Journal of Insect Physiology 46 (2000) 1433–1439 www.elsevier.com/locate/jinsphys Caste-specific differences in ecdysteroid titers in early larval stages of the bumblebee Bombus terrestris Klaus Hartfelder a,* , Jonathan Cnaani b, c , Abraham Hefetz b a Departamento de Biologia, FFCLRP–Universidade de Sa ˜o Paulo, 14040-901 Ribeirao Preto, SP, Brazil b G.S. Wise Faculty of Life Sciences, Department of Zoology, Tel Aviv University, 69978 Tel Aviv, Israel c Carl Hayden Bee Research Center, USDA-ARS, 2000 E. Allen Road, Tucson, AZ 85719-1596, USA Received 15 November 1999; received in revised form 30 March 2000; accepted 8 April 2000 Abstract Mounting evidence implicates ecdysteroids in queen–worker differentiation during the last larval instars of highly social insects. In the present study, we analyzed ecdysteroid titers in queen and worker larvae of the bumblebee Bombus terrestris from the second to the early fourth instar. B. terrestris is of particular interest because caste is already determined in the second instar, presumably by a pheromonal signal emitted by the egg-laying queen. Caste differences in the adults, however, are only expressed at the physiological and not at the morphological level, except for the distinctly larger size of the queen. In the second and third instar, ecdysteroid titers in queen larvae were generally higher than those of workers. These early caste-specific differences, however, were abolished in the fourth instar. In the early fourth instar we could detect two small ecdysteroid peaks, with the one preceding the cocoon-spinning phase presenting the characteristics of a pupal commitment peak. The synchrony of caste differences in ecdys- teroid and juvenile hormone titers suggests a synergistic action of these hormones in caste determination.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Ecdysteroids; Radioimmunoassay; Juvenile hormone; Caste development; Bumblebee 1. Introduction Reproductive division of labor or monopolization of reproduction by one or few individuals is a core charac- teristic of social insect colonies. In the highly eusocial insects this is accompanied by the evolution of pheno- typic differences between the highly fertile queens and the functionally or factually sterile workers. As caste development, with rare exceptions, is not based on gen- etic differences between these morphs, the phenotypic differences between queens and workers have to be accomplished by flexible developmental programs. The observed flexibility in preimaginal development is a prime example of phenotypic plasticity (West-Eberhard, 1989) and has been explored in many of the highly social Hymenoptera. Most bumblebees have an annual cycle of colony * Corresponding author: Tel.: + 55-16-602-3077; fax: + 55-16-633- 6482. E-mail address: khartfel@rgm.fmrp.usp.br (K. Hartfelder). 0022-1910/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII:S0022-1910(00)00067-6 development. An overwintered, fertilized queen selects a suitable nest site and rears a first round of worker brood. These aid the queen in rearing several more brood cycles. In Bombus terrestris, queens start to lay haploid, male-producing eggs at some point in colony social development (switch point), and shortly thereafter some of the workers challenge the queen’s reproductive domi- nance (competition point) and start to lay haploid eggs themselves (Duchateau and Velthuis, 1988). This is also the time when new queens are reared from diploid eggs laid by the queen, and soon after, the colony begins to disintegrate. These virgin queens are considerably larger than their worker sisters but do not exhibit any further morphological differences. Physiologically, however, virgin queens are clearly distinct from workers, as they are attractive to males, and capable of overwintering and forming a new colony. There is a high correlation between the onset of the competition point and queen rearing, suggesting that social signals affect larval com- mitment to the queen pathway (Cnaani et al., 2000). The observed incipient caste differentiation in bumblebees is attractive for studying initial steps of