Polydnaviruses: potent mediators of host insect immune dysfunction

Reviews 9 Sturm, N. and Simpson, L. (1991) Nucleic Acids Res. 19, 6277-6281 10 Bhat, G.J. et al. (1990) Cell 61, 885-894 11 Pollard, V.W. et al. (1990) Cell 63,783-790 12 van der Spek, H. et al. (1991) EMBO J. 10,1217-1224 13 Abraham, J.M. et al. (1988) Cell 55,267-272 14 Sturm, N.R. and Simpson, L. (1990) Cell 61,871-878 15 Maslov, D.A. and Simpson, L. (1992) Cell 70,459-467 16 Missel, A. and Goringer, U.H. (1994) Nucleic Acids Res. 22, 405m56 17 Souza, A.E. et ~2. (1992) Mol. Cell. Biol. 12,2100-2107 18 Decker, CJ. and Sollner-Webb, B. (1990) Cell 61,1001-1011 19 Riley, G.R. et aZ. (1994) 1. Biol. Chem. 269,61014108 20 Kosiowsky, D.J. et al. (i991) Cell 67,537-546 21 Maslov. D.A. et al. (19921 Mol. Cell. Biol. 12. 56-67 22 Sturm, N.R. et uZ.(i992) ?eZZ70,469-476 23 Maslov, D.A. et al. (1994) Mol. Biochem. Parusitol. 68, 155159 24 Corell, R.A. et al. (1993) Nucleic Acids Res. 21,4313-4320 25 Seiwert, S.D. and Stuart, K. (1994) Science 266,llP117 26 Cech, T.R. (1991) Cell 64,667-669 27 Blum, B. et al. (l&l) Cell 65,543-550 28 Peris. M. et al. 119941 EMBO 1.13.1664-1672 29 Blud, B. and Simpsbn, L. (l&O)CeZZ 62,391-397 30 White, T.C. and Borst, P. (1987) NucZeic Acids Res. 15, 32753289 31 Bakalara, N. et al. (1989) J. Biol. Chem. 264,18679-18686 32 Harris, M.E. and Hajduk, S.L. (1992) Cell 68,1091-1099 33 Harris, M. et al. (1992) Mol. Cell. BioZ. 12,2591-2598 34 Read, L.K. et al. (1992) Nucleic Acids Res. 20, 2341-2347 35 Arts, G.J. et UZ.(1993) EMBO 1. 12,1523-1532 36 Koslowsky, D.J. et al. (1992) Nature 356,807-809 37 Blum, B. and Simpson, L. (1992) Proc. NutZ Acud. Sci. USA 89 11944-11948

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Riley, G.R. et al. (1995) Nucleic Acids Res. 23,708-712 Pollard, V.W. et al. (1992) EMBO 1. 11,4429-4438 Sontheimer, E.J. and Steitz, J.A. (1994) Science 262,1989-1996 Harris, M.E. et al. (1990) 1. BioZ. Chem. 265,11368-11376 Read, L.K. et al. (1994) MoZ. CeZZ.Bid. 14,2629-2639 Keller, J. et uZ.(1994) Ir]ucZeicAcids Res. 22, 1988-1995 Bass. B. (1993) in The RNA World (Gesteland, R.F. and Atkins, J.F., eds),‘pp 3318, Cold Spring Harbor Laboratory Press Adler, B.K. and Hajduk, S.L. (1994) Curr. @in. Genet. Dev. 4, 316-322 Sogin, M.L. (1994) in Early Life on Earth (Bengtson, S., ed.), pp 181-192, Columbia University Press Femandes, AI’. et UI. (1993) Proc. Nut2 Acud. Sci. USA 90, 11608-11612 Landweber, L.F. and Gilbert, W. (1994) Proc. NutZ Ad. Sci. USA 91,918-921 Landweber, L.F. and Gilbert, W. (1993) Nature 363,179-182 Maslov, D.A. et ~2. (1994) Nature 368,345-348 Lukes, J. et a2. (1994) EMBO ].13,5086-5098 Maslov, D. and Simpson, L. (1994) Mol. CeZZ. BioZ. 14, 8174-8182 Gabriel, A. and Boeke, J-D. (1991) Proc. NutZ Acud. Sci. USA 88, 9794-979s Gray, M.W. (1994) Nature 368,288 Gilbert, W. (1986) Nature 319,618 Benne, R. (1990) Trends Genet. 6,177-181 Priest, J.W. and Hajduk, S.L. (1994) 1. Bioenerg. Biomembrunes 26, 179-191 Landweber, L.F. et al. (1993) Proc. NufZ Acud. Sci. USA 90, 9242-9246 Read, L.K. et al. (1994) Nucleic Acids Res. 22, 1489-1495 Thiemann, O.H. et al. (1994) EMBO ].13,5689-5700

Polydnaviruses: Potent Mediators of Host Insect Immune Dysfunction M.D. Lavine and N.E. Beckage Endoparasitic insects are used as biological control agents to kill many species of insect pest. One key to the success of parasitoids that develop in the hemocoel of their host is their ability to knock out the host’s immune system, inducing a decline in the responsiveness of a variety of cellular and humoral components so that parasitoid eggs are not encapsulated. In many species parasitized by braconid and ichneumonid wasps, host immunosuppression appears to be mediated by polydnaviruses (PDVs) injected by the female parasitoid into the host hemocoel. The viruses exhibit a complex and intimate genetic relationship with the wasp, since viral sequences are integrated within the wasp’s chromosomal DNA. Here Mark Lavine and Nancy Beckage summarize the current evidence for mechanisms of virally induced host immunosuppression in parasitized insects, as well as the roles of other factors including wasp ovarian proteins and venom components, in suppressing hemocytemediated and humoral immune responses. Interestingly, in some species, the PDV-induced host immunosuppression appears transitory, with older parasitoid larvae probably exploiting other mechanisms to protect themselves from the Mark Lavlne IS at the Department of Biology, and Nancy Beckage IS at the Department of Entomology 5419 Boyce Hall, Univenlty of Califomla-Rivewde, Rivenlde, CA 9252 I-03 14, USA. Tel: + I 909 787 3521, Fax: + I 909 787 3087, e-mail: [email protected]

host’s immune system during thefinal stages of parasitism. During the final stages of parasitism, the parasitoids likely exploit other mechanisms of immunoaasion via antigen masking, antigen mimicry, or production of active inhibitors of the hemocyte-mediated encapsulation response as well as inhibiting melanization. Many members of the insect order Hymenoptera are endoparasitoids, which are parasites that develop within, and always kill, their insect hosts. For endoparasitoids to develop successfully, they must avoid or suppress their host’s natural defense mechanisms, and hence have evolved complex mechanisms for modulating the host insect’s cellular and humoral immune system.G. Parasitoids exhibit sophisticated mechanisms of interaction with their host insect’s immune, endocrine and metabolic pathways, and, frequently, even modify the development and behavior of their host; the outcome is the successful maturation of the parasitoid accompanied simultaneously by the host’s demise. Endoparasitic wasps in the families Braconidae and Ichneumonidae have developed unique associations with a family of polydisperse DNA viruses (polydnaviruses, or PDVs). These viruses replicate exclusively in the female wasp’s ovaries, and each species of wasp is presumed to have a unique PDV; the

Reviews Fig. I. Life cycle of the hymenopteran parasitoid Cotesio congregate and its host, Manduca sexta. The cycle of transmission and replication of the wasp’s polydnavirus is illustrated in the inner circle.

Mature female wasp injects eggs bathed in calyx fluid containing polydnavirus virions (and in some species, other virus-like particles), ovarian proteins, and venom into host hemocoel.

Virus enters host cells, including hemocytes and fat body; viral genes are expressed by host cellular machinery.

Virus passed vertically to next wasp generation as integrated sequences of wasp chromosomes.

Dunng wasp metamorphosis, polydnavirus replicates in ovarian calyx cells.

Wasp larvae develop within host, feeding on QINIX3 hemolymph components QXUB2 (gregarious *3%333Q species), or host tissues such as fat body in addition to hemolymph (solispecies). /

Para&id( s) emerges from host and pupates. Host may die shortly after emergence (solitary parasitoids) or persist for several weeks in a state of developmental arrest (gregarious parasitoids). viruses therefore have potential utility as taxonomic tool&4 in parasitoid systematics. While the males carry integrated viral sequences dispersed in their genome, the PDVs are not observed to replicate in males; obviously, a complex genetic relationship with the wasp exists, giving rise to speculation about the origin of these viruses. Did the parasitoids evolve the capacity selectively to replicate and encapsidate part of their genome (ie. subsets of their genes) in a virus-like particle? Or did progenitor viruses invade these two lineages of wasps, then coevolve with their respective hymenopteran ‘host’? While certain strains of human and veterinary parasites (eg. protozoa, amoebae) have viruses associated with them5, none of these have viral sequences integrated in the parasites’ genomic DNA similar to the PDVs, and their presence is variable. Moreover, their correlation with parasite virulence mechanisms or successful infection remains unclarified in most cases. Some parasitoids have other types of virus-like elements (eg. pox or baculovirus-like particles), instead of, or in addition to, polydnaviruses, suggesting that multiple lines of these wasps have evolved intimate relationships with viruses, all of which appear to be injected into the host insect. The tissues giving rise to Porostoiogy

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the virus particles [such as the ovary and the venom gland (also known as the accessory gland)] as well as the nature of the particles themselves differ according to the wasp’s taxnomic affiliation, usually varying on a wasp genus-specific level. Our goal here is to summarize how PDVs disarm various components of the host insect immune system, allowing the parasitoid to develop in the hemocoel and escape the hemocytemediated defense reactions of its host. Interestingly, the strategy of these hymenopteran parasitoids appears distinctly different from that of the major group of dipteran parasitoids, ie. tachinids, which activate and successfully exploit the cellular immune response of their host by inducing host hemocytes to form a respiratory sheath encircling the larva to facilitate oxygen transfer to the fly from host tracheae. The parasitoid and polydnavirus life cycles The adult female braconid and ichneumonid wasps oviposit their egg(s) directly into the body cavities of their lepidopteran or dipteran hosts (Fig. 1). Solitary species inject a single egg per host, whereas gregarious parasitoids inject multiple eggs during oviposition. The eggs hatch and the wasp larvae develop in the host hemocoel, feeding on host 369

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Fig. 2. Transmission electron micrograph of ovarian calyx fluid from an adult female Cotesio congregota showing abundant polydnavirus virions (PDV) and less numerous virus-like long particles (LP). Scale bar = 2OOnm. (Reproduced, with permission, from Ref. IO.)

Fig. 3. Photomicrograph of developing Cotesio congregata egg dissected 72 h after oviposition into a fourth instar Monduco sexta larva, showing the serosal cells (arrow) that will dissociate upon emergence of the neonate larva, giving rise to teratocytes. Scale bar = 40p.m. (Photograph by M.D. Lavine and N.E. Beckage.)

hemolymph (gregarious species) or a combination of hemolymph and host tissues (solitary species). Upon reaching the appropriate developmental stage, the wasp larvae emerge from their host and spin cocoons and pupate on the host’s external surface. Some species actually leave the host and pupate in the near vicinity, rather than on the host directly; yet the latter partner is invariably developmentally arrested. With solitary species, the host’s tissues are usually consumed prior to parasitoid pupation, but with gregarious species only the hemolymph is ingested, and the host may linger for several weeks after the parasitoids have emerged, pupated and eclosed as adult wasps. In tobacco hornworm larvae parasitized by the gregarious braconid parasitoid Cot&u congregata, host death occurs due to multiple endocrine and neuroendocrine disturbances which interfere with molting and pupation6. The host is developmentally arrested because of a combination of factors including lack of 370

release of prothoracicotropic hormone which results in a ecdysteroid deficiency which prevents molting6. Arrest is due in part to action of the PDV on the host endocrine system (see below) or hormone metabolism. During oviposition, eggs are not the sole component introduced by the wasp into the host. The eggs in the wasp’s reproductive tract are first transported through a neck-like region called the calyx, which joins the ovary and the lateral oviduct. The calyx fluid contains PDV virions7, ovarian-secreted proteins8 and, in some species, other assorted virus-like particles that differ morphologically from PDVs+ii (Fig. 2). This fluid and its contents, along with eggs and venom from the venom gland, are injected into the host via the wasp’s common oviduct. In the braconids, a serosal membrane surrounding the wasp embryo dissociates upon hatching of the neonate, releasing serosal cells, called teratocytes, into the host hemocoel (Fig. 3); these cells grow in size and persist in the hemolymph for the duration of parasitismn. It is likely that all these factors play a role in ensuring proper parasitoid development in the host, and they have been implicated in regulating various aspects of the host’s immune (and endocrine) physiology. In particular, parasitoid venom components have been documented as playing major roles during endoparasitism, acting both independently and synergistically with PDV7Ja. PDV genomes are composed of multiple nonconcatenated circles of double-stranded DNA7J4. Bracoviruses (from Braconidae) and ichnoviruses (from Ichneumonidae) show morphological differences in packaging of their multiple DNA segments. The nucleocapsids of ichnoviruses are fusiform and bound by a double-membrane envelope (one nucleocapsid /envelope), while bracoviruses have rodshaped nucleocapsids embedded in a protein matrix and packaged within a single virion (with a single unit membrane)i4. PDVs replicate only in specialized cells localized in the female wasp’s ovarian calyx. To gain entry and accumulate in the calyx lumen, packaged virions bud from (ichnoviruses) or lyse (bracoviruses) the calyx cells in which they replicatei4Js. In Cmnpoletis sonorensis, viral replication is stimulated by ecdysteroids produced during adult development of the wasp, suggesting replication is regulated by hormonal stimulii6. In the wasp, assembled virions are not found anywhere outside the female reproductive tract; however, viral DNA sequences are dispersed throughout the genomes of both male and female waspsl7. The PDV exists as a provirus, permanently integrated in the wasp chromosomes, and is passed vertically with each fertilizationl7J8. No evidence has yet been found for PDV replication in the wasps’ lepidopteran hosts9JQo. However, PDV DNA is transcriptionally active in lepidopteran tissues, and, following parasitization, several viral mRNAs are rapidly producedl7J9-26. Transcription and translation of virally encoded mRNAs has been demonstrated in the fat body, hemocytes, gut, Malpighian tubules and even the nervous systems of permissive hosts20,25,26, suggesting that several tissues participate in viral gene expression. Different genes are expressed in the moth and in the wasp; other constitutive genes are expressed in both speciesi7. Porasrtoiogy

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Reviews Parasitism-induced host immunosuppression Insect cellular and humoral responses. The insect immune response comprises both humoral and cellular components*. The humoral arm seems to consist primarily of induction of bactericidal and other defense proteins, such as agglutinin$-30. Defense molecules include intermediates produced during the phenoloxidase cascade reaction, which have been implicated in both recognition of ‘foreignness’ and killing of invading organism+M; the killing activity may reflect toxic effects of melanin and its precursors, or other molecules such as cytokines or superoxide1,34-36 that are also induced. The cellular arm involves three responses: phagocytosis, encapsulation and nodulationx. Small (40 Km diameter) abiotic and biotic particles, such as bacteria and yeast, are phagocytosed by hemocytes. Encapsulation is triggered in response to particles too large to be phagocytosed. Hemocytes adhere to and flatten against the target, eventually walling it off from the rest of hemocoel, then the melanized capsule adheres to host tissues including the fat body, Malpighian tubules, or tracheal epithelium36-38. Nodulation is characterized by formation of microaggregates of hemocytes and bacteria which ultimately are encased in hemocytes, melanized and removed from circulation; the nodulation response, and other cell-mediated defense mechanisms such as phagocytosis, appear to be mediated by eicosanoids39,a. In the Lepidoptera, the two most numerous and immunologically active classes of hemocytes are the granulocytes and plasmatocyte+41. Encapsulation involves initial recognition of ‘foreignness’ by granulocytes, which release intracytoplasmic granules in response to contact with the foreign body. This serves as a signal for recruitment and adherence of plasmatocytes, which flatten out and form the bulk of in hemocyte classifithe capsule 36,4*. Inconsistencies cation have often complicated identification of the behavior and function(s) of individual classes, although the use of gradient separation methods43 and monoclonal antibodies capable of recognizing specific hemocyte morphotypes& should help resolve these problems. Suppression of encapsulation and nodulation. Following oviposition by the wasp, the lepidopteran host does not exhibit any encapsulation response to the parasitoid egg(s). Moreover, the rate of encapsulation of abiotic targets, such as glass rods and Sephadex beads, also is commonly suppressed relative to responses of nonparasitized control larvae (Fig. 4a,b)37,4%50 suggesting a complete inhibition of the host’s encapsulation response occurs. However, in several cases the suppression of encapsulation seems specifically directed to the parasitoid egg, which remains well protected while abiotic targets are efficiently encapsulated45,46. In addition to suppressing encapsulation, parasitism may also inhibit nodulation. Injection of calyx fluid has been shown to alter nodulation of carbonyl iron particles47, and naturally parasitized hosts showed reduced nodulation and clearance of microorganisms such as yeasP,” and bacteria5*,52. This phenomenon probably explains why parasitized insects are frequently rendered more susceptible to pathogenic infection53. PoraaroiogyTo&r, voj. i

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Fig. 4. Photomicrographs of Sephadex A-25 beads recovered 24 h after injection into the hemocoels of fourth instar Manduca sexta larvae: from nonparasitized larva (note prominent hemocytic capsule and surface melanization) (a); from larva 24 h after oviposition by Cotesia congregata (b); from larva eight days after oviposition by C. congregata (note capsule and melanization as in panel a, and that the adjacent parasitoid larva has escaped the encapsulation and melanization response) (c). Scale bar = 4Okm. (Reproduced, with permission, from Ref. 37.)

The observed absence of encapsulation is accompanied by major alterations in host hemocyte appearance (Fig. 5a,b) and function. Plasmatocytes exhibit obvious abnormalities, with most studies reporting a reduction or elimination of in vitro spreading behavior by plasmatocytes isolated from parasitized or PDV-injected nonparasitized 1arvae37,46-48JoJ4. It is likely that this represents an in vitro manifestation of 371

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Fig. 5. Photomicrographs (Nomarski optics) of hemocytes from fourth instar Manduca sexta larvae: from nonparasitized larva (a), from larva 24 h after oviposition by Cotesia congregata (b), from larva eight days after oviposition by C. congregata (c), from nonparasitized larva 24 h after injection of filter-purified C. congregata polydnavirus (d). Note clumped hemocytes (large arrowheads) and apparent surface blebbing (small arrowheads) in (b) and (d), and adherent and spreading hemocytes (arrows) in (a) and (c). Blebbing and other symptoms seen in hemocytes of the parasitized and PDV-injected larvae are characteristic of apoptosis. Scale bar = 40 km. (Reproduced, with permission, from Ref. 37.)

the observed in vivo reduction in the occurrence of encapsulation and nodulation. There is also commonly a decrease in the absolutes4 or relative4@8,“’ number of circulating plasmatocytes in these hosts; this decrease in the number of functional plasmatocytes may contribute signficantly to host immunosuppression. Parasitism or virus-induced alterations have also been documented in other classes of hemocytes, and total hemocyte counts have been reported to increase48,50 or decrease”6,51,“4 following oviposition or injection of purified polydnavirus. In addition to effects on plasmatocytes, there are also many parasitism-induced alterations in granulocytes, which exhibit reduced spreading4@“, reduction of degranulation55, massive lysis when exposed in vitro to wasp calyx fluid and venom”6, and PDV-induced apoptosis as demonstrated both in zjivo and in vitrolJ7 (see below). Reduced spreading of granulocytes may be due to a disruption of actin polymerization, as indicated by the diffuse immunofluorescence staining pattern noted in the cells using anti-actin antibodies8 (Fig. 6). Plasmatocytes also show this type of cytoskeletal disruption, although to a lesser degree. Guzo and Stoltz5l observed nuclear pycnosis in, and the apparent destruction of, both prohemocytes 372

(hemocyte stem cell.+) and the putative hemopoietic tissue associated with the imaginal wing discs, with effects being seen as early as 24 h after parasitization. Prevost et al.54 also noted the disappearance of prohemocytes from host hemolymph. Large amounts of hemolymph ‘debris’ (presumably damaged or fragmented cells) have also been reported in hemolymph from parasitized larvae3”@,j’. Suppression ofphugocytosis. Parasitism-induced suppression of phagocytosis has been less-intensively investigated than suppression of encapsulation. Several authors47,50,51 all found that, despite inhibition of nodulation, phagocytosis of both biotic and abiotic targets was unaffected. However, Ross and Dunn52 concluded that since M. sexta parasitized by C. congredata were unable to clear even small numbers (l-5 cells) of Pseudomonas ueruginosu bacteria, which were otherwise rapidly cleared by nonparasitized M. sextu, they concluded that phagocytosis, in addition to nodulation, was inhibited during parasitism. Suppression or ulterution of humorul responses. Although the role of the prophenoloxidase cascade in the insect immune response is far from clear3*,34,57, a reduction in tyrosinase or diphenoloxidase activity may play a part in parasitoid avoidance of encapsulation. A decrease in phenoloxidase activity (measured Pormtoiogy

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Reviews by rate of conversion of intermediates in the cascade) occurs in both naturally parasitized larvae and nonparasitized ‘hosts’ injected with PDVa,58. Stoltz and Guzos observed notably slower rates of melanization of hemolymph from host Malacosoma disstria parasitized by Hyposoter fugitiuus, and correlated this phenomenon with an enhanced stability of the normally labile oenocytoids. These cells appear to be involved in the production and storage of prophenoloxidase, although their precise roles may vary among specie+. Baculoviruses have also been reported to reduce hemolymph phenoloxidase activity59, suggesting that inhibition of hemolymph melanization may be a common mechanism of viral interference with the insect immune response. Other humoral regulators of host immunity may also be affected. Hayakawa60 isolated a 470 kDa hexamerit protein that was found at higher levels in Pseudaletia separata parasitized by Cotesia kariyai than in nonparasitized P. separata. This protein, which shows sequence homology and antigenic similarity to the storage protein arylphorin, prevents degranulation by P. separata hemocytes in uitro, and thus may terminate encapsulation at the early recognition and recruitment stages. Suprisingly, effects of parasitization on the synthesis of common insect antibacterial proteins (eg. lysozyme, attacins, cecropins) have not been well investigated. Additional, virally induced effects on hemolymph characteristics include a decrease in host hemolymph viscosity4648, and decreases in total hemolymph protein content, including a reduction in the level of production of arylphorin61+3; interestingly, its expression appears to be translationally blocked, at least in some species6*,63. Agents invoking immunosuppression Polydnaviruses. The specific mechanisms whereby PDVs cause host immunosuppression remain elusive. Parasite eggs injected into habitual hosts in the absence of virus are invariably encapsulated, while recombination with calyx fluid or purified virus affords protection to virus-free eggs45-48,sOJi@i (Fig. 7). Most immunosuppressive effects of parasitization can be duplicated by manual injection of suitable hosts with calyx fluid or PDV isolated and purified from female wasps. Observed variations from effects of natural parasitism are largely a matter of degree, and may well be due to dosage differences between the amount transferred during natural parasitism versus artificial PDV injections. Indeed, exposure to PDV has a dose-dependent effect on hemocyte spreading behavior and phenoloxidase activity, with higher doses of virus having more pronounced and prolonged immunosuppressive effects48,@,65. Interestingly, doses of PDV too low to cause immunosuppressive effects induce other changes in the host characteristic of natural parasitism, such as arrested development and pigmentation anomalies@. Although PDVs do not appear to replicate in the lepidopteran host, treatment of PDV with psoralen and ultraviolet light abrogates any immunosuppressive effect of virus on caterpillar defense mechafisms50,51,58,66indicating that viral genes must be expressed to induce immunosuppression. Viral mRNAs, some of which may be found in the lepidopteran host as early as 30min post-virus introduction, are

Fig. 6. Photomicrographs of F-actin specific BodipylFLTM phallicidin fluorescent staining pattern for hemocytes (PL, plasmatocytes; GR, granulocytes) from Heliothis virescens larvae after I h spreading in vitro. Hemocytes from nonparasitized larva showing characteristic actin fiber organization (a). Hemocytes from l-l. virescens larva 30min after oviposition by Campoletis sonorensis showing altered actin organization (b). Arrowheads point to actin-ring surrounding the endoplasm, suggestive of a collapse of the actin cytoskeleton. (Reproduced, with permission. from Ref. 8.)

detectable for several days in a variety of host tissues, often most abundantly in host hemocytes*9JsJ6,50. In Pseudoplusia includens parasitized by Microplitis demolitor, all hemocyte classes transcribe viral DNA, and more than 90% of host hemocytes may initiate transcription by 4 h post-oviposition65. In addition, granulocyte and plasmatocyte spreading is reduced by direct in vitro exposure to I’DW. Granulocytes undergo blebbing and apoptosis in response to PDV1,“7,67;virally mediated apoptosis appears critically important in causing an immediate ‘knockout’ of host hemocyte defenses. Translation of viral mRNAs produces several de noz~oproteins that show different temporal patterns of expression in different tissues, including hemocytes. At least one polydnaviral gene product, a 30 kDa protein encoded by the Campoletis sonorensis associated polydnavirus VHvl.1 gene, has immunosuppressive action@. Host Heliothis virescens infected with a recombinant baculovirus carrying the VHvl. 1 gene show a 50% reduction in encapsulation of washed C. sonorensis eggs relative to larvae injected with the or saline-injected controls. wild-type baculovirus 373

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Fig. 7. Photomicrographs of eggs of Cotesia congregate 24 h after natural parasitization (a) or injection (b) into hemocoels of fourth instar Monduca sexto larvae. C. congregata egg 24 h after oviposition (a); note the host has not mounted an encapsulation response. In contrast, note that the C. congregoto egg washed free of any polydnavirus (see Ref. 64) and examined 24 h after manual injection into M. sexto larva was enclosed in a thick hemocytic capsule (b) (arrowheads point to surface of egg). Scale bar = 40 urn. (Photograph by M.D. Lavine and N.E. Beckage.)

Immunocytochemical analysis indicates that the VHv1.1 protein, which is found in parasitized H. virescens larvae as little as 5 h post-oviposition, binds to the surface of host plasmatocytes and to the interior of host granulocytes, suggesting a direct interaction of the 30 kDa protein with host hemocytes. In M. sexta parasitized by C. congregata, a 31 kDa virally encoded polypeptide is rapidly expressed in permissive hosts of the parasitoid, but its role, if any, in modulating the host immune system remains speculative2526. Other studies have indicated that a soluble hemolymph-borne factor, possibly a viral gene product, is responsible for alterations in normal plasmatocyte function. Davies et al .46 found that hemolymph plasma from C. sonorensis PDV-infected H. virescens would suppress spreading behavior of plasmatocytes from uninfected H. zlirescens larvae in vitro. Likewise, normal plasma had a ‘rescue’ effect on non-spreading plasmatocytes from PDV-exposed larvae, eliciting spreading of these cells. Strand65 also found a suppressive effect of cell-free plasma from Pseudopllrsin 374

includens larvae parasitzed by Microplitis demolitor on control hemocytes. However, effects varied depending on the time post-oviposition that plasma was isolated. Davies et aZ.46could not reproduce their observations by direct in vitro exposure of hemocytes to PDV. Thus, they speculated that a hemolymph-borne factor may be first expressed primarily in cells other than hemocytes in which viral gene transcription occurs, which then targets hemocytes. In the ichneumonid Venturia canescens, virus-like particles play a role in the parasitoid’s escape of the host immune response, possibly by adhering to the surface of the parasitoid egg and mimicking surface characteristics of host tissues@. However, no nucleic acids have been recovered from these particles, suggesting their mode of action differs markedly from PDVs”. Other virus-like long filamentous particles have been observed in calyx fluid of several members of the genus Cotesia9-11. They do not appear to adhere to the egg surface, however, and it is unclear what role, if any, they play in the successful development of the parasitoids. Diuchasmu (=Biosteres) longicuudutus, a braconid endoparasitoid of dipterans, injects into its host two types of viral-like particles which replicate in the venom gland: rod-shaped particles that can ultimately be found infecting host epidermal cells; and entomopoxvirus-like particles that infect host hemocytes 71. It is not known if either of these virus-like particles plays an immunological role similar to that of a PDV. Venom. The specific actions of PDVs may be further complicated by the role of venom, which is also injected into the host during oviposition. Although venom alone generally does not show immunosuppressive effects, in some host/parasitoid systems it may act as a viral synergist, allowing successful suppression of the host response to parasitoids at lower levels of PDV48,si and in some cases may be obligatory45. In vitro observations by Stoltz et al.72 indicate that venom may facilitate PDV entry into lepidopteran cells. Interestingly, wasp venom components show antigenic crossreactivity with PDV virionassociated proteinGJ4. Webb and Summers73 showed that Cumpoletis sonorensis venom gland mRNAs hybridized to genomic DNA from its corresponding PDV, suggesting that the PDV may have evolved as an amplifier of venom genes critical for effecting successful parasitization. Thus far, the synergistic effects of venom and PDV on host immunity have only been demonstrated for some braconid parasitoids. In other systems, including the ichneumonid C. sonorensis46 and the braconid Cotesiu congreguta74, parasitoid venom has no observable effect on host physiology and exhibits no obvious synergism with the PDV. Indeed, C. sonorensis larvae develop normally following oviposition by wasps whose venom glands have been surgically removeds. The lack of requirement for venom in some systems may be due to duplication of its function(s) by PDVs or other virus-like particles or biochemical factorsiJ1. Soluble ovarian proteins. Although transcripts of viral mRNAs may appear in parasitized hosts as rapidly as 30min post-oviposition, it may be several hours before the mature protein encoded by the PDV appears in the hemolymphz. The process of encapsulation may occur extremely rapidly, with adherent Pormtoiogy

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Reviews granulocytes observable within 5 min of introduction of a foreign body into the hemocoeP6, and thus viral gene products alone may not be able to account for the absence of encapsulation of parasitoid eggs. Indeed, Webb and LuckharP found reduction and alteration of granulocyte and plasmatocyte spreading by 30 min post-parasitization. They then investigated the presence of non-viral ovarian proteins introduced into the caterpillar host during oviposition. Wasp ovarian proteins can be found bound to caterpillar hemocytes (plasmatocytes and especially granulocytes) by 30 min post-oviposition, and may persist for four days, suggesting they may be responsible for the observed alterations in hemocyte actin polymerization and the cells’ lack of spreading behavior. These ovarian proteins are, in some cases, antigenically related to the wasp’s PDV, and they may prove to share both evolutionary origins and functions with the PDV. The parasitoid egg. The complete absence of the normally rapid encapsulation response to the parasitoid egg, coupled with some at least partial responses to contemporaneous abiotic challenges, points to the possibility that surface characteristics of the egg itself may contribute to its lack of encapsulation. It is well known that surface characteristics of foreign bodies are important elicitors or inhibitors of immune responses75,76, and insects often show a reduction or absence of encapsulation responses to negatively charged objects 37.49Js. The eggs of most of the PDVcarrying parasitoids do not appear capable of passively avoiding host immunity without the intervention of the PDV, except for the eggs of Caydiochiles nigricqs, which possess a fibrous coat that prevents encapsulation by host Heliofhis virescens hemocytes until translation of viral proteins or other immunosuppressive mechanisms can take effect”. The eggs of Cofesia rubeculu, while traversing the ovarian calyx, are coated with factors that have been implicated in protecting the eggs from encapsulation in its host, Pieris rupae 78. This protective coating appears to be antigenitally related to viral proteins, but does not consist of adherent virions. Intact PDV does not protect C. rubeculu eggs washed of the protective coating, providing evidence that this layer affords protection to the eggs before synthesis of virally encoded proteins commences. Surface charge and other egg-surface characteristics (either passive constituents or active secretions) may prove to be important contributors to the eggs’ complete avoidance of encapsulation. Terutocyfes. Teratocytes have also been hypothesized to play a role in altering the immune response of caterpillar hosts. In both Pseudulefia separafu larvae parasitized by Cofesiu kuriyui79 and Pieris rupae CYUcivoru larvae parasitized by Cofesiu glomerufu80, inhibition of the phenoloxidase cascade is hypothesized to be caused by factors secreted by teratocytes. However, when Microplitis demolifor teratocytes are injected into Pseudoplusia includens host larvae in the absence of calyx fluid and venom, the teratocytes are encapsulated, indicating they alone are not fully responsible for host immunosuppressiorG*. Teratocytes synthesize and release proteins into host hemolymphl2JJ2, and, in some parasitoids, teratocyte secretions may have fungicidalss or even digestive actions, due to the production of enzymes such as

Fig. 8. Appearance of parasitoid larvae that were excised from a fourth instar host eight days post-oviposition, killed by brief immersion in ethanol, and implanted into a newly ecdysed fourth instar ‘surrogate host.’ Note that dense, agglutinated, and melanized hemocyte capsules surrounded the larvae 24 h after injection. Since living parasitoid surfaces are not encapsulated (Fig. 4c), these observations suggest that at this stage the parasitoids actively evade the host’s immune response by antigen mimicry, secretion of host immunosuppressive molecules, or other mechanisms. (Photograph by M.D. Lavine and N.E. Beckage.)

acid phosphatases, esterases and leucine aminopepti dases%. However, it is not clear to what extent terato cyte secretions are involved in immunosuppression Since teratocytes do not occur in the Ichneumonidae, any potential function they may have in braconid parasitoids must be effected by an alternative mechanism during parasitism by ichneumonids. Temporal trends Several studies show that host immunosuppression is at least to some degree nonspecific, and that parasitized hosts do not successfully respond to, and are in fact frequently killed by, foreign organisms that are usually nonpathogenic in immunocompetent hosts?-52. This presents an interesting evolutionary paradox: immunosuppressive effects needed to protect the parasitoid(s) from encapsulation may also lead to premature host (and thus parasitoid) death following other immunological challenges. Some immune functions in parasitized hosts do exhibit at least a partial recovery. Relative numbers of normally spreading hemocytes may gradually return to normal control levels during natural parasitism48 and in larvae injected with purified PDV37,46s4s. In addition, newly or recently parasitized hosts show significantly less clearing of, and greater mortality due to, normally nonpathogenic biotic challenges 375

Components introduced during oviposition by wasp into host’s hemocoei

in host ceils (17-26)

Facilitation (72)

I

v interference

with host immune function( 1,2,7,8, 45-5264-67)

f

Duplicatioi of PDV functions, or generation of additional mechanisms (8,45,48,51)

Humoral fsuppressive protein (60)

Role in recognition and/or killing of

cascade

Destruction (51,54)

JI Granulocytes 4

inhibition of degranulation

Prohemocfles+ 7 and 7 . hemopoietic tissue

__)

Disruption of actin cytosketeton (8) +

+

Reduced adherence and spreading (43,49) Cell lysis (apoptosis?) (1,37,56,67)

Continuing suppression or avoidance of immune response despite possible return of encapsulation capability (37.50.52)

3;

Developing parasitoid

376

Destruction or reduced production? (37,46,48,51,54)

initial adherence and degranuiation (recognition?) by granuiocytes (36,37,42,43,94)

f----------------

than do hosts parasitized earlier. This is true for both fungaP and bacterial52 challenge@. Our own observations indicate that, after an initial lack of response to injected Sephadex beads following parasitization, host caterpillars recover their immune

Disruption of actin cytoskeleton (8) ?

?

PD V encoded or parasitoid released factors?

Plasmatocytes

+

Reduced adherence and spreading (46-48, 50,54)

Hemocyte recruitment and formation of capsule by plasmatocytes (36,37,42,43)

I Passive role of f--egg surface and $ factors adhering to it Parasitoid egg (69,70,77,78)

function, and by eight days post-parasitization they encapsulate beads at rates similar to nonparasitized controls (Fig. 4a-c) 37. This temporal variation in encapsulation ability superficially correlates with both alterations of host hemocyte morphology (Fig. 5a-c) Parasrtoiogy

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Reviews and levels of expression of some virally encoded proteins (such as El’1253 ), suggesting the cellular events and appearance of this protein may be functionally related. When beads are implanted into parasitized larvae eight days post-oviposition, they are efficiently encapsulated, yet the parasitoid itself escapes immunorecognition (Fig. 4c)37. In contrast, when the parasitoid larvae are killed (by cold treatment or immersion in ethanol), then implanted into a nonparasitized surrogate ‘host’ they are encapsulated and melanized (Fig. 8), and the capsules are agglutinated (Fig. 6b) and removed from circulation. Whether their strategy involves immunoevasion or active immunosuppression remains unclear at this point. Since these lepidopteran hosts do recover some immune function, the immune response to developing parasitoids must continue to be suppressed by an alternative method compared to the early ‘knockout’ of hemocyte function (ie. altered behaviour and apoptosis), or the wasps must no longer be recognized as foreign. How this is achieved is not known, although the developing parasitoid larvae are known to release proteins into the hemocoel of their hostQfi. Such proteins may correspond to ‘late-expressed’ parasitization specific proteins, which do not appear to be a result of virally directed expressionsss9. Conclusions and future perspectives Polydnaviruses have evolved as potent regulators of lepidopteran physiology, particularly the host immune response, allowing successful development of an invader in an environment that would otherwise prove lethal. The process whereby a parasitoid escapes the host immune response is a complex one, involving venom components, ovarian proteins, egg characteristics, teratocytes, and temporal variations in immunocompetence, in addition to host expression of viral proteins and viral regulation of host protein production (Fig. 9). Little is known about the specific mechanisms whereby such factors alter the host immune response. Future research will surely focus on molecules which are nov being revealed as important modulators of invertebrate immune responses, such as lectins and other non-bactericidal inducible proteins*a3, eicosanoids3K39,94 FAD-glucose dehydrogenasegl, octopamine92,93, cytokines and integrinlike recognition sequence+4. In vertebrates95 and plants%, the mechanisms of resistance, the subversion of the immune system by pathogens and parasites, and killing by nitric oxide9’ and other mediators are much better understood. Mechanisms of gene-forgene co-evolution are are also more clearly delineated in other species 98. Since the DNA viruses infecting vertebrates produce a variety of virokines and related immune modulators99, the same may be true for insect-associated DNA viruses, including PDVs, leaving open the possibility that the viruses themselves produce molecular manipulators of the host immune response. PDV-carrying parasitoids and their regulation of the host immune response have generated a high degree of interest, and the continuing investigations of these systems will probably advance our knowledge of the immunology of insects and other invertebrates, with attendant applications in more applied areas such as agricultural pest and vector control. Porasltolo~

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Acknowledgements Research on the tobacco homworm system has been supported by grants to N.E. Beckage from the Natlonal Science Foundation (IBN9006003-04 and IBN-9420638), and US Department of Agnculture (NRI 92-37302-7470). We are indebted to Jason Odell for assistance with computer graphics and text revisions. We also sincerely thank two anonymous reviewers and Darcy Reed, Shelley Adamo and Manannevan Laarhoven for helpful comments. Luis Villarreal (Department of Molecular Biology and Blochemistty University of CallfomiaIrvine) drew our attention to helpful vertebrate references. References 1 Strand, M.R. and Pech, L.L. (1995) Annu. Rev. Entomol. 40,31-56 2 Summers, M.D. and Dib-Hajj, SD. (1995) Proc. Nat2 Acad. Sci. USA 92,29-36 3 Stoltz, D.B. and Whitfield, J.B. (1992) 1. Hymenoptera Res. 1, 125-139 4 Whitfield, J.B. (1990) Parasitolo,qy-.. Today 6,381-384 5 Wang, A.L. and Wang, CC. (1991) P&s&logy Today 7,76-80 6 Zitnan, D. et al. (1995) 1. Coma. Neural. 356, 83-100 7 Stoltz, D.B. (1993) in ‘Par&es and Pathogens of Insects (Vol. 1) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 167-187, Academic Press 8 Webb, B.A. and Luckhart, S. (1994) Arch. Insect Biochem. Physiol. 26, 147-163 9 Stoltz, D.B. et al. (1988) 1. Gen. Viral. 69,903-907 10 de Buron, I. and Beckage, N.E. (1992) J. Invertebr. Pathol. 59, 315-327 11 Hamm, J.J. et al. (1990) 1. Invertebr. Pafhol. 55, 357-374 12 Dahlman, D.L. and Vinson, S.B. (1993) in Parasites and Pathogens of Insects (Vol. 1) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 145-165, Academic Press 13 Jones, D. and Coudron, T. (1993) in Parasites and Pathogens of Insects (Vol. 1) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 227-244, Academic Press 14 Stoltz, D.B. et al. (1984) Intervirology 21, l-4 15 Stoltz, D.B. and Vinson, S.B. (1979) Adz!. Virus Res. 24, 125-171 16 Webb, B.A. and Summers, M.D. (1992) Experientia 48,1018-1022 17 Fleming, J.G.W. and Krell, P.J. (1993) in Parasites and Pathogens of Insects (Vol. 1) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 189-225, Academic Press 18 Stoltz, D.B. (1990) J. Gen. Viral. 71, 1051-1056 19 Blissard, G.W. et al. (1989) Virology 169,78-89 20 Strand, M.R. et al. (1992) 1. Gen. Viral. 73, 1627-1635 21 Blissard, G.W. et al. (1986)]. Viral. 57,318-327 22 Blissard, G.W. et al. (1986) 1. Insect Physiol. 32,351-359 23 Theilmann, D.A. and Summers, M.D. (1986) J. Gen. Viral. 67, 1961-1969 24 Theilmann, D.A. and Summers, M.D. (1988) Virology 167, 329,341 25 Harwood, S.H. and Beckage, N.E. (1994) Znsect Biochem. Mol. Biol. 24, 685-698 26 Harwood, S.H. et al. (1994) Virology 205,381-392 27 Cociancich, S. et al. (1994) Parasitology Today 10, 132-139 28 Faye, I. and H&mark, D. (1993) % Parakes and Pathogens of Insects (Vol. 2) (Beckage, N.E. ThomDson. S.N. and Federici. B.A., edi), pp i5:53, Acidemic Press ’ 29 Kanost, M.R. et al. (1994) Arch. Insect Biochem. Physiol. 27, 123-136 30 Spence, K.D. and Minnick, M.F. (1991) in Immunology of Insects and Other Arthropods (Gupta, AI’., ed.), pp 273-287, CRC Press 31 Christensen, B.M. and Severson, D.W. (1993) in Parasites and Pathogens ofInsects(Vol. 1) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 245-266, Academic Press 32 Dunphy, G.B. and Bouchier, R.S. (1992) 1. Invertebr. Pathol. 60, 26-32 33 Brookman, J.L. et al. (1989) Insect Biochem. 19,47-57 34 Sugumaran, M. and Kanost, M.R. (1993) in Parasites and Pathogens ofInsects(Vol. 1) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 2317-2342, Academic Press 35 Arakawa, T. (1995) Insect Biochem. Mol. BioI. 25,247-253 36 Ratcliffe, N. (1993) in Parasites and Pathogens of Insects (Vol. 1) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 267-304, Academic Press 37 Lavine, M.D. and Beckage, N.E. I. Insect Physiol. (in press) 38 Miller, J.S., Nguyen, TT and Stanley-Sa&el&, ‘D.W.’ (1994) Proc. Nat1 Acad. Sci. USA 91.12418-12422 39 Stanley-Samuelson, D.W. (i994) Adu. Insect Physiol. 24, 115-212 40 Sun, S.C. et of. (1990) Science 250, 1729-1732 377

Reviews 41 Gupta, A.P. (1991) in lmmunolo~y oflnsects And Other Arthropods (Gupta, AI’., ed.), pp 19-118, CRC Press 42 Davies, D.H. and Siva-Jothy, M.T. (1991) in Immunology qf Insects and Other Arthropods (Gupta, A.P., ed.), pp 119-132, CRC Press 43 F’ech, L.L. et al. (1994) Cell Tissue Res. 277, 159-167 44 Willott, E. et al. (1994) Eur. 1. Cell Biol. 65, 417-423 45 Tanaka, T. (1987) J. Insect Physiol. 33,413-420 46 Davies, D.H. et al. (1987) 1. Insect Physiol. 33,143-153 47 Davies, D.H. and Vinson, S.B. (1988) Ce21 Tissue Res. 251, 467-475 48 Strand, M.R. and Noda, T. (1991) 1. Insect Physiol. 37, 839-850 49 Vinson, S.B. (1974) Parasitology 68, 27-33 50 Stoltz, D.B. and Guzo, D. (1986) 7. Insect Physiol. 32,377-388 51 Guzo, D. and Stoltz, D.B. (1987) I. Insect Physiol. 33, 1931 52 Ross, D.R. and DUM, P.E. (1989) Dev. Comp. hnmunol. 13, 205-216 53 Brooks, W.M. (1993) in Purusites nnd Pathogens of Insects (Vol. 2) (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 231-272, Academic Press 54 Prevost, G. et al. (1990) Ent. Exp. Appt. 55, l-10 55 Rizki, R.M. and Rizki, T.M. (1990) I. Insect Physiol. 36, 523-529 56 Wago, H. and Tanaka, T. (1989) Zool. SCI. 6, 691-696 57 Dunphy, G.B. and Webster, J.M. (1991) 1. Invertebr. Pufhol. 58, 40-51 58 Beckage, N.E. et al. (1990) Insect Btochem. 20, 285-294 59 Anderson, D. et a/. (1990) insect Btochem. 20,537-543 60 Hayakawa, Y. (1994) J. Biol. Chem. 269,14536-14540 61 Beckage, N.E. and Kanost, M.R. (1993) Insect Biochem. Mol. Btol. 23,643-653 62 Fathpour, H. and Dahlman, D.L. (1995) Arch. Insect Biochem. Physiol. 28, 3348 63 Shelby, KS. and Webb, B.A. (1994) 1. Gen. I;lrol. 75,2285-2292 64 Dushay, M.S. and Beckage, N.E. (1993) 1. Insect Physiol. 39, 1029-1040 65 Strand, M.R. (1994) J. Gen. Viral. 75, 3007-3020 66 Beckage, N.E. et al. (1987) Insect Biochem. 17,439-455 67 Strand, M.R. and Pech, L.L. (1995) 7. Gen. Vtrol. 76,283-291 68 Li, X. and Webb, B.A. (1994) J. Viral. 68,7482-7489 69 Feddersen, I. et al. (1986) Elperientiu 42, 1278-1281 70 Theopold, U. et al. (1994) Arch. Insect Biochem. Physiol. 26, 137-145 71 Lawrence, P.O. and Akin, D. (1988) Cair. 1. Zoo/. 68, 539-546

72 Stoltz, D.B. et al. (1988) 1 Gen. Viral. 69,90>907 73 Webb, B.A. and Summers, M.D. (1990) Proc. Nutl Acad. Sci. USA 87,49614965 74 Beckage, N.E. et al. (1994) Arch. Insect Biochem. Physiol. 26, 165-195 75 Lackie, A.M. (1983)J. Cell Sci. 63, 181-190 76 Wiesner, A. (1992) J. Insect Physiol. 38,533-541 77 Davies, D.H. and Vinson, S.B. (1986) . ,, r. Insect Physiol. 32, 1003-1010 78 Asgari, S. and Schmidt, 0. (1994) 1. insect Physiol. 40,789-795 79 Tanaka, T. and Wago, H. (1990) Arch. Insect Biochem. Physiol. 13, 187-197 80 Kitano, H. et al. (1990) Arch. Insect Biochem. Physiol. 13,177-185 81 Strand, M.R. and Wong, E.A. (1991) J. Insect Physiol. 37,50%515 82 Vinson, S.B. et at. (1994) Arch. Insect Biochem. Physiol. 26,197-209 83 Furher, E. and Elsufty, R. (1979) Z. Purusitenkd. 59,21-25 84 Strand, M.R. et al. (1986) J. Insect Physiol. 32,389-402 Insecfs(Vol. 1) 85 Beckage, N.E. (1993) in Parasites and Pathogens (Beckage, N.E., Thompson, S.N. and Federici, B.A., eds), pp 23-57, Academic Press 86 Beckage, N.E. et al. (1989) Arch. lnsecf Biochem. Physiol. lo,2945 87 Beckage, N.E. and Kanost, M.R. (1993) Insect Biochem. Mol. Biol. 23,643-653 88 Soldevila, AI. and Jones, D. (1993) Arch. Insect Biochem. Physiol. 24,149-169 89 Rolle, R.S. and Lawrence, P.O. (1994) Arch. Insect Biochem. Physiol. 27, 265-285 90 Stanley-Samuelson, D.W. et al. (1991) Proc. Nutl Acad. Sci. USA 88,1064-1068 91 Cox-Foster, D.L. and Stehr, J.E. (1994) 1. Insect Physiol. 40, 235-249 92 Dunphy, G.B. and Downer, R.G.H. (1994) 1. Insect Physiol. 40, 267-272 93 Baines, D. and Downer, R.G.H. (1994) Arch. Insect Biochem. Physiol. 26, 249-261 94 Pech, L.L. and Strand, M.R. (1995)J. Insect Physiol. 41,481488 95 Marrack, P. and Kappler, J. (1994) Cell 76,325332 96 Staskawica, B.J. et al. (1995) Science 268,661-667 97 Oswald, II’. et al. (1994) Camp. Biochem. Physiol. C 108, 11-18 98 Frank, S.A. (1994) Philos. Trans. R. Sot. London Ser. B 346, 283-293 99 McFadden, G. (1995) Viroreceptors, Virokines, and Related hnmune Modulators Encoded by DNA Viruses, R.G. Landes Co.

of

The Sticky Secrets of Sequestration I.W. Sherman, I.E. Crandall, N. Guthrie Sequestration, the adherence of infected erythrocytes containing the more mature stages of parasite development (trophozoites and schizonts) to the endothelial cells lining the capillaries and post-capillary venules, is characteristic of Plasmodium falciparum infections. In this review, Irwin Sherman and his colleagues discuss recent advances in the characterization of the adhesive molecules on the surface of malaria-infected erythocytes and the receptors on the endothelium to which they bind. The absence of erythrocytes containing mature, pigmented stages (trophozoites and schizonts) of malaria parasites in the peripheral blood, due to their attachment to the endothelial cells of capillaries and postcapillary venules in the deep tissues, was first described in individuals who had died from

Irwin Sherman, Ian Crandall, Nell Guthrte and KIrkwood Land are at the Department of Biology Unlvenlty of Callfomla, Riverside, Callfomla 9252 I USA. Tel: + I 909 787 5905, Fax: + I 909 787 4286, e-mail: sherman@ucrac I .ucr.edu 378

L UY, I <,_,(.,

and K,M. Land

aestivo-autumnal fever. Although this century-old observation1 of sequestration provided a simple and rational explanation for the lack of mature parasites in the blood of individuals infected with Plasmodium falciparum (the causative agent of these fevers), it did not explain why red blood cells bearing the young, unpigmented ring-stage parasites or gametocytes circulated freely. Much later, by examination of P. falciparum-infected red cells by electron microscopy Z-4, it became clear that sequestration was related to morphologic changes in the infected red blood cell surface: erythrocytes bearing either trophozoites or schizonts have a distorted shape and their surface is covered with small (
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