infections and bolster the effectiveness of antifungal medications in both animal models and clinical investigations. G-CSF, M-CSF, and GM-CSF have been employed in the management of invasive fungal infections stemming from Candida (61, 62) and Aspergillus (63–66), especially in patients undergoing chemotherapy or after stem cell transplantation (67–69). As such, the fact that these proteins also play a potential role in fungal infection in insects suggests supports their attractiveness as potential research models.

In mammals, two subpopulations of T helper cells are involved in the response to fungal infection: ‘protective’ type‐1 (Th1) and ‘non‐protective’ type‐2 (Th2). The type‐1 response involves the production of Th1 cytokines, such as IFN‐γ and IL‐2, IL‐12, and IL‐18; these stimulate macrophage activation, cytotoxic CD4+ T cell generation, opsonizing antibody production and delayed-type hypersensitivity. The type‐2 response entails the generation of Th2 cytokines, like IL‐4, IL‐5, and IL‐13, which trigger the production of non‐opsonizing antibodies and allergic reactions, while also suppressing the pronounced inflammatory response induced by Th1 cytokines. Several other cytokines contribute to the immune response against fungal pathogens, including IL‐1, IL‐6, IL‐8, IL‐10, IL‐15, TNF‐α, and transforming growth factor (TGF)‐β (70).

Our research with the G. mellonella insect model showed that C. coronatus infection did not appear to increase the levels of IFN-gamma-like protein and TNF‐α-like protein, as these proteins were produced by hemocytes from both healthy and infected insects. Nevertheless, as in mammals, it is possible that the IL-1-β, IL-6 and IL-15-like proteins may play an important role in fungal infection, as their levels increased in infected insects; however, a better understanding of the role of these proteins during infection is needed.

IL-1β signaling promotes monocyte, macrophage and neutrophil recruitment in mammals, as well as enhanced phagocytosis and killing, and increased production of reactive oxygen species/nitrogen oxide synthase (ROS/NOS) (71). IL-15 plays a role in innate immunity against fungal infections by boosting the antifungal activity of polymorphonuclear or monocyte cells (72); it also recently has been found to play a part in NK cell activation (73). IL-6 can stimulate the secretion of various chemokines, resulting in the recruitment of monocytes and/or macrophages and the resolution of inflammation (74).

Many similarities have been noted between the immune systems of mammals and insects, particularly regarding the functioning of immunocompetent cells, immune pathways and factors. This has been confirmed by our present findings, indicating that certain cytokine-like molecules are present in the insect hemocyte subpopulation and that their level correlates with the degree of fungal infection. These findings may also be valuable in contributing to further research concerning insect physiology, parasitology and immunology, as well as pest biocontrol.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because The research was conducted on invertebrates.

AW: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft. AK: Data curation, Formal analysis, Investigation, Writing – review & editing. JS: Investigation, Visualization, Writing – review & editing. MB: Methodology, Supervision, Writing – review & editing.