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(M. Bate and A. Martinez Arias, eds.), pp. 843-897. Cold Spring Harbor Laboratory Press, Plainview, NY.

Kim, C.-W. (1959). The differentiation centre inducing development from larval to adult leg in Pieris brassicae (Lepidoptera). J. Embryol. Exp. Morphol. 7, 572-582.

Svacha, P. (1992). What are and what are not imaginal discs: Reevaluation of some basic concepts (Insecta, Holometabola). Dev. Biol. 154, 101-117.

Tower, W. L. (1903). The origin and development of the wings of Coleoptera. Zool. Jahrb. Anat. Ontog. Tiere 17, 517-572.

Truman, J. W., and Riddiford, L. M. (1999). The origins of insect metamorphosis. Nature 401, 447-452.

apterous

FIGURE 4 Signaling and dorsal-ventral boundary cells in the wing disc of D. melanogaster.

levels of boundary signal they detect. Thus, these boundaries define important axes used to pattern the entire disc.

The subdivision of the wing into dorsal and ventral compartments by the dorsal expression of apterous plays a very similar role. This subdivision establishes reciprocal signaling between dorsal and ventral cells, this time via the Notch pathway, which results in the specification of cells on either side of the dorsal-ventral boundary (Fig. 4). These cells secrete Wingless, which helps pattern the wing blade along the proximodistal axis.

Do the other Holometabola share these axis-defining patterning mechanisms? There have been to date only a few descriptions of gene expression in the imaginal discs of other taxa, and no experimental tests of the type that verified these patterning mechanisms in Drosophila. However, it seems likely that at least some of the fundamental features are shared. All insect appendages so far examined (both larval and adult) are subdivided into apparent anterior and posterior compartments by the posterior expression of engrailed-like transcription factors, including the wing discs of the lepidopteran Precis coenia. Similarly, the wing blade of P. coenia is subdivided into dorsal and ventral domains by the dorsal expression of an apterous-like molecule. As in D. melanogaster, a wingless-like molecule is expressed along the dorsal-ventral compartment boundary in P. coenia.

See Also the Following Articles

Development, Hormonal Control of • Drosophila melanogaster • Embryogenesis

Further Reading

Blair, S. S. (1999). Drosophila imaginal disc development: Patterning the adult fly. In "Development-Genetics, Epigenetics and Environmental Regulation" (V. E. A. Russo, D. Cove, L. Edgar, R. Jaenisch, and F. Salamini, eds.), pp. 347-370. Springer-Verlag, Heidelberg. Carroll, S. B., Gates, J., Keys, D. N., Paddock, S. W., Panganiban, G. E. F., Selegue, J. E., and Williams, J. A. (1994). Pattern formation and eyespot determination in butterfly wings. Science 265, 109-114. Fristrom, D. K., and Fristrom, J. W. (1993). The metamorphic development of the adult epidermis. In "The Development of Drosophila melanogaster'

Nancy E. Beckage

University of California, Riverside

Insects lack immunoglobulins but nevertheless mount a variety of effective immune responses to parasites and pathogens. In permissive or susceptible hosts, either no response is mobilized or the responses induced fail to counter the invader, so that the host is successfully infected. In resistant (also called refractory) hosts, the parasite or pathogen is thwarted, and infection is prevented. Regardless of the outcome, the result is a dynamic interplay of host and parasite genes, with the products of host resistance/susceptibility genes being counterbalanced by the virulence/avirulence characteristics of the invader (Fig. 1).

In insects, both cellular and humoral immune responses figure prominently in host defense, with many parasitic and pathogenic infections resulting in the deployment of defenses of both types. Insect hemocytes mobilize the cellular defenses, which include phagocytosis, nodulation, and encapsulation. In the main classes of humoral defenses, the following substances are produced: antibacterial, antifungal, and (presumably) antiviral molecules, the melanizing enzyme phenoloxidase, and agglutinins or clotting factors.

Although much recent progress has been made in deciphering the cellular and humoral aspects of insect immune responses, much less is known about the recognition mechanisms responsible for the initial discimination of "nonself" material at the host-invader interface that sets the stage for the immune response. This is especially true for the recognition of multicellular parasites, which seem to use a combination of active and passive strategies to avoid being detected as foreign.

CELLULAR IMMUNE RESPONSES Insect Hemocytes

Initially, hemocytes were classified on the basis of morphological criteria alone, resulting in the publication of numerous

FIGURE 1 (A) The parasitoid wasp, L. boulardi, preparing to oviposit in D. melanogaster host larvae. If the host strain is of the susceptible genotype, and the wasp is of the virulent strain of this parasitoid species, the wasp will develop normally in the host (B) and successfully emerge while the host dies. However, if the host expresses resistance genes, the parasitoid will be encapsulated and melanized (C), and the host will survive to adulthood. (Photographs courtesy of Dr. Yves Carton, CNRS, Gif-sur-Yvette, France.)

FIGURE 1 (A) The parasitoid wasp, L. boulardi, preparing to oviposit in D. melanogaster host larvae. If the host strain is of the susceptible genotype, and the wasp is of the virulent strain of this parasitoid species, the wasp will develop normally in the host (B) and successfully emerge while the host dies. However, if the host expresses resistance genes, the parasitoid will be encapsulated and melanized (C), and the host will survive to adulthood. (Photographs courtesy of Dr. Yves Carton, CNRS, Gif-sur-Yvette, France.)

conflicting classification schemes, even for the same species. The different hemocyte types display marked inter-, and even intra-, species variability in appearance and behavior, further complicating classification. Additionally, hemocytes that are examined before they have attached to a substrate are difficult to identify in the unspread state, in contrast to spreading cells (e.g., plasmatocytes). Today, tools such as monoclonal antibodies, which bind cell-type-specific epitopes, are used in combination with other biochemical markers to facilitate identification of the different hemocyte classes. In addition, techniques such as density gradient centrifugation have been employed to purify homogeneous populations of the individual hemocyte morphotypes, facilitating in vitro studies of their biochemistry and behavior.

In the Lepidoptera, the two most abundant hemocyte classes are plasmatocytes and granulocytes, which are the primary phagocytic and encapsulative cells. In the higher

Diptera including Drosophila melanogaster, the multifunctional lamellocytes play these roles, and crystal cells carry the melaniz-ing enzyme phenoloxidase. In contrast, in lepidopterans including the tobacco hornworm, Manduca sexta, the bulk of this enzyme is carried in the plasma. Taxonomic variations are seen in the total hemocyte count, with the Lepidoptera having abundant hemocytes in contrast to dipterans (e.g., mosquitoes), which have many fewer cells per microliter of hemolymph.

The fat body is also an immunoresponsive organ and synthesizes a variety of antimicrobial peptides, enzymes (e.g., lysozyme), and other immunoreactive molecules. Indeed, the fat body represents the primary source of hemolymph-borne macromolecules and is the most metabolically active tissue in the insect. The gut, which is constantly assaulted with pathogens ingested by the insect, also produces a battery of antibacterial and antifungal agents to counter these invaders. During molting, when the newly synthesized cuticle is most fragile and the cuticular linings of the fore- and hindguts are shed, bacteria are released into the lumen of the gut, resulting in enhanced production of antimicrobial peptides by the gut when the animal is most vulnerable to infection.

Hemocyte-Mediated Immune Responses

Phagocytosis is the process by which pathogens such as bacteria and small particles (< 1 |lm in diameter) are engulfed by host hemocytes, culminating in death of the invader. The membrane of the cell invaginates, and the pathogen is engulfed in a membrane-bound vesicle into which lytic enzymes are released, causing the pathogen's demise. This process appears to be mediated by prostaglandins (eicosanoids) produced by the hemocytes. The phagocytic cells include plasmatocytes and granulocytes, although other cell types may also participate in this response to a lesser degree. Large numbers of bacteria-laden hemocytes may clump together to form nodules, which attach to the host's internal tissues and are removed from circulation. Frequently these nodules are melanized and deposited on lobes of fat body, the Malpighian tubules, or gut tissues.

To counter eukaryotic invaders (i.e., parasites) that are too large to be phagocytosed by a single cell, multiple classes of hemocytes cooperate in the mobilization of the multiphasic encapsulation reaction. Usually the capsule is formed from several hundred to several thousand cells that form a dense capsule enclosing the parasite. In the initial phases of encapsulation of parasitoid eggs/larvae, or encapsulation of abiotic (nonliving) implants such as Sephadex beads, granulocytes are the first cells to make contact with the target. These cells, which contain large numbers of refractile granules in their cytoplasm, attach to the target surface and then release their contents in a degranulation reaction, forming a sticky matrix on the surface of the target. The degranulation event triggers the attachment of multiple layers of plasmatocytes, which are fibroblast-like cells that spread, flatten out, and encase the parasite in a multilayered sheath of cells. Sometimes multiple

FIGURE 2 Light micrograph series showing fully encapsulated C. congregata eggs recovered from the nonpermissive host P. occidental 72 h postparasitization of the host larva. Multiple eggs are sometimes engulfed in a single capsule (A), which frequently shows signs of partial or complete melanization (B). [Reprinted from Harwood, S. H., etal. (1998). Production of early expressed parasitism-specific proteins in alternate sphingid hosts of the braconid wasp Cotesia congregata. J. Inv. Pathol. 71, 271—279, with permission from Academic Press.]

FIGURE 2 Light micrograph series showing fully encapsulated C. congregata eggs recovered from the nonpermissive host P. occidental 72 h postparasitization of the host larva. Multiple eggs are sometimes engulfed in a single capsule (A), which frequently shows signs of partial or complete melanization (B). [Reprinted from Harwood, S. H., etal. (1998). Production of early expressed parasitism-specific proteins in alternate sphingid hosts of the braconid wasp Cotesia congregata. J. Inv. Pathol. 71, 271—279, with permission from Academic Press.]

targets are enclosed in a single capsule, as occurs with eggs of the parasitoid Cotesia congregata, in the nonpermissive host Pachyshpinx occidentalis (Fig. 2A). The encapsulation reaction is terminated by the adherence of additional granular cells to form a thin envelope around the completed capsule. Like nodules, fully formed capsules frequently adhere to the host's internal tissues (including fat bodies, Malpighian tubules, gut, or salivary glands) and are thereby removed from circulation. In rare instances, the encapsulated parasite may actually be extruded from the hemocoel and pass through the epidermis, to be shed with the host's exuvial cuticle in a molt during "cuticular encystment," which occurs when a parasitoid develops in a nonpermissive host.

During the encapsulation process or upon its completion, the innermost layer of cells deposits melanin or its toxic quionone precursors over the surface of the invader, regardless of whether the foreign material is an abiotic or biotic target (Figs. 2B, 3A,B). However, the occurrence of melanization is variable, and sometimes capsules persist for the remaining life span of the host, showing no signs of melanization. Some species of hosts with encapsulated parasitoids undergo metamorphosis and live to the adult stage (e.g., D. melanogaster with encapsulated Leptipolina boulardi parasitoid egg; Fig. 1), whereas other hosts die prematurely, frequently showing symptoms of endocrine disruption. When the host dies prematurely, the parasitoid fails to survive. When the parasitoid C. congregata is encapsulated in host larvae of the tobacco hornworm, which is normally permissive for this parastoid, the "hosts" with encapsulated parasitoids molt to supernumerary instars and then to nonviable larval—pupal intermediates. These developmental symptoms suggest that the host's juvenile hormone titer is elevated to a level high enough to interfere with normal pupation. In these instances, endocrine disruption seems to be mediated by the factors (i.e., polydisperse DNA viruses: polydnaviruses) injected by the wasp into the host during parasitization and thus even in the absence of developing wasps, host development is arrested prematurely.

During parasitization, endoparasitoids belonging to the hymenopteran families Braconidae and Ichneumonidae inject into their lepidopteran hosts polydnaviruses that subsequently play a critical role in suppressing the host immune response. These viruses, which are integrated in the genomic DNA of the wasp and undergo replication only in the female's ovary, rapidly enter host hemocytes following parasitization, and viral genes are expressed. Depending on the host—parasitoid combination, the host's hemocytes either alter their behavior and fail to spread (thereby inhibiting the encapsulation response) or, alternatively, undergo fragmentation and programmed cell death. In M. sexta, larvae parasitized by C. congregata, massive numbers of dead and dying hemocytes undergo clumping and then are removed from circulation soon after parasitization, resulting in a dramatic drop in the host's total hemocyte count. By 8 days postparasiti-zation, new cells have differentiated from prohemocytes, and the host regains its ability to encapsulate Sephadex beads. However, the living parasitoid larvae remain unencapsulated (Fig. 3A), suggesting that they escape being detected as foreign by another mechanism. However, if second-instar parasitoids are dissected from a host, killed, then implanted into a surrogate "host" caterpillar, the parasitoids are avidly encapsulated and melanized (Fig. 3B), suggesting that something unique about the living parasitoid surface suppresses a host immune response. These simple observations suggest that the living parasitoid larvae either escape being detected as foreign by mechanisms that may involve host antigen mimicry (or masking) or by the presence of specific as yet unidentified surface molecules that prevent their recognition as "nonself" by hemocytes.

The final biochemical events that culminate in death of the parasite remain unclear for the most part. Although melanin and its precursors are toxic, much recent evidence points to the potential role of other toxic molecules such as reactive intermediates of nitrogen (nitric oxide) or oxygen (superoxide), released by cells localized in the innermost layers of the capsule, in causing lethality. Although it was formerly presumed that death was induced by asphyxiation of the parasite or parasitoid inside the capsule, biochemically

FIGURE 3 (A) Evidence that living C. congregata parasitoids are not encapsulated larvae of the host, M. sexta, which are permissive for this parasitoid, even though the host may mobilize hemocytes to encapsulate abiotic targets such as Sephadex beads. The bead shows hemocytic encapsulation and associated melanization, whereas the parasitoid larva has avoided this response, suggesting that the living parasitoids actively evade the host's immune response by antigen mimicry, secretion of immunosuppressive molecules, or other mechanisms. (B) Encapsulation of first-instar C. congregata that had been dissected from a fifth-instar host tobacco hornworm, killed by immersion in ethanol, then implanted into the hemocoel of a "surrogate host." Thick, agglutinated, and melanized capsules surround the larvae 24 h after implantation. [Photographs from Lavine, M., and Beckage, N. (1995). Polydnaviruses: Potent mediators of host insect immune dysfunction. ParasitoL Today 11, 368—378, with permission from Elsevier Science.]

FIGURE 3 (A) Evidence that living C. congregata parasitoids are not encapsulated larvae of the host, M. sexta, which are permissive for this parasitoid, even though the host may mobilize hemocytes to encapsulate abiotic targets such as Sephadex beads. The bead shows hemocytic encapsulation and associated melanization, whereas the parasitoid larva has avoided this response, suggesting that the living parasitoids actively evade the host's immune response by antigen mimicry, secretion of immunosuppressive molecules, or other mechanisms. (B) Encapsulation of first-instar C. congregata that had been dissected from a fifth-instar host tobacco hornworm, killed by immersion in ethanol, then implanted into the hemocoel of a "surrogate host." Thick, agglutinated, and melanized capsules surround the larvae 24 h after implantation. [Photographs from Lavine, M., and Beckage, N. (1995). Polydnaviruses: Potent mediators of host insect immune dysfunction. ParasitoL Today 11, 368—378, with permission from Elsevier Science.]

mediated parasite-killing strategies now seem to be important in causing the ultimate death of the invader. Phagocytosis is followed by the generation of these cytotoxic intermediates in the phagolysozome, indicating that this pathway is also important to the function of phagocytic cells.

The antiviral defenses of insects are just now beginning to be deciphered, although recent observations made in parasitized insects have yielded some insights. Larvae of M. sexta that are parasitized by C. congregata are dramatically more susceptible to the Autographa californica nucleopolyhedrovirus than nonparasitized larvae of the same age, which are normally semipermissive hosts of this pathogenic baculovirus. Parasitized larvae die faster and at higher rates than nonparasitized larvae infected with the same dose of the occluded form of the baculovirus. This enhanced susceptibility appears because of the inactivation of the cellular immune response of the host induced by the wasp's polydnavirus. Thus, there is likely to be a cellular immune response to the baculovirus, which is suppressed in parasitized larvae. In nonparasitized larvae, cellular plaques composed of hemocytes clump on cells localized in the host's tracheal epithelium that harbor the virus. These plaques do not form in parasitized larvae, and the virus is rapidly disseminated throughout the body cavity via the tracheolar epithelium, uninhibited by the hemocytic response. Although the polydnavirus genes that render the host more susceptible to the baculovirus have yet to be isolated, they offer promise for formulation of baculovirus biopesticides with enhanced potency and a broader host range for control of lepiopteran insect pests.

HUMORAL IMMUNE RESPONSES Plasma-Borne Factors

In addition to hemocyte-mediated immune reactions, insects possess a variety of potent plasma-borne defense molecules that are toxic to parasites and pathogens. Usually these are synthesized by the fat body or the hemocytes and secreted into the plasma, where they act either on the invader directly or via the hemocytes in altering their behavior to enhance the immune response. A battery of antibacterial proteins are produced by many insects, including defensins, drosocin, cecropins, attacins, and diptericins, depending on the species, and, in addition, the ubiquitous lysozyme family of antibacterial proteins. These proteins differ in their specificity for gram-positive versus gram-negative bacteria, with some acting on both types of bacteria with varying degrees of potency. Often the proteins disrupt the bacterial cell membrane function by inducing pore formation, causing lysis of the cell. Antifungal molecules are also produced by insects, providing a first line of defense against fungal invaders that often infect the insect via its cuticle, which is penetrated by the fungus. Although insects are not known to produce antiviral interferon-like molecules, the mobilization of biochemical defenses against viruses seems to be a likely component contributing to the evolution of viral resistance in insect populations treated with viral biopesticides. Although antiviral resistance has been characterized at the insect population level, the cellular and molecular mechanisms contributing to resistance remain relatively ill-defined.

Fortuitously, several antimicrobial peptides such as defensins have also been shown to have antiparasite activity, killing malaria parasites and filarial nematodes in insects that are injected with these molecules. Hence, molecular geneticists are now exploiting defensin genes in the production of trans-genic mosquitoes that show up-regulation of defensin gene expression under regulation of tissue-specific promoters either in the gut (malaria) or flight muscle (filaria) where the parasites develop.

In Drosophila, mosquitoes, and other insects, the activation of transmembrane toll receptors mediates the physiological response to microbial ligands or septic injury, leading to activation of killing mechanisms such as production of nitric oxide, resulting in death of the invader. The toll receptors are conserved across a wide range of animal phyla including Mammalia, indicating that this pathway, which insects share with a variety of species, is likely of ancient evolutionary origin.

The phenoloxidase pathway is activated by the synthesis of DOPA from tyrosine via the action of the monophenoloxidase enzyme (also called tyrosinase). Then DOPA is converted to DOPA quinone by diphenoloxidase, and thence to melanin via a series of toxic intermediates. Phenoloxidase activity may be associated with hemocytes, as occurs in mosquitoes, or as in lepidopterans (e.g., M. sexta), it may be secreted into the plasma. The first step is the activation of prophenoloxidase by a serine protease, which cleaves a peptide from the proenzyme, generating the active phenoloxidase molecule.

In many species of parasitized lepidopterans, including M. sexta larvae parasitized by C. congregata, levels of hemolymph phenoloxidase activity have been found to be suppressed following parasitization, which benefits the parasitoid by inhibiting this immunoreaction. This effect seems to be expression of polydnavirus genes that inhibit translation of the phenoloxidase mRNA, thereby suppressing levels of this enzyme in the blood.

In refractory strains of mosquitoes, melanization of the malaria ookinete occurs in the midgut wall, apparently without the intervention of phenoloxidase derived from hemocytes. Disease transmission stops because melanized parasites die trapped in the gut without ever moving to the hemocoel and salivary gland. Hence, there is widespread interest in using phenoloxidase genes to bioengineer refractory transgenic mosquitoes to halt malaria transmission. One approach has been to link this gene to the vitellogenin promoter, which is activated when the mosquito takes a blood meal in preparation for production of eggs.

Cross-Talk between the Cellular and Humoral Immune Response Networks

Blood cells in vertebrates produce many cytokines, which act at close range on other immunocompetent cells. In insects, the characterization of cytokines is less well documented, but recent evidence indicates that factors such as plasmatocyte-spreading peptide (which was first isolated in the moth Pseudoplusia includens) are produced by plasmatocytes and act to stimulate spreading of the hemocyte over the surface of the parasite. Other cytokines, which likely play a role in the cell-to-cell communication events that accompany encapsulation, have yet to be characterized. In parasitized lepidopterans, these cytokines may include plasmatocyte and granulocyte depletion factors. Without these cell types, parasite encapsulation cannot occur, and thus the number of viable circulating cells available to mount the encapsulation response is drastically reduced.

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