Institute of Cell Biology, University of Berne, Baltzerstrasse 4, CH-3012 Bern, Switzerland
Correspondence
Beatrice Lanzrein
beatrice.lanzrein{at}izb.unibe.ch
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession numbers for the sequences described in this paper are AJ278677, Z58828 and Z31378.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We are studying the bracovirus of the egglarval parasitoid Chelonus inanitus (CiV) and its role in parasitism of one of its natural hosts, Spodoptera littoralis. The genome of CiV consists of at least 1012 segments with sizes between 7 and 31 kb, which appear to be singly encapsidated (Albrecht et al., 1994). Five segments (CiV12, CiV14, CiV14.5, CiV16.8 and CiV21) have been fully sequenced up to now (Wyder et al., 2002
; GenBank accession no. AJ627175) and all of them appear to contain at least one gene. The wasp oviposits into the egg stage of the host and it was found that viral DNA then enters the cells of the host embryo within the first day after parasitization (Wyder et al., 2003
). CiV prevents encapsulation of the parasitoid (Pfister-Wilhelm & Lanzrein, 1996
; Stettler et al., 1998
) and, along with the parasitoid larva, greatly influences development of the host. Metamorphosis is induced precociously in the fifth instar, followed by a developmental arrest in the prepupal stage (Grossniklaus-Bürgin et al., 1994
). CiV, synergized by venom, was seen to be responsible for the developmental arrest (Soller & Lanzrein, 1996
), whilst for the precocious onset of metamorphosis, the parasitoid larva, in the presence of CiV/venom, has been shown to be responsible (Pfister-Wilhelm & Lanzrein, 1996
). Analysis of expression of eight CiV genes in the course of parasitization revealed four patterns, namely early, late, persistent and early and late, whereby highest amounts of transcripts were seen for the late-expressed genes (Bonvin et al., 2004
). Here, we describe the partial or complete cloning and analysis of three CiV genes that had been predicted from sequencing segments CiV12 and CiV16.8. Furthermore, we report on the use of RNA interference (RNAi) to analyse the function of late-expressed viral genes in parasitized and in CiV/venom only-containing hosts.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Screening and cloning.
Two existing cDNA libraries in gt11, one from fifth-instar parasitized larvae and the other from sixth-instar X-ray-parasitized larvae at the early cell-formation stage, were used (Johner & Lanzrein, 2002
). Production of digoxigenin-labelled probes with primers on predicted exons of 12g2, library screening, cDNA cloning into pBluescript and sequencing were done as described previously (Johner & Lanzrein, 2002
). For partial cloning of 12g1, primers 12g1forw (5'-GAGTCCATGCCGAATGTCAC-3') and 12g1rev (5'-CTTCTTGCACAGCGACGAAC-3') were set to amplify the middle region of 12g1, as predicted with FGENESH 1.0 and Drosophila settings. For partial cloning of 16.8g1, primers 16.8g1forw (5'-CGAAACTTCTTTCCAGATCCAG-3') and 16.8g1rev (5'-CCGGACGCCAGTAATAATAAG-3') were set to amplify the middle region of 16.8g1, as predicted with FGENESH 1.0 and Drosophila settings. For both genes, the reaction was carried out with 250 ng cDNA of X-ray-parasitized first-instar larvae and a Taq core kit (Qiagen). Gel-purified PCR products were cloned into a pCR II TOPO vector (Invitrogen) and sequencing was done on an ABI 3000 sequencer (Applied Biosystems).
Southern and Northern blot analyses.
For Southern blots, calyx fluid was collected and purified as described previously (Albrecht et al., 1994). Undigested and HindIII-digested calyx DNA (13 µg) was separated on a 0·8 % agarose gel by field-inversion gel electrophoresis (FIGE), blotted and hybridized as described previously (Wyder et al., 2002
). High-stringency washes were done in 0·2x SSC [1x SSC is 0·15 M NaCl, 15 mM sodium citrate (pH 7·0)], 0·1 % SDS, at 65 °C twice for 15 min. cDNAs of 12g1, 12g2 and 16.8g1 were used as probes after two gel-purification steps and labelling with Ready-to-Go DNA-labelling beads (Amersham Biosciences) in the presence of [
-32P]dCTP. Northern blots were performed as described previously (Johner & Lanzrein, 2002
). As a probe, 12g2 cDNA radiolabelled as described above was used. As a control, a fragment of S. littoralis actin (GenBank accession no. Z46873) was used.
Preparation of dsRNA.
To synthesize both sense and antisense RNAs from CiV genes, two clones containing the insert in opposite directions served as template, or a single clone was transcribed with two RNA polymerases. The following plasmids, which were first linearized, were used. On CiV14, cloning of 14g1 and 14g2 has been reported (Johner & Lanzrein, 2002); clone 6.5.1.#16 contains the 5' end of 14g1 cDNA (1555 bp) and was transcribed with either T7 or T3 RNA polymerase. Clones 6.5.1.#17 and
7.3#5 contain the full 14g2 cDNA in opposite directions (615 bp). On CiV12, 12g1 was partially and 12g2 fully cloned (Fig. 1
). In the case of 12g1, clones 12g1#2 and 12g1#4 contain the middle part of the ORF in opposite directions (862 bp) and, in the case of 12g2, clones 12g2#11 and 12g2Z1T7 contain the insert in opposite directions. T7 RNA polymerase was used with all of these clones. For the partially cloned 16.8g1 (Fig. 1
), clone Ro#7 (762 bp) was used with either T7 or SP6 RNA polymerase. RNA strands were synthesized in a 100 µl reaction volume, containing 5 µg plasmid DNA, 5x HEPES/KOH/DDT [1x HEPES/KOH/DDT is 1 M HEPES/KOH (pH 7·5), 0·5 M dithiothreitol], 5 mM each NTP, 100 µg BSA ml1, 20 U RNAsin and 300 U RNA polymerase. For T3 polymerase, 10x transcription buffer (Boehringer Mannheim) was added and for T7 polymerase, 5x MgCl2/spermidine buffer (1x MgCl2/spermidine buffer is 1 M MgCl2, 1 M spermidine) was used. After 2 h incubation at 37 °C, an additional 300 U polymerase was added and the mix was incubated for another 2 h. Thereafter, 200 µl diethyl pyrocarbonate (DEPC) H2O was added and the solution was centrifuged briefly. The RNA in the supernatant was purified by acidic phenol extraction and ethanol precipitation in the presence of 100 µg glycogen. The pellet was diluted in DEPC H2O and the yield was measured in a spectrophotometer. Quality of the transcripts was checked on a denaturing gel. For annealing of the complementary strands, approximately 1 µg of each was dissolved in 10 mM Tris and 1 mM EDTA, pH 8, in a 50 µl reaction volume and the tube was placed in a PCR machine (Mastercycler gradient; Eppendorf). At first, the temperature was set for 10 min at 95 °C, then it was reduced by 0·5 °C every 2 min until 70 °C was reached, followed by cooling down to room temperature. The annealed RNA was analysed on a gel (1 % agarose/TBE) and was only used for injection when most of the RNA was of the expected length. Before injection, the dsRNA in TE was diluted with DEPC H2O. Initially, various concentrations were tested (0·1, 1 and 10 µM), but 0·2 µM was used most often.
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequencing of 12g1, comparison with genomic CiV12 and predictions with FGENESH revealed that all four splice sites were predicted correctly, again illustrating the high accuracy of FGENESH 1.0. The sequence of the partial cDNA thus reflects the predicted ORF1 on CiV12 and encompasses positions 77408914 on the genomic clone (Fig. 1; GenBank accession no. Z58828). Only at position 630 of the cDNA (position 8682 of the genomic clone), we found an A, whereas genomic clone 1G10 has a T. This mutation possibly occurred in the cloning process. Database searches revealed that 12g1 has high similarity to 14g1, but not to other known proteins.
12g2 is expressed very late in parasitization (Bonvin et al., 2004) and this gene could be cloned from the cDNA library of last-instar X-ray-parasitized larvae (Fig. 1a, b
). The two sequenced clones were identical, with the exception of the 5' untranslated region (UTR), where one clone was 2 bp longer (611 and 609 bp). Comparison with the genomic clone CiV12 and the FGENESH predictions revealed that all three exons and both introns had been predicted correctly. A polyadenylation signal (AATAAA) was found 19 nt upstream of the poly(A) tail (Fig. 1b
). 12g2 cDNA contains one putative ORF of 243 bp, encoding a 80 aa putative peptide. Two stop codons were found in-frame in the 5' UTR, indicating that the 5' end is complete. The hypothetical peptide contains two potential N-glycosylation sites and three potential protein kinase C phosphorylation sites (Fig. 2b
). The peptide is predicted to be localized in the nucleus. No significant similarity to known proteins was found, with the exception of CiV14g2 putative protein (Johner & Lanzrein, 2002
), to which 57 % identity and 76 % similarity were found.
|
RNAi with X-ray-parasitized S. littoralis
For the CiV genes 14g1 and 14g2, it had been shown that they are upregulated in last-instar X-ray-parasitized larvae when developmental arrest is induced (Johner & Lanzrein, 2002). Here, it was attempted to rescue X-ray-parasitized larvae from developmental arrest by silencing these two genes. S. littoralis eggs were parasitized with X-ray-irradiated wasps and thereafter, single-stranded (ss) RNA or TE (controls) or dsRNA of 14g1 and 14g2 alone or in combination was injected; a portion of the eggs was left uninjected. The larvae were observed and, after pupation or developmental arrest, the resulting phenotypes were scored by using the pp system of Soller & Lanzrein (1996)
. Fig. 3(a)
shows pictures of a normal pupa (0 pp) and several arrested larvalpupal intermediates, as observed after injection of calyx fluid/venom; after X-ray parasitization, the majority score 12 pp and about 20 % score 14 pp (Soller & Lanzrein, 1996
). Fig. 3(b)
shows typical examples of X-ray-parasitized larvae being partially rescued by RNAi with 14g1 and 14g2: a deformed pupa scoring 2 pp or larvalpupal intermediates scoring 9 or 11 pp. Injections of various concentrations from 0·1 to 10 µM gave comparable effects and thus results of the scores of all injections and controls are shown in Fig. 4
. After control injections or no injection, all larvae became developmentally arrested and scored mainly 1214 pp (Fig. 4a
). When 14g1 dsRNA was injected, a small percentage of the larvae were rescued almost fully (04 pp) and approximately 10 % were partially rescued and scored 9 pp (Fig. 4b
). These larvalpupal intermediates had larval legs and their abdomen and the ventral part of the body was covered with the partially ecdysed larval cuticle; however, the dorsal part of thorax and head displayed a pupa-like cuticle (Fig. 3b
). When 14g2 dsRNA was injected, only very few scored 03 pp, but around 30 % were partially rescued and scored 11 pp (Fig. 4c
); these larvalpupal intermediates were more pupa-like in the abdomen than X-ray-parasitized larvae (Fig. 3b
). Thus, 14g1 and 14g2 dsRNA injection led to slightly different phenotypes. When 14g1 and 14g2 dsRNAs were injected together, a large portion scored 911 pp and some scored 13 pp (Fig. 4d
). For statistical evaluation, three classes of pp scores were defined: 1217 pp (no rescue effect), 611 pp (intermediate rescue effect) and 15 pp (strong rescue effect). Normal pupae scoring 0 pp were not included in this presentation, as it is possible that single eggs were not parasitized and these would then erroneously be valued as a strong rescue effect. Prepupae scoring 18 pp were also not included, as they are not the result of X-ray parasitization, but rather of sickness. Statistical analyses by a
2 test revealed that the effect of dsRNA injection was significant for both genes (for
2 and P values, see legend to Fig. 4
). These data suggest that, to some extent, it is possible to silence polydnavirus genes in vivo and that 14g1 and 14g2 play a role in inducing developmental arrest in the prepupal stage.
|
|
|
The mean hatching rate of injected eggs was 43 %, but the differences between egg clutches ranged from 10 to 65 %. Also, non-injected eggs can display great mortality and we know from earlier experiments that the manipulation of the eggs (brushing, attaching on glass slide etc.) already reduces the hatching rate of the host (Lanzrein et al., 2001). Parasitoid mortality was also occasionally considerable and varied greatly between egg clutches; from studies on nutritional physiology, we know that the parasitoid larva and pupa are very sensitive to the nutritional status of the host larva (M. Kaeslin, personal communication). Thus, some parasitoids fail to develop fully, even without experimental disturbance. In the dsRNA experiments with parasitized eggs, we thus only evaluated egg clutches in which over 70 % successful parasitoid development was observed. After injection of 14g1 and 14g2 dsRNA together, fewer adult wasps emerged than in controls (
2=8·593, P=0·003) and the majority were in category IV (Fig. 7
); most scored 79 dfp, which indicates that the parasitoid did not manage to emerge from the host. Also, separate injection of 14g1 and 14g2 dsRNA increased the number of parasitoid larvae not emerging from the host, but the effect was not statistically significant (data not shown). Injection of 12g1 dsRNA significantly reduced the number of adult wasps (
2=7·52, P=0·006) and increased the proportion of parasitoids that died before or at emergence from the host (Fig. 7b
). With 12g2 dsRNA, no effect was seen and with 16.8g1 dsRNA, the number of adult wasps was only weakly reduced (data not shown). These data show that injection of dsRNA of viral genes into parasitized eggs can reduce the developmental success of the parasitoid; however, this approach is even more difficult than injection into X-ray-parasitized eggs, as parasitoid larvae are very vulnerable under experimental conditions.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Measurement of relative amounts of transcripts showed that 14g1, 14g2 and 12g2 are transcribed only towards the end of parasitization or X-ray parasitization, when the CiV-dependent developmental arrest in the prepupal stage is induced and 12g1 is expressed early and late (Johner & Lanzrein, 2002; Bonvin et al., 2004
). Injection of dsRNA of 14g1 and 14g2 alone or in combination into X-ray-parasitized eggs could partially reverse the developmental arrest in a way such that several individuals became more pupa-like; interestingly, dsRNA of 14g1 affected mainly the anterior part and that of 14g2, mainly the posterior part (Figs 3 and 4
). These observations indicate a role of these viral genes in preventing pupation of the host. This is the first in vivo analysis of polydnavirus gene function by RNAi, but only 1015 % of the individuals showed a strong and up to 40 % a weak effect. This could be due to poor penetration of host cells, dilution in the course of host growth or degradation of the dsRNA. It has to be kept in mind that these dsRNA molecules must be taken up into cells of the embryo and maintained for over 10 days, possibly in the intermediary form of small interfering RNAs, before any target mRNA is synthesized. Attempts to measure the extent of gene silencing by real-time PCR with reverse-transcribed RNA of potentially affected last-instar larvae were inconclusive until now. With the early- and late-expressed gene 12g1 (Bonvin et al., 2004
), the rescue effect after injection of dsRNA into X-ray-parasitized eggs was only very weak, and no effect was seen with 12g2 or 16.8g1 (Fig. 5
). RNAi has recently been applied successfully to a lepidopteran cell line to study the role of polydnavirus genes in immunosuppression (Beck & Strand, 2003
). With respect to comparable in vivo applications of RNAi, an effectiveness of approximately 50 % was observed after injection of dsRNA of a developmental gene into early embryos of a spider (Schoppmeier & Damen, 2001
). Injection of dsRNA into syncytial blastoderm-stage embryos was very efficient in Drosophila, Tribolium, Bombyx and Gryllus (Brown et al., 1999
; Quan et al., 2002
; Miyawaki et al., 2004
) but, in Drosophila, it was not effective in later embryonic stages (Brown et al., 1999
). It is thus conceivable that the low and variable effectiveness observed here has to do with the time point of injection and possibly also with the site of deposition of the dsRNA in the egg. Interestingly, injection of dsRNA was effective in adult Drosophila (Goto et al., 2003
), adult Anopheles (Blandin et al., 2004
) and planarians (Sánchez Alvarado & Newmark, 1999
). Parental RNAi was applied successfully in Hyalophora (Bettencourt et al., 2002
). It is still unclear how the dsRNA penetrates the cells. Silencing by RNAi of baculovirus genes that are essential for virus propagation was seen to protect Tenebrio larvae from infection (Valdes et al., 2003
) and it was found that this effect is strongly dose-dependent (Flores-Jasso et al., 2004
). Comparison of the expression patterns of the eight CiV genes analysed up to now reveals four different patterns and indicates a stage-dependent regualtion (Bonvin et al., 2004
). We assume the existence of at least 2035 CiV genes and it is thus very likely that the CiV-induced developmental arrest results from the combined effect of several known and unknown viral genes. This would mean that dsRNA of all CiV genes involved in developmental arrest would have to be introduced successfully to fully reverse the developmental arrest.
With parasitized eggs, the situation is even more complex. The experimental procedures cause some mortality and we know that this parasitoid is susceptible to various stress conditions: developmental failures as described in Table 1 can occasionally even occur in the rearing colony. Variability in parasitoid developmental success has also been observed in other systems (Boulétreau, 1986
). The combination of limited efficiency of the dsRNA application with a variable background mortality hampered the RNAi approach in parasitized eggs. Nevertheless, the injection of 14g1 and 14g2 dsRNA together or 12g1 alone significantly reduced parasitoid survival; the majority of affected parasitoid larvae could not emerge from the host (Fig. 7
). These three late-expressed CiV genes could thus play a role in keeping the host cuticle soft to allow egression of the parasitoid and feeding on the host. In non-parasitized larvae, the cuticle becomes tougher in the stage of pupal cell formation and the delay of this process by virus/venom can be considered the initial phase of the developmental arrest. In this stage, ecdysteroid titres are reduced in both parasitized (R. Pfister-Wilhelm & B. Lanzrein, unpublished data) or in X-ray-parasitized (Grossniklaus-Bürgin et al., 1998
) larvae and it is conceivable that these three viral genes act through inhibition of ecdysone production. Induction of host developmental arrest before pupation by polydnaviruses is very common (Lawrence & Lanzrein, 1993
) but, in many cases, the parasitoid larva emerges from the host before it approaches pupation. Interestingly, Campoletis sonorensis parasitization or ichnovirus injection induces degeneration of the host's prothoracic gland only in the last instar (Dover et al., 1988
), but not if parasitization occurred in the penultimate instar; under natural conditions, C. sonorensis hardly attacks last-instar larvae (Dover & Vinson, 1990
). It thus appears that the developmental arrest comes mainly into effect in the case of parasitoid larvae not having reached maturity when the host approaches pupation. It could thus be considered a means of extending successful parasitoid development and egression. Alternatively, these three viral genes might play a role in parasitoid survival by manipulating the nutritional milieu of the host to the parasitoid's benefit. Work in progress aims at increasing the effectiveness of RNAi in vivo, and we are also attempting to study the role of early- and constitutively expressed CiV genes with this method.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beck, M. & Strand, M. R. (2003). RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells. Virology 314, 521535.[CrossRef][Medline]
Bettencourt, R., Terenius, O. & Faye, I. (2002). Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos. Insect Mol Biol 11, 267271.[CrossRef][Medline]
Blandin, S., Shiao, S.-H., Moita, L. F., Janse, C. J., Waters, A. P., Kafatos, F. C. & Levashina, E. A. (2004). Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116, 661670.[CrossRef][Medline]
Bonvin, M., Kojic, D., Blank, F., Annaheim, M., Wehrle, I., Wyder, S., Kaeslin, M. & Lanzrein, B. (2004). Stage-dependent expression of Chelonus inanitus polydnavirus genes in the host and the parasitoid. J Insect Physiol 50, 10151026.[CrossRef][Medline]
Boulétreau, M. (1986). The genetic and coevolutionary interactions between parasitoids and their hosts. In Insect Parasitoids, pp. 169200. Edited by J. Waage & D. Greathead. London: Academic Press.
Brown, S. J., Mahaffey, J. P., Lorenzen, M. D., Denell, R. E. & Mahaffey, J. W. (1999). Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects. Evol Dev 1, 1115.[CrossRef][Medline]
Chen, Y. P. & Gundersen-Rindal, D. E. (2003). Morphological and genomic characterization of the polydnavirus associated with the parasitoid wasp Glyptapanteles indiensis (Hymenoptera: Braconidae). J Gen Virol 84, 20512060.
Chen, Y. P., Taylor, P. B., Shapiro, M. & Gundersen-Rindal, D. E. (2003). Quantitative expression analysis of a Glyptapanteles indiensis polydnavirus protein tyrosine phosphatase gene in its natural lepidopteran host, Lymantria dispar. Insect Mol Biol 12, 271280.[CrossRef][Medline]
Dover, B. A. & Vinson, S. B. (1990). Stage-specific effects of Campoletis sonorensis parasitism on Heliothis virescens development and prothoracic glands. Physiol Entomol 15, 405414.
Dover, B. A., Davies, D. H. & Vinson, S. B. (1988). Degeneration of last instar Heliothis virescens prothoracic glands by Campoletis sonorensis polydnavirus. J Invertebr Pathol 51, 8091.[CrossRef]
Falabella, P., Varricchio, P., Gigliotti, S., Tranfaglia, A., Pennacchio, F. & Malva, C. (2003). Toxoneuron nigriceps polydnavirus encodes a putative aspartyl protease highly expressed in parasitized host larvae. Insect Mol Biol 12, 917.[CrossRef][Medline]
Flores-Jasso, C. F., Valdes, V. J., Sampieri, A., Valadez-Graham, V., Recillas-Targa, F. & Vaca, L. (2004). Silencing structural and nonstructural genes in baculovirus by RNA interference. Virus Res 102, 7584.[CrossRef][Medline]
Goto, A., Blandin, S., Royet, J., Reichhart, J.-M. & Levashina, E. A. (2003). Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies. Nucleic Acids Res 31, 66196623.
Grossniklaus-Bürgin, C., Wyler, T., Pfister-Wilhelm, R. & Lanzrein, B. (1994). Biology and morphology of the parasitoid Chelonus inanitus (Braconidae, Hymenoptera) and effects on the development of its host Spodoptera littoralis (Noctuidae, Lepidoptera). Invertebr Reprod Dev 25, 143158.
Grossniklaus-Bürgin, C., Pfister-Wilhelm, R., Meyer, V., Treiblmayr, K. & Lanzrein, B. (1998). Physiological and endocrine changes associated with polydnavirus/venom in the parasitoidhost system Chelonus inanitus-Spodoptera littoralis. J Insect Physiol 44, 305321.[CrossRef][Medline]
Johner, A. & Lanzrein, B. (2002). Characterization of two genes of the polydnavirus of Chelonus inanitus and their stage-specific expression in the host Spodoptera littoralis. J Gen Virol 83, 10751085.
Kroemer, J. A. & Webb, B. A. (2004). Polydnavirus genes and genomes: emerging gene families and new insights into polydnavirus replication. Annu Rev Entomol 49, 431456.[CrossRef][Medline]
Lanzrein, B., Pfister-Wihelm, R. & von Niederhäusern, F. (2001). Effects of an egg-larval parasitoid and its polydnavirus on development and the endocrine system of the host. In Endocrine Interactions of Insect Parasites and Pathogens, pp. 95109. Edited by J. P. Edwards & R. J. Weaver. Oxford: BIOS Scientific Publishers.
Lawrence, P. O. & Lanzrein, B. (1993). Hormonal interactions between insect endoparasites and their host insects. In Parasites and Pathogens of Insects, vol. 1, pp. 5986. Edited by N. E. Beckage, S. N. Thompson & B. A. Federici. San Diego: Academic Press.
Lewis, B. P., Green, R. E. & Brenner, S. E. (2003). Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc Natl Acad Sci U S A 100, 189192.
Malva, C., Varricchio, P., Falabella, P., La Scaleia, R., Graziani, F. & Pennacchio, F. (2004). Physiological and molecular interaction in the hostparasitoid system Heliothis virescensToxoneuron nigriceps: current status and future perspectives. Insect Biochem Mol Biol 34, 177183.[CrossRef][Medline]
Miyawaki, K., Mito, T., Sarashina, I., Zhang, H., Shinmyo, Y., Ohuchi, H. & Noji, S. (2004). Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis. Mech Dev 121, 119130.[CrossRef][Medline]
Pfister-Wilhelm, R. & Lanzrein, B. (1996). Precocious induction of metamorphosis in Spodoptera littoralis (Noctuidae) by the parasitic wasp Chelonus inanitus (Braconidae): identification of the parasitoid larva as the key element and the host corpora allata as a main target. Arch Insect Biochem Physiol 32, 511525.
Quan, G. X., Kanda, T. & Tamura, T. (2002). Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene. Insect Mol Biol 11, 217222.[CrossRef][Medline]
Rosén, M., Castillejo-López, C. & Edström, J.-E. (2002). Telomere terminating with centromere-specific repeats is closely associated with a transposon derived gene in Chironomus pallidivitatus. Chromosoma 110, 532541.[Medline]
Sánchez Alvarado, A. & Newmark, P. A. (1999). Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc Natl Acad Sci U S A 96, 50495054.
Schmidt, O., Theopold, U. & Strand, M. (2001). Innate immunity and its evasion and suppression by hymenopteran endoparasitoids. Bioessays 23, 344351.[CrossRef][Medline]
Schoppmeier, M. & Damen, W. G. M. (2001). Double-stranded RNA interference in the spider Cupiennus salei: the role of Distal-less is evolutionarily conserved in arthropod appendage formation. Dev Genes Evol 211, 7682.[CrossRef][Medline]
Soller, M. & Lanzrein, B. (1996). Polydnavirus and venom of the egg-larval parasitoid Chelonus inanitus (Braconidae) induce developmental arrest in the prepupa of its host Spodoptera littoralis (Noctuidae). J Insect Physiol 42, 471481.[CrossRef]
Stettler, P., Trenczek, T., Wyler, T., Pfister-Wilhelm, R. & Lanzrein, B. (1998). Overview of parasitism associated effects on host haemocytes in larval parasitoids and comparison with effects of the egg-larval parasitoid Chelonus inanitus on its host Spodoptera littoralis. J Insect Physiol 44, 817831.[CrossRef][Medline]
Tillinger, N. A., Hoch, G. & Schopf, A. (2004). Effects of parasitoid associated factors of the endoparasitoid Glyptapanteles liparidis (Hymenoptera: Braconidae). Eur J Entomol 101, 243249.
Turnbull, M. & Webb, B. A. (2002). Perspectives on polydnavirus origins and evolution. Adv Virus Res 58, 203254.[Medline]
Valdes, V. J., Sampieri, A., Sepulveda, J. & Vaca, L. (2003). Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus. J Biol Chem 278, 1931719324.
Whitfield, J. B. & Asgari, S. (2003). Virus or not? Phylogenetics of polydnaviruses and their wasp carriers. J Insect Physiol 49, 397405.[CrossRef][Medline]
Wyder, S., Tschannen, A., Hochuli, A., Gruber, A., Saladin, V., Zumbach, S. & Lanzrein, B. (2002). Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering. J Gen Virol 83, 247256.
Wyder, S., Blank, F. & Lanzrein, B. (2003). Fate of polydnavirus DNA of the egglarval parasitoid Chelonus inanitus in the host Spodoptera littoralis. J Insect Physiol 49, 491500.[CrossRef][Medline]
Received 16 December 2004;
accepted 20 December 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |