Diseased Larvae Research Articles

Alternative Routes for the Horizontal Transmission of a Nucleopolyhedrovirus

Two alternative routes for nucleopolyhedrovirus transmission were investigated usingMamestra brassicaelarvae: cannibalism of infected larvae and the release of virus prior to the death of the diseased host. The low frequency of cannibalism observed suggests that this is not a relevant transmission route in this species. However, larvae were shown to transfer viable virus to the environment before death through either defecation or regurgitation. Over 40% mortality was observed in larvae reared in diet containers where infected hosts had previously fed. Virus transfer to cabbages also occurred before death of the infected host, as revealed by plant bioassay. Virus was not detected in the feces or regurgitate of diseased larvae until the late stages of infection.

Biological Control of the Potato Tuber Moth (Phthorimaea operculella Zeller) in the Republic of Yemen Using Granulosis Virus: Biochemical Characterization, Pathogenicity and Stability of the Virus

A granulosis virus infecting the potato tuber moth, Phthorimaea operculella, has been identified, isolated and purified from diseased potato tuber moth larvae collected from potato fields in the highland basin Qa al-Boun of the Republic of Yemen. The granulosis virus was propagated by feeding potato tuber moth larvae with potatoes treated with a concentration of one granulosis virus-infected fourth instar larva to 5l of water (corresponding to an occlusion body (OB) concentration of 2 106 OB ml-1). Restriction enzyme analysis of viral DNA revealed that the isolated virus seemed to correspond to an isolate from the Lima region of Peru. A median LC 50 value of 7.3 104 OB ml-1 was calculated for a purified virus preparation. Different preparations of the granulosis virus were investigated for their persistence in the field on tubers and leaves. A purified virus preparation (PoGV) applied to potato tubers and exposed in the open had a half-life of 1.3 days. On leaves, the activity of granulosis virus spray de...

Prey selection and baculovirus dissemination by carabid predators of Lepidoptera

Abstract. The interaction between coleopteran predators and baculovirus‐infected larvae was studied in the laboratory and the field in order to assess the potential role of predators in the dissemination of a nucleopolyhedrovirus (NPV). Preference tests using three carabid species, Harpalus rufipes De Geer, Pterostichus melanarius Illiger and Agonum dorsale Pont, showed no evidence of discrimination between healthy and diseased larvae of the cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae) as prey items. Virus infectivity was maintained after passage through the predator's gut. NPV mortality ranged from 97% to 20% when test larvae were exposed to faeces collected immediately after and 15 days post‐infected meal respectively. The potential for transfer of inoculum in the environment was estimated in the laboratory by soil bioassay. Carabids continuously passed infective virus to the soil for at least 15 days after feeding on infected larvae. Field experiments showed that carabids which had previously fed on diseased larvae transferred sufficient virus to the soil to cause low levels of mortality in larval populations of the cabbage moth at different instars.

Isolation of Multiple Subspecies of Bacillus thuringiensis from a Population of the European Sunflower Moth, Homoeosoma nebulella

Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of H. nebulella showed that the isolates of B. thuringiensis subspp. aizawai, kurstaki, and thuringiensis were the most toxic, with 50% lethal concentrations (LC(inf50)s) of 0.15, 0.17, and 0.26 (mu)g/ml, respectively. The isolates of B. thuringiensis subspp. morrisoni and thompsoni had LC(inf50)s of 2.62 and 37.5 (mu)g/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of B. thuringiensis producing different insecticidal proteins.

Characterization and partial cloning of the genomic DNA of a baculovirus from Penaeus japonicus (PjNOB = BMNV)

Many particles of a non-occluded baculovirus with a membrane 70 × 330 nm in size were observed in the nuclei of the mid-gut glands of larval Kuruma shrimp ( Penaeus japonicus) with mid-gut necrosis using an electron microscope. Nucleotides of the baculovirus were isolated from the diseased larvae in the presence of Triton X-100. The nucleocapsid was a bacilliform virion, approximately 50 nm in width and 260 nm in length. From the purified preparations of nucleocapsids of the virus, two major proteins with molecular weights of 35 and 14 kDa were identified by SDS-polyacrylamide gel electrophoretic analysis. The viral genomic DNA extracted from the nucleocapsids was digested with BamHI and Sau3AI, but not with EcoRI, PstI, XhoI and SalI. The molecular weight of the DNA was estimated to be approximately 85.1 × 10 6 from the agarose gel electrophoretic pattern of the BamHI fragments. A part of the BamHI fragments was cloned, labeled with 32P-CTP and used for Southern blot analysis as a probe. The fragment hybridized to DNA isolated from the diseased larvae, but not to DNA isolated from healthy larvae.

Amber Disease Reduces Trypsin Activity in Midgut of Costeltyra zealandica (Coleoptera; Scarabaeidae) Larvae

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.

Vibriosis of Swimming Crab Portunus trituberculatus in Larviculture

AbstractSince 1985, frequent mass mortalities due to a bacterial infection have occurred in zoeal larvae of swimming crab Portunus trituberculatus reared at the Tamano Station of the Japan Sea‐Farming Association. A bacterium was predominantly isolated from diseased larvae and was demonstrated by experimental infection to be the causative agent of the disease. Based on morphological, physiological, biochemical, serological and genetic characteristics of the pathogen, it was thought to be a new species and tentatively named Vibrio sp. Zoea. Challenge experiments with the bacterium in several aquatic animals indicate that Vibrio sp. Zoea appears to be a pathogen specific for crustaceans.

Pathogenicity of the causative agent of viral nervous necrosis disease in striped jack, Pseudocaranx dentex (Bloch & Schneider)

Abstract. The pathogenicity of the agent causing viral nervous necrosis (VNN) of striped jack, Pseudocaranx dentex (Bloch & Schneider), was examined in striped jack and other selected marine fish species. Fish were exposed to purified striped jack nervous necrosis virus (SJNNV) (0·1–100 ng ml−1) or homogenates of diseased striped jack larvae. Striped jack larvae (3·5 and 4·4 mm total length) were susceptible to the virus, but juveniles (78 mm) were not. The viral antigens were detected by indirect ELISA and the characteristic pathological changes, i.e. vacuolation in the retina and brain, were reproduced in the affected larvae. The infection was also established in healthy larvae by cohabitation with the diseased larvae. Larvae of red sea bream, Pagrus major Temminck & Schlegel, yellowtail, Seriola quinqueradiata Temminck & Schlegel, and goldstriped amberjack, Seriola lalandi Valenciennes, were not susceptible to SJNNV.

Isolation of Bacillus thuringiensis subsp. israelensis from diseased field-collected larvae of the saturniid moth, Hylesia metabus, in French Guiana

Isolation of Bacillus thuringiensis subsp. israelensis from diseased field-collected larvae of the saturniid moth, Hylesia metabus, in French Guiana

Mechanisms of Transmission of the Gypsy Moth (Lepidoptera: Lymantriidae) Fungus, Entomophaga maimaiga (Entomphthorales: Entomophthoraceae) and Effects of Site Conditions on Its Prevalence

Sampling for the gypsy moth fungus, Entomophaga maimaiga, was carried out in 10 plots in Connecticut to determine the importance of overwintering fungal load, rainfall, gypsy moth abundance, and physiographic site conditions to fungal prevalence. At one of the plots, gypsy moths were also exposed daily in cages on the soil and at 2 m in shrubs to examine the pathogen transmission potential of germinating resting spores and of conidia produced by diseased larvae. Gypsy moth population levels and an index of site moisture (as determined by tree composition) were marginally related to fungus prevalence. However, the relationship between fungal prevalence and population was an inverse one, opposite that found in a previous study. Resting spore load did not appear to affect prevalence; it is possible that a threshold effect occurs. Daily sampling showed that germination of resting Spores is necessary for infection of early-stage larvae, but that conidia produced from diseased larvae are mainly responsible for infection of late-stage caterpillars. Maximum germination of resting spores occurred 1 or 2 d after significant precipitation, whereas the greatest production of conidia from diseased caterpillars took place on the day of rainfall. We hypothesize that in areas where overwintering resting spores are abundant, the most important determinant of fungal effectiveness will be rainfall amount and pattern.

Movement of Nuclear Polyhedrosis Virus into Velvetbean Caterpillar (Lepidoptera: Noctuidae) Larval Populations on Soybean by Nabis roseipennis (Heteroptera: Nabidae) Nymphs

Nabis roseipennis Reuter nymphs that preyed on larvae of the nuclear polyhedrosis virus (NPV) infected velvetbean caterpillar, Anticarsia gemmatalis (Hübner), excreted the virus (AgNPV) for several days thereafter. Based on bioassays, fifth instar and second instar nymphs excreted 84.7 × 105 and 9.7 × 105 polyhedral inclusion bodies (PIB) per nymph, respectively. The AgNPV-contaminated nymphs effectively disseminated the virus via the feces over soybean plants where it served as inoculum to initiate disease in larval populations of A. gemmatalis caged in the field. Larval mortality from AgNPV ranged from 11.4 to 48.5% over treatments in two tests. Larval mortality in treatments where the source of virus inoculum was AgNPV-contaminated fifth instar nymphs was similar to that in treatments where the source of viral inoculum was diseased larvae. Larval mortality resulting from AgNPV dissemination by the nymphs was usually higher in treatments containing fifth instar nymphs than in those with second instar nymphs. Dissemination of NPV by fifth instar nymphs was higher in mixed-age than in uniformed-age A. gemmatalis larval populations. This was not the case with the smaller second instar nymphs.

Hirsutella stylophoraMains, a pathogen ofCostelytra zealandica(Coleoptera: Scarabaeidae) in New Zealand

The entomopathogenic fungus, Hirsutella stylophora, is described as a pathogen of larvae of the New Zealand grass grub, Costelytra zealandica, for the first time. This species is the anamorph of Cordyceps stylophora. Infected grass grub larvae were dull brown in colour, usually with a distinctive stromatal growth extending from the head capsule. Despite the frequent production of stromata on diseased larvae, no ascospores (sexual stage) were found. Collections of diseased C. zealandica were made from several sites in the southern half of the South Island.

Drosophila C virus cycle during the development of two Dorosphila melanogaster strains (Charolles and Champetie`res) after larval contamination by food

Dorosphila C virus (DCV) cycle during Drosophila melanogaster development was studied after feeding contamination at the first, most sensitive, instar (L1). Two Drosophila strains were examined and compared. Presence of DCV C in apparently healthy animals (L3 larvae bred on a contaminated rearing medium and adults coming from larvae which were grown on medium containing DCV C) was demonstrated by biological tests. Using the immunofluorescence technique, DCV was exhibited in the diseased Charolles larvae, in the lumen of the digestive tract and in the basal part of gut cells which is in contact with the haemolymph. On the contrary, in Charolles larvae which seemed ‘healthy’, DCV was exhibited only in the lumen of the digestive tract at the apical boundary of the gut cells. But DCV typical protein capsid was not shown in the tissue of Drosophila L3 and adults. However, C virus remained in Drosophila tissues even after host metamorphosis and would seem to interact with Drosophila cells. Hypotheses are proposed concerning the intracellular state of Drosophila C virus in this case.

Effects of an isolate of gypsy moth, Porthetria (Lymantria) dispar, nuclear polyhedrosis virus on tobacco hornworm, Manduca sexta, larvae

An isolate of the gypsy moth, Porthetria (Lymantria) dispar, nuclear polyhedrosis virus infected larvae of the tobacco hornworm, Manduca sexta, when it was fed to first instar (neonate) individuals. The virus caused unusually heavy proliferation of cytoplasmic polyhedra in the midgut cells very soon after larvae began feeding and growth was markedly reduced. However, noticeable mortality did not occur until after 25 days when most of the control larvae had begun to pupate. About 65% of the treated larvae had died by day 31 and none had pupated. Electron micrographs showed both cytoplasmic and nuclear polyhedrosis virions in diseased larvae. Those in nuclei were rod-shaped (baculovirus) and they were sparsely distributed. Polyhedra in these nuclei were often poorly formed or absent.

Bioencapsulation of Therapeutic Quantities of the Antibacterial Romet‐30 in Nauplii of the Brine Shrimp Artemia and in the Nematode Panagrellus redivivus

AbstractNauplii of the brine shrimp, Artemia, and cultures of the soil nematode, Panagrellus redivivus, were fed a suspension of the water‐insoluble antibacterial Romet‐30 (sulfadimethoxine plus ormetoprim) to determine if they could be used as a mechanism for delivery of the drug to larval penaeid shrimp.Romet‐30 was added to groups of brine shrimp nauplii and nematodes in 1 L beakers to achieve a concentration of 3 mg of drug per ml of seawater. After a 4 h uptake period, predetermined numbers of organisms were assayed for antibiotic content using the micro‐plate diffusion method and a Romet‐30 sensitive strain of Vibrio alginolyticus as the indicator. The resulting zones of inhibition were compared to that of a standard Kirby‐Bauer sensitivity disc containing 25 μg of the drug that was placed on each plate as standard control. Negative controls consisted of equal numbers of nauplii and nematodes that were not exposed to the antibiotic. Artemia nauplii and nematodes accumulated approximately 0.1 μg of the drug per nauplius and approximately 0.25 μg per nematode. They exhibited no signs of toxicity from exposure to the drug and contained the antibiotic in sufficient quantity to provide a theoretical therapeutic dose of the drug when fed to penaeid larvae.In subsequent tests, normal and diseased larval Penaeus stylirostris readily accepted Romet‐30 enriched Artemia. Diseased larvae fed the drug in this manner exhibited a statistically greater overall survival and a greater survival to the post‐larval stage by Student's t‐test at the 5% significance level.

Nutritional requirements for extracellular accumulation of toxin produced by a pathogenic Vibrio sp.

Abstract. A Vibrio species isolated from hatchery‐reared, diseased oyster larvae, Crassostrea virginica Gmelin, was shown to produce a teratogenic metabolite when grown in a complex, undefined medium. Nutritional studies determined that the pathogen required several inorganic salts, glucose and asparagine for growth. Bioassays demonstrated that toxin did not accumulate in this chemically‐defined medium. However, toxin accumulation did occur when the medium was supplemented with hypoxanthine and either glutamic acid, histidine or thiosulphate.

Persistence and Movement of Nuclear Polyhedrosis Virus on Soybean Plants After Death of Infected Anticarsia gemmatalis (Lepidoptera: Noctuidae)

The persistence of a nuclear polyhedrosis virus (NPV) on soybean after death and lysis of NPV-killed (AgNPV) Anticarsia gemmatalis (Hubner) larvae on the plants was examined. Bioassays of virus on leaflets (which had no direct contact with the diseased larvae) collected from these plants showed that AgNPV activity persisted on the plants for the entire 7-wk test period, with up to 62.7% larval mortality in bioassays conducted after the seventh week. Regardless of the canopy location (higher or lower) of the virus-killed cadavers, AgNPV activity was higher on the leaflets collected from the lower canopy after 2 wk and remained so for the duration of the test. This may be related to movement of AgNPV into the lower canopy by rainfall and wind where it can be better shielded from sunlight inactivation by the foliage. A separate test was conducted in which uninfected larvae were caged for 5 d on soybeans at weekly intervals after death of AgNPV-infected larvae. The mean larval mortality during the 4-wk period of this test was higher in treatments in which AgNPV-killed cadavers had died in the upper canopy. This may be explained by the preference of the uninfected larvae for the upper canopy.

A Viral Disease in Larvae and Juveniles of the Japanese Flounder Paralichthys olivaceus

From 1985 to 1987, outbreaks of a disease resulting in mass mortality occurred in larvae and juveniles of the Japanese flounder Paralichthys olivaceus cultured at prefectural and private hatcheries in Hiroshima Prefecture, Japan. The disease occurred in 10-30-d-old fish that were reared at about 18–20°C, and mortality usually reached 80–90% in a few weeks. The affected fish had opaque fins and a hyperplastic epidermis on the fins and skin. Electron microscopy revealed hexagonal virus particles in the nucleus (100–140 nm in diameter without an envelope) and cytoplasm (190–230 nm with an envelope) of the affected epidermal cells. Although isolation of the causative agent by the use of five fish-cell cultures was not successful, the disease was transmitted to healthy larval flounder by exposing them to a 0.45-μm filtrate of diseased fish homogenate. The agent's morphological features and its sensitivity to ether, to a pH of 3, and to a 30min exposure to 50°C indicate it is a herpesvirus.

Nutritional physiology of the eastern spruce budworm, Choristoneura fumiferana, infected with Nosema fumiferanae, and interactions with dietary nitrogen.

Female eastern spruce budworm larvae, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae), inoculated with a medium lethal spore dosage of the microsporidium Nosema fumiferanae (Thomson) exhibited significant reductions in consumptive index (CI), nitrogen consumptive index (NCI), relative growth rate (RGR), and gross (ECI) and net (ECD) production effeciencies when compared to microsporidian-free larvae. Diseased larvae also exhibited significant increases in approximate digestibility (AD), N utilization efficiency (NUE), and larval moisture content. Both healthy and diseased insects were reared on 2.5% N and 4.5% N diets. Those on the 2.5% N diet showed significant increases in CI, although NCI was still lower than NCI measured for larvae reared on 4.5% N. NUE was also higher on the 2.5% N diet. Diseased cohorts reared on 2.5% N diet had significantly greater mortality than those reared on 4.5% N diet. Pupal weight and development time of infected individuals did not respond to dietary N concentration. However, healthy insects achieved greater pupal weights in a shorter time on the 4.5% N diet than those on the 2.5% N diet. Mortality of healthy insects was unaffected by dietary N.

Egg contamination as a method for the inoculative release of exotic microsporidia of the gypsy moth

Two species of microsporidia, a Nosema sp. and a Vavraia sp., obtained from gypsy moths in Portugal, were released in a central Maryland woodlot. Each woodlot had low but increasing gypsy moth populations. Both species of microsporidia were introduced by placing gypsy moth egg masses that were contaminated by microsporidian spores on trees centrally located in each woodlot. Three different sampling methods, one for early instar and two for late instar larvae, were used to determine the incidence of microsporidia infection and the extent of dispersal of diseased larvae from the woodlot center. The early larval samples had 10.5% Nosema sp. and 21.1% Varaia sp. infections. Infected larvae had dispersed throughout the sample area. The two late instar larval samples had infection rates of 6.3 and 7.8% for Nosema sp. and 9.4 and 16.9% for Vavraia sp. Late instar collections also indicated that infestations had spread throughout the sampling plots. Our studies indicate that contamination of egg masses is an acceptable technique for introducing these microsporidia into gypsy moth populations.