Gypsy moth cell lines divergent in viral susceptibility

Summary A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus. This research was funded in part through a reimbursable agreement with the U.S. Forest Service.     

Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses

Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3-6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2-10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5,000-75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.     

Lepidopteran cell lines after long-term culture in alternative media: Comparison of growth rates and baculovirus replication

Summary Three insect cell lines, IPLB-LdFB and IPLB-LdEIta from gypsy moth fat body and embryos and UFL-AG-286 from velvetbean caterpillar embryos, have been concurrently maintained for 1 to 12 yr on two media formulations, modified TC-100 containing 9% fetal bovine serum and Ex-cell 400, a commercial serum-free medium (SFM). Cells grown in each medium were tested for susceptibility to and productivity of various multiply embedded nucleopolyhedroviruses. The three lines chosen for these experiments fall into three categories of relative growth in SFM versus TC-100: LdFB cells grew similarly in each medium, LdEIta grew better in Ex-Cell than in TC-100, and AG-286 grew better in TC-100 than in Ex-Cell. The susceptibility of cells to infection also varies, although without any apparent correlation to which medium was best for supporting growth. Endpoint assays suggested that LdFB cells grown in serum-containing medium are more susceptible to virus infection than their SFM counterparts, while the opposite is true for LdEIta cells. Production of virus, based on numbers of occlusion bodies, showed fewer differences with only AcMNPV production with AG-286 in TC-100 being statistically higher than production of the same virus in Ex-cell 400. These studies suggest that long-term passage in alternative media may impact the ability of cells to support virus infection and replication, but the effects on each cell line and virus system need to be determined.     

Pathogen-Driven Outbreaks in Forest Defoliators Revisited: Building Models from Experimental Data

Models of outbreaks in forest-defoliating insects are typically built from a priori considerations and tested only with long time series of abundances. We instead present a model built from experimental data on the gypsy moth and its nuclear polyhedrosis virus, which has been extensively tested with epidemic data. These data have identified key details of the gypsy moth-virus interaction that are missing from earlier models, including seasonality in host reproduction, delays between host infection and death, and heterogeneity among hosts in their susceptibility to the virus. Allowing for these details produces models in which annual epidemics are followed by bouts of reproduction among surviving hosts and leads to quite different conclusions than earlier models. First, these models suggest that pathogen-driven outbreaks in forest defoliators occur partly because newly hatched insect larvae have higher average susceptibility than do older larvae. Second, the models show that a combination of seasonality and delays between infection and death can lead to unstable cycles in the absence of a stabilizing mechanism; these cycles, however, are stabilized by the levels of heterogeneity in susceptibility that we have observed in our experimental data. Moreover, our experimental estimates of virus transmission rates and levels of heterogeneity in susceptibility in gypsy moth populations give model dynamics that closely approximate the dynamics of real gypsy moth populations. Although we built our models from data for gypsy moth, our models are, nevertheless, quite general. Our conclusions are therefore likely to be true, not just for other defoliator-pathogen interactions, but for many host-pathogen interactions in which seasonality plays an important role. Our models thus give qualitative insight into the dynamics of host-pathogen interactions, while providing a quantitative interpretation of our gypsy moth-virus data.     

CO2-mediated changes in aspen chemistry: effects on gypsy moth performance and susceptibility to virus

We investigated the effects of long-term CO₂ enrichment on foliar chemistry of quaking aspen ( Populus tremuloides ) and the consequences of chemical changes for performance of the gypsy moth ( Lymantria dispar ) and susceptibility of the gypsy moth to a nucleopolyhedrosis virus (NPV). Foliage was collected from outdoor open-top chambers and fed to insects in a quarantine rearing facility. Under enriched CO₂, levels of leaf nitrogen declined marginally, levels of starch and phenolic glycosides did not change, and levels of condensed tannins increased. Long-term bioassays revealed reduced growth (especially females), prolonged development and increased consumption in larvae fed high-CO₂ foliage but no significant differences in final pupal weights or female fecundity. Short-term bioassays showed weaker, and sex-specific, effects of CO₂ treatment on larval performance. Correlation analyses revealed strong, negative associations between insect performance and phenolic glycoside concentrations, independent of CO₂ treatment. Larval susceptibility to NPV did not differ between CO₂ treatments, suggesting that effects of this natural enemy on gypsy moths are buffered from CO₂-induced changes in foliar chemistry. Our results emphasize that the impact of enriched CO₂ on plant–insect interactions will be determined not only by how concentrations of plant compounds are altered, but also by the relevance of particular compounds for insect fitness. This work also underscores the need for studies of genetic variation in plant responses to enriched CO₂ and long-term population-level responses of insects to CO₂-induced changes in host quality.     

The effects of cations on the activity of the gypsy moth (Lepidoptera: Lymantriidae) nuclear polyhedrosis virus

Fourteen cations were tested at a 1% concentration (wt:wt), as chlorides, for their effects on the biological activity of the gypsy moth, Lymantria dispar (L.), nuclear polyhedrosis virus (LdMNPV). Cupric chloride was toxic to gypsy moth larvae. Ferrous and ferric chloride were inhibitory to larval growth and development as well as to virus activity. Strontium chloride was inhibitory to virus activity but had no apparent effects on gypsy moth larvae. Six cations had little or no effect on virus activity (i.e., calcium, lanthanum, magnesium, nickel, potassium, sodium), whereas four cations (i.e., cobalt, manganese, ruthenium, zinc) acted as viral enhancers, as indicated by reductions in LC50s.     

Production of recombinant proteins by baculovirus-infected gypsy moth cells.

An experimental study was undertaken to evaluate alternative insect cell lines to Sf9 [from Spodoptera frugiperda (fall armyworm)] for the production of recombinant proteins. Insect cell lines from two different organisms were considered: IPLB-LdEIta (LdEIta) from Lymantria dispar (gypsy moth) and IPLB-HvT1 (HvT1) from Heliothis virescens (tobacco budworm). Both LdEIta and HvT1 produced higher total activity levels of recombinant beta-galactosidase in monolayer culture than Sf9 after infection with the Autographa californica nuclear polyhedrosis virus (AcMNPV). However, only LdEIta generated a product yield (activity per milligram of total protein) which exceeded that of Sf9 (by 25%), so its growth and production characteristics were investigated in depth. LdEIta generated production levels and yields of a recombinant rotaviral protein, VP4, which exceeded those of Sf9 by 84 and 38%, respectively. In suspension culture, the LdEIta cells grew as aggregates with a doubling time several hours longer than Sf9, but the recombinant product yields of LdEIta were still higher than Sf9 by 38% in this culture environment. beta-Galactosidase expression rates and cell death rates suggested that the difference in productivity between the two hosts was due to the ability of LdEIta to survive the baculovirus infection and produce recombinant proteins longer than Sf9. The presence of LdEIta aggregates in suspension culture may be used as a method to separate live cells from dead cells, labile product, and spent medium in recombinant protein production processes.     

Induced plant defenses breached? Phytochemical induction protects an herbivore from disease

Although wound-induced responses in plants are widespread, neither the ecological nor the evolutionary significance of phytochemical induction is clear. Several studies have shown, for example, that induced responses can act against both plant pathogens and herbivores simultaneously. We present the first evidence that phytochemical induction can inhibit a pathogen of the herbivore responsible for the defoliation. In 1990, we generated leaf damage by enclosing gypsy moth larvae on branches of red oak trees. We then inoculated a second cohort of larvae with a nuclear polyhedrosis virus (LdNPV) on foliage from the damaged branches. Larvae were less susceptible to virus consumed on foliage from branches with increasing levels of defoliation, and with higher concentrations of gallotannin. Defoliation itself was not related to any of our chemistry measures. Field sampling supported the results of our experiments: death from virus among feral larvae collected from unmanipulated trees was also negatively correlated with defoliation. In 1991, defoliation and gallotannin were again found to inhibit the virus. In addition, gallotannin concentrations were found to be positively correlated with defoliation the previous year. Compared with previous results that demonstrated a delecterious effect of induction on gypsy moth pupal weight and fecundity, the inhibition of the virus should confer an advantage to the gypsy moth. Since leaf damage levels increase as gypsy moth density increases, and since leaf damage inhibits the gypsy moth virus, there is the potential for positive feedback in the system. If phytochemical induction in red oak can inhibit an animal pathogen such as LdNPV, it suggests to us that induction in red oak is a generalized response to tissue damage rather than an adaptive defense against herbivores.     

Semipermissive Replication of a Nuclear Polyhedrosis Virus of Autographa californica in a Gypsy Moth Cell Line

Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, we demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with [(35)S]methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early alpha proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.     

Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA

Summary Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R², IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.     

Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication

Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged over five times in Ex-Cell 400 to which serum was added; both cell lines produced as much virus as that in SFM. These results suggest that it is not the serum per se but rather some other components which differ between the SFM and the SCM formulations that are responsible for the varied virus production obtained in these studies. The results of these studies suggest that a maintenance and virus production protocol can be developed with Ld cells which could improve overall efficiency of virus production. These studies also suggest that long-term maintenance of cells in SFM was not detrimental to their ability to produce baculoviruses.     

Comparative Replication of Lymantria dispar Nuclear Polyhedrosis Virus Strains in Three Continuous-Culture Cell Lines

We compared the replication of the gypsy moth (Lymantria dispar) nuclear polyhedrosis virus in two new cell lines, from embryos and fat body of L. dispar, and in a previously available ovarian cell line. Three virus isolates (the Hamden strain [LDP-67] used commercially as GYPCHEK, a plaque-purified clone of Hamden [5-7d], and an isolate from Abington, Mass. [Ab]) were each tested on the three cell lines. The fat-body-derived cell line proved best in terms of occlusion body production for all three virus strains, with the highest yield produced by the Abington strain. On the basis of these results, we conclude that a more efficient in vitro production of gypsy moth virus can be obtained by using the fat body cell line in conjunction with the Abington strain of the virus.     

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest. This revised version was published online in August 2006 with corrections to the Cover Date.     

Fluorescent brightener inhibits apoptosis in baculovirus-infected gypsy moth larval midgut cells in vitro

Fluorescent brighteners significantly lower the LC₅₀ and LT₅₀ in a variety of nucleopolyhedrovirus-insect host systems. In larvae of the gypsy moth, Lymantria dispar (L.), a European NPV strain of virus (LdMNPV) does not normally replicate in the midgut, but addition of a fluorescent brightener (Calcofluor M2R) to the virus suspension results in productive infections. In the current study, we show that LdMNPV also does not replicate in a larval midgut primary cell culture system unless a fluorescent brightener (Blankophor P167) is added. Morphological and cellular changes characteristic of apoptotic cell death were noted in infected midgut cells in vitro. We used the TUNEL assay to measure apoptosis in virus-challenged midgut cell cultures at 24-48 h post-inoculation. A significant decrease in apoptotic midgut cells was noted in the presence of 0.01 M brightener. The inhibition of apoptosis and presumptive inhibition of shedding of infected midgut cells in the presence of fluorescent brightener in the insect midgut appeared to promote virus replication and are likely to be partly responsible for enhancement of LdMNPV activity that is observed in gypsy moth larvae.     

中国舞毒蛾六个地理种群的RAPD分析及SCAR标记构建

舞毒蛾Lymantria dispar L.是世界性农林害虫, 包含不同的亚种, 其中亚洲舞毒蛾的雌蛾具有较强的飞行能力, 已成为国际性的重要检疫性有害生物。然而, 不同舞毒蛾亚种及种群间形态难辨, 因此采用传统的手段鉴别舞毒蛾亚种种群是很困难的。本研究首先采用RAPD标记分析了中国舞毒蛾6个地理种群的遗传多态性。结果表明, 所检测的舞毒蛾种群的遗传分化系数G_st为0.7571, 由此推算出的平均有效迁移数（基因流参数）N_em为0.1604, 说明不同舞毒蛾种群间的遗传分化程度较高, 缺乏广泛的基因流动。本研究在RAPD遗传分析基础之上, 筛选出了4个舞毒蛾种群的特异性遗传位点, 然后对这些特异性位点进行了克隆测序、 序列分析和位点特异性引物设计。结果表明, 其中2个舞毒蛾种群的位点特异性引物可产生序列特征性扩增区域（SCAR）标记。经验证, 这些标记可被用来鉴别特定的舞毒蛾地理种群, 因此有助于对这些舞毒蛾地理种群的分布与扩散进行监测。     

用免疫金颗粒标记鉴定舞毒蛾核型多角体病毒的抗原

用免疫电镜金颗粒标记技术准确、快速地对舞毒蛾(Lymantria dispar)核型多角体病毒抗原进行了定位和鉴定.舞毒娥病毒的多克隆抗体与同源的多角体抗原之间存在着强烈的亲和性,但与不同源的松柏锯角叶蜂(Neodiprion sertifer)病毒多角体抗原仅有极微弱的交叉反应.舞毒蛾病毒粒子和核衣壳抗原也能与同源的多克隆抗体作用.在被病毒感染的舞毒蛾脂肪体细胞核中,成熟的多角体被金颗粒重重标记,其外缘的游离病毒粒子和核衣壳亦被标记,但亲和力较弱.在被感染的脂肪体细胞核和质内发现一种与多角体蛋白晶体不同源的菱形结晶体.     

Effects of SPLAT® GM sprayable pheromone formulation on gypsy moth mating success

Several integrated pest management programs rely on the use of mating disruption tactics to control insect pests. Some programs specifically target non‐native species, such as the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). We evaluated SPLAT^® GM, a new sprayable formulation of the gypsy moth sex pheromone disparlure, for its ability to disrupt gypsy moth mating. The study was conducted in 2006, 2007, and 2008 in forested areas in Virginia, USA. Mating success of gypsy moth females was reduced by >99% and male moth catches in pheromone‐baited traps by >90%, in plots treated with SPLAT^® GM at dosages ranging from 15 to 75 g of active ingredient (a.i.) ha^?1. Dosage‐response tests conducted in 2008 indicated that SPLAT^® GM applied at a dosage of 7.5 g a.i. ha^?1 was as effective as a 15 g a.i. ha^?1 dosage.     

Establishment and dominance of an introduced herbivore has limited impact on native host-parasitoid food webs

The gypsy moth is considered one of the most harmful invasive forest insects in North America. It has been suggested that gypsy moth may indirectly impact native caterpillar communities via shared parasitoids. However, the impact of gypsy moth on forest insect food webs in general remains unstudied. Here we assess such potential impacts by surveying forest insect food webs in Ontario, Canada. We systematically collected caterpillars using burlap bands at sites with and without histories of gypsy moth outbreak, and then reared these caterpillars until potential parasitoid emergence. This procedure allowed us to generate quantitative food webs describing caterpillar-parasitoid interactions. We estimated the degree of parasitoid sharing between gypsy moth and native caterpillars. We also statistically modeled the effect of gypsy moth outbreak history and current gypsy moth abundance on standard indices of quantitative food web structure and the diversity of parasitoid communities. Rates of gypsy moth parasitism were very low and gypsy moth shared very few parasitoids with native caterpillars, suggesting limited potential for indirect interactions. We did not detect any significant effects of gypsy moth on either food web structure or parasitoid diversity, and the small amount of parasitoid sharing strongly implies that this lack of significance is not merely due to low statistical power. Our study suggests that gypsy moth has limited impact on native host-parasitoid food webs, at least for species that use burlap bands. Our results emphasize that extrapolations of theoretical and experimental conclusions on the impacts of invasive species should be tested in natural settings.     

Asynchrony between Host Plant and Insects-Defoliator within a Tritrophic System: The Role of Herbivore Innate Immunity

The effects of asynchrony in the phenology of spring-feeding insect-defoliators and their host plants on insects’ fitness, as well as the importance of this effect for the population dynamics of outbreaking species of insects, is a widespread and well-documented phenomenon. However, the spreading of this phenomenon through the food chain, and especially those mechanisms operating this spreading, are still unclear. In this paper, we study the effect of seasonally declined leafquality (estimated in terms of phenolics and nitrogen content) on herbivore fitness, immune parameters and resistance against pathogen by using the silver birch Betula pendula—gypsy moth Lymantria dispar—nucleopolyhedrovirus as the tritrophic system. We show that a phenological mismatch induced by the delay in the emergence of gypsy moth larvae and following feeding on mature leaves has negative effects on the female pupal weight, on the rate of larval development and on the activity of phenoloxidase in the plasma of haemolymph. In addition, the larval susceptibility to exogenous nucleopolyhydrovirus infection as well as covert virus activation were both enhanced due to the phenological mismatch. The observed effects of phenological mismatch on insect-baculovirus interaction may partially explain the strong and fast fluctuations in the population dynamics of the gypsy moth that is often observed in the studied part of the defoliator area. This study also reveals some indirect mechanisms of effect related to host plant quality, which operate through the insect innate immune status and affect resistance to both exogenous and endogenous virus.