Donald B. Stoltz

Viruses and Parasitism in Insects

Publisher Summary This chapter focuses on viruses and parasitism in insects. In recent years, virus-like particles have been observed in the reproductive tracts of a large number of endoparasitic hymenoptera (i.e., wasps whose larvae develop inside host larvae), belonging to the families of braconidae and ichneumonidae. Some of these agents have now been characterized to the extent that one does not hesitate in referring to them as viruses, in which context they are discussed in the chapter; indeed, the most prevalent type of particle observed in braconid wasps appears to represent a highly unusual form of baculovirus. A unique feature of all parasitoid viruses, thus far discovered, is that they replicate only in the ovary (of wasps), from which they are transmitted to host larvae or eggs during oviposition. In affected species, large concentrations of virions can always be observed in either cell nuclei of the calyx, an epithelium situated between the ovarioles and oviduct, or within the lumen of calyces and oviducts. It is now apparent that the presence of virus particles in the ovaries of parasitoid wasps is not an isolatcd phenomenon. Indeed, it may represent the rule rather than the exception among certain groups of endoparasitic hymenoptera. The invariable presence of particulate calyx fluids in some parasitoid species suggests that these particles represent an integral component of the parasitoid life cycle.

Apparent haemocytic transformations associated with parasitoid-induced inhibition of immunity in Malacosoma disstria larvae

Abstract The icheneumonid wasp, Hyposoter fugitivus , is an habitual parasitoid of tent caterpillar ( Malacosoma disstria ) larvae. Successful parasitism is associated with an apparent behavioural transformation of certain host haemocytes, and is in addition characterized by a suppression of immunity to foreign objects such as parasitoid eggs, yeast, and Sephadex beads; it is suggested that transformation of haemocytes renders them incapable of participating in normal immune responses. Inhibition of some aspects of cellular immunity in host insects can also be induced by manual injection of a polydnavirus isolated from the ovaries of the parasitoid. It was observed that haemolymph from parasitized larvae does not melanize in vitro; this phenomenon may be associated with a greatly increased stability of oencytoids.

Venom promotes uncoating in vitro and persistence in vivo of DNA from a braconid polydnavirus

Summary Earlier studies have suggested that successful parasitism by certain braconid parasitoids may depend on the presence in host insect larvae of both polydnavirus and venom. We have shown that venom from the braconid parasitoid, Cotesia melanoscela, was required for in vivo persistence of polydnavirus DNA in host larvae. In parallel studies using an in vitro system, we observed that in the presence of venom nucleocapsids were released into the cytoplasm and subsequently uncoated at nuclear pores; in the absence of venom, this sequence of events was not observed.

Evidence for chromosomal transmission of polydnavirus DNA.

Heterogeneity in polydnavirus DNA was exploited as a means of following the transmission of viral genomes in parasitoid populations. Parasitoid lines isogenic for viral DNA markers were established from both a braconid (Cotesia melanoscela) and an ichneumonid (Hyposoter fugitivus) species. In crossing experiments these markers routinely segregated in Mendelian (chromosomal) fashion, suggesting that the structure of polydnavirus genomes is probably determined by the integrated form of viral DNA, rather than by extrachromosomal molecules.

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.

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.

Induction of a new haemolymph glycoprotein in larvae of permissive hosts parasitized by Campoletis sonorensis

Abstract Parasitization by Campoletis sonorensis consistently results in the appearance of a new polypeptide, a glycoprotein, in several habitual host species. This occurs prior to the hatching of parasitoid eggs, and can be duplicated by the injection of either calyx fluid or purified C. sonorensis virus. Oviposition in two non-permissive hosts. Anticarsia gemmatalis and Mamestra configurata, leaves haemolymph polypeptide profiles unchanged.

Virus-like particles in the ovary of an ichneumonid wasp: Purification and preliminary characterization

Virus-like particles have been isolated from the oviducts of a parasitoid wasp, Hyposoter exiguae (Hymenoptera: Ichneumonidae). Particles are readily purified by centrifugation on either Ficoll or sucrose gradients. Double-stranded circular DNA isolated from purified particles is heterodisperse in terms of molecular weight; none of the molecules are sufficiently large to code for the aggregate of structural proteins comprising the particles. Preliminary Southern blot hybridization data suggest that there is minimal sequence homology between the different size classes of DNA examined.

An Unusual Virus from the Parasitic Wasp Cotesia melanoscela

Certain strains of the braconid parasitoid Cotesia melanoscela carry two different viruses within their ovaries, one of which (here designated CmV2) is apparently not a polydnavirus. Virus replication occurs in the ovarian calyx and in some other tissues of both male and female parasitoids; as yet, no replication has been observed in the testis, however. In addition, CmV2 is one of only two parasitoid viruses known to replicate in host insect larvae, and we not show that this virus is also capable of replicating in vitro; the virus is nevertheless nonpathogenic for gypsy moth larvae. The virus is not transmissible per os, either to host animals or to larvae of parasitoid strains lacking it. CmV2 is stably maintained within strains carrying it apparently by a vertical transmission mode involving the maternal line; transmission via the male germ line could not be demonstrated. While purification of the virus was not achieved, preliminary work allows us to suggest the genome consists of a single double-stranded DNA molecule of approximately 125 kb.

Three related TrIV genes: comparative sequence analysis and expression in host larvae and Cf-124T cells.

We report on the cloning and sequencing of two Tranosema rostrale ichnovirus (TrIV) genes, and assess their relatedness to TrV1, the gene encoding the most abundant TrIV transcript in last-instar Choristoneura fumiferana larvae parasitized by T. rostrale. One of the two newly isolated genes, TrV2, features an organization similar to that of TrV1, with one intron flanked by two exons; it encodes a 102 amino acid protein showing 79% similarity to TrV1. The third gene, TrV4, encodes a larger protein (143 aa) displaying similarity to the other two only over the first approximately 50 amino acid residues of its sequence; the remaining portion contains an imperfect octad repeat. Although the TrV4 gene contains only one exon, it has an intron similar in size and sequence to that of TrV1 and TrV2; in fact, the non-coding regions of all three genes show higher sequence identity than the coding regions, pointing to their common origin. Southern analysis suggests that each gene maps to a different TrIV genome segment, with homologous sequences apparently present on other segments. TrV1 and TrV4 transcription in penultimate (5th) instar hosts, parasitized shortly after the molt, was strong for both genes 1 and 2 days p.p., with transcript abundance decreasing after the final molt; thus, neither of these genes is upregulated during induction of developmental arrest in last-instar hosts. Cf-124T cells inoculated with T. rostrale calyx fluid showed significant levels of apoptosis 24-72 h p.i.; TrV1 was detected in the culture medium, suggesting that this and/or other TrIV-encoded proteins may be responsible for the observed cytopathic effect. Southern and Northern analyses, using DNA and RNA extracted from infected Cf-124T cells, revealed the presence of both TrV1- and TrV4-carrying genome segments and transcripts, but neither DNA, at least in episomal form, nor mRNA persisted for more than a few days p.i. This in vitro system may provide a suitable starting point for the study of TrIV gene functions.