Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum
© Etebari et al; licensee BioMed Central Ltd. 2011
Received: 9 March 2011
Accepted: 9 September 2011
Published: 9 September 2011
Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (Ds IV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported.
De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the Ds IV genes.
This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, Ds IV genes expressed in the host and also improves our current understanding of this host-parasitoid interaction.
Endoparasitoids of the insect order Hymenoptera inject their eggs inside a host insect where they hatch and subsequently feed on the host until its death. For successful parasitism, endoparasitoids bring about a change in their hosts' conditions in favour of the developing parasitoid larvae. For this purpose, female wasps introduce secretions such as venom or ovary fluids, which may contain symbiotic viruses (polydnavirus (PDV) and/or virus-like particles), and other maternal factors, into the host . In addition to suppression of the host immune system to protect the developing parasitoid, several studies have shown that parasitoids and their introduced maternal factors have significant effects on host metabolism and development such as plasma protein composition, food consumption, endocrine system activity and even on regulatory microRNA levels [2–8].
Diadegma semiclausum Hellén is an ichneumonid endoparasitoid that carries a PDV with a circular, double-stranded and segmented DNA genome encoding proteins that suppress the host immune response and cause developmental arrest/delay . PDVs are the most highly characterized of the known mutualistic viruses , replicating only within the calyx cells of the reproductive tract of female wasps . No virus replication occurs in parasitized larvae; however expression of encapsidated PDV genes induces different physiological modifications such as interruption of the larval endocrine system and suppression of the host immune system [12–15].
PDVs are classified into two genera based on their host wasp families; Ichnovirus containing ichnoviruses (IVs) and Bracovirus containing bracoviruses (BVs) [11, 16]. These genera are morphologically distinct and their gene functions vary. Analysis of virion structural components from IVs indicates that the set of structural genes is conserved among wasps associated with IVs and might originate from an ancestral virus . Recently, Bigot et al. suggested that IVs originated from ascoviruses by lateral transfer of ascoviral genes into wasp genomes . Interestingly, the DNA that is encapsidated within PDVs appears more similar to that of eukaryotes than that of other viral genomes . It has been demonstrated that less than 2% of encapsidated PDV genes have homologs in other viruses in contrast to the proviral or structural genes . Most of the PDV DNA appear to be non-coding, except for some groups of genes that are involved in host immune suppression pathways . IV genomes generally encompass more than 20 circular DNA segments ranging in size from 2-28 Kb with estimated total genome sizes ranging from 75 Kb to greater than 250 Kb .
Research so far has concentrated mainly on individual genes or small defined groups of host or PDV genes, to explore their function or differential expression following parasitization. Deep sequencing data can provide extensive information about host-parasitoid interactions at the transcriptome level. The large amount of sequencing data that can readily be produced by next-generation sequencing platforms, such as the Illumina GAII, reduces the need for prior sequence knowledge for gene expression profiling and are now making direct sequencing approaches the method of choice for whole transcriptome analysis in many species [22–26]. The large numbers of short reads produced by next-generation sequencers provide opportunities for development of new applications where sequencing only a portion of a molecule is sufficient. However, the analysis of transcriptome data produced by these technologies for organisms with limited genomic information still presents challenges because the sequenced fragments must be aligned against existing good quality reference genomes .
In this study, we used deep sequencing to explore the impact of D. semiclausum parasitization on its host, Diamondback moth Plutella xylostella L. (Lepidoptera, Plutellidae), a notorious pest of cruciferous plants. P. xylostella has developed resistance to many groups of chemical insecticides and also Bacillus thuringiensis endotoxin [28, 29]. This has made P. xylostella one of the world's most destructive insect pests and the estimated global cost of controlling this insect is around US$1 billion annually . This emphasizes the necessity for the continued development of innovative alternative control measures and resistance management strategies. Parasitoids or parasitoid-produced regulatory molecules can be used to improve conventional pest control strategies in sustainable agriculture. We have used a deep sequencing approach to give a comprehensive view of immune- and metabolic-related genes that are differentially expressed in parasitized versus non-parasitized P. xylostella larvae, revealing a significant number of D. semiclausum ichnovirus genes (Ds IV) which have not been reported previously. This type of study may facilitate new controls for pest larvae by identifying molecules that are crucial for larval immune defence, development, pesticide resistance and other important metabolic regulatory functions.
Results and Discussion
P. xylostella transcriptome profile
A transcriptome is the complete set of expressed RNA transcripts in one or more cells. Transcriptome profiling of organisms under stress or parasitization challenge helps us to obtain a better understanding of subsequent related cellular activities in organisms including growth, development, and immune defence. Recently, the newly developed deep sequencing approaches have significantly changed how the functional complexity of the transcriptome can be investigated [22, 25, 30].
Summary of contig statistics resulting from Illumina deep sequencing of parasitized and non-parasitized P. xylostella larvae
Total de novo assembled contigs with CLC software
Contigs used for BLAST (cut-off: above 200 bp length)
Best BLAST matches (cut-off: E-value > E-6)
No Hits (contigs without any BLAST match)
Not mapped with any Gene Ontology (GO) database
Annotated sequences (cut off: GO weight 5E value > E-6)
Overall, from the assembled contigs of over 200 bp in length, 44% showed similarity with genes or proteins in the NCBI database. The rest may represent unknown genes, non-coding RNA or misassembled contigs that are expected due to the presence of large repetitive or duplicated regions. David et al. (2010) suggested that a significant proportion of transcript signatures detected outside predicted genes represent regulatory non-coding RNAs, because these large numbers of non-coding RNA can be antisense, intergenic or overlapping with protein-coding genes .
Effects of parasitism on the transcription of host immune-related genes
A list of P .xylostella immune-related genes that were differentially transcribed after parasitization by D. semiclausum
TSA Accession No.
Moricin-like peptide C2
Serine proteinase inhibitor
Beta-1,3-glucan recognition protein 3
Odorant binding protein
Samia cynthia ricini
Cecropin 1 (antibacterial peptide)
Thrombin inhibitor infestin
Peptidoglycan recognition protein
Peptidoglycan recognition protein S6
Prophenoloxidase-activating proteinase 3
Trypsin-like serine proteinase 1
Bifunctional protein folD
Nucleotide excision repair protein
Beta-1,3-glucan recognition protein 2
Prophenoloxidase activating factor 3
Serine protease 33
Serine protease inhibitor 7
Lysosomal acid lipase
Pattern recognition serine proteinase
Serine protease inhibitor (pxSerpin 3)
Serine protease inhibitor (Serpin 13)
Broad-Complex isoform Z2
Heat Shock Protein (HSP70)
Beta-1,3-glucan recognition protein 2a
Serine protease 1
Peripheral-type benzodiazepine receptor
Hemolymph proteinase 8
Heat Shock Protein (HSP90)
Putative defense protein Hdd11
Developmental- and non-immune metabolism-related transcripts of P. xylostella, which were differentially expressed after D. semiclausum parasitization
TSA Accession No.
Glucose dehydrogenase precursor
Pediculus humanus corporis
Methionine-rich storage protein 1
Putative RecQ Helicase
Methionine-rich storage protein 2
44 kDa zymogen (serine protease)
Methionine-rich storage protein
Leucine-rich transmembrane protein
Pediculus humanus corporis
Sugar transporter 4
Cathepsin L precursor
Hemolymph proteinase 5
Juvenile hormone binding protein
Imaginal disk growth factor
Cathepsin B-like cysteine proteinase
Cathepsin D isoform 1
DNA-binding protein Ewg putative
Pediculus humanus corporis
Pediculus humanus corporis
Cytochrome P450 monooxygenase
Trypsin alkaline B
Cuticular protein glycine-rich 20
Voltage & ligand gated potassium channel
Multidrug resistance protein 2
Cyclin-dependent kinase 2-like
Juvenile hormone epoxide hydrolase
Cuticular protein RR-1 motif 10
37-kDa serine protease
Ecdysteroid regulated protein
In addition to post-transcriptional effects of PDV infection, there may also be variations in different host-parasitoid interactions with regard to host transcripts affected by parasitization/PDV infection. In a very recent study, it was shown that in Spodoptera frugiperda larvae injected with Hyposoter didymator ichnovirus (Hd IV) or Microplitis demolitor bracovirus (Md BV), the differentially expressed transcripts in hemocytes and fat body largely differed depending on the PDV injected suggesting that the tissues responded differently to the different viruses . However, there were a number of host genes that responded similarly when infected with Hd IV or Md BV. Based on this study, Hd IV affected transcript levels in both hemocytes and fat body, whereas Md BV mostly affected gene expression in the fat body.
Other related genes which were up-regulated in parasitized larvae included a serine protease and serine protease inhibitor (pxSerpin 3) (Table 2). Serine proteases are major immune regulatory proteins, which are found in a wide range of species from insects to mammals. In contrast, a proteomics analysis showed that pxSerpin 2 was suppressed in P. xylostella larval plasma during parasitism by C. plutellae. Beck et al. demonstrated that the ovarian calyx fluid of the ichneumonid endoparasitoid Venturia canescens has the potential to suppress the host immune system due to a putative serpin activity . Here, we found that serine protease inhibitors (JL943746, JL943748, JL943775, JL943864) were over-expressed two- to seven-fold, after parasitism in P. xylostella. Thus, the induction of serine protease inhibitors upon immune challenge in parasitized larvae could be part of an endoparasitoid immune suppressive strategy. Many insect protease inhibitors are known to inactivate enzymes isolated from entomopathogenic fungi, and their involvement in insect-pathogen interactions has been widely postulated . Aguilar et al. reported that five serine proteases involved in a single metabolic cascade were up-regulated in Anopheles gambiae upon microbial challenge, but they suggested a role for the proteases in protecting the mosquito from detrimental effects of an uncontrolled spread of immune reaction .
Serine proteases also play a significant role in the activation of the prophenoloxidase (proPO) cascade. In insects, proPO is activated upon injury or invasion, which results in localized melanization of the wound area and/or melanotic capsules capturing invading microorganisms and parasites . In the current study, proPO activating protease (PAP) transcription was up-regulated in parasitized larvae of P. xylostella. In a genome-wide microarray study of D. melanogaster, several genes encoding enzymes of the melanization cascade were found to be up-regulated by L. boulardi parasitization , consistent with this study. Asgari et al. (2003) reported that venom protein (Vn50) from Cotesia rubecula is homologous to serine protease homologs . It is likely that the injection of this protein (or putative homologs thereof) by parasitoid wasps into the host body, may interfere with the proteolytic cascade that leads to the activation of proPO. cDNA microarray analysis of S. frugiperda hemocytes and fat body 24 hours after injection of Hd IV revealed differential expression of several host genes . Among these, eight immune-related genes showed differential expression in hemocytes with proPO-1 and proPO-2 showing up-regulation, while PAP transcript levels declined. Other immune-related genes that were differentially expressed in the hemocytes were galectin, which showed up-regulation, whereas scavenger R, immulectin-2, lysozyme and calreticulin showed down-regulation . A recent study confirmed up-regulation of proPOs in S. frugiperda larvae injected with Hd IV; however, in the same host injected with Md BV, proPO transcript levels declined  which suggested differential responses of the host to different PDVs.
Transcripts of most proteins, which are involved in the Toll pathway such as Relish, Dorsal, Pelle, Cactus and Toll receptor, were found in our deep sequencing analysis, but only transcription levels of proteins that showed similarity to the Toll receptor were up-regulated (2.5 fold; Table 2). In D. melanogaster larvae parasitized by Asobara tabida or L. boulardi, components of the Toll/Imd (Immune deficiency) pathways were up-regulated, and antimicrobial peptide expression was increased [44, 46]. In addition, in Drosophila, Toll and Imd pathways are required for activation and stimulation of NF-κBs signal transduction and also responsible for innate immune response in parasitized Drosophila. NF-κB proteins are a family of proteins in eukaryotes that are involved in the control of a large number of cellular and organismal processes, such as immune responses, developmental processes, cellular growth, and apoptosis. Furthermore, NF-κB signalling is important in immune inducibility of pathogen-associated-molecular-patterns, and it is widely assumed that it plays a conserved role in invertebrate immune regulation [48, 49].
It has been suggested that PDV-expressed vankyrin proteins may interfere with NF-κB-mediated signalling during immune response and development in parasitized larvae [15, 50]. Fath-Goodin et al. (2009) reported that Cs IV vankyrin genes also encode proteins with sequence homology to the inhibitory domains of NF-κB transcription factor inhibitors .
The results of our transcriptome analysis also indicated that genes known to be involved in insecticide resistance/detoxification are up-regulated following parasitism (Table 2). In agreement, it has previously been reported that cytochrome P450 (CYP) and glutathione-S-transferase (GST) activities increased in parasitized P. xylostella larvae . Takeda et al. (2006) suggested that parasitoid larvae contributed to CYP activity enhancement since the parasitoid hatched two days after oviposition and the CYP activity was significantly increased three days after parasitization .
Our analyses also detected a considerable number of other immune-related genes whose transcription levels altered after parasitization by D. semiclausum. However, only a small group affected by parasitoid attack were found to be altered enough to be statistically biologically relevant (i.e. showing greater than two-fold change).
Transcription levels of host development-related genes
Generally, larval endoparasitoids lay their eggs into the host hemocoel, and their progenies develop by consuming host hemolymph and tissues. As a consequence, the parasitoid's larval growth also fully depends on the host's development [1, 51, 52]. In P. xylostella larvae parasitized by D. semiclausum, development is arrested at the prepupal stage . Developmental arrest before pupation is one of the most common effects of PDVs and/or other maternal factors injected by many endoparasitoids into their hosts [14, 54–56]. In these interactions, the parasitoid larvae and PDVs are responsible for increasing the juvenile hormone (JH) titre in host larvae and preventing ecdysteroid levels from rising sufficiently to allow host pupation [57–61].
Based on our transcriptome data, parasitism by D. semiclausum leads to down-regulation of genes associated with ecdysteroid activities; for example, the transcription level of ecdysteroid regulated protein was down-regulated more than 26 times in parasitized larvae (Table 3). Considering that ecdysteroids are required to trigger expression of ecdysteroid regulated protein , and that parasitism in general leads to reductions in ecdysteroid titres [54, 63–66], down-regulation of ecdysteroid regulated protein is expected.
Juvenile hormone binding protein (JHBP) may protect JH from non-specific degradation and adsorption by preventing exposure of JH to epoxide hydration by JH epoxide hydrolase (JHEH), which generates the hormonally inactive JH diol . In agreement, JHBP transcript levels were up-regulated more than 2 times and interestingly, transcription levels of JHEH were down-regulated more than 2 times (Table 3). In all the reported host-parasitoid systems, it seems that JH is maintained at high levels during parasitoid larval development [58, 66, 68]. Therefore, an increase in JHBP, and corresponding decrease in JHEH transcription levels, after parasitism, seems logical to maintain higher JH levels during parasitism.
As indicated above, previous studies have shown that PDVs may inhibit translation of specific storage or growth-associated proteins despite up-regulation (or steady-state) of transcript levels of the encoding genes, following parasitism or injection of PDVs [69–71]. In this study, we found that arylphorin and methionine-rich storage proteins were over-transcribed in parasitized larvae (Table 3); however, their translation may be affected similarly to mechanisms in the reports discussed above, which needs to be experimentally shown.
Quantitative RT-PCR validation of transcriptome analysis
Diadegma semiclausum ichnovirus genes
PDV genes are divided into three groups based on whether they are expressed in the carrier wasp (class I), the infected host larvae (class II) or both (class III) . Among these genes, class II genes have received the greatest attention and have been studied more than other groups . The genomes of some IVs, such as those found in the wasps C. sonorensis, C. chlorideae, Hyposoter fugitivus, H. didymator, and Tranosema rostrale, have been sequenced and resultant data are available on public databases [21, 72, 74]. Six conserved gene families: repeat element, cysteine motif, viral innexin, viral ankyrin, N-family and the polar-residue-rich proteins (a newly defined putative family), have been reported in most IV genomes .
D. semiclausum IV transcripts which were detected in parasitized P. xylostella larvae
Similarity (Protein/virus/Accession No.)
Nt. ID %
vankyrin-b17 (HfIV) AAS90270.1
vankyrin-d8.3 (HfIV) BAF45734.1
hypothetical protein 2 (HdIV) AAR99845.1
Viral Innexin 1
viral innexin-b5.1 (HfIV) BAF45654.1
Viral Innexin 2
viral innexin-c16 (HfIV) AAS58041.1
Repeat element 1
repeat element protein 7 (HdIV) AAR89179.1
Repeat element 2
repeat element protein-d10.1 (HfIV) BAF45740.1
Repeat element 3
repeat element protein-c18.1 (HfIV) BAF45697.1
Repeat element 4
repeat element protein-e2.3 (HfIV) BAF45758.1
Repeat element 5
repeat element protein (HdIV) AAO16959.1
Repeat element 6
repeat element protein-d11.2 (HfIV) BAF45744.1
Cysteine rich motif
cysteine motif gene-d9.1(HfIV) BAF45736.1
Polar residue rich protein
polar residue rich protein-b13.2 (HfIV) BAF45664.1
c7-2.1 (TrIV) BAF45599.1
c12.1 (HfIV) AAS68099.1
P12 (HdIV) AAS83461.1
c10.1 (HfIV) AAS90272.1
b5.3 (HfIV) BAF45655.1
Overall, this study provides the first comprehensive analysis of the impact of a parasitoid wasp on its host at the transcriptomic level, using RNA deep sequencing technique. The results showed differential expression of a large number of P. xylostella genes, including immune-related genes, upon parasitization by D. semiclausum. In addition, although presence of Ds IV particles has been reported in parasitized larvae, our results provide evidence for expression of 19 Ds IV genes expressed in the host, which have not been previously reported. Analysis of these sequences indicated the presence of conserved genes that belong to major IV class II genes. The transcriptome profiling data sets obtained in this study provide a basis for future research in this under-explored host-parasitoid interaction. In addition, the identified immune-, development- and detoxification-related genes may be targets for P. xylostella control and allow manipulation of host-parasite interactions.
Insects and parasitization
P. xylostella and the parasitoid wasp (D. semiclausum) were raised on cabbage plants and host larvae, respectively, at 25°C. Twenty five 3rd and 4th instar P. xylostella larvae each were exposed to wasps until parasitization was observed. Individual larvae that had been attacked by the parasitoid were collected and fed on fresh cabbage leaves. Larval samples were taken at four different time intervals after parasitization (6, 12, 24 and 48 hrs post-parasitization) and the samples were kept at -80°C until RNA isolation. The same numbers of mixed larval instars (3rd and 4th) of non-parasitized larvae were collected as the control treatment. It is worth mentioning that P. xylostella larvae parasitized at 3rd instar continue to develop to 4th instar.
Sample preparation, deep sequencing and de novo transcriptome assembly
Total RNA was extracted from all larval samples using Tri-Reagent™ (Molecular Research Center Inc.). RNA extracted from larvae at various time points post-parasitization were pooled and therefore temporal expression data was lost. This was also performed for non-parasitized samples. The pooled RNA sample concentrations were measured using a spectrophotometer and integrity was ensured through analysis on a 1% (w/v) agarose gel. The samples with total concentration of 3.9 and 4.1 μg/μl for parasitized and non-parasitized larvae, respectively, were used for cDNA library production.
The cDNA library was prepared by using 5 μg of starting material for the Illumina mRNA Sequencing Sample preparation procedure (kit RS-930-1001). This involved purification and fragmentation of mRNA, first strand cDNA synthesis, second strand cDNA synthesis, end repair, addition of "A" bases to 3' ends, ligation of adapters, purification of ligated products, and PCR amplification to enrich cDNA templates. The library was validated, quantified and subjected to deep sequencing using a Genome Analyzer IIx Next generation sequencer on a 66 cycle single-end sequencing run, following the supplier's instructions (Geneworks, Adelaide). The GAII analyzer data were output as sequence tags of 65 bases. Sequence.txt files (in FASTQ format) were generated using Illumina Pipeline version 1.5.1. The CLC Genome Workbench (version 4.0.2)  algorithm for de novo sequence assembly was used to assemble contigs from a pooling of all the short-read data, using default parameters (similarity = 0.8, length fraction = 0.5, insertion/deletion cost = 3, mismatch cost = 3).
RNA sequence analysis
The contigs arising from the de novo assembly were then used as a reference set of transcripts for RNAseq analysis. Short-read sequence data from parasitized and non-parasitized larvae were separately mapped against the reference set of assembled transcripts using the CLC Genome Workbench RNAseq function (min. length fraction = 0.9, maximum mismatches = 2). The relative transcript levels were output as RPKM (Reads Per Kilobase of exon model per Million mapped reads) values, which take into account the relative size of the transcripts and only uses the mapped-read datasets (i.e. excludes the non-mapped reads), to determine relative transcript abundance. In this way, the output for each dataset can be directly compared as the number of mapped reads per dataset and transcript size has already been taken into account.
Reads from parasitized and non-parasitized larvae were cleaned and combined, before de novo transcriptome assembly was carried out using Oases 0.1.18 . The individual sets of reads were then mapped back to the transcripts using BWA 0.5.8a . The average read depth (proportional to expression level) for each transcript was then calculated using SAMtools 0.1.8 . The transcripts that had a greater than two-fold average read depth difference between the parasitized and non-parasitized sets were counted as being statistically biologically relevant and were selected for annotation. We used both CLC and Oases to compare assembly of contigs. In general, Oases produced similar contigs to CLC, although contig lengths produced by Oases were in some instances longer.
BLAST homology search and annotation
BLASTX algorithm  with an E-value cut off of 10-6 was applied to the National Centre for Biotechnology Information (NCBI) non-redundant protein sequence database, to determine the homology of sequences with known genes. In the absence of P. xylostella and D. semiclausum genome sequences, we discarded annotations that showed similarity to hymenopteran genes and tried to use annotations that showed the highest similarity to lepidopteran genes. Gene ontology and annotation were performed on all assembled contigs greater than 200 bp length by BLAST2GO software http://www.blast2go.org. For gene ontology mapping, Blast2GO (which performs four different mapping strategies) was used, and the program defaults were applied for all annotation steps . BLAST2GO allows the selection of a significance level for the False Discovery Rate (FDR), which was used as a cut-off at the 0.05% probability level. The data from InterPro terms , enzyme classification codes (EC), and metabolic pathways (KEGG, Kyoto Encyclopedia of Genes and Genomes) were merged with GO terms for a wide functional range cover in annotation.
For some of the identified D. semiclausum ichnovirus (Ds IV) genes, ORFs were predicted and identified by using ORF finder at NCBI http://www.ncbi.nlm.nih.gov/gorf/gorf.html. Predicted ORFs with highest BLASTp E-values in internal comparisons involving other IV genes, were accepted for further analyses.
Quantitative RT-PCR (qRT-PCR) validation of deep sequencing data
Quantitative RT-PCR technique was used on the same RNA samples which were used for transcriptome profiling to verify deep sequencing results using three replicates, each obtained from a pool of 10 larvae. In addition, to observe gene expression levels at different time points after parasitization for a selected group of genes, another experiment was performed by parasitizing 2nd instar P. xylostella larvae. For each time point after parasitization, a pool of 10 larvae was used. The RNA samples were extracted from larvae at 16, 24 and 48 hrs after parasitization.
Primers used for qRT-PCR analyses to validate deep sequencing data
Storage protein 1
JH epoxide hydrolase
Ecdysteroid regulated protein
This work was supported by a UQ Research Higher Degree scholarship to Etebari, an ARC Discovery grant (DP110102112) to Asgari and a Horticulture Australia Ltd grant (VG08048) to Glatz and Asgari.
- Asgari S: Venom proteins from polydnavirus-producing endoparasitoids: Their role in host-parasite interactions. Arch Insect Biochem Physiol. 2006, 61 (3): 146-156. 10.1002/arch.20109.PubMedGoogle Scholar
- Bae S, Kim Y: Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Comp Biochem Physiol a-Mol Integ Physiol. 2004, 138 (1): 39-44. 10.1016/j.cbpb.2004.02.018.Google Scholar
- Bai SF, Cai DZ, Li X, Chen XX: Parasitic castration of Plutella xylostella larvae induced by polydnaviruses and venom of Cotesia vestalis and Diadegma semiclausum. Arch Insect Biochem Physiol. 2009, 70 (1): 30-43. 10.1002/arch.20279.PubMedGoogle Scholar
- Gundersen-Rindal DE, Pedroni MJ: Larval stage Lymantria dispar microRNAs differentially expressed in response to parasitization by Glyptapanteles flavicoxis parasitoid. Arch Virol. 2010, 155 (5): 783-787. 10.1007/s00705-010-0616-1.PubMedGoogle Scholar
- Kaeslin M, Reinhard M, Buhler D, Roth T, Pfister-Wilhelm R, Lanzrein B: Venom of the egg-larval parasitoid Chelonus inanitus is a complex mixture and has multiple biological effects. J Insect Physiol. 2010, 56 (7): 686-694. 10.1016/j.jinsphys.2009.12.005.PubMedGoogle Scholar
- Shi M, Huang F, Chen YF, Meng XF, Chen XX: Characterization of midgut trypsinogen-like cDNA and enzymatic activity in Plutella xylostella parasitized by Cotesia vestalis or Diadegma semiclausum. Arch Insect Biochem Physiol. 2009, 70 (1): 3-17. 10.1002/arch.20249.PubMedGoogle Scholar
- Song KH, Jung MK, Eum JH, Hwang IC, Han SS: Proteomic analysis of parasitized Plutella xylostella larvae plasma. J Insect Physiol. 2008, 54 (8): 1271-1280. 10.1016/j.jinsphys.2008.06.010.Google Scholar
- Zhu JY, Ye GY, Dong SZ, Fang Q, Hu C: Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host, Pieris rapae. Arch Insect Biochem Physiol. 2009, 71 (1): 45-53. 10.1002/arch.20304.PubMedGoogle Scholar
- Huang F, Shi M, Chen YF, Cao TT, Chen XX: Oogenesis of Diadegma semiclausum (Hymenoptera:Ichneumonidae) and its associated polydnavirus. Microsc Res Tech. 2008, 71 (9): 676-683. 10.1002/jemt.20594.PubMedGoogle Scholar
- Roossinck MJ: The good viruses: viral mutualistic symbioses. Nat Rev Microbiol. 2011, 9 (2): 99-108. 10.1038/nrmicro2491.PubMedGoogle Scholar
- Luckhart S, Webb BA: Interaction of a wasp ovarian protein and polydnavirus in host immune suppression. Dev Comp Immunol. 1996, 20 (1): 1-21. 10.1016/0145-305X(95)00040-Z.PubMedGoogle Scholar
- Barandoc KP, Kim J, Kim Y: Cotesia plutellae bracovirus suppresses expression of an antimicrobial peptide, Cecropin, in the Diamondback Moth, Plutella xylostella, challenged by bacteria. J Microbiol. 2010, 48 (1): 117-123. 10.1007/s12275-009-9261-3.PubMedGoogle Scholar
- Fath-Goodin A, Kroemer JA, Webb BA: The Campoletis sonorensis ichnovirus vankyrin protein P-vank-1 inhibits apoptosis in insect Sf9 cells. Insect Mol Biol. 2009, 18 (4): 497-506. 10.1111/j.1365-2583.2009.00892.x.PubMedGoogle Scholar
- Kwon B, Song S, Choi JY, Je YH, Kim Y: Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella. J Insect Physiol. 2010, 56 (6): 650-658. 10.1016/j.jinsphys.2010.01.013.PubMedGoogle Scholar
- Tian SP, Zhang JH, Wang CZ: Cloning and characterization of two Campoletis chlorideae ichnovirus vankyrin genes expressed in parasitized host Helicoverpa armigera. J Insect Physiol. 2007, 53 (7): 699-707. 10.1016/j.jinsphys.2007.03.015.PubMedGoogle Scholar
- Beckage NE: Parasitoids and polydnaviruses - An unusual mode of symbiosis in which a DNA virus causes host insect immunosuppression and allows the parasitoid to develop. Bioscience. 1998, 48 (4): 305-311. 10.2307/1313357.Google Scholar
- Volkoff AN, Jouan V, Urbach S, Samain S, Bergoin M, Wincker P, Demettre E, Cousserans F, Provost B, Coulibaly F, Legeai F, Beliveau C, Cusson M, Gyapay G, Drezen JM: Analysis of virion structural components reveals vestiges of the ancestral Ichnovirus genome. PLoS Pathog. 2010, 6 (5): e1000923-10.1371/journal.ppat.1000923.PubMedPubMed CentralGoogle Scholar
- Bigot Y, Samain S, Auge-Gouillou C, Federici BA: Molecular evidence for the evolution of ichnoviruses from ascoviruses by symbiogenesis. BMC Evo Biol. 2008, 8: 253-10.1186/1471-2148-8-253.Google Scholar
- Webb B, Fisher T, Nusawardani T: The Natural Genetic Engineering of Polydnaviruses. Natural Genetic Engineering and Natural Genome Editing. Edited by: Witzany G. 2009, 1178: 146-156.Google Scholar
- Webb BA: Polydnavirus biology, genome structure, and evolution. The insect viruses. Edited by: Miller LK, Ball LA. 1998, New York: Plenum Press, 105-139.Google Scholar
- Webb BA, Strand MR, Dickey SE, Beck MH, Hilgarth RS, Barney WE, Kadash K, Kroemer JA, Lindstrom KG, Rattanadechakul W, Shelby KS, Thoetkiattikul H, Turnbull MW, Witherell RA: Polydnavirus genomes reflect their dual roles as mutualists and pathogens. Virology. 2006, 347 (1): 160-174. 10.1016/j.virol.2005.11.010.PubMedGoogle Scholar
- David JP, Coissac E, Melodelima C, Poupardin R, Riaz MA, Chandor-Proust A, Reynaud S: Transcriptome response to pollutants and insecticides in the dengue vector Aedes aegypti using next-generation sequencing technology. BMC Genomics. 2010, 11: 216-10.1186/1471-2164-11-216.PubMedPubMed CentralGoogle Scholar
- Hegedus Z, Zakrzewska A, Agoston VC, Ordas A, Racz P, Mink M, Spaink HP, Meijer AH: Deep sequencing of the zebrafish transcriptome response to mycobacterium infection. Mol Immunol. 2009, 46 (15): 2918-2930. 10.1016/j.molimm.2009.07.002.PubMedGoogle Scholar
- Morozova O, Marra MA: Applications of next-generation sequencing technologies in functional genomics. Genomics. 2008, 92 (5): 255-264. 10.1016/j.ygeno.2008.07.001.PubMedGoogle Scholar
- Xiang L, He D, Dong W, Zhang Y, Shao J: Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish. BMC Genomics. 2010, 11: 472-10.1186/1471-2164-11-472.PubMedPubMed CentralGoogle Scholar
- Xiao SQ, Jia JY, Mo DL, Wang QW, Qin LM, He ZY, Zhao XA, Huang YK, Li AN, Yu JW, Niu YN, Liu XH, Chen YS: Understanding PRRSV infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing. PLoS One. 2010, 5 (6): e11377-10.1371/journal.pone.0011377.PubMedPubMed CentralGoogle Scholar
- Scheibye-Alsing K, Hoffmann S, Frankel A, Jensen P, Stadler PF, Mang Y, Tommerup N, Gilchrist MJ, Nygard AB, Cirera S, Jorgensen CB, Fredholm M, Gorodkin J: Sequence assembly. Comput Biol Chem. 2009, 33 (2): 121-136. 10.1016/j.compbiolchem.2008.11.003.PubMedGoogle Scholar
- Chen M, Zhao J-Z, Collins HL, Earle ED, Cao J, Shelton AM: A critical assessment of the effects of Bt transgenic plants on parasitoids. PLoS One. 2008, 3 (5): e2284-10.1371/journal.pone.0002284.PubMedPubMed CentralGoogle Scholar
- Takeda T, Nakamatsu Y, Tanaka T: Parasitization by Cotesia plutellae enhances detoxifying enzyme activity in Plutella xylostella. Pestic Biochem Physiol. 2006, 86 (1): 15-22. 10.1016/j.pestbp.2005.11.012.Google Scholar
- Mu YN, Ding F, Cui P, Ao JQ, Hu SN, Chen XH: Transcriptome and expression profiling analysis revealed changes of multiple signaling pathways involved in immunity in the large yellow croaker during Aeromonas hydrophila infection. BMC Genomics. 2010, 11: 506-10.1186/1471-2164-11-506.PubMedPubMed CentralGoogle Scholar
- Brent MR: Steady progress and recent breakthroughs in the accuracy of automated genome annotation. Nat Rev Gen. 2008, 9 (1): 62-73. 10.1038/nrg2220.Google Scholar
- Oases: a transcriptome assembler for very short reads. [http://www.ebi.ac.uk/~zerbino/oases]
- Shelby KS, Cui L, Webb BA: Polydnavirus-mediated inhibition of lysozyme gene expression and the antibacterial response. Insect Mol Biol. 1998, 7 (3): 265-272.PubMedGoogle Scholar
- Barandoc KP, Kim Y: Translation inhibitory factors encoded in Cotesia plutellae bracovirus require the 5 '-UTR of a host mRNA target. Comp Biochem Physiol B-Biochem Mol Biol. 2010, 156 (2): 129-136. 10.1016/j.cbpb.2010.03.001.PubMedGoogle Scholar
- Nicolas E, Nappi AJ, Lemaitre B: Expression of antimicrobial peptide genes after infection by parasitoid wasps in Drosophila. Dev Comp Immunol. 20 (3): 175-181.
- Provost B, Jouan V, Hilliou F, Delobel P, Bernardo P, Ravallec M, Cousserans F, Wajnberg E, Darboux I, Fournier P, Strand MR, Volkof AN: Lepidopteran transcriptome analysis following infection by phylogenetically unrelated polydnaviruses highlights differential and common responses. Insect Biochem Mol Biolo. 2011, 41 (8): 582-591. 10.1016/j.ibmb.2011.03.010.Google Scholar
- Eum JH, Seo YR, Yoe SM, Kang SW, Han SS: Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray. Dev Comp Immunol. 2007, 31 (11): 1107-1120. 10.1016/j.dci.2007.02.002.PubMedGoogle Scholar
- Abdel-Latief M, Hilker M: Innate immunity: Eggs of Manduca sexta are able to respond to parasitism by Trichogramma evanescens. Insect Biochem Mol Biol. 2008, 38 (2): 136-145. 10.1016/j.ibmb.2007.10.001.PubMedGoogle Scholar
- Kim J, Kim Y: Transient expression of a viral histone H4, CpBV-H4, suppresses immune-associated genes of Plutella xylostella and Spodoptera exigua. J Asia-Pacific Entomol. 2010, 13 (4): 313-318. 10.1016/j.aspen.2010.05.003.Google Scholar
- Beck M, Theopold U, Schmidt O: Evidence for serine protease inhibitor activity in the ovarian calyx fluid of the endoparasitoid Venturia canescens. J Insect Physiol. 2000, 46 (9): 1275-1283. 10.1016/S0022-1910(00)00048-2.PubMedGoogle Scholar
- Rai S, Aggarwal KK, Mitra B, Das TK, Babu CR: Purification, characterization and immunolocalization of a novel protease inhibitor from hemolymph of tasar silkworm, Antheraea mylitta. Peptides. 2010, 31 (3): 474-481. 10.1016/j.peptides.2009.08.021.PubMedGoogle Scholar
- Aguilar R, Jedlicka AE, Mintz M, Mahairaki V, Scott AL, Dimopoulos G: Global gene expression analysis of Anopheles gambiae responses to microbial challenge. Insect Biochem Mol Biol. 2005, 35 (7): 709-719. 10.1016/j.ibmb.2005.02.019.PubMedGoogle Scholar
- Asgari S, Zareie R, Zhang GM, Schmidt O: Isolation and characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym:Braconidae). Arch Insect Biochem Physiol. 2003, 53 (2): 92-100. 10.1002/arch.10088.PubMedGoogle Scholar
- Schlenke TA, Morales J, Govind S, Clark AG: Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster. PLoS Pathog. 2007, 3: 1486-1501.PubMedGoogle Scholar
- Barat-Houari M, Hilliou F, Jousset FX, Sofer L, Deleury E, Rocher J, Ravallec M, Galibert L, Delobel P, Feyereisen R, Fournier P, Volkoff AN: Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels. BMC Genomics. 2006, 7 (160): 1-20.Google Scholar
- Wertheim B, Kraaijeveld AR, Schuster E, Blanc E, Hopkins M, Pletcher SD, Strand MR, Partridge L, Godfray HCJ: Genome-wide gene expression in response to parasitoid attack in Drosophila. Genome Biol. 2005, 6 (11): R94-10.1186/gb-2005-6-11-r94.PubMedPubMed CentralGoogle Scholar
- Govind S: Innate immunity in Drosophila: Pathogens and pathways. Insect Science. 2008, 15 (1): 29-43. 10.1111/j.1744-7917.2008.00185.x.PubMedPubMed CentralGoogle Scholar
- Antonova Y, Alvarez KS, Kim YJ, Kokoza V, Raikhel AS: The role of NF-kappa B factor REL2 in the Aedes aegypti immune response. Insect Biochem Mol Biol. 2009, 39 (4): 303-314. 10.1016/j.ibmb.2009.01.007.PubMedPubMed CentralGoogle Scholar
- Shrestha S, Kim HH, Kim Y: An inhibitor of NF-kB encoded in Cotesia plutella bracovirus inhibits expression of antimicrobial peptides and enhances pathogenicity of Bacillus thuringiensis. J Asia-Pacific Entomol. 2009, 12 (4): 277-283. 10.1016/j.aspen.2009.06.004.Google Scholar
- Thoetkiattikul H, Beck MH, Strand MR: Inhibitor kappa B-like proteins from a polydnavirus inhibit NF-kappa B activation and suppress the insect immune response. Proc Natl Acad Sci USA. 2005, 102 (32): 11426-11431. 10.1073/pnas.0505240102.PubMedPubMed CentralGoogle Scholar
- Asgari S: Endoparasitoid venom proteins as modulators of host immunity and development. Recent advances in the biochemistry, toxicity and mode of action of parasitic wasp venom. Edited by: Rivers D, Yoder J. 2007, Kerala: Research Signpost, 57-73.Google Scholar
- Pennacchio F, Strand MR: Evolution of developmental strategies in parasitic hymenoptera. Annu Rev Entomol. 2006, 51: 233-258. 10.1146/annurev.ento.51.110104.151029.PubMedGoogle Scholar
- Li X, Cui L, Bai S, Yuan G: The capacity of regulation on development and host diamondback moth, Plutella xylostella, by endoparasitoids. J Henan Agric Univ. 2005, 39: 308-311.Google Scholar
- Dover BA, Davies DH, Strand MR, Gray RS, Keeley LL, Vinson SB: Ecdysteroid-titre reduction and developmental arrest of last-instar Heliothis virescens larvae by calyx fluid from the parasitoid Campoletis sonorensis. J Insect Physiol. 1987, 33 (5): 333-338. 10.1016/0022-1910(87)90121-1.Google Scholar
- Soller M, Lanzrein B: 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. 1996, 42 (5): 471-481. 10.1016/0022-1910(95)00132-8.Google Scholar
- Webb BA, Dahlman DL: Developmental pathology of Heliothis virescns larvae parasitized by Microplitis croceipes parasite mediated host developmental arrest. Arch Insect Biochem Physiol. 1985, 2 (2): 131-143. 10.1002/arch.940020203.Google Scholar
- Lee S, Kim Y: Juvenile hormone esterase of diamondback moth, Plutella xylostella, and parasitism of Cotesia plutellae. J Asia-Pacific Entomol. 2004, 7 (3): 283-287. 10.1016/S1226-8615(08)60228-9.Google Scholar
- Li S, Falabella P, Kuriachan I, Vinson SB, Borst DW, Malva C, Pennacchio F: Juvenile hormone synthesis, metabolism, and resulting haemolymph titre in Heliothis virescens larvae parasitized by Toxoneuron nigriceps. J Insect Physiol. 2003, 49 (11): 1021-1030. 10.1016/S0022-1910(03)00185-9.PubMedGoogle Scholar
- Pfister-Wilhelm R, Lanzrein B: Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids. J Insect Physiol. 2009, 55 (8): 707-715. 10.1016/j.jinsphys.2009.04.018.PubMedGoogle Scholar
- Schafellner C, Marktl RC, Schopf A: Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae). J Insect Physiol. 2007, 53 (8): 858-868. 10.1016/j.jinsphys.2007.05.010.PubMedGoogle Scholar
- Zhu JY, Ye GY, Dong SZ, Fang Q, Hu C: Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host, Pieris rapae (Lepidoptera:Pieridae). Arch Insect Biochem Physiol. 2009, 71 (1): 45-53. 10.1002/arch.20304.PubMedGoogle Scholar
- Meszaros M, Morton DB: Isolation and prtial caracterization of a gene from trachea of Manduca sexta that requires and is negatively regulated by ecdysteroids. Deve Biol. 1994, 162 (2): 618-630. 10.1006/dbio.1994.1115.Google Scholar
- Dahlman DL, Coar DL, Koller CN, Neary TJ: Contributing factors to reduced ecdysteroid titers in Heliothis virescens parasitized by Microplitis croceipes. Arch Insect Biochem Physiol. 1990, 13 (1-2): 29-39. 10.1002/arch.940130104.Google Scholar
- Gelman DB, Reed DA, Beckage NE: Manipulation of fifth-instar host (Manduca sexta) ecdysteroid levels by the parasitoid wasp Cotesia congregata. J Insect Physiol. 1998, 44 (9): 833-843. 10.1016/S0022-1910(98)00015-8.PubMedGoogle Scholar
- Pruijssers AJ, Falabella P, Eum JH, Pennacchio F, Brown MR, Strand MR: Infection by a symbiotic polydnavirus induces wasting and inhibits metamorphosis of the moth Pseudoplusia includens. J Exp Biol. 2009, 212 (18): 2998-3006. 10.1242/jeb.030635.PubMedPubMed CentralGoogle Scholar
- Strand MR, Dover BA, Johnson JA: Alterations in the ecdysteroid and juvenile hormone esterase profiles of Trichoplusia ni parasitized by the polyembryonic wasp Copidosoma floridanum. Arch Insect Biochem Physiol. 1990, 13 (1-2): 41-51. 10.1002/arch.940130105.Google Scholar
- Touhara K, Prestwich GD: Juvenile hormone epoxide hydrolase, photoaffinity labeling, purification and characterization from tobacco hornworm eggs. J Biol Chem. 1993, 268 (26): 19604-19609.PubMedGoogle Scholar
- Dover BA, Menon A, Brown RC, Strand MR: Suppression of juvenile hormone estrase in Heliothis virescens by Microplitis demolitor calyx fluid. J Insect Physiol. 1995, 41 (9): 809-817. 10.1016/0022-1910(95)00017-O.Google Scholar
- Nalini M, Kim Y: A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior. J Insect Physiol. 2007, 53 (12): 1283-1292. 10.1016/j.jinsphys.2007.07.004.PubMedGoogle Scholar
- Shelby KS, Webb BA: Polydnavirus infection inhibits synthesis of an insect plasma protein arylphorin. J Gen Virol. 1994, 75: 2285-2292. 10.1099/0022-1317-75-9-2285.PubMedGoogle Scholar
- Shelby KS, Webb BA: Polydnavirus infection inhibits translation of specific growth-associated host proteins. Insect Biochem Mol Biol. 1997, 27 (3): 263-270. 10.1016/S0965-1748(96)00095-1.PubMedGoogle Scholar
- Tanaka K, Lapointe R, Barney WE, Makkay AM, Stoltz D, Cusson M, Webb BA: Shared and species-specific features among ichnovirus genomes. Virology. 2007, 363 (1): 26-35. 10.1016/j.virol.2006.11.034.PubMedGoogle Scholar
- Strand MR: Polydnaviruses. Insect Virology. Edited by: Asgari S, Janson K. 2010, Norfolk: Caister Academic Press, 171-197.Google Scholar
- Lapointe R, Tanaka K, Barney WE, Whitfield JB, Banks JC, Beliveau C, Stoltz D, Webb BA, Cusson M: Genomic and morphological features of a banchine polydnavirus: Comparison with bracoviruses and ichnoviruses. J Virol. 2007, 81 (12): 6491-6501. 10.1128/JVI.02702-06.PubMedPubMed CentralGoogle Scholar
- CLC Bio: CLC genomics workbench. [http://www.clcbio.com]
- Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009, 25 (14): 1754-1760. 10.1093/bioinformatics/btp324.PubMedPubMed CentralGoogle Scholar
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data P: The Sequence alignment/map format and SAMtools. Bioinformatics. 2009, 25 (16): 2078-2079. 10.1093/bioinformatics/btp352.PubMedPubMed CentralGoogle Scholar
- Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nuc Acids Res. 1997, 25 (17): 3389-3402. 10.1093/nar/25.17.3389.Google Scholar
- Conesa A, Gotz S: Blast2GO: A comprehensive suite for functional analysis in plant genomics. Int J Plant Genomics. 2008, 2008: 619832-PubMedGoogle Scholar
- Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L, Finn RD, Gough J, Haft D, Hulo N, Kahn D, Kelly E, Laugraud A, Letunic I, Lonsdale D, Lopez R, Madera M, Maslen J, McAnulla C, McDowall J, Mistry J, Mitchell A, Mulder N, Natale D, Orengo C, Quinn AF, Selengut JD, Sigrist CJA, Thimma M, Thomas PD, Valentin F, Wilson D, Wu CH, Yeats C: InterPro: the integrative protein signature database. Nuc Acids Res. 2009, 37: D211-D215. 10.1093/nar/gkn785.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.