Insect Immunology

Yali Friedman

March 6, 1998

Introduction

Approximately 1 million species of insects in 25 orders and more than 600 families have been described. It is estimated that as many as 30 million species may exist. Insects occupy nearly all terrestrial and freshwater habitats and use nearly all species of plants and animals as food sources^[1]. In light of this diversity, it is difficult to summarize the immunology of even a portion of the insects in a single paper. This review will illustrate examples from selected insect species to give a flavour of insect immunology as a whole.

Because of their short life spans relative to most mammals and the diversity of their habitats, insects face unique immunological challenges. Insects also serve as vectors for human diseases such as malaria and trypanosomiasis. Knowledge of the insect immune system can therefore yield information important in the prevention of human disease and the study of immunology in general.

Insect and mammal lineages diverged more than 580 million years ago^[2]; thus one would expect many differences in the respective metabolic processes of insects and mammals. The study of insect immunology may demonstrate alternative antimicrobial strategies compared with our current paradigms. A better knowledge of insect immunology can improve both our basic and applied sciences in several areas.

Acquired Immunity

In vertebrates, acquired immunity can take days or weeks to develop. While most insect species show no evidence of acquired immunity, it has been proposed that the short life span of these insects makes a lengthy training system as found in vertebrates impractical^[3]. This argument does not hold for cockroaches, as they can live 3-4 years in the lab - longer than a laboratory mouse^[4]. To test for immunological memory, cockroaches were immunized with heat killed Pseudomonas aeruginosa, rested, and challenged with an LD₁₀₀ dose of living P. aeruginosa. An initial non-specific response was followed 5 days post-immunization by a more specific and longer-lasting response (Faulhaber and Karp, unpublished). Thus, longer-living insects demonstrate characteristics consistent with an acquired immune response - specificity and memory^[5].

Innate Immunity

The current model of innate insect immunity in higher insects, using Dipteran examples, involves three interconnected reactions: 1) the induction of proteolytic cascades by wounding (even sterile wounding); 2) cellular defence reactions and/or encapsidation of invading microorganisms; 3) rapid and transient synthesis of antimicrobial (poly)peptides by the fat body^[6] and midgut^[7]. Close to 100 antimicrobial (poly)peptides have been characterized and are divided into two major classes: cyclic peptides with disulfide bridges and linear (poly)peptides without disulfides^[6]. Lysozymes have also been identified and are closely related to c-type lysozymes in vertebrates^[8].

Cyclic Antimicrobial Peptides

The most prominent peptides in this class are the 4 kDa anti-Gram- positive defensins and the 5 kDa anti-fungal peptide drosomycin. The three dimensional structure of defensin demonstrates a central amphipathic à- helix linked via two disulfide bridges to an antiparallel-sheet. Defensins are structurally similar to some scorpion venom toxins (scorpions appeared 150 million years before higher insects^[5]). Defensin homology among Drosophila and members of the orders Coleptera, Hymenoptera and Hemiptera shows high degrees of homology and strict structure conservation^[6]. Drosomycin shows homology to plant antifungal peptides^[8].

Linear antimicrobial (poly)peptides

The linear immune-inducible peptides are grouped into three families: cecropins, proline-rich peptides, and glycine-rich polypeptides. Cecropins are 4 kDa, strongly cationic, contain amphipathic à-helices, and cause instantaneous lysis of bacterial cells through disintegration of their cytoplasmic membranes. Although cecropins can form channels, it is thought that they instead act as detergents. Cecropin-like molecules have also been found in mammals^[6].

The proline-rich peptides comprise a family of 2-3 kDa, predominantly anti-Gram negative, antibacterial peptides. The mode of action of proline-rich peptides is not understood at present. Originally isolated from honey bee, apidaecin is the prototypic member of this family. Apidaecin, which inhibits the viablility of many Gram-negative bacteria at nanomolar doses, but does not affect Gram-negative bacteria nor fungi, exhibits a near immediate lethal activity that is not due to conventional lytic activity. It has been proposed that the proline-rich peptides derive from a common precursor, based upon the sequence similarity identified in the known members^[6].

The glycine-rich family of inducible antibacterial molecules includes several 9 to 30 kDa polypeptides, all with a 10-22% glycine content. These polypeptides are predominantly active on Gram-negative bacteria. The mode of action of glycine-rich antimicrobial polypeptides is not well understood^[6].

Self vs. Non-Self

One of the most important characteristics of an immune system is the ability to recognize and destroy foreign material while sparing native tissues. While most organisms may not expect to be infiltrated by tissue from non-genetically identical same species organisms (allogeneic tissue), allograft rejection may be indicative of sensitivity of the host immune system to mutated or virally transformed cells^[5]. Following conflicting experimental results on the existence of insect allograft rejection, Howcraft and Karp^[9] conducted an experiment to test for allograft rejection in cockroaches. Filter paper fragments were implanted into donor animals to facilitate coating with host hemocytes. After in vivo incubation, the filter papers were removed and incubated in vitro with ³H and implanted into recipient cockroaches. Increased loss of radioactivity in allogeneic implants vs. autogeneic implants after 3-5 days was taken as an indication of increased cytotoxic activity. A second experiment^[10] showed short-term memory: in previously grafted animals, 94% of second-set allografts were rejected compared to 53% rejection of third-party allografts after 3 days. This response shows memory and specificity - both attributed to an adaptive immune response.

The Immune Response

Studies involving Drosophila melanogaster demonstrate that two reactions are immediately triggered in insects by wounding or introduction of foreign objects: phenoloxidase activation and hemolymph clotting. Phenoloxidase catalyses the key steps in the formation of melanin, resulting in a dark layer around wounds and encapsidated parasites. Phenoloxidase is present in the hemolymph as an inactive pro-enzyme that is converted to its active form by a serine protease cascade. The mechanism of blood clotting in insects is poorly understood. In Limulus, another arthropod, clotting has been shown to be activated by a serine protease cascade^[8].

The presence of microorganisms in the body of Drosophila can give rise to profound changes in physiology and behaviour. The animals may stop feeding, development may be delayed, and the animals may seek a higher temperature if placed in a thermal gradient. This malaise reaction is also reflected by altered expression of lysozyme in the midgut. In adaptation to a diet rich in microorganisms, Drosophila expresses highly specialized lysozymes in the midgut. If bacteria are injected into the body cavity, expression of these specialized lysozymes in the midgut is specifically repressed^[8].

Tissues involved in the immune response

Inside the body cavity, microorganisms are attacked by hemocytes, and some hemocytes are also involved in the production of antibacterial peptides. The main site of synthesis of induced antibacterial vpeptides, however, is the fat body - an adipose tissue with somewhat analogous metabolic function to the mammalian liver. It is hypothesized that peptidoglycan fragments and lipopolysaccharide molecules released from digested bacteria, as well as other putative endogenous signals, act to signal the fat body to begin antibacterial peptide synthesis. Classically regarded as providing passive immunity, the epithelial and cuticular layers of the integument and intestinal tracts play an active role by a localized secretion of cecropin following abrasion, as seen in the silk moth, Bombyx^[8].

Control of Gene Expression

Comparison of several cloned Drosophila immune genes shows a shared upstream motif (GGGRAYYYYY) that is very similar to the binding motif for the transcription factor NF-kB (GGGRNNYYCC) that is a regulator of immune and acute-phase responses in mammals. Cloned insect factors compete with NF-kB for binding to the same DNA template, and antibodies against NF-kB prevent binding of the insect factors. In Drosophila, deletion of the NF-kB - like element destroys the promoter in fusion constructs, and a trimer of the element is sufficient to allow induced expression from a minimal promoter in mbn-2 cell lines^[8].

Recognition Molecules

Since the Drosophila system is not adaptive, it is likely that the specificity of the immune response is controlled by a fixed number of recognition molecules that are specific for common microbial epitopes and perhaps also for substances released by damaged tissues. The only purified insect proteins that can definitely be assigned a function as recognition molecules are those involved in the activation of the phenoloxidase pathway. Soluble proteins have been found in the hemolymph of silk moth that are specific for peptidoglycan or B-1,3 glucans and can activate the phenoloxidase system in vitro upon binding their respective ligands.

Induction of the immune response in higher insects shows characteristics of the innate response in mammals: recognition of geretic microbial epitopes, synthesis of antibiotic peptides at the site of insult and in specialized tissues, and gross physiological adaptations. The remarkable similarity in promoter elements and promoter binding proteins in insects and mammals is even more interesting in the face of the dissimilarity of the induced immune molecules.

Conclusion

The discovery of novel antimicrobial peptides in insects suggests several applications. Two possible uses are as food preservatives and antibiotics against otherwise resistant bacterial strains. The continued search for novel antimicrobial agents from insects may also provide insights into the design of synthetic antibiotics of greater efficacy, and elucidate previously unknown metabolic pathways in pathogens.

Genetic engineering of insect vectors of human diseases with agents designed to destroy parasites^[11], along with appropriate selection determinants, may reduce or eliminate the prevalence of the human disease. The cloning of genes for antimicrobial peptides into plants under appropriate promoters can augment natural plant defenses to confer greater resistance to microbes^[12].

Because the immune system is essential to protect both insects and mammals from infection, selection for a functional immune system over evolutionary time was presumable quite strong. Thus, differences and similarities between human and insect immune systems over the 580 plus million years since their lineages diverged can tell us more about the immune system of the common progenitor as well elucidating additional paradigms of immune defence.

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