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Invertebrate organisms utilise a very diverse range of habitats and they are found in a wide spectrum of areas. The success of these organisms is largely due to an effective series of defence mechanisms that provide protection from the micro-organisms coexisting alongside them. The essential question is why do insects require such elaborate defence mechanisms? Insects are comparatively small, rapidly reproducing animals and it may be logical to reason that although a specific individual may die from infection the species overall could survive by the huge reproductive capacity of the species overall. However when the life cycles of both Drosophila melanogaster and micro-organisms like Escherichia coli are compared we see that insects must have a powerful immune system to counteract micro-organisms. Given optimal conditions Escherichia coli have a generation time of 20 minutes and even the most rapidly reproducing insect model, Drosophila melanogaster requires around 14 days to produce offspring (). Insects that lacked a highly evolved immune system would never survive long enough to produce the subsequent generation. Studies into insect immunity have become very popular in recent years as numerous similarities between the immune system of Drosophila melanogaster and humans have been discovered. Therefore Drosophila melanogaster have become very useful models to investigate parts of human immunity.
Evolutionary history of immunity
Immunity may originate from the earliest competition for resources. The best way to secure a limited resource is to eliminate all other competition by infection. Bacteria produce antibacterial peptides to kill other bacterial species and thus succeed in securing a resource. As multicellular organisms began to evolve the requirement to recognise assess and react to micro-organisms was vital. Mechanisms of defence evolved early in metazoan history () and the most effective mechanisms have been conserved across species explaining the similarities between human and Drosophila immune defence (). Cellular machinery, molecular components and general signalling pathways are shared to combat the invasion of pathogens as some plants utilise the same detection mechanisms ().
The three most important features of an immune system are microbial detection, rapid response and effective elimination. Micro-organisms are detected through cell surface receptors that recognise components of the pathogen cell wall (). Components of the pathogen differ significantly from host cells therefore confirming the cells are non-self (). In order to initiate immune defense immediately, receptors are produced by the cells involved in the defensive frontline of the cell. The cellular reaction results in the production of natural killer cells and macrophages coupled with the expression of antimicrobial peptides (). Insect immunity only involves innate responses as they lack adaptive immunity because they do not contain the infectious memory of T-cells. 45,000 vertebrates utilise both innate and acquired immunity in response to micro-organisms (). Acquired immunity offers two main advantageous features; clonal selection which drives exceptional pathogen response specificity and immune memory. This enables rapid response to reinfection. However the clonally specific acquired response requires time and is dependant on the evolutionary selected innate defense. The innate response ensures the immune memory of the species is retained and acquired immunity retains the individual's immune response (). Drosophila has reignited the immunological interest in innate immunity in the last few decades.
Overview of insect immunity
The immune response in Drosophila melanogaster is a multifaceted system. The epithelial surfaces that line the epidermis, digestive and genital tracts all express antimicrobial peptides as the first line of defense against invading pathogens. Pathogens that succeed in evading the first line of defense and enter the haemocoele (the body cavity of the Drosophila) are counteracted by both hummoral and cellular immune responses.
The humoral response involves the rapid production of antimicrobial peptides (AMPs) in the body, subsequently released into the Drosophila haemolymph. These AMPs can also be secreted from the circulating haemocytes and epithelial cells but in much smaller concentrations. Proteolytic enzymes are also secreted into the haemolymph inducing melanisation around wounds and invading objects ().
Several types of haemocytes have been identified in various literature using histochemical, functional and morphological characteristics to distinguish cell types (). However in recent years genetic and antibody markers have successfully been used to identify these cell types and distinguish different haemocytes in specific species (). Prohaemocytes, plasmatocytes, spherule cells, granular cells and oenocytoids are the most frequently discussed haemocytes in literature. They occur in a diverse range of species including Coleoptera, Hymenoptera, Lepidoptera, Blattaria, Collemboda, Hemiptera and Diptera (). Drosophila haemocytes are described using different names than most other insects, including other insects from the Diptera order such as simuliids and culicids (). The only haemocytes present during Drosophila embryogenesis are plasmatocytes. In the larval stages of Drosophila development the most abundant circling haemocyte are plasmatocytes however both crystal cells and lamellocytes also exist in larvae haemocoele.
The cellular defence mechanisms of Drosophila are executed by three different types of haemocytes circulating in the haemolymph. Plasmatocytes phagocytise invading pathogens and this process involves the plasmatocyte adhering to a target pathogen then modifying the cytoskeleton, internalising the pathogen and destroying the engulfed target (). Adhering to the target particle involves receptors that either recognises micro-organisms directly or through binding to intermediate opsonising molecules. Plasmatocytes are responsible for the recognition and engulfment of micro-organisms however they also dispose of apoptotic cells. Several potential plasmatocyte receptors have been identified. The croquemort gene has been identified as a requirement for the destruction of apoptotic cells in the embryonic stages (). Drosophila scavenger receptor CI (dSk-CI) was identified by Pearson et al (1995). This scavenger receptor displays very broad ligand recognition which is similar to mammalian class A scavenger receptor. Other recognition proteins found within insects are GNBPs, Gram-negative bacteria binding proteins. All GNBPs contain glucanase- like domains.
Lamellocytes and crystal cells:
Two other Drosophila haemocytes are involved in encapsulating micro-organisms; and the process of encapsulation appears to be an immune response entirely restricted to invertebrates and is executed by specialised haemocytes that differ significantly between species. In Drosophila, encapsulation is undertaken by the lamellocytes and crystal cells. There are in excess of 50 species of hymenopteran that parasitize Drosophila. Hymenopteran females often lay their eggs in the haemocoel of Drosophila larvae (). The parasitic egg is detected by plasmatocytes circulating in the haemocoel and they tether to the egg chorion (). After the egg tethers a massive cellular response is observed in the fat body with a significant increase in the number of crystal cells (), greater haemocyte proliferation and huge levels of differentiation of lamellocytes (). The increased numbers of lamellocytes are then able to form a multi-layered capsule around the pathogen. This encapsulation is subsequently accompanied by the melanisation and eventual blackening of the capsule. The pathogen within the capsules killed by cytotoxic free radicals, quinones and semi-quinones and the eventual asphyxiation of the parasite ().
Haemocytic-mediated effector responses
The process of phagocytosis is widely conserved cellular reaction to invading pathogens occurring in all metazoan and most protozoa phyla. Phagocytosis has been observed most extensively in mammalian leukocytes such as macrophages (). A target binds to its cognate receptor and this activates signalling cascades which regulate the formation of a phagosome and ingestion of the pathogen is triggered by an actin polymerization-dependant mechanism. Vesicle fusion events produce effector molecules that mature the phagosome into a phagolysosome and the target is then degenerated by the effector molecules. Phagocytosis in insect haemocytes has not been as extensively studied as mammalian systems however similarities between haemocyte lysate proteins in the fruit fly C. capitata and mammalian phagocytosis regulating signalling molecules have been discovered. The cross-reaction of these molecules indicates that the signal transduction cascades that regulate this process are homologous between mammals and insects ().
Encapsulation and nodule formation:
Recognition of a target is followed by the formation of a capsule or nodule. Encapsulation requires haemocytes to change from non-adhesive circulating cells to adhesive cells with the capacity to bind to the recognised target and other adhesive circulating haemocytes. These haemocytes then aggregate to the surface of the parasitoid adhering to it and each other forming a multilayered, melanotic capsule which is coupled with the deposition of the black pigment eumelanin on the surface of the capsule and among the inner most layers (). Cytotoxic quinoid intermediates of eumelanin have been discovered that bind to nucleophiles present on the surface of parasitoids. This binding forms cross linking complexes which could effectively immobilise the invading parasite. During melanogenesis ROS (reactive oxygen species) are produced which are cytotoxic to parasitioids. Levels of the ROS superoxide anion were measured in Drosophila parasitized by L. boulardi. Increased levels of this ROS were detected in immune-reactive flies during encapsulation. However in immune susceptible hosts elevated levels of super oxide anion were not identified and the parasite egg developed uninterrupted (). Elevated levels of H2O2 and nitrite were also discovered in resistant Drosophila compared to susceptible strains. Once encapsulated the parasite almost always dies and other factors potentially leading to its eradication include asphyxiation, RNI and antibacterial peptides (). Superoxide anion and H2O2 are not toxic to L. boulardi and it remains to be confirmed that significantly increased levels of these compounds actually exist inside the capsule. Capsules formed in Drosophila do undergo melanization reactions but many insects produce capsules that do melanise. The melanization of capsules within the Drosophila immune system indicates that it is the production of cytotoxic quinoid intermediates produced during the process of melanisation that definitely act as killing agents to invading parasites ().
Cellular Response to Parasite Infection in Larvae
Previous studies had focused primarily on observing the role of circulating haemocytes in response to parasite infection (). However recent investigations into the parasite immune response have suggested that the lymph gland may be a vital part of the encapsulation process. Parasitization by the virulent wasp species' Ganaspis xanthopoda and L. heterotoma correlated significantly with a reduction in the size of lymph glands in third-instar host Drosophila (). This evidence indicates a significant release of lymph gland haemocytes consistent with an immune response or the destruction of Drosophila immune effectors by the parasitoid wasps. Lamellocytes were observed in the lymph gland 24 hours after parasitisation by L. boulardi. This was followed by the anterior lobes dispersal and lamellocytes appearing in the posterior lobes indicating that lamellocytes needed for encapsulation originate from the lymph glands (). Second-instar wild-type Drosophila were infected with L. boulardi and lymph gland haemocyte proliferation increased in the early stages of developing third-instar larvae. However this increase was not detected in second-instar larvae. The quantity of lamellocytes and crystal cells increase in the third-instar hosts and lymph gland dispersal correlates significantly with parasite infection confirming the link between cellular immune capacity in larvae and lymph gland ontogeny ().
Two Signalling Pathways in Response to infection
It was established in the 1990s that Drosophila produced six entirely different AMPs to resist various fungal, Gram-negative and Gram-positive bacterial infections. The AMP promoter genes contain sequence motifs with high similarity to NF-«B response factors in the mammalian immune system (). These sequence motifs were discovered to be crucial to induce an immune response in the AMP genes (). High similarity was discovered between the activation of Dorsal by the Drosophila Toll pathway and the mammalian cytokine-induced activation of NF-«B (). In 1996 Drosomycin and general resistance to fungal infections was shown to require a Toll transmembrane receptor and numerous other components of the Toll signalling pathway (). Conversely the AMP Diptericin was mostly independent of the Toll pathway and instead needed a wild-type copy of an undiscovered gene named immune deficiency (imd) (). imd mutants responded solely to Gram-negative infections leading to the discovery of a second signalling pathway in the Drosophila immune response.
Gram-positive and fungal infections- The Toll pathway
Fungal infections and Gram-positive bacterial infections are combated by components of the Toll pathway and this was discovered in the early embryonic stages through mutations affecting dorsoventral patterning (). The transmembrane receptor Toll has leucine-rich repeats in the extracellular domain. The intracytoplasmic domain demonstrates high sequence similarity with the equivalent section of the interleukin 1 receptor (IL- IR). Collectively the Toll-IL-IR domain is referred to as TIR. TIR interacts with numerous intracytoplasmic regions containing death domains. Two of the intracytoplasmic regions are adaptor proteins, MyD88, containing a TIR domain like Toll () and Tube (). Pelle is a third death domain protein containing a serine-threonine kinase region (). When challenged with a fungal or Gram-positive bacterial infection, mutants of MyD88, Tube and Pelle can not produce a normal immune response (). Once activated the Toll receptor adaptor complex sends a signal to a transcriptional factor of the NF-«B-Rel transactivators which are inducible. A complex is formed from the factor binds to the ankyrin-repeat inhibitor protein Cactus (). Cactus then dissociates from the Rel protein due to Toll signalling (). Three Rel proteins are encoded by the Drosophila genome and each of these proteins share common homology in the domain responsible for DNA binding and dimerization. These Rel proteins are Dorsal (), Relish () and Dorsal-related immunity factor (DIF) (). DIF is the primary Rel protein involved in the response to fungal and Gram-positive bacteria in adult Drosophila (). A study by Rutschmann et al demonstrated that in larvae Dorsal can substitute for DIF (2000). The dissociation of Cactus from Dorsal and DIF is controlled by the phosphorylation cactus (). A genomic analysis of Drosophila indicated that DIF activates the transcription of several hundred genes involved in the immune response (). AMPs such as Drosomycin and Metchnikowin are encoded for by these genes transcribed from DIF. The products of many of these genes do act in conjunction to fight off both Gram-positive bacterial and fungal infections but the ability to resist these infections is not exclusively based on the production of these AMPs.
Gram-negative Infection- The Imd Pathway
The Imd pathway reacts exclusively to Gram-negative bacterial infection and is not induced by fungi or Gram-positive bacteria. Once a Gram-negative infection has occurred transcription of several genes encoding the AMPs Cecropins, Drosocin, Diptericins and Attacins is induced. In the Toll signalling pathway induction of genes encoding AMPs requires a member of the Rel inducible transactivators' family. Contrastingly the Rel protein Relish involved in the Imd pathway is not inhibited by Cactus as it is in the Toll pathway. Relish is inhibited by several COOH-terminally located ankyrin repeat domains within its own structure (). An endoproteolytic clevage induced by a signal is needed to activate Relish; this clevage then liberates the Rel domain from the domain containing ankyrin repeats. After the Rel domain has been freed then nuclear translocation can occur (). A signalosome containing proteins with high sequence homology to I«B kinase
¢ (IKK¢) in the mammalian immune system and IKK (NEMO) which is a protein required for structural formations, is needed for commencing activation of Relish and the induction of required target genes after its activation. Signalosome mutant's kenny and ird5 have significantly increased infections from Gram-negative bacteria than wild-type flies but they are able to combat invasion of Gram-positive bacteria and fungi as efficiently as wild-type flies. This process leads to the eventual cleavage of Relish due to its phosphorylation and is triggered by bacterial LPS (lipopolysaccharide) (). It is thought that a product of the imd gene has been identified as a death domain protein with a high sequence similarity to receptor-interacting protein (RIP) of TNF-¡, a tumor necrois factor-¡ in mammals (). The imd gene also acts upstream of the gene encoding DREDD which is a caspase-8 homolog also essential for Gram-negative bacterial resistance (). MAP3 which is a mitogen-activated protein 3 kinase is located downstream of the imd gene and this is necessary for gene expression associated with Relish and Drosophila mutants that lack this kinase. Mutants lacking MAP3 demonstrate inhibited production of AMPs and a decreased level of Gram-negative bacterial resistance (). In a similar way to imd, MAP3 shows high sequence similarity with mammalian TAK1 which is the transforming growth factor-activated kinase 1. IMD is almost certainly involved in the detection of Gram-negative bacteria using a receptor-adaptor complex most likely activated by the final product of a proteolytic cascade similar to that of Toll.
Activation of the Toll and imd pathways by the introduction of pathogens
Both pathways have been investigated using mutants deficient in component genes in either of the pathways however Drosophila wounded with a sterile needle demonstrated activation in both pathways in low levels. An injury with either Gram-negative or Gram-positive initiates the production of the full range of Drosophila AMPs gene products. However Drosophila do have a degree of specificity as a significant increase in gene expression for AMPs with response to Gram-negative bacterial infections when challenged with those Gram-negative bacteria. Experiments coating the exterior of the Drosophila with fungal spores evoked a strong but slow production of antifungal peptides yet antimicrobial peptide expression remains switched off (). The whole range of AMPs are induced in reaction to the injection of fungal spores. Many microorganisms contain many distinct structural patterns on their exterior cell walls and when they are administered internally to the flies both the Toll and Imd pathways are activated by diverse structural components. Exchange of information between the two separate pathways may potentially occur due to the interaction of transcription factors. Expression of antimicrobial peptide genes could be influenced by the heterodimerization of numerous Rel proteins (Han et al, 1999). Some AMP genes contain promoter regions that respond to both the Imd and Toll pathways one such antimicrobial peptide gene is the one encoding Drosomycin and it can potentially be induced by either or both pathways. Investigations into the existence of a third diverse pathway that accounts for the induction of these AMPs used double mutant Drosophila that lacked the components for both pathways. However these flies lacked expression of any AMPs and fail to resist any microbial challenge ().
Epithelial AMP induction
The breadth and quantity of AMPs induced by the fat body in response to a microbial challenge is huge. In contrast to the AMPs initiated by the fat body, the AMPs produced by the Drosophila epithelial barrier seem limited. AMP prodution in the epithelial barrier has been perceived in many other insects and in mammals such as humans leading to the theory that epithelial AMP response is the shared ancestral defense against microorganisms. Experiments showed that the epithelial response was not compromised in Toll pathway mutants and it was only deficient in Imd mutants (). Drosomycin is the antifungal peptide induced by the Toll pathway but in mutant flies lacking a sufficient Toll pathway Drosomycin is induced by the epithelial response.
Challenge with Gram-negative bacteria
Live E.coli challenge up-regulated gene expression
Numerous genes have been found to be up-regulated in response to infection of Drosophila with live E.coli. Genes involved in both the cellular and humoral response like actin bound/cytoskeleton proteins and Attacin A are up-regulated. Pvf2 which is one of the three ligands to the Drosophila receptor protein homologous to the mammalian receptor PDGF/VEGF and this up-regulation suggests enhanced cell proliferation. In larvae the increased expression of Pvf2 leads to increased levels of haemocytes circulating in the haematocele (). This suggests that the introduction of a significant bacterial infection activates the differentiation or maturation of haemocytes from pro-haemocytes and therefore increases the Drosophila cellular response.
Induction of genes in response to complete E.coli bacteria
The most abundant proteins with increased gene expression in response to E.coli were those required for modification of tissue and stress signalling. Est21C a transcription factor and Mmp1 a matrix metalloproteinase 1 were found to be up-regulated in a study carried out by (2005) injecting live E.coli bacteria. This confirmed results from an earlier microarray study by (2002) although Johansson et al found that Mmp 1 expression only increased after exposure to whole E.coli bacteria and not in response to introduction of crude LPS alone. In Drosophila matrix metalloproteinases are involved in tissue alteration () and in the mammalian immune system they are involved in wound repair (). The mammalian equivalent of transcription factor Est21C is ERG and it is associated with the central nervous system development (). Genes encoding proteins that are linked to cytoskeletal structure demonstrated high levels of up-regulation when challenged with complete E.coli bacteria. The proteins D-SCAR and D-WASP are essential for correct phagocytosis in Drosophila () and one protein (CG13503) with an actin binding domain is significantly up-regulated in response to immune challenge. A significant up-regulation of Relish was observed signifying an activation of the Toll signalling pathway. The up-regulation of Relish in response to bacterial challenge is indicative of cell proliferation in the mbn-2 cell line.
Down-regulation of protein expression after E.coli immune challenge
The up-regulation of Relish and increased level of proteins associated with this pathway after a large bacterial infection seems in accordance with most previous studies. In contrast to this (2005) found that numerous AMPs are actually down-regulated in response to complete bacterial challenge, AMPs such as Drosocin, Drosomycin, Diptericin, Attacin-A and C however these AMPs can be induced by the injection of crude LPS alone. PGRPs, -SD, -SB1, -SA are all immune genes with down-regulated expression following bacterial infection (). CG30086 and CG4259 which are serine-type end peptidases were previously unidentified as immune genes were seen to be down-regulated in response to E.coli. Significant decrease of P450 appears to be indicative of immune response in Drosophila (). Finally the transcription factor Bigmax was also found to be down-regulated; this transcription factor regulates cell death during development.
Unlike previous studies that have focused primarily on gene regulation and overall expression in response to bacterial challenge I want to observe the fluctuations in haemocyte number in adult Drosophila using the bacterial strain M15pREP4 Escherichia coli. Using varying concentrations of E.coli to infect both male and female Drosophila I will examine the relationship between the degree of pathogenic challenge and the gender of the samples over a period of 240 minutes.