The Defensive Secretion of Carabus lefebvrei Dejean 1826 Pupa (Coleoptera, Carabidae): Gland Ultrastructure and Chemical Identification ANITA GIGLIO, 1 * PIETRO BRANDMAYR, 1 RENATO DALPOZZO, 2 GIOVANNI SINDONA, 2 ANTONIO TAGARELLI, 2 FEDERICA TALARICO, 1 TULLIA ZETTO BRANDMAYR, 1 AND ENRICO A. FERRERO 3 1 Department of Ecology, University of Calabria, I-87036 Arcavacata di Rende (CS), Calabria, Italy 2 Department of Chemistry, University of Calabria , I-87036 Arcavacata di Rende (CS), Calabria, Italy 3 Department of Life Sciences, University of Trieste, I-34127 Trieste, Calabria, Italy KEY WORDS abdominal glands; ultrastructure; chemical defense; monoterpenes ABSTRACT This study documents the defensive function of flavored humor secreted by the ab- dominal glands of Carabus lefebvrei pupae. The morphology and the ultrastructure of these glands were described and the volatile compounds of glands secretion were identified by gas chromatogra- phy/mass spectrometry. The ultrastructure analysis shows an acinose complex formed by about 50 clusters. Each cluster has 20 glandular units and the unit—composed of one secretory and one canal cell lying along a duct—belongs to the class 3 cell type of Quennedey (1998). In the cyto- plasm, the secretory cell contains abundant rough endoplasmatic reticula, glycogen granules, numerous mitochondria, and many well-developed Golgi complexes producing electron-dense se- cretory granules. Mitochondria are large, elongated, and often adjoining electronlucent vesicles. The kind and the origin of secretory granules varying in size and density were discussed. The chemical analysis of the gland secretion revealed the presence of a mixture of low molecular weight terpenes, ketones, aldehydes, alcohols, esters, and carboxylic acids. Monoterpenes, especially linal- ool, were the major products. We supposed that ketones, aldehydes, alcohols, esters, and carboxylic acids have a deterrent function against the predators and monoterpenes provide a prophylaxis function against pathogens. Microsc. Res. Tech. 72:351–361, 2009. V V C 2008 Wiley-Liss, Inc. INTRODUCTION A wide variety of insect species have evolved chemi- cal defense mechanisms—involved in interspecific interactions—that contributed to their widespread suc- cess in ecosystems (Thiele, 1977). In general, insect de- fensive substances are relatively low in molecular weight and are highly volatile, such as alkanes, ter- penes, alcohols, ketones, esters, aldehydes, organic acids, phenols, and quinones. They are secreted by exo- crine glands as blends and have repellent, toxicant, or antimicrobic function. The products of exocrine glands are well known in Isoptera, Thysanoptera, Hymenop- tera, Hemiptera, Lepidoptera, and Coleoptera (Burse et al., 2007; Chapman, 1998; Dettner, 1987; Whitman et al., 1990). According to our current knowledge, the order Coleoptera shows the largest number of species with the greatest variety of defensive glands. Allo- mones are extensively investigated in coleopteran adult beetles, their larvae and eggs but no attention has been paid to the pupae (Chapman, 1998; Dettner, 1987). Defensive chemicals have a high adaptive value in the pupal stage because they do not show antipreda- tion strategies such as behavior or structural modifica- tions to protect themselves against natural enemies. The ground beetles (Coleoptera, Carabidae) make up the largest adephagan family with about 40,000 species and their distribution is cosmopolitan. They are well known as bioindicators and natural enemies of agricul- tural pests. Because of their bio-indication and econom- ical importance, the role of biotic and abiotic factors in carabid population dynamics has been studied exten- sively. Abiotic factors and predation represent the main causes of mortality for all life-cycle stages of cara- bid beetles; pathogens and parasites can be important for some developmental stages (Lo ¨vei and Sunderland, 1996). Therefore, in response to predation pressure they have evolved morphological, behavioral, and chemical mechanisms against predation. Carabid bee- tles produce a large variety of defensive allomones (Andrew et al., 2000; Moore, 1979). Although exocrine glands (pygidial glands) and their defensive secretion were well investigated in adults, no information is available about larvae and pupae which are the most vulnerable stages of life cycle. This study is focused on gland secretion at the pupal stage of carabid beetle Carabus lefebvrei Dejean, 1826. We chose this species because it is easy to rear under laboratory conditions and easy to manipulate during experimental protocols. C. lefebvrei is a snail eating predatory Italian endemic species that lives in the beech, oak, chestnut, and pine forests of the Central and Southern Apennines, from lower altitudes to about *Correspondence to: Anita Giglio, Department of Ecology, University of Cala- bria, via P. Bucci cubo 4B, I-87036 Arcavacata di Rende (CS), Calabria, Italy. E-mail: anita.giglio@unical.it Received 10 July 2008; accepted in revised form 18 October 2008 DOI 10.1002/jemt.20660 Published online 9 December 2008 in Wiley InterScience (www.interscience. wiley.com). V V C 2008 WILEY-LISS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 72:351–361 (2009)