Intrahemocoelic Injection Research Articles

An insect cell line persistently infected with a baculovirus-like particle.

A persistent infection by a baculovirus-like particle was found in the established lepidopteran (Heliothis zea) cell line, IMC-HZ-1. The virus caused CPE in less than 1% of the IMC-HZ-1 cells, as measured by phase-contrast and electron microscopy. Transmission tests showed that four lepidopteran cell lines were susceptible to the persistent virus (designated as IMC-HZ-I-NOV). In inoculated TN-368 cell cultures, 90--100% infection was achieved. The ultrastructure and development of IMC-HZ-I-NOV in cell cultures were similar to known baculoviruses, and it is probable that this persistent virus is a member of the family Baculoviridae. Two lepidopterous species (Estigmene acrea and H. zea) inoculated with IMC-HZ-1-NOV by intrahemocoelic injection and/or per os feeding of larvae were not susceptible.

Host-parasite Interactions between Larvae of the Gypsy Moth, Lymantria dispar (L.) (Lepidoptera : Lymantridae) and the Entomogenous Fungus, Nomuraea rileyi (FARLOW) SAMSON (Moniliales : Moniliaceae)

Second-instar larvae of the gypsy moth, Lymantria dispar were exposed to sporulating cultures of Nomuraea rileyi and fixed for histopathogical examination to study the host-parasite relationship. Penetration of the cuticle occurred within 42 hours post-inoculation and by 68 hours, the interior of the host, including the gut was completely filled with hyphae. By 48 hours post-inoculation, 100% mortality was recorded in all the infected larvae. Toxin production by N. rileyi was observed and the extract of the mycelium of N. rileyi was applied to gypsy moth larvae via topical application and by intra-haemocoelic injection which produced high levels of mortality in the larvae.

Present Status of Milky Disease of Japanese and Oriental Beetles in Connecticut1

A survey of Japanese beetle, Papillia japonica Newman, and Oriental beetle, Anomala orientalis Waterhouse, grubs from 53 sites in the spring and 62 sites in the fall of 1974, showed that incidence of infection with Bacillus popilliae Dutky, causal agent of milky disease, was remarkably low in Connecticut. Laboratory tests conducted with B. popilliae from field collected Connecticut and New York grubs determined that spores were especially low in infectivity both by ingestion and by intrahemocoelic injection when compared to infection data from earlier published reports. A. orientalis and P. japonica seemed comparable in their sensitivity to B. papilliae by injection. Spores from diseased grubs collected at different locations in the state varied in their rates of infectivity.

Purification and properties of a nuclear polyhedrosis virus from Choristoneura fumiferana.

The polyhedral inclusion bodies of a nuclear polyhedrosis virus from the spruce budworm Choristoneura fumiferana were purified by fluorocarbon treatment, sucrose columns, sucrose density gradients, and equilibrium centrifugation. The preparation yields a single and homogeneous band in potassium tartrate gradients at a density of 1.193 g/ml. The virions were released from the polyhedra by alkali treatment and purified by either rate zonal or equilibrium centrifugation in sucrose gradients. The purified virions, which band at a density of 1.265-1.300 g/ml in sucrose, were infectious to the spruce budworm larvae and their mean LD50 was 1.09 +/- 0.1 mug of virus. However, viral nucleocapsids, released by a nonionic detergent, Nonidet P-40, had a density of 1.315 g/ml and were noninfectious by either per os inoculation or intrahemocoelic injection.

Milky disease (Bacillusspp.) occurrence and experimental infection in larvae ofCostelytra zealandicaand other Scarabaeidae

Milky disease was transmitted experimentally to larvae of Costelytra zealandica (White), Costleya (= Chlorochiton) suturalis Fabr., and Odontria striata White (Melolon‐thinae) by feeding and by intra‐haemocoelic injection of spores of Bacillus popilliae and two unnamed New Zealand Bacillus strains, and also to Pericoptus truncatus Fabr. (Dynastinae) by intrahaemocoelic injection and to C. zealandica by housing the larvae in soil containing the spores. The infection rate resulting from any of these methods rarely exceeded 5 %, and was usually much less. Monthly records from a 2‐acre (approx. 0.8 ha) field plot showed an initial infection rate of up to 62% in lst‐and 2nd‐instar C. zealandica larvae. Most of these were transient, non‐lethal infections which did not develop to a visibly milky condition. Evidence from field records and from the survival of diseased larvae held in the laboratory suggested a total mortality from milky disease in this population, throughout the season, of more than 20%. Records from 21 other sites are summarised.

Infection of the boll weevil by Chilo iridescent virus

When infections with Chilo iridescent virus (CIV) were induced in larvae of boll weevils, Anthonomus grandis, by intrahemocoelic injection or by feeding, and in adults by feeding, the typical blue coloration of adipose tissue developed at 3–7 days postinfection, and mortality occurred after 3 days. The symptomatology and the pathological expressions depended on the initial infectious titer. The virus remained viable when it was added to the feeding stimulant bait used to infect weevils with protozoan pathogens in the field, and weevils feeding on the formulation became infected when it had been exposed 1–3 days in nature.

Spontaneous formation of protoplasts by a species of Entomophthora

Conidia of a species of Entomophthora isolated from the eastern hemlock looper, Lambdina fiscellaria fiscellaria, were germinated in Grace's insect tissue culture medium. Two germ tubes were normally formed per conidium, and when these reached a length of approximately 100–150 μ, the protoplasm was spontaneously released from the tip of the germ tube as a viable protoplast. The subsequent development of the protoplast is described. Two-day-old protoplasts were highly pathogenic to a number of lepidopterous larvae upon intrahemocoelic injection, death of the insects ensuing within 4–5 days. The Entomophthora species could be reisolated from the killed insects.

Inducement of cytoplasmic polyhedrosis by the intrahemocoelic injection of freed viral particles into the silkworm, Bombyx mori

Cytoplasmic polyhedrosis was induced 1 1 In this paper the words “induce” and “inducement” are used in the general sense of causing to bring about, and not in the sense of meaning the activation of an occult virus or the provoked transformation of a provirus into a virulent virus, as defined by Steinhaus and Martignoni (1967). by the intrahemocoelic injection of freed viral particles into the silkworm, Bombyx mori. Examination of tissues of the infected insects revealed that formation of the typical tetragonal type of polyhedron was restricted to the gut epithelial cells. The LD 50's obtained with preparations of whole intact polyhedra and freed viral particles injected per os and into the hemocoel are compared.

Histochemical changes in Melolontha melolontha (Linnaeus) (Coleoptera) infected by a new virus.

Changes in DNA, RNA, protein, and glycogen in the fat body of a beetle, Melolontha melolontha (Linnaeus), were studied histochemically at intervals of 15, 23, 75, and 195 days after intrahemocoelic injection of a cytoplasmic polyhedrosis virus, Vagoiavirus melolonthae. As the disease progressed, the average nuclear diameter increased considerably without any apparent change in DNA content. Cytoplasmic RNA increased progressively for about 75 days and then decreased slightly. Nuclear protein content increased during the first 23 days after virus inoculation. Cytoplasmic protein content progressively increased throughout the disease process. There was no significant change in glycogen reserves 23 days after inoculation; thereafter, glycogen was drastically reduced.Three morphologically different types of cytoplasmic inclusions all staining intensely with naphthol yellow S (thus establishing their proteinaceous nature) were observed. The pyroninophilic "round" type is considered to be "mature" inclusions containing the virus particles demonstrated by electron microscopy. The resemblances between this virus and some viruses of higher animals are discussed.

Toxins from the entomogenous fungus Metarrhizium anisopliae: II. Symptoms and detection in moribund hosts

Intrahemocoelic injection of filtrates of Metarrhizium anisopliae cultures induced tetanic paralysis in larvae of Galleria mellonella and Bombyx mori. Bombyx mori larvae infected with M. anisopliae underwent tetanic paralysis immediately prior to death, and blood from insects heat-treated (56° to 58°C) while in this condition was toxic to Galleria larvae when administered intrahemocoelically. Thus, it is possible that toxins produced by the fungus play an important role in this mycosis.

Artificial infection of Aedes aegypti with semliki forest virus by intrahaemocoelic injection

A method is described for the intrahaemocoelic inoculation of Semliki Forest virus in the form of infected mouse brain suspension into Aedes aegypti. Infectivity titrations carried out on the insects at intervals after infection showed rapid rise in virus level in the first 24 hours. The raised virus titre was maintained for at least 18 days in one experiment, and was associated with biological transmission of the virus on the 4th, 10th and 18th days of the experiment.