brief communications Entomology the result of monitoring cues from worker bees, as in the density-dependent prophy- Control Challenge 13 s.e.) Immune defence in s.e.) 1,200 laxis observed in other insects . ± ± 12 Yannick Moret, Paul Schmid-Hempel 11 max 1,000 bumble-bee offspring V 10 Eidgenössische Technische Hochschule Zürich, Ecology and Evolution, ETH-Zentrum NW, mmune-challenged vertebrate females 9 800 transfer specific antibodies to their off- 8092 Zürich, Switzerland 8 600 spring1–3, but this gratuitous immunity e-mail: [email protected] I 7 4 cannot operate in invertebrates . Here we 400 6 1. Hanson, L. A. Ann. Allergy Asthma Immunol. 81, 523–537 show that constitutive immune defence is (1998). 5 200 enhanced in sexual offspring of the bumble- Workers Males Workers Males 2. Coste, A., Sirard, J. C., Johansen, K., Cohen, J. & Kraehenbuhl, J. P. J. Virol. 74, 8966–8971 (2000). Phenol oxidase activity ( bee Bombus terrestris L. when the parental Zone inhibition diameter (mm Antibacterial Enzyme activity activity 3. Rollier, C., Charollois, C., Jamard, C., Trepo, C. & Cova, L. colony is immune-challenged. Our findings Vaccine 18, 3091–3096 (2000). indicate that invertebrates may use a differ- Figure 1 Antibacterial and phenol oxidase activities in the 4. Hoffmann, J. A., Reichhart, J. M. & Hetru, C. Curr. Opin. ent component of the immune system to haemolymph of workers and males from control (orange bars) Immunol. 8, 8–13 (1996). generate a facultative trans-generational and challenged (blue bars) groups from 11 bumble-bee colonies. 5. Söderhäll, K. & Cerenius, L. Curr. Opin. Immunol. 10, 23–28 (1998). increase in the immune response. In workers, antibacterial activity was higher in challenged 6. Nappi, A. J. & Vass, E. Pigment Cell Res. 6, 117–126 (1993). Insect immunity is characterized by the groups than in controls (Wilcoxon’s paired signed-rank test: 7. Thörnqvist, P.-O. & Söderhäll, K. in Diseases in Asian Aquaculture vol. 3 (eds Flegel, T. W. & MacRae, I. H.) inducible expression of a large array of T&46, n411, P*0.02), but phenol oxidase activity was lower 203–218 (Fish Health Section, Asian Fisheries Soc., Manila, (T 43, n411, P40.005). In males, antibacterial activity was antimicrobial peptides and by the constitu- 1 Philippines, 1996). tive melanization–encapsulation response, the same (T&431, n411, NS) but phenol oxidase activity was 8. Nigam, Y., Maudlin, I., Welburn, S. & Rattcliffe, N. A. which is based on a cascade involving an higher (T&46, n411, P*0.02) in the challenged side than in J. Invertebr. Pathol. 69, 279–281 (1997). inactive precursor of the enzyme phenol oxi- controls. Further experiments showed that increased activity in 9. Schmid-Hempel, P. Parasites in Social Insects (Princeton Univ. 4,5 Press, Princeton, New Jersey, 1998). dase . Antibacterial activity can be induced, males correlates with an increased encapsulation response 10.Schmid-Hempel, R. & Schmid-Hempel, P. Funct. Ecol. 12, 4 4 for example, by lipopolysaccharide (LPS) against an invader (F1, 23 5.25, P 0.031). Vmax is measured as 22–30 (1998). extracted from bacterial surfaces. The opera- the maximum change in optical density21013 per minute. 11.Kato, Y. et al. Insect Biochem. Mol. Biol. 24, 539–545 (1994). tion of the cascade is indicated by the phenol 12.Moret, Y. & Schmid-Hempel, P. Science 290, 1166–1168 (2000). 6,7 12 13 13.Barnes, A. I. & Siva-Jothy, M. T. Proc. R. Soc. Lond. B 267, oxidase activity in the insect haemolymph antibacterial and phenol oxidase activity 177–182 (2000). and can be monitored by measuring the rate (1/20 dilution). of conversion of a phenol substrate into As expected, workers in the challenged quinone, which then polymerizes to form groups showed more antibacterial activity melanin. Because both quinone and melanin than controls (Fig. 1). Their phenol oxidase Nanotechnology are toxic to microorganisms5, hosts with high activity, however, was lower (Fig. 1), phenol oxidase activity are less susceptible to indicating that there could be a possible Synthesis of carbon microbial infection8. trade-off between these two immune ‘onions’ in water Social insects cooperate in brood care responses in challenged workers. Haemo- and make a considerable investment in cyte counts were similar between the he fabrication of carbon nanomateri- their offspring. In annual species such as two groups (Wilcoxon’s paired signed-rank als usually calls for expensive vacuum 4 4 1,2 bumble-bees, reproduction occurs at the test, T- 29, n 11, NS). Immune- Tsystems to generate plasmas and end of the colony cycle, when sexuals challenged groups had lower reproductive yields are disappointingly low. Here we (daughter queens and males) emerge — output (repeated measures-MANOVA for describe a simple method for producing here the term ‘trans-generational’ distin- log-transformed number of males and high-quality spherical carbon nano- 4 4 guishes the queen and workers from sexual queens: Hotelling’s T 1.297, F2,9 5.839, ‘onions’ in large quantities without the use offspring. Unlike daughter queens, males P40.024), notably producing fewer queens of vacuum equipment. The nanoparticles, 4 4 do not hibernate, so their reproductive suc- (F1,10 12.082, P 0.006), indicating a pos- which have C60 cores surrounded by onion- cess depends on post-emergence survival sible trade-off between reproductive output like nested particles, are generated by an arc after they leave the parental colony and are and immune response. Male offspring from discharge between two graphite electrodes exposed to parasites in the same habitat9. challenged groups showed higher phenol submerged in water. This technique is eco- Assuming facultative adjustment of off- oxidase activity than controls, but anti- nomical and environmentally benign, and spring immunity, we investigated whether bacterial activity (Fig. 1) and haemocyte produces uncontaminated nanoparticles parasite-challenged parental colonies could counts were comparable between the two which may be useful in many applications. 4 4 enhance their males’ immunocompetence. groups (T- 29, n 11, NS). Nanoparticles have previously been pre- We used a split-colony design10 with As insects do not produce antibodies, pared using a range of vacuum and non- 11 colonies, each equally split into treat- they cannot transfer specific immunity as vacuum methods3–5. Ours is a non-vacuum ment and control groups. In the immune- mammals do1. Male bumble-bees from method in which the carbon arc is sus- challenged group, 70–80% of workers were immune-challenged groups have increased tained in deionized water. The apparatus injected weekly with LPS (Sigma L-2755, constitutive immunity relative to controls, consists of two submerged graphite elec- 0.5 mg ml11 in Ringer’s solution (5 ml)), which both enhances encapsulation (Fig. 1) trodes, and the arc discharge is initiated by which activates the immune system for long and protects against microorganisms5,8. As contacting a pure grounded graphite anode periods11. Control workers were treated in the phenol oxidase enzyme cascade pro- (tip diameter, 5 mm) with the carbon cath- the same way, but with the omission of LPS. vides a broader immunity than the costly ode (tip diameter, 12 mm) of similar puri- Colonies completed their life cycle in the antibacterial immune response12, males ty; the discharge voltage and current were laboratory under standard conditions may benefit by enhancing their most gener- 16–17 V and 30 A, respectively. The nano- (24 7C, 60% relative humidity). We counted al means of prophylaxis. Although the phys- onions are mostly found floating on the the number of sexuals and haemocytes iological mechanism by which this water surface, with the rest falling to the (Neubauer haemocytometer, 1/6 dilution) trans-generational transfer is achieved is bottom of the beaker through natural and used standard protocols to measure unknown, the enhanced immunity could be segregation, giving material of high purity. 506 © 2001 Macmillan Magazines Ltd NATURE | VOL 414 | 29 NOVEMBER 2001 | www.nature.com brief communications ab Cretaceous Gansus (possibly a shore- dwelling bird), from Gansu Province in c the continental interior of China, and Liaononingornis, perhaps the oldest known Onions ornithurine bird from the famous Early Cretaceous Confuciusornis locality2. As hesperornithiform birds (highly derived ornithurines) were already well adapted for foot-propelled diving by the Aptian epoch (the latter part of the Early Figure 1 Carbon nano-‘onions’ created by arc discharge in water. a, Image of a carbon arc discharge in water. Scale bar, 12 mm. b, c, Low- Cretaceous), their divergence must have been and high-magnification electron micrographs of carbon nano-onions floating on the water surface after their production. Scale bars, 10 nm. from even earlier ornithurine birds. By the Late Cretaceous, numerous taxa of hesper- A digital image of the arc discharge in water 3. Cabioch, T., Riviere, J. P. & Delafond, J. J. Mater. Res. 30, ornithiform birds, together with the volant is shown in Fig. 1a. 4787–4792 (1995). ornithurines Ichthyornis and Apatornis, are We investigated the unpurified material 4. Ishigami, M., Cumings, J., Zettl, A. & Chen, S. Chem. Phys. Lett. known from many marine localities in both 319, 457–459 (2000). from the water surface using a JEOL 5. Hsu, W. K. et al. Nature 377, 687 (1995). the Northern and Southern Hemispheres. 4000EX transmission electron microscope. 6. Hsin, Y. L. et al. Adv. Mater. 13, 830–833 (2001). What correlation is there between the dis- A typical high-resolution micrograph of the 7. Tenne, R., Homyonfer, M. & Feldman, Y. Chem. Mater. 10, tributions of shore birds (waders; Charadri- material is shown in Fig. 1b — several 3225–3238 (1998). iformes) and the marine, shoreline habitats spherical carbon nano-onions are evident, of the continental margins? These birds are as well as polyhedral, nested onion-like par- quite cosmopolitan, occurring in shallow ticles.