Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.

Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens

Publisher Summary This chapter discusses the structure and function of major groups of hemolymph proteins that are common to all insects, storage proteins, lipoproteins, vitellogenins, and inducible antibacterial proteins. It also discusses some proteins and peptides that are present in smaller amounts, sometimes occurring only in a few insect species. With the development of microsequencing techniques that can provide information about the sequence of amino acids at the N-terminal end of an intact protein on a sample of less than 20 picomoles, it has become feasible to use one- or two-dimensional polyacrylamide gel electrophoresis (PAGE) a the protein purification method of choice. In many cases, proteins can be transferred directly from gels to derivatized paper or other media and spots cut from the medium can be inserted directly into the sequencer. If necessary, proteins on the medium can be cleaved to peptides, which can be separated by high-pressure liquid chromatography (HPLC) or by PAGE.

Insect Haemolymph Proteins

Parasite transmission generally exhibits some form of positive density dependence. Thus, as population density increases, so too does the per capita risk of becoming infected. Under such circumstances, natural selection should favor individuals that use cues associated with population density to determine the optimal allocation of resources to disease resistance mechanisms. As a consequence, individuals experiencing crowded conditions are predicted to be more resistant to parasites and pathogens than those experiencing low-density conditions. This phenomenon (termed "density-dependent prophylaxis") [Wilson, K. & Reeson, A. F. (1998) Ecol. Entomol. 23, 100-101] is predicted to be particularly prevalent in outbreak pest species and in species exhibiting density-dependent phase polyphenism, such as the desert locust, Schistocerca gregaria. Here we show that, as predicted, desert locusts reared under crowded conditions are significantly more resistant than solitary locusts to the entomopathogenic fungus, Metarhizium anisopliae var. acridum, a key natural disease of acridids and an important agent in locust and grasshopper biocontrol. Moreover, enhanced pathogen resistance in crowded locusts is associated with elevated antimicrobial activity, but not with any difference in thermal preferences or behavioral fever response. These results have implications for understanding the development and biocontrol of locust plagues.

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Coping with crowds: density-dependent disease resistance in desert locusts.

Lepidopteran insects provide important model systems for innate immunity of insects, particularly for cell biology of hemocytes and biochemical analyses of plasma proteins. Caterpillars are also among the most serious agricultural pests, and understanding of their immune systems has potential practical significance. An early response to infection in lepidopteran larvae is the activation of hemocyte adhesion, leading to phagocytosis, nodule formation, or encapsulation. Plasmatocytes and granular cells are the hemocyte types involved in these responses. Infectious microorganisms are recognized by binding of hemolymph plasma proteins to microbial surface components. This "pattern recognition" triggers phagocytosis and nodule formation, activation of prophenoloxidase and melanization and the synthesis of antimicrobial proteins that are secreted into the hemolymph. Many hemolymph proteins that function in such innate immune responses of insects were first discovered in lepidopterans. Microbial proteinases and nucleic acids released from lysed host cells may also activate lepidopteran immune responses. Hemolymph antimicrobial peptides and proteins can reach high concentrations and may have activity against a broad spectrum of microorganisms, contributing significantly to clearing of infections. Serine proteinase cascade pathways triggered by microbial components interacting with pattern recognition proteins stimulate activation of the cytokine Spatzle, which initiates the Toll pathway for expression of antimicrobial peptides. A proteinase cascade also results inproteolytic activation of phenoloxidase and production of melanin coatings that trap and kill parasites and pathogens. The proteinases in hemolymph are regulated by specific inhibitors, including members of the serpin superfamily. New developments in lepidopteran functional genomics should lead to much more complete understanding of the immune systems of this insect group.

Immunity in lepidopteran insects.

All higher organisms can in some way defend or protect themselves against their natural flora of bacteria This applies also to insects which have highly effective immune systems, both cellular and humoral Many insects respond to an injection of live, non-pathogenic bacteria with the production of a potent cell-free antibacterial activity (for a review see Boman and Hultmark 1987) This phenomenon was first analysed at the molecular level using as a model system the Cecropia moth, Hyalophora cecropia (hereafter referred to as Cecropia) When a diapausing pupa is immunized it turns on the genes for immunity while the rest of the animal remains in a dormant state Immunized pupae of Cecropia are therefore a system for biological enrichment of the RNA and the proteins which are synthesized from the genes for immunity We have taken advantage of this fact both in the purification of 15 inducible immune proteins and in the isolation of immune RNA, used for the preparation of a cDNA library After a short period of RNA synthesis, the insects respond to live bacteria by the production of a potent antibacterial activity which is due to the synthesis of 15–20 immune proteins

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1991.tb16252.x

Cell-free immunity in Cecropia

1. 1. Bacteriolytic activity develops in haemolymph of immature stages of Bomyx mori and Galleria mellonella after injection with microorganisms but not after saline injection. 2. 2. The purified insect lytic enzymes are small basic proteins with properties, such as stability to heat, optima for pH and ionic strength, similar to those of egg white lysozyme, but with specific activities about six times that of lysozyme. 3. 3. The Galleria enzyme releases from bacterial cell walls reducing sugar end groups which were identified as acetylmuramic acid. The properties of the insect proteins permit their classification as mucopeptide N-aacetylmuramyl- hydrolase (E.C. 3.2.1.17).

Studies on insect bacteriolytic enzymes-I. Lysozyme in haemolymph of Galleria mellonella and Bombyx mori

1. 1. Purified Galleria mellonella lysozyme generally resembles the avian lysozymes in amino acid composition but differs from goose egg-white lysozyme, particularly in cystine and tryptophane content. 2. 2. Complete inactivation followed oxidation of tryptophane in Galleria lysozyme, and reduction of the protein disulphide bonds caused an inhibition which was reversed in oxidising conditions. 3. 3. Transglycosylation is catalysed by Galleria lysozyme. Binding of bacterial cell walls, chitin oligosaccharides and Biebrich scarlet is weaker with Galleria lysozyme than with hen lysozyme. The association of substrate with Galleria lysozyme may involve tryptophane, but no detailed structural similarity with hen lysozyme was established for the active centres.

Studies on insect bacteriolytic enzymes--II. Some physical and enzymatic properties of lysozyme from haemolymph of Galleria mellonella.

1.	
1. Bacteriolytic activity derived from plant materials may provide insects with a useful protection from bacteria if the conditions in the insect gut are suitable.

2.	
2. Significant bacteriolytic activity was measured in seeds of Brassicaceae, Caesalpineaceae, Apiaceae and Fabaceae, with very high activity in kidney beans (Phaseolus vulgaris L.).

3.	
3. Maximal lytic activity in all seed preparations was obtained only in conditions of low ionic strength and pH. In addition, a purified bean enzyme had a molecular weight of 25,000, an isoelectric point of 6.1 and was slightly inhibited by acetylglucosamine. This plant lytic enzyme is an endo-N-acetylglucosaminidase.

W.J. Davidson

Papers

Studies on insect bacteriolytic enzymes-I. Lysozyme in haemolymph of Galleria mellonella and Bombyx mori

Studies on insect bacteriolytic enzymes--II. Some physical and enzymatic properties of lysozyme from haemolymph of Galleria mellonella.

Studies on insect bacteriolytic enzymes—III. Lytic activities in some plant materials of possible benefit to insects