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AMER. ZOOL., 23:185-194 (1983) Phagocytosis and Encapsulation: Cellular Immune Responses in Arthropoda1 STUART RATNER AND S. BRADLEIGH VINSON Department of Entomology, Texas A&M University, College Station, Texas 77843 SYNOPSIS. The least understood aspects of the cellular immune reactions of arthropods are the earliest events: the initial recognition of foreignness, and the resulting changes in hemocyte behavior and morphology. There are indications that the recognition of a surface as foreign is based primarily upon its electrostatic charge, but more specific criteria may be utilized in some situations. Phagocytes are capable of recognizing foreignness without the intervention of soluble opsonins but, in some arthropods, there is in vitro evidence that opsonins can increase the efficiency of phagocytosis. It has been hypothesized, on the basis of ultrastructural evidence that encapsulation, a multicellular immune response, is induced when labile hemocytes rupture upon encountering a foreign object. The released products may promote the formation of a sheath of ameboid hemocytes around the object. This hypothesis is now supported by the results of experiments performed on an in vitro encapsulation system. This system may prove useful in the purification of encapsulation—promoting factors; in determining the mechanism of their release from hemocytes; and in investigating the possibility that the various cellular immune reactions of arthropods have a common underlying mechanism. unicellular defense reaction which clears the hemolymph of bacteria and other The reactions of arthropodan hemo- microscopic foreign objects; and cellular cytes against foreign bodies can be consid- encapsulation, one of the multicellular ered to constitute an "immune system" only reactions, which is directed against larger in the most general sense of the term: they invaders of the hemocoel (i.e., those greater aid in keeping the animals free of parasites than 10 Mm in diameter [Salt, 1970]). The and pathogens. The connotations which morphological aspects of these reactions have become irreversibly bound to the term are becoming increasingly clear, but our "immunity" as it has been used by verte- understanding of their physiology and biobrate-oriented immunologists—specific- chemistry lags far behind (see recent ity, memory, the involvement of immu- reviews: Schapiro, 1975; Ratcliffe and noglobulins—do not apply to the situation Rowley, 1979; Baerwald, 1979; Lackie, in arthropods. No immunoglobulins are 1980). Some of the most difficult unrepresent, nor do the hemocytes possess solved questions deal with the earliest receptors for them (Anderson, 1977). Dis- events in the cellular reactions: the actual crimination is generally so poor that allo- recognition of foreignness and the prografts, and sometimes even xenografts, are cesses directly triggered by this recognitolerated (Lackie, 1981a). Although there tion. In this review we will survey the proare indications of specific memory in some gress which has been made toward humoral reactions (Karp and Rheins, 1980), answering some of these questions. We will no convincing evidence of cellular immune also consider the possibility that phagocymemory has been produced. Nevertheless, tosis, encapsulation, and other cellular the cellular immune system evidently does immune responses share common underan adequate job of eliminating foreign lying mechanisms. Recent findings we have material from the arthropodal hemocoel, made using an in vitro encapsulation system judging from the success of the phylum. will be incorporated wherever necessary. Arthropodan hemolymph contains an This review concerns phagocytosis, the array of humoral immune factors, many of which are antimicrobial (see Boman and 1 From the Symposium on Comparative Aspects of Hultmark, 1981). In this review, only Inflammation presented at the Annual Meeting of the humoral factors associated with cellular American Society of Zoologists, 27-30 December immunity will be considered. 1981, at Dallas, Texas. INTRODUCTION 185 186 S. RATNER AND S. B. VINSON classes, as suggested by Scott (1971); or it Arthropodan phagocytes are morpho- may be merely a reflection of the use of logically diverse, and have been granted different techniques, in a small number of such names as granulocyte, amebocyte, experiments, upon a limited range of lamellocyte and, perhaps most commonly, species. Recently, there have been preliminary plasmatocyte. For convenience, the term "plasmatocyte" will be used in this paper indications of humoral mediators of to denote all ameboid, phagocytic hemo- phagocytosis in insects which could operate by means other than opsonization. cytes. Mohrig and Schittek (1979) have isolated Are humoral factors involved in phagocytosis'? from the hemolymph of Galleria mellonella Opsonins are serum substances which, (L.) a complex of factors induced by the when attached to foreign objects, increase injection of readily encapsulated particles their susceptibility to phagocytosis. In ver- (latex beads). These factors confer upon a tebrates, immunoglobulin G and the third naive larva the ability to clear its hemocomponent of complement act as opsonins. lymph of the normally unphagocytizable Arthropod serum lacks immunoglobulins Bacillus thuringiensis subtoxicus. It remains and complement (Whitcomb et al., 1974), to be proven, however, that this clearance is accomplished by phagocytosis. Wago but does it contain opsonic factors? The standard test for serum opsonins is (1980) found that a component of Bombyx the addition of serum-treated or untreated mori (L.) serum causes the elongation of test particles (often mammalian erythro- hemocytic filipodia, an action which could cytes) to hemocyte suspensions or mono- facilitate phagocytosis. Regardless of the role of serum factors layers. If an opsonin is present, serum-pretreatment will increase the binding or it seems certain, from the evidence preuptake of the particles by the hemocytes. sented above, that phagocytosis in arthroThis test has revealed the presence of poda can proceed in their absence. Recopsonins in some crustaceans (McKay et al., ognition of foreignness must be made by 1969; McKay and Jenkin, 1970; Paterson a component of the plasmatocyte memand Stewart, 1974; Schapiro et al., 1977). brane. The possible mechanisms of this There is also evidence that a component recognition will be discussed in a later secof the serum of the crayfish Parachaeraps tion. bicarinatus aids in the elimination of bacCELLULAR ENCAPSULATION teria in vivo (Tyson and Jenkin, 1973) Most arthropods have the ability to form although it was not definitely determined that phagocytosis was the means of elimi- a tight, multilayered hemocytic capsule nation. In most cases, some degree of around macroscopic parasites and pathophagocytosis or binding can occur even in gens. This process, called encapsulation, often kills the offending organism, or at the absence of serum. In contrast to the situation in Crustacea, least restricts its growth and movement. all reported attempts to demonstrate Theories advanced to explain the mortalopsonins in insects have been unsuccessful ity of encapsulated organisms include (Rabinovitch and DeStefano, 1970; Scott, asphyxiation, buildup of wastes, and toxic 1971; Anderson et al., 1972; Wago and action of quinones (Salt, 1963;Nappi, 1977; Ichikawa, 1979; Rowley and Ratcliffe, Poinar et al., 1979). The quinones are pre1980). This is true even in insects whose cursors of melanin which often forms in serum agglutinates the test particles the capsule. The structure of hemocytic capsules is (agglutinins are opsonic in some molluscs [Hardy et al., 1977]). The discrepancy in fairly consistent in all arthropods in which opsonin occurrence between crustaceans it has been described. A typical capsule and insects may be the result of different consists of 5-30 layers of hemocytes packed selective pressures exerted on the two tightly together with few or no intercellular spaces. Desmosomes and gap and sepPHAGOCYTOSIS CELLULAR IMMUNE RESPONSES IN ARTHROPODA tate junctions are sometimes observed between the cells (Baerwald, 1979). The innermost cell layer adheres tightly to the foreign surface, and is often extremely flattened against it. Many of the overlying layers are similarly flattened, so that the inner portions of some capsules resemble a myelin sheath. Capsule morphology varies from species to species in relatively minor aspects: extent of cell flattening, cell necrosis, presence or absence of melanin, and amount of extracellular material (Crompton, 1967; Grimstone et al., 1967; Norton and Vinson, 1977; Poinar and Hess, 1977; Schmit and Ratcliffe, 1977). Capsule development The cells which comprise a capsule are mainly ameboid phagocytic cells which resemble (and may actually be) plasmatocytes. Ultrastructural studies of the early phases of encapsulation, however, indicate that a different class of hemocyte is responsible for the actual recognition of foreignness and initiation of encapsulation. These labile, generally nonphagocytic hemocytes have been termed hyaline cells, cystocytes, spherule cells, or, as they will be referred to in this paper, granulocytes. The use of a single term is for convenience; these cells may not all be homologous (Price and Ratcliffe, 1974). Ultrastructural evidence indicates that granulocytes rupture upon encountering a foreign surface. The released granular and/or electron-dense flocculent material seems to cause plasmatocytes to adhere to the foreign surface, and may contain the chemical signal which induces them to flatten and conjoin to form the capsule. The material may also contain enzymes and precursors for melanin synthesis (Reik, 1968, as summarized by Salt, 1970; Nappi and Streams, 1969; Unestam and Nylund, 1972; Brehelin et al., 1975; Schmit and Ratcliffe, 1977, 1978). The material released by the granulocytes will thus be referred to as "encapsulation-promoting factors" (EPF). What is the nature of EPF? Despite the important effects of EPF upon cellular behavior, their composition 187 has been characterized in only the most general terms. The labile hemocytes of arthropoda have been demonstrated histochemically to contain acid or neutral mucopolysaccharides and glyco- or mucoproteins (Ashhurst and Richards, 1964; Dumont et al., 1966; Gupta and Sutherland, 1967; Wood?/al., 1971). Reik (1968; summarized by Salt, 1970) implicated mucopolysaccharides in the early stages of capsule formation in Ephestia kuehniella Zeller. A precise determination of the nature of EPF and their action upon target cells awaits purification of EPF, a feat which would require an unambiguous bioassay. We have recently developed a technique which may fill this requirement, and would like to digress briefly in order to describe it. An in vitro encapsulation system Short-term culture of fresh hemolymph of Heliothis virescens (F.) and H. zea (Boddie) can be employed in the study of cellular encapsulation in vitro. The hemolymph is diluted 3-1 OX in Grace's insect tissue culture medium containing phenylthiourea at 8% of saturation. If foreign objects (e.g., cotton thread) are present in the medium, flocculent hemocytic aggregations form around them almost immediately. The aggregations become progressively denser and more sharply defined until, at ca. 2 hr, a classic hemocytic capsule surrounds the foreign object. At 5 hr, the capsule bears strong ultrastructural resemblance to that formed in vivo by H. virescens (Ratner and Vinson, in preparation). When no foreign object is present, the hemocytes aggregate spontaneously, eventually forming smooth, rounded nodules. This response may be triggered by the foreign surface of the culture dish, or by factors exuded from injured tissues during the bleeding process (see Cherbas, 1973). If the addition of a foreign object is delayed until after nodule formation is well under way (ca. 30 min), the object is not encapsulated (Fig. 1 A). Thus, the system rapidly becomes exhausted, probably because of the commitment of cells, and perhaps other components, to the nodules. Both encapsulation and nodule forma- 188 S. RATNER AND S. B. VINSON ADD FOREIGN OBJECT FRESHLY DRAWN HEMOLYMPH + MEDIUM WITHHOLD FOREIGN OBJECT AGGREGATION OF HEMOCYTES BEGINS ENCAPSULATION OF OBJECT BEGINS ADD FOREIGN , .OBJECT OBJECT NOT ENCAPSULATED sulation. Neuraminidase and hyaluronidase do not prevent encapsulation so, if the in vitro system can be taken as a guide, the crucial components of Heliothis EPF are proteins, not mucopolysaccharides. It should be possible to assay hemolymph fractions for EPF activity, and eventually purify an EPF, using trypsin-inactivated in vitro systems. What is the mechanism of EPF release? It is desirable to know what cellular processes cause granulocytes to rupture after the initial recognition of foreignness. This knowledge might be used in devising a method of inducing inappropriate EPF release in pest arthropods, in effect turning their own immune systems against them. Some preliminary findings about the process have emerged from the in vitro Heliothis system. When the hemolymph culture is held at 4°C, the usual aggregation of hemocytes still forms and clings to a foreign object, but it will not mature into a tight, smoothFIG. 1. In vitro demonstration of an encapsulation- contoured capsule unless the culture is subpromoting factor released from hemocytes of Heliothis sequently warmed to room temperature. virescens. A. Behavior of an untreated culture. B. Inhibitors of glycolysis and oxidative phosBehavior of a gently trypsinized culture (Ratner and phorylation also prevent capsule maturaVinson, in preparation). tion, but not the initial aggregation of hemocytes. It seems, then, that the rection can be prevented, however, by a brief, ognition of foreignness and consequent mild trypsinization of the culture (soybean release of EPF are passive processes, while trypsin inhibitor is used to control the the subsequent consolidation of the capduration of trypsinization) (Fig. IB). The sule requires metabolic energy. ability to encapsulate can subsequently be Capsule initiation is also unaffected by restored to the culture by the addition of the presence of membrane-stabilizing drugs lysate prepared by the ultrasonic disrup- such as corticosteroids and local anestion of fresh, whole hemolymph (Fig. IB). thetics (Ratner and Vinson, in preparaThe addition of freshly prepared serum or tion). Direct microscopic observation conGrace's medium has no restorative effect. firms that these compounds are ineffective It therefore appears that mild trypsiniza- in preserving granulocyte structure in vitro. tion prevents encapsulation not by altering Since the drugs are thought to act by discomponents of the hemocyte membrane, placing membrane-bound calcium and but by digesting a protein released by the ATP-ase (Seeman, 1970), these substances hemocytes—an EPF (stronger trypsiniza- are apparently not involved in granulocyte tion does appear to damage the hemocyte rupture in Heliothis. Obviously, our knowlmembrane, since it renders the culture edge of the mechanisms of EPF release are unable to encapsulate, even after the addi- at present rudimentary. Much may be tion of lysate). This is, to our knowledge, revealed by tests of other membrane-active the first experimental evidence that such compounds. Such tests are best done in vitro, a factor can actually play a role in encap- to ensure that the compounds reach the FRESHLY DRAWN HEMOLYMPH + MEDIUM +0.05% TRYPS1N + FOREIGN OBJECT ADO LYSATE ADO SERUM OR MEDIUM OBJECT NOT ENCAPSULATED INCUBATE 15 M1N I ADDTRVPSIN I INHIBITOR ENCAPSULATION OF OBJECT BEGINS I INCUBATE I 2 KR OBJECT NOT ENCAPSULATED ] ' INCUBATE 12 HR OBJECT NOT ENCAPSULATED CELLULAR IMMUNE RESPONSES IN ARTHROPODA 189 Nolan, 1980). This finding is consistent with Salt's hypothesis since, at physiological pH, basement membrane is negatively charged (Vinson, 1974). Also negatively WHAT ARE THE PRINCIPLES OF NON-SELF charged are some of the coatings and memRECOGNITION IN ARTHROPODA? branes which protect parasites from encapWe have seen evidence that two types of sulation (e.g., Brennan and Cheng, 1979). arthropodan hemocytes react to a foreign It is not known whether surface charge surface: the phagocytic plasmatocytes and similarly affects phagocytosis by arthrothe labile granulocytes. What aspects of a podan hemocytes. It is interesting that catsurface do these cells actually recognize as ionic substances increase the phagocytosis foreign? Salt (1970) hypothesized that, to of bacteria by human neutrophils in an an insect immune system, "non-self is opsonin-free system (Pruzanski and Sato, equivalent to anything other than intact, 1978). In a different system, bacteria with native basement membrane. His hypothe- surfaces more hydrophobic than neutrosis stemmed from the fact that, except for phils were phagocytized more readily than parasites and pathogens which have devel- those with more hydrophilic surfaces (Van oped specific counteracting strategies, few Oss and Gilman, 1972). It should be posforeign surfaces prove immunologically sible to use similar experiments to deterinert to arthropods. Even materials such as mine the effect of surface charge on phagoglass and polyfluorocarbons elicit a cytosis by arthropodan hemocytes. response. Normally tolerated allografts, A model of non-self discrimination based however, are encapsulated if their baseon nonspecific physicochemical factors can ment membrane is damaged. Salt further adequately account for all known properhypothesized that the immunologically important components of basement mem- ties of arthropodan cellular immune sysbrane are acid mucopolysaccharides. There tems. For example, in a single host, the have been some criticisms (Crossley, 1975), severity of encapsulation of transplanted but Salt's hypothesis is still the best avail- tissue can vary according to the species of able. It does not, however, address the spe- the donor (Lackie, 1981a; S. E.Jones, percific molecular principles of non-self rec- sonal communication). These differences in severity could be related to the number ognition. of granulocyte ruptures provoked by each transplant. This in turn could be a reflecInitial recognition offoreignness tion of different distributions and strengths Since the arthropodan immune system of charged groups on the basement memcan react against inorganic surfaces, it is branes of the various donors (Lackie, obvious that the initial recognition of for- 1981a). Recognition in a nonspecific syseignness cannot be explained completely tem need not occur only at the hemocyte by models involving lock-and-key interac- membrane. Soluble factors could stick to tions between complex biogenic molecules surfaces with foreign physicochemical (e.g., the glycosyl transferases of Roseman, characteristics, and bind to hemocytes by 1974, and Parish, 1977). Such highly spe- cytophilic groups. cific factors may come into play in secondThe fact remains, though, that arthroary reactions, or in special cases, as we shall podan hemolymph does contain molecules see below; but the initial recognition of which have highly specific binding affiniforeignness must involve less specific, ties. Hemagglutinins, with their saccha"physicochemical" properties of a surface. ride-specific binding, are an example (YeaElectrostatic charge seems to be such a ton, 1981). What role could such molecules property. A growing body of evidence sug- play in an immune system which shows so gests that hemocytes form capsules on pos- little in vivo specificity? One possiblity is itively charged surfaces, but not neutral or that they are secondary factors, released negatively charged ones (Walters and Wil- or activated only after the initial recogniliams, 1966; Vinson, 1974; Dunphy and tion of foreignness. For example, one would hemocytes in known forms and concentrations. 190 S. RATNER AND S. B. VINSON expect that EPF must bind specifically to some components of the plasmatocyte membrane, since capsules are composed almost exclusively of these cells. This specific binding could not occur, however, until the EPF is released by granulocyte rupture, i.e., after foreignness has been recognized. Since granulocytes are extremely fragile, it would not be surprising if their released factors showed up as serum components in extracted hemolymph. Another possibility is raised by Lackie (1981a), who suggests that specifically binding factors actually can participate in the initial recognition of foreignness. In Lackie's "two-tier" hypothesis, the nonspecific recognition system—the first "tier"—could be supplemented by factors which trigger cellular responses against more specific stimuli. A two-tier system is an entirely plausible concept. It might arise if the physicochemical surface properties of a damaging parasite were close enough to those of the host to prevent recognition by host granulocytes. One possible consequence of the resulting selective pressure upon the host might be the development of a receptor which could recognize other, possibly more specific, aspects of the parasite's surface. This new receptor could be soluble or bound to hemocyte membranes. What components of arthropodan hemolymph might act as specific recognition factors in the type of system proposed by Lackie? One possibility is a group of enzymes, the phenoloxidases, which catalyze the oxidation of tyrosine and other phenolic compounds into dopa, a precursor of melanin. A connection between phenoloxidases and encapsulation has long been suspected, since inhibitors of melanin formation have sometimes proven inhibitory to encapsulation as well (Salt, 1956; Brewer and Vinson, 1971; Nappi, 1973; Beresky and Hall, 1977). The hemolymph of some arthropods contains an inactive prophenoloxidase which can be activated by fairly specific stimuli such as fungal wall polysaccharides and yeast zymosan (Pye, 1974; Soderhall and Unestam, 1979: Ashida and Dohke, 1980). Interestingly, the activated enzyme adheres readily to fungal cell walls and abiotic surfaces such as glass. The inactive proenzyme is not adhesive (Soderhall et al, 1979). Phenoloxidases, then, exhibit the type of behavior one would expect from an arthorpodan nonself recognition factor, although it remains to be proven whether they actually perform this function. The fact that the phenoloxidases and their substrates are often found localized within hemocytes may mean that they come into play only after granulocyte rupture. Another class of molecules which might act as the "specific tier" in a two-tier system is the hemolymph agglutinins. Many agglutinins bind polyvalently to specific oligosaccharide residues, which is why they are often detected by their ability to clump mammalian erythrocytes and bacteria. In some molluscs, agglutinins have been proven to act as opsonins (Hardy et al., 1977). In arthropoda, only circumstantial evidence links agglutinins with cellular immune response. The most convincing evidence was obtained by Lackie (19816, c). Comparing an insect with low xenograft tolerance (Periplaneta americana [L.]) to one with greater tolerance (Schistocerca gregaria Forsk.), Lackie found that the insect with better non-self discrimination possesses a wider spectrum of specific agglutinins. Even more intriguing was the finding that the ability to agglutinate Hymenolepis diminuta oncospheres in vitro is correlated with the ability to encapsulate them in vivo. Lackie suggests that the agglutinins might act as hemocyte-bound receptors, an idea partly inspired by the immunofluorescent localization of Leucophaea maderae Fabr. agglutinin on hemocyte membranes (Amirante and Mazzalai, 1978). Definitive proof of the involvement of arthropodan agglutinins in the recognition of foreignness is still lacking. The blocking of phagocytosis by antibody to purified agglutinins would constitute such a proof. A third possibility must be considered: The specifically binding hemolymph factors may not be involved in cellular immunity at all. Instead, they may constitute a strictly humoral antimicrobial system, or they may perform presently unsuspected functions unrelated to immunity. CELLULAR IMMUNE RESPONSES IN ARTHROPODA 191 question, for if distinctions between the reactions are only superficial, then coordination between experimenters is needArthropodan hemocytes participate in a lessly inhibited. Experimental designs may variety of multicellular reactions other than even be distorted. For example, in the in encapsulation. In nodule formation, con- vitro Heliothis system, there seems to be glomerates of bacteria or other particu- equivalence between the immune and lates are sealed into a hemocytic sheath hemostatic reactions. If no foreign object (Ratcliffe and Gagen, 1977). In certain is present, a nebulous clot forms and eventypes of coagulation, hemocytes and extra- tually contracts into a nodular mass. If a cellular materials form a mass which can foreign object is present, the clot forms be an early stage in the process of wound around it and matures into a capsule. Thus, repair (Clark and Harvey, 1965; Gregoire, if the "clot"were allowed to form before 1974). The formation of so-called "tumors" the start of an experiment (a practice not in insects is, in most cases, the walling off unknown in in vitro studies of hemocyte of pathological tissues by hemocytes (Pe- behavior), the remaining culture would be seriously depleted of immunoreactive cells. rotti and Bairati, 1968). It has often been suggested that these One way to test for similarities among reactions are interrelated, since they all the mechanisms of the cellular responses show similar patterns of development is to look for similarities among the extra(reviewed in insects by Ratcliffe and Row- cellular factors associated with those ley, 1979; for crustaceans, see Schapiro, responses. Two types of factors which have 1975, and Ghidalia et al., 1981). The first often been analyzed are the clottable proevent is usually the release of the contents teins and the hemagglutinins, so they might of labile hemocytes at or near the site of be good subjects for comparison. At presthe triggering stimulus. Mucopolysaccha- ent, though, it is hard to draw comparirides and mucoproteins have been detected sons, because of the diverse analytical histochemically in the released material. methods used by different groups of experNonlabile hemocytes are subsequently imenters. The only generalization that ensnared in, or perhaps attracted to, this emerges from a survey of the literature is material. These hemocytes eventually that both types of factor tend to be proteins become organized into a multi-layered of molecular weight 200,000-500,000, sheath. There are exceptions (Crossley, composed of noncovalently linked subunits 1975), but that is the general pattern, based with weights of 20,000-50,000 (Marchalprimarily on electron microscopic obser- onis and Edelman, 1968; Doolittle and vations. There are also experimental results Fuller, 1972; Hall and Rowlands, 1974; which suggest that a variety of cellular Donlon and Wemyss, 1976; Shishikura et immune reactions are all potentiated by al., 1977; Brehelin, 1979). Obviously, substances liberated from hemocytes. meaningful comparisons are impossible at Hemocyte lysate promotes encapsulation present. They await the purification of in the in vitro Heliothis system (Ratner and more factors, including EPF, as well as a Vinson, in preparation); simulates in vitro coordination of analytical techniques. clotting of the hemolymph of the crab CarAnother way to approach the question anus maena (Bang, 1971); and, when coated of interrelationships between cellular on bacteria, speeds their clearance (pre- immune response mechanisms is to comsumably phagocytic) from the circulation pare the metabolic activities associated with of the crayfish P. bicarinatus (Tyson and them, especially the activation of biochemJenkin, 1973). ical pathways. This strategy was employed None of this information provides an by Anderson et al. (1973) in their comparanswer to a fundamental question: To what ison of phagocytotic processes in inverteextent do all the cellular reactions, includ- brates and mammals. It has not yet been ing phagocytosis, share a common under- applied to a comparison of arthropodan lying mechanism? This is an important multicellular reactions. INTERRELATIONSHIPS BETWEEN CELLULAR IMMUNE RESPONSES 192 S. RATNER AND S. B. VINSON PROSPECTS FOR FUTURE RESEARCH It must by now be obvious to the reader that our present understanding of the early events in arthropodan immune response is rudimentary. We have a fairly good ultrastructural picture of what is going on, but tentativeness creeps into even the most general statements about the molecular nature of non-self recognition, and consequent hemocytic changes. Tools now becoming available can and should be used to deepen our knowledge of the mechanisms of arthropodan immune response. A variety of charged groups can now easily be attached to inert substrates (see, for example, Carlsson et al., 1979). This should make possible a finer determination of the effect of surface charge on phagocytosis and encapsulation. Investigations of reactions against transplants are growing more numerous, and more subtle. It would be helpful if biochemical analyses of the transplant surfaces could be coordinated with these investigations. Another research opportunity is afforded by the viruses associated with some hymenopterous parasitoids of insects. These viruses suppress the host's immune response to the parasitoid's egg (Edson et al., 1981). The determination of their mode of action may reveal underlying mechanisms of that response. In vitro systems have proven valuable in the detection and study of the serum and cellular factors involved in phagocytosis. A high priority should now be placed upon the improvement of methods of delaying and controlling the multicellular immune reactions in vitro, so that their associated factors, such as EPF, can be isolated, purified, and tested. Such in vitro systems will also make it possible to determine whether substances which inhibit multicellular reactions in vivo (e.g., phenoloxidase inhibitors and viruses) act directly upon the hemocytes, or by some indirect mechanism. ACKNOWLEDGMENTS Approved as TA 16787 by the Director, Texas Agricultural Experiment Station. All programs and information of the Texas Agricultural Experiment Station are avail- able without regard to race, ethnic origin, sex, or age. Supported in part by NSF Grant PCM-8021646. REFERENCES Amirante, G. A. and F. G. Mazzalai. 1978. Synthesis and localization of hemoagglutinins in hemocytes of the cockroach Leucophaea maderae L. Devel. Comp. Immunol. 2:735-740. Anderson, R. S. 1977. Biochemistry and physiology of invertebrate macrophages in vitro. In L. A. Bulla, Jr. and T. C. Cheng (eds.), Comparative pathobiology, Vol. 3, pp. 1-20. Plenum Press, New York. Anderson, R. S., N. K. B. Day, and R. A. Good. 1972. 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