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4. Fuhrer, M. S. et al. Science 288, 494–497 (2000).

5. Bockrath, M. et al. Science 291, 283–285 (2001).

6. Hong, S. & Mirkin, C. A. Science 288, 1808–1811 (2000).

7. Manandhar, P. et al. Phys. Rev. Lett. 90, 115505 (2003).

8. Zhang, M. et al. Nanotechnology 13, 212–217 (2002).

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3274–3275 (1995).

10.Liu, J. et al. Chem. Phys. Lett. 303, 125–129 (1999).

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630–633 (2001).

Supplementary information accompanies this communication on

Nature’s website.

Competing financial interests: declared none.

Innate defence

Evidence for memory ininvertebrate immunity

Acquired immunity in vertebrates ischaracterized by immunologicalmemory and specificity, whereas the

innate defence systems of invertebrates areassumed to have no specific memory1–3.Here we use a model system of a copepod,which is a minute crustacean, and a para-sitic tapeworm to show that the success ofreinfection depends on the antigenic resem-blance between the consecutively encoun-tered parasites. This finding indicates thatan invertebrate defence system may becapable of specific memory.

The degree of specificity and memory in invertebrate immunity is unclear3–5.Although a defence reaction can be inducedin invertebrates against pathogens6, theresponses may not be specific as they are ableto distinguish only between different classesof pathogen2,3. However, genotype-specificinteractions between invertebrate hosts anddifferent lines of parasites are possible4,7.Such coevolutionary phenomena are diffi-cult to explain unless there is some specificityin the immune system.

We investigated the line-specific memoryof the defence system of an invertebrate host,the copepod Macrocyclops albidus, against a natural parasite, the tapeworm Schisto-cephalus solidus. To vary the antigenic fea-tures of the pathogen presented to the host, we exposed each copepod to three tapeworm larvae and then either to three sibling parasites or to unrelated parasites of adifferent sib-group three days later (Fig. 1a).All tapeworms used for the second exposurewere fluorescently labelled to distinguishthem from the parasites used for primaryinfection8.

If there is a specific memory inherent inthe defence by the copepod hosts, we wouldexpect a reduction in the success of reinfec-tion by the sibling parasites. Indeed, wefound that prior exposure to related para-sites resulted in less secondary infection thanoccurred after exposure to unrelated para-

sites (Fig. 1b). On average, the reinfectionsuccess was reduced from 59.5�3.5% to47.6�4.8% of copepods infected (paired t-test, t�3.236, n�24, P�0.0037). In addi-tion, the average intensity of reinfectiondecreased from 0.81�0.06 to 0.66�0.07tapeworms per host (t�2.723, n�24,P�0.0121). This effect should increase withthe antigenic similarity between the consec-utively encountered parasites.

Because the tapeworms are simultaneoushermaphrodites whose self-fertilization(selfing) leads to homogeneous offspring,wecompared the size of the host’s ‘memory’effect towards tapeworms produced by self-ing against that for tapeworms produced byoutcrossing. As predicted, the reduction inreinfection increased for selfed worms (Fig.1c; t-test,n�8; n�16 for outcrossed worms;reinfection success, t�1.361, P�0.187;intensity, t�2.230,P�0.036).

Could this reduced reinfection be due tofactors other than the host defence system?Neither host mortality nor primary infec-tion (determined in 16 of the 24 sibships)

differed between treatments (nominal logis-tic models, P�0.2). Could the parasitesthemselves cause the reduction in reinfec-tion? This is unlikely because cooperationbetween kin should facilitate rather thanreduce reinfection.

If within-host competition between sib-lings is particularly strong, only those hoststhat did not clear the primary infectionwould be affected. Excluding hosts with aresident primary infection from our sampledid not diminish the effect: prior exposure tosib-group parasites still reduced reinfectionfrom 64.1% to 45.6% in this smaller sample(in a nominal logistic model, including sibship, the effect of previous exposure was �2

1�7.728, n�128, P�0.0054). Weconclude that parasite-derived effects areunlikely to explain the observed reduction inreinfection after consecutive exposure of thehost to related parasites.

Our results show that the defence systemof copepods can react more efficiently after it has previously encountered antigenicallysimilar parasites. To our knowledge, this

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NATURE | VOL 425 | 4 SEPTEMBER 2003 | www.nature.com/nature 37

Figure 1 Exposure of a copepod to a tapeworm parasite reduces the chances of reinfection by antigenically similar tapeworm larvae.

a, Experimental design. Two parasite sib-groups are shown (denoted by white and black larvae). There were n�24 sib-groups, which

varied in infectivity (nominal logistic model, �2

23�95.95, n�684, r 2�0.102, P�0.001). The experiment was reproduced in two rounds,

both of which gave similar results and so were combined. Comparisons are made within the same parasite sib-group to control for varia-

tion in infectivity (further details are available from the authors). b, Reduction in reinfection due to prior exposure of the copepod host to

sibling parasites, compared with exposure to unrelated tapeworms, for each of the 24 tapeworm sib-groups (arranged in rank order of

difference). Sample sizes (number of copepods) are indicated above each bar. c, Difference in the success (left) and intensity (right) of

copepod reinfection due to the degree of antigenic resemblance between tapeworm larvae used for reinfection and those used for primary

infection. Left bars, selfed larvae; right bars, outcrossed larvae. Standard errors are indicated.

is the first demonstration of line-specificmemory in individual invertebrates. In astudy based on only two bacterial strains,maternally transferred immunity seemed tobe strain-specific9. Invertebrates are occa-sionally capable of a specific response in thecontext of allogenic recognition10, but speci-ficity in these cases is based on compatibilityfactors that have evolved within the speciesand so is not directly comparable withdefence against a parasite.

The mechanism responsible for the line-specific memory in this copepod remains tobe investigated, although binding by lectinsis one possibility11. If specific memory ininvertebrate defence is more widespreadthan originally thought, it would have impli-cations for our understanding of innateimmunity, host–parasite coevolution, andinvertebrate-vector diseases such as malaria.Joachim Kurtz, Karoline FranzDepartment of Evolutionary Ecology, Max PlanckInstitute of Limnology, August-Thienemann Strasse 2,24306 Plön, Germanyemail: kurtz@mpil-ploen.mpg.de1. Janeway, C. A., Travers, P., Walport, M. & Capra, J. D.

Immunobiology: The Immune System in Health and Disease

(Current Biology, London, 1999).

2. Hoffmann, J. A. & Reichhart, J. M. Nature Immunol. 3,

121–126 (2002).

3. Klein, J. Scand. J. Immunol. 29, 499–505 (1989).

4. Schmid-Hempel, P. & Ebert, D. Trends Ecol. Evol. 18,

27–32 (2003).

5. Rolff, J. & Siva-Jothy, M. T. Science 301, 472–475 (2003).

6. Faulhaber, L. M. & Karp, R. D. Immunology 75, 378–381 (1992).

7. Carius, H. J., Little, T. J. & Ebert, D. Evolution 55,

1136–1145 (2001).

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36–43 (2002).

9. Little, T. J., O’Connor, B., Colegrave, N., Watt, K. & Read, A. F.

Curr. Biol. 13, 489–492 (2003).

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I. L. Nature 295, 499–502 (1982).

11.Marques, M. R. F. & Barracco, M. A. Aquaculture 191,

23–44 (2000).

Competing financial interests: declared none.

Microfluidic systems

High radial accelerationin microvortices

Microfluidic systems can convenientlybe used for rapid analysis of biol-ogical samples1–5. Here we describe a

single re-circulating flow, or microvortex,that can generate a maximum fluid rota-tional velocity of up to 12 m s�1 and a cor-responding radial acceleration in excess of106g. Such microvortices may be exploitedin centrifugal microdevices to investigatethe effects of high radial acceleration onbiological and chemical processes.

A microvortex was generated in a dia-mond-shaped microchamber (Fig. 1a),which was visualized by using fluorescentpolystyrene beads (diameter, 2 �m; Fig. 1b,left). The average linear velocity of the pressure-driven flow in the 30-�m-widechannel was 3 m s�1. The diamond-shaped

notch along the side wall of an otherwisesmooth channel causes flow detachment atthe opening of the notch.

Unlike macroscale lid-driven cavity flow6,the combination of extremely tight rotationradii (r�50 �m) and high rotational velocity(v�10 m s�1) of fluid flow from constrictedmicrochannels can lead to generation of radi-al accelerations (v2/r) as high as 107 m s�2. Asthe average linear velocity in the 30-�m-widechannel was increased from 3 to 45 m s�1

(Reynolds number (Re)�245 at themicrochamber opening), the polystyrenebeads (density, �1.05 g cm�3) in water werespun out of the microchamber owing to highcentrifugal acceleration (Fig.1b,right).

To illustrate the potential applications ofthis microvortex system, we flowed red poly-styrene beads ( �1.05 g cm�3) and greensilica beads ( , 1.8–2.0 g cm�3) in a solutionof 50 % (w/w) of CsCl ( �1.56 g cm�3;

Fig. 1c). As the average flow velocity in the30-�m-wide channel was increased from1.5 m s�1 (Re�13 at the cavity’s opening) to20 m s�1 (Re�122 at the cavity’s opening),the red fluorescent beads were concentratedtowards the centre of the vortex, whereas thegreen beads were spun towards the chamber’souter edge. Although the linear flow velocitywas 20 m s�1, the corresponding volumetricflow rate was only about 18 �l s�1, owing tothe small cross-sectional area of the 30-�m-wide channel.

A simple calculation of the radial accelera-tion from the average flow rates in themicrochannel overestimates the accelerationthat we observed experimentally, mainlybecause of the damping of the flow velocitynear the walls of the microchannels for pres-sure-driven flows.Because the flow that drivesthe vortex at the opening of the diamond-shaped cavity is near the 30-�m-wide channelwall, the maximum fluid rotational velocityinside the cavity in Fig.1b,c is only about 20%of the average flow velocity in the channel.

To attain ultrahigh rotational velocities in the microvortex,we designed a trapezoidalmicrochamber connected to a constrictedmicrochannel that was placed asymmetricallyalong the width of a 200-�m200-�mchannel (see supplementary information).Using this design, we measured maximumrotational velocities as high as 12 m s�1 at adistance of 10 �m from the vortex core,which corresponds to a radial acceleration of1.4107 m s�2 or 1.4106g (Fig. 1d). Wemeasured rotation rates (Fig.1d),both by thetime taken for a photolysed (uncaged) plug offluorescein dye to travel a calibrated distance(5 �m) from a focused nitrogen photolysislaser beam to an argon laser probe volume(Fig. 1d, squares), and by the time taken forthe plug of dye to travel completely aroundthe microvortex (Fig. 1d, open circles)7 (andsee supplementary information).

The ability to use fluid flow with no mov-ing parts to generate large centrifugal forcesshould prove useful for integrated micro-analytical separations — for example, insample preparation.J. Patrick Shelby, David S.W. Lim,Jason S. Kuo, Daniel T. ChiuDepartment of Chemistry, University of Washington,Seattle, Washington 98195-1700, USAe-mail: chiu@chem.washington.edu1. Reyes, D. R., Iossifidis, D., Auroux, P.-A. & Manz, A. Anal. Chem.

74, 2623–2636 (2002).

2. Auroux, P.-A., Iossifidis, D., Reyes, D. R. & Manz, A. Anal.

Chem. 74, 2637–2652 (2002).

3. Thorsen, T., Maerkl, S. J. & Quake, S. R. Science 298,

580–584 (2002).

4. Beebe, D. J., Mensing, G. A. & Walker, G. M. Annu. Rev. Biomed.

Eng. 4, 261–286 (2002).

5. Ng, J. M. K., Gitlin, I., Stroock, A. D. & Whitesides, G. M.

Electrophoresis 23, 3461–3473 (2002).

6. Shankar, P. N. & Deshpande, M. D. Annu. Rev. Fluid Mech. 32,

93–136 (2000).

7. Shelby, J. P. & Chiu, D. T. Anal. Chem. 75, 1387–1392 (2003).

Supplementary information accompanies this communication on

Nature’s website.

Competing financial interests: declared none.

brief communications

38 NATURE | VOL 425 | 4 SEPTEMBER 2003 | www.nature.com/nature

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Figure 1 Generation and application of microvortices. a, Micro-

channel used to create the microvortex shown in b, c; dimensions

are in �m. b, Fluorescent beads are spun out of the chamber as

the flow velocity increases (from left to right). c, Separation of

low-density red beads from high-density green beads in the

microvortex as the flow velocity increases (from left to right). Scale

bar, 25 �m. d, The experimentally measured rotational rates

(squares and open circles) show excellent agreement with the

vorticity results (filled circles) extracted from our simulations

(some symbols overlap).