Effect of the venom of the endoparasitoid,Apanteles kariyai Watanabe,on the cellular defence reaction of the host,Pseudaletia separata Walker

Although a venom apparatus is present in all female braconid wasps, the function of the venom injected into the host at the time of oviposition by the endophagous members of this group is unknown. Eggs laid by females of Apanteles kariyai, from which the venom apparatus had been removed, were encapsulated, which suggests that the fluid is necessary to enable the parasitoid eggs to escape the cellular defence reaction of the host. Studies with anti-venom serum demonstrated that the venom is attached to the surface of the egg. However, injection of DEAE-Sephadex A-50 particles (Sephadex particles) revealed that the venom alone is insufficient to inhibit the encapsulation reaction. Calyx and venom fluids together seem to be essential for evasion of the host defence reaction by the parasitoid eggs. Eighty-five % Sephadex particles, injected together with calyx and venom fluids, fail to become fully encapsulated, whereas 46% of particles injected with only the calyx fluid avoided encapsulation. Furthermore, when eggs from the lateral oviduct were injected into unparasitized larvae, together with the calyx and venom fluids, a few eggs developed successfully although they had undergone no mechanical distortion.     

Resistance of Apanteles eggs to the haemocytic encapsulation by their habitual host, Pieris

The calyx fluid in the lateral oviduct of a gregarious parasitoid, Apanteles glomeratus contained ellipsoid particles of ca. 130 × 200 nm. These calyx fluid particles did not appear to be embedded in a fibrous outer layer on the surface of eggs in the lateral oviduct. They were not observed on the surfaces of the eggs 3 to 4 hr after being deposited into the host haemocoele. Oviposition experiments indicated that the occurrence of haemocytic defence reactions of the late 2nd instar larvae of the Pieris rapae crucivora against 1 st instar larvae of the parasitoid increased with a decreasing number of the parasitoid eggs introduced into a host, and that more than 5 to 9 parasitoid eggs were needed for suppressing the ability of the host to encapsulate its parasitoid larvae immediately after hatching. When eggs with calyx fluid obtained from egg reservoir were injected into the host, they were found to be encapsulated 1 to 2 days after the injection. They could not start their embryonic development. When calyx fluid-free 3-hr-old eggs were injected in a number of more than 5 eggs into a 5th instar larva of Pieris, 58% of 31 eggs injected had normally hatched without evoking encapsulation reactions by the host. Both electron microscopic observations of parasitoid eggs in the host haemocoele and the experimental results suggested that calyx fluid or calyx fluid particles of the parasitoid might not be involved in the encapsulation-inhibiting activity of the parasitoid eggs. Rather it was anticipated that a substance (or substances) might be secreted by the parasitoid eggs into the haemocoele of the host, which suppressed defence reactions of the host.     

Deadly venom of Asobara japonica parasitoid needs ovarian antidote to regulate host physiology

Asobara japonica (Braconidae) is an endophagous parasitoid developing in Drosophila larvae. The present study shows that A. japonica was never encapsulated in Drosophila melanogaster, and that it caused an overall inhibition of the host encapsulation reaction since injected foreign bodies were never encapsulated in parasitized hosts. Both the number of circulating hemocytes and the phenoloxidase activity decreased in parasitized larvae, and the hematopoietic organ appeared highly disrupted. We also found that A. japonica venom secretions had atypical effects on hosts compared to other braconid wasps. A. japonica venom secretions induced permanent paralysis followed by death of D. melanogaster larvae, whether injected by the female wasp during an interrupted oviposition, or manually injected into unparasitized larvae. More remarkably, these effects could be reversed by injection of ovarian extracts from female wasps. This is the first report that the venom of an endophagous braconid parasitoid can have a deadly effect on hosts, and moreover, that ovarian extracts can act as an antidote to reverse the effects of the wasp's venom. These results also demonstrate that A. japonica secretions from both venom gland and ovary are required to regulate synergistically the host physiology for the success of the parasitoid.     

Venom of the egg-larval parasitoid Chelonus inanitus is a complex mixture and has multiple biological effects

The egg-larval parasitoid Chelonus inanitus injects bracoviruses (BVs) and venom along with the egg into the host egg; both components are essential for successful parasitoid development. All stages of eggs of its natural host, Spodoptera littoralis, can be successfully parasitized, i.e. from mainly a yolk sphere to a fully developed embryo. Here, we show that the venom contains at least 25 proteins with masses from 14 kDa to over 300 kDa ranging from acidic to basic. The majority is glycosylated and their persistence in the host is short when old eggs are parasitized and much longer when young eggs are parasitized. Physiological experiments indicated three different functions. (1) Venom synergized the effect of BVs in disrupting host development when injected into third instar larvae. (2) Venom had a transient paralytic effect when injected into sixth instar larvae. (3) In vitro experiments with haemocytes of fourth instar larvae suggested that venom alters cell membrane permeability. We propose that venom promotes entry of BVs into host cells and facilitates placement of the egg in the embryo's haemocoel when old eggs are parasitized. The multifunctionality of the venom might thus be essential in enabling parasitization of all stages of host eggs.     

Encapsulation of a parasitoid egg within its habitual host: An ultrastructural investigation

Eggs of the ichneumonid parasitoid Campoletis sonorensis oviposited within its natural host Heliothis virescens are rarely encapsulated. The chorion of eggs deposited within the natural host were ultrastructurally similar to eggs within the lateral oviduct. Less than 5% of eggs incubated in invertase or saline and injected into the host evoked a hemocytic response within 24 hr; however, all the eggs incubated in either protease or lipase prior to injection evoked a hemocytic reaction in the host within 24 hr. It would appear that encapsulation of eggs incubated in either lipase or protase resulted from modification of the chorion surface. Eggs incubated in invertase or saline were encapsulated within 72 to 96 hr while eggs incubated in the fluid from the female parasitoid oviduct were not. The ultrastructural development of the hemocytic capsule is presented and the possible role of cell contacts in capsule regulation is discussed.     

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.     

Development of the braconid wasp Cotesia flavipes in two Crambids, Diatraea saccharalis and Eoreuma loftini: Evidence of host developmental disruption

Cotesia flavipes is an important gregarious larval endoparasitoid of several crambid stem borers, including Diatraea saccharalis. The suitability of two crambid species, Eoreuma loftini and D. saccharalis, pests of sugarcane and rice in Texas, for C. flavipes development was tested. The effect of parasitization by C. flavipes on encapsulation response was assessed in vivo in both D. saccharalis and E. loftini. The results indicated that the parasitoid developed and emerged successfully in D. saccharalis larvae. Although E. loftini larvae were readily parasitized by C. flavipes parasitoids, no wasp larvae hatched from the eggs in this host because eggs were encapsulated by the host's hemocytes. The developmental fate of the E. loftini larvae with encapsulated parasitoids was variable. Most died as abnormal fifth instars or as post-wandering prepupae, while a few developed normally to the pupal stage. In vivo experiments, there was a significant reduction in the percent of beads encapsulated in parasitized larvae in both hosts. However, the percent of beads showing melanization decreased significantly in parasitized D. saccharalis larvae but did not differ significantly in parasitized or unparasitized E. loftini larvae. Our results showed that D. saccharalis is a suitable host for C. flavipes whereas E. loftini is an unsuitable host. This study indicated that lepidopteran stem borers that are taxonomically, behaviorally, and ecologically very similar can differ in their ability to encapsulate a parasitoid species.     

Overview of parasitism associated effects on host haemocytes in larval parasitoids and comparison with effects of the egg-larval parasitoid Chelonus inanitus on its host Spodoptera littoralis

In the first part we review the effects of larval endoparasitoids and their polydnavirus and venom on the immune system of their hosts. In all systems investigated, haemocyte spreading and encapsulation activity was reduced; in some cases effects on total (THC) or differential (DHC) haemocyte count as well as modification of haemocyte morphology and ultrastructure were also documented. In many cases polydnavirus (and venom) were shown to play a major role in abrogation of the host's immune reaction. In the second part we present the first investigation of effects of parasitism and polydnavirus/venom on the immune system of the host for an egg-larval parasitoid, Chelonus inanitus. We observed that in 4th and 5th instar larvae, i.e. 7 to 10 days after parasitization, neither haemocyte spreading and encapsulation activity, nor DHC, nor haemocyte ultrastructure were altered. After parasitization with X-ray irradiated wasps, which inject polydnavirus and venom and infertile eggs, there was no alteration of the above mentioned parameters. Nevertheless, parasitoid larvae implanted into 4th instar larvae which developed from eggs parasitized with X-ray irradiated wasps were not encapsulated, whereas co-injected latex beads were. These results show that parasitism by this egg-larval parasitoid does not generally suppress the host's immune system but that polydnavirus/venom injected at oviposition prevent, by, as yet unknown mechanisms, encapsulation of the parasitoid larva.     

Regulation of wing formation and adult development in an aphid host, Aphis fabae, by the parasitoid Aphidius colemani

Nymphs of presumptive winged gynoparae of Aphis fabae (Hemiptera: Aphididae), were exposed to female parasitoids, Aphidius colemani (Hymenoptera: Aphidiidae) and stung once with the ovipositor. Wing development was inhibited and, when aphids were parasitised during the early stages, they did not reach the adult stage but mummies with rudimentary or no wingbuds are observed in the host's fourth-stadium. These and previous studies have suggested that wing development may be inhibited by factor(s) from the maternal parasitoid injected into the host at the time of oviposition. In an attempt to identify such factor(s), saline extracts of whole female parasitoids, abdomens, ovaries and venom glands were prepared. When a saline extract of venom glands was injected into late-second-stadium aphids, many develop to fourth-stadium nymphs with rudimentary wingbuds, indicating an effect on wing formation but also showed developmental arrest and often died when attempting to moult to the adult stage. It appears that host death may be related to physiological/biochemical interactions of parasitoid and host rather than just late stage parasitoid larvae ingesting the host's vital organs. Injections with extracts into later host stadia gave similar results with regard to development to the adult, although aphids injected in the late-fourth-stadium develop normally to the adult stage with no effect on wing formation. The results indicate that the earlier the injection before the final moult the greater the effect of the injected extract on preventing adult development.Extracts prepared from head + thorax do not affect aphid development and the results indicate that there is an active factor(s) - likely a protein - in the female parasitoid's venom that disrupts wing development and/or inhibits development to the adult stage. Surprisingly, injections of extracts from male parasitoids have similar effects but the location and function of such a factor(s) in males are unknown.     

Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum

Encapsulation of eggs inserted by Metaphycus stanleyi (Hymenoptera: Encyrtidae) into the brown soft scale Coccus hesperidum (Homoptera: Coccidae) became more frequent as the host matured. This occurred with both laboratory reared and field-collected parasites. After parasitism for 24 hr at 27°C, encapsulation frequency did not differ in hosts reared at 20° or at 27°C, but significantly increased in hosts reared at 33°C. When parasitism and rearing were carried out at the same temperature, the percentage of eggs encapsulated increased from 48.7% at 27°C to 94.1% at 33°C. With M. helvolus, the percentage of eggs encapsulated was considerably higher than with M. stanleyi; e.g., 99.3 vs 48.7%, respectively, at 27°C. At 20° and 27°C, some M. helvolus development occurred in the larvae of brown soft scale but none at 33°C; the adult stages of the host encapsulated all the parasite eggs at these temperatures.     

Host refractoriness of the tobacco hornworm, Manduca sexta, to the braconid endoparasitoid Cotesia flavipes

Cotesia flavipes (Hymenoptera:Braconidae) is a gregarious endoparasitoid of several pyralid stemborer larvae of economic significance including the sugarcane borer, Diatraea saccharalis. In this study, the ability of this parasitoid to develop in a sphingid host, Manduca sexta, was tested. First, second, third, fourth, and even pharate fifth instar host tobacco hornworm larvae were readily parasitized by the female C. flavipes parasitoids but no wasp larvae hatched from the eggs in this refractory host. Instead, the parasitoid eggs were invariably encapsulated by the host's hemocytes and, ultimately, no parasitoids emerged from tobacco hornworm hosts. The first stages of encapsulation were evident at 2 h post-parasitization of the host M. sexta larvae, when the beginning stages of capsule formation were seen. The developmental fate of the host larvae with encapsulated parasitoids was variable. Most succumbed as abnormally small fifth instars or as post-wandering prepupal animals, while a few developed normally to the pupal stage. Dissection of all the larvae or pupae with encapsulated wasp eggs showed evidence of hemocytic encapsulation and melanization of the C. flavipes eggs. This report describes the association between C. flavipes and M. sexta, which appears to be an excellent model system for studying the physiological processes accompanying wasp egg encapsulation that result in death of the host as well as the parasitoid. Since the parasitoid egg never hatches, the system offers an excellent opportunity to identify and study the effects of parasitoid-injected polydnavirus and venom on host physiology.     

黄腹潜蝇茧蜂寄生因子的特性及其对寄主的生理效应

初步研究了黄腹潜蝇茧蜂Opius caricivorae Fischer寄生因子的特性及其对寄主美洲斑潜蝇Liriomyza sativae Blanchard幼虫的生理效应。黄腹潜蝇茧蜂携带的主要因子是毒液。毒液器官是由一个土黄色的锥形毒囊和7个透明的椭圆形的毒腺及导管构成的;毒液的电泳图谱显示约有12条蛋白带,其中绝大多数低于100 kD,含量最高的3条蛋白带为43.5、25.9和20.1 kD;杜氏腺约有15条左右蛋白质条带,其中有5条含量很高（121.4、77.0、51.5、42.7和36.5 kD）。通过透射电镜观察,在黄腹潜蝇茧蜂毒腺分泌细胞和卵巢表皮细胞中新发现存在一种类病毒颗粒,这些球状颗粒直径大约为50 nm。雌蜂经Co60辐射处理后再寄生（即假寄生）3龄寄主幼虫,被寄生后的寄主依然能正常化蛹,但不能羽化;7 h后寄生体壁开始出现红斑;脂肪体形态结构无显著变化;绝大多数的蜂卵没有被包囊。推测在正常寄生的情况下可能是毒液抑制了寄主的包囊作用,而新发现的类病毒颗粒是否参与了这一过程目前还不清楚。     

Particles containing DNA associated with the oocyte of an insect parasitoid

Viruslike DNA-containing particles are generated in the nucleus of cells located in the calyx region of the reproductive tract of the Hymenopterous parasitoid, Cardiochiles nigriceps. These particles are ca. 130 nm in diameter with a central core of DNA surrounded by an amorphous material enclosed by a unit membrane. These particles are secreted into the calyx lumen and compose the calyx fluid which is injected into hosts along with the eggs. The calyx fluid has been implicated in host regulation and in protection of the parasitoid from the immune response.     

Encapsulation of Aspidogaster conchicola (Trematoda: Aspidogastrea) by unionid mussels

Of 334 mussel specimens representing 13 species, 143 individuals of seven species were found infected with Aspidogaster conchicola; encapsulation of worms was seen in six molluscan species. Encapsulated worms were most abundant anterior to the pericardium and were surrounded by inner fibroblastic and outer fibrocytic/fibrous walls, with occasional adjoining compressed host connective tissues. The inner wall contained acid mucins and phospholipids, while the outer wall contained reticulum fibers, neutral mucins, and phospholipids. Capsule structure was compared to the molluscan encapsulation classification system of T. C. Cheng and E. Rifkin (1970, Amer. Fish. Soc. Spec. Pub., No. 5, pp. 443–496) and to G. B. Pauley and C. D. Becker's (1968, J. Parasitol., 54, 917–920) account of A. conchicola encapsulation. Capsule contents included living or moribund adult worms, viable eggs or empty egg shells produced by disintegrated worms, juveniles hatched from eggs deposited by encapsulated adults, and host cells of which “brown cells” were most abundant. Because of the high frequency of encapsulated moribund worms observed in this study (more than 60%), we infer that this host reaction probably contributed to the parasite's death. Worm eggs individually encapsulated by hemocytes in hemal spaces also were observed. Life cycle implications of juvenile A. conchicola within digestive gland diverticula are discussed.     

Effects of the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) parasitism,venom, and calyx fluid on cellular and humoral immunity of its host Chilo suppressalis (Lepidoptera: Crambidae) larvae

The larval endoparasitoid Cotesia chilonis injects venom and bracoviruses into its host Chilo suppressalis during oviposition. Here we study the effects of the polydnavirus (PDV)-carrying endoparasitoid C. chilonis (Hymenoptera: Braconidae) parasitism, venom and calyx fluid on host cellular and humoral immunity, specifically hemocyte composition, cellular spreading, encapsulation and melanization. Total hemocyte counts (THCs) were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization. While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts, in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development. C. chilonis parasitism suppressed the encapsulation response and melanization in the early stages. Venom alone did not alter cellular immune responses, including effects on THCs, mortality, hemocyte composition, cell spreading and encapsulation, but venom did inhibit humoral immunity by reducing melanization within 6 h after injection. In contrast to venom, calyx fluid had a significant effect on cell spreading, encapsulation and melanization from 6 h after injection. Dose–response injection studies indicated the effects of venom and calyx fluid synergized, showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.     

Passive evasion of encapsulation in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae), a polyembryonic parasitoid of Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

The hymenopteran Macrocentrus cingulum usually deposits one egg into the larval body cavity of lepidopteran Ostrinia furnacalis, and the egg subsequently splits into several dozens of embryos during its development. How the parasitoid eggs and embryos avoid encapsulation by the host's immune response remains unknown. We compared hemocyte counts, morphologies and behaviors between unparasitized O. furnacalis larvae, and larvae parasitized by M. cingulum. No distinct differences were observed. Sephadex A-25 beads elicited a strong encapsulation response when injected into the parasitized host larvae, which indicates that parasitism by M. cingulum does not affect host's cellular immunity. However, there were significant differences in the host's encapsulation reactions towards injected eggs from different sources. Injected M. cingulum mature eggs excised from the lateral oviducts of the female wasps were not encapsulated, while immature eggs or driselase treated mature ones provoked an encapsulation response within 2 h after injection. Inspection of eggs by transmission electron microscopy revealed that the driselase collapsed the surface fibrous layer of the eggs, indicating that surface fibrous layer may play a role in protecting eggs from host's immune attack.     

Levels of encapsulation and melanization in two larval instars of Ostrinia furnacalis Guenée (Lep., Pyralidae) during simulation of parasitization by Macrocentrus cingulum Brischke (Hym., Braconidae)

Abstract:  The hymenopteran Macrocentrus cingulum is a polyembryonic endoparasitoid that uses larvae of the lepidopteran Ostrinia furnacalis as one of its host insects. Previous studies indicated that although this parasitoid does not transmit polydnaviruses when it lays its eggs, a layer of fibrous tissue on the surface of the eggs helps them to avoid being encapsulated by the immune system of the host insect. However, as eggs of M. cingulum that are laid into late instar larvae of O . furnacalis often do not survive, there is a tendency for the adults to lay their eggs in earlier instar larvae. We studied the amounts of encapsulation and melanization around beads of DEAE-Sephadex A-25 injected into the haemoceol of fourth and fifth larval instars of O. furnacalis . The beads were injected to simulate the presence of eggs of the parasitoid M. cingulum . We found that the levels of encapsulation and melanization around the beads increased with the age of the O. furnacalis larvae. Likewise, the total counts of the haemocytes circulating within the haemolymph increased with the age of the O. furnacalis larvae and were correlated negatively with the percentage of larvae parasitized by M. cingulum . It appears that young O. furnacalis possess a weak cellular defence, and as a result are more susceptible to being parasitized. Hence, the correlation between the levels of encapsulation and the age of the host insect appears simply to reflect passive evasion.     

Reciprocal influences and costs of parasitism on the development of Corcyra cephalonica and its endoparasitoid Venturia canescens

Many endoparasitoids develop successfully within a range of host instars. Parasitoid survival is highest when parasitism is initiated in earlier host instars, due to age-related changes in internal (physiological) host defences. Most studies examining fitness-related costs associated with differences in host instar have concentrated on the parasitoid, ignoring the effects of parasitism on the development of surviving hosts that have encapsulated parasitoid eggs. A laboratory experiment was undertaken examining fitness-related costs associated with encapsulation of Venturia canescens (Hymenoptera: Ichneumonidae) eggs by fifth (L5) instar larvae of Corcyra cephalonica (Lepidoptera: Pyralidae). Growth and development of both host and parasitoid were monitored in C. cephalonica larvae containing 0, 1, 2, or 4 parasitoid eggs. Adult size and fecundity of C. cephalonica did not vary with the number of eggs per host. However, there was a distinct increase in host mortality with egg number, although most parasitoids emerged from hosts containing a single egg. The most dramatic effect on the host was a highly significant increase in development time from parasitism to adult eclosion, with hosts containing 4 parasitoid eggs taking over 2.5 days longer to complete development than unparasitized larvae. The egg-to-adult development time and size of adult V. canescens did not vary with egg number per host, as demonstrated in a previous experiment using a different host (Plodia interpunctella). The results described here show that there are fitness-related costs to the host associated with resistance to parasitism.     

Immune response of the hibiscus mealybug, Maconellicoccus hirsutus Green (Homoptera: Pseudococcidae), to oviposition of the parasitoid Anagyrus kamali Moursi (Hymenoptera: Encyrtidae)

Anagyrus kamali Moursi has been recently introduced into the Caribbean as a biological agent against the hibiscus mealybug, Maconellicoccus hirsutus Green. This host has a cellular defense reaction that involves encapsulation and melanization of the endoparasitoid egg. The impact of this immune response on the parasitoid progeny was assessed, as well as the response of the parasitoid countermeasures to overcome it. Under laboratory conditions, significant differences in the immune response were found for different developmental stages of M. hirsutus. The intensity of the immune response varied between second instar, third instar and adult mealybugs. After 30 h, the level of encapsulation was the highest for eggs oviposited in adults: 58% of eggs were encapsulated, followed by third (32%) and second (4%) instars. Three days after oviposition 23, 44 and 86% of the parasitoid eggs oviposited, respectively, in adult, third and second instars were not encapsulated. The unencapsulated parasitoid eggs could hatch and continue their development. Adult mealybugs required 30 h to encapsulate 50% of the eggs, whereas in second and third instars, 50% level encapsulation was never reached. Superparasitism had a saturating effect on the immune system; reduced levels of encapsulation occurred when more than 10 eggs were oviposited in a single mealybug. Wasp larvae were never encapsulated by M. hirsutus.