Effect of the venom of the gregarious parasitoid Apanteles glomeratus on its hemocytic encapsulation by the host, Pieris

When eggs from the lateral oviduct of the gregarious parasitoid Apanteles glomeratus were injected with calyx fluid and venom apparatus material into host larvae, Pieris rapae crucivora, most of the eggs were not encapsulated. Apanteles eggs deposited by the parasitoid from which the venom apparatus was removed were usually encapsulated by the host. These results indicate that the parasitoid venom apparatus material is an important factor in suppressing the encapsulation of 1- or 2-day-old eggs in the host. In order to clearly demonstrate that the venom suppresses egg encapsulation but not the encapsulation of other foreign objects, DEAE-Sephadex A-50 ion-exchange particles stained with 0.001% (w/v) Congo Red solution were injected into hosts together with venom apparatus material. The Sephadex particles were encapsulated by host hemocytes. The results suggest that the venom does not inhibit the encapsulation ability of the host.     

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.     

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.     

Observations on cellular immunity and parasitism in the tussock moth

Cellular responses to the introduction of foreign objects into the haemocoele of both control and parasitized tussock moth larvae were examined. In normal larvae, the response to large foreign objects such as Hyposoter fugitivus eggs and Sephadex beads was encapsulation, accompanied by a rapid and sustained increase in the total haemocyte count. Smaller objects such as yeast cells were cleared into nodules within a matter of minutes; nodulation too was accompanied by an increased total haemocyte count.In larvae parasitized by the braconid wasp Cotesia melanoscela, both encapsulation and nodulation were permanently suppressed. Inhibition of these normal cellular defence reactions was accompanied by a reduction in the total haemocyte count, the appearance of debris in the haemocoele, and by nuclear pycnosis in an unidentified population of cells; however, since extensive nuclear pycnosis also occurred in haemopoietic tissue, it is assumed that prohaemocytes may represent a target cell population. All of the observed effects required the presence of both C. melanoscela calyx fluid and venom in the host animal. Phagocytosis as an immune response remained essentially intact, and was capable of completely clearing both yeast and Escherichia coli cells injected into the haemocoele.     

菜蛾盘绒茧蜂多分DNA病毒的特性及其对小菜蛾幼虫的生理效应

对菜蛾盘绒茧蜂Cotesia plutellae多分DNA病毒的特性及其对寄主小菜蛾Plutella xylostella幼虫的生理效应进行了研究。结果表明：菜蛾盘绒茧蜂雌蜂输卵管萼中含有大量的多分DNA病毒（polydnavirus, PDV）;一个PDV内含多个核衣壳,最多可达16个;核衣壳长40～168 nm,直径39～40 nm;PDV仅在输卵管萼细胞内复制;雌蜂产卵时,随蜂卵将PDV注入寄主血腔,并扩散到寄主的许多组织中;PDV可能先通过脱膜再侵染寄主组织。雌蜂经Co⁶⁰辐射处理后再寄生（即假寄生）小菜蛾2龄、3龄和4龄初期的幼虫,被寄生后的寄主幼虫几乎全部不能化蛹,但末龄（即4龄）幼虫期显著延长,并在寄生后期,幼虫胸部有褐色的短翅芽出现;即将化蛹的4龄末小菜蛾幼虫被假寄生后,即使每头寄主被过寄生9次,依然能正常化蛹,但不能羽化。假寄生与正常寄生后寄主的脂肪体数量和形态结构有明显的不同,推测在正常寄生的情况下蜂卵孵化时释放的畸形细胞及随后的幼蜂可能对脂肪体的结构产生了作用。     

Effects of parasitization or injection of parasitoid-derived factors from the endoparasitic wasp Glyptapanteles porthetriae (Hym., Braconidae) on the development of the larval host, Lymantria dispar (Lep., Lymantriidae)

Second instar larvae of Lymantria dispar were parasitized or injected with parasitoid-derived factors such as venom, calyx fluid or parasitoid eggs from Glyptapanteles porthetriae . Growth and development of the host larvae were affected in all different groups compared to control larvae of the same age, injected with Ringer solution. The greatest impact on host growth and on the duration of the 3rd instar was caused by injecting parasitoid eggs. Treated larvae showed melanized capsules or nodules in the hemocoel. While the wasp age had no effect on parasitization efficiency or on the percentage of melanized particles in the hemocoel, significantly more encapsulations were found in larvae parasitized by old wasps as opposed to young wasps. Superparasitization (double or quadruple oviposition) increased the parasitization efficiency markedly. While none of the control larvae showed melanized particles, in the groups of single and superparasitized (2× and 4×) hosts a high percentage of melanized particles (capsules and nodules) occurred.     

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.     

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.     

Ultrastructure of host tissues exposed to the calyx fluid of the parasitoid,Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae)

Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) is a solitary endoparasitoid of Heliothis virescens. The lateral oviducts of the female parasitoid contain a particulate suspension called calyx fluid. The particles in calyx fluid are a polydnavirus (CsV) which, when injected into last-instar H. virescens, stimulates degeneration of the host's prothoracic glands. In order to determine if CsV-induced degeneration is specific to prothoracic glands, last-instar H. virescens larvae were injected with C. sonorensis calyx fluid. After 4 days, a variety of host tissues were dissected from both calyx fluid-injected and uninjected control larvae and fixed for transmission electron microscopy. Prothoracic glands from injected larvae were ultrastructurally degenerated by 4 days post-injection, whereas control glands remained intact. Other tissues from calyx fluid-injected larvae (tracheal epithelia, corpora allata, Malpighian tubules, fat body, skeletal muscle, and the brain) showed no signs of ultrastructural degeneration or gross abnormalities as compared with control tissues. These observations suggested that CsV-induced degeneration is specific to the host's prothoracic glands.     

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.     

Parasitism-induced hemolymph polypeptides in Manduca sexta (L.) larvae parasitized by the braconid wasp Cotesia congregata (Say)

Parasitization of newly ecdysed third, fourth, or fifth instar Manduca sexta larvae by the gregarious braconid wasp Cotesia congregata induces synthesis of new hemolymph proteins in the host. Analysis of hemolymph from parasitized and unparasitized control larvae using SDS gel electrophoresis showed that a major 33 kd band, plus several minor bands, were synthesized in parasitized but not control larvae; autoradiograms of proteins labeled in vivo for 1 hr with [³⁵S]methionine indicated that synthesis of the 33 kd polypeptide began a few hours following oviposition by the wasp. Synthesis of the 33 kd parasitism-specific polypeptide was induced in unparasitized larvae by the injection of ovarian calyx fluid from adult female wasps; this fluid is known to contain two morphologically distinct types of virus particles that are normally injected into the host along with eggs during parasitization. Exposure of calyx fluid to psoralen in the presence of long-wave u.v. light destroyed its capacity to induce synthesis of the 33 kd protein, suggesting that synthesis of this polypeptide may be mediated by viral nucleic acid.     

Developmental interaction between suboptimal instars of Spodoptera littoralis (Lepidoptera:Noctuidae) and its parasitoid Microplitis rufiventris (Hymenoptera:Braconidae)

Relative effects of parasitism by Microplitis rufiventris on the development of the third instar Spodoptera littoralis (preferable, optimal host) with the development of penultimate (5th) and last (6th) instars (suboptimal hosts) were investigated. Newly molted 6th instar hosts were more acceptable for parasitization by the wasp female than older hosts. In singly parasitized 3rd instar hosts, 82.0 +/- 3.9% of the parasitoid eggs developed to full-grown instar wasp larvae. However, parasitoid eggs deposited singly in 73.9 +/- 3.3% of 5th and 100% of 6th instar hosts failed to develop. Superparasitization in the 3rd instar hosts reduced the production of pseudoparasitized larvae and, conversely, all parasitized hosts yielded viable parasitoid offspring. In suboptimal hosts, the development interaction between the parasitoid and its host larvae was highly influenced by the age of hosts at parasitism, load of deposited eggs, and other parasitoid factors. The latter factors, e.g., mainly calyx fluid particles, might be involved in establishing parasitoid eggs in the suboptimal hosts. In the last two host instars, superparasitization significantly increased the number of parasitoid larvae successfully reaching their final instar. Variation in host quality, e.g., physiological status, might be attributed, in part, to the partial breakdown of the solitary habit observed in the earlier instars. More parasitoid eggs developed to mature parasitoid larvae in hosts superparasitized as 6th instar than parasitoid eggs laid in 5th instar hosts. Superparasitization significantly lengthened the developmental period of 5th and 6th host instars and inhibited their development to the pupal stage. Studying parasitoid development in suboptimal instars of its habitual host provided physiological insight, as shown here. The results may have implication for biological control and in vitro mass rearing programs with solitary parasitoids.     

Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus

Campoletis sonorensis is a habitual parasitoid of 3rd-instar larvae of Heliothis virescens. C. sonorensis eggs and small glass rods were encapsulated in 5th-instar host larvae implanted in the absence of wasp calyx fluid; prior injection of calyx fluid into larvae suppressed the encapsulation response. Within 8 h of calyx fluid injection there was a removal of approx. 75% of the circulating capsule-forming haemocytes (plasmatocytes). The remaining subpopulation of plasmatocytes, in addition to being incapable of encapsulating targets in vivo, spread at a significantly reduced rate in vitro. Identical changes in plasmatocyte count and behaviour were observed after injection of virus purified from calyx fluid. Additionally, the activity of calyx fluid was abolished after ultraviolet irradiation. The onset of haemocytic abnormalities occurred more rapidly after natural parasitism of 3rd-instar host larvae. The cell-free haemolymph of calyx fluid-injected 5th-instar larvae also retarded the spreading of plasmatocytes from non-injected control larvae in vitro. We conclude that the abnormalities induced in H. virescens plasmatocytes by C. sonorensis virus contribute to the suppression of encapsulation.     

The rôle of the calyx and poison gland of Cardiochiles nigriceps in the host-parasitoid relationship

Parasitism of Heliothis virescens by Cardiochiles nigriceps reduced the growth of the host. Both the poison gland and the calyx of the female parasitoid were important in reducing the growth of the parasitized host. Injections of poison gland contents (0·04 gl/larva) or calyx fluid (0·04 gl/larva) into H. virescens larvae did not affect their growth. However, a mixture of the two glands (1:1) at this low dosage significantly reduced the weight gained by Heliothis larvae.     

Effect of a virus associated with the reproductive system of the parasitoid wasp,Campoletis sonorensis,on host weight gain

The particulate fraction of the calyx fluid of the endoparasitoid, Campoletis sonorensis, reduces host weight gain when manually injected into healthy Heliothis virescens larvae. Reduced weight gain of the host, H. virescens, is normally associated with parasitism by C. sonorensis. Electron microscopy has confirmed that the particulate fraction of the calyx fluid is composed of virus particles and it appears that this virus, injected with the egg at oviposition, actually reduces host weight gain. The effect of the virus is negated when the calyx fluid is exposed to ultraviolet light prior to injection. Furthermore, the calyx fluid is effective only if injected into hosts; there is no effect on host weight gain when hosts are fed or topically treated with the virus-containing calyx fluid.     

中红侧沟茧蜂雌蜂输卵管萼中病毒样纤丝的特征和功能

中红侧沟茧蜂Microplitis mediator雌蜂输卵管萼中有一种病毒样纤丝（virus-like filaments, VLFs）。在蜂卵从卵巢管通过输卵管萼产出的过程中,VLF包裹在蜂卵的表面,随蜂卵进入寄主体内。透射电镜显示,VLF中心是电子致密物质,外有单层膜包被,直径约35 nm。负染技术表明,VLF是具有左螺旋结构的纤丝,负染时的直径约25 nm。不含VLF的蜂卵进入3龄初寄主后,全部被寄主血细胞包囊;含有VLF的蜂卵进入同样的寄主后,88.2%受到保护。VLF对蜂卵的保护作用在不同发育期的寄主中不同,在3龄初的粘虫体内,平均有64.7%的初产卵不被包囊,而在4龄初的粘虫体内,只有9.5%的初产卵受到保护。这一结果说明,VLF只能为蜂卵提供部分的保护作用,需配合其它寄生蜂因子（萼液、蜂毒等）共同作用于寄主的免疫系统。     

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.     

Biosteres longicaudatus: Developmental dependence on host (Anastrepha suspensa) physiology

Larvae of Anastrepha suspensa that were in the first day of the third instar were parasitized by females of the solitary endoparasitoid, Biosteres longicaudatus. At the end of the 6-hr oviposition period, larvae were ligated posterior to the ring gland so that some larvae had parasitoids anterior to the ligature while in others, the parasitoids were in the abdomen, posterior to the ligature. Ninety-two percent of the parasitoids anterior to the ligature hatched to the first through third instars. Parasitoids posterior to the ligature had a 75% egg hatch to the first instar only. No larval molts to the second or subsequent instars occurred in these parasitoids. Upon parabiosis to 3-day-old, unparasitized host pupae, the ligated larvae pupated and 97% of the first-instar parasitoids in these parabiosed larval abdomens molted to the second instar. Newly laid parasitoid eggs transplanted to 3-day-old pupal hosts had less than one-third of the egg hatch of those transplanted to first-day third-instar hosts. The data implicate the physiological state of the host (vis-a-vis pupation and associated events) as being an important factor in the development of the endoparasitoid.     

Interference with function of plasmatocytes of Heliothis virescens in vivo by calyx fluid of the parasitoid Campoletis sonorensis

Summary Immature stages of the ichneumonid parasitoid, Campoletis sonorensis, develop within the haemocoel of its noctuid host, Heliothis virescens. The host cannot encapsulate the parasitoid egg owing to the suppressive effect of the polydnavirus-laden calyx fluid injected by the female parasitoid during oviposition. We have examined the effects of injection of calyx fluid on the following haemocytic manifestations of the immune system of 5th-instar larvae of H. virescens: encapsulation, nodulation, phagocytosis, erythrocyte rosetting and coagulation. Of these phenomena, only those requiring the formation of a multicellular sheath of plasmatocytes were affected. In general, encapsulation was fully suppressed; all of the C. sonorensis eggs and most of the glass rods implanted as targets were devoid of attached haemocytes 3 days after implantation although a few of the latter were coated by a sparsely distributed layer of granulocytes. Plasmatocytes also appeared to be present in thicker depositions of haemocytes. In nodulation, only the second, encapsulation-like phase was inhibited. The resistant first stage, involving the entrapment of particles by haemocytes, only resulted in the formation of amorphous, disorganized nodules. Granulocyte-dependent aspects of the immune system (phagocytosis, rosetting and possibly coagulation and the first stage of encapsulation and nodulation) occurred normally. The data suggest that in 5th-instar hosts injection of calyx fluid acts specifically on plasmatocyte function.