Explosive Backpacks in Old Termite Workers J. Š obotník, 1 * T. Bourguignon, 2,3 * R. Hanus, 1,2 † Z. Demianová, 1 J. Pytelková, 1 M. Mare š , 1 P. Foltynová, 4 J. Preisler, 4 J. Cvac ˇka, 1 J. Krasulová, 1,5 Y. Roisin 2 W e send our young men to war; ants send their old ladies (1). This meta- phor illustrates a long-known feature of insect societies: Older workers generally as- sume a larger share of defense, which may involve self-sacrifice ( 2, 3). In some termites, suicidal defense by bursting is performed by soldiers (4), but, because defense is their sole function, there is little potential for age dependence. How- ever, in a few species, workers are known to rupture and release a sticky fluid (4). Because workers perform a variety of tasks, this readiness for suicidal defense would be expected to increase as their aptitude for other tasks declines. Neocapritermes taracua is a neo- tropical termite commonly found in decayed wood on which it feeds (5). Close examination of workers reveals in many (hereafter blue workers) a pair of dark blue, elongated dorsal spots at the thorax-abdomen junction (Fig. 1A). These spots are variously developed among workers and are lacking in some individuals (white workers). During ag- gressive encounters with other termites, blue workers actively bite and, when hampered, burst and emit a drop of fluid, which in a few seconds becomes sticky as the blue color fades out (Fig. 1B and movie S1). The blue spots are a pair of crystal- like structures (Fig. 1C) enclosed within pouches formed by posterior outgrowths of the metanotum over the first abdom- inal segment. The crystals are produced by a pair of glands (crystal glands) located below the epidermal cell layer at the anterior part of each pouch (Fig. 1D). The dorsal apparatus also comprises the salivary glands, producing se- cretion granules stored below the dorsal wall (Fig. 1D). Mandibles of Neocapritermes workers wear out with time and usage because they cannot be renewed by molting (6) (fig. S1A). Mature work- ers showed a positive correlation between fresh weight and mandible sharpness index and a neg- ative relationship between crystal weight and mandible sharpness index, indicating that, as the feeding efficiency of workers decreases, they build up their backpacks for suicidal fighting (fig. S1, B and C). Blue workers were more aggressive than white workers toward other soil-feeding ter- mites, such as Labiotermes labralis, and they burst sooner after being seized by their oppo- nent (fig. S2). Bursting liquid from blue work- ers was more effective than liquid from white workers against L. labralis (table S1). Removing the blue crystals reduced the toxicity of blue workers to the level of white workers. Recip- rocally, the toxicity of white worker bursting fluid was enhanced by the addition of blue crys- tals but remained lower than that of blue work- ers (table S1). This pinpoints the importance of the reaction between blue crystals and salivary secretion to achieve toxicity. An important component of the blue crystal is a 76-kD protein (hereafter called BP76) as judged from the protein pattern on a 15% SDS– polyacrylamide gel electrophoresis (PAGE), and by a single blue band on the 10% native PAGE gel (fig. S3A). By using mass spectrometric meth- ods, we confirmed the molecular weight of BP76 (fig. S3B) and detected the presence of 9 T 2 ng (SD) of copper per crystal (fig. S3, C and D). Its molecular weight, color, and copper content sug- gest that BP76 is an oxygen-binding type III copper protein of the hemocyanin/phenoloxidase family, known to occur in arthropods (7). The defensive apparatus of N. taracua is exceptional. First, it involves a previously un- known exocrine gland, unusual in its product (the copper-containing blue crystal protein) and its storage (the external “backpack” pouches). Second, its efficacy derives from the reaction, upon autothysis, of the labial gland secretion and the blue crystals. Consistent with theory, workers develop this weaponry and increase their prone- ness to use it as their feeding efficiency decreases when their mandibles become worn. References and Notes 1. B. Hölldobler, E. O. Wilson, The Ants (Springer, Berlin, 1990), p. 179. 2. G. E. Robinson, Annu. Rev. Entomol. 37, 637 (1992). 3. J. R. Shorter, O. Rueppell, Insectes Soc. 59,1 (2012). 4. J. Š obotník, A. Jirosová, R. Hanus, J. Insect Physiol. 56, 1012 (2010). 5. T. Bourguignon et al., Ecol. Entomol. 36, 261 (2011). 6. P. Kaiser, Mitt. Hamb. Zool. Mus. Inst. 54, 129 (1956). 7. H. Decker, F. Tuczek, Trends Biochem. Sci. 25, 392 (2000). Acknowledgments: We are grateful to P. Cerdan and the staff of the Hydreco lab at Petit Saut for logistical help. Support was provided by the Fund for Scientific Research (F.R.S.-FNRS, Belgium); the Japan Society for Promotion of Science (no. 22-00393); the Grant Agency of the Czech Republic (P600/525/09, P206/12/0538); the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic (Z40550506); and the project CEITEC (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund. The data reported in this paper are presented in the supplementary materials. Supplementary Materials www.sciencemag.org/cgi/content/full/337/6093/436/DC1 Materials and Methods Figs. S1 to S3 Table S1 References (8–11) Movie S1 13 January 2012; accepted 12 April 2012 10.1126/science.1219129 BREVIA 1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic. 2 Evolutionary Biology and Ecology, Université Libre de Bruxelles, 1050 Brussels, Belgium. 3 Graduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan. 4 CEITEC (Central European Institute of Technology) and Department of Chemistry, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic. 5 Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague, Czech Republic. *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: robert@uochb.cas.cz Fig. 1. Anatomy of the defensive apparatus of N. taracua. (A) Soldier (s) with two blue (bw) and two white workers (ww). (B) Blue worker after autothysis triggered by grasping it with tweezers. (C) Blue worker after removal of the blue crystals (placed next to it). (D) Section of anterior abdomen of a blue worker. Asterisk marks crystal-bearing pouch (crystal dissolved); arrows mark crystal gland cells. Scale bar, 200 mm. 1a, first abdominal segment; 2t, mesothorax; aa, anterior acini (clumps of secretory cells); fb, fat body; m, muscles; p, dorsal part of the crystal-bearing pouch; pa, posterior acini; sg, aggregates of secretion granules budding off posterior acini. 27 JULY 2012 VOL 337 SCIENCE www.sciencemag.org 436 on July 26, 2012 www.sciencemag.org Downloaded from