The humoral immune response of insects consists of the processes of melanisation, haemolymph clotting and wound healing in response to injury . In addition, the humoral response also involves the synthesis of a range of anti-microbial peptides and heat shock proteins . Although insects do not produce antibodies they are capable of generating a series of proteins which confer a degree of non-specific immunity to a range of microorganisms.
The process of melanisation is key to the defence against a wide range of pathogens and results in the deposition of melanin on the microbe within the haemolymph . The formation of melanin is catalysed by phenoloxidase-monophenyl-l-dopa: oxygen oxidoreductase (EC 18.104.22.168) . PO is found in insects in its inactive form pro-phenoloxidase (ProPO) located in the haemocytes as a zymogen. It is released from haemocytes by rupture and is either actively transported to the cuticle or deposited around wounds or encapsulated parasite. PO catalyses the o-hydroxylation of monophenols and oxidation of phenols to quinines which then polymerise non-enzymatically to form melanin . Insect ProPO has a sequence similar to the thiol-ester region of the vertebrate complement proteins C3 and C4. In vertebrates following cleavage by an activating protease, the thiol-ester region becomes active and can react with hydroxyl or amino groups on biological surfaces leading to immobilisation of the molecule on the foreign material.
The process of melanisation is initiated by soluble pattern recognition receptors that bind target surfaces thus initiating the serine protease cascade leading to cleavage of ProPO to PO and ultimately the cross-linking and melanisation of proteins. This process is frequently referred to as the Prophenoloxidase activating system (ProPO-AS). Pattern recognition receptors (PRRs) activate the complement system in vertebrates and the PrpPO-AS system in insects. A number of PRRs are present in insects (Table 2) and include: the C-type lectins which bind bacterial LPS, the peptidoglycan recognition protein which is expressed in the fat body, haemocytes and epithelial cells and the β-1,3 glucan binding protein which recognises fungal glucan and activates the ProPO-AS pathway.
Table 2. A comparison of humoral and cellular PPRs, and anti-microbial peptides and enzymes in humans and insects
|Humoral PRRs||Macrophage mannose receptor (175 kDa).||LPS binding protein|
| ||f-Met–Leu–Phe receptor (binds to N-formyl peptide).|| |
| ||c-type lectins||Lectins|
| ||C2-type immunoglobulin domain||Hemolin|
| || ||β-1,3 glucan binding protein|
| || ||Gram (−ve) bacterial recognition protein|
| || ||Peptidoglycan recognition protein|
| ||Complement/α2 macroglobulin||αTEPI|
| ||von Willebrand platelet aggregation factor||Hemocytin|
| ||Scavenger receptor|| |
| || || |
|Cellular PRRs||Toll-like receptors||Toll|
| || ||Toll 3–8|
| || ||18-wheeler|
| || ||Immune deficiency (imd)|
| ||Integrins (CD11b/(CD18) and LFA-1.||Integrins (α,β)|
| || ||heterodimeric proteins|
| || || |
|Cationic proteins||Elastase (29–31 kDa) AB, AF||Attractin/|
| ||Cathepsin G (25–29 kDa) AB, AF||Sarcotoxin (20–28 kDa) AB|
| ||BPI (55–60 kDa) AB|| |
| ||Lactoferrin (78 kDa) AB|| |
| ||Proteinase 3|| |
| ||Azurocidine (29 kDa) AB, AF|| |
| ||Lysozyme (14.4 kDa) AB, AF||Lysozyme AB, AF|
| ||MPO/H2O2(150 kDa) AB, AF|| |
| || || |
|Metalloproteinases||Collagenase||Metalloproteinase (297, 198 and 95 kDa)|
| ||Gelatinase|| |
| || || |
|Peptides||Defensins (4 kDa) AB, AF||Defensins AB|
| || ||Cepropins (4 kDa) AF|
| || ||Diptericins (9 kDa) AB|
| || ||Drosocin AB|
| || ||Metchnikowin AB, AF|
| || ||Proline-rich anti-microbial peptides AB|
| || ||Drosomycin AF|
| || ||AFP AF|
The serine protease cascade controlling the cleavage of PPO or PO is highly controlled and counter-balanced by protease inhibitors since the reaction must be maintained near the site of invasion because active PO can produce highly reactive and detrimental oxygen intermediates .
Mammalian immune cells express several Toll-like receptors (TLR) that are considered cellular PPRs, because they directly recognise LPS and other microbial products. Within the Drosophila genome, Toll and several additional Toll-related genes (Toll-3-8, 18-wheeler) and the gene immune deficiency (imd)  have been identified. In 1997, Medzhitov et al.  cloned the human homologue of Toll (TLR4) and to date 10 members of the TLR family have been identified in humans .
Drosophila Toll is a transmembrane protein with an extracellular leucine-rich domain and a cytoplasmic domain  (Fig. 3) that resembles the cytoplasmic domain of the interleukin (IL)-1 receptor. Toll was originally identified as a critical factor for the dorsoventral polarity of the Drosophila embryo . Toll induces the activation of Dorsal, a member of the Rel family, of rapid inducible transcription factors through the degradation of the protein cactus (Fig. 3). The latter causes retention of Dorsal in the cytoplasm, and the complex Toll/Tube/Pelle mediates its phosphorylation and degradation. Spaetzle, the extracellular Toll ligand, controls the generation of microbicidal peptides and involves the gene cassette spaetzle/Toll/cactus but instead of Dorsal, the Dorsal-related immunity factor (Dif) is involved. As illustrated in Fig. 3, there are remarkable structural and functional similarities between the systems mediating Drosophila Toll and mammalian IL-1 receptor-mediated signalling . Nuclear factor NF-κB, inhibitory I-κB and the serine threonine kinase IRAK are mammalian homologues of the Drosophila Dorsal, cactus and Pelle, respectively.
Figure 3. A diagrammatic representation of the similarities between Drosophilia and human Toll cascades. Signalling through Toll and Cactus proteins resulting in activation of Dorsal or Dif, parallels signalling induced by Toll/I-κB and activation of NF-κB. In insects, microbicidal peptide synthesis is controlled by spaetzle, the extracellular Toll ligand, involving the gene cassette spaetzle/Toll/cactus/Dif (illustrated by dashed line).TLr2 and TLr4 are the best studied in humans as the co-receptor for LPS, the other co-receptor being CD14 leading to the expression of pro-inflammatory cytokines. Abbreviations used: LBP, lipopolysaccharide binding protein; I-κB, inhibitory kappa B; NF-κB, nuclear factor kappa B.
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Signalling processes leading to production of anti-microbial peptides have been studied by subtractive hybridisation and RACE PCR with the positive identification of immunorelevant genes and LPS-induced defensin-like anti-microbial molecules in G. mellonella. In vivo knock-out studies revealed that adult flies lacking Toll had compromising levels of drosomycin resulting in reduced resistance to fungal infection . In addition mutants of the other Toll family member, 18-wheeler are more susceptible to bacterial infection .
Although the cellular and humoral responses so far described are effective in combating microbial invasion they are unable to totally clear the haemocoel if a large number of microorganisms enter. The last line of defence is the synthesis of a range of anti-microbial peptides, which are released into the haemolymph where they attack elements of the bacterial or fungal cell wall . These peptides play a crucial role in combating infection and similar classes of proteins are found in vertebrates, invertebrates and plants [2,6] (Table 2).
The main sites of synthesis of anti-microbial peptides in the insect are the fat body, haemocytes, the digestive tract, salivary glands and the reproductive tract. The fat body functions as a biosynthetic organ, is analogous to the liver in mammals and is also a site of binding for many haemocytes . A number of peptides are produced and all are amphipatic basic molecules that act in a detergent-like manner on cell membranes causing the death of the microorganism by lysis. Anti-microbial peptides are synthesised as pre-proproteins at a rate up to 100 times faster than IgM in mammals . Their small size allows diffusion through the haemolymph to counteract invading pathogens.
In humans the anti-microbial neutrophil proteins are located within intracellular granules which are released into newly formed phagocytic vacuoles. The proteins and peptides stored in the granules are of two kinds: (1) those with cytotoxic properties, including bactericidal/permeability-increasing protein, azurocidin and defensins, and (2) a range of enzymes, capable of contributing to the destruction of killed bacteria by digesting their macromolecules [66,67]. Among these are lysozyme, proteinases, some with independent anti-microbial activity (elastase and cathepsin G), nucleases and saccharidases (Table 2). Enzymes degrading bacterial phospholipids  and lipopolysaccharides (LPS)  are also known to be granule associated.
Attacins display a relatively narrow spectrum of anti-bacterial and anti-fungal activity and are believed to act on the outer membranes of microbial cells . It appears that the primary function of attacins may be to facilitate the action of lysozyme and cecropins thereby allowing the three immune proteins to work in consort.
Proline-rich peptides, glycine-rich peptides and diptericins. Proline-rich peptides are small, 15–34 residues and between 2 and 4 kDa . These were first isolated in larvae of Phormia terranovae. Other examples of such peptides include abaecin and the apidaecins from honey bees and other hymenoptera and drosocin from Drosophila. These peptides appear to function by increasing membrane permeability of bacteria and lyse gram-negative bacteria. Glycine-rich peptides are 9–30 kDa and are active against gram-negative bacteria. Diptericins are only found in dipterin species and are induced by and active against gram-negative bacteria.
Lysozyme is a 14.4 kDa (pI > 10) cationic protein with the ability to kill a wide range of gram-positive bacteria, by virtue of its ability to hydrolyse cell wall components. It is present in both azurophilic and specific granules of human neutrophils and is also found in the granules of monocytes and macrophages, in blood plasma, tears, saliva and airway secretions. Lysozyme is extremely active against such bacteria as Bacillus subtilis, Bacillus megaterium[71,72] and Micrococcus lysodeikticus, indeed, the susceptibility of this latter organism to lysozyme forms the basis of a laboratory assay for this enzyme . Bacterial cell walls consist in general of linear polysaccharide chains containing repeating units of N-acetylglucosamine and N-acetylmuramic acid residues in β-1-4 linkage. Lysozyme hydrolyses β-(1,4) glycosidic bonds in peptidoglucan of bacterial cell wall, is proteinaceous in nature with insect lysozyme possessing a high degree of similarity with mammalian lysozymes . Lysozymes in insects are 14 kDa proteins , may be found in haemostatic cells  and were the first anti-bacterial factor purified from insect haemolymph . Lysozyme has been located in the gut of several insects, in haemocytes of Spodoptera eridania and Locusta and in haemocyte cell lines . While lysozyme displays anti-bacterial activity it appears to work in combination with cecropins and attacins .
LPS-binding proteins. In insects, bacterial LPS-binding protein facilitates the clearance of bacteria by promoting nodule formation. Smooth strains of E. coli are cleared slowly from larvae of Bomyx mori since they possess 0-specifc polysaccharides which protect the lipid A binding site. In contrast rough strains are cleared within 30 min by nodule formation and have no polysaccharides protecting the relevant binding site .
Transferrin. In insects transferrin has an iron binding domain in the N-terminal region and may function by sequestering iron from pathogens thus inhibiting their growth . Human lactoferrin is a member of the transferrin family and displays anti-microbial properties against gram-positive and gram-negative bacteria  by limiting the availability of environmental iron . However, since iron-saturated lactoferrin is also able to kill certain bacteria, mechanisms other than iron depletion are involved .
Defensins. Another important group of anti-microbial peptides is the group of β-sheet defensins that comprise four members in humans: HNPI–HNP4 . Defensins in insects are anti-microbial cationic peptides of 4 kDa and 40 residues long which play an important role in innate and adaptive immunity . In insects, defensins show activity against gram-positive bacteria and some gram-negative species . Defensins are cysteine-rich cationic peptides containing three or four disulphide bridges and represent an early defence against invading microorganisms. Defensins may be produced within 3 h of infection and their level declines 12–36 h post-infection, suggesting a correlation between expression and presence of bacteria. Defensins act on the cytoplasmic membrane of bacteria and lyse cells by forming voltage-dependent ion channels, which lead to leakage of potassium and other ions . The widespread occurrence of defensins in higher animals and more distant defensin relatives in plants  and insects  is consistent with an early evolutionary origin [79,83]. In vitro studies reveal the microbicidal activity of defensins against a variety of bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa and E. coli, many fungi and some viruses [84–86].
Cecropins are active against gram positive and negative bacteria  and are approximately 4 kDa with 35–39 residues. Cecropins are amphipathic molecules that penetrate bacterial cell walls resulting in pore formation and subsequent ion leakage .