CHAPTER THIRTEEN

 

INSECT

IMMUNOLOGY:

 

HUMORAL IMMUNITY

 

One question entomologists and university administrators might wonder about now and again would be along the lines of "What are some of the emerging areas of insect physiology that are likely to have great impact on how entomology might be carried out in the future? Several topics come to mind, including understanding the roles of peptide hormones, the significance of biogenic amines, more detailed appreciation of sensory physiology, and intracellular signal transduction mechanisms.

However, above all of these topics, and this is certainly not to be taken as an exhaustive list, the study of insect immunology may well develop into one of the most active and most important areas of insect physiology.

 

INSECT DISEASES

 

The idea that insects can have diseases goes well back to antiquity. The idea predates the science of microbiology by centuries. The earliest records come from descriptions of a pest of honey bees by Aristotle. In the infancy of microbiology, Pasteur showed that two diseases of silkworms were caused by microbes. As you might expect, much of Pasteur's work was motivated by economic concern: the silk industry suffered considerable losses caused by diseased silkworms.

In more recent times insect pathogens have used microbes as agents of biological control of insects. Although many microorganisms have been studied as actually or potentially useful control agents, none have so far received as much attention as Bacillus thuringeinsis. The potential of various microbial forms, including bacteria, fungi and protistans, as insect control agents has driven an impressive body of work on insect microbiology and pathology.

The study of insect immunity went along with much of the very early work on the general question immunology. There was a period of considerable activity in the 1920's. A second surge of work on insect immunity started in the late 1950's, when it was discovered that a certain immunity could be induced in some insects. This induced immunity can be compared to a sort of standing, or constitutive, immunity. The most recent period of research on insect immunity began in 1980 with the first isolation and purification of an induced antibacterial factor. Modern work on insect immunity is particularly satisfactory because it is one of the areas of insect biology where molecular biological tools have yielded a rich harvest of information that would not otherwise have been generated.

It is appropriate to define immunity. Immunity is "resistance to or protection against a specified disease". This definition draws attention to the phenomonology, and it is independent of specific immunological mechanisms. The distinction is important because the contemporary wisdom in some circles holds that insects do not have an immune system because they do not produce antibodies of the mammalian sort.

 

THE MAJOR IMMUNITY MECHANISMS

 

There are two major areas of insect immunity, cell-free immunity and hemocytic immunity. These are sometimes called humoral immunity and cellular immunity, respectively. These two immunological responses are complimentary, and both may be seen in response to the same infection. Please note that immunity in insects and in other invertebrates differs from the immune systems of mammals in fundamental ways. First, insects produce no lymphocytes, of which there are many sub-types including eosinophils and neutrophils, no T-cells and no helper cells. So the cellular immunity of insects is not to be compared to the cellular immunity of mammals, although some people have tried to make that sort of comparison. Second, insects do not biosynthesize immunoglobulins, such as gamma-globulin. The important point here is that the immunity of insects lacks the molecular specifity of antibodies from mammals.

 

INDUCED ANTIBACTERIAL PROTEINS

 

Wounding or injection of certain bacteria some insects induces the biosynthesis of antibacterial proteins called cecropins. These proteins were first isolated from the hemolymph of Hyalophora cecropia, from whence the term cecropin was coined. Molecules with similar structure have since been isolated from the waxmoth Galleria, the tobacco horn worm Manduca sexta, several species of Diptera and from a beetle. Not all insects appear to produce cecropins, because a cricket, a cockroach and a locuts do not produce them. The blood-sucking bug Rhodnius produces antibacterial proteins that differ from cecropins. H. cecropia produces cecropins A, B and D as well as several minor cecropins, C, E, and F, which differ from the major cecropin by only a single amino acid substitution. All of these proteins are about 4000 in molecular weight, each with 35 to 37 amino acids. There is a very high level of homology among these proteins, all of which feature long hydrophobic reaches in the C-terminal portions of the molecule. The very close homology among these proteins is consistent with the idea that they have arisen through gene duplication.

The cecropins are not active against many, but not all, bacteria. Cecropins A and B are active against a large number of gram-positive and gram-negative species, including some that are occasional insect pathogens and some that are obligate insect pathogens and others that are non-pathogenic species. None of the cecropins are active against B. theringeinsis.

 

 

 

 

ANTIBACTERIAL ACTIONS

 

Cecropins lyse bacterial cell membranes; they also inhibit proline uptake and cause leaky membranes. These actions at the level of procaryotic membranes are consistent with the structures of cecropins. They are all cylindrical, amphipathic molecules with long hydrophobic regions on one end. In effect, these protein act like detergents. Synthesis of proteins that are analoges to cecropins show that activity against a broad spectrum of bacteria species is lost when amino acid substitutions that disrupt the alpha-helix structure are made.

The genes that code for cecropins have been cloned. These molecular biological approaches give insight into the biosynthesis of the cecropins. The gene for cecropin B, for example, codes for 26 amino acids in the N-terminus of the protein that are removed during post-transcriptional processing to produce the mature cecropin B. These are removed in 3 steps, first 22 residues are removed in one cleavage, which produces a pro-cecropin. The pro-cecropin is further processed by two sequential cleavages that yield the final product.

 

ATTACINS

 

A second set of antibacterial proteins are called attacins. These also have been isolated from H. cecropia and three other moth species. Attacins are considerably larger than cecropins, made up with over 180 amino acids. The antibacterial activity of these proteins covers a rather narrow spectrum compared to the cecropins, with high activity only against a few species of bacteria in the alimentary canal. Their site of action is at the outer bacterial membrane. These proteins appear to work in synergism with the cecropins and with lysozyme so that the three types of immune proteins can work together.

 

 

LYSOZYME

 

Peptidoglycan is a large, bag-shaped macromolecule that encases bacterial membranes. It is universally present in bacteria. Minor variations of peptidoglycan occur in among different bacterial species. Lysozyme is an enzyme that hydrolyzes the glycosidic bonds of peptidoglycan, and thereby causes bacterial cell lysis. This enzyme constitutively occurs in low levels in hemolymph of most insects. Lysozyme is rapidly induced immediately upon bacterial infection.

 

STILL OTHER ANTIBACTERIAL PROTEINS

 

Antibacterial proteins widely occur in vertebrate and invertebrate animals. It is not too surprizing, then, to learn that representatives of various insect orders produce different antibactieral proteins. The attacins and cecropins are known mostly from lepidopterans, although they have been detected in other insects and even in intestine of pigs.

Honeybees produce another family of such proteins that are known as apidaecins. These are small peptides, molecular weight about 2100. Apidaecins do not appear to disturb bacterial membranes, and it is not yet clear how they do work. It is speculated that these are bacteriostatic, rather than bacteriocidal, proteins. Royalisin is another antibacterial peptide is found in the royal jelly of honeybees. This is also an amphipathic protein, and its mode of action may be similar to cecropins. It is a small molecule, molecular weight estimated at 5523.

Dipterans also produce induced antibacterial proteins. Two peptides from Phormia are called defensin and diptericin. Another dipteran peptide is a male-specific antibacterial product from Drosophila melanogaster. This peptide is known as andropin, and it thought to protect seminal fluid and the male reproductive tract against microbial infections.

One of the most exciting findings in insect immunology is the discovery of an insect immune protein called hemolin. This protein belongs to the immunoglobulin superfamily, that is, it shares sequence homologies to mammalian immunoglobulins. It is thought that this protein is one of the first proteins to appear in the hemolymph of the giant silkmoth H. cecropia. This protein binds to the surface of bacteria, and it is likely that the binding may be the first step in the insect immune response.

 

BACTERIAL RESISTANCE TO INSECT IMMUNITY

 

With respect to evolutionary ecology, our discussion of insect immunity offers a sort of physiological perspective on host-parasite relationships. Within the context of these relationships, we can imagine strong evolutionary pressure on the microbes to develop mechanisms that circumvent immunity reactions with hosts. Two such mechanisms are known in some of the bacteria that invade insects. These are called passive resistence and active resistence. Please note that resistence in this context refers to the parasite side of host-parasite relationships.

Passive resistence is related to the structures of some bacterial envelopes. These envelopes happen to be resistent to antibacterial activity of immune proteins. In these cases the resistence is a feature of outer membranes, rather than something bacteria might actively produce. There appear to be mutants of insect-pathogenic bacteria that have lost this sort of passive protection and are comcominantly less virulent.

Active resistence refers to active biochemical breakdown of immunity, rather than a sort of side-stepping as seen in passive resistence. B. theringeinsis, for example, produces an immune inhibitor called InA that works by proteolytic breakdown of cecropins and attacins. A beautifully complex relationship occurs in a nematode with a symbiotic bacterium appropriately named Xenorhabdus nematophilus. This bacterium is sensitive to two cecropins, A and B. However, the nematode produces an immune inhibitor enzyme that digests cecropins and attacins, thereby rendering them without activity.

One co-evolutionary response to these bacterial proteases that inactivate the insect anti-bacterial proteins may be another protein produced by some insects that inhibit the bacterial proteases. A molecule known as alpha2-macroglobulin has been identified in the hemolymph of some arthropods, but not yet from insects. Alpha2-macroglobulins are protease inhibitors, well known from mammals. These inhibitors do not work by competitively inhibiting the proteases, which is the most general mechanism of inhibiting proteases. Instead, these molecules form "cages" around protease enzymes. The cages inhibit protease activity by screening larger molecules out. Smaller peptides are not screened out, so they can get into the active sites of the proteases, where they can be hydrolyzed. This forms a sort of selective inhibition of proteases. It is speculated that the alpha2-macroglobulins are a regular feature of arthropod hemolymph. They are thought to clear protease activity from hemolymph by collecting the proteases, then delivering them to hemocytes where they participate in a endocytotic protease clearance pathway that is similar to protease clearance pathways known in mammals.

 

THE BIOLOGICAL SIGNIFICANCE OF IMMUNITY

 

There are over two hundred species of bacteria that live in one or another association with insects. These can be categorized with repect to their pathogenicity:

harmless forms that are not pathogenic

occasional pathogens

obligate pathogens

Induced insect immune systems do not have antibacterial activity against all, or even, most pathogenic species. B. theringensis is a good example of a pathogenic species that is not troubled by immune systems. On the other hand, as far as can be known, insect immune sytems are effective against all non-pathogenic systems. What sense can we make of this? One suggestion is that insect immunity has more to do with regulating population dynamics of the non-pathogenic bacteria that naturally occur in insect bodies. My speculation is that many pathogenic bacterial species have secondarily lost protective and resistence mechanisms such that they have become labile to the induced proteins.

 

OTHER HUMORAL IMMUNITY FACTORS

 

We most often regard phenyloxidases in terms of sclerotization, or tanning, of cuticle. These enzymes are found within cuticle and in hemolymph of insects. In hemolymph phenyloxidases are involved in melanin synthesis by way of quinones. Phenyloxidases are very active enzymes, and the intermediates and products of phenyloxidase reactions can be harmful to animals. These enzymes are nearly always found in a pro-phenyloxidase form. Phenyloxidases are activated in response to bacterial infections, and melanin, a tanned insoluble material is deposited around clumps of bacteria. A similar reaction occurs at the site of wounding in insect integument.

Activation of phenyloxidase has been studied in a few insects. This usually involves a signal from the surface of bacterial walls, such as the putative beta-1,3-glucan receptor. Conformational changes in this receptor activate a serine protease called prophenyloxidase activating enzyme. The activating enzyme removes a 5-kD peptide from prophenyloxidase, which yields phenyloxidase. These sort of cascade reactions are common biochemical activating mechanisms. Another important reaction cascade is the blood-clotting reaction in mammals.

Lectins are agglutinating molecules that are well known from plants and many classes of animals. Lectins have been purified from the hemolymph of many insect species, including a meat fly, a cricket and a grasshopper. A possible function of some lectins may be agglutination of microbes, but the this has been clearly shown in only a single case.


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