A Tipula paludosa population with a high incidence of two pathogens

The incidence of lethal parasites in the larvae of a Tipula paludosa population was monitored for two seasons. The proportions of larvae infected with Tipula iridescent virus (TIV) and a tachinid insect were similar to those in previously studied populations, whereas the proportions of larvae infected with Tipula nuclear polyhedrosis virus (NPV) and a spore-forming bacterium (SFB) were higher. Conservative estimates of mortality due to these four agents were 10.7% in 1977–1978 and 7.7% in 1978–1979. The mean population density and the proportion of SFB-infected larvae were lower in 1978–1979 than in 1977–1978, while the proportion of NPV-infected larvae was higher. In 1979 the proportion of NPV-infected larvae was positively correlated with population density, which was highest in the wettest part of the study area. In both seasons the proportion of SFB-infected larvae was negatively correlated with population density. Larvae infected with the NPV or the SFB became pallid at an advanced stage of infection, but, although infected larvae were found throughout the larval period, pallid larvae were only found in the later part. It is suggested that larvae become infected in an early instar, then the infections slowly develop throughout the remainder of the larval period. Five larvae were found with mixed infections; four were infected with the SFB and NPV, while the fifth was infected with the SFB and TIV.     

The mode of transmission of Tipula iridescent virus: II. Route of infection

Tipula iridescent virus (TIV) was inoculated into Tipula oleracea larvae via different routes. It was found to be much more infective when it was injected into the hemocoel than when it was ingested by the larvae.T. oleracea embryos did not become infected when eggs were laid in agar containing TIV, and there was no evidence that larvae can become infected via the spiracles or that transovum transmission occurs. It is suggested that TIV is transmitted principally by cannibalism, including killing and ingesting infected larvae, and finding dead infected larvae and ingesting them. It is proposed that a new generation becomes infected by first-instar larvae feeding upon infected fourth-instar larvae which have survived from the previous generation.     

Tipula iridescent virus infection in the developmental stages of Tipula oleracea

Tipula iridescent virus (TIV) is infective to all four larval instars, pupae, and adults of both sexes of Tipula oleracea, and iridescence has been observed in infected insects at all these stages. Third- and fourth-instar larvae were more resistant to ingested TIV than first and second instars. When TIV was injected into the hemocoel, the results suggested a possible decrease in resistance from the third larval instar to the pupa. Incubation periods (times from injection of TIV to appearance of iridescence) were significantly shorter in older fourth-instar larvae than in younger fourth-instar or thirdinstar larvae, but variability in incubation period was significantly greater in younger fourth-instar larvae than in the other two stages. Many insects which were inoculated with TIV in one stage developed iridescence and died in later stages. The amounts of infective TIV in two infected adults were estimated.     

The mode of transmission of Tipula iridescent virus: I. Source of infection

A virus was isolated from a diseased tipulid larva and identified as Tipula iridescent virus (TIV) on the basis of the size and morphology of the virion, the production of iridescence in vitro and in infected tipulid larvae, and a serological reaction between antiserum against the virus and an isolate of TIV.A stock of Tipula oleracea was bred in the laboratory. Subjection of larvae to several stress factors did not result in any evidence for activation of a latent virus. Healthy T. oleracea larvae did not develop iridescence when confined in petri dishes with either live TIV-infected larvae or with large amounts of their feces, although these feces were found to contain infective virus by injecting extracts into healthy larvae. It appears that the concentration of virus in the feces of infected larvae is not high enough for them to serve as a source of infection. It was shown that the cadavers of TIV-infected larvae can serve as a source of infection for healthy first- and fourth-instar larvae.     

Field trials withTipula iridescent virus againstTipula spp. larvae in grassland

Field trials withTipula iridescent virus (TIV) were carried out to determine whether the infection can be introduced into populations ofTipula spp. in grassland. The virus was introduced into plots in live and deadTipula oleracea L. larvae, in a bran bait and in sprayed aqueous suspensions. Trials were conducted at 1 site in 3 successive years and at 5 further sites in the 3rd year. Tipulid larval populations in the plots were sampled at intervals of approximately 2 months. The majority of sampled larvae were not iridescent and did not become iridescent when they were incubated at 20°C for 30 days. In plots where iridescent larvae were found they generally comprised between 1 and 17% of the tipulid population. The identity of the virus infecting these insects was confirmed by the latex agglutination test. The results suggest that all the treatments introduced the virus infection into one or more of the tipulid populations; they all did so, however, with low efficiencies.

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Résumé Des essais en parcelles avec le virus irisant deTipula (TIV) ont été effectués pour déterminer si l'on peut introduire l'infection dans des populations deTipula spp. en prairie. Le virus a été utilisé sous forme de larves vivantes ou mortes deTipula oleracea L., d'appat de son, et en suspensions aqueuses. L'expérimentation a été réalisée dans un emplacement pendant 3 ans successifs et dans 5 sites complémentaires pendant la 3^e année. L'échantillonnage des populations de larves de tipules a eu lieu tous les 2 mois. La majorité des larves récoltées n'etaient pas irisantes et elles ne le sont pas devenues après un élevage à 20°C pendant 30 jours. Dans les parcelles où l'on a trouvé des larves irisantes, celles-ci représentaient 1 à 17% de la population de tipules. L'identité du virus dans ces insectes a été confirmée par agglutination au latex. Les résultats suggèrent que tous les traitements ont introduit l'infection virale dans les populations de tipules mais avec une faible efficacité.

     

The effect of sodium butyrate on Tipula iridescent virus synthesis in insect suspension cell cultures

The effect of sodium butyrate on Tipula iridescent virus (TIV) synthesis in suspension-cultured cells of Estigmene acrea was investigated. Sodium butyrate reduces viral-induced cell fusion but this is reversible with the removal of butyrate. At 7 mM sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 mm butyrate in the cell medium; cells so treated contain large vesicles with inoculum. Upon removal of the inhibitor, TIV replication appears normal, but large inoculum vesicles can still be found in the cytoplasm, and many infected cells have highly condensed chromatin in their nuclei. Sodium butyrate causes a lag of at least 2 hr in viral DNA synthesis as detected by [³H]thymidine incorporation into viroplasmic centres and at 7 mm butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 mm concentration does not inhibit host DNA synthesis, but at 15 and 20 mm, nuclear DNA synthesis is markedly reduced.     

In vivo insect hemocyte destruction by UV-irradiated Chilo iridescent virus

Large inoculum of infective or UV-irradiated Chilo iridescent virus (CIV) caused an early drastic cytopathy of the larval hemocytes of the greater wax moth, Galleria mellonella. Cytopathy of the hemocyte could be detected 2 hr after virus injection at 25°C and most hemocytes were completely destroyed within 8 hr after virus injection. Heat-treated CIV did not produce any cytopathy of Galleria hemocytes. Antiserum to CIV neutralized this cytopathic effect, while that to Tipula iridescent virus did not demonstrate any inhibitory effect. Actinomycin D, mitomycin C, and puromycin did not inhibit hemocyte destruction by UV-irradiated CIV. Galleria larva injected with a large dose of UV-irradiated CIV progressively became stunted with a decrease of body weight and did not regain its hemocyte numbers. Hemocytes infected with CIV were not destroyed by any additive injection of a large dose of UV-irradiated CIV.     

Metabolic changes in diseased insects. IV. Radioautographic studies on protein changes in nuclear polyhedrosis, densonucleosis, and Tipula iridescent virus infections

Changes in glutamic acid, leucine, arginine, and tyrosine in Lepidoptera and Hymenoptera tissues infected with nuclear polyhedrosis (NPV), densonucleosis (DNV), or Tipula iridescent (TIV) viruses were studied by radioautography with a view to determining the effect of the viruses on protein metabolism.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

Mitotic responses of the anterior pericardial wall of Biomphalaria glabrata (Mollusca) subjected to challenge

Using light microscope autoradiography and electron microscopy we studied the effect of juvenile hormone III (JHIII) and β-ecdysone insect molting hormone (β-ecd) on the replication of Tipula iridescent virus (TIV) in suspension cultured cells of Estigmene acrea. JHIII at a concentration of 87.5 μg/ml completely inhibited viral DNA synthesis, but upon removal of JHIII, [³H]thymidine was incorporated into the cytoplasm as detected by autoradiography and virions in developmental stages from the same cell samples were-readily seen by electron microscopy. β-ecd at a concentration of 17.5 μg/ml, unlike JHIII, permitted viral DNA synthesis in the presence of the hormone although at a reduced level when compared to TIV-infected cells. But the presence of β-ecd seemed to prevent capsid formation, although islands similar in fine structure to those of viroplastic centers were seen by electron microscopy. Once β-ecd was removed from the medium, TIV-inoculated cells appeared to synthesize new virions in a normal pattern. Both hormones inhibited host cell DNA synthesis in noninfected cells.     

Physicochemical properties of tipula iridescent virus

The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 × 10⁸, with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 ± 0.2%. At 260 mμ, the virus gave an uncorrected absorbance of 18.2 cm²/mg of virus and a light-scattering corrected absorbance of 9.8 cm²/mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.     

The effect of temperature on the growth,development and survival of Macropetasma africanus (Balss) (Penaeoidea:Penaeidae) larvae reared in the laboratory

Macropetasma africanus (Balss) has been successfully spawned and its larvae reared under controlled laboratory conditions. The relationship between egg number (E) and female total length (L) was E = 18.59 L^2.11. An experiment was designed to test the effect of temperature on larval development, survival and growth. Temperature effected larval development time, from 13–15 days at 25°C, to 25 days at 15°C (nauplius 1 to post-larva). Mortality was low for the naupliar stages at 25, 22 and 18°C, while at 15°C only 52% of the larvae reached nauplius 6. Mortality was highest from nauplius 6 to protozoea 1 (17, 21, and 18% at 25, 22, and 18°C, respectively), but decreased considerably for all temperatures once the mysis stage was reached. Overall survival rates from nauplius 1 to post-larva decreased with decreasing temperature (65, 54, 48, and 39% at 25, 22, 18, and 15°C respectively). Temperature also significantly affected larval growth. At 25°C mean total length was significantly (P < 0.05) larger than at 15°C (protozoea 2 to post-larva), while from protozoea 3 to post-larva total length differences were significantly different (P < 0.05) between 18 and 25°C. M. africanus has a major spawning peak in summer, suggesting that there may be a selective advantage to reproducing during the warmer months.     

Culex pipiens affected by joint infection of a mosquito iridescent virus and Strelkovimermis spiculatus

Dual infections with a mosquito iridescent virus (MIV) and the mermithid nematode, Strelkovimermis spiculatus were recorded in natural Culex pipiens populations around La Plata city, Argentina. S. spiculatus was detected in 82% of samples that were positive for MIV infection. Dissected larvae of Cx. pipiens with patent MIV infection presented 42% infection with S. spiculatus. Larvae of Cx. pipiens exposed to MIV and S. spiculatus under laboratory conditions produced a high joint infection rate (82.5%) while no infection was recorded on larvae exposed to virus suspension only. Field and laboratory results suggest a strong association between S. spiculatus and MIV in natural populations of Cx. pipiens, in which S. spiculatus could be a mode of entry for the virus into the mosquito hemocele.     

Structure of aggregates of Tipula iridescent virus and polystyrene latex particles

Tipula iridescent virus aggregated with polystyrene latex particles 126 nm in diameter. There was a region of optimal proportion of the two particles. The aggregation proceeded faster and the amount of resultant aggregates was larger at the higher concentrations of the two particles, but the size of the individual aggregates was smaller. The aggregates consisted of clusters of the two particles with vacant spaces interspersed among them. A hypothetical model of the particle arrangements was presented. The aggregates were reversibly dissolved in 0.03% sodium dodecyl sulfate and 30% n-propanol and isopropanol. In the presence of lower concentrations of these solvents, the aggregation occurred at high temperatures but not at low temperatures. These results were interpreted as implicating hydrophobic interactions in the formation and stability of the aggregates.     

Dose dependency of time to death in single and mixed infections with a wildtype and egt deletion strain of Helicoverpa armigera nucleopolyhedrovirus

Recombinant insect nucleopolyhedroviruses lacking the egt gene generally kill their hosts faster than wild-type strains, but the response of insects to mixtures of virus genotypes is less well known. Here, we compared the survival time, lethal dose and occlusion body yield in third instar larvae of Helicoverpa armigera (Hübner) after challenge with wild-type H. armigera SNPV (HaSNPV-wt), a strain with a deletion of the egt gene, HaSNPV-LM2, and a 1:1 mixture of these two virus strains. A range of doses was used to determine whether the total number of OBs influenced the response to challenge with a mixture of virus strains versus single strains. At high virus doses, HaSNPV-LM2 killed H. armigera larvae significantly faster (ca. 20 h) than HaSNPV-wt, but at low doses, there was no significant difference in survival time between the viruses. The survival time after challenge with mixed virus inoculum was significantly different from and intermediate between that of the single viruses at high doses, and not different from that of the single viruses at low doses. No differences in lethal dose were found between single and mixed infections or between virus genotypes. The number of occlusion bodies produced per larva increased with time to death and decreased with virus dose, but no significant differences among virus types were found.     

Isolation and characterization of a granulosis virus from the tomato moth,Lacanobia oleracea,and its potential as a control agent

A disease causing death in Lacanobia oleracea (Lepidoptera: Noctuidae) occurring in glasshouses in Scotland was shown to be caused by a granulosis virus (GV). Structural properties of the virus were examined by electron microscopy, immunodiffusion, polyacrylamide gel electrophoresis, and restriction endonuclease analysis and compared with an isolate of GV from L. oleracea obtained from France. The two isolates were structurally very similar but could be distinguished by analysis of EcoRI digests of their DNAs. Bioassays of the virus gave LD₅₀ values from 10^4.3 capsules for second-instar larvae to 10^6.6 capsules for fifth-instar larvae. The French isolate was bioassayed in third-instar larvae and was not found to differ significantlyfrom the Scottish isolate. Two small glasshouse trials using the virus to control artificial infestations of L. oleracea indicated that high-volume sprays of virus at 10⁸ to 10⁹ capsules/ml achieved good control. An alternative strategy using much smaller amounts of virus to control the insect is discussed.     

Some effects of time,temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae

Dalgliesh R. J. and Stewart N. P. 1982. Some effects of time, temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae. International Journal for Parasitology12: 323–326. Percentages of larval ticks in which Babesia bovis and B. bigemina parasites could be detected (infection rates) were determined after the larvae had been exposed to temperatures between 9°C and 27°C for periods of 1–35 days and then either fed on calves or heated at 37°C to stimulate babesial development. Infection rates with both species increased during 2–4 weeks after the larvae hatched, regardless of the temperature of exposure. Infection rates with B. bovis were higher after exposure of larvae to 14°C than to 27°C. This effect was less pronounced with B. bigemina. Infection rates were higher in fed larvae than in unfed, ‘heat stimulated’ larvae. The findings indicate that infected larval ticks become more efficient vectors of Babesia during the first 2–4 weeks after hatching and that repeated sampling of a tick population is necessary to determine valid infection rates.     

Influence of starvation on larval development of Carcinus maenas L. (Decapoda : Portunidae)

Lethal and sublethal effects of particular starvation events were investigated in larvae of Carcinusmaenas L. Mean survival times of continuously starved zoeae-1 were approximately twice the normal stage duration (12, 18, 25°C), and both increased with falling temperatures. At 6°C zoea-1 was unable to develop to stage-2. No larva retained the ability for successful further development if starved for half the stage duration time and was then refed. The zoea-1 larvae had to feed for at least 20 % of the normal stage duration for some larvae to moult to zoea-2. Some initial feeding was necessary to start zoea-1 development. Beyond a certain point of energy and accumulation of reserves development of the larvae seems to continue regardless of feeding rates. The demands for larval feeding correspond very well with the larval moulting cycle. Larvae of C. maenas proved to be well adapted to natural shortage of food.