largely because the Sulston score takes into account total band number as well as thenumber of bands matching between two clones. Consequently, white leghorn cloneswere added to the map based only on their fingerprint matches to RJF clones. Thecriteria for adding white leghorn clones to RJF contigs were Sulston score threshold andnumber of RJF contig clones matching at that threshold. We examined the number ofmarker position inconsistencies introduced after adding white leghorn clones to chooseappropriate parameters. The heuristically determined requirement of fingerprintmatches to a minimum of five RJF contig clones at a Sulston score threshold of1 £ 1026 limited marker inconsistencies yet added a significant number of whiteleghorn clones with positional information to contigs. Manual comparisons of whiteleghorn fingerprints to surrounding clones were limited to the clones that have assignedSTS markers.

Fingerprint map linkage to the draft sequence assemblyThe threshold of BES links used to declare regions of the fingerprint map and draftsequence collinear is subjective. Our choice of parameters was guided by their impacton the frequency of marker inconsistencies and number of topologically impossiblecontig combinations. A minimum of six BES links were required before aligning an FPCcontig to a sequenced region, provided that 70% of all links were consistent and notopological constraints were violated. With these parameters, the overall frequency ofspurious BES links from an FPC contig to non-collinear sequence contigs was 0.10 andthe frequency of BES links to sequence contigs that were not aligned to the FPC mapwas 0.05. Discrepant BES links reveal regions of the FPC map where manual reviewmight be useful. Each FPC contig was scanned using a sliding window of ten BES links,which corresponds to roughly 109 kb (assuming a genome size of 1.06 Gb and a randomdistribution of BES links). If four or more BES links were to an unaligned sequencecontig, that FPC region was flagged for review. Of the 195 flagged regions, 14 containedcontaminated clones. The remaining number could be a reflection of the reliability ofBES links, errors in fingerprint map not revealed by fingerprints, or errors in thesequence assembly.

Received 4 August; accepted 17 September 2004; doi:10.1038/nature03030.

1. Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse

genome. Nature 420, 520–562 (2002).

2. Rat Genome Sequencing Project Consortium. Genome sequence of the brown Norway rat yields

insights into mammalian evolution. Nature 428, 493–521 (2004).

3. Krzywinski, M. et al. A set of BAC clones spanning the human genome. Nucleic Acids Res. 32,

3651–3660 (2004).

4. International Chicken Genome Sequencing Consortium. Sequence and comparative analysis of the

chicken genome provide unique perspectives on vertebrate evolution. Nature doi:10.1038/

nature03154 (this issue).

5. Gregory, S. G. et al. A physical map of the mouse genome. Nature 418, 743–750 (2002).

6. Krzywinski, M. et al. Integrated and sequence-ordered BAC and YAC-based physical maps for the rat

genome. Genome Res. 14, 766–779 (2004).

7. Ren, C. et al. A BAC-based physical map of the chicken genome. Genome Res. 13, 2754–2758

(2003).

8. Aerts, J. et al. Integration of chicken genomic resources to enable whole-genome sequencing.

Cytogenet. Genome Res. 102, 297–303 (2003).

9. Groenen, M. A. M. et al. A consensus linkage map of the chicken genome. Genome Res. 10, 137–147

(2000).

10. Groenen, M. A. M. & Crooijmans, R. P. M. A. in Poultry Genetics, Breeding and Technology (eds Muir,

W. M. & Aggrey, S. E.) 497–536 (CAB International, Wallingford, UK, 2003).

11. Fuhrmann, D. R. et al. Software for automated analysis of DNA fingerprinting gels. Genome Res. 13,

940–953 (2003).

12. Soderlund, C. H. S., Dunham, A. & French, L. Contigs built with fingerprints, markers, and FPC V4.7.

Genome Res. 10, 1772–1787 (2000).

13. Sulston, J., Mallett, F., Durbin, R. & Horsnell, T. Image analysis of restriction enzyme fingerprint

autoradiograms. Comput. Appl. Biosci. 5, 101–106 (1989).

14. Flibotte, S. et al. Automated ordering of fingerprinted clones. Bioinformatics 20, 1264–1271

(2004).

15. Romanov, M. N., Price, J. A. & Dodgson, J. B. Integration of animal linkage and BAC contig maps

using overgo oligo hybridization. Cytogenet. Genome Res. 102, 277–281 (2003).

16. Cai, W. et al. Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays.

Nature Biotechnol. 20, 393–396 (2002).

17. Meyers, B. C., Scalabrin, S. & Morgante, M. Mapping and sequencing complex genomes: let’s get

physical! Nature Rev. Genet. 5, 578–588 (2004).

18. Marra, M. A. et al. High throughput fingerprint analysis of large-insert clones. Genome Res. 7,

1072–1084 (1997).

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125–132 (1988).

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1539–1546 (1999).

Acknowledgements We thank J. C. Eissenberg, P. Cliften and M. Wendl for critically reading the

manuscript. We also thank S. Cornelissen, K. Hemmatian, J. van der Poel, T. Veenendaal and

R. Dijkhof for their help fingerprinting the WAG BAC library and, along with J. Price and

C. Rondelli, linking BAC clones to markers from the genetic linkage map.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to W.C.W.

(wwarren@watson.wustl.edu).

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Early blastomere determines embryoproliferation and caste fate in apolyembryonic waspVladimir Zhurov*, Tomislav Terzin* & Miodrag Grbic

Department of Biology, The University of Western Ontario, London N6A 5B7,Canada

* These authors contributed equally to this work.

.............................................................................................................................................................................

Polyembryonic development is a unique mode of metazoandevelopment in which a single zygote generates multiple embryosby clonal proliferation1. The polyembryonic parasitic insectCopidosoma floridanum shows one of the most extreme casesof polyembryony, producing up to 2,000 embryos from a singleegg. In addition, this wasp exhibits an unusual polyphenism,producing two morphologically distinct larval castes, termedprecocious and reproductive, that develop clonally from thesame zygote2. This form of development seems incompatiblewith a model of insect development in which maternal pre-patterning of the egg specifies embryonic axial polarity3. Herewe show that maternal pre-patterning in the form of germplasm creates cellular asymmetry at the four-cell stage embryoof Copidosoma that is perpetuated throughout development.Laser ablations of cells show that the cell inheriting thegerm plasm regulates both the fate and proliferation of thereproductive caste. Thus, we have uncovered a new mechanismof caste specification, mediated by the regulatory capacity of asingle cell. This study shows that the evolution of mammalian-like regulative development of an insect embryo relies on a novelcellular context that might ultimately enhance developmentalplasticity.

Polyphenism is a phylogenetically widespread feature of someplants and animals, in which organisms with complex life historiesform different phenotypes from a single genome4. Because modelorganisms do not exhibit polyphenism, the developmental basisof this marked genetic plasticity remains largely undetermined.Traditional polyphenic eusocial species such as ants and beesproduce reproductive (queen) and sterile (worker) castes from thesame genome when separate zygotes are exposed to differenthormonal or environmental influences5. However, one of themost extreme examples of polyphenism coupled with a complexlife history strategy is the clonal production of two differentmorphs from the same zygote in the polyembryonic parasiticwasp Copidosoma floridanum.

Copidosoma parasitizes eggs of the moth Trichoplusia ni (Fig. 1a).In contrast with other insects that undergo syncytial early cleavagesin which nuclear divisions do not follow the division of cytoplasm6,the Copidosoma egg undergoes total cleavage forming individualcells7. During the first two days, the wasp embryo emerges from itschorion and forms a primary morula surrounded by the extra-embryonic membrane derived from the polar body region (Fig. 1a,grey)8. The primary morula then undergoes a series of embryonicmovements to ‘implant’ itself into the developing host embryo(Supplementary movie). After parasitization, the host larva emergesand undergoes five larval instars. During the first to third host larvalinstars, Copidosoma enters the proliferative phase generating manyproliferative morulae, each containing thousands of round andapparently non-differentiated cells8. This phase represents thedevelopmental novelty initiating the clonal production of up to2,000 embryos. In the proliferative phase the developmental pro-gramme bifurcates to produce two terminal larval phenotypes.During the first to third larval instars some of the morulae initiate

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morphogenesis, producing sequentially up to 100 long, slenderlarvae with strong mandibles, called precocious larvae. These larvaehave a complex parasitic function, performing workers’ tasksincluding a defensive role against interspecific competitors9 andaltering the brood sex ratio10. The precocious larvae never moult,and die when their reproductive siblings consume the host. Thereminder of the proliferating morulae synchronously initiate thede novo formation of up to 2,000 embryonic axes in the fourth hostinstar, ultimately producing reproductive embryos8,11. Theseembryos develop into short, compact larvae that consume thehost, moult, pupate and form adult wasps.

Animal embryos studied up to now specify their embryonic axesat the beginning of development12. In contrast, de novo specificationof thousands of embryonic axes in Copidosoma late in developmentchallenges the current model of early axial specification in meta-zoans. Drosophila is an example of animal development in whichextensive axial pre-patterning relies on maternal determinantssupplied in the form of specialized cytoplasm containing infor-mation directing anterior, posterior and germline development13.

At the other end of the continuum is the mouse egg, in whichneither a pre-pattern nor examples of specialized cytoplasm suchas germ plasm have been demonstrated14. Mathematical modelsindicate that it is implausible that maternal pre-patterning has arole in Copidosoma development, because maintenance of pre-pattern would be impossible during the extensive proliferativegrowth3. However, in 1906 Silvestri described a specific region ofegg cytoplasm called the oosome that he believed to representthe germ plasm15. We determined previously that at the four-cellstage, the smallest cell that inherits the oosome becomes dye-uncoupled from other cells7 and could be stained with anti-Drosophila Vasa antibody11, corroborating Silvestri’s proposalthat early asymmetries might exist in the Copidosoma embryo.To determine the role of pre-patterning in polyembryonic insectembryogenesis we established a molecular marker for theoosome during Copidosoma embryogenesis and developed atransplantation protocol that allowed us to map the fate of theoosome-containing cell of the early embryo with the use of laserablation.

Figure 1 Life cycle of Copidosoma, alignment and phylogram of CfVasa amino acid

sequences. a, Copidosoma life cycle. b, Predicted amino acid sequences and alignment

of DEAD-box region of C. floridanum, C. elegans, H. magnipapillata, E. fluviatilis,

D. dorotocephala, D. melanogaster, B. mori, C. intestinalis, D. rerio, O. mykiss, X. laevis,

G. gallus, M. musculus and R. norvegicus Vasa proteins; for complete sequence

alignment see Supplementary Information. c, Phylogram of Vasa amino acid sequences.

Numbers at branches represent bootstrap values. Branch-length scale bar represents 0.1

amino acid substitutions per site.

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We cloned Copidosoma vasa (Cfvasa) mRNA, a ubiquitous germ-line marker in metazoans16 (Fig. 1b). Phylogenetic analysis ofCfVasa protein (Fig. 1c) indicates that it clusters with other insectVASA homologues, representing a VASA orthologue in Copidosoma.Cfvasa mRNA was expressed in nurse cells of early ovarial eggs

(Fig. 2a). In late ovarial eggs where unique cytoplasm (oosome) isvisible (Fig. 2b, arrowhead), unlocalized Cfvasa mRNA was detectedin the egg chamber. However, staining with anti-Vasa antibody co-localized to the oosome (Fig. 2c), indicating that CfVasa protein is acomponent of the oosome. In the uncleaved egg, CfVasa stainingcontinued to mark the oosome that becomes positioned on one sideof the spindle apparatus (Fig. 2d). The oosome was then inheritedby one large cell in the two-cell stage (not shown) and sequesteredinvariably to a small cell that stains with anti-Vasa antibody at thefour-cell stage (Fig. 2e). The small cell undergoes equal cleavage;both daughter cells inherit an antigen recognized by anti-Vasaantibody (Fig. 2f) and exhibit delayed cleavage relative to theprogeny of large cells. In the primary morula, six to eight cellsexpress Cfvasa mRNA and protein (Fig. 2g, h). During the pro-liferative period, Vasa-positive cells are distributed in individualproliferative morulae together with Vasa-negative cells (Fig. 2i, j).Both daughters of Vasa-positive cells inherit Vasa protein during theproliferative period (Fig. 2j, inset). However, some proliferativemorulae do not contain Vasa-positive cells (Fig. 2j, arrow). At theonset of reproductive morphogenesis, two Vasa-positive cells areparcelled to each individual embryonic primordium together withabout 20–30 Vasa-negative cells (Fig. 2k, n). These cells remain atthe posterior of the embryos undergoing morphogenesis (Fig. 2l, o)and become incorporated into paired gonads (Fig. 2m, p). Thus,the distribution of Cfvasa mRNA and protein demonstrates thatthe small cell at the four-cell stage that inherits the oosome iscontinuous with the germ cells of reproductive larvae. This suggeststhat the unique cytoplasm forming the oosome represents germplasm, and that the Copidosoma egg exhibits a maternally specifiedcellular asymmetry that is maintained throughout the proliferativeperiod.

Caste formation in eusocial hymenoptera is a complex series ofevents in which individual larvae or embryos develop towards thereproductive or sterile caste fate in response to environmentalstimuli4. However, both sterile and reproductive castes initiate theformation of gonads, which later degenerate in sterile workers andsoldiers17. Even though precocious Copidosoma larvae nevermature, it has been unclear whether they represent a true sterilemorph or whether they simply do not complete their developmentalprogramme because of their premature death. Both Cfvasa mRNAand Vasa protein were absent during the course of precocious larvamorphogenesis (more than 600 embryos analysed; Fig. 3) eventhough Vasa protein was expressed in adjacent reproductive

Figure 3 Cfvasa mRNA and protein expression patterns during Copidosoma precocious

larvae morphogenesis (purple, mRNA; red, Vasa protein; green, tubulin). a, d, Early

embryonic primordium. b, e, Extended germband stage. c, f, Completely formed larva,

arrow points to reproductive embryo with germ cells. Scale bar, 30 mm.

Figure 2 Cfvasa mRNA and protein expression patterns during Copidosoma

embryogenesis. Purple, Cfvasa mRNA; red, Vasa protein; green, tubulin. a–c, Ovarial

eggs. a, Early ovarial egg; white arrow points to nurse cells and black arrow points to

oocyte. b, Late ovarial egg; black arrowhead points to oosome. c, 1, Vasa protein

expression; 2, tubulin; 3, transmission differential interference contrast image, with

arrowhead pointing to oosome; 4, merged image. d–f, Early cleavages. d, Uncleaved egg.

e, Second cleavage. f, Third cleavage. g, h, Primary morula. i, j, Proliferative phase. Inset:

cleavage of Vasa-positive cell; arrow points to the proliferative morula without germ cells.

k–p, Reproductive morphogenesis. k, n, Early embryonic primordium. l, o, Extended

germband stage. m, p, Completely formed larva. Scale bar 25, mm.

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embryos (Fig. 3f, arrow). Thus, precocious morphs originate frommorulae that do not contain Vasa-positive cells. The lack of germcells suggests that precocious morphs represent true sterile morphsand that the distinction between reproductive and precociousmorphs is determined by a differential distribution of germlineprecursor cells.

Because of extensive pre-patterning in many invertebrate eggs,the ablation of early cells or removal of special cytoplasm containinglocalized determinants results in significant developmentaldefects12. In contrast, mammalian embryos show extensive regula-tive abilities, attributed to a lack of pre-patterning where theremoval of early cells results in the formation of normal embryos18.To determine the role of the Vasa-positive cell in the embryonicdevelopment of Copidosoma, we laser-ablated cells at the four-cellstage and analysed the development of sham-treated and manipu-lated embryos (Fig. 4a). To exclude the possibility that embryos failto initiate development after our treatments, we first culturedsubpopulations of embryos in vitro to confirm normal developmentand to assess Vasa expression. Short-term cultured embryos thatwere sham-treated, Vasa-positive cells that were ablated, andembryos with ablated large cells (Fig. 4b–d) all emerged from thechorion and formed primary morulae, indicating that our treat-ments did not disrupt normal development. Whereas sham-treatedand large-cell ablated embryos showed staining of an expectednumber of Vasa-expressing cells in the morula stage, no cellsshowing Vasa staining were observed in Vasa-positive-cell ablatedembryos, showing that laser ablation eliminates the progeny ofthe Vasa-positive cell. In addition, the sham-treated embryostransplanted into a host egg developed along a normal time-line, producing reproductive and precocious larvae (Fig. 4e) in

proportions and numbers almost identical to those of waspsovipositing naturally10. These embryos developed into viable adultwasps (not shown), indicating that our transplantation protocolrecapitulates the normal life cycle of the parasite.

Germ cells specified by the maternally preformed germ plasm inDrosophila and some crustaceans show lineage restriction, exclu-sively forming the germ line19,20. Consequently, the removal ofgerm-cell precursors results in a cell-autonomous defect: failureto form gonads. However, mouse and urodele amphibians lackpreformed germ plasm, and germ cells are induced later in develop-ment in a pluripotent cell population that can give rise to bothprimordial germ cells and somatic cells14,21. If a Vasa-positive cellis lineage restricted to reproductive embryos in Copidosoma, asindicated by our antibody staining, it could have a cell-autonomousrole in specifying the germ line. Thus, its ablation would result in theproduction of both precocious and germ-cell-deficient reproduc-tive larvae. Alternatively, a cell with early asymmetry could imposeorganizational properties on the embryo12, exerting non-cell-auton-omous influences. In this model, cell ablation should cause broadereffects, not limited to the germ line. Dissection and counting oflarval morphs at the fifth host instar revealed that total embryoproliferation decreased by almost 95% in embryos in which theVasa-positive cell was ablated at the four-cell stage. The populationof larvae dissected from the host consisted exclusively of precociousmorphs in numbers similar to those found in the sham-treatedembryos (t ¼ 0.24; P ¼ 0.81). This indicates that the Vasa-positivecell might have a role in the development of reproductive embryos,by regulating their proliferation and specifying reproductive casteidentity. To test whether this is a unique property of the Vasa-positive cell, we individually ablated the other three large cells.

Figure 4 Cell ablation of four-cell stage Copidosoma embryo. Red, Vasa protein; green,

tubulin. a, Experimental design. Copidosoma embryos were dissected from the host

egg at the four-cell stage. Embryos were either sham-treated, or a small cell (red) was

ablated (yellow arrow, upper row), or one of large cells was ablated (yellow arrow,

lower row). Embryos were transplanted to the host egg and host larvae were dissected

when they reached the fifth larval i star. We counted numbers and examined the

morphology of precocious and reproductive larvae to determine the effect of cell

ablation on embryo proliferation and development. b–d, Short-term culture of

Copidosoma embryos in vitro. b, Sham-treated embryo. c, Vasa-positive cell ablated.

d, Large cell ablated. Scale bars 25mm. e, Effect of cell ablation on the development

of precocious and reproductive larvae. Results are means ^ s.e.m. Solid bars, sham

treatment; dark-hatched bars, Vasa-positive cell ablation; light dotted bars, large cell

ablation.

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When progeny of large-cell-ablated embryos were dissected, wefound that both precocious and reproductive larval morphs wereproduced, although with reduced numbers of reproductive larvae(38%) compared with sham-treated embryos (t ¼ 6.6; P , 0.001).Cumulatively, these manipulations show that the Vasa-positive cellat the four-cell stage specifies the fate of reproductive larvae andhas a major effect on embryo proliferation in Copidosoma. Theother cells modulate the level of proliferation of reproductivelarvae. Surprisingly, the development of precocious larvae wasnot different between treatments (P . 0.81; three pairwise com-parisons), indicating that they develop independently of thereproductive lineage and possess a mechanism to compensate forablated cells.

In contrast with monoembryonic eusocial Hymenoptera, Copi-dosoma castes form clonally from the same zygote in the same hostenvironment. Our data reveal that this unique polyphenism is dueto the evolution of a novel developmental process for specificationof the sterile and reproductive castes. Instead of environmentalcues for the determination of caste sterility, Copidosoma relies ona developmental process that distributes germ-cell precursors toreproductive embryos but not to the sterile precocious embryos.This process could rely on a mechanism that uses the differentialsorting of Vasa-positive cells to future reproductive embryos;alternatively, differential distribution of germ-cell precursorscould be achieved by early downregulation of the germlinedevelopmental programme in a subpopulation of Vasa-positivecells in proliferative morulae destined to become precociousembryos.

Our previous work documented many similarities between thedevelopment of Copidosoma and mammalian embryos, indicatingconvergent morphological evolution as a result of development in anutritive host environment8. The present results functionallydemonstrate that Copidosoma combines pre-patterning withregulative abilities in early embryonic development. Our findingparallels recent evidence that mammals might also use pre-pattern-ing in early development22,23. Copidosoma, like many invertebrateembryos, segregates determinants that function to specify the germline. However, early cleavage cells in Copidosoma are unusual in thatthey segregate determinants for cellular proliferation and caste faterather than determinants for embryo axial polarity. Our data arguefor a model in which reproductive embryos represent a mixedlineage of large cells and Vasa-positive cell progeny. The non-cell-autonomous function of Vasa-positive cells is consistent with themodel in which these cells serve as signalling centres inducingthe proliferation of reproductive embryos. Our data show thatspecies with complex development might have evolved previouslyunsuspected cellular mechanisms that contribute to developmentalplasticity, underscoring a cellular context in the evolution ofdevelopmental programmes. A

MethodsIsolation of vasa mRNA, phylogenetic analysis and immunocytochemistryA Cfvasa genomic fragment was isolated by polymerase chain reaction (PCR) with thefollowing degenerate primers: 5 0 -CAGACGGGITCIGGIAARAC-3 0 and 5 0 -TGGAGACGRTCICCRTGDAT-3

0. A Copidosoma morphogenetic complementary DNA library was

screened with a 32P-labelled PCR fragment to isolate a 2,550-base-pair vasa cDNA(GenBank accession number AY611624). The sequences were aligned with a CLUSTAL Walgorithm24 using the Gonnet protein matrix, a gap-opening penalty of 10 and a gap-extension penalty of 0.2, using CLUSTALW v. 1.81 (Linux). An unrooted dendrogram wasgenerated with the maximum-likelihood method25 with 10,000 bootstrap iterations andthe computation of clock-like maximum-likelihood branch lengths with TREE-PUZZLEv. 5.1 (Linux). The following Vasa protein sequences were used to create the dendrogram:Caenorhabditis elegans: GLH-1 (P34689), GLH-2 (AAB03337), GLH-3 (AAC28388) andGLH-4 (AAC28387); Hydra magnipapillata: CnVAS1 (BAB13307) and CnVAS2(BAB13308); Ephydatia fluviatilis: PoVAS1 (BAB13310); Dugesia dorotocephala: PlVAS1(BAB13310); Drosophila melanogaster: VASA (P09052); Bombyx mori: BmVLG(BAA19572); Ciona intestinalis: CiDEAD1A (BAA36711); Danio rerio: VLG (CAA72735);Oncorhyncus mykiss: OmVASA (BAA88059); Xenopus laevis: XVLG1 (AAC03114); Gallusgallus: CVH (BAB12337); Mus musculus: MVH (Q61496); Rattus norvegicus: RVLG(Q64060); Copidosoma floridanum: CfVAS (AY611624). In situ hybridization was

performed with a fragment of the Cfvasa cDNA spanning nucleotides 991–1920 asdescribed in ref. 26. Antibody staining, using anti-Drosophila Vasa antibody27 (donated byP. Lasko) at a dilution of 1:3 was performed as described in ref. 7.

Laser ablation, embryo culture and embryo transplantationCopidosoma embryos at the four-cell stage were dissected from the host egg in Ringer’ssolution28 and transferred to a depression slide. Embryos were sham-treated or individualcells were laser-ablated under a 63 £ water-immersion objective on a Zeiss Axioplan IImicroscope fitted with a Micropoint laser ablation system. After treatment, the embryoswere transferred to a bevelled glass microsyringe (60 mm diameter) and transplanted into0–4-hour-old T. ni eggs with a Narashige micromanipulator fitted with an Eppendorfmanual air microinjector. Emerged host larvae were reared on an artificial diet as describedin ref. 29. Success rates of transplantation were as follows: sham-treated, 27% (241 hostlarvae developed after transplantation; 65 contained Copidosoma embryos); Vasa-positivecell ablated, 20% (251 host larvae developed after transplantation; 50 containedCopidosoma embryos); large cell ablated, 20% (299 host larvae developed aftertransplantation; 60 contained Copidosoma embryos). Embryos were cultured in vitro asdescribed in ref. 30.

Received 18 May; accepted 2 November 2004; doi:10.1038/nature03171.

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30. Grbic, M., Rivers, D. & Strand, M. R. Caste formation in the polyembryonic wasp Copidosoma

floridanum (Hymenoptera: Encyrtidae): In vivo and in vitro analysis. J. Insect Physiol. 43, 553–565

(1997).

Supplementary Information accompanies the paper on www.nature.com/nature.

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Acknowledgements We are thankful to L. Nagy for the idea of performing embryo

transplantation into host eggs; E. Zhurov for expert embryo transplantation; P. Lasko, L. Nagy,

L. Dickinson, T. Reavell, T. Drysdale, P. Wigge and G. Velicer for critical reading of the

manuscript; E. Zhurov and I. Craig for the artwork; J. Whistlecraft, L. Verdon and N. Terzin for

Copidosoma rearing; and R. Kulperger for the statistical analysis. M.G. thanks V. Grbic and Z. Srdic

for introducing him to parasitic wasps. This work was supported by grants from the Canadian

Foundation of Innovation, Premier’s Research Excellence Award and National Science and

Engineering Research Council to M.G.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to M.G. (mgrbic@uwo.ca).

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Dominant influence of HLA-Bin mediating the potentialco-evolution of HIV and HLAPhotini Kiepiela1, Alasdair J. Leslie2, Isobella Honeyborne1,2,Danni Ramduth1, Christina Thobakgale1, Senica Chetty1,Prinisha Rathnavalu1, Corey Moore3, Katja J. Pfafferott2, Louise Hilton2,Peter Zimbwa2, Sarah Moore4, Todd Allen5, Christian Brander5,Marylyn M. Addo5, Marcus Altfeld5, Ian James3, Simon Mallal3,Michael Bunce6, Linda D. Barber7, James Szinger8, Cheryl Day2,Paul Klenerman2, James Mullins4, Bette Korber8, Hoosen M. Coovadia1,Bruce D. Walker1,5,9 & Philip J. R. Goulder1,2,5

1HIV Pathogenesis Programme, The Doris Duke Medical Research Institute,University of KwaZuluNatal, 719 Umbilo Road, Durban 4015, South Africa2Department of Paediatrics, Nuffield Department of Medicine, Peter MedawarBuilding for Pathogen Research, South Parks Road, Oxford OX1 3SY, UK3Centre for Clinical Immunology and Biomedical Statistics, Royal Perth Hospitaland Murdoch University, Level 2 North Block, Royal Perth Hospital, WellingtonStreet, Western Australia 6000, Australia4Department of Microbiology, University of Washington, Seattle, Washington98195-8070, USA5Partners AIDS Research Center, Massachusetts General Hospital, 149 13th Street,Charlestown, Massachusetts 02129, USA6Dynal Biotech Ltd, 11 Bassendale Road, Wirral CH62 3QL, UK7Anthony Nolan Research Institute, Royal Free Hospital, Pond Street, LondonNW3 2QG, UK8Santa Fe Institute, Santa Fe, New Mexico 87501, USA, and Los Alamos NationalLaboratory, Los Alamos, New Mexico 87545, USA9Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA.............................................................................................................................................................................

The extreme polymorphism in the human leukocyte antigen(HLA) class I region of the human genome is suggested toprovide an advantage in pathogen defence mediated by CD81 Tcells1–3. HLA class I molecules present pathogen-derived pep-tides on the surface of infected cells for recognition by CD81 Tcells. However, the relative contributions of HLA-A and -Balleles have not been evaluated. We performed a comprehensiveanalysis of the class I restricted CD81 T-cell responses againsthuman immunodeficiency virus (HIV-1), immune control ofwhich is dependent upon virus-specific CD81 T-cell activity4,5.In 375 HIV-1-infected study subjects from southern Africa, asignificantly greater number of CD81 T-cell responses are HLA-B-restricted, compared to HLA-A (2.5-fold; P 5 0.0033). Herewe show that variation in viral set-point, in absolute CD4 countand, by inference, in rate of disease progression in the cohort, isstrongly associated with particular HLA-B but not HLA-A alleleexpression (P < 0.0001 and P 5 0.91, respectively). Moreover,substantially greater selection pressure is imposed on HIV-1 byHLA-B alleles than by HLA-A (4.4-fold, P 5 0.0003). These data

indicate that the principal focus of HIV-specific activity is atthe HLA-B locus. Furthermore, HLA-B gene frequencies in thepopulation are those likely to be most influenced by HIVdisease, consistent with the observation that B alleles evolvemore rapidly than A alleles6–8. The dominant involvement ofHLA-B in influencing HIV disease outcome is of specificrelevance to the direction of HIV research and to vaccinedesign.

Despite apparently similar roles in pathogen defence, there arewell-established differences between the class I loci. HLA-C allelesare expressed at lower levels on the cell surface than HLA-A andHLA-B (ref. 9) and thus it is not unexpected that HLA-C showsthe least diversity of the three classical HLA class I loci. However,an unexplained observation is that greater diversity exists at theHLA-B locus than at the HLA-A locus. Overall, there are 563HLA-B alleles described, compared with 309 HLA-A alleles and167 HLA-C alleles (http://www.ebi.ac.uk/imgt/hla/docs/relea-se.html). It appears that the HLA-B locus is diversifying morerapidly than the HLA-A locus; the significance of which remainsunknown.

In order to test the hypothesis that the observed differentialdiversity at the class I loci reflects functional differences betweenthe HLA-A, -B and -C-restricted CD8þ cytotoxic T lymphocyte(CTL) responses, we investigated the contribution of differentHLA class I molecules within the CD8þ T-cell activity directedagainst HIV—a pathogen whose control is strongly influenced byCD8þ T cells.

We used an empirical approach to determine the contributions ofindividual HLA class I molecules in anti-HIV host defence in 375infected, treatment-naive persons in southern Africa, a regionburdened with more HIV infections than any other (http://www.unaids.org). This avoids the heavy biases inherent in the use ofpreviously defined optimal epitopes that largely favour particular,well-studied, HLA class I molecules. We employed a panel of 410overlapping synthetic peptides, spanning the entire expressed HIVgenome, and characterized the T-cell responses to these peptides ininterferon-g enzyme-linked immunospot (elispot) assays. Previousstudies have validated this approach, and have shown that theresponses detected by this method are, with few exceptions, theresult of CD8þ T-cell activity10.

This analysis revealed marked differences in the frequency oftargeting of individual peptides; 132 peptides being targeted by nosubjects, and five peptides targeted by .25% of persons. For manytargeted peptides, we noted a strong association with specific class Iallele expression: 180 peptides exhibited a strong allele-specificassociation (P , 0.05), of which 111 remained significant(P , 0.001) following correction for multiple comparisons (seeSupplementary Tables 1 and 2). These included 58 previouslydefined epitopes (http://www.hiv.lanl.gov/content/immunology/tables/ctl_summary.html). To demonstrate further that the HLA–peptide associations reflect true HLA class I restricted CD8þ T-cellresponses, we experimentally defined the restriction elements for 12additional, randomly selected epitopes (Fig. 1, Supplementary Fig. 1and Supplementary Tables 1 and 2). In each case the restricting allelewas precisely that predicted by the statistical association. Thisanalysis therefore affords a stringent approach to define the HLAclass I restriction for the large majority of CD8þ T-cell responsesdetected in this population.

We next used this approach to determine the relative contri-butions of the different class I alleles to immune recognition ofHIV-1. The association of HLA-B alleles with peptide-specificresponses far exceeded that of HLA-A alleles (67 versus 25/111,P ¼ 0.0033, two-tailed unpaired t-test; mean peptide associationsper allele, 3.94 versus 1.56) and of HLA-C alleles (19/111, P ¼ 0.004;Fig. 2a and b). Of the 30 most highly targeted peptides, 67%were HLA-B-restricted responses (Fig. 2c). Thus, HLA-B allelescontribute significantly more towards the total HIV-specific

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