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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Arthropod Structure & Development 37 (2008) 273e286 www.elsevier.com/locate/asd Distribution of corazonin and pigment-dispersing factor in the cephalic ganglia of termites Radka Za´vodska´ a,b, Chih-Jen Wen c, Ivan Hrdy´ d, Ivo Sauman b, How-Jing Lee c, Frantisˇek Sehnal b,* a Pedagogical Faculty, University of South Bohemia, Jerony´mova 10, 371 15 Ceske ´ Budeˇjovice, Czech Republic b Biology Centre AVCR, Institute of Entomology, Branisˇovska´ 31, 370 05 Ceske ´ Budeˇjovice, Czech Republic c Department of Entomology, National Taiwan University, Roosevelt Rd. 1, Sec. 4, Taipei 106, Taiwan d Institute of Organic Chemistry and Biochemistry AVCR, Flemingovo na´m. 2, 160 00 Praha, Czech Republic Received 9 May 2007; received in revised form 24 January 2008; accepted 24 January 2008 Abstract Distribution of neurones detectable with antisera to the corazonin (Crz) and the pigment-dispersing factor (PDF) was mapped in the workers or pseudergates of 10 species representing six out of seven termite families. All species contained two triads of Crz-immunoreactive (Crz-ir) neurones in the protocerebrum. Their fibres were linked to the opposite hemisphere, formed a network in the fronto-lateral protocerebrum, and projected to the corpora cardiaca (CC); in most species the fibres also supplied the deuto- and tritocerebrum and the frontal ganglion. Some species possessed additional Crz-ir perikarya in the protocerebrum and the suboesophageal ganglion (SOG). The PDF-ir somata were primarily located in the optic lobe (OL) and SOG. OL harboured a group (3 groups in Coptotermes)of2e6 PDF-ir cells with processes extend- ing to the medulla, connecting to the contralateral OL, forming 1e2 networks in the protocerebrum, and in most species running also to CC. Such a PDF-ir system associated with the OL was missing in Reticulitermes. Except for Mastotermes, the termites contained 1e2 PDF-ir cell pairs in the SOG and two species had additional perikarya in the protocerebrum. The results are consistent with the view of a monophyletic termite origin and demonstrate how the Crz-ir and PDF-ir systems diversified in the course of termite phylogeny. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Brain; Insect nervous system; Neuropeptides; Suboesophageal ganglion; Termite evolution 1. Introduction recently identified in a crustacean without any functional as- signment (Billimoria et al., 2005). In insects, Crz is involved The neuropeptide corazonin (Crz) (pGlueThrePheeGlne in the control of heartbeat (Sla´ma, 2004), pigmentation (Taw- TyreSereArgeGlyeTrpeThreAsn-amide) was first isolated fik et al., 1999), behaviour (Hua et al., 2000), and ecdysis as a cardioaccelerator in cockroaches (Veenstra, 1989) and (Truman, 2005). Crz functions as one of the early ecdysial sig- later found in a cricket, in Drosophila, and in two moth spe- nals may represent a link between hormonal regulations and cies (Veenstra, 1994; Hua et al., 2000; Hansen et al., 2001). the neuronal circadian clock (Kim et al., 2004). The clock, A [His7] variant was detected in three species of locusts and which orchestrates diurnal rhythms in various body functions, a stick insect (Veenstra, 1991; Predel et al., 1994; Tawfik rests on a central pacemaker that consists of a network of neu- et al., 1999), and [Thr4, His7]-Crz in the honeybee (Roller rones located mostly in the brain. Circadian cycles in the ex- et al., 2006; Verleyen et al., 2006). A Crz homologue was pression of specific ‘‘clock’’ proteins, which are driven by several interlocked transcriptional feedback loops, are trans- duced to the body periphery by little understood mechanisms * Coresponding author. Tel.: þ420 38 777 5220; fax: þ420 38 531 0354. that include neuropeptides (Taghert and Shafer, 2006). A link- E-mail address: [email protected] (F. Sehnal). age of Crz to the circadian clock is indicated in Lepidoptera by 1467-8039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2008.01.005 Author's personal copy 274 R. Za´vodska´ et al. / Arthropod Structure & Development 37 (2008) 273e286 Crz-immunoreactivity (Crz-ir) in neurones that express clock wing pads), nymphs (immature individuals possessing wing proteins (Wise et al., 2002; Qi-Miao et al., 2003). In cock- pads), workers, and soldiers (Roisin, 2000; Hanus and Sˇobot- roaches (Petri et al., 1995), locusts (Roller et al., 2003), and nı´k, 2004). Larvae can develop into any of the specialised crickets (Sehadova´ et al., 2007), Crz-ir fibres ramify in the castes (winged reproductives, wingless neotenic reproductives, accessory medulla of optic lobes in proximity to the cells soldiers, and workers), while the developmental pathways of expressing the clock protein period (Lupien et al., 2003). nymphs are restricted to the production of reproductives and The pigment dispersing hormone (AsneSereGlueLeue in some species also soldiers. Several termite families do not IleeAsneSereIleeLeueGlyeLeueProeLyseValeMeteAsne have a specialised worker caste but use larva-like pseudergates AspeAla-amide) was identified by Rao et al. (1985) as a reg- that undergo stationary moults and under certain conditions ulator of pigmentation in crustaceans. Related compounds can develop into reproductives or soldiers. The appearance with up to four amino acid replacements in positions 4, 8, and the function of the pseudergates and true workers are 14, and 15 were isolated from diverse insect species and usu- alike. Since these non-reproductive individuals (helpers) dom- ally referred to as pigment dispersing factor (PDF) (for refer- inate the colonies, they were chosen for our comparative study. ences see Hamasaka et al., 2005). A single species may contain several PDF isoforms (Honda et al., 2006). Termites 2. Materials and methods are likely to contain [Leu8, Leu15]-PDF that was found in two species from their sister group, the cockroaches (Mohrherr 2.1. Animals et al., 1991; Hamasaka et al., 2005), and in a cricket (Singar- avel et al., 2003). The distribution of PDF-ir neurones in the The termites (Isoptera) are categorised into seven families, brain of polyneopteran insects indicates that it is a component of which Serritermitidae include only a few species that were of the circadian clock (Homberg et al., 1991; Sehadova´ et al., not available for our study. The examined species, their affil- 2003). PDF injections into the brain of cockroaches (Petri and iations to families and subfamilies, and the occurrence of Stengl, 1997) or genetic suppression (Renn et al., 1999) and/or workers versus pseudergates in their colonies, are listed in ectopic PDF expression in Drosophila (Helfrich-Fo¨rster et al., Table 1. The specimens of Mastotermes darwiniensis, Neo- 2000) affected circadian behavioural rhythms of these insects. termes castaneus, Zootermopsis angusticollis, Prorhinotermes It has been proposed that PDF might be a component of the simplex, Reticulitermes flavipes, Coptotermes formosanus, clock pacemaker (Petri and Stengl, 1997) but it is more likely and Nasutitermes costalis came from laboratory stock colonies that it functions as an output signal from the clock (reviewed kept at 27 C in a room under dim light (8e10 Lux) with nat- by Taghert and Shafer, 2006). ural day/night cycles. Any manipulation with the colonies was The conservation of Crz and PDF molecules facilitated im- done during the photophase at artificial light of 36e40 Lux. munohistochemical mapping of antigens identical or closely Colonies were kept in glass containers supplied with regularly related to these neuropeptides in various insects. Crz-ir neuro- moistened fine sand and piled sticks (ca 5 mm thick) of spruce, nes in the lateral protocerebrum with fibres projecting into the pine or poplar wood. Except for the following three species, ipsilateral corpora cardiaca and into the ventral nerve cord the colonies had been reared under these conditions for were found in all examined insect orders except the beetles many years at sustained caste ratios. The colony of C. formo- (Roller et al., 2003). PDF-ir cells were also detected in all in- sanus was abnormal by containing an extremely high number vestigated species, typically as a group of neurones in the of soldiers. Two orphan colonies consisting of the workers, proximal frontoventral (Pfv) position of the optic lobe (Za´vod- nymphs, and soldiers of M. darwiniensis were separated ska´ et al., 2003). Most Polyneoptera harbour also one distal from a big and reproducing termite nest in the Institute for Ma- posteriodorsal (Dpd), and one distal posterioventral (Dpv) terial Testing in Berlin (Germany) and kept for 3 months in cluster of PDF-ir perikarya (Homberg et al., 1991; Sehadova´ our cultures before the workers were examined (all of them et al., 2003). were at least 3 months old). The culture of N. costalis was We used antibodies to Crz and PDF to investigate the dis- based on termites collected in Florida by Dr. Jan Krˇecek one tribution of the Crz-like and PDF-like antigens in the cephalic month before our study. ganglia of diverse termites. The aim of our work was to find Groups of at least 20 termites were selected for our inves- out how much are the positions of immunoreactive neurons tigations from each species. They were transferred to Petri conserved across this insect order. The history of termites dishes, which contained moist sand and thin (2 mm) wood goes back 300 million years when they apparently evolved chops.