Norman A. Ratcliffe

Prophenoloxidase Activation: Nonself Recognition and Cell Cooperation in Insect Immunity

The mechanism of nonself recognition by the immune system of insects is unknown. In this report the activation of the prophenoloxidase system in the wax moth Galleria mellonella by a microbial product is shown to enhance the recognition of nonself material. These results explain previous observations of the interaction of two different blood cell populations in the cellular defense reactions of insects.

Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razor-shell (Ensis siliqua)

Much of the current knowledge concerning bivalve immunology and immunotoxicology has come from studies on the mussel genus, Mytilus, or from the American oyster, Crassostrea virginica. Following a major oil spill, it was observed that the marine mussel, Mytilus edulis, underwent significant immunosuppression but no oil-induced mortalities, while in contrast, mass mortalities were noted in the edible cockle, Cerastoderma edule, and the razor-shell, Ensis siliqua. A study comparing immune cells and functions in these three species was initiated (i) to assess whether M. edulis was a representative model species and (ii) to provide baseline data on immunity in two common species, which had previously received little or no attention in this respect. While all three species shared similar cell types, their lectin-binding and enzyme cytochemistry differed considerably. M. edulis had significantly different proportions of haemocytes binding with the lectins concanavalin A, wheatgerm agglutinin and Helix pomatia agglutinin and stained positive for eight enzymes, compared with only five in C. edule and three in E. siliqua. In terms of immune function, M. edulis haemocytes were much more active in phagocytosis and superoxide generation than haemocytes of the other two species. The results show that immune cells and functions differed extensively in these three closely related species, with M. edulis showing a much higher level of immunological vigour that may be linked to its considerable resilience to adverse environmental conditions. This suggests that M. edulis may not be particularly representative of the bivalves in terms of immune reactivity and that a wider range of species should be included in studies of molluscan immunotoxicology.

Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes

Using a citrate-EDTA buffer as an anticoagulant it was possible to isolate intact haemocytes from the insect, Blaberus craniifer, without causing extensive degranulation and subsequent clotting. A haemocyte lysate from this insect contained prophenoloxidase (proPO), which could be activated by β 1,3-glucans. The activation process was dependent upon Ca2+ ions and seemed to occur by a limited proteolysis, since several serine protease inhibitors such as soybean trypsin inhibitor, benzamidine and p-nitrophenyl-p′-guanidobenzoate blocked convertion of proPO to the active enzyme. Treatment of proPO with urea or heat also caused proPO activation but probably without the intervention of serine proteases, since the protease inhibitors used failed to block the activation. Within the haemocyte lysate, several endopeptidases were present, which were enhanced in activity by prior treatment with β 1,3-glucans. These endopeptidases were inhibited in activity when the haemocyte lysate was incubated with benzamidine prior to the addition of β 1,3-glucan. This provides further indications that the activation of proPO involves a limited proteolytic attack. The active phenoloxidase enzyme became strongly bound to foreign surfaces and this phenomenon may assist in providing opsonic properties for the proPO cascade.

A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1,3-Glucan Pattern Recognition and Cellular Encapsulation Reactions

Lipoproteins and molecules for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study we describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide production in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study we show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and β-1,3-glucan and therefore may act as a pattern recognition molecule for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiological levels, and, with the notable exception of β-glucan binding, are only observed with the lipid-associated form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition molecule and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunology, particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.

The role of prophenoloxidase activation in non-self recognition and phagocytosis by insect blood cells

Abstract Experiments indicate that the prophenoloxidase activating system, which is responsible for melanin production, is also involved in immunorecognition in insects. Using haemocyte monolayer preparations of Blaberus craniifer, Galleria mellonella and Leucophae maderae , it was shown that laminarin, a β 1,3-glucan extracted from fungal cell walls and an activator of the prophenoloxidase system, enhanced the phagocytosis of test bacteria. Scanning electron microscopy of haemocyte monolayers showed that incubation of test bacteria with laminarin significantly increased the number of microorganisms attached to both the plasmatocytes and the granular cells. Furthermore with the granular cells, these bacteria became entrapped in an amorphous matrix. This material probably consists of the “sticky” proteins previously reported to be produced by crustacean haemocytes following prophenoloxidase activation. Pretreatment of haemocytes with laminarin abolished the stimulatory effect on ingestion, indicating that these “sticky” proteins are opsonic, since they would have been discharged from the haemocytes onto the glass monolayer leaving few molecules available for subsequent coating of the test particles. Preliminary biochemical studies on the G. mellonella prophenoloxidase system demonstrated that it was activated by trypsin, laminarin and laminarin G, a highly purified β 1,3-glucan, but not by dextran. Serine protease activities were also enhanced by adding laminarin to a haemocyte lysate supernatant, suggesting that the stimulatory mechanism may involve the proteolytic activity of such enzymes.

Studies on the in vivo cellular reactions of insects: an ultrastructural analysis of nodule formation in Galleria mellonella.

Cellular aggregates or nodules which formed in Galleria mellonella larvae in response to injections of killed Bacillus cereus were examined during their first 24 hr of development. The initial aggregation stage was very rapid, occurring within 1 min, and was brought about by the release of an adhesive flocculent material by granular haemocytes in contact with the bacteria. This material, which subsequently became increasingly electron dense owing to melanin deposition, consolidated and by 5 min ramified all the intercellular spaces. The granular cells degenerated, releasing portions of cytoplasm form their peripheries, and eventurally appeared in the matrix as voids containing loosely flocculent material. No bacterial breakdown was seen, however. From 2-4 hr, plasmatocytes began to encapsulate the necrotic mass, and by 24 hr a complete sheath, composed of three discrete regions of cells, was formed. These findings are discussed in relation to the various proposed mechanisms of nodule and capsule formation.

The separation of insect haemocyte types on Percoll gradients; methodology and problems

Abstract Techniques for obtaining stable, relatively pure, haemocyte populations from the Lepidopterans, Manduca sexta and Galleria mellonella , and the Dictyopteran, Blaberus craniifer , have been developed. Using preformed, continuous, density gradients of Percoll containing an anticoagulant, the main haemocyte types of these species have been successfully separated and recovered. The methodology is given in detail and the problems encountered are discussed.

Insect natural products and processes: New treatments for human disease

In this overview, some of the more significant recent developments in bioengineering natural products from insects with use or potential use in modern medicine are described, as well as in utilisation of insects as models for studying essential mammalian processes such as immune responses to pathogens. To date, insects have been relatively neglected as sources of modern drugs although they have provided valuable natural products, including honey and silk, for at least 4-7000 years, and have featured in folklore medicine for thousands of years. Particular examples of Insect Folk Medicines will briefly be described which have subsequently led through the application of molecular and bioengineering techniques to the development of bioactive compounds with great potential as pharmaceuticals in modern medicine. Insect products reviewed have been derived from honey, venom, silk, cantharidin, whole insect extracts, maggots, and blood-sucking arthropods. Drug activities detected include powerful antimicrobials against antibiotic-resistant bacteria and HIV, as well as anti-cancer, anti-angiogenesis and anti-coagulant factors and wound healing agents. Finally, the many problems in developing these insect products as human therapeutic drugs are considered and the possible solutions emerging to these problems are described.

The properties and purification of a Blaberus craniifer plasma protein which enhances the activation of haemocyte prophenoloxidase by a β 1,3-glucan

Abstract A plasma factor has been detected in the cockroach, Blaberus craniifer , which, in haemocyte lysates, enhances the activation of a peptidase and prophenoloxidase (proPO) by laminarin (a β 1,3-glucan). The factor was isolated by affinity chromatography on laminarin-Sepharose and FPLC ion-exchange chromatography. It is a glycoprotein with a molecular weight ( M w ), as determined by SDS-electrophoresis, of ca 90,000. Amino acid analysis showed a very high content ( ca 65%) of hydrophilic amino acids. No peptidase or phenoloxidase (PO) activity was detected in the isolated plasma protein. After removal of the proPO-activating protease by chromatography on Blue Sepharose, the resulting partially purified proPO could no longer be activated by laminarin or laminarin plus purified plasma factor.

Comparisons of PAH-induced immunomodulation in three bivalve molluscs

There is growing evidence that contaminants may be partly responsible for the observed increase in disease in marine organisms by adversely affecting their immunity. Bivalve molluscs are common sentinels used in invertebrate immunotoxicology, however, to date, studies have been restricted to a few resilient species. This present study is a comparative investigation into the effects of the polycyclic aromatic hydrocarbon, phenanthrene, on the immunocompetence of three bivalve species. The commonly-studied marine mussel, Mytilus edulis, was compared with two species that have never been studied with respect to immunomodulation, namely, the edible cockle, Cerastoderma edule and the razor shell, Ensis siliqua. Animals were exposed to a range of phenanthrene concentrations (50, 100, 200 or 400 microg l(-1)) and haemocyte immune parameters, including haemocyte counts, phagocytosis, superoxide generation, lysosomal enzymes and lectin-binding, were monitored. Aims were not only to extend existing knowledge of bivalve immunotoxicology, but also to establish whether contaminant-induced immunomodulation in the sentinel species, M. edulis, is comparable to that observed in other bivalves. Results showed that the immune response of the three species was differentially affected by phenanthrene exposure, with immunomodulation in M. edulis not reflecting the immunological changes observed in the other two species. This suggests M. edulis may not be a suitable sentinel bivalve, and that other species, such as C. edule, may more accurately reflect the general immunological response of this group of marine animals.