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 effect of temperature upon Tipula iridescent virus infection in Tipula oleracea

The mean incubation period (time from inoculation with virus to first appearance of iridescence) was used as an indication of the rate of replication of Tipula iridescent virus (TIV) in Tipula oleracea larvae. The mean incubation period and survival time (time from inoculation with virus to death) were compared with the mean instar duration at a series of temperatures. In most stages of the insect the optimum temperature for the replication of TIV and the temperature for the shortest mean survival time coincided with the peak temperature (the temperature for the fastest development of the insect stage). The peak temperature for T. oleracea does not appear to be the same for each stage, and the optimum temperature for TIV replication appears to be closely linked to the peak temperature of the infected stage. The optimum temperature (the temperature at which most individuals survived from hatching to the adult stage) of the insect was 20°C. Tipula iridescent virus replicated in T. oleracea larvae and pupae at 3° and 27°C, which are near the temperature limits for the insect. Incubation periods and survival times in TIV-inoculated larvae incubated in the field were much longer in winter than in summer.     

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.     

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.

     

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.     

Thermal therapy of the flacherie virus disease in the silkworm,Bombyx mori

An effective method of thermal therapy to fifth-instar silkworm larva (Bombyx mori) has been developed for the control of the flacherie virus disease. Fifth-instar larvae, which were infected with the flacherie virus in their fourth instar, were reared at 27°C for 5 days and then transferred to 37°C for 1–3 days. Such larvae were able to form normal cocoons. The basis for the thermal therapy appeared to be: (1) the discharge of the virus-infected goblet cells into the midgut lumen and out with the feces and (2) the escape of the newly regenerated goblet cells from infection and virus multiplication.     

Transmission of a baculovirus in populations of Oryctes rhinoceros

The transmission of the baculovirus of Oryctes rhinoceros, previously called Rhabdionvirus oryctes, was studied. O. rhinoceros adults became infected with the virus when kept in a mixture of sawdust and ground-up virus-killed larvae or together with other virus-infected adults. In the field, mated females were more frequently infected than unmated females. Adults developing from larvae that had survived exposure to various dosages of the virus were not infected. No virus infections occurred in larvae hatching from eggs surface-contaminated with the virus. Larvae hatching from eggs laid by virus-infected females very rarely were infected.In the O. rhinoceros population the virus is transmitted most frequently during mating, possibly when the uninfected partner contacts virus material excreted by the infected partner. The virus can be transmitted in a similar way when infected and healthy beetles feed together in palm trees. Beetles visiting larval breeding sites containing freshly virus-killed larvae can become infected. Virus-infected beetles can pass the infection to healthy larvae when visiting a breeding site.     

Modeling horizontal transmission of microsporidia infecting gypsy moth,Lymantria dispar (L.), larvae

We developed a simulation model that describes the horizontal transmission of three different microsporidia, Endoreticulatus schubergi, Nosema lymantriae and Vairimorpha disparis and their insect host, the gypsy moth, Lymantria dispar. The model describes the stage specific development and mortality of uninfected, latently infected or infectious hosts, the food consumption, the infection by spore-laden feces of E. schubergi and N. lymantriae and by spore-laden cadaver of N. lymantriae and V. disparis. Model results were compared to percent infection of L. dispar test larvae published in earlier studies using caged oak trees and potted oak-plants. When feces were selected as the source of spores for transmission of E. schubergi or N. lymantriae, the model estimated a percent infection in susceptible larvae that was in the range of the experimental studies. When spore-laden cadavers were the source of spores of N. lymantriae or V. disparis, the model did not correctly predict the experimentally measured percent infection in susceptible larvae. The most critical points of the simulation model are exact calculation of spore release, mortality and exact determination of the transmission coefficients when cadavers were included as a source for microsporidian infection.     

Parasitoid-mediated transmission of an iridescent virus

We examined the interaction between an invertebrate iridescent virus (IIV) isolated from Spodoptera frugiperda (J.E. Smith) and the solitary ichneumonid endoparasitoid Eiphosoma vitticolle Cresson. In choice tests, parasitoids examined and stung significantly more virus infected than healthy larvae, apparently due to a lack of defense reaction in virus infected hosts. Parasitoid-mediated virus transmission was observed in 100% of the female parasitoids that stung a virus infected host in the laboratory. Each female parasitoid transmitted the virus to an average (+/-SE) of 3.7+/-0.3 larvae immediately after stinging an infected larva. Caged field experiments supported this result; virus transmission to healthy larvae only occurred in cages containing infected hosts (as inoculum) and parasitoids (as vectors). The virus was highly detrimental to parasitoid development because of premature host death and lethal infection of the developing endoparasitoid. Female parasitoids that emerged from virus infected hosts did not transmit the virus to healthy hosts. We suggest that the polyphagous habits of many noctuid parasitoids combined with the catholic host range of most IIVs may represent a mechanism for the transmission of IIVs between different host species in the field.     

Acid and alkaline phosphatase activity in the fat body and midgut of the beet armyworm,Spodoptera exigua (Lepidoptera: Noctuidae), infected with nuclear polyhedrosis virus

Changes in the activity of acid and alkaline phosphatase in Spodoptera exigua larvae infected with nuclear polyhedrosis virus have been investigated. Three days after per os infection, the activity of acid phosphatase in the fat body and midgut of infected larvae was significantly higher than that in normal larvae. Alkaline phosphatase activity did not show such significant changes. There were differences in the phosphatase patterns depending on whether their activities were expressed as enzyme units per milligram of fresh organ weight or per milligram of homogenate protein. The literature relevant to the subject allows us to conclude that the increase in phosphatase activities in S. exigua larvae is not specifically associated with virus infection itself, but, rather, is a reaction of the insect organism to the diminishing supply of energy sources.     

An iridescent virus fromPopillia japonica (Col.: Scarabaeidae)

A very low incidence (<0.01%) of a blue iridovirus (IV) was found in larvae of the Japanese beetle,Popillia japonica Newman, that were sampled over a two year period on Terceira Island (Azores, Portugal). In the most heavily infected larvae, a deep blue iridescence was observed, particularly in the fat body. Transmission electron microscopy revealed the characteristic crystalline arrays of the hexagonal virus particles in the cytoplasm of fat body cells, tracheal matrix, muscle, hypodermis and blood cells. Crystals of the virus particles were also observed freely circulating in the hemolymph. The average diameter of negatively stained purified virus particles was 157 nm. Similarities and differences with other IVs found in the Scarabaeidae are discussed. Considering the broad host range of some of the iridescent viruses, the relatively recent invasion of Terceira byP. japonica, and the rarity of the virus in the beetle, it is probable that the infection was the result of transmission from another species of soil-inhabiting arthropod. Its value as a potential biological control agent ofP. japonica is negligible.     

A Histological Procedure to Study Fungal Infection in the Wax Moth Galleria Mellonella

The invertebrate model Galleria mellonella is a widely used factitious host to study the microbial pathogenesis in vivo. However, a specific procedure for the recovery and the processing of the infected tissues, important for a better understanding of the host-pathogen interactions, has not been reported to our knowledge. In the present study we describe a new procedure of fixation and processing of larval tissue that allows studying the larval topographic anatomy and assessing the morphological changes due to the fungal infection. Lepidopteran larvae were infected with Candida albicans strains displaying various biofilm-forming abilities. The whole larvae were then examined for tissue changes by histological techniques. We show that comparing cutting planes, serial transversal sections of paraffin-embedded larva result in better accuracy and information recovering. Using this technique, it was possible to preserve the integrity of G. mellonella internal structures allowing the detailed analysis of morphological differences in different experimental groups (i.e., healthy vs infected larvae). We were also able to study strain-related differences in the pathogenesis of C. albicans by observing the immune response elicited and the invasiveness of two isolates within the larval tissues.In general, by processing the whole larva and optimizing routinely histochemical stainings, it is possible to visualize and analyse infected tissues. Various degrees of pathogenicity (strain- or inoculum-related), and the infection time course can be described in details. Moreover, the host immune response events can be followed throughout the infectious process leading to a comprehensive picture of the studied phenomenon.Key words: Galleria mellonella, Candida albicans, host-pathogen interaction     

The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.     

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.     

Effect of a nuclear polyhedrosis virus on the relationship between Trichoplusia ni (Lepidoptera: Noctuidae) and the parasite, Hyposoter exiguae (hymenoptera: Ichneumonidae)

The effect of a nuclear polyhedrosis virus on the relationship between Trichoplusia ni and the parasite, Hyposoter exiguae, was investigated to determine if the virus could invade and multiply in the tissues of the parasites, if parasites which emerged from virus-infected T. ni larvae had normal emergence, fecundity, and longevity, and if the parasite could serve as a vector for the virus. Light microscopy revealed particles which appeared to be polyhedra within the lumen of the midgut of parasite larvae from virus-infected hosts. Transmission electron microscopy confirmed the presence of polyhedra and free virions within the midgut of the larvae. Polyhedra or free virions were never found within any parasite tissues. Parasite larvae within hosts exposed to virus before parasitization perished when their hosts died of virus infection. Parasite larvae in hosts exposed to virus after parasitization completed their development before their hosts died of virus infection. The proportion of parasites which survived increased as the time between host parasitization and host virus exposure increased. Parasite larvae which developed in hosts exposed to the virus soon after parasitization spent significantly less time in their hosts than did parasites which developed in noninfected hosts. There was no significant difference in time spent in the pupal stage, percent adult emergence, adult longevity with and without food and water, and fecundity of parasites which developed in virus-infected hosts and those which developed in noninfected hosts. Female parasites laid as many eggs in virus-infected hosts as they did in noninfected hosts. Sixty percent of the female parasites which oviposited in virus-infected hosts vectored infective doses of virus to an average of 6% of the healthy hosts subsequently exposed to them. None of the healthy host larvae exposed to male parasites which had been exposed to virus-infected host larvae became infected with the virus. Forty percent of the female parasites which developed in virus-infected hosts transmitted infective doses of the virus to an average of 65% of the healthy host larvae exposed to them. Ninety percent of the male parasites which developed in virus-infected hosts transferred infective doses of the virus to an average of 21% of the healthy host larvae exposed to them.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

Effects of an ascovirus (HvAV-3e) on diamondback moth, Plutella xylostella, and evidence for virus transmission by a larval parasitoid

Ascoviruses (AVs) are pathogenic to lepidopteran larvae, and most commonly attack species in the Noctuidae. The unique pathology includes cleavage of host cells into virion-containing vesicles which leads to the milky white colouration of the hemolymph as opposed to the clear hemolymph of healthy larvae. Recently, we showed that a Heliothis virescens AV (HvAV-3e) isolate is able to induce disease in Crocidolomia pavonana F. (Lepidoptera: Crambidae), affecting feeding, growth and survival of infected larvae. In this study, we investigated the effect of different variants of HvAV-3e on diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) larvae, another non-noctuid host. In hemolymph inoculation bioassays fourth instar larvae showed a significant dose response to each of the HvAV-3e variants and significant differences between the virulence of the three variants were detected. Both second and fourth instars were readily infected with the virus and infected individuals demonstrated significant reductions in food consumption and growth. The majority of infected individuals died at the larval or pupal stage and individuals which developed into adults were usually deformed, less fecund than non-infected controls and died shortly after emergence. In transmission studies, Diadegmasemiclausum (Hymenoptera: Ichneumonidae), a key parasitoid of diamondback moth, infected healthy host larvae during oviposition following previous attack of HvAV-3e infected hosts. In choice tests D. semiclausum did not discriminate between infected individuals but host infection had no detectable impact on the development of immature D. semiclausum or on subsequent adults.     

Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus following infection with Lagenidium giganteum

The effect of infection of larvae of the mosquito Culex pipiens quinquefasciatus by the fungus Lagenidium giganteum has been studied from a biochemical standpoint. Methods were developed to analyze larval extracts that were essentially free of parasitic material. Biochemical parameters investigated on a per larva basis were protein, and the enzymes o-diphenol oxidase, glutamate transaminase, alkaline phosphatase, trehalase, and chitobiase. The behavior of the total amino acid and sugar pools were also examined. In general it was found that the infected larvae exhibited a significant decrease in rate of synthesis of most of the parameters studied and that the rates eventually fell to zero when compared to healthy control organisms. The progress of mycosis was correlated with visual evidence and it was concluded that the larvae were dying from starvation as a consequence of the utilization of their endogenous reserves by the parasite. Death of the larvae occurred as a general rule, in laboratory conditions, some 48 hr after initiation of infection by the fungus.     

保幼激素类似物对斜纹夜蛾核多角体病毒增殖的影响

分别用5×10⁶,1×10⁷,5×10⁷,1×10⁸ PIBs/mL 4种浓度的斜纹夜蛾核多角体病毒(SpltNPV)感染斜纹夜蛾末龄幼虫（第6龄）,并于同龄期以10μg/头的保幼激素类似物(JHA) methoprene分别对各感染组进行点滴处理,以研究JHA对SpltNPV增殖的影响。研究表明,与对照组相比各不同浓度处理组病毒总产量分别提高227.06%、128.71%、52.62%、33.15%,平均单头病毒含量分别提高49.15%、48.40%、36.40%和31.11%,病毒感染死亡率分别提高119.21%、52.72%、12.64%和1.12%。其中以1×10.7 PIBs/mL感染再经JHA处理的病毒总产量和平均单头病毒含量最高,分别为1 701.8×10⁸PIBs和60.1×10⁸ PIBs。各处理组的病毒总产量和平均单头病毒含量均显著高于其对照组。在此基础上,进一步研究了JHA对染毒与未染毒宿主消化生理的影响。结果表明,JHA处理不仅延长了6龄幼虫的寿命,增加了其取食量,而且还显著提高了幼虫的食物转化率和病毒产量。     

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 3^rd — and 4^th — 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.