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.     

Parasitism of Lacanobia oleracea (lepidoptera) by the ectoparasitic wasp, Eulophus pennicornis, disrupts the cytoskeleton of host haemocytes and suppresses encapsulation in vivo

Parasitism of Lacanobia oleracea larvae by the ectoparasitic wasp Eulophus pennicornis suppressed host haemocyte-mediated encapsulation of Sephadex DEAE A-25 beads in vivo. Beads dissected out of parasitized larvae had fewer haemocytes associated with them. Moreover, those haemocytes that were associated with the beads tended to retain a rounded configuration and rarely flattened. Similar results were obtained using in vitro encapsulation assays. SDS PAGE indicated that for parasitized and PBS injected larvae, there were some differences in the plasma proteins that bound to Sephadex DEAE A-25 beads, suggesting that parasitism-mediated changes to host plasma proteins might contribute to the differences in the encapsulation response occurring in these larvae. However, in vitro encapsulation assays using beads that had been pre-incubated in plasma from parasitized and unparasitized larvae, demonstrated that major differences in the extent of encapsulation did not occur. These results, plus in vitro haemocyte attachment and spreading assays, suggest that parasitism-mediated suppression of encapsulation is primarily due to reductions in the ability of host haemocytes to attach to (i.e., recognize) and flatten over non-self surfaces and other haemocytes. This proposal is corroborated by staining of actin in the haemocyte cytoskeleton by FITC-labelled phalloidin, which indicated that parasitism disrupts the formation of stress fibers and focal adhesions in plasmatocytes. By contrast, experimental injection of adult female wasp venom into unparasitized L. oleracea larvae had no significant effect on in vivo encapsulation responses or the haemocyte cytoskeleton. Arch. Insect Biochem. Physiol. 49:108-124, 2002. Published 2002 Wiley-Liss, Inc.     

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.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

颈双缘姬蜂毒液对寄主小菜蛾的免疫抑制作用

对颈双缘姬蜂Diadromus collaris (Gravenhorst)及其毒液引起寄主小菜蛾Plutella xylostella的一些生理效应进行了研究。结果表明,颈双缘姬蜂寄生寄主后可引起寄主小菜蛾蛹总血细胞及浆血细胞和颗粒血细胞数量的上升。寄生后1天观察,血细胞延展行为受到影响,表现在颗粒血细胞放射状丝的产生及浆血细胞伪足的形成受到抑制。通过毒液对寄主离体幼虫血细胞延展行为、形态及活性影响的研究,发现毒液抑制了寄主离体浆血细胞的延展,但对颗粒血细胞的影响不明显;毒液引起寄主浆血细胞和颗粒血细胞的破裂和死亡,毒液对寄主幼虫血淋巴酚氧化酶活性有一定的抑制作用,当反应至40、60及80 min时,毒液处理和未经毒液处理的寄主血淋巴在490 nm处的吸光值差异比较明显。对毒液蛋白成分的聚丙烯酰胺凝胶电泳分析发现,毒液中有9种多肽,分子量介于9～50.2 kD,其中50.2、30.5、28.2、25.1 和12.6 kD的多肽含量较高, 与其他蜂毒液的一些作用已知的蛋白条带相似,因而推测它们同样具有免疫及发育抑制作用。结果证明颈双缘姬蜂毒液能破坏寄主细胞及体液因子调节的免疫反应。     

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.     

Haemocytes of the pink bollworm, Pectinophora gossypiella, during larval development and diapause

Seven types of haemocytes were observed in the last larval instar of the pink bollworm, Pectinophora gossypiella (Saunders): prohaemocytes, plasmatocytes, granular haemocytes, spherule cells, adipohaemocytes, oenocytoids, and podocytes. Total and differential haemocyte counts made from diapausing and non-diapausing larvae showed that during diapause there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. There were no significant differences in the level of haemocytes in the pharate pupae that developed from diapause or non-diapause type larvae, except in the case of adipohaemocytes, which were three times as prevalent in pharate pupae from diapausing larvae. Functional aspects of various types of haemocytes are discussed, and it is suggested that the lower haemocyte level observed during diapause is the result of lower metabolic activity.     

The ectoparasitic wasp Eulophus pennicornis (Hymenoptera: Eulophidae) uses instar-specific endocrine disruption strategies to suppress the development of its host Lacanobia oleracea (Lepidoptera: Noctuidae)

To successfully complete its development, the gregarious ectoparasitoid Eulophus pennicornis must inhibit the moult of its host, Lacanobia oleracea. In the present study, we examined the possibility that moult- and metamorphosis-associated endocrine events may be disrupted in caterpillars parasitized as newly moulted last (sixth) instars. Juvenile hormone (JH) titres on days 2 and 5 of the final stadium were significantly higher (> 100 fold) in parasitized than in non-parasitized hosts, in which JH was essentially absent. Elevated JH levels were associated with reduced haemolymph JH esterase (JHE) activity (down by 99.8%) and enhanced in vitro JH biosynthesis by the corpora allata (CA) (up to 4.5 fold). Wasp adults and/or larvae, in which we measured high levels of JH III (up to 2.7 ng/g), but little or no JH I or JH II, were not seen as likely sources of JH in parasitized hosts, in which we found mostly JH I and JH II. In addition, removal of parasitoid eggs or larvae after oviposition did not prevent the rise in JH titres seen in parasitoid-laden hosts, suggesting that wasp venom may be responsible for the observed hormonal dysfunction. Host haemolymph 20-hydroxyecdysone (20-E) levels were largely unaffected by parasitism during the final stadium although they were observed to increase earlier and decrease more rapidly in parasitized insects. We compare these results with those reported earlier for L. oleracea larvae parasitized by E. pennicornis as penultimate (fifth) instars, which display significantly depressed 20-E titres relative to control larvae. We conclude that E. pennicornis employs host endocrine-disruption strategies that differ according to whether the host is parasitized as a penultimate or final-stadium larva.     

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.     

Proteins synthesized and secreted by larvae of the ectoparasitic wasp, Eulophus pennicornis

A microscopic examination of Eulophus pennicornis larvae on their host Lacanobia oleracea, revealed that peristaltic waves travelled from the anterior to posterior end of the feeding wasp larvae, and vice versa. In addition, when wasp larvae were immersed in PBS in vitro, they released a variety of proteins, with molecular weights ranging from (at least) 14 to 200 kDa. Amongst these was a protein with an estimated molecular weight similar to that of the 27 kDa parasitism-specific protein (PSP) detected in plasma from parasitized L. oleracea [Richards and Edwards, Insect Biochem Mol Biol 29:557-569 (1999)]. Similar results were obtained when the wasp larvae were incubated on balls of cotton wool soaked in tissue culture medium or sucrose, i.e., conditions that resemble their natural feeding behaviour. These results (and others) indicate that the wasp larvae release proteins, putatively through their mouth. Protein synthesis studies using (35)S-methionine indicated that the wasp larvae synthesize and secrete a variety of proteins in vitro, including one with a molecular weight corresponding to that of the L. oleracea 27 kDa PSP. As expected, only a portion of the total proteins synthesized by the parasitoid larvae were subsequently secreted. In addition, the autoradiogram of secreted proteins contained significantly fewer bands than silver-stained SDS gels of proteins released into PBS or onto cotton wool. Thus, some of the additional bands detected on the latter gels are thought to represent proteins that were not of wasp origin. Instead, these proteins released by the wasp larvae are speculated to be derived from their gut and, as such, probably represent proteins derived from host haemolymph and ingested during feeding. This possibility was supported by an electrophoretic analysis of homogenate supernatants prepared from wasp larvae with or without their gut contents. These studies indicated that the gut contents of the larval parasitoid contributes several distinct bands to the total protein profile. The ability of E. pennicornis larvae to synthesize, secrete, and release proteins is discussed with reference to those produced by endoparasitoid larvae. Published 2001 Wiley-Liss, Inc.     

Venom from the ectoparasitoid wasp Eulophus pennicornis disrupts host ecdysteroid production by regulating host prothoracic gland activity

Abstract. Attack by the ectoparasitoid Eulophus pennicornis Nees (Hymenoptera: Eulophidae) prevents larvae of Lacanobia oleracea L. (Lepidoptera: Noctuidae) from moulting. Prothoracic glands (PGs) excised from parasitized or artificially envenomated hosts show a reduced basal level of ecdysteroid release at a time when non-parasitized caterpillars produce an ecdysteroid surge (48 h post moult to 5th stadium = penultimate stadium in non-venomated hosts). By contrast, PGs from similarly parasitized or envenomated caterpillars release comparatively high levels of ecdysteroid at 120 h post-moult. Temporary inactivation of PGs cannot be attributed solely to a parasitoid-induced reduction in cell viability, and incubation in E. pennicornis venom in vitro does not exert any direct effect on either PG cell viability or ecdysteroid release. However, inactivated PGs are not stimulated by forskolin, which may indicate that the absence of the required pre-moult ecdysteroid surge in developmentally arrested L. oleracea is due to insensitivity to a prothoracicotropic hormone. Even though parasitized caterpillars never moult, reversed-phase HPLC separations and radioimmunoassay confirm that they produce active moulting hormone (20-hydroxyecdysone) at 120 h post-moult. These results suggest that E. pennicornis arrests host development through the indirect effects on their hosts' PGs. This effect is not achieved through the destruction of gland cells, but more likely reflects the interruption of an innate cycle in PG activity, such that they lose their ability to respond to a normal cue to produce an essential hormone peak at a crucial point in development.     

The effect of snowdrop lectin (GNA) delivered via artificial diet and transgenic plants on Eulophus pennicornis (Hymenoptera: Eulophidae), a parasitoid of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae)

Snowdrop lectin (Galanthus nivalis agglutinin, GNA) has previously been shown to confer significant levels of protection against the lepidopteran pest Lacanobia oleracea when expressed in transgenic potato. The effect of GNA on the parasitism of L. oleracea by the gregarious ectoparasitoid Eulophus pennicornis was investigated. Maize-based, and potato leaf-based diets containing GNA, and excised transgenic potato leaves expressing GNA, were fed to L. oleracea larvae from the beginning of either the third or fourth larval instar. Lacanobia oleracea larvae were individually exposed to single mated adult female E. pennicornis parasitoids from the fifth instar onwards.The success of the wasp was not reduced by the presence of GNA in any of the diets, or by the length of feeding of the host prior to parasitism. However, the mean number of wasps that developed on L. oleracea reared from the third instar on the GNA-containing maize diet was significantly higher than on the controls (20.6 and 9.3 adults/host respectively). In all other cases differences were not significant. Eulophus pennicornis progeny that developed on L. oleracea reared on GNA-containing diets showed little or no alteration in size, longevity, egg load and fecundity when compared with wasps that had developed on hosts fed the respective control diets.The results suggest that expression of GNA in transgenic crops to confer resistance to lepidopteran pests will not adversely affect the ability of the ectoparasitoid E. pennicornis to utilise the pest species as a host.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by a pupal endoparasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae): effects of parasitization and venom on host hemocytes

In contrast to the situation with egg-larval and larval endoparasitic wasps, little is known about the effects of pupal endoparasitoids and their secretions on the hemocytes of their insect hosts. This study focuses on the pupal endoparasitoid, Pteromalus puparum, and its host, the small white butterfly, Pieris rapae. Parasitism by P. puparum, resulted in a significant increase in the total number of host hemocytes up to day five after parasitization. From day one to day four after parasitization, the percentage of plasmatocytes significantly decreased, and the proportion of granular cells increased. Moreover, from 12 h to day three after parasitization, hemocyte mortality in parasitized pupae was noticeably higher. When P. rapae pupae were parasitized by adult females of P. puparum irradiated by gamma-ray (pseudoparasitization), it was clear that the treated wasps could induce similar hemocyte changes. However, such phenomena did not occur in punctured host pupae (mimic-parasitization). After treatment with P. puparum venom, both the percentages of spreading plasmatocytes and encapsulated Sephadex G-25 beads were lessened significantly in vitro. Electron microscopy analysis and visualization of hemocyte F-actin with phalloidin-FITC showed that hemocytes treated with venom had a rounded configuration and neither spread nor extended pseudopods, while there was no marked alteration of hemocyte cytoskeletons after venom treatment. The results suggested that venom of P. puparum could actively suppress the hemocyte immune response of its host, but not by destroying the host hemocyte cytoskeleton.     

Partial amino acid sequence and physiological effects of a 27 kDa parasitism-specific protein present in the plasma of parasitized Lacanobia oleracea (Noctuidae)

Abstract.  Parasitization of larvae of the tomato moth, Lacanonbia oleracea , by the ectoparasitic wasp, Eulophus pennicornis , results in the appearance of a 27 kDa parasitism-specific protein (PSP) in the plasma of the host. After isolation of this protein by native discontinuous polyacrylamide gel electrophoresis, whole gel elution and electroblotting, the N-terminal sequence of the 27 kDa PSP is determined by Edman degradation. The 20 amino acid residues obtained reveal 70% identity with a female-specific fat body protein from the moths Antheraea pernyi and Antheraea yamamai , 60% identity with a glutathione S-transferase (GST) isolated from Orthosia gothica , and a low level of identity with the N-termini of proteins belonging to the GST superfamily. Injection of the 27 kDa PSP into L. oleracea larvae has no significant effect on their ability to gain weight or the time at which they pupate. Furthermore, assays performed in vitro demonstrate that the 27 kDa PSP does not affect the ability of L. oleracea haemocytes to form aggregates. The precise source of the 27 kDa PSP remains unclear, although the current results suggest that it is most likely synthesized by host larvae in response to parasitism. The possible role(s) of the 27 kDa PSP are discussed with regard to the physiological effects of parasitism on the host.     

Presence of IL-1- and TNF-like molecules in Galleria mellonella (Lepidoptera) haemocytes and in an insect cell line Fromestigmene acraea (Lepidoptera).

In this study the authors give immunocytochemical evidence for the presence of interleukin (IL)-1alpha- and tumour necrosis factor (TNF) alpha-like molecules in the haemocytes of last instar larvae from the greater wax moth Galleria mellonella. Similar results are demonstrated in a continuous haemocyte line (BTI-EA-1174-A) from the salt marsh caterpillar Estigmene acraea. In Galleria mellonella larvae granular cells show a strong positive reaction with both primary antibodies, whereas plasmatocytes are stained to a lesser extent. Cell line haemocytes also react positively with both antibodies. After activating the cells with lipopolysaccharide (LPS) staining of Estigmene acraea cells is decreased, whereas Galleria mellonella haemocytes show no visible reaction in comparison to non-activated cells.     

Impact of insecticides on the haemogram of Rhynocoris kumarii Ambrose and Livingstone (Hem., Reduviidae)

Abstract:  The haemogram of Rhynocoris kumarii Ambrose and Livingstone comprises prohaemocytes, plasmatocytes, granular haemocytes, cystocytes and oenocytoids. The impact of five insecticides, viz. monocrotophos, dimethoate, methylparathion, quinalphos and endosulfan on the total haemocyte count (THC) and differential haemocyte counts (DHC) was studied. All of the insecticides except endosulfan initially reduced both prohaemocytes and plasmatocytes, increased the granular haemocytes, altered the percentage of cystocytes and oenocytoids and increased the total haemocyte count (THC). On the contrary, endosulfan initially increased the prohaemocytes and plasmatocytes, decreased the granular haemocytes and also the THC. The highest impact on the DHC and THC was caused by methylparathion and monocrotophos and the least impact by endosulfan. Hence, endosulfan is considered as the safest insecticide followed by dimethoate and quinalphos among these five insecticides to use with R. kumarii .     

Haemocyte population and ultrastructural changes during the immune response of the mosquito Culex quinquefasciatus to microfilariae of Wuchereria bancrofti

Abstract Haemocytes circulating in the haemolymph protect insects against pathogens that enter the haemocoel. Changes in haemocyte morphology and differences in haemocyte counts during the immune response of Culex quinquefasciatus Say (Diptera: Culicidae) to microfilariae of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) were investigated in the present study. The mean number of total haemocytes was significantly elevated in infected mosquitoes (P < 0.001), reaching a peak on the third day post‐infection. Differential counts show that mean numbers of prohaemocytes, plasmatocytes, granular cells and oenocytoids increased significantly after infection with microfilariae granulocytes compared to the control and näive groups of Cx. quinquefasciatus (P < 0.05). Changes in proportional counts of haemocytes were also analysed in haemolymph perfusates of Cx. quinquefasciatus infected with W. bancrofti. On the first day post‐infection, infected mosquitoes showed an increase in the proportion of prohaemocytes (18.8% compared to 9.6% for the control) and of oenocytoids (7.1% compared to 4.7% control); however, they exhibited lower levels of plasmatocytes (36.6% compared to 42.1% control) and granular cells (36.1% compared to 41.4% control). On day 14 post‐infection, similar changes were observed for these haemocyte types, except that the proportion of granular cells was significantly greater than the control (41.2% compared to 31.3% control). Although an enhancement of prohaemocyte numbers was observed, this cellular type did not show any ultrastructural alteration. On the other hand, granular cells, plasmatocytes and oenocytoids presented morphological alterations indicative of innate immunological activation in mosquitoes infected with W. bancrofti.     

Morphological characterization of Anticarsia gemmatalis M nucleopolyhedrovirus infection in haemocytes from its natural larval host, the velvet bean caterpillar Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

For a better understanding of virus x host interactions, transmission electron microscopy was used to characterize the intrahaemocoelic infection of Anticarsia gemmatalis larval haemocytes by A. gemmatalis M nucleopolyhedrovirus (AgMNPV). At 12 h post-infection (h p.i.), we observed nuclear hypertrophy, budded virus assembling, and protrusion towards the cytoplasm, virion envelopment, and accumulation of fibrillar aggregates in the cytoplasm. Around 24 h p.i., fibrillar aggregates also appeared inside nuclei of infected cells. By 48 h p.i., virogenic stroma and polyhedra were visualised in nuclei and at 72 h p.i., widespread infection in haemocytes was observed. Cell remnants and free polyhedra were phagocytosed by granular haemocyte 1 and plasmatocytes. Entire cells were phagocytosed only by plasmatocytes. Necrosis of infected cells was quite common, suggesting a putative cytotoxic response. Granular haemocyte 1 presented a more exuberant protrusion of budded viruses in comparison to other haemocytes. All types of haemocytes were shown to be infected, and the intense virus replication in some of these cells reveals the importance of haemolymph for AgMNPV spread in its natural host, a critical factor for permissiveness.     

Altered actin polymerization of Plutella xylostella (L.) in response to ovarian calyx components of an endoparasitoid Cotesia plutellae (Kurdjumov)

Abstract Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a solitary braconid endoparasitoid wasp, parasitizes the diamondback moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) by suppressing the host defense response, thereby resulting in successful parasitization. During parasitization, ovarian calyx fluid is also delivered into the haemocoel of the host along with the wasp egg. The effect of calyx fluid constituents on haemocyte‐spreading behaviour of P. xylostella is analysed by measuring F‐actin development in the haemocytes. For this purpose, the calyx fluid of C. plutellae is separated into ovarian protein and C. plutellae bracovirus (CpBV). The ovarian protein consists of a wide range of molecular weight proteins, which are apparently different from those of CpBV. When nonparasitized P. xylostella haemocytes are incubated with either ovarian protein or CpBV for 1 or 2 h, haemocytes lose their responsiveness to a cytokine, plasmatocyte‐spreading peptide, in a dose‐dependent manner for each calyx component and fail to exhibit haemocyte‐spreading behaviour. Some CpBV genes are expressed within 1 h of parasitization. The inhibition of haemocyte‐spreading could be explained by measuring F‐actin contents, in which parasitization by C. plutellae inhibits F‐actin development in the haemocytes of P. xylostella. Either ovarian protein or CpBV could inhibit F‐actin development in the nonparasitized haemocytes. In addition, co‐incubation of ovarian protein and CpBV results in significant additive inhibition of both haemocyte‐spreading and F‐actin development in the haemocytes in response to cytokine. These results suggest that both components of C. plutellae calyx fluid function in a synergistic manner, leading to immunosuppression during the early stage of parasitization.     

Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae

Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides. The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria. The total number of bacteria adhering to all the haemocytes on the slides declined. Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides. Purified apoLp-III bound to B. subtilis. ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min. ApoLp-III bound to B. subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.