Elaine H. Richards

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect hosts immune system.

Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae).

Venom from the endoparasitic wasp, Pimpla hypochondriaca, is composed of a mixture of high and low molecular weight proteins, possesses phenoloxidase activity, has immunosuppressive properties, and induces paralysis in several insect species. In the present study we demonstrate that P. hypochondriaca venom also contains antibacterial and proteolytic activity. Antibacterial activity was detected against the Gram-negative bacteria Escherichia coli and Xanthamonas campestris but not against Pseudomonas syringae nor against two Gram-positive bacteria, Bacillus cereus and Bacillus subtilis. Endopeptidase and aminopeptidase activity in venom was detected using the synthetic fluorogenic substrates N-t-BOC-Phe-Ser-Arg-AMC, Arg-AMC and Leu-Arg. The aminopeptidase activity towards Arg-AMC was sensitive to amastatin (70% inhibition), an aminopeptidase inhibitor. Angiotensin-converting enzyme (ACE)-like enzyme activity was detected, by reverse-phase HPLC using the synthetic tripeptide Hip-His-Leu as a substrate. This activity was sensitive to captopril, an ACE inhibitor (IC(50) 3.8 x 10(-8) M). Using an antiserum raised against recombinant Drosophila melanogaster ACE-like enzyme, (rAnce), Western blot analysis revealed an immunoreactive protein, with a molecular weight estimate of 74 kDa, in P. hypochondriaca venom. The possibility that the endopeptidase, aminopeptidase and ACE are involved in the processing of peptide precursors in the venom sac is discussed.

Parasitization of Lacanobia oleracea (Lepidoptera: Noctuidae) by the ectoparasitc wasp, Eulophus pennicornis. Effects of parasitization, venom and starvation on host haemocytes.

In contrast to the situation with endoparasitic wasps, little is known about the effects of ectoparasitoids and their secretions on the haemocytes of their insect hosts. To address this deficit, a study has been made of the ectoparasitic wasp, Eulophus pennicornis, and its host, the tomato moth, Lacanobia oleracea. Using light microscopy, it was determined that L. oleracea has five main haemocyte types, namely, plasmatocytes, granular cells, spherule cells, oenocytoids and pro-haemocytes, representing 56%, 30%, 10%, 2% and 2% of the population, respectively. Parasitization by E. pennicornis, resulted in an increase in the number of circulating haemocytes up to day three, followed by a decrease towards day eight; the latter being associated with changes to the morphology and viability of the cells. For example, on day five after parasitization, plasmatocytes and granular cells had become more rounded and put out pseudopods less readily compared with those from non-parasitized controls, whilst from day seven onwards there was a significant decrease in haemocyte viability and by day nine, extensive haemocyte damage and disintegration was evident. These changes were not observed when larvae were injected with E. pennicornis venom, or when haemocytes were exposed directly to venom in vitro, neither did they occur in starved larvae. Thus, although the observed effects on L. oleracea haemocytes are definitely associated with parasitization they are not due to wasp venom components, nor are they a non-specific effect resulting from nutritional deprivation. The possibility that the feeding wasp larvae produce factors which perturb host haemocytes in order to help condition the host to ensure that successful parasitization occurs, is discussed.

Biochemical isolation of an insect haemocyte anti-aggregation protein from the venom of the endoparasitic wasp, Pimpla hypochondriaca, and identification of its gene.

Pimpla hypochondriaca venom is complex and contains a number of different proteins and polypeptides that exert a variety of effects on insect physiology. In particular, it possesses factors with potent anti-haemocyte and immunosuppressive properties. In the current work, we describe the biochemical isolation of a single venom factor with insect haemocyte anti-aggregation properties. The protein was isolated using gel filtration and ion exchange chromatography, in conjunction with a qualitative in vitro haemocyte anti-aggregation assay to monitor activity and confirm identity. The protein has a molecular weight estimate of 33kDa (determined by SDS PAGE under reducing conditions), and an N-terminal sequence of Asp-Ser-Asp-Ile-Tyr-Leu-Leu. The biochemically isolated protein has been demonstrated to inhibit haemocyte aggregation and to suppress encapsulation responses, using in vitro and in vivo assays, respectively. Furthermore, its gene has been identified as vpr3. The work is presented within the context of the role of P. hypochondriaca venom and the isolated protein in host immune suppression.

Direct binding and lectin-mediated binding of erythrocytes to haemocytes of the insect, Extatosoma tiaratum.

Treatment of Extatosoma haemocytes on a monolayer with fixed rabbit erythrocytes (RE) in the presence of Ca2+ results in ca. 11% plasmatocytes (PL) and 58% spreading granular (SG) cells rosetting. Pretreatment of haemocytes with lactose, D-galactose, or asialofetuin reduces rosetting, suggesting that the membrane-associated receptors involved may be lectins. The percentage of rosetting PL and SG cells is increased by a number of heterologous lectins (indicating the presence of D-galactose, mannose, and Glu Nac receptors on these haemocytes) and a D-galactose specific lectin isolated from the serum of the insect. The latter acts as a bridging molecule and requires Ca2+ to bind to specific receptors on both haemocytes and fixed RE.

Parasitism of Lacanobia oleracea (Lepidoptera) by the ectoparasitoid, Eulophus pennicornis, is associated with a reduction in host haemolymph phenoloxidase activity

When haemolymph from fifth instar Lacanobia oleracea was incubated in vitro, rapid melanization occurred. Similar levels of melanization occurred in haemolymph from larvae that had been experimentally injected with venom from the ectoparasitic wasp, Eulophus pennicornis. In contrast, haemolymph from larvae parasitized by this wasp melanized more slowly and less extensively. Phenoloxidase assays indicated that enzyme activity was present in haemocyte lysate supernatants, serum and plasma from L. oleracea and that on day 5 post-parasitization, fractions prepared from parasitized larvae had significantly less phenoloxidase activity than similar fractions from untreated or experimentally envenomated larvae. In addition, no PO activity was detectable in wasp venom, and the venom had no effect on L. oleracea plasma phenoloxidase activity in vitro. These results indicate that parasitism of L. oleracea by E. pennicornis suppresses host haemolymph phenoloxidase activity and that this suppression is not induced by adult wasp venom. The results are discussed with reference to the survival advantages of suppressing the activity of this host enzyme, and to the possible source(s) of putative suppressive factors.

Parasitization of Lacanobia oleracea (Lepidoptera) by the ectoparasitic wasp, Eulophus pennicornis, suppresses haemocyte-mediated recognition of non-self and phagocytosis

Although many endoparasitic wasps suppress the haemocyte-mediated immune defences of their insect hosts, the effects of ectoparasitoids are virtually unknown. In view of this, a study has been made of the ectoparasitic wasp, Eulophus pennicornis, and its host, the tomato moth, Lacanobia oleracea. For unparasitized insects, in vitro assays indicated that less than 3.0% of L. oleracea haemocytes on a monolayer formed rosettes with yeast cells or fresh rabbit erythrocytes (rbc), and virtually no phagocytosis of these particles occurred. In addition, although fixed rbc formed rosettes with 51.21% of haemocytes, only about 3.0% of the haemocytes ingested one or more of these particles. In contrast to this, B. cereus and E. coli were readily phagocytosed by 14.75% and 53.70% of haemocytes, respectively. These results indicate that L. oleracea haemocytes can recognise different types of non-self particles and demonstrate that ingestion does not necessarily follow attachment. When rosetting and phagocytosis assays were performed with fixed rbc and FITC-labelled E. coli, and haemocytes from starved L. oleracea, PBS injected L. oleracea, and experimentally envenomated insects on day five of treatment, there was no significant difference in the percentage of rosetting or phagocytosis occurring. When haemocytes from parasitized insects on day five of treatment were utilised, however, rosetting and phagocytosis were reduced by 31.41% and 34.94%, respectively. Thus, the effects of parasitization and experimental envenomation are not the same. In addition, suppression of host haemocyte-mediated recognition and phagocytosis was not a secondary effect of nutritional deprivation and was not due to ectoparasitoid venom components, rather it was a direct result of parasitization of L. oleracea by E. pennicornis. The putative nature and source of the immunosuppressive factor(s) involved is discussed with reference to those produced by endoparasitic wasps.

Parasitism of Lacanobia oleracea (Lepidoptera, noctuidae) by the ectoparasitic wasp Eulophus pennicornis, results in the appearance of a 27 kDa parasitism-specific protein in host plasma

Abstract Little is known about the effects of ectoparasitoids and their secretions on the plasma protein profiles of their insect hosts. To address this deficit, a study has been made of the interactions between an ectoparasitic wasp, Eulophus pennicornis , and its host, the tomato moth, Lacanobia oleracea . In particular, the quantitative and qualitative effects of parasitism or the experimental injection of wasp venom on host plasma proteins were investigated. Results demonstrated that both treatments caused an initial increase in L. oleracea total plasma protein concentration up to day 5 of treatment, but whereas the protein concentration remained high in the experimentally envenomated group, a decrease towards day 8 occurred in parasitized insects. Parasitism was also associated with the appearance of a protein with an estimated molecular weight of 27 kDa. This protein first appeared on day 3 after parasitization and its levels subsequently increased. The protein was not detected in any of the unparasitized larvae (including all the various control groups) or in experimentally envenomated L. oleracea larvae. In addition, the appearance of this protein was not a non-specific result of nutritional deprivation, nor was it a general injury, stress, or infection induced protein. Its appearance was strictly associated with parasitism of L. oleracea by E. pennicornis and thus, it may be described as a parasitism-specific protein (PSP). The PSP has been partially purified using whole gel elution. Gel filtration and SDS PAGE indicated that it has a native molecular weight of 27 kDa and that it does not appear to aggregate to produce higher molecular weight molecules, nor dissociate into lower molecular weight subunits held together by disulphide or covalent bonds. The precise site of synthesis of the 27 kDa PSP is not yet known but some evidence leads us to speculate that it may be synthesised by the feeding E. pennicornis larvae and introduced into their host. This possibility is discussed in relation to previous work detailing the effects of parasitism on L. oleracea haemocyte morphology, function and viability, and the effects of endoparasitoids on host plasma proteins.

Larvae of the ectoparasitic wasp, Eulophus pennicornis, release factors which adversely affect haemocytes of their host, Lacanobia oleracea.

When larvae of the ectoparasitic wasp Eulophus pennicornis were incubated for 4 h on balls of cotton wool soaked in tissue culture medium (TC-100), they released a variety of factors. Subsequent incubation of these larval wasp secretions with monolayers of haemocytes from their host, Lacanobia oleracea, demonstrated that they adversely affect haemocyte morphology, behaviour and viability. For instance, when monolayers of haemocytes were incubated for 18 h in TC-100, approximately 73% of the cells present, attached firmly to and spread over the tissue culture surface by extending pseudopods. By contrast, when incubated in TC-100 containing larval wasp secretions, only about 27% of the haemocytes present remained attached to the tissue culture surface after washing. The majority of these had a rounded configuration and neither spread nor extended pseudopods. Furthermore, viability assays indicated that approximately 36% of the attached haemocytes were dead, as opposed to 11-12% in the controls. The E. pennicornis secretions also significantly reduced the ability of L. oleracea haemocytes to move across the surface of the slide and form clumps (p</=0.0005) and to phagocytose FITC-labelled Escherichia coli in vitro (p</=0.0005). These results indicate that secretions from E. pennicornis larvae contain an anti-haemocyte factor(s) that can kill and/or alter the behaviour of host haemocytes. As a result, the ability of the haemocytes to execute important immune responses is compromised. Preliminary data suggest that the active molecules are proteins, and that their mechanism of action may involve inhibition of polymerization and/or disorganization of the haemocyte cytoskeleton.

The mode of action of venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae) involves Ca+2-dependent cell death pathways.

The endoparasitoid Pimpla hypochondriaca injects venom during oviposition to condition its lepidopteran hosts. Venom is a complex mixture of proteins and polypeptides, many of which have been identified as enzymes, including phenoloxidase, endopeptidase, aminopeptidase, hydrolase, and angiotensin-converting enzyme. Constituents of the venom have been shown to possess cytolytic and paralytic activity, but the modes of action of factor(s) responsible for exerting such effects have not been deciphered. In this study, we examined the mode of action of isolated venom using cultured cells (BTI-TN-5B1-4). A series of blockage and inhibition assays were performed using a potent inhibitor (phenylthiourea, PTU) of venom phenoloxidase, and anti-calreticulin antibodies. Monolayers exposed to venom alone were highly susceptible with more than 84.6+/-2.3% dead within 15 min. Susceptible cells displayed a retraction of cytoplasmic extensions, rounding, and swelling prior to lysis in more than half (55.7+/-1.7%) of the dying cells. Within 15 min of exposure to venom, cells displayed qualitative increases in [Ca(+2)](i) as evidenced by staining with the calcium-sensitive probe fluo-4 AM, and mitochondrial membrane potential (DeltaPsi(m)) was undetectable by 5 min post-treatment with venom. These venom-mediated changes occurred regardless of whether an external source of calcium was present, or whether venom was pre-treated with PTU. In contrast, venom toxicity was attenuated by treatment with anti-calreticulin antibodies. Not only did fewer cells die when exposed to antibody-treated venom but also cell swelling diminished and no increases in intracellular calcium were detected. A possible mode of action for the venom is discussed.