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dependence on dose, whereas only a slight shoulder is found formonogononts. Beyond their respective shoulders, reproductionfalls off rapidly, with no indication of a resistant fraction. Thereproduction of daughters of irradiated rotifers is also radiosen-sitive, with a dose–response relation similar to that of theirparents but shifted to somewhat higher dose. Again, P. roseolaappears to be a little more radiosensitive than A. vaga.

DNA Breakage. Fig. 2 shows the gel electrophoretic distribution ofDNA found after exposing A. vaga to doses in the range of 0 to1,120 Gy. The average molecular sizes at 560, 840, and 1,120 Gyare 350, 250, and 190 kbp, respectively, with an average of 0.005double-strand breaks (DSBs) Gy!1!Mbp!1, a value within therange of 4 to 7 " 10!3 DSBs Gy!1!Mbp!1 measured by pulsed-field gel electrophoresis or sucrose sedimentation analysis forEscherichia coli, Saccharomyces cerevisiae, and diverse mamma-lian cell lines (9–12). Some of the DSBs we observed may havearisen from non-DSB lesions as a result of high-temperaturedigestion, an effect estimated to cause about a 30% overestimatein !-irradiated mammalian cells (13).

DiscussionReproduction of Irradiated Rotifers. We find that reproduction ofthe bdelloid rotifers A. vaga and P. roseola is much more resistantto 137Cs !-radiation than that of the monogonont rotifer E.dilatata. A dose of #200 Gy reduced the number of daughtersproduced by the monogonont by #90%, whereas a correspond-ing reduction in bdelloid fecundity required a dose five timeshigher. The principal difference between the bdelloid and mono-gonont dose–response relations lies in the extended shoulderover which bdelloid fertility and fecundity remain high beforefalling steeply, whereas only a much less extended shoulder isfound for the monogonont. The sterilizing effect of IR extends

to the daughters of irradiated parents, with dose–responserelations shifted to somewhat higher doses. Taking the size of theA. vaga genome as #180 Mbp (R. Gregory, personal commu-nication), the average molecular size of 350 kbp observed aftera dose of 560 Gy, causing a reduction in fecundity of only 20%,corresponds to #500 DSBs per genome.

The reproduction of A. vaga and P. roseola is more resistantto IR than that of any other metazoan for which we have foundrelevant data. Such resistance to IR is likely to be characteristicof the Bdelloidea generally, as A. vaga and P. roseola representfamilies that diverged millions of years ago (14) and becauseof the probable association, discussed below, of bdelloidradiation resistance with anhydrobiosis. Doses of IR or high-energy electrons required to prevent reproduction have beenmeasured for many arthropod species at various life stages,mainly for purposes of pest control. In a tabulation of suchsterilizing doses for male arthropods, which are generally moreradiosensitive than females, including 285 species representing61 families of insects and other arthropods, irradiated mainlyas pupae or nymphs and generally achieving at least 90%parental or F1 sterility, the average dose is #110 Gy (ref. 15and www-ididas.iaea.org/ididas/). For the most radio-resistantspecies in the tabulation, the lepidopteran Spilosoma (Diacra-sia) obliqua, a dose of 200 Gy to pupal or adult males, the mostresistant sex, resulted in $99% F1 sterilization.§ Among otherinvertebrates, sensitivity to IR has been measured for X-irradiation of Caenorhabditis elegans eggs in buffer, for which#30 Gy reduced the number developing into adults by 90%

§Rahman R, Rahman MM, Islam S, Huque R, Food and Agriculture Organization/Interna-tional Atomic Energy Agency Final Research Coordination Meeting, Evaluation of Popu-lation Suppression by Irradiated Lepidoptera and Their Progeny, May 28–30, 1998, Pen-ang, Malasia.

Table 1. Reproductive performance of bdelloid and monogont rotifers exposed to IR

SpeciesDose,

h

Parental wells Daughter wells

Wellswith eggs,

n

Wellswith F1,

nTotal

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Relativeparentalfecundity

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n

Wellswith F2,

nTotal eggshatched, n

RelativeF1

fertility

RelativeF1

fecundity

E. dilatata 0 81 68 290 249 1.00 1.00 58 58 1.001 82 26 272 35 0.38 0.14 9 6 0.272 78 0 266 0 0.00 0.00 n/a n/a n/a3 84 0 278 0 0.00 0.00 n/a n/a n/a

E. dilatata 0 88 86 324 291 1.00 1.00 65 64 1.000.5 87 80 302 160 0.93 0.55 45 40 0.671 87 24 278 58 0.28 0.20 15 10 0.562 85 2 316 2 0.02 0.01 1 0 0.00

E. dilatata 0 83 83 613 588 1.00 1.00 78 78 1,085 1.00 1.000.25 86 86 577 461 1.04 0.78 74 72 977 0.89 0.870.5 88 82 602 360 0.99 0.61 73 54 612 0.70 0.571.5 82 14 538 24 0.17 0.04 4 1 9 0.08 0.05

A. vaga 0 89 89 985 866 1.00 1.00 87 87 1.002 91 90 1,063 845 1.01 0.98 87 86 0.984 88 84 1,240 672 0.94 0.78 80 78 0.956 93 62 1,002 187 0.70 0.22 54 50 0.838 92 6 1,169 12 0.07 0.01 3 2 0.34

P. roseola 0 95 95 1,914 1,569 1.00 1.00 92 92 2,356 1.00 1.002 96 95 1,956 1,134 1.00 0.72 91 89 1,877 0.97 0.804 93 76 1,873 638 0.80 0.41 73 67 1,201 0.91 0.646 89 46 1,690 217 0.48 0.14 36 29 240 0.65 0.218 89 9 1,711 13 0.09 0.01 3 1 5 0.11 0.02

Rotifers were irradiated on ice for various times with a 137Cs source delivering 140 Gy per h. Parental and F1 relative fecundity and relative fertility are definedin Materials and Methods. The number of daughters transferred to new wells was in each case equal to the number of parental wells in which daughters wereproduced. For E. dilatata the number of hatched eggs was counted directly. For bdelloids it was taken as the number of progeny, as described in Materials andMethods.

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Fluorescent in situ hybridization (FISH) with aprobe from Av240A confirmed that these CDSreside in the A. vaga genome (Fig. 1E). Theirhybridization pattern resembles that of known telo-meric fosmids (15), suggesting proximity to telo-meres. The appearance of two hybridizing sites insome nuclei is consistentwith the genome structureof bdelloids in which chromosomes occur as co-linear pairs (16, 17). Indeed, we found colinear part-ners of both Av240A and Av212A (Av240B andAv212B) with overall pairwise divergence (4%)similar to that in other regions of bdelloid genomes.

The cluster of foreign genes near the Juno1LTR in Av240 includes two divergently orientedgenes for enzymes involved in bacterial cell wallpeptidoglycan biosynthesis—Alr, encoding ala-nine racemase, and Ddl, encoding D-Ala-D-Alaligase—adjacent to a uridine diphosphate (UDP)–glycosyltransferase apparently of plant origin(Fig. 1A). Reverse transcription polymerase chainreaction (RT-PCR) shows that all three genes aretranscribed and that their introns are properlyspliced (Fig. 1B). 5′ RACE (rapid amplificationof cDNA ends) demonstrates that the UDP-glycosyltransferase mRNA is trans-spliced, as arenumerous bdelloid genes (18), and thatAlr andDdltranscription initiates at oppositely oriented pro-

moters located between them (Fig. 1C). Further-more, the purified A. vaga AvDdl protein over-expressed in E. coli catalyzes the synthesis of theD-Ala-D-Ala dipeptide from D-Ala in vitro (Fig. 1D).

In addition to ubiquitous bacterial genes, suchas Alr and Ddl, we identified genes occurring inonly a few species of bacteria and fungi. TheXynB-like gene (figs. S1A and S2B) apparentlyrepresents a fusion between two different con-served domains and is found in only 10 bacterialspecies. Next to the Alr-Ddl pseudo-operon, thereis a HemK-like methyltransferase and a putativeadenosine triphosphatase. These two genes are alsorare: They are found in only four genera of proteo-bacteria and three genera of filamentous fungi. Inthe bacterium Methylobacillus flagellatus and inthe fungus Phaeosphaeria nodorum they areadjacent and in the same relative orientation as inA. vaga, indicating that they might have been ac-quired from a single source.

We also found genes with similarity to thosepresent in both metazoans and nonmetazoans.We characterized the foreignness of such geneswith an alien index (AI), which measures by howmany orders of magnitude the BLAST E-valuefor the best metazoan hit differs from that for thebest nonmetazoan hit (Table 1 and table S1) (19).We tested the AI validity by phylogenetic analysesof all CDS with AI > 0 yielding metazoan hits,excluding those with repetitive sequences. Wefound that AI ≥ 45, corresponding to a differenceof ≥20 orders of magnitude between the bestnonmetazoan and metazoan hits, was a good

indicator of foreign origin, as judged by phyloge-netic assignment to bacterial, plant, or fungalclades (Fig. 2 and fig. S2A). Genes with 0 < AI <45 were designated indeterminate, because theirphylogenetic analysis may or may not confidentlysupport a foreign origin. Four FabG-like genes forshort-chain dehydrogenases/reductases (SDH),from two different SDH subfamilies, are mostlikely of bacterial origin (AI = 98/92/88/45; Fig.2A). The A. vaga galacturonidase (AI = 212) ap-pears to be of fungal origin (Fig. 2B), and theUDP-glycosyltransferase in Av240 (AI = 28,indeterminate) belongs to a plant clade (Fig. 2C).Two genes, XynB and MviM, had sufficientnucleotide sequence similarity (~70%) to bacte-rial homologs for phylogenetic reconstructionand determination of nonsynonymous and syn-onymous divergence (fig. S2B).

We extended our search for foreign genes totwo pairs of contigs ending with telomeric repeats(telomeres K and L) (15) (fig. S1, B and C). In ad-dition to various TEs, telomeric repeats, and Athenaretroelements characteristic of bdelloid telomeric re-gions, we found additional examples of foreigngenes, including a pseudogene of fungal origin (pu-tative urea transporter; Table 1) with three frame-shifts and two in-frame stop codons in one of thetwo colinear homologs of telomere L. Additionally,we identified foreign genes sandwiched betweenshort stretches of telomeric repeats, suggesting theiraddition todeprotectedchromosomeends(fig.S1C).

We also observed examples of possible lossof genes and TEs from telomeres (fig. S3A), such

Table 1. Representative bdelloid CDS of foreign origin homologous to genes with known function. For a complete list and additional information oneach CDS, see table S1. Data are from BLASTP similarity searches, as described in (19). Asterisks indicate putative pseudogenes.

Gene ID, name Contig ID Introns AI % Identityto best hit

Best hit,E-value

Best hit,metazoan Best hit, taxonomy Definition

AV10027_XynB Av212A 0 460 63 0.00E+00 No hits Bacteria; Bacteroidetes Xylosidase/arabinosidaseAV10001_NRPS Av110A 10 460 32 0.00E+00 No hits Bacteria; (Proteobacteria/

Cyanobacteria)Nonribosomal peptide synthetase

AV10134_PheA 161F07 0 400 61 1.00E-174 No hits (Fungi; Bacteria) Monooxygenase, FAD dependentAV10002_TrkA Av110A 0 379 54 1.00E-175 4.00E-11 Bacteria; Proteobacteria Monooxygenase, NAD dependentPR10002_MviM 182F10 0 327 67 1.00E-149 2.00E-07 Bacteria; (Acidobacteria/

Chloroflexi)Oxidoreductase

PR10010_DAP2 182F10 0 310 27 1.00E-140 1.00E-05 Bacteria; (Acidobacteria/Proteobacteria)

Prolyl oligopeptidase*

AV10104_Dur3 AvTelL.B 1 243 44 1.00E-132 4.00E-27 Eukaryota; Fungi Urea active transporter*PR10012_RamA 182J17 0 246 31 1.00E-107 No hits (Bacteria; Fungi) a-L-RhamnosidaseAV10121_NRPS 99O7 4 237 30 1.00E-103 No hits Bacteria; Cyanobacteria Nonribosomal peptide synthetaseAV10153_XghA 210B3 0 212 50 1.00E-108 2.00E-16 Eukaryota; Fungi Endo-xylogalacturonan hydrolaseAV10042_HemK Av240B 1 199 56 2.00E-91 1.00E-04 Bacteria; Proteobacteria HemK-like methyltransferaseAV10092_b-Gal AvTelL.A 0 153 33 1.00E-105 4.00E-39 Eukaryota; Viridiplantae b-D-GalactosidaseAV10044_Alr Av240B 1 152 38 1.00E-67 No hits Bacteria; Bacteroidetes Alanine racemaseAV10025_AMH Av212A 1 150 52 8.00E-77 2.00E-11 Eukaryota; Fungi AmidohydrolaseAV10045_Ddl Av240B 1 138 40 1.00E-60 No hits Bacteria; Bacteroidetes D-Alanine-D-alanine ligaseAV10140_PLDc 193E18 2 126 31 1.00E-55 No hits Eukaryota; Fungi Phospholipase-D active site motif

protein*AV10016_FabG Av212A 0 98 58 1.00E-74 8.00E-32 Bacteria Short-chain dehydrogenase/reductaseAV10109_FabG AvTelL.B 0 92 57 4.00E-73 5.00E-33 Bacteria Short-chain dehydrogenase/reductase*AV10011_FabG Av212A 0 88 54 6.00E-67 2.00E-28 Bacteria Short-chain dehydrogenase/reductaseAV10071_HAL AvTelK.A 0 77 48 2.00E-61 1.00E-27 Bacteria Histidine ammonia-lyaseAV10095_GCN5 AvTelL.A 0 59 35 2.00E-27 No hits Bacteria; Proteobacteria GCN5-related N-acetyltransferase+/*AV10158_FabG 210B3 2 46 41 2.00E-39 2.00E-19 Bacteria Short-chain dehydrogenase/reductase

1Department of Molecular and Cellular Biology, HarvardUniversity, Cambridge, MA 02138, USA. 2Josephine Bay PaulCenter for Comparative Molecular Biology and Evolution,Marine Biological Laboratory, Woods Hole, MA 02543, USA.

*To whom correspondence should be addressed. E-mail:msm@wjh.harvard.edu (M.M.); iarkhipova@mbl.edu (I.R.A.)

www.sciencemag.org SCIENCE VOL 320 30 MAY 2008 1211

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