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.     

Influence of the parasitoid Chelonus inanitus and its polydnavirus on host nutritional physiology and implications for parasitoid development

Chelonus inanitus is a solitary egg-larval endoparasitoid, which feeds on host haemolymph during its internal phase. Parasitization induces in the host Spodoptera littoralis a precocious onset of metamorphosis and a developmental arrest in the prepupal stage. At this stage the parasitoid larva emerges from the host and consumes it. We show here that parasitization and the co-injected polydnaviruses affect the nutritional physiology of the host mainly in the last larval instar. Polydnaviruses cause a reduced uptake of food and an increase in the concentration of free sugars in the haemolymph and of glycogen in whole body. The parasitoid larva, along with polydnaviruses, causes a reduction of proteins in the host's plasma and an accumulation of lipids in whole body. Dilution of host haemolymph led to a reduced concentration of lipid in parasitoid larvae and a reduced survival rate. Thus, a sufficient concentration of nutrients in the host's haemolymph appears to be crucial for successful parasitoid development. Altogether, the data show that the parasitoid and the polydnavirus differentially influence host nutritional physiology and that the accumulated lipids and glycogen are taken up by the parasitoid in its haematophagous stage as well as through the subsequent external host feeding.     

Changes in the haemolymph proteome of Spodoptera littoralis induced by the parasitoid Chelonus inanitus or its polydnavirus and physiological implications

The egg-larval parasitoid Chelonus inanitus induces in its host Spodoptera littoralis two major developmental effects, namely a precocious onset of metamorphosis followed by a developmental arrest in the prepupal stage. Along with each egg, the wasp injects polydnavirus and venom into the host egg. The polydnavirus has been shown to play a major role in inducing the developmental arrest while the parasitoid larva is instrumental in inducing the precocious onset of metamorphosis. Here we report that experimental dilution of haemolymph of polydnavirus-containing larvae can partially prevent the developmental arrest while injection of native, but not of heat-treated, haemolymph or plasma from polydnavirus-containing larvae into nonparasitized larvae could induce developmental arrest in 14-15% of the larvae. This illustrates that heat-labile factors present in haemolymph play a role in causing developmental arrest. Injection of parasitoid medium increased the proportion of larvae entering metamorphosis precociously while injection of antibodies against a parasitoid-released protein had the opposite effect; this indicates that this protein and possibly other parasitoid-released substances are involved in inducing the precocious onset of metamorphosis. Analysis of the plasma proteome of nonparasitized, parasitized and polydnavirus-containing larvae revealed that the developmental effects are associated with only minor differences: eleven low abundant viral or virus-induced proteins and five parasitoid-released proteins were seen at specific stages of the host.     

Titres of juvenile hormone I,II and III in Spodoptera littoralis (Noctuidae) from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus (Braconidae)

Physico-chemical analysis of juvenile hormones (JHs) of Spodoptera littoralis revealed highest quantities in the second half of embryonic development and in newly hatched 1st instar larvae. At these stages, mostly JH II, JH I and little JH III were found, while in later stages only JH II and JH III were found. Titres fluctuated in a similar manner in all larval instars, being lowest during the moults. In last (=6th) instar larvae, JHs disappeared in the late feeding-digging stage and again increased in the early prepupal stage. Parasitisation with Chelonus inanitus, a solitary egg-larval parasitoid which induces in its host the precocious onset of metamorphosis in the 5th instar, did not alter JH homologue composition but led to a disappearance of JHs in the 5th instar. Implantation of a parasitoid larva into early 5th instar larvae containing polydnavirus/venom caused a drop in the JH titre which indicates that the parasitoid larva plays an important role in the manipulation of the host's JH titre. In the parasitoid larva, only JH III was found; titres were highest in the 2nd larval instar, a stage when the host is in the 5th instar and contains almost no JHs. Thus, JHs of the parasitoid and the host fluctuate in an independent manner.     

Characterization of a 212kD protein,released into the host by the larva of the endoparasitoid Chelonus inanitus (Hymenoptera,Braconidae)

Previous studies have shown that the larva of Chelonus inanitus plays an essential role in inducing the precocious onset of metamorphosis in polydnavirus/venom-containing Spodoptera littoralis, and that this effect might be due to proteins released by the parasitoid larva into the host. Here we show that a 212kD protein is the predominant protein released in vitro by late first instar parasitoids. This protein does not accumulate anywhere in the parasitoid body. Polyclonal antibodies specific for the 212kD protein were produced and immunoblot analyses revealed that this protein begins to be released by parasitoid larvae when the precocious onset of metamorphosis is induced in the host. Amino acid sequences of the N-terminus and of internal fragments of the 212kD protein were determined and glycoprotein detection showed that this protein is glycosylated. The availability of specific antibodies and the amino acid sequence information should now allow us to clone and express the 212kD protein and to directly study its functional significance.     

Stage-dependent strategies of host invasion in the egg-larval parasitoid Chelonus inanitus

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid which lays its eggs into eggs of Spodoptera littoralis (Noctuidae); the parasitoid larva then develops in the haemocoel of the host larva. Host embryonic development lasts approx. 3.5 days while parasitoid embryonic development lasts approx. 16 h. All stages of host eggs can be successfully parasitized, and we show here that either the parasitoid larva or the wasp assures that the larva eventually is located in the host's haemocoel. (1) When freshly laid eggs, up to almost 1-day-old, are parasitized, the parasitoid hatches while still in the yolk and enters the host either after waiting or immediately through the dorsal opening. (2) When 1-2-day-old eggs are parasitized, the host embryo has accomplished final dorsal closure and is covered by an embryonic cuticle when the parasitoid hatches; in this case the parasitoid larva bores with its moving abdominal tip into the host. (3) When 2.5-3.5-day-old eggs are parasitized, the wasp oviposits directly into the haemocoel of the host embryo; from day 2 to 2.5 the embryo is still very small and the wasps, after probing, often restrain from oviposition for a few hours.     

Analysis of endoparasitoid-released proteins and their effects on host development in the system Chelonus inanitus (Braconidae)-Spodoptera littoralis (Noctuidae)

Having shown earlier that the larva of C. inanitus is essential in inducing the precocious onset of metamorphosis in polydnavirus/venom containing S. littoralis, we here analysed release of proteins by parasitoid larvae and their effects on host development. Parasitoid larvae released proteins in vivo and in vitro in a stage dependent manner. An approximately 212 kD protein was released from the mid 1st instar onwards and additional smaller proteins could be associated mainly with the 2nd and 3rd instar. When parasitoids were implanted into S. littoralis larvae, parasitoid-released proteins were seen 6 hr later. When parasitoids were removed from hosts, parasitoid-released proteins persisted in the host haemolymph for some time. Injection of antiserum against parasitoid-released proteins after removal of the parasitoid larva accelerated the disappearance of the 212 kD protein and reduced the number of larvae entering metamorphosis precociously. Repeated injections of concentrated parasitoid medium into polydnavirus/venom containing larvae caused a reduction of the head capsule width and formation of miniature 6th instar larvae; this effect was not seen in the absence of polydnavirus/venom. These observations suggest that proteins released by the parasitoid might play a role in modifying host metamorphosis in the presence of polydnavirus/venom, and the temporal appearance of the 212 kD protein makes it the most interesting candidate for being involved in such an effect.     

Parasitoid polydnaviruses: evolution,pathology and applications

One of the more unusual groups of insect pathogens consists of members of the family Polydnaviridae, insect DNA viruses that live in mutual symbioses with their associated parasitoid wasp (Hymentoptera) carriers until they are injected into specific lepidopteran hosts. Once inside this secondary host, polydnaviruses cause a wide variety of negative effects that ultimately ensure the survival of the parasitoid larvae. Because of their unusual life strategy and genetic features, it had been difficult to fully characterise polydnaviruses in terms of evolutionary history, replication cycle and functions in the host that might normally be well characterised for more conventional viruses. Recently, our understanding of polydnavirus evolutionary origins, gene content, genome organisation and functions in parasitism has greatly increased. Key findings are summarised in this review with emphasis on evolution of polydnavirus genes and genomes, their functional roles in insect pathology and their potential applications in insect biological control and biotechnology.     

Isolation of an imaginal disc growth factor homologue from Pieris rapae and its expression following parasitization by Cotesia rubecula

Endoparasitoid insects introduce maternal factors into the body of their host at oviposition to suppress cellular defences for the protection of the developing parasitoid. We have shown that transient expression of polydnavirus genes from a hymenopteran parasitoid Cotesia rubecula (CrPDV) is responsible for the inactivation of hemocytes from the lepidopteran host Pieris rapae. Since the observed downregulation of CrPDV genes in infected host tissues is not due to cis-regulatory elements at the CrV1 gene locus, we speculated that the termination of CrPDV gene expression may be due to cellular inactivation caused by the CrV1-mediated immune suppression of infected tissues. To test this assumption, we isolated an imaginal disc growth factor (IDGF) that is expressed in fat body and hemocytes, the target of viral infection and expression of CrPDV genes. Time-course experiments showed that the level of P. rapae IDGF is not affected by parasitization and polydnavirus infection. However, the amount of highly expressed genes, such as storage proteins, arylphorin and lipophorin, are significantly reduced following parasitization.     

Physiological and endocrine changes associated with polydnavirus/venom in the parasitoid-host system Chelonus inanitus-Spodoptera littoralis

As shown earlier, parasitization by the egg-larval parasitoid C. inanitus causes in its host the precocious onset of metamorphosis in the 5th instar followed by developmental arrest in the prepupal stage. Polydnavirus/venom were shown to be responsible for the developmental arrest. We investigated how polydnavirus/venom affect growth of the host larvae and found that head capsule widths were smaller from the 4th to 6th stadium and weights were lower in the 6th stadium in polydnavirus/venom-containing larvae than in non-parasitized larvae. In an attempt to identify endocrine parameters that are modified by polydnavirus/venom and might be responsible for the developmental arrest in the prepupa, we compared juvenile hormones, juvenile hormone esterase and ecdysteroids between non-parasitized and polydnavirus/venom-containing larvae from the 4th instar until pupation or developmental arrest, respectively. Obvious differences became manifest only in the 6th instar at the pupal cell formation stage, i.e. 12 days after entry of polydnavirus/venom into the host egg. Then, prothoracic glands of polydnavirus/venom-containing larvae released less ecdysteroids and ecdysteroid titres were lower than in non-parasitized larvae; this was followed by a delayed, reduced and desynchronized increase in prepupal juvenile hormones and juvenile hormone esterase and a slightly modified metabolism of ecdysone. This indicates that polydnavirus/venom affects the endocrine system of the host only after pupal commitment and that inhibition of prothoracic gland activity is the first detectable effect.     

Viral cystatin evolution and three-dimensional structure modelling: A case of directional selection acting on a viral protein involved in a host-parasitoid interaction

Background  

In pathogens, certain genes encoding proteins that directly interact with host defences coevolve with their host and are subject to positive selection. In the lepidopteran host-wasp parasitoid system, one of the most original strategies developed by the wasps to defeat host defences is the injection of a symbiotic polydnavirus at the same time as the wasp eggs. The virus is essential for wasp parasitism success since viral gene expression alters the immune system and development of the host. As a wasp mutualist symbiont, the virus is expected to exhibit a reduction in genome complexity and evolve under wasp phyletic constraints. However, as a lepidopteran host pathogenic symbiont, the virus is likely undergoing strong selective pressures for the acquisition of new functions by gene acquisition or duplication. To understand the constraints imposed by this particular system on virus evolution, we studied a polydnavirus gene family encoding cyteine protease inhibitors of the cystatin superfamily.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Characterization of a cDNA encoding a putative chitinase from teratocytes of the endoparasitoid Toxoneuron nigriceps

Females of insect endoparasitoids inject substances along with the egg at oviposition, which can regulate or induce changes in a number of host physiological processes to benefit the developing parasitoid. These changes can be caused by substances such as venoms, calyx fluids, or symbiotic‐associated virus particles (polydnavirus), which are injected by females along with their eggs, and by substances secreted by parasitoid‐derived tissues (teratocytes) or the developing parasitoid larvae. Teratocytes (dissociated cells of the serosal membrane after parasitoid eclosion) release substances that have roles (i) in parasitoid nutrition, (ii) in the digestion of host tissues, and (iii) in the regulation of host development. Teratocytes of Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) have been implicated in the arrestment of the host development and in the regulation of circulating levels of host ecdysteroids. Here we describe the cDNA of a teratocyte‐secreted chitinase and its expression during parasitoid development, and discuss its putative role in this host–parasitoid association.     

寄生蜂多分DNA病毒的基因表达产物及对寄主昆虫的生理作用

最近有关由多分DNA病毒介导的寄生蜂与寄主关系的研究主要集中在多分DNA病毒基因的表达和伴随寄主生理功能失常的分子机理上 ,本文介绍在寄生蜂作用于寄主时一些重要的PDV基因表达产物 ,包括CsPDV ,MdPDV ,CcPDV ,TrPDV ,HdPDV ,CrPDV和其它PDV的基因表达产物 ,并简叙这些基因表达产物对寄主的生理影响。     

Cardiochiles nigriceps polydnavirus: molecular characterization and gene expression in parasitized Heliothis virescens larvae

Cardiochiles nigriceps Viereck is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.). This hymenopteran parasitoid, belonging to the family Braconidae, is associated with a polydnavirus (CnPDV), injected at oviposition along with the egg. The infection of various tissues by CnPDV determines the suppression of the host immune system and the developmental arrest of mature host larvae. In this study, CnPDV has been characterized at the structural and molecular level. The negatively stained nucleocapsids show evident ‘end structures’ and a tail-like appendage. The CnPDV genome is typically segmented, with circular dsDNA molecules, ranging in size from 2.5 kb to more than 23 kb. The early expression pattern of CnPDV in parasitized hosts has been analysed and viral clones, genomic and cDNAs, identifying genes expressed within 48 h after parasitization have been isolated. The molecular organization of one of these genes, named CnPDV1, and its putative protein product have been determined. Significant sequence homologies with other known proteins were not detected. In situ hybridization experiments indicated that this gene is expressed in the prothoracic glands of parasitized host mature larvae. A functional analysis of CnPDV1 gene product is required to assess its possible role in the regulation of parasitoid-induced alterations of host larvae.     

Physiological and molecular interaction in the host-parasitoid system Heliothis virescens-Toxoneuron nigriceps: current status and future perspectives

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is an endophagous parasitoid of the tobacco budworm Heliothis virescens (F.) (Lepidoptera, Noctuidae). Parasitized H. virescens larvae are developmentally arrested and show a complex array of pathological symptoms ranging from the suppression of the immune response to an alteration of ecdysone biosynthesis and metabolism. Most of these pathological syndromes are induced by the polydnavirus associated with T. nigriceps (TnBV). An overview of our recent research work on this system is described herein. The mechanisms involved in the disruption of the host hormonal balance have been further investigated, allowing to better define the physiological model previously proposed. A functional genomic approach has been undertaken to identify TnBV genes expressed in the host and to assess their role in the major parasitoid-induced pathologies. Some TnBV genes cloned so far are novel and do not show any similarity with genes already available in genomic databases, while others code for proteins having conserved domains, such as aspartic proteases and tyrosine phosphatases. Sequencing of the entire TnBV genome is in progress and will considerably contribute to the understanding of the molecular bases of parasitoid-induced host alterations.     

The UGA-CiE1 cell line from Chrysodeixis includens exhibits characteristics of granulocytes and is permissive to infection by two viruses

The soybean looper, Chrysodeixis (Pseudoplusia) includens (Lepidoptera: Noctuidae) is an economically important insect pest and a highly permissive host for the parasitoid Microplitis demolitor and its associated polydnavirus M. demolitor bracovirus (MdBV). Here we established a cell line from C. includens embryos designated UGA-CiE1 cells. CiE1 cells morphologically resemble granulocytes, which are a subpopulation of C. includens hemocytes. Antibody and RT-PCR analyses indicated that CiE1 cells express several molecular and functional markers that identify granulocytes. We further determined that CiE1 cells are permissive to infection by MdBV, exhibiting alterations very similar to MdBV-infected granulocytes, and Autographa californica multiple nucleopolyhedrosis virus (AcMNPV). Combined with the ability to transfect CiE1 cells with high efficiency and knock down expression of viral genes by RNA interference, we conclude this cell line has several attributes of value for studying immune interactions with polydnaviruses and potentially other pathogens.