Infected Pupae Research Articles

Studies on the Life History of a Neogregarine Parasite Found in Leptothorax Ants from North America

Pupal stages of Leptothorax ants collected near West Yellowstone, MT, USA, displayed striking signs and symptoms of disease, i.e., grey to black coloration, irregular pigmentation of compound eyes and toothless mandibles. Light microscope studies revealed heavy infections by a neogregarine, the life history of which is described. The life cycle of the pathogen includes micronuclear and macronuclear schizogonies, gametogony and sporogony. Schizonts of both types vary in size depending on the number of nuclei which is usually defined by doubling, thus giving rise to 8, 16, 32, 64 or even 128 uninucleate merozoites. In smears and sections, micronuclear merozoites are typically arranged in rosettes. In the early transformation of zygotes, sickle-shaped developmental stages have been encountered, so far undescribed from neogregarines. Two spores (oocysts), each developing eight sporozoites, evolve from each gametocyst, as is typical of the genus Mattesia. Mature lemon-shaped spores measure 13.8 9.3 μm in fresh preparations. Infections can be readily transmitted to healthy colonies and to other Leptothorax species by feeding crushed infected pupae. Vegetative life cycle stages grow and multiply in the haemocoel, only to some extent they infect fat body cells. Macronuclear merozoites invade the hypodermis and the fat body but also settle extracellularly in the haemocoel. The disease process terminates with the death of the pupae that harbour abundant spores. Infections of adults have not been observed. Despite some minor differences that may result from development of the pathogen in this host, from the type, sequence and morphology of life cycle stages and from the signs and symptoms of disease, this Mattesia species is identified with M. geminata, first discovered in the tropical fire ant, Solenopsis geminata (Fabricius).

Protein synthesis in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus: resistance to viral infection acquired during pupal period.

Protein synthesis has been studied in pupae of the silkworm Bombyx mori (Bm) infected with nuclear polyhedrosis virus (BmNPV) at various stages of the pupal period. Nascent proteins were labelled by injection of [35S]methionine into pupae and then analysed by SDS-PAGE. Temporal regulation of synthesis of infected cell-specific proteins (ICSPs) in pupae was demonstrated by electrophoretic analysis of the proteins labelled at different times post-infection (p.i.). The rate of ICSP synthesis reached a maximum at 4 to 5 days p.i., exceeding the rate of synthesis of cellular proteins in uninfected pupae by about twofold. The viral proteins p10 and polyhedrin were the most abundant products synthesized late in the infection. Both proteins were found to be associated with the nuclear matrix after fractionation of nuclei from infected pupae. Two virus-induced phosphoproteins, pp35 and ppB, were found to be the major acceptors of labelled phosphate from [gamma-32P]ATP during in vitro phosphorylation of proteins in pupal homogenates, nuclei and nuclear extracts. These proteins had electrophoretic mobilities comparable to those of structural phosphoproteins of BmNPV virions with M(r)s of 35K and 11K to 16K, respectively. The latter polypeptide was identified as the major DNA-binding protein of the virus. The susceptibility of silkworms to BmNPV decreased markedly during the pupal period. Following injection of BmNPV all young pupae acquired polyhedrosis and finally died whereas most of the older pupae did not exhibit disease and completed metamorphosis normally. Moreover, the later in the pupal period the silkworms were infected, the lower the production of polyhedrin in diseased pupae.

Induction of a novel protein kinase in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus.

Protein kinases induced by Bombyx mori nuclear polyhedrosis virus in pupae of the silkworm B. mori were examined by activity gel analysis using phosvitin as a protein substrate. The method involved PAGE of the soluble fraction from pupae under native conditions and in the presence of SDS, followed by in situ renaturation of proteins and recovery of protein kinase activity in the intact gel. A novel protein kinase able to phosphorylate phosvitin was detected in the infected pupae from 2 days post-infection. This enzyme was not present in uninfected silkworms at any stage of the pupal period. The novel kinase activity was found by SDS-PAGE to be associated with a single polypeptide with an apparent M(r) of 50K. However, on electrophoresis under native conditions its activity was associated with a set of polypeptides with similar but not identical electrophoretic mobilities. Microheterogeneity of the catalytically active polypeptides suggests that the virus-induced protein kinase undergoes post-translational modification during the course of infection.

Effects of Ascogregarina barretti (Eugregarinida: Lecudinidae) infection on Aedes triseriatus (Diptera: Culicidae) in Illinois.

Ascogregarina barretti (Vavra), a gregarine protozoan parasite, infected 23.6 and 5.0% of Aedes triseriatus (Say) adults that emerged from pupae collected from an east-central Illinois tire dump in 1990 and 1991, respectively. Infection significantly reduced the wing length of males and females in both years, but its effect was greatest in 1991. Females were approximately 1.7 times as likely to be infected as males. Infected pupae were 3.53 and 2.76 times more likely to die than uninfected pupae in 1990 and 1991, respectively. Wing length also was affected by location within the tire dump and by tire water volume. We conclude that the A. barretti infection was deleterious to Ae. triseriatus and that the effects of this pathogen may be moderated by environmental factors.

Influence of temperature on developmental parameters of the parasite/host system Edhazardia aedis (Microsporida: Amblyosporidae) and Aedes aegypti (Diptera: Culicidae)

Larvae of Aedes aegypti, transovarially infected with Edhazardia aedis, were reared between 20 and 36°C to determine the influence of temperature on the development of the parasite and the infected host. Development of the parasite was evaluated based on spore yield and size. The predicted optimum temperature for maximum spore production of E. aedis in A. aegypti was 30.8°C. The results demonstrate that the E. aedis-A. aegypti system has a wide temperature tolerance; whereas spore yield will be lower at unfavorable temperatures, the host will remain infected. Additionally, spores were significantly smaller from individuals reared at 34°C than those reared at either 20 or 27°C. Development of the infected host was evaluated based on pupal weight and time of pupation. Infected pupae were significantly larger than uninfected pupae. There was also a significant difference in the pupation rate between controls and infected A. aegypti larvae. Controls had a 50% cumulative pupation time (CPT 50) of 65.7 degree days and infected individuals a CPT 50 of 76.6 degree days.

Selection for an Increased Rate of Vertical Transmission of Spodoptera frugiperda (Lepidoptera: Noctuidae) Nuclear Polyhedrosis Virus

In a series of laboratory experiments, larvae of Spodoptera frugiperda (J. E. Smith) were exposed to median lethal concentrations of S. frugiperda nuclear polyhedrosis virus (NPV), and the survivors were reared to the adult stage and mated. Larvae, pupae, and, in later experiments, adults of the second-generation (F1) insects were then examined for signs of nuclear polyhedrosis. When natural isolates of NPV were fed to third instars in the first generation, total infection rates in F1 S. frugiperda larvae and pupae ranged from o to 0.89%. When fifth instars were fed virus in the first generation, infection rates in F1 S. frugiperda ranged from 3.35 to 4.32% in larvae, from 0.75 to 2.08% in pupae, and from 8.05 to 9.92% in adults. NPV was selected for an increased rate of vertical transmission by isolation of virus from infected F1 pupae. The selected NPV exhibited significantly ( P < 0.05) better vertical transmission than did the wild isolate. When fifth instars were fed selected virus in the first generation, infection rates in F1 S. frugiperda were 7.85–12.73% in larvae, 0.73–5.93% in pupae, and 9.72–10.45% in adults. Vertically transmitted NPV caused F1 mortality mainly in early-instar larvae and in prepupae and pupae; there was little F1 mortality in late-instar larvae or adults.

Interaction between the tachinid parasite,Sturmiopsis inferens and granulosis virus in the sugarcane shoot borer,Chilo infuscatellus [Lep.: Crambidae

Competition between granulosis virus (GV) and the larval parasite,Sturmiopsis inferens Tns. (Tachinidae: Diptera), was studied in 3rd — and 4th — instar larvae of the sugarcane shoot borer,Chilo infuscatellus Snellen (Crambidae: Lepidoptera), under laboratory conditions. Mortality due to GV infection and parasitization was 76.8 and 47.6 per cent, respectively, when they were tested separately. But when hosts were infected simultaneously with microfeeding of GV and larval parasite, a significantly low parasitism (5.5%) was obtained compared to 74.8 per cent mortality by GV infection. When the larvae were microfed with the GV 6 days after inoculation with parasitic maggots, mortality due to the virus was reduced significantly to 20.5 per cent, but when the maggot inoculation was preceded by virus microfeeding 6 days before, parasitization was unsuccessful, while 75% of larvae died of virus. Results obtained from field — collected larvae also showed that significantly more parasite puparia were recovered from healthy larvae than from virus — infected larvae. Similar differences in parasitization were not obtained in the case of healthy or virus — infected pupae.

Transpuparial transmission of Kashmir bee virus and sacbrood virus in the honey bee (Apis mellifera)

SummaryWhen particles of Kashmir bee virus (KBV) and sacbrood virus (SBV) were fed to larvae of the honey bee, Apis mellifera, in Australian colonies, the resulting pupae became in apparently infected. There was no statistically significant difference in the susceptibility of 1, 2, 3 or 4‐day‐old larvae for either virus, but 5‐day‐old larvae were significantly less susceptible to SBV than younger larvae. There was no significant difference in the proportions of pupae that became in apparently infected when, as larvae, they were fed various concentrations of each virus, but significantly more larvae were removed from their cells when fed concentrated preparations of each virus than when fed diluted preparations. Susceptible larvae that became in apparently infected with KBV and SBV developed normally into in apparently infected pupae and later, emerged as in apparently infected worker bees.

Paecilomyces farinosus, a potential biocontrol agent of some lepidopterous tree pests in India

Paecilomyces farinosus was isolated from naturally infected pupae of Eligma narcissus (Lepidoptera: Noctuidae), a major pest of Ailanthus triphysa . Field surveys during 1983–6 showed ca 40 % pupal mortality due to P. farinosus . Inoculation of larvae of E. narcissus and Atteva fabriciella (Lepidoptera: Yponomeutidae), another major pest of Ailanthus , with this fungus caused mortality within 48–72 h of incubation.

Multiplication of Kashmir bee virus in pupae of the honeybee, Apis mellifera

Laboratory investigation has shown that Kashmir bee virus (KBV) is a virulent pathogen of honeybee pupae. Inoculation with 1 × 10 −8 ng of purified virus was sufficient to cause infection in some pupae, and at doses of 1 × 10 −5 ng every inoculated individual became infected (ID 100). At completion of virus multiplication, between 200 and 500 μg of KBV could be recovered from each pupa. No significant relationship was found between final quantity of virus per pupa and inoculum size when the latter was greater than or equal to an ID 100. Observation by electron microscopy showed that KBV multiplied in fore- and hindgut epithelial tissue, alimentary canal musculature, epidermis and tracheal epithelium, and in hemocytes, oenocytes, and tracheal end cells. Infected cells showed a marked pathological response which included condensation of chromatin, disruption of cytoplasmic organelles, and sloughing of lobules of cytoplasm. Virus multiplication apparently took place within cytoplasmic membrane-bound vesicles. The ultrastructure of these vesicles shows little similarity to those previously described for other small RNA viruses. No evidence of KBV multiplication was found in tissues of the nervous system, even though degeneration of glial cells was observed. Electron microscopy showed that the cytoplasm of affected glial cells contained large numbers of membrane-bound vacuoles. Coalescence of these vacuoles apparently caused a separation of the nucleus and cytoplasm of cells and was followed by the progressive degeneration of each. KBV infection caused significant changes in the hemolymph osmolality of infected pupae, but no discernible changes could be seen in pupal brain morphology following induction of identical osmolality levels by inoculation with concentrated salt solutions.

Structure and properties of protein P4, the major bacteria-inducible protein in pupae of Hyalophora cecropia

Protein P4 is the major protein induced in pupae of Hyalophora cecropia as a response to a bacterial infection. The bacterial induction of protein P4 synthesis was quantitated. It was also shown that the immune system can be transiently turned on by an injection of protein P4 itself. There exist at least two forms of protein P4 one of which was purified to homogeneity. Its amino acid composition was determined as well as the sequence of the 25 N-terminal amino acid residues. The peptide backbone is largely folded as β-structures. The biological role of protein P4 remains an open question. However, we have found that there was no rapid turn-over of protein P4 in the course of an infection, but rather an accumulation in the hemolymph. Immunostaining showed that protein P4 exists in fat body basal membranes as well as in certain hemocytes and nodules from infected pupae.

Natural microsporidian infection in laboratory colonies of Spodoptera spp

Nosema sp. naturally occurred in both Spodoptera littoralis (Boisd.) and S. exigua (Hubner) laboratory colonies in Alexandria, Egypt (since November 1984). It was responsible for the dramatic suppression of their laboratory populations. Both young (3-day-old) and old (9-day-old) larvae of S. littoralis were susceptible to Nosema sp. However, the pathogen was more effective against the young larvae, especially at high doses, than the old ones. The estimated LC 50 was 4.0 × 10 7 spores'ml, for 3-day-old larvae. The slope value was low (1.2) as typical of protozoan insect pathogens. Heavily infected 9-day-old larvae tended to die as larvae; while less infected ones either pupate or produce larval-pupal intermediate forms (12–23%). The developmental period of both infected larvae and pupae was increased by 4–6 and 3–5 days, respectively. The number of infected insects that reached the adult stage was decreased as the concentration of Nosema sp. spores was increased. Furthermore, infection was shown to reduce adult longevity and fecundity, and viability of the eggs. Larval faecal pellets harboured large numbers of viable spores. Transovum transmission of infection occurred. Two dipterous species, Culex pipiens and Aedes aegypti , were not susceptible to Nosema sp.

Changes in DNA Polymerase Activities in Pupae of the Silkworm Bombyx mori after Infection with Nuclear Polyhedrosis Virus

Summary Changes in DNA polymerase activities were studied in pupae of the silkworm Bombyx mori upon infection with nuclear polyhedrosis virus (BmNPV). Two effects were observed in male and female pupae after inoculation of BmNPV: a marked increase in cellular α-polymerase activity and the induction of a new DNA polymerase (BmNPV-polymerase). Activities of both DNA polymerases, α and BmNPV, in the infected pupae increased in parallel, reached a maximum at 72 h post-inoculation and decreased subsequently. The virus infection did not affect the activity of γ-polymerase in the pupae. An increased activity of β-polymerase in the pupae was detected at the late stage of the infection cycle prior to pupal death. The possible roles of the DNA polymerases in BmNPV replication are discussed.

The influence of starvation on mortality, development, and protein content in parasitized and unparasitized Tribolium castaneum

The effects of starvation on mortality, development, and protein content in Nosema whitei-infected and uninfected Tribolium castaneum were investigated. T. castaneum larvae, starved for 4, 6, 8, and 10 days postinfection, showed an increase in larval mortality. Pupal mortality also increased, producing a decrease in adult emergence. Starvation of larvae for 6 days or more delayed development and the average time to adult emergence increased. These effects tended to be more marked in Nosema-infected larvae. No consistent pattern of weight changes was observed in either the uninfected or infected pupae and adults. Infected T. castaneum larvae showed a significant decrease in protein compared to controls. Starvation apparently does not aggravate this condition nor does it have any significant effects on the total hemolymph protein content of uninfected and infected larvae.

Histopathology of the tropical fire ant, Solenopsis geminata, infected with Burenella dimorpha (Microspora: Microsporida)

Pupae of the tropical and fire ant, Solenopsis geminata, infected by the microspordium Burenella dimorpha develop clear, blister-like areas in the vertex of the head and in the petiole. In sexual pupae, clearing may also develop in the dorsal thorax. The developing eyes of both worker and sexual pupae appear sunken and irregular in outline, with deranged facits. These pathognomonic signs are the result of destruction or inhibition of development of the cuticle. The clear areas results from tissue fluids seeping into the space between the denuded hypodermal tissue and the pupal sheath (old larval integrument). The fact body is progressively diminished and the brain shrinks as the disease progresses. These organs, which are covered by a layer of hypodermal tissues, decrease in mass and recede from the pupal sheath, which is kept extended by tissue fluids. The malformation of the eyes is also the results of destruction of the cuticle. The lenses, which are cuticular, are destroyed, leaving the ommatidia unanchored distally. In advanced infection, the pupae rupture and are cannibalized by adult workers. The workers do not ingest the spores into the crop, but divert them (with other particulate matter) to the infrabuccal cavity. An infrabuccal pellet is formed which is subsequently expelled and fed to fourth instar larvae only. The intracolonial dissemination of spores is thus facilitated by the destruction of the cuticle of infected pupae.

Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia.

Diapausing pupae of the Cecropia moth (Hyalophora cecropia) respond to an injection of live bacteria by the selective synthesis of certain types of RNA and immune proteins (designated P1-P9). The in vitro translation products of RNA from both injured and infected pupae showed specific patterns with a defined number of extra bands. Some proteins characteristic of the normal RNA were reduced in the immune RNA translation products. Antibody reaction was used to show the selective synthesis of immune proteins P4 and P5 with mRNA from pupae subjected to injury or infection. The protein synthesized in vitro, which cross-reacted with P5 antibodies, is most likely a precursor of the attacins described in the preceding paper. A cDNA clone bank was prepared and two clones were isolated and shown to contain 750 bp corresponding to P4 and 250 bp of attacin information. These clones were used to estimate the sizes of the mRNAs by Northern blotting and to estimate, by RNA/DNA hybridization, the levels of P4 and P5 mRNA. In vivo incorporation of [35S]methionine into attacins and P4 during different conditions was compared with the levels of the corresponding mRNA.

The effects of Farinocystis tribolii on the growth and development of the flour beetle Tribolium castaneum

Farinocystis tribolii multiplied only in the fatbody of Tribolium castaneum larvae. In the advanced stages of infection, the fatbody was destroyed and the albuminoid granules were completely depleted. Larvae in late stages of infection were “C” shaped. Diseased pupae were flat and straight with the head and mouth parts not completely flexed with the pupal body. Infected pupae failed to develop into adults. Heavily infected adults were distended. Larvae of third, fourth, and fifth instars, although succumbing to the infection ultimately, continued to live even after the healthy larvae of the same age had entered pupation, indicating a prolongation of larval period. Molting was either affected or delayed in infected insects. F. tribolii infection induced a juvenile hormonal effect producing larval-pupal and pupal-adult intermediate forms. Ether extract of spores of F. tribolii when applied on pupae of T. castaneum and final instar nymphs of Dysdercus cingulatus produced adultoids.

Monitoring the course of bacterial infections by hemolymph pressure pulses in insects

Changes in rhythmic pulsations of hemolymph pressure have been continuously monitored from several hours before injection of the pathogenic bacteria until death of the infected Tenebrio pupae. Lethality induced by five different species of entomopathogenic bacteria was associated with specific changes in the hemolymph pressure pattern. During the initial stages of infection (incubation period) the pupae continued to produce regular and synchronized series of normal pulsations. The pathophysiological symptoms of the acute disease became manifested by successive desynchronisation of the frequency and simultaneous depression of the amplitude of the pulsations. This irreversibly proceeded, with species specific modifications, until death of the pupae when all peaks in hemolymph pressure disappeared. Duration of the incubation period was inversely proportional to the injected dose, while the later period characterized by development of the pathophysiological symptoms had more or less constant course in infection with each bacterial species. Certain toxic enzymes, protease and phospholipase C, had similar action on hemolymph pressure pulses. Application of the tensometric method to pathophysiological studies in insects has been discussed.

Fatty acid and glycogen requirement of Heliothis virescens infected with cytoplasmic polyhedrosis virus

1. 1. Five-day-old larvae of Heliothis virescens infected with cytoplasmic polyhedrosis virus (CPV) had 66% more fatty acid calories than healthy larvae of the same age. 2. 2. Fatty acid caloric values of healthy and CPV-infected pupae were not significantly different. 3. 3. Infected pupae had six times the fatty acid calories of 10-day-old larvae; healthy pupae had a three-fold increase. 4. 4. Infected pupae had an average loss of 50% weight during metamorphosis to adults, 10% due to loss of fatty acids. 5. 5. Calculated on fresh weight basis, infected adult males had 31% less fatty acid calories than healthy adult males; infected females had 34% less than the healthy female. 6. 6. Infected males and females had 31 and 49% less glycogen, respectively, than healthy adults. 7. 7. During emergence of pupae to adults, approx 60 and 30 glycogen cal/insect were used by healthy and infected insects, respectively.

Effects of Nosema heliothidis on fatty and amino acids in larvae and pupae of the bollworm, Heliothis zea

1. 1. Nosema heliothidis had a total fatty acid calorie value of 9.92 × 10 −9/spore; H. zea larvae (4th and 5th instar), male pupae, and female pupae had averages of 0.64, 1.36, and 0.75 calories, respectively. 2. 2. The change in weight of healthy and infected larvae with age was significantly different ( P < 0.01). 3. 3. The change of fatty acid caloric value of healthy and infected H. zea larvae with age was significantly different ( P < 0.01, r = 0.40). 4. 4. The change in fatty add caloric value of healthy and infected male pupae with age was not significantly different, but the change in age was significant at the 99% level. 5. 5. Fresh weight was constant in healthy and infected pupae, but the fatty acid caloric value was significantly different ( P < 0.01). 6. 6. The difference in the fatty acid caloric value of healthy male and female pupae was highly significant ( P < 0.01). The infected male and female showed a significant difference at the 99% level. 7. 7. Amino acids n healthy H. zea were not significantly different from those in infected H. zea.