There are different forms of hemocytes documented in insects, including prohemocytes, plasmatocytes, crystal cells, oenocytoids, and granular cells (38). These hemocytes possess adhesion and phagocytosis capabilities (39). While some forms of hemocytes such as oenocytoids, may produce prophenoloxidase (proPO) (40). The usual morphology-based categorization of hemocytes does not necessarily correspond with cell function (41). As a result, considerable effort has been put into classifying different hemocytes. Flow cytometry permits the grouping of three primary kinds of hemocytes: big granular cells, tiny semi granular cells and small hyaline cells (42). Additionally, some monoclonal antibodies can differentiate hemocytes based on their immunogenicity rather than their physical attributes (43). Also, these antibodies may also be able to inhibit other types of cellular responses (44). Three distinct hemocyte types in D. melanogaster have been characterized in further details: plasmatocytes, lamellocytes, and crystal cells (45). The large cells, known as crystal cells, earn their moniker from the crystalline inclusions they contain. During melanization, they release proPO, a zymogen that is essential for various physiological processes, including the closure of parasite eggs and the repair of damaged skin (22). About 95% of the hemocyte pool is comprised of plasmatocytes. They have relatively tiny (10 µm in diameter) cells, yet they produce enormous lamellipodial protrusions and active filopodia (46). Plasmatocytes are the persistent cells, that appear to remain throughout a fly’s life (47). Mature plasmatocytes were reported to express scavenger receptor ortholog Croquemort (Crq), phagocytic receptors, and the extracellular Peroxidasin (48). The lamellocytes were reported to only show up in the parasitized larval stages. One of the principal purposes of a hemocyte is to encase the parasitic wasp egg (49). With infection or sterile damage to wasp eggs, lamellocytes appear to develop from a pool of plasmatocytes that served as their predecessors (50). The first stage of the immune response in insects is the adhesion of granular hemocytes and plasmatocytes to the surface of the invading organism or to other cells (51). Phagocytosis, nodule formation, and encapsulation all result from hemocyte adhesion. Further the function of these innate cellular mechanisms are also explained.

Macrophages are a type of white blood cell that play a crucial role in the immune system, as they engulf and digest harmful particles and cells in the body (52). Recent advances in research have shed light on the evolution of macrophages, particularly the transition from invertebrate to vertebrate organisms (53). It is now believed that macrophages evolved from primitive, sessile cells found in invertebrates, which were then modified into motile cells in vertebrates (54). Additionally, studies have shown that different types of macrophages have evolved in different organisms, and that they play different roles in immune function (55). Understanding the evolution and function of macrophages can help improve our understanding of the immune system and the development of therapies for diseases (56). The principal role of macrophages entails the process of phagocytosis, which involves the engulfment and subsequent digestion of extraneous particles, including bacteria, viruses, and cellular remnants. This mechanism facilitates the eradication of pathogens by macrophages and their involvement in tissue repair processes. Moreover, macrophages are responsible for the synthesis and secretion of signaling molecules known as cytokines, which play a crucial role in the regulation of immune responses and facilitate communication among various immune cells (57). Gaining a comprehensive understanding of the complex functions of macrophages and their interactions with other immune cells yields significant insights into the pathogenesis and advancement of diverse pathological conditions. The dysregulation of macrophage activity has been linked to a variety of conditions, such as infectious diseases, autoimmune disorders, cancer, and chronic inflammatory diseases (58). Through the analysis of the molecular mechanisms that govern macrophage function, researchers have the opportunity to devise precise therapeutic interventions aimed at regulating macrophage activity. This approach holds promise for the treatment and prevention of various diseases. The phagocytosis of melanized bacteria and other small pathogens is common ( Figure 1 ) (59). Phagocytosis is a cellular immunological mechanism that has been used by both vertebrate and invertebrate species throughout evolutionary history to prevent the spread of disease-causing microorganisms (60). It may phagocytose hundreds of bacteria at once and hydrolyze foreign bodies in a matter of seconds (61). Phagocytes including plasmatocytes and granulocytes, which can be either circulating or sessile are responsible for identifying foreign matter in Hemiptera, mosquitoes, Lepidoptera (granulocytes) and fruit flies (plasmatocytes) (62). The latter is taken up by a membrane-bound phagosome, which further fuses with a lysosome before being destroyed by enzymatic hydrolysis (46). The intracellular mechanisms driving phagocytosis are poorly understood, but it all starts with the binding of a cell-surface and humoral PRR on a PAMP (63). Nimrod proteins, Thioester-containing proteins, β-integrins, DSCAM and PGRPs are PRRs that have been experimentally shown to be associated with phagocytosis (64). There are several specifics among PRRs. For instance, NimC1 mediates the phagocytosis of S. aureus and, to a lesser degree, E. coli, whereas in D. melanogaster PGRP-LC mediates the phagocytosis of E. coli but not S. aureus (65).

Encapsulation, a cellular immunological response, is deployed by insects to combat infections that are too huge to be phagocytosed ( Figure 1 ) (66). Insects were found to exhibit two forms of encapsulation: melanotic humoral encapsulation (Diptera) and cellular encapsulation (Lepidoptera) (67). The latter may take place even in the absence of melanization (68). Contrarily, phenoloxidase (PO) activity is necessary for melanotic encapsulation, which may take place with or without hemocyte support (40). Granulocytes and plasmatocytes play a key role in encapsulation in Lepidoptera, whereas plasmatocytes and lamellocytes do so in Drosophila (69). In Lepidoptera, encapsulated substances were enclosed by both an external layer of plasmatocytes and an internal layer of granulocytes (36). Insects, such as lepidopteran and dipteran larvae, often utilize this response when infected with parasitoid wasp eggs (70). Encapsulation in Lepidoptera begins with the integrin dependent attachments of granulocytes to certain sites designated by an Arg-Gly-Asp (RGD) sequence (31). The granulocyte cells surrounding the pathogen are covered by several layers of plasmatocytes, and these plasmatocytes are further enclosed by an adhesive layer of more granulocytes (71). In contrast, plasmatocytes and lamellocytes are the cells engaged in a similar process in Drosophila (72). The capsule may subsequently get melanized depending on the infection and insect.

The insect immune system relies in part on melanization, an enzymatic process that serves a number of purposes ( Figure 1 ) (73). To achieve this, serine proteases, its inhibitors, pattern recognition receptors, and enzymes involved in melanin synthesis should act together (36). In response to recognition of PAMPs by PRRs (C-type lectins, β-1,3 glucan recognition proteins and Gram-negative binding proteins), the serine protease cascade is activated, resulting in the conversion of pro-phenoloxidase (PPO) to phenoloxidase (PO), which in turn leads to melanotic capsule formation (74, 75). The pathogen’s proteinaceous capsule, in conjunction with damage, oxidative stress, or starvation, serves as a mediator in its annihilation (1). Additionally, melanization aids in the removal of infections (76). Oenocytoids, which are the main producers of PPO, are one kind of hemocytes that produce a number of enzymes and PRRs that trigger the process of melanization (1). Another crucial component of the insect’s immune system’s resistance against fungal infection is through melanization (77). Melanin, a dark pigment, is deposited at the site of infection. Upon detecting fungal pathogens, insects initiate their immune response, resulting in the release of specialized immune cells known as hemocytes. Hemocytes identify and phagocytose the invading fungi, initiating subsequent biochemical reactions (78). One reaction involves the activation of phenoloxidase, an enzyme that converts phenolic compounds to quinones. Quinones are subsequently polymerized and oxidized to generate melanin. Melanin deposition restricts the dissemination of fungal pathogens and hinders their capacity to induce harm (79). Moreover, melanin produces harmful by-products that negatively affect fungal cells. It is essential for containing and delaying the development and spread of the invasive mosquito pathogen Beauveria bassiana (80).

Although, the detailed molecular mechanisms defining this defensive process are still incompletely understood, nodulation depends on eicosanoid-based communication and the protein Noduler, which resembles an extracellular matrix (81). The first step in this procedure is the adhesion of granulocytes to one another to form layers that enclose many bacterial or fungal spores (1). When the granulocytes discharge their contents, a flocculent substance is created that traps the microorganisms. The nodule’s surface is then covered with an accumulation of plasmatocytes. Melanization often occurs after this stage (82).

As a result of biochemical analysis of the hemolymph of the fruit fly Drosophila melanogaster and other Dipterans, seven different types of AMPs have been revealed in insects (89). Based on their primary biological targets, they may be divided into three categories and exhibit a broad range of effects against microbes (90). Drosocin, attacins, cecropins and diptericin may all be used to combat Gram-negative bacteria. While, metchnikowin and drosomycin are antifungal medications (91). A characteristic feature of insect defensins is the presence of three or four stabilizing intramolecular disulfide bonds (92). The term derives from their chemical resemblance to mammalian α and β defensins (93). Insect defensins fall into two categories: those that include peptides with mixed α/β-helix-sheet structures and those that have triple-stranded antiparallel β-sheets (94). It has been revealed that numerous Lepidopteran species contain defensins with antibacterial and antifungal properties (95). Likewise, cecropins are short, basic peptides having an amphipathic α-helix shape, ranging in size from 31 to 37 amino acids (96). Cecropin is the first insect-derived amphipathic antimicrobial peptide discovered in the hemolymph of the silkworm Hyalophora cecropia (93). Cecropin was also observed to decrease proline absorption and cause membrane permeability by disrupting pathogen cell membranes and breaking the amphipathic peptides (97). Numerous Lepidopteran species have cecropin-family genes that have been identified. 13 cecropin genes have been discovered in Bombyx mori (98). Another category of amphipathic -helical antimicrobial peptides, moricins were initially identified in the silkworm, B. mori (99). Furthermore, nine moricin genes were found in the B. mori genome, while eight moricin homologs with antibacterial, antiyeast, and antifilamentous fungal activity were found in the G. mellonella genome (100, 101). Different terms, such as bactericidin, lepidopterin, and sarcotoxin, have been given to cecropins derived from insects besides H. cecropia (102). D. melanogaster produces the antimicrobial peptide drosocin, which is 19 amino acids long (93). The peptide’s antibacterial action has been traced to an O-glycosylated threonine residue since its absence significantly reduces the peptide’s potency compared to the original molecule (96).

The first known source of the 20 kDa AMPs called attacins, which are glycine-rich, was the hemolymph of H. cecropia. The acidic and basic isoforms of attacin have been cloned from H. cecropia. By primarily binding to lipo-polysaccharide (LPS), these attacins increase the permeability of the bacterial outer membrane (103, 104) and these AMPs were also reported to inhibit the synthesis of outer-membrane proteins by preventing the bacterial transcription (105). As a result, basic attacin was found to be more effective against E. coli than acidic attacin. Spodoptera exigua, the beet armyworm, is one of the several species of Lepidoptera whose attacins have been cloned (106). The Lepidopterans also have glycine-rich AMPs known as globerins and lebocins (107). These peptides prevent outer membrane protein production, which is essential for bacterial growth (108). The effectiveness of glomerins against bacteria, fungi, and even viruses has been demonstrated, and it has been hypothesized that they may also be able to inhibit viral replication (31).

Diptericin is a glycine-rich AMP that insects produce in response to bacterial infection or injury (109). It is basically a heat-stable peptide with an 8.6 kDa molecular weight with high concentrations of Asx, Pro and Gly (93). Although this treatment appears to work by rupturing the cytoplasmic membrane of growing bacteria, only a small subset of Gram-negative bacteria are susceptible to it (18). In addition to inhibiting bacterial growth, studies claim that diptericin also prevents oxidative stress (110). Through an increase in antioxidant enzyme activity in D. melanogaster, diptericin may “scavenge” or trap free radical anions as well as reduce oxygen toxicity (111). Drosomycin is a 44-residue antifungal peptide that was originally isolated from D. melanogaster exposed to microorganisms (112). It has been observed to be secreted from insect’s fat body into the hemolymph. It is quite effective against fungi but has little effect on bacteria. Drosomycin is a member of the cysteine-stabilized α‐helical and β‐(CSαβ) superfamily. It consists of a three-stranded α‐helix and a β‐sheet stabilized by four disulfide bridges (113). Moreover, it is highly similar to a class of 5 kDa cysteine-rich plant antifungal peptides identified in the seeds of diverse Brassicaceae species (114). Drosomycin has a narrow antibacterial range and is effective against just a few numbers of filamentous fungi (115). In E. coli, recombinant drosomycin was found to have antiyeast and antiparasitic activities (93). Drosophila produces the proline-rich peptide metchnikowin (26 residues) in response to infection (116). This peptide is produced in the adipose tissue in response to an immune challenge and its production may be initiated by the Toll or Imd pathways (117). Metchnikowin, a Drosophila based antimicrobial peptide (116) was recently demonstrated to have higher potential against Gram-positive bacteria and fungi. Further, transgenic barley containing the metchnikowin gene was successful in suppressing Fusarium head blight and powdery mildew like diverse ascomycetes fungal infections (118).

After pattern recognition receptors (PRRs) identify a microorganism, a series of signaling chemicals within the cells trigger various actions ( Figure 2 ) (93). The eventual cellular response is determined by the molecule’s interactions with certain signaling pathways (119). Production of AMPs by the fat body through the Toll, immunological deficiency (Imd), and JAKSTAT pathways predominantly mediates humoral immune responses (120). The Toll signaling pathway is typically activated by gram-positive bacteria and fungi, while the Imd signaling system is generally triggered by gram-negative bacteria (121).

Peptidoglycan recognition proteins (PGRPs), which play a key role in inflammation and antibacterial defense by recognizing bacterial peptidoglycan, are essential proteins associated with innate immunity ( Figure 3 ) (152). They are polymorphonuclear leukocyte-expressed (PGRP1), liver-expressed (PGRP2) or secreted proteins (PGRP3 and PGRP4) (93). Up to 19 PGRPs exist in insects, categorized as short (S) and long (L) variants (153). The short versions are found in hemolymph, cuticle and fatbody cells, while the long forms are mostly expressed in hemocytes (154). Insect PGRP expression is frequently elevated when subjected to bacterial exposure. These receptors cause proteolytic cascades that result in the production of antimicrobial compounds, activate the Toll or Imd signal transduction pathways, or both (155). The following are PGRPs in Drosophila that have known uses: The hemolymph’s PGRP-SA binds to Lys-type peptidoglycan, and together with the PGRP-SD and GNBP-1, this causes the Toll pathway to be activated (28). In reaction to yeast, GNBP3 also causes the Toll pathway to be activated (156). These pattern recognition proteins trigger a series of serine protease cascades that ultimately activate the Spatzle-processing enzyme (SPE), which cleaves proSpatzle to create free Spatzle, the ligand for Toll (157). In a similar way, the Imd pathway is triggered by the binding of DAP-type polymeric peptidoglycan to the PGRP-LCx homodimer complex or DAP-type monomeric peptidoglycan to the PGRP-LCx/PGRP-LCa heterodimer (158). Both monomeric and polymeric DAP-type peptidoglycans are capable of being bound by PGRP-LE (159). It has been shown that extracellular PGRP-LE activates the Imd pathway through PGRPLC transmembrane receptors and is also important in activating the prophenoloxidase (proPO) cascade ahead of the proPO-activating enzyme (PPAE) (160). By binding to the Imd adaptor protein, PGRP-LE inside the cell is able to activate the Imd pathway in response to the recognition of DAP-type peptidoglycan from bacteria within the cell. Furthermore, PGRP-LE produced within the cell may stimulate autophagy in a way that is independent of the Imd signaling pathway (161). PGRP-LF inhibits the Imd pathway because it binds to PGRP-LCx instead of peptidoglycan. By doing so, it inhibits the development of an active dimer of PGRP-LC. DAP-type peptidoglycan is cleaved into inactive pieces by PGRPLB and SC1a/1b/2, which prevents Imd pathway activation (162). As a result of its specialized amidase activity on DAP-type peptidoglycans, PGRP-SB1 is very lethal to bacteria (163).

A family of plasma proteins known as insect β‐1,3-glucan recognition proteins (GRPs) and Gram-negative bacteria binding proteins (GNBPs) have glucan-binding domains at their amino and carboxyl ends that are comparable to those of β‐1,3-glucanases (164). All βGRPs may trigger the proPO cascade ( Figure 3 ) by attaching to β-1,3-glucans on bacterial surfaces. Manduca sexta βGRP1 gene expression is constant in the fat body, but βGRP2 gene expression is elevated during the initial wandering stage just before pupation or after an immunological attack (165). These βGRPs bind to the hemolymph proteinase-14 precursor (proHP14), which causes HP14 to autoactivate and start a proteinase cascade that activates proPO (166). Helicoverpa armigera larval midgut extract was used to identify a βGRP with glucanase activity. This enzyme likely serves as a digestive enzyme rather than an immune stimulator since it hydrolyzes β-1,3-glucan but not β-1,4-glucan (167).

The plasma protein known as hemolin, which is frequently found in the adhesion molecules of vertebrates, contains four immunoglobulin (Ig) domains (168). Hemolin is present in many Lepidopteran species, especially B. mori (101), Antheraea mylitta (169), Plutella xylostella (170) and Samia cynthia (171), yet it hasn’t been seen in insects from other orders. Lipoteichoic acid and LPS from bacteria are bound by hemolin (172). Hemolin also binds to hemocytes, acting as a link between hemocytes and microorganisms and triggering phagocytosis or nodulation (37).

Animal C-type lectins (CTLs) are a vast class of molecules that recognize carbohydrates and bind ligands in a calcium-dependent way (173). Lepidopterans have been reported to include a number of C-type lectins, including immulectins 1, 2, 3 and 4 as well as LPS-binding protein (also known as CTL20), CTL10, CTL11, CTL19 and CTL21 (174). These lectins all contain two carbohydrate recognition domains, and based on their genes, it seems that Lepidoptera is the only insect group that has these particular sorts of lectins (175). The majority of Lepidopteran CTLs bind to lipoteichoic acid and bacterial LPS, causing bacterial and yeast agglutination. This is likely because each of the two carbohydrate-binding domains binds to sugar residues on the surface of nearby microbial cells (176). This microbial aggregation may help hemocytes fight off pathogens by phagocytosing them and forming nodules. By phagocytosing the pathogens and forming nodules, this microbial aggregation could aid hemocytes in their fight against them.

The RNA interference (RNAi) pathway provides the most potent insect response to viral infection. Dicer2 (an endoribonuclease belonging to the RNase III family) and the protein R2D2 collaborate to detect double-stranded viral RNA (182). Later, Dicer2 snips the dsRNA into manageable duplex DNA pieces (of around 21 nucleotides) (183). The duplex is unwound, and a guide strand is chosen according to its complementarity with the other strands. The RNA-induced silencing complex (RISC), which has the RNase Argonaute as part of it, is subsequently loaded with the siRNA guide strand (184). Argonaut destroys target viral RNA by disintegrating the complementary guide strand. Several viruses produce RNAi suppressor proteins (1A proteins in Nodaviridae or B2 proteins in Dicistroviridae) that limit the action of the RISC during infection, thus indicating the significance of the RNAi pathway in the regulation of viral infections (185). The FHV B2 protein is a dimer that binds to dsRNA to inhibit Dicer2 from digesting it (186). DCV A1 protein functions similarly to FHV B2 by attaching to dsRNA (187); while Argonaute suppresses the RNAse activity of the 1A protein of the cricket paralysis virus (CrPV) by binding to it (188). When a virus lacks these proteins, the reproduction is inhibited thus enabling the insects to easily remove the infectious microbe propagule. Clearly, the flavivirus NS4B protein of Dengue virus 2 (DENV2) inhibits siRNA pathways in human and insect (Sf21) cells as well (93).

Insects also use autophagy as an antiviral strategy; unlike Toll, Imd, and JAK-STAT pathways, this one does not rely on them (1). During autophagy, double-membrane vesicles called autophagosomes are formed inside of cells (189). Newly synthesized membranes, including fragmented organelles and protein clumps, are used to form these vesicles (190). Lysosomes then work along with the autophagosome to digest its cargo. Autophagy is activated in response to a wide variety of stress signals, including as food deprivation, infection, and the need for cellular repair (191). Consequently, autophagy, a form of cellular degradation, aids in nutrient recycling and keeps cells in balance (192). The autophagy signaling system includes the phosphoinositide 3-kinase (PI3K)-Akt pathway, which elevates levels of the autophagy inhibitor target of rapamycin (TOR) (193). Under normal development conditions, TOR is active and phosphorylates the Autophagy-Related (Atg) 13 protein several times. Reduced Atg1 kinase activity prevents Atg13 from interacting with Atg1, preventing this crucial autophagy regulator from doing its job (194). In the presence of hunger, Atg13 is rapidly dephosphorylated, allowing it to form a complex with Atg1 and activate it; this in turn reduces TOR activity. Atg1 then binds to more Atg proteins to form the PAS, which in turn initiates autophagy (195). Under normal development settings, different Atg proteins assemble at the PAS to generate cytoplasm to vacuole targeting (Cvt) vesicles, but under famine conditions, different Atg proteins assemble at the PAS to generate autophagosomes (196). When infected with vesicular stomatitis virus (VSV), Drosophila exhibit a reduction in the PI3K-Akt-TOR signaling pathway (197). Dueto the increased autophagy, viral replication is inhibited (198). The viral surface glycoprotein (VSV-G) was hypothesized to be the PAMP that induced this cellular response (199). Toll7, the Drosophila TLR ortholog, was found to detect VSV on the cell surface, according to recent studies (200). Toll7 signaling was triggered by VSV infection, and blocking this signaling led to elevated viral protein levels in vitro and pathogenicity in vivo (201).

Apoptosis may be thought of as a sort of programmed cell death (202). A molecular complex is formed between the adaptor protein Ark and the caspase Dronc (203). Effector caspases like Drice and Dcp1 are activated by Dronc, and when they cleave proteins, they ultimately cause programmed cell death (1). Apoptosis has a role in the defense against baculoviruses in Lepidoptera (204). This process seems to be important in mosquitoe’s defense mechanisms against the West Nile Virus and the Sindbis virus (205), Additionally, phagocytosis of apoptotic cells was found to impart defense against the Drosophila C virus (129).

The interactions between a host and a pathogen are significantly influenced by miRNAs. Insects rely on the dynamic miRNA-mRNA for immunological response to pathogen attacks ( Figure 4 ), as it is responsible for regulating the potent insect signaling pathways that either enhance or suppress innate immune responses ensuring homeostasis (1). For instance, an extensive variety of signaling pathways were enhanced after infection of Drosophila with Micrococcus luteus due to differentially produced miRNAs and mRNAs. These pathways included Toll and Imd (207).