Cell Tissue Res (1994) 278:145-151 Cell&Tissue Research 9 Springer-Verlag 1994 Immunogold labelling of serotonin-like and FMRFamide-like immunoreaetive material on abdominal nerves of Rhodnius prolixus Sharon Miksys, Ian Orchard Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 1A1 Received: 8 December 1993 / Accepted: 1 March 1994 in neurohaemal areas Abstract. The ultrastructure of neurohaemal areas on ab- dominal nerves of the blood-sucking bug Rhodnius pro- lixus was investigated. Four types of axon terminals were found, distinguished by the morphology of their neu- rosecretory granules. By use of post-embedding im- munogold labelling, granules in Type I axon terminals were shown to contain serotonin-like immunoreactive material, and granules in Type II axon terminals were shown to contain FMRFamide-like immunoreactive ma- terial. There was no colocalization of these materials. It is suggested that Type III terminals contain peptidergic di- uretic hormone, which has previously been reported to be present in electron-dense neurosecretory granules in this neurohaemal area. The identity of material in Type IV terminals is unknown. Key words: Abdominal nerve neurohaemal area FMRFamide - Immunogold-labelling - Serotonin - U1- trastructure - Rhodnius prolixus (Insecta) Introduction With the wide availability of antisera specific for neuro- physiologically active antigens, there is now a wealth of information on the distribution of such materials throughout the nervous systems of insects (e.g., N/issel 1993). This information is based mainly on studies at the level of the light microscope, with relatively few reports on the ultrastructural immunocytochemical localization of neurosecretory antigens. The use of colloidal gold as an immunolabel allows both good ultrastructural resolu- tion, and subcellular localization of the antigen, and as such is a useful tool to correlate data from light-micro- scopic immunocytochemistry with the fine structure of neurones, their axons and axon terminals, both neuro- haemal and synaptic. Correspondence to: S. Miksys In the blood-sucking bug Rhodnius prolixus the mesothoracic ganglionic mass (MTGM) gives rise to sev- eral peripheral nerves including five pairs of abdominal nerves. In whole-mount preparations these nerves appear to be covered by an elaborate neurohaemal plexus, which shows both serotonin-like (Lange et al. 1988) and FMRFamide-like immunoreactivity (Tsang and Orchard 1991). Serotonin is thought to co-ordinate feeding-relat- ed functions in Rhodnius prolixus (Lange et al. 1989; Cook and Orchard 1990; Barrett et al. 1993), acting in part as a neurohormone, released into the haemolymph from the abdominal nerves. FMRFamide-related pep- tides (FaRPs) are also thought to be involved in feeding in this bug (Tsang and Orchard t991), and may be neuro- hormones, with the abdominal nerves as a possible re- lease site (Elia et al. 1993). A third neurohormone, a pep- tidergic diuretic hormone, is thought to be released from the same site during feeding (Maddrell et al. 1991). There have been numerous reports of colocalization of more than one physiologically active neurosecretory product in single neurones in both vertebrates and invertebrates (e.g., Scheller et al. 1984; H6kfelt et al. 1986; Homberg et al. 1990). In some cases neuropeptides are Colocalized with more conventional neurotransmitters, in others with peptides or amines. In Rhodnius prolixus there are five serotoninergic dorsal unpaired median (DUM) neurones which release serotonin from their axon endings on the five pairs of abdominal nerves (Orchard et al. 1989). The cellular source of FMRFamide-like material released in the same area is unknown. One of the purposes of the present study, in addition to providing a detailed descrip- tion of the fine structure of the abdominal nerve neuro- haemal plexus, was to clarify whether serotonin- and FMRFamide-like immunoreactive materials are colocal- ized in the same axons, by use of immunogold labelling. Materials and methods Unfed filth instar male and female Rhodniusprolixus, from an estab- lished colony at the University of Toronto, were used between 10 and 20 days following ecdysis.