A novel milky disease organism from Australian scarabaeids: Field occurrence,isolation, and infectivity

A novel milky disease organism has been found causing disease in Aphodius tasmaniae and other scarabaeid larvae in the field in Australia. The sporangium is exceptionally long, measuring 10.5 × 1.5 μm, with a small central spore, measuring 1.0 × 0.6 μm. The vegetative cell is about half the size of the sporangium. The disease was easily transmitted by injection of spores into the hemocoel, with typically milky symptoms developing in 2–4 weeks. Spores will form in vivo at temperatures down to 12°C. For A. tasmaniae third-instar larvae, the ID₅₀ by injection was 3 × 10² spores/larva, yet no infection resulted when larvae were reared in peat containing up to 10⁸ spores/g, i.e., the disease was not successfully transmitted per os. All 10 species of scarabaeids tested were susceptible to the disease when spores were injected; however, all attempts to infect larvae per os were unsuccessful. In vitro culture was also unsuccessful.     

Hemolymph responses in Heliothis zea to inoculation with Bacillus thuringiensis or Micrococcus lysodeikticus

Direct injection into the hemolymph of Heliothis zea of either an entomopathogen (Bacillus thuringiensis subsp. kurstaki) or a nonpathogen (Micrococcus lysodeikticus) is followed by a rapid phagocytosis and extensive removal of the organisms within 2 hr. The bacteria that survive this initial clearance initiate a new round of growth that is clearly evident 6–8 hr after injection. When the infecting organism is M. lysodeikticus, a second period of clearance occurs 8–12 hr after injection and nearly complete removal (many by lysis) is evident by the 12th hr. Larvae usually survive infection with this organism. When B. thuringiensis is the infecting organism, 60–80% of the phagocytized bacteria are lysed, however, the second wave of clearance seen with M. lysodeikticus does not occur; instead, the bacteria multiply extensively and death of the larvae results 12–16 hr after injection. This death does not appear to be caused either by crystalline protein or by the β-exotoxin. Analysis of hemolymph proteins using one-dimensional polyacrylamide gel electrophoresis indicated that although some quantitative changes were observed in some experiments, in the faster moving proteins when the infecting agent was B. thuringiensis, they were not consistent enough to support the idea that hemolymph proteins were either synthesized or used up during the time larvae were responding to the infectious agent. Dramatic changes were evident when the larvae were near death. No changes were ever observed when M. lysodeikticus was used as the infecting organism. A rapid response to infection using free spores of B. thuringiensis (sickness within 2–4 hr followed by death at 6–8 hr) may indicate that the spore germinating process is accompanied by release of a highly toxic material.     

Brugia pahangi: infections and their effect on the lymphatic system of Mongolian jirds (Meriones unguiculatus)

Brugia pahangi has been found to be primarily a lymphatic-dwelling parasite in jirds when infections are induced by the subcutaneous injection of infective larvae or by allowing infected Aedes aegypti to feed.Migration to the regional lymphatics occurred as early as 1–4 days. Although some injected larvae remained in the skin for as long as 30 days and some became localized in the heart, lungs, pleural cavity, or peritoneal cavity, about three-fourths of the recovered filariae were found in the regional lymphatics. In contrast, when larvae were injected peritoneally they remained largely in the peritoneal cavity for at least 30 days.The relevant lymphatics and their drainage patterns in jirds have been described.The major pathological changes noted in jirds involved the regional lymphatic vessels and nodes, which were severely affected when they contained dead worms. Pulmonary granulomas due to dead microfilariae and occasionally to dead larvae or adult worms were noted.Observations are included on the susceptibility and course of B. pahangi infections in jirds.     

Induction of ovarian maturation and development of eggs,larvae and juveniles of the tonguefish,Cynoglossus abbreviates,reared in the laboratory

Cynoglossus abbreviatus spawns from mid-March to mid-April in the Sea of Shimabara in Kyushu. During the spawning season ovarian maturation was successfully induced by injection of the pituitary homogenate ofHypophthalmichthys molitrix. The dose of the aceton-dried pituitary homogenate was 6.5 mg/kg body weight ofC. abbreviatus. It took about 2 days for ovulation after injection at a water temperature of 14 to 16°C. Artificial fertilizations were accomplished on March 29, 1974 and again on April 7, 1984, using the females matured by hormone injection in the latter case only. The larvae were reared on the rotifers,Artemia nauplii,Tigriopus japonicus and copepods collected from the sea over a period of 113 days in 1974 and 58 days in 1984. The eggs were pelagic, spherical, 1.19–1.23 mm in diameter and had 30–50 oilglobules of 0.068–0.095 mm in diameter, and the perivitelline space was narrow. The incubation period was 90–98 hours at a water temperature of 14 to 16°C. The newly hatched larvae were 3.18–3.45 mm TL and had 61–64 myomeres. The larvae had many melanophores and xanthophores on the body, forming three bands on the caudal region, but were lacking chromatophores on the finfolds. The yolk was completely absorbed when the larvae attained a size of 4.7–5.6 mm TL 8 days after hatching. A single elongated dosai fin ray developed on the head in the 8-day old larvae. The ray was reduced in size as long as the other rays 1 or 2 days after metamorphosis. The rudiment of pectoral fins were found on the both sides of the body in the 2-day old larvae, but two of them disappeared after metamorphosis. A pelvic fin first appeared as a ventral bud just anterior to the gut in the larva of 8.39 mm TL. The full count of 4 rays was observed on the larva of 10.83 mm TL. Metamorphosis began 22 days after hatching when the larvae were 11.20 mm TL. The right eye began to shift the left side of the head at night and reached to the final place after 8.5 hours. It took about 36 hours to complete the metamorphosis, including the eye movement and fusion of the hole in the rostral beak. At the last stage of metamorphosis, the dosal, caudal, anal and ventral fins became confluent. The larvae reached the juvenile stage at a size of 13.5–14.0 mm TL, approximately 28 days after hatchling. The growth of larvae reared in 1974 is expressed by the following equations: Y₁ = 3.448 · 1.0507^x (8≦X≦28) Y₂ = 6.3322 · 1.0275^x (28≦X≦75) where Y is the total length (mm) and X is the number of days after hatching. Growth rate changed after metamorphosis.     

Juvenile hormone esterase is a biochemical anti-juvenile hormone agent

Juvenile hormone esterase, purified by affinity chromatography from the larval hemolymph of Manduca sexta in the fifth stadium, was injected into larvae of the same species in the earlier stadia resulting in a blackening of the cuticle following ecdysis to the next larval stadium. This anti-juvenile hormone response was dose-dependent for an injection in the second, third or fourth stadium. Cuticular blackening was prevented by treating larvae with the juvenoid epofenonane. Larval response to injected juvenile hormone esterase also varied with the time of injection within a single stadium, having a maximum effect for injections at the time of head capsule slippage. Juvenile hormone esterase activity measured from the hemolymph after injection of larvae in the second stadium decreased over an 11 h time-course. Because the anti-juvenile hormone effects resulting from a single injection of juvenile hormone esterase were dependent on the time of injection, it appears that when juvenile hormone biosynthesis is active in the insect, the duration of enzyme activity limits the anti-juvenile effects that can be induced.     

Response of Galleria mellonella (Lepidoptera: Pyralidae) and Tenebrio molitor (Coleoptera: Tenebrionidae) to Spiroplasma citri inoculation

Injections into 4th instar larvae of Galleria mellonella using either in vitro or in vivo inoculum of the BR-6 isolate of Spiroplasma citri, propagated for one to nine passages, caused 5.7 to 24.7% mortality. Weight gain of the larvae injected at their 4th, 5th, and 6th instar was reduced in the first 4 days after inoculation but final pupal weight of the survivors was not significantly affected. Fourth instar larvae pupated within 10 days after the injection, but more larvae (6–13%) injected with 5th- to 9th-passage cultures pupated 5 or more days later than did larvae injected with 1st to 4th-passage cultures (0–3%). As many as one-third of the injected larvae developed into deformed pupae, with some external appendages missing or with a reduced and distorted thorax or abdomen with uneven tanning of the integument. Spiroplasma multiplied to dense concentrations (10⁸ to 10⁹/ml) in hemolymph smears from injected larvae incubated under oil. Larvae of Tenebrio molitor were not susceptible to S. citri by injection or feeding and G. mellonella were not susceptible by feeding. Transmissibility of S. citri by leafhopper vector to celery and periwinkle plants was retained after propagation for nine successive passages during 7 months in a nonhost insect such as Galleria.     

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.     

Ecdysteroid-titre reduction and developmental arrest of last-instar Heliothis virescens larvae by calyx fluid from the parasitoid Campoletis sonorensis

Fifth-instar Heliothis virescens larvae did not pupate after injections of Campoletis sonorensis calyx fluid in or before the burrow-digging stage of development. Arrested development occurred in 40% of larvae injected at the cell-formation stage. Further experiments showed that the particles in calyx fluid were responsible for developmental arrest. Arrested development due to calyx fluid could be reversed by injecting 10 μg of either ecdysone or 20-hydroxyecdysone, although a second injection of 20-hydroxyecdysone was needed for some larvae 3 days after the first treatment. Ecdysteroid production ceased for up to 10 days in 5th-instar H. virescens after calyx-fluid injection. After 10 days, some experimental larvae began to produce ecdysteroids again but remained developmentally arrested. The head, thorax, or abdomen of larvae were isolated by ligations and calyx fluid injected into the isolated body region. After 24 h, ligatures were released and the larvae observed for developmental arrest. Only injections into the isolated thorax stopped development. This, along with ecdysteroid data, indicated that C. sonorensis calyx fluid may directly affect the prothoracic glands of 5th-instar H. virescens.     

Streptococcus faecalis,Bacillus alvei, and sacbrood virus in European foulbrood of the honey bee

Contrary to reports from elsewhere, Streptococcus faecalis or Bacillus alvei did not cause European foulbrood in bee larvae also inoculated with sacbrood virus. The larvae died of sacbrood, by which time S. faecalis had mostly disappeared, although B. alvei multiplied saprophytically, as in European foulbrood, in some of the remains. Larvae that died of sacbrood already contained much sacbrood virus before they were sealed in their cells, when they appeared unaffected by the virus, but when they are most likely to die of European foulbrood, which is caused by Streptococcus pluton, often accompanied by secondary invaders, such as S. faecalis. Therefore, larvae killed by European foulbrood can be expected sometimes to contain much sacbrood virus, particularly as this virus is common.     

A Pathogenic Bacterium,Enterococcus faecalis,to the Beet Armyworm,Spodoptera exigua

A bacterial disease was found in the beet armyworm, Spodoptera exigua (Hübner). Blackened body of the infected larvae was a typical symptom of the epizootic disease especially at the intersegmental areas. We isolated the bacteria from the hemolymph of the infected 5th instar larvae and identified the isolate as a gram-positive bacterium, Enterococcus faecalis. When the 4th instar larvae were injected with the bacteria, half lethal dose of the bacteria was estimated as 22,593 colony-forming units (cfu) per larva and half lethal time of the bacteria was estimated as 2 days at 10⁷ cfu injection and 6 days at 10⁸ cfu injection. The bacteria were strongly resistant to each 1,000 ppm of ampicillin, kanamycin, and streptomycin. They were, however, relatively susceptible to mixture (1,000 ppm) of different combinations of the three antibiotics.     

Resistance to Taenia pisiformis larvae in rabbits: immunization against infection using non-living antigens from in vitro culture.

Heath D. D. 1976. Resistance to Taenia pisiformis larvae in rabbits: Immunization against infection using non-living antigens from in vitro culture. International Journal for Parasitology6: 19–24. A 97 % protection of rabbits against infection with Taenia pisiformis larvae was stimulated by subcutaneous injections of killed larvae cultured in vitro for 6 or 9 days, combined with the concentrated culture media in which the larvae grew. Larvae cultured in vitro for 3 days or less stimulated only 60% protective immunity.Exogenous antigens produced by 10-day old larvae in vitro were collected free of contaminating macromolecules, and were partially characterized. There appeared to be 6 exogenous antigens. Rabbits were immunized with either frozen larvae, or the exogenous complex, or both, using one subcutaneous injection of antigens adsorbed on aluminium phosphate. Exogenous antigens stimulated an 88% protection against challenge infection 14 days later, while only 52% protection was stimulated by somatic antigens from frozen larvae. The effects of the two antigen complexes were not additive. The protective ability of exogenous antigens was destroyed by exposure to air.     

C75, a Fatty Acid Synthase Inhibitor,Inhibits Feeding Activity and Pheromone Production in a Moth,Helicoverpa zea

Fatty acid synthesis produces long-chain fatty acids that are principal forms of stored energy and essential constituents of cellular membrane lipids. In animals fatty acid synthesis is catalyzed by fatty acid synthase (FAS) from acetyl-coenyzyme A (CoA) and malonyl-CoA. Cerulenin and C75, potent FAS inhibitors, can inhibit feeding in mammals.Using these inhibitors we examined the effect of feeding inhibition during H. zea larval stage. Growth of larvae injected (30 μg/g body weight) with C75 or cerulenin was significantly delayed during the first 8 hrs after injection, but recovered to normal levels within 20 hrs. During the first 8 hr period, the amount of consumed diet in the inhibitor treated larvae was significantly less than the control group. The retardation of larval development could be caused from the reduction of food intake after injection of the inhibitor. The result indicates that C75 or cerulenin inhibits fatty acid synthesis, resulting in feeding suppression in the larval moth as demonstrated in vertebrates.Pheromone production was significantly decreased in the isolated pheromone gland of H. zea females treated with FAS inhibitors. Pheromone production was inhibited by blocking fatty acid synthesis, even though PBAN stimulated pheromone biosynthesis. After topical application of D3-16: Acid to pheromone glands the relative labeled pheromone amount was increased when the gland was incubated with C75. This result indicates that a part of the pheromone amount could be synthesized from 16: Acid directly when fatty acid synthesis was blocked. These results indicate that the inhibitors have a potential possibility to control insect feeding activity and inhibit pheromone biosynthesis in moths.     

Interactions between a braconid,Microplitis croceipes,and a fungus,Nomuraea rileyi,in laboratory-reared bollworm larvae

The parasite Microplitis croceipes required 1.1 days longer at 26°C to complete development in Heliothis zea larvae than was required for the fungus Nomuraea rileyi to kill the host larvae and sporulate. Host larvae parasitized by M. croceipes or infected with N. rileyi failed to complete a fifth larval molt or pupate. Of the remaining healthy larvae, one-half completed six larval stadia before popation. Larvae parasitized by M. croceipes were predisposed to infection by N. rileyi, but the fungus inhibited development of M. croceipes if host larvae were infected with N. rileyi within 1 day after parasitization.     

Examining the relationship between hemolymph phenoloxidase and resistance to a DNA virus, Plodia interpunctella granulosis virus (PiGV)

We have a detailed understanding of invertebrate immune responses to bacteria and fungal pathogens, but we know less about how insects respond to virus challenge. Phenoloxidase (PO) functions as an important immune response against many parasites and pathogens and is routinely used as a measure of immune competance. We examine the role of haemolymph PO activity in Plodia interpuncetella's response to its natural granulosis virus (PiGV). Larvae were challenged with virus by both oral inoculation of occluded virus (the natural infection route) and direct intrahaemocoelic injection of budded virus. Haemolymph was collected at time points post-viral challenge using a novel method that allows the volume of haemolymph to be quanitified. The haemolmyph was collected without killing the larvae so that haemolymph samples from individuals that developed viral disease could be distinguished from samples collected from those that fought off infection. The level of haemolymph PO activity in resistant larvae did not differ from control larvae. Therefore we have no evidence that PO is involved in resistance to virus in the haemocoel whether larvae are challenged naturally by oral innoculation or directly by intraheamocoelic injection. Phenoloxidase may therefore not be a relevant metric of immunocompetence for viral infection.     

Sound and vibration are not obligatory host finding stimuli for the bark beetle parasite,Coeloides brunneri [Hymenoptera: Braconidae]

Previous researchers, have speculated or concluded thatCoeloides brunneri Viereck females detect their bark beetle hosts by perception of the vibrations or sound made by boring larvae. However, when placed on logs containing various actively mining stages ofDendroctonus pseudotsugae Hopkins,C. brunneri females actively searched for the host only on logs infested with young or maturing brood larvae, and oviposited only in logs with maturing brood larvae. Moreover, when offered larvae in logs that had been frozen at approximately ? 50°C, and then allowed to thaw at room temperature for 2 days, they found the motionless, dead larvae, and oviposited through the bark on to them. Therefore,C. brunneri is able to find ist host by perception of some stimulus (or stimuli) other than sound or vibration.     

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.     

Control of Codling Moth,Cydia pomonella (Lepidoptera: Tortricidae), with Steinernema carpocapsae: Effects of Supplemental Wetting and Pupation Site on Infection Rate

Infection of cocooned codling moth (cydia pomonella) larvae by the entomopathogenic nematode Steinernema carpocapsae was studied in three field experiments. Factors that varied within or between experiments included method of application, type of substrate containing cocooned larvae, time when nematodes were applied, seasonal effects, and supplemental wetting before or after nematode application. Conventional air-blast sprayer applications of 0.5–5.0 million infective juveniles (IJs)/tree in fall resulted in ca. 30% mortality of larvae in cardboard trap bands, whereas hand-gun application (2 million IJs/tree) produced mortality of ca. 70%. Application in the evening caused higher larval mortality than application in the morning when no supplemental wetting was used after treatments. Morning and evening applications caused equivalent larval mortality when a postwetting treatment was included. In a trial conducted in midsummer, supplemental wetting, either before or after hand-gun application of 1 million IJs/tree, enhanced nematode-produced mortality. Mortality approached 100% if both pre- and postwetting was used. Larvae in exposed cocoons on apple wood were infected at a higher rate (86%) than those on wood in less exposed positions (73%) or in nonperforated cardboard (72%). Mortality rates for larvae in perforated cardboard were intermediate (77%). Application volumes used to deliver nematodes slightly enhanced infection rate of larvae in some substrates but not others. In one trial, parasitism of codling moth by the wasp Mastrus ridibundus (Ichneumonidae) was negatively correlated with nematode infection of codling moth larvae. Dissections showed that ca. 10% of larvae infected by nematodes had been attacked by the wasp.     

Infection of the corn earworm, Heliothis zea, with Nosema acridophagus and Nosema cuneatum from grasshoppers: Relative virulence and production of spores

Per os inoculations of 4- to 6-day-old larvae of the corn earworm, Heliothis zea, with suspensions containing 10⁶ spores of Nosema acridophagus or 10⁴, 10⁵, and 10⁶ spores of Nosema cuneatum retarded the growth and development of the larvae. Migratory grasshoppers, Melanoplus sanguinipes, inoculated with N. acridophagus produced fewer spores than similarly inoculated corn earworms, but spore production was similar in these insects when they were inoculated with N. cuneatum. Standard bioassay procedures showed that spores of both microsporidians were some-what more virulent when they were produced in corn earworms than when they were produced in grasshoppers. Spores of these microsporidians might be produced more efficiently in corn earworm larvae than in grasshoppers.     

Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth,Galleria mellonella

The effects of beauverolide L and cyclosporin A, cyclic peptidic metabolites, produced by several genera of entomopathogenic fungi on immune responses of last instar larvae of the greater wax moth Galleria mellonella have been examined. Intrahemocoelic injection of either metabolite-coated silica particles or dissolved metabolites in a concentrations ranging between 10 and 30 μg per larva caused no mortality but activated humoral responses in G. mellonella larvae. The challenge induced a significant release of lysozyme and cecropin-like activity into the hemolymph, suggesting stimulatory activity on humoral immune responses. Injected metabolite-coated particles were rapidly surrounded by hemocytes which subsequently accomplished formation of melanized nodules, which increased in size and number compared with controls. In vitro assays with dissolved metabolites indicated no adverse effects of beauverolide L or cyclosporin A on attachment or spreading of isolated plasmatocytes but dose-dependent inhibition of their phagocytic activity. Isolated plasmatocytes incubated with cyclosporin A or beauverolide L exhibited cytoskeleton alterations that differed from those observed in plasmatocytes from infected G. mellonella larvae or reported from other fungal secondary metabolites. The experiments provided further data to elucidate the role of fungal secondary metabolites in development of mycoses in insects.