Cell Tissue Res (1993) 273:327-333 Cell Tissue Research 9 Springer-Verlag 1993 Diffuse serotoninergic neurohemal systems associated with cerebral and suboesophageal nerves in the head of the Colorado potato beetle Leptinotarsa decemlineata T. van Haeften 1,2. H. Schooneveld 2 1Department of Anatomy and Embryology, Faculty of Medicine, Vrije Universiteit, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands 2 Department of Entomology, Wageningen Agricultural University, Binnenhaven 7, NL-6709 PD Wageningen, The Netherlands Received: 9 November 1992/Accepted: 22 January 1993 Abstract. We analyzed the anatomy of two diffuse neuro- hemal systems for serotonin in the head of the Colorado potato beetle Leptinotarsa decemIineata by means of im- munohistochemistry. One system is formed by axons from two bilateral pairs of neurons in the frontal margin of the suboesophageal ganglion that enter the ipsilateral mandibular nerve, emerge from this nerve at some dis- tance from the suboesophageal ganglion, and cover all branches of the mandibular nerve with a dense plexus of immunoreactive axon swellings. The other system is formed by axons from two large neurons in the frontal ganglion that enter the ipsilateral frontal connectives, emerge from these connectives, and form a network of axon swellings on the labrofrontal, pharyngeal, and an- tennal nerves and on the surface of the frontal ganglion. Immunohistochemical electron microscopy demonstrat- ed that the axon swellings are located outside the neural sheaths of the nerves and hence in close contact with the hemolymph. We therefore suggest that these plexuses represent extensive neurohemal systems for serotonin. Most immunoreactive terminals are in direct contact with the hemolymph, and other terminals are closely as- sociated with the muscles of the mandibles, labrum, and anterior pharynx, as well as with the salivary glands, indi- cating that these organs are under serotoninergic control. Key words: Serotonin (5-HT) - Neurohemal systems - Immunohistochemistry - Electron microscopy - Target- ed release - Leptinotarsa decemlineata (Insecta) Introduction Serotonin (5-hydroxytryptamine) has been demon- strated biochemically and immunohistochemically in the nervous systems of many insect species (Nfissel 1988). This neurochemical is usually assumed to be a neu- * Present address and address for correspondence: Department of Anatomy and Embryology, Faculty of Medicine, Vrije Universiteit, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Nether- lands rotransmitter or neuromodulator in the central nervous system, but it also has a peripheral function, for instance on visceral muscles (Cook et al. 1969; Banner et al. 1987; T. van Haeften, H.M. Smid, H. Schooneveld, un- published observations), salivary glands (Trimmer 1985), Malpighian tubules (Morgan and Mordue 1984; Veen- stra 1988), and heart (Tublitz and Truman 1985). Some of these physiological effects can be explained only by assuming that serotonin is released into the hemolymph as a neurohormone. In the context of our studies on the control of feeding physiology of the Colorado potato beetle Leptinotarsa decemlineata, we are investigating the central and peripheral distributions of serotonin. We have shown that it is rather abundant in the ventral nerve cord (van Haeften and Schooneveld 1992) and in the brain (T. van Haeften, unpublished observations) and that it is carried to the foregut and hindgut and associated areas (T. van Haeften, H.M. Staid, H. Schooneveld, unpublished observations). Release of serotonin as a neurohormone into the he- molymph of insects typically occurs through specialized neurohemal organs, usually the corpora cardiaca, and in many species also through the perisympathetic organs (Raabe 1989). This is not the case, however, in L. decem- lineata. The corpora cardiaca could never be shown by immunohistochemical methods to contain serotonin, and no perisympathetic organs appear to be present (T. van Haeften, unpublished observations). Instead, a pilot study indicated that another release system for serotonin is in operation: a diffuse system of serotoninergic axons carried by several nerves in the head. We here report on immunohistochemical studies to describe the sources of serotonin-like immunoreactive (SLI) material in the major ganglia of the head and the spatial organization of peripheral axon branches. Structural evidence for neu- rohemal axon specializations was sought by electron mi- croscopy. Materials and methods Sexually mature male and female Colorado potato beetles (Leptino- tarsa decemlineata Say) from a laboratory culture (16-h photo-